WorldWideScience

Sample records for terpenoid biosynthesis pathway

  1. Terpenoid biosynthesis in prokaryotes.

    Science.gov (United States)

    Boronat, Albert; Rodríguez-Concepción, Manuel

    2015-01-01

    Prokaryotic organisms (archaea and eubacteria) are found in all habitats where life exists on our planet. This would not be possible without the astounding biochemical plasticity developed by such organisms. Part of the metabolic diversity of prokaryotes was transferred to eukaryotic cells when endosymbiotic prokaryotes became mitochondria and plastids but also in a large number of horizontal gene transfer episodes. A group of metabolites produced by all free-living organisms is terpenoids (also known as isoprenoids). In prokaryotes, terpenoids play an indispensable role in cell-wall and membrane biosynthesis (bactoprenol, hopanoids), electron transport (ubiquinone, menaquinone), or conversion of light into chemical energy (chlorophylls, bacteriochlorophylls, rhodopsins, carotenoids), among other processes. But despite their remarkable structural and functional diversity, they all derive from the same metabolic precursors. Here we describe the metabolic pathways producing these universal terpenoid units and provide a complete picture of the main terpenoid compounds found in prokaryotic organisms.

  2. Simulation of a Petri net-based model of the terpenoid biosynthesis pathway.

    Science.gov (United States)

    Hawari, Aliah Hazmah; Mohamed-Hussein, Zeti-Azura

    2010-02-09

    The development and simulation of dynamic models of terpenoid biosynthesis has yielded a systems perspective that provides new insights into how the structure of this biochemical pathway affects compound synthesis. These insights may eventually help identify reactions that could be experimentally manipulated to amplify terpenoid production. In this study, a dynamic model of the terpenoid biosynthesis pathway was constructed based on the Hybrid Functional Petri Net (HFPN) technique. This technique is a fusion of three other extended Petri net techniques, namely Hybrid Petri Net (HPN), Dynamic Petri Net (HDN) and Functional Petri Net (FPN). The biological data needed to construct the terpenoid metabolic model were gathered from the literature and from biological databases. These data were used as building blocks to create an HFPNe model and to generate parameters that govern the global behaviour of the model. The dynamic model was simulated and validated against known experimental data obtained from extensive literature searches. The model successfully simulated metabolite concentration changes over time (pt) and the observations correlated with known data. Interactions between the intermediates that affect the production of terpenes could be observed through the introduction of inhibitors that established feedback loops within and crosstalk between the pathways. Although this metabolic model is only preliminary, it will provide a platform for analysing various high-throughput data, and it should lead to a more holistic understanding of terpenoid biosynthesis.

  3. Terpenoids and Their Biosynthesis in Cyanobacteria

    Directory of Open Access Journals (Sweden)

    Bagmi Pattanaik

    2015-01-01

    Full Text Available Terpenoids, or isoprenoids, are a family of compounds with great structural diversity which are essential for all living organisms. In cyanobacteria, they are synthesized from the methylerythritol-phosphate (MEP pathway, using glyceraldehyde 3-phosphate and pyruvate produced by photosynthesis as substrates. The products of the MEP pathway are the isomeric five-carbon compounds isopentenyl diphosphate and dimethylallyl diphosphate, which in turn form the basic building blocks for formation of all terpenoids. Many terpenoid compounds have useful properties and are of interest in the fields of pharmaceuticals and nutrition, and even potentially as future biofuels. The MEP pathway, its function and regulation, and the subsequent formation of terpenoids have not been fully elucidated in cyanobacteria, despite its relevance for biotechnological applications. In this review, we summarize the present knowledge about cyanobacterial terpenoid biosynthesis, both regarding the native metabolism and regarding metabolic engineering of cyanobacteria for heterologous production of non-native terpenoids.

  4. Biosynthesis of terpenoid natural products in fungi.

    Science.gov (United States)

    Schmidt-Dannert, Claudia

    2015-01-01

    Tens of thousands of terpenoid natural products have been isolated from plants and microbial sources. Higher fungi (Ascomycota and Basidiomycota) are known to produce an array of well-known terpenoid natural products, including mycotoxins, antibiotics, antitumor compounds, and phytohormones. Except for a few well-studied fungal biosynthetic pathways, the majority of genes and biosynthetic pathways responsible for the biosynthesis of a small number of these secondary metabolites have only been discovered and characterized in the past 5-10 years. This chapter provides a comprehensive overview of the current knowledge on fungal terpenoid biosynthesis from biochemical, genetic, and genomic viewpoints. Enzymes involved in synthesizing, transferring, and cyclizing the prenyl chains that form the hydrocarbon scaffolds of fungal terpenoid natural products are systematically discussed. Genomic information and functional evidence suggest differences between the terpenome of the two major fungal phyla--the Ascomycota and Basidiomycota--which will be illustrated for each group of terpenoid natural products.

  5. De novo assembly of Eugenia uniflora L. transcriptome and identification of genes from the terpenoid biosynthesis pathway.

    Science.gov (United States)

    Guzman, Frank; Kulcheski, Franceli Rodrigues; Turchetto-Zolet, Andreia Carina; Margis, Rogerio

    2014-12-01

    Pitanga (Eugenia uniflora L.) is a member of the Myrtaceae family and is of particular interest due to its medicinal properties that are attributed to specialized metabolites with known biological activities. Among these molecules, terpenoids are the most abundant in essential oils that are found in the leaves and represent compounds with potential pharmacological benefits. The terpene diversity observed in Myrtaceae is determined by the activity of different members of the terpene synthase and oxidosqualene cyclase families. Therefore, the aim of this study was to perform a de novo assembly of transcripts from E. uniflora leaves and to annotation to identify the genes potentially involved in the terpenoid biosynthesis pathway and terpene diversity. In total, 72,742 unigenes with a mean length of 1048bp were identified. Of these, 43,631 and 36,289 were annotated with the NCBI non-redundant protein and Swiss-Prot databases, respectively. The gene ontology categorized the sequences into 53 functional groups. A metabolic pathway analysis with KEGG revealed 8,625 unigenes assigned to 141 metabolic pathways and 40 unigenes predicted to be associated with the biosynthesis of terpenoids. Furthermore, we identified four putative full-length terpene synthase genes involved in sesquiterpenes and monoterpenes biosynthesis, and three putative full-length oxidosqualene cyclase genes involved in the triterpenes biosynthesis. The expression of these genes was validated in different E. uniflora tissues. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  6. Comparative analysis of the terpenoid biosynthesis pathway in Azadirachta indica and Melia azedarach by RNA-seq.

    Science.gov (United States)

    Wang, Yuwei; Chen, Xiang; Wang, Jin; Xun, Hang; Sun, Jia; Tang, Feng

    2016-01-01

    Azadirachta indica (neem) is the only source of azadirachtin, which is known for its insecticide activity. Melia azedarach is a related species of A. indica, widely distributed in the south of China. In this study, the leaf transcriptomes of these two Meliaceae plants were sequenced. More than 40 million clean reads were generated from each library. About 80 % of A. indica reads were mapped to the neem genome, while 93 % of M. azedarach reads were mapped to its assembled transcripts and unigenes dateset. After mapping and assembly, 225,972 transcripts and 91,607 unigenes of M. azedarach were obtained and 1179 new genes of A. indica were detected. Comparative analysis of the annotated differentially expressed genes (DEG) showed that all six DEGs involved in terpenoid backbone biosynthesis were up-regulated in A. indica. Chemical analysis of the two plants revealed A. indica leaves contained 2.45 % total terpenoid and nearly 20-50 µg azadirachtin per gram, whereas azadirachtin was not detected in M. azedarach and total terpenoid content was reached 1.67 %. These results give us a better insight into the transcriptomes differences between A. indica and M. azedarach, and help us to understand the terpenoid biosynthesis pathway in vivo.

  7. Metabolic cross-talk between pathways of terpenoid backbone biosynthesis in spike lavender.

    Science.gov (United States)

    Mendoza-Poudereux, Isabel; Kutzner, Erika; Huber, Claudia; Segura, Juan; Eisenreich, Wolfgang; Arrillaga, Isabel

    2015-10-01

    The metabolic cross-talk between the mevalonate (MVA) and the methylerythritol phosphate (MEP) pathways in developing spike lavender (Lavandula latifolia Med) was analyzed using specific inhibitors and on the basis of (13)C-labeling experiments. The presence of mevinolin (MEV), an inhibitor of the MVA pathway, at concentrations higher than 0.5 μM significantly reduced plant development, but not the synthesis of chlorophylls and carotenoids. On the other hand, fosmidomycin (FSM), an inhibitor of the MEP pathway, at concentrations higher than 20 μM blocked the synthesis of chlorophyll, carotenoids and essential oils, and significantly reduced stem development. Notably, 1.2 mM MVA could recover the phenotype of MEV-treated plants, including the normal growth and development of roots, and could partially restore the biosynthesis of photosynthetic pigments and, to a lesser extent, of the essential oils in plantlets treated with FSM. Spike lavender shoot apices were also used in (13)C-labeling experiments, where the plantlets were grown in the presence of [U-(13)C6]glucose. GC-MS-analysis of 1,8-cineole and camphor indicated that the C5-precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) of both monoterpenes are predominantly biosynthesized via the methylerythritol phosphate (MEP) pathway. However, on the basis of the isotopologue profiles, a minor contribution of the MVA pathway was evident that was increased in transgenic spike lavender plants overexpressing the 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), the first enzyme of the MVA pathway. Together, these findings provide evidence for a transport of MVA-derived precursors from the cytosol to the plastids in leaves of spike lavender. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  8. Biosynthesis and biological functions of terpenoids in plants.

    Science.gov (United States)

    Tholl, Dorothea

    2015-01-01

    Terpenoids (isoprenoids) represent the largest and most diverse class of chemicals among the myriad compounds produced by plants. Plants employ terpenoid metabolites for a variety of basic functions in growth and development but use the majority of terpenoids for more specialized chemical interactions and protection in the abiotic and biotic environment. Traditionally, plant-based terpenoids have been used by humans in the food, pharmaceutical, and chemical industries, and more recently have been exploited in the development of biofuel products. Genomic resources and emerging tools in synthetic biology facilitate the metabolic engineering of high-value terpenoid products in plants and microbes. Moreover, the ecological importance of terpenoids has gained increased attention to develop strategies for sustainable pest control and abiotic stress protection. Together, these efforts require a continuous growth in knowledge of the complex metabolic and molecular regulatory networks in terpenoid biosynthesis. This chapter gives an overview and highlights recent advances in our understanding of the organization, regulation, and diversification of core and specialized terpenoid metabolic pathways, and addresses the most important functions of volatile and nonvolatile terpenoid specialized metabolites in plants.

  9. The biosynthesis of C5-C25 terpenoid compounds.

    Science.gov (United States)

    Dewick, Paul M

    2002-04-01

    This review covers recently-published experimental information on the biosynthesis of terpenoids in the range C5-C25. In addition to sections on the mevalonate and mevalonate-independent (deoxyxylulose phosphate) pathways, the review considers in turn hemiterpenoids, polyprenyl diphosphate synthases, monoterpenoids, sesquiterpenoids, diterpenoids, and sesterterpenoids. The literature from January 1998 to December 2000 is reviewed, with 248 references cited.

  10. Studies of plant terpenoid biosynthesis using 13C stable isotope labeling techniques (KIT Scientific Reports ; 7583)

    OpenAIRE

    Ghirardo, Andrea

    2011-01-01

    This thesis aims to deepen our understanding of plant terpenoid biosynthesis and the regulation of the 2-C-methylerythritol-4-phosphate pathway (MEP) with respect to isoprene and monoterpene biosynthesis in plant. For this purposes, stable isotope techniques were employed, providing information on the carbon sources for terpenoids biosynthesis and C fluxes within the MEP pathway at various spatial and temporal scales, as well as giving insight into the regulation of enzymes from the MEP pathway.

  11. An overview of the non-mevalonate pathway for terpenoid ...

    Indian Academy of Sciences (India)

    Terpenoids are known to have many important biological and physiological functions. Some of them are also known for their pharmaceutical significance. In the late nineties after the discovery of a novel non-mevalonate (non-MVA) pathway, the whole concept of terpenoid biosynthesis has changed. In higher plants, the ...

  12. The Terpenoid Biosynthesis Toolkit of Trichoderma.

    Science.gov (United States)

    Bansal, Ravindra; Mukherjee, Prasun Kumar

    2016-04-01

    The widely used biotechnologically important fungi belonging to the genus Trichoderma are rich sources of secondary metabolites. Even though the genomes of several Trichoderma spp. have been published, and data are available on the genes involved in biosynthesis of non-ribosomal peptide synthetases and polyketide synthases, no genome-wide data are available for the terpenoid biosynthesis machinery in these organisms. In the present study, we have identified the genes involved in terpene biosynthesis in the genomes of three Trichoderma spp., viz., T. virens, T. atroviride and T. reesei. While the genes involved in the condensation steps are highly conserved across the three species, these fungi differed in the number and organization of terpene cyclases. T. virens genome harbours eleven terpene cyclases, while T. atroviride harbours seven, and T. reeseisix in their genomes; seven, three and two being part of putative secondary metabolism related gene clusters.

  13. A Glimpse into the Biosynthesis of Terpenoids

    NARCIS (Netherlands)

    Abdallah, Ingy; Quax, Wim J.

    Terpenoids represent the largest class of natural products with a diverse array of structures and functions. Many terpenoids have reported therapeutic properties such as antimicrobial, anti-inflammatory, immunomodulatory and chemotherapeutic properties making them of great interest in the medical

  14. Transcriptome and metabolite analyses reveal the complex metabolic genes involved in volatile terpenoid biosynthesis in garden sage (Salvia officinalis).

    Science.gov (United States)

    Ali, Mohammed; Li, Penghui; She, Guangbiao; Chen, Daofu; Wan, Xiaochun; Zhao, Jian

    2017-11-22

    A large number of terpenoid compounds have been extracted from different tissues of S. officinalis. However, the molecular genetic basis of terpene biosynthesis pathways is virtually unknown. In this study, approximately 6.6 Gb of raw data were generated from the transcriptome of S. officinalis leaves using Illumina HiSeq 2000 sequencing. After filtering and removing the adapter sequences from the raw data, the number of reads reached 21 million, comprising 98 million of high-quality nucleotide bases. 48,671 unigenes were assembled de novo and annotated for establishing a valid database for studying terpenoid biosynthesis. We identified 135 unigenes that are putatively involved in terpenoid metabolism, including 70 mevalonate and methyl-erythritol phosphate pathways, terpenoid backbone biosynthesis genes, and 65 terpene synthase genes. Moreover, five terpene synthase genes were studied for their functions in terpenoid biosynthesis by using transgenic tobacco; most transgenic tobacco plants expressing these terpene synthetic genes produced increased amounts of terpenoids compared with wild-type control. The combined data analyses from the transcriptome and metabolome provide new insights into our understanding of the complex metabolic genes in terpenoid-rich sage, and our study paves the way for the future metabolic engineering of the biosynthesis of useful terpene compounds in S. officinalis.

  15. Transcriptome sequencing and expression analysis of terpenoid biosynthesis genes in Litsea cubeba.

    Science.gov (United States)

    Han, Xiao-Jiao; Wang, Yang-Dong; Chen, Yi-Cun; Lin, Li-Yuan; Wu, Qing-Ke

    2013-01-01

    Aromatic essential oils extracted from fresh fruits of Litsea cubeba (Lour.) Pers., have diverse medical and economic values. The dominant components in these essential oils are monoterpenes and sesquiterpenes. Understanding the molecular mechanisms of terpenoid biosynthesis is essential for improving the yield and quality of terpenes. However, the 40 available L. cubeba nucleotide sequences in the public databases are insufficient for studying the molecular mechanisms. Thus, high-throughput transcriptome sequencing of L. cubeba is necessary to generate large quantities of transcript sequences for the purpose of gene discovery, especially terpenoid biosynthesis related genes. Using Illumina paired-end sequencing, approximately 23.5 million high-quality reads were generated. De novo assembly yielded 68,648 unigenes with an average length of 834 bp. A total of 38,439 (56%) unigenes were annotated for their functions, and 35,732 and 25,806 unigenes could be aligned to the GO and COG database, respectively. By searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG), 16,130 unigenes were assigned to 297 KEGG pathways, and 61 unigenes, which contained the mevalonate and 2-C-methyl-D-erythritol 4-phosphate pathways, could be related to terpenoid backbone biosynthesis. Of the 12,963 unigenes, 285 were annotated to the terpenoid pathways using the PlantCyc database. Additionally, 14 terpene synthase genes were identified from the transcriptome. The expression patterns of the 16 genes related to terpenoid biosynthesis were analyzed by RT-qPCR to explore their putative functions. RNA sequencing was effective in identifying a large quantity of sequence information. To our knowledge, this study is the first exploration of the L. cubeba transcriptome, and the substantial amount of transcripts obtained will accelerate the understanding of the molecular mechanisms of essential oils biosynthesis. The results may help improve future genetic and genomics

  16. Transcriptome sequencing and expression analysis of terpenoid biosynthesis genes in Litsea cubeba.

    Directory of Open Access Journals (Sweden)

    Xiao-Jiao Han

    Full Text Available BACKGROUND: Aromatic essential oils extracted from fresh fruits of Litsea cubeba (Lour. Pers., have diverse medical and economic values. The dominant components in these essential oils are monoterpenes and sesquiterpenes. Understanding the molecular mechanisms of terpenoid biosynthesis is essential for improving the yield and quality of terpenes. However, the 40 available L. cubeba nucleotide sequences in the public databases are insufficient for studying the molecular mechanisms. Thus, high-throughput transcriptome sequencing of L. cubeba is necessary to generate large quantities of transcript sequences for the purpose of gene discovery, especially terpenoid biosynthesis related genes. RESULTS: Using Illumina paired-end sequencing, approximately 23.5 million high-quality reads were generated. De novo assembly yielded 68,648 unigenes with an average length of 834 bp. A total of 38,439 (56% unigenes were annotated for their functions, and 35,732 and 25,806 unigenes could be aligned to the GO and COG database, respectively. By searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG, 16,130 unigenes were assigned to 297 KEGG pathways, and 61 unigenes, which contained the mevalonate and 2-C-methyl-D-erythritol 4-phosphate pathways, could be related to terpenoid backbone biosynthesis. Of the 12,963 unigenes, 285 were annotated to the terpenoid pathways using the PlantCyc database. Additionally, 14 terpene synthase genes were identified from the transcriptome. The expression patterns of the 16 genes related to terpenoid biosynthesis were analyzed by RT-qPCR to explore their putative functions. CONCLUSION: RNA sequencing was effective in identifying a large quantity of sequence information. To our knowledge, this study is the first exploration of the L. cubeba transcriptome, and the substantial amount of transcripts obtained will accelerate the understanding of the molecular mechanisms of essential oils biosynthesis. The

  17. Metabolic engineering of terpenoid biosynthesis in plants

    NARCIS (Netherlands)

    Aharoni, A.; Jongsma, M.A.; Kim, T.Y.; Ri, M.B.; Giri, A.P.; Verstappen, F.W.A.; Schwab, W.; Bouwmeester, H.J.

    2006-01-01

    Metabolic engineering of terpenoids in plants is a fascinating research topic from two main perspectives. On the one hand, the various biological activities of these compounds make their engineering a new tool for improving a considerable number of traits in crops. These include for example enhanced

  18. Biosynthetic pathway of terpenoid indole alkaloids in Catharanthus roseus.

    Science.gov (United States)

    Zhu, Xiaoxuan; Zeng, Xinyi; Sun, Chao; Chen, Shilin

    2014-09-01

    Catharanthus roseus is one of the most extensively investigated medicinal plants, which can produce more than 130 alkaloids, including the powerful antitumor drugs vinblastine and vincristine. Here we review the recent advances in the biosynthetic pathway of terpenoid indole alkaloids (TIAs) in C. roseus, and the identification and characterization of the corresponding enzymes involved in this pathway. Strictosidine is the central intermediate in the biosynthesis of different TIAs, which is formed by the condensation of secologanin and tryptamine. Secologanin is derived from terpenoid (isoprenoid) biosynthetic pathway, while tryptamine is derived from indole biosynthetic pathway. Then various specific end products are produced by different routes during downstream process. Although many genes and corresponding enzymes have been characterized in this pathway, our knowledge on the whole TIA biosynthetic pathway still remains largely unknown up to date. Full elucidation of TIA biosynthetic pathway is an important prerequisite to understand the regulation of the TIA biosynthesis in the medicinal plant and to produce valuable TIAs by synthetic biological technology.

  19. Chrysolina herbacea modulates terpenoid biosynthesis of Mentha aquatica L.

    Science.gov (United States)

    Atsbaha Zebelo, Simon; Bertea, Cinzia M; Bossi, Simone; Occhipinti, Andrea; Gnavi, Giorgio; Maffei, Massimo E

    2011-03-09

    Interactions between herbivorous insects and plants storing terpenoids are poorly understood. This study describes the ability of Chrysolina herbacea to use volatiles emitted by undamaged Mentha aquatica plants as attractants and the plant's response to herbivory, which involves the production of deterrent molecules. Emitted plant volatiles were analyzed by GC-MS. The insect's response to plant volatiles was tested by Y-tube olfactometer bioassays. Total RNA was extracted from control plants, mechanically damaged leaves, and leaves damaged by herbivores. The terpenoid quantitative gene expressions (qPCR) were then assayed. Upon herbivory, M. aquatica synthesizes and emits (+)-menthofuran, which acts as a deterrent to C. herbacea. Herbivory was found to up-regulate the expression of genes involved in terpenoid biosynthesis. The increased emission of (+)-menthofuran was correlated with the upregulation of (+)-menthofuran synthase.

  20. Chrysolina herbacea modulates terpenoid biosynthesis of Mentha aquatica L.

    Directory of Open Access Journals (Sweden)

    Simon Atsbaha Zebelo

    Full Text Available Interactions between herbivorous insects and plants storing terpenoids are poorly understood. This study describes the ability of Chrysolina herbacea to use volatiles emitted by undamaged Mentha aquatica plants as attractants and the plant's response to herbivory, which involves the production of deterrent molecules. Emitted plant volatiles were analyzed by GC-MS. The insect's response to plant volatiles was tested by Y-tube olfactometer bioassays. Total RNA was extracted from control plants, mechanically damaged leaves, and leaves damaged by herbivores. The terpenoid quantitative gene expressions (qPCR were then assayed. Upon herbivory, M. aquatica synthesizes and emits (+-menthofuran, which acts as a deterrent to C. herbacea. Herbivory was found to up-regulate the expression of genes involved in terpenoid biosynthesis. The increased emission of (+-menthofuran was correlated with the upregulation of (+-menthofuran synthase.

  1. Mitochondrial acetyl-CoA utilization pathway for terpenoid productions.

    Science.gov (United States)

    Yuan, Jifeng; Ching, Chi-Bun

    2016-11-01

    Acetyl-CoA is a central molecule in the metabolism of the cell, which is also a precursor molecule to a variety of value-added products such as terpenoids and fatty acid derived molecules. Considering subcellular compartmentalization of metabolic pathways allows higher concentrations of enzymes, substrates and intermediates, and bypasses competing pathways, mitochondrion-compartmentalized acetyl-CoA utilization pathways might offer better pathway activities with improved product yields. As a proof-of-concept, we sought to explore a mitochondrial farnesyl pyrophosphate (FPP) biosynthetic pathway for the biosynthesis of amorpha-4,11-diene in budding yeast. In the present study, the eight-gene FPP biosynthetic pathway was successfully expressed inside yeast mitochondria to enable high-level amorpha-4,11-diene production. In addition, we also found the mitochondrial compartment serves as a partial barrier for the translocation of FPP from mitochondria into the cytosol, which would potentially allow minimized loss of FPP to cytosolic competing pathways. To our best knowledge, this is the first report to harness yeast mitochondria for terpenoid productions from the mitochondrial acetyl-CoA pool. We envision subcellular metabolic engineering might also be employed for an efficient production of other bio-products from the mitochondrial acetyl-CoA in other eukaryotic organisms. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  2. Over-expression of DXS gene enhances terpenoidal secondary metabolite accumulation in rose-scented geranium and Withania somnifera: active involvement of plastid isoprenogenic pathway in their biosynthesis.

    Science.gov (United States)

    Jadaun, Jyoti Singh; Sangwan, Neelam S; Narnoliya, Lokesh K; Singh, Neha; Bansal, Shilpi; Mishra, Bhawana; Sangwan, Rajender Singh

    2017-04-01

    Rose-scented geranium (Pelargonium spp.) is one of the most important aromatic plants and is well known for its diverse perfumery uses. Its economic importance is due to presence of fragrance rich essential oil in its foliage. The essential oil is a mixture of various volatile phytochemicals which are mainly terpenes (isoprenoids) in nature. In this study, on the geranium foliage genes related to isoprenoid biosynthesis (DXS, DXR and HMGR) were isolated, cloned and confirmed by sequencing. Further, the first gene of 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway, 1-deoxy-d-xylulose-5-phosphate synthase (GrDXS), was made full length by using rapid amplification of cDNA ends strategy. GrDXS contained a 2157 bp open reading frame that encoded a polypeptide of 792 amino acids having calculated molecular weight 77.5 kDa. This study is first report on heterologous expression and kinetic characterization of any gene from this economically important plant. Expression analysis of these genes was performed in different tissues as well as at different developmental stages of leaves. In response to external elicitors, such as methyl jasmonate, salicylic acid, light and wounding, all the three genes showed differential expression profiles. Further GrDXS was over expressed in the homologous (rose-scented geranium) as well as in heterologous (Withania somnifera) plant systems through genetic transformation approach. The over-expression of GrDXS led to enhanced secondary metabolites production (i.e. essential oil in rose-scented geranium and withanolides in W. somnifera). To the best of our knowledge, this is the first report showing the expression profile of the three genes related to isoprenoid biosynthesis pathways operated in rose-scented geranium as well as functional characterization study of any gene from rose-scented geranium through a genetic transformation system. © 2016 Scandinavian Plant Physiology Society.

  3. De Novo Transcriptome Analysis of Warburgia ugandensis to Identify Genes Involved in Terpenoids and Unsaturated Fatty Acids Biosynthesis.

    Science.gov (United States)

    Wang, Xin; Zhou, Chen; Yang, Xianpeng; Miao, Di; Zhang, Yansheng

    2015-01-01

    The bark of Warburgia ugandensis (Canellaceae family) has been used as a medicinal source for a long history in many African countries. The presence of diverse terpenoids and abundant polyunsaturated fatty acids (PUFAs) in this organ contributes to its broad range of pharmacological properties. Despite its medicinal and economic importance, the knowledge on the biosynthesis of terpenoid and unsaturated fatty acid in W. ugandensis bark remains largely unknown. Therefore, it is necessary to construct a genomic and/or transcriptomic database for the functional genomics study on W. ugandensis. The chemical profiles of terpenoids and fatty acids between the bark and leaves of W. ugandensis were compared by gas chromatography-mass spectrometry (GC-MS) analysis. Meanwhile, the transcriptome database derived from both tissues was created using Illumina sequencing technology. In total, about 17.1 G clean nucleotides were obtained, and de novo assembled into 72,591 unigenes, of which about 38.06% can be aligned to the NCBI non-redundant protein database. Many candidate genes in the biosynthetic pathways of terpenoids and unsaturated fatty acids were identified, including 14 unigenes for terpene synthases. Furthermore, 2,324 unigenes were discovered to be differentially expressed between both tissues; the functions of those differentially expressed genes (DEGs) were predicted by gene ontology enrichment and metabolic pathway enrichment analyses. In addition, the expression of 12 DEGs with putative roles in terpenoid and unsaturated fatty acid metabolic pathways was confirmed by qRT-PCRs, which was consistent with the data of the RNA-sequencing. In conclusion, we constructed a comprehensive transcriptome dataset derived from the bark and leaf of W. ugandensis, which forms the basis for functional genomics studies on this plant species. Particularly, the comparative analysis of the transcriptome data between the bark and leaf will provide critical clues to reveal the regulatory

  4. De Novo Transcriptome Analysis of Warburgia ugandensis to Identify Genes Involved in Terpenoids and Unsaturated Fatty Acids Biosynthesis

    Science.gov (United States)

    Wang, Xin; Zhou, Chen; Yang, Xianpeng; Miao, Di; Zhang, Yansheng

    2015-01-01

    The bark of Warburgia ugandensis (Canellaceae family) has been used as a medicinal source for a long history in many African countries. The presence of diverse terpenoids and abundant polyunsaturated fatty acids (PUFAs) in this organ contributes to its broad range of pharmacological properties. Despite its medicinal and economic importance, the knowledge on the biosynthesis of terpenoid and unsaturated fatty acid in W. ugandensis bark remains largely unknown. Therefore, it is necessary to construct a genomic and/or transcriptomic database for the functional genomics study on W. ugandensis. The chemical profiles of terpenoids and fatty acids between the bark and leaves of W. ugandensis were compared by gas chromatography-mass spectrometry (GC-MS) analysis. Meanwhile, the transcriptome database derived from both tissues was created using Illumina sequencing technology. In total, about 17.1 G clean nucleotides were obtained, and de novo assembled into 72,591 unigenes, of which about 38.06% can be aligned to the NCBI non-redundant protein database. Many candidate genes in the biosynthetic pathways of terpenoids and unsaturated fatty acids were identified, including 14 unigenes for terpene synthases. Furthermore, 2,324 unigenes were discovered to be differentially expressed between both tissues; the functions of those differentially expressed genes (DEGs) were predicted by gene ontology enrichment and metabolic pathway enrichment analyses. In addition, the expression of 12 DEGs with putative roles in terpenoid and unsaturated fatty acid metabolic pathways was confirmed by qRT-PCRs, which was consistent with the data of the RNA-sequencing. In conclusion, we constructed a comprehensive transcriptome dataset derived from the bark and leaf of W. ugandensis, which forms the basis for functional genomics studies on this plant species. Particularly, the comparative analysis of the transcriptome data between the bark and leaf will provide critical clues to reveal the regulatory

  5. Novel bioassay for the discovery of inhibitors of the 2-C-methyl-D-erythritol 4-phosphate (MEP and terpenoid pathways leading to carotenoid biosynthesis.

    Directory of Open Access Journals (Sweden)

    Natália Corniani

    Full Text Available The 2-C-methyl-D-erythritol 4-phosphate (MEP pathway leads to the synthesis of isopentenyl diphosphate in plastids. It is a major branch point providing precursors for the synthesis of carotenoids, tocopherols, plastoquinone and the phytyl chain of chlorophylls, as well as the hormones abscisic acid and gibberellins. Consequently, disruption of this pathway is harmful to plants. We developed an in vivo bioassay that can measure the carbon flow through the carotenoid pathway. Leaf cuttings are incubated in the presence of a phytoene desaturase inhibitor to induce phytoene accumulation. Any compound reducing the level of phytoene accumulation is likely to interfere with either one of the steps in the MEP pathway or the synthesis of geranylgeranyl diphosphate. This concept was tested with known inhibitors of steps of the MEP pathway. The specificity of this in vivo bioassay was also verified by testing representative herbicides known to target processes outside of the MEP and carotenoid pathways. This assay enables the rapid screen of new inhibitors of enzymes preceding the synthesis of phytoene, though there are some limitations related to the non-specific effect of some inhibitors on this assay.

  6. Novel bioassay for the discovery of inhibitors of the 2-C-methyl-D-erythritol 4-phosphate (MEP) and terpenoid pathways leading to carotenoid biosynthesis.

    Science.gov (United States)

    Corniani, Natália; Velini, Edivaldo D; Silva, Ferdinando M L; Nanayakkara, N P Dhammika; Witschel, Matthias; Dayan, Franck E

    2014-01-01

    The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway leads to the synthesis of isopentenyl diphosphate in plastids. It is a major branch point providing precursors for the synthesis of carotenoids, tocopherols, plastoquinone and the phytyl chain of chlorophylls, as well as the hormones abscisic acid and gibberellins. Consequently, disruption of this pathway is harmful to plants. We developed an in vivo bioassay that can measure the carbon flow through the carotenoid pathway. Leaf cuttings are incubated in the presence of a phytoene desaturase inhibitor to induce phytoene accumulation. Any compound reducing the level of phytoene accumulation is likely to interfere with either one of the steps in the MEP pathway or the synthesis of geranylgeranyl diphosphate. This concept was tested with known inhibitors of steps of the MEP pathway. The specificity of this in vivo bioassay was also verified by testing representative herbicides known to target processes outside of the MEP and carotenoid pathways. This assay enables the rapid screen of new inhibitors of enzymes preceding the synthesis of phytoene, though there are some limitations related to the non-specific effect of some inhibitors on this assay.

  7. Novel bioassay for the discovery of inhibitors of the 2-C-Methyl-D-Erythritol 4-Phosphate (MEP) and terpenoid pathways leading to carotenoid biosynthesis

    Science.gov (United States)

    The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway leads to the synthesis of isopentenyl-phosphate (IPP) in plastids. It is a major branch point providing precursors for the synthesis of carotenoids, tocopherols, plastoquinone and the phytyl chain of chlorophylls, as well as the hormones abscisi...

  8. Analysis of iridoids content and expression studies of genes encoding early enzymes in the indol terpenoid biosynthesis pathway in Catharanthus roseus Análisis de iridoides y expresión de genes que codifican enzimas tempranas en la síntesis de alcaloides indol terpenoicos en Catharanthus roseus

    OpenAIRE

    Leech Mark; Palacios-Rojas Natalia

    2004-01-01

    Terpenoid indole alkaloids (TIA) are of pharmaceutical importance, however the industrial use of these compouds is very limited because its accumulation is very low in plant tissues. TIA are derived f rom the shikimate and terpenoid pathways, which supply secologanin and tryptamine, the indole and iridoid moieties, respectively. Secololganin is a terpenoid which is belived to be synthesised the MEP pathway rather than by the acetate/mevalonic acid pathway. Secologanin is thought to be a limit...

  9. Terpenoid biosynthesis in Euphorbia lathyris and Copaifera spp

    Energy Technology Data Exchange (ETDEWEB)

    Skrukrud, C.L.

    1987-07-01

    Biosynthesis of triterpenoids by isolated latex of Euphorbia lathyris was investigated. The rate of in vitro incorporation of mevalonic acid into triterpenoids was thirty times greater than acetate incorporation indicating that the rate-limiting step in the pathway occurs prior to mevalonate. Both HMG-CoA reductase (EC 1.1.1.34) and HMG-CoA lyase (EC 4.1.3.4) activities were detected in isolated latex. HMG-CoA reductase was localized to a membrane-bound fraction of a 5000g pellet of latex. The rate of conversion of HMG-CoA to mevalonate by this enzyme is comparable to the overall rate of acetate incorporation into the triterpenoids suggesting that this enzyme is rate-determining in the biosynthesis of triterpenoids in E. lathyris latex. HMG-CoA reductase of E. lathyris vegetative tissue was localized to the membrane-bound portion of a particulate fraction (18,000g), and was solubilized by treatment with 2% polyoxyethylene ether W-1. Differences in the optimal pH for activity of HMG-CoA reductase from the latex and vegetative tissue suggest that isozymes of the enzyme may be present in the two tissue types. Studies of the incorporation of various precursors into leaf discs and cuttings taken from Copaifera spp. show differences in the rate of incorporation into Copaifera sesquiterpenes suggesting that the site of sesquiterpene biosynthesis may differ in its accessibility to the different substrates and/or reflecting the metabolic controls on carbon allocation to the terpenes. Mevalonate incorporation by Copaifera langsdorfii cuttings into sesquiterpenes was a hundred-fold greater than either acetate or glucose incorporation, however, its incorporation into squalene and triterpenoids was also a hundred-fold greater than the incorporation into sesquiterpenes. 119 refs., 58 figs., 16 tabs.

  10. Estrogenic terpenes and terpenoids: Pathways, functions and applications.

    Science.gov (United States)

    Kiyama, Ryoiti

    2017-11-15

    Terpenes are made of the isoprene unit (C 5 ), and along with their derivatives, terpenoids, they are widely distributed in plants as active ingredients involved in anti-inflammation, anti-carcinogenesis and neuroprotection. Estrogenic terpenes and terpenoids are an important category of phytoestrogens and have been used as traditional medicines. The comprehensive list of estrogenic terpenes and terpenoids includes hemi-, mono-, sesqui-, di-, tri-, tetra- and polyterpenes, their derivatives, and meroterpenes, along with the signaling pathways and cellular functions on which their estrogenicity is exerted. Signaling pathways are further classified as bidirectional or unidirectional, the latter being further divided into two types depending upon the presence of both ligands, or the absence of one or both ligands. Although estrogenic activity of terpenes and terpenoids was evaluated by ligand-binding assays, yeast two-hybrid assays, reporter-gene assays, transcription assays, protein assays, cell assays and animal testing, the mechanism of estrogenic activity is still not fully understood. Applications of estrogenic terpenes and terpenoids are categorized into cancer treatment and prevention, cardioprotection, endocrine toxicity/reproductive dysfunction, food/supplement/traditional medicine, immunology/inflammation, menopausal syndromes and neuroprotection, where their benefits are discussed based on their availability, stability and variations. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Volatile terpenoids: multiple functions, biosynthesis, modulation and manipulation by genetic engineering.

    Science.gov (United States)

    Abbas, Farhat; Ke, Yanguo; Yu, Rangcai; Yue, Yuechong; Amanullah, Sikandar; Jahangir, Muhammad Muzammil; Fan, Yanping

    2017-11-01

    Terpenoids play several physiological and ecological functions in plant life through direct and indirect plant defenses and also in human society because of their enormous applications in the pharmaceutical, food and cosmetics industries. Through the aid of genetic engineering its role can by magnified to broad spectrum by improving genetic ability of crop plants, enhancing the aroma quality of fruits and flowers and the production of pharmaceutical terpenoids contents in medicinal plants. Terpenoids are structurally diverse and the most abundant plant secondary metabolites, playing an important role in plant life through direct and indirect plant defenses, by attracting pollinators and through different interactions between the plants and their environment. Terpenoids are also significant because of their enormous applications in the pharmaceutical, food and cosmetics industries. Due to their broad distribution and functional versatility, efforts are being made to decode the biosynthetic pathways and comprehend the regulatory mechanisms of terpenoids. This review summarizes the recent advances in biosynthetic pathways, including the spatiotemporal, transcriptional and post-transcriptional regulatory mechanisms. Moreover, we discuss the multiple functions of the terpene synthase genes (TPS), their interaction with the surrounding environment and the use of genetic engineering for terpenoid production in model plants. Here, we also provide an overview of the significance of terpenoid metabolic engineering in crop protection, plant reproduction and plant metabolic engineering approaches for pharmaceutical terpenoids production and future scenarios in agriculture, which call for sustainable production platforms by improving different plant traits.

  12. Genome-wide identification and characterization of novel genes involved in terpenoid biosynthesis in Salvia miltiorrhiza.

    Science.gov (United States)

    Ma, Yimian; Yuan, Lichai; Wu, Bin; Li, Xian'en; Chen, Shilin; Lu, Shanfa

    2012-04-01

    Terpenoids are the largest class of plant secondary metabolites and have attracted widespread interest. Salvia miltiorrhiza, belonging to the largest and most widely distributed genus in the mint family, is a model medicinal plant with great economic and medicinal value. Diterpenoid tanshinones are the major lipophilic bioactive components in S. miltiorrhiza. Systematic analysis of genes involved in terpenoid biosynthesis has not been reported to date. Searching the recently available working draft of the S. miltiorrhiza genome, 40 terpenoid biosynthesis-related genes were identified, of which 27 are novel. These genes are members of 19 families, which encode all of the enzymes involved in the biosynthesis of the universal isoprene precursor isopentenyl diphosphate and its isomer dimethylallyl diphosphate, and two enzymes associated with the biosynthesis of labdane-related diterpenoids. Through a systematic analysis, it was found that 20 of the 40 genes could be involved in tanshinone biosynthesis. Using a comprehensive approach, the intron/exon structures and expression patterns of all identified genes and their responses to methyl jasmonate treatment were analysed. The conserved domains and phylogenetic relationships among the deduced S. miltiorrhiza proteins and their homologues isolated from other plant species were revealed. It was discovered that some of the key enzymes, such as 1-deoxy-D-xylulose 5-phosphate synthase, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, hydroxymethylglutaryl-CoA reductase, and geranylgeranyl diphosphate synthase, are encoded by multiple gene members with different expression patterns and subcellular localizations, and both homomeric and heteromeric geranyl diphosphate synthases exist in S. miltiorrhiza. The results suggest the complexity of terpenoid biosynthesis and the existence of metabolic channels for diverse terpenoids in S. miltiorrhiza and provide useful information for improving tanshinone production through genetic

  13. Engineering the MEP pathway enhanced ajmalicine biosynthesis.

    Science.gov (United States)

    Chang, Kai; Qiu, Fei; Chen, Min; Zeng, Lingjiang; Liu, Xiaoqiang; Yang, Chunxian; Lan, Xiaozhong; Wang, Qiang; Liao, Zhihua

    2014-01-01

    The 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway genes encoding DXR and MECS from Taxus species and STR from Catharanthus roseus were used to genetically modify the ajmalicine biosynthetic pathway in hairy root cultures of C. roseus. As expected, the STR-overexpressed root cultures showed twofold higher accumulation of ajmalicine than the control. It was important to discover that overexpression of the single DXR or MECS gene from the MEP pathway also remarkably enhanced ajmalicine biosynthesis in transgenic hairy root cultures, and this suggested that engineering the MEP pathway by overexpression of DXR or MECS promoted the metabolic flux into ajmalicine biosynthesis. The transgenic hairy root cultures with co-overexpression of DXR and STR or MECS and STR had higher levels of ajmalicine than those with overexpression of a single gene alone such as DXR, MECS, and STR. It could be concluded that transgenic hairy root cultures harboring both DXR/MECS and STR possessed an increased flux in the terpenoid indole alkaloid biosynthetic pathway that enhanced ajmalicine yield, which was more efficient than cultures harboring only one of the three genes. © 2013 International Union of Biochemistry and Molecular Biology, Inc.

  14. Terpenoid biosynthesis in Arabidopsis attacked by caterpillars and aphids

    NARCIS (Netherlands)

    Kroes, Anneke; Weldegergis, Berhane T.; Cappai, Francesco; Dicke, Marcel; Loon, van Joop J.A.

    2017-01-01

    One of the responses of plants to insect attack is the production of volatile organic compounds that mediate indirect defence of plants by attracting natural enemies of the attacking herbivores. Herbivore-induced plant volatiles (HIPVs) include terpenoids that play key roles in the attraction of

  15. Identification of novel knockout targets for improving terpenoids biosynthesis in Saccharomyces cerevisiae.

    Science.gov (United States)

    Sun, Zhiqiang; Meng, Hailin; Li, Jing; Wang, Jianfeng; Li, Qian; Wang, Yong; Zhang, Yansheng

    2014-01-01

    Many terpenoids have important pharmacological activity and commercial value; however, application of these terpenoids is often limited by problems associated with the production of sufficient amounts of these molecules. The use of Saccharomyces cerevisiae (S. cerevisiae) for the production of heterologous terpenoids has achieved some success. The objective of this study was to identify S. cerevisiae knockout targets for improving the synthesis of heterologous terpeniods. On the basis of computational analysis of the S. cerevisiae metabolic network, we identified the knockout sites with the potential to promote terpenoid production and the corresponding single mutant was constructed by molecular manipulations. The growth rates of these strains were measured and the results indicated that the gene deletion had no adverse effects. Using the expression of amorphadiene biosynthesis as a testing model, the gene deletion was assessed for its effect on the production of exogenous terpenoids. The results showed that the dysfunction of most genes led to increased production of amorphadiene. The yield of amorphadiene produced by most single mutants was 8-10-fold greater compared to the wild type, indicating that the knockout sites can be engineered to promote the synthesis of exogenous terpenoids.

  16. Chrysolina herbacea Modulates Terpenoid Biosynthesis of Mentha aquatica L.

    OpenAIRE

    Simon Atsbaha Zebelo; Bertea, Cinzia M.; Simone Bossi; Andrea Occhipinti; Giorgio Gnavi; Maffei, Massimo E.

    2011-01-01

    Interactions between herbivorous insects and plants storing terpenoids are poorly understood. This study describes the ability of Chrysolina herbacea to use volatiles emitted by undamaged Mentha aquatica plants as attractants and the plant's response to herbivory, which involves the production of deterrent molecules. Emitted plant volatiles were analyzed by GC-MS. The insect's response to plant volatiles was tested by Y-tube olfactometer bioassays. Total RNA was extracted from control plants,...

  17. Digital Gene Expression Profiling to Explore Differentially Expressed Genes Associated with Terpenoid Biosynthesis during Fruit Development in Litsea cubeba

    Directory of Open Access Journals (Sweden)

    Ming Gao

    2016-09-01

    Full Text Available Mountain pepper (Litsea cubeba (Lour. Pers. (Lauraceae is an important industrial crop as an ingredient in cosmetics, pesticides, food additives and potential biofuels. These properties are attributed to monoterpenes and sesquiterpenes. However, there is still no integrated model describing differentially expressed genes (DEGs involved in terpenoid biosynthesis during the fruit development of L. cubeba. Here, we performed digital gene expression (DGE using the Illumina NGS platform to evaluated changes in gene expression during fruit development in L. cubeba. DGE generated expression data for approximately 19354 genes. Fruit at 60 days after flowering (DAF served as the control, and a total of 415, 1255, 449 and 811 up-regulated genes and 505, 1351, 1823 and 1850 down-regulated genes were identified at 75, 90, 105 and 135 DAF, respectively. Pathway analysis revealed 26 genes involved in terpenoid biosynthesis pathways. Three DEGs had continued increasing or declining trends during the fruit development. The quantitative real-time PCR (qRT-PCR results of five differentially expressed genes were consistent with those obtained from Illumina sequencing. These results provide a comprehensive molecular biology background for research on fruit development, and information that should aid in metabolic engineering to increase the yields of L. cubeba essential oil.

  18. Transcriptome Analysis of Thapsia laciniata Rouy Provides Insights into Terpenoid Biosynthesis and Diversity in Apiaceae

    Directory of Open Access Journals (Sweden)

    Henrik Toft Simonsen

    2013-04-01

    Full Text Available Thapsia laciniata Rouy (Apiaceae produces irregular and regular sesquiterpenoids with thapsane and guaiene carbon skeletons, as found in other Apiaceae species. A transcriptomic analysis utilizing Illumina next-generation sequencing enabled the identification of novel genes involved in the biosynthesis of terpenoids in Thapsia. From 66.78 million HQ paired-end reads obtained from T. laciniata roots, 64.58 million were assembled into 76,565 contigs (N50: 1261 bp. Seventeen contigs were annotated as terpene synthases and five of these were predicted to be sesquiterpene synthases. Of the 67 contigs annotated as cytochromes P450, 18 of these are part of the CYP71 clade that primarily performs hydroxylations of specialized metabolites. Three contigs annotated as aldehyde dehydrogenases grouped phylogenetically with the characterized ALDH1 from Artemisia annua and three contigs annotated as alcohol dehydrogenases grouped with the recently described ADH1 from A. annua. ALDH1 and ADH1 were characterized as part of the artemisinin biosynthesis. We have produced a comprehensive EST dataset for T. laciniata roots, which contains a large sample of the T. laciniata transcriptome. These transcriptome data provide the foundation for future research into the molecular basis for terpenoid biosynthesis in Thapsia and on the evolution of terpenoids in Apiaceae.

  19. Genomics-Based Discovery of Plant Genes for Synthetic Biology of Terpenoid Fragrances: A Case Study in Sandalwood oil Biosynthesis.

    Science.gov (United States)

    Celedon, J M; Bohlmann, J

    2016-01-01

    Terpenoid fragrances are powerful mediators of ecological interactions in nature and have a long history of traditional and modern industrial applications. Plants produce a great diversity of fragrant terpenoid metabolites, which make them a superb source of biosynthetic genes and enzymes. Advances in fragrance gene discovery have enabled new approaches in synthetic biology of high-value speciality molecules toward applications in the fragrance and flavor, food and beverage, cosmetics, and other industries. Rapid developments in transcriptome and genome sequencing of nonmodel plant species have accelerated the discovery of fragrance biosynthetic pathways. In parallel, advances in metabolic engineering of microbial and plant systems have established platforms for synthetic biology applications of some of the thousands of plant genes that underlie fragrance diversity. While many fragrance molecules (eg, simple monoterpenes) are abundant in readily renewable plant materials, some highly valuable fragrant terpenoids (eg, santalols, ambroxides) are rare in nature and interesting targets for synthetic biology. As a representative example for genomics/transcriptomics enabled gene and enzyme discovery, we describe a strategy used successfully for elucidation of a complete fragrance biosynthetic pathway in sandalwood (Santalum album) and its reconstruction in yeast (Saccharomyces cerevisiae). We address questions related to the discovery of specific genes within large gene families and recovery of rare gene transcripts that are selectively expressed in recalcitrant tissues. To substantiate the validity of the approaches, we describe the combination of methods used in the gene and enzyme discovery of a cytochrome P450 in the fragrant heartwood of tropical sandalwood, responsible for the fragrance defining, final step in the biosynthesis of (Z)-santalols. © 2016 Elsevier Inc. All rights reserved.

  20. Differential microRNA Analysis of Glandular Trichomes and Young Leaves in Xanthium strumarium L. Reveals Their Putative Roles in Regulating Terpenoid Biosynthesis.

    Directory of Open Access Journals (Sweden)

    Rongyan Fan

    Full Text Available The medicinal plant Xanthium strumarium L. (X. strumarium is covered with glandular trichomes, which are the sites for synthesizing pharmacologically active terpenoids such as xanthatin. MicroRNAs (miRNAs are a class of 21-24 nucleotide (nt non-coding RNAs, most of which are identified as regulators of plant growth development. Identification of miRNAs involved in the biosynthesis of plant secondary metabolites remains limited. In this study, high-throughput Illumina sequencing, combined with target gene prediction, was performed to discover novel and conserved miRNAs with potential roles in regulating terpenoid biosynthesis in X. strumarium glandular trichomes. Two small RNA libraries from leaves and glandular trichomes of X. strumarium were established. In total, 1,185 conserved miRNAs and 37 novel miRNAs were identified, with 494 conserved miRNAs and 18 novel miRNAs being differentially expressed between the two tissue sources. Based on the X. strumarium transcriptome data that we recently constructed, 3,307 annotated mRNA transcripts were identified as putative targets of the differentially expressed miRNAs. KEGG (Kyoto Encyclopedia of Genes and Genomes pathway analysis suggested that some of the differentially expressed miRNAs, including miR6435, miR5021 and miR1134, might be involved in terpenoid biosynthesis in the X. strumarium glandular trichomes. This study provides the first comprehensive analysis of miRNAs in X. strumarium, which forms the basis for further understanding of miRNA-based regulation on terpenoid biosynthesis.

  1. Differential microRNA Analysis of Glandular Trichomes and Young Leaves in Xanthium strumarium L. Reveals Their Putative Roles in Regulating Terpenoid Biosynthesis.

    Science.gov (United States)

    Fan, Rongyan; Li, Yuanjun; Li, Changfu; Zhang, Yansheng

    2015-01-01

    The medicinal plant Xanthium strumarium L. (X. strumarium) is covered with glandular trichomes, which are the sites for synthesizing pharmacologically active terpenoids such as xanthatin. MicroRNAs (miRNAs) are a class of 21-24 nucleotide (nt) non-coding RNAs, most of which are identified as regulators of plant growth development. Identification of miRNAs involved in the biosynthesis of plant secondary metabolites remains limited. In this study, high-throughput Illumina sequencing, combined with target gene prediction, was performed to discover novel and conserved miRNAs with potential roles in regulating terpenoid biosynthesis in X. strumarium glandular trichomes. Two small RNA libraries from leaves and glandular trichomes of X. strumarium were established. In total, 1,185 conserved miRNAs and 37 novel miRNAs were identified, with 494 conserved miRNAs and 18 novel miRNAs being differentially expressed between the two tissue sources. Based on the X. strumarium transcriptome data that we recently constructed, 3,307 annotated mRNA transcripts were identified as putative targets of the differentially expressed miRNAs. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis suggested that some of the differentially expressed miRNAs, including miR6435, miR5021 and miR1134, might be involved in terpenoid biosynthesis in the X. strumarium glandular trichomes. This study provides the first comprehensive analysis of miRNAs in X. strumarium, which forms the basis for further understanding of miRNA-based regulation on terpenoid biosynthesis.

  2. Silencing the Transcriptional Repressor, ZCT1, Illustrates the Tight Regulation of Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus Hairy Roots.

    Directory of Open Access Journals (Sweden)

    Noreen F Rizvi

    Full Text Available The Catharanthus roseus plant is the source of many valuable terpenoid indole alkaloids (TIAs, including the anticancer compounds vinblastine and vincristine. Transcription factors (TFs are promising metabolic engineering targets due to their ability to regulate multiple biosynthetic pathway genes. To increase TIA biosynthesis, we elicited the TIA transcriptional activators (ORCAs and other unidentified TFs with the plant hormone, methyl jasmonate (MJ, while simultaneously silencing the expression of the transcriptional repressor ZCT1. To silence ZCT1, we developed transgenic hairy root cultures of C. roseus that expressed an estrogen-inducible Zct1 hairpin for activating RNA interference. The presence of 17β-estradiol (5μM effectively depleted Zct1 in hairy root cultures elicited with MJ dosages that either optimize or inhibit TIA production (250 or 1000μM. However, silencing Zct1 was not sufficient to increase TIA production or the expression of the TIA biosynthetic genes (G10h, Tdc, and Str, illustrating the tight regulation of TIA biosynthesis. The repression of the TIA biosynthetic genes at the inhibitory MJ dosage does not appear to be solely regulated by ZCT1. For instance, while Zct1 and Zct2 levels decreased through activating the Zct1 hairpin, Zct3 levels remained elevated. Since ZCT repressors have redundant yet distinct functions, silencing all three ZCTs may be necessary to relieve their repression of alkaloid biosynthesis.

  3. Silencing the Transcriptional Repressor, ZCT1, Illustrates the Tight Regulation of Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus Hairy Roots.

    Science.gov (United States)

    Rizvi, Noreen F; Weaver, Jessica D; Cram, Erin J; Lee-Parsons, Carolyn W T

    2016-01-01

    The Catharanthus roseus plant is the source of many valuable terpenoid indole alkaloids (TIAs), including the anticancer compounds vinblastine and vincristine. Transcription factors (TFs) are promising metabolic engineering targets due to their ability to regulate multiple biosynthetic pathway genes. To increase TIA biosynthesis, we elicited the TIA transcriptional activators (ORCAs and other unidentified TFs) with the plant hormone, methyl jasmonate (MJ), while simultaneously silencing the expression of the transcriptional repressor ZCT1. To silence ZCT1, we developed transgenic hairy root cultures of C. roseus that expressed an estrogen-inducible Zct1 hairpin for activating RNA interference. The presence of 17β-estradiol (5μM) effectively depleted Zct1 in hairy root cultures elicited with MJ dosages that either optimize or inhibit TIA production (250 or 1000μM). However, silencing Zct1 was not sufficient to increase TIA production or the expression of the TIA biosynthetic genes (G10h, Tdc, and Str), illustrating the tight regulation of TIA biosynthesis. The repression of the TIA biosynthetic genes at the inhibitory MJ dosage does not appear to be solely regulated by ZCT1. For instance, while Zct1 and Zct2 levels decreased through activating the Zct1 hairpin, Zct3 levels remained elevated. Since ZCT repressors have redundant yet distinct functions, silencing all three ZCTs may be necessary to relieve their repression of alkaloid biosynthesis.

  4. Flax terpenoid pathway as a source of health promoting compounds.

    Science.gov (United States)

    Styrczewska, Monika; Kulma, Anna; Kostyn, Kamil; Hasiewicz-Derkacz, Karolina; Szopa, Jan

    2013-03-01

    Flax is an important crop plant grown mainly for its fiber and seeds, which are also rich in omega-3 fatty acids and valuable antioxidants derived from the terpenoid pathways including carotenoids, tocochromanols and sterols. Many of those components found in flax have been recently shown to positively influence human health. Although terpenes vary greatly in their chemical structure, mainly two mechanisms of their biological activity can be considered: direct antioxidation, and a recently explored one, connected to specific receptor and cell signaling pathway activation. Recent studies show that many of the health promoting agents derived from flax act through both of the mentioned mechanisms, resulting in synergistic physiological effects. The work summarizes the two mechanisms, focusing mainly on the one involving cell signaling, as a promising target for medicine and pharmacotherapy.

  5. Transcriptome analysis of distinct Lindera glauca tissues revealed the differences in the unigenes related to terpenoid biosynthesis.

    Science.gov (United States)

    Niu, Jun; Hou, Xinyu; Fang, Chengliang; An, Jiyong; Ha, Denglong; Qiu, Lin; Ju, Yuxi; Zhao, Haiyan; Du, WenZhi; Qi, Ji; Zhang, Zhixiang; Liu, Genan; Lin, Shanzhi

    2015-03-15

    The Lindera glauca, an economically and ecologically important tree species, has emerged as a novel potential plant for the intensive studies of essential oil owing to its characteristic aroma and medicinal property in distinct tissues. However, the transcriptome information and molecular research on this species is still unknown to date. To reveal the formation and accumulation mechanism of essential oil in distinct L. glauca tissues, it is crucial to analyze transcriptome and to identify the full repertoire of potential unigenes involved in terpenoid biosynthesis. In this paper, the transcriptomes of the roots, sarcocarps, stems, leaves and kernels of L. glauca were analyzed for the first time by using short-read sequencing technology (Illumina). A total of 27.2GB valid reads (the average length=92.7bp) was obtained from distinct L. glauca tissues, and then assembled de novo into 264,831 unigenes by Trinity strategy (mean size=560.2bp). The resulting 98,141 unigenes (38%) of all the assembled unigenes were annotated in multiple public databases, of which 114 potential unigenes were identified to be involved in the terpenoid biosynthetic accumulation in L. glauca. Additionally, the differential expression profiles revealed 675, 697, 432, 1702 and 844 high tissue-specificity expressions of unigenes in the roots, sarcocarps, stems, leaves and kernels of L. glauca, respectively. Overall, these obtained comprehensive unigene resources will contribute to advance the research regarding the specific plant and more specifically discovery of genes participating in the terpenoid pathway and its regulation in specific tissues of the L. glauca, but also could help the understanding of the differential accumulation of secondary metabolites in distinct plant tissues. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Large-Scale Evolutionary Analysis of Genes and Supergene Clusters from Terpenoid Modular Pathways Provides Insights into Metabolic Diversification in Flowering Plants

    Science.gov (United States)

    Hofberger, Johannes A.; Ramirez, Aldana M.; van den Bergh, Erik; Zhu, Xinguang; Bouwmeester, Harro J.; Schuurink, Robert C.; Schranz, M. Eric

    2015-01-01

    An important component of plant evolution is the plethora of pathways producing more than 200,000 biochemically diverse specialized metabolites with pharmacological, nutritional and ecological significance. To unravel dynamics underlying metabolic diversification, it is critical to determine lineage-specific gene family expansion in a phylogenomics framework. However, robust functional annotation is often only available for core enzymes catalyzing committed reaction steps within few model systems. In a genome informatics approach, we extracted information from early-draft gene-space assemblies and non-redundant transcriptomes to identify protein families involved in isoprenoid biosynthesis. Isoprenoids comprise terpenoids with various roles in plant-environment interaction, such as pollinator attraction or pathogen defense. Combining lines of evidence provided by synteny, sequence homology and Hidden-Markov-Modelling, we screened 17 genomes including 12 major crops and found evidence for 1,904 proteins associated with terpenoid biosynthesis. Our terpenoid genes set contains evidence for 840 core terpene-synthases and 338 triterpene-specific synthases. We further identified 190 prenyltransferases, 39 isopentenyl-diphosphate isomerases as well as 278 and 219 proteins involved in mevalonate and methylerithrol pathways, respectively. Assessing the impact of gene and genome duplication to lineage-specific terpenoid pathway expansion, we illustrated key events underlying terpenoid metabolic diversification within 250 million years of flowering plant radiation. By quantifying Angiosperm-wide versatility and phylogenetic relationships of pleiotropic gene families in terpenoid modular pathways, our analysis offers significant insight into evolutionary dynamics underlying diversification of plant secondary metabolism. Furthermore, our data provide a blueprint for future efforts to identify and more rapidly clone terpenoid biosynthetic genes from any plant species. PMID

  7. Large-Scale Evolutionary Analysis of Genes and Supergene Clusters from Terpenoid Modular Pathways Provides Insights into Metabolic Diversification in Flowering Plants.

    Science.gov (United States)

    Hofberger, Johannes A; Ramirez, Aldana M; Bergh, Erik van den; Zhu, Xinguang; Bouwmeester, Harro J; Schuurink, Robert C; Schranz, M Eric

    2015-01-01

    An important component of plant evolution is the plethora of pathways producing more than 200,000 biochemically diverse specialized metabolites with pharmacological, nutritional and ecological significance. To unravel dynamics underlying metabolic diversification, it is critical to determine lineage-specific gene family expansion in a phylogenomics framework. However, robust functional annotation is often only available for core enzymes catalyzing committed reaction steps within few model systems. In a genome informatics approach, we extracted information from early-draft gene-space assemblies and non-redundant transcriptomes to identify protein families involved in isoprenoid biosynthesis. Isoprenoids comprise terpenoids with various roles in plant-environment interaction, such as pollinator attraction or pathogen defense. Combining lines of evidence provided by synteny, sequence homology and Hidden-Markov-Modelling, we screened 17 genomes including 12 major crops and found evidence for 1,904 proteins associated with terpenoid biosynthesis. Our terpenoid genes set contains evidence for 840 core terpene-synthases and 338 triterpene-specific synthases. We further identified 190 prenyltransferases, 39 isopentenyl-diphosphate isomerases as well as 278 and 219 proteins involved in mevalonate and methylerithrol pathways, respectively. Assessing the impact of gene and genome duplication to lineage-specific terpenoid pathway expansion, we illustrated key events underlying terpenoid metabolic diversification within 250 million years of flowering plant radiation. By quantifying Angiosperm-wide versatility and phylogenetic relationships of pleiotropic gene families in terpenoid modular pathways, our analysis offers significant insight into evolutionary dynamics underlying diversification of plant secondary metabolism. Furthermore, our data provide a blueprint for future efforts to identify and more rapidly clone terpenoid biosynthetic genes from any plant species.

  8. Methyl Jasmonate Induces Traumatic Resin Ducts, Terpenoid Resin Biosynthesis, and Terpenoid Accumulation in Developing Xylem of Norway Spruce Stems

    National Research Council Canada - National Science Library

    Diane Martin; Dorothea Tholl; Jonathan Gershenzon; Jörg Bohlmann

    2002-01-01

    Norway spruce (Picea abies L. Karst) produces an oleoresin characterized by a diverse array of terpenoids, monoterpenoids, sesquiterpenoids, and diterpene resin acids that can protect conifers against potential herbivores and pathogens...

  9. Genetic engineering approach using early Vinca alkaloid biosynthesis genes led to increased tryptamine and terpenoid indole alkaloids biosynthesis in differentiating cultures of Catharanthus roseus.

    Science.gov (United States)

    Sharma, Abhishek; Verma, Priyanka; Mathur, Archana; Mathur, Ajay Kumar

    2017-08-14

    Catharanthus roseus today occupies the central position in ongoing metabolic engineering efforts in medicinal plants. The entire multi-step biogenetic pathway of its very expensive anticancerous alkaloids vinblastine and vincristine is fairly very well dissected at biochemical and gene levels except the pathway steps leading to biosynthesis of monomeric alkaloid catharanthine and tabersonine. In order to enhance the plant-based productivity of these pharma molecules for the drug industry, cell and tissue cultures of C. roseus are being increasingly tested to provide their alternate production platforms. However, a rigid developmental regulation and involvement of different cell, tissues, and organelles in the synthesis of these alkaloids have restricted the utility of these cultures. Therefore, the present study was carried out with pushing the terpenoid indole alkaloid pathway metabolic flux towards dimeric alkaloids vinblastine and vincristine production by over-expressing the two upstream pathway genes tryptophan decarboxylase and strictosidine synthase at two different levels of cellular organization viz. callus and leaf tissues. The transformation experiments were carried out using Agrobacterium tumefaciens LBA1119 strain having tryptophan decarboxylase and strictosidine synthase gene cassette. The callus transformation reported a maximum of 0.027% dry wt vindoline and 0.053% dry wt catharanthine production, whereas, the transiently transformed leaves reported a maximum of 0.30% dry wt vindoline, 0.10% catharanthine, and 0.0027% dry wt vinblastine content.

  10. Transcriptomic insight into terpenoid and carbazole alkaloid biosynthesis, and functional characterization of two terpene synthases in curry tree (Murraya koenigii).

    Science.gov (United States)

    Meena, Seema; Rajeev Kumar, Sarma; Dwivedi, Varun; Kumar Singh, Anup; Chanotiya, Chandan S; Akhtar, Md Qussen; Kumar, Krishna; Kumar Shasany, Ajit; Nagegowda, Dinesh A

    2017-03-08

    Curry tree (Murraya koenigii L.) is a rich source of aromatic terpenes and pharmacologically important carbazole alkaloids. Here, M. koenigii leaf transcriptome was generated to gain insight into terpenoid and alkaloid biosynthesis. Analysis of de novo assembled contigs yielded genes for terpene backbone biosynthesis and terpene synthases. Also, gene families possibly involved in carbazole alkaloid formation were identified that included polyketide synthases, prenyltransferases, methyltransferases and cytochrome P450s. Further, two genes encoding terpene synthases (MkTPS1 and MkTPS2) with highest in silico transcript abundance were cloned and functionally characterized to determine their involvement in leaf volatile formation. Subcellular localization using GFP fusions revealed the plastidial and cytosolic localization of MkTPS1 and MkTPS2, respectively. Enzymatic characterization demonstrated the monoterpene synthase activity of recombinant MkTPS1, which produced primarily (-)-sabinene from geranyl diphosphate (GPP). Recombinant MkTPS2 exhibited sesquiterpene synthase activity and formed (E,E)-α-farnesene as the major product from farnesyl diphosphate (FPP). Moreover, mRNA expression and leaf volatile analyses indicated that MkTPS1 accounts for (-)-sabinene emitted by M. koenigii leaves. Overall, the transcriptome data generated in this study will be a great resource and the start point for characterizing genes involved in the biosynthetic pathway of medicinally important carbazole alkaloids.

  11. Proteomics of terpenoid biosynthesis and secretion in trichomes of higher plant species.

    Science.gov (United States)

    Champagne, Antoine; Boutry, Marc

    2016-08-01

    Among the specialized (secondary) plant metabolites, terpenoids represent the most diverse family and are often involved in the defense against pathogens and herbivores. Terpenoids can be produced both constitutively and in response to the environment. At the front line of this defense strategy are the glandular trichomes, which are organs dedicated primarily to the production of specialized metabolites. Mass spectrometry-based proteomics is a powerful tool, which is very useful to investigate enzymes involved in metabolic pathways, such as the synthesis and secretion of terpenoids in glandular trichomes. Here we review the strategies used to investigate the specific roles of these particular organs from non-model plant species, mainly belonging to the Lamiaceae, Solanaceae, and Cannabaceae families. We discuss how proteomics helps to accurately pinpoint candidate proteins to be functionally characterized, and how technological progresses create opportunities for studying low-abundance proteins, such as the ones related to the synthesis and transport of specialized metabolites. This article is part of a Special Issue entitled: Plant Proteomics--a bridge between fundamental processes and crop production, edited by Dr. Hans-Peter Mock. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. The ORCA2 transcription factor plays a key role in regulation of the terpenoid indole alkaloid pathway.

    Science.gov (United States)

    Li, Chun Yao; Leopold, Alex L; Sander, Guy W; Shanks, Jacqueline V; Zhao, Le; Gibson, Susan I

    2013-10-08

    The terpenoid indole alkaloid (TIA) pathway leads to the production of pharmaceutically important drugs, such as the anticancer compounds vinblastine and vincristine. Unfortunately, these drugs are produced in trace amounts, causing them to be very costly. To increase production of these drugs, an improved understanding of the TIA regulatory pathway is needed. Towards this end, transgenic Catharanthus roseus hairy roots that overexpress the ORCA2 TIA transcriptional activator were generated and characterized. Transcriptional profiling experiments revealed that overexpression of ORCA2 results in altered expression of key genes from the indole and terpenoid pathways, which produce precursors for the TIA pathway, and from the TIA pathway itself. In addition, metabolite-profiling experiments revealed that overexpression of ORCA2 significantly affects the levels of several TIA metabolites. ORCA2 overexpression also causes significant increases in transcript levels of several TIA regulators, including TIA transcriptional repressors. Results presented here indicate that ORCA2 plays a critical role in regulation of TIA metabolism. ORCA2 regulates expression of key genes from both feeder pathways, as well as the genes (STR and SGD) encoding the enzymes that catalyze the first two steps in TIA biosynthesis. ORCA2 may play an especially important role in regulation of the downstream branches of the TIA pathway, as it regulates four out of five genes characterized from this part of the pathway. Regulation of TIA transcriptional repressors by ORCA2 may provide a mechanism whereby increases in TIA metabolite levels in response to external stimuli are transient and limited in magnitude.

  13. Differential expressed analysis of Tripterygium wilfordii unigenes involved in terpenoid backbone biosynthesis.

    Science.gov (United States)

    Zhang, Jing; Huo, Yan-Bo; Liu, Yan; Feng, Jun-Tao; Ma, Zhi-Qing; Zhu, Chuan-Shu; Zhang, Xing

    2017-08-01

    Tripterygium wilfordii Hook. f. is the traditional medicinal plants in China. Triptolide, wilforgine, and wilforine are the bioactive compounds in T. wilfordii. In this study, the contents of three metabolites and transcription levels of 21 genes involved in three metabolites biosynthesis in T. wilfordii were examined using high-performance liquid chromatography and reverse transcription PCR after application of methyl jasmonate (MeJA) on hairy roots in time course experiment (3-24 h). The results indicated that application of MeJA inhibited triptolide accumulation and promoted wilforgine and wilforine metabolites biosynthesis. In hairy roots, wilforgine content reached 693.36 μg/g at 6 h after adding MeJA, which was 2.23-fold higher than control. The accumulation of triptolide and wilforine in hairy roots increased the maximum at 9 h, which was 1.3- and 1.6-folds more than the control. Most of the triptolide secretes into the medium, but wilforgine and wilforine cannot secrete into the medium. The expression levels of unigenes which involved terpenoid backbone biosynthesis exist the correlation with marker metabolites (triptolide, wilforgine and wilforine) after induction by MeJA, and can be then used to infer flux bottlenecks in T. wilfordii secondary metabolites accumulation. These results showed that these genes may have potential applications in the metabolic engineering of T. wilfordii metabolites production.

  14. Clade IVa Basic Helix-Loop-Helix Transcription Factors Form Part of a Conserved Jasmonate Signaling Circuit for the Regulation of Bioactive Plant Terpenoid Biosynthesis.

    Science.gov (United States)

    Mertens, Jan; Van Moerkercke, Alex; Vanden Bossche, Robin; Pollier, Jacob; Goossens, Alain

    2016-12-01

    Plants produce many bioactive, specialized metabolites to defend themselves when facing various stress situations. Their biosynthesis is directed by a tightly controlled regulatory circuit that is elicited by phytohormones such as jasmonate (JA). The basic helix-loop-helix (bHLH) transcription factors (TFs) bHLH iridoid synthesis 1 (BIS1) and Triterpene Saponin Activating Regulator (TSAR) 1 and 2, from Catharanthus roseus and Medicago truncatula, respectively, all belong to clade IVa of the bHLH protein family and activate distinct terpenoid pathways, thereby mediating monoterpenoid indole alkaloid (MIA) and triterpene saponin (TS) accumulation, respectively, in these two species. In this study, we report that promoters of the genes encoding the enzymes involved in the specific terpenoid pathway of one of these species can be transactivated by the orthologous bHLH factor from the other species through recognition of the same cis-regulatory elements. Accordingly, ectopic expression of CrBIS1 in M. truncatula hairy roots up-regulated the expression of all genes required for soyasaponin production, resulting in strongly increased levels of soyasaponins in the transformed roots. Likewise, transient expression of MtTSAR1 and MtTSAR2 in C. roseus petals led to up-regulation of the genes involved in the iridoid branch of the MIA pathway. Together, our data illustrate the functional similarity of these JA-inducible TFs and indicate that recruitment of defined cis-regulatory elements constitutes an important aspect of the evolution of conserved regulatory modules for the activation of species-specific terpenoid biosynthesis pathways by common signals such as the JA phytohormones. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  15. Analysis of the transcriptome of Isodon rubescens and key enzymes involved in terpenoid biosynthesis

    Directory of Open Access Journals (Sweden)

    Xiuhong Su

    2016-05-01

    Full Text Available Isodon rubescens is an important medicinal plant in China that has been shown to reduce tumour growth due to the presence of the compound oridonin. In an effort to facilitate molecular research on oridonin biosynthesis, we reported the use of next generation massively parallel sequencing technologies and de novo transcriptome assembly to gain a comprehensive overview of I. rubescens transcriptome. In our study, a total of 50,934,276 clean reads, 101,640 transcripts and 44,626 unigenes were generated through de novo transcriptome assembly. A number of unigenes – 23,987, 10,263, 7359, 18,245, 17,683, 19,485, 9361 – were annotated in the National Center for Biotechnology Information (NCBI non-redundant protein (Nr, NCBI nucleotide sequences (Nt, Kyoto Encyclopedia of Genes and Genomes (KEGG Orthology (KO, Swiss-Prot, protein family (Pfam, gene ontology (GO, eukaryotic ortholog groups (KOG databases, respectively. Furthermore, the annotated unigenes were functionally classified according to the GO, KOG and KEGG. Based on these results, candidate genes encoding enzymes involved in terpenoids backbone biosynthesis were detected. Our data provided the most comprehensive sequence resource available for the study on I. rubescens, as well as demonstrated the effective use of Illumina sequencing and de novo transcriptome assembly on a species lacking genomic information.

  16. Plant metabolic engineering strategies for the production of pharmaceutical terpenoids

    Directory of Open Access Journals (Sweden)

    Xu Lu

    2016-11-01

    Full Text Available Pharmaceutical terpenoids belong to the most diverse class of natural products. They have significant curative effects on a variety of diseases, such as cancer, cardiovascular diseases, malaria and Alzheimer’s disease. Nowadays, elicitors, including biotic and abiotic elicitors, are often used to activate the pathway of secondary metabolism and enhance the production of target terpenoids. Based on Agrobacterium-mediated genetic transformation, several plant metabolic engineering strategies hold great promise to regulate the biosynthesis of pharmaceutical terpenoids. Overexpressing terpenoids biosynthesis pathway genes in homologous and ectopic plants is an effective strategy to enhance the yield of pharmaceutical terpenoids. Another strategy is to suppress the expression of competitive metabolic pathways. In addition, global regulation which includes regulating the relative transcription factors, endogenous phytohormones and primary metabolism could also markedly increase their yield. All these strategies offer great opportunities to enhance the supply of scarce terpenoids drugs, reduce the price of expensive drugs and improve people's standards of living.

  17. Fungal endophytes of Catharanthus roseus enhance vindoline content by modulating structural and regulatory genes related to terpenoid indole alkaloid biosynthesis.

    Science.gov (United States)

    Pandey, Shiv S; Singh, Sucheta; Babu, C S Vivek; Shanker, Karuna; Srivastava, N K; Shukla, Ashutosh K; Kalra, Alok

    2016-05-25

    Not much is known about the mechanism of endophyte-mediated induction of secondary metabolite production in Catharanthus roseus. In the present study two fungal endophytes, Curvularia sp. CATDLF5 and Choanephora infundibulifera CATDLF6 were isolated from the leaves of the plant that were found to enhance vindoline content by 229-403%. The isolated endophytes did not affect the primary metabolism of the plant as the maximum quantum efficiency of PSII, net CO2 assimilation, plant biomass and starch content of endophyte-inoculated plants was similar to endophyte-free control plants. Expression of terpenoid indole alkaloid (TIA) pathway genes, geraniol 10-hydroxylase (G10H), tryptophan decarboxylase (TDC), strictosidine synthase (STR), 16-hydoxytabersonine-O-methyltransferase (16OMT), desacetoxyvindoline-4-hydroxylase (D4H), deacetylvindoline-4-O-acetyltransferase (DAT) were upregulated in endophyte-inoculated plants. Endophyte inoculation upregulated the expression of the gene for transcriptional activator octadecanoid-responsive Catharanthus AP2-domain protein (ORCA3) and downregulated the expression of Cys2/His2-type zinc finger protein family transcriptional repressors (ZCTs). The gene for the vacuolar class III peroxidase (PRX1), responsible for coupling vindoline and catharanthine, was upregulated in endophyte-inoculated plants. These endophytes may enhance vindoline production by modulating the expression of key structural and regulatory genes of vindoline biosynthesis without affecting the primary metabolism of the host plant.

  18. Fungal endophytes of Catharanthus roseus enhance vindoline content by modulating structural and regulatory genes related to terpenoid indole alkaloid biosynthesis

    Science.gov (United States)

    Pandey, Shiv S.; Singh, Sucheta; Babu, C. S. Vivek; Shanker, Karuna; Srivastava, N. K.; Shukla, Ashutosh K.; Kalra, Alok

    2016-01-01

    Not much is known about the mechanism of endophyte-mediated induction of secondary metabolite production in Catharanthus roseus. In the present study two fungal endophytes, Curvularia sp. CATDLF5 and Choanephora infundibulifera CATDLF6 were isolated from the leaves of the plant that were found to enhance vindoline content by 229–403%. The isolated endophytes did not affect the primary metabolism of the plant as the maximum quantum efficiency of PSII, net CO2 assimilation, plant biomass and starch content of endophyte-inoculated plants was similar to endophyte-free control plants. Expression of terpenoid indole alkaloid (TIA) pathway genes, geraniol 10-hydroxylase (G10H), tryptophan decarboxylase (TDC), strictosidine synthase (STR), 16-hydoxytabersonine-O-methyltransferase (16OMT), desacetoxyvindoline-4-hydroxylase (D4H), deacetylvindoline-4-O-acetyltransferase (DAT) were upregulated in endophyte-inoculated plants. Endophyte inoculation upregulated the expression of the gene for transcriptional activator octadecanoid-responsive Catharanthus AP2-domain protein (ORCA3) and downregulated the expression of Cys2/His2-type zinc finger protein family transcriptional repressors (ZCTs). The gene for the vacuolar class III peroxidase (PRX1), responsible for coupling vindoline and catharanthine, was upregulated in endophyte-inoculated plants. These endophytes may enhance vindoline production by modulating the expression of key structural and regulatory genes of vindoline biosynthesis without affecting the primary metabolism of the host plant. PMID:27220774

  19. Methyl Jasmonate Induces Traumatic Resin Ducts, Terpenoid Resin Biosynthesis, and Terpenoid Accumulation in Developing Xylem of Norway Spruce Stems1

    Science.gov (United States)

    Martin, Diane; Tholl, Dorothea; Gershenzon, Jonathan; Bohlmann, Jörg

    2002-01-01

    Norway spruce (Picea abies L. Karst) produces an oleoresin characterized by a diverse array of terpenoids, monoterpenoids, sesquiterpenoids, and diterpene resin acids that can protect conifers against potential herbivores and pathogens. Oleoresin accumulates constitutively in resin ducts in the cortex and phloem (bark) of Norway spruce stems. De novo formation of traumatic resin ducts (TDs) is observed in the developing secondary xylem (wood) after insect attack, fungal elicitation, and mechanical wounding. Here, we characterize the methyl jasmonate-induced formation of TDs in Norway spruce by microscopy, chemical analyses of resin composition, and assays of terpenoid biosynthetic enzymes. The response involves tissue-specific differentiation of TDs, terpenoid accumulation, and induction of enzyme activities of both prenyltransferases and terpene synthases in the developing xylem, a tissue that constitutively lacks axial resin ducts in spruce. The induction of a complex defense response in Norway spruce by methyl jasmonate application provides new avenues to evaluate the role of resin defenses for protection of conifers against destructive pests such as white pine weevils (Pissodes strobi), bark beetles (Coleoptera, Scolytidae), and insect-associated tree pathogens. PMID:12114556

  20. Method for determining heterologous biosynthesis pathways

    KAUST Repository

    Gao, Xin

    2017-08-10

    The present invention relates to a method and system for dynamically analyzing, determining, predicting and displaying ranked suitable heterologous biosynthesis pathways for a specified host. The present invention addresses the problem of finding suitable pathways for the endogenous metabolism of a host organism because the efficacy of heterologous biosynthesis is affected by competing endogenous pathways. The present invention is called MRE (Metabolic Route Explorer), and it was conceived and developed to systematically and dynamically search for, determine, analyze, and display promising heterologous pathways while considering competing endogenous reactions in a given host organism.

  1. The transcription factor CrWRKY1 positively regulates the terpenoid indole alkaloid biosynthesis in Catharanthus roseus.

    Science.gov (United States)

    Suttipanta, Nitima; Pattanaik, Sitakanta; Kulshrestha, Manish; Patra, Barunava; Singh, Sanjay K; Yuan, Ling

    2011-12-01

    Catharanthus roseus produces a large array of terpenoid indole alkaloids (TIAs) that are an important source of natural or semisynthetic anticancer drugs. The biosynthesis of TIAs is tissue specific and induced by certain phytohormones and fungal elicitors, indicating the involvement of a complex transcriptional control network. However, the transcriptional regulation of the TIA pathway is poorly understood. Here, we describe a C. roseus WRKY transcription factor, CrWRKY1, that is preferentially expressed in roots and induced by the phytohormones jasmonate, gibberellic acid, and ethylene. The overexpression of CrWRKY1 in C. roseus hairy roots up-regulated several key TIA pathway genes, especially Tryptophan Decarboxylase (TDC), as well as the transcriptional repressors ZCT1 (for zinc-finger C. roseus transcription factor 1), ZCT2, and ZCT3. However, CrWRKY1 overexpression repressed the transcriptional activators ORCA2, ORCA3, and CrMYC2. Overexpression of a dominant-repressive form of CrWRKY1, created by fusing the SRDX repressor domain to CrWRKY1, resulted in the down-regulation of TDC and ZCTs but the up-regulation of ORCA3 and CrMYC2. CrWRKY1 bound to the W box elements of the TDC promoter in electrophoretic mobility shift, yeast one-hybrid, and C. roseus protoplast assays. Up-regulation of TDC increased TDC activity, tryptamine concentration, and resistance to 4-methyl tryptophan inhibition of CrWRKY1 hairy roots. Compared with control roots, CrWRKY1 hairy roots accumulated up to 3-fold higher levels of serpentine. The preferential expression of CrWRKY1 in roots and its interaction with transcription factors including ORCA3, CrMYC2, and ZCTs may play a key role in determining the root-specific accumulation of serpentine in C. roseus plants.

  2. Melanin biosynthesis pathway in Agaricus bisporus mushrooms

    NARCIS (Netherlands)

    Weijn, A.; Bastiaan-Net, S.; Wichers, H.J.; Mes, J.J.

    2013-01-01

    With the full genome sequence of Agaricus bisporus available, it was possible to investigate the genes involved in the melanin biosynthesis pathway of button mushrooms. Based on different BLAST and alignments, genes were identified in the genome which are postulated to be involved in this pathway.

  3. A differentially regulated AP2/ERF transcription factor gene cluster acts downstream of a MAP kinase cascade to modulate terpenoid indole alkaloid biosynthesis in Catharanthus roseus.

    Science.gov (United States)

    Paul, Priyanka; Singh, Sanjay K; Patra, Barunava; Sui, Xueyi; Pattanaik, Sitakanta; Yuan, Ling

    2017-02-01

    Catharanthus roseus produces bioactive terpenoid indole alkaloids (TIAs), including the chemotherapeutics, vincristine and vinblastine. Transcriptional regulation of TIA biosynthesis is not fully understood. The jasmonic acid (JA)-responsive AP2/ERF transcription factor (TF), ORCA3, and its regulator, CrMYC2, play key roles in TIA biosynthesis. ORCA3 forms a physical cluster with two uncharacterized AP2/ERFs, ORCA4 and 5. Here, we report that (1) the ORCA gene cluster is differentially regulated; (2) ORCA4, while overlapping functionally with ORCA3, modulates an additional set of TIA genes. Unlike ORCA3, ORCA4 overexpression resulted in dramatic increase of TIA accumulation in C. roseus hairy roots. In addition, CrMYC2 is capable of activating ORCA3 and co-regulating TIA pathway genes concomitantly with ORCA3. The ORCA gene cluster and CrMYC2 act downstream of a MAP kinase cascade that includes a previously uncharacterized MAP kinase kinase, CrMAPKK1. Overexpression of CrMAPKK1 in C. roseus hairy roots upregulated TIA pathways genes and increased TIA accumulation. This work provides detailed characterization of a TF gene cluster and advances our understanding of the transcriptional and post-translational regulatory mechanisms that govern TIA biosynthesis in C. roseus. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  4. Terpenoid biosynthesis off the beaten track: unconventional cyclases and their impact on biomimetic synthesis.

    Science.gov (United States)

    Baunach, Martin; Franke, Jakob; Hertweck, Christian

    2015-02-23

    Terpene and terpenoid cyclizations are counted among the most complex chemical reactions occurring in nature and contribute crucially to the tremendous structural diversity of this largest family of natural products. Many studies were conducted at the chemical, genetic, and biochemical levels to gain mechanistic insights into these intriguing reactions that are catalyzed by terpene and terpenoid cyclases. A myriad of these enzymes have been characterized. Classical textbook knowledge divides terpene/terpenoid cyclases into two major classes according to their structure and reaction mechanism. However, recent discoveries of novel types of terpenoid cyclases illustrate that nature's enzymatic repertoire is far more diverse than initially thought. This Review outlines novel terpenoid cyclases that are out of the ordinary. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Positive Darwinian selection is a driving force for the diversification of terpenoid biosynthesis in the genus Oryza.

    Science.gov (United States)

    Chen, Hao; Li, Guanglin; Köllner, Tobias G; Jia, Qidong; Gershenzon, Jonathan; Chen, Feng

    2014-09-16

    Terpenoids constitute the largest class of secondary metabolites made by plants and display vast chemical diversity among and within species. Terpene synthases (TPSs) are the pivotal enzymes for terpenoid biosynthesis that create the basic carbon skeletons of this class. Functional divergence of paralogous and orthologous TPS genes is a major mechanism for the diversification of terpenoid biosynthesis. However, little is known about the evolutionary forces that have shaped the evolution of plant TPS genes leading to terpenoid diversity. The orthologs of Oryza Terpene Synthase 1 (OryzaTPS1), a rice terpene synthase gene involved in indirect defense against insects in Oryza sativa, were cloned from six additional Oryza species. In vitro biochemical analysis showed that the enzymes encoded by these OryzaTPS1 genes functioned either as (E)-β-caryophyllene synthases (ECS), or (E)-β-caryophyllene & germacrene A synthases (EGS), or germacrene D & germacrene A synthases (DAS). Because the orthologs of OryzaTPS1 in maize and sorghum function as ECS, the ECS activity was inferred to be ancestral. Molecular evolutionary detected the signature of positive Darwinian selection in five codon substitutions in the evolution from ECS to DAS. Homology-based structure modeling and the biochemical analysis of laboratory-generated protein variants validated the contribution of the five positively selected sites to functional divergence of OryzaTPS1. The changes in the in vitro product spectra of OryzaTPS1 proteins also correlated closely to the changes in in vivo blends of volatile terpenes released from insect-damaged rice plants. In this study, we found that positive Darwinian selection is a driving force for the functional divergence of OryzaTPS1. This finding suggests that the diverged sesquiterpene blend produced by the Oryza species containing DAS may be adaptive, likely in the attraction of the natural enemies of insect herbivores.

  6. Artemisinic Acid Serves as a Novel ORCA3 Inducer to Enhance Biosynthesis of Terpenoid Indole Alkaloids in Catharanthus roseus Cambial Meristematic Cells.

    Science.gov (United States)

    Wang, Mingxuan; Zi, Jiachen; Zhu, Jianhua; Chen, Shan; Wang, Pu; Song, Liyan; Yu, Rongmin

    2016-06-01

    To investigate the effect of artemisinic acid (AA) on improving the production of terpenoid indole alkaloids (TIAs) of Catharanthus roseus cambial meristematic cells (CMCs), feeding AA to C. roseus CMCs caused 2.35-fold and 2.51-fold increases in the production of vindoline and catharanthine, respectively, compared with those of the untreated CMCs. qRT-PCR experiments showed that AA resulted in a 1.36-8.52 fold increase in the transcript levels of several related genes, including octadecanoid-derivative responsive Catharanthus AP2-domain protein 3 (ORCA3), tryptophan decarboxylase (TDC), strictosidine synthase (STR) and desacetoxyvindoline 4-hydroxylase (D4H). However, no effect was observed on the concentration of either jasmonic acid (JA), or the octadecanoid-pathway inhibitors block TIA accumulation caused by AA. The results indicated that AA might serve as a novel ORCA3 inducer to manipulate biosynthesis of TIAs in C. roseus CMCs via an unknown mechanism.

  7. The miRNAome of Catharanthus roseus: identification, expression analysis, and potential roles of microRNAs in regulation of terpenoid indole alkaloid biosynthesis

    Science.gov (United States)

    Shen, Ethan M.; Singh, Sanjay K.; Ghosh, Jayadri S.; Patra, Barunava; Paul, Priyanka; Yuan, Ling; Pattanaik, Sitakanta

    2017-01-01

    MicroRNAs (miRNAs) regulate numerous crucial biological processes in plants. However, information is limited on their involvement in the biosynthesis of specialized metabolites in plants, including Catharanthus roseus that produces a number of pharmaceutically valuable, bioactive terpenoid indole alkaloids (TIAs). Using small RNA-sequencing, we identified 181 conserved and 173 novel miRNAs (cro-miRNAs) in C. roseus seedlings. Genome-wide expression analysis revealed that a set of cro-miRNAs are differentially regulated in response to methyl jasmonate (MeJA). In silico target prediction identified 519 potential cro-miRNA targets that include several auxin response factors (ARFs). The presence of cleaved transcripts of miRNA-targeted ARFs in C. roseus cells was confirmed by Poly(A) Polymerase-Mediated Rapid Amplification of cDNA Ends (PPM-RACE). We showed that auxin (indole acetic acid, IAA) repressed the expression of key TIA pathway genes in C. roseus seedlings. Moreover, we demonstrated that a miRNA-regulated ARF, CrARF16, binds to the promoters of key TIA pathway genes and repress their expression. The C. roseus miRNAome reported here provides a comprehensive account of the cro-miRNA populations, as well as their abundance and expression profiles in response to MeJA. In addition, our findings underscore the importance of miRNAs in posttranscriptional control of the biosynthesis of specialized metabolites. PMID:28223695

  8. Diversity of ABBA Prenyltransferases in Marine Streptomyces sp. CNQ-509: Promiscuous Enzymes for the Biosynthesis of Mixed Terpenoid Compounds.

    Science.gov (United States)

    Leipoldt, Franziska; Zeyhle, Philipp; Kulik, Andreas; Kalinowski, Jörn; Heide, Lutz; Kaysser, Leonard

    2015-01-01

    Terpenoids are arguably the largest and most diverse family of natural products, featuring prominently in e.g. signalling, self-defence, UV-protection and electron transfer. Prenyltransferases are essential players in terpenoid and hybrid isoprenoid biosynthesis that install isoprene units on target molecules and thereby often modulate their bioactivity. In our search for new prenyltransferase biocatalysts we focused on the marine-derived Streptomyces sp. CNQ-509, a particularly rich source of meroterpenoid chemistry. Sequencing and analysis of the genome of Streptomyces sp. CNQ-509 revealed seven putative phenol/phenazine-specific ABBA prenyltransferases, and one putative indole-specific ABBA prenyltransferase. To elucidate the substrate specificity of the ABBA prenyltransferases and to learn about their role in secondary metabolism, CnqP1 -CnqP8 were produced in Escherichia coli and incubated with various aromatic and isoprenoid substrates. Five of the eight prenyltransferases displayed enzymatic activity. The efficient conversion of dihydroxynaphthalene derivatives by CnqP3 (encoded by AA958_24325) and the co-location of AA958_24325 with genes characteristic for the biosynthesis of THN (tetrahydroxynaphthalene)-derived natural products indicates that the enzyme is involved in the formation of debromomarinone or other naphthoquinone-derived meroterpenoids. Moreover, CnqP3 showed high flexibility towards a range of aromatic and isoprenoid substrates and thus represents an interesting new tool for biocatalytic applications.

  9. Diversity of ABBA Prenyltransferases in Marine Streptomyces sp. CNQ-509: Promiscuous Enzymes for the Biosynthesis of Mixed Terpenoid Compounds.

    Directory of Open Access Journals (Sweden)

    Franziska Leipoldt

    Full Text Available Terpenoids are arguably the largest and most diverse family of natural products, featuring prominently in e.g. signalling, self-defence, UV-protection and electron transfer. Prenyltransferases are essential players in terpenoid and hybrid isoprenoid biosynthesis that install isoprene units on target molecules and thereby often modulate their bioactivity. In our search for new prenyltransferase biocatalysts we focused on the marine-derived Streptomyces sp. CNQ-509, a particularly rich source of meroterpenoid chemistry. Sequencing and analysis of the genome of Streptomyces sp. CNQ-509 revealed seven putative phenol/phenazine-specific ABBA prenyltransferases, and one putative indole-specific ABBA prenyltransferase. To elucidate the substrate specificity of the ABBA prenyltransferases and to learn about their role in secondary metabolism, CnqP1 -CnqP8 were produced in Escherichia coli and incubated with various aromatic and isoprenoid substrates. Five of the eight prenyltransferases displayed enzymatic activity. The efficient conversion of dihydroxynaphthalene derivatives by CnqP3 (encoded by AA958_24325 and the co-location of AA958_24325 with genes characteristic for the biosynthesis of THN (tetrahydroxynaphthalene-derived natural products indicates that the enzyme is involved in the formation of debromomarinone or other naphthoquinone-derived meroterpenoids. Moreover, CnqP3 showed high flexibility towards a range of aromatic and isoprenoid substrates and thus represents an interesting new tool for biocatalytic applications.

  10. Amino Acid Biosynthesis Pathways in Diatoms

    Directory of Open Access Journals (Sweden)

    Mariusz A. Bromke

    2013-04-01

    Full Text Available Amino acids are not only building blocks for proteins but serve as precursors for the synthesis of many metabolites with multiple functions in growth and other biological processes of a living organism. The biosynthesis of amino acids is tightly connected with central carbon, nitrogen and sulfur metabolism. Recent publication of genome sequences for two diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum created an opportunity for extensive studies on the structure of these metabolic pathways. Based on sequence homology found in the analyzed diatomal genes, the biosynthesis of amino acids in diatoms seems to be similar to higher plants. However, one of the most striking differences between the pathways in plants and in diatomas is that the latter possess and utilize the urea cycle. It serves as an important anaplerotic pathway for carbon fixation into amino acids and other N-containing compounds, which are essential for diatom growth and contribute to their high productivity.

  11. Bacterial exopolysaccharides: biosynthesis pathways and engineering strategies.

    Science.gov (United States)

    Schmid, Jochen; Sieber, Volker; Rehm, Bernd

    2015-01-01

    Bacteria produce a wide range of exopolysaccharides which are synthesized via different biosynthesis pathways. The genes responsible for synthesis are often clustered within the genome of the respective production organism. A better understanding of the fundamental processes involved in exopolysaccharide biosynthesis and the regulation of these processes is critical toward genetic, metabolic and protein-engineering approaches to produce tailor-made polymers. These designer polymers will exhibit superior material properties targeting medical and industrial applications. Exploiting the natural design space for production of a variety of biopolymer will open up a range of new applications. Here, we summarize the key aspects of microbial exopolysaccharide biosynthesis and highlight the latest engineering approaches toward the production of tailor-made variants with the potential to be used as valuable renewable and high-performance products for medical and industrial applications.

  12. Terpenoid pathway discovery in Tripterygium wilfordii

    DEFF Research Database (Denmark)

    Hansen, Nikolaj Lervad

    . To achieve this feat requires knowledge of the underlying biosynthetic pathways that give rise to the metabolites of interest in the native plants. This thesis presents an investigation of the biosynthetic pathways of medicinal compounds in Tripterygium wilfordii (Celastraceae), a widely used plant...

  13. High-throughput testing of terpenoid biosynthesis candidate genes using transient expression in Nicotiana benthamiana.

    Science.gov (United States)

    Bach, Søren Spanner; Bassard, Jean-Étienne; Andersen-Ranberg, Johan; Møldrup, Morten Emil; Simonsen, Henrik Toft; Hamberger, Björn

    2014-01-01

    To respond to the rapidly growing number of genes putatively involved in terpenoid metabolism, a robust high-throughput platform for functional testing is needed. An in planta expression system offers several advantages such as the capacity to produce correctly folded and active enzymes localized to the native compartments, unlike microbial or prokaryotic expression systems. Two inherent drawbacks of plant-based expression systems, time-consuming generation of transgenic plant lines and challenging gene-stacking, can be circumvented by transient expression in Nicotiana benthamiana. In this chapter we describe an expression platform for rapid testing of candidate terpenoid biosynthetic genes based on Agrobacterium mediated gene expression in N. benthamiana leaves. Simultaneous expression of multiple genes is facilitated by co-infiltration of leaves with several engineered Agrobacterium strains, possibly making this the fastest and most convenient system for the assembly of plant terpenoid biosynthetic routes. Tools for cloning of expression plasmids, N. benthamiana culturing, Agrobacterium preparation, leaf infiltration, metabolite extraction, and automated GC-MS data mining are provided. With all steps optimized for high throughput, this in planta expression platform is particularly suited for testing large panels of candidate genes in all possible permutations.

  14. Toward a photosynthetic microbial platform for terpenoid engineering.

    Science.gov (United States)

    Davies, Fiona K; Jinkerson, Robert E; Posewitz, Matthew C

    2015-03-01

    Plant terpenoids are among the most diverse group of naturally-occurring organic compounds known, and several are used in contemporary consumer products. Terpene synthase enzymes catalyze complex rearrangements of carbon skeleton precursors to yield thousands of unique chemical structures that range in size from the simplest five carbon isoprene unit to the long polymers of rubber. Such chemical diversity has established plant terpenoids as valuable commodity chemicals with applications in the pharmaceutical, neutraceutical, cosmetic, and food industries. More recently, terpenoids have received attention as a renewable alternative to petroleum-derived fuels and as the building blocks of synthetic biopolymers. However, the current plant- and petrochemical-based supplies of commodity terpenoids have major limitations. Photosynthetic microorganisms provide an opportunity to generate terpenoids in a renewable manner, employing a single consolidated host organism that is able to use solar energy, H2O and CO2 as the primary inputs for terpenoid biosynthesis. Advances in synthetic biology have seen important breakthroughs in microbial terpenoid engineering, traditionally via fermentative pathways in yeast and Escherichia coli. This review draws on the knowledge obtained from heterotrophic microbial engineering to propose strategies for the development of microbial photosynthetic platforms for industrial terpenoid production. The importance of utilizing the wealth of genetic information provided by nature to unravel the regulatory mechanisms of terpenoid biosynthesis is highlighted.

  15. Melanin biosynthesis pathway in Agaricus bisporus mushrooms.

    Science.gov (United States)

    Weijn, A; Bastiaan-Net, S; Wichers, H J; Mes, J J

    2013-06-01

    With the full genome sequence of Agaricus bisporus available, it was possible to investigate the genes involved in the melanin biosynthesis pathway of button mushrooms. Based on different BLAST and alignments, genes were identified in the genome which are postulated to be involved in this pathway. Seven housekeeping genes were tested of which 18S rRNA was the only housekeeping gene that was stably expressed in various tissues of different developmental stages. Gene expression was determined for most gene homologs (26 genes) involved in the melanin pathway. Of the analysed genes, those encoding polyphenol oxidase (PPO), the PPO co-factor L-chain (unique for A. bisporus), and a putative transcription factor (photoregulator B) were among the highest expressed in skin tissue. An in depth look was taken at the clustering of several PPO genes and the PPO co-factor gene on chromosome 5, which showed that almost 25% of the protein encoding genes in this cluster have a conserved NACHT and WD40 domain or a P-loop nucleoside triphosphate hydrolase. This article will be the start for an in depth study of the melanin pathway and its role in quality losses of this economically important product. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Coregulation of terpenoid pathway genes and prediction of isoprene production in Bacillus subtilis using transcriptomics

    Energy Technology Data Exchange (ETDEWEB)

    Hess, Becky M.; Xue, Junfeng; Markillie, Lye Meng; Taylor, Ronald C.; Wiley, H. S.; Ahring, Birgitte K.; Linggi, Bryan E.

    2013-06-19

    The isoprenoid pathway converts pyruvate to isoprene and related isoprenoid compounds in plants and some bacteria. Currently, this pathway is of great interest because of the critical role that isoprenoids play in basic cellular processes as well as the industrial value of metabolites such as isoprene. Although the regulation of several pathway genes has been described, there is a paucity of information regarding the system level regulation and control of the pathway. To address this limitation, we examined Bacillus subtilis grown under multiple conditions and then determined the relationship between altered isoprene production and the pattern of gene expression. We found that terpenoid genes appeared to fall into two distinct subsets with opposing correlations with respect to the amount of isoprene produced. The group whose expression levels positively correlated with isoprene production included dxs, the gene responsible for the commitment step in the pathway, as well as ispD, and two genes that participate in the mevalonate pathway, yhfS and pksG. The subset of terpenoid genes that inversely correlated with isoprene production included ispH, ispF, hepS, uppS, ispE, and dxr. A genome wide partial least squares regression model was created to identify other genes or pathways that contribute to isoprene production. This analysis showed that a subset of 213 regulated genes was sufficient to create a predictive model of isoprene production under different conditions and showed correlations at the transcriptional level. We conclude that gene expression levels alone are sufficiently informative about the metabolic state of a cell that produces increased isoprene and can be used to build a model which accurately predicts production of this secondary metabolite across many simulated environmental conditions.

  17. Transcriptome sequence analysis of an ornamental plant, Ananas comosus var. bracteatus, revealed the potential unigenes involved in terpenoid and phenylpropanoid biosynthesis.

    Science.gov (United States)

    Ma, Jun; Kanakala, S; He, Yehua; Zhang, Junli; Zhong, Xiaolan

    2015-01-01

    Ananas comosus var. bracteatus (Red Pineapple) is an important ornamental plant for its colorful leaves and decorative red fruits. Because of its complex genome, it is difficult to understand the molecular mechanisms involved in the growth and development. Thus high-throughput transcriptome sequencing of Ananas comosus var. bracteatus is necessary to generate large quantities of transcript sequences for the purpose of gene discovery and functional genomic studies. The Ananas comosus var. bracteatus transcriptome was sequenced by the Illumina paired-end sequencing technology. We obtained a total of 23.5 million high quality sequencing reads, 1,555,808 contigs and 41,052 unigenes. In total 41,052 unigenes of Ananas comosus var. bracteatus, 23,275 unigenes were annotated in the NCBI non-redundant protein database and 23,134 unigenes were annotated in the Swiss-Port database. Out of these, 17,748 and 8,505 unigenes were assigned to gene ontology categories and clusters of orthologous groups, respectively. Functional annotation against Kyoto Encyclopedia of Genes and Genomes Pathway database identified 5,825 unigenes which were mapped to 117 pathways. The assembly predicted many unigenes that were previously unknown. The annotated unigenes were compared against pineapple, rice, maize, Arabidopsis, and sorghum. Unigenes that did not match any of those five sequence datasets are considered to be Ananas comosus var. bracteatus unique. We predicted unigenes encoding enzymes involved in terpenoid and phenylpropanoid biosynthesis. The sequence data provide the most comprehensive transcriptomic resource currently available for Ananas comosus var. bracteatus. To our knowledge; this is the first report on the de novo transcriptome sequencing of the Ananas comosus var. bracteatus. Unigenes obtained in this study, may help improve future gene expression, genetic and genomics studies in Ananas comosus var. bracteatus.

  18. The Transcription Factor CrWRKY1 Positively Regulates the Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus1[W][OA

    Science.gov (United States)

    Suttipanta, Nitima; Pattanaik, Sitakanta; Kulshrestha, Manish; Patra, Barunava; Singh, Sanjay K.; Yuan, Ling

    2011-01-01

    Catharanthus roseus produces a large array of terpenoid indole alkaloids (TIAs) that are an important source of natural or semisynthetic anticancer drugs. The biosynthesis of TIAs is tissue specific and induced by certain phytohormones and fungal elicitors, indicating the involvement of a complex transcriptional control network. However, the transcriptional regulation of the TIA pathway is poorly understood. Here, we describe a C. roseus WRKY transcription factor, CrWRKY1, that is preferentially expressed in roots and induced by the phytohormones jasmonate, gibberellic acid, and ethylene. The overexpression of CrWRKY1 in C. roseus hairy roots up-regulated several key TIA pathway genes, especially Tryptophan Decarboxylase (TDC), as well as the transcriptional repressors ZCT1 (for zinc-finger C. roseus transcription factor 1), ZCT2, and ZCT3. However, CrWRKY1 overexpression repressed the transcriptional activators ORCA2, ORCA3, and CrMYC2. Overexpression of a dominant-repressive form of CrWRKY1, created by fusing the SRDX repressor domain to CrWRKY1, resulted in the down-regulation of TDC and ZCTs but the up-regulation of ORCA3 and CrMYC2. CrWRKY1 bound to the W box elements of the TDC promoter in electrophoretic mobility shift, yeast one-hybrid, and C. roseus protoplast assays. Up-regulation of TDC increased TDC activity, tryptamine concentration, and resistance to 4-methyl tryptophan inhibition of CrWRKY1 hairy roots. Compared with control roots, CrWRKY1 hairy roots accumulated up to 3-fold higher levels of serpentine. The preferential expression of CrWRKY1 in roots and its interaction with transcription factors including ORCA3, CrMYC2, and ZCTs may play a key role in determining the root-specific accumulation of serpentine in C. roseus plants. PMID:21988879

  19. CRISPR interference-guided balancing of a biosynthetic mevalonate pathway increases terpenoid production.

    Science.gov (United States)

    Kim, Seong Keun; Han, Gui Hwan; Seong, Wonjae; Kim, Haseong; Kim, Seon-Won; Lee, Dae-Hee; Lee, Seung-Goo

    2016-11-01

    Methods for simple and efficient regulation of metabolic pathway genes are essential for maximizing product titers and conversion yields, and for minimizing the metabolic burden caused by heterologous expression of multiple genes often in the operon context. Clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) is emerging as a promising tool for transcriptional modulation. In this study, we developed a regulatable CRISPRi system for fine-tuning biosynthetic pathways and thus directing carbon flux toward target product synthesis. By exploiting engineered Escherichia coli harboring a biosynthetic mevalonate (MVA) pathway and plant-derived terpenoid synthases, the CRISPRi system successfully modulated the expression of all the MVA pathway genes in the context of operon and blocked the transcription of the acetoacetyl-CoA thiolase enzyme that catalyzes the first step in the MVA pathway. This CRISPRi-guided balancing of expression of MVA pathway genes led to enhanced production of (-)-α-bisabolol (C15) and lycopene (C40) and alleviation of cell growth inhibition that may be caused by expression of multiple enzymes or production of toxic intermediate metabolites in the MVA pathway. Coupling CRISPRi to cell growth by regulating an endogenous essential gene (ispA) increased isoprene (C5) production. The regulatable CRISPRi system proved to be a robust platform for systematic modulation of biosynthetic and endogenous gene expression, and can be used to tune biosynthetic metabolic pathways. Its application can enable the development of microbial 'smart cell' factories that can produce other industrially valuable products in the future. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  20. Expression of MEP Pathway Genes and Non-volatile Sequestration Are Associated with Circadian Rhythm of Dominant Terpenoids Emission in Osmanthus fragrans Lour. Flowers

    Directory of Open Access Journals (Sweden)

    Riru Zheng

    2017-10-01

    Full Text Available Osmanthus fragrans Lour. is one of the top 10 traditional ornamental flowers in China famous for its unique fragrance. Preliminary study proved that the terpenoids including ionone, linalool, and ocimene and their derivatives are the dominant aroma-active compounds that contribute greatly to the scent bouquet. Pollination observation implies the emission of aromatic terpenoids may follow a circadian rhythm. In this study, we investigated the variation of volatile terpenoids and its potential regulators. The results showed that both volatile and non-volatile terpenoids presented circadian oscillation with high emission or accumulation during the day and low emission or accumulation during the night. The volatile terpenoids always increased to reach their maximum values at 12:00 h, while free and glycosylated compounds continued increasing throughout the day. The depletion of non-volatile pool might provide the substrates for volatile emission at 0:00–6:00, suggesting the sequestration of non-volatile compounds acted like a buffer regulating emission of terpenoids. Further detection of MEP pathway genes demonstrated that their expressions increased significantly in parallel with the evident increase of both volatile and non-volatile terpenoids during the day, indicating that the gene expressions were also closely associated with terpenoid formation. Thus, the expression of MEP pathway genes and internal sequestration both played crucial roles in modulating circadian rhythm of terpenoid emission in O. fragrans.

  1. Expression of MEP Pathway Genes and Non-volatile Sequestration Are Associated with Circadian Rhythm of Dominant Terpenoids Emission in Osmanthus fragrans Lour. Flowers.

    Science.gov (United States)

    Zheng, Riru; Liu, Cai; Wang, Yanli; Luo, Jing; Zeng, Xiangling; Ding, Haiqin; Xiao, Wei; Gan, Jianping; Wang, Caiyun

    2017-01-01

    Osmanthus fragrans Lour. is one of the top 10 traditional ornamental flowers in China famous for its unique fragrance. Preliminary study proved that the terpenoids including ionone, linalool, and ocimene and their derivatives are the dominant aroma-active compounds that contribute greatly to the scent bouquet. Pollination observation implies the emission of aromatic terpenoids may follow a circadian rhythm. In this study, we investigated the variation of volatile terpenoids and its potential regulators. The results showed that both volatile and non-volatile terpenoids presented circadian oscillation with high emission or accumulation during the day and low emission or accumulation during the night. The volatile terpenoids always increased to reach their maximum values at 12:00 h, while free and glycosylated compounds continued increasing throughout the day. The depletion of non-volatile pool might provide the substrates for volatile emission at 0:00-6:00, suggesting the sequestration of non-volatile compounds acted like a buffer regulating emission of terpenoids. Further detection of MEP pathway genes demonstrated that their expressions increased significantly in parallel with the evident increase of both volatile and non-volatile terpenoids during the day, indicating that the gene expressions were also closely associated with terpenoid formation. Thus, the expression of MEP pathway genes and internal sequestration both played crucial roles in modulating circadian rhythm of terpenoid emission in O. fragrans.

  2. Engineering yeast metabolism for production of terpenoids for use as perfume ingredients, pharmaceuticals and biofuels.

    Science.gov (United States)

    Zhang, Yueping; Nielsen, Jens; Liu, Zihe

    2017-12-01

    Terpenoids represent a large class of natural products with significant commercial applications. These chemicals are currently mainly obtained through extraction from plants and microbes or through chemical synthesis. However, these sources often face challenges of unsustainability and low productivity. In order to address these issues, Escherichia coli and yeast have been metabolic engineered to produce non-native terpenoids. With recent reports of engineering yeast metabolism to produce several terpenoids at high yields, it has become possible to establish commercial yeast production of terpenoids that find applications as perfume ingredients, pharmaceuticals and advanced biofuels. In this review, we describe the strategies to rewire the yeast pathway for terpenoid biosynthesis. Recent advances will be discussed together with challenges and perspectives of yeast as a cell factory to produce different terpenoids. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  3. Both methylerythritol phosphate and mevalonate pathways contribute to biosynthesis of each of the major isoprenoid classes in young cotton seedlings.

    Science.gov (United States)

    Opitz, Stefan; Nes, W David; Gershenzon, Jonathan

    2014-02-01

    In higher plants, both the methylerythritol phosphate (MEP) and mevalonate (MVA) pathways contribute to the biosynthesis of isoprenoids. However, despite a significant amount of research on the activity of these pathways under different conditions, the relative contribution of each to the biosynthesis of diverse isoprenoids remains unclear. In this work, we examined the formation of several classes of isoprenoids in cotton (Gossypium hirsutum L.). After feeding [5,5-(2)H2]-1-deoxy-D-xylulose ([5,5-(2)H2]DOX) and [2-(13)C]MVA to intact cotton seedlings hydroponically, incorporation into isoprenoids was analyzed by MS and NMR. The predominant pattern of incorporation followed the classical scheme in which C5 units from the MEP pathway were used to form monoterpenes (C10), phytol side chains (C20) and carotenoids (C40) while C5 units from the MVA pathway were used to form sesquiterpenes (C15), terpenoid aldehydes (C15 and C25) and steroids/triterpenoids (C30). However, both pathways contributed to all classes of terpenoids, sometimes substantially. For example, the MEP pathway provided up to 20% of the substrate for sterols and the MVA pathway provided as much as 50% of the substrate for phytol side chains and carotenoids. Incorporation of C5 units from the MEP pathway was highest in cotyledons, compared to true leaves, and not observed at all in the roots. Incorporation of C5 units from the MVA pathway was highest in the roots (into sterols) and more prominent in the first true leaves than in other above-ground organs. The relative accumulation of label in intermediates vs. end products of phytosterol metabolism confirmed previous identification of slow steps in this pathway. Copyright © 2013. Published by Elsevier Ltd.

  4. Bacterial transformation of terpenoids

    Science.gov (United States)

    Grishko, V. V.; Nogovitsina, Y. M.; Ivshina, I. B.

    2014-04-01

    Data on the bacterial transformation of terpenoids published in the literature in the past decade are analyzed. Possible pathways for chemo-, regio- and stereoselective modifications of terpenoids are discussed. Considerable attention is given to new technological approaches to the synthesis of terpenoid derivatives suitable for the use in the perfume and food industry and promising as drugs and chiral intermediates for fine organic synthesis. The bibliography includes 246 references.

  5. Análisis de iridoides y expresión de genes que codifican enzimas tempranas en la síntesis de alcaloides indol terpenoicos en Catharanthus roseus Analysis of iridoids content and expression studies of genes encoding early enzymes in the indol terpenoid biosynthesis pathway in Catharanthus roseus

    OpenAIRE

    Palacios-Rojas Natalia; Leech Mark

    2004-01-01

    Los alcaloides indol terpenoicos (TIA) son metabolitos secundarios de importancia medicinal por sus propiedades como agentes anticancerígenos, entre otras. Sin embargo, su explotación en la industria farmacéutica se ha visto limitada, ya que la acumulación de estos compuestos en las plantas que los producen es mínima. Dichos alcaloides son biosintetizados por la vía del shikimato y de los terpenoides, los cuales proveen los precursores: secologanina y triptamina, respectivamente. La secologan...

  6. Targeting apoptosis pathway with natural terpenoids: implications for treatment of breast and prostate cancer.

    Science.gov (United States)

    Yang, Huanjie; Dou, Q Ping

    2010-06-01

    Terpenoids represent a large and diverse class of naturally occurring compounds found in a variety of fruits, vegetables and medicinal plants. Structurally some of the terpenoids are similar to human hormones. A diet rich in terpenoids is inversely related with the risk of chronic diseases including cancers. Breast and prostate cancers are hormone-related diseases and the second leading cause of female and male cancer mortality. Diterpenoid paclitaxel, and its semi-synthetic analogue docetaxel, have entered clinical use against established breast and prostate cancers. Here we reviewed potential molecular targets and biological properties of natural terpenoids, including monoterpenoids, diterpenoids, triterpenoids and tetraterpenoids, and their applications in treatment of human breast and prostate cancers. These terpenoids are able to inhibit tumor cell proliferation and induce tumor cell death by inhibiting multiple cancer-specific targets including the proteasome, NF-kappaB, and antiapoptotic protein Bcl-2. The efficacy of these terpenoids against breast or prostate cancer cells, as demonstrated in pre-clinical studies support clinical application of these naturally occurring terpenoids in treatment of hormone-related human cancers.

  7. Yeast metabolic engineering--targeting sterol metabolism and terpenoid formation.

    Science.gov (United States)

    Wriessnegger, Tamara; Pichler, Harald

    2013-07-01

    Terpenoids comprise various structures conferring versatile functions to eukaryotes, for example in the form of prenyl-anchors they attach proteins to membranes. The physiology of eukaryotic membranes is fine-tuned by another terpenoid class, namely sterols. Evidence is accumulating that numerous membrane proteins require specific sterol structural features for function. Moreover, sterols are intermediates in the synthesis of steroids serving as hormones in higher eukaryotes. Like steroids many compounds of the terpenoid family do not contribute to membrane architecture, but serve as signalling, protective or attractant/repellent molecules. Particularly plants have developed a plenitude of terpenoid biosynthetic routes branching off early in the sterol biosynthesis pathway and, thereby, forming one of the largest groups of naturally occurring organic compounds. Many of these aromatic and volatile molecules are interesting for industrial application ranging from foods to pharmaceuticals. Combining the fortunate situation that sterol biosynthesis is highly conserved in eukaryotes with the amenability of yeasts to genetic and metabolic engineering, basically all naturally occurring terpenoids might be produced involving yeasts. Such engineered yeasts are useful for the study of biological functions and molecular interactions of terpenoids as well as for the large-scale production of high-value compounds, which are unavailable in sufficient amounts from natural sources due to their low abundance. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Methylerythritol Phosphate Pathway of Isoprenoid Biosynthesis

    Science.gov (United States)

    Zhao, Lishan; Chang, Wei-chen; Xiao, Youli; Liu, Hung-wen; Liu, Pinghua

    2016-01-01

    Isoprenoids are a class of natural products with more than 50,000 members. All isoprenoids are constructed from two precursors, isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). Two of the most important discoveries in isoprenoid biosynthetic studies in recent years are the elucidation of a second isoprenoid biosynthetic pathway (the methylerythritol phosphate (MEP) pathway) and a modified mevalonate (MVA) pathway. In this review, mechanistic insights on the MEP pathway enzymes are summarized. Since many isoprenoids have important biological activities, the need to produce them in sufficient quantities for downstream research efforts or commercial application is apparent. Recent advances in both the MVA and MEP pathway-based synthetic biology efforts are also illustrated by reviewing the landmark work of artemisinic acid and taxadien-5α-ol production through microbial fermentations. PMID:23746261

  9. The pyrroloquinoline quinone biosynthesis pathway revisited: A structural approach

    Directory of Open Access Journals (Sweden)

    Schwarzenbacher Robert

    2008-03-01

    Full Text Available Abstract Background The biosynthesis pathway of Pyrroloquinoline quinone, a bacterial redox active cofactor for numerous alcohol and aldose dehydrogenases, is largely unknown, but it is proven that at least six genes in Klebsiella pneumoniae (PqqA-F are required, all of which are located in the PQQ-operon. Results New structural data of some PQQ biosynthesis proteins and their homologues provide new insights and functional assignments of the proteins in the pathway. Based on sequence analysis and homology models we propose the role and catalytic function for each enzyme involved in this intriguing biosynthesis pathway. Conclusion PQQ is derived from the two amino acids glutamate and tyrosine encoded in the precursor peptide PqqA. Five reactions are necessary to form this quinone cofactor. The PqqA peptide is recognised by PqqE, which links the C9 and C9a, afterwards it is accepted by PqqF which cuts out the linked amino acids. The next reaction (Schiff base is spontaneous, the following dioxygenation is catalysed by an unknown enzyme. The last cyclization and oxidation steps are catalysed by PqqC. Taken together the known facts of the different proteins we assign a putative function to all six proteins in PQQ biosynthesis pathway.

  10. Spectral Dependence of Chlorophyll Biosynthesis Pathways in Plant Leaves.

    Science.gov (United States)

    Belyaeva, O B; Litvin, F F

    2015-12-01

    This review covers studies on the dependence of chlorophyll photobiosynthesis reactions from protochlorophyllide on the spectral composition of actinic light. A general scheme of the reaction sequence for the photochemical stage in chlorophyll biosynthesis for etiolated plant leaves is presented. Comparative analysis of the data shows that the use of light with varied wavelengths for etiolated plant illumination reveals parallel transformation pathways of different protochlorophyllide forms into chlorophyllide, including a pathway for early photosystem II reaction center P-680 pigment formation.

  11. Antimicrobial mechanism of epigallocatechin gallate and gallocatechin gallate: They target 1-deoxy-d-xylulose 5-phosphate reductoisomerase, the key enzyme of the MEP terpenoid biosynthetic pathway.

    Science.gov (United States)

    Hui, Xian; Hua, Shui-Hong; Wu, Qian-Qian; Li, Heng; Gao, Wen-Yun

    2017-05-15

    The catechins EGCG and GCG show a variety of pharmacological activities, especially an antibacterial capacity, but their modes of antimicrobial action have not been fully elucidated. 1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), the first key enzyme in the MEP pathway for terpenoid biosynthesis, is a recently validated antimicrobial target. In order to disclose the antibacterial mechanism of EGCG and GCG, the DXR inhibitory activity of them was investigated in this study. The data show that EGCG and GCG both could specifically suppress the activity of DXR, with EGCG exhibiting relatively low effect against DXR (IC 50 about 210 μM) and GCG displaying strong activity (IC 50 27.5 μM). In addition, studies on inhibition kinetics of the catechins against DXR demonstrate that they are competitive inhibitors of DXR against DXP and uncompetitive inhibitors with respect to NADPH. Meanwhile, the possible interactions between DXR and the catechine, esyth onlols were simulated via docking experiments. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Terpenoids in plant and arbuscular mycorrhiza-reinforced defence against herbivorous insects.

    Science.gov (United States)

    Sharma, Esha; Anand, Garima; Kapoor, Rupam

    2017-03-01

    Plants, though sessile, employ various strategies to defend themselves against herbivorous insects and convey signals of an impending herbivore attack to other plant(s). Strategies include the production of volatiles that include terpenoids and the formation of symbiotic associations with fungi, such as arbuscular mycorrhiza (AM). This constitutes a two-pronged above-ground/below-ground attack-defence strategy against insect herbivores. Terpenoids represent an important constituent of herbivore-induced plant volatiles that deter herbivores and/or attract their predators. Terpenoids serve as airborne signals that can induce defence responses in systemic undamaged parts of the plant and also prime defence responses in neighbouring plants. Colonization of roots by AM fungi is known to influence secondary metabolism in plants; this includes alteration of the concentration and composition of terpenoids, which can boost both direct and indirect plant defence against herbivorous insects. Enhanced nutrient uptake facilitated by AM, changes in plant morphology and physiology and increased transcription levels of certain genes involved in the terpenoid biosynthesis pathway result in alterations in plant terpenoid profiles. The common mycorrhizal networks of external hyphae have added a dimension to the two-pronged plant defence strategy. These act as conduits to transfer defence signals and terpenoids. Improved understanding of the roles of terpenoids in plant and AM defences against herbivory and of interplant signalling in natural communities has significant implications for sustainable management of pests in agricultural ecosystems.

  13. Evaluation of Biosynthetic Pathway and Engineered Biosynthesis of Alkaloids

    Directory of Open Access Journals (Sweden)

    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. [Salidroside biosynthesis pathway: the initial reaction and glycosylation of tyrosol].

    Science.gov (United States)

    Ma, Lanqing; Liu, Chunmei; Yu, Hansong; Zhang, Jixing; Gao, Dongyao; Li, Yanfang; Wang, Younian

    2012-03-01

    Salidroside, the 8-O-beta-D-glucoside of tyrosol, is a novel adaptogenic drug extracted from the medicinal plant Rhodiola sachalinensis A. Bor. Due to the scarcity of R. sachalinensis and its low yield of salidroside, there is great interest in enhancing the production of salidroside by biotechnological process. Glucosylation of tyrosol is thought to be the final step in salidroside biosynthesis. In our related works, three UGT clones were isolated from the roots and the cultured cells. Our intention was to combine the catalytic specificity of these UGTs in vitro in order to change the level of salidroside in vivo by over-expression of the above UGTs. However, as the aglycone substrate of salidroside, the biosynthetic pathway of tyrosol and its regulation are less well understood. The results of related studies revealed that there are two different possibilities for the tyrosol biosynthetic pathway. One possibility is that tyrosol is produced from a p-coumaric acid precursor, which is derived mainly from phenylalanine. The second possibility is that the precursor of tyrosol might be tyramine, which is synthesized from tyrosine. Our previous work demonstrated that over-expression of the endogenous phenylalanine ammonia-lyase gene (PALrs1) and accumulation of p-coumaric acid did not facilitate tyrosol biosynthesis. In contrast, the data presented in our recent work provide in vitro and in vivo evidence that the tyrosine decarboxylase (RsTyrDC) is most likely to have an important function in the initial reaction of the salidroside biosynthesis pathway in R. Sachalinensis.

  15. An account of cloned genes of Methyl-erythritol-4-phosphate pathway of isoprenoid biosynthesis in plants.

    Science.gov (United States)

    Ganjewala, Deepak; Kumar, Shiv; Luthra, Rajesh

    2009-01-01

    Isoprenoids, also known as terpenoids, are biosynthesized by the condensation of the two C5 unit isopentenyl diphosphate (IPP) and isomer dimethylallyl diphosphate (DMAPP). Generally, plants use two separate pathways plastidial Methyl-erythritol-4-phosphate (MEP) and cytosolic acetate-mevalonate (MVA) pathways for formation of IPP. The genes, enzymes and intermediates of the MEP pathway have been unravelled in plants over the past few years. Interestingly, MEP pathway enzymes are encoded by nuclear genes but they function in plastids to produce precursors for isoprenes, monoterpenes, carotenoids, abscisic acid, gibberellins, and the side chain of chlorophylls, tocopherols, phylloquinones, and plastoquinone. In Arabidopsis thaliana, a complete set of genes of MEP pathway homologous to the E. coli MEP pathway genes have been identified. Although, these genes have been cloned and characterized from several other plants but overall information about them at one place is not available so far. Though, a range of reviews are available about their roles in isoprenoid biosynthesis and regulation. Therefore, we decided to compile the data on cloned and characterized genes of MEP pathway in plants. Also, we summarize the results of the previously published reports, particularly those which were based on incorporation of 13C-glucose or by application of specific inhibitors such as mevinolin and fosmidomycin to look into the MEP pathway in plants. In addition, we searched for the two key enzymes DXS and HMGR that could be assigned for the acetate-MVA and MEP pathway with the help of bioinformatics tools. Presence or absence of these enzymes can be correlated with respective isoprenoid biosynthetic pathways in plants.

  16. Cytokinin and ethylene control indole alkaloid production at the level of the MEP/terpenoid pathway in Catharanthus roseus suspension cells.

    Science.gov (United States)

    Papon, Nicolas; Bremer, Jennifer; Vansiri, Amérin; Andreu, Françoise; Rideau, Marc; Crèche, Joël

    2005-06-01

    The Madagascar periwinkle Catharanthus roseus accumulates a number of terpenoid indole alkaloids, some of which have high therapeutic interest. The biotechnological approach with cells in vitro remains an alternative to the field culture of periwinkle for the production of such compounds. We previously reported that two phytohormones, cytokinin and ethylene, remarkably enhanced the accumulation of alkaloids in periwinkle cell suspensions. In this work, we investigated the effects of these hormones on the regulation of several genes of the indole alkaloid biosynthetic pathway. We show that cytokinin and/or ethylene greatly enhanced the expression of the geraniol 10-hydroxylase gene. When given together, these hormones also increased the expression of three genes belonging to the methyl-erythritol pathway. These results make it possible to consider elements of cytokinin and ethylene signalling pathways as tools for improving terpenoid indole alkaloid production through metabolic engineering.

  17. LIL3, a Light-Harvesting Complex Protein, Links Terpenoid and Tetrapyrrole Biosynthesis inArabidopsis thaliana.

    Science.gov (United States)

    Hey, Daniel; Rothbart, Maxi; Herbst, Josephine; Wang, Peng; Müller, Jakob; Wittmann, Daniel; Gruhl, Kirsten; Grimm, Bernhard

    2017-06-01

    The LIL3 protein of Arabidopsis ( Arabidopsis thaliana ) belongs to the light-harvesting complex (LHC) protein family, which also includes the light-harvesting chlorophyll-binding proteins of photosystems I and II, the early-light-inducible proteins, PsbS involved in nonphotochemical quenching, and the one-helix proteins and their cyanobacterial homologs designated high-light-inducible proteins. Each member of this family is characterized by one or two LHC transmembrane domains (referred to as the LHC motif) to which potential functions such as chlorophyll binding, protein interaction, and integration of interacting partners into the plastid membranes have been attributed. Initially, LIL3 was shown to interact with geranylgeranyl reductase (CHLP), an enzyme of terpene biosynthesis that supplies the hydrocarbon chain for chlorophyll and tocopherol. Here, we show another function of LIL3 for the stability of protochlorophyllide oxidoreductase (POR). Multiple protein-protein interaction analyses suggest the direct physical interaction of LIL3 with POR but not with chlorophyll synthase. Consistently, LIL3-deficient plants exhibit substantial loss of POR as well as CHLP, which is not due to defective transcription of the POR and CHLP genes but to the posttranslational modification of their protein products. Interestingly, in vitro biochemical analyses provide novel evidence that LIL3 shows high binding affinity to protochlorophyllide, the substrate of POR. Taken together, this study suggests a critical role for LIL3 in the organization of later steps in chlorophyll biosynthesis. We suggest that LIL3 associates with POR and CHLP and thus contributes to the supply of the two metabolites, chlorophyllide and phytyl pyrophosphate, required for the final step in chlorophyll a synthesis. © 2017 American Society of Plant Biologists. All Rights Reserved.

  18. Transcriptome and metabolite profiling reveals that prolonged drought modulates the phenylpropanoid and terpenoid pathway in white grapes (Vitis vinifera L.).

    Science.gov (United States)

    Savoi, Stefania; Wong, Darren C J; Arapitsas, Panagiotis; Miculan, Mara; Bucchetti, Barbara; Peterlunger, Enrico; Fait, Aaron; Mattivi, Fulvio; Castellarin, Simone D

    2016-03-21

    Secondary metabolism contributes to the adaptation of a plant to its environment. In wine grapes, fruit secondary metabolism largely determines wine quality. Climate change is predicted to exacerbate drought events in several viticultural areas, potentially affecting the wine quality. In red grapes, water deficit modulates flavonoid accumulation, leading to major quantitative and compositional changes in the profile of the anthocyanin pigments; in white grapes, the effect of water deficit on secondary metabolism is still largely unknown. In this study we investigated the impact of water deficit on the secondary metabolism of white grapes using a large scale metabolite and transcript profiling approach in a season characterized by prolonged drought. Irrigated grapevines were compared to non-irrigated grapevines that suffered from water deficit from early stages of berry development to harvest. A large effect of water deficit on fruit secondary metabolism was observed. Increased concentrations of phenylpropanoids, monoterpenes, and tocopherols were detected, while carotenoid and flavonoid accumulations were differentially modulated by water deficit according to the berry developmental stage. The RNA-sequencing analysis carried out on berries collected at three developmental stages-before, at the onset, and at late ripening-indicated that water deficit affected the expression of 4,889 genes. The Gene Ontology category secondary metabolic process was overrepresented within up-regulated genes at all the stages of fruit development considered, and within down-regulated genes before ripening. Eighteen phenylpropanoid, 16 flavonoid, 9 carotenoid, and 16 terpenoid structural genes were modulated by water deficit, indicating the transcriptional regulation of these metabolic pathways in fruit exposed to water deficit. An integrated network and promoter analyses identified a transcriptional regulatory module that encompasses terpenoid genes, transcription factors, and enriched

  19. Proteolytic pathways induced by herbicides that inhibit amino acid biosynthesis.

    Directory of Open Access Journals (Sweden)

    Amaia Zulet

    Full Text Available The herbicides glyphosate (Gly and imazamox (Imx inhibit the biosynthesis of aromatic and branched-chain amino acids, respectively. Although these herbicides inhibit different pathways, they have been reported to show several common physiological effects in their modes of action, such as increasing free amino acid contents and decreasing soluble protein contents. To investigate proteolytic activities upon treatment with Gly and Imx, pea plants grown in hydroponic culture were treated with Imx or Gly, and the proteolytic profile of the roots was evaluated through fluorogenic kinetic assays and activity-based protein profiling.Several common changes in proteolytic activity were detected following Gly and Imx treatment. Both herbicides induced the ubiquitin-26 S proteasome system and papain-like cysteine proteases. In contrast, the activities of vacuolar processing enzymes, cysteine proteases and metacaspase 9 were reduced following treatment with both herbicides. Moreover, the activities of several putative serine protease were similarly increased or decreased following treatment with both herbicides. In contrast, an increase in YVADase activity was observed under Imx treatment versus a decrease under Gly treatment.These results suggest that several proteolytic pathways are responsible for protein degradation upon herbicide treatment, although the specific role of each proteolytic activity remains to be determined.

  20. Comparative transcriptome analysis of different chemotypes elucidates withanolide biosynthesis pathway from medicinal plant Withania somnifera.

    Science.gov (United States)

    Gupta, Parul; Goel, Ridhi; Agarwal, Aditya Vikram; Asif, Mehar Hasan; Sangwan, Neelam Singh; Sangwan, Rajender Singh; Trivedi, Prabodh Kumar

    2015-12-21

    Withania somnifera is one of the most valuable medicinal plants synthesizing secondary metabolites known as withanolides. Despite pharmaceutical importance, limited information is available about the biosynthesis of withanolides. Chemo-profiling of leaf and root tissues of Withania suggest differences in the content and/or nature of withanolides in different chemotypes. To identify genes involved in chemotype and/or tissue-specific withanolide biosynthesis, we established transcriptomes of leaf and root tissues of distinct chemotypes. Genes encoding enzymes for intermediate steps of terpenoid backbone biosynthesis with their alternatively spliced forms and paralogous have been identified. Analysis suggests differential expression of large number genes among leaf and root tissues of different chemotypes. Study also identified differentially expressing transcripts encoding cytochrome P450s, glycosyltransferases, methyltransferases and transcription factors which might be involved in chemodiversity in Withania. Virus induced gene silencing of the sterol ∆7-reductase (WsDWF5) involved in the synthesis of 24-methylene cholesterol, withanolide backbone, suggests role of this enzyme in biosynthesis of withanolides. Information generated, in this study, provides a rich resource for functional analysis of withanolide-specific genes to elucidate chemotype- as well as tissue-specific withanolide biosynthesis. This genomic resource will also help in development of new tools for functional genomics and breeding in Withania.

  1. A Stilbenoid-Specific Prenyltransferase Utilizes Dimethylallyl Pyrophosphate from the Plastidic Terpenoid Pathway.

    Science.gov (United States)

    Yang, Tianhong; Fang, Lingling; Rimando, Agnes M; Sobolev, Victor; Mockaitis, Keithanne; Medina-Bolivar, Fabricio

    2016-08-01

    Prenylated stilbenoids synthesized in some legumes exhibit plant pathogen defense properties and pharmacological activities with potential benefits to human health. Despite their importance, the biosynthetic pathways of these compounds remain to be elucidated. Peanut (Arachis hypogaea) hairy root cultures produce a diverse array of prenylated stilbenoids upon treatment with elicitors. Using metabolic inhibitors of the plastidic and cytosolic isoprenoid biosynthetic pathways, we demonstrated that the prenyl moiety on the prenylated stilbenoids derives from a plastidic pathway. We further characterized, to our knowledge for the first time, a membrane-bound stilbenoid-specific prenyltransferase activity from the microsomal fraction of peanut hairy roots. This microsomal fraction-derived resveratrol 4-dimethylallyl transferase utilizes 3,3-dimethylallyl pyrophosphate as a prenyl donor and prenylates resveratrol to form arachidin-2. It also prenylates pinosylvin to chiricanine A and piceatannol to arachidin-5, a prenylated stilbenoid identified, to our knowledge, for the first time in this study. This prenyltransferase exhibits strict substrate specificity for stilbenoids and does not prenylate flavanone, flavone, or isoflavone backbones, even though it shares several common features with flavonoid-specific prenyltransferases. © 2016 American Society of Plant Biologists. All Rights Reserved.

  2. LOCALIZATION OF THE PATHWAY OF THE PENICILLIN BIOSYNTHESIS IN PENICILLIUM-CHRYSOGENUM

    NARCIS (Netherlands)

    MULLER, WH; VANDERKRIFT, TP; KROUWER, AJJ; WOSTEN, HAB; VANDERVOORT, LHM; SMAAL, EB; VERKLEIJ, AJ

    The localization of the enzymes involved in penicillin biosynthesis in Penicillium chrysogenum hyphae has been studied by immunological detection methods in combination with electron microscopy and cell fractionation. The results suggest a complicated pathway involving different intracellular

  3. The tomato B-type cyclin gene, SlCycB2, plays key roles in reproductive organ development, trichome initiation, terpenoids biosynthesis and Prodenia litura defense.

    Science.gov (United States)

    Gao, Shenghua; Gao, Yanna; Xiong, Cheng; Yu, Gang; Chang, Jiang; Yang, Qihong; Yang, Changxian; Ye, Zhibiao

    2017-09-01

    Cyclins exist extensively in various plant species. Among them, B-type cyclins play important roles in the transition of G2-to-M. However, few B-type cyclins have been reported to participate in reproductive organ development and trichome formation. In this study, transgene analysis showed that SlCycB2 overexpression caused abnormal flower with the unclosed stamen, shortened style and aberrant pollen. In addition, nearly all non-glandular trichomes, as well as the glandular ones were disappeared. On the contrary, suppression of SlCycB2 could promote type III and type V trichomes formation. Detection of secondary metabolites indicated that the production of monoterpene and sesquiterpene were significantly decreased in SlCycB2-OE plants, which thus resulted in the reduction of the defense against Prodenia litura. Transcriptome profile demonstrated that the differentially expressed genes mainly participate in the biosynthesis of terpenes, cutin, suberine and wax. Furthermore, we identified several homologs of SlCycB2, SlCycB3, NtCycB2, AtCycB2, which have similar regulatory functions in trichome formation. These results indicate that SlCycB2 plays a critical role in reproductive organ development, multicellular trichome initiation, secondary metabolite biosynthesis and Prodenia litura defense in tomato. The similar roles of its homologs in multicellular trichome formation suggest that Solanaceous species may share common regulatory pathway. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Molecular Genetic Characterization of a Cluster in A. terreus for Biosynthesis of the Meroterpenoid Terretonin

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Chun-Jun; Knox, Benjamin P.; Chiang, Yi Ming; Lo, Hsien-Chun; Sanchez, James F.; Lee, Kuan-Han; Oakley, Berl R.; Bruno, Kenneth S.; Wang, Clay C.

    2012-11-01

    Meroterpenoids are natural products produced from polyketide and terpenoid precursors. A gene targeting system for A. terreus NIH2624 was developed, and a gene cluster for terretonin biosynthesis was characterized. The intermediates and shunt products were isolated from the mutant strains, and a pathway for terretonin biosynthesis is proposed. Analysis of two meroterpenoid pathways corresponding to terretonin in A. terreus and austinol in A. nidulans reveals that they are closely related evolutionarily.

  5. A chloroplast pathway for the de novo biosynthesis of triacylglycerol in Chlamydomonas reinhardtii

    Energy Technology Data Exchange (ETDEWEB)

    Fan, J.; Xu, C.; Andre, C.

    2011-06-23

    Neutral lipid metabolism has been extensively studied in yeast, plants and mammals. In contrast, little information is available regarding the biochemical pathway, enzymes and regulatory factors involved in the biosynthesis of triacylglycerol (TAG) in microalgae. In the conventional TAG biosynthetic pathway widely accepted for yeast, plants and mammals, TAG is assembled in the endoplasmic reticulum (ER) from its immediate precursor diacylglycerol (DAG) made by ER-specific acyltransferases, and is deposited exclusively in lipid droplets in the cytosol. Here, we demonstrated that the unicellular microalga Chlamydomonas reinhardtii employs a distinct pathway that uses DAG derived almost exclusively from the chloroplast to produce TAG. This unique TAG biosynthesis pathway is largely dependent on de novo fatty acid synthesis, and the TAG formed in this pathway is stored in lipid droplets in both the chloroplast and the cytosol. These findings have wide implications for understanding TAG biosynthesis and storage and other areas of lipid metabolism in microalgae and other organisms.

  6. Biosynthetic studies on terpenoids produced by Streptomyces.

    Science.gov (United States)

    Kuzuyama, Tomohisa

    2017-07-01

    Terpenoids are a large and highly diverse group of natural products. All terpenoids are biosynthesized from isoprenyl diphosphate formed by the consecutive condensation of the five-carbon monomer isopentenyl diphosphate (IPP) to its isomer dimethylallyl diphosphate (DMAPP). Two distinct biosynthetic pathways produce the essential primary metabolites IPP and DMAPP: the 2-C-methylerythritol 4-phosphate pathway and the mevalonate pathway. The isoprenyl substrates can be cyclized by terpene cyclase into single-ring or multi-ring products, which can be further diversified by subsequent modification reactions, such as hydroxylation and glycosylation. This review article describes the biosynthetic pathways of terpenoids produced by Streptomyces and their related novel enzymes.

  7. Withanolide biosynthesis recruits both mevalonate and DOXP pathways of isoprenogenesis in Ashwagandha Withania somnifera L. (Dunal).

    Science.gov (United States)

    Chaurasiya, Narayan D; Sangwan, Neelam S; Sabir, Farzana; Misra, Laxminarain; Sangwan, Rajender S

    2012-10-01

    Withanolides are pharmaceutically important C(28)-phytochemicals produced in most prodigal amounts and diversified forms by Withania somnifera. Metabolic origin of withanolides from triterpenoid pathway intermediates implies that isoprenogenesis could significantly govern withanolide production. In plants, isoprenogenesis occurs via two routes: mevalonate (MVA) pathway in cytosol and non-mevalonate or DOXP/MEP pathway in plastids. We have investigated relative carbon contribution of MVA and DOXP pathways to withanolide biosynthesis in W. somnifera. The quantitative NMR-based biosynthetic study involved tracing of (13)C label from (13)C(1)-D-glucose to withaferin A in withanolide producing in vitro microshoot cultures of the plant. Enrichment of (13)C abundance at each carbon of withaferin A from (13)C(1)-glucose-fed cultures was monitored by normalization and integration of NMR signal intensities. The pattern of carbon position-specific (13)C enrichment of withaferin A was analyzed by a retro-biosynthetic approach using a squalene-intermediated metabolic model of withanolide (withaferin A) biosynthesis. The pattern suggested that both DOXP and MVA pathways of isoprenogenesis were significantly involved in withanolide biosynthesis with their relative contribution on the ratio of 25:75, respectively. The results have been discussed in a new conceptual line of biosynthetic load-driven model of relative recruitment of DOXP and MVA pathways for biosynthesis of isoprenoids. Key message The study elucidates significant contribution of DOXP pathway to withanolide biosynthesis. A new connotation of biosynthetic load-based role of DOXP/MVA recruitment in isoprenoid biosynthesis has been proposed.

  8. Agrobacterium Mediated Transient Gene Silencing (AMTS) in Stevia rebaudiana: Insights into Steviol Glycoside Biosynthesis Pathway

    Science.gov (United States)

    Guleria, Praveen; Yadav, Sudesh Kumar

    2013-01-01

    Background Steviol glycoside biosynthesis pathway has emerged as bifurcation from ent-kaurenoic acid, substrate of methyl erythritol phosphate pathway that also leads to gibberellin biosynthesis. However, the genetic regulation of steviol glycoside biosynthesis has not been studied. So, in present study RNA interference (RNAi) based Agrobacterium mediated transient gene silencing (AMTS) approach was followed. SrKA13H and three SrUGTs (SrUGT85C2, SrUGT74G1 and SrUGT76G1) genes encoding ent-kaurenoic acid-13 hydroxylase and three UDP glycosyltransferases of steviol glycoside biosynthesis pathway were silenced in Stevia rebaudiana to understand its molecular mechanism and association with gibberellins. Methodology/Principal Findings RNAi mediated AMTS of SrKA13H and three SrUGTs has significantly reduced the expression of targeted endogenous genes as well as total steviol glycoside accumulation. While gibberellins (GA3) content was significantly enhanced on AMTS of SrUGT85C2 and SrKA13H. Silencing of SrKA13H and SrUGT85C2 was found to block the metabolite flux of steviol glycoside pathway and shifted it towards GA3 biosynthesis. Further, molecular docking of three SrUGT proteins has documented highest affinity of SrUGT76G1 for the substrates of alternate pathways synthesizing steviol glycosides. This could be a plausible reason for maximum reduction in steviol glycoside content on silencing of SrUGT76G1 than other genes. Conclusions SrKA13H and SrUGT85C2 were identified as regulatory genes influencing carbon flux between steviol glycoside and gibberellin biosynthesis. This study has also documented the existence of alternate steviol glycoside biosynthesis route. PMID:24023961

  9. Agrobacterium mediated transient gene silencing (AMTS in Stevia rebaudiana: insights into steviol glycoside biosynthesis pathway.

    Directory of Open Access Journals (Sweden)

    Praveen Guleria

    Full Text Available BACKGROUND: Steviol glycoside biosynthesis pathway has emerged as bifurcation from ent-kaurenoic acid, substrate of methyl erythritol phosphate pathway that also leads to gibberellin biosynthesis. However, the genetic regulation of steviol glycoside biosynthesis has not been studied. So, in present study RNA interference (RNAi based Agrobacterium mediated transient gene silencing (AMTS approach was followed. SrKA13H and three SrUGTs (SrUGT85C2, SrUGT74G1 and SrUGT76G1 genes encoding ent-kaurenoic acid-13 hydroxylase and three UDP glycosyltransferases of steviol glycoside biosynthesis pathway were silenced in Stevia rebaudiana to understand its molecular mechanism and association with gibberellins. METHODOLOGY/PRINCIPAL FINDINGS: RNAi mediated AMTS of SrKA13H and three SrUGTs has significantly reduced the expression of targeted endogenous genes as well as total steviol glycoside accumulation. While gibberellins (GA3 content was significantly enhanced on AMTS of SrUGT85C2 and SrKA13H. Silencing of SrKA13H and SrUGT85C2 was found to block the metabolite flux of steviol glycoside pathway and shifted it towards GA3 biosynthesis. Further, molecular docking of three SrUGT proteins has documented highest affinity of SrUGT76G1 for the substrates of alternate pathways synthesizing steviol glycosides. This could be a plausible reason for maximum reduction in steviol glycoside content on silencing of SrUGT76G1 than other genes. CONCLUSIONS: SrKA13H and SrUGT85C2 were identified as regulatory genes influencing carbon flux between steviol glycoside and gibberellin biosynthesis. This study has also documented the existence of alternate steviol glycoside biosynthesis route.

  10. In vivo kinetic analysis of the penicillin biosynthesis pathway using PAA stimulus response experiments.

    Science.gov (United States)

    Deshmukh, Amit T; Verheijen, Peter J T; Maleki Seifar, Reza; Heijnen, Joseph J; van Gulik, Walter M

    2015-11-01

    In this study we combined experimentation with mathematical modeling to unravel the in vivo kinetic properties of the enzymes and transporters of the penicillin biosynthesis pathway in a high yielding Penicillium chrysogenum strain. The experiment consisted of a step response experiment with the side chain precursor phenyl acetic acid (PAA) in a glucose-limited chemostat. The metabolite data showed that in the absence of PAA all penicillin pathway enzymes were expressed, leading to the production of a significant amount of 6-aminopenicillanic acid (6APA) as end product. After the stepwise perturbation with PAA, the pathway produced PenG within seconds. From the extra- and intracellular metabolite measurements, hypotheses for the secretion mechanisms of penicillin pathway metabolites were derived. A dynamic model of the penicillin biosynthesis pathway was then constructed that included the formation and transport over the cytoplasmic membrane of pathway intermediates, PAA and the product penicillin-G (PenG). The model parameters and changes in the enzyme levels of the penicillin biosynthesis pathway under in vivo conditions were simultaneously estimated using experimental data obtained at three different timescales (seconds, minutes, hours). The model was applied to determine changes in the penicillin pathway enzymes in time, calculate fluxes and analyze the flux control of the pathway. This led to a reassessment of the in vivo behavior of the pathway enzymes and in particular Acyl-CoA:Isopenicillin N Acyltransferase (AT). Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  11. Insect-Induced Conifer Defense. White Pine Weevil and Methyl Jasmonate Induce Traumatic Resinosis, de Novo Formed Volatile Emissions, and Accumulation of Terpenoid Synthase and Putative Octadecanoid Pathway Transcripts in Sitka Spruce1[w

    Science.gov (United States)

    Miller, Barbara; Madilao, Lufiani L.; Ralph, Steven; Bohlmann, Jörg

    2005-01-01

    Stem-boring insects and methyl jasmonate (MeJA) are thought to induce similar complex chemical and anatomical defenses in conifers. To compare insect- and MeJA-induced terpenoid responses, we analyzed traumatic oleoresin mixtures, emissions of terpenoid volatiles, and expression of terpenoid synthase (TPS) genes in Sitka spruce (Picea sitchensis) following attack by white pine weevils (Pissodes strobi) or application of MeJA. Both insects and MeJA caused traumatic resin accumulation in stems, with more accumulation induced by the weevils. Weevil-induced terpenoid emission profiles were also more complex than emissions induced by MeJA. Weevil feeding caused a rapid release of a blend of monoterpene olefins, presumably by passive evaporation of resin compounds from stem feeding sites. These compounds were not found in MeJA-induced emissions. Both weevils and MeJA caused delayed, diurnal emissions of (−)-linalool, indicating induced de novo biosynthesis of this compound. TPS transcripts strongly increased in stems upon insect attack or MeJA treatment. Time courses and intensity of induced TPS transcripts were different for monoterpene synthases, sesquiterpene synthases, and diterpene synthases. Increased levels of weevil- and MeJA-induced TPS transcripts accompanied major changes in terpenoid accumulation in stems. Induced TPS expression profiles in needles were less complex than those in stems and matched induced de novo emissions of (−)-linalool. Overall, weevils and MeJA induced similar, but not identical, terpenoid defense responses in Sitka spruce. Findings of insect- and MeJA-induced accumulation of allene oxide synthase-like and allene oxide cyclase-like transcripts are discussed in the context of traumatic resinosis and induced volatile emissions in this gymnosperm system. PMID:15618433

  12. GROWTH RETARDANTS: Effects on Gibberellin Biosynthesis and Other Metabolic Pathways.

    Science.gov (United States)

    Rademacher, Wilhelm

    2000-06-01

    Plant growth retardants are applied in agronomic and horticultural crops to reduce unwanted longitudinal shoot growth without lowering plant productivity. Most growth retardants act by inhibiting gibberellin (GA) biosynthesis. To date, four different types of such inhibitors are known: (a) Onium compounds, such as chlormequat chloride, mepiquat chloride, chlorphonium, and AMO-1618, which block the cyclases copalyl-diphosphate synthase and ent-kaurene synthase involved in the early steps of GA metabolism. (b) Compounds with an N-containing heterocycle, e.g. ancymidol, flurprimidol, tetcyclacis, paclobutrazol, uniconazole-P, and inabenfide. These retardants block cytochrome P450-dependent monooxygenases, thereby inhibiting oxidation of ent-kaurene into ent-kaurenoic acid. (c) Structural mimics of 2-oxoglutaric acid, which is the co-substrate of dioxygenases that catalyze late steps of GA formation. Acylcyclohexanediones, e.g. prohexadione-Ca and trinexapac-ethyl and daminozide, block particularly 3ss-hydroxylation, thereby inhibiting the formation of highly active GAs from inactive precursors, and (d) 16,17-Dihydro-GA5 and related structures act most likely by mimicking the GA precursor substrate of the same dioxygenases. Enzymes, similar to the ones involved in GA biosynthesis, are also of importance in the formation of abscisic acid, ethylene, sterols, flavonoids, and other plant constituents. Changes in the levels of these compounds found after treatment with growth retardants can mostly be explained by side activities on such enzymes.

  13. The biosynthesis of the chromophore of phycocyanin. Pathway of reduction of biliverdin to phycocyanobilin.

    Science.gov (United States)

    Brown, S B; Holroyd, J A; Vernon, D I; Shim, Y K; Smith, K M

    1989-01-01

    The later stages in the pathway of biosynthesis of phycocyanobilin, the chromophore of phycocyanin, were studied by using radiolabelled intermediates. Three possible pathways from biliverdin IX-alpha to phycocyanobilin were considered. 14C-labelled samples of key intermediates in two of the pathways, 3-vinyl-18-ethyl biliverdin IX-alpha and 3-ethyl-18-vinyl biliverdin IX-alpha, were synthesized chemically and were administered to cultures of Cyanidium caldarium that were actively synthesizing photosynthetic pigments in the light. Neither of these two compounds was apparently incorporated into the phycobiliprotein chromophore, suggesting that two of the three pathways were not operative. By elimination, the results imply that the third possible pathway, which involves phytochromobilin, the chromophore of phytochrome, represents the route for biosynthesis of phycocyanobilin. Unfortunately, since 14C-labelled phytochromobilin is not available, no direct proof of this pathway could be obtained. However, if correct, the present interpretation represents a unified pathway for biosynthesis of all plant bilins, via the intermediacy of phytochromobilin. PMID:2505754

  14. Ecdysteroid biosynthesis in varroa mites: identification of halloween genes from the biosynthetic pathway

    Science.gov (United States)

    Biosynthesis of ecdysteroids involves sequential enzymatic hydroxylations by microsomal enzymes and mitochondrial cytochrome P450’s. Enzymes of the pathway are collectively known as Halloween genes. Complete sequences for three Halloween genes, spook (Vdspo), disembodied (Vddib) and shade (Vdshd), w...

  15. Developing fermentative terpenoid production for commercial usage.

    Science.gov (United States)

    Leavell, Michael D; McPhee, Derek J; Paddon, Chris J

    2016-02-01

    Terpenoids comprise a large (>55000) family of compounds, very few of which have been used commercially due to low and economically unpractical production in their native hosts (generally plants and microorganisms). Two examples of natural terpenoid production are described (rubber and astaxanthin), but the advent of metabolic engineering has allowed the development of fermentative production processes using heterologous microorganisms. The two biochemical pathways responsible for terpenoid production are described, along with manipulations that enable production of terpenoids at economically viable levels. Finally, this article reviews some terpenoids that are currently in commercial production or development, ranging from semisynthetic production of the antimalarial drug artemisinin, through fragrance molecules, to commodity chemicals such as isoprene and β-farnesene. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Quantitation of NAD+ biosynthesis from the salvage pathway in Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Sporty, J; Lin, S; Kato, M; Ognibene, T; Stewart, B; Turteltaub, K; Bench, G

    2009-02-18

    Nicotinamide adenine dinucleotide (NAD{sup +}) is synthesized via two major pathways in prokaryotic and eukaryotic systems: the de novo biosynthesis pathway from tryptophan precursors, or by the salvage biosynthesis pathway from either extracellular nicotinic acid or various intracellular NAD{sup +} decomposition products. NAD{sup +} biosynthesis via the salvage pathway has been linked to an increase in yeast replicative lifespan under calorie restriction (CR). However, the relative contribution of each pathway to NAD{sup +} biosynthesis under both normal and CR conditions is not known. Here, we have performed lifespan, NAD{sup +} and NADH (the reduced form of NAD{sup +}) analyses on BY4742 wild type, NAD+ salvage pathway knockout (npt1{Delta}), and NAD+ de novo pathway knockout (qpt1{Delta}) yeast strains cultured in media containing either 2% glucose (normal growth) or 0.5% glucose (CR). We have utilized {sup 14}C labeled nicotinic acid in the culture media combined with HPLC speciation and both UV and {sup 14}C detection to quantitate the total amounts of NAD{sup +} and NADH and the amounts derived from the salvage pathway. We observe that wild type and qpt1{Delta} yeast exclusively utilize extracellular nicotinic acid for NAD{sup +} and NADH biosynthesis under both the 2% and 0.5% glucose growth conditions suggesting that the de novo pathway plays little role if a functional salvage pathway is present. We also observe that NAD{sup +} concentrations decrease in all three strains under CR. However, unlike the wild type strain, NADH concentrations do not decrease and NAD{sup +}:NADH ratios do not increase under CR for either knockout strain. Lifespan analyses reveal that CR results in a lifespan increase of approximately 25% for the wild type and qpt1{Delta} strains, while no increase in lifespan is observed for the npt1{Delta} strain. In combination these data suggest that having a functional salvage pathway is more important than the absolute levels of NAD

  17. Microbial conversions of terpenoids

    OpenAIRE

    Parshikov, Igor A

    2015-01-01

    The monograph describes examples of the application of microbial technology for obtaining of derivatives of terpenoids. Obtaining new derivatives of terpenoids, including artemisinin derivatives with increased antimalarial activity, is an important goal of research in microbial biotechnology and medicinal chemistry.

  18. Novel tryptophan metabolic pathways in auxin biosynthesis in silkworm.

    Science.gov (United States)

    Yokoyama, Chiaki; Takei, Mami; Kouzuma, Yoshiaki; Nagata, Shinji; Suzuki, Yoshihito

    2017-08-01

    In the course of our study of the biosynthetic pathway of auxin, a class of phytohormones, in insects, we proposed the biosynthetic pathway tryptophan (Trp)→indole-3-acetaldoxime (IAOx)→indole-3-acetadehyde (IAAld)→indole-3-acetic acid (IAA). In this study, we identified two branches in the metabolic pathways in the silkworm, possibly affecting the efficiency of IAA production: Trp→indole-3-pyruvic acid→indole-3-lactic acid and IAAld→indole-3-ethanol. We also determined the apparent conversion activities (2.05×10-7UmL-1 for Trp→IAA, 1.30×10-5UmL-1 for IAOx→IAA, and 3.91×10-1UmL-1 for IAAld→IAA), which explain why IAOx and IAAld are barely detectable as either endogenous compounds or metabolites of their precursors. The failure to detect IAAld, even in the presence of an inhibitor of the conversion IAAld→IAA, is explained by a switch in the conversion from IAAld→IAA to IAAld→IEtOH. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Evolution of the Phosphatidylcholine Biosynthesis Pathways in Green Algae: Combinatorial Diversity of Methyltransferases.

    Science.gov (United States)

    Hirashima, Takashi; Toyoshima, Masakazu; Moriyama, Takashi; Sato, Naoki

    2018-01-12

    Phosphatidylcholine (PC) is one of the most common phospholipids in eukaryotes, although some green algae such as Chlamydomonas reinhardtii are known to lack PC. Recently, we detected PC in four species in the genus Chlamydomonas: C. applanata NIES-2202, C. asymmetrica NIES-2207, C. debaryana NIES-2212, and C. sphaeroides NIES-2242. To reveal the PC biosynthesis pathways in green algae and the evolutionary scenario involved in their diversity, we analyzed the PC biosynthesis genes in these four algae using draft genome sequences. Homology searches suggested that PC in these species is synthesized by phosphoethanolamine-N-methyltransferase (PEAMT) and/or phosphatidylethanolamine-N-methyltransferase (PEMT), both of which are absent in C. reinhardtii. Recombinant PEAMTs from these algae showed methyltransferase activity for phosphoethanolamine but not for monomethyl phosphoethanolamine in vitro, in contrast to land plant PEAMT, which catalyzes the three methylations from phosphoethanolamine to phosphocholine. This suggested an involvement of other methyltransferases in PC biosynthesis. Here, we characterized the putative phospholipid-N-methyltransferase (PLMT) genes of these species by genetic and phylogenetic analysis. Complementation assays using a PC biosynthesis-deficient yeast suggested that the PLMTs of these algae can synthesize PC from phosphatidylethanolamine. These results indicated that the PC biosynthesis pathways in green algae differ from those of land plants, although the enzymes involved are homologous. Phylogenetic analysis suggested that the PEAMTs and PLMTs in these algae were inherited from the common ancestor of green algae. The absence of PC biosynthesis in many Chlamydomonas species is likely a result of parallel losses of PEAMT and PLMT in this genus.

  20. Transcriptome Analysis of Manganese-deficient Chlamydomonas reinhardtii Provides Insight on the Chlorophyll Biosynthesis Pathway

    Energy Technology Data Exchange (ETDEWEB)

    Lockhart, Ainsley; Zvenigorodsky, Natasha; Pedraza, Mary Ann; Lindquist, Erika

    2011-08-11

    The biosynthesis of chlorophyll and other tetrapyrroles is a vital but poorly understood process. Recent genomic advances with the unicellular green algae Chlamydomonas reinhardtii have created opportunity to more closely examine the mechanisms of the chlorophyll biosynthesis pathway via transcriptome analysis. Manganese is a nutrient of interest for complex reactions because of its multiple stable oxidation states and role in molecular oxygen coordination. C. reinhardtii was cultured in Manganese-deplete Tris-acetate-phosphate (TAP) media for 24 hours and used to create cDNA libraries for sequencing using Illumina TruSeq technology. Transcriptome analysis provided intriguing insight on possible regulatory mechanisms in the pathway. Evidence supports similarities of GTR (Glutamyl-tRNA synthase) to its Chlorella vulgaris homolog in terms of Mn requirements. Data was also suggestive of Mn-related compensatory up-regulation for pathway proteins CHLH1 (Manganese Chelatase), GUN4 (Magnesium chelatase activating protein), and POR1 (Light-dependent protochlorophyllide reductase). Intriguingly, data suggests possible reciprocal expression of oxygen dependent CPX1 (coproporphyrinogen III oxidase) and oxygen independent CPX2. Further analysis using RT-PCR could provide compelling evidence for several novel regulatory mechanisms in the chlorophyll biosynthesis pathway.

  1. Biosynthesis of salvinorin A proceeds via the deoxyxylulose phosphate pathway

    Science.gov (United States)

    Kutrzeba, Lukasz; Dayan, Franck E.; Howell, J’Lynn; Feng, Ju; Giner, José-Luis; Zjawiony, Jordan K.

    2007-01-01

    Salvinorin A, a neoclerodane diterpenoid, isolated from the Mexican hallucinogenic plant, Salvia divinorum is a potent kappa-opioid receptor agonist. Its biosynthetic route was studied by NMR and HR-ESI-MS analysis of the products of the incorporation of [1-13C]-glucose, [Me-13C]-methionine, and [1-13C; 3,4-2H2]-1-deoxy-d-xylulose into its structure. The use of cuttings and direct stem injection were unsuccessful, however, incorporation of 13C into salvinorin A was achieved using in vitro sterile culture of microshoots. NMR analysis of salvinorin A (2.7 mg) isolated from 200 microshoots grown in the presence of [1-13C]-glucose established that this pharmacologically important diterpene is biosynthesized via the 1-deoxy-d-xylulose-5-phosphate pathway, instead of the classic mevalonic acid pathway. This was confirmed in plants grown in the presence of [1-13C; 3,4-2H2]-1-deoxy-d-xylulose. In addition, analysis of salvinorin A produced by plants grown in the presence of [Me-13C]-methionine indicates that the methylation of the C-4 carboxyl group is catalyzed by a type III S-adenosyl-l-methionine-dependent O-methyltransferase. PMID:17574635

  2. Biosynthesis of promatrix metalloproteinase-9/chondroitin sulphate proteoglycan heteromer involves a Rottlerin-sensitive pathway.

    Directory of Open Access Journals (Sweden)

    Nabin Malla

    Full Text Available BACKGROUND: Previously we have shown that a fraction of the matrix metalloproteinase-9 (MMP-9 synthesized by the macrophage cell line THP-1 was bound to a chondroitin sulphate proteoglycan (CSPG core protein as a reduction sensitive heteromer. Several biochemical properties of the enzyme were changed when it was bound to the CSPG. METHODOLOGY/PRINCIPAL FINDINGS: By use of affinity chromatography, zymography, and radioactive labelling, various macrophage stimulators were tested for their effect on the synthesis of the proMMP-9/CSPG heteromer and its components by THP-1 cells. Of the stimulators, only PMA largely increased the biosynthesis of the heteromer. As PMA is an activator of PKC, we determined which PKC isoenzymes were expressed by performing RT-PCR and Western Blotting. Subsequently specific inhibitors were used to investigate their involvement in the biosynthesis of the heteromer. Of the inhibitors, only Rottlerin repressed the biosynthesis of proMMP-9/CSPG and its two components. Much lower concentrations of Rottlerin were needed to reduce the amount of CSPG than what was needed to repress the synthesis of the heteromer and MMP-9. Furthermore, Rottlerin caused a minor reduction in the activation of the PKC isoenzymes δ, ε, θ and υ (PKD3 in both control and PMA exposed cells. CONCLUSIONS/SIGNIFICANCE: The biosynthesis of the proMMP-9/CSPG heteromer and proMMP-9 in THP-1 cells involves a Rottlerin-sensitive pathway that is different from the Rottlerin sensitive pathway involved in the CSPG biosynthesis. MMP-9 and CSPGs are known to be involved in various physiological and pathological processes. Formation of complexes may influence both the specificity and localization of the enzyme. Therefore, knowledge about biosynthetic pathways and factors involved in the formation of the MMP-9/CSPG heteromer may contribute to insight in the heteromers biological function as well as pointing to future targets for therapeutic agents.

  3. BnWRI1 coordinates fatty acid biosynthesis and photosynthesis pathways during oil accumulation in rapeseed.

    Science.gov (United States)

    Wu, Xue-Long; Liu, Zhi-Hong; Hu, Zhang-Hua; Huang, Rui-Zhi

    2014-06-01

    Photosynthesis in "green" seeds, such as rapeseed, soybean, and Arabidopsis, plays a substantial role in the improved efficiency of oil accumulation. However, the molecular mechanism underpinning the coordinated expression of fatty acid (FA) biosynthesis- and photosynthesis-related genes in such developing seeds remains to be elucidated. Here, we found that seed-specific overexpression of BnWRI1, a WRI1 homolog from rapeseed (Brassica napus cv. ZGY2), results in enhanced chlorophyll content in developing seeds and increased oil content and seed mass in matured seeds. BnWRI1 was co-expressed with BnBCCP and BnCAB, two marker genes of FA biosynthesis and photosynthesis during seed development, respectively. Overexpression of BnWRI1 increased expression of both marker genes. Further, the nuclear-localized BnWRI1 protein was found to act as a transcription activator. It could bind to the GT1-element and/or GCC-box, which are widespread in the upstream regions of genes involved in FA biosynthesis and photosynthesis pathways. Accordingly, BnWRI1 could interact with promoters of BCCP2 and LHB1B2 in vivo. These results suggested that BnWRI1 may coordinate FA biosynthesis and photosynthesis pathways in developing seeds via directly stimulating expression of GT1-element and/or GCC-box containing genes. © 2014 Institute of Botany, Chinese Academy of Sciences.

  4. RNA-Seq analysis for indigo biosynthesis pathway genes in Indigofera tinctoria and Polygonum tinctorium

    Directory of Open Access Journals (Sweden)

    Bijaya K. Sarangi

    2015-12-01

    Full Text Available Natural indigo is the most important blue dye for textile dyeing and valuable secondary metabolite biosynthesized in Indigofera tinctoria and Polygonum tinctorium plants. Present investigation is made to generation of gene resource for pathway enrichment and to understand possible gene expression involved in indigo biosynthesis. The data about raw reads and the transcriptome assembly project has been deposited at GenBank under the accessions SRA180766 and SRX692542 for I. tinctoria and P. tinctorium, respectively.

  5. Essential role of Bordetella NadC in a quinolinate salvage pathway for NAD biosynthesis.

    Science.gov (United States)

    Brickman, Timothy J; Suhadolc, Ryan J; McKelvey, Pamela J; Armstrong, Sandra K

    2017-02-01

    Nicotinamide adenine dinucleotide (NAD) is produced via de novo biosynthesis pathways and by salvage or recycling routes. The classical Bordetella bacterial species are known to be auxotrophic for nicotinamide or nicotinic acid. This study confirmed that Bordetella bronchiseptica, Bordetella pertussis and Bordetella parapertussis have the recycling/salvage pathway genes pncA and pncB, for use of nicotinamide or nicotinic acid, respectively, for NAD synthesis. Although these Bordetellae lack the nadA and nadB genes needed for de novo NAD biosynthesis, remarkably, they have one de novo pathway gene, nadC, encoding quinolinate phosphoribosyltransferase. Genomic analyses of taxonomically related Bordetella and Achromobacter species also indicated the presence of an 'orphan' nadC and the absence of nadA and nadB. When supplied as the sole NAD precursor, quinolinate promoted B. bronchiseptica growth, and the ability to use it required nadC. Co-expression of Bordetella nadC with the nadB and nadA genes of Paraburkholderia phytofirmans allowed B. bronchiseptica to grow in the absence of supplied pyridines, indicative of de novo NAD synthesis and functional confirmation of Bordetella NadC activity. Expression of nadC in B. bronchiseptica was influenced by nicotinic acid and by a NadQ family transcriptional repressor, indicating that these organisms prioritize their use of pyridines for NAD biosynthesis. © 2016 John Wiley & Sons Ltd.

  6. Filling gaps in bacterial amino acid biosynthesis pathways with high-throughput genetics.

    Science.gov (United States)

    Price, Morgan N; Zane, Grant M; Kuehl, Jennifer V; Melnyk, Ryan A; Wall, Judy D; Deutschbauer, Adam M; Arkin, Adam P

    2018-01-01

    For many bacteria with sequenced genomes, we do not understand how they synthesize some amino acids. This makes it challenging to reconstruct their metabolism, and has led to speculation that bacteria might be cross-feeding amino acids. We studied heterotrophic bacteria from 10 different genera that grow without added amino acids even though an automated tool predicts that the bacteria have gaps in their amino acid synthesis pathways. Across these bacteria, there were 11 gaps in their amino acid biosynthesis pathways that we could not fill using current knowledge. Using genome-wide mutant fitness data, we identified novel enzymes that fill 9 of the 11 gaps and hence explain the biosynthesis of methionine, threonine, serine, or histidine by bacteria from six genera. We also found that the sulfate-reducing bacterium Desulfovibrio vulgaris synthesizes homocysteine (which is a precursor to methionine) by using DUF39, NIL/ferredoxin, and COG2122 proteins, and that homoserine is not an intermediate in this pathway. Our results suggest that most free-living bacteria can likely make all 20 amino acids and illustrate how high-throughput genetics can uncover previously-unknown amino acid biosynthesis genes.

  7. Inhibition of pyrimidine biosynthesis pathway suppresses viral growth through innate immunity.

    Directory of Open Access Journals (Sweden)

    Marianne Lucas-Hourani

    Full Text Available Searching for stimulators of the innate antiviral response is an appealing approach to develop novel therapeutics against viral infections. Here, we established a cell-based reporter assay to identify compounds stimulating expression of interferon-inducible antiviral genes. DD264 was selected out of 41,353 compounds for both its immuno-stimulatory and antiviral properties. While searching for its mode of action, we identified DD264 as an inhibitor of pyrimidine biosynthesis pathway. This metabolic pathway was recently identified as a prime target of broad-spectrum antiviral molecules, but our data unraveled a yet unsuspected link with innate immunity. Indeed, we showed that DD264 or brequinar, a well-known inhibitor of pyrimidine biosynthesis pathway, both enhanced the expression of antiviral genes in human cells. Furthermore, antiviral activity of DD264 or brequinar was found strictly dependent on cellular gene transcription, nuclear export machinery, and required IRF1 transcription factor. In conclusion, the antiviral property of pyrimidine biosynthesis inhibitors is not a direct consequence of pyrimidine deprivation on the virus machinery, but rather involves the induction of cellular immune response.

  8. Diverse pathways of phosphatidylcholine biosynthesis in algae as estimated by labeling studies and genomic sequence analysis.

    Science.gov (United States)

    Sato, Naoki; Mori, Natsumi; Hirashima, Takashi; Moriyama, Takashi

    2016-08-01

    Phosphatidylcholine (PC) is an almost ubiquitous phospholipid in eukaryotic algae and plants but is not found in a few species, for example Chlamydomonas reinhardtii. We recently found that some species of the genus Chlamydomonas possess PC. In the universal pathway, PC is synthesized de novo by methylation of phosphatidylethanolamine (PE) or transfer of phosphocholine from cytidine diphosphate (CDP)-choline to diacylglycerol. Phosphocholine, the direct precursor to CDP-choline, is synthesized either by methylation of phosphoethanolamine or phosphorylation of choline. Here we analyzed the mechanism of PC biosynthesis in two species of Chlamydomonas (asymmetrica and sphaeroides) as well as in a red alga, Cyanidioschyzon merolae. Comparative genomic analysis of enzymes involved in PC biosynthesis indicated that C. merolae possesses only the PE methylation pathway. Radioactive tracer experiments using [(32) P]phosphate showed delayed labeling of PC with respect to PE, which was consistent with the PE methylation pathway. In Chlamydomonas asymmetrica, labeling of PC was detected from the early time of incubation with [(32) P]phosphate, suggesting the operation of phosphoethanolamine methylation pathway. Genomic analysis indeed detected the genes for the phosphoethanolamine methylation pathway. In contrast, the labeling of PC in C. sphaeroides was slow, suggesting that the PE methylation pathway was at work. These results as well as biochemical and computational results uncover an unexpected diversity of the mechanisms for PC biosynthesis in algae. Based on these results, we will discuss plausible mechanisms for the scattered distribution of the ability to biosynthesize PC in the genus Chlamydomonas. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  9. Improving yeast strains using recyclable integration cassettes, for the production of plant terpenoids

    Directory of Open Access Journals (Sweden)

    Johnson Christopher B

    2011-01-01

    Full Text Available Abstract Background Terpenoids constitute a large family of natural products, attracting commercial interest for a variety of uses as flavours, fragrances, drugs and alternative fuels. Saccharomyces cerevisiae offers a versatile cell factory, as the precursors of terpenoid biosynthesis are naturally synthesized by the sterol biosynthetic pathway. Results S. cerevisiae wild type yeast cells, selected for their capacity to produce high sterol levels were targeted for improvement aiming to increase production. Recyclable integration cassettes were developed which enable the unlimited sequential integration of desirable genetic elements (promoters, genes, termination sequence at any desired locus in the yeast genome. The approach was applied on the yeast sterol biosynthetic pathway genes HMG2, ERG20 and IDI1 resulting in several-fold increase in plant monoterpene and sesquiterpene production. The improved strains were robust and could sustain high terpenoid production levels for an extended period. Simultaneous plasmid-driven co-expression of IDI1 and the HMG2 (K6R variant, in the improved strain background, maximized monoterpene production levels. Expression of two terpene synthase enzymes from the sage species Salvia fruticosa and S. pomifera (SfCinS1, SpP330 in the modified yeast cells identified a range of terpenoids which are also present in the plant essential oils. Co-expression of the putative interacting protein HSP90 with cineole synthase 1 (SfCinS1 also improved production levels, pointing to an additional means to improve production. Conclusions Using the developed molecular tools, new yeast strains were generated with increased capacity to produce plant terpenoids. The approach taken and the durability of the strains allow successive rounds of improvement to maximize yields.

  10. Improving yeast strains using recyclable integration cassettes, for the production of plant terpenoids

    Science.gov (United States)

    2011-01-01

    Background Terpenoids constitute a large family of natural products, attracting commercial interest for a variety of uses as flavours, fragrances, drugs and alternative fuels. Saccharomyces cerevisiae offers a versatile cell factory, as the precursors of terpenoid biosynthesis are naturally synthesized by the sterol biosynthetic pathway. Results S. cerevisiae wild type yeast cells, selected for their capacity to produce high sterol levels were targeted for improvement aiming to increase production. Recyclable integration cassettes were developed which enable the unlimited sequential integration of desirable genetic elements (promoters, genes, termination sequence) at any desired locus in the yeast genome. The approach was applied on the yeast sterol biosynthetic pathway genes HMG2, ERG20 and IDI1 resulting in several-fold increase in plant monoterpene and sesquiterpene production. The improved strains were robust and could sustain high terpenoid production levels for an extended period. Simultaneous plasmid-driven co-expression of IDI1 and the HMG2 (K6R) variant, in the improved strain background, maximized monoterpene production levels. Expression of two terpene synthase enzymes from the sage species Salvia fruticosa and S. pomifera (SfCinS1, SpP330) in the modified yeast cells identified a range of terpenoids which are also present in the plant essential oils. Co-expression of the putative interacting protein HSP90 with cineole synthase 1 (SfCinS1) also improved production levels, pointing to an additional means to improve production. Conclusions Using the developed molecular tools, new yeast strains were generated with increased capacity to produce plant terpenoids. The approach taken and the durability of the strains allow successive rounds of improvement to maximize yields. PMID:21276210

  11. Isoprenoid biosynthesis in Artemisia annua: Cloning and heterologous expression of a germacrene A synthase from a glandular trichome cDNA library

    NARCIS (Netherlands)

    Bertea, C.M.; Voster, A.; Verstappen, F.W.A.; Maffei, M.; Beekwilder, M.J.; Bouwmeester, H.J.

    2006-01-01

    Artemisia annua (Asteraceae) is the source of the anti-malarial compound artemisinin. To elucidate the biosynthetic pathway and to isolate and characterize genes involved in the biosynthesis of terpenoids including artemisinin in A. annua, glandular trichomes were used as an enriched source for

  12. Transcriptome and biochemical analyses revealed a detailed proanthocyanidin biosynthesis pathway in brown cotton fiber.

    Directory of Open Access Journals (Sweden)

    Yue-Hua Xiao

    Full Text Available Brown cotton fiber is the major raw material for colored cotton industry. Previous studies have showed that the brown pigments in cotton fiber belong to proanthocyanidins (PAs. To clarify the details of PA biosynthesis pathway in brown cotton fiber, gene expression profiles in developing brown and white fibers were compared via digital gene expression profiling and qRT-PCR. Compared to white cotton fiber, all steps from phenylalanine to PA monomers (flavan-3-ols were significantly up-regulated in brown fiber. Liquid chromatography mass spectrometry analyses showed that most of free flavan-3-ols in brown fiber were in 2, 3-trans form (gallocatechin and catechin, and the main units of polymeric PAs were trihydroxylated on B ring. Consistent with monomeric composition, the transcript levels of flavonoid 3', 5'-hydroxylase and leucoanthocyanidin reductase in cotton fiber were much higher than their competing enzymes acting on the same substrates (dihydroflavonol 4-reductase and anthocyanidin synthase, respectively. Taken together, our data revealed a detailed PA biosynthesis pathway wholly activated in brown cotton fiber, and demonstrated that flavonoid 3', 5'-hydroxylase and leucoanthocyanidin reductase represented the primary flow of PA biosynthesis in cotton fiber.

  13. Transcriptome analysis of bitter acid biosynthesis and precursor pathways in hop (Humulus lupulus

    Directory of Open Access Journals (Sweden)

    Clark Shawn M

    2013-01-01

    Full Text Available Abstract Background Bitter acids (e.g. humulone are prenylated polyketides synthesized in lupulin glands of the hop plant (Humulus lupulus which are important contributors to the bitter flavour and stability of beer. Bitter acids are formed from acyl-CoA precursors derived from branched-chain amino acid (BCAA degradation and C5 prenyl diphosphates from the methyl-D-erythritol 4-phosphate (MEP pathway. We used RNA sequencing (RNA-seq to obtain the transcriptomes of isolated lupulin glands, cones with glands removed and leaves from high α-acid hop cultivars, and analyzed these datasets for genes involved in bitter acid biosynthesis including the supply of major precursors. We also measured the levels of BCAAs, acyl-CoA intermediates, and bitter acids in glands, cones and leaves. Results Transcripts encoding all the enzymes of BCAA metabolism were significantly more abundant in lupulin glands, indicating that BCAA biosynthesis and subsequent degradation occurs in these specialized cells. Branched-chain acyl-CoAs and bitter acids were present at higher levels in glands compared with leaves and cones. RNA-seq analysis showed the gland-specific expression of the MEP pathway, enzymes of sucrose degradation and several transcription factors that may regulate bitter acid biosynthesis in glands. Two branched-chain aminotransferase (BCAT enzymes, HlBCAT1 and HlBCAT2, were abundant, with gene expression quantification by RNA-seq and qRT-PCR indicating that HlBCAT1 was specific to glands while HlBCAT2 was present in glands, cones and leaves. Recombinant HlBCAT1 and HlBCAT2 catalyzed forward (biosynthetic and reverse (catabolic reactions with similar kinetic parameters. HlBCAT1 is targeted to mitochondria where it likely plays a role in BCAA catabolism. HlBCAT2 is a plastidial enzyme likely involved in BCAA biosynthesis. Phylogenetic analysis of the hop BCATs and those from other plants showed that they group into distinct biosynthetic (plastidial and

  14. Processing of Kansui Roots Stir-Baked with Vinegar Reduces Kansui-Induced Hepatocyte Cytotoxicity by Decreasing the Contents of Toxic Terpenoids and Regulating the Cell Apoptosis Pathway

    Directory of Open Access Journals (Sweden)

    Xiaojing Yan

    2014-06-01

    Full Text Available Euphorbia kansui is a Traditional Chinese Medicine widely used for the treatment of oedema, ascites and asthma. However, its serious hepatotoxicity hinders its safe clinical application. The process of stir-baking with vinegar is regularly used to reduce the toxicity of kansui. Up till now, the exact mechanism of the reduction in hepatotoxicity of kansui stir-baked with vinegar has been poorly defined. In this study, decreased  contents of five diterpene and one triterpene in kansui (GS-1 after stir-baking with vinegar (GS-2 was investigated by UPLC-QTOF/MS. Flow cytometry and Hoechst staining were used to show that the stir-baking with vinegar process reduces kansui-induced cell apoptosis. Furthermore, the result also indicated that kansui stir-baked with vinegar protects LO2 cells from apoptosis by increasing the cell mitochondrial membrane potential (ΔΨm, decreasing the release of cytochrome c and inhibiting the activities of caspase-9 and caspase-3 as evidenced by means of high content screening (HCS, ELISA and western blotting. These results suggested that the stir-baking vinegar could reduce the hepatotoxicity of kansui by effectively decreasing the contents of toxic terpenoids and inhibiting the intrinsic pathway of hepatocyte cell apoptosis. In conclusion, the study provided significant data for promoting safer and better clinical use of this herb.

  15. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Antimicrobial mechanism of theaflavins: They target 1-deoxy-D-xylulose 5-phosphate reductoisomerase, the key enzyme of the MEP terpenoid biosynthetic pathway.

    Science.gov (United States)

    Hui, Xian; Yue, Qiao; Zhang, Dan-Dan; Li, Heng; Yang, Shao-Qing; Gao, Wen-Yun

    2016-12-12

    1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is the first committed enzyme in the 2-methyl-D-erythritol 4-phosphate (MEP) terpenoid biosynthetic pathway and is also a validated antimicrobial target. Theaflavins, which are polyphenolic compounds isolated from fermented tea, possess a wide range of pharmacological activities, especially an antibacterial effect, but little has been reported on their modes of antimicrobial action. To uncover the antibacterial mechanism of theaflavins and to seek new DXR inhibitors from natural sources, the DXR inhibitory activity of theaflavins were investigated in this study. The results show that all four theaflavin compounds could specifically suppress the activity of DXR, with theaflavin displaying the lowest effect against DXR (IC50 162.1 μM) and theaflavin-3,3'-digallate exhibiting the highest (IC50 14.9 μM). Moreover, determination of inhibition kinetics of the theaflavins demonstrates that they are non-competitive inhibitors of DXR against 1-deoxy-D-xylulose 5-phosphate (DXP) and un-competitive inhibitors with respect to NADPH. The possible interactions between DXR and the theaflavins were simulated via docking experiments.

  17. Fatty Acid Biosynthesis Pathways in Methylomicrobium buryatense 5G(B1)

    Science.gov (United States)

    Demidenko, Aleksandr; Akberdin, Ilya R.; Allemann, Marco; Allen, Eric E.; Kalyuzhnaya, Marina G.

    2017-01-01

    Methane utilization by methanotrophic bacteria is an attractive application for biotechnological conversion of natural or biogas into high-added-value products. Haloalcaliphilic methanotrophic bacteria belonging to the genus Methylomicrobium are among the most promising strains for methane-based biotechnology, providing easy and inexpensive cultivation, rapid growth, and the availability of established genetic tools. A number of methane bioconversions using these microbial cultures have been discussed, including the derivation of biodiesel, alkanes, and OMEGA-3 supplements. These compounds are derived from bacterial fatty acid pools. Here, we investigate fatty acid biosynthesis in Methylomicrobium buryatense 5G(B1). Most of the genes homologous to typical Type II fatty acid biosynthesis pathways could be annotated by bioinformatics analyses, with the exception of fatty acid transport and regulatory elements. Different approaches for improving fatty acid accumulation were investigated. These studies indicated that both fatty acid degradation and acetyl- and malonyl-CoA levels are bottlenecks for higher level fatty acid production. The best strain generated in this study synthesizes 111 ± 2 mg/gDCW of extractable fatty acids, which is ~20% more than the original strain. A candidate gene for fatty acid biosynthesis regulation, farE, was identified and studied. Its deletion resulted in drastic changes to the fatty acid profile, leading to an increased pool of C18-fatty acid methyl ester. The FarE-regulon was further investigated by RNA-seq analysis of gene expression in farE-knockout mutants and farE-overexpressing strains. These gene profiles highlighted a novel set of enzymes and regulators involved in fatty acid biosynthesis. The gene expression and fatty acid profiles of the different farE-strains support the hypothesis that metabolic fluxes upstream of fatty acid biosynthesis restrict fatty acid production in the methanotroph. PMID:28119683

  18. Yeast glucose pathways converge on the transcriptional regulation of trehalose biosynthesis

    Directory of Open Access Journals (Sweden)

    Apweiler Eva

    2012-06-01

    Full Text Available Abstract Background Cellular glucose availability is crucial for the functioning of most biological processes. Our understanding of the glucose regulatory system has been greatly advanced by studying the model organism Saccharomyces cerevisiae, but many aspects of this system remain elusive. To understand the organisation of the glucose regulatory system, we analysed 91 deletion mutants of the different glucose signalling and metabolic pathways in Saccharomyces cerevisiae using DNA microarrays. Results In general, the mutations do not induce pathway-specific transcriptional responses. Instead, one main transcriptional response is discerned, which varies in direction to mimic either a high or a low glucose response. Detailed analysis uncovers established and new relationships within and between individual pathways and their members. In contrast to signalling components, metabolic components of the glucose regulatory system are transcriptionally more frequently affected. A new network approach is applied that exposes the hierarchical organisation of the glucose regulatory system. Conclusions The tight interconnection between the different pathways of the glucose regulatory system is reflected by the main transcriptional response observed. Tps2 and Tsl1, two enzymes involved in the biosynthesis of the storage carbohydrate trehalose, are predicted to be the most downstream transcriptional components. Epistasis analysis of tps2Δ double mutants supports this prediction. Although based on transcriptional changes only, these results suggest that all changes in perceived glucose levels ultimately lead to a shift in trehalose biosynthesis.

  19. Catabolism of citronellol and related acyclic terpenoids in pseudomonads.

    Science.gov (United States)

    Förster-Fromme, Karin; Jendrossek, Dieter

    2010-07-01

    Terpenes are a huge group of natural compounds characterised by their predominantly pleasant smell. They are built up by isoprene units in cyclic or acyclic form and can be functionalised by carbonyl, hydroxyl or carboxyl groups and by presence of additional carbon-carbon double bonds (terpenoids). Currently, much more than 10,000 terpenoid compounds are known, and many thereof are present in different iso- and stereoforms. Terpenoids are secondary metabolites and can have important biological functions in living organisms. In many cases, the biological functions of terpenoids are not known at all. Nevertheless, terpenoids are used in large quantities as perfumes and aroma compounds for food additives. Terpenoids can be also precursors and building blocks for synthesis of complex chiral compounds in chemical and pharmaceutical industry. Unfortunately, only few terpenoids are available in large quantities at reasonable costs. Therefore, characterisation of suited biocatalysts specific for terpenoid compounds and development of biotransformation processes of abundant terpenoids to commercially interesting derivates becomes more and more important. This minireview summarises knowledge on catabolic pathways and biotransformations of acyclic monoterpenes that have received only little attention. Terpenoids with 20 or more carbon atoms are not a subject of this study.

  20. Transcription factors in alkaloid biosynthesis.

    Science.gov (United States)

    Yamada, Yasuyuki; Sato, Fumihiko

    2013-01-01

    Higher plants produce a large variety of low-molecular weight secondary compounds. Among them, nitrogen-containing alkaloids are the most biologically active and are often used pharmaceutically. Whereas alkaloid chemistry has been intensively investigated, alkaloid biosynthesis, including the relevant biosynthetic enzymes, genes and their regulation, and especially transcription factors, is largely unknown, as only a limited number of plant species produce certain types of alkaloids and they are difficult to study. Recently, however, several groups have succeeded in isolating the transcription factors that are involved in the biosynthesis of several types of alkaloids, including bHLH, ERF, and WRKY. Most of them show Jasmonate (JA) responsiveness, which suggests that the JA signaling cascade plays an important role in alkaloid biosynthesis. Here, we summarize the types and functions of transcription factors that have been isolated in alkaloid biosynthesis, and characterize their similarities and differences compared to those in other secondary metabolite pathways, such as phenylpropanoid and terpenoid biosyntheses. The evolution of this biosynthetic pathway and regulatory network, as well as the application of these transcription factors to metabolic engineering, is discussed. © 2013, Elsevier Inc. All Rights Reserved.

  1. A mutation of EPT1 (SELENOI) underlies a new disorder of Kennedy pathway phospholipid biosynthesis.

    Science.gov (United States)

    Ahmed, Mustafa Y; Al-Khayat, Aisha; Al-Murshedi, Fathiya; Al-Futaisi, Amna; Chioza, Barry A; Pedro Fernandez-Murray, J; Self, Jay E; Salter, Claire G; Harlalka, Gaurav V; Rawlins, Lettie E; Al-Zuhaibi, Sana; Al-Azri, Faisal; Al-Rashdi, Fatma; Cazenave-Gassiot, Amaury; Wenk, Markus R; Al-Salmi, Fatema; Patton, Michael A; Silver, David L; Baple, Emma L; McMaster, Christopher R; Crosby, Andrew H

    2017-03-01

    Mutations in genes involved in lipid metabolism have increasingly been associated with various subtypes of hereditary spastic paraplegia, a highly heterogeneous group of neurodegenerative motor neuron disorders characterized by spastic paraparesis. Here, we report an unusual autosomal recessive neurodegenerative condition, best classified as a complicated form of hereditary spastic paraplegia, associated with mutation in the ethanolaminephosphotransferase 1 (EPT1) gene (now known as SELENOI), responsible for the final step in Kennedy pathway forming phosphatidylethanolamine from CDP-ethanolamine. Phosphatidylethanolamine is a glycerophospholipid that, together with phosphatidylcholine, constitutes more than half of the total phospholipids in eukaryotic cell membranes. We determined that the mutation defined dramatically reduces the enzymatic activity of EPT1, thereby hindering the final step in phosphatidylethanolamine synthesis. Additionally, due to central nervous system inaccessibility we undertook quantification of phosphatidylethanolamine levels and species in patient and control blood samples as an indication of liver phosphatidylethanolamine biosynthesis. Although this revealed alteration to levels of specific phosphatidylethanolamine fatty acyl species in patients, overall phosphatidylethanolamine levels were broadly unaffected indicating that in blood EPT1 inactivity may be compensated for, in part, via alternate biochemical pathways. These studies define the first human disorder arising due to defective CDP-ethanolamine biosynthesis and provide new insight into the role of Kennedy pathway components in human neurological function. © The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain.

  2. Terpenoids from Endophytic Fungi

    Directory of Open Access Journals (Sweden)

    Jucimar Jorgeane de Souza

    2011-12-01

    Full Text Available This work reviews the production of terpenoids by endophytic fungi and their biological activities, in period of 2006 to 2010. Sixty five sesquiterpenes, 45 diterpenes, five meroterpenes and 12 other terpenes, amounting to 127 terpenoids were isolated from endophytic fungi.

  3. Aromatic glucosinolate biosynthesis pathway in Barbarea vulgaris and its response to Plutella xylostella infestation

    Directory of Open Access Journals (Sweden)

    Tongjin eLiu

    2016-02-01

    Full Text Available The inducibility of the glucosinolate resistance mechanism is an energy-saving strategy for plants, but whether induction would still be triggered by glucosinolate-tolerant Plutella xylostella (diamondback moth, DBM after a plant had evolved a new resistance mechanism (e.g. saponins in Barbara vulgaris was unknown. In B. vulgaris, aromatic glucosinolates derived from homo-phenylalanine are the dominate glucosinolates, but their biosynthesis pathway are unclear in this plant. In this study, we used G-type (pest-resistant and P-type (pest-susceptible B. vulgaris to compare glucosinolate levels and the expression profiles of their biosynthesis genes before and after infestation by DBM larvae. Two different stereoisomers of hydroxylated aromatic glucosinolates are dominant in G- and P-type B. vulgaris, respectively, and are induced by DBM. The transcripts of genes in the glucosinolate biosynthesis pathway and their corresponding transcription factors were identified from an Illumina dataset of G- and P-type B. vulgaris. Many genes involved or potentially involved in glucosinolate biosynthesis were induced in both plant types. The expression patterns of six DBM induced genes were validated by quantitative PCR (qPCR, while six long-fragment genes were validated by molecular cloning. The core structure biosynthetic genes showed high sequence similarities between the two genotypes. In contrast, the sequence identity of two apparent side chain modification genes, the SHO gene in the G-type and the RHO in P-type plants, showed only 77.50% identity in coding DNA sequences and 65.48% identity in deduced amino acid sequences. The homology to GS-OH in Arabidopsis, DBM induction of the transcript and a series of qPCR and glucosinolate analyses of G-type, P-type and F1 plants indicated that these genes control the production of S and R isomers of 2-hydroxy-2-phenylethyl glucosinolate. These glucosinolates were significantly induced by P. xylostella larvae in

  4. Nematicidal activity of terpenoids.

    Science.gov (United States)

    Abdel-Rahman, Fawzia H; Alaniz, Nina M; Saleh, Mahmoud A

    2013-01-01

    Thirty four phytoterpenoids were evaluated for their nematicidal effect using the model nematode Caenorhabditis elegans. Nematicidal activities of the tested compounds at concentrations of 50 μg/mL showed wide variation in their effects ranging from no effect, weak, moderate and strong effects. Terpenoids exerting 50% or higher mortality at 50 μg/mL were further tested at five different concentrations to calculate the concentration that will kill 50% of the nematode population (LC(50)). Among the most effective terpenoids were carvacrol, thymol, nerolidol, α-terpinene, geraniol, citronellol, farnesol, limonene, pseudoionone and eugenol in a descending order. These compounds exhibited a dose-dependent effect. The results suggest that the selected monoterpenoids and essential oils with a high concentration of these compounds mayprovide potential natural nematicides and merit further study as botanical nematicides for the control of both plant and animal parasitic nematodes. In general, oxygenated terpenoids and phenolic terpenoids exhibited higher nematicidal activity than hydrocarbons terpenoids.

  5. Unravelling the architecture and dynamics of tropane alkaloid biosynthesis pathways using metabolite correlation networks.

    Science.gov (United States)

    Nguyen, Thi-Kieu-Oanh; Jamali, Arash; Lanoue, Arnaud; Gontier, Eric; Dauwe, Rebecca

    2015-08-01

    The tropane alkaloid spectrum in Solanaceae is highly variable within and between species. Little is known about the topology and the coordination of the biosynthetic pathways leading to the variety of tropine and pseudotropine derived esters in the alkaloid spectrum, or about the metabolic dynamics induced by tropane alkaloid biosynthesis stimulating conditions. A good understanding of the metabolism, including all ramifications, is however necessary for the development of strategies to increase the abundance of pharmacologically interesting compounds such as hyoscyamine and scopolamine. The present study explores the tropane alkaloid metabolic pathways in an untargeted approach involving a correlation-based network analysis. Using GC-MS metabolite profiling, the variation and co-variation among tropane alkaloids and primary metabolites was monitored in 60 Datura innoxia Mill. individuals, of which half were exposed to tropane alkaloid biosynthesis stimulating conditions by co-culture with Agrobacterium rhizogenes. Considerable variation was evident in the relative proportions of the tropane alkaloids. Remodeling of the tropane alkaloid spectrum under co-culture with A. rhizogenes involved a specific and strong increase of hyoscyamine production and revealed that the accumulation of hyoscyamine, 3-tigloyloxy-6,7-epoxytropane, and 3-methylbutyryloxytropane was controlled independently of the majority of tropane alkaloids. Based on correlations between metabolites, we propose a biosynthetic origin of hygrine, the order of esterification of certain di-oxygenated tropanes, and that the rate of acetoxylation contributes to control of hyoscyamine production. Overall, this study shows that the biosynthesis of tropane alkaloids may be far more complex and finely controlled than previously expected. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. MRE: a web tool to suggest foreign enzymes for the biosynthesis pathway design with competing endogenous reactions in mind

    KAUST Repository

    Kuwahara, Hiroyuki

    2016-04-29

    To rationally design a productive heterologous biosynthesis system, it is essential to consider the suitability of foreign reactions for the specific endogenous metabolic infrastructure of a host. We developed a novel web server, called MRE, which, for a given pair of starting and desired compounds in a given chassis organism, ranks biosynthesis routes from the perspective of the integration of new reactions into the endogenous metabolic system. For each promising heterologous biosynthesis pathway, MRE suggests actual enzymes for foreign metabolic reactions and generates information on competing endogenous reactions for the consumption of metabolites. These unique, chassis-centered features distinguish MRE from existing pathway design tools and allow synthetic biologists to evaluate the design of their biosynthesis systems from a different angle. By using biosynthesis of a range of high-value natural products as a case study, we show that MRE is an effective tool to guide the design and optimization of heterologous biosynthesis pathways. The URL of MRE is http://www.cbrc.kaust.edu.sa/mre/.

  7. MRE: a web tool to suggest foreign enzymes for the biosynthesis pathway design with competing endogenous reactions in mind.

    Science.gov (United States)

    Kuwahara, Hiroyuki; Alazmi, Meshari; Cui, Xuefeng; Gao, Xin

    2016-07-08

    To rationally design a productive heterologous biosynthesis system, it is essential to consider the suitability of foreign reactions for the specific endogenous metabolic infrastructure of a host. We developed a novel web server, called MRE, which, for a given pair of starting and desired compounds in a given chassis organism, ranks biosynthesis routes from the perspective of the integration of new reactions into the endogenous metabolic system. For each promising heterologous biosynthesis pathway, MRE suggests actual enzymes for foreign metabolic reactions and generates information on competing endogenous reactions for the consumption of metabolites. These unique, chassis-centered features distinguish MRE from existing pathway design tools and allow synthetic biologists to evaluate the design of their biosynthesis systems from a different angle. By using biosynthesis of a range of high-value natural products as a case study, we show that MRE is an effective tool to guide the design and optimization of heterologous biosynthesis pathways. The URL of MRE is http://www.cbrc.kaust.edu.sa/mre/. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. Steviol glycosides from Stevia: biosynthesis pathway review and their application in foods and medicine.

    Science.gov (United States)

    Yadav, Sudesh Kumar; Guleria, Praveen

    2012-01-01

    Stevia rebaudiana, a perennial herb from the Asteraceae family, is known to the scientific world for its sweetness and steviol glycosides (SGs). SGs are the secondary metabolites responsible for the sweetness of Stevia. They are synthesized by SG biosynthesis pathway operating in the leaves. Most of the genes encoding the enzymes of this pathway have been cloned and characterized from Stevia. Out of various SGs, stevioside and rebaudioside A are the major metabolites. SGs including stevioside have also been synthesized by enzymes and microbial agents. These are non-mutagenic, non-toxic, antimicrobial, and do not show any remarkable side-effects upon consumption. Stevioside has many medical applications and its role against diabetes is most important. SGs have made Stevia an important part of the medicinal world as well as the food and beverage industry. This article presents an overview on Stevia and the importance of SGs.

  9. Reconstruction of the carnitine biosynthesis pathway from Neurospora crassa in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Franken, Jaco; Burger, Anita; Swiegers, Jan H; Bauer, Florian F

    2015-08-01

    Industrial synthesis of L-carnitine is currently performed by whole-cell biotransformation of industrial waste products, mostly D-carnitine and cronobetaine, through specific bacterial species. No comparable system has been established using eukaryotic microorganisms, even though there is a significant and growing international demand for either the pure compound or carnitine-enriched consumables. In eukaryotes, including the fungus Neurospora crassa, L-carnitine is biosynthesized through a four-step metabolic conversion of trimethyllysine to L-carnitine. In contrast, the industrial yeast, Saccharomyces cerevisiae lacks the enzymes of the eukaryotic biosynthesis pathway and is unable to synthesize carnitine. This study describes the cloning of all four of the N. crassa carnitine biosynthesis genes and the reconstruction of the entire pathway in S. cerevisiae. The engineered yeast strains were able to catalyze the synthesis of L-carnitine, which was quantified using hydrophilic interaction liquid chromatography electrospray ionization mass spectrometry (HILIC-ESI-MS) analyses, from trimethyllysine. Furthermore, the yeast threonine aldolase Gly1p was shown to effectively catalyze the second step of the pathway, fulfilling the role of a serine hydroxymethyltransferase. The analyses also identified yeast enzymes that interact with the introduced pathway, including Can1p, which was identified as the yeast transporter for trimethyllysine, and the two yeast serine hydroxymethyltransferases, Shm1p and Shm2p. Together, this study opens the possibility of using an engineered, carnitine-producing yeast in various industrial applications while providing insight into possible future strategies aimed at tailoring the production capacity of such strains.

  10. RNA-seq analysis for secondary metabolite pathway gene discovery in Polygonum minus

    Directory of Open Access Journals (Sweden)

    Kok-Keong Loke

    2016-03-01

    Full Text Available Polygonum minus plant is rich in secondary metabolites, especially terpenoids and flavonoids. Present study generates transcriptome resource for P. minus to decipher its secondary metabolite biosynthesis pathways. Raw reads and the transcriptome assembly project have been deposited at GenBank under the accessions SRX313492 (root and SRX669305 (leaf respectively.

  11. Ginger and turmeric expressed sequence tags identify signature genes for rhizome identity and development and the biosynthesis of curcuminoids, gingerols and terpenoids

    Science.gov (United States)

    2013-01-01

    Background Ginger (Zingiber officinale) and turmeric (Curcuma longa) accumulate important pharmacologically active metabolites at high levels in their rhizomes. Despite their importance, relatively little is known regarding gene expression in the rhizomes of ginger and turmeric. Results In order to identify rhizome-enriched genes and genes encoding specialized metabolism enzymes and pathway regulators, we evaluated an assembled collection of expressed sequence tags (ESTs) from eight different ginger and turmeric tissues. Comparisons to publicly available sorghum rhizome ESTs revealed a total of 777 gene transcripts expressed in ginger/turmeric and sorghum rhizomes but apparently absent from other tissues. The list of rhizome-specific transcripts was enriched for genes associated with regulation of tissue growth, development, and transcription. In particular, transcripts for ethylene response factors and AUX/IAA proteins appeared to accumulate in patterns mirroring results from previous studies regarding rhizome growth responses to exogenous applications of auxin and ethylene. Thus, these genes may play important roles in defining rhizome growth and development. Additional associations were made for ginger and turmeric rhizome-enriched MADS box transcription factors, their putative rhizome-enriched homologs in sorghum, and rhizomatous QTLs in rice. Additionally, analysis of both primary and specialized metabolism genes indicates that ginger and turmeric rhizomes are primarily devoted to the utilization of leaf supplied sucrose for the production and/or storage of specialized metabolites associated with the phenylpropanoid pathway and putative type III polyketide synthase gene products. This finding reinforces earlier hypotheses predicting roles of this enzyme class in the production of curcuminoids and gingerols. Conclusion A significant set of genes were found to be exclusively or preferentially expressed in the rhizome of ginger and turmeric. Specific

  12. The Expression Analysis of Genes in Fatty Acid Biosynthesis Pathway during the Seed Development of Tung Tree

    Directory of Open Access Journals (Sweden)

    Guixiong Jiang

    2013-07-01

    Full Text Available Tung tree (Vernicia fordii is one of the important oil plants in China. The current researches on tung tree are mainly focused on the cultivation and breeding while the molecular mechanisms hidden in the back of tung tree's phenotypes are still uncovered. This research compared the transcriptome of three different stages during tung tree's seed development using RNA-Seq and obtained a lot of differentially expressed Unigenes. Through GO classification and pathway enrichment analysis, all of these differentially expressed Unigenes were classified into 128 signaling pathways including fatty acid biosynthesis. Fourteen homologous proteins were obtained when the sequences of 54 Unigenes within fatty acid biosynthesis pathway were aligned against KEGG database and the expression profiles of the genes encoding these proteins during seed development were analyzed. This research provides necessary data platform to elucidate the molecular mechanism underlies fatty acid biosynthesis of tung tree's seeds and theoretical guidance of tung tree varieties' improvement to increase output of tung oil.

  13. Transcriptional Regulation and Transport of Terpenoid Indole Alkaloid in Catharanthus roseus: Exploration of New Research Directions.

    Science.gov (United States)

    Liu, Jiaqi; Cai, Junjun; Wang, Rui; Yang, Shihai

    2016-12-28

    As one of the model medicinal plants for exploration of biochemical pathways and molecular biological questions on complex metabolic pathways, Catharanthus roseus synthesizes more than 100 terpenoid indole alkaloids (TIAs) used for clinical treatment of various diseases and for new drug discovery. Given that extensive studies have revealed the major metabolic pathways and the spatial-temporal biosynthesis of TIA in C. roseus plant, little is known about subcellular and inter-cellular trafficking or long-distance transport of TIA end products or intermediates, as well as their regulation. While these transport processes are indispensable for multi-organelle, -tissue and -cell biosynthesis, storage and their functions, great efforts have been made to explore these dynamic cellular processes. Progress has been made in past decades on transcriptional regulation of TIA biosynthesis by transcription factors as either activators or repressors; recent studies also revealed several transporters involved in subcellular and inter-cellular TIA trafficking. However, many details and the regulatory network for controlling the tissue-or cell-specific biosynthesis, transport and storage of serpentine and ajmalicine in root, catharanthine in leaf and root, vindoline specifically in leaf and vinblastine and vincristine only in green leaf and their biosynthetic intermediates remain to be determined. This review is to summarize the progress made in biosynthesis, transcriptional regulation and transport of TIAs. Based on analysis of organelle, tissue and cell-type specific biosynthesis and progresses in transport and trafficking of similar natural products, the transporters that might be involved in transport of TIAs and their synthetic intermediates are discussed; according to transcriptome analysis and bioinformatic approaches, the transcription factors that might be involved in TIA biosynthesis are analyzed. Further discussion is made on a broad context of transcriptional and

  14. Transcriptional Regulation and Transport of Terpenoid Indole Alkaloid in Catharanthus roseus: Exploration of New Research Directions

    Directory of Open Access Journals (Sweden)

    Jiaqi Liu

    2016-12-01

    Full Text Available As one of the model medicinal plants for exploration of biochemical pathways and molecular biological questions on complex metabolic pathways, Catharanthus roseus synthesizes more than 100 terpenoid indole alkaloids (TIAs used for clinical treatment of various diseases and for new drug discovery. Given that extensive studies have revealed the major metabolic pathways and the spatial-temporal biosynthesis of TIA in C. roseus plant, little is known about subcellular and inter-cellular trafficking or long-distance transport of TIA end products or intermediates, as well as their regulation. While these transport processes are indispensable for multi-organelle, -tissue and -cell biosynthesis, storage and their functions, great efforts have been made to explore these dynamic cellular processes. Progress has been made in past decades on transcriptional regulation of TIA biosynthesis by transcription factors as either activators or repressors; recent studies also revealed several transporters involved in subcellular and inter-cellular TIA trafficking. However, many details and the regulatory network for controlling the tissue-or cell-specific biosynthesis, transport and storage of serpentine and ajmalicine in root, catharanthine in leaf and root, vindoline specifically in leaf and vinblastine and vincristine only in green leaf and their biosynthetic intermediates remain to be determined. This review is to summarize the progress made in biosynthesis, transcriptional regulation and transport of TIAs. Based on analysis of organelle, tissue and cell-type specific biosynthesis and progresses in transport and trafficking of similar natural products, the transporters that might be involved in transport of TIAs and their synthetic intermediates are discussed; according to transcriptome analysis and bioinformatic approaches, the transcription factors that might be involved in TIA biosynthesis are analyzed. Further discussion is made on a broad context of

  15. Enhanced levels of S-linalool by metabolic engineering of the terpenoid pathway in spike lavender leaves.

    Science.gov (United States)

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

    2014-05-01

    Transgenic Lavandula latifolia plants overexpressing the linalool synthase (LIS) gene from Clarkia breweri, encoding the LIS enzyme that catalyzes the synthesis of linalool were generated. Most of these plants increased significantly their linalool content as compared to controls, especially in the youngest leaves, where a linalool increase up to a 1000% was observed. The phenotype of increased linalool content observed in young leaves was maintained in those T1 progenies that inherit the LIS transgene, although this phenotype was less evident in the flower essential oil. Cross-pollination of transgenic spike lavender plants allowed the generation of double transgenic plants containing the DXS (1-deoxy-d-xylulose-5-P synthase), coding for the first enzyme of the methyl-d-erythritol-4-phosphate pathway, and LIS genes. Both essential oil yield and linalool content in double DXS-LIS transgenic plants were lower than that of their parentals, which could be due to co-suppression effects linked to the structures of the constructs used. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  16. Identification of Candidate Genes and Biosynthesis Pathways Related to Fertility Conversion by Wheat KTM3315A Transcriptome Profiling

    Directory of Open Access Journals (Sweden)

    Lingli Zhang

    2017-04-01

    Full Text Available The Aegilops kotschyi thermo-sensitive cytoplasmic male sterility (K-TCMS system may facilitate hybrid wheat (Triticum aestivum L. seed multiplication and production. The K-TCMS line is completely male sterile during the normal wheat-growing season, whereas its fertility can be restored in a high-temperature environment. To elucidate the molecular mechanisms responsible for male sterility/fertility conversion and candidate genes involved with pollen development in K-TCMS, we employed RNA-seq to sequence the transcriptomes of anthers from K-TCMS line KTM3315A during development under sterile and fertile conditions. We identified 16840 differentially expressed genes (DEGs in different stages including15157 known genes (15135 nuclear genes and 22 plasmagenes and 1683 novel genes. Bioinformatics analysis identified possible metabolic pathways involved with fertility based on KEGG pathway enrichment of the DEGs expressed in fertile and sterile plants. We found that most of the genes encoding key enzyme in the phenylpropanoid biosynthesis and jasmonate biosynthesis pathways were significant upregulated in uninucleate, binuclate or trinucleate stage, which both interact with MYB transcription factors, and that link between all play essential roles in fertility conversion. The relevant DEGs were verified by quantitative RT-PCR. Thus, we suggested that phenylpropanoid biosynthesis and jasmonate biosynthesis pathways were involved in fertility conversion of K-TCMS wheat. This will provide a new perspective and an effective foundation for the research of molecular mechanisms of fertility conversion of CMS wheat. Fertility conversion mechanism in thermo-sensitive cytoplasmic male sterile/fertile wheat involves the phenylpropanoid biosynthesis pathway, jasmonate biosynthesis pathway, and MYB transcription factors.

  17. A terpenoid phytoalexin plays a role in basal defense of Nicotiana benthamiana against Potato virus X.

    Science.gov (United States)

    Li, Ran; Tee, Chuan-Sia; Jiang, Yu-Lin; Jiang, Xi-Yuan; Venkatesh, Prasanna Nori; Sarojam, Rajani; Ye, Jian

    2015-05-20

    Terpenoid phytoalexins function as defense compound against a broad spectrum of pathogens and pests in the plant kingdom. However, the role of phytoalexin in antiviral defense is still elusive. In this study, we identified the biosynthesis pathway of a sesquiterpenoid phytoalexin, capsidiol 3-acetate as an antiviral response against RNA virus Potato Virus X (PVX) in Nicotiana benthamiana. NbTPS1 and NbEAH genes were found strongly induced by PVX-infection. Enzymatic activity and genetic evidence indicated that both genes were involved in the PVX-induced biosynthesis of capsidiol 3-acetate. NbTPS1- or NbEAH-silenced plant was more susceptible to PVX. The accumulation of capsidiol 3-acetate in PVX-infected plant was partially regulated by jasmonic acid signaling receptor COI1. These findings provide an insight into a novel mechanism of how plant uses the basal arsenal machinery to mount a fight against virus attack even in susceptible species.

  18. Norovirus contamination and the glycosphingolipid biosynthesis pathway in Pacific oyster: A transcriptomics study.

    Science.gov (United States)

    Ma, Liping; Su, Laijin; Liu, Hui; Zhao, Feng; Zhou, Deqing; Duan, Delin

    2017-07-01

    Noroviruses are the primary pathogens associated with shellfish-borne gastroenteritis outbreaks. These viruses remain stable in oysters, suggesting an active mechanism for virus concentration. In this study, a deep RNA sequencing technique was used to analyze the transcriptome profiles of Pacific oysters at different time points after inoculation with norovirus (GII.4). We obtained a maximum of 65, 294, 698 clean sample reads. When aligned to the reference genome, the average mapping ratio of clean data was approximately 65%. In the samples harvested at 12, 24, and 48 h after contamination, 2,223, 2,990, and 2020 genes, respectively, were differentially expressed in contaminated and non-contaminated oyster digestive tissues, including 500, 1748, and 1039 up-regulated and 1723, 1242, and 981 down-regulated genes, respectively. In particular, FUT2 and B3GNT4, genes encoding the signaling components of glycosphingolipid biosynthesis, were significantly up-regulated in contaminated samples. In addition, we found up-regulation of some immune- and disease-related genes in the MHC I pathway (PA28, HSP 70, HSP90, CANX, BRp57, and CALR) and MHC II pathway (GILT, CTSBLS, RFX, and NFY), although NoVs did not cause diseases in the oysters. We detected two types of HBGA-like molecules with positive-to-negative ratios similar to type A and H1 HBGA-like molecules in digestive tissues that were significantly higher in norovirus-contaminated than in non-contaminated oysters. Thus, our transcriptome data analysis indicated that a human pathogen (GII.4 Norovirus) was likely concentrated in the digestive tissues of oysters via HBGA-like molecules that were synthesized by the glycosphingolipid biosynthesis pathway. The identified differentially expressed genes also provide potential candidates for functional analysis to identify genes involved in the accumulation of noroviruses in oysters. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Gene transcript profiles of the TIA biosynthetic pathway in response to ethylene and copper reveal their interactive role in modulating TIA biosynthesis in Catharanthus roseus.

    Science.gov (United States)

    Pan, Ya-Jie; Liu, Jia; Guo, Xiao-Rui; Zu, Yuan-Gang; Tang, Zhong-Hua

    2015-05-01

    Research on transcriptional regulation of terpenoid indole alkaloid (TIA) biosynthesis of the medicinal plant, Catharanthus roseus, has largely been focused on gene function and not clustering analysis of multiple genes at the transcript level. Here, more than ten key genes encoding key enzyme of alkaloid synthesis in TIA biosynthetic pathways were chosen to investigate the integrative responses to exogenous elicitor ethylene and copper (Cu) at both transcriptional and metabolic levels. The ethylene-induced gene transcripts in leaves and roots, respectively, were subjected to principal component analysis (PCA) and the results showed the overall expression of TIA pathway genes indicated as the Q value followed a standard normal distribution after ethylene treatments. Peak gene expression was at 15-30 μM of ethephon, and the pre-mature leaf had a higher Q value than the immature or mature leaf and root. Treatment with elicitor Cu found that Cu up-regulated overall TIA gene expression more in roots than in leaves. The combined effects of Cu and ethephon on TIA gene expression were stronger than their separate effects. It has been documented that TIA gene expression is tightly regulated by the transcriptional factor (TF) ethylene responsive factor (ERF) and mitogen-activated protein kinase (MAPK) cascade. The loading plot combination with correlation analysis for the genes of C. roseus showed that expression of the MPK gene correlated with strictosidine synthase (STR) and strictosidine b-D-glucosidase(SGD). In addition, ERF expression correlated with expression of secologanin synthase (SLS) and tryptophan decarboxylase (TDC), specifically in roots, whereas MPK and myelocytomatosis oncogene (MYC) correlated with STR and SGD genes. In conclusion, the ERF regulates the upstream pathway genes in response to heavy metal Cu mainly in C. roseus roots, while the MPK mainly participates in regulating the STR gene in response to ethylene in pre-mature leaf. Interestingly, the

  20. Largely different contents of terpenoids in beef red-flesh tangerine and its wild type.

    Science.gov (United States)

    Li, Wenyun; Liu, Cuihua; He, Min; Li, Jinqiang; Cai, Yongqiang; Ma, Yuhua; Xu, Juan

    2017-02-03

    Niurouhong (Citrus reticulata Blanco. Niurouhong) (NRH) is a spontaneous beef-red flesh mutant with distinctive flavor compared with its wild type orange-red flesh Zhuhongju (ZHJ). To illustrate the biochemical mechanism of its special flesh color and flavor, fruits at commercial mature stage were used to profile the volatiles in the flavedo and determine the levels of carotenoids, limonoid aglycones and phytohormones in the juice sacs in two seasons. Our results showed the content of total volatile terpenoids in NRH was 1.27-fold that in ZHJ. The components of volatiles were found to be common between the two tangerines. This result indicates that the distinctive flavor of NRH might not be derived from the presence/absence of specific volatiles; instead, it was derived from the altered concentrations or balance of α-citral, β-citral, 2-cyclohexen-1-one, (S)-3-methyl-6-(1-methylethenyl) and n-hexadecanoic acid. Analyses of the contents of total and specific carotenoids indicated that the beef-red color of NRH flesh might be largely attributed to the over accumulation of β-cryptoxanthin and β-carotene. However, lower ABA level was found in NRH than in ZHJ, reflecting a possible feedback regulation of ABA biosynthesis on carotenogenesis and the balance in the metabolism among terpenoids. Collectively, our study suggested that the MEP pathway was enhanced in NRH tangerine. However, a certain unknown co-regulatory mechanism might be present in the metabolism pathway of secondary metabolites (especially terpenoids) in beef-red flesh mutant. Our study provides new insights into the regulatory network of terpenoid metabolism and mutation mechanism of red-fleshed citrus.

  1. Terpenoids for medicine

    NARCIS (Netherlands)

    Fischedick, Justin

    2013-01-01

    This thesis is concerns research on monoterpenoids, sesquiterpenoids, and diterpenoids with medicinal properties. Terpenoids from commond herbs as well as Cannabis sativa, Inula britannica, Tanacetum parthenium, and Salvia officinalis were investigated

  2. Evaluation of tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) hairy roots for the production of geraniol, the first committed step in terpenoid indole alkaloid pathway

    NARCIS (Netherlands)

    Ritala, A.; Dong, L.; Imseng, N.; Seppanen-Laakso, T.; Vasilev, N.; Krol, van der A.R.; Rischer, H.; Maaheimo, H.; Virkki, A.; Brandli, J.; Schillberg, S.; Eibl, R.; Bouwmeester, H.J.; Oksman-Caldentey, K.M.

    2014-01-01

    The terpenoid indole alkaloids are one of the major classes of plant-derived natural products and are well known for their many applications in the pharmaceutical, fragrance and cosmetics industries. Hairy root cultures are useful for the production of plant secondary metabolites because of their

  3. Host and Pathway Engineering for Enhanced Lycopene Biosynthesis in Yarrowia lipolytica

    Directory of Open Access Journals (Sweden)

    Cory Schwartz

    2017-11-01

    Full Text Available Carotenoids are a class of molecules with commercial value as food and feed additives with nutraceutical properties. Shifting carotenoid synthesis from petrochemical-based precursors to bioproduction from sugars and other biorenewable carbon sources promises to improve process sustainability and economics. In this work, we engineered the oleaginous yeast Yarrowia lipolytica to produce the carotenoid lycopene. To enhance lycopene production, we tested a series of strategies to modify host cell physiology and metabolism, the most successful of which were mevalonate pathway overexpression and alleviating auxotrophies previously engineered into the PO1f strain of Y. lipolytica. The beneficial engineering strategies were combined into a single strain, which was then cultured in a 1-L bioreactor to produce 21.1 mg/g DCW. The optimized strain overexpressed a total of eight genes including two copies of HMG1, two copies of CrtI, and single copies of MVD1, EGR8, CrtB, and CrtE. Recovering leucine and uracil biosynthetic capacity also produced significant enhancement in lycopene titer. The successful engineering strategies characterized in this work represent a significant increase in understanding carotenoid biosynthesis in Y. lipolytica, not only increasing lycopene titer but also informing future studies on carotenoid biosynthesis.

  4. Two alternative pathways for docosahexaenoic acid (DHA, 22:6n-3) biosynthesis are widespread among teleost fish.

    Science.gov (United States)

    Oboh, Angela; Kabeya, Naoki; Carmona-Antoñanzas, Greta; Castro, L Filipe C; Dick, James R; Tocher, Douglas R; Monroig, Oscar

    2017-06-20

    Docosahexaenoic acid (DHA) plays important physiological roles in vertebrates. Studies in rats and rainbow trout confirmed that DHA biosynthesis proceeds through the so-called "Sprecher pathway", a biosynthetic process requiring a Δ6 desaturation of 24:5n-3 to 24:6n-3. Alternatively, some teleosts possess fatty acyl desaturases 2 (Fads2) that enable them to biosynthesis DHA through a more direct route termed the "Δ4 pathway". In order to elucidate the prevalence of both pathways among teleosts, we investigated the Δ6 ability towards C24 substrates of Fads2 from fish with different evolutionary and ecological backgrounds. Subsequently, we retrieved public databases to identify Fads2 containing the YXXN domain responsible for the Δ4 desaturase function, and consequently enabling these species to operate the Δ4 pathway. We demonstrated that, with the exception of Δ4 desaturases, fish Fads2 have the ability to operate as Δ6 desaturases towards C24 PUFA enabling them to synthesise DHA through the Sprecher pathway. Nevertheless, the Δ4 pathway represents an alternative route in some teleosts and we identified the presence of putative Δ4 Fads2 in a further 11 species and confirmed the function as Δ4 desaturases of Fads2 from medaka and Nile tilapia. Our results demonstrated that two alternative pathways for DHA biosynthesis exist in teleosts.

  5. Ecdysteroid biosynthesis in varroa mites: identification of halloween genes from the biosynthetic pathway and their regulation during reproduction

    Science.gov (United States)

    Biosynthesis of ecdysteroids involves sequential enzymatic hydroxylations by microsomal enzymes and mitochondrial cytochrome P450’s. Enzymes of the pathway are collectively known as Halloween genes. Complete sequences for three Halloween genes, spook (Vdspo), disembodied (Vddib) and shade (Vdshd), w...

  6. Genetic Profiling of the Isoprenoid and Sterol Biosynthesis Pathway Genes of Trypanosoma cruzi

    Science.gov (United States)

    Cosentino, Raúl O.; Agüero, Fernán

    2014-01-01

    In Trypanosoma cruzi the isoprenoid and sterol biosynthesis pathways are validated targets for chemotherapeutic intervention. In this work we present a study of the genetic diversity observed in genes from these pathways. Using a number of bioinformatic strategies, we first identified genes that were missing and/or were truncated in the T. cruzi genome. Based on this analysis we obtained the complete sequence of the ortholog of the yeast ERG26 gene and identified a non-orthologous homolog of the yeast ERG25 gene (sterol methyl oxidase, SMO), and we propose that the orthologs of ERG25 have been lost in trypanosomes (but not in Leishmanias). Next, starting from a set of 16 T. cruzi strains representative of all extant evolutionary lineages, we amplified and sequenced ∼24 Kbp from 22 genes, identifying a total of 975 SNPs or fixed differences, of which 28% represent non-synonymous changes. We observed genes with a density of substitutions ranging from those close to the average (∼2.5/100 bp) to some showing a high number of changes (11.4/100 bp, for the putative lathosterol oxidase gene). All the genes of the pathway are under apparent purifying selection, but genes coding for the sterol C14-demethylase, the HMG-CoA synthase, and the HMG-CoA reductase have the lowest density of missense SNPs in the panel. Other genes (TcPMK, TcSMO-like) have a relatively high density of non-synonymous SNPs (2.5 and 1.9 every 100 bp, respectively). However, none of the non-synonymous changes identified affect a catalytic or ligand binding site residue. A comparative analysis of the corresponding genes from African trypanosomes and Leishmania shows similar levels of apparent selection for each gene. This information will be essential for future drug development studies focused on this pathway. PMID:24828104

  7. Arbuscular mycorrhizal fungi: effects on plant terpenoid accumulation.

    Science.gov (United States)

    Welling, M T; Liu, L; Rose, T J; Waters, D L E; Benkendorff, K

    2016-07-01

    Arbuscular mycorrhizal fungi (AMF) are a diverse group of soil-dwelling fungi that form symbiotic associations with land plants. AMF-plant associations promote the accumulation of plant terpenoids beneficial to human health, although how AMF mediate terpenoid accumulation is not fully understood. A critical assessment and discussion of the literature relating to mechanisms by which AMF influence plant terpenoid accumulation, and whether this symbiosis can be harnessed in horticultural ecosystems was performed. Modification of plant morphology, phosphorus availability and gene transcription involved with terpenoid biosynthetic pathways were identified as key mechanisms associated with terpenoid accumulation in AMF-colonised plants. In order to exploit AMF-plant symbioses in horticultural ecosystems it is important to consider the specificity of the AMF-plant association, the predominant factor affecting terpenoid accumulation, as well as the end use application of the harvested plant material. Future research should focus on resolving the relationship between ecologically matched AMF genotypes and terpenoid accumulation in plants to establish if these associations are effective in promoting mechanisms favourable for plant terpenoid accumulation. © 2015 German Botanical Society and The Royal Botanical Society of the Netherlands.

  8. Multi-domain terpenoid cyclase architecture and prospects for proximity in bifunctional catalysis.

    Science.gov (United States)

    Chen, Mengbin; Harris, Golda G; Pemberton, Travis A; Christianson, David W

    2016-12-01

    Crystal structures of terpenoid cyclases reveal assemblies of three basic domains designated α, β, and γ. While the biosynthesis of cyclic monoterpenes (C 10 ) and sesquiterpenes (C 15 ) most often involves enzymes with α or αβ domain architecture, the biosynthesis of cyclic diterpenes (C 20 ), sesterterpenes (C 25 ), and triterpenes (C 30 ) can involve enzymes with α, αα, βγ, or αβγ domain architecture. Indeed, some enzymes of terpenoid biosynthesis are bifunctional, with distinct active sites that catalyze sequential reactions. Interestingly, some of these enzymes oligomerize to form dimers, tetramers, and hexamers. Not only can such assemblies enable enzyme regulation by allostery, but they can also provide a modest enhancement of terpenoid product flux through proximity channeling or cluster channeling. The mixing and matching of functional terpenoid cyclase domains through tertiary and/or quaternary structure may also comprise an evolutionary strategy for facile product diversification. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Genetic analysis of pathway regulation for enhancing branched-chain amino acid biosynthesis in plants

    KAUST Repository

    Chen, Hao

    2010-08-01

    The branched-chain amino acids (BCAAs) valine, leucine and isoleucine are essential amino acids that play critical roles in animal growth and development. Animals cannot synthesize these amino acids and must obtain them from their diet. Plants are the ultimate source of these essential nutrients, and they synthesize BCAAs through a conserved pathway that is inhibited by its end products. This feedback inhibition has prevented scientists from engineering plants that accumulate high levels of BCAAs by simply over-expressing the respective biosynthetic genes. To identify components critical for this feedback regulation, we performed a genetic screen for Arabidopsis mutants that exhibit enhanced resistance to BCAAs. Multiple dominant allelic mutations in the VALINE-TOLERANT 1 (VAT1) gene were identified that conferred plant resistance to valine inhibition. Map-based cloning revealed that VAT1 encodes a regulatory subunit of acetohydroxy acid synthase (AHAS), the first committed enzyme in the BCAA biosynthesis pathway. The VAT1 gene is highly expressed in young, rapidly growing tissues. When reconstituted with the catalytic subunit in vitro, the vat1 mutant-containing AHAS holoenzyme exhibits increased resistance to valine. Importantly, transgenic plants expressing the mutated vat1 gene exhibit valine tolerance and accumulate higher levels of BCAAs. Our studies not only uncovered regulatory characteristics of plant AHAS, but also identified a method to enhance BCAA accumulation in crop plants that will significantly enhance the nutritional value of food and feed. © 2010 Blackwell Publishing Ltd.

  10. Structure, biosynthesis, and function of bacterial capsular polysaccharides synthesized by ABC transporter-dependent pathways.

    Science.gov (United States)

    Willis, Lisa M; Whitfield, Chris

    2013-08-30

    Bacterial capsules are formed primarily from long-chain polysaccharides with repeat-unit structures. A given bacterial species can produce a range of capsular polysaccharides (CPSs) with different structures and these help distinguish isolates by serotyping, as is the case with Escherichia coli K antigens. Capsules are important virulence factors for many pathogens and this review focuses on CPSs synthesized via ATP-binding cassette (ABC) transporter-dependent processes in Gram-negative bacteria. Bacteria utilizing this pathway are often associated with urinary tract infections, septicemia, and meningitis, and E. coli and Neisseria meningitidis provide well-studied examples. CPSs from ABC transporter-dependent pathways are synthesized at the cytoplasmic face of the inner membrane through the concerted action of glycosyltransferases before being exported across the inner membrane and translocated to the cell surface. A hallmark of these CPSs is a conserved reducing terminal glycolipid composed of phosphatidylglycerol and a poly-3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) linker. Recent discovery of the structure of this conserved lipid terminus provides new insights into the early steps in CPS biosynthesis. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. Biosynthesis of vitamin B12. Different pathways in some aerobic and anaerobic microorganisms.

    Science.gov (United States)

    Höllriegl, V; Lamm, L; Rowold, J; Hörig, J; Renz, P

    1982-08-01

    Radioactivity from [1'-14C]riboflavin was incorporated into the 5,6-dimethylbenzimidazole moiety of Vitamin B12 in the aerobes Bacillus megaterium, Nocardia rugosa and Streptomyces sp. as well as in the aerotolerant anaerobe Propionibacterium freudenreichii, but not in the anaerobe Eubacterium limosum. As recently published for E. limosum, also in the anaerobe Clostridium barkeri radioactivity from [1-14C]glycine and [2-14C]glycine was found in the 5,6-dimethylbenzimidazole moiety, but not in the corrin moiety. The addition of L-[methyl-14C]methionine to C. barkeri led to the labeling of the corrin moiety and the 5,6-dimethylbenzimidazole moiety, showing that the seven "extra" methyl groups in the corrin ring as well as the two methyl groups of the base part originate from this precursor. In Clostridium thermoaceticum, forming the vitamin B12 analog 5-methoxybenzimidazolylcobamide, [1-14C]glycine and [2-14C]glycine were also incorporated into the 5-methoxybenzimidazole moiety, but not into the corrin ring. In E. limosum L-[U-14C]glutamate led to the labeling of the corrin ring of vitamin B12, but not of its base moiety. These results together with data from the literature indicate that a common biosynthetic pathway might exist for the corrinoid biosynthesis in aerobic microorganisms, and in those aerotolerant anaerobes like the Propionibacteria, which form the 5,6-dimethylbenzimidazole moiety of vitamin B12 only under aerobic conditions. They also show that this pathway differs from the pathway found in anaerobic bacteria.

  12. Biosynthesis Pathway of ADP-l-glycero-β-d-manno-Heptose in Escherichia coli

    Science.gov (United States)

    Kneidinger, Bernd; Marolda, Cristina; Graninger, Michael; Zamyatina, Alla; McArthur, Fiona; Kosma, Paul; Valvano, Miguel A.; Messner, Paul

    2002-01-01

    The steps involved in the biosynthesis of the ADP-l-glycero-β-d-manno-heptose (ADP-l-β-d-heptose) precursor of the inner core lipopolysaccharide (LPS) have not been completely elucidated. In this work, we have purified the enzymes involved in catalyzing the intermediate steps leading to the synthesis of ADP-d-β-d-heptose and have biochemically characterized the reaction products by high-performance anion-exchange chromatography. We have also constructed a deletion in a novel gene, gmhB (formerly yaeD), which results in the formation of an altered LPS core. This mutation confirms that the GmhB protein is required for the formation of ADP-d-β-d-heptose. Our results demonstrate that the synthesis of ADP-d-β-d-heptose in Escherichia coli requires three proteins, GmhA (sedoheptulose 7-phosphate isomerase), HldE (bifunctional d-β-d-heptose 7-phosphate kinase/d-β-d-heptose 1-phosphate adenylyltransferase), and GmhB (d,d-heptose 1,7-bisphosphate phosphatase), as well as ATP and the ketose phosphate precursor sedoheptulose 7-phosphate. A previously characterized epimerase, formerly named WaaD (RfaD) and now renamed HldD, completes the pathway to form the ADP-l-β-d-heptose precursor utilized in the assembly of inner core LPS. PMID:11751812

  13. Biosynthesis of hemiketal eicosanoids by cross-over of the 5-lipoxygenase and cyclooxygenase-2 pathways

    Science.gov (United States)

    Griesser, Markus; Suzuki, Takashi; Tejera, Noemi; Mont, Stacey; Boeglin, William E.; Pozzi, Ambra; Schneider, Claus

    2011-01-01

    The prostaglandin and leukotriene families of lipid mediators are formed via two distinct biosynthetic pathways that are initiated by the oxygenation of arachidonic acid by either cyclooxygenase-2 (COX-2) or 5-lipoxygenase (5-LOX), respectively. The 5-LOX product 5S-hydroxyeicosatetraenoic acid, however, can also serve as an efficient substrate for COX-2, forming a bicyclic diendoperoxide with structural similarities to the arachidonic acid-derived prostaglandin endoperoxide PGH2 [Schneider C, et al. (2006) J Am Chem Soc 128:720–721]. Here we identify two cyclic hemiketal (HK) eicosanoids, HKD2 and HKE2, as the major nonenzymatic rearrangement products of the diendoperoxide using liquid chromatography–mass spectrometry analyses as well as UV and NMR spectroscopy. HKD2 and HKE2 are furoketals formed by spontaneous cyclization of their respective 8,9-dioxo-5S,11R,12S,15S-tetrahydroxy- or 11,12-dioxo-5S,8S,9S,15S-tetrahydroxy-eicosadi-6E,13E-enoic acid precursors, resulting from opening of the 9S,11R- and 8S,12S-peroxide rings of the diendoperoxide. Furthermore, the diendoperoxide is an efficient substrate for the hematopoietic type of prostaglandin D synthase resulting in formation of HKD2, equivalent to the enzymatic transformation of PGH2 to PGD2. HKD2 and HKE2 were formed in human blood leukocytes activated with bacterial lipopolysaccharide and calcium ionophore A23187, and biosynthesis was blocked by inhibitors of 5-LOX or COX-2. HKD2 and HKE2 stimulated migration and tubulogenesis of microvascular endothelial cells, implicating a proangiogenic role of the hemiketals in inflammatory sites that involve expression of 5-LOX and COX-2. Identification of the highly oxygenated hemiketal eicosanoids provides evidence for a previously unrecognized biosynthetic cross-over of the 5-LOX and COX-2 pathways. PMID:21482803

  14. Plant Metabolic Engineering Strategies for the Production of Pharmaceutical Terpenoids

    OpenAIRE

    Lu, Xu; Tang, Kexuan; Li, Ping

    2016-01-01

    Pharmaceutical terpenoids belong to the most diverse class of natural products. They have significant curative effects on a variety of diseases, such as cancer, cardiovascular diseases, malaria and Alzheimer’s disease. Nowadays, elicitors, including biotic and abiotic elicitors, are often used to activate the pathway of secondary metabolism and enhance the production of target terpenoids. Based on Agrobacterium-mediated genetic transformation, several plant metabolic engineering strategies ho...

  15. Mechanistic Insights on the Reductive Dehydroxylation Pathway for the Biosynthesis of Isoprenoids Promoted by the IspH Enzyme

    KAUST Repository

    Abdel-Azeim, Safwat

    2015-06-22

    Here, we report an integrated quantum mechanics/molecular mechanics (QM/MM) study of the bio-organometallic reaction pathway of the 2H+/2e- reduction of (E)-4-hydroxy-3-methylbut-2-enyl pyrophosphate (HMBPP) into the so called universal terpenoids precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), promoted by the IspH enzyme. Our results support the viability of the bio-organometallic pathway from rotation of the OH group of HMBPP away from the [Fe4S4] cluster at the core of the catalytic site, to be engaged in a H-bond with Glu126. This rotation is synchronous with π-coordination of the C2=C3 double bond of HMBPP to the apical Fe atom of the [Fe4S4] cluster. Dehydroxylation of HMBPP is triggered by a proton transfer from Glu126 to the OH group of HMBPP. The reaction pathway is completed by competitive proton transfer from the terminal phosphate group to the C2 or C4 atom of HMBPP.

  16. Metabolic engineering of Bacillus subtilis for terpenoid production.

    Science.gov (United States)

    Guan, Zheng; Xue, Dan; Abdallah, Ingy I; Dijkshoorn, Linda; Setroikromo, Rita; Lv, Guiyuan; Quax, Wim J

    2015-11-01

    Terpenoids are the largest group of small-molecule natural products, with more than 60,000 compounds made from isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). As the most diverse group of small-molecule natural products, terpenoids play an important role in the pharmaceutical, food, and cosmetic industries. For decades, Escherichia coli (E. coli) and Saccharomyces cerevisiae (S. cerevisiae) were extensively studied to biosynthesize terpenoids, because they are both fully amenable to genetic modifications and have vast molecular resources. On the other hand, our literature survey (20 years) revealed that terpenoids are naturally more widespread in Bacillales. In the mid-1990s, an inherent methylerythritol phosphate (MEP) pathway was discovered in Bacillus subtilis (B. subtilis). Since B. subtilis is a generally recognized as safe (GRAS) organism and has long been used for the industrial production of proteins, attempts to biosynthesize terpenoids in this bacterium have aroused much interest in the scientific community. This review discusses metabolic engineering of B. subtilis for terpenoid production, and encountered challenges will be discussed. We will summarize some major advances and outline future directions for exploiting the potential of B. subtilis as a desired "cell factory" to produce terpenoids.

  17. Evidence for a Saponin Biosynthesis Pathway in the Body Wall of the Commercially Significant Sea Cucumber Holothuria scabra

    Directory of Open Access Journals (Sweden)

    Shahida Akter Mitu

    2017-11-01

    Full Text Available The sea cucumber (phylum Echinodermata body wall is the first line of defense and is well known for its production of secondary metabolites; including vitamins and triterpenoid glycoside saponins that have important ecological functions and potential benefits to human health. The genes involved in the various biosynthetic pathways are unknown. To gain insight into these pathways in an echinoderm, we performed a comparative transcriptome analysis and functional annotation of the body wall and the radial nerve of the sea cucumber Holothuria scabra; to define genes associated with body wall metabolic functioning and secondary metabolite biosynthesis. We show that genes related to signal transduction mechanisms were more highly represented in the H. scabra body wall, including genes encoding enzymes involved in energy production. Eight of the core triterpenoid biosynthesis enzymes were found, however, the identity of the saponin specific biosynthetic pathway enzymes remains unknown. We confirm the body wall release of at least three different triterpenoid saponins using solid phase extraction followed by ultra-high-pressure liquid chromatography-quadrupole time of flight-mass spectrometry. The resource we have established will help to guide future research to explore secondary metabolite biosynthesis in the sea cucumber.

  18. Cloning and expression analysis of terpenoid metabolism-related gene NsyCMS in Nicotiana sylvestris.

    Science.gov (United States)

    Lv, J; Liu, G S; Li, W; Hussian, Z; Sun, Y H

    2015-04-13

    Terpenoids constitute the main class of secondary metabolites produced in plants with industrial, pharmacological, and agricultural interests. Nicotiana sylvestris has been widely adopted as a diploid model system in plant biology for studies of terpenoid biosynthesis. In this paper, we report the isolation and analysis of the 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (CMS) gene of the MEP (methylerythritol 4-phosphate) pathway from N. sylvestris. We used homologous-based cloning with a RACE method to obtain the full-length coding sequence of the NsCMS. Then, the physical and chemical properties, function, and three-dimensional structure of the NsyCMS protein were predicted. Fluorogenic quantitative PCR was used to conduct an expression analysis at different developmental stages of various tissues of the NsyCMS. The sequence of the NsyCMS consists of a 954-bp open reading frame and encodes a predicted protein of 317 amino acids, with a molecular weight of approximately 49.6 kDa and pi of 6.92. The in vivo localization of the encoded protein was cytoplasmic with no signal peptide, whereas 2 transmembrane regions were found in NsyCMS. The conserved domains of typical 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase, aminotransferase, and pyridoxal phosphate-dependent transferase were found in NsyCMS. Differential expression patterns of the NsyCMS were observed throughout the different developmental stages and tissues. NsyCMS messenger RNA was expressed in all tissues, with the highest level of expression in the seedling leaves. NsyMK was expressed at a higher level in the resettling roots. The results from our study set the foundation for exploring the terpenoid biosynthetic pathways in N. sylvestris.

  19. Altered activity of heme biosynthesis pathway enzymes in individuals chronically exposed to arsenic in Mexico

    Energy Technology Data Exchange (ETDEWEB)

    Hernandez-Zavala, A.; Del Razo, L.M.; Garcia-Vargas, G.G.; Aguilar, C.; Borja, V.H.; Albores, A.; Cebrian, M.E. [CINVESTAV-IPN, Mexico (Mexico). Dept. de Farmacologia y Toxicologica

    1999-03-01

    Our objective was to evaluate the activities of some enzymes of the heme biosynthesis pathway and their relationship with the profile of urinary porphyrin excretion in individuals exposed chronically to arsenic (As) via drinking water in Region Lagunera, Mexico. We selected 17 individuals from each village studied: Benito Juarez, which has current exposure to 0.3 mg As/l; Santa Ana, where individuals have been exposed for more than 35 years to 0.4 mg As/l, but due to changes in the water supply (in 1992) exposure was reduced to its current level (0.1 mg As/l), and Nazareno, with 0.014 mg As/l. Average arsenic concentrations in urine were 2058, 398, and 88 {mu}g As/g creatinine, respectively. The more evident alterations in heme metabolism observed in the highly exposed individuals were: (1) small but significant increases in porphobilinogen deaminase (PBG-D) and uroporphyrinogen decarboxylase (URO-D) activities in peripheral blood erythrocytes; (2) increases in the urinary excretion of total porphyrins, mainly due to coproporphyrin III (COPROIII) and uroporphyrin III (UROIII); and (3) increases in the COPRO/URO and COPROIII/COPROI ratios. No significant changes were observed in uroporphyrinogen III synthetase (UROIII-S) activity. The direct relationships between enzyme activities and urinary porphyrins, suggest that the increased porphyrin excretion was related to PBG-D, whereas the increased URO-D activity would enhance coproporphyrin synthesis and excretion at the expense of uroporphyrin. None of the human studies available have reported the marked porphyric response and enzyme inhibition observed in rodents. In conclusion, chronic As exposure alters human heme metabolism; however the severity of the effects appears to depend on characteristics of exposure not yet fully characterized. (orig.) With 1 fig., 3 tabs., 20 refs.

  20. Horizontal acquisition of a hypoxia-responsive molybdenum cofactor biosynthesis pathway contributed to Mycobacterium tuberculosis pathoadaptation.

    Science.gov (United States)

    Levillain, Florence; Poquet, Yannick; Mallet, Ludovic; Mazères, Serge; Marceau, Michael; Brosch, Roland; Bange, Franz-Christoph; Supply, Philip; Magalon, Axel; Neyrolles, Olivier

    2017-11-01

    The unique ability of the tuberculosis (TB) bacillus, Mycobacterium tuberculosis, to persist for long periods of time in lung hypoxic lesions chiefly contributes to the global burden of latent TB. We and others previously reported that the M. tuberculosis ancestor underwent massive episodes of horizontal gene transfer (HGT), mostly from environmental species. Here, we sought to explore whether such ancient HGT played a part in M. tuberculosis evolution towards pathogenicity. We were interested by a HGT-acquired M. tuberculosis-specific gene set, namely moaA1-D1, which is involved in the biosynthesis of the molybdenum cofactor. Horizontal acquisition of this gene set was striking because homologues of these moa genes are present all across the Mycobacterium genus, including in M. tuberculosis. Here, we discovered that, unlike their paralogues, the moaA1-D1 genes are strongly induced under hypoxia. In vitro, a M. tuberculosis moaA1-D1-null mutant has an impaired ability to respire nitrate, to enter dormancy and to survive in oxygen-limiting conditions. Conversely, heterologous expression of moaA1-D1 in the phylogenetically closest non-TB mycobacterium, Mycobacterium kansasii, which lacks these genes, improves its capacity to respire nitrate and grants it with a marked ability to survive oxygen depletion. In vivo, the M. tuberculosis moaA1-D1-null mutant shows impaired survival in hypoxic granulomas in C3HeB/FeJ mice, but not in normoxic lesions in C57BL/6 animals. Collectively, our results identify a novel pathway required for M. tuberculosis resistance to host-imposed stress, namely hypoxia, and provide evidence that ancient HGT bolstered M. tuberculosis evolution from an environmental species towards a pervasive human-adapted pathogen.

  1. Developmental changes in skin collagen biosynthesis pathway in posthatch male and female chickens

    Science.gov (United States)

    Pines, M.; Schickler, M.; Hurwitz, S.; Yamauchi, M.

    1996-01-01

    The developmental changes in skin collagen biosynthesis pathway in male and female chickens were evaluated. Concentration of collagen, levels of mRNA for collagen type I subunits and for lysyl hydroxylase, and the level of three lysyl oxidase-derived cross-links: dehydro-dihydroxylysinonorleucine (DHLNL), dehydro-hydroxylysinonorleucine (HLNL), and dehydro-histidinohydroxymerodesmosine (HHMD) were determined during 4 wk posthatching. Skin collagen content increased with age and was higher in males than in females. In both sexes, the expression of the genes coding for alpha 1 and alpha 2 of collagen type I decreased with age: alpha 1(I) gene expression decreased from Day 3 onwards, whereas the reduction in alpha 2(I) gene expression started 1 wk later. At all ages examined, the expression of both genes was higher in male than in female skin. Males and females lysyl hydroxylase gene expression remained low until Day 16, after which an increase in the enzyme gene expression was observed. An increase in skin HLNL content was observed from Day 3 in both sexes reaching a peak in males at Day 9 and in females 1 wk later. The DHLNL content, which was higher in males than in females at all ages tested, dramatically decreased in both male and female skin from 3 d of age, reaching its lowest level at Day 16, and remained at that low level thereafter. The skin content of HHMD in males and females followed an oscillatory behavior with higher peaks in the male skin. The results suggest that the higher tensile strength of male skin than female skin may be due to the elevated skin collagen content that resulted from increased expression in collagen type I genes on the one hand, and from the higher amounts of various collagen cross-links on the other.

  2. In silico profiling of Escherichia coli and Saccharomyces cerevisiae as terpenoid factories

    OpenAIRE

    Gruchattka, Evamaria; H?dicke, Oliver; Klamt, Steffen; Sch?tz, Verena; Kayser, Oliver

    2013-01-01

    Background Heterologous microbial production of rare plant terpenoids of medicinal or industrial interest is attracting more and more attention but terpenoid yields are still low. Escherichia coli and Saccharomyces cerevisiae are the most widely used heterologous hosts; a direct comparison of both hosts based on experimental data is difficult though. Hence, the terpenoid pathways of E. coli (via 1-deoxy-D-xylulose 5-phosphate, DXP) and S. cerevisiae (via mevalonate, MVA), the impact of the re...

  3. De Novo Transcriptome and Expression Profile Analysis to Reveal Genes and Pathways Potentially Involved in Cantharidin Biosynthesis in the Blister Beetle Mylabris cichorii.

    Directory of Open Access Journals (Sweden)

    Yi Huang

    Full Text Available The dried body of Mylabris cichorii is well-known Chinese traditional medicine. The sesquiterpenoid cantharidin, which is secreted mostly by adult male beetles, has recently been used as an anti-cancer drug. However, little is known about the mechanisms of cantharidin biosynthesis. Furthermore, there is currently no genomic or transcriptomic information for M. cichorii. In this study, we performed de novo assembly transcriptome of M. cichorii using the Illumina Hiseq2000. A single run produced 9.19 Gb of clean nucleotides comprising 29,247 sequences, including 23,739 annotated sequences (about 81%. We also constructed two expression profile libraries (20-25 day-old adult males and 20-25 day-old adult females and discovered 2,465 significantly differentially-expressed genes. Putative genes and pathways involved in the biosynthesis of cantharidin were then characterized. We also found that cantharidin biosynthesis in M. cichorii might only occur via the mevalonate (MVA pathway, not via the methylerythritol 4-phosphate/deoxyxylulose 5-phosphate (MEP/DOXP pathway or a mixture of these. Besides, we considered that cantharidin biosynthesis might be related to the juvenile hormone (JH biosynthesis or degradation. The results of transcriptome and expression profiling analysis provide a comprehensive sequence resource for M. cichorii that could facilitate the in-depth study of candidate genes and pathways involved in cantharidin biosynthesis, and may thus help to improve our understanding of the mechanisms of cantharidin biosynthesis in blister beetles.

  4. Biosynthesis of vitamin K1 (phylloquinone) by plant peroxisomes and its integration into signaling molecule synthesis pathways.

    Science.gov (United States)

    Reumann, Sigrun

    2013-01-01

    Vitamin K1 (phylloquinone) is a substituted membrane-anchored naphthoquinone that functions as an essential electron carrier in photosystem I in photosynthetic organisms. While plants can synthesize phylloquinone de novo, humans rely on vitamin K1 uptake from green leafy vegetables as a precursor for the synthesis of its structural derivative, menaquinone-4 (vitamin K2). In vertebrates, menaquinone-4 serves as an enzymatic co-factor that is required for posttranslational protein modification, i.e. the γ-carboxylation of glutamate residues in specific proteins involved in blood coagulation, bone metabolism and vascular biology. Comprehensive knowledge of the subcellular compartmentalization of vitamin K biosynthesis in plants, pathway regulation and its integration in cellular metabolic networks is important to design functional food with elevated vitamin levels and health benefits to human consumers. It had long been assumed that plants obtained all enzymes for phylloquinone biosynthesis from the ancient cyanobacterial endosymbiont and that, upon gene transfer to the nucleus, all biosynthetic enzymes were re-directed to the plastid. This view, however, has been recently challenged by the exclusive localization of the 6th pathway enzyme (MenB/NS) to peroxisomes in Arabidopsis. Soon afterwards, not only the preceding enzyme, acyl-activating enzyme 14 (MenE/AAE14), but also the succeeding thioesterase (DHNAT) were also shown to be peroxisomal. Phylogenetic analysis revealed a heterogeneous evolutionary origin of the peroxisomal enzymes. Phylloquinone biosynthesis reveals several branching points leading to the synthesis of important defence signalling molecules, such as salicylic acid and benzoic acid derivatives. Recent research data demonstrate that, of the two phenylalanine-dependent pathways for benzoic and salicylic acid biosynthesis, the CoA-dependent β-oxidative pathway, which is peroxisomal, is the major route. Hence, peroxisomes emerge as an important cell

  5. Critical importance of the de novo pyrimidine biosynthesis pathway for Trypanosoma cruzi growth in the mammalian host cell cytoplasm

    Energy Technology Data Exchange (ETDEWEB)

    Hashimoto, Muneaki, E-mail: muneaki@juntendo.ac.jp [Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Morales, Jorge; Fukai, Yoshihisa; Suzuki, Shigeo; Takamiya, Shinzaburo; Tsubouchi, Akiko; Inoue, Syou [Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Inoue, Masayuki [Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (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); Harada, Shigeharu [Department of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Tanaka, Akiko [Systems and Structural Biology Center, RIKEN, Tsurumi, Yokohama 230-0045 (Japan); Aoki, Takashi [Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Nara, Takeshi, E-mail: tnara@juntendo.ac.jp [Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan)

    2012-01-20

    Highlights: Black-Right-Pointing-Pointer We established Trypanosoma cruzi lacking the gene for carbamoyl phosphate synthetase II. Black-Right-Pointing-Pointer Disruption of the cpsII gene significantly reduced the growth of epimastigotes. Black-Right-Pointing-Pointer In particular, the CPSII-null mutant severely retarded intracellular growth. Black-Right-Pointing-Pointer The de novo pyrimidine pathway is critical for the parasite growth in the host cell. -- Abstract: The intracellular parasitic protist Trypanosoma cruzi is the causative agent of Chagas disease in Latin America. In general, pyrimidine nucleotides are supplied by both de novo biosynthesis and salvage pathways. While epimastigotes-an insect form-possess both activities, amastigotes-an intracellular replicating form of T. cruzi-are unable to mediate the uptake of pyrimidine. However, the requirement of de novo pyrimidine biosynthesis for parasite growth and survival has not yet been elucidated. Carbamoyl-phosphate synthetase II (CPSII) is the first and rate-limiting enzyme of the de novo biosynthetic pathway, and increased CPSII activity is associated with the rapid proliferation of tumor cells. In the present study, we showed that disruption of the T. cruzicpsII gene significantly reduced parasite growth. In particular, the growth of amastigotes lacking the cpsII gene was severely suppressed. Thus, the de novo pyrimidine pathway is important for proliferation of T. cruzi in the host cell cytoplasm and represents a promising target for chemotherapy against Chagas disease.

  6. Synthetic biology for production of natural and new-to-nature terpenoids in photosynthetic organisms.

    Science.gov (United States)

    Arendt, Philipp; Pollier, Jacob; Callewaert, Nico; Goossens, Alain

    2016-07-01

    With tens of thousands of characterized members, terpenoids constitute the largest class of natural compounds that are synthesized by all living organisms. Several terpenoids play primary roles in the maintenance of cell membrane fluidity, as pigments or as phytohormones, but most of them function as specialized metabolites that are involved in plant resistance to herbivores or plant-environment interactions. Terpenoids are an essential component of human nutrition, and many are economically important pharmaceuticals, aromatics and potential next-generation biofuels. Because of the often low abundance in their natural source, as well as the demand for novel terpenoid structures with new or improved bioactivities, terpenoid biosynthesis has become a prime target for metabolic engineering and synthetic biology projects. In this review we focus on the creation of new-to-nature or tailor-made plant-derived terpenoids in photosynthetic organisms, in particular by means of combinatorial biosynthesis and the activation of silent metabolism. We reflect on the characteristics of different potential photosynthetic host organisms and recent advances in synthetic biology and discuss their utility for the (heterologous) production of (novel) terpenoids. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  7. The Arabidopsis thiamin-deficient mutant pale green1 lacks thiamin monophosphate phosphatase of the vitamin B1 biosynthesis pathway.

    Science.gov (United States)

    Hsieh, Wei-Yu; Liao, Jo-Chien; Wang, Hsin-Tzu; Hung, Tzu-Huan; Tseng, Ching-Chih; Chung, Tsui-Yun; Hsieh, Ming-Hsiun

    2017-07-01

    Thiamin diphosphate (TPP, vitamin B1 ) is an essential coenzyme present in all organisms. Animals obtain TPP from their diets, but plants synthesize TPPde novo. We isolated and characterized an Arabidopsis pale green1 (pale1) mutant that contained higher concentrations of thiamin monophosphate (TMP) and less thiamin and TPP than the wild type. Supplementation with thiamin, but not the thiazole and pyrimidine precursors, rescued the mutant phenotype, indicating that the pale1 mutant is a thiamin-deficient mutant. Map-based cloning and whole-genome sequencing revealed that the pale1 mutant has a mutation in At5g32470 encoding a TMP phosphatase of the TPP biosynthesis pathway. We further confirmed that the mutation of At5g32470 is responsible for the mutant phenotypes by complementing the pale1 mutant with constructs overexpressing full-length At5g32470. Most plant TPP biosynthetic enzymes are located in the chloroplasts and cytosol, but At5g32470-GFP localized to the mitochondrion of the root, hypocotyl, mesophyll and guard cells of the 35S:At5g32470-GFP complemented plants. The subcellular localization of a functional TMP phosphatase suggests that the complete vitamin B1 biosynthesis pathway may involve the chloroplasts, mitochondria and cytosol in plants. Analysis of PALE1 promoter-uidA activity revealed that PALE1 is mainly expressed in vascular tissues of Arabidopsis seedlings. Quantitative RT-PCR analysis of TPP biosynthesis genes and genes encoding the TPP-dependent enzymes pyruvate dehydrogenase, α-ketoglutarate dehydrogenase and transketolase revealed that the transcript levels of these genes were upregulated in the pale1 mutant. These results suggest that endogenous levels of TPP may affect the expression of genes involved in TPP biosynthesis and TPP-dependent enzymes. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  8. Biosynthesis and transport of terpenes

    NARCIS (Netherlands)

    Ting, H.M.

    2014-01-01

    Terpenoids are the largest class of natural product that are produced by plants, with functions that range from a role in plant development to direct defence against pathogens and indirect defence against insects through the attraction of natural enemies. While terpene biosynthesis genes have been

  9. The methylerythritol phosphate (MEP) pathway for isoprenoid biosynthesis as a target for the development of new drugs against tuberculosis.

    Science.gov (United States)

    Obiol-Pardo, C; Rubio-Martinez, J; Imperial, S

    2011-01-01

    Tuberculosis remains a major infectious disease to humans. It accounts for approximately 8-9 million new cases worldwide and an estimated 1.6 million deaths annually. Effective treatments for tuberculosis consist of a combination of several drugs administered over long periods of time. Since Mycobacterium tuberculosis often acquires multiple drug resistant mechanisms, development of new drugs with innovative actions is urgently required. The 2C-methyl-D-erythritol 4-phosphate (MEP) pathway, in charge of the essential biosynthesis of isoprenoids, represents a promising and selective target for developing new drugs against tuberculosis. To date, only fosmidomycin, a molecule that targets the second enzyme of the MEP pathway, has reached clinical trials but recent advances elucidating the structure and kinetics of the MEP enzymes are likely to change this scenario. This review describes the structure, mechanism of action and inhibitors of the seven enzymes of the MEP pathway, with special attention to the reported studies in M. tuberculosis.

  10. In silico profiling of Escherichia coli and Saccharomyces cerevisiae as terpenoid factories.

    Science.gov (United States)

    Gruchattka, Evamaria; Hädicke, Oliver; Klamt, Steffen; Schütz, Verena; Kayser, Oliver

    2013-09-23

    Heterologous microbial production of rare plant terpenoids of medicinal or industrial interest is attracting more and more attention but terpenoid yields are still low. Escherichia coli and Saccharomyces cerevisiae are the most widely used heterologous hosts; a direct comparison of both hosts based on experimental data is difficult though. Hence, the terpenoid pathways of E. coli (via 1-deoxy-D-xylulose 5-phosphate, DXP) and S. cerevisiae (via mevalonate, MVA), the impact of the respective hosts metabolism as well as the impact of different carbon sources were compared in silico by means of elementary mode analysis. The focus was set on the yield of isopentenyl diphosphate (IPP), the general terpenoid precursor, to identify new metabolic engineering strategies for an enhanced terpenoid yield. Starting from the respective precursor metabolites of the terpenoid pathways (pyruvate and glyceraldehyde-3-phosphate for the DXP pathway and acetyl-CoA for the MVA pathway) and considering only carbon stoichiometry, the two terpenoid pathways are identical with respect to carbon yield. However, with glucose as substrate, the MVA pathway has a lower potential to supply terpenoids in high yields than the DXP pathway if the formation of the required precursors is taken into account, due to the carbon loss in the formation of acetyl-CoA. This maximum yield is further reduced in both hosts when the required energy and reduction equivalents are considered. Moreover, the choice of carbon source (glucose, xylose, ethanol or glycerol) has an effect on terpenoid yield with non-fermentable carbon sources being more promising. Both hosts have deficiencies in energy and redox equivalents for high yield terpenoid production leading to new overexpression strategies (heterologous enzymes/pathways) for an enhanced terpenoid yield. Finally, several knockout strategies are identified using constrained minimal cut sets enforcing a coupling of growth to a terpenoid yield which is higher than any

  11. In silico profiling of Escherichia coli and Saccharomyces cerevisiae as terpenoid factories

    Science.gov (United States)

    2013-01-01

    Background Heterologous microbial production of rare plant terpenoids of medicinal or industrial interest is attracting more and more attention but terpenoid yields are still low. Escherichia coli and Saccharomyces cerevisiae are the most widely used heterologous hosts; a direct comparison of both hosts based on experimental data is difficult though. Hence, the terpenoid pathways of E. coli (via 1-deoxy-D-xylulose 5-phosphate, DXP) and S. cerevisiae (via mevalonate, MVA), the impact of the respective hosts metabolism as well as the impact of different carbon sources were compared in silico by means of elementary mode analysis. The focus was set on the yield of isopentenyl diphosphate (IPP), the general terpenoid precursor, to identify new metabolic engineering strategies for an enhanced terpenoid yield. Results Starting from the respective precursor metabolites of the terpenoid pathways (pyruvate and glyceraldehyde-3-phosphate for the DXP pathway and acetyl-CoA for the MVA pathway) and considering only carbon stoichiometry, the two terpenoid pathways are identical with respect to carbon yield. However, with glucose as substrate, the MVA pathway has a lower potential to supply terpenoids in high yields than the DXP pathway if the formation of the required precursors is taken into account, due to the carbon loss in the formation of acetyl-CoA. This maximum yield is further reduced in both hosts when the required energy and reduction equivalents are considered. Moreover, the choice of carbon source (glucose, xylose, ethanol or glycerol) has an effect on terpenoid yield with non-fermentable carbon sources being more promising. Both hosts have deficiencies in energy and redox equivalents for high yield terpenoid production leading to new overexpression strategies (heterologous enzymes/pathways) for an enhanced terpenoid yield. Finally, several knockout strategies are identified using constrained minimal cut sets enforcing a coupling of growth to a terpenoid yield which

  12. Convergent Evolution of Syringyl Lignin Biosynthesis via Distinct Pathways in the Lycophyte Selaginella and Flowering Plants[C][W

    Science.gov (United States)

    Weng, Jing-Ke; Akiyama, Takuya; Bonawitz, Nicholas D.; Li, Xu; Ralph, John; Chapple, Clint

    2010-01-01

    Phenotypic convergence in unrelated lineages arises when different organisms adapt similarly under comparable selective pressures. In an apparent example of this process, syringyl lignin, a fundamental building block of plant cell walls, occurs in two major plant lineages, lycophytes and angiosperms, which diverged from one another more than 400 million years ago. Here, we show that this convergence resulted from independent recruitment of lignin biosynthetic cytochrome P450-dependent monooxygenases that route cell wall monomers through related but distinct pathways in the two lineages. In contrast with angiosperms, in which syringyl lignin biosynthesis requires two phenylpropanoid meta-hydroxylases C3′H and F5H, the lycophyte Selaginella employs one phenylpropanoid dual meta-hydroxylase to bypass several steps of the canonical lignin biosynthetic pathway. Transgenic expression of the Selaginella hydroxylase in Arabidopsis thaliana dramatically reroutes its endogenous lignin biosynthetic pathway, yielding a novel lignin composition not previously identified in nature. Our findings demonstrate a unique case of convergent evolution via distinct biochemical strategies and suggest a new way to genetically reconstruct lignin biosynthesis in higher plants. PMID:20371642

  13. Regulation of the cholesterol biosynthetic pathway and its integration with fatty acid biosynthesis in the oleaginous microalga Nannochloropsis oceanica.

    Science.gov (United States)

    Lu, Yandu; Zhou, Wenxu; Wei, Li; Li, Jing; Jia, Jing; Li, Fei; Smith, Steven M; Xu, Jian

    2014-01-01

    Sterols are vital structural and regulatory components in eukaryotic cells; however, their biosynthetic pathways and functional roles in microalgae remain poorly understood. In the oleaginous microalga Nannochloropsis oceanica, the sterol biosynthetic pathway produces phytosterols as minor products and cholesterol as the major product. The evidence together with their deduced biosynthetic pathways suggests that N. oceanica exhibits features of both higher plants and mammals. Temporal tracking of sterol profiles and sterol-biosynthetic transcripts in response to changes in light intensity and nitrogen supply reveal that sterols play roles in cell proliferation, chloroplast differentiation, and photosynthesis. Furthermore, the dynamics of fatty acid (FA) and FA-biosynthetic transcripts upon chemical inhibitor-induced sterol depletion reveal possible co-regulation of sterol production and FA synthesis, in that the squalene epoxidase inhibitor terbinafine reduces sterol content yet significantly elevates free FA production. Thus, a feedback regulation of sterol and FA homeostasis is proposed, with the 1-deoxy-D-xylulose 5-phosphate synthase (DXS, the committed enzyme in isoprenoid and sterol biosynthesis) gene potentially subject to feedback regulation by sterols. These findings reveal features of sterol function and biosynthesis in microalgae and suggest new genetic engineering or chemical biology approaches for enhanced oil production in microalgae.

  14. Heterologous stable expression of terpenoid biosynthetic genes using the moss Physcomitrella patens.

    Science.gov (United States)

    Bach, Søren Spanner; King, Brian Christopher; Zhan, Xin; Simonsen, Henrik Toft; Hamberger, Björn

    2014-01-01

    Heterologous and stable expression of genes encoding terpenoid biosynthetic enzymes in planta is an important tool for functional characterization and is an attractive alternative to expression in microbial hosts for biotechnological production. Despite improvements to the procedure, such as streamlining of large scale Agrobacterium infiltration and upregulation of the upstream pathways, transient in planta heterologous expression quickly reaches limitations when used for production of terpenoids. Stable integration of transgenes into the nuclear genome of the moss Physcomitrella patens has already been widely recognized as a viable alternative for industrial-scale production of biopharmaceuticals. For expression of terpenoid biosynthetic genes, and reconstruction of heterologous pathways, Physcomitrella has unique attributes that makes it a very promising biotechnological host. These features include a high native tolerance to terpenoids, a simple endogenous terpenoid profile, convenient genome editing using homologous recombination, and cultivation techniques that allow up-scaling from single cells in microtiter plates to industrial photo-bioreactors. Beyond its use for functional characterization of terpenoid biosynthetic genes, engineered Physcomitrella can be a green biotechnological platform for production of terpenoids. Here, we describe two complementary and simple procedures for stable nuclear transformation of Physcomitrella with terpenoid biosynthetic genes, selection and cultivation of transgenic lines, and metabolite analysis of terpenoids produced in transgenic moss lines. We also provide tools for metabolic engineering through genome editing using homologous recombination.

  15. Orthologs of the archaeal isopentenyl phosphate kinase regulate terpenoid production in plants.

    Science.gov (United States)

    Henry, Laura K; Gutensohn, Michael; Thomas, Suzanne T; Noel, Joseph P; Dudareva, Natalia

    2015-08-11

    Terpenoids, compounds found in all domains of life, represent the largest class of natural products with essential roles in their hosts. All terpenoids originate from the five-carbon building blocks, isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), which can be derived from the mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathways. The absence of two components of the MVA pathway from archaeal genomes led to the discovery of an alternative MVA pathway with isopentenyl phosphate kinase (IPK) catalyzing the final step, the formation of IPP. Despite the fact that plants contain the complete classical MVA pathway, IPK homologs were identified in every sequenced green plant genome. Here, we show that IPK is indeed a member of the plant terpenoid metabolic network. It is localized in the cytosol and is coexpressed with MVA pathway and downstream terpenoid network genes. In planta, IPK acts in parallel with the MVA pathway and plays an important role in regulating the formation of both MVA and MEP pathway-derived terpenoid compounds by controlling the ratio of IP/DMAP to IPP/DMAPP. IP and DMAP can also competitively inhibit farnesyl diphosphate synthase. Moreover, we discovered a metabolically available carbon source for terpenoid formation in plants that is accessible via IPK overexpression. This metabolite reactivation approach offers new strategies for metabolic engineering of terpenoid production.

  16. De Novo transcriptome assembly (NGS) of Curcuma longa L. rhizome reveals novel transcripts related to anticancer and antimalarial terpenoids.

    Science.gov (United States)

    Annadurai, Ramasamy S; Neethiraj, Ramprasad; Jayakumar, Vasanthan; Damodaran, Anand C; Rao, Sudha Narayana; Katta, Mohan A V S K; Gopinathan, Sreeja; Sarma, Santosh Prasad; Senthilkumar, Vanitha; Niranjan, Vidya; Gopinath, Ashok; Mugasimangalam, Raja C

    2013-01-01

    Herbal remedies are increasingly being recognised in recent years as alternative medicine for a number of diseases including cancer. Curcuma longa L., commonly known as turmeric is used as a culinary spice in India and in many Asian countries has been attributed to lower incidences of gastrointestinal cancers. Curcumin, a secondary metabolite isolated from the rhizomes of this plant has been shown to have significant anticancer properties, in addition to antimalarial and antioxidant effects. We sequenced the transcriptome of the rhizome of the 3 varieties of Curcuma longa L. using Illumina reversible dye terminator sequencing followed by de novo transcriptome assembly. Multiple databases were used to obtain a comprehensive annotation and the transcripts were functionally classified using GO, KOG and PlantCyc. Special emphasis was given for annotating the secondary metabolite pathways and terpenoid biosynthesis pathways. We report for the first time, the presence of transcripts related to biosynthetic pathways of several anti-cancer compounds like taxol, curcumin, and vinblastine in addition to anti-malarial compounds like artemisinin and acridone alkaloids, emphasizing turmeric's importance as a highly potent phytochemical. Our data not only provides molecular signatures for several terpenoids but also a comprehensive molecular resource for facilitating deeper insights into the transcriptome of C. longa.

  17. De Novo Transcriptome Assembly (NGS) of Curcuma longa L. Rhizome Reveals Novel Transcripts Related to Anticancer and Antimalarial Terpenoids

    Science.gov (United States)

    Jayakumar, Vasanthan; Damodaran, Anand C.; Rao, Sudha Narayana; Katta, Mohan A. V. S. K.; Gopinathan, Sreeja; Sarma, Santosh Prasad; Senthilkumar, Vanitha; Niranjan, Vidya; Gopinath, Ashok; Mugasimangalam, Raja C.

    2013-01-01

    Herbal remedies are increasingly being recognised in recent years as alternative medicine for a number of diseases including cancer. Curcuma longa L., commonly known as turmeric is used as a culinary spice in India and in many Asian countries has been attributed to lower incidences of gastrointestinal cancers. Curcumin, a secondary metabolite isolated from the rhizomes of this plant has been shown to have significant anticancer properties, in addition to antimalarial and antioxidant effects. We sequenced the transcriptome of the rhizome of the 3 varieties of Curcuma longa L. using Illumina reversible dye terminator sequencing followed by de novo transcriptome assembly. Multiple databases were used to obtain a comprehensive annotation and the transcripts were functionally classified using GO, KOG and PlantCyc. Special emphasis was given for annotating the secondary metabolite pathways and terpenoid biosynthesis pathways. We report for the first time, the presence of transcripts related to biosynthetic pathways of several anti-cancer compounds like taxol, curcumin, and vinblastine in addition to anti-malarial compounds like artemisinin and acridone alkaloids, emphasizing turmeric's importance as a highly potent phytochemical. Our data not only provides molecular signatures for several terpenoids but also a comprehensive molecular resource for facilitating deeper insights into the transcriptome of C. longa. PMID:23468859

  18. De Novo transcriptome assembly (NGS of Curcuma longa L. rhizome reveals novel transcripts related to anticancer and antimalarial terpenoids.

    Directory of Open Access Journals (Sweden)

    Ramasamy S Annadurai

    Full Text Available Herbal remedies are increasingly being recognised in recent years as alternative medicine for a number of diseases including cancer. Curcuma longa L., commonly known as turmeric is used as a culinary spice in India and in many Asian countries has been attributed to lower incidences of gastrointestinal cancers. Curcumin, a secondary metabolite isolated from the rhizomes of this plant has been shown to have significant anticancer properties, in addition to antimalarial and antioxidant effects. We sequenced the transcriptome of the rhizome of the 3 varieties of Curcuma longa L. using Illumina reversible dye terminator sequencing followed by de novo transcriptome assembly. Multiple databases were used to obtain a comprehensive annotation and the transcripts were functionally classified using GO, KOG and PlantCyc. Special emphasis was given for annotating the secondary metabolite pathways and terpenoid biosynthesis pathways. We report for the first time, the presence of transcripts related to biosynthetic pathways of several anti-cancer compounds like taxol, curcumin, and vinblastine in addition to anti-malarial compounds like artemisinin and acridone alkaloids, emphasizing turmeric's importance as a highly potent phytochemical. Our data not only provides molecular signatures for several terpenoids but also a comprehensive molecular resource for facilitating deeper insights into the transcriptome of C. longa.

  19. Evaluation of tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) hairy roots for the production of geraniol, the first committed step in terpenoid indole alkaloid pathway.

    Science.gov (United States)

    Ritala, Anneli; Dong, Lemeng; Imseng, Nicole; Seppänen-Laakso, Tuulikki; Vasilev, Nikolay; van der Krol, Sander; Rischer, Heiko; Maaheimo, Hannu; Virkki, Arho; Brändli, Johanna; Schillberg, Stefan; Eibl, Regine; Bouwmeester, Harro; Oksman-Caldentey, Kirsi-Marja

    2014-04-20

    The terpenoid indole alkaloids are one of the major classes of plant-derived natural products and are well known for their many applications in the pharmaceutical, fragrance and cosmetics industries. Hairy root cultures are useful for the production of plant secondary metabolites because of their genetic and biochemical stability and their rapid growth in hormone-free media. Tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) hairy roots, which do not produce geraniol naturally, were engineered to express a plastid-targeted geraniol synthase gene originally isolated from Valeriana officinalis L. (VoGES). A SPME-GC-MS screening tool was developed for the rapid evaluation of production clones. The GC-MS analysis revealed that the free geraniol content in 20 hairy root clones expressing VoGES was an average of 13.7 μg/g dry weight (DW) and a maximum of 31.3 μg/g DW. More detailed metabolic analysis revealed that geraniol derivatives were present in six major glycoside forms, namely the hexose and/or pentose conjugates of geraniol and hydroxygeraniol, resulting in total geraniol levels of up to 204.3 μg/g DW following deglycosylation. A benchtop-scale process was developed in a 20-L wave-mixed bioreactor eventually yielding hundreds of grams of biomass and milligram quantities of geraniol per cultivation bag. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Biochemical pathways supporting beta-lactam biosynthesis in the springtail Folsomia candida

    NARCIS (Netherlands)

    Suring, Wouter; Marien, Janine; Broekman, R.A.; van Straalen, N.M.; Roelofs, D.

    2016-01-01

    Recently, an active set of beta-lactam biosynthesis genes was reported in the genome of the arthropod springtail Folsomia candida (Collembola). Evidence was provided that these genes were acquired through horizontal gene transfer. However, successful integration of fungal- or bacterial-derived

  1. Engineering PQS biosynthesis pathway for enhancement of bioelectricity production in Pseudomonas aeruginosa microbial fuel cells

    DEFF Research Database (Denmark)

    Wang, Victor Bochuan; Chua, Song-Lin; Cao, Bin

    2013-01-01

    The biosynthesis of the redox shuttle, phenazines, in Pseudomonas aeruginosa, an ubiquitous microorganism in wastewater microflora, is regulated by the 2-heptyl-3,4-dihydroxyquinoline (PQS) quorum-sensing system. However, PQS inhibits anaerobic growth of P. aeruginosa. We constructed a P. aerugin...... by genetic engineering is a suitable technique to improve power output of bioelectrochemical systems....

  2. The putrescine biosynthesis pathway in Lactococcus lactis is transcriptionally regulated by carbon catabolic repression, mediated by CcpA.

    Science.gov (United States)

    Linares, Daniel M; del Río, Beatriz; Ladero, Victor; Redruello, Begoña; Martín, María Cruz; Fernández, María; Alvarez, Miguel A

    2013-07-01

    Lactococcus lactis is the lactic acid bacterium most widely used by the dairy industry as a starter for the manufacture of fermented products such as cheese and buttermilk. However, some strains produce putrescine from agmatine via the agmatine deiminase (AGDI) pathway. The proteins involved in this pathway, including those necessary for agmatine uptake and conversion into putrescine, are encoded by the aguB, aguD, aguA and aguC genes, which together form an operon. This paper reports the mechanism of regulation of putrescine biosynthesis in L. lactis. It is shown that the aguBDAC operon, which contains a cre site at the promoter of aguB (the first gene of the operon), is transcriptionally regulated by carbon catabolic repression (CCR) mediated by the catabolite control protein CcpA. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Genomic and transcriptomic analyses reveal differential regulation of diverse terpenoid and polyketides secondary metabolites in Hericium erinaceus.

    Science.gov (United States)

    Chen, Juan; Zeng, Xu; Yang, Yan Long; Xing, Yong Mei; Zhang, Qi; Li, Jia Mei; Ma, Ke; Liu, Hong Wei; Guo, Shun Xing

    2017-08-31

    The lion's mane mushroom Hericium erinaceus is a famous traditional medicinal fungus credited with anti-dementia activity and a producer of cyathane diterpenoid natural products (erinacines) useful against nervous system diseases. To date, few studies have explored the biosynthesis of these compounds, although their chemical synthesis is known. Here, we report the first genome and tanscriptome sequence of the medicinal fungus H. erinaceus. The size of the genome is 39.35 Mb, containing 9895 gene models. The genome of H. erinaceus reveals diverse enzymes and a large family of cytochrome P450 (CYP) proteins involved in the biosynthesis of terpenoid backbones, diterpenoids, sesquiterpenes and polyketides. Three gene clusters related to terpene biosynthesis and one gene cluster for polyketides biosynthesis (PKS) were predicted. Genes involved in terpenoid biosynthesis were generally upregulated in mycelia, while the PKS gene was upregulated in the fruiting body. Comparative genome analysis of 42 fungal species of Basidiomycota revealed that most edible and medicinal mushroom show many more gene clusters involved in terpenoid and polyketide biosynthesis compared to the pathogenic fungi. None of the gene clusters for terpenoid or polyketide biosynthesis were predicted in the poisonous mushroom Amanita muscaria. Our findings may facilitate future discovery and biosynthesis of bioactive secondary metabolites from H. erinaceus and provide fundamental information for exploring the secondary metabolites in other Basidiomycetes.

  4. Modeling the flux of metabolites in the juvenile hormone biosynthesis pathway using generalized additive models and ordinary differential equations.

    Science.gov (United States)

    Martínez-Rincón, Raúl O; Rivera-Pérez, Crisalejandra; Diambra, Luis; Noriega, Fernando G

    2017-01-01

    Juvenile hormone (JH) regulates development and reproductive maturation in insects. The corpora allata (CA) from female adult mosquitoes synthesize fluctuating levels of JH, which have been linked to the ovarian development and are influenced by nutritional signals. The rate of JH biosynthesis is controlled by the rate of flux of isoprenoids in the pathway, which is the outcome of a complex interplay of changes in precursor pools and enzyme levels. A comprehensive study of the changes in enzymatic activities and precursor pool sizes have been previously reported for the mosquito Aedes aegypti JH biosynthesis pathway. In the present studies, we used two different quantitative approaches to describe and predict how changes in the individual metabolic reactions in the pathway affect JH synthesis. First, we constructed generalized additive models (GAMs) that described the association between changes in specific metabolite concentrations with changes in enzymatic activities and substrate concentrations. Changes in substrate concentrations explained 50% or more of the model deviances in 7 of the 13 metabolic steps analyzed. Addition of information on enzymatic activities almost always improved the fitness of GAMs built solely based on substrate concentrations. GAMs were validated using experimental data that were not included when the model was built. In addition, a system of ordinary differential equations (ODE) was developed to describe the instantaneous changes in metabolites as a function of the levels of enzymatic catalytic activities. The results demonstrated the ability of the models to predict changes in the flux of metabolites in the JH pathway, and can be used in the future to design and validate experimental manipulations of JH synthesis.

  5. 1,2-Diacylglycerol choline phosphotransferase catalyzes the final step in the unique Treponema denticola phosphatidylcholine biosynthesis pathway.

    Science.gov (United States)

    Vences-Guzmán, Miguel Ángel; Paula Goetting-Minesky, M; Guan, Ziqiang; Castillo-Ramirez, Santiago; Córdoba-Castro, Luz América; López-Lara, Isabel M; Geiger, Otto; Sohlenkamp, Christian; Christopher Fenno, J

    2017-03-01

    Treponema denticola synthesizes phosphatidylcholine through a licCA-dependent CDP-choline pathway identified only in the genus Treponema. However, the mechanism of conversion of CDP-choline to phosphatidylcholine remained unclear. We report here characterization of TDE0021 (herein designated cpt) encoding a 1,2-diacylglycerol choline phosphotransferase homologous to choline phosphotransferases that catalyze the final step of the highly conserved Kennedy pathway for phosphatidylcholine synthesis in eukaryotes. T. denticola Cpt catalyzed in vitro phosphatidylcholine formation from CDP-choline and diacylglycerol, and full activity required divalent manganese. Allelic replacement mutagenesis of cpt in T. denticola resulted in abrogation of phosphatidylcholine synthesis. T. denticola Cpt complemented a Saccharomyces cerevisiae CPT1 mutant, and expression of the entire T. denticola LicCA-Cpt pathway in E. coli resulted in phosphatidylcholine biosynthesis. Our findings show that T. denticola possesses a unique phosphatidylcholine synthesis pathway combining conserved prokaryotic choline kinase and CTP:phosphocholine cytidylyltransferase activities with a 1,2-diacylglycerol choline phosphotransferase that is common in eukaryotes. Other than in a subset of mammalian host-associated Treponema that includes T. pallidum, this pathway is found in neither bacteria nor Archaea. Molecular dating analysis of the Cpt gene family suggests that a horizontal gene transfer event introduced this gene into an ancestral Treponema well after its divergence from other spirochetes. © 2016 John Wiley & Sons Ltd.

  6. Evolutionarily conserved Δ25(27)-olefin ergosterol biosynthesis pathway in the alga Chlamydomonas reinhardtii

    OpenAIRE

    Miller, Matthew B.; Haubrich, Brad A; Wang, Qian; Snell, William J.; Nes, W. David

    2012-01-01

    Ergosterol is the predominant sterol of fungi and green algae. Although the biosynthetic pathway for sterol synthesis in fungi is well established and is known to use C24-methylation-C24 (28)-reduction (Δ24(28)-olefin pathway) steps, little is known about the sterol pathway in green algae. Previous work has raised the possibility that these algae might use a novel pathway because the green alga Chlamydomonas reinhardtii was shown to possess a mevalonate-independent methylerythritol 4-phosphat...

  7. Comprehensive assessment of the genes involved in withanolide biosynthesis from Withania somnifera: chemotype-specific and elicitor-responsive expression.

    Science.gov (United States)

    Agarwal, Aditya Vikram; Gupta, Parul; Singh, Deeksha; Dhar, Yogeshwar Vikram; Chandra, Deepak; Trivedi, Prabodh Kumar

    2017-07-01

    Withania somnifera (L.) Dunal (Family, Solanaceae), is among the most valuable medicinal plants used in Ayurveda owing to its rich reservoir of pharmaceutically active secondary metabolites known as withanolides. Withanolides are C28-steroidal lactones having a triterpenoidal metabolic origin synthesised via mevalonate (MVA) pathway and methyl-D-erythritol-4-phosphate (MEP) pathway involving metabolic intermediacy of 24-methylene (C30-terpenoid) cholesterol. Phytochemical studies suggest differences in the content and/or nature of withanolides in different tissues of different chemotypes. Though development of genomic resources has provided information about putative genes encoding enzymes for biosynthesis of intermediate steps of terpenoid backbone, not much is known about their regulation and response to elicitation. In this study, we generated detailed molecular information about genes catalysing key regulatory steps of withanolide biosynthetic pathway. The full-length sequences of genes encoding enzymes for intermediate steps of terpenoid backbone biosynthesis and their paralogs have been characterized for their functional and structural properties as well as phylogeny using bioinformatics approach. The expression analysis suggests that these genes are differentially expressed in different tissues (with maximal expression in young leaf), chemotypes and in response to salicylic acid (SA) and methyl jasmonate (MJ) treatments. Sub-cellular localization studies suggest that both paralogs of sterol ∆-7 reductase (WsDWF5-1 and WsDWF5-2) are localized in the endoplasmic reticulum (ER) thus supporting their indispensible role in withanolide biosynthesis. Comprehensive information developed, in this study, will lead to elucidation of chemotype- as well as tissue-specific withanolide biosynthesis and development of new tools for functional genomics in this important medicinal plant.

  8. DCEO Biotechnology: Tools To Design, Construct, Evaluate, and Optimize the Metabolic Pathway for Biosynthesis of Chemicals

    DEFF Research Database (Denmark)

    Chen, Xiulai; Gao, Cong; Guo, Liang

    2017-01-01

    Chemical synthesis is a well established route for producing many chemicals on a large scale, but some drawbacks still exist in this process, such as unstable intermediates, multistep reactions, complex process control, etc. Biobased production provides an attractive alternative to these challenges......, but how to make cells into efficient factories is challenging. As a key enabling technology to develop efficient cell factories, design-construction-evaluation-optimization (DCEO) biotechnology, which incorporates the concepts and techniques of pathway design, pathway construction, pathway evaluation......, and pathway optimization at the systems level, offers a conceptual and technological framework to exploit potential pathways, modify existing pathways and create new pathways for the optimal production of desired chemicals. Here, we summarize recent progress of DCEO biotechnology and examples of its...

  9. Identification of genes in Thuja plicata foliar terpenoid defenses.

    Science.gov (United States)

    Foster, Adam J; Hall, Dawn E; Mortimer, Leanne; Abercromby, Shelley; Gries, Regine; Gries, Gerhard; Bohlmann, Jörg; Russell, John; Mattsson, Jim

    2013-04-01

    Thuja plicata (western redcedar) is a long-lived conifer species whose foliage is rarely affected by disease or insect pests, but can be severely damaged by ungulate browsing. Deterrence to browsing correlates with high foliar levels of terpenoids, in particular the monoterpenoid α-thujone. Here, we set out to identify genes whose products may be involved in the production of α-thujone and other terpenoids in this species. First, we generated a foliar transcriptome database from which to draw candidate genes. Second, we mapped the storage of thujones and other terpenoids to foliar glands. Third, we used global expression profiling to identify more than 600 genes that are expressed at high levels in foliage with glands, but can either not be detected or are expressed at low levels in a natural variant lacking foliar glands. Fourth, we used in situ RNA hybridization to map the expression of a putative monoterpene synthase to the epithelium of glands and used enzyme assays with recombinant protein of the same gene to show that it produces sabinene, the monoterpene precursor of α-thujone. Finally, we identified candidate genes with predicted enzymatic functions for the conversion of sabinene to α-thujone. Taken together, this approach generated both general resources and detailed functional characterization in the identification of genes of foliar terpenoid biosynthesis in T. plicata.

  10. Construction of a synthetic metabolic pathway for biosynthesis of the non-natural methionine precursor 2,4-dihydroxybutyric acid.

    Science.gov (United States)

    Walther, Thomas; Topham, Christopher M; Irague, Romain; Auriol, Clément; Baylac, Audrey; Cordier, Hélène; Dressaire, Clémentine; Lozano-Huguet, Luce; Tarrat, Nathalie; Martineau, Nelly; Stodel, Marion; Malbert, Yannick; Maestracci, Marc; Huet, Robert; André, Isabelle; Remaud-Siméon, Magali; François, Jean Marie

    2017-06-20

    2,4-Dihydroxybutyric acid (DHB) is a molecule with considerable potential as a versatile chemical synthon. Notably, it may serve as a precursor for chemical synthesis of the methionine analogue 2-hydroxy-4-(methylthio)butyrate, thus, targeting a considerable market in animal nutrition. However, no natural metabolic pathway exists for the biosynthesis of DHB. Here we have therefore conceived a three-step metabolic pathway for the synthesis of DHB starting from the natural metabolite malate. The pathway employs previously unreported malate kinase, malate semialdehyde dehydrogenase and malate semialdehyde reductase activities. The kinase and semialdehyde dehydrogenase activities were obtained by rational design based on structural and mechanistic knowledge of candidate enzymes acting on sterically cognate substrates. Malate semialdehyde reductase activity was identified from an initial screening of several natural enzymes, and was further improved by rational design. The pathway was expressed in a minimally engineered Escherichia coli strain and produces 1.8 g l(-1) DHB with a molar yield of 0.15.

  11. Large-Scale Evolutionary Analysis of Genes and Supergene Clusters from Terpenoid Modular Pathways Provides Insights into Metabolic Diversification in Flowering Plants

    NARCIS (Netherlands)

    Hofberger, J.A.; Ramirez, A.M.; van den Bergh, E.; Zhu, X.; Bouwmeester, H.J.; Schuurink, R.C.; Schranz, M.E.

    2015-01-01

    An important component of plant evolution is the plethora of pathways producing more than 200,000 biochemically diverse specialized metabolites with pharmacological, nutritional and ecological significance. To unravel dynamics underlying metabolic diversification, it is critical to determine

  12. Large-scale evolutionary analysis of genes and supergene clusters from terpenoid modular pathways provides insights into metabolic diversification in flowering plants.

    NARCIS (Netherlands)

    Hofberger, J.A.; Ramirez, A.M.; Bergh, van den E.; Zhu, X.; Bouwmeester, H.J.; Schuurink, R.C.; Schranz, M.E.

    2015-01-01

    An important component of plant evolution is the plethora of pathways producing more than 200,000 biochemically diverse specialized metabolites with pharmacological, nutritional and ecological significance. To unravel dynamics underlying metabolic diversification, it is critical to determine

  13. Transcriptional profiling of three key genes of terpenoid indole ...

    African Journals Online (AJOL)

    The response of three key genes: strictosidine synthase (str1), tryptophan decarboxylase (tdc) and secologanin synthase (cyp72A1) of the wild plant species, Catharanthus roseus to different plant tissue culture treatments was studied. These genes encode enzymes acting early in the biosynthetic pathway of terpenoid ...

  14. Genome Mining for antibiotics biosynthesis pathways with antiSMASH 3

    DEFF Research Database (Denmark)

    Weber, Tilmann; Kim, Hyun Uk; Blin, Kai

    2014-01-01

    aiming directly to the substances or to putative targets were the only possibility to identify and isolate such compounds. With the recent progress of sequencing technologies, genome mining has become a very important method to complement the laborious and expensive experimental approach and to broaden...... are searched compared against different integrated databases to identify homologous (often uncharacterized) gene clusters in other microorganisms, genes encoding the biosynthesis of conserved precursors or related experimentally validated gene clusters. A new module of antiSMASH3 now also provides a direct...

  15. Lactate Racemization as a Rescue Pathway for Supplying d-Lactate to the Cell Wall Biosynthesis Machinery in Lactobacillus plantarum

    Science.gov (United States)

    Goffin, Philippe; Deghorain, Marie; Mainardi, Jean-Luc; Tytgat, Isabelle; Champomier-Vergès, Marie-Christine; Kleerebezem, Michiel; Hols, Pascal

    2005-01-01

    Lactobacillus plantarum is a lactic acid bacterium that produces d- and l-lactate using stereospecific NAD-dependent lactate dehydrogenases (LdhD and LdhL, respectively). However, reduction of glycolytic pyruvate by LdhD is not the only pathway for d-lactate production since a mutant defective in this activity still produces both lactate isomers (T. Ferain, J. N. Hobbs, Jr., J. Richardson, N. Bernard, D. Garmyn, P. Hols, N. E. Allen, and J. Delcour, J. Bacteriol. 178:5431-5437, 1996). Production of d-lactate in this species has been shown to be connected to cell wall biosynthesis through its incorporation as the last residue of the muramoyl-pentadepsipeptide peptidoglycan precursor. This particular feature leads to natural resistance to high concentrations of vancomycin. In the present study, we show that L. plantarum possesses two pathways for d-lactate production: the LdhD enzyme and a lactate racemase, whose expression requires l-lactate. We report the cloning of a six-gene operon, which is involved in lactate racemization activity and is positively regulated by l-lactate. Deletion of this operon in an L. plantarum strain that is devoid of LdhD activity leads to the exclusive production of l-lactate. As a consequence, peptidoglycan biosynthesis is affected, and growth of this mutant is d-lactate dependent. We also show that the growth defect can be partially restored by expression of the d-alanyl-d-alanine-forming Ddl ligase from Lactococcus lactis, or by supplementation with various d-2-hydroxy acids but not d-2-amino acids, leading to variable vancomycin resistance levels. This suggests that L. plantarum is unable to efficiently synthesize peptidoglycan precursors ending in d-alanine and that the cell wall biosynthesis machinery in this species is specifically dedicated to the production of peptidoglycan precursors ending in d-lactate. In this context, the lactate racemase could thus provide the bacterium with a rescue pathway for d-lactate production upon

  16. Mutations in Four Glycosyl Hydrolases Reveal a Highly Coordinated Pathway for Rhodopsin Biosynthesis and N-Glycan Trimming in Drosophila melanogaster

    Science.gov (United States)

    Rosenbaum, Erica E.; Vasiljevic, Eva; Brehm, Kimberley S.; Colley, Nansi Jo

    2014-01-01

    As newly synthesized glycoproteins move through the secretory pathway, the asparagine-linked glycan (N-glycan) undergoes extensive modifications involving the sequential removal and addition of sugar residues. These modifications are critical for the proper assembly, quality control and transport of glycoproteins during biosynthesis. The importance of N-glycosylation is illustrated by a growing list of diseases that result from defects in the biosynthesis and processing of N-linked glycans. The major rhodopsin in Drosophila melanogaster photoreceptors, Rh1, is highly unique among glycoproteins, as the N-glycan appears to be completely removed during Rh1 biosynthesis and maturation. However, much of the deglycosylation pathway for Rh1 remains unknown. To elucidate the key steps in Rh1 deglycosylation in vivo, we characterized mutant alleles of four Drosophila glycosyl hydrolases, namely α-mannosidase-II (α-Man-II), α-mannosidase-IIb (α-Man-IIb), a β-N-acetylglucosaminidase called fused lobes (Fdl), and hexosaminidase 1 (Hexo1). We have demonstrated that these four enzymes play essential and unique roles in a highly coordinated pathway for oligosaccharide trimming during Rh1 biosynthesis. Our results reveal that α-Man-II and α-Man-IIb are not isozymes like their mammalian counterparts, but rather function at distinct stages in Rh1 maturation. Also of significance, our results indicate that Hexo1 has a biosynthetic role in N-glycan processing during Rh1 maturation. This is unexpected given that in humans, the hexosaminidases are typically lysosomal enzymes involved in N-glycan catabolism with no known roles in protein biosynthesis. Here, we present a genetic dissection of glycoprotein processing in Drosophila and unveil key steps in N-glycan trimming during Rh1 biosynthesis. Taken together, our results provide fundamental advances towards understanding the complex and highly regulated pathway of N-glycosylation in vivo and reveal novel insights into the

  17. Mutations in four glycosyl hydrolases reveal a highly coordinated pathway for rhodopsin biosynthesis and N-glycan trimming in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Erica E Rosenbaum

    2014-05-01

    Full Text Available As newly synthesized glycoproteins move through the secretory pathway, the asparagine-linked glycan (N-glycan undergoes extensive modifications involving the sequential removal and addition of sugar residues. These modifications are critical for the proper assembly, quality control and transport of glycoproteins during biosynthesis. The importance of N-glycosylation is illustrated by a growing list of diseases that result from defects in the biosynthesis and processing of N-linked glycans. The major rhodopsin in Drosophila melanogaster photoreceptors, Rh1, is highly unique among glycoproteins, as the N-glycan appears to be completely removed during Rh1 biosynthesis and maturation. However, much of the deglycosylation pathway for Rh1 remains unknown. To elucidate the key steps in Rh1 deglycosylation in vivo, we characterized mutant alleles of four Drosophila glycosyl hydrolases, namely α-mannosidase-II (α-Man-II, α-mannosidase-IIb (α-Man-IIb, a β-N-acetylglucosaminidase called fused lobes (Fdl, and hexosaminidase 1 (Hexo1. We have demonstrated that these four enzymes play essential and unique roles in a highly coordinated pathway for oligosaccharide trimming during Rh1 biosynthesis. Our results reveal that α-Man-II and α-Man-IIb are not isozymes like their mammalian counterparts, but rather function at distinct stages in Rh1 maturation. Also of significance, our results indicate that Hexo1 has a biosynthetic role in N-glycan processing during Rh1 maturation. This is unexpected given that in humans, the hexosaminidases are typically lysosomal enzymes involved in N-glycan catabolism with no known roles in protein biosynthesis. Here, we present a genetic dissection of glycoprotein processing in Drosophila and unveil key steps in N-glycan trimming during Rh1 biosynthesis. Taken together, our results provide fundamental advances towards understanding the complex and highly regulated pathway of N-glycosylation in vivo and reveal novel insights

  18. Coordinated Activation of Cellulose and Repression of Lignin Biosynthesis Pathways in Rice1[C][W][OA

    Science.gov (United States)

    Ambavaram, Madana M.R.; Krishnan, Arjun; Trijatmiko, Kurniawan R.; Pereira, Andy

    2011-01-01

    Cellulose from plant biomass is the largest renewable energy resource of carbon fixed from the atmosphere, which can be converted into fermentable sugars for production into ethanol. However, the cellulose present as lignocellulosic biomass is embedded in a hemicellulose and lignin matrix from which it needs to be extracted for efficient processing. Here, we show that expression of an Arabidopsis (Arabidopsis thaliana) transcription factor, SHINE (SHN), in rice (Oryza sativa), a model for the grasses, causes a 34% increase in cellulose and a 45% reduction in lignin content. The rice AtSHN lines also exhibit an altered lignin composition correlated with improved digestibility, with no compromise in plant strength and performance. Using a detailed systems-level analysis of global gene expression in rice, we reveal the SHN regulatory network coordinating down-regulation of lignin biosynthesis and up-regulation of cellulose and other cell wall biosynthesis pathway genes. The results thus support the development of nonfood crops and crop wastes with increased cellulose and low lignin with good agronomic performance that could improve the economic viability of lignocellulosic crop utilization for biofuels. PMID:21205614

  19. Microbial production strategies and applications of lycopene and other terpenoids.

    Science.gov (United States)

    Ma, Tian; Deng, Zixin; Liu, Tiangang

    2016-01-01

    Terpenoids are a large class of compounds that have far-reaching applications and economic value, particularly those most commonly found in plants; however, the extraction and synthesis of these compounds is often expensive and technically challenging. Recent advances in microbial metabolic engineering comprise a breakthrough that may enable the efficient, cost-effective production of these limited natural resources. Via the engineering of safe, industrial microorganisms that encode product-specific enzymes, and even entire metabolic pathways of interest, microbial-derived semisynthetic terpenoids may soon replace plant-derived terpenoids as the primary source of these valuable compounds. Indeed, the recent metabolic engineering of an Escherichia coli strain that produces the precursor to lycopene, a commercially and medically important compound, with higher yields than those in tomato plants serves as a successful example. Here, we review the recent developments in the metabolic engineering of microbes for the production of certain terpenoid compounds, particularly lycopene, which has been increasingly used in pharmaceuticals, nutritional supplements, and cosmetics. Furthermore, we summarize the metabolic engineering strategies used to achieve successful microbial production of some similar compounds. Based on this overview, there is a reason to believe that metabolic engineering comprises an optimal approach for increasing the production of lycopene and other terpenoids.

  20. Osmotic regulation and tissue localization of the myo-inositol biosynthesis pathway in tilapia (Oreochromis mossambicus) larvae.

    Science.gov (United States)

    Sacchi, Romina; Gardell, Alison M; Chang, Nicole; Kültz, Dietmar

    2014-10-01

    The myo-inositol biosynthesis (MIB) pathway converts glucose-6-phosphate to the compatible osmolyte myo-inositol, which protects cells from salinity stress. We exposed tilapia larvae just after yolk sac resorption to various hypersaline environments and recorded robust induction of the enzymes that constitute the MIB pathway, myo-inositol-phosphate synthase (MIPS), and inositol monophosphatase 1 (IMPA1). Strong up-regulation of these enzymes is evident at both mRNA (quantitative real-time PCR) and protein (densitometric analysis of Western blots) levels. The highest level of induction of these enzymes occurs at the highest salinity that larvae were exposed to (90 ppt). Less severe salinity stress causes a proportionately reduced induction of the MIB pathway. Two distinct MIPS mRNA variants are present in tilapia larvae and both are induced at comparable levels for all the salinity challenges tested (34, 70, and 90 ppt). Immunohistochemical localization of IMPA1 protein in sagittal sections of salinity stressed and control larvae identified tissues that are particularly potent in inducing the MIB pathway. These tissues include the skin (epidermis), gills, eye (ciliary epithelium) and heart. In particular, the epidermis directly facing the external milieu showed a very strong induction of IMPA1 immunoreactivity. IMPA1 induction in response to salinity stress was not observed in other tissues suggesting that tilapia larvae may also utilize compatible organic osmolytes other than solely myo-inositol for osmoprotection. We conclude that the MIB pathway plays an important role in protecting multiple (but not all) tissues of tilapia larvae from hyperosmotic salinity stress. © 2014 Wiley Periodicals, Inc.

  1. Effect of yeast species on the terpenoids profile of Chinese light-style liquor.

    Science.gov (United States)

    Wu, Qun; Zhu, Weian; Wang, Wei; Xu, Yan

    2015-02-01

    Terpenoids are important trace flavour constituents in Chinese light-style liquors, and are formed by the various yeast species present during fermentation of liquor from cereal and legume materials. Saccharomyces cerevisiae, Pichia kudriavzevii and Wickerhamomyces anomalus are three such yeast species, and we found S. cerevisiae capable of generating thirteen different terpenoids in cereal and legume extract fermentation, by both de novo and biotransformation pathways. We also found that cereals such as sorghum and barley, and legumes such as peas, contained different terpenoids precursors, which differentially affected the formation and profile of terpenoids mixtures. This work gives new insights into the role of yeast species in generating the various terpenoids mixtures found in Chinese light-style liquors. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Evolutionarily conserved Delta(25(27))-olefin ergosterol biosynthesis pathway in the alga Chlamydomonas reinhardtii.

    Science.gov (United States)

    Miller, Matthew B; Haubrich, Brad A; Wang, Qian; Snell, William J; Nes, W David

    2012-08-01

    Ergosterol is the predominant sterol of fungi and green algae. Although the biosynthetic pathway for sterol synthesis in fungi is well established and is known to use C24-methylation-C24 (28)-reduction (Δ(24(28))-olefin pathway) steps, little is known about the sterol pathway in green algae. Previous work has raised the possibility that these algae might use a novel pathway because the green alga Chlamydomonas reinhardtii was shown to possess a mevalonate-independent methylerythritol 4-phosphate not present in fungi. Here, we report that C. reinhardtii synthesizes the protosterol cycloartenol and converts it to ergosterol (C24β-methyl) and 7-dehydroporiferasterol (C24β-ethyl) through a highly conserved sterol C24- methylation-C25-reduction (Δ(25(27))-olefin) pathway that is distinct from the well-described acetate-mevalonate pathway to fungal lanosterol and its conversion to ergosterol by the Δ(24(28))-olefin pathway. We isolated and characterized 23 sterols by a combination of GC-MS and proton nuclear magnetic resonance spectroscopy analysis from a set of mutant, wild-type, and 25-thialanosterol-treated cells. The structure and stereochemistry of the final C24-alkyl sterol side chains possessed different combinations of 24β-methyl/ethyl groups and Δ(22(23))E and Δ(25(27))-double bond constructions. When incubated with [methyl-(2)H(3)]methionine, cells incorporated three (into ergosterol) or five (into 7-dehydroporiferasterol) deuterium atoms into the newly biosynthesized 24β-alkyl sterols, consistent only with a Δ(25(27))-olefin pathway. Thus, our findings demonstrate that two separate isoprenoid-24-alkyl sterol pathways evolved in fungi and green algae, both of which converge to yield a common membrane insert ergosterol.

  3. Molecular dissection of the pathogen-inducible 3-deoxyanthocyanidin biosynthesis pathway in sorghum.

    Science.gov (United States)

    Liu, Hongjia; Du, Yegang; Chu, Hung; Shih, Chun Hat; Wong, Yu Wai; Wang, Mingfu; Chu, Ivan K; Tao, Yuezhi; Lo, Clive

    2010-07-01

    3-Deoxyanthocyanidins are the unique phytoalexins synthesized by sorghum in response to fungal inoculation. They are structurally related to anthocyanins but the final steps of their pathogen-inducible biosynthesis are not fully understood. We have identified new flavonoid structural genes from the recently completed sorghum BTx623 genome sequence. The biochemical functions of the different expressed sorghum genes were established in planta by complementation in the appropriate Arabidopsis transparent testa mutants. There is a family of nine chalcone synthase genes which are all inducible by fungal inoculation in sorghum seedlings. Specific dihydroflavonol 4-reductase (DFR) genes responsive to conditions which stimulated anthocyanin accumulation (SbDFR1) or 3-deoxyanthocyanidin production (SbDFR3) were identified. Recombinant SbDFR1 and SbDFR3 were found to function as typical DFRs by accepting dihydroflavonol substrates. On the other hand, both DFRs showed substantially lower but detectable NADPH-dependent activities toward flavanones. Reduction of flavanones to flavan-4-ols is a reaction step required for 3-deoxyanthocyanidin production. Flavanone 3-hydroxylase (F3H) converts flavanones to dihydroflavonols for anthocyanin biosynthesis. In sorghum seedlings, expression of two F3H genes was either absent or strongly suppressed during the accumulation of 3-deoxyanthocyanidins. Under such conditions, most flavanones are expected to be reduced by the pathogen-induced SbDFR3 for the formation of flavan-4-ols. Our work also revealed that 3-deoxyanthocyanidin accumulation and SbDFR3 expression were induced by methyl jasmonate treatment in sorghum roots but the stimulation effects were antagonized by salicylic acid.

  4. Orm protein phosphoregulation mediates transient sphingolipid biosynthesis response to heat stress via the Pkh-Ypk and Cdc55-PP2A pathways.

    Science.gov (United States)

    Sun, Yidi; Miao, Yansong; Yamane, Yukari; Zhang, Chao; Shokat, Kevan M; Takematsu, Hiromu; Kozutsumi, Yasunori; Drubin, David G

    2012-06-01

    Sphingoid intermediates accumulate in response to a variety of stresses, including heat, and trigger cellular responses. However, the mechanism by which stress affects sphingolipid biosynthesis has yet to be identified. Recent studies in yeast suggest that sphingolipid biosynthesis is regulated through phosphorylation of the Orm proteins, which in humans are potential risk factors for childhood asthma. Here we demonstrate that Orm phosphorylation status is highly responsive to sphingoid bases. We also demonstrate, by monitoring temporal changes in Orm phosphorylation and sphingoid base production in cells inhibited for yeast protein kinase 1 (Ypk1) activity, that Ypk1 transmits heat stress signals to the sphingolipid biosynthesis pathway via Orm phosphorylation. Our data indicate that heat-induced sphingolipid biosynthesis in turn triggers Orm protein dephosphorylation, making the induction transient. We identified Cdc55-protein phosphatase 2A (PP2A) as a key phosphatase that counteracts Ypk1 activity in Orm-mediated sphingolipid biosynthesis regulation. In total, our study reveals a mechanism through which the conserved Pkh-Ypk kinase cascade and Cdc55-PP2A facilitate rapid, transient sphingolipid production in response to heat stress through Orm protein phosphoregulation. We propose that this mechanism serves as the basis for how Orm phosphoregulation controls sphingolipid biosynthesis in response to stress in a kinetically coupled manner.

  5. Twinfilin 1 enhances milk bio-synthesis and proliferation of bovine mammary epithelial cells via the mTOR signaling pathway.

    Science.gov (United States)

    Li, Lu; Liu, Lijie; Qu, Bo; Li, Xueying; Gao, Xuejun; Zhang, Minghui

    2017-10-21

    Twinfilin1 (TWF1) is an actin monomer-binding protein, which biological function has not yet been fully uncovered. In our previous study, we found by mass spectrometry analysis that TWF1 might be one of the major proteins responsible for milk bio-synthesis and proliferation of bovine mammary epithelial cells (BMECs). The purpose of this study was to explore the possible mechanism by which TWF1 regulates signaling pathways that enhance milk bio-synthesis and proliferation of BMECs. We first explored the effects of TWF1 on milk bio-synthesis and cell proliferation, and analyzed the role of TWF1 on the protein levels of signaling molecules (mTOR, SREBP-1c and Cyclin D1) related to milk bio-synthesis and cell proliferation. Then we determinate the impacts of amino acids (methionine and leucine) and hormones (estrogen and prolactin) on the expressions of TWF1. These results reveal that TWF1 is highly induced by the stimulation of amino acids and hormones and involved in regulation of milk bio-synthesis and cell proliferation via the mTOR pathway in BMECs. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Differential selection on carotenoid biosynthesis genes as a function of gene position in the metabolic pathway: a study on the carrot and dicots.

    Directory of Open Access Journals (Sweden)

    Jérémy Clotault

    Full Text Available BACKGROUND: Selection of genes involved in metabolic pathways could target them differently depending on the position of genes in the pathway and on their role in controlling metabolic fluxes. This hypothesis was tested in the carotenoid biosynthesis pathway using population genetics and phylogenetics. METHODOLOGY/PRINCIPAL FINDINGS: Evolutionary rates of seven genes distributed along the carotenoid biosynthesis pathway, IPI, PDS, CRTISO, LCYB, LCYE, CHXE and ZEP, were compared in seven dicot taxa. A survey of deviations from neutrality expectations at these genes was also undertaken in cultivated carrot (Daucus carota subsp. sativus, a species that has been intensely bred for carotenoid pattern diversification in its root during its cultivation history. Parts of sequences of these genes were obtained from 46 individuals representing a wide diversity of cultivated carrots. Downstream genes exhibited higher deviations from neutral expectations than upstream genes. Comparisons of synonymous and nonsynonymous substitution rates between genes among dicots revealed greater constraints on upstream genes than on downstream genes. An excess of intermediate frequency polymorphisms, high nucleotide diversity and/or high differentiation of CRTISO, LCYB1 and LCYE in cultivated carrot suggest that balancing selection may have targeted genes acting centrally in the pathway. CONCLUSIONS/SIGNIFICANCE: Our results are consistent with relaxed constraints on downstream genes and selection targeting the central enzymes of the carotenoid biosynthesis pathway during carrot breeding history.

  7. Biosynthesis of β-carotene in engineered E. coli using the MEP and MVA pathways.

    Science.gov (United States)

    Yang, Jianming; Guo, Lizhong

    2014-11-18

    β-carotene is a carotenoid compound that has been widely used not only in the industrial production of pharmaceuticals but also as nutraceuticals, animal feed additives, functional cosmetics, and food colorants. Currently, more than 90% of commercial β-carotene is produced by chemical synthesis. Due to the growing public concern over food safety, the use of chemically synthesized β-carotene as food additives or functional cosmetic agents has been severely controlled in recent years. This has reignited the enthusiasm for seeking natural β-carotene in large-scale fermentative production by microorganisms. To increase β-carotene production by improving the isopentenyl pyrophosphate (IPP) and geranyl diphospate (GPP) concentration in the cell, the optimized MEP (methylerythritol 4-phosphate) pathway containing 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and isopentenyl pyrophosphate isomerase (FNI) from Bacillus subtilis, geranyl diphosphate synthase (GPPS2) from Abies grandis have been co-expressed in an engineered E. coli strain. To further enhance the production of β-carotene, the hybrid MVA (mevalonate) pathway has been introduced into an engineered E. coli strain, co-expressed with the optimized MEP pathway and GPPS2. The final genetically modified strain, YJM49, can accumulate 122.4±6.2 mg/L β-carotene in flask culture, approximately 113-fold and 1.7 times greater than strain YJM39, which carries the native MEP pathway, and YJM45, which harbors the MVA pathway and the native MEP pathway, respectively. Subsequently, the fermentation process was optimized to enhance β-carotene production with a maximum titer of 256.8±10.4 mg/L. Finally, the fed-batch fermentation of β-carotene was evaluated using the optimized culture conditions. After induction for 56 h, the final engineered strain YJM49 accumulated 3.2 g/L β-carotene with a volumetric productivity of 0.37 mg/(L · h · OD600) in aerobic fed-batch fermentation, and the conversion efficiency

  8. Tracing metabolic pathways of lipid biosynthesis in ectomycorrhizal fungi from position-specific 13C-labelling in glucose.

    Science.gov (United States)

    Scandellari, Francesca; Hobbie, Erik A; Ouimette, Andrew P; Stucker, Valerie K

    2009-12-01

    Six position-specific (13)C-labelled isotopomers of glucose were supplied to the ectomycorrhizal fungi Suillus pungens and Tricholoma flavovirens. From the resulting distribution of (13)C among fungal PLFAs, the overall order and contribution of each glucose atom to fatty acid (13)C enrichment was: C6 (approximately 31%) > C5 (approximately 25%) > C1 (approximately 18%) > C2 (approximately 18%) > C3 (approximately 8%) > C4 (approximately 1%). These data were used to parameterize a metabolic model of the relative fluxes from glucose degradation to lipid synthesis. Our data revealed that a higher amount of carbon is directed to glycolysis than to the oxidative pentose phosphate pathway (60% and 40% respectively) and that a significant part flows through these pathways more than once (73%) due to the reversibility of some glycolysis reactions. Surprisingly, 95% of carbon cycled through glyoxylate prior to incorporation into lipids, possibly to consume the excess of acetyl-CoA produced during fatty acid turnover. Our approach provides a rigorous framework for analysing lipid biosynthesis in fungi. In addition, this approach could ultimately improve the interpretation of isotopic patterns at natural abundance in field studies.

  9. HBV core protein allosteric modulators differentially alter cccDNA biosynthesis from de novo infection and intracellular amplification pathways

    Science.gov (United States)

    Guo, Fang; Zhao, Qiong; Cheng, Junjun; Qi, Yonghe; Su, Qing; Wei, Lai; Li, Wenhui; Chang, Jinhong

    2017-01-01

    Hepatitis B virus (HBV) core protein assembles viral pre-genomic (pg) RNA and DNA polymerase into nucleocapsids for reverse transcriptional DNA replication to take place. Several chemotypes of small molecules, including heteroaryldihydropyrimidines (HAPs) and sulfamoylbenzamides (SBAs), have been discovered to allosterically modulate core protein structure and consequentially alter the kinetics and pathway of core protein assembly, resulting in formation of irregularly-shaped core protein aggregates or “empty” capsids devoid of pre-genomic RNA and viral DNA polymerase. Interestingly, in addition to inhibiting nucleocapsid assembly and subsequent viral genome replication, we have now demonstrated that HAPs and SBAs differentially modulate the biosynthesis of covalently closed circular (ccc) DNA from de novo infection and intracellular amplification pathways by inducing disassembly of nucleocapsids derived from virions as well as double-stranded DNA-containing progeny nucleocapsids in the cytoplasm. Specifically, the mistimed cuing of nucleocapsid uncoating prevents cccDNA formation during de novo infection of hepatocytes, while transiently accelerating cccDNA synthesis from cytoplasmic progeny nucleocapsids. Our studies indicate that elongation of positive-stranded DNA induces structural changes of nucleocapsids, which confers ability of mature nucleocapsids to bind CpAMs and triggers its disassembly. Understanding the molecular mechanism underlying the dual effects of the core protein allosteric modulators on nucleocapsid assembly and disassembly will facilitate the discovery of novel core protein-targeting antiviral agents that can more efficiently suppress cccDNA synthesis and cure chronic hepatitis B. PMID:28945802

  10. Activation of glycerol metabolic pathway by evolutionary engineering of Rhizopus oryzae to strengthen the fumaric acid biosynthesis from crude glycerol.

    Science.gov (United States)

    Huang, Di; Wang, Ru; Du, Wenjie; Wang, Guanyi; Xia, Menglei

    2015-11-01

    Rhizopus oryzae is strictly inhibited by biodiesel-based by-product crude glycerol, which results in low fumaric acid production. In this study, evolutionary engineering was employed to activate the glycerol utilization pathway for fumaric acid production. An evolved strain G80 was selected, which could tolerate and utilize high concentrations of crude glycerol to produce 14.9g/L fumaric acid with a yield of 0.248g/g glycerol. Key enzymes activity analysis revealed that the evolved strain displayed a significant upregulation in glycerol dissimilation, pyruvate consumption and reductive tricarboxylic acid pathways, compared with the parent strain. Subsequently, intracellular metabolic profiling analysis showed that amino acid biosynthesis, tricarboxylic acid cycle, fatty acid and stress response metabolites accounted for metabolic difference between two strains. Moreover, a glycerol fed-batch strategy was optimized to obtain the highest fumaric acid production of 25.5g/L, significantly increased by 20.9-fold than that of the parent strain of 1.2g/L. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Modification of targets related to the Entner-Doudoroff/pentose phosphate pathway route for methyl-D-erythritol 4-phosphate-dependent carotenoid biosynthesis in Escherichia coli.

    Science.gov (United States)

    Li, Chun; Ying, Lan-Qing; Zhang, Sha-Sha; Chen, Nan; Liu, Wei-Feng; Tao, Yong

    2015-08-12

    In engineered strains of Escherichia coli, bioconversion efficiency is determined by not only metabolic flux but also the turnover efficiency of relevant pathways. Methyl-D-erythritol 4-phosphate (MEP)-dependent carotenoid biosynthesis in E. coli requires efficient turnover of precursors and balanced flux among precursors, cofactors, and cellular energy. However, the imbalanced supply of glyceraldehyde 3-phosphate (G3P) and pyruvate precursors remains the major metabolic bottleneck. To address this problem, we manipulated various genetic targets related to the Entner-Doudoroff (ED)/pentose phosphate (PP) pathways. Systematic target modification was conducted to improve G3P and pyruvate use and rebalance the precursor and redox fluxes. Carotenoid production was improved to different degrees by modifying various targets in the Embden-Meyerhof-Parnas (EMP) and ED pathways, which directed metabolic flux from the EMP pathway towards the ED pathway. The improvements in yield were much greater when the MEP pathway was enhanced. The coordinated modification of ED and MEP pathway targets using gene expression enhancement and protein coupling strategies in the pgi deletion background further improved carotenoid synthesis. The fine-tuning of flux at the branch point between the ED and PP pathways was important for carotenoid biosynthesis. Deletion of pfkAB instead of pgi reduced the carotenoid yield. This suggested that anaplerotic flux of G3P and pyruvate might be necessary for carotenoid biosynthesis. Improved carotenoid yields were accompanied by increased biomass and decreased acetate overflow. Therefore, efficient use of G3P and pyruvate precursors resulted in a balance among carotenoid biosynthesis, cell growth, and by-product metabolism. An efficient and balanced MEP-dependent carotenoid bioconversion strategy involving both the ED and PP pathways was implemented by the coordinated modification of diverse central metabolic pathway targets. In this strategy, enhancement

  12. Amino acids biosynthesis and nitrogen assimilation pathways: a great genomic deletion during eukaryotes evolution

    Science.gov (United States)

    2011-01-01

    Background Besides being building blocks for proteins, amino acids are also key metabolic intermediates in living cells. Surprisingly a variety of organisms are incapable of synthesizing some of them, thus named Essential Amino Acids (EAAs). How certain ancestral organisms successfully competed for survival after losing key genes involved in amino acids anabolism remains an open question. Comparative genomics searches on current protein databases including sequences from both complete and incomplete genomes among diverse taxonomic groups help us to understand amino acids auxotrophy distribution. Results Here, we applied a methodology based on clustering of homologous genes to seed sequences from autotrophic organisms Saccharomyces cerevisiae (yeast) and Arabidopsis thaliana (plant). Thus we depict evidences of presence/absence of EAA biosynthetic and nitrogen assimilation enzymes at phyla level. Results show broad loss of the phenotype of EAAs biosynthesis in several groups of eukaryotes, followed by multiple secondary gene losses. A subsequent inability for nitrogen assimilation is observed in derived metazoans. Conclusions A Great Deletion model is proposed here as a broad phenomenon generating the phenotype of amino acids essentiality followed, in metazoans, by organic nitrogen dependency. This phenomenon is probably associated to a relaxed selective pressure conferred by heterotrophy and, taking advantage of available homologous clustering tools, a complete and updated picture of it is provided. PMID:22369087

  13. Nitrogen treatment enhances sterols and withaferin A through transcriptional activation of jasmonate pathway, WRKY transcription factors, and biosynthesis genes in Withania somnifera (L.) Dunal.

    Science.gov (United States)

    Pal, Shaifali; Yadav, Akhilesh Kumar; Singh, Anup Kumar; Rastogi, Shubhra; Gupta, Madan Mohan; Verma, Rajesh Kumar; Nagegowda, Dinesh A; Pal, Anirban; Shasany, Ajit Kumar

    2017-01-01

    The medicinal plant Withania somnifera is researched extensively to increase the quantity of withanolides and specifically withaferin A, which finds implications in many pharmacological activities. Due to insufficient knowledge on biosynthesis and unacceptability of transgenic approach, it is preferred to follow alternative physiological methods to increase the yield of withanolides. Prior use of elicitors like salicylic acid, methyl jasmonate, fungal extracts, and even mechanical wounding have shown to increase the withanolide biosynthesis with limited success; however, the commercial viability and logistics of application are debatable. In this investigation, we tested the simple nitrogeneous fertilizers pertaining to the enhancement of withaferin A biosynthesis. Application of ammonium sulfate improved the sterol contents required for the withanolide biosynthesis and correlated to higher expression of pathway genes like FPPS, SMT1, SMT2, SMO1, SMO2, and ODM. Increased expression of a gene homologous to allene oxide cyclase, crucial in jasmonic acid biosynthetic pathway, suggested the involvement of jasmonate signaling. High levels of WRKY gene transcripts indicated transcriptional regulation of the pathway genes. Increase in transcript level could be correlated with a corresponding increase in the protein levels for WsSMT1 and WsWRKY1. The withaferin A increase was also demonstrated in the potted plants growing in the glasshouse and in the open field. These results implicated simple physiological management of nitrogen fertilizer signal to improve the yield of secondary metabolite through probable involvement of jasmonate signal and WRKY transcription factor for the first time, in W. somnifera besides improving the foliage.

  14. Identification and characterization of a complete carnitine biosynthesis pathway in Candida albicans

    NARCIS (Netherlands)

    Strijbis, Karin; van Roermund, Carlo W. T.; Hardy, Guy P.; van den Burg, Janny; Bloem, Karien; de Haan, Jolanda; van Vlies, Naomi; Wanders, Ronald J. A.; Vaz, Frédéric M.; Distel, Ben

    2009-01-01

    Carnitine is an essential metabolite that enables intracellular transport of fatty acids and acetyl units. Here we show that the yeast Candida albicans can synthesize carnitine de novo, and we identify the 4 genes of the pathway. Null mutants of orf19.4316 (trimethyllysine dioxygenase), orf19.6306

  15. A fragrant introduction to terpenoid chemistry

    National Research Council Canada - National Science Library

    Royal Society of Chemistry (Great Britain); Sell, Charles

    2003-01-01

    ... expertise, we will concentrate on the monoand sesquiterpenoids and primarily those of interest as fragrance ingredients. Higher terpenoids will be mentioned and the reader will be able to extrapolate the basic principles of terpenoid chemistry from the more detailed examples using lower terpenoids to these higher homologues. Chemistry i...

  16. Neoproterozoic Oxygenation of Earth Surface Environments Reflected in the Late Evolution of the O2-Dependent Vitamin B12 Biosynthesis Pathway

    Science.gov (United States)

    Saito, M. A.; Bertrand, E. M.; Anbar, A.

    2008-12-01

    There are multiple lines of evidence for a significant rise of O2 in the Earth's atmosphere ~2.4 Ga. A second oxygenation event in the Neoproterozoic is not as well constrained. These changes in environmental redox affected the abundances of bioessential elements. Trace elements such as Co, Fe, and Ni were likely favored in the early evolution of metalloenzymes, prior to the first oxidation event. Consistent with this expectation, vitamin B12 is a Co-containing biomolecule whose biosynthesis is thought to have evolved prior to the origin of oxygenic photosynthesis and the first rise in O2. However, biochemical characterization of the many enzymes involved in B12 biosynthesis has revealed two distinct pathways: an O2-independent pathway and an O2-dependant pathway. The major difference between these pathways involves the timing of the insertion of Co. We examined the amino acid sequences of enzymes in the B12 biosynthesis pathway from a set of 100 phylogenetically diverse microbial genomes, focusing on enzymes exclusive to each pathway as well as enzymes shared by both. Molecular clock and phylogenetic analyses were performed on alignments of the sequences obtained from these study genomes. This approach focused on functional genes rather than the phylogeny of microbes in an attempt to understand the evolution of the pathway itself, rather than its presence in individual phylogenetic groups. Clear differences in age are apparent between representatives of each pathway. The O2-independent pathway and enzymes shared in both pathways show the most ancient last common ancestors. In contrast, the enzymes associated exclusively with the O2-dependent pathway diverged from a common ancestor less than a billion years ago. Phylogenetic analysis suggests that these enzymes were recruited from other biochemical pathways. From these results it seems likely that the evolution of the O2-dependent pathway occurred long after the initial evolution of the B12 biosynthesis. This

  17. Physiological function of IspE, a plastid MEP pathway gene for isoprenoid biosynthesis, in organelle biogenesis and cell morphogenesis in Nicotiana benthamiana.

    Science.gov (United States)

    Ahn, Chang Sook; Pai, Hyun-Sook

    2008-03-01

    Isoprenoid biosynthesis in plants occurs by two independent pathways: the cytosolic mevalonate (MVA) pathway and the plastidic methylerythritol phosphate (MEP) pathway. In this study, we investigated the cellular effects of depletion of IspE, a protein involved in the MEP pathway, using virus-induced gene silencing (VIGS). The IspE gene is preferentially expressed in young tissues, and induced by light and methyl jasmonate. The GFP fusion protein of IspE was targeted to chloroplasts. Reduction of IspE expression by VIGS resulted in a severe leaf yellowing phenotype. At the cellular level, depletion of IspE severely affected chloroplast development, dramatically reducing both the number and size of chloroplasts. Interestingly, mitochondrial development was also impaired, suggesting a possibility that the plastidic MEP pathway contributes to mitochondrial isoprenoid biosynthesis in leaves. A deficiency in IspE activity decreased cellular levels of the metabolites produced by the MEP pathway, such as chlorophylls and carotenoids, and stimulated expression of some of the downstream MEP pathway genes, particularly IspF and IspG. Interestingly, the IspE VIGS lines had significantly increased numbers of cells of reduced size in all leaf layers, compared with TRV control and other VIGS lines for the MEP pathway genes. The increased cell division in the IspE VIGS lines was particularly pronounced in the abaxial epidermal layer, in which the over-proliferated cells bulged out of the plane, making the surface uneven. In addition, trichome numbers dramatically increased and the stomata size varied in the affected tissues. Our results show that IspE deficiency causes novel developmental phenotypes distinct from the phenotypes of other MEP pathway mutants, indicating that IspE may have an additional role in plant development besides its role in isoprenoid biosynthesis.

  18. Biosynthesis of isoprene in Escherichia coli via methylerythritol phosphate (MEP) pathway.

    Science.gov (United States)

    Zhao, Yaru; Yang, Jianming; Qin, Bo; Li, Yonghao; Sun, Yuanzhang; Su, Sizheng; Xian, Mo

    2011-06-01

    Isoprene is an aviation fuel of high quality and an important polymer building block in the synthetic chemistry industry. In light of high oil prices, sustained availability, and environmental concerns, isoprene from renewable materials is contemplated as a substitute for petroleum-based product. Escherichia coli with advantages over other wild microorganisms, is considered as a powerful host for biofuels and chemicals. Here, we constructed a synthetic pathway of isoprene in E. coli by introducing an isoprene synthase (ispS) gene from Populus nigra, which catalyzes the conversion of dimethylallyl diphosphate (DMAPP) to isoprene. To improve the isoprene production, we overexpressed the native 1-deoxy-D: -xylulose-5-phosphate (DXP) synthase gene (dxs) and DXP reductoisomerase gene (dxr) in E. coli, which catalyzed the first step and the second step of MEP pathway, respectively. The fed-batch fermentation results showed that overexpression of DXS is helpful for the improvement of isoprene production. Surprisingly, heterologous expression of dxs and dxr from Bacillus subtilis in the E. coli expressing ispS resulted in a 2.3-fold enhancement of isoprene production (from 94 to 314 mg/L). The promising results showed that dxs and dxr from B. subtilis functioned more efficiently on the enhancement of isoprene production than native ones. This could be caused by the consequence of great difference in protein structures of the two original DXSs. It could be practical to produce isoprene in E. coli via MEP pathway through metabolic engineering. This work provides an alternative way for production of isoprene by engineered E. coli via MEP pathway through metabolic engineering.

  19. Aromatic glucosinolate biosynthesis pathway in Barbarea vulgaris and its response to Plutella xylostella infestation

    DEFF Research Database (Denmark)

    Liu, Tongjin; Zhang, Xiaohui; Yang, Haohui

    2016-01-01

    and the expression profiles of their biosynthesis genes before and after infestation by DBM larvae. Two different stereoisomers of hydroxylated aromatic glucosinolates are dominant in G- and P-type B. vulgaris, respectively, and are induced by DBM. The transcripts of genes in the glucosinolate biosynthesis pathway...... and their corresponding transcription factors were identified from an Illumina dataset of G- and P-type B. vulgaris. Many genes involved or potentially involved in glucosinolate biosynthesis were induced in both plant types. The expression patterns of six DBM induced genes were validated by quantitative PCR (qPCR), while......, showed only 77.50% identity in coding DNA sequences and 65.48% identity in deduced amino acid sequences. The homology to GS-OH in Arabidopsis, DBM induction of the transcript and a series of qPCR and glucosinolate analyses of G-type, P-type and F1 plants indicated that these genes control the production...

  20. Isoprenoid biosynthesis in dandelion latex is enhanced by the overexpression of three key enzymes involved in the mevalonate pathway.

    Science.gov (United States)

    Pütter, Katharina M; van Deenen, Nicole; Unland, Kristina; Prüfer, Dirk; Schulze Gronover, Christian

    2017-05-22

    Latex from the dandelion species Taraxacum brevicorniculatum contains many high-value isoprenoid end products, e.g. triterpenes and polyisoprenes such as natural rubber. The isopentenyl pyrophosphate units required as precursors for these isoprenoids are provided by the mevalonate (MVA) pathway. The key enzyme in this pathway is 3-hydroxy-methyl-glutaryl-CoA reductase (HMGR) and its activity has been thoroughly characterized in many plant species including dandelion. However, two enzymes acting upstream of HMGR have not been characterized in dandelion latex: ATP citrate lyase (ACL), which provides the acetyl-CoA utilized in the MVA pathway, and acetoacetyl-CoA thiolase (AACT), which catalyzes the first step in the pathway to produce acetoacetyl-CoA. Here we isolated ACL and AACT genes from T. brevicorniculatum latex and characterized their expression profiles. We also overexpressed the well-characterized HMGR, ACL and AACT genes from Arabidopsis thaliana in T. brevicorniculatum to determine their impact on isoprenoid end products in the latex. The spatial and temporal expression profiles of T. brevicorniculatum ACL and AACT revealed their pivotal role in the synthesis of precursors necessary for isoprenoid biosynthesis in latex. The overexpression of A. thaliana ACL and AACT and HMGR in T. brevicorniculatum latex resulted in the accumulation of all three enzymes, increased the corresponding enzymatic activities and ultimately increased sterol levels by ~5-fold and pentacyclic triterpene and cis-1,4-isoprene levels by ~2-fold. Remarkably high levels of the triterpene precursor squalene were also detected in the triple-transgenic lines (up to 32 mg/g root dry weight) leading to the formation of numerous lipid droplets which were observed in root cross-sections. We could show the effective expression of up to three transgenes in T. brevicorniculatum latex which led to increased enzymatic activity and resulted in high level squalene accumulation in the dandelion roots

  1. Transcriptional profiling analysis of Penicillium digitatum, the causal agent of citrus green mold, unravels an inhibited ergosterol biosynthesis pathway in response to citral.

    Science.gov (United States)

    OuYang, Qiuli; Tao, Nengguo; Jing, Guoxing

    2016-08-11

    Green mold caused by Penicillium digitatum is the most damaging postharvest diseases of citrus fruit. Previously, we have observed that citral dose-dependently inhibited the mycelial growth of P. digitatum, with the minimum inhibitory concentration (MIC) of 1.78 mg/mL, but the underlying molecular mechanism is barely understood. In this study, the transcriptional profiling of the control and 1/2MIC-citral treated P. digitatum mycelia after 30 min of exposure were analyzed by RNA-Seq. A total of 6355 genes, including 2322 up-regulated and 4033 down-regulated genes, were found to be responsive to citral. These genes were mapped to 155 KEGG pathways, mainly concerning mRNA surveillance, RNA polymerase, RNA transport, aminoacyl-tRNA biosynthesis, ABC transporter, glycolysis/gluconeogenesis, citrate cycle, oxidative phosphorylation, sulfur metabolism, nitrogen metabolism, inositol phosphate metabolism, fatty acid biosynthesis, unsaturated fatty acids biosynthesis, fatty acid metabolism, and steroid biosynthesis. Particularly, citral exposure affected the expression levels of five ergosterol biosynthetic genes (e.g. ERG7, ERG11, ERG6, ERG3 and ERG5), which corresponds well with the GC-MS results, the reduction in ergosterol content, and accumulation of massive lanosterol. In addition, ERG11, the gene responsible for lanosterol 14α-demethylase, was observed to be the key down-regulated gene in response to citral. Our present finding suggests that citral could exhibit its antifungal activity against P. digitatum by the down-regulation of ergosterol biosynthesis.

  2. Bioinspired total syntheses of terpenoids.

    Science.gov (United States)

    Hugelshofer, Cedric L; Magauer, Thomas

    2016-12-20

    Nature's highly efficient routes for constructing natural products have inspired chemists to mimic these processes in a laboratory setting. This Perspective presents some recent examples of conceptually different bioinspired total syntheses of complex terpenoids and thereby aims to highlight the vast benefits offered by bioinspired strategies.

  3. Propiconazole-enhanced hepatic cell proliferation is associated with dysregulation of the cholesterol biosynthesis pathway leading to activation of Erk1/2 through Ras farnesylation

    Energy Technology Data Exchange (ETDEWEB)

    Murphy, Lynea A.; Moore, Tanya; Nesnow, Stephen, E-mail: nesnow.stephen@epa.gov

    2012-04-15

    Propiconazole is a mouse hepatotumorigenic fungicide designed to inhibit CYP51, a key enzyme in the biosynthesis of ergosterol in fungi and is widely used in agriculture to prevent fungal growth. Metabolomic studies in mice revealed that propiconazole increased levels of hepatic cholesterol metabolites and bile acids, and transcriptomic studies revealed that genes within the cholesterol biosynthesis, cholesterol metabolism and bile acid biosyntheses pathways were up-regulated. Hepatic cell proliferation was also increased by propiconazole. AML12 immortalized hepatocytes were used to study propiconazole's effects on cell proliferation focusing on the dysregulation of cholesterol biosynthesis and resulting effects on Ras farnesylation and Erk1/2 activation as a primary pathway. Mevalonate, a key intermediate in the cholesterol biosynthesis pathway, increases cell proliferation in several cancer cell lines and tumors in vivo and serves as the precursor for isoprenoids (e.g. farnesyl pyrophosphate) which are crucial in the farnesylation of the Ras protein by farnesyl transferase. Farnesylation targets Ras to the cell membrane where it is involved in signal transduction, including the mitogen-activated protein kinase (MAPK) pathway. In our studies, mevalonic acid lactone (MVAL), a source of mevalonic acid, increased cell proliferation in AML12 cells which was reduced by farnesyl transferase inhibitors (L-744,832 or manumycin) or simvastatin, an HMG-CoA reductase inhibitor, indicating that this cell system responded to alterations in the cholesterol biosynthesis pathway. Cell proliferation in AML12 cells was increased by propiconazole which was reversed by co-incubation with L-744,832 or simvastatin. Increasing concentrations of exogenous cholesterol muted the proliferative effects of propiconazole and the inhibitory effects of L-733,832, results ascribed to reduced stimulation of the endogenous cholesterol biosynthesis pathway. Western blot analysis of subcellular

  4. Heterologous stable expression of terpenoid biosynthetic genes using the moss Physcomitrella patens

    DEFF Research Database (Denmark)

    Bach, Søren Spanner; King, Brian Christopher; Zhan, Xin

    2014-01-01

    , such as streamlining of large scale Agrobacterium infiltration and upregulation of the upstream pathways, transient in planta heterologous expression quickly reaches limitations when used for production of terpenoids. Stable integration of transgenes into the nuclear genome of the moss Physcomitrella patens has...... and cultivation of transgenic lines, and metabolite analysis of terpenoids produced in transgenic moss lines. We also provide tools for metabolic engineering through genome editing using homologous recombination....

  5. Evidence for a universal pathway of abscisic acid biosynthesis in higher plants from sup 18 O incorporation patterns

    Energy Technology Data Exchange (ETDEWEB)

    Zeevaart, J.A.D.; Heath, T.G.; Gage, D.A. (Michigan State University, East Lansing (USA))

    1989-12-01

    Previous labeling studies of abscisic acid (ABA) with {sup 18}O{sub 2} have been mainly conducted with water-stressed leaves. In this study, {sup 18}O incorporation into ABA of stressed leaves of various species was compared with {sup 18}O labeling of ABA of turgid leaves and of fruit tissue in different stages of ripening. In stressed leaves of all six species investigated, avocado (Persea americana), barley (Hordeum vulgare), bean (Phaseolus vulgaris), cocklebur (Xanthium strumarium), spinach (Spinacia oleracea), and tobacco (Nicotiana tabacum), {sup 18}O was most abundant in the carboxyl group, whereas incorporation of a second and third {sup 18}O in the oxygen atoms on the ring of ABA was much less prominent after 24 h in {sup 18}O{sub 2}. ABA from turgid bean leaves showed significant {sup 18}O incorporation, again with highest {sup 18}O enrichment in the carboxyl group. On the basis of {sup 18}O-labeling patterns observed in ABA from different tissues it is concluded that, despite variations in precusor pool sizes and intermediate turnover rates, there is a universal pathway of ABA biosynthesis in higher plants which involves cleavage of a larger precursor molecule, presumably an oxygenated carotenoid.

  6. Terpene synthases of oregano (Origanum vulgare L.) and their roles in the pathway and regulation of terpene biosynthesis.

    Science.gov (United States)

    Crocoll, Christoph; Asbach, Julia; Novak, Johannes; Gershenzon, Jonathan; Degenhardt, Jörg

    2010-08-01

    The aroma, flavor and pharmaceutical value of cultivated oregano (Origanum vulgare L.) is a consequence of its essential oil which consists mostly of monoterpenes and sesquiterpenes. To investigate the biosynthetic pathway to oregano terpenes and its regulation, we identified and characterized seven terpene synthases, key enzymes of terpene biosynthesis, from two cultivars of O. vulgare. Heterologous expression of these enzymes showed that each forms multiple mono- or sesquiterpene products and together they are responsible for the direct production of almost all terpenes found in O. vulgare essential oil. The correlation of essential oil composition with relative and absolute terpene synthase transcript concentrations in different lines of O. vulgare demonstrated that monoterpene synthase activity is predominantly regulated on the level of transcription and that the phenolic monoterpene alcohol thymol is derived from gamma-terpinene, a product of a single monoterpene synthase. The combination of heterologously-expressed terpene synthases for in vitro assays resulted in blends of mono- and sesquiterpene products that strongly resemble those found in vivo, indicating that terpene synthase expression levels directly control the composition of the essential oil. These results will facilitate metabolic engineering and directed breeding of O. vulgare cultivars with higher quantity of essential oil and improved oil composition.

  7. Expanding metabolic pathway for de novo biosynthesis of the chiral pharmaceutical intermediate L-pipecolic acid in Escherichia coli.

    Science.gov (United States)

    Ying, Hanxiao; Tao, Sha; Wang, Jing; Ma, Weichao; Chen, Kequan; Wang, Xin; Ouyang, Pingkai

    2017-03-27

    The six-carbon circular non-proteinogenic compound L-pipecolic acid is an important chiral drug intermediate with many applications in the pharmaceutical industry. In the present study, we developed a metabolically engineered strain of Escherichia coli for the overproduction of L-pipecolic acid from glucose. The metabolic pathway from L-lysine to L-pipecolic acid was constructed initially by introducing lysine cyclodeaminase (LCD). Next, L-lysine metabolic flux from glucose was amplified by the plasmid-based overexpression of dapA, lysC, and lysA under the control of the strong trc promoter to increase the biosynthetic pool of the precursor L-lysine. Additionally, since the catalytic efficiency of the key enzyme LCD is limited by the cofactor NAD(+), the intracellular pyridine nucleotide concentration was rebalanced by expressing the pntAB gene encoding the transhydrogenase, which elevated the proportion of LCD with bound NAD(+) and enhanced L-pipecolic acid production significantly. Further, optimization of Fe(2+) and surfactant in the fermentation process resulted in 5.33 g/L L-pipecolic acid, with a yield of 0.13 g/g of glucose via fed-batch cultivation. We expanded the metabolic pathway for the synthesis of the chiral pharmaceutical intermediate L-pipecolic acid in E. coli. Using the engineered E. coli, a fast and efficient fermentative production of L-pipecolic acid was achieved. This strategy could be applied to the biosynthesis of other commercially and industrially important chiral compounds containing piperidine rings.

  8. Dehydroepiandrosterone-Regulated Testosterone Biosynthesis via Activation of the ERK1/2 Signaling Pathway in Primary Rat Leydig Cells

    Directory of Open Access Journals (Sweden)

    Lin Liu

    2015-07-01

    Full Text Available Background: Dehydroepiandrosterone decreases with age and this reduction has been shown to be associated with physical health in human. Some studies have suggested that the effects of DHEA are exerted after it is biotransformed into more biologically-active hormones in peripheral target cells. This study investigated the effects of DHEA on the testosterone biosynthesis and possible signaling pathway mechanism underlying these DHEA effects were also explored in primary rat Leydig cells. Methods: Primary Leydig cells were treated with DHEA and then detected testosterone content by RIA and steroidogenic enzymes, ERK1/2 signal pathway factors protein expression level by Western blot. Results: Incubation of primary Leydig cells with DHEA significantly increased testosterone content and 3β-HSD and 17β-HSD protein expression levels, while aromatase protein expression levels were decreased. Compared with the control group, p-ERK1/2 and p-CREB protein levels were significantly increased in DHEA-treated groups. Testosterone content was significantly decreased in the DHEA-treated group pre-incubated with U0126 (p-ERK1/2 inhibitor. Additionally, the rise in p-ERK1/2, 3β-HSD and 17β-HSD protein levels induced by DHEA was reversed when cells were pre-incubated with U0126. Interestingly, no significant difference was found in aromatase protein expression level in cells pretreated with U0126. Conclusion: These findings demonstrate that (a exogenous DHEA might preferentially convert to testosterone rather than estradiol due to the up-regulation of 3β-HSD and 17β-HSD protein levels and the down-regulation of aromatase protein level in primary Leydig cells, and (b the action of DHEA is at least partly associated with the elevation of p-ERK1/2 and p-CREB protein levels.

  9. Dehydroepiandrosterone-Regulated Testosterone Biosynthesis via Activation of the ERK1/2 Signaling Pathway in Primary Rat Leydig Cells.

    Science.gov (United States)

    Liu, Lin; Kang, Jian; Ding, Xiao; Chen, Di; Zhou, Yingqiao; Ma, Haitian

    2015-01-01

    Dehydroepiandrosterone decreases with age and this reduction has been shown to be associated with physical health in human. Some studies have suggested that the effects of DHEA are exerted after it is biotransformed into more biologically-active hormones in peripheral target cells. This study investigated the effects of DHEA on the testosterone biosynthesis and possible signaling pathway mechanism underlying these DHEA effects were also explored in primary rat Leydig cells. Primary Leydig cells were treated with DHEA and then detected testosterone content by RIA and steroidogenic enzymes, ERK1/2 signal pathway factors protein expression level by Western blot. Incubation of primary Leydig cells with DHEA significantly increased testosterone content and 3β-HSD and 17β-HSD protein expression levels, while aromatase protein expression levels were decreased. Compared with the control group, p-ERK1/2 and p-CREB protein levels were significantly increased in DHEA-treated groups. Testosterone content was significantly decreased in the DHEA-treated group pre-incubated with U0126 (p-ERK1/2 inhibitor). Additionally, the rise in p-ERK1/2, 3β-HSD and 17β-HSD protein levels induced by DHEA was reversed when cells were pre-incubated with U0126. Interestingly, no significant difference was found in aromatase protein expression level in cells pretreated with U0126. These findings demonstrate that (a) exogenous DHEA might preferentially convert to testosterone rather than estradiol due to the up-regulation of 3β-HSD and 17β-HSD protein levels and the down-regulation of aromatase protein level in primary Leydig cells, and (b) the action of DHEA is at least partly associated with the elevation of p-ERK1/2 and p-CREB protein levels. Copyright © 2015 S. Karger AG, Basel

  10. The chitin biosynthesis pathway in Entamoeba and the role of glucosamine-6-P isomerase by RNA interference.

    Science.gov (United States)

    Samanta, Sintu Kumar; Ghosh, Sudip K

    2012-11-01

    Entamoeba histolytica, the causative agent of amoebiasis, infects through its cyst form. A thick chitin wall protects the cyst from the harsh environment outside of the body. It is known that chitin is synthesized only during encystation, but the chitin synthesis pathway (CSP) of Entamoeba is not well characterized. In this report, we have identified the genes involved in chitin biosynthesis from the Entamoeba genome database and verified their expression profile at the transcriptional level in encysting Entamoeba invadens. Semi-quantitative RT-PCR (sqRT-PCR) analysis showed that all the chitin pathway genes are entirely absent or transcribed at low levels in trophozoites. The mRNA expression of most of the CSP genes reached their maximum level between 9 and 12h after the in vitro initiation of encystation. Double-stranded RNA-mediated silencing of glucosamine-6-P isomerase (Gln6Pi) reduced chitin synthesis to 62-64%, which indicates that Gln6Pi might be a key enzyme for regulating chitin synthesis in Entamoeba. The study of different enzymes involved in glycogen metabolism revealed that stored glycogen is converted to glucose during encystation. It is clear from the sqRT-PCR analysis that the rate of glycolysis decreases as encystation proceeds. Encystation up-regulates the expression of glycogen phosphorylase, which is responsible for glycogen degradation. The significant decrease in chitin synthesis in encysting cells treated with a specific inhibitor of glycogen phosphorylase indicates that the glucose obtained from the degradation of stored glycogen in trophozoites might be one of the major sources of glucose for chitin synthesis. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Inhibition of tumor progression by naturally occurring terpenoids.

    Science.gov (United States)

    Kuttan, Girija; Pratheeshkumar, Poyil; Manu, Kanjoormana Aryan; Kuttan, Ramadasan

    2011-10-01

    Cancer is a major public health problem in India and many other parts of the world. Its two main characteristics are uncontrolled cell growth and metastasis. Natural products represent a rich source of compounds that have found many applications in various fields of medicines and therapy including cancer therapy. Effective ingredients in several plant-derived medicinal extracts are terpenoid compounds and many terpenes have biological activities and are used for the treatment of human diseases. This review attempted to collect all available published scientific literature of eight naturally occurring terpenoids and their effect on inhibition of tumor progression. The present review is about eight potent naturally occurring terpenoids that have been studied for their pharmacological properties in our lab and this review includes 130 references compiled from all major databases. Literature survey revealed that triterpenoids, such as glycyrrhizic acid, ursolic acid, oleanolic acid, and nomilin, the diterpene andrographolide, and the monoterpenoids like limonene and perillic acid had shown immunomodulatory and antitumor activities. All of them could induce apoptosis in various cancer cells by activating various proapoptotic signaling cascades. Many of these terpenoids found to inhibit metastatic progression and tumor-induced angiogenesis. The molecular mechanisms that involved in these activities include inhibition of various oncogenic and anti-apoptotic signaling pathways and suppression or nuclear translocation of various transcription factors including nuclear factor kappa B (NF-κB). The chemopreventive and chemoprotective effects of these compounds point toward their possible role in modern anticancer therapies.

  12. Development of a Terpenoid-Production Platform in Streptomyces reveromyceticus SN-593.

    Science.gov (United States)

    Khalid, Ammara; Takagi, Hiroshi; Panthee, Suresh; Muroi, Makoto; Chappell, Joe; Osada, Hiroyuki; Takahashi, Shunji

    2017-12-15

    Terpenoids represent the largest class of natural products, some of which are resources for pharmaceuticals, fragrances, and fuels. Generally, mass production of valuable terpenoid compounds is hampered by their low production levels in organisms and difficulty of chemical synthesis. Therefore, the development of microbial biosynthetic platforms represents an alternative approach. Although microbial terpenoid-production platforms have been established in Escherichia coli and yeast, an optimal platform has not been developed for Streptomyces species, despite the large capacity to produce secondary metabolites, such as polyketide compounds. To explore this potential, we constructed a terpenoid-biosynthetic platform in Streptomyces reveromyceticus SN-593. This strain is unique in that it harbors the mevalonate gene cluster enabling the production of furaquinocin, which can be controlled by the pathway specific regulator Fur22. We simultaneously expressed the mevalonate gene cluster and subsequent terpenoid-biosynthetic genes under the control of Fur22. To achieve improved fur22 gene expression, we screened promoters from S. reveromyceticus SN-593. Our results showed that the promoter associated with rvr2030 gene enabled production of 212 ± 20 mg/L botryococcene to levels comparable to those previously reported for other microbial hosts. Given that the rvr2030 gene encodes for an enzyme involved in the primary metabolism, these results suggest that optimized expression of terpenoid-biosynthetic genes with primary and secondary metabolism might be as important for high yields of terpenoid compounds as is the absolute expression level of a target gene(s).

  13. Biosynthesis of rice seed alpha-amylase: two pathways of amylase secretion by the scutellum.

    Science.gov (United States)

    Mitsui, T; Akazawa, T; Christeller, J T; Tartakoff, A M

    1985-08-15

    The alpha-amylase molecule secreted from the scutellar tissues of rice seedlings bears asparagine-linked oligosaccharides which include both (modified) complex-type and high-mannose-type structures. On the basis of their sensitivity to endo-beta-N-acetylglucosaminidase (Endo-beta-H), they are designated as R and S types. When labeled with [3H]fucose a typical R-type alpha-amylase is labeled. By contrast, [3H]mannose-labeled alpha-amylase can be partly digested by Endo-beta-H; hence, it contains both R and S molecules. The role of the Golgi complex in the post-translational oligosaccharide maturation of alpha-amylase was explored by use of the carboxylic ionophore, monensin (10(-7)M), a known perturbant of the structure and function of the Golgi complex. The monensin sensitivity of alpha-amylase transport and acquisition of terminal sugars as well as the morphologic consequences of monensin treatment point to a similarity between the Golgi complex of plant and animal cells. In order to elucidate the relationship between the secretion of two different forms of alpha-amylase and the partial inhibitory effect exerted by monensin, the possible role of Ca2+ in the secretory pathway was examined. The secretion of the R form was stimulated by Ca2+, whereas that of the S form was not affected by the external concentration of Ca2+. In pulse-chase experiments, we found that R-type alpha-amylase accumulates intracellularly under Ca2+-free conditions. These results indicate that there is both Ca2+-dependent and Ca2+-independent secretion of alpha-amylase in the rice scutellar epithelium cells.

  14. Effects of Exogenous Salicylic Acid on Ganoderic Acid Biosynthesis and the Expression of Key Genes in the Ganoderic Acid Biosynthesis Pathway in the Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes).

    Science.gov (United States)

    Cao, Peng-Fei; Wu, Chen-Gao; Dang, Zhi-Hao; Shi, Liang; Jiang, Ai-Liang; Ren, Ang; Zhao, Ming-Wen

    2017-01-01

    We demonstrate herein that salicylic acid (SA) can enhance ganoderic acid (GA) accumulation in the lingzhi or reishi medicinal mushroom Ganoderma lucidum. Following treatment with different concentrations of SA, the GA content was increased 22.72% to 43.04% compared with the control group. When the fungi were treated with 200 μmol/L SA at different times, the GA content was improved 10.21% to 35.24% compared with the control group. By choosing the optimum point based on response surface methodology, the GA content could be increased up to 229.03 μg/100 mg, which was improved 66.38% compared with the control group. When the fungi were treated with 200 μmol/L SA, the transcription levels of key genes in the GA biosynthesis pathway-squalene (SQ) synthase (sqs), lanosterol (Lano; osc), and hydroxy-3-methylglutaryl-coenzyme A reductase (hmgr)-were improved 119.6-, 3.2-, and 4.2-fold, respectively. In addition, following treatment with 100 μmol/L SA, the levels of Lano and SQ, which are intermediate metabolites of GA biosynthesis, were increased 2.8- and 1.4-fold, respectively. These results indicate that SA can regulate the expression of genes related to GA biosynthesis and increases the metabolic levels of Lano and SQ, thereby resulting in the accumulation of GA.

  15. A distinct difference in the metabolic stimulus-response coupling pathways for regulating proinsulin biosynthesis and insulin secretion that lies at the level of a requirement for fatty acyl moieties

    National Research Council Canada - National Science Library

    Skelly, R H; Bollheimer, L C; Wicksteed, B L; Corkey, B E; Rhodes, C J

    1998-01-01

    ... of divergence in the respective metabolic stimulus-response coupling pathways must exist. A parallel examination of the metabolic regulation of proinsulin biosynthesis and insulin secretion was undertaken in the same beta-cells...

  16. Transcriptome Analysis Reveals Putative Genes Involved in Iridoid Biosynthesis in Rehmannia glutinosa

    Directory of Open Access Journals (Sweden)

    Xianen Li

    2012-10-01

    Full Text Available Rehmannia glutinosa, one of the most widely used herbal medicines in the Orient, is rich in biologically active iridoids. Despite their medicinal importance, no molecular information about the iridoid biosynthesis in this plant is presently available. To explore the transcriptome of R. glutinosa and investigate genes involved in iridoid biosynthesis, we used massively parallel pyrosequencing on the 454 GS FLX Titanium platform to generate a substantial EST dataset. Based on sequence similarity searches against the public sequence databases, the sequences were first annotated and then subjected to Gene Ontology (GO and Kyoto Encyclopedia of Genes and Genomes (KEGG based analysis. Bioinformatic analysis indicated that the 454 assembly contained a set of genes putatively involved in iridoid biosynthesis. Significantly, homologues of the secoiridoid pathway genes that were only identified in terpenoid indole alkaloid producing plants were also identified, whose presence implied that route II iridoids and route I iridoids share common enzyme steps in the early stage of biosynthesis. The gene expression patterns of four prenyltransferase transcripts were analyzed using qRT-PCR, which shed light on their putative functions in tissues of R. glutinosa. The data explored in this study will provide valuable information for further studies concerning iridoid biosynthesis.

  17. Investigating sesquiterpene biosynthesis in Ginkgo biloba: molecular cloning and functional characterization of (E,E)-farnesol and α-bisabolene synthases.

    Science.gov (United States)

    Parveen, Iffat; Wang, Mei; Zhao, Jianping; Chittiboyina, Amar G; Tabanca, Nurhayat; Ali, Abbas; Baerson, Scott R; Techen, Natascha; Chappell, Joe; Khan, Ikhlas A; Pan, Zhiqiang

    2015-11-01

    Ginkgo biloba is one of the oldest living tree species and has been extensively investigated as a source of bioactive natural compounds, including bioactive flavonoids, diterpene lactones, terpenoids and polysaccharides which accumulate in foliar tissues. Despite this chemical diversity, relatively few enzymes associated with any biosynthetic pathway from ginkgo have been characterized to date. In the present work, predicted transcripts potentially encoding enzymes associated with the biosynthesis of diterpenoid and terpenoid compounds, including putative terpene synthases, were first identified by mining publicly-available G. biloba RNA-seq data sets. Recombinant enzyme studies with two of the TPS-like sequences led to the identification of GbTPS1 and GbTPS2, encoding farnesol and bisabolene synthases, respectively. Additionally, the phylogenetic analysis revealed the two terpene synthase genes as primitive genes that might have evolved from an ancestral diterpene synthase.

  18. Role of the Colletotrichum acutatum sesquiterpene synthase CaTPS in the biosynthesis of sesquiterpenoids

    DEFF Research Database (Denmark)

    Amby, Daniel Buchvaldt; Manczak, Tom; Petersen, Mikael Agerlin

    2016-01-01

    biosynthesis is performed by sesquiterpene synthases (TPS). Only a few TPSs have been functionally characterized from filamentous fungi and none from the genus Colletotrichum. Despite being an important fungal pathogen to agriculture, it is poorly understood at the molecular and chemical levels. The terpenoid...... characterization of TPS in Colletotrichum spp. and terpenoid profiles of Coll. acutatum, which could facilitate studies on the role of terpenoids in the ecology of Coll. acutatum....

  19. Isoprenoid biosynthesis via 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (DOXP/MEP) pathway.

    Science.gov (United States)

    Wanke, M; Skorupinska-Tudek, K; Swiezewska, E

    2001-01-01

    Higher plants, several algae, bacteria, some strains of Streptomyces and possibly malaria parasite Plasmodium falciparum contain the novel, plastidic DOXP/MEP pathway for isoprenoid biosynthesis. This pathway, alternative with respect to the classical mevalonate pathway, starts with condensation of pyruvate and glyceraldehyde-3-phosphate which yields 1-deoxy-D-xylulose 5-phosphate (DOXP); the latter product can be converted to isopentenyl diphosphate (IPP) and eventually to isoprenoids or thiamine and pyridoxal. Subsequent reactions of this pathway involve transformation of DOXP to 2-C-methyl-D-erythritol 4-phosphate (MEP) which after condensation with CTP forms 4-diphosphocytidyl-2-amethyl-D-erythritol (CDP-ME). Then CDP-ME is phosphorylated to 4-diphosphocytidyl-2-amethyl-D-erythritol 2-phosphate (CDP-ME2P) and to 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (ME-2,4cPP) which is the last known intermediate of the DOXP/MEP pathway. For- mation of IPP and dimethylallyl diphosphate (DMAPP) from ME-2,4cPP still requires clarification. This novel pathway appears to be involved in biosynthesis of carotenoids, phytol (side chain of chlorophylls), isoprene, mono-, di-, tetraterpenes and plastoquinone whereas the mevalonate pathway is responsible for formation of sterols, sesquiterpenes and triterpenes. Several isoprenoids were found to be of mixed origin suggesting that some exchange and/or cooperation exists between these two pathways of different biosynthetic origin. Contradictory results described below could indicate that these two pathways are operating under different physiological conditions of the cell and are dependent on the developmental state of plastids.

  20. RNA-seq based transcriptomic analysis uncovers α-linolenic acid and jasmonic acid biosynthesis pathways respond to cold acclimation in Camellia japonica.

    Science.gov (United States)

    Li, Qingyuan; Lei, Sheng; Du, Kebing; Li, Lizhi; Pang, Xufeng; Wang, Zhanchang; Wei, Ming; Fu, Shao; Hu, Limin; Xu, Lin

    2016-11-07

    Camellia is a well-known ornamental flower native to Southeast of Asia, including regions such as Japan, Korea and South China. However, most species in the genus Camellia are cold sensitive. To elucidate the cold stress responses in camellia plants, we carried out deep transcriptome sequencing of 'Jiangxue', a cold-tolerant cultivar of Camellia japonica, and approximately 1,006 million clean reads were generated using Illumina sequencing technology. The assembly of the clean reads produced 367,620 transcripts, including 207,592 unigenes. Overall, 28,038 differentially expressed genes were identified during cold acclimation. Detailed elucidation of responses of transcription factors, protein kinases and plant hormone signalling-related genes described the interplay of signal that allowed the plant to fine-tune cold stress responses. On the basis of global gene regulation of unsaturated fatty acid biosynthesis- and jasmonic acid biosynthesis-related genes, unsaturated fatty acid biosynthesis and jasmonic acid biosynthesis pathways were deduced to be involved in the low temperature responses in C. japonica. These results were supported by the determination of the fatty acid composition and jasmonic acid content. Our results provide insights into the genetic and molecular basis of the responses to cold acclimation in camellia plants.

  1. De novo assembly and transcriptome analysis of the rubber tree (Hevea brasiliensis) and SNP markers development for rubber biosynthesis pathways.

    Science.gov (United States)

    Mantello, Camila Campos; Cardoso-Silva, Claudio Benicio; da Silva, Carla Cristina; de Souza, Livia Moura; Scaloppi Junior, Erivaldo José; de Souza Gonçalves, Paulo; Vicentini, Renato; de Souza, Anete Pereira

    2014-01-01

    Hevea brasiliensis (Willd. Ex Adr. Juss.) Muell.-Arg. is the primary source of natural rubber that is native to the Amazon rainforest. The singular properties of natural rubber make it superior to and competitive with synthetic rubber for use in several applications. Here, we performed RNA sequencing (RNA-seq) of H. brasiliensis bark on the Illumina GAIIx platform, which generated 179,326,804 raw reads on the Illumina GAIIx platform. A total of 50,384 contigs that were over 400 bp in size were obtained and subjected to further analyses. A similarity search against the non-redundant (nr) protein database returned 32,018 (63%) positive BLASTx hits. The transcriptome analysis was annotated using the clusters of orthologous groups (COG), gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Pfam databases. A search for putative molecular marker was performed to identify simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs). In total, 17,927 SSRs and 404,114 SNPs were detected. Finally, we selected sequences that were identified as belonging to the mevalonate (MVA) and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways, which are involved in rubber biosynthesis, to validate the SNP markers. A total of 78 SNPs were validated in 36 genotypes of H. brasiliensis. This new dataset represents a powerful information source for rubber tree bark genes and will be an important tool for the development of microsatellites and SNP markers for use in future genetic analyses such as genetic linkage mapping, quantitative trait loci identification, investigations of linkage disequilibrium and marker-assisted selection.

  2. Evaluation of biosynthetic pathways to delta-aminolevulinic acid in Propionibacterium shermanii based on biosynthesis of vitamin B12 from D-[1-13C]glucose.

    Science.gov (United States)

    Iida, K; Kajiwara, M

    2000-04-04

    Analysis of the (13)C nuclear magnetic resonance (NMR) spectrum of (13)C-labeled vitamin B(12) biosynthesized from D-[1-(13)C]glucose by Propionibacterium shermanii provided evidence suggesting that delta-aminolevulinic acid (ALA) incorporated in the (13)C-labeled vitamin B(12) may have been synthesized via both the Shemin pathway and the C5 pathway under anaerobic conditions in the ratio of 1 < [(ratio of ALA biosynthesis from the Shemin pathway)/(that from the C5 pathway)] < 1.8. The D-ribose moiety of vitamin B(12) was labeled with (13)C at R-1, R-3, and R-5. The aminopropanol moiety of vitamin B(12) was labeled on Pr-1 and Pr-2, but not Pr-3.

  3. A comprehensive proteome map of glandular trichomes of hop (Humulus lupulus L.) female cones: Identification of biosynthetic pathways of the major terpenoid-related compounds and possible transport proteins.

    Science.gov (United States)

    Champagne, Antoine; Boutry, Marc

    2017-04-01

    Female flowers of hop (Humulus lupulus) are an essential source of terpenoid-related compounds, which are mainly used as flavoring in the beer brewing process. The compounds involved are bitter acids, terpenophenolics, as well as mono- and sesquiterpenoids. In this work, we analyzed the proteome of purified glandular trichomes (lupulin glands) from female flowers, which produce and accumulate these compounds. An extensive 2D-LC-MS/MS analysis identified 1015 proteins. Of these, most correspond to housekeeping and primary metabolism-related proteins, albeit predominantly including amino acid and lipid metabolism, which feeds the specialized (secondary) metabolism. Indeed, 75 proteins belong to the specialized metabolism. No less than 40 enzymes are involved in the synthesis of terpenoid-derived compounds and 21 are predicted transporters, some of which might be involved in the transport of specialized metabolites. We discuss the possible routes involved in the intra- and intercellular translocation of terpenoids and their precursors. This comprehensive proteomic map of the glandular trichomes of hop female flowers represents a valuable resource to improve our knowledge on the function of glandular trichomes. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. A model of flux regulation in the cholesterol biosynthesis pathway: Immune mediated graduated flux reduction versus statin-like led stepped flux reduction.

    Science.gov (United States)

    Watterson, Steven; Guerriero, Maria Luisa; Blanc, Mathieu; Mazein, Alexander; Loewe, Laurence; Robertson, Kevin A; Gibbs, Holly; Shui, Guanghou; Wenk, Markus R; Hillston, Jane; Ghazal, Peter

    2013-03-01

    The cholesterol biosynthesis pathway has recently been shown to play an important role in the innate immune response to viral infection with host protection occurring through a coordinate down regulation of the enzymes catalysing each metabolic step. In contrast, statin based drugs, which form the principle pharmaceutical agents for decreasing the activity of this pathway, target a single enzyme. Here, we build an ordinary differential equation model of the cholesterol biosynthesis pathway in order to investigate how the two regulatory strategies impact upon the behaviour of the pathway. We employ a modest set of assumptions: that the pathway operates away from saturation, that each metabolite is involved in multiple cellular interactions and that mRNA levels reflect enzyme concentrations. Using data taken from primary bone marrow derived macrophage cells infected with murine cytomegalovirus or treated with IFNγ, we show that, under these assumptions, coordinate down-regulation of enzyme activity imparts a graduated reduction in flux along the pathway. In contrast, modelling a statin-like treatment that achieves the same degree of down-regulation in cholesterol production, we show that this delivers a step change in flux along the pathway. The graduated reduction mediated by physiological coordinate regulation of multiple enzymes supports a mechanism that allows a greater level of specificity, altering cholesterol levels with less impact upon interactions branching from the pathway, than pharmacological step reductions. We argue that coordinate regulation is likely to show a long-term evolutionary advantage over single enzyme regulation. Finally, the results from our models have implications for future pharmaceutical therapies intended to target cholesterol production with greater specificity and fewer off target effects, suggesting that this can be achieved by mimicking the coordinated down-regulation observed in immunological responses. Copyright © 2012

  5. Complexity generation in fungal polyketide biosynthesis: a spirocycle-forming P450 in the concise pathway to the antifungal drug griseofulvin.

    Science.gov (United States)

    Cacho, Ralph A; Chooi, Yit-Heng; Zhou, Hui; Tang, Yi

    2013-10-18

    Griseofulvin (1) is a spirocyclic fungal natural product used in treatment of fungal dermatophytes. Formation of the spirocycle, or the grisan scaffold, from a benzophenone precursor is critical for the activity of 1. In this study, we have systematically characterized each of the biosynthetic enzymes related to the biogenesis of 1, including the characterization of a new polyketide synthase GsfA that synthesizes the benzophenone precursor and a cytochrome P450 GsfF that performs oxidative coupling between the orcinol and the phloroglucinol rings to yield the grisan structure. Notably, the finding of GsfF is in sharp contrast to the copper-dependent dihydrogeodin oxidase that performs a similar reaction in the geodin biosynthetic pathway. The biosynthetic knowledge enabled the in vitro total biosynthesis of 1 from malonyl-CoA using all purified enzyme components. This work therefore completely maps out the previously unresolved enzymology of the biosynthesis of a therapeutically relevant natural product.

  6. Proteomic analysis of conidia germination in Fusarium oxysporum f. sp. cubense tropical race 4 reveals new targets in ergosterol biosynthesis pathway for controlling Fusarium wilt of banana.

    Science.gov (United States)

    Deng, Gui-Ming; Yang, Qiao-Song; He, Wei-Di; Li, Chun-Yu; Yang, Jing; Zuo, Cun-Wu; Gao, Jie; Sheng, Ou; Lu, Shao-Yun; Zhang, Sheng; Yi, Gan-Jun

    2015-09-01

    Conidial germination is a crucial step of the soilborne fungus Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), a most important lethal disease of banana. In this study, a total of 3659 proteins were identified by isobaric tags for relative and absolute quantitation (iTRAQ)-based comparative proteomic approach, of which 1009 were differentially expressed during conidial germination of the fungus at 0, 3, 7, and 11 h. Functional classification and bioinformatics analysis revealed that the majority of the differentially expressed proteins are involved in six metabolic pathways. Particularly, all differential proteins involved in the ergosterol biosynthesis pathway were significantly upregulated, indicating the importance of the ergosterol biosynthesis pathway to the conidial germination of Foc TR4. Quantitative RT-PCR, western blotting, and in vitro growth inhibition assay by several categories of fungicides on the Foc TR4 were used to validate the proteomics results. Four enzymes, C-24 sterol methyltransferase (ERG6), cytochrome P450 lanosterol C-14α-demethylase (EGR11), hydroxymethylglutaryl-CoA synthase (ERG13), and C-4 sterol methyl oxidase (ERG25), in the ergosterol biosynthesis pathway were identified and verified, and they hold great promise as new targets for effective inhibition of Foc TR4 early growth in controlling Fusarium wilt of banana. To the best of our knowledge, this report represents the first comprehensive study on proteomics profiling of conidia germination in Foc TR4. It provides new insights into a better understanding of the developmental processes of Foc TR4 spores. More importantly, by host plant-induced gene silencing (HIGS) technology, the new targets reported in this work allow us to develop novel transgenic banana leading to high protection from Fusarium wilt and to explore more effective antifungal drugs against either individual or multiple target proteins of Foc TR4.

  7. Regulation of melanin biosynthesis via the dihydroxynaphthalene pathway is dependent on sexual development in the ascomycete Sordaria macrospora.

    Science.gov (United States)

    Engh, Ines; Nowrousian, Minou; Kück, Ulrich

    2007-10-01

    The filamentous ascomycete Sordaria macrospora accumulates melanin during sexual development. The four melanin biosynthesis genes pks, teh, sdh and tih were isolated and their homology to genes involved in 1,8 dihydroxynaphthalene (DHN) melanin biosynthesis was shown. The presence of DHN melanin in S. macrospora was further confirmed by disrupting the pks gene encoding a putative polyketide synthase and by RNA interference-mediated silencing of the sdh gene encoding a putative scytalone dehydratase. Because melanin occurs in fruiting bodies that develop through several intermediate stages within 7 days of growth, a Northern analysis of a developmental time-course was conducted. These data revealed a time-dependent regulation of teh and sdh transcript levels. Comparing the transcriptional expression by real-time PCR of melanin biosynthesis genes in the wild type under conditions allowing or repressing sexual development, a significant downregulation during vegetative growth was detected. Quantitative real-time PCR and Northern blot analysis of melanin biosynthesis gene expression in different developmental mutants confirmed that melanin biosynthesis is linked to fruiting body development and is under the control of specific regulatory genes that participate in sexual differentiation.

  8. Cytokinin and auxin effect on the terpenoid profile of the essential oil and morphological characteristics of shoot cultures of Artemisia alba.

    Science.gov (United States)

    Danova, Kalina; Todorova, Milka; Trendafilova, Antoaneta; Evstatieva, Ljuba

    2012-08-01

    The influence of plant growth regulators (PGR) on the essential oil composition and in vitro development of A. alba shoot cultures was studied. Two types of oils were determined, based on their terpenoid content. Close relations between the morphogenetic effects of PGR and the essential oil profile of the species were observed Predominance of root over shoot development was connected with a drop in the amounts of sesquiterpenoids and the direction of biosynthesis towards oxygenated monoterpenoids in the PGR-free control medium, as well in those with 1.0 mg L(-1) indole-3-butyric acid (IBA) supplementation. On the contrary, lack of root formation and stimulated callusogenesis caused by the addition of 0.2 mg L(-1) 6-benzyladenine (BA), irrespectively of the presence of IBA (either 0.5 mg L(-1) or 1.0 mg L(-1)), resulted in the strong prevalence of sesquiterpenoids in the oils. These results are indicative that the morphological development ofA. alba shoot cultures affects the terpenoid biosynthetic pathway bringing out the hypothesis for a possible root to shoot signaling.

  9. Combined effect of water loss and wounding stress on gene activation of metabolic pathways associated with phenolic biosynthesis in carrot

    Directory of Open Access Journals (Sweden)

    Alejandro eBecerra-Moreno

    2015-10-01

    Full Text Available Abstract: The application of postharvest abiotic stresses is an effective strategy to activate the primary and secondary metabolism of plants inducing the accumulation of antioxidant phenolic compounds. In the present study, the effect of water stress applied alone and in combination with wounding stress on the activation of primary (shikimic acid and secondary (phenylpropanoid metabolic pathways related with the accumulation of phenolic compound in plants was evaluated. Carrot (Daucus carota was used as model system for this study, and the effect of abiotic stresses was evaluated at the gene expression level and on the accumulation of metabolites. As control of the study, whole carrots were stored under the same conditions. Results demonstrated that water stress activated the primary and secondary metabolism of carrots, favoring the lignification process. Likewise, wounding stress induced higher activation of the primary and secondary metabolism of carrots as compared to water stress alone, leading to higher accumulation of shikimic acid, phenolic compounds and lignin. Additional water stress applied on wounded carrots exerted a synergistic effect on the wound-response at the gene expression level. For instance, when wounded carrots were treated with water stress, the tissue showed 20- and 14-fold increases in the relative expression of 3-deoxy-D-arabino-heptulosanate synthase and phenylalanine ammonia-lyase genes, respectively. However, since lignification was increased, lower accumulation of phenolic compounds was detected. Indicatively, at 48 h of storage, wounded carrots treated with water stress showed ~31% lower levels of phenolic compounds and ~23% higher lignin content as compared with wounded controls. In the present study, it was demonstrated that water stress is one of the pivotal mechanism of the wound-response in carrot. Results allowed the elucidation of strategies to induce the accumulation of specific primary or secondary

  10. Location of chlorogenic acid biosynthesis pathway and polyphenol oxidase genes in a new interspecific anchored linkage map of eggplant.

    Science.gov (United States)

    Gramazio, Pietro; Prohens, Jaime; Plazas, Mariola; Andújar, Isabel; Herraiz, Francisco Javier; Castillo, Elena; Knapp, Sandra; Meyer, Rachel S; Vilanova, Santiago

    2014-12-10

    Eggplant is a powerful source of polyphenols which seems to play a key role in the prevention of several human diseases, such as cancer and diabetes. Chlorogenic acid is the polyphenol most present in eggplant, comprising between the 70% and 90% of the total polyphenol content. Introduction of the high chlorogenic acid content of wild relatives, such as S. incanum, into eggplant varieties will be of great interest. A potential side effect of the increased level polyphenols could be a decrease on apparent quality due to browning caused by the polyphenol oxidase enzymes mediated oxidation of polyphenols. We report the development of a new interspecific S. melongena × S. incanum linkage map based on a first backcross generation (BC1) towards the cultivated S. melongena as a tool for introgressing S. incanum alleles involved in the biosynthesis of chlorogenic acid in the genetic background of S. melongena. The interspecific genetic linkage map of eggplant developed in this work anchor the most informative previously published genetic maps of eggplant using common markers. The 91 BC1 plants of the mapping population were genotyped with 42 COSII, 99 SSRs, 88 AFLPs, 9 CAPS, 4 SNPs and one morphological polymorphic markers. Segregation marker data resulted in a map encompassing 1085 cM distributed in 12 linkage groups. Based on the syntheny with tomato, the candidate genes involved in the core chlorogenic acid synthesis pathway in eggplant (PAL, C4H, 4CL, HCT, C3'H, HQT) as well as five polyphenol oxidase (PPO1, PPO2, PPO3, PPO4, PPO5) were mapped. Except for 4CL and HCT chlorogenic acid genes were not linked. On the contrary, all PPO genes clustered together. Candidate genes important in domestication such as fruit shape (OVATE, SISUN1) and prickliness were also located. The achievements in location of candidate genes will allow the search of favorable alleles employing marker-assisted selection in order to develop new varieties with higher chlorogenic content

  11. Biosynthesis, elicitation and roles of monocot terpenoid phytoalexins

    Science.gov (United States)

    A long standing goal in plant defense research is to optimize the protective function of biochemicals that impede pathogen and pest attack. Nearly 40 years ago pathogen-inducible non-volatile diterpenoids were described in rice and demonstrated to function as potent antimicrobial phytoalexins. Using...

  12. Bioconversion and combinatorial biosynthesis of selected terpenoids and lignans

    NARCIS (Netherlands)

    Julsing, Mattijs Kamiel

    2006-01-01

    Een aanzienlijk deel van de huidige geneesmiddelen is oorspronkelijk een natuurstof of een afgeleide hiervan. Hoewel eenvoudige verbindingen chemisch kunnen worden nagemaakt, is voor complexe verbindingen de chemische synthese lastig en economisch niet rendabel, waardoor we afhankelijk zijn van de

  13. The phosphorylated pathway of serine biosynthesis is essential both for male gametophyte and embryo development and for root growth in Arabidopsis.

    Science.gov (United States)

    Cascales-Miñana, Borja; Muñoz-Bertomeu, Jesús; Flores-Tornero, María; Anoman, Armand Djoro; Pertusa, José; Alaiz, Manuel; Osorio, Sonia; Fernie, Alisdair R; Segura, Juan; Ros, Roc

    2013-06-01

    This study characterizes the phosphorylated pathway of Ser biosynthesis (PPSB) in Arabidopsis thaliana by targeting phosphoserine phosphatase (PSP1), the last enzyme of the pathway. Lack of PSP1 activity delayed embryo development, leading to aborted embryos that could be classified as early curled cotyledons. The embryo-lethal phenotype of psp1 mutants could be complemented with PSP1 cDNA under the control of Pro35S (Pro35S:PSP1). However, this construct, which was poorly expressed in the anther tapetum, did not complement mutant fertility. Microspore development in psp1.1/psp1.1 Pro35S:PSP1 arrested at the polarized stage. The tapetum from these lines displayed delayed and irregular development. The expression of PSP1 in the tapetum at critical stages of microspore development suggests that PSP1 activity in this cell layer is essential in pollen development. In addition to embryo death and male sterility, conditional psp1 mutants displayed a short-root phenotype, which was reverted in the presence of Ser. A metabolomic study demonstrated that the PPSB plays a crucial role in plant metabolism by affecting glycolysis, the tricarboxylic acid cycle, and the biosynthesis of amino acids. We provide evidence of the crucial role of the PPSB in embryo, pollen, and root development and suggest that this pathway is an important link connecting primary metabolism with development.

  14. Selected terpenoids from medicinal plants modulate endoplasmic reticulum stress in metabolic disorders.

    Science.gov (United States)

    Beukes, Natasha; Levendal, Ruby-Ann; Frost, Carminita L

    2014-11-01

    The majority of research performed on cellular stress and apoptosis focuses on mitochondrial dysfunction; however, the importance of the endoplasmic reticulum dysfunction and the link to metabolic diseases has gained a substantial interest. This review focuses on the potential of terpenoids to influence endoplasmic reticulum stress and the possible role terpenoids play as the treatment of metabolic diseases. Metabolic diseases develop as a result of a cascade of cellular pathways. In most cases, cells are able to compensate for the disruption of the cellular homeostasis although the initiation of response pathways; however, chronic stress initiates apoptotic pathways. This reviewed (1) showed the importance of phytoterpenoids to influence endoplasmic reticulum (ER) stress and homeostasis, (2) showed how regulating ER stress affect the cell survival and death, and (3) highlighted some examples of how the progression of metabolic diseases can be influenced by ER. Due to the substantial number of terpenoids that have been identified in literature, this review gave examples of 21 terpenoids that have been documented to have an effect on the different proteins associated with ER stress, how these plant terpenoids influence ER dysfunction and metabolic diseases such as diabetes, cancer, liver, and neurological diseases and parasitic infections. © 2014 Royal Pharmaceutical Society.

  15. Induction of SA-signaling pathway and ethylene biosynthesis in Trichoderma harzianum-treated tomato plants after infection of the root-knot nematode Meloidogyne incognita.

    Science.gov (United States)

    Leonetti, Paola; Zonno, Maria Chiara; Molinari, Sergio; Altomare, Claudio

    2017-04-01

    Salicylic acid-signaling pathway and ethylene biosynthesis were induced in tomato treated with Trichoderma harzianum when infected by root-knot nematodes and limited the infection by activation of SAR and ethylene production. Soil pre-treatment with Trichoderma harzianum (Th) strains ITEM 908 (T908) and T908-5 decreased susceptibility of tomato to Meloidogyne incognita, as assessed by restriction in nematode reproduction and development. The effect of T. harzianum treatments on plant defense was detected by monitoring the expression of the genes PR-1/PR-5 and JERF3/ACO, markers of the SA- and JA/ET-dependent signaling pathways, respectively. The compatible nematode-plant interaction in absence of fungi caused a marked suppression of PR-1, PR-5, and ACO gene expressions, either locally or systemically, whilst expression of JERF3 gene resulted unaffected. Conversely, when plants were pre-treated with Th-strains, over-expression of PR-1, PR-5, and ACO genes was observed in roots 5 days after nematode inoculation. JERF3 gene expression did not change in Th-colonized plants challenged with nematodes. In the absence of nematodes, Trichoderma-root interaction was characterized by the inhibition of both SA-dependent signaling pathway and ET biosynthesis, and, in the case of PR-1 and ACO genes, this inhibition was systemic. JERF3 gene expression was systemically restricted only at the very early stages of plant-fungi interaction. Data presented indicate that Th-colonization primed roots for Systemic Acquired Resistance (SAR) against root-knot nematodes and reacted to nematode infection more efficiently than untreated plants. Such a response probably involves also activation of ET production, through an augmented transcription of the ACO gene, which encodes for the enzyme catalyzing the last step of ET biosynthesis. JA signaling and Induced Systemic Resistance (ISR) do not seem to be involved in the biocontrol action of the tested Th-strains against RKNs.

  16. Down-regulation of the strawberry Bet v 1-homologous allergen in concert with the flavonoid biosynthesis pathway in colorless strawberry mutant

    DEFF Research Database (Denmark)

    Hjernø, Karin; Alm, Rikard; Canbäck, Björn

    2006-01-01

    to the nomenclature for allergen proteins, showed sequence identity of 54 and 77%, respectively, with corresponding allergens from birch and apple. Differential expression, as evaluated by 2-D DIGE, occurred in 10% of protein spots when red strawberries were compared to a colorless (white) strawberry mutant. White...... strawberries, known to be tolerated by individuals affected by allergy, were found to be virtually free from the strawberry allergen. Also several enzymes in the pathway for biosynthesis of flavonoids, to which the red color pelargonidin belongs, were down-regulated. This approach to assess differential...

  17. Bioengineering of plant (tri)terpenoids: from metabolic engineering of plants to synthetic biology in vivo and in vitro.

    Science.gov (United States)

    Moses, Tessa; Pollier, Jacob; Thevelein, Johan M; Goossens, Alain

    2013-10-01

    Terpenoids constitute a large and diverse class of natural products that serve many functions in nature. Most of the tens of thousands of the discovered terpenoids are synthesized by plants, where they function as primary metabolites involved in growth and development, or as secondary metabolites that optimize the interaction between the plant and its environment. Several plant terpenoids are economically important molecules that serve many applications as pharmaceuticals, pesticides, etc. Major challenges for the commercialization of plant-derived terpenoids include their low production levels in planta and the continuous demand of industry for novel molecules with new or superior biological activities. Here, we highlight several synthetic biology methods to enhance and diversify the production of plant terpenoids, with a foresight towards triterpenoid engineering, the least engineered class of bioactive terpenoids. Increased or cheaper production of valuable triterpenoids may be obtained by 'classic' metabolic engineering of plants or by heterologous production of the compounds in other plants or microbes. Novel triterpenoid structures can be generated through combinatorial biosynthesis or directed enzyme evolution approaches. In its ultimate form, synthetic biology may lead to the production of large amounts of plant triterpenoids in in vitro systems or custom-designed artificial biological systems. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

  18. Identification of a trichothecene gene cluster and description of the harzianum A biosynthesis pathway in the fungus Trichoderma arundinaceum

    Science.gov (United States)

    Trichothecenes are sesquiterpenes that act like mycotoxins. Their biosynthesis has been mainly studied in the fungal genera Fusarium, where most of the biosynthetic genes (tri) are grouped in a cluster regulated by ambient conditions and regulatory genes. Unexpectedly, few studies are available abou...

  19. Harnessing Yeast Peroxisomes for Biosynthesis of Fatty-Acid-Derived Biofuels and Chemicals with Relieved Side-Pathway Competition

    DEFF Research Database (Denmark)

    Zhou, Yongjin J.; Buijs, Nicolaas A; Zhu, Zhiwei

    2016-01-01

    to peroxisomes can increase the production of fatty-acid-derived fatty alcohols, alkanes and olefins up to 700%. In addition, we demonstrate that biosynthesis of these chemicals in the peroxisomes results in significantly decreased accumulation of byproducts formed by competing enzymes. We further demonstrate...

  20. Exploring two plant hosts for expression of diterpenoid pathway genes

    DEFF Research Database (Denmark)

    Bach, Søren Spanner

    ), but have been shared with a number of labs around the globe already. The content of this thesis will enable esearchers to explore the benefits of these plant expression hosts for characterization of terpenoid genes and contribute to a better understanding of terpenoid biosynthesis in planta....

  1. Contrasting ontogenetic trajectories for phenolic and terpenoid defences in Eucalyptus froggattii

    Science.gov (United States)

    Goodger, Jason Q. D.; Heskes, Allison M.; Woodrow, Ian E.

    2013-01-01

    Background and Aims Plant defence metabolites are considered costly due to diversion of energy and nutrients away from growth. These costs combined with changes in resource availability and herbivory throughout plant ontogeny are likely to promote changes in defence metabolites. A comprehensive understanding of plant defence strategy requires measurement of lifetime ontogenetic trajectories – a dynamic component largely overlooked in plant defence theories. This study aimed to compare ontogenetic trajectories of foliar phenolics and terpenoids. Phenolics are predicted to be inexpensive to biosynthesize, whereas expensive terpenoids also require specialized, non-photosynthetic secretory structures to avoid autotoxicity. Based on these predicted costs, it is hypothesized that phenolics would be maximally deployed early in ontogeny, whereas terpenoids would be maximally deployed later, once the costs of biosynthesis and foregone photosynthesis could be overcome by enhanced resource acquisition. Methods Leaves were harvested from a family of glasshouse-grown Eucalyptus froggattii seedlings, field-grown saplings and the maternal parent tree, and analysed for total terpenoids and phenolics. Key Results Foliar phenolics were highest in young seedlings and lowest in the adult tree. Indeed the ratio of total phenolics to total terpenoids decreased in a significantly exponential manner with plant ontogeny. Most individual terpene constituents increased with plant ontogeny, but some mono- and sesquiterpenes remained relatively constant or even decreased in concentration as plants aged. Conclusions Plant ontogeny can influence different foliar defence metabolites in directionally opposite ways, and the contrasting trajectories support our hypothesis that phenolics would be maximally deployed earlier than terpenoids. The results highlight the importance of examining ontogenetic trajectories of defence traits when developing and testing theories of plant defence, and

  2. The pathway via D-galacturonate/L-galactonate is significant for ascorbate biosynthesis in Euglena gracilis: identification and functional characterization of aldonolactonase.

    Science.gov (United States)

    Ishikawa, Takahiro; Nishikawa, Hitoshi; Gao, Youngshun; Sawa, Yoshihiro; Shibata, Hitoshi; Yabuta, Yukinori; Maruta, Takanori; Shigeoka, Shigeru

    2008-11-07

    We have previously proposed that Euglena gracilis possesses a pathway for the production of ascorbate (AsA) through d-galacturonate/L-galactonate as representative intermediates ( Shigeoka, S., Nakano, Y., and Kitaoka, S. (1979) J. Nutr. Sci. Vitaminol. 25, 299-307 ). However, genetic evidence proving that the pathway exists has not been obtained yet. We report here the identification of a gene encoding aldonolactonase, which catalyzes a penultimate step of the biosynthesis of AsA in Euglena. By a BLAST search, we identified one candidate for the enzyme having significant sequence identity with rat gluconolactonase, a key enzyme for the production of AsA via d-glucuronate in animals. The purified recombinant aldonolactonase expressed in Escherichia coli catalyzed the reversible reaction of L-galactonate and L-galactono-1,4-lactone with zinc ion as a cofactor. The apparent K(m) values for L-galactonate and L-galactono-1,4-lactone were 1.55 +/- 0.3 and 1.67 +/- 0.39 mm, respectively. The cell growth of Euglena was arrested by silencing the expression of aldonolactonase through RNA interference and then restored to the normal state by supplementation with L-galactono-1,4-lactone. Euglena cells accumulated more AsA on supplementation with d-galacturonate than d-glucuronate. The present results indicate that aldonolactonase is significant for the biosynthesis of AsA in Euglena cells, which predominantly utilize the pathwayviad-galacturonate/L-galactonate. The identification of aldonolactonase provides the first insight into the biosynthesis of AsA via uronic acids as the intermediate in photosynthetic algae including Euglena.

  3. AP2/ERF Transcription Factor, Ii049, Positively Regulates Lignan Biosynthesis in Isatis indigotica through Activating Salicylic Acid Signaling and Lignan/Lignin Pathway Genes.

    Science.gov (United States)

    Ma, Ruifang; Xiao, Ying; Lv, Zongyou; Tan, Hexin; Chen, Ruibing; Li, Qing; Chen, Junfeng; Wang, Yun; Yin, Jun; Zhang, Lei; Chen, Wansheng

    2017-01-01

    Lignans, such as lariciresinol and its derivatives, have been identified as effective antiviral ingredients in Isatis indigotica. Evidence suggests that the APETALA2/ethylene response factor (AP2/ERF) family might be related to the biosynthesis of lignans in I. indigotica. However, the special role played by the AP2/ERF family in the metabolism and its underlying putative mechanism still need to be elucidated. One novel AP2/ERF gene, named Ii049, was isolated and characterized from I. indigotica in this study. The quantitative real-time PCR analysis revealed that Ii049 was expressed highest in the root and responded to methyl jasmonate, salicylic acid (SA) and abscisic acid treatments to various degrees. Subcellular localization analysis indicated that Ii049 protein was localized in the nucleus. Knocking-down the expression of Ii049 caused a remarkable reduction of lignan/lignin contents and transcript levels of genes involved in the lignan/lignin biosynthetic pathway. Ii049 bound to the coupled element 1, RAV1AAT and CRTAREHVCBF2 motifs of genes IiPAL and IiCCR, the key structural genes in the lignan/lignin pathway. Furthermore, Ii049 was also essential for SA biosynthesis, and SA induced lignan accumulation in I. indigotica. Notably, the transgenic I. indigotica hairy roots overexpressing Ii049 showed high expression levels of lignan/lignin biosynthetic genes and SA content, resulting in significant accumulation of lignan/lignin. The best-engineered line (OVX049-10) produced 425.60 μg·g(-1) lariciresinol, an 8.3-fold increase compared with the wild type production. This study revealed the function of Ii049 in regulating lignan/lignin biosynthesis, which had the potential to increase the content of valuable lignan/lignin in economically significant medicinal plants.

  4. AP2/ERF Transcription Factor, Ii049, Positively Regulates Lignan Biosynthesis in Isatis indigotica through Activating Salicylic Acid Signaling and Lignan/Lignin Pathway Genes

    Directory of Open Access Journals (Sweden)

    Ruifang Ma

    2017-08-01

    Full Text Available Lignans, such as lariciresinol and its derivatives, have been identified as effective antiviral ingredients in Isatis indigotica. Evidence suggests that the APETALA2/ethylene response factor (AP2/ERF family might be related to the biosynthesis of lignans in I. indigotica. However, the special role played by the AP2/ERF family in the metabolism and its underlying putative mechanism still need to be elucidated. One novel AP2/ERF gene, named Ii049, was isolated and characterized from I. indigotica in this study. The quantitative real-time PCR analysis revealed that Ii049 was expressed highest in the root and responded to methyl jasmonate, salicylic acid (SA and abscisic acid treatments to various degrees. Subcellular localization analysis indicated that Ii049 protein was localized in the nucleus. Knocking-down the expression of Ii049 caused a remarkable reduction of lignan/lignin contents and transcript levels of genes involved in the lignan/lignin biosynthetic pathway. Ii049 bound to the coupled element 1, RAV1AAT and CRTAREHVCBF2 motifs of genes IiPAL and IiCCR, the key structural genes in the lignan/lignin pathway. Furthermore, Ii049 was also essential for SA biosynthesis, and SA induced lignan accumulation in I. indigotica. Notably, the transgenic I. indigotica hairy roots overexpressing Ii049 showed high expression levels of lignan/lignin biosynthetic genes and SA content, resulting in significant accumulation of lignan/lignin. The best-engineered line (OVX049-10 produced 425.60 μg·g−1 lariciresinol, an 8.3-fold increase compared with the wild type production. This study revealed the function of Ii049 in regulating lignan/lignin biosynthesis, which had the potential to increase the content of valuable lignan/lignin in economically significant medicinal plants.

  5. Regulation of FA and TAG biosynthesis pathway genes in endosperms and embryos of high and low oil content genotypes of Jatropha curcas L.

    Science.gov (United States)

    Sood, Archit; Chauhan, Rajinder Singh

    2015-09-01

    The rising demand for biofuels has raised concerns about selecting alternate and promising renewable energy crops which do not compete with food supply. Jatropha (Jatropha curcas L.), a non-edible energy crop of the family euphorbiaceae, has the potential of providing biodiesel feedstock due to the presence of high proportion of unsaturated fatty acids (75%) in seed oil which is mainly accumulated in endosperm and embryo. The molecular basis of seed oil biosynthesis machinery has been studied in J. curcas, however, what genetic differences contribute to differential oil biosynthesis and accumulation in genotypes varying for oil content is poorly understood. We investigated expression profile of 18 FA and TAG biosynthetic pathway genes in different developmental stages of embryo and endosperm from high (42%) and low (30%) oil content genotypes grown at two geographical locations. Most of the genes showed relatively higher expression in endosperms of high oil content genotype, whereas no significant difference was observed in endosperms versus embryos of low oil content genotype. The promoter regions of key genes from FA and TAG biosynthetic pathways as well as other genes implicated in oil accumulation were analyzed for regulatory elements and transcription factors specific to oil or lipid accumulation in plants such as Dof, CBF (LEC1), SORLIP, GATA and Skn-1_motif etc. Identification of key genes from oil biosynthesis and regulatory elements specific to oil deposition will be useful not only in dissecting the molecular basis of high oil content but also improving seed oil content through transgenic or molecular breeding approaches. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  6. Terpenoids: natural products for cancer therapy.

    Science.gov (United States)

    Huang, Min; Lu, Jin-Jian; Huang, Ming-Qing; Bao, Jiao-Lin; Chen, Xiu-Ping; Wang, Yi-Tao

    2012-12-01

    Terpenoids constitute the largest class of natural products and are a rich reservoir of candidate compounds for drug discovery. Recent efforts into the research and development of anti-cancer drugs derived from natural products have led to the identification of a variety of terpenoids that inhibit cancer cell proliferation and metastasis via various mechanisms. Despite the increasing number of research reports, there lacks a comprehensive review of anti-cancer activity of terpenoids. The present article provides an overview of the recent progress in the anti-cancer studies on terpenoids. Over a dozen naturally originated terpenoid compounds, in particular those derived from traditional Chinese medicine, were classified into five categories according to the structures, namely monoterpenoids, sesquiterpenoids, diterpenoids, triterpenoids and tetraterpenoids. The anti-cancer activities and relevant mechanistic insights of these compounds are discussed in this review. The anti-cancer activity of terpenoids appears promising and will potentially open more opportunities for cancer therapy. However, current studies are restricted to descriptive findings and lack mechanistic insights and systematic structure-activity relationship (SAR) studies. Future efforts into the systematic identification of the targets of terpenoids are believed to increase chances of gaining breakthrough insights in the field.

  7. Structural and Chemical Biology of Terpenoid Cyclases.

    Science.gov (United States)

    Christianson, David W

    2017-09-13

    The year 2017 marks the twentieth anniversary of terpenoid cyclase structural biology: a trio of terpenoid cyclase structures reported together in 1997 were the first to set the foundation for understanding the enzymes largely responsible for the exquisite chemodiversity of more than 80000 terpenoid natural products. Terpenoid cyclases catalyze the most complex chemical reactions in biology, in that more than half of the substrate carbon atoms undergo changes in bonding and hybridization during a single enzyme-catalyzed cyclization reaction. The past two decades have witnessed structural, functional, and computational studies illuminating the modes of substrate activation that initiate the cyclization cascade, the management and manipulation of high-energy carbocation intermediates that propagate the cyclization cascade, and the chemical strategies that terminate the cyclization cascade. The role of the terpenoid cyclase as a template for catalysis is paramount to its function, and protein engineering can be used to reprogram the cyclization cascade to generate alternative and commercially important products. Here, I review key advances in terpenoid cyclase structural and chemical biology, focusing mainly on terpenoid cyclases and related prenyltransferases for which X-ray crystal structures have informed and advanced our understanding of enzyme structure and function.

  8. The flavonoid biosynthetic enzyme chalcone isomerase modulates terpenoid production in glandular trichomes of tomato.

    Science.gov (United States)

    Kang, Jin-Ho; McRoberts, John; Shi, Feng; Moreno, Javier E; Jones, A Daniel; Howe, Gregg A

    2014-03-01

    Flavonoids and terpenoids are derived from distinct metabolic pathways but nevertheless serve complementary roles in mediating plant interactions with the environment. Here, we show that glandular trichomes of the anthocyanin free (af) mutant of cultivated tomato (Solanum lycopersicum) fail to accumulate both flavonoids and terpenoids. This pleiotropic metabolic deficiency was associated with loss of resistance to native populations of coleopteran herbivores under field conditions. We demonstrate that Af encodes an isoform (SlCHI1) of the flavonoid biosynthetic enzyme chalcone isomerase (CHI), which catalyzes the conversion of naringenin chalcone to naringenin and is strictly required for flavonoid production in multiple tissues of tomato. Expression of the wild-type SlCHI1 gene from its native promoter complemented the anthocyanin deficiency in af. Unexpectedly, the SlCHI1 transgene also complemented the defect in terpenoid production in glandular trichomes. Our results establish a key role for SlCHI1 in flavonoid production in tomato and reveal a link between CHI1 and terpenoid production. Metabolic coordination of the flavonoid and terpenoid pathways may serve to optimize the function of trichome glands in dynamic environments.

  9. New terpenoids from Resina Commiphora.

    Science.gov (United States)

    Dong, Lu; Luo, Qi; Cheng, Li-Zhi; Yan, Yong-Ming; Cheng, Yong-Xian; Wang, Shu-Mei

    2017-03-01

    Three new eudesmane sesquiterpenoids, commiphoranes E1-E3 (1-3), a new podocarpane diterpenoid, commiphorane F (4), three new triterpenoids, commiphoranes G1-G3 (5-7), and fourteen known terpenoids (8-21) were isolated from Resina Commiphora. The structures and relative configurations of new compounds were mainly assigned by spectroscopic methods. In particular, the absolute configuration of 3 was clarified by computational methods. Renoprotective effects of all these isolates were evaluated by using an ELISA assay in TGF-β1-induced rat kidney tubular epithelial cells. The present findings add new facets for chemical profiling of plant resins. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Biosynthesis and biodegradation of wood components

    Energy Technology Data Exchange (ETDEWEB)

    Higuchi, T. (ed.)

    1985-01-01

    A textbook containing 22 chapters by various authors covers the structure of wood, the localization of polysaccharides and lignins in wood cell walls, metabolism and synthetic function of cambial tissue, cell organelles and their function in the biosynthesis of cell wall components, biosynthesis of plant cell wall polysaccharides, lignin, cutin, suberin and associated waxes, phenolic acids and monolignols, quinones, flavonoids, tannins, stilbenes and terpenoid wood extractives, the occurrence of extractives, the metabolism of phenolic acids, wood degradation by micro-organisms and fungi, and biodegradation of cellulose, hemicelluloses, lignin, and aromatic extractives of wood. An index is included.

  11. Transcriptome analysis of medicinal plant Salvia miltiorrhiza and identification of genes related to tanshinone biosynthesis.

    Directory of Open Access Journals (Sweden)

    Lei Yang

    Full Text Available Salvia miltiorrhiza Bunge, a perennial plant of Lamiaceae, accumulates abietane-type diterpenoids of tanshinones in root, which have been used as traditional Chinese medicine to treat neuroasthenic insomnia and cardiovascular diseases. However, to date the biosynthetic pathway of tanshinones is only partially elucidated and the mechanism for their root-specific accumulation remains unknown. To identify enzymes and transcriptional regulators involved in the biosynthesis of tanshinones, we conducted transcriptome profiling of S. miltiorrhiza root and leaf tissues using the 454 GS-FLX pyrosequencing platform, which generated 550,546 and 525,292 reads, respectively. RNA sequencing reads were assembled and clustered into 64,139 unigenes (29,883 isotigs and 34,256 singletons. NCBI non-redundant protein databases (NR and Swiss-Prot database searches anchored 32,096 unigenes (50% with functional annotations based on sequence similarities. Further assignments with Gene Ontology (GO terms and KEGG biochemical pathways identified 168 unigenes referring to the terpenoid backbone biosynthesis (including 144 MEP and MVA pathway genes and 24 terpene synthases. Comparative analysis of the transcriptomes identified 2,863 unigenes that were highly expressed in roots, including those encoding enzymes of early steps of tanshinone biosynthetic pathway, such as copalyl diphosphate synthase (SmCPS, kaurene synthase-like (SmKSL and CYP76AH1. Other differentially expressed unigenes predicted to be related to tanshinone biosynthesis fall into cytochrome P450 monooxygenases, dehydrogenases and reductases, as well as regulatory factors. In addition, 21 P450 genes were selectively confirmed by real-time PCR. Thus we have generated a large unigene dataset which provides a valuable resource for further investigation of the radix development and biosynthesis of tanshinones.

  12. Cryptic indole hydroxylation by a non-canonical terpenoid cyclase parallels bacterial xenobiotic detoxification

    Science.gov (United States)

    Kugel, Susann; Baunach, Martin; Baer, Philipp; Ishida-Ito, Mie; Sundaram, Srividhya; Xu, Zhongli; Groll, Michael; Hertweck, Christian

    2017-06-01

    Terpenoid natural products comprise a wide range of molecular architectures that typically result from C-C bond formations catalysed by classical type I/II terpene cyclases. However, the molecular diversity of biologically active terpenoids is substantially increased by fully unrelated, non-canonical terpenoid cyclases. Their evolutionary origin has remained enigmatic. Here we report the in vitro reconstitution of an unusual flavin-dependent bacterial indoloterpenoid cyclase, XiaF, together with a designated flavoenzyme-reductase (XiaP) that mediates a key step in xiamycin biosynthesis. The crystal structure of XiaF with bound FADH2 (at 2.4 Å resolution) and phylogenetic analyses reveal that XiaF is, surprisingly, most closely related to xenobiotic-degrading enzymes. Biotransformation assays show that XiaF is a designated indole hydroxylase that can be used for the production of indigo and indirubin. We unveil a cryptic hydroxylation step that sets the basis for terpenoid cyclization and suggest that the cyclase has evolved from xenobiotics detoxification enzymes.

  13. Identification of Genes in Thuja plicata Foliar Terpenoid Defenses1[C][W][OA

    Science.gov (United States)

    Foster, Adam J.; Hall, Dawn E.; Mortimer, Leanne; Abercromby, Shelley; Gries, Regine; Gries, Gerhard; Bohlmann, Jörg; Russell, John; Mattsson, Jim

    2013-01-01

    Thuja plicata (western redcedar) is a long-lived conifer species whose foliage is rarely affected by disease or insect pests, but can be severely damaged by ungulate browsing. Deterrence to browsing correlates with high foliar levels of terpenoids, in particular the monoterpenoid α-thujone. Here, we set out to identify genes whose products may be involved in the production of α-thujone and other terpenoids in this species. First, we generated a foliar transcriptome database from which to draw candidate genes. Second, we mapped the storage of thujones and other terpenoids to foliar glands. Third, we used global expression profiling to identify more than 600 genes that are expressed at high levels in foliage with glands, but can either not be detected or are expressed at low levels in a natural variant lacking foliar glands. Fourth, we used in situ RNA hybridization to map the expression of a putative monoterpene synthase to the epithelium of glands and used enzyme assays with recombinant protein of the same gene to show that it produces sabinene, the monoterpene precursor of α-thujone. Finally, we identified candidate genes with predicted enzymatic functions for the conversion of sabinene to α-thujone. Taken together, this approach generated both general resources and detailed functional characterization in the identification of genes of foliar terpenoid biosynthesis in T. plicata. PMID:23388118

  14. Terpenoids in plant signaling, chemical ecology

    NARCIS (Netherlands)

    Kappers, I.F.; Dicke, M.; Bouwmeester, H.J.

    2008-01-01

    Terpenoids constitute the largest class of secondary metabolites in the plant kingdom. Because of their immense structural diversity and the resulting diversity in physiochemical properties, these molecules are particularly important for plant communication with other organisms. In this article, we

  15. Reconstruction of the astaxanthin biosynthesis pathway in rice endosperm reveals a metabolic bottleneck at the level of endogenous β-carotene hydroxylase activity.

    Science.gov (United States)

    Bai, Chao; Berman, Judit; Farre, Gemma; Capell, Teresa; Sandmann, Gerhard; Christou, Paul; Zhu, Changfu

    2017-02-01

    Astaxanthin is a high-value ketocarotenoid rarely found in plants. It is derived from β-carotene by the 3-hydroxylation and 4-ketolation of both ionone end groups, in reactions catalyzed by β-carotene hydroxylase and β-carotene ketolase, respectively. We investigated the feasibility of introducing an extended carotenoid biosynthesis pathway into rice endosperm to achieve the production of astaxanthin. This allowed us to identify potential metabolic bottlenecks that have thus far prevented the accumulation of this valuable compound in storage tissues such as cereal grains. Rice endosperm does not usually accumulate carotenoids because phytoene synthase, the enzyme responsible for the first committed step in the pathway, is not present in this tissue. We therefore expressed maize phytoene synthase 1 (ZmPSY1), Pantoea ananatis phytoene desaturase (PaCRTI) and a synthetic Chlamydomonas reinhardtii β-carotene ketolase (sCrBKT) in transgenic rice plants under the control of endosperm-specific promoters. The resulting grains predominantly accumulated the diketocarotenoids canthaxanthin, adonirubin and astaxanthin as well as low levels of monoketocarotenoids. The predominance of canthaxanthin and adonirubin indicated the presence of a hydroxylation bottleneck in the ketocarotenoid pathway. This final rate-limiting step must therefore be overcome to maximize the accumulation of astaxanthin, the end product of the pathway.

  16. Mutations in Escherichia coli aceE and ribB genes allow survival of strains defective in the first step of the isoprenoid biosynthesis pathway.

    Directory of Open Access Journals (Sweden)

    Jordi Perez-Gil

    Full Text Available A functional 2-C-methyl-D-erythritol 4-phosphate (MEP pathway is required for isoprenoid biosynthesis and hence survival in Escherichia coli and most other bacteria. In the first two steps of the pathway, MEP is produced from the central metabolic intermediates pyruvate and glyceraldehyde 3-phosphate via 1-deoxy-D-xylulose 5-phosphate (DXP by the activity of the enzymes DXP synthase (DXS and DXP reductoisomerase (DXR. Because the MEP pathway is absent from humans, it was proposed as a promising new target to develop new antibiotics. However, the lethal phenotype caused by the deletion of DXS or DXR was found to be suppressed with a relatively high efficiency by unidentified mutations. Here we report that several mutations in the unrelated genes aceE and ribB rescue growth of DXS-defective mutants because the encoded enzymes allowed the production of sufficient DXP in vivo. Together, this work unveils the diversity of mechanisms that can evolve in bacteria to circumvent a blockage of the first step of the MEP pathway.

  17. Transcriptome analysis reveals the genetic basis underlying the biosynthesis of volatile oil, gingerols, and diarylheptanoids in ginger (Zingiber officinale Rosc.).

    Science.gov (United States)

    Jiang, Yusong; Liao, Qinhong; Zou, Yong; Liu, Yiqing; Lan, Jianbin

    2017-10-23

    Ginger (Zingiber officinale Rosc.) is a popular flavoring that widely used in Asian, and the volatile oil in ginger rhizomes adds a special fragrance and taste to foods. The bioactive compounds in ginger, such as gingerols, diarylheptanoids, and flavonoids, are of significant value to human health because of their anticancer, anti-oxidant, and anti-inflammatory properties. However, as a non-model plant, knowledge about the genome sequences of ginger is extremely limited, and this limits molecular studies on this plant. In this study, de novo transcriptome sequencing was performed to investigate the expression of genes associated with the biosynthesis of major bioactive compounds in matured ginger rhizome (MG), young ginger rhizome (YG), and fibrous roots of ginger (FR). A total of 361,876 unigenes were generated by de novo assembly. The expression of genes involved in the pathways responsible for the biosynthesis of major bioactive compounds differed between tissues (MG, YG, and FR). Two pathways that give rise to volatile oil, gingerols, and diarylheptanoids, the "terpenoid backbone biosynthesis" and "stilbenoid, diarylheptanoid and gingerol biosynthesis" pathways, were significantly enriched (adjusted P value < 0.05) for differentially expressed genes (DEGs) (FDR < 0.005) both between the FR and YG libraries, and the FR and MG libraries. Most of the unigenes mapped in these two pathways, including curcumin synthase, phenylpropanoylacetyl-CoA synthase, trans-cinnamate 4-monooxygenase, and 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase, were expressed to a significantly higher level (log2 (fold-change) ≥ 1) in FR than in YG or MG. This study provides the first insight into the biosynthesis of bioactive compounds in ginger at a molecular level and provides valuable genome resources for future molecular studies on ginger. Moreover, our results establish that bioactive compounds in ginger may predominantly synthesized in the root and then transported to

  18. The Δ4-desaturation pathway for DHA biosynthesis is operative in the human species: Differences between normal controls and children with the Zellweger syndrome

    Directory of Open Access Journals (Sweden)

    Martinez Manuela

    2010-09-01

    Full Text Available Abstract Background Docosahexaenoic acid (DHA, 22:6ω3 is a fundamental component of cell membranes, especially in the brain and retina. In the experimental animal, DHA deficiency leads to suboptimal neurological performance and visual deficiencies. Children with the Zellweger syndrome (ZS have a profound DHA deficiency and symptoms that can be attributed to their extremely low DHA levels. These children seem to have a metabolic defect in DHA biosynthesis, which has never been totally elucidated. Treatment with DHA ethyl ester greatly improves these patients, but if we could normalize their endogenous DHA production we could get additional benefits. We examined whether DHA biosynthesis by Δ4-desaturation could be enhanced in the human species by transfecting the enzyme, and if this could normalize the DHA levels in cells from ZS patients. Results We showed that the Δ4-desaturase gene (Fad4 from Thraustochytrium sp, which can be expressed by heterologous transfection in other plant and yeast cells, can also be transfected into human lymphocytes, and that it expresses the enzyme (FAD4, Δ4-desaturase by producing DHA from direct Δ4-desaturation of 22:5ω3. We also found that the other substrate for Δ4-desaturase, 22:4ω6, was parallely desaturated to 22:5ω6. Conclusions The present "in vitro" study demonstrates that Δ4-desaturase can be transfected into human cells and synthesize DHA (as well as 22:5ω6, DPA from 22:5ω3 and 22:4ω6, respectively, by putative Δ4-desaturation. Even if this pathway may not be the physiological route for DHA biosynthesis "in vivo", the present study opens new perspectives for the treatment of patients within the ZS spectrum.

  19. The Δ4-desaturation pathway for DHA biosynthesis is operative in the human species: differences between normal controls and children with the Zellweger syndrome.

    Science.gov (United States)

    Martinez, Manuela; Ichaso, Natalia; Setien, Fernando; Durany, Nuria; Qiu, Xiao; Roesler, William

    2010-09-09

    Docosahexaenoic acid (DHA, 22:6ω3) is a fundamental component of cell membranes, especially in the brain and retina. In the experimental animal, DHA deficiency leads to suboptimal neurological performance and visual deficiencies. Children with the Zellweger syndrome (ZS) have a profound DHA deficiency and symptoms that can be attributed to their extremely low DHA levels. These children seem to have a metabolic defect in DHA biosynthesis, which has never been totally elucidated. Treatment with DHA ethyl ester greatly improves these patients, but if we could normalize their endogenous DHA production we could get additional benefits. We examined whether DHA biosynthesis by Δ4-desaturation could be enhanced in the human species by transfecting the enzyme, and if this could normalize the DHA levels in cells from ZS patients. We showed that the Δ4-desaturase gene (Fad4) from Thraustochytrium sp, which can be expressed by heterologous transfection in other plant and yeast cells, can also be transfected into human lymphocytes, and that it expresses the enzyme (FAD4, Δ4-desaturase) by producing DHA from direct Δ4-desaturation of 22:5ω3. We also found that the other substrate for Δ4-desaturase, 22:4ω6, was parallely desaturated to 22:5ω6. The present "in vitro" study demonstrates that Δ4-desaturase can be transfected into human cells and synthesize DHA (as well as 22:5ω6, DPA) from 22:5ω3 and 22:4ω6, respectively, by putative Δ4-desaturation. Even if this pathway may not be the physiological route for DHA biosynthesis "in vivo", the present study opens new perspectives for the treatment of patients within the ZS spectrum.

  20. Optimization of fatty alcohol biosynthesis pathway for selectively enhanced production of C12/14 and C16/18 fatty alcohols in engineered Escherichia coli.

    Science.gov (United States)

    Zheng, Yan-Ning; Li, Ling-Ling; Liu, Qiang; Yang, Jian-Ming; Wang, Xiang-Wei; Liu, Wei; Xu, Xin; Liu, Hui; Zhao, Guang; Xian, Mo

    2012-05-20

    With the increasing stress from oil price and environmental pollution, aroused attention has been paid to the microbial production of chemicals from renewable sources. The C12/14 and C16/18 alcohols are important feedstocks for the production of surfactants and detergents, which are widely used in the most respected consumer detergents, cleaning products and personal care products worldwide. Though bioproduction of fatty alcohols has been carried out in engineered E. coli, several key problems have not been solved in earlier studies, such as the quite low production of C16/18 alcohol, the lack of optimization of the fatty alcohol biosynthesis pathway, and the uncharacterized performance of the engineered strains in scaled-up system. We improved the fatty alcohol production by systematically optimizing the fatty alcohol biosynthesis pathway, mainly targeting three key steps from fatty acyl-acyl carrier proteins (ACPs) to fatty alcohols, which are sequentially catalyzed by thioesterase, acyl-coenzyme A (CoA) synthase and fatty acyl-CoA reductase. By coexpression of thioesterase gene BTE, acyl-CoA synthase gene fadD and fatty acyl-CoA reductase gene acr1, 210.1 mg/L C12/14 alcohol was obtained. A further optimization of expression level of BTE, fadD and acr1 increased the C12/14 alcohol production to 449.2 mg/L, accounting for 75.0% of the total fatty alcohol production (598.6 mg/L). In addition, by coexpression of thioesterase gene 'tesA, acyl-CoA synthase gene fadD and fatty acyl-CoA reductase gene FAR, 101.5 mg/L C16/18 alcohol was obtained, with C16/18 alcohol accounting for 89.2% of the total fatty alcohol production. To our knowledge, this is the first report on selective production of C12/14 and C16/18 alcohols by microbial fermentation. This work achieved high-specificity production of both C12/14 and C16/18 alcohols. The encouraging 598.6 mg/L of fatty alcohols represents the highest titer reported so far. In addition, the 101.5 mg/L 89.2%

  1. Optimization of fatty alcohol biosynthesis pathway for selectively enhanced production of C12/14 and C16/18 fatty alcohols in engineered Escherichia coli

    Directory of Open Access Journals (Sweden)

    Zheng Yan-Ning

    2012-05-01

    Full Text Available Abstract Background With the increasing stress from oil price and environmental pollution, aroused attention has been paid to the microbial production of chemicals from renewable sources. The C12/14 and C16/18 alcohols are important feedstocks for the production of surfactants and detergents, which are widely used in the most respected consumer detergents, cleaning products and personal care products worldwide. Though bioproduction of fatty alcohols has been carried out in engineered E. coli, several key problems have not been solved in earlier studies, such as the quite low production of C16/18 alcohol, the lack of optimization of the fatty alcohol biosynthesis pathway, and the uncharacterized performance of the engineered strains in scaled-up system. Results We improved the fatty alcohol production by systematically optimizing the fatty alcohol biosynthesis pathway, mainly targeting three key steps from fatty acyl-acyl carrier proteins (ACPs to fatty alcohols, which are sequentially catalyzed by thioesterase, acyl-coenzyme A (CoA synthase and fatty acyl-CoA reductase. By coexpression of thioesterase gene BTE, acyl-CoA synthase gene fadD and fatty acyl-CoA reductase gene acr1, 210.1 mg/L C12/14 alcohol was obtained. A further optimization of expression level of BTE, fadD and acr1 increased the C12/14 alcohol production to 449.2 mg/L, accounting for 75.0% of the total fatty alcohol production (598.6 mg/L. In addition, by coexpression of thioesterase gene ‘tesA, acyl-CoA synthase gene fadD and fatty acyl-CoA reductase gene FAR, 101.5 mg/L C16/18 alcohol was obtained, with C16/18 alcohol accounting for 89.2% of the total fatty alcohol production. Conclusions To our knowledge, this is the first report on selective production of C12/14 and C16/18 alcohols by microbial fermentation. This work achieved high-specificity production of both C12/14 and C16/18 alcohols. The encouraging 598.6 mg/L of fatty alcohols represents the highest

  2. Targeting Nuclear Thymidylate Biosynthesis

    Science.gov (United States)

    Chon, James; Stover, Patrick J.; Field, Martha S.

    2016-01-01

    Thymidylate (dTMP) biosynthesis plays an essential and exclusive function in DNA synthesis and proper cell division, and therefore has been an attractive therapeutic target. Folate analogues, known as antifolates, and nucleotide analogs that inhibit the enzymatic action of the de novo thymidylate biosynthesis pathway and are commonly used in cancer treatment. In this review, we examine the mechanisms by which the antifolate 5-fluorouracil, as well as other dTMP synthesis inhibitors, function in cancer treatment in light of emerging evidence that dTMP synthesis occurs in the nucleus. Nuclear localization of the de novo dTMP synthesis pathway requires modification of the pathway enzymes by the small ubiquitin-like modifier (SUMO) protein. SUMOylation is required for nuclear localization of the de novo dTMP biosynthesis pathway, and disruption in the SUMO pathway inhibits cell proliferation in several cancer models. We summarize evidence that the nuclear localization of the dTMP biosynthesis pathway is a critical factor in the efficacy of antifolate-based therapies that target dTMP synthesis. PMID:27876557

  3. Insights into the mechanism of natural terpenoids as NF-κB inhibitors: an overview on their anticancer potential.

    Science.gov (United States)

    Jain, H; Dhingra, N; Narsinghani, T; Sharma, R

    2016-09-01

    The transcription factor, nuclear factor kappa B (NF-kB) is one of the principal inducible protein in mammals known to control the gene expression in many critical physiological responses such as oxidative stress, inflammation etc. and has been shown to play an important role in the pathogenesis of cancer. Terpenoids are major constituents present in nutritionally used fruits, vegetables and different spices which possess various pharmacological action including anticancer activity. Various terpenoids, viz. monoterpenoids, sesquiterpenoids, diterpenoids, sesterterpenoids, triterpenoids, tetraterpenoids and polyterpenoids inhibit NF-kB signa-ling pathway through IkB phosphorylation, DNA binding, p65 translocation etc. Keeping in mind these facts, the present review revealed the anti-cancer potential of naturally occurring terpenoids highlighting their mechanism of NF-kB inhibition. This review also focuses on some of the naturally occurring terpenoids belonging to various chemical categories with potential inhibitory effects on NF-kB and their role in the treatment of cancer.

  4. Discovering the role of the apolipoprotein gene and the genes in the putative pullulan biosynthesis pathway on the synthesis of pullulan, heavy oil and melanin in Aureobasidium pullulans.

    Science.gov (United States)

    Guo, Jian; Huang, Siyao; Chen, Yefu; Guo, Xuewu; Xiao, Dongguang

    2017-12-18

    Pullulan produced by Aureobasidium pullulans presents various applications in food manufacturing and pharmaceutical industry. However, the pullulan biosynthesis mechanism remains unclear. This work proposed a pathway suggesting that heavy oil and melanin may correlate with pullulan production. The effects of overexpression or deletion of genes encoding apolipoprotein, UDPG-pyrophosphorylase, glucosyltransferase, and α-phosphoglucose mutase on the production of pullulan, heavy oil, and melanin were examined. Pullulan production increased by 16.93 and 8.52% with the overexpression of UDPG-pyrophosphorylase and apolipoprotein genes, respectively. Nevertheless, the overexpression or deletion of other genes exerted little effect on pullulan biosynthesis. Heavy oil production increased by 146.30, 64.81, and 33.33% with the overexpression of UDPG-pyrophosphorylase, α-phosphoglucose mutase, and apolipoprotein genes, respectively. Furthermore, the syntheses of pullulan, heavy oil, and melanin can compete with one another. This work may provide new guidance to improve the production of pullulan, heavy oil, and melanin through genetic approach.

  5. Amino Acids Attenuate Insulin Action on Gluconeogenesis and Promote Fatty Acid Biosynthesis via mTORC1 Signaling Pathway in trout Hepatocytes

    Directory of Open Access Journals (Sweden)

    Weiwei Dai

    2015-06-01

    Full Text Available Background/Aims: Carnivores exhibit poor utilization of dietary carbohydrates and glucose intolerant phenotypes, yet it remains unclear what are the causal factors and underlying mechanisms. We aimed to evaluate excessive amino acids (AAs-induced effects on insulin signaling, fatty acid biosynthesis and glucose metabolism in rainbow trout and determine the potential involvement of mTORC1 and p38 MAPK pathway. Methods: We stimulated trout primary hepatocytes with different AA levels and employed acute administration of rapamycin to inhibit mTORC1 activation. Results: Increased AA levels enhanced the phosphorylation of ribosomal protein S6 kinase (S6K1, S6, and insulin receptor substrate 1 (IRS-1 on Ser302 but suppressed Akt and p38 phosphorylation; up-regulated the expression of genes related to gluconeogenesis and fatty acid biosynthesis. mTORC1 inhibition not only inhibited the phosphorylation of mTORC1 downstream targets, but also blunted IRS-1 Ser302 phosphorylation and restored excessive AAs-suppressed Akt phosphorylation. Rapamycin also inhibited fatty acid biosynthetic and gluconeogenic gene expression. Conclusion: High levels of AAs up-regulate hepatic fatty acid biosynthetic gene expression through an mTORC1-dependent manner, while attenuate insulin-mediated repression of gluconeogenesis through elevating IRS-1 Ser302 phosphorylation, which in turn impairs Akt activation and thereby weakening insulin action. We propose that p38 MAPK probably also involves in these AAs-induced metabolic changes.

  6. Analysis of the isoprenoid biosynthesis pathways in Listeria monocytogenes reveals a role for the alternative 2-C-methyl-D-erythritol 4-phosphate pathway in murine infection.

    Science.gov (United States)

    Begley, Máire; Bron, Peter A; Heuston, Sinead; Casey, Pat G; Englert, Nadine; Wiesner, Jochen; Jomaa, Hassan; Gahan, Cormac G M; Hill, Colin

    2008-11-01

    Most bacteria synthesize isoprenoids through one of two essential pathways which provide the basic building block, isopentyl diphosphate (IPP): either the classical mevalonate pathway or the alternative non-mevalonate 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. However, postgenomic analyses of the Listeria monocytogenes genome revealed that this pathogen possesses the genetic capacity to produce the complete set of enzymes involved in both pathways. The nonpathogenic species Listeria innocua naturally lacks the last two genes (gcpE and lytB) of the MEP pathway, and bioinformatic analyses strongly suggest that the genes have been lost through evolution. In the present study we show that heterologous expression of gcpE and lytB in L. innocua can functionally restore the MEP pathway in this organism and confer on it the ability to induce Vgamma9 Vdelta2 T cells. We have previously confirmed that both pathways are functional in L. monocytogenes and can provide sufficient IPP for normal growth in laboratory media (M. Begley, C. G. Gahan, A. K. Kollas, M. Hintz, C. Hill, H. Jomaa, and M. Eberl, FEBS Lett. 561:99-104, 2004). Here we describe a targeted mutagenesis strategy to create a double pathway mutant in L. monocytogenes which cannot grow in the absence of exogenously provided mevalonate, confirming the requirement for at least one intact pathway for growth. In addition, murine studies revealed that mutants lacking the MEP pathway were impaired in virulence relative to the parent strain during intraperitoneal infection, while mutants lacking the classical mevalonate pathway were not impaired in virulence potential. In vivo bioluminescence imaging also confirmed in vivo expression of the gcpE gene (MEP pathway) during murine infection.

  7. A decade of molecular understanding of withanolide biosynthesis and in vitro studies in Withania somnifera (L. Dunal: Prospects and perspectives for pathway engineering

    Directory of Open Access Journals (Sweden)

    Niha eDhar

    2015-11-01

    Full Text Available Withania somnifera, a multipurpose medicinal plant is a rich reservoir of pharmaceutically active triterpenoids that are steroidal lactones known as withanolides. Though the plant has been well characterized in terms of phytochemical profiles as well as pharmaceutical activities, limited attempts have been made to decipher the biosynthetic route and identification of key regulatory genes involved in withanolide biosynthesis. This scenario limits biotechnological interventions for enhanced production of bioactive compounds. Nevertheless, recent emergent trends vis-à-vis, the exploration of genomic, transcriptomic, proteomic, metabolomic and in vitro studies have opened new vistas regarding pathway engineering of withanolide production. During recent years, various strategic pathway genes have been characterized with significant amount of regulatory studies which allude towards development of molecular circuitries for production of key intermediates or end products in heterologous hosts. Another pivotal aspect covering redirection of metabolic flux for channelizing the precursor pool towards enhanced withanolide production has also been attained by deciphering decisive branch point(s as robust targets for pathway modulation. With these perspectives, the current review provides a detailed overview of various studies undertaken by the authors and collated literature related to molecular and in vitro approaches employed in W. Somnifera for understanding various molecular network interactions in entirety.

  8. A Decade of Molecular Understanding of Withanolide Biosynthesis and In vitro Studies in Withania somnifera (L.) Dunal: Prospects and Perspectives for Pathway Engineering.

    Science.gov (United States)

    Dhar, Niha; Razdan, Sumeer; Rana, Satiander; Bhat, Wajid W; Vishwakarma, Ram; Lattoo, Surrinder K

    2015-01-01

    Withania somnifera, a multipurpose medicinal plant is a rich reservoir of pharmaceutically active triterpenoids that are steroidal lactones known as withanolides. Though the plant has been well-characterized in terms of phytochemical profiles as well as pharmaceutical activities, limited attempts have been made to decipher the biosynthetic route and identification of key regulatory genes involved in withanolide biosynthesis. This scenario limits biotechnological interventions for enhanced production of bioactive compounds. Nevertheless, recent emergent trends vis-à-vis, the exploration of genomic, transcriptomic, proteomic, metabolomics, and in vitro studies have opened new vistas regarding pathway engineering of withanolide production. During recent years, various strategic pathway genes have been characterized with significant amount of regulatory studies which allude toward development of molecular circuitries for production of key intermediates or end products in heterologous hosts. Another pivotal aspect covering redirection of metabolic flux for channelizing the precursor pool toward enhanced withanolide production has also been attained by deciphering decisive branch point(s) as robust targets for pathway modulation. With these perspectives, the current review provides a detailed overview of various studies undertaken by the authors and collated literature related to molecular and in vitro approaches employed in W. somnifera for understanding various molecular network interactions in entirety.

  9. Magnolol Affects Cellular Proliferation, Polyamine Biosynthesis and Catabolism-Linked Protein Expression and Associated Cellular Signaling Pathways in Human Prostate Cancer Cells in vitro

    Directory of Open Access Journals (Sweden)

    Brendan T. McKeown

    2015-01-01

    Full Text Available Background: Prostate cancer is the most commonly diagnosed form of cancer in men in Canada and the United States. Both genetic and environmental factors contribute to the development and progression of many cancers, including prostate cancer. Context and purpose of this study: This study investigated the effects of magnolol, a compound found in the roots and bark of the magnolia tree Magnolia officinalis, on cellular proliferation and proliferation-linked activities of PC3 human prostate cancer cells in vitro. Results: PC3 cells exposed to magnolol at a concentration of 80 μM for 6 hours exhibited decreased protein expression of ornithine decarboxylase, a key regulator in polyamine biosynthesis, as well as affecting the expression of other proteins involved in polyamine biosynthesis and catabolism. Furthermore, protein expression of the R2 subunit of ribonucleotide reductase, a key regulatory protein associated with DNA synthesis, was significantly decreased. Finally, the MAPK (mitogen-activated protein kinase, PI3K (phosphatidylinositol 3-kinase, NFκB (nuclear factor of kappa-light-chain-enhancer of activated B cells and AP-1 (activator protein 1 cellular signaling pathways were assayed to determine which, if any, of these pathways magnolol exposure would alter. Protein expressions of p-JNK-1 and c-jun were significantly increased while p-p38, JNK-1/2, PI3Kp85, p-PI3Kp85, p-Akt, NFκBp65, p-IκBα and IκBα protein expressions were significantly decreased. Conclusions: These alterations further support the anti-proliferative effects of magnolol on PC3 human prostate cancer cells in vitro and suggest that magnolol may have potential as a novel anti-prostate cancer agent.

  10. The Effects of Herbicides Targeting Aromatic and Branched Chain Amino Acid Biosynthesis Support the Presence of Functional Pathways in Broomrape

    Directory of Open Access Journals (Sweden)

    Evgenia Dor

    2017-05-01

    Full Text Available It is not clear why herbicides targeting aromatic and branched-chain amino acid biosynthesis successfully control broomrapes—obligate parasitic plants that obtain all of their nutritional requirements, including amino acids, from the host. Our objective was to reveal the mode of action of imazapic and glyphosate in controlling the broomrape Phelipanche aegyptiaca and clarify if this obligatory parasite has its own machinery for the amino acids biosynthesis. P. aegyptiaca callus was studied to exclude the indirect influence of the herbicides on the parasite through the host plant. Using HRT – tomato plants resistant to imidazolinone herbicides, it was shown that imazapic is translocated from the foliage of treated plants to broomrape attachments on its roots and controls the parasite. Both herbicides inhibited P. aegyptiaca callus growth and altered the free amino acid content. Blasting of Arabidopsis thaliana 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS and acetolactate synthase (ALS cDNA against the genomic DNA of P. aegyptiaca yielded a single copy of each homolog in the latter, with about 78 and 75% similarity, respectively, to A. thaliana counterparts at the protein level. We also show for the first time that both EPSPS and ALS are active in P. aegyptiaca callus and flowering shoots and are inhibited by glyphosate and imazapic, respectively. Thus leading to deficiency of those amino acids in the parasite tissues and ultimately, death of the parasite, indicating the ability of P. aegyptiaca to synthesize branched-chain and aromatic amino acids through the activity of ALS and EPSPS, respectively.

  11. Molecular cloning and functional characterization of Catharanthus roseus hydroxymethylbutenyl 4-diphosphate synthase gene promoter from the methyl erythritol phosphate pathway.

    Science.gov (United States)

    Ginis, Olivia; Courdavault, Vincent; Melin, Céline; Lanoue, Arnaud; Giglioli-Guivarc'h, Nathalie; St-Pierre, Benoit; Courtois, Martine; Oudin, Audrey

    2012-05-01

    The Madagascar periwinkle produces monoterpenoid indole alkaloids (MIA) of high interest due to their therapeutical values. The terpenoid moiety of MIA is derived from the methyl erythritol phosphate (MEP) and seco-iridoid pathways. These pathways are regarded as the limiting branch for MIA biosynthesis in C. roseus cell and tissue cultures. In previous studies, we demonstrated a coordinated regulation at the transcriptional and spatial levels of genes from both pathways. We report here on the isolation of the 5'-flanking region (1,049 bp) of the hydroxymethylbutenyl 4-diphosphate synthase (HDS) gene from the MEP pathway. To investigate promoter transcriptional activities, the HDS promoter was fused to GUS reporter gene. Agrobacterium-mediated transformation of young tobacco leaves revealed that the cloned HDS promoter displays a tissue-specific GUS staining restricted to the vascular region of the leaves and limited to a part of the vein that encompasses the phloem in agreement with the previous localization of HDS transcripts in C. roseus aerial organs. Further functional characterizations in stably or transiently transformed C. roseus cells allowed us to identify the region that can be consider as the minimal promoter and to demonstrate the induction of HDS promoter by several hormonal signals (auxin, cytokinin, methyljasmonate and ethylene) leading to MIA production. These results, and the bioinformatic analysis of the HDS 5'-region, suggest that the HDS promoter harbours a number of cis-elements binding specific transcription factors that would regulate the flux of terpenoid precursors involved in MIA biosynthesis.

  12. Extraction and Analysis of Terpenes/Terpenoids.

    Science.gov (United States)

    Jiang, Zuodong; Kempinski, Chase; Chappell, Joe

    2016-01-01

    Terpenes/terpenoids constitute one of the largest classes of natural products, this is due to the incredible chemical diversity that can arise from the biochemical transformations of the relatively simple prenyl diphosphate starter units. All terpenes/terpenoids comprise a hydrocarbon backbone that is generated from the various length prenyl diphosphates (a polymer chain of prenyl units). Upon ionization (removal) of the diphosphate group, the remaining allylic carbocation intermediates can be coaxed down complex chemical cascades leading to diverse linear and cyclized hydrocarbon backbones, which can then be further modified with a wide range of functional groups ( e.g . alcohol, ketones, etc .) and substituent additions ( e.g . sugars, fatty acids). Because of this chemical diversity, terpenes/terpenoids have great industrial uses as flavors, fragrances, high grade lubricants, biofuels, agricultural chemicals and medicines. The protocols presented here focus on the extraction of terpenes/terpenoids from various plant sources and have been divided into extraction methods for terpenes/terpenoids with various levels of chemical decoration, from the relative small, nonpolar, volatile hydrocarbons to substantially large molecules with greater physical complexity due to their chemical modifications.

  13. Genes of the de novo and salvage biosynthesis pathways of vitamin B6 are regulated under oxidative stress in the plant pathogen Rhizoctonia solani

    Directory of Open Access Journals (Sweden)

    Jamil eSamsatly

    2016-01-01

    Full Text Available B6 is recognized as an important cofactor required for numerous metabolic enzymes, and has been shown to act as an antioxidant and play a role in stress responses. It can be synthesized through two different routes: salvage and de novo pathways. However, little is known about the possible function of the vitamin B6 pathways in the fungal plant pathogen Rhizoctonia solani. Using genome walking, the de novo biosynthetic pathway genes; RsolPDX1 and RsolPDX2 and the salvage biosynthetic pathway gene, RsolPLR were sequenced. The predicted amino acid sequences of the three genes had high degree of similarity to other fungal PDX1, PDX2, and PLR proteins and are closely related to other R. solani anastomosis groups. We also examined their regulation when subjected to ROS stress inducers, the superoxide generator paraquat, or H2O2, and compared it to the well-known antioxidant genes, catalase and glutathione-S-transferase (GST. The genes were differentially regulated with substantial transcript levels as high as 33 fold depending on the gene and type of stress reflecting that differences in the type of damage induced by ROS. Exogenous addition of the vitamers PN or PLP in culture medium significantly induced the transcription of the vitamin B6 de novo encoding genes as early as 0.5 hour post treatment (HPT. On the other hand, transcription of RsolPLR was vitamer-specific; a down regulation upon supplementation of PN and upregualtion with PLP. Our results suggest that accumulation of ROS in R. solani mycelia was linked to transcriptional regulation of the three genes and R. solani vitamin B6 biosynthesis machinery could be implicated similar to catalases and GST as an antioxidant stress protector against oxidative stress.

  14. Ion-pairing liquid chromatography-tandem mass spectrometry-based quantification of uridine diphosphate-linked intermediates in the Staphylococcus aureus cell wall biosynthesis pathway.

    Science.gov (United States)

    Vemula, Harika; Bobba, Sudheer; Putty, Sandeep; Barbara, Joanna E; Gutheil, William G

    2014-11-15

    Bacterial cell wall biosynthesis is the target of several antibiotics and is of interest as a target for new inhibitor development. The cytoplasmic steps of this pathway involve a series of uridine diphosphate (UDP)-linked peptidoglycan intermediates. Quantification of these intermediates is essential for studies of current agents targeting this pathway and for the development of new agents targeting this pathway. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for quantification of these intermediates in Staphylococcus aureus. To address the problem of poor retention of UDP-linked intermediates on reverse phase media, an ion-pairing (IP) approach using N,N-dimethylhexylamine was developed. MS/MS detection in negative mode was optimized for UDP-GlcNAc, UDP-MurNAc, UDP-MurNAc-L-Ala, UDP-MurNAc-L-Ala-D-Glu, UDP-MurNAc-L-Ala-D-Glu-L-Lys, and UDP-MurNAc-L-Ala-D-Glu-L-Lys-D-Ala-D-Ala. The lower limits of quantification (LLOQs) for these analytes were 1.8, 1.0, 0.8, 2.2, 0.6, and 0.5 pmol, respectively, which correspond to LLOQs of 6, 3, 3, 7, 2, and 2 nmol/g bacteria, respectively. This method was demonstrated for quantification of in vivo levels of these intermediates from S. aureus (0.3mg dry weight analyzed) treated with fosfomycin, D-boroAla, D-cycloserine, and vancomycin. Metabolite accumulation is consistent with the known targets of these antibiotics and indicates potential regulatory loops within this pathway. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. De Novo Biosynthesis of Glutarate via α-Keto Acid Carbon Chain Extension and Decarboxylation Pathway in Escherichia coli.

    Science.gov (United States)

    Wang, Jian; Wu, Yifei; Sun, Xinxiao; Yuan, Qipeng; Yan, Yajun

    2017-10-20

    Microbial based bioplastics are promising alternatives to petroleum based synthetic plastics due to their renewability and economic feasibility. Glutarate is one of the most potential building blocks for bioplastics. The recent biosynthetic routes for glutarate were mostly based on the l-lysine degradation pathway from Pseudomonas putida that required lysine either by feeding or lysine overproduction via genetic manipulations. Herein, we established a novel glutarate biosynthetic pathway by incorporation of a "+1" carbon chain extension pathway from α-ketoglutarate (α-KG) in combination with α-keto acid decarboxylation pathway in Escherichia coli. Introduction of homocitrate synthase (HCS), homoaconitase (HA) and homoisocitrate dehydrogenase (HICDH) from Saccharomyces cerevisiae into E. coli enabled "+1" carbon extension from α-KG to α-ketoadipate (α-KA), which was subsequently converted into glutarate by a promiscuous α-keto acid decarboxylase (KivD) and a succinate semialdehyde dehydrogenase (GabD). The recombinant E. coli coexpressing all five genes produced 0.3 g/L glutarate from glucose. To further improve the titers, α-KG was rechanneled into carbon chain extension pathway via the clustered regularly interspersed palindromic repeats system mediated interference (CRISPRi) of essential genes sucA and sucB in tricarboxylic acid (TCA) cycle. The final strain could produce 0.42 g/L glutarate, which was increased by 40% compared with the parental strain.

  16. Trinuclear Metal Clusters in Catalysis by Terpenoid Synthases

    OpenAIRE

    Aaron, Julie A.; Christianson, David. W.

    2010-01-01

    Terpenoid synthases are ubiquitous enzymes that catalyze the formation of structurally and stereochemically diverse isoprenoid natural products. Many isoprenoid coupling enzymes and terpenoid cyclases from bacteria, fungi, protists, plants, and animals share the class I terpenoid synthase fold. Despite generally low amino acid sequence identity among these examples, class I terpenoid synthases contain conserved metal binding motifs that coordinate to a trinuclear metal cluster. This cluster n...

  17. Isoprenoid biosynthesis in eukaryotic phototrophs: A spotlight on algae

    Energy Technology Data Exchange (ETDEWEB)

    Lohr M.; Schwender J.; Polle, J. E. W.

    2012-04-01

    Isoprenoids are one of the largest groups of natural compounds and have a variety of important functions in the primary metabolism of land plants and algae. In recent years, our understanding of the numerous facets of isoprenoid metabolism in land plants has been rapidly increasing, while knowledge on the metabolic network of isoprenoids in algae still lags behind. Here, current views on the biochemistry and genetics of the core isoprenoid metabolism in land plants and in the major algal phyla are compared and some of the most pressing open questions are highlighted. Based on the different evolutionary histories of the various groups of eukaryotic phototrophs, we discuss the distribution and regulation of the mevalonate (MVA) and the methylerythritol phosphate (MEP) pathways in land plants and algae and the potential consequences of the loss of the MVA pathway in groups such as the green algae. For the prenyltransferases, serving as gatekeepers to the various branches of terpenoid biosynthesis in land plants and algae, we explore the minimal inventory necessary for the formation of primary isoprenoids and present a preliminary analysis of their occurrence and phylogeny in algae with primary and secondary plastids. The review concludes with some perspectives on genetic engineering of the isoprenoid metabolism in algae.

  18. Divergent Isoprenoid Biosynthesis Pathways in Staphylococcus Species Constitute a Drug Target for Treating Infections in Companion Animals

    Science.gov (United States)

    Cain, Christine L.; Morris, Daniel O.; Rankin, Shelley C.

    2016-01-01

    ABSTRACT Staphylococcus species are a leading cause of skin and soft tissue infections in humans and animals, and the antibiotics used to treat these infections are often the same. Methicillin- and multidrug-resistant staphylococcal infections are becoming more common in human and veterinary medicine. From a “One Health” perspective, this overlap in antibiotic use and resistance raises concerns over the potential spread of antibiotic resistance genes. Whole-genome sequencing and comparative genomics analysis revealed that Staphylococcus species use divergent pathways to synthesize isoprenoids. Species frequently associated with skin and soft tissue infections in companion animals, including S. schleiferi and S. pseudintermedius, use the nonmevalonate pathway. In contrast, S. aureus, S. epidermidis, and S. lugdunensis use the mevalonate pathway. The antibiotic fosmidomycin, an inhibitor of the nonmevalonate pathway, was effective in killing canine clinical staphylococcal isolates but had no effect on the growth or survival of S. aureus and S. epidermidis. These data identify an essential metabolic pathway in Staphylococcus that differs among members of this genus and suggest that drugs such as fosmidomycin, which targets enzymes in the nonmevalonate pathway, may be an effective treatment for certain staphylococcal infections. IMPORTANCE Drug-resistant Staphylococcus species are a major concern in human and veterinary medicine. There is a need for new antibiotics that exhibit a selective effect in treating infections in companion and livestock animals and that would not be used to treat human bacterial infections. We have identified fosmidomycin as an antibiotic that selectively targets certain Staphylococcus species that are often encountered in skin infections in cats and dogs. These findings expand our understanding of Staphylococcus evolution and may have direct implications for treating staphylococcal infections in veterinary medicine. PMID:27704053

  19. Domestication and the storage starch biosynthesis pathway: signatures of selection from a whole sorghum genome sequencing strategy.

    Science.gov (United States)

    Campbell, Bradley C; Gilding, Edward K; Mace, Emma S; Tai, Shuaishuai; Tao, Yongfu; Prentis, Peter J; Thomelin, Pauline; Jordan, David R; Godwin, Ian D

    2016-12-01

    Next-generation sequencing of complete genomes has given researchers unprecedented levels of information to study the multifaceted evolutionary changes that have shaped elite plant germplasm. In conjunction with population genetic analytical techniques and detailed online databases, we can more accurately capture the effects of domestication on entire biological pathways of agronomic importance. In this study, we explore the genetic diversity and signatures of selection in all predicted gene models of the storage starch synthesis pathway of Sorghum bicolor, utilizing a diversity panel containing lines categorized as either 'Landraces' or 'Wild and Weedy' genotypes. Amongst a total of 114 genes involved in starch synthesis, 71 had at least a single signal of purifying selection and 62 a signal of balancing selection and others a mix of both. This included key genes such as STARCH PHOSPHORYLASE 2 (SbPHO2, under balancing selection), PULLULANASE (SbPUL, under balancing selection) and ADP-glucose pyrophosphorylases (SHRUNKEN2, SbSH2 under purifying selection). Effectively, many genes within the primary starch synthesis pathway had a clear reduction in nucleotide diversity between the Landraces and wild and weedy lines indicating that the ancestral effects of domestication are still clearly identifiable. There was evidence of the positional rate variation within the well-characterized primary starch synthesis pathway of sorghum, particularly in the Landraces, whereby low evolutionary rates upstream and high rates downstream in the metabolic pathway were expected. This observation did not extend to the wild and weedy lines or the minor starch synthesis pathways. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  20. Decreased Biosynthesis of Jasmonic Acid via Lipoxygenase Pathway Compromised Caffeine-Induced Resistance to Colletotrichum gloeosporioides Under Elevated CO2 in Tea Seedlings.

    Science.gov (United States)

    Li, Xin; Ahammed, Golam Jalal; Li, Zhixin; Tang, Meijun; Yan, Peng; Han, Wenyan

    2016-11-01

    Caffeine, the major purine alkaloid in tea has long been known for its role in plant defense. However, its effect on Colletotrichum gloeosporioides that causes brown blight disease in tea is largely unknown especially under elevated CO2. Here we show that elevated CO2 reduced endogenous caffeine content in tea leaves, but sharply increased susceptibility of tea to C. gloeosporioides. The expression of C. gloeosporioides actin gene was gradually increased during the postinoculation period. In contrast, foliar application of caffeine decreased the C. gloeosporioides-induced necrotic lesions and the expression of C. gloeosporioides actin. Analysis of endogenous jasmonic acid (JA) content revealed that exogenous caffeine could induce JA content under both CO2 conditions in absence of fungal infection; however, in presence of fungal infection, caffeine increased JA content only under elevated CO2. Furthermore, exogenous caffeine enhanced lipoxygenase (LOX) activity and its biosynthetic gene expression under both CO2 conditions, indicating that increased JA biosynthesis via LOX pathway by caffeine might strengthen plant defense only under elevated CO2, while caffeine-induced defense under ambient CO2 might be associated with JA-independent LOX pathway in tea. These results provide novel insights into caffeine-induced plant defense mechanisms that might help to develop an eco-friendly approach for disease control.

  1. Isoprenoid biosynthesis in plant chloroplasts via the MEP pathway: direct thylakoid/ferredoxin-dependent photoreduction of GcpE/IspG.

    Science.gov (United States)

    Seemann, Myriam; Tse Sum Bui, Bernadette; Wolff, Murielle; Miginiac-Maslow, Myroslawa; Rohmer, Michel

    2006-03-06

    In the methylerythritol phosphate pathway for isoprenoid biosynthesis, the GcpE/IspG enzyme catalyzes the conversion of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate into (E)-4-hydroxy-3-methylbut-2-enyl diphosphate. This reaction requires a double one-electron transfer involving a [4Fe-4S] cluster. A thylakoid preparation from spinach chloroplasts was capable in the presence of light to act as sole electron donor for the plant GcpE Arabidopsis thaliana in the absence of any pyridine nucleotide. This is in sharp contrast with the bacterial Escherichia coli GcpE, which requires flavodoxin/flavodoxin reductase and NADPH as reducing system and represents the first proof that the electron flow from photosynthesis can directly act in phototrophic organisms as reducer in the 2-C-methyl-d-erythritol 4-phosphate pathway, most probably via ferredoxin, in the absence of any reducing cofactor. In the dark, the plant GcpE catalysis requires in addition of ferredoxin NADP(+)/ferredoxin oxido-reductase and NADPH as electron shuttle.

  2. Biosynthesis of Trehangelin in Polymorphospora rubra K07-0510: Identification of Metabolic Pathway to Angelyl-CoA.

    Science.gov (United States)

    Inahashi, Yuki; Shiraishi, Taro; Palm, Kaia; Takahashi, Yoko; Ōmura, Satoshi; Kuzuyama, Tomohisa; Nakashima, Takuji

    2016-08-03

    Trehangelins are trehalose angelates discovered from endophytic actinomycete Polymorphospora rubra K07-0510. We identified the trehangelin biosynthetic gene cluster, including genes that encode a glycoside hydrolase-like protein (thgC), α-amylase (thgD), 3-ketoacyl-ACP synthase III (thgI), 3-ketoacyl-ACP reductase (thgK), enoyl-CoA hydratase (thgH) and acyl transferase (thgJ). Heterologous expression of thgH, thgI, thgJ and thgK confirmed the importance of these genes in the biosynthesis of trehangelin A. Enzymatic activity studies showed that ThgI catalyses the condensation of acetyl-CoA and methylmalonyl-CoA to 2-methylacetoacetyl-CoA (MAA-CoA), ThgK catalyses NADPH-dependent reduction of MAA-CoA to 3-hydroxy-2-methylbutyryl-CoA (HMB-CoA) and ThgH catalyses the dehydration of HMB-CoA to angelyl-CoA (AN-CoA). This is the first report on the elucidation of the enzymatic formation of AN-CoA. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Organ-specific transcriptome profiling of metabolic and pigment biosynthesis pathways in the floral ornamental progenitor species Anthurium amnicola Dressler.

    Science.gov (United States)

    Suzuki, Jon Y; Amore, Teresita D; Calla, Bernarda; Palmer, Nathan A; Scully, Erin D; Sattler, Scott E; Sarath, Gautam; Lichty, Joanne S; Myers, Roxana Y; Keith, Lisa M; Matsumoto, Tracie K; Geib, Scott M

    2017-05-04

    Anthurium amnicola Dressler possesses a number of desirable and novel ornamental traits such as a purple-colored upright spathe, profuse flowering, and floral scent, some of which have been introgressed into modern Anthurium cultivars. As a first step in identifying genes associated with these traits, the transcriptome from root, leaf, spathe, and spadix from an accession of A. amnicola was assembled, resulting in 28,019 putative transcripts representing 19,458 unigenes. Genes involved in pigmentation, including those for the metabolism of chlorophyll and the biosynthesis of carotenoids, phenylpropanoids, and flavonoids were identified. The expression levels of one MYB transcription factor was highly correlated with naringenin 3-dioxygenase (F3H) and dihydroflavonol-4-reductase (DFR) in leaves, whereas a bHLH transcription factor was highly correlated with flavonoid 3'-monooxygenase (F3'H) and a DFR in spathes, suggesting that these two transcription factors might regulate flavonoid and anthocyanin synthesis in A. amnicola. Gene sequence and expression data from four major organs of A. amnicola provide novel basal information for understanding the genetic bases of ornamental traits and the determinants and evolution of form and function in the Araceae.

  4. Integrated transcriptome sequencing and dynamic analysis reveal carbon source partitioning between terpenoid and oil accumulation in developing Lindera glauca fruits.

    Science.gov (United States)

    Niu, Jun; Chen, Yinlei; An, Jiyong; Hou, Xinyu; Cai, Jian; Wang, Jia; Zhang, Zhixiang; Lin, Shanzhi

    2015-10-08

    Lindera glauca fruits (LGF) with the abundance of terpenoid and oil has emerged as a novel specific material for industrial and medicinal application in China, but the complex regulatory mechanisms of carbon source partitioning into terpenoid biosynthetic pathway (TBP) and oil biosynthetic pathway (OBP) in developing LGF is still unknown. Here we perform the analysis of contents and compositions of terpenoid and oil from 7 stages of developing LGF to characterize a dramatic difference in temporal accumulative patterns. The resulting 3 crucial samples at 50, 125 and 150 days after flowering (DAF) were selected for comparative deep transcriptome analysis. By Illumina sequencing, the obtained approximately 81 million reads are assembled into 69,160 unigenes, among which 174, 71, 81 and 155 unigenes are implicated in glycolysis, pentose phosphate pathway (PPP), TBP and OBP, respectively. Integrated differential expression profiling and qRT-PCR, we specifically characterize the key enzymes and transcription factors (TFs) involved in regulating carbon allocation ratios for terpenoid or oil accumulation in developing LGF. These results contribute to our understanding of the regulatory mechanisms of carbon source partitioning between terpenoid and oil in developing LGF, and to the improvement of resource utilization and molecular breeding for L. glauca.

  5. An antagonist treatment in combination with tracer experiments revealed isocitrate pathway dominant to oxalate biosynthesis in Rumex obtusifolius L

    Science.gov (United States)

    Oxalate accumulates in leaves of certain plants such as Rumex species (Polygonaceae). Oxalate plays important roles in defense to predator, detoxification of metallic ions, and in hydroxyl peroxide formation upon wounding/senescence. However, biosynthetic pathways of soluble oxalate are largely unkn...

  6. Salicylate-mediated suppression of jasmonate-responsive gene expression in Arabidopsis is targeted downstream of the jasmonate biosynthesis pathway

    NARCIS (Netherlands)

    Leon Reyes, H.A.; Does, D. van der; Lange, E.S. de; Delker, C.; Wasternack, C.; Wees, A.C.M. van; Ritsema, T.; Pieterse, C.M.J.

    2010-01-01

    Jasmonates (JAs) and salicylic acid (SA) are plant hormones that play pivotal roles in the regulation of induced defenses against microbial pathogens and insect herbivores. Their signaling pathways cross-communicate providing the plant with a regulatory potential to finely tune its defense response

  7. Terpenoids from the Octocoral Sinularia gaweli

    Directory of Open Access Journals (Sweden)

    Wun-Jie Lin

    2015-08-01

    Full Text Available Two eudesmane sesquiterpenoids, verticillatol (1 and 5α-acetoxy-4(14-eudesmene-1β-ol (2 and two cembrane diterpenoids, (–-leptodiol acetate (3 and sinulacembranolide A (4 were isolated from the octocoral Sinularia gaweli and compounds 2–4 are new isolates. The structures of new terpenoids 2–4 were elucidated by spectroscopic methods and by comparison the spectral data with those of known analogues. Terpenoid 4 was found to inhibit the accumulation of the pro-inflammatory inducible nitric oxide synthase (iNOS protein of the lipopolysaccharide (LPS-stimulated RAW264.7 marcophage cells.

  8. Methods and materials for production of terpenoids

    DEFF Research Database (Denmark)

    2015-01-01

    The invention provide novel methods for production of terpenoids. The methods take advantage of combinations of enzymes, which are not present in nature. Thus the methods involves use of host organisms expressing a diTPS of class II, diTPS of class I, and one or more CYPs.......The invention provide novel methods for production of terpenoids. The methods take advantage of combinations of enzymes, which are not present in nature. Thus the methods involves use of host organisms expressing a diTPS of class II, diTPS of class I, and one or more CYPs....

  9. Flavonoids and terpenoids from Croton muscicarpa (Euphorbiaceae)

    Energy Technology Data Exchange (ETDEWEB)

    Barreto, Milena B.; Gomes, Clerton L.; Freitas, Joao Vito B. de; Pinto, Francisco das Chagas L.; Silveira, Edilberto R.; Gramosa, Nilce V., E-mail: nilce@dqoi.ufc.br [Departamento de Quimica Organica e Inorganica, Centro de Ciencias, Universidade Federal do Ceara, Fortaleza (Brazil); Torres, Daniela S. Carneiro [Departamento de Ciencias Biologicas Jequie, Universidade Estadual do Sudoeste da Bahia, Jequie, BA (Brazil)

    2013-09-01

    A new sesquiterpene and twelve known compounds comprising eight flavonoids and four terpenoids, were isolated from the leaves, stems, roots and exudate of Croton muscicarpa Muell.. Arg.. Their structures were identified as the terpenoids 6{alpha}-methoxy-cyperene, dammaradienol, squalene, acetyl aleuritolic acid and spathulenol, and as the flavonoids retusin, 3,7,4'-trimethoxy kaempferol, ombuine, pachipodol, kaempferol, casticin, 5-hydroxy-3,6,7,4'-tetramethoxyflavone and artemetin. All isolated compounds were characterized based on IR, MS, {sup 1}H and {sup 13}C NMR, including 2D analyses (COSY, HSQC, HMBC, NOESY) and comparison with data from the literature. (author)

  10. Co-expression of three MEP pathway genes and geraniol 10-hydroxylase in internal phloem parenchyma of Catharanthus roseus implicates multicellular translocation of intermediates during the biosynthesis of monoterpene indole alkaloids and isoprenoid-derived primary metabolites.

    Science.gov (United States)

    Burlat, Vincent; Oudin, Audrey; Courtois, Martine; Rideau, Marc; St-Pierre, Benoit

    2004-04-01

    In higher plants, isopentenyl diphosphate (IPP) is synthesised both from the plastidic 2-C-methyl-d-erythritol 4-phosphate (MEP) and from the cytosolic mevalonate (MVA) pathways. Primary metabolites, such as phytol group of chlorophylls, carotenoids and the plant hormones abscisic acid (ABA) and gibberellins (GAs) are derived directly from the MEP pathway. Many secondary metabolites, such as monoterpene indole alkaloids (MIAs) in Catharanthus roseus, are also synthesised from this source of IPP. Using Northern blot and in situ hybridisation experiments, we show that three MEP pathway genes (1-deoxy-d-xylulose 5-phosphate synthase (DXS), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) and 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (MECS)) and the gene encoding geraniol 10-hydroxylase (G10H), a cytochrome P450 monooxygenase involved in the first committed step in the formation of iridoid monoterpenoids display identical cell-specific expression patterns. The co-localisation of these four transcripts to internal phloem parenchyma of young aerial organs of C. roseus adds a new level of complexity to the multicellular nature of MIA biosynthesis. We predict the translocation of pathway intermediates from the internal phloem parenchyma to the epidermis and, ultimately, to laticifers and idioblasts during MIA biosynthesis. Similarly, the translocation of intermediates from the phloem parenchyma is probably also required during the biosynthesis of hormones and photosynthetic primary metabolites derived from the MEP pathway.

  11. Triterpenoid Biosynthesis and Engineering in Plants

    Science.gov (United States)

    Sawai, Satoru; Saito, Kazuki

    2011-01-01

    Triterpenoid saponins are a diverse group of natural products in plants and are considered defensive compounds against pathogenic microbes and herbivores. Because of their various beneficial properties for humans, saponins are used in wide-ranging applications in addition to medicinally. Saponin biosynthesis involves three key enzymes: oxidosqualene cyclases, which construct the basic triterpenoid skeletons; cytochrome P450 monooxygenases, which mediate oxidations; and uridine diphosphate-dependent glycosyltransferases, which catalyze glycosylations. The discovery of genes committed to saponin biosynthesis is important for the stable supply and biotechnological application of these compounds. Here, we review the identified genes involved in triterpenoid biosynthesis, summarize the recent advances in the biotechnological production of useful plant terpenoids, and discuss the bioengineering of plant triterpenoids. PMID:22639586

  12. De novo transcriptome assembly and characterization of nine tissues of Lonicera japonica to identify potential candidate genes involved in chlorogenic acid, luteolosides, and secoiridoid biosynthesis pathways.

    Science.gov (United States)

    Rai, Amit; Kamochi, Hidetaka; Suzuki, Hideyuki; Nakamura, Michimi; Takahashi, Hiroki; Hatada, Tomoki; Saito, Kazuki; Yamazaki, Mami

    2017-01-01

    Lonicera japonica is one of the most important medicinal plants with applications in traditional Chinese and Japanese medicine for thousands of years. Extensive studies on the constituents of L. japonica extracts have revealed an accumulation of pharmaceutically active metabolite classes, such as chlorogenic acid, luteolin and other flavonoids, and secoiridoids, which impart characteristic medicinal properties. Despite being a rich source of pharmaceutically active metabolites, little is known about the biosynthetic enzymes involved, and their expression profile across different tissues of L. japonica. In this study, we performed de novo transcriptome assembly for L. japonica, representing transcripts from nine different tissues. A total of 22 Gbps clean RNA-seq reads from nine tissues of L. japonica were used, resulting in 243,185 unigenes, with 99,938 unigenes annotated based on a homology search using blastx against the NCBI-nr protein database. Unsupervised principal component analysis and correlation studies using transcript expression data from all nine tissues of L. japonica showed relationships between tissues, explaining their association at different developmental stages. Homologs for all genes associated with chlorogenic acid, luteolin, and secoiridoid biosynthesis pathways were identified in the L. japonica transcriptome assembly. Expression of unigenes associated with chlorogenic acid was enriched in stems and leaf-2, unigenes from luteolin were enriched in stems and flowers, while unigenes from secoiridoid metabolic pathways were enriched in leaf-1 and shoot apex. Our results showed that different tissues of L. japonica are enriched with sets of unigenes associated with specific pharmaceutically important metabolic pathways and, therefore, possess unique medicinal properties. The present study will serve as a resource for future attempts for functional characterization of enzyme coding genes within key metabolic processes.

  13. Homology modeling of Mycobacterium tuberculosis 2C-methyl-D-erythritol-4-phosphate cytidylyltransferase, the third enzyme in the MEP pathway for isoprenoid biosynthesis.

    Science.gov (United States)

    Obiol-Pardo, Cristian; Cordero, Alex; Rubio-Martinez, Jaime; Imperial, Santiago

    2010-06-01

    Tuberculosis is one of the leading infectious diseases in humans. Discovering new treatments for this disease is urgently required, especially in view of the emergence of multiple drug resistant organisms and to reduce the total duration of current treatments. The synthesis of isoprenoids in Mycobacterium tuberculosis has been reported as an interesting pathway to target, and particular attention has been focused on the methylerythritol phosphate (MEP) pathway comprising the early steps of isoprenoid biosynthesis. In this context we have studied the enzyme 2C-methyl-D-erythritol-4-phosphate cytidylyltransferase (CMS), the third enzyme in the MEP pathway, since the lack of a resolved structure of this protein in M. tuberculosis has seriously limited its use as a drug target. We performed homology modeling of M. tuberculosis CMS in order to provide a reliable model for use in structure-based drug design. After evaluating the quality of the model, we performed a thorough study of the catalytic site and the dimerization interface of the model, which suggested the most important sites (conserved and non-conserved) that could be useful for drug discovery and mutagenesis studies. We found that the metal coordination of CDP-methylerythritol in M. tuberculosis CMS differs substantially with respect to the Escherichia coli variant, consistent with the fact that the former is able to utilize several metal ions for catalysis. Moreover, we propose that electrostatic interactions could explain the higher affinity of the MEP substrate compared with the cytosine 5'-triphosphate substrate in the M. tuberculosis enzyme as reported previously.

  14. Transcriptome and Metabolite analysis reveal candidate genes of the cardiac glycoside biosynthetic pathway from Calotropis procera.

    Science.gov (United States)

    Pandey, Akansha; Swarnkar, Vishakha; Pandey, Tushar; Srivastava, Piush; Kanojiya, Sanjeev; Mishra, Dipak Kumar; Tripathi, Vineeta

    2016-10-05

    Calotropis procera is a medicinal plant of immense importance due to its pharmaceutical active components, especially cardiac glycosides (CG). As genomic resources for this plant are limited, the genes involved in CG biosynthetic pathway remain largely unknown till date. Our study on stage and tissue specific metabolite accumulation showed that CG's were maximally accumulated in stems of 3 month old seedlings. De novo transcriptome sequencing of same was done using high throughput Illumina HiSeq platform generating 44074 unigenes with average mean length of 1785 base pair. Around 66.6% of unigenes were annotated by using various public databases and 5324 unigenes showed significant match in the KEGG database involved in 133 different pathways of plant metabolism. Further KEGG analysis resulted in identification of 336 unigenes involved in cardenolide biosynthesis. Tissue specific expression analysis of 30 putative transcripts involved in terpenoid, steroid and cardenolide pathways showed a positive correlation between metabolite and transcript accumulation. Wound stress elevated CG levels as well the levels of the putative transcripts involved in its biosynthetic pathways. This result further validated the involvement of identified transcripts in CGs biosynthesis. The identified transcripts will lay a substantial foundation for further research on metabolic engineering and regulation of cardiac glycosides biosynthesis pathway genes.

  15. [Effects of arbuscular mycorrhizal fungi on terpenoids in Isodon adenantha].

    Science.gov (United States)

    Han, Bing-Yang; Wang, Ke-Dong; Zhou, Nong; Jiang, Bei

    2013-02-01

    To observe the effects of Arbuscular Mycorrhizal (AM) fungi on terpenoids in Isodon adenantha. Three different treated plant groups were divided as followings: indoor inoculation with AM fungi, non-inoculation with AM fungi, and outdoor natural growth, the effects of AM fungi on the major terpenoids in Isodon adenantha were evaluated. AM fungi had little influence on the content of terpenoids in underground part of Isodon adenantha, but they showed great impact on the content of terpenoids in the aerial part of the plant. The content of terpenoids in the aerial part of Isodon adenantha is positively correlated with AM fungi.

  16. Merochlorins A–D, cyclic meroterpenoid antibiotics biosynthesized in divergent pathways with vanadium-dependent chloroperoxidases

    Science.gov (United States)

    Kaysser, Leonard; Bernhardt, Peter; Nam, Sang-Jip; Loesgen, Sandra; Ruby, J. Graham; Skewes-Cox, Peter; Jensen, Paul R.; Fenical, William; Moore, Bradley S.

    2012-01-01

    Meroterpenoids are mixed polyketide-terpenoid natural products with a broad range of biological activities. Herein, we present the structures of four new meroterpenoid antibiotics, merochlorins A–D, produced by the marine bacterium Streptomyces sp. strain CNH-189, which possess novel chemical skeletons unrelated to known bacterial agents. Draft genome sequencing, mutagenesis and heterologous biosynthesis in the genome-minimized model actinomycete Streptomyces coelicolor provided a 57.6 kb gene cluster that contains two genes encoding rare bacterial vanadium-dependent haloperoxidase (VHPO) genes. Pathway expression of two different fosmid clones that differ largely by the presence or absence of the VHPO gene mcl40, resulted in the differential biosynthesis of merochlorin C, suggesting that Mcl40 catalyzes an unprecedented 15-membered chloronium-induced macrocyclization reaction converting merochlorin D to merochlorin C. PMID:22784372

  17. Merochlorins A-D, cyclic meroterpenoid antibiotics biosynthesized in divergent pathways with vanadium-dependent chloroperoxidases.

    Science.gov (United States)

    Kaysser, Leonard; Bernhardt, Peter; Nam, Sang-Jip; Loesgen, Sandra; Ruby, J Graham; Skewes-Cox, Peter; Jensen, Paul R; Fenical, William; Moore, Bradley S

    2012-07-25

    Meroterpenoids are mixed polyketide-terpenoid natural products with a broad range of biological activities. Herein, we present the structures of four new meroterpenoid antibiotics, merochlorins A-D, produced by the marine bacterium Streptomyces sp. strain CNH-189, which possess novel chemical skeletons unrelated to known bacterial agents. Draft genome sequencing, mutagenesis, and heterologous biosynthesis in the genome-minimized model actinomycete Streptomyces coelicolor provided the 57.6 kb merochlorin gene cluster that contains two genes encoding rare bacterial vanadium-dependent haloperoxidase (VHPO) genes. Pathway expression of two different fosmid clones that differ largely by the presence or absence of the VHPO gene mcl40 resulted in the differential biosynthesis of merochlorin C, suggesting that Mcl40 catalyzes an unprecedented 15-membered chloronium-induced macrocyclization reaction converting merochlorin D to merochlorin C.

  18. Bio-crude transcriptomics: Gene discovery and metabolic network reconstruction for the biosynthesis of the terpenome of the hydrocarbon oil-producing green alga, Botryococcus braunii race B (Showa*

    Directory of Open Access Journals (Sweden)

    Molnár István

    2012-10-01

    Full Text Available Abstract Background Microalgae hold promise for yielding a biofuel feedstock that is sustainable, carbon-neutral, distributed, and only minimally disruptive for the production of food and feed by traditional agriculture. Amongst oleaginous eukaryotic algae, the B race of Botryococcus braunii is unique in that it produces large amounts of liquid hydrocarbons of terpenoid origin. These are comparable to fossil crude oil, and are sequestered outside the cells in a communal extracellular polymeric matrix material. Biosynthetic engineering of terpenoid bio-crude production requires identification of genes and reconstruction of metabolic pathways responsible for production of both hydrocarbons and other metabolites of the alga that compete for photosynthetic carbon and energy. Results A de novo assembly of 1,334,609 next-generation pyrosequencing reads form the Showa strain of the B race of B. braunii yielded a transcriptomic database of 46,422 contigs with an average length of 756 bp. Contigs were annotated with pathway, ontology, and protein domain identifiers. Manual curation allowed the reconstruction of pathways that produce terpenoid liquid hydrocarbons from primary metabolites, and pathways that divert photosynthetic carbon into tetraterpenoid carotenoids, diterpenoids, and the prenyl chains of meroterpenoid quinones and chlorophyll. Inventories of machine-assembled contigs are also presented for reconstructed pathways for the biosynthesis of competing storage compounds including triacylglycerol and starch. Regeneration of S-adenosylmethionine, and the extracellular localization of the hydrocarbon oils by active transport and possibly autophagy are also investigated. Conclusions The construction of an annotated transcriptomic database, publicly available in a web-based data depository and annotation tool, provides a foundation for metabolic pathway and network reconstruction, and facilitates further omics studies in the absence of a genome

  19. Bio-crude transcriptomics: gene discovery and metabolic network reconstruction for the biosynthesis of the terpenome of the hydrocarbon oil-producing green alga, Botryococcus braunii race B (Showa).

    Science.gov (United States)

    Molnár, István; Lopez, David; Wisecaver, Jennifer H; Devarenne, Timothy P; Weiss, Taylor L; Pellegrini, Matteo; Hackett, Jeremiah D

    2012-10-30

    Microalgae hold promise for yielding a biofuel feedstock that is sustainable, carbon-neutral, distributed, and only minimally disruptive for the production of food and feed by traditional agriculture. Amongst oleaginous eukaryotic algae, the B race of Botryococcus braunii is unique in that it produces large amounts of liquid hydrocarbons of terpenoid origin. These are comparable to fossil crude oil, and are sequestered outside the cells in a communal extracellular polymeric matrix material. Biosynthetic engineering of terpenoid bio-crude production requires identification of genes and reconstruction of metabolic pathways responsible for production of both hydrocarbons and other metabolites of the alga that compete for photosynthetic carbon and energy. A de novo assembly of 1,334,609 next-generation pyrosequencing reads form the Showa strain of the B race of B. braunii yielded a transcriptomic database of 46,422 contigs with an average length of 756 bp. Contigs were annotated with pathway, ontology, and protein domain identifiers. Manual curation allowed the reconstruction of pathways that produce terpenoid liquid hydrocarbons from primary metabolites, and pathways that divert photosynthetic carbon into tetraterpenoid carotenoids, diterpenoids, and the prenyl chains of meroterpenoid quinones and chlorophyll. Inventories of machine-assembled contigs are also presented for reconstructed pathways for the biosynthesis of competing storage compounds including triacylglycerol and starch. Regeneration of S-adenosylmethionine, and the extracellular localization of the hydrocarbon oils by active transport and possibly autophagy are also investigated. The construction of an annotated transcriptomic database, publicly available in a web-based data depository and annotation tool, provides a foundation for metabolic pathway and network reconstruction, and facilitates further omics studies in the absence of a genome sequence for the Showa strain of B. braunii, race B. Further

  20. Interspecies and Intraspecies Analysis of Trehalose Contents and the Biosynthesis Pathway Gene Family Reveals Crucial Roles of Trehalose in Osmotic-Stress Tolerance in Cassava

    Directory of Open Access Journals (Sweden)

    Bingying Han

    2016-07-01

    Full Text Available Trehalose is a nonreducing α,α-1,1-disaccharide in a wide range of organisms, and has diverse biological functions that range from serving as an energy source to acting as a protective/signal sugar. However, significant amounts of trehalose have rarely been detected in higher plants, and the function of trehalose in the drought-tolerant crop cassava (Manihot esculenta Crantz is unclear. We measured soluble sugar concentrations of nine plant species with differing levels of drought tolerance and 41 cassava varieties using high-performance liquid chromatography with evaporative light-scattering detector (HPLC-ELSD. Significantly high amounts of trehalose were identified in drought-tolerant crops cassava, Jatropha curcas, and castor bean (Ricinus communis. All cassava varieties tested contained high amounts of trehalose, although their concentrations varied from 0.23 to 1.29 mg·g−1 fresh weight (FW, and the trehalose level was highly correlated with dehydration stress tolerance of detached leaves of the varieties. Moreover, the trehalose concentrations in cassava leaves increased 2.3–5.5 folds in response to osmotic stress simulated by 20% PEG 6000. Through database mining, 24 trehalose pathway genes, including 12 trehalose-6-phosphate synthases (TPS, 10 trehalose-6-phosphate phosphatases (TPP, and two trehalases were identified in cassava. Phylogenetic analysis indicated that there were four cassava TPS genes (MeTPS1–4 that were orthologous to the solely active TPS gene (AtTPS1 and OsTPS1 in Arabidopsis and rice, and a new TPP subfamily was identified in cassava, suggesting that the trehalose biosynthesis activities in cassava had potentially been enhanced in evolutionary history. RNA-seq analysis indicated that MeTPS1 was expressed at constitutionally high level before and after osmotic stress, while other trehalose pathway genes were either up-regulated or down-regulated, which may explain why cassava accumulated high level of trehalose

  1. Secondary organic aerosol formation from ozone reactions with single terpenoids and terpenoid mixtures

    Science.gov (United States)

    Waring, Michael S.; Wells, J. Raymond; Siegel, Jeffrey A.

    2011-08-01

    Ozone reacts with indoor-emitted terpenoids to form secondary organic aerosol (SOA). Most SOA research has focused on ozone reactions with single terpenoids or with consumer products, and this paper reports the results from an investigation of SOA formation from ozone reactions with both single terpenoids and mixtures of D-limonene, α-pinene, and α-terpineol. Transient experiments were conducted at low (25 ppb) and high (100 ppb) initial concentrations of ozone. The three terpenoids were tested singly and in combinations in a manner that controlled for their different reaction rates with ozone. The SOA formation was assessed by examining the evolution in time of the resulting number size-distributions and estimates of the mass concentrations. The results suggest that at higher ozone and terpenoid concentrations, SOA number formation follows a linear trend as a function of the initial rate of reaction. This finding was valid for both single terpenoids and mixtures. Generally speaking, higher ozone and terpenoid concentrations also led to larger geometric mean diameters and smaller geometric standard deviations of fitted lognormal distributions of the formed SOA. By assuming a density, mass concentrations were also assessed and did not follow as consistent of a trend. At low ozone concentration conditions, reactions with only D-limonene yielded the largest number concentrations of any experiment, even more than experiments with mixtures containing D-limonene and much higher overall terpenoid concentrations. This finding was not seen for high ozone concentrations. These experiments demonstrate quantifiable trends for SOA forming reactions of ozone and mixtures, and this work provides a framework for expanding these results to more complex mixtures and consumer products.

  2. Sterols and terpenoids from Melia azedarach.

    Science.gov (United States)

    Tan, Qin-Gang; Li, Xiao-Ning; Chen, Hao; Feng, Tao; Cai, Xiang-Hai; Luo, Xiao-Dong

    2010-04-23

    Three new sterols (1-3) including an unprecedented ring A-seco natural product (1), five new terpenoids (4-8), and 15 known compounds were isolated from the bark of Melia azedarach. Their structures were elucidated by means of spectroscopic data, and the structure of 1 was confirmed by X-ray crystallography.

  3. Volatile terpenoids from aeciospores of Cronartium fusiforme.

    Science.gov (United States)

    Laseter, J. L.; Weete, J. D.; Walkinshaw, C. H.

    1973-01-01

    Identification of the terpenoids present in the volatile fraction from aeciospores of the gall rust fungus Cronartium fusiforme. The major monoterpenoid hydrocarbons found to be present with only traces of camphene include alpha-pinene, beta-pinene, delta(3)-carene, myrcene, linonene, beta-phellandrene, and delta-terpinene. A number of monoterpenoid alcohols, acyclic sesquiterpenes, and aromatic compounds were also present.

  4. Natural terpenoids as a promising source for modulation of GABAergic system and treatment of neurological diseases.

    Science.gov (United States)

    Manayi, Azadeh; Nabavi, Seyed Mohammad; Daglia, Maria; Jafari, Samineh

    2016-08-01

    γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter reducing neural excitability in the mammalian central nervous system (CNS) with three subclasses of receptors. Several conventional drugs and compounds modulate the GABAergic system, demonstrating different pharmacological effects. In this review, interactions of natural terpenoids with the GABAergic system are highlighted with relation to disorders like anxiety, insomnia, convulsion, pain, and cognitive deficits. Terpenoids with various structures affect the function of the GABAergic system via dissimilar mechanisms. Most of the discussed compounds interact with GABA receptors, but especially with the GABAA subtype. This may be due to the fact that researchers tend to assess the interaction of compounds using GABAA receptors. However, bilobalide, a sesquiterpene, showed anticonvulsant properties through the activation of glutamic acid decarboxylase (GAD) enzyme, which is a key enzyme in biosynthesis of GABA. Therefore, further studies evaluating and comparing terpenoids of different classes and their interaction with the GABA system, along with their pharmacokinetic properties, could be worthwhile in future studies. Copyright © 2016 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  5. Treponema pallidum putative novel drug target identification and validation: rethinking syphilis therapeutics with plant-derived terpenoids.

    Science.gov (United States)

    Dwivedi, Upendra N; Tiwari, Sameeksha; Singh, Priyanka; Singh, Swati; Awasthi, Manika; Pandey, Veda P

    2015-02-01

    Syphilis, a slow progressive and the third most common sexually transmitted disease found worldwide, is caused by a spirochete gram negative bacteria Treponema pallidum. Emergence of antibiotic resistant T. pallidum has led to a search for novel drugs and their targets. Subtractive genomics analyses of pathogen T. pallidum and host Homo sapiens resulted in identification of 126 proteins essential for survival and viability of the pathogen. Metabolic pathway analyses of these essential proteins led to discovery of nineteen proteins distributed among six metabolic pathways unique to T. pallidum. One hundred plant-derived terpenoids, as potential therapeutic molecules against T. pallidum, were screened for their drug likeness and ADMET (absorption, distribution, metabolism, and toxicity) properties. Subsequently the resulting nine terpenoids were docked with five unique T. pallidum targets through molecular modeling approaches. Out of five targets analyzed, D-alanine:D-alanine ligase was found to be the most promising target, while terpenoid salvicine was the most potent inhibitor. A comparison of the inhibitory potential of the best docked readily available natural compound, namely pomiferin (flavonoid) with that of the best docked terpenoid salvicine, revealed that salvicine was a more potent inhibitor than that of pomiferin. To the best of our knowledge, this is the first report of a terpenoid as a potential therapeutic molecule against T. pallidum with D-alanine:D-alanine ligase as a novel target. Further studies are warranted to evaluate and explore the potential clinical ramifications of these findings in relation to syphilis that has public health importance worldwide.

  6. The Flavonoid Biosynthetic Enzyme Chalcone Isomerase Modulates Terpenoid Production in Glandular Trichomes of Tomato1[C][W][OPEN

    Science.gov (United States)

    Kang, Jin-Ho; McRoberts, John; Shi, Feng; Moreno, Javier E.; Jones, A. Daniel; Howe, Gregg A.

    2014-01-01

    Flavonoids and terpenoids are derived from distinct metabolic pathways but nevertheless serve complementary roles in mediating plant interactions with the environment. Here, we show that glandular trichomes of the anthocyanin free (af) mutant of cultivated tomato (Solanum lycopersicum) fail to accumulate both flavonoids and terpenoids. This pleiotropic metabolic deficiency was associated with loss of resistance to native populations of coleopteran herbivores under field conditions. We demonstrate that Af encodes an isoform (SlCHI1) of the flavonoid biosynthetic enzyme chalcone isomerase (CHI), which catalyzes the conversion of naringenin chalcone to naringenin and is strictly required for flavonoid production in multiple tissues of tomato. Expression of the wild-type SlCHI1 gene from its native promoter complemented the anthocyanin deficiency in af. Unexpectedly, the SlCHI1 transgene also complemented the defect in terpenoid production in glandular trichomes. Our results establish a key role for SlCHI1 in flavonoid production in tomato and reveal a link between CHI1 and terpenoid production. Metabolic coordination of the flavonoid and terpenoid pathways may serve to optimize the function of trichome glands in dynamic environments. PMID:24424324

  7. Gene expression profiling of lymphoblasts from autistic and nonaffected sib pairs: altered pathways in neuronal development and steroid biosynthesis.

    Directory of Open Access Journals (Sweden)

    Valerie W Hu

    2009-06-01

    Full Text Available Despite the identification of numerous autism susceptibility genes, the pathobiology of autism remains unknown. The present "case-control" study takes a global approach to understanding the molecular basis of autism spectrum disorders based upon large-scale gene expression profiling. DNA microarray analyses were conducted on lymphoblastoid cell lines from over 20 sib pairs in which one sibling had a diagnosis of autism and the other was not affected in order to identify biochemical and signaling pathways which are differentially regulated in cells from autistic and nonautistic siblings. Bioinformatics and gene ontological analyses of the data implicate genes which are involved in nervous system development, inflammation, and cytoskeletal organization, in addition to genes which may be relevant to gastrointestinal or other physiological symptoms often associated with autism. Moreover, the data further suggests that these processes may be modulated by cholesterol/steroid metabolism, especially at the level of androgenic hormones. Elevation of male hormones, in turn, has been suggested as a possible factor influencing susceptibility to autism, which affects approximately 4 times as many males as females. Preliminary metabolic profiling of steroid hormones in lymphoblastoid cell lines from several pairs of siblings reveals higher levels of testosterone in the autistic sibling, which is consistent with the increased expression of two genes involved in the steroidogenesis pathway. Global gene expression profiling of cultured cells from ASD probands thus serves as a window to underlying metabolic and signaling deficits that may be relevant to the pathobiology of autism.

  8. Quercetin protects Saccharomyces cerevisiae against oxidative stress by inducing trehalose biosynthesis and the cell wall integrity pathway.

    Directory of Open Access Journals (Sweden)

    Rita Vilaça

    Full Text Available BACKGROUND: Quercetin is a naturally occurring flavonol with antioxidant, anticancer and anti-ageing properties. In this study we aimed to identify genes differentially expressed in yeast cells treated with quercetin and its role in oxidative stress protection. METHODS: A microarray analysis was performed to characterize changes in the transcriptome and the expression of selected genes was validated by RT-qPCR. Biological processes significantly affected were identified by using the FUNSPEC software and their relevance in H(2O(2 resistance induced by quercetin was assessed. RESULTS: Genes associated with RNA metabolism and ribosome biogenesis were down regulated in cells treated with quercetin, whereas genes associated with carbohydrate metabolism, endocytosis and vacuolar proteolysis were up regulated. The induction of genes related to the metabolism of energy reserves, leading to the accumulation of the stress protectant disaccharide trehalose, and the activation of the cell wall integrity pathway play a key role in oxidative stress resistance induced by quercetin. CONCLUSIONS: These results suggest that quercetin may act as a modulator of cell signaling pathways related to carbohydrate metabolism and cell integrity to exert its protective effects against oxidative stress.

  9. Detection of phytohormones in temperate forest fungi predicts consistent abscisic acid production and a common pathway for cytokinin biosynthesis.

    Science.gov (United States)

    Morrison, Erin N; Knowles, Sarah; Hayward, Allison; Thorn, R Greg; Saville, Barry J; Emery, R J N

    2015-01-01

    The phytohormones, abscisic acid and cytokinin, once were thought to be present uniquely in plants, but increasing evidence suggests that these hormones are present in a wide variety of organisms. Few studies have examined fungi for the presence of these "plant" hormones or addressed whether their levels differ based on the nutrition mode of the fungus. This study examined 20 temperate forest fungi of differing nutritional modes (ectomycorrhizal, wood-rotting, saprotrophic). Abscisic acid and cytokinin were present in all fungi sampled; this indicated that the sampled fungi have the capacity to synthesize these two classes of phytohormones. Of the 27 cytokinins analyzed by HPLC-ESI MS/MS, seven were present in all fungi sampled. This suggested the existence of a common cytokinin metabolic pathway in fungi that does not vary among different nutritional modes. Predictions regarding the source of isopentenyl, cis-zeatin and methylthiol CK production stemming from the tRNA degradation pathway among fungi are discussed. © 2015 by The Mycological Society of America.

  10. Salicylic acid-induced changes in physiological parameters and genes of the flavonoid biosynthesis pathway in Artemisia vulgaris and Dendranthema nankingense during aphid feeding.

    Science.gov (United States)

    Sun, Y; Xia, X L; Jiang, J F; Chen, S M; Chen, F D; Lv, G S

    2016-02-19

    Phloem-feeding aphids cause serious damage to plants. The mechanisms of plant-aphid interactions are only partially understood and involve multiple pathways, including phytohormones. In order to investigate whether salicylic acid (SA) is involved and how it plays a part in the defense response to the aphid Macrosiphoniella sanbourni, physiological changes and gene expression profiles in response to aphid inoculation with or without SA pretreatment were compared between the aphid-resistant Artemisia vulgaris 'Variegata' and the susceptible chrysanthemum, Dendranthema nankingense. Changes in levels of reactive oxygen species, malondialdehyde (MDA), and flavonoids, and in the expression of genes involved in flavonoid biosynthesis, including PAL (phenylalanine ammonia-lyase), CHS (chalcone synthase), CHI (chalcone isomerase), F3H (flavanone 3-hydroxylase), F3'H (flavanone 3'-hydroxylase), and DFR (dihydroflavonol reductase), were investigated. Levels of hydrogen peroxide, superoxide anions, MDA, and flavonoids, and their related gene expression, increased after aphid infestation and SA pretreatment followed by aphid infestation; the aphid-resistant A. vulgaris exhibited a more rapid response than the aphid-susceptible D. nankingense to SA treatment and aphid infestation. Taken together, our results suggest that SA could be used to increase aphid resistance in the chrysanthemum.

  11. Upregulated mRNA expression of desaturase and elongase, two enzymes involved in highly unsaturated fatty acids biosynthesis pathways during follicle maturation in zebrafish

    Directory of Open Access Journals (Sweden)

    Enyu Yee-Ling

    2008-11-01

    Full Text Available Abstract Background Although unsaturated fatty acids such as eicosapentaenoic acid (EPA, C20:5n-3, docosahexaenoic acid (DHA, C22:6n-3 and arachidonic acid (ARA, C20:4n-6, collectively known as the highly unsaturated fatty acids (HUFA, play pivotal roles in vertebrate reproduction, very little is known about their synthesis in the ovary. The zebrafish (Danio rerio display capability to synthesize all three HUFA via pathways involving desaturation and elongation of two precursors, the linoleic acid (LA, C18:2n-6 and linolenic acid (LNA, C18:3n-3. As a prerequisite to gain full understanding on the importance and regulation of ovarian HUFA synthesis, we described here the mRNA expression pattern of two enzymes; desaturase (fadsd6 and elongase (elovl5, involved in HUFA biosynthesis pathway, in different zebrafish ovarian follicle stages. Concurrently, the fatty acid profile of each follicle stage was also analyzed. Methods mRNA levels of fadsd6 and elovl5 in different ovarian follicle stages were determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR assays. For analysis of the ovarian follicular fatty acid composition, gas chromatography was used. Results Our results have shown that desaturase displayed significant upregulation in expression during the oocyte maturation stage. Expression of elongase was significantly highest in pre-vitellogenic follicles, followed by maturation stage. Fatty acid composition analysis of different ovarian follicle stages also showed that ARA level was significantly highest in pre-vitellogenic and matured follicles. DHA level was highest in both late vitellogenic and maturation stage. Conclusion Collectively, our findings seem to suggest the existence of a HUFA synthesis system, which could be responsible for the synthesis of HUFA to promote oocyte maturation and possibly ovulation processes. The many advantages of zebrafish as model system to understand folliculogenesis will be

  12. The Transcript Profile of a Traditional Chinese Medicine, Atractylodes lancea, Revealing Its Sesquiterpenoid Biosynthesis of the Major Active Components.

    Directory of Open Access Journals (Sweden)

    Shakeel Ahmed

    Full Text Available Atractylodes lancea (Thunb. DC., named "Cangzhu" in China, which belongs to the Asteraceae family. In some countries of Southeast Asia (China, Thailand, Korea, Japan etc. its rhizome, commonly called rhizoma atractylodis, is used to treat many diseases as it contains a variety of sesquiterpenoids and other components of medicinal importance. Despite its medicinal value, the information of the sesquiterpenoid biosynthesis is largely unknown. In this study, we investigated the transcriptome analysis of different tissues of non-model plant A. lancea by using short read sequencing technology (Illumina. We found 62,352 high quality unigenes with an average sequence length of 913 bp in the transcripts of A. Lancea. Among these, 43,049 (69.04%, 30,264 (48.53%, 26,233 (42.07%, 17,881 (28.67% and 29,057(46.60% unigenes showed significant similarity (E-value<1e(-5 to known proteins in Nr, KEGG, SWISS-PROT, GO, and COG databases, respectively. Of the total 62,352 unigenes, 43,049 (Nr Database open reading frames were predicted. On the basis of different bioinformatics tools we identify all the enzymes that take part in the terpenoid biosynthesis as well as five different known sesquiterpenoids via cytosolic mevalonic acid (MVA pathway and plastidal methylerythritol phosphate (MEP pathways. In our study, 6, 864 Simple Sequence Repeats (SSRs were also found as great potential markers in A. lancea. This transcriptomic resource of A. lancea provides a great contribution in advancement of research for this specific medicinal plant and more specifically for the gene mining of different classes of terpenoids and other chemical compounds that have medicinal as well as economic importance.

  13. The gene PatG involved in the biosynthesis pathway of patulin, a food-borne mycotoxin, encodes a 6-methylsalicylic acid decarboxylase.

    Science.gov (United States)

    Snini, Selma P; Tadrist, Souria; Laffitte, Joelle; Jamin, Emilien L; Oswald, Isabelle P; Puel, Olivier

    2014-02-03

    -methylsalicylic acid decarboxylase. With this study, the four genes involved in the four first steps of patulin biosynthesis pathway (acetate→gentisyl alcohol) are now identified. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. NaCl Induces Flavonoid Biosynthesis through a Putative Novel Pathway in Post-harvest Ginkgo Leaves

    Science.gov (United States)

    Ni, Jun; Hao, Juan; Jiang, Zhifang; Zhan, Xiaori; Dong, Lixiang; Yang, Xiuli; Sun, Zhehang; Xu, Wenya; Wang, Zhikun; Xu, Maojun

    2017-01-01

    The flavonoids in the extracts of Ginkgo leaves have been shown to have great medical value: thus, a method to increase the flavonoid contents in these extracts is of significant importance for human health. In the present study, we investigated the changes in flavonoid contents and the corresponding gene expression levels in post-harvest Ginkgo leaves after various treatments. We found that both ultraviolet-B and NaCl treatment induced flavonoid accumulation. However, gene expression analysis showed that the increases in flavonoid contents were achieved by different pathways. Furthermore, post-harvest Ginkgo leaves responded differently to NaCl treatment compared with naturally grown leaves in both flavonoid contents and corresponding gene expression. In addition, combined treatment with ultraviolet-B and NaCl did not further increase the flavonoid contents compared with ultraviolet-B or NaCl treatment alone. Our results indicate the existence of a novel mechanism in response to NaCl treatment in post-harvest Ginkgo leaves, and provide a technique to increase flavonoid content in the pharmaceutical industry. PMID:28659935

  15. Golden Rice: introducing the beta-carotene biosynthesis pathway into rice endosperm by genetic engineering to defeat vitamin A deficiency.

    Science.gov (United States)

    Beyer, Peter; Al-Babili, Salim; Ye, Xudong; Lucca, Paola; Schaub, Patrick; Welsch, Ralf; Potrykus, Ingo

    2002-03-01

    To obtain a functioning provitamin A (beta-carotene) biosynthetic pathway in rice endosperm, we introduced in a single, combined transformation effort the cDNA coding for phytoene synthase (psy) and lycopene beta-cyclase (beta-lcy) both from Narcissus pseudonarcissus and both under the control of the endosperm-specific glutelin promoter together with a bacterial phytoene desaturase (crtI, from Erwinia uredovora under constitutive 35S promoter control). This combination covers the requirements for beta-carotene synthesis and, as hoped, yellow beta-carotene-bearing rice endosperm was obtained in the T(0)-generation. Additional experiments revealed that the presence of beta-lcy was not necessary, because psy and crtI alone were able to drive beta-carotene synthesis as well as the formation of further downstream xanthophylls. Plausible explanations for this finding are that these downstream enzymes are constitutively expressed in rice endosperm or are induced by the transformation, e.g., by enzymatically formed products. Results using N. pseudonarcissus as a model system led to the development of a hypothesis, our present working model, that trans-lycopene or a trans-lycopene derivative acts as an inductor in a kind of feedback mechanism stimulating endogenous carotenogenic genes. Various institutional arrangements for disseminating Golden Rice to research institutes in developing countries also are discussed.

  16. Alterations of Mitochondrial Protein Assembly and Jasmonic Acid Biosynthesis Pathway in Honglian (HL)-type Cytoplasmic Male Sterility Rice*

    Science.gov (United States)

    Liu, Gai; Tian, Han; Huang, Yun-Qing; Hu, Jun; Ji, Yan-Xiao; Li, Shao-Qing; Feng, Yu-Qi; Guo, Lin; Zhu, Ying-Guo

    2012-01-01

    It has been suggested that the mitochondrial chimeric gene orfH79 is the cause for abortion of microspores in Honglian cytoplasmic male sterile rice, yet little is known regarding its mechanism of action. In this study, we used a mass spectrometry-based quantitative proteomics strategy to compare the mitochondrial proteome between the sterile line Yuetai A and its fertile near-isogenic line Yuetai B. We discovered a reduced quantity of specific proteins in mitochondrial complexes in Yuetai A compared with Yuetai B, indicating a defect in mitochondrial complex assembly in the sterile line. Western blotting showed that ORFH79 protein and ATP1 protein, an F1 sector component of complex V, are both associated with large protein complexes of similar size. Respiratory complex activity assays and transmission electron microscopy revealed functional and morphological defects in the mitochondria of Yuetai A when compared with Yuetai B. In addition, we identified one sex determination TASSELSEED2-like protein increased in Yuetai A, leading to the discovery of an aberrant variation of the jasmonic acid pathway during the development of microspores. PMID:23027867

  17. Unusual cyclic terpenoids with terminal pendant prenyl moieties: from occurrence to synthesis.

    Science.gov (United States)

    Kulcitki, Veaceslav; Harghel, Petru; Ungur, Nicon

    2014-12-01

    The paper reviews the known examples of cyclic terpenoids produced from open chain polyenic precursors by an "unusual" biosynthetic pathway, involving selective electrophilic attack on an internal double bond followed by cyclization. The resulting compounds possess cyclic backbones with pendant terminal prenyl groups. Synthetic approaches applied for the synthesis of such specifically functionalized compounds are also discussed, as well as biological activity of reported representatives.

  18. Characterization of three novel isoprenyl diphosphate synthases from the terpenoid rich mango fruit.

    Science.gov (United States)

    Kulkarni, Ram; Pandit, Sagar; Chidley, Hemangi; Nagel, Raimund; Schmidt, Axel; Gershenzon, Jonathan; Pujari, Keshav; Giri, Ashok; Gupta, Vidya

    2013-10-01

    Mango (cv. Alphonso) is popular due to its highly attractive, terpenoid-rich flavor. Although Alphonso is clonally propagated, its fruit-flavor composition varies when plants are grown in different geo-climatic zones. Isoprenyl diphosphate synthases catalyze important branch-point reactions in terpenoid biosynthesis, providing precursors for common terpenoids such as volatile terpenes, sterols and carotenoids. Two geranyl diphosphate synthases and a farnesyl diphosphate synthase were isolated from Alphonso fruits, cloned for recombinant expression and found to produce the respective products. Although, one of the geranyl diphosphate synthases showed high sequence similarity to the geranylgeranyl diphosphate synthases, it did not exhibit geranylgeranyl diphosphate synthesizing activity. When modeled, this geranyl diphosphate synthase and farnesyl diphosphate synthase structures were found to be homologous with the reference structures, having all the catalytic side chains appropriately oriented. The optimum temperature for both the geranyl diphosphate synthases was 40 °C and that for farnesyl diphosphate synthase was 25 °C. This finding correlated well with the dominance of monoterpenes in comparison to sesquiterpenes in the fruits of Alphonso mango in which the mesocarp temperature is higher during ripening than development. The absence of activity of these enzymes with the divalent metal ion other than Mg(2+) indicated their adaptation to the Mg(2+) rich mesocarp. The typical expression pattern of these genes through the ripening stages of fruits from different cultivation localities depicting the highest transcript levels of these genes in the stage preceding the maximum terpene accumulation indicated the involvement of these genes in the biosynthesis of volatile terpenes. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  19. Plant terpenoid synthases: Molecular biology and phylogenetic analysis

    OpenAIRE

    Bohlmann, Jörg; Meyer-Gauen, Gilbert; Croteau, Rodney

    1998-01-01

    This review focuses on the monoterpene, sesquiterpene, and diterpene synthases of plant origin that use the corresponding C10, C15, and C20 prenyl diphosphates as substrates to generate the enormous diversity of carbon skeletons characteristic of the terpenoid family of natural products. A description of the enzymology and mechanism of terpenoid cyclization is followed by a discussion of molecular cloning and heterologous expression of terpenoid synthases. Sequence relatedness and phylogeneti...

  20. Cytochrome P450 promiscuity leads to a bifurcating biosynthetic pathway for tanshinones.

    Science.gov (United States)

    Guo, Juan; Ma, Xiaohui; Cai, Yuan; Ma, Ying; Zhan, Zhilai; Zhou, Yongjin J; Liu, Wujun; Guan, Mengxin; Yang, Jian; Cui, Guanghong; Kang, Liping; Yang, Lei; Shen, Ye; Tang, Jinfu; Lin, Huixin; Ma, Xiaojing; Jin, Baolong; Liu, Zhenming; Peters, Reuben J; Zhao, Zongbao K; Huang, Luqi

    2016-04-01

    Cytochromes P450 (CYPs) play a key role in generating the structural diversity of terpenoids, the largest group of plant natural products. However, functional characterization of CYPs has been challenging because of the expansive families found in plant genomes, diverse reactivity and inaccessibility of their substrates and products. Here we present the characterization of two CYPs, CYP76AH3 and CYP76AK1, which act sequentially to form a bifurcating pathway for the biosynthesis of tanshinones, the oxygenated diterpenoids from the Chinese medicinal plant Danshen (Salvia miltiorrhiza). These CYPs had similar transcription profiles to that of the known gene responsible for tanshinone production in elicited Danshen hairy roots. Biochemical and RNA interference studies demonstrated that both CYPs are promiscuous. CYP76AH3 oxidizes ferruginol at two different carbon centers, and CYP76AK1 hydroxylates C-20 of two of the resulting intermediates. Together, these convert ferruginol into 11,20-dihydroxy ferruginol and 11,20-dihydroxy sugiol en route to tanshinones. Moreover, we demonstrated the utility of these CYPs by engineering yeast for heterologous production of six oxygenated diterpenoids, which in turn enabled structural characterization of three novel compounds produced by CYP-mediated oxidation. Our results highlight the incorporation of multiple CYPs into diterpenoid metabolic engineering, and a continuing trend of CYP promiscuity generating complex networks in terpenoid biosynthesis. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  1. Towards Elucidating Carnosic Acid Biosynthesis in Lamiaceae: Functional Characterization of the Three First Steps of the Pathway in Salvia fruticosa and Rosmarinus officinalis.

    Directory of Open Access Journals (Sweden)

    Dragana Božić

    Full Text Available Carnosic acid (CA is a phenolic diterpene with anti-tumour, anti-diabetic, antibacterial and neuroprotective properties that is produced by a number of species from several genera of the Lamiaceae family, including Salvia fruticosa (Cretan sage and Rosmarinus officinalis (Rosemary. To elucidate CA biosynthesis, glandular trichome transcriptome data of S. fruticosa were mined for terpene synthase genes. Two putative diterpene synthase genes, namely SfCPS and SfKSL, showing similarities to copalyl diphosphate synthase and kaurene synthase-like genes, respectively, were isolated and functionally characterized. Recombinant expression in Escherichia coli followed by in vitro enzyme activity assays confirmed that SfCPS is a copalyl diphosphate synthase. Coupling of SfCPS with SfKSL, both in vitro and in yeast, resulted in the synthesis miltiradiene, as confirmed by 1D and 2D NMR analyses (1H, 13C, DEPT, COSY H-H, HMQC and HMBC. Coupled transient in vivo assays of SfCPS and SfKSL in Nicotiana benthamiana further confirmed production of miltiradiene in planta. To elucidate the subsequent biosynthetic step, RNA-Seq data of S. fruticosa and R. officinalis were searched for cytochrome P450 (CYP encoding genes potentially involved in the synthesis of the first phenolic compound in the CA pathway, ferruginol. Three candidate genes were selected, SfFS, RoFS1 and RoFS2. Using yeast and N. benthamiana expression systems, all three where confirmed to be coding for ferruginol synthases, thus revealing the enzymatic activities responsible for the first three steps leading to CA in two Lamiaceae genera.

  2. Two Chitin Biosynthesis Pathway Genes in Bactrocera dorsalis (Diptera: Tephritidae): Molecular Characteristics, Expression Patterns, and Roles in Larval-Pupal Transition.

    Science.gov (United States)

    Yang, Wen-Jia; Wu, Yi-Bei; Chen, Li; Xu, Kang-Kang; Xie, Yi-Fei; Wang, Jin-Jun

    2015-10-01

    Glucose-6-phosphate isomerase (G6PI) and UDP-N-acetylglucosamine pyrophosphorylase (UAP), two key components in the chitin biosynthesis pathway, are critical for insect growth and metamorphosis. In this study, we identified the genes BdG6PI and BdUAP from the oriental fruit fly, Bactrocera dorsalis (Hendel). The open reading frames (ORFs) of BdG6PI (1,491 bp) and BdUAP (1,677 bp) encoded 496 and 558 amino acid residues, respectively. Multiple sequence alignments showed that BdG6PI and BdUAP had high amino acid sequence identity with other insect homologues. Quantitative real-time polymerase chain reaction (qPCR) analysis indicated that BdG6PI was mainly expressed in the early stages of third-instar larvae and adults, while significantly higher expression of BdUAP was observed in adults. Both transcripts were expressed highly in the Malpighian tubules, but only slightly in the tracheae. The expression of both BdG6PI and BdUAP was significantly up-regulated by 20-hydroxyecdysone exposure and down-regulated by starvation. Moreover, injection of double-stranded RNAs of BdG6PI and BdUAP into third-instar larvae significantly reduced the corresponding gene expressions. Additionally, silencing of BdUAP resulted in 65% death and abnormal phenotypes of larvae, while silencing of BdG6PI had a slight effect on insect molting. These findings provide some data on the roles of BdG6PI and BdUAP in B. dorsalis and demonstrate the potential role for BdUAP in larval-pupal transition. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  3. Gene modification of the acetate biosynthesis pathway in Escherichia coli and implementation of the cell recycling technology to increase L-tryptophan production.

    Science.gov (United States)

    Xu, Qingyang; Bai, Fang; Chen, Ning; Bai, Gang

    2017-01-01

    The implementation of a novel cell recycling technology based on a special disk centrifuge during microbial fermentation process can continuously separate the product and harmful intermediates, while maintaining the cell viability owing to the installed cooling system. Acetate accumulation is an often encountered problem in L-tryptophan fermentation by Escherichia coli. To extend our previous studies, the current study deleted the key genes underlying acetate biosynthesis to improve l-tryptophan production. The deletion of the phosphotransacetylase (pta)-acetate kinase (ackA) pathway in a gltB (encoding glutamate synthase) mutant of E. coli TRTHB, led to the highest production of l-tryptophan (47.18 g/L) and glucose conversion rate (17.83%), with a marked reduction in acetate accumulation (1.22 g/L). This strain, TRTHBPA, was then used to investigate the effects of the cell recycling process on L-tryptophan fermentation. Four different strategies were developed concerning two issues, the volume ratio of the concentrated cell solution and clear solution and the cell recycling period. With strategy I (concentrated cell solution: clear solution, 1: 1; cell recycling within 24-30 h), L-tryptophan production and the glucose conversion rate increased to 55.12 g/L and 19.75%, respectively, 17.55% and 10.77% higher than those without the cell recycling. In addition, the biomass increased by 13.52% and the fermentation period was shortened from 40 h to 32 h. These results indicated that the cell recycling technology significantly improved L-tryptophan production by E. coli.

  4. Gene modification of the acetate biosynthesis pathway in Escherichia coli and implementation of the cell recycling technology to increase L-tryptophan production.

    Directory of Open Access Journals (Sweden)

    Qingyang Xu

    Full Text Available The implementation of a novel cell recycling technology based on a special disk centrifuge during microbial fermentation process can continuously separate the product and harmful intermediates, while maintaining the cell viability owing to the installed cooling system. Acetate accumulation is an often encountered problem in L-tryptophan fermentation by Escherichia coli. To extend our previous studies, the current study deleted the key genes underlying acetate biosynthesis to improve l-tryptophan production. The deletion of the phosphotransacetylase (pta-acetate kinase (ackA pathway in a gltB (encoding glutamate synthase mutant of E. coli TRTHB, led to the highest production of l-tryptophan (47.18 g/L and glucose conversion rate (17.83%, with a marked reduction in acetate accumulation (1.22 g/L. This strain, TRTHBPA, was then used to investigate the effects of the cell recycling process on L-tryptophan fermentation. Four different strategies were developed concerning two issues, the volume ratio of the concentrated cell solution and clear solution and the cell recycling period. With strategy I (concentrated cell solution: clear solution, 1: 1; cell recycling within 24-30 h, L-tryptophan production and the glucose conversion rate increased to 55.12 g/L and 19.75%, respectively, 17.55% and 10.77% higher than those without the cell recycling. In addition, the biomass increased by 13.52% and the fermentation period was shortened from 40 h to 32 h. These results indicated that the cell recycling technology significantly improved L-tryptophan production by E. coli.

  5. Towards Elucidating Carnosic Acid Biosynthesis in Lamiaceae: Functional Characterization of the Three First Steps of the Pathway in Salvia fruticosa and Rosmarinus officinalis

    Science.gov (United States)

    Božić, Dragana; Papaefthimiou, Dimitra; Brückner, Kathleen; de Vos, Ric C. H.; Tsoleridis, Constantinos A.; Katsarou, Dimitra; Papanikolaou, Antigoni; Pateraki, Irini; Chatzopoulou, Fani M.; Dimitriadou, Eleni; Kostas, Stefanos; Manzano, David; Scheler, Ulschan; Ferrer, Albert; Tissier, Alain; Makris, Antonios M.; Kampranis, Sotirios C.; Kanellis, Angelos K.

    2015-01-01

    Carnosic acid (CA) is a phenolic diterpene with anti-tumour, anti-diabetic, antibacterial and neuroprotective properties that is produced by a number of species from several genera of the Lamiaceae family, including Salvia fruticosa (Cretan sage) and Rosmarinus officinalis (Rosemary). To elucidate CA biosynthesis, glandular trichome transcriptome data of S. fruticosa were mined for terpene synthase genes. Two putative diterpene synthase genes, namely SfCPS and SfKSL, showing similarities to copalyl diphosphate synthase and kaurene synthase-like genes, respectively, were isolated and functionally characterized. Recombinant expression in Escherichia coli followed by in vitro enzyme activity assays confirmed that SfCPS is a copalyl diphosphate synthase. Coupling of SfCPS with SfKSL, both in vitro and in yeast, resulted in the synthesis miltiradiene, as confirmed by 1D and 2D NMR analyses (1H, 13C, DEPT, COSY H-H, HMQC and HMBC). Coupled transient in vivo assays of SfCPS and SfKSL in Nicotiana benthamiana further confirmed production of miltiradiene in planta. To elucidate the subsequent biosynthetic step, RNA-Seq data of S. fruticosa and R. officinalis were searched for cytochrome P450 (CYP) encoding genes potentially involved in the synthesis of the first phenolic compound in the CA pathway, ferruginol. Three candidate genes were selected, SfFS, RoFS1 and RoFS2. Using yeast and N. benthamiana expression systems, all three where confirmed to be coding for ferruginol synthases, thus revealing the enzymatic activities responsible for the first three steps leading to CA in two Lamiaceae genera. PMID:26020634

  6. Towards Elucidating Carnosic Acid Biosynthesis in Lamiaceae: Functional Characterization of the Three First Steps of the Pathway in Salvia fruticosa and Rosmarinus officinalis.

    Science.gov (United States)

    Božić, Dragana; Papaefthimiou, Dimitra; Brückner, Kathleen; de Vos, Ric C H; Tsoleridis, Constantinos A; Katsarou, Dimitra; Papanikolaou, Antigoni; Pateraki, Irini; Chatzopoulou, Fani M; Dimitriadou, Eleni; Kostas, Stefanos; Manzano, David; Scheler, Ulschan; Ferrer, Albert; Tissier, Alain; Makris, Antonios M; Kampranis, Sotirios C; Kanellis, Angelos K

    2015-01-01

    Carnosic acid (CA) is a phenolic diterpene with anti-tumour, anti-diabetic, antibacterial and neuroprotective properties that is produced by a number of species from several genera of the Lamiaceae family, including Salvia fruticosa (Cretan sage) and Rosmarinus officinalis (Rosemary). To elucidate CA biosynthesis, glandular trichome transcriptome data of S. fruticosa were mined for terpene synthase genes. Two putative diterpene synthase genes, namely SfCPS and SfKSL, showing similarities to copalyl diphosphate synthase and kaurene synthase-like genes, respectively, were isolated and functionally characterized. Recombinant expression in Escherichia coli followed by in vitro enzyme activity assays confirmed that SfCPS is a copalyl diphosphate synthase. Coupling of SfCPS with SfKSL, both in vitro and in yeast, resulted in the synthesis miltiradiene, as confirmed by 1D and 2D NMR analyses (1H, 13C, DEPT, COSY H-H, HMQC and HMBC). Coupled transient in vivo assays of SfCPS and SfKSL in Nicotiana benthamiana further confirmed production of miltiradiene in planta. To elucidate the subsequent biosynthetic step, RNA-Seq data of S. fruticosa and R. officinalis were searched for cytochrome P450 (CYP) encoding genes potentially involved in the synthesis of the first phenolic compound in the CA pathway, ferruginol. Three candidate genes were selected, SfFS, RoFS1 and RoFS2. Using yeast and N. benthamiana expression systems, all three where confirmed to be coding for ferruginol synthases, thus revealing the enzymatic activities responsible for the first three steps leading to CA in two Lamiaceae genera.

  7. Heterologous expression of the isopimaric acid pathway in Nicotiana benthamiana and the effect of N-terminal modifications of the involved cytochrome P450 enzyme

    DEFF Research Database (Denmark)

    Gnanasekaran, Thiyagarajan; Vavitsas, Konstantinos; Andersen-Ranberg, Johan

    2015-01-01

    BACKGROUND: Plant terpenoids are known for their diversity, stereochemical complexity, and their commercial interest as pharmaceuticals, food additives, and cosmetics. Developing biotechnology approaches for the production of these compounds in heterologous hosts can increase their market...... to photosynthesis paves the way for light-driven biosynthesis of valuable terpenoids....

  8. Terpenoid antifeedants against insects : a behavioural and sensory study

    NARCIS (Netherlands)

    Messchendorp, L.

    1998-01-01

    This thesis describes a study on the behavioural and sensory effects of terpenoid antifeedants on several insect species. The main aim was to elucidate the mechanisms of action of terpenoid antifeedants. From a fundamental point of view, this will yield insight in the role of these

  9. Metabolic engineering of Bacillus subtilis for terpenoid production

    NARCIS (Netherlands)

    Guan, Zheng; Xue, Dan; Abdallah, Ingy I; Dijkshoorn, Linda; Setroikromo, Rita; Lv, Guiyuan; Quax, Wim J

    2015-01-01

    Terpenoids are the largest group of small-molecule natural products, with more than 60,000 compounds made from isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). As the most diverse group of small-molecule natural products, terpenoids play an important role in the

  10. Volatile science? Metabolic engineering of terpenoids in plants

    NARCIS (Netherlands)

    Aharoni, A.; Jongsma, M.A.; Bouwmeester, H.J.

    2005-01-01

    Terpenoids are important for plant survival and also possess biological properties that are beneficial to humans. Here, we describe the state of the art in terpenoid metabolic engineering, showing that significant progress has been made over the past few years. Subcellular targeting of enzymes has

  11. Molecular Cloning, Characterization, and Functional Analysis of Acetyl-CoA C-Acetyltransferase and Mevalonate Kinase Genes Involved in Terpene Trilactone Biosynthesis from Ginkgo biloba

    Directory of Open Access Journals (Sweden)

    Qiangwen Chen

    2017-01-01

    Full Text Available Ginkgolides and bilobalide, collectively termed terpene trilactones (TTLs, are terpenoids that form the main active substance of Ginkgo biloba. Terpenoids in the mevalonate (MVA biosynthetic pathway include acetyl-CoA C-acetyltransferase (AACT and mevalonate kinase (MVK as core enzymes. In this study, two full-length (cDNAs encoding AACT (GbAACT, GenBank Accession No. KX904942 and MVK (GbMVK, GenBank Accession No. KX904944 were cloned from G. biloba. The deduced GbAACT and GbMVK proteins contain 404 and 396 amino acids with the corresponding open-reading frame (ORF sizes of 1215 bp and 1194 bp, respectively. Tissue expression pattern analysis revealed that GbAACT was highly expressed in ginkgo fruits and leaves, and GbMVK was highly expressed in leaves and roots. The functional complementation of GbAACT in AACT-deficient Saccharomyces cerevisiae strain Δerg10 and GbMVK in MVK-deficient strain Δerg12 confirmed that GbAACT mediated the conversion of mevalonate acetyl-CoA to acetoacetyl-CoA and GbMVK mediated the conversion of mevalonate to mevalonate phosphate. This observation indicated that GbAACT and GbMVK are functional genes in the cytosolic mevalonate (MVA biosynthesis pathway. After G. biloba seedlings were treated with methyl jasmonate and salicylic acid, the expression levels of GbAACT and GbMVK increased, and TTL production was enhanced. The cloning, characterization, expression and functional analysis of GbAACT and GbMVK will be helpful to understand more about the role of these two genes involved in TTL biosynthesis.

  12. Molecular Cloning, Characterization, and Functional Analysis of Acetyl-CoA C-Acetyltransferase and Mevalonate Kinase Genes Involved in Terpene Trilactone Biosynthesis from Ginkgo biloba.

    Science.gov (United States)

    Chen, Qiangwen; Yan, Jiaping; Meng, Xiangxiang; Xu, Feng; Zhang, Weiwei; Liao, Yongling; Qu, Jinwang

    2017-01-02

    Ginkgolides and bilobalide, collectively termed terpene trilactones (TTLs), are terpenoids that form the main active substance of Ginkgo biloba . Terpenoids in the mevalonate (MVA) biosynthetic pathway include acetyl-CoA C -acetyltransferase (AACT) and mevalonate kinase (MVK) as core enzymes. In this study, two full-length (cDNAs) encoding AACT ( GbAACT , GenBank Accession No. KX904942) and MVK ( GbMVK , GenBank Accession No. KX904944) were cloned from G. biloba . The deduced GbAACT and GbMVK proteins contain 404 and 396 amino acids with the corresponding open-reading frame (ORF) sizes of 1215 bp and 1194 bp, respectively. Tissue expression pattern analysis revealed that GbAACT was highly expressed in ginkgo fruits and leaves, and GbMVK was highly expressed in leaves and roots. The functional complementation of GbAACT in AACT-deficient Saccharomyces cerevisiae strain Δerg10 and GbMVK in MVK-deficient strain Δerg12 confirmed that GbAACT mediated the conversion of mevalonate acetyl-CoA to acetoacetyl-CoA and GbMVK mediated the conversion of mevalonate to mevalonate phosphate. This observation indicated that GbAACT and GbMVK are functional genes in the cytosolic mevalonate (MVA) biosynthesis pathway. After G. biloba seedlings were treated with methyl jasmonate and salicylic acid, the expression levels of GbAACT and GbMVK increased, and TTL production was enhanced. The cloning, characterization, expression and functional analysis of GbAACT and GbMVK will be helpful to understand more about the role of these two genes involved in TTL biosynthesis.

  13. Plant terpenoid synthases: Molecular biology and phylogenetic analysis

    Science.gov (United States)

    Bohlmann, Jörg; Meyer-Gauen, Gilbert; Croteau, Rodney

    1998-01-01

    This review focuses on the monoterpene, sesquiterpene, and diterpene synthases of plant origin that use the corresponding C10, C15, and C20 prenyl diphosphates as substrates to generate the enormous diversity of carbon skeletons characteristic of the terpenoid family of natural products. A description of the enzymology and mechanism of terpenoid cyclization is followed by a discussion of molecular cloning and heterologous expression of terpenoid synthases. Sequence relatedness and phylogenetic reconstruction, based on 33 members of the Tps gene family, are delineated, and comparison of important structural features of these enzymes is provided. The review concludes with an overview of the organization and regulation of terpenoid metabolism, and of the biotechnological applications of terpenoid synthase genes. PMID:9539701

  14. Draft genome of Omphalotus olearius provides a predictive framework for sesquiterpenoid natural product biosynthesis in Basidiomycota.

    Science.gov (United States)

    Wawrzyn, Grayson T; Quin, Maureen B; Choudhary, Swati; López-Gallego, Fernando; Schmidt-Dannert, Claudia

    2012-06-22

    The secondary metabolome of Basidiomycota represents a largely uncharacterized source of pharmaceutically relevant natural products. Terpenoids are the primary class of bioactive compounds isolated from mushrooms. The Jack O'Lantern mushroom Omphalotus olearius was identified 50 years ago as a prolific producer of anticancer illudin sesquiterpenoids; however, to date there have been exceptionally few studies into the biosynthesis of these important compounds. Here, we report the draft genome sequence of O. olearius, which reveals a diverse network of sesquiterpene synthases and two metabolic gene clusters associated with illudin biosynthesis. Characterization of the sesquiterpene synthases enabled a comprehensive survey of all currently available Basidiomycota genomes, thereby creating a predictive resource for terpenoid natural product biosynthesis in these organisms. Our results will facilitate discovery and biosynthetic production of unique pharmaceutically relevant bioactive compounds from Basidiomycota. Copyright © 2012 Elsevier Ltd. All rights reserved.

  15. DIETARY TERPENOIDS AND PROSTATE CANCER CHEMOPREVENTION

    Science.gov (United States)

    Rabi, Thangaiyan; Gupta, Sanjay

    2014-01-01

    Cancer chemoprevention by phytochemicals may be one of the most feasible approaches for cancer control. Phytochemicals obtained from vegetables, fruits, spices, teas, herbs and medicinal plants, such as terpenoids and other phenolic compounds, have been proven to suppress experimental carcinogenesis in various organs in pre-clinical models. Recent studies have indicated that mechanisms underlying chemopreventive potential may be a combination of antioxidant, anti-inflammatory, immune-enhancing, and hormone modulation effects, with modification of drug metabolizing enzymes, influence on cell cycle and cell differentiation, induction of apoptosis, suppression of proliferation and angiogenesis playing roles in the initiation and secondary modification stages of neoplastic development. Specific features of prostate cancer, such as high prevalence and long latency period provides ample opportunities for chemopreventive agents to work at various stages of disease progression. Finally, suitable populations with appropriate risk factors, including the presence of pre-malignant lesions and genetic predispositions, need to be well characterized for future chemopreventive interventions. Here we review naturally occurring dietary terpenoids as useful agents for prostate cancer chemoprevention with reference to their classes and sources. PMID:18508447

  16. Terpenes and terpenoids in chemical sensitivity.

    Science.gov (United States)

    Rea, William J; Restrepo, Carolina; Pan, Yaqin

    2015-01-01

    CONTEXT : Terpenes and terpenoids are a diverse class of organic compounds produced by a variety of plants, particularly conifers. Chemically sensitive patients can be targeted by terpenes and terpenoids, resulting in a triggering of symptoms and pathology. Often patients cannot clear their symptoms from exposure to chemicals unless terpenes and terpenoids are avoided and neutralized along with chemical avoidance and treatment. This article evaluates the presence, diagnosis, and treatment of terpenes exposure in chemically sensitive patients. A double-blind, placebo-controlled, 2-part study was designed to establish the chemically sensitive state of the patients in part 1, followed by a second set of challenges to determine each patient's concurrent sensitivity to terpenes and terpenoids in part 2. In all of the challenges, normal saline was used as a control. A case report illustrates the history of 1 patient and describes the authors' treatment methods. The study was developed and conducted at the Environmental Health Center of Dallas (EHC-D) because the environment within the center is 5 times less polluted than the surrounding environments, as determined by quantitative air analysis and particulate counts. A total of 45 chemically sensitive patients at EHC-D with odor sensitivity to terpenes. The cohort included 18 males and 27 females, aged 24-62 y.Intervention • Patients were deadapted (4 d) and evaluated in a 5-times-less-polluted environment, which was evaluated using air analysis and particulate counts. After deadaptation, the patients were challenged by inhalation in a controlled, less-polluted glass steel booth inside an environmentally controlled room with an ambient air dose of the toxics in the order of parts per billion (PPB) and parts per million (PPM). These toxics included formaldehyde, pesticide, cigarette smoke, ethanol, phenol, chlorine, new sprint, perfume, and placebo. They were also challenged intradermally with extracts of volatile

  17. Structures, biological activities and phylogenetic relationships of terpenoids from marine ciliates of the genus Euplotes.

    Science.gov (United States)

    Guella, Graziano; Skropeta, Danielle; Di Giuseppe, Graziano; Dini, Fernando

    2010-07-08

    In the last two decades, large scale axenic cell cultures of the marine species comprising the family Euplotidae have resulted in the isolation of several new classes of terpenoids with unprecedented carbon skeletons including the (i) euplotins, highly strained acetylated sesquiterpene hemiacetals; (ii) raikovenals, built on the bicyclo[3.2.0]heptane ring system; (iii) rarisetenolides and focardins containing an octahydroazulene moiety; and (iv) vannusals, with a unique C30 backbone. Their complex structures have been elucidated through a combination of nuclear magnetic resonance spectroscopy, mass spectrometry, molecular mechanics and quantum chemical calculations. Despite the limited number of biosynthetic experiments having been performed, the large diversity of ciliate terpenoids has facilitated the proposal of biosynthetic pathways whereby they are produced from classical linear precursors. Herein, the similarities and differences emerging from the comparison of the classical chemotaxonomy approach based on secondary metabolites, with species phylogenesis based on genetic descriptors (SSU-rDNA), will be discussed. Results on the interesting ecological and biological properties of ciliate terpenoids are also reported.

  18. Terpenoids as anti-colon cancer agents - A comprehensive review on its mechanistic perspectives.

    Science.gov (United States)

    Sharma, Sharada H; Thulasingam, Senthilkumar; Nagarajan, Sangeetha

    2017-01-15

    Multistep model of colon carcinogenesis has provided the framework to advance our understanding of the molecular basis of colon cancer. This multistage process of carcinogenesis takes a long period to transform from a normal epithelial cell to invasive carcinoma. Thus, it provides enough time to intervene the process of carcinogenesis especially through dietary modification. In spite of the in-depth understanding of the colon cancer etiology and pathophysiology and its association with diet, colon cancer remains a major cause of cancer mortality worldwide. Phytochemicals and their derivatives are gaining attention in cancer prevention and treatment strategies because of cancer chemotherapy associated adverse effects. Being the largest group of phytochemicals traditionally used for medicinal purpose in India and China, terpenoids are recently being explored as anticancer agents. Anticancer properties of terpenoids are associated with various mechanisms like counteraction of oxidative stress, potentiating endogenous antioxidants, improving detoxification potential, disrupting cell survival pathways and inducing apoptosis. This review gives a comprehensive idea of naturally occurring terpenoids as useful agents for the prevention of colon cancer with reference to their classes, sources and molecular targets. Based on the explored molecular targets further research in colon cancer chemoprevention is warranted. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. The border sequence of the balhimycin biosynthesis gene cluster from Amycolatopsis balhimycina contains bbr, encoding a StrR-like pathway-specific regulator

    NARCIS (Netherlands)

    Shawky, Riham M.; Puk, Oliver; Wietzorrek, Andreas; Pelzer, Stefan; Takano, Eriko; Wohlleben, Wolfgang; Stegmann, Efthimia

    2007-01-01

    Balhimycin, produced by the actinomycete Amycolatopsis balhimycina DSM5908, is a glycopeptide antibiotic highly similar to vancomycin, the antibiotic of 'last resort' used for the treatment of resistant Gram-positive pathogenic bacteria. Partial sequence of the balhimycin biosynthesis gene cluster

  20. Cyclic terpenoids of contemporary resinous plant detritus and of fossil woods, ambers and coals

    Science.gov (United States)

    Simoneit, B.R.T.; Grimalt, J.O.; Wang, T.-G.; Cox, R.E.; Hatcher, P.G.; Nissenbaum, A.

    1986-01-01

    transformation of terrestrial cyclic terpenoids during diagenesis, constituting a general pathway for all terpenoids. ?? 1986 Pergamon Journals Ltd.

  1. Evidence that biosynthesis of the second and third sugars of the archaellin Tetrasaccharide in the archaeon Methanococcus maripaludis occurs by the same pathway used by Pseudomonas aeruginosa to make a di-N-acetylated sugar.

    Science.gov (United States)

    Siu, Sarah; Robotham, Anna; Logan, Susan M; Kelly, John F; Uchida, Kaoru; Aizawa, Shin-Ichi; Jarrell, Ken F

    2015-05-01

    Methanococcus maripaludis has two surface appendages, archaella and type IV pili, which are composed of glycoprotein subunits. Archaellins are modified with an N-linked tetrasaccharide with the structure Sug-1,4-β-ManNAc3NAmA6Thr-1,4-β-GlcNAc3NAcA-1,3-β-GalNAc, where Sug is (5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-L-erythro-hexos-5-ulo-1,5-pyranose. The pilin glycan has an additional hexose attached to GalNAc. In this study, genes located in two adjacent, divergently transcribed operons (mmp0350-mmp0354 and mmp0359-mmp0355) were targeted for study based on annotations suggesting their involvement in biosynthesis of N-glycan sugars. Mutants carrying deletions in mmp0350, mmp0351, mmp0352, or mmp0353 were nonarchaellated and synthesized archaellins modified with a 1-sugar glycan, as estimated from Western blots. Mass spectroscopy analysis of pili purified from the Δmmp0352 strain confirmed a glycan with only GalNAc, suggesting mmp0350 to mmp0353 were all involved in biosynthesis of the second sugar (GlcNAc3NAcA). The Δmmp0357 mutant was archaellated and had archaellins with a 2-sugar glycan, as confirmed by mass spectroscopy of purified archaella, indicating a role for MMP0357 in biosynthesis of the third sugar (ManNAc3NAmA6Thr). M. maripaludis mmp0350, mmp0351, mmp0352, mmp0353, and mmp0357 are proposed to be functionally equivalent to Pseudomonas aeruginosa wbpABEDI, involved in converting UDP-N-acetylglucosamine to UDP-2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, an O5-specific antigen sugar. Cross-domain complementation of the final step of the P. aeruginosa pathway with mmp0357 supports this hypothesis. This work identifies a series of genes in adjacent operons that are shown to encode the enzymes that complete the entire pathway for generation of the second and third sugars of the N-linked tetrasaccharide that modifies archaellins of Methanococcus maripaludis. This posttranslational modification of archaellins is important, as it is necessary for

  2. Analysis of Polygala tenuifolia Transcriptome and Description of Secondary Metabolite Biosynthetic Pathways by Illumina Sequencing

    Directory of Open Access Journals (Sweden)

    Hongling Tian

    2015-01-01

    Full Text Available Radix polygalae, the dried roots of Polygala tenuifolia and P. sibirica, is one of the most well-known traditional Chinese medicinal plants. Radix polygalae contains various saponins, xanthones, and oligosaccharide esters and these compounds are responsible for several pharmacological properties. To provide basic breeding information, enhance molecular biological analysis, and determine secondary metabolite biosynthetic pathways of P. tenuifolia, we applied Illumina sequencing technology and de novo assembly. We also applied this technique to gain an overview of P. tenuifolia transcriptome from samples with different years. Using Illumina sequencing, approximately 67.2% of unique sequences were annotated by basic local alignment search tool similarity searches against public sequence databases. We classified the annotated unigenes by using Nr, Nt, GO, COG, and KEGG databases compared with NCBI. We also obtained many candidates CYP450s and UGTs by the analysis of genes in the secondary metabolite biosynthetic pathways, including putative terpenoid backbone and phenylpropanoid biosynthesis pathway. With this transcriptome sequencing, future genetic and genomics studies related to the molecular mechanisms associated with the chemical composition of P. tenuifolia may be improved. Genes involved in the enrichment of secondary metabolite biosynthesis-related pathways could enhance the potential applications of P. tenuifolia in pharmaceutical industries.

  3. An overview of the non-mevalonate pathway for terpenoid ...

    Indian Academy of Sciences (India)

    Unknown

    methyl-2-(E)-butenyl-4-diphosphate; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase ... functional roles in plants, human health and commerce. ... done in E. coli and demonstration of orthologous genes in various plants, encoding such enzymes that catalyze the same reaction.

  4. Arabinogalactan biosynthesis

    DEFF Research Database (Denmark)

    Poulsen, Christian Peter; Dilokpimol, Adiphol; Geshi, Naomi

    2015-01-01

    Arabinogalactan proteins are abundant cell surface proteoglycans in plants and are implicated to act as developmental markers during plant growth. We previously reported that AtGALT31A, AtGALT29A, and AtGLCAT14A-C, which are involved in the biosynthesis of arabinogalactan proteins, localize......), from Arabidopsis thaliana that co-localize with AtGALT31A in the small compartments when expressed transiently in Nicotiana benthamiana. These proteins may play roles in nucleotide sugar metabolism in the small compartments together with arabinogalactan glycosyltransferases....

  5. Isopentenyl diphosphate isomerase: A checkpoint to isoprenoid biosynthesis.

    Science.gov (United States)

    Berthelot, Karine; Estevez, Yannick; Deffieux, Alain; Peruch, Frédéric

    2012-08-01

    Even if the isopentenyl diphosphate (IPP) isomerases have been discovered in the 50s, it is only in the last decade that the genetical, enzymatical, structural richness and cellular importance of this large family of crucial enzymes has been uncovered. Present in all living kingdoms, they can be classified in two subfamilies: type 1 and type 2 IPP isomerases, which show clearly distinct characteristics. They all perform the regulatory isomerization of isopentenyl diphosphate into dimethylallyl diphosphate, a key rate-limiting step of the terpenoid biosynthesis, via a protonation/deprotonation mechanism. Due to their importance in the isoprenoid metabolism and the increasing interest of industry devoted to terpenoid production, it is foreseen that the biotechnological development of such enzymes should be under intense scrutiny in the near future. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  6. Sesamol decreases melanin biosynthesis in melanocyte cells and zebrafish: Possible involvement of MITF via the intracellular cAMP and p38/JNK signalling pathways

    OpenAIRE

    Baek, Seung Hwa; Lee, Sang Han

    2015-01-01

    Abstract The development of antimelanogenic agents is important for the prevention of serious aesthetic problems such as melasma, freckles, age spots and chloasma. The aim of this study was to investigate the antimelanogenic effect of sesamol, an active lignan isolated from Sesamum indicum, in melan?a cells. Sesamol strongly inhibited melanin biosynthesis and the activity of intracellular tyrosinase by decreasing cyclic adenosine monophosphate (cAMP) accumulation. Sesamol significantly decrea...

  7. The Δ4-desaturation pathway for DHA biosynthesis is operative in the human species: Differences between normal controls and children with the Zellweger syndrome

    OpenAIRE

    Martinez Manuela; Ichaso Natalia; Setien Fernando; Durany Nuria; Qiu Xiao; Roesler William

    2010-01-01

    Abstract Background Docosahexaenoic acid (DHA, 22:6ω3) is a fundamental component of cell membranes, especially in the brain and retina. In the experimental animal, DHA deficiency leads to suboptimal neurological performance and visual deficiencies. Children with the Zellweger syndrome (ZS) have a profound DHA deficiency and symptoms that can be attributed to their extremely low DHA levels. These children seem to have a metabolic defect in DHA biosynthesis, which has never been totally elucid...

  8. Genetic mapping and characterization of the globe artichoke (+)-germacrene A synthase gene, encoding the first dedicated enzyme for biosynthesis of the bitter sesquiterpene lactone cynaropicrin.

    Science.gov (United States)

    Menin, Barbara; Comino, Cinzia; Portis, Ezio; Moglia, Andrea; Cankar, Katarina; Bouwmeester, Harro J; Lanteri, Sergio; Beekwilder, Jules

    2012-07-01

    Globe artichoke (Cynara cardunculus var. scolymus L., Asteraceae) is a perennial crop traditionally consumed as a vegetable in the Mediterranean countries and rich in nutraceutically and pharmaceutically active compounds, including phenolic and terpenoid compounds. Its bitter taste is caused by its high content of sesquiterpene lactones (STLs), such as cynaropicrin. The biosynthetic pathway responsible for STL biosynthesis in globe artichoke is unknown, but likely proceeds through germacrene A, as has been shown for other Asteraceae species. Here, we investigated the accumulation of cynaropicrin in different tissues of globe artichoke, and compared it to accumulation of phenolic compounds. Cynaropicrin concentration was highest in old leaves. A putative germacrene A synthase (GAS) gene was identified in a set of ~19,000 globe artichoke unigenes. When heterologously expressed in Escherichia coli, the putative globe artichoke GAS converted farnesyl diphosphate (FPP) into (+)-germacrene A. Among various tissues assayed, the level of globe artichoke GAS expression was highest in mature (six week old) leaves. A sequence polymorphism within a mapping population parent allowed the corresponding GAS gene to be positioned on a genetic map. This study reports the isolation, expression and mapping of a key gene involved in STL biosynthesis in C. cardunculus. This is a good basis for further investigation of this pathway. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  9. The influence of abscisic acid on the ethylene biosynthesis pathway in the functioning of the flower abscission zone in Lupinus luteus.

    Science.gov (United States)

    Wilmowicz, Emilia; Frankowski, Kamil; Kućko, Agata; Świdziński, Michał; de Dios Alché, Juan; Nowakowska, Anna; Kopcewicz, Jan

    2016-11-01

    Flower abscission is a highly regulated developmental process activated in response to exogenous (e.g. changing environmental conditions) and endogenous stimuli (e.g. phytohormones). Ethylene (ET) and abscisic acid (ABA) are very effective stimulators of flower abortion in Lupinus luteus, which is a widely cultivated species in Poland, Australia and Mediterranean countries. In this paper, we show that artificial activation of abscission by flower removal caused an accumulation of ABA in the abscission zone (AZ). Moreover, the blocking of that phytohormone's biosynthesis by NDGA (nordihydroguaiaretic acid) decreased the number of abscised flowers. However, the application of NBD - an inhibitor of ET action - reversed the stimulatory effect of ABA on flower abscission, indicating that ABA itself is not sufficient to turn on the organ separation. Our analysis revealed that exogenous ABA significantly accelerated the transcriptional activity of the ET biosynthesis genes ACC synthase (LlACS) and oxidase (LlACO), and moreover, strongly increased the level of 1-aminocyclopropane-1-carboxylic acid (ACC) - ET precursor, which was specifically localized within AZ cells. We cannot exclude the possibility that ABA mediates flower abscission processes by enhancing the ET biosynthesis rate. The findings of our study will contribute to the overall basic knowledge on the phytohormone-regulated generative organs abscission in L. luteus. Copyright © 2016 Elsevier GmbH. All rights reserved.

  10. Sesamol decreases melanin biosynthesis in melanocyte cells and zebrafish: Possible involvement of MITF via the intracellular cAMP and p38/JNK signalling pathways.

    Science.gov (United States)

    Baek, Seung-hwa; Lee, Sang-Han

    2015-10-01

    The development of antimelanogenic agents is important for the prevention of serious aesthetic problems such as melasma, freckles, age spots and chloasma. The aim of this study was to investigate the antimelanogenic effect of sesamol, an active lignan isolated from Sesamum indicum, in melan-a cells. Sesamol strongly inhibited melanin biosynthesis and the activity of intracellular tyrosinase by decreasing cyclic adenosine monophosphate (cAMP) accumulation. Sesamol significantly decreased the expression of melanogenesis-related genes, such as tyrosinase, tyrosinase-related protein-1,2 (TRP-1,2), microphthalmia-associated transcription factor (MITF) and melanocortin 1 receptor (MC1R). In addition, sesamol also induces phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK). Moreover, sesamol dose-dependently decreased zebrafish pigment formation, tyrosinase activity and expression of melanogenesis-related genes. These findings indicate that sesamol inhibited melanin biosynthesis by down-regulating tyrosinase activity and melanin production via regulation of gene expression of melanogenesis-related proteins through modulation of MITF activity, which promoted phosphorylation of p38 and JNK in melan-a cells. Together, these results suggest that sesamol strongly inhibits melanin biosynthesis, and therefore, sesamol represents a new skin-whitening agent for use in cosmetics. © 2015 The Authors. Experimental Dermatology Published by John Wiley & Sons Ltd.

  11. Terpenoid marker compounds derived from biogenic precursors in volcanic ash from Mount St. Helens, Washington

    Science.gov (United States)

    Pereira, W.E.; Rostad, C.E.

    1983-01-01

    A volcanic-ash sample obtained after the 1980 eruption of Mount St. Helens, Washington, was analyzed for cyclic terpenoid organic compounds and polycyclic aromatic hydrocarbons using capillary gas chromatography-mass spectrometry-computer techniques. Various tricyclic diterpenoid acids and hydrocarbons were identified including dehydroabietic acid, dehydroabietin, dehydroabietane, simonellite, and retene. Preliminary evidence indicates that these compounds were derived from forest soils or atmospheric aerosols or both in the vicinity of coniferous forests. A diagenetic scheme involving three possible pathways for the conversion of abietic acid to retene is presented. ?? 1983.

  12. Auxin biosynthesis and storage forms.

    Science.gov (United States)

    Korasick, David A; Enders, Tara A; Strader, Lucia C

    2013-06-01

    The plant hormone auxin drives plant growth and morphogenesis. The levels and distribution of the active auxin indole-3-acetic acid (IAA) are tightly controlled through synthesis, inactivation, and transport. Many auxin precursors and modified auxin forms, used to regulate auxin homeostasis, have been identified; however, very little is known about the integration of multiple auxin biosynthesis and inactivation pathways. This review discusses the many ways auxin levels are regulated through biosynthesis, storage forms, and inactivation, and the potential roles modified auxins play in regulating the bioactive pool of auxin to affect plant growth and development.

  13. Integrated metabolome and transcriptome analysis of Magnolia champaca identifies biosynthetic pathways for floral volatile organic compounds.

    Science.gov (United States)

    Dhandapani, Savitha; Jin, Jingjing; Sridhar, Vishweshwaran; Sarojam, Rajani; Chua, Nam-Hai; Jang, In-Cheol

    2017-06-14

    Magnolia champaca, commonly known as champak is a well-known tree due to its highly fragrant flowers. Champak floral scent is attributed to a complex mix of volatile organic compounds (VOCs). These aromatic flowers are widely used in flavors and fragrances industry. Despite its commercial importance, the VOC biosynthesis pathways in these flowers are largely unknown. Here, we combine metabolite and RNA sequencing (RNA-seq) analyses of fully opened champak flowers to discover the active VOC biosynthesis pathways as well as floral scent-related genes. Volatile collection by headspace method and analysis by gas chromatography-mass spectrometry (GC-MS) identified a total of 43 VOCs from fully opened champak flowers, of which 46.9% were terpenoids, 38.9% were volatile esters and 5.2% belonged to phenylpropanoids/benzenoids. Sequencing and de novo assembly of champak flower transcriptome yielded 47,688 non-redundant unigenes. Transcriptome assembly was validated using standard polymerase chain reaction (PCR) based approach for randomly selected unigenes. The detailed profiles of VOCs led to the discovery of pathways and genes involved in floral scent biosynthesis from RNA-seq data. Analysis of expression levels of many floral-scent biosynthesis-related unigenes in flowers and leaves showed that most of them were expressed higher in flowers than in leaf tissues. Moreover, our metabolite-guided transcriptomics, in vitro and in vivo enzyme assays and transgenic studies identified (R)-linalool synthase that is essential for the production of major VOCs of champak flowers, (R)-linalool and linalool oxides. As our study is the first report on transcriptome analysis of Magnolia champaca, this transcriptome dataset that serves as an important public information for functional genomics will not only facilitate better understanding of ecological functions of champak floral VOCs, but also provide biotechnological targets for sustainable production of champak floral scent.

  14. Characterization of Flavan-3-ols and Expression of MYB and Late Pathway Genes Involved in Proanthocyanidin Biosynthesis in Foliage of Vitis bellula

    Directory of Open Access Journals (Sweden)

    Ke-Gang Li

    2013-03-01

    Full Text Available Proanthocyanidins (PAs are fundamental nutritional metabolites in different types of grape products consumed by human beings. Although the biosynthesis of PAs in berry of Vitis vinifera has gained intensive investigations, the understanding of PAs in other Vitis species is limited. In this study, we report PA formation and characterization of gene expression involved in PA biosynthesis in leaves of V. bellula, a wild edible grape species native to south and south-west China. Leaves are collected at five developmental stages defined by sizes ranging from 0.5 to 5 cm in length. Analyses of thin layer chromatography (TLC and high performance liquid chromatography-photodiode array detector (HPLC-PAD show the formation of (+-catechin, (−-epicatechin, (+-gallocatechin and (−-epigallocatechin during the entire development of leaves. Analyses of butanol-HCl boiling cleavage coupled with spectrometry measurement at 550 nm show a temporal trend of extractable PA levels, which is characterized by an increase from 0.5 cm to 1.5 cm long leaves followed by a decrease in late stages. TLC and HPLC-PAD analyses identify cyanidin, delphinidin and pelargonidin produced from the cleavage of PAs in the butanol-HCl boiling, showing that the foliage PAs of V. bellula include three different types of extension units. Four cDNAs, which encode VbANR, VbDFR, VbLAR1 and VbLAR2, respectively, are cloned from young leaves. The expression patterns of VbANR and VbLAR2 but not VbLAR1 and VbDFR follow a similar trend as the accumulation patterns of PAs. Two cDNAs encoding VbMYBPA1 and VbMYB5a, the homologs of which have been demonstrated to regulate the expression of both ANR and LAR in V. vinifera, are also cloned and their expression profiles are similar to those of VbANR and VbLAR2. In contrast, the expression profiles of MYBA1 and 2 homologs involved in anthocyanin biosynthesis are different from those of VbANR and VbLAR2. Our data show that both ANR and LAR branches are

  15. Analysis of the Staphylococcus aureus capsule biosynthesis pathway in vitro: characterization of the UDP-GlcNAc C6 dehydratases CapD and CapE and identification of enzyme inhibitors.

    Science.gov (United States)

    Li, Wenjin; Ulm, Hannah; Rausch, Marvin; Li, Xue; O'Riordan, Katie; Lee, Jean C; Schneider, Tanja; Müller, Christa E

    2014-11-01

    Polysaccharide capsules significantly contribute to virulence of invasive pathogens, and inhibition of capsule biosynthesis may offer a valuable strategy for novel anti-infective treatment. We purified and characterized the enzymes CapD and CapE of the Staphylococcus aureus serotype 5 biosynthesis cluster, which catalyze the first steps in the synthesis of the soluble capsule precursors UDP-D-FucNAc and UDP-L-FucNAc, respectively. CapD is an integral membrane protein and was obtained for the first time in a purified, active form. A capillary electrophoresis (CE)-based method applying micellar electrokinetic chromatography (MEKC) coupled with UV detection at 260 nm was developed for functional characterization of the enzymes using a fused-silica capillary, electrokinetic injection, and dynamic coating with polybrene at pH 12.4. The limits of detection for the CapD and CapE products UDP-2-acetamido-2,6-dideoxy-α-D-xylo-hex-4-ulose and UDP-2-acetamido-2,6-dideoxy-β-L-arabino-hex-4-ulose, respectively, were below 1 μM. Using this new, robust and sensitive method we performed kinetic studies for CapD and CapE and screened a compound library in search for enzyme inhibitors. Several active compounds were identified and characterized, including suramin (IC50 at CapE 1.82 μM) and ampicillin (IC50 at CapD 40.1 μM). Furthermore, the cell wall precursors UDP-D-MurNAc-pentapeptide and lipid II appear to function as inhibitors of CapD enzymatic activity, suggesting an integrated mechanism of regulation for cell envelope biosynthesis pathways in S. aureus. Corroborating the in vitro findings, staphylococcal cells grown in the presence of subinhibitory concentrations of ampicillin displayed drastically reduced CP production. Our studies contribute to a profound understanding of the capsule biosynthesis in pathogenic bacteria. This approach may lead to the identification of novel anti-virulence and antibiotic drugs. Copyright © 2014 Elsevier GmbH. All rights reserved.

  16. Pyrosequencing of the Camptotheca acuminata transcriptome reveals putative genes involved in camptothecin biosynthesis and transport

    Directory of Open Access Journals (Sweden)

    Sun Yongzhen

    2011-10-01

    Full Text Available Abstract Background Camptotheca acuminata is a Nyssaceae plant, often called the "happy tree", which is indigenous in Southern China. C. acuminata produces the terpenoid indole alkaloid, camptothecin (CPT, which exhibits clinical effects in various cancer treatments. Despite its importance, little is known about the transcriptome of C. acuminata and the mechanism of CPT biosynthesis, as only few nucleotide sequences are included in the GenBank database. Results From a constructed cDNA library of young C. acuminata leaves, a total of 30,358 unigenes, with an average length of 403 bp, were obtained after assembly of 74,858 high quality reads using GS De Novo assembler software. Through functional annotation, a total of 21,213 unigenes were annotated at least once against the NCBI nucleotide (Nt, non-redundant protein (Nr, Uniprot/SwissProt, Kyoto Encyclopedia of Genes and Genomes (KEGG, and Arabidopsis thaliana proteome (TAIR databases. Further analysis identified 521 ESTs representing 20 enzyme genes that are involved in the backbone of the CPT biosynthetic pathway in the library. Three putative genes in the upstream pathway, including genes for geraniol-10-hydroxylase (CaPG10H, secologanin synthase (CaPSCS, and strictosidine synthase (CaPSTR were cloned and analyzed. The expression level of the three genes was also detected using qRT-PCR in C. acuminata. With respect to the branch pathway of CPT synthesis, six cytochrome P450s transcripts were selected as candidate transcripts by detection of transcript expression in different tissues using qRT-PCR. In addition, one glucosidase gene was identified that might participate in CPT biosynthesis. For CPT transport, three of 21 transcripts for multidrug resistance protein (MDR transporters were also screened from the dataset by their annotation result and gene expression analysis. Conclusion This study produced a large amount of transcriptome data from C. acuminata by 454 pyrosequencing. According to

  17. Isoprenoid biosynthesis via the MEP pathway: in vivo Mössbauer spectroscopy identifies a [4Fe-4S]2+ center with unusual coordination sphere in the LytB protein.

    Science.gov (United States)

    Seemann, Myriam; Janthawornpong, Karnjapan; Schweizer, Julia; Böttger, Lars H; Janoschka, Adam; Ahrens-Botzong, Anne; Tambou, Erasmienne Ngouamegne; Rotthaus, Olaf; Trautwein, Alfred X; Rohmer, Michel

    2009-09-23

    The MEP pathway for the biosynthesis of isoprene units is present in most pathogenic bacteria, in the parasite responsible for malaria, and in plant plastids. This pathway is absent in animals and is accordingly a target for the development of antimicrobial drugs. LytB, also called IspH, the last enzyme of this pathway catalyzes the conversion of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) into a mixture of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) using an oxygen sensitive iron sulfur cluster. The exact nature of this iron sulfur cluster is still a matter of debate. We have used (57)Fe Mössbauer spectroscopy to investigate the LytB cluster in whole E. coli cells and in the anaerobically purified enzyme: In LytB an unusual [4Fe-4S](2+) cluster is attached to the protein by three conserved cysteines and contains a hexacoordinated iron linked to three sulfurs of the cluster and three additional oxygen or nitrogen ligands.

  18. Draft genome of Omphalotus olearius provides a predictive framework for sesquiterpenoid natural product biosynthesis in Basidiomycota

    OpenAIRE

    Wawrzyn, Grayson T.; Quin, Maureen B.; Choudhary, Swati; López-Gallego, Fernando; Schmidt-Dannert, Claudia

    2012-01-01

    The secondary metabolome of Basidiomycota represents a largely uncharacterized source of pharmaceutically relevant natural products. Terpenoids are the primary class of bioactive compounds isolated from mushrooms. The Jack O’Lantern mushroom Omphalotus olearius was identified 50 years ago as a prolific producer of anticancer illudin sesquiterpenoids, however to date there have been exceptionally few studies into the biosynthesis of these important compounds. Here we report the draft genome se...

  19. Engineering microorganisms for improving polyhydroxyalkanoate biosynthesis.

    Science.gov (United States)

    Chen, Guo-Qiang; Jiang, Xiao-Ran

    2017-11-20

    Biosynthesis of polyhydroxyalkanoates (PHA) has been studied since the 1920s. The biosynthesis pathways have been well understood and various attempts have been made to improve the PHA biosynthesis efficiency. Recent progresses have been focused on systematic improvements on PHA biosynthesis including changing growth pattern for rapid proliferation, engineering to enlarge cell sizes for more PHA accumulation space, reprogramming the PHA synthesis pathways using optimized RBS and promoter, redirecting metabolic flux to PHA synthesis using CRISPR/Cas9 tools, and very importantly, the employment of non-traditional host such as halophiles for reduced complexity on PHA production. All of the efforts should lead to ultrahigh PHA accumulation, controllable PHA compositions and molecular weights, open and continuous PHA production with gravity separation processes, resulting in competitive PHA production cost. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Hairy root biotechnology of Rauwolfia serpentina: a potent approach for the production of pharmaceutically important terpenoid indole alkaloids.

    Science.gov (United States)

    Mehrotra, Shakti; Goel, Manoj K; Srivastava, Vikas; Rahman, Laiq Ur

    2015-02-01

    Hairy root cultures of Rauwolfia serpentina induced by Agrobacterium rhizogenes have been investigated extensively for the production of terpenoid indole alkaloids. Various biotechnological developments, such as scaling up in bioreactors, pathway engineering etc., have been explored to improve their metabolite production potential. These hairy roots are competent for regenerating into complete plants and show survival and unaltered biosynthetic potential during storage at low temperature. This review provides a comprehensive account of the hairy root cultures of R. serpentina, their biosynthetic potential and various biotechnological methods used to explore the production of pharmaceutically important terpenoid indole alkaloids. The review also indicates how biotechnological endeavors might improve the future progress of research for production of alkaloids using Rauwolfia hairy roots.

  1. Salviprzols A and B, C₂₁- and C₂₂-terpenoids from the roots of Salvia przewalskii Maxim.

    Science.gov (United States)

    Xue, Yongbo; Wu, Ye; Zhu, Hucheng; Li, Xiao-Nian; Qian, Jin-Fu; Lai, Yongji; Chen, Chunmei; Yao, Guangmin; Luo, Zengwei; Li, Yan; Zhang, Yonghui

    2014-12-01

    Two C₂₁- and C₂₂-terpenoids, salviprzols A (1) and B (2), together with 24 known compounds including 17 diterpenoids (3-19), a triterpenoid (20), and 6 phenolic derivatives (21-26), were isolated from the roots of Salvia przewalskii Maxim. Salviprzols A and B represented a new subtype of C₂₃-terpenoids featured by an additional 2-oxopropyl moiety at C-12 and a rare γ-hydroxyl-α-methyl-α,β-unsaturated-γ-lactone ring system. Their structures were elucidated by extensive spectroscopic analyses, and the structure of 2 was confirmed by a single-crystal X-ray diffraction crystallography. The cytotoxic activities of the new isolates were tested. A plausible biosynthetic pathway for 1 and 2 was also proposed. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Sulforaphane, a cancer chemopreventive agent, induces pathways associated with membrane biosynthesis in response to tissue damage by aflatoxin B{sub 1}

    Energy Technology Data Exchange (ETDEWEB)

    Techapiesancharoenkij, Nirachara [Laboratory of Environmental Toxicology, Chulabhorn Research Institute, Bangkok 10210 (Thailand); Fiala, Jeannette L.A. [Department of Biological Engineering and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Navasumrit, Panida [Laboratory of Environmental Toxicology, Chulabhorn Research Institute, Bangkok 10210 (Thailand); Croy, Robert G.; Wogan, Gerald N. [Department of Biological Engineering and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Groopman, John D. [Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205 (United States); Ruchirawat, Mathuros [Laboratory of Environmental Toxicology, Chulabhorn Research Institute, Bangkok 10210 (Thailand); Essigmann, John M., E-mail: jessig@mit.edu [Department of Biological Engineering and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States)

    2015-01-01

    Aflatoxin B{sub 1} (AFB{sub 1}) is one of the major risk factors for liver cancer globally. A recent study showed that sulforaphane (SF), a potent inducer of phase II enzymes that occurs naturally in widely consumed vegetables, effectively induces hepatic glutathione S-transferases (GSTs) and reduces levels of hepatic AFB{sub 1}-DNA adducts in AFB{sub 1}-exposed Sprague Dawley rats. The present study characterized the effects of SF pre-treatment on global gene expression in the livers of similarly treated male rats. Combined treatment with AFB{sub 1} and SF caused reprogramming of a network of genes involved in signal transduction and transcription. Changes in gene regulation were observable 4 h after AFB{sub 1} administration in SF-pretreated animals and may reflect regeneration of cells in the wake of AFB{sub 1}-induced hepatotoxicity. At 24 h after AFB{sub 1} administration, significant induction of genes that play roles in cellular lipid metabolism and acetyl-CoA biosynthesis was detected in SF-pretreated AFB{sub 1}-dosed rats. Induction of this group of genes may indicate a metabolic shift toward glycolysis and fatty acid synthesis to generate and maintain pools of intermediate molecules required for tissue repair, cell growth and compensatory hepatic cell proliferation. Collectively, gene expression data from this study provide insights into molecular mechanisms underlying the protective effects of SF against AFB{sub 1} hepatotoxicity and hepatocarcinogenicity, in addition to the chemopreventive activity of this compound as a GST inducer. - Highlights: • This study revealed sulforaphane (SF)-deregulated gene sets in aflatoxin B{sub 1} (AFB{sub 1})-treated rat livers. • SF redirects biochemical networks toward lipid biosynthesis in AFB{sub 1}-dosed rats. • SF enhanced gene sets that would be expected to favor cell repair and regeneration.

  3. Engineering Microbial Cells for the Biosynthesis of Natural Compounds of Pharmaceutical Significance

    Directory of Open Access Journals (Sweden)

    Philippe Jeandet

    2013-01-01

    Full Text Available Microbes constitute important platforms for the biosynthesis of numerous molecules of pharmaceutical interest such as antitumor, anticancer, antiviral, antihypertensive, antiparasitic, antioxidant, immunological agents, and antibiotics as well as hormones, belonging to various chemical families, for instance, terpenoids, alkaloids, polyphenols, polyketides, amines, and proteins. Engineering microbial factories offers rich opportunities for the production of natural products that are too complex for cost-effective chemical synthesis and whose extraction from their originating plants needs the use of many solvents. Recent progresses that have been made since the millennium beginning with metabolic engineering of microorganisms for the biosynthesis of natural products of pharmaceutical significance will be reviewed.

  4. Stable heterologous expression of biologically active terpenoids in green plant cells

    DEFF Research Database (Denmark)

    Binti Khairul Ikram, Nur Kusaira; Zhan, Xin; Pan, Xiwu

    2015-01-01

    Plants biosynthesize a great diversity of biologically active small molecules of interest for fragrances, flavors, and pharmaceuticals. Among specialized metabolites, terpenoids represent the greatest molecular diversity. Many terpenoids are very complex, and total chemical synthesis often requires...... heterologous expression for production of valuable plant-derived natural products. In this review, our primary focus is on small terpenoids and we discuss the benefits of plant expression platforms and provide several successful examples of stable production of small terpenoids in plants....

  5. (-)-Menthol biosynthesis and molecular genetics

    Science.gov (United States)

    Croteau, Rodney B.; Davis, Edward M.; Ringer, Kerry L.; Wildung, Mark R.

    2005-12-01

    (-)-Menthol is the most familiar of the monoterpenes as both a pure natural product and as the principal and characteristic constituent of the essential oil of peppermint ( Mentha x piperita). In this paper, we review the biosynthesis and molecular genetics of (-)-menthol production in peppermint. In Mentha species, essential oil biosynthesis and storage is restricted to the peltate glandular trichomes (oil glands) on the aerial surfaces of the plant. A mechanical method for the isolation of metabolically functional oil glands, has provided a system for precursor feeding studies to elucidate pathway steps, as well as a highly enriched source of the relevant biosynthetic enzymes and of their corresponding transcripts with which cDNA libraries have been constructed to permit cloning and characterization of key structural genes. The biosynthesis of (-)-menthol from primary metabolism requires eight enzymatic steps, and involves the formation and subsequent cyclization of the universal monoterpene precursor geranyl diphosphate to the parent olefin (-)-(4 S)-limonene as the first committed reaction of the sequence. Following hydroxylation at C3, a series of four redox transformations and an isomerization occur in a general “allylic oxidation-conjugate reduction” scheme that installs three chiral centers on the substituted cyclohexanoid ring to yield (-)-(1 R, 3 R, 4 S)-menthol. The properties of each enzyme and gene of menthol biosynthesis are described, as are their probable evolutionary origins in primary metabolism. The organization of menthol biosynthesis is complex in involving four subcellular compartments, and regulation of the pathway appears to reside largely at the level of gene expression. Genetic engineering to up-regulate a flux-limiting step and down-regulate a side route reaction has led to improvement in the composition and yield of peppermint oil.

  6. Terpenoids from Euphorbia soongarica and Their Multidrug Resistance Reversal Activity.

    Science.gov (United States)

    Gao, Jie; Aisa, Haji A

    2017-06-23

    Ten new terpenoids, including five diterpenoids (1-5), three nortriterpenoids (6-8), and two triterpenoids (9, 10), and 15 known terpenoids (11-25) were isolated from an acetone extract of Euphorbia soongarica. Sooneuphoramine (1) is the first example of a euphoractine B-type diterpenoid alkaloid, while sooneuphanones A-C (6-8) are rare nortriterpenoids from the Euphorbia genus. The isolated terpenoids were tested for their cytotoxicity and multidrug resistance (MDR) reversal activity, 10 of which showed moderate cytotoxicity against the KB and KBv200 cell lines, while 11 compounds exhibited P-gp modulating potential. The triterpenoid sooneuphanone D (9) possessed a remarkable MDR reversal activity much higher than the positive control, verapamil.

  7. Turmeric (Curcuma longa): miRNAs and their regulating targets are involved in development and secondary metabolite pathways.

    Science.gov (United States)

    Singh, Noopur; Sharma, Ashok

    Turmeric has been used as a therapeutic herb over centuries in traditional medicinal systems due to the presence of several secondary metabolite compounds. microRNAs are known to regulate gene expression at the post-transcriptional level by transcriptional cleavage or translation repression. miRNAs have been demonstrated to play an active role in secondary metabolism regulation. The present work was focused on the identification of the miRNAs involved in the regulation of secondary metabolite and development process of turmeric. Eighteen miRNA families were identified for turmeric. Sixteen miRNA families were observed to regulate 238 target transcripts. LncRNAs targets of the putative miRNA candidates were also predicted. Our results indicated their role in binding, reproduction, stress, and other developmental processes. Gene annotation and pathway analysis illustrated the biological function of the targets regulated by the putative miRNAs. The miRNA-mediated gene regulatory network also revealed co-regulated targets that were regulated by two or more miRNA families. miR156 and miR5015 were observed to be involved in rhizome development. miR5021 showed regulation for terpenoid backbone biosynthesis and isoquinoline alkaloid biosynthesis pathways. The flavonoid biosynthesis pathway was observed to be regulated by miR2919. The analysis revealed the probable involvement of three miRNAs (miR1168.2, miR156b and miR1858) in curcumin biosynthesis. Other miRNAs were found to be involved in the growth and developmental process of turmeric. Phylogenetic analysis of selective miRNAs was also performed. Copyright © 2017 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.

  8. A new family of enzymes catalyzing the first committed step of the methylerythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in bacteria.

    Science.gov (United States)

    Sangari, Félix J; Pérez-Gil, Jordi; Carretero-Paulet, Lorenzo; García-Lobo, Juan M; Rodríguez-Concepción, Manuel

    2010-08-10

    Isoprenoids are a large family of compounds with essential functions in all domains of life. Most eubacteria synthesize their isoprenoids using the methylerythritol 4-phosphate (MEP) pathway, whereas a minority uses the unrelated mevalonate pathway and only a few have both. Interestingly, Brucella abortus and some other bacteria that only use the MEP pathway lack deoxyxylulose 5-phosphate (DXP) reductoisomerase (DXR), the enzyme catalyzing the NADPH-dependent production of MEP from DXP in the first committed step of the pathway. Fosmidomycin, a specific competitive inhibitor of DXR, inhibited growth of B. abortus cells expressing the Escherichia coli GlpT transporter (required for fosmidomycin uptake), confirming that a DXR-like (DRL) activity exists in these bacteria. The B. abortus DRL protein was found to belong to a family of uncharacterized proteins similar to homoserine dehydrogenase. Subsequent experiments confirmed that DRL and DXR catalyze the same biochemical reaction. DRL homologues shown to complement a DXR-deficient E. coli strain grouped within the same phylogenetic clade. The scattered taxonomic distribution of sequences from the DRL clade and the occurrence of several paralogues in some bacterial strains might be the result of lateral gene transfer and lineage-specific gene duplications and/or losses, similar to that described for typical mevalonate and MEP pathway genes. These results reveal the existence of a novel class of oxidoreductases catalyzing the conversion of DXP into MEP in prokaryotic cells, underscoring the biochemical and genetic plasticity achieved by bacteria to synthesize essential compounds such as isoprenoids.

  9. The bHLH transcription factor BIS1 controls the iridoid branch of the monoterpenoid indole alkaloid pathway in Catharanthus roseus.

    Science.gov (United States)

    Van Moerkercke, Alex; Steensma, Priscille; Schweizer, Fabian; Pollier, Jacob; Gariboldi, Ivo; Payne, Richard; Vanden Bossche, Robin; Miettinen, Karel; Espoz, Javiera; Purnama, Purin Candra; Kellner, Franziska; Seppänen-Laakso, Tuulikki; O'Connor, Sarah E; Rischer, Heiko; Memelink, Johan; Goossens, Alain

    2015-06-30

    Plants make specialized bioactive metabolites to defend themselves against attackers. The conserved control mechanisms are based on transcriptional activation of the respective plant species-specific biosynthetic pathways by the phytohormone jasmonate. Knowledge of the transcription factors involved, particularly in terpenoid biosynthesis, remains fragmentary. By transcriptome analysis and functional screens in the medicinal plant Catharanthus roseus (Madagascar periwinkle), the unique source of the monoterpenoid indole alkaloid (MIA)-type anticancer drugs vincristine and vinblastine, we identified a jasmonate-regulated basic helix-loop-helix (bHLH) transcription factor from clade IVa inducing the monoterpenoid branch of the MIA pathway. The bHLH iridoid synthesis 1 (BIS1) transcription factor transactivated the expression of all of the genes encoding the enzymes that catalyze the sequential conversion of the ubiquitous terpenoid precursor geranyl diphosphate to the iridoid loganic acid. BIS1 acted in a complementary manner to the previously characterized ethylene response factor Octadecanoid derivative-Responsive Catharanthus APETALA2-domain 3 (ORCA3) that transactivates the expression of several genes encoding the enzymes catalyzing the conversion of loganic acid to the downstream MIAs. In contrast to ORCA3, overexpression of BIS1 was sufficient to boost production of high-value iridoids and MIAs in C. roseus suspension cell cultures. Hence, BIS1 might be a metabolic engineering tool to produce sustainably high-value MIAs in C. roseus plants or cultures.

  10. Escherichia coli phnN, encoding ribose 1,5-bisphosphokinase activity (phosphoribosyl diphosphate forming): dual role in phosphonate degradation and NAD biosynthesis pathways

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Rosenkrantz, Tina J; Haldimann, Andreas

    2003-01-01

    An enzymatic pathway for synthesis of 5-phospho-D-ribosyl alpha-1-diphosphate (PRPP) without the participation of PRPP synthase was analyzed in Escherichia coli. This pathway was revealed by selection for suppression of the NAD requirement of strains with a deletion of the prs gene, the gene...... encoding PRPP synthase (B. Hove-Jensen, J. Bacteriol. 178:714-722, 1996). The new pathway requires three enzymes: phosphopentomutase, ribose 1-phosphokinase, and ribose 1,5-bisphosphokinase. The latter activity is encoded by phnN; the product of this gene is required for phosphonate degradation, but its......-phosphorus (C-P) bond by a C-P lyase. The phnN gene was manipulated in vitro to encode a variant of ribose 1,5-bisphosphokinase with a tail consisting of six histidine residues at the carboxy-terminal end. PhnN was purified almost to homogeneity and characterized. The enzyme accepted ATP but not GTP...

  11. Bioconversion of methanol to value-added mevalonate by engineered Methylobacterium extorquens AM1 containing an optimized mevalonate pathway.

    Science.gov (United States)

    Zhu, Wen-Liang; Cui, Jin-Yu; Cui, Lan-Yu; Liang, Wei-Fan; Yang, Song; Zhang, Chong; Xing, Xin-Hui

    2016-03-01

    Methylotrophic biosynthesis using methanol as a feedstock is a promising and attractive method to solve the over-dependence of the bioindustry on sugar feedstocks derived from grains that are used for food. In this study, we introduced and engineered the mevalonate pathway into Methylobacterium extorquens AM1 to achieve high mevalonate production from methanol, which could be a platform for terpenoid synthesis. We first constructed a natural operon (MVE) harboring the mvaS and mvaE genes from Enterococcus faecalis as well as an artificial operon (MVH) harboring the hmgcs1 gene from Blattella germanica and the tchmgr gene from Trypanosoma cruzi that encoded enzymes with the highest reported activities. We achieved mevalonate titers of 56 and 66 mg/L, respectively, in flask cultivation. Introduction of the phaA gene from Ralstonia eutropha into the operon MVH increased the mevalonate titer to 180 mg/L, 3.2-fold higher than that of the natural operon MVE. Further modification of the expression level of the phaA gene by regulating the strength of the ribosomal binding site resulted in an additional 20 % increase in mevalonate production to 215 mg/L. A fed-batch fermentation of the best-engineered strain yielded a mevalonate titer of 2.22 g/L, which was equivalent to an overall yield and productivity of 28.4 mg mevalonate/g methanol and 7.16 mg/L/h, respectively. The production of mevalonate from methanol, which is the initial, but critical step linking methanol with valuable terpenoids via methylotrophic biosynthesis, represents a proof of concept for pathway engineering in M. extorquens AM1.

  12. Identification and characterization of biosynthetic components involved in Vitex agnus-castus diterpenes biosynthesis

    DEFF Research Database (Denmark)

    Sundram, Tamil Chelvan Meenakshi

    as host platforms for biosynthesis of several high-value terpenoids (Chapter 2). Here, in this PhD project we initially investigated the biosynthesis of Vitex agnus-castus diterpenoids. Bioactive diterpenoids detected in Vitex agnus-castus were reported to be beneficial for the treatment of menstrual...... variants of plastidial targeting sequence truncated cDNAs. We next attempted to engineer a green platform for V. agnus-castus diterpenes biosynthesis by expressing the characterized (VacTPSs) in Synechocystis sp. PCC 6803. To this extend, we only managed to synthesize syn-copalyl diphosphate by expressing...... the VacTPS3 in Synechocystis cells (Chapter 4). In summary, through this study we discovered and characterized VacTPSs and P450 enzymes involved in the first steps of Vitex agnus-castus diterpenoids biosynthesis. Besides, we explored the alternative possibility of producing V. agnus -castus diterpenes...

  13. Quantitative structure-activity relationship of terpenoid aphid antifeedants.

    Science.gov (United States)

    Wang, Zongde; Song, Jie; Han, Zhaojiu; Jiang, Zhikuan; Zheng, Weiqing; Chen, Jinzhu; Song, Zhanqian; Shang, Shibin

    2008-12-10

    A series of terpenoid compounds containing a six-member-ring were synthesized from alpha- and beta-pinenes. Antifeedant activity of these terpenoid compounds were tested on the aphid, Lipaphis erysimi (Kalt.), with promising results. Stepwise regression was applied to study the quantitative structure-activity relationship of these compounds. The statistically best model showed that the relative number of O atoms, molecular volume, HOMO-LUMO energy gap, and total charge on the positively charged fragments were the most statistically significant descriptors to predict the antifeedant activity. The possible mechanism of interaction between the antifeedant and aphid chemoreceptor was discussed.

  14. Variations in terrigenous matter transport evaluated by plant terpenoid analysis in the Neogene eastern equatorial Pacific sediment

    Science.gov (United States)

    Nakamura, H.; Sawada, K.; Yamamoto, S.; Kobayashi, M.

    2012-12-01

    We analyze land plant biomarkers in sediments recovered during the IODP Expeditions 320/321, the Pacific Equatorial Age Transect (PEAT), to reconstruct variations in terrestrial input in the eastern equatorial Pacific Ocean, as well as to evaluate transport systems of terrigenous matter from land to ocean. We used the Neogene sediments (23-0.23 Ma) recovered from site U1337. We could identify cadalene (sesquiterpenoid), as well as series of aromatic abietane type diterpenoids (e.g. retene) from most samples. These terpenoids are derived from biomolecules that constitute plant resin and supportive tissues. Plant-derived compounds in deep-sea sediments are considered that initially transported via fluvial process, followed by redistribution by gravity flow to the deeper part. Although, the typical pelagic setting of site U1337, even far from land across the mid-ocean-ridge, exclude such pathway. Therefore, the variations in mass accumulation rate (MAR) of plant terpenoids in this site depend eolian transportation and can be used as an indicator of change in atmospheric circulation. The increasing spikes in MAR of plant terpenoids were observed in the horizons of 16 Ma, 14 Ma, 12 Ma, 10 Ma, 9 - 8 Ma, 7 Ma and 2.5 - 1Ma. It is found that some of the spikes, 14 Ma, 7 Ma, 2.5 - 1 Ma correspond to the East Antarctica Ice Sheet event (EAIS), the Biogenic bloom event, and the Northern Hemisphere Glaciation (NHG), respectively. Furthermore, the degree of aromatization in diterpenoids, which represented by retene to the sum of abietane type diterpenoids, tends to increase concurrently with the MAR of total plant terpenoids. The aromatization of plant terpenoids take place under thermal maturation after deposition as well as in earlier processes such as biomass burning. In the U1337 sediments, the degree of aromatization of diterpenoids was uncorrelated with the age or depositional depth, suggesting its variations retain potential for paleoclimatic application. For example

  15. Adrenal C11-oxy C21 steroids contribute to the C11-oxy C19 steroid pool via the backdoor pathway in the biosynthesis and metabolism of 21-deoxycortisol and 21-deoxycortisone.

    Science.gov (United States)

    Barnard, Lise; Gent, Rachelle; van Rooyen, Desmaré; Swart, Amanda C

    2017-11-01

    21-Hydroxylase deficiency presents with increased levels of cytochrome P450 21-hydroxylase substrates, progesterone and 17α-hydroxyprogesterone, which have been implicated in the production of androgens via the backdoor pathway. This study shows the biosynthesis of C11-oxy C21 steroids, 21-deoxycortisol and 21-deoxycortisone, and their metabolism by steroidogenic enzymes in the backdoor pathway yielding novel steroid metabolites: 5α-pregnan-11β,17α-diol-3,20-dione; 5α-pregnan-17α-ol-3,11,20-trione; 5α-pregnan-3α,11β,17α-triol-20-one and 5α-pregnan-3α,17α-diol-11,20-dione. The metabolism of 21-deoxycortisol was validated in LNCaP cells expressing the relevant steroidogenic enzymes showing for the first time that the steroid, produced at high levels in 21OHD, is metabolised via the C11-oxy derivatives of 5α-pregnan-17α-ol-3,20-dione and 5α-pregnan-3α,17α-diol-20-one to substrates for the lyase activity of CYP17A1, leading to the production of C11-oxy C19 steroids. 21-Deoxycortisol thus contributes to the pool of potent androgens in 21OHD, with novel steroid metabolites also presenting possible biomarkers in disease identification. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Manipulation of carbon flux into fatty acid biosynthesis pathway in Dunaliella salina using AccD and ME genes to enhance lipid content and to improve produced biodiesel quality

    Directory of Open Access Journals (Sweden)

    Ahmad Farhad Talebi

    2014-08-01

    Full Text Available Advanced generations of biofuels basically revolve around non-agricultural energy crops. Among those, microalgae owing to its unique characteristics i.e. natural tolerance to waste and saline water, sustainable biomass production and high lipid content (LC, is regarded by many as the ultimate choice for the production of various biofuels such as biodiesel. In the present study, manipulation of carbon flux into fatty acid biosynthesis pathway in Dunaliella salina was achieved using pGH plasmid harboring AccD and ME genes to enhance lipid content and to improve produced biodiesel quality. The stability of transformation was confirmed by PCR after several passages. Southern hybridization of AccD probe with genomic DNA revealed stable integration of the cassette in the specific positions in the chloroplast genome with no read through transcription by indigenous promoters. Comparison of the LC and fatty acid profile of the transformed algal cell line and the control revealed the over-expression of the ME/AccD genes in the transformants leading to 12% increase in total LC and significant improvements in biodiesel properties especially by increasing algal oil oxidation stability. The whole process successfully implemented herein for transforming algal cells by genes involved in lipid production pathway could be helpful for large scale biodiesel production from microalgae.

  17. Glyphosate’s Suppression of Cytochrome P450 Enzymes and Amino Acid Biosynthesis by the Gut Microbiome: Pathways to Modern Diseases

    Directory of Open Access Journals (Sweden)

    Anthony Samsel

    2013-04-01

    Full Text Available Glyphosate, the active ingredient in Roundup®, is the most popular herbicide used worldwide. The industry asserts it is minimally toxic to humans, but here we argue otherwise. Residues are found in the main foods of the Western diet, comprised primarily of sugar, corn, soy and wheat. Glyphosate's inhibition of cytochrome P450 (CYP enzymes is an overlooked component of its toxicity to mammals. CYP enzymes play crucial roles in biology, one of which is to detoxify xenobiotics. Thus, glyphosate enhances the damaging effects of other food borne chemical residues and environmental toxins. Negative impact on the body is insidious and manifests slowly over time as inflammation damages cellular systems throughout the body. Here, we show how interference with CYP enzymes acts synergistically with disruption of the biosynthesis of aromatic amino acids by gut bacteria, as well as impairment in serum sulfate transport. Consequences are most of the diseases and conditions associated with a Western diet, which include gastrointestinal disorders, obesity, diabetes, heart disease, depression, autism, infertility, cancer and Alzheimer’s disease. We explain the documented effects of glyphosate and its ability to induce disease, and we show that glyphosate is the “textbook example” of exogenous semiotic entropy: the disruption of homeostasis by environmental toxins.

  18. Lipoteichoic acid induces surfactant protein-A biosynthesis in human alveolar type II epithelial cells through activating the MEK1/2-ERK1/2-NF-κB pathway

    Directory of Open Access Journals (Sweden)

    Liu Feng-Lin

    2012-10-01

    Full Text Available Abstract Background Lipoteichoic acid (LTA, a gram-positive bacterial outer membrane component, can cause septic shock. Our previous studies showed that the gram-negative endotoxin, lipopolysaccharide (LPS, could induce surfactant protein-A (SP-A production in human alveolar epithelial (A549 cells. Objectives In this study, we further evaluated the effect of LTA on SP-A biosynthesis and its possible signal-transducing mechanisms. Methods A549 cells were exposed to LTA. Levels of SP-A, nuclear factor (NF-κB, extracellular signal-regulated kinase 1/2 (ERK1/2, and mitogen-activated/extracellular signal-regulated kinase kinase (MEK1 were determined. Results Exposure of A549 cells to 10, 30, and 50 μg/ml LTA for 24 h did not affect cell viability. Meanwhile, when exposed to 30 μg/ml LTA for 1, 6, and 24 h, the biosynthesis of SP-A mRNA and protein in A549 cells significantly increased. As to the mechanism, LTA enhanced cytosolic and nuclear NF-κB levels in time-dependent manners. Pretreatment with BAY 11–7082, an inhibitor of NF-κB activation, significantly inhibited LTA-induced SP-A mRNA expression. Sequentially, LTA time-dependently augmented phosphorylation of ERK1/2. In addition, levels of phosphorylated MEK1 were augmented following treatment with LTA. Conclusions Therefore, this study showed that LTA can increase SP-A synthesis in human alveolar type II epithelial cells through sequentially activating the MEK1-ERK1/2-NF-κB-dependent pathway.

  19. Differential contribution of the first two enzymes of the MEP pathway to the supply of metabolic precursors for carotenoid and chlorophyll biosynthesis in carrot (Daucus carota

    Directory of Open Access Journals (Sweden)

    Kevin Simpson

    2016-08-01

    Full Text Available Carotenoids and chlorophylls are photosynthetic pigments synthesized in plastids from metabolic precursors provided by the methylerythritol 4-phosphate (MEP pathway. The first two steps in the MEP pathway are catalyzed by the deoxyxylulose 5-phosphate synthase (DXS and reductoisomerase (DXR enzymes. While DXS has been recently shown to be the main flux-controlling step of the MEP pathway, both DXS and DXR enzymes have been proven to be able to promote an increase in MEP-derived products when overproduced in diverse plant systems. Carrot (Daucus carota produces photosynthetic pigments (carotenoids and chlorophylls in leaves and in light-exposed roots, whereas only carotenoids (mainly α- and β-carotene accumulate in the storage root in darkness. To evaluate whether DXS and DXR activities influence the production of carotenoids and chlorophylls in carrot leaves and roots, the corresponding Arabidopsis thaliana genes were constitutively expressed in transgenic carrot plants. Our results suggest that DXS is limiting for the production of both carotenoids and chlorophylls in roots and leaves, whereas the regulatory role of DXR appeared to be minor. Interestingly, increased levels of DXS (but not of DXR resulted in higher transcript abundance of endogenous carrot genes encoding phytoene synthase, the main rate-determining enzyme of the carotenoid pathway. These results support a central role for DXS on modulating the production of MEP-derived precursors to synthesize carotenoids and chlorophylls in carrot, confirming the pivotal relevance of this enzyme to engineer healthier, carotenoid-enriched products.

  20. Differential Contribution of the First Two Enzymes of the MEP Pathway to the Supply of Metabolic Precursors for Carotenoid and Chlorophyll Biosynthesis in Carrot (Daucus carota).

    Science.gov (United States)

    Simpson, Kevin; Quiroz, Luis F; Rodriguez-Concepción, Manuel; Stange, Claudia R

    2016-01-01

    Carotenoids and chlorophylls are photosynthetic pigments synthesized in plastids from metabolic precursors provided by the methylerythritol 4-phosphate (MEP) pathway. The first two steps in the MEP pathway are catalyzed by the deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR) enzymes. While DXS has been recently shown to be the main flux-controlling step of the MEP pathway, both DXS and DXR enzymes have been proven to be able to promote an increase in MEP-derived products when overproduced in diverse plant systems. Carrot (Daucus carota) produces photosynthetic pigments (carotenoids and chlorophylls) in leaves and in light-exposed roots, whereas only carotenoids (mainly α- and β-carotene) accumulate in the storage root in darkness. To evaluate whether DXS and DXR activities influence the production of carotenoids and chlorophylls in carrot leaves and roots, the corresponding Arabidopsis thaliana genes were constitutively expressed in transgenic carrot plants. Our results suggest that DXS is limiting for the production of both carotenoids and chlorophylls in roots and leaves, whereas the regulatory role of DXR appeared to be minor. Interestingly, increased levels of DXS (but not of DXR) resulted in higher transcript abundance of endogenous carrot genes encoding phytoene synthase, the main rate-determining enzyme of the carotenoid pathway. These results support a central role for DXS on modulating the production of MEP-derived precursors to synthesize carotenoids and chlorophylls in carrot, confirming the pivotal relevance of this enzyme to engineer healthier, carotenoid-enriched products.

  1. A single nucleotide mutation of IspF gene involved in the MEP pathway for isoprenoid biosynthesis causes yellow-green leaf phenotype in rice.

    Science.gov (United States)

    Huang, Rui; Wang, Yang; Wang, Pingrong; Li, Chunmei; Xiao, Fuliang; Chen, Nenggang; Li, Na; Li, Caixia; Sun, Changhui; Li, Lihua; Chen, Rongjun; Xu, Zhengjun; Zhu, Jianqing; Deng, Xiaojian

    2017-11-15

    We identified IspF gene through yellow-green leaf mutant 505ys in rice. OsIspF was expressed in all tissues detected, and its encoded protein was targeted to the chloroplast. On expression levels of genes in this mutant, OsIspF itself and the genes encoding other enzymes of the MEP pathway and chlorophyll synthase were all up-regulated, however, among eight genes associated with photosynthesis, only psaA, psaN and psbA genes for three reaction center subunits of photosystem obviously changed. Isoprenoids are the most abundant natural compounds in all organisms, which originate from the basic five-carbon units isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In plants, IPP and DMAPP are synthesized through two independent pathways, the mevalonic acid pathway in cytoplasm and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway in plastids. The MEP pathway comprises seven enzymatic steps, in which IspF is the fifth enzyme. So far, no IspF gene has been identified in monocotyledonous plants. In this study, we isolated a leaf-color mutant, 505ys, in rice (Oryza sativa). The mutant displayed yellow-green leaf phenotype, reduced level of photosynthetic pigments, and arrested development of chloroplasts. By map-based cloning of this mutant, we identified OsIspF gene (LOC_Os02g45660) showing significant similarity to IspF gene of Arabidopsis, in which a missense mutation occurred in the mutant, resulting in an amino acid change in the encoded protein. OsIspF gene was expressed in all tissues detected, and its encoded protein was targeted to the chloroplast. Further, the mutant phenotype of 505ys was complemented by transformation with the wild-type OsIspF gene. Therefore, we successfully identified an IspF gene in monocotyledonous plants. In addition, real-time quantitative RT-PCR implied that a positive regulation could exist between the OsIspF gene and the genes encoding other enzymes of the MEP pathway and chlorophyll synthase. At the same time, it

  2. Outlining eicosanoid biosynthesis in the crustacean Daphnia

    Directory of Open Access Journals (Sweden)

    Timmermans Martijn JTN

    2008-07-01

    Full Text Available Abstract Background Eicosanoids are biologically active, oxygenated metabolites of three C20 polyunsaturated fatty acids. They act as signalling molecules within the autocrine or paracrine system in both vertebrates and invertebrates mainly functioning as important mediators in reproduction, the immune system and ion transport. The biosynthesis of eicosanoids has been intensively studied in mammals and it is known that they are synthesised from the fatty acid, arachidonic acid, through either the cyclooxygenase (COX pathway; the lipoxygenase (LOX pathway; or the cytochrome P450 epoxygenase pathway. However, little is still known about the synthesis and structure of the pathway in invertebrates. Results Here, we show transcriptomic evidence from Daphnia magna (Crustacea: Branchiopoda together with a bioinformatic analysis of the D. pulex genome providing insight on the role of eicosanoids in these crustaceans as well as outlining a putative pathway of eicosanoid biosynthesis. Daphnia appear only to have one copy of the gene encoding the key enzyme COX, and phylogenetic analysis reveals that the predicted protein sequence of Daphnia COX clusters with other invertebrates. There is no current evidence of an epoxygenase pathway in Daphnia; however, LOX products are most certainly synthesised in daphnids. Conclusion We have outlined the structure of eicosanoid biosynthesis in Daphnia, a key genus in freshwater ecosystems. Improved knowledge of the function and synthesis of eicosanoids in Daphnia and other invertebrates could have important implications for several areas within ecology. This provisional overview of daphnid eicosanoid biosynthesis provides a guide on where to focus future research activities in this area.

  3. Outlining eicosanoid biosynthesis in the crustacean Daphnia.

    Science.gov (United States)

    Heckmann, Lars-Henrik; Sibly, Richard M; Timmermans, Martijn Jtn; Callaghan, Amanda

    2008-07-14

    Eicosanoids are biologically active, oxygenated metabolites of three C20 polyunsaturated fatty acids. They act as signalling molecules within the autocrine or paracrine system in both vertebrates and invertebrates mainly functioning as important mediators in reproduction, the immune system and ion transport. The biosynthesis of eicosanoids has been intensively studied in mammals and it is known that they are synthesised from the fatty acid, arachidonic acid, through either the cyclooxygenase (COX) pathway; the lipoxygenase (LOX) pathway; or the cytochrome P450 epoxygenase pathway. However, little is still known about the synthesis and structure of the pathway in invertebrates. Here, we show transcriptomic evidence from Daphnia magna (Crustacea: Branchiopoda) together with a bioinformatic analysis of the D. pulex genome providing insight on the role of eicosanoids in these crustaceans as well as outlining a putative pathway of eicosanoid biosynthesis. Daphnia appear only to have one copy of the gene encoding the key enzyme COX, and phylogenetic analysis reveals that the predicted protein sequence of Daphnia COX clusters with other invertebrates. There is no current evidence of an epoxygenase pathway in Daphnia; however, LOX products are most certainly synthesised in daphnids. We have outlined the structure of eicosanoid biosynthesis in Daphnia, a key genus in freshwater ecosystems. Improved knowledge of the function and synthesis of eicosanoids in Daphnia and other invertebrates could have important implications for several areas within ecology. This provisional overview of daphnid eicosanoid biosynthesis provides a guide on where to focus future research activities in this area.

  4. Integration of Residue Attributes for Sequence Diversity Characterization of Terpenoid Enzymes

    Science.gov (United States)

    Ikeda, Shun; Ono, Naoaki; Altaf-Ul-Amin, Md.; Kanaya, Shigehiko

    2014-01-01

    Progress in the “omics” fields such as genomics, transcriptomics, proteomics, and metabolomics has engendered a need for innovative analytical techniques to derive meaningful information from the ever increasing molecular data. KNApSAcK motorcycle DB is a popular database for enzymes related to secondary metabolic pathways in plants. One of the challenges in analyses of protein sequence data in such repositories is the standard notation of sequences as strings of alphabetical characters. This has created lack of a natural underlying metric that eases amenability to computation. In view of this requirement, we applied novel integration of selected biochemical and physical attributes of amino acids derived from the amino acid index and quantified in numerical scale, to examine diversity of peptide sequences of terpenoid synthases accumulated in KNApSAcK motorcycle DB. We initially generated a reduced amino acid index table. This is a set of biochemical and physical properties obtained by random forest feature selection of important indices from the amino acid index. Principal component analysis was then applied for characterization of enzymes involved in synthesis of terpenoids. The variance explained was increased by incorporation of residue attributes for analyses. PMID:24900985

  5. High-throughput testing of terpenoid biosynthesis candidate genes using transient expression in Nicotiana benthamiana

    DEFF Research Database (Denmark)

    Bach, Søren Spanner; Bassard, Jean-Étienne André; Andersen-Ranberg, Johan

    2014-01-01

    to the native compartments, unlike microbial or prokaryotic expression systems. Two inherent drawbacks of plant-based expression systems, time-consuming generation of transgenic plant lines and challenging gene-stacking, can be circumvented by transient expression in Nicotiana benthamiana. In this chapter we...

  6. Volatile non-terpenoid hydrocarbons from Ligusticum grayi roots

    Science.gov (United States)

    The root essential oil of Ligusticum grayi Coult. & Rose (Apiaceae) contains three volatile non-terpenoid hydrocarbons: the known C11 compound viridene, whose structure is hereby corrected to 1-[(2Z)-pent-2-en-1-yl]cyclohexa-1,3-diene; and the heretofore unreported C10 compounds 1-[(2Z)-but-2-en-1-y...

  7. Iodine, a Mild Reagent for the Aromatization of Terpenoids.

    Science.gov (United States)

    Domingo, Victoriano; Prieto, Consuelo; Silva, Lucia; Rodilla, Jesús M L; Quílez del Moral, José F; Barrero, Alejandro F

    2016-04-22

    Efficient procedures based on the use of iodine for the aromatization of a series of terpenoids possessing diene and homoallylic or allylic alcohol functionalities are described. Different examples are reported as a proof-of-concept study. Furthermore, iodine also proved to mediate the dehydrogenation of testosterone.

  8. Terpenoids of Sinularia soft corals: chemistry and bioactivity

    Directory of Open Access Journals (Sweden)

    Wen-ting Chen

    2012-06-01

    Full Text Available Soft corals of the genus Sinularia are one of the most widespread soft corals. They are a rich source of bioactive substances with intriguing and unique structural features. The present paper reviews the latest progress in the chemistry and pharmacological activities of terpenoids from Sinularia soft corals and provides a perspective on future areas of research interest.

  9. Terpenoid composition and class of Tertiary resins from India

    Energy Technology Data Exchange (ETDEWEB)

    Dutta, Suryendu; Mallick, Monalisa; Mathews, Runcie Paul [Department of Earth Sciences, Indian Institute of Technology Bombay, Powai, Mumbai-400076 (India); Bertram, Norbert [LTA-Labor fuer Toxikologie und Analytik, Friedrichshoeher Str. 28, D-53639 Koenigswinter (Germany); Greenwood, Paul F. [John De Laeter Mass Spectrometry and WA Biogeochemitry Centres (M090), The University of Western Australia, 35 Stirling Hwy, Crawley, WA, 6009 (Australia); WA - Organic and Isotope Geochemistry Centre, Curtin University of Technology, Kent St., Bentley 6102 (Australia)

    2009-10-01

    The terpenoid composition and class of Tertiary resins preserved within lignites of Cambay, Kutch and Cauvery Basins of India have been characterized using Pyrolysis-Gas Chromatography-Mass Spectrometry (Py-GC-MS) and Fourier Transform Infrared (FTIR) Spectroscopy. Major pyrolysis products include cadalene-based C{sub 15}-bicyclic sesquiterpenoids with some C{sub 30} and C{sub 31} bicadinanes and bicadinenes typical of Class II or dammar resin. The occurrence of these terpenoids in Early Eocene sediments may extend the first appearance of Dipterocarpaceae angiosperms, the predominant source of this resin class, back to the Early Eocene epoch in India. The same terpenoid biomarkers have been detected in many SE Asian oils reflecting a close source relationship with these resins. Strong CH{sub 3} (1377 cm{sup -} {sup 1}) and other CH{sub x} (3000-2800 and 1460-1450 cm{sup -} {sup 1}) aliphatic absorptions of much larger intensity than the aromatic C = C (1560-1650 cm{sup -} {sup 1}) absorption were detected in the Indian resins by FTIR Spectroscopy, confirming the quantitative significance of the terpenoid pyrolysates. (author)

  10. Terpenoid compounds from the latex of Euphorbia drupifera ...

    African Journals Online (AJOL)

    This study aimed at the isolation and characterization of terpenoid compounds from the latex of Euphorbia drupifera. Methylated spirit extract of the latex was suspended in aqueous methanol and partitioned with petroleum spirit. The aqueous methanol residue was washed with ether. The ethereal fraction was subjected to ...

  11. Auxin biosynthesis and its role in plant development

    Science.gov (United States)

    Zhao, Yunde

    2011-01-01

    Indole-3-acetic acid (IAA), the main auxin in higher plants, has profound effects on plant growth and development. Both plants and some plant pathogens can produce IAA to modulate plant growth. While the genes and biochemical reactions for auxin biosynthesis in some plant pathogens are well understood, elucidation of the mechanisms by which plants produce auxin has proven to be difficult. So far, no complete pathway of de novo auxin biosynthesis in plants has been firmly established. However, recent studies have led to the discoveries of several genes in tryptophan dependent auxin biosynthesis pathways. Recent findings have also revealed that local auxin biosynthesis plays essential roles in many developmental processes including gametogenesis, embryogenesis, seedling growth, vascular patterning, and flower development. In this review, I summarize the recent advances in dissecting auxin biosynthetic pathways and how the understanding of auxin biosynthesis provides a different angle for analyzing the mechanisms of plant development. PMID:20192736

  12. Random mutagenesis in Corynebacterium glutamicum ATCC 13032 using an IS6100-based transposon vector identified the last unknown gene in the histidine biosynthesis pathway

    Directory of Open Access Journals (Sweden)

    Gaigalat Lars

    2006-08-01

    Full Text Available Abstract Background Corynebacterium glutamicum, a Gram-positive bacterium of the class Actinobacteria, is an industrially relevant producer of amino acids. Several methods for the targeted genetic manipulation of this organism and rational strain improvement have been developed. An efficient transposon mutagenesis system for the completely sequenced type strain ATCC 13032 would significantly advance functional genome analysis in this bacterium. Results A comprehensive transposon mutant library comprising 10,080 independent clones was constructed by electrotransformation of the restriction-deficient derivative of strain ATCC 13032, C. glutamicum RES167, with an IS6100-containing non-replicative plasmid. Transposon mutants had stable cointegrates between the transposon vector and the chromosome. Altogether 172 transposon integration sites have been determined by sequencing of the chromosomal inserts, revealing that each integration occurred at a different locus. Statistical target site analyses revealed an apparent absence of a target site preference. From the library, auxotrophic mutants were obtained with a frequency of 2.9%. By auxanography analyses nearly two thirds of the auxotrophs were further characterized, including mutants with single, double and alternative nutritional requirements. In most cases the nutritional requirement observed could be correlated to the annotation of the mutated gene involved in the biosynthesis of an amino acid, a nucleotide or a vitamin. One notable exception was a clone mutagenized by transposition into the gene cg0910, which exhibited an auxotrophy for histidine. The protein sequence deduced from cg0910 showed high sequence similarities to inositol-1(or 4-monophosphatases (EC 3.1.3.25. Subsequent genetic deletion of cg0910 delivered the same histidine-auxotrophic phenotype. Genetic complementation of the mutants as well as supplementation by histidinol suggests that cg0910 encodes the hitherto unknown

  13. Metabolic plasticity for isoprenoid biosynthesis in bacteria.

    Science.gov (United States)

    Pérez-Gil, Jordi; Rodríguez-Concepción, Manuel

    2013-05-15

    Isoprenoids are a large family of compounds synthesized by all free-living organisms. In most bacteria, the common precursors of all isoprenoids are produced by the MEP (methylerythritol 4-phosphate) pathway. The MEP pathway is absent from archaea, fungi and animals (including humans), which synthesize their isoprenoid precursors using the completely unrelated MVA (mevalonate) pathway. Because the MEP pathway is essential in most bacterial pathogens (as well as in the malaria parasites), it has been proposed as a promising new target for the development of novel anti-infective agents. However, bacteria show a remarkable plasticity for isoprenoid biosynthesis that should be taken into account when targeting this metabolic pathway for the development of new antibiotics. For example, a few bacteria use the MVA pathway instead of the MEP pathway, whereas others possess the two full pathways, and some parasitic strains lack both the MVA and the MEP pathways (probably because they obtain their isoprenoids from host cells). Moreover, alternative enzymes and metabolic intermediates to those of the canonical MVA or MEP pathways exist in some organisms. Recent work has also shown that resistance to a block of the first steps of the MEP pathway can easily be developed because several enzymes unrelated to isoprenoid biosynthesis can produce pathway intermediates upon spontaneous mutations. In the present review, we discuss the major advances in our knowledge of the biochemical toolbox exploited by bacteria to synthesize the universal precursors for their essential isoprenoids.

  14. Non-additive phenotypic and transcriptomic inheritance in a citrus allotetraploid somatic hybrid between C. reticulata and C. limon: the case of pulp carotenoid biosynthesis pathway.

    Science.gov (United States)

    Bassene, Jean Baptiste; Froelicher, Yann; Dhuique-Mayer, Claudie; Mouhaya, Waffa; Ferrer, Rosa Mar; Ancillo, Gema; Morillon, Raphael; Navarro, Luis; Ollitrault, Patrick

    2009-11-01

    Allopolyploidy is known to induce novel patterns of gene expression and often gives rise to new phenotypes. Here we report on the first attempt to relate phenotypic inheritance in an allotetraploid somatic hybrid with gene expression. Carotenoid compounds in the fruit pulp of the two parental species and the hybrid were evaluated quantitatively by HPLC. Only very low levels of beta-carotene and beta-cryptoxanthin were observed in Citrus limon, while beta-cryptoxanthin was a major component of C. reticulata, which also displayed high levels of phytoene, phytofluene, beta-carotene, lutein, zeaxantin and violaxanthin. Total carotenoid content in mandarin juice sacs was 60 times greater than that in lemon. The allotetraploid hybrid produced all the same compounds as mandarin but at very low levels. Transgressive concentration of abscisic acid (ABA) was observed in the somatic hybrid. Real-time RT-PCR of total RNA from juice sacs was used to study expression of seven genes (CitDxs, CitPsy, CitPds, CitZds, CitLcy-b, CitChx-b, and CitZep) of the carotenoid biosynthetic pathway and two genes (CitNced1 and CitNced2) involved in abscisic acid synthesis from carotenoid. Gene expression was significantly higher for mandarin than lemon for seven of the nine genes analyzed. Lemon under expression was partially dominant in the somatic hybrid for three upstream steps of the biosynthetic pathway, particularly for CitDxs. Transgressive over expression was observed for the two CitNced genes. A limitation of the upstream steps of the pathway and a downstream higher consumption of carotenoids may explain the phenotype of the somatic hybrid.

  15. Metabolic Characterization of the Anthocyanidin Reductase Pathway Involved in the Biosynthesis of Flavan-3-ols in Elite Shuchazao Tea (Camellia sinensis Cultivar in the Field

    Directory of Open Access Journals (Sweden)

    Lei Zhao

    2017-12-01

    Full Text Available Anthocyanidin reductase (ANR is a key enzyme in the ANR biosynthetic pathway of flavan-3-ols and proanthocyanidins (PAs in plants. Herein, we report characterization of the ANR pathway of flavan-3-ols in Shuchazao tea (Camellia sinesis, which is an elite and widely grown cultivar in China and is rich in flavan-3-ols providing with high nutritional value to human health. In our study, metabolic profiling was preformed to identify two conjugates and four aglycones of flavan-3-ols: (−-epigallocatechin-gallate [(−-EGCG], (−-epicatechin-gallate [(−-ECG], (−-epigallocatechin [(−-EGC], (−-epicatechin [(−-EC], (+-catechin [(+-Ca], and (+-gallocatechin [(+-GC], of which (−-EGCG, (−-ECG, (−-EGC, and (−-EC accounted for 70–85% of total flavan-3-ols in different tissues. Crude ANR enzyme was extracted from young leaves. Enzymatic assays showed that crude ANR extracts catalyzed cyanidin and delphinidin to (−-EC and (−-Ca and (−-EGC and (−-GC, respectively, in which (−-EC and (−-EGC were major products. Moreover, two ANR cDNAs were cloned from leaves, namely CssANRa and CssANRb. His-Tag fused recombinant CssANRa and CssANRb converted cyanidin and delphinidin to (−-EC and (−-Ca and (−-EGC and (−-GC, respectively. In addition, (+-EC was observed from the catalysis of recombinant CssANRa and CssANRb. Further overexpression of the two genes in tobacco led to the formation of PAs in flowers and the reduction of anthocyanins. Taken together, these data indicate that the majority of leaf flavan-3-ols in Shuchazao’s leaves were produced from the ANR pathway.

  16. Biosynthesis and role of 3-methylbutanal in cheese by lactic acid bacteria: Major metabolic pathways, enzymes involved, and strategies for control.

    Science.gov (United States)

    Afzal, Muhammad Inam; Ariceaga, Citlalli Celeste González; Boulahya, Kenza-Amel; Jacquot, Muriel; Delaunay, Stéphane; Cailliez-Grimal, Catherine

    2017-01-22

    Branched chain aldehyde, 3-methylbutanal is associated as a key flavor compound with many hard and semi-hard cheese varieties. The presence and impact of this flavor compound in bread, meat, and certain beverages has been recently documented, however its presence and consequences regarding cheese flavor were not clearly reported. This paper gives an overview of the role of 3-methylbutanal in cheese, along with the major metabolic pathways and key enzymes leading to its formation. Moreover, different strategies are highlighted for the control of this particular flavor compound in specific cheese types.

  17. Bile acid biosynthesis and its regulation

    Directory of Open Access Journals (Sweden)

    Areta Hebanowska

    2010-10-01

    Full Text Available Bile acid biosynthesis is the main pathway of cholesterol catabolism. Bile acids are more soluble than cholesterol so are easier to excrete. As amphipathic molecules they participate in lipid digestion and absorption in the intestine and they help to excrete free cholesterol with bile. They are also ligands for nuclear receptors regulating the expression of genes involved in cholesterol metabolism. Interconversion of cholesterol into bile acids is an important point of its homeostasis. Seventeen enzymes are engaged in this process and many of them are cytochromes P450. Bile acid synthesis initiation may proceed with the “classical” pathway (starting with cholesterol hydroxylation at the C7α position or the “alternative” pathway (starting with cholesterol hydroxylation at the C27 position. Two additional pathways are possible, though their quantitative significance is small (initiated with cholesterol hydroxylations of C24 and C25 positions. Oxysterols produced are not only intermediates of bile acid biosynthesis but also important regulators of metabolism. Bile acid biosynthesis takes place in the liver, but some enzymes are also present in other organs, where they participate in regulation of cholesterol metabolism. Those enzymes are potential targets for new drugs against cholesterol metabolism disturbances. This article is a brief description of the bile acid biosynthesis pathway and participating enzymes.

  18. Analysis of the ergosterol biosynthesis pathway cloning, molecular characterization and phylogeny of lanosterol 14α-demethylase (ERG11 gene of Moniliophthora perniciosa

    Directory of Open Access Journals (Sweden)

    Geruza de Oliveira Ceita

    2014-12-01

    Full Text Available The phytopathogenic fungus Moniliophthora perniciosa (Stahel Aime & Philips-Mora, causal agent of witches' broom disease of cocoa, causes countless damage to cocoa production in Brazil. Molecular studies have attempted to identify genes that play important roles in fungal survival and virulence. In this study, sequences deposited in the M. perniciosa Genome Sequencing Project database were analyzed to identify potential biological targets. For the first time, the ergosterol biosynthetic pathway in M. perniciosa was studied and the lanosterol 14α-demethylase gene (ERG11 that encodes the main enzyme of this pathway and is a target for fungicides was cloned, characterized molecularly and its phylogeny analyzed. ERG11 genomic DNA and cDNA were characterized and sequence analysis of the ERG11 protein identified highly conserved domains typical of this enzyme, such as SRS1, SRS4, EXXR and the heme-binding region (HBR. Comparison of the protein sequences and phylogenetic analysis revealed that the M. perniciosa enzyme was most closely related to that of Coprinopsis cinerea.

  19. Metabolic and transcriptional elucidation of the carotenoid biosynthesis pathway in peel and flesh tissue of loquat fruit during on-tree development.

    Science.gov (United States)

    Hadjipieri, Margarita; Georgiadou, Egli C; Marin, Alicia; Diaz-Mula, Huertas M; Goulas, Vlasios; Fotopoulos, Vasileios; Tomás-Barberán, Francisco A; Manganaris, George A

    2017-06-14

    Carotenoids are the main colouring substances found in orange-fleshed loquat fruits. The aim of this study was to unravel the carotenoid biosynthetic pathway of loquat fruit (cv. 'Obusa') in peel and flesh tissue during distinct on-tree developmental stages through a targeted analytical and molecular approach. Substantial changes regarding colour parameters, both between peel and flesh and among the different developmental stages, were monitored, concomitant with a significant increment in carotenoid content. Key genes and individual compounds that are implicated in the carotenoid biosynthetic pathway were further dissected with the employment of molecular (RT-qPCR) and advanced analytical techniques (LC-MS). Results revealed significant differences in carotenoid composition between peel and flesh. Thirty-two carotenoids were found in the peel, while only eighteen carotenoids were identified in the flesh. Trans-lutein and trans-β-carotene were the major carotenoids in the peel; the content of the former decreased with the progress of ripening, while the latter registered a 7.2-fold increase. However, carotenoid profiling of loquat flesh indicated trans-β-cryptoxanthin, followed by trans-β-carotene and 5,8-epoxy-β-carotene to be the most predominant carotenoids. High amounts of trans-β-carotene in both tissues were supported by significant induction in a chromoplast-specific lycopene β-cyclase (CYCB) transcript levels. PSY1, ZDS, CYCB and BCH were up-regulated and CRTISO, LCYE, ECH and VDE were down-regulated in most of the developmental stages compared with the immature stage in both peel and flesh tissue. Overall, differential regulation of expression levels with the progress of on-tree fruit development was more evident in the middle and downstream genes of carotenoid biosynthetic pathway. Carotenoid composition is greatly affected during on-tree loquat development with striking differences between peel and flesh tissue. A link between gene up- or down

  20. Cadmium and copper induced changes in growth, oxidative metabolism and terpenoids of Tanacetum parthenium.

    Science.gov (United States)

    Hojati, Mostafa; Modarres-Sanavy, Seyed Ali Mohammad; Enferadi, Sattar Tahmasebi; Majdi, Mohammad; Ghanati, Faezeh; Farzadfar, Soudeh; Pazoki, Alireza

    2017-05-01

    Morphological and biochemical responses of feverfew plants exposed to low (5 μM) and high (35 and 70 μM) levels of Cd or Cu were investigated. Increasing metal supply notably reduced the plant biomass. Elevated Cd and Cu levels also resulted in an increase in the leaf proline content. Besides, decrease in ascorbic acid (AsA) and glutathione (GSH) contents was similar in the leaves of Cd- and Cu-treated plants, indicating altered biosynthesis of AsA and GSH under metal excess. High metal doses stimulated increase in antioxidative enzyme activities that could be related to elevated hydrogen peroxide (H 2 O 2 ) content and subsequent lipid peroxidation. Cd was typically more accumulated in shoots and roots than Cu, leading to higher translocation factor at high Cd doses. In terms of essential oil content, it seems that Cd had an inhibitory effect during the experiment, whereas Cu was found to stimulate it only at 5 μM. Furthermore, high Cd supply enhanced the relative proportion of monoterpene hydrocarbons, while Cu increased the proportion of sesquiterpenes, especially at 5 μM. This result provides the first evidence of the response of feverfew plants to Cd or Cu by associating stress-related responses with changes in terpenoids.

  1. The Terpene Synthase Gene Family of Carrot (Daucus carota L.: Identification of QTLs and Candidate Genes Associated with Terpenoid Volatile Compounds

    Directory of Open Access Journals (Sweden)

    Jens Keilwagen

    2017-11-01

    Full Text Available Terpenes are an important group of secondary metabolites in carrots influencing taste and flavor, and some of them might also play a role as bioactive substances with an impact on human physiology and health. Understanding the genetic and molecular basis of terpene synthases (TPS involved in the biosynthesis of volatile terpenoids will provide insights for improving breeding strategies aimed at quality traits and for developing specific carrot chemotypes possibly useful for pharmaceutical applications. Hence, a combination of terpene metabolite profiling, genotyping-by-sequencing (GBS, and genome-wide association study (GWAS was used in this work to get insights into the genetic control of terpene biosynthesis in carrots and to identify several TPS candidate genes that might be involved in the production of specific monoterpenes. In a panel of 85 carrot cultivars and accessions, metabolite profiling was used to identify 31 terpenoid volatile organic compounds (VOCs in carrot leaves and roots, and a GBS approach was used to provide dense genome-wide marker coverage (>168,000 SNPs. Based on this data, a total of 30 quantitative trait loci (QTLs was identified for 15 terpenoid volatiles. Most QTLs were detected for the monoterpene compounds ocimene, sabinene, β-pinene, borneol and bornyl acetate. We identified four genomic regions on three different carrot chromosomes by GWAS which are both associated with high significance (LOD ≥ 5.91 to distinct monoterpenes and to TPS candidate genes, which have been identified by homology-based gene prediction utilizing RNA-seq data. In total, 65 TPS candidate gene models in carrot were identified and assigned to known plant TPS subfamilies with the exception of TPS-d and TPS-h. TPS-b was identified as largest subfamily with 32 TPS candidate genes.

  2. Spatial organization of the vindoline biosynthetic pathway in Catharanthus roseus.

    Science.gov (United States)

    Guirimand, Grégory; Guihur, Anthony; Poutrain, Pierre; Héricourt, François; Mahroug, Samira; St-Pierre, Benoit; Burlat, Vincent; Courdavault, Vincent

    2011-04-15

    Vindoline constitutes the main terpenoid indole alkaloid accumulated in leaves of Catharanthus roseus, and four genes involved in its biosynthesis have been identified. However, the spatial organization of the tabersonine-to-vindoline biosynthetic pathway is still incomplete. To pursue the characterization of this six-step conversion, we illustrated, with in situ hybridization, that the transcripts of the second biosynthetic enzyme, 16-hydroxytabersonine 16-O-methyltransferase (16OMT), are specifically localized to the aerial organ epidermis. At the subcellular level, by combining GFP imaging, bimolecular fluorescence complementation assays and yeast two-hybrid analysis, we established that the first biosynthetic enzyme, tabersonine 16-hydroxylase (T16H), is anchored to the ER as a monomer via a putative N-terminal helix that we cloned using a PCR approach. We also showed that 16OMT homodimerizes in the cytoplasm, allowing its exclusion from the nucleus and thus facilitating the uptake of T16H conversion product, although no T16H/16OMT interactions occur. Moreover, the two last biosynthetic enzymes, desacetoxyvindoline-4-hydroxylase (D4H) and deacetylvindoline-4-O-acetyltransferase (DAT), were shown to operate as monomers that reside in the nucleocytoplasmic compartment following passive diffusion to the nucleus allowed by the protein size. No D4H/DAT interactions were detected, suggesting the absence of metabolic channeling in the vindoline biosynthetic pathway. Finally, these results highlight the importance of the inter- and intracellular translocations of intermediates during the vindoline biosynthesis and their potential regulatory role. Copyright © 2010 Elsevier GmbH. All rights reserved.

  3. Probenazole treatment inhibits anthocyanins biosynthesis via ...

    African Journals Online (AJOL)

    ELO

    2012-01-05

    Jan 5, 2012 ... chalcone synthase (CHS), chalcone isomerase (CHI), dihydroflavonol reductase (DFR) and flavonoid 3- hydroxylase (F3H) genes have been found to be the essential components of the anthocyanins biosynthesis pathway (Boss et al., 1996; Romero et al., 2008; Xu and. Li, 2006). Probenazole (3-allyloxy-1, ...

  4. Combinatorial biosynthesis of medicinal plant secondary metabolites

    NARCIS (Netherlands)

    Julsing, Mattijs K.; Koulman, Albert; Woerdenbag, Herman J.; Quax, Wim J.; Kayser, Oliver

    2006-01-01

    Combinatorial biosynthesis is a new tool in the generation of novel natural products and for the production of rare and expensive natural products. The basic concept is combining metabolic pathways in different organisms on a genetic level. As a consequence heterologous organisms provide precursors

  5. Expression of important pathway genes involved in withanolides biosynthesis in hairy root culture of Withania somnifera upon treatment with Gracilaria edulis and Sargassum wightii.

    Science.gov (United States)

    Sivanandhan, Ganeshan; Arunachalam, Chinnathambi; Selvaraj, Natesan; Sulaiman, Ali Alharbi; Lim, Yong Pyo; Ganapathi, Andy

    2015-06-01

    The investigation of seaweeds, Gracilaria edulis and Sargassum wightii extracts was carried out for the estimation of growth characteristics and major withanolides production in hairy root culture of Withania somnifera. The extract of G. edulis (50%) in MS liquid basal medium enabled maximum production of dry biomass (5.46 g DW) and withanolides contents (withanolide A 5.23 mg/g DW; withaferin A 2.24 mg/g DW and withanone 4.83 mg/g DW) in hairy roots after 40 days of culture with 48 h contact time. The obtained withanolides contents were significantly higher (2.32-fold-2.66-fold) in hairy root culture when compared to the control. RT PCR analysis of important pathway genes such as SE, SS, HMGR and FPPS exhibited substantial higher expression upon the seaweed extracts treatment in hairy root culture. This experiment would paw a platform for withanolides production in hairy root culture with the influence of sea weed extracts for pharmaceutical companies in the future. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  6. The Sorghum Gene for Leaf Color Changes upon Wounding (P Encodes a Flavanone 4-Reductase in the 3-Deoxyanthocyanidin Biosynthesis Pathway

    Directory of Open Access Journals (Sweden)

    Hiroyuki Kawahigashi

    2016-05-01

    Full Text Available Upon wounding or pathogen invasion, leaves of sorghum [Sorghum bicolor (L. Moench] plants with the P gene turn purple, whereas leaves with the recessive allele turn brown or tan. This purple phenotype is determined by the production of two 3-deoxyanthocyanidins, apigeninidin and luteolinidin, which are not produced by the tan-phenotype plants. Using map-based cloning in progeny from a cross between purple Nakei-MS3B (PP and tan Greenleaf (pp cultivars, we isolated this gene, which was located in a 27-kb genomic region around the 58.1 Mb position on chromosome 6. Four candidate genes identified in this region were similar to the maize leucoanthocyanidin reductase gene. None of them was expressed before wounding, and only the Sb06g029550 gene was induced in both cultivars after wounding. The Sb06g029550 protein was detected in Nakei-MS3B, but only slightly in Greenleaf, in which it may be unstable because of a Cys252Tyr substitution. A recombinant Sb06g029550 protein had a specific flavanone 4-reductase activity, and converted flavanones (naringenin or eriodictyol to flavan-4-ols (apiforol or luteoforol in vitro. Our data indicate that the Sb06g029550 gene is involved in the 3-deoxyanthocyanidin synthesis pathway.

  7. DEVELOPING A YEAST CELL FACTORY FOR THE PRODUCTION OF TERPENOIDS

    Directory of Open Access Journals (Sweden)

    Sotirios C. Kampranis

    2012-10-01

    Full Text Available Technological developments over the past century have made microbes the work-horses of large scale industrial production processes. Current efforts focus on the metabolic engineering of microbial strains to produce high levels of desirable end-products. The arsenal of the contemporary metabolic engineer contains tools that allow either targeted rational interventions or global screens that combine classical approaches with –omics technologies. Production of terpenoids in S. cerevisiae presents a characteristic example of contemporary biotechnology that integrates all the variety of novel approaches used in metabolic engineering. Terpenoids have attracted significant interest as pharmaceuticals, flavour and fragrance additives, and, more recently, biofuels. The ongoing metabolic engineering efforts, combined with the continuously increasing number of terpene biosynthetic enzymes discovered will enable the economical and environmentally friendly production of a wide range of compounds.

  8. Developing a yeast cell factory for the production of terpenoids

    Directory of Open Access Journals (Sweden)

    Sotirios C Kampranis

    2012-10-01

    Full Text Available Technological developments over the past century have made microbes the work-horses of large scale industrial production processes. Current efforts focus on the metabolic engineering of microbial strains to produce high levels of desirable end-products. The arsenal of the contemporary metabolic engineer contains tools that allow either targeted rational interventions or global screens that combine classical approaches with –omics technologies. Production of terpenoids in S. cerevisiae presents a characteristic example of contemporary biotechnology that integrates all the variety of novel approaches used in metabolic engineering. Terpenoids have attracted significant interest as pharmaceuticals, flavour and fragrance additives, and, more recently, biofuels. The ongoing metabolic engineering efforts, combined with the continuously increasing number of terpene biosynthetic enzymes discovered will enable the economical and environmentally friendly production of a wide range of compounds.

  9. Progress in renewable polymers from natural terpenes, terpenoids, and rosin.

    Science.gov (United States)

    Wilbon, Perry A; Chu, Fuxiang; Tang, Chuanbing

    2013-01-11

    The development of sustainable renewable polymers from natural resources has increasingly gained attention from scientists, engineers as well as the general public and government agencies. This review covers recent progress in the field of renewable bio-based monomers and polymers from natural resources: terpenes, terpenoids, and rosin, which are a class of hydrocarbon-rich biomass with abundance and low cost, holding much potential for utilization as organic feedstocks for green plastics and composites. This review details polymerization and copolymerization of terpenes such as pinene, limonene, and myrcene and their derivatives, terpenoids including carvone and menthol, and rosin-derived monomers. The future direction on the utilization of these natural resources is discussed. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Structural Diversity and Biological Activity of the Genus Pieris Terpenoids.

    Science.gov (United States)

    Li, Chun-Huan; Yan, Xi-Tao; Zhang, An-Ling; Gao, Jin-Ming

    2017-11-22

    Secondary metabolites, particularly the grayanane diterpenoids produced by the members of genus Pieris, have been investigated in past decades for their remarkable antifeedant and insecticidal activities and toxicity. Grayanoids exhibit diverse biological properties such as antifeedant, insecticidal, cAMP regulatory, and sodium-channel-modulating activities. Structural complexity and diverse bioactivity of grayanoids have made them attractive targets for chemical, biological, and synthetic purposes. The current review synthesized findings published from 1966 to 2017, which include 135 reports that describe 130 terpenoids, including 103 grayanoids. The distribution, structure, skeleton, as well as the antifeedant and insecticidal activity of terpenoids, particularly the grayanane diterpenoids, are discussed in detail in this review. In cases where sufficient information is available, the structure-activity relationships of their antifeedant activity are also presented. We hope that this contribution will prompt more scientists to pay attention to these diterpenoids, which may be potentially applied in the agricultural field.

  11. Terpenoids as Potential Anti-Alzheimer’s Disease Therapeutics

    Directory of Open Access Journals (Sweden)

    So-Young Park

    2012-03-01

    Full Text Available Alzheimer’s disease (AD is one of the most well-known neurodegenerative diseases and explains 50–60% of dementia in patients. The prevalence rate of AD is positively correlated with age and AD affects ≥ 40% of those over 85 years old. The major AD therapeutics available on the market are acetylcholinesterase inhibitors, such as tacrine and donepezil. New therapeutic agents that can block the disease-inducing mechanisms are essential. Diverse efforts have been made to discover anti-AD agents from natural sources. In this review article, we describe some representative terpenoids such as ginsenosides, gingkolides, and canabinoids as potential anti-AD agents. These compounds exhibit promising in vitro and in vivo biological activities, but are still waiting clinical trials. Additionally, we also discuss some terpenoids including cornel iridoid glycoside, oleanolic acid, tenuifolin, cryptotanshinone, and ursolic acid, which are under investigation for their in vitro and in vivo animal studies.

  12. Production of Aromatic Plant Terpenoids in Recombinant Baker's Yeast.

    Science.gov (United States)

    Emmerstorfer-Augustin, Anita; Pichler, Harald

    2016-01-01

    Plant terpenoids are high-value compounds broadly applied as food additives or fragrances in perfumes and cosmetics. Their biotechnological production in yeast offers an attractive alternative to extraction from plants. Here, we provide two optimized protocols for the production of the plant terpenoid trans-nootkatol with recombinant S. cerevisiae by either (I) converting externally added (+)-valencene with resting cells or (II) cultivating engineered self-sufficient production strains. By synthesis of the hydrophobic compounds in self-sufficient production cells, phase transfer issues can be avoided and the highly volatile products can be enriched in and easily purified from n-dodecane, which is added to the cell broth as second phase.

  13. Estimating terpene and terpenoid emissions from conifer oleoresin composition

    Science.gov (United States)

    Flores, Rosa M.; Doskey, Paul V.

    2015-07-01

    The following algorithm, which is based on the thermodynamics of nonelectrolyte partitioning, was developed to predict emission rates of terpenes and terpenoids from specific storage sites in conifers: Ei =xoriγoripi∘ where Ei is the emission rate (μg C gdw-1 h-1) and pi∘ is the vapor pressure (mm Hg) of the pure liquid terpene or terpenoid, respectively, and xori and γori are the mole fraction and activity coefficient (on a Raoult's law convention), respectively, of the terpene and terpenoid in the oleoresin. Activity coefficients are calculated with Hansen solubility parameters that account for dispersive, polar, and H-bonding interactions of the solutes with the oleoresin matrix. Estimates of pi∘ at 25 °C and molar enthalpies of vaporization are made with the SIMPOL.1 method and are used to estimate pi∘ at environmentally relevant temperatures. Estimated mixing ratios of terpenes and terpenols were comparatively higher above resin-acid- and monoterpene-rich oleoresins, respectively. The results indicated a greater affinity of terpenes and terpenols for the non-functionalized and carboxylic acid containing matrix through dispersive and H-bonding interactions, which are expressed in the emission algorithm by the activity coefficient. The correlation between measured emission rates of terpenes and terpenoids for Pinus strobus and emission rates predicted with the algorithm were very good (R = 0.95). Standard errors for the range and average of monoterpene emission rates were ±6 - ±86% and ±54%, respectively, and were similar in magnitude to reported standard deviations of monoterpene composition of foliar oils (±38 - ±51% and ±67%, respectively).

  14. Hyperjapones A-E, Terpenoid Polymethylated Acylphloroglucinols from Hypericum japonicum.

    Science.gov (United States)

    Yang, Xing-Wei; Li, Yan-Ping; Su, Jia; Ma, Wei-Guang; Xu, Gang

    2016-04-15

    Hyperjapones A-E (1-5), novel terpenoid polymethylated acylphloroglucinols (TPAPs) with unusual architectures, were characterized from Hypericum japonicum. Their structures and absolute configurations were determined by comprehensive spectroscopic data and X-ray diffractions. Compound 1 was obtained as a racemic mixture and was separated by a column coated with cellulose tris(4-methylbenzoate) after attempts with various chiral materials. Compounds 1, 2, and 4 exhibited moderate antitumor activities in vitro.

  15. Suppression of terpenoid synthesis in plants by a virus promotes its mutualism with vectors.

    Science.gov (United States)

    Luan, Jun-Bo; Yao, Dan-Mei; Zhang, Tong; Walling, Linda L; Yang, Mei; Wang, Yu-Jun; Liu, Shu-Sheng

    2013-03-01

    Vectors often perform better on plants infected with pathogens, and this promotes the spread of pathogens. However, few studies have examined how plant defensive compounds mediate such mutualistic relationships. Although tobacco plants are relatively poor host plants for the whitefly Bemisia tabaci, tobacco's suitability to the whitefly was substantially increased when infected by the begomovirus Tomato yellow leaf curl China virus. The change in suitability was associated with induced terpenoid synthesis in whitefly-infested plants and repressed terpenoid synthesis in virus-infected plants. Elevation of terpenoid levels via exogenous stem applications reduced the performance of whiteflies. In contrast, suppression of terpenoid synthesis via gene silencing improved whitefly fitness. By integrating genomics, transcriptomics and metabolomics, this study demonstrated that virus infection depleted the terpenoid-mediated plant defence against whiteflies, thereby favouring vector-virus mutualism. These data suggest that plant terpenoids play a key role in shaping vector-pathogen relationships. © 2012 Blackwell Publishing Ltd/CNRS.

  16. General base-general acid catalysis by terpenoid cyclases.

    Science.gov (United States)

    Pemberton, Travis A; Christianson, David W

    2016-07-01

    Terpenoid cyclases catalyze the most complex reactions in biology, in that more than half of the substrate carbon atoms often undergo changes in bonding during the course of a multistep cyclization cascade that proceeds through multiple carbocation intermediates. Many cyclization mechanisms require stereospecific deprotonation and reprotonation steps, and most cyclization cascades are terminated by deprotonation to yield an olefin product. The first bacterial terpenoid cyclase to yield a crystal structure was pentalenene synthase from Streptomyces exfoliatus UC5319. This cyclase generates the hydrocarbon precursor of the pentalenolactone family of antibiotics. The structures of pentalenene synthase and other terpenoid cyclases reveal predominantly nonpolar active sites typically lacking amino acid side chains capable of serving general base-general acid functions. What chemical species, then, enables the Brønsted acid-base chemistry required in the catalytic mechanisms of these enzymes? The most likely candidate for such general base-general acid chemistry is the co-product inorganic pyrophosphate. Here, we briefly review biological and nonbiological systems in which phosphate and its derivatives serve general base and general acid functions in catalysis. These examples highlight the fact that the Brønsted acid-base activities of phosphate derivatives are comparable to the Brønsted acid-base activities of amino acid side chains.

  17. Estimation of total Terpenoids concentration in plant tissues using a monoterpene, Linalool as standard reagent.

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Narayan Ghorai, Sondipon Chakraborty, Shamik Gucchait, Samir Kumar Saha & Suman Biswas ### Abstract Terpenes and terpenoids are primary constituents of essential oils of different type of plants and flowers. Some qualitative estimation methods of terpenoids in plant tissue have been previously described but there is no protocol of estimating the same quantitatively till date. In the present study a protocol has been attempted to estimate the total terpenoids concentrati...

  18. BIOSYNTHESIS OF NANOPARTICLES

    OpenAIRE

    S Sen

    2011-01-01

    Biosynthesis of nanoparticles is reviewed in detail in this study. Comparison of different synthesis methods, namely physical, chemical and green methods giving emphasis to biological synthesis is documented here. This study also details limitations of the present techniques and envisages the future scope of nanoparticle biosynthesis. Important applications of nanoparticles are also discussed briefly in the present report.

  19. Terpenoids of plant origin inhibit morphogenesis, adhesion, and biofilm formation by Candida albicans.

    Science.gov (United States)

    Raut, Jayant S; Shinde, Ravikumar B; Chauhan, Nitin M; Karuppayil, S Mohan

    2013-01-01

    Biofilm-related infections caused by Candida albicans and associated drug resistant micro-organisms are serious problems for immunocompromised populations. Molecules which can prevent or remove biofilms are needed. Twenty-eight terpenoids of plant origin were analysed for their activity against growth, virulence attributes, and biofilms of C. albicans. Eighteen molecules exhibited minimum inhibitory concentrations of terpenoids resulted in significant (p terpenoids were identified as inhibitors of mature biofilms. This study demonstrated the antibiofilm potential of terpenoids, which need to be further explored as therapeutic strategy against biofilm associated infections of C. albicans.

  20. Increased Terpenoid Accumulation in Cotton (Gossypium hirsutum) Foliage is a General Wound Response

    Science.gov (United States)

    Kunert, Grit; Gershenzon, Jonathan

    2008-01-01

    The subepidermal pigment glands of cotton accumulate a variety of terpenoid products, including monoterpenes, sesquiterpenes, and terpenoid aldehydes that can act as feeding deterrents against a number of insect herbivore species. We compared the effect of herbivory by Spodoptera littoralis caterpillars, mechanical damage by a fabric pattern wheel, and the application of jasmonic acid on levels of the major representatives of the three structural classes of terpenoids in the leaf foliage of 4-week-old Gossypium hirsutum plants. Terpenoid levels increased successively from control to mechanical damage, herbivory, and jasmonic acid treatments, with E-β-ocimene and heliocide H1 and H4 showing the highest increases, up to 15-fold. Herbivory or mechanical damage to older leaves led to terpenoid increases in younger leaves. Leaf-by-leaf analysis of terpenes and gland density revealed that higher levels of terpenoids were achieved by two mechanisms: (1) increased filling of existing glands with terpenoids and (2) the production of additional glands, which were found to be dependent on damage intensity. As the relative response of individual terpenoids did not differ substantially among herbivore, mechanical damage, and jasmonic acid treatments, the induction of terpenoids in cotton foliage appears to represent a non-specific wound response mediated by jasmonic acid. Electronic supplementary material The online version of this article (doi:10.1007/s10886-008-9453-z) contains supplementary material, which is available to authorized users. PMID:18386096

  1. Terpenoids in Buddleja: relevance to chemosystematics, chemical ecology and biological activity.

    Science.gov (United States)

    Houghton, Peter J; Mensah, Abraham Y; Iessa, Noha; Hong, Liao Yong

    2003-09-01

    The terpenoids reported from Buddleja species are described. The antifungal activity of chloroform extracts of B. cordata and B. davidii stembark against the soil fungi Fusarium culmorum and Sordari fimicola is reported, with buddledin A shown to be the major compound responsible. The terpenoids present support the view that the Buddlejaceae should be classified in a taxon with Scrophulariaceae rather than Loganiaceae. Ecological aspects of the terpenoids are considered in relation to insects and soil fungi and the role of terpenoids in the chemical basis of the use of Buddleja in traditional medicine is also discussed, especially with regard to their anti-inflammatory properties.

  2. Nucleoside antibiotics: biosynthesis, regulation, and biotechnology.

    Science.gov (United States)

    Niu, Guoqing; Tan, Huarong

    2015-02-01

    The alarming rise in antibiotic-resistant pathogens has coincided with a decline in the supply of new antibiotics. It is therefore of great importance to find and create new antibiotics. Nucleoside antibiotics are a large family of natural products with diverse biological functions. Their biosynthesis is a complex process through multistep enzymatic reactions and is subject to hierarchical regulation. Genetic and biochemical studies of the biosynthetic machinery have provided the basis for pathway engineering and combinatorial biosynthesis to create new or hybrid nucleoside antibiotics. Dissection of regulatory mechanisms is leading to strategies to increase the titer of bioactive nucleoside antibiotics. Copyright © 2014. Published by Elsevier Ltd.

  3. Taxadiene Synthase Structure and Evolution of Modular Architecture in Terpene Biosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    M Köksal; Y Jin; R Coates; R Croteau; D Christianson

    2011-12-31

    With more than 55,000 members identified so far in all forms of life, the family of terpene or terpenoid natural products represents the epitome of molecular biodiversity. A well-known and important member of this family is the polycyclic diterpenoid Taxol (paclitaxel), which promotes tubulin polymerization and shows remarkable efficacy in cancer chemotherapy. The first committed step of Taxol biosynthesis in the Pacific yew (Taxus brevifolia) is the cyclization of the linear isoprenoid substrate geranylgeranyl diphosphate (GGPP) to form taxa-4(5),11(12)diene, which is catalysed by taxadiene synthase. The full-length form of this diterpene cyclase contains 862 residues, but a roughly 80-residue amino-terminal transit sequence is cleaved on maturation in plastids. We now report the X-ray crystal structure of a truncation variant lacking the transit sequence and an additional 27 residues at the N terminus, hereafter designated TXS. Specifically, we have determined structures of TXS complexed with 13-aza-13,14-dihydrocopalyl diphosphate (1.82 {angstrom} resolution) and 2-fluorogeranylgeranyl diphosphate (2.25 {angstrom} resolution). The TXS structure reveals a modular assembly of three {alpha}-helical domains. The carboxy-terminal catalytic domain is a class I terpenoid cyclase, which binds and activates substrate GGPP with a three-metal ion cluster. The N-terminal domain and a third 'insertion' domain together adopt the fold of a vestigial class II terpenoid cyclase. A class II cyclase activates the isoprenoid substrate by protonation instead of ionization, and the TXS structure reveals a definitive connection between the two distinct cyclase classes in the evolution of terpenoid biosynthesis.

  4. The Spatial Organization of Glucosinolate Biosynthesis

    DEFF Research Database (Denmark)

    Nintemann, Sebastian

    . However, questions concerning the spatial arrangement of the glucosinolate biosynthetic machinery and the consequential distribution of the metabolites remain. Different types of glucosinolates require specialized enzymes for certain steps in their biosynthetic pathways and whether these act in the same...... cells is an open question. Likewise, it is not known how glucosinolate biosynthesis is orchestrated at the subcellular level. These open questions were addressed with several approaches in this project, with the aim of shedding light on the spatial organization of glucosinolate biosynthesis from...... between the individual classes of glucosinolates under constitutive and induced conditions and identified the source tissues of these defense compounds. Protein-protein interaction studies were carried out to investigate the subcellular organization of glucosinolate biosynthesis. We identified a family...

  5. Quantifying the Metabolites of the Methylerythritol 4-Phosphate (MEP) Pathway in Plants and Bacteria by Liquid Chromatography-Triple Quadrupole Mass Spectrometry.

    Science.gov (United States)

    González-Cabanelas, D; Hammerbacher, A; Raguschke, B; Gershenzon, J; Wright, L P

    2016-01-01

    The 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway occurs in the plastids of higher plants and in most economically important prokaryotes where it is responsible for the biosynthesis of the isoprenoid building blocks, isopentenyl diphosphate and dimethylallyl diphosphate. These five-carbon compounds are the substrates for the enormous variety of terpenoid products, including many essential metabolites and substances of commercial value.