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Sample records for monoterpene biosynthetic enzymes

  1. Domain swapping of Citrus limon monoterpene synthases: impact on enzymatic activity and product specifity.

    NARCIS (Netherlands)

    Tamer, el M.K.; Lucker, J.; Bosch, D.; Verhoeven, H.A.; Verstappen, F.W.A.; Schwab, W.; Tunen, van A.J.; Voragen, A.G.J.; Maagd, de R.A.; Bouwmeester, H.J.

    2003-01-01

    Monoterpene cyclases are the key enzymes in the monoterpene biosynthetic pathway, as they catalyze the cyclization of the ubiquitous geranyl diphosphate (GDP) to the specific monoterpene skeletons. From Citrus limon, four monoterpene synthase-encoding cDNAs for a P-pinene synthase named

  2. Domain swapping of Citrus limon monoterpene synthases: impact on enzymatic activity and product specifity.

    NARCIS (Netherlands)

    Tamer, el M.K.; Lucker, J.; Bosch, D.; Verhoeven, H.A.; Verstappen, F.W.A.; Schwab, W.; Tunen, van A.J.; Voragen, A.G.J.; Maagd, de R.A.; Bouwmeester, H.J.

    2003-01-01

    Monoterpene cyclases are the key enzymes in the monoterpene biosynthetic pathway, as they catalyze the cyclization of the ubiquitous geranyl diphosphate (GDP) to the specific monoterpene skeletons. From Citrus limon, four monoterpene synthase-encoding cDNAs for a P-pinene synthase named Cl(-)betaPIN

  3. Endoplasmic reticulum localization and activity of maize auxin biosynthetic enzymes.

    Science.gov (United States)

    Kriechbaumer, Verena; Seo, Hyesu; Park, Woong June; Hawes, Chris

    2015-09-01

    Auxin is a major growth hormone in plants and the first plant hormone to be discovered and studied. Active research over >60 years has shed light on many of the molecular mechanisms of its action including transport, perception, signal transduction, and a variety of biosynthetic pathways in various species, tissues, and developmental stages. The complexity and redundancy of the auxin biosynthetic network and enzymes involved raises the question of how such a system, producing such a potent agent as auxin, can be appropriately controlled at all. Here it is shown that maize auxin biosynthesis takes place in microsomal as well as cytosolic cellular fractions from maize seedlings. Most interestingly, a set of enzymes shown to be involved in auxin biosynthesis via their activity and/or mutant phenotypes and catalysing adjacent steps in YUCCA-dependent biosynthesis are localized to the endoplasmic reticulum (ER). Positioning of auxin biosynthetic enzymes at the ER could be necessary to bring auxin biosynthesis in closer proximity to ER-localized factors for transport, conjugation, and signalling, and allow for an additional level of regulation by subcellular compartmentation of auxin action. Furthermore, it might provide a link to ethylene action and be a factor in hormonal cross-talk as all five ethylene receptors are ER localized.

  4. Redox Impact on Starch Biosynthetic Enzymes in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Skryhan, Katsiaryna

    Summary The thesis provides new insight into the influence of the plant cell redox state on the transient starch metabolism in Arabidopsis thaliana with a focus on starch biosynthetic enzymes. Two main hypotheses forms the basis of this thesis: 1) photosynthesis and starch metabolism are coordina......Summary The thesis provides new insight into the influence of the plant cell redox state on the transient starch metabolism in Arabidopsis thaliana with a focus on starch biosynthetic enzymes. Two main hypotheses forms the basis of this thesis: 1) photosynthesis and starch metabolism...... are coordinated by the redox state of the cell via post-translational modification of the starch metabolic enzymes containing redox active cysteine residues and these cysteine residues became cross-linked upon oxidation providing a conformational change leading to activity loss; 2) cysteine residues...... of chloroplast enzymes can play a role not only in enzyme activity and redox sensitivity but also in protein folding and stability upon oxidation. Several redox sensitive enzymes identified in this study can serve as potential targets to control the carbon flux to and from starch during the day and night...

  5. Redox Impact on Starch Biosynthetic Enzymes in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Skryhan, Katsiaryna

    Summary The thesis provides new insight into the influence of the plant cell redox state on the transient starch metabolism in Arabidopsis thaliana with a focus on starch biosynthetic enzymes. Two main hypotheses forms the basis of this thesis: 1) photosynthesis and starch metabolism...... are coordinated by the redox state of the cell via post-translational modification of the starch metabolic enzymes containing redox active cysteine residues and these cysteine residues became cross-linked upon oxidation providing a conformational change leading to activity loss; 2) cysteine residues...... of chloroplast enzymes can play a role not only in enzyme activity and redox sensitivity but also in protein folding and stability upon oxidation. Several redox sensitive enzymes identified in this study can serve as potential targets to control the carbon flux to and from starch during the day and night...

  6. Effect of photoperiod on gibberellin biosynthetic enzymes in spinach

    Energy Technology Data Exchange (ETDEWEB)

    Gilmour, S.J.; Bleecker, A.B.; Zeevaart, J.A.D.

    1986-04-01

    The photoperiodic control of stem elongation in spinach, a long day (LD) rosette plant, is mediated by gibberellins (GAs). The early 13-hydroxylated GA biosynthetic pathway from GA/sub 12/ to GA/sub 20/ operates in spinach: GA/sub 12/ ..-->.. GA/sub 53/ ..-->.. GA/sub 44/ ..-->.. GA/sub 19/ ..-->.. GA/sub 20/. Two enzymes of this pathway, those converting GA/sub 53/ to GA/sub 44/ (GA/sub 53/ oxidase) and GA/sub 19/ to GA/sub 20/ (GA/sub 19/ oxidase), are regulated by light. The enzyme converting GA/sub 44/ to GA/sub 19/ (GA/sub 44/ oxidase) is not light-regulated. In the light GA/sub 53/ and GA/sub 18/ oxidase activities are increased, therefore causing the GA biosynthetic pathway to be turned on. This leads to the production of an active GA in LD, which causes an increase in stem elongation. Two the enzymes, GA/sub 44/ and GA/sub 53/ oxidases, can be separated from one another by anion exchange HPLC. Estimates of the molecular weights of these two enzymes based on gel filtration HPLC will be reported.

  7. Signal transduction and metabolic flux of beta-thujaplicin and monoterpene biosynthesis in elicited Cupressus lusitanica cell cultures.

    Science.gov (United States)

    Zhao, Jian; Matsunaga, Yoko; Fujita, Koki; Sakai, Kokki

    2006-01-01

    beta-Thujaplicin is an antimicrobial tropolone derived from geranyl pyrophosphate(GPP) and monoterpene intermediate. Yeast elicitor-treated Cupressus lusitanica cell cultures accumulate high levels of beta-thujaplicin at early stages and other monoterpenes at later stages post-elicitation. The different regulation of beta-thujaplicin and monoterpene biosynthesis and signal transduction directing metabolic flux to beta-thujaplicin firstly and then shifting metabolic flow from beta-thujaplicin to other monoterpene biosynthesis were investigated. The earlier rapid induction of beta-thujaplicin accumulation and a later stimulation of monoterpene biosynthesis by yeast elicitor are in well agreement with elicitor-induced changes in activity of three monoterpene biosynthetic enzymes including isopentenyl pyrophosphate isomerase, GPP synthase, and monoterpene synthase. Yeast elicitor induces an earlier and stronger beta-thujaplicin production and monoterpene biosynthetic enzyme activity than methyl jasmonate (MeJA) does. Profiling all monoterpenes produced by C. lusitanica cell cultures under different conditions reveals that beta-thujaplicin biosynthesis parallels with other monoterpenes and competes for common precursor pools. Yet beta-thujaplicin is produced pre-dominantly at early stage of elicitation whereas other monoterpenes are mainly accumulated at late stage while beta-thujaplicin is metabolized. It is suggested that yeast elicitor-treated C. lusitanica cells preferentially accumulate beta-thujaplicin as a primary defense and other monoterpenes as a secondary defense. Inhibitor treatments suggest that immediate production of beta-thujaplicin post-elicitation largely depends on pre-existing enzymes and translation of pre-existing transcripts as well as recruitment of precursor pools from both the cytosol and plastids. The later beta-thujaplicin and other monoterpene accumulation strictly depends on active transcription and translation. Induction of beta

  8. Nanolipoprotein particles comprising a natural rubber biosynthetic enzyme complex and related products, methods and systems

    Energy Technology Data Exchange (ETDEWEB)

    Hoeprich, Paul D.; Whalen, Maureen

    2016-04-05

    Provided herein are nanolipoprotein particles that comprise a biosynthetic enzyme more particularly an enzyme capable of catalyzing rubber or other rubbers polymerization, and related assemblies, devices, methods and systems.

  9. Metabolism of monoterpenes in cell cultures of common sage (Salvia officinalis)

    Energy Technology Data Exchange (ETDEWEB)

    Falk, K.L.; Gershenzon, J.; Croteau, R. (Washington State Univ., Pullman (USA))

    1990-08-01

    Leaves of common sage (Salvia officinalis) accumulate monoterpenes in glandular trichomes at levels exceeding 15 milligrams per gram fresh weight at maturity, whereas sage cells in suspension culture did not accumulate detectable levels of monoterpenes (<0.3 nanograms per gram fresh weight) at any stage of the growth cycle, even in the presence of a polystyrene resin trap. Monoterpene biosynthesis from (U-{sup 14}C)sucrose was also virtually undetectable in this cell culture system. In vitro assay of each of the enzymes required for the sequential conversion of the ubiquitous isoprenoid precursor geranyl pyrophosphate to (+){minus}camphor (a major monoterpene product of sage) in soluble extracts of the cells revealed the presence of activity sufficient to produce (+){minus}camphor at a readily detectable level (>0.3 micrograms per gram fresh weight) at the late log phase of growth. Other monoterpene synthetic enzymes were present as well. In vivo measurement of the ability to catabolize (+){minus}camphor in these cells indicated that degradative capability exceeded biosynthetic capacity by at least 1,000-fold. Therefore, the lack of monoterpene accumulation in undifferentiated sage cultures could be attributed to a low level of biosynthetic activity (relative to the intact plant) coupled to a pronounced capacity for monoterpene catabolism.

  10. Characterization of the promoter region of biosynthetic enzyme genes involved in berberine biosynthesis in Coptis japonica

    Directory of Open Access Journals (Sweden)

    Yasuyuki Yamada

    2016-09-01

    Full Text Available The presence of alkaloids is rather specific to certain plant species. However, berberine, an isoquinoline alkaloid, is relatively broadly distributed in the plant kingdom. Thus, berberine biosynthesis has been intensively investigated, especially using Coptis japonica cell cultures. Almost all biosynthetic enzyme genes have already been characterized at the molecular level. Particularly, two transcription factors (TFs, a plant-specific WRKY-type transcription factor, CjWRKY1, and a basic helix-loop-helix (bHLH transcription factor, CjbHLH1, were shown to comprehensively regulate berberine biosynthesis in C. japonica cells. In this study, we characterized the promoter region of some biosynthetic enzyme genes and associated cis-acting elements involved in the transcriptional regulation via two TFs. The promoter regions of three berberine biosynthetic enzyme genes (CYP80B2, 4’OMT and CYP719A1 were isolated, and their promoter activities were dissected by a transient assay involving the sequentially truncated promoter::luciferase (LUC reporter constructs. Furthermore, transactivation activities of CjWRKY1 were determined using the truncated promoter::LUC reporter constructs or constructs with mutated cis-elements. These results suggest the involvement of a putative W-box in the regulation of biosynthetic enzyme genes. Direct binding of CjWRKY1 to the W-box DNA sequence was also confirmed by an electrophoresis mobility shift assay (EMSA and by a chromatin immunoprecipitation (ChIP assay. In addition, CjbHLH1 also activated transcription from truncated 4’OMT and CYP719A1 promoters independently of CjWRKY1, suggesting the involvement of a putative E-box. Unexpected transcriptional activation of biosynthetic enzyme genes via a non-W-box sequence and by CjWRKY1 as well as the possible involvement of a GCC-box in berberine biosynthesis in C. japonica are discussed.

  11. Inhibitors of Testosterone Biosynthetic and Metabolic Activation Enzymes

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    Leping Ye

    2011-12-01

    Full Text Available The Leydig cells of the testis have the capacity to biosynthesize testosterone from cholesterol. Testosterone and its metabolically activated product dihydrotestosterone are critical for the development of male reproductive system and spermatogenesis. At least four steroidogenic enzymes are involved in testosterone biosynthesis: Cholesterol side chain cleavage enzyme (CYP11A1 for the conversion of cholesterol into pregnenolone within the mitochondria, 3β-hydroxysteroid dehydrogenase (HSD3B, for the conversion of pregnenolone into progesterone, 17α-hydroxylase/17,20-lyase (CYP17A1 for the conversion of progesterone into androstenedione and 17β-hydroxysteroid dehydrogenase (HSD17B3 for the formation of testosterone from androstenedione. Testosterone is also metabolically activated into more potent androgen dihydrotestosterone by two isoforms 5α-reductase 1 (SRD5A1 and 2 (SRD5A2 in Leydig cells and peripheral tissues. Many endocrine disruptors act as antiandrogens via directly inhibiting one or more enzymes for testosterone biosynthesis and metabolic activation. These chemicals include industrial materials (perfluoroalkyl compounds, phthalates, bisphenol A and benzophenone and pesticides/biocides (methoxychlor, organotins, 1,2-dibromo-3-chloropropane and prochloraz and plant constituents (genistein and gossypol. This paper reviews these endocrine disruptors targeting steroidogenic enzymes.

  12. Designing universal primers for the isolation of DNA sequences encoding Proanthocyanidins biosynthetic enzymes in Crataegus aronia.

    Science.gov (United States)

    Zuiter, Afnan Saeid; Sawwan, Jammal; Al Abdallat, Ayed

    2012-08-10

    Hawthorn is the common name of all plant species in the genus Crataegus, which belongs to the Rosaceae family. Crataegus are considered useful medicinal plants because of their high content of proanthocyanidins (PAs) and other related compounds. To improve PAs production in Crataegus tissues, the sequences of genes encoding PAs biosynthetic enzymes are required. Different bioinformatics tools, including BLAST, multiple sequence alignment and alignment PCR analysis were used to design primers suitable for the amplification of DNA fragments from 10 candidate genes encoding enzymes involved in PAs biosynthesis in C. aronia. DNA sequencing results proved the utility of the designed primers. The primers were used successfully to amplify DNA fragments of different PAs biosynthesis genes in different Rosaceae plants. To the best of our knowledge, this is the first use of the alignment PCR approach to isolate DNA sequences encoding PAs biosynthetic enzymes in Rosaceae plants.

  13. Designing universal primers for the isolation of DNA sequences encoding Proanthocyanidins biosynthetic enzymes in Crataegus aronia

    Directory of Open Access Journals (Sweden)

    Zuiter Afnan

    2012-08-01

    Full Text Available Abstract Background Hawthorn is the common name of all plant species in the genus Crataegus, which belongs to the Rosaceae family. Crataegus are considered useful medicinal plants because of their high content of proanthocyanidins (PAs and other related compounds. To improve PAs production in Crataegus tissues, the sequences of genes encoding PAs biosynthetic enzymes are required. Findings Different bioinformatics tools, including BLAST, multiple sequence alignment and alignment PCR analysis were used to design primers suitable for the amplification of DNA fragments from 10 candidate genes encoding enzymes involved in PAs biosynthesis in C. aronia. DNA sequencing results proved the utility of the designed primers. The primers were used successfully to amplify DNA fragments of different PAs biosynthesis genes in different Rosaceae plants. Conclusion To the best of our knowledge, this is the first use of the alignment PCR approach to isolate DNA sequences encoding PAs biosynthetic enzymes in Rosaceae plants.

  14. Producing the Ethylene Signal: Regulation and Diversification of Ethylene Biosynthetic Enzymes.

    Science.gov (United States)

    Booker, Matthew A; DeLong, Alison

    2015-09-01

    Strictly controlled production of ethylene gas lies upstream of the signaling activities of this crucial regulator throughout the plant life cycle. Although the biosynthetic pathway is enzymatically simple, the regulatory circuits that modulate signal production are fine tuned to allow integration of responses to environmental and intrinsic cues. Recently identified posttranslational mechanisms that control ethylene production converge on one family of biosynthetic enzymes and overlay several independent reversible phosphorylation events and distinct mediators of ubiquitin-dependent protein degradation. Although the core pathway is conserved throughout seed plants, these posttranslational regulatory mechanisms may represent evolutionarily recent innovations. The evolutionary origins of the pathway and its regulators are not yet clear; outside the seed plants, numerous biochemical and phylogenetic questions remain to be addressed.

  15. exo-Brevicomin biosynthetic pathway enzymes from the Mountain Pine Beetle, Dendroctonus ponderosae.

    Science.gov (United States)

    Song, Minmin; Delaplain, Patrick; Nguyen, Trang T; Liu, Xibei; Wickenberg, Leah; Jeffrey, Christopher; Blomquist, Gary J; Tittiger, Claus

    2014-10-01

    exoBrevicomin (exo-7-ethyl-5-methyl-6,8-dioxabicyclo[3.2.1]octane) is an important semiochemical for a number of beetle species, including the highly destructive Mountain Pine Beetle (Dendroctonus ponderosae). It is also found in other insects and the African elephant. Despite its significance, very little is known about its biosynthesis. A recent microarray analysis implicated a small cluster of three D. ponderosae genes in exo-brevicomin biosynthesis, two of which had identifiable open reading frames (Aw et al., 2010; BMC Genomics 11:215). Here we report further expression profiling of two genes in that cluster and functional analysis of their recombinantly-produced enzymes. One encodes a short-chain dehydrogenase that used NAD(P)(+) as a co-factor to catalyze the oxidation of (Z)-6-nonen-2-ol to (Z)-6-nonen-2-one. We therefore named the enzyme (Z)-6-nonen-2-ol dehydrogenase (ZnoDH). The other encodes the cytochrome P450, CYP6CR1, which epoxidized (Z)-6-nonen-2-one to 6,7-epoxynonan-2-one with very high specificity and substrate selectivity. Both the substrates and products of the two enzymes are intermediates in the exo-brevicomin biosynthetic pathway. Thus, ZnoDH and CYP6CR1 are enzymes that apparently catalyze the antepenultimate and penultimate steps in the exo-brevicomin biosynthetic pathway, respectively. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Treadmill exercise does not change gene expression of adrenal catecholamine biosynthetic enzymes in chronically stressed rats

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    LJUBICA GAVRILOVIC

    2013-09-01

    Full Text Available ABSTRACT Chronic isolation of adult animals represents a form of psychological stress that produces sympatho-adrenomedullar activation. Exercise training acts as an important modulator of sympatho-adrenomedullary system. This study aimed to investigate physical exercise-related changes in gene expression of catecholamine biosynthetic enzymes (tyrosine hydroxylase, dopamine-ß-hydroxylase and phenylethanolamine N-methyltransferase and cyclic adenosine monophosphate response element-binding (CREB in the adrenal medulla, concentrations of catecholamines and corticosterone (CORT in the plasma and the weight of adrenal glands of chronically psychosocially stressed adult rats exposed daily to 20 min treadmill running for 12 weeks. Also, we examined how additional acute immobilization stress changes the mentioned parameters. Treadmill running did not result in modulation of gene expression of catecholamine synthesizing enzymes and it decreased the level of CREB mRNA in the adrenal medulla of chronically psychosocially stressed adult rats. The potentially negative physiological adaptations after treadmill running were recorded as increased concentrations of catecholamines and decreased morning CORT concentration in the plasma, as well as the adrenal gland hypertrophy of chronically psychosocially stressed rats. The additional acute immobilization stress increases gene expression of catecholamine biosynthetic enzymes in the adrenal medulla, as well as catecholamines and CORT levels in the plasma. Treadmill exercise does not change the activity of sympatho-adrenomedullary system of chronically psychosocially stressed rats.

  17. Metabolism of Monoterpenes: Acetylation of (-)-Menthol by a Soluble Enzyme Preparation from Peppermint (Mentha piperita) Leaves.

    Science.gov (United States)

    Croteau, R; Hooper, C L

    1978-05-01

    The essential oil from mature leaves of flowering peppermint (Mentha piperita L.) contains up to 15% (-)-menthyl acetate, and leaf discs converted exogenous (-)-[G-(3)H]menthol into this ester in approximately 15% yield of the incorporated precursor. Leaf extracts catalyzed the acetyl coenzyme A-dependent acetylation of (-)-[G-(3)H]menthol and the product of this transacetylase reaction was identified by radiochromatographic techniques. Transacetylase activity was located mainly in the 100,000g supernatant fraction, and the preparation was partially purified by combination of Sephadex G-100 gel filtration and chromatography on O-diethylaminoethyl-cellulose. The transacetylase had a molecular weight of about 37,000 as judged by Sephadex G-150 gel filtration, and a pH optimum near 9. The apparent K(m) and velocity for (-)-menthol were 0.3 mm and 16 nmol/hr. mg of protein, respectively. The saturation curve for acetyl coenzyme A was sigmoidal, showing apparent saturation near 0.1 mm. Dithioerythritol was required for maximum activity and stability of the enzyme, and the enzyme was inhibited by thiol directed reagents such as p-hydroxymercuribenzoate. Diisopropylfluorophosphate also inhibited transacylation suggesting the involvement of a serine residue in catalysis. The transacylase was highly specific for acetyl coenzyme A; propionyl coenzyme A and butyryl coenzyme A were not nearly as efficient as acyl donors (11% and 2%, respectively). However, the enzyme was much less selective with regard to the alcohol substrate, suggesting that the nature of the acetate ester synthesized in mint is more dependent on the type of alcohol available than on the specificity of the transacetylase. This is the first report on an enzyme involved in monoterpenol acetylation in plants. A very similar enzyme, catalyzing this key reaction in the metabolism of menthol, was also isolated from the flowers of peppermint.

  18. Limonene Synthase, the Enzyme Responsible for Monoterpene Biosynthesis in Peppermint, Is Localized to Leucoplasts of Oil Gland Secretory Cells

    National Research Council Canada - National Science Library

    Glenn Turner; Jonathan Gershenzon; Erik E. Nielson; John E. Froehlich; Rodney Croteau

    1999-01-01

    ...)-Limonene synthase, which is responsible for the first dedicated step of monoterpene biosynthesis in mint species, appears to be translated as a preprotein bearing a long plastidial transit peptide...

  19. A novel approach for the characterisation of proteoglycans and biosynthetic enzymes in a snail model.

    Science.gov (United States)

    Gesteira, Tarsis F; Coulson-Thomas, Vivien Jane; Ogata, Fernando T; Farias, Eduardo H C; Cavalheiro, Renan P; de Lima, Marcelo A; Cunha, Gabriel L A; Nakayasu, Ernesto S; Almeida, Igor C; Toma, Leny; Nader, Helena B

    2011-12-01

    Proteoglycans encompass a heterogeneous group of glycoconjugates where proteins are substituted with linear, highly negatively charged glycosaminoglycan chains. Sulphated glycosaminoglycans are ubiquitous to the animal kingdom of the Eukarya domain. Information on the distribution and characterisation of proteoglycans in invertebrate tissues is limited and restricted to a few species. By the use of multidimensional protein identification technology and immunohistochemistry, this study shows for the first time the presence and tissue localisation of different proteoglycans, such as perlecan, aggrecan, and heparan sulphate proteoglycan, amongst others, in organs of the gastropoda Achatina fulica. Through a proteomic analysis of Golgi proteins and immunohistochemistry of tissue sections, we detected the machinery involved in glycosaminoglycan biosynthesis, related to polymer formation (polymerases), as well as secondary modifications (sulphation and uronic acid epimerization). Therefore, this work not only identifies both the proteoglycan core proteins and glycosaminoglycan biosynthetic enzymes in invertebrates but also provides a novel method for the study of glycosaminoglycan and proteoglycan evolution.

  20. Functional characterization of the vitamin K2 biosynthetic enzyme UBIAD1.

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    Yoshihisa Hirota

    Full Text Available UbiA prenyltransferase domain-containing protein 1 (UBIAD1 plays a significant role in vitamin K2 (MK-4 synthesis. We investigated the enzymological properties of UBIAD1 using microsomal fractions from Sf9 cells expressing UBIAD1 by analysing MK-4 biosynthetic activity. With regard to UBIAD1 enzyme reaction conditions, highest MK-4 synthetic activity was demonstrated under basic conditions at a pH between 8.5 and 9.0, with a DTT ≥0.1 mM. In addition, we found that geranyl pyrophosphate and farnesyl pyrophosphate were also recognized as a side-chain source and served as a substrate for prenylation. Furthermore, lipophilic statins were found to directly inhibit the enzymatic activity of UBIAD1. We analysed the aminoacid sequences homologies across the menA and UbiA families to identify conserved structural features of UBIAD1 proteins and focused on four highly conserved domains. We prepared protein mutants deficient in the four conserved domains to evaluate enzyme activity. Because no enzyme activity was detected in the mutants deficient in the UBIAD1 conserved domains, these four domains were considered to play an essential role in enzymatic activity. We also measured enzyme activities using point mutants of the highly conserved aminoacids in these domains to elucidate their respective functions. We found that the conserved domain I is a substrate recognition site that undergoes a structural change after substrate binding. The conserved domain II is a redox domain site containing a CxxC motif. The conserved domain III is a hinge region important as a catalytic site for the UBIAD1 enzyme. The conserved domain IV is a binding site for Mg2+/isoprenyl side-chain. In this study, we provide a molecular mapping of the enzymological properties of UBIAD1.

  1. Structural basis for acyl acceptor specificity in the achromobactin biosynthetic enzyme AcsD.

    Science.gov (United States)

    Schmelz, Stefan; Botting, Catherine H; Song, Lijiang; Kadi, Nadia F; Challis, Gregory L; Naismith, James H

    2011-09-23

    Siderophores are known virulence factors, and their biosynthesis is a target for new antibacterial agents. A non-ribosomal peptide synthetase-independent siderophore biosynthetic pathway in Dickeya dadantii is responsible for production of the siderophore achromobactin. The D. dadantii achromobactin biosynthesis protein D (AcsD) enzyme has been shown to enantioselectively esterify citric acid with l-serine in the first committed step of achromobactin biosynthesis. The reaction occurs in two steps: stereospecific activation of citric acid by adenylation, followed by attack of the enzyme-bound citryl adenylate by l-serine to produce the homochiral ester. We now report a detailed characterization of the substrate profile and mechanism of the second (acyl transfer) step of AcsD enzyme. We demonstrate that the enzyme catalyzes formation of not only esters but also amides from the citryl-adenylate intermediate. We have rationalized the substrate utilization profile for the acylation reaction by determining the first X-ray crystal structure of a product complex for this enzyme class. We have identified the residues that are important for both recognition of l-serine and catalysis of ester formation. Our hypotheses were tested by biochemical analysis of various mutants, one of which shows a reversal of specificity from the wild type with respect to non-natural substrates. This change can be rationalized on the basis of our structural data. That this change in specificity is accompanied by no loss in activity suggests that AcsD and other members of the non-ribosomal peptide synthetase-independent siderophore superfamily may have biotransformation potential.

  2. Structural Basis for Acyl Acceptor Specificity in the Achromobactin Biosynthetic Enzyme AcsD

    Science.gov (United States)

    Schmelz, Stefan; Botting, Catherine H.; Song, Lijiang; Kadi, Nadia F.; Challis, Gregory L.; Naismith, James H.

    2011-01-01

    Siderophores are known virulence factors, and their biosynthesis is a target for new antibacterial agents. A non-ribosomal peptide synthetase-independent siderophore biosynthetic pathway in Dickeya dadantii is responsible for production of the siderophore achromobactin. The D. dadantii achromobactin biosynthesis protein D (AcsD) enzyme has been shown to enantioselectively esterify citric acid with l-serine in the first committed step of achromobactin biosynthesis. The reaction occurs in two steps: stereospecific activation of citric acid by adenylation, followed by attack of the enzyme-bound citryl adenylate by l-serine to produce the homochiral ester. We now report a detailed characterization of the substrate profile and mechanism of the second (acyl transfer) step of AcsD enzyme. We demonstrate that the enzyme catalyzes formation of not only esters but also amides from the citryl-adenylate intermediate. We have rationalized the substrate utilization profile for the acylation reaction by determining the first X-ray crystal structure of a product complex for this enzyme class. We have identified the residues that are important for both recognition of l-serine and catalysis of ester formation. Our hypotheses were tested by biochemical analysis of various mutants, one of which shows a reversal of specificity from the wild type with respect to non-natural substrates. This change can be rationalized on the basis of our structural data. That this change in specificity is accompanied by no loss in activity suggests that AcsD and other members of the non-ribosomal peptide synthetase-independent siderophore superfamily may have biotransformation potential. PMID:21835184

  3. Isobutanol production in engineered Saccharomyces cerevisiae by overexpression of 2-ketoisovalerate decarboxylase and valine biosynthetic enzymes.

    Science.gov (United States)

    Lee, Won-Heong; Seo, Seung-Oh; Bae, Yi-Hyun; Nan, Hong; Jin, Yong-Su; Seo, Jin-Ho

    2012-11-01

    Engineering of Saccharomyces cerevisiae to produce advanced biofuels such as isobutanol has received much attention because this yeast has a natural capacity to produce higher alcohols. In this study, construction of isobutanol production systems was attempted by overexpression of effective 2-keto acid decarboxylase (KDC) and combinatorial overexpression of valine biosynthetic enzymes in S. cerevisiae D452-2. Among the six putative KDC enzymes from various microorganisms, 2-ketoisovalerate decarboxylase (Kivd) from L. lactis subsp. lactis KACC 13877 was identified as the most suitable KDC for isobutanol production in the yeast. Isobutanol production by the engineered S. cerevisiae was assessed in micro-aerobic batch fermentations using glucose as a sole carbon source. 93 mg/L isobutanol was produced in the Kivd overexpressing strain, which corresponds to a fourfold improvement as compared with the control strain. Isobutanol production was further enhanced to 151 mg/L by additional overexpression of acetolactate synthase (Ilv2p), acetohydroxyacid reductoisomerase (Ilv5p), and dihydroxyacid dehydratase (Ilv3p) in the cytosol.

  4. Structure, function and regulation of the enzymes in the starch biosynthetic pathway.

    Energy Technology Data Exchange (ETDEWEB)

    Geiger, Jim

    2013-11-30

    structure of ADP- Glucose pyrophosphorylase from potato in its inhibited conformation, and bound to both ATP and ADP-glucose. In addition, we have determined the first structure of glycogen synthase in its "closed", catalytically active conformation bound to ADP-glucose. We also determined the structure of glycogen synthase bound to malto-oligosaccharides, showing for the first time that an enzyme in the starch biosynthetic pathway recognizes glucans not just in its active site but on binding sites on the surface of the enzyme ten’s of Angstroms from the active site. In addition our structure of a glycogen branching enzyme bound to malto-oligosaccharides identified seven distinct binding sites distributed about the surface of the enzyme. We will now determine the function of these sites to get a molecular-level picture of exactly how these enzymes interact with their polymeric substrates and confer specificity leading to the complex structure of the starch granule. We will extend our studies to other isoforms of the enzymes, to understand how their structures give rise to their distinct function. Our goal is to understand what accounts for the various functional differences between SS and SBE isoforms at a molecular level.

  5. Dual Role of a Biosynthetic Enzyme, CysK, in Contact Dependent Growth Inhibition in Bacteria.

    Directory of Open Access Journals (Sweden)

    Soni Kaundal

    Full Text Available Contact dependent growth inhibition (CDI is the phenomenon where CDI+ bacterial strain (inhibitor inhibits the growth of CDI-strain (target by direct cell to cell contact. CDI is mediated by cdiBAI gene cluster where CdiB facilitates the export of CdiA, an exotoxin, on the cell surface and CdiI acts as an immunity protein to protect CDI+ cells from autoinhibition. CdiA-CT, the C-terminal region of the toxin CdiA, from uropathogenic Escherichia coli strain 536 (UPEC536 is a latent tRNase that requires binding of a biosynthetic enzyme CysK (O-acetylserine sulfyhydrylase for activation in the target cells. CdiA-CT can also interact simultaneously with CysK and immunity protein, CdiI, to form a ternary complex in UPEC536. But the role of CysK in the ternary complex is not clear. We studied the hydrodynamic, thermodynamic and kinetic parameters of binary and ternary complexes using AUC, ITC and SPR respectively, to investigate the role of CysK in UPEC536. We report that CdiA-CT binds CdiI and CysK with nanomolar range affinity. We further report that binding of CysK to CdiA-CT improves its affinity towards CdiI by ~40 fold resulting in the formation of a more stable complex with over ~130 fold decrease in dissociation rate. Thermal melting experiments also suggest the role of CysK in stabilizing CdiA-CT/CdiI complex as Tm of the binary complex shifts ~10°C upon binding CysK. Hence, CysK acts a modulator of CdiA-CT/CdiI interactions by stabilizing CdiA-CT/CdiI complex and may play a crucial role in preventing autoinhibition in UPEC536. This study reports a new moonlighting function of a biosynthetic enzyme, CysK, as a modulator of toxin/immunity interactions in UPEC536 inhibitor cells.

  6. Enzymes of the taurine biosynthetic pathway are expressed in rat mammary gland.

    Science.gov (United States)

    Ueki, Iori; Stipanuk, Martha H

    2007-08-01

    Taurine is the most abundant free amino acid in the body and is present at high concentrations during development and in the early milk. It is synthesized from cysteine via oxidation of cysteine to cysteinesulfinate by the enzyme cysteine dioxygenase (CDO), followed by the decarboxylation of cysteinesulfinate to hypotaurine, catalyzed by cysteine sulfinic acid decarboxylase (CSAD). To determine whether the taurine biosynthetic pathway is present in mammary gland and whether it is differentially expressed during pregnancy and lactation, and also to further explore the possible regulation of hepatic taurine synthesis during pregnancy and lactation, we measured mammary and hepatic CDO and CSAD mRNA and protein concentrations and tissue, plasma and milk taurine concentrations. CDO and CSAD mRNA and protein were expressed in mammary gland and liver regardless of physiological state. Immunohistochemistry demonstrated the expression of CDO in ductal cells of pregnant rats, but not in other mammary epithelial cells or in ductal cells of nonpregnant rats. CDO was also present in stromal adipocytes in mammary glands of both pregnant and nonpregnant rats. Our findings support an upregulation of taurine synthetic capacity in the mammary gland of pregnant rats, based on mammary taurine and hypotaurine concentrations and the intense immunohistochemical staining for CDO in ductal cells of pregnant rats. Hepatic taurine synthetic capacity, particularly CSAD, and taurine concentrations were highest in rats during the early stages of lactation, suggesting the liver may also play a role in the synthesis of taurine to support lactation or repletion of maternal reserves.

  7. Production of wax esters in plant seed oils by oleosomal cotargeting of biosynthetic enzymes[S

    Science.gov (United States)

    Heilmann, Mareike; Iven, Tim; Ahmann, Katharina; Hornung, Ellen; Stymne, Sten; Feussner, Ivo

    2012-01-01

    Wax esters are neutral lipids exhibiting desirable properties for lubrication. Natural sources have traditionally been whales. Additionally some plants produce wax esters in their seed oil. Currently there is no biological source available for long chain length monounsaturated wax esters that are most suited for industrial applications. This study aimed to identify enzymatic requirements enabling their production in oilseed plants. Wax esters are generated by the action of fatty acyl-CoA reductase (FAR), generating fatty alcohols and wax synthases (WS) that esterify fatty alcohols and acyl-CoAs to wax esters. Based on their substrate preference, a FAR and a WS from Mus musculus were selected for this study (MmFAR1 and MmWS). MmWS resides in the endoplasmic reticulum (ER), whereas MmFAR1 associates with peroxisomes. The elimination of a targeting signal and the fusion to an oil body protein yielded variants of MmFAR1 and MmWS that were cotargeted and enabled wax ester production when coexpressed in yeast or Arabidopsis. In the fae1 fad2 double mutant, rich in oleate, the cotargeted variants of MmFAR1 and MmWS enabled formation of wax esters containing >65% oleyl-oleate. The data suggest that cotargeting of unusual biosynthetic enzymes can result in functional interplay of heterologous partners in transgenic plants. PMID:22878160

  8. Chronic physical stress changes gene expression of catecholamine biosynthetic enzymes in the adrenal medulla of adult rats

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    Gavrilović Ljubica

    2012-01-01

    Full Text Available In this study we examined how chronic forced running (CFR affects the expression of catecholamine biosynthetic enzymes and cAMP response element-binding (CREB in the adrenal medulla and the weight of adrenal glands of rats. Also, we examined how CFR and additional acute immobilization stress affect the expression of catecholamine biosynthetic enzymes in the adrenal medulla and the concentration of catecholamines and corticosterone (CORT in the blood plasma. In this experiment we used as a model forced exercise in rats (treadmill running. We used the most advanced method for determining the level of gene expression, Real-time PCR with TaqMan probes, as well as Western blot analysis (ECL. We found that CFR decreases tyrosine hydroxylase (TH, and dopamine-β-hydroxylase (DBH mRNA and protein levels in the adrenal medulla. The decreased TH and DBH mRNA levels coincide with the reduced expression of CREB in the adrenal medulla and with the reduced plasma CORT level. Additionally, CFR reduces the level of phenylethanolamine N-methyltransferase (PNMT mRNA, but elevates its protein level in the adrenal medulla and increases the concentration of adrenaline (A in the plasma. Reduced level of PNMT mRNA in the adrenal medulla coincides with reduced plasma CORT level. The additional acute immobilization stress increases gene expression of catecholamine biosynthetic enzymes in the adrenal medulla, as well as catecholamines and CORT levels in the plasma. The increased synthesis of PNMT enzyme in the adrenal medulla may result in an increased biosynthesis of A under chronic stress conditions. Additionally, increased level of catecholamines in the plasma after chronic physical stress is the allostatic load that may induce numerous diseases and pathological conditions.

  9. Molecular interaction of the first 3 enzymes of the de novo pyrimidine biosynthetic pathway of Trypanosoma cruzi

    Energy Technology Data Exchange (ETDEWEB)

    Nara, Takeshi, E-mail: tnara@juntendo.ac.jp [Department of Molecular and Cellular Parasitology, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Hashimoto, Muneaki; Hirawake, Hiroko [Department of Molecular and Cellular Parasitology, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Liao, Chien-Wei [Department of Molecular and Cellular Parasitology, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Department of Parasitology, Taipei Medical University, 250 Wu-Xing Street, Taipei 110, Taiwan, ROC (China); Fukai, Yoshihisa; Suzuki, Shigeo; Tsubouchi, Akiko; Morales, Jorge; Takamiya, Shinzaburo [Department of Molecular and Cellular Parasitology, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Fujimura, Tsutomu; Taka, Hikari; Mineki, Reiko [Division of Proteomics and Biomolecular Science, Biomedical Research Center, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Fan, Chia-Kwung [Department of Parasitology, Taipei Medical University, 250 Wu-Xing Street, Taipei 110, Taiwan, ROC (China); Inaoka, Daniel Ken [Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Inoue, Masayuki [Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Tanaka, Akiko [Systems and Structural Biology Center, RIKEN, Tsurumi, Yokohama 230-0045 (Japan); Harada, Shigeharu [Department of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Kita, Kiyoshi [Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); and others

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer An Escherichia coli strain co-expressing CPSII, ATC, and DHO of Trypanosoma cruzi was constructed. Black-Right-Pointing-Pointer Molecular interactions between CPSII, ATC, and DHO of T. cruzi were demonstrated. Black-Right-Pointing-Pointer CPSII bound with both ATC and DHO. Black-Right-Pointing-Pointer ATC bound with both CPSII and DHO. Black-Right-Pointing-Pointer A functional tri-enzyme complex might precede the establishment of the fused enzyme. -- Abstract: The first 3 reaction steps of the de novo pyrimidine biosynthetic pathway are catalyzed by carbamoyl-phosphate synthetase II (CPSII), aspartate transcarbamoylase (ATC), and dihydroorotase (DHO), respectively. In eukaryotes, these enzymes are structurally classified into 2 types: (1) a CPSII-DHO-ATC fusion enzyme (CAD) found in animals, fungi, and amoebozoa, and (2) stand-alone enzymes found in plants and the protist groups. In the present study, we demonstrate direct intermolecular interactions between CPSII, ATC, and DHO of the parasitic protist Trypanosoma cruzi, which is the causative agent of Chagas disease. The 3 enzymes were expressed in a bacterial expression system and their interactions were examined. Immunoprecipitation using an antibody specific for each enzyme coupled with Western blotting-based detection using antibodies for the counterpart enzymes showed co-precipitation of all 3 enzymes. From an evolutionary viewpoint, the formation of a functional tri-enzyme complex may have preceded-and led to-gene fusion to produce the CAD protein. This is the first report to demonstrate the structural basis of these 3 enzymes as a model of CAD. Moreover, in conjunction with the essentiality of de novo pyrimidine biosynthesis in the parasite, our findings provide a rationale for new strategies for developing drugs for Chagas disease, which target the intermolecular interactions of these 3 enzymes.

  10. Structure of Nampt/PBEF/visfatin, a mammalian NAD[superscript +]biosynthetic enzyme

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Tao; Zhang, Xiangbin; Bheda, Poonam; Revollo, Javier R.; Imai, Shin-ichiro; Wolberger, Cynthia (JHU-MED); (WU-MED)

    2010-07-22

    Nicotinamide phosphoribosyltransferase (Nampt) synthesizes nicotinamide mononucleotide (NMN) from nicotinamide in a mammalian NAD{sup +} biosynthetic pathway and is required for SirT1 activity in vivo. Nampt has also been presumed to be a cytokine (PBEF) or a hormone (visfatin). The crystal structure of Nampt in the presence and absence of NMN shows that Nampt is a dimeric type II phosphoribosyltransferase and provides insights into the enzymatic mechanism.

  11. Co-ordinated synthesis of phytoalexin biosynthetic enzymes in biologically-stressed cells of bean (Phaseolus vulgaris L.).

    Science.gov (United States)

    Cramer, C L; Bell, J N; Ryder, T B; Bailey, J A; Schuch, W; Bolwell, G P; Robbins, M P; Dixon, R A; Lamb, C J

    1985-02-01

    Changes in the rates of synthesis of three enzymes of phenyl-propanoid biosynthesis in Phaseolus vulgaris L. (dwarf French bean) have been investigated by immunoprecipitation of [S]methionine-labeled enzyme subunits with mono-specific antisera. Elicitor causes marked, rapid but transient co-ordinated increases in the rate of synthesis of phenyl-alanine ammonia-lyase, chalcone synthase and chalcone isomerase concomitant with the phase of rapid increase in enzyme activity at the onset of accumulation of phenyl-propanoid-derived phytoalexin antibiotics in suspension cultures of P. vulgaris. Co-ordinate induction of enzyme synthesis is also observed in hypocotyl tissue during race:cultivar-specific interactions with Colletotrichum lindemuthianum, causal agent of anthracnose. In an incompatible interaction (host resistant) there are early increases apparently localized to the initial site of infection prior to the onset of phytoalexin accumulation and expression of hypersensitive resistance. In contrast, in a compatible interaction (host susceptible) there is no induction of synthesis in the early stages of infection, but a delayed widespread response at the onset of lesion formation associated with attempted lesion limitation. It is concluded that expression of the phytoalexin defense response in biologically stressed cells of P. vulgaris characteristically involves co-ordinate induction of synthesis of phytoalexin biosynthetic enzymes.

  12. Fructan Biosynthetic and Breakdown Enzymes in Dicots Evolved From Different Invertases. Expression of Fructan Genes Throughout Chicory Development

    Directory of Open Access Journals (Sweden)

    Wim Van den Ende

    2002-01-01

    Full Text Available Fructans are fructose-based oligo- and polymers that serve as reserve carbohydrates in many plant species. The biochemistry of fructan biosynthesis in dicots has been resolved, and the respective cDNAs have been cloned. Recent progress has now succeeded in elucidating the biochemistry and molecular biology of fructan biodegradation in chicory, an economically important species used for commercial inulin extraction. Unlike fructan biosynthetic genes that originated from vacuolar-type invertase, fructan exohydrolases (FEHs seem to have evolved from a cell-wall invertase ancestor gene that later obtained a low iso-electric point and a vacuolar targeting signal. Expression analysis reveals that fructan enzymes are controlled mainly at the transcriptional level. Using chicory as a model system, northern analysis was consistent with enzymatic activity measurements and observed carbohydrate changes throughout its development.

  13. Strategies for transgenic manipulation of monoterpene biosynthesis in plants.

    Science.gov (United States)

    Mahmoud, Soheil S; Croteau, Rodney B

    2002-08-01

    Monoterpenes, the C(10) isoprenoids, are a large family of natural products that are best known as constituents of the essential oils and defensive oleoresins of aromatic plants. In addition to ecological roles in pollinator attraction, allelopathy and plant defense, monoterpenes are used extensively in the food, cosmetic and pharmaceutical industries. The importance of these plant products has prompted the definition of many monoterpene biosynthetic pathways, the cloning of the relevant genes and the development of genetic transformation techniques for agronomically significant monoterpene-producing plants. Metabolic engineering of monoterpene biosynthesis in the model plant peppermint has resulted in yield increase and compositional improvement of the essential oil, and also provided strategies for manipulating flavor and fragrance production, and plant defense.

  14. Staphylococcus aureus mevalonate kinase: isolation and characterization of an enzyme of the isoprenoid biosynthetic pathway.

    Science.gov (United States)

    Voynova, Natalya E; Rios, Sandra E; Miziorko, Henry M

    2004-01-01

    It has been proposed that isoprenoid biosynthesis in several gram-positive cocci depends on the mevalonate pathway for conversion of acetyl coenzyme A to isopentenyl diphosphate. Mevalonate kinase catalyzes a key reaction in this pathway. In this study the enzyme from Staphylococcus aureus was expressed in Escherichia coli, isolated in a highly purified form, and characterized. The overall amino acid sequence of this enzyme was very heterologous compared with the sequences of eukaryotic mevalonate kinases. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical gel filtration chromatography suggested that the native enzyme is a monomer with a molecular mass of approximately 33 kDa. The specific activity was 12 U/mg, and the pH optimum was 7.0 to 8.5. The apparent K(m) values for R,S-mevalonate and ATP were 41 and 339 micro M, respectively. There was substantial substrate inhibition at millimolar levels of mevalonate. The sensitivity to feedback inhibition by farnesyl diphosphate and its sulfur-containing analog, farnesyl thiodiphosphate, was characterized. These compounds were competitive inhibitors with respect to ATP; the K(i) values were 46 and 45 micro M for farnesyl diphosphate and its thio analog, respectively. Parallel measurements with heterologous eukaryotic mevalonate kinases indicated that S. aureus mevalonate kinase is much less sensitive to feedback inhibition (K(i) difference, 3 orders of magnitude) than the human enzyme. In contrast, both enzymes tightly bound trinitrophenyl-ATP, a fluorescent substrate analog, suggesting that there are similarities in structural features that are important for catalytic function.

  15. Effect of immobilization stress on gene expression of catecholamine biosynthetic enzymes in heart auricles of socially isolated rats

    Directory of Open Access Journals (Sweden)

    L. Gavrilovic

    2009-12-01

    Full Text Available Chronic stress is associated with the development of cardiovascular diseases. The sympathoneural system plays an important role in the regulation of cardiac function both in health and disease. In the present study, the changes in gene expression of the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH, dopamine-β-hydroxylase (DBH and phenylethanolamine N-methyltransferase (PNMT and protein levels in the right and left heart auricles of naive control and long-term (12 weeks socially isolated rats were investigated by Taqman RT-PCR and Western blot analysis. The response of these animals to additional immobilization stress (2 h was also examined. Long-term social isolation produced a decrease in TH mRNA level in left auricles (about 70% compared to the corresponding control. Expression of the DBH gene was markedly decreased both in the right (about 62% and left (about 81% auricles compared to the corresponding control, group-maintained rats, whereas PNMT mRNA levels remained unchanged. Exposure of group-housed rats to acute immobilization for 2 h led to a significant increase of mRNA levels of TH (about 267%, DBH (about 37% and PNMT (about 60% only in the right auricles. Additional 2-h immobilization of individually housed rats did not affect gene expression of these enzymes in either the right or left auricle. Protein levels of TH, DBH and PNMT in left and right heart auricles were unchanged either in both individually housed and immobilized rats. The unchanged mRNA levels of the enzymes examined after short-term immobilization suggest that the catecholaminergic system of the heart auricles of animals previously exposed to chronic psychosocial stress was adapted to maintain appropriate cardiovascular homeostasis.

  16. Effects of overexpressing individual lignin biosynthetic enzymes on feeding and growth of corn earworms and fall armyworms

    Science.gov (United States)

    Lignin is an important insect resistance component of plants. Enhancing or disrupting the lignin biosynthetic pathway for different bioenergy uses may alter pest resistance. The lignin biosynthetic pathway is complex, and a number of pathway compounds are also involved in the biosynthesis of simpler...

  17. Characterization of the De Novo Biosynthetic Enzyme of Platelet Activating Factor, DDT-Insensitive Cholinephosphotransferase, of Human Mesangial Cells

    Directory of Open Access Journals (Sweden)

    Constantinos Alexandros Demopoulos

    2007-06-01

    Full Text Available Platelet activating factor (PAF, a potent inflammatory mediator, is implicated in several proinflammatory/inflammatory diseases such as glomerulonephritis, glomerulosclerosis, atherosclerosis, cancer, allergy, and diabetes. PAF can be produced by several renal cells under appropriate stimuli and it is thought to be implicated in renal diseases. The aim of this study is the characterization of DTT-insensitive cholinephosphotransferase (PAF-CPT of human mesangial cell (HMC, the main regulatory enzyme of PAF de novo biosynthetic pathway. Microsomal fractions of mesangial cells were isolated and enzymatic activity and kinetic parameters were determined by TLC and in vitro biological test in rabbit washed platelets. The effect of bovine serum albumin (BSA, dithiothreitol (DTT, divalent cations (Mg2+ and Ca2+, EDTA, and various chemicals on the activity of PAF-CPT of HMC was also studied. Moreover, preliminary in vitro tests have been performed with several anti-inflammatory factors such as drugs (simvastatin, IFNa, rupatadine, tinzaparin, and salicylic acid and bioactive compounds of Mediterranean diet (resveratrol and lipids of olive oil, olive pomace, sea bass “Dicentrarchus labrax,” and gilthead sea bream “Sparus aurata”. The results indicated that the above compounds can influence PAF-CPT activity of HMC.

  18. GO-PROMTO illuminates protein membrane topologies of glycan biosynthetic enzymes in the Golgi apparatus of living tissues.

    Science.gov (United States)

    Søgaard, Casper; Stenbæk, Anne; Bernard, Sophie; Hadi, Masood; Driouich, Azeddine; Scheller, Henrik Vibe; Sakuragi, Yumiko

    2012-01-01

    The Golgi apparatus is the main site of glycan biosynthesis in eukaryotes. Better understanding of the membrane topology of the proteins and enzymes involved can impart new mechanistic insights into these processes. Publically available bioinformatic tools provide highly variable predictions of membrane topologies for given proteins. Therefore we devised a non-invasive experimental method by which the membrane topologies of Golgi-resident proteins can be determined in the Golgi apparatus in living tissues. A Golgi marker was used to construct a series of reporters based on the principle of bimolecular fluorescence complementation. The reporters and proteins of interest were recombinantly fused to split halves of yellow fluorescent protein (YFP) and transiently co-expressed with the reporters in the Nicotiana benthamiana leaf tissue. Output signals were binary, showing either the presence or absence of fluorescence with signal morphologies characteristic of the Golgi apparatus and endoplasmic reticulum (ER). The method allows prompt and robust determinations of membrane topologies of Golgi-resident proteins and is termed GO-PROMTO (for GOlgi PROtein Membrane TOpology). We applied GO-PROMTO to examine the topologies of proteins involved in the biosynthesis of plant cell wall polysaccharides including xyloglucan and arabinan. The results suggest the existence of novel biosynthetic mechanisms involving transports of intermediates across Golgi membranes.

  19. GO-PROMTO illuminates protein membrane topologies of glycan biosynthetic enzymes in the Golgi apparatus of living tissues.

    Directory of Open Access Journals (Sweden)

    Casper Søgaard

    Full Text Available The Golgi apparatus is the main site of glycan biosynthesis in eukaryotes. Better understanding of the membrane topology of the proteins and enzymes involved can impart new mechanistic insights into these processes. Publically available bioinformatic tools provide highly variable predictions of membrane topologies for given proteins. Therefore we devised a non-invasive experimental method by which the membrane topologies of Golgi-resident proteins can be determined in the Golgi apparatus in living tissues. A Golgi marker was used to construct a series of reporters based on the principle of bimolecular fluorescence complementation. The reporters and proteins of interest were recombinantly fused to split halves of yellow fluorescent protein (YFP and transiently co-expressed with the reporters in the Nicotiana benthamiana leaf tissue. Output signals were binary, showing either the presence or absence of fluorescence with signal morphologies characteristic of the Golgi apparatus and endoplasmic reticulum (ER. The method allows prompt and robust determinations of membrane topologies of Golgi-resident proteins and is termed GO-PROMTO (for GOlgi PROtein Membrane TOpology. We applied GO-PROMTO to examine the topologies of proteins involved in the biosynthesis of plant cell wall polysaccharides including xyloglucan and arabinan. The results suggest the existence of novel biosynthetic mechanisms involving transports of intermediates across Golgi membranes.

  20. Microbial monoterpene transformations – A review

    Directory of Open Access Journals (Sweden)

    Robert eMarmulla

    2014-07-01

    Full Text Available Isoprene and monoterpenes constitute a significant fraction of new plant biomass. Emission rates into the atmosphere alone are estimated to be over 500 Tg per year. These natural hydrocarbons are mineralized annually in similar quantities. In the atmosphere, abiotic photochemical processes cause lifetimes of minutes to hours. Microorganisms encounter isoprene, monoterpenes and other volatiles of plant origin while living in and on plants, in the soil and in aquatic habitats. Below toxic concentrations, the compounds can serve as carbon and energy source for aerobic and anaerobic microorganisms. Besides these catabolic reactions, transformations may occur as part of detoxification processes. Initial transformations of monoterpenes involve the introduction of functional groups, oxidation reactions and molecular rearrangements catalyzed by various enzymes. Pseudomonas and Rhodococcus strains and members of the genera Castellaniella and Thauera have become model organisms for the elucidation of biochemical pathways. We review here the enzymes and their genes together with microorganisms known for a monoterpene metabolism, with a strong focus on microorganisms that are taxonomically validly described and currently available from culture collections. Metagenomes of microbiomes with a monoterpene-rich diet confirmed the ecological relevance of monoterpene metabolism and raised concerns on the quality of our insights based on the limited biochemical knowledge.

  1. Suppression of rat and human androgen biosynthetic enzymes by apigenin: Possible use for the treatment of prostate cancer.

    Science.gov (United States)

    Wang, Xiudi; Wang, Guimin; Li, Xiaoheng; Liu, Jianpeng; Hong, Tingting; Zhu, Qiqi; Huang, Ping; Ge, Ren-Shan

    2016-06-01

    Apigenin is a natural flavone. It has recently been used as a chemopreventive agent. It may also have some beneficial effects to treat prostate cancer by inhibiting androgen production. The objective of the present study was to investigate the effects of apigenin on the steroidogenesis of rat immature Leydig cells and some human testosterone biosynthetic enzyme activities. Rat immature Leydig cells were incubated for 3h with 100μM apigenin without (basal) or with 1ng/ml luteinizing hormone (LH), 10mM 8-bromoadenosine 3',5'-cyclic monophosphate (8BR), and 20μM of the following steroid substrates: 22R-hydroxychloesterol (22R), pregnenolone (P5), progesterone (P4), and androstenedione (D4). The medium levels of 5α-androstane-3α, 17β-diol (DIOL), the primary androgen produced by rat immature Leydig cells, were measured. Apigenin significantly inhibited basal, 8BR, 22R, PREG, P4, and D4 stimulated DIOL production in rat immature Leydig cells. Further study showed that apigenin inhibited rat 3β-hydroxysteroid dehydrogenase, 17α-hydroxylase/17, 20-lyase, and 17β-hydroxysteroid dehydrogenase 3 with IC50 values of 11.41±0.7, 8.98±0.10, and 9.37±0.07μM, respectively. Apigenin inhibited human 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase 3 with IC50 values of 2.17±0.04 and 1.31±0.09μM, respectively. Apigenin is a potent inhibitor of rat and human steroidogenic enzymes, being possible use for the treatment of prostate cancer.

  2. Enhanced production of n-alkanes in Escherichia coli by spatial organization of biosynthetic pathway enzymes.

    Science.gov (United States)

    Rahmana, Ziaur; Sung, Bong Hyun; Yi, Ji-Yeun; Bui, Le Minh; Lee, Jun Hyoung; Kim, Sun Chang

    2014-12-20

    Alkanes chemically mimic hydrocarbons found in petroleum, and their demand as biofuels is steadily increasing. Biologically, n-alkanes are produced from fatty acyl-ACPs by acyl-ACP reductases (AARs) and aldehyde deformylating oxygenases (ADOs). One of the major impediments in n-alkane biosynthesis is the low catalytic turnover rates of ADOs. Here, we studied n-alkane biosynthesis in Escherichia coli using a chimeric ADO-AAR fusion protein or zinc finger protein-guided ADO/AAR assembly on DNA scaffolds to control their stoichiometric ratios and spatial arrangements. Bacterial production of n-alkanes with the ADO-AAR fusion protein was increased 4.8-fold (24 mg/L) over a control strain expressing ADO and AAR separately. Optimal n-alkane biosynthesis was achieved when the ADO:AAR binding site ratio on a DNA scaffold was 3:1, yielding an 8.8-fold increase (44 mg/L) over the control strain. Our findings indicate that the spatial organization of alkane-producing enzymes is critical for efficient n-alkane biosynthesis in E. coli.

  3. Structure of PqsD, a Pseudomonas Quinolone Signal Biosynthetic Enzyme, in Complex with Anthranilate

    Energy Technology Data Exchange (ETDEWEB)

    Bera, A.; Atanasova, V; Robinson, H; Eisenstein, E; Coleman, J; Pesci, E; Parsons, J

    2009-01-01

    Here we present a structural and biophysical characterization of PqsD that includes several crystal structures of the enzyme, including that of the PqsD-anthranilate covalent intermediate and the inactive Cys112Ala active site mutant in complex with anthranilate. The structure reveals that PqsD is structurally similar to the FabH and chalcone synthase families of fatty acid and polyketide synthases. The crystallographic asymmetric unit contains a PqsD dimer. The PqsD monomer is composed of two nearly identical 170-residue ????? domains. The structures show anthranilate-liganded Cys112 is positioned deep in the protein interior at the bottom of an 15 A long channel while a second anthraniloyl-CoA molecule is waiting in the cleft leading to the protein surface. Cys112, His257, and Asn287 form the FabH-like catalytic triad of PqsD. The C112A mutant is inactive, although it still reversibly binds anthraniloyl-CoA. The covalent complex between anthranilate and Cys112 clearly illuminates the orientation of key elements of the PqsD catalytic machinery and represents a snapshot of a key point in the catalytic cycle.

  4. Identification of mRNA for endocannabinoid biosynthetic enzymes within hippocampal pyramidal cells and CA1 stratum radiatum interneuron subtypes using quantitative real-time polymerase chain reaction.

    Science.gov (United States)

    Merrill, C B; McNeil, M; Williamson, R C; Poole, B R; Nelson, B; Sudweeks, S; Edwards, J G

    2012-08-30

    The hippocampus is required for short-term memory and contains both excitatory pyramidal cells and inhibitory interneurons. These cells exhibit various forms of synaptic plasticity, the mechanism underlying learning and memory. More recently, endocannabinoids were identified to be involved in synaptic plasticity. Our goal was to describe the distribution of endocannabinoid biosynthetic enzymes within CA1 stratum radiatum interneurons and CA3/CA1 pyramidal cells. We extracted mRNA from single interneurons and pyramidal cells and used real-time quantitative polymerase chain reaction (RT-PCR) to detect the presence of 12-lipoxygenase, N-acyl-phosphatidylethanolamine-specific phospholipase D, diacylglycerol lipase α, and type I metabotropic glutamate receptors, all known to be involved in endocannabinoid production and plasticity. We observed that the expression of endocannabinoid biosynthetic enzyme mRNA does occur within interneurons and that it is coexpressed with type I metabotropic glutamate receptors, suggesting interneurons have the potential to produce endocannabinoids. We also identified that CA3 and CA1 pyramidal cells express endocannabinoid biosynthetic enzyme mRNA. Our data provide the first molecular biological evidence for putative endocannabinoid production in interneurons, suggesting their potential ability to regulate endocannabinoid-mediated processes, such as synaptic plasticity.

  5. The sub-cellular localisation of the potato (Solanum tuberosum L.) carotenoid biosynthetic enzymes, CrtRb2 and PSY2.

    Science.gov (United States)

    Pasare, Stefania; Wright, Kathryn; Campbell, Raymond; Morris, Wayne; Ducreux, Laurence; Chapman, Sean; Bramley, Peter; Fraser, Paul; Roberts, Alison; Taylor, Mark

    2013-12-01

    Carotenoids are isoprenoids with important biological roles both for plants and animals. The yellow flesh colour of potato (Solanum tuberosum L.) tubers is a quality trait dependent on the types and levels of carotenoids that accumulate. The carotenoid biosynthetic pathway is well characterised, facilitating the successful engineering of carotenoid content in numerous crops including potato. However, a clear understanding concerning the factors regulating carotenoid accumulation and localisation in plant storage organs, such as tubers, is lacking. In the present study, the localisation of key carotenoid biosynthetic enzymes was investigated, as one of the unexplored factors that could influence the accumulation of carotenoids in potato tubers. Stable transgenic potato plants were generated by over-expressing β-CAROTENE HYDROXYLASE 2 (CrtRb2) and PHYTOENE SYNTHASE 2 (PSY2) genes, fused to red fluorescent protein (RFP). Gene expression and carotenoid levels were both significantly increased, confirming functionality of the fluorescently tagged proteins. Confocal microscopy studies revealed different sub-organellar localisations of CrtRb2-RFP and PSY2-RFP within amyloplasts. CrtRb2 was detected in small vesicular structures, inside amyloplasts, whereas PSY2 was localised in the stroma of amyloplasts. We conclude that it is important to consider the location of biosynthetic enzymes when engineering the carotenoid metabolic pathway in storage organs such as tubers.

  6. Seasonal alteration in amounts of lignans and their glucosides and gene expression of the relevant biosynthetic enzymes in the Forsythia suspense leaf.

    Science.gov (United States)

    Morimoto, Kinuyo; Satake, Honoo

    2013-01-01

    Lignans of Forsythia spp. are essential components of various Chinese medicines and health diets. However, the seasonal alteration in lignan amounts and the gene expression profile of lignan-biosynthetic enzymes has yet to be investigated. In this study, we have assessed seasonal alteration in amounts of major lignans, such as pinoresinol, matairesinol, and arctigenin, and examined the gene expression profile of pinoresinol/lariciresinol reductase (PLR), pinoresinol-glucosylating enzyme (UGT71A18), and secoisolariciresinol dehydrogenase (SIRD) in the leaf of Forsythia suspense from April to November. All of the lignans in the leaf continuously increased from April to June, reached the maximal level in June, and then decreased. Ninety percent of pinoresinol and matairesinol was converted into glucosides, while approximately 50% of arctigenin was aglycone. PLR was stably expressed from April to August, whereas the PLR expression was not detected from September to November. In contrast, the UGT71A18 expression was found from August to November, but not from April to July. The SIRD expression was prominent from April to May, not detected in June to July, and then increased again from September to November. These expression profiles of the lignan-synthetic enzymes are largely compatible with the alteration in lignan contents. Furthermore, such seasonal lignan profiles are in good agreement with the fact that the Forsythia leaves for Chinese medicinal tea are harvested in June. This is the first report on seasonal alteration in lignans and the relevant biosynthetic enzyme genes in the leaf of Forsythia species.

  7. Effective use of heterologous hosts for characterization of biosynthetic enzymes allows production of natural products and promotes new natural product discovery.

    Science.gov (United States)

    Watanabe, Kenji

    2014-01-01

    In the past few years, there has been impressive progress in elucidating the mechanism of biosynthesis of various natural products accomplished through the use of genetic, molecular biological and biochemical techniques. Here, we present a comprehensive overview of the current results from our studies on fungal natural product biosynthetic enzymes, including nonribosomal peptide synthetase and polyketide synthase-nonribosomal peptide synthetase hybrid synthetase, as well as auxiliary enzymes, such as methyltransferases and oxygenases. Specifically, biosynthesis of the following compounds is described in detail: (i) Sch210972, potentially involving a Diels-Alder reaction that may be catalyzed by CghA, a functionally unknown protein identified by targeted gene disruption in the wild type fungus; (ii) chaetoglobosin A, formed via multi-step oxidations catalyzed by three redox enzymes, one flavin-containing monooxygenase and two cytochrome P450 oxygenases as characterized by in vivo biotransformation of relevant intermediates in our engineered Saccharomyces cerevisiae; (iii) (-)-ditryptophenaline, formed by a cytochrome P450, revealing the dimerization mechanism for the biosynthesis of diketopiperazine alkaloids; (iv) pseurotins, whose variations in the C- and O-methylations and the degree of oxidation are introduced combinatorially by multiple redox enzymes; and (v) spirotryprostatins, whose spiro-carbon moiety is formed by a flavin-containing monooxygenase or a cytochrome P450 as determined by heterologous de novo production of the biosynthetic intermediates and final products in Aspergillus niger. We close our discussion by summarizing some of the key techniques that have facilitated the discovery of new natural products, production of their analogs and identification of biosynthetic mechanisms in our study.

  8. Recent advances in Cannabis sativa research: biosynthetic studies and its potential in biotechnology.

    Science.gov (United States)

    Sirikantaramas, Supaart; Taura, Futoshi; Morimoto, Satoshi; Shoyama, Yukihiro

    2007-08-01

    Cannabinoids, consisting of alkylresorcinol and monoterpene groups, are the unique secondary metabolites that are found only in Cannabis sativa. Tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabichromene (CBC) are well known cannabinoids and their pharmacological properties have been extensively studied. Recently, biosynthetic pathways of these cannabinoids have been successfully established. Several biosynthetic enzymes including geranylpyrophosphate:olivetolate geranyltransferase, tetrahydrocannabinolic acid (THCA) synthase, cannabidiolic acid (CBDA) synthase and cannabichromenic acid (CBCA) synthase have been purified from young rapidly expanding leaves of C. sativa. In addition, molecular cloning, characterization and localization of THCA synthase have been recently reported. THCA and cannabigerolic acid (CBGA), its substrate, were shown to be apoptosis-inducing agents that might play a role in plant defense. Transgenic tobacco hairy roots expressing THCA synthase can produce THCA upon feeding of CBGA. These results open the way for biotechnological production of cannabinoids in the future.

  9. Starch biosynthetic genes and enzymes are expressed and active in the absence of starch accumulation in sugar beet tap-root

    Science.gov (United States)

    2014-01-01

    Background Starch is the predominant storage compound in underground plant tissues like roots and tubers. An exception is sugar beet tap-root (Beta vulgaris ssp altissima) which exclusively stores sucrose. The underlying mechanism behind this divergent storage accumulation in sugar beet is currently not fully known. From the general presence of starch in roots and tubers it could be speculated that the lack in sugar beet tap-roots would originate from deficiency in pathways leading to starch. Therefore with emphasis on starch accumulation, we studied tap-roots of sugar beet using parsnip (Pastinaca sativa) as a comparator. Results Metabolic and structural analyses of sugar beet tap-root confirmed sucrose as the exclusive storage component. No starch granules could be detected in tap-roots of sugar beet or the wild ancestor sea beet (Beta vulgaris ssp. maritima). Analyses of parsnip showed that the main storage component was starch but tap-root tissue was also found to contain significant levels of sugars. Surprisingly, activities of four main starch biosynthetic enzymes, phosphoglucomutase, ADP-glucose pyrophosphorylase, starch synthase and starch branching enzyme, were similar in sugar beet and parsnip tap-roots. Transcriptional analysis confirmed expression of corresponding genes. Additionally, expression of genes involved in starch accumulation such as for plastidial hexose transportation and starch tuning functions could be determined in tap-roots of both plant species. Conclusion Considering underground storage organs, sugar beet tap-root upholds a unique property in exclusively storing sucrose. Lack of starch also in the ancestor sea beet indicates an evolved trait of biological importance. Our findings in this study show that gene expression and enzymatic activity of main starch biosynthetic functions are present in sugar beet tap-root during storage accumulation. In view of this, the complete lack of starch in sugar beet tap-roots is enigmatic. PMID

  10. Structure of the D-alanylgriseoluteic acid biosynthetic protein EhpF, an atypical member of the ANL superfamily of adenylating enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Bera, A.K.; Robinson, H.; Atanasova, V.; Gamage, S.; Parsons, J. F.

    2010-06-01

    The structure of EhpF, a 41 kDa protein that functions in the biosynthetic pathway leading to the broad-spectrum antimicrobial compound D-alanylgriseoluteic acid (AGA), is reported. A cluster of approximately 16 genes, including ehpF, located on a 200 kbp plasmid native to certain strains of Pantoea agglomerans encodes the proteins that are required for the conversion of chorismic acid to AGA. Phenazine-1,6-dicarboxylate has been identified as an intermediate in AGA biosynthesis and deletion of ehpF results in accumulation of this compound in vivo. The crystallographic data presented here reveal that EhpF is an atypical member of the acyl-CoA synthase or ANL superfamily of adenylating enzymes. These enzymes typically catalyze two-step reactions involving adenylation of a carboxylate substrate followed by transfer of the substrate from AMP to coenzyme A or another phosphopantetheine. EhpF is distinguished by the absence of the C-terminal domain that is characteristic of enzymes from this family and is involved in phosphopantetheine binding and in the second half of the canonical two-step reaction that is typically observed. Based on the structure of EhpF and a bioinformatic analysis, it is proposed that EhpF and EhpG convert phenazine-1,6-dicarboxylate to 6-formylphenazine-1-carboxylate via an adenylyl intermediate.

  11. Antioxidant Potential and Inhibition of Key Enzymes Linked to Alzheimer’s Diseases and Diabetes Mellitus by Monoterpene-Rich Essential Oil from Sideritis galatica Bornm. Endemic to Turkey

    Directory of Open Access Journals (Sweden)

    Gökhan Zengin

    2016-03-01

    Full Text Available The present study was designated to (1 characterize the essential oil from S. galatica (SGEOs and (2 evaluate its antioxidant and enzyme inhibitory activities. Antioxidant capacity was tested different methods including free radical scavenging (DPPH, ABTS and NO, reducing power (FRAP and CUPRAC, metal chelating and phosphomolybdenum. Inhibitory activities were analyzed on acetylcholiesterase, butrylcholinesterase, α-amylase and α-glucosidase. SGEOs were chemically analyzed and identified by gas chromatography and gas chromatography/mass spectrophotometry. 23 components, representing 98.1% of SGEOs were identified. Monoterpene hydrocarbons (74.1%, especially α- (23.0% and β-pinene (32.2%, were the main constituents in SGEOs. The main sesquiterpene hydrocarbons were β-caryophyllene (16.9%, germacrene-D (1.2% and caryophyllene oxide (1.2%, respectively. Generally, SGEOs has shown moderate free radical, reducing power, metal chelating and enzyme inhibitory activities. These activities related to chemical profile in SGEOs. Our findings supported that the possible utility of SGEOs is a source of natural agents for food or pharmaceutical industries.

  12. Hypoxia decreases the expression of the two enzymes responsible for producing linear and cyclic tetrapyrroles in the heme biosynthetic pathway.

    Science.gov (United States)

    Vargas, Patrick D; Furuyama, Kazumichi; Sassa, Shigeru; Shibahara, Shigeki

    2008-12-01

    Heme is synthesized in all cell types in aerobic organisms. Hydroxymethylbilane synthase (HMBS) and uroporphyrinogen III synthase (UROS) catalyze two consecutive reactions in the heme biosynthetic pathway, generating the first linear and the first cyclic tetrapyrroles, respectively. Each of the HMBS and UROS genes contains the two separate promoters that generate ubiquitous and erythroid-specific mRNAs. Despite the functional significance of HMBS and UROS, regulation of their gene expression remains to be investigated. Here, we showed that hypoxia (1% O(2)) decreased the expression of ubiquitous mRNAs for HMBS and UROS by three- and twofold, respectively, in human hepatic cells (HepG2 and Hep3B), whereas the expression of ubiquitous and erythroid HMBS and UROS mRNAs remained unchanged in erythroid cells (YN-1 and K562). Unexpectedly, hypoxia did not decrease the half-life of HMBS mRNA (8.4 h under normoxia versus 9.1 h under hypoxia) or UROS mRNA (9.0 versus 10.4 h) in hepatic cells. It is therefore unlikely that a change in mRNA stability is responsible for the hypoxia-mediated decrease in the expression levels of these mRNAs. Furthermore, expression levels of HMBS and UROS mRNAs were decreased under normoxia by treatment with deferoxamine or cobalt chloride in hepatic cells, while hypoxia-inducible factor 1alpha was accumulated. Thus, the decrease in the expression of ubiquitous HMBS and UROS mRNAs is associated with accumulation of hypoxia-inducible factor 1alpha protein. In conclusion, the expression of HMBS and UROS mRNAs may be coordinately regulated, which represents a newly identified mechanism that is important for heme homeostasis.

  13. Characterization of the endosperm starch and the pleiotropic effects of biosynthetic enzymes on their properties in novel mutant rice lines with high resistant starch and amylose content.

    Science.gov (United States)

    Itoh, Yuuki; Crofts, Naoko; Abe, Misato; Hosaka, Yuko; Fujita, Naoko

    2017-05-01

    Resistant starch (RS) is beneficial to human health. In order to reduce the current prevalence of diabetes and obesity, several transgenic and mutant crops containing high RS content are being developed. RS content of steamed rice with starch-branching enzyme (BE)IIb-deficient mutant endosperms is considerably high. To understand the mechanisms of RS synthesis and to increase RS content, we developed novel mutant rice lines by introducing the gene encoding starch synthase (SS)IIa and/or granule-bound starch synthase (GBSS)I from an indica rice cultivar into a japonica rice-based BEIIb-deficient mutant line, be2b. Introduction of SSIIa from an indica rice cultivar produced higher levels of amylopectin chains with degree of polymerization (DP) 11-18 than those in be2b; the extent of the change was slight due to the shortage of donor chains for SSIIa (DP 6-12) owing to BEIIb deficiency. The introduction of GBSSI from an indica rice cultivar significantly increased amylose content (by approximately 10%) in the endosperm starch. RS content of the new mutant lines was the same as or slightly higher than that of the be2b parent line. The relationship linking starch structure, RS content, and starch biosynthetic enzymes in the new mutant lines has also been discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Method development and analysis of free HS and HS in proteoglycans from pre- and postmenopausal women: evidence for biosynthetic pathway changes in sulfotransferase and sulfatase enzymes.

    Science.gov (United States)

    Wei, Wei; Miller, Rebecca L; Leary, Julie A

    2013-06-18

    Heparan sulfate (HS) is one of the most complex and informative biopolymers found on the cell surface or in the extracellular matrix as either free HS fragments or constituents of HS proteoglycans (HSPGs). Analysis of free HS and HSPG sugar chains in human serum at the disaccharide level has great potential for early disease diagnosis and prognosis; however, the low concentration of HS in human serum, together with the complexity of the serum matrix, limits the information on HS. In this study, we present and validate the development of a new sensitive method for in-depth compositional analysis of free HS and HSPG sugar chains. This protocol involved several steps including weak anion exchange chromatography, ultrafiltration, and solid-phase extraction for enhanced detection prior to LC-MS/MS analysis. Using this protocol, a total of 51 serum samples from 26 premenopausal and 25 postmenopausal women were analyzed. Statistically significant differences in heparin/HS disaccharide profiles were observed. The proportion of N-acetylation and N-sulfation in both free HS and HSPG sugar chains were significantly different between pre- and postmenopausal women, indicating changes in N-deacetylase/N-sulfotransferases (NDSTs), the enzymes involved in the initial step of the biosynthetic pathway. Differences in the proportion of 6-O-sulfation suggest that 6-O-sulfotransferase and/or 6-O-sulfatase enzymes may also be implicated.

  15. Role of oxidative stress and the activity of ethylene biosynthetic enzymes on the formation of spongy tissue in 'Alphonso' mango.

    Science.gov (United States)

    Nagamani, J E; Shivashankara, K S; Roy, T K

    2010-06-01

    Spongy tissue formation in 'Alphonso' mangoes (Mangifera indica L) is a major national problem leading to loss for farmers and traders. Spongy tissue is whitish sponge like tissue formed near the seed with insipid taste and off odour. Lipid peroxidation of membranes as studied by malondialdehyde formation was significantly higher in spongy tissue. Activities of antioxidative enzymes like superoxide dismutase, catalase, peroxidase and polyphenol oxidase were lower in spongy tissue. Among the antioxidative enzymes, activities of catalase and peroxidases were severely reduced leading to membrane damage in spongy tissue. A significant reduction in 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase and accumulation of ACC was also observed in spongy tissue. However, ACC synthase activity in spongy tissue was more compared to healthy tissue. Results indicate that the membrane peroxidation leading to lower activity of ACC oxidase might lead to the formation of spongy tissue in 'Alphonso' mango.

  16. Structure of the d-alanylgriseoluteic acid biosynthetic protein EhpF, an atypical member of the ANL superfamily of adenylating enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Bera, Asim K.; Atanasova, Vesna [Center for Advanced Research in Biotechnology, The University of Maryland Biotechnology Institute, 9600 Gudelsky Drive, Rockville, MD 20850 (United States); Gamage, Swarna [Auckland Cancer Society Research Centre, School of Medicine, Faculty of Medical and Health Sciences, University of Auckland, Auckland (New Zealand); Robinson, Howard [Biology Department, Brookhaven National Laboratory, Upton, NY 11973 (United States); Parsons, James F., E-mail: parsonsj@umbi.umd.edu [Center for Advanced Research in Biotechnology, The University of Maryland Biotechnology Institute, 9600 Gudelsky Drive, Rockville, MD 20850 (United States)

    2010-06-01

    The structure of EhpF from P. agglomerans has been solved alone and in complex with phenazine-1,6-dicarboxylate. Apo EhpF was solved and refined in two different space groups at 1.95 and 2.3 Å resolution and the EhpF–phenazine-1,6-dicarboxylate complex structure was determined at 2.8 Å resolution. The structure of EhpF, a 41 kDa protein that functions in the biosynthetic pathway leading to the broad-spectrum antimicrobial compound d-alanylgriseoluteic acid (AGA), is reported. A cluster of approximately 16 genes, including ehpF, located on a 200 kbp plasmid native to certain strains of Pantoea agglomerans encodes the proteins that are required for the conversion of chorismic acid to AGA. Phenazine-1,6-dicarboxylate has been identified as an intermediate in AGA biosynthesis and deletion of ehpF results in accumulation of this compound in vivo. The crystallographic data presented here reveal that EhpF is an atypical member of the acyl-CoA synthase or ANL superfamily of adenylating enzymes. These enzymes typically catalyze two-step reactions involving adenylation of a carboxylate substrate followed by transfer of the substrate from AMP to coenzyme A or another phosphopantetheine. EhpF is distinguished by the absence of the C-terminal domain that is characteristic of enzymes from this family and is involved in phosphopantetheine binding and in the second half of the canonical two-step reaction that is typically observed. Based on the structure of EhpF and a bioinformatic analysis, it is proposed that EhpF and EhpG convert phenazine-1,6-dicarboxylate to 6-formylphenazine-1-carboxylate via an adenylyl intermediate.

  17. Demonstration that limonene is the first cyclic intermediate in the biosynthesis of oxygenated p-menthane monoterpenes in Mentha piperita and other Mentha species.

    Science.gov (United States)

    Kjonaas, R; Croteau, R

    1983-01-01

    The volatile oil of mature Mentha piperita (peppermint) leaves contains as major components the oxygenated p-menthane monoterpenes l-menthol (47%) and l-menthone (24%) as well as very low levels of the monoterpene olefins limonene (1%) and terpinolene (0.1%), which are considered to be probable precursors of the oxygenated derivatives. Immature leaves, which are actively synthesizing monoterpenes, produce an oil with comparatively higher levels of limonene (approximately 3%), and isolation of the pure olefin showed this compound to consist of approximately 80% of the l-(4S)-enantiomer and approximately 20% of the d-(4R)-enantiomer. The time course of incorporation of [U-14C]sucrose into the monoterpenes of M. piperita shoot tips was consistent with the initial formation of limonene and its subsequent conversion to menthone via pulegone. d,l-[9-3H]Limonene and [9,10-3H]terpinolene were prepared and tested directly as precursors of oxygenated p-menthane monoterpenes in M. piperita shoot tips. Limonene was readily incorporated into pulegone, menthone, and other oxygenated derivatives, whereas terpinolene was not appreciably incorporated into these compounds. Similarly, d,l-[9-3H]limonene was specifically incorporated into pulegone in Mentha pulegium and into the C-2-oxygenated derivative carvone in Mentha spicata, confirming the role of this olefin as the essential precursor of oxygenated p-menthane monoterpenes. Soluble enzyme preparations from the epidermis of immature M. piperita leaves converted the acyclic terpenoid precursor [1-3H]geranyl pyrophosphate to limonene as the major cyclic product, providing a further indication that this olefin plays a central role in the formation of oxygenated monoterpenes in Mentha. No free intermediates were detected in the cyclization of geranyl pyrophosphate to limonene, suggesting that the olefin is the first cyclic intermediate to arise in the pathway, and resolution of the biosynthetic limonene, by crystallization of the

  18. Effect of exogenous hormones on transcription levels of pyridoxal 5'-phosphate biosynthetic enzymes in the silkworm (Bombyx mori).

    Science.gov (United States)

    Huang, ShuoHao; Yang, HuanHuan; Yao, LiLi; Zhang, JianYun; Huang, LongQuan

    2016-01-01

    Vitamin B6 includes 6 pyridine derivatives, among which pyridoxal 5'-phosphate is a coenzyme for over 140 enzymes. Animals acquire their vitamin B6 from food. Through a salvage pathway, pyridoxal 5'-phosphate is synthesized from pyridoxal, pyridoxine or pyridoxamine, in a series of reactions catalyzed by pyridoxal kinase and pyridoxine 5'-phosphate oxidase. The regulation of pyridoxal 5'-phospahte biosynthesis and pyridoxal 5'-phospahte homeostasis are at the center of study for vitamin B6 nutrition. How pyridoxal 5'-phosphate biosynthesis is regulated by hormones has not been reported so far. Our previous studies have shown that pyridoxal 5'-phosphate level in silkworm larva displays cyclic developmental changes. In the current study, effects of exogenous juvenile hormone and molting hormone on the transcription level of genes coding for the enzymes involved in the biosynthesis of pyridoxal 5'-phospahte were examined. Results show that pyridoxal kinase and pyridoxine 5'-phosphate oxidase are regulated at the transcription level by development and are responsive to hormones. Molting hormone stimulates the expression of genes coding for pyridoxal kinase and pyridoxine 5'-phosphate oxidase, and juvenile hormone appears to work against molting hormone. Whether pyridoxal 5'-phosphate biosynthesis is regulated by hormones in general is an important issue for further studies.

  19. New N-Acetyltransferase Fold in the Structure and Mechanism of the Phosphonate Biosynthetic Enzyme FrbF

    Energy Technology Data Exchange (ETDEWEB)

    Bae, Brian; Cobb, Ryan E.; DeSieno, Matthew A.; Zhao, Huimin; Nair, Satish K. (UIUC)

    2015-10-15

    The enzyme FrbF from Streptomyces rubellomurinus has attracted significant attention due to its role in the biosynthesis of the antimalarial phosphonate FR-900098. The enzyme catalyzes acetyl transfer onto the hydroxamate of the FR-900098 precursors cytidine 5'-monophosphate-3-aminopropylphosphonate and cytidine 5'-monophosphate-N-hydroxy-3-aminopropylphosphonate. Despite the established function as a bona fide N-acetyltransferase, FrbF shows no sequence similarity to any member of the GCN5-like N-acetyltransferase (GNAT) superfamily. Here, we present the 2.0 {angstrom} resolution crystal structure of FrbF in complex with acetyl-CoA, which demonstrates a unique architecture that is distinct from those of canonical GNAT-like acetyltransferases. We also utilized the co-crystal structure to guide structure-function studies that identified the roles of putative active site residues in the acetyltransferase mechanism. The combined biochemical and structural analyses of FrbF provide insights into this previously uncharacterized family of N-acetyltransferases and also provide a molecular framework toward the production of novel N-acyl derivatives of FR-900098.

  20. Inhibition of green tea and the catechins against 1-deoxy-d-xylulose 5-phosphate reductoisomerase, the key enzyme of the MEP terpenoid biosynthetic pathway.

    Science.gov (United States)

    Hui, Xian; Liu, Hui; Tian, Fang-Lin; Li, Fei-Fei; Li, Heng; Gao, Wen-Yun

    2016-09-01

    1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) is the first committed enzyme in the MEP terpenoid biosynthetic pathway and also a validated antimicrobial target. Green tea which is rich in polyphenolic components such as the catechins, possesses a plenty of pharmacological activities, in particular an antibacterial effect. To uncover the antibacterial mechanism of green tea and to seek new DXR inhibitors from natural sources, the DXR inhibitory activity of green tea and its main antimicrobial catechins were investigated in this study. The results show that the raw extract of green tea and its ethyl acetate fraction are able to suppress DXR activity explicitly. Further determination of the DXR inhibitory capacity of eight catechin compounds demonstrates that the most active compound is gallocatechin gallate that is able to inhibit around 50% activity of DXR at 25μM. Based on these data, the primary structure-activity relationship of the catechins against DXR is discussed. This study would be very helpful to elucidate the antimicrobial mechanism of green tea and the catechins and also would be very useful to direct the rational utilization of them as food additives.

  1. Evolutionary diversification and characterization of the eubacterial gene family encoding DXR type II, an alternative isoprenoid biosynthetic enzyme.

    Science.gov (United States)

    Carretero-Paulet, Lorenzo; Lipska, Agnieszka; Pérez-Gil, Jordi; Sangari, Félix J; Albert, Victor A; Rodríguez-Concepción, Manuel

    2013-09-03

    Isoprenoids constitute a vast family of natural compounds performing diverse and essential functions in all domains of life. In most eubacteria, isoprenoids are synthesized through the methylerythritol 4-phosphate (MEP) pathway. The production of MEP is usually catalyzed by deoxyxylulose 5-phosphate reductoisomerase (DXR-I) but a few organisms use an alternative DXR-like enzyme (DXR-II). Searches through 1498 bacterial complete proteomes detected 130 sequences with similarity to DXR-II. Phylogenetic analysis identified three well-resolved clades: the DXR-II family (clustering 53 sequences including eleven experimentally verified as functional enzymes able to produce MEP), and two previously uncharacterized NAD(P)-dependent oxidoreductase families (designated DLO1 and DLO2 for DXR-II-like oxidoreductases 1 and 2). Our analyses identified amino acid changes critical for the acquisition of DXR-II biochemical function through type-I functional divergence, two of them mapping onto key residues for DXR-II activity. DXR-II showed a markedly discontinuous distribution, which was verified at several levels: taxonomic (being predominantly found in Alphaproteobacteria and Firmicutes), metabolic (being mostly found in bacteria with complete functional MEP pathways with or without DXR-I), and phenotypic (as no biological/phenotypic property was found to be preferentially distributed among DXR-II-containing strains, apart from pathogenicity in animals). By performing a thorough comparative sequence analysis of GC content, 3:1 dinucleotide frequencies, codon usage and codon adaptation indexes (CAI) between DXR-II sequences and their corresponding genomes, we examined the role of horizontal gene transfer (HGT), as opposed to an scenario of massive gene loss, in the evolutionary origin and diversification of the DXR-II subfamily in bacteria. Our analyses support a single origin of the DXR-II family through functional divergence, in which constitutes an exceptional model of

  2. The Vibrio cholerae quorum-sensing autoinducer CAI-1: analysis of the biosynthetic enzyme CqsA

    Energy Technology Data Exchange (ETDEWEB)

    Kelly, R.; Bolitho, M; Higgins, D; Lu, W; Ng, W; Jeffrey, P; Rabinowitz, J; Semmelhack, M; Hughson, F; Bassler, B

    2009-01-01

    Vibrio cholerae, the bacterium that causes the disease cholera, controls virulence factor production and biofilm development in response to two extracellular quorum-sensing molecules, called autoinducers. The strongest autoinducer, called CAI-1 (for cholera autoinducer-1), was previously identified as (S)-3-hydroxytridecan-4-one. Biosynthesis of CAI-1 requires the enzyme CqsA. Here, we determine the CqsA reaction mechanism, identify the CqsA substrates as (S)-2-aminobutyrate and decanoyl coenzyme A, and demonstrate that the product of the reaction is 3-aminotridecan-4-one, dubbed amino-CAI-1. CqsA produces amino-CAI-1 by a pyridoxal phosphate-dependent acyl-CoA transferase reaction. Amino-CAI-1 is converted to CAI-1 in a subsequent step via a CqsA-independent mechanism. Consistent with this, we find cells release {ge}100 times more CAI-1 than amino-CAI-1. Nonetheless, V. cholerae responds to amino-CAI-1 as well as CAI-1, whereas other CAI-1 variants do not elicit a quorum-sensing response. Thus, both CAI-1 and amino-CAI-1 have potential as lead molecules in the development of an anticholera treatment.

  3. CYP82Y1 is N-methylcanadine 1-hydroxylase, a key noscapine biosynthetic enzyme in opium poppy.

    Science.gov (United States)

    Dang, Thu-Thuy T; Facchini, Peter J

    2014-01-24

    Noscapine is a phthalideisoquinoline alkaloid investigated for its potent pharmacological properties. Although structurally elucidated more than a century ago, the biosynthesis of noscapine has not been established. Radiotracer studies have shown that noscapine is derived from the protoberberine alkaloid (S)-scoulerine and has been proposed to proceed through (S)-N-methylcanadine. However, pathway intermediates involved in the conversion of N-methylcanadine to noscapine have not been identified. We report the isolation and characterization of the cytochrome P-450 CYP82Y1, which catalyzes the 1-hydroxylation of N-methylcanadine to 1-hydroxy-N-methylcanadine. Comparison of transcript and metabolite profiles of eight opium poppy chemotypes revealed four cytochrome P-450s, three from the CYP82 and one from the CYP719 families, that were tightly correlated with noscapine accumulation. Recombinant CYP82Y1 was the only enzyme that accepted (R,S)-N-methylcanadine as a substrate with strict specificity and high affinity. As expected, CYP82Y1 was abundantly expressed in opium poppy stems where noscapine accumulation is highest among plant organs. Suppression of CYP82Y1 using virus-induced gene silencing caused a significant reduction in the levels of noscapine, narcotoline, and a putative downstream secoberbine intermediate and also resulted in increased accumulation of the upstream pathway intermediates scoulerine, tetrahydrocolum-bamine, canadine, and N-methylcanadine. The combined biochemical and physiological data support the 1-hydroxylation of (S)-N-methylcanadine catalyzed by CYP82Y1 as the first committed step in the formation of noscapine in opium poppy.

  4. Diurnal variations of mouse plasma and hepatic bile acid concentrations as well as expression of biosynthetic enzymes and transporters.

    Directory of Open Access Journals (Sweden)

    Yu-Kun Jennifer Zhang

    Full Text Available BACKGROUND: Diurnal fluctuation of bile acid (BA concentrations in the enterohepatic system of mammals has been known for a long time. Recently, BAs have been recognized as signaling molecules beyond their well-established roles in dietary lipid absorption and cholesterol homeostasis. METHODS AND RESULTS: The current study depicted diurnal variations of individual BAs detected by ultra-performance liquid chromatography/mass spectrometry (UPLC/MS in serum and livers collected from C57BL/6 mice fed a regular chow or a chow containing cholestyramine (resin. Circadian rhythms of mRNA of vital BA-related nuclear receptors, enzymes, and transporters in livers and ilea were determined in control- and resin-fed mice, as well as in farnesoid X receptor (FXR null mice. The circadian profiles of BAs showed enhanced bacterial dehydroxylation during the fasting phase and efficient hepatic reconjugation of BAs in the fed phase. The resin removed more than 90% of BAs with β-hydroxy groups, such as muricholic acids and ursodeoxycholic acid, from serum and livers, but did not exert as significant influence on CA and CDCA in both compartments. Both resin-fed and FXR-null mouse models indicate that BAs regulate their own biosynthesis through the FXR-regulated ileal fibroblast growth factor 15. BA flux also influences the daily mRNA levels of multiple BA transporters. CONCLUSION: BA concentration and composition exhibit circadian variations in mouse liver and serum, which influences the circadian rhythms of BA metabolizing genes in liver and ileum. The diurnal variations of BAs appear to serve as a signal that coordinates daily nutrient metabolism in mammals.

  5. Elevation of the Yields of Very Long Chain Polyunsaturated Fatty Acids via Minimal Codon Optimization of Two Key Biosynthetic Enzymes.

    Science.gov (United States)

    Xia, Fei; Li, Xueying; Li, Xinzheng; Zheng, Desong; Sun, Quanxi; Liu, Jiang; Li, Yaxiao; Hua, Jinping; Qi, Baoxiu

    2016-01-01

    Eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17) and Docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19) are nutritionally beneficial to human health. Transgenic production of EPA and DHA in oilseed crops by transferring genes originating from lower eukaryotes, such as microalgae and fungi, has been attempted in recent years. However, the low yield of EPA and DHA produced in these transgenic crops is a major hurdle for the commercialization of these transgenics. Many factors can negatively affect transgene expression, leading to a low level of converted fatty acid products. Among these the codon bias between the transgene donor and the host crop is one of the major contributing factors. Therefore, we carried out codon optimization of a fatty acid delta-6 desaturase gene PinD6 from the fungus Phytophthora infestans, and a delta-9 elongase gene, IgASE1 from the microalga Isochrysis galbana for expression in Saccharomyces cerevisiae and Arabidopsis respectively. These are the two key genes encoding enzymes for driving the first catalytic steps in the Δ6 desaturation/Δ6 elongation and the Δ9 elongation/Δ8 desaturation pathways for EPA/DHA biosynthesis. Hence expression levels of these two genes are important in determining the final yield of EPA/DHA. Via PCR-based mutagenesis we optimized the least preferred codons within the first 16 codons at their N-termini, as well as the most biased CGC codons (coding for arginine) within the entire sequences of both genes. An expression study showed that transgenic Arabidopsis plants harbouring the codon-optimized IgASE1 contained 64% more elongated fatty acid products than plants expressing the native IgASE1 sequence, whilst Saccharomyces cerevisiae expressing the codon optimized PinD6 yielded 20 times more desaturated products than yeast expressing wild-type (WT) PinD6. Thus the codon optimization strategy we developed here offers a simple, effective and low-cost alternative to whole gene synthesis for high expression of

  6. Elevation of the Yields of Very Long Chain Polyunsaturated Fatty Acids via Minimal Codon Optimization of Two Key Biosynthetic Enzymes.

    Directory of Open Access Journals (Sweden)

    Fei Xia

    Full Text Available Eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17 and Docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19 are nutritionally beneficial to human health. Transgenic production of EPA and DHA in oilseed crops by transferring genes originating from lower eukaryotes, such as microalgae and fungi, has been attempted in recent years. However, the low yield of EPA and DHA produced in these transgenic crops is a major hurdle for the commercialization of these transgenics. Many factors can negatively affect transgene expression, leading to a low level of converted fatty acid products. Among these the codon bias between the transgene donor and the host crop is one of the major contributing factors. Therefore, we carried out codon optimization of a fatty acid delta-6 desaturase gene PinD6 from the fungus Phytophthora infestans, and a delta-9 elongase gene, IgASE1 from the microalga Isochrysis galbana for expression in Saccharomyces cerevisiae and Arabidopsis respectively. These are the two key genes encoding enzymes for driving the first catalytic steps in the Δ6 desaturation/Δ6 elongation and the Δ9 elongation/Δ8 desaturation pathways for EPA/DHA biosynthesis. Hence expression levels of these two genes are important in determining the final yield of EPA/DHA. Via PCR-based mutagenesis we optimized the least preferred codons within the first 16 codons at their N-termini, as well as the most biased CGC codons (coding for arginine within the entire sequences of both genes. An expression study showed that transgenic Arabidopsis plants harbouring the codon-optimized IgASE1 contained 64% more elongated fatty acid products than plants expressing the native IgASE1 sequence, whilst Saccharomyces cerevisiae expressing the codon optimized PinD6 yielded 20 times more desaturated products than yeast expressing wild-type (WT PinD6. Thus the codon optimization strategy we developed here offers a simple, effective and low-cost alternative to whole gene synthesis for high

  7. Elevation of the Yields of Very Long Chain Polyunsaturated Fatty Acids via Minimal Codon Optimization of Two Key Biosynthetic Enzymes

    Science.gov (United States)

    Zheng, Desong; Sun, Quanxi; Liu, Jiang; Li, Yaxiao; Hua, Jinping

    2016-01-01

    Eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17) and Docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19) are nutritionally beneficial to human health. Transgenic production of EPA and DHA in oilseed crops by transferring genes originating from lower eukaryotes, such as microalgae and fungi, has been attempted in recent years. However, the low yield of EPA and DHA produced in these transgenic crops is a major hurdle for the commercialization of these transgenics. Many factors can negatively affect transgene expression, leading to a low level of converted fatty acid products. Among these the codon bias between the transgene donor and the host crop is one of the major contributing factors. Therefore, we carried out codon optimization of a fatty acid delta-6 desaturase gene PinD6 from the fungus Phytophthora infestans, and a delta-9 elongase gene, IgASE1 from the microalga Isochrysis galbana for expression in Saccharomyces cerevisiae and Arabidopsis respectively. These are the two key genes encoding enzymes for driving the first catalytic steps in the Δ6 desaturation/Δ6 elongation and the Δ9 elongation/Δ8 desaturation pathways for EPA/DHA biosynthesis. Hence expression levels of these two genes are important in determining the final yield of EPA/DHA. Via PCR-based mutagenesis we optimized the least preferred codons within the first 16 codons at their N-termini, as well as the most biased CGC codons (coding for arginine) within the entire sequences of both genes. An expression study showed that transgenic Arabidopsis plants harbouring the codon-optimized IgASE1 contained 64% more elongated fatty acid products than plants expressing the native IgASE1 sequence, whilst Saccharomyces cerevisiae expressing the codon optimized PinD6 yielded 20 times more desaturated products than yeast expressing wild-type (WT) PinD6. Thus the codon optimization strategy we developed here offers a simple, effective and low-cost alternative to whole gene synthesis for high expression of

  8. Qualitative and quantitative variation in monoterpene co-occurrence and composition in the essential oil of Thymus vulgaris chemotypes.

    Science.gov (United States)

    Thompson, John D; Chalchat, Jean-Claude; Michet, André; Linhart, Yan B; Ehlers, Bodil

    2003-04-01

    Thymus vulgaris has a chemical polymorphism with six different chemotypes that show marked spatial segregation in nature. Although some populations have a single chemotype in majority, many have two or three chemotypes. In this study we analyze the quantitative variation among T. vulgaris populations in the percentage of oil composed of the dominant monoterpene(s) for each chemotype. In general, phenolic chemotypes (thymol and carvacrol), which occur at the end of the biosynthetic chain, have a significantly lower proportion of their oil composed of their dominant monoterpene than nonphenolic chemotypes (geraniol, alpha-terpineol, and linalool). This is due to the presence of high amounts of precursors (gamma-terpinene and paracymene) in the oil of phenolic chemotypes. The essential oil of the nonphenolic thuyanol chemotype has four characteristic monoterpenes that together make up a lower proportion of the oil than the single dominant monoterpene of the other nonphenolic chemotypes. For all chemotypes, the percentage composition of the dominant monoterpene decreased significantly at sites where the chemotype is not the majority type. This decrease is correlated with a significant increase in either the proportion of the two precursors for the thymol chemotype or the monoterpenes characteristic of the other chemotypes at the site. The latter result suggests that a plant with dominant genes is responsible for the production of different monoterpenes can produce several molecules.

  9. Determination and identification of estrogenic compounds generated with biosynthetic enzymes using hyphenated screening assays, high resolution mass spectrometry and off-line NMR

    NARCIS (Netherlands)

    Vlieger, de J.S.B.; Kolkman, A.J.; Ampt, K.A.M.; Commandeur, J.N.M.; Vermeulen, N.P.E.; Kool, J.; Wijmenga, S.S.; Niessen, W.M.A.; Irth, H.; Honing, M.

    2010-01-01

    This paper describes the determination and identification of active and inactive estrogenic compounds produced by biosynthetic methods. A hyphenated screening assay towards the human estrogen receptor ligand binding domain (hER)α and hERβ integrating target–ligand interactions and liquid chromatogra

  10. In vivo roles of fatty acid-biosynthetic enzymes in biosynthesis of biotin and α-lipoic acid in Corynebacterium glutamicum.

    Science.gov (United States)

    Ikeda, Masato; Nagashima, Takashi; Nakamura, Eri; Kato, Ryosuke; Ohshita, Masakazu; Hayashi, Mikiro; Takeno, Seiki

    2017-07-28

    For fatty acid biosynthesis, Corynebacterium glutamicum uses two type I fatty acid synthases (FAS-I), FasA and FasB, in addition to acetyl-CoA carboxylase (ACC) consisting of AccBC, AccD1, and AccE. The in vivo roles of the enzymes in supplying precursors for biotin and α-lipoic acid remain unclear. Here, we report genetic evidence demonstrating that the biosynthesis of these cofactors is linked to fatty acid biosynthesis through the FAS-I pathway. For this study, we used wild-type C. glutamicum and its derived biotin-vitamer producer BFI-5, which was engineered to express Escherichia coli bioBF and Bacillus subtilis bioI Disruption of either fasA or fasB in strain BFI-5 led to decreased production of biotin-vitamers, whereas its amplification contributed to increased production, with a larger impact of fasA in both cases. Double disruptions of fasA and fasB resulted in no biotin-vitamer production. The acc genes showed a positive effect on production when amplified simultaneously. Augmented fatty acid biosynthesis was also reflected on pimelic acid production when carbon flow was blocked at the BioF reaction. These results indicate that carbon flow down the FAS-I pathway is destined for channeling into the biotin-biosynthetic pathway, and that FasA in particular has a significant impact on precursor supply. In contrast, fasB disruption resulted in auxotrophy for lipoic acid or its precursor octanoic acid in both wild-type and BFI-5 strains. The phenotypes were fully complemented by plasmid-mediated expression of fasB, but not fasA These results reveal that FasB plays a specific physiological role in lipoic acid biosynthesis in C. glutamicumIMPORTANCE For the de novo biosynthesis of fatty acids, C. glutamicum exceptionally uses eukaryotic, multifunctional type I fatty acid synthase (FAS-I) system comprising FasA and FasB, in contrast to most bacteria, such as E. coli and B. subtilis, which use individual, nonaggregating type II fatty acid synthase (FAS-II) system

  11. Genetic and biochemical characterization of a novel monoterpene epsilon-lactone hydrolase from Rhodococcus erythropolis DCL14

    NARCIS (Netherlands)

    Vlugt-Bergmans, van der C.J.B.; Werf, van der M.J.

    2001-01-01

    A monoterpene ε-lactone hydrolase (MLH) from Rhodococcus erythropolis DCL14, catalyzing the ring opening of lactones which are formed during degradation of several monocyclic monoterpenes, including carvone and menthol, was purified to apparent homogeneity. It is a monomeric enzyme of 31 kDa that is

  12. Genetic and biochemical characterization of a novel monoterpene e-lactone hydrolase from Rhodococcus erythropolis DCL14

    NARCIS (Netherlands)

    Vlugt-Bergmans, C.J.B. van der; Werf, M.J. van der

    2001-01-01

    A monoterpene ε-lactone hydrolase (MLH) from Rhodococcus erythropolis DCL14, catalyzing the ring opening of lactones which are formed during degradation of several monocyclic monoterpenes, including carvone and menthol, was purified to apparent homogeneity. It is a monomeric enzyme of 31 kDa that is

  13. Monoterpene emission from ponderosa pine

    Science.gov (United States)

    Lerdau, Manual; Dilts, Stephen B.; Westberg, Hal; Lamb, Brian K.; Allwine, Eugene J.

    1994-01-01

    We explore the variability in monoterpene emissions from ponderosa pine beyond that which can be explained by temperature alone. Specifically, we examine the roles that photosynthesis and needle monoterpene concentrations play in controlling emissions. We measure monoterpene concentrations and emissions, photosynthesis, temperature, and light availability in the late spring and late summer in a ponderosa pine forest in central Oregon. We use a combination of measurements from cuvettes and Teflon bag enclosures to show that photosynthesis is not correlated with emissions in the short term. We also show that needle monoterpene concentrations are highly correlated with emissions for two compounds, alpha-pinene and beta-pinene, but that Delta-carene concentrations are not correlated with emissions. We suggest that direct effects of light and photosynthesis do not need to be included in emission algorithms. Our results indicate that the role of needle concentration bears further investigation; our results for alpha-pinene and beta-pinene are explainable by a Raoult's law relationship, but we cannot yet explain the cause of our results with Delta-carene.

  14. Adaptive evolution of the chrysanthemyl diphosphate synthase gene involved in irregular monoterpene metabolism

    Directory of Open Access Journals (Sweden)

    Liu Ping-Li

    2012-11-01

    Full Text Available Abstract Background Chrysanthemyl diphosphate synthase (CDS is a key enzyme in biosynthetic pathways producing pyrethrins and irregular monoterpenes. These compounds are confined to plants of the tribe Anthemideae of the Asteraceae, and play an important role in defending the plants against herbivorous insects. It has been proposed that the CDS genes arose from duplication of the farnesyl diphosphate synthase (FDS gene and have different function from FDSs. However, the duplication time toward the origin of CDS and the evolutionary force behind the functional divergence of the CDS gene are still unknown. Results Two duplication events were detected in the evolutionary history of the FDS gene family in the Asteraceae, and the second duplication led to the origin of CDS. CDS occurred after the divergence of the tribe Mutisieae from other tribes of Asteraceae but before the birth of the Anthemideae tribe. After its origin, CDS accumulated four mutations in sites homologous to the substrate-binding and catalysis sites of FDS. Of these, two sites were involved in the binding of the nucleophilic substrate isopentenyl diphosphate in FDS. Maximum likelihood analyses showed that some sites in CDS were under positive selection and were scattered throughout primary sequences, whereas in the three-dimensional structure model they clustered in the large central cavity. Conclusion Positive selection associated with gene duplication played a major role in the evolution of CDS.

  15. Monoterpenes in essential oils. Biosynthesis and properties.

    Science.gov (United States)

    Loza-Tavera, H

    1999-01-01

    Monoterpenes are compounds found in the essential oils extracted from many plants, including fruits, vegetables, spices and herbs. These compounds contribute to the flavor and aroma of plant from which they are extracted. Monoterpenes are acyclic, monocyclic, or bicyclic C30 compounds synthesized by monoterpene synthases using geranyl pyrophosphate (GPP) as substrate. GPP is also the precursor in the synthesis of farnesyl pyrophosphate (FPP) and geranyl-geranyl pyrophosphate (GGPP), two important compounds in cell metabolism of animals, plants and yeast. Monoterpene cyclases produce cyclic monoterpenes through a multistep mechanism involving a universal intermediate, a terpinyl cation which can be transformed to several compounds. Experimental studies, using animal cancer models, have demonstrated that some monoterpenes possess anticarcinogenic properties, acting at different cellular and molecular levels. From these discoveries it seems clear that monoterpenes could be considered as effective, nontoxic dietary antitumorigenic agents that hold promise as a novel class of anticancer drugs.

  16. Monoterpene synthases of loblolly pine (Pinus taeda) produce pinene isomers and enantiomers.

    Science.gov (United States)

    Phillips, M A; Savage, T J; Croteau, R

    1999-12-01

    The turpentine fraction of conifer oleoresin is a complex mixture of monoterpene olefins and plays important roles in defense and in the mediation of chemical communication between conifer hosts and insect predators. The stereochemistry of the turpentine monoterpenes is critical in these interactions, influencing host recognition, toxicity, and potency of derived pheromones, and the stereochemical composition of these compounds lends insight into their biogenetic origin, with implications for the numbers and types of enzymes responsible and their corresponding genes. Analysis of the oleoresin from several tissues of loblolly pine (Pinus taeda) showed the derived turpentine to consist mainly of (+)-(3R:5R)-alpha-pinene and (-)-(3S:5S)-beta-pinene. Cell-free extracts from xylem tissue yielded three monoterpene synthases which together account for the monoterpene isomer and enantiomer content of the turpentine of this tissue. The major products of these enzymes, produced from the universal precursor of monoterpenes, geranyl diphosphate, were shown to be (+)-alpha-pinene, (-)-alpha-pinene, and (-)-beta-pinene, respectively. In most properties (molecular mass of approximately 60 kDa, K(m) for geranyl diphosphate of 3 microM, requirement for monovalent and divalent cations), these enzymes resemble other monoterpene synthases from conifer species.

  17. Monoterpene Indole Alkaloids from the Fruit of Tabernaemontana litoralis and Differential Alkaloid Composition in Various Fruit Components.

    Science.gov (United States)

    Qu, Yang; Simonescu, Razvan; De Luca, Vincenzo

    2016-12-23

    Two new monoterpene indole alkaloids, isoakuammiline (1) and 18-hydroxypseudovincadifformine (2), and five known alkaloids, coronaridine (3), heyneanine (4), 3,19-oxidocoronaridine (5), tabersonine, and strictosidine, were identified from the fruit of Tabernaemontana litoralis. The structures of the alkaloids were determined using NMR and MS data analyses. While 18-hydroxypseudovincadifformine (2) showed a new hydroxylation pattern, isoakuammiline (1) revealed a novel skeleton for monoterpene indole alkaloids. In spite of the isolation of stemmadenine from the fruit tissues in other Tabernaemontana species, this vital biosynthetic precursor of iboga, aspidosperma, and pseudoaspidosperma skeletons was not found in T. litoralis.

  18. Biotransformations of monoterpenes by photoautotrophic micro-organisms.

    Science.gov (United States)

    Balcerzak, L; Lipok, J; Strub, D; Lochyński, S

    2014-12-01

    Monoterpenes are widely used in food technology, cosmetic and pharmaceutical industries and as compounds of agricultural importance. It is known that compounds comprising this class can be transformed by a variety of organisms, namely by: bacteria, fungi, yeasts, plants or isolated enzymes. Biotransformations, as one of the most important tools of green chemistry, allow obtaining new products using whole cells of micro-organisms or isolated enzymes in mild reaction conditions. Therefore, biotransformations of monoterpenes, by different type of reaction such as: epoxidation, oxidation and stereoselective hydroxylation, resulted in the production of so desired, enantiomerically defined compounds that can be advised as natural seem to be interesting. Bearing in mind that such processes are carried out also by easy to maintain, photoautotrophic micro-organisms cultivated at large scale, this paper is focused on biotransformations of acyclic, monocyclic and bicyclic monoterpenes by freshwater or haliphylic cyanobacteria and microalgae on the way of mainly stereoselective hydroxylation. Moreover, aspects of potential industrial application of obtained products in medicine, perfume, cosmetics and food industry are discussed.

  19. Aedes aegypti juvenile hormone acid methyl transferase, the ultimate enzyme in the biosynthetic pathway of juvenile hormone III, exhibits substrate control

    Science.gov (United States)

    We report on the cloning, sequencing, characterization, 3D modeling and docking of Aedes aegypti juvenile hormone acid methyl transferase (AeaJHAMT), the enzyme that converts juvenile hormone acid (JHA) into juvenile hormone (JH). Purified recombinant AeaJHAMT was extensively characterized for enzym...

  20. Metabolic engineering of monoterpene biosynthesis in plants

    NARCIS (Netherlands)

    Lücker, J.

    2002-01-01

    Monoterpenes are a large group of compounds that belong to the terpenoid family of natural compounds in plants. They are small, volatile, lipophilic substances of which around one thousand different structures have been identified. Monoterpenes are

  1. Isolation and characterization of three new monoterpene synthases from Artemisia annua

    Directory of Open Access Journals (Sweden)

    Ju-Xin eRuan

    2016-05-01

    Full Text Available Artemisia annua, an annual herb used in traditional Chinese medicine, produces a wealth of monoterpenes and sesquiterpenes, including the well-known sesquiterpene lactone artemisinin, an active ingredient in the treatment for malaria. Here we report three new monoterpene synthases of A. annua. From a glandular trichome cDNA library, monoterpene synthases of AaTPS2, AaTPS5 and AaTPS6, were isolated and characterized. The recombinant proteins of AaTPS5 and AaTPS6 produced multiple products with camphene and 1,8-cineole as major products, respectively, and AaTPS2 produced a single product, β-myrcene. Although both Mg2+ and Mn2+ were able to support their catalytic activities, altered product spectrum was observed in the presence of Mn2+ for AaTPS2 and AaTPS5. Analysis of extracts of aerial tissues and root of A. annua with gas chromatography-mass spectrometry (GC-MS detected more than 20 monoterpenes, of which the three enzymes constituted more than 1/3 of the total. Mechanical wounding induced the expression of all three monoterpene synthase genes, and transcript levels of AaTPS5 and AaTPS6 were also elevated after treatments with phytohormones of methyl jasmonate (MeJA, salicylic acid (SA and gibberellin (GA, suggesting a role of these monoterpene synthases in plant-environment interactions. The three new monoterpene synthases reported here further our understanding of molecular basis of monoterpene biosynthesis and regulation in plant.

  2. Potential of Synechocystis PCC 6803 as a novel cyanobacterial chassis for heterologous expression of enzymes in the trans-resveratrol biosynthetic pathway.

    Science.gov (United States)

    Tantong, Supaluk; Incharoensakdi, Aran; Sirikantaramas, Supaart; Lindblad, Peter

    2016-05-01

    Selected model strains of phototrophic cyanobacteria have been genetically engineered for heterologous expression of numerous enzymes. In the present study, we initially explored the heterologous expression of enzymes involved in trans-resveratrol production, namely, the production of tyrosine ammonia-lyase, coumaroyl CoA-ligase, and stilbene synthase, in the unicellular cyanobacterium Synechocystis PCC 6803. Under the promoters Ptrc1Ocore and Ptrc1O, the respective genes were transcribed and translated into the corresponding soluble proteins at concentrations of 16-34 μg L(-1). The expression levels of these enzymes did not affect the growth rate of the cyanobacterial cells. Interestingly, coumaroyl CoA-ligase expression slightly increased the chlorophyll a content of the cells. Overall, our results suggest that the complete pathway of trans-resveratrol production can be engineered in Synechocystis PCC 6803. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Monoterpene biosynthesis potential of plant subcellular compartments

    NARCIS (Netherlands)

    Dong, L.; Jongedijk, E.J.; Bouwmeester, H.J.; Krol, van der A.R.

    2016-01-01

    Subcellular monoterpene biosynthesis capacity based on local geranyl diphosphate (GDP) availability or locally boosted GDP production was determined for plastids, cytosol and mitochondria. A geraniol synthase (GES) was targeted to plastids, cytosol, or mitochondria. Transient expression in Nicotiana

  4. Alleviating monoterpene toxicity using a two-phase extractive fermentation for the bioproduction of jet fuel mixtures in Saccharomyces cerevisiae.

    Science.gov (United States)

    Brennan, Timothy C R; Turner, Christopher D; Krömer, Jens O; Nielsen, Lars K

    2012-10-01

    Monoterpenes are a diverse class of compounds with applications as flavors and fragrances, pharmaceuticals and more recently, jet fuels. Engineering biosynthetic pathways for monoterpene production in microbial hosts has received increasing attention. However, monoterpenes are highly toxic to many microorganisms including Saccharomyces cerevisiae, a widely used industrial biocatalyst. In this work, the minimum inhibitory concentration (MIC) for S. cerevisiae was determined for five monoterpenes: β-pinene, limonene, myrcene, γ-terpinene, and terpinolene (1.52, 0.44, 2.12, 0.70, 0.53 mM, respectively). Given the low MIC for all compounds tested, a liquid two-phase solvent extraction system to alleviate toxicity during fermentation was evaluated. Ten solvents were tested for biocompatibility, monoterpene distribution, phase separation, and price. The solvents dioctyl phthalate, dibutyl phthalate, isopropyl myristate, and farnesene showed greater than 100-fold increase in the MIC compared to the monoterpenes in a solvent-free system. In particular, the MIC for limonene in dibutyl phthalate showed a 702-fold (308 mM, 42.1 g L(-1) of limonene) improvement while cell viability was maintained above 90%, demonstrating that extractive fermentation is a suitable tool for the reduction of monoterpene toxicity. Finally, we estimated that a limonane to farnesane ratio of 1:9 has physicochemical properties similar to traditional Jet-A aviation fuel. Since farnesene is currently produced in S. cerevisiae, its use as a co-product and extractant for microbial terpene-based jet fuel production in a two-phase system offers an attractive bioprocessing option. Copyright © 2012 Wiley Periodicals, Inc.

  5. Gene Coexpression Analysis Reveals Complex Metabolism of the Monoterpene Alcohol Linalool in Arabidopsis FlowersW

    NARCIS (Netherlands)

    Ginglinger, J.F.; Boachon, B.; Hofer, R.; Paetz, C.; Kollner, T.G.; Miesch, L.; Lugan, R.; Baltenweck, R.; Mutterer, J.; Ullman, P.; Verstappen, F.W.A.; Bouwmeester, H.J.

    2013-01-01

    The cytochrome P450 family encompasses the largest family of enzymes in plant metabolism, and the functions of many of its members in Arabidopsis thaliana are still unknown. Gene coexpression analysis pointed to two P450s that were coexpressed with two monoterpene synthases in flowers and were thus

  6. Gene Coexpression Analysis Reveals Complex Metabolism of the Monoterpene Alcohol Linalool in Arabidopsis FlowersW

    NARCIS (Netherlands)

    Ginglinger, J.F.; Boachon, B.; Hofer, R.; Paetz, C.; Kollner, T.G.; Miesch, L.; Lugan, R.; Baltenweck, R.; Mutterer, J.; Ullman, P.; Verstappen, F.W.A.; Bouwmeester, H.J.

    2013-01-01

    The cytochrome P450 family encompasses the largest family of enzymes in plant metabolism, and the functions of many of its members in Arabidopsis thaliana are still unknown. Gene coexpression analysis pointed to two P450s that were coexpressed with two monoterpene synthases in flowers and were thus

  7. (—) S-adenosyl-L-methionine-magnesium Protoporphyrin Methyltransferase, an Enzyme in the Biosynthetic Pathway of Chlorophyll in Zea mays 1

    Science.gov (United States)

    Radmer, Richard J.; Bogorad, Lawrence

    1967-01-01

    The enzyme (—) S-adenosyl-L-methionine-magnesium protoporphyrin methyltransferase, which catalyzes the transfer of the methyl group from (—) S-adenosyl-L-methionine to magnesium protoporphyrin to form magnesium protoporphyrin monomethyl ester, has been detected in chloroplasts isolated from Zea mays. Zinc protoporphyrin and free protoporphyrin also act as substrates in the system, although neither one is as active as magnesium protoporphyrin. The following scheme of chlorophyll synthesis in higher plants is proposed: δ-aminolevulinic acid → → → protoporphyrin → magnesium protoporphyrin → magnesium protoporphyrin monomethyl ester → → → chlorophyll a. PMID:6045301

  8. Enzyme

    Science.gov (United States)

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  9. A comparison of new measurements of total monoterpene flux with improved measurements of speciated monoterpene flux

    Directory of Open Access Journals (Sweden)

    A. Lee

    2004-12-01

    Full Text Available Many monoterpenes have been identified in forest emissions using gas chromatography (GC. Until now, it has been impossible to determine whether all monoterpenes are appropriately measured using GC techniques. We used a proton transfer reaction mass spectrometer (PTR-MS coupled with the eddy covariance (EC technique to measure mixing ratios and fluxes of total monoterpenes above a ponderosa pine plantation. We compared PTR-MS-EC results with simultaneous measurements of eight speciated monoterpenes, β-pinene, α-pinene, 3-carene, d-limonene, β-phellandrene, α-terpinene, camphene, and terpinolene, made with an automated, in situ gas chromatograph with flame ionization detectors (GC-FID, coupled to a relaxed eddy accumulation system (REA. Monoterpene mixing ratios and fluxes measured by PTR-MS averaged 30±2.3% and 31±9.2% larger than by GC-FID, with larger differences at night than during the day. Four unidentified peaks that correlated with β-pinene were resolved in the chromatograms and completely accounted for the daytime difference and reduced the nighttime difference to 19±3.4%. Measurements of total monoterpenes by PTR-MS-EC indicated that GC-FID-REA measured the common, longer-lived monoterpenes well, but that additional monoterpenes were emitted from the ecosystem that represented an important contribution to the total mixing ratio above the forest at night, and that must have been oxidized during the day before they escaped the forest canopy.

  10. Monoterpenes in the glandular trichomes of tomato are synthesized from a neryl diphosphate precursor rather than geranyl diphosphate.

    Science.gov (United States)

    Schilmiller, Anthony L; Schauvinhold, Ines; Larson, Matthew; Xu, Richard; Charbonneau, Amanda L; Schmidt, Adam; Wilkerson, Curtis; Last, Robert L; Pichersky, Eran

    2009-06-30

    We identified a cis-prenyltransferase gene, neryl diphosphate synthase 1 (NDPS1), that is expressed in cultivated tomato (Solanum lycopersicum) cultivar M82 type VI glandular trichomes and encodes an enzyme that catalyzes the formation of neryl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. mRNA for a terpene synthase gene, phellandrene synthase 1 (PHS1), was also identified in these glands. It encodes an enzyme that uses neryl diphosphate to produce beta-phellandrene as the major product as well as a variety of other monoterpenes. The profile of monoterpenes produced by PHS1 is identical with the monoterpenes found in type VI glands. PHS1 and NDPS1 map to chromosome 8, and the presence of a segment of chromosome 8 derived from Solanum pennellii LA0716 causes conversion from the M82 gland monoterpene pattern to that characteristic of LA0716 plants. The data indicate that, contrary to the textbook view of geranyl diphosphate as the "universal" substrate of monoterpene synthases, in tomato glands neryl diphosphate serves as a precursor for the synthesis of monoterpenes.

  11. Monoterpene metabolism. Cloning, expression, and characterization of menthone reductases from peppermint.

    Science.gov (United States)

    Davis, Edward M; Ringer, Kerry L; McConkey, Marie E; Croteau, Rodney

    2005-03-01

    (-)-Menthone is the predominant monoterpene produced in the essential oil of maturing peppermint (Mentha x piperita) leaves during the filling of epidermal oil glands. This early biosynthetic process is followed by a second, later oil maturation program (approximately coincident with flower initiation) in which the C3-carbonyl of menthone is reduced to yield (-)-(3R)-menthol and (+)-(3S)-neomenthol by two distinct NADPH-dependent ketoreductases. An activity-based in situ screen, by expression in Escherichia coli of 23 putative redox enzymes from an immature peppermint oil gland expressed sequence tag library, was used to isolate a cDNA encoding the latter menthone:(+)-(3S)-neomenthol reductase. Reverse transcription-PCR amplification and RACE were used to acquire the former menthone:(-)-(3R)-menthol reductase directly from mRNA isolated from the oil gland secretory cells of mature leaves. The deduced amino acid sequences of these two reductases share 73% identity, provide no apparent subcellular targeting information, and predict inclusion in the short-chain dehydrogenase/reductase family of enzymes. The menthone:(+)-(3S)-neomenthol reductase cDNA encodes a 35,722-D protein, and the recombinant enzyme yields 94% (+)-(3S)-neomenthol and 6% (-)-(3R)-menthol from (-)-menthone as substrate, and 86% (+)-(3S)-isomenthol and 14% (+)-(3R)-neoisomenthol from (+)-isomenthone as substrate, has a pH optimum of 9.3, and K(m) values of 674 mum, > 1 mm, and 10 mum for menthone, isomenthone, and NADPH, respectively, with a k(cat) of 0.06 s(-1). The recombinant menthone:(-)-(3R)-menthol reductase has a deduced size of 34,070 D and converts (-)-menthone to 95% (-)-(3R)-menthol and 5% (+)-(3S)-neomenthol, and (+)-isomenthone to 87% (+)-(3R)-neoisomenthol and 13% (+)-(3S)-isomenthol, displays optimum activity at neutral pH, and has K(m) values of 3.0 mum, 41 mum, and 0.12 mum for menthone, isomenthone, and NADPH, respectively, with a k(cat) of 0.6 s(-1). The respective activities of

  12. Branched-chain amino acids inhibit the TGF-beta-induced down-regulation of taurine biosynthetic enzyme cysteine dioxygenase in HepG2 cells.

    Science.gov (United States)

    Hagiwara, Asami; Ishizaki, Sonoko; Takehana, Kenji; Fujitani, Shoji; Sonaka, Ichiro; Satsu, Hideo; Shimizu, Makoto

    2014-05-01

    Taurine deficiency has been suggested to contribute to the pathogenesis and complications of advanced hepatic diseases. The molecular basis for a low level of taurine associated with hepatic failure is largely unknown. Using carbon tetrachloride (CCl4)-induced cirrhotic rat model, we found that the activity and expression of cysteine dioxygenase (CDO), a rate-limiting enzyme in taurine synthesis, were significantly decreased in the liver of these rats. To investigate the underlying mechanisms for the suppression, we examined the effects of pathological cytokines on CDO expression in human hepatoma HepG2 cells. Among the several cytokines, transforming growth factor-β (TGF-β), one of the key mediators of fibrogenesis, suppressed Cdo1 gene transcription through the MEK/ERK pathway. Finally, we further examined potential effects of branched-chain amino acids (BCAA) on CDO expression, as it has been reported that oral BCAA supplementation increased plasma taurine level in the patients with liver cirrhosis. BCAA, especially leucine, promoted Cdo1 gene transcription, and attenuated TGF-β-mediated suppression of Cdo1 gene expression. These results indicate that the low plasma level of taurine in advanced hepatic disease is due to decreased hepatic CDO expression, which can be partly attributed to suppressive effect of TGF-β on Cdo1 gene transcription. Furthermore, our observation that BCAA promotes Cdo1 expression suggests that BCAA may be therapeutically useful to improve hepatic taurine metabolism and further suppress dysfunctions associated with low level of taurine in hepatic diseases.

  13. Evaluation of Biosynthetic Pathway and Engineered Biosynthesis of Alkaloids

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    Shinji Kishimoto

    2016-08-01

    Full Text Available Varieties of alkaloids are known to be produced by various organisms, including bacteria, fungi and plants, as secondary metabolites that exhibit useful bioactivities. However, understanding of how those metabolites are biosynthesized still remains limited, because most of these compounds are isolated from plants and at a trace level of production. In this review, we focus on recent efforts in identifying the genes responsible for the biosynthesis of those nitrogen-containing natural products and elucidating the mechanisms involved in the biosynthetic processes. The alkaloids discussed in this review are ditryptophenaline (dimeric diketopiperazine alkaloid, saframycin (tetrahydroisoquinoline alkaloid, strictosidine (monoterpene indole alkaloid, ergotamine (ergot alkaloid and opiates (benzylisoquinoline and morphinan alkaloid. This review also discusses the engineered biosynthesis of these compounds, primarily through heterologous reconstitution of target biosynthetic pathways in suitable hosts, such as Escherichia coli, Saccharomyces cerevisiae and Aspergillus nidulans. Those heterologous biosynthetic systems can be used to confirm the functions of the isolated genes, economically scale up the production of the alkaloids for commercial distributions and engineer the biosynthetic pathways to produce valuable analogs of the alkaloids. In particular, extensive involvement of oxidation reactions catalyzed by oxidoreductases, such as cytochrome P450s, during the secondary metabolite biosynthesis is discussed in details.

  14. The Simultaneous Repression of CCR and CAD, Two Enzymes of the Lignin Biosynthetic Pathway, Results in Sterility and Dwarfism in Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    Johanne Thévenin; Brigitte Pollet; Bruno Letarnec; Luc Saulnier; Lionel Gissot; Alessandra Maia-Grondard; Catherine Lapierre; Lise Jouanina

    2011-01-01

    Cinnamoyl CoA reductase(CCR)and cinnamyl alcohol dehydrogenase(CAD)catalyze the last steps of monolignol biosynthesis.In Arabidopsis,one CCR gene(CCR1,At1g15950)and two CAD genes(CAD C At3g19450 and CAD D At4g34230)are involved in this pathway.A triple cad c cad d ccr1 mutant,named ccc,was obtained.This mutant displays a severe dwarf phenotype and male sterility.The lignin content in ccc mature stems is reduced to 50% of the wild-type level.In addition,stem lignin structure is severely affected,as shown by the dramatic enrichment in resistant inter-unit bonds and incorporation into the polymer of monolignol precursors such as coniferaldehyde,sinapaldehyde,and ferulic acid.Male sterility is due to the lack of lignification in the anther endothecium,which causes the failure of anther dehiscence and of pollen release.The ccc hypolignified stems accumulate higher amounts of flavonol glycosides,sinapoyl malate and feruloyl malate,which suggests a redirection of the phenolic pathway.Therefore,the absence of CAD and CCR,key enzymes of the monolignol pathway,has more severe consequences on the phenotype than the individual absence of each of them.Induction of another CCR(CCR2,At1g80820)and another CAD(CAD1,At4g39330)does not compensate the absence of the main CCR and CAD activities.This lack of CCR and CAD activities not only impacts lignification,but also severely affects the development of the plants.These consequences must be carefully considered when trying to reduce the lignin content of plants in order to facilitate the lignocellulose-to-bioethanol conversion process.

  15. Cardiovascular effects of monoterpenes: a review

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    Márcio R. V. Santos

    2011-08-01

    Full Text Available The monoterpenes are secondary metabolites of plants. They have various pharmacological properties including antifungal, antibacterial, antioxidant, anticancer, anti-spasmodic, hypotensive, and vasorelaxant. The purpose of this research was to review the cardiovascular effects of monoterpenes. The data in this resarch were collected using the Internet portals Pubmed, Scopus, and ISI Web of Knowledge between the years 1987 and 2010. In the study 33 monoterpenes were included, which were related to each of the thirteen individual words: artery, cardiovascular, heart, myocyte, vasorelaxant, vessel, hypotension, hypotensive, cardiomyocyte, ventricular, vasodilatory, aorta, and aortic. The research utilized 22 articles published mainly in the journals Phytomedicine, Fundamental Clinical Pharmacology, Planta Medica, Life Science, European Journal of Pharmacology, and Brazilian Journal of Medical and Biological Research. Of the 33 monoterpenes studied surveyed, sixteen of them had already been studied for their effects on the cardiovascular system: carvacrol, citronellol, p-cymene, eucalyptol (1,8-cineole, linalool, menthol, myrtenal, myrtenol, α-pinene, rotundifolone (piperitenone oxide, sobrerol, thymol, α-limonene, α-terpinen-4-ol, α-terpineol, and perillyl alcohol. The main effects observed were vasorelaxation, decreased heart rate and blood pressure. This review showed that the monoterpenes may be considered promising agents for prevention or treatment of diseases of the cardiovascular system.

  16. A comparison of new measurements of total monoterpene flux with improved measurements of speciated monoterpene flux

    Directory of Open Access Journals (Sweden)

    A. Lee

    2005-01-01

    Full Text Available Many monoterpenes have been identified in forest emissions using gas chromatography (GC. Until now, it has been impossible to determine whether all monoterpenes are appropriately measured using GC techniques. We used a proton transfer reaction mass spectrometer (PTR-MS coupled with the eddy covariance (EC technique to measure mixing ratios and fluxes of total monoterpenes above a ponderosa pine plantation. We compared PTR-MS-EC results with simultaneous measurements of eight speciated monoterpenes, β-pinene, α-pinene, 3-carene, d-limonene, β-phellandrene, α-terpinene, camphene, and terpinolene, made with an automated, in situ gas chromatograph with flame ionization detectors (GC-FID, coupled to a relaxed eddy accumulation system (REA. Monoterpene mixing ratios and fluxes measured by PTR-MS averaged 30±2.3% and 31±9.2% larger than by GC-FID, with larger mixing ratio discrepancies between the two techniques at night than during the day. Two unidentified peaks that correlated with β-pinene were resolved in the chromatograms and completely accounted for the daytime difference and reduced the nighttime mixing ratio difference to 20±2.9%. Measurements of total monoterpenes by PTR-MS-EC indicated that GC-FID-REA measured the common, longer-lived monoterpenes well, but that additional terpenes were emitted from the ecosystem that represented an important contribution to the total mixing ratio above the forest at night.

  17. Isotopically sensitive branching in the formation of cyclic monoterpenes: proof that (-)-alpha-pinene and (-)-beta-pinene are synthesized by the same monoterpene cyclase via deprotonation of a common intermediate

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, R.B.; Wheeler, C.J.; Cane, D.E.; Ebert, R.; Ha, H.J.

    1987-08-25

    To determine whether the bicyclic monoterpene olefins (-)-alpha-pinene and (-)-beta-pinene arise biosynthetically from the same monoterpene cyclase by alternate deprotonations of a common carbocationic intermediate, the product distributions arising from the acyclic precursor (10-/sup 2/H/sub 3/,1-/sup 3/H)geranyl pyrophosphate were compared with those resulting from incubation of (1-3H)geranyl pyrophosphate with (-)-pinene cyclase from Salvia officinalis. Alteration in proportions of the olefinic products generated by the partially purified pinene cyclase resulted from the suppression of the formation of (-)-beta-pinene (C10 deprotonation) by a primary deuterium isotope effect with a compensating stimulation of the formation of (-)-alpha-pinene (C4 deprotonation). (-)-Pinene cyclase as well as (+)-pinene cyclase also exhibited a decrease in the proportion of the acyclic olefin myrcene generated from the deuteriated substrate, accompanied by a corresponding increase in the commitment to cyclized products. The observation of isotopically sensitive branching, in conjunction with quantitation of the magnitude of the secondary deuterium isotope effect on the overall rate of product formation by the (+)- and (-)-pinene cyclases as well as two other monoterpene cyclases from the same tissue, supports the biosynthetic origin of (-)-alpha-pinene and (-)-beta-pinene by alternative deprotonations of a common enzymatic intermediate. A biogenetic scheme consistent with these results is presented, and alternate proposals for the origin of the pinenes are addressed.

  18. Inhibitory effects of monoterpenes on seed germination and seedling growth.

    Science.gov (United States)

    Kordali, Saban; Cakir, Ahmet; Sutay, Sunay

    2007-01-01

    Monoterpenes, the chemical constituents of essential oils found in plants, are known biologically active compounds. The present study was conducted to investigate the inhibitory effects of 30 monoterpenes including monoterpene hydrocarbons and oxygenated monoterpenes on seed germination and seedling growth of Amaranthus retroflexus, Chenopodium album and Rumex crispus under laboratory conditions. The monoterpenes were applied at contents of 10 and 20 microl for liquid compounds and 10 and 20 microg for solid compounds. The results show that most of the monoterpenes significantly inhibited seed germination and seedling growth of the tested plants. Oxygenated monoterpenes including beta-citronellol, nerol and terpinen-4-ol completely inhibited seed germination and seedling growth of all tested plants. Their inhibitory effects were also stronger than that of the herbicide 2,4-D. In general, monoterpenes were less effective against seed germination and seedling growth of C. album as compared with R. crispus and A. retroflexus. Phytotoxic effects of monoterpene hydrocarbons were found to be lower than those of oxygenated monoterpenes. The alcohol derivatives of oxygenated monoterpenes were also found to be more phytotoxic as compared with their acetate derivatives. Based on the present results, it can be concluded that the oxygenated monoterpenes can be used as potential bio-herbicides.

  19. Biosynthetic inorganic chemistry.

    Science.gov (United States)

    Lu, Yi

    2006-08-25

    Inorganic chemistry and biology can benefit greatly from each other. Although synthetic and physical inorganic chemistry have been greatly successful in clarifying the role of metal ions in biological systems, the time may now be right to utilize biological systems to advance coordination chemistry. One such example is the use of small, stable, easy-to-make, and well-characterized proteins as ligands to synthesize novel inorganic compounds. This biosynthetic inorganic chemistry is possible thanks to a number of developments in biology. This review summarizes the progress in the synthesis of close models of complex metalloproteins, followed by a description of recent advances in using the approach for making novel compounds that are unprecedented in either inorganic chemistry or biology. The focus is mainly on synthetic "tricks" learned from biology, as well as novel structures and insights obtained. The advantages and disadvantages of this biosynthetic approach are discussed.

  20. Highly oxygenated monoterpenes from Chenopodium ambrosioides.

    Science.gov (United States)

    Ahmed, A A

    2000-07-01

    Three new monoterpenes (3-5) were isolated from an organic extract of the aerial parts of Chenopodium ambrosioides. Structures were established on the basis of MS and NMR spectroscopic ((1)H, (13)C, (1)H-(1)H COSY, HMQC and HMBC) data.

  1. Structural relationships and vasorelaxant activity of monoterpenes

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    Cardoso Lima Tamires

    2012-09-01

    Full Text Available Abstract Background and purpose of the study The hypotensive activity of the essential oil of Mentha x villosa and its main constituent, the monoterpene rotundifolone, have been reported. Therefore, our objective was to evaluate the vasorelaxant effect of monoterpenes found in medicinal plants and establish the structure-activity relationship of rotundifolone and its structural analogues on the rat superior mesenteric artery. Methods Contractions of the vessels were induced with 10 μM of phenylephine (Phe in rings with endothelium. During the tonic phase of the contraction, the monoterpenes (10-8 - 10-3, cumulatively were added to the organ bath. The extent of relaxation was expressed as the percentage of Phe-induced contraction. Results The results from the present study showed that both oxygenated terpenes (rotundifolone, (+-limonene epoxide, pulegone epoxide, carvone epoxide, and (+-pulegone and non-oxygenated terpene ((+-limonene exhibit relaxation activity. The absence of an oxygenated molecular structure was not a critical requirement for the molecule to be bioactive. Also it was found that the position of ketone and epoxide groups in the monoterpene structures influence the vasorelaxant potency and efficacy. Major conclusion The results suggest that the presence of functional groups in the chemical structure of rotundifolone is not essential for its vasorelaxant activity.

  2. Structural Relationships and Vasorelaxant Activity of Monoterpenes

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    Tamires Cardoso Lima

    2012-09-01

    Full Text Available Background and purpose of the study The hypotensive activity of the essential oil of Mentha x villosa and its main constituent, the monoterpene rotundifolone, have been reported. Therefore, our objective was to evaluate the vasorelaxant effect of monoterpenes found in medicinal plants and establish the structureactivity relationship of rotundifolone and its structural analogues on the rat superior mesenteric artery. Methods:Contractions of the vessels were induced with 10 muM of phenylephine (Phe in rings with endothelium. During the tonic phase of the contraction, the monoterpenes (10-8 - 10-3, cumulatively were added to the organ bath. The extent of relaxation was expressed as the percentage of Phe-induced contraction. Results:The results from the present study showed that both oxygenated terpenes (rotundifolone, (+- limonene epoxide, pulegone epoxide, carvone epoxide, and (+-pulegone and nonoxygenated terpene ((+-limonene exhibit relaxation activity. The absence of an oxygenated molecular structure was not a critical requirement for the molecule to be bioactive. Also it was found that the position of ketone and epoxide groups in the monoterpene structures influence the vasorelaxant potency and efficacy. Major conclusion The results suggest that the presence of functional groups in the chemical structure of rotundifolone is not essential for its vasorelaxant activity.

  3. 2,2-Diphenyl-1-picrylhydrazyl as a screening tool for recombinant monoterpene biosynthesis

    Science.gov (United States)

    2013-01-01

    Background Monoterpenes are a class of natural C10 compounds with a range of potential applications including use as fuel additives, fragrances, and chemical feedstocks. Biosynthesis of monoterpenes in heterologous systems is yet to reach commercially-viable levels, and therefore is the subject of strain engineering and fermentation optimization studies. Detection of monoterpenes typically relies on gas chromatography/mass spectrometry; this represents a significant analytical bottleneck which limits the potential to analyse combinatorial sets of conditions. To address this, we developed a high-throughput method for pre-screening monoterpene biosynthesis. Results An optimised DPPH assay was developed for detecting monoterpenes from two-phase microbial cultures using dodecane as the extraction solvent. The assay was useful for reproducible qualitative ranking of monoterpene concentrations, and detected standard preparations of myrcene and γ-terpinene dissolved in dodecane at concentrations as low as 10 and 15 μM, respectively, and limonene as low as 200 μM. The assay could not be used quantitatively due to technical difficulties in capturing the initial reaction rate in a multi-well plate and the presence of minor DPPH-reactive contaminants. Initially, limonene biosynthesis in Saccharomyces cerevisiae was tested using two different limonene synthase enzymes and three medium compositions. The assay indicated that limonene biosynthesis was enhanced in a supplemented YP medium and that the Citrus limon limonene synthase (CLLS) was more effective than the Mentha spicata limonene synthase (MSLS). GC-MS analysis revealed that the DPPH assay had correctly identified the best limonene synthase (CLLS) and culture medium (supplemented YP medium). Because only traces of limonene were detected in SD medium, we subsequently identified medium components that improved limonene production and developed a defined medium based on these findings. The best limonene titres obtained

  4. Product Variability of the ‘Cineole Cassette'Monoterpene Synthases of Related Nicotiana Species

    Institute of Scientific and Technical Information of China (English)

    Anke F(a)hnrich; Katrin Krause; Birgit Piechulla

    2011-01-01

    Nicotiana species of the section Alatae characteristically emit the floral scent compounds of the ‘cineole cassere' comprising 1,8-cineole,limonene,myrcene,α-pinene,β-pinene,sabinene,and α-terpineol.We successfully isolated genes of Nicotiana alata and Nicotiana langsdorfii that encoded enzymes,which produced the characteristic monoterpenes of this ‘cineole cassette' with α-terpineol being most abundant in the volatile spectra.The amino acid sequences of both terpineol synthases were 99% identical.The enzymes cluster in a monophyletic branch together with the closely related cineole synthase of Nicotiana suaveolens and monoterpene synthase 1 of Solanum lycopersicum.The cyclization reactions (α-terpineol to 1,8-cineole) of the terpineol synthases of N.alata and N.langsdorfii were less efficient compared to the ‘cineole cassette′ monoterpene synthases of Arabidopsis thaliana,N.suaveolens,Salvia fruticosa,Salvia officinalis,and Citrus unshiu.The terpineol synthases of N.alata and N.langsdorfii were localized in pistils and in the adaxial and abaxial epidermis of the petals.The enzyme activities reached their maxima at the second day after anthesis when flowers were fully opened and the enzyme activity in N.alata was highest at the transition from day to night (diurnal rhythm).

  5. Stereochemical mechanism of two sabinene hydrate synthases forming antipodal monoterpenes in thyme (Thymus vulgaris).

    Science.gov (United States)

    Krause, Sandra T; Köllner, Tobias G; Asbach, Julia; Degenhardt, Jörg

    2013-01-15

    The essential oil of Thymus vulgaris consists of a complex blend of mono- and sesquiterpenes that provides the plant with its characteristic aromatic odor. Several chemotypes have been described for thyme. In this study, we identified two enzymes of the sabinene hydrate chemotype which are responsible for the biosynthesis of its major monoterpene alcohols, (1S,2R,4S)-(Z)-sabinene hydrate and (1S,2S,4R)-(E)-sabinene hydrate. Both TPS6 and TPS7 are multiproduct enzymes that formed 16 monoterpenes and thus cover almost the whole monoterpene spectrum of the chemotype. Although the product spectra of both enzymes are similar, they form opposing enantiomers of their chiral products. Incubation of the enzymes with the potential reaction intermediates revealed that the stereospecificity of TPS6 and TPS7 is determined by the formation of the first intermediate, linalyl diphosphate. Since TPS6 and TPS7 shared an amino acid sequence identity of 85%, a mutagenesis study was employed to identify the amino acids that determine the stereoselectivity. One amino acid position had a major influence on the stereochemistry of the formed products. Based on comparative models of TPS6 and TPS7 protein structures with the GPP substrate docked in the active site pocket, the influence of this amino acid residue on the reaction mechanism is discussed.

  6. Capturing of the monoterpene olefin limonene produced in Saccharomyces cerevisiae.

    Science.gov (United States)

    Jongedijk, Esmer; Cankar, Katarina; Ranzijn, Jorn; van der Krol, Sander; Bouwmeester, Harro; Beekwilder, Jules

    2015-01-01

    Monoterpene olefins such as limonene are plant compounds with applications as flavouring and fragrance agents, as solvents and potentially also in polymer and fuel chemistry. We engineered baker's yeast Saccharomyces cerevisiae to express a (-)-limonene synthase from Perilla frutescens and a (+)-limonene synthase from Citrus limon. Both proteins were expressed either with their native plastid targeting signal or in a truncated form in which the plastidial sorting signal was removed. The yeast host strain for expression was AE9 K197G, which expresses a mutant Erg20 enzyme. This enzyme catalyses the formation of geranyl diphosphate, which is the precursor for monoterpenes. Several methods were tested to capture limonene produced by the yeast. Extraction from the culture medium by pentane, or by the addition of CaCl2 followed by solid-phase micro-extraction, did not lead to detectable limonene, indicating that limonene is rapidly lost from the culture medium. Volatile terpenes such as limonene may also be trapped in a dodecane phase added to the medium during fermentation. This method resulted in recovery of 0.028 mg/l (+)-limonene and 0.060 mg/l (-)-limonene in strains using the truncated Citrus and Perilla synthases, respectively. Trapping the headspace during culture of the limonene synthase-expressing strains resulted in higher titres, at 0.12 mg/l (+)-limonene and 0.49 mg/l (-)-limonene. These results show that the volatile properties of the olefins produced require specific methods for efficient recovery of these molecules from biotechnological production systems.

  7. Highly Oxygenated Monoterpenes from Eupatorium fortunei

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    A pair of epimers of highly-oxygenated monoterpenes were isolated from the traditional Chinese medicine Eupatorium fortunei. Their structures were elucidated on the basis of the spectral analysis as (1R*, 2S*, 3R*, 4R*, 6S*)-1, 2, 3, 6-tetrehydroxy-p-menthane (1) and (1S*,2S*, 3S*, 4R*, 6R*)-1, 2, 3, 6-tetrehydroxy-p-menthane (2).

  8. Organization of monoterpene biosynthesis in Mentha. Immunocytochemical localizations of geranyl diphosphate synthase, limonene-6-hydroxylase, isopiperitenol dehydrogenase, and pulegone reductase.

    Science.gov (United States)

    Turner, Glenn W; Croteau, Rodney

    2004-12-01

    We present immunocytochemical localizations of four enzymes involved in p-menthane monoterpene biosynthesis in mint: the large and small subunits of peppermint (Mentha x piperita) geranyl diphosphate synthase, spearmint (Mentha spicata) (-)-(4S)-limonene-6-hydroxylase, peppermint (-)-trans-isopiperitenol dehydrogenase, and peppermint (+)-pulegone reductase. All were localized to the secretory cells of peltate glandular trichomes with abundant labeling corresponding to the secretory phase of gland development. Immunogold labeling of geranyl diphosphate synthase occurred within secretory cell leucoplasts, (-)-4S-limonene-6-hydroxylase labeling was associated with gland cell endoplasmic reticulum, (-)-trans-isopiperitenol dehydrogenase labeling was restricted to secretory cell mitochondria, while (+)-pulegone reductase labeling occurred only in secretory cell cytoplasm. We discuss this pathway compartmentalization in relation to possible mechanisms for the intracellular movement of monoterpene metabolites, and for monoterpene secretion into the extracellular essential oil storage cavity.

  9. An NPF transporter exports a central monoterpene indole alkaloid intermediate from the vacuole.

    Science.gov (United States)

    Payne, Richard M E; Xu, Deyang; Foureau, Emilien; Teto Carqueijeiro, Marta Ines Soares; Oudin, Audrey; Bernonville, Thomas Dugé de; Novak, Vlastimil; Burow, Meike; Olsen, Carl-Erik; Jones, D Marc; Tatsis, Evangelos C; Pendle, Ali; Ann Halkier, Barbara; Geu-Flores, Fernando; Courdavault, Vincent; Nour-Eldin, Hussam Hassan; O'Connor, Sarah E

    2017-01-13

    Plants sequester intermediates of metabolic pathways into different cellular compartments, but the mechanisms by which these molecules are transported remain poorly understood. Monoterpene indole alkaloids, a class of specialized metabolites that includes the anticancer agent vincristine, antimalarial quinine and neurotoxin strychnine, are synthesized in several different cellular locations. However, the transporters that control the movement of these biosynthetic intermediates within cellular compartments have not been discovered. Here we present the discovery of a tonoplast localized nitrate/peptide family (NPF) transporter from Catharanthus roseus, CrNPF2.9, that exports strictosidine, the central intermediate of this pathway, into the cytosol from the vacuole. This discovery highlights the role that intracellular localization plays in specialized metabolism, and sets the stage for understanding and controlling the central branch point of this pharmacologically important group of compounds.

  10. Metabolic engineering of the carotenoid biosynthetic pathway in the yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

    NARCIS (Netherlands)

    Verdoes, J.C.; Sandmann, G.; Visser, H.; Diaz, M.; Mossel, van M.; Ooyen, van A.J.J.

    2003-01-01

    The crtYB locus was used as an integrative platform for the construction of specific carotenoid biosynthetic mutants in the astaxanthin-producing yeast Xanthophyllomyces dendrorhous. The crtYB gene of X. dendrorhous, encoding a chimeric carotenoid biosynthetic enzyme, could be inactivated by both si

  11. Metabolic engineering of the carotenoid biosynthetic pathway in the yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

    NARCIS (Netherlands)

    Verdoes, J.C.; Sandmann, G.; Visser, H.; Diaz, M.; Mossel, van M.; Ooyen, van A.J.J.

    2003-01-01

    The crtYB locus was used as an integrative platform for the construction of specific carotenoid biosynthetic mutants in the astaxanthin-producing yeast Xanthophyllomyces dendrorhous. The crtYB gene of X. dendrorhous, encoding a chimeric carotenoid biosynthetic enzyme, could be inactivated by both

  12. High-resolution MALDI mass spectrometry imaging of gallotannins and monoterpene glucosides in the root of Paeonia lactiflora

    Science.gov (United States)

    Li, Bin; Bhandari, Dhaka Ram; Römpp, Andreas; Spengler, Bernhard

    2016-10-01

    High-resolution atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI) at 10 μm pixel size was performed to unravel the spatio-chemical distribution of major secondary metabolites in the root of Paeonia lactiflora. The spatial distributions of two major classes of bioactive components, gallotannins and monoterpene glucosides, were investigated and visualized at the cellular level in tissue sections of P. lactiflora roots. Accordingly, other primary and secondary metabolites were imaged, including amino acids, carbohydrates, lipids and monoterpenes, indicating the capability of untargeted localization of metabolites by using high-resolution MSI platform. The employed AP-SMALDI MSI system provides significant technological advancement in the visualization of individual molecular species at the cellular level. In contrast to previous histochemical studies of tannins using unspecific staining reagents, individual gallotannin species were accurately localized and unequivocally discriminated from other phenolic components in the root tissues. High-quality ion images were obtained, providing significant clues for understanding the biosynthetic pathway of gallotannins and monoterpene glucosides and possibly helping to decipher the role of tannins in xylem cells differentiation and in the defence mechanisms of plants, as well as to investigate the interrelationship between tannins and lignins.

  13. Influence of gibberellin and daminozide on the expression of terpene synthases and on monoterpenes in common sage (Salvia officinalis).

    Science.gov (United States)

    Schmiderer, Corinna; Grausgruber-Gröger, Sabine; Grassi, Paolo; Steinborn, Ralf; Novak, Johannes

    2010-07-01

    Common sage (Salvia officinalis L., Lamiaceae) is one of the most important medicinal and aromatic plants, with antioxidant, antimicrobial, spasmolytic, astringent, antihidrotic and specific sensorial properties. The essential oil of the plant, composed mainly of the monoterpenes 1,8-cineole, alpha-thujone, beta-thujone and camphor, is responsible for some of these effects. Gibberellins regulate diverse physiological processes in plants, such as seed germination, shoot elongation and cell division. In this study, we analyzed the effect of exogenously applied plant growth regulators, namely gibberellic acid (GA(3)) and daminozide, on leaf morphology and essential oil formation of two leaf stages during the period of leaf expansion. Essential oil content increased with increasing levels of gibberellins and decreased when gibberellin biosynthesis was blocked with daminozide. With increasing levels of gibberellins, 1,8-cineole and camphor contents increased. Daminozide blocked the accumulation of alpha- and beta-thujone. GA(3) at the highest level applied also led to a significant decrease of alpha- and beta-thujone. Monoterpene synthases are a class of enzymes responsible for the first step in monoterpene biosynthesis, competing for the same substrate geranylpyrophosphate. The levels of gene expression of the three most important monoterpene synthases in sage were investigated, 1,8-cineole synthase leading directly to 1,8-cineole, (+)-sabinene synthase responsible for the first step in the formation of alpha- and beta-thujone, and (+)-bornyl diphosphate synthase, the first step in camphor biosynthesis. The foliar application of GA(3) increased, while daminozide significantly decreased gene expression of the monoterpene synthases. The amounts of two of the end products, 1,8-cineole and camphor, were directly correlated with the levels of gene expression of the respective monoterpene synthases, indicating transcriptional control, while the formation of alpha- and beta

  14. Pyrimidine biosynthetic pathway of Baccillus subtilis.

    Science.gov (United States)

    Potvin, B W; Kelleher, R J; Gooder, H

    1975-08-01

    Biochemical and genetic data were obtained from a series of 51 Pyr- strains of Bacillus subtilis. The observed enzymatic deficiencies allowed the mutants to be placed into 12 clases, some of which represent defects in more than one of the six known pyrimidine biosynthetic enzymes. Mapping analysis by transformation has shown that all the Pyr- mutations are located in a single small area of the B. subtilis genome. A correlation of the biochemical defects and the genetic data has been made. Those mutations conferring similar enzymatic deficiencies were found to be contiguous on the B. subtilis map. Regulatory aspects of the pyrimidine pathway have also been investigated and are compared to previously reported results from other organisms. Evidence is presented which bears upon the possible physical association of the first three enzymes and the association of at least some of the enzymes of this pathway with particulate elements of the cell. A model for the organization of the enzymes is presented with dihydroorotate dehydrogenase as the central enzyme in a proposed aggregate.

  15. Monoterpenes from thyme (Thymus vulgaris) as potential mosquito repellents.

    Science.gov (United States)

    Park, Byeoung-Soo; Choi, Won-Sik; Kim, Jeong-Han; Kim, Kap-Ho; Lee, Sung-Eun

    2005-03-01

    Five monoterpenes (carvacrol, p-cymene, linalool, alpha-terpinene, and thymol) derived from the essential oil of thyme (Thymus vulgaris) were examined for their repellency against the mosquito Culex pipiens pallens. All 5 monoterpenes effectively repelled mosquitoes based on a human forearm bioassay. Alpha-terpinene and carvacrol showed significantly greater repellency than a commercial formulation, N,N-diethyl-m-methylbenzamide (deet), whereas thymol showed similar repellency to that of deet. The duration of repellency after application for all these monoterpenes was equal to or higher than that of deet. These findings indicate that a spray-type solution containing 2% alpha-terpinene may serve as an alternative mosquito repellent.

  16. Typical Monoterpenes as Insecticides and Repellents against Stored Grain Pests

    Directory of Open Access Journals (Sweden)

    Suelen L. Reis

    2016-02-01

    Full Text Available Five monoterpenes naturally occurring in essential oils were tested for their insecticidal and repellent activities against the bruchid beetle Callosobruchus maculatus and the maize weevil Sitophilus zeamais. The monoterpenes were highly efficient as inducers of mortality or repellency against both insect species. They were more efficient in their fumigant activity against C. maculatus than against S. zeamais, while this profile of action was inverted when considering the repellent activities. Eugenol was one the most effective fumigants against both insects and one the most effective repellent against C. maculatus, while citronellal and geranial were one the most effective repellents against S. zeamais. Functional and positional isomerism of the monoterpenes pairs appears to exert little or no influence on theirs effects, especially in case of repellency. The validation of the insecticidal/repellent efficacy of isolated monoterpenes may permit a more advantageous, rapid, economic and optimized approach to the identification of promising oils for commercial formulations when combined with ethnobotanical strategies.

  17. Two New Monoterpene Diglycosides from Winchia calophylla A. DC

    Institute of Scientific and Technical Information of China (English)

    Wei Ming ZHU; Bin Gui WANG; Wen Yi KANG; Xin HONG; Jun ZHOU; Xiao Jiang HAO

    2003-01-01

    Two new monoterpene diglycosides, wincaloside A (1) and wincaloside B (2), alongwith loganin (3) were isolated from the stem barks of Winchia calophylla A. DC. The structuresof 1 and 2 were established by spectroscopic and chemical methods.

  18. Antitumor Activity of Monoterpenes Found in Essential Oils

    Directory of Open Access Journals (Sweden)

    Marianna Vieira Sobral

    2014-01-01

    Full Text Available Cancer is a complex genetic disease that is a major public health problem worldwide, accounting for about 7 million deaths each year. Many anticancer drugs currently used clinically have been isolated from plant species or are based on such substances. Accumulating data has revealed anticancer activity in plant-derived monoterpenes. In this review the antitumor activity of 37 monoterpenes found in essential oils is discussed. Chemical structures, experimental models, and mechanisms of action for bioactive substances are presented.

  19. New biosynthetic pathway for pink pigments from uncultured oceanic viruses.

    Science.gov (United States)

    Ledermann, Benjamin; Béjà, Oded; Frankenberg-Dinkel, Nicole

    2016-12-01

    The pink open-chain tetrapyrrole pigment phycoerythrobilin (PEB) is employed by marine cyanobacteria, red algae and cryptophytes as a light-harvesting chromophore in phycobiliproteins. Genes encoding biosynthesis proteins for PEB have also been discovered in cyanophages, viruses that infect cyanobacteria, and mimic host pigment biosynthesis with the exception of PebS which combines the enzymatic activities of two host enzymes. In this study, we have identified novel members of the PEB biosynthetic enzyme families, heme oxygenases and ferredoxin-dependent bilin reductases. Encoding genes were found in metagenomic datasets and could be traced back to bacteriophage but not cyanophage origin. While the heme oxygenase exhibited standard activity, a new bilin reductase with highest homology to the teal pigment producing enzyme PcyA revealed PEB biosynthetic activity. Although PcyX possesses PebS-like activity both enzymes share only 9% sequence identity and likely catalyze the reaction via two independent mechanisms. Our data point towards the presence of phycobilin biosynthetic genes in phages that probably infect alphaproteobacteria and, therefore, further support a role of phycobilins outside oxygenic phototrophs.

  20. Monoterpene metabolism. Cloning, expression, and characterization of (-)-isopiperitenol/(-)-carveol dehydrogenase of peppermint and spearmint.

    Science.gov (United States)

    Ringer, Kerry L; Davis, Edward M; Croteau, Rodney

    2005-03-01

    The essential oils of peppermint (Mentha x piperita) and spearmint (Mentha spicata) are distinguished by the oxygenation position on the p-menthane ring of the constitutive monoterpenes that is conferred by two regiospecific cytochrome P450 limonene-3- and limonene-6-hydroxylases. Following hydroxylation of limonene, an apparently similar dehydrogenase oxidizes (-)-trans-isopiperitenol to (-)-isopiperitenone in peppermint and (-)-trans-carveol to (-)-carvone in spearmint. Random sequencing of a peppermint oil gland secretory cell cDNA library revealed a large number of clones that specified redox-type enzymes, including dehydrogenases. Full-length dehydrogenase clones were screened by functional expression in Escherichia coli using a recently developed in situ assay. A single full-length acquisition encoding (-)-trans-isopiperitenol dehydrogenase (ISPD) was isolated. The (-)-ISPD cDNA has an open reading frame of 795 bp that encodes a 265-residue enzyme with a calculated molecular mass of 27,191. Nondegenerate primers were designed based on the (-)-trans-ISPD cDNA sequence and employed to screen a spearmint oil gland secretory cell cDNA library from which a 5'-truncated cDNA encoding the spearmint homolog, (-)-trans-carveol-dehydrogenase, was isolated. Reverse transcription-PCR amplification and RACE were used to acquire the remaining 5'-sequence from RNA isolated from oil gland secretory cells of spearmint leaf. The full-length spearmint dehydrogenase shares >99% amino acid identity with its peppermint homolog and both dehydrogenases are capable of utilizing (-)-trans-isopiperitenol and (-)-trans-carveol. These isopiperitenol/carveol dehydrogenases are members of the short-chain dehydrogenase/reductase superfamily and are related to other plant short-chain dehydrogenases/reductases involved in secondary metabolism (lignan biosynthesis), stress responses, and phytosteroid biosynthesis, but they are quite dissimilar (approximately 13% identity) to the monoterpene

  1. Reducing AsA leads to leaf lesion and defence response in knock-down of the AsA biosynthetic enzyme GDP-D-mannose pyrophosphorylase gene in tomato plant.

    Science.gov (United States)

    Zhang, Chanjuan; Ouyang, Bo; Yang, Changxian; Zhang, Xiaohui; Liu, Hui; Zhang, Yuyang; Zhang, Junhong; Li, Hanxia; Ye, Zhibiao

    2013-01-01

    As a vital antioxidant, L-ascorbic acid (AsA) affects diverse biological processes in higher plants. Lack of AsA in cell impairs plant development. In the present study, we manipulated a gene of GDP-mannose pyrophosphorylase which catalyzes the conversion of D-mannose-1-P to GDP-D-mannose in AsA biosynthetic pathway and found out the phenotype alteration of tomato. In the tomato genome, there are four members of GMP gene family and they constitutively expressed in various tissues in distinct expression patterns. As expected, over-expression of SlGMP3 increased total AsA contents and enhanced the tolerance to oxidative stress in tomato. On the contrary, knock-down of SlGMP3 significantly decreased AsA contents below the threshold level and altered the phenotype of tomato plants with lesions and further senescence. Further analysis indicated the causes for this symptom could result from failing to instantly deplete the reactive oxygen species (ROS) as decline of free radical scavenging activity. More ROS accumulated in the leaves and then triggered expressions of defence-related genes and mimic symptom occurred on the leaves similar to hypersensitive responses against pathogens. Consequently, the photosynthesis of leaves was dramatically fallen. These results suggested the vital roles of AsA as an antioxidant in leaf function and defence response of tomato.

  2. Reducing AsA leads to leaf lesion and defence response in knock-down of the AsA biosynthetic enzyme GDP-D-mannose pyrophosphorylase gene in tomato plant.

    Directory of Open Access Journals (Sweden)

    Chanjuan Zhang

    Full Text Available As a vital antioxidant, L-ascorbic acid (AsA affects diverse biological processes in higher plants. Lack of AsA in cell impairs plant development. In the present study, we manipulated a gene of GDP-mannose pyrophosphorylase which catalyzes the conversion of D-mannose-1-P to GDP-D-mannose in AsA biosynthetic pathway and found out the phenotype alteration of tomato. In the tomato genome, there are four members of GMP gene family and they constitutively expressed in various tissues in distinct expression patterns. As expected, over-expression of SlGMP3 increased total AsA contents and enhanced the tolerance to oxidative stress in tomato. On the contrary, knock-down of SlGMP3 significantly decreased AsA contents below the threshold level and altered the phenotype of tomato plants with lesions and further senescence. Further analysis indicated the causes for this symptom could result from failing to instantly deplete the reactive oxygen species (ROS as decline of free radical scavenging activity. More ROS accumulated in the leaves and then triggered expressions of defence-related genes and mimic symptom occurred on the leaves similar to hypersensitive responses against pathogens. Consequently, the photosynthesis of leaves was dramatically fallen. These results suggested the vital roles of AsA as an antioxidant in leaf function and defence response of tomato.

  3. Polyol monoterpenes isolated from Chenopodium ambrosioides.

    Science.gov (United States)

    Hou, Shu-Qun; Li, Yan-Hong; Huang, Xiang-Zhong; Li, Rong; Lu, Huai; Tian, Kai; Ruan, Rong-Sheng; Li, Yu-Kui

    2017-04-12

    Phytochemical study on the 95% ethyl alcohol extract of stems of Chenopodium ambrosioides resulted in the isolation of two new polyol monoterpenes, 4-hydroxy-4(α or β)-isopropyl-2-methyl-2-cyclohexen-1-one (1) and 1-methyl-4β- isopropyl-1-cyclohexene-4α,5α,6α-triol (2), together with five known compounds, (1S,2S,3R,4S)-1-methyl-4-(propan-2-yl)cyclohexane-1,2,3,4-tetrol (3), (1R,2S,3S,4S)- 1,2,3,4-tetrahydroxy-p-menthane (4), (1R,2S)-3-p-menthen-1,2-diol (5), (1R,4S)-p- menth-2-en-1-ol (6) and 1,4-dihydroxy-p-menth-2-ene (7). The structures of the new compounds were established on the basis of detailed spectroscopic evidence including extensive 1D and 2D NMR techniques. Compounds 1-7 were evaluated for their anti-inflammatory activity, and compound 1 showed moderate ability to inhibit NO production of LPS-stimulated RAW 264.7 macrophages with an IC50 value of 16.83 μM.

  4. Complete Genome Sequence of the Filamentous Fungus Aspergillus westerdijkiae Reveals the Putative Biosynthetic Gene Cluster of Ochratoxin A

    Science.gov (United States)

    Chakrabortti, Alolika; Li, Jinming

    2016-01-01

    Ochratoxin A (OTA) is a common mycotoxin that contaminates food and agricultural products. Sequencing of the complete genome of Aspergillus westerdijkiae, a major producer of OTA, reveals more than 50 biosynthetic gene clusters, including a putative OTA biosynthetic gene cluster that encodes a dozen of enzymes, transporters, and regulatory proteins. PMID:27635003

  5. Organization of Monoterpene Biosynthesis in Mentha. Immunocytochemical Localizations of Geranyl Diphosphate Synthase, Limonene-6-Hydroxylase, Isopiperitenol Dehydrogenase, and Pulegone Reductase1

    Science.gov (United States)

    Turner, Glenn W.; Croteau, Rodney

    2004-01-01

    We present immunocytochemical localizations of four enzymes involved in p-menthane monoterpene biosynthesis in mint: the large and small subunits of peppermint (Mentha x piperita) geranyl diphosphate synthase, spearmint (Mentha spicata) (−)-(4S)-limonene-6-hydroxylase, peppermint (−)-trans-isopiperitenol dehydrogenase, and peppermint (+)-pulegone reductase. All were localized to the secretory cells of peltate glandular trichomes with abundant labeling corresponding to the secretory phase of gland development. Immunogold labeling of geranyl diphosphate synthase occurred within secretory cell leucoplasts, (−)-4S-limonene-6-hydroxylase labeling was associated with gland cell endoplasmic reticulum, (−)-trans-isopiperitenol dehydrogenase labeling was restricted to secretory cell mitochondria, while (+)-pulegone reductase labeling occurred only in secretory cell cytoplasm. We discuss this pathway compartmentalization in relation to possible mechanisms for the intracellular movement of monoterpene metabolites, and for monoterpene secretion into the extracellular essential oil storage cavity. PMID:15542490

  6. Isolation and expression of cytochrome P450 genes in the antennae and gut of pine beetle Dendroctonus rhizophagus (Curculionidae: Scolytinae) following exposure to host monoterpenes.

    Science.gov (United States)

    Cano-Ramírez, Claudia; López, María Fernanda; Cesar-Ayala, Ana K; Pineda-Martínez, Verónica; Sullivan, Brian T; Zúñiga, Gerardo

    2013-05-10

    Bark beetles oxidize the defensive monoterpenes of their host trees both to detoxify them and convert them into components of their pheromone system. This oxidation is catalyzed by cytochrome P450 enzymes and occurs in different tissues of the insect, including the gut (i.e., the site where the beetle's pheromones are produced and accumulated) and the antennae (i.e., the olfactory organs used for perception of airborne defensive monoterpenes as well as other host-associated compounds and pheromones). We identified ten new CYP genes in the pine beetle Dendroctonus rhizophagus in either antennae or gut tissue after stimulation with the vapors of major host monoterpenes α-pinene, β-pinene and 3-carene. Five genes belong to the CYP4 family, four to the CYP6 family and one to the CYP9 family. Differential expression of almost all of the CYP genes was observed between sexes, and within these significant differences among time, stimuli, anatomical region, and their interactions were found upon exposure to host monoterpenes. Increased expression of cytochrome P450 genes suggests that they play a role in the detoxification of monoterpenes released by this insect's host trees. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Monoterpene synthases from common sage (Salvia officinalis)

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce (Pullman, WA); Wise, Mitchell Lynn (Pullman, WA); Katahira, Eva Joy (Pullman, WA); Savage, Thomas Jonathan (Christchurch 5, NZ)

    1999-01-01

    cDNAs encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase from common sage (Salvia officinalis) have been isolated and sequenced, and the corresponding amino acid sequences has been determined. Accordingly, isolated DNA sequences (SEQ ID No:1; SEQ ID No:3 and SEQ ID No:5) are provided which code for the expression of (+)-bornyl diphosphate synthase (SEQ ID No:2), 1,8-cineole synthase (SEQ ID No:4) and (+)-sabinene synthase SEQ ID No:6), respectively, from sage (Salvia officinalis). In other aspects, replicable recombinant cloning vehicles are provided which code for (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase, or for a base sequence sufficiently complementary to at least a portion of (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant monoterpene synthases that may be used to facilitate their production, isolation and purification in significant amounts. Recombinant (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase may be used to obtain expression or enhanced expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase in plants in order to enhance the production of monoterpenoids, or may be otherwise employed for the regulation or expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase, or the production of their products.

  8. Expression of spearmint limonene synthase in transgenic spike lavender results in an altered monoterpene composition in developing leaves.

    Science.gov (United States)

    Muñoz-Bertomeu, Jesús; Ros, Roc; Arrillaga, Isabel; Segura, Juan

    2008-01-01

    We generated transgenic spike lavender (Lavandula latifolia) plants constitutively expressing the limonene synthase (LS) gene from spearmint (Mentha spicata), encoding the LS enzyme that catalyzes the synthesis of limonene from geranyl diphosphate. Overexpression of the LS transgene did not consistently affect monoterpene profile in pooled leaves or flowers from transgenic T(0) plants. Analyses from cohorts of leaves sampled at different developmental stages showed that essential oil accumulation in transgenic and control plants was higher in developing than in mature leaves. Furthermore, developing leaves of transgenic plants contained increased limonene contents (more than 450% increase compared to controls) that correlated with the highest transcript accumulation of the LS gene. The levels of other monoterpene pathway components were also significantly altered. T(0) transgenic plants were grown for 2 years, self-pollinated, and the T(1) seeds obtained. The increased limonene phenotype was maintained in the progenies that inherited the LS transgene.

  9. Structures of Bacterial Biosynthetic Arginine Decarboxylases

    Energy Technology Data Exchange (ETDEWEB)

    F Forouhar; S Lew; J Seetharaman; R Xiao; T Acton; G Montelione; L Tong

    2011-12-31

    Biosynthetic arginine decarboxylase (ADC; also known as SpeA) plays an important role in the biosynthesis of polyamines from arginine in bacteria and plants. SpeA is a pyridoxal-5'-phosphate (PLP)-dependent enzyme and shares weak sequence homology with several other PLP-dependent decarboxylases. Here, the crystal structure of PLP-bound SpeA from Campylobacter jejuni is reported at 3.0 {angstrom} resolution and that of Escherichia coli SpeA in complex with a sulfate ion is reported at 3.1 {angstrom} resolution. The structure of the SpeA monomer contains two large domains, an N-terminal TIM-barrel domain followed by a {beta}-sandwich domain, as well as two smaller helical domains. The TIM-barrel and {beta}-sandwich domains share structural homology with several other PLP-dependent decarboxylases, even though the sequence conservation among these enzymes is less than 25%. A similar tetramer is observed for both C. jejuni and E. coli SpeA, composed of two dimers of tightly associated monomers. The active site of SpeA is located at the interface of this dimer and is formed by residues from the TIM-barrel domain of one monomer and a highly conserved loop in the {beta}-sandwich domain of the other monomer. The PLP cofactor is recognized by hydrogen-bonding, {pi}-stacking and van der Waals interactions.

  10. Biosynthetic labeling of hypusine in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Park, M.H.; Folk, J.E.

    1986-05-01

    Using a dual-label technique in which /sup 3/H - and /sup 14/C-labeled forms of putrescine and of spermidine were employed as biosynthetic precursors of hypusine, two -C-H bond cleavages were detected during production of this unique amino acid in Chinese hamster ovary cells. One of these cleavages occurs at the C-1 position of the 4-aminobutyl group during its transfer from the secondary amine nitrogen of spermidine to the nitrogen at the upsilon-position of a specific lysine residue in the polypeptide precursor of eukaryotic initiation factor 4D. Breakage of the other -C-H bond takes place at the C-2 position in this aminobutyl segment after it has been coupled to lysine to form the intermediate deoxyhypusine residue. Hydroxylation at this carbon atom, which constitutes the last step in hypusine biosynthesis, is the cause of bond cleavage. The data obtained are consistent with a notion that no additional -C-H bond fissions occur during hypusine biosynthesis. The authors findings permit a suggestion of a mechanism for enzymic aminobutyl group transfer in which 4-amino-butyraldehyde produced by oxidative cleavage of spermidine is coupled with the upsilon-amino group of a specific lysine residue to form an enzyme-bound imine intermediate.

  11. Factors affecting the emission of monoterpenes from red pine (Pinus densiflora)

    Energy Technology Data Exchange (ETDEWEB)

    Yokouchi, Y.; Ambe, Y.

    1984-08-01

    The mechanism of monoterpene emission from Pinus densiflora was studied using an environmentally controlled gas cabinet. It was found that monoterpene emission rate increases exponentially with temperature and is also influenced by light. These observations were explained reasonably by a mechanism whereby monoterpene emission rate depends on the monoterpene amount in the leaf oil and its saturated vapor pressure. 14 references, 2 figures, 1 table.

  12. Identification and expression analysis of chitin synthase and related enzymes in the chitin biosynthetic pathway genes of Cnaphalocrocis medinalis%稻纵卷叶螟几丁质合成酶及合成通路相关酶基因的鉴定及表达分析

    Institute of Scientific and Technical Information of China (English)

    余海中; 黄克慧; 汪婉玲; 刘明辉; 杨鑫; 张彦; 徐家萍

    2015-01-01

    [Objectives] The rice leaf folder, Cnaphalocrocis medinalis (Guenee), is one of four rice pest insects that cause serious crop damage. In recent years, chitin synthesis and metabolism has become a focus of pest control research. Cloning and spatio-temporal expression of two chitin synthases, and other two key enzymes in the chitin biosynthetic pathway encoding genes in C. medinalis, were conducted to reveal the function of these genes. [Methods] Based on transcriptome data, we used the PCR and RACE techniques to clone the full length cDNA sequences of 4 key enzymes in the chitin biosynthetic pathway. Prediction of the structure, sequence alignment and phylogenetic analysis of the products of these 4 genes were performed using different bioinformatics software. The relative expression levels of the 4 genes in different developmental stages and larval tissues of C. medinalis were determined with quantitative Real-time PCR. [Results] Two full-length cDNA sequences encoding chitin synthase, and two full-length cDNA sequences encoding other two key enzymes related to the chitin biosynthetic pathway, were obtained. These were; Chitin Synthase A (CHSA), Chitin Synthase B (CHSB), Phosphoacetylglucosamine Mutase (PGM) and UDP-N-acetylglucosamine pyrophosphorylase (UAP) (hereafter CmCHSA, CmCHSB, CmPGM and CmUAP, respectively). Sequence analysis shows that the full length of the CmCHSA gene is 4 868 bp, which encodes a polypeptide of 1 564 amino acids, the full length of the CmCHSB gene is 4 651 bp, which encodes a polypeptide of 1 525 amino acids, the full length of CmPGM gene is 1 934 bp, which encodes a polypeptide of 548 amino acids, and the full length of CmUAP gene is 1 837 bp, which encodes a polypeptide of 487 amino acids. The results of RT-qPCR indicate that CmUAP and CmPGM had higher expression in hemolymph, whereas CmCHSA was more highly expressed in the head and integument than the midgut and CmCHSB was more highly expressed in the midgut than in other tissues

  13. Do monoterpenes released from feverfew (Tanacetum parthenium) plants cause airborne Compositae dermatitis?

    DEFF Research Database (Denmark)

    Paulsen, E.; Christensen, Lars Porskjær; Andersen, K.E.

    2002-01-01

    and parthenolide were tested with 15 selected monoterpenes and 2 sesquiterpenes. Of the 17 persons, 13 had positive and/or doubtful positive reactions to 1 or more monoterpenes. Only 1 person was allergic to several monoterpenes. Her history of gradually worsening Compositae dermatitis culminating in a probable...

  14. Monoterpene synthases from grand fir (Abies grandis). cDNA isolation, characterization, and functional expression of myrcene synthase, (-)-(4S)-limonene synthase, and (-)-(1S,5S)-pinene synthase.

    Science.gov (United States)

    Bohlmann, J; Steele, C L; Croteau, R

    1997-08-29

    Grand fir (Abies grandis) has been developed as a model system for studying defensive oleoresin formation in conifers in response to insect attack or other injury. The turpentine fraction of the oleoresin is a complex mixture of monoterpene (C10) olefins in which (-)-limonene and (-)-alpha- and (-)-beta-pinene are prominent components; (-)-limonene and (-)-pinene synthase activities are also induced upon stem wounding. A similarity based cloning strategy yielded three new cDNA species from a wounded stem cDNA library that appeared to encode three distinct monoterpene synthases. After expression in Escherichia coli and enzyme assay with geranyl diphosphate as substrate, subsequent analysis of the terpene products by chiral phase gas chromatography and mass spectrometry showed that these sequences encoded a (-)-limonene synthase, a myrcene synthase, and a (-)-pinene synthase that produces both alpha-pinene and beta-pinene. In properties and reaction stereochemistry, the recombinant enzymes resemble the corresponding native monoterpene synthases of wound-induced grand fir stem. The deduced amino acid sequences indicated the limonene synthase to be 637 residues in length (73.5 kDa), the myrcene synthase to be 627 residues in length (72.5 kDa), and the pinene synthase to be 628 residues in length (71.5 kDa); all of these monoterpene synthases appear to be translated as preproteins bearing an amino-terminal plastid targeting sequence. Sequence comparison revealed that these monoterpene synthases from grand fir resemble sesquiterpene (C15) synthases and diterpene (C20) synthases from conifers more closely than other monoterpene synthases from angiosperm species. This similarity between extant monoterpene, sesquiterpene, and diterpene synthases of gymnosperms is surprising since functional diversification of this enzyme class is assumed to have occurred over 300 million years ago. Wound-induced accumulation of transcripts for monoterpene synthases was demonstrated by RNA

  15. Bioinformatic analysis of an unusual gene-enzyme relationship in the arginine biosynthetic pathway among marine gamma proteobacteria: implications concerning the formation of N-acetylated intermediates in prokaryotes

    Directory of Open Access Journals (Sweden)

    Labedan Bernard

    2006-01-01

    Full Text Available Abstract Background The N-acetylation of L-glutamate is regarded as a universal metabolic strategy to commit glutamate towards arginine biosynthesis. Until recently, this reaction was thought to be catalyzed by either of two enzymes: (i the classical N-acetylglutamate synthase (NAGS, gene argA first characterized in Escherichia coli and Pseudomonas aeruginosa several decades ago and also present in vertebrates, or (ii the bifunctional version of ornithine acetyltransferase (OAT, gene argJ present in Bacteria, Archaea and many Eukaryotes. This paper focuses on a new and surprising aspect of glutamate acetylation. We recently showed that in Moritella abyssi and M. profunda, two marine gamma proteobacteria, the gene for the last enzyme in arginine biosynthesis (argH is fused to a short sequence that corresponds to the C-terminal, N-acetyltransferase-encoding domain of NAGS and is able to complement an argA mutant of E. coli. Very recently, other authors identified in Mycobacterium tuberculosis an independent gene corresponding to this short C-terminal domain and coding for a new type of NAGS. We have investigated the two prokaryotic Domains for patterns of gene-enzyme relationships in the first committed step of arginine biosynthesis. Results The argH-A fusion, designated argH(A, and discovered in Moritella was found to be present in (and confined to marine gamma proteobacteria of the Alteromonas- and Vibrio-like group. Most of them have a classical NAGS with the exception of Idiomarina loihiensis and Pseudoalteromonas haloplanktis which nevertheless can grow in the absence of arginine and therefore appear to rely on the arg(A sequence for arginine biosynthesis. Screening prokaryotic genomes for virtual argH-X 'fusions' where X stands for a homologue of arg(A, we retrieved a large number of Bacteria and several Archaea, all of them devoid of a classical NAGS. In the case of Thermus thermophilus and Deinococcus radiodurans, the arg(A-like sequence

  16. Demonstration that limonene is the first cyclic intermediate in the biosynthesis of oxygenated p-menthane monoterpenes in Mentha piperita and other Mentha species

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, R.; Kjonaas, R.

    1983-01-01

    The volatile oil of mature Mentha piperita (peppermint) leaves contains as major components the oxygenated p-menthane monoterpenes l-menthol (47%) and l-menthone (24%) as well as very low levels of the monoterpene olefins limonene (1%) and terpinolene (0.1%), which are considered to be probable precursors of the oxygenated derivatives. Immature leaves, which are actively synthesizing monoterpenes, produce an oil with comparatively higher levels of limonene approx.3%), and isolation of the pure olefin showed this compound to consist of approx.80% of the l-(4S)-enantiomer and approx.20% of the d-(4R)-enantiomer. The time course of incorporation of (U-/sup 14/C)sucrose into the monoterpenes of M. piperita shoot tips was consistent with the inital formation of limonene and its subsequent conversion to menthone via pulegone. d,l-(9-/sup 3/H)Limonene and (9,10-/sup 3/H)terpinolene were prepared and tested directly as precursors of oxygenated p-menthane monoterpenes in M. piperita shoot tips. Limonene was readily incorporated into pulegone, menthone, and other oxygenated derivatives, whereas terpinolene was not appreciably incorporated into these compounds. Similarly, d,l-(9-/sup 3/H)limonene was specifically incorporated into pulegone in Mentha pulegium and into the C-2-oxygenated derivative carvone in Mentha spicata, confirming the role of this olefin as the essential precursor of oxygenated p-menthane monoterpenes. Soluble enzyme preparations from the epidermis of immature M. piperita leaves converted the acyclic terpenoid precursor (1-/sup 3/H)geranyl pyrophosphate to limonene as the major cyclic product.

  17. Monoterpene Glycosides from the Roots of Paeonia lactiflora

    Institute of Scientific and Technical Information of China (English)

    Xiao Ling WANG; Wei JIAO; Xun LIAO; Shu Ling PENG; Li Sheng DING

    2006-01-01

    A new monoterpene glycoside, together with nine known ones, 3-O-methylpaeoniflorin,mudanpioside J, paeoniflorin, benzoylpaeoniflorin, oxypaeoniflorin, benzoyloxypaeoniflorin,oxybenzoylpaeoniflorin, albiflorin and lactiflorin, was isolated from the roots of Paeonia lactiflora Pall.. The structure of the new compound was elucidated as galloylalbiflorin by the spectroscopic evidence including ESI-MS, 1D- and 2D-NMR spectra.

  18. A Concise Synthesis of Monoterpene Pyridine Alkaloid Aucubinine B

    Institute of Scientific and Technical Information of China (English)

    杨晓霞; 赵景瑞; 贾学顺; 杨力维; 翟宏斌

    2003-01-01

    Aucubinine B (4), a monoterpene alkaloid obtained from the metabolites of aucubin in the presence of human intestinal bacteria, has been synthesized from 3-bromo-4-pyridinecarboxaldehyde (5) in four steps with 39% overall yield. The construction of the cyclopenta[c]pyridine intermediate (7) was realized by an intramolecular Heck reaction.

  19. Investigation of Monoterpene Degradation in the Atmospheric Simulation Chamber SAPHIR

    Science.gov (United States)

    Kaminski, Martin; Acir, Ismail-Hakki; Bohn, Birger; Brauers, Theo; Dorn, Hans-Peter; Fuchs, Hendrik; Haeseler, Rolf; Hofzumahaus, Andreas; Li, Xin; Lutz, Anna; Nehr, Sascha; Rohrer, Franz; Tillmann, Ralf; Wegener, Robert; Wahner, Andreas

    2013-04-01

    Monoterpenes are the volatile organic compound (VOC) species with the highest emission rates on a global scale beside isoprene. In the atmosphere these compounds are rapidly oxidized. Due to their high reactivity towards hydroxyl radicals (OH) they determine the radical chemistry under biogenic conditions if monoterpene concentration is higher than isoprene concentration. Recent field campaigns showed large discrepancies between measured and modeled OH concentration at low NOx conditions together with high reactivity of VOC towards OH (Hofzumahaus et al. 2009) especially in tropical forest areas (Lelieveld et al. 2008). These discrepancies were partly explained by new reaction pathways in the isoprene degradation mechanism (Whalley et al 2011). However, even an additional recycling rate of 2.7 was insufficient to explain the measured OH concentration. So other VOC species could be involved in a nonclassical OH recycling. Since the discrepancies in OH also occurred in the morning hours when the OH chemistry was mainly dominated by monoterpenes, it was assumed that also the degradation of monoterpenes may lead to OH recycling in the absence of NO. (Whalley et al 2011). The photochemical degradation of four monoterpene species was studied under high VOC reactivity and low NOx conditions in a dedicated series of experiments in the atmospheric simulation chamber SAPHIR from August to September 2012 to overcome the lack of mechanistic information for monoterpene degradation schemes. α-Pinene, β-pinene and limonene were chosen as most prominent representatives of this substance class. Moreover the degradation of myrcene was investigated due to its structural analogy to isoprene. The SAPHIR chamber was equipped with instrumentation to measure all important OH precursors (O3, HONO, HCHO), the parent VOC and their main oxidation products, radicals (OH, HO2, RO2), the total OH reactivity, and photolysis frequencies to investigate the degradation mechanism of monoterpenes in

  20. Identification and functional expression of a type 2 acyl-CoA:diacylglycerol acyltransferase (DGAT2) in developing castor bean seeds which has high homology to the major triglyceride biosynthetic enzyme of fungi and animals.

    Science.gov (United States)

    Kroon, Johan T M; Wei, Wenxue; Simon, William J; Slabas, Antoni R

    2006-12-01

    Seed oil from castor bean (Ricinus communis) contains high amounts of hydroxy fatty acid rich triacylglycerols (TAGs) that can serve as raw material for production of bio-based products such as nylon, cosmetics, lubricants, foams, and surfactants. Diacylglycerol acyltransferase (DGAT) catalyses the terminal reaction in the acyl-CoA dependent Kennedy pathway of triglyceride biosynthesis. There is still some debate whether there are three or four enzymes in yeast that have DGAT activity and catalyse the synthesis of TAG but of these the DGAT2 homologue Dga1 contributes in a major way to TAG biosynthesis. Here we report on the cloning of a cDNA for DGAT2 from castor bean and prove its biological activity following expression in yeast and enzymatic assays using diricinolein as the acceptor and ricinoleoyl-CoA as the donor. Previous reports of DGAT in castor have focussed on DGAT1 which has little amino acid sequence homology to DGAT2. Expressional studies demonstrate that DGAT2 is 18-fold more highly expressed in seeds than in leaves and shows temporal specific expression during seed development. In contrast, DGAT1 shows little difference in expression in seeds versus leaves. We conclude that in castor bean DGAT2 is more likely to play a major role in seed TAG biosynthesis than DGAT1.

  1. Monoterpene biosynthesis in lemon (Citrus limon) cDNA isolation and functional analysis of four monoterpene synthases

    NARCIS (Netherlands)

    Lücker, J.; Tamer, El M.K.; Schwab, W.; Verstappen, F.W.A.; Plas, van der L.H.W.; Bouwmeester, H.J.; Verhoeven, H.A.

    2002-01-01

    Citrus limon possesses a high content and large variety of monoterpenoids, especially in the glands of the fruit flavedo. The genes responsible for the production of these monoterpenes have never been isolated. By applying a random sequencing approach to a cDNA library from mRNA isolated from the

  2. Monoterpene biosynthesis in lemon (Citrus limon) cDNA isolation and functional analysis of four monoterpene synthases

    NARCIS (Netherlands)

    Lücker, J.; El Tamer, M.K.; Schwab, W.; Verstappen, F.W.A.; Plas, van der L.H.W.; Bouwmeester, H.J.; Verhoeven, H.A.

    2002-01-01

    Citrus limon possesses a high content and large variety of monoterpenoids, especially in the glands of the fruit flavedo. The genes responsible for the production of these monoterpenes have never been isolated. By applying a random sequencing approach to a cDNA library from mRNA isolated from the pe

  3. Intensive sampling identifies previously unknown chemotypes, population divergence and biosynthetic connections among terpenoids in Eucalyptus tricarpa.

    Science.gov (United States)

    Andrew, Rose L; Keszei, Andras; Foley, William J

    2013-10-01

    Australian members of the Myrtaceae produce large quantities of ecologically and economically important terpenes and display abundant diversity in both yield and composition of their oils. In a survey of the concentrations of leaf terpenes in Eucalyptus tricarpa (L.A.S. Johnson) L.A.S. Johnson & K.D. Hill, which were previously known from few samples, exceptional variability was found in composition. The aim was to characterize the patterns of variation and covariation among terpene components in this species and to use this information to enhance our understanding of their biosynthesis. There were marked discontinuities in the distributions of numerous compounds, including the overall proportions of mono- and sesquiterpenes, leading us to delineate three distinct chemotypes. Overall, positive covariation predominated, but negative covariation suggested competitive interactions involved in monoterpene synthesis. Two groups of covarying monoterpenes were found, each of which was positively correlated with a group of sesquiterpenes and negatively correlated with the alternate sesquiterpene group. These results imply substantial cross-talk between mono- and sesquiterpene biosynthesis pathways. However, only those compounds hypothesized to share final carbocation intermediates or post-processing steps were strongly positively correlated within chemotypes. This suggests that the broader patterns of covariation among groups of compounds may result from co-regulation of multiple biosynthetic genes, controlling the complex terpene profiles of the chemotypes of Eucalyptus.

  4. In situ localization of gene transcriptions for monoterpene synthesis in irregular parenchymic cells surrounding the secretory cavities in rough lemon (Citrus jambhiri).

    Science.gov (United States)

    Yamasaki, Yumiko; Akimitsu, Kazuya

    2007-11-01

    A cDNA (RlemispF) encoding 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase, an enzyme of the methyl erythritol phosphate (MEP) pathway, and two homologs (RlemTPS1 and RlemTPS2) of citrus monoterpene synthase cDNA were isolated from the rough lemon (Citrus jambhiri). Transient localization of all or a part of RlemispF fused to a green fluorescence protein using particle gun-mediated DNA delivery localized RlemispF in the chloroplast. Transcripts of RlemispF and other monoterpene synthase genes are constitutively expressed in leaves of rough lemon. Transcript accumulations of RlemispF and RlemTPS1 were not induced by microbe attacks, but microbe attack weakly induced RlemTPS2 expression. Wounding decreased RlemispF expression. RlemispF and two different monoterpene synthase genes were specifically expressed in the epithelial tissue cells with dense cytoplasm that surround secretory cavities, which form a broadly round package containing a large volume of essential oils composed of monoterpenes. Interestingly, although expressions of RlemTPS1 and RlemTPS2 were detected at both mature and developing secretory cavities, the RlemispF-expressing cells were found more at around developing secretory cavities.

  5. Discovery of Unclustered Fungal Indole Diterpene Biosynthetic Pathways through Combinatorial Pathway Reassembly in Engineered Yeast.

    Science.gov (United States)

    Tang, Man-Cheng; Lin, Hsiao-Ching; Li, Dehai; Zou, Yi; Li, Jian; Xu, Wei; Cacho, Ralph A; Hillenmeyer, Maureen E; Garg, Neil K; Tang, Yi

    2015-11-01

    The structural diversity and biological activities of fungal indole diterpenes (IDTs) are generated in large part by the IDT cyclases (IDTCs). Identifying different IDTCs from IDT biosynthetic pathways is therefore important toward understanding how these enzymes introduce chemical diversity from a common linear precursor. However, IDTCs involved in the cyclization of the well-known aflavinine subgroup of IDTs have not been discovered. Here, using Saccharomyces cerevisiae as a heterologous host and a phylogenetically guided enzyme mining approach, we combinatorially assembled IDT biosynthetic pathways using IDTCs homologues identified from different fungal hosts. We identified the genetically standalone IDTCs involved in the cyclization of aflavinine and anominine and produced new IDTs not previously isolated. The cyclization mechanisms of the new IDTCs were proposed based on the yeast reconstitution results. Our studies demonstrate heterologous pathway assembly is a useful tool in the reconstitution of unclustered biosynthetic pathways.

  6. Single cell genome amplification accelerates identification of the apratoxin biosynthetic pathway from a complex microbial assemblage.

    Directory of Open Access Journals (Sweden)

    Rashel V Grindberg

    Full Text Available Filamentous marine cyanobacteria are extraordinarily rich sources of structurally novel, biomedically relevant natural products. To understand their biosynthetic origins as well as produce increased supplies and analog molecules, access to the clustered biosynthetic genes that encode for the assembly enzymes is necessary. Complicating these efforts is the universal presence of heterotrophic bacteria in the cell wall and sheath material of cyanobacteria obtained from the environment and those grown in uni-cyanobacterial culture. Moreover, the high similarity in genetic elements across disparate secondary metabolite biosynthetic pathways renders imprecise current gene cluster targeting strategies and contributes sequence complexity resulting in partial genome coverage. Thus, it was necessary to use a dual-method approach of single-cell genomic sequencing based on multiple displacement amplification (MDA and metagenomic library screening. Here, we report the identification of the putative apratoxin. A biosynthetic gene cluster, a potent cancer cell cytotoxin with promise for medicinal applications. The roughly 58 kb biosynthetic gene cluster is composed of 12 open reading frames and has a type I modular mixed polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS organization and features loading and off-loading domain architecture never previously described. Moreover, this work represents the first successful isolation of a complete biosynthetic gene cluster from Lyngbya bouillonii, a tropical marine cyanobacterium renowned for its production of diverse bioactive secondary metabolites.

  7. Production of aromas and fragrances through microbial oxidation of monoterpenes

    Directory of Open Access Journals (Sweden)

    H. F. Rozenbaum

    2006-09-01

    Full Text Available Aromas and fragrances can be obtained through the microbial oxidation of monoterpenes. Many microorganisms can be used to carry out extremely specific conversions using substrates of low commercial value. However, for many species, these substrates are highly toxic, consequently inhibiting their metabolism. In this work, the conversion ability of Aspergillus niger IOC-3913 for terpenic compounds was examined. This species was preselected because of its high resistance to toxic monoterpenic substrates. Though it has been grown in media containing R-limonene (one of the cheapest monoterpenic hydrocarbons, which is widely available on the market, the species has not shown the ability to metabolize it, since biotransformation products were not detected in high resolution gas chromatography analyses. For this reason, other monoterpenes (alpha-pinene, beta-pinene and camphor were used as substrates. These compounds were shown to be metabolized by the selected strain, producing oxidized compounds. Four reaction systems were used: a biotransformation in a liquid medium with cells in growth b with pre-grown cultures c with cells immobilized in a synthetic polymer network and d in a solid medium to which the substrate was added via the gas phase. The main biotransformation products were found in all the reaction systems, although the adoption of previously cultivated cells seemed to favor biotransformation. Cell immobilization seemed to be a feasible strategy for alleviating the toxic effect of the substrate. Through mass spectrometry it was possible to identify verbenone and alpha-terpineol as the biotransformation products of alpha-pinene and beta-pinene, respectively. The structures of the other oxidation products are described.

  8. A Review on Anti-Inflammatory Activity of Monoterpenes

    Directory of Open Access Journals (Sweden)

    Damião Pergentino de Sousa

    2013-01-01

    Full Text Available Faced with the need to find new anti-inflammatory agents, great effort has been expended on the development of drugs for the treatment of inflammation. This disorder reduces the quality of life and overall average productivity, causing huge financial losses. In this review the anti-inflammatory activity of 32 bioactive monoterpenes found in essential oils is discussed. The data demonstrate the pharmacological potential of this group of natural chemicals to act as anti-inflammatory drugs.

  9. The Antigerminative Activity of Twenty-Seven Monoterpenes

    Directory of Open Access Journals (Sweden)

    Laura De Martino

    2010-09-01

    Full Text Available Monoterpenes, the main constituents of essential oils, are known for their many biological activities. The present work studied the potential biological activity of twenty-seven monoterpenes, including monoterpene hydrocarbons and oxygenated ones, against seed germination and subsequent primary radicle growth of Raphanus sativus L. (radish and Lepidium sativum L. (garden cress, under laboratory conditions. The compounds, belonging to different chemical classes, showed different potency in affecting both parameters evaluated. The assayed compounds demonstrated a good inhibitory activity in a dose-dependent way. In general, radish seed is more sensitive than garden cress and its germination appeares more inhibited by alcohols; at the highest concentration tested, the more active substances were geraniol, borneol, (±-β-citronellol and α-terpineol. Geraniol and carvone inhibited, in a significant way, the germination of garden cress, at the highest concentration tested. Radicle elongation of two test species was inhibited mainly by alcohols and ketones. Carvone inhibited the radicle elongation of both seeds, at almost all concentrations assayed, while 1,8-cineole inhibited their radicle elongation at the lowest concentrations (10−5 M, 10−6 M.

  10. The antigerminative activity of twenty-seven monoterpenes.

    Science.gov (United States)

    De Martino, Laura; Mancini, Emilia; de Almeida, Luiz Fernando Rolim; De Feo, Vincenzo

    2010-09-21

    Monoterpenes, the main constituents of essential oils, are known for their many biological activities. The present work studied the potential biological activity of twenty-seven monoterpenes, including monoterpene hydrocarbons and oxygenated ones, against seed germination and subsequent primary radicle growth of Raphanus sativus L. (radish) and Lepidium sativum L. (garden cress), under laboratory conditions. The compounds, belonging to different chemical classes, showed different potency in affecting both parameters evaluated. The assayed compounds demonstrated a good inhibitory activity in a dose-dependent way. In general, radish seed is more sensitive than garden cress and its germination appeares more inhibited by alcohols; at the highest concentration tested, the more active substances were geraniol, borneol, (±)-β-citronellol and α-terpineol. Geraniol and carvone inhibited, in a significant way, the germination of garden cress, at the highest concentration tested. Radicle elongation of two test species was inhibited mainly by alcohols and ketones. Carvone inhibited the radicle elongation of both seeds, at almost all concentrations assayed, while 1,8-cineole inhibited their radicle elongation at the lowest concentrations (10(-5) M, 10(-6) M).

  11. Dynamics of monoterpene emissions in Pinus sylvestris during early spring

    Energy Technology Data Exchange (ETDEWEB)

    Baeck, J.; Hari, P.; Juurola, E. [Department of Forest Ecology, University of Helsinki (Finland); Hakola, H. [Finnish Meteorological Institute, Research and Development, Helsinki (Finland); Kulmala, M. [Department of Physical Sciences, University of Helsinki (Finland)

    2005-07-01

    The seasonal dynamics of biogenic volatile organic compound (BVOC) emissions, which can be related to the formation and growth of secondary organic aerosols, represent an important but at the present poorly understood linkage between vegetation activity and climate. Although a close relationship between photosynthesis and terpenoid emissions has been proposed, high monoterpene emission rates for Scots pine shoots (Pinus sylvestris) are frequently recorded during spring, in times when photosynthetic activity is strongly inhibited due to inherent seasonal restrictions. We suggest that terpenoid emissions are related to either photosynthesis or photorespiration for precursors for terpenoid biosynthesis. We developed two dynamic models describing temporal fluctuations in Scots pine monoterpene emissions, calculating the emissions by using CO{sub 2} exchange and ambient climate data. The models accurately predicted the measured monoterpene flux, and especially in March-April, during the time when photosynthesis was negligible and ambient temperatures were between -5 and +15 C, a good agreement was found with measured emissions and the model involving photorespiration. (orig.)

  12. Oxygenated VOC and monoterpene emissions from a boreal coniferous forest

    Science.gov (United States)

    Taipale, R.; Rantala, P.; Kajos, M. K.; Patokoski, J.; Ruuskanen, T. M.; Aalto, J.; Kolari, P.; Bäck, J.; Hari, P.; Kulmala, M.; Rinne, J.

    2012-04-01

    Compared with terpenoids, emissions of oxygenated volatile organic compounds (VOCs) from boreal ecosystems have been poorly characterized. We measured ecosystem scale emissions of three oxygenated compounds (methanol, acetaldehyde, and acetone) and monoterpenes from a Scots pine dominated forest in southern Finland during the summers 2006-2008. The measurements were conducted using the disjunct eddy covariance method combined with proton transfer reaction mass spectrometry. The contribution of the three oxygenated compounds to the measured total emissions was 40-60 %. The highest oxygenated VOC emissions were those of methanol, comprising 20-30 % of the total, followed by acetone with a share of 10-20 %. The acetaldehyde emissions were 5-10 % of the total. This emission composition will be compared with that obtained from shoot enclosure measurements. Methanol showed deposition during some periods although its overall flux was towards the atmosphere. The monoterpene emissions had a light dependent component, suggesting that part of the emissions originated directly from monoterpene biosynthesis. Diurnal, seasonal, and inter-annual variations in the emissions, along with temperature and light dependencies, will be discussed.

  13. Monoterpene Metabolism. Cloning, Expression, and Characterization of (−)-Isopiperitenol/(−)-Carveol Dehydrogenase of Peppermint and Spearmint1

    Science.gov (United States)

    Ringer, Kerry L.; Davis, Edward M.; Croteau, Rodney

    2005-01-01

    The essential oils of peppermint (Mentha x piperita) and spearmint (Mentha spicata) are distinguished by the oxygenation position on the p-menthane ring of the constitutive monoterpenes that is conferred by two regiospecific cytochrome P450 limonene-3- and limonene-6-hydroxylases. Following hydroxylation of limonene, an apparently similar dehydrogenase oxidizes (−)-trans-isopiperitenol to (−)-isopiperitenone in peppermint and (−)-trans-carveol to (−)-carvone in spearmint. Random sequencing of a peppermint oil gland secretory cell cDNA library revealed a large number of clones that specified redox-type enzymes, including dehydrogenases. Full-length dehydrogenase clones were screened by functional expression in Escherichia coli using a recently developed in situ assay. A single full-length acquisition encoding (−)-trans-isopiperitenol dehydrogenase (ISPD) was isolated. The (−)-ISPD cDNA has an open reading frame of 795 bp that encodes a 265-residue enzyme with a calculated molecular mass of 27,191. Nondegenerate primers were designed based on the (−)-trans-ISPD cDNA sequence and employed to screen a spearmint oil gland secretory cell cDNA library from which a 5′-truncated cDNA encoding the spearmint homolog, (−)-trans-carveol-dehydrogenase, was isolated. Reverse transcription-PCR amplification and RACE were used to acquire the remaining 5′-sequence from RNA isolated from oil gland secretory cells of spearmint leaf. The full-length spearmint dehydrogenase shares >99% amino acid identity with its peppermint homolog and both dehydrogenases are capable of utilizing (−)-trans-isopiperitenol and (−)-trans-carveol. These isopiperitenol/carveol dehydrogenases are members of the short-chain dehydrogenase/reductase superfamily and are related to other plant short-chain dehydrogenases/reductases involved in secondary metabolism (lignan biosynthesis), stress responses, and phytosteroid biosynthesis, but they are quite dissimilar (approximately 13

  14. Reconstructing fungal natural product biosynthetic pathways.

    Science.gov (United States)

    Lazarus, C M; Williams, K; Bailey, A M

    2014-10-01

    Large scale fungal genome sequencing has revealed a multitude of potential natural product biosynthetic pathways that remain uncharted. Here we describe some of the methods that have been used to explore them via heterologous gene expression. We focus on filamentous fungal hosts and discuss the technological challenges and successes behind the reconstruction of fungal natural product pathways. Optimised, efficient heterologous expression of reconstructed biosynthetic pathways promises progress in the discovery of novel compounds that could be utilised by the pharmaceutical and agrochemical industries.

  15. Development of transcriptomic resources for interrogating the biosynthesis of monoterpene indole alkaloids in medicinal plant species.

    Directory of Open Access Journals (Sweden)

    Elsa Góngora-Castillo

    Full Text Available The natural diversity of plant metabolism has long been a source for human medicines. One group of plant-derived compounds, the monoterpene indole alkaloids (MIAs, includes well-documented therapeutic agents used in the treatment of cancer (vinblastine, vincristine, camptothecin, hypertension (reserpine, ajmalicine, malaria (quinine, and as analgesics (7-hydroxymitragynine. Our understanding of the biochemical pathways that synthesize these commercially relevant compounds is incomplete due in part to a lack of molecular, genetic, and genomic resources for the identification of the genes involved in these specialized metabolic pathways. To address these limitations, we generated large-scale transcriptome sequence and expression profiles for three species of Asterids that produce medicinally important MIAs: Camptotheca acuminata, Catharanthus roseus, and Rauvolfia serpentina. Using next generation sequencing technology, we sampled the transcriptomes of these species across a diverse set of developmental tissues, and in the case of C. roseus, in cultured cells and roots following elicitor treatment. Through an iterative assembly process, we generated robust transcriptome assemblies for all three species with a substantial number of the assembled transcripts being full or near-full length. The majority of transcripts had a related sequence in either UniRef100, the Arabidopsis thaliana predicted proteome, or the Pfam protein domain database; however, we also identified transcripts that lacked similarity with entries in either database and thereby lack a known function. Representation of known genes within the MIA biosynthetic pathway was robust. As a diverse set of tissues and treatments were surveyed, expression abundances of transcripts in the three species could be estimated to reveal transcripts associated with development and response to elicitor treatment. Together, these transcriptomes and expression abundance matrices provide a rich resource

  16. The effects of increasing atmospheric ozone on biogenic monoterpene profiles and the formation of secondary aerosols

    Science.gov (United States)

    Pinto, Delia M.; Tiiva, Päivi; Miettinen, Pasi; Joutsensaari, Jorma; Kokkola, Harri; Nerg, Anne-Marja; Laaksonen, Ari; Holopainen, Jarmo K.

    Monoterpenes are biogenic volatile organic compounds (BVOCs) which play an important role in plant adaptation to stresses, atmospheric chemistry, plant-plant and plant-insect interactions. In this study, we determined whether ozonolysis can influence the monoterpenes in the headspace of cabbage. The monoterpenes were mixed with an air-flow enriched with 100, 200 or 400 ppbv of ozone (O 3) in a Teflon chamber. The changes in the monoterpene and O 3 concentrations, and the formation of secondary organic aerosols (SOA) were determined during ozonolysis. Furthermore, the monoterpene reactions with O 3 and OH were modelled using reaction kinetics equations. The results showed that all of the monoterpenes were unequally affected: α-thujene, sabinene and D-limonene were affected to the greatest extend, whereas the 1,8-cineole concentration did not change. In addition, plant monoterpene emissions reduced the O 3 concentration by 12-24%. The SOA formation was dependent on O 3 concentration. At 100 ppbv of O 3, virtually no new particles were formed but clear SOA formation was observed at the higher ozone concentrations. The modelled results showed rather good agreements for α-pinene and 1,8-cineole, whereas the measured concentrations were clearly lower compared to modelled values for sabinene and limonene. In summary, O 3-quenching by monoterpenes occurs beyond the boundary layer of leaves and results in a decreased O 3 concentration, altered monoterpene profiles and SOA formation.

  17. Volatile and non-volatile monoterpenes produced by elicitor-stimulated Cupressus lusitanica cultured cells.

    Science.gov (United States)

    De Alwis, Ransika; Fujita, Koki; Ashitani, Tatsuya; Kuroda, Ken'ichi

    2009-05-01

    Elicitor treatment initiates defense responses in cultured Cupressus lusitanica cells. In order to investigate the defense mechanism with a yeast extract elicitor, we carried out solid-phase microextraction coupled with gas chromatography for monoterpene analysis. Ten hydrocarbon monoterpenes, including high amounts of sabinene and limonene, were detected in the gas phase of the elicitor-treated cell cultures. Six oxidized monoterpenes including beta-thujaplicin were also detected in the ether extract of the cells and the medium. Time-course profiles of volatile monoterpenes showed that one group of hydrocarbon monoterpenes was maximized on the second day after elicitation, while the other group was maximized on the third day. There were no oxidized monoterpenes that are structurally related to sabinene and limonene in the gas phase or cell extracts, suggesting that these compounds are produced exclusively for emission. Other monoterpenes, which are produced during later stages of elicitation, are metabolized into more complex compounds such as oxidized monoterpenes, including beta-thujaplicin. Although terpinolene synthase was the principal monoterpene synthase in these cell cultures, terpinolene was detected only as a minor compound in the gas phase. The time course for terpinolene synthase activity coincided with beta-thujaplicin biosynthesis. Thus, most of the terpinolene is metabolized rapidly to oxidized terpenes such as beta-thujaplicin rather than emitted.

  18. Metabolic profiling of alternative NAD biosynthetic routes in mouse tissues.

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    Valerio Mori

    Full Text Available NAD plays essential redox and non-redox roles in cell biology. In mammals, its de novo and recycling biosynthetic pathways encompass two independent branches, the "amidated" and "deamidated" routes. Here we focused on the indispensable enzymes gating these two routes, i.e. nicotinamide mononucleotide adenylyltransferase (NMNAT, which in mammals comprises three distinct isozymes, and NAD synthetase (NADS. First, we measured the in vitro activity of the enzymes, and the levels of all their substrates and products in a number of tissues from the C57BL/6 mouse. Second, from these data, we derived in vivo estimates of enzymes'rates and quantitative contributions to NAD homeostasis. The NMNAT activity, mainly represented by nuclear NMNAT1, appears to be high and nonrate-limiting in all examined tissues, except in blood. The NADS activity, however, appears rate-limiting in lung and skeletal muscle, where its undetectable levels parallel a relative accumulation of the enzyme's substrate NaAD (nicotinic acid adenine dinucleotide. In all tissues, the amidated NAD route was predominant, displaying highest rates in liver and kidney, and lowest in blood. In contrast, the minor deamidated route showed higher relative proportions in blood and small intestine, and higher absolute values in liver and small intestine. Such results provide the first comprehensive picture of the balance of the two alternative NAD biosynthetic routes in different mammalian tissues under physiological conditions. This fills a gap in the current knowledge of NAD biosynthesis, and provides a crucial information for the study of NAD metabolism and its role in disease.

  19. Volatile terpenes from actinomycetes: a biosynthetic study correlating chemical analyses to genome data.

    Science.gov (United States)

    Rabe, Patrick; Citron, Christian A; Dickschat, Jeroen S

    2013-11-25

    The volatile terpenes of 24 actinomycetes whose genomes have been sequenced (or are currently being sequenced) were collected by use of a closed-loop stripping apparatus and identified by GC/MS. The analytical data were compared against a phylogenetic analysis of all 192 currently available sequences of bacterial terpene cyclases (excluding geosmin and 2-methylisoborneol synthases). In addition to the several groups of terpenes with known biosynthetic origin, selinadienes were identified as a large group of biosynthetically related sesquiterpenes that are produced by several streptomycetes. The detection of a large number of previously unrecognised side products of known terpene cyclases proved to be particularly important for an in depth understanding of biosynthetic pathways to known terpenes in actinomycetes. Interpretation of the chemical analytical data in the context of the phylogenetic tree of bacterial terpene cyclases pointed to the function of three new enzymes: (E)-β-caryophyllene synthase, selina-3,7(11)-diene synthase and aristolochene synthase.

  20. Nonlinear biosynthetic gene cluster dose effect on penicillin production by Penicillium chrysogenum.

    Science.gov (United States)

    Nijland, Jeroen G; Ebbendorf, Bjorg; Woszczynska, Marta; Boer, Rémon; Bovenberg, Roel A L; Driessen, Arnold J M

    2010-11-01

    Industrial penicillin production levels by the filamentous fungus Penicillium chrysogenum increased dramatically by classical strain improvement. High-yielding strains contain multiple copies of the penicillin biosynthetic gene cluster that encodes three key enzymes of the β-lactam biosynthetic pathway. We have analyzed the gene cluster dose effect on penicillin production using the high-yielding P. chrysogenum strain DS17690 that was cured from its native clusters. The amount of penicillin V produced increased with the penicillin biosynthetic gene cluster number but was saturated at high copy numbers. Likewise, transcript levels of the biosynthetic genes pcbAB [δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase], pcbC (isopenicillin N synthase), and penDE (acyltransferase) correlated with the cluster copy number. Remarkably, the protein level of acyltransferase, which localizes to peroxisomes, was saturated already at low cluster copy numbers. At higher copy numbers, intracellular levels of isopenicillin N increased, suggesting that the acyltransferase reaction presents a limiting step at a high gene dose. Since the number and appearance of the peroxisomes did not change significantly with the gene cluster copy number, we conclude that the acyltransferase activity is limiting for penicillin biosynthesis at high biosynthetic gene cluster copy numbers. These results suggest that at a high penicillin production level, productivity is limited by the peroxisomal acyltransferase import activity and/or the availability of coenzyme A (CoA)-activated side chains.

  1. Process-based modelling of biogenic monoterpene emissions combining production and release from storage

    NARCIS (Netherlands)

    Schurgers, G.; Arneth, A.; Holzinger, R.; Goldstein, A.H.

    2009-01-01

    Monoterpenes, primarily emitted by terrestrial vegetation, can influence atmospheric ozone chemistry, and can form precursors for secondary organic aerosol. The short-term emissions of monoterpenes have been well studied and understood, but their long-term variability, which is particularly importan

  2. Monoterpene fluxes measured above a Japanese red pine forest at Oshiba plateau, Japan

    Science.gov (United States)

    Tani, A.; Nozoe, S.; Aoki, M.; Hewitt, C. N.

    Monoterpene fluxes above a Japanese red pine ( Pinus densiflora) forest in Japan were measured with a heat balance method from May to November 2000. The most abundant monoterpenes were α-pinene and limonene+ β-phellandrene. Degradation losses of the major monoterpenes by the reactions with ozone and OH during transfer between the two sampling heights were estimated to be negligibly small. The highest values of average fluxes were observed in June measurement period, with values for α-pinene and limonene+ β-phellandrene of 0.6 and 0.5 nmol m -2 s -1. Their average fluxes in September, October and November measurement periods were almost the same and lowest. Vertical profiles of monoterpene concentrations inside the forest suggest that large amounts of monoterpenes are accumulated in the aerial space in the forest and transferred to the atmosphere above. The difference between logarithms of measured and calculated total monoterpene fluxes, ln F mea-ln F cal, had positive values in many morning measurements and negative values in most late afternoon measurements, indicating that monoterpenes accumulated during the night were transported to the upper atmosphere the next morning and they began to accumulate again in the late afternoon, following a decrease of turbulent mixing. Leaf wetness effect was also considered and, finally, a simple model was proposed to explain controlling parameters for monoterpene flux above the forest.

  3. Host-tree monoterpenes and biosynthesis of aggregation pheromones in the bark beetle ips paraconfusus

    Science.gov (United States)

    In the 1970-80s, vapors of the common conifer tree monoterpenes, myrcene and a-pinene, were shown to serve as precursors of ipsenol, ipsdienol and cis-verbenol, aggregation pheromone components of Ips paraconfusus. A paradigm developed that Ips bark beetles utilize pre-formed monoterpene precursors ...

  4. Process-based modelling of biogenic monoterpene emissions combining production and release from storage

    NARCIS (Netherlands)

    Schurgers, G.; Arneth, A.; Holzinger, R.|info:eu-repo/dai/nl/337989338; Goldstein, A.H.

    2009-01-01

    Monoterpenes, primarily emitted by terrestrial vegetation, can influence atmospheric ozone chemistry, and can form precursors for secondary organic aerosol. The short-term emissions of monoterpenes have been well studied and understood, but their long-term variability, which is particularly importan

  5. Monoterpene emissions from boreal tree species: Determination of de novo and pool emissions

    Science.gov (United States)

    Ghirardo, Andrea; Koch, Kristine; Taipale, Risto; Zimmer, Ina; Schnitzler, Joerg-Peter; Rinne, Janne

    2010-05-01

    Boreal forests emit a large amount of monoterpenes into the atmosphere. Traditionally these emissions are assumed to originate as evaporation from large storage pools. Thus their diurnal cycle would depend mostly on temperature. However, there is indication that a significant part of the monoterpene emission would originate directly from de novo synthesis. By applying 13CO2 fumigation and analyzing the isotope fractions with proton transfer reaction mass spectrometry (PTR-MS) and classical GC-MS we studied the origin of monoterpene emissions from some major Eurasian boreal and alpine tree species. We determined the fractions originating from de novo biosynthesis and from large internal monoterpene storages for three coniferous tree species with specialized monoterpene storage structures and one dicotyledon species without such structures. The emission from dicotyledon species Betula pendula originated solely from the de novo synthesis. The origin of the emissions from coniferous species was mixed with varying fraction originating from de novo synthesis (Pinus sylvestris 58 %, Picea abies 33.5 %, Larix decidua 9.8 %) and the rest from large internal monoterpene storage pools. We have also measured the ecosystem scale monoterpene emission fluxes from a boreal Pinus sylvestris forest by disjunct eddy covariance technique. Application of the observed fraction of emission originating from de novo synthesis and large storage pools in a hybrid emission algorithm resulted in a better description of ecosystem scale monoterpene emissions, as compared to the measured fluxes.

  6. Global isoprene and monoterpene emissions under changing climate, vegetation, CO2 and land use

    DEFF Research Database (Denmark)

    Hantson, Stijn; Knorr, Wolfgang; Schurgers, Guy

    2017-01-01

    .). It is therefore necessary to know how isoprene and monoterpene emissions have changed over the past and how future changes in climate, land-use and other factors will impact them. Here we present emission estimates of isoprene and monoterpenes over the period 1901–2 100 based on the dynamic global vegetation...... model LPJ-GUESS, including the effects of all known important drivers. We find that both isoprene and monoterpene emissions at the beginning of the 20th century were higher than at present. While anthropogenic land-use change largely drives the global decreasing trend for isoprene over the 20th century......, changes in natural vegetation composition caused a decreasing trend for monoterpene emissions. Future global isoprene and monoterpene emissions depend strongly on the climate and land-use scenarios considered. Over the 21st century, global isoprene emissions are simulated to either remain stable (RCP 4...

  7. Seasonal variations of monoterpene emissions from Pinus densiflora in East Asia.

    Science.gov (United States)

    Lim, Jun-Ho; Kim, Jo-Chun; Kim, Ki-Joon; Son, Youn-Suk; Sunwoo, Young; Han, Jin-Seok

    2008-09-01

    The emission rates and compositions of monoterpene from Pinus densiflora were investigated in the Gumsung (GM) and Worak (WM) mountains. The standard emission rates (ERs: ERs is the monoterpene emission rate at standard temperature, 30 degrees C) from P. densiflora ranged from 0.817 to 1.704 (microgC/gdw-h). The ERs and beta-values of total monoterpene were measured at the two study sites (GM and WM). In the spring and summer, the ERs were the highest, while relatively low values (densiflora were alpha-pinene, myrcene, beta-phellandrene, d-limonene and alpha-terpinene. The fractional compositions of individual monoterpene compounds were significantly different between the two test sites in the summer and winter. The ERs of the older group (31-40 years) were higher than those in the younger group (21-30 years). However, the monoterpene compositions were similar between the two age groups.

  8. Monoterpene hydroperoxides with trypanocidal activity from Chenopodium ambrosioides.

    Science.gov (United States)

    Kiuchi, Fumiyuki; Itano, Yoshiaki; Uchiyama, Nahoko; Honda, Gisho; Tsubouchi, Akiko; Nakajima-Shimada, Junko; Aoki, Takashi

    2002-04-01

    Four monoterpene hydroperoxides were isolated from aerial parts of Chenopodium ambrosioides along with ascaridole (1), the anthelmintic principle of this plant, as anti-trypanosomal compounds. The structures of these monoterpenes were determined to be (-)-(2S,4S)- and (-)-(2R,4S)-p-mentha-1(7),8-dien-2-hydroperoxide (2a and 3a) and (-)-(1R,4S)- and (-)-(1S,4S)-p-mentha-2,8-dien-1-hydroperoxide (4a and 5a) on the basis of spectroscopic methods and chemical correlations. In vitro trypanocidal activities of ascaridole (1) and these hydroperoxides (2a-5a) against epimastigotes of Trypanosoma cruzi were 23, 1.2, 1.6, 3.1, and 0.8 microM, respectively. Fresh leaves of C. ambrosioides also contained isomeric hydroperoxides 6a and 7a, and the content ratio of 2a-7a suggested that these hydroperoxides were formed through the singlet-oxygen oxidation of limonene.

  9. Do multiple herbivores maintain chemical diversity of Scots pine monoterpenes?

    Science.gov (United States)

    Iason, Glenn R; O'Reilly-Wapstra, Julianne M; Brewer, Mark J; Summers, Ron W; Moore, Ben D

    2011-05-12

    A central issue in our understanding of the evolution of the diversity of plant secondary metabolites (PSMs) is whether or not compounds are functional, conferring an advantage to the plant, or non-functional. We examine the hypothesis that the diversity of monoterpene PSMs within a plant species (Scots pine Pinus sylvestris) may be explained by different compounds acting as defences against high-impact herbivores operating at different life stages. We also hypothesize that pairwise coevolution, with uncorrelated interactions, is more likely to result in greater PSM diversity, than diffuse coevolution. We tested whether up to 13 different monoterpenes in Scots pine were inhibitory to herbivory by slugs (Arion ater), bank voles (Clethrionomys glareolus), red deer (Cervus elaphus) and capercaillie (Tetrao urogallus), each of which attack trees at a different life stage. Plants containing more α-pinene were avoided by both slugs and capercaillie, which may act as reinforcing selective agents for this dominant defensive compound. Herbivory by red deer and capercaillie were, respectively, weakly negatively associated with δ(3)-carene, and strongly negatively correlated with the minor compound β-ocimene. Three of the four herbivores are probably contributory selective agents on some of the terpenes, and thus maintain some, but by no means all, of the phytochemical diversity in the species. The correlated defensive function of α-pinene against slugs and capercaillie is consistent with diffuse coevolutionary processes.

  10. Evaluation of light dependence of monoterpene emission and its effect on surface ozone concentration

    Science.gov (United States)

    Nishimura, Hiroshi; Shimadera, Hikari; Kondo, Akira; Bao, Hai; Shrestha, Kundan Lal; Inoue, Yoshio

    2015-03-01

    This study evaluated the effect of light intensity on monoterpene emission from the three dominant coniferous tree species in Japan (Cryptomeria japonica, Chamaecyparis obtusa and Pinus densiflora). Monoterpene emission experiments were conducted by using a growth chamber where temperature and light intensity can be controlled. In the experiments, air temperature was set at 30 °C and light intensity was set at 0, 500, 700, 850, 1200, and 1400 μmol m-2s-1. Because monoterpene emissions from the three tree species similarly increased with increasing light intensity, a new empirical equation considering light dependence was proposed to estimate monoterpene emission. In addition, monoterpene emission in the Kinki region of Japan was estimated with and without light dependence using meteorological field produced by the Weather Research and Forecasting model (WRF) in summer 2010. The monoterpene emissions estimated with light dependence were larger than those without light dependence in the daytime under clear sky conditions and consistently smaller in the nighttime. In order to evaluate the effect of light dependence of monoterpene emission on ozone concentration in the Kinki region, two cases of air quality simulations by the Community Multiscale Air Quality model (CMAQ) were conducted using the monoterpene emission data estimated with and without light dependence. Comparisons of the two cases showed that the monoterpene emission changes due to light dependence slightly but systematically affected ozone concentrations. Monoterpene generally played a role of reducing ozone concentration in the CMAQ simulations. Consequently, because of the light dependence, the mean daily maximum ozone concentrations decreased by 0.3 ppb on average with a maximum of 2.2 ppb, and the mean daily minimum values increased by 0.4 ppb on average with a maximum of 1.8 ppb in the Kinki region in summer 2010.

  11. A novel synthetic pathway for tropolone ring formation via the olefin monoterpene intermediate terpinolene in cultured Cupressus lusitanica cells.

    Science.gov (United States)

    Fujita, Koki; Bunyu, Yasufumi; Kuroda, Ken'ich; Ashitani, Tatsuya; Shigeto, Jun; Tsutsumi, Yuji

    2014-05-01

    β-Thujaplicin is a wood monoterpene and tropolone compound with a unique conjugated 7-membered ring. Because of its strong antifungal and antitumor activities, β-thujaplicin is used in several fields. The biosynthesis pathway of β-thujaplicin has not yet been elucidated. Using Cupressus lusitanica cell cultures in a radioisotope feeding experiment, our group previously demonstrated that geranyl pyrophosphate (GPP) is the starting material of β-thujaplicin biosynthesis. The results of our previous terpene synthase assay suggested that terpinolene is the first olefin terpenoid intermediate from GPP to β-thujaplicin, although there was no experimental evidence of this at that time. In the present study, we fed deuterium-labeled terpinolene to cultured C. lusitanica cells to determine whether terpinolene is an intermediate metabolite of β-thujaplicin biosynthesis. A gas chromatography-mass spectroscopy analysis of the cell extracts from labeled terpinolene cultures revealed a peak of labeled β-thujaplicin that was not observed after treatment with non-labeled terpinolene. The identification of labeled β-thujaplicin was also performed by mass spectrum assignment. The outcome indicated that terpinolene is indeed an intermediate metabolite of β-thujaplicin biosynthesis. To the best of our knowledge, there has been no prior report that tropolone compounds are biosynthesized via a terpene biosynthesis system, and our results thus suggest the existence of a novel biosynthetic pathway that produces the conjugated 7-membered ring.

  12. Substrate specificity of the sialic acid biosynthetic pathway

    Energy Technology Data Exchange (ETDEWEB)

    Jacobs, Christina L.; Goon, Scarlett; Yarema, Kevin J.; Hinderlich, Stephan; Hang, Howard C.; Chai, Diana H.; Bertozzi, Carolyn R.

    2001-07-18

    Unnatural analogs of sialic acid can be delivered to mammalian cell surfaces through the metabolic transformation of unnatural N-acetylmannosamine (ManNAc) derivatives. In previous studies, mannosamine analogs bearing simple N-acyl groups up to five carbon atoms in length were recognized as substrates by the biosynthetic machinery and transformed into cell-surface sialoglycoconjugates [Keppler, O. T., et al. (2001) Glycobiology 11, 11R-18R]. Such structural alterations to cell surface glycans can be used to probe carbohydrate-dependent phenomena. This report describes our investigation into the extent of tolerance of the pathway toward additional structural alterations of the N-acyl substituent of ManNAc. A panel of analogs with ketone-containing N-acyl groups that varied in the lengthor steric bulk was chemically synthesized and tested for metabolic conversion to cell-surface glycans. We found that extension of the N-acyl chain to six, seven, or eight carbon atoms dramatically reduced utilization by the biosynthetic machinery. Likewise, branching from the linear chain reduced metabolic conversion. Quantitation of metabolic intermediates suggested that cellular metabolism is limited by the phosphorylation of the N-acylmannosamines by ManNAc 6-kinase in the first step of the pathway. This was confirmed by enzymatic assay of the partially purified enzyme with unnatural substrates. Identification of ManNAc 6-kinase as a bottleneck for unnatural sialic acid biosynthesis provides a target for expanding the metabolic promiscuity of mammalian cells.

  13. The biosynthetic pathway of vitamin C in higher plants.

    Science.gov (United States)

    Wheeler, G L; Jones, M A; Smirnoff, N

    1998-05-28

    Vitamin C (L-ascorbic acid) has important antioxidant and metabolic functions in both plants and animals, but humans, and a few other animal species, have lost the capacity to synthesize it. Plant-derived ascorbate is thus the major source of vitamin C in the human diet. Although the biosynthetic pathway of L-ascorbic acid in animals is well understood, the plant pathway has remained unknown-one of the few primary plant metabolic pathways for which this is the case. L-ascorbate is abundant in plants (found at concentrations of 1-5 mM in leaves and 25 mM in chloroplasts) and may have roles in photosynthesis and transmembrane electron transport. We found that D-mannose and L-galactose are efficient precursors for ascorbate synthesis and are interconverted by GDP-D-mannose-3,5-epimerase. We have identified an enzyme in pea and Arabidopsis thaliana, L-galactose dehydrogenase, that catalyses oxidation of L-galactose to L-galactono-1,4-lactone. We propose an ascorbate biosynthesis pathway involving GDP-D-mannose, GDP-L-galactose, L-galactose and L-galactono-1,4-lactone, and have synthesized ascorbate from GDP-D-mannose by way of these intermediates in vitro. The definition of this biosynthetic pathway should allow engineering of plants for increased ascorbate production, thus increasing their nutritional value and stress tolerance.

  14. Characterization of the biosynthetic gene cluster of rebeccamycin from Lechevalieria aerocolonigenes ATCC 39243.

    Science.gov (United States)

    Onaka, Hiroyasu; Taniguchi, Shin-ichi; Igarashi, Yasuhiro; Furumai, Tamotsu

    2003-01-01

    The biosynthetic gene cluster for rebeccamycin, an indolocarbazole antibiotic, from Lechevalieria aerocolonigenes ATCC 39243 has 11 ORFs. To clarify their functions, mutants with rebG, rebD, rebC, rebP, rebM, rebR, rebH, rebT, or orfD2 disrupted were constructed, and the gene products were examined. rebP disruptants produced 11,11'-dichlorochromopyrrolic acid, found to be a biosynthetic intermediate by a bioconversion experiment. Other genes encoded N-glycosyltransferase (rebG), monooxygenase (rebC), methyltransferase (rebM), a transcriptional activator (rebR), and halogenase (rebH). rebT disruptants produced rebeccamycin as much as the wild strain, so rebT was probably not involved in rebeccamycin production. Biosynthetic genes of staurosporine, an another indolocarbazole antibiotic, were cloned from Streptomyces sp. TP-A0274. staO, staD, and staP were similar to rebO, rebD, and rebP, respectively, all of which are responsible for indolocarbazole biosynthesis, But a rebC homolog, encoding a putative enzyme oxidizing the C-7 site of pyrrole rings, was not found in the staurosporine biosynthetic gene cluster. These results suggest that indolocarbazole is constructed by oxidative decarboxylation of chromopyrrolic acid (11,11'-dichlorochromopyrrolic acid in rebeccamycin) generated from two molecules of tryptophan by coupling and that the oxidation state at the C-7 position depends on the additional enzyme(s) encoded by the biosynthetic genes.

  15. Impact of lead ions on biosynthetic capacity of Streptomyces recifensis var. lyticus 2p-15 strain

    Directory of Open Access Journals (Sweden)

    Т. P. Kilochok

    2006-01-01

    Full Text Available The influence of different concentrations of Pb ions on biosynthetical ability of Streptomyces recifensis var. lyticus 2P-15, which is the producer of compound complex of extracellular enzymes and growth stimulators, was studied. It has been showed, that Pb ions introduced in agar medium have had a stimulative effect on production of surface and depth mycelia. The Pb ions, which have been inoculated into liquid fermentative medium in concentration of 1,0–2,0 mg/l realized directed synthesis of bacterio- and proteolytic enzymes, had an influence on qualitative and quantitative composition of produced enzymes.

  16. Alanylclavam Biosynthetic Genes Are Clustered Together with One Group of Clavulanic Acid Biosynthetic Genes in Streptomyces clavuligerus▿ §

    Science.gov (United States)

    Zelyas, Nathan J.; Cai, Hui; Kwong, Thomas; Jensen, Susan E.

    2008-01-01

    Streptomyces clavuligerus produces at least five different clavam metabolites, including clavulanic acid and the methionine antimetabolite, alanylclavam. In vitro transposon mutagenesis was used to analyze a 13-kb region upstream of the known paralogue gene cluster. The paralogue cluster includes one group of clavulanic acid biosynthetic genes in S. clavuligerus. Twelve open reading frames (ORFs) were found in this area, and mutants were generated in each using either in vitro transposon or PCR-targeted mutagenesis. Mutants with defects in any of the genes orfA, orfB, orfC, or orfD were unable to produce alanylclavam but could produce all of the other clavams, including clavulanic acid. orfA encodes a predicted hydroxymethyltransferase, orfB encodes a YjgF/YER057c/UK114-family regulatory protein, orfC encodes an aminotransferase, and orfD encodes a dehydratase. All of these types of proteins are normally involved in amino acid metabolism. Mutants in orfC or orfD also accumulated a novel clavam metabolite instead of alanylclavam, and a complemented orfC mutant was able to produce trace amounts of alanylclavam while still producing the novel clavam. Mass spectrometric analyses, together with consideration of the enzymes involved in its production, led to tentative identification of the novel clavam as 8-OH-alanylclavam, an intermediate in the proposed alanylclavam biosynthetic pathway. PMID:18931110

  17. Minimum Information about a Biosynthetic Gene cluster

    NARCIS (Netherlands)

    Medema, M.H.; Kottmann, Renzo; Yilmaz, Pelin; Cummings, Matthew; Biggins, J.B.; Blin, Kai; Bruijn, De Irene; Chooi, Yit Heng; Claesen, Jan; Coates, R.C.; Cruz-Morales, Pablo; Duddela, Srikanth; Düsterhus, Stephanie; Edwards, Daniel J.; Fewer, David P.; Garg, Neha; Geiger, Christoph; Gomez-Escribano, Juan Pablo; Greule, Anja; Hadjithomas, Michalis; Haines, Anthony S.; Helfrich, Eric J.N.; Hillwig, Matthew L.; Ishida, Keishi; Jones, Adam C.; Jones, Carla S.; Jungmann, Katrin; Kegler, Carsten; Kim, Hyun Uk; Kötter, Peter; Krug, Daniel; Masschelein, Joleen; Melnik, Alexey V.; Mantovani, Simone M.; Monroe, Emily A.; Moore, Marcus; Moss, Nathan; Nützmann, Hans Wilhelm; Pan, Guohui; Pati, Amrita; Petras, Daniel; Reen, F.J.; Rosconi, Federico; Rui, Zhe; Tian, Zhenhua; Tobias, Nicholas J.; Tsunematsu, Yuta; Wiemann, Philipp; Wyckoff, Elizabeth; Yan, Xiaohui; Yim, Grace; Yu, Fengan; Xie, Yunchang; Aigle, Bertrand; Apel, Alexander K.; Balibar, Carl J.; Balskus, Emily P.; Barona-Gómez, Francisco; Bechthold, Andreas; Bode, Helge B.; Borriss, Rainer; Brady, Sean F.; Brakhage, Axel A.; Caffrey, Patrick; Cheng, Yi Qiang; Clardy, Jon; Cox, Russell J.; Mot, De René; Donadio, Stefano; Donia, Mohamed S.; Donk, Van Der Wilfred A.; Dorrestein, Pieter C.; Doyle, Sean; Driessen, Arnold J.M.; Ehling-Schulz, Monika; Entian, Karl Dieter; Fischbach, Michael A.; Gerwick, Lena; Gerwick, William H.; Gross, Harald; Gust, Bertolt; Hertweck, Christian; Höfte, Monica; Jensen, Susan E.; Ju, Jianhua; Katz, Leonard; Kaysser, Leonard; Klassen, Jonathan L.; Keller, Nancy P.; Kormanec, Jan; Kuipers, Oscar P.; Kuzuyama, Tomohisa; Kyrpides, Nikos C.; Kwon, Hyung Jin; Lautru, Sylvie; Lavigne, Rob; Lee, Chia Y.; Linquan, Bai; Liu, Xinyu; Liu, Wen; Luzhetskyy, Andriy; Mahmud, Taifo; Mast, Yvonne; Méndez, Carmen; Metsä-Ketelä, Mikko; Micklefield, Jason; Mitchell, Douglas A.; Moore, Bradley S.; Moreira, Leonilde M.; Müller, Rolf; Neilan, Brett A.; Nett, Markus; Nielsen, Jens; O'Gara, Fergal; Oikawa, Hideaki; Osbourn, Anne; Osburne, Marcia S.; Ostash, Bohdan; Payne, Shelley M.; Pernodet, Jean Luc; Petricek, Miroslav; Piel, Jörn; Ploux, Olivier; Raaijmakers, Jos M.; Salas, José A.; Schmitt, Esther K.; Scott, Barry; Seipke, Ryan F.; Shen, Ben; Sherman, David H.; Sivonen, Kaarina; Smanski, Michael J.; Sosio, Margherita; Stegmann, Evi; Süssmuth, Roderich D.; Tahlan, Kapil; Thomas, Christopher M.; Tang, Yi; Truman, Andrew W.; Viaud, Muriel; Walton, Jonathan D.; Walsh, Christopher T.; Weber, Tilmann; Wezel, Van Gilles P.; Wilkinson, Barrie; Willey, Joanne M.; Wohlleben, Wolfgang; Wright, Gerard D.; Ziemert, Nadine; Zhang, Changsheng; Zotchev, Sergey B.; Breitling, Rainer; Takano, Eriko; Glöckner, Frank Oliver

    2015-01-01

    A wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants are known to be encoded in biosynthetic gene clusters. Information about these clusters, pathways and metabolites is currently dispersed throughout the literature, making it difficult to exploi

  18. Changes in monoterpene mixing ratios during summer storms in rural New Hampshire (USA

    Directory of Open Access Journals (Sweden)

    K. B. Haase

    2011-11-01

    Full Text Available Monoterpenes are an important class of biogenic hydrocarbons that influence ambient air quality and are a principle source of secondary organic aerosol (SOA. Emitted from vegetation, monoterpenes are a product of photosynthesis and act as a response to a variety of environmental factors. Most parameterizations of monoterpene emissions are based on clear weather models that do not take into account episodic conditions that can drastically change production and release rates into the atmosphere. Here, the monoterpene dataset from the rural Thompson Farm measurement site in Durham, New Hampshire is examined in the context of a set of known severe storm events. While some storm systems had a negligible influence on ambient monoterpene mixing ratios, the average storm event increased mixing ratios by 0.59 ± 0.21 ppbv, a factor of 93% above pre-storm levels. In some events, mixing ratios reached the 10's of ppbv range and persisted overnight. These mixing ratios correspond to increases in the monoterpene emission rate, ranging from 120 to 1240 g km−2 h−1 compared to an estimated clear weather rate of 116 to 193 g km−2 h−1. Considering the regularity of storm events over most forested areas, this could be an important factor to consider when modeling global monoterpene emissions and their resulting influence on the formation of organic aerosols.

  19. Changes in monoterpene mixing ratios during summer storms in rural New Hampshire (USA)

    Science.gov (United States)

    Haase, K.B.; Jordan, C.; Mentis, E.; Cottrell, L.; Mayne, H.R.; Talbot, R.; Sive, B.C.

    2011-01-01

    Monoterpenes are an important class of biogenic hydrocarbons that influence ambient air quality and are a principle source of secondary organic aerosol (SOA). Emitted from vegetation, monoterpenes are a product of photosynthesis and act as a response to a variety of environmental factors. Most parameterizations of monoterpene emissions are based on clear weather models that do not take into account episodic conditions that can drastically change production and release rates into the atmosphere. Here, the ongoing monoterpene dataset from the rural Thompson Farm measurement site in Durham, New Hampshire is examined in the context of a set of known severe storm events. While some storm systems had a negligible influence on ambient monoterpene mixing ratios, the average storm event increased mixing ratios by 0.59 ?? 0.21 ppbv, a factor of 93 % above pre-storm levels. In some events, mixing ratios reached the 10's of ppbv range and persisted overnight. These mixing ratios correspond to increases in the monoterpene emission rate, ranging from 120 to 1240 g km-2 h -1 compared to an estimated clear weather rate of 116 to 193 g km-2 h-1. Considering the regularity of storm events over most forested areas, this could be an important factor to consider when modeling global monoterpene emissions and their resulting influence on the formation of organic aerosols. ?? 2011 Author(s).

  20. Acyclic monoterpenes in tree essential oils as a shrinking agent for waste-expanded polystyrene.

    Science.gov (United States)

    Shimotori, Yasutaka; Hattori, Kazuyuki; Aoyama, Masakazu; Miyakoshi, Tetsuo

    2011-01-01

    We examined the dissolution of polystyrene (PS) into acyclic monoterpenes present in tree essential oils, to develop an environmentally friendly shrinking agent for waste-expanded polystyrene (EPS). The dissolving powers of geranyl acetate, geranylacetone, and geranyl formate [221.8-241.2 g PS (100 g solvent)(-1)] compared favorably with that of (R)-limonene [181.7 g PS (100 g solvent)(-1)]. Their favorable dissolving powers for PS can be explained by their flexible linear structures, which may be more accessible to the inside of bulk PS compared with cyclic monoterpenes. These acyclic monoterpenes and PS were recovered almost quantitatively by simple steam distillation of the PS solution.

  1. The Enzymes of the Ammonia Assimilation in Pseudomonas aeruginosa

    NARCIS (Netherlands)

    Janssen, Dick B.; Camp, Huub J.M. op den; Leenen, Pieter J.M.; Drift, Chris van der

    1980-01-01

    Glutamine synthetase from Pseudomonas aeruginosa is regulated by repression/derepression of enzyme synthesis and by adenylylation/deadenylylation control. High levels of deadenylylated biosynthetically active glutamine synthetase were observed in cultures growing with limiting amounts of nitrogen wh

  2. The Enzymes of the Ammonia Assimilation in Pseudomonas aeruginosa

    NARCIS (Netherlands)

    Janssen, Dick B.; Camp, Huub J.M. op den; Leenen, Pieter J.M.; Drift, Chris van der

    1980-01-01

    Glutamine synthetase from Pseudomonas aeruginosa is regulated by repression/derepression of enzyme synthesis and by adenylylation/deadenylylation control. High levels of deadenylylated biosynthetically active glutamine synthetase were observed in cultures growing with limiting amounts of nitrogen wh

  3. An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae (on linr)

    NARCIS (Netherlands)

    Wang, Kui-Lin; Bolitho, Karen; Grafton, Karryn; Kortstee, A.J.; Karunairetnam, Sakuntala; McGhie, T.K.; Espley, R.V.; Hellens, R.P.; Allan, A.C.

    2010-01-01

    Background - The control of plant anthocyanin accumulation is via transcriptional regulation of the genes encoding the biosynthetic enzymes. A key activator appears to be an R2R3 MYB transcription factor. In apple fruit, skin anthocyanin levels are controlled by a gene called MYBA or MYB1, while the

  4. Emission of monoterpenes from European beech (Fagus sylvatica L. as a function of light and temperature

    Directory of Open Access Journals (Sweden)

    J. Kesselmeier

    2005-01-01

    Full Text Available Using a dynamic branch enclosure technique European beech (Fagus sylvatica L. was characterised as a strong emitter of monoterpenes, with sabinene being the predominant compound released. Since monoterpene emission was demonstrated to be a function of light and temperature, application of light and temperature dependent algorithms resulted in reasonable agreement with the measured data. Furthermore, during high temperature periods the depression of net CO2 exchange during midday (midday depression was accompanied by a depression of monoterpene emission on one occasion. The species dependent standard emission factor and the light and temperature regulated release of monoterpenes is of crucial importance for European VOC emissions. All measurements were performed within the framework of the ECHO project (Emission and CHemical transformation of biogenic volatile Organic compounds during two intensive field campaigns in the summers of 2002 and 2003.

  5. A New Monoterpene Glucoside from the Leaves of Betula platyphylla Suk.

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A new monoterpene glucoside, (2E,6Z)-2,6-dimethyl-8-b-D-glucosyloxy-2,6-octadien- oic acid, was isolated from the leaves of Betula platyphylla Suk. The structure was established by spectroscopic data.

  6. Isotope ratio analysis by HRGC-MS of monoterpene hydrocarbons from citrus essential oils.

    Science.gov (United States)

    Satake, Atsushi; Une, Akitoshi; Ueno, Takao; Ukeda, Hiroyuki; Sawamura, Masayoshi

    2003-03-01

    The isotope ratio of monoterpene hydrocarbons in citrus essential oils of different origins was measured by ordinary high-resolution gas chromatography-mass spectrometry (HRGC-MS). The isotope ratio (Ir) was determined by the ratio of the isotope peak intensity (m/z 137) to the molecular mass peak intensity (m/z 136) of the monoterpene hydrocarbons. The accuracy of Ir was examined by measuring monoterpene hydrocarbon standards and 13C-labeled compounds. The isotope fingerprints based on the values of monoterpene hydrocarbons from lemon, lime and yuzu essential oils were determined. These citrus essential oils were also discriminated by a principal component analysis of their Ir data. The characteristic vectors showed that alpha-terpinene, beta-pinene and beta-phellandrene were important components for distinguishing between the citrus species. It is suggested that this technique will be applicable to evaluate the quality, genuineness and origin of citrus fruits and their products.

  7. Distinct mechanisms for spiro-carbon formation reveal biosynthetic pathway crosstalk.

    Science.gov (United States)

    Tsunematsu, Yuta; Ishikawa, Noriyasu; Wakana, Daigo; Goda, Yukihiro; Noguchi, Hiroshi; Moriya, Hisao; Hotta, Kinya; Watanabe, Kenji

    2013-12-01

    Spirotryprostatins, an indole alkaloid class of nonribosomal peptides isolated from Aspergillus fumigatus, are known for their antimitotic activity in tumor cells. Because spirotryprostatins and many other chemically complex spiro-carbon-bearing natural products exhibit useful biological activities, identifying and understanding the mechanism of spiro-carbon biosynthesis is of great interest. Here we report a detailed study of spiro-ring formation in spirotryprostatins from tryprostatins derived from the fumitremorgin biosynthetic pathway, using reactants and products prepared with engineered yeast and fungal strains. Unexpectedly, FqzB, an FAD-dependent monooxygenase from the unrelated fumiquinazoline biosynthetic pathway, catalyzed spiro-carbon formation in spirotryprostatin A via an epoxidation route. Furthermore, FtmG, a cytochrome P450 from the fumitremorgin biosynthetic pathway, was determined to catalyze the spiro-ring formation in spirotryprostatin B. Our results highlight the versatile role of oxygenating enzymes in the biosynthesis of structurally complex natural products and indicate that cross-talk of different biosynthetic pathways allows product diversification in natural product biosynthesis.

  8. Quantitative and enantioselective analysis of monoterpenes from plant chambers and in ambient air using SPME

    Directory of Open Access Journals (Sweden)

    N. Yassaa

    2010-11-01

    Full Text Available A headspace solid-phase microextraction (HS-SPME and gas chromatography/mass spectrometry (GC/MS system has been developed for quantifying enantiomeric and nonenantiomeric monoterpenes in plant chamber studies and ambient air. Performance of this system was checked using a capillary diffusion system to produce monoterpene standards. The adsorption efficiency, competitive adsorption and chromatographic peak resolution of monoterpene enantiomer pairs were compared for three SPME fibre coatings: 75 μm Carboxen-PDMS (CAR-PDMS, 50/30 μm divinylbenzene-carboxen-polydimethylsiloxane (DVB-CAR-PDMS and 65 μm divinylbenzene-polydimethylsiloxane (DVB-PDMS. Key parameters such as the linearity and reproducibility of the SPME system have been investigated in this work. The best compromise between the enantiomeric separation of monoterpenes and competitive adsorption of the isoprenoids on the solid SPME fibre coating was found for DVB-PDMS fibres. The optimum conditions using DVB-PDMS fibres were applied to measure the exchange rates of monoterpenes in the emission of Quercus ilex using a laboratory whole plant enclosure under light and dark conditions, as well as in ambient air. With 592 and 223 ng m−2 s−1 respectively, β-myrcene and limonene were the predominant monoterpenes in the emission of Q. ilex. These values were closely comparable to those obtained using a zNose and cartridge GC-FID systems.

  9. Quantitative and enantioselective analysis of monoterpenes from plant chambers and in ambient air using SPME

    Directory of Open Access Journals (Sweden)

    N. Yassaa

    2010-08-01

    Full Text Available A solid-phase microextraction (HS-SPME and gas chromatography/mass spectrometry (GC/MS system has been developed for quantifying enantiomeric and nonenantiomeric monoterpenes in plant chamber studies and ambient air. Performance of this system was checked using a capillary diffusion system to produce monoterpene standards. The adsorption efficiency, competitive adsorption and chromatographic peak resolution of monoterpene enantiomer pairs were compared for three SPME fibre coatings: 75 μm Carboxen-PDMS (CAR-PDMS, 50/30 μm, divinylbenzene-carboxen-polydimethylsiloxane (DVB-CAR-PDMS and 65 μm divinylbenzene-polydimethyl-siloxane (DVB-PDMS. Key parameters such as the linearity and reproducibility of the SPME system have been investigated in this work. The best compromise between the enantiomeric separation of monoterpenes and competitive adsorption of the isoprenoids on the solid SPME fibre coating was found for DVB-PDMS fibres. The optimum conditions using DVB-PDMS fibres were applied to measure the exchange rates of monoterpenes in the emission of Quercus ilex using a laboratory whole plant enclosure under light and dark conditions, as well as in ambient air. With 592 and 223 ng m−2 s−1, respectively, β-myrcene and limonene were the predominant monoterpenes in the emission of Q. ilex. These values were closely comparable to those obtained using a zNose and cartridge GC-FID systems.

  10. Neurosteroid biosynthetic pathways changes in prefrontal cortex in Alzheimer's disease.

    Science.gov (United States)

    Luchetti, Sabina; Bossers, Koen; Van de Bilt, Saskia; Agrapart, Vincent; Morales, Rafael Ramirez; Frajese, Giovanni Vanni; Swaab, Dick F

    2011-11-01

    Expression of the genes for enzymes involved in neurosteroid biosynthesis was studied in human prefrontal cortex (PFC) in the course of Alzheimer's disease (AD) (n=49). Quantitative RT-PCR (qPCR) revealed that mRNA levels of diazepam binding inhibitor (DBI), which is involved in the first step of steroidogenesis and in GABAergic transmission, were increased, as were mRNA levels for several neurosteroid biosynthetic enzymes. Aromatase, 17β-hydroxysteroid dehydrogenase type 1 (HSD17B1) and aldo-keto reductase 1C2 (AKR1C2), were all increased in the late stages of AD. Several GABA-A subunits were significantly reduced in AD. Increased expression of aromatase in the PFC was confirmed by immunohistochemistry and was found to be localized predominantly in astrocytes. These data suggest a role for estrogens and allopregnanolone produced by astrocytes in the PFC in AD, possibly as part of a rescue program. The reduced gene expression of some synaptic and extra-synaptic GABA-A subunits may indicate a deficit of modulation of GABA-A receptors by neuroactive steroids, which may contribute to the neuropsychiatric characteristics of this disease.

  11. Isoprene and monoterpene emissions from an Inner Mongolia grassland

    Science.gov (United States)

    Bai, Jianhui; Baker, Bradly; Liang, Baosheng; Greenberg, James; Guenther, Alex

    Terpenoid emissions were measured at a grassland site in Inner Mongolia, China during four campaigns over a 2-year period. Emissions were strongly correlated with light and temperature and the variations could be simulated using a canopy emission model. Substantial seasonal and interannual variations in isoprene emissions were also observed. Area averaged isoprene emissions normalized to standard above-canopy temperature and light conditions (30 °C and 1500 μmol m -2 s -1) ranged from about 50 to 500 μg m -2 h -1. These rates are more than an order of magnitude higher than those previously reported for temperate grasslands but are lower than emission rates observed from ground cover vegetation at higher latitudes. Isoprene emissions from this Inner Mongolia grassland may be dominated by emissions from sedges, e.g. Carex appendiculata. Total monoterpene emissions normalized to a standard temperature of 30 °C were only about 3 μg m -2 h -1 and consisted primarily of carene and limonene with smaller contributions of α-pinene and β-pinene. A model sensitivity study showed that grass and other herbaceous ground cover can contribute >10% of the total isoprene emission from certain regions, such as Inner Mongolia, but are<4% on a global annual scale.

  12. Monoterpenes Released from Fruit, Plant, and Vegetable Systems

    Science.gov (United States)

    Iqbal, Mohammad Asif; Kim, Ki-Hyun; Ahn, Jeong Hyeon

    2014-01-01

    To quantify the emission rate of monoterpenes (MTs) from diverse natural sources, the sorbent tube (ST)-thermal desorption (TD) method was employed to conduct the collection and subsequent detection of MTs by gas chromatography. The calibration of MTs, when made by both mass spectrometric (MS) and flame ionization detector (FID), consistently exhibited high coefficient of determination values (R2 > 0.99). This approach was employed to measure their emission rate from different fruit/plant/vegetable (F/P/V) samples with the aid of an impinger-based dynamic headspace sampling system. The results obtained from 10 samples (consisting of carrot, pine needle (P. sylvestris), tangerine, tangerine peel, strawberry, sepals of strawberry, plum, apple, apple peel, and orange juice) marked α-pinene, β-pinene, myrcene, α-terpinene, R-limonene, γ-terpinene, and p-cymene as the most common MTs. R-limonene was the major species emitted from citrus fruits and beverages with its abundance exceeding 90%. In contrast, α-pinene was the most abundant MT (37%) for carrot, while it was myrcene (31%) for pine needle. The overall results for F/P/V samples confirmed α-pinene, β-pinene, myrcene, α-terpinene, and γ-terpinene as common MTs. Nonetheless, the types and magnitude of MTs released from fruits were distinguished from those of vegetables and plants. PMID:25268921

  13. Monoterpenes Released from Fruit, Plant, and Vegetable Systems

    Directory of Open Access Journals (Sweden)

    Mohammad Asif Iqbal

    2014-09-01

    Full Text Available To quantify the emission rate of monoterpenes (MTs from diverse natural sources, the sorbent tube (ST-thermal desorption (TD method was employed to conduct the collection and subsequent detection of MTs by gas chromatography. The calibration of MTs, when made by both mass spectrometric (MS and flame ionization detector (FID, consistently exhibited high coefficient of determination values (R2 > 0.99. This approach was employed to measure their emission rate from different fruit/plant/vegetable (F/P/V samples with the aid of an impinger-based dynamic headspace sampling system. The results obtained from 10 samples (consisting of carrot, pine needle (P. sylvestris, tangerine, tangerine peel, strawberry, sepals of strawberry, plum, apple, apple peel, and orange juice marked α-pinene, β-pinene, myrcene, α-terpinene, R-limonene, γ-terpinene, and p-cymene as the most common MTs. R-limonene was the major species emitted from citrus fruits and beverages with its abundance exceeding 90%. In contrast, α-pinene was the most abundant MT (37% for carrot, while it was myrcene (31% for pine needle. The overall results for F/P/V samples confirmed α-pinene, β-pinene, myrcene, α-terpinene, and γ-terpinene as common MTs. Nonetheless, the types and magnitude of MTs released from fruits were distinguished from those of vegetables and plants.

  14. Emergent biosynthetic capacity in simple microbial communities.

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    Hsuan-Chao Chiu

    2014-07-01

    Full Text Available Microbes have an astonishing capacity to transform their environments. Yet, the metabolic capacity of a single species is limited and the vast majority of microorganisms form complex communities and join forces to exhibit capabilities far exceeding those achieved by any single species. Such enhanced metabolic capacities represent a promising route to many medical, environmental, and industrial applications and call for the development of a predictive, systems-level understanding of synergistic microbial capacity. Here we present a comprehensive computational framework, integrating high-quality metabolic models of multiple species, temporal dynamics, and flux variability analysis, to study the metabolic capacity and dynamics of simple two-species microbial ecosystems. We specifically focus on detecting emergent biosynthetic capacity--instances in which a community growing on some medium produces and secretes metabolites that are not secreted by any member species when growing in isolation on that same medium. Using this framework to model a large collection of two-species communities on multiple media, we demonstrate that emergent biosynthetic capacity is highly prevalent. We identify commonly observed emergent metabolites and metabolic reprogramming patterns, characterizing typical mechanisms of emergent capacity. We further find that emergent secretion tends to occur in two waves, the first as soon as the two organisms are introduced, and the second when the medium is depleted and nutrients become limited. Finally, aiming to identify global community determinants of emergent capacity, we find a marked association between the level of emergent biosynthetic capacity and the functional/phylogenetic distance between community members. Specifically, we demonstrate a "Goldilocks" principle, where high levels of emergent capacity are observed when the species comprising the community are functionally neither too close, nor too distant. Taken together

  15. Preparation of Ecofriendly Formulations Containing Biologically Active Monoterpenes with Their Fumigant and Residual Toxicities against Adults of Culex pipiens

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    Mohamed E. I. Badawy

    2016-01-01

    Full Text Available Different mixtures of monoterpenes (ketone, alcohol, and alkene were loaded on paper discs and wax and their knockdown activities were evaluated against Culex pipiens adults. Some individual monoterpenes were also evaluated by residual toxicity technique. Citronella oil as a reference was also loaded separately or in combination with monoterpenes on paper discs and wax. The ketone monoterpenes mixture (camphor, menthone, carvone, and fenchone on paper discs was the most active (KT50 = 17.20 min followed by ketone monoterpenes with citronella oil (KT50 = 20.79 min and citronella oil alone (KT50 = 28.72 min. Wax formulations proved that the ketone and alcohol (geraniol, thymol, and menthol monoterpenes gave the most activity as knockdown (KT50 = 31.79 and 43.39 min, resp.. Alcohol monoterpenes formulation recorded KT50 = 43.39 min. Residual activity of tested individual monoterpenes reported that the menthol was more toxic than camphor and camphene. Generally, this study suggests that the monoterpenes have the properties, which make them used as eco-friendly compounds in the control programs of Cx. pipiens adult. The use of paper discs is more applicable than wax in the adulticidal formulations.

  16. Biosynthetic Relationship between Acutumine and Dechloroacutumine in Menispermum dauricum Root Cultures.

    Science.gov (United States)

    Babiker, H A; Sugimoto, Y; Saisho, T; Inanaga, S; Hashimoto, M; Isogai, A

    1999-01-01

    The biosynthetic relationship between acutumine 1 and dechloroacutumine 2 was studied using (13)C-labeled tyrosine and (3)H-labeled 2 as tracers. (13)C-NMR spectra of (13)C-labeled 1 and 2 showed that the alkaloids, each composed of two molecules of tyrosine, are derived from the same biosynthetic pathway. Feeding Menispermum dauricum (Menispermaceae) roots, cultured in a chloride-enriched medium, with (3)H-labeled 2 demonstrated that 1 is the only alkaloid metabolite of 2. Conversion (5%) of the exogenously applied 2, taken up by the roots, into 1 showed that 2 is the precursor of 1. Incomplete conversion of 2 into 1 suggests accumulation of the exogenously applied 2 in cell organelles and/or compartmentation of the enzymes involved in the biosynthesis of 1.

  17. [Effect of drying methods on monoterpenes, phenolic acids and flavonoids in Mentha haplocalyx].

    Science.gov (United States)

    Zhu, Shao-qing; Zhu, Zhen-hua; Guo, Sheng; Zhao, Yu-yang; Lu, Xue-jun; Sha, Xiu-xiu; Qian, Da-wei; Duan, Jin-ao

    2015-12-01

    To provide a scientific basis for the selection of the appropriate drying method for Mentha Haplocalyx Herba (MHH), determine 2 monoterpenes, 4 phenolic acids and 5 flavonoids in MHH by GC-MS and UPLC-TQ-MS methods, and investigate the effects of the drying methods on the changes in contents of these analytes. The qualities of products obtained with different drying methods were evaluated by the multivariate statistical method of Technique for Order Preference by Similarity to Ideal Solution (TOPSIS). Results showed that the drying methods had the greatest impact on menthol, caffeic acid, and rosemary acid, which were followed by chlorogenic acid and diosmetin-7-O-glucoside. The contents in these analytes processed with hot-air-drying method were higher than those with microwave-drying and infrared-drying methods at the same temperatures. The contents in these analytes processed under low temperature (40-45 °C) were higher than those under higher temperature (60-70 °C). Above all, the contents in phenolic acids processed with microwave fixation (exposed under microwave at 100 °C for several minutes) were obviously higher than those of not being processed, showing an inhibition of some enzymes in samples after fixation. The TOPSIS evaluation showed that the variable temperature drying method of 'Hot-Air 45-60 °C' was the most suitable approach for the primary drying processing of MHH. The results could provide the scientific basis for the selection of appropriate drying method for MHH, and helpful reference for the primary drying proces of herbs containing volatile chemical components.

  18. Menthol: a simple monoterpene with remarkable biological properties.

    Science.gov (United States)

    Kamatou, Guy P P; Vermaak, Ilze; Viljoen, Alvaro M; Lawrence, Brian M

    2013-12-01

    Menthol is a cyclic monoterpene alcohol which possesses well-known cooling characteristics and a residual minty smell of the oil remnants from which it was obtained. Because of these attributes it is one of the most important flavouring additives besides vanilla and citrus. Due to this reason it is used in a variety of consumer products ranging from confections such as chocolate and chewing gum to oral-care products such as toothpaste as well as in over-the-counter medicinal products for its cooling and biological effects. Its cooling effects are not exclusive to medicinal use. Approximately one quarter of the cigarettes on the market contain menthol and small amounts of menthol are even included in non-mentholated cigarettes. Natural menthol is isolated exclusively from Mentha canadensis, but can also be synthesised on industrial scale through various processes. Although menthol exists in eight stereoisomeric forms, (-)-menthol from the natural source and synthesised menthol with the same structure is the most preferred isomer. The demand for menthol is high and it was previously estimated that the worldwide use of menthol was 30-32,000 metric tonnes per annum. Menthol is not a predominant compound of the essential oils as it can only be found as a constituent of a limited number of aromatic plants. These plants are known to exhibit biological activity in vitro and in vivo such as antibacterial, antifungal, antipruritic, anticancer and analgesic effects, and are also an effective fumigant. In addition, menthol is one of the most effective terpenes used to enhance the dermal penetration of pharmaceuticals. This review summarises the chemical and biological properties of menthol and highlights its cooling effects and toxicity. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Larvicidal efficacy of monoterpenes against the larvae of Anopheles gambiae

    Institute of Scientific and Technical Information of China (English)

    Eliningaya J.Kweka; Tamires Cardoso Lima; Chrian M.Marciale; Dami?o Pergentino de Sousa

    2016-01-01

    Objective:To evaluate the larvicidal efficacy of eight volatile components of essential oils against 3rd instar larvae of Anopheles gambiae s.s.Methods:Larvicidal effects of each compound were evaluated in both laboratory and semi-field trials.Stock solution was prepared and serial dilutions were made in six concentrations for each compound.A total of 20 larvae were exposed to larvicides for each replicate and monitored at intervals of 12,24,48 and 72 h.Larvae monitoring was done on basis of dead and live larvae in all intervals.Results:All assayed compounds were larvicides and presented varying degrees of larval toxicity,with LC50 values ranging from 1.28 to 1 938.92 mg/L depending on the treatment time(12,24,48 or 72 h).(-)-Perillyl alcohol presented the strongest larvicidal activity towards Anopheles gambiae larvae,with LC50 values of 73.60,18.36,1.72 and1.28 mg/L after 12,24,48 and 72 h of exposure,respectively.The next strongest were(-)-isopulegol(LC50= 135.10,49.39,34.39 and 20.22 mg/L) and(-)-carvone epoxide(LC50= 168.86,124.74,80.84 and 23.46 mg/L).After 12,24 and 48 h of treatment,hydroxydihydrocarvone was the least toxic compound,with LC50 values of 1 938.92,1 172.18 and 401.03 mg/L,respectively.Conclusions:The data obtained in this study suggest that all evaluated monoterpenes,especially(-)-perillyl alcohol,have remarkable larvicidal effects and may be considered as potential sources for the development of suitable natural larvicides for mosquito management programs.Further small-scale field trials should be conducted.

  20. Gastroprotective Mechanisms of the Monoterpene 1,8-Cineole (Eucalyptol.

    Directory of Open Access Journals (Sweden)

    Germana Freire Rocha Caldas

    Full Text Available Recently, our research group identified and reported 1,8-cineole (CIN, a monoterpene that naturally occur in many aromatic plants, as one of the major constituent of the essential oil from leaves of Hyptis martiusii (EOHM, as well as characterized the gastroprotective action of this oil. The aim of this study was to investigate the mechanisms of action involved in the antiulcer and healing activity of CIN, in order to confirm its correlation with the gastroprotective effect of EOHM. Wistar rats were exposed to different protocols (acute ulceration, gastrointestinal motility and antisecretory activity. In addition, were determinated the involvement of nitric oxide and sulphydryl groups; the levels of gastric mucus, lipid peroxidation, sulphydryl groups and myeloperoxidase activity. The healing ability was evaluated by acetic acid-induced chronic ulcer and histological and immunohistochemical analysis (PCNA, Ki-67 and BrdU. The treatment with CIN inhibited ethanol-, ethanol/HCl- and indomethacin-induced gastric lesions. The highest doses of CIN inhibited gastric emptying, but did not affect intestinal transit. CIN (100 mg/kg reduced the volume of basal but not stimulated acid secretion. CIN increased levels of mucus (89.3%, prevented depletion of -SH groups (62.6% and reduced the level of lipid peroxidation (55.3% and myeloperoxidase activity (59.4% in the gastric mucosa. In chronic ulcer model, CIN reduced in 43.1% the gastric area lesion, promoted significant regeneration and restoration of the levels of mucus in glandular cells as confirmed by histological analysis; and promoted increase in cell proliferation as evidenced by reactivity for PCNA, Ki-67 and BrdU. This findings demonstrate the role of 1,8-cineole as an important ulcer healing agent and indicate the involvement of antioxidant and cytoprotective mechanisms in the gastroprotective effect of compound. This study also provides evidence that 1,8-cineole is related to the

  1. Antiviral activity of monoterpenes beta-pinene and limonene against herpes simplex virus in vitro.

    Directory of Open Access Journals (Sweden)

    Akram Astani

    2014-06-01

    Full Text Available Essential oils are complex mixtures containing compounds of several different functional- group classes. Depending on the structure, we can distinguish monoterpenes, phenylpropanes, and other components. Here in this study two monoterpene compounds of essential oils, i.e. β-pinene and limonene were examined for their antiviral activity against herpes simplex virus type 1 (HSV-1 in vitro.All antiviral assays were performed using RC-37 cells. Cytotoxicity was determined in a neutral red assay, antiviral assays were performed with HSV-1 strain KOS. The mode of antiviral action was evaluated at different periods during the viral replication cycle. Acyclovir was used as positive antiviral control.Beta-pinenene and limonenen reduced viral infectivity by 100 %. The mode of antiviral action has been determined, only moderate antiviral effects were revealed by monoterpenes when these drugs were added to host cells prior infection or after entry of HSV into cells. However, both monoterpenes exhibited high anti-HSV-1 activity by direct interaction with free virus particles. Both tested drugs interacted with HSV-1 in a dose-dependent manner thereby inactivating viral infection.These results suggest that monoterpenes in essential oils exhibit antiherpetic activity in the early phase of viral multiplication and might be used as potential antiviral agents.

  2. Seasonal variability of monoterpene emission factors for a ponderosa pine plantation in California

    Directory of Open Access Journals (Sweden)

    R. Holzinger

    2006-01-01

    Full Text Available Monoterpene fluxes have been measured over an 11 month period from June 2003 to April 2004. During all seasons ambient air temperature was the environmental factor most closely related to the measured emission rates. The monoterpene flux was modeled using a basal emission rate multiplied by an exponential function of a temperature, following the typical practice for modelling temperature dependent biogenic emissions. A basal emission of 1.0 μmol h−1 m−2 (at 30°C, based on leaf area and a temperature dependence (β of 0.12°C−1 reproduced measured summer emissions well but underestimated spring and winter measured emissions by 60–130%. The total annual monoterpene emission may be underestimated by ~50% when using a model optimized to reproduce monoterpene emissions in summer. The long term dataset also reveals an indirect connection between non-stomatal ozone and monoterpene flux beyond the dependence on temperature that has been shown for both fluxes.

  3. A Systematic Review of the Wound-Healing Effects of Monoterpenes and Iridoid Derivatives

    Directory of Open Access Journals (Sweden)

    Rosana S.S. Barreto

    2014-01-01

    Full Text Available The search for more effective and lower cost therapeutic approaches for wound healing remains a challenge for modern medicine. In the search for new therapeutic options, plants and their metabolites are a great source of novel biomolecules. Among their constituents, the monoterpenes represent 90% of essential oils, and have a variety of structures with several activities such as antimicrobial, anti-inflammatory, antioxidant and wound healing. Based on that, and also due to the lack of reviews concerning the wound-healing activity of monoterpenes, we performed this systematic review—which provides an overview of their characteristics and mechanisms of action. In this search, the terms “terpenes”, “monoterpenes”, “wound healing” and “wound closure techniques” were used to retrieve articles published in LILACS, PUBMED and EMBASE until May 2013. Seven papers were found concerning the potential wound healing effect of five compouds (three monoterpenes and two iridoid derivatives in preclinical studies. Among the products used for wound care, the films were the most studied pharmaceutical form. Monoterpenes are a class of compounds of great diversity of biological activities and therapeutic potential. The data reviewed here suggest that monoterpenes, although poorly studied in this context, are promising compounds for the treatment of chronic wound conditions.

  4. Tree water relations trigger monoterpene emissions from Scots pine stem during spring recovery

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    A. Vanhatalo

    2015-05-01

    Full Text Available Tree canopies are known to emit large amounts of VOCs (volatile organic compounds such as monoterpenes to the surrounding air. The main source for these is considered to be the green biomass, i.e. foliage, but emissions from the woody compartments have not been quantified. A VOC emission anomaly has been observed during transition from winter to summer activity. We analyzed if non-foliar components could partially explain the anomaly. We measured the VOC emissions from Scots pine (Pinus sylvestris L. stems and shoots during the dehardening phase of trees in field conditions in two consecutive springs. We observed a large, transient monoterpene burst from stems, while the shoot monoterpene emissions and transpiration remained low. The burst lasted about 12 h. Simultaneously, an unusual night-time sap flow and an anomalous diurnal pattern of tree diameter were detected. Hence, we suggest that the monoterpene burst was a consequence of the recovery of the stem from winter-time. This indicates that the dominant processes and environmental drivers triggering the monoterpene emissions are different between stems and foliage.

  5. Production of beta-thujaplicin in Cupressus lusitanica suspension cultures fed with organic acids and monoterpenes.

    Science.gov (United States)

    Zhao, J; Fujita, K; Sakai, K

    2001-05-01

    Effects of some organic acids and monoterpenes on production of beta-thujaplicin were studied in Cupressus lusitanica suspension cultures. The fungal elicitor-induced biosynthesis of beta-thujaplicin was promoted by the feedings of malate, pyruvate, fumarate, succinate, and acetate. These results suggest some relationships between acetate/pyruvate metabolism and beta-thujaplicin biosynthesis, or between tricarboxylic acid cycle and beta-thujaplicin biosynthesis. Feedings of C. lusitanica suspension cultures with some monoterpenes inhibited elicitor-triggered beta-thujaplicin biosynthesis, but 2-carene and terpinyl acetate feedings significantly improved the beta-thujaplicin production of C. lusitanica suspension cultures. These results indicate a possible involvement of terpinyl acetate and 2-carene in beta-thujaplicin biosynthesis, as well as potential uses of these monoterpenes in large-scale beta-thujaplicin production.

  6. Monoterpene derivatives with anti-allergic activity from red peony root, the root of Paeonia lactiflora.

    Science.gov (United States)

    Shi, Yan-Hong; Zhu, Shu; Ge, Yue-Wei; He, Yu-Min; Kazuma, Kohei; Wang, Zhengtao; Yoshimatsu, Kayo; Komatsu, Katsuko

    2016-01-01

    The methanolic extract and its subfractions from red peony root, the dried roots of Paeonia lactiflora Pallas showed potent antiallergic effects, as inhibition of immunoglobulin E (IgE)-mediated degranulation in rat basophil leukemia (RBL)-2H3 cells. Bioassay-guided fractionation led to the isolation of 16 monoterpene derivatives, including 3 new compounds, paeoniflorol (1), 4'-hydroxypaeoniflorigenone (2) and 4-epi-albiflorin (3), together with 13 known ones (4-16). The chemical structures of the new compounds were elucidated on the basis of spectroscopic and chemical evidences. Among the isolated monoterpene derivatives, nine compounds showed potent anti-allergic effects and compound 1 was the most effective. A primary structure-activity relationship of monoterpene derivatives was discussed.

  7. Expanding the product profile of a microbial alkane biosynthetic pathway.

    Science.gov (United States)

    Harger, Matthew; Zheng, Lei; Moon, Austin; Ager, Casey; An, Ju Hye; Choe, Chris; Lai, Yi-Ling; Mo, Benjamin; Zong, David; Smith, Matthew D; Egbert, Robert G; Mills, Jeremy H; Baker, David; Pultz, Ingrid Swanson; Siegel, Justin B

    2013-01-18

    Microbially produced alkanes are a new class of biofuels that closely match the chemical composition of petroleum-based fuels. Alkanes can be generated from the fatty acid biosynthetic pathway by the reduction of acyl-ACPs followed by decarbonylation of the resulting aldehydes. A current limitation of this pathway is the restricted product profile, which consists of n-alkanes of 13, 15, and 17 carbons in length. To expand the product profile, we incorporated a new part, FabH2 from Bacillus subtilis , an enzyme known to have a broader specificity profile for fatty acid initiation than the native FabH of Escherichia coli . When provided with the appropriate substrate, the addition of FabH2 resulted in an altered alkane product profile in which significant levels of n-alkanes of 14 and 16 carbons in length are produced. The production of even chain length alkanes represents initial steps toward the expansion of this recently discovered microbial alkane production pathway to synthesize complex fuels. This work was conceived and performed as part of the 2011 University of Washington international Genetically Engineered Machines (iGEM) project.

  8. antiSMASH 3.0-a comprehensive resource for the genome mining of biosynthetic gene clusters.

    Science.gov (United States)

    Weber, Tilmann; Blin, Kai; Duddela, Srikanth; Krug, Daniel; Kim, Hyun Uk; Bruccoleri, Robert; Lee, Sang Yup; Fischbach, Michael A; Müller, Rolf; Wohlleben, Wolfgang; Breitling, Rainer; Takano, Eriko; Medema, Marnix H

    2015-07-01

    Microbial secondary metabolism constitutes a rich source of antibiotics, chemotherapeutics, insecticides and other high-value chemicals. Genome mining of gene clusters that encode the biosynthetic pathways for these metabolites has become a key methodology for novel compound discovery. In 2011, we introduced antiSMASH, a web server and stand-alone tool for the automatic genomic identification and analysis of biosynthetic gene clusters, available at http://antismash.secondarymetabolites.org. Here, we present version 3.0 of antiSMASH, which has undergone major improvements. A full integration of the recently published ClusterFinder algorithm now allows using this probabilistic algorithm to detect putative gene clusters of unknown types. Also, a new dereplication variant of the ClusterBlast module now identifies similarities of identified clusters to any of 1172 clusters with known end products. At the enzyme level, active sites of key biosynthetic enzymes are now pinpointed through a curated pattern-matching procedure and Enzyme Commission numbers are assigned to functionally classify all enzyme-coding genes. Additionally, chemical structure prediction has been improved by incorporating polyketide reduction states. Finally, in order for users to be able to organize and analyze multiple antiSMASH outputs in a private setting, a new XML output module allows offline editing of antiSMASH annotations within the Geneious software.

  9. Monoterpene Compositions of Three Forested Ecosystems in the Central Amazon Basin

    Science.gov (United States)

    Jardine, A.; Fuentes, J. D.; Manzi, A. O.; Higuchi, N.; Chambers, J. Q.; Jardine, K.

    2014-12-01

    Monoterpenes play fundamental roles as secondary metabolites in forested ecosystems and as gas and liquid phase secondary organic aerosol (SOA) precursors in their surrounding atmospheres. While the chemical pathways involved in ozonolysis driven SOA formation from individual monoterpene precursors is known, local and regional chemical transport models are still lacking observations of speciated monoterpenes from forested atmospheres. Here, we present high vertically resolved mixing ratio profiles of speciated monoterpenes from the ambient air of three neighboring forested ecosystems in the central Amazon Basin. Two well-drained plateau primary forests and one seasonally flooded valley forest were sampled during the afternoon hours (13:00 - 16:30) on walkup towers from the initiation of the 2013-14 wet season through the onset of the 2014 dry season (Nov 2013 - Jul 2014). Ambient mixing ratios in all three ecosystems were greatest in the upper canopy with secondary sources of some monoterpenes within the sub-canopies. Relative vertical compositions of monoterpenes did not change significantly throughout the seasons for either ecosystem type. Both ecosystem types were dominated by d-limonene (up to 1.6 ppb) with equally strong mixing ratios of alpha-pinene in the valley compared to the much weaker a-pinene mixing ratios on the plateaus (up to 200 ppt). The highly reactive cis- and trans-beta-ocimene were consistently present in both ecosystems (up to 250 ppt) with the addition of equally high camphene mixing ratios in the valley forest (up to 200 ppt) which is present in the plateau ecosystems in low quantities (50 ppt). With respect to clean atmosphere mixing ratios of 10 ppb ozone, lifetimes are below 2 hours for camphene and below 30 minutes for ocimene, suggesting a potentially large impact on local and possibly regional ozonolysis and subsequent SOA composition.

  10. Process-based modelling of biogenic monoterpene emissions combining production and release from storage

    Directory of Open Access Journals (Sweden)

    G. Schurgers

    2009-05-01

    Full Text Available Monoterpenes, primarily emitted by terrestrial vegetation, can influence atmospheric ozone chemistry, and can form precursors for secondary organic aerosol. The short-term emissions of monoterpenes have been well studied and understood, but their long-term variability, which is particularly important for atmospheric chemistry, has not. This understanding is crucial for the understanding of future changes.

    In this study, two algorithms of terrestrial biogenic monoterpene emissions, the first one based on the short-term volatilization of monoterpenes, as commonly used for temperature-dependent emissions, and the second one based on long-term production of monoterpenes (linked to photosynthesis combined with emissions from storage, were compared and evaluated with measurements from a Ponderosa pine plantation (Blodgett Forest, California. The measurements were used to parameterize the long-term storage of monoterpenes, which takes place in specific storage organs and which determines the temporal distribution of the emissions over the year. The difference in assumptions between the first (emission-based method and the second (production-based method, which causes a difference in upscaling from instantaneous to daily emissions, requires roughly a doubling of emission capacities to bridge the gap to production capacities. The sensitivities to changes in temperature and light were tested for the new methods, the temperature sensitivity was slightly higher than that of the short-term temperature dependent algorithm.

    Applied on a global scale, the first algorithm resulted in annual total emissions of 29.6 Tg C a−1, the second algorithm resulted in 31.8 Tg C a−1 when applying the correction factor 2 between emission capacities and production capacities. However, the exact magnitude of such a correction is spatially varying and hard to determine as a global average.

  11. Process-based modelling of biogenic monoterpene emissions: sensitivity to temperature and light

    Directory of Open Access Journals (Sweden)

    G. Schurgers

    2009-01-01

    Full Text Available Monoterpenes, primarily emitted by terrestrial vegetation, can influence atmospheric ozone chemistry, and can form precursors for secondary organic aerosol. The short-term emissions of monoterpenes have been well studied and understood, but their long-term variability, which is particularly important for atmospheric chemistry, has not. This understanding is crucial for the understanding of future changes.

    In this study, two algorithms of terrestrial biogenic monoterpene emissions, the first one based on the short-term volatilization of monoterpenes, as commonly used for temperature-dependent emissions, and the second one based on long-term production of monoterpenes (linked to photosynthesis combined with emissions from storage, were compared and evaluated with measurements from a Ponderosa pine plantation (Blodgett Forest, California. The measurements were used to parameterize the long-term storage of monoterpenes, which takes place in specific storage organs and which determines the temporal distribution of the emissions over the year. The difference in assumptions between the first (emission-based method and the second (production-based method, which causes a difference in upscaling from instantaneous to daily emissions, requires roughly a doubling of emission capacities to bridge the gap to production capacities. The sensitivities to changes in temperature and light were tested for the new methods, the temperature sensitivity was slightly higher than that of the short-term temperature dependent algorithm.

    Applied on a global scale, the first algorithm resulted in annual total emissions of 29.6 Tg C a−1, the second algorithm resulted in 31.8 Tg C a−1 when applying the correction factor 2 between emission capacities and production capacities. However, the exact magnitude of such a correction is spatially varying and hard to determine as a global average.

  12. Toxicity of Monoterpene Structure, Diversity and Concentration to Mountain Pine Beetles, Dendroctonus ponderosae: Beetle Traits Matter More.

    Science.gov (United States)

    Reid, Mary L; Sekhon, Jagdeep K; LaFramboise, Lanielle M

    2017-03-03

    A high diversity of plant defenses may be a response to herbivore diversity or may be collectively more toxic than single compounds, either of which may be important for understanding insect-plant associations. Monoterpenes in conifers are particularly diverse. We tested the fumigant toxicity of four monoterpenes, alone and in combination, to mountain pine beetles, Dendroctonus ponderosae, in the context of the beetles' individual body traits. Chemical structures of tested monoterpene hydrocarbons had modest effects on beetle survival, mass loss, water content and fat content, with (R)-(+)-limonene tending to be more toxic than (-)-α-pinene, (-)-β-pinene, and (+)-3-carene. Monoterpene diversity (all qualitative combinations of one to four monoterpenes) did not affect toxicity. Concentration (0 to 1200 ppm) of individual monoterpenes was a strong determinant of toxicity. Beetle body size and body condition index strongly and positively affected survival during monoterpene treatments. Larger beetles in better condition lost proportionally less mass during exposure, where proportion mass loss negatively affected survivorship. Toxicity was much more associated with water loss than with fat loss, suggesting that a main cost of detoxification is excretion, a process that has received little attention. These results provide insight into the determinants of beetle success in historic and novel hosts that differ in monoterpene composition and concentration. We also suggest that water availability will affect beetle success directly through their ability to tolerate detoxification as well as indirectly through host responses to drought.

  13. Biosynthetic Polypeptides as Templates in Materials Design

    Science.gov (United States)

    Kiick, Kristi

    2007-03-01

    Biosynthetic routes to protein-based polymeric materials offer important opportunities for the production of well-defined macromolecular templates, owing to the control of sequence and molecular weight inherent in the biosynthesis of proteins. In particular, the biosynthesis of polypeptides with controlled presentation of functional groups in multiple positions, coupled with their subsequent chemical modification with biologically relevant ligands, will permit the production of well-defined, bioactive macromolecules that may provide insight into biological binding events in which multivalent binding is important. Modification of the well-defined macromolecules with ligands such as saccharides has application in the study of events such as toxin neutralization and mediation of the immune and inflammatory responses. In this work, alanine-rich polypeptides of both random coil and helical conformations, equipped with glutamic acid residues to impart chemical versatility, have been produced via biosynthetic strategies. Analysis via spectroscopic and calorimetric methods indicates that the polypeptides adopt helical, beta-sheet, or random-coil conformations that can be controlled with variations in temperature, pH, and salt concentration; the conformational behavior of the polypeptides is not compromised upon chemical modification with saccharides. The binding of these macromolecules to bacterial toxins has been characterized via immunochemical and spectroscopic methods; results indicate that specific architectural features of the glycopolymer scaffold cause changes in the binding of these molecules to multivalent receptors. Given the chemical flexibility in the design of such scaffolds, they can be modified with many different moieties in addition to saccharides, so multiple opportunities exist for their application in areas where control of active side chains is important, such as in biomaterials, electronic devices, and bioinorganic structures.

  14. Assembly of a novel biosynthetic pathway for production of the plant flavonoid fisetin in Escherichia coli.

    Science.gov (United States)

    Stahlhut, Steen G; Siedler, Solvej; Malla, Sailesh; Harrison, Scott J; Maury, Jérôme; Neves, Ana Rute; Forster, Jochen

    2015-09-01

    Plant secondary metabolites are an underutilized pool of bioactive molecules for applications in the food, pharma and nutritional industries. One such molecule is fisetin, which is present in many fruits and vegetables and has several potential health benefits, including anti-cancer, anti-viral and anti-aging activity. Moreover, fisetin has recently been shown to prevent Alzheimer's disease in mice and to prevent complications associated with diabetes type I. Thus far the biosynthetic pathway of fisetin in plants remains elusive. Here, we present the heterologous assembly of a novel fisetin pathway in Escherichia coli. We propose a novel biosynthetic pathway from the amino acid, tyrosine, utilizing nine heterologous enzymes. The pathway proceeds via the synthesis of two flavanones never produced in microorganisms before--garbanzol and resokaempferol. We show for the first time a functional biosynthetic pathway and establish E. coli as a microbial platform strain for the production of fisetin and related flavonols. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  15. Molecular evolution of paclitaxel biosynthetic genes TS and DBAT of Taxus species.

    Science.gov (United States)

    Hao, Da Cheng; Yang, Ling; Huang, Beili

    2009-03-01

    Evolutionary patterns of sequence divergence were analyzed in genes from the conifer genus Taxus (yew), encoding paclitaxel biosynthetic enzymes taxadiene synthase (TS) and 10-deacetylbaccatin III-10 beta-O-acetyltransferase (DBAT). N-terminal fragments of TS, full-length DBAT and internal transcribed spacer (ITS) were amplified from 15 closely related Taxus species and sequenced. Premature stop codons were not found in TS and DBAT sequences. Codon usage bias was not found, suggesting that synonymous mutations are selectively neutral. TS and DBAT gene trees are not consistent with the ITS tree, where species formed monophyletic clades. In fact, for both genes, alleles were sometimes shared across species and parallel amino acid substitutions were identified. While both TS and DBAT are, overall, under purifying selection, we identified a number of amino acids of TS under positive selection based on inference using maximum likelihood models. Positively selected amino acids in the N-terminal region of TS suggest that this region might be more important for enzyme function than previously thought. Moreover, we identify lineages with significantly elevated rates of amino acid substitution using a genetic algorithm. These findings demonstrate that the pattern of adaptive paclitaxel biosynthetic enzyme evolution can be documented between closely related Taxus species, where species-specific taxane metabolism has evolved recently.

  16. Stimulation of nicotinamide adenine dinucleotide biosynthetic pathways delays axonal degeneration after axotomy.

    Science.gov (United States)

    Sasaki, Yo; Araki, Toshiyuki; Milbrandt, Jeffrey

    2006-08-16

    Axonal degeneration occurs in many neurodegenerative diseases and after traumatic injury and is a self-destructive program independent from programmed cell death. Previous studies demonstrated that overexpression of nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1) or exogenous application of nicotinamide adenine dinucleotide (NAD) can protect axons of cultured dorsal root ganglion (DRG) neurons from degeneration caused by mechanical or neurotoxic injury. In mammalian cells, NAD can be synthesized from multiple precursors, including tryptophan, nicotinic acid, nicotinamide, and nicotinamide riboside (NmR), via multiple enzymatic steps. To determine whether other components of these NAD biosynthetic pathways are capable of delaying axonal degeneration, we overexpressed each of the enzymes involved in each pathway and/or exogenously administered their respective substrates in DRG cultures and assessed their capacity to protect axons after axotomy. Among the enzymes tested, Nmnat1 had the strongest protective effects, whereas nicotinamide phosphoribosyl transferase and nicotinic acid phosphoribosyl transferase showed moderate protective activity in the presence of their substrates. Strong axonal protection was also provided by Nmnat3, which is predominantly located in mitochondria, and an Nmnat1 mutant localized to the cytoplasm, indicating that the subcellular location of NAD production is not crucial for protective activity. In addition, we showed that exogenous application of the NAD precursors that are the substrates of these enzymes, including nicotinic acid mononucleotide, nicotinamide mononucleotide, and NmR, can also delay axonal degeneration. These results indicate that stimulation of NAD biosynthetic pathways via a variety of interventions may be useful in preventing or delaying axonal degeneration.

  17. Simple proxies for estimating the concentrations of monoterpenes and their oxidation products at a boreal forest site

    Science.gov (United States)

    Kontkanen, Jenni; Paasonen, Pauli; Aalto, Juho; Bäck, Jaana; Rantala, Pekka; Petäjä, Tuukka; Kulmala, Markku

    2016-10-01

    The oxidation products of monoterpenes likely have a crucial role in the formation and growth of aerosol particles in boreal forests. However, the continuous measurements of monoterpene concentrations are usually not available on decadal timescales, and the direct measurements of the concentrations of monoterpene oxidation product have so far been scarce. In this study we developed proxies for the concentrations of monoterpenes and their oxidation products at a boreal forest site in Hyytiälä, southern Finland. For deriving the proxies we used the monoterpene concentration measured with a proton transfer reaction mass spectrometer (PTR-MS) during 2006-2013. Our proxies for the monoterpene concentration take into account the temperature-controlled emissions from the forest ecosystem, the dilution caused by the mixing within the boundary layer and different oxidation processes. All the versions of our proxies captured the seasonal variation of the monoterpene concentration, the typical proxy-to-measurements ratios being between 0.8 and 1.3 in summer and between 0.6 and 2.6 in winter. In addition, the proxies were able to describe the diurnal variation of the monoterpene concentration rather well, especially in summer months. By utilizing one of the proxies, we calculated the concentration of oxidation products of monoterpenes by considering their production in the oxidation and their loss due to condensation on aerosol particles. The concentration of oxidation products was found to have a clear seasonal cycle, with a maximum in summer and a minimum in winter. The concentration of oxidation products was lowest in the morning or around noon and highest in the evening. In the future, our proxies for the monoterpene concentration and their oxidation products can be used, for example, in the analysis of new particle formation and growth in boreal environments.

  18. plantiSMASH: automated identification, annotation and expression analysis of plant biosynthetic gene clusters

    DEFF Research Database (Denmark)

    Kautsar, Satria A.; Suarez Duran, Hernando G.; Blin, Kai

    2017-01-01

    of predicted biosynthetic enzyme-coding genes, and facilitates comparative genomic analysis to study the evolutionary conservation of each cluster. Applied on 48 high-quality plant genomes, plantiSMASH identifies a rich diversity of candidate plant BGCs. These results will guide further experimental...... exploration of the nature and dynamics of gene clustering in plant metabolism. Moreover, spurred by the continuing decrease in costs of plant genome sequencing, they will allow genome mining technologies to be applied to plant natural product discovery. The plantiSMASH web server, precalculated results...

  19. Deoxyxylulose 5-phosphate reductoisomerase is not a rate-determining enzyme for essential oil production in spike lavender.

    Science.gov (United States)

    Mendoza-Poudereux, Isabel; Muñoz-Bertomeu, Jesús; Arrillaga, Isabel; Segura, Juan

    2014-11-01

    Spike lavender (Lavandula latifolia) is an economically important aromatic plant producing essential oils, whose components (mostly monoterpenes) are mainly synthesized through the plastidial methylerythritol 4-phosphate (MEP) pathway. 1-Deoxy-D-xylulose-5-phosphate (DXP) synthase (DXS), that catalyzes the first step of the MEP pathway, plays a crucial role in monoterpene precursors biosynthesis in spike lavender. To date, however, it is not known whether the DXP reductoisomerase (DXR), that catalyzes the conversion of DXP into MEP, is also a rate-limiting enzyme for the biosynthesis of monoterpenes in spike lavender. To investigate it, we generated transgenic spike lavender plants constitutively expressing the Arabidopsis thaliana DXR gene. Although two out of the seven transgenic T0 plants analyzed accumulated more essential oils than the controls, this is hardly imputable to the DXR transgene effect since a clear correlation between transcript accumulation and monoterpene production could not be established. Furthermore, these increased essential oil phenotypes were not maintained in their respective T1 progenies. Similar results were obtained when total chlorophyll and carotenoid content in both T0 transgenic plants and their progenies were analyzed. Our results then demonstrate that DXR enzyme does not play a crucial role in the synthesis of plastidial monoterpene precursors, suggesting that the control flux of the MEP pathway in spike lavender is primarily exerted by the DXS enzyme.

  20. Role of de novo biosynthesis in ecosystem scale monoterpene emissions from a boreal Scots pine forest

    Directory of Open Access Journals (Sweden)

    R. Taipale

    2011-08-01

    Full Text Available Monoterpene emissions from Scots pine have traditionally been assumed to originate as evaporation from specialized storage pools. More recently, the significance of de novo emissions, originating directly from monoterpene biosynthesis, has been recognized. To study the role of biosynthesis at the ecosystem scale, we measured monoterpene emissions from a Scots pine dominated forest in southern Finland using the disjunct eddy covariance method combined with proton transfer reaction mass spectrometry. The interpretation of the measurements was based on a correlation analysis and a hybrid emission algorithm describing both de novo and pool emissions. During the measurement period May–August 2007, the monthly medians of daytime emissions were 200, 290, 180, and 200 μg m−2 h−1. The emissions were partly light dependent, probably due to de novo biosynthesis. The emission potential for both de novo and pool emissions exhibited a decreasing summertime trend. The ratio of the de novo emission potential to the total emission potential varied between 30 % and 46 %. Although the monthly changes were not significant, the ratio always differed statistically from zero, suggesting that the role of de novo biosynthesis was observable. Given the uncertainties in this study, we conclude that more accurate estimates of the contribution of de novo emissions are required for improving monoterpene emission algorithms for Scots pine dominated forests.

  1. The monoterpenes of Artemisia tridentata ssp. vaseyana, Artemisia cana ssp. viscidula and Artemisia tridentata ssp. spiciformis.

    Science.gov (United States)

    Gunawardena, K; Rivera, S B; Epstein, W W

    2002-01-01

    Monoterpenes from three different members of the Anthemideae family, Artemisia tridentata ssp. vaseyana, Artemisia cana ssp. viscidula and Artemisia tridentata ssp. spiciformis were isolated and their structures determined using spectroscopic techniques. A total of 26 irregular and regular monoterpenes were identified. Among these, 20 had previously been identified in the Anthemideae family. Of the remaining six, four were known, but previously unidentified in this family. 2,2-Dimethyl-6-isopropenyl-2H-pyran, 2,3-dimethyl-6-isopropyl-4H-pyran and 2-isopropenyl-5-methylhexa-trans-3,5-diene-1-ol were isolated from both A. tridentata ssp. vaseyana and A. cana ssp. viscidula. The irregular monoterpene 2,2-dimethyl-6-isopropenyl-2H-pyran has a carbon skeleton analogous to the biologically important triterpene squalene. Two additional irregular monoterpenes, artemisia triene and trans-chrysanthemal were isolated from A. cana ssp. viscidula and lavandulol was isolated from A. tridentata ssp. spiciformis. This is the first time a compound possessing a lavandulyl-skeletal type has been found in the Anthemideae family.

  2. Comparative study of the antitumor effect of natural monoterpenes: relationship to cell cycle analysis

    Directory of Open Access Journals (Sweden)

    Abdeslam Jaafari

    2012-06-01

    Full Text Available Monoterpenes have been identified as responsible of important therapeutic effects of plant-extracts. In this work, we try to compare the cytotoxic effect of six monoterpenes (carvacrol, thymol, carveol, carvone, eugenol and isopulegol as well as their molecular mechanisms. The in vitro antitumor activity of the tested products, evaluated against five tumor cell lines, show that the carvacrol is the most cytotoxic monoterpene. The investigation of an eventual synergistic effect of the six natural monoterpenes with two anticancer drugs revealed that there is a significant synergy between them (p<5%. On the other hand, the effect of the tested products on cell cycle progression was examined by flow cytometry after DNA staining in order to investigate the molecular mechanism of their cytotoxic activity. The results revealed that carvacrol and carveol stopped the cell cycle progression in S phase; however, thymol and isopulegol stopped it in G0/G1 phase. Regarding carvone and eugenol, no effect on cell cycle was observed.

  3. Xylem monoterpenes of some hard pines of Western North America: three studies

    Science.gov (United States)

    Richard H. Smith

    1982-01-01

    Monoterpene composition was studied in a number of hard pine species and results were compared with earlier work. (1) Intratree measurements showed strong constancy of composition in both single-stemmed and forked trees of ponderosa, Jeffrey, Coulter, and Jeffrey x ponderosa pines. In grafts of these and other pines, the scion influenced the root stock, but not the...

  4. Introducing Organic Chemistry Students to Natural Product Isolation Using Steam Distillation and Liquid Phase Extraction of Thymol, Camphor, and Citral, Monoterpenes Sharing a Unified Biosynthetic Precursor

    Science.gov (United States)

    McLain, Katherine A.; Miller, Kenneth A.; Collins, William R.

    2015-01-01

    Plants have provided and continue to provide the inspiration and foundation for modern medicines. Natural product isolation is a key component of the process of drug discovery from plants. The purpose of this experiment is to introduce first semester undergraduate organic chemistry students, who have relatively few lab techniques at their…

  5. Introducing Organic Chemistry Students to Natural Product Isolation Using Steam Distillation and Liquid Phase Extraction of Thymol, Camphor, and Citral, Monoterpenes Sharing a Unified Biosynthetic Precursor

    Science.gov (United States)

    McLain, Katherine A.; Miller, Kenneth A.; Collins, William R.

    2015-01-01

    Plants have provided and continue to provide the inspiration and foundation for modern medicines. Natural product isolation is a key component of the process of drug discovery from plants. The purpose of this experiment is to introduce first semester undergraduate organic chemistry students, who have relatively few lab techniques at their…

  6. Biosynthesis of ascaridole: iodide peroxidase-catalyzed synthesis of a monoterpene endoperoxide in soluble extracts of Chenopodium ambrosioides fruit.

    Science.gov (United States)

    Johnson, M A; Croteau, R

    1984-11-15

    Ascaridole, an asymmetric monoterpene endoperoxide with anthelmintic properties, occurs as a major constituent (60-80%) in the volatile oil of American wormseed fruit (Chenopodium ambrosioides: Chenopodiaceae), and as a lesser component in the leaf pocket oil of the boldo tree (Peumus boldus: Monimiaceae). Determination of optical activity and chromatographic resolution of naturally occurring ascaridole, and several synthetic derivatives, showed that both wormseed and boldo produce ascaridole in racemic form. The biosynthesis of ascaridole from the conjugated, symmetrical diene alpha-terpinene (a major component of the oil from wormseed) was shown to be catalyzed by a soluble iodide peroxidase isolated from homogenates of C. ambrosioides fruit and leaves. The enzymatic synthesis of ascaridole was confirmed by capillary gas-liquid chromatography and mass spectrometry of the product, which was also shown to be racemic. Optimal enzymatic activity occurred at pH 4.0 in the presence of 2.5 mM H2O2 and 1 mM NaI. Soluble enzyme extracts were fractionated by gel filtration on both Sephacryl S-300 and Sephadex G-100, and were shown to consist of a high-molecular-weight peroxidase component (Mr greater than 1,000,000, 30% of total activity) and two other peroxidase species having apparent molecular weights of 62,000 and 45,000 (major component). Peroxidase activity was susceptible to proteolytic destruction only after periodate treatment, suggesting an association of the enzyme(s) with polysaccharide material. Ascaridole biosynthesis from alpha-terpinene was inhibited by cyanide, catalase, and reducing agents, but not by compounds that trap superoxide or quench singlet oxygen. A peroxide transfer reaction initiated by peroxidase-generated I+ is proposed for the conversion of alpha-terpinene to ascaridole.

  7. The biosynthetic basis of adult lactase deficiency.

    OpenAIRE

    Witte, J; Lloyd, M.; Lorenzsonn, V; Korsmo, H.; Olsen, W.

    1990-01-01

    The intestinal brush-border enzyme lactase splits lactose into its component monosaccharides, glucose and galactose. Relative deficiency of the enzyme during adulthood is a common condition worldwide and is frequently associated with symptoms of lactose intolerance. We studied the synthesis and processing of lactase in normal and adult hypolactasic subjects using human intestinal explants in organ culture. Metabolic labeling experiments in our control subjects with [35S]methionine followed by...

  8. Monoterpenes produced by Cupressus lusitanica cultured cells including a novel monoterpene (1R, 2S, 6S)-(+)-1,6-epoxy-4(8)-p-menthen-2-ol [corrected].

    Science.gov (United States)

    Matsunaga, Yoko; Fujita, Koki; Yamada, Junko; Ashitani, Tatsuya; Sakai, Kokki

    2003-12-01

    A novel monoterpene,(1R, 2S, 6S)-(+)-1,6-epoxy-4(8)-p-menthen-2-ol [corrected], was isolated from an elicitor-treated cell culture of Cupressus lusitanica (Mexican cypress). Ten known monoterpenes--limonene, myrcene, beta-ocimene, sabinene, terpinolene, 4-terpineol, alpha-terpineol, 4(8)-p-menthen-1,2-diol, 4-hydroxyphellandric acid methylester and beta-thujaplicin methylether--were also identified. Regioselective metabolisms of monoterpenes were observed in this culture.

  9. Origin of monoterpene emissions from boreal tree species: Determination of de novo and pool emissions by 13CO2 labeling

    Science.gov (United States)

    Rinne, J.; Ghirardo, A.; Koch, K.; Taipale, R.; Zimmer, I.; Schnitzler, J.

    2009-12-01

    Boreal forests emit a large amount of monoterpenes into the atmosphere. Traditionally these emissions are assumed to originate as evaporation from large storage pools. Thus their diurnal cycle would depend mostly on temperature. However, there is indication that a significant part of the monoterpene emission would originate directly from de novo synthesis. By applying 13CO2 fumigation and analyzing the isotope fractions with proton transfer reaction mass spectrometry (PTR-MS) and classical GC-MS we studied the origin of monoterpene emissions from some major Eurasian boreal and alpine tree species. We determined the fractions originating from de novo biosynthesis and from large internal monoterpene storages for three coniferous tree species with specialized monoterpene storage structures and one dicotyledon species without such structures. The emission from dicotyledon species Betula pendula originated solely from the de novo synthesis. The origin of the emissions from coniferous species was mixed with varying fraction originating from de novo synthesis (Pinus sylvestris 58%, Picea abies 33.5%, Larix decidua 9.8%) and the rest from large internal monoterpene storage pools. Application of the observed fractions of emission originating from de novo synthesis and large storage pools in a hybrid emission algorithm resulted in a better description of ecosystem scale monoterpene emissions from a boreal Scots pine forest stand.

  10. Quantifying environmental stress-induced emissions of algal isoprene and monoterpenes using laboratory measurements

    Science.gov (United States)

    Meskhidze, N.; Sabolis, A.; Reed, R.; Kamykowski, D.

    2015-02-01

    We report here production rates of isoprene and monoterpene compounds (α-pinene, β-pinene, camphene and d-limonene) from six phytoplankton monocultures as a function of irradiance and temperature. Irradiance experiments were carried out for diatom strains (Thalassiosira weissflogii and Thalassiosira pseudonana), prymnesiophyte strains (Pleurochrysis carterae), dinoflagellate strains (Karenia brevis and Prorocentrum minimum), and cryptophyte strains (Rhodomonas salina), while temperature experiments were carried out for diatom strains (Thalassiosira weissflogii and Thalassiosira pseudonana). Phytoplankton species, incubated in a climate-controlled room, were subject to variable light (90 to 900 μmol m-2 s-1) and temperature (18 to 30 °C) regimes. Compared to isoprene, monoterpene emissions were an order of magnitude lower at all light and temperature levels. Emission rates are normalized by cell count and Chlorophyll a (Chl a) content. Diatom strains were the largest emitters, with ~ 2 × 10-17 g(cell)-1h-1 (~ 35 μg (g Chl a)-1 h-1) for isoprene and ~ 5 × 10-19 g (cell)-1 h-1 (~ 1 μg (g Chl a)-1) h-1) for α-pinene. The contribution to the total monoterpene production was ~ 70% from α-pinene, ~ 20% for d-limonene, and 250 μmol m-2 s-1) irradiance. Measurements revealed different patterns for time-averaged emissions rates over two successive days. On the first day, most of the species showed a distinct increase in production rates within the first 4 h while, on the second day, the emission rates were overall higher, but less variable. The data suggest that enhanced amounts of isoprene and monoterpenes are emitted from phytoplankton as a result of perturbations in environmental conditions that cause imbalance in chloroplasts and force primary producers to acclimate physiologically. This relationship could be a valuable tool for development of dynamic ecosystem modeling approaches for global marine isoprene and monoterpene emissions based on phytoplankton

  11. Quantifying environmental stress induced emissions of algal isoprene and monoterpenes using laboratory measurements

    Science.gov (United States)

    Meskhidze, N.; Sabolis, A.; Reed, R.; Kamykowski, D.

    2014-09-01

    We report here production rates of isoprene and monoterpene compounds (α-pinene, β-pinene, camphene and d-limonene) from six phytoplankton monocultures as a function of irradiance and temperature. Irradiance experiments were carried out for diatom strains - Thalassiosira weissflogii and Thalassiosira pseudonana; prymnesiophyte strains - Pleurochrysis carterae; dinoflagellate strains - Karenia brevis and Prorocentrum minimum; cryptophyte strains - Rhodomonas salina, while temperature experiments were carried out for diatom strains - Thalassiosira weissflogii and Thalassiosira pseudonana. Phytoplankton species, incubated in a climate-controlled room, were subject to variable light (90 to 900 μmol m-2s-1) and temperature (18 to 30 °C) regimes. Compared to isoprene, monoterpene emissions were an order of magnitude lower at all light and temperature levels. Emission rates are normalized by cell count and Chlorophyll a (Chl a) content. Diatom strains were the largest emitters, with ~2x1017g (cell)-1h-1 (~35 μg (g Chl a)-1h-1) for isoprene and ~5x10-19 g (cell)-1h-1 (~1μg (g Chl a)-1) h-1) for α-pinene. The contribution to the total monoterpene production was ~70% from α-pinene, ~20% for d-limonene, and 250 μmol m-2s-1) irradiance. Measurements revealed different patterns for time-averaged emissions rates over two successive days. On the first day most of the species showed distinct increase in production rates within the first four hours, while on the second day the emission rates were overall higher, but less variable. The data suggest that enhanced amounts of isoprene and monoterpenes are emitted from phytoplankton as a result of perturbations in environmental conditions that cause disbalance in chloroplasts and forces primary producers to acclimate physiologically. This relationship could be a valuable tool for development of dynamic ecosystem modeling approaches for global marine isoprene and monoterpene emissions based on phytoplankton physiological

  12. Laboratory and field measurements of enantiomeric monoterpene emissions as a function of chemotype, light and temperature

    Science.gov (United States)

    Song, W.; Staudt, M.; Bourgeois, I.; Williams, J.

    2014-03-01

    Plants emit significant amounts of monoterpenes into the earth's atmosphere, where they react rapidly to form a multitude of gas phase species and particles. Many monoterpenes exist in mirror-image forms or enantiomers. In this study the enantiomeric monoterpene profile for several representative plants (Quercus ilex L., Rosmarinus officinalis L., and Pinus halepensis Mill.) was investigated as a function of chemotype, light and temperature both in the laboratory and in the field. Analysis of enantiomeric monoterpenes from 19 Quercus ilex individuals from Southern France and Spain revealed four regiospecific chemotypes (genetically fixed emission patterns). In agreement with previous work, only Quercus ilex emissions increased strongly with light. However, for all three plant species no consistent enantiomeric variation was observed as a function of light, and the enantiomeric ratio of α-pinene was found to vary by less than 20% from 100 and 1000 μmol m-2 s-1 PAR (photosynthetically active radiation). The rate of monoterpene emission increased with temperature from all three plant species, but little variation in the enantiomeric distribution of α-pinene was observed with temperature. There was more enantiomeric variability between individuals of the same species than could be induced by either light or temperature. Field measurements of α-pinene enantiomer mixing ratios in the air, taken at a Quercus ilex forest in Southern France, and several other previously reported field enantiomeric ratio diel cycle profiles are compared. All show smoothly varying diel cycles (some positive and some negative) even over changing wind directions. This is surprising in comparison with variations of enantiomeric emission patterns shown by individuals of the same species.

  13. Mutational analysis of a monoterpene synthase reaction: altered catalysis through directed mutagenesis of (-)-pinene synthase from Abies grandis.

    Science.gov (United States)

    Hyatt, David C; Croteau, Rodney

    2005-07-15

    Two monoterpene synthases, (-)-pinene synthase and (-)-camphene synthase, from grand fir (Abies grandis) produce different product mixtures despite having highly homologous amino acid sequences and, presumably, very similar three-dimensional structures. The major product of (-)-camphene synthase, (-)-camphene, and the major products of (-)-pinene synthase, (-)-alpha-pinene, and (-)-beta-pinene, arise through distinct mechanistic variations of the electrophilic reaction cascade that is common to terpenoid synthases. Structural modeling followed by directed mutagenesis in (-)-pinene synthase was used to replace selected amino acid residues with the corresponding residues from (-)-camphene synthase in an effort to identify the amino acids responsible for the catalytic differences. This approach produced an enzyme in which more than half of the product was channeled through an alternative pathway. It was also shown that several (-)-pinene synthase to (-)-camphene synthase amino acid substitutions were necessary before catalysis was significantly altered. The data support a model in which the collective action of many key amino acids, located both in and distant from the active site pocket, regulate the course of the electrophilic reaction cascade.

  14. Role of the hexosamine biosynthetic pathway in diabetic nephropathy.

    Science.gov (United States)

    Schleicher, E D; Weigert, C

    2000-09-01

    The hexosamine biosynthetic pathway has been hypothesized to be involved in the development of insulin resistance and diabetic vascular complications. In particular, it was demonstrated that hyperglycemia-induced production of transforming growth factor-beta (TGF-beta1), a prosclerotic cytokine causally involved in the development of diabetic nephropathy. Several lines of evidence indicate that TGF-beta1 induction is mediated by the hexosamine pathway. In cultured mesangial cells, high glucose levels induce TGF-beta1 production. This effect is eliminated by inhibition of glutamine: fructose-6-phosphate-amidotransferase (GFAT), the rate-limiting enzyme of this pathway. Furthermore, stable overexpression of GFAT increased levels of TGF-beta1 protein, mRNA, and promoter activity. Inasmuch as stimulation or inhibition of GFAT increased or decreased high glucose-stimulated activity of protein kinase C (PKC), respectively, the observed effects appear to be transduced by PKC. In similar experiments, involvement of the hexosamine pathway in hyperglycemia-induced production of cytokines (TGF-alpha and basic fibroblast growth factor [bFGF]) was demonstrated in vascular smooth muscle cells. These studies also revealed a rapid increase in GFAT activity by treatment with agents that elevated levels of cyclic adenosine 3',5' monophosphate (cAMP), thus indicating that GFAT activity is tightly regulated by cAMP-dependent phosphorylation. Using immunohistochemistry and in situ hybridization, high expression of GFAT was found in human adipocytes, skeletal muscle, vascular smooth muscle cells, and renal tubular epithelial cells. whereas glomerular cells remained essentially unstained. However, significant staining occurred in glomerular cells of patients with diabetic nephropathy. Current data indicate that the flux through the hexosamine pathway, regulated by GFAT, may be causally involved in the development of diabetic vascular disease, particularly diabetic nephropathy.

  15. Identification of a novel sesquiterpene biosynthetic machinery involved in astellolide biosynthesis

    Science.gov (United States)

    Shinohara, Yasutomo; Takahashi, Shunji; Osada, Hiroyuki; Koyama, Yasuji

    2016-01-01

    Esterified drimane-type sesquiterpene lactones such as astellolides display various biological activities and are widely produced by plants and fungi. Given their low homology to known sesquiterpene cyclases, the genes responsible for their biosynthesis have not been uncovered yet. Here, we identified the astellolide gene cluster from Aspergillus oryzae and discovered a novel sesquiterpene biosynthetic machinery consisting of AstC, AstI, and AstK. All these enzymes are annotated as haloacid dehalogenase-like hydrolases, whereas AstC also contains a DxDTT motif conserved in class II diterpene cyclases. Based on enzyme reaction analyses, we found that AstC catalysed the protonation-initiated cyclisation of farnesyl pyrophosphate into drimanyl pyrophosphate. This was successively dephosphorylated by AstI and AstK to produce drim-8-ene-11-ol. Moreover, we also identified and characterised a unique non-ribosomal peptide synthetase, AstA, responsible for esterifying aryl acids to drimane-type sesquiterpene lactones. In this study, we highlight a new biosynthetic route for producing sesquiterpene and its esterified derivative. Our findings shed light on the identification of novel sesquiterpenes via genome mining. PMID:27628599

  16. Modulation of guanosine nucleotides biosynthetic pathways enhanced GDP-L-fucose production in recombinant Escherichia coli.

    Science.gov (United States)

    Lee, Won-Heong; Shin, So-Yeon; Kim, Myoung-Dong; Han, Nam Soo; Seo, Jin-Ho

    2012-03-01

    Guanosine 5'-triphosphate (GTP) is the key substrate for biosynthesis of guanosine 5'-diphosphate (GDP)-L-fucose. In this study, improvement of GDP-L-fucose production was attempted by manipulating the biosynthetic pathway for guanosine nucleotides in recombinant Escherichia coli-producing GDP-L-fucose. The effects of overexpression of inosine 5'-monophosphate (IMP) dehydrogenase, guanosine 5'-monophosphate (GMP) synthetase (GuaB and GuaA), GMP reductase (GuaC) and guanosine-inosine kinase (Gsk) on GDP-L-fucose production were investigated in a series of fed-batch fermentations. Among the enzymes tested, overexpression of Gsk led to a significant improvement of GDP-L-fucose production. Maximum GDP-L-fucose concentration of 305.5 ± 5.3 mg l(-1) was obtained in the pH-stat fed-batch fermentation of recombinant E. coli-overexpressing Gsk, which corresponds to a 58% enhancement in the GDP-L-fucose production compared with the control strain overexpressing GDP-L-fucose biosynthetic enzymes. Such an enhancement of GDP-L-fucose production could be due to the increase in the intracellular level of GMP.

  17. Metabolic engineering of the carotenoid biosynthetic pathway in the yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma).

    Science.gov (United States)

    Verdoes, Jan C; Sandmann, Gerhard; Visser, Hans; Diaz, Maria; van Mossel, Minca; van Ooyen, Albert J J

    2003-07-01

    The crtYB locus was used as an integrative platform for the construction of specific carotenoid biosynthetic mutants in the astaxanthin-producing yeast Xanthophyllomyces dendrorhous. The crtYB gene of X. dendrorhous, encoding a chimeric carotenoid biosynthetic enzyme, could be inactivated by both single and double crossover events, resulting in non-carotenoid-producing transformants. In addition, the crtYB gene, linked to either its homologous or a glyceraldehyde-3-phosphate dehydrogenase promoter, was overexpressed in the wild type and a beta-carotene-accumulating mutant of X. dendrorhous. In several transformants containing multiple copies of the crtYB gene, the total carotenoid content was higher than in the control strain. This increase was mainly due to an increase of the beta-carotene and echinone content, whereas the total content of astaxanthin was unaffected or even lower. Overexpression of the phytoene synthase-encoding gene (crtI) had a large impact on the ratio between mono- and bicyclic carotenoids. Furthermore, we showed that in metabolic engineered X. dendrorhous strains, the competition between the enzymes phytoene desaturase and lycopene cyclase for lycopene governs the metabolic flux either via beta-carotene to astaxanthin or via 3,4-didehydrolycopene to 3-hydroxy-3'-4'-didehydro-beta-psi-caroten-4-one (HDCO). The monocylic carotenoid torulene and HDCO, normally produced as minority carotenoids, were the main carotenoids produced in these strains.

  18. Genome sequence of Thermofilum pendens reveals an exceptional loss of biosynthetic pathways without genome reduction

    Energy Technology Data Exchange (ETDEWEB)

    Kyrpides, Nikos; Anderson, Iain; Rodriguez, Jason; Susanti, Dwi; Porat, Iris; Reich, Claudia; Ulrich, Luke E.; Elkins, James G.; Mavromatis, Kostas; Lykidis, Athanasios; Kim, Edwin; Thompson, Linda S.; Nolan, Matt; Land, Miriam; Copeland, Alex; Lapidus, Alla; Lucas, Susan; Detter, Chris; Zhulin, Igor B.; Olsen, Gary J.; Whitman, William; Mukhopadhyay, Biswarup; Bristow, James; Kyrpides, Nikos

    2008-01-01

    We report the complete genome of Thermofilum pendens, a deep-branching, hyperthermophilic member of the order Thermoproteales within the archaeal kingdom Crenarchaeota. T. pendens is a sulfur-dependent, anaerobic heterotroph isolated from a solfatara in Iceland. It is an extracellular commensal, requiring an extract of Thermoproteus tenax for growth, and the genome sequence reveals that biosynthetic pathways for purines, most amino acids, and most cofactors are absent. In fact T. pendens has fewer biosynthetic enzymes than obligate intracellular parasites, although it does not display other features common among obligate parasites and thus does not appear to be in the process of becoming a parasite. It appears that T. pendens has adapted to life in an environment rich in nutrients. T. pendens was known to utilize peptides as an energy source, but the genome reveals substantial ability to grow on carbohydrates. T. pendens is the first crenarchaeote and only the second archaeon found to have a transporter of the phosphotransferase system. In addition to fermentation, T. pendens may gain energy from sulfur reduction with hydrogen and formate as electron donors. It may also be capable of sulfur-independent growth on formate with formate hydrogenlyase. Additional novel features are the presence of a monomethylamine:corrinoid methyltransferase, the first time this enzyme has been found outside of Methanosarcinales, and a presenilin-related protein. Predicted highly expressed proteins do not include housekeeping genes, and instead include ABC transporters for carbohydrates and peptides, and CRISPR-associated proteins.

  19. Regulation of polyamine biosynthetic activity by spermidine and spermine analogs--a novel antiproliferative strategy.

    Science.gov (United States)

    Porter, C W; Bergeron, R J

    1988-01-01

    Interference with polyamine biosynthesis by analog-mediated regulatory mechanisms represents a viable alternative to the use of specific enzyme inhibitors as an antiproliferative strategy. The approach is unique among antimetabolite approaches and is made possible by unusual characteristics inherent to the polyamines and their biosynthetic pathway. Current antitumor data obtained with these analogs provides indication of their potential usefulness as antitumor agents but, at the same time, demonstrates the need for improvement. This latter might be attained by the rational design of analogs which (a) bind more tightly at enzyme regulatory sites, (b) which are less able to substitute for natural polyamines in growth related functions and (c) which are eliminated less rapidly from tumor-bearing animals. At the same time, the continued preclinical development of available analogs might proceed most productively by targeting large cell lung carcinoma and melanoma and by examining the generality of the relationship between oncogene expression and the accompanying sensitivity to regulatory analogs.

  20. Subcellular Compartmentalization and Trafficking of the Biosynthetic Machinery for Fungal Melanin.

    Science.gov (United States)

    Upadhyay, Srijana; Xu, Xinping; Lowry, David; Jackson, Jennifer C; Roberson, Robert W; Lin, Xiaorong

    2016-03-22

    Protection by melanin depends on its subcellular location. Although most filamentous fungi synthesize melanin via a polyketide synthase pathway, where and how melanin biosynthesis occurs and how it is deposited as extracellular granules remain elusive. Using a forward genetic screen in the pathogen Aspergillus fumigatus, we find that mutations in an endosomal sorting nexin abolish melanin cell-wall deposition. We find that all enzymes involved in the early steps of melanin biosynthesis are recruited to endosomes through a non-conventional secretory pathway. In contrast, late melanin enzymes accumulate in the cell wall. Such subcellular compartmentalization of the melanin biosynthetic machinery occurs in both A. fumigatus and A. nidulans. Thus, fungal melanin biosynthesis appears to be initiated in endosomes with exocytosis leading to melanin extracellular deposition, much like the synthesis and trafficking of mammalian melanin in endosomally derived melanosomes.

  1. Origin of saxitoxin biosynthetic genes in cyanobacteria.

    Directory of Open Access Journals (Sweden)

    Ahmed Moustafa

    Full Text Available BACKGROUND: Paralytic shellfish poisoning (PSP is a potentially fatal syndrome associated with the consumption of shellfish that have accumulated saxitoxin (STX. STX is produced by microscopic marine dinoflagellate algae. Little is known about the origin and spread of saxitoxin genes in these under-studied eukaryotes. Fortuitously, some freshwater cyanobacteria also produce STX, providing an ideal model for studying its biosynthesis. Here we focus on saxitoxin-producing cyanobacteria and their non-toxic sisters to elucidate the origin of genes involved in the putative STX biosynthetic pathway. METHODOLOGY/PRINCIPAL FINDINGS: We generated a draft genome assembly of the saxitoxin-producing (STX+ cyanobacterium Anabaena circinalis ACBU02 and searched for 26 candidate saxitoxin-genes (named sxtA to sxtZ that were recently identified in the toxic strain Cylindrospermopsis raciborskii T3. We also generated a draft assembly of the non-toxic (STX- sister Anabaena circinalis ACFR02 to aid the identification of saxitoxin-specific genes. Comparative phylogenomic analyses revealed that nine putative STX genes were horizontally transferred from non-cyanobacterial sources, whereas one key gene (sxtA originated in STX+ cyanobacteria via two independent horizontal transfers followed by fusion. In total, of the 26 candidate saxitoxin-genes, 13 are of cyanobacterial provenance and are monophyletic among the STX+ taxa, four are shared amongst STX+ and STX-cyanobacteria, and the remaining nine genes are specific to STX+ cyanobacteria. CONCLUSIONS/SIGNIFICANCE: Our results provide evidence that the assembly of STX genes in ACBU02 involved multiple HGT events from different sources followed presumably by coordination of the expression of foreign and native genes in the common ancestor of STX+ cyanobacteria. The ability to produce saxitoxin was subsequently lost multiple independent times resulting in a nested relationship of STX+ and STX- strains among Anabaena

  2. Synthesis of both enantiomers of isorobinal, a novel cyclic monoterpene isolated from the astigmatid mite, Rhizoglyphus sp.

    Science.gov (United States)

    Liang, Ting; Kuwahara, Shigefumi

    2002-11-01

    Both enantiomers of isorobinal, a cyclic monoterpene isolated from the astigmatid mite (Rhizoglyphus sp.), were synthesized from the enantiomers of perillaldehyde in four steps by using PCC-oxidation of a tertiary allylic alcohol intermediate as the key step.

  3. Drought reduced monoterpene emissions from the evergreen Mediterranean oak Quercus ilex: results from a throughfall displacement experiment

    Directory of Open Access Journals (Sweden)

    S. Rambal

    2009-07-01

    Full Text Available The effects of water limitations on the emission of biogenic volatile organic compounds are not well understood. Experimental approaches studying drought effects in natural conditions are still missing. To address this question, a throughfall displacement experiment was set up in a natural forest of Quercus ilex, an evergreen Mediterranean oak emitting monoterpenes. Mature trees were exposed in 2005 and 2006 either to an additional drought, to irrigation or to natural drought (untreated control. In both years, absolute monoterpene emission rates as well as the respective standard factors of the trees exposed to normal and additional drought strongly declined during the drought periods. Monoterpene emissions were lower in year 2006 than in year 2005 (factor 2 due to a more pronounced summer drought period in this respective year. We observed a significant difference between the irrigation and additional drought or control treatment: irrigated trees emitted 82% more monoterpenes during the drought period 2006 than the trees of the other treatments. However, no significant effect on monoterpene emission was observed between normal and additional drought treatments, despite a significant effect on leaf water potential and photochemical efficiency. During the development of drought, monoterpene emissions responded exponentially rather than linearly to decreasing leaf water potential. Emissions rapidly declined when the water potential dropped below −2 MPa and photosynthesis was persistently inhibited. Monoterpene synthase activities measured in vitro showed no clear reduction during the same period. From our results we conclude that drought significantly reduces monoterpene fluxes of Mediterranean Holm oak forest into the atmosphere due to a lack of primary substrates coming from photosynthetic processes.

  4. Synthesis and Biological Evaluation of Novel Phosphatidylcholine Analogues Containing Monoterpene Acids as Potent Antiproliferative Agents.

    Directory of Open Access Journals (Sweden)

    Anna Gliszczyńska

    Full Text Available The synthesis of novel phosphatidylcholines with geranic and citronellic acids in sn-1 and sn-2 positions is described. The structured phospholipids were obtained in high yields (59-87% and evaluated in vitro for their cytotoxic activity against several cancer cell lines of different origin: MV4-11, A-549, MCF-7, LOVO, LOVO/DX, HepG2 and also towards non-cancer cell line BALB/3T3 (normal mice fibroblasts. The phosphatidylcholines modified with monoterpene acid showed a significantly higher antiproliferative activity than free monoterpene acids. The highest activity was observed for the terpene-phospholipids containing the isoprenoid acids in sn-1 position of phosphatidylcholine and palmitic acid in sn-2.

  5. Synthesis and Biological Evaluation of Novel Phosphatidylcholine Analogues Containing Monoterpene Acids as Potent Antiproliferative Agents

    Science.gov (United States)

    Gliszczyńska, Anna; Niezgoda, Natalia; Gładkowski, Witold; Czarnecka, Marta; Świtalska, Marta; Wietrzyk, Joanna

    2016-01-01

    The synthesis of novel phosphatidylcholines with geranic and citronellic acids in sn-1 and sn-2 positions is described. The structured phospholipids were obtained in high yields (59–87%) and evaluated in vitro for their cytotoxic activity against several cancer cell lines of different origin: MV4-11, A-549, MCF-7, LOVO, LOVO/DX, HepG2 and also towards non-cancer cell line BALB/3T3 (normal mice fibroblasts). The phosphatidylcholines modified with monoterpene acid showed a significantly higher antiproliferative activity than free monoterpene acids. The highest activity was observed for the terpene-phospholipids containing the isoprenoid acids in sn-1 position of phosphatidylcholine and palmitic acid in sn-2. PMID:27310666

  6. Selective removal of monoterpenes from bergamot oil by inclusion in deoxycholic acid.

    Science.gov (United States)

    Fantin, Giancarlo; Fogagnolo, Marco; Maietti, Silvia; Rossetti, Stefano

    2010-05-12

    A new approach for removing monoterpenes (MTs) from bergamot oil by selective inclusion in deoxycholic acid (DCA) is proposed. The inclusion process is very efficient, the included fraction being composed mainly of limonene (71.7%) and gamma-terpinene (19.8%). On the other hand, the deterpenated bergamot oil fraction showed for the linalool and linalyl acetate derivatives significant increases from 16.6 and 21.4% to 18.3 and 42.2%, respectively. The major advantages of this methodology are its simplicity, the mild conditions employed, and the quantitative recovery of both host (DCA) and guest (monoterpenes) compounds. Differential scanning calorimetry (DSC), thermal gravimetry (TG), powder X-ray diffractometry (XRPD), infrared spectroscopy (IR), and proton magnetic resonance ((1)H NMR) analysis were used to investigate and characterize the inclusion compounds.

  7. Biosynthesis of monoterpenes: Stereochemistry of the coupled isomerization and cyclization of geranyl pyrophosphate to camphane and isocamphane monoterpenes

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, R.; Gershenzon, J.; Wheeler, C.J.; Satterwhite, D.M. (Washington State Univ., Pullman (USA))

    1990-03-01

    The conversion of geranyl pyrophosphate to (+)-bornyl pyrophosphate and (+)-camphene is considered to proceed by the initial isomerization of the substrate to (-)-(3R)-linalyl pyrophosphate and the subsequent cyclization of this bound intermediate. In the case of (-)-bornyl pyrophosphate and (-)-camphene, isomerization of the substrate to the (+)-(3S)-linalyl intermediate precedes cyclization. The geranyl and linalyl precursors were shown to be mutually competitive substrates (inhibitors) of the relevant cyclization enzymes isolated from Salvia officinalis (sage) and Tanacetum vulgare (tansy) by the mixed substrate analysis method, demonstrating that isomerization and cyclization take place at the same active site. Incubation of partially purified enzyme preparations with (3R)-(1Z-3H)linalyl pyrophosphate plus (1-14C)geranyl pyrophosphate gave rise to double-labeled (+)-bornyl pyrophosphate and (+)-camphene, whereas incubation of enzyme preparations catalyzing the antipodal cyclizations with (3S)-(1Z-3H)-linalyl pyrophosphate plus (1-14C)geranyl pyrophosphate yielded double-labeled (-)-bornyl pyrophosphate and (-)-camphene. Each product was then transformed to the corresponding (+)- or (-)-camphor without change in the 3H:14C isotope ratio, and the location of the tritium label was deduced in each case by stereoselective, base-catalyzed exchange of the exo-alpha-hydrogen of the derived ketone. The finding that the 1Z-3H of the linalyl precursor was positioned at the endo-alpha-hydrogen of the corresponding camphor in all cases, coupled to the previously demonstrated retention of configuration at C1 of the geranyl substrate in these transformations, confirmed the syn-isomerization of geranyl pyrophosphate to linalyl pyrophosphate and the cyclization of the latter via the anti,endo- conformer.

  8. Seasonal influence on gene expression of monoterpene synthases in Salvia officinalis (Lamiaceae).

    Science.gov (United States)

    Grausgruber-Gröger, Sabine; Schmiderer, Corinna; Steinborn, Ralf; Novak, Johannes

    2012-03-01

    Garden sage (Salvia officinalis L., Lamiaceae) is one of the most important medicinal and aromatic plants and possesses antioxidant, antimicrobial, spasmolytic, astringent, antihidrotic and specific sensorial properties. The essential oil of the plant, formed mainly in very young leaves, is in part responsible for these activities. It is mainly composed of the monoterpenes 1,8-cineole, α- and β-thujone and camphor synthesized by the 1,8-cineole synthase, the (+)-sabinene synthase and the (+)-bornyl diphosphate synthase, respectively, and is produced and stored in epidermal glands. In this study, the seasonal influence on the formation of the main monoterpenes in young, still expanding leaves of field-grown sage plants was studied in two cultivars at the level of mRNA expression, analyzed by qRT-PCR, and at the level of end-products, analyzed by gas chromatography. All monoterpene synthases and monoterpenes were significantly influenced by cultivar and season. 1,8-Cineole synthase and its end product 1,8-cineole remained constant until August and then decreased slightly. The thujones increased steadily during the vegetative period. The transcript level of their corresponding terpene synthase, however, showed its maximum in the middle of the vegetative period and declined afterwards. Camphor remained constant until August and then declined, exactly correlated with the mRNA level of the corresponding terpene synthase. In summary, terpene synthase mRNA expression and respective end product levels were concordant in the case of 1,8-cineole (r=0.51 and 0.67 for the two cultivars, respectively; p<0.05) and camphor (r=0.75 and 0.82; p<0.05) indicating basically transcriptional control, but discordant for α-/β-thujone (r=-0.05 and 0.42; p=0.87 and 0.13, respectively).

  9. Evaluating the Potential Importance of Monoterpene Degradation for Global Acetone Production

    Science.gov (United States)

    Kelp, M. M.; Brewer, J.; Keller, C. A.; Fischer, E. V.

    2015-12-01

    Acetone is one of the most abundant volatile organic compounds (VOCs) in the atmosphere, but estimates of the global source of acetone vary widely. A better understanding of acetone sources is essential because acetone serves as a source of HOx in the upper troposphere and as a precursor to the NOx reservoir species peroxyacetyl nitrate (PAN). Although there are primary anthropogenic and pyrogenic sources of acetone, the dominant acetone sources are thought to be from direct biogenic emissions and photochemical production, particularly from the oxidation of iso-alkanes. Recent work suggests that the photochemical degradation of monoterpenes may also represent a significant contribution to global acetone production. We investigate that hypothesis using the GEOS-Chem chemical transport model. In this work, we calculate the emissions of eight terpene species (α-pinene, β-pinene, limonene, Δ3-carene, myrcene, sabinene, trans-β-ocimene, and an 'other monoterpenes' category which contains 34 other trace species) and couple these with upper and lower bound literature yields from species-specific chamber studies. We compare the simulated acetone distributions against in situ acetone measurements from a global suite of NASA aircraft campaigns. When simulating an upper bound on yields, the model-to-measurement comparison improves for North America at both the surface and in the upper troposphere. The inclusion of acetone production from monoterpene degradation also improves the ability of the model to reproduce observations of acetone in East Asian outflow. However, in general the addition of monoterpenes degrades the model comparison for the Southern Hemisphere.

  10. European emissions of isoprene and monoterpenes from the Last Glacial Maximum to present

    OpenAIRE

    Schurgers, G.; Hickler, T.; Miller, P. A.; Arneth, A.

    2009-01-01

    Biogenic volatile organic compounds (BVOC), such as isoprene and monoterpenes, play an important role in atmospheric processes. BVOC species are oxidized in the atmosphere and influence levels of ozone. The less volatile amongst the BVOC and their oxidation products are important for the formation and growth of secondary biogenic aerosol. In this way, the earth's radiation balance is affected.

    Geographic distribution and temporal changes in BVOC emissions are highly uncer...

  11. Improving monoterpene geraniol production through geranyl diphosphate synthesis regulation in Saccharomyces cerevisiae.

    Science.gov (United States)

    Zhao, Jianzhi; Bao, Xiaoming; Li, Chen; Shen, Yu; Hou, Jin

    2016-05-01

    Monoterpenes have wide applications in the food, cosmetics, and medicine industries and have recently received increased attention as advanced biofuels. However, compared with sesquiterpenes, monoterpene production is still lagging in Saccharomyces cerevisiae. In this study, geraniol, a valuable acyclic monoterpene alcohol, was synthesized in S. cerevisiae. We evaluated three geraniol synthases in S. cerevisiae, and the geraniol synthase Valeriana officinalis (tVoGES), which lacked a plastid-targeting peptide, yielded the highest geraniol production. To improve geraniol production, synthesis of the precursor geranyl diphosphate (GPP) was regulated by comparing three specific GPP synthase genes derived from different plants and the endogenous farnesyl diphosphate synthase gene variants ERG20 (G) (ERG20 (K197G) ) and ERG20 (WW) (ERG20 (F96W-N127W) ), and controlling endogenous ERG20 expression, coupled with increasing the expression of the mevalonate pathway by co-overexpressing IDI1, tHMG1, and UPC2-1. The results showed that overexpressing ERG20 (WW) and strengthening the mevalonate pathway significantly improved geraniol production, while expressing heterologous GPP synthase genes or down-regulating endogenous ERG20 expression did not show positive effect. In addition, we constructed an Erg20p(F96W-N127W)-tVoGES fusion protein, and geraniol production reached 66.2 mg/L after optimizing the amino acid linker and the order of the proteins. The best strain yielded 293 mg/L geraniol in a fed-batch cultivation, a sevenfold improvement over the highest titer previously reported in an engineered S. cerevisiae strain. Finally, we showed that the toxicity of geraniol limited its production. The platform developed here can be readily used to synthesize other monoterpenes.

  12. Remote sensing estimation of isoprene and monoterpene emissions generated by natural vegetation in Monterrey, Mexico.

    Science.gov (United States)

    Gastelum, Sandra L; Mejía-Velázquez, G M; Lozano-García, D Fabián

    2016-06-01

    In addition to oxygen, hydrocarbons are the most reactive chemical compounds produced by plants into the atmosphere. These compounds are part of the family of volatile organic compounds (VOCs) and are discharged in a great variety of forms. Among the VOCs produced by natural sources such as vegetation, the most studied until today are the isoprene and monoterpene. These substances can play an important role in the chemical balance of the atmosphere of a region. In this project, we develop a methodology to estimate the natural (vegetation) emission of isoprene and monoterpenes and applied it to the Monterrey Metropolitan Area, Mexico and its surrounding areas. Landsat-TM data was used to identify the dominant vegetation communities and field work to determine the foliage biomass density of key species. The studied communities were submontane scrub, oak, and pine forests and a combination of both. We carried out the estimation of emissions for isoprene and monoterpenes compounds in the different plant communities, with two different criteria: (1) taking into account the average foliage biomass density obtained from the various sample point in each vegetation community, and (2) using the foliage biomass density obtained for each transect, associated to an individual spectral class within a particular vegetation type. With this information, we obtained emission maps for each case. The results show that the main producers of isoprene are the communities that include species of the genus Quercus, located mainly on the Sierra Madre Oriental and Sierra de Picachos, with average isoprene emissions of 314.6 ton/day and 207.3 ton/day for the two methods utilized. The higher estimates of monoterpenes were found in the submontane scrub areas distributed along the valley of the metropolitan zone, with an estimated average emissions of 47.1 ton/day and 181.4 tons for the two methods respectively.

  13. Aspergillus nidulans as a platform for discovery and characterization of complex biosynthetic pathways

    DEFF Research Database (Denmark)

    Anyaogu, Diana Chinyere

    of secondary metabolites and 2) Developing A. nidulans as a model systemfor protein production with human-like glycan structure.  The first part of this study resulted in the development of a method for the transfer and expression ofintact biosynthetic gene clusters to A. nidulans to facilitate pathway...... of geodin. Expression of the enzymes inthe pathway was validated by transcription analysis and the functions of specific genes were investigatedby gene deletions. This proved that this method is a fast and easy way to transfer biosynthetic gene clustersregardless of size and characterize them. Furthermore......, a different approach to activate silent clusters wasdemonstrated, as the heterologous expression of a putative transcription factor from A. niger in A. nidulansinduced the synthesis of insect juvenile hormones in A. nidulans, which had previously not been reportedas fungal metabolites.  The second part...

  14. Genome mining of the hitachimycin biosynthetic gene cluster: involvement of a phenylalanine-2,3-aminomutase in biosynthesis.

    Science.gov (United States)

    Kudo, Fumitaka; Kawamura, Koichi; Uchino, Asuka; Miyanaga, Akimasa; Numakura, Mario; Takayanagi, Ryuichi; Eguchi, Tadashi

    2015-04-13

    Hitachimycin is a macrolactam antibiotic with (S)-β-phenylalanine (β-Phe) at the starter position of its polyketide skeleton. To understand the incorporation mechanism of β-Phe and the modification mechanism of the unique polyketide skeleton, the biosynthetic gene cluster for hitachimycin in Streptomyces scabrisporus was identified by genome mining. The identified gene cluster contains a putative phenylalanine-2,3-aminomutase (PAM), five polyketide synthases, four β-amino-acid-carrying enzymes, and a characteristic amidohydrolase. A hitA knockout mutant showed no hitachimycin production, but antibiotic production was restored by feeding with (S)-β-Phe. We also confirmed the enzymatic activity of the HitA PAM. The results suggest that the identified gene cluster is responsible for the biosynthesis of hitachimycin. A plausible biosynthetic pathway for hitachimycin, including a unique polyketide skeletal transformation mechanism, is proposed.

  15. The contribution of wine-derived monoterpene glycosides to retronasal odour during tasting.

    Science.gov (United States)

    Parker, Mango; Black, Cory A; Barker, Alice; Pearson, Wes; Hayasaka, Yoji; Francis, I Leigh

    2017-10-01

    This study investigated the sensory significance of monoterpene glycosides during tasting, by retronasal perception of odorant aglycones released in-mouth. Monoterpene glycosides were isolated from Gewürztraminer and Riesling juices and wines, chemically characterised and studied using sensory time-intensity methodology, together with a synthesised monoterpene glucoside. When assessed in model wine at five times wine-like concentration, Gewürztraminer glycosides and geranyl glucoside gave significant fruity flavour, although at wine-like concentrations, or in the presence of wine volatiles, the effect was not significant. Gewürztraminer glycosides, geranyl glucoside and guaiacyl glucoside were investigated using a sensory panel (n=39), revealing large inter-individual variability, with 77% of panellists responding to at least one glycoside. The study showed for the first time that grape-derived glycosides can contribute perceptible fruity flavour, providing a means of enhancing flavour in wines, and confirms the results of previous studies that the effect is highly variable across individuals. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. In situ measurements of isoprene and monoterpenes within a south-east Asian tropical rainforest

    Directory of Open Access Journals (Sweden)

    C. E. Jones

    2011-07-01

    Full Text Available Biogenic volatile organic compounds (BVOCs emitted from tropical rainforests comprise a substantial fraction of global atmospheric VOC emissions, however there are only relatively limited measurements of these species in tropical rainforest regions. We present observations of isoprene, α-pinene, camphene, Δ-3-carene, γ-terpinene and limonene, as well as oxygenated VOCs (OVOCs of biogenic origin such as methacrolein, in ambient air above a tropical rainforest in Malaysian Borneo during the Oxidant and Particle Photochemical Processes above a south-east Asian tropical rainforest (OP3 project in 2008. Daytime composition was dominated by isoprene, with an average mixing ratio of the order of ~1 ppb. γ-terpinene, limonene and camphene were the most abundant monoterpenes, with average daytime mixing ratios of 102, 71 and 66 ppt respectively, and with an average monoterpene toisoprene ratio of 0.3 during sunlit hours, compared to 2.0 at night. Limonene and camphene abundances were seen to be related to both temperature and light conditions. In contrast, γ-terpinene emission continued into the late afternoon/evening, under relatively low temperature and light conditions. The contributions of isoprene, monoterpenes and other classes of VOC to the volatile carbon budget and OH reactivity have been summarised for this rainforest location. We observe good agreement between surface and aircraft measurements of boundary layer isoprene and methacrolein above the natural rainforest, suggesting that the ground-level observations are broadly representative of isoprene emissions from this region.

  17. Biocontrol potential of essential oil monoterpenes against housefly, Musca domestica (Diptera: Muscidae).

    Science.gov (United States)

    Kumar, Peeyush; Mishra, Sapna; Malik, Anushree; Satya, Santosh

    2014-02-01

    Housefly (Musca domestica L.), one of the most common insects in human settlements, has been associated as vectors for various food-borne pathogens, causing food spoilage and disease transmission. The control of housefly was attempted using plant monoterpenes; menthone, menthol, menthyl acetate, limonene, citral and 1,8-cineole, against different life stages of housefly. Bioefficacy against housefly adults revealed highest repellent activity by menthol (95.6 percent) and menthone (83.3 percent). Against housefly larvae, menthol with an LC90 of 0.02 µl/cm(2) in contact toxicity assay and menthone with a LC90 value of 5.4 µl/L in fumigation assay were found to be most effective control agent. With respect to pupicidal activity, superior performance was shown by menthol, citral and 1,8-cineole in contact toxicity assay and citral and 1,8-cineole in fumigation assay. Limonene was found to be the poorest performer in all the assays. Overall, highest efficacy observed for menthol and menthone in various bioassays was in agreement with the results of essential oil activity obtained previously. Significant activity of monoterpenes against various life stages of housefly demonstrates their potential as excellent insecticides with prospects of monoterpenes being developed into eco-friendly and acceptable products for housefly control.

  18. Emissions of isoprene, monoterpene and short-chained carbonyl compounds from Eucalyptus spp. in southern Australia

    Science.gov (United States)

    Winters, Anthony J.; Adams, Mark A.; Bleby, Tim M.; Rennenberg, Heinz; Steigner, Dominik; Steinbrecher, Rainer; Kreuzwieser, Jürgen

    Eucalypts are among the highest emitters of biogenic volatile organic compounds, yet there is relatively little data available from field studies of this genus. Emissions of isoprene, monoterpenes and the short-chained carbonyls formaldehyde, acetaldehyde and acetone were determined from four species ( Eucalyptus camaldulensis, Eucalyptus globulus, Eucalyptus grandis, and Eucalytpus viminalis) in Australia. A smaller comparative study was conducted on E. camaldulensis in south-eastern Australia. Carbonyl emissions, reported here for the first time from eucalypts, were generally comparable with rates reported for other species, with diurnal emissions peaking at about 4, 75 and 34 nmol m -2 min -1 for acetone, formaldehyde and acetaldehyde respectively. There was wide variation in diurnal isoprene and monoterpene emissions between species, but under standard conditions, isoprene emissions were much lower than previous reports. Conversely, standard emission rates of monoterpenes were as much as six times greater than previous reports for some species. Emission of each carbonyl was correlated with its ambient concentration across different species, but more weakly related to temperature. Acetaldehyde emission in particular was significantly correlated with transpiration, but not with sap flow or with ethanol concentrations in xylem sap, suggesting fermentation within the leaf and stomatal conductance are primary controlling processes. Differences in acetaldehyde exchange velocities between sites, in addition to transpiration differences, suggest stomata may indeed exert long term emission regulation, in contrast to compounds for which no biological sink exists.

  19. Ochratoxin A Producing Fungi, Biosynthetic Pathway and Regulatory Mechanisms.

    Science.gov (United States)

    Wang, Yan; Wang, Liuqing; Liu, Fei; Wang, Qi; Selvaraj, Jonathan Nimal; Xing, Fuguo; Zhao, Yueju; Liu, Yang

    2016-03-21

    Ochratoxin A (OTA), mainly produced by Aspergillus and Penicillum species, is one of the most important mycotoxin contaminants in agricultural products. It is detrimental to human health because of its nephrotoxicity, hepatotoxicity, carcinogenicity, teratogenicity, and immunosuppression. OTA structurally consists of adihydrocoumarin moiety linked with l-phenylalanine via an amide bond. OTA biosynthesis has been putatively hypothesized, although several contradictions exist on some processes of the biosynthetic pathway. We discuss recent information on molecular studies of OTA biosynthesis despite insufficient genetic background in detail. Accordingly, genetic regulation has also been explored with regard to the interaction between the regulators and the environmental factors. In this review, we focus on three aspects of OTA: OTA-producing strains, OTA biosynthetic pathway and the regulation mechanisms of OTA production. This can pave the way to assist in protecting food and feed from OTA contamination by understanding OTA biosynthetic pathway and regulatory mechanisms.

  20. Natural Product Biosynthetic Diversity and Comparative Genomics of the Cyanobacteria.

    Science.gov (United States)

    Dittmann, Elke; Gugger, Muriel; Sivonen, Kaarina; Fewer, David P

    2015-10-01

    Cyanobacteria are an ancient lineage of slow-growing photosynthetic bacteria and a prolific source of natural products with intricate chemical structures and potent biological activities. The bulk of these natural products are known from just a handful of genera. Recent efforts have elucidated the mechanisms underpinning the biosynthesis of a diverse array of natural products from cyanobacteria. Many of the biosynthetic mechanisms are unique to cyanobacteria or rarely described from other organisms. Advances in genome sequence technology have precipitated a deluge of genome sequences for cyanobacteria. This makes it possible to link known natural products to biosynthetic gene clusters but also accelerates the discovery of new natural products through genome mining. These studies demonstrate that cyanobacteria encode a huge variety of cryptic gene clusters for the production of natural products, and the known chemical diversity is likely to be just a fraction of the true biosynthetic capabilities of this fascinating and ancient group of organisms.

  1. Ochratoxin A Producing Fungi, Biosynthetic Pathway and Regulatory Mechanisms

    Science.gov (United States)

    Wang, Yan; Wang, Liuqing; Liu, Fei; Wang, Qi; Selvaraj, Jonathan Nimal; Xing, Fuguo; Zhao, Yueju; Liu, Yang

    2016-01-01

    Ochratoxin A (OTA), mainly produced by Aspergillus and Penicillum species, is one of the most important mycotoxin contaminants in agricultural products. It is detrimental to human health because of its nephrotoxicity, hepatotoxicity, carcinogenicity, teratogenicity, and immunosuppression. OTA structurally consists of adihydrocoumarin moiety linked with l-phenylalanine via an amide bond. OTA biosynthesis has been putatively hypothesized, although several contradictions exist on some processes of the biosynthetic pathway. We discuss recent information on molecular studies of OTA biosynthesis despite insufficient genetic background in detail. Accordingly, genetic regulation has also been explored with regard to the interaction between the regulators and the environmental factors. In this review, we focus on three aspects of OTA: OTA-producing strains, OTA biosynthetic pathway and the regulation mechanisms of OTA production. This can pave the way to assist in protecting food and feed from OTA contamination by understanding OTA biosynthetic pathway and regulatory mechanisms. PMID:27007394

  2. CCN activity and droplet growth kinetics of fresh and aged monoterpene secondary organic aerosol

    Directory of Open Access Journals (Sweden)

    G. J. Engelhart

    2008-01-01

    Full Text Available The ability of secondary organic aerosol (SOA produced from the ozonolysis of α-pinene and monoterpene mixtures (α-pinene, β-pinene, limonene and 3-carene to become cloud droplets was investigated. Monoterpene SOA is quite active and would likely be a good source of cloud condensation nuclei (CCN in the atmosphere. A static CCN counter and a Scanning Mobility CCN Analyser (a Scanning Mobility Particle Sizer coupled with a Continuous Flow counter were used for the CCN measurements. A decrease in CCN activation diameter for α-pinene SOA of approximately 3 nm h−1 was observed as the aerosol continued to react with oxidants. Hydroxyl radicals further oxidize the SOA particles thereby enhancing the particle CCN activity with time. The initial concentrations of ozone and monoterpene precursor (for concentrations lower than 40 ppb do not appear to affect the activity of the resulting SOA. Köhler Theory Analysis (KTA is used to infer the molar mass of the SOA sampled online and offline from atomized filter samples. KTA suggests that the aged aerosol (both from α-pinene and the mixed monoterpene oxidation is primarily water-soluble (around 70–80%, with an estimated average molar mass of 180±55 g mol−1 (consistent with existing SOA speciation studies. CCN activity measurements of the SOA mixed with (NH42SO4 suggest that the organic can depress surface tension by as much as 10 nM m−1 (with respect to pure water. The droplet growth kinetics of SOA samples are similar to (NH42SO4, except at low supersaturation, where SOA tends to grow more slowly. The CCN activity of α-pinene and mixed monoterpene SOA can be modelled by a very simple implementation of Köhler theory, assuming complete dissolution of the particles, no dissociation into ions, molecular weight of 180 g mol−1, density of 1.5 g cm−3, and surface tension to

  3. CCN activity and droplet growth kinetics of fresh and aged monoterpene secondary organic aerosol

    Directory of Open Access Journals (Sweden)

    G. J. Engelhart

    2008-07-01

    Full Text Available The ability of secondary organic aerosol (SOA produced from the ozonolysis of α-pinene and monoterpene mixtures (α-pinene, β-pinene, limonene and 3-carene to become cloud droplets was investigated. A static CCN counter and a Scanning Mobility CCN Analyser (a Scanning Mobility Particle Sizer coupled with a Continuous Flow counter were used for the CCN measurements. Consistent with previous studies monoterpene SOA is quite active and would likely be a good source of cloud condensation nuclei (CCN in the atmosphere. A decrease in CCN activation diameter for α-pinene SOA of approximately 3 nm hr−1 was observed as the aerosol continued to react with oxidants. Hydroxyl radicals further oxidize the SOA particles thereby enhancing the particle CCN activity with time. The initial concentrations of ozone and monoterpene precursor (for concentrations lower than 40 ppb do not appear to affect the activity of the resulting SOA. Köhler Theory Analysis (KTA is used to infer the molar mass of the SOA sampled online and offline from atomized filter samples. The estimated average molar mass of online SOA was determined to be 180±55 g mol−1 (consistent with existing SOA speciation studies assuming complete solubility. KTA suggests that the aged aerosol (both from α-pinene and the mixed monoterpene oxidation is primarily water-soluble (around 65%. CCN activity measurements of the SOA mixed with (NH42SO4 suggest that the organic can depress surface tension by as much as 10 N m−1 (with respect to pure water. The droplet growth kinetics of SOA samples are similar to (NH42SO4, except at low supersaturation, where SOA tends to grow more slowly. The CCN activation diameter of α-pinene and mixed monoterpene SOA can be modelled to within 10–15% of experiments by a simple implementation of Köhler theory, assuming complete dissolution of the particles, no

  4. Quantifying environmental stress induced emissions of algal isoprene and monoterpenes using laboratory measurements

    Directory of Open Access Journals (Sweden)

    N. Meskhidze

    2014-09-01

    Full Text Available We report here production rates of isoprene and monoterpene compounds (α-pinene, β-pinene, camphene and d-limonene from six phytoplankton monocultures as a function of irradiance and temperature. Irradiance experiments were carried out for diatom strains – Thalassiosira weissflogii and Thalassiosira pseudonana; prymnesiophyte strains – Pleurochrysis carterae; dinoflagellate strains – Karenia brevis and Prorocentrum minimum; cryptophyte strains – Rhodomonas salina, while temperature experiments were carried out for diatom strains – Thalassiosira weissflogii and Thalassiosira pseudonana. Phytoplankton species, incubated in a climate-controlled room, were subject to variable light (90 to 900 μmol m−2s−1 and temperature (18 to 30 °C regimes. Compared to isoprene, monoterpene emissions were an order of magnitude lower at all light and temperature levels. Emission rates are normalized by cell count and Chlorophyll a (Chl a content. Diatom strains were the largest emitters, with ~2x1017g (cell−1h−1 (~35 μg (g Chl a−1h−1 for isoprene and ~5x10−19 g (cell−1h−1 (~1μg (g Chl a−1 h−1 for α-pinene. The contribution to the total monoterpene production was ~70% from α-pinene, ~20% for d-limonene, and −2s−1 and a gradual increase at high (>250 μmol m−2s−1 irradiance. Measurements revealed different patterns for time-averaged emissions rates over two successive days. On the first day most of the species showed distinct increase in production rates within the first four hours, while on the second day the emission rates were overall higher, but less variable. The data suggest that enhanced amounts of isoprene and monoterpenes are emitted from phytoplankton as a result of perturbations in environmental conditions that cause disbalance in chloroplasts and forces primary producers to acclimate physiologically. This relationship could be a valuable tool for development of dynamic ecosystem modeling approaches for global

  5. Secondary organic aerosol formation from hydroxyl radical oxidation and ozonolysis of monoterpenes

    Science.gov (United States)

    Zhao, D. F.; Kaminski, M.; Schlag, P.; Fuchs, H.; Acir, I.-H.; Bohn, B.; Häseler, R.; Kiendler-Scharr, A.; Rohrer, F.; Tillmann, R.; Wang, M. J.; Wegener, R.; Wildt, J.; Wahner, A.; Mentel, Th. F.

    2015-01-01

    Oxidation by hydroxyl radical (OH) and ozonolysis are the two major pathways of daytime biogenic volatile organic compound (BVOC) oxidation and secondary organic aerosol (SOA) formation. In this study, we investigated the particle formation of several common monoterpenes (α-pinene, β-pinene and limonene) by OH-dominated oxidation, which has seldom been investigated. OH oxidation experiments were carried out in the SAPHIR (Simulation of Atmospheric PHotochemistry In a large Reaction) chamber in Jülich, Germany, at low NOx (0.01 ~ 1 ppbV) and low ozone (O3) concentration (< 20 ppbV). OH concentration and total OH reactivity (kOH) were measured directly, and through this the overall reaction rate of total organics with OH in each reaction system was quantified. Multi-generation reaction process, particle growth, new particle formation (NPF), particle yield and chemical composition were analyzed and compared with that of monoterpene ozonolysis. Multi-generation products were found to be important in OH-dominated SOA formation. The relative role of functionalization and fragmentation in the reaction process of OH oxidation was analyzed by examining the particle mass and the particle size as a function of OH dose. We developed a novel method which quantitatively links particle growth to the reaction rate of OH with total organics in a reaction system. This method was also used to analyze the evolution of functionalization and fragmentation of organics in the particle formation by OH oxidation. It shows that functionalization of organics was dominant in the beginning of the reaction (within two lifetimes of the monoterpene) and fragmentation started to play an important role after that. We compared particle formation from OH oxidation with that from pure ozonolysis. In individual experiments, growth rates of the particle size did not necessarily correlate with the reaction rate of monoterpene with OH and O3. Comparing the size growth rates at the similar reaction rates

  6. Pectic enzymes

    NARCIS (Netherlands)

    Benen, J.A.E.; Voragen, A.G.J.; Visser, J.

    2003-01-01

    The pectic enzymes comprise a diverse group of enzymes. They consist of main-chain depolymerases and esterases active on methyl- and acetylesters of galacturonosyl uronic acid residues. The depolymerizing enzymes comprise hydrolases as wel as lyases

  7. Pectic enzymes

    NARCIS (Netherlands)

    Benen, J.A.E.; Voragen, A.G.J.; Visser, J.

    2003-01-01

    The pectic enzymes comprise a diverse group of enzymes. They consist of main-chain depolymerases and esterases active on methyl- and acetylesters of galacturonosyl uronic acid residues. The depolymerizing enzymes comprise hydrolases as wel as lyases

  8. Evolutionary systems biology of amino acid biosynthetic cost in yeast.

    Directory of Open Access Journals (Sweden)

    Michael D Barton

    Full Text Available Every protein has a biosynthetic cost to the cell based on the synthesis of its constituent amino acids. In order to optimise growth and reproduction, natural selection is expected, where possible, to favour the use of proteins whose constituents are cheaper to produce, as reduced biosynthetic cost may confer a fitness advantage to the organism. Quantifying the cost of amino acid biosynthesis presents challenges, since energetic requirements may change across different cellular and environmental conditions. We developed a systems biology approach to estimate the cost of amino acid synthesis based on genome-scale metabolic models and investigated the effects of the cost of amino acid synthesis on Saccharomyces cerevisiae gene expression and protein evolution. First, we used our two new and six previously reported measures of amino acid cost in conjunction with codon usage bias, tRNA gene number and atomic composition to identify which of these factors best predict transcript and protein levels. Second, we compared amino acid cost with rates of amino acid substitution across four species in the genus Saccharomyces. Regardless of which cost measure is used, amino acid biosynthetic cost is weakly associated with transcript and protein levels. In contrast, we find that biosynthetic cost and amino acid substitution rates show a negative correlation, but for only a subset of cost measures. In the economy of the yeast cell, we find that the cost of amino acid synthesis plays a limited role in shaping transcript and protein expression levels compared to that of translational optimisation. Biosynthetic cost does, however, appear to affect rates of amino acid evolution in Saccharomyces, suggesting that expensive amino acids may only be used when they have specific structural or functional roles in protein sequences. However, as there appears to be no single currency to compute the cost of amino acid synthesis across all cellular and environmental

  9. Isoprene and Monoterpene Emissions from Duke Forest: A Comparison of Ambient and Elevated CO2 Environments

    Science.gov (United States)

    Sive, B. C.; Varner, R. K.; Neilsen, C.; Russo, R. S.; Zhou, Y.; White, M. L.; Csakai, A.; Beckman, P.; Ambrose, J.; Wingenter, O. W.; Mao, H.; Talbot, R. W.

    2005-12-01

    elevated CO2 ring. The isoprene oxidation products methyl vinyl ketone (MVK) and methacrolein (MACR) followed the same trend as isoprene, with levels being enhanced at Ring 2 compared to Ring 1. Significant gradients were observed from the PTR-MS measurements for the total monoterpenes in addition to the speciated monoterpenes from the canister samples at both rings. However, in contrast to isoprene, monoterpene mixing ratios were lower in the elevated CO2 environment. Canopy fluxes calculated using the measured CO2 gradient and the eddy covariance data show that isoprene fluxes were higher in Ring 2 while monoterpene fluxes were higher in Ring 1. We returned to the FACTS-1 Research Facility in June 2005 to conduct direct branch flux measurements of isoprene and monoterpenes. Teflon bag enclosures were placed on branches of Pinus taeda (loblolly pine) and Liquidambar styraciflua (sweetgum) over two 48-hour sampling periods. Ambient and post-branch enclosure samples were collected in 2-L electropolished stainless steel canisters at both Rings 1 and 2 approximately every 2 hours for each tree species. The canister analysis revealed significantly enhanced mixing ratios of isoprene and monoterpenes from ambient to post-branch enclosure. Flux calculations based on the emission per leaf area of the branch confirm our 2004 results of enhanced isoprene and suppressed monoterpene mixing ratios in an elevated CO2 environment.

  10. Enzyme assays.

    Science.gov (United States)

    Reymond, Jean-Louis; Fluxà, Viviana S; Maillard, Noélie

    2009-01-07

    Enzyme assays are analytical tools to visualize enzyme activities. In recent years a large variety of enzyme assays have been developed to assist the discovery and optimization of industrial enzymes, in particular for "white biotechnology" where selective enzymes are used with great success for economically viable, mild and environmentally benign production processes. The present article highlights the aspects of fluorogenic and chromogenic substrates, sensors, and enzyme fingerprinting, which are our particular areas of interest.

  11. Differential hexosamine biosynthetic pathway gene expression with type 2 diabetes

    Directory of Open Access Journals (Sweden)

    Megan Coomer

    2014-01-01

    Full Text Available The hexosamine biosynthetic pathway (HBP culminates in the attachment of O-linked β-N-acetylglucosamine (O-GlcNAc onto serine/threonine residues of target proteins. The HBP is regulated by several modulators, i.e. O-linked β-N-acetylglucosaminyl transferase (OGT and β-N-acetylglucosaminidase (OGA catalyze the addition and removal of O-GlcNAc moieties, respectively; while flux is controlled by the rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase (GFPT, transcribed by two genes, GFPT1 and GFPT2. Since increased HBP flux is glucose-responsive and linked to insulin resistance/type 2 diabetes onset, we hypothesized that diabetic individuals exhibit differential expression of HBP regulatory genes. Volunteers (n = 60; n = 20 Mixed Ancestry, n = 40 Caucasian were recruited from Stellenbosch and Paarl (Western Cape, South Africa and classified as control, pre- or diabetic according to fasting plasma glucose and HbA1c levels, respectively. RNA was purified from leukocytes isolated from collected blood samples and OGT, OGA, GFPT1 and GFPT2 expressions determined by quantitative real-time PCR. The data reveal lower OGA expression in diabetic individuals (P < 0.01, while pre- and diabetic subjects displayed attenuated OGT expression vs. controls (P < 0.01 and P < 0.001, respectively. Moreover, GFPT2 expression decreased in pre- and diabetic Caucasians vs. controls (P < 0.05 and P < 0.01, respectively. We also found ethnic differences, i.e. Mixed Ancestry individuals exhibited a 2.4-fold increase in GFPT2 expression vs. Caucasians, despite diagnosis (P < 0.01. Gene expression of HBP regulators differs between diabetic and non-diabetic individuals, together with distinct ethnic-specific gene profiles. Thus differential HBP gene regulation may offer diagnostic utility and provide candidate susceptibility genes for different ethnic groupings.

  12. Expression of phenazine biosynthetic genes during the arbuscular mycorrhizal symbiosis of Glomus intraradices

    Directory of Open Access Journals (Sweden)

    Dionicia Gloria León-Martínez

    2012-06-01

    Full Text Available To explore the molecular mechanisms that prevail during the establishment of the arbuscular mycorrhiza symbiosis involving the genus Glomus, we transcriptionally analysed spores of Glomus intraradices BE3 during early hyphal growth. Among 458 transcripts initially identified as being expressed at presymbiotic stages, 20% of sequences had homology to previously characterized eukaryotic genes, 30% were homologous to fungal coding sequences, and 9% showed homology to previously characterized bacterial genes. Among them, GintPbr1a encodes a homolog to Phenazine Biosynthesis Regulator (Pbr of Burkholderia cenocepacia, an pleiotropic regulatory protein that activates phenazine production through transcriptional activation of the protein D isochorismatase biosynthetic enzyme phzD (Ramos et al., 2010. Whereas GintPbr1a is expressed during the presymbiotic phase, the G. intraradices BE3 homolog of phzD (BGintphzD is transcriptionally active at the time of the establishment of the arbuscular mycorrhizal symbiosis. DNA from isolated bacterial cultures found in spores of G. intraradices BE3 confirmed that both BGintPbr1a and BGintphzD are present in the genome of its potential endosymbionts. Taken together, our results indicate that spores of G. intraradices BE3 express bacterial phenazine biosynthetic genes at the onset of the fungal-plant symbiotic interaction.

  13. Bacterial Long-Chain Polyunsaturated Fatty Acids: Their Biosynthetic Genes, Functions, and Practical Use.

    Science.gov (United States)

    Yoshida, Kiyohito; Hashimoto, Mikako; Hori, Ryuji; Adachi, Takumi; Okuyama, Hidetoshi; Orikasa, Yoshitake; Nagamine, Tadashi; Shimizu, Satoru; Ueno, Akio; Morita, Naoki

    2016-05-12

    The nutritional and pharmaceutical values of long-chain polyunsaturated fatty acids (LC-PUFAs) such as arachidonic, eicosapentaenoic and docosahexaenoic acids have been well recognized. These LC-PUFAs are physiologically important compounds in bacteria and eukaryotes. Although little is known about the biosynthetic mechanisms and functions of LC-PUFAs in bacteria compared to those in higher organisms, a combination of genetic, bioinformatic, and molecular biological approaches to LC-PUFA-producing bacteria and some eukaryotes have revealed the notably diverse organization of the pfa genes encoding a polyunsaturated fatty acid synthase complex (PUFA synthase), the LC-PUFA biosynthetic processes, and tertiary structures of the domains of this enzyme. In bacteria, LC-PUFAs appear to take part in specific functions facilitating individual membrane proteins rather than in the adjustment of the physical fluidity of the whole cell membrane. Very long chain polyunsaturated hydrocarbons (LC-HCs) such as hentriacontanonaene are considered to be closely related to LC-PUFAs in their biosynthesis and function. The possible role of LC-HCs in strictly anaerobic bacteria under aerobic and anaerobic environments and the evolutionary relationships of anaerobic and aerobic bacteria carrying pfa-like genes are also discussed.

  14. Bacterial Long-Chain Polyunsaturated Fatty Acids: Their Biosynthetic Genes, Functions, and Practical Use

    Directory of Open Access Journals (Sweden)

    Kiyohito Yoshida

    2016-05-01

    Full Text Available The nutritional and pharmaceutical values of long-chain polyunsaturated fatty acids (LC-PUFAs such as arachidonic, eicosapentaenoic and docosahexaenoic acids have been well recognized. These LC-PUFAs are physiologically important compounds in bacteria and eukaryotes. Although little is known about the biosynthetic mechanisms and functions of LC-PUFAs in bacteria compared to those in higher organisms, a combination of genetic, bioinformatic, and molecular biological approaches to LC-PUFA-producing bacteria and some eukaryotes have revealed the notably diverse organization of the pfa genes encoding a polyunsaturated fatty acid synthase complex (PUFA synthase, the LC-PUFA biosynthetic processes, and tertiary structures of the domains of this enzyme. In bacteria, LC-PUFAs appear to take part in specific functions facilitating individual membrane proteins rather than in the adjustment of the physical fluidity of the whole cell membrane. Very long chain polyunsaturated hydrocarbons (LC-HCs such as hentriacontanonaene are considered to be closely related to LC-PUFAs in their biosynthesis and function. The possible role of LC-HCs in strictly anaerobic bacteria under aerobic and anaerobic environments and the evolutionary relationships of anaerobic and aerobic bacteria carrying pfa-like genes are also discussed.

  15. The pyrimidine nucleotide biosynthetic pathway modulates production of biofilm determinants in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Marco Garavaglia

    Full Text Available Bacteria are often found in multicellular communities known as biofilms, which constitute a resistance form against environmental stresses. Extracellular adhesion and cell aggregation factors, responsible for bacterial biofilm formation and maintenance, are tightly regulated in response to physiological and environmental cues. We show that, in Escherichia coli, inactivation of genes belonging to the de novo uridine monophosphate (UMP biosynthetic pathway impairs production of curli fibers and cellulose, important components of the bacterial biofilm matrix, by inhibiting transcription of the csgDEFG operon, thus preventing production of the biofilm master regulator CsgD protein. Supplementing growth media with exogenous uracil, which can be converted to UMP through the pyrimidine nucleotide salvage pathway, restores csgDEFG transcription and curli production. In addition, however, exogenous uracil triggers cellulose production, particularly in strains defective in either carB or pyrB genes, which encode enzymes catalyzing the first steps of de novo UMP biosynthesis. Our results indicate the existence of tight and complex links between pyrimidine metabolism and curli/cellulose production: transcription of the csgDEFG operon responds to pyrimidine nucleotide availability, while cellulose production is triggered by exogenous uracil in the absence of active de novo UMP biosynthesis. We speculate that perturbations in the UMP biosynthetic pathways allow the bacterial cell to sense signals such as starvation, nucleic acids degradation, and availability of exogenous pyrimidines, and to adapt the production of the extracellular matrix to the changing environmental conditions.

  16. Deciphering the sugar biosynthetic pathway and tailoring steps of nucleoside antibiotic A201A unveils a GDP-l-galactose mutase.

    Science.gov (United States)

    Zhu, Qinghua; Chen, Qi; Song, Yongxiang; Huang, Hongbo; Li, Jun; Ma, Junying; Li, Qinglian; Ju, Jianhua

    2017-05-09

    Galactose, a monosaccharide capable of assuming two possible configurational isomers (d-/l-), can exist as a six-membered ring, galactopyranose (Galp), or as a five-membered ring, galactofuranose (Galf). UDP-galactopyranose mutase (UGM) mediates the conversion of pyranose to furanose thereby providing a precursor for d-Galf Moreover, UGM is critical to the virulence of numerous eukaryotic and prokaryotic human pathogens and thus represents an excellent antimicrobial drug target. However, the biosynthetic mechanism and relevant enzymes that drive l-Galf production have not yet been characterized. Herein we report that efforts to decipher the sugar biosynthetic pathway and tailoring steps en route to nucleoside antibiotic A201A led to the discovery of a GDP-l-galactose mutase, MtdL. Systematic inactivation of 18 of the 33 biosynthetic genes in the A201A cluster and elucidation of 10 congeners, coupled with feeding and in vitro biochemical experiments, enabled us to: (i) decipher the unique enzyme, GDP-l-galactose mutase associated with production of two unique d-mannose-derived sugars, and (ii) assign two glycosyltransferases, four methyltransferases, and one desaturase that regiospecifically tailor the A201A scaffold and display relaxed substrate specificities. Taken together, these data provide important insight into the origin of l-Galf-containing natural product biosynthetic pathways with likely ramifications in other organisms and possible antimicrobial drug targeting strategies.

  17. Biosynthetic arginine decarboxylase in phytopathogenic fungi.

    Science.gov (United States)

    Khan, A J; Minocha, S C

    1989-01-01

    It has been reported that while bacteria and higher plants possess two different pathways for the biosynthesis of putrescine, via ornithine decarboxylase (ODC) and arginine decarboxylase (ADC); the fungi, like animals, only use the former pathway. We found that contrary to the earlier reports, two of the phytopathogenic fungi (Ceratocystis minor and Verticillium dahliae) contain significant levels of ADC activity with very little ODC. The ADC in these fungi has high pH optimum (8.4) and low Km (0.237 mM for C. minor, 0.103 mM for V. dahliae), and is strongly inhibited by alpha-difluoromethylarginine (DFMA), putrescine and spermidine, further showing that this enzyme is probably involved in the biosynthesis of polyamines and not in the catabolism of arginine as in Escherichia coli. The growth of these fungi is strongly inhibited by DFMA while alpha-difluoromethylornithine (DFMO) has little effect.

  18. Mycotoxin fumonisins: Health impacts and biosynthetic mechanism

    Institute of Scientific and Technical Information of China (English)

    WANG Qiaomei; WANG Jiansheng; YU Fengan; ZHU Xiangcheng; Zaleta-Rivera Kathia; DU Liangcheng

    2006-01-01

    Fumonisins are one of the most important groups of mycotoxins in agriculture and the food industry. They are produced by several widespread fungal pathogens of corn. Fumonisin contamination in maize-derived food and feeds causes several fatal diseases in livestock and poses a significant cancer risk to humans (Group 2B carcinogen). In the recent years, fumonisins have become a hot area in mycotoxin research. This review attempts to highlight the progress in the studies of molecular mechanisms for fumonisin biosynthesis and mode of action. The current understandings in the molecular basis for the cellular effects induced by fumonisins are discussed. Special attentions are paid to introduce the progress in the characterization of individual genes and enzymes involved in the biosynthesis of fumonisins.

  19. Transformation with Oncogenic Ras and the Simian Virus 40 T Antigens Induces Caspase-Dependent Sensitivity to Fatty Acid Biosynthetic Inhibition

    Science.gov (United States)

    Xu, Shihao; Spencer, Cody M.

    2015-01-01

    ABSTRACT Oncogenesis is frequently accompanied by the activation of specific metabolic pathways. One such pathway is fatty acid biosynthesis, whose induction is observed upon transformation of a wide variety of cell types. Here, we explored how defined oncogenic alleles, specifically the simian virus 40 (SV40) T antigens and oncogenic Ras12V, affect fatty acid metabolism. Our results indicate that SV40/Ras12V-mediated transformation of fibroblasts induces fatty acid biosynthesis in the absence of significant changes in the concentration of fatty acid biosynthetic enzymes. This oncogene-induced activation of fatty acid biosynthesis was found to be mammalian target of rapamycin (mTOR) dependent, as it was attenuated by rapamycin treatment. Furthermore, SV40/Ras12V-mediated transformation induced sensitivity to treatment with fatty acid biosynthetic inhibitors. Pharmaceutical inhibition of acetyl-coenzyme A (CoA) carboxylase (ACC), a key fatty acid biosynthetic enzyme, induced caspase-dependent cell death in oncogene-transduced cells. In contrast, isogenic nontransformed cells were resistant to fatty acid biosynthetic inhibition. This oncogene-induced sensitivity to fatty acid biosynthetic inhibition was independent of the cells' growth rates and could be attenuated by supplementing the medium with unsaturated fatty acids. Both the activation of fatty acid biosynthesis and the sensitivity to fatty acid biosynthetic inhibition could be conveyed to nontransformed breast epithelial cells through transduction with oncogenic Ras12V. Similar to what was observed in the transformed fibroblasts, the Ras12V-induced sensitivity to fatty acid biosynthetic inhibition was independent of the proliferative status and could be attenuated by supplementing the medium with unsaturated fatty acids. Combined, our results indicate that specific oncogenic alleles can directly confer sensitivity to inhibitors of fatty acid biosynthesis. IMPORTANCE Viral oncoproteins and cellular mutations

  20. Elucidation and in planta reconstitution of the parthenolide biosynthetic pathway

    NARCIS (Netherlands)

    Liu, Q.; Manzano, D.; Tanic, N.; Pesic, M.; Bankovic, J.; Pateraki, I.; Ricard, L.; Ferrer, A.; Vos, de R.C.H.; Krol, van der A.R.; Bouwmeester, H.J.

    2014-01-01

    Parthenolide, the main bioactive compound of the medicinal plant feverfew (Tanacetum parthenium), is a promising anti-cancer drug. However, the biosynthetic pathway of parthenolide has not been elucidated yet. Here we report on the isolation and characterization of all the genes from feverfew that a

  1. The preliminary research for biosynthetic engineering by radiation fusion technology

    Energy Technology Data Exchange (ETDEWEB)

    Roh, Chang Hyun; Jung, U Hee; Park, Hae Ran [KAERI, Daejeon (Korea, Republic of)

    2012-01-15

    The purpose of this project is to elucidate the solution to the production of bioactive substance using biotransformation process from core technology of biosynthetic engineering by radiation fusion technology. And, this strategy will provide core technology for development of drugs as new concept and category. Research scopes and contents of project include 1) The development of mutant for biosynthetic engineering by radiation fusion technology 2) The development of host for biosynthetic engineering by radiation fusion technology 3) The preliminary study for biosynthetic engineering of isoflavone by radiation fusion technology. The results are as follows. Isoflavone compounds(daidzein, hydroxylated isoflavone) were analyzed by GC-MS. The study of radiation doses and p-NCA high-throughput screening for mutant development were elucidated. And, it was carried out the study of radiation doses for host development. Furthermore, the study of redox partner and construction of recombinant strain for region-specific hydroxylation(P450, redox partner). In addition, the biological effect of 6,7,4'-trihydroxyisoflavone as an anti-obesity agent was elucidated in this study.

  2. Minimum Information about a Biosynthetic Gene cluster : commentary

    NARCIS (Netherlands)

    Medema, Marnix H; Kottmann, Renzo; Yilmaz, Pelin; Cummings, Matthew; Biggins, John B; Blin, Kai; de Bruijn, Irene; Chooi, Yit Heng; Claesen, Jan; Coates, R Cameron; Cruz-Morales, Pablo; Duddela, Srikanth; Dusterhus, Stephanie; Edwards, Daniel J; Fewer, David P; Garg, Neha; Geiger, Christoph; Gomez-Escribano, Juan Pablo; Greule, Anja; Hadjithomas, Michalis; Haines, Anthony S; Helfrich, Eric J N; Hillwig, Matthew L; Ishida, Keishi; Jones, Adam C; Jones, Carla S; Jungmann, Katrin; Kegler, Carsten; Kim, Hyun Uk; Kotter, Peter; Krug, Daniel; Masschelein, Joleen; Melnik, Alexey V; Mantovani, Simone M; Monroe, Emily A; Moore, Marcus; Moss, Nathan; Nutzmann, Hans-Wilhelm; Pan, Guohui; Pati, Amrita; Petras, Daniel; Reen, F Jerry; Rosconi, Federico; Rui, Zhe; Tian, Zhenhua; Tobias, Nicholas J; Tsunematsu, Yuta; Wiemann, Philipp; Wyckoff, Elizabeth; Yan, Xiaohui; Yim, Grace; Yu, Fengan; Xie, Yunchang; Aigle, Bertrand; Apel, Alexander K; Balibar, Carl J; Balskus, Emily P; Barona-Gomez, Francisco; Bechthold, Andreas; Bode, Helge B; Borriss, Rainer; Brady, Sean F; Brakhage, Axel A; Caffrey, Patrick; Cheng, Yi-Qiang; Clardy, Jon; Cox, Russell J; De Mot, Rene; Donadio, Stefano; Donia, Mohamed S; van der Donk, Wilfred A; Dorrestein, Pieter C; Doyle, Sean; Driessen, Arnold J M; Ehling-Schulz, Monika; Entian, Karl-Dieter; Fischbach, Michael A; Gerwick, Lena; Gerwick, William H; Gross, Harald; Gust, Bertolt; Hertweck, Christian; Hofte, Monica; Jensen, Susan E; Ju, Jianhua; Katz, Leonard; Kaysser, Leonard; Klassen, Jonathan L; Keller, Nancy P; Kormanec, Jan; Kuipers, Oscar P; Kuzuyama, Tomohisa; Kyrpides, Nikos C; Kwon, Hyung-Jin; Lautru, Sylvie; Lavigne, Rob; Lee, Chia Y; Linquan, Bai; Liu, Xinyu; Liu, Wen; Luzhetskyy, Andriy; Mahmud, Taifo; Mast, Yvonne; Mendez, Carmen; Metsa-Ketela, Mikko; Micklefield, Jason; Mitchell, Douglas A; Moore, Bradley S; Moreira, Leonilde M; Muller, Rolf; Neilan, Brett A; Nett, Markus; Nielsen, Jens; O'Gara, Fergal; Oikawa, Hideaki; Osbourn, Anne; Osburne, Marcia S; Ostash, Bohdan; Payne, Shelley M; Pernodet, Jean-Luc; Petricek, Miroslav; Piel, Jorn; Ploux, Olivier; Raaijmakers, Jos M; Salas, Jose A; Schmitt, Esther K; Scott, Barry; Seipke, Ryan F; Shen, Ben; Sherman, David H; Sivonen, Kaarina; Smanski, Michael J; Sosio, Margherita; Stegmann, Evi; Sussmuth, Roderich D; Tahlan, Kapil; Thomas, Christopher M; Tang, Yi; Truman, Andrew W; Viaud, Muriel; Walton, Jonathan D; Walsh, Christopher T; Weber, Tilmann; van Wezel, Gilles P; Wilkinson, Barrie; Willey, Joanne M; Wohlleben, Wolfgang; Wright, Gerard D; Ziemert, Nadine; Zhang, Changsheng; Zotchev, Sergey B; Breitling, Rainer; Takano, Eriko; Glockner, Frank Oliver

    2015-01-01

    A wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants are known to be encoded in biosynthetic gene clusters. Information about these clusters, pathways and metabolites is currently dispersed throughout the literature, making it difficult to exploit.

  3. Biosynthetic pathway of the phytohormone auxin in insects and screening of its inhibitors.

    Science.gov (United States)

    Suzuki, Hiroyoshi; Yokokura, Junpei; Ito, Tsukasa; Arai, Ryoma; Yokoyama, Chiaki; Toshima, Hiroaki; Nagata, Shinji; Asami, Tadao; Suzuki, Yoshihito

    2014-10-01

    Insect galls are abnormal plant tissues induced by galling insects. The galls are used for food and habitation, and the phytohormone auxin, produced by the insects, may be involved in their formation. We found that the silkworm, a non-galling insect, also produces an active form of auxin, indole-3-acetic acid (IAA), by de novo synthesis from tryptophan (Trp). A detailed metabolic analysis of IAA using IAA synthetic enzymes from silkworms indicated an IAA biosynthetic pathway composed of a three-step conversion: Trp → indole-3-acetaldoxime → indole-3-acetaldehyde (IAAld) → IAA, of which the first step is limiting IAA production. This pathway was shown to also operate in gall-inducing sawfly. Screening of a chemical library identified two compounds that showed strong inhibitory activities on the conversion step IAAld → IAA. The inhibitors can be efficiently used to demonstrate the importance of insect-synthesized auxin in gall formation in the future.

  4. Transcription factor TnrA inhibits the biosynthetic activity of glutamine synthetase in Bacillus subtilis.

    Science.gov (United States)

    Fedorova, Ksenia; Kayumov, Airat; Woyda, Kathrin; Ilinskaja, Olga; Forchhammer, Karl

    2013-05-02

    The Bacillus subtilis glutamine synthetase (GS) plays a dual role in cell metabolism by functioning as catalyst and regulator. GS catalyses the ATP-dependent synthesis of glutamine from glutamate and ammonium. Under nitrogen-rich conditions, GS becomes feedback-inhibited by high intracellular glutamine levels and then binds transcription factors GlnR and TnrA, which control the genes of nitrogen assimilation. While GS-bound TnrA is no longer able to interact with DNA, GlnR-DNA binding is shown to be stimulated by GS complex formation. In this paper we show a new physiological feature of the interaction between glutamine synthetase and TnrA. The transcription factor TnrA inhibits the biosynthetic activity of glutamine synthetase in vivo and in vitro, while the GlnR protein does not affect the activity of the enzyme.

  5. Identification of a new diterpene biosynthetic gene cluster that produces O-methylkolavelool in Herpetosiphon aurantiacus.

    Science.gov (United States)

    Nakano, Chiaki; Oshima, Misaki; Kurashima, Nodoka; Hoshino, Tsutomu

    2015-03-23

    Diterpenoids are usually found in plants and fungi, but are rare in bacteria. We have previously reported new diterpenes, named tuberculosinol and isotuberculosinol, which are generated from the Mycobacterium tuberculosis gene products Rv3377c and Rv3378c. No homologous gene was found at that time, but we recently found highly homologous proteins in the Herpetosiphon aurantiacus ATCC 23779 genome. Haur_2145 was a class II diterpene cyclase responsible for the conversion of geranylgeranyl diphosphate into kolavenyl diphosphate. Haur_2146, homologous to Rv3378c, synthesized (+)-kolavelool through the nucleophilic addition of a water molecule to the incipient cation formed after the diphosphate moiety was released. Haur_2147 afforded (+)-O-methylkolavelool from (+)-kolavelool, so this enzyme was an O-methyltransferase. This new diterpene was indeed detected in H. aurantiacus cells. This is the first report of the identification of a (+)-O-methylkolavelool biosynthetic gene cluster.

  6. Nutrient shortage triggers the hexosamine biosynthetic pathway via the GCN2-ATF4 signalling pathway.

    Science.gov (United States)

    Chaveroux, Cédric; Sarcinelli, Carmen; Barbet, Virginie; Belfeki, Sofiane; Barthelaix, Audrey; Ferraro-Peyret, Carole; Lebecque, Serge; Renno, Toufic; Bruhat, Alain; Fafournoux, Pierre; Manié, Serge N

    2016-06-03

    The hexosamine biosynthetic pathway (HBP) is a nutrient-sensing metabolic pathway that produces the activated amino sugar UDP-N-acetylglucosamine, a critical substrate for protein glycosylation. Despite its biological significance, little is known about the regulation of HBP flux during nutrient limitation. Here, we report that amino acid or glucose shortage increase GFAT1 production, the first and rate-limiting enzyme of the HBP. GFAT1 is a transcriptional target of the activating transcription factor 4 (ATF4) induced by the GCN2-eIF2α signalling pathway. The increased production of GFAT1 stimulates HBP flux and results in an increase in O-linked β-N-acetylglucosamine protein modifications. Taken together, these findings demonstrate that ATF4 provides a link between nutritional stress and the HBP for the regulation of the O-GlcNAcylation-dependent cellular signalling.

  7. Diversification of the monoterpene synthase gene family (TPSb) in Protium, a highly diverse genus of tropical trees.

    Science.gov (United States)

    Zapata, Felipe; Fine, Paul V A

    2013-09-01

    Plant monoterpenes are a diverse class of secondary metabolites mediating biotic and abiotic interactions with direct effects on plant fitness. To evaluate the hypothesis that monoterpene diversity is related to functional diversification after gene duplication, we reconstructed the evolutionary history of monoterpene synthases (TPSb)--the genes underlying monoterpene synthesis--in Protium, a taxonomically and chemically diverse genus of tropical trees. We isolated multiple copies of TPSb genes from chemically divergent Protium species, reconstructed the phylogeny of this gene family, used maximum-likelihood estimation of selection coefficients, and inferred residues evolving under positive selection. We found evidence for one ancient and multiple more recent duplication events giving rise to three, and potentially five, copies of TPSb genes currently present in Protium. There was evidence for adaptive evolution in one copy with a positively selected residue likely involved in protein folding and product specificity. All other copies were inferred to be evolving under a combination of stabilizing and/or relaxed selection. Although gene copy number is consistent with the extensive phenotypic diversity in monoterpenes shown in Protium, selection analyses suggest that not all copies are undergoing divergent selection consistent with a coevolutionary arms race with enemies, but instead may be under stabilizing and relaxed selection consistent with signaling or physiological stress functionality.

  8. Polymorphisms in monolignol biosynthetic genes are associated with biomass yield and agronomic traits in European maize (Zea mays L.).

    Science.gov (United States)

    Chen, Yongsheng; Zein, Imad; Brenner, Everton Alen; Andersen, Jeppe Reitan; Landbeck, Mathias; Ouzunova, Milena; Lübberstedt, Thomas

    2010-01-15

    Reduced lignin content leads to higher cell wall digestibility and, therefore, better forage quality and increased conversion of lignocellulosic biomass into ethanol. However, reduced lignin content might lead to weaker stalks, lodging, and reduced biomass yield. Genes encoding enzymes involved in cell wall lignification have been shown to influence both cell wall digestibility and yield traits. In this study, associations between monolignol biosynthetic genes and plant height (PHT), days to silking (DTS), dry matter content (DMC), and dry matter yield (DMY) were identified by using a panel of 39 European elite maize lines. In total, 10 associations were detected between polymorphisms or tight linkage disequilibrium (LD) groups within the COMT, CCoAOMT2, 4CL1, 4CL2, F5H, and PAL genomic fragments, respectively, and the above mentioned traits. The phenotypic variation explained by these polymorphisms or tight LD groups ranged from 6% to 25.8% in our line collection. Only 4CL1 and F5H were found to have polymorphisms associated with both yield and forage quality related characters. However, no pleiotropic polymorphisms affecting both digestibility of neutral detergent fiber (DNDF), and PHT or DMY were discovered, even under less stringent statistical conditions. Due to absence of pleiotropic polymorphisms affecting both forage yield and quality traits, identification of optimal monolignol biosynthetic gene haplotype(s) combining beneficial quantitative trait polymorphism (QTP) alleles for both quality and yield traits appears possible within monolignol biosynthetic genes. This is beneficial to maximize forage and bioethanol yield per unit land area.

  9. Druggability of the enzymes of the non-mevalonate-pathway

    NARCIS (Netherlands)

    Masini, Tiziana; Kroezen, Blijke S.; Hirsch, Anna K.H.

    2013-01-01

    The non-mevalonate pathway constitutes a source of novel drug targets. This biosynthetic route is essential for pathogens but is absent in humans. Our systematic evaluation of the druggability of all enzymes provides a convenient way of selecting targets that should be most easily inhibited by small

  10. The biosynthetic pathway for myxol-2' fucoside (myxoxanthophyll) in the cyanobacterium Synechococcus sp. strain PCC 7002.

    Science.gov (United States)

    Graham, Joel E; Bryant, Donald A

    2009-05-01

    Synechococcus sp. strain PCC 7002 produces a variety of carotenoids, which comprise predominantly dicylic beta-carotene and two dicyclic xanthophylls, zeaxanthin and synechoxanthin. However, this cyanobacterium also produces a monocyclic myxoxanthophyll, which was identified as myxol-2' fucoside. Compared to the carotenoid glycosides produced by diverse microorganisms, cyanobacterial myxoxanthophyll and closely related compounds are unusual because they are glycosylated on the 2'-OH rather than on the 1'-OH position of the psi end of the molecule. In this study, the genes encoding two enzymes that modify the psi end of myxoxanthophyll in Synechococcus sp. strain PCC 7002 were identified. Mutational and biochemical studies showed that open reading frame SynPCC7002_A2032, renamed cruF, encodes a 1',2'-hydroxylase [corrected] and that open reading frame SynPCC7002_A2031, renamed cruG, encodes a 2'-O-glycosyltransferase. The enzymatic activity of CruF was verified by chemical characterization of the carotenoid products synthesized when cruF was expressed in a lycopene-producing strain of Escherichia coli. Database searches showed that homologs of cruF and cruG occur in the genomes of all sequenced cyanobacterial strains that are known to produce myxol or the acylic xanthophyll oscillaxanthin. The genomes of many other bacteria that produce hydroxylated carotenoids but do not contain crtC homologs also contain cruF orthologs. Based upon observable intermediates, a complete biosynthetic pathway for myxoxanthophyll is proposed. This study expands the suite of enzymes available for metabolic engineering of carotenoid biosynthetic pathways for biotechnological applications.

  11. Process-based simulation of seasonality and drought stress in monoterpene emission models

    Directory of Open Access Journals (Sweden)

    R. Grote

    2009-09-01

    Full Text Available Canopy emissions of volatile hydrocarbons such as isoprene and monoterpenes play an important role in air chemistry. They depend on various environmental conditions, are highly species-specific and are expected to be affected by global change. In order to estimate future emissions of these isoprenoids, differently complex models are available. However, seasonal dynamics driven by phenology, enzymatic activity, or drought stress strongly modify annual ecosystem emissions. Although these impacts depend themselves on environmental conditions, they have yet received little attention in mechanistic modelling.

    In this paper we propose the application of a mechanistic method for considering the seasonal dynamics of emission potential using the ''Seasonal Isoprenoid synthase Model'' (Lehning et al., 2001. We test this approach with three different models (GUENTHER, Guenther et al., 1993; NIINEMETS, Niinemets et al., 2002a; BIM2, Grote et al., 2006 that are developed for simulating light-dependent monoterpene emission. We also suggest specific drought stress representations for each model. Additionally, the proposed model developments are compared with the approach realized in the MEGAN (Guenther et al., 2006 emission model. Models are applied to a Mediterranean Holm oak (Quercus ilex site with measured weather data.

    The simulation results demonstrate that the consideration of a dynamic emission potential has a strong effect on annual monoterpene emission estimates. The investigated models, however, show different sensitivities to the procedure for determining this seasonality impact. Considering a drought impact reduced the differences between the applied models and decreased emissions at the investigation site by approximately 33% on average over a 10 year period. Although this overall reduction was similar in all models, the sensitivity to weather conditions in specific years was different. We conclude that the proposed

  12. Location, formation and biosynthetic regulation of cellulases in the gliding bacteria Cytophaga hutchinsonii

    Directory of Open Access Journals (Sweden)

    Elijah Johnson

    2006-01-01

    Full Text Available An analysis of the recently published genome sequence of Cytophagahutchinsonii revealed an unusual collection of genes for an organism that can attackcrystalline cellulose. Consequently, questions were being raised by cellulase scientists, as towhat mechanism this organism uses to degrade its insoluble substrates. Cellulose, being ahighly polymeric compound and insoluble in water, cannot enter the cell walls ofmicroorganisms. Cellulose-degrading enzymes have therefore to be located on the surface ofthe cell wall or released extracellularly. The location of most cellulase enzymes has beenstudied. However, basic information on C. hutchinsonii cellulases is almost non-existent. Inthe present study, the location, formation and biosynthetic regulation of cellulases in C.hutchinsonii were demonstrated on different substrates. Various fractions isolated from C.hutchinsonii after cell rupture were assayed for carboxymethyl-cellulase activity (CMC.The cellulases were found to be predominantly cell-free during active growth on solka-flok,although 30% of activity was recorded on cell-bound enzymes. Relatively little CM-cellulase was formed when cells were grown on glucose and cellobiose. Apparently glucoseor labile substrates such as cellobiose seem to repress the formation of CM-cellulase. Thesefindings should provide some insight into possible hydrolysis mechanisms by C.hutchinsonii.

  13. Hydroxycinnamic acid functional ingredients and their biosynthetic genes in tubers of Solanum tuberosum Group Phureja

    Directory of Open Access Journals (Sweden)

    Liyao Ji

    2016-12-01

    Full Text Available Potato is an ideal candidate for the delivery of functional ingredients due to its high worldwide consumption. The metabolites in cooked tubers of eight diploid potato genotypes from Colombia were explored. Potato tubers were harvested, cooked,lyophilized, and then stored at −80°C. Metabolites were extracted from flesh samples and analyzed using liquid chromatography and high-resolution mass spectrometry. A total of 294 metabolites were putatively identified, of which 87 metabolites were associated with health-benefiting roles for humans, such as anticancer and anti-inflammatory properties. Two metabolites, chlorogenic acid and N-Feruloyltyramine were detected in high abundance and were mapped on to the potato metabolic pathways to predict the related biosynthetic enzymes: hydroxycinnamoyl-CoA quinate transferase (HQT and tyramine hydroxycinnamoyl transferase (THT, respectively. The coding genes of these enzymes identified nonsynonymous single-nucleotide polymorphisms (nsSNPs in AC09, AC64, and Russet Burbank, with the highest enzyme stability found in AC09. This is consistent with the highest presence of hydroxycinnamic acids in the AC09 genotype. The metabolites detected at high fold change, their functional ingredient properties, and their enhancement through breeding to improve health of the indigenous communities’ of Colombia are discussed.

  14. Purine biosynthetic genes are required for cadmium tolerance in Schizosaccharomyces pombe

    Energy Technology Data Exchange (ETDEWEB)

    Speiser, D.M.; Ortiz, D.F.; Kreppel, L.; Scheel, G.; McDonald, G.; Ow, D.W. (Dept. of Agriculture, Albany, CA (United States) Univ. of California, Berkeley (United States))

    1992-12-01

    Phytochelatins (PCs) are metal-chelating peptides produced in plants and some fungi in response to heavy metal exposure. A Cd-sensitive mutant of the fission yeast Schizosaccharomyces pombe, defective in production of a PC-Cd-sulfide complex essential for metal tolerance, was found to harbor mutations in specific genes of the purine biosynthetic pathway. Genetic analysis of the link between metal complex accumulation and purine biosynthesis enzymes revealed that genetic lesions blocking two segments of the pathway, before and after the IMP branchpoint, are required to produce the Cd-sensitive phenotype. The biochemical functions of these two segments of the pathway are similar, and a model based on the alternate use of a sulfur analog substrate is presented. The novel participation of purine biosynthesis enzymes in the conversion of the PC-Cd complex to the PC-Cd-sulfide complex in the fission yeast raises an intriguing possibility that these same enzymes might have a role in sulfur metabolism in the fission yeast S. pombe, and perhaps in other biological systems. 41 refs., 8 figs., 2 tabs.

  15. Insights into the pyrimidine biosynthetic pathway of human malaria parasite Plasmodium falciparum as chemotherapeutic target.

    Science.gov (United States)

    Krungkrai, Sudaratana R; Krungkrai, Jerapan

    2016-06-01

    Malaria is a major cause of morbidity and mortality in humans. Artemisinins remain as the first-line treatment for Plasmodium falciparum (P. falciparum) malaria although drug resistance has already emerged and spread in Southeast Asia. Thus, to fight this disease, there is an urgent need to develop new antimalarial drugs for malaria chemotherapy. Unlike human host cells, P. falciparum cannot salvage preformed pyrimidine bases or nucleosides from the extracellular environment and relies solely on nucleotides synthesized through the de novo biosynthetic pathway. This review presents significant progress on understanding the de novo pyrimidine pathway and the functional enzymes in the human parasite P. falciparum. Current knowledge in genomics and metabolomics are described, particularly focusing on the parasite purine and pyrimidine nucleotide metabolism. These include gene annotation, characterization and molecular mechanism of the enzymes that are different from the human host pathway. Recent elucidation of the three-dimensional crystal structures and the catalytic reactions of three enzymes: dihydroorotate dehydrogenase, orotate phosphoribosyltransferase, and orotidine 5'-monophosphate decarboxylase, as well as their inhibitors are reviewed in the context of their therapeutic potential against malaria.

  16. CYP345E2, an antenna-specific cytochrome P450 from the mountain pine beetle, Dendroctonus ponderosae Hopkins, catalyses the oxidation of pine host monoterpene volatiles.

    Science.gov (United States)

    Keeling, Christopher I; Henderson, Hannah; Li, Maria; Dullat, Harpreet K; Ohnishi, Toshiyuki; Bohlmann, Jörg

    2013-12-01

    The mountain pine beetle (MPB, Dendroctonus ponderosae Hopkins) is a significant pest of western North American pine forests. This beetle responds to pheromones and host volatiles in order to mass attack and thus overcome the terpenoid chemical defences of its host. The ability of MPB antennae to rapidly process odorants is necessary to avoid odorant receptor saturation and thus the enzymes responsible for odorant clearance are an important aspect of host colonization. An antenna-specific cytochrome P450, DponCYP345E2, is the most highly expressed transcript in adult MPB antenna. In in vitro assays with recombinant enzyme, DponCYP345E2 used several pine host monoterpenes as substrates, including (+)-(3)-carene, (+)-β-pinene, (-)-β-pinene, (+)-limonene, (-)-limonene, (-)-camphene, (+)-α-pinene, (-)-α-pinene, and terpinolene. The substrates were epoxidized or hydroxylated, depending upon the substrate. To complement DponCYP345E2, we also functionally characterized the NADPH-dependent cytochrome P450 reductase and the cytochrome b5 from MPB. DponCYP345E2 is the first cytochrome P450 to be functionally characterized in insect olfaction and in MPB.

  17. Purification and characterization of a Baeyer-Villiger mono-oxygenase from Rhodococcus erythropolis DCL14 involved in three different monoterpene degradation pathways

    NARCIS (Netherlands)

    Werf, van der M.J.

    2000-01-01

    A Baeyer-Villiger mono-oxygenase (BVMO), catalysing the NADPH- and oxygen-dependent oxidation of the monocyclic monoterpene ketones 1-hydroxy-2-oxolimonene, dihydrocarvone and menthone, was purified to homogeneity from Rhodococcus erythropolis DCL14. Monocyclic monoterpene ketone mono-oxygenase (MMK

  18. Purification and characterization of a Baeyer-Villiger mono-oxygenase from Rhodococcus erythropolis DCL14 involved in three different monocyclic monoterpene degradation pathways

    NARCIS (Netherlands)

    Werf, M.J. van der

    2000-01-01

    A Baeyer-Villiger mono-oxygenase (BVMO), catalysing the NADPH- and oxygen-dependent oxidation of the monocyclic monoterpene ketones 1-hydroxy-2-oxolimonene, dihydrocarvone and menthone, was purified to homogeneity from Rhodococcus erythropolis DCL14. Monocyclic monoterpene ketone mono-oxygenase (MMK

  19. Pondering the monoterpene composition of Pinus serotina Michx.: can limonene be used as a chemotaxonomic marker for the identification of old turpentine stumps?

    Science.gov (United States)

    Thomas L. Eberhardt; Jolie M. Mahfouz; Philip M. Sheridan

    2010-01-01

    Wood samples from old turpentine stumps in Virginia were analyzed by GC-MS to determine if the monoterpene compositions could be used for species identification. Given that limonene is reported to be the predominant monoterpene for pond pine (Pinus serotina Michx.), low relative proportions of limonene in these samples appeared to suggest that these...

  20. Host-Tree Monoterpenes and Biosynthesis of Aggregation Pheromones in the Bark Beetle Ips paraconfusus

    Directory of Open Access Journals (Sweden)

    John A. Byers

    2012-01-01

    Full Text Available A paradigm developed in the 1970s that Ips bark beetles biosynthesize their aggregation pheromone components ipsenol and ipsdienol by hydroxylating myrcene, a host tree monoterpene. Similarly, host α-pinene was hydroxylated to a third pheromone component cis-verbenol. In 1990, however, we reported that amounts of ipsenol and ipsdienol produced by male Ips paraconfusus (Coleoptera: Scolytinae feeding in five host pine species were nearly the same, even though no detectable myrcene precursor was detected in one of these pines (Pinus sabiniana. Subsequent research showed ipsenol and ipsdienol are also biosynthesized from smaller precursors such as acetate and mevalonate, and this de novo pathway is the major one, while host tree myrcene conversion by the beetle is the minor one. We report concentrations of myrcene, α-pinene and other major monoterpenes in five pine hosts (Pinus ponderosa, P. lambertiana, P. jeffreyi, P. sabiniana, and P. contorta of I. paraconfusus. A scheme for biosynthesis of ipsdienol and ipsenol from myrcene and possible metabolites such as ipsenone is presented. Mass spectra and quantities of ipsenone are reported and its possible role in biosynthesis of aggregation pheromone. Coevolution of bark beetles and host trees is discussed in relation to pheromone biosynthesis, host plant selection/suitability, and plant resistance.

  1. Monoterpene variation mediated attack preference evolution of the bark beetle Dendroctonus valens.

    Science.gov (United States)

    Liu, Zhudong; Wang, Bo; Xu, Bingbing; Sun, Jianghua

    2011-01-01

    Several studies suggest that some bark beetle like to attack large trees. The invasive red turpentine beetle (RTB), Dendroctonus valens LeConte, one of the most destructive forest pests in China, is known to exhibit this behavior. Our previous study demonstrated that RTBs preferred to attack large-diameter trees (diameter at breast height, DBH ≥30 cm) over small-diameter trees (DBH ≤10 cm) in the field. In the current study, we studied the attacking behavior and the underlying mechanisms in the laboratory. Behavioral assays showed that RTBs preferred the bark of large-DBH trees and had a higher attack rate on the bolts of these trees. Y-tube assays showed that RTBs preferred the volatiles released by large-DBH trees to those released by small-DBH trees. Subsequent analysis revealed that both large- and small-DBH trees had the same composition of monoterpenes, but the concentration of each component differed; thus it appeared that the concentrations acted as cues for RTBs to locate the right-sized host which was confirmed by further behavioral assays. Moreover, large-DBH pine trees provided more spacious habitat and contained more nutrients, such as nitrogen, than did small-DBH pine trees, which benefited RTBs' fecundity and larval development. RTBs seem to have evolved mechanisms to locate those large hosts that will allow them to maximize their fitness. Monoterpene variation mediated attack preference implies the potential for the management of RTB.

  2. Induction of senescence and identification of differentially expressed genes in tomato in response to monoterpene.

    Directory of Open Access Journals (Sweden)

    Sumit Ghosh

    Full Text Available Monoterpenes, which are among the major components of plant essential oils, are known for their ecological roles as well for pharmaceutical properties. Geraniol, an acyclic monoterpene induces cell cycle arrest and apoptosis/senescence in various cancer cells and plants; however, the genes involved in the process and the underlying molecular mechanisms are not well understood. In this study, we demonstrate that treatment of tomato plants with geraniol results in induction of senescence due to a substantial alteration in transcriptome. We have identified several geraniol-responsive protein encoding genes in tomato using suppression subtractive hybridization (SSH approach. These genes comprise of various components of signal transduction, cellular metabolism, reactive oxygen species (ROS, ethylene signalling, apoptosis and DNA damage response. Upregulation of NADPH oxidase and antioxidant genes, and increase in ROS level after geraniol treatment point towards the involvement of ROS in geraniol-mediated senescence. The delayed onset of seedling death and induced expression of geraniol-responsive genes in geraniol-treated ethylene receptor mutant (Nr suggest that geraniol-mediated senescence involves both ethylene dependent and independent pathways. Moreover, expression analysis during tomato ripening revealed that geraniol-responsive genes are also associated with the natural organ senescence process.

  3. Development and Evaluation of Stability of a Gel Formulation Containing the Monoterpene Borneol

    Directory of Open Access Journals (Sweden)

    Milla Gabriela Belarmino Dantas

    2016-01-01

    Full Text Available Borneol is a bicyclic monoterpenoid alcohol commonly used in traditional Chinese and Indian medicine. It is extracted from the essential oil of various medicinal plants. It has antibacterial, analgesic, and anti-inflammatory action proven in studies that used oral and intraperitoneal applications of this monoterpene in mice. The current study was designed to develop a topical gel formulation containing the monoterpene borneol using carbopol as gel base and to evaluate its stability. The prepared formulation was subjected to physical characterization and physical-chemistry assessment. The gel was prepared from carbopol and 5% of borneol. The prepared gel was subjected to pharmacotechnical tests such as its pH, viscosity, conductivity, spreadability, centrifugation, and accelerated stability with freezing-thaw cycle. The borneol was successfully incorporated into the carbopol formulation. Borneol gel (BG5 showed good stability after eight months of its development and after 12 days in the freeze-thaw cycle, not showing statistical difference in pH value, conductivity, and viscosity before and after test. Furthermore, the formulation showed a good spreadability. Therefore, it was concluded that the formulation could be very promising alternative for the topical or transdermal treatment of skin diseases.

  4. Impact of diffuse light on isoprene and monoterpene emissions from a mixed temperate forest

    Science.gov (United States)

    Laffineur, Q.; Aubinet, M.; Schoon, N.; Amelynck, C.; Müller, J.-F.; Dewulf, J.; Steppe, K.; Heinesch, B.

    2013-08-01

    This study investigated the impact of diffuse light on canopy scale emission of isoprene and monoterpenes measured continuously above a mixed temperate forest, using the disjunct eddy-covariance by mass scanning technique with a proton transfer reaction-mass spectrometer (PTR-MS) instrument. To assess this impact, the relationship between emissions/radiation and emissions/gross primary production (GPP) under clear sky and cloudy conditions were analysed. Under cloudy conditions (high proportion of diffuse radiation), the isoprene and monoterpene fluxes were enhanced compared to clear sky conditions (low proportion of diffuse radiation) at equivalent temperature and above-canopy total radiation. The whole-canopy enzymatic activity of the metabolic isoprene production pathway, however, was suggested to be lower under cloudy conditions than under clear sky conditions at equivalent temperature. The mechanisms behind these observations are probably linked to the better penetration of diffuse radiation in the canopy. Shade leaves/needles receive more radiation in cloudy conditions than in clear sky conditions, thereby inducing the observed effects.

  5. In situ measurements of isoprene and monoterpenes within a South-East Asian tropical rainforest

    Directory of Open Access Journals (Sweden)

    C. E. Jones

    2011-01-01

    Full Text Available Biogenic volatile organic compounds (BVOCs emitted from tropical rainforests comprise a substantial fraction of global atmospheric VOC emissions, however there are only relatively limited measurements of these species in tropical rainforest regions. We present observations of isoprene, α-pinene, camphene, Δ-3-carene, γ-terpinene and limonene, and oxygenated VOCs (OVOCs of biogenic origin such as methacrolein, in ambient air above a~tropical rainforest in Malaysian Borneo. Daytime composition was dominated by isoprene, with an average mixing ratio of the order of ~1 ppb. γ-terpinene, limonene and camphene were the most abundant monoterpenes, with average daytime mixing ratios of 102, 71 and 66 ppt, respectively, and with an average monoterpene to isoprene ratio of 0.3 during sunlight hours, compared to 2.0 at night. Limonene and camphene abundances were seen to be related to both temperature and light conditions. In contrast, γ-terpinene emission occurred into the late afternoon/evening, under relatively low temperature and light conditions. We observe good agreement between surface and aircraft measurements of boundary layer isoprene and methacrolein above the natural rainforest, suggesting that the ground-level observations are broadly representative of isoprene emissions from this region.

  6. Monoterpene synthase from Dracocephalum kotschyi and SPME-GC-MS analysis of its aroma profile

    Directory of Open Access Journals (Sweden)

    S. Saeidnia

    2014-04-01

    Full Text Available Dracocephalum kotschyi (Lamiaceae, as one of the remarkable aromatic plants, widely grows and also is cultivated in various temperate regions of Iran. There are diverse reports about the composition of the oil of this plant representing limonene derivatives as its major compounds. There is no report on cloning of mono- or sesquiterpene synthases from this plant. In the present study, the aroma profile of D. kotschyi has been extracted and analyzed via Headspace Solid-Phase Microextraction technique coupled with Gas Chromatography- Mass Spectroscopy. In order to determine the sequence of the active terpene synthase in this plant, first mRNA was prepared and cloning was performed by 3’ and 5’-RACEs-PCR method, then cDNA was sequenced and finally aligned with other recognized terpene synthases. The results showed that the plant leaves mainly comprised geranial (37.2%, limonene-10-al (28.5%, limonene (20.1% and 1,1-dimethoxy decane (14.5%. Sequencing the cDNA cloned from this plant revealed the presence of a monoterpene synthase absolutely similar to limonene synthase, responsible in formation of limonene, terpinolene, camphene and some other cyclic monoterpenes in its young leaves.

  7. Monoterpene and sesquiterpene emissions of three Mediterranean species through calcareous and siliceous soils in natural conditions

    Science.gov (United States)

    Ormeño, E.; Fernandez, C.; Bousquet-Mélou, A.; Greff, S.; Morin, E.; Robles, C.; Vila, B.; Bonin, G.

    Little is known about terpene emissions released by plants in response to abiotic factors, except for climate-related factors. Standard emissions ( ES) of monoterpenes ( ESM) and sesquiterpenes ( ESS) of Rosmarinus officinalis, Pinus halepensis and Cistus albidus in siliceous and calcareous sites were examined. Their dependency on some nutrients in these soils was also analyzed. The study was carried out in the south of France at the end of March, when C. albidus exhibited a leaf growth state, while the other two species exhibited a pre-budbreak state. The results revealed that ES of all major monoterpenes released by R. officinalis and ES of α-pinene and α-humulene of P. halepensis were higher in plants growing in calcareous soils. In contrast, for C. albidus, ESM and ES of β-bourbonene and α-humulene were higher in siliceous soils. ESM of all species was mainly correlated with nitrogen ( N) and available phosphorous (P A), while dependency on Ca 2+ or K + was variable. None of these nutrients was significantly correlated with ESS, suggesting that sesquiterpene synthesis pathway requires different nutrient supplies. While higher soil nutrient content stimulated ESM of R. officinalis and P. halepensis, it had a negative effect on ESM of C. albidus, probably because C. albidus exhibited a different phenological state. Considering the soil nature, and particularly N and P A as inputs in plant terpene inventories could hence contribute to obtain more accurate terpene estimates.

  8. Anxiolytic-like effect of the monoterpene 1,4-cineole in mice.

    Science.gov (United States)

    Gomes, Patrícia Bezerra; Feitosa, Mariana Lima; Silva, Maria Izabel Gomes; Noronha, Emmanuelle Coelho; Moura, Brinell Arcanjo; Venâncio, Edith Teles; Rios, Emiliano Ricardo Vasconcelos; de Sousa, Damião Pergentino; de Vasconcelos, Silvânia Maria Mendes; Fonteles, Marta Maria de França; de Sousa, Francisca Cléa Florenço

    2010-09-01

    Recent studies have shown that some monoterpenes exert anxiolytic- and depressant-like actions, however, these effects from monoterpene 1,4-cineole are still unknown. This work aimed to study the effects of 1,4-cineole in classic animal models for depression- and anxiety-like behavior, specifically the elevated plus maze (EPM), hole board, open field, pentobarbital sleeping time, forced swimming, tail suspension and rota rod tests. 1,4-Cineole was administered orally to mice (100, 200 and 400 mg/kg), while diazepam (1 or 2 mg/kg) and imipramine (10 or 30 mg/kg) were used as standard drugs. 1,4-Cineole (400 mg/kg) modified all parameters observed in the EPM, while no significant variation was observed on general motor activity in the open-field test. In the hole-board assay, 1,4-cineole induced increase on the number of head dips. Forced swimming and tail suspension tests showed that cineole (200 and/or 400 mg/kg) was able to promote significant increase on the immobility time, while a decreased sleep latency was observed (200 and 400 mg/kg ) on the pentobarbital sleeping time. Cineole had no effect on the motor coordination of animals in the rota rod test. The results suggest that 1,4-cineole presents potential anxiolytic-like action consistent with possible general depression of the CNS.

  9. Suffruyabiosides A and B, Two New Monoterpene Diglycosides from Moutan Cortex

    Directory of Open Access Journals (Sweden)

    Hideyuki Shigemori

    2012-04-01

    Full Text Available Two new monoterpene diglycosides, suffruyabiosides A (1 and B (2, and seven known compounds 39 were isolated from Moutan Cortex (root cortex of Paeonia suffruticosa Andrews. The structures were elucidated on the basis of 2D NMR spectral data. Suffruyabiosides A (1 and B (2 are rare monoterpene diglycosides, including a cellobiose in the molecules. Salicylpaeoniflorin (4 had a antiproliferation effect similar to paeoniflorin (3 on human lung adenocarcinoma epitherial A549 cells. Galloylpaeoniflorin (8 and salicylpaeoniflorin (4 revealed a more pronounced radical scavenging effect than a-tocopherol (positive control. An increase in the number of phenolic hydroxyl groups produced a more effective radical scavenging effect [8 > mudanpioside E (6 > oxypaeoniflorin (5]. Comparison of the effects of 4 and 5 showed that o-substitution with a phenolic hydroxyl group was more effective than p-substitution. The results suggest that salicylpaeoniflorin (4 may be useful as a cytotoxic and a radical scavenging agent.

  10. Monoterpene variation mediated attack preference evolution of the bark beetle Dendroctonus valens.

    Directory of Open Access Journals (Sweden)

    Zhudong Liu

    Full Text Available Several studies suggest that some bark beetle like to attack large trees. The invasive red turpentine beetle (RTB, Dendroctonus valens LeConte, one of the most destructive forest pests in China, is known to exhibit this behavior. Our previous study demonstrated that RTBs preferred to attack large-diameter trees (diameter at breast height, DBH ≥30 cm over small-diameter trees (DBH ≤10 cm in the field. In the current study, we studied the attacking behavior and the underlying mechanisms in the laboratory. Behavioral assays showed that RTBs preferred the bark of large-DBH trees and had a higher attack rate on the bolts of these trees. Y-tube assays showed that RTBs preferred the volatiles released by large-DBH trees to those released by small-DBH trees. Subsequent analysis revealed that both large- and small-DBH trees had the same composition of monoterpenes, but the concentration of each component differed; thus it appeared that the concentrations acted as cues for RTBs to locate the right-sized host which was confirmed by further behavioral assays. Moreover, large-DBH pine trees provided more spacious habitat and contained more nutrients, such as nitrogen, than did small-DBH pine trees, which benefited RTBs' fecundity and larval development. RTBs seem to have evolved mechanisms to locate those large hosts that will allow them to maximize their fitness. Monoterpene variation mediated attack preference implies the potential for the management of RTB.

  11. Biosynthetic Modularity Rules in the Bisintercalator Family of Antitumor Compounds

    Directory of Open Access Journals (Sweden)

    Javier Fernández

    2014-05-01

    Full Text Available Diverse actinomycetes produce a family of structurally and biosynthetically related non-ribosomal peptide compounds which belong to the chromodepsipeptide family. These compounds act as bisintercalators into the DNA helix. They give rise to antitumor, antiparasitic, antibacterial and antiviral bioactivities. These compounds show a high degree of conserved modularity (chromophores, number and type of amino acids. This modularity and their high sequence similarities at the genetic level imply a common biosynthetic origin for these pathways. Here, we describe insights about rules governing this modular biosynthesis, taking advantage of the fact that nowadays five of these gene clusters have been made public (thiocoraline, triostin, SW-163 and echinomycin/quinomycin. This modularity has potential application for designing and producing novel genetic engineered derivatives, as well as for developing new chemical synthesis strategies. These would facilitate their clinical development.

  12. Elucidation and in planta reconstitution of the parthenolide biosynthetic pathway

    DEFF Research Database (Denmark)

    Liu, Qing; Manzano, David; Tanić, Nikola

    2014-01-01

    Parthenolide, the main bioactive compound of the medicinal plant feverfew (Tanacetum parthenium), is a promising anti-cancer drug. However, the biosynthetic pathway of parthenolide has not been elucidated yet. Here we report on the isolation and characterization of all the genes from feverfew...... that are required for the biosynthesis of parthenolide, using a combination of 454 sequencing of a feverfew glandular trichome cDNA library, co-expression analysis and metabolomics. When parthenolide biosynthesis was reconstituted by transient co-expression of all pathway genes in Nicotiana benthamiana, up to 1.......4μgg-1 parthenolide was produced, mostly present as cysteine and glutathione conjugates. These relatively polar conjugates were highly active against colon cancer cells, with only slightly lower activity than free parthenolide. In addition to these biosynthetic genes, another gene encoding...

  13. YUCCA auxin biosynthetic genes are required for Arabidopsis shade avoidance

    Directory of Open Access Journals (Sweden)

    Patricia Müller-Moulé

    2016-10-01

    Full Text Available Plants respond to neighbor shade by increasing stem and petiole elongation. Shade, sensed by phytochrome photoreceptors, causes stabilization of PHYTOCHROME INTERACTING FACTOR proteins and subsequent induction of YUCCA auxin biosynthetic genes. To investigate the role of YUCCA genes in phytochrome-mediated elongation, we examined auxin signaling kinetics after an end-of-day far-red (EOD-FR light treatment, and found that an auxin responsive reporter is rapidly induced within 2 hours of far-red exposure. YUCCA2, 5, 8, and 9 are all induced with similar kinetics suggesting that they could act redundantly to control shade-mediated elongation. To test this hypothesis we constructed a yucca2, 5, 8, 9 quadruple mutant and found that the hypocotyl and petiole EOD-FR and shade avoidance responses are completely disrupted. This work shows that YUCCA auxin biosynthetic genes are essential for detectable shade avoidance and that YUCCA genes are important for petiole shade avoidance.

  14. YUCCA auxin biosynthetic genes are required for Arabidopsis shade avoidance

    Science.gov (United States)

    Müller-Moulé, Patricia; Nozue, Kazunari; Pytlak, Melissa L.; Palmer, Christine M.; Covington, Michael F.; Wallace, Andreah D.; Harmer, Stacey L.

    2016-01-01

    Plants respond to neighbor shade by increasing stem and petiole elongation. Shade, sensed by phytochrome photoreceptors, causes stabilization of PHYTOCHROME INTERACTING FACTOR proteins and subsequent induction of YUCCA auxin biosynthetic genes. To investigate the role of YUCCA genes in phytochrome-mediated elongation, we examined auxin signaling kinetics after an end-of-day far-red (EOD-FR) light treatment, and found that an auxin responsive reporter is rapidly induced within 2 hours of far-red exposure. YUCCA2, 5, 8, and 9 are all induced with similar kinetics suggesting that they could act redundantly to control shade-mediated elongation. To test this hypothesis we constructed a yucca2, 5, 8, 9 quadruple mutant and found that the hypocotyl and petiole EOD-FR and shade avoidance responses are completely disrupted. This work shows that YUCCA auxin biosynthetic genes are essential for detectable shade avoidance and that YUCCA genes are important for petiole shade avoidance. PMID:27761349

  15. Identification of the Scopularide Biosynthetic Gene Cluster in Scopulariopsis brevicaulis

    Directory of Open Access Journals (Sweden)

    Mie Bech Lukassen

    2015-07-01

    Full Text Available Scopularide A is a promising potent anticancer lipopeptide isolated from a marine derived Scopulariopsis brevicaulis strain. The compound consists of a reduced carbon chain (3-hydroxy-methyldecanoyl attached to five amino acids (glycine, l-valine, d-leucine, l-alanine, and l-phenylalanine. Using the newly sequenced S. brevicaulis genome we were able to identify the putative biosynthetic gene cluster using genetic information from the structurally related emericellamide A from Aspergillus nidulans and W493-B from Fusarium pseudograminearum. The scopularide A gene cluster includes a nonribosomal peptide synthetase (NRPS1, a polyketide synthase (PKS2, a CoA ligase, an acyltransferase, and a transcription factor. Homologous recombination was low in S. brevicaulis so the local transcription factor was integrated randomly under a constitutive promoter, which led to a three to four-fold increase in scopularide A production. This indirectly verifies the identity of the proposed biosynthetic gene cluster.

  16. Enzyme inhibitor studies reveal complex control of methyl-D-erythritol 4-phosphate (MEP pathway enzyme expression in Catharanthus roseus.

    Directory of Open Access Journals (Sweden)

    Mei Han

    Full Text Available In Catharanthus roseus, the monoterpene moiety exerts a strong flux control for monoterpene indole alkaloid (MIA formation. Monoterpene synthesis depends on the methyl-D-erythritol 4-phosphate (MEP pathway. Here, we have explored the regulation of this pathway in response to developmental and environmental cues and in response to specific enzyme inhibitors. For the MEP pathway entry enzyme 1-deoxy-D-xylulose 5-phosphate synthase (DXS, a new (type I DXS isoform, CrDXS1, has been cloned, which, in contrast to previous reports on type II CrDXS, was not transcriptionally activated by the transcription factor ORCA3. Regulation of the MEP pathway in response to metabolic perturbations has been explored using the enzyme inhibitors clomazone (precursor of 5-ketochlomazone, inhibitor of DXS and fosmidomycin (inhibitor of deoxyxylulose 5-phosphate reductoisomerase (DXR, respectively. Young leaves of non-flowering plants were exposed to both inhibitors, adopting a non-invasive in vivo technique. Transcripts and proteins of DXS (3 isoforms, DXR, and hydroxymethylbutenyl diphosphate synthase (HDS were monitored, and protein stability was followed in isolated chloroplasts. Transcripts for DXS1 were repressed by both inhibitors, whereas transcripts for DXS2A&B, DXR and HDS increased after clomazone treatment but were barely affected by fosmidomycin treatment. DXS protein accumulated in response to both inhibitors, whereas DXR and HDS proteins were less affected. Fosmidomycin-induced accumulation of DXS protein indicated substantial posttranscriptional regulation. Furthermore, fosmidomycin effectively protected DXR against degradation in planta and in isolated chloroplasts. Thus our results suggest that DXR protein stability may be affected by substrate binding. In summary, the present results provide novel insight into the regulation of DXS expression in C. roseus in response to MEP-pathway perturbation.

  17. Enzyme inhibitor studies reveal complex control of methyl-D-erythritol 4-phosphate (MEP) pathway enzyme expression in Catharanthus roseus.

    Science.gov (United States)

    Han, Mei; Heppel, Simon C; Su, Tao; Bogs, Jochen; Zu, Yuangang; An, Zhigang; Rausch, Thomas

    2013-01-01

    In Catharanthus roseus, the monoterpene moiety exerts a strong flux control for monoterpene indole alkaloid (MIA) formation. Monoterpene synthesis depends on the methyl-D-erythritol 4-phosphate (MEP) pathway. Here, we have explored the regulation of this pathway in response to developmental and environmental cues and in response to specific enzyme inhibitors. For the MEP pathway entry enzyme 1-deoxy-D-xylulose 5-phosphate synthase (DXS), a new (type I) DXS isoform, CrDXS1, has been cloned, which, in contrast to previous reports on type II CrDXS, was not transcriptionally activated by the transcription factor ORCA3. Regulation of the MEP pathway in response to metabolic perturbations has been explored using the enzyme inhibitors clomazone (precursor of 5-ketochlomazone, inhibitor of DXS) and fosmidomycin (inhibitor of deoxyxylulose 5-phosphate reductoisomerase (DXR)), respectively. Young leaves of non-flowering plants were exposed to both inhibitors, adopting a non-invasive in vivo technique. Transcripts and proteins of DXS (3 isoforms), DXR, and hydroxymethylbutenyl diphosphate synthase (HDS) were monitored, and protein stability was followed in isolated chloroplasts. Transcripts for DXS1 were repressed by both inhibitors, whereas transcripts for DXS2A&B, DXR and HDS increased after clomazone treatment but were barely affected by fosmidomycin treatment. DXS protein accumulated in response to both inhibitors, whereas DXR and HDS proteins were less affected. Fosmidomycin-induced accumulation of DXS protein indicated substantial posttranscriptional regulation. Furthermore, fosmidomycin effectively protected DXR against degradation in planta and in isolated chloroplasts. Thus our results suggest that DXR protein stability may be affected by substrate binding. In summary, the present results provide novel insight into the regulation of DXS expression in C. roseus in response to MEP-pathway perturbation.

  18. Establishment of pomegranate (Punica granatum) hairy root cultures for genetic interrogation of the hydrolyzable tannin biosynthetic pathway.

    Science.gov (United States)

    Ono, Nadia N; Bandaranayake, Pradeepa C G; Tian, Li

    2012-09-01

    In contrast to the numerous reports on the human therapeutic applications of hydrolyzable tannins (HTs), genes involved in their biosynthesis have not been identified at the molecular level from any plant species. Although we have previously identified candidate HT biosynthetic genes in pomegranate using transcriptomic and bioinformatic analyses, characterization of in planta enzyme function remains a critical step in biochemical pathway elucidation. We here report the establishment of a pomegranate (Punica granatum) hairy root culture system that produces HTs. Agrobacterium rhizogenes strains transformed with a binary vector harboring a yellow fluorescent protein (YFP) gene were used for hairy root induction, allowing visual, non-destructive, detection of transgene incorporation. It also demonstrated that the pomegranate hairy root culture system is suitable for expressing heterologous genes (YFP in this case). Expression of 26 putative UDP-glycosyltransferase (UGT) genes, obtained from a pomegranate fruit peel (a tissue highly abundant in HTs) RNA-Seq library, were verified in wild type and hairy roots. In addition, two candidate UGTs for HT biosynthesis were identified based on HPLC and differential gene expression analyses of various pomegranate tissues. Together with in vitro enzyme activity assays, the hairy root culture system holds great promise for revealing the undivulged HT biosynthetic pathway using pomegranate as a model system.

  19. Targeting of the polyhydroxybutyrate biosynthetic pathway to the plastids of Arabidopsis thaliana results in high levels of polymer accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Nawrath, C.; Poirier, Y.; Somerville, C. (Carnegie Institution of Washington, Stanford, CA (United States))

    1994-12-20

    In the bacterium Alcaligenes eutrophus, three genes encode the enzymes necessary to catalyze the synthesis of poly[(R)-(-)-3-hydroxybutyrate] (PHB) from acetyl-CoA. In order to target these enzymes into the plastids of higher plants, the genes were modified by addition of DNA fragments encoding a pea chloroplast transit peptide, a constitutive plant promoter, and a poly(A) addition sequence. Each of the modified bacterial genes was introduced into Arabidopsis thaliana by Agrobacterium-mediated transformation, and plants containing all three genes were obtained by sexual crosses. These plans accumulated PHB up to 14% of the dry weight as 0.2- to 0.7-[mu]m granules within plastids. In contrast to earlier experiments in which expression of the PHB biosynthetic pathway in the cytoplasm led to a deleterious effect on growth, expression of the PHB biosynthetic pathway in plastids had no obvious effect on the growth or fertility of the transgenic plants and resulted in a 100-fold increase in the amount of PHB in higher plants. The high level of PHB accumulation also suggests that the synthesis of plastid acetyl-CoA is regulated by a mechanism which responds to metabolic demand. 20 refs., 6 figs.

  20. Structural genes for thiamine biosynthetic enzymes (thiCEFGH) in Escherichia coli K-12.

    OpenAIRE

    Vander Horn, P B; Backstrom, A D; Stewart, V; Begley, T. P.

    1993-01-01

    Escherichia coli K-12 synthesizes thiamine pyrophosphate (vitamin B1) de novo. Two precursors [4-methyl-5-(beta-hydroxyethyl)thiazole monophosphate and 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate] are coupled to form thiamine monophosphate, which is then phosphorylated to make thiamine pyrophosphate. Previous studies have identified two classes of thi mutations, clustered at 90 min on the genetic map, which result in requirements for the thiazole or the hydroxymethylpryimidine. W...

  1. Garlic γ-glutamyl transpeptidases that catalyze deglutamylation of biosynthetic intermediate of alliin

    Directory of Open Access Journals (Sweden)

    Naoko eYoshimoto

    2015-01-01

    Full Text Available S-Alk(enyl-L-cysteine sulfoxides are pharmaceutically important secondary metabolites produced by plants that belong to the genus Allium. Biosynthesis of S-alk(enyl-L-cysteine sulfoxides is initiated by S-alk(enylation of glutathione, which is followed by the removal of glycyl and γ-glutamyl groups and S-oxygenation. However, most of the enzymes involved in the biosynthesis of S-alk(enyl-L-cysteine sulfoxides in Allium plants have not been identified. In this study, we identified three genes, AsGGT1, AsGGT2, and AsGGT3, from garlic (Allium sativum that encode γ-glutamyl transpeptidases catalyzing the removal of the γ-glutamyl moiety from a putative biosynthetic intermediate of S-allyl-L-cysteine sulfoxide (alliin. The recombinant proteins of AsGGT1, AsGGT2, and AsGGT3 exhibited considerable deglutamylation activity toward a putative alliin biosynthetic intermediate, γ-glutamyl-S-allyl-L-cysteine, whereas these proteins showed very low deglutamylation activity toward another possible alliin biosynthetic intermediate, γ-glutamyl-S-allyl-L-cysteine sulfoxide. The deglutamylation activities of AsGGT1, AsGGT2, and AsGGT3 toward γ-glutamyl-S-allyl-L-cysteine were elevated in the presence of the dipeptide glycylglycine as a γ-glutamyl acceptor substrate, although these proteins can act as hydrolases in the absence of a proper acceptor substrate, except water. The apparent Km values of AsGGT1, AsGGT2, and AsGGT3 for γ-glutamyl-S-allyl-L-cysteine were 86 μM, 1.1 mM, and 9.4 mM, respectively. Subcellular distribution of GFP-fusion proteins transiently expressed in onion cells suggested that AsGGT2 localizes in the vacuole, whereas AsGGT1 and AsGGT3 possess no apparent transit peptide for localization to intracellular organelles. The different kinetic properties and subcellular localizations of AsGGT1, AsGGT2, and AsGGT3 suggest that these three GGTs may contribute differently to the biosynthesis of alliin in garlic.

  2. Isoprene and monoterpene emission from the coniferous species Abies Borisii-regis - implications for regional air chemistry in Greece

    Energy Technology Data Exchange (ETDEWEB)

    Harrison, D.; Hunter, M.C.; Lewis, A.C.; Seakins, P.W. [Leeds Univ., School of Chemistry, Leeds (United Kingdom); Nunes, T.V.; Pio, C.A. [Universidade de Aveiro, Dept. de Ambiente e Ordenamento, Aveiro (Portugal)

    2001-07-01

    The emission of isoprene has been studied from a forest of Abies Borisii-regis, a Mediterranean fir species previously thought to emit only monoterpenes. Emission studies from two independent enclosure experiments indicated a standardised isoprene emission rate of (18.4{+-}3.8){mu}gg{sub dry-weight} {sup -1}h{sup -1}, similar in magnitude to species such as eucalyptus and oak which are considered to be strong isoprene emitters. Isoprene emission depended strongly on both leaf temperature (2degC-34degC) and photosynthetically active radiation (PAR) below 250{mu}molm{sup -2}s{sup -1}, becoming saturated with respect to PAR above this value. The annual isoprene emission rate was estimated to be (132{+-}29) kTyr{sup -1} for those trees growing within Greece, comparable to current estimates of the total isoprene budget of Greece as a whole, and contributing significantly to regional ozone and carbon monoxide budgets. Monoterpene emission exhibited exponential temperature dependence, with 1,8-cineole, {alpha}-pinene, {beta}-pinene and limonene forming the primary emissions. A standardised total monoterpene emission rate of (2.7{+-}1.1){mu}gg{sub dry-weight} {sup -1}h{sup -1} was calculated, corresponding to an annual monoterpene emission rate of (24{+-}12) kTyr{sup -1}. Research was conducted as part of the AEROBIC'97 (AEROsol formation from Biogenic organic Carbon) series of field campaigns. (Author)

  3. Bark Beetles, Pityogenes bidentatus, Orienting to Aggregation Pheromone Avoid Conifer Monoterpene Odors When Flying but Not When Walking

    Directory of Open Access Journals (Sweden)

    John A. Byers

    2012-01-01

    Full Text Available Previous studies and data presented here suggest that odors from healthy host Scotch pine (Pinus sylvestris and nonhost Norway spruce (Picea abies, as well as major monoterpenes of these trees at natural release rates, significantly reduce the attraction of flying bark beetles, Pityogenes bidentatus, of both sexes to their aggregation pheromone components grandisol and cis-verbenol in the field, as tested by slow rotation of trap pairs. In contrast, P. bidentatus males and females walking in an open-arena olfactometer in the laboratory did not avoid monoterpene vapors at release rates spanning several orders of magnitude in combination with aggregation pheromone. The bark beetle may avoid monoterpenes when flying as a mechanism for avoiding nonhost species, vigorous and thus unsuitable host trees, as well as harmful resinous areas of hosts. Inhibition of this flight avoidance response in beetles after landing would allow them to initiate, or to find and enter, gallery holes with high monoterpene vapor concentrations in order to feed and reproduce.

  4. Dose-Dependent and Species-Specific Responses of Pine Bark Beetles (Coeoptera: Scolytidae) to Monoterpenes in Association with Phermones

    Science.gov (United States)

    Daniel R. Miller; John H. Borden

    2000-01-01

    Monoterpenes affected the attraction of three sympatric species of bark beetles (Coleoptera: Scolytidae) to pheromone-baited multiple-funnel traps in stands of lodgepole pine. Catches of Ips pini(Say) in traps baited with its pheromone, ipsdienol, were directly related to the release rates of 3-carene, ß-pphellandrene, and ß-pinene. Catches of

  5. Stress and developmental responses of terpenoid biosynthetic genes in Cistus creticus subsp. creticus.

    Science.gov (United States)

    Pateraki, Irene; Kanellis, Angelos K

    2010-06-01

    Plants, and specially species adapted in non-friendly environments, produce secondary metabolites that help them to cope with biotic or abiotic stresses. These metabolites could be of great pharmaceutical interest because several of those show cytotoxic, antibacterial or antioxidant activities. Leaves' trichomes of Cistus creticus ssp. creticus, a Mediterranean xerophytic shrub, excrete a resin rich in several labdane-type diterpenes with verified in vitro and in vivo cytotoxic and cytostatic activity against human cancer cell lines. Bearing in mind the properties and possible future exploitation of these natural products, it seemed interesting to study their biosynthesis and its regulation, initially at the molecular level. For this purpose, genes encoding enzymes participating in the early steps of the terpenoids biosynthetic pathways were isolated and their gene expression patterns were investigated in different organs and in response to various stresses and defence signals. The genes studied were the CcHMGR from the mevalonate pathway, CcDXS and CcDXR from the methylerythritol 4-phosphate pathway and the two geranylgeranyl diphosphate synthases (CcGGDPS1 and 2) previously characterized from this species. The present work indicates that the leaf trichomes are very active biosynthetically as far as it concerns terpenoids biosynthesis, and the terpenoid production from this tissue seems to be transcriptionally regulated. Moreover, the CcHMGR and CcDXS genes (the rate-limiting steps of the isoprenoids' pathways) showed an increase during mechanical wounding and application of defence signals (like meJA and SA), which is possible to reflect an increased need of the plant tissues for the corresponding metabolites.

  6. Secondary Organic Aerosol (SOA) Formation from Hydroxyl Radical Oxidation and Ozonolysis of Monoterpenes

    Science.gov (United States)

    Zhao, Defeng; Kaminski, Martin; Schlag, Patrick; Fuchs, Hendrik; Acir, Ismail-Hakki; Bohn, Birger; Haeseler, Rolf; Kiendler-Scharr, Astrid; Rohrer, Franz; Tillmann, Ralf; Wang, Mingjin; Wegner, Robert; Wahner, Andreas; Mentel, Thomas

    2014-05-01

    Hydroxyl radical (OH) oxidation and ozonolysis are the two major pathways of daytime biogenic volatile organic compounds (VOCs) oxidation and secondary organic aerosol (SOA) formation. The pure OH oxidation of monoterpenes, an important biogenic VOC class, has seldom been investigated. In order to elucidate the importance of the reaction pathyways of the OH oxidation and ozonolysis and their roles in particle formation and growth, we investigated the particle formation of several common monoterpenes (alpha-pinene, beta-pinene, and limonene) in the large atmosphere simulation chamber SAPHIR in Juelich, Germany. The experiments were conducted for both OH dominant and pure ozonolysis case (in the presence of CO as OH scavenger) at ambient relevant conditions (low OA, low VOC and low NOx concentration). OH and ozone (O3) concentrations were measured so that the oxidation rates of OH and O3 with precursors were quantified. The particle formation and growth, aerosol yield, multi-generation reaction process and aerosol composition were analyzed. Pure ozonolysis generated a large amount of particles indicating ozonolysis plays an important role in particle formation as well as OH oxidation. In individual experiments, particle growth rates did not necessarily correlate with OH or O3 oxidation rates. However, comparing the growth rates at similar OH or O3 oxidation rates shows that generally, OH oxidation and ozonolysis have similar efficiency in particle growth. Multi-generation products are shown to be important in the OH oxidation experiment based on aerosol yield "growth curve" (Ng et al., 2006). The reaction process of OH oxidation experiments was analyzed as a function of OH dose to elucidate the role of functionalization and fragmentation. A novel analysis was developed to link the particle formation with the reaction with OH, which was also used to examine the role of functionalization and fragmentation in the particle formation by OH oxidation. These analyses show

  7. Molecular basis of the evolution of alternative tyrosine biosynthetic routes in plants

    Energy Technology Data Exchange (ETDEWEB)

    Schenck, Craig A.; Holland, Cynthia K.; Schneider, Matthew R.; Men, Yusen; Lee, Soon Goo; Jez, Joseph M.; Maeda, Hiroshi A.

    2017-06-26

    L-Tyrosine (Tyr) is essential for protein synthesis and is a precursor of numerous specialized metabolites crucial for plant and human health. Tyr can be synthesized via two alternative routes by different key regulatory TyrA family enzymes, prephenate dehydrogenase (PDH, also known as TyrAp) or arogenate dehydrogenase (ADH, also known as TyrAa), representing a unique divergence of primary metabolic pathways. The molecular foundation underlying the evolution of these alternative Tyr pathways is currently unknown. Here we characterized recently diverged plant PDH and ADH enzymes, obtained the X-ray crystal structure of soybean PDH, and identified a single amino acid residue that defines TyrA substrate specificity and regulation. Structures of mutated PDHs co-crystallized with Tyr indicate that substitutions of Asn222 confer ADH activity and Tyr sensitivity. Reciprocal mutagenesis of the corresponding residue in divergent plant ADHs further introduced PDH activity and relaxed Tyr sensitivity, highlighting the critical role of this residue in TyrA substrate specificity that underlies the evolution of alternative Tyr biosynthetic pathways in plants.

  8. Reconstitution and Minimization of a Micrococcin Biosynthetic Pathway in Bacillus subtilis

    Science.gov (United States)

    Bennallack, Philip R.; Bewley, Kathryn D.; Burlingame, Mark A.; Robison, Richard A.; Miller, Susan M.

    2016-01-01

    ABSTRACT Thiopeptides represent one of several families of highly modified peptide antibiotics that hold great promise for natural product engineering. These macrocyclic peptides are produced by a combination of ribosomal synthesis and extensive posttranslational modification by dedicated processing enzymes. We previously identified a compact, plasmid-borne gene cluster for the biosynthesis of micrococcin P1 (MP1), an archetypal thiopeptide antibiotic. In an effort to genetically dissect this pathway, we have reconstituted it in Bacillus subtilis. Successful MP1 production required promoter engineering and the reassembly of essential biosynthetic genes in a modular plasmid. The resulting system allows for rapid pathway manipulation, including protein tagging and gene deletion. We find that 8 processing proteins are sufficient for the production of MP1 and that the tailoring enzyme TclS catalyzes a C-terminal reduction step that distinguishes MP1 from its sister compound micrococcin P2. IMPORTANCE The emergence of antibiotic resistance is one of the most urgent human health concerns of our day. A crucial component in an integrated strategy for countering antibiotic resistance is the ability to engineer pathways for the biosynthesis of natural and derivatized antimicrobial compounds. In this study, the model organism B. subtilis was employed to reconstitute and genetically modularize a 9-gene system for the biosynthesis of micrococcin, the founding member of a growing family of thiopeptide antibiotics. PMID:27381911

  9. Alteration of the coenzyme A biosynthetic pathway in neurodegeneration with brain iron accumulation syndromes.

    Science.gov (United States)

    Venco, Paola; Dusi, Sabrina; Valletta, Lorella; Tiranti, Valeria

    2014-08-01

    NBIA (neurodegeneration with brain iron accumulation) comprises a heterogeneous group of neurodegenerative diseases having as a common denominator, iron overload in specific brain areas, mainly basal ganglia and globus pallidus. In the past decade a bunch of disease genes have been identified, but NBIA pathomechanisms are still not completely clear. PKAN (pantothenate kinase-associated neurodegeneration), an autosomal recessive disorder with progressive impairment of movement, vision and cognition, is the most common form of NBIA. It is caused by mutations in the PANK2 (pantothenate kinase 2) gene, coding for a mitochondrial enzyme that phosphorylates vitamin B5 in the first reaction of the CoA (coenzyme A) biosynthetic pathway. A distinct form of NBIA, denominated CoPAN (CoA synthase protein-associated neurodegeneration), is caused by mutations in the CoASY (CoA synthase) gene coding for a bifunctional mitochondrial enzyme, which catalyses the final steps of CoA biosynthesis. These two inborn errors of CoA metabolism further support the concept that dysfunctions in CoA synthesis may play a crucial role in the pathogenesis of NBIA.

  10. Biosynthetic labeling of hypusine in mammalian cells. Carbon-hydrogen bond fissions revealed by dual labeling

    Energy Technology Data Exchange (ETDEWEB)

    Park, M.H.; Folk, J.E.

    1986-10-25

    Using a dual-label technique in which /sup 3/H- and /sup 14/C-labeled forms of putrescine and of spermidine were employed as biosynthetic precursors of hypusine, two -C-H bond cleavages were detected during production of this unique amino acid in Chinese hamster ovary cells. One of these cleavages occurs at C-1 of the 4-aminobutyl group during its transfer from the secondary amine nitrogen of spermidine to the nitrogen at the epsilon-position of a specific lysine residue in the polypeptide precursor of eukaryotic initiation factor 4D. Breakage of the other -C-H bond takes place at C-2 in this aminobutyl segment after it has been coupled to lysine to form the intermediate deoxyhypusine residue. Hydroxylation at this carbon atom, which constitutes the last step in hypusine biosynthesis, is the cause of bond cleavage. The data obtained are consistent with a notion that no additional -C-H bond fissions occur during hypusine biosynthesis. Our findings permit suggestion of a mechanism for enzymic aminobutyl group transfer in which 4-aminobutyraldehyde produced by oxidative cleavage of spermidine is coupled with the epsilon-amino group of a specific lysine residue to form an enzyme-bound imine intermediate.

  11. Elucidation of the complete ferrichrome A biosynthetic pathway in Ustilago maydis.

    Science.gov (United States)

    Winterberg, Britta; Uhlmann, Stefanie; Linne, Uwe; Lessing, Franziska; Marahiel, Mohamed A; Eichhorn, Heiko; Kahmann, Regine; Schirawski, Jan

    2010-03-01

    Iron is an important element for many essential processes in living organisms. To acquire iron, the basidiomycete Ustilago maydis synthesizes the iron-chelating siderophores ferrichrome and ferrichrome A. The chemical structures of these siderophores have been elucidated long time ago but so far only two enzymes involved in their biosynthesis have been described. Sid1, an ornithine monoxygenase, is needed for the biosynthesis of both siderophores, and Sid2, a non-ribosomal peptide synthetase (NRPS), is involved in ferrichrome generation. In this work we identified four novel enzymes, Fer3, Fer4, Fer5 and Hcs1, involved in ferrichrome A biosynthesis in U. maydis. By HPLC-MS analysis of siderophore accumulation in culture supernatants of deletion strains, we show that Fer3, an NRPS, Fer4, an enoyl-coenzyme A (CoA)-hydratase, and Fer5, an acylase, are required for ferrichrome A production. We demonstrate by conditional expression of the hydroxymethyl glutaryl (HMG)-CoA synthase Hcs1 in U. maydis that HMG-CoA is an essential precursor for ferrichrome A. In addition, we heterologously expressed and purified Hcs1, Fer4 and Fer5, and demonstrated the enzymatic activities by in vitro experiments. Thus, we describe the first complete fungal siderophore biosynthetic pathway by functionally characterizing four novel genes responsible for ferrichrome A biosynthesis in U. maydis.

  12. Fate and transport of monoterpenes through soils. Part II: calculation of the effect of soil temperature, water saturation and organic carbon content.

    Science.gov (United States)

    van Roon, André; Parsons, John R; Krap, Lenny; Govers, Harrie A J

    2005-09-01

    This theoretical study was performed to investigate the influence of soil temperature, soil water content and soil organic carbon fraction on the mobility of monoterpenes (C10HnOn') applied as pesticides to a top soil layer. This mobility was expressed as the amount volatilized and leached from the contaminated soil layer after a certain amount of time. For this, (slightly modified) published analytical solutions to a one dimensional, homogeneous medium, diffusion/advection/biodegradation mass balance equation were used. The required input-parameters were determined in a preceding study. Because the monoterpenes studied differ widely in the values for their physico-chemical properties, the relative importance of the various determinants also differed widely. Increasing soil water saturation reduced monoterpene vaporization and leaching losses although a modest increase was usually observed at high soil water contents. Organic matter served as the major retention domain, reducing volatilization and leaching losses. Increasing temperature resulted in higher volatilization and leaching losses. Monoterpene mobility was influenced by vertical water flow. Volatilization losses could be reduced by adding a clean soil layer on top of the contaminated soil. Detailed insight into the specific behaviour of different monoterpenes was obtained by discussing intermediate calculation results; the transport retardation factors and effective soil diffusion coefficients. One insight was that the air-water interface compartment is probably not an important partitioning domain for monoterpenes in most circumstances. The results further indicated that biodegradation is an important process for monoterpenes in soil.

  13. The impact of bark beetle infestation on monoterpene emissions and secondary organic aerosol formation in Western North America

    Directory of Open Access Journals (Sweden)

    A. R. Berg

    2012-11-01

    Full Text Available Over the last decade, extensive beetle outbreaks in Western North America have destroyed over 100 000 km2 of forest throughout British Columbia and the Western United States. Beetle infestations impact monoterpene emissions through both decreased emissions as trees are killed (mortality effect and increased emissions in trees under attack (attack effect. We use 14 yr of beetle mortality data together with beetle-induced monoterpene concentration data in the National Center for Atmospheric Research (NCAR Community Earth System Model (CESM to investigate the impact of beetle mortality and attack on monoterpene emissions and secondary organic aerosol (SOA formation in Western North America.

    Regionally, beetle infestations may have a significant impact on monoterpene emissions and SOA concentrations, with up to a 4-fold increase in monoterpene emissions and up to a 40% increase in SOA concentrations in some years (following a scenario where the attack effect is based on observed lodgepole pine response. Responses to beetle attack depend on the extent of previous mortality and the number of trees under attack in a given year, which can vary greatly over space and time. Simulated enhancements peak in 2004 (British Columbia and 2008 (US. Responses to beetle attack are shown to be substantially larger (up to a 3-fold localized increase in SOA concentrations when following a scenario based on bark-beetle attack in spruce trees. Placed in the context of observations from the IMPROVE network, the changes in SOA concentrations due to beetle attack are in most cases small compared to the large annual and interannual variability in total organic aerosol which is driven by wildfire activity in Western North America. This indicates that most beetle-induced SOA changes are not likely detectable in current observation networks; however these changes may impede efforts to achieve natural visibility conditions in the national parks and

  14. The impact of bark beetle infestations on monoterpene emissions and secondary organic aerosol formation in western North America

    Directory of Open Access Journals (Sweden)

    A. R. Berg

    2013-03-01

    Full Text Available Over the last decade, extensive beetle outbreaks in western North America have destroyed over 100 000 km2 of forest throughout British Columbia and the western United States. Beetle infestations impact monoterpene emissions through both decreased emissions as trees are killed (mortality effect and increased emissions in trees under attack (attack effect. We use 14 yr of beetle-induced tree mortality data together with beetle-induced monoterpene emission data in the National Center for Atmospheric Research (NCAR Community Earth System Model (CESM to investigate the impact of beetle-induced tree mortality and attack on monoterpene emissions and secondary organic aerosol (SOA formation in western North America. Regionally, beetle infestations may have a significant impact on monoterpene emissions and SOA concentrations, with up to a 4-fold increase in monoterpene emissions and up to a 40% increase in SOA concentrations in some years (in a scenario where the attack effect is based on observed lodgepole pine response. Responses to beetle attack depend on the extent of previous mortality and the number of trees under attack in a given year, which can vary greatly over space and time. Simulated enhancements peak in 2004 (British Columbia and 2008 (US. Responses to beetle attack are shown to be substantially larger (up to a 3-fold localized increase in summertime SOA concentrations in a scenario based on bark-beetle attack in spruce trees. Placed in the context of observations from the IMPROVE network, the changes in SOA concentrations due to beetle attack are in most cases small compared to the large annual and interannual variability in total organic aerosol which is driven by wildfire activity in western North America. This indicates that most beetle-induced SOA changes are not likely detectable in current observation networks; however, these changes may impede efforts to achieve natural visibility conditions in the national parks and wilderness

  15. Supercritical Fluid Extraction (SFE of Monoterpenes from the Leaves of Melaleuca alternifolia (Tea Tree

    Directory of Open Access Journals (Sweden)

    Deidre Tronson

    2001-01-01

    Full Text Available The technique of supercritical fluid extraction (SFE was applied to various sample matrices under a range of supercritical carbon dioxide (scCO2 densities and chamber temperatures. The purpose was to develop an effective extraction condition for the removal of eight target monoterpenes from Australian tea tree (Melaleuca alternifolia Cheel leaves. The optimum conditions for extraction were found to be 0.25 g/mL scCO2 density at a chamber temperature of 110oC. These condition were most effective when applied to whole fresh and rehydrated whole dried leaves, where it yielded maximum recovery of target analytes with minimum change in oil composition for the extractor system employed. This study demonstrates the importance of the type of sample matrix used in SFE work, and that a different extraction protocol would need to be developed for each matrix.

  16. New fatty acid, aromatic ester and monoterpenic benzyl glucoside from the fruits of Withania coagulans Dunal.

    Science.gov (United States)

    Ali, Abuzer; Jameel, Mohammad; Ali, Mohammed

    2015-01-01

    The fruits of Withania coagulans Dunal (family: Solanaceae) are sweet, sedative, emetic, alterative and diuretic; used to treat asthma, biliousness, strangury, wounds, dyspepsia, flatulent colic, liver complaints and intestinal infections in the indigenous system of medicine. Phytochemical investigation of the methanolic extract of W. coagulans fruits led to the isolation of a new fatty acid, an aromatic ester and a monoterpenic benzyl glucoside characterised as n-octatriacont-17-enoic acid (3), geranilan-10-olyl dihydrocinnamoate (4) and geranilan-8-oic acid-10-olyl salicyloxy-2-O-β-D-glucofuranosyl-(6″→1‴)-O-β-D-glucofuranosyl-6‴-n-octadec-9‴',11‴'-dienoate (5) along with two known fatty acids, n-dotriacont-21-enoic acid (1) and n-tetratriacontanoic acid (2). The structures of isolated phytoconstituents were established on the basis of 1D and 2D NMR, FT-IR, UV, and MS data and chemical means.

  17. Bioefficacy of acyclic monoterpenes and their saturated derivatives against the West Nile vector Culex pipiens.

    Science.gov (United States)

    Michaelakis, Antonios; Vidali, Veroniki P; Papachristos, Dimitrios P; Pitsinos, Emmanuel N; Koliopoulos, George; Couladouros, Elias A; Polissiou, Moschos G; Kimbaris, Athanasios C

    2014-02-01

    Twenty acyclic monoterpenes with different functional groups (acetoxy, hydroxyl, carbonyl and carboxyl) bearing a variable number of carbon double bonds were assayed as repellent and larvicidal agents against the West Nile vector Culex pipiens. Seven of them were derivatives that were synthesized through either hydrogenation or oxidation procedures. All repellent compounds were tested at the dose of 1mgcm(-2) and only neral and geranial were also tested at a 4-fold lower dose (0.25mgcm(-2)). Repellency results revealed that geranial, neral, nerol, citronellol, geranyl acetate and three more derivatives dihydrolinalool (3), dihydrocitronellol (5) and dihydrocitronellyl acetate (6) resulted in no landings. Based on the LC50 values the derivative dihydrocitronellyl acetate (6) was the most active of all, resulting in an LC50 value of 17.9mgL(-1). Linalyl acetate, citronellyl acetate, neryl acetate, geranyl acetate, dihydrocitronellol (5), dihydrocitronellal (7), citronellol, dihydrolinalyl acetate (2), citronellic acid and tetrahydrolinalyl acetate (1) were also toxic with LC50 values ranging from 23 to 45mgL(-1). Factors modulating toxicity have been identified, thus providing information on structural requirements for the selected acyclic monoterpenes. The acetoxy group enhanced toxicity, without being significantly affected by the unsaturation degree. Within esters, reduction of the vinyl group appears to decrease potency. Presence of a hydroxyl or carbonyl group resulted in increased activity but only in correlation to saturation degree. Branched alcohols proved ineffective compared to the corresponding linear isomers. Finally, as it concerns acids, data do not allow generalizations or correlations to be made.

  18. Contributions of organic peroxides to secondary aerosol formed from reactions of monoterpenes with O3.

    Science.gov (United States)

    Docherty, Kenneth S; Wu, Wilbur; Lim, Yong Bin; Ziemann, Paul J

    2005-06-01

    The role of organic peroxides in secondary organic aerosol (SOA) formation from reactions of monoterpenes with O3 was investigated in a series of environmental chamber experiments. Reactions were performed with endocyclic (alpha-pinene and delta3-carene) and exocyclic (beta-pinene and sabinene) alkenes in dry and humid air and in the presence of the OH radical scavengers: cyclohexane, 1-propanol, and formaldehyde. A thermal desorption particle beam mass spectrometer was used to probe the identity and volatility of SOA components, and an iodometric-spectrophotometric method was used to quantify organic peroxides. Thermal desorption profiles and mass spectra showed that the most volatile SOA components had vapor pressures similar to pinic acid and that much of the SOA consisted of less volatile species that were probably oligomeric compounds. Peroxide analyses indicated that the SOA was predominantly organic peroxides, providing evidence that the oligomers were mostly peroxyhemiacetals formed by heterogeneous reactions of hydroperoxides and aldehydes. For example, it was estimated that organic peroxides contributed approximately 47 and approximately 85% of the SOA mass formed in the alpha- and beta-pinene reactions, respectively. Reactions performed with different OH radical scavengers indicated that most of the hydroperoxides were formed through the hydroperoxide channel rather than by reactions of stabilized Criegee intermediates. The effect of the OH radical scavenger on the SOA yield was also investigated, and the results were consistent with results of recent experiments and model simulations that support a mechanism based on changes in the [HO2]/[RO2] ratios. These are the first measurements of organic peroxides in monoterpene SOA, and the results have important implications for understanding the mechanisms of SOA formation and the potential effects of atmospheric aerosol particles on the environment and human health.

  19. Transcriptome profiling, and cloning and characterization of the main monoterpene synthases of Coriandrum sativum L.

    Science.gov (United States)

    Galata, Mariana; Sarker, Lukman S; Mahmoud, Soheil S

    2014-06-01

    Terpenoids are a large and diverse class of specialized metabolites that are essential for the growth and development of plants, and have tremendous industrial applications. The mericarps of Coriandrum sativum L. (coriander) produce an essential oil (EO) rich in monoterpenes, volatile C10 terpenoids. To investigate EO metabolism, the transcriptome of coriander mericarps, at three developmental stages (early, mid, late) was sequenced via Illumina technology and a transcript library was produced. To validate the usability of the transcriptome sequences, two terpene synthase candidate genes, CsγTRPS and CsLINS, encoding 558 and 562 amino acid proteins were expressed in bacteria, and the recombinant proteins purified by Ni-NTA affinity chromatography. The 65.16 (CsγTRPS) and 65.91 (CsLINS)kDa recombinant proteins catalyzed the conversion of geranyl diphosphate, the precursor to monoterpenes, to γ-terpinene and (S)-linalool, respectively, with apparent Vmax and Km values of 2.24±0.16 (CsγTRPS); 19.63±1.05 (CsLINS)pkat/mg and 66.25±13 (CsγTRPS); 2.5±0.6 (CsLINS)μM, respectively. Together, CsγTRPS and CsLINS account for the majority of EO constituents in coriander mericarps. Investigation of the coriander transcriptome, and knowledge gained from these experiments will facilitate future studies concerning essential and fatty acid oil production in coriander. They also enable efforts to improve the coriander oils through metabolic engineering or plant breeding.

  20. A kinetic model for the penicillin biosynthetic pathway in

    DEFF Research Database (Denmark)

    Nielsen, Jens; Jørgensen, Henrik

    1996-01-01

    A kinetic model for the first two steps in the penicillin biosynthetic pathway, i.e. the ACV synthetase (ACVS) and the isopenicillin N synthetase (IPNS) is proposed. The model is based on Michaelis-Menten type kinetics with non-competitive inhibition of the ACVS by ACV, and competitive inhibition...... of the IPNS by glutathione. The model predicted flux through the pathway corresponds well with the measured rate of penicillin biosynthesis. From the kinetic model the elasticity coefficients and the flux control coefficients are calculated throughout a fed-batch cultivation, and it is found...

  1. Survey of volatile oxylipins and their biosynthetic precursors in bryophytes.

    Science.gov (United States)

    Croisier, Emmanuel; Rempt, Martin; Pohnert, Georg

    2010-04-01

    Oxylipins are metabolites which are derived from the oxidative fragmentation of polyunsaturated fatty acids. These metabolites play central roles in plant hormonal regulation and defense. Here we survey the production of volatile oxylipins in bryophytes and report the production of a high structural variety of C5, C6, C8 and C9 volatiles of mosses. In liverworts and hornworts oxylipin production was not as pronounced as in the 23 screened mosses. A biosynthetic investigation revealed that both, C18 and C20 fatty acids serve as precursors for the volatile oxylipins that are mainly produced after mechanical wounding of the green tissue of mosses.

  2. Carotenoid biosynthetic genes in Brassica rapa: comparative genomic analysis, phylogenetic analysis, and expression profiling

    OpenAIRE

    Li, Peirong; Zhang, Shujiang; Zhang, Shifan; Li, Fei; Zhang, Hui; Cheng, Feng; Wu, Jian; Wang, Xiaowu; Sun, Rifei

    2015-01-01

    Background Carotenoids are isoprenoid compounds synthesized by all photosynthetic organisms. Despite much research on carotenoid biosynthesis in the model plant Arabidopsis thaliana, there is a lack of information on the carotenoid pathway in Brassica rapa. To better understand its carotenoid biosynthetic pathway, we performed a systematic analysis of carotenoid biosynthetic genes at the genome level in B. rapa. Results We identified 67 carotenoid biosynthetic genes in B. rapa, which were ort...

  3. [Certain properties of "biosynthetic" L-threonine dehydratase from subcellular structures of brewers' yeast Saccharomyces carlsbergensis].

    Science.gov (United States)

    Kovaleva, S V; Korozhko, A I; Beliaeva, N F; Kagan, Z S

    1981-01-01

    The paper is concerned with kinetic properties of the "biosynthetic" L-threonine dehydratase (EC 4.2.1.16) solubilized from subcellular structures of brewers' yeast Saccharomyces carlsbergensis in the absence and presence of the allosteric inhibitor, L-isoleucine, at three pH-values (pH 6.5, 7.8 and 9.5). The curve of the initial reaction rate versus initial substrate concentration in the absence of L-isoleucine at pH 6.5 was of hyperbolic character (Km = 5.5.10(-2) M), and at pH 7.8 and 9.5 the kinetic curve had a weakly sigmoidal pattern with a sharp going into the saturation plateaux; the values of [S] 0.5 are 1.10(-2) and 8.7.10(-3) M, respectively. An addition of L-isoleucine to the reaction mixture led to the appearance (at pH 6.5) or to an increase (at pH 7.8 and 9.5) of the sigmoidality of these kinetic curves and to a decrease in values of the maximum reaction rate V. The enzyme sensibility to the inhibitory effect of L-isoleucine decreased with an increase in pH values. Low L-isoleucine concentrations at low substrate concentrations activated the enzyme. The pH optimum for L-threonine dehydratase under study was 9.5-10.0. The enzyme molecular weight is about 300 000.

  4. Biosynthetic Studies and Genetic Engineering of Pactamycin Analogs with Improved Selectivity toward Malarial Parasites

    National Research Council Canada - National Science Library

    Lu, Wanli; Roongsawang, Niran; Mahmud, Taifo

    2011-01-01

    .... However, through extensive biosynthetic studies and genetic engineering, we were able to produce analogs of pactamycin that show potent antimalarial activity, but lack significant antibacterial...

  5. Polymorphisms in monolignol biosynthetic genes are associated with biomass yield and agronomic traits in European maize (Zea mays L.

    Directory of Open Access Journals (Sweden)

    Andersen Jeppe

    2010-01-01

    Full Text Available Abstract Background Reduced lignin content leads to higher cell wall digestibility and, therefore, better forage quality and increased conversion of lignocellulosic biomass into ethanol. However, reduced lignin content might lead to weaker stalks, lodging, and reduced biomass yield. Genes encoding enzymes involved in cell wall lignification have been shown to influence both cell wall digestibility and yield traits. Results In this study, associations between monolignol biosynthetic genes and plant height (PHT, days to silking (DTS, dry matter content (DMC, and dry matter yield (DMY were identified by using a panel of 39 European elite maize lines. In total, 10 associations were detected between polymorphisms or tight linkage disequilibrium (LD groups within the COMT, CCoAOMT2, 4CL1, 4CL2, F5H, and PAL genomic fragments, respectively, and the above mentioned traits. The phenotypic variation explained by these polymorphisms or tight LD groups ranged from 6% to 25.8% in our line collection. Only 4CL1 and F5H were found to have polymorphisms associated with both yield and forage quality related characters. However, no pleiotropic polymorphisms affecting both digestibility of neutral detergent fiber (DNDF, and PHT or DMY were discovered, even under less stringent statistical conditions. Conclusion Due to absence of pleiotropic polymorphisms affecting both forage yield and quality traits, identification of optimal monolignol biosynthetic gene haplotype(s combining beneficial quantitative trait polymorphism (QTP alleles for both quality and yield traits appears possible within monolignol biosynthetic genes. This is beneficial to maximize forage and bioethanol yield per unit land area.

  6. Living with high putrescine: expression of ornithine and arginine biosynthetic pathway genes in high and low putrescine producing poplar cells.

    Science.gov (United States)

    Page, Andrew F; Minocha, Rakesh; Minocha, Subhash C

    2012-01-01

    Arginine (Arg) and ornithine (Orn), both derived from glutamate (Glu), are the primary substrates for polyamine (PA) biosynthesis, and also play important roles as substrates and intermediates of overall N metabolism in plants. Their cellular homeostasis is subject to multiple levels of regulation. Using reverse transcription quantitative PCR (RT-qPCR), we studied changes in the expression of all genes of the Orn/Arg biosynthetic pathway in response to up-regulation [via transgenic expression of mouse Orn decarboxylase (mODC)] of PA biosynthesis in poplar (Populus nigra × maximowiczii) cells grown in culture. Cloning and sequencing of poplar genes involved in the Orn/Arg biosynthetic pathway showed that they have high homology with similar genes in other plants. The expression of the genes of Orn, Arg and PA biosynthetic pathway fell into two hierarchical clusters; expression of one did not change in response to high putrescine, while members of the other cluster showed a shift in expression pattern during the 7-day culture cycle. Gene expression of branch point enzymes (N-acetyl-Glu synthase, Orn aminotransferase, Arg decarboxylase, and spermidine synthase) in the sub-pathways, constituted a separate cluster from those involved in intermediary reactions of the pathway (N-acetyl-Glu kinase, N-acetyl-Glu-5-P reductase, N-acetyl-Orn aminotransferase, N (2)-acetylOrn:N-acetyl-Glu acetyltransferase, N (2)-acetyl-Orn deacetylase, Orn transcarbamylase, argininosuccinate synthase, carbamoylphosphate synthetase, argininosuccinate lyase, S-adenosylmethionine decarboxylase, spermine synthase). We postulate that expression of all genes of the Glu-Orn-Arg pathway is constitutively coordinated and is not influenced by the increase in flux rate through this pathway in response to increased utilization of Orn by mODC; thus the pathway involves mostly biochemical regulation rather than changes in gene expression. We further suggest that Orn itself plays a major role in the

  7. Biosynthetic multitasking facilitates thalassospiramide structural diversity in marine bacteria

    KAUST Repository

    Ross, Avena C.

    2013-01-23

    Thalassospiramides A and B are immunosuppressant cyclic lipopeptides first reported from the marine α-proteobacterium Thalassospira sp. CNJ-328. We describe here the discovery and characterization of an extended family of 14 new analogues from four Tistrella and Thalassospira isolates. These potent calpain 1 protease inhibitors belong to six structure classes in which the length and composition of the acylpeptide side chain varies extensively. Genomic sequence analysis of the thalassospiramide-producing microbes revealed related, genus-specific biosynthetic loci encoding hybrid nonribosomal peptide synthetase/polyketide synthases consistent with thalassospiramide assembly. The bioinformatics analysis of the gene clusters suggests that structural diversity, which ranges from the 803.4 Da thalassospiramide C to the 1291.7 Da thalassospiramide F, results from a complex sequence of reactions involving amino acid substrate channeling and enzymatic multimodule skipping and iteration. Preliminary biochemical analysis of the N-terminal nonribosomal peptide synthetase module from the Thalassospira TtcA megasynthase supports a biosynthetic model in which in cis amino acid activation competes with in trans activation to increase the range of amino acid substrates incorporated at the N terminus. © 2012 American Chemical Society.

  8. Chrysanthemyl diphosphate synthase: Isolation of the gene and characterization of the recombinant non-head-to-tail monoterpene synthase from Chrysanthemum cinerariaefolium

    OpenAIRE

    Rivera, Susan B.; Swedlund, Bradley D.; King, Gretchen J.; Bell, Russell N.; Hussey, Charles E.; Shattuck-Eidens, Donna M.; Wrobel, Wislawa M.; Peiser, Galen D.; Poulter, C. Dale

    2001-01-01

    Chrysanthemyl diphosphate synthase (CPPase) catalyzes the condensation of two molecules of dimethylallyl diphosphate to produce chrysanthemyl diphosphate (CPP), a monoterpene with a non-head-to-tail or irregular c1′-2-3 linkage between isoprenoid units. Irregular monoterpenes are common in Chrysanthemum cinerariaefolium and related members of the Asteraceae family. In C. cinerariaefolium, CPP is an intermediate in the biosynthesis of the pyrethrin ester insecticides. CPPa...

  9. Unique actinomycetes from marine caves and coral reef sediments provide novel PKS and NRPS biosynthetic gene clusters.

    Science.gov (United States)

    Hodges, Tyler W; Slattery, Marc; Olson, Julie B

    2012-06-01

    In the ever-expanding search for novel bioactive molecules and enzymes, marine actinomycetes have proven to be a productive source. While open reef sediment and sponge-associated actinomycetes have been extensively examined, their marine cave counterparts remain unevaluated. Anchialine cave systems in the Bahamas offered an ideal setting to evaluate the occurrence and variation within sediment-associated actinomycete communities. While in close geographical proximity to open reef environments, these systems provide a specialized environmental niche devoid of light and direct exposure to nutrient input. In the present study, selective isolation techniques and molecular methods were used to test the hypothesis that variable distribution of actinomycetes and secondary metabolite gene clusters occur between open reef and marine cave systems. The results indicated that differences exist within the culturable sediment-associated actinomycete communities between marine caves and open reef systems, with members of the genus Streptomyces dominating cultures from open reef sediments and a more diverse suite of actinomycetes isolated from marine cave sediment samples. Within the cave isolates, members of the proposed genus Solwaraspora were the most represented. Based on PKS- and NRPS-gene-targeted PCR amplification and sequencing, geographic variation in the occurrence of these biosynthetic pathways was also observed. These findings indicate that marine cave systems are a lucrative source in the search for novel secondary metabolite producers with biotechnological applications and that environmental and geographic factors likely affect the occurrence of these biosynthetic pathways.

  10. Wide Distribution of Foxicin Biosynthetic Gene Clusters in Streptomyces Strains - An Unusual Secondary Metabolite with Various Properties.

    Science.gov (United States)

    Greule, Anja; Marolt, Marija; Deubel, Denise; Peintner, Iris; Zhang, Songya; Jessen-Trefzer, Claudia; De Ford, Christian; Burschel, Sabrina; Li, Shu-Ming; Friedrich, Thorsten; Merfort, Irmgard; Lüdeke, Steffen; Bisel, Philippe; Müller, Michael; Paululat, Thomas; Bechthold, Andreas

    2017-01-01

    Streptomyces diastatochromogenes Tü6028 is known to produce the polyketide antibiotic polyketomycin. The deletion of the pokOIV oxygenase gene led to a non-polyketomycin-producing mutant. Instead, novel compounds were produced by the mutant, which have not been detected before in the wild type strain. Four different compounds were identified and named foxicins A-D. Foxicin A was isolated and its structure was elucidated as an unusual nitrogen-containing quinone derivative using various spectroscopic methods. Through genome mining, the foxicin biosynthetic gene cluster was identified in the draft genome sequence of S. diastatochromogenes. The cluster spans 57 kb and encodes three PKS type I modules, one NRPS module and 41 additional enzymes. A foxBII gene-inactivated mutant of S. diastatochromogenes Tü6028 ΔpokOIV is unable to produce foxicins. Homologous fox biosynthetic gene clusters were found in more than 20 additional Streptomyces strains, overall in about 2.6% of all sequenced Streptomyces genomes. However, the production of foxicin-like compounds in these strains has never been described indicating that the clusters are expressed at a very low level or are silent under fermentation conditions. Foxicin A acts as a siderophore through interacting with ferric ions. Furthermore, it is a weak inhibitor of the Escherichia coli aerobic respiratory chain and shows moderate antibiotic activity. The wide distribution of the cluster and the various properties of the compound indicate a major role of foxicins in Streptomyces strains.

  11. Wide Distribution of Foxicin Biosynthetic Gene Clusters in Streptomyces Strains – An Unusual Secondary Metabolite with Various Properties

    Science.gov (United States)

    Greule, Anja; Marolt, Marija; Deubel, Denise; Peintner, Iris; Zhang, Songya; Jessen-Trefzer, Claudia; De Ford, Christian; Burschel, Sabrina; Li, Shu-Ming; Friedrich, Thorsten; Merfort, Irmgard; Lüdeke, Steffen; Bisel, Philippe; Müller, Michael; Paululat, Thomas; Bechthold, Andreas

    2017-01-01

    Streptomyces diastatochromogenes Tü6028 is known to produce the polyketide antibiotic polyketomycin. The deletion of the pokOIV oxygenase gene led to a non-polyketomycin-producing mutant. Instead, novel compounds were produced by the mutant, which have not been detected before in the wild type strain. Four different compounds were identified and named foxicins A–D. Foxicin A was isolated and its structure was elucidated as an unusual nitrogen-containing quinone derivative using various spectroscopic methods. Through genome mining, the foxicin biosynthetic gene cluster was identified in the draft genome sequence of S. diastatochromogenes. The cluster spans 57 kb and encodes three PKS type I modules, one NRPS module and 41 additional enzymes. A foxBII gene-inactivated mutant of S. diastatochromogenes Tü6028 ΔpokOIV is unable to produce foxicins. Homologous fox biosynthetic gene clusters were found in more than 20 additional Streptomyces strains, overall in about 2.6% of all sequenced Streptomyces genomes. However, the production of foxicin-like compounds in these strains has never been described indicating that the clusters are expressed at a very low level or are silent under fermentation conditions. Foxicin A acts as a siderophore through interacting with ferric ions. Furthermore, it is a weak inhibitor of the Escherichia coli aerobic respiratory chain and shows moderate antibiotic activity. The wide distribution of the cluster and the various properties of the compound indicate a major role of foxicins in Streptomyces strains. PMID:28270798

  12. Molecular cloning and expression of phosphoglycerate dehydrogenase and phosphoserine aminotransferase in the serine biosynthetic pathway from Acanthamoeba castellanii.

    Science.gov (United States)

    Deng, Yihong; Wu, Duo; Tachibana, Hiroshi; Cheng, Xunjia

    2015-04-01

    Free-living amoebae of the genus Acanthamoeba are widespread protozoans that can cause serious infectious diseases. This study characterised phosphoglycerate dehydrogenase (PGDH) and phosphoserine aminotransferase (PSAT) in the phosphorylated serine biosynthetic pathway of Acanthamoeba castellanii. The PGDH gene encodes a protein of 442 amino acids with a calculated molecular weight of 47.7 kDa and an isoelectric point (pI) of 7.64. Meanwhile, the PSAT gene encodes a protein of 394 amino acids with a calculated molecular weight of 43.8 kDa and a pI of 5.80. Confocal microscopy suggests that PGDH is mainly diffused in the cytoplasm, whereas PSAT is located in the inner part of the cell membrane. The messenger RNA (mRNA) expression levels of PGDH and PSAT vary depending on growth state under consecutive culture conditions. No significant changes in the mRNA expression levels of both PGDH and PSAT occur after the incubation of L-serine with Acanthamoeba. This result indicates that exogenous serine exerts no influence on the expression of these genes and that the so-called feedback inhibition of both PGDH and PSAT in Acanthamoeba differs from that in bacteria or other organisms. We propose that the enzymes in the phosphorylated serine biosynthetic pathway function in amoeba growth and proliferation.

  13. Capture of micrococcin biosynthetic intermediates reveals C-terminal processing as an obligatory step for in vivo maturation

    Science.gov (United States)

    Bewley, Kathryn D.; Bennallack, Philip R.; Burlingame, Mark A.; Robison, Richard A.; Griffitts, Joel S.

    2016-01-01

    Thiopeptides, including micrococcins, are a growing family of bioactive natural products that are ribosomally synthesized and heavily modified. Here we use a refactored, modular in vivo system containing the micrococcin P1 (MP1) biosynthetic genes (TclIJKLMNPS) from Macrococcus caseolyticus str 115 in a genetically tractable Bacillus subtilis strain to parse the processing steps of this pathway. By fusing the micrococcin precursor peptide to an affinity tag and coupling it with catalytically defective enzymes, biosynthetic intermediates were easily captured for analysis. We found that two major phases of molecular maturation are separated by a key C-terminal processing step. Phase-I conversion of six Cys residues to thiazoles (TclIJN) is followed by C-terminal oxidative decarboxylation (TclP). This TclP-mediated oxidative decarboxylation is a required step for the peptide to progress to phase II. In phase II, Ser/Thr dehydration (TclKL) and peptide macrocycle formation (TclM) occurs. A C-terminal reductase, TclS, can optionally act on the substrate peptide, yielding MP1, and is shown to act late in the pathway. This comprehensive characterization of the MP1 pathway prepares the way for future engineering efforts. PMID:27791142

  14. Two Cytochrome P450 Monooxygenases Catalyze Early Hydroxylation Steps in the Potato Steroid Glycoalkaloid Biosynthetic Pathway1[OPEN

    Science.gov (United States)

    Nakayasu, Masaru; Ohyama, Kiyoshi; Saito, Kazuki

    2016-01-01

    α-Solanine and α-chaconine, steroidal glycoalkaloids (SGAs) found in potato (Solanum tuberosum), are among the best-known secondary metabolites in food crops. At low concentrations in potato tubers, SGAs are distasteful; however, at high concentrations, SGAs are harmful to humans and animals. Here, we show that POTATO GLYCOALKALOID BIOSYNTHESIS1 (PGA1) and PGA2, two genes that encode cytochrome P450 monooxygenases (CYP72A208 and CYP72A188), are involved in the SGA biosynthetic pathway, respectively. The knockdown plants of either PGA1 or PGA2 contained very little SGA, yet vegetative growth and tuber production were not affected. Analyzing metabolites that accumulated in the plants and produced by in vitro enzyme assays revealed that PGA1 and PGA2 catalyzed the 26- and 22-hydroxylation steps, respectively, in the SGA biosynthetic pathway. The PGA-knockdown plants had two unique phenotypic characteristics: The plants were sterile and tubers of these knockdown plants did not sprout during storage. Functional analyses of PGA1 and PGA2 have provided clues for controlling both potato glycoalkaloid biosynthesis and tuber sprouting, two traits that can significantly impact potato breeding and the industry. PMID:27307258

  15. Lignin chemistry: biosynthetic study and structural characterisation of coniferyl alcohol oligomers formed in vitro in a micellar environment.

    Science.gov (United States)

    Reale, Samantha; Attanasio, Francesca; Spreti, Nicoletta; De Angelis, Francesco

    2010-05-25

    Model coniferyl alcohol lignin (the so-called dehydrogenative polymerisate, DHP) was produced in water under homogeneous conditions guaranteed by the presence of a micellised cationic surfactant. A complete study of the activity of the enzymatic system peroxidase/H(2)O(2) under our reaction conditions was reported and all the reaction products up to the pentamer were characterised by (1)H NMR spectroscopy and ESI mass spectrometry. Our system, and the molecules that have been generated in it, represent a closer mimicry of the natural microenvironment since an enzyme, under micellar conditions, reproduces the cell system better than in buffer alone. On the basis of the oligomers structures a new biosynthetic perspective was proposed that focused attention on a coniferyl alcohol dimeric quinone methide as the key intermediate of the reaction. A formal, strictly alternate sequence of a radical and an ionic step underlines the reaction, thus generating ordered oligolignols structures. Alternatively to other model lignins, our olignols present a lower degree of radical coupling between oligomeric units. This offers a closer biosynthetic situation to the observation of a low rate of radical generation in the cell wall.

  16. Microbial modulation of bacoside A biosynthetic pathway and systemic defense mechanism in Bacopa monnieri under Meloidogyne incognita stress

    Science.gov (United States)

    Gupta, Rupali; Singh, Akanksha; Srivastava, Madhumita; Singh, Vivek; Gupta, M. M.; Pandey, Rakesh

    2017-01-01

    Plant-associated beneficial microbes have been explored to fulfill the imperative function for plant health. However, their impact on the host secondary metabolite production and nematode disease management remains elusive. Our present work has shown that chitinolytic microbes viz., Chitiniphilus sp. MTN22 and Streptomyces sp. MTN14 singly as well as in combination modulated the biosynthetic pathway of bacoside A and systemic defense mechanism against Meloidogyne incognita in Bacopa monnieri. Interestingly, expression of bacoside biosynthetic pathway genes (3-Hydroxy-3-methylglutaryl coenzyme A reductase, mevalonate diphosphate decarboxylase, and squalene synthase) were upregulated in plants treated with the microbial combination in the presence as well as in absence of M. incognita stress. These microbes not only augmented bacoside A production (1.5 fold) but also strengthened host resistance via enhancement in chlorophyll a, defense enzymes and phenolic compounds like gallic acid, syringic acid, ferulic acid and cinnamic acid. Furthermore, elevated lignification and callose deposition in the microbial combination treated plants corroborate well with the above findings. Overall, the results provide novel insights into the underlying mechanisms of priming by beneficial microbes and underscore their capacity to trigger bacoside A production in B. monnieri under biotic stress. PMID:28157221

  17. Monoterpene emissions from needles of hybrid larch F1 (Larix gmelinii var. japonica × Larix kaempferi) grown under elevated carbon dioxide and ozone

    Science.gov (United States)

    Mochizuki, Tomoki; Watanabe, Makoto; Koike, Takayoshi; Tani, Akira

    2017-01-01

    We measured monoterpene emissions from needles of hybrid larch F1 (Larix gmelinii var. japonica × Larix kaempferi) to evaluate the response of monoterpene emission rates and their composition to elevated CO2 (600 μmol mol-1) and O3 (60 nmol mol-1) conditions. The dominant monoterpenes were α-pinene and β-pinene. The emission rate of total monoterpenes significantly decreased under elevated CO2 conditions (P photosynthetically fixed carbon also significantly decreased under elevated CO2 conditions. By contrast, elevated O3 did not significantly affect the emission rate of total monoterpenes. The ratios of α-pinene/β-pinene, limonene/β-pinene, and myrcene/β-pinene were all significantly decreased by O3 exposure (P < 0.05). High reactivity of α-pinene, limonene, and myrcene when combining with O3 may be able to mitigate oxidative damage inside the larch needles. No significant combined effects of elevated CO2 and O3 on individual or total monoterpene emissions were detected.

  18. Irreversible impacts of heat on the emissions of monoterpenes, sesquiterpenes, phenolic BVOC and green leaf volatiles from several tree species

    Directory of Open Access Journals (Sweden)

    E. Kleist

    2012-12-01

    Full Text Available Climate change will induce extended heat waves to parts of the vegetation more frequently. High temperatures may act as stress (thermal stress on plants changing emissions of biogenic volatile organic compounds (BVOCs. As BVOCs impact the atmospheric oxidation cycle and aerosol formation, it is important to explore possible alterations of BVOC emissions under high temperature conditions. Applying heat to European beech, Palestine oak, Scots pine, and Norway spruce in a laboratory setup either caused the well-known exponential increases of BVOC emissions or induced irreversible changes of BVOC emissions. Considering only irreversible changes of BVOC emissions as stress impacts, we found that high temperatures decreased the de novo emissions of monoterpenes, sesquiterpenes and phenolic BVOC. This behaviour was independent of the tree species and whether the de novo emissions were constitutive or induced by biotic stress.

    In contrast, application of thermal stress to conifers amplified the release of monoterpenes stored in resin ducts of conifers and induced emissions of green leaf volatiles. In particular during insect attack on conifers, the plants showed de novo emissions of sesquiterpenes and phenolic BVOCs, which exceeded constitutive monoterpene emissions from pools. The heat-induced decrease of de novo emissions was larger than the increased monoterpene release caused by damage of resin ducts. For insect-infested conifers the net effect of thermal stress on BVOC emissions could be an overall decrease.

    Global change-induced heat waves may put hard thermal stress on plants. If so, we project that BVOC emissions increase is more than predicted by models only in areas predominantly covered with conifers that do not emit high amounts of sesquiterpenes and phenolic BVOCs. Otherwise overall effects of high temperature stress will be lower increases of BVOC emissions than predicted by algorithms that do

  19. European emissions of isoprene and monoterpenes from the Last Glacial Maximum to present

    Directory of Open Access Journals (Sweden)

    G. Schurgers

    2009-12-01

    Full Text Available Biogenic volatile organic compounds (BVOC, such as isoprene and monoterpenes, play an important role in atmospheric processes. BVOC species are oxidized in the atmosphere and influence levels of ozone. The less volatile amongst the BVOC and their oxidation products are important for the formation and growth of secondary biogenic aerosol. In this way, the Earth's radiation balance is affected.

    Geographic distribution and temporal changes in BVOC emissions are highly uncertain. Here we assessed changes in emission patterns across Europe since the Last Glacial Maximum (LGM with a dynamic vegetation model. This model reproduces European tree species distribution and includes a process-based algorithm for terpenoid production. In a set of simulations the model was driven with paleoclimate anomalies and reconstructed CO2 concentrations. We quantified three main driving factors for the changes in emissions of isoprene and monoterpenes since the LGM: (1 the changes in climate, with temperature changes as the most important factor affecting plant physiology and terpenoid production in all plant species, (2 a change in species distribution related to the changes in climate, causing local shifts in emission characteristics of the vegetation, and (3 a change in CO2 concentration, causing opposing effects on the availability of different substrates for terpenoid production. The effect of atmospheric CO2 concentration is particularly uncertain, but sensitivity simulations showed an increase in European BVOC emissions in all sensitivity experiments irrespective of the use of a direct inhibition of terpenoid production by CO2. The effects of climate change on physiology and terpenoid production resulted in an overall relatively uniform increase of emissions in Europe over the simulation period, but regionally the effect of changes in species distribution and the related changes in emission capacities resulted

  20. European emissions of isoprene and monoterpenes from the Last Glacial Maximum to present

    Directory of Open Access Journals (Sweden)

    G. Schurgers

    2009-09-01

    Full Text Available Biogenic volatile organic compounds (BVOC, such as isoprene and monoterpenes, play an important role in atmospheric processes. BVOC species are oxidized in the atmosphere and influence levels of ozone. The less volatile amongst the BVOC and their oxidation products are important for the formation and growth of secondary biogenic aerosol. In this way, the earth's radiation balance is affected.

    Geographic distribution and temporal changes in BVOC emissions are highly uncertain. Here we assessed changes in emission patterns across Europe since the Last Glacial Maximum (LGM with a dynamic vegetation model that reproduces European tree species distribution and in which a process-based algorithm for terpenoid production was incorporated. In a set of simulations the model was driven with paleoclimate anomalies and reconstructed CO2 concentrations. We quantified three main driving factors for the changes in emissions of isoprene and monoterpenes since the LGM: (1 the changes in climate, with temperature changes as the most important factor affecting plant physiology and terpenoid production in all plant species, (2 a change in species distribution related to the changes in climate, causing local shifts in emission characteristics of the vegetation, and (3 a change in CO2 concentration, causing opposing effects on the availability of different substrates for terpenoid production. The effect of atmospheric CO2 concentration is particularly uncertain, but sensitivity simulations showed an increase in European BVOC emissions in all sensitivity experiments irrespective of the use of a direct inhibition of terpenoid production by CO2. The effects of climate change on physiology and terpenoid production resulted in an overall relatively uniform increase of emissions in Europe over the simulation period, but regionally the effect of changes in species distribution and the related changes in emission

  1. Hygroscopicity of secondary organic aerosols formed by oxidation of cycloalkenes, monoterpenes, sesquiterpenes, and related compounds

    Directory of Open Access Journals (Sweden)

    V. Varutbangkul

    2006-01-01

    Full Text Available A series of experiments has been conducted in the Caltech indoor smog chamber facility to investigate the water uptake properties of aerosol formed by oxidation of various organic precursors. Secondary organic aerosol (SOA from simple and substituted cycloalkenes (C5-C8 is produced in dark ozonolysis experiments in a dry chamber (RH~5%. Biogenic SOA from monoterpenes, sesquiterpenes, and oxygenated terpenes is formed by photooxidation in a humid chamber (~50% RH. Using the hygroscopicity tandem differential mobility analyzer (HTDMA, we measure the diameter-based hygroscopic growth factor (GF of the SOA as a function of time and relative humidity. All SOA studied is found to be slightly hygroscopic, with smaller water uptake than that of typical inorganic aerosol substances. The aerosol water uptake increases with time early in the experiments for the cycloalkene SOA, but decreases with time for the sesquiterpene SOA. This behavior could indicate competing effects between the formation of more highly oxidized polar compounds (more hygroscopic, and formation of longer-chained oligomers (less hygroscopic. All SOA also exhibit a smooth water uptake with RH with no deliquescence or efflorescence. The water uptake curves are found to be fitted well with an empirical three-parameter functional form. The measured pure organic GF values at 85% RH are between 1.09–1.16 for SOA from ozonolysis of cycloalkenes, 1.01–1.04 for sesquiterpene photooxidation SOA, and 1.06–1.10 for the monoterpene and oxygenated terpene SOA. The GF of pure SOA (GForg in experiments in which inorganic seed aerosol is used is determined by assuming volume-weighted water uptake (Zdanovskii-Stokes-Robinson or 'ZSR' approach and using the size-resolved organic mass fraction measured by the Aerodyne Aerosol Mass Spectrometer. Knowing the water content associated with the inorganic fraction yields GForg values. However, for each precursor, the GForg values computed from different

  2. Assessment of the repellent effect of Lippia alba essential oil and major monoterpenes on the cattle tick Rhipicephalus microplus.

    Science.gov (United States)

    Lima, A da Silva; Carvalho, J F de; Peixoto, M G; Blank, A F; Borges, L M F; Costa Junior, L M

    2016-03-01

    The control of Rhipicephalus microplus (Ixodida: Ixodidae) is achieved using synthetic acaricides. However, resistant tick populations are widespread around the world. Plant essential oils can act as repellents, keeping ticks away from hosts and decreasing the selection pressure on synthetic acaricides. The aim of this study was to evaluate the in vitro repellent effect of Lippia alba essential oil on R. microplus larvae. Leaves from two L. alba genotypes maintained under the same agronomic and environmental conditions were collected. Essential oil was extracted by hydrodistillation and analysed by gas chromatography-mass spectrometry (GC-MS). The major monoterpenes detected in the chemical analysis were commercially acquired and tested. For the repellency test, a glass rod was vertically fixed to measure active climbing of approximately 30 R. microplus larvae aged 14-21 days in response to essential oils and monoterpenes. Repellency was evaluated at 1 h, 3 h and 5 h after treatment. Variation in repellent action was detected between the genotypes. The major monoterpenes identified in the essential oils (limonene and carvone) showed low repellent effects in comparison with intact essential oils. Thus, the present results showed that L. alba essential oil contains bioactive compounds with great repellent activity against ticks that varies according to the plant genotype.

  3. Examination of Triacylglycerol Biosynthetic Pathways via De Novo Transcriptomic and Proteomic Analyses in an Unsequenced Microalga

    Science.gov (United States)

    2011-10-17

    Examination of Triacylglycerol Biosynthetic Pathways via De Novo Transcriptomic and Proteomic Analyses in an Unsequenced Microalga Michael T...dependent upon available genomic sequence data, and the lack of these data has hindered the pursuit of such analyses for many oleaginous microalgae . In order...to examine the triacylglycerol biosynthetic pathway in the unsequenced oleaginous microalga , Chlorella vulgaris, we have established a strategy with

  4. Detection of additional genes of the patulin biosynthetic pathway in Penicillium griseofulvum

    Science.gov (United States)

    Genes in the patulin biosynthetic pathway are likely to be arranged in a cluster as has been found for biosynthetic pathways of other mycotoxins. The mycotoxin patulin, common in apples and apple juice, is most often associated with Penicillium expansum. However, of 15 fungal species capable of sy...

  5. Cloning and expression of anthocyanin biosynthetic genes in red and white pomegranate.

    Science.gov (United States)

    Zhao, Xueqing; Yuan, Zhaohe; Feng, Lijuan; Fang, Yanming

    2015-07-01

    Exterior fruit color is an important trait for the evaluation of pomegranate fruit quality, but the molecular mechanism underlying the variation in color between red- and white-fruited pomegranate is poorly understood. In this study, full-length cDNA clones encoding enzymes involved in anthocyanin biosynthesis-such as chalcone synthase, chalcone isomerase, flavanone 3-hydoxylase, dihydroflavonol 4-reductase, anthocyanidin synthase (ANS), UDP-glucose-flavonoid 3-O-glucosyltransferase, and the R2R3 MYB transcription factor PgMYB-were isolated from fruit peels. In addition, transcript levels of anthocyanin biosynthetic genes were quantitatively measured by real-time PCR in red and white fruits. In both cultivars, two expression peaks for structural genes were detected during fruit development, whereas only one peak was observed-during early development-for PgMYB. While PgMYB is important for flavonoid biosynthesis, other transcription factors appear to also be necessary for the regulation of anthocyanin biosynthesis. No anthocyanins were detected in the white cultivar. Peels of white fruits contained transcripts of all identified genes except for PgANS, suggesting that the lack of PgANS expression may be the main factor responsible for the absence of anthocyanins in white pomegranate. PgANS may be the key gene involved in anthocyanin biosynthesis in pomegranate fruit.

  6. Spliced X-box binding protein 1 couples the unfolded protein response to hexosamine biosynthetic pathway.

    Science.gov (United States)

    Wang, Zhao V; Deng, Yingfeng; Gao, Ningguo; Pedrozo, Zully; Li, Dan L; Morales, Cyndi R; Criollo, Alfredo; Luo, Xiang; Tan, Wei; Jiang, Nan; Lehrman, Mark A; Rothermel, Beverly A; Lee, Ann-Hwee; Lavandero, Sergio; Mammen, Pradeep P A; Ferdous, Anwarul; Gillette, Thomas G; Scherer, Philipp E; Hill, Joseph A

    2014-03-13

    The hexosamine biosynthetic pathway (HBP) generates uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) for glycan synthesis and O-linked GlcNAc (O-GlcNAc) protein modifications. Despite the established role of the HBP in metabolism and multiple diseases, regulation of the HBP remains largely undefined. Here, we show that spliced X-box binding protein 1 (Xbp1s), the most conserved signal transducer of the unfolded protein response (UPR), is a direct transcriptional activator of the HBP. We demonstrate that the UPR triggers HBP activation via Xbp1s-dependent transcription of genes coding for key, rate-limiting enzymes. We further establish that this previously unrecognized UPR-HBP axis is triggered in a variety of stress conditions. Finally, we demonstrate a physiologic role for the UPR-HBP axis by showing that acute stimulation of Xbp1s in heart by ischemia/reperfusion confers robust cardioprotection in part through induction of the HBP. Collectively, these studies reveal that Xbp1s couples the UPR to the HBP to protect cells under stress. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. BmPAH catalyzes the initial melanin biosynthetic step in Bombyx mori.

    Directory of Open Access Journals (Sweden)

    Ping Chen

    Full Text Available Pigmentation during insect development is a primal adaptive requirement. In the silkworm, melanin is the primary component of larval pigments. The rate limiting substrate in melanin synthesis is tyrosine, which is converted from phenylalanine by the rate-limiting enzyme phenylalanine hydroxylase (PAH. While the role of tyrosine, derived from phenylalanine, in the synthesis of fiber proteins has long been known, the role of PAH in melanin synthesis is still unknown in silkworm. To define the importance of PAH, we cloned the cDNA sequence of BmPAH and expressed its complete coding sequence using the Bac-to-Bac baculovirus expression system. Purified recombinant protein had high PAH activity, some tryptophan hydroxylase activity, but no tyrosine hydroxylase activity, which are typical properties of PAH in invertebrates. Because melanin synthesis is most robust during the embryonic stage and larval integument recoloring stage, we injected BmPAH dsRNA into silkworm eggs and observed that decreasing BmPAH mRNA reduced neonatal larval tyrosine and caused insect coloration to fail. In vitro cultures and injection of 4(th instar larval integuments with PAH inhibitor revealed that PAH activity was essential for larval marking coloration. These data show that BmPAH is necessary for melanin synthesis and we propose that conversion of phenylalanine to tyrosine by PAH is the first step in the melanin biosynthetic pathway in the silkworm.

  8. Calmodulin-mediated suppression of 2-ketoisovalerate reductase in Beauveria bassiana beauvericin biosynthetic pathway.

    Science.gov (United States)

    Kim, Jiyoung; Yoon, Deok-Hyo; Oh, Junsang; Hyun, Min-Woo; Han, Jae-Gu; Sung, Gi-Ho

    2016-11-01

    Ketoisovalerate reductase (KIVR, E.C. 1.2.7.7) mediates the specific reduction of 2-ketoisovalerate (2-Kiv) to d-hydroxyisovalerate (d-Hiv), a precursor for beauvericin biosynthesis. Beauvericin, a famous mycotoxin produced by many fungi, is a cyclooligomer depsipeptide, which has insecticidal, antimicrobial, antiviral, and cytotoxic activities. In this report, we demonstrated that Beauveria bassiana 2-ketoisovalerate reductase (BbKIVR) acts as a typical KIVR enzyme in the entomopathogenic fungus B. bassiana. In addition, we found that BbKIVR interacts with calmodulin (CaM) in vitro and in vivo. The functional role of CaM-binding to BbKIVR was to negatively regulate the BbKIVR activity in B. bassiana. Environmental stimuli such as light and salt stress suppressed BbKIVR activity in B. bassiana. Interestingly, this negative effect of BbKIVR activity by light and salt stress was recovered by CaM inhibitors, suggesting that the inhibitory mechanism is mediated through stimulation of CaM activity. Therefore, this work suggests that BbKIVR plays an important role in the beauvericin biosynthetic pathway mediated by environmental stimuli such as light and salt stress via the CaM signaling pathway.

  9. Engineered Streptomyces avermitilis host for heterologous expression of biosynthetic gene cluster for secondary metabolites.

    Science.gov (United States)

    Komatsu, Mamoru; Komatsu, Kyoko; Koiwai, Hanae; Yamada, Yuuki; Kozone, Ikuko; Izumikawa, Miho; Hashimoto, Junko; Takagi, Motoki; Omura, Satoshi; Shin-ya, Kazuo; Cane, David E; Ikeda, Haruo

    2013-07-19

    An industrial microorganism, Streptomyces avermitilis, which is a producer of anthelmintic macrocyclic lactones, avermectins, has been constructed as a versatile model host for heterologous expression of genes encoding secondary metabolite biosynthesis. Twenty of the entire biosynthetic gene clusters for secondary metabolites were successively cloned and introduced into a versatile model host S. avermitilis SUKA17 or 22. Almost all S. avermitilis transformants carrying the entire gene cluster produced metabolites as a result of the expression of biosynthetic gene clusters introduced. A few transformants were unable to produce metabolites, but their production was restored by the expression of biosynthetic genes using an alternative promoter or the expression of a regulatory gene in the gene cluster that controls the expression of biosynthetic genes in the cluster using an alternative promoter. Production of metabolites in some transformants of the versatile host was higher than that of the original producers, and cryptic biosynthetic gene clusters in the original producer were also expressed in a versatile host.

  10. Limiting Cholesterol Biosynthetic Flux Spontaneously Engages Type I IFN Signaling.

    Science.gov (United States)

    York, Autumn G; Williams, Kevin J; Argus, Joseph P; Zhou, Quan D; Brar, Gurpreet; Vergnes, Laurent; Gray, Elizabeth E; Zhen, Anjie; Wu, Nicholas C; Yamada, Douglas H; Cunningham, Cameron R; Tarling, Elizabeth J; Wilks, Moses Q; Casero, David; Gray, David H; Yu, Amy K; Wang, Eric S; Brooks, David G; Sun, Ren; Kitchen, Scott G; Wu, Ting-Ting; Reue, Karen; Stetson, Daniel B; Bensinger, Steven J

    2015-12-17

    Cellular lipid requirements are achieved through a combination of biosynthesis and import programs. Using isotope tracer analysis, we show that type I interferon (IFN) signaling shifts the balance of these programs by decreasing synthesis and increasing import of cholesterol and long chain fatty acids. Genetically enforcing this metabolic shift in macrophages is sufficient to render mice resistant to viral challenge, demonstrating the importance of reprogramming the balance of these two metabolic pathways in vivo. Unexpectedly, mechanistic studies reveal that limiting flux through the cholesterol biosynthetic pathway spontaneously engages a type I IFN response in a STING-dependent manner. The upregulation of type I IFNs was traced to a decrease in the pool size of synthesized cholesterol and could be inhibited by replenishing cells with free cholesterol. Taken together, these studies delineate a metabolic-inflammatory circuit that links perturbations in cholesterol biosynthesis with activation of innate immunity.

  11. Functional Analysis of the Fusarielin Biosynthetic Gene Cluster

    Directory of Open Access Journals (Sweden)

    Aida Droce

    2016-12-01

    Full Text Available Fusarielins are polyketides with a decalin core produced by various species of Aspergillus and Fusarium. Although the responsible gene cluster has been identified, the biosynthetic pathway remains to be elucidated. In the present study, members of the gene cluster were deleted individually in a Fusarium graminearum strain overexpressing the local transcription factor. The results suggest that a trans-acting enoyl reductase (FSL5 assists the polyketide synthase FSL1 in biosynthesis of a polyketide product, which is released by hydrolysis by a trans-acting thioesterase (FSL2. Deletion of the epimerase (FSL3 resulted in accumulation of an unstable compound, which could be the released product. A novel compound, named prefusarielin, accumulated in the deletion mutant of the cytochrome P450 monooxygenase FSL4. Unlike the known fusarielins from Fusarium, this compound does not contain oxygenized decalin rings, suggesting that FSL4 is responsible for the oxygenation.

  12. Extending the biosynthetic repertoires of cyanobacteria and chloroplasts

    DEFF Research Database (Denmark)

    Nielsen, Agnieszka Janina Zygadlo; Mellor, Silas Busck; Vavitsas, Konstantinos

    2016-01-01

    derived from the photosynthetic light reactions, appears to be non-limiting, but redirection of the fixed carbon via precursor molecules presents a challenge. We also discuss the synthetic biology tools available and the need to expand the molecular toolbox to facilitate cellular reprogramming......The chloroplasts found in plants and algae, and photosynthetic microorganisms such as cyanobacteria, are emerging hosts for sustainable production of valuable biochemicals, using only inorganic nutrients, water, CO2 and light as inputs. In the past decade, many bioengineering efforts have focused...... of chloroplasts and cyanobacteria as biosynthetic compartments and hosts, and we estimate the production levels to be expected from photosynthetic hosts in light of the fraction of electrons and carbon that can potentially be diverted from photosynthesis. The supply of reducing power, in the form of electrons...

  13. Alkaloids from Pandanus amaryllifolius: Isolation and Their Plausible Biosynthetic Formation.

    Science.gov (United States)

    Tsai, Yu-Chi; Yu, Meng-Lun; El-Shazly, Mohamed; Beerhues, Ludger; Cheng, Yuan-Bin; Chen, Lei-Chin; Hwang, Tsong-Long; Chen, Hui-Fen; Chung, Yu-Ming; Hou, Ming-Feng; Wu, Yang-Chang; Chang, Fang-Rong

    2015-10-23

    Pandanus amaryllifolius Roxb. (Pandanaceae) is used as a flavor and in folk medicine in Southeast Asia. The ethanolic crude extract of the aerial parts of P. amaryllifolius exhibited antioxidant, antibiofilm, and anti-inflammatory activities in previous studies. In the current investigation, the purification of the ethanolic extract yielded nine new compounds, including N-acetylnorpandamarilactonines A (1) and B (2); pandalizines A (3) and B (4); pandanmenyamine (5); pandamarilactones 2 (6) and 3 (7), and 5(E)-pandamarilactonine-32 (8); and pandalactonine (9). The isolated alkaloids, with either a γ-alkylidene-α,β-unsaturated-γ-lactone or γ-alkylidene-α,β-unsaturated-γ-lactam system, can be classified into five skeletons including norpandamarilactonine, indolizinone, pandanamine, pandamarilactone, and pandamarilactonine. A plausible biosynthetic route toward 1-5, 7, and 9 is proposed.

  14. Metabolic engineering of biosynthetic pathway for production of renewable biofuels.

    Science.gov (United States)

    Singh, Vijai; Mani, Indra; Chaudhary, Dharmendra Kumar; Dhar, Pawan Kumar

    2014-02-01

    Metabolic engineering is an important area of research that involves editing genetic networks to overproduce a certain substance by the cells. Using a combination of genetic, metabolic, and modeling methods, useful substances have been synthesized in the past at industrial scale and in a cost-effective manner. Currently, metabolic engineering is being used to produce sufficient, economical, and eco-friendly biofuels. In the recent past, a number of efforts have been made towards engineering biosynthetic pathways for large scale and efficient production of biofuels from biomass. Given the adoption of metabolic engineering approaches by the biofuel industry, this paper reviews various approaches towards the production and enhancement of renewable biofuels such as ethanol, butanol, isopropanol, hydrogen, and biodiesel. We have also identified specific areas where more work needs to be done in the future.

  15. Resorbable biosynthetic mesh for crural reinforcement during hiatal hernia repair.

    Science.gov (United States)

    Alicuben, Evan T; Worrell, Stephanie G; DeMeester, Steven R

    2014-10-01

    The use of mesh to reinforce crural closure during hiatal hernia repair is controversial. Although some studies suggest that using synthetic mesh can reduce recurrence, synthetic mesh can erode into the esophagus and in our opinion should be avoided. Studies with absorbable or biologic mesh have not proven to be of benefit for recurrence. The aim of this study was to evaluate the outcome of hiatal hernia repair with modern resorbable biosynthetic mesh in combination with adjunct tension reduction techniques. We retrospectively analyzed all patients who had crural reinforcement during repair of a sliding or paraesophageal hiatal hernia with Gore BioA resorbable mesh. Objective follow-up was by videoesophagram and/or esophagogastroduodenoscopy. There were 114 patients. The majority of operations (72%) were laparoscopic primary repairs with all patients receiving a fundoplication. The crura were closed primarily in all patients and reinforced with a BioA mesh patch. Excessive tension prompted a crural relaxing incision in four per cent and a Collis gastroplasty in 39 per cent of patients. Perioperative morbidity was minor and unrelated to the mesh. Median objective follow-up was one year, but 18 patients have objective follow-up at two or more years. A recurrent hernia was found in one patient (0.9%) three years after repair. The use of crural relaxing incisions and Collis gastroplasty in combination with crural reinforcement with resorbable biosynthetic mesh is associated with a low early hernia recurrence rate and no mesh-related complications. Long-term follow-up will define the role of these techniques for hiatal hernia repair.

  16. Effect of Mentha x piperita essential oil and monoterpenes on cucumber root membrane potential.

    Science.gov (United States)

    Maffei, M; Camusso, W; Sacco, S

    2001-11-01

    Peppermint (Mentha piperita L.) essential oil and its main components were assessed for their ability to interfere with plant plasma membrane potentials. Tests were conducted on root segments isolated from etiolated seedlings of cucumber (Cucumis sativus L.). Increasing the concentration of peppermint essential oil from 5 to 50 ppm caused a decrease in membrane potential (Vm) hyperpolarization of 10-3 mV, whereas concentrations from 100 up to 900 ppm caused an increasing depolarization of Vm (from 5 to 110 mV). When tested at 300 ppm, (+)-menthyl acetate, (-)-limonene and 1,8-cineole did not exert any significant effect on V(m), whereas (+)-menthofuran (73 mV), (+)-pulegone (85 mV), (+)-neomenthol (96 mV), (-)-menthol (105 mV) and (-)-menthone (111 mV) showed increased ability to depolarize V(m). A plot of log of octanol-water partition coefficient (K(ow)) against their depolarizing effect showed a significant negative correlation, suggesting that among all monoterpenoids increased membrane depolarization depends on lower K(ow). However, among monoterpene ketones, alcohols and furans, increased membrane depolarization is associated with a decline in water solubility. The possible effect of monoterpenoids on membrane ion fluxes is also discussed, since changes in the bioelectric potential of cells imply changes in the flux of ions across the plasma membrane

  17. Biochemical and Histochemical Localization of Monoterpene Biosynthesis in the Glandular Trichomes of Spearmint (Mentha spicata) 12

    Science.gov (United States)

    Gershenzon, Jonathan; Maffei, Massimo; Croteau, Rodney

    1989-01-01

    The primary monoterpene accumulated in the glandular trichomes of spearmint (Mentha spicata) is the ketone (−)-carvone which is formed by cyclization of the C10 isoprenoid intermediate geranyl pyrophosphate to the olefin (−)-limonene, hydroxylation to (−)-trans-carveol and subsequent dehydrogenation. Selective extraction of the contents of the glandular trichomes indicated that essentially all of the cyclase and hydroxylase activities resided in these structures, whereas only about 30% of the carveol dehydrogenase was located here with the remainder located in the rest of the leaf. This distribution of carveol dehydrogenase activity was confirmed by histochemical methods. Electrophoretic analysis of the partially purified carveol dehydrogenase from extracts of both the glands and the leaves following gland removal indicated the presence of a unique carveol dehydrogenase species in the glandular trichomes, suggesting that the other dehydrogenase found throughout the leaf probably utilizes carveol only as an adventitious substrate. These results demonstrate that carvone biosynthesis takes place exclusively in the glandular trichomes in which this natural product accumulates. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:16666709

  18. Biochemical and histochemical localization of monoterpene biosynthesis in the glandular trichomes of spearmint (Mentha spicata)

    Energy Technology Data Exchange (ETDEWEB)

    Gershenzon, J.; Maffei, M.; Croteau, R. (Washington State Univ., Pullman (USA))

    1989-04-01

    The primary monoterpene accumulated in the glandular trichomes of spearmint (Mentha spicata) is the ketone (-)-carvone which is formed by cyclization of the C{sub 10} isoprenoid intermediate geranyl pyrophosphate to the olefin (-)-limonene, hydroxylation to (-)-trans-carveol and subsequent dehydrogenation. Selective extraction of the contents of the glandular trichomes indicated that essentially all of the cyclase and hydroxylase activities resided in these structures, whereas only about 30% of the carveol dehydrogenase was located here with the remainder located in the rest of the leaf. This distribution of carveol dehydrogenase activity was confirmed by histochemical methods. Electrophoretic analysis of the partially purified carveol dehydrogenase from extracts of both the glands and the leaves following gland removal indicated the presence of a unique carveol dehydrogenase species in the glandular trichomes, suggesting that the other dehydrogenase found throughout the leaf probably utilizes carveol only as an adventitious substrate. These results demonstrate that carvone biosynthesis takes place exclusively in the glandular trichomes in which this natural product accumulates.

  19. Antihypertensive effect of auraptene, a monoterpene coumarin from the genus Citrus, upon chronic administration

    Directory of Open Access Journals (Sweden)

    Bibi Marjan Razavi

    2015-02-01

    Full Text Available Objective(s: Auraptene, a monoterpene coumarin from Citrus species, exhibits cardioprotective effects.In this study, the effects of auraptene administration were investigated on blood pressure of normotensive and desoxycorticosterone acetate (DOCA salt induced hypertensive rats. Materials and Methods: Five weeks administration of auraptene (2, 4, 8 and 16 mg/kg/day and nifedipine (0.25, 0.5, 1, 2 and 4 mg/kg/day in different groups of normotensive and hypertensive rats (at the end of 3 weeks treatment by DOCA salt was carried out and their effects on mean systolic blood pressure (MSBP and mean heart rate (MHR were evaluated using tail cuff method. Results: Our results indicated that chronic administration of auraptene (2, 4, 8 and 16 mg/kg/day significantly reduced the MSBP in DOCA salt treated rats in a dose and time dependent manner. The percent of decreases in MSBP levels by the highest dose of auraptene (16 mg/kg at the end of 4 th to 8 th weeks, were 7.00%, 10.78%, 16.07%, 21.28% and 27.54% respectively(P

  20. Induction of allergic contact dermatitis by astigmatid mite-derived monoterpene, alpha-acaridial.

    Science.gov (United States)

    Sasai, Toshio; Hirano, Yunosuke; Maeda, Sayaka; Matsunaga, Isamu; Otsuka, Atsushi; Morita, Daisuke; Nishida, Ritsuo; Nakayama, Hideo; Kuwahara, Yasumasa; Sugita, Masahiko; Mori, Naoki

    2008-10-24

    alpha-Acaridial [2(E)-(4-methyl-3-pentenyl)butenedial] is a novel monoterpene secreted from the house dust mites. Because of its molecular nature of a highly reactive, small lipidic compound, we addressed whether alpha-acaridial might function as a haptenic allergen that induced allergic contact dermatitis. Mice sensitized with alpha-acaridial were challenged by the same antigen on the ear skin. After 2 days, significant ear swelling with a prominent infiltration of CD4(+) T lymphocytes was observed. In vitro, alpha-acaridial exhibited an outstanding ability to quickly interact with and chemically modify a reference protein. Virtually all cysteine residues and a sizable fraction of lysine residues were found to be selectively modified, suggesting that alpha-acaridial could potentially interact with any proteins. Previously, numerous mite-derived proteinaceous allergens have been associated with contact dermatitis. Our study now emphasizes that small lipidic compounds released from mites comprise a new class of mite allergens, and therefore, is of significant medical implications.

  1. Borneol, a Bicyclic Monoterpene Alcohol, Reduces Nociceptive Behavior and Inflammatory Response in Mice

    Directory of Open Access Journals (Sweden)

    Jackson Roberto Guedes da Silva Almeida

    2013-01-01

    Full Text Available Borneol, a bicyclic monoterpene, has been evaluated for antinociceptive and anti-inflammatory activities. Antinociceptive and anti-inflammatory activities were studied by measuring nociception by acetic acid, formalin, hot plate, and grip strength tests, while inflammation was prompted by carrageenan-induced peritonitis. The rotarod test was used to evaluate motor coordination. Borneol produced a significant (P<0.01 reduction of the nociceptive behavior at the early and late phases of paw licking and reduced the writhing reflex in mice (formalin and writhing tests, resp.. When the hot plate test was conducted, borneol (in higher dose produced an inhibition (P<0.05 of the nociceptive behavior. Such results were unlikely to be provoked by motor abnormality. Additionally, borneol-treated mice reduced the carrageenan-induced leukocytes migration to the peritoneal cavity. Together, our results suggest that borneol possess significant central and peripheral antinociceptive activity; it has also anti-inflammatory activity. In addition, borneol did not impair motor coordination.

  2. Optically active conjugated polymer from solvent chirality transfer polymerization in monoterpenes.

    Science.gov (United States)

    Kim, Hyojin; Lee, Daehoon; Lee, Seul; Suzuki, Nozomu; Fujiki, Michiya; Lee, Chang-Lyoul; Kwak, Giseop

    2013-09-01

    Disubstituted acetylene monomers [1,2-diphenylacetylenes (DPAs: DPA-pC1, DPA-mC1, DPA-pC8); 1-phenyl-2-hexylacetylene (PHA-pC1)] are tested for asymmetric polymerization in chiral monoterpenes used as solvents and compared with the corresponding monosubstituted acetylene monomer [1-phenylacetylene (PA-pC1)]. DPA-pC1 containing a trimethylsilyl group in the para-position of the phenyl ring produces an optically active polymer with a large Cotton effect, despite the absence of a stereogenic center. The polymer sample obtained by polymerization in 87% ee (-)-α-pinene shows the strongest CD signal (gCD value at 385 nm: ∼3.2 × 10⁻³). The Cotton bands of the polymers obtained in (-)- and (+)-α-pinenes show the opposite sign in the CD signals. Theoretical calculations show that only the cis-cisoid model adopts a helical conformation. A time-correlated single photon counting experiment shows that the emission of the chiral polymer originates from a virtually single excited species with a 98% component fraction. This polymer solution does not show any significant decrease in gCD value over a wide temperature range of 20 to 80 °C. No noticeable decrease in the gCD value is detected when the polymer solution is kept at relatively low temperatures for a prolonged period (35 d). In contrast, the other polymers show no CD signal.

  3. Active monoterpene ketones isolated from Rosmarinus officinalis with fumigant and contact action against Tyrophagus putrescentiae (Schrank).

    Science.gov (United States)

    Jeon, Ju-Hyun; Park, Jun-Hwan; Chung, Namhyun; Lee, Hoi-Seon

    2014-08-01

    The acaricidal activities of an active material derived from Rosmarinus officinalis oil and its relative monoterpene ketones were determined using fumigant and contact toxicity bioassays against Tyrophagus putrescentiae and were compared with that of a commercial acaricide (benzyl benzoate). The active component of R. officinalis oil, isolated by silica gel column chromatography and high-performance liquid chromatography, was identified as camphor, based on various spectroscopic analyses. In the fumigant toxicity bioassay, camphor (2.25 μg/cm(3)) was 5.58 times more active than benzyl benzoate (12.56 μg/cm(3)) against T. putrescentiae, followed by (+)-camphor (3.89 μg/cm(3)) and (-)-camphor (5.61 μg/cm(3)). In the contact toxicity bioassay, camphor (1.34 μg/cm(2)) was 6.74 times more toxic than benzyl benzoate (9.03 μg/cm(2)) against T. putrescentiae, followed by (+)-camphor (2.23 μg/cm(2)) and (-)-camphor (2.94 μg/cm(2)). These results indicate that camphor and its derivatives are very useful as potential control agents against stored food mites regardless of the application method.

  4. Inhibition of N-nitrosodiethylamine carcinogenesis in mice by naturally occurring organosulfur compounds and monoterpenes.

    Science.gov (United States)

    Wattenberg, L W; Sparnins, V L; Barany, G

    1989-05-15

    Naturally occurring compounds belonging to two chemical groups were studied for their capacities to inhibit N-nitrosodiethylamine (NDEA)-induced carcinogenesis in female A/J mice. One group consists of organosulfur compounds found in Allium species, including garlic, onions, leeks, and shallots, and the other, two monoterpenes, i.e., D-limonene and D-carvone. In an initial experiment, in which organosulfur compounds were investigated, diallyl disulfide, allyl mercaptan, and allyl methyl disulfide were found to produce a marked inhibition of NDEA-induced neoplasia of the forestomach when the test compounds were administered p.o. 96 and 48 h prior to NDEA. The most potent was diallyl disulfide which reduced forestomach tumor formation by more than 90%. Pulmonary adenoma formation also was inhibited but to a considerably lesser extent, i.e., about 30%. In three additional experiments, test compounds were given p.o. either 15 min or 1 h prior to NDEA. Under these conditions diallyl disulfide and allyl mercaptan again inhibited forestomach tumor formation substantially, i.e., greater than 75%, and pulmonary adenoma formation marginally, i.e., less than 20%. In these experiments D-limonene and D-carvone were tested and reduced forestomach tumor formation by slightly over 60% and pulmonary adenoma formation by about 35%. The results of these studies provide evidence of an increasing diversity of naturally occurring compounds having the capacity to inhibit nitrosamine carcinogenesis.

  5. Phylogenomics of the benzoxazinoid biosynthetic pathway of Poaceae: gene duplications and origin of the Bx cluster

    Directory of Open Access Journals (Sweden)

    Dutartre Leslie

    2012-05-01

    Full Text Available Abstract Background The benzoxazinoids 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA and 2,4-dihydroxy-7- methoxy-1,4-benzoxazin-3-one (DIMBOA, are key defense compounds present in major agricultural crops such as maize and wheat. Their biosynthesis involves nine enzymes thought to form a linear pathway leading to the storage of DI(MBOA as glucoside conjugates. Seven of the genes (Bx1-Bx6 and Bx8 form a cluster at the tip of the short arm of maize chromosome 4 that includes four P450 genes (Bx2-5 belonging to the same CYP71C subfamily. The origin of this cluster is unknown. Results We show that the pathway appeared following several duplications of the TSA gene (α-subunit of tryptophan synthase and of a Bx2-like ancestral CYP71C gene and the recruitment of Bx8 before the radiation of Poaceae. The origins of Bx6 and Bx7 remain unclear. We demonstrate that the Bx2-like CYP71C ancestor was not committed to the benzoxazinoid pathway and that after duplications the Bx2-Bx5 genes were under positive selection on a few sites and underwent functional divergence, leading to the current specific biochemical properties of the enzymes. The absence of synteny between available Poaceae genomes involving the Bx gene regions is in contrast with the conserved synteny in the TSA gene region. Conclusions These results demonstrate that rearrangements following duplications of an IGL/TSA gene and of a CYP71C gene probably resulted in the clustering of the new copies (Bx1 and Bx2 at the tip of a chromosome in an ancestor of grasses. Clustering favored cosegregation and tip chromosomal location favored gene rearrangements that allowed the further recruitment of genes to the pathway. These events, a founding event and elongation events, may have been the key to the subsequent evolution of the benzoxazinoid biosynthetic cluster.

  6. Phylogenomics of the benzoxazinoid biosynthetic pathway of Poaceae: gene duplications and origin of the Bx cluster.

    Science.gov (United States)

    Dutartre, Leslie; Hilliou, Frédérique; Feyereisen, René

    2012-05-11

    The benzoxazinoids 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and 2,4-dihydroxy-7- methoxy-1,4-benzoxazin-3-one (DIMBOA), are key defense compounds present in major agricultural crops such as maize and wheat. Their biosynthesis involves nine enzymes thought to form a linear pathway leading to the storage of DI(M)BOA as glucoside conjugates. Seven of the genes (Bx1-Bx6 and Bx8) form a cluster at the tip of the short arm of maize chromosome 4 that includes four P450 genes (Bx2-5) belonging to the same CYP71C subfamily. The origin of this cluster is unknown. We show that the pathway appeared following several duplications of the TSA gene (α-subunit of tryptophan synthase) and of a Bx2-like ancestral CYP71C gene and the recruitment of Bx8 before the radiation of Poaceae. The origins of Bx6 and Bx7 remain unclear. We demonstrate that the Bx2-like CYP71C ancestor was not committed to the benzoxazinoid pathway and that after duplications the Bx2-Bx5 genes were under positive selection on a few sites and underwent functional divergence, leading to the current specific biochemical properties of the enzymes. The absence of synteny between available Poaceae genomes involving the Bx gene regions is in contrast with the conserved synteny in the TSA gene region. These results demonstrate that rearrangements following duplications of an IGL/TSA gene and of a CYP71C gene probably resulted in the clustering of the new copies (Bx1 and Bx2) at the tip of a chromosome in an ancestor of grasses. Clustering favored cosegregation and tip chromosomal location favored gene rearrangements that allowed the further recruitment of genes to the pathway. These events, a founding event and elongation events, may have been the key to the subsequent evolution of the benzoxazinoid biosynthetic cluster.

  7. Engineering the leucine biosynthetic pathway for isoamyl alcohol overproduction in Saccharomyces cerevisiae.

    Science.gov (United States)

    Yuan, Jifeng; Mishra, Pranjul; Ching, Chi Bun

    2017-01-01

    Isoamyl alcohol can be used not only as a biofuel, but also as a precursor for various chemicals. Saccharomyces cerevisiae inherently produces a small amount of isoamyl alcohol via the leucine degradation pathway, but the yield is very low. In the current study, several strategies were devised to overproduce isoamyl alcohol in budding yeast. The engineered yeast cells with the cytosolic isoamyl alcohol biosynthetic pathway produced significantly higher amounts of isobutanol over isoamyl alcohol, suggesting that the majority of the metabolic flux was diverted to the isobutanol biosynthesis due to the broad substrate specificity of Ehrlich pathway enzymes. To channel the key intermediate 2-ketosiovalerate (KIV) towards α-IPM biosynthesis, we introduced an artificial protein scaffold to pull dihydroxyacid dehydratase and α-IPM synthase into the close proximity, and the resulting strain yielded more than twofold improvement of isoamyl alcohol. The best isoamyl alcohol producer yielded 522.76 ± 38.88 mg/L isoamyl alcohol, together with 540.30 ± 48.26 mg/L isobutanol and 82.56 ± 8.22 mg/L 2-methyl-1-butanol. To our best knowledge, our work represents the first study to bypass the native compartmentalized α-IPM biosynthesis pathway for the isoamyl alcohol overproduction in budding yeast. More importantly, artificial protein scaffold based on the feature of quaternary structure of enzymes would be useful in improving the catalytic efficiency and the product specificity of other enzymatic reactions.

  8. Evolutionary origins and functions of the carotenoid biosynthetic pathway in marine diatoms.

    Directory of Open Access Journals (Sweden)

    Sacha Coesel

    Full Text Available Carotenoids are produced by all photosynthetic organisms, where they play essential roles in light harvesting and photoprotection. The carotenoid biosynthetic pathway of diatoms is largely unstudied, but is of particular interest because these organisms have a very different evolutionary history with respect to the Plantae and are thought to be derived from an ancient secondary endosymbiosis between heterotrophic and autotrophic eukaryotes. Furthermore, diatoms have an additional xanthophyll-based cycle for dissipating excess light energy with respect to green algae and higher plants. To explore the origins and functions of the carotenoid pathway in diatoms we searched for genes encoding pathway components in the recently completed genome sequences of two marine diatoms. Consistent with the supplemental xanthophyll cycle in diatoms, we found more copies of the genes encoding violaxanthin de-epoxidase (VDE and zeaxanthin epoxidase (ZEP enzymes compared with other photosynthetic eukaryotes. However, the similarity of these enzymes with those of higher plants indicates that they had very probably diversified before the secondary endosymbiosis had occurred, implying that VDE and ZEP represent early eukaryotic innovations in the Plantae. Consequently, the diatom chromist lineage likely obtained all paralogues of ZEP and VDE genes during the process of secondary endosymbiosis by gene transfer from the nucleus of the algal endosymbiont to the host nucleus. Furthermore, the presence of a ZEP gene in Tetrahymena thermophila provides the first evidence for a secondary plastid gene encoded in a heterotrophic ciliate, providing support for the chromalveolate hypothesis. Protein domain structures and expression analyses in the pennate diatom Phaeodactylum tricornutum indicate diverse roles for the different ZEP and VDE isoforms and demonstrate that they are differentially regulated by light. These studies therefore reveal the ancient origins of several

  9. Supercritical CO2 Extraction of Lavandula angustifolia Mill. Flowers: Optimisation of Oxygenated Monoterpenes, Coumarin and Herniarin Content.

    Science.gov (United States)

    Jerković, Igor; Molnar, Maja; Vidović, Senka; Vladić, Jelena; Jokić, Stela

    2017-07-14

    Lavandula angustifolia is good source of oxygenated monoterpenes containing coumarins as well, which are all soluble in supercritical CO2 (SC-CO2 ). The study objective is to investigate SC-CO2 extraction parameters on: the total yield; GC-MS profile of the extracts; relative content of oxygenated monoterpenes; the amount of coumarin and herniarin; and to determine optimal SC-CO2 extraction conditions by response surface methodology (RSM). SC-CO2 extraction was performed under different pressure, temperature and CO2 flow rate determined by Box-Behnken design (BBD). The sample mass and the extraction time were kept constant. The chemical profiles and relative content of oxygenated monoterpenes (as coumarin equivalents, CE) were determined by GC-MS. Coumarin and herniarin concentrations were dosed by HPLC. SC-CO2 extracts contained linalool (57.4-217.9 mg CE/100 g), camphor (10.6-154.4 mg CE/100 g), borneol (6.2-99.9 mg CE/100 g), 1,8-cineole (5.0-70.4 mg CE/100 g), linalyl acetate (86.1-267.9 mg CE/100 g), coumarin (0.95-18.16 mg/100 g), and herniarin (0.95-13.63 mg/100 g). The interaction between the pressure and CO2 flow rate as well as between the temperature and CO2 flow rate showed statistically significant influence on the extraction yield. Applying BBD, the optimum extraction conditions for higher monoterpenes and lower coumarin content were at 10 MPa, 41°C and CO2 flow rate 2.3 kg/h, and at 30 MPa, 50°C and CO2 flow rate 3 kg/h for higher monoterpenes and coumarin content. SC-CO2 extraction is a viable technique for obtaining lavender extracts with desirable flavour components. The second-order model based on BBD predicts the results for SC-CO2 extraction quite satisfactorily. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  10. How have both cultivation and warming influenced annual global isoprene and monoterpene emissions since the preindustrial era?

    Directory of Open Access Journals (Sweden)

    K. Tanaka

    2012-10-01

    Full Text Available To examine the influence of both crop cultivation and surface air temperatures (SATs on annual global isoprene and monoterpene emissions, which can lead to the formation of secondary organic aerosols (SOAs, we simulated, on a monthly basis, the annual emissions of volatile organic compounds (VOCs during the period 1854–2000. The model estimates were based on historical climate data such as SATs, and downward solar radiation (DSR reproduced with an atmospheric-ocean circulation model, as well as a time series of the global distribution of cropland (to test the hypothesis that conversion of forests into croplands lowers emissions. The simulations demonstrated that global SAT, DSR, the combination of SAT and DSR, and the expansion of cropland all affected emissions. The effect of cropland expansion (i.e., forest conversion on annual emissions during this period was larger for isoprene (~7% reduction on a global scale than for monoterpenes (~2% reduction, mainly because of the reduction in broadleaf evergreen forests (BEFs in Southeast Asia, which have the highest and most constant emissions of isoprene and where both temperature and radiation are high all year round. The reduction in the Amazon region and in parts of Africa, which are other primary sources of annual global isoprene emissions, but where the conversion of BEF to cropland has been much smaller than in Southeast Asia, was less remarkable, probably because the broadleaf deciduous forests and C4 grasslands in these areas have lower and seasonal emissions; hence, their conversion has less effect. On the other hand, the difference in the emission factors (ε between cropland and the other vegetation types was much lower for monoterpenes than for isoprene, although the ε for cropland was generally the lowest for both compounds. Thus, the expansion of cropland also contributed to the reduction in monoterpene emissions to some degree, but had less effect. A ~5% increase in emissions due to

  11. Engineering the central biosynthetic and secondary metabolic pathways of Pseudomonas aeruginosa strain PA1201 to improve phenazine-1-carboxylic acid production.

    Science.gov (United States)

    Jin, Kaiming; Zhou, Lian; Jiang, Haixia; Sun, Shuang; Fang, Yunling; Liu, Jianhua; Zhang, Xuehong; He, Ya-Wen

    2015-11-01

    The secondary metabolite phenazine-1-carboxylic acid (PCA) is an important component of the newly registered biopesticide Shenqinmycin. We used a combined method involving gene, promoter, and protein engineering to modify the central biosynthetic and secondary metabolic pathways in the PCA-producing Pseudomonas aeruginosa strain PA1201. The PCA yield of the resulting strain PA-IV was increased 54.6-fold via the following strategies: (1) blocking PCA conversion and enhancing PCA efflux pumping; (2) increasing metabolic flux towards the PCA biosynthetic pathway through the over-production of two DAHP synthases and blocking the synthesis of 21 secondary metabolites; (3) increasing the PCA precursor supply through the engineering of five chorismate-utilizing enzymes; (4) engineering the promoters of two PCA biosynthetic gene clusters. Strain PA-IV produced 9882 mg/L PCA in fed-batch fermentation, which is twice as much as that produced by the current industrial strain. Strain PA-IV was also genetically stable and comparable to Escherichia coli in cytotoxicity.

  12. Food Enzymes

    Science.gov (United States)

    McBroom, Rachel; Oliver-Hoyo, Maria T.

    2007-01-01

    Many students view biology and chemistry as two unrelated, separate sciences; how these courses are generally taught in high schools may do little to change that impression. The study of enzymes provide a great opportunity for both biology and chemistry teachers to share with students the interdisciplinary nature of science. This article describes…

  13. Food Enzymes

    Science.gov (United States)

    McBroom, Rachel; Oliver-Hoyo, Maria T.

    2007-01-01

    Many students view biology and chemistry as two unrelated, separate sciences; how these courses are generally taught in high schools may do little to change that impression. The study of enzymes provide a great opportunity for both biology and chemistry teachers to share with students the interdisciplinary nature of science. This article describes…

  14. Enzyme immunoassay

    DEFF Research Database (Denmark)

    Feldt-Rasmussen, B; Dinesen, B; Deckert, M

    1985-01-01

    An enzyme linked immunoadsorbent assay for urinary albumin using commercially available reagents is described. The assay range is 2.5-120 micrograms/l. When samples are analysed in two standard dilutions, the assayable albumin concentration range is 2.5-240 mg/l, covering the clinical range from...

  15. Biotechnological potential of insect fatty acid-modifying enzymes.

    Science.gov (United States)

    Tupec, Michal; Buček, Aleš; Valterová, Irena; Pichová, Iva

    2017-07-25

    There are more than one million described insect species. This species richness is reflected in the diversity of insect metabolic processes. In particular, biosynthesis of secondary metabolites, such as defensive compounds and chemical signals, encompasses an extraordinarily wide range of chemicals that are generally unparalleled among natural products from other organisms. Insect genomes, transcriptomes and proteomes thus offer a valuable resource for discovery of novel enzymes with potential for biotechnological applications. Here, we focus on fatty acid (FA) metabolism-related enzymes, notably the fatty acyl desaturases and fatty acyl reductases involved in the biosynthesis of FA-derived pheromones. Research on insect pheromone-biosynthetic enzymes, which exhibit diverse enzymatic properties, has the potential to broaden the understanding of enzyme specificity determinants and contribute to engineering of enzymes with desired properties for biotechnological production of FA derivatives. Additionally, the application of such pheromone-biosynthetic enzymes represents an environmentally friendly and economic alternative to the chemical synthesis of pheromones that are used in insect pest management strategies.

  16. The Halloween genes code for cytochrome P450 enzymes mediating synthesis of the insect molting hormone

    DEFF Research Database (Denmark)

    Rewitz, Kim; Rybczynski, Robert; Warren, James T.;

    2006-01-01

    during the fifth instar. Transcript levels of shd in the fat body and midgut closely parallel the enzyme activity measured in vitro. The data indicate that these Halloween genes are transcriptionally regulated to support the high biosynthetic activity that produces the cyclic ecdysteroid pulses...

  17. Significant light and temperature dependent monoterpene emissions from European beech (Fagus sylvatica L.) and their potential impact on the European volatile organic compound budget

    Science.gov (United States)

    Dindorf, T.; Kuhn, U.; Ganzeveld, L.; Schebeske, G.; Ciccioli, P.; Holzke, C.; KöBle, R.; Seufert, G.; Kesselmeier, J.

    2006-08-01

    By using a dynamic branch enclosure system the emission of monoterpenes from European beech (Fagus sylvatica L.) was investigated during two consecutive summer vegetation periods in the years of 2002 and 2003 in Germany. All measurements were performed under field conditions within the framework of the ECHO project (Emission and Chemical Transformation of Biogenic Volatile Organic Compounds, AFO 2000). European beech was characterized as a substantial emitter of monoterpenes, with sabinene being the predominant compound released. The monoterpene emission from European beech was shown to be a function of light and temperature and agreed well to emission algorithms that consider a light and temperature dependent release of volatile organics. Standard emission factors that were measured from these sunlit leaves of European beech ranged up to 4-13 μg g-1 h-1 (normalized to 1000 μmol m-2 s-1, 30°C) in the years of 2003 and 2002, respectively. The nighttime emission of monoterpene compounds was negligible. Also the artificial darkening of the sunlit branch during daylight conditions led to an immediate cessation of monoterpene emission. European beech is the dominating deciduous tree species in Europe. To demonstrate the effect of an updated monoterpene emission factor for European beech in combination with the consideration of a light and temperature dependent monoterpene emission, we applied a species based model simulation on a European scale. With respect to conventional estimates of the European volatile organic compound budget, the latter simulation resulted in relative increases of 16% by taking solely this tree species into account. On local scales these increases exceeded even more than 100% depending on the respective vegetation area coverage of European beech.

  18. Cloning and characterization of the gene encoding β-amyrin synthase in the glycyrrhizic acid biosynthetic pathway in Glycyrrhiza uralensis

    Directory of Open Access Journals (Sweden)

    Honghao Chen

    2013-12-01

    Full Text Available Glycyrrhiza uralensis is considered to be one of the most important herbs in traditional Chinese medicine due to its numerous pharmacological effects particularly its ability to relieve cough and act as a mucolytic. Based on previous research, these effects are mediated by a number of active ingredients, especially glycyrrhizic acid (GA. In the present study, a gene encoding β-amyrin synthase (β-AS involved in GA biosynthesis in G. uralensis has been cloned and expressed in Saccharomyces cerevisiae. The cloned enzyme showed similar activity to native enzymes isolated from other Glycyrrhiza species to catalyze the conversion of 2,3-oxidosqualene into β-amyrin. In fact the β-AS gene is particularly important in the GA biosynthetic pathway in G. uralensis. The complete sequence of the enzyme was determined and a phylogenetic tree based on the β-AS gene of G. uralensis and 20 other species was created. This showed that Glycyrrhiza glabra had the closest kinship with G. uralensis. The results of this work will be useful in determining how to improve the efficacy of G. uralensis by improving its GA content and in exploring the biosynthesis of GA in vitro.

  19. A retro-biosynthetic approach to the prediction of biosynthetic pathways from position-specific isotope analysis as shown for tramadol

    Science.gov (United States)

    Romek, Katarzyna M.; Nun, Pierrick; Remaud, Gérald S.; Silvestre, Virginie; Taïwe, Germain Sotoing; Lecerf-Schmidt, Florine; Boumendjel, Ahcène; De Waard, Michel; Robins, Richard J.

    2015-01-01

    Tramadol, previously only known as a synthetic analgesic, has now been found in the bark and wood of roots of the African medicinal tree Nauclea latifolia. At present, no direct evidence is available as to the biosynthetic pathway of its unusual skeleton. To provide guidance as to possible biosynthetic precursors, we have adopted a novel approach of retro-biosynthesis based on the position-specific distribution of isotopes in the extracted compound. Relatively recent developments in isotope ratio monitoring by 13C NMR spectrometry make possible the measurement of the nonstatistical position-specific natural abundance distribution of 13C (δ13Ci) within the molecule with better than 1‰ precision. Very substantial variation in the 13C positional distribution is found: between δ13Ci = −11 and −53‰. Distribution is not random and it is argued that the pattern observed can substantially be interpreted in relation to known causes of isotope fractionation in natural products. Thus, a plausible biosynthetic scheme based on sound biosynthetic principals of precursor–substrate relationships can be proposed. In addition, data obtained from the 18O/16O ratios in the oxygen atoms of the compound add support to the deductions made from the carbon isotope analysis. This paper shows how the use of 13C NMR at natural abundance can help with proposing a biosynthetic route to compounds newly found in nature or those difficult to tackle by conventional means. PMID:26106160

  20. β-Citronellol, an alcoholic monoterpene with inhibitory properties on the contractility of rat trachea

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    T.B. Vasconcelos

    2016-01-01

    Full Text Available β-Citronellol is an alcoholic monoterpene found in essential oils such Cymbopogon citratus (a plant with antihypertensive properties. β-Citronellol can act against pathogenic microorganisms that affect airways and, in virtue of the popular use of β-citronellol-enriched essential oils in aromatherapy, we assessed its pharmacologic effects on the contractility of rat trachea. Contractions of isolated tracheal rings were recorded isometrically through a force transducer connected to a data-acquisition device. β-Citronellol relaxed sustained contractions induced by acetylcholine or high extracellular potassium, but half-maximal inhibitory concentrations (IC50 for K+-elicited stimuli were smaller than those for cholinergic contractions. It also inhibited contractions induced by electrical field stimulation or sodium orthovanadate with pharmacologic potency equivalent to that seen against acetylcholine-induced contractions. When contractions were evoked by selective recruitment of Ca2+ from the extracellular medium, β-citronellol preferentially inhibited contractions that involved voltage-operated (but not receptor-operated pathways. β-Citronellol (but not verapamil inhibited contractions induced by restoration of external Ca2+ levels after depleting internal Ca2+ stores with the concomitant presence of thapsigargin and recurrent challenge with acetylcholine. Treatment of tracheal rings with L-NAME, indomethacin or tetraethylammonium did not change the relaxing effects of β-citronellol. Inhibition of transient receptor potential vanilloid subtype 1 (TRPV1 or transient receptor potential ankyrin 1 (TRPA1 receptors with selective antagonists caused no change in the effects of β-citronellol. In conclusion, β-citronellol exerted inhibitory effects on rat tracheal rings, with predominant effects on contractions that recruit Ca2+ inflow towards the cytosol by voltage-gated pathways, whereas it appears less active against contractions elicited by

  1. Antihypertensive effect of auraptene, a monoterpene coumarin from the genus Citrus, upon chronic administration

    Science.gov (United States)

    Razavi, Bibi Marjan; Arasteh, Ebrahim; Imenshahidi, Mohsen; Iranshahi, Mehrdad

    2015-01-01

    Objective(s): Auraptene, a monoterpene coumarin from Citrus species, exhibits cardioprotective effects. In this study, the effects of auraptene administration were investigated on blood pressure of normotensive and desoxycorticosterone acetate (DOCA) salt induced hypertensive rats. Materials and Methods: Five weeks administration of auraptene (2, 4, 8 and 16 mg/kg/day) and nifedipine (0.25, 0.5, 1, 2 and 4 mg/kg/day) in different groups of normotensive and hypertensive rats (at the end of 3 weeks treatment by DOCA salt) was carried out and their effects on mean systolic blood pressure (MSBP) and mean heart rate (MHR) were evaluated using tail cuff method. Results: Our results indicated that chronic administration of auraptene (2, 4, 8 and 16 mg/kg/day) significantly reduced the MSBP in DOCA salt treated rats in a dose and time dependent manner. The percent of decreases in MSBP levels by the highest dose of auraptene (16 mg/kg) at the end of 4 th to 8 th weeks, were 7.00%, 10.78%, 16.07%, 21.28% and 27.54% respectively (P<0.001). Moreover the antihypertensive effect of auraptene was less than nifedipine (ED50 value of nifedipine = 0.7 mg/kg at 8th week and ED50 value of auraptene = 5.64 mg/kg at 8 week). Conclusion: Auraptene considerably reduced MSBP in hypertensive rats, but not in normotensive (normal saline treated) rats. The results of MHR measurement showed that the increase in MHR was not significant in comparison with DOCA treated rats. PMID:25810889

  2. Structurally Related Monoterpenes p-Cymene, Carvacrol and Thymol Isolated from Essential Oil from Leaves of Lippia sidoides Cham. (Verbenaceae Protect Mice against Elastase-Induced Emphysema

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    Ellen Games

    2016-10-01

    Full Text Available Background: Chronic obstructive pulmonary disease (COPD is characterized by irreversible airflow obstruction and inflammation. Natural products, such as monoterpenes, displayed anti-inflammatory and anti-oxidant activities and can be used as a source of new compounds to COPD treatment. Our aim was to evaluate, in an elastase-induced pulmonary emphysema in mice, the effects of and underlying mechanisms of three related natural monoterpenes (p-cymene, carvacrol and thymol isolated from essential oil from leaves Lippia sidoides Cham. (Verbenaceae. Methods: Mices received porcine pancreatic elastase (PPE and were treated with p-cymene, carvacrol, thymol or vehicle 30 min later and again on 7th, 14th and 28th days. Lung inflammatory profile and histological sections were evaluated. Results: In the elastase-instilled animals, the tested monoterpenes reduced alveolar enlargement, macrophages and the levels of IL-1β, IL-6, IL-8 and IL-17 in bronchoalveolar lavage fluid (BALF, and collagen fibers, MMP-9 and p-65-NF-κB-positive cells in lung parenchyma (p < 0.05. All treatments attenuated levels of 8-iso-PGF2α but only thymol was able to reduced exhaled nitric oxide (p < 0.05. Conclusion: Monoterpenes p-cymene, carvacrol and thymol reduced lung emphysema and inflammation in mice. No significant differences among the three monoterpenes treatments were found, suggesting that the presence of hydroxyl group in the molecular structure of thymol and carvacrol do not play a central role in the anti-inflammatory effects.

  3. Molecular cloning and expression levels of the monoterpene synthase gene (ZMM1 in Cassumunar ginger (Zingiber montanum (Koenig Link ex Dietr.

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    Bua-In Saowaluck

    2014-01-01

    Full Text Available Cassumunar ginger (Zingiber montanum (Koenig Link ex Dietr. is a native Thai herb with a high content and large variety of terpenoids in its essential oil. Improving the essential oil content and quality of cassumunar ginger is difficult for a breeder due to its clonally propagated nature. In this research, we describe the isolation and expression level of the monoterpene synthase gene that controls the key step of essential oil synthesis in this plant and evaluate the mechanical wounding that may influence the transcription level of the monoterpene synthase gene. To isolate the gene, the selected clones from DNA derived from young leaves were sequenced and analyzed and the monoterpene synthase gene from cassumunar ginger (ZMM1 was identified. The ZMM1 CDS containing 1 773 bp (KF500399 is predicted to encode a protein of 590 amino acids. The deduced amino acid sequence is 40-74% identical with known sequences of other angiosperm monoterpene synthases belonging to the isoprenoid biosynthesis C1 superfamily. A transcript of ZMM1 was detected almost exclusively in the leaves and was related to leaf wounding. The results of this research offer insight into the control of monoterpene synthesis in this plant. This finding can be applied to breeding programs or crop management of cassumunar ginger for better yield and quality of essential oil.

  4. Sequence diversity and differential expression of major phenylpropanoid-flavonoid biosynthetic genes among three mango varieties.

    Science.gov (United States)

    Hoang, Van L T; Innes, David J; Shaw, P Nicholas; Monteith, Gregory R; Gidley, Michael J; Dietzgen, Ralf G

    2015-07-30

    Mango fruits contain a broad spectrum of phenolic compounds which impart potential health benefits; their biosynthesis is catalysed by enzymes in the phenylpropanoid-flavonoid (PF) pathway. The aim of this study was to reveal the variability in genes involved in the PF pathway in three different mango varieties Mangifera indica L., a member of the family Anacardiaceae: Kensington Pride (KP), Irwin (IW) and Nam Doc Mai (NDM) and to determine associations with gene expression and mango flavonoid profiles. A close evolutionary relationship between mango genes and those from the woody species poplar of the Salicaceae family (Populus trichocarpa) and grape of the Vitaceae family (Vitis vinifera), was revealed through phylogenetic analysis of PF pathway genes. We discovered 145 SNPs in total within coding sequences with an average frequency of one SNP every 316 bp. Variety IW had the highest SNP frequency (one SNP every 258 bp) while KP and NDM had similar frequencies (one SNP every 369 bp and 360 bp, respectively). The position in the PF pathway appeared to influence the extent of genetic diversity of the encoded enzymes. The entry point enzymes phenylalanine lyase (PAL), cinnamate 4-mono-oxygenase (C4H) and chalcone synthase (CHS) had low levels of SNP diversity in their coding sequences, whereas anthocyanidin reductase (ANR) showed the highest SNP frequency followed by flavonoid 3'-hydroxylase (F3'H). Quantitative PCR revealed characteristic patterns of gene expression that differed between mango peel and flesh, and between varieties. The combination of mango expressed sequence tags and availability of well-established reference PF biosynthetic genes from other plant species allowed the identification of coding sequences of genes that may lead to the formation of important flavonoid compounds in mango fruits and facilitated characterisation of single nucleotide polymorphisms between varieties. We discovered an association between the extent of sequence variation and

  5. Biosynthetic pathways of plastid-derived organelles as potential drug targets against parasitic apicomplexa.

    Science.gov (United States)

    Seeber, Frank

    2003-06-01

    Apicomplexan parasites are a large phylum of unicellular and obligate intracellular organisms of great medical importance. They include the human pathogens Plasmodium spp., the causative agent of malaria, and Toxoplasma gondii, an opportunistic parasite of immunosuppressed individuals and a common cause of congenital disease, together affecting several hundred million people worldwide. The search for new and effective drugs against these pathogens has been boosted during the last years by an unexpected finding. Through molecular and cell biological analysis it was realized that probably most members of this phylum harbor a plastid-like organelle, called the apicoplast, which probably is derived from the engulfment of a red alga in ancient times. Although the apicoplast itself contains a small circular genome, most of the proteome of this organelle is encoded in the nuclear genome, and the proteins are subsequently transported to the apicoplast. It is assumed to contain a number of unique metabolic pathways not found in the vertebrate host, making it an ideal "playground" for those interested in drug targets. Recent reports have shown that the rationale of this approach is valid and that new drugs which are urgently needed especially for plasmodial infections, might be developed in the near future based on these targets. Amongst them are three enzymes of the plant-like fatty acid synthesis machinery and enzymes of the non-mevalonat isoprenoid biosynthesis pathway. From their presence in the apicoplast it can be concluded that fatty acid and lipid biosynthesis seems to be a major function of the apicoplast. Another recently described apicoplast enzyme, ferredoxin-NADP(+)-reductase and its redox partner, ferredoxin, points to another interesting organelle-specific biosynthetic pathway, namely [Fe-S] cluster biosynthesis. In the present review, the fundamental aspects of the apicoplast as drug target will be described, together with the specific pathways and their

  6. Overexpression of the riboflavin biosynthetic pathway in Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Mattanovich Diethard

    2008-07-01

    Full Text Available Abstract Background High cell density cultures of Pichia pastoris grown on methanol tend to develop yellow colored supernatants, attributed to the release of free flavins. The potential of P. pastoris for flavin overproduction is therefore given, but not pronounced when the yeast is grown on glucose. The aim of this study is to characterize the relative regulatory impact of each riboflavin synthesis gene. Deeper insight into pathway control and the potential of deregulation is established by overexpression of the single genes as well as a combined deregulation of up to all six riboflavin synthesis genes. Results Overexpression of the first gene of the riboflavin biosynthetic pathway (RIB1 is already sufficient to obtain yellow colonies and the accumulation of riboflavin in the supernatant of shake flask cultures growing on glucose. Sequential deregulation of all the genes, by exchange of their native promoter with the strong and constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP increases the riboflavin accumulation significantly. Conclusion The regulation of the pathway is distributed over more than one gene. High cell density cultivations of a P. pastoris strain overexpressing all six RIB genes allow the accumulation of 175 mg/L riboflavin in the supernatant. The basis for rational engineering of riboflavin production in P. pastoris has thus been established.

  7. A biosynthetic pathway for hexanoic acid production in Kluyveromyces marxianus.

    Science.gov (United States)

    Cheon, Yuna; Kim, Jun-Seob; Park, Jun-Bum; Heo, Paul; Lim, Jae Hyung; Jung, Gyoo Yeol; Seo, Jin-Ho; Park, Jin Hwan; Koo, Hyun Min; Cho, Kwang Myung; Park, Jin-Byung; Ha, Suk-Jin; Kweon, Dae-Hyuk

    2014-07-20

    Hexanoic acid can be used for diverse industrial applications and is a precursor for fine chemistry. Although some natural microorganisms have been screened and evolved to produce hexanoic acid, the construction of an engineered biosynthetic pathway for producing hexanoic acid in yeast has not been reported. Here we constructed hexanoic acid pathways in Kluyveromyces marxianus by integrating 5 combinations of seven genes (AtoB, BktB, Crt, Hbd, MCT1, Ter, and TES1), by which random chromosomal sites of the strain are overwritten by the new genes from bacteria and yeast. One recombinant strain, H4A, which contained AtoB, BktB, Crt, Hbd, and Ter, produced 154mg/L of hexanoic acid from galactose as the sole substrate. However, the hexanoic acid produced by the H4A strain was re-assimilated during the fermentation due to the reverse activity of AtoB, which condenses two acetyl-CoAs into a single acetoacetyl-CoA. This product instability could be overcome by the replacement of AtoB with a malonyl CoA-acyl carrier protein transacylase (MCT1) from Saccharomyces cerevisiae. Our results suggest that Mct1 provides a slow but stable acetyl-CoA chain elongation pathway, whereas the AtoB-mediated route is fast but unstable. In conclusion, hexanoic acid was produced for the first time in yeast by the construction of chain elongation pathways comprising 5-7 genes in K. marxianus.

  8. High-resolution MALDI mass spectrometry imaging of gallotannins and monoterpene glucosides in the root of Paeonia lactiflora

    DEFF Research Database (Denmark)

    Li, Bin; Bhandari, Dhaka Ram; Römpp, Andreas

    2016-01-01

    High-resolution atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI) at 10 μm pixel size was performed to unravel the spatio-chemical distribution of major secondary metabolites in the root of Paeonia lactiflora. The spatial...... distributions of two major classes of bioactive components, gallotannins and monoterpene glucosides, were investigated and visualized at the cellular level in tissue sections of P. lactiflora roots. Accordingly, other primary and secondary metabolites were imaged, including amino acids, carbohydrates, lipids...

  9. Comparison of carotenoid accumulation and biosynthetic gene expression between Valencia and Rohde Red Valencia sweet oranges

    Science.gov (United States)

    Carotenoid accumulation and biosynthetic gene expression levels during fruit maturation were compared between ordinary Valencia (VAL) and its more deeply colored mutant Rohde Red Valencia orange (RRV). The two cultivars exhibited different carotenoid profiles and regulatory mechanisms in flavedo and...

  10. Biosynthetic Pathway and Health Benefits of Fucoxanthin, an Algae-Specific Xanthophyll in Brown Seaweeds

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    Masashi Hosokawa

    2013-07-01

    Full Text Available Fucoxanthin is the main carotenoid produced in brown algae as a component of the light-harvesting complex for photosynthesis and photoprotection. In contrast to the complete elucidation of the carotenoid biosynthetic pathways in red and green algae, the biosynthetic pathway of fucoxanthin in brown algae is not fully understood. Recently, two models for the fucoxanthin biosynthetic pathway have been proposed in unicellular diatoms; however, there is no such information for the pathway in brown seaweeds to date. Here, we propose a biosynthetic pathway for fucoxanthin in the brown seaweed, Ectocarpus siliculosus, derived from comparison of carotenogenic genes in its sequenced genome with those in the genomes of two diatoms, Thalassiosira pseudonana and Phaeodactylum tricornutum. Currently, fucoxanthin is receiving attention, due to its potential benefits for human health. Therefore, new knowledge regarding the medical and nutraceutical properties of fucoxanthin from brown seaweeds is also summarized here.

  11. Reconstitution of Biosynthetic Machinery for the Synthesis of the Highly Elaborated Indole Diterpene Penitrem

    DEFF Research Database (Denmark)

    Liu, Chengwei; Tagami, Koichi; Minami, Atsushi;

    2015-01-01

    KULNJ). Importantly, without conventional gene disruption, reconstitution of the biosynthetic machinery provided sufficient data to determine the pathway. It was thus demonstrated that the Aspergillus oryzae reconstitution system is a powerful method for studying the biosynthesis of complex natural products....

  12. Comparative genomic analysis of secondary metabolite biosynthetic gene clusters in 207 isolates of Fusarium

    Science.gov (United States)

    Fusarium species are known for their ability to produce secondary metabolites (SMs), including plant hormones, pigments, mycotoxins, and other compounds with potential agricultural, pharmaceutical, and biotechnological impact. Understanding the distribution of SM biosynthetic gene clusters across th...

  13. Monoterpene emissions in response to long-term night-time warming, elevated CO2 and extended summer drought in a temperate heath ecosystem

    DEFF Research Database (Denmark)

    Tiiva, Päivi; Tang, Jing; Michelsen, Anders

    2017-01-01

    Monoterpenes emitted from plants have an important role in atmospheric chemistry through changing atmospheric oxidative capacity, forming new particles and secondary organic aerosols. The emission rates and patterns can be affected by changing climate. In this study, emission responses to six years...... and limonene. The emissions of these three compounds were positively correlated with light, chamber temperature and litter abundance, but negatively correlated with soil temperature. Elevated CO2 tended to decrease the average monoterpene emissions by 40% over the whole growing season, and significantly...... by drought. The inhibition effects of elevated CO2 on emissions were diminished when the treatment was combined with drought or warming. The emission responses to different treatments were not explained by vegetation changes, and the monoterpene emission profile was only moderately related to plant species...

  14. Dormancy removal in apple embryos by nitric oxide or cyanide involves modifications in ethylene biosynthetic pathway.

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    Gniazdowska, Agnieszka; Krasuska, Urszula; Bogatek, Renata

    2010-11-01

    The connection between classical phytohormone-ethylene and two signaling molecules, nitric oxide (NO) and hydrogen cyanide (HCN), was investigated in dormancy removal and germination "sensu stricto" of apple (Malus domestica Borkh.) embryos. Deep dormancy of apple embryos was removed by short-term (3-6 h) pre-treatment with NO or HCN. NO- or HCN-mediated stimulation of germination was associated with enhanced emission of ethylene by the embryos, coupled with transient increase in ROS concentration in embryos. Ethylene vapors stimulated germination of dormant apple embryos and eliminated morphological anomalies characteristic for young seedlings developed from dormant embryos. Inhibitors of ethylene receptors completely impeded beneficial effect of NO and HCN on embryo germination. NO- and HCN-induced ethylene emission by apple embryo was only slightly reduced by inhibitor of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase activity during first 4 days of germination. Short-term pre-treatment of the embryos with NO and HCN modified activity of both key enzymes of ethylene biosynthetic pathway: ACC synthase and ACC oxidase. Activity of ACC synthase declined during first 4 days of germination, while activity of ACC oxidase increased markedly at that time. Additional experiments point to non-enzymatic conversion of ACC to ethylene in the presence of ROS (H(2)O(2)). The results indicate that NO and HCN may alleviate dormancy of apple embryos "via" transient accumulation of ROS, leading to enhanced ethylene emission which is required to terminate germination "sensu stricto". Therefore, ethylene seems to be a trigger factor in control of apple embryo dormancy removal and germination.

  15. Mutational studies of putative biosynthetic genes for the cyanobacterial sunscreen scytonemin in Nostoc punctiforme ATCC 29133

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    Daniela eFerreira

    2016-05-01

    Full Text Available The heterocyclic indole-alkaloid scytonemin is a sunscreen found exclusively among cyanobacteria. An 18-gene cluster is responsible for scytonemin production in Nostoc punctiforme ATCC 29133. The upstream genes scyABCDEF in the cluster are proposed to be responsible for scytonemin biosynthesis from aromatic amino acid substrates. In vitro studies of ScyA, ScyB and ScyC proved that these enzymes indeed catalyze initial pathway reactions. Here we characterize the role of ScyD, ScyE and ScyF, which were logically predicted to be responsible for late biosynthetic steps, in the biological context of N. punctiforme. In-frame deletion mutants of each were constructed (∆scyD, ∆scyE and ∆scyF and their phenotypes studied. Expectedly, ∆scyE presents a scytoneminless phenotype, but no accumulation of the predicted intermediaries. Surprisingly, ∆scyD retains scytonemin production, implying that it is not required for biosynthesis. Indeed, scyD presents an interesting evolutionary paradox: it likely originated in a duplication event from scyE, and unlike other genes in the operon, it has not been subjected to purifying selection. This would suggest that it is a pseudogene, and yet scyD is highly conserved in the scytonemin operon of cyanobacteria. ∆scyF also retains scytonemin production, albeit exhibiting a reduction of the production yield compared with the wild-type. This indicates that ScyF is not essential but may play an adjuvant role for scytonemin synthesis. Altogether, our findings suggest that these downstream genes are not responsible, as expected, for the late steps of scytonemin synthesis and we must look for those functions elsewhere. These findings are particularly important for biotechnological production of this sunscreen through heterologous expression of its genes in more tractable organisms.

  16. Characterization of the CDP-D-mannitol biosynthetic pathway in Streptococcus pneumoniae 35A.

    Science.gov (United States)

    Wang, Quan; Xu, Yanli; Perepelov, Andrei V; Knirel, Yuriy A; Reeves, Peter R; Shashkov, Alexander S; Ding, Peng; Guo, Xi; Feng, Lu

    2012-12-01

    Streptococcus pneumoniae is a major human pathogen associated with diseases worldwide. The capsular polysaccharides (CPSs) are considered a major virulence factor and are targets for a vaccine. d-Mannitol was found to be present in the CPS of several S. pneumoniae serotypes. Two genes, mnp1 and mnp2, which are located in the CPS gene cluster, were proposed to be responsible for the synthesis of NDP-d-mannitol (the nucleotide activated form of d-mannitol). However, the pathway has never been identified by experimental methods and we aimed to characterize it in the present study. To achieve this, the two genes, mnp1 and mnp2, were cloned and the gene products were overexpressed, purified, and analyzed in vitro for their respective enzymatic activities. Products of reactions catalyzed by Mnp1 and Mnp2 were detected by capillary electrophoresis and validated using electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. We show that Mnp1 is responsible for the transfer of CMP from CTP to d-fructose-6-phosphate (Fru-6-P) to form CDP-d-fructose, whereas Mnp2 catalyzed the conversion of CDP-d-fructose to CDP-d-mannitol. Therefore, Mnp1 (renamed as mnpA) was identified as Fru-6-P cytidylyltransferase-encoding gene, and mnp2 (renamed as mnpB) as a CDP-d-fructose reductase-encoding gene. The kinetics of Mnp1 for the substrate (Fru-6-P and CTP) and of Mnp2 for the substrate (CDP-d-fructose) and the cofactor NADH or NADPH fitted the Michaelis-Menten model. The effects of temperature, pH and cations on the two enzymes were analyzed. This is the first time that the biosynthetic pathway of CDP-d-mannitol has been identified biochemically.

  17. Metabolic engineering of the astaxanthin-biosynthetic pathway of Xanthophyllomyces dendrorhous.

    Science.gov (United States)

    Visser, Hans; van Ooyen, Albert J J; Verdoes, Jan C

    2003-12-01

    This review describes the different approaches that have been used to manipulate and improve carotenoid production in Xanthophyllomyces dendrorhous. The red yeast X. dendrorhous (formerly known as Phaffia rhodozyma) is one of the microbiological production systems for natural astaxanthin. Astaxanthin is applied in food and feed industry and can be used as a nutraceutical because of its strong antioxidant properties. However, the production levels of astaxanthin in wild-type isolates are rather low. To increase the astaxanthin content in X. dendrorhous, cultivation protocols have been optimized and astaxanthin-hyperproducing mutants have been obtained by screening of classically mutagenized X. dendrorhous strains. The knowledge about the regulation of carotenogenesis in X. dendrorhous is still limited in comparison to that in other carotenogenic fungi. The X. dendrorhous carotenogenic genes have been cloned and a X. dendrorhous transformation system has been developed. These tools allowed the directed genetic modification of the astaxanthin pathway in X. dendrorhous. The crtYB gene, encoding the bifunctional enzyme phytoene synthase/lycopene cyclase, was inactivated by insertion of a vector by single and double cross-over events, indicating that it is possible to generate specific carotenoid-biosynthetic mutants. Additionally, overexpression of crtYB resulted in the accumulation of beta-carotene and echinone, which indicates that the oxygenation reactions are rate-limiting in these recombinant strains. Furthermore, overexpression of the phytoene desaturase-encoding gene (crtI) showed an increase in monocyclic carotenoids such as torulene and HDCO (3-hydroxy-3',4'-didehydro-beta,-psi-carotene-4-one) and a decrease in bicyclic carotenoids such as echinone, beta-carotene and astaxanthin.

  18. Enhancement of sterol synthesis by the monoterpene perillyl alcohol is unaffected by competitive 3-hydroxy-3-methylglutaryl-CoA reductase inhibition.

    Science.gov (United States)

    Cerda, S R; Wilkinson, J; Branch, S K; Broitman, S A

    1999-06-01

    Monoterpenes such as limonene and perillyl alcohol (PA) are currently under investigation for their chemotherapeutic properties which have been tied to their ability to affect protein isoprenylation. Because PA affects the synthesis of isoprenoids, such as ubiquinone, and cholesterol is the end product of the synthetic pathway from which this isoprenoid pathway branches, we investigated the effects of this compound upon cholesterol metabolism in the colonic adenocarcinoma cell line SW480. PA (1 mM) inhibited incorporation of 14C-mevalonate into 21-26 kDa proteins by 25% in SW480 cells. Cholesterol (CH) biosynthesis was assessed by measuring the incorporation of 14C-acetate and 14C-mevalonate into 27-carbon-sterols. Cells treated with PA (1 mM) exhibited a fourfold increase in the incorporation of 14C-acetate but not 14C-mevalonate into cholesterol. Mevinolin (lovastatin), an inhibitor of 3-hydroxy-3-methylglutaryl-CoA(HMG-CoA) reductase, at 2 microM concentration, inhibited CH synthesis from 14C-acetate by 80%. Surprisingly, concurrent addition of mevinolin and PA did not significantly alter the stimulatory effects of PA. As observed differences in 14C-acetate and 14C-mevalonate precursor labeling could indicate PA affects early pathway events, the effects of this monoterpene on HMG-CoA reductase activity were evaluated. Unexpectedly, 1 mM PA did not stimulate activity of this enzyme. Consistent with its action as a reversibly bound inhibitor, in washed microsomes, 2 microM mevinolin pretreatment increased reductase protein expression causing a 12.7 (+/- 2.4)-fold compensatory HMG-CoA reductase activity increase; concurrent treatment with 1 mM PA attenuated this to a 5.3 (+/- 0.03)-fold increase. Gas chromatographic analysis confirmed CH was the major lipid present in the measured thin-layer chromatography spot. Since 14C-acetate incorporation into free fatty acid and phospholipid pools was not significantly affected by PA treatment, nonspecific changes in whole

  19. Functions of genes and enzymes involved in phenalinolactone biosynthesis.

    Science.gov (United States)

    Daum, Martina; Schnell, Hans-Jörg; Herrmann, Simone; Günther, Andreas; Murillo, Renato; Müller, Rolf; Bisel, Philippe; Müller, Michael; Bechthold, Andreas

    2010-07-05

    Phenalinolactones are novel terpene glycoside antibiotics produced by Streptomyces sp. Tü6071. Inactivation of three oxygenase genes (plaO2, plaO3 and plaO5), two dehydrogenase genes (plaU, plaZ) and one putative acetyltransferase gene (plaV) led to the production of novel phenalinolactone derivatives (PL HS6, PL HS7, PL HS2 and PL X1). Furthermore, the exact biosynthetic functions of two enzymes were determined, and their in vitro activities were demonstrated. PlaO1, an Fe(II)/alpha-ketoglutarate-dependent dioxygenase, is responsible for the key step in gamma-butyrolactone formation, whereas PlaO5, a cytochrome P450-dependent monooxygenase, catalyses the 1-C-hydroxylation of phenalinolactone D. In addition, stable isotope feeding experiments with biosynthetic precursors shed light on the origin of the carbons in the gamma-butyrolactone moiety.

  20. Arabidopsis brassinosteroid biosynthetic mutant dwarf7-1 exhibits slower rates of cell division and shoot induction

    Directory of Open Access Journals (Sweden)

    Schulz Burkhard

    2010-12-01

    Full Text Available Abstract Background Plant growth depends on both cell division and cell expansion. Plant hormones, including brassinosteroids (BRs, are central to the control of these two cellular processes. Despite clear evidence that BRs regulate cell elongation, their roles in cell division have remained elusive. Results Here, we report results emphasizing the importance of BRs in cell division. An Arabidopsis BR biosynthetic mutant, dwarf7-1, displayed various characteristics attributable to slower cell division rates. We found that the DWARF4 gene which encodes for an enzyme catalyzing a rate-determining step in the BR biosynthetic pathways, is highly expressed in the actively dividing callus, suggesting that BR biosynthesis is necessary for dividing cells. Furthermore, dwf7-1 showed noticeably slower rates of callus growth and shoot induction relative to wild-type control. Flow cytometric analyses of the nuclei derived from either calli or intact roots revealed that the cell division index, which was represented as the ratio of cells at the G2/M vs. G1 phases, was smaller in dwf7-1 plants. Finally, we found that the expression levels of the genes involved in cell division and shoot induction, such as PROLIFERATING CELL NUCLEAR ANTIGEN2 (PCNA2 and ENHANCER OF SHOOT REGENERATION2 (ESR2, were also lower in dwf7-1 as compared with wild type. Conclusions Taken together, results of callus induction, shoot regeneration, flow cytometry, and semi-quantitative RT-PCR analysis suggest that BRs play important roles in both cell division and cell differentiation in Arabidopsis.

  1. The carnitine biosynthetic pathway in Arabidopsis thaliana shares similar features with the pathway of mammals and fungi.

    Science.gov (United States)

    Rippa, Sonia; Zhao, Yingjuan; Merlier, Franck; Charrier, Aurélie; Perrin, Yolande

    2012-11-01

    Carnitine is an essential quaternary ammonium amino acid that occurs in the microbial, plant and animal kingdoms. The role and synthesis of this compound are very well documented in bacteria, fungi and mammals. On the contrary, although the presence of carnitine in plant tissue has been reported four decades ago and information about its biological implication are available, nothing is known about its synthesis in plants. We designed experiments to determine if the carnitine biosynthetic pathway in Arabidopsis thaliana is similar to the pathway in mammals and in the fungi Neurospora crassa and Candida albicans. We first checked for the presence of trimetyllysine (TML) and γ-butyrobetaine (γ-BB), two precursors of carnitine in fungi and in mammals, using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Both compounds were shown to be present in plant extracts at concentrations in the picomole range per mg of dry weight. We next synthesized deuterium-labeled TML and transferred A. thaliana seedlings on growth medium supplemented with 1 mM of the deuterated precursor. LC-ESI-MS/MS analysis of plant extracts clearly highlighted the synthesis of deuterium labeled γ-BB and labeled carnitine in deuterated-TML fed plants. The similarities between plant, fungal and mammalian pathways provide very useful information to search homologies between genomes. As a matter of fact the analysis of A. thaliana protein database provides homology for several enzymes responsible for carnitine synthesis in fungi and mammals. The study of mutants affected in the corresponding genes would be very useful to elucidate the plant carnitine biosynthetic pathway and to investigate further the role of carnitine in plant physiology.

  2. Generation of the natamycin analogs by gene engineering of natamycin biosynthetic genes in Streptomyces chattanoogensis L10.

    Science.gov (United States)

    Liu, Shui-Ping; Yuan, Peng-Hui; Wang, Yue-Yue; Liu, Xiao-Fang; Zhou, Zhen-Xing; Bu, Qing-ting; Yu, Pin; Jiang, Hui; Li, Yong-Quan

    2015-04-01

    The polyene antibiotic natamycin is widely used as an antifungal agent in both human therapy and the food industry. Here we obtained four natamycin analogs with high titers, including two new compounds, by engineering of six post-polyketide synthase (PKS) tailoring enzyme encoding genes in a natamycin industrial producing strain, Streptomyces chattanoogensis L10. Precise analysis of S. chattanoogensis L10 culture identified natamycin and two natamycin analogs, 4,5-deepoxy-natamycin and 4,5-deepoxy-natamycinolide. The scnD deletion mutant of S. chattanoogensis L10 did not produce natamycin but increased the titer of 4,5-deepoxy-natamycin. Inactivation of each of scnK, scnC, and scnJ in S. chattanoogensis L10 abolished natamycin production and accumulated 4,5-deepoxy-natamycinolide. Deletion of scnG in S. chattanoogensis L10 resulted in production of two new compounds, 4,5-deepoxy-12-decarboxyl-12-methyl-natamycin and its dehydration product without natamycin production. Inactivation of the ScnG-associated ferredoxin ScnF resulted in impaired production of natamycin. Bioassay of these natamycin analogs showed that three natamycin analogs remained antifungal activities. We found that homologous glycosyltransferases genes including amphDI and nysDI can partly complement the ΔscnK mutant. Our results here also support that ScnG, ScnK, and ScnD catalyze carboxylation, glycosylation, and epoxidation in turn in the natamycin biosynthetic pathway. Thus this paper provided a method to generate natamycin analogs and shed light on the natamycin biosynthetic pathway.

  3. The heme biosynthetic pathway of the obligate Wolbachia endosymbiont of Brugia malayi as a potential anti-filarial drug target.

    Directory of Open Access Journals (Sweden)

    Bo Wu

    Full Text Available BACKGROUND: Filarial parasites (e.g., Brugia malayi, Onchocerca volvulus, and Wuchereria bancrofti are causative agents of lymphatic filariasis and onchocerciasis, which are among the most disabling of neglected tropical diseases. There is an urgent need to develop macro-filaricidal drugs, as current anti-filarial chemotherapy (e.g., diethylcarbamazine [DEC], ivermectin and albendazole can interrupt transmission predominantly by killing microfilariae (mf larvae, but is less effective on adult worms, which can live for decades in the human host. All medically relevant human filarial parasites appear to contain an obligate endosymbiotic bacterium, Wolbachia. This alpha-proteobacterial mutualist has been recognized as a potential target for filarial nematode life cycle intervention, as antibiotic treatments of filarial worms harboring Wolbachia result in the loss of worm fertility and viability upon antibiotic treatments both in vitro and in vivo. Human trials have confirmed this approach, although the length of treatments, high doses required and medical counter-indications for young children and pregnant women warrant the identification of additional anti-Wolbachia drugs. METHODS AND FINDINGS: Genome sequence analysis indicated that enzymes involved in heme biosynthesis might constitute a potential anti-Wolbachia target set. We tested different heme biosynthetic pathway inhibitors in ex vivo B. malayi viability assays and report a specific effect of N-methyl mesoporphyrin (NMMP, which targets ferrochelatase (FC, the last step. Our phylogenetic analysis indicates evolutionarily significant divergence between Wolbachia heme genes and their human homologues. We therefore undertook the cloning, overexpression and analysis of several enzymes of this pathway alongside their human homologues, and prepared proteins for drug targeting. In vitro enzyme assays revealed a approximately 600-fold difference in drug sensitivities to succinyl acetone (SA between

  4. VOC measurements within a boreal forest during spring 2005: the role of monoterpenes and sulphuric acid in selected intense nucleation events

    Directory of Open Access Journals (Sweden)

    G. Eerdekens

    2009-06-01

    Full Text Available In this study we present measurements of selected trace gases and aerosol made in a boreal forest during the BACCI-QUEST IV intensive field campaign in Hyytiälä, Finland in April 2005. Several clear aerosol "nucleation events" were observed and characterized. One type of "event" occurred synchronously with huge increases in monoterpenes, while a second event type correlated instead with sulphuric acid. Here we elucidate the origin of these two distinct forms of aerosol production at the Hyytiälä site using the measurement data, airmass back trajectories and the optical stereoisomery of monoterpenes.

  5. Solanapyrone synthase, a possible Diels-Alderase and iterative type I polyketide synthase encoded in a biosynthetic gene cluster from Alternaria solani.

    Science.gov (United States)

    Kasahara, Ken; Miyamoto, Takanori; Fujimoto, Takashi; Oguri, Hiroki; Tokiwano, Tetsuo; Oikawa, Hideaki; Ebizuka, Yutaka; Fujii, Isao

    2010-06-14

    The solanapyrone biosynthetic gene cluster was cloned from Alternaria solani. It consists of six genes-sol1-6-coding for a polyketide synthase, an O-methyltransferase, a dehydrogenase, a transcription factor, a flavin-dependent oxidase, and cytochrome P450. The prosolanapyrone synthase (PSS) encoded by sol1 was expressed in Aspergillus oryzae and its product was identified as desmethylprosolanapyrone I (8). Although PSS is closely related to the PKSs/Diels-Alderases LovB and MlcA of lovastatin and compactin biosynthesis, it did not catalyze cycloaddition. Sol5, encoding a flavin-dependent oxidase (solanapyrone synthase, SPS), was expressed in Pichia pastoris and purified. The purified recombinant SPS showed activity for the formation of (-)-solanapyrone A (1) from achiral prosolanapyrone II (2), establishing that this single enzyme catalyzes both the oxidation and the subsequent cycloaddition reaction, possibly as a Diels-Alder enzyme.

  6. Identification and Functional Characterization of Genes Encoding Omega-3 Polyunsaturated Fatty Acid Biosynthetic Activities from Unicellular Microalgae

    Directory of Open Access Journals (Sweden)

    Royah Vaezi

    2013-12-01

    Full Text Available In order to identify novel genes encoding enzymes involved in the biosynthesis of nutritionally important omega-3 long chain polyunsaturated fatty acids, a database search was carried out in the genomes of the unicellular photoautotrophic green alga Ostreococcus RCC809 and cold-water diatom Fragilariopsis cylindrus. The search led to the identification of two putative “front-end” desaturases (Δ6 and Δ4 from Ostreococcus RCC809 and one Δ6-elongase from F. cylindrus. Heterologous expression of putative open reading frames (ORFs in yeast revealed that the encoded enzyme activities efficiently convert their respective substrates: 54.1% conversion of α-linolenic acid for Δ6-desaturase, 15.1% conversion of 22:5n-3 for Δ4-desaturase and 38.1% conversion of γ-linolenic acid for Δ6-elongase. The Δ6-desaturase from Ostreococcus RCC809 displays a very strong substrate preference resulting in the predominant synthesis of stearidonic acid (C18:4Δ6,9,12,15. These data confirm the functional characterization of omega-3 long chain polyunsaturated fatty acid biosynthetic genes from these two species which have until now not been investigated for such activities. The identification of these new genes will also serve to expand the repertoire of activities available for metabolically engineering the omega-3 trait in heterologous hosts as well as providing better insights into the synthesis of eicosapentaenoic acid (EPA and docosahexaenoic acid (DHA in marine microalgae.

  7. Formulation of sage essential oil (Salvia officinalis, L.) monoterpenes into chitosan hydrogels and permeation study with GC-MS analysis.

    Science.gov (United States)

    Kodadová, Alexandra; Vitková, Zuzana; Herdová, Petra; Ťažký, Anton; Oremusová, Jarmila; Grančai, Daniel; Mikuš, Peter

    2015-01-01

    This study deals with the formulation of natural drugs into hydrogels. For the first time, compounds from the sage essential oil were formulated into chitosan hydrogels. A sample preparation procedure for hydrophobic volatile analytes present in a hydrophilic water matrix along with an analytical method based on the gas chromatography coupled with the mass spectrometry (GC-MS) was developed and applied for the evaluation of the identity and quantity of essential oil components in the hydrogels and saline samples. The experimental results revealed that the chitosan hydrogels are suitable for the formulation of sage essential oil. The monoterpene release can be effectively controlled by both chitosan and caffeine concentration in the hydrogels. Permeation experiment, based on a hydrogel with the optimized composition [3.5% (w/w) sage essential oil, 2.0% (w/w) caffeine, 2.5% (w/w) chitosan and 0.1% (w/w) Tween-80] in donor compartment, saline solution in acceptor compartment, and semi-permeable cellophane membrane, demonstrated the useful permeation selectivity. Here, (according to lipophilicity) an enhanced permeation of the bicyclic monoterpenes with antiflogistic and antiseptic properties (eucalyptol, camphor and borneol) and, at the same time, suppressed permeation of toxic thujone (not exceeding its permitted applicable concentration) was observed. These properties highlight the pharmaceutical importance of the developed chitosan hydrogel formulating sage essential oil in the dermal applications.

  8. In vitro inhibition of the bovine viral diarrhoea virus by the essential oil of Ocimum basilicum (basil) and monoterpenes.

    Science.gov (United States)

    Kubiça, Thaís F; Alves, Sydney H; Weiblen, Rudi; Lovato, Luciane T

    2014-01-01

    The bovine viral diarrhoea virus (BVDV) is suggested as a model for antiviral studies of the hepatitis C virus (HCV). The antiviral activity of the essential oil of Ocimum basilicum and the monoterpenes camphor, thymol and 1,8-cineole against BVDV was investigated. The cytotoxicities of the compounds were measured by the MTT (3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide) test, and the antiviral activities were tested by the plaque reduction assay. The oil or compounds were added to the assay in three different time points: a) pre-treatment of the virus (virucidal assay); b) pre-treatment of the cells; or c) post-treatment of the cells (after virus inoculation). The percentage of plaques inhibition for each compound was determined based on the number of plaques in the viral control. The results were expressed by CC50 (50% cytotoxic concentration), IC50 (inhibitory concentration for 50% of plaques) and SI (selectivity index = CC50/IC50). Camphor (CC50 = 4420.12 μg mL(-1)) and 1,8-cineole (CC50 = 2996.10 μg mL(-1)) showed the lowest cytotoxicities and the best antiviral activities (camphor SI = 13.88 and 1,8-cineol SI = 9.05) in the virucidal assay. The higher activities achieved by the monoterpenes in the virucidal assay suggest that these compounds act directly on the viral particle.

  9. Tailoring enzyme-rich environmental DNA clones: a source of enzymes for generating libraries of unnatural natural products.

    Science.gov (United States)

    Banik, Jacob J; Craig, Jeffrey W; Calle, Paula Y; Brady, Sean F

    2010-11-10

    A detailed bioinformatics analysis of six glycopeptide biosynthetic gene clusters isolated from soil environmental DNA (eDNA) megalibraries indicates that a subset of these gene clusters contains collections of tailoring enzymes that are predicted to result in the production of new glycopeptide congeners. In particular, sulfotransferases appear in eDNA-derived gene clusters at a much higher frequency than would be predicted from the characterization of glycopeptides from cultured Actinomycetes . Enzymes found on tailoring-enzyme-rich eDNA clones associated with these six gene clusters were used to produce a series of new sulfated glycopeptide derivatives in both in vitro and in vivo derivatization studies. The derivatization of known natural products with eDNA-derived tailoring enzymes is likely to be a broadly applicable strategy for generating libraries of new natural product variants.

  10. Evolutionary Diversification of Alanine Transaminases in Yeast: Catabolic Specialization and Biosynthetic Redundancy

    Directory of Open Access Journals (Sweden)

    Ximena Escalera-Fanjul

    2017-06-01

    Full Text Available Gene duplication is one of the major evolutionary mechanisms providing raw material for the generation of genes with new or modified functions. The yeast Saccharomyces cerevisiae originated after an allopolyploidization event, which involved mating between two different ancestral yeast species. ScALT1 and ScALT2 codify proteins with 65% identity, which were proposed to be paralogous alanine transaminases. Further analysis of their physiological role showed that while ScALT1 encodes an alanine transaminase which constitutes the main pathway for alanine biosynthesis and the sole pathway for alanine catabolism, ScAlt2 does not display alanine transaminase activity and is not involved in alanine metabolism. Moreover, phylogenetic studies have suggested that ScALT1 and ScALT2 come from each one of the two parental strains which gave rise to the ancestral hybrid. The present work has been aimed to the understanding of the properties of the ancestral type Lacchancea kluyveri LkALT1 and Kluyveromyces lactis KlALT1, alanine transaminases in order to better understand the ScALT1 and ScALT2 evolutionary history. These ancestral -type species were chosen since they harbor ALT1 genes, which are related to ScALT2. Presented results show that, although LkALT1 and KlALT1 constitute ScALT1 orthologous genes, encoding alanine transaminases, both yeasts display LkAlt1 and KlAlt1 independent alanine transaminase activity and additional unidentified alanine biosynthetic and catabolic pathway(s. Furthermore, phenotypic analysis of null mutants uncovered the fact that KlAlt1 and LkAlt1 have an additional role, not related to alanine metabolism but is necessary to achieve wild type growth rate. Our study shows that the ancestral alanine transaminase function has been retained by the ScALT1 encoded enzyme, which has specialized its catabolic character, while losing the alanine independent role observed in the ancestral type enzymes. The fact that ScAlt2 conserves 64

  11. Enzyme toolbox: novel enantiocomplementary imine reductases.

    Science.gov (United States)

    Scheller, Philipp N; Fademrecht, Silvia; Hofelzer, Sebastian; Pleiss, Jürgen; Leipold, Friedemann; Turner, Nicholas J; Nestl, Bettina M; Hauer, Bernhard

    2014-10-13

    Reducing reactions are among the most useful transformations for the generation of chiral compounds in the fine-chemical industry. Because of their exquisite selectivities, enzymatic approaches have emerged as the method of choice for the reduction of C=O and activated C=C bonds. However, stereoselective enzymatic reduction of C=N bonds is still in its infancy-it was only recently described after the discovery of enzymes capable of imine reduction. In our work, we increased the spectrum of imine-reducing enzymes by database analysis. By combining the currently available knowledge about the function of imine reductases with the experimentally uncharacterized diversity stored in protein sequence databases, three novel imine reductases with complementary enantiopreference were identified along with amino acids important for catalysis. Furthermore, their reducing capability was demonstrated by the reduction of the pharmaceutically relevant prochiral imine 2-methylpyrroline. These novel enzymes exhibited comparable to higher catalytic efficiencies than previously described enzymes, and their biosynthetic potential is highlighted by the full conversion of 2-methylpyrroline in whole cells with excellent selectivities. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Components of complex lipid biosynthetic pathways in developing castor (Ricinus communis) seeds identified by MudPIT analysis of enriched endoplasmic reticulum.

    Science.gov (United States)

    Brown, Adrian P; Kroon, Johan T M; Topping, Jennifer F; Robson, Joanne L; Simon, William J; Slabas, Antoni R

    2011-08-05

    Ricinoleic acid is a feedstock for nylon-11 (N11) synthesis which is currently obtained from castor (Ricinus communis) oil. Production of this fatty acid in a temperate oilseed crop is of great commercial interest, but the highest reported level in transgenic plant oils is 30%, below the 90% observed in castor and insufficient for commercial exploitation. To identify castor oil-biosynthetic enzymes and inform strategies to improve ricinoleic acid yields, we performed MudPIT analysis on endoplasmic reticulum (ER) purified from developing castor bean endosperm. Candidate enzymes for all steps of triacylglycerol synthesis were identified among 72 proteins in the data set related to complex-lipid metabolism. Previous reported proteomic data from oilseeds had not included any membrane-bound enzyme that might incorporate ricinoleic acid into oil. Analysis of enriched ER enabled determination of which protein isoforms for these enzymes were in developing castor seed. To complement this data, quantitative RT-PCR experiments with castor seed and leaf RNA were performed for orthologues of Arabidopsis oil-synthetic enzymes, determining which were highly expressed in the seed. These data provide important information for further manipulation of ricinoleic acid content in oilseeds and peptide data for future quantification strategies.

  13. Survey of foliar monoterpenes across the range of jack pine reveal three widespread chemotypes: implications to host expansion of invasive mountain pine beetle

    Directory of Open Access Journals (Sweden)

    Spencer eTaft

    2015-05-01

    Full Text Available The secondary compounds of pines (Pinus can strongly affect the physiology, ecology and behaviors of the bark beetles (Coleoptera: Curculionidae, Scolytinae that feed on sub-cortical tissues of hosts. Jack pine (Pinus banksiana has a wide natural distribution range in North America (Canada and USA and thus variations in its secondary compounds, particularly monoterpenes, could affect the host expansion of invasive mountain pine beetle (Dendroctonus ponderosae, which has recently expanded its range into the novel jack pine boreal forest. We investigated monoterpene composition of 601 jack pine trees from natural and provenance forest stands representing 63 populations from Alberta to the Atlantic coast. Throughout its range, jack pine exhibited three chemotypes characterized by high proportions of α-pinene, β-pinene, or limonene. The frequency with which the α-pinene and β-pinene chemotypes occurred at individual sites was correlated to climatic variables, such as continentality and mean annual precipitation, as were the individual α-pinene and β-pinene concentrations. However, other monoterpenes were generally not correlated to climatic variables or geographic distribution. Finally, while the enantiomeric ratios of β-pinene and limonene remained constant across jack pine’s distribution, (‒:(+-α-pinene exhibited two separate trends, thereby delineating two α-pinene phenotypes, both of which occurred across jack pine’s range. These significant variations in jack pine monoterpene composition may have cascading effects on the continued eastward spread and success of D. ponderosae in the Canadian boreal forest.

  14. Significant light and temperature dependent monoterpene emissions from European beech (fagus sylvatiga L.) and their potential impact on the European VOC budget

    NARCIS (Netherlands)

    Dindorf, T.; Kuhn, U.; Ganzeveld, L.N.; Schebeske, G.; Ciccioli, P.; Holzke, C.; Köble, R.; Seufert, G.; Kesselmeier, J.

    2006-01-01

    By using a dynamic branch enclosure system the emission of monoterpenes from European beech (Fagus sylvatica L.) was investigated during two consecutive summer vegetation periods in the years of 2002 and 2003 in Germany. All measurements were performed under field conditions within the framework of

  15. The Differential Effects of the Blue-Stain Fungus Leptographium qinlingensis on Monoterpenes and Sesquiterpenes in the Stem of Chinese White Pine (Pinus armandi Saplings

    Directory of Open Access Journals (Sweden)

    Thanh Pham

    2014-11-01

    Full Text Available When conifers such as Chinese white pine (Pinus armandi Fr. are attacked by insects or pathogens, they respond by increasing their content of monoterpenes and sesquiterpenes. In this study, we determined the effects of the blue-stain fungus Leptographium qinlingensis Tang and Chen on monoterpenes and sesquiterpenes in the phloem and xylem of the stem of P. armandi saplings. We found that the total monoterpene concentrations in the phloem and xylem of the stem and the total sesquiterpene concentrations in the xylem of the stem were significantly higher in L. qinlingensis-inoculated saplings than in control (mechanically wounded saplings or untreated saplings. Additionally, the proportions of β-pinene in the xylem of the stem and limonene + β-phellandrene in the phloem and xylem of the stem were significantly higher in L. qinlingensis-inoculated saplings than in both control and untreated saplings. The proportions of individual sesquiterpenes in the phloem and xylem of the stem were significantly greater in L. qinlingensis-inoculated saplings than in untreated saplings. Based on the results of this study, we suggest that increases in total monoterpene and sesquiterpene concentrations, as well as increases in the concentrations of β-pinene and limonene + β-phellandrene, may play an important defensive role against blue-stain fungus L. qinlingensis inoculation.

  16. WET AND DRY SEASON ECOSYSTEM LEVEL FLUXES OF ISOPRENE AND MONOTERPENES FROM A SOUTHEAST ASIAN SECONDARY FOREST AND RUBBER TREE PLANTATION

    Science.gov (United States)

    Canopy scale fluxes of isoprene and monoterpenes were investigated in both wet and dry seasons above a rubber tree (Hevea brasiliensis)/secondary tropical forest in the Yunnan province of southwestern China. Drought conditions were unusually high during the dry season experiment....

  17. Challenges in modelling isoprene and monoterpene emission dynamics of Arctic plants: a case study from a subarctic tundra heath

    Science.gov (United States)

    Tang, Jing; Schurgers, Guy; Valolahti, Hanna; Faubert, Patrick; Tiiva, Päivi; Michelsen, Anders; Rinnan, Riikka

    2016-12-01

    The Arctic is warming at twice the global average speed, and the warming-induced increases in biogenic volatile organic compounds (BVOCs) emissions from Arctic plants are expected to be drastic. The current global models' estimations of minimal BVOC emissions from the Arctic are based on very few observations and have been challenged increasingly by field data. This study applied a dynamic ecosystem model, LPJ-GUESS, as a platform to investigate short-term and long-term BVOC emission responses to Arctic climate warming. Field observations in a subarctic tundra heath with long-term (13-year) warming treatments were extensively used for parameterizing and evaluating BVOC-related processes (photosynthesis, emission responses to temperature and vegetation composition). We propose an adjusted temperature (T) response curve for Arctic plants with much stronger T sensitivity than the commonly used algorithms for large-scale modelling. The simulated emission responses to 2 °C warming between the adjusted and original T response curves were evaluated against the observed warming responses (WRs) at short-term scales. Moreover, the model responses to warming by 4 and 8 °C were also investigated as a sensitivity test. The model showed reasonable agreement to the observed vegetation CO2 fluxes in the main growing season as well as day-to-day variability of isoprene and monoterpene emissions. The observed relatively high WRs were better captured by the adjusted T response curve than by the common one. During 1999-2012, the modelled annual mean isoprene and monoterpene emissions were 20 and 8 mg C m-2 yr-1, with an increase by 55 and 57 % for 2 °C summertime warming, respectively. Warming by 4 and 8 °C for the same period further elevated isoprene emission for all years, but the impacts on monoterpene emissions levelled off during the last few years. At hour-day scale, the WRs seem to be strongly impacted by canopy air T, while at the day-year scale, the WRs are a combined

  18. Why are estimates of global terrestrial isoprene emissions so similar (and why is this not so for monoterpenes)?

    Science.gov (United States)

    Arneth, A.; Monson, R. K.; Schurgers, G.; Niinemets, É.; Palmer, P. I.

    2008-08-01

    Emissions of biogenic volatile organic compounds (BVOC) are a chief uncertainty in calculating the burdens of important atmospheric compounds like tropospheric ozone or secondary organic aerosol, reflecting either imperfect chemical oxidation mechanisms or unreliable emission estimates, or both. To provide a starting point for a more systematic discussion we review here global isoprene and monoterpene emission estimates to-date. We note a surprisingly small variation in the predictions of global isoprene emission rate that is in stark contrast with our lack of process understanding and the small number of observations for model parameterisation and evaluation. Most of the models are based on similar emission algorithms, using fixed values for the emission capacity of various plant functional types. In some cases, these values are very similar but differ substantially in other models. The similarities with regard to the global isoprene emission rate would suggest that the dominant parameters driving the ultimate global estimate, and thus the dominant determinant of model sensitivity, are the specific emission algorithm and isoprene emission capacity. But the models also differ broadly with regard to their representation of net primary productivity, method of biome coverage determination and climate data. Contrary to isoprene, monoterpene estimates show significantly larger model-to-model variation although variation in terms of leaf algorithm, emission capacities, the way of model upscaling, vegetation cover or climatology used in terpene models are comparable to those used for isoprene. From our summary of published studies there appears to be no evidence that the terrestrial modelling community has been any more successful in "resolving unknowns" in the mechanisms that control global isoprene emissions, compared to global monoterpene emissions. Rather, the proliferation of common parameterization schemes within a large variety of model platforms lends the illusion

  19. Why are estimates of global isoprene emissions so similar (and why is this not so for monoterpenes)?

    Science.gov (United States)

    Arneth, A.; Monson, R. K.; Schurgers, G.; Niinemets, Ü.; Palmer, P. I.

    2008-04-01

    Emissions of biogenic volatile organic compounds (BVOC) are a chief uncertainty in calculating the burdens of important atmospheric compounds like tropospheric ozone or secondary organic aerosol, reflecting either imperfect chemical oxidation mechanisms or unreliable emission estimates, or both. To provide a starting point for a more systematic discussion we review here global isoprene and monoterpene emission estimates to-date. We note a surprisingly small variation in the predictions of global isoprene emission rate that is in stark contrast with our lack of process understanding and the small number of observations for model parameterisation and evaluation. Most of the models are based on similar emission algorithms, using fixed values for the emission capacity of various plant functional types. In some studies these values are very similar, but they differ substantially in others. The models differ also broadly with regard to their representation of net primary productivity, method of biome coverage determination and climate data. Their similarities with regard to the global isoprene emission rate would suggest that the dominant parameters driving the ultimate global estimate, and thus the dominant determinant of model sensitivity, are the specific emission algorithm and isoprene emission capacity. Contrary to isoprene, monoterpene estimates show significantly larger model-to-model variation although variation in terms of leaf algorithm, emission capacities, the way of model upscaling, vegetation cover or climatology used in terpene models are comparable to those used for isoprene. From our summary of published studies there appears to be no evidence that the terrestrial modelling community has been any more successful in "resolving unknowns" in the mechanisms that control global isoprene emissions, compared to global monoterpene emissions. Rather, the proliferation of common parameterization schemes within a large variety of model platforms lends the illusion

  20. Why are estimates of global isoprene emissions so similar (and why is this not so for monoterpenes?

    Directory of Open Access Journals (Sweden)

    A. Arneth

    2008-04-01

    Full Text Available Emissions of biogenic volatile organic compounds (BVOC are a chief uncertainty in calculating the burdens of important atmospheric compounds like tropospheric ozone or secondary organic aerosol, reflecting either imperfect chemical oxidation mechanisms or unreliable emission estimates, or both. To provide a starting point for a more systematic discussion we review here global isoprene and monoterpene emission estimates to-date. We note a surprisingly small variation in the predictions of global isoprene emission rate that is in stark contrast with our lack of process understanding and the small number of observations for model parameterisation and evaluation. Most of the models are based on similar emission algorithms, using fixed values for the emission capacity of various plant functional types. In some studies these values are very similar, but they differ substantially in others. The models differ also broadly with regard to their representation of net primary productivity, method of biome coverage determination and climate data. Their similarities with regard to the global isoprene emission rate would suggest that the dominant parameters driving the ultimate global estimate, and thus the dominant determinant of model sensitivity, are the specific emission algorithm and isoprene emission capacity. Contrary to isoprene, monoterpene estimates show significantly larger model-to-model variation although variation in terms of leaf algorithm, emission capacities, the way of model upscaling, vegetation cover or climatology used in terpene models are comparable to those used for isoprene. From our summary of published studies there appears to be no evidence that the terrestrial modelling community has been any more successful in "resolving unknowns" in the mechanisms that control global isoprene emissions, compared to global monoterpene emissions. Rather, the proliferation of common parameterization schemes within a large variety of model platforms

  1. Why are estimates of global terrestrial isoprene emissions so similar (and why is this not so for monoterpenes?

    Directory of Open Access Journals (Sweden)

    A. Arneth

    2008-08-01

    Full Text Available Emissions of biogenic volatile organic compounds (BVOC are a chief uncertainty in calculating the burdens of important atmospheric compounds like tropospheric ozone or secondary organic aerosol, reflecting either imperfect chemical oxidation mechanisms or unreliable emission estimates, or both. To provide a starting point for a more systematic discussion we review here global isoprene and monoterpene emission estimates to-date. We note a surprisingly small variation in the predictions of global isoprene emission rate that is in stark contrast with our lack of process understanding and the small number of observations for model parameterisation and evaluation. Most of the models are based on similar emission algorithms, using fixed values for the emission capacity of various plant functional types. In some cases, these values are very similar but differ substantially in other models. The similarities with regard to the global isoprene emission rate would suggest that the dominant parameters driving the ultimate global estimate, and thus the dominant determinant of model sensitivity, are the specific emission algorithm and isoprene emission capacity. But the models also differ broadly with regard to their representation of net primary productivity, method of biome coverage determination and climate data. Contrary to isoprene, monoterpene estimates show significantly larger model-to-model variation although variation in terms of leaf algorithm, emission capacities, the way of model upscaling, vegetation cover or climatology used in terpene models are comparable to those used for isoprene. From our summary of published studies there appears to be no evidence that the terrestrial modelling community has been any more successful in "resolving unknowns" in the mechanisms that control global isoprene emissions, compared to global monoterpene emissions. Rather, the proliferation of common parameterization schemes within a large variety of model platforms

  2. Effects of Acute Ozone Exposure and Methyl Jasmonate Treatment on White Pine Monoterpene and Sesquiterpene Emission Rates

    Science.gov (United States)

    Faiola, C. L.; Wagner, D.; Allwine, E.; Harley, P. C.; Vanreken, T. M.

    2010-12-01

    Biogenic volatile organic compounds (BVOCs) are produced by plants and include monoterpenes, sesquiterpenes, and their oxygenated derivatives. These BVOCs are one of the principal factors influencing the oxidative capacity of the atmosphere in forested regions, and impact both ozone concentration and secondary organic aerosol formation. Under unstressed conditions, the release of BVOCs to the atmosphere is primarily controlled by the vapor pressure of the relevant compounds within the plant tissue, which is in turn dependent on temperature as well as complex biochemical production processes. However, various natural and anthropogenic stressors can alter both the quantity and composition of the BVOCs emitted by plants. Many potential stressors are expected to become stronger as climate change effects escalate. The impacts of most stressors on BVOC emissions have not been well characterized, particularly in a field setting where changes in BVOC emissions could have influential feedbacks with climate. This study investigated the effects of two stressors on monoterpene and sesquiterpene emission rates at a field site in northern Michigan: acute ozone exposure and treatment with methyl jasmonate, an herbivory proxy. The study included six repetitions of the same experiment, each time using a new set of sub-canopy eastern white pine specimens. For each experiment, dynamic branch enclosures were simultaneously used on three specimens for sample collection: one ozone treatment tree, one methyl jasmonate treatment tree, and one control tree. Sampling lines were placed in each enclosure and VOCs were collected onto cartridges packed with Tenax GR adsorbent. Samples were collected several times per day for at least two days before treatment and for five days after treatment. Cartridges were analyzed via thermodesorption with an Agilent GC/MS/FID. This analysis allowed the identification and quantification of several monoterpene and sesquiterpene species in the samples

  3. An intercomparison of GC-FID and PTR-MS toluene measurements in ambient air under conditions of enhanced monoterpene loading

    Directory of Open Access Journals (Sweden)

    J. L. Ambrose

    2010-01-01

    Full Text Available Toluene was measured using both a gas chromatographic system (GC, with a flame ionization detector (FID, and a proton transfer reaction-mass spectrometer (PTR-MS at the AIRMAP atmospheric monitoring station Thompson Farm (THF in rural Durham, NH during the summer of 2004. Simultaneous measurements of monoterpenes, including α- and β-pinene, camphene, Δ3-carene, and d-limonene, by GC-FID demonstrated large enhancements in monoterpene mixing ratios relative to toluene, with median and maximum enhancement ratios of ~2 and ~30, respectively. A detailed intercomparison among the GC-FID and PTR-MS toluene measurements was conducted to test the specificity of PTR-MS for atmospheric toluene measurements under conditions often dominated by biogenic emissions. We derived quantitative estimates of potential interferences in the PTR-MS toluene measurements related to sampling and analysis of monoterpenes, including fragmentation of the monoterpenes and some of their primary carbonyl oxidation products via reactions with H3O+, O2+ and NO+ in the PTR-MS drift tube. The PTR-MS and GC-FID toluene measurements were in good quantitative agreement and the two systems tracked one another well from the instrumental limits of detection to maximum mixing ratios of ~0.5 ppbv. Discrepancies in the measured mixing ratios were not well correlated with enhancements in the monoterpenes. Better quantitative agreement between the two systems was obtained by correcting the PTR-MS measurements for contributions from monoterpene fragmentation in the PTR-MS drift tube; however, the improvement was minor. Interferences in the PTR-MS measurements from fragmentation of the monoterpene oxidation products pinonaldehyde, caronaldehyde and α-pinene oxide were also likely negligible. The results from THF suggest that toluene can be reliably quantified by PTR-MS using our operating conditions under the ambient

  4. The Biosynthesis of Capuramycin-type Antibiotics: IDENTIFICATION OF THE A-102395 BIOSYNTHETIC GENE CLUSTER, MECHANISM OF SELF-RESISTANCE, AND FORMATION OF URIDINE-5'-CARBOXAMIDE.

    Science.gov (United States)

    Cai, Wenlong; Goswami, Anwesha; Yang, Zhaoyong; Liu, Xiaodong; Green, Keith D; Barnard-Britson, Sandra; Baba, Satoshi; Funabashi, Masanori; Nonaka, Koichi; Sunkara, Manjula; Morris, Andrew J; Spork, Anatol P; Ducho, Christian; Garneau-Tsodikova, Sylvie; Thorson, Jon S; Van Lanen, Steven G

    2015-05-29

    A-500359s, A-503083s, and A-102395 are capuramycin-type nucleoside antibiotics that were discovered using a screen to identify inhibitors of bacterial translocase I, an essential enzyme in peptidoglycan cell wall biosynthesis. Like the parent capuramycin, A-500359s and A-503083s consist of three structural components: a uridine-5'-carboxamide (CarU), a rare unsaturated hexuronic acid, and an aminocaprolactam, the last of which is substituted by an unusual arylamine-containing polyamide in A-102395. The biosynthetic gene clusters for A-500359s and A-503083s have been reported, and two genes encoding a putative non-heme Fe(II)-dependent α-ketoglutarate:UMP dioxygenase and an l-Thr:uridine-5'-aldehyde transaldolase were uncovered, suggesting that C-C bond formation during assembly of the high carbon (C6) sugar backbone of CarU proceeds from the precursors UMP and l-Thr to form 5'-C-glycyluridine (C7) as a biosynthetic intermediate. Here, isotopic enrichment studies with the producer of A-503083s were used to indeed establish l-Thr as the direct source of the carboxamide of CarU. With this knowledge, the A-102395 gene cluster was subsequently cloned and characterized. A genetic system in the A-102395-producing strain was developed, permitting the inactivation of several genes, including those encoding the dioxygenase (cpr19) and transaldolase (cpr25), which abolished the production of A-102395, thus confirming their role in biosynthesis. Heterologous production of recombinant Cpr19 and CapK, the transaldolase homolog involved in A-503083 biosynthesis, confirmed their expected function. Finally, a phosphotransferase (Cpr17) conferring self-resistance was functionally characterized. The results provide the opportunity to use comparative genomics along with in vivo and in vitro approaches to probe the biosynthetic mechanism of these intriguing structures.

  5. Bacterial molybdoenzymes: old enzymes for new purposes.

    Science.gov (United States)

    Leimkühler, Silke; Iobbi-Nivol, Chantal

    2016-01-01

    Molybdoenzymes are widespread in eukaryotic and prokaryotic organisms where they play crucial functions in detoxification reactions in the metabolism of humans and bacteria, in nitrate assimilation in plants and in anaerobic respiration in bacteria. To be fully active, these enzymes require complex molybdenum-containing cofactors, which are inserted into the apoenzymes after folding. For almost all the bacterial molybdoenzymes, molybdenum cofactor insertion requires the involvement of specific chaperones. In this review, an overview on the molybdenum cofactor biosynthetic pathway is given together with the role of specific chaperones dedicated for molybdenum cofactor insertion and maturation. Many bacteria are involved in geochemical cycles on earth and therefore have an environmental impact. The roles of molybdoenzymes in bioremediation and for environmental applications are presented.

  6. A novel mechanism of sulfur transfer catalyzed by O-acetylhomoserine sulfhydrylase in the methionine-biosynthetic pathway of Wolinella succinogenes

    Energy Technology Data Exchange (ETDEWEB)

    Tran, Timothy H. [Cornell University, Ithaca, New York 14853-1301 (United States); Krishnamoorthy, Kalyanaraman; Begley, Tadhg P., E-mail: begley@tamu.edu [Texas A& M University, College Station, TX 77842 (United States); Ealick, Steven E., E-mail: begley@tamu.edu [Cornell University, Ithaca, New York 14853-1301 (United States)

    2011-10-01

    MetY is the first reported structure of an O-acetylhomoserine sulfhydrylase that utilizes a protein thiocarboxylate intermediate as the sulfur source in a novel methionine-biosynthetic pathway instead of catalyzing a direct sulfhydrylation reaction. O-Acetylhomoserine sulfhydrylase (OAHS) is a pyridoxal 5′-phosphate (PLP) dependent sulfide-utilizing enzyme in the l-cysteine and l-methionine biosynthetic pathways of various enteric bacteria and fungi. OAHS catalyzes the conversion of O-acetylhomoserine to homocysteine using sulfide in a process known as direct sulfhydrylation. However, the source of the sulfur has not been identified and no structures of OAHS have been reported in the literature. Here, the crystal structure of Wolinella succinogenes OAHS (MetY) determined at 2.2 Å resolution is reported. MetY crystallized in space group C2 with two monomers in the asymmetric unit. Size-exclusion chromatography, dynamic light scattering and crystal packing indicate that the biological unit is a tetramer in solution. This is further supported by the crystal structure, in which a tetramer is formed using a combination of noncrystallographic and crystallographic twofold axes. A search for structurally homologous proteins revealed that MetY has the same fold as cystathionine γ-lyase and methionine γ-lyase. The active sites of these enzymes, which are also PLP-dependent, share a high degree of structural similarity, suggesting that MetY belongs to the γ-elimination subclass of the Cys/Met metabolism PLP-dependent family of enzymes. The structure of MetY, together with biochemical data, provides insight into the mechanism of sulfur transfer to a small molecule via a protein thiocarboxylate intermediate.

  7. Exploiting the Biosynthetic Potential of Type III Polyketide Synthases

    Directory of Open Access Journals (Sweden)

    Yan Ping Lim

    2016-06-01

    Full Text Available Polyketides are structurally and functionally diverse secondary metabolites that are biosynthesized by polyketide synthases (PKSs using acyl-CoA precursors. Recent studies in the engineering and structural characterization of PKSs have facilitated the use of target enzymes as biocatalysts to produce novel functionally optimized polyketides. These compounds may serve as potential drug leads. This review summarizes the insights gained from research on type III PKSs, from the discovery of chalcone synthase in plants to novel PKSs in bacteria and fungi. To date, at least 15 families of type III PKSs have been characterized, highlighting the utility of PKSs in the development of natural product libraries for therapeutic development.

  8. Polyketide synthase chemistry does not direct biosynthetic divergence between 9- and 10-membered enediynes.

    Science.gov (United States)

    Horsman, Geoff P; Chen, Yihua; Thorson, Jon S; Shen, Ben

    2010-06-22

    Enediynes are potent antitumor antibiotics that are classified as 9- or 10-membered according to the size of the enediyne core structure. However, almost nothing is known about enediyne core biosynthesis, and the determinants of 9- versus 10-membered enediyne core biosynthetic divergence remain elusive. Previous work identified enediyne-specific polyketide synthases (PKSEs) that can be phylogenetically distinguished as being involved in 9- versus 10-membered enediyne biosynthesis, suggesting that biosynthetic divergence might originate from differing PKSE chemistries. Recent in vitro studies have identified several compounds produced by the PKSE and associated thioesterase (TE), but condition-dependent product profiles make it difficult to ascertain a true catalytic difference between 9- and 10-membered PKSE-TE systems. Here we report that PKSE chemistry does not direct 9- versus 10-membered enediyne core biosynthetic divergence as revealed by comparing the products from three 9-membered and two 10-membered PKSE-TE systems under identical conditions using robust in vivo assays. Three independent experiments support a common catalytic function for 9- and 10-membered PKSEs by the production of a heptaene metabolite from: (i) all five cognate PKSE-TE pairs in Escherichia coli; (ii) the C-1027 and calicheamicin cognate PKSE-TEs in Streptomyces lividans K4-114; and (iii) selected native producers of both 9- and 10-membered enediynes. Furthermore, PKSEs and TEs from different 9- and 10-membered enediyne biosynthetic machineries are freely interchangeable, revealing that 9- versus 10-membered enediyne core biosynthetic divergence occurs beyond the PKSE-TE level. These findings establish a starting point for determining the origins of this biosynthetic divergence.

  9. Enzyme detection by microfluidics

    DEFF Research Database (Denmark)

    2013-01-01

    Microfluidic-implemented methods of detecting an enzyme, in particular a DNA-modifying enzyme, are provided, as well as methods for detecting a cell, or a microorganism expressing said enzyme. The enzyme is detected by providing a nucleic acid substrate, which is specifically targeted...... by that enzyme...

  10. Apicoplast Biosynthetic Pathways as Possible Targetsfor Combination Therapy of Malaria

    Institute of Scientific and Technical Information of China (English)

    Solomon Tesfaye; Bhanu Prakash; Prati Pal Singh

    2015-01-01

    The emergence of malaria parasite strains resistant to practically all the antimalarial drugs in clinical use is now making itnecessary to discover and develop both new antimalarial drugs and treatments. Recent advances in molecular techniques along withthe availability of genome sequence ofPlasmodiumfalciparum may provide a wide range of novel targets in metabolic pathways likeisoprenoid biosynthesis, fatty acid biosynthesis and heme biosynthesis in the apicoplast of Plasmodiurn. On the other hand, thecombination therapy approach (currently used to retard the selection of parasite strains resistant to individual components of acombination of drugs) has proved to be a success in the combination of sulphadoxine and pyrimethamine, which targets two differentsteps in the folate pathway of malaria parasite. However, after the success of this therapeutic combination, the efficacy of othercombinations of drugs which target different enzymes in a particular metabolic pathway has, apparently, not been reported. Therefore,herein, we review various drug targets so far discovered in apicoplast-related anabolic pathways, especially, with a sharper focus onthe possibility to target more than one enzyme at a time in a particular metabolic pathway of malaria parasites.

  11. HPLC-SPE-NMR for combinatorial biosynthetic investigations – expanding the landscape of diterpene structural diversity

    DEFF Research Database (Denmark)

    Kongstad, Kenneth Thermann; Andersen-Ranberg, Johan; Hamberger, Björn Robert

    In this work, the analytical technique, HPLC-HRMS-SPE-NMR was used for the first time in combination with combinatorial biosynthetic investigations in N. benthamiana. This efficient setup allowed for identification of several diterpene synthase (diTPS) combinations responsible for stereospecific ......In this work, the analytical technique, HPLC-HRMS-SPE-NMR was used for the first time in combination with combinatorial biosynthetic investigations in N. benthamiana. This efficient setup allowed for identification of several diterpene synthase (diTPS) combinations responsible for...

  12. An Integrated Metabolomic and Genomic Mining Workflow to Uncover the Biosynthetic Potential of Bacteria

    DEFF Research Database (Denmark)

    Månsson, Maria; Vynne, Nikolaj Grønnegaard; Klitgaard, Andreas

    2016-01-01

    considerable diversity: only 2% of the chemical features and 7% of the biosynthetic genes were common to all strains, while 30% of all features and 24% of the genes were unique to single strains. The list of chemical features was reduced to 50 discriminating features using a genetic algorithm and support...... vector machines. Features were dereplicated by tandem mass spectrometry (MS/MS) networking to identify molecular families of the same biosynthetic origin, and the associated pathways were probed using comparative genomics. Most of the discriminating features were related to antibacterial compounds...

  13. Assembly of a novel biosynthetic pathway for production of the plant flavonoid fisetin in Escherichia coli

    DEFF Research Database (Denmark)

    Stahlhut, Steen Gustav; Siedler, Solvej; Malla, Sailesh

    2015-01-01

    Plant secondary metabolites are an underutilized pool of bioactive molecules for applications in the food, pharma and nutritional industries. One such molecule is fisetin, which is present in many fruits and vegetables and has several potential health benefits, including anti-cancer, anti......-viral and anti-aging activity. Moreover, fisetin has recently been shown to prevent Alzheimer׳s disease in mice and to prevent complications associated with diabetes type I. Thus far the biosynthetic pathway of fisetin in plants remains elusive. Here, we present the heterologous assembly of a novel fisetin...... biosynthetic pathway and establish E. coli as a microbial platform strain for the production of fisetin and related flavonols....

  14. Structure of ThiM from Vitamin B1 biosynthetic pathway of Staphylococcus aureus - Insights into a novel pro-drug approach addressing MRSA infections

    Science.gov (United States)

    Drebes, Julia; Künz, Madeleine; Windshügel, Björn; Kikhney, Alexey G.; Müller, Ingrid B.; Eberle, Raphael J.; Oberthür, Dominik; Cang, Huaixing; Svergun, Dmitri I.; Perbandt, Markus; Betzel, Christian; Wrenger, Carsten

    2016-03-01

    Infections caused by the methicillin-resistant Staphylococcus aureus (MRSA) are today known to be a substantial threat for global health. Emerging multi-drug resistant bacteria have created a substantial need to identify and discover new drug targets and to develop novel strategies to treat bacterial infections. A promising and so far untapped antibiotic target is the biosynthesis of vitamin B1 (thiamin). Thiamin in its activated form, thiamin pyrophosphate, is an essential co-factor for all organisms. Therefore, thiamin analogous compounds, when introduced into the vitamin B1 biosynthetic pathway and further converted into non-functional co-factors by the bacterium can function as pro-drugs which thus block various co-factor dependent pathways. We characterized one of the key enzymes within the S. aureus vitamin B1 biosynthetic pathway, 5-(hydroxyethyl)-4-methylthiazole kinase (SaThiM; EC 2.7.1.50), a potential target for pro-drug compounds and analyzed the native structure of SaThiM and complexes with the natural substrate 5-(hydroxyethyl)-4-methylthiazole (THZ) and two selected substrate analogues.

  15. Elevated Liver Enzymes

    Science.gov (United States)

    Symptoms Elevated liver enzymes By Mayo Clinic Staff Elevated liver enzymes may indicate inflammation or damage to cells in the liver. Inflamed or ... than normal amounts of certain chemicals, including liver enzymes, into the bloodstream, which can result in elevated ...

  16. Genistein: a novel anthocyanin synthesis promoter that directly regulates biosynthetic genes in red cabbage in a light-dependent way

    Directory of Open Access Journals (Sweden)

    Na Zhang

    2016-12-01

    Full Text Available Genistein (GNT, an isoflavone, is used in the clinical treatment of various health disorders. GNT is found in primary food source plants and some medical plants. However, studies on the functions of GNT in plants are rarely reported. In this study, we demonstrated that GNT plays an important role in promoting anthocyanin accumulation in red cabbage. GNT solutions (10, 20, 30, 40, and 50 mg/L as foliar fertilizers were applied to red cabbage. Consequently, anthocyanin accumulation in red cabbage increased in a light-dependent manner. GNT solution at 30 mg/L exhibited the optimal effect on anthocyanin accumulation, which was twice that of the control. Quantitative real-time PCR analysis indicated that GNT application upregulated the expression of all structural genes, contributing to anthocyanin biosynthesis under light conditions. Under dark conditions, GNT exerted no significant promotive effect on anthocyanin accumulation; only early biosynthetic genes of anthocyanin biosynthesis responded to GNT. The promotive effect of GNT on anthocyanin biosynthesis is directly attributable to the regulation of structural gene expression. Transcription factors exhibited no response to GNT. The levels of anthocyanin in red cabbage positively correlated with the enzyme activities of antioxidant systems. This finding correlation suggested that the promotive effect of GNT on anthocyanin levels was correlated with improved antioxidant activity in the red cabbage.

  17. Discovery of putative capsaicin biosynthetic genes by RNA-Seq and digital gene expression analysis of pepper

    Science.gov (United States)

    Zhang, Zi-Xin; Zhao, Shu-Niu; Liu, Gao-Feng; Huang, Zu-Mei; Cao, Zhen-Mu; Cheng, Shan-Han; Lin, Shi-Sen

    2016-01-01

    The Indian pepper ‘Guijiangwang’ (Capsicum frutescens L.), one of the world’s hottest chili peppers, is rich in capsaicinoids. The accumulation of the alkaloid capsaicin and its analogs in the epidermal cells of the placenta contribute to the pungency of Capsicum fruits. To identify putative genes involved in capsaicin biosynthesis, RNA-Seq was used to analyze the pepper’s expression profiles over five developmental stages. Five cDNA libraries were constructed from the total RNA of placental tissue and sequenced using an Illumina HiSeq 2000. More than 19 million clean reads were obtained from each library, and greater than 50% of the reads were assignable to reference genes. Digital gene expression (DGE) profile analysis using Solexa sequencing was performed at five fruit developmental stages and resulted in the identification of 135 genes of known function; their expression patterns were compared to the capsaicin accumulation pattern. Ten genes of known function were identified as most likely to be involved in regulating capsaicin synthesis. Additionally, 20 new candidate genes were identified related to capsaicin synthesis. We use a combination of RNA-Seq and DGE analyses to contribute to the understanding of the biosynthetic regulatory mechanism(s) of secondary metabolites in a nonmodel plant and to identify candidate enzyme-encoding genes. PMID:27756914

  18. Influence of the dissolved oxygen concentration on the penicillin biosynthetic pathway in steady-state cultures of Penicillium chrysogenum

    DEFF Research Database (Denmark)

    Henriksen, Claus Maxel; Nielsen, Jens Bredal; Villadsen, John

    1997-01-01

    The influence the of dissolved oxygen concentration on penicillin biosynthesis was studied in steady-state continuous cultures of a high-yielding strain of Penicillium chrysogenum operated at a dilution rate of 0.05 h-l. The dissolved oxygen concentration was varied between 0.019 and 0.344 mM (co...... and cysteine decreased at low dissolved oxygen concentrations. On the basis of the intracellular pool measurements, metabolic control analysis is performed, and the flux control coefficients for the first two enzymes in the penicillin biosynthetic pathway, i.e., delta......The influence the of dissolved oxygen concentration on penicillin biosynthesis was studied in steady-state continuous cultures of a high-yielding strain of Penicillium chrysogenum operated at a dilution rate of 0.05 h-l. The dissolved oxygen concentration was varied between 0.019 and 0.344 m......M (corresponding to 7% and 131% air saturation at 1 bar) solely through manipulations of the inlet gas composition. At dissolved oxygen concentrations above 0.06-0.08 mM, a constant specific penicillin productivity of around 22 (mu mol/g of DW)/h is maintained. At lower oxygen concentrations, the specific...

  19. Genistein: A Novel Anthocyanin Synthesis Promoter that Directly Regulates Biosynthetic Genes in Red Cabbage in a Light-Dependent Way

    Science.gov (United States)

    Zhang, Na; Qi, Yan; Zhang, Hai-Jun; Wang, Xiaoyun; Li, Hongfei; Shi, Yantong; Guo, Yang-Dong

    2016-01-01

    Genistein (GNT), an isoflavone, is used in the clinical treatment of various health disorders. GNT is found in primary food source plants and some medical plants. However, studies on the functions of GNT in plants are rarely reported. In this study, we demonstrated that GNT plays an important role in promoting anthocyanin accumulation in red cabbage. GNT solutions (10, 20, 30, 40, and 50 mg/L) as foliar fertilizers were applied to red cabbage. Consequently, anthocyanin accumulation in red cabbage increased in a light-dependent manner. GNT solution at 30 mg/L exhibited the optimal effect on anthocyanin accumulation, which was twice that of the control. Quantitative real-time PCR analysis indicated that GNT application upregulated the expression of all structural genes, contributing to anthocyanin biosynthesis under light conditions. Under dark conditions, GNT exerted no significant promotive effect on anthocyanin accumulation; only early biosynthetic genes of anthocyanin biosynthesis responded to GNT. The promotive effect of GNT on anthocyanin biosynthesis is directly attributable to the regulation of structural gene expression. Transcription factors exhibited no response to GNT. The levels of anthocyanin in red cabbage positively correlated with the enzyme activities of antioxidant systems. This finding correlation suggested that the promotive effect of GNT on anthocyanin levels was correlated with improved antioxidant activity in the red cabbage. PMID:27990149

  20. Porphyrin Binding to Gun4 Protein, Facilitated by a Flexible Loop, Controls Metabolite Flow through the Chlorophyll Biosynthetic Pathway.

    Science.gov (United States)

    Kopečná, Jana; Cabeza de Vaca, Israel; Adams, Nathan B P; Davison, Paul A; Brindley, Amanda A; Hunter, C Neil; Guallar, Victor; Sobotka, Roman

    2015-11-20

    In oxygenic phototrophs, chlorophylls, hemes, and bilins are synthesized by a common branched pathway. Given the phototoxic nature of tetrapyrroles, this pathway must be tightly regulated, and an important regulatory role is attributed to magnesium chelatase enzyme at the branching between the heme and chlorophyll pathway. Gun4 is a porphyrin-binding protein known to stimulate in vitro the magnesium chelatase activity, but how the Gun4-porphyrin complex acts in the cell was unknown. To address this issue, we first performed simulations to determine the porphyrin-docking mechanism to the cyanobacterial Gun4 structure. After correcting crystallographic loop contacts, we determined the binding site for magnesium protoporphyrin IX. Molecular modeling revealed that the orientation of α6/α7 loop is critical for the binding, and the magnesium ion held within the porphyrin is coordinated by Asn-211 residue. We also identified the basis for stronger binding in the Gun4-1 variant and for weaker binding in the W192A mutant. The W192A-Gun4 was further characterized in magnesium chelatase assay showing that tight porphyrin binding in Gun4 facilitates its interaction with the magnesium chelatase ChlH subunit. Finally, we introduced the W192A mutation into cells and show that the Gun4-porphyrin complex is important for the accumulation of ChlH and for channeling metabolites into the chlorophyll biosynthetic pathway.

  1. Multiple GCD genes required for repression of GCN4, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae.

    Science.gov (United States)

    Harashima, S; Hinnebusch, A G

    1986-11-01

    GCN4 encodes a positive regulator of multiple unlinked genes encoding amino acid biosynthetic enzymes in Saccharomyces cerevisiae. Expression of GCN4 is coupled to amino acid availability by a control mechanism involving GCD1 as a negative effector and GCN1, GCN2, and GCN3 as positive effectors of GCN4 expression. We used reversion of a gcn2 gcn3 double mutation to isolate new alleles of GCD1 and mutations in four additional GCD genes which we designate GCD10, GCD11, GCD12, and GCD13. All of the mutations lead to constitutive derepression of HIS4 transcription in the absence of the GCN2+ and GCN3+ alleles. By contrast, the gcd mutations require the wild-type GCN4 allele for their derepressing effect, suggesting that each acts by influencing the level of GCN4 activity in the cell. Consistent with this interpretation, mutations in each GCD gene lead to constitutive derepression of a GCN4::lacZ gene fusion. Thus, at least five gene products are required to maintain the normal repressed level of GCN4 expression in nonstarvation conditions. Interestingly, the gcd mutations are pleiotropic and also affect growth rate in nonstarvation conditions. In addition, certain alleles lead to a loss of M double-stranded RNA required for the killer phenotype. This pleiotropy suggests that the GCD gene products contribute to an essential cellular function, in addition to, or in conjunction with, their role in GCN4 regulation.

  2. Mutations in Biosynthetic Enzymes for the Protein Linker Region of Chondroitin/Dermatan/Heparan Sulfate Cause Skeletal and Skin Dysplasias

    Directory of Open Access Journals (Sweden)

    Shuji Mizumoto

    2015-01-01

    Full Text Available Glycosaminoglycans, including chondroitin, dermatan, and heparan sulfate, have various roles in a wide range of biological events such as cell signaling, cell proliferation, tissue morphogenesis, and interactions with various growth factors. Their polysaccharides covalently attach to the serine residues on specific core proteins through the common linker region tetrasaccharide, -xylose-galactose-galactose-glucuronic acid, which is produced through the stepwise addition of respective monosaccharides by four distinct glycosyltransferases. Mutations in the human genes encoding the glycosyltransferases responsible for the biosynthesis of the linker region tetrasaccharide cause a number of genetic disorders, called glycosaminoglycan linkeropathies, including Desbuquois dysplasia type 2, spondyloepimetaphyseal dysplasia, Ehlers-Danlos syndrome, and Larsen syndrome. This review focused on recent studies on genetic diseases caused by defects in the biosynthesis of the common linker region tetrasaccharide.

  3. Accumulation of Rutin and Betulinic Acid and Expression of Phenylpropanoid and Triterpenoid Biosynthetic Genes in Mulberry (Morus alba L.).

    Science.gov (United States)

    Zhao, Shicheng; Park, Chang Ha; Li, Xiaohua; Kim, Yeon Bok; Yang, Jingli; Sung, Gyoo Byung; Park, Nam Il; Kim, Soonok; Park, Sang Un

    2015-09-30

    Mulberry (Morus alba L.) is used in traditional Chinese medicine and is the sole food source of the silkworm. Here, 21 cDNAs encoding phenylpropanoid biosynthetic genes and 21 cDNAs encoding triterpene biosynthetic genes were isolated from mulberry. The expression levels of genes involved in these biosynthetic pathways and the accumulation of rutin, betulin, and betulinic acid, important secondary metabolites, were investigated in different plant organs. Most phenylpropanoid and triterpene biosynthetic genes were highly expressed in leaves and/or fruit, and most genes were downregulated during fruit ripening. The accumulation of rutin was more than fivefold higher in leaves than in other organs, and higher levels of betulin and betulinic acid were found in roots and leaves than in fruit. By comparing the contents of these compounds with gene expression levels, we speculate that MaUGT78D1 and MaLUS play important regulatory roles in the rutin and betulin biosynthetic pathways.

  4. Characterization of the fumonisin B2 biosynthetic gene cluster in Aspergillus niger and A. awamori.

    Science.gov (United States)

    Aspergillus niger and A. awamori strains isolated from grapes cultivated in Mediterranean basin were examined for fumonisin B2 (FB2) production and presence/absence of sequences within the fumonisin biosynthetic gene (fum) cluster. Presence of 13 regions in the fum cluster was evaluated by PCR assay...

  5. Intertidal marine sediment harbours Actinobacteria with promising bioactive and biosynthetic potential.

    Science.gov (United States)

    Jose, Polpass Arul; Jha, Bhavanath

    2017-08-30

    Actinobacteria are the major source of bioactive natural products that find their value in research and drug discovery programmes. Antimicrobial resistance and the resulting high demand for novel antibiotics underscore the need for exploring novel sources of these bacteria endowed with biosynthetic potential. Intertidal ecosystems endure regular periods of immersion and emersion, and represent an untapped source of Actinobacteria. In this study, we studied the diversity and biosynthetic potential of cultivable Actinobacteria from intertidal sediments of Diu Island in the Arabian Sea. A total of 148 Actinobacteria were selectively isolated using a stamping method with eight isolation media. Isolates were grouped into OTUs based on their 16S rRNA gene sequence, and categorized within actinobacterial families such as Glycomycetaceae, Micromonosporaceae, Nocardiaceae, Nocardiopsaceae, Pseudonocardiaceae, Streptomycetaceae, and Thermomonosporaceae. The biosynthetic potential of the Actinobacteria, necessary for secondary metabolite biosynthesis, was screened and confirmed by extensive fingerprinting approaches based on genes coding for polyketide synthases and nonribosomal peptide synthetases. The observed biosynthetic potential was correlated with the antibacterial activity exhibited by these isolates in laboratory conditions. Ultimately, the results demonstrate that intertidal sediment is a rich source of diverse cultivable Actinobacteria with high potential to synthesize novel bioactive compounds in their genomes.

  6. Sugars as the optimal biosynthetic carbon substrate of aqueous life throughout the universe

    Science.gov (United States)

    Weber, A. L.

    2000-01-01

    Our previous analysis of the energetics of metabolism showed that both the biosynthesis of amino acids and lipids from sugars, and the fermentation of organic substrates, were energetically driven by electron transfer reactions resulting in carbon redox disproportionation (Weber, 1997). Redox disproportionation--the spontaneous (energetically favorable) direction of carbon group transformation in biosynthesis--is brought about and driven by the energetically downhill transfer of electron pairs from more oxidized carbon groups (with lower half-cell reduction potentials) to more reduced carbon groups (with higher half-cell reduction potentials). In this report, we compare the redox and kinetic properties of carbon groups in order to evaluate the relative biosynthetic capability of organic substrates, and to identify the optimal biosubstrate. This analysis revealed that sugars (monocarbonyl alditols) are the optimal biosynthetic substrate because they contain the maximum number of biosynthetically useful high energy electrons/carbon atom while still containing a single carbonyl group needed to kinetically facilitate their conversion to useful biosynthetic intermediates. This conclusion applies to aqueous life throughout the Universe because it is based on invariant aqueous carbon chemistry--primarily, the universal reduction potentials of carbon groups.

  7. Lactococcus lactis as expression host for the biosynthetic incorporation of tryptophan analogues into recombinant proteins

    NARCIS (Netherlands)

    El Khattabi, Mohamed; van Roosmalen, Maarten L.; Jager, Dennis; Metselaar, Heidi; Permentier, Hjalmar; Leenhouts, Kees; Broos, Jaap

    2008-01-01

    Incorporation of Trp (tryptophan) analogues into a protein may facilitate its structural analysis by spectroscopic techniques. Development of a biological system for the biosynthetic incorporation of such analogues into proteins is of considerable importance. The Gram-negative Escherichia coli is th

  8. Phytochemical and Biosynthetic Studies of Lignans, with a Focus on Indonesian Medicinal Plants

    NARCIS (Netherlands)

    Elfahmi, [No Value

    2006-01-01

    In this thesis phytochemical and biosynthetic studies of lignans are described. The focus is on the Indonesian medicinal plants Phyllanthus niruri and Piper cubeba and on two Linum species, Linum flavum and L. leonii, native to European countries. Both Indonesian plants are used in jamu. Jamu is the

  9. Phytochemical and Biosynthetic Studies of Lignans, with a Focus on Indonesian Medicinal Plants

    NARCIS (Netherlands)

    Elfahmi, [No Value

    2006-01-01

    In this thesis phytochemical and biosynthetic studies of lignans are described. The focus is on the Indonesian medicinal plants Phyllanthus niruri and Piper cubeba and on two Linum species, Linum flavum and L. leonii, native to European countries. Both Indonesian plants are used in jamu. Jamu is the

  10. Design-based re-engineering of biosynthetic gene clusters : plug-and-play in practice

    NARCIS (Netherlands)

    Frasch, Hans-Jörg; Medema, Marnix H.; Takano, Eriko; Breitling, Rainer; Gago, Federico; Parayil, Ajikumar

    2013-01-01

    Synthetic biology is revolutionizing the way in which the biosphere is explored for natural products. Through computational genome mining, thousands of biosynthetic gene clusters are being identified in microbial genomes, which constitute a rich source of potential novel pharmaceuticals. New methods

  11. SELECTIVE SEPARATION OF BIOSYNTHETIC PRODUCTS BY PERTRACTION - CHALLENGE FOR THE “WHITE BIOTECHNOLOGY”

    OpenAIRE

    Dan Cascaval; Anca-Irina Galaction

    2010-01-01

    This review presents our original results on selective separation of some biosynthetic products (antibiotics, carboxylic acids, amino acids) by free or facilitated pertraction (extraction and transport through liquid membranes). Selecting the optimum conditions, for all studied cases these pertraction technique simplify the technologies applied at industrial scale for separation and purification, allows to reaching high selectivity and reducing the overall cost of the products.

  12. HPLC-SPE-NMR for combinatorial biosynthetic investigations – Expanding the landscape of diterpene structural diversity

    DEFF Research Database (Denmark)

    Kongstad, Kenneth Thermann; Andersen-Ranberg, Johan; Hamberger, Björn Robert;

    In this work, the analytical technique, HPLC-HRMS-SPE-NMR was used for the first time in combination with combinatorial biosynthetic investigations in N. benthamiana. This efficient setup allowed for identification of several diterpene synthase (diTPS) combinations responsible for stereospecific...

  13. Design-based re-engineering of biosynthetic gene clusters : plug-and-play in practice

    NARCIS (Netherlands)

    Frasch, Hans-Jörg; Medema, Marnix H.; Takano, Eriko; Breitling, Rainer; Gago, Federico; Parayil, Ajikumar

    2013-01-01

    Synthetic biology is revolutionizing the way in which the biosphere is explored for natural products. Through computational genome mining, thousands of biosynthetic gene clusters are being identified in microbial genomes, which constitute a rich source of potential novel pharmaceuticals. New methods

  14. Production of Odd-Carbon Dicarboxylic Acids in Escherichia coli Using an Engineered Biotin–Fatty Acid Biosynthetic Pathway

    Energy Technology Data Exchange (ETDEWEB)

    Haushalter, Robert W. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Physical Bioscience Division; Phelan, Ryan M. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Physical Bioscience Division; Hoh, Kristina M. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Physical Bioscience Division; Su, Cindy [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Physical Bioscience Division; Wang, George [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Physical Bioscience Division; Baidoo, Edward E. K. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Physical Bioscience Division; Keasling, Jay D. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Physical Bioscience Division

    2017-03-14

    Dicarboxylic acids are commodity chemicals used in the production of plastics, polyesters, nylons, fragrances, and medications. Bio-based routes to dicarboxylic acids are gaining attention due to environmental concerns about petroleum-based production of these compounds. Some industrial applications require dicarboxylic acids with specific carbon chain lengths, including odd-carbon species. Biosynthetic pathways involving cytochrome P450-catalyzed oxidation of fatty acids in yeast and bacteria have been reported, but these systems produce almost exclusively even-carbon species. Here in this paper we report a novel pathway to odd-carbon dicarboxylic acids directly from glucose in Escherichia coli by employing an engineered pathway combining enzymes from biotin and fatty acid synthesis. Optimization of the pathway will lead to industrial strains for the production of valuable odd-carbon diacids.

  15. Biosynthetic consequences of multiple sequential post-transition-state bifurcations

    Science.gov (United States)

    Hong, Young Joo; Tantillo, Dean J.

    2014-02-01

    Selectivity in chemical reactions that form complex molecular architectures from simpler precursors is usually rationalized by comparing competing transition-state structures that lead to different possible products. Herein we describe a system for which a single transition-state structure leads to the formation of many isomeric products via pathways that feature multiple sequential bifurcations. The reaction network described connects the pimar-15-en-8-yl cation to miltiradiene, a tricyclic diterpene natural product, and isomers via cyclizations and/or rearrangements. The results suggest that the selectivity of the reaction is controlled by (post-transition-state) dynamic effects, that is, how the carbocation structure changes in response to the distribution of energy in its vibrational modes. The inherent dynamical effects revealed herein (characterized through quasiclassical direct dynamics calculations using density functional theory) have implications not only for the general principles of selectivity prediction in systems with complex potential energy surfaces, but also for the mechanisms of terpene synthase enzymes and their evolution. These findings redefine the challenges faced by nature in controlling the biosynthesis of complex natural products.

  16. Structural basis for biosynthetic programming of fungal aromatic polyketide cyclization.

    Science.gov (United States)

    Crawford, Jason M; Korman, Tyler P; Labonte, Jason W; Vagstad, Anna L; Hill, Eric A; Kamari-Bidkorpeh, Oliver; Tsai, Shiou-Chuan; Townsend, Craig A

    2009-10-22

    Polyketides are a class of natural products with diverse structures and biological activities. The structural variability of aromatic products of fungal nonreducing, multidomain iterative polyketide synthases (NR-PKS group of IPKSs) results from regiospecific cyclizations of reactive poly-beta-keto intermediates. How poly-beta-keto species are synthesized and stabilized, how their chain lengths are determined, and, in particular, how specific cyclization patterns are controlled have been largely inaccessible and functionally unknown until recently. A product template (PT) domain is responsible for controlling specific aldol cyclization and aromatization of these mature polyketide precursors, but the mechanistic basis is unknown. Here we present the 1.8 A crystal structure and mutational studies of a dissected PT monodomain from PksA, the NR-PKS that initiates the biosynthesis of the potent hepatocarcinogen aflatoxin B(1) in Aspergillus parasiticus. Despite having minimal sequence similarity to known enzymes, the structure displays a distinct 'double hot dog' (DHD) fold. Co-crystal structures with palmitate or a bicyclic substrate mimic illustrate that PT can bind both linear and bicyclic polyketides. Docking and mutagenesis studies reveal residues important for substrate binding and catalysis, and identify a phosphopantetheine localization channel and a deep two-part interior binding pocket and reaction chamber. Sequence similarity and extensive conservation of active site residues in PT domains suggest that the mechanistic insights gleaned from these studies will prove general for this class of IPKSs, and lay a foundation for defining the molecular rules controlling NR-PKS cyclization specificity.

  17. Enzyme detection by microfluidics

    DEFF Research Database (Denmark)

    2013-01-01

    Microfluidic-implemented methods of detecting an enzyme, in particular a DNA-modifying enzyme, are provided, as well as methods for detecting a cell, or a microorganism expressing said enzyme. The enzyme is detected by providing a nucleic acid substrate, which is specifically targeted...

  18. Monoterpene composition of essential oil from peppermint (Mentha x piperita L.) with regard to leaf position using solid-phase microextraction and gas chromatography/mass spectrometry analysis.

    Science.gov (United States)

    Rohloff, J

    1999-09-01

    Monoterpene compounds of leaf pairs and flowers of Mentha x piperita have been studied by direct headspace sampling using solid-phase microextraction coupled with gas chromatography/mass spectrometry (SPME-GC/MS). The content of peppermint-characteristic compounds such as menthol, menthyl acetate, and neomenthol increased in a basipetal direction (older plant parts), whereas menthone and isomenthone showed higher levels in the acropetal direction (younger plant parts). Higher levels of menthofuran were found in peppermint flowers in contrast to the leaves. SPME sampling resulted in relatively higher amounts of high-volatile monoterpenes and lower detection of less volatile compounds such as menthol and menthone, compared to solvent-based samples from essential oil distillation.

  19. Comparative study on the in vitro human skin permeation of monoterpenes and phenylpropanoids applied in rose oil and in form of neat single compounds.

    Science.gov (United States)

    Schmitt, S; Schaefer, U; Sporer, F; Reichling, J

    2010-02-01

    Essential oils are ingredients of cosmetic and health care products as well as massage oil used in aromatherapy. There is no doubt that essential oils and their components are able to permeate human skin. But information is rare dealing with percutanous absorption of essential oils in more detail. In this paper we investigated the in vitro skin permeation of monoterpenes and phenylpropanoids applied in pure rose oil and in form of neat single substances. We found that the application form had an exceeding influence on the skin permeation behaviour of the compounds. For substances applied in rose oil a clear relationship between their lipophilic character, chemical structure, and skin permeation could be confirmed. Regarding the P(app)-values the substances are ranked in the order: monoterpene hydrocarbons rose oil than in their neat form. This suggests that co-operative interactions between essential oil components may promote skin permeation behaviour of essential oil and its components.

  20. Genes and enzymes of ectoine biosynthesis in halotolerant methanotrophs.

    Science.gov (United States)

    Reshetnikov, Alexander S; Khmelenina, Valentina N; Mustakhimov, Ildar I; Trotsenko, Yuri A

    2011-01-01

    Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) is a widely distributed compatible solute accumulated by halophilic and halotolerant microorganisms to prevent osmotic stress in highly saline environments. Ectoine as a highly water keeping compound stabilizing biomolecules and whole cells can be used in scientific work, cosmetics, and medicine. Detailed understanding of the organization/regulation of the ectoine biosynthetic pathway in various producers is an active area of research. Here we review current knowledge on some genetic and enzymatic aspects of ectoine biosynthesis in halophilic and halotolerant methanotrophs. By using PCR methodology, the genes coding for the specific enzymes of ectoine biosynthesis, diaminobutyric acid (DABA) aminotransferase (EctB), DABA acetyltransferase (EctA), and ectoine synthase (EctC), were identified in several methanotrophic species. Organization of these genes in either ectABC or ectABC-ask operons, the latter additionally encoding aspartate kinase isozyme (Ask), correlated well with methanotroph halotolerance and intracellular ectoine level. A new gene, ectR1 encoding the MarR-like transcriptional regulatory protein EctR1, negatively controlling transcription of ectoine biosynthetic genes was found upstream of ectABC-ask operon in Methylomicrobium alcaliphilum 20Z. The ectR-like genes were also found in halotolerant methanol utilizers Methylophaga alcalica and Methylophaga thalassica as well as in several genomes of nonmethylotrophic species. The His(6)-tagged DABA acetyltransferases from Mm. alcaliphilum, M. alcalica, and M. thalassica were purified and the enzyme properties were found to correlate with the ecophysiologies of these bacteria. All these discoveries should be very helpful for better understanding the biosynthetic mechanism of this important natural compound, and for the targeted metabolic engineering of its producers.

  1. A new monoterpene acid from Marrubium vulgare with potential antihepatotoxic activity.

    Science.gov (United States)

    Ahmed, Bahar; Masoodi, Mubashir H; Siddique, Anwarul H; Khan, Shamshir

    2010-11-01

    The whole plant of Marrubium vulgare L. afforded a new terpenoid, characterised as p-menthane-5,6-dihydroxy-3-carboxylic acid (1), which has been designated as marrubic acid. Its structure has been elucidated on the basis of spectral and chemical analyses. The compound (1) also exhibited a significant antihepatotoxic activity by reducing the elevated levels of serum enzymes such as serum glutamate oxaloacetate transaminase (SGOT) by 40.16%, serum glutamate pyruvate oxaloacetate transaminase (SGPT) by 35.06%, and alkaline phosphatase (ALP) by 30.51%. On the other hand, total protein (TP) levels were increased by 34.07%, as compared to the standard drug silymarin, which decreased SGOT by 53.04%, SGPT by 55.96%, ALP by 35.87% and increased TP levels by 59.59%. These biochemical observations were also supplemented by histopathological examinations of liver sections.

  2. Structural investigation of the β-cyclodextrin complexes with chiral bicyclic monoterpenes - Influence of the functionality group on the host-guest stoichiometry

    Science.gov (United States)

    Ceborska, Magdalena

    2017-10-01

    The crystal structures of the complexes of β-cyclodextrin with (+)- and (-)-camphors are presented. The comparison of the obtained crystal structures with available data for other complexes of β-cyclodextrin with chiral bicyclic monoterpenes (hydrocarbon (+)-fenchene and alcohols: (-)-isopinocampheol, and (+)-, and (-)-borneols) obtained from Cambridge Structural Database (CSD) shows the trend of alcohols to form dimeric complexes of 2:3 stoichiometry, while hydrocarbons and ketones prefer to form 2:2 host-guest inclusion complexes.

  3. Monoterpene isolated from the essential oil of Trachyspermum ammi is cytotoxic to multidrug-resistant Pseudomonas aeruginosa and Staphylococcus aureus strains

    Directory of Open Access Journals (Sweden)

    Faride Hosseinkhani

    2016-04-01

    Full Text Available Abstract INTRODUCTION: The aim of this study was to determine whether an herbal extract containing monoterpene exhibited activity against multidrug-resistant Staphylococcus aureus and Pseudomonas aeruginosa isolated from clinical infection samples. METHODS: The essential oil of Trachyspermum ammi (L. Sprague ex Turrill (Apiaceae fruit was extracted by hydrodistillation. Fruit residues were treated with hydrochloric acid and re-hydrodistilled to obtain volatile compounds. Compounds in the distilled oil were identified using gas-chromatography (GC and GC-mass spectrometry (MS. The antibiotic susceptibility of all bacterial isolates was analyzed using both the disc diffusion method and determination of the minimum inhibitory concentration (MIC. The sensitivity of antibiotic-resistant isolates to essential oil was also determined by using the disc diffusion method and MIC determination. RESULTS: Of 26 clinical isolates, 92% were multidrug-resistant (MDR. Aromatic monoterpenes (thymol, paracymene, and gamma-terpinene were the major (90% components of the oil. Growth of S. aureus strains was successfully inhibited by the oil, with an inhibitory zone diameter (IZD between 30-60mm and MIC <0.02μL/mL. The oil had no antimicrobial activity against clinical isolates of P. aeruginosa; rather, it prevented pigment production in these isolates. CONCLUSIONS: This study revealed that the essential oil of Trachyspermum ammi, which contains monoterpene, has good antibacterial potency. Monoterpenes could thus be incorporated into antimicrobial ointment formulas in order to treat highly drug-resistant S. aureus infections. Our findings also underscore the utility of research on natural products in order to combat bacterial multidrug resistance.

  4. Ice core records of monoterpene- and isoprene-SOA tracers from Aurora Peak in Alaska since 1660s: Implication for climate change variability in the North Pacific Rim

    Science.gov (United States)

    Pokhrel, Ambarish; Kawamura, Kimitaka; Ono, Kaori; Seki, Osamu; Fu, Pingqing; Matoba, Sumio; Shiraiwa, Takayuki

    2016-04-01

    Monoterpene and isoprene secondary organic aerosol (SOA) tracers are reported for the first time in an Alaskan ice core to better understand the biological source strength before and after the industrial revolution in the Northern Hemisphere. We found significantly high concentrations of monoterpene- and isoprene-SOA tracers (e.g., pinic, pinonic, and 2-methylglyceric acids, 2-methylthreitol and 2-methylerythritol) in the ice core, which show historical trends with good correlation to each other since 1660s. They show positive correlations with sugar compounds (e.g., mannitol, fructose, glucose, inositol and sucrose), and anti-correlations with α-dicarbonyls (glyoxal and methylglyoxal) and fatty acids (e.g., C18:1) in the same ice core. These results suggest similar sources and transport pathways for monoterpene- and isoprene-SOA tracers. In addition, we found that concentrations of C5-alkene triols (e.g., 3-methyl-2,3,4-trihydroxy-1-butene, cis-2-methyl 1,3,4-trihydroxy-1-butene and trans-2-methyl-1,3,4-trihydroxy-1-butene) in the ice core have increased after the Great Pacific Climate Shift (late 1970s). They show positive correlations with α-dicarbonyls and fatty acids (e.g., C18:1) in the ice core, suggesting that enhanced oceanic emissions of biogenic organic compounds through the marine boundary layer are recorded in the ice core from Alaska. Photochemical oxidation process for these monoterpene- and isoprene-/sesquiterpene-SOA tracers are suggested to be linked with the periodicity of multi-decadal climate oscillations and retreat of sea ice in the Northern Hemisphere.

  5. Evaluation of fast enantioselective multidimensional gas chromatography methods for monoterpenic compounds: Authenticity control of Australian tea tree oil.

    Science.gov (United States)

    Wong, Yong Foo; West, Rachel N; Chin, Sung-Tong; Marriott, Philip J

    2015-08-07

    This work demonstrates the potential of fast multiple heart-cut enantioselective multidimensional gas chromatography (GC-eGC) and enantioselective comprehensive two-dimensional gas chromatography (eGC×GC), to perform the stereoisomeric analysis of three key chiral monoterpenes (limonene, terpinen-4-ol