WorldWideScience

Sample records for heterologous gene expression

  1. Heterologous gene expression in filamentous fungi.

    Science.gov (United States)

    Su, Xiaoyun; Schmitz, George; Zhang, Meiling; Mackie, Roderick I; Cann, Isaac K O

    2012-01-01

    Filamentous fungi are critical to production of many commercial enzymes and organic compounds. Fungal-based systems have several advantages over bacterial-based systems for protein production because high-level secretion of enzymes is a common trait of their decomposer lifestyle. Furthermore, in the large-scale production of recombinant proteins of eukaryotic origin, the filamentous fungi become the vehicle of choice due to critical processes shared in gene expression with other eukaryotic organisms. The complexity and relative dearth of understanding of the physiology of filamentous fungi, compared to bacteria, have hindered rapid development of these organisms as highly efficient factories for the production of heterologous proteins. In this review, we highlight several of the known benefits and challenges in using filamentous fungi (particularly Aspergillus spp., Trichoderma reesei, and Neurospora crassa) for the production of proteins, especially heterologous, nonfungal enzymes. We review various techniques commonly employed in recombinant protein production in the filamentous fungi, including transformation methods, selection of gene regulatory elements such as promoters, protein secretion factors such as the signal peptide, and optimization of coding sequence. We provide insights into current models of host genomic defenses such as repeat-induced point mutation and quelling. Furthermore, we examine the regulatory effects of transcript sequences, including introns and untranslated regions, pre-mRNA (messenger RNA) processing, transcript transport, and mRNA stability. We anticipate that this review will become a resource for researchers who aim at advancing the use of these fascinating organisms as protein production factories, for both academic and industrial purposes, and also for scientists with general interest in the biology of the filamentous fungi. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. Combinatorial engineering for heterologous gene expression.

    Science.gov (United States)

    Zwick, Friederike; Lale, Rahmi; Valla, Svein

    2013-01-01

    Tools for strain engineering with predictable outcome are of crucial importance for the nascent field of synthetic biology. The success of combining different DNA biological parts is often restricted by poorly understood factors deriving from the complexity of the systems. We have previously identified variants for different regulatory elements of the expression cassette XylS/Pm. When such elements are combined they act in a manner consistent with their individual behavior, as long as they affect different functions, such as transcription and translation. Interestingly, sequence context does not seem to influence the final outcome significantly. Expression of reporter gene bla could be increased up to 75 times at the protein level by combining three variants in one cassette. For other tested reporter genes similar results were obtained, except that the stimulatory effect was quantitatively less. Combination of individually characterized DNA parts thus stands as suitable method to achieve a desired phenotype.

  3. Recombinant cells that highly express chromosomally-integrated heterologous gene

    Science.gov (United States)

    Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

    2007-03-20

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

  4. Improved heterologous gene expression in Trichoderma reesei by cellobiohydrolase I gene (cbh1) promoter optimization

    Institute of Scientific and Technical Information of China (English)

    Ti Liu; Tianhong Wang; Xian Li; Xuan Liu

    2008-01-01

    To improve heterologous gene expression in Trichoderma reesei, a set of optimal artificial cellobiohydrolase I gene (cbh1) promoters was obtained. The region from-677 to -724 with three potential glucose repressor binding sites was deleted. Then the region from-620 to-820 of the modified cbh1 promoter, including the CCAAT box and the Ace2 binding site, was repeatedly inserted into the modified cbh1 promoter, obtaining promoters with copy numbers 2, 4,and 6. The results showed that the glucose repression effects were abolished and the expression level of the glucuronidase (gus) reporter gene regulated by these multi-copy promoters was markedly enhanced as the copy number increased simultaneously. The data showed the great promise of using the promoter artificial modification strategy to increase heterologous gene expression in filamentous fungi and provided a set of optional high-expression vectors for gene function investigation and strain modification.

  5. Heterologous gene expression driven by carbonic anhydrase gene promoter in Dunaliella salina

    Institute of Scientific and Technical Information of China (English)

    CHAI Yurong; LU Yumin; WANG Tianyun; HOU Weihong; XUE Lexun

    2006-01-01

    Dunaliella salina, a halotolerant unicellular green alga without a rigid cell wall, can live in salinities ranging from 0.05 to 5 mol/L NaCl. These features of D. salina make it an ideal host for the production of antibodies, oral vaccine, and commercially valuable polypeptides. To produce high level of heterologous proteins from D. salina, highly efficientpromoters are required to drive expression of target genes under controlled condition. In the present study, we cloned a 5' franking region of 1.4 kb from the carbonic anhydrase (CAH) gene of D. salina by genomic walking and PCR. The fragment was ligated to the pMD18-T vector and characterized. Sequence analysis indicated that this region contained conserved motifs, including a TATA- like box and CAAT-box. Tandem (GT)n repeats that had a potential role of transcriptional control, were also found in this region. The transcription start site (TSS) of the CAH gene was determined by 5' RACE and nested PCR method. Transformation assays showed that the 1.4 kb fragment was able to drive expression of the selectable bar (bialaphos resistance) gene when the fusion was transformed into D. salina by biolistics.Northern blotting hybridizations showed that the bar transcript was most abundant in cells grown in 2 mol/L NaCl, and less abundant in 0.5 mol/L NaCl, indicating that expression of the bar gene was induced at high salinity. These results suggest the potential use of the CAH gene promoter to induce the expression of heterologous genes in D. salina under varied salt condition.

  6. Expression of heterologous genes from an IRES translational cassette in replication competent murine leukemia virus vectors

    DEFF Research Database (Denmark)

    Jespersen, Thomas; Duch, Mogens R.; Carrasco, M L

    1999-01-01

    We describe replication competent retroviruses capable of expressing heterologous genes during multiple rounds of infection. An internal ribosome entry site (IRES) from encephalomyocarditis virus was inserted in the U3 region of Akv- and SL3-3-murine leukemia viruses (MLV) to direct translation o...

  7. Engineered Streptomyces avermitilis host for heterologous expression of biosynthetic gene cluster for secondary metabolites.

    Science.gov (United States)

    Komatsu, Mamoru; Komatsu, Kyoko; Koiwai, Hanae; Yamada, Yuuki; Kozone, Ikuko; Izumikawa, Miho; Hashimoto, Junko; Takagi, Motoki; Omura, Satoshi; Shin-ya, Kazuo; Cane, David E; Ikeda, Haruo

    2013-07-19

    An industrial microorganism, Streptomyces avermitilis, which is a producer of anthelmintic macrocyclic lactones, avermectins, has been constructed as a versatile model host for heterologous expression of genes encoding secondary metabolite biosynthesis. Twenty of the entire biosynthetic gene clusters for secondary metabolites were successively cloned and introduced into a versatile model host S. avermitilis SUKA17 or 22. Almost all S. avermitilis transformants carrying the entire gene cluster produced metabolites as a result of the expression of biosynthetic gene clusters introduced. A few transformants were unable to produce metabolites, but their production was restored by the expression of biosynthetic genes using an alternative promoter or the expression of a regulatory gene in the gene cluster that controls the expression of biosynthetic genes in the cluster using an alternative promoter. Production of metabolites in some transformants of the versatile host was higher than that of the original producers, and cryptic biosynthetic gene clusters in the original producer were also expressed in a versatile host.

  8. Heterologous expression of cellulase genes in natural Saccharomyces cerevisiae strains.

    Science.gov (United States)

    Davison, Steffi A; den Haan, Riaan; van Zyl, Willem Heber

    2016-09-01

    Enzyme cost is a major impediment to second-generation (2G) cellulosic ethanol production. One strategy to reduce enzyme cost is to engineer enzyme production capacity in a fermentative microorganism to enable consolidated bio-processing (CBP). Ideally, a strain with a high secretory phenotype, high fermentative capacity as well as an innate robustness to bioethanol-specific stressors, including tolerance to products formed during pre-treatment and fermentation of lignocellulosic substrates should be used. Saccharomyces cerevisiae is a robust fermentative yeast but has limitations as a potential CBP host, such as low heterologous protein secretion titers. In this study, we evaluated natural S. cerevisiae isolate strains for superior secretion activity and other industrially relevant characteristics needed during the process of lignocellulosic ethanol production. Individual cellulases namely Saccharomycopsis fibuligera Cel3A (β-glucosidase), Talaromyces emersonii Cel7A (cellobiohydrolase), and Trichoderma reesei Cel5A (endoglucanase) were utilized as reporter proteins. Natural strain YI13 was identified to have a high secretory phenotype, demonstrating a 3.7- and 3.5-fold higher Cel7A and Cel5A activity, respectively, compared to the reference strain S288c. YI13 also demonstrated other industrially relevant characteristics such as growth vigor, high ethanol titer, multi-tolerance to high temperatures (37 and 40 °C), ethanol (10 % w/v), and towards various concentrations of a cocktail of inhibitory compounds commonly found in lignocellulose hydrolysates. This study accentuates the value of natural S. cerevisiae isolate strains to serve as potential robust and highly productive chassis organisms for CBP strain development.

  9. Novel terpenes generated by heterologous expression of bacterial terpene synthase genes in an engineered Streptomyces host.

    Science.gov (United States)

    Yamada, Yuuki; Arima, Shiho; Nagamitsu, Tohru; Johmoto, Kohei; Uekusa, Hidehiro; Eguchi, Tadashi; Shin-ya, Kazuo; Cane, David E; Ikeda, Haruo

    2015-06-01

    Mining of bacterial genome data has revealed numerous presumptive terpene synthases. Heterologous expression of several putative terpene synthase genes in an engineered Streptomyces host has revealed 13 newly discovered terpenes whose GC-MS and NMR data did not match with any known compounds in spectroscopic databases. Each of the genes encoding the corresponding terpene synthases were silent in their parent microorganisms. Heterologous expression and detailed NMR spectroscopic analysis allowed assignment of the structures of 13 new cyclic terpenes. Among these newly identified compounds, two were found to be linear triquinane sesquiterpenes that have never previously been isolated from bacteria or any other source. The remaining 11 new compounds were shown to be diterpene hydrocarbons and alcohol, including hydropyrene (1), hydropyrenol (2), tsukubadiene (11) and odyverdienes A (12) and B (13) each displaying a novel diterpene skeleton that had not previously been reported.

  10. Heterologous expression of a plant arginine decarboxylase gene in Trypanosoma cruzi.

    Science.gov (United States)

    Carrillo, Carolina; Serra, María P; Pereira, Claudio A; Huber, Alejandra; González, Nélida S; Algranati, Israel D

    2004-11-01

    Wild-type Trypanosoma cruzi epimastigotes lack arginine decarboxylase (ADC) enzymatic activity. However, the transformation of these parasites with a recombinant plasmid containing the oat ADC cDNA coding region gave rise to the transient heterologous expression of the enzyme, suggesting the absence of endogenous mechanisms that could inhibit the expression of a hypothetical own ADC gene or the assay used to measure its enzymatic activity. The foreign ADC enzyme expressed in the transgenic T. cruzi was characterized by identification of the products, the stoichiometry of the catalysed reaction, the specific inhibition by alpha-difluoromethylarginine (DFMA) and the study of its metabolic turnover. The half-life of the heterologous ADC activity in T. cruzi was about 150 min. Bioinformatics studies and polymerase chain reaction (PCR) analyses seem to indicate the absence of ADC-like DNA sequences in the wild-type T. cruzi genome.

  11. Characterization of cell lysis in Pseudomonas putida induced upon expression of heterologous killing genes

    DEFF Research Database (Denmark)

    Ronchel, M.C.; Molina, L.; Witte, A.;

    1998-01-01

    Active biological containment systems are based on the controlled expression of killing genes. These systems are of interest for the Pseudomonadaceae because of the potential applications of these microbes as bioremediation agents and biopesticides, The physiological effects that lead to cell death...... upon the induction of expression of two different heterologous killing genes in nonpathogenic Pseudomonas putida KT2440 derivatives have been analyzed, P. putida CMC4 and CMC12 carry in their chromosomes a fusion of the PAl-04/03 promoter to the Escherichia coli gef gene and the phi X174 lysis gene E......, respectively. Expression of the killing genes is controlled by the LacI protein, whose expression is initiated from the XylS-dependent Pm promoter. Under induced conditions, killing of P. putida CMC12 cells mediated by phi X174 lysis protein E was faster than that observed for P. putida CMC4, for which the Gef...

  12. Intron-Mediated Enhancement: A Tool for Heterologous Gene Expression in Plants?

    Science.gov (United States)

    Laxa, Miriam

    2017-01-01

    Many plant promoters were characterized and used for transgene expression in plants. Even though these promoters drive high levels of transgene expression in plants, the expression patterns are rarely constitutive but restricted to some tissues and developmental stages. In terms of crop improvement not only the enhancement of expression per se but, in particular, tissue-specific and spatial expression of genes plays an important role. Introns were used to boost expression in transgenic plants in the field of crop improvement for a long time. However, the mechanism behind this so called intron-mediated enhancement (IME) is still largely unknown. This review highlights the complexity of IME on the levels of its regulation and modes of action and gives an overview on IME methodology, examples in fundamental research and models of proposed mechanisms. In addition, the application of IME in heterologous gene expression is discussed. PMID:28111580

  13. Heterologous expression of Streptomyces clavuligerus ATCC 27064 cephamycin C gene cluster.

    Science.gov (United States)

    Martínez-Burgo, Y; Álvarez-Álvarez, R; Pérez-Redondo, R; Liras, P

    2014-09-30

    The Streptomyces clavuligerus cephamycin C gene cluster has been subcloned in a SuperCos-derived cosmid and introduced in Streptomyces flavogriseus ATCC 33331, Streptomyces coelicolor M1146 and Streptomyces albus J1074. The exconjugant strains were supplemented with an additional copy of the S. clavuligerus cephamycin regulatory activator gene, ccaRC, expressed from the constitutive Pfur promoter. Only S. flavogriseus-derived exconjugants produced a compound active against Escherichia coli ESS22-31 that was characterized by HPLC-MS as cephamycin C. The presence of an additional ccaR copy resulted in about 40-fold increase in cephamycin C production. Optimal heterologous cephamycin C production was in the order of 9% in relation to that of S. clavuligerus ATCC 27064. RT-qPCR studies indicated that ccaRC expression in S. flavogriseus::[SCos-CF] was 7% of that in S. clavuligerus and increased to 47% when supplemented with a copy of Pfur-ccaR. The effect on cephamycin biosynthesis gene expression was thus improved but not in an uniform manner for every gene. In heterologous strains, integration of the cephamycin cluster results in a ccaR-independent increased resistance to penicillin, cephalosporin and cefoxitin, what corresponds well to the strong expression of the pcbR and pbpA genes in S. flavogriseus-derived strains.

  14. Enhancing hydrogen production of Enterobacter aerogenes by heterologous expression of hydrogenase genes originated from Synechocystis sp.

    Science.gov (United States)

    Song, Wenlu; Cheng, Jun; Zhao, Jinfang; Zhang, Chuanxi; Zhou, Junhu; Cen, Kefa

    2016-09-01

    The hydrogenase genes (hoxEFUYH) of Synechocystis sp. PCC 6803 were cloned and heterologously expressed in Enterobacter aerogenes ATCC13408 for the first time in this study, and the hydrogen yield was significantly enhanced using the recombinant strain. A recombinant plasmid containing the gene in-frame with Glutathione-S-Transferase (GST) gene was transformed into E. aerogenes ATCC13408 to produce a GST-fusion protein. SDS-PAGE and western blot analysis confirm the successful expression of the hox genes. The hydrogenase activity of the recombinant strain is 237.6±9.3ml/(g-DW·h), which is 152% higher than the wild strain. The hydrogen yield of the recombinant strain is 298.3ml/g-glucose, which is 88% higher than the wild strain. During hydrogen fermentation, the recombinant strain produces more acetate and butyrate, but less ethanol. This is corresponding to the NADH metabolism in the cell due to the higher hydrogenase activity with the heterologous expression of hox genes.

  15. Cloning and heterologous expression of the antibiotic peptide (ABP) genes from Rhizopus oligosporus NBRC 8631.

    Science.gov (United States)

    Yamada, Osamu; Sakamoto, Kazutoshi; Tominaga, Mihoko; Nakayama, Tasuku; Koseki, Takuya; Fujita, Akiko; Akita, Osamu

    2005-03-01

    We carried out protein sequencing of purified Antibiotic Peptide (ABP), and cloned two genes encoding this peptide as abp1 and abp2, from Rhizopus oligosporus NBRC 8631. Both genes contain an almost identical 231-bp segment, with only 3 nucleotide substitutions, encoding a 77 amino acid peptide. The abp gene product comprises a 28 amino acid signal sequence and a 49 amino acid mature peptide. Northern blot analysis showed that at least one of the abp genes is transcribed in R. oligosporus NBRC 8631. A truncated form of abp1 encoding only the mature peptide was fused with the alpha-factor signal peptide and engineered for expression in Pichia pastoris SMD1168H. Culture broth of the recombinant Pichia displayed ABP activity against Bacillus subtilis NBRC 3335 after induction of heterologous gene expression. This result indicates that mature ABP formed the active structure without the aid of other factors from R. oligosporus, and was secreted.

  16. Reduced expression of Autographa californica nucleopolyhedrovirus ORF34, an essential gene, enhances heterologous gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Salem, Tamer Z. [Department of Entomology, Michigan State University, East Lansing, MI 48824 (United States); Department of Microbial Molecular Biology, AGERI, Agricultural Research Center, Giza 12619 (Egypt); Division of Biomedical Sciences, Zewail University, Zewail City of Science and Technology, Giza 12588 (Egypt); Zhang, Fengrui [Department of Entomology, Michigan State University, East Lansing, MI 48824 (United States); Thiem, Suzanne M., E-mail: smthiem@msu.edu [Department of Entomology, Michigan State University, East Lansing, MI 48824 (United States); Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824 (United States)

    2013-01-20

    Autographa californica multiple nucleopolyhedrovirus ORF34 is part of a transcriptional unit that includes ORF32, encoding a viral fibroblast growth factor (FGF) and ORF33. We identified ORF34 as a candidate for deletion to improve protein expression in the baculovirus expression system based on enhanced reporter gene expression in an RNAi screen of virus genes. However, ORF34 was shown to be an essential gene. To explore ORF34 function, deletion (KO34) and rescue bacmids were constructed and characterized. Infection did not spread from primary KO34 transfected cells and supernatants from KO34 transfected cells could not infect fresh Sf21 cells whereas the supernatant from the rescue bacmids transfection could recover the infection. In addition, budded viruses were not observed in KO34 transfected cells by electron microscopy, nor were viral proteins detected from the transfection supernatants by western blots. These demonstrate that ORF34 is an essential gene with a possible role in infectious virus production.

  17. Development of a site-directed integration plasmid for heterologous gene expression in Mycoplasma gallisepticum.

    Directory of Open Access Journals (Sweden)

    Isolde Nieszner

    Full Text Available Deciphering the molecular basis of the interactions between the parasite Mycoplasma gallisepticum and its avian hosts suffers from the lack of genetic tools available for the pathogen. In the absence of well established methods for targeted disruption of relevant M. gallisepticum genes, we started to develop suicide vectors and equipped them with a short fragment of M. gallisepticum origin or replication (oriC MG. We failed to create a disruption vector, although by adding a further short fragment of the M. gallisepticum tufB upstream region we created a "Trojan horse" plasmid. This is fully integrated into the genomic DNA of M. gallisepticum, always at the same site, oriC MG, and is able to carry and express any gene of interest in the genetic background of M. gallisepticum. Successful expression of a heterologous gene was shown with the lacZ gene of E. coli. When used for gene complementation or expression of hybrid genes in M. gallisepticum, a site-specific combined integration/expression vector constitutes an improvement on randomly integrating transposons, which might have unexpected effects on the expression of chromosomal genes.

  18. Function-Based Metagenomic Library Screening and Heterologous Expression Strategy for Genes Encoding Phosphatase Activity.

    Science.gov (United States)

    Villamizar, Genis A Castillo; Nacke, Heiko; Daniel, Rolf

    2017-01-01

    The release of phosphate from inorganic and organic phosphorus compounds can be mediated enzymatically. Phosphate-releasing enzymes, comprising acid and alkaline phosphatases, are recognized as useful biocatalysts in applications such as plant and animal nutrition, bioremediation and diagnostic analysis. Metagenomic approaches provide access to novel phosphatase-encoding genes. Here, we describe a function-based screening approach for rapid identification of genes conferring phosphatase activity from small-insert and large-insert metagenomic libraries derived from various environments. This approach bears the potential for discovery of entirely novel phosphatase families or subfamilies and members of known enzyme classes hydrolyzing phosphomonoester bonds such as phytases. In addition, we provide a strategy for efficient heterologous phosphatase gene expression.

  19. Laccase Gene Family in Cerrena sp. HYB07: Sequences, Heterologous Expression and Transcriptional Analysis

    Directory of Open Access Journals (Sweden)

    Jie Yang

    2016-08-01

    Full Text Available Laccases are a class of multi-copper oxidases with industrial potential. In this study, eight laccases (Lac1–8 from Cerrena sp. strain HYB07, a white-rot fungus with high laccase yields, were analyzed. The laccases showed moderate identities to each other as well as with other fungal laccases and were predicted to have high redox potentials except for Lac6. Selected laccase isozymes were heterologously expressed in the yeast Pichia pastoris, and different enzymatic properties were observed. Transcription of the eight laccase genes was differentially regulated during submerged and solid state fermentation, as shown by quantitative real-time polymerase chain reaction and validated reference genes. During 6-day submerged fermentation, Lac7 and 2 were successively the predominantly expressed laccase gene, accounting for over 95% of all laccase transcripts. Interestingly, accompanying Lac7 downregulation, Lac2 transcription was drastically upregulated on days 3 and 5 to 9958-fold of the level on day 1. Consistent with high mRNA abundance, Lac2 and 7, but not other laccases, were identified in the fermentation broth by LC-MS/MS. In solid state fermentation, less dramatic differences in transcript abundance were observed, and Lac3, 7 and 8 were more highly expressed than other laccase genes. Elucidating the properties and expression profiles of the laccase gene family will facilitate understanding, production and commercialization of the fungal strain and its laccases.

  20. GAL promoter-driven heterologous gene expression in Saccharomyces cerevisiae Δ strain at anaerobic alcoholic fermentation.

    Science.gov (United States)

    Ahn, Jungoh; Park, Kyung-Min; Lee, Hongweon; Son, Yeo-Jin; Choi, Eui-Sung

    2013-02-01

    The removal of Gal80 protein by gene disruption turned into efficient GAL promoter-driven heterologous gene expression under anaerobic alcoholic fermentation of Saccharomyces cerevisiae. Using lipase B from Candida antarctica as a reporter, the relative strength of GAL10 promoter (P(GAL10) ) in Δgal80 mutant that does not require galactose as an inducer was compared to those of ADH1, PDC1, and PGK promoters, which have been known to work well anaerobically in actively fermenting yeast cells under high glucose concentration. P(GAL10) in the Δgal80 mutant showed 0.8-fold (ADH1), fourfold (PDC1), and 50-fold (PGK) in promoter strength.

  1. Heterologous expression of leader-less pga gene in Pichia pastoris: intracellular production of prokaryotic enzyme

    Directory of Open Access Journals (Sweden)

    Kyslík Pavel

    2010-02-01

    Full Text Available Abstract Background Penicillin G acylase of Escherichia coli (PGAEc is a commercially valuable enzyme for which efficient bacterial expression systems have been developed. The enzyme is used as a catalyst for the hydrolytic production of β-lactam nuclei or for the synthesis of semi-synthetic penicillins such as ampicillin, amoxicillin and cephalexin. To become a mature, periplasmic enzyme, the inactive prepropeptide of PGA has to undergo complex processing that begins in the cytoplasm (autocatalytic cleavage, continues at crossing the cytoplasmic membrane (signal sequence removing, and it is completed in the periplasm. Since there are reports on impressive cytosolic expression of bacterial proteins in Pichia, we have cloned the leader-less gene encoding PGAEc in this host and studied yeast production capacity and enzyme authenticity. Results Leader-less pga gene encoding PGAEcunder the control of AOX1 promoter was cloned in Pichia pastoris X-33. The intracellular overproduction of heterologous PGAEc(hPGAEc was evaluated in a stirred 10 litre bioreactor in high-cell density, fed batch cultures using different profiles of transient phases. Under optimal conditions, the average volumetric activity of 25900 U l-1 was reached. The hPGAEc was purified, characterized and compared with the wild-type PGAEc. The α-subunit of the hPGAEc formed in the cytosol was processed aberrantly resulting in two forms with C- terminuses extended to the spacer peptide. The enzyme exhibited modified traits: the activity of the purified enzyme was reduced to 49%, the ratios of hydrolytic activities with cephalexin, phenylacetamide or 6-nitro-3-phenylacetylamidobenzoic acid (NIPAB to penicillin G increased and the enzyme showed a better synthesis/hydrolysis ratio for the synthesis of cephalexin. Conclusions Presented results provide useful data regarding fermentation strategy, intracellular biosynthetic potential, and consequences of the heterologous expression of PGAEc

  2. Heterologous expression of the filarial nematode alt gene products reveals their potential to inhibit immune function

    Directory of Open Access Journals (Sweden)

    Aebischer Toni

    2005-03-01

    Full Text Available Abstract Background Parasites exploit sophisticated strategies to evade host immunity that require both adaptation of existing genes and evolution of new gene families. We have addressed this question by testing the immunological function of novel genes from helminth parasites, in which conventional transgenesis is not yet possible. We investigated two such novel genes from Brugia malayi termed abundant larval transcript (alt, expression of which reaches ~5% of total transcript at the time parasites enter the human host. Results To test the hypothesis that ALT proteins modulate host immunity, we adopted an alternative transfection strategy to express these products in the protozoan parasite Leishmania mexicana. We then followed the course of infection in vitro in macrophages and in vivo in mice. Expression of ALT proteins, but not a truncated mutant, conferred greater infectivity of macrophages in vitro, reaching 3-fold higher parasite densities. alt-transfected parasites also caused accelerated disease in vivo, and fewer mice were able to clear infection of organisms expressing ALT. alt-transfected parasites were more resistant to IFN-γ-induced killing by macrophages. Expression profiling of macrophages infected with transgenic L. mexicana revealed consistently higher levels of GATA-3 and SOCS-1 transcripts, both associated with the Th2-type response observed in in vivo filarial infection. Conclusion Leishmania transfection is a tractable and informative approach to determining immunological functions of single genes from heterologous organisms. In the case of the filarial ALT proteins, our data suggest that they may participate in the Th2 bias observed in the response to parasite infection by modulating cytokine-induced signalling within immune system cells.

  3. Enhancement of heterologous gene expression in Flammulina velutipes using polycistronic vectors containing a viral 2A cleavage sequence.

    Directory of Open Access Journals (Sweden)

    Yu-Ju Lin

    Full Text Available Agrobacterium tumefaciens-mediated transformation for edible mushrooms has been previously established. However, the enhancement of heterologous protein production and the expression of multi-target genes remains a challenge. In this study, heterologous protein expression in the enoki mushroom Flammulina velutipes was notably enhanced using 2A peptide-mediated cleavage to co-express multiple copies of single gene. The polycistronic expression vectors were constructed by connecting multi copies of the enhanced green fluorescent protein (egfp gene using 2A peptides derived from porcine teschovirus-1. The P2A peptides properly self-cleaved as shown by the formation of the transformants with antibiotic resistant capacity and exciting green fluorescence levels after introducing the vectors into F. velutipes mycelia. The results of western blot analysis, epifluorescent microscopy and EGFP production showed that heterologous protein expression in F. velutipes using the polycistronic strategy increased proportionally as the gene copy number increased from one to three copies. In contrast, much lower EGFP levels were detected in the F. velutipes transformants harboring four copies of the egfp gene due to mRNA instability. The polycistronic strategy using 2A peptide-mediated cleavage developed in this study can not only be used to express single gene in multiple copies, but also to express multiple genes in a single reading frame. It is a promising strategy for the application of mushroom molecular pharming.

  4. Enhancement of heterologous gene expression in Flammulina velutipes using polycistronic vectors containing a viral 2A cleavage sequence.

    Science.gov (United States)

    Lin, Yu-Ju; Huang, Li-Hsin; Huang, Ching-Tsan

    2013-01-01

    Agrobacterium tumefaciens-mediated transformation for edible mushrooms has been previously established. However, the enhancement of heterologous protein production and the expression of multi-target genes remains a challenge. In this study, heterologous protein expression in the enoki mushroom Flammulina velutipes was notably enhanced using 2A peptide-mediated cleavage to co-express multiple copies of single gene. The polycistronic expression vectors were constructed by connecting multi copies of the enhanced green fluorescent protein (egfp) gene using 2A peptides derived from porcine teschovirus-1. The P2A peptides properly self-cleaved as shown by the formation of the transformants with antibiotic resistant capacity and exciting green fluorescence levels after introducing the vectors into F. velutipes mycelia. The results of western blot analysis, epifluorescent microscopy and EGFP production showed that heterologous protein expression in F. velutipes using the polycistronic strategy increased proportionally as the gene copy number increased from one to three copies. In contrast, much lower EGFP levels were detected in the F. velutipes transformants harboring four copies of the egfp gene due to mRNA instability. The polycistronic strategy using 2A peptide-mediated cleavage developed in this study can not only be used to express single gene in multiple copies, but also to express multiple genes in a single reading frame. It is a promising strategy for the application of mushroom molecular pharming.

  5. Altered Phenotypes in Saccharomyces cerevisiae by Heterologous Expression of Basidiomycete Moniliophthora perniciosa SOD2 Gene

    Directory of Open Access Journals (Sweden)

    Sônia C. Melo

    2015-06-01

    Full Text Available Heterologous expression of a putative manganese superoxide dismutase gene (SOD2 of the basidiomycete Moniliophthora perniciosa complemented the phenotypes of a Saccharomyces cerevisiae sod2Δ mutant. Sequence analysis of the cloned M. perniciosa cDNA revealed an open reading frame (ORF coding for a 176 amino acid polypeptide with the typical metal-binding motifs of a SOD2 gene, named MpSOD2. Phylogenetic comparison with known manganese superoxide dismutases (MnSODs located the protein of M. perniciosa (MpSod2p in a clade with the basidiomycete fungi Coprinopsis cinerea and Laccaria bicolor. Haploid wild-type yeast transformants containing a single copy of MpSOD2 showed increased resistance phenotypes against oxidative stress-inducing hydrogen peroxide and paraquat, but had unaltered phenotype against ultraviolet–C (UVC radiation. The same transformants exhibited high sensitivity against treatment with the pro-mutagen diethylnitrosamine (DEN that requires oxidation to become an active mutagen/carcinogen. Absence of MpSOD2 in the yeast sod2Δ mutant led to DEN hyper-resistance while introduction of a single copy of this gene restored the yeast wild-type phenotype. The haploid yeast wild-type transformant containing two SOD2 gene copies, one from M. perniciosa and one from its own, exhibited DEN super-sensitivity. This transformant also showed enhanced growth at 37 °C on the non-fermentable carbon source lactate, indicating functional expression of MpSod2p. The pro-mutagen dihydroethidium (DHE-based fluorescence assay monitored basal level of yeast cell oxidative stress. Compared to the wild type, the yeast sod2Δ mutant had a much higher level of intrinsic oxidative stress, which was reduced to wild type (WT level by introduction of one copy of the MpSOD2 gene. Taken together our data indicates functional expression of MpSod2 protein in the yeast S. cerevisiae.

  6. Heterologous Expression of Pantoea Agglomerans Phytase Gene Optimized for Plant-Host Expression

    Directory of Open Access Journals (Sweden)

    N.N. Khabipova

    2016-04-01

    Full Text Available Here we report expression and characterization of recombinant bacterial phytase PaPhyC from Pantoea sp. Codon-optimized phytase gene was expressed E.coli BL21 pLysS and protein expression was confirmed by Western blotting. Recombinant protein expressed in E.coli has high phytase activity. We show that PaPhyC recombinant phytase has different molecular masses when expressed in bacteria and plants, suggesting that possible protein glycosylation in plants may influence its overall size.

  7. Development of a novel plasmid as a shuttle vector for heterologous gene expression in Mycoplasma yeatsii.

    Science.gov (United States)

    Kent, Bethany N; Foecking, Mark F; Calcutt, Michael J

    2012-10-01

    A circular plasmid, pMyBK1, was detected in Mycoplasma yeatsii strain GIH(T). Analysis of the sequence of the 3432-bp replicon identified two predicted open reading frames (ORFs), one with sequence similarity to multiple plasmid mobilization proteins and one that matches only to hypothetical ORFs encoded by integrated chromosomal elements in the sequenced genomes of two Mycoplasma species. Shuttle vectors were constructed in Escherichia coli which could be introduced into M. yeatsii at high efficiency (10(4)-10(5) per μg DNA) by electroporation. Independent deletion analysis of the two ORFs disclosed that whereas mob was dispensable, orf2 was necessary for plasmid replication or maintenance. The absence of plasmid-encoded database matches for ORF2 indicates that pMyBK1 represents a novel plasmid family. One shuttle vector was used to demonstrate heterologous expression of the Mycoplasma fermentans malp gene and was stable during multiple passages. The host-plasmid system described has potential application for genetic manipulation in a genus for which few replicative vectors are available.

  8. Identification and heterologous expression of a Δ4-fatty acid desaturase gene from Isochrysis sphaerica.

    Science.gov (United States)

    Guo, Bing; Jiang, Mulan; Wan, Xia; Gong, Yangmin; Liang, Zhuo; Hu, Chuanjiong

    2013-10-28

    The marine microalga Isochrysis sphaerica is rich in the very-long-chain polyunsaturated fatty acids, including eicosapentaenoic acid (EPA, C20:5ω-3) and docosahexaenoic acid (DHA, C22:6ω-3) that are important to human health. Here, we report a functional characterization of a Δ4-fatty acid desaturase gene (FAD4) from I. sphaerica. IsFAD4 contains a 1,284 bp open reading frame encoding a 427 amino acid polypeptide. The deduced amino sequence comprises three conserved histidine motifs and a cytochrome b5 domain at its N-terminus. Phylogenetic analysis indicated that IsFad4 formed a unique Isochrysis clade distinct from the counterparts of other eukaryotes. Heterologous expression of IsFAD4 in Pichia pastoris showed that IsFad4 was able to desaturate docosapentaenoic acid (DPA) to form DHA, and the rate of converting DPA to DHA was 79.8%. These results throw light on the potential industrial production of specific polyunsaturated fatty acids through IsFAD4 transgenic yeast or oil crops.

  9. Heterologous expression in transgenic mosquitoes

    Institute of Scientific and Technical Information of China (English)

    Santhosh P K; Yu hua Deng; Weidong Gu; Xiaoguang Chen

    2010-01-01

    Arthropod-borne diseases such as malaria and dengue virus afflict billions of people worldwide imposing major economic and social burdens. Control of such pathogens is mainly performed by vector management and treatment of affected individuals with drugs. The failure of these conventional approaches due to emergence of insecticide-resistant insects and drug-resistant parasites demonstrate the need of novel and efficacious control strategies to combat these diseases. Genetic modification(GM) of mosquito vectors to impair their ability to be infected and transmit pathogens has emerged as a new strategy to reduce transmission of many vector-borne diseases and deliver public health gains. Several advances in developing transgenic mosquitoes unable to transmit pathogens have gained support, some of them attempt to manipulate the naturally occurring endogenous refractory mechanisms, while others initiate the identification of an exogenous foreign gene which disrupt the pathogen development in insect vectors. Heterologous expression of transgenes under a native or heterologous promoter is important for the screening and effecting of the transgenic mosquitoes. The effect of the transgene on mosquito fitness is a crucial parameter influencing the success of this transgenic approach. This review examines these two aspects and describes the basic research work that has been accomplished towards understanding the complex relation between the parasite and its vector and focuses on recent advances and perspectives towards construction of transgenic mosquitoes refractory to vector-borne disease transmission.

  10. Expression of heterologous sigma factors enables functional screening of metagenomic and heterologous genomic libraries.

    Science.gov (United States)

    Gaida, Stefan M; Sandoval, Nicholas R; Nicolaou, Sergios A; Chen, Yili; Venkataramanan, Keerthi P; Papoutsakis, Eleftherios T

    2015-05-06

    A key limitation in using heterologous genomic or metagenomic libraries in functional genomics and genome engineering is the low expression of heterologous genes in screening hosts, such as Escherichia coli. To overcome this limitation, here we generate E. coli strains capable of recognizing heterologous promoters by expressing heterologous sigma factors. Among seven sigma factors tested, RpoD from Lactobacillus plantarum (Lpl) appears to be able of initiating transcription from all sources of DNA. Using the promoter GFP-trap concept, we successfully screen several heterologous and metagenomic DNA libraries, thus enlarging the genomic space that can be functionally sampled in E. coli. For an application, we show that screening fosmid-based Lpl genomic libraries in an E. coli strain with a chromosomally integrated Lpl rpoD enables the identification of Lpl genetic determinants imparting strong ethanol tolerance in E. coli. Transcriptome analysis confirms increased expression of heterologous genes in the engineered strain.

  11. Enhancement of crystallinity of cellulose produced by Escherichia coli through heterologous expression of bcsD gene from Gluconacetobacter xylinus.

    Science.gov (United States)

    Sajadi, Elaheh; Babaipour, Valiollah; Deldar, Ali Asghar; Yakhchali, Bagher; Fatemi, Seyed Safa-Ali

    2017-09-01

    To evaluate the crystallinity index of the cellulose produced by Escherichia coli Nissle 1917 after heterologous expression of the cellulose synthase subunit D (bcsD) gene of Gluconacetobacter xylinus BPR2001. The bcsD gene of G. xylinus BPR2001 was expressed in E. coli and its protein product was visualized using SDS-PAGE. FTIR analysis showed that the crystallinity index of the cellulose produced by the recombinants was 0.84, which is 17% more than that of the wild type strain. The increased crystallinity index was also confirmed by X-ray diffraction analysis. The cellulose content was not changed significantly after over-expressing the bcsD. The bcsD gene can improve the crystalline structure of the bacterial cellulose but there is not any significant difference between the amounts of cellulose produced by the recombinant and wild type E. coli Nissle 1917.

  12. Cloning and Heterologous Expression of a Large-sized Natural Product Biosynthetic Gene Cluster in Streptomyces Species

    Science.gov (United States)

    Nah, Hee-Ju; Pyeon, Hye-Rim; Kang, Seung-Hoon; Choi, Si-Sun; Kim, Eung-Soo

    2017-01-01

    Actinomycetes family including Streptomyces species have been a major source for the discovery of novel natural products (NPs) in the last several decades thanks to their structural novelty, diversity and complexity. Moreover, recent genome mining approach has provided an attractive tool to screen potentially valuable NP biosynthetic gene clusters (BGCs) present in the actinomycetes genomes. Since many of these NP BGCs are silent or cryptic in the original actinomycetes, various techniques have been employed to activate these NP BGCs. Heterologous expression of BGCs has become a useful strategy to produce, reactivate, improve, and modify the pathways of NPs present at minute quantities in the original actinomycetes isolates. However, cloning and efficient overexpression of an entire NP BGC, often as large as over 100 kb, remain challenging due to the ineffectiveness of current genetic systems in manipulating large NP BGCs. This mini review describes examples of actinomycetes NP production through BGC heterologous expression systems as well as recent strategies specialized for the large-sized NP BGCs in Streptomyces heterologous hosts. PMID:28360891

  13. Use of homologous and heterologous gene expression profiling tools to characterize transcription dynamics during apple fruit maturation and ripening.

    Science.gov (United States)

    Costa, Fabrizio; Alba, Rob; Schouten, Henk; Soglio, Valeria; Gianfranceschi, Luca; Serra, Sara; Musacchi, Stefano; Sansavini, Silviero; Costa, Guglielmo; Fei, Zhangjun; Giovannoni, James

    2010-10-25

    Fruit development, maturation and ripening consists of a complex series of biochemical and physiological changes that in climacteric fruits, including apple and tomato, are coordinated by the gaseous hormone ethylene. These changes lead to final fruit quality and understanding of the functional machinery underlying these processes is of both biological and practical importance. To date many reports have been made on the analysis of gene expression in apple. In this study we focused our investigation on the role of ethylene during apple maturation, specifically comparing transcriptomics of normal ripening with changes resulting from application of the hormone receptor competitor 1-methylcyclopropene. To gain insight into the molecular process regulating ripening in apple, and to compare to tomato (model species for ripening studies), we utilized both homologous and heterologous (tomato) microarray to profile transcriptome dynamics of genes involved in fruit development and ripening, emphasizing those which are ethylene regulated.The use of both types of microarrays facilitated transcriptome comparison between apple and tomato (for the later using data previously published and available at the TED: tomato expression database) and highlighted genes conserved during ripening of both species, which in turn represent a foundation for further comparative genomic studies. The cross-species analysis had the secondary aim of examining the efficiency of heterologous (specifically tomato) microarray hybridization for candidate gene identification as related to the ripening process. The resulting transcriptomics data revealed coordinated gene expression during fruit ripening of a subset of ripening-related and ethylene responsive genes, further facilitating the analysis of ethylene response during fruit maturation and ripening. Our combined strategy based on microarray hybridization enabled transcriptome characterization during normal climacteric apple ripening, as well as

  14. Use of homologous and heterologous gene expression profiling tools to characterize transcription dynamics during apple fruit maturation and ripening

    Directory of Open Access Journals (Sweden)

    Sansavini Silviero

    2010-10-01

    Full Text Available Abstract Background Fruit development, maturation and ripening consists of a complex series of biochemical and physiological changes that in climacteric fruits, including apple and tomato, are coordinated by the gaseous hormone ethylene. These changes lead to final fruit quality and understanding of the functional machinery underlying these processes is of both biological and practical importance. To date many reports have been made on the analysis of gene expression in apple. In this study we focused our investigation on the role of ethylene during apple maturation, specifically comparing transcriptomics of normal ripening with changes resulting from application of the hormone receptor competitor 1-Methylcyclopropene. Results To gain insight into the molecular process regulating ripening in apple, and to compare to tomato (model species for ripening studies, we utilized both homologous and heterologous (tomato microarray to profile transcriptome dynamics of genes involved in fruit development and ripening, emphasizing those which are ethylene regulated. The use of both types of microarrays facilitated transcriptome comparison between apple and tomato (for the later using data previously published and available at the TED: tomato expression database and highlighted genes conserved during ripening of both species, which in turn represent a foundation for further comparative genomic studies. The cross-species analysis had the secondary aim of examining the efficiency of heterologous (specifically tomato microarray hybridization for candidate gene identification as related to the ripening process. The resulting transcriptomics data revealed coordinated gene expression during fruit ripening of a subset of ripening-related and ethylene responsive genes, further facilitating the analysis of ethylene response during fruit maturation and ripening. Conclusion Our combined strategy based on microarray hybridization enabled transcriptome characterization

  15. Heterologous expression of Mus musculus immunoresponsive gene 1 (irg1 in Escherichia coli results in itaconate production

    Directory of Open Access Journals (Sweden)

    Kiira S Vuoristo

    2015-08-01

    Full Text Available Itaconic acid, a C5-dicarboxylic acid, is a potential biobased building block for the polymer industry. It is obtained from the citric acid cycle by decarboxylation of cis-aconitic acid. This reaction is catalysed by CadA in the native itaconic acid producer Aspergillus terreus. Recently, another enzyme encoded by the mammalian immunoresponsive gene 1 (irg1, was found to decarboxylate cis-aconitate to itaconate in vitro. We show that heterologous expression of irg1 enabled itaconate production in E. coli with production titres up to 560 mg/L.

  16. A simple and accurate two-step long DNA sequences synthesis strategy to improve heterologous gene expression in pichia.

    Science.gov (United States)

    Yang, Jiang-Ke; Chen, Fang-Yuan; Yan, Xiang-Xiang; Miao, Li-Hong; Dai, Jiang-Hong

    2012-01-01

    In vitro gene chemical synthesis is a powerful tool to improve the expression of gene in heterologous system. In this study, a two-step gene synthesis strategy that combines an assembly PCR and an overlap extension PCR (AOE) was developed. In this strategy, the chemically synthesized oligonucleotides were assembled into several 200-500 bp fragments with 20-25 bp overlap at each end by assembly PCR, and then an overlap extension PCR was conducted to assemble all these fragments into a full length DNA sequence. Using this method, we de novo designed and optimized the codon of Rhizopus oryzae lipase gene ROL (810 bp) and Aspergillus niger phytase gene phyA (1404 bp). Compared with the original ROL gene and phyA gene, the codon-optimized genes expressed at a significantly higher level in yeasts after methanol induction. We believe this AOE method to be of special interest as it is simple, accurate and has no limitation with respect to the size of the gene to be synthesized. Combined with de novo design, this method allows the rapid synthesis of a gene optimized for expression in the system of choice and production of sufficient biological material for molecular characterization and biotechnological application.

  17. A simple and accurate two-step long DNA sequences synthesis strategy to improve heterologous gene expression in pichia.

    Directory of Open Access Journals (Sweden)

    Jiang-Ke Yang

    Full Text Available In vitro gene chemical synthesis is a powerful tool to improve the expression of gene in heterologous system. In this study, a two-step gene synthesis strategy that combines an assembly PCR and an overlap extension PCR (AOE was developed. In this strategy, the chemically synthesized oligonucleotides were assembled into several 200-500 bp fragments with 20-25 bp overlap at each end by assembly PCR, and then an overlap extension PCR was conducted to assemble all these fragments into a full length DNA sequence. Using this method, we de novo designed and optimized the codon of Rhizopus oryzae lipase gene ROL (810 bp and Aspergillus niger phytase gene phyA (1404 bp. Compared with the original ROL gene and phyA gene, the codon-optimized genes expressed at a significantly higher level in yeasts after methanol induction. We believe this AOE method to be of special interest as it is simple, accurate and has no limitation with respect to the size of the gene to be synthesized. Combined with de novo design, this method allows the rapid synthesis of a gene optimized for expression in the system of choice and production of sufficient biological material for molecular characterization and biotechnological application.

  18. Comparisons of Ribosomal Protein Gene Promoters Indicate Superiority of Heterologous Regulatory Sequences for Expressing Transgenes in Phytophthora infestans.

    Science.gov (United States)

    Poidevin, Laetitia; Andreeva, Kalina; Khachatoorian, Careen; Judelson, Howard S

    2015-01-01

    Molecular genetics approaches in Phytophthora research can be hampered by the limited number of known constitutive promoters for expressing transgenes and the instability of transgene activity. We have therefore characterized genes encoding the cytoplasmic ribosomal proteins of Phytophthora and studied their suitability for expressing transgenes in P. infestans. Phytophthora spp. encode a standard complement of 79 cytoplasmic ribosomal proteins. Several genes are duplicated, and two appear to be pseudogenes. Half of the genes are expressed at similar levels during all stages of asexual development, and we discovered that the majority share a novel promoter motif named the PhRiboBox. This sequence is enriched in genes associated with transcription, translation, and DNA replication, including tRNA and rRNA biogenesis. Promoters from the three P. infestans genes encoding ribosomal proteins S9, L10, and L23 and their orthologs from P. capsici were tested for their ability to drive transgenes in stable transformants of P. infestans. Five of the six promoters yielded strong expression of a GUS reporter, but the stability of expression was higher using the P. capsici promoters. With the RPS9 and RPL10 promoters of P. infestans, about half of transformants stopped making GUS over two years of culture, while their P. capsici orthologs conferred stable expression. Since cross-talk between native and transgene loci may trigger gene silencing, we encourage the use of heterologous promoters in transformation studies.

  19. Novel polyoxins generated by heterologously expressing polyoxin biosynthetic gene cluster in the sanN inactivated mutant of Streptomyces ansochromogenes

    Directory of Open Access Journals (Sweden)

    Li Jine

    2012-10-01

    Full Text Available Abstract Background Polyoxins are potent inhibitors of chitin synthetases in fungi and insects. The gene cluster responsible for biosynthesis of polyoxins has been cloned and sequenced from Streptomyces cacaoi and tens of polyoxin analogs have been identified already. Results The polyoxin biosynthetic gene cluster from Streptomyces cacaoi was heterologously expressed in the sanN inactivated mutant of Streptomyces ansochromogenes as a nikkomycin producer. Besides hybrid antibiotics (polynik A and polyoxin N and some known polyoxins, two novel polyoxin analogs were accumulated. One of them is polyoxin P that has 5-aminohexuronic acid with N-glycosidically bound thymine as the nucleoside moiety and dehydroxyl-carbamoylpolyoxic acid as the peptidyl moiety. The other analog is polyoxin O that contains 5-aminohexuronic acid bound thymine as the nucleoside moiety, but recruits polyoximic acid as the sole peptidyl moiety. Bioassay against phytopathogenic fungi showed that polyoxin P displayed comparatively strong inhibitory activity, whereas the inhibitory activity of polyoxin O was weak under the same testing conditions. Conclusion Two novel polyoxin analogs (polyoxin P and O were generated by the heterologous expression of polyoxin biosynthetic gene cluster in the sanN inactivated mutant of Streptomyces ansochromogenes. Polyoxin P showed potent antifungal activity,while the activity of polyoxin O was weak. The strategy presented here may be available for other antibiotics producers.

  20. Enhancement of welan gum production in Sphingomonas sp. HT-1 via heterologous expression of Vitreoscilla hemoglobin gene.

    Science.gov (United States)

    Liu, Xiaoliu; Zhu, Ping; Jiang, Ruifan; Wu, Lingtian; Feng, Xiaohai; Li, Sha; Xu, Hong

    2017-01-20

    Welan gum is a microbial polysaccharide produced by Sphingomonas sp. Its production is limited by the dissolved oxygen levels in the highly viscous fermentation. A strategy of heterologous expression of the Vitreoscilla hemoglobin gene in Sphingomonas sp. HT-1 was investigated to alleviate oxygen limitation and improve the yield of welan gum. Ultimately, the welan gum production increased from 25.3g/L to 34.6g/L, whereas the rheological behavior of welan gum solutions remained virtually unchanged. The transcriptional levels of the key genes in the electron transfer chain, TCA cycle and welan gum synthesis pathway, as well as ATP level revealed that the VHb expression in Sphingomonas sp. HT-1 enhanced welan gum biosynthesis by improving respiration and ATP supply. This study would pave the genetic manipulation way for enhancing welan gum yield, and it's also of great importance for the industrial applications of welan gum under harsh conditions.

  1. The Issue of Secretion in Heterologous Expression of Clostridium cellulolyticum Cellulase-Encoding Genes in Clostridium acetobutylicum ATCC 824▿

    Science.gov (United States)

    Mingardon, Florence; Chanal, Angélique; Tardif, Chantal; Fierobe, Henri-Pierre

    2011-01-01

    The genes encoding the cellulases Cel5A, Cel8C, Cel9E, Cel48F, Cel9G, and Cel9M from Clostridium cellulolyticum were cloned in the C. acetobutylicum expression vector pSOS952 under the control of a Gram-positive constitutive promoter. The DNA encoding the native leader peptide of the heterologous cellulases was maintained. The transformation of the solventogenic bacterium with the corresponding vectors generated clones in the cases of Cel5A, Cel8C, and Cel9M. Analyses of the recombinant strains indicated that the three cellulases are secreted in an active form to the medium. A large fraction of the secreted cellulases, however, lost the C-terminal dockerin module. In contrast, with the plasmids pSOS952-cel9E, pSOS952-cel48F, and pSOS952-cel9G no colonies were obtained, suggesting that the expression of these genes has an inhibitory effect on growth. The deletion of the DNA encoding the leader peptide of Cel48F in pSOS952-cel48F, however, generated strains of C. acetobutylicum in which mature Cel48F accumulates in the cytoplasm. Thus, the growth inhibition observed when the wild-type cel48F gene is expressed seems related to the secretion of the cellulase. The weakening of the promoter, the coexpression of miniscaffoldin-encoding genes, or the replacement of the native signal sequence of Cel48F by that of secreted heterologous or endogenous proteins failed to generate strains secreting Cel48F. Taken together, our data suggest that a specific chaperone(s) involved in the secretion of the key family 48 cellulase, and probably Cel9G and Cel9E, is missing or insufficiently synthesized in C. acetobutylicum. PMID:21378034

  2. Improvement of a Sulfolobus-E. coli shuttle vector for heterologous gene expression in Sulfolobus acidocaldarius.

    Science.gov (United States)

    Hwang, Sungmin; Choi, Kyoung-Hwa; Yoon, Naeun; Cha, Jaeho

    2015-02-01

    A Sulfolobus-E. coli shuttle vector for an efficient expression of the target gene in S. acidocaldarius strain was constructed. The plasmid-based vector pSM21 and its derivative pSM21N were generated based on the pUC18 and Sulfolobus cryptic plasmid pRN1. They carried the S. solfataricus P2 pyrEF gene for the selection marker, a multiple cloning site (MCS) with C-terminal histidine tag, and a constitutive promoter of the S. acidocaldarius gdhA gene for strong expression of the target gene, as well as the pBR322 origin and ampicillin-resistant gene for E. coli propagation. The advantage of pSM21 over other Sulfolobus shuttle vectors is that it contains a MCS and a histidine tag for the simple and easy cloning of a target gene as well as one-step purification by histidine affinity chromatography. For successful expression of the foreign genes, two genes from archaeal origins (PH0193 and Ta0298) were cloned into pSM21N and the functional expression was examined by enzyme activity assay. The recombinant PH0193 was successfully expressed under the control of the gdhA promoter and purified from the cultures by His-tag affinity chromatography. The yield was approximately 1 mg of protein per liter of cultures. The enzyme activity measurements of PH0913 and Ta0298 revealed that both proteins were expressed as an active form in S. acidocaldarius. These results indicate that the pSM21N shuttle vector can be used for the functional expression of foreign archaeal genes that form insoluble aggregates in the E. coli system.

  3. Recombination and Heterologous Expression of Allophycocyanin Gene in the Chloroplast of Chlamydomonas reinhardtii

    Institute of Scientific and Technical Information of China (English)

    Zhong-Liang SU; Kai-Xian QIAN; Cong-Ping TAN; Chun-Xiao MENG; Song QIN

    2005-01-01

    Heterogeneous expression of multiple genes in the nucleus of transgenic plants requires the introduction of an individual gene and the subsequent backcross to reconstitute multi-subunit proteins or metabolic pathways. In order to accomplish the expression of multiple genes in a single transformation event, we inserted both large and small subunits of allophycocyanin gene (apcA and apcB) into Chlamydomonas reinhardtii chloroplast expression vector, resulting in papc-S. The constructed vector was then introduced into the chloroplast of C. reinhardtii by micro-particle bombardment. Polymerase chain reaction and Southern blot analysis revealed that the two genes had integrated into the chloroplast genome. Western blot and enzyme-linked immunosorbent assay showed that the two genes from the prokaryotic cyanobacteria could be correctly expressed in the chloroplasts of C. reinhardtii. The expressed foreign protein in transformants accounted for about 2%-3% of total soluble proteins. These findings pave the way to the reconstitution of multi-subunit proteins or metabolic pathways in transgenic C. reinhardtii chloroplasts in a single transformation event.

  4. A novel platform for heterologous gene expression in Trichoderma reesei (Teleomorph Hypocrea jecorina)

    DEFF Research Database (Denmark)

    Jørgensen, Mikael Skaanning; Skovlund, Dominique Aubert; Johannesen, Pia Francke;

    2014-01-01

    ABSTRACT: BACKGROUND: The industrially applied filamentous fungus Trichoderma reesei has received substantial interest due to its highly efficient synthesis apparatus of cellulytic enzymes. However, the production of heterologous enzymes in T. reesei still remains low mainly due to lack of tools...

  5. Inactivation, complementation, and heterologous expression of encP, a novel bacterial phenylalanine ammonia-lyase gene.

    Science.gov (United States)

    Xiang, Longkuan; Moore, Bradley S

    2002-09-06

    The enzyme phenylalanine ammonia-lyase, which catalyzes the nonoxidative deamination of l-phenylalanine to trans-cinnamic acid, is ubiquitously distributed in plants. We now report its characterization for the first time in a bacterium. The phenylalanine ammonia-lyase homologous gene encP from the "Streptomyces maritimus" enterocin biosynthetic gene cluster was functionally characterized and shown to encode the first enzyme in the pathway to the enterocin polyketide synthase starter unit benzoyl-coenzyme A. The disruption of the encP gene completely inhibited the production of cinnamate and enterocin, whereas complementation of the mutant with benzoyl-coenzyme A pathway intermediates or with the wild-type gene encP restored the formation of the benzoate-primed polyketide antibiotic enterocin. Heterologous expression of the encP gene under the control of the ermE* promoter in Streptomyces coelicolor furthermore led to the production of cinnamic acid in the fermented cultures, confirming that the encP gene indeed encodes a novel bacterial phenylalanine ammonia-lyase.

  6. Characterization of heterologously expressed transporter genes by patch- and voltage-clamp methods: Application to cyclic nucleotide-dependent responses

    KAUST Repository

    Lemtiri-Chlieh, Fouad

    2013-09-03

    The application of patch- and voltage-clamp methods to study ion transport can be limited by many hurdles: the size of the cells to be patched and/or stabbed, the subcellular localization of the molecule of interest, and its density of expression that could be too low even in their own native environment. Functional expression of genes using recombinant DNA technology not only overcomes those hurdles but also affords additional and elegant investigations such as single-point mutation studies and subunit associations/regulations. In this chapter, we give a step-by-step description of two electrophysiological methods, patch clamp and two-electrode voltage clamp (TEVC), that are routinely used in combination with heterologous gene expression to assist researchers interested in the identification and characterization of ion transporters. We describe how to (1) obtain and maintain the cells suitable for the use with each of the above-mentioned methods (i.e., HEK-293 cells and yeast spheroplasts to use with the patch-clamp methodology and Xenopus laevis oocytes with TEVC), (2) transfect/inject them with the gene of interest, and (3) record ion transport activities. © Springer Science+Business Media New York 2013.

  7. MOLECULAR CLONING AND HETEROLOGOUS EXPRESSION OF HUMAN INTERFERON ALPHA2b GENE

    Directory of Open Access Journals (Sweden)

    I. Made Artika

    2013-01-01

    Full Text Available Human alpha Interferons (hIFNα have been shown to have antiviral, antiproliferative and immunomodulatory activities. The human interferon alpha2b (hIFNα2b, is one of the human interferon alpha2 sub variants, naturally synthesized as a polypeptide of 188 amino acid residues, the first 23 residues of which represents a signal peptide. In the present study, the hIFNα2b gene was expressed after being fused with Glutathione S-Transferase (GST gene. The hIFNα2b gene was amplified from human genomic DNA by using a pair of specific primers, cloned into an Escherichia coli expression vector and expressed in E. coli cells under the direction of the tac promoter. The expressed protein was purified using a one-step affinity chromatography column containing immobilized gluthatione-bound resin. The purified protein was shown to react specifically with anti-human-interferon-alpha antibody, confirming that the protein was the human interferon alpha molecule. This strategy has the potential to be used as an alternative mean for production of pure human interferon α proteins for therapeutic purposes and for further studies on their molecular characterization and mechanism of action.

  8. Cloning and heterologous expression of ectoine biosynthesis genes from Bacillus halodurans in Escherichia coli.

    Science.gov (United States)

    Anbu Rajan, Lawrance; Joseph, Toms C; Thampuran, Nirmala; James, Roswin; Ashok Kumar, Kesavan; Viswanathan, Chinnusamy; Bansal, Kailash C

    2008-08-01

    The genes involved in the biosynthetic pathway of ectoine (2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid) from Bacillus halodurans were cloned as an operon and expressed in E. coli. Analysis of the deduced ectoine biosynthesis cluster amino acid sequence revealed that the ectoine operon contain 2,389 bp, encoded by three genes; ectA, ectB and ectC that encode proteins of 189, 427 and 129 amino acids with deduced molecular masses of 21,048, 47,120 and 14,797 Da respectively. Extracts of induced cells showed two bands at 41 kDa and 17 kDa, possibly corresponding to the products of the later two genes. However the expression of ectA gene could not be ascertained by SDS-PAGE. The activity of the ectA protein was confirmed by an acylation assay. The transgenic E. coli accumulated upto 4.6 mg ectoine/l culture. This is the first report of an engineered E. coli strain carrying the ectoine genes of the alkaliphilic bacterium, B. halodurans.

  9. Transcriptional analysis of heterologous gene expression using the endogenous sD promoter from Bacillus halodurans

    CSIR Research Space (South Africa)

    Crampton, Michael C

    2010-07-01

    Full Text Available (2006). Trends in Microbiology 14: 151-155. Slide 7 © CSIR 2006 www.csir.co.za Evaluation of different genetic backgrounds on peptide secretion during log and stationary phase Berger et al (2009) Applied and Environmental... • Transcriptional analysis Fermentation • Micro-array analysis of global gene expression during transition between exponential and stationary phase • Directed evolution of σD promoter Acknowledgements Maureen Louw Eldie Berger Erika Du Plessis Nolwandle...

  10. Characterization of Saccharomyces cerevisiae promoters for heterologous gene expression in Kluyveromyces marxianus.

    Science.gov (United States)

    Lee, Ki-Sung; Kim, Jun-Seob; Heo, Paul; Yang, Tae-Jun; Sung, Young-Je; Cheon, Yuna; Koo, Hyun Min; Yu, Byung Jo; Seo, Jin-Ho; Jin, Yong-Su; Park, Jae Chan; Kweon, Dae-Hyuk

    2013-03-01

    Kluyveromyces marxianus is now considered one of the best choices of option for industrial applications of yeast because the strain is able to grow at high temperature, utilizes various carbon sources, and grows fast. However, the use of K. marxianus as a host for industrial applications is still limited. This limitation is largely due to a lack of knowledge on the characteristics of the promoters since the time and amount of protein expression is strongly dependent on the promoter employed. In this study, four well-known constitutive promoters (P(CYC), P(TEF), P(GPD), and P(ADH)) of Saccharomyces cerevisiae were characterized in K. marxianus in terms of protein expression level and their stochastic behavior. After constructing five URA3-auxotrophic K. marxianus strains and a plasmid vector, four cassettes each comprising one of the promoters--the gene for the green fluorescence protein (GFP)--CYC1 terminator (T(CYC)) were inserted into the vector. GFP expression under the control of each one of the promoters was analyzed by reverse transcription PCR, fluorescence microscopy, and flow cytometer. Using these combined methods, the promoter strength was determined to be in the order of P(GPD) > P(ADH) ∼ P(TEF) > P(CYC). All promoters except for the P(CYC) exhibited three distinctive populations, including non-expressing cells, weakly expressing cells, and strongly expressing cells. The relative ratios between populations were strongly dependent on the promoter and culture time. Forward scattering was independent of GFP fluorescence intensity, indicating that the different fluorescence intensities were not just due to different cell sizes derived from budding. It also excluded the possibility that the non-expressing cells resulted from plasmid loss because plasmid stability was maintained at almost 100 % over the culture time. The same cassettes, cloned into a single copy plasmid pRS416 and transformed into S. cerevisiae, showed only one population. When the

  11. Co-expression of two heterologous lactate dehydrogenases genes in Kluyveromyces marxianus for l-lactic acid production.

    Science.gov (United States)

    Lee, Jae Won; In, Jung Hoon; Park, Joon-Bum; Shin, Jonghyeok; Park, Jin Hwan; Sung, Bong Hyun; Sohn, Jung-Hoon; Seo, Jin-Ho; Park, Jin-Byoung; Kim, Soo Rin; Kweon, Dae-Hyuk

    2017-01-10

    Lactic acid (LA) is a versatile compound used in the food, pharmaceutical, textile, leather, and chemical industries. Biological production of LA is possible by yeast strains expressing a bacterial gene encoding l-lactate dehydrogenase (LDH). Kluyveromyces marxianus is an emerging non-conventional yeast with various phenotypes of industrial interest. However, it has not been extensively studied for LA production. In this study, K. marxianus was engineered to express and co-express various heterologous LDH enzymes that were reported to have different pH optimums. Specifically, three LDH enzymes originating from Staphylococcus epidermidis (SeLDH; optimal at pH 5.6), Lactobacillus acidophilus (LaLDH; optimal at pH 5.3), and Bos taurus (BtLDH; optimal at pH 9.8) were functionally expressed individually and in combination in K. marxianus, and the resulting strains were compared in terms of LA production. A strain co-expressing SeLDH and LaLDH (KM5 La+SeLDH) produced 16.0g/L LA, whereas the strains expressing those enzymes individually produced only 8.4 and 6.8g/L, respectively. This co-expressing strain produced 24.0g/L LA with a yield of 0.48g/g glucose in the presence of CaCO3. Our results suggest that co-expression of LDH enzymes with different pH optimums provides sufficient LDH activity under dynamic intracellular pH conditions, leading to enhanced production of LA compared to individual expression of the LDH enzymes. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Cloning and characterization of Pangasianodon hypophthalmus growth hormone gene and its heterologous expression.

    Science.gov (United States)

    Sekar, Megarajan; Singh, Shiva Dhar; Gupta, Subodh

    2014-07-01

    Pangasianodon hypophthalmus is one of the fast-growing catfish of freshwater origin, and its growth is attributed by the action of growth hormone (GH). In this study, the growth hormone gene (PhGH) of 3.0 kb was characterized, and it is composed of five exons and four introns and having characteristics of an upstream region that contains TATA, CAAT boxes, and binding sites of important transcription factors like Pit-1a, CRE, CREB, CREBP, Ap-1, SP1, and TBP. The full-length cDNA sequence of 1,069 bp was isolated using RACE technique, and it is composed of untranslated regions of 60 and 403 bp at 5' and 3', respectively, with an open reading frame of 603 bp that encodes a putative polypeptide of 200 amino acids with an estimated molecular mass of 22.57 kDa. The precursor of PhGH is composed of 22 amino acid signal peptides and 178 amino acid mature peptides. Five conserved Cys residues (Cys(71), Cys(135), Cys(173), Cys(190), and Cys(198)) and two possible sites of N-glycosylation (145th and 197th) were detected on GH polypeptide. The PhGH gene showed more than 90 % sequence similarity with other catfishes, and the phylogeny constructed revealed the close proximity of Siluriformes fishes with Cypriniformes fishes. The PhGH gene was observed to be expressed predominantly in pituitary tissues while weekly expressed in extrapituitary tissues. Further, the recombinant PhGH was expressed in Escherichia coli using His-tag expression vector pET 32(a), and the recombinant protein of ~23 kDa was confirmed by western blotting. Our findings suggest that the identified functional GH gene would provide basic information in transgenic studies aiming for faster growth rate. This recombinant growth hormone (GH) may be produced in large scale to exploit its growth-promoting function in other cultured fishes.

  13. Heterologous expression of Avermectins biosynthetic gene cluster by construction of a Bacterial Artificial Chromosome library of the producers

    Directory of Open Access Journals (Sweden)

    Qian Deng

    2017-03-01

    Full Text Available Avermectins, a group of polyketide natural products, are widely used as anthelmintics in agriculture. Metabolic engineering and combinatorial biosynthesis were extensively employed to improve Avermectins production and create novel Avermectin derivatives, including Ivermectin and Doramectin. It is labor intensive and time cost to genetically manipulate Avermectins producer Streptomyces avermitilis in vivo. Cloning and heterologous expression of Avermectins biosynthetic gene cluster will make it possible to tailor the cluster in vitro. We constructed a Bacterial Artificial Chromosome (BAC library of S. avermitilis ATCC 31267 with inserted DNA fragments ranged from 100 to 130 Kb. Five recombinant BAC clones which carried the Avermectins biosynthetic gene cluster ave (81 Kb in size were screened out from the library. Then, ave was hetero-expressed in S. lividans. Three Avermectin components, A2a, B1a and A1a were detected from the cell extracts of recombinant strains. It will facilitate the development of Avermectin derivatives by polyketide synthase domain swapping and provide functional element for Avermectins synthetic biology study.

  14. Heterologous expression and activity studying of a nisin resistance gene in Lactobacillus delbrueckii subsp. bulgaricus

    Directory of Open Access Journals (Sweden)

    Fanghong Gong

    2013-04-01

    Full Text Available Background: Lactic acid bacteria are generally regarded as safe microorganisms and widely used in industry and medicine. We are trying to add additional properties to them by gene engineering. However, the genetically modified bacteria are not acceptable to use in food and medicine due to the presence of antibiotic resistance genes in plasmids. Thus, it is necessary to develop food-grade selection markers. Methods: To evaluate the activity of nisin resistance gene (nsr cloned from Lactococcus lactis as a food-grade selective marker in transformant bacterial strains, we isolated L.lactis nisin resistant strains from fresh milk, and cloned the nsr gene to Lactobacillus delbrueckii subsp. bulgaricus, then studied the activity of nsr in positive transformant bacterial strains. Results: Six nisin resistant strains were isolated from fresh milk on M17 plates supplemented with nisin at a final concentration of 400 μg/ml. All of these isolates were identified as L.lactis by morphological, physiological, biochemical and 16S rDNA sequence analysis. A pair of primers was designed on the basis of the DNA sequences of reported nsr. PCR amplification was carried out with chromosome and plasmid DNA as templates from these six strains respectively, and an expected PCR product was obtained from one chromosome. After the amplicon was confirmed as nsr gene, it was cloned into E.coli-Lactobacillus shuttle vector pMG36e, resulting in pMG36e-nsr. The construct was obtained when the recombinant plasmid pMG36e-nsr was electroporated into L.delbrueckii subsp. bulgaricus L6032 competent cells. When the medium contained a maximum of 400 μg/ml nisin, the construct carrying pMG36e-nsr showed the same growth curve as that cultured in medium containing 10 μg/ml erythromycin. Conclusions: The nsr gene was cloned and successfully expressed in L.delbrueckii subsp. bulgaricus L6032. This study suggests that nsr gene could be used as a potential food-grade selective marker to

  15. Fungal His-tagged nitrilase from Gibberella intermedia: gene cloning, heterologous expression and biochemical properties.

    Directory of Open Access Journals (Sweden)

    Jin-Song Gong

    Full Text Available BACKGROUND: Nitrilase is an important member of the nitrilase superfamiliy. It has attracted substantial interest from academia and industry for its function of converting nitriles directly into the corresponding carboxylic acids in recent years. Thus nitrilase has played a crucial role in production of commercial carboxylic acids in chemical industry and detoxification of nitrile-contaminated wastes. However, conventional studies mainly focused on the bacterial nitrilase and the potential of fungal nitrilase has been far from being fully explored. Research on fungal nitrilase gene expression will advance our understanding for its biological function of fungal nitrilase in nitrile hydrolysis. METHODOLOGY/PRINCIPAL FINDINGS: A fungal nitrilase gene from Gibberella intermedia was cloned through reverse transcription-PCR. The open reading frame consisted of 963 bp and potentially encoded a protein of 320 amino acid residues with a theoretical molecular mass of 35.94 kDa. Furthermore, the catalytic triad (Glu-45, Lys-127, and Cys-162 was proposed and confirmed by site-directed mutagenesis. The encoding gene was expressed in Escherichia coli Rosetta-gami (DE3 and the recombinant protein with His(6-tag was purified to electrophoretic homogeneity. The purified enzyme exhibited optimal activity at 45°C and pH 7.8. This nitrilase was specific towards aliphatic and aromatic nitriles. The kinetic parameters V(max and K(m for 3-cyanopyridine were determined to be 0.81 µmol/min·mg and 12.11 mM through Hanes-Woolf plot, respectively. 3-Cyanopyridine (100 mM could be thoroughly hydrolyzed into nicotinic acid within 10 min using the recombinant strain with the release of about 3% nicotinamide and no substrate was detected. CONCLUSIONS/SIGNIFICANCE: In the present study, a fungal nitrilase was cloned from the cDNA sequence of G. intermedia and successfully expressed in E. coli Rosetta-gami (DE3. The recombinant strain displayed good 3-cyanopyridine

  16. Heterologous Expression of Amylase Gene from Saccharomycopsis fibuligera in an Industrial Strain of Saccharomyces cerevisiae

    Institute of Scientific and Technical Information of China (English)

    LIU Zeng-ran; ZHANG Guang-yi; LONG Zhang-fu; LIU Shi-gui

    2005-01-01

    An α-amylase encoding gene was amplified by polymerase chain reaction from Saccharomycopsis fibuligera and inserted into a shuttle vector YEp352,together with the yeast phosphoglycerate kinase 1 promoter and α-factor signal gene. The recombinant expression plasmid pLA8α was transformed into an industrial strain of Saccharomyces cerevisiae Sc-11. The activity of the α-amylase produced by the transformant Sc-11-pLA8α was 6.3 U/mL and the starch utilization rate in YPS medium was 42 %. The purified amylase was analyzed by SDS-PAGE,showing a molecular weight of 55×103 protein band. Furthermore, the residual sugar, ethanol and some volatile compounds in the fermented worts under simulating brewing conditions were determined by chromatographic analyses. The fermentation characteristics of Sc-11-pLA8α were similar to that of Sc-11 and only minor changes in the concentration of flavor compounds could be observed.

  17. Heterologous expression of an insecticidal gene from the armed spider (Phoneutria nigriventer

    Directory of Open Access Journals (Sweden)

    J. E. F. Figueiredo

    2008-01-01

    Full Text Available Insect-pests are global problems that cause severe damage to crop plants, and their control is commonly based on chemical insecticides. However, negative effects of pesticides on the environment and human health emphasize the necessity to develop alternative methods for insect-pest control. In the present study, a gene coding for the insecticidal peptide TX4(6-1 of the Brazilian armed spider (Phoneutria nigriventer was cloned in fusion with maltose binding protein (MBP and expressed in Escherichia coli. The affinity purified protein MBP-GlyTX4 was cleaved with the Xa factor and used for a bioassay against Spodoptera frugiperda and rabbit immunization. Five micrograms GlyTX4 protein injected into the hemocoel of larvae and abdominal cavity of adults produced trembling and uncoordinated movements immediately after injection and all adult insects died after 12h. After two days, larvae became paralyzed and the epidermal color changed to dark brown. Furthermore, the development stage was prolonged for two weeks. Alternatively, slices of maize leaves were imbibed with 15 micrograms of the recombinant protein cleaved with the Xa factor and used as diet for larvae. In this experiment, all larvae died in about 30 minutes. Polyclonal antibodies anti-MBP-GlyTX4 were effective for recognizing MBP and GlyTX4 in whole cell extract from E. coli expressing the recombinant protein.

  18. Heterologous expression of the Pleurotus ostreatus MnP3 gene by the laccase gene promoter in Lentinula edodes.

    Science.gov (United States)

    Sato, Toshitsugu; Irie, Toshikazu; Yoshino, Fumihiko

    2017-08-01

    Lentinula edodes (shiitake), which have a powerful ligninolytic system, is one of the most important edible mushrooms in Asia. In this study, we introduced the manganese peroxidase (MnP, EC 1.11.1.13) gene from Pleurotus ostreatus driven by L. edodes laccase 1 gene promoter into L. edodes for expression. The resulting transformant expressed the recombinant gene and showed a higher level of MnP activity than that of the wild-type strain.

  19. Heterologous expression of the AtDREB1A gene in chrysanthemum increases drought and salt stress tolerance

    Institute of Scientific and Technical Information of China (English)

    HONG; Bo; TONG; Zheng; MA; Nan; LI; Jianke; KASUGA; Mie; YAMAGUCHI-SHINOZAKI; Kazuko; GAO; Junping

    2006-01-01

    DNA cassette containing an AtDREB1A cDNA and a nos terminator, driven by a cauliflower mosaic 35S promoter, or a stress-inducible rd29A promoter, was transformed into the ground cover chrysanthemum (Dendranthema grandiflorum) 'Fall Color' genome. Compared with wild type plants, severe growth retardation was observed in 35S:DREB1A plants, but not in rd29A:DREB1A plants. RT-PCR analysis revealed that, under stress conditions, the DREB1A gene was over-expressed constitutively in 35S:DREB1A plants, but was over-expressed inductively in rd29A:DREB1A plants. The transgenic plants exhibited tolerance to drought and salt stress, and the tolerance was significantly stronger in rd29A:DREB1A plants than tn 35S:DREB1A plants. Proline content and SOD activity were increased inductively in rd29A:DREB1A plants than in 35S:DREB1A plants under stress conditions. These results indicate that heterologous AtDREB1A can confer drought and salt tolerance in transgenic chrysanthemum, and improvement of the stress tolerance may be related to enhancement of proline content and SOD activity.

  20. Effect of Genome Position on Heterologous Gene Expression in Bacillus subtilis: An Unbiased Analysis

    NARCIS (Netherlands)

    Sauer, C.; Syvertsson, S.; Bohorquez, L.C.; Cruz, R.; Harwood, C.R.; van Rij, T.; Hamoen, L.W.

    2016-01-01

    A fixed gene copy number is important for the in silico construction of engineered synthetic networks. However, the copy number of integrated genes depends on their genomic location. This gene dosage effect is rarely addressed in synthetic biology. Two studies in Escherichia coli presented conflicti

  1. Heterologous expression of oxytetracycline biosynthetic gene cluster in Streptomyces venezuelae WVR2006 to improve production level and to alter fermentation process.

    Science.gov (United States)

    Yin, Shouliang; Li, Zilong; Wang, Xuefeng; Wang, Huizhuan; Jia, Xiaole; Ai, Guomin; Bai, Zishang; Shi, Mingxin; Yuan, Fang; Liu, Tiejun; Wang, Weishan; Yang, Keqian

    2016-12-01

    Heterologous expression is an important strategy to activate biosynthetic gene clusters of secondary metabolites. Here, it is employed to activate and manipulate the oxytetracycline (OTC) gene cluster and to alter OTC fermentation process. To achieve these goals, a fast-growing heterologous host Streptomyces venezuelae WVR2006 was rationally selected among several potential hosts. It shows rapid and dispersed growth and intrinsic high resistance to OTC. By manipulating the expression of two cluster-situated regulators (CSR) OtcR and OtrR and precursor supply, the OTC production level was significantly increased in this heterologous host from 75 to 431 mg/l only in 48 h, a level comparable to the native producer Streptomyces rimosus M4018 in 8 days. This work shows that S. venezuelae WVR2006 is a promising chassis for the production of secondary metabolites, and the engineered heterologous OTC producer has the potential to completely alter the fermentation process of OTC production.

  2. Heterologous expression of tyrosinase recapitulates the misprocessing and mistrafficking in oculocutaneous albinism type 2: effects of altering intracellular pH and pink-eyed dilution gene expression.

    Science.gov (United States)

    Ni-Komatsu, Li; Orlow, Seth J

    2006-03-01

    The processing and trafficking of tyrosinase, a melanosomal protein essential for pigmentation, was investigated in a human epithelial 293 cell line that stably expresses the protein. The effects of the pink-eyed dilution (p) gene product, in which mutations result in oculocutaneous albinism type 2 (OCA2), on the processing and trafficking of tyrosinase in this cell line were studied. The majority of tyrosinase was retained in the endoplasmic reticulum-Golgi intermediate compartment and the early Golgi compartment in the 293 cells expressing the protein. Coexpression of p could partially correct the mistrafficking of tyrosinase in 293 cells. Tyrosinase was targeted to the late endosomal and lysosomal compartments after treatment of the cells with compounds that correct the tyrosinase mistrafficking in albino melanocytes, most likely through altering intracellular pH, while the substrate tyrosine had no effect on the processing of tyrosinase. Remarkably, this heterologous expression system recapitulates the defective processing and mistrafficking of tyrosinase observed in OCA2 albino melanocytes and certain amelanotic melanoma cells. Coexpression of other melanosomal proteins in this heterologous system may further aid our understanding of the details of normal and pathologic processing of melanosomal proteins.

  3. Development of dengue virus replicons expressing HIV-1 gp120 and other heterologous genes: a potential future tool for dual vaccination against dengue virus and HIV

    Directory of Open Access Journals (Sweden)

    Dayton Andrew I

    2001-11-01

    Full Text Available Abstract Background Toward the goals of providing an additional vector to add to the armamentarium available to HIV vaccinologists and of creating a bivalent vaccine effective against dengue virus and HIV, we have attempted to create vectors which express dengue virus non-structural proteins and HIV immunogens. Previously we reported the successful construction of dengue virus replicons which lack structural genes necessary for virion release and spreading infection in culture but which can replicate intracellularly and abundantly produce dengue non-structural proteins. Here we attempted to express heterologous genetic material from these replicons. Results We cloned into a Δpre-M/E dengue virus replicon genes for either green fluorescent protein (GFP, HIV gp160 or HIV gp120 and tested the ability of these constructs to express dengue virus proteins as well as the heterologous proteins in tissue culture after transfection of replicon RNA. Conclusions Heterologous proteins were readily expressed from these constructs. GFP and gp120 demonstrated minimal or no toxicity. Gp160 expressing replicons were found to express proteins abundantly at 36 hours post transfection, but after 50 hrs of transfection, few replicon positive cells could be found despite the presence of cellular debris positive for replicon proteins. This suggested that gp160 expressed from dengue virus replicons is considerably more toxic than either GFP or gp120. The successful expression of heterologous proteins, including HIV gp120 for long periods in culture suggests this vector system may be useful as a vaccine vector, given appropriate delivery methods.

  4. The S-layer gene of Lactobacillus helveticus CNRZ 892 : cloning, sequence and heterologous expression

    NARCIS (Netherlands)

    Callegari, M.L.; Riboli, B.; Sanders, J.W; Cocconcelli, P.S.; Kok, J.; Venema, G; Morelli, L.

    1998-01-01

    Lactobacillus helveticus CNRZ 892 contains a surface layer (S-layer) composed of protein monomers of 43 kDa organized in regular arrays. The gene encoding this protein (slpH) has been cloned in Escherichia coli and sequenced. slpH consists of 440 codons and is preceded by a ribosome-binding site (RB

  5. Heterologous gene expression and functional analysis of a type III polyketide synthase from Aspergillus niger NRRL 328

    Energy Technology Data Exchange (ETDEWEB)

    Kirimura, Kohtaro, E-mail: kkohtaro@waseda.jp; Watanabe, Shotaro; Kobayashi, Keiichi

    2016-05-13

    Type III polyketide synthases (PKSs) catalyze the formation of pyrone- and resorcinol-types aromatic polyketides. The genomic analysis of the filamentous fungus Aspergillus niger NRRL 328 revealed that this strain has a putative gene (chr-8-2: 2978617–2979847) encoding a type III PKS, although its functions are unknown. In this study, for functional analysis of this putative type III PKS designated as An-CsyA, cloning and heterologous expression of the An-CsyA gene (An-csyA) in Escherichia coli were performed. Recombinant His-tagged An-CsyA was successfully expressed in E. coli BL21 (DE3), purified by Ni{sup 2+}-affinity chromatography, and used for in vitro assay. Tests on the substrate specificity of the His-tagged An-CsyA with myriad acyl-CoAs as starter substrates and malonyl-CoA as extender substrate showed that His-tagged An-CsyA accepted fatty acyl-CoAs (C2-C14) and produced triketide pyrones (C2-C14), tetraketide pyrones (C2-C10), and pentaketide resorcinols (C10-C14). Furthermore, acetoacetyl-CoA, malonyl-CoA, isobutyryl-CoA, and benzoyl-CoA were also accepted as starter substrates, and both of triketide pyrones and tetraketide pyrones were produced. It is noteworthy that the His-tagged An-CsyA produced polyketides from malonyl-CoA as starter and extender substrates and produced tetraketide pyrones from short-chain fatty acyl-CoAs as starter substrates. Therefore, this is the first report showing the functional properties of An-CsyA different from those of other fungal type III PKSs. -- Highlights: •Type III PKS from Aspergillus niger NRRL 328, An-CsyA, was cloned and characterized. •An-CsyA produced triketide pyrones, tetraketide pyrones and pentaketide resorcinols. •Functional properties of An-CsyA differs from those of other fungal type III PKSs.

  6. Heterologous Expression and Characterization of Mimosinase from Leucaena leucocephala.

    Science.gov (United States)

    Negi, Vishal Singh; Borthakur, Dulal

    2016-01-01

    Heterologous expression of eukaryotic genes in bacterial system is an important method in synthetic biology to characterize proteins. It is a widely used method, which can be sometimes quite challenging, as a number of factors that act along the path of expression of a transgene to mRNA, and mRNA to protein, can potentially affect the expression of a transgene in a heterologous system. Here, we describe a method for successful cloning and expression of mimosinase-encoding gene from Leucaena leucocephala (leucaena) in E. coli as the heterologous host. Mimosinase is an important enzyme especially in the context of metabolic engineering of plant secondary metabolite as it catalyzes the degradation of mimosine, which is a toxic secondary metabolite found in all Leucaena and Mimosa species. We also describe the methods used for characterization of the recombinant mimosinase.

  7. Direct cloning and heterologous expression of the salinomycin biosynthetic gene cluster from Streptomyces albus DSM41398 in Streptomyces coelicolor A3(2).

    Science.gov (United States)

    Yin, Jia; Hoffmann, Michael; Bian, Xiaoying; Tu, Qiang; Yan, Fu; Xia, Liqiu; Ding, Xuezhi; Stewart, A Francis; Müller, Rolf; Fu, Jun; Zhang, Youming

    2015-10-13

    Linear plus linear homologous recombination-mediated recombineering (LLHR) is ideal for obtaining natural product biosynthetic gene clusters from pre-digested bacterial genomic DNA in one or two steps of recombineering. The natural product salinomycin has a potent and selective activity against cancer stem cells and is therefore a potential anti-cancer drug. Herein, we separately isolated three fragments of the salinomycin gene cluster (salO-orf18) from Streptomyces albus (S. albus) DSM41398 using LLHR and assembled them into intact gene cluster (106 kb) by Red/ET and expressed it in the heterologous host Streptomyces coelicolor (S. coelicolor) A3(2). We are the first to report a large genomic region from a Gram-positive strain has been cloned using LLHR. The successful reconstitution and heterologous expression of the salinomycin gene cluster offer an attractive system for studying the function of the individual genes and identifying novel and potential analogues of complex natural products in the recipient strain.

  8. Identification of the subgenomic promoter of the coat protein gene of cucumber fruit mottle mosaic virus and development of a heterologous expression vector.

    Science.gov (United States)

    Rhee, Sun-Ju; Jang, Yoon Jeong; Lee, Gung Pyo

    2016-06-01

    Heterologous gene expression using plant virus vectors enables research on host-virus interactions and the production of useful proteins, but the host range of plant viruses limits the practical applications of such vectors. Here, we aimed to develop a viral vector based on cucumber fruit mottle mosaic virus (CFMMV), a member of the genus Tobamovirus, whose members infect cucurbits. The subgenomic promoter (SGP) in the coat protein (CP) gene, which was used to drive heterologous expression, was mapped by analyzing deletion mutants from a CaMV 35S promoter-driven infectious CFMMV clone. The region from nucleotides (nt) -55 to +160 relative to the start codon of the open reading frame (ORF) of CP was found to be a fully active promoter, and the region from nt -55 to +100 was identified as the active core promoter. Based on these SGPs, we constructed a cloning site in the CFMMV vector and successfully expressed enhanced green fluorescent protein (EGFP) in Nicotiana benthamiana and watermelon (Citrullus lanatus). Co-inoculation with the P19 suppressor increased EGFP expression and viral replication by blocking degradation of the viral genome. Our CFMMV vector will be useful as an expression vector in cucurbits.

  9. Genome mining of the Streptomyces avermitilis genome and development of genome-minimized hosts for heterologous expression of biosynthetic gene clusters.

    Science.gov (United States)

    Ikeda, Haruo; Kazuo, Shin-ya; Omura, Satoshi

    2014-02-01

    To date, several actinomycete genomes have been completed and annotated. Among them, Streptomyces microorganisms are of major pharmaceutical interest because they are a rich source of numerous secondary metabolites. S. avermitilis is an industrial microorganism used for the production of an anthelmintic agent, avermectin, which is a commercially important antiparasitic agent in human and veterinary medicine, and agricultural pesticides. Genome analysis of S. avermitilis provides significant information for not only industrial applications but also understanding the features of this genus. On genome mining of S. avermitilis, the microorganism has been found to harbor at least 38 secondary metabolic gene clusters and 46 insertion sequence (IS)-like sequences on the genome, which have not been searched so far. A significant use of the genome data of Streptomyces microorganisms is the construction of a versatile host for heterologous expression of exogenous biosynthetic gene clusters by genetic engineering. Since S. avermitilis is used as an industrial microorganism, the microorganism is already optimized for the efficient supply of primary metabolic precursors and biochemical energy to support multistep biosynthesis. The feasibility of large-deletion mutants of S. avermitilis has been confirmed by heterologous expression of more than 20 exogenous biosynthetic gene clusters.

  10. Development of a new vector using Soybean yellow common mosaic virus for gene function study or heterologous protein expression in soybeans.

    Science.gov (United States)

    Lim, Seungmo; Nam, Moon; Kim, Kil Hyun; Lee, Su-Heon; Moon, Jung-Kyung; Lim, Hyoun-Sub; Choung, Myoung-Gun; Kim, Sang-Mok; Moon, Jae Sun

    2016-02-01

    A new vector using Soybean yellow common mosaic virus (SYCMV) was constructed for gene function study or heterologous protein expression in soybeans. The in vitro transcript with a 5' cap analog m7GpppG from an SYCMV full-length infectious vector driven by a T7 promoter infected soybeans (pSYCMVT7-full). The symptoms observed in the soybeans infected with either the sap from SYCMV-infected leaves or pSYCMVT7-full were indistinguishable, suggesting that the vector exhibits equivalent biological activity as the virus itself. To utilize the vector further, a DNA-based vector driven by the Cauliflower mosaic virus (CaMV) 35S promoter was constructed. The complete sequence of the SYCMV genome was inserted into a binary vector flanked by a CaMV 35S promoter at the 5' terminus of the SYCMV genome and a cis-cleaving ribozyme sequence followed by a nopaline synthase terminator at the 3' terminus of the SYCMV genome (pSYCMV-full). The SYCMV-derived vector was tested for use as a virus-induced gene silencing (VIGS) vector for the functional analysis of soybean genes. VIGS constructs containing either a fragment of the Phytoene desaturase (PDS) gene (pSYCMV-PDS1) or a fragment of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RbcS) gene (pSYCMV-RbcS2) were constructed. Plants infiltrated with each vector using the Agrobacterium-mediated inoculation method exhibited distinct symptoms, such as photo-bleaching in plants infiltrated with pSYCMV-PDS1 and yellow or pale green coloring in plants infiltrated with pSYCMV-RbcS2. In addition, down-regulation of the transcripts of the two target genes was confirmed via northern blot analysis. Particle bombardment and direct plasmid DNA rubbing were also confirmed as alternative inoculation methods. To determine if the SYCMV vector can be used for the expression of heterologous proteins in soybean plants, the vector encoding amino acids 135-160 of VP1 of Foot-and-mouth disease virus (FMDV) serotype O1 Campos (O1C

  11. Heterologous expression of phaC2 gene and poly-3-hydroxyalkanoate production by recombinant Cupriavidus necator strains using canola oil as carbon source.

    Science.gov (United States)

    Valdés, J; Kutralam-Muniasamy, G; Vergara-Porras, B; Marsch, R; Pérez-Guevara, F; López-Cuellar, M R

    2017-08-19

    Many heterologous transformation studies have been carried out using the Cupriavidus necator PHB(-4) strain to investigate the expression characteristics of various polyhydroxyalkanoate (PHA) synthase enzymes. In this study, we generated a recombinant C. necator PHB(-4) strain by transforming a plasmid (pMRC03) harbouring the synthetic phaC2 gene of Pseudomonas putida CA-3. Under conditions favourable for expression of the phaC2 P.putCA-3 gene, canola oil was used as carbon source for the synthesis of PHAs. The expressed synthase polymerised monomers of 3-hydroxybutyrate (3-HB), 3-hydroxyvalerate (3-HV) and 3-hydroxyhexanoate (3-HHx) in the recombinant C. necator PHB(-4) (pMRC03) strain. We then co-expressed the phaC2P.putCA-3 gene with the native phaC1C.ne gene in wild type Cupriavidus necator H16 (C. necator H16 (pMRC03)). This co-expression produced a PHA blend of 3-HB, 3-HV, 3-HHx and 3-hydroxyoctanoate (3-HO) monomers in the presence of canola oil. Gas chromatography analysis revealed the presence of 94mol% 3-HB, 1mol% 3-HV, 4mol% 3-HHx and 1mol% 3-HO in a tetra-polymer. Thus, we confirmed that a synthetic phaC2 gene encoding the synthase enzyme is functionally active with substrates ranging from short to medium chain length PHAs. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Molecular Characterization of Heterologous HIV-1gp120 Gene Expression Disruption in Mycobacterium bovis BCG Host Strain: A Critical Issue for Engineering Mycobacterial Based-Vaccine Vectors

    Directory of Open Access Journals (Sweden)

    Joan Joseph

    2010-01-01

    Full Text Available Mycobacterium bovis Bacillus Calmette-Guérin (BCG as a live vector of recombinant bacterial vaccine is a promising system to be used. In this study, we evaluate the disrupted expression of heterologous HIV-1gp120 gene in BCG Pasteur host strain using replicative vectors pMV261 and pJH222. pJH222 carries a lysine complementing gene in BCG lysine auxotrophs. The HIV-1 gp120 gene expression was regulated by BCG hsp60 promoter (in plasmid pMV261 and Mycobacteria spp. α-antigen promoter (in plasmid pJH222. Among 14 rBCG:HIV-1gp120 (pMV261 colonies screened, 12 showed a partial deletion and two showed a complete deletion. However, deletion was not observed in all 10 rBCG:HIV-1gp120 (pJH222 colonies screened. In this study, we demonstrated that E. coli/Mycobacterial expression vectors bearing a weak promoter and lysine complementing gene in a recombinant lysine auxotroph of BCG could prevent genetic rearrangements and disruption of HIV 1gp120 gene expression, a key issue for engineering Mycobacterial based vaccine vectors.

  13. Definition of culture conditions for Arxula adeninivorans, a rational basis for studying heterologous gene expression in this dimorphic yeast.

    Science.gov (United States)

    Stöckmann, Christoph; Palmen, Thomas G; Schroer, Kirsten; Kunze, Gotthard; Gellissen, Gerd; Büchs, Jochen

    2014-06-01

    The yeast Arxula adeninivorans is considered to be a promising producer of recombinant proteins. However, growth characteristics are poorly investigated and no industrial process has been established yet. Though of vital interest for strain screening and production processes, rationally defined culture conditions remain to be developed. A cultivation system was evolved based on targeted sampling and mathematical analysis of rationally designed small-scale cultivations in shake flasks. The oxygen and carbon dioxide transfer rates were analyzed as conclusive online parameters. Oxygen limitation extended cultivation and led to ethanol formation in cultures supplied with glucose. Cultures were inhibited at pH-values below 2.8. The phosphorus demand was determined as 1.55 g phosphorus per 100 g cell dry weight. Synthetic SYN6 medium with 20 g glucose l(-1) was optimized for cultivation in shake flasks by buffering at pH 6.4 with 140 mmol MES l(-1). Optimized SYN6 medium and operating conditions provided non-limited cultivations without by-product formation. A maximal specific growth rate of 0.32 h(-1) and short fermentations of 15 h were achieved. A pH optimum curve was derived from the oxygen transfer rates of differently buffered cultures, showing maximal growth between pH 2.8 and 6.5. Furthermore, it was shown that the applied medium and cultivation conditions were also suitable for non-limiting growth and product formation of a genetically modified A. adeninivorans strain expressing a heterologous phytase.

  14. Heterologous expression of lcc1 gene from Trametes trogii in Pichia pastoris and characterization of the recombinant enzyme

    Directory of Open Access Journals (Sweden)

    Buonocore Vincenzo

    2006-10-01

    Full Text Available Abstract Background Fungal laccases are useful enzymes for industrial applications; they exhibit broad substrate specificity and thus are able to oxidize a variety of xenobiotic compounds including chlorinated phenolics, synthetic dyes, pesticides and polycyclic aromatic hydrocarbons. Unfortunately, the biotechnological exploitation of laccases can be hampered by the difficulties concerning the enzyme production by the native hosts. Results In order to obtain a simple and efficient source of laccase, the lcc1 cDNA isolated from the white-rot fungus Trametes trogii has been successfully expressed in the methylotrophic yeast Pichia pastoris under the control of the methanol induced alcohol oxidase promoter PAOX1. The recombinant Lcc1 was produced as a secreted protein with the native N-terminal prepropeptide for signal trafficking, and thus easily recovered from the culture medium. At the 1-liter scale, as calculated on the basis of the specific activity, the recombinant protein was produced at a yield of 17 mg/l. The highest production level obtained in fed-batch culture was 2520 U/l, corresponding to a specific productivity of 31.5 U/g biomass. The purified recombinant laccase exhibited a behaviour similar to the main laccase produced by T. trogii. Lcc1 showed high activity in the presence of organic solvents and a high decolourization capacity towards azo, triarylmethane, indigo carmine and anthraquinonic dyes, that could be significantly enhanced in the presence of the redox mediators 1-hydroxybenzotriazole and violuric acid. Conclusion Heterologous expression of T. trogii laccase lcc1 in the methylotrophic yeast P. pastoris was successfully achieved. The biochemical and kinetic characterization of the recombinant protein suggests potential technological applications for this enzyme.

  15. PCR-based amplification and heterologous expression of Pseudomonas alcohol dehydrogenase genes from the soil metagenome for biocatalysis.

    Science.gov (United States)

    Itoh, Nobuya; Isotani, Kentaro; Makino, Yoshihide; Kato, Masaki; Kitayama, Kouta; Ishimota, Tuyoshi

    2014-02-05

    The amplification of useful genes from metagenomes offers great biotechnological potential. We employed this approach to isolate alcohol dehydrogenase (adh) genes from Pseudomonas to aid in the synthesis of optically pure alcohols from various ketones. A PCR primer combination synthesized by reference to the adh sequences of known Pseudomonas genes was used to amplify full-length adh genes directly from 17 samples of DNA extracted from soil. Three such adh preparations were used to construct Escherichia coli plasmid libraries. Of the approximately 2800 colonies obtained, 240 putative adh-positive clones were identified by colony-PCR. Next, 23 functional adh genes named using the descriptors HBadh and HPadh were analyzed. The adh genes obtained via this metagenomic approach varied in their DNA and amino acid sequences. Expression of the gene products in E. coli indicated varying substrate specificity. Two representative genes, HBadh-1 and HPadh-24, expressed in E. coli and Pseudomonas putida, respectively, were purified and characterized in detail. The enzyme products of these genes were confirmed to be useful for producing anti-Prelog chiral alcohols.

  16. High-flavonol tomatoes resulting from the heterologous expression of the maize transcription factor genes LC and C1.

    Science.gov (United States)

    Bovy, Arnaud; de Vos, Ric; Kemper, Mark; Schijlen, Elio; Almenar Pertejo, Maria; Muir, Shelagh; Collins, Geoff; Robinson, Sue; Verhoeyen, Martine; Hughes, Steve; Santos-Buelga, Celestino; van Tunen, Arjen

    2002-10-01

    Flavonoids are a group of polyphenolic plant secondary metabolites important for plant biology and human nutrition. In particular flavonols are potent antioxidants, and their dietary intake is correlated with a reduced risk of cardiovascular diseases. Tomato fruit contain only in their peel small amounts of flavonoids, mainly naringenin chalcone and the flavonol rutin, a quercetin glycoside. To increase flavonoid levels in tomato, we expressed the maize transcription factor genes LC and C1 in the fruit of genetically modified tomato plants. Expression of both genes was required and sufficient to upregulate the flavonoid pathway in tomato fruit flesh, a tissue that normally does not produce any flavonoids. These fruit accumulated high levels of the flavonol kaempferol and, to a lesser extent, the flavanone naringenin in their flesh. All flavonoids detected were present as glycosides. Anthocyanins, previously reported to accumulate upon LC expression in several plant species, were present in LC/C1 tomato leaves but could not be detected in ripe LC/C1 fruit. RNA expression analysis of ripening fruit revealed that, with the exception of chalcone isomerase, all of the structural genes required for the production of kaempferol-type flavonols and pelargonidin-type anthocyanins were induced strongly by the LC/C1 transcription factors. Expression of the genes encoding flavanone-3'-hydroxylase and flavanone-3'5'-hydroxylase, which are required for the modification of B-ring hydroxylation patterns, was not affected by LC/C1. Comparison of flavonoid profiles and gene expression data between tomato leaves and fruit indicates that the absence of anthocyanins in LC/C1 fruit is attributable primarily to an insufficient expression of the gene encoding flavanone-3'5'-hydroxylase, in combination with a strong preference of the tomato dihydroflavonol reductase enzyme to use the flavanone-3'5'-hydroxylase reaction product dihydromyricetin as a substrate.

  17. Heterologous expression systems for lipases: a review.

    Science.gov (United States)

    Valero, Francisco

    2012-01-01

    The production of heterologous lipases is one of the most promising strategies to increase the productivity of the bioprocesses and to reduce costs, with the final objective that more industrial lipase applications could be implemented. In this chapter, an overview of the most common microbial expression systems for the overproduction of microbial lipases is presented. Prokaryotic system as Escherichia coli and eukaryotic systems as Saccharomyces cerevisiae and Pichia pastoris are analyzed and compared in terms of productivity, operational, and downstream processing facilities. Finally, an overview of heterologous Candida rugosa and Rhizopus oryzae lipases, two of the most common lipases used in biocatalysis, is presented. In both cases, P. pastoris has been shown as the most promising host system.

  18. The varicella-zoster virus-mediated delayed host shutoff: open reading frame 17 has no major function, whereas immediate-early 63 protein represses heterologous gene expression.

    Science.gov (United States)

    Desloges, Nathalie; Rahaus, Markus; Wolff, Manfred H

    2005-12-01

    We reported that varicella-zoster virus (VZV) causes a delayed host shutoff during its replicative cycle. VZV open reading frame 17 (ORF17) is the homologue of the herpes simplex virus (HSV) UL41 gene encoding the virion host shutoff (vhs) protein which is responsible for the shutoff effect observed in HSV-infected cells. In the present study, we demonstrated that ORF17 is expressed as a late protein during the VZV replicative cycle in different infected permissive cell lines which showed a delayed shutoff of cellular RNA. A cell line with stable expression of VZV ORF17 was infected with VZV. In these cells, VZV replication and delayed host shutoff remained unchanged when compared to normal infected cells. ORF17 was not capable of repressing the expression of the beta-gal reporter gene under the control of the human cytomegalovirus immediate-early gene promoter or to inhibit the expression of a CAT reporter gene under the control of the human GAPDH promoter, indicating that ORF17 has no major function in the VZV-mediated delayed host shutoff. To determine whether other viral factors are involved in the host shutoff, a series of cotransfection assays was performed. We found that the immediate-early 63 protein (IE63) was able to downregulate the expression of reporter genes under the control of the two heterologous promoters, indicating that this viral factor can be involved in the VZV-mediated delayed host shutoff. Other factors can be also implicated to modulate the repressing action of IE63 to achieve a precise balance between the viral and cellular gene expression.

  19. Structure and heterologous expression of the gene encoding the cell surface glycoprotein from Haloarcula japonica strain TR-1.

    Science.gov (United States)

    Wakai, H; Takada, K; Nakamura, S; Horikoshi, K

    1995-01-01

    The gene encoding the cell surface glycoprotein (CSG) of Haloarcula japonica strain TR-1 was cloned and sequenced. The structural gene consisted from an open reading frame of 2,586 bp. A potential promoter sequence was found about 150 bp upstream of the ATG initiation codon. N-terminal amino acid sequence of the Ha. japonica CSG revealed that the mature CSG consisted of 828 amino acids. Five potential N-glycosylation sites were found in the mature sequence. The cloned CSG gene of Ha. japonica was expressed in closely-related halophilic archaea.

  20. Inducible amplification of gene copy number and heterologous protein production in the yeast Kluyveromyces lactis.

    Science.gov (United States)

    Morlino, G B; Tizzani, L; Fleer, R; Frontali, L; Bianchi, M M

    1999-11-01

    Heterologous protein production can be doubled by increasing the copy number of the corresponding heterologous gene. We constructed a host-vector system in the yeast Kluyveromyces lactis that was able to induce copy number amplification of pKD1 plasmid-based vectors upon expression of an integrated copy of the plasmid recombinase gene. We increased the production and secretion of two heterologous proteins, glucoamylase from the yeast Arxula adeninivorans and mammalian interleukin-1beta, following gene dosage amplification when the heterologous genes were carried by pKD1-based vectors. The choice of the promoters for expression of the integrated recombinase gene and of the episomal heterologous genes are critical for the mitotic stability of the host-vector system.

  1. Heterologous Gene Expression in Lactococcus lactis subsp. lactis : Synthesis, Secretion, and Processing of the Bacillus subtilis Neutral Protease

    NARCIS (Netherlands)

    Guchte, Maarten van de; Kodde, Jan; Vossen, Jos M.B.M. van der; Kok, Jan; Venema, Gerard

    1990-01-01

    The Bacillus subtilis nprE gene lacking its own promoter sequence was inserted in the lactococcal expression vector pMG36e. Upon introduction of the recombinant plasmid into Lactococcus lactis subsp. lactis strain MG1363, neutral protease activity could be visualized by the appearance of large clear

  2. Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: modular construction of multiple cDNA expression elements using recombinant cloning.

    Science.gov (United States)

    Sone, Takefumi; Yahata, Kazuhide; Sasaki, Yukari; Hotta, Junko; Kishine, Hiroe; Chesnut, Jonathan D; Imamoto, Fumio

    2008-09-10

    Much attention has been focused on manipulating multiple genes in living cells for analyzing protein function. In order to perform high-throughput generation of multi-gene expression clones, gateway cloning technology (which represents a high-throughput DNA transfer from vector to vector) can be anticipated. In the conventional strategy for gateway cloning, the construction of two or more expression elements into tandem elements on a single plasmid requires the recombination of multiple entry clones with a destination vector in a single reaction mixture. Use of increasing numbers of entry clones in a single reaction is inefficient due to the difficulty in successfully recognizing multiple pairs of matched att signals simultaneously. To address this problem, a "Modular Destination" vector has been devised and constructed, whereby cDNA inserts are sequentially introduced, resulting in a tandem structure with multiple inserts. Whereas the standard destination vector contains only Cm(R) and ccdB genes flanked by two attR signals, this destination vector contains, in addition, one or two cDNA expression elements. Here, we show the rapid construction of expression vectors containing three or four tandemly arrayed cDNA expression elements and their expression in mammalian cells.

  3. Heterologous expression of glucose oxidase in the yeast Kluyveromyces marxianus

    Directory of Open Access Journals (Sweden)

    Gombert Andreas K

    2010-01-01

    Full Text Available Abstract Background In spite of its advantageous physiological properties for bioprocess applications, the use of the yeast Kluyveromyces marxianus as a host for heterologous protein production has been very limited, in constrast to its close relative Kluyveromyces lactis. In the present work, the model protein glucose oxidase (GOX from Aspergillus niger was cloned into K. marxianus CBS 6556 and into K. lactis CBS 2359 using three different expression systems. We aimed at verifying how each expression system would affect protein expression, secretion/localization, post-translational modification, and biochemical properties. Results The highest GOX expression levels (1552 units of secreted protein per gram dry cell weight were achieved using an episomal system, in which the INU1 promoter and terminator were used to drive heterologous gene expression, together with the INU1 prepro sequence, which was employed to drive secretion of the enzyme. In all cases, GOX was mainly secreted, remaining either in the periplasmic space or in the culture supernatant. Whereas the use of genetic elements from Saccharomyces cerevisiae to drive heterologous protein expression led to higher expression levels in K. lactis than in K. marxianus, the use of INU1 genetic elements clearly led to the opposite result. The biochemical characterization of GOX confirmed the correct expression of the protein and showed that K. marxianus has a tendency to hyperglycosylate the protein, in a similar way as already observed for other yeasts, although this tendency seems to be smaller than the one of e.g. K. lactis and S. cerevisiae. Hyperglycosylation of GOX does not seem to affect its affinity for the substrate, nor its activity. Conclusions Taken together, our results indicate that K. marxianus is indeed a good host for the expression of heterologous proteins, not only for its physiological properties, but also because it correctly secretes and folds these proteins.

  4. [Improvement of natamycin production in an industrial strain by heterologous expression of the afsRS(cla) global regulatory genes].

    Science.gov (United States)

    Tao, Zhengsheng; Wang, Yemin; Zheng, Hualiang; Tao, Meifeng

    2015-05-01

    The afsRS(cla) global regulatory genes from Streptomyces clavuligerus activate the production of two antibiotics in Streptomyces lividans. In this study, we gained an increase of 38% in the production of natamycin (3.56 g/L) in an industrial strain Streptomyces gilvosporeus TZ1401 through the integration of pHL851 that bears the afsRS(cla) global regulatory genes into its genome. We discovered by quantitive real-time reverse transcription PCR (qRT-PCR) that the expression of 6 genes of the natamycin biosynthetic gene cluster were improved from 1.9 to 2.7 times. This suggests that afsRS(cla) improve the production of natamycin through increased transcription. This study provides a good example for applying afsRS(cla) in high yield breeding of industrial antibiotic producers.

  5. Improved polysaccharide production in a submerged culture of Ganoderma lucidum by the heterologous expression of Vitreoscilla hemoglobin gene.

    Science.gov (United States)

    Li, Huan-Jun; Zhang, De-Huai; Yue, Tong-Hui; Jiang, Lu-Xi; Yu, Xuya; Zhao, Peng; Li, Tao; Xu, Jun-Wei

    2016-01-10

    Expression of Vitreoscilla hemoglobin (VHb) gene was used to improve polysaccharide production in Ganoderma lucidum. The VHb gene, vgb, under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase gene promoter was introduced into G. lucidum. The activity of expressed VHb was confirmed by the observation of VHb specific CO-difference spectrum with a maximal absorption at 419 nm for the transformant. The effects of VHb expression on intracellular polysaccharide (IPS) content, extracellular polysaccharide (EPS) production and transcription levels of three genes encoding the enzymes involved in polysaccharide biosynthesis, including phosphoglucomutase (PGM), uridine diphosphate glucose pyrophosphorylase (UGP), and β-1,3-glucan synthase (GLS), were investigated. The maximum IPS content and EPS production in the vgb-bearing G. lucidum were 26.4 mg/100mg dry weight and 0.83 g/L, respectively, which were higher by 30.5% and 88.2% than those of the wild-type strain. The transcription levels of PGM, UGP and GLS were up-regulated by 1.51-, 1.55- and 3.83-fold, respectively, in the vgb-bearing G. lucidum. This work highlights the potential of VHb to enhance G. lucidum polysaccharide production by large scale fermentation.

  6. Evaluating Light-Induced Promoters for the Control of Heterologous Gene Expression in Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Albers, Stevan C; Peebles, Christie A M

    2017-01-01

    Cyanobacteria are enticing microbial factories, but little is understood how their gene control elements respond to the periodic availability to light. This research tested the capability of PpsbAII to control gene expression during light/dark conditions when moved to a neutral location within the Synechocystis sp. PCC 6803 genome. When the eYFP reporter gene was run by PpsbAII in the promoter's native genomic location, mutants exposed to 12-hour light conditions experienced a 15.8× increase in transcript abundance over that observed from the same construct exposed to 12-hour dark conditions. When this same construct was moved to the hypothetical coding region slr0168 in the genome, transcripts generated during 12 hour light conditions accumulated to 1.67X of the levels of transcripts generated by the same construct during 12 hour dark conditions. Three additional promoter constructs, PpsbAIII , PgroEL2 , and PsigD were also tested for differential expression in light and dark conditions within the neutral region slr0168. While low amounts of transcript accumulation were observed from PgroEL2 and PsigD , the PpsbAIII construct accumulated 5.79× more transcripts when compared to transcript abundance during dark conditions, which highlights the potential of this promoter to control gene expression during diel-cycle light conditions. Additionally, nucleotide mutations were made to regions within PpsbAII . Mutations to the cis-acting hexo-nucleotide region increased expression 3.71× over that of the native promoter, while the addition of the "HLR" nucleotide region to the PpsbAII::ΔHex construct increased expression 2.76× over that of the native promoter. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:45-53, 2017.

  7. L-lactic acid production from D-xylose with Candida sonorensis expressing a heterologous lactate dehydrogenase encoding gene.

    Science.gov (United States)

    Koivuranta, Kari T; Ilmén, Marja; Wiebe, Marilyn G; Ruohonen, Laura; Suominen, Pirkko; Penttilä, Merja

    2014-08-08

    Bioplastics, like polylactic acid (PLA), are renewable alternatives for petroleum-based plastics. Lactic acid, the monomer of PLA, has traditionally been produced biotechnologically with bacteria. With genetic engineering, yeast have the potential to replace bacteria in biotechnological lactic acid production, with the benefits of being acid tolerant and having simple nutritional requirements. Lactate dehydrogenase genes have been introduced to various yeast to demonstrate this potential. Importantly, an industrial lactic acid producing process utilising yeast has already been implemented. Utilisation of D-xylose in addition to D-glucose in production of biochemicals such as lactic acid by microbial fermentation would be beneficial, as it would allow lignocellulosic raw materials to be utilised in the production processes. The yeast Candida sonorensis, which naturally metabolises D-xylose, was genetically modified to produce L-lactic acid from D-xylose by integrating the gene encoding L-lactic acid dehydrogenase (ldhL) from Lactobacillus helveticus into its genome. In microaerobic, CaCO3-buffered conditions a C. sonorensis ldhL transformant having two copies of the ldhL gene produced 31 g l-1 lactic acid from 50 g l-1 D-xylose free of ethanol.Anaerobic production of lactic acid from D-xylose was assessed after introducing an alternative pathway of D-xylose metabolism, i.e. by adding a xylose isomerase encoded by XYLA from Piromyces sp. alone or together with the xylulokinase encoding gene XKS1 from Saccharomyces cerevisiae. Strains were further modified by deletion of the endogenous xylose reductase encoding gene, alone or together with the xylitol dehydrogenase encoding gene. Strains of C. sonorensis expressing xylose isomerase produced L-lactic acid from D-xylose in anaerobic conditions. The highest anaerobic L-lactic acid production (8.5 g l-1) was observed in strains in which both the xylose reductase and xylitol dehydrogenase encoding genes had been

  8. Heterologous expression of a new manganese-dependent peroxidase gene from Peniophora incarnata KUC8836 and its ability to remove anthracene in Saccharomyces cerevisiae.

    Science.gov (United States)

    Lee, Aslan Hwanhwi; Kang, Chang-Min; Lee, Young Min; Lee, Hanbyul; Yun, Cheol-Won; Kim, Gyu-Hyeok; Kim, Jae-Jin

    2016-12-01

    The white rot fungus Peniophora incarnata KUC8836 has received an attention as the greatest degrader of polycyclic aromatic hydrocarbons (PAHs), which are hazardous xenobiotics and recalcitrant pollutants. To characterize the mechanisms through which MnP degrades PAHs, heterologous expression of manganese-dependent peroxidase (MnP) gene pimp1 was performed in Saccharomyces cerevisiae BY4741 via the pGEM-T Easy vector, resulting in the recombinant plasmid pESC-URA/pimp1 containing the MnP signal peptide. MnP was significantly secreted into the culture medium with galactose as an active protein with higher efficiency (3.58 U mL(-1)) by transformants than by the wild-type S. cerevisiae. The recombinant MnP protein was shown to have a molecular weight of 44 kDa by western blotting analysis. With regard to enhancing the bioremediation of PAHs in the environment, anthracene was effectively degraded by the MnP encoded by pimp1, with a degradation rate of 6.5% when Tween 80 was added. In addition, the MnP activity of the transformant exhibited the highest efficiency (2.49 U mL(-1)) during the degradation. These results show that pimp1 might be useful for biodegradation and gene expression technologies at a transcriptional level, and genetic approaches can be improved by incorporating the highly ligninolytic gene pimp1 and the fungus P. incarnata KUC8836.

  9. Heterologous expression and in-silico characterization of Pathogenesis related protein1 (CsPR1 gene from Camellia sinensis.

    Directory of Open Access Journals (Sweden)

    Niraj Agarwala*

    2014-01-01

    Full Text Available Pathogenesis related protein1 gene induced after pathogen infection in plantshave been frequently used as marker gene for systemic acquired resistance. We have carried out isolation, annotation and expression of CsPR1, a potential disease resistance gene. The full length cDNA consist of 671 bp in length containing 162 amino acids with a signal peptide of 22 amino acids and 17.92 kDa predicted molecular weight. Recombinant CsPR1 was successfully expressed in BL21(DE3pLysS cells using pET 43.1 EK LIC vector system and was purified. Three dimensional models weregenerated using Phyre2 and I-TASSER and built a compact structureconsisting beta sheets surrounded by alpha helixes. The models werevalidated by MolProbity and RAMPAGE servers. Validation of modelledstructures based on Ramachandran plot, revealed I-TASSER producebetter quality and reliable 3D model. Purified recombinant CsPR1 and insilico generated 3D models from this study provide foundation forcomprehensive functional and structural characterization of CsPR1protein.

  10. Steroidogenesis and early response gene expression in MA-10 Leydig tumor cells following heterologous receptor down-regulation and cellular desensitization

    Directory of Open Access Journals (Sweden)

    Tsuey-Ming Chen

    2016-03-01

    Full Text Available The Leydig tumor cell line, MA-10, expresses the luteinizing hormone receptor, a G protein-coupled receptor that, when activated with luteinizing hormone or chorionic gonadotropin (CG, stimulates cAMP production and subsequent steroidogenesis, notably progesterone. These cells also respond to epidermal growth factor (EGF and phorbol esters with increased steroid biosynthesis. In order to probe the intracellular pathways along with heterologous receptor down-regulation and cellular desensitization, cells were preincubated with EGF or phorbol esters and then challenged with CG, EGF, dibutryl-cyclic AMP, and a phorbol ester. Relative receptor numbers, steroid biosynthesis, and expression of the early response genes, JUNB and c-FOS, were measured. It was found that in all cases but one receptor down-regulation and decreased progesterone production were closely coupled under the conditions used; the exception involved preincubation of the cells with EGF followed by addition of CG where the CG-mediated stimulation of steroidogenesis was considerably lower than the level of receptor down-regulation. In a number of instances JUNB and c-FOS expression paralleled the decreases in receptor number and progesterone production, while in some cases these early response genes were affected little if at all by the changes in receptor number. This finding may indicate that even low levels of activated signaling kinases, e.g. protein kinase A, protein kinase C, or receptor tyrosine kinase, may suffice to yield good expression of JUNB and c-FOS, or it may suggest alternative pathways for regulating expression of these two early response genes.

  11. Heterologous expression of Anabaena PCC 7120 all3940 (a Dps family gene) protects Escherichia coli from nutrient limitation and abiotic stresses

    Energy Technology Data Exchange (ETDEWEB)

    Narayan, Om Prakash; Kumari, Nidhi [Molecular Biology Section, Laboratory of Algal Biology, Center of Advanced Study in Botany, Banaras Hindu University, Varanasi-22 1005 (India); Rai, Lal Chand, E-mail: lcraibhu@gmail.com [Molecular Biology Section, Laboratory of Algal Biology, Center of Advanced Study in Botany, Banaras Hindu University, Varanasi-22 1005 (India)

    2010-03-26

    This study presents first hand data on the cloning and heterologous expression of Anabaena PCC 7120 all3940 (a dps family gene) in combating nutrients limitation and multiple abiotic stresses. The Escherichia coli transformed with pGEX-5X-2-all3940 construct when subjected to iron, carbon, nitrogen, phosphorus limitation and carbofuron, copper, UV-B, heat, salt and cadmium stress registered significant increase in growth over the cells transformed with empty vector under iron (0%), carbon (0.05%), nitrogen (3.7 mM) and phosphorus (2 mM) limitation and carbofuron (0.025 mg ml{sup -1}), CuCl{sub 2} (1 mM), UV-B (10 min), heat (47 {sup o}C), NaCl (6% w/v) and CdCl{sub 2} (4 mM) stress. Enhanced expression of all3940 gene measured by semi-quantitative RT-PCR at different time points under above mentioned treatments clearly demonstrates its role in tolerance against aforesaid abiotic stresses. This study opens the gate for developing transgenic cyanobacteria capable of growing successfully under above mentioned stresses.

  12. Heterology expression of the sweet pepper CBF3 gene confers elevated tolerance to chilling stress in transgenic tobacco.

    Science.gov (United States)

    Yang, Sha; Tang, Xian-Feng; Ma, Na-Na; Wang, Li-Yan; Meng, Qing-Wei

    2011-10-15

    Various studies have confirmed that the CBF (C-repeat binding factor) family of transcription factors has a key role in regulating many plants' responses to cold stress. Here we isolated CBF3 from sweet pepper (Capsicum frutescens). Green fluorescent protein (GFP) fusion protein of CfCBF3 was targeted to the nucleus of the onion epidermis cell. RNA gel blot analysis indicated that CfCBF3 was expressed in leaves of sweet pepper and the expression was induced by low temperature, drought and salinity stresses but not by ABA. Overexpression of CfCBF3 under the control of the CaMV35S promoter in tobacco induced expression of orthologs of CBF3-targeted genes and increased chilling tolerance without a dwarf phenotype. Indeed it also led to multiple biochemical and physiological changes associated with chilling stress. Higher levels of proline (Pro) and soluble sugars and lower content of reactive oxygen species (ROS) were observed in transgenic plants. Our results demonstrated that the increase in total unsaturated fatty acids, especially in phosphatidylglycerol (PG) was detected by overexpression of CfCBF3. During exposure to chilling stress, the transgenic lines were less susceptible to chilling-induced photoinhibition than wild-type (WT) plants. These results suggest that overexpression of CfCBF3 led to modification of the fatty acid unsaturation and alleviated the injuries under chilling stress.

  13. Effects of simultaneous expression of heterologous genes involved in phytochelatin biosynthesis on thiol content and cadmium accumulation in tobacco plants.

    Science.gov (United States)

    Wawrzynski, Adam; Kopera, Edyta; Wawrzynska, Anna; Kaminska, Jolanta; Bal, Wojciech; Sirko, Agnieszka

    2006-01-01

    Transgenic tobacco (Nicotiana tabacum cv. LA Burley 21) lines expressing three genes encoding enzymes thought to be critical for the efficient production of phytochelatins, (i) serine acetyltransferase (EC 2.3.1.30) involved in the production of O-acetylserine, the cysteine precursor, (ii) gamma-glutamylcysteine synthetase (EC 6.3.2.2) involved in the production of gamma-glutamylcysteine, the precursor of glutathione, and (iii) phytochelatin synthase (EC 2.3.2.15), were obtained and analysed for non-protein thiol content and cadmium accumulation. After a 3 week exposure to 15 microM CdCl2, plants expressing transgenes (either separately or in combination) had increased cadmium concentration in roots but not in shoots compared with the wild type. Nearly all transgenic lines analysed had more non-protein thiols than the wild type. The greatest effects (about 8-fold elevation of thiols) were found in one of the lines simultaneously expressing the three transgenes. Despite the fact that a multi-transgene strategy described in this work resulted in a strong increase in the levels of several classes of non-protein thiols in transgenic plants, other factors appeared to restrict cadmium accumulation in shoots.

  14. Heterologous expression and biochemical characterization of an α1,2-mannosidase encoded by the Candida albicans MNS1 gene

    Directory of Open Access Journals (Sweden)

    Héctor M Mora-Montes

    2008-11-01

    Full Text Available Protein glycosylation pathways, commonly found in fungal pathogens, offer an attractive new area of study for the discovery of antifungal targets. In particular, these post-translational modifications are required for virulence and proper cell wall assembly in Candida albicans, an opportunistic human pathogen. The C. albicans MNS1 gene is predicted to encode a member of the glycosyl hydrolase family 47, with 1,2-mannosidase activity. In order to characterise its activity, we first cloned the C. albicans MNS1 gene into Escherichia coli, then expressed and purified the enzyme. The recombinant Mns1 was capable of converting a Man9GlcNAc2 N-glycan core into Man8GlcNAc2 isomer B, but failed to process a Man5GlcNAc2-Asn N-oligosaccharide. These properties are similar to those displayed by Mns1 purified from C. albicansmembranes and strongly suggest that the enzyme is an ±1,2-mannosidase that is localised to the endoplasmic reticulum and involved in the processing of N-linked mannans. Polyclonal antibodies specifically raised against recombinant Mns1 also immunoreacted with the soluble ±1,2-mannosidases E-I and E-II, indicating that Mns1 could share structural similarities with both soluble enzymes. Due to the high degree of similarity between the members of family 47, it is conceivable that these antibodies may recognise ±1,2-mannosidases in other biological systems as well.

  15. Heterologous expression of cellobiohydrolases in filamentous fungi

    DEFF Research Database (Denmark)

    Zoglowek, Marta; Lübeck, Peter S.; Ahring, Birgitte K.

    2015-01-01

    Cellobiohydrolases are among the most important enzymes functioning in the hydrolysis of crystalline cellulose, significantly contributing to the efficient biorefining of recalcitrant lignocellulosic biomass into biofuels and bio-based products. Filamentous fungi are recognized as both well...... into valuable products. However, due to low cellobiohydrolase activities, certain fungi might be deficient with regard to enzymes of value for cellulose conversion, and improving cellobiohydrolase expression in filamentous fungi has proven to be challenging. In this review, we examine the effects of altering...... promoters, signal peptides, culture conditions and host post-translational modifications. For heterologous cellobiohydrolase production in filamentous fungi to become an industrially feasible process, the construction of site-integrating plasmids, development of protease-deficient strains and glycosylation...

  16. Identification by heterologous expression and gene disruption of VisA as L-lysine 2-aminotransferase essential for virginiamycin S biosynthesis in Streptomyces virginiae.

    Science.gov (United States)

    Namwat, Wises; Kinoshita, Hiroshi; Nihira, Takuya

    2002-09-01

    The visA gene of Streptomyces virginiae has been thought to be a part of the virginiamycin S (VS) biosynthetic gene cluster based on its location in the middle of genes that encode enzymes highly similar to those participating in the biosynthesis of streptogramin-type antibiotics. Heterologous expression of the visA gene was achieved in Escherichia coli by an N-terminal fusion with thioredoxin (TrxA), and the intact recombinant VisA protein (rVisA) was purified after cleavage with enterokinase to remove the TrxA moiety. The purified rVisA showed clear L-lysine 2-aminotransferase activity with an optimum pH of around 8.0 and an optimum temperature at 35 degrees C, with 2-oxohexanoate as the best amino acceptor, indicating that VisA converts L-lysine into Delta(1)-piperidine 2-carboxylic acid. A visA deletion mutant of S. virginiae was created by homologous recombination, and the in vivo function of the visA gene was studied by phenotypic comparison between the wild type and the visA deletion mutant. No differences in growth in liquid media or in morphological behavior on solid media were observed, indicating that visA is not involved in primary metabolism or morphological differentiation. However, the visA mutant failed to produce VS while maintaining the production of virginiamycin M(1) at a level comparable to that of the parental wild-type strain, demonstrating that visA is essential to VS biosynthesis. These results, together with the observed recovery of the defect in VS production by the external addition of 3-hydroxypicolinic acid (3-HPA), a starter molecule in VS biosynthesis, suggest that VisA is the first enzyme of the VS biosynthetic pathway and that it supplies 3-HPA from L-lysine.

  17. Effects of codon optimization on the mRNA levels of heterologous genes in filamentous fungi.

    Science.gov (United States)

    Tanaka, Mizuki; Tokuoka, Masafumi; Gomi, Katsuya

    2014-05-01

    Filamentous fungi, particularly Aspergillus species, have recently attracted attention as host organisms for recombinant protein production. Because the secretory yields of heterologous proteins are generally low compared with those of homologous proteins or proteins from closely related fungal species, several strategies to produce substantial amounts of recombinant proteins have been conducted. Codon optimization is a powerful tool for improving the production levels of heterologous proteins. Although codon optimization is generally believed to improve the translation efficiency of heterologous genes without affecting their mRNA levels, several studies have indicated that codon optimization causes an increase in the steady-state mRNA levels of heterologous genes in filamentous fungi. However, the mechanism that determines the low mRNA levels when native heterologous genes are expressed was poorly understood. We recently showed that the transcripts of heterologous genes are polyadenylated prematurely within the coding region and that the heterologous gene transcripts can be stabilized significantly by codon optimization, which is probably attributable to the prevention of premature polyadenylation in Aspergillus oryzae. In this review, we describe the detailed mechanism of premature polyadenylation and the rapid degradation of mRNA transcripts derived from heterologous genes in filamentous fungi.

  18. Gene cloning, heterologous expression, and characterization of a high maltose-producing α-amylase of Rhizopus oryzae.

    Science.gov (United States)

    Li, Song; Zuo, Zhirui; Niu, Dandan; Singh, Suren; Permaul, Kugenthiren; Prior, Bernard A; Shi, Guiyang; Wang, Zhengxiang

    2011-07-01

    A putative α-amylase gene, designated as RoAmy, was cloned from Rhizopus oryzae. The deduced amino acid sequence showed the highest (42.8%) similarity to the α-amylase from Trichoderma viride. The RoAmy gene was successfully expressed in Pichia pastoris GS115 under the induction of methanol. The molecular weight of the purified RoAmy determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis was approximately 48 kDa. The optimal pH and temperature were 4-6 and 60 °C, respectively. The enzyme was stable at pH ranges of 4.5-6.5 and temperatures below 50 °C. Purified RoAmy had a K(m) and V(max) of 0.27 mg/ml and 0.068 mg/min, respectively, with a specific activity of 1,123 U/mg on soluble starch. Amylase activity was strongly inhibited by 5 mM Cu(2+) and 5 mM Fe(2+), whereas 5 mM Ca(2+) showed no significant effect. The RoAmy hydrolytic activity was the highest on wheat starch but showed only 55% activity on amylopectin relative to soluble corn starch, while the pullulanase activity was negligible. The main end products of the polysaccharides tested were glucose and maltose. Maltose reached a concentration of 74% (w/w) with potato starch as the substrate. The enzyme had an extremely high affinity (K(m) = 0.22 mM) to maltotriose. A high ratio of glucose/maltose of 1:4 was obtained when maltotriose was used at an initial concentration of 40 mM.

  19. Cell surface expression system for the display of heterologous gene products using chimeric flagellin fusions of bacillus halodurans isolate

    CSIR Research Space (South Africa)

    Du Plessis, A

    2006-10-01

    Full Text Available N-terminal sequencing gave rise to homology to flagellin protein, product of the hag gene. protein, product of the hag gene. Gene was cloned by using degenerate primers and inverse PCR. The gene sequence as well as the up- and down- stream regions...

  20. Heterologous expression of human H1 histones in yeast.

    Science.gov (United States)

    Albig, W; Runge, D M; Kratzmeier, M; Doenecke, D

    1998-09-18

    The complete set of seven human H1 histone subtype genes was heterologously expressed in yeast. Since Saccharomyces cerevisiae lacks standard histone H1 we could isolate each recombinantly expressed human H1 subtype in pure form without contamination by endogenous H I histones. For isolation of the H1 histones in this expression system no tagging was needed and the isoforms could be extracted with the authentic primary structure by a single extraction step with 5%(0.74 M) perchloric acid. The isolated H1 histone proteins were used to assign the subtype genes to the corresponding protein spots or peaks after two-dimensional gel electrophoresis and capillary zone electrophoresis, respectively. This allowed us to correlate transcriptional data with protein data, which was barely possible until now.

  1. Ligninolytic peroxidase genes in the oyster mushroom genome: heterologous expression, molecular structure, catalytic and stability properties, and lignin-degrading ability

    Science.gov (United States)

    2014-01-01

    Background The genome of Pleurotus ostreatus, an important edible mushroom and a model ligninolytic organism of interest in lignocellulose biorefineries due to its ability to delignify agricultural wastes, was sequenced with the purpose of identifying and characterizing the enzymes responsible for lignin degradation. Results Heterologous expression of the class II peroxidase genes, followed by kinetic studies, enabled their functional classification. The resulting inventory revealed the absence of lignin peroxidases (LiPs) and the presence of three versatile peroxidases (VPs) and six manganese peroxidases (MnPs), the crystal structures of two of them (VP1 and MnP4) were solved at 1.0 to 1.1 Å showing significant structural differences. Gene expansion supports the importance of both peroxidase types in the white-rot lifestyle of this fungus. Using a lignin model dimer and synthetic lignin, we showed that VP is able to degrade lignin. Moreover, the dual Mn-mediated and Mn-independent activity of P. ostreatus MnPs justifies their inclusion in a new peroxidase subfamily. The availability of the whole POD repertoire enabled investigation, at a biochemical level, of the existence of duplicated genes. Differences between isoenzymes are not limited to their kinetic constants. Surprising differences in their activity T50 and residual activity at both acidic and alkaline pH were observed. Directed mutagenesis and spectroscopic/structural information were combined to explain the catalytic and stability properties of the most interesting isoenzymes, and their evolutionary history was analyzed in the context of over 200 basidiomycete peroxidase sequences. Conclusions The analysis of the P. ostreatus genome shows a lignin-degrading system where the role generally played by LiP has been assumed by VP. Moreover, it enabled the first characterization of the complete set of peroxidase isoenzymes in a basidiomycete, revealing strong differences in stability properties and providing

  2. Heterologous expression of Gaeumannomyces graminis lipoxygenase in Aspergillus nidulans.

    Science.gov (United States)

    Heshof, Ruud; van Schayck, J Paul; Tamayo-Ramos, Juan Antonio; de Graaff, Leo H

    2014-01-01

    Aspergillus sp. contain ppo genes coding for Ppo enzymes that produce oxylipins from polyunsaturated fatty acids. These oxylipins function as signal molecules in sporulation and influence the asexual to sexual ratio of Aspergillus sp. Fungi like Aspergillus nidulans and Aspergillus niger contain just ppo genes where the human pathogenic Aspergillus flavus and Aspergillus fumigatus contain ppo genes as well as lipoxygenases. Lipoxygenases catalyze the synthesis of oxylipins and are hypothesized to be involved in quorum-sensing abilities and invading plant tissue. In this study we used A. nidulans WG505 as an expression host to heterologously express Gaeumannomyces graminis lipoxygenase. The presence of the recombinant LOX induced phenotypic changes in A. nidulans transformants. Also, a proteomic analysis of an A. nidulans LOX producing strain indicated that the heterologous protein was degraded before its glycosylation in the secretory pathway. We observed that the presence of LOX induced the specific production of aminopeptidase Y that possibly degrades the G. graminis lipoxygenase intercellularly. Also the presence of the protein thioredoxin reductase suggests that the G. graminis lipoxygenase is actively repressed in A. nidulans.

  3. Rationally designed, heterologous S. cerevisiae transcripts expose novel expression determinants

    Science.gov (United States)

    Ben-Yehezkel, Tuval; Atar, Shimshi; Zur, Hadas; Diament, Alon; Goz, Eli; Marx, Tzipy; Cohen, Rafael; Dana, Alexandra; Feldman, Anna; Shapiro, Ehud; Tuller, Tamir

    2015-01-01

    Deducing generic causal relations between RNA transcript features and protein expression profiles from endogenous gene expression data remains a major unsolved problem in biology. The analysis of gene expression from heterologous genes contributes significantly to solving this problem, but has been heavily biased toward the study of the effect of 5′ transcript regions and to prokaryotes. Here, we employ a synthetic biology driven approach that systematically differentiates the effect of different regions of the transcript on gene expression up to 240 nucleotides into the ORF. This enabled us to discover new causal effects between features in previously unexplored regions of transcripts, and gene expression in natural regimes. We rationally designed, constructed, and analyzed 383 gene variants of the viral HRSVgp04 gene ORF, with multiple synonymous mutations at key positions along the transcript in the eukaryote S. cerevisiae. Our results show that a few silent mutations at the 5′UTR can have a dramatic effect of up to 15 fold change on protein levels, and that even synonymous mutations in positions more than 120 nucleotides downstream from the ORF 5′end can modulate protein levels up to 160%–300%. We demonstrate that the correlation between protein levels and folding energy increases with the significance of the level of selection of the latter in endogenous genes, reinforcing the notion that selection for folding strength in different parts of the ORF is related to translation regulation. Our measured protein abundance correlates notably(correlation up to r = 0.62 (p=0.0013)) with mean relative codon decoding times, based on ribosomal densities (Ribo-Seq) in endogenous genes, supporting the conjecture that translation elongation and adaptation to the tRNA pool can modify protein levels in a causal/direct manner. This report provides an improved understanding of transcript evolution, design principles of gene expression regulation, and suggests simple

  4. Use of the EGP-2/Ep-CAM promoter for targeted expression of heterologous genes in carcinoma derived cell lines

    NARCIS (Netherlands)

    McLaughlin, PMJ; Trzpis, M; Kroesen, BJ; Helfrich, W; Terpstra, P; Dokter, WHA; Ruiters, MHJ; de Leij, LF; Harmsen, MC

    2004-01-01

    EGP-2, also known as Ep-CAM, is expressed at high levels on the surface of most carcinomas and is therefore considered an attractive target for anticancer strategies. To explore the mechanisms regulating the expression of EGP-2, sequences 3.4 kb upstream of the transcription start site were isolated

  5. Production of novel antioxidative phenolic amides through heterologous expression of the plant’s chlorogenic acid biosynthesis genes in yeast

    NARCIS (Netherlands)

    Moglia, A.; Comino, C.; Lanteri, S.; Vos, de C.H.; Waard, de P.; Beek, van T.A.; Goitre, L.; Retta, S.F.; Beekwilder, M.J.

    2010-01-01

    Phenolic esters like chlorogenic acid play an important role in therapeutic properties of many plant extracts. We aimed to produce phenolic esters in baker’s yeast, by expressing tobacco 4CL and globe artichoke HCT. Indeed yeast produced phenolic esters. However, the primary product was identified a

  6. Heterologous expression and biochemical characterization of an α1,2-mannosidase encoded by the Candida albicans MNS1 gene

    OpenAIRE

    2008-01-01

    Protein glycosylation pathways, commonly found in fungal pathogens, offer an attractive new area of study for the discovery of antifungal targets. In particular, these post-translational modifications are required for virulence and proper cell wall assembly in Candida albicans, an opportunistic human pathogen. The C. albicans MNS1 gene is predicted to encode a member of the glycosyl hydrolase family 47, with 1,2-mannosidase activity. In order to characterise its activity, we first cloned the ...

  7. Secretion, purification, and characterisation of barley alpha-amylase produced by heterologous gene expression in Aspergillus niger.

    Science.gov (United States)

    Juge, N; Svensson, B; Williamson, G

    1998-04-01

    Efficient production of recombinant barley alpha-amylase has been achieved in Aspergillus niger. The cDNA encoding alpha-amylase isozyme 1 (AMY1) and its signal peptide was placed under the control of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter and the A. nidulans trpC gene terminator. Secretion yields up to 60 mg/l were obtained in media optimised for alpha-amylase activity and low protease activity. The recombinant AMY1 (reAMY1) was purified to homogeneity and found to be identical to native barley AMY1 with respect to size, pI, and immunoreactivity. N-terminal sequence analysis of the recombinant protein indicated that the endogenous plant signal peptide is correctly processed in A. niger. Electrospray ionisation/mass spectrometry gave a molecular mass for the dominant form of 44,960 Da, in accordance with the loss of the LQRS C-terminal residues; glycosylation apparently did not occur. The activities of recombinant and native barley alpha-amylases are very similar towards insoluble and soluble starch as well as 2-chloro-4-nitrophenol beta-D-maltoheptaoside and amylose (degree of polymerisation = 17). Barley alpha-amylase is the first plant protein efficiently secreted and correctly processed by A. niger using its own signal sequence.

  8. Development of an efficient genetic manipulation strategy for sequential gene disruption and expression of different heterologous GFP genes in Candida tropicalis.

    Science.gov (United States)

    Zhang, Lihua; Chen, Xianzhong; Chen, Zhen; Wang, Zezheng; Jiang, Shan; Li, Li; Pötter, Markus; Shen, Wei; Fan, You

    2016-11-01

    The diploid yeast Candida tropicalis, which can utilize n-alkane as a carbon and energy source, is an attractive strain for both physiological studies and practical applications. However, it presents some characteristics, such as rare codon usage, difficulty in sequential gene disruption, and inefficiency in foreign gene expression, that hamper strain improvement through genetic engineering. In this work, we present a simple and effective method for sequential gene disruption in C. tropicalis based on the use of an auxotrophic mutant host defective in orotidine monophosphate decarboxylase (URA3). The disruption cassette, which consists of a functional yeast URA3 gene flanked by a 0.3 kb gene disruption auxiliary sequence (gda) direct repeat derived from downstream or upstream of the URA3 gene and of homologous arms of the target gene, was constructed and introduced into the yeast genome by integrative transformation. Stable integrants were isolated by selection for Ura(+) and identified by PCR and sequencing. The important feature of this construct, which makes it very attractive, is that recombination between the flanking direct gda repeats occurs at a high frequency (10(-8)) during mitosis. After excision of the URA3 marker, only one copy of the gda sequence remains at the recombinant locus. Thus, the resulting ura3 strain can be used again to disrupt a second allelic gene in a similar manner. In addition to this effective sequential gene disruption method, a codon-optimized green fluorescent protein-encoding gene (GFP) was functionally expressed in C. tropicalis. Thus, we propose a simple and reliable method to improve C. tropicalis by genetic manipulation.

  9. Heterologous Protein Expression by Lactococcus lactis

    NARCIS (Netherlands)

    Villatoro-Hernández, J.; Kuipers, O.P.; Saucedo-Cárdenas, O.; Montes-de-Oca-Luna, R.

    2012-01-01

    This chapter describes the use of Lactococcus lactis as a safe and efficient cell factory to produce heterologous proteins of medical interest. The relevance of the use of this lactic acid bacterium (LAB) is that it is a noncolonizing, nonpathogenic microorganism that can be delivered in vivo at a m

  10. Heterologous expression of plant virus genes that suppress post-transcriptional gene silencing results in suppression of RNA interference in Drosophila cells

    Directory of Open Access Journals (Sweden)

    Canto Tomas

    2004-08-01

    Full Text Available Abstract Background RNA interference (RNAi in animals and post-transcriptional gene silencing (PTGS in plants are related phenomena whose functions include the developmental regulation of gene expression and protection from transposable elements and viruses. Plant viruses respond by expressing suppressor proteins that interfere with the PTGS system. Results Here we demonstrate that both transient and constitutive expression of the Tobacco etch virus HC-Pro silencing suppressor protein, which inhibits the maintenance of PTGS in plants, prevents dsRNA-induced RNAi of a lacZ gene in cultured Drosophila cells. Northern blot analysis of the RNA present in Drosophila cells showed that HC-Pro prevented degradation of lacZ RNA during RNAi but that there was accumulation of the short (23nt RNA species associated with RNAi. A mutant HC-Pro that does not suppress PTGS in plants also does not affect RNAi in Drosophila. Similarly, the Cucumber mosaic virus 2b protein, which inhibits the systemic spread of PTGS in plants, does not suppress RNAi in Drosophila cells. In addition, we have used the Drosophila system to demonstrate that the 16K cysteine-rich protein of Tobacco rattle virus, which previously had no known function, is a silencing suppressor protein. Conclusion These results indicate that at least part of the process of RNAi in Drosophila and PTGS in plants is conserved, and that plant virus silencing suppressor proteins may be useful tools to investigate the mechanism of RNAi.

  11. Identification and characterization of ectoine biosynthesis genes and heterologous expression of the ectABC gene cluster from Halomonas sp. QHL1, a moderately halophilic bacterium isolated from Qinghai Lake.

    Science.gov (United States)

    Zhu, Derui; Liu, Jian; Han, Rui; Shen, Guoping; Long, Qifu; Wei, Xiaoxing; Liu, Deli

    2014-02-01

    The moderately halophilic bacterium Halomonas sp. QHL1 was identified as a member of the genus Halomonas by 16S rRNA gene sequencing. HPLC analysis showed that strain QHL1 synthesizes ectoine in its cytoplasm. The genes involved in the ectoine biosynthesis pathway were identified on the chromosome in the order ectABC. Subsequently, the ectB gene from this strain was amplified by PCR, and the entire ectABC gene cluster (3,580 bp) was cloned using genome walking. Analysis showed that the ectA (579 bp), ectB (1269 bp), and ectC (390 bp) genes were organized in a single transcriptional unit and were predicted to encode three peptides of 21.2 kDa, 46.4 kDa, and 14.7 kDa, respectively. Two putative promoters, a δ(70)-dependent promoter and a δ(38)-controlled promoter, as well as several conserved motifs with unknown function were identified. Individual ectA, ectB, and ectC genes, and the entire ectABC gene cluster were inserted into the expression plasmid pET-28a(+) to generate the recombinant plasmids pET-28a(+)-ectA, pET-28a(+)-ectB, pET-28a(+)-ectC and pET-28a(+)-ectABC, respectively. Heterologous expression of these proteins in Escherichia coli BL21 (DE3) was confirmed by SDS-PAGE. The recombinant E. coli strain BL21 (pET-28a (+)-ectABC) displayed a higher salt tolerance than native E. coli cells but produced far less ectoine than the wild-type QHL1 strain.

  12. Hordeum vulgare cysteine protease heterologous expressed in yeast

    DEFF Research Database (Denmark)

    Rosenkilde, Anne Lind; Dionisio, Giuseppe; Holm, Preben Bach

    , (Hordeum vulgare) endoprotease B2 (HvEPB2) was cloned with and without the 5 amino acid C-terminal sequence into the Pichia pastoris expression vector pPICZ Aα and electrotransformed into Pichia pastoris strain SDM1163. Heterologous protein production was induced with 2% MeOH and the protein expression...... were monitered during induction by collecting 1 ml samples every hr for 24 hrs. After 4 days, the supernatant were harvested and analyzed by SDS-PAGE, activity assay and Western blot. A significant amount of functional, heterologous protein was produced and the protein production was highest after 4...

  13. Heterologous expression of Saccharomyces cerevisiae MPR1 gene confers tolerance to ethanol and L: -azetidine-2-carboxylic acid in Hansenula polymorpha.

    Science.gov (United States)

    Ishchuk, Olena P; Abbas, Charles A; Sibirny, Andriy A

    2010-02-01

    Hansenula polymorpha is a naturally xylose-fermenting yeast; however, both its ethanol yield from xylose and ethanol resistance have to be improved before this organism can be used for industrial high-temperature simultaneous saccharification and fermentation of lignocellulosic materials. In the current research, we checked if the expression of the Saccharomyces cerevisiae MPR1 gene encoding N-acetyltransferase can increase the ethanol tolerance of H. polymorpha. The S. cerevisiae MPR1 gene was cloned in the H. polymorpha expression vector under the control of the H. polymorpha strong constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH). H. polymorpha recombinant strains harboring 1-3 copies of the S. cerevisiae MPR1 gene showed enhanced tolerance to L: -azetidine-2-carboxylic acid and ethanol. The obtained results suggest that the expression of the S. cerevisiae MPR1 gene in H. polymorpha can be a useful approach in the construction of H. polymorpha strains with improved ethanol resistance.

  14. Heterologous expression in Tritrichomonas foetus of functional Trichomonas vaginalis AP65 adhesin

    Science.gov (United States)

    Kucknoor, Ashwini S; Mundodi, Vasanthakrishna; Alderete, JF

    2005-01-01

    Background Trichomonosis, caused by Trichomonas vaginalis, is the number one, nonviral sexually transmitted infection that has adverse consequences for the health of women and children. The interaction of T. vaginalis with vaginal epithelial cells (VECs), a step preparatory to infection, is mediated in part by the prominent surface protein AP65. The bovine trichomonad, Tritrichomonas foetus, adheres poorly to human VECs. Thus, we established a transfection system for heterologous expression of the T. vaginalis AP65 in T. foetus, as an alternative approach to confirm adhesin function for this virulence factor. Results In this study, we show stable transfection and expression of the T. vaginalis ap65 gene in T. foetus from an episomal pBS-ap65-neo plasmid. Expression of the gene and protein was confirmed by RT-PCR and immunoblots, respectively. AP65 in transformed T. foetus bound to host cells. Specific mAbs revealed episomally-expressed AP65 targeted to the parasite surface and hydrogenosome organelles. Importantly, surface-expression of AP65 in T. foetus paralleled increased levels of adherence of transfected bovine trichomonads to human VECs. Conclusion The T. vaginalis AP65 adhesin was stably expressed in T. foetus, and the data obtained using this heterologous system strongly supports the role of AP65 as a prominent adhesin for T. vaginalis. In addition, the heterologous expression in T. foetus of a T. vaginalis gene offers an important, new approach for confirming and characterizing virulence factors. PMID:15748280

  15. Heterologous expression in Tritrichomonas foetus of functional Trichomonas vaginalis AP65 adhesin

    Directory of Open Access Journals (Sweden)

    Alderete JF

    2005-03-01

    Full Text Available Abstract Background Trichomonosis, caused by Trichomonas vaginalis, is the number one, nonviral sexually transmitted infection that has adverse consequences for the health of women and children. The interaction of T. vaginalis with vaginal epithelial cells (VECs, a step preparatory to infection, is mediated in part by the prominent surface protein AP65. The bovine trichomonad, Tritrichomonas foetus, adheres poorly to human VECs. Thus, we established a transfection system for heterologous expression of the T. vaginalis AP65 in T. foetus, as an alternative approach to confirm adhesin function for this virulence factor. Results In this study, we show stable transfection and expression of the T. vaginalis ap65 gene in T. foetus from an episomal pBS-ap65-neo plasmid. Expression of the gene and protein was confirmed by RT-PCR and immunoblots, respectively. AP65 in transformed T. foetus bound to host cells. Specific mAbs revealed episomally-expressed AP65 targeted to the parasite surface and hydrogenosome organelles. Importantly, surface-expression of AP65 in T. foetus paralleled increased levels of adherence of transfected bovine trichomonads to human VECs. Conclusion The T. vaginalis AP65 adhesin was stably expressed in T. foetus, and the data obtained using this heterologous system strongly supports the role of AP65 as a prominent adhesin for T. vaginalis. In addition, the heterologous expression in T. foetus of a T. vaginalis gene offers an important, new approach for confirming and characterizing virulence factors.

  16. Heterologous expression of biologically active chicken granulocyte ...

    African Journals Online (AJOL)

    African Journal of Biotechnology ... In this study, we investigated the function of recombinant chicken GM-CSF (rchGM-CSF). ... The recombinant Pichia pastoris expression vector pPICZαA-rchGM-CSF was constructed by inserting the reformed ...

  17. Heterologous viral expression systems in fosmid vectors increase the functional analysis potential of metagenomic libraries

    OpenAIRE

    2013-01-01

    The extraordinary potential of metagenomic functional analyses to identify activities of interest present in uncultured microorganisms has been limited by reduced gene expression in surrogate hosts. We have developed vectors and specialized E. coli strains as improved metagenomic DNA heterologous expression systems, taking advantage of viral components that prevent transcription termination at metagenomic terminators. One of the systems uses the phage T7 RNA-polymerase to drive metagenomic ge...

  18. Heterologous expression of membrane proteins: choosing the appropriate host.

    Directory of Open Access Journals (Sweden)

    Florent Bernaudat

    Full Text Available BACKGROUND: Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. METHODOLOGY/PRINCIPAL FINDINGS: The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals, functions (transporters, receptors, enzymes and topologies (between 0 and 13 transmembrane segments. The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. CONCLUSIONS/SIGNIFICANCE: Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein.

  19. Heterologous expression of membrane proteins: choosing the appropriate host.

    Science.gov (United States)

    Bernaudat, Florent; Frelet-Barrand, Annie; Pochon, Nathalie; Dementin, Sébastien; Hivin, Patrick; Boutigny, Sylvain; Rioux, Jean-Baptiste; Salvi, Daniel; Seigneurin-Berny, Daphné; Richaud, Pierre; Joyard, Jacques; Pignol, David; Sabaty, Monique; Desnos, Thierry; Pebay-Peyroula, Eva; Darrouzet, Elisabeth; Vernet, Thierry; Rolland, Norbert

    2011-01-01

    Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein. © 2011 Bernaudat et al.

  20. Heterologous expression of mannanase and developing a new Reporter gene system in Lactobacillus casei and Escherichia coli

    DEFF Research Database (Denmark)

    Lin, Jinzhong; Zou, Yexia; Ma, Chengjie

    2015-01-01

    from the S-layer of L. acidophilus (pELWH). The secretion of ManB was detected in the supernatant of the pELSH-ManB transformants and in the S-layer of the cell surface of the pELWH-ManB transformants. This is the first report demonstrating that the B. pumilus manB gene is a useful reporter gene in L......Reporter gene systems are useful for studying bacterial molecular biology, including the regulation of gene expression and the histochemical analysis of protein products. Here, two genes, β-1,4-mannanase (manB) from Bacillus pumilus and β-glucuronidase (gusA) from Escherichia coli K12, were cloned...... into the expression vector pELX1. The expression patterns of these reporter genes in Lactobacillus casei were investigated by measuring their enzymatic activities and estimating their recombinant protein yields using western blot analysis. Whereas mannanase activity was positively correlated with the accumulation...

  1. Production of Avaroferrin and Putrebactin by Heterologous Expression of a Deep-Sea Metagenomic DNA

    Directory of Open Access Journals (Sweden)

    Masaki J. Fujita

    2014-09-01

    Full Text Available The siderophore avaroferrin (1, an inhibitor of Vibrio swarming that was recently identified in Shewanella algae B516, was produced by heterologous expression of the biosynthetic gene cluster cloned from a deep-sea sediment metagenomic DNA, together with two analogues, bisucaberin (2 and putrebactin (3. Avaroferrin (1 is a macrocyclic heterodimer of N-hydroxy-N-succinyl cadaverine (4 and N-hydroxy-N-succinyl-putrescine (5, whereas analogues 2 and 3 are homodimers of 4 and 5, respectively. Heterologous expression of two other related genes from culturable marine bacteria resulted in production of compounds 1–3, but in quite different proportions compared with production through expression of the metagenomic DNA.

  2. Industrial-scale production and purification of a heterologous protein in Lactococcus lactis using the nisin-controlled gene expression system NICE: The case of lysostaphin

    Directory of Open Access Journals (Sweden)

    Floris Esther

    2005-05-01

    Full Text Available Abstract Background The NIsin-Controlled gene Expression system NICE of Lactococcus lactis is one of the most widespread used expression systems of Gram-positive bacteria. It is used in more than 100 laboratories for laboratory-scale gene expression experiments. However, L. lactis is also a micro-organism with a large biotechnological potential. Therefore, the aim of this study was to test whether protein production in L. lactis using the NICE system can also effectively be performed at the industrial-scale of fermentation. Results Lysostaphin, an antibacterial protein (mainly against Staphylococcus aureus from S. simulans biovar. Staphylolyticus, was used as a model system. Food-grade lysostaphin expression constructs in L. lactis were grown at 1L-, 300-L and 3000-L scale and induced with nisin for lysostaphin production. The induction process was equally effective at all scales and yields of about 100 mg/L were obtained. Up-scaling was easy and required no specific effort. Furthermore, we describe a simple and effective way of downstream processing to obtain a highly purified lysostaphin, which has been used for clinical phase I trials. Conclusion This is the first example that shows that nisin-regulated gene expression in L. lactis can be used at industrial scale to produce large amounts of a target protein, such as lysostaphin. Downstream processing was simple and in a few steps produced a highly purified and active enzyme.

  3. The MsPRP2 promoter enables strong heterologous gene expression in a root-specific manner and is enhanced by overexpression of Alfin 1.

    Science.gov (United States)

    Winicov, Ilga; Valliyodan, Babu; Xue, Lingru; Hoober, J Kenneth

    2004-10-01

    Promoter specificity and efficiency of utilization are essential for endogenous and transgene expression. Selective root expression remains to be defined in terms of both promoter elements and transcription factors that provide high levels of ubiquitous expression. We characterized expression from the MsPRP2 promoter with the green fluorescent protein (GFP) reporter transgene in alfalfa (Medicago sativa) and found that a promoter fragment (+1 to -652 bp) retained the root and callus specificity of the endogenous MsPRP2 gene and hence this promoter fragment contains elements necessary for root-specific expression. The strong ubiquitous expression obtained from this promoter was comparable to that of the CaMV 35S promoter in roots and was enhanced by transgenic overexpression of Alfin 1, a root- and callus-specific transcription factor in alfalfa. No transgenic expression was obtained in leaves with this promoter in the presence or absence of Alfin 1. The increased expression of GFP in alfalfa containing the Alfin 1 transgene confirms the function of Alfin 1 binding sites in the MsPRP2 promoter fragment and also indicates that Alfin 1 concentrations are limiting for maximal expression in calli and roots. These findings characterize the MsPRP2 promoter as a novel root- and callus-specific promoter of plant origin that can be used as an effective tool for strong root-directed gene expression. In addition, we have demonstrated that the signal sequence of MsPRP2 can be used for efficient secretion of transgene products from callus and roots.

  4. Characterization of cycP gene expression in Achromobacter xylosoxidans NCIMB 11015 and high-level heterologous synthesis of cytochrome c' in Escherichia coli.

    Science.gov (United States)

    Harris, Roger L; Barbieri, Sonia; Paraskevopoulos, Kostas; Murphy, Loretta M; Eady, Robert R; Hasnain, S Samar; Sawers, R Gary

    2010-01-01

    The cycP gene encoding a periplasmic cytochrome c' from the denitrifying beta-proteobacterium Achromobacter xylosoxidans was characterized. The genes flanking cycP encode components of a mobile genetic element characteristic of the beta-proteobacteria, suggesting that cycP has inserted within a transposon or insertion element. The gene therefore does not form part of a denitrification operon or gene cluster. The level of expression of the cycP gene and the level of synthesis of its corresponding gene product were found to increase by maximally 3-fold anaerobically. Expression of cycP appears to occur mainly by non-specific read-through transcription from portions of the insertion element. Conditions were developed for high-level overproduction of cytochrome c' in Escherichia coli, which resulted in signal peptide cleavage concomitant with secretion of the protein into the periplasm. Using a single-step purification, 20-30 mg of pure protein were isolated from a 1-litre culture. Based on UV-visible spectrophotometry the dimeric protein was shown to have a full complement of haem and to be indistinguishable from the native protein purified from A. xylosoxidans. This system provides an excellent platform to facilitate biochemical and structural dissection of the mechanism underlying the novel specificity of NO binding to the proximal face of the haem.

  5. Cloning, DNA sequencing and heterologous expression of the gene for thermostable N-acylamino acid racemase from Amycolatopsis sp. TS-1-60 in Escherichia coli.

    Science.gov (United States)

    Tokuyama, S; Hatano, K

    1995-03-01

    The gene encoding the novel enzyme N-acylamino acid racemase (AAR) was cloned in recombinant phage lambda-4 from the DNA library of Amycolatopsis sp. TS-1-60, a rare actinomycete, using antiserum against the enzyme. The cloned gene was subcloned and transformed in Escherichia coli JM105 using pUC118 as a vector. The AAR gene consists of an open-reading frame of 1104 nucleotides, which specifies a 368-amino-acid protein with a molecular mass of 39411Da. The molecular mass deduced from the AAR gene is in good agreement with the subunit molecular mass (40kDa) of AAR from Amycolatopsis sp. TS-1-60. The guanosine plus cytosine content of the AAR gene was about 70%. Although the AAR gene uses the unusual initiation codon GTG, the gene was expressed in Escherichia coli using the lac promoter of pUC118. The amount of the enzyme produced by the transformant was 16 times that produced by Amycolatopsis sp. TS-1-60. When the unusual initiation codon GTG was changed to ATG, the enzyme productivity of the transformant increased to more than 37 times that of Amycolatopsis sp. TS-1-60. In the comparison of the DNA sequence and the deduced amino acid sequence of AAR with those of known racemases and epimerases in data bases, no significant sequence homology was found. However, AAR resembles mandelate racemase in that requires metal ions for enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Expression of the Anabaena sp. strain PCC 7120 xisA gene from a heterologous promoter results in excision of the nifD element.

    Science.gov (United States)

    Brusca, J S; Chastain, C J; Golden, J W

    1990-01-01

    An 11-kilobase-pair element interrupts the nifD gene in vegetative cells of Anabaena sp. strain PCC 7120. The nifD element normally excises only from the chromosomes of cells that differentiate into nitrogen-fixing heterocysts. The xisA gene contained within the element is required for the excision. Shuttle vectors containing the Escherichia coli tac consensus promoter fused to various 5' deletions of the xisA gene were constructed and conjugated into Anabaena sp. strain PCC 7120 cells. Some of the expression plasmids resulted in excision of the nifD element in a high proportion of vegetative cells. Excision of the element required deletion of an xisA 5' regulatory region which presumably blocks expression in Anabaena sp. strain PCC 7120 vegetative cells but not in E. coli. Strains lacking the nifD element grew normally in medium containing a source of combined nitrogen and showed normal growth and heterocyst development in medium lacking combined nitrogen. The xisA gene was shown to be the only Anabaena gene required for the proper rearrangement in E. coli of a plasmid containing the borders of the nifD element. Images PMID:2113913

  7. Heterologous expression of the gourd E3 ubiquitin ligase gene LsRZF1 compromises the drought stress tolerance in Arabidopsis thaliana.

    Science.gov (United States)

    Min, Ji-Hee; Ju, Hyun-Woo; Yang, Kwang-Yeol; Chung, Jung-Sung; Cho, Baik-Ho; Kim, Cheol Soo

    2014-04-01

    Protein ubiquitination is one of the major regulatory processes used by eukaryotic cells. The ubiquitin E3 ligase acts as a main determinant of substrate specificity. However, the precise roles of E3 ligase in plants to drought stress are poorly understood. In this study, a gourd family (Lagenaria siceraria) ortholog of Arabidopsis thaliana RING Zinc Finger 1 (AtRZF1) gene, designated LsRZF1, was identified and characterized. LsRZF1 was reduced by abscisic acid (ABA), osmotic stress, and drought conditions. Compared to wild type, transgenic Arabidopsis plants ectopic expressing LsRZF1 were hypersensitive to ABA and osmotic stress during early seedling development, indicating that LsRZF1 negatively regulates drought-mediated control of early seedling development. Moreover, the ectopic expression of the LsRZF1 gene was very influential in drought sensitive parameters including proline content, water loss, and the expression of dehydration stress-related genes. Furthermore, ubiquitin E3 ligase activity and genetic data indicate that AtRZF1 and LsRZF1 function in similar pathway to control proline metabolism in Arabidopsis under drought condition. Together, these results suggest that the E3 ligase LsRZF1 is an important regulator of water deficit stress during early seedling development.

  8. Construction and heterologous expression of a synthetic copy of the cutinase cDNA from Fusarium solani pisi

    NARCIS (Netherlands)

    Gemeren, I.A. van; Musters, W.; Hondel, C.A.M.J.J. van den; Verrips, C.T.

    1995-01-01

    A copy of the cutinase cDNA from Fusarium solani pisi was constructed starting from synthetic oligonucleotides. For this construction three separate cassettes were made, which were subsequently assembled to form the cutinase gene. Heterologous expression of the synthetic cutinase gene and the subseq

  9. Construction and heterologous expression of a synthetic copy of the cutinase cDNA from Fusarium solani pisi

    NARCIS (Netherlands)

    Gemeren, I.A. van; Musters, W.; Hondel, C.A.M.J.J. van den; Verrips, C.T.

    1995-01-01

    A copy of the cutinase cDNA from Fusarium solani pisi was constructed starting from synthetic oligonucleotides. For this construction three separate cassettes were made, which were subsequently assembled to form the cutinase gene. Heterologous expression of the synthetic cutinase gene and the

  10. Construction of a novel cell-surface display system for heterologous gene expression in Escherichia coli by using an outer membrane protein of Zymomonas mobilis as anchor motif.

    Science.gov (United States)

    He, Ming-Xiong; Feng, Hong; Zhang, Yi-Zheng

    2008-12-01

    A novel bacterial cell-surface display system was developed in Escherichia coli using omp1, a hypothetical outer membrane protein of Zymomonas mobilis. By using this system, we successfully expressed beta-amylase gene of sweet potato in E. coli. The display of enzyme on the membrane surface was also confirmed. The recombinant beta-amylase showed to significantly increase hydrolytic activity toward soluble starch. Our results provide a basis for constructing an engineered Z. mobilis strain directly fermenting raw starch to produce ethanol.

  11. Heterologous expression of the glucose oxidase gene in Trichoderma atroviride leads enhanced ability to attack phytopathogenic fungi and induction of plant systemic disease resistance

    Institute of Scientific and Technical Information of China (English)

    Robert L Mach; Brunner Kurt; Matteo Lorito; Susanne Zeilinger; Rosalia Ciliento; Sheridan Woo

    2004-01-01

    @@ A transgenic strain of Trichoderma atroviride that expresses the Aspergillus niger glucose oxidase gene goxA under a homologous pathogen-inducible promoter (nag1) has been constructed, with the aim of increasing the ability of this biocontrol agent (BCA) to attack phytopathogenic fungi and enhance plant systemic disease resistance. The sporulation and growth rate of the transgenic progenies were similar to the wild-type strain Pl. goxA expression occurred immediately after contact with the plant pathogen,and the glucose oxidase formed was secreted extracellularly. The transformed strain SJ3 4, containing 12-14 copies of the transgene, produced significantly less N-acetyl-glucosaminidase and endochitinase then wild type. However, the ability of its culture filtrate to inhibit the germination of Botrytis cinerea spores was increased by about 3-fold. In comparison to P1, the transgenic strain more quickly overgrew and lysed in vitro the pathogens Rhizoctonia solani and Pythium ultimum.

  12. Cestrum yellow leaf curling virus (CmYLCV) promoter: a new strong constitutive promoter for heterologous gene expression in a wide variety of crops.

    Science.gov (United States)

    Stavolone, Livia; Kononova, Maria; Pauli, Sandra; Ragozzino, Antonio; de Haan, Peter; Milligan, Steve; Lawton, Kay; Hohn, Thomas

    2003-11-01

    Appropriately regulated gene expression requires a suitable promoter. A number of promoters have been isolated and shown to be functional in plants, but only a few of them activate transcription of transgenes at high levels constitutively. We report here the cloning and characterization of a novel, constitutively expressed promoter isolated from Cestrum yellow leaf curling virus (CmYLCV), a double-stranded DNA plant pararetrovirus belonging to the Caulimoviridae family. The CmYLCV promoter is highly active in callus, meristems and vegetative and reproductive tissues in Arabidopsis thaliana, Nicotiana tabacum, Lycopersicon esculentum, Zea mays and Oryza sativa. Furthermore, the level of expression is comparable to, or higher than, that from the CaMV 35S, the 'super-promoter' or the maize ubiquitin 1 promoters, three frequently used promoters in agricultural biotechnology. The heritable, strong and constitutive activity in both monocotyledonous and dicotyledonous plants, combined with the extremely narrow CmYLCV host range, makes the CmYLCV promoter an attractive tool for regulating transgene expression in a wide variety of plant species.

  13. Heterologous expression of ApGSMT2 and ApDMT2 genes from Aphanothece halophytica enhanced drought tolerance in transgenic tobacco.

    Science.gov (United States)

    He, Ying; He, Chunmei; Li, Lihua; Liu, Zhili; Yang, Aifang; Zhang, Juren

    2011-01-01

    The glycine-methylation biosynthetic pathway of glycinebetaine (GB) has been investigated, but only a few studies on GB accumulation in transgenic higher plants have utilized this pathway. In this study, two methyltransferase genes named ApGSMT2 and ApDMT2, encoding proteins catalyzing GB biosynthesis from glycine, were cloned from a relative strain of Aphanothece halophytica. The potential roles of ApGSMT2 and ApDMT2 in GB synthesis were first examined in transgenic Escherichia coli, which had increased levels of GB and improved salt tolerance. Then ApGSMT2 and ApDMT2 were transferred into tobacco. Compared with transgenic tobacco expressing betA, transgenic tobacco co-expressing ApGSMT2 and ApDMT2 accumulated more GB and exhibited enhanced drought resistance with better germination performance, higher relative water content, less cell membrane damage and better photosynthetic capacity under drought stress. We concluded that the ApGSMT2 and ApDMT2 genes cloned in this study will be very useful for engineering GB-accumulating transgenic plants with enhanced drought resistance.

  14. Heterologous co-expression of accA, fabD, and thioesterase genes for improving long-chain fatty acid production in Pseudomonas aeruginosa and Escherichia coli.

    Science.gov (United States)

    Lee, Sunhee; Jeon, Eunyoung; Jung, Yeontae; Lee, Jinwon

    2012-05-01

    The goal of the present study was to increase the content of intracellular long-chain fatty acids in two bacterial strains, Pseudomonas aeruginosa PA14 and Escherichia coli K-12 MG1655, by co-overexpressing essential enzymes that are involved in the fatty acid synthesis metabolic pathway. Recently, microbial fatty acids and their derivatives have been receiving increasing attention as an alternative source of fuel. By introducing two genes (accA and fabD) of P. aeruginosa into the two bacterial strains and by co-expressing with them the fatty acyl-acyl carrier protein thioesterase gene of Streptococcus pyogenes (strain MGAS10270), we have engineered recombinant strains that are efficient producers of long-chain fatty acids (C16 and C18). The recombinant strains exhibit a 1.3-1.7-fold increase in the production of long-chain fatty acids over the wild-type strains. To enhance the production of total long-chain fatty acids, we researched the carbon sources for optimized culture conditions and results were used for post-culture incubation period. E. coli SGJS17 (containing the accA, fabD, and thioesterase genes) produced the highest content of intracellular total fatty acids; in particular, the unsaturated fatty acid content was about 20-fold higher than that in the wild-type E. coli.

  15. Modification of a salmonid alphavirus replicon vector for enhanced expression of heterologous antigens.

    Science.gov (United States)

    Guo, Tz-Chun; Johansson, Daniel X; Liljeström, Peter; Evensen, Øystein; Haugland, Øyvind

    2015-03-01

    A salmonid alphavirus (SAV) replicon has been developed to express heterologous antigens but protein production was low to modest compared with terrestrial alphavirus replicons. In this study, we have compared several modifications to a SAV replicon construct and analysed their influence on foreign gene expression. We found that an insertion of a translational enhancer consisting of the N-terminal 102 nt of the capsid gene, together with a nucleotide sequence encoding the foot-and-mouth disease virus (FMDV) 2A peptide, caused a significant increase in EGFP reporter gene expression. The importance of fusing a hammerhead (HH) ribozyme sequence at the 5' end of the viral genome was also demonstrated. In contrast, a hepatitis D virus ribozyme (HDV-RZ) sequence placed at the 3' end did not augment expression of inserted genes. Taken together, we have developed a platform for optimized antigen production, which can be applied for immunization of salmonid fish in the future.

  16. Heterologous expression of a Penicillium purpurogenum exo-arabinanase in Pichia pastoris and its biochemical characterization.

    Science.gov (United States)

    Mardones, Wladimir; Callegari, Eduardo; Eyzaguirre, Jaime

    2015-12-01

    Arabinan is a component of pectin, which is one of the polysaccharides present in lignocelluose. The enzymes degrading the main chain of arabinan are the endo- (EC 3.2.1.99) and exo-arabinanases (3.2.1.-). Only three exo-arabinanases have been biochemically characterized; they belong to glycosyl hydrolase family 93. In this work, the cDNA of an exo-arabinanase (Arap2) from Penicillium purpurogenum has been heterologously expressed in Pichia pastoris. The gene is 1310 bp long, has three introns and codes for a protein of 380 amino acid residues; the mature protein has a calculated molecular mass of 39 823 Da. The heterologously expressed Arap2 has a molecular mass in the range of 60-80 kDa due to heterogeneous glycosylation. The enzyme is active on debranched arabinan with optimum pH of 4-5.5 and optimal temperature of 40 °C, and has an exo-type action mode, releasing arabinobiose from its substrates. The expression profile of arap2 in corncob and sugar beet pulp follows a different pattern and is not related to the presence of arabinan. This is the first exo-arabinanase studied from P. purpurogenum and the first expressed in yeast. The availability of heterologous Arap2 may be useful for biotechnological applications requiring acidic conditions. Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  17. Heterologous expression of pentalenene synthase (PSS) from Streptomyces UC5319 in Xanthophyllomyces dendrorhous.

    Science.gov (United States)

    Melillo, Elena; Muntendam, Remco; Quax, Wim J; Kayser, Oliver

    2012-10-31

    For the first time, the pentalenene synthase (PSS) gene from Streptomyces UC5319 was expressed in Xanthophyllomyces dendrorhous, a native producer of astaxanthin. For the expression of the gene and the concurrent knock out of the native crtE or crtYB genes, two new vectors were engineered and used for the transformation of the wild-type strain of X. dendrorhous. The transformations resulted in white colonies, showing a complete shutdown of the carotenoid production. Furthermore, an additional vector was constructed for the insertion of the PSS gene in the rDNA of the yeast. All the mutant strains produce the sesquiterpene pentalenene and show no difference in growth when compared to the wild-type strain. In this report, we demonstrate that X. dendrorhous is a suitable host for the expression of heterologous terpene cyclases and for the production of foreign terpene compounds.

  18. Natural products from filamentous fungi and production by heterologous expression.

    Science.gov (United States)

    Alberti, Fabrizio; Foster, Gary D; Bailey, Andy M

    2017-01-01

    Filamentous fungi represent an incredibly rich and rather overlooked reservoir of natural products, which often show potent bioactivity and find applications in different fields. Increasing the naturally low yields of bioactive metabolites within their host producers can be problematic, and yield improvement is further hampered by such fungi often being genetic intractable or having demanding culturing conditions. Additionally, total synthesis does not always represent a cost-effective approach for producing bioactive fungal-inspired metabolites, especially when pursuing assembly of compounds with complex chemistry. This review aims at providing insights into heterologous production of secondary metabolites from filamentous fungi, which has been established as a potent system for the biosynthesis of bioactive compounds. Numerous advantages are associated with this technique, such as the availability of tools that allow enhanced production yields and directing biosynthesis towards analogues of the naturally occurring metabolite. Furthermore, a choice of hosts is available for heterologous expression, going from model unicellular organisms to well-characterised filamentous fungi, which has also been shown to allow the study of biosynthesis of complex secondary metabolites. Looking to the future, fungi are likely to continue to play a substantial role as sources of new pharmaceuticals and agrochemicals-either as producers of novel natural products or indeed as platforms to generate new compounds through synthetic biology.

  19. Heterologous viral expression systems in fosmid vectors increase the functional analysis potential of metagenomic libraries.

    Science.gov (United States)

    Terrón-González, L; Medina, C; Limón-Mortés, M C; Santero, E

    2013-01-01

    The extraordinary potential of metagenomic functional analyses to identify activities of interest present in uncultured microorganisms has been limited by reduced gene expression in surrogate hosts. We have developed vectors and specialized E. coli strains as improved metagenomic DNA heterologous expression systems, taking advantage of viral components that prevent transcription termination at metagenomic terminators. One of the systems uses the phage T7 RNA-polymerase to drive metagenomic gene expression, while the other approach uses the lambda phage transcription anti-termination protein N to limit transcription termination. A metagenomic library was constructed and functionally screened to identify genes conferring carbenicillin resistance to E. coli. The use of these enhanced expression systems resulted in a 6-fold increase in the frequency of carbenicillin resistant clones. Subcloning and sequence analysis showed that, besides β-lactamases, efflux pumps are not only able contribute to carbenicillin resistance but may in fact be sufficient by themselves to convey carbenicillin resistance.

  20. Heterologous expression of the Aspergillus nidulans alcR-alcA system in Aspergillus niger.

    Science.gov (United States)

    Nikolaev, I; Mathieu, M; van de Vondervoort, P; Visser, J; Felenbok, B

    2002-10-01

    The inducible and strongly expressed alcA gene encoding alcohol dehydrogenase I from Aspergillus nidulans was transferred together with the activator gene alcR, in the industrial fungus Aspergillus niger. This latter organism does not possess an inducible alc system but has an endogenously constitutive lowly expressed alcohol dehydrogenase activity. The overall induced expression of the alcA gene was of the same order in both fungi, as monitored by alcA transcription, alcohol dehydrogenase activity and heterologous expression of the reporter enzyme, beta-glucuronidase. However, important differences in the pattern of alcA regulation were observed between the two fungi. A high basal level of alcA transcription was observed in A. niger resulting in a lower ratio of alcA inducibility. This may be due to higher levels of the physiological inducer of the alc regulon, acetaldehyde, from general metabolism in A. niger which differs from that of A. nidulans.

  1. Heterologous expression of the Treponema pallidum laminin-binding adhesin Tp0751 in the culturable spirochete Treponema phagedenis.

    Science.gov (United States)

    Cameron, Caroline E; Kuroiwa, Janelle M Y; Yamada, Mitsunori; Francescutti, Teresa; Chi, Bo; Kuramitsu, Howard K

    2008-04-01

    Treponema pallidum subsp. pallidum, the causative agent of syphilis, is an unculturable, genetically intractable bacterium. Here we report the use of the shuttle vector pKMR4PEMCS for the expression of a previously identified T. pallidum laminin-binding adhesin, Tp0751, in the nonadherent, culturable spirochete Treponema phagedenis. Heterologous expression of Tp0751 in T. phagedenis was confirmed via reverse transcriptase PCR analysis with tp0751 gene-specific primers and immunofluorescence analysis with Tp0751-specific antibodies; the latter assay verified the expression of the laminin-binding adhesin on the treponemal surface. Expression of Tp0751 within T. phagedenis was functionally confirmed via laminin attachment assays, in which heterologous Tp0751 expression conferred upon T. phagedenis the capacity to attach to laminin. Further, specific inhibition of the attachment of T. phagedenis heterologously expressing Tp0751 to laminin was achieved by using purified antibodies raised against recombinant T. pallidum Tp0751. This is the first report of heterologous expression of a gene from an unculturable treponeme in T. phagedenis. This novel methodology will significantly advance the field of syphilis research by allowing targeted investigations of T. pallidum proteins purported to play a role in pathogenesis, and specifically host cell attachment, in the nonadherent spirochete T. phagedenis.

  2. Identification and expression profiling of microRNAs during bovine oocyte maturation using heterologous approach.

    Science.gov (United States)

    Tesfaye, Dawit; Worku, Dagnachew; Rings, Franca; Phatsara, Chirawath; Tholen, Ernst; Schellander, Karl; Hoelker, Michael

    2009-07-01

    The accumulation of maternal mRNA and protein during oogenesis for supporting oocyte maturation and the newly fertilised zygote marks the beginning of developmental process in mammals. MicroRNAs (approximately 18-22 nt long) which are known for post-transcriptional gene regulation are evidenced for their essential role during animal development. We, therefore, aimed to investigate the expression of miRNAs in immature and in vitro matured bovine oocytes, using heterologous miRNA array platform. To attain this, we used a mercury locked nucleic acids (LNA) array (Exiqon, Vedbaek, Denmark) microarray that consist of 454 capture probes for human, mouse and rat miRNAs as registered and annotated in the miRBase release 8.0 at The Wellcome Trust Sanger Institute. Our result revealed the differential expression of 59 miRNAs, of which 31 and 28 miRNAs were found to be preferentially expressed in immature and matured oocytes, respectively. Here, we also report the identification of 32 orthologous miRNAs using a heterologous approach. Expression profiling of selected miRNAs during preimplantation stage embryos showed a distinct temporal expression pattern. After target prediction for selected candidate miRNAs high ranking target mRNA were quantified in immature and matured oocytes and showed a reciprocal expression pattern between the miRNA and the predicted targets suggesting a cause and effect relationship.

  3. 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

    many steps and difficult chemical reactions, resulting in a low final yield or incorrect stereochemistry. Several drug candidates with terpene skeletons are difficult to obtain by chemical synthesis due to their large number of chiral centers. Thus, biological production remains the preferred method...... in heterologous hosts. Although there are many examples of successful engineering of microbes such as yeast or bacteria to produce these compounds, this often requires extensive changes to the host organism's metabolism. Optimization of plant gene expression, post-translational protein modifications, subcellular...

  4. A Stranger in a Strange Land: The Utility and Interpretation of Heterologous Expression

    Directory of Open Access Journals (Sweden)

    Elena M Kramer

    2015-09-01

    Full Text Available One of the major goals of the modern study of evodevo is to understand the evolution of gene function across a range of contexts, including sub/neofunctionalization, co-option of genetic modules, and the evolution of morphological novelty. To these ends, comparative studies of gene expression can be useful for constructing hypotheses, but cannot provide direct evidence of functional evolution. Unfortunately, determining endogenous gene function in non-model species is often not an option. Faced with this dilemma, a common approach is to use heterologous expression (HE in genetically tractable model species as a proxy for functional analyses. Such experiments have important limitations, however, and require caution in the interpretation of their results. How do we dissociate biochemical function from its original genomic context? In the end, what does HE actually tell us? Here, I argue that HE only sheds light on specific types of biochemical conservation, but can be useful when experiments are carefully interpreted.

  5. Cloning and Heterologous Expression of Insecticidal-Protein-Encoding Genes from Photorhabdus luminescens TT01 in Enterobacter cloacae for Termite Control▿

    OpenAIRE

    Zhao, Ruihua; Han, Richou; Qiu, Xuehong; Yan, Xun; Cao, Li; Liu, Xiuling

    2008-01-01

    Enterobacter cloacae, one of the indigenous gut bacteria of the Formosan subterranean termite (Coptotermes formosanus), was genetically modified with a transposon Tn5 vector containing genes (tcdA1 and tcdB1) encoding orally insecticidal proteins from the entomopathogenic bacterium Photorhabdus luminescens subsp. laumondii TT01, a symbiont of the entomopathogenic nematode Heterorhabditis bacteriophora, for termite control. In the laboratory, termites were fed filter paper inoculated with the ...

  6. An extracytoplasmic function sigma factor-mediated cell surface signaling system in Pseudomonas syringae pv. tomato DC3000 regulates gene expression in response to heterologous siderophores.

    Science.gov (United States)

    Markel, Eric; Maciak, Charlene; Butcher, Bronwyn G; Myers, Christopher R; Stodghill, Paul; Bao, Zhongmeng; Cartinhour, Sam; Swingle, Bryan

    2011-10-01

    The diversity of regulatory systems encoded by bacteria provides an indication of the variety of stresses and interactions that these organisms encounter in nature. We have been investigating how the plant pathogen Pseudomonas syringae pv. tomato DC3000 responds to iron limitation and have focused on the iron starvation (IS) sigma factors to identify regulon members and to explore the mechanistic details of genetic control for this class of regulators. In the study described in this report, we used chromatin immunoprecipitation paired with high-throughput sequencing (ChIP-Seq) to screen the genome for locations associated with binding of the P. syringae IS sigma factor PSPTO_1203. We used multiple methods to demonstrate differential regulation of two genes identified in the ChIP-Seq screen and characterize the promoter elements that facilitate PSPTO_1203-dependent regulation. The genes regulated by PSPTO_1203 encode a TonB-dependent transducer (PSPTO_1206) and a cytoplasmic membrane protein (PSPTO_2145), which is located in the P. syringae pyoverdine cluster. Additionally, we identified siderophores that induce the activity of PSPTO_1203 and used this information to investigate the functional components of the signal transduction cascade.

  7. Engineering Fungal Nonreducing Polyketide Synthase by Heterologous Expression and Domain Swapping

    Energy Technology Data Exchange (ETDEWEB)

    Yeh, Hsu-Hua; Chang, Shu-Lin; Chiang, Yi-Ming; Bruno, Kenneth S.; Oakley, Berl R.; Wu, Tung-Kung; Wang, Clay C. C.

    2013-02-15

    Heterologous expression of the A. niger NR-PKS gene, e_gw1_19.204 and the adjacent stand-alone R domain gene, est_GWPlus_C_190476 in A. nidulans demonstrated that they belong to a single gene named dtbA. The DtbA protein produces two polyketides, 2,4-dihydroxy-3,5,6-trimethylbenzaldehyde 1 and 2-ethyl-4,6-dihydroxy-3,5-dimethylbenzaldehyde 2. Generation of DtbA+R-TE chimeric PKSs by swapping the DtbA R domain with the AusA (austinol biosynthesis) or ANID_06448 TE domain enabled the production of two metabolites with carboxylic acids replacing the corresponding aldehydes.

  8. Heterologous expression of an algal hydrogenase in a heterocystous cyanobacterium

    Energy Technology Data Exchange (ETDEWEB)

    Thorsten Heidorn; Peter Lindblad [Dept. of Physiological Botany, Uppsala University, Villavogen 6, SE-752 36 Uppsala, (Sweden)

    2006-07-01

    For the expression of an active algal [FeFe] hydrogenase in the heterocystous cyanobacterium Nostoc punctiforme A TCC 29133 the Chlamydomonas reinhardtii hydrogenase gene hydA1 and the accessory genes hydEF and hydG are to be introduced into the cyanobacteria cells. The genes were amplified by PCR from EST clones, cloned into the cloning vector pBluescript SK+ and sequenced. An expression vector for multi-cistronic cloning, based on pSCR202, was constructed and for a functional test GFP was inserted as a reporter gene. The GFP construct was transformed into Nostoc punctiforme A TCC 29133 by electroporation and expression of GFP was visualized by fluorescence microscopy. (authors)

  9. Heterologous expression of the Monilinia fructicola CYP51 (MfCYP51 gene in Pichia pastoris confirms the mode of action of the novel fungicide, SYP-Z048

    Directory of Open Access Journals (Sweden)

    Fengping eChen

    2015-05-01

    Full Text Available The novel agricultural fungicide 3-[5-(4-chlorophenyl-2,3-dimethyl-3-isoxazolidinyl] pyridine (SYP-Z048 developed by China Shenyang Research Institute of Chemical Industry has been confirmed to be an ergosterol biosynthesis inhibitor (EBI. Previous studies have shown that EBIs target the proteins from a range of genes, including CYP51, ERG2 and/or ERG24, and ERG27, which are involved in the ergosterol biosynthesis pathway. In the current study the ERG2, ERG24, and ERG27 genes were cloned from wild type and resistant mutants of Monilinia fructicola in an attempt to clarify the target site of SYP-Z048. Comparative analysis of the deduced aa sequence of these genes, as well as CYP51, revealed several point mutations that resulted in amino acid variation among the sensitive and resistant isolates. However, sensitivity assays indicated that only one, the substitution of phenylalanine (F for the tyrosine (Y at 136 in CYP51, was correlated with reduced sensitivity to SYP-Z048. Heterologous expression of MfCYP51-136Y (MfCYP136Y and MfCYP51-136F (MfCYP136F in Pichia pastoris revealed that MfCYP136F significantly reduced sensitivity to SYP-Z048, increasing the average EC50 of the transformants 11-fold relative to those carrying MfCYP136Y. However, neither the additional copy of MfCYP136Y nor multiple copies of MfCYP136F were found to reduce sensitivity relative to the empty vector control or single copy transformants, respectively. Molecular docking experiments using SYP-Z048 with HsCYP145Y and the mutated version HsCYP145F as substitutes for MfCYP136Y and MfCYP136F, respectively, indicated that the reduced affinity of HsCYP145F for SYP-Z048 resulted from the loss of a hydrogen bond between the fungicide and the active site. Taken together these results indicate that MfCYP51 is the major target site of SYP-Z048 in M. fructicola, which has important implications for the resistance management of this fungicide in the field.

  10. Xenobiotic Compounds Degradation by Heterologous Expression of a Trametes sanguineus Laccase in Trichoderma atroviride.

    Directory of Open Access Journals (Sweden)

    Edgar Balcázar-López

    Full Text Available Fungal laccases are enzymes that have been studied because of their ability to decolorize and detoxify effluents; they are also used in paper bleaching, synthesis of polymers, bioremediation, etc. In this work we were able to express a laccase from Trametes (Pycnoporus sanguineus in the filamentous fungus Trichoderma atroviride. For this purpose, a transformation vector was designed to integrate the gene of interest in an intergenic locus near the blu17 terminator region. Although monosporic selection was still necessary, stable integration at the desired locus was achieved. The native signal peptide from T. sanguineus laccase was successful to secrete the recombinant protein into the culture medium. The purified, heterologously expressed laccase maintained similar properties to those observed in the native enzyme (Km and kcat and kcat/km values for ABTS, thermostability, substrate range, pH optimum, etc. To determine the bioremediation potential of this modified strain, the laccase-overexpressing Trichoderma strain was used to remove xenobiotic compounds. Phenolic compounds present in industrial wastewater and bisphenol A (an endocrine disruptor from the culture medium were more efficiently removed by this modified strain than with the wild type. In addition, the heterologously expressed laccase was able to decolorize different dyes as well as remove benzo[α]pyrene and phenanthrene in vitro, showing its potential for xenobiotic compound degradation.

  11. Xenobiotic Compounds Degradation by Heterologous Expression of a Trametes sanguineus Laccase in Trichoderma atroviride

    Science.gov (United States)

    Balcázar-López, Edgar; Méndez-Lorenzo, Luz Helena; Batista-García, Ramón Alberto; Esquivel-Naranjo, Ulises; Ayala, Marcela; Kumar, Vaidyanathan Vinoth; Savary, Olivier; Cabana, Hubert; Herrera-Estrella, Alfredo; Folch-Mallol, Jorge Luis

    2016-01-01

    Fungal laccases are enzymes that have been studied because of their ability to decolorize and detoxify effluents; they are also used in paper bleaching, synthesis of polymers, bioremediation, etc. In this work we were able to express a laccase from Trametes (Pycnoporus) sanguineus in the filamentous fungus Trichoderma atroviride. For this purpose, a transformation vector was designed to integrate the gene of interest in an intergenic locus near the blu17 terminator region. Although monosporic selection was still necessary, stable integration at the desired locus was achieved. The native signal peptide from T. sanguineus laccase was successful to secrete the recombinant protein into the culture medium. The purified, heterologously expressed laccase maintained similar properties to those observed in the native enzyme (Km and kcat and kcat/km values for ABTS, thermostability, substrate range, pH optimum, etc). To determine the bioremediation potential of this modified strain, the laccase-overexpressing Trichoderma strain was used to remove xenobiotic compounds. Phenolic compounds present in industrial wastewater and bisphenol A (an endocrine disruptor) from the culture medium were more efficiently removed by this modified strain than with the wild type. In addition, the heterologously expressed laccase was able to decolorize different dyes as well as remove benzo[α]pyrene and phenanthrene in vitro, showing its potential for xenobiotic compound degradation. PMID:26849129

  12. Heterologously Expressed Family 51 α-l-Arabinofuranosidases from Oenococcus oeni and Lactobacillus brevis▿

    OpenAIRE

    Michlmayr, Herbert; Schümann, Christina; Kulbe, Klaus D.; del Hierro, Andrés M

    2010-01-01

    Putative α-l-arabinofuranosidases of Oenococcus oeni and Lactobacillus brevis were heterologously expressed and characterized. We report the basic functional properties of the recombinant enzymes in comparison to those of a commercial family 51 arabinosidase of Aspergillus niger.

  13. Heterologous expression of active human uridine diphosphate glucuronosyltransferase 1A3 in Chinese hamster lung cells

    Institute of Scientific and Technical Information of China (English)

    Ya-Kun Chen; Xin Li; Shu-Qing Chen; Su Zeng

    2005-01-01

    AIM: To obtain the active human recombinant uridine diphosphate glucuronosyltransferase 1A3 (UGT1A3) enzyme from Chinese hamster lung (CHL) cells.METHODS: The full-length UGT1A3 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR)using total RNA from human liver as template. The correct fragment confirmed by sequencing was subcloned into the mammalian expression vector pcDNA3.1 (+), and the recombinant vector was transfected into CHL cells using a calcium phosphate method. Expressed UGT1A3 protein was prepared from CHL cells resistant to neomycin (G418). Then the protein was added into a reaction mixture for glucuronidation of quercetin. The glucuronidation activity of UGT1A3 was determined by reverse phase-high performance liquid chromatography (RP-HPLC) coupled with a diode array detector (DAD). The quercetin glucuronide was confirmed by hydrolysis with β-glucuronidase. Control experiments were performed in parallel. The transcriptions of recombinants were also determined by RT-PCR.RESULTS: The gene was confirmed to be an allele (UGT1A3-3) of UGT1A3 by DNA sequencing. The fragment was introduced into pcDNA3.1 (+) successfully. Several colonies were obtained under the selection pressure of G418.The result of RT-PCR showed transcription of recombinants in mRNA level. Glucuronidation assay and HPLC analysis indicated UGT1A3 expressed heterologously in CHL cells was in an active form, and one of the gulcuronides corresponding to quercetin was also detected.CONCLUSION: Correct sequence of UGT1A3 gene can be obtained, and active UGT1A3 enzyme is expressed heterologously in CHL cells.

  14. Assessment of Anabaena sp. Strain PCC 7120 as a Heterologous Expression Host for Cyanobacterial Natural Products: Production of Lyngbyatoxin A.

    Science.gov (United States)

    Videau, Patrick; Wells, Kaitlyn N; Singh, Arun J; Gerwick, William H; Philmus, Benjamin

    2016-09-16

    Cyanobacteria are well-known producers of natural products of highly varied structure and biological properties. However, the long doubling times, difficulty in establishing genetic methods for marine cyanobacteria, and low compound titers have hindered research into the biosynthesis of their secondary metabolites. While a few attempts to heterologously express cyanobacterial natural products have occurred, the results have been of varied success. Here, we report the first steps in developing the model freshwater cyanobacterium Anabaena sp. strain PCC 7120 (Anabaena 7120) as a general heterologous expression host for cyanobacterial secondary metabolites. We show that Anabaena 7120 can heterologously synthesize lyngbyatoxin A in yields comparable to those of the native producer, Moorea producens, and detail the design and use of replicative plasmids for compound production. We also demonstrate that Anabaena 7120 recognizes promoters from various biosynthetic gene clusters from both free-living and obligate symbiotic marine cyanobacteria. Through simple genetic manipulations, the titer of lyngbyatoxin A can be improved up to 13-fold. The development of Anabaena 7120 as a general heterologous expression host enables investigation of interesting cyanobacterial biosynthetic reactions and genetic engineering of their biosynthetic pathways.

  15. Isolation, characterization and evaluation of the Pichia pastoris sorbitol dehydrogenase promoter for expression of heterologous proteins.

    Science.gov (United States)

    Periyasamy, Sankar; Govindappa, Nagaraj; Sreenivas, Suma; Sastry, Kedarnath

    2013-11-01

    Sorbitol is used as a non-repressive carbon source to develop fermentation process for Mut(s) recombinant clones obtained using the AOX1 promoter in Pichia pastoris. Sorbitol dehydrogenase is an enzyme in the carbohydrate metabolism that catalyzes reduction of D-fructose into D-sorbitol in the presence of NADH. The small stretch of 211bps upstream region of sorbitol dehydrogenase coding gene has all the promoter elements like CAAT box, GC box, etc. It is able to promote protein production under repressive as well as non-repressive carbon sources. In this study, the strength of the sorbitol dehydrogenase promoter was evaluated by expression of two heterologous proteins: human serum albumin and erythrina trypsin inhibitor. Sorbitol dehydrogenase promoter allowed constitutive expression of recombinant proteins in all carbon sources that were tested to grow P. pastoris and showed activity similar to GAP promoter. The sorbitol dehydrogenase promoter was active in all the growth phases of the P. pastoris.

  16. [Effects of seven RNA silencing suppressors on heterologous expression of green fluorescence protein expression mediated by a plant virus-based system in Nicotiana benthamiana].

    Science.gov (United States)

    Wang, Sheng; Dong, Jie; Cao, Min; Mu, Hongzhen; Ding, Guoping; Zhang, Hong

    2012-11-01

    To test the effects of 7 virus-encoded RNA silencing suppressors (RSSs) for enhancement of a plant virus-based vector system-mediated heterologous expression of green fluorescence protein (GFP) in Nicotiana benthamiana. Seven transient expression vectors for the 7 RSSs were constructed and co-inoculated on the leaves of Nicotiana benthamiana with PVXdt-GFP vector, a novel Potato virus X-based plant expression vector, through agroinfiltration. The protein and mRNA expression levels of the reporter gene GFP in the co-inoculated Nicotiana leaves were examined by Western blotting, ELISA and RT-qPCR to assess the effect of the RSSs for GFP expression enhancement. The 7 RSSs differed in the degree and duration of enhancement of heterologous GFP expression, and the p19 protein of Tomato bushy stunt virus (TBSV) induced the highest expression of GFP. African cassava mosaic virus AC2 protein and Rice yellow mettle virus P1 protein produced no obvious enhancement GFP expression. Transient co-expression of RSSs suppresses host silencing response to allow high-level and long-term expression of heterologous genes in plant, but the optimal RSS has to be identified for each plant virus-based expression vector system.

  17. Functional expression of a heterologous nickel-dependent, ATP-independent urease in Saccharomyces cerevisiae.

    Science.gov (United States)

    Milne, N; Luttik, M A H; Cueto Rojas, H F; Wahl, A; van Maris, A J A; Pronk, J T; Daran, J M

    2015-07-01

    In microbial processes for production of proteins, biomass and nitrogen-containing commodity chemicals, ATP requirements for nitrogen assimilation affect product yields on the energy producing substrate. In Saccharomyces cerevisiae, a current host for heterologous protein production and potential platform for production of nitrogen-containing chemicals, uptake and assimilation of ammonium requires 1 ATP per incorporated NH3. Urea assimilation by this yeast is more energy efficient but still requires 0.5 ATP per NH3 produced. To decrease ATP costs for nitrogen assimilation, the S. cerevisiae gene encoding ATP-dependent urease (DUR1,2) was replaced by a Schizosaccharomyces pombe gene encoding ATP-independent urease (ure2), along with its accessory genes ureD, ureF and ureG. Since S. pombe ure2 is a Ni(2+)-dependent enzyme and Saccharomyces cerevisiae does not express native Ni(2+)-dependent enzymes, the S. pombe high-affinity nickel-transporter gene (nic1) was also expressed. Expression of the S. pombe genes into dur1,2Δ S. cerevisiae yielded an in vitro ATP-independent urease activity of 0.44±0.01 µmol min(-1) mg protein(-1) and restored growth on urea as sole nitrogen source. Functional expression of the Nic1 transporter was essential for growth on urea at low Ni(2+) concentrations. The maximum specific growth rates of the engineered strain on urea and ammonium were lower than those of a DUR1,2 reference strain. In glucose-limited chemostat cultures with urea as nitrogen source, the engineered strain exhibited an increased release of ammonia and reduced nitrogen content of the biomass. Our results indicate a new strategy for improving yeast-based production of nitrogen-containing chemicals and demonstrate that Ni(2+)-dependent enzymes can be functionally expressed in S. cerevisiae.

  18. Characterization of myotonic dystrophy kinase (DMK) in heterologous expression systems

    Energy Technology Data Exchange (ETDEWEB)

    Waring, J.D.; Haq, R.; Mahadevan, M.S. [Children`s Hospital of Eastern Ontario, Ottawa (Canada)] [and others

    1994-09-01

    Myotonic dystrophy is caused by expansion of a (CTG){sub n} repeat within the 3{prime} untranslated region of the DMK gene. This gene encodes a product with a predicted M.W. of {approximately}69 kDa which has homology to cAMP-regulated serine-threonine protein kinases. In addition, there is a domain with similarity to coiled-coil regions found in myofibrillar proteins and a predicted transmembrane domain found at the extreme C-terminus. As an approach to identifying the function of this gene, we have expressed various forms of DMK by both in vitro translation and in insect cells using a recombinant baculovirus system. These forms include one corresponding to a cDNA isoform which results in a C-terminal truncation, as well as constructs containing varying CTG repeat lengths in their transcripts. Affinity-purified immunoglobulin elicited to a GST fusion protein (including amino acids corresponding to exons 11 and 15 of DMK) specifically recognizes products close to the predicted size. The products have been analyzed for their levels of expression, post-translational modifications, subcellular localization, and kinase activity.

  19. Improved heterologous protein expression in the chloroplast of Chlamydomonas reinhardtii through promoter and 5' untranslated region optimization.

    Science.gov (United States)

    Rasala, Beth A; Muto, Machiko; Sullivan, Joseph; Mayfield, Stephen P

    2011-08-01

    Microalgae have the potential to be a valuable biotechnological platform for the production of recombinant proteins. However, because of the complex regulatory network that tightly controls chloroplast gene expression, heterologous protein accumulation in a wild-type, photosynthetic-competent algal chloroplast remains low. High levels of heterologous protein accumulation have been achieved using the psbA promoter/5' untranslated region (UTR), but only in a psbA-deficient genetic background, because of psbA/D1-dependent auto-attenuation. Here, we examine the effect of fusing the strong 16S rRNA promoter to the 5' UTR of the psbA and atpA genes on transgene expression in the chloroplast of Chlamydomonas reinhardtii. We show that fusion of the 16S promoter had little impact on protein accumulation from the psbA 5' UTR in a psbA-deficient genetic background. Furthermore, the 16S/psbA promoter/UTR fusion was silenced in the presence of wild-type levels of D1 protein, confirming that the psbA 5' UTR is the primary target for D1-dependent auto-repression. However, fusion of the 16S promoter to the atpA 5' UTR significantly boosts mRNA levels and supports high levels of heterologous protein accumulation in photosynthetic-competent cells. The 16S/atpA promoter/UTR drove LUXCT protein accumulation to levels close to that of psbA in a psbA- background, and drove expression of a human therapeutic protein to levels only twofold lower than the psbA 5' UTR. The 16S/atpA promoter/UTR combination should have utility for heterologous protein production when expression from a photosynthetic-competent microalgal strain is required.

  20. Hydrophobins in ectomycorrhizas: heterologous transcription of the Pisolithus HydPt-1 gene in yeast and Hebeloma cylindrosporum

    Directory of Open Access Journals (Sweden)

    D Tagu

    2009-12-01

    Full Text Available Hydrophobins are fungal cell wall proteins involved in aggregation of hyphae. Upon the development of the ectomycorrhizal symbiosis between tree roots and fungal hyphae, the transcripts of hydrophobin genes markedly accumulated. As the precise role of these proteins in symbiosis is not yet known, we develop heterologous expression system of the Pisolithus hydrophobin HYDPt-1. This gene has been introduced in Saccharomyces cerevisiae and in the ectomycorrhizal basidiomycete Hebeloma cylindrosporum. Introns were required for hydPt-1 transcript accumulation in the basidiomycete H. cylindrosporum. Heterologous transcript accumulation did not alter the phenotype of either species. The lack of altered phenotype resulted from the absence of HYDPt-1 polypeptide accumulation in transformed strains.

  1. Mapping of heterologous expressed sequence tags as an alternative to microarrays for study of defense responses in plants

    Directory of Open Access Journals (Sweden)

    Postnikova Olga A

    2009-06-01

    Full Text Available Abstract Background Microarray technology helped to accumulate an immense pool of data on gene expression changes in response to different environmental factors. Yet, computer- generated gene profiling using expressed sequence tags (EST represents a valuable alternative to microarrays, which allows efficient discovery of homologous sequences in evolutionarily different species and comparison of gene sets on the whole genome scale. In this study, we used publicly available EST database derived from different plant species infected with a variety of pathogens, to generate an expression profile of homologous genes involved in defense response of a model organism, Arabidopsis thaliana. Results EST-driven prediction identified 4,935 genes (16% of the total Arabidopsis genome which, according to the origin of EST sets, were associated with defense responses in the reference genome. Profiles of defense-related genes, obtained by mapping of heterologous EST, represent putative Arabidopsis homologs of the corresponding species. Comparison of these profiles in pairs and locating common genes allowed estimating similarity between defense-related gene sets of different plant species. To experimentally support computer data, we arbitrarily selected a number of transcription factor genes (TF detected by EST mapping. Their expression levels were examined by real-time polymerase chain reaction during infection with yellow strain of Cucumber mosaic virus, a compatible virus systemically infecting Arabidopsis. We observed that 65% of the designated TF were upregulated in accordance with the EST-generated profile. Conclusion We demonstrated that heterologous EST mapping may be efficiently used to reveal genes involved in host defense responses to pathogens. Upregulated genes identified in this study substantially overlap with those previously obtained by microarrays.

  2. Barley (Hordeum vulgare L.) cysteine proteases: heterologous expression, purification and characterization

    DEFF Research Database (Denmark)

    Rosenkilde, Anne Lind; Dionisio, Giuseppe; Holm, Preben Bach

    2011-01-01

    B2 (HvEPB2) was ligated into the Pichia pastoris expression vector pPICZ Aα and electrotransformed into Pichia pastoris strain KM71H. Heterologous protein production was induced with 2% MeOH and maximum yield were obtained after 4 days where the supernatant was harvested. Purification of HvEPB2 from...

  3. Improved cellulolytic efficacy in Penicilium decumbens via heterologous expression of Hypocrea jecorina endoglucanase II

    Directory of Open Access Journals (Sweden)

    Qin Yuqi

    2013-01-01

    Full Text Available Hypocrea jecorina endoglucanase II (Hjegl2 was heterologously expressed in Penicillium decumbens (yielding strain Pd::Hjegl2. After induction in cellulose containing media, strain Pd::Hjeg2 displayed increased carboxymethylcellulase activity (CMCase, 5.77 IU/ml, representing a 21% increase and cellulose degradation determined with a filter paper assay (FPA, 0.40 IU/ml, 67% increase, as compared to the parent strain. In media supplemented with glucose (2%, Pd::Hjegl2, displayed 51.2-fold and 3-fold higher CMCase and FPA activities, respectively, as compared to the parent strain. No changes in the expression levels of the four main native cellulase genes of P. decumbens (Pdegl1, Pdegl2, Pdcbh1, and Pdcbh2 were noted between the transformant and wild-type strains. These data support the idea that Hjegl2 cleaves both internal and terminal glycosidic residues, in a relatively random and processive manner. In situ polyacrylamide gelactivity staining of extracts derived from wild-type and Pd::Hjegl2 revealed two additional active fractions in the latter strain; one with a molecular mass ~50-65 KDa and another ~80-116 kDa.

  4. Heterologous expression of DsRed2 in young sponges (Porifera).

    Science.gov (United States)

    Pfannkuchen, Martin; Brümmer, Franz

    2009-01-01

    Sponges (Porifera) are currently considered to be the first branch off the Urmetazoa, common ancestors of all multicellular animals or metazoa. Research in the field of the developmental biology of sponges was restricted to morphological observations. Nowadays, research is mainly concentrated on larval development, primarily dealing with tissue formation. Already since 1907, methods for developing functional sponges from stem cells have been at hand. Functional freshwater sponges can be grown from stem cell populations originating from gemmulae. A number of poriferan sequences with high similarity to regulative genes in higher metazoa have already been found. We have now succeeded in heterologously expressing the red fluorescent protein DsRedN1 under the control of the cytomegalovirus promoter in young specimens of the freshwater sponge Spongilla lacustris. The protein folded correctly, polymerized and subsequently was detected by fluorescence microscopy. Reporting this expression system, we now consider this appealing system for early meatazoan development to be ready for molecular developmental biology and functional genetics research.

  5. Diversification of echinomycin molecular structure by way of chemoenzymatic synthesis and heterologous expression of the engineered echinomycin biosynthetic pathway.

    Science.gov (United States)

    Watanabe, Kenji; Oguri, Hiroki; Oikawa, Hideaki

    2009-04-01

    Echinomycin, a bis-intercalator antitumor cyclic peptide, is biosynthesized by a unique nonribosomal peptide synthetase (NRPS). Successful heterologous expression of the whole gene cluster of echinomycin in Escherichia coli let us to investigate a further application of echinomycin NRPS. To construct a cyclic peptide library, our approach through both chemoenzymatic and rational genetic engineering has been successfully demonstrated. These achievements provided the further support that E. coli-based system can serve as a flexible yet robust platform for producing complex natural products and their analogs.

  6. Heterologous expression of Gaeumannomyces graminis lipoxygenase in Aspergillus nidulans

    OpenAIRE

    Heshof, R.; Schayck, van, J.P.; Tamayo Ramos, J.A.; Graaff, de, R.

    2014-01-01

    Aspergillus sp. contain ppo genes coding for Ppo enzymes that produce oxylipins from polyunsaturated fatty acids. These oxylipins function as signal molecules in sporulation and influence the asexual to sexual ratio of Aspergillus sp. Fungi like Aspergillus nidulans and Aspergillus niger contain just ppo genes where the human pathogenic Aspergillus flavus and Aspergillus fumigatus contain ppo genes as well as lipoxygenases. Lipoxygenases catalyze the synthesis of oxylipins and are hypothesize...

  7. Broad-Host-Range Expression Reveals Native and Host Regulatory Elements That Influence Heterologous Antibiotic Production in Gram-Negative Bacteria.

    Science.gov (United States)

    Zhang, Jia Jia; Tang, Xiaoyu; Zhang, Michelle; Nguyen, Darlene; Moore, Bradley S

    2017-09-05

    Heterologous expression has become a powerful tool for studying microbial biosynthetic gene clusters (BGCs). Here, we extend the transformation-associated recombination cloning and heterologous expression platform for microbial BGCs to include Gram-negative proteobacterial expression hosts. Using a broad-host-range expression platform, we test the implicit assumption that biosynthetic pathways are more successfully expressed in more closely related heterologous hosts. Cloning and expression of the violacein BGC from Pseudoalteromonas luteoviolacea 2ta16 revealed robust production in two proteobacterial hosts, Pseudomonas putida KT2440 and Agrobacterium tumefaciens LBA4404, but very little production of the antibiotic in various laboratory strains of Escherichia coli, despite their closer phylogenetic relationship. We identified a nonclustered LuxR-type quorum-sensing receptor from P. luteoviolacea 2ta16, PviR, that increases pathway transcription and violacein production in E. coli by ∼60-fold independently of acyl-homoserine lactone autoinducers. Although E. coli harbors the most similar homolog of PviR identified from all of the hosts tested, overexpression of various E. coli transcription factors did not result in a statistically significant increase in violacein production, while overexpression of two A. tumefaciens PviR homologs significantly increased production. Thus, this work not only introduces a new genetic platform for the heterologous expression of microbial BGCs, it also challenges the assumption that host phylogeny is an accurate predictor of host compatibility.IMPORTANCE Although Gram-positive heterologous hosts such as Streptomyces have been developed and optimized to support diverse secondary metabolic reactions, there has been comparatively less work on Gram-negative hosts, some of which grow faster and are easier to work with. This work presents a new genetic platform for direct cloning and broad-host-range heterologous expression of BGCs

  8. Kinetic behaviour of recombinant Fusarium solani lipases using monomolecular films: Effect of the heterologous expression.

    Science.gov (United States)

    Jallouli, Raida; Bouali, Madiha; Gargouri, Youssef; Bezzine, Sofiane

    2017-01-01

    Two lipases from Fusarium solani, FSL and FSL2, were efficiently expressed in Pichia pastoris. To check the influence of the expression on interfacial properties of FSL and to study kinetic properties of FSL2, interfacial parameters of FSL2, native FSL, untagged recombinant and tagged recombinant forms of FSL were compared using the monomolecular film technique. Kinetic study on the dependence of the stereoselectivity of these lipases on the surface pressure was performed using three dicaprin isomers spread in the form of monomolecular films at the air-water interface. The FSL2 seems to have an important penetration power with a preference for adjacent ester groups and the heterologous expression accompanied or not with the N-His-tag extension on the FSL were found to modify the pressure preference and increase the catalytic hydrolysis rate of three dicaprin isomers. The heterologous expression was found to preserve the FSL regioselectivity without affecting its stereospecificity at high and low surface pressure. The evaluation of the recombinant expression Effects on Catalysis (REC), the N-Tag Effects on Catalysis (TEC), and the N-Tag and Recombinant expression Effects on Catalysis (TREC) showed that the heterologous expression was more efficient than the presence of the N-terminal tag extension on the FSL. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Recombinant measles AIK-C vaccine strain expressing heterologous virus antigens.

    Science.gov (United States)

    Nakayama, Tetsuo; Sawada, Akihito; Yamaji, Yoshiaki; Ito, Takashi

    2016-01-04

    Further attenuated measles vaccines were developed more than 50 years ago and have been used throughout the world. Recombinant measles vaccine candidates have been developed and express several heterologous virus protective antigens. Immunogenicity and protective actions were confirmed using experimental animals: transgenic mice, cotton rats, and primates. The recent development of measles vaccine-based vectored vaccine candidates has been reviewed and some information on recombinant measles vaccines expressing respiratory syncytial virus proteins has been shown and discussed.

  10. Phaseolin expression in tobacco chloroplast reveals an autoregulatory mechanism in heterologous protein translation.

    Science.gov (United States)

    De Marchis, Francesca; Bellucci, Michele; Pompa, Andrea

    2016-02-01

    Plastid DNA engineering is a well-established research area of plant biotechnology, and plastid transgenes often give high expression levels. However, it is still almost impossible to predict the accumulation rate of heterologous protein in transplastomic plants, and there are many cases of unsuccessful transgene expression. Chloroplasts regulate their proteome at the post-transcriptional level, mainly through translation control. One of the mechanisms to modulate the translation has been described in plant chloroplasts for the chloroplast-encoded subunits of multiprotein complexes, and the autoregulation of the translation initiation of these subunits depends on the availability of their assembly partners [control by epistasy of synthesis (CES)]. In Chlamydomonas reinhardtii, autoregulation of endogenous proteins recruited in the assembly of functional complexes has also been reported. In this study, we revealed a self-regulation mechanism triggered by the accumulation of a soluble recombinant protein, phaseolin, in the stroma of chloroplast-transformed tobacco plants. Immunoblotting experiments showed that phaseolin could avoid this self-regulation mechanism when targeted to the thylakoids in transplastomic plants. To inhibit the thylakoid-targeted phaseolin translation as well, this protein was expressed in the presence of a nuclear version of the phaseolin gene with a transit peptide. Pulse-chase and polysome analysis revealed that phaseolin mRNA translation on plastid ribosomes was repressed due to the accumulation in the stroma of the same soluble polypeptide imported from the cytosol. We suggest that translation autoregulation in chloroplast is not limited to heteromeric protein subunits but also involves at least some of the foreign soluble recombinant proteins, leading to the inhibition of plastome-encoded transgene expression in chloroplast.

  11. Molecular cloning, heterologous expression, and characterization of Ornithine decarboxylase from Oenococcus oeni.

    Science.gov (United States)

    Bonnin-Jusserand, Maryse; Grandvalet, Cosette; David, Vanessa; Alexandre, Hervé

    2011-08-01

    Ornithine decarboxylase (ODC) is responsible for the production of putrescine, the major biogenic amine found in wine. Oenococcus oeni is the most important lactic acid bacterium in the winemaking process and is involved in malolactic fermentation. We report here the characterization of ODC from an O. oeni strain isolated from wine. Screening of 263 strains isolated from wine and cider from all over the world revealed that the presence of the odc gene appears to be strain specific in O. oeni. After cloning, heterologous expression in Escherichia coli, and characterization, the enzyme was found to have a molecular mass of 85 kDa and a pI of 6.2 and revealed maximal activity at pH 5.5 and an optimum temperature of 35°C. Kinetic studies showed that O. oeni ODC is specific for L-ornithine with a K(m) value of 1 mM and a V(max) of 0.57 U·mg(-1). The hypothesis that cadaverine, which results from lysine decarboxylation, may be linked to putrescine production is not valid since O. oeni ODC cannot decarboxylate L-lysine. As no lysine decarboxylase was detected in any of the O. oeni genomes sequenced, cadaverine synthesis may result from another metabolic pathway. This work is the first characterization of an ODC from a lactic acid bacterium isolated from a fermented product.

  12. Modulating effect of ginseng saponins on heterologously expressed HERG currents in Xenopus oocytes

    Institute of Scientific and Technical Information of China (English)

    Cuk-seong KIM; Sook-jin SON; Hyo-shin KIM; Yong-duk KIM; Kyu-seung LEE; Byeong-hwa JEON; Kwang-jin KIM; Jin-kyu PARK; Jin-bong PARK

    2005-01-01

    Aim: To examine the effects of ginseng saponins on the heterologously expressed human ether-a-go-go related gene (HERG) that encodes the rapid component of the delayed rectifier K+ channel. Methods: A two-electrode voltage clamp tech nique was used. HERG currents were recorded in Xenopus oocytes injected with HERG cRNA. Results: Crude saponins of Korean red ginseng (GS) induced a minimal increase of the maximal HERG conductance without changes in the voltage-dependent HERG current activation and inactivation curves. GS, however,decelerated HERG current deactivation in a concentration-dependent manner,which was more noticeable with panaxitriol (PT) than panaxidiol (PD). Consistently,ginseng saponins increased the HERG deactivation time constants with the order of potency of Rg1 (a major component of PT)>Rf 1>Rb1 (a major component of PD).Re had little effect on HERG deactivation. During a cardiac action Potential, GS increased the outward HERG current. Conclusion: Ginseng saponins enhance HERG currents, which could be in part a possible mechanism of the shortening cardiac action potential of ginseng saponins.

  13. [Heterologous expression, purification and characterization of exo-inulinase from Kluyveromyces marxianus YX01].

    Science.gov (United States)

    Li, Yimin; Gao, Jiaoqi; Yuan, Wenjie; Xiang, Ruijuan; Hou, Shengbo

    2015-05-01

    To improve the inulinase application in biotechnology, the characteristic of inulinase from Kluyveromyces marxianus YX01 was investigated. The inu gene of K. marxianus YX01 was transformed into Pichiapastoris GS115 host cells with molecular biology techniques. Then we achieved the heterologous expression of exo-inulinase whose molecular mass was about 86.0 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Furthermore, six His-tag was added to the inulinase and a two-step method was applied in the purification of inulinase, including concentration via dialysis by polyethylene glycol 20 000 and metal Ni-NTA Agarose affinity adsorption. The purification factor of purified protein was 3.6 and the recovery rate of enzyme activity was 33.1%. We characterized the purified inulinase. The optimum temperature was 60 degrees C and pH was 4.62. When inulin and sucrose were used as substrates, the K(m) and V(max) values were 80.53 g/L vs 4.49 g/(L x min) and 183.10 g/L vs 20.20 g/(L x min), respectively. In addition, metal ions including Mn2+, Ca2+, Cu2+, Zn2+ and Fe2+ exhibited different degrees of inhibition on the enzyme activity, and Cu2+, Zn2+ and Fe2+ exhibited the most significant inhibition. Our findings might lay a good foundation for industrial application of inulinase.

  14. Heterologous expression of Gaeumannomyces graminis lipoxygenase in Aspergillus nidulans

    NARCIS (Netherlands)

    Heshof, R.; Schayck, van J.P.; Tamayo Ramos, J.A.; Graaff, de L.H.

    2014-01-01

    Aspergillus sp. contain ppo genes coding for Ppo enzymes that produce oxylipins from polyunsaturated fatty acids. These oxylipins function as signal molecules in sporulation and influence the asexual to sexual ratio of Aspergillus sp. Fungi like Aspergillus nidulans and Aspergillus niger contain jus

  15. Adaption of Saccharomyces cerevisiae expressing a heterologous protein

    DEFF Research Database (Denmark)

    Krogh, Astrid Mørkeberg; Beck, Vibe; Højlund Christensen, Lars

    2008-01-01

    specific growth rate of the strains was reduced by 54% and 33%, respectively, upon the introduction of the gene encoding aprotinin. Growing the strains in sequential shake flask cultivations for 250 generations led to an increased maximal specific growth rate and a decrease in the yield of aprotinin...

  16. Heterologous expression and enzymatic characterization of γ-glutamyltranspeptidase from Bacillus amyloliquefaciens.

    Science.gov (United States)

    Lee, Jung-Min; Lee, Jaejung; Nam, Gyeong-Hwa; Son, Byung-Sam; Jang, Myoung-Uoon; Lee, So-Won; Hurh, Byung-Serk; Kim, Tae-Jip

    2017-02-01

    γ-Glutamyltranspeptidase (GGT) catalyzes the cleavage of γ-glutamyl compounds and the transfer of γ-glutamyl moiety to water or to amino acid/peptide acceptors. GGT can be utilized for the generation of γ-glutamyl peptides or glutamic acid, which are used as food taste enhancers. In the present study, Bacillus amyloliquefaciens SMB469 with high GGT activity was isolated from Doenjang, a traditional fermented soy food of Korea. The gene encoding GGT from B. amyloliquefaciens SMB469 (BaGGT469) was cloned from the isolate, and heterologously expressed in E. coli and B. subtilis. For comparison, three additional GGT genes were cloned from B. subtilis 168, B. licheniformis DSM 13, and B. amyloliquefaciens FZB42. The BaGGT469 protein was composed of 591 amino acids. The final protein comprises two separate polypeptide chains of 45.7 and 19.7 kDa, generated via autocatalytic cleavage. The specific activity of BaGGT469 was determined to be 17.8 U/mg with γ-L-glutamyl-p-nitroanilide as the substrate and diglycine as the acceptor. GGTs from B. amyloliquefaciens showed 1.4- and 1.7-fold higher transpeptidase activities than those from B. subtilis and B. licheniformis, respectively. Especially, recombinant B. subtilis expressing BaGGT469 demonstrated 11- and 23-fold higher GGT activity than recombinant E. coli and the native B. amyloliquefaciens, respectively, did. These results suggest that BaGGT469 can be utilized for the enzymatic production of various γ-glutamyl compounds.

  17. Expression of the endogenous and heterologous clavulanic acid cluster in Streptomyces flavogriseus: why a silent cluster is sleeping.

    Science.gov (United States)

    Alvarez-Álvarez, R; Martínez-Burgo, Y; Pérez-Redondo, R; Braña, A F; Martín, J F; Liras, P

    2013-11-01

    Clusters for clavulanic acid (CA) biosynthesis are present in the actinomycetes Streptomyces flavogriseus ATCC 33331 and Saccharomonospora viridis DSM 43017. These clusters, which are silent, contain blocks of conserved genes in the same order as those of the Streptomyces clavuligerus CA cluster but assembled in a different organization. S. flavogriseus was grown in nine different media, but clavulanic acid production was undetectable using bioassays or by high-performance liquid chromatography analyses. Reverse-transcriptase polymerase chain reaction (RT-PCR) of S. flavogriseus CA biosynthesis genes showed that the regulatory genes ccaR and claR and some biosynthetic genes were expressed whereas expression of cyp, orf12, orf13, and oppA2 was undetectable. The ccaR gene of S. clavuligerus was unable to switch on CA production in S. flavogriseus::[Pfur-ccaR C], but insertion of a cosmid carrying the S. clavuligerus CA cluster (not including the ccaR gene) conferred clavulanic acid production on S. flavogriseus::[SCos-CA] particularly in TBO and YEME media; these results suggests that some of the S. flavogriseus CA genes are inactive. The known heptameric sequences recognized by CcaR in S. clavuligerus are poorly or not conserved in S. flavogriseus. Quantitative RT-PCR analysis of the CA gene clusters of S. clavuligerus and S. flavogriseus showed that the average expression value of the expressed genes in the former strain was in the order of 1.68-fold higher than in the later. The absence of CA production by S. flavogriseus can be traced to the lack of expression of the essential genes cyp, orf12, orf13, orf14, and oppA2. Heterologous expression of S. clavuligerus CA gene cluster in S. flavogriseus::[SCos-CA] was 11- to 14-fold lower than in the parental strain, suggesting that the genetic background of the host strain is important for optimal production of CA in Streptomyces.

  18. Homologous and heterologous expression of grapevine E-(β)-caryophyllene synthase (VvGwECar2).

    Science.gov (United States)

    Salvagnin, Umberto; Carlin, Silvia; Angeli, Sergio; Vrhovsek, Urska; Anfora, Gianfranco; Malnoy, Mickael; Martens, Stefan

    2016-11-01

    E-(β)-caryophyllene is a sesquiterpene volatile emitted by plants and involved in many ecological interactions within and among trophic levels and it has a kairomonal activity for many insect species. In grapevine it is a key compound for host-plant recognition by the European grapevine moth, Lobesia botrana, together with other two sesquiterpenes. In grapevine E-(β)-caryophyllene synthase is coded by the VvGwECar2 gene, although complete characterization of the corresponding protein has not yet been achieved. Here we performed the characterization of the enzyme after heterologous expression in E. coli, which resulted to produce in vitro also minor amounts of the isomer α-humulene and of germacrene D. The pH optimum was estimated to be 7.8, and the Km and Kcat values for farnesyl pyrophosphate were 31.4 μM and 0.19 s(-1) respectively. Then, we overexpressed the gene in the cytoplasm of two plant species, Arabidopsis thaliana and the native host Vitis vinifera. In Arabidopsis the enzyme changed the plant head space release, showing a higher selectivity for E-(β)-caryophyllene, but also the production of thujopsene instead of germacrene D. Overall plants increased the E-(β)-caryophyllene emission in the headspace collection by 8-fold compared to Col-0 control plants. In grapevine VvGwECar2 overexpression resulted in higher E-(β)-caryophyllene emissions, although there was no clear correlation between gene activity and sesquiterpene quantity, suggesting a key role by the plant regulation machinery.

  19. A Western Blot Protocol for Detection of Proteins Heterologously Expressed in Xenopus laevis Oocytes.

    Science.gov (United States)

    Jørgensen, Morten Egevang; Nour-Eldin, Hussam Hassan; Halkier, Barbara Ann

    2016-01-01

    Oocytes of the African clawed frog, Xenopus laevis, are often used for expression and biochemical characterization of transporter proteins as the oocytes are particularly suitable for uptake assays and electrophysiological recordings. Assessment of the expression level of expressed transporters at the individual oocyte level is often desirable when comparing properties of wild type and mutant transporters. However, a large content of yolk platelets in the oocyte cytoplasm makes this a challenging task. Here we report a method for fast and easy, semiquantitative Western blot analysis of proteins heterologously expressed in Xenopus oocytes.

  20. Functional expression and evaluation of heterologous phosphoketolases in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Bergman, Alexandra; Siewers, Verena; Nielsen, Jens;

    2016-01-01

    Phosphoketolases catalyze an energy-and redox-independent cleavage of certain sugar phosphates. Hereby, the two-carbon (C2) compound acetyl-phosphate is formed, which enzymatically can be converted into acetyl-CoA-a key precursor in central carbon metabolism. Saccharomyces cerevisiae does...... C5 and C6 sugars towards C2-synthesis. Nine phosphoketolase candidates were expressed in S. cerevisiae of which seven produced significant amounts of acetyl-phosphate after provision of sugar phosphate substrates in vitro. The candidates showed differing substrate specificities, and some...... demonstrated activity levels significantly exceeding those of candidates previously expressed in yeast. The conducted studies also revealed that S. cerevisiae contains endogenous enzymes capable of breaking down acetyl-phosphate, likely into acetate, and that removal of the phosphatases Gpp1 and Gpp2 could...

  1. GmSARK启动子驱动IPT基因在拟南芥中的表达研究%Heterologous Expression of Isopentenyl Transferase(Ipt) Gene Directed by Senescence Associated Receptor Protein Kinase(SARK) Promoter in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    霍巍巍; 闫璇玲; 孙晓文; 李海燕

    2012-01-01

    [Objective] This study aimed to investigate the expression of IPT gene directed by GmSARK promoter in Arabidopsis. [ Method] IPT gene and GmSARK promoter were respectively cloned to construct plant expression vector and transform Arabidopsis. T, transgenic plant lines were detected by PPT(phosphinothricin) herbicide selection and treated under drought and dark conditions for semi-quantitive RT-PCR analysis. [ Result] GmSARK and IPT fusion gene was successfully cloned and the plant expression vector p3301 -GmSAHK-IPT was constructed. RT-PCR analysis showed that the target gene was expressed in T1 transgenic plant lines at the mRNA level. [ Conclusion] This study laid the foundation for further investigating the roles of IPT gene directed by GmSARK promoter in plant stress resistance.%[目的]对GmSARK启动子驱动IPT基因在拟南芥中的表达进行研究.[方法]分别克隆IPT基因及GmSARK基因启动子,构建它们的植物表达栽体并进行拟南芥的转化,利用PPT(phosphinothricin)除草剂筛选,检测T1代转基因植株;对T1代转基因植株进行黑暗避光和干旱处理后进行半定量RT-PCR分析.[结果]成功克隆得到了IPT基因及GmSARK基因,并构建了它们的p3301 -GmSARK-IPT植物表达载体;对T代转基因植株的RT-PCR表明,目的基因mRNA水平上有所表达.[结论]为进一步研究GmSARK启动子驱动的IPT基因在抗逆中的作用奠定了基础.

  2. Heterologous expression and purification of wheat storage proteins in the yeast Saccharomyces cerevisiae

    OpenAIRE

    2007-01-01

    Im Rahmen des Teilprojektes “Expression und Produktion von Weizenspeicherproteinen in der Hefe Saccharomyces cerevisiae“ des BMBF-Leitprojektes „Entwicklung von Weizen-, Roggen- und Gerstenproteinen ohne Zöliakietoxizität und deren Verwendung zur Herstellung von Lebensmitteln“ (Förderkennzeichen 0312246C) sollten die Ausbeute der heterolog in S. cerevisiae exprimierten Weizenproteine gesteigert werden, um sie für Zöliakietoxizitätsteste einzusetzen. Durch Optimierungsstrategien des Substrates...

  3. Heterologous expression and purification of membrane-bound pyrophosphatases

    DEFF Research Database (Denmark)

    Kellosalo, J.; Kajander, T.; Palmgren, Michael Broberg

    2011-01-01

    Membrane-bound pyrophosphatases (M-PPases) are enzymes that couple the hydrolysis of inorganic pyrophosphate to pumping of protons or sodium ions. In plants and bacteria they are important for relieving stress caused by low energy levels during anoxia, drought, nutrient deficiency, cold and low...... to obtain structural information for drug design. We have tested the expression of eight integral membrane pyrophosphatases in Saccharomyces cerevisiae, six from bacterial and archaeal sources and two from protozoa. Two proteins originating from hyperthermophilic organisms were purified in dimeric...

  4. Heterologous Expression of Three Plant Serpins with Distinct Inhibitory Specificities

    DEFF Research Database (Denmark)

    Dahl, Søren Weis; Rasmussen, Søren Kjærsgård; Hejgaard, Jørn

    1996-01-01

    For the first time, inhibitory plant serpins, including WSZ1 from wheat, BSZ4, and the previously unknown protein BSZx from barley, have been expressed in Escherichia coli, and a procedure for fast purification of native plant serpins has been developed, BSZx, BSZ4, and WSZ1 were assayed for inhi...... favorable P-2 Leu. BSZ4 inhibited cathepsin G (k(a) = 2.7 x 10(4) M(-1) s(-1)) at P-1 Met but was hydrolyzed by trypsin and chymotrypsin. The three plant serpins formed stable SDS-resistant complexes with the proteinases in accordance with the kinetic data.......For the first time, inhibitory plant serpins, including WSZ1 from wheat, BSZ4, and the previously unknown protein BSZx from barley, have been expressed in Escherichia coli, and a procedure for fast purification of native plant serpins has been developed, BSZx, BSZ4, and WSZ1 were assayed......, the apparent rate constant for chymotrypsin inhibition at P-2 (k(a) = 9.4 x 10(5) M(-1) s(-1)) was only four times lower than for trypsin at P-1 (k(a) = 3.9 X 10(6) M(-1) s(-1)), and the apparent inhibition stoichiometries were close to 1. Furthermore, our data suggest that cathepsin G was inhibited by BSZx (k...

  5. Heterologous Expression of the Carrot Hsp17.7 gene Increased Growth, Cell Viability, and Protein Solubility in Transformed Yeast (Saccharomyces cerevisiae) under Heat, Cold, Acid, and Osmotic Stress Conditions.

    Science.gov (United States)

    Ko, Eunhye; Kim, Minhye; Park, Yunho; Ahn, Yeh-Jin

    2017-08-01

    In industrial fermentation of yeast (Saccharomyces cerevisiae), culture conditions are often modified from the optimal growth conditions of the cells to maintain large-scale cultures and/or to increase recombinant protein production. However, altered growth conditions can be stressful to yeast cells resulting in reduced cell growth and viability. In this study, a small heat shock protein gene from carrot (Daucus carota L.), Hsp17.7, was inserted into the yeast genome via homologous recombination to increase tolerance to stress conditions that can occur during industrial culture. A DNA construct, Translational elongation factor gene promoter-carrot Hsp17.7 gene-Phosphoribosyl-anthranilate isomerase gene (an auxotrophic marker), was generated by a series of PCRs and introduced into the chromosome IV of the yeast genome. Immunoblot analysis showed that carrot Hsp17.7 accumulated in the transformed yeast cell lines. Growth rates and cell viability of these cell lines were higher than control cell lines under heat, cold, acid, and hyperosmotic stress conditions. Soluble protein levels were higher in the transgenic cell lines than control cell lines under heat and cold conditions, suggesting the molecular chaperone function of the recombinant Hsp17.7. This study showed that a recombinant DNA construct containing a HSP gene from carrot was successfully expressed in yeast by homologous recombination and increased tolerances to abiotic stress conditions.

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

    Science.gov (United States)

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

    2016-05-01

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

  7. Xylose reductase from the thermophilic fungus Talaromyces emersonii: cloning and heterologous expression of the native gene (Texr) and a double mutant (TexrK271R+N273D) with altered coenzyme specificity

    Indian Academy of Sciences (India)

    Sara Fernandes; Maria G Tuohy; Patrick G Murray

    2009-12-01

    Xylose reductase is involved in the first step of the fungal pentose catabolic pathway. The gene encoding xylose reductase (Texr) was isolated from the thermophilic fungus Talaromyces emersonii, expressed in Escherichia coli and purified to homogeneity. Texr encodes a 320 amino acid protein with a molecular weight of 36 kDa, which exhibited high sequence identity with other xylose reductase sequences and was shown to be a member of the aldoketoreductase (AKR) superfamily with a preference for reduced nicotinamide adenine dinucleotide phosphate (NADPH) as coenzyme. Given the potential application of xylose reductase enzymes that preferentially utilize the reduced form of nicotinamide adenine dinucleotide (NADH) rather than NADPH in the fermentation of five carbon sugars by genetically engineered microorganisms, the coenzyme selectivity of TeXR was altered by site-directed mutagenesis. The TeXRK271R+N273D double mutant displayed an altered coenzyme preference with a 16-fold improvement in NADH utilization relative to the wild type and therefore has the potential to reduce redox imbalance of xylose fermentation in recombinant S. cerevisiae strains. Expression of Texr was shown to be inducible by the same carbon sources responsible for the induction of genes encoding enzymes relevant to lignocellulose hydrolysis, suggesting a coordinated expression of intracellular and extracellular enzymes relevant to hydrolysis and metabolism of pentose sugars in T. emersonii in adaptation to its natural habitat. This indicates a potential advantage in survival and response to a nutrient-poor environment.

  8. Heterologous expression of a membrane-spanning auxin importer: implications for functional analyses of auxin transporters.

    Science.gov (United States)

    Carrier, David John; Abu Bakar, Norliza Tendot; Lawler, Karen; Dorrian, James Matthew; Haider, Ameena; Bennett, Malcolm John; Kerr, Ian Derek

    2009-01-01

    Biochemical studies of plant auxin transporters in vivo are made difficult by the presence of multiple auxin transporters and auxin-interacting proteins. Furthermore, the expression level of most such transporters in plants is likely to be too low for purification and downstream functional analysis. Heterologous expression systems should address both of these issues. We have examined a number of such systems for their efficiency in expressing AUX1 from Arabidopsis thaliana. We find that a eukaryotic system based upon infection of insect cells with recombinant baculovirus provides a high level, easily scalable expression system capable of delivering a functional assay for AUX1. Furthermore, a transient transfection system in mammalian cells enables localization of AUX1 and AUX1-mediated transport of auxin to be investigated. In contrast, we were unable to utilise P. pastoris or L. lactis expression systems to reliably express AUX1.

  9. Streptococcus thermophilus, an emerging and promising tool for heterologous expression: Advantages and future trends.

    Science.gov (United States)

    Lecomte, Xavier; Gagnaire, Valérie; Lortal, Sylvie; Dary, Annie; Genay, Magali

    2016-02-01

    Streptococcus thermophilus is the second most used bacterium in dairy industry. It is daily consumed by millions of people through the worldwide consumption of yogurts, cheeses and fermented milks. S. thermophilus presents many features that make it a good candidate for the production of heterologous proteins. First, its ability to be naturally transformable allows obtaining swiftly and easily recombinant strains using various genetic tools available. Second, its Generally Recognised As Safe status and its ability to produce beneficial molecules or to liberate bioactive peptides from milk proteins open up the way for the development of new functional foods to maintain health and well-being of consumers. Finally, its ability to survive the intestinal passage and to be metabolically active in gastrointestinal tract allows considering S. thermophilus as a potential tool for delivering various biological molecules to the gastrointestinal tract. The aim of this review is therefore to take stock of various genetic tools which can be employed in S. thermophilus to produce heterologous proteins and to highlight the advantages and future trends of use of this bacterium as a heterologous expression host.

  10. The elicitor-inducible alfalfa isoflavone reductase promoter confers different patterns of developmental expression in homologous and heterologous transgenic plants.

    Science.gov (United States)

    Oommen, A; Dixon, R A; Paiva, N L

    1994-01-01

    In legumes, the synthesis of infection- and elicitor-inducible antimicrobial phytoalexins occurs via the isoflavonoid branch of the phenylpropanoid pathway. To study transcriptional regulation of isoflavonoid pathway-specific genes, we have isolated the gene encoding isoflavone reductase (IFR), which is the enzyme that catalyzes the penultimate step in the synthesis of the phytoalexin medicarpin in alfalfa. Chimeric gene fusions were constructed between 765- and 436-bp promoter fragments of the IFR gene and the beta-glucuronidase reporter gene and transferred to alfalfa and tobacco by Agrobacterium-mediated transformation. Both promoter fragments conferred elicitor-mediated expression in cell suspension cultures derived from transgenic plants of both species and fungal infection-mediated expression in leaves of transgenic alfalfa. Developmental expression directed by both promoter fragments in transgenic alfalfa was observed only in the root meristem, cortex, and nodules, which is consistent with the accumulation of endogenous IFR transcripts. However, in transgenic tobacco, expression from the 765-bp promoter was observed in vegetative tissues (root meristem and cortex, inner vascular tissue of stems and petioles, leaf tips, and stem peripheries adjacent to petioles) and in reproductive tissues (stigma, placenta, base of the ovary, receptacle, seed, tapetal layer, and pollen grains), whereas the 436-bp promoter was expressed only in fruits, seed, and pollen. These data indicate that infection/elicitor inducibility of the IFR promoter in both species and developmental expression in alfalfa are determined by sequences downstream of position -436, whereas sequences between -436 and -765 confer a complex pattern of strong ectopic developmental expression in the heterologous species that lacks the isoflavonoid pathway. PMID:7866024

  11. Four disulfide-bridged scorpion beta neurotoxin CssII: heterologous expression and proper folding in vitro.

    Science.gov (United States)

    Estrada, Georgina; Garcia, Blanca I; Schiavon, Emanuele; Ortiz, Ernesto; Cestele, Sandrine; Wanke, Enzo; Possani, Lourival D; Corzo, Gerardo

    2007-08-01

    The gene of the four disulfide-bridged Centruroides suffusus suffusus toxin II was cloned into the expression vector pQE30 containing a 6His-tag and a FXa proteolytic cleavage region. This recombinant vector was transfected into Escherichia coli BL21 cells and expressed under induction with isopropyl thiogalactoside (IPTG). The level of expression was 24.6 mg/l of culture medium, and the His tagged recombinant toxin (HisrCssII) was found exclusively in inclusion bodies. After solubilization the HisrCssII peptide was purified by affinity and hydrophobic interaction chromatography. The reverse-phase HPLC profile of the HisrCssII product obtained from the affinity chromatography step showed several peptide fractions having the same molecular mass of 9392.6 Da, indicating that HisrCssII was oxidized forming several distinct disulfide bridge arrangements. The multiple forms of HisrCssII after reduction eluted from the column as a single protein component of 9400.6 Da. Similarly, an in vitro folding of the reduced HisrCssII generated a single oxidized component of HisrCssII, which was cleaved by the proteolytic enzyme FXa to the recombinant CssII (rCssII). The molecular mass of rCssII was 7538.6 Da as expected. Since native CssII (nCssII) is amidated at the C-terminal residue whereas the rCssII is heterologously expressed in the format of free carboxyl end, there is a difference of 1 Da, when comparing both peptides (native versus heterologously expressed). Nevertheless, they show similar toxicity when injected intracranially into mice, and both nCssII and rCssII show the typical electrophysiological properties of beta-toxins in Na(v)1.6 channels, which is for the first time demonstrated here. Binding and displacement experiments conducted with radiolabelled CssII confirms the electrophysiological results. Several problems associated with the heterologously expressed toxins containing four disulfide bridges are discussed.

  12. Heterologous expression of the BABY BOOM AP2/ERF transcription factor enhances the regeneration capacity of tobacco (Nicotiana tabacum L.).

    Science.gov (United States)

    Srinivasan, Chinnathambi; Liu, Zongrang; Heidmann, Iris; Supena, Ence Darmo Jaya; Fukuoka, Hiro; Joosen, Ronny; Lambalk, Joep; Angenent, Gerco; Scorza, Ralph; Custers, Jan B M; Boutilier, Kim

    2007-01-01

    Gain-of-function studies have shown that ectopic expression of the BABY BOOM (BBM) AP2/ERF domain transcription factor is sufficient to induce spontaneous somatic embryogenesis in Arabidopsis (Arabidopsis thaliana (L.) Heynh) and Brassica napus (B. napus L.) seedlings. Here we examined the effect of ectopic BBM expression on the development and regenerative capacity of tobacco (Nicotiana tabacum L.) through heterologous expression of Arabidopsis and B. napus BBM genes. 35S::BBM tobacco lines exhibited a number of the phenotypes previously observed in 35S::BBM Arabidopsis and B. napus transgenics, including callus formation, leaf rumpling, and sterility, but they did not undergo spontaneous somatic embryogenesis. 35S::BBM plants with severe ectopic expression phenotypes could not be assessed for enhanced regeneration at the seedling stage due to complete male and female sterility of the primary transformants, therefore fertile BBM ectopic expression lines with strong misexpression phenotypes were generated by expressing a steroid-inducible, post-translationally controlled BBM fusion protein (BBM:GR) under the control of a 35S promoter. These lines exhibited spontaneous shoot and root formation, while somatic embryogenesis could be induced from in-vitro germinated seedling hypocotyls cultured on media supplemented with cytokinin. Together these results suggest that ectopic BBM expression in transgenic tobacco also activates cell proliferation pathways, but differences exist between Arabidopsis/B. napus and N. tabacum with respect to their competence to respond to the BBM signalling molecule.

  13. An Approach to Heterologous Expression of Membrane Proteins. The Case of Bacteriorhodopsin.

    Science.gov (United States)

    Bratanov, Dmitry; Balandin, Taras; Round, Ekaterina; Shevchenko, Vitaly; Gushchin, Ivan; Polovinkin, Vitaly; Borshchevskiy, Valentin; Gordeliy, Valentin

    2015-01-01

    Heterologous overexpression of functional membrane proteins is a major bottleneck of structural biology. Bacteriorhodopsin from Halobium salinarum (bR) is a striking example of the difficulties in membrane protein overexpression. We suggest a general approach with a finite number of steps which allows one to localize the underlying problem of poor expression of a membrane protein using bR as an example. Our approach is based on constructing chimeric proteins comprising parts of a protein of interest and complementary parts of a homologous protein demonstrating advantageous expression. This complementary protein approach allowed us to increase bR expression by two orders of magnitude through the introduction of two silent mutations into bR coding DNA. For the first time the high quality crystals of bR expressed in E. Coli were obtained using the produced protein. The crystals obtained with in meso nanovolume crystallization diffracted to 1.67 Å.

  14. An Approach to Heterologous Expression of Membrane Proteins. The Case of Bacteriorhodopsin.

    Directory of Open Access Journals (Sweden)

    Dmitry Bratanov

    Full Text Available Heterologous overexpression of functional membrane proteins is a major bottleneck of structural biology. Bacteriorhodopsin from Halobium salinarum (bR is a striking example of the difficulties in membrane protein overexpression. We suggest a general approach with a finite number of steps which allows one to localize the underlying problem of poor expression of a membrane protein using bR as an example. Our approach is based on constructing chimeric proteins comprising parts of a protein of interest and complementary parts of a homologous protein demonstrating advantageous expression. This complementary protein approach allowed us to increase bR expression by two orders of magnitude through the introduction of two silent mutations into bR coding DNA. For the first time the high quality crystals of bR expressed in E. Coli were obtained using the produced protein. The crystals obtained with in meso nanovolume crystallization diffracted to 1.67 Å.

  15. Heterologous Expression and Purification of S-layer Protein Genes of Bacillus thuringiensis%苏云金芽胞杆菌S-层蛋白基因的异源表达及分离纯化

    Institute of Scientific and Technical Information of China (English)

    杨晓琳; 郭刚; 张凤娟; 孙士锋; 朱朝华; 曾会才

    2013-01-01

      S-层(S-layer)普遍存在于古菌、G+、G-菌中,由S-层蛋白所构成细菌S-层结构的生物体中,其功能引起了科学家的广泛关注,但目前S-层的功能大都处于推测阶段.究其原因,是因为外源S-层基因可以造成宿主大肠杆菌的致死效应.本研究将一株苏云金芽胞杆菌的两个S-层蛋白基因(GenBank登录号为AJ012290和AY460125)的3'端序列在大肠杆菌BL21(DE3)中成功进行异源表达,在0.8 mmol/L IPTG诱导培养6 h时,菌体中包涵体表达量达到最高;并对表达蛋白进行了初步分离纯化,以期为后期利用该纯化蛋白进行抗血清制备,进而对该菌的S-层蛋白在宿主菌中的定位、功能分析打下基础.%Surface layer, S-layer, is a part of the cell envelope commonly found in bacteria (both Gram-positive bac-teria and Gram-negative bacteria), as well as among archaea. The functions of S-layer, attracting wide attention from scientists, are not clearly studied yet due to the lethal effect of exogenous S-layer protein gene to Escherichia coli host strains. Most of the functions we knew currently are deduced and have not been certified. In this study, the 3' end sequence from two S-layer protein gene of a Bacillus thuringiensis strain (GenBank accession number AJ012290 and AY460125) have been successfully expressed in Escherichia coli BL21 (DE3). The expression of the inclusion body in the mycelium reached the highest level after culturing for 6 h in 0.8 mmol/L IPTG. The expressed proteins were preliminary separated and purified, in order to lay a foundation for the preparation of antiserum, and for the localiza-tion and function analysis of S-layer protein in the host.

  16. Heterologous Expression of Tulip Petal Plasma Membrane Aquaporins in Pichia pastoris for Water Channel Analysis▿

    Science.gov (United States)

    Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

    2009-01-01

    Water channels formed by aquaporins (AQPs) play an important role in the control of water homeostasis in individual cells and in multicellular organisms. Plasma membrane intrinsic proteins (PIPs) constitute a subclass of plant AQPs. TgPIP2;1 and TgPIP2;2 from tulip petals are members of the PIP family. In this study, we overexpressed TgPIP2;1 and TgPIP2;2 in Pichia pastoris and monitored their water channel activity (WCA) either by an in vivo spheroplast-bursting assay performed after hypo-osmotic shock or by growth assay. Osmolarity, pH, and inhibitors of AQPs, protein kinases (PKs), and protein phosphatases (PPs) affect the WCA of heterologous AQPs in this expression system. The WCA of TgPIP2;2-expressing spheroplasts was affected by inhibitors of PKs and PPs, which indicates that the water channel of this homologue is regulated by phosphorylation in P. pastoris. From the results reported herein, we suggest that P. pastoris can be employed as a heterologous expression system to assay the WCA of PIPs and to monitor the AQP-mediated channel gating mechanism, and it can be developed to screen inhibitors/effectors of PIPs. PMID:19251885

  17. Heterologous expression of tulip petal plasma membrane aquaporins in Pichia pastoris for water channel analysis.

    Science.gov (United States)

    Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

    2009-05-01

    Water channels formed by aquaporins (AQPs) play an important role in the control of water homeostasis in individual cells and in multicellular organisms. Plasma membrane intrinsic proteins (PIPs) constitute a subclass of plant AQPs. TgPIP2;1 and TgPIP2;2 from tulip petals are members of the PIP family. In this study, we overexpressed TgPIP2;1 and TgPIP2;2 in Pichia pastoris and monitored their water channel activity (WCA) either by an in vivo spheroplast-bursting assay performed after hypo-osmotic shock or by growth assay. Osmolarity, pH, and inhibitors of AQPs, protein kinases (PKs), and protein phosphatases (PPs) affect the WCA of heterologous AQPs in this expression system. The WCA of TgPIP2;2-expressing spheroplasts was affected by inhibitors of PKs and PPs, which indicates that the water channel of this homologue is regulated by phosphorylation in P. pastoris. From the results reported herein, we suggest that P. pastoris can be employed as a heterologous expression system to assay the WCA of PIPs and to monitor the AQP-mediated channel gating mechanism, and it can be developed to screen inhibitors/effectors of PIPs.

  18. Heterologous expression of IbMYB1a by different promoters exhibits different patterns of anthocyanin accumulation in tobacco.

    Science.gov (United States)

    An, Chul Han; Lee, Ki-Won; Lee, Sang-Hoon; Jeong, Yu Jeong; Woo, Su Gyoung; Chun, Hyokon; Park, Youn-Il; Kwak, Sang-Soo; Kim, Cha Young

    2015-04-01

    We previously reported that the transient and stable expression of IbMYB1a produced anthocyanin pigmentation in tobacco leaves and transgenic Arabidopsis plants, respectively. To further determine the effects of different promoters on the expression of IbMYB1a and anthocyanin production, we generated and characterized stably transformed tobacco (Nicotiana tabacum SR1) plants expressing IbMYB1a under the control of three different promoters. We compared the differences in anthocyanin accumulation patterns and phenotypic features of the leaves of these transgenic tobacco plants during growth. Expression of IbMYB1a under the control of these three different promoters led to a remarkable variation in anthocyanin pigmentation in tobacco leaves. The anthocyanin contents of the leaves of the SPO-IbMYB1a-OX (SPO-M) line were higher than those of the SWPA2-IbMYB1a-OX (SPA-M) and 35S-IbMYB1a-OX (35S-M) lines. High levels of anthocyanin pigments negatively affected plant growth in the SPO-M lines, resulting delayed growth and, occasionally, a stunted phenotype. Furthermore, HPLC analysis revealed that transcriptional regulation of IbMYB1a led to the production of cyanidin-based anthocyanins in the tobacco plants. In addition, RT-PCR analysis revealed that IbMYB1a expression induced the up-regulation of several structural genes in the anthocyanin biosynthetic pathway, including DFR and ANS. Differential expression levels of IbMYB1a under the control of different promoters were highly correlated with the expression levels of the structural genes, thereby affecting anthocyanin production levels. These results indicate that IbMYB1a positively controls the expression of multiple anthocyanin biosynthetic genes and anthocyanin accumulation in heterologous tobacco plants.

  19. Heterologous expression of chloroplast-localized geranylgeranyl pyrophosphate synthase confers fast plant growth, early flowering and increased seed yield.

    Science.gov (United States)

    Tata, Sandeep Kumar; Jung, Jihye; Kim, Yoon-Ha; Choi, Jun Young; Jung, Ji-Yul; Lee, In-Jung; Shin, Jeong Sheop; Ryu, Stephen Beungtae

    2016-01-01

    Geranylgeranyl pyrophosphate synthase (GGPS) is a key enzyme for a structurally diverse class of isoprenoid biosynthetic metabolites including gibberellins, carotenoids, chlorophylls and rubber. We expressed a chloroplast-targeted GGPS isolated from sunflower (Helianthus annuus) under control of the cauliflower mosaic virus 35S promoter in tobacco (Nicotiana tabacum). The resulting transgenic tobacco plants expressing heterologous GGPS showed remarkably enhanced growth (an increase in shoot and root biomass and height), early flowering, increased number of seed pods and greater seed yield compared with that of GUS-transgenic lines (control) or wild-type plants. The gibberellin levels in HaGGPS-transgenic plants were higher than those in control plants, indicating that the observed phenotype may result from increased gibberellin content. However, in HaGGPS-transformant tobacco plants, we did not observe the phenotypic defects such as reduced chlorophyll content and greater petiole and stalk length, which were previously reported for transgenic plants expressing gibberellin biosynthetic genes. Fast plant growth was also observed in HaGGPS-expressing Arabidopsis and dandelion plants. The results of this study suggest that GGPS expression in crop plants may yield desirable agronomic traits, including enhanced growth of shoots and roots, early flowering, greater numbers of seed pods and/or higher seed yield. This research has potential applications for fast production of plant biomass that provides commercially valuable biomaterials or bioenergy. © 2015 Korea Research Institute of Bioscience & Biotechnology. Plant Biotechnology Journal published by John Wiley & Sons Ltd and Society for Experimental Biology, Association of Applied Biologists.

  20. Heterologous Expression in Remodeled C. elegans: A Platform for Monoaminergic Agonist Identification and Anthelmintic Screening.

    Directory of Open Access Journals (Sweden)

    Wenjing Law

    2015-04-01

    Full Text Available Monoamines, such as 5-HT and tyramine (TA, paralyze both free-living and parasitic nematodes when applied exogenously and serotonergic agonists have been used to clear Haemonchus contortus infections in vivo. Since nematode cell lines are not available and animal screening options are limited, we have developed a screening platform to identify monoamine receptor agonists. Key receptors were expressed heterologously in chimeric, genetically-engineered Caenorhabditis elegans, at sites likely to yield robust phenotypes upon agonist stimulation. This approach potentially preserves the unique pharmacologies of the receptors, while including nematode-specific accessory proteins and the nematode cuticle. Importantly, the sensitivity of monoamine-dependent paralysis could be increased dramatically by hypotonic incubation or the use of bus mutants with increased cuticular permeabilities. We have demonstrated that the monoamine-dependent inhibition of key interneurons, cholinergic motor neurons or body wall muscle inhibited locomotion and caused paralysis. Specifically, 5-HT paralyzed C. elegans 5-HT receptor null animals expressing either nematode, insect or human orthologues of a key Gαo-coupled 5-HT1-like receptor in the cholinergic motor neurons. Importantly, 8-OH-DPAT and PAPP, 5-HT receptor agonists, differentially paralyzed the transgenic animals, with 8-OH-DPAT paralyzing mutant animals expressing the human receptor at concentrations well below those affecting its C. elegans or insect orthologues. Similarly, 5-HT and TA paralyzed C. elegans 5-HT or TA receptor null animals, respectively, expressing either C. elegans or H. contortus 5-HT or TA-gated Cl- channels in either C. elegans cholinergic motor neurons or body wall muscles. Together, these data suggest that this heterologous, ectopic expression screening approach will be useful for the identification of agonists for key monoamine receptors from parasites and could have broad application for

  1. An aryl-alcohol oxidase of Pleurotus sapidus: heterologous expression, characterization, and application in a 2-enzyme system.

    Science.gov (United States)

    Galperin, Ilya; Javeed, Aysha; Luig, Hanno; Lochnit, Günter; Rühl, Martin

    2016-09-01

    Aryl-alcohol oxidases (AAOs) are enzymes supporting the degradation of lignin by fungal derived class II peroxidases produced by white-rot fungi. AAOs are able to generate H2O2 as a by-product via oxidation of an aryl-alcohol into its correspondent aldehyde. In this study, an AAO was heterologously expressed in a basidiomycete host for the first time. The gene for an AAO of the white-rot fungus Pleurotus sapidus, a close relative to the oyster mushroom Pleurotus ostreatus, was cloned into an expression vector and put under control of the promotor of the glyceraldehyde-3-phosphate dehydrogenase gene 2 (gpdII) of the button mushroom Agaricus bisporus. The expression vector was transformed into the model basidiomycete Coprinopsis cinerea, and several positive transformants were obtained. The best producing transformants were grown in shake-flasks and in a stirred tank reactor reaching enzymatic activities of up to 125 U L(-1) using veratryl alcohol as a substrate. The purified AAO was biochemically characterized and compared to the previously described native and recombinant AAOs from other Pleurotus species. In addition, a two-enzyme system comprising a dye-decolorizing peroxidase (DyP) from Mycetinis scorodonius and the P. sapidus AAO was successfully employed to bleach the anthraquinone dye Reactive Blue 5.

  2. Enhancing functional expression of codon-optimized heterologous enzymes in Escherichia coli BL21(DE3) by selective introduction of synonymous rare codons.

    Science.gov (United States)

    Zhong, Chao; Wei, Ping; Zhang, Yi-Heng Percival

    2017-05-01

    Rare codon in a heterologous gene may cause premature termination of protein synthesis, misincorporation of amino acids, and/or slow translation of mRNA, decreasing the heterologous protein expression. However, its hypothetical function pertaining to functional protein folding has been barely reported. Here, we investigated the effects of selective introduction of synonymous rare codons (SRCs) to two codon-optimized (i.e., rare codon-free) genes sucrose phosphorylase (SP) gene from Thermoanaerobacterium thermosaccharolyticum and amidohydrolase gene from Streptomyces caatingaensis on their expression levels in Escherichia coli BL21(DE3). We investigated the introduction of a single SRC to the coding regions of alpha-helix, beta-strand, or linker in the first half of rare codon-free sp and ah gene. The introduction of a single SRC in the beginning of the coding regions of beta-strand greatly enhanced their soluble expression levels as compared to the other regions. Also, we applied directed evolution to test multi-SRC-containing sp gene mutants for enhanced soluble SP expression levels. To easily identify the soluble SP expression level of colonies growing on Petri dishes, mCherry fluorescent protein was used as a SP-folding reporter when it was fused to the 3' end of the sp gene mutant libraries. After three rounds of screening, the best sp gene mutant containing nine SRCs exhibited an approximately six-fold enhancement in soluble protein expression level as compared to the wild-type and rare codon-free sp control. This study suggests that the selective introduction of SRCs can attenuate translation at specific points and such discontinuous attenuation can temporally separate the translation of segments of the peptide chains and actively coordinates their co-translational folding, resulting in enhanced functional protein expression. Biotechnol. Bioeng. 2017;114: 1054-1064. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  3. Heterologous expression of a Streptomyces cyaneus laccase for biomass modification applications.

    Science.gov (United States)

    Ece, Selin; Lambertz, Camilla; Fischer, Rainer; Commandeur, Ulrich

    2017-12-01

    Laccases are used for the conversion of biomass into fermentable sugars but it is difficult to produce high yields of active laccases in heterologous expression systems. We overcame this challenge by expressing Streptomyces cyaneus CECT 3335 laccase in Escherichia coli (ScLac) and we achieved a yield of up to 104 mg L(-1) following purification by one-step affinity chromatography. Stability and activity assays using simple lignin model substrates showed that the purified enzyme preparation was active over a broad pH range and at high temperatures, suggesting it would be suitable for biomass degradation. The reusability of ScLac was also demonstrated by immobilizing the enzyme on agarose beads with a binding yield of 33%, and by the synthesis of cross-linked enzyme aggregates with an initial activity recovery of 72%.

  4. Heterologous Expression of Mycobacterium tuberculosis Ag85B in Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    LIU Yan; PENG Jian-hong; MA Li-xin; JU Wei; QU Xue-ju; LIU Jun-yan

    2005-01-01

    The DNA fragment encoding mature Mycobacterium tuberculosis major secretory protein Ag85B was inserted into the Pichia pastoris secretory expression vector pHBM905A, under the control of the AOX1 promoter. The recombinant plasmid pHBM905A-85B linearized by Sal Ⅰ was introduced into Pichia pastoris strain GS115 by PEG1000 transformation method. After phenotype screening and PCR identification, the resulting GSll5-pHBM905A-85B strain was cultivated and induced with methanol. The recombinant Ag85B protein in secreted form was attained with molecular weight of 35×103 approximately detected by SDS-PAGE and Western blot. ELISA experiment proved that the protein had good antigen specificity. Secretory expression of recombinant M. tuberculosis Ag85B in P. pastoris will open a door to mass production of the protein in heterologous host and allow ready evaluation of its immunological function.

  5. Challenges in the Heterologous Production of Antibiotics in Streptomyces.

    Science.gov (United States)

    Bekiesch, Paulina; Basitta, Patrick; Apel, Alexander K

    2016-08-01

    The fast growing genome databases provide us with a large number of so far unknown secondary metabolite biosynthetic gene clusters. A key method to study these gene clusters is their heterologous expression in an engineered host strain. Gene clusters derived from actinomycetes are usually expressed in a Streptomyces host strain to identify and investigate the corresponding compounds. However, heterologous expression is often accompanied with some challenges affecting the production rates of secondary metabolites. The first step is therefore the selection of a suitable expression vector and host strain. Once production has been established, there are several possibilities to improve compound yields either by media screens, by overexpression of regulatory or transport genes or by introduction of constitutive or inducible promoters. A surely important, but hitherto little studied factor is also the regulation of a heterologously expressed gene cluster by its host strain. This review gives a short overview on the chances and challenges provided by heterologous production of secondary metabolites in Streptomyces.

  6. Heterologous expression of pentalenene synthase (PSS) from Streptomyces UC5319 in Xanthophyllomyces dendrorhous

    NARCIS (Netherlands)

    Melillo, Elena; Muntendam, Remco; Quax, Wim J.; Kayser, Oliver

    2012-01-01

    For the first time, the pentalenene synthase (PSS) gene from Streptomyces UC5319 was expressed in Xanthophyllomyces dendrorhous, a native producer of astaxanthin. For the expression of the gene and the concurrent knock out of the native crtE or crtYB genes, two new vectors were engineered and used f

  7. A simple and rapid method for the preparation of homologous DNA oligonucleotide hybridization probes from heterologous gene sequences and probes.

    Science.gov (United States)

    Maxwell, E S; Sarge, K D

    1988-11-30

    We describe a simple and rapid method for the preparation of homologous DNA oligonucleotide probes for hybridization analysis and/or cDNA/genomic library screening. With this method, a synthetic DNA oligonucleotide derived from a known heterologous DNA/RNA/protein sequence is annealed to an RNA preparation containing the gene transcript of interest. Any unpaired 3'-terminal oligonucleotides of the heterologous DNA primer are then removed using the 3' exonuclease activity of the DNA Polymerase I Klenow fragment before primer extension/dideoxynucleotide sequencing of the annealed RNA species with AMV reverse transcriptase. From the determined RNA sequence, a completely homologous DNA oligonucleotide probe is then prepared. This approach has been used to prepare a homologous DNA oligonucleotide probe for the successful library screening of the yeast hybRNA gene starting with a heterologous mouse hybRNA DNA oligonucleotide probe.

  8. Heterologous Proteins Production in Escherichia coli: An Investigation on the Effect of Codon Usage and Expression Host Optimization

    Directory of Open Access Journals (Sweden)

    Hasan Mirzahoseini

    2011-01-01

    Full Text Available Objective: The production of heterologous proteins in Escherichia coli is strongly affectedby codon bias. This phenomenon occurs when the codon usage of mRNA coding for theforeign protein differs from that of the bacterium. The ribosome pauses upon encounteringa rare codon and may detach from mRNA, thereby the yield of recombinant protein productionreduces. The aim of this study is to investigate the effect of these codon numbersreductions on the recombinant protein production.Materials and Methods: Since most amino acids are encoded by more than one codon,codons were changed in order to their usage in a special host such as E. coli without anytransformation in amino acids sequence. Silent mutations in 5' codons of human basicfibroblast growth factor cDNA carried out by site-directed mutagenesis and the expressionlevel of the recombinant protein is analyzed by means of sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE and Western blot.Results: Expression level in mutant and wild-type genes indicated a considerable difference.In contrast with the remarkable bands of wild-type gene in all the strains particularly in codonplus strain, there were no significant bands related to mutant gene in SDS-PAGE analysis.Conclusion: Because of the same conditions of mutant and wild-type genes during thetranslation and transcription, this significant difference may relate to mRNA efficiency fortranslation. Our results indicate that increased stability of 5' mRNA secondary structuresin E. coli prevents efficient translation initiation. Furthermore, wild-type gene significantbands in codon plus strain support the hypothesis that the possible elimination of translationalpauses that increase translation rate leads to over expression.

  9. Heterologous expression of Translocated promoter region protein, Tpr, identified as a transcription factor from Rattus norvegicus.

    Science.gov (United States)

    Agarwal, Shivani; Yadav, Sunita Kumari; Dixit, Aparna

    2011-05-01

    Our earlier studies have demonstrated that the 35 kDa isoform of Translocated promoter region protein (Tpr) of Rattus norvegicus was able to augment c-jun transcription efficiently. Identification of direct targets that may in part downregulate c-jun transcription might prove to be an ideal target to curtail the proliferation of normal cells under pathophysiological conditions. In order to evaluate its potential as a pharmaceutical target, the protein must be produced and purified in sufficiently high yields. In the present study, we report the high level expression of Tpr protein of R. norvegicus employing heterologous host, Escherichia coli, to permit its structural characterization in great detail. We here demonstrate that the Tpr protein was expressed in soluble form and approximately 90 mg/L of the purified protein at the shake flask level could be achieved to near homogeneity using single step-metal chelate affinity chromatography. The amino acid sequence of the protein was confirmed by mass spectroscopic analysis. The highly unstable and disordered Tpr protein was imparted structural and functional stability by the addition of glycerol and it has been shown that the natively unfolded Tpr protein retains DNA binding ability under these conditions only. Thus, the present study emphasizes the significance of an efficient prokaryotic system, which results in a high level soluble expression of a DNA binding protein of eukaryotic origin. Thus, the present strategy employed for purification of the R. norvegicus Tpr protein bypasses the need for the tedious expression strategies associated with the eukaryotic expression systems.

  10. Heterologous expression of functional G-protein-coupled receptors in Caenorhabditis elegans.

    Science.gov (United States)

    Salom, David; Cao, Pengxiu; Sun, Wenyu; Kramp, Kristopher; Jastrzebska, Beata; Jin, Hui; Feng, Zhaoyang; Palczewski, Krzysztof

    2012-02-01

    New strategies for expression, purification, functional characterization, and structural determination of membrane-spanning G-protein-coupled receptors (GPCRs) are constantly being developed because of their importance to human health. Here, we report a Caenorhabditis elegans heterologous expression system able to produce milligram amounts of functional native and engineered GPCRs. Both bovine opsin [(b)opsin] and human adenosine A(2A) subtype receptor [(h)A(2A)R] expressed in neurons or muscles of C. elegans were localized to cell membranes. Worms expressing these GPCRs manifested changes in motor behavior in response to light and ligands, respectively. With a newly devised protocol, 0.6-1 mg of purified homogenous 9-cis-retinal-bound bovine isorhodopsin [(b)isoRho] and ligand-bound (h)A(2A)R were obtained from C. elegans from one 10-L fermentation at low cost. Purified recombinant (b)isoRho exhibited its signature absorbance spectrum and activated its cognate G-protein transducin in vitro at a rate similar to native rhodopsin (Rho) obtained from bovine retina. Generally high expression levels of 11 native and mutant GPCRs demonstrated the potential of this C. elegans system to produce milligram quantities of high-quality GPCRs and possibly other membrane proteins suitable for detailed characterization.

  11. Specificity of Ocimum basilicum geraniol synthase modified by its expression in different heterologous systems.

    Science.gov (United States)

    Fischer, Marc J C; Meyer, Sophie; Claudel, Patricia; Perrin, Mireille; Ginglinger, Jean François; Gertz, Claude; Masson, Jean E; Werck-Reinhardt, Danièle; Hugueney, Philippe; Karst, Francis

    2013-01-10

    Numerous aromatic plant species produce high levels of monoterpenols, using geranyl diphosphate (GPP) as a precursor. Sweet basil (Ocimum basilicum) geraniol synthase (GES) was used to evaluate the monoterpenol profiles arising from heterologous expressions in various plant models. Grapevine (Vitis vinifera) calli were transformed using Agrobacterium tumefasciens and the plants were regenerated. Thale cress (Arabidopsis thaliana) was transformed using the floral dip method. Tobacco (Nicotiana benthamiana) leaves were agro-infiltrated for transient expression. Although, as expected, geraniol was the main product detected in the leaves, different minor products were observed in these plants (V. vinifera: citronellol and nerol; N. benthamiana: linalool and nerol; A. thaliana: none). O. basilicum GES expression was also carried out with microbial system yeasts (Saccharomyces cerevisiae) and Escherichia coli. These results suggest that the functional properties of a monoterpenol synthase depend not only on the enzyme's amino-acidic sequence, but also on the cellular background. They also suggest that some plant species or microbial expression systems could induce the simultaneous formation of several carbocations, and could thus have a natural tendency to produce a wider spectrum of monoterpenols.

  12. Heterologous Expression of Membrane and Soluble Proteins Derepresses GCN4 mRNA Translation in the Yeast Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Steffensen, L.; Pedersen, P. A.

    2006-01-01

    of the membrane-bound 1ß1 Na,K-ATPase from pig kidney, the rat pituitary adenylate cyclase seven-transmembrane-domain receptor, or a 401-residue soluble part of the Na,K-ATPase 1 subunit derepressed GCN4 mRNA translation up to 70-fold. GCN4 translation was very sensitive to the presence of heterologous protein......This paper describes the first physiological response at the translational level towards heterologous protein production in Saccharomyces cerevisiae. In yeast, the phosphorylation of eukaryotic initiation factor 2 (eIF-2 ) by Gcn2p protein kinase mediates derepression of GCN4 mRNA translation. Gcn4......, as a density of 1 of heterologous membrane protein derepressed translation maximally. Translational derepression of GCN4 was not triggered by misfolding in the endoplasmic reticulum, as expression of the wild type or temperature-sensitive folding mutants of the Na,K-ATPase increased GCN4 translation...

  13. Molecular characterization and heterologous expression of a Xanthophyllomyces dendrorhous α-glucosidase with potential for prebiotics production.

    Science.gov (United States)

    Gutiérrez-Alonso, Patricia; Gimeno-Pérez, María; Ramírez-Escudero, Mercedes; Plou, Francisco J; Sanz-Aparicio, Julia; Fernández-Lobato, María

    2016-04-01

    Basidiomycetous yeast Xanthophyllomyces dendrorhous expresses an α-glucosidase with strong transglycosylation activity producing prebiotic sugars such as panose and an unusual tetrasaccharides mixture including α-(1-6) bonds as major products, which makes it of biotechnological interest. Initial analysis pointed to a homodimeric protein of 60 kDa subunit as responsible for this activity. In this study, the gene Xd-AlphaGlu was characterized. The 4131-bp-long gene is interrupted by 13 short introns and encodes a protein of 990 amino acids (Xd-AlphaGlu). The N-terminal sequence of the previously detected 60 kDa protein resides in this larger protein at residues 583-602. Functionality of the gene was proved in Saccharomyces cerevisiae, which produced a protein of about 130 kDa containing Xd-AlphaGlu sequences. All properties of the heterologously expressed protein, including thermal and pH profiles, activity on different substrates, and ability to produce prebiotic sugars were similar to that of the α-glucosidase produced in X. dendrorhous. No activity was detected in S. cerevisiae containing exclusively the 1256-bp from gene Xd-AlphaGlu that would encode synthesis of the 60 kDa protein previously detected. Data were compatible with an active monomeric α-glucosidase of 990 amino acids and an inactive hydrolysis product of 60 kDa. Protein Xd-AlphaGlu contained most of the elements characteristic of α-glucosidases included in the glycoside hydrolases family GH31 and its structural model based on the homologous human maltase-glucoamylase was obtained. Remarkably, the Xd-AlphaGlu C-terminal domain presents an unusually long 115-residue insertion that could be involved in this enzyme's activity against long-size substrates such as maltoheptaose and soluble starch.

  14. New recombinant bacterium comprises a heterologous gene encoding glycerol dehydrogenase and/or an up-regulated native gene encoding glycerol dehydrogenase, useful for producing ethanol

    DEFF Research Database (Denmark)

    2010-01-01

    from Geobacillus. It is selected from SEQ ID NO. 1-17. Sequences not defined here may be found at ftp://ftp.wipo.int/pub/publishedpctsequences/publication. The heterologous gene encoding glycerol dehydrogenase has been incorporated into the chromosome of the bacterium, or is inserted into a lactate...... glycerol dehydrogenase; and/or (ii) up-regulating a native gene encoding glycerol dehydrogenase; and (b) obtaining the recombinant bacterium. Preferred Bacterium: In the recombinant bacterium above, the inserted heterologous gene and/or the up-regulated native gene is encoding a glycerol dehydrogenase...... selected from glycerol dehydrogenase (E.C 1.1.1.6); glycerol dehydrogenase (NADP(+)) (E.C. 1.1.1.72); glycerol 2-dehydrogenase (NADP(+)) (E.C. 1.1.1.156); and glycerol dehydrogenase (acceptor) (E.C. 1.1.99.22). The heterologous gene encoding a glycerol dehydrogenase is derived from Thermotoga or is derived...

  15. Engineering genes for predictable protein expression.

    Science.gov (United States)

    Gustafsson, Claes; Minshull, Jeremy; Govindarajan, Sridhar; Ness, Jon; Villalobos, Alan; Welch, Mark

    2012-05-01

    The DNA sequence used to encode a polypeptide can have dramatic effects on its expression. Lack of readily available tools has until recently inhibited meaningful experimental investigation of this phenomenon. Advances in synthetic biology and the application of modern engineering approaches now provide the tools for systematic analysis of the sequence variables affecting heterologous expression of recombinant proteins. We here discuss how these new tools are being applied and how they circumvent the constraints of previous approaches, highlighting some of the surprising and promising results emerging from the developing field of gene engineering.

  16. Isolation and characterization of a thermotolerant ene reductase from Geobacillus sp. 30 and its heterologous expression in Rhodococcus opacus.

    Science.gov (United States)

    Tsuji, Naoto; Honda, Kohsuke; Wada, Mayumi; Okano, Kenji; Ohtake, Hisao

    2014-07-01

    Rhodococcus opacus B-4 cells are adhesive to and even dispersible in water-immiscible hydrocarbons owing to their highly lipophilic nature. In this study, we focused on the high operational stability of thermophilic enzymes and applied them to a biocatalytic conversion in an organic reaction medium using R. opacus B-4 as a lipophilic capsule of enzymes to deliver them into the organic medium. A novel thermo- and organic-solvent-tolerant ene reductase, which can catalyze the enantioselective reduction of ketoisophorone to (6R)-levodione, was isolated from Geobacillus sp. 30, and the gene encoding the enzyme was heterologously expressed in R. opacus B-4. Another thermophilic enzyme which catalyzes NAD(+)-dependent dehydrogenation of cyclohexanol was identified from the gene-expression library of Thermus thermophilus and the gene was coexpressed in R. opacus B-4 for cofactor regeneration. While the recombinant cells were not viable in the mixture due to high reaction temperature, 634 mM of (6R)-levodione could be produced with an enantiopurity of 89.2 % ee by directly mixing the wet cells of the recombinant R. opacus with a mixture of ketoisophorone and cyclohexanol at 50 °C. The conversion rate observed with the heat-killed recombinant cells was considerably higher than that obtained with a cell-free enzyme solution, demonstrating that the accessibility between the substrates and enzymes could be improved by employing R. opacus cells as a lipophilic enzyme capsule. These results imply that a combination of thermophilic enzymes and lipophilic cells can be a promising approach for the biocatalytic production of water-insoluble chemicals.

  17. Molecular cloning, heterologous expression and functional characterization of gamma tocopherol methyl transferase (γ-TMT) from Glycine max.

    Science.gov (United States)

    Tewari, Kalpana; Dahuja, Anil; Sachdev, Archana; Kumar, Vaibhav; Ali, Kishwar; Kumar, Amresh; Kumari, Sweta

    2017-12-01

    γ-Tocopherol methyltransferase (γ-TMT) (EC 2.1.1.95) is the last enzyme in the tocopherol biosynthetic pathway and it catalyzes the conversion of γ-tocopherol into α-tocopherol, the nutritionally significant and most bioactive form of vitamin E. Although the γ-TMT gene has been successfully overexpressed in many crops to enhance their α-tocopherol content but still only few attempts have been made to uncover its structural, functional and regulation aspects at protein level. In this study, we have cloned the complete 909bp coding sequence of Glycine max γ-TMT (Gm γ-TMT) gene that encodes the corresponding protein comprising of 302 amino acid residues. The deduced Gm γ-TMT protein showed 74-87% sequence identity with other characterized plant γ-TMTs. Gm γ-TMT belongs to Class I Methyl Transferases that have a Rossmann-like fold which consists of a seven-stranded β sheet joined by α helices. Heterologous expression of Gm γ-TMT in pET29a expression vector under the control of bacteriophage T7 promoter produced a 37.9 kDa recombinant Gm γ-TMT protein with histidine hexamer tag at its C-terminus. The expression of recombinant Gm γ-TMT protein was confirmed by western blotting using anti-His antibody. The recombinant protein was purified by Ni(2+)-NTA column chromatography. The purified protein showed SAM dependent methyltransferase activity. The α-tocopherol produced in the in-vitro reaction catalyzed by the purified enzyme was detected using reverse phase HPLC. This study has laid the foundation to unveil the biochemical understanding of Gm γ-TMT enzyme which can be further explored by studying its kinetic behaviour, substrate specificity and its interaction with other biomolecules. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Induction of antimicrobial activities in heterologous streptomycetes using alleles of the Streptomyces coelicolor gene absA1.

    Science.gov (United States)

    McKenzie, Nancy L; Thaker, Maulik; Koteva, Kalinka; Hughes, Donald W; Wright, Gerard D; Nodwell, Justin R

    2010-04-01

    The bacterial genus Streptomyces is endowed with a remarkable secondary metabolism that generates an enormous number of bioactive small molecules. Many of these genetically encoded small molecules are used as antibiotics, anticancer agents and as other clinically relevant therapeutics. The rise of resistant pathogens has led to calls for renewed efforts to identify antimicrobial activities, including expanded screening of streptomycetes. Indeed, it is known that most strains encode >20 secondary metabolites and that many, perhaps most of these, have not been considered for their possible therapeutic use. One roadblock is that many strains do not express their secondary metabolic gene clusters efficiently under laboratory conditions. As one approach to this problem, we have used alleles of a pleiotropic regulator of secondary metabolism from Streptomyces coelicolor to activate secondary biosynthetic gene clusters in heterologous streptomycetes. In one case, we demonstrate the activation of pulvomycin production in S. flavopersicus, a metabolite not previously attributed to this species. We find that the absA1-engineered strains produced sufficient material for purification and characterization. As a result, we identified new, broad-spectrum antimicrobial activities for pulvomycin, including a potent antimicrobial activity against highly antibiotic-resistant Gram-negative and Gram-positive pathogens.

  19. Electrophysiological effect of fluoxetine on Xenopus oocytes heterologously expressing human serotonin transporter

    Institute of Scientific and Technical Information of China (English)

    Hong-wei WANG; Ci-zhen LI; Zhi-fang YANG; Yan-qian ZHENG; Ying ZHANG; Yuan-mou LIU

    2006-01-01

    Aim:To investigate the electrophysiological effect of fluoxetine on serotonin transporter.Methods:A heterologous expression system was used to introduce human serotonin transporter(hSERT)into Xenopus oocytes.A 2-electrode voltage clamp technique was used to study the pharmacological properties of fluoxetine.Results:hSERT-expressing oocytes were perfused with 10 μmol/L serotonin (5-HT)to induce hSERT-current.The 5-HT-induced hSERT currents were dose-dependently reversed by fluoxetine.The RC50(concentration that achieved a 50% reversal)was approximately 3.12 μmol/L.Fluoxetine took more time to combine with hSERT than 5-HT did,and it was also slow to dissociate from hSERRT. This long-lasting effect of fluoxetine affected normal 5-HT transport.Fluoxetine significantly prolonged the time constant for 5-HT-induced hSERT current.These results might be used to explain the long-lasting anti-anxiety effect of fluoxetine in clinical practice,because it increases the concentration of 5-HT in the synaptic cleft by its enduring suppression of the function of 5-HT transporters.Conclusion:Fluoxetine inhibits 5-HT reuptake by competing with 5-HT and changing the normal dynamics of hSERT.

  20. Heterologous expression of Pycnoporus cinnabarinus cellobiose dehydrogenase in Pichia pastoris and involvement in saccharification processes

    Directory of Open Access Journals (Sweden)

    Bey Mathieu

    2011-12-01

    Full Text Available Abstract Background Cellobiose dehydrogenase (CDH is an extracellular hemoflavoenzyme produced by lignocellulose-degrading fungi including Pycnoporus cinnabarinus. We investigated the cellulolytic system of P. cinnabarinus, focusing on the involvement of CDH in the deconstruction of lignocellulosic biomass. Results First, P. cinnabarinus growth conditions were optimized for CDH production. Following growth under cellulolytic conditions, the main components secreted were cellulases, xylanases and CDH. To investigate the contribution of P. cinnabarinus secretome in saccharification processes, the Trichoderma reesei enzymatic cocktail was supplemented with the P. cinnabarinus secretome. A significant enhancement of the degradation of wheat straw was observed with (i the production of a large amount of gluconic acid, (ii increased hemicellulose degradation, and (iii increased overall degradation of the lignocellulosic material. P. cinnabarinus CDH was heterologously expressed in Pichia pastoris to obtain large amounts of pure enzyme. In a bioreactor, the recombinant CDH (rCDH expression level reached 7800 U/L. rCDH exhibited values of biochemical parameters similar to those of the natural enzyme, and was able to bind cellulose despite the absence of a carbohydrate-binding module (CBM. Following supplementation of purified rCDH to T. reesei enzymatic cocktail, formation of gluconic acid and increased hemicellulose degradation were observed, thus confirming the previous results observed with P. cinnabarinus secretome. Conclusions We demonstrate that CDH offers an attractive tool for saccharification process enhancement due to gluconic acid production from raw lignocellulosic material.

  1. Heterologously expressed formyl peptide receptor 2 (FPR2/ALX) does not respond to lipoxin A₄.

    Science.gov (United States)

    Hanson, Julien; Ferreirós, Nerea; Pirotte, Bernard; Geisslinger, Gerd; Offermanns, Stefan

    2013-06-15

    Lipoxin A₄ (LXA₄) has been described as an anti-inflammatory mediator, which exerts its effects through the formyl peptide receptor FPR2, also known as ALX. However, there has been a controversy whether or not cells expressing FPR2/ALX, such as neutrophils, respond to LXA₄. We, therefore, systematically examined the ability of the human and murine forms of the receptor to respond to LXA₄. We show that both receptor orthologues responded to the FPR2/ALX peptide agonist WKYMVM when expressed heterologously. In contrast, LXA₄ from different sources neither increased [Ca²⁺](i) and extracellular-signal-regulated kinase (ERK) phosphorylation, nor did it induce a decrease in cAMP levels or a translocation of β-arrestin. Also, several LXA₄ analogs were found to be unable to signal through FPR2/ALX. We conclude that FPR2/ALX is not activated by LXA₄ and that the molecular mechanism by which LXA₄ functions still needs to be identified. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Heterologous Expression of Plant Cell Wall Degrading Enzymes for Effective Production of Cellulosic Biofuels

    Science.gov (United States)

    Jung, Sang-Kyu; Parisutham, Vinuselvi; Jeong, Seong Hun; Lee, Sung Kuk

    2012-01-01

    A major technical challenge in the cost-effective production of cellulosic biofuel is the need to lower the cost of plant cell wall degrading enzymes (PCDE), which is required for the production of sugars from biomass. Several competitive, low-cost technologies have been developed to produce PCDE in different host organisms such as Escherichia coli, Zymomonas mobilis, and plant. Selection of an ideal host organism is very important, because each host organism has its own unique features. Synthetic biology-aided tools enable heterologous expression of PCDE in recombinant E. coli or Z. mobilis and allow successful consolidated bioprocessing (CBP) in these microorganisms. In-planta expression provides an opportunity to simplify the process of enzyme production and plant biomass processing and leads to self-deconstruction of plant cell walls. Although the future of currently available technologies is difficult to predict, a complete and viable platform will most likely be available through the integration of the existing approaches with the development of breakthrough technologies. PMID:22911272

  3. An "instant gene bank" method for heterologous gene cloning: complementation of two Aspergillus nidulans mutants with Gaeumannomyces graminis DNA.

    Science.gov (United States)

    Bowyer, P; Osbourn, A E; Daniels, M J

    1994-02-01

    We present a novel technique for gene cloning by complementation of mutations in Aspergillus nidulans with DNA from a heterologous organism, Gaeumannomyces graminis. This technique bypasses the time-consuming and difficult construction of gene libraries, making it both rapid and simple. The method relies on recombination between a fungal replicating vector pHELP1 and linear G. graminis genomic DNA during co-transformation. We were able to complement two out of seven A. nidulans mutants tested and to rescue transforming DNA from both in Escherichia coli. Complementation of the A. nidulans argB mutation resulted from integration of 8-10 kb segments of G. graminis DNA into pHELP1. The complementation of the A. nidulans pyrG mutation resulted from a complex rearrangement. Complementing DNA was shown to originate from G. graminis, and was capable of retransforming the original mutants to give the expected phenotype.

  4. (+)-Pinoresinol/(+)-lariciresinol reductase from Forsythia intermedia. Protein purification, cDNA cloning, heterologous expression and comparison to isoflavone reductase.

    Science.gov (United States)

    Dinkova-Kostova, A T; Gang, D R; Davin, L B; Bedgar, D L; Chu, A; Lewis, N G

    1996-11-15

    Lignans are a widely distributed class of natural products, whose functions and distribution suggest that they are one of the earliest forms of defense to have evolved in vascular plants; some, such as podophyllotoxin and enterodiol, have important roles in cancer chemotherapy and prevention, respectively. Entry into lignan enzymology has been gained by the approximately 3000-fold purification of two isoforms of (+)-pinoresinol/(+)-lariciresinol reductase, a pivotal branchpoint enzyme in lignan biosynthesis. Both have comparable ( approximately 34.9 kDa) molecular mass and kinetic (Vmax/Km) properties and catalyze sequential, NADPH-dependent, stereospecific, hydride transfers where the incoming hydride takes up the pro-R position. The gene encoding (+)-pinoresinol/(+)-lariciresinol reductase has been cloned and the recombinant protein heterologously expressed as a functional beta-galactosidase fusion protein. Its amino acid sequence reveals a strong homology to isoflavone reductase, a key branchpoint enzyme in isoflavonoid metabolism and primarily found in the Fabaceae (angiosperms). This is of great evolutionary significance since both lignans and isoflavonoids have comparable plant defense properties, as well as similar roles as phytoestrogens. Given that lignans are widespread from primitive plants onwards, whereas the isoflavone reductase-derived isoflavonoids are mainly restricted to the Fabaceae, it is tempting to speculate that this branch of the isoflavonoid pathway arose via evolutionary divergence from that giving the lignans.

  5. Inhibitory Action of Antidepressants on Mouse Betaine/GABA Transporter (BGT1 Heterologously Expressed in Cell Cultures

    Directory of Open Access Journals (Sweden)

    Shigeo Kitayama

    2012-02-01

    Full Text Available Betaine/γ-aminobutyric acid (GABA transporter (BGT1, SLC6A12 is a member of the Na+- and Cl−-dependent neurotransmitter transporter gene family with a homology to the GABA transporters (GATs, GAT1 (SLC6A1, GAT2 (SLC6A13 and GAT3 (SLC6A11 (HUGO nomenclature. Since antidepressants have been reported to inhibit GABA uptake, we examined those effects on mouse BGT1 (mBGT1 in comparison with other mouse GAT (mGAT subtypes in the heterologously expressed cell cultures. All antidepressants tested here inhibited the [3H]GABA uptake through mBGT1 and mGATs in a rank order of potency with mBGT1 > mGAT1-3. Kinetic analyses for maprotilline, mianserine and trimipramine revealed that they inhibited mBGT1 and mGAT1 noncompetitively, except that mianserine competitively inhibited mBGT1. These results provided a clue to investigate the structure-function relationship of mBGT1 using antidepressants as a tool, leading to the identification of potential candidates for selective and specific inhibitors of mBGT1.

  6. Cloning, heterologous expression and biochemical characterization of plastidial sn-glycerol-3-phosphate acyltransferase from Helianthus annuus.

    Science.gov (United States)

    Payá-Milans, Miriam; Venegas-Calerón, Mónica; Salas, Joaquín J; Garcés, Rafael; Martínez-Force, Enrique

    2015-03-01

    The acyl-[acyl carrier protein]:sn-1-glycerol-3-phosphate acyltransferase (GPAT; E.C. 2.3.1.15) catalyzes the first step of glycerolipid assembly within the stroma of the chloroplast. In the present study, the sunflower (Helianthus annuus, L.) stromal GPAT was cloned, sequenced and characterized. We identified a single ORF of 1344base pairs that encoded a GPAT sharing strong sequence homology with the plastidial GPAT from Arabidopsis thaliana (ATS1, At1g32200). Gene expression studies showed that the highest transcript levels occurred in green tissues in which chloroplasts are abundant. The corresponding mature protein was heterologously overexpressed in Escherichia coli for purification and biochemical characterization. In vitro assays using radiolabelled acyl-ACPs and glycerol-3-phosphate as substrates revealed a strong preference for oleic versus palmitic acid, and weak activity towards stearic acid. The positional fatty acid composition of relevant chloroplast phospholipids from sunflower leaves did not reflect the in vitro GPAT specificity, suggesting a more complex scenario with mixed substrates at different concentrations, competition with other acyl-ACP consuming enzymatic reactions, etc. In summary, this study has confirmed the affinity of this enzyme which would partly explain the resistance to cold temperatures observed in sunflower plants.

  7. Functional expression of a heterologous nickel-dependent, ATP-independent urease in Saccharomyces cerevisiae

    NARCIS (Netherlands)

    Milne, N.; Luttik, M.A.H.; Cueto Rojas, H.F.; Wahl, A.; Van Maris, A.J.A.; Pronk, J.T.; Daran, J.G.

    2015-01-01

    In microbial processes for production of proteins, biomass and nitrogen-containing commodity chemicals, ATP requirements for nitrogen assimilation affect product yields on the energy producing substrate. In Saccharomyces cerevisiae, a current host for heterologous protein production and potential pl

  8. Production of 2-ketoisocaproate with Corynebacterium glutamicum strains devoid of plasmids and heterologous genes.

    Science.gov (United States)

    Vogt, Michael; Haas, Sabine; Polen, Tino; van Ooyen, Jan; Bott, Michael

    2015-03-01

    2-Ketoisocaproate (KIC), the last intermediate in l-leucine biosynthesis, has various medical and industrial applications. After deletion of the ilvE gene for transaminase B in l-leucine production strains of Corynebacterium glutamicum, KIC became the major product, however, the strains were auxotrophic for l-isoleucine. To avoid auxotrophy, reduction of IlvE activity by exchanging the ATG start codon of ilvE by GTG was tested instead of an ilvE deletion. The resulting strains were indeed able to grow in glucose minimal medium without amino acid supplementation, but at the cost of lowered growth rates and KIC production parameters. The best production performance was obtained with strain MV-KICF1, which carried besides the ilvE start codon exchange three copies of a gene for a feedback-resistant 2-isopropylmalate synthase, one copy of a gene for a feedback-resistant acetohydroxyacid synthase and deletions of ltbR and iolR encoding transcriptional regulators. In the presence of 1 mM l-isoleucine, MV-KICF1 accumulated 47 mM KIC (6.1 g l(-1)) with a yield of 0.20 mol/mol glucose and a volumetric productivity of 1.41 mmol KIC l(-1)  h(-1). Since MV-KICF1 is plasmid free and lacks heterologous genes, it is an interesting strain for industrial application and as platform for the production of KIC-derived compounds, such as 3-methyl-1-butanol.

  9. Gram negative shuttle BAC vector for heterologous expression of metagenomic libraries.

    Science.gov (United States)

    Kakirde, Kavita S; Wild, Jadwiga; Godiska, Ronald; Mead, David A; Wiggins, Andrew G; Goodman, Robert M; Szybalski, Waclaw; Liles, Mark R

    2011-04-15

    Bacterial artificial chromosome (BAC) vectors enable stable cloning of large DNA fragments from single genomes or microbial assemblages. A novel shuttle BAC vector was constructed that permits replication of BAC clones in diverse Gram-negative species. The "Gram-negative shuttle BAC" vector (pGNS-BAC) uses the F replicon for stable single-copy replication in E. coli and the broad-host-range RK2 mini-replicon for high-copy replication in diverse Gram-negative bacteria. As with other BAC vectors containing the oriV origin, this vector is capable of an arabinose-inducible increase in plasmid copy number. Resistance to both gentamicin and chloramphenicol is encoded on pGNS-BAC, permitting selection for the plasmid in diverse bacterial species. The oriT from an IncP plasmid was cloned into pGNS-BAC to enable conjugal transfer, thereby allowing both electroporation and conjugation of pGNS-BAC DNA into bacterial hosts. A soil metagenomic library was constructed in pGNS-BAC-1 (the first version of the vector, lacking gentamicin resistance and oriT), and recombinant clones were demonstrated to replicate in diverse Gram-negative hosts, including Escherichia coli, Pseudomonas spp., Salmonella enterica, Serratia marcescens, Vibrio vulnificus and Enterobacter nimipressuralis. This shuttle BAC vector can be utilized to clone genomic DNA from diverse sources, and then transfer it into diverse Gram-negative bacterial species to facilitate heterologous expression of recombinant pathways.

  10. Expression of lactococcin A and pediocin PA-1 in heterologous hosts

    NARCIS (Netherlands)

    Chikindas, M.L.; Venema, K.; Ledeboer, A.M.; Venema, G.; Kok, Jan

    Pediocin PA-1 production, immunity and secretion are specified by a cluster of four genes in Pediococcus acidilactici PAC1.0. The production by, secretion of, and immunity to lactococcin A of Lactococcus lactis are also determined by four genes. Here, expression of the pediocin operon in Lactococcus

  11. Heterologous expression of the Aspergillus nidulans alcR-alcA system in Aspergillus niger

    NARCIS (Netherlands)

    Nikolaev, I.; Mathieu, M.; Vondervoort, van de P.J.I.; Visser, J.; Felenbok, B.

    2002-01-01

    The inducible and strongly expressed alcA gene encoding alcohol dehydrogenase I from Aspergillus nidulans was transferred together with the activator gene alcR, in the industrial fungus Aspergillus niger. This latter organism does not possess an inducible alc system but has an endogenously constitut

  12. Heterologous Expression of Panax ginseng PgTIP1 Confers Enhanced Salt Tolerance of Soybean Cotyledon Hairy Roots, Composite, and Whole Plants

    Directory of Open Access Journals (Sweden)

    Jing An

    2017-07-01

    Full Text Available The Panax ginseng TIP gene PgTIP1 was previously demonstrated to have high water channel activity by its heterologous expression in Xenopus laevis oocytes and in yeast; it also plays a significant role in growth of PgTIP1-transgenic Arabidopsis plants under favorable conditions and has enhanced tolerance toward salt and drought treatment. In this work, we first investigated the physiological effects of heterologous PgTIP1 expression in soybean cotyledon hairy roots or composite plants mediated by Agrobacterium rhizogenes toward enhanced salt tolerance. The PgTIP1-transgenic soybean plants mediated by the pollen tube pathway, represented by the lines N and J11, were analyzed at the physiological and molecular levels for enhanced salt tolerance. The results showed that in terms of root-specific heterologous expression, the PgTIP1-transformed soybean cotyledon hairy roots or composite plants displayed superior salt tolerance compared to the empty vector-transformed ones according to the mitigatory effects of hairy root growth reduction, drop in leaf RWC, and rise in REL under salt stress. Additionally, declines in K+ content, increases in Na+ content and Na+/K+ ratios in the hairy roots, stems, or leaves were effectively alleviated by PgTIP1-transformation, particularly the stems and leaves of composite soybean plants. At the whole plant level, PgTIP1-trasgenic soybean lines were found to possess stronger root vigor, reduced root and leaf cell membrane damage, increased SOD, POD, CAT, and APX activities, steadily increased leaf Tr, RWC, and Pn values, and smaller declines in chlorophyll and carotenoid content when exposed to salt stress compared to wild type. Moreover, the distribution patterns of Na+, K+, and Cl- in the roots, stems, and leaves of salt-stressed transgenic plants were readjusted, in that the absorbed Na+ and Cl- were mainly restricted to the roots to reduce their transport to the shoots, and the transport of root-absorbed K+ to the

  13. Comparison of plant-based expression platforms for the heterologous production of geraniol

    NARCIS (Netherlands)

    Vasilev, N.; Schmitz, C.; Dong, L.; Ritala, A.; Imseng, N.; Hakkinen, S.T.; Krol, van der A.R.; Eibl, R.; Oksman-Caldentey, K.M.; Bouwmeester, H.J.; Fischer, R.; Schillberg, S.

    2014-01-01

    We compared the ability of different plant-based expression platforms to produce geraniol, a key metabolite in the monoterpenoid branch of the terpenoid indole alkaloid biosynthesis pathway. A geraniol synthase gene isolated from Valeriana officinalis (VoGES) was stably expressed in different

  14. Production of L-lactic acid by the yeast Candida sonorensis expressing heterologous bacterial and fungal lactate dehydrogenases.

    Science.gov (United States)

    Ilmén, Marja; Koivuranta, Kari; Ruohonen, Laura; Rajgarhia, Vineet; Suominen, Pirkko; Penttilä, Merja

    2013-05-25

    Polylactic acid is a renewable raw material that is increasingly used in the manufacture of bioplastics, which offers a more sustainable alternative to materials derived from fossil resources. Both lactic acid bacteria and genetically engineered yeast have been implemented in commercial scale in biotechnological production of lactic acid. In the present work, genes encoding L-lactate dehydrogenase (LDH) of Lactobacillus helveticus, Bacillus megaterium and Rhizopus oryzae were expressed in a new host organism, the non-conventional yeast Candida sonorensis, with or without the competing ethanol fermentation pathway. Each LDH strain produced substantial amounts of lactate, but the properties of the heterologous LDH affected the distribution of carbon between lactate and by-products significantly, which was reflected in extra-and intracellular metabolite concentrations. Under neutralizing conditions C. sonorensis expressing L. helveticus LDH accumulated lactate up to 92 g/l at a yield of 0.94 g/g glucose, free of ethanol, in minimal medium containing 5 g/l dry cell weight. In rich medium with a final pH of 3.8, 49 g/l lactate was produced. The fermentation pathway was modified in some of the strains studied by deleting either one or both of the pyruvate decarboxylase encoding genes, PDC1 and PDC2. The deletion of both PDC genes together abolished ethanol production and did not result in significantly reduced growth characteristic to Saccharomyces cerevisiae deleted of PDC1 and PDC5. We developed an organism without previous record of genetic engineering to produce L-lactic acid to a high concentration, introducing a novel host for the production of an industrially important metabolite, and opening the way for exploiting C. sonorensis in additional biotechnological applications. Comparison of metabolite production, growth, and enzyme activities in a representative set of transformed strains expressing different LDH genes in the presence and absence of a functional

  15. Heterologous expression of an algal hydrogenase in a hetero-cystous cyanobacterium

    Energy Technology Data Exchange (ETDEWEB)

    Thorsten Heidorn; Peter Lindblad [Dept. of Physiological Botany, Uppsala University, V illavagen 6, SE-752 36 Uppsala, (Sweden)

    2006-07-01

    For the expression of an active algal [FeFe] hydrogenase in the hetero-cystous cyanobacterium Nostoc punctiforme A TCC 29133 the Chlamydomonas reinhardtii hydrogenase gene hydA1 and the accessory genes hydEF and hydG are to be introduced into the cyano-bacterial cells. The genes were amplified by PCR from EST clones, cloned into the cloning vector pBluescript SK+ and sequenced. An expression vector for multi-cistronic cloning, based on pSCR202, was constructed and for a functional test GFP was inserted as a reporter gene. The GFP construct was transformed into Nostoc punctiforme A TCC 29133 by electroporation and expression of GFP was visualized by fluorescence microscopy. (authors)

  16. Heterologous Expression and Biochemical Characterization of Two Lipoxygenases in Oriental Melon, Cucumis melo var. makuwa Makino.

    Science.gov (United States)

    Cao, Songxiao; Chen, Hao; Zhang, Chong; Tang, Yufan; Liu, Jieying; Qi, Hongyan

    2016-01-01

    Lipoxygenases (LOXs) are a class of non-heme iron-containing dioxygenases that catalyse oxidation of polyunsaturated fatty acids to produce hydroperoxidation that are in turn converted to oxylipins. Although multiple isoforms of LOXs have been detected in several plants, LOXs in oriental melon have not attracted much attention. Two full-length LOX cDNA clones, CmLOX10 and CmLOX13 which have been isolated from oriental melon (Cucumis melo var. makuwa Makino) cultivar "Yumeiren", encode 902 and 906 amino acids, respectively. Bioinformatics analysis showed that CmLOX10 and CmLOX13 included all of the typical LOX domains and shared 58.11% identity at the amino acid level with each other. The phylogenetic analysis revealed that CmLOX10 and CmLOX13 were members of the type 2 13-LOX subgroup which are known to be involved in biotic and abiotic stress. Heterologous expression of the full-length CmLOX10 and truncated CmLOX13 in Escherichia coli revealed that the encoded exogenous proteins were identical to the predicted molecular weights and possessed the lipoxygenase activities. The purified CmLOX10 and CmLOX13 recombinant enzymes exhibited maximum activity at different temperature and pH and both had higher affinity for linoleic acid than linolenic acid. Chromatogram analysis of reaction products from the CmLOX10 and CmLOX13 enzyme reaction revealed that both enzymes produced 13S-hydroperoxides when linoleic acid was used as substrate. Furthermore, the subcellular localization analysis by transient expression of the two LOX fusion proteins in tobacco leaves showed that CmLOX10 and CmLOX13 proteins were located in plasma membrane and chloroplasts respectively. We propose that the two lipoxygenases may play different functions in oriental melon during plant growth and development.

  17. Inhibitory effects of coronary vasodilator papaverine on heterologously-expressed HERG currents in Xenopus oocytes

    Institute of Scientific and Technical Information of China (English)

    Cuk-seong KIM; Jin-bong PARK; Nam LEE; Sook-jin SON; Kyu-seung LEE; Hyo-shin KIM; Yong-geun KWAK; Soo-wan CHAE; Sang-do LEE; Byeong-hwa JEON

    2007-01-01

    Aim: To characterize the effects of papaverine on HERG channels expressed in Xenopus oocytes as well as cardiac action potential in rabbit ventricular myocytes.Methods: Conventional microelectrodes were used to record action potential in rabbit ventricular myocytes. HERG currents were recorded by 2-electrode voltage clamp technique in Xenopus oocytes injected with HERG cRNA. Results: Papa-verine increased the cardiac action potential duration in rabbit ventricular myocytes.It blocked heterologously-expressed HERG currents in a concentration-depen-dent manner (IC50 71.03±4.75 μmol/L, NH 0.80, n=6), whereas another phosphodi-esterase inhibitor, theophyUine (500 μmol/L), did not. The blockade of papaverine on HERG currents was not voltage-dependent. The slope conductance measured as a slope of the fully activated HERG current-voltage curves decreased from 78.03±4.25 μS of the control to 56.84±5.33, 36.06±6.53, and 27.09±5.50 μS (n=4) by 30, 100, and 300 μmol/L of papaverine, respectively. Papaverine (100 μmol/L)caused a 9 mV hyperpolarizing shift in the voltage-dependence of steady-state inactivation, but there were no changes in the voltage-dependence of HERG cur-rent activation. Papaverine blocked HERG channels in the closed, open, and inactivated states. Conclusion: These results showed that papaverine blocked HERG channels in a voltage- and state-independent manner, which may most likely be the major mechanism of papaverine-induced cardiac arrhythmia reported in humans.

  18. Heterologous expression and characterization of two chitinase 5 enzymes from the migratory locust Locusta migratoria.

    Science.gov (United States)

    Li, Ying-Long; Song, Hui-Fang; Zhang, Xue-Yao; Li, Da-Qi; Zhang, Ting-Ting; Ma, En-Bo; Zhang, Jian-Zhen

    2016-06-01

    Insect chitinases are involved in degradation of chitin from the exoskeleton or peritrophic metrix of midgut. In Locusta migratoria, two duplicated Cht5s (LmCht5-1 and LmCht5-2) have been shown to have distinct molecular characteristics and biological roles. To explore the protein properties of the two LmCht5s, we heterologously expressed both enzymes using baculovirus expression system in SF9 cells, and characterized kinetic and carbohydrate-binding properties of purified enzymes. LmCht5-1 and LmCht5-2 exhibited similar pH and temperature optimums. LmCht5-1 has lower Km value for the oligomeric substrate (4MU-(GlcNAc)3 ), and higher Km value for the longer substrate (CM-Chitin-RBV) compared with LmCht5-2. A comparison of amino acids and homology modeling of catalytic domain presented similar TIM barrel structures and differentiated amino acids between two proteins. LmCht5-1 has a chitin-binding domain (CBD) tightly bound to colloidal chitin, but LmCht5-2 does not have a CBD for binding to colloidal chitin. Our results suggested both LmCht5-1 and LmCht5-2, which have the critical glutamate residue in region II of catalytic domain, exhibited chitinolytic activity cleaving both polymeric and oligomeric substrates. LmCht5-1 had relatively higher activity against the oligomeric substrate, 4MU-(GlcNAc)3 , whereas LmCht5-2 exhibited higher activity toward the longer substrate, CM-Chitin-RBV. These findings are helpful for further research to clarify their different roles in insect growth and development.

  19. Comparison of Voltage Gated K(+) Currents in Arterial Myocytes with Heterologously Expressed K v Subunits.

    Science.gov (United States)

    Cox, Robert H; Fromme, Samantha

    2016-12-01

    We have shown that three components contribute to functional voltage gated K(+) (K v) currents in rat small mesenteric artery myocytes: (1) Kv1.2 plus Kv1.5 with Kvβ1.2 subunits, (2) Kv2.1 probably associated with Kv9.3 subunits, and (3) Kv7.4 subunits. To confirm and address subunit stoichiometry of the first two, we have compared the biophysical properties of K v currents in small mesenteric artery myocytes with those of Kv subunits heterologously expressed in HEK293 cells using whole cell voltage clamp methods. Selective inhibitors of Kv1 (correolide, COR) and Kv2 (stromatoxin, ScTx) channels were used to separate these K v current components. Conductance-voltage and steady state inactivation data along with time constants of activation, inactivation, and deactivation of native K v components were generally well represented by those of Kv1.2-1.5-β1.2 and Kv2.1-9.3 channels. The slope of the steady state inactivation-voltage curve (availability slope) proved to be the most sensitive measure of accessory subunit presence. The availability slope curves exhibited a single peak for both native K v components. Availability slope curves for Kv1.2-1.5-β1.2 and Kv2.1-9.3 channels expressed in human embryonic kidney cells also exhibited a single peak that shifted to more depolarized voltages with increasing accessory to α subunit transfection ratio. Availability slope curves for SxTc-insensitive currents were similar to those of Kv1.2-1.5 expressed with Kvβ1.2 at a 1:5 molar ratio while curves for COR-insensitive currents closely resembled those of Kv2.1 expressed with Kv9.3 at a 1:1 molar ratio. These results support the suggested Kv subunit combinations in small mesenteric artery, and further suggest that Kv1 α and Kvβ1.2 but not Kv2.1 and Kv9.3 subunits are present in a saturated (4:4) stoichiometry.

  20. Shewanella oneidensis: a new and efficient System for Expression and Maturation of heterologous [Fe-Fe] Hydrogenase from Chlamydomonas reinhardtii

    Directory of Open Access Journals (Sweden)

    Sybirna Kateryna

    2008-09-01

    Full Text Available Abstract Background The eukaryotic green alga, Chlamydomonas reinhardtii, produces H2 under anaerobic conditions, in a reaction catalysed by a [Fe-Fe] hydrogenase HydA1. For further biochemical and biophysical studies a suitable expression system of this enzyme should be found to overcome its weak expression in the host organism. Two heterologous expression systems used up to now have several advantages. However they are not free from some drawbacks. In this work we use bacterium Shewanella oneidensis as a new and efficient system for expression and maturation of HydA1 from Chlamydomonas reinhardtii. Results Based on codon usage bias and hydrogenase maturation ability, the bacterium S. oneidensis, which possesses putative [Fe-Fe] and [Ni-Fe] hydrogenase operons, was selected as the best potential host for C. reinhardtii [Fe-Fe] hydrogenase expression. Hydrogen formation by S. oneidensis strain AS52 (ΔhydAΔhyaB transformed with a plasmid bearing CrHydA1 and grown in the presence of six different substrates for anaerobic respiration was determined. A significant increase in hydrogen evolution was observed for cells grown in the presence of trimethylamine oxide, dimethylsulfoxide and disodium thiosulfate, showing that the system of S. oneidensis is efficient for heterologous expression of algal [Fe-Fe] hydrogenase. Conclusion In the present work a new efficient system for heterologous expression and maturation of C. reinhardtii hydrogenase has been developed. HydA1 of C. reinhardtii was purified and shown to contain 6 Fe atoms/molecule of protein, as expected. Using DMSO, TMAO or thiosulfate as substrates for anaerobic respiration during the cell growth, 0.4 – 0.5 mg l-1(OD600 = 1 of catalytically active HydA1 was obtained with hydrogen evolution rate of ~700 μmol H2 mg-1 min-1.

  1. Antigen production using heterologous expression of dengue virus-2 non-structural protein 1 (NS1) in Nicotiana tabacum (Havana) for immunodiagnostic purposes.

    Science.gov (United States)

    Amaro, Marilane O F; Xisto, Mariana F; Dias, Ana Carolina F; Versiani, Alice F; Cardoso, Silvia A; Otoni, Wagner C; da Silva, Cynthia C; De Paula, Sérgio O

    2015-06-01

    Expression of dengue-2 virus NS1 protein in Nicotiana tabacum plants for development of dengue immunodiagnostic kits. Dengue is one of the most important diseases caused by arboviruses in the world. A significant increase in its geographical distribution has been noticed over the last 20 years, with continuous transmission of several serotypes and emergence of the hemorrhagic fever in areas where the disease was previously not prevalent. Although the methodological processes for dengue diagnosis are in deep development and improvement, a limitation for the realization of dengue diagnostic tests is the difficulty of large-scale production of the antigen to be used in diagnostic tests. Due to this demand, the purpose of this study was to obtain the non-structural protein 1 (NS1) from dengue-2 serotype by heterologous expression in Nicotiana tabacum (Havana). After confirmation of the NS1 protein gene integration in the plant genome, the heterologous protein was characterized using SDS-PAGE and immunoblotting. In an immunoenzymatic test, the recombinant NS1 protein presents an antigen potential for development of dengue immunodiagnostic kits.

  2. Heterologous expression of a Tpo1 homolog from Arabidopsis thaliana confers resistance to the herbicide 2,4-D and other chemical stresses in yeast.

    Science.gov (United States)

    Cabrito, Tânia R; Teixeira, Miguel C; Duarte, Alexandra A; Duque, Paula; Sá-Correia, Isabel

    2009-10-01

    The understanding of the molecular mechanisms underlying acquired herbicide resistance is crucial in dealing with the emergence of resistant weeds. Saccharomyces cerevisiae has been used as a model system to gain insights into the mechanisms underlying resistance to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The TPO1 gene, encoding a multidrug resistance (MDR) plasma membrane transporter of the major facilitator superfamily (MFS), was previously found to confer resistance to 2,4-D in yeast and to be transcriptionally activated in response to the herbicide. In this work, we demonstrate that Tpo1p is required to reduce the intracellular concentration of 2,4-D. ScTpo1p homologs encoding putative plasma membrane MFS transporters from the plant model Arabidopsis thaliana were analyzed for a possible role in 2,4-D resistance. At5g13750 was chosen for further analysis, as its transcript levels were found to increase in 2,4-D stressed plants. The functional heterologous expression of this plant open reading frame in yeast was found to confer increased resistance to the herbicide in Deltatpo1 and wild-type cells, through the reduction of the intracellular concentration of 2,4-D. Heterologous expression of At5g13750 in yeast also leads to increased resistance to indole-3-acetic acid (IAA), Al(3+) and Tl(3+). At5g13750 is the first plant putative MFS transporter to be suggested as possibly involved in MDR.

  3. Titer improvement of iso-migrastatin in selected heterologous Streptomyces hosts and related analysis of mRNA expression by quantitative RT–PCR

    Science.gov (United States)

    Yang, Dong; Zhu, Xiangcheng; Wu, Xueyun; Feng, Zhiyang; Huang, Lei; Shen, Ben; Xu, Zhinan

    2011-01-01

    iso-Migrastatin (iso-MGS) has been actively pursued recently as an outstanding candidate of antimetastasis agents. Having characterized the iso-MGS biosynthetic gene cluster from its native producer Streptomyces platensis NRRL 18993, we have recently succeeded in producing iso-MGS in five selected heterologous Streptomyces hosts, albeit the low titers failed to meet expectations and cast doubt on the utility of this novel technique for large-scale production. To further explore and capitalize on the production capacity of these hosts, a thorough investigation of these five engineered strains with three fermentation media for iso-MGS production was undertaken. Streptomyces albus J1074 and Streptomyces lividans K4-114 were found to be preferred heterologous hosts, and subsequent analysis of carbon and nitrogen sources revealed that sucrose and yeast extract were ideal for iso-MGS production. After the initial optimization, the titers of iso-MGS in all five hosts were considerably improved by 3–18-fold in the optimized R2YE medium. Furthermore, the iso-MGS titer of S. albus J1074 (pBS11001) was significantly improved to 186.7 mg/L by a hybrid medium strategy. Addition of NaHCO3 to the latter finally afforded an optimized iso-MGS titer of 213.8 mg/L, about 5-fold higher than the originally reported system. With S. albus J1074 (pBS11001) as a model host, the expression of iso-MGS gene cluster in four different media was systematically studied via the quantitative RT–PCR technology. The resultant comparison revealed the correlation of gene expression and iso-MGS production for the first time; synchronous expression of the whole gene cluster was crucial for optimal iso-MGS production. These results reveal new insights into the iso-MGS biosynthetic machinery in heterologous hosts and provide the primary data to realize large-scale production of iso-MGS for further preclinical studies. PMID:21132287

  4. Heterologously expressed bacterial and human multidrug resistance proteins confer cadmium resistance to Escherichia coli

    NARCIS (Netherlands)

    Achard-Joris, M; van Saparoea, HBV; Driessen, AJM; Bourdineaud, JP; Bourdineaud, Jean-Paul

    2005-01-01

    The human MDR1 gene is induced by cadmium exposure although no resistance to this metal is observed in human cells overexpressing hMDR1. To access the role of MDR proteins in cadmium resistance, human MDR1, Lactococcus lactis lmrA, and Oenococcus oeni omrA were expressed in an Escherichia coli tolC

  5. Heterologous expression of rat testis GABAA receptor βt variant in Chinese hamster ovary cells

    Institute of Scientific and Technical Information of China (English)

    Shi-FengLi; Yu-GuangChen; Yuan-ChangYan; Yi-PingLi

    2004-01-01

    Aim: To study the characteristics and possible retention functionof specific sequence in the 5'-end of rat testis GABAA receptor β 3t variant, Methods: Rat testis GABAA receptor β 3t variant cDNA was cloned and inserted into two eukaryotic expression vectors of pEGFP-N1 and pEGFP-C1 respectively, which have EGFP reporter gene.

  6. Recombinant phytochrome of the moss Ceratodon purpureus: heterologous expression and kinetic analysis of Pr-->Pfr conversion.

    Science.gov (United States)

    Zeidler, M; Lamparter, T; Hughes, J; Hartmann, E; Remberg, A; Braslavsky, S; Schaffner, K; Gärtner, W

    1998-12-01

    The phytochrome-encoding gene Cerpu;PHY;2 (CP2) of the moss Ceratodon purpureus was heterologously expressed in Saccharomyces cerevisiae as a polyhistidine-tagged apoprotein and assembled with phytochromobilin (P phi B) and phycocyanobilin (PCB). Nickel-affinity chromatography yielded a protein fraction containing approximately 80% phytochrome. The holoproteins showed photoreversibility with both chromophores. Difference spectra gave maxima at 644/716 nm (red-absorbing phytochrome [Pr]/far-red-absorbing phytochrome [Pfr]) for the PCB adduct, and 659/724 nm for the P phi B-adduct, the latter in close agreement with values for phytochrome extracted from Ceratodon itself, implying that P phi B is the native chromophore in this moss species. Immunoblots stained with the antiphytochrome antibody APC1 showed that the recombinant phytochrome had the same molecular size as phytochrome from Ceratodon extracts. Further, the mobility of recombinant CP2 holophytochrome on native size-exclusion chromatography was similar to that of native oat phytochrome, implying that CP2 forms a dimer. Kinetics of absorbance changes during the Pr-->Pfr photoconversion of the PCB adduct, monitored between 620 and 740 nm in the microsecond range, revealed the rapid formation of a red-shifted intermediate (I700), decaying with a time constant of approximately 110 microseconds. This is similar to the behavior of phytochromes from higher plants when assembled with the same chromophore. When following the formation of the Pfr state, two major processes were identified (with time constants of 3 and 18 ms) that are followed by slow reactions in the range of 166 ms and 8 s, respectively, albeit with very small amplitudes.

  7. Identification of suitable grapevine reference genes for qRT-PCR derived from heterologous species.

    Science.gov (United States)

    Tashiro, Rebecca M; Philips, Joshua G; Winefield, Christopher S

    2016-02-01

    Identification and validation of suitable reference genes that exhibit robust transcriptional stability across many sample types is an absolute requirement of all qRT-PCR experiments. Often, however, only small numbers of reference genes, validated across limited sample types, are available for non-model species. This points to a clear need to assess and validate a wider range of potential reference genes than is currently available. We therefore looked to test and validate a large number of potential reference genes across a wide range of tissue types and treatments to determine the applicability of these reference genes for use in grapevine and other non-model plant species. Potential reference genes were selected based on stability of gene transcription in the model plant species Arabidopsis or due to their common use in the grapevine community. The selected reference genes were analyzed across two datasets consisting of a range of either 'Sauvignon blanc' or 'Pinot noir' tissues. A total of 11 potential reference genes were screened across the two datasets. Gene stability was analyzed by GeNorm, a widely used Excel application, or an ANOVA-based method developed in red clover. Both analysis methods showed that all 11 potential reference genes are stably expressed in the datasets tested, but the rankings of gene stability differed based on the datasets and analysis method used. Furthermore, the transcript stability of these genes, initially identified in Arabidopsis and now validated in grapevine, suggests applicability across a wide range of non-model plant species in addition to their utility in grapevine.

  8. Evaluation of different expression systems for the heterologous expression of pyranose 2-oxidase from Trametes multicolor in E. coli

    Directory of Open Access Journals (Sweden)

    Ludwig Roland

    2010-03-01

    Full Text Available Abstract The heterologous production of the industrially relevant fungal enzyme pyranose 2-oxidase in the prokaryotic host E. coli was investigated using 3 different expression systems, i.e. the well-studied T7 RNA polymerase based pET21d+, the L-arabinose inducible pBAD and the pCOLD system. Preliminary experiments were done in shaking flasks at 25°C and optimized induction conditions to compare the productivity levels of the different expression systems. The pET21d+ and the pCOLD system gave 29 U/L·h and 14 U/L·h of active pyranose 2-oxidase, respectively, whereas the pBAD system only produced 6 U/L·h. Process conditions for batch fermentations were optimized for the pET21d+ and the pCOLD systems in order to reduce the formation of inactive inclusion bodies. The highest productivity rate with the pET21d+ expression system in batch fermentations was determined at 25°C with 32 U/L·h. The pCOLD system showed the highest productivity rate (19 U/L·h at 25°C and induction from the start of the cultivation. Using the pCOLD system in a fed batch fermentation at 25°C with a specific growth rate of μ = 0.15 h-1resulted in the highest productivity rate of active pyranose oxidase with 206 U/L·h.

  9. Heterologous expression of the Bacillus subtilis (natto) alanine dehydrogenase in Escherichia coli and Lactococcus lactis.

    Science.gov (United States)

    Ye, Wei; Huo, Guicheng; Chen, Junliang; Liu, Fei; Yin, Jingyuan; Yang, Lijie; Ma, Xiaolong

    2010-05-30

    The major objective of the present study is to change the alanine production of Lactic acid bacteria by expression of Bacillus subtilis (natto) alanine dehydrogenase (AlaDH), the gene that is not present in Lactic acid. B. subtilis AlaDH gene (ald) was cloned into a pGEX6p-1 and expressed in E. coli JM109. Its enzyme activity was 48.3U/mg at 30 degrees C and 45.2U/mg at 42 degrees C. This ald gene was then cloned into a vector pNZ8148 to generate a vector pNZ8148/ald. The same ald gene was placed downstream of the ldh promoter from Streptococcus thermophilus to generate pNZ273/ldhp/ald. The pNZ8148/ald and pNZ273/ldhp/ald were introduced separately in Lactococcus lactis NZ9000. As a result of over-expressed ald, the production of alanine detected by HPLC in L. lactis NZ9000 carrying pNZ273/ldhp/ald reached 52mug/ml, an approximately 26-fold increase compared to the parent strain L. lactis NZ9000, but not in L. lactis NZ9000 carrying pNZ8148/ald. This study would help strain improvement to be used in dairy fermentation for developing healthy yogurts with sweet taste or other fermented dairy foods. Copyright 2009 Elsevier GmbH. All rights reserved.

  10. Gene Expression Omnibus (GEO)

    Data.gov (United States)

    U.S. Department of Health & Human Services — Gene Expression Omnibus is a public functional genomics data repository supporting MIAME-compliant submissions of array- and sequence-based data. Tools are provided...

  11. An influence of the copy number of biosynthetic gene clusters on the production level of antibiotics in a heterologous host.

    Science.gov (United States)

    Manderscheid, Niko; Bilyk, Bohdan; Busche, Tobias; Kalinowski, Jörn; Paululat, Thomas; Bechthold, Andreas; Petzke, Lutz; Luzhetskyy, Andriy

    2016-08-20

    Streptomyces albus J1074 is a well-known host for heterologous expression of secondary metabolites. To further increase its potential and to study the influence of cluster multiplication, additional φC31-attachment site was integrated into its genome using a system for transposon mutagenesis. Four secondary metabolite clusters were expressed in strains with different numbers of attachment sites, ranging from one to three copies of the site. Secondary metabolite production was examined and a new compound could be detected, purified and its structure was elucidated.

  12. Improved silencing suppression and enhanced heterologous protein expression are achieved using an engineered viral helper component proteinase.

    Science.gov (United States)

    Haikonen, T; Rajamäki, M-L; Valkonen, J P T

    2013-11-01

    RNA silencing limits transient expression of heterologous proteins in plants. Co-expression of viral silencing suppressor proteins can increase and prolong protein expression, but highly efficient silencing suppressors may stress plant tissue and be detrimental to protein yields. Little is known whether silencing suppression could be improved without harm to plant tissues. This study reports development of enhanced silencing suppressors by engineering the helper component proteinase (HCpro) of Potato virus A (PVA). Mutations were introduced to a short region of HCpro (positions 330-335 in PVA HCpro), which is hypervariable among potyviruses. Three out of the four HCpro mutants suppressed RNA silencing more efficiently and sustained expression of co-expressed jellyfish green fluorescent protein for a longer time than wild-type HCpro in agroinfiltrated leaves of Nicotiana benthamiana. Leaf tissues remained healthy-looking without any visible signs of stress.

  13. Heterologous Expression of PA8FAD9 and Functional Characterization of a Δ9-Fatty Acid Desaturase from a Cold-Tolerant Pseudomonas sp. A8.

    Science.gov (United States)

    Garba, Lawal; Ali, Mohd Shukuri Mohamad; Oslan, Siti Nurbaya; Rahman, Raja Noor Zaliha Raja Abdul

    2016-11-01

    Fatty acid desaturase enzymes are capable of inserting double bonds between carbon atoms of saturated fatty acyl-chains to produce unsaturated fatty acids. A gene coding for a putative Δ9-fatty acid desaturase-like protein was isolated from a cold-tolerant Pseudomonas sp. A8, cloned and heterologously expressed in Escherichia coli. The gene named as PA8FAD9 has an open reading frame of 1185 bp and codes for 394 amino acids with a predicted molecular weight of 45 kDa. The enzyme showed high Δ9-fatty acid desaturase-like protein activity and increased overall levels of cellular unsaturated fatty acids in the recombinant E. coli cells upon expression at different temperatures. The results showed that the ratio of palmitoleic to palmitic acid in the recombinant E. coli cells increased by more than twice the amount observed in the control cells at 20 °C using 0.4 mM IPTG. GCMS analysis confirmed the ability of this enzyme to convert exogenous stearic acid to oleic acid incorporated into the recombinant E. coli membrane phospholipids. It may be concluded that the PA8FAD9 gene from Pseudomonas sp. A8 codes for a putative Δ9-fatty acid desaturase protein actively expressed in E. coli under the influence of temperature and an inducer.

  14. RNA viral vectors for improved Agrobacterium-mediated transient expression of heterologous proteins in Nicotiana benthamiana cell suspensions and hairy roots

    Directory of Open Access Journals (Sweden)

    Larsen Jeffrey S

    2012-05-01

    Full Text Available Abstract Background Plant cell suspensions and hairy root cultures represent scalable protein expression platforms. Low protein product titers have thus far limited the application of transient protein expression in these hosts. The objective of this work was to overcome this limitation by harnessing A. tumefaciens to deliver replicating and non-replicating RNA viral vectors in plant tissue co-cultures. Results Replicating vectors derived from Potato virus X (PVX and Tobacco rattle virus (TRV were modified to contain the reporter gene β-glucuronidase (GUS with a plant intron to prevent bacterial expression. In cell suspensions, a minimal PVX vector retaining only the viral RNA polymerase gene yielded 6.6-fold more GUS than an analogous full-length PVX vector. Transient co-expression of the minimal PVX vector with P19 of Tomato bushy stunt virus or HC-Pro of Tobacco etch virus to suppress post-transcriptional gene silencing increased GUS expression by 44 and 83%, respectively. A non-replicating vector containing a leader sequence from Cowpea mosaic virus (CPMV-HT modified for enhanced translation led to 70% higher transient GUS expression than a control treatment. In hairy roots, a TRV vector capable of systemic movement increased GUS accumulation by 150-fold relative to the analogous PVX vector. Histochemical staining for GUS in TRV-infected hairy roots revealed the capacity for achieving even higher productivity per unit biomass. Conclusions For the first time, replicating PVX vectors and a non-replicating CPMV-HT vector were successfully applied toward transient heterologous protein expression in cell suspensions. A replicating TRV vector achieved transient GUS expression levels in hairy roots more than an order of magnitude higher than the highest level previously reported with a viral vector delivered by A. tumefaciens.

  15. RNA viral vectors for improved Agrobacterium-mediated transient expression of heterologous proteins in Nicotiana benthamiana cell suspensions and hairy roots

    Science.gov (United States)

    2012-01-01

    Background Plant cell suspensions and hairy root cultures represent scalable protein expression platforms. Low protein product titers have thus far limited the application of transient protein expression in these hosts. The objective of this work was to overcome this limitation by harnessing A. tumefaciens to deliver replicating and non-replicating RNA viral vectors in plant tissue co-cultures. Results Replicating vectors derived from Potato virus X (PVX) and Tobacco rattle virus (TRV) were modified to contain the reporter gene β-glucuronidase (GUS) with a plant intron to prevent bacterial expression. In cell suspensions, a minimal PVX vector retaining only the viral RNA polymerase gene yielded 6.6-fold more GUS than an analogous full-length PVX vector. Transient co-expression of the minimal PVX vector with P19 of Tomato bushy stunt virus or HC-Pro of Tobacco etch virus to suppress post-transcriptional gene silencing increased GUS expression by 44 and 83%, respectively. A non-replicating vector containing a leader sequence from Cowpea mosaic virus (CPMV-HT) modified for enhanced translation led to 70% higher transient GUS expression than a control treatment. In hairy roots, a TRV vector capable of systemic movement increased GUS accumulation by 150-fold relative to the analogous PVX vector. Histochemical staining for GUS in TRV-infected hairy roots revealed the capacity for achieving even higher productivity per unit biomass. Conclusions For the first time, replicating PVX vectors and a non-replicating CPMV-HT vector were successfully applied toward transient heterologous protein expression in cell suspensions. A replicating TRV vector achieved transient GUS expression levels in hairy roots more than an order of magnitude higher than the highest level previously reported with a viral vector delivered by A. tumefaciens. PMID:22559055

  16. A riboswitch-based inducible gene expression system for mycobacteria.

    Directory of Open Access Journals (Sweden)

    Jessica C Seeliger

    Full Text Available Research on the human pathogen Mycobacterium tuberculosis (Mtb would benefit from novel tools for regulated gene expression. Here we describe the characterization and application of a synthetic riboswitch-based system, which comprises a mycobacterial promoter for transcriptional control and a riboswitch for translational control. The system was used to induce and repress heterologous protein overexpression reversibly, to create a conditional gene knockdown, and to control gene expression in a macrophage infection model. Unlike existing systems for controlling gene expression in Mtb, the riboswitch does not require the co-expression of any accessory proteins: all of the regulatory machinery is encoded by a short DNA segment directly upstream of the target gene. The inducible riboswitch platform has the potential to be a powerful general strategy for creating customized gene regulation systems in Mtb.

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

    DEFF Research Database (Denmark)

    Bach, Søren Spanner

    by humanity in biopharmaceuticals or as industrial bioproducts. Yields and purity of diterpenoids purified from natural sources or made by chemical synthesis are generally insufficient for large-volume or high-end applications, thus alternative sources are needed. Synthetic biology, where heterologous pathways...... have been reconstructed in host production organisms is an attractive lternative, which holds the promise to enable a scalable, costeffective and table supply of natural products. Knowledge about the genes and mechanisms nvolved in the original pathway is a prerequisite for such heterologous production...... is compatible with native codon usage, and through the conserved mechanisms of protein targeting and posttranslational odifications, has the capacity to produce functional enzymes. To further explore plant based expression and characterization of diterpenoid pathway genes, two different plant expression hosts...

  18. Heterologous expression, biochemical characterization, and overproduction of alkaline α-amylase from Bacillus alcalophilus in Bacillus subtilis

    Directory of Open Access Journals (Sweden)

    Li Jianghua

    2011-10-01

    Full Text Available Abstract Background Alkaline α-amylases have potential applications for hydrolyzing starch under high pH conditions in the starch and textile industries and as ingredients in detergents for automatic dishwashers and laundries. While the alkaline α-amylase gains increased industrial interest, the yield of alkaline α-amylases from wild-type microbes is low, and the combination of genetic engineering and process optimization is necessary to achieve the overproduction of alkaline α-amylase. Results The alkaline α-amylase gene from Bacillus alcalophilus JN21 (CCTCC NO. M 2011229 was cloned and expressed in Bacillus subtilis strain WB600 with vector pMA5. The recombinant alkaline α-amylase was stable at pH from 7.0 to 11.0 and temperature below 40°C. The optimum pH and temperature of alkaline α-amylase was 9.0 and 50°C, respectively. Using soluble starch as the substrate, the Km and Vmax of alkaline α-amylase were 9.64 g/L and 0.80 g/(L·min, respectively. The effects of medium compositions (starch, peptone, and soybean meal and temperature on the recombinant production of alkaline α-amylase in B. subtilis were investigated. Under the optimal conditions (starch concentration 0.6% (w/v, peptone concentration 1.45% (w/v, soybean meal concentration 1.3% (w/v, and temperature 37°C, the highest yield of alkaline α-amylase reached 415 U/mL. The yield of alkaline α-amylase in a 3-L fermentor reached 441 U/mL, which was 79 times that of native alkaline α-amylase from B. alcalophilus JN21. Conclusions This is the first report concerning the heterologous expression of alkaline α-amylase in B. subtilis, and the obtained results make it feasible to achieve the industrial production of alkaline α-amylase with the recombinant B. subtilis.

  19. Heterologous protein production using euchromatin-containing expression vectors in mammalian cells

    Science.gov (United States)

    Zboray, Katalin; Sommeregger, Wolfgang; Bogner, Edith; Gili, Andreas; Sterovsky, Thomas; Fauland, Katharina; Grabner, Beatrice; Stiedl, Patricia; Moll, Herwig P.; Bauer, Anton; Kunert, Renate; Casanova, Emilio

    2015-01-01

    Upon stable cell line generation, chromosomal integration site of the vector DNA has a major impact on transgene expression. Here we apply an active gene environment, rather than specified genetic elements, in expression vectors used for random integration. We generated a set of Bacterial Artificial Chromosome (BAC) vectors with different open chromatin regions, promoters and gene regulatory elements and tested their impact on recombinant protein expression in CHO cells. We identified the Rosa26 BAC as the most efficient vector backbone showing a nine-fold increase in both polyclonal and clonal production of the human IgG-Fc. Clonal protein production was directly proportional to integrated vector copy numbers and remained stable during 10 weeks without selection pressure. Finally, we demonstrated the advantages of BAC-based vectors by producing two additional proteins, HIV-1 glycoprotein CN54gp140 and HIV-1 neutralizing PG9 antibody, in bioreactors and shake flasks reaching a production yield of 1 g/l. PMID:25977298

  20. Heterologous expression and characterization of the hydrophobin HFBI in Pichia pastoris and evaluation of its contribution to the food industry.

    Science.gov (United States)

    Niu, Baolong; Wang, Dandan; Yang, Yanyan; Xu, Haijin; Qiao, Mingqiang

    2012-08-01

    The class II hydrophobin HFBI from Trichoderma reesei was heterologously expressed by Pichia pastoris using pPIC9 vector under the control of the promoter AOX1. The recombinant HFBI (rHFBI) was purified by ultrafiltration and reverse-phase high performance liquid chromatography. Tricine-SDS-PAGE and Western blotting demonstrated that rHFBI with the expected molecular weight of 7.5 kDa was secreted into the culture medium. X-ray photoelectron spectroscopy and water contact angle measurements indicated that rHFBI could lead to the conversion of the wettability of the hydrophobic siliconized glass and hydrophilic mica surfaces relying on the self-assembly membrane on hydrophobic/hydrophilic interfaces. It was demonstrated that rHFBI had the ability to stabilize oil droplets, which was far excess of the class I hydrophobin HGFI heterologously expressed in P. pastoris (rHGFI) and the typical food emulsifier sodium caseinate. In gushing experiments, it was shown that rHFBI was a strong gushing inducer in beer, whereas rHGFI did not display any signs of gushing. This provided the potential of rHFBI to be used as a novel emulsifying agent and a predictor of gushing risk.

  1. Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications

    Directory of Open Access Journals (Sweden)

    Chew Chieng Yeo

    2016-02-01

    Full Text Available Toxin-antitoxin (TA systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies.

  2. Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications.

    Science.gov (United States)

    Yeo, Chew Chieng; Abu Bakar, Fauziah; Chan, Wai Ting; Espinosa, Manuel; Harikrishna, Jennifer Ann

    2016-02-19

    Toxin-antitoxin (TA) systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies.

  3. Heterologous Expression and Delivery of Biologically Active Exendin-4 by Lactobacillus paracasei L14

    Science.gov (United States)

    Zeng, Zhu; Yu, Rui; Zuo, Fanglei; Zhang, Bo; Peng, Deju; Ma, Huiqin; Chen, Shangwu

    2016-01-01

    Exendin-4, a glucagon-like protein-1 (GLP-1) receptor agonist, is an excellent therapeutic peptide drug for type 2 diabetes due to longer lasting biological activity compared to GLP-1. This study explored the feasibility of using probiotic Lactobacillus paracasei as an oral vector for recombinant exendin-4 peptide delivery, an alternative to costly chemical synthesis and inconvenient administration by injection. L. paracasei transformed with a plasmid encoding the exendin-4 gene (L. paracasei L14/pMG76e-exendin-4) with a constitutive promotor was successfully constructed and showed efficient secretion of exendin-4. The secreted exendin-4 significantly enhanced insulin secretion of INS-1 β-cells, along with an increment in their proliferation and inhibition of their apoptosis, corresponding to the effect of GLP-1 on these cells. The transcription level of the pancreatic duodenal homeobox-1 gene (PDX-1), a key transcription factor for cellular insulin synthesis and secretion, was upregulated by the treatment with secreted exendin-4, paralleling the upregulation of insulin gene expression. Caco-2 cell monolayer permeability assay showed a 34-fold increase in the transport of exendin-4 delivered by L. paracasei vs. that of free exendin-4 (control), suggesting effective facilitation of exendin-4 transport across the intestinal barrier by this delivery system. This study demonstrates that the probiotic Lactobacillus can be engineered to secrete bioactive exendin-4 and facilitate its transport through the intestinal barrier, providing a novel strategy for oral exendin-4 delivery using this lactic acid bacterium. PMID:27764251

  4. Identifying promoters for gene expression in Clostridium thermocellum

    Directory of Open Access Journals (Sweden)

    Daniel G. Olson

    2015-12-01

    Full Text Available A key tool for metabolic engineering is the ability to express heterologous genes. One obstacle to gene expression in non-model organisms, and especially in relatively uncharacterized bacteria, is the lack of well-characterized promoters. Here we test 17 promoter regions for their ability to drive expression of the reporter genes β-galactosidase (lacZ and NADPH-alcohol dehydrogenase (adhB in Clostridium thermocellum, an important bacterium for the production of cellulosic biofuels. Only three promoters have been commonly used for gene expression in C. thermocellum, gapDH, cbp and eno. Of the new promoters tested, 2638, 2926, 966 and 815 showed reliable expression. The 2638 promoter showed relatively higher activity when driving adhB (compared to lacZ, and the 815 promoter showed relatively higher activity when driving lacZ (compared to adhB.

  5. Heterologous Expression of AtWRKY57 Confers Drought Tolerance in Oryza sativa.

    Science.gov (United States)

    Jiang, Yanjuan; Qiu, Yuping; Hu, Yanru; Yu, Diqiu

    2016-01-01

    Drought stress is a severe environmental factor that greatly restricts plant distribution and crop production. Recently, we have found that overexpressing AtWRKY57 enhanced drought tolerance in Arabidopsis thaliana. In this study, we further reported that the Arabidopsis WRKY57 transcription factor was able to confer drought tolerance to transgenic rice (Oryza sativa) plants. The enhanced drought tolerance of transgenic rice was resulted from the lower water loss rates, cell death, malondialdehyde contents and relative electrolyte leakage while a higher proline content and reactive oxygen species-scavenging enzyme activities was observed during stress conditions. Moreover, further investigation revealed that the expression levels of several stress-responsive genes were up-regulated in drought-tolerant transgenic rice plants, compared with those in wild-type plants. In addition to the drought tolerance, the AtWRKY57 over-expressing plants also had enhanced salt and PEG stress tolerances. Taken together, our study indicates that over-expressing AtWRKY57 in rice improved not only drought tolerance but also salt and PEG tolerance, demonstrating its potential role in crop improvement.

  6. Heterologous expression of AtWRKY57 confers drought tolerance in Oryza sativa

    Directory of Open Access Journals (Sweden)

    Yanjuan eJiang

    2016-02-01

    Full Text Available Drought stress is a severe environmental factor that greatly restricts plant distribution and crop production. Recently, we have found that overexpressing AtWRKY57 enhanced drought tolerance in Arabidopsis thaliana. In this study, we further reported that the Arabidopsis WRKY57 transcription factor was able to confer drought tolerance to transgenic rice (Oryza sativa plants. The enhanced drought tolerance of transgenic rice was resulted from the lower water loss rates, cell death, malondialdehyde contents and relative electrolyte leakage while a higher proline content and reactive oxygen species-scavenging enzyme activities was observed during stress conditions. Moreover, further investigation revealed that the expression levels of several stress-responsive genes were up-regulated in drought-tolerant transgenic rice plants, compared with those in wild-type plants. In addition to the drought tolerance, the AtWRKY57 over-expressing plants also had enhanced salt and PEG stress tolerances. Taken together, our study indicates that over-expressing AtWRKY57 in rice improved not only drought tolerance but also salt and PEG tolerance, demonstrating its potential role in crop improvement.

  7. Folate biosynthesis in higher plants. cDNA cloning, heterologous expression, and characterization of dihydroneopterin aldolases.

    Science.gov (United States)

    Goyer, Aymeric; Illarionova, Victoria; Roje, Sanja; Fischer, Markus; Bacher, Adelbert; Hanson, Andrew D

    2004-05-01

    Dihydroneopterin aldolase (EC 4.1.2.25) is one of the enzymes of folate synthesis that remains to be cloned and characterized from plants. This enzyme catalyzes conversion of 7,8-dihydroneopterin (DHN) to 6-hydroxymethyl-7,8-dihydropterin, and is encoded by the folB gene in Escherichia coli. The E. coli FolB protein also mediates epimerization of DHN to 7,8-dihydromonapterin. Searches of the Arabidopsis genome detected three genes encoding substantially diverged FolB homologs (AtFolB1-3, sharing 57%-73% identity), for which cDNAs were isolated. A fourth cDNA specifying a FolB-like protein (LeFolB1) was obtained from tomato (Lycopersicon esculentum) by reverse transcription-PCR. When overproduced in E. coli, recombinant AtFolB1, AtFolB2, and LeFolB1 proteins all had both dihydroneopterin aldolase and epimerase activities, and carried out the aldol cleavage reaction on the epimerization product, 7,8-dihydromonapterin, as well as on DHN. AtFolB3, however, could not be expressed in active form. Size exclusion chromatography indicated that the plant enzyme is an octamer, like the bacterial enzyme. Quantifying expression of the Arabidopsis genes by real-time reverse transcription-PCR showed that AtFolB1 and AtFolB2 messages occur at low levels throughout the plant, whereas the AtFolB3 mRNA was detected only in siliques and only with an extremely low abundance. Sequence comparisons and phylogenetic analysis of FolB homologs from 16 plants indicated that their N-terminal regions are highly variable, and that most species have a small number of FolB genes that diverged after separation of the lineages leading to families. The substantial divergence of FolB homologs in Arabidopsis and other plants suggests that some of them may act on substrates other than DHN.

  8. Heterologous expression and characterization of a laccase from Laccaria bicolor in Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Bo Wang

    2016-01-01

    Full Text Available Synthetic dyes are known to be highly toxic to mammalian cells and mutagenic and carcinogenic to humans and, therefore, should be detoxified and removed from industrial effluents. Different approaches for removal and detoxication are extensively sought. Biochemical methods are considered the most economical and effective method of dye decolourization. In this research, the laccase gene from Laccaria bicolor was modified and expressed in Pichia pastoris. The properties of the recombinant laccase and its ability to degrade synthetic dyes were studied. The laccase activity was optimal at pH 2.2 and 50 °C. Its Km value was 0.187 mmol/L for ABTS [2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid]. The laccase obtained was shown to decolorize the synthetic dyes, malachite green, crystal violet and orange G, with ABTS as a mediator. These results indicated that the laccase obtained may be used to treat industrial effluents containing artificial dyes.

  9. Heterologous expression of plant cell wall glycosyltransferases in Pichia, pea and tobacco

    DEFF Research Database (Denmark)

    Petersen, Bent Larsen; Damager, Iben; Faber, Kirsten

    to participate in plant CW biosynthesis, has been achieved in only a few cases. We have previously reported the characterisation of two highly homologous plant-specific membrane-bound GTs, which when expressed as secreted tagged soluble proteins in the baculo virus system, catalysed the transfer of xylose from......The plant cell wall (CW) consists of numerous complex and uniqe carbohydrate polymer structures. Although the structure (sugar composition) of the various plant CW components are known in some detail, functional characterisation of of the more than 300 glycosyltransferases (GTs), that are believed...... UDP-xylose on to the monosaccharide sugar fucose. Partly based on these data, the two genes were proposed to function in the biosynthesis of pectic rhamnogalacturonan II (RG-II) and designated RhamnoGalacturonan XylosylTransferase 1 and -2 (RGXT1 and -2), accordingly (Egelund et al. 2006, The Plant...

  10. Enhanced succinic acid production in Aspergillus saccharolyticus by heterologous expression of fumarate reductase from Trypanosoma brucei.

    Science.gov (United States)

    Yang, Lei; Lübeck, Mette; Ahring, Birgitte K; Lübeck, Peter S

    2016-02-01

    Aspergillus saccharolyticus exhibits great potential as a cell factory for industrial production of dicarboxylic acids. In the analysis of the organic acid profile, A. saccharolyticus was cultivated in an acid production medium using two different pH conditions. The specific activities of the enzymes, pyruvate carboxylase (PYC), malate dehydrogenase (MDH), and fumarase (FUM), involved in the reductive tricarboxylic acid (rTCA) branch, were examined and compared in cells harvested from the acid production medium and a complete medium. The results showed that ambient pH had a significant impact on the pattern and the amount of organic acids produced by A. saccharolyticus. The wild-type strain produced higher amount of malic acid and succinic acid in the pH buffered condition (pH 6.5) compared with the pH non-buffered condition. The enzyme assays showed that the rTCA branch was active in the acid production medium as well as the complete medium, but the measured enzyme activities were different depending on the media. Furthermore, a soluble NADH-dependent fumarate reductase gene (frd) from Trypanosoma brucei was inserted and expressed in A. saccharolyticus. The expression of the frd gene led to an enhanced production of succinic acid in frd transformants compared with the wild-type in both pH buffered and pH non-buffered conditions with highest amount produced in the pH buffered condition (16.2 ± 0.5 g/L). This study demonstrates the feasibility of increasing succinic acid production through the cytosolic reductive pathway by genetic engineering in A. saccharolyticus.

  11. Molecular cloning, characterization and heterologous expression of bile salt hydrolase (Bsh) from Lactobacillus fermentum NCDO394.

    Science.gov (United States)

    Kumar, Rajesh; Rajkumar, Hemalatha; Kumar, Manoj; Varikuti, Sudarshan Reddy; Athimamula, Ramakrishna; Shujauddin, Mohd; Ramagoni, Ramesh; Kondapalli, Narendrababu

    2013-08-01

    Bile salt hydrolase (Bsh) active probiotic strains hydrolyze bile acid amino conjugates in vivo, which triggers cholesterol consumption in liver to synthesize new bile leading to consequential cholesterol lowering. Hence, bile salt hydrolyzing potential was the criterion to select L. fermentum NCDO394 for this study and its gene encoding Bsh was identified and cloned. The resulting nucleotide sequence of bsh gene contained an open reading frame (ORF) of 978 nucleotides encoding a predicted protein of 325 amino acids with a theoretical pI of 6.39. Moreover, deduced Bsh protein had high similarity with the Bshs of L. fermentum only and also exhibited significant similarity to the Pencillin V amidases of other Lactobacillus spp. Five catalytically important amino acids were highly conserved in L. fermentum Bsh while four amino acid motifs around these active sites, were not as consistent as in other Bsh proteins. Furthermore, L. fermentum bsh gene was sub-cloned into pET-28b(+) vector, and its expression was induced with 0.05 mM isopropylthiogalactopyranoside (IPTG) in Escherichia coli BL21(DE3). The recombinant Bsh (rBsh) was purified with homogeneity using Ni+2-NTA column and characterized for substrate specificity, pH and temperature. The rBsh hydrolyzed six major human bile salts with a slight preference towards glycine-conjugated bile salts. The optimum pH of rBsh was six, and its enzymatic activity declined below pH 5 and above pH 7. The enzyme was stable and functional even at 65 °C while showed its maximum activity at 37 °C. In conclusion, L. fermentum NCDO394 may be a promising candidate probiotic which may affect cholesterol metabolism in vivo.

  12. An efficient strategy for heterologous expression and purification of active peptide hainantoxin-IV.

    Directory of Open Access Journals (Sweden)

    Hui Zhang

    Full Text Available Hainantoxin-IV (HNTX-IV from the venom of the spider Selenocosmia hainana is a potent antagonist that specifically inhibits the tetrodotoxin-sensitive (TTX-S sodium channels. The toxin peptide consists of 35 amino acids and adopts a typical inhibitory cystine knot (ICK motif. To obtain adequate HNTX-IV peptides for further insight into the structure-activity relationships of the toxin, a novel strategy including cloning, expression and purification was developed in an E. coli expression system. For this purpose, a seamless restriction-free (RF cloning method was employed for the construction of an expression vector to avoid introducing unwanted sequences into the target gene. Furthermore, the solubility of recombinant HNTX-IV could be promoted efficiently by the combination of a glutathione S-transferase (GST tag and a small ubiquitin-related modifier (SUMO tag. Finally, an affinity-chromatography-free purification strategy was developed by cut-off dialysis tubing combined with trichloroacetic acid (TCA extraction. Further HPLC purification yielded recombinant, tag-free HNTX-IV with high yield and purity. The molecular weight of recombinant HNTX-IV (rHNTX-IV is identical to its theoretical value according to Matrix-Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS analysis. The recombinant toxin has similar activity (IC50 value of 120 nM on the tetrodotoxin-sensitive (TTX-S sodium channels in adult rat dorsal root ganglion (DRG neurons to native toxins. In the report, an efficient and cost-effective strategy for producing rHNTX-IV was developed, which paved the way for the further study of structure-activity relationships of rHNTX-IV and its pharmaceutical applications.

  13. A more desirable balanced polyunsaturated fatty acid composition achieved by heterologous expression of Δ15/Δ4 desaturases in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Guiming Zhu

    Full Text Available Arachidonic (ARA, eicosapentaenoic (EPA and docosahexaenoic (DHA acids are the most biologically active polyunsaturated fatty acids, but their biosyntheses in mammals are very limited. The biosynthesis of DHA is the most difficult, because this undergoes the Sprecher pathway--a further elongation step from docosapentaenoic acid (DPA, a Δ6-desaturase acting on a C24 fatty acid substrate followed by a peroxisomal chain shortening step. This paper reports the successful heterologous expression of two non-mammalian genes (with modification of codon usage, coding for Euglena gracilis Δ4-desaturase and Siganus canaliculatus Δ4-desaturase respectively, in mammalian cells (HEK293 cell line. Both of the Δ4-desaturases can efficiently function, directly converting DPA into DHA. Moreover, the cooperation of the E. gracilis Δ4-desaturase with C. elegans Δ15-desaturase (able to convert a number of n-6 PUFAs to their corresponding n-3 PUFAs in transgenic HEK293 cells made a more desirable fatty acid composition--a drastically reduced n-6/n-3 PUFAs ratio and a high level of DHA as well as EPA and ARA. Our findings provide a basis for potential applications of the gene constructs for expression of Δ15/Δ4-desaturases in transgenic livestock to produce such a fatty acid profile in the related products, which certainly will bring benefit to human health.

  14. Heterologous expression of a plant RelA-SpoT homologue results in increased stress tolerance in Saccharomyces cerevisiae by accumulation of the bacterial alarmone ppGpp.

    Science.gov (United States)

    Ochi, Kozo; Nishizawa, Tomoyasu; Inaoka, Takashi; Yamada, Akiyo; Hashimoto, Kohsuke; Hosaka, Takeshi; Okamoto, Susumu; Ozeki, Yoshihiro

    2012-08-01

    The bacterial alarmone ppGpp is present only in bacteria and the chloroplasts of plants, but not in mammalian cells or eukaryotic micro-organisms such as yeasts and fungi. The importance of the ppGpp signalling system in eukaryotes has therefore been largely overlooked. Here, we demonstrated that heterologous expression of a relA-spoT homologue (Sj-RSH) isolated from the halophilic plant Suaeda japonica in the yeast Saccharomyces cerevisiae results in accumulation of ppGpp, accompanied by enhancement of tolerance against various stress stimuli, such as osmotic stress, ethanol, hydrogen peroxide, high temperature and freezing. Unlike bacterial ppGpp accumulation, ppGpp was accumulated in the early growth phase but not in the late growth phase. Moreover, nutritional downshift resulted in a decrease in ppGpp level, suggesting that the observed Sj-RSH activity to synthesize ppGpp is not starvation-dependent, contrary to our expectations based on bacteria. Accumulated ppGpp was found to be present solely in the cytosolic fraction and not in the mitochondrial fraction, perhaps reflecting the ribosome-independent ppGpp synthesis in S. cerevisiae cells. Unlike bacterial inosine monophosphate (IMP) dehydrogenases, the IMP dehydrogenase of S. cerevisiae was insensitive to ppGpp. Microarray analysis showed that ppGpp accumulation gave rise to marked changes in gene expression, with both upregulation and downregulation, including changes in mitochondrial gene expression. The most prominent upregulation (38-fold) was detected in the hypothetical gene YBR072C-A of unknown function, followed by many other known stress-responsive genes. S. cerevisiae may provide new opportunities to uncover and analyse the ppGpp signalling system in eukaryotic cells.

  15. Isolation, characterization and heterologous expression of a novel chitosanase from Janthinobacterium sp. strain 4239

    Directory of Open Access Journals (Sweden)

    Stougaard Peter

    2010-01-01

    Full Text Available Abstract Background Chitosanases (EC 3.2.1.132 hydrolyze the polysaccharide chitosan, which is composed of partially acetylated β-(1,4-linked glucosamine residues. In nature, chitosanases are produced by a number of Gram-positive and Gram-negative bacteria, as well as by fungi, probably with the primary role of degrading chitosan from fungal and yeast cell walls for carbon metabolism. Chitosanases may also be utilized in eukaryotic cell manipulation for intracellular delivery of molecules formulated with chitosan as well as for transformation of filamentous fungi by temporal modification of the cell wall structures. However, the chitosanases used so far in transformation and transfection experiments show optimal activity at high temperature, which is incompatible with most transfection and transformation protocols. Thus, there is a need for chitosanases, which display activity at lower temperatures. Results This paper describes the isolation of a chitosanase-producing, cold-active bacterium affiliated to the genus Janthinobacterium. The 876 bp chitosanase gene from the Janthinobacterium strain was isolated and characterized. The chitosanase was related to the Glycosyl Hydrolase family 46 chitosanases with Streptomyces chitosanase as the closest related (64% amino acid sequence identity. The chitosanase was expressed recombinantly as a periplasmic enzyme in Escherichia coli in amounts about 500 fold greater than in the native Janthinobacterium strain. Determination of temperature and pH optimum showed that the native and the recombinant chitosanase have maximal activity at pH 5-7 and at 45°C, but with 30-70% of the maximum activity at 10°C and 30°C, respectively. Conclusions A novel chitosanase enzyme and its corresponding gene was isolated from Janthinobacterium and produced recombinantly in E. coli as a periplasmic enzyme. The Janthinobacterium chitosanase displayed reasonable activity at 10°C to 30°C, temperatures that are preferred in

  16. Heterologous expression, purification and characterization of nitrilase from Aspergillus niger K10

    Directory of Open Access Journals (Sweden)

    Felsberg Jürgen

    2011-01-01

    Full Text Available Abstract Background Nitrilases attract increasing attention due to their utility in the mild hydrolysis of nitriles. According to activity and gene screening, filamentous fungi are a rich source of nitrilases distinct in evolution from their widely examined bacterial counterparts. However, fungal nitrilases have been less explored than the bacterial ones. Nitrilases are typically heterogeneous in their quaternary structures, forming short spirals and extended filaments, these features making their structural studies difficult. Results A nitrilase gene was amplified by PCR from the cDNA library of Aspergillus niger K10. The PCR product was ligated into expression vectors pET-30(+ and pRSET B to construct plasmids pOK101 and pOK102, respectively. The recombinant nitrilase (Nit-ANigRec expressed in Escherichia coli BL21-Gold(DE3(pOK101/pTf16 was purified with an about 2-fold increase in specific activity and 35% yield. The apparent subunit size was 42.7 kDa, which is approx. 4 kDa higher than that of the enzyme isolated from the native organism (Nit-ANigWT, indicating post-translational cleavage in the enzyme's native environment. Mass spectrometry analysis showed that a C-terminal peptide (Val327 - Asn356 was present in Nit-ANigRec but missing in Nit-ANigWT and Asp298-Val313 peptide was shortened to Asp298-Arg310 in Nit-ANigWT. The latter enzyme was thus truncated by 46 amino acids. Enzymes Nit-ANigRec and Nit-ANigWT differed in substrate specificity, acid/amide ratio, reaction optima and stability. Refolded recombinant enzyme stored for one month at 4°C was fractionated by gel filtration, and fractions were examined by electron microscopy. The late fractions were further analyzed by analytical centrifugation and dynamic light scattering, and shown to consist of a rather homogeneous protein species composed of 12-16 subunits. This hypothesis was consistent with electron microscopy and our modelling of the multimeric nitrilase, which supports an

  17. A simple, highly efficient method for heterologous expression in mammalian primary neurons using cationic lipid-mediated mRNA transfection

    Directory of Open Access Journals (Sweden)

    Damian J Williams

    2010-11-01

    Full Text Available Expression of heterologous proteins in adult mammalian neurons is a valuable technique for the study of neuronal function. The postmitotic nature of mature neurons prevents effective DNA transfection using simple, cationic lipid-based methods. Adequate heterologous protein expression is often only achievable using complex techniques that, in many cases, are associated with substantial toxicity. Here, a simple method for high efficiency transfection of mammalian primary neurons using in vitro-transcribed mRNA and the cationic lipid transfection reagent Lipofectamine 2000 is described. Optimal transfection conditions were established in adult mouse dissociated dorsal root ganglion (DRG neurons using a 96-well based luciferase activity assay. Using these conditions, a transfection efficiency of 25% was achieved in DRG neurons transfected with EGFP mRNA. High transfection efficiencies were also obtained in dissociated rat superior cervical ganglion (SCG neurons and mouse cortical and hippocampal cultures. Endogenous Ca2+ currents in EGFP mRNA-transfected SCG neurons were not significantly different from untransfected neurons, which suggested that this technique is well suited for heterologous expression in patch clamp recording experiments. Functional expression of a cannabinoid receptor (CB1R, a G protein inwardly-rectifying K+ channel (GIRK4 and a dominant-negative G protein α-subunit mutant (GoA G203T indicate that the levels of heterologous protein expression attainable using mRNA transfection are suitable for most functional protein studies. This study demonstrates that mRNA transfection is a straightforward and effective method for heterologous expression in neurons and is likely to have many applications in neuroscience research.

  18. Effects of gene orientation and use of multiple promoters on the expression of XYL1 and XYL2 in Saccharomyces cerevisiae

    Science.gov (United States)

    Ju Yun Bae; Jose Laplaza; Thomas W. Jeffries

    2008-01-01

    Orientation of adjacent genes has been reported to affect their expression in eukaryotic systems, and metabolic engineering also often makes repeated use of a few promoters to obtain high expression. To improve transcriptional control in heterologous expression, we examined how these factors affect gene expression and enzymatic activity in Saccharomyces cerevisiae. We...

  19. Recombinant vectors construction for cellobiohydrolase encoding gene constitutive expression

    Directory of Open Access Journals (Sweden)

    Leontina GURGU

    2012-12-01

    Full Text Available Cellobiohydrolases (EC 3.2.1.91 are important exo enzymes involved in cellulose hydrolysis alongside endoglucanases (EC 3.2.1.4 and β-glucosidases (EC 3.2.1.21. Heterologous cellobiohydrolase gene expression under constitutive promoter control using Saccharomyces cerevisiae as host system is of great importance for a successful SSF process. From this point of view, the main objective of the work was to use Yeplac181 expression vector as a recipient for cellobiohdrolase - cbhB encoding gene expression under the control of the actin promoter, in Saccharomyces cerevisiae. Two hybridvectors, YEplac-Actp and YEplac-Actp-CbhB, were generated usingEscherichia coli XLI Blue for the cloning experiments. Constitutive cbhB gene expression was checked by proteine gel electrophoresis (SDS-PAGE after insertion of these constructs into Saccharomyces cerevisiae.

  20. Analysis of the role of the gene bipA, encoding the major endoplasmic reticulum chaperone protein in the secretion of homologous and heterologous proteins in black Aspergilli

    NARCIS (Netherlands)

    Punt, P.J.; Gemeren, I.A. van; Drint-Kuijvenhoven, J.; Hessing, J.G.M.; Muijlwijk van - Harteveld, G.M.; Beijersbergen, A.; Verrips, C.T.; Hondel, C.A.M.J.J. van den

    1998-01-01

    The function of the endoplasmic-reticulum-localized chaperone binding protein (BiP) in relation to protein secretion in filamentous fungi was studied. It was shown that the overproduction of several homologous and heterologous recombinant proteins by Aspergillus strains induces the expression of bip

  1. A new lactobacilli in vivo expression system for the production and delivery of heterologous proteins at mucosal surfaces.

    Science.gov (United States)

    Allain, Thibault; Mansour, Nahla M; Bahr, May M A; Martin, Rebeca; Florent, Isabelle; Langella, Philippe; Bermúdez-Humarán, Luis G

    2016-07-01

    Food-grade lactic acid bacteria, such as lactobacilli, represent good candidates for the development of mucosal vectors. Indeed, they are generally recognized as safe microorganisms and some strains display beneficial effects (probiotics). In this study, we described a new lactobacilli in vivo expression (LIVE) system for the production and delivery of therapeutic molecules at mucosal surfaces. The versatility and functionality of this system was successfully validated in several lactobacilli species; furthermore, we assessed in vivo LIVE system in two different mouse models of human pathologies: (i) a model of therapy against intestinal inflammation (inflammatory bowel diseases) and (ii) a model of vaccination against dental caries. We demonstrated that Lactobacillus gasseri expressing the anti-inflammatory cytokine IL-10 under LIVE system efficiently delivered the recombinant protein at mucosal surfaces and display anti-inflammatory effects. In the vaccination model against caries, LIVE system allowed the heterologous expression of Streptococcus mutans antigen GbpB by L. gasseri, leading to a stimulation of the host immune response.

  2. Aspergillus nidulans Synthesize Insect Juvenile Hormones upon Expression of a Heterologous Regulatory Protein and in Response to Grazing by Drosophila melanogaster Larvae

    DEFF Research Database (Denmark)

    Nielsen, Morten Thrane; Klejnstrup, Marie Louise; Rohlfs, Marko;

    2013-01-01

    Secondary metabolites are known to serve a wide range of specialized functions including communication, developmental control and defense. Genome sequencing of several fungal model species revealed that the majority of predicted secondary metabolite related genes are silent in laboratory strains......, indicating that fungal secondary metabolites remain an underexplored resource of bioactive molecules. In this study, we combine heterologous expression of regulatory proteins in Aspergillus nidulans with systematic variation of growth conditions and observe induced synthesis of insect juvenile hormone...... induced synthesis of juvenile hormone in A. nidulans indicating a possible role of juvenile hormone biosynthesis in affecting fungal-insect antagonisms....

  3. Tumor-specific gene expression patterns with gene expression profiles

    Institute of Scientific and Technical Information of China (English)

    RUAN Xiaogang; LI Yingxin; LI Jiangeng; GONG Daoxiong; WANG Jinlian

    2006-01-01

    Gene expression profiles of 14 common tumors and their counterpart normal tissues were analyzed with machine learning methods to address the problem of selection of tumor-specific genes and analysis of their differential expressions in tumor tissues. First, a variation of the Relief algorithm, "RFE_Relief algorithm" was proposed to learn the relations between genes and tissue types. Then, a support vector machine was employed to find the gene subset with the best classification performance for distinguishing cancerous tissues and their counterparts. After tissue-specific genes were removed, cross validation experiments were employed to demonstrate the common deregulated expressions of the selected gene in tumor tissues. The results indicate the existence of a specific expression fingerprint of these genes that is shared in different tumor tissues, and the hallmarks of the expression patterns of these genes in cancerous tissues are summarized at the end of this paper.

  4. Efficient Heterologous Transformation of Chlamydomonas reinhardtii npq2 Mutant with the Zeaxanthin Epoxidase Gene Isolated and Characterized from Chlorella zofingiensis

    Directory of Open Access Journals (Sweden)

    Herminia Rodríguez

    2012-09-01

    Full Text Available In the violaxanthin cycle, the violaxanthin de-epoxidase and zeaxanthin epoxidase catalyze the inter-conversion between violaxanthin and zeaxanthin in both plants and green algae. The zeaxanthin epoxidase gene from the green microalga Chlorella zofingiensis (Czzep has been isolated. This gene encodes a polypeptide of 596 amino acids. A single copy of Czzep has been found in the C. zofingiensis genome by Southern blot analysis. qPCR analysis has shown that transcript levels of Czzep were increased after zeaxanthin formation under high light conditions. The functionality of Czzep gene by heterologous genetic complementation in the Chlamydomonas mutant npq2, which lacks zeaxanthin epoxidase (ZEP activity and accumulates zeaxanthin in all conditions, was analyzed. The Czzep gene was adequately inserted in the pSI105 vector and expressed in npq2. The positive transformants were able to efficiently convert zeaxanthin into violaxanthin, as well as to restore their maximum quantum efficiency of the PSII (Fv/Fm. These results show that Chlamydomonas can be an efficient tool for heterologous expression and metabolic engineering for biotechnological applications.

  5. Functional Expression of the Ectoine Hydroxylase Gene (thpD) from Streptomyces chrysomallus in Halomonas elongata

    OpenAIRE

    Prabhu, Julia; Schauwecker, Florian; Grammel, Nicolas; Keller, Ullrich; Bernhard, Michael

    2004-01-01

    The formation of hydroxyectoine in the industrial ectoine producer Halomonas elongata was improved by the heterologous expression of the ectoine hydroxylase gene, thpD, from Streptomyces chrysomallus. The efficient conversion of ectoine to hydroxyectoine was achieved by the concerted regulation of thpD by the H. elongata ectA promoter.

  6. Heterologous expression of C. elegans fat-1 decreases the n-6/n-3 fatty acid ratio and inhibits adipogenesis in 3T3-L1 cells

    Energy Technology Data Exchange (ETDEWEB)

    An, Lei, E-mail: anleim@yahoo.com.cn [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China); Pang, Yun-Wei, E-mail: yunweipang@126.com [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China); Gao, Hong-Mei, E-mail: Gaohongmei_123@yahoo.cn [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China); Research Unit for Animal Life Sciences, Animal Resource Science Center, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Ibaraki-Iwama 319-0206 (Japan); Tao, Li, E-mail: Eunice8023@yahoo.cn [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China); College of Animal Science and Technology, Jilin Agricultural University, Changchun, Jilin 130118 (China); Miao, Kai, E-mail: miaokai7@163.com [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China); Wu, Zhong-Hong, E-mail: wuzhh@cau.edu.cn [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China); and others

    2012-11-23

    Highlights: Black-Right-Pointing-Pointer Expression of C. elegans fat-1 reduces the n-6/n-3 PUFA ratio in 3T3-L1 cells. Black-Right-Pointing-Pointer fat-1 inhibits the proliferation and differentiation of 3T3-L1 preadipocytes. Black-Right-Pointing-Pointer fat-1 reduces lipid deposition in 3T3-L1 adipocytes. Black-Right-Pointing-Pointer The lower n-6/n-3 ratio induces apoptosis in 3T3-L1 adipocytes. -- Abstract: In general, a diet enriched in polyunsaturated fatty acids (PUFAs) inhibits the development of obesity and decreases adipose tissue. The specific impacts of n-3 and n-6 PUFAs on adipogenesis, however, have not been definitively determined. Traditional in vivo and in vitro supplementation studies have yielded inconsistent or even contradictory results, which likely reflect insufficiently controlled experimental systems. Caenorhabditiselegans fat-1 gene encodes an n-3 fatty acid desaturase, and its heterologous expression represents an effective method both for altering the n-6/n-3 PUFA ratio and for evaluating the biological effects of n-3 and n-6 PUFAs. We sought to determine whether a reduced n-6/n-3 ratio could influence adipogenesis in 3T3-L1 cells. Lentivirus-mediated introduction of the fat-1 gene into 3T3-L1 preadipocytes significantly reduced the n-6/n-3 ratio and inhibited preadipocyte proliferation and differentiation. In mature adipocytes, fat-1 expression reduced lipid deposition, as measured by Oil Red O staining, and induced apoptosis. Our results indicate that a reduced n-6/n-3 ratio inhibits adipogenesis through several mechanisms and that n-3 PUFAs more effectively inhibit adipogenesis (but not lipogenesis) than do n-6 PUFAs.

  7. Heterologous expression of a fungal sterol esterase/lipase in different hosts: Effect on solubility, glycosylation and production.

    Science.gov (United States)

    Vaquero, María Eugenia; Barriuso, Jorge; Medrano, Francisco Javier; Prieto, Alicia; Martínez, María Jesús

    2015-12-01

    Ophiostoma piceae secretes a versatile sterol-esterase (OPE) that shows high efficiency in both hydrolysis and synthesis of triglycerides and sterol esters. This enzyme produces aggregates in aqueous solutions, but the recombinant protein, expressed in Komagataella (synonym Pichia) pastoris, showed higher catalytic efficiency because of its higher solubility. This fact owes to a modification in the N-terminal sequence of the protein expressed in Pichia pastoris, which incorporated 4-8 additional amino acids, affecting its aggregation behavior. In this study we present a newly engineered P. pastoris strain with improved protein production. We also produced the recombinant protein in the yeast Saccharomyces cerevisiae and in the prokaryotic host Escherichia coli, corroborating that the presence of these N-terminal extra amino acids affected the protein's solubility. The OPE produced in the new P. pastoris strain presented the same physicochemical properties than the old one. An inactive form of the enzyme was produced by the bacterium, but the recombinant esterase from both yeasts was active even after its enzymatic deglycosylation, suggesting that the presence of N-linked carbohydrates in the mature protein is not essential for enzyme activity. Although the yield in S. cerevisiae was lower than that obtained in P. pastoris, this work demonstrates the importance of the choice of the heterologous host for successful production of soluble and active recombinant protein. In addition, S. cerevisiae constitutes a good engineering platform for improving the properties of this biocatalyst.

  8. The generation of successive unmarked mutations and chromosomal insertion of heterologous genes in Actinobacillus pleuropneumoniae using natural transformation.

    Directory of Open Access Journals (Sweden)

    Janine T Bossé

    Full Text Available We have developed a simple method of generating scarless, unmarked mutations in Actinobacillus pleuropneumoniae by exploiting the ability of this bacterium to undergo natural transformation, and with no need to introduce plasmids encoding recombinases or resolvases. This method involves two successive rounds of natural transformation using linear DNA: the first introduces a cassette carrying cat (which allows selection by chloramphenicol and sacB (which allows counter-selection using sucrose flanked by sequences to either side of the target gene; the second transformation utilises the flanking sequences ligated directly to each other in order to remove the cat-sacB cassette. In order to ensure efficient uptake of the target DNA during transformation, A. pleuropneumoniae uptake sequences are added into the constructs used in both rounds of transformation. This method can be used to generate multiple successive deletions and can also be used to introduce targeted point mutations or insertions of heterologous genes into the A. pleuropneumoniae chromosome for development of live attenuated vaccine strains. So far, we have applied this method to highly transformable isolates of serovars 8 (MIDG2331, which is the most prevalent in the UK, and 15 (HS143. By screening clinical isolates of other serovars, it should be possible to identify other amenable strains.

  9. Genome mining of astaxanthin biosynthetic genes from Sphingomonas sp. ATCC 55669 for heterologous overproduction in Escherichia coli.

    Science.gov (United States)

    Ma, Tian; Zhou, Yuanjie; Li, Xiaowei; Zhu, Fayin; Cheng, Yongbo; Liu, Yi; Deng, Zixin; Liu, Tiangang

    2016-02-01

    As a highly valued keto-carotenoid, astaxanthin is widely used in nutritional supplements and pharmaceuticals. Therefore, the demand for biosynthetic astaxanthin and improved efficiency of astaxanthin biosynthesis has driven the investigation of metabolic engineering of native astaxanthin producers and heterologous hosts. However, microbial resources for astaxanthin are limited. In this study, we found that the α-Proteobacterium Sphingomonas sp. ATCC 55669 could produce astaxanthin naturally. We used whole-genome sequencing to identify the astaxanthin biosynthetic pathway using a combined PacBio-Illumina approach. The putative astaxanthin biosynthetic pathway in Sphingomonas sp. ATCC 55669 was predicted. For further confirmation, a high-efficiency targeted engineering carotenoid synthesis platform was constructed in E. coli for identifying the functional roles of candidate genes. All genes involved in astaxanthin biosynthesis showed discrete distributions on the chromosome. Moreover, the overexpression of exogenous E. coli idi in Sphingomonas sp. ATCC 55669 increased astaxanthin production by 5.4-fold. This study described a new astaxanthin producer and provided more biosynthesis components for bioengineering of astaxanthin in the future.

  10. A putative amino acid ABC transporter substrate-binding protein, NMB1612, from Neisseria meningitidis, induces murine bactericidal antibodies against meningococci expressing heterologous NMB1612 proteins.

    Science.gov (United States)

    Hung, Miao-Chiu; Humbert, María Victoria; Laver, Jay R; Phillips, Renee; Heckels, John E; Christodoulides, Myron

    2015-08-26

    The nmb1612 (NEIS1533) gene encoding the ~27-kDa putative amino acid ATP-binding cassette (ABC) transporter, periplasmic substrate-binding protein from Neisseria meningitidis serogroup B (MenB) strain MC58 was cloned and expressed in Escherichia coli, and the purified recombinant (r)NMB1612 was used for animal immunization studies. Immunization of mice with rNMB1612 adsorbed to Al(OH)3 and in liposomes with and without MPLA, induced antiserum with bactericidal activity in an assay using baby rabbit complement, against the homologous strain MC58 (encoding protein representative of Allele 62) and killed heterologous strains encoding proteins of three other alleles (representative of Alleles 1, 64 and 68), with similar SBA titres. However, strain MC58 was not killed (titre protein was killed (median titres of 16-64 in the hSBA). Analysis of the NMB1612 amino acid sequences from 4351 meningococcal strains in the pubmlst.org/Neisseria database and a collection of 13 isolates from colonized individuals and from patients, showed that antibodies raised against rNMB1612 could potentially kill at least 72% of the MenB strains in the complete sequence database. For MenB disease occurring specifically in the UK from 2013 to 2015, >91% of the isolates causing disease in this recent period expressed NMB1612 protein encoded by Allele 1 and could be potentially killed by sera raised to the recombinant antigen in the current study. The NMB1612 protein was surface-accessible and expressed by different meningococcal strains. In summary, the properties of (i) NMB1612 protein conservation and expression, (ii) limited amino acid sequence variation between proteins encoded by different alleles, and (iii) the ability of a recombinant protein to induce cross-strain bactericidal antibodies, would all suggest a promising antigen for consideration for inclusion in new meningococcal vaccines.

  11. Glutathione S-transferase Ya subunit gene: identification of regulatory elements required for basal level and inducible expression.

    OpenAIRE

    Telakowski-Hopkins, C A; King, R. G.; Pickett, C B

    1988-01-01

    The function of the 5'-flanking region of a rat glutathione S-transferase Ya subunit structural gene has been examined in homologous and heterologous cells. By using the 5'-flanking region of the Ya subunit gene fused to the structural gene encoding chloramphenicol acetyltransferase, we have identified two cis-acting regulatory elements in the upstream region of the Ya gene. One element is required for maximum basal level expression in homologous cells, whereas maximum basal level expression ...

  12. Heterologous expression and characterization of a sigma glutathione S-transferase involved in carbaryl detoxification from oriental migratory locust, Locusta migratoria manilensis (Meyen).

    Science.gov (United States)

    Qin, Guohua; Jia, Miao; Liu, Ting; Zhang, Xueyao; Guo, Yaping; Zhu, Kun Yan; Ma, Enbo; Zhang, Jianzhen

    2012-02-01

    Glutathione S-transferases (GSTs) play a major role in detoxification of xenobiotics and resistance to insecticides in insects. In the present study, a sigma-class GST gene (LmGSTs3) was identified from the locust, Locusta migratoria manilensis. Its full-length cDNA sequence is 828 bp containing an open reading frame (ORF) of 612 bp that encodes 204 amino acid residues. The predicted protein molecular mass and pI are 23.4 kDa and 7.62, respectively. Recombinant LmGSTs3 was heterologously expressed in Escherichia coli as a soluble fusion protein. Its optimal activity was observed at pH 8.0. Incubation for 30 min at temperatures below 40 °C scarcely affected activity. The LmGSTs3 at pH values between 4.0 and 11.0 retained more than 80% of its original activity. Ethacrynic acid and cibacron blue were very effective inhibitors of LmGSTs3 with I50-values 1.7 and 3.7 μM, respectively. In response to heavy metal (CuSO4, CdCl2) exposure there was a concentration-dependent and time-dependent decrease in activity. The nymph mortalities after carbaryl treatment increased 38.7% after LmGSTs3 were silenced. These results suggest that LmGSTs3 may be involved in carbaryl detoxification in L. migratoria manilensis.

  13. Heterologous expression of plant cell wall glycosyltransferases in Pichia, pea and tobacco

    DEFF Research Database (Denmark)

    Petersen, Bent Larsen; Damager, Iben; Faber, Kirsten

    UDP-xylose on to the monosaccharide sugar fucose. Partly based on these data, the two genes were proposed to function in the biosynthesis of pectic rhamnogalacturonan II (RG-II) and designated RhamnoGalacturonan XylosylTransferase 1 and -2 (RGXT1 and -2), accordingly (Egelund et al. 2006, The Plant...

  14. Heterologous expression and characterization of recombinant Lactococcus lactis neutral endopeptidase (Neprilysin)

    NARCIS (Netherlands)

    Lian, W; Wu, D; Konings, W.N; Mierau, I; Hersh, L.B

    1996-01-01

    A neutral endopeptidase (NEP) from Lactococcus lactis has recently been cloned and shown to contain high sequence homology with the human neutral endopeptidase, endopeptidase 24.11 (I. Mierau et al., J. Bacteriol. 175, 2087-2096, 1993). The gene for the neutral endopeptidase from L. lactis was clone

  15. Heterologous expression of plasmodial proteins for structural studies and functional annotation

    CSIR Research Space (South Africa)

    Birkholtz, LM

    2008-01-01

    Full Text Available of interest is cloned into a transfer vector, which is co-transfected with viral DNA into Spodoptera frugiperda (usually Sf9 or Sf21) insect cells. In a homologous recombination event, the foreign gene, under the control of a strong viral promoter...

  16. A chronic high fat diet alters the homologous and heterologous control of appetite regulating peptide receptor expression.

    Science.gov (United States)

    Kentish, Stephen J; Wittert, Gary A; Blackshaw, L Ashley; Page, Amanda J

    2013-08-01

    Leptin, ghrelin and neuropeptide W (NPW) modulate vagal afferent activity, which may underlie their appetite regulatory actions. High fat diet (HFD)-induced obesity induces changes in the plasma levels of these peptides and alters the expression of receptors on vagal afferents. We investigated homologous and heterologous receptor regulation by leptin, ghrelin and NPW. Mice were fed (12 weeks) a standard laboratory diet (SLD) or HFD. Nodose ganglia were cultured overnight in the presence or absence of each peptide. Leptin (LepR), ghrelin (GHS-R), NPW (GPR7) and cholecystokinin type-1 (CCK1R) receptor mRNA, and the plasma leptin, ghrelin and NPW levels were measured. SLD: leptin reduced LepR, GPR7, increased GHS-R and CCK1R mRNA; ghrelin increased LepR, GPR7, CCK1R, and decreased GHS-R. HFD: leptin decreased GHS-R and GPR7, ghrelin increased GHS-R and GPR7. NPW decreased all receptors except GPR7 which increased with HFD. Plasma leptin was higher and NPW lower in HFD. Thus, HFD-induced obesity disrupts inter-regulation of appetite regulatory receptors in vagal afferents.

  17. Cloning and heterologous expression of two aryl-aldehyde dehydrogenases from the white-rot basidiomycete Phanerochaete chrysosporium

    Energy Technology Data Exchange (ETDEWEB)

    Nakamura, Tomofumi [Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan); Fukuoka Institute of Health and Environmental Sciences, 39 Mukaizano, Dazaifu-shi, Fukuoka 818-0135 (Japan); Ichinose, Hirofumi [Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan); Wariishi, Hiroyuki, E-mail: hirowari@agr.kyushu-u.ac.jp [Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan); Bio-Architecture Center, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan); Innovation Center for Medical Redox Navigation, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan)

    2010-04-09

    We identified two aryl-aldehyde dehydrogenase proteins (PcALDH1 and PcALDH2) from the white-rot basidiomycete Phanerochaete chrysosporium. Both PcALDHs were translationally up-regulated in response to exogenous addition of vanillin, one of the key aromatic compounds in the pathway of lignin degradation by basidiomycetes. To clarify the catalytic functions of PcALDHs, we isolated full-length cDNAs encoding these proteins and heterologously expressed the recombinant enzymes using a pET/Escherichia coli system. The open reading frames of both PcALDH1 and PcALDH2 consisted of 1503 nucleotides. The deduced amino acid sequences of both proteins showed high homologies with aryl-aldehyde dehydrogenases from other organisms and contained ten conserved domains of ALDHs. Moreover, a novel glycine-rich motif 'GxGxxxG' was located at the NAD{sup +}-binding site. The recombinant PcALDHs catalyzed dehydrogenation reactions of several aryl-aldehyde compounds, including vanillin, to their corresponding aromatic acids. These results strongly suggested that PcALDHs metabolize aryl-aldehyde compounds generated during fungal degradation of lignin and various aromatic xenobiotics.

  18. Identification of a protein glycosylation operon from Campylobacter jejuni JCM 2013 and its heterologous expression in Escherichia coli.

    Science.gov (United States)

    Srichaisupakit, Akkaraphol; Ohashi, Takao; Fujiyama, Kazuhito

    2014-09-01

    Campylobacter jejuni is a human enteropathogenic bacterium possessing an N-glycosylation system. In this work, a protein glycosylation (pgl) operon conferring prokaryotic N-glycosylation in C. jejuni JCM 2013 was cloned and identified. Fourteen open reading frames (ORFs) were found in the pgl operon. The operon organization was similar to that of C. jejuni NCTC 11168, with 98% and 99% identities in overall nucleotide sequence and amino acid sequence, respectively. The pgl operon was heterologously co-expressed with model protein CmeA in the Escherichia coli BL21 ΔwaaL mutant. The immuno- and lectin-blotting analysis indicated the protein glycosylation on the recombinant CmeA. In addition, to analyze the glycan composition, the recombinant CmeA was purified and subjected to in-gel trypsin digestion followed by mass spectrometry analysis. The mass spectrometry analysis showed the presence of the N-acetylhexosamine residue at the reducing end but not the predicted di-N-acetylbacillosamine (diNAcBac) residue. Further glycan structural study using the conventional fluorophore-labeling method revealed the GalNAcα-GalNAcα-(Hex-)HexNAc-HexNAc-HexNAc-HexNAc structure. Transcriptional analysis showed that UDP-diNAcBac synthases and diNAcBac transferase are transcribed but might not function in the constructed system. In conclusion, a pgl operon from C. jejuni JCM 2013 successfully functioned in E. coli, resulting in the observed prokaryotic glycosylation.

  19. Cofactor engineering through heterologous expression of an NADH oxidase and its impact on metabolic flux redistribution in Klebsiella pneumoniae

    Directory of Open Access Journals (Sweden)

    Ji Xiao-Jun

    2013-01-01

    Full Text Available Abstract Background Acetoin is an important bio-based platform chemical. However, it is usually existed as a minor byproduct of 2,3-butanediol fermentation in bacteria. Results The present study reports introducing an exogenous NAD+ regeneration sysytem into a 2,3-butanediol producing strain Klebsiella pneumoniae to increse the accumulation of acetoin. Batch fermentation suggested that heterologous expression of the NADH oxidase in K. pneumoniae resulted in large decreases in the intracellular NADH concentration (1.4 fold and NADH/NAD+ ratio (2.0 fold. Metabolic flux analysis revealed that fluxes to acetoin and acetic acid were enhanced, whereas, production of lactic acid and ethanol were decreased, with the accumualation of 2,3-butanediol nearly unaltered. By fed-batch culture of the recombinant, the highest reported acetoin production level (25.9 g/L by Klebsiella species was obtained. Conclusions The present study indicates that microbial production of acetoin could be improved by decreasing the intracellular NADH/NAD+ ratio in K. pneumoniae. It demonstrated that the cofactor engineering method, which is by manipulating the level of intracellular cofactors to redirect cellular metabolism, could be employed to achieve a high efficiency of producing the NAD+-dependent microbial metabolite.

  20. 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

    for industrial production for many of these compounds. However, because these chemicals are often found in low abundance in the native plant, or are produced in plants which are difficult to cultivate, there is great interest in engineering increased production or expression of the biosynthetic pathways...

  1. Heterologous Expression of Rat Testis GABAA Receptor β3t Splicing Variant in CHO Cells

    Institute of Scientific and Technical Information of China (English)

    Shi-feng LI; Yu-guang CHEN; Yuan-chang YAN; Yi-ping LI

    2004-01-01

    Objective To characterize a possible retention function of unique sequence in the 5'end of rat testis GABAA receptor β3t splicing variantMethods Rat testis GABAA receptor β3t splicing variant cDNA was cloned and two eukaryotic expression recombinant plasmids of pEGFP-N1 and pEGFP-C1 were constructed respectively by fusing green fluorescent protein to the N or C-terminus of β3t isoform. The recombinant plasmids were transfected into CHO cells by calcium phosphate co-precipitation method. Fluorescence microscope and laser confocal microscope were used to analyze localization of β3t in the transfected cells. ConA-Texas-Red was used to label cell ER and the localization of rat testis β3t splicing variant in CHO cells was determined.Results When rat testis β3t splicing variant was expressed in CHO cells, two expression patterns were delineated, the distributions of uniform and mainly discrete intracellular compartments respectively. The chimera product failed to be translocated into the cell surface when expressed in CHO cells; whereas the β3 subunit of rat brain was incorporated into the plasma membrane.Conclusion The inability of β3t to target into the ER may be a consequence of the unique 25 specific amino acid segments in the N terminus.

  2. Engineering Cowpea Mosaic Virus RNA-2 into a vector to express heterologous proteins in plants

    NARCIS (Netherlands)

    Kodetham Gopinath,; Wellink, J.; Porta, C.; Taylor, K.M.; Lomonossoff, G.P.; Kammen, van A.

    2000-01-01

    series of new cowpea mosaic virus (CPMV) RNA-2-based expression vectors were designed. The jellyfish green fluorescent protein (GFP) was introduced between the movement protein (MP) and the large (L) coat protein or downstream of the small (S) coat protein. Release of the GFP inserted between the MP

  3. Functional heterologous protein expression by genetically engineered probiotic yeast Saccharomyces boulardii.

    Directory of Open Access Journals (Sweden)

    Lauren E Hudson

    Full Text Available Recent studies have suggested the potential of probiotic organisms to be adapted for the synthesis and delivery of oral therapeutics. The probiotic yeast Saccharomyces boulardii would be especially well suited for this purpose due to its ability, in contrast to probiotic prokaryotes, to perform eukaryotic post translational modifications. This probiotic yeast thus has the potential to express a broad array of therapeutic proteins. Currently, however, use of wild type (WT S. boulardii relies on antibiotic resistance for the selection of transformed yeast. Here we report the creation of auxotrophic mutant strains of S. boulardii that can be selected without antibiotics and demonstrate that these yeast can express functional recombinant protein even when recovered from gastrointestinal immune tissues in mice. A UV mutagenesis approach was employed to generate three uracil auxotrophic S. boulardii mutants that show a low rate of reversion to wild type growth. These mutants can express recombinant protein and are resistant in vitro to low pH, bile acid salts, and anaerobic conditions. Critically, oral gavage experiments using C57BL/6 mice demonstrate that mutant S. boulardii survive and are taken up into gastrointestinal immune tissues on a similar level as WT S. boulardii. Mutant yeast recovered from gastrointestinal immune tissues furthermore retain expression of functional recombinant protein. These data show that auxotrophic mutant S. boulardii can safely express recombinant protein without antibiotic selection and can deliver recombinant protein to gastrointestinal immune tissues. These auxotrophic mutants of S. boulardii pave the way for future experiments to test the ability of S. boulardii to deliver therapeutics and mediate protection against gastrointestinal disorders.

  4. Functional coupling between heterologously expressed dopamine D(2) receptors and KCNQ channels

    DEFF Research Database (Denmark)

    Ljungstrom, Trine; Grunnet, Morten; Jensen, Bo Skaaning

    2003-01-01

    Activation of KCNQ potassium channels by stimulation of co-expressed dopamine D(2) receptors was studied electrophysiologically in Xenopus laevis oocytes and in mammalian cells. To address the specificity of the interaction between D(2)-like receptors and KCNQ channels, combinations of KCNQ1...... activation of the KCNQ channels was confirmed by co-expression of other neuronal K(+) channels (BK, K(V)1.1, and K(V)4.3) with the D(2L) receptor in Xenopus oocytes. None of these K(+) channels responded to stimulation of the D(2L) receptor. In the mammalian brain, dopamine D(2) receptors and KCNQ channels...... co-localise postsynaptically in several brain regions, so modulation of neuronal excitability by dopamine release could in part be mediated via an effect on KCNQ channels....

  5. Heterologous expression of cellobiohydrolase II (Cel6A) in maize endosperm.

    Science.gov (United States)

    Devaiah, Shivakumar Pattada; Requesens, Deborah Vicuna; Chang, Yeun-Kyung; Hood, Kendall R; Flory, Ashley; Howard, John A; Hood, Elizabeth E

    2013-06-01

    The technology of converting lignocellulose to biofuels has advanced swiftly over the past few years, and enzymes are a significant constituent of this technology. In this regard, cost effective production of cellulases has been the focus of research for many years. One approach to reach cost targets of these enzymes involves the use of plants as bio-factories. The application of this technology to plant biomass conversion for biofuels and biobased products has the potential for significantly lowering the cost of these products due to lower enzyme production costs. Cel6A, one of the two cellobiohydrolases (CBH II) produced by Hypocrea jecorina, is an exoglucanase that cleaves primarily cellobiose units from the non-reducing end of cellulose microfibrils. In this work we describe the expression of Cel6A in maize endosperm as part of the process to lower the cost of this dominant enzyme for the bioconversion process. The enzyme is active on microcrystalline cellulose as exponential microbial growth was observed in the mixture of cellulose, cellulases, yeast and Cel6A, Cel7A (endoglucanase), and Cel5A (cellobiohydrolase I) expressed in maize seeds. We quantify the amount accumulated and the activity of the enzyme. Cel6A expressed in maize endosperm was purified to homogeneity and verified using peptide mass finger printing.

  6. Heat shock response improves heterologous protein secretion in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hou, Jin; Österlund, Tobias; Liu, Zihe

    2013-01-01

    The yeast Saccharomyces cerevisiae is a widely used platform for the production of heterologous proteins of medical or industrial interest. However, heterologous protein productivity is often low due to limitations of the host strain. Heat shock response (HSR) is an inducible, global, cellular...... stress response, which facilitates the cell recovery from many forms of stress, e.g., heat stress. In S. cerevisiae, HSR is regulated mainly by the transcription factor heat shock factor (Hsf1p) and many of its targets are genes coding for molecular chaperones that promote protein folding and prevent...... the accumulation of mis-folded or aggregated proteins. In this work, we over-expressed a mutant HSF1 gene HSF1-R206S which can constitutively activate HSR, so the heat shock response was induced at different levels, and we studied the impact of HSR on heterologous protein secretion. We found that moderate and high...

  7. Physiological relation between respiration activity and heterologous expression of selected benzoylformate decarboxylase variants in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Pohl Martina

    2010-10-01

    Full Text Available Abstract Background The benzoylformate decarboxylase (BFD from Pseudomonas putida is a biotechnologically interesting biocatalyst. It catalyses the formation of chiral 2-hydroxy ketones, which are important building blocks for stereoselective syntheses. To optimise the enzyme function often the amino acid composition is modified to improve the performance of the enzyme. So far it was assumed that a relatively small modification of the amino acid composition of a protein does not significantly influence the level of expression or media requirements. To determine, which effects these modifications might have on cultivation and product formation, six different BFD-variants with one or two altered amino acids and the wild type BFD were expressed in Escherichia coli SG13009 pKK233-2. The oxygen transfer rate (OTR as parameter for growth and metabolic activity of the different E. coli clones was monitored on-line in LB, TB and modified PanG mineral medium with the Respiratory Activity MOnitoring System (RAMOS. Results Although the E. coli clones were genetically nearly identical, the kinetics of their metabolic activity surprisingly differed in the standard media applied. Three different types of OTR curves could be distinguished. Whereas the first type (clones expressing Leu476Pro-Ser181Thr or Leu476Pro had typical OTR curves, the second type (clones expressing the wild type BFD, Ser181Thr or His281Ala showed an early drop of OTR in LB and TB medium and a drastically reduced maximum OTR in modified PanG mineral medium. The third type (clone expressing Leu476Gln behaved variable. Depending on the cultivation conditions, its OTR curve was similar to the first or the second type. It was shown, that the kinetics of the metabolic activity of the first type depended on the concentration of thiamine, which is a cofactor of BFD, in the medium. It was demonstrated that the cofactor binding strength of the different BFD-variants correlated with the differences

  8. Screening and expression of genes from metagenomes.

    Science.gov (United States)

    Leis, Benedikt; Angelov, Angel; Liebl, Wolfgang

    2013-01-01

    Microorganisms are the most abundant and widely spread organisms on earth. They colonize a huge variety of natural and anthropogenic environments, including very specialized ecological niches and even extreme habitats, which are made possible by the immense metabolic diversity and genetic adaptability of microbes. As most of the organisms from environmental samples defy cultivation, cultivation-independent metagenomics approaches have been applied since more than one decade to access and characterize the phylogenetic diversity in microbial communities as well as their metabolic potential and ecological functions. Thereby, metagenomics has fully emerged as an own scientific field for mining new biocatalysts for many industrially relevant processes in biotechnology and pharmaceutics. This review summarizes common metagenomic approaches ranging from sampling, isolation of nucleic acids, construction of metagenomic libraries and their evaluation. Sequence-based screenings implement next-generation sequencing platforms, microarrays or PCR-based methods, while function-based analysis covers heterologous expression of metagenomic libraries in diverse screening setups. Major constraints and advantages of each strategy are described. The importance of alternative host-vector systems is discussed, and in order to underline the role of phylogenetic and physiological distance from the gene donor and the expression host employed, a case study is presented that describes the screening of a genomic library from an extreme thermophilic bacterium in both Escherichia coli and Thermus thermophilus. Metatranscriptomics, metaproteomics and single-cell-based methods are expected to complement metagenomic screening efforts to identify novel biocatalysts from environmental samples.

  9. Heterologous expression and purification of barley (Hordeum vulgare L.) cysteine protease in yeast

    DEFF Research Database (Denmark)

    Rosenkilde, Anne Lind; Dionisio, Giuseppe; Holm, Preben Bach

    2011-01-01

    The mobilization of protein during germination of barley seeds is essential and Cysteine Proteases accounts for more than 90 % of the total proteolytic activity in the degradation of barley seed storage proteins [1]. Cysteine proteases exist as pro-enzyme until activated through reduction...... of the active site cysteines and via removal of the pro-domain. The complement of cysteine proteases is comprehensive and for detailed studies of the individual components of this complement, a fast and efficient eukaryotic expression platform is highly desirable. The barley key cysteine protease, endoprotease...

  10. Heterologous expression and characterisation of the Aspergillus aspartic protease involved in the hydrolysis and decolorisation of red-pigmented proteins.

    Science.gov (United States)

    Takenaka, Shinji; Umeda, Mayo; Senba, Hisanori; Koyama, Dai; Tanaka, Kosei; Yoshida, Ken-Ichi; Doi, Mikiharu

    2017-01-01

    Aspergillus repens strain MK82 produces an aspartic protease (PepA_MK82) that efficiently decolorises red-pigmented proteins during dried bonito fermentation. However, further expansion of the industrial applications of PepA_MK82 requires the high-level production and efficient preparation of the recombinant enzyme. The genomic DNA and cDNA fragments encoding the protease were cloned from strain MK82 and sequenced. Phylogenetic analysis of PepA_MK82 and comparisons with previously reported fungal aspartic proteases showed that PepA_MK 82 clusters with different groups of these enzymes. Heterologous expression of PepA_MK82 in Pichia pastoris yielded preparations of higher purity than obtained with an Escherichia coli expression system. Total protease activity in a 100-mL culture of the P. pastoris transformant was 14 times higher than that from an equivalent culture of A. repense MK82. The recombinant PepA_MK82 was easily obtained via acetone precipitation; the final recovery was 83%. PepA_MK82 and its recombinant had similar characteristics in terms of their optimal pH, thermostability, and decolorisation activity. The recombinant was also able to decolorise flaked, dried bonito and to bleach a blood-stained cloth. Given its ability to hydrolyse and decolorise red-pigmented proteins, recombinant PepA_MK8 can be exploited in the food industry and as a stain-removal agent in laundry applications. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  11. High diversity of genes for nonhost resistance of barley to heterologous rust fungi

    NARCIS (Netherlands)

    Jafary, H.; Albertazzi, G.; Marcel, T.C.; Niks, R.E.

    2008-01-01

    Inheritance studies on the nonhost resistance of plants would normally require interspecific crosses that suffer from sterility and abnormal segregation. Therefore, we developed the barley¿Puccinia rust model system to study, using forward genetics, the specificity, number, and diversity of genes in

  12. Engineering and Validation of a Vector for Concomitant Expression of Rare Transfer RNA (tRNA) and HIV-1 nef Genes in Escherichia coli.

    Science.gov (United States)

    Mualif, Siti Aisyah; Teow, Sin-Yeang; Omar, Tasyriq Che; Chew, Yik Wei; Yusoff, Narazah Mohd; Ali, Syed A

    2015-01-01

    Relative ease in handling and manipulation of Escherichia coli strains make them primary candidate to express proteins heterologously. Overexpression of heterologous genes that contain codons infrequently used by E. coli is related with difficulties such as mRNA instability, early termination of transcription and/or translation, deletions and/or misincorporation, and cell growth inhibition. These codon bias -associated problems are addressed by co-expressing ColE1-compatible, rare tRNA expressing helper plasmids. However, this approach has inadequacies, which we have addressed by engineering an expression vector that concomitantly expresses the heterologous protein of interest, and rare tRNA genes in E. coli. The expression vector contains three (argU, ileY, leuW) rare tRNA genes and a useful multiple cloning site for easy in-frame cloning. To maintain the overall size of the parental plasmid vector, the rare tRNA genes replaced the non-essential DNA segments in the vector. The cloned gene is expressed under the control of T7 promoter and resulting recombinant protein has a C-terminal 6His tag for IMAC-mediated purification. We have evaluated the usefulness of this expression vector by expressing three HIV-1 genes namely HIV-1 p27 (nef), HIV-1 p24 (ca), and HIV-1 vif in NiCo21(DE3) E.coli and demonstrated the advantages of using expression vector that concomitantly expresses rare tRNA and heterologous genes.

  13. The flow of gene expression.

    Science.gov (United States)

    Misteli, Tom

    2004-03-01

    Gene expression is a highly interconnected multistep process. A recent meeting in Iguazu Falls, Argentina, highlighted the need to uncover both the molecular details of each single step as well as the mechanisms of coordination among processes in order to fully understand the expression of genes.

  14. Heterologous expression and functional characterization of avian mu-class glutathione S-transferases.

    Science.gov (United States)

    Bunderson, Brett R; Kim, Ji Eun; Croasdell, Amanda; Mendoza, Kristelle M; Reed, Kent M; Coulombe, Roger A

    2013-08-01

    Hepatic glutathione S-transferases (GSTs: EC2.5.1.1.8) catalyze the detoxification of reactive electrophilic compounds, many of which are toxic and carcinogenic intermediates, via conjugation with the endogenous tripeptide glutathione (GSH). Glutathione S-transferase (GST)-mediated detoxification is a critical determinant of species susceptibility to the toxic and carcinogenic mycotoxin aflatoxin B1 (AFB1), which in resistant animals efficiently detoxifies the toxic intermediate produced by hepatic cytochrome P450 bioactivation, the exo-AFB1-8,9-epoxide (AFBO). Domestic turkeys (Meleagris gallopavo) are one of the most sensitive animals known to AFB1, a condition associated with a deficiency of hepatic GST-mediated detoxification of AFBO. We have recently shown that unlike their domestic counterparts, wild turkeys (Meleagris gallopavo silvestris), which are relatively resistant, express hepatic GST-mediated detoxification activity toward AFBO. Because of the importance of GSTs in species susceptibility, and to explore possible GST classes involved in AFB1 detoxification, we amplified, cloned, expressed and functionally characterized the hepatic mu-class GSTs tGSTM3 (GenBank accession no. JF340152), tGSTM4 (JF340153) from domestic turkeys, and a GSTM4 variant (ewGSTM4, JF340154) from Eastern wild turkeys. Predicted molecular masses of tGSTM3 and two tGSTM4 variants were 25.6 and 25.8kDa, respectively. Multiple sequence comparisons revealed four GSTM motifs and the mu-loop in both proteins. tGSTM4 has 89% amino acid sequence identity to chicken GSTM2, while tGSTM3 has 73% sequence identity to human GSTM3 (hGSTM3). Specific activities of Escherichia coli-expressed tGSTM3 toward 1-chloro-2,4-dinitrobenzene (CDNB) and peroxidase activity toward cumene hydroperoxide were five-fold greater than tGSTM4 while tGSTM4 possessed more than three-fold greater activity toward 1,2-dichloro-4-nitrobenzene (DCNB). The two enzymes displayed equal activity toward ethacrynic acid (ECA

  15. Effect of culture temperature on the heterologous expression of Pleurotus eryngii versatile peroxidase in Aspergillus hosts.

    Science.gov (United States)

    Eibes, G M; Lú-Chau, T A; Ruiz-Dueñas, F J; Feijoo, G; Martínez, M J; Martínez, A T; Lema, J M

    2009-01-01

    Production of recombinant versatile peroxidase in Aspergillus hosts was optimized through the modification of temperature during bioreactor cultivations. To further this purpose, the cDNA encoding a versatile peroxidase of Pleurotus eryngii was expressed under control of the alcohol dehydrogenase (alcA) promoter of Aspergillus nidulans. A dependence of recombinant peroxidase production on cultivation temperature was found. Lowering the culture temperature from 28 to 19 degrees C enhanced the level of active peroxidase 5.8-fold and reduced the effective proteolytic activity twofold. Thus, a maximum peroxidase activity of 466 U L(-1) was reached. The same optimization scheme was applied to a recombinant Aspergillus niger that bore the alcohol dehydrogenase regulator (alcR), enabling transformation with the peroxidase cDNA under the same alcA promoter. However, with this strain, the peroxidase activity was not improved, while the effective proteolytic activity was increased between 3- and 11-fold compared to that obtained with A. nidulans.

  16. Increasing the stearate content in seed oil of Brassica juncea by heterologous expression of MlFatB affects lipid content and germination frequency of transgenic seeds.

    Science.gov (United States)

    Bhattacharya, Surajit; Sinha, Saheli; Das, Natasha; Maiti, Mrinal K

    2015-11-01

    Fatty acids from dietary lipids can impart both beneficial and harmful health effects. The compositional balance between saturated and unsaturated fatty acids plays a decisive role in maintaining the physiological harmony, proper growth and development in the human system. In case of Brassica juncea seed oil, the level of saturated fatty acid, especially desirable stearate is very much lower than the recommended value, along with a high content of nutritionally undesirable erucic acid. Therefore, in order to shift the carbon flux towards the production of stearate at the expense of erucate, the MlFatB gene encoding a FatB thioesterase from Madhuca longifolia (latifolia) was expressed heterologously in seed tissues of B. juncea. The functional MlFatB competed with the highly active endogenous BjFatA thioesterase, and the transgenic B. juncea lines showed noteworthy changes in their seed fatty acid profiles. The proportion of stearate increased up to 16-fold, constituting almost 31% of the total fatty acids along with the production of arachidic acid in significant amount (up to ∼11%). Moreover, the content of erucate was reduced up to 71% in the seed oils of transgenic lines. Although a nutritionally desirable fatty acid profile was achieved, the transgenic seeds exhibit reduction or abolition of seed germination in addition to a decrease in seed lipid content. The findings of the present study revealing the stearoyl-ACP thioesterase-mediated enhancement of the stearate content that is associated with reduced germination frequency of transgenic B. juncea seeds, may explain why no natural or induced stearate-rich Brassica has been found or developed. Furthermore, this study also suggests that the newly characterized MlFatB is a potential candidate gene for refined metabolic engineering strategy in B. juncea or other plant species for increasing stearate content in seed oil.

  17. Enhanced production of butanol and acetoin by heterologous expression of an acetolactate decarboxylase in Clostridium acetobutylicum.

    Science.gov (United States)

    Shen, Xiaoning; Liu, Dong; Liu, Jun; Wang, Yanyan; Xu, Jiahui; Yang, Zhengjiao; Guo, Ting; Niu, Huanqing; Ying, Hanjie

    2016-09-01

    Butanol is an important industrial chemical and an attractive transportation fuel. However, the deficiency of reducing equivalents NAD(P)H in butanol fermentation results in a large quantity of oxidation products, which is a major problem limiting the atom economy and economic viability of bio-butanol processes. Here, we integrated the butanol fermentation process with a NADH-generating, acetoin biosynthesis process to improve the butanol production. By overexpressing the α-acetolactate decarboxylase gene alsD from Bacillus subtilis in Clostridium acetobutylicum, acetoin yield was significantly increased at the cost of acetone. After optimization of fermentation conditions, butanol (12.9g/L), acetoin (6.5g/L), and ethanol (1.9g/L) were generated by the recombinant strain, with acetone no more than 1.8g/L. Thus, both mass yield and product value were greatly improved. This study demonstrates that reducing power compensation is effective to improve the atom economy of butanol fermentation, and provides a novel approach to improve the economic viability of bio-butanol production. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Viral promoters can initiate expression of toxin genes introduced into Escherichia coli.

    Science.gov (United States)

    Lewin, Astrid; Mayer, Martin; Chusainow, Janet; Jacob, Daniela; Appel, Bernd

    2005-06-20

    The expression of recombinant proteins in eukaryotic cells requires the fusion of the coding region to a promoter functional in the eukaryotic cell line. Viral promoters are very often used for this purpose. The preceding cloning procedures are usually performed in Escherichia coli and it is therefore of interest if the foreign promoter results in an expression of the gene in bacteria. In the case molecules toxic for humans are to be expressed, this knowledge is indispensable for the specification of safety measures. We selected five frequently used viral promoters and quantified their activity in E. coli with a reporter system. Only the promoter from the thymidine kinase gene from HSV1 showed no activity, while the polyhedrin promoter from baculovirus, the early immediate CMV promoter, the early SV40 promoter and the 5' LTR promoter from HIV-1 directed gene expression in E. coli. The determination of transcription start sites in the immediate early CMV promoter and the polyhedrin promoter confirmed the existence of bacterial -10 and -35 consensus sequences. The importance of this heterologous gene expression for safety considerations was further supported by analysing fusions between the aforementioned promoters and a promoter-less cytotoxin gene. According to our results a high percentage of viral promoters have the ability of initiating gene expression in E. coli. The degree of such heterologous gene expression can be sufficient for the expression of toxin genes and must therefore be considered when defining safety measures for the handling of corresponding genetically modified organisms.

  19. Ascidian gene-expression profiles

    OpenAIRE

    Jeffery, William R.

    2002-01-01

    With the advent of gene-expression profiling, a large number of genes can now be investigated simultaneously during critical stages of development. This approach will be particularly informative in studies of ascidians, basal chordates whose genomes and embryology are uniquely suited for mapping developmental gene networks.

  20. Heterologous expression and biochemical characterization of glucose isomerase from Thermobifida fusca.

    Science.gov (United States)

    Deng, Hui; Chen, Sheng; Wu, Dan; Chen, Jian; Wu, Jing

    2014-06-01

    Glucose isomerase (GIase) catalyzes the isomerization of D-glucose to D-fructose. The GIase from Thermobifida fusca WSH03-11 was expressed in Escherichia coli BL21(DE3), and the purified enzyme took the form of a tetramer in solution and displayed a pI value of 5.05. The temperature optimum of GIase was 80 °C and its half life was about 2 h at 80 °C or 15 h at 70 °C. The pH optimum of GIase was 10 and the enzyme retained 95 % activity over the pH range of 5-10 after incubating at 4 °C for 24 h. Kinetic studies showed that the K m and K cat values of the enzyme are 197 mM and 1,688 min(-1), respectively. The maximum conversion yield of glucose (45 %, w/v) to fructose of the enzyme was 53 % at pH 7.5 and 70 °C. The present study provides the basis for the industrial application of recombinant T. fusca GIase in the production of high fructose syrup.

  1. Human glucocerebrosidase: heterologous expression of active site mutants in murine null cells.

    Science.gov (United States)

    Fabrega, S; Durand, P; Codogno, P; Bauvy, C; Delomenie, C; Henrissat, B; Martin, B M; McKinney, C; Ginns, E I; Mornon, J P; Lehn, P

    2000-11-01

    Using bioinformatics methods, we have previously identified Glu235 and Glu340 as the putative acid/base catalyst and nucleophile, respectively, in the active site of human glucocerebrosidase. Thus, we undertook site-directed mutagenesis studies to obtain experimental evidence supporting these predictions. Recombinant retroviruses were used to express wild-type and E235A and E340A mutant proteins in glucocerebrosidase-deficient murine cells. In contrast to wild-type enzyme, the mutants were found to be catalytically inactive. We also report the results of various studies (Western blotting, glycosylation analysis, subcellular fractionation, and confocal microscopy) indicating that the wild-type and mutant enzymes are identically processed and sorted to the lysosomes. Thus, enzymatic inactivity of the mutant proteins is not the result of incorrect folding/processing. These findings indicate that Glu235 plays a key role in the catalytic machinery of human glucocerebrosidase and may indeed be the acid/base catalyst. As concerns Glu340, the results both support our computer-based predictions and confirm, at the biological level, previous identification of Glu340 as the nucleophile by use of active site labeling techniques. Finally, our findings may help to better understand the molecular basis of Gaucher disease, the human lysosomal disease resulting from deficiency in glucocerebrosidase.

  2. Four disulfide-bridged scorpion beta neurotoxin CssII: heterologous expression and proper folding in vitro.

    OpenAIRE

    Estrada, Georgina; Garcia, Blanca,; Schiavon, Emanuele; Ortiz, Ernesto; Cestèle, Sandrine; Wanke, Enzo; Possani, Lourival,; Corzo, Gerardo

    2007-01-01

    International audience; The gene of the four disulfide-bridged Centruroides suffusus suffusus toxin II was cloned into the expression vector pQE30 containing a 6His-tag and a FXa proteolytic cleavage region. This recombinant vector was transfected into Escherichia coli BL21 cells and expressed under induction with isopropyl thiogalactoside (IPTG). The level of expression was 24.6 mg/l of culture medium, and the His tagged recombinant toxin (HisrCssII) was found exclusively in inclusion bodies...

  3. Heterologous expression of Paranosema (Antonospora) locustae hexokinase in lepidopteran, Sf9, cells is followed by accumulation of the microsporidian protein in insect cell nuclei.

    Science.gov (United States)

    Timofeev, Sergey A; Senderskiy, Igor V; Tsarev, Alexander A; Tokarev, Yuri S; Dolgikh, Viacheslav V

    2017-02-01

    Paranosema (Nosema, Antonospora) locustae is the only microsporidium produced as a commercial product for biological control. Molecular mechanisms of the effects of this pathogen and other invertebrate microsporidia on host cells remain uncharacterized. Previously, we immunolocalized P. locustae hexokinase in nuclei of Locusta migratoria infected adipocytes. Here, the microsporidian protein was expressed in the yeast Pichia pastoris and in lepidopteran Sf9 cells. During heterologous expression, P. locustae hexokinase was accumulated in the nuclei of insect cells but not in yeast cell nuclei. This confirms nuclear localization of hexokinase secreted by microsporidia into infected host cells and suggests convenient model for its further study.

  4. Human Lacrimal Gland Gene Expression

    Science.gov (United States)

    Aakalu, Vinay Kumar; Parameswaran, Sowmya; Maienschein-Cline, Mark; Bahroos, Neil; Shah, Dhara; Ali, Marwan; Krishnakumar, Subramanian

    2017-01-01

    Background The study of human lacrimal gland biology and development is limited. Lacrimal gland tissue is damaged or poorly functional in a number of disease states including dry eye disease. Development of cell based therapies for lacrimal gland diseases requires a better understanding of the gene expression and signaling pathways in lacrimal gland. Differential gene expression analysis between lacrimal gland and other embryologically similar tissues may be helpful in furthering our understanding of lacrimal gland development. Methods We performed global gene expression analysis of human lacrimal gland tissue using Affymetrix ® gene expression arrays. Primary data from our laboratory was compared with datasets available in the NLM GEO database for other surface ectodermal tissues including salivary gland, skin, conjunctiva and corneal epithelium. Results The analysis revealed statistically significant difference in the gene expression of lacrimal gland tissue compared to other ectodermal tissues. The lacrimal gland specific, cell surface secretory protein encoding genes and critical signaling pathways which distinguish lacrimal gland from other ectodermal tissues are described. Conclusions Differential gene expression in human lacrimal gland compared with other ectodermal tissue types revealed interesting patterns which may serve as the basis for future studies in directed differentiation among other areas. PMID:28081151

  5. Agonist-induced hump current production in heterologously-expressed human α4β2-nicotinic acetylcholine receptors

    Institute of Scientific and Technical Information of China (English)

    Qiang LIE; Ke-wei YU; Yong-chang CHANG; Ronald J LUKAS; Jie WU

    2008-01-01

    Aim:To characterize the functional and pharmacological features of agonist-induced hump currents in human α4β2-nicotinic acetylcholine receptors (nAChR).Methods:Whole-cell and outside-out patch recordings were performed using human α4β2-nAChR heterologously expressed in stably-transfected,native nAChR-null subclonal human epithelial 1 (SH-EP1) cells.RT-PCR was used to test the mRNA expression of transfected nAChR.Homology modeling and ace-tylcholine (Ach) docking were applied to show the possible Ach-binding site in the channel pore.Results:The rapid exposure of 10 mmol/L Ach induced an inward current with a decline from peak to steady-state.However,after the re-moval of Ach,an additional inward current,called "hump" current,reoccurred.The ability of agonists to produce these hump currents cannot be easily explained based on drug size,charge,acute potency,or actions as full or partial agonists.Hump currents were associated with a rebound increase in whole-cell conductance,and they had voltage dependence-like peak currents induced by agonist action.Hump currents blocked by the α4β2-nAChR antagonist dihydro-β-erythroidine were reduced when α4β2-nAChR were desensitized,and were more pronounced in the absence of external Ca2+.Outside-out single-channel recordings demon-strated that compared to 1 μmol/L nicotine,100 μmol/L nicotine reduced channel current amplitude,shortened the channel mean open time,and prolonged the channel mean closed time,supporting an agonist-induced open-channel block before hump current production.A docking model also simulated the agonist-binding site in the channel pore.Conclusion:These results support the hypoth-esis that hump currents reflect a rapid release of agonists from the α4β2-nAChR channel pore and a rapid recovery from desensitized α4β2-nAChR.

  6. Identification of growth phenotype-related genes in Aspergillus oryzae by heterologous macroarray and suppression subtractive hybridization

    NARCIS (Netherlands)

    Biesebeke, R. te; Levin, A.; Sagt, C.; Bartels, J.; Goosen, T.; Ram, A.; Hondel, C. van den; Punt, P.

    2005-01-01

    Aspergillus oryzae requires polarized growth for colonization of solid substrates, and this growth phenotype differs from that seen in liquid medium. Various experimental approaches were used to identify genes that are differentially expressed when A. oryzae is grown on wheat kernels and in a wheat-

  7. Identification of growth phenotype-related genes in Aspergillus oryzae by heterologous macroarray and suppression subtractive hybridization

    NARCIS (Netherlands)

    Biesebeke, R. te; Levin, A.; Sagt, C.; Bartels, J.; Goosen, T.; Ram, A.; Hondel, C. van den; Punt, P.

    2005-01-01

    Aspergillus oryzae requires polarized growth for colonization of solid substrates, and this growth phenotype differs from that seen in liquid medium. Various experimental approaches were used to identify genes that are differentially expressed when A. oryzae is grown on wheat kernels and in a wheat-

  8. Identification of growth phenotype-related genes in Aspergillus oryzae by heterologous macroarray and suppression subtractive hybridization

    NARCIS (Netherlands)

    Biesebeke, R. te; Levin, A.; Sagt, C.; Bartels, J.; Goosen, T.; Ram, A.; Hondel, C. van den; Punt, P.

    2005-01-01

    Aspergillus oryzae requires polarized growth for colonization of solid substrates, and this growth phenotype differs from that seen in liquid medium. Various experimental approaches were used to identify genes that are differentially expressed when A. oryzae is grown on wheat kernels and in a

  9. Cloning and expression of lipoxygenase genes and enzyme activity in ripening persimmon fruit in response to GA and ABA treatment

    Science.gov (United States)

    Two genes of the lipoxygenase (LOX) family, DkLox1 and DkLox3 (GenBank accession No. JF436951 and JF436950), were cloned from persimmon fruit (Diospyros kaki L. ‘Fuping Jianshi’). Sequence analysis indicated that they belong to the 9-LOX sub-group. Heterologous expression of DkLox1 in E. coli produc...

  10. Heterologous expression of hydrophobins RodA and RodB from Aspergillus fumigatus in host Pichia pastoris

    DEFF Research Database (Denmark)

    Pedersen, Mona Højgaard; Borodina, Irina; Frisvad, Jens Christian

    and investigation using the expression host Pichia pastoris. Methods and materials: The genes encoding hydrophobins RodA and RodB were amplified by RT-PCR with gene-specific primers from the total RNA isolated from the spores of A. fumigatus (AF296 strain). The resulting cDNA was cloned into TOPO vectors using TOPO...... proteins with the signal sequence of alfa-mating factor from Saccharomyces cerevisia known to work well for protein secretion from P. pastoris and the pPICZB plasmids had proteins cloned with their native signal sequences. The plasmids were linearized, transformed into P. pastoris strain X33...... of the purification of hydrophobins and functional investigations are being carried out at the moment. Conclusion: Hydrophobins RodA and RodB from Aspergillus fumigatus were successfully expressed and secreted by yeast host Pichia pastoris....

  11. Heterologous Transformation and Expression of Hericium erinaceum Manganese Peroxidase 1 Gene in Aspergillus nidulans%猴头菌锰过氧化物酶1基因在构巢曲霉的异源转化与表达

    Institute of Scientific and Technical Information of China (English)

    尹立伟; 池玉杰

    2013-01-01

    The recombinant plasmid pLB01/He-mnp1 which contains a gene encoding for manganese peroxidase (He-mnp1) from Hericium erinaceum CB1 was transformated into protoplasts of auxotrophic stain TN02A7 of Aspergillus nidulans by means of protoplast transformation method mediated by PEG/CaC12so as to enhance MnP production.A transformant stain TN02A7-He-mnp1 of A.nidulans was gained,the gene He-mnp1 was expressed under the control of alcohol dehydrogenase alcA (p) promoter.The transformant stain TN02A7-He-mnp1,auxotrophic stain TN02A7,wild stain of A.nidulans WJA01,and H.erinaceum CB1 were cultured under the same lignin condition and detected the MnP activity.The results showed that TN02A7-He-mnp1 could produce MnP activity in the absence and presence of heme,but the MnP activity was up to 38.31 U · L-1 on 96h with 0.05 g · L-1 heme which was 8.64 times higher than that without heme but less than that of H.erinaceum CB1,whereas TN02A7 and WJA01 could not produce MnP activity at any time,indicating that the gene He-mnp1 had been successfully transformed into TN02A7-He-mnp1 and expressed in lignin environment,and the heme was one of the restrictive factors for rescombinant mnp gene to express in A.nidulans.The study provides a new method to produce MnP and enhance MnP yield.%为提高猴头菌菌株CB1锰过氧化物酶(MnP)基因的表达产量,采用PEG/CaCl2介导的原生质体转化方法,将携带有He-mnp1的重组质粒pLB01/He-mnp1转入到构巢曲霉尿嘧啶尿苷营养缺陷菌株TN02A7的原生质体中,获得了转化子菌株TN02A 7-He-mnp1,并在乙醇脱氢酶启动子alcA(P)控制下实现了异源表达.将TN02A7-He-mnp1、TN02A7、构巢曲霉野生型菌株WJA01、猴头菌菌株CB1在相同的木质素环境下进行培养并检测MnP酶活性,结果表明:转化子菌株TN02A7-He-mnp1在0.05 g· L-1血红素的情况下、诱导96 h后酶活性最高为38.31 U·L-1,比不添加血红素的酶活力高8.64倍,但比猴头菌菌株CB1

  12. Gene expression in colorectal cancer

    DEFF Research Database (Denmark)

    Birkenkamp-Demtroder, Karin; Christensen, Lise Lotte; Olesen, Sanne Harder

    2002-01-01

    Understanding molecular alterations in colorectal cancer (CRC) is needed to define new biomarkers and treatment targets. We used oligonucleotide microarrays to monitor gene expression of about 6,800 known genes and 35,000 expressed sequence tags (ESTs) on five pools (four to six samples in each p...... with a high frequency of loss of heterozygosity. The genes and ESTs presented in this study encode new potential tumor markers as well as potential novel therapeutic targets for prevention or therapy of CRC.......Understanding molecular alterations in colorectal cancer (CRC) is needed to define new biomarkers and treatment targets. We used oligonucleotide microarrays to monitor gene expression of about 6,800 known genes and 35,000 expressed sequence tags (ESTs) on five pools (four to six samples in each...... pool) of total RNA from left-sided sporadic colorectal carcinomas. We compared normal tissue to carcinoma tissue from Dukes' stages A-D (noninvasive to distant metastasis) and identified 908 known genes and 4,155 ESTs that changed remarkably from normal to tumor tissue. Based on intensive filtering 226...

  13. Targeting gene expression to the female larval fat body of transgenic Aedes aegypti mosquitoes.

    Science.gov (United States)

    Totten, D C; Vuong, M; Litvinova, O V; Jinwal, U K; Gulia-Nuss, M; Harrell, R A; Beneš, H

    2013-02-01

    As the fat body is a critical tissue for mosquito development, metamorphosis, immune and reproductive system function, the characterization of regulatory modules targeting gene expression to the female mosquito fat body at distinct life stages is much needed for multiple, varied strategies for controlling vector-borne diseases such as dengue and malaria. The hexameric storage protein, Hexamerin-1.2, of the mosquito Aedes atropalpus is female-specific and uniquely expressed in the fat body of fourth instar larvae and young adults. We have identified in the Hex-1.2 gene, a short regulatory module that directs female-, tissue-, and stage-specific lacZ reporter gene expression using a heterologous promoter in transgenic lines of the dengue vector Aedes aegypti. Male transgenic larvae and pupae of one line expressed no Escherichia coli β-galactosidase or transgene product; in two other lines reporter gene activity was highly female-biased. All transgenic lines expressed the reporter only in the fat body; however, lacZ mRNA levels were no different in males and females at any stage examined, suggesting that the gene regulatory module drives female-specific expression by post-transcriptional regulation in the heterologous mosquito. This regulatory element from the Hex-1.2 gene thus provides a new molecular tool for transgenic mosquito control as well as functional genetic analysis in aedine mosquitoes.

  14. Development of a plant viral-vector-based gene expression assay for the screening of yeast cytochrome p450 monooxygenases.

    Science.gov (United States)

    Hanley, Kathleen; Nguyen, Long V; Khan, Faizah; Pogue, Gregory P; Vojdani, Fakhrieh; Panda, Sanjay; Pinot, Franck; Oriedo, Vincent B; Rasochova, Lada; Subramanian, Mani; Miller, Barbara; White, Earl L

    2003-02-01

    Development of a gene discovery tool for heterologously expressed cytochrome P450 monooxygenases has been inherently difficult. The activity assays are labor-intensive and not amenable to parallel screening. Additionally, biochemical confirmation requires coexpression of a homologous P450 reductase or complementary heterologous activity. Plant virus gene expression systems have been utilized for a diverse group of organisms. In this study we describe a method using an RNA vector expression system to phenotypically screen for cytochrome P450-dependent fatty acid omega-hydroxylase activity. Yarrowia lipolytica CYP52 gene family members involved in n-alkane assimilation were amplified from genomic DNA, cloned into a plant virus gene expression vector, and used as a model system for determining heterologous expression. Plants infected with virus vectors expressing the yeast CYP52 genes (YlALK1-YlALK7) showed a distinct necrotic lesion phenotype on inoculated plant leaves. No phenotype was detected on negative control constructs. YlALK3-, YlALK5-, and YlALK7-inoculated plants all catalyzed the terminal hydroxylation of lauric acid as confirmed using thin-layer and gas chromatography/mass spectrometry methods. The plant-based cytochrome P450 phenotypic screen was tested on an n-alkane-induced Yarrowia lipolytica plant virus expression library. A subset of 1,025 random library clones, including YlALK1-YlALK7 constructs, were tested on plants. All YlALK gene constructs scored positive in the randomized screen. Following nucleotide sequencing of the clones that scored positive using a phenotypic screen, approximately 5% were deemed appropriate for further biochemical analysis. This report illustrates the utility of a plant-based system for expression of heterologous cytochrome P450 monooxygenases and for the assignment of gene function.

  15. Molecular cloning and heterologous expression of a true lipase in Pichia pastoris isolated via a metagenomic approach.

    Science.gov (United States)

    Zheng, Jianhua; Liu, Liguo; Liu, Cuina; Jin, Qi

    2012-01-01

    Lipases are important enzymes for various biotechnological applications. By using functional expression screening, lipZ03, a novel lipase gene, was isolated from a soil-derived metagenomic library. The gene was supposed to encode a protein of 617 amino acids with a C-terminal targeting signal region and four potential N-linked glycosylation sites. The protein sequence shared a conserved GXSXG motif (X represents any amino acid residue) with other microbial lipases. Gene lipZ03 was expressed in Pichia pastoris and the molecular weight was estimated to be approximately 65 kDa by electrophoresis. The optimum reaction temperature and pH value for LipZ03 was 50°C and 9.0, respectively. The enzyme was highly stable in the temperature range of 40-60°C and under alkaline conditions (pH 8-10). Lipolytic activity was significantly enhanced by Ca(2+) and Mg(2+) ions, but dramatically inhibited by Cu(2+), Ni(2+) and Hg(2+) ions and EDTA. The purified enzyme preferentially hydrolyzed relatively long-chain triacylglycerols and was a true lipase rather than an esterase. Using a multi-stepwise methanol supply, the purified LipZ03 achieved a conversion yield of biodiesel production up to 74% after 36 h. Some interesting characteristics described here showed that the recombinant lipase may have potential to be a useful enzyme in industrial applications.

  16. Genome mining of astaxanthin biosynthetic genes from Sphingomonas sp. ATCC 55669 for heterologous overproduction in Escherichia coli

    OpenAIRE

    Ma, Tian; Zhou, Yuanjie; Li, Xiaowei; Zhu, Fayin; Cheng, Yongbo; Liu, Yi; Deng, Zixin; Liu, Tiangang

    2015-01-01

    Abstract As a highly valued keto‐carotenoid, astaxanthin is widely used in nutritional supplements and pharmaceuticals. Therefore, the demand for biosynthetic astaxanthin and improved efficiency of astaxanthin biosynthesis has driven the investigation of metabolic engineering of native astaxanthin producers and heterologous hosts. However, microbial resources for astaxanthin are limited. In this study, we found that the α‐Proteobacterium Sphingomonas sp. ATCC 55669 could produce astaxanthin n...

  17. Zipf's Law in Gene Expression

    CERN Document Server

    Furusawa, C; Furusawa, Chikara; Kaneko, Kunihiko

    2002-01-01

    Using data from gene expression databases on various organisms and tissues, including yeast, nematodes, human normal and cancer tissues, and embryonic stem cells, we found that the abundances of expressed genes exhibit a power-law distribution with an exponent close to -1, i.e., they obey Zipf's law. Furthermore, by simulations of a simple model with an intra-cellular reaction network, we found that Zipf's law of chemical abundance is a universal feature of cells where such a network optimizes the efficiency and faithfulness of self-reproduction. These findings provide novel insights into the nature of the organization of reaction dynamics in living cells.

  18. Zipf's Law in Gene Expression

    Science.gov (United States)

    Furusawa, Chikara; Kaneko, Kunihiko

    2003-02-01

    Using data from gene expression databases on various organisms and tissues, including yeast, nematodes, human normal and cancer tissues, and embryonic stem cells, we found that the abundances of expressed genes exhibit a power-law distribution with an exponent close to -1; i.e., they obey Zipf’s law. Furthermore, by simulations of a simple model with an intracellular reaction network, we found that Zipf’s law of chemical abundance is a universal feature of cells where such a network optimizes the efficiency and faithfulness of self-reproduction. These findings provide novel insights into the nature of the organization of reaction dynamics in living cells.

  19. Correction of gene expression data

    DEFF Research Database (Denmark)

    Darbani Shirvanehdeh, Behrooz; Stewart, C. Neal, Jr.; Noeparvar, Shahin;

    2014-01-01

    This report investigates for the first time the potential inter-treatment bias source of cell number for gene expression studies. Cell-number bias can affect gene expression analysis when comparing samples with unequal total cellular RNA content or with different RNA extraction efficiencies...... an analytical approach to examine the suitability of correction methods by considering the inter-treatment bias as well as the inter-replicate variance, which allows use of the best correction method with minimum residual bias. Analyses of RNA sequencing and microarray data showed that the efficiencies...

  20. 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

    in the chloroplast and subsequently oxidized by a cytochrome P450, CYP720B4. RESULTS: We transiently expressed the isopimaric acid pathway in Nicotiana benthamiana leaves and enhanced its productivity by the expression of two rate-limiting steps in the pathway (providing the general precursor of diterpenes). This co...... enzymes. CONCLUSIONS: It is possible to localize a diterpenoid pathway from spruce fully within the chloroplast of N. benthamiana and a few modifications of the N-terminal sequences of the CYP720B4 can facilitate the expression of plant P450s in the plastids. The coupling of terpene biosynthesis closer......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...

  1. 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

    in the chloroplast and subsequently oxidized by a cytochrome P450, CYP720B4. RESULTS: We transiently expressed the isopimaric acid pathway in Nicotiana benthamiana leaves and enhanced its productivity by the expression of two rate-limiting steps in the pathway (providing the general precursor of diterpenes). This co...... enzymes. CONCLUSIONS: It is possible to localize a diterpenoid pathway from spruce fully within the chloroplast of N. benthamiana and a few modifications of the N-terminal sequences of the CYP720B4 can facilitate the expression of plant P450s in the plastids. The coupling of terpene biosynthesis closer......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...

  2. Heterologous expression and characterization of bacterial 2-C-methyl-d-erythritol-4-phosphate pathway in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Carlsen, Simon; Ajikumar, Parayil Kumaran; Formenti, Luca Riccardo;

    2013-01-01

    the engineering of Escherichia coli genes encoding the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway into the genome of Saccharomyces cerevisiae and the characterization of intermediate metabolites synthesized by the MEP pathway in yeast. Our UPLC-MS analysis of the MEP pathway metabolites from engineered...... yeast showed that the pathway is active until the synthesis of 2-C-methyl-d-erythritol-2,4-cyclodiphosphate, but appears to lack functionality of the last two steps of the MEP pathway, catalyzed by the [4Fe–4S] iron sulfur cluster proteins encoded by ispG and ispH. In order to functionalize the last two...... steps of the MEP pathway, we co-expressed the genes for the E. coli iron sulfur cluster (ISC) assembly machinery. By deleting ERG13, thereby incapacitating the mevalonate pathway, in conjunction with labeling experiments with U–13C6 glucose and growth experiments, we found that the ISC assembly...

  3. Heterologous expression and maturation of an NADP-dependent [NiFe]-hydrogenase: a key enzyme in biofuel production.

    Directory of Open Access Journals (Sweden)

    Junsong Sun

    Full Text Available Hydrogen gas is a major biofuel and is metabolized by a wide range of microorganisms. Microbial hydrogen production is catalyzed by hydrogenase, an extremely complex, air-sensitive enzyme that utilizes a binuclear nickel-iron [NiFe] catalytic site. Production and engineering of recombinant [NiFe]-hydrogenases in a genetically-tractable organism, as with metalloprotein complexes in general, has met with limited success due to the elaborate maturation process that is required, primarily in the absence of oxygen, to assemble the catalytic center and functional enzyme. We report here the successful production in Escherichia coli of the recombinant form of a cytoplasmic, NADP-dependent hydrogenase from Pyrococcus furiosus, an anaerobic hyperthermophile. This was achieved using novel expression vectors for the co-expression of thirteen P. furiosus genes (four structural genes encoding the hydrogenase and nine encoding maturation proteins. Remarkably, the native E. coli maturation machinery will also generate a functional hydrogenase when provided with only the genes encoding the hydrogenase subunits and a single protease from P. furiosus. Another novel feature is that their expression was induced by anaerobic conditions, whereby E. coli was grown aerobically and production of recombinant hydrogenase was achieved by simply changing the gas feed from air to an inert gas (N2. The recombinant enzyme was purified and shown to be functionally similar to the native enzyme purified from P. furiosus. The methodology to generate this key hydrogen-producing enzyme has dramatic implications for the production of hydrogen and NADPH as vehicles for energy storage and transport, for engineering hydrogenase to optimize production and catalysis, as well as for the general production of complex, oxygen-sensitive metalloproteins.

  4. High-level intracellular expression of heterologous proteins in Brevibacillus choshinensis SP3 under the control of a xylose inducible promoter

    Directory of Open Access Journals (Sweden)

    D’Urzo Nunzia

    2013-02-01

    Full Text Available Abstract Background In past years research has focused on the development of alternative Gram positive bacterial expression systems to produce industrially relevant proteins. Brevibacillus choshinensis is an easy to handle non-sporulating bacterium, lacking extracellular proteases, that has been already shown to provide a high level of recombinant protein expression. One major drawback, limiting the applicability of the Brevibacillus expression system, is the absence of expression vectors based on inducible promoters. Here we used the PxylA inducible promoter, commonly employed in other Bacillae expression systems, in Brevibacillus. Results Using GFP, α-amylase and TcdA-GT as model proteins, high level of intracellular protein expression (up to 250 mg/L for the GFP was achieved in Brevibacillus, using the pHis1522 vector carrying the B. megaterium xylose-inducible promoter (PxylA. The GFP expression yields were more than 25 fold higher than those reported for B. megaterium carrying the same vector. All the tested proteins show significant increment in their expression levels (2-10 folds than those obtained using the available plasmids based on the P2 constitutive promoter. Conclusion Combining the components of two different commercially available Gram positive expression systems, such as Brevibacillus (from Takara Bio and B. megaterium (from Mobitec, we demonstrate that vectors based on the B. megaterium PxylA xylose inducible promoter can be successfully used to induce high level of intracellular expression of heterologous proteins in Brevibacillus.

  5. Heterologous expression of MlcE in Saccharomyces cerevisiae provides resistance to natural and semi-synthetic statins

    DEFF Research Database (Denmark)

    Ley, Ana; Coumou, Hilde Cornelijne; Frandsen, Rasmus John Normand

    2015-01-01

    -encoding gene mlcE from the mevastatin-producing Penicillium citrinum into the S. cerevisiae genome. The resulting strain showed increased resistance to both natural statins (mevastatin and lovastatin) and semi-synthetic statin (simvastatin) when compared to the wild type strain. Expression of RFP-tagged mlc......E showed that MlcE is localized to the yeast plasma and vacuolar membranes. We provide a possible engineering strategy for improvement of future yeast based production of natural and semi-synthetic statins....

  6. Heterologous expression, chaperone mediated solubilization and purification of parasitic nematode-specific growth factor-like protein of Setaria digitata

    Institute of Scientific and Technical Information of China (English)

    W WP Rodrigo; R S Dassanayake; E H Karunanayake; YIN Silva Gunawardene; O VDS J Weerasena

    2014-01-01

    Objective:To clone, express and purify a putative parasitic nematode specific protein of Setaria digitata (S. digitata), filarial nematode that infects livestock and cause significant economic losses inFarEast andAsia to be used for structural and functional analyses. Methods:To characterize uncharacterized gene ofS. digitata(SDUG), the herterologous expression ofSDUG was carried out in the pET [cloned into pET45b(+)] expression system initially and co-expression ofSDUG using chaperone plasmids pG-KJE8, pGro7, pKJE7, pG-Tf2 and pTf16 containing chaperone proteins of dnaK-dnaJ-grpE-groES-gro-E, groES-groEL, dnaK-dnaJ-grpE, groES-groEL-tig, and tig respectively, was carried out subsequently.Results:Expression ofSDUG was seen whenEscherichia coli strainBL21(DE3) is used, while concentrating protein largely into the insoluble fraction.The co-expression ofSDUG using chaperone plasmid mediated system indicated a significant increase of the protein in the soluble fraction.Of the chaperon plasmid sets, the highest amount of recombinantSDUP in the soluble fraction was seen when pGro7 was used in the presence of 2 mg/mLL-arabinose and0.6MIPTG concentration in the culture medium and for3 h of incubation at the temperature of28 ℃.RecombinantSDUG was purified both from soluble and insoluble fractions usingNi affinity chromatography.SDS-PAGE and western blot analyses of these proteins revealed a single band having expected size of ~24 kDa.Conclusions:SDUG seems to be more aggregate-prone and hydrophobic in nature and such protein can make soluble by correct selecting the inducer concentrations and induction temperature and its duration.

  7. Directed natural product biosynthesis gene cluster capture and expression in the model bacterium Bacillus subtilis

    Science.gov (United States)

    Li, Yongxin; Li, Zhongrui; Yamanaka, Kazuya; Xu, Ying; Zhang, Weipeng; Vlamakis, Hera; Kolter, Roberto; Moore, Bradley S.; Qian, Pei-Yuan

    2015-03-01

    Bacilli are ubiquitous low G+C environmental Gram-positive bacteria that produce a wide assortment of specialized small molecules. Although their natural product biosynthetic potential is high, robust molecular tools to support the heterologous expression of large biosynthetic gene clusters in Bacillus hosts are rare. Herein we adapt transformation-associated recombination (TAR) in yeast to design a single genomic capture and expression vector for antibiotic production in Bacillus subtilis. After validating this direct cloning ``plug-and-play'' approach with surfactin, we genetically interrogated amicoumacin biosynthetic gene cluster from the marine isolate Bacillus subtilis 1779. Its heterologous expression allowed us to explore an unusual maturation process involving the N-acyl-asparagine pro-drug intermediates preamicoumacins, which are hydrolyzed by the asparagine-specific peptidase into the active component amicoumacin A. This work represents the first direct cloning based heterologous expression of natural products in the model organism B. subtilis and paves the way to the development of future genome mining efforts in this genus.

  8. Directed natural product biosynthesis gene cluster capture and expression in the model bacterium Bacillus subtilis

    KAUST Repository

    Li, Yongxin

    2015-03-24

    Bacilli are ubiquitous low G+C environmental Gram-positive bacteria that produce a wide assortment of specialized small molecules. Although their natural product biosynthetic potential is high, robust molecular tools to support the heterologous expression of large biosynthetic gene clusters in Bacillus hosts are rare. Herein we adapt transformation-associated recombination (TAR) in yeast to design a single genomic capture and expression vector for antibiotic production in Bacillus subtilis. After validating this direct cloning plug-and-playa approach with surfactin, we genetically interrogated amicoumacin biosynthetic gene cluster from the marine isolate Bacillus subtilis 1779. Its heterologous expression allowed us to explore an unusual maturation process involving the N-acyl-asparagine pro-drug intermediates preamicoumacins, which are hydrolyzed by the asparagine-specific peptidase into the active component amicoumacin A. This work represents the first direct cloning based heterologous expression of natural products in the model organism B. subtilis and paves the way to the development of future genome mining efforts in this genus.

  9. Homeobox gene expression in Brachiopoda

    DEFF Research Database (Denmark)

    Altenburger, Andreas; Martinez, Pedro; Wanninger, Andreas

    2011-01-01

    The molecular control that underlies brachiopod ontogeny is largely unknown. In order to contribute to this issue we analyzed the expression pattern of two homeobox containing genes, Not and Cdx, during development of the rhynchonelliform (i.e., articulate) brachiopod Terebratalia transversa. Not...

  10. Heterologous production of fungal secondary metabolites in Aspergilli

    DEFF Research Database (Denmark)

    Anyaogu, Diana Chinyere; Mortensen, Uffe Hasbro

    2015-01-01

    Fungal natural products comprise a wide range of compounds. Some are medically attractive as drugs and drug leads, some are used as food additives, while others are harmful mycotoxins. In recent years the genome sequence of several fungi has become available providing genetic information of a large...... number of putative biosynthetic pathways. However, compound discovery is difficult as the genes required for the production of the compounds often are silent or barely expressed under laboratory conditions. Furthermore, the lack of available tools for genetic manipulation of most fungal species hinders...... pathway discovery. Heterologous expression of the biosynthetic pathway in model systems or cell factories facilitates product discovery, elucidation, and production. This review summarizes the recent strategies for heterologous expression of fungal biosynthetic pathways in Aspergilli....

  11. Vascular Gene Expression: A Hypothesis

    Directory of Open Access Journals (Sweden)

    Angélica Concepción eMartínez-Navarro

    2013-07-01

    Full Text Available The phloem is the conduit through which photoassimilates are distributed from autotrophic to heterotrophic tissues and is involved in the distribution of signaling molecules that coordinate plant growth and responses to the environment. Phloem function depends on the coordinate expression of a large array of genes. We have previously identified conserved motifs in upstream regions of the Arabidopsis genes, encoding the homologs of pumpkin phloem sap mRNAs, displaying expression in vascular tissues. This tissue-specific expression in Arabidopsis is predicted by the overrepresentation of GA/CT-rich motifs in gene promoters. In this work we have searched for common motifs in upstream regions of the homologous genes from plants considered to possess a primitive vascular tissue (a lycophyte, as well as from others that lack a true vascular tissue (a bryophyte, and finally from chlorophytes. Both lycophyte and bryophyte display motifs similar to those found in Arabidopsis with a significantly low E-value, while the chlorophytes showed either a different conserved motif or no conserved motif at all. These results suggest that these same genes are expressed coordinately in non- vascular plants; this coordinate expression may have been one of the prerequisites for the development of conducting tissues in plants. We have also analyzed the phylogeny of conserved proteins that may be involved in phloem function and development. The presence of CmPP16, APL, FT and YDA in chlorophytes suggests the recruitment of ancient regulatory networks for the development of the vascular tissue during evolution while OPS is a novel protein specific to vascular plants.

  12. Neighboring Genes Show Correlated Evolution in Gene Expression

    Science.gov (United States)

    Ghanbarian, Avazeh T.; Hurst, Laurence D.

    2015-01-01

    When considering the evolution of a gene’s expression profile, we commonly assume that this is unaffected by its genomic neighborhood. This is, however, in contrast to what we know about the lack of autonomy between neighboring genes in gene expression profiles in extant taxa. Indeed, in all eukaryotic genomes genes of similar expression-profile tend to cluster, reflecting chromatin level dynamics. Does it follow that if a gene increases expression in a particular lineage then the genomic neighbors will also increase in their expression or is gene expression evolution autonomous? To address this here we consider evolution of human gene expression since the human-chimp common ancestor, allowing for both variation in estimation of current expression level and error in Bayesian estimation of the ancestral state. We find that in all tissues and both sexes, the change in gene expression of a focal gene on average predicts the change in gene expression of neighbors. The effect is highly pronounced in the immediate vicinity (genes increasing their expression in humans tend to avoid nuclear lamina domains and be enriched for the gene activator 5-hydroxymethylcytosine, we conclude that, most probably owing to chromatin level control of gene expression, a change in gene expression of one gene likely affects the expression evolution of neighbors, what we term expression piggybacking, an analog of hitchhiking. PMID:25743543

  13. DsdA (D-serine deaminase): a new heterologous MX cassette for gene disruption and selection in Saccharomyces cerevisiae.

    Science.gov (United States)

    Vorachek-Warren, Mara K; McCusker, John H

    2004-01-30

    Dominant drug resistance markers offer experimental flexibility in the study of Saccharomyces cerevisiae by eliminating the dependence on auxotrophic mutations and, because they are phenotypically neutral, avoid the deleterious effects of auxotrophic mutations. We have developed a new dominant resistance marker, dsdAMX4, for use in the genetic manipulation of S. cerevisiae. The dsdA gene, which is derived from Escherichia coli and encodes a D-serine deaminase, confers to S. cerevisiae resistance to D-serine and the ability to use D-serine as a nitrogen source. Here we describe the construction of a dsdAMX4 cassette, capable of expression in S. cerevisiae, and the characterization of this new marker for use in chromosomal gene disruption. The unique selection properties of the dsdAMX4 cassette make it an important addition to the existing array of S. cerevisiae genetic tools. Copyright 2004 John Wiley & Sons, Ltd.

  14. Heterologous Reconstitution of the Intact Geodin Gene Cluster in Aspergillus nidulans through a Simple and Versatile PCR Based Approach

    DEFF Research Database (Denmark)

    Nielsen, Morten Thrane; Nielsen, Jakob Blæsbjerg; Anyaogu, Dianna Chinyere;

    2013-01-01

    Fungal natural products are a rich resource for bioactive molecules. To fully exploit this potential it is necessary to link genes to metabolites. Genetic information for numerous putative biosynthetic pathways has become available in recent years through genome sequencing. However, the lack...... was transferred in a two step procedure to an expression platform in A. nidulans. The individual cluster fragments were generated by PCR and assembled via efficient USER fusion prior to ransformation and integration via re-iterative gene targeting. A total of 13 open reading frames contained in 25 kb of DNA were...... in characterizing the many exciting pathways for secondary metabolite production that are currently being uncovered by the fungal genome sequencing projects....

  15. [Heterologous extracellular expression and initial characterization of the peroxisomal catalase from the methylotrophic yeast Hansenula polymorpha in Pichia pastoris].

    Science.gov (United States)

    Tian, Y -S; Xu, H; Peng, R -H; Yao, Q -H

    2013-01-01

    Catalase is well known to eliminate H2O2 in cells and reduces the toxicity of peroxide compounds. A catalase gene HpCat1 of methylotrophic yeast Hansenula polymorpha without the part coding the native signal peptide was cloned into expression vector pYM3165 and then integrated into genome of Pichia pastoris GS115 by electroporation. The result of the enzyme activity assay and SDS-PAGE demonstrated that the recombinant protein (HpCAT1) of H. polymorpha was extracellularly expressed in P. pastoris. The expressed catalase was recovered from the culture supernatant of P. pastoris GS 115 and purified by (NH4) 2SO4 fractionation and Ni-NTA affinity chromatography. The main biochemical properties of the recombinant protein HpCAT1, such as thermodependence and thermostability, pH optimum and pH stability, as well as the effect of metal ions and chemicals, were characterized. With H2O2 as the substrate, HpCAT1 displayed pH and tem- perature optima of approximately 2.6 and 45°C,respectively. The recombinant HpCAT1 activity was inhibited by 1 mM Hg2+ and Cu2+, but was highly enhanced by 1.0 mM Fe2+.

  16. Heterologous expression of equine CYP3A94 and investigation of a tunable system to regulate co-expressed NADPH P450 oxidoreductase levels.

    Directory of Open Access Journals (Sweden)

    Ramona Dettwiler

    Full Text Available The activity of cytochrome P450 enzymes depends on the enzyme NADPH P450 oxidoreductase (POR. The aim of this study was to investigate the activity of the equine CYP3A94 using a system that allows to regulate the POR protein levels in mammalian cells. CYP3A94 and the equine POR were heterologously expressed in V79 cells. In the system used, the POR protein regulation is based on a destabilizing domain (DD that transfers its instability to a fused protein. The resulting fusion protein is therefore degraded by the ubiquitin-proteasome system (UPS. Addition of "Shield-1" prevents the DD fusion protein from degradation. The change of POR levels at different Shield-1 concentrations was demonstrated by cytochrome c reduction, Western immunoblot analysis, and immunocytochemistry. The alteration of CYP3A94 activity was investigated using a substrate (BFC known to detect CYP3A4 activity. Equine CYP3A94 was demonstrated to be metabolically active and its activity could be significantly elevated by co-expression of POR. Cytochrome c reduction was significantly increased in V79-CYP3A94/DD-POR cells compared to V79-CYP3A94 cells. Surprisingly, incubation with different Shield-1 concentrations resulted in a decrease in POR protein shown by Western immunoblot analysis. Cytochrome c reduction did not change significantly, but the CYP3A94 activity decreased more than 4-fold after incubation with 500 nM and 1 µM Shield-1 for 24 hours. No differences were obtained when V79-CYP3A94 POR cells with and without Shield-1 were compared. The basal activity levels of V79-CYP3A94/DD-POR cells were unexpectedly high, indicating that DD/POR is not degraded without Shield-1. Shield-1 decreased POR protein levels and CYP3A94 activity suggesting that Shield-1 might impair POR activity by an unknown mechanism. Although regulation of POR with the pPTuner system could not be obtained, the cell line V79-CYP3A94/DD-POR system can be used for further experiments to characterize the

  17. Gene Expression in Trypanosomatid Parasites

    Directory of Open Access Journals (Sweden)

    Santiago Martínez-Calvillo

    2010-01-01

    Full Text Available The parasites Leishmania spp., Trypanosoma brucei, and Trypanosoma cruzi are the trypanosomatid protozoa that cause the deadly human diseases leishmaniasis, African sleeping sickness, and Chagas disease, respectively. These organisms possess unique mechanisms for gene expression such as constitutive polycistronic transcription of protein-coding genes and trans-splicing. Little is known about either the DNA sequences or the proteins that are involved in the initiation and termination of transcription in trypanosomatids. In silico analyses of the genome databases of these parasites led to the identification of a small number of proteins involved in gene expression. However, functional studies have revealed that trypanosomatids have more general transcription factors than originally estimated. Many posttranslational histone modifications, histone variants, and chromatin modifying enzymes have been identified in trypanosomatids, and recent genome-wide studies showed that epigenetic regulation might play a very important role in gene expression in this group of parasites. Here, we review and comment on the most recent findings related to transcription initiation and termination in trypanosomatid protozoa.

  18. Synthetic promoter libraries- tuning of gene expression

    DEFF Research Database (Denmark)

    Hammer, Karin; Mijakovic, Ivan; Jensen, Peter Ruhdal

    2006-01-01

    The study of gene function often requires changing the expression of a gene and evaluating the consequences. In principle, the expression of any given gene can be modulated in a quasi-continuum of discrete expression levels but the traditional approaches are usually limited to two extremes: gene ...

  19. Isolation and heterologous expression of a polygalacturonase produced by Fusarium oxysporum f. sp. cubense race 1 and 4.

    Science.gov (United States)

    Dong, Zhangyong; Wang, Zhenzhong

    2015-04-03

    Fusarium wilt (Panama disease) caused by Fusarium oxysporum f. sp. cubense (FOC) represents a significant threat to banana (Musa spp.) production. Musa AAB is susceptible to Race 1 (FOC1) and Race 4 (FOC4), while Cavendish Musa AAA is found to be resistant to FOC1 but still susceptible to Race 4. A polygalacturonase (PGC3) was purified from the supernatant of Fusarium oxysporum f. sp. cubense race 4 (FOC4), which is the pathogen of Fusarium wilt. PGC3 had an apparent molecular weight of 45 kDa according to SDS-PAGE. The enzyme hydrolyzed polygalacturonic acid in an exo-manner, as demonstrated by analysis of degradation products. The Km and Vmax values of PGC3 from FOC4 were determined to be 0.70 mg·mL-1 and 101.01 Units·mg·protein-1·min-1, respectively. Two pgc3 genes encoding PGC3 from FOC4 and FOC1, both genes of 1368 bp in length encode 456 amino-acid residues with a predicted signal peptide sequence of 21 amino acids. There are 16 nucleotide sites difference between FOC4-pgc3 and FOC1-pgc3, only leading to four amino acid residues difference. In order to obtain adequate amounts of protein required for functional studies, two genes were cloned into the expression vector pPICZaA and then expressed in Pichia pastoris strains of SMD1168. The recombinant PGC3, r-FOC1-PGC3 and r-FOC4-PGC3, were expressed and purified as active proteins. The optimal PGC3 activity was observed at 50 °C and pH 4.5. Both recombinant PGC3 retained >40% activity at pH 3-7 and >50% activity in 10-50 °C. Both recombinant PGC3 proteins could induce a response but with different levels of tissue maceration and necrosis in banana plants. In sum, our results indicate that PGC3 is an exo-PG and can be produced with full function in P. pastoris.

  20. Transient gene expression in tobacco using Gibson assembly and the Gene Gun.

    Science.gov (United States)

    Mattozzi, Matthew D; Voges, Mathias J; Silver, Pamela A; Way, Jeffrey C

    2014-04-18

    In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5' mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work(11), and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

  1. Heterologous protein production in Streptomyces lividans

    DEFF Research Database (Denmark)

    Rattleff, Stig

    Alternative hosts for expressing heterologous proteins have recently gained an increased attention. Actinomycetes, and among these especially the Streptomycetes are considered promising candidates, since they through their saprophytic lifestyle are capable of excreting large amounts of proteins...

  2. Optogenetic switches for light-controlled gene expression in yeast.

    Science.gov (United States)

    Salinas, Francisco; Rojas, Vicente; Delgado, Verónica; Agosin, Eduardo; Larrondo, Luis F

    2017-04-01

    Light is increasingly recognized as an efficient means of controlling diverse biological processes with high spatiotemporal resolution. Optogenetic switches are molecular devices for regulating light-controlled gene expression, protein localization, signal transduction and protein-protein interactions. Such molecular components have been mainly developed through the use of photoreceptors, which upon light stimulation undergo conformational changes passing to an active state. The current repertoires of optogenetic switches include red, blue and UV-B light photoreceptors and have been implemented in a broad spectrum of biological platforms. In this review, we revisit different optogenetic switches that have been used in diverse biological platforms, with emphasis on those used for light-controlled gene expression in the budding yeast Saccharomyces cerevisiae. The implementation of these switches overcomes the use of traditional chemical inducers, allowing precise control of gene expression at lower costs, without leaving chemical traces, and positively impacting the production of high-value metabolites and heterologous proteins. Additionally, we highlight the potential of utilizing this technology beyond laboratory strains, by optimizing it for use in yeasts tamed for industrial processes. Finally, we discuss how fungal photoreceptors could serve as a source of biological parts for the development of novel optogenetic switches with improved characteristics. Although optogenetic tools have had a strong impact on basic research, their use in applied sciences is still undervalued. Therefore, the invitation for the future is to utilize this technology in biotechnological and industrial settings.

  3. High-level gene expression in Aedes albopictus cells using a baculovirus Hr3 enhancer and IE1 transactivator

    Directory of Open Access Journals (Sweden)

    Gray Christine E

    2004-07-01

    Full Text Available Abstract Background Aedes aegypti is the key vector of both the Yellow Fever and Dengue Fever viruses throughout many parts of the world. Low and variable transgene expression levels due to position effect and position effect variegation are problematic to efforts to create transgenic laboratory strains refractory to these viruses. Transformation efficiencies are also less than optimal, likely due to failure to detect expression from all integrated transgenes and potentially due to limited expression of the transposase required for transgene integration. Results Expression plasmids utilizing three heterologous promoters and three heterologous enhancers, in all possible combinations, were tested. The Hr3/IE1 enhancer-transactivator in combination with each of the constitutive heterologous promoters tested increased reporter gene expression significantly in transiently transfected Aedes albopictus C7-10 cells. Conclusions The addition of the Hr3 enhancer to expression cassettes and concomitant expression of the IE1 transactivator gene product is a potential method for increasing the level of transgene expression in insect systems. This mechanism could also potentially be used to increase the level of transiently-expressed transposase in order to increase the number of integration events in transposon-mediated transformation experiments.

  4. Molecular cloning and heterologous expression in Pichia pastoris of X-prolyl-dipeptidyl aminopeptidase from basidiomycete Ustilago maydis.

    Science.gov (United States)

    Juárez-Montiel, Margarita; Ibarra, J Antonio; Chávez-Camarillo, Griselda; Hernández-Rodríguez, César; Villa-Tanaca, Lourdes

    2014-03-01

    Dipeptidyl aminopeptidases are enzymes involved in the posttranslational control of bioactive peptides. Here we identified the gene dapUm in Ustilago maydis by homology with other fungal dipeptidyl aminopeptidases. Analysis of the dapUm-deduced amino acid sequence indicated that it encodes for membrane-type serine protease with a characteristic prolyl oligopeptidase catalytic motif triad: Ser, Asp, His. In order to overexpress the DapUm, the gene encoding for it was cloned and transformed into Pichia. Using this system, we observed a ∼ 125-kDa recombinant protein with an optimal enzymatic activity at pH 6.0 and at 40 °C for the Ala-Pro-p-nitroanilide substrate and an experimental pH of 6.9. U. maydis DapUm was specifically inhibited by phenylmethylsulfonyl fluoride and Pefabloc, confirming the presence of a serine residue in the active site. To our knowledge, this study is the first report on the cloning and expression of a DPP IV dipeptidyl aminopeptidase from a basidiomycete organism. Moreover, the use of recombinant DapUm will allow us to further study and characterize this enzyme, in addition to testing chemical compounds for pharmaceutical purposes.

  5. Classification with binary gene expressions

    OpenAIRE

    Tuna, Salih; Niranjan, Mahesan

    2009-01-01

    Microarray gene expression measurements are reported, used and archived usually to high numerical precision. However, properties of mRNA molecules, such as their low stability and availability in small copy numbers, and the fact that measurements correspond to a population of cells, rather than a single cell, makes high precision meaningless. Recent work shows that reducing measurement precision leads to very little loss of information, right down to binary levels. In this paper we show how p...

  6. The Gene Expression Omnibus database

    Science.gov (United States)

    Clough, Emily; Barrett, Tanya

    2016-01-01

    The Gene Expression Omnibus (GEO) database is an international public repository that archives and freely distributes high-throughput gene expression and other functional genomics data sets. Created in 2000 as a worldwide resource for gene expression studies, GEO has evolved with rapidly changing technologies and now accepts high-throughput data for many other data applications, including those that examine genome methylation, chromatin structure, and genome–protein interactions. GEO supports community-derived reporting standards that specify provision of several critical study elements including raw data, processed data, and descriptive metadata. The database not only provides access to data for tens of thousands of studies, but also offers various Web-based tools and strategies that enable users to locate data relevant to their specific interests, as well as to visualize and analyze the data. This chapter includes detailed descriptions of methods to query and download GEO data and use the analysis and visualization tools. The GEO homepage is at http://www.ncbi.nlm.nih.gov/geo/. PMID:27008011

  7. Exploring the potential of the glycerol-3-phosphate dehydrogenase 2 (GPD2) promoter for recombinant gene expression in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Knudsen, Jan Dines; Johanson, Ted; Eliasson Lantz, Anna

    2015-01-01

    by placing it in strains with different ability to reoxidise NADH, and applying different environmental conditions. Flow cytometric analysis of reporter strains expressing green fluorescent protein (GFP) under the control of the GPD2 promoter was used to determine the promoter activity at the single...... mapping revealed conditions where the GPD2 promoter was either completely inactive or hyperactive, which has implications for its implementation in future biotechnological applications such as for process control of heterologous gene expression....

  8. Viral promoters can initiate expression of toxin genes introduced into Escherichia coli

    Directory of Open Access Journals (Sweden)

    Jacob Daniela

    2005-06-01

    Full Text Available Abstract Background The expression of recombinant proteins in eukaryotic cells requires the fusion of the coding region to a promoter functional in the eukaryotic cell line. Viral promoters are very often used for this purpose. The preceding cloning procedures are usually performed in Escherichia coli and it is therefore of interest if the foreign promoter results in an expression of the gene in bacteria. In the case molecules toxic for humans are to be expressed, this knowledge is indispensable for the specification of safety measures. Results We selected five frequently used viral promoters and quantified their activity in E. coli with a reporter system. Only the promoter from the thymidine kinase gene from HSV1 showed no activity, while the polyhedrin promoter from baculovirus, the early immediate CMV promoter, the early SV40 promoter and the 5' LTR promoter from HIV-1 directed gene expression in E. coli. The determination of transcription start sites in the immediate early CMV promoter and the polyhedrin promoter confirmed the existence of bacterial -10 and -35 consensus sequences. The importance of this heterologous gene expression for safety considerations was further supported by analysing fusions between the aforementioned promoters and a promoter-less cytotoxin gene. Conclusion According to our results a high percentage of viral promoters have the ability of initiating gene expression in E. coli. The degree of such heterologous gene expression can be sufficient for the expression of toxin genes and must therefore be considered when defining safety measures for the handling of corresponding genetically modified organisms.

  9. Gene expression throughout a vertebrate's embryogenesis

    Directory of Open Access Journals (Sweden)

    Hinton David E

    2011-02-01

    Full Text Available Abstract Background Describing the patterns of gene expression during embryonic development has broadened our understanding of the processes and patterns that define morphogenesis. Yet gene expression patterns have not been described throughout vertebrate embryogenesis. This study presents statistical analyses of gene expression during all 40 developmental stages in the teleost Fundulus heteroclitus using four biological replicates per stage. Results Patterns of gene expression for 7,000 genes appear to be important as they recapitulate developmental timing. Among the 45% of genes with significant expression differences between pairs of temporally adjacent stages, significant differences in gene expression vary from as few as five to more than 660. Five adjacent stages have disproportionately more significant changes in gene expression (> 200 genes relative to other stages: four to eight and eight to sixteen cell stages, onset of circulation, pre and post-hatch, and during complete yolk absorption. The fewest differences among adjacent stages occur during gastrulation. Yet, at stage 16, (pre-mid-gastrulation the largest number of genes has peak expression. This stage has an over representation of genes in oxidative respiration and protein expression (ribosomes, translational genes and proteases. Unexpectedly, among all ribosomal genes, both strong positive and negative correlations occur. Similar correlated patterns of expression occur among all significant genes. Conclusions These data provide statistical support for the temporal dynamics of developmental gene expression during all stages of vertebrate development.

  10. Impact of cell type and epitope tagging on heterologous expression of G protein-coupled receptor: a systematic study on angiotensin type II receptor.

    Directory of Open Access Journals (Sweden)

    Lili Jiang

    Full Text Available Despite heterologous expression of epitope-tagged GPCR is widely adopted for functional characterization, there is lacking of systematic analysis of the impact of expression host and epitope tag on GPCR expression. Angiotensin type II (AT2 receptor displays agonist-dependent and -independent activities, coupling to a spectrum of signaling molecules. However, consensus has not been reached on the subcellular distributions, signaling cascades and receptor-mediated actions. To examine the contributions of host cell and epitope tag on receptor expression and activity, epitope-tagged AT2 receptor variants were transiently or stably expressed in HEK293, CHO-K1 and PC12 cells. The epitope-tagged AT2 receptor variants were detected both on the cell membrane and in the perinuclear region. In transiently transfected HEK293 cells, Myc-AT2 existed predominantly as monomer. Additionally, a ladder of ubiquitinated AT2 receptor proteins was detected. By contrast, stably expressed epitope-tagged AT2 receptor variants existed as both monomer and high molecular weight complexes, and the latter was enriched in cell surface. Glycosylation promoted cell surface expression of Myc-AT2 but had no effect on AT2-GFP in HEK293 cells. In cells that stably expressed Myc-AT2, serum starvation induced apoptosis in CHO-K1 cells but not in HEK293 or PC12 cells. Instead, HEK293 and PC12 cells stably expressing Myc-AT2 exhibited partial cell cycle arrest with cells accumulating at G1 and S phases, respectively. Taken together, these results suggest that expression levels, subcellular distributions and ligand-independent constitutive activities of AT2 receptor were cell type-dependent while posttranslational processing of nascent AT2 receptor protein was modulated by epitope tag and mode of expression.

  11. Antisense expression increases gene expression variability and locus interdependency

    OpenAIRE

    Xu, Zhenyu; Wei, Wu; Gagneur, Julien; Clauder-Münster, Sandra; Smolik, Miłosz; Huber, Wolfgang; Steinmetz, Lars M.

    2011-01-01

    Genome-wide transcription profiling has revealed extensive expression of non-coding RNAs antisense to genes, yet their functions, if any, remain to be understood. In this study, we perform a systematic analysis of sense–antisense expression in response to genetic and environmental changes in yeast. We find that antisense expression is associated with genes of larger expression variability. This is characterized by more ‘switching off' at low levels of expression for genes with antisense compa...

  12. Gene expression analysis identifies global gene dosage sensitivity in cancer

    DEFF Research Database (Denmark)

    Fehrmann, Rudolf S. N.; Karjalainen, Juha M.; Krajewska, Malgorzata;

    2015-01-01

    expression. We reanalyzed 77,840 expression profiles and observed a limited set of 'transcriptional components' that describe well-known biology, explain the vast majority of variation in gene expression and enable us to predict the biological function of genes. On correcting expression profiles...... for these components, we observed that the residual expression levels (in 'functional genomic mRNA' profiling) correlated strongly with copy number. DNA copy number correlated positively with expression levels for 99% of all abundantly expressed human genes, indicating global gene dosage sensitivity. By applying...

  13. Noise in eukaryotic gene expression

    Science.gov (United States)

    Blake, William J.; KÆrn, Mads; Cantor, Charles R.; Collins, J. J.

    2003-04-01

    Transcription in eukaryotic cells has been described as quantal, with pulses of messenger RNA produced in a probabilistic manner. This description reflects the inherently stochastic nature of gene expression, known to be a major factor in the heterogeneous response of individual cells within a clonal population to an inducing stimulus. Here we show in Saccharomyces cerevisiae that stochasticity (noise) arising from transcription contributes significantly to the level of heterogeneity within a eukaryotic clonal population, in contrast to observations in prokaryotes, and that such noise can be modulated at the translational level. We use a stochastic model of transcription initiation specific to eukaryotes to show that pulsatile mRNA production, through reinitiation, is crucial for the dependence of noise on transcriptional efficiency, highlighting a key difference between eukaryotic and prokaryotic sources of noise. Furthermore, we explore the propagation of noise in a gene cascade network and demonstrate experimentally that increased noise in the transcription of a regulatory protein leads to increased cell-cell variability in the target gene output, resulting in prolonged bistable expression states. This result has implications for the role of noise in phenotypic variation and cellular differentiation.

  14. Identification of four soybean reference genes for gene expression normalization

    Science.gov (United States)

    Gene expression analysis requires the use of reference genes stably expressed independently of specific tissues or environmental conditions. Housekeeping genes (e.g., actin, tubulin, ribosomal, polyubiquitin and elongation factor 1-alpha) are commonly used as reference genes with the assumption tha...

  15. Activating the expression of bacterial cryptic genes by rpoB mutations in RNA polymerase or by rare earth elements.

    Science.gov (United States)

    Ochi, Kozo; Tanaka, Yukinori; Tojo, Shigeo

    2014-02-01

    Since bacteria were found to contain genes encoding enzymes that synthesize a plethora of potential secondary metabolites, interest has grown in the activation of these cryptic pathways. Homologous and heterologous expression of these cryptic secondary metabolite-biosynthetic genes, often "silent" under ordinary laboratory fermentation conditions, may lead to the discovery of novel secondary metabolites. We review current progress on this topic, describing concepts for activating silent genes. We especially focus on genetic manipulation of transcription and translation, as well as the utilization of rare earth elements as a novel method to activate the silent genes. The possible roles of silent genes in bacterial physiology are also discussed.

  16. d-2,3-Butanediol Production Due to Heterologous Expression of an Acetoin Reductase in Clostridium acetobutylicum ▿ †

    Science.gov (United States)

    Siemerink, Marco A. J.; Kuit, Wouter; López Contreras, Ana M.; Eggink, Gerrit; van der Oost, John; Kengen, Servé W. M.

    2011-01-01

    Acetoin reductase (ACR) catalyzes the conversion of acetoin to 2,3-butanediol. Under certain conditions, Clostridium acetobutylicum ATCC 824 (and strains derived from it) generates both d- and l-stereoisomers of acetoin, but because of the absence of an ACR enzyme, it does not produce 2,3-butanediol. A gene encoding ACR from Clostridium beijerinckii NCIMB 8052 was functionally expressed in C. acetobutylicum under the control of two strong promoters, the constitutive thl promoter and the late exponential adc promoter. Both ACR-overproducing strains were grown in batch cultures, during which 89 to 90% of the natively produced acetoin was converted to 20 to 22 mM d-2,3-butanediol. The addition of a racemic mixture of acetoin led to the production of both d-2,3-butanediol and meso-2,3-butanediol. A metabolic network that is in agreement with the experimental data is proposed. Native 2,3-butanediol production is a first step toward a potential homofermentative 2-butanol-producing strain of C. acetobutylicum. PMID:21335380

  17. Heterologous expression of MlcE in Saccharomyces cerevisiae provides resistance to natural and semi-synthetic statins

    Directory of Open Access Journals (Sweden)

    Ana Ley

    2015-12-01

    Full Text Available Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the key enzyme in cholesterol biosynthesis. Their extensive use in treatment and prevention of cardiovascular diseases placed statins among the best selling drugs. Construction of Saccharomyces cerevisiae cell factory for the production of high concentrations of natural statins will require establishment of a non-destructive self-resistance mechanism to overcome the undesirable growth inhibition effects of statins. To establish active export of statins from yeast, and thereby detoxification, we integrated a putative efflux pump-encoding gene mlcE from the mevastatin-producing Penicillium citrinum into the S. cerevisiae genome. The resulting strain showed increased resistance to both natural statins (mevastatin and lovastatin and semi-synthetic statin (simvastatin when compared to the wild type strain. Expression of RFP-tagged mlcE showed that MlcE is localized to the yeast plasma and vacuolar membranes. We provide a possible engineering strategy for improvement of future yeast based production of natural and semi-synthetic statins.

  18. Engineering and Validation of a Vector for Concomitant Expression of Rare Transfer RNA (tRNA and HIV-1 nef Genes in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Siti Aisyah Mualif

    Full Text Available Relative ease in handling and manipulation of Escherichia coli strains make them primary candidate to express proteins heterologously. Overexpression of heterologous genes that contain codons infrequently used by E. coli is related with difficulties such as mRNA instability, early termination of transcription and/or translation, deletions and/or misincorporation, and cell growth inhibition. These codon bias -associated problems are addressed by co-expressing ColE1-compatible, rare tRNA expressing helper plasmids. However, this approach has inadequacies, which we have addressed by engineering an expression vector that concomitantly expresses the heterologous protein of interest, and rare tRNA genes in E. coli. The expression vector contains three (argU, ileY, leuW rare tRNA genes and a useful multiple cloning site for easy in-frame cloning. To maintain the overall size of the parental plasmid vector, the rare tRNA genes replaced the non-essential DNA segments in the vector. The cloned gene is expressed under the control of T7 promoter and resulting recombinant protein has a C-terminal 6His tag for IMAC-mediated purification. We have evaluated the usefulness of this expression vector by expressing three HIV-1 genes namely HIV-1 p27 (nef, HIV-1 p24 (ca, and HIV-1 vif in NiCo21(DE3 E.coli and demonstrated the advantages of using expression vector that concomitantly expresses rare tRNA and heterologous genes.

  19. Gene cloning and soluble expression of Aspergillus niger phytase in E. coli cytosol via chaperone co-expression.

    Science.gov (United States)

    Ushasree, Mrudula Vasudevan; Vidya, Jalaja; Pandey, Ashok

    2014-01-01

    A phytase gene from Aspergillus niger was isolated and two Escherichia coli expression systems, based on T7 RNA polymerase promoter and tac promoter, were used for its recombinant expression. Co-expression of molecular chaperone, GroES/EL, aided functional cytosolic expression of the phytase in E. coli BL21 (DE3). Untagged and maltose-binding protein-tagged recombinant phytase showed an activity band of ~49 and 92 kDa, respectively, on a zymogram. Heterologously-expressed phytase was fractionated from endogenous E. coli phytase by (NH4)2SO4 precipitation. The enzyme had optimum activity at 50 °C and pH 6.5.

  20. MRI of Transgene Expression: Correlation to Therapeutic Gene Expression

    Directory of Open Access Journals (Sweden)

    Tomotsugu Ichikawa

    2002-01-01

    Full Text Available Magnetic resonance imaging (MRI can provide highresolution 3D maps of structural and functional information, yet its use of mapping in vivo gene expression has only recently been explored. A potential application for this technology is to noninvasively image transgene expression. The current study explores the latter using a nonregulatable internalizing engineered transferrin receptor (ETR whose expression can be probed for with a superparamagnetic Tf-CLIO probe. Using an HSV-based amplicon vector system for transgene delivery, we demonstrate that: 1 ETR is a sensitive MR marker gene; 2 several transgenes can be efficiently expressed from a single amplicon; 3 expression of each transgene results in functional gene product; and 4 ETR gene expression correlates with expression of therapeutic genes when the latter are contained within the same amplicon. These data, taken together, suggest that MRI of ETR expression can serve as a surrogate for measuring therapeutic transgene expression.

  1. Flexible tools for gene expression and silencing in tomato.

    Science.gov (United States)

    Fernandez, Ana I; Viron, Nicolas; Alhagdow, Moftah; Karimi, Mansour; Jones, Matthew; Amsellem, Ziva; Sicard, Adrien; Czerednik, Anna; Angenent, Gerco; Grierson, Donald; May, Sean; Seymour, Graham; Eshed, Yuval; Lemaire-Chamley, Martine; Rothan, Christophe; Hilson, Pierre

    2009-12-01

    As a genetic platform, tomato (Solanum lycopersicum) benefits from rich germplasm collections and ease of cultivation and transformation that enable the analysis of biological processes impossible to investigate in other model species. To facilitate the assembly of an open genetic toolbox designed to study Solanaceae, we initiated a joint collection of publicly available gene manipulation tools. We focused on the characterization of promoters expressed at defined time windows during fruit development, for the regulated expression or silencing of genes of interest. Five promoter sequences were captured as entry clones compatible with the versatile MultiSite Gateway format: PPC2, PG, TPRP, and IMA from tomato and CRC from Arabidopsis (Arabidopsis thaliana). Corresponding transcriptional fusions were made with the GUS gene, a nuclear-localized GUS-GFP reporter, and the chimeric LhG4 transcription factor. The activity of the promoters during fruit development and in fruit tissues was confirmed in transgenic tomato lines. Novel Gateway destination vectors were generated for the transcription of artificial microRNA (amiRNA) precursors and hairpin RNAs under the control of these promoters, with schemes only involving Gateway BP and LR Clonase reactions. Efficient silencing of the endogenous phytoene desaturase gene was demonstrated in transgenic tomato lines producing a matching amiRNA under the cauliflower mosaic virus 35S or PPC2 promoter. Lastly, taking advantage of the pOP/LhG4 two-component system, we found that well-characterized flower-specific Arabidopsis promoters drive the expression of reporters in patterns generally compatible with heterologous expression. Tomato lines and plasmids will be distributed through a new Nottingham Arabidopsis Stock Centre service unit dedicated to Solanaceae resources.

  2. Correlating Expression Data with Gene Function Using Gene Ontology

    Institute of Scientific and Technical Information of China (English)

    LIU,Qi; DENG,Yong; WANG,Chuan; SHI,Tie-Liu; LI,Yi-Xue

    2006-01-01

    Clustering is perhaps one of the most widely used tools for microarray data analysis. Proposed roles for genes of unknown function are inferred from clusters of genes similarity expressed across many biological conditions.However, whether function annotation by similarity metrics is reliable or not and to what extent the similarity in gene expression patterns is useful for annotation of gene functions, has not been evaluated. This paper made a comprehensive research on the correlation between the similarity of expression data and of gene functions using Gene Ontology. It has been found that although the similarity in expression patterns and the similarity in gene functions are significantly dependent on each other, this association is rather weak. In addition, among the three categories of Gene Ontology, the similarity of expression data is more useful for cellular component annotation than for biological process and molecular function. The results presented are interesting for the gene functions prediction research area.

  3. Adaptive Evolution of Gene Expression in Drosophila

    Directory of Open Access Journals (Sweden)

    Armita Nourmohammad

    2017-08-01

    Full Text Available Gene expression levels are important quantitative traits that link genotypes to molecular functions and fitness. In Drosophila, population-genetic studies have revealed substantial adaptive evolution at the genomic level, but the evolutionary modes of gene expression remain controversial. Here, we present evidence that adaptation dominates the evolution of gene expression levels in flies. We show that 64% of the observed expression divergence across seven Drosophila species are adaptive changes driven by directional selection. Our results are derived from time-resolved data of gene expression divergence across a family of related species, using a probabilistic inference method for gene-specific selection. Adaptive gene expression is stronger in specific functional classes, including regulation, sensory perception, sexual behavior, and morphology. Moreover, we identify a large group of genes with sex-specific adaptation of expression, which predominantly occurs in males. Our analysis opens an avenue to map system-wide selection on molecular quantitative traits independently of their genetic basis.

  4. Natural and Heterologous Production of Bacteriocins

    Science.gov (United States)

    Cintas, Luis M.; Herranz, Carmen; Hernández, Pablo E.

    Bacteriocins are ribosomally synthesized antimicrobial peptides produced by bacteria, and their use as natural and nontoxic food preservatives has been the source of considerable interest for the research community. In addition, bacteriocins have been investigated for their potential use in human and veterinary applications and in the animal production field. In the native bacterial strain, most bacteriocins are synthesized as biologically inactive precursors, with N-terminal extensions, that are cleaved concomitantly during export of the bacteriocin by dedicated ABC transporters, or the general secretory pathway (GSP) or Sec-dependent pathway. However, a few bacteriocins are synthesized without an N-terminal extension, and others are circularized through a head-to-tail peptide bond, complicating the elucidation of their processing and transport across the cytoplasmic membrane. The high cost of synthetic bacteriocin synthesis and their low yields from many natural producers recommends the exploration of recombinant microbial systems for the heterologous production of bacteriocins. Other advantages of such systems include production of bacteriocins in safer hosts, increased bacteriocin production, control of bacteriocin gene expression, production of food ingredients with antimicrobial activity, construction of multibacteriocinogenic strains with a wider antagonistic spectrum, a better adaptation of the selected hosts to food environments, and providing antagonistic properties to lactic acid bacteria (LAB) used as starter, protective, or probiotic cultures. The recombinant production of bacteriocins mostly relies on the use of expression vectors that replicate in Gram-negative bacteria, Gram-positive bacteria, and yeasts, whereas the production of bacteriocins in heterologous LAB hosts may be essentially based on the expression of native biosynthetic genes, by exchanging or replacing leader peptides and/or dedicated processing and secretion systems (ABC transporters

  5. Development of a novel thermostable Newcastle disease virus vaccine vector for expression of a heterologous gene

    Science.gov (United States)

    The thermostable Newcastle disease virus (NDV) vaccines have been used widely to control Newcastle disease (ND) for village flocks, due to their independence of cold chains for delivery and storage. To explore the potential use of the thermostable NDV as a vaccine vector, an infectious clone of the...

  6. 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

    . 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...... 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....

  7. CASCADE, a platform for controlled gene amplification for high, tunable and selection-free gene expression in yeast

    Science.gov (United States)

    Strucko, Tomas; Buron, Line Due; Jarczynska, Zofia Dorota; Nødvig, Christina Spuur; Mølgaard, Louise; Halkier, Barbara Ann; Mortensen, Uffe Hasbro

    2017-01-01

    Over-expression of a gene by increasing its copy number is often desirable in the model yeast Saccharomyces cerevisiae. It may facilitate elucidation of enzyme functions, and in cell factory design it is used to increase production of proteins and metabolites. Current methods are typically exploiting expression from the multicopy 2 μ-derived plasmid or by targeting genes repeatedly into sequences like Ty or rDNA; in both cases, high gene expression levels are often reached. However, with 2 μ-based plasmid expression, the population of cells is very heterogeneous with respect to protein production; and for integration into repeated sequences it is difficult to determine the genetic setup of the resulting strains and to achieve specific gene doses. For both types of systems, the strains often suffer from genetic instability if proper selection pressure is not applied. Here we present a gene amplification system, CASCADE, which enables construction of strains with defined gene copy numbers. One or more genes can be amplified simultaneously and the resulting strains can be stably propagated on selection-free medium. As proof-of-concept, we have successfully used CASCADE to increase heterologous production of two fluorescent proteins, the enzyme β-galactosidase the fungal polyketide 6-methyl salicylic acid and the plant metabolite vanillin glucoside. PMID:28134264

  8. Methods for monitoring multiple gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Berka, Randy (Davis, CA); Bachkirova, Elena (Davis, CA); Rey, Michael (Davis, CA)

    2012-05-01

    The present invention relates to methods for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells using microarrays containing Trichoderma reesei ESTs or SSH clones, or a combination thereof. The present invention also relates to computer readable media and substrates containing such array features for monitoring expression of a plurality of genes in filamentous fungal cells.

  9. Methods for monitoring multiple gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Berka, Randy; Bachkirova, Elena; Rey, Michael

    2013-10-01

    The present invention relates to methods for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells using microarrays containing Trichoderma reesei ESTs or SSH clones, or a combination thereof. The present invention also relates to computer readable media and substrates containing such array features for monitoring expression of a plurality of genes in filamentous fungal cells.

  10. Methods for monitoring multiple gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Berka, Randy [Davis, CA; Bachkirova, Elena [Davis, CA; Rey, Michael [Davis, CA

    2012-05-01

    The present invention relates to methods for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells using microarrays containing Trichoderma reesei ESTs or SSH clones, or a combination thereof. The present invention also relates to computer readable media and substrates containing such array features for monitoring expression of a plurality of genes in filamentous fungal cells.

  11. Methods for monitoring multiple gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Berka, Randy; Bachkirova, Elena; Rey, Michael

    2013-10-01

    The present invention relates to methods for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells using microarrays containing Trichoderma reesei ESTs or SSH clones, or a combination thereof. The present invention also relates to computer readable media and substrates containing such array features for monitoring expression of a plurality of genes in filamentous fungal cells.

  12. Expanding the molecular toolbox for Lactococcus lactis: construction of an inducible thioredoxin gene fusion expression system

    LENUS (Irish Health Repository)

    Douillard, Francois P

    2011-08-09

    Abstract Background The development of the Nisin Inducible Controlled Expression (NICE) system in the food-grade bacterium Lactococcus lactis subsp. cremoris represents a cornerstone in the use of Gram-positive bacterial expression systems for biotechnological purposes. However, proteins that are subjected to such over-expression in L. lactis may suffer from improper folding, inclusion body formation and\\/or protein degradation, thereby significantly reducing the yield of soluble target protein. Although such drawbacks are not specific to L. lactis, no molecular tools have been developed to prevent or circumvent these recurrent problems of protein expression in L. lactis. Results Mimicking thioredoxin gene fusion systems available for E. coli, two nisin-inducible expression vectors were constructed to over-produce various proteins in L. lactis as thioredoxin fusion proteins. In this study, we demonstrate that our novel L. lactis fusion partner expression vectors allow high-level expression of soluble heterologous proteins Tuc2009 ORF40, Bbr_0140 and Tuc2009 BppU\\/BppL that were previously insoluble or not expressed using existing L. lactis expression vectors. Over-expressed proteins were subsequently purified by Ni-TED affinity chromatography. Intact heterologous proteins were detected by immunoblotting analyses. We also show that the thioredoxin moiety of the purified fusion protein was specifically and efficiently cleaved off by enterokinase treatment. Conclusions This study is the first description of a thioredoxin gene fusion expression system, purposely developed to circumvent problems associated with protein over-expression in L. lactis. It was shown to prevent protein insolubility and degradation, allowing sufficient production of soluble proteins for further structural and functional characterization.

  13. Amplification of kinetic oscillations in gene expression

    Science.gov (United States)

    Zhdanov, V. P.

    2008-10-01

    Because of the feedbacks between the DNA transcription and mRNA translation, the gene expression in cells may exhibit bistability and oscillations. The deterministic and stochastic calculations presented illustrate how the bistable kinetics of expression of one gene in a cell can be influenced by the kinetic oscillations in the expression of another gene. Due to stability of the states of the bistable kinetics of gene 1 and the relatively small difference between the maximum and minimum protein amounts during the oscillations of gene 2, the induced oscillations of gene 1 are found to typically be related either to the low-or high-reactive state of this gene. The quality of the induced oscillations may be appreciably better than that of the inducing oscillations. This means that gene 1 can serve as an amplifier of the kinetic oscillations of gene 2.

  14. cis sequence effects on gene expression

    Directory of Open Access Journals (Sweden)

    Jacobs Kevin

    2007-08-01

    Full Text Available Abstract Background Sequence and transcriptional variability within and between individuals are typically studied independently. The joint analysis of sequence and gene expression variation (genetical genomics provides insight into the role of linked sequence variation in the regulation of gene expression. We investigated the role of sequence variation in cis on gene expression (cis sequence effects in a group of genes commonly studied in cancer research in lymphoblastoid cell lines. We estimated the proportion of genes exhibiting cis sequence effects and the proportion of gene expression variation explained by cis sequence effects using three different analytical approaches, and compared our results to the literature. Results We generated gene expression profiling data at N = 697 candidate genes from N = 30 lymphoblastoid cell lines for this study and used available candidate gene resequencing data at N = 552 candidate genes to identify N = 30 candidate genes with sufficient variance in both datasets for the investigation of cis sequence effects. We used two additive models and the haplotype phylogeny scanning approach of Templeton (Tree Scanning to evaluate association between individual SNPs, all SNPs at a gene, and diplotypes, with log-transformed gene expression. SNPs and diplotypes at eight candidate genes exhibited statistically significant (p cis sequence effects in our study, respectively. Conclusion Based on analysis of our results and the extant literature, one in four genes exhibits significant cis sequence effects, and for these genes, about 30% of gene expression variation is accounted for by cis sequence variation. Despite diverse experimental approaches, the presence or absence of significant cis sequence effects is largely supported by previously published studies.

  15. D-2,3-Butanediol Production Due to Heterologous Expression of an Acetoin Reductase in Clostridium acetobutylicum

    NARCIS (Netherlands)

    Siemerink, M.A.J.; Kuit, W.; Lopez Contreras, A.M.; Eggink, G.; Oost, van der J.; Kengen, S.W.M.

    2011-01-01

    Acetoin reductase (ACR) catalyzes the conversion of acetoin to 2,3-butanediol. Under certain conditions Clostridium acetobutylicum ATCC 824 (and derived strains) generates both D- and L-stereoisomers of acetoin, but due to the lack of an ACR enzyme, does not produce 2,3-butanediol. A gene encoding

  16. Constitutive knox1 gene expression in dandelion (Taraxacum officinale, Web.) changes leaf morphology from simple to compound.

    Science.gov (United States)

    Müller, Kai J; He, Xinqiang; Fischer, Rainer; Prüfer, Dirk

    2006-10-01

    Seed plants with compound leaves constitute a polyphyletic group, but studies of diverse taxa show that genes of the class 1 KNOTTED-LIKE HOMEOBOX (KNOX1) family are often involved in compound leaf development. This suggests that knox1 genes have been recruited on multiple occasions during angiosperm evolution (Bharathan et al. in Science 296:1858-1860, 2002). In agreement with this, we demonstrate that the simple leaf of dandelion (Taraxacum officinale Web.) can be converted into a compound leaf by the constitutive expression of heterologous knox1 genes. Dandelion is a rosette plant of the family Asteraceae, characterised by simple leaves with deeply lobed margins and endogenous knox1 gene expression. Transgenic dandelion plants constitutively expressing the barley (Hordeum vulgare L.) hooded gene (bkn3, barley knox3) or the related bkn1 gene, developed compound leaves featuring epiphyllous rosettes. We discuss these results in the context of two current models of compound leaf formation.

  17. Heterologous expression of surface-active proteins from barley and filamentous fungi in Pichia pastoris and characterization of their contribution to beer gushing.

    Science.gov (United States)

    Lutterschmid, Georg; Muranyi, Monika; Stübner, Matthias; Vogel, Rudi F; Niessen, Ludwig

    2011-05-14

    The spontaneous over-foaming of beer upon opening, i.e. beer gushing, is an unwanted phenomenon for the brewing industry. Currently, surface-active proteins from filamentous fungi and non-specific lipid transfer proteins (nsLTP1) from barley are discussed as gushing inducers. In our study the class I hydrophobin FcHyd3p from Fusarium culmorum, the class II hydrophobin Hfb2 from Trichoderma reesei, the alkaline foam protein A (AfpA) from F. graminearum and nsLTP1 from Hordeum vulgare cv. Marnie (barley) were heterologously expressed in Pichia pastoris and used in gushing tests. The class I hydrophobin FcHyd3p was unable to induce gushing in beer. The class II hydrophobin Hfb2 was able to induce gushing in beer, but proved to be inhibited by heat treatment as well as by the presence of enriched hop compounds. Both resulted in a reduced gushing potential. AfpA and nsLTP1 exhibited no gushing-inducing potential at the amounts added to beer. Addition of these proteins to beer or carbonated water previously treated with class II hydrophobins revealed a gushing reducing character.

  18. Molecular cloning, heterologous expression and functional characterization of a novel translationally-controlled tumor protein (TCTP) family member from Loxosceles intermedia (brown spider) venom.

    Science.gov (United States)

    Sade, Youssef B; Bóia-Ferreira, Marianna; Gremski, Luiza H; da Silveira, Rafael B; Gremski, Waldemiro; Senff-Ribeiro, Andrea; Chaim, Olga M; Veiga, Silvio S

    2012-01-01

    Envenoming with brown spiders (Loxosceles genus) is common throughout the world. Cutaneous symptoms following spider bite accidents include dermonecrosis, erythema, itching and pain. In some cases, accidents can cause hypersensibility or even allergic reactions. These responses could be associated with histaminergic events, such as an increase in vascular permeability and vasodilatation. A protein that may be related to the effects of spider venom was identified from a previously obtained cDNA library of the L. intermedia venom gland. The amino acid sequence of this protein is homologous to proteins from the TCTP (translationally-controlled tumor protein) family, which are extracellular histamine-releasing factors (HRF) that are associated with the allergic reactions to parasites. Herein, we described the cloning, heterologous expression, purification and functional characterization of a novel member of the TCTP family from the Loxosceles intermedia venom gland. This recombinant protein, named LiRecTCTP, causes edema, enhances vascular permeability and is likely related to the inflammatory activity of the venom. Moreover, LiRecTCTP presents an immunological relationship with mammalian TCTPs.

  19. Biochemical characterization of Paracoccidioides brasiliensis α-1,3-glucanase Agn1p, and its functionality by heterologous Expression in Schizosaccharomyces pombe.

    Directory of Open Access Journals (Sweden)

    Héctor Villalobos-Duno

    Full Text Available α-1,3-Glucan is present as the outermost layer of the cell wall in the pathogenic yeastlike (Y form of Paracoccidioides brasiliensis. Based on experimental evidence, this polysaccharide has been proposed as a fungal virulence factor. To degrade α-1,3-glucan and allow remodeling of the cell wall, α-1,3-glucanase is required. Therefore, the study of this enzyme, its encoding gene, and regulatory mechanisms, might be of interest to understand the morphogenesis and virulence process in this fungus. A single gene, orthologous to other fungal α-1,3-glucanase genes, was identified in the Paracoccidioides genome, and labeled AGN1. Transcriptional levels of AGN1 and AGS1 (α-1,3-glucan synthase-encoding gene increased sharply when the pathogenic Y phase was cultured in the presence of 5% horse serum, a reported booster for cell wall α-1,3-glucan synthesis in this fungus. To study the biochemical properties of P. brasiliensis Agn1p, the enzyme was heterologously overexpressed, purified, and its activity profile determined by means of the degradation of carboxymethyl α-1,3-glucan (SCMG, chemically modified from P. brasiliensis α-1,3-glucan, used as a soluble substrate for the enzymatic reaction. Inhibition assays, thin layer chromatography and enzymatic reactions with alternative substrates (dextran, starch, chitin, laminarin and cellulose, showed that Agn1p displays an endolytic cut pattern and high specificity for SCMG. Complementation of a Schizosaccharomyces pombe agn1Δ strain with the P. brasiliensis AGN1 gene restored the wild type phenotype, indicating functionality of the gene, suggesting a possible role of Agn1p in the remodeling of P. brasiliensis Y phase cell wall. Based on amino acid sequence, P. brasiliensis Agn1p, groups within the family 71 of fungal glycoside hydrolases (GH-71, showing similar biochemical characteristics to other members of this family. Also based on amino acid sequence alignments, we propose a subdivision of fungal

  20. Deriving Trading Rules Using Gene Expression Programming

    Directory of Open Access Journals (Sweden)

    Adrian VISOIU

    2011-01-01

    Full Text Available This paper presents how buy and sell trading rules are generated using gene expression programming with special setup. Market concepts are presented and market analysis is discussed with emphasis on technical analysis and quantitative methods. The use of genetic algorithms in deriving trading rules is presented. Gene expression programming is applied in a form where multiple types of operators and operands are used. This gives birth to multiple gene contexts and references between genes in order to keep the linear structure of the gene expression programming chromosome. The setup of multiple gene contexts is presented. The case study shows how to use the proposed gene setup to derive trading rules encoded by Boolean expressions, using a dataset with the reference exchange rates between the Euro and the Romanian leu. The conclusions highlight the positive results obtained in deriving useful trading rules.

  1. Heterologous expression of the BABY BOOM AP2/ERF transcription factor enhances the regeneration capacity of tobacco (Nicotiana tabacum L.)

    NARCIS (Netherlands)

    Srinivasan, C.; Liu, Z.; Heidmann, I.; Supena, E.D.J.; Fukuoka, H.; Joosen, R.V.L.; Lambalk, J.; Angenent, G.C.; Scorza, R.; Custers, J.B.M.; Boutilier, K.A.

    2007-01-01

    Gain-of-function studies have shown that ectopic expression of the BABY BOOM (BBM) AP2/ERF domain transcription factor is sufficient to induce spontaneous somatic embryogenesis in Arabidopsis (Arabidopsis thaliana (L.) Heynh) and Brassica napus (B. napus L.) seedlings. Here we examined the effect of

  2. Gene Expression Profiling of Gastric Cancer

    Science.gov (United States)

    Marimuthu, Arivusudar; Jacob, Harrys K.C.; Jakharia, Aniruddha; Subbannayya, Yashwanth; Keerthikumar, Shivakumar; Kashyap, Manoj Kumar; Goel, Renu; Balakrishnan, Lavanya; Dwivedi, Sutopa; Pathare, Swapnali; Dikshit, Jyoti Bajpai; Maharudraiah, Jagadeesha; Singh, Sujay; Sameer Kumar, Ghantasala S; Vijayakumar, M.; Veerendra Kumar, Kariyanakatte Veeraiah; Premalatha, Chennagiri Shrinivasamurthy; Tata, Pramila; Hariharan, Ramesh; Roa, Juan Carlos; Prasad, T.S.K; Chaerkady, Raghothama; Kumar, Rekha Vijay; Pandey, Akhilesh

    2015-01-01

    Gastric cancer is the second leading cause of cancer death worldwide, both in men and women. A genomewide gene expression analysis was carried out to identify differentially expressed genes in gastric adenocarcinoma tissues as compared to adjacent normal tissues. We used Agilent’s whole human genome oligonucleotide microarray platform representing ~41,000 genes to carry out gene expression analysis. Two-color microarray analysis was employed to directly compare the expression of genes between tumor and normal tissues. Through this approach, we identified several previously known candidate genes along with a number of novel candidate genes in gastric cancer. Testican-1 (SPOCK1) was one of the novel molecules that was 10-fold upregulated in tumors. Using tissue microarrays, we validated the expression of testican-1 by immunohistochemical staining. It was overexpressed in 56% (160/282) of the cases tested. Pathway analysis led to the identification of several networks in which SPOCK1 was among the topmost networks of interacting genes. By gene enrichment analysis, we identified several genes involved in cell adhesion and cell proliferation to be significantly upregulated while those corresponding to metabolic pathways were significantly downregulated. The differentially expressed genes identified in this study are candidate biomarkers for gastric adenoacarcinoma. PMID:27030788

  3. Optimization of a yeast RNA interference system for controlling gene expression and enabling rapid metabolic engineering.

    Science.gov (United States)

    Crook, Nathan C; Schmitz, Alexander C; Alper, Hal S

    2014-05-16

    Reduction of endogenous gene expression is a fundamental operation of metabolic engineering, yet current methods for gene knockdown (i.e., genome editing) remain laborious and slow, especially in yeast. In contrast, RNA interference allows facile and tunable gene knockdown via a simple plasmid transformation step, enabling metabolic engineers to rapidly prototype knockdown strategies in multiple strains before expending significant cost to undertake genome editing. Although RNAi is naturally present in a myriad of eukaryotes, it has only been recently implemented in Saccharomyces cerevisiae as a heterologous pathway and so has not yet been optimized as a metabolic engineering tool. In this study, we elucidate a set of design principles for the construction of hairpin RNA expression cassettes in yeast and implement RNA interference to quickly identify routes for improvement of itaconic acid production in this organism. The approach developed here enables rapid prototyping of knockdown strategies and thus accelerates and reduces the cost of the design-build-test cycle in yeast.

  4. Modulation of gene expression made easy

    DEFF Research Database (Denmark)

    Solem, Christian; Jensen, Peter Ruhdal

    2002-01-01

    A new approach for modulating gene expression, based on randomization of promoter (spacer) sequences, was developed. The method was applied to chromosomal genes in Lactococcus lactis and shown to generate libraries of clones with broad ranges of expression levels of target genes. In one example...... beta-glucuronidase, resulting in an operon structure in which both genes are transcribed from a common promoter. We show that there is a linear correlation between the expressions of the two genes, which facilitates screening for mutants with suitable enzyme activities. In a second example, we show......, overexpression was achieved by introducing an additional gene copy into a phage attachment site on the chromosome. This resulted in a series of strains with phosphofructokinase activities from 1.4 to 11 times the wild-type activity level. In this example, the pfk gene was cloned upstream of a gusA gene encoding...

  5. Analysis of pea HMG-I/Y expression suggests a role in defence gene regulation.

    Science.gov (United States)

    Klosterman, Steven J; Choi, Jane J; Hadwiger, Lee A

    2003-07-01

    SUMMARY HMG-I/Y proteins are characterized by the presence of AT-hook motifs, DNA binding domains that recognize AT-rich tracts of DNA. By facilitating protein:protein and protein:DNA interactions in the vicinity of these AT-rich binding sites, HMG-I/Y positively or negatively regulates gene expression. Several pea defence gene promoters have AT-rich tracts of DNA that are potential targets for modulation via HMG-I/Y. In this study, a comparison of the expression of a pea defence gene (DRR206) mRNA relative to the expression of HMG-I/Y mRNA was monitored by Northern analysis following the inoculation of a fungal pathogen, Fusarium solani or treatment with chitosan and a F. solani DNase (Fsph DNase). In pea pod endocarp tissue, HMG-I/Y expression was observed at high levels in untreated tissue and at lower levels 6 h following inoculation or wounding of the tissue. Western blots with an antipea HMG-I/Y polyclonal antibody also revealed that pea HMG-I/Y is expressed at decreased levels 6 h following inoculation or elicitor treatment. HMG-I/Y extracted from pea caused alterations in the gel migration of radio-labelled AT-rich sequences from the pea DRR206 promoter, suggesting that similar interactions could exist in vivo. Agroinfiltration was utilized to express the pea HMG-I/Y gene in tobacco containing a chimeric gene fusion of a promoter from the PR gene, DRR206, and the beta-glucuronidase (GUS) reporter gene. Transient expression of pea HMG-I/Y led to a decrease in GUS reporter gene activity in the heterologous tobacco system. These data implicate pea HMG-I/Y abundance in the down-regulation of DRR206 gene expression, and possibly HMG-I/Y depletion in the expression of defence genes in pea.

  6. Gene expression profiling during murine tooth development

    Directory of Open Access Journals (Sweden)

    Maria A dos Santos silva Landin

    2012-07-01

    Full Text Available The aim of this study was to describe the expression of genes, including ameloblastin (Ambn, amelogenin X chromosome (Amelx and enamelin (Enam during early (pre-secretory tooth development. The expression of these genes has predominantly been studied at post-secretory stages. Deoxyoligonucleotide microarrays were used to study gene expression during development of the murine first molar tooth germ at 24h intervals, starting at the eleventh embryonic day (E11.5 and up to the seventh day after birth (P7. The profile search function of Spotfire software was used to select genes with similar expression profile as the enamel genes (Ambn, Amelx and Enam. Microarray results where validated using real-time Reverse Transcription-Polymerase Chain Reaction (real-time RT-PCR, and translated proteins identified by Western blotting. In situ localisation of the Ambn, Amelx and Enam mRNAs were monitored from E12.5 to E17.5 using deoxyoligonucleotide probes. Bioinformatics analysis was used to associate biological functions with differentially (p ≤0.05 expressed (DE genes.Microarray results showed a total of 4362 genes including Ambn, Amelx and Enam to be significant differentially expressed throughout the time-course. The expression of the three enamel genes was low at pre-natal stages (E11.5-P0 increasing after birth (P1-P7. Profile search lead to isolation of 87 genes with significantly similar expression to the three enamel proteins. The mRNAs expressed in dental epithelium and epithelium derived cells. Although expression of Ambn, Amelx and Enam were lower during early tooth development compared to secretory stages enamel proteins were detectable by Western blotting. Bioinformatic analysis associated the 87 genes with multiple biological functions. Around thirty-five genes were associated with fifteen transcription factors.

  7. Glycoprotein is enough for sindbis virus-derived DNA vector to express heterogenous genes

    Directory of Open Access Journals (Sweden)

    Fu Juanjuan

    2011-07-01

    Full Text Available Abstract To investigate the necessity and potential application of structural genes for expressing heterogenous genes from Sindbis virus-derived vector, the DNA-based expression vector pVaXJ was constructed by placing the recombinant genome of sindbis-like virus XJ-160 under the control of the human cytomegalovirus (CMV promoter of the plasmid pVAX1, in which viral structural genes were replaced by a polylinker cassette to allow for insertion of heterologous genes. The defect helper plasmids pVaE or pVaC were developed by cloning the gene of glycoprotein E3E26KE1 or capsid protein of XJ-160 virus into pVAX1, respectively. The report gene cassette pVaXJ-EGFP or pV-Gluc expressing enhanced green fluorescence protein (EGFP or Gaussia luciferase (G.luc were constructed by cloning EGFP or G.luc gene into pVaXJ. EGFP or G.luc was expressed in the BHK-21 cells co-transfected with report gene cassettes and pVaE at levels that were comparable to those produced by report gene cassettes, pVaC and pVaE and were much higher than the levels produced by report gene cassette and pVaC, suggesting that glycoprotein is enough for Sindbis virus-derived DNA vector to express heterogenous genes in host cells. The method of gene expression from Sindbis virus-based DNA vector only co-transfected with envelop E gene increase the conveniency and the utility of alphavirus-based vector systems in general.

  8. Glycoprotein is enough for sindbis virus-derived DNA vector to express heterogenous genes.

    Science.gov (United States)

    Zhu, Wuyang; Li, Jiangjiao; Tang, Li; Wang, Huanqin; Li, Jia; Fu, Juanjuan; Liang, Guodong

    2011-07-10

    To investigate the necessity and potential application of structural genes for expressing heterogenous genes from Sindbis virus-derived vector, the DNA-based expression vector pVaXJ was constructed by placing the recombinant genome of sindbis-like virus XJ-160 under the control of the human cytomegalovirus (CMV) promoter of the plasmid pVAX1, in which viral structural genes were replaced by a polylinker cassette to allow for insertion of heterologous genes. The defect helper plasmids pVaE or pVaC were developed by cloning the gene of glycoprotein E3E26KE1 or capsid protein of XJ-160 virus into pVAX1, respectively. The report gene cassette pVaXJ-EGFP or pV-Gluc expressing enhanced green fluorescence protein (EGFP) or Gaussia luciferase (G.luc) were constructed by cloning EGFP or G.luc gene into pVaXJ. EGFP or G.luc was expressed in the BHK-21 cells co-transfected with report gene cassettes and pVaE at levels that were comparable to those produced by report gene cassettes, pVaC and pVaE and were much higher than the levels produced by report gene cassette and pVaC, suggesting that glycoprotein is enough for Sindbis virus-derived DNA vector to express heterogenous genes in host cells. The method of gene expression from Sindbis virus-based DNA vector only co-transfected with envelop E gene increase the conveniency and the utility of alphavirus-based vector systems in general.

  9. Heterologous expression of human cytochrome P450 2E1 in HepG2 cell line

    Institute of Scientific and Technical Information of China (English)

    Jian Zhuge; Ye Luo; Ying-Nian Yu

    2003-01-01

    AIM: Human cytochrome P-450 2E1 (CYP2E1) takes part in the biotransformation of ethanol, acetone, many smallmolecule substrates and volatile anesthetics. CYP2E1 is involved in chemical activation of many carcinogens,procarcinogens, and toxicants. To assess the metabolic and toxicological characteristics of CYP2E1, we cloned CYP2E1 cDNA and established a HepG2 cell line stably expressing recombinant CYP 2E1.METHODS: Human CYP2E1 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR)from total RNAs extracted from human liver and cloned into pGEM-T vector. The cDNA segment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant plasmid of pREP9-CYP2E1 to HepG2 cells. The expression of CYP2E1 mRNA was validated by RT-PCR. The enzyme activity of CYP2E1 catalyzing oxidation of 4-nitrophenol in postmitochondrial supernate (S9) fraction of the cells was determined by spectrophotometry. The metabolic activation of HepG2-CYP2E1 cells was assayed by N-nitrosodiethylamine (NDEA)cytotoxicity and micronucleus test.RESULTS: The cloned CYP2E1 cDNA segment was identical to that reported by Umeno et al(GenBank access No.J02843). HepG2-CYP2E1 cells expressed CYP2E1 mRNA and had 4-nitrophenol hydroxylase activity (0.162±0.025nmol.min-1.mg-1 S9 protein), which were undetectable in parent HepG2 cells. HepG2-CYP2E1 cells increased the cytotoxicity and micronucleus rate of NDEA in comparison with those of HepG2 cells.CONCLUSION: The cDNA of human CYP2E1 can be successfully cloned, and a cell line, HepG2-CYP2E1, which can efficiently express mRNA and has CYP2E1 activity, is established. The cell line is useful for testing the cytotoxicity,mutagenicity and metabolism of xenobiotics, which may possibly be activated or metabolized by CYP2E1.

  10. Improved production of biohydrogen in light-powered Escherichia coli by co-expression of proteorhodopsin and heterologous hydrogenase.

    Science.gov (United States)

    Kim, Jaoon Y H; Jo, Byung Hoon; Jo, Younghwa; Cha, Hyung Joon

    2012-01-04

    Solar energy is the ultimate energy source on the Earth. The conversion of solar energy into fuels and energy sources can be an ideal solution to address energy problems. The recent discovery of proteorhodopsin in uncultured marine γ-proteobacteria has made it possible to construct recombinant Escherichia coli with the function of light-driven proton pumps. Protons that translocate across membranes by proteorhodopsin generate a proton motive force for ATP synthesis by ATPase. Excess protons can also be substrates for hydrogen (H(2)) production by hydrogenase in the periplasmic space. In the present work, we investigated the effect of the co-expression of proteorhodopsin and hydrogenase on H(2) production yield under light conditions. Recombinant E. coli BL21(DE3) co-expressing proteorhodopsin and [NiFe]-hydrogenase from Hydrogenovibrio marinus produced ~1.3-fold more H(2) in the presence of exogenous retinal than in the absence of retinal under light conditions (70 μmole photon/(m2·s)). We also observed the synergistic effect of proteorhodopsin with endogenous retinal on H(2) production (~1.3-fold more) with a dual plasmid system compared to the strain with a single plasmid for the sole expression of hydrogenase. The increase of light intensity from 70 to 130 μmole photon/(m(2)·s) led to an increase (~1.8-fold) in H(2) production from 287.3 to 525.7 mL H(2)/L-culture in the culture of recombinant E. coli co-expressing hydrogenase and proteorhodopsin in conjunction with endogenous retinal. The conversion efficiency of light energy to H(2) achieved in this study was ~3.4%. Here, we report for the first time the potential application of proteorhodopsin for the production of biohydrogen, a promising alternative fuel. We showed that H(2) production was enhanced by the co-expression of proteorhodopsin and [NiFe]-hydrogenase in recombinant E. coli BL21(DE3) in a light intensity-dependent manner. These results demonstrate that E. coli can be applied as light

  11. Improved production of biohydrogen in light-powered Escherichia coli by co-expression of proteorhodopsin and heterologous hydrogenase

    Directory of Open Access Journals (Sweden)

    Kim Jaoon YH

    2012-01-01

    Full Text Available Abstract Background Solar energy is the ultimate energy source on the Earth. The conversion of solar energy into fuels and energy sources can be an ideal solution to address energy problems. The recent discovery of proteorhodopsin in uncultured marine γ-proteobacteria has made it possible to construct recombinant Escherichia coli with the function of light-driven proton pumps. Protons that translocate across membranes by proteorhodopsin generate a proton motive force for ATP synthesis by ATPase. Excess protons can also be substrates for hydrogen (H2 production by hydrogenase in the periplasmic space. In the present work, we investigated the effect of the co-expression of proteorhodopsin and hydrogenase on H2 production yield under light conditions. Results Recombinant E. coli BL21(DE3 co-expressing proteorhodopsin and [NiFe]-hydrogenase from Hydrogenovibrio marinus produced ~1.3-fold more H2 in the presence of exogenous retinal than in the absence of retinal under light conditions (70 μmole photon/(m2·s. We also observed the synergistic effect of proteorhodopsin with endogenous retinal on H2 production (~1.3-fold more with a dual plasmid system compared to the strain with a single plasmid for the sole expression of hydrogenase. The increase of light intensity from 70 to 130 μmole photon/(m2·s led to an increase (~1.8-fold in H2 production from 287.3 to 525.7 mL H2/L-culture in the culture of recombinant E. coli co-expressing hydrogenase and proteorhodopsin in conjunction with endogenous retinal. The conversion efficiency of light energy to H2 achieved in this study was ~3.4%. Conclusion Here, we report for the first time the potential application of proteorhodopsin for the production of biohydrogen, a promising alternative fuel. We showed that H2 production was enhanced by the co-expression of proteorhodopsin and [NiFe]-hydrogenase in recombinant E. coli BL21(DE3 in a light intensity-dependent manner. These results demonstrate that E. coli

  12. Gene Expression Patterns in Ovarian Carcinomas

    Science.gov (United States)

    Schaner, Marci E.; Ross, Douglas T.; Ciaravino, Giuseppe; Sørlie, Therese; Troyanskaya, Olga; Diehn, Maximilian; Wang, Yan C.; Duran, George E.; Sikic, Thomas L.; Caldeira, Sandra; Skomedal, Hanne; Tu, I-Ping; Hernandez-Boussard, Tina; Johnson, Steven W.; O'Dwyer, Peter J.; Fero, Michael J.; Kristensen, Gunnar B.; Børresen-Dale, Anne-Lise; Hastie, Trevor; Tibshirani, Robert; van de Rijn, Matt; Teng, Nelson N.; Longacre, Teri A.; Botstein, David; Brown, Patrick O.; Sikic, Branimir I.

    2003-01-01

    We used DNA microarrays to characterize the global gene expression patterns in surface epithelial cancers of the ovary. We identified groups of genes that distinguished the clear cell subtype from other ovarian carcinomas, grade I and II from grade III serous papillary carcinomas, and ovarian from breast carcinomas. Six clear cell carcinomas were distinguished from 36 other ovarian carcinomas (predominantly serous papillary) based on their gene expression patterns. The differences may yield insights into the worse prognosis and therapeutic resistance associated with clear cell carcinomas. A comparison of the gene expression patterns in the ovarian cancers to published data of gene expression in breast cancers revealed a large number of differentially expressed genes. We identified a group of 62 genes that correctly classified all 125 breast and ovarian cancer specimens. Among the best discriminators more highly expressed in the ovarian carcinomas were PAX8 (paired box gene 8), mesothelin, and ephrin-B1 (EFNB1). Although estrogen receptor was expressed in both the ovarian and breast cancers, genes that are coregulated with the estrogen receptor in breast cancers, including GATA-3, LIV-1, and X-box binding protein 1, did not show a similar pattern of coexpression in the ovarian cancers. PMID:12960427

  13. Microanalysis of gene expression in cultured cells

    NARCIS (Netherlands)

    E. van der Veer (Eveliene)

    1982-01-01

    textabstractIn this thesis two aspects of gene expression in cultured cells have been studied: the heterogeneity in gene expression in relation with the development and application of microchemical techniques for the prenatal diagnosis of inborn errors of metabolism and the possibility of inducing g

  14. Arabidopsis gene expression patterns during spaceflight

    Science.gov (United States)

    Paul, A.-L.; Ferl, R. J.

    The exposure of Arabidopsis thaliana (Arabidopsis) plants to spaceflight environments resulted in the differential expression of hundreds of genes. A 5 day mission on orbiter Columbia in 1999 (STS-93) carried transgenic Arabidopsis plants engineered with a transgene composed of the alcohol dehydrogenase (Adh) gene promoter linked to the β -Glucuronidase (GUS) reporter gene. The plants were used to evaluate the effects of spaceflight on two fronts. First, expression patterns visualized with the Adh/GUS transgene were used to address specifically the possibility that spaceflight induces a hypoxic stress response, and to assess whether any spaceflight response was similar to control terrestrial hypoxia-induced gene expression patterns. (Paul et al., Plant Physiol. 2001, 126:613). Second, genome-wide patterns of native gene expression were evaluated utilizing the Affymetrix ATH1 GeneChip? array of 8,000 Arabidopsis genes. As a control for the veracity of the array analyses, a selection of genes identified with the arrays was further characterized with quantitative Real-Time RT PCR (ABI - TaqmanTM). Comparison of the patterns of expression for arrays of hybridized with RNA isolated from plants exposed to spaceflight compared to the control arrays revealed hundreds of genes that were differentially expressed in response to spaceflight, yet most genes that are hallmarks of hypoxic stress were unaffected. These results will be discussed in light of current models for plant responses to the spaceflight environment, and with regard to potential future flight opportunities.

  15. Molecular Characterization of Native and Recom­binant Ionotrophic Glutamate Receptors Expressed in Neurons and Heterologous Systems

    DEFF Research Database (Denmark)

    Drasbek, Kim Ryun

    2005-01-01

    but one construct mediated GluR2 protein knockdown four days after transfection. Due to the low transfection efficiency of cultured primary neurons, GluR2 knockdown was estimated at the single cell level. Rectification properties in transfected neurons were analyzed followed by single cell RTPCR....... No inward rectification was seen in control neurons, while several neurons transfected with the same construct showed inward rectification, indicating GluR2 knockdown in surface expressed AMPARs. In addition, because no mEPSCs were observed at positive holding potentials in these neurons, synaptic AMPARs...

  16. Expression of Sox genes in tooth development.

    Science.gov (United States)

    Kawasaki, Katsushige; Kawasaki, Maiko; Watanabe, Momoko; Idrus, Erik; Nagai, Takahiro; Oommen, Shelly; Maeda, Takeyasu; Hagiwara, Nobuko; Que, Jianwen; Sharpe, Paul T; Ohazama, Atsushi

    2015-01-01

    Members of the Sox gene family play roles in many biological processes including organogenesis. We carried out comparative in situ hybridization analysis of seventeen sox genes (Sox1-14, 17, 18, 21) during murine odontogenesis from the epithelial thickening to the cytodifferentiation stages. Localized expression of five Sox genes (Sox6, 9, 13, 14 and 21) was observed in tooth bud epithelium. Sox13 showed restricted expression in the primary enamel knots. At the early bell stage, three Sox genes (Sox8, 11, 17 and 21) were expressed in pre-ameloblasts, whereas two others (Sox5 and 18) showed expression in odontoblasts. Sox genes thus showed a dynamic spatio-temporal expression during tooth development.

  17. Expression of Sox genes in tooth development

    Science.gov (United States)

    KAWASAKI, KATSUSHIGE; KAWASAKI, MAIKO; WATANABE, MOMOKO; IDRUS, ERIK; NAGAI, TAKAHIRO; OOMMEN, SHELLY; MAEDA, TAKEYASU; HAGIWARA, NOBUKO; QUE, JIANWEN; SHARPE, PAUL T.; OHAZAMA, ATSUSHI

    2017-01-01

    Members of the Sox gene family play roles in many biological processes including organogenesis. We carried out comparative in situ hybridization analysis of seventeen sox genes (Sox1-14, 17, 18, 21) during murine odontogenesis from the epithelial thickening to the cytodifferentiation stages. Localized expression of five Sox genes (Sox6, 9, 13, 14 and 21) was observed in tooth bud epithelium. Sox13 showed restricted expression in the primary enamel knots. At the early bell stage, three Sox genes (Sox8, 11, 17 and 21) were expressed in pre-ameloblasts, whereas two others (Sox5 and 18) showed expression in odontoblasts. Sox genes thus showed a dynamic spatio-temporal expression during tooth development. PMID:26864488

  18. Heterologous expression of tyrosinase (MelC2) from Streptomyces avermitilis MA4680 in E. coli and its application for ortho-hydroxylation of resveratrol to produce piceatannol.

    Science.gov (United States)

    Lee, Nahum; Lee, Sang-Hyuk; Baek, Kiheon; Kim, Byung-Gee

    2015-10-01

    Recombinant tyrosinase from Streptomyces avermitilis MA4680, MelC2 (gi:499291317), was heterologously expressed in Escherichia coli BL21 (DE3). The expression level of active MelC2 was increased by the codon-optimized MelC1 caddie protein (KP198295.1). By performing saturation mutagenesis of the Y91 residue of MelC1, it was found that aromatic residues such as Y, F, and W at the 91st position help produce a correctly folded conformation of MelC2. The recombinant MelC2 was utilized as a biocatalyst to convert trans-resveratrol into piceatannol. In order to improve the product yield through suppression of the formation of melanin, a by-product, an increase in the ratio of monooxygenation (k 1) to dioxygenation (k 2) of MelC2 is desirable. This was achieved by a combination of protein engineering and regeneration of NADH with glucose dehydrogenase (GDH). Saturation mutagenesis was performed at 15 residues within 8-Å radius from copper ions of MelC2. A total of 2760 mutants were examined (99.7 % probability for NNK codon) and I41Y, a mutant, was screened. The ratio of k 1 to k 2 of the mutant increased sevenfold on tyrosine and fivefold on resveratrol, when compared to wild-type MelC2. As a result, the overall product yield from 500 μM resveratrol in 50-mL reaction was 15.4 % (77.4 μM piceatannol), 1.7 times higher than wild type. When I41Y was incorporated with the NADH regeneration system, the total product yield was 58.0 %, an eightfold increase (290.2 μM of piceatannol).

  19. Gene set analysis for longitudinal gene expression data

    Directory of Open Access Journals (Sweden)

    Piepho Hans-Peter

    2011-07-01

    Full Text Available Abstract Background Gene set analysis (GSA has become a successful tool to interpret gene expression profiles in terms of biological functions, molecular pathways, or genomic locations. GSA performs statistical tests for independent microarray samples at the level of gene sets rather than individual genes. Nowadays, an increasing number of microarray studies are conducted to explore the dynamic changes of gene expression in a variety of species and biological scenarios. In these longitudinal studies, gene expression is repeatedly measured over time such that a GSA needs to take into account the within-gene correlations in addition to possible between-gene correlations. Results We provide a robust nonparametric approach to compare the expressions of longitudinally measured sets of genes under multiple treatments or experimental conditions. The limiting distributions of our statistics are derived when the number of genes goes to infinity while the number of replications can be small. When the number of genes in a gene set is small, we recommend permutation tests based on our nonparametric test statistics to achieve reliable type I error and better power while incorporating unknown correlations between and within-genes. Simulation results demonstrate that the proposed method has a greater power than other methods for various data distributions and heteroscedastic correlation structures. This method was used for an IL-2 stimulation study and significantly altered gene sets were identified. Conclusions The simulation study and the real data application showed that the proposed gene set analysis provides a promising tool for longitudinal microarray analysis. R scripts for simulating longitudinal data and calculating the nonparametric statistics are posted on the North Dakota INBRE website http://ndinbre.org/programs/bioinformatics.php. Raw microarray data is available in Gene Expression Omnibus (National Center for Biotechnology Information with

  20. Molecular cloning of amphioxus uncoupling protein and assessment of its uncoupling activity using a yeast heterologous expression system

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Kun [Jiangsu Diabetes Research Center, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu (China); Sun, Guoxun [Department of Hematology, Fourth Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150001 (China); Lv, Zhiyuan; Wang, Chen [Jiangsu Diabetes Research Center, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu (China); Jiang, Xueyuan, E-mail: xueyuanjiang@yahoo.com.cn [Jiangsu Diabetes Research Center, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu (China); Li, Donghai, E-mail: lidonghai@gmail.com [Jiangsu Diabetes Research Center, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu (China); Zhang, Chenyu, E-mail: cyzhang@nju.edu.cn [Jiangsu Diabetes Research Center, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu (China)

    2010-10-01

    Research highlights: {yields} Invertebrates, for example amphioxus, do express uncoupling proteins. {yields} Both the sequence and the uncoupling activity of amphioxus UCP resemble UCP2. {yields} UCP1 is the only UCP that can form dimer on yeast mitochondria. -- Abstract: The present study describes the molecular cloning of a novel cDNA fragment from amphioxus (Branchiostoma belcheri) encoding a 343-amino acid protein that is highly homologous to human uncoupling proteins (UCP), this protein is therefore named amphioxus UCP. This amphioxus UCP shares more homology with and is phylogenetically more related to mammalian UCP2 as compared with UCP1. To further assess the functional similarity of amphioxus UCP to mammalian UCP1 and -2, the amphioxus UCP, rat UCP1, and human UCP2 were separately expressed in Saccharomyces cerevisiae, and the recombinant yeast mitochondria were isolated and assayed for the state 4 respiration rate and proton leak, using pYES2 empty vector as the control. UCP1 increased the state 4 respiration rate by 2.8-fold, and the uncoupling activity was strongly inhibited by GDP, while UCP2 and amphioxus UCP only increased the state 4 respiration rate by 1.5-fold and 1.7-fold in a GDP-insensitive manner, moreover, the proton leak kinetics of amphioxus UCP was very similar to UCP2, but much different from UCP1. In conclusion, the amphioxus UCP has a mild, unregulated uncoupling activity in the yeast system, which resembles mammalian UCP2, but not UCP1.

  1. Heterologous expression of Ceratophyllum demersum phytochelatin synthase, CdPCS1, in rice leads to lower arsenic accumulation in grain.

    Science.gov (United States)

    Shri, Manju; Dave, Richa; Diwedi, Sanjay; Shukla, Devesh; Kesari, Ravi; Tripathi, Rudra Deo; Trivedi, Prabodh Kumar; Chakrabarty, Debasis

    2014-07-22

    Recent studies have identified rice (Oryza sativa) as a major dietary source of inorganic arsenic (As) and poses a significant human health risk. The predominant model for plant detoxification of heavy metals is complexation of heavy metals with phytochelatins (PCs), synthesized non-translationally by PC synthase (PCS) and compartmentalized in vacuoles. In this study, in order to restrict As in the rice roots as a detoxification mechanism, a transgenic approach has been followed through expression of phytochelatin synthase, CdPCS1, from Ceratophyllum demersum, an aquatic As-accumulator plant. CdPCS1 expressing rice transgenic lines showed marked increase in PCS activity and enhanced synthesis of PCs in comparison to non-transgenic plant. Transgenic lines showed enhanced accumulation of As in root and shoot. This enhanced metal accumulation potential of transgenic lines was positively correlated to the content of PCs, which also increased several-fold higher in transgenic lines. However, all the transgenic lines accumulated significantly lower As in grain and husk in comparison to non-transgenic plant. The higher level of PCs in transgenic plants relative to non-transgenic presumably allowed sequestering and detoxification of higher amounts of As in roots and shoots, thereby restricting its accumulation in grain.

  2. Identification, cloning and heterologous expression of active [NiFe]-hydrogenase 2 from Citrobacter sp. SG in Escherichia coli.

    Science.gov (United States)

    Maier, Johannes A H; Ragozin, Sergey; Jeltsch, Albert

    2015-04-10

    Hydrogen (H2) is a potential alternative energy carrier which only produces water and heat upon combustion. Today, industrial hydrogen production mainly uses thermochemical processes based on fossil fuels or electrolysis of water. Therefore, biotechnological approaches to produce H2 from biomass are an interesting alternative. We introduce here a novel direct hydrogen measurement system using a semiconducting device specific for hydrogen detection. Using this device, a bacterium producing considerable amounts of hydrogen under aerobic cultivation was isolated and identified by 16S ribosomal DNA sequencing as Citrobacter sp. The enzyme responsible for the observed hydrogenase activity was partially purified by 3 chromatographic purification steps and could be identified by peptide mass fingerprinting to be a type 2 [NiFe]-hydrogenase. Expression of the [NiFe]-hydrogenase 2 containing operon from Citrobacter sp. SG in Escherichia coli allowed recombinant hydrogen production. The [NiFe]-hydrogenase 2 identified here may be useful for biotechnological hydrogen production. We speculate that the expression of the hydrogenase in Citrobacter may be an adaptation to growth in acidic conditions.

  3. FARO server: Meta-analysis of gene expression by matching gene expression signatures to a compendium of public gene expression data

    DEFF Research Database (Denmark)

    Manijak, Mieszko P.; Nielsen, Henrik Bjørn

    2011-01-01

    BACKGROUND: Although, systematic analysis of gene annotation is a powerful tool for interpreting gene expression data, it sometimes is blurred by incomplete gene annotation, missing expression response of key genes and secondary gene expression responses. These shortcomings may be partially...... circumvented by instead matching gene expression signatures to signatures of other experiments. FINDINGS: To facilitate this we present the Functional Association Response by Overlap (FARO) server, that match input signatures to a compendium of 242 gene expression signatures, extracted from more than 1700...

  4. Heterologous prime-boost-boost immunisation of Chinese cynomolgus macaques using DNA and recombinant poxvirus vectors expressing HIV-1 virus-like particles

    Directory of Open Access Journals (Sweden)

    Anson Donald S

    2011-09-01

    Full Text Available Abstract Background There is renewed interest in the development of poxvirus vector-based HIV vaccines due to the protective effect observed with repeated recombinant canarypox priming with gp120 boosting in the recent Thai placebo-controlled trial. This study sought to investigate whether a heterologous prime-boost-boost vaccine regimen in Chinese cynomolgus macaques with a DNA vaccine and recombinant poxviral vectors expressing HIV virus-like particles bearing envelopes derived from the most prevalent clades circulating in sub-Saharan Africa, focused the antibody response to shared neutralising epitopes. Methods Three Chinese cynomolgus macaques were immunised via intramuscular injections using a regimen composed of a prime with two DNA vaccines expressing clade A Env/clade B Gag followed by boosting with recombinant fowlpox virus expressing HIV-1 clade D Gag, Env and cholera toxin B subunit followed by the final boost with recombinant modified vaccinia virus Ankara expressing HIV-1 clade C Env, Gag and human complement protein C3d. We measured the macaque serum antibody responses by ELISA, enumerated T cell responses by IFN-γ ELISpot and assessed seroneutralisation of HIV-1 using the TZM-bl β-galactosidase assay with primary isolates of HIV-1. Results This study shows that large and complex synthetic DNA sequences can be successfully cloned in a single step into two poxvirus vectors: MVA and FPV and the recombinant poxviruses could be grown to high titres. The vaccine candidates showed appropriate expression of recombinant proteins with the formation of authentic HIV virus-like particles seen on transmission electron microscopy. In addition the b12 epitope was shown to be held in common by the vaccine candidates using confocal immunofluorescent microscopy. The vaccine candidates were safely administered to Chinese cynomolgus macaques which elicited modest T cell responses at the end of the study but only one out of the three macaques

  5. Heterologous expression, purification, and enzymatic characterization of the acyclic carotenoid 1,2-hydratase from Rubrivivax gelatinosus.

    Science.gov (United States)

    Steiger, Sabine; Mazet, Andreas; Sandmann, Gerhard

    2003-06-01

    The carotenoid 1,2-hydratase CrtC from Rubrivivax gelatinosus has been expressed in Escherichia coli in an active form and purified by affinity chromatography. The enzyme catalyzes the conversion of various acyclic carotenes including 1-hydroxy derivatives. This broad substrate specificity reflects the participation of CrtC in 1'-HO-spheroidene and in spirilloxanthin biosynthesis. Enzyme kinetic studies including the determination of substrate specificity constants indicate that among the alternative biosynthetic routes to 1'-HO-spheroidene the one via spheroidene is the dominating pathway. In contrast to CrtC from Rvi. gelatinosus, the equivalent enzyme from Rhodobacter capsulatus, a closely related bacterium which lacks the biosynthetic branch to spirilloxanthin and accumulates spheroidene instead of substantial amounts of 1'-HO-spheroidene, is extremely poor in converting 1-HO-carotenoids. The individual catalytic properties of both carotenoid 1,2-hydratases reflect the in situ carotenogenic pathways in both purple photosynthetic bacteria.

  6. Effects of seven RNA silencing suppressors on heterologous expression of green fluorescence protein expression mediated by a plant virus-based system in Nicotiana benthamiana%7种RNA沉默抑制子对植物病毒载体表达系统表达水平的影响

    Institute of Scientific and Technical Information of China (English)

    王盛; 董洁; 曹慜; 穆红珍; 丁国平; 张虹

    2012-01-01

    Objective To test the effects of 7 virus-encoded RNA silencing suppressors (RSSs) for enhancement of a plant virus-based vector system-mediated heterologous expression of green fluorescence protein (GFP) in Nicotiana benthamiana. Methods Seven transient expression vectors for the 7 RSSs were constructed and co-inoculated on the leaves of Nicotiana benthamiana with PVXdt-GFP vector, a novel Potato virus X-based plant expression vector, through agroinfiltration. The protein and mRNA expression levels of the reporter gene GFP in the co-inoculated Nicotiana leaves were examined by Western blotting, ELISA and RT-qPCR to assess the effect of the RSSs for GFP expression enhancement. Results The 7 RSSs differed in the degree and duration of enhancement of heterologous GFP expression, and the pl9 protein of Tomato bushy stunt virus (TBSV) induced the highest expression of GFP. African cassava mosaic virus AC2 protein and Rice yellow mettle virus PI protein produced no obvious enhancement GFP expression. Conclusion Transient co-expression of RSSs suppresses host silencing response to allow high-level and long-term expression of heterologous genes in plant, but the optimal RSS has to be identified for each plant virus-based expression vector system.%目的 探索不同植物病毒RNA沉默抑制子对植物病毒载体表达系统重组蛋白表达水平的作用,为合理高效地利用这一新型的外源基因表达平台奠定基础.方法 构建了7种不同的RNA沉默抑制子瞬时表达载体,以农杆菌渗滤法,与马铃薯X病毒表达载体PVXdt-GFP共侵染寄主植物本明烟,通过对报告基因绿色荧光蛋白(GFP)的荧光观察,并以Western blotting、ELISA 和RT-qPCR等测定GFP在烟草中的表达情况,分析不同RNA沉默抑制子对植物中外源基因表达水平的作用和特点.结果 7种病毒RNA沉默抑制子对外源基因GFP在烟草中表达水平的作用效果和持续时间存在差异,其中,番茄丛矮病毒的P19蛋白的

  7. Functional studies of TcRjl, a novel GTPase of Trypanosoma cruzi, reveals phenotypes related with MAPK activation during parasite differentiation and after heterologous expression in Drosophila model system

    Energy Technology Data Exchange (ETDEWEB)

    Reis Monteiro dos-Santos, Guilherme Rodrigo [Laboratório de Parasitologia Molecular, Instituto de Biofísica Carlos Chagas Filho, CCS, UFRJ, Rio de Janeiro (Brazil); Fontenele, Marcio Ribeiro [Laboratório de Biologia Molecular do Desenvolvimento Instituto de Ciências Biomédicas, CCS, UFRJ, Rio de Janeiro (Brazil); Dias, Felipe de Almeida [Laboratório de Bioquímica de Artrópodes Hematófagos, Instituto de Bioquímica Médica, CCS, UFRJ, Rio de Janeiro (Brazil); Oliveira, Pedro Lagerblad de [Laboratório de Bioquímica de Artrópodes Hematófagos, Instituto de Bioquímica Médica, CCS, UFRJ, Rio de Janeiro (Brazil); Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCT-EM) (Brazil); Nepomuceno-Silva, José Luciano [Laboratório Integrado de Bioquímica Hatisaburo Masuda, NUPEM/UFRJ, Pólo Barreto, Universidade Federal do Rio de Janeiro, Campus Macaé, Macaé (Brazil); and others

    2015-11-06

    The life cycle of the protozoan parasite Trypanosoma cruzi comprises rounds of proliferative cycles and differentiation in distinct host environments. Ras GTPases are molecular switches that play pivotal regulatory functions in cell fate. Rjl is a novel GTPase with unknown function. Herein we show that TcRjl blocks in vivo cell differentiation. The forced expression of TcRjl leads to changes in the overall tyrosine protein phosphorylation profile of parasites. TcRjl expressing parasites sustained DNA synthesis regardless the external stimuli for differentiation. Heterologous expression in the Drosophila melanogaster genetic system strongly suggests a role from TcRjl protein in RTK-dependent pathways and MAPK activation.

  8. Functional studies of TcRjl, a novel GTPase of Trypanosoma cruzi, reveals phenotypes related with MAPK activation during parasite differentiation and after heterologous expression in Drosophila model system.

    Science.gov (United States)

    dos-Santos, Guilherme Rodrigo Reis Monteiro; Fontenele, Marcio Ribeiro; Dias, Felipe de Almeida; de Oliveira, Pedro Lagerblad; Nepomuceno-Silva, José Luciano; de Melo, Luiz Dione Barbosa; Araujo, Helena Maria Marcolla; Lopes, Ulisses Gazos

    2015-11-06

    The life cycle of the protozoan parasite Trypanosoma cruzi comprises rounds of proliferative cycles and differentiation in distinct host environments. Ras GTPases are molecular switches that play pivotal regulatory functions in cell fate. Rjl is a novel GTPase with unknown function. Herein we show that TcRjl blocks in vivo cell differentiation. The forced expression of TcRjl leads to changes in the overall tyrosine protein phosphorylation profile of parasites. TcRjl expressing parasites sustained DNA synthesis regardless the external stimuli for differentiation. Heterologous expression in the Drosophila melanogaster genetic system strongly suggests a role from TcRjl protein in RTK-dependent pathways and MAPK activation.

  9. Two heterologously expressed Planobispora rosea proteins cooperatively induce Streptomyces lividans thiostrepton uptake and storage from the extracellular medium

    Directory of Open Access Journals (Sweden)

    Süssmuth Roderich D

    2010-06-01

    Full Text Available Abstract Background A bacterial artificial chromosomal library of Planobispora rosea, a genetically intractable actinomycete strain, was constructed using Escherichia coli-Streptomyces artificial chromosome (ESAC and screened for the presence of genes known to be involved in the biosynthesis of antibiotics. Results One clone with a 40 kb insert showed antimicrobial activity against Gram positive bacteria. Insert sequence analysis and subcloning experiments revealed that the bioactivity was due to a 3.5 kb DNA fragment containing two open reading frames. These orfs encode two proteins with high similarity to a putative membrane protein of Streptomyces coelicolor and to the nogalamycin resistance protein SnorO of Streptomyces nogalater, respectively. The role of these two Orfs is unknown in Planobispora. Disruption and complementation experiments revealed that both proteins are necessary for the antibacterial activity and chemical analysis demonstrated that the antibiotic activity was due to thiostrepton, antibiotic used as recombinant clone selection marker. Conclusion Two Planobispora rosea orfs are responsible for increasing intracellular amounts and storage of thiostrepton in Streptomyces lividans.

  10. The impact of heterologous catalase expression and superoxide dismutase overexpression on enhancing the oxidative resistance in Lactobacillus casei.

    Science.gov (United States)

    Lin, Jinzhong; Zou, Yexia; Cao, Kunlin; Ma, Chengjie; Chen, Zhengjun

    2016-05-01

    Two heme-dependent catalase genes were amplified from genomic DNA of Lactobacillus plantarum WCFS1 (KatE1) and Lactobacillus brevis ATCC 367 (KatE2), respectively, and a manganese-containing superoxide dismutase from Lactobacillus casei MCJΔ1 (MnSOD) were cloned into plasmid pELX1, yielding pELX1-KatE1, pELX1-KatE2 and pELX1-MnSOD, then the recombinant plasmids were transferred into L. casei MCJΔ1. The strains of L. casei MCJΔ1/pELX1-KatE1 and L. casei MCJΔ1/pELX1-KatE2 were tolerant at 2 mM H2O2. The survival rates of L. casei MCJΔ1/pELX1-KatE1 and L. casei MCJΔ1/pELX1-KatE2 were 270-fold and 300-fold higher than that of the control strain on a short-term H2O2 exposure, and in aerated condition, the survival cells counts were 146- and 190-fold higher than that of the control strain after 96 h of incubation. Furthermore, L. casei MCJΔ1/pELX1-MnSOD was the best in three recombinants which was superior in the living cell viability during storage when co-storage with Lactobacillus delbrueckii subsp. lactis LBCH-1.

  11. Biosynthesis, isolation and characterization of {sup 57}Fe-enriched Phaseolus vulgaris ferritin after heterologous expression in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Hoppler, Matthias [ETH Zurich, Laboratory of Human Nutrition, Zurich (Switzerland); Meile, Leo [ETH Zurich, Laboratory of Food Biotechnology, Zurich (Switzerland); Walczyk, Thomas [National University of Singapore, Department of Chemistry and Department of Biochemistry, Singapore (Singapore)

    2008-01-15

    Ferritin is the major iron storage protein in the biosphere. Iron stores of an organism are commonly assessed by measuring the concentration of the protein shell of the molecule in fluids and tissues. The amount of ferritin-bound iron, the more desirable information, still remains inaccessible owing to the lack of suitable techniques. Iron saturation of ferritin is highly variable, with a maximum capacity of 4,500 iron atoms per molecule. This study describes the direct isotopic labeling of a complex metalloprotein in vivo by biosynthesis, in order to measure ferritin-bound iron by isotope dilution mass spectrometry. [{sup 57}Fe]ferritin was produced by cloning and overexpressing the Phaseolus vulgaris ferritin gene pfe in Escherichia coli in the presence of {sup 57}FeCl{sub 2}. Recombinant ferritin was purified in a fully assembled form and contained approximately 1,000 iron atoms per molecule at an isotopic enrichment of more than 95% {sup 57}Fe. We did not find any evidence of species conversion of the isotopic label for at least 5 months of storage at -20 C. Transfer efficiency of enriched iron into [{sup 57}Fe]ferritin of 20% was sufficient to be economically feasible. Negligible amounts of non-ferritin-bound iron in the purified [{sup 57}Fe]ferritin solution allows for use of this spike for quantification of ferritin-bound iron by isotope dilution mass spectrometry. (orig.)

  12. The functional landscape of mouse gene expression

    Directory of Open Access Journals (Sweden)

    Zhang Wen

    2004-12-01

    Full Text Available Abstract Background Large-scale quantitative analysis of transcriptional co-expression has been used to dissect regulatory networks and to predict the functions of new genes discovered by genome sequencing in model organisms such as yeast. Although the idea that tissue-specific expression is indicative of gene function in mammals is widely accepted, it has not been objectively tested nor compared with the related but distinct strategy of correlating gene co-expression as a means to predict gene function. Results We generated microarray expression data for nearly 40,000 known and predicted mRNAs in 55 mouse tissues, using custom-built oligonucleotide arrays. We show that quantitative transcriptional co-expression is a powerful predictor of gene function. Hundreds of functional categories, as defined by Gene Ontology 'Biological Processes', are associated with characteristic expression patterns across all tissues, including categories that bear no overt relationship to the tissue of origin. In contrast, simple tissue-specific restriction of expression is a poor predictor of which genes are in which functional categories. As an example, the highly conserved mouse gene PWP1 is widely expressed across different tissues but is co-expressed with many RNA-processing genes; we show that the uncharacterized yeast homolog of PWP1 is required for rRNA biogenesis. Conclusions We conclude that 'functional genomics' strategies based on quantitative transcriptional co-expression will be as fruitful in mammals as they have been in simpler organisms, and that transcriptional control of mammalian physiology is more modular than is generally appreciated. Our data and analyses provide a public resource for mammalian functional genomics.

  13. Molecular cloning of soluble trehalase from Chironomus riparius larvae, its heterologous expression in Escherichia coli and bioinformatic analysis.

    Science.gov (United States)

    Forcella, Matilde; Mozzi, Alessandra; Bigi, Alessandra; Parenti, Paolo; Fusi, Paola

    2012-10-01

    Trehalase is involved in the control of trehalose concentration, the main blood sugar in insects. Here, we describe the molecular cloning of the cDNA encoding for the soluble form of the trehalase from the midge larvae of Chironomus riparius, a well-known bioindicator of the quality of freshwater environments. Molecular cloning was achieved through multiple alignment of Diptera trehalase sequences, allowing the synthesis of internal homology-based primers; the complete open reading frame(ORF) was subsequently obtained through RACE-PCR(where RACE is rapid amplification of cDNA ends). The cDNA contained the 5' untranslated region (UTR), the 3' UTR including a poly(A) tail and the ORF of 1,725 bp consisting of 574 amino acid residues with a predicted molecular mass of 65,778 Da. Recombinant trehalase was successfully expressed in Escherichia coli as a His-tagged protein and purified on Ni-NTA affinity chromatography. Primary structure analysis showed a series of characteristic features shared by all insect trehalases, while three-dimensional structure prediction yielded the typical glucosidase fold, the two key residues involved in the catalytic mechanism being conserved. Production of recombinant insect trehalases opens the way to structural characterizations of the catalytic site, which might represent, among others, an element for reconsidering the enzyme as a target in pest insects' control.

  14. Heterologous expression of proteorhodopsin enhances H2 production in Escherichia coli when endogenous Hyd-4 is overexpressed.

    Science.gov (United States)

    Kuniyoshi, Taís M; Balan, Andrea; Schenberg, Ana Clara G; Severino, Divinomar; Hallenbeck, Patrick C

    2015-07-20

    Proteorhodopsin (PR) is a light harvesting protein widely distributed among bacterioplankton that plays an integral energetic role in a new pathway of marine light capture. The conversion of light into chemical energy in non-chlorophyll-based bacterial systems could contribute to overcoming thermodynamic and metabolic constraints in biofuels production. In an attempt to improve biohydrogen production yields, H2 evolution catalyzed by endogenous hydrogenases, Hyd-3 and/or Hyd-4, was measured when recombinant proteorhodopsin (PR) was concomitantly expressed in Escherichia coli cells. Higher amounts of H2 were obtained with recombinant cells in a light and chromophore dependent manner. This effect was only observed when HyfR, the specific transcriptional activator of the hyf operon encoding Hyd-4 was overexpressed in E. coli, suggesting that an excess of protons generated by PR activity could increase hydrogen production by Hyd-4 but not by Hyd-3. Although many of the subunits of Hyd-3 and Hyd-4 are very similar, Hyd-4 possesses three additional proton-translocating NADH-ubiquinone oxidoreductase subunits, suggesting that it is dependent upon ΔμH(+). Altogether, these results suggest that protons generated by proteorhodopsin in the periplasm can only enhance hydrogen production by hydrogenases with associated proton translocating subunits.

  15. A xylanase from Streptomyces sp. FA1: heterologous expression, characterization, and its application in Chinese steamed bread.

    Science.gov (United States)

    Xu, Yang; Wu, Jing; Zheng, Kaixuan; Wu, Dan

    2016-05-01

    Xylanases (EC 3.2.1.8) are hydrolytic enzymes that have found widespread application in the food, feed, and paper-pulp industries. Streptomyces sp. FA1 xynA was expressed as a secreted protein in Pichia pastoris, and the xylanase was applied to the production of Chinese steamed bread for the first time. The optimal pH and the optimal temperature of XynA were 5.5 and 60 °C, respectively. Using beechwood as substrate, the K m and V max were 2.408 mg mL(-1) and 299.3 µmol min(-1) mg(-1), respectively. Under optimal conditions, a 3.6-L bioreactor produced 1374 U mL(-1) of XynA activity at a protein concentration of 6.3 g L(-1) after 132 h of fermentation. Use of recombinant XynA led to a greater increase in the specific volume of the CSB than could be achieved using commercial xylanase under optimal conditions. This study provides the basis for the application of the enzyme in the baking industry.

  16. Crystallization and preliminary characterization of three different crystal forms of human saposin C heterologously expressed in Pichia pastoris

    Energy Technology Data Exchange (ETDEWEB)

    Schultz-Heienbrok, Robert [Institut für Chemie und Biochemie/Kristallographie, Freie Universität Berlin (Germany); Remmel, Natascha; Klingenstein, R. [Kekule-Institut für Organische Chemie und Biochemie, Rheinische Friedrich-Wilhelms-Universität Bonn (Germany); Rossocha, Maksim [Institut für Chemie und Biochemie/Kristallographie, Freie Universität Berlin (Germany); Sandhoff, Konrad [Kekule-Institut für Organische Chemie und Biochemie, Rheinische Friedrich-Wilhelms-Universität Bonn (Germany); Saenger, Wolfram [Institut für Chemie und Biochemie/Kristallographie, Freie Universität Berlin (Germany); Maier, Timm, E-mail: timm.maier@mol.biol.ethz.ch [Institut für Chemie und Biochemie/Kristallographie, Freie Universität Berlin (Germany); Institute for Molecular Biology and Biophysics, Swiss Federal Institute of Technology, ETH Zürich (Switzerland)

    2006-02-01

    Three different crystal forms were obtained of human saposin C. The structures could not be determined by molecular replacement using known solution structures of the protein as search models, supporting the notion of a highly flexible protein. The amphiphilic saposin proteins (A, B, C and D) act at the lipid–water interface in lysosomes, mediating the hydrolysis of membrane building blocks by water-soluble exohydrolases. Human saposin C activates glucocerebrosidase and β-galactosylceramidase. The protein has been expressed in Pichia pastoris, purified and crystallized in three different crystal forms, diffracting to a maximum resolution of 2.5 Å. Hexagonal crystals grew from 2-propanol-containing solution and contain a single molecule in the asymmetric unit according to the Matthews coefficient. Orthorhombic and tetragonal crystals were both obtained with pentaerythritol ethoxylate and are predicted to contain two molecules in the asymmetric unit. Attempts to determine the respective crystal structures by molecular replacement using either the known NMR structure of human saposin C or a related crystal structure as search models have so far failed. The failure of the molecular-replacement method is attributed to conformational changes of the protein, which are known to be required for its biological activity. Crystal structures of human saposin C therefore might be the key to mapping out the conformational trajectory of saposin-like proteins.

  17. Heterologous expression and biochemical characterization of an endo-β-1,4-glucanase from Thermobifida fusca.

    Science.gov (United States)

    Yan, Peng'an; Su, Lingqia; Chen, Jian; Wu, Jing

    2013-01-01

    The endoglucanase Cel5A from Thermobifida fusca was cloned and expressed in Escherichia coli BL21(DE3). The carboxymethyl cellulase (CMCase) activity in shake flasks and 3-L fermentation scale reached 46.8 and 656.6 IU/mL, respectively. The CMCase activity in 3-L fermentation scale represented the highest yield of T. fusca Cel5A reported so far. Recombinant Cel5A was purified and characterized in detail. The optimum temperature of recombinant enzyme was 80 °C, and the half-life of the enzyme was 132 H at 50 °C and 65 H at 60 °C. The activity of recombinant Cel5A was retained more than 90% over the range of pH 5.0-10.0 with maximal activity at pH 5.5. Using carboxymethyl cellulose as the substrate, the Km and Vmax values were 5.1 mg/mL and 48.7 IU/mg, respectively. The enzyme showed superstability in surfactants and was retained above 90% activity after treatment with sodium dodecyl sulfate, linear alkyl benzene sulfonate, fatty alcohol polyoxyethylene (9) ether, and polyoxyethylene (10) nonyl phenyl ether at 25 °C for 1 H, indicating that the enzyme could be a valuable component in detergents. The potential mechanism of this stability was investigated by analysis of the electrostatic potential of the surface of the enzyme.

  18. Heterologous expression of the hydrophobin FcHyd5p from Fusarium culmorum in Pichia pastoris and evaluation of its surface activity and contribution to gushing of carbonated beverages.

    Science.gov (United States)

    Stübner, Matthias; Lutterschmid, Georg; Vogel, Rudi F; Niessen, Ludwig

    2010-06-30

    The class II hydrophobin FcHyd5p from Fusarium culmorum was heterologously expressed by Pichia pastoris. Transcription of the recombinant gene was confirmed by RT-PCR and expression of FcHyd5p was demonstrated using SDS-PAGE and immuno-staining with 6 x His-tag antibodies. FcHyd5p was purified and concentrated by dialysis, isoelectric focussing and the use of ultra filtration. It was demonstrated that FcHyd5p is able to change the hydrophopic properties of surfaces rendering them wettable after coating with the supernatant of recombinant P. pastoris cultures. Furthermore, due to its surface activity, FcHyd5p was able to stabilise air bubbles in aqueous solutions. The supernatant of a culture medium containing a FcHyd5p producing P. pastoris clone remained turbid for 24h and the foam stable for more than 72 h after the treatment with a homogeniser, whereas the liquid of the wild type strain clarified after 10 min and the foam disintegrated after 2h. Finally it was demonstrated, that FcHyd5p can induce spontaneous overfoaming of carbonated liquids, referred to as gushing. Addition of 2 mg freeze-dried culture supernatant from a FcHyd5p producing P. pastoris clone resulted in a lost volume of 252 ml +/- 20 per 500 ml of beer (50%) and 179 ml +/- 7 per 330 ml of carbonated water (54%), respectively. Neither untreated beer/water, nor beer/water treated with freeze-dried culture supernatant from the wild type strain showed any gushing. Furthermore, addition of 215 microg highly purified FcHyd5p resulted in a lost volume of 77 ml +/- 40 per 500 ml beer (15%). 2010 Elsevier B.V. All rights reserved.

  19. Simultaneous stable expression of neomycin phosphotransferase and green fluorescence protein genes in Trypanosoma cruzi.

    Science.gov (United States)

    dos Santos, W G; Buck, G A

    2000-12-01

    The ribosomal RNA (rRNA) gene promoter was used to construct plasmid vectors that simultaneously express multiple exogenous genes in Trypanosoma cruzi. Vector pBSPANEO expresses neomycin phosphotransferase, and pPAGFPAN expresses both green fluorescent protein and neomycin phosphotransferase from a single promoter. Both vectors require the presence of the rRNA promoter for stable transfection; epimastigotes transfected with pPAGFPAN strongly fluoresced due to green fluorescent protein expression. Intact plasmids were rescued from the T. cruzi-transfected population after >8 mo of culture, indicating stable replication of these vectors. Vectors were integrated into the rRNA locus by homologous recombination and into other loci, presumably by illegitimate recombination. Parasites bearing tandem concatamers of plasmids were also found among the transfectants. Transfectants expressing green fluorescent protein showed a bright green fluorescence distributed throughout the cell. Fluorescence was also detected in amastigotes after infection of mammalian cells with transfected parasites, indicating that the rRNA promoter can drive efficient expression of these reporter genes in multiple life-cycle stages of the parasite. Expression of the heterologous genes was detected after passage in mice or in the insect vector. These vectors will be useful for the genetic dissection of T. cruzi biology and pathogenesis.

  20. Differential gene expression during Trypanosoma cruzi metacyclogenesis

    Directory of Open Access Journals (Sweden)

    Marco Aurelio Krieger

    1999-09-01

    Full Text Available The transformation of epimastigotes into metacyclic trypomastigotes involves changes in the pattern of expressed genes, resulting in important morphological and functional differences between these developmental forms of Trypanosoma cruzi. In order to identify and characterize genes involved in triggering the metacyclogenesis process and in conferring to metacyclic trypomastigotes their stage specific biological properties, we have developed a method allowing the isolation of genes specifically expressed when comparing two close related cell populations (representation of differential expression or RDE. The method is based on the PCR amplification of gene sequences selected by hybridizing and subtracting the populations in such a way that after some cycles of hybridization-amplification genes specific to a given population are highly enriched. The use of this method in the analysis of differential gene expression during T. cruzi metacyclogenesis (6 hr and 24 hr of differentiation and metacyclic trypomastigotes resulted in the isolation of several clones from each time point. Northern blot analysis showed that some genes are transiently expressed (6 hr and 24 hr differentiating cells, while others are present in differentiating cells and in metacyclic trypomastigotes. Nucleotide sequencing of six clones characterized so far showed that they do not display any homology to gene sequences available in the GeneBank.

  1. Multivariate search for differentially expressed gene combinations

    Directory of Open Access Journals (Sweden)

    Klebanov Lev

    2004-10-01

    Full Text Available Abstract Background To identify differentially expressed genes, it is standard practice to test a two-sample hypothesis for each gene with a proper adjustment for multiple testing. Such tests are essentially univariate and disregard the multidimensional structure of microarray data. A more general two-sample hypothesis is formulated in terms of the joint distribution of any sub-vector of expression signals. Results By building on an earlier proposed multivariate test statistic, we propose a new algorithm for identifying differentially expressed gene combinations. The algorithm includes an improved random search procedure designed to generate candidate gene combinations of a given size. Cross-validation is used to provide replication stability of the search procedure. A permutation two-sample test is used for significance testing. We design a multiple testing procedure to control the family-wise error rate (FWER when selecting significant combinations of genes that result from a successive selection procedure. A target set of genes is composed of all significant combinations selected via random search. Conclusions A new algorithm has been developed to identify differentially expressed gene combinations. The performance of the proposed search-and-testing procedure has been evaluated by computer simulations and analysis of replicated Affymetrix gene array data on age-related changes in gene expression in the inner ear of CBA mice.

  2. Gene Expression Profiling in Porcine Fetal Thymus

    Institute of Scientific and Technical Information of China (English)

    Yanjiong Chen; Shengbin Li; Lin Ye; Jianing Geng; Yajun Deng; Songnian Hu

    2003-01-01

    obtain an initial overview of gene diversity and expression pattern in porcinethymus, 11,712 ESTs (Expressed Sequence Tags) from 100-day-old porcine thymus(FTY) were sequenced and 7,071 cleaned ESTs were used for gene expressionanalysis. Clustered by the PHRAP program, 959 contigs and 3,074 singlets wereobtained. Blast search showed that 806 contigs and 1,669 singlets (totally 5,442ESTs) had homologues in GenBank and 1,629 ESTs were novel. According to theGene Ontology classification, 36.99% ESTs were cataloged into the gene expressiongroup, indicating that although the functional gene (18.78% in defense group) ofthymus is expressed in a certain degree, the 100-day-old porcine thymus still existsin a developmental stage. Comparative analysis showed that the gene expressionpattern of the 100-day-old porcine thymus is similar to that of the human infantthymus.

  3. Phytochrome-regulated Gene Expression

    Institute of Scientific and Technical Information of China (English)

    Peter H. Quail

    2007-01-01

    Identification of all genes involved in the phytochrome (phy)-mediated responses of plants to their light environment is an important goal in providing an overall understanding of light-regulated growth and development. This article highlights and integrates the central findings of two recent comprehensive studies in Arabidopsis that have identified the genome-wide set of phy-regulated genes that respond rapidly to red-light signals upon first exposure of dark-grown seedlings, and have tested the functional relevance to normal seedling photomorphogenesis of an initial subset of these genes. The data: (a) reveal considerable complexity in the channeling of the light signals through the different phy-family members (phyA to phyE) to responsive genes; (b) identify a diversity of transcription-factor-encoding genes as major early, if not primary, targets of phy signaling, and, therefore, as potentially important regulators in the transcriptional-network hierarchy; and (c) identify auxin-related genes as the dominant class among rapidly-regulated, hormone-related genes. However, reverse-genetic functional profiling of a selected subset of these genes reveals that only a limited fraction are necessary for optimal phy-induced seedling deetiolation.

  4. Antisense RNA decreases AP33 gene expression and cytoadherence by T. vaginalis

    Science.gov (United States)

    Mundodi, V; Kucknoor, AS; Alderete, JF

    2007-01-01

    Background Host parasitism by Trichomonas vaginalis is complex. Adherence to vaginal epithelial cells (VECs) is mediated by surface proteins. We showed before that antisense down-regulation of expression of adhesin AP65 decreased amounts of protein, which lowered levels of T. vaginalis adherence to VECs. We now perform antisense down-regulation of expression of the ap33 gene to evaluate and confirm a role for AP33 in adherence by T. vaginalis. We also used an established transfection system for heterologous expression of AP33 in T. foetus as an additional confirmatory approach. Results We successfully select stable trichomonads with sense (S) and antisense (AS) plasmids. RT-PCR confirmed decreased amounts of ap33 mRNA in AS-transfected parasites, and decreased amounts of AP33 had no effect on growth and viability when compared to wild-type (wt) trichomonads. Immunoblots of proteins from AS-transfectants gave significant decreased amounts of functional AP33 capable of binding to host cells compared to wt- and S-transfected trichomonads. As expected, AS-transfectants had lower levels of adherence to VECs, which was related to reduction in surface expression of AP33. Stable expression of T. vaginalis AP33::HA fusion in T. foetus was confirmed by immunoblots and fluorescence. The episomally-expressed surface AP33::HA fusion increased adherence of trichomonads to human VECs, which was abrogated with anti-AP33 serum. Conclusion These results using both antisense inhibition of gene expression and AP33 synthesis and the heterologous expression of AP33 in T. foetus confirms a role for this protein as an adhesin in T. vaginalis. PMID:17608941

  5. Antisense RNA decreases AP33 gene expression and cytoadherence by T. vaginalis

    Directory of Open Access Journals (Sweden)

    Alderete JF

    2007-07-01

    Full Text Available Abstract Background Host parasitism by Trichomonas vaginalis is complex. Adherence to vaginal epithelial cells (VECs is mediated by surface proteins. We showed before that antisense down-regulation of expression of adhesin AP65 decreased amounts of protein, which lowered levels of T. vaginalis adherence to VECs. We now perform antisense down-regulation of expression of the ap33 gene to evaluate and confirm a role for AP33 in adherence by T. vaginalis. We also used an established transfection system for heterologous expression of AP33 in T. foetus as an additional confirmatory approach. Results We successfully select stable trichomonads with sense (S and antisense (AS plasmids. RT-PCR confirmed decreased amounts of ap33 mRNA in AS-transfected parasites, and decreased amounts of AP33 had no effect on growth and viability when compared to wild-type (wt trichomonads. Immunoblots of proteins from AS-transfectants gave significant decreased amounts of functional AP33 capable of binding to host cells compared to wt- and S-transfected trichomonads. As expected, AS-transfectants had lower levels of adherence to VECs, which was related to reduction in surface expression of AP33. Stable expression of T. vaginalis AP33::HA fusion in T. foetus was confirmed by immunoblots and fluorescence. The episomally-expressed surface AP33::HA fusion increased adherence of trichomonads to human VECs, which was abrogated with anti-AP33 serum. Conclusion These results using both antisense inhibition of gene expression and AP33 synthesis and the heterologous expression of AP33 in T. foetus confirms a role for this protein as an adhesin in T. vaginalis.

  6. Nucleosome repositioning underlies dynamic gene expression.

    Science.gov (United States)

    Nocetti, Nicolas; Whitehouse, Iestyn

    2016-03-15

    Nucleosome repositioning at gene promoters is a fundamental aspect of the regulation of gene expression. However, the extent to which nucleosome repositioning is used within eukaryotic genomes is poorly understood. Here we report a comprehensive analysis of nucleosome positions as budding yeast transit through an ultradian cycle in which expression of >50% of all genes is highly synchronized. We present evidence of extensive nucleosome repositioning at thousands of gene promoters as genes are activated and repressed. During activation, nucleosomes are relocated to allow sites of general transcription factor binding and transcription initiation to become accessible. The extent of nucleosome shifting is closely related to the dynamic range of gene transcription and generally related to DNA sequence properties and use of the coactivators TFIID or SAGA. However, dynamic gene expression is not limited to SAGA-regulated promoters and is an inherent feature of most genes. While nucleosome repositioning occurs pervasively, we found that a class of genes required for growth experience acute nucleosome shifting as cells enter the cell cycle. Significantly, our data identify that the ATP-dependent chromatin-remodeling enzyme Snf2 plays a fundamental role in nucleosome repositioning and the expression of growth genes. We also reveal that nucleosome organization changes extensively in concert with phases of the cell cycle, with large, regularly spaced nucleosome arrays being established in mitosis. Collectively, our data and analysis provide a framework for understanding nucleosome dynamics in relation to fundamental DNA-dependent transactions.

  7. Digital gene expression signatures for maize development.

    Science.gov (United States)

    Eveland, Andrea L; Satoh-Nagasawa, Namiko; Goldshmidt, Alexander; Meyer, Sandra; Beatty, Mary; Sakai, Hajime; Ware, Doreen; Jackson, David

    2010-11-01

    Genome-wide expression signatures detect specific perturbations in developmental programs and contribute to functional resolution of key regulatory networks. In maize (Zea mays) inflorescences, mutations in the RAMOSA (RA) genes affect the determinacy of axillary meristems and thus alter branching patterns, an important agronomic trait. In this work, we developed and tested a framework for analysis of tag-based, digital gene expression profiles using Illumina's high-throughput sequencing technology and the newly assembled B73 maize reference genome. We also used a mutation in the RA3 gene to identify putative expression signatures specific to stem cell fate in axillary meristem determinacy. The RA3 gene encodes a trehalose-6-phosphate phosphatase and may act at the interface between developmental and metabolic processes. Deep sequencing of digital gene expression libraries, representing three biological replicate ear samples from wild-type and ra3 plants, generated 27 million 20- to 21-nucleotide reads with frequencies spanning 4 orders of magnitude. Unique sequence tags were anchored to 3'-ends of individual transcripts by DpnII and NlaIII digests, which were multiplexed during sequencing. We mapped 86% of nonredundant signature tags to the maize genome, which associated with 37,117 gene models and unannotated regions of expression. In total, 66% of genes were detected by at least nine reads in immature maize ears. We used comparative genomics to leverage existing information from Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) in functional analyses of differentially expressed maize genes. Results from this study provide a basis for the analysis of short-read expression data in maize and resolved specific expression signatures that will help define mechanisms of action for the RA3 gene.

  8. The heterologous overexpression of hsp23, a small heat-shock protein gene from Trichoderma virens, confers thermotolerance to T. harzianum.

    Science.gov (United States)

    Montero-Barrientos, Marta; Cardoza, Rosa E; Gutiérrez, Santiago; Monte, Enrique; Hermosa, Rosa

    2007-07-01

    An EST showing high values of identity with genes coding for small heat shock proteins (sHSPs) was selected from an EST library collection of Trichoderma virens T59. The cDNA gene (hsp23) with a sequence size of 645 bp long was amplified by PCR. The expression of this gene was evaluated in cultures grown at temperatures ranging from 4 to 41 degrees C. An increased level of expression was detected when the fungus was grown at extreme temperatures (4, 10 or 41 degrees C). A high-expression level was also observed when the fungus was grown in 10% ethanol for 4 h. The hsp23 gene was present as a unique copy in the T. virens genome, and a homologous gene was also present in other five investigated Trichoderma species. Strain T. harzianum T34 was transformed with the hsp23 gene from T. virens T59 under the control of the pki (pyruvate kinase) promoter from T. reesei and the ble (phleomycin resistance) gene as selection marker. Statistically significant differences were detected between the strains T34 and two selected transformants in the biomass quantities obtained after heat shock treatment and in the colony diameters after incubation at 4 degrees C for 2 months.

  9. Gene expression profile of sprinter's muscle.

    Science.gov (United States)

    Yoshioka, M; Tanaka, H; Shono, N; Shindo, M; St-Amand, J

    2007-12-01

    We have characterized the global gene expression profile in left vastus lateralis muscles of sprinters and sedentary men. The gene expression profile was analyzed by using serial analysis of gene expression (SAGE) method. The abundantly expressed transcripts in the sprinter's muscle were mainly involved in contraction and energy metabolism, whereas six transcripts were corresponding to potentially novel transcripts. Thirty-eight transcripts were differentially expressed between the sprinter and sedentary individuals. Moreover, sprinters showed higher expressions of both uncharacterized and potentially novel transcripts. Sprinters also highly expressed seven transcripts, such as glycine-rich protein, myosin heavy polypeptide (MYH) 2, expressed sequence tag similar to (EST) fructose-bisphosphate aldolase 1 isoform A (ALDOA), glyceraldehyde-3-phosphate dehydrogenase and ATP synthase F0 subunit 6. On the other hand, 20 transcripts such as MYH1, tropomyosin 2 and 3, troponin C slow, C2 fast, I slow, T1 slow and T3 fast, myoglobin, creatine kinase, ALDOA, glycogen phosphorylase, cytochrome c oxidase II and III, and NADH dehydrogenase 1 and 2 showed lower expression levels in the sprinters than the sedentary controls. The current study has characterized the global gene expressions in sprinters and identified a number of transcripts that can be subjected to further mechanistic analysis.

  10. Widespread ectopic expression of olfactory receptor genes

    Directory of Open Access Journals (Sweden)

    Yanai Itai

    2006-05-01

    Full Text Available Abstract Background Olfactory receptors (ORs are the largest gene family in the human genome. Although they are expected to be expressed specifically in olfactory tissues, some ectopic expression has been reported, with special emphasis on sperm and testis. The present study systematically explores the expression patterns of OR genes in a large number of tissues and assesses the potential functional implication of such ectopic expression. Results We analyzed the expression of hundreds of human and mouse OR transcripts, via EST and microarray data, in several dozens of human and mouse tissues. Different tissues had specific, relatively small OR gene subsets which had particularly high expression levels. In testis, average expression was not particularly high, and very few highly expressed genes were found, none corresponding to ORs previously implicated in sperm chemotaxis. Higher expression levels were more common for genes with a non-OR genomic neighbor. Importantly, no correlation in expression levels was detected for human-mouse orthologous pairs. Also, no significant difference in expression levels was seen between intact and pseudogenized ORs, except for the pseudogenes of subfamily 7E which has undergone a human-specific expansion. Conclusion The OR superfamily as a whole, show widespread, locus-dependent and heterogeneous expression, in agreement with a neutral or near neutral evolutionary model for transcription control. These results cannot reject the possibility that small OR subsets might play functional roles in different tissues, however considerable care should be exerted when offering a functional interpretation for ectopic OR expression based only on transcription information.

  11. Codon optimization of genes for efficient protein expression in mammalian cells by selection of only preferred human codons.

    Science.gov (United States)

    Inouye, Satoshi; Sahara-Miura, Yuiko; Sato, Jun-ichi; Suzuki, Takahiro

    2015-05-01

    A simple design method for codon optimization of genes to express a heterologous protein in mammalian cells is described. Codon optimization was performed by choosing only codons preferentially used in humans and with over 60% GC content, and the method was named the "preferred human codon-optimized method." To test our simple rule for codon optimization, the preferred human codon-optimized genes for six proteins containing photoproteins (aequorin and clytin II) and luciferases (Gaussia luciferase, Renilla luciferase, and firefly luciferases from Photinus pyralis and Luciola cruciata) were chemically synthesized and transiently expressed in Chinese hamster ovary-K1 cells. All preferred human codon-optimized genes showed higher luminescence activity than the corresponding wild-type genes. Our simple design method could be used to improve protein expression in mammalian cells efficiently. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Regulation of Gene Expression in Protozoa Parasites

    Directory of Open Access Journals (Sweden)

    Consuelo Gomez

    2010-01-01

    Full Text Available Infections with protozoa parasites are associated with high burdens of morbidity and mortality across the developing world. Despite extensive efforts to control the transmission of these parasites, the spread of populations resistant to drugs and the lack of effective vaccines against them contribute to their persistence as major public health problems. Parasites should perform a strict control on the expression of genes involved in their pathogenicity, differentiation, immune evasion, or drug resistance, and the comprehension of the mechanisms implicated in that control could help to develop novel therapeutic strategies. However, until now these mechanisms are poorly understood in protozoa. Recent investigations into gene expression in protozoa parasites suggest that they possess many of the canonical machineries employed by higher eukaryotes for the control of gene expression at transcriptional, posttranscriptional, and epigenetic levels, but they also contain exclusive mechanisms. Here, we review the current understanding about the regulation of gene expression in Plasmodium sp., Trypanosomatids, Entamoeba histolytica and Trichomonas vaginalis.

  13. Expression of polarity genes in human cancer.

    Science.gov (United States)

    Lin, Wan-Hsin; Asmann, Yan W; Anastasiadis, Panos Z

    2015-01-01

    Polarity protein complexes are crucial for epithelial apical-basal polarity and directed cell migration. Since alterations of these processes are common in cancer, polarity proteins have been proposed to function as tumor suppressors or oncogenic promoters. Here, we review the current understanding of polarity protein functions in epithelial homeostasis, as well as tumor formation and progression. As most previous studies focused on the function of single polarity proteins in simplified model systems, we used a genomics approach to systematically examine and identify the expression profiles of polarity genes in human cancer. The expression profiles of polarity genes were distinct in different human tissues and classified cancer types. Additionally, polarity expression profiles correlated with disease progression and aggressiveness, as well as with identified cancer types, where specific polarity genes were commonly altered. In the case of Scribble, gene expression analysis indicated its common amplification and upregulation in human cancer, suggesting a tumor promoting function.

  14. Regulation of meiotic gene expression in plants

    Directory of Open Access Journals (Sweden)

    Adele eZhou

    2014-08-01

    Full Text Available With the recent advances in genomics and sequencing technologies, databases of transcriptomes representing many cellular processes have been built. Meiotic transcriptomes in plants have been studied in Arabidopsis thaliana, rice (Oryza sativa, wheat (Triticum aestivum, petunia (Petunia hybrida, sunflower (Helianthus annuus, and maize (Zea mays. Studies in all organisms, but particularly in plants, indicate that a very large number of genes are expressed during meiosis, though relatively few of them seem to be required for the completion of meiosis. In this review, we focus on gene expression at the RNA level and analyze the meiotic transcriptome datasets and explore expression patterns of known meiotic genes to elucidate how gene expression could be regulated during meiosis. We also discuss mechanisms, such as chromatin organization and non-coding RNAs, that might be involved in the regulation of meiotic transcription patterns.

  15. The development of an efficient multipurpose bean pod mottle virus viral vector set for foreign gene expression and RNA silencing.

    Science.gov (United States)

    Zhang, Chunquan; Bradshaw, Jeffrey D; Whitham, Steven A; Hill, John H

    2010-05-01

    Plant viral vectors are valuable tools for heterologous gene expression, and because of virus-induced gene silencing (VIGS), they also have important applications as reverse genetics tools for gene function studies. Viral vectors are especially useful for plants such as soybean (Glycine max) that are recalcitrant to transformation. Previously, two generations of bean pod mottle virus (BPMV; genus Comovirus) vectors have been developed for overexpressing and silencing genes in soybean. However, the design of the previous vectors imposes constraints that limit their utility. For example, VIGS target sequences must be expressed as fusion proteins in the same reading frame as the viral polyprotein. This requirement limits the design of VIGS target sequences to open reading frames. Furthermore, expression of multiple genes or simultaneous silencing of one gene and expression of another was not possible. To overcome these and other issues, a new BPMV-based vector system was developed to facilitate a variety of applications for gene function studies in soybean as well as in common bean (Phaseolus vulgaris). These vectors are designed for simultaneous expression of multiple foreign genes, insertion of noncoding/antisense sequences, and simultaneous expression and silencing. The simultaneous expression of green fluorescent protein and silencing of phytoene desaturase shows that marker gene-assisted silencing is feasible. These results demonstrate the utility of this BPMV vector set for a wide range of applications in soybean and common bean, and they have implications for improvement of other plant virus-based vector systems.

  16. Use of expression-enhancing terminators in Saccharomyces cerevisiae to increase mRNA half-life and improve gene expression control for metabolic engineering applications.

    Science.gov (United States)

    Curran, Kathleen A; Karim, Ashty S; Gupta, Akash; Alper, Hal S

    2013-09-01

    Control of gene and protein expression of both endogenous and heterologous genes is a key component of metabolic engineering. While a large amount of work has been published characterizing promoters for this purpose, less effort has been exerted to elucidate the role of terminators in yeast. In this study, we characterize over 30 terminators for use in metabolic engineering applications in Saccharomyces cerevisiae and determine mRNA half-life changes to be the major cause of the varied protein and transcript expression level. We demonstrate that the difference in transcript level can be over 6.5-fold even for high strength promoters. The influence of terminator selection is magnified when coupled with a low-expression promoter, with a maximum difference in protein expression of 11-fold between an expression-enhancing terminator and the parent plasmid terminator and over 35-fold difference when compared with a no-terminator baseline. This is the first time that terminators have been investigated in the context of multiple promoters spanning orders of magnitude in activity. Finally, we demonstrate the utility of terminator selection for metabolic engineering by using a mutant xylose isomerase gene as a proof-of-concept. Through pairing an expression-enhancing terminator with a low-expression promoter, we were able to achieve the same phenotypic result as with a promoter considerably higher in strength. Moreover, we can further boost the phenotype of the high-strength promoter by pairing it with an expression-enhancing terminator. This work highlights how terminator elements can be used to control metabolic pathways in the same way that promoters are traditionally used in yeast. Together, this work demonstrates that terminators will be an important part of heterologous gene expression and metabolic engineering for yeast in the future.

  17. Optimal Reference Genes for Gene Expression Normalization in Trichomonas vaginalis.

    Science.gov (United States)

    dos Santos, Odelta; de Vargas Rigo, Graziela; Frasson, Amanda Piccoli; Macedo, Alexandre José; Tasca, Tiana

    2015-01-01

    Trichomonas vaginalis is the etiologic agent of trichomonosis, the most common non-viral sexually transmitted disease worldwide. This infection is associated with several health consequences, including cervical and prostate cancers and HIV acquisition. Gene expression analysis has been facilitated because of available genome sequences and large-scale transcriptomes in T. vaginalis, particularly using quantitative real-time polymerase chain reaction (qRT-PCR), one of the most used methods for molecular studies. Reference genes for normalization are crucial to ensure the accuracy of this method. However, to the best of our knowledge, a systematic validation of reference genes has not been performed for T. vaginalis. In this study, the transcripts of nine candidate reference genes were quantified using qRT-PCR under different cultivation conditions, and the stability of these genes was compared using the geNorm and NormFinder algorithms. The most stable reference genes were α-tubulin, actin and DNATopII, and, conversely, the widely used T. vaginalis reference genes GAPDH and β-tubulin were less stable. The PFOR gene was used to validate the reliability of the use of these candidate reference genes. As expected, the PFOR gene was upregulated when the trophozoites were cultivated with ferrous ammonium sulfate when the DNATopII, α-tubulin and actin genes were used as normalizing gene. By contrast, the PFOR gene was downregulated when the GAPDH gene was used as an internal control, leading to misinterpretation of the data. These results provide an important starting point for reference gene selection and gene expression analysis with qRT-PCR studies of T. vaginalis.

  18. Role of RNA splicing in mediating lineage-specific expression of the von Willebrand factor gene in the endothelium.

    Science.gov (United States)

    Yuan, Lei; Janes, Lauren; Beeler, David; Spokes, Katherine C; Smith, Joshua; Li, Dan; Jaminet, Shou-Ching; Oettgen, Peter; Aird, William C

    2013-05-23

    We previously demonstrated that the first intron of the human von Willebrand factor (vWF) is required for gene expression in the endothelium of transgenic mice. Based on this finding, we hypothesized that RNA splicing plays a role in mediating vWF expression in the vasculature. To address this question, we used transient transfection assays in human endothelial cells and megakaryocytes with intron-containing and intronless human vWF promoter-luciferase constructs. Next, we generated knockin mice in which LacZ was targeted to the endogenous mouse vWF locus in the absence or presence of the native first intron or heterologous introns from the human β-globin, mouse Down syndrome critical region 1, or hagfish coagulation factor X genes. In both the in vitro assays and the knockin mice, the loss of the first intron of vWF resulted in a significant reduction of reporter gene expression in endothelial cells but not megakaryocytes. This effect was rescued to varying degrees by the introduction of a heterologous intron. Intron-mediated enhancement of expression was mediated at a posttranscriptional level. Together, these findings implicate a role for intronic splicing in mediating lineage-specific expression of vWF in the endothelium.

  19. Regulation of glnB gene promoter expression in Azospirillum brasilense by the NtrC protein.

    Science.gov (United States)

    Huergo, Luciano F; Souza, Emanuel M; Steffens, M Berenice R; Yates, M Geoffrey; Pedrosa, Fabio O; Chubatsu, Leda S

    2003-06-01

    In Azospirillum brasilense the glnB and glnA genes are clustered in an operon regulated by three different promoters: two located upstream of glnB (glnBp1-sigma(70), and glnBp2-sigma(N)) and one as yet unidentified promoter, in the glnBA intergenic region. We have investigated the expression of the glnB gene promoter using glnB-lacZ gene fusions, mutation analysis, heterologous expression and DNA band-shift assays. Deletion of the glnB promoter region showed that NtrC-binding sequences were essential for glnB expression under nitrogen limitation. The A. brasilense NtrC protein activated transcription of glnB-lacZ fusions in the heterologous genetic background of Escherichia coli. Expression of glnB-lacZ fusions in two A. brasilense ntrC mutants differed from that in the wild-type strain. In vitro studies also indicated that the purified NtrC protein from E. coli was able to bind to the glnB promoter region of A. brasilense. Our results show that the NtrC protein activates glnBglnA expression under nitrogen limitation in A. brasilense.

  20. Gene expression profiling in autoimmune diseases

    DEFF Research Database (Denmark)

    Bovin, Lone Frier; Brynskov, Jørn; Hegedüs, Laszlo

    2007-01-01

    A central issue in autoimmune disease is whether the underlying inflammation is a repeated stereotypical process or whether disease specific gene expression is involved. To shed light on this, we analysed whether genes previously found to be differentially regulated in rheumatoid arthritis (RA...

  1. Bayesian modeling of differential gene expression.

    Science.gov (United States)

    Lewin, Alex; Richardson, Sylvia; Marshall, Clare; Glazier, Anne; Aitman, Tim

    2006-03-01

    We present a Bayesian hierarchical model for detecting differentially expressing genes that includes simultaneous estimation of array effects, and show how to use the output for choosing lists of genes for further investigation. We give empirical evidence that expression-level dependent array effects are needed, and explore different nonlinear functions as part of our model-based approach to normalization. The model includes gene-specific variances but imposes some necessary shrinkage through a hierarchical structure. Model criticism via posterior predictive checks is discussed. Modeling the array effects (normalization) simultaneously with differential expression gives fewer false positive results. To choose a list of genes, we propose to combine various criteria (for instance, fold change and overall expression) into a single indicator variable for each gene. The posterior distribution of these variables is used to pick the list of genes, thereby taking into account uncertainty in parameter estimates. In an application to mouse knockout data, Gene Ontology annotations over- and underrepresented among the genes on the chosen list are consistent with biological expectations.

  2. Perspectives: Gene Expression in Fisheries Management

    Science.gov (United States)

    Nielsen, Jennifer L.; Pavey, Scott A.

    2010-01-01

    Functional genes and gene expression have been connected to physiological traits linked to effective production and broodstock selection in aquaculture, selective implications of commercial fish harvest, and adaptive changes reflected in non-commercial fish populations subject to human disturbance and climate change. Gene mapping using single nucleotide polymorphisms (SNPs) to identify functional genes, gene expression (analogue microarrays and real-time PCR), and digital sequencing technologies looking at RNA transcripts present new concepts and opportunities in support of effective and sustainable fisheries. Genomic tools have been rapidly growing in aquaculture research addressing aspects of fish health, toxicology, and early development. Genomic technologies linking effects in functional genes involved in growth, maturation and life history development have been tied to selection resulting from harvest practices. Incorporating new and ever-increasing knowledge of fish genomes is opening a different perspective on local adaptation that will prove invaluable in wild fish conservation and management. Conservation of fish stocks is rapidly incorporating research on critical adaptive responses directed at the effects of human disturbance and climate change through gene expression studies. Genomic studies of fish populations can be generally grouped into three broad categories: 1) evolutionary genomics and biodiversity; 2) adaptive physiological responses to a changing environment; and 3) adaptive behavioral genomics and life history diversity. We review current genomic research in fisheries focusing on those that use microarrays to explore differences in gene expression among phenotypes and within or across populations, information that is critically important to the conservation of fish and their relationship to humans.

  3. Gene Expression Profiles of Inflammatory Myopathies

    Directory of Open Access Journals (Sweden)

    J Gordon Millichap

    2002-11-01

    Full Text Available The simultaneous expression of 10,000 genes was measured, using Affymetrix GeneChip microarrays, in muscle specimens from 45 patients with various myopathies (dystrophy, congenital myopathy, and inflammatory myopathy examined at Brigham and Women’s Hospital, and Children’s Hospital, Harvard Medical School, Boston, MA.

  4. Translational control of gene expression and disease

    NARCIS (Netherlands)

    Calkhoven, Cornelis F; Müller, Christine; Leutz, Achim

    2002-01-01

    In the past decade, translational control has been shown to be crucial in the regulation of gene expression. Research in this field has progressed rapidly, revealing new control mechanisms and adding constantly to the list of translationally regulated genes. There is accumulating evidence that trans

  5. Inferring gene networks from discrete expression data

    KAUST Repository

    Zhang, L.

    2013-07-18

    The modeling of gene networks from transcriptional expression data is an important tool in biomedical research to reveal signaling pathways and to identify treatment targets. Current gene network modeling is primarily based on the use of Gaussian graphical models applied to continuous data, which give a closedformmarginal likelihood. In this paper,we extend network modeling to discrete data, specifically data from serial analysis of gene expression, and RNA-sequencing experiments, both of which generate counts of mRNAtranscripts in cell samples.We propose a generalized linear model to fit the discrete gene expression data and assume that the log ratios of the mean expression levels follow a Gaussian distribution.We restrict the gene network structures to decomposable graphs and derive the graphs by selecting the covariance matrix of the Gaussian distribution with the hyper-inverse Wishart priors. Furthermore, we incorporate prior network models based on gene ontology information, which avails existing biological information on the genes of interest. We conduct simulation studies to examine the performance of our discrete graphical model and apply the method to two real datasets for gene network inference. © The Author 2013. Published by Oxford University Press. All rights reserved.

  6. A novel PR10 promoter from Erianthus arundinaceus directs high constitutive transgene expression and is enhanced upon wounding in heterologous plant systems.

    Science.gov (United States)

    Chakravarthi, M; Syamaladevi, Divya P; Harunipriya, P; Augustine, Sruthy Maria; Subramonian, N

    2016-01-01

    In genetic engineering, inducible promoters play an important role as the expression of genes driven by them can be turned on or off under situations like biotic or abiotic factors. There are few reports on inducible promoters that can be employed in the development of transgenic plants, particularly in sugarcane. In the present study, four wound inducible genes (Chitinase, PR1A, PR10 and HRGP) were selected and were amplified from Erianthus arundinaceus, a distant relative of sugarcane. In order to determine the gene that is highly induced upon wounding, RT-qPCR was performed, which showed that PR10 gene expression was instantaneous and higher upon wounding when compared to the other three genes. Using the random amplification of genomic ends technique, a 592 bp promoter sequence was obtained and in silico analysis of the upstream regulatory region revealed a 469 bp promoter and 123 bp of 5' untranslated region (UTR). Functional analyses of the promoter sequence (with and without 5' UTR) in tobacco, rice and sugarcane using β-glucuronidase (GUS) as the reporter gene revealed the constitutive and inducible nature of the PR10 promoter. Our studies have demonstrated that the PR10 promoter, though highly constitutive, was quickly induced upon wounding as well as on treatment with abscisic acid and methyl jasmonate hormones. This is the first report on the isolation and characterization of a PR10 promoter from a wild grass and is expected to have application for development of transgenic plants.

  7. Genetic transformation of novel isolates of chicken Lactobacillus bearing probiotic features for expression of heterologous proteins: a tool to develop live oral vaccines

    Directory of Open Access Journals (Sweden)

    Neumann Elisabeth

    2006-01-01

    Full Text Available Abstract Background The use of lactic acid bacteria as vehicles to delivery antigens to immunize animals is a promising issue. When genetically modified, these bacteria can induce a specific local and systemic immune response against selected pathogens. Gastric acid and bile salts tolerance, production of antagonistic substances against pathogenic microorganisms, and adhesive ability to gut epithelium are other important characteristics that make these bacteria useful for oral immunization. Results Bacteria isolated on de Man, Rogosa and Sharpe medium (MRS from different gastrointestinal portions of broiler chicks were evaluated for their resistance to artificial gastric acid and bile salts, production of hydrogen peroxide, and cell surface hydrophobicity. Thirty-eight isolates were first typed at species level by PCR amplification of 16S-23S rRNA intergenic spacers using universal primers that anneal within 16S and 23S genes, followed by restriction digestion analyses of PCR amplicons (PCR-ARDRA. An expression cassette was assembled onto the pCR2.1-Topo vector by cloning the promoter, leader peptide, cell wall anchor and terminator sequences derived from the laminin binding S-layer protein gene of L. crispatus strain F5.7 (lbs gene. A sequence encoding the green fluorescent protein (GFP was inserted as reporter gene, and an erythromycin resistance gene was added as selective marker. All constructs were able to express GFP in the cloning host E. coli XL1-Blue and different Lactobacillus strains as verified by FACS and laser scanning confocal microscopy. Conclusion Lactobacillus isolated from gastrointestinal tract of broiler chickens and selected for probiotic characteristics can be genetically modified by introducing an expression cassette into the lbs locus. The transformed bacteria expressed on its cell wall surface different fluorescent proteins used as reporters of promoter function. It is possible then that similar bacterial model

  8. Genetic transformation of novel isolates of chicken Lactobacillus bearing probiotic features for expression of heterologous proteins: a tool to develop live oral vaccines

    Science.gov (United States)

    Mota, Rodrigo M; Moreira, João Luiz S; Souza, Marcelo R; Fátima Horta, M; Teixeira, Santuza MR; Neumann, Elisabeth; Nicoli, Jacques R; Nunes, Álvaro C

    2006-01-01

    Background The use of lactic acid bacteria as vehicles to delivery antigens to immunize animals is a promising issue. When genetically modified, these bacteria can induce a specific local and systemic immune response against selected pathogens. Gastric acid and bile salts tolerance, production of antagonistic substances against pathogenic microorganisms, and adhesive ability to gut epithelium are other important characteristics that make these bacteria useful for oral immunization. Results Bacteria isolated on de Man, Rogosa and Sharpe medium (MRS) from different gastrointestinal portions of broiler chicks were evaluated for their resistance to artificial gastric acid and bile salts, production of hydrogen peroxide, and cell surface hydrophobicity. Thirty-eight isolates were first typed at species level by PCR amplification of 16S-23S rRNA intergenic spacers using universal primers that anneal within 16S and 23S genes, followed by restriction digestion analyses of PCR amplicons (PCR-ARDRA). An expression cassette was assembled onto the pCR2.1-Topo vector by cloning the promoter, leader peptide, cell wall anchor and terminator sequences derived from the laminin binding S-layer protein gene of L. crispatus strain F5.7 (lbs gene). A sequence encoding the green fluorescent protein (GFP) was inserted as reporter gene, and an erythromycin resistance gene was added as selective marker. All constructs were able to express GFP in the cloning host E. coli XL1-Blue and different Lactobacillus strains as verified by FACS and laser scanning confocal microscopy. Conclusion Lactobacillus isolated from gastrointestinal tract of broiler chickens and selected for probiotic characteristics can be genetically modified by introducing an expression cassette into the lbs locus. The transformed bacteria expressed on its cell wall surface different fluorescent proteins used as reporters of promoter function. It is possible then that similar bacterial model expressing pathogen antigens can

  9. Gene expression studies using microarrays

    NARCIS (Netherlands)

    Burgess, Janette

    2001-01-01

    1. The rapid progression of the collaborative sequencing programmes that are unravelling the complete genome sequences of many organisms are opening pathways for new approaches to gene analysis. As the sequence data become available, the bottleneck in biological research will shift to understanding

  10. Insulin gene: organisation, expression and regulation.

    Science.gov (United States)

    Dumonteil, E; Philippe, J

    1996-06-01

    Insulin, a major hormone of the endocrine pancreas, plays a key role in the control of glucose homeostasis. This review discusses the mechanisms of cell-specific expression and regulation of the insulin gene. Whereas expression is restricted to islet beta-cells in adults, the insulin gene is more widely expressed at several embryonic stages, although the role of extrapancreatic expression is still unclear. beta-cell-specific expression relies on the interactions of 5'-flanking sequence motifs of the promoter with a number of ubiquitous and islet-specific transcription factors. IEF1 and IPF-1, by their binding to the E and A boxes, respectively, of the insulin gene promoter, appear to be the major determinants of beta-cell-specific expression. IEF1 is a heterodimer of the basic helix-loop-helix family of transcription factors, whereas IPF-1 belongs to the homeodomain-containing family. beta-cell specific determinants are conserved throughout evolution, although the human insulin gene 5'-flanking sequence also contains a polymorphic minisatellite which is unique to primates and may play a role in insulin gene regulation. Glucose modulates insulin gene transcription, with multiple elements of the promoter involved in glucose responsiveness. Remarkably, IPF-1 and IEF1 are involved in both beta-cell-specific expression and glucose regulation of the insulin gene. cAMP also regulates insulin gene transcription through a CRE, in response to various hormonal stimuli. On the whole, recent studies have provided a better understanding of beta-cell differentiation and function.

  11. Application of multidisciplinary analysis to gene expression.

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Xuefel (University of New Mexico, Albuquerque, NM); Kang, Huining (University of New Mexico, Albuquerque, NM); Fields, Chris (New Mexico State University, Las Cruces, NM); Cowie, Jim R. (New Mexico State University, Las Cruces, NM); Davidson, George S.; Haaland, David Michael; Sibirtsev, Valeriy (New Mexico State University, Las Cruces, NM); Mosquera-Caro, Monica P. (University of New Mexico, Albuquerque, NM); Xu, Yuexian (University of New Mexico, Albuquerque, NM); Martin, Shawn Bryan; Helman, Paul (University of New Mexico, Albuquerque, NM); Andries, Erik (University of New Mexico, Albuquerque, NM); Ar, Kerem (University of New Mexico, Albuquerque, NM); Potter, Jeffrey (University of New Mexico, Albuquerque, NM); Willman, Cheryl L. (University of New Mexico, Albuquerque, NM); Murphy, Maurice H. (University of New Mexico, Albuquerque, NM)

    2004-01-01

    Molecular analysis of cancer, at the genomic level, could lead to individualized patient diagnostics and treatments. The developments to follow will signal a significant paradigm shift in the clinical management of human cancer. Despite our initial hopes, however, it seems that simple analysis of microarray data cannot elucidate clinically significant gene functions and mechanisms. Extracting biological information from microarray data requires a complicated path involving multidisciplinary teams of biomedical researchers, computer scientists, mathematicians, statisticians, and computational linguists. The integration of the diverse outputs of each team is the limiting factor in the progress to discover candidate genes and pathways associated with the molecular biology of cancer. Specifically, one must deal with sets of significant genes identified by each method and extract whatever useful information may be found by comparing these different gene lists. Here we present our experience with such comparisons, and share methods developed in the analysis of an infant leukemia cohort studied on Affymetrix HG-U95A arrays. In particular, spatial gene clustering, hyper-dimensional projections, and computational linguistics were used to compare different gene lists. In spatial gene clustering, different gene lists are grouped together and visualized on a three-dimensional expression map, where genes