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Sample records for addition heterologous expression

  1. Streptomyces coelicolor as an expression host for heterologous gene clusters.

    PubMed

    Gomez-Escribano, Juan Pablo; Bibb, Mervyn J

    2012-01-01

    The expression of a gene or a set of genes from one organism in a different species is known as "heterologous expression." In actinomycetes, prolific producers of natural products, heterologous gene expression has been used to confirm the clustering of secondary metabolite biosynthetic genes, to analyze natural product biosynthesis, to produce variants of natural products by genetic engineering, and to discover new compounds by screening genomic libraries. Recent advances in DNA sequencing have enabled the rapid and affordable sequencing of actinomycete genomes and revealed a large number of secondary metabolite gene clusters with no known products. Heterologous expression of these cryptic gene clusters combined with comparative metabolic profiling provides an important means to identify potentially novel compounds. In this chapter, the methods and strategies used to heterologously express actinomycete gene clusters, including the techniques used for cloning secondary metabolite gene clusters, the Streptomyces hosts used for their expression, and the techniques employed to analyze their products by metabolic profiling, are described.

  2. Recent Advances in Artemisinin Production Through Heterologous Expression

    PubMed Central

    Arsenault, Patrick R.; Wobbe, Kristin K.; Weathers, Pamela J.

    2010-01-01

    Artemisinin the sesquiterpene endoperoxide lactone extracted from the herb Artemisia annua, remains the basis for the current preferred treatment against the malaria parasite Plasmodium falciparum. In addition, artemisinin and its derivatives show additional anti-parasite, anti-cancer, and anti-viral properties. Widespread use of this valuable secondary metabolite has been hampered by low production in vivo and high cost of chemical synthesis in vitro. Novel production methods are required to accommodate the ever-growing need for this important drug. Past work has focused on increasing production through traditional breeding approaches, with limited success, and on engineering cultured plants for high production in bioreactors. New research is focusing on heterologous expression systems for this unique biochemical pathway. Recently discovered genes, including a cytochrome P450 and its associated reductase, have been shown to catalyze multiple steps in the biochemical pathway leading to artemisinin. This has the potential to make a semi-synthetic approach to production both possible and cost effective. Artemisinin precursor production in engineered Saccharomyces cerevisiae is about two orders of magnitude higher than from field-grown A. annua. Efforts to increase flux through engineered pathways are on-going in both E. coli and S. cerevisiae through combinations of engineering precursor pathways and downstream optimization of gene expression. This review will compare older approaches to overproduction of this important drug, and then focus on the results from the newer approaches using heterologous expression systems and how they might meet the demands for treating malaria and other diseases. PMID:18991643

  3. Recent advances in artemisinin production through heterologous expression.

    PubMed

    Arsenault, Patrick R; Wobbe, Kristin K; Weathers, Pamela J

    2008-01-01

    Artemisinin the sesquiterpene endoperoxide lactone extracted from the herb Artemisia annua, remains the basis for the current preferred treatment against the malaria parasite Plasmodium falciparum. In addition, artemisinin and its derivatives show additional anti-parasite, anti-cancer, and anti-viral properties. Widespread use of this valuable secondary metabolite has been hampered by low production in vivo and high cost of chemical synthesis in vitro. Novel production methods are required to accommodate the ever-growing need for this important drug. Past work has focused on increasing production through traditional breeding approaches, with limited success, and on engineering cultured plants for high production in bioreactors. New research is focusing on heterologous expression systems for this unique biochemical pathway. Recently discovered genes, including a cytochrome P450 and its associated reductase, have been shown to catalyze multiple steps in the biochemical pathway leading to artemisinin. This has the potential to make a semi-synthetic approach to production both possible and cost effective. Artemisinin precursor production in engineered Saccharomyces cerevisiae is about two orders of magnitude higher than from field-grown A. annua. Efforts to increase flux through engineered pathways are on-going in both E. coli and S. cerevisiae through combinations of engineering precursor pathways and downstream optimization of gene expression. This review will compare older approaches to overproduction of this important drug, and then focus on the results from the newer approaches using heterologous expression systems and how they might meet the demands for treating malaria and other diseases. PMID:18991643

  4. Alternative Sigma Factor Over-Expression Enables Heterologous Expression of a Type II Polyketide Biosynthetic Pathway in Escherichia coli

    PubMed Central

    Stevens, David Cole; Conway, Kyle R.; Pearce, Nelson; Villegas-Peñaranda, Luis Roberto; Garza, Anthony G.; Boddy, Christopher N.

    2013-01-01

    Background Heterologous expression of bacterial biosynthetic gene clusters is currently an indispensable tool for characterizing biosynthetic pathways. Development of an effective, general heterologous expression system that can be applied to bioprospecting from metagenomic DNA will enable the discovery of a wealth of new natural products. Methodology We have developed a new Escherichia coli-based heterologous expression system for polyketide biosynthetic gene clusters. We have demonstrated the over-expression of the alternative sigma factor σ54 directly and positively regulates heterologous expression of the oxytetracycline biosynthetic gene cluster in E. coli. Bioinformatics analysis indicates that σ54 promoters are present in nearly 70% of polyketide and non-ribosomal peptide biosynthetic pathways. Conclusions We have demonstrated a new mechanism for heterologous expression of the oxytetracycline polyketide biosynthetic pathway, where high-level pleiotropic sigma factors from the heterologous host directly and positively regulate transcription of the non-native biosynthetic gene cluster. Our bioinformatics analysis is consistent with the hypothesis that heterologous expression mediated by the alternative sigma factor σ54 may be a viable method for the production of additional polyketide products. PMID:23724102

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

    PubMed

    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. PMID:26843166

  6. The molecular toolbox for chromosomal heterologous multiprotein expression in Escherichia coli.

    PubMed

    Richter, Katrin; Gescher, Johannes

    2012-12-01

    Heterologous multiprotein expression is the tool to answer a number of questions in basic science as well as to convert strains into producers and/or consumers of certain compounds in applied sciences. Multiprotein expression can be driven by plasmids with the disadvantages that the gene dosage might, in some cases, lead to toxic effects and that the continuous addition of antibiotics is undesirable. Stable genomic expression of proteins can forgo these problems and is a helpful and promising tool in synthetic biology. In the present paper, we provide an extract of methods from the toolbox for chromosome-based heterologous expression in Escherichia coli. PMID:23176458

  7. Cloning and Heterologous Expression of the Grecocycline Biosynthetic Gene Cluster

    PubMed Central

    Bilyk, Oksana; Sekurova, Olga N.; Zotchev, Sergey B.; Luzhetskyy, Andriy

    2016-01-01

    Transformation-associated recombination (TAR) in yeast is a rapid and inexpensive method for cloning and assembly of large DNA fragments, which relies on natural homologous recombination. Two vectors, based on p15a and F-factor replicons that can be maintained in yeast, E. coli and streptomycetes have been constructed. These vectors have been successfully employed for assembly of the grecocycline biosynthetic gene cluster from Streptomyces sp. Acta 1362. Fragments of the cluster were obtained by PCR and transformed together with the “capture” vector into the yeast cells, yielding a construct carrying the entire gene cluster. The obtained construct was heterologously expressed in S. albus J1074, yielding several grecocycline congeners. Grecocyclines have unique structural moieties such as a dissacharide side chain, an additional amino sugar at the C-5 position and a thiol group. Enzymes from this pathway may be used for the derivatization of known active angucyclines in order to improve their desired biological properties. PMID:27410036

  8. Cloning and Heterologous Expression of the Grecocycline Biosynthetic Gene Cluster.

    PubMed

    Bilyk, Oksana; Sekurova, Olga N; Zotchev, Sergey B; Luzhetskyy, Andriy

    2016-01-01

    Transformation-associated recombination (TAR) in yeast is a rapid and inexpensive method for cloning and assembly of large DNA fragments, which relies on natural homologous recombination. Two vectors, based on p15a and F-factor replicons that can be maintained in yeast, E. coli and streptomycetes have been constructed. These vectors have been successfully employed for assembly of the grecocycline biosynthetic gene cluster from Streptomyces sp. Acta 1362. Fragments of the cluster were obtained by PCR and transformed together with the "capture" vector into the yeast cells, yielding a construct carrying the entire gene cluster. The obtained construct was heterologously expressed in S. albus J1074, yielding several grecocycline congeners. Grecocyclines have unique structural moieties such as a dissacharide side chain, an additional amino sugar at the C-5 position and a thiol group. Enzymes from this pathway may be used for the derivatization of known active angucyclines in order to improve their desired biological properties. PMID:27410036

  9. Recombinant cells that highly express chromosomally-integrated heterologous genes

    DOEpatents

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

    1998-01-01

    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.

  10. Recombinant cells that highly express chromosomally-integrated heterologous genes

    DOEpatents

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

    2000-08-22

    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.

  11. Recombinant cells that highly express chromosomally-integrated heterologous genes

    DOEpatents

    Ingram, L.O.; Ohta, Kazuyoshi; Wood, B.E.

    1998-10-13

    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. 13 figs.

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

    DOEpatents

    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.

  13. Plant biofarming: novel insights for peptide expression in heterologous systems.

    PubMed

    Viana, Antônio Américo Barbosa; Pelegrini, Patrícia Barbosa; Grossi-de-Sá, Maria Fátima

    2012-01-01

    Peptide expression methods have been widely studied and developed from many different biological sources. The cultivation ofprokaryotic and eukaryotic cells has proven to be efficient for the expression of foreign peptides in several heterologous systems, including bacteria, insects, yeasts, and mammals. Earlier reports brought up new insights for the improvement of expressed products to not only increase the production rate of desired peptides but also reproduce desirable post-translational modifications and even to reduce the risk of allergenicity when those products are aimed for human use. The development of bioreactor systems provided the optimization of cell growth conditions to scale up the amounts of expressed peptides. On the other hand, different cell systems and mutants provided a plethora of possible peptide modifications. Hence, in this report, we describe the many organisms and systems used for the large scale production of several macromolecules with relevance in health and agriculture. We also bring into discussion plant biofarming in the moss Physcomitrella patens and its recent adaptations, as a cost-effective and efficient approach in the production of more complex heterologous proteins, given the fact that its glycosylation pattern can be engineered to avoid allergenicity to humans (common to plant-derived glycoproteins). PMID:23193604

  14. RNA viruses as vectors for the expression of heterologous proteins.

    PubMed

    Schlesinger, S

    1995-04-01

    RNA viruses comprise a wide variety of infectious agents, some of which are the cause of disease in humans, animals, and plants. Recombinant DNA technology is now making it feasible to modify these genomes and engineer them to express heterologous proteins. Several different schemes are being employed that depend on the genome organization of the virus and on the strategy of replication of the particular virus. Several different examples are illustrated and potential uses as well as possible problems are discussed. In the future reverse genetics may convert some of these viruses from agents of disease to agents of cure. PMID:7620976

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

    PubMed Central

    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

  16. Expression of Heterologous Cellulases in Thermotoga sp. Strain RQ2

    PubMed Central

    Xu, Hui; Han, Dongmei; Xu, Zhaohui

    2015-01-01

    The ability of Thermotoga spp. to degrade cellulose is limited due to a lack of exoglucanases. To address this deficiency, cellulase genes Csac_1076 (celA) and Csac_1078 (celB) from Caldicellulosiruptor saccharolyticus were cloned into T. sp. strain RQ2 for heterologous overexpression. Coding regions of Csac_1076 and Csac_1078 were fused to the signal peptide of TM1840 (amyA) and TM0070 (xynB), resulting in three chimeric enzymes, namely, TM1840-Csac_1078, TM0070-Csac_1078, and TM0070-Csac_1076, which were carried by Thermotoga-E. coli shuttle vectors pHX02, pHX04, and pHX07, respectively. All three recombinant enzymes were successfully expressed in E. coli DH5α and T. sp. strain RQ2, rendering the hosts with increased endo- and/or exoglucanase activities. In E. coli, the recombinant enzymes were mainly bound to the bacterial cells, whereas in T. sp. strain RQ2, about half of the enzyme activities were observed in the culture supernatants. However, the cellulase activities were lost in T. sp. strain RQ2 after three consecutive transfers. Nevertheless, this is the first time heterologous genes bigger than 1 kb (up to 5.3 kb in this study) have ever been expressed in Thermotoga, demonstrating the feasibility of using engineered Thermotoga spp. for efficient cellulose utilization. PMID:26273605

  17. Heterologous gene expression in the hyperthermophilic archaeon Sulfolobus solfataricus.

    PubMed

    Angelov, Angel; Liebl, Wolfgang

    2010-01-01

    One of the few available systems for gene expression in (hyper)thermophilic Archaea is the virus-based shuttle vector pMJ05 for Sulfolobus solfataricus. Although it is still not fully developed and there are some difficulties arising from the large size of the vector (>20 kb), it has successfully been used for the production of foreign and own proteins in S. solfataricus. Most often, the development of genetic tools for Archaea is held back by the lack of an efficient transformation system. In the case of pMJ05, this difficulty has been alleviated by using the Sulfolobus virus SSV1 as the vector backbone. The ability of the pMJ05 plasmid to spread in the culture as a virus, the availability of an effective selection marker (pyr) and of tunable promoters (araS and tf55α) make this system one of the first choices for heterologous gene expression in (hyper)thermophilic Archaea.

  18. Heterologous expression of G-protein-coupled receptors in yeast.

    PubMed

    Bertheleme, Nicolas; Singh, Shweta; Dowell, Simon; Byrne, Bernadette

    2015-01-01

    Heterologous yeast expression systems have been successfully used for the production of G-protein-coupled receptors (GPCRs) for both structural and functional studies. Yeast combine comparatively low cost and short culture times with straightforward generation of expression clones. They also perform some key posttranslational modifications not possible in bacterial systems. There are two major yeast expression systems, Pichia pastoris and Saccharomyces cerevisiae, both of which have been used for the production of GPCRs. P. pastoris has a proven track record for the production of large amounts of GPCR for structural studies. High-resolution crystal structures of both the adenosine A2A and the histamine H1 receptors have been obtained using protein expressed in this system. S. cerevisiae is relatively easy to engineer and this has resulted in the development of sophisticated tools for the functional characterization of GPCRs. In this chapter, we provide protocols for both large-scale receptor expression in P. pastoris for structural studies and small-scale receptor expression in S. cerevisiae for functional characterization. In both cases, the receptor used is the human adenosine A2A receptor. The results that both we and others have obtained using these protocols show the wide utility of the yeast expression systems for the production of GPCRs.

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

    PubMed Central

    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

  20. Engineering Streptomyces coelicolor for heterologous expression of secondary metabolite gene clusters.

    PubMed

    Gomez-Escribano, Juan Pablo; Bibb, Mervyn J

    2011-03-01

    We have constructed derivatives of Streptomyces coelicolor M145 as hosts for the heterologous expression of secondary metabolite gene clusters. To remove potentially competitive sinks of carbon and nitrogen, and to provide a host devoid of antibiotic activity, we deleted four endogenous secondary metabolite gene clusters from S. coelicolor M145--those for actinorhodin, prodiginine, CPK and CDA biosynthesis. We then introduced point mutations into rpoB and rpsL to pleiotropically increase the level of secondary metabolite production. Introduction of the native actinorhodin gene cluster and of gene clusters for the heterologous production of chloramphenicol and congocidine revealed dramatic increases in antibiotic production compared with the parental strain. In addition to lacking antibacterial activity, the engineered strains possess relatively simple extracellular metabolite profiles. When combined with liquid chromatography and mass spectrometry, we believe that these genetically engineered strains will markedly facilitate the discovery of new compounds by heterologous expression of cloned gene clusters, particularly the numerous cryptic secondary metabolic gene clusters that are prevalent within actinomycete genome sequences.

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

    PubMed Central

    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

  2. Heterologous Expression of a Bioactive β-Hexosyltransferase, an Enzyme Producer of Prebiotics, from Sporobolomyces singularis

    PubMed Central

    Dagher, Suzanne F.; Azcarate-Peril, M. Andrea

    2013-01-01

    Galacto-oligosaccharides (GOS) are indigestible dietary fibers that are able to reach the lower gastrointestinal tract to be selectively fermented by health-promoting bacteria. In this report, we describe the heterologous expression of an optimized synthetically produced version of the β-hexosyltransferase gene (Bht) from Sporobolomyces singularis. The Bht gene encodes a glycosyl hydrolase (EC 3.2.1.21) that acts as galactosyltransferase, able to catalyze a one-step conversion of lactose to GOS. Expression of the enzyme in Escherichia coli yielded an inactive insoluble protein, while the methylotrophic yeast Pichia pastoris GS115 produced a bioactive β-hexosyltransferase (rBHT). The enzyme exhibited faster kinetics at pHs between 3.5 and 6 and at temperatures between 40 and 50°C. Enzyme stability improved at temperatures lower than 40°C, and glucose was found to be a competitive inhibitor of enzymatic activity. P. pastoris secreted a fraction of the bioactive rBHT into the fermentation broth, while the majority of the enzyme remained associated with the outer membrane. Both the secreted and the membrane-associated forms were able to efficiently convert lactose to GOS. Additionally, resting cells with membrane-bound enzyme converted 90% of the initial lactose into GOS at 68% yield (g/g) (the maximum theoretical is 75%) with no secondary residual (glucose or galactose) products. This is the first report of a bioactive BHT from S. singularis that has been heterologously expressed. PMID:23241974

  3. Cloning, functional characterization and heterologous expression of TaLsi1, a wheat silicon transporter gene.

    PubMed

    Montpetit, Jonatan; Vivancos, Julien; Mitani-Ueno, Namiki; Yamaji, Naoki; Rémus-Borel, Wilfried; Belzile, François; Ma, Jian Feng; Bélanger, Richard R

    2012-05-01

    Silicon (Si) is known to be beneficial to plants, namely in alleviating biotic and abiotic stresses. The magnitude of such positive effects is associated with a plant's natural ability to absorb Si. Many grasses can accumulate as much as 10% on a dry weight basis while most dicots, including Arabidopsis, will accumulate less than 0.1%. In this report, we describe the cloning and functional characterization of TaLsi1, a wheat Si transporter gene. In addition, we developed a heterologous system for the study of Si uptake in plants by introducing TaLsi1 and OsLsi1, its ortholog in rice, into Arabidopsis, a species with a very low innate Si uptake capacity. When expressed constitutively under the control of the CaMV 35S promoter, both TaLsi1 and OsLsi1 were expressed in cells of roots and shoots. Such constitutive expression of TaLsi1 or OsLsi1 resulted in a fourfold to fivefold increase in Si accumulation in transformed plants compared to WT. However, this Si absorption caused deleterious symptoms. When the wheat transporter was expressed under the control of a root-specific promoter (a boron transporter gene (AtNIP5;1) promoter), a similar increase in Si absorption was noted but the plants did not exhibit symptoms and grew normally. These results demonstrate that TaLsi1 is indeed a functional Si transporter as its expression in Arabidopsis leads to increased Si uptake, but that this expression must be confined to root cells for healthy plant development. The availability of this heterologous expression system will facilitate further studies into the mechanisms and benefits of Si uptake. PMID:22351076

  4. Polysaccharide-Degrading Thermophiles Generated by Heterologous Gene Expression in Geobacillus kaustophilus HTA426

    PubMed Central

    Yoshida, Ken-ichi; Ohshima, Toshihisa

    2013-01-01

    Thermophiles have important advantages over mesophiles as host organisms for high-temperature bioprocesses, functional production of thermostable enzymes, and efficient expression of enzymatic activities in vivo. To capitalize on these advantages of thermophiles, we describe here a new inducible gene expression system in the thermophile Geobacillus kaustophilus HTA426. Six promoter regions in the HTA426 genome were identified and analyzed for expression profiles using β-galactosidase reporter assay. This analysis identified a promoter region upstream of a putative amylose-metabolizing gene cluster that directed high-level expression of the reporter gene. The expression was >280-fold that without a promoter and was further enhanced 12-fold by maltose addition. In association with a multicopy plasmid, this promoter region was used to express heterologous genes. Several genes, including a gene whose product was insoluble when expressed in Escherichia coli, were successfully expressed as soluble proteins, with yields of 0.16 to 59 mg/liter, and conferred new functions to G. kaustophilus strains. Remarkably, cellulase and α-amylase genes conferred the ability to degrade cellulose paper and insoluble starch at high temperatures, respectively, generating thermophiles with the potential to degrade plant biomass. Our results demonstrate that this novel expression system expands the potential applications of G. kaustophilus. PMID:23793634

  5. Heterologous expression of mitochondria-targeted microbial nitrilase enzymes increases cyanide tolerance in Arabidopsis.

    PubMed

    Molojwane, E; Adams, N; Sweetlove, L J; Ingle, R A

    2015-07-01

    Anthropogenic activities have resulted in cyanide (CN) contamination of both soil and water in many areas of the globe. While plants possess a detoxification pathway that serves to degrade endogenously generated CN, this system is readily overwhelmed, limiting the use of plants in bioremediation. Genetic engineering of additional CN degradation pathways in plants is one potential strategy to increase their tolerance to CN. Here we show that heterologous expression of microbial nitrilase enzymes targeted to the mitochondria increases CN tolerance in Arabidopsis. Root length in seedlings expressing either a CN dihydratase from Bacillus pumilis or a CN hydratase from Neurospora crassa was increased by 45% relative in wild-type plants in the presence of 50 μm KCN. We also demonstrate that in contrast to its strong inhibitory effects on seedling establishment, seed germination of the Col-0 ecotype of Arabidopsis is unaffected by CN.

  6. Heterologous Expression of Fungal Secondary Metabolite Pathways in the Aspergillus nidulans Host System.

    PubMed

    van Dijk, J W A; Wang, C C C

    2016-01-01

    Heterologous expression of fungal secondary metabolite genes allows for the product formation of otherwise silent secondary metabolite biosynthesis pathways. It also allows facile expression of mutants or combinations of genes not found in nature. This capability makes model fungi an ideal platform for synthetic biology. In this chapter a detailed description is provided of how to heterologously express any fungal secondary metabolite gene(s) in a well-developed host strain of Aspergillus nidulans. It covers all the necessary steps from identifying a gene(s) of interest to culturing mutant strains to produce secondary metabolites.

  7. Heterologous Expression of Fungal Secondary Metabolite Pathways in the Aspergillus nidulans Host System.

    PubMed

    van Dijk, J W A; Wang, C C C

    2016-01-01

    Heterologous expression of fungal secondary metabolite genes allows for the product formation of otherwise silent secondary metabolite biosynthesis pathways. It also allows facile expression of mutants or combinations of genes not found in nature. This capability makes model fungi an ideal platform for synthetic biology. In this chapter a detailed description is provided of how to heterologously express any fungal secondary metabolite gene(s) in a well-developed host strain of Aspergillus nidulans. It covers all the necessary steps from identifying a gene(s) of interest to culturing mutant strains to produce secondary metabolites. PMID:27417927

  8. Heterologous expression of human interleukin-6 in Streptomyces lividans TK24 using novel secretory expression vectors.

    PubMed

    Zhu, Yuanjun; Wang, Lifei; Du, Yu; Wang, Songmei; Yu, Tengfei; Hong, Bin

    2011-02-01

    Streptomyces is an attractive host for heterologous protein secretion. To further optimize its expression capacity, better expression vectors will be helpful. Here, based on pSGL1, a high copy number plasmid present in Streptomyces globisporus C-1027, we constructed a series of novel E. coli-Streptomyces shuttle expression vectors pIMB2-4. These vectors, which are compatible with pIJ-derived vectors, contain the strong promoter ermE*p and signal sequence SP (MelC1) of the first ORF of melanin operon in S. antibiotics (pIMB2), SP (CagA) of C-1027 apoprotein in S. globisporus C-1027 (pIMB3 and pIMB4). Using these vectors, human interleukin-6 (IL-6) could successfully be expressed and secreted using S. lividans TK24 as host. Furthermore, replacement of a rare leucine codon TTA with CTG in SP (CagA) enhanced IL-6 production.

  9. Heterologous Expression and Characterization of the Manganese-Oxidizing Protein from Erythrobacter sp. Strain SD21

    PubMed Central

    Nakama, Katherine; Medina, Michael; Lien, Ahn; Ruggieri, Jordan; Collins, Krystle

    2014-01-01

    The manganese (Mn)-oxidizing protein (MopA) from Erythrobacter sp. strain SD21 is part of a unique enzymatic family that is capable of oxidizing soluble Mn(II). This enzyme contains two domains, an animal heme peroxidase domain, which contains the catalytic site, followed by a C-terminal calcium binding domain. Different from the bacterial Mn-oxidizing multicopper oxidase enzymes, little is known about MopA. To gain a better understanding of MopA and its role in Mn(II) oxidation, the 238-kDa full-length protein and a 105-kDa truncated protein containing only the animal heme peroxidase domain were cloned and heterologously expressed in Escherichia coli. Despite having sequence similarity to a peroxidase, hydrogen peroxide did not stimulate activity, nor was activity significantly decreased in the presence of catalase. Both pyrroloquinoline quinone (PQQ) and hemin increased Mn-oxidizing activity, and calcium was required. The Km for Mn(II) of the full-length protein in cell extract was similar to that of the natively expressed protein, but the Km value for the truncated protein in cell extract was approximately 6-fold higher than that of the full-length protein, suggesting that the calcium binding domain may aid in binding Mn(II). Characterization of the heterologously expressed MopA has provided additional insight into the mechanism of bacterial Mn(II) oxidation, which will aid in understanding the role of MopA and Mn oxidation in bioremediation and biogeochemical cycling. PMID:25172859

  10. Direct capture and heterologous expression of Salinispora natural product genes for the biosynthesis of enterocin.

    PubMed

    Bonet, Bailey; Teufel, Robin; Crüsemann, Max; Ziemert, Nadine; Moore, Bradley S

    2015-03-27

    Heterologous expression of secondary metabolic pathways is a promising approach for the discovery and characterization of bioactive natural products. Herein we report the first heterologous expression of a natural product from the model marine actinomycete genus Salinispora. Using the recently developed method of yeast-mediated transformation-associated recombination for natural product gene clusters, we captured a type II polyketide synthase pathway from Salinispora pacifica with high homology to the enterocin pathway from Streptomyces maritimus and successfully produced enterocin in two different Streptomyces host strains. This result paves the way for the systematic interrogation of Salinispora's promising secondary metabolome.

  11. Direct Capture and Heterologous Expression of Salinispora Natural Product Genes for the Biosynthesis of Enterocin

    PubMed Central

    2015-01-01

    Heterologous expression of secondary metabolic pathways is a promising approach for the discovery and characterization of bioactive natural products. Herein we report the first heterologous expression of a natural product from the model marine actinomycete genus Salinispora. Using the recently developed method of yeast-mediated transformation-associated recombination for natural product gene clusters, we captured a type II polyketide synthase pathway from Salinispora pacifica with high homology to the enterocin pathway from Streptomyces maritimus and successfully produced enterocin in two different Streptomyces host strains. This result paves the way for the systematic interrogation of Salinispora’s promising secondary metabolome. PMID:25382643

  12. Expression systems for heterologous production of antimicrobial peptides.

    PubMed

    Parachin, Nádia Skorupa; Mulder, Kelly Cristina; Viana, Antônio Américo Barbosa; Dias, Simoni Campos; Franco, Octávio Luiz

    2012-12-01

    Antimicrobial peptides (AMPs) consist of molecules that act on the defense systems of numerous organisms toward multiple pathogens such as bacteria, fungi, parasites and viruses. These compounds have become extremely significant due to the increasing resistance of microorganisms to common antibiotics. However, the low quantity of peptides obtained from direct purification is, to date, still a remarkable bottleneck for scientific and industrial research development. Therefore, this review describes the main heterologous systems currently used for AMP production, including bacteria, fungi and plants, and also the related strategies for reaching greater functional peptide production. The main difficulties of each system are also described in order to provide some directions for AMP production. In summary, data revised here indicate that large-scale production of AMPs can be obtained using biotechnological tools, and the products may be applied in the pharmaceutical industry as well as in agribusiness.

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

    PubMed

    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.

  14. Heterologous protein expression in Hypocrea jecorina: a historical perspective and new developments.

    PubMed

    Singh, Arjun; Taylor, Larry E; Vander Wall, Todd A; Linger, Jeffrey; Himmel, Michael E; Podkaminer, Kara; Adney, William S; Decker, Stephen R

    2015-01-01

    Hypocrea jecorina, the sexual teleomorph of Trichoderma reesei, has long been favored as an industrial cellulase producer, first utilizing its native cellulase system and later augmented by the introduction of heterologous enzymatic activities or improved variants of native enzymes. Expression of heterologous proteins in H. jecorina was once considered difficult when the target was an improved variant of a native cellulase. Developments over the past nearly 30 years have produced strains, vectors, and selection mechanisms that have continued to simplify and streamline heterologous protein expression in this fungus. More recent developments in fungal molecular biology have pointed the way toward a fundamental transformation in the ease and efficiency of heterologous protein expression in this important industrial host. Here, 1) we provide a historical perspective on advances in H. jecorina molecular biology, 2) outline host strain engineering, transformation, selection, and expression strategies, 3) detail potential pitfalls when working with this organism, and 4) provide consolidated examples of successful cellulase expression outcomes from our laboratory. PMID:25479282

  15. Isoprenoid-Based Biofuels: Homologous Expression and Heterologous Expression in Prokaryotes.

    PubMed

    Phulara, Suresh Chandra; Chaturvedi, Preeti; Gupta, Pratima

    2016-10-01

    Enthusiasm for mining advanced biofuels from microbial hosts has increased remarkably in recent years. Isoprenoids are one of the highly diverse groups of secondary metabolites and are foreseen as an alternative to petroleum-based fuels. Most of the prokaryotes synthesize their isoprenoid backbone via the deoxyxylulose-5-phosphate pathway from glyceraldehyde-3-phosphate and pyruvate, whereas eukaryotes synthesize isoprenoids via the mevalonate pathway from acetyl coenzyme A (acetyl-CoA). Microorganisms do not accumulate isoprenoids in large quantities naturally, which restricts their application for fuel purposes. Various metabolic engineering efforts have been utilized to overcome the limitations associated with their natural and nonnatural production. The introduction of heterologous pathways/genes and overexpression of endogenous/homologous genes have shown a remarkable increase in isoprenoid yield and substrate utilization in microbial hosts. Such modifications in the hosts' genomes have enabled researchers to develop commercially competent microbial strains for isoprenoid-based biofuel production utilizing a vast array of substrates. The present minireview briefly discusses the recent advancement in metabolic engineering efforts in prokaryotic hosts for the production of isoprenoid-based biofuels, with an emphasis on endogenous, homologous, and heterologous expression strategies.

  16. Isoprenoid-Based Biofuels: Homologous Expression and Heterologous Expression in Prokaryotes.

    PubMed

    Phulara, Suresh Chandra; Chaturvedi, Preeti; Gupta, Pratima

    2016-10-01

    Enthusiasm for mining advanced biofuels from microbial hosts has increased remarkably in recent years. Isoprenoids are one of the highly diverse groups of secondary metabolites and are foreseen as an alternative to petroleum-based fuels. Most of the prokaryotes synthesize their isoprenoid backbone via the deoxyxylulose-5-phosphate pathway from glyceraldehyde-3-phosphate and pyruvate, whereas eukaryotes synthesize isoprenoids via the mevalonate pathway from acetyl coenzyme A (acetyl-CoA). Microorganisms do not accumulate isoprenoids in large quantities naturally, which restricts their application for fuel purposes. Various metabolic engineering efforts have been utilized to overcome the limitations associated with their natural and nonnatural production. The introduction of heterologous pathways/genes and overexpression of endogenous/homologous genes have shown a remarkable increase in isoprenoid yield and substrate utilization in microbial hosts. Such modifications in the hosts' genomes have enabled researchers to develop commercially competent microbial strains for isoprenoid-based biofuel production utilizing a vast array of substrates. The present minireview briefly discusses the recent advancement in metabolic engineering efforts in prokaryotic hosts for the production of isoprenoid-based biofuels, with an emphasis on endogenous, homologous, and heterologous expression strategies. PMID:27422837

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

    PubMed

    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. PMID:27470141

  18. Construction of a Shuttle Vector for Heterologous Expression of a Novel Fungal α-Amylase Gene in Aspergillus oryzae.

    PubMed

    Yin, Yanchen; Mao, Youzhi; Yin, Xiaolie; Gao, Bei; Wei, Dongzhi

    2015-07-01

    The filamentous fungus Aspergillus oryzae is a well-known expression host used to express homologous and heterologous proteins in a number of industrial applications. To facilitate higher yields of proteins of interest, we constructed the pAsOP vector to express heterologous proteins in A. oryzae. pAsOP carries a selectable marker, pyrG, derived from Aspergillus nidulans, and a strong promoter and a terminator of the amyB gene derived from A. oryzae. pAsOP transformed A. oryzae efficiently via the PEG-CaCl2-mediated transformation method. As proof of concept, green fluorescent protein (GFP) was successfully expressed in A. oryzae transformed by pAsOP-GFP. Additionally, we identified a novel fungal α-amylase (PcAmy) gene from Penicillium sp. and cloned the gene into the vector. After transformation by pAsOPPcAmy, the α-amylase PcAmy from Penicillium sp. was successfully expressed in a heterologous host system for the first time. The α-amylase activity in the A. oryzae transformant was increased by 62.3% compared with the untransformed A. oryzae control. The PcAmy protein produced in the system had an optimum pH of 5.0 and optimum temperature of 30°C. As a cold-adapted enzyme, PcAmy shows potential value in industrial applications because of its high catalytic activity at low temperature. Furthermore, the expression vector reported in this study provides promising utility for further scientific research and biotechnological applications.

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

    PubMed

    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.

  20. Secretory expression of a heterologous nattokinase in Lactococcus lactis.

    PubMed

    Liang, Xiaobo; Zhang, Lixin; Zhong, Jin; Huan, Liandong

    2007-05-01

    Nattokinase has been reported as an oral health product for the prevention of atherosclerosis. We developed a novel strategy to express a nattokinase from Bacillus subtilis in a live delivery vehicle, Lactococcus lactis. Promoter P( nisZ) and signal peptide SP(Usp) were used for inducible and secretory expression of nattokinase in L. lactis. Western blotting analysis demonstrated that nattokinase was successfully expressed, and about 94% of the enzyme was secreted to the culture. The recombinant nattokinase showed potent fibrinolytic activity, equivalent to 41.7 urokinase units per milliliter culture. Expression and delivery of such a fibrinolytic enzyme in the food-grade vehicle L. lactis would facilitate the widespread application of nattokinase in the control and prevention of thrombosis diseases.

  1. Transient expression directed by homologous and heterologous promoter and enhancer sequences in fish cells.

    PubMed Central

    Friedenreich, H; Schartl, M

    1990-01-01

    In order to construct fish specific expression vectors for studies on gene regulation in vitro and in vivo a variety of heterologous enhancers and promoters from mammals and from viruses of higher vertebrate cells were tested for expression of the bacterial chloramphenicol acetyl transferase reporter gene in three teleost fish cell lines. Several viral enhancers were found to be constitutively active at high levels. The human metallothionein promoter showed inducible expression in the presence of heavy metal ions. A fish sequence was isolated that can be used as a homologous constitutively active promoter for expression of foreign genes. Using the human growth hormone gene with an active promoter in fish cells for transient expression insufficient splicing and lack of translation were observed, pointing to limitations in the use of heterologous genes in gene transfer experiments. On the contrary, some heterologous promoters and enhancers functioned in fish cells as well as in their cell type of origin, indicating that corresponding transcription factors are sufficiently conserved between fish and human over a period of 900 million years of independent evolution. Images PMID:2356120

  2. Heterologous expression of the mevalonic acid pathway in cyanobacteria enhances endogenous carbon partitioning to isoprene.

    PubMed

    Bentley, Fiona K; Zurbriggen, Andreas; Melis, Anastasios

    2014-01-01

    Heterologous expression of the isoprene synthase gene in the cyanobacterium Synechocystis PCC 6803 conferred upon these microorganisms the property of photosynthetic isoprene (C₅H₈) hydrocarbons production. Continuous production of isoprene from CO₂ and H₂O was achieved in the light, occurring via the endogenous methylerythritol-phosphate (MEP) pathway, in tandem with the growth of Synechocystis. This work addressed the issue of photosynthetic carbon partitioning between isoprene and biomass in Synechocystis. Evidence is presented to show heterologous genomic integration and cellular expression of the mevalonic acid (MVA) pathway genes in Synechocystis endowing a non-native pathway for carbon flux amplification to isopentenyl-diphosphate (IPP) and dimethylallyl-diphosphate (DMAPP) precursors of isoprene. Heterologous expression of the isoprene synthase in combination with the MVA pathway enzymes resulted in photosynthetic isoprene yield improvement by approximately 2.5-fold, compared with that measured in cyanobacteria transformed with the isoprene synthase gene only. These results suggest that the MVA pathway introduces a bypass in the flux of endogenous cellular substrate in Synechocystis to IPP and DMAPP, overcoming flux limitations of the native MEP pathway. The work employed a novel chromosomal integration and expression of synthetic gene operons in Synechocystis, comprising up to four genes under the control of a single promoter, and expressing three operons simultaneously. This is the first time an entire biosynthetic pathway with seven recombinant enzymes has been heterologously expressed in a photosynthetic microorganism. It constitutes contribution to the genetic engineering toolkit of photosynthetic microorganisms and a paradigm in the pursuit of photosynthetic approaches for the renewable generation of high-impact products.

  3. Strategies for increasing heterologous expression of a thermostable esterase from Archaeoglobus fulgidus in Escherichia coli.

    PubMed

    Kim, Jinyeong; Kim, Seul I; Hong, Eunsoo; Ryu, Yeonwoo

    2016-11-01

    Heterologous proteins expressed in bacteria are used for numerous biotechnological applications. Escherichia coli is the most commonly used host for heterologous protein expression because of its many advantages. Researchers have been studying proteins from extremophiles heterologously expressed in E. coli because the proteins of extremophiles are strongly resistant to extreme conditions. In a previous study, a thermostable esterase Est-AF was isolated from Archaeoglobus fulgidus and expressed in E. coli. However, further studies of Est-AF were difficult owing to its low expression levels in E. coli. In this study, we used various strategies, such as changing the expression vector and host strain, codon optimization, and optimization of induction conditions, to increase the expression of Est-AF. Through codon optimization and by changing the vector and host strain, Est-AF expression was increased from 31.50 ± 0.35 mg/L to 61.75 ± 0.28 mg/L. The optimized expression system consisted of a codon-optimized Est-AF gene in a pET28a(+)-based expression plasmid in E. coli Rosetta cells. The expression level was further increased by optimizing the induction conditions. The optimized conditions were induction with 0.4 mM isopropyl-b-d-1-thiogalactoside (IPTG) at 37 °C for 5 h. Under these conditions, the expression level of Est-AF was increased from 31.5 ± 0.35 mg/L to 119.52 ± 0.34 mg/L. PMID:27449918

  4. Expression and secretion of heterologous proteases by Corynebacterium glutamicum.

    PubMed Central

    Billman-Jacobe, H; Wang, L; Kortt, A; Stewart, D; Radford, A

    1995-01-01

    Genes encoding the basic protease of Dichelobacter nodosus (bprV) and the subtilisin of Bacillus subtilis (aprE) were cloned and expressed in Corynebacterium glutamicum. In each case, enzymatically active protein was detected in the supernatants of liquid cultures. While the secretion of subtilisin was directed by its own signal peptide, the natural signal peptide of the bprV basic protease did not facilitate secretion. A hybrid aprE-bprV gene in which the promoter and signal peptide coding sequences of subtilisin replaced those of bprV could be expressed, and basic protease was secreted by C. glutamicum. Expression of these proteases in C. glutamicum provides an opportunity to compare protein secretion from this gram-positive host with that from other gram-positive and gram-negative bacteria. PMID:7747974

  5. Reconstitution of acetosyringone-mediated Agrobacterium tumefaciens virulence gene expression in the heterologous host Escherichia coli.

    PubMed

    Lohrke, S M; Yang, H; Jin, S

    2001-06-01

    The ability to utilize Escherichia coli as a heterologous system in which to study the regulation of Agrobacterium tumefaciens virulence genes and the mechanism of transfer DNA (T-DNA) transfer would provide an important tool to our understanding and manipulation of these processes. We have previously reported that the rpoA gene encoding the alpha subunit of RNA polymerase is required for the expression of lacZ gene under the control of virB promoter (virBp::lacZ) in E. coli containing a constitutively active virG gene [virG(Con)]. Here we show that an RpoA hybrid containing the N-terminal 247 residues from E. coli and the C-terminal 89 residues from A. tumefaciens was able to significantly express virBp::lacZ in E. coli in a VirG(Con)-dependent manner. Utilization of lac promoter-driven virA and virG in combination with the A. tumefaciens rpoA construct resulted in significant inducer-mediated expression of the virBp::lacZ fusion, and the level of virBp::lacZ expression was positively correlated to the copy number of the rpoA construct. This expression was dependent on VirA, VirG, temperature, and, to a lesser extent, pH, which is similar to what is observed in A. tumefaciens. Furthermore, the effect of sugars on vir gene expression was observed only in the presence of the chvE gene, suggesting that the glucose-binding protein of E. coli, a homologue of ChvE, does not interact with the VirA molecule. We also evaluated other phenolic compounds in induction assays and observed significant expression with syringealdehyde, a low level of expression with acetovanillone, and no expression with hydroxyacetophenone, similar to what occurs in A. tumefaciens strain A348 from which the virA clone was derived. These data support the notion that VirA directly senses the phenolic inducer. However, the overall level of expression of the vir genes in E. coli is less than what is observed in A. tumefaciens, suggesting that additional gene(s) from A. tumefaciens may be required for

  6. Heterologous expression of xylanase enzymes in lipogenic yeast Yarrowia lipolytica

    DOE PAGES

    Wang, Wei; Wei, Hui; Alahuhta, Markus; Chen, Xiaowen; Hyman, Deborah; Johnson, David K.; Zhang, Min; Himmel, Michael E.

    2014-12-02

    In order to develop a direct microbial sugar conversion platform for the production of lipids, drop-in fuels and chemicals from cellulosic biomass substrate, we chose Yarrowia lipolytica as a viable demonstration strain. Y. lipolytica is known to accumulate lipids intracellularly and is capable of metabolizing sugars to produce lipids; however, it lacks the lignocellulose-degrading enzymes needed to break down biomass directly. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica. The XynII and XlnD expressing Yarrowia strains exhibited an abilitymore » to grow on xylan mineral plates. This was shown by Congo Red staining of halo zones on xylan mineral plates. Enzymatic activity tests further demonstrated active expression of XynII and XlnD in Y. lipolytica. Furthermore, synergistic action in converting xylan to xylose was observed when XlnD acted in concert with XynII. Finally, the successful expression of these xylanases in Yarrowia further advances us toward our goal to develop a direct microbial conversion process using this organism.« less

  7. Heterologous Expression of Xylanase Enzymes in Lipogenic Yeast Yarrowia lipolytica

    PubMed Central

    Alahuhta, Markus; Chen, Xiaowen; Hyman, Deborah; Johnson, David K.; Zhang, Min; Himmel, Michael E.

    2014-01-01

    To develop a direct microbial sugar conversion platform for the production of lipids, drop-in fuels and chemicals from cellulosic biomass substrate, we chose Yarrowia lipolytica as a viable demonstration strain. Y. lipolytica is known to accumulate lipids intracellularly and is capable of metabolizing sugars to produce lipids; however, it lacks the lignocellulose-degrading enzymes needed to break down biomass directly. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica. The XynII and XlnD expressing Yarrowia strains exhibited an ability to grow on xylan mineral plates. This was shown by Congo Red staining of halo zones on xylan mineral plates. Enzymatic activity tests further demonstrated active expression of XynII and XlnD in Y. lipolytica. Furthermore, synergistic action in converting xylan to xylose was observed when XlnD acted in concert with XynII. The successful expression of these xylanases in Yarrowia further advances us toward our goal to develop a direct microbial conversion process using this organism. PMID:25462572

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

    PubMed

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

    2014-01-01

    Heterologous and stable expression of genes encoding terpenoid biosynthetic enzymes in planta is an important tool for functional characterization and is an attractive alternative to expression in microbial hosts for biotechnological production. Despite improvements to the procedure, such as streamlining of large scale Agrobacterium infiltration and upregulation of the upstream pathways, transient in planta heterologous expression quickly reaches limitations when used for production of terpenoids. Stable integration of transgenes into the nuclear genome of the moss Physcomitrella patens has already been widely recognized as a viable alternative for industrial-scale production of biopharmaceuticals. For expression of terpenoid biosynthetic genes, and reconstruction of heterologous pathways, Physcomitrella has unique attributes that makes it a very promising biotechnological host. These features include a high native tolerance to terpenoids, a simple endogenous terpenoid profile, convenient genome editing using homologous recombination, and cultivation techniques that allow up-scaling from single cells in microtiter plates to industrial photo-bioreactors. Beyond its use for functional characterization of terpenoid biosynthetic genes, engineered Physcomitrella can be a green biotechnological platform for production of terpenoids. Here, we describe two complementary and simple procedures for stable nuclear transformation of Physcomitrella with terpenoid biosynthetic genes, selection and cultivation of transgenic lines, and metabolite analysis of terpenoids produced in transgenic moss lines. We also provide tools for metabolic engineering through genome editing using homologous recombination. PMID:24777804

  9. Stable heterologous expression of biologically active terpenoids in green plant cells

    PubMed Central

    Ikram, N. Kusaira B. K.; Zhan, Xin; Pan, Xi-Wu; King, Brian C.; Simonsen, Henrik T.

    2015-01-01

    Plants biosynthesize a great diversity of biologically active small molecules of interest for fragrances, flavors, and pharmaceuticals. Among specialized metabolites, terpenoids represent the greatest molecular diversity. Many terpenoids are very complex, and total chemical synthesis often requires 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 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 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 localization, and other factors often present challenges. To address the future demand for natural products used as drugs, new platforms are being established that are better suited for heterologous production of plant metabolites. Specifically, direct metabolic engineering of plants can provide effective heterologous expression for production of valuable plant-derived natural products. In this review, our primary focus is on small terpenoids and we discuss the benefits of plant expression platforms and provide several successful examples of stable production of small terpenoids in plants. PMID:25852702

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

    PubMed

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

    2014-01-01

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

  11. Heterologous expression of Aspergillus terreus fructosyltransferase in Kluyveromyces lactis.

    PubMed

    Spohner, Sebastian C; Czermak, Peter

    2016-06-25

    Fructo-oligosaccharides are prebiotic and hypocaloric sweeteners that are usually extracted from chicory. They can also be produced from sucrose using fructosyltransferases, but the only commercial enzyme suitable for this purpose is Pectinex Ultra, which is produced with Aspergillus aculeatus. Here we used the yeast Kluyveromyces lactis to express a secreted recombinant fructosyltransferase from the inulin-producing fungus Aspergillus terreus. A synthetic codon-optimised version of the putative β-fructofuranosidase ATEG 04996 (XP 001214174.1) from A. terreus NIH2624 was secreted as a functional protein into the extracellular medium. At 60°C, the purified A. terreus enzyme generated the same pattern of oligosaccharides as Pectinex Ultra, but at lower temperatures it also produced oligomers with up to seven units. We achieved activities of up to 986.4U/mL in high-level expression experiments, which is better than previous reports of optimised Aspergillus spp. fermentations. PMID:27084521

  12. Enhanced heterologous gene expression in novel rpoH mutants of Escherichia coli.

    PubMed

    Obukowicz, M G; Staten, N R; Krivi, G G

    1992-05-01

    Extragenic temperature-resistant suppressor mutants of an rpoD800 derivative of Escherichia coli W3110 were selected at 43.5 degrees C. Two of the mutants were shown to have a phenotype of enhanced accumulation of heterologous proteins. Genetic mapping of the two mutants showed that the mutation conferring temperature resistance resided in the rpoH gene. P1-mediated transduction of the rpoD+ gene into both of the rpoD800 rpoH double mutants resulted in viable rpoH mutants, MON102 and MON105, that retained temperature resistance at 46 degrees C, the maximum growth temperature of W3110. The complete rpoH gene, including the regulatory region, from MON102, MON105, and the parental W3110 was cloned and sequenced. Sequencing results showed that a single C----T transition at nucleotide 802 was present in both MON102 and MON105, resulting in an Arg(CGC)----Cys(TGC) substitution at amino acid residue 268 (R-268-C; this gene was designated rpoH358). Heterologous protein accumulation levels in both MON102 and MON105, as well as in rpoH358 mutants constructed in previously unmanipulated W3110 and JM101, were assessed and compared with parental W3110 and JM101 levels. Expression studies utilizing the recA or araBAD promoter and the phage T7 gene 10L ribosome-binding site (g10L) showed that increased accumulation levels of a number of representative heterologous proteins (i.e., human or bovine insulin-like growth factor-1, bovine insulin-like growth factor-2, prohormone of human atrial natriuretic factor, bovine placental lactogen, and/or bovine prolactin) were obtained in the rpoH358 mutants compared with the levels in the parental W3110 and JM101. The mechanism of enhanced heterologous protein accumulation in MON102 and MON105 was unique compared with those of previously described rpoH mutants. Pulse-chase and Northern (RNA) blot analyses showed that the enhanced accumulation of heterologous proteins was not due to decreased proteolysis but was instead due to increased levels

  13. Heterologous expression of five disulfide-bonded insecticidal spider peptides.

    PubMed

    Estrada, Georgina; Silva, Anita O; Villegas, Elba; Ortiz, Ernesto; Beirão, Paulo S L; Corzo, Gerardo

    2016-09-01

    The genes of the five disulfide-bonded peptide toxins 1 and 2 (named Oxytoxins or Oxotoxins) from the spider Oxyopes lineatus were cloned into the expression vector pQE30 containing a 6His-tag and a Factor Xa proteolytic cleavage region. These two recombinant vectors were transfected into Escherichia coli BL21 cells and expressed under induction with isopropyl thiogalactoside (IPTG). The product of each gene was named HisrOxyTx1 or HisrOxyTx2, and the protein expression was ca 14 and 6 mg/L of culture medium, respectively. Either recombinant toxin HisrOxyTx1 or HisrOxyTx2 were found exclusively in inclusion bodies, which were solubilized using a chaotropic agent, and then, purified using affinity chromatography and reverse-phase HPLC (RP-HPLC). The HisrOxyTx1 and HisrOxyTx2 products, obtained from the affinity chromatographic step, showed several peptide fractions having the same molecular mass of 9913.1 and 8030.1 Da, respectively, indicating that both HisrOxyTx1 and HisrOxyTx2 were oxidized forming several distinct disulfide bridge arrangements. The isoforms of both HisrOxyTx1 and HisrOxyTx2 after DTT reduction eluted from the column as a single protein component of 9923 and 8040 Da, respectively. In vitro folding of either HisrOxyTx1 or HisrOxyTx2 yielded single oxidized components, which were cleaved independently by the proteolytic enzyme Factor Xa to give the recombinant peptides rOxyTx1 and rOxyTx2. The experimental molecular masses of rOxyTx1 and rOxyTx2 were 8059.0 and 6176.4 Da, respectively, which agree with their expected theoretical masses. The recombinant peptides rOxyTx1 and rOxyTx2 showed lower but comparable toxicity to the native toxins when injected into lepidopteran larvae; furthermore, rOxyTx1 was able to inhibit calcium ion currents on dorsal unpaired median (DUM) neurons from Periplaneta americana. PMID:27263806

  14. Heterologous expression of five disulfide-bonded insecticidal spider peptides.

    PubMed

    Estrada, Georgina; Silva, Anita O; Villegas, Elba; Ortiz, Ernesto; Beirão, Paulo S L; Corzo, Gerardo

    2016-09-01

    The genes of the five disulfide-bonded peptide toxins 1 and 2 (named Oxytoxins or Oxotoxins) from the spider Oxyopes lineatus were cloned into the expression vector pQE30 containing a 6His-tag and a Factor Xa proteolytic cleavage region. These two recombinant vectors were transfected into Escherichia coli BL21 cells and expressed under induction with isopropyl thiogalactoside (IPTG). The product of each gene was named HisrOxyTx1 or HisrOxyTx2, and the protein expression was ca 14 and 6 mg/L of culture medium, respectively. Either recombinant toxin HisrOxyTx1 or HisrOxyTx2 were found exclusively in inclusion bodies, which were solubilized using a chaotropic agent, and then, purified using affinity chromatography and reverse-phase HPLC (RP-HPLC). The HisrOxyTx1 and HisrOxyTx2 products, obtained from the affinity chromatographic step, showed several peptide fractions having the same molecular mass of 9913.1 and 8030.1 Da, respectively, indicating that both HisrOxyTx1 and HisrOxyTx2 were oxidized forming several distinct disulfide bridge arrangements. The isoforms of both HisrOxyTx1 and HisrOxyTx2 after DTT reduction eluted from the column as a single protein component of 9923 and 8040 Da, respectively. In vitro folding of either HisrOxyTx1 or HisrOxyTx2 yielded single oxidized components, which were cleaved independently by the proteolytic enzyme Factor Xa to give the recombinant peptides rOxyTx1 and rOxyTx2. The experimental molecular masses of rOxyTx1 and rOxyTx2 were 8059.0 and 6176.4 Da, respectively, which agree with their expected theoretical masses. The recombinant peptides rOxyTx1 and rOxyTx2 showed lower but comparable toxicity to the native toxins when injected into lepidopteran larvae; furthermore, rOxyTx1 was able to inhibit calcium ion currents on dorsal unpaired median (DUM) neurons from Periplaneta americana.

  15. Heterologous Expression of Two Ferulic Acid Esterases from Penicillium funiculosum

    NASA Astrophysics Data System (ADS)

    Knoshaug, Eric P.; Selig, Michael J.; Baker, John O.; Decker, Stephen R.; Himmel, Michael E.; Adney, William S.

    Two recombinant ferulic acid esterases from Penicillium funiculosum produced in Aspergillus awamori were evaluated for their ability to improve the digestibility of pretreated corn stover. The genes, faeA and faeB, were cloned from P. funiculosum and expressed in A. awamori using their native signal sequences. Both enzymes contain a catalytic domain connected to a family 1 carbohydrate-binding module by a threonine-rich linker peptide. Interestingly, the carbohydrate binding-module is N-terminal in FaeA and C-terminal in FaeB. The enzymes were purified to homogeneity using column chromatography, and their thermal stability was characterized by differential scanning microcalorimetry. We evaluated both enzymes for their potential to enhance the cellulolytic activity of purified Trichoderma reesei Cel7A on pretreated corn stover.

  16. Heterologous Expression of Two Ferulic Acid Esterases from Penicillium Funiculosum

    SciTech Connect

    Knoshaug, E. P.; Selig, M. J.; Baker, J. O.; Decker, S. R.; Himmel, M. E.; Adney, W. S.

    2008-01-01

    Two recombinant ferulic acid esterases from Penicillium funiculosum produced in Aspergillus awamori were evaluated for their ability to improve the digestibility of pretreated corn stover. The genes, faeA and faeB, were cloned from P. funiculosum and expressed in A. awamori using their native signal sequences. Both enzymes contain a catalytic domain connected to a family 1 carbohydrate-binding module by a threonine-rich linker peptide. Interestingly, the carbohydrate binding-module is N-terminal in FaeA and C-terminal in FaeB. The enzymes were purified to homogeneity using column chromatography, and their thermal stability was characterized by differential scanning microcalorimetry. We evaluated both enzymes for their potential to enhance the cellulolytic activity of purified Trichoderma reesei Cel7A on pretreated corn stover.

  17. Generation of New Complestatin Analogues by Heterologous Expression of the Complestatin Biosynthetic Gene Cluster from Streptomyces chartreusis AN1542.

    PubMed

    Park, Ok-Kyung; Choi, Ha-Young; Kim, Geon-Woo; Kim, Won-Gon

    2016-09-15

    The heterologous expression of the biosynthetic gene cluster (BGC) of natural products enables the production of complex metabolites in a well-characterized host, and facilitates the generation of novel analogues by the manipulation of the genes. However, the BGCs of glycopeptides such as vancomycin, teicoplanin, and complestatin are usually too large to be directly cloned into a single cosmid. Here, we describe the heterologous expression of the complestatin BGC. The 54.5 kb cluster was fully reconstituted from two overlapping cosmids into one cosmid by λ-RED recombination-mediated assembly. Heterologous expression of the assembled gene cluster in Streptomyces lividans TK24 resulted in the production of complestatin. Deletion of cytochrome P450 monooxygenase genes (open reading frames 10 and 11) and heterologous expression of the modified clusters led to the production of two new monocyclic and linear derivatives, complestatins M55 and S56. PMID:27383040

  18. Yarrowia lipolytica: recent achievements in heterologous protein expression and pathway engineering.

    PubMed

    Madzak, Catherine

    2015-06-01

    The oleaginous yeast Yarrowia lipolytica has become a recognized system for expression/secretion of heterologous proteins. This non-conventional yeast is currently being developed as a workhorse for biotechnology by several research groups throughout the world, especially for single-cell oil production, whole cell bioconversion and upgrading of industrial wastes. This mini-review presents established tools for protein expression in Y. lipolytica and highlights novel developments in the areas of promoter design, surface display, and host strain or metabolic pathway engineering. An overview of the industrial and commercial biotechnological applications of Y. lipolytica is also presented. PMID:25947247

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

    PubMed Central

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

    2012-01-01

    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 A2A subtype receptor [(h)A2AR] 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)A2AR 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.—Salom, D., Cao, P., Sun, W., Kramp, K., Jastrzebska, B., Jin, H., Feng, Z., Palczewski, K. Heterologous expression of functional G-protein-coupled receptors in Caenorhabditis elegans. PMID:22090314

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed

    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. PMID:26611164

  3. Robust expression of heterologous genes by selection marker fusion system in improved Chlamydomonas strains.

    PubMed

    Kong, Fantao; Yamasaki, Tomohito; Kurniasih, Sari Dewi; Hou, Liyuan; Li, Xiaobo; Ivanova, Nina; Okada, Shigeru; Ohama, Takeshi

    2015-09-01

    Chlamydomonas is a very attractive candidate plant cell factory. However, its main drawback is the difficulty to find the transformants that robustly express heterologous genes randomly inserted in the nuclear genome. We previously showed that domestic squalene synthase (SQS) gene of Chlamydomonas was much more efficiently overexpressed in a mutant strain [UV-mediated mutant (UVM) 4] than in wild type. In this study, we evaluated the possibility of a new mutant strain, met1, which contains a tag in the maintenance type methyltransferase gene that is expected to play a key role in the maintenance of transcriptional gene silencing. The versatile usefulness of the UVM4 strain to express heterologous genes was also analyzed. We failed to overexpress CrSSL3 cDNA, which is the codon-adjusted squalene synthase-like gene originated from Botryococcus braunii, using the common expression cassette in the wild-type CC-1690 and UVM4 strains. However, we succeeded in isolating western blot-positive transformants through the combinational use of the UVM4 strain and ble2A expression system of which expression cassette bears a fused ORF of the target gene and the antibiotic resistance gene ble via the foot-and-mouth disease virus (FMDV) self-cleaving 2A sequence. It is noteworthy that even with this system, huge deviations in the accumulated protein levels were still observed among the UVM4 transformants. PMID:25660568

  4. Functional Heterologous Expression of an Engineered Full Length CipA from Clostridium thermocellum in Thermoanaerobacterium saccharolyticum

    SciTech Connect

    Currie, Devin; Herring, Christopher; Guss, Adam M; Olson, Daniel G.; Hogsett, David; Lynd, Lee R

    2013-01-01

    BACKGROUND: Cellulose is highly recalcitrant and thus requires a specialized suite of enzymes to solubilize it into fermentable sugars. In C. thermocellum, these extracellular enzymes are present as a highly active multi-component system known as the cellulosome. This study explores the expression of a critical C. thermocellum cellulosomal component in T. saccharolyticum as a step toward creating a thermophilic bacterium capable of consolidated bioprocessing by employing heterologously expressed cellulosomes. RESULTS: We developed an inducible promoter system based on the native T. saccharolyticum xynA promoter, which was shown to be induced by xylan and xylose. The promoter was used to express the cellulosomal component cipA*, an engineered form of the wild-type cipA from C. thermocellum. Expression and localization to the supernatant were both verified for CipA*. When a cipA mutant C. thermocellum strain was cultured with a CipA*-expressing T. saccharolyticum strain, hydrolysis and fermentation of 10 grams per liter SigmaCell 101, a highly crystalline cellulose, were observed. This trans-species complementation of a cipA deletion demonstrated the ability for CipA* to assemble a functional cellulosome. CONCLUSION: This study is the first example of an engineered thermophile heterologously expressing a structural component of a cellulosome. To achieve this goal we developed and tested an inducible promoter for controlled expression in T. saccharolyticum as well as a synthetic cipA. In addition, we demonstrate a high degree of hydrolysis (up to 93%) on microcrystalline cellulose.

  5. Ligand-selective activation of heterologously-expressed mammalian olfactory receptor.

    PubMed

    Ukhanov, K; Bobkov, Y; Corey, E A; Ache, B W

    2014-10-01

    Mammalian olfactory receptors (ORs) appear to have the capacity to couple to multiple G protein-coupled signaling pathways in a ligand-dependent selective manner. To better understand the mechanisms and molecular range of such ligand selectivity, we expressed the mouse eugenol OR (mOR-EG) in HEK293T cells together with Gα15 to monitor activation of the phospholipase-C (PLC) signaling pathway and/or Gαolf to monitor activation of the adenylate cyclase (AC) signaling pathway, resulting in intracellular Ca(2+) release and/or Ca(2+) influx through a cyclic nucleotide-gated channel, respectively. PLC-dependent responses differed dynamically from AC-dependent responses, allowing them to be distinguished when Gα15 and Gαolf were co-expressed. The dynamic difference in readout was independent of the receptor, the heterologous expression system, and the ligand concentration. Of 17 reported mOR-EG ligands tested, including eugenol, its analogs, and structurally dissimilar compounds (mousse cristal, nootkatone, orivone), some equally activated both signaling pathways, some differentially activated both signaling pathways, and some had no noticeable effect even at 1-5mM. Our findings argue that mOR-EG, when heterologously expressed, can couple to two different signaling pathways in a ligand selective manner. The challenge now is to determine the potential of mOR-EG, and perhaps other ORs, to activate multiple signaling pathways in a ligand selective manner in native ORNs. PMID:25149566

  6. Ligand-selective activation of heterologously-expressed mammalian olfactory receptor

    PubMed Central

    Ukhanov, K.; Bobkov, Y.; Corey, E.A.; Ache, B.W.

    2014-01-01

    Mammalian olfactory receptors (ORs) appear to have the capacity to couple to multiple G protein-coupled signaling pathways in a ligand-dependent selective manner. To better understand the mechanisms and molecular range of such ligand selectivity, we expressed the mouse eugenol OR (mOR-EG) in HEK293T cells together with Gα15 to monitor activation of the phospholipase-C (PLC) signaling pathway and/or Gαolf to monitor activation of the adenylate cyclase (AC) signaling pathway, resulting in intracellular Ca2+ release and/or Ca2+ influx through a cyclic nucleotide-gated channel, respectively. PLC-dependent responses differed dynamically from AC-dependent responses, allowing them to be distinguished when Gα15 and Gαolf were co-expressed. The dynamic difference in readout was independent of the receptor, the heterologous expression system, and the ligand concentration. Of 17 reported mOR-EG ligands tested, including eugenol, its analogs, and structurally dissimilar compounds (mousse cristal, nootkatone, orivone), some equally activated both signaling pathways, some differentially activated both signaling pathways, and some had no noticeable effect even at 1-5 mM. Our findings argue that mOR-EG, when heterologously expressed, can couple to two different signaling pathways in a ligand selective manner. The challenge now is to determine the potential of mOR-EG, and perhaps other ORs, to activate multiple signaling pathways in a ligand selective manner in native ORNs. PMID:25149566

  7. Heterologous Expression of Fluostatin Gene Cluster Leads to a Bioactive Heterodimer.

    PubMed

    Yang, Chunfang; Huang, Chunshuai; Zhang, Wenjun; Zhu, Yiguang; Zhang, Changsheng

    2015-11-01

    The biosynthesis gene cluster (fls) for atypical angucycline fluostatins was identified from the marine derived Micromonospora rosaria SCSIO N160 and was confirmed by gene knockouts and the biochemical characterization of a bifunctional oxygenase FlsO2. The absolute configuration of the key biosynthetic intermediate prejadomycin was determined for the first time by Cu Kα X-ray analysis. Heterologous expression of the intact fls-gene cluster in Streptomyces coelicolor YF11 in the presence of 3% sea salts led to the isolation of two new compounds: fluostatin L (1) and difluostatin A (2). Difluostatin A (2), an unusual heterodimer, exhibited antibacterial activities.

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

    PubMed

    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. PMID:25395591

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

    PubMed Central

    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. PMID:23346364

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

    PubMed

    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.

  11. A self-inducible heterologous protein expression system in Escherichia coli

    PubMed Central

    Briand, L.; Marcion, G.; Kriznik, A.; Heydel, J. M.; Artur, Y.; Garrido, C.; Seigneuric, R.; Neiers, F.

    2016-01-01

    Escherichia coli is an important experimental, medical and industrial cell factory for recombinant protein production. The inducible lac promoter is one of the most commonly used promoters for heterologous protein expression in E. coli. Isopropyl-β-D-thiogalactoside (IPTG) is currently the most efficient molecular inducer for regulating this promoter’s transcriptional activity. However, limitations have been observed in large-scale and microplate production, including toxicity, cost and culture monitoring. Here, we report the novel SILEX (Self-InducibLe Expression) system, which is a convenient, cost-effective alternative that does not require cell density monitoring or IPTG induction. We demonstrate the broad utility of the presented self-inducible method for a panel of diverse proteins produced in large amounts. The SILEX system is compatible with all classical culture media and growth temperatures and allows protein expression modulation. Importantly, the SILEX system is proven to be efficient for protein expression screening on a microplate scale. PMID:27611846

  12. A self-inducible heterologous protein expression system in Escherichia coli.

    PubMed

    Briand, L; Marcion, G; Kriznik, A; Heydel, J M; Artur, Y; Garrido, C; Seigneuric, R; Neiers, F

    2016-01-01

    Escherichia coli is an important experimental, medical and industrial cell factory for recombinant protein production. The inducible lac promoter is one of the most commonly used promoters for heterologous protein expression in E. coli. Isopropyl-β-D-thiogalactoside (IPTG) is currently the most efficient molecular inducer for regulating this promoter's transcriptional activity. However, limitations have been observed in large-scale and microplate production, including toxicity, cost and culture monitoring. Here, we report the novel SILEX (Self-InducibLe Expression) system, which is a convenient, cost-effective alternative that does not require cell density monitoring or IPTG induction. We demonstrate the broad utility of the presented self-inducible method for a panel of diverse proteins produced in large amounts. The SILEX system is compatible with all classical culture media and growth temperatures and allows protein expression modulation. Importantly, the SILEX system is proven to be efficient for protein expression screening on a microplate scale. PMID:27611846

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

    PubMed

    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.

  14. Combined protein construct and synthetic gene engineering for heterologous protein expression and crystallization using Gene Composer

    SciTech Connect

    Raymond, Amy; Lovell, Scott; Lorimer, Don; Walchli, John; Mixon, Mark; Wallace, Ellen; Thompkins, Kaitlin; Archer, Kimberly; Burgin, Alex; Stewart, Lance

    2009-12-01

    With the goal of improving yield and success rates of heterologous protein production for structural studies we have developed the database and algorithm software package Gene Composer. This freely available electronic tool facilitates the information-rich design of protein constructs and their engineered synthetic gene sequences, as detailed in the accompanying manuscript. In this report, we compare heterologous protein expression levels from native sequences to that of codon engineered synthetic gene constructs designed by Gene Composer. A test set of proteins including a human kinase (P38{alpha}), viral polymerase (HCV NS5B), and bacterial structural protein (FtsZ) were expressed in both E. coli and a cell-free wheat germ translation system. We also compare the protein expression levels in E. coli for a set of 11 different proteins with greatly varied G:C content and codon bias. The results consistently demonstrate that protein yields from codon engineered Gene Composer designs are as good as or better than those achieved from the synonymous native genes. Moreover, structure guided N- and C-terminal deletion constructs designed with the aid of Gene Composer can lead to greater success in gene to structure work as exemplified by the X-ray crystallographic structure determination of FtsZ from Bacillus subtilis. These results validate the Gene Composer algorithms, and suggest that using a combination of synthetic gene and protein construct engineering tools can improve the economics of gene to structure research.

  15. Heterologous expression of the lipid transfer protein CERT increases therapeutic protein productivity of mammalian cells.

    PubMed

    Florin, Lore; Pegel, Antje; Becker, Eric; Hausser, Angelika; Olayioye, Monilola A; Kaufmann, Hitto

    2009-04-20

    Recent studies have demonstrated that the introduction of transgenes regulating protein transport or affecting post-translational modifications can further improve industrial processes for the production of therapeutic proteins in mammalian cells. Our study on improving therapeutic protein production in CHO cells by heterologous expression of the ceramide transfer protein (CERT) was initiated by the recent discovery that CERT is involved in protein kinase D (PKD)-dependent protein transport from the Golgi to the plasma membrane. We generated a set of CHO DG44 cell lines by stable integration of constructs expressing either CERT wild-type or CERT S132A, a mutant conferring increased lipid transfer activity, or a mock plasmid. CHO cells expressing heterologous CERT demonstrated significantly higher specific productivities of the therapeutic protein HSA when grown in inoculum suspension cultures. This effect translated into significantly increased overall HSA titers in a fed-batch format where cells are grown in chemically defined serum-free media. Furthermore, we could show that CERT also enhanced monoclonal antibody secretion in two IgG production cell lines with different basal productivities. The data demonstrate the potential of CERT engineering to improve mammalian cell culture production processes to yield high amounts of a therapeutic protein product of desired quality. To our knowledge, this is the first study showing a bottle neck in recombinant protein secretion at the Golgi complex in mammalian cells. PMID:19428735

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

    PubMed

    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. PMID:27343449

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed

    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.

  20. Characterization and heterologous expression of a PR-1 protein from traps of the carnivorous plant Nepenthes mirabilis.

    PubMed

    Buch, Franziska; Pauchet, Yannick; Rott, Matthias; Mithöfer, Axel

    2014-04-01

    Carnivorous plants capture and digest prey to obtain additional nutrients. Therefore, different trapping mechanisms were developed in different species. Plants of the genus Nepenthes possess pitfall-traps filled with a digestive fluid, which is secreted by the plants themselves. This pitcher fluid is composed of various enzymes to digest the captured prey. Besides hydrolytic enzymes, defense-related proteins have been identified in the fluid. The present study describes the identification and heterologous expression of a pathogenesis-related protein, NmPR-1, from pitchers of Nepenthes mirabilis with features that are unusual for PR-1 proteins. In particular, it was proven to be highly glycosylated and, furthermore, it exhibited antibacterial instead of antifungal activities. These properties are probably due to the specific environment of the pitcher fluid.

  1. Characterization and heterologous expression of a PR-1 protein from traps of the carnivorous plant Nepenthes mirabilis.

    PubMed

    Buch, Franziska; Pauchet, Yannick; Rott, Matthias; Mithöfer, Axel

    2014-04-01

    Carnivorous plants capture and digest prey to obtain additional nutrients. Therefore, different trapping mechanisms were developed in different species. Plants of the genus Nepenthes possess pitfall-traps filled with a digestive fluid, which is secreted by the plants themselves. This pitcher fluid is composed of various enzymes to digest the captured prey. Besides hydrolytic enzymes, defense-related proteins have been identified in the fluid. The present study describes the identification and heterologous expression of a pathogenesis-related protein, NmPR-1, from pitchers of Nepenthes mirabilis with features that are unusual for PR-1 proteins. In particular, it was proven to be highly glycosylated and, furthermore, it exhibited antibacterial instead of antifungal activities. These properties are probably due to the specific environment of the pitcher fluid. PMID:24534104

  2. Production of antibacterial peptide from bee venom via a new strategy for heterologous expression.

    PubMed

    Hou, Chunsheng; Guo, Liqiong; Lin, Junfang; You, Linfeng; Wu, Wuhua

    2014-12-01

    Honey bee is important economic insect that not only pollinates fruits and crops but also provides products with various physiological activities. Bee venom is a functional agent that is widely applied in clinical treatment and pharmacy. Secapin is one of these agents that have a significant role in therapy. The functions of secapin from the bee venom have been documented, but little information is known about its heterologous expression under natural condition. Moreover, few scholars verified experimentally the functions of secapin from bee venom in vitro. In this study, we successfully constructed a heterologous expression vector, which is different from conventional expression system. A transgenic approach was established for transformation of secapin gene from the venom of Apis mellifera carnica (Ac-sec) into the edible fungi, Coprinus cinereus. Ac-sec was encoded by a 234 bp nucleotide that contained a signal peptide domain and two potential phosphorylation sites. The sequence exhibited highly homology with various secapins characterized from honey bee and related species. Southern blot data indicated that Ac-sec was present as single or multiple copy loci in the C. cinereus genome. By co-transformation and double-layer active assay, Ac-sec was expressed successfully in C. cinereus and the antibacterial activity of the recombinants was identified, showing notable antibacterial activities on different bacteria. Although Ac-sec is from the venom of Apidae, phylogenetic analysis demonstrated that Ac-sec was more closely related to that of Vespid than to bee species from Apidae. The molecular characteristics of Ac-sec and the potential roles of small peptides in biology were discussed. PMID:25189650

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

    SciTech Connect

    Salem, Tamer Z.; Zhang, Fengrui; Thiem, Suzanne M.

    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.

  4. Deciphering activation of olfactory receptors using heterologous expression in Saccharomyces cerevisiae and bioluminescence resonance energy transfer.

    PubMed

    Sanz, Guenhaël; Pajot-Augy, Edith

    2013-01-01

    Hetero- and homo-oligomerization of G protein-coupled receptors (GPCRs) has been addressed in the past years using various approaches such as co-immunoprecipitation, fluorescence resonance energy transfer and bioluminescence resonance energy transfer (BRET). Here, we report the methodological details from a previously published study to investigate the relationships between oligomerization and activation states of olfactory receptors (ORs). This methodology combines heterologous expression of ORs in Saccharomyces cerevisiae and BRET assays on membrane fractions, in particular, upon odorant stimulation. We have demonstrated that ORs constitutively homodimerize at the plasma membrane and that high odorant concentrations promote a conformational change of the dimer, which becomes inactive. We proposed a model in which one odorant molecule binding the dimer would induce activation, while two odorant molecules, each binding one protomer of the dimer, would blunt signaling.

  5. Identification and Heterologous Expression of the Chaxamycin Biosynthesis Gene Cluster from Streptomyces leeuwenhoekii.

    PubMed

    Castro, Jean Franco; Razmilic, Valeria; Gomez-Escribano, Juan Pablo; Andrews, Barbara; Asenjo, Juan A; Bibb, Mervyn J

    2015-09-01

    Streptomyces leeuwenhoekii, isolated from the hyperarid Atacama Desert, produces the new ansamycin-like compounds chaxamycins A to D, which possess potent antibacterial activity and moderate antiproliferative activity. We report the development of genetic tools to manipulate S. leeuwenhoekii and the identification and partial characterization of the 80.2-kb chaxamycin biosynthesis gene cluster, which was achieved by both mutational analysis in the natural producer and heterologous expression in Streptomyces coelicolor A3(2) strain M1152. Restoration of chaxamycin production in a nonproducing ΔcxmK mutant (cxmK encodes 3-amino-5-hydroxybenzoic acid [AHBA] synthase) was achieved by supplementing the growth medium with AHBA, suggesting that mutasynthesis may be a viable approach for the generation of novel chaxamycin derivatives.

  6. Identification and Heterologous Expression of the Chaxamycin Biosynthesis Gene Cluster from Streptomyces leeuwenhoekii

    PubMed Central

    Castro, Jean Franco; Razmilic, Valeria; Gomez-Escribano, Juan Pablo; Andrews, Barbara; Asenjo, Juan A.

    2015-01-01

    Streptomyces leeuwenhoekii, isolated from the hyperarid Atacama Desert, produces the new ansamycin-like compounds chaxamycins A to D, which possess potent antibacterial activity and moderate antiproliferative activity. We report the development of genetic tools to manipulate S. leeuwenhoekii and the identification and partial characterization of the 80.2-kb chaxamycin biosynthesis gene cluster, which was achieved by both mutational analysis in the natural producer and heterologous expression in Streptomyces coelicolor A3(2) strain M1152. Restoration of chaxamycin production in a nonproducing ΔcxmK mutant (cxmK encodes 3-amino-5-hydroxybenzoic acid [AHBA] synthase) was achieved by supplementing the growth medium with AHBA, suggesting that mutasynthesis may be a viable approach for the generation of novel chaxamycin derivatives. PMID:26092459

  7. Heterologously expressed serotonin 1A receptors couple to muscarinic K+ channels in heart.

    PubMed Central

    Karschin, A; Ho, B Y; Labarca, C; Elroy-Stein, O; Moss, B; Davidson, N; Lester, H A

    1991-01-01

    In cardiac atrial cells, muscarinic acetylcholine receptors activate a K+ current directly via a guanine nucleotide-binding protein (G protein). Serotonin type 1A receptors may activate a similar pathway in hippocampal neurons. To develop a system in which receptor/G protein/K+ channel coupling can be experimentally manipulated, we have used a highly efficient recombinant vaccinia virus vector system to express human serotonin 1A receptors in primary cultures of rat atrial myocytes. The expressed 1A receptors activated the inwardly rectifying K+ conductance that is normally activated by the endogenous muscarinic acetylcholine receptors. Maximal responses to either agonist occluded further activation by the other agonist. The average activation time constants for serotonin were about 5 times slower than for acetylcholine. The data support suggestions that the intracellular signaling pathway from seven-helix receptors to G proteins and directly to ion channels is widespread in excitable cells. After a fraction of the G proteins are activated irreversibly by guanosine 5'-[gamma-thio]triphosphate, subsequent transduction proceeds more efficiently. One possible interpretation is that multiple G-protein molecules are required to activate each channel. Vaccinia virus expression vectors are thus useful for expressing seven-helix receptors in primary cultures of postmitotic cells and have provided a heterologous expression system for the signaling pathway from seven-helix receptors to G proteins and directly to ion channels. Images PMID:1905814

  8. Investigation of Proposed Ladderane Biosynthetic Genes from Anammox Bacteria by Heterologous Expression in E. coli.

    PubMed

    Javidpour, Pouya; Deutsch, Samuel; Mutalik, Vivek K; Hillson, Nathan J; Petzold, Christopher J; Keasling, Jay D; Beller, Harry R

    2016-01-01

    Ladderanes are hydrocarbon chains with three or five linearly concatenated cyclobutane rings that are uniquely produced as membrane lipid components by anammox (anaerobic ammonia-oxidizing) bacteria. By virtue of their angle and torsional strain, ladderanes are unusually energetic compounds, and if produced biochemically by engineered microbes, could serve as renewable, high-energy-density jet fuel components. The biochemistry and genetics underlying the ladderane biosynthetic pathway are unknown, however, previous studies have identified a pool of 34 candidate genes from the anammox bacterium, Kuenenia stuttgartiensis, some or all of which may be involved with ladderane fatty acid biosynthesis. The goal of the present study was to establish a systematic means of testing the candidate genes from K. stuttgartiensis for involvement in ladderane biosynthesis through heterologous expression in E. coli under anaerobic conditions. This study describes an efficient means of assembly of synthesized, codon-optimized candidate ladderane biosynthesis genes in synthetic operons that allows for changes to regulatory element sequences, as well as modular assembly of multiple operons for simultaneous heterologous expression in E. coli (or potentially other microbial hosts). We also describe in vivo functional tests of putative anammox homologs of the phytoene desaturase CrtI, which plays an important role in the hypothesized ladderane pathway, and a method for soluble purification of one of these enzymes. This study is, to our knowledge, the first experimental effort focusing on the role of specific anammox genes in the production of ladderanes, and lays the foundation for future efforts toward determination of the ladderane biosynthetic pathway. Our substantial, but far from comprehensive, efforts at elucidating the ladderane biosynthetic pathway were not successful. We invite the scientific community to take advantage of the considerable synthetic biology resources and

  9. Investigation of Proposed Ladderane Biosynthetic Genes from Anammox Bacteria by Heterologous Expression in E. coli.

    PubMed

    Javidpour, Pouya; Deutsch, Samuel; Mutalik, Vivek K; Hillson, Nathan J; Petzold, Christopher J; Keasling, Jay D; Beller, Harry R

    2016-01-01

    Ladderanes are hydrocarbon chains with three or five linearly concatenated cyclobutane rings that are uniquely produced as membrane lipid components by anammox (anaerobic ammonia-oxidizing) bacteria. By virtue of their angle and torsional strain, ladderanes are unusually energetic compounds, and if produced biochemically by engineered microbes, could serve as renewable, high-energy-density jet fuel components. The biochemistry and genetics underlying the ladderane biosynthetic pathway are unknown, however, previous studies have identified a pool of 34 candidate genes from the anammox bacterium, Kuenenia stuttgartiensis, some or all of which may be involved with ladderane fatty acid biosynthesis. The goal of the present study was to establish a systematic means of testing the candidate genes from K. stuttgartiensis for involvement in ladderane biosynthesis through heterologous expression in E. coli under anaerobic conditions. This study describes an efficient means of assembly of synthesized, codon-optimized candidate ladderane biosynthesis genes in synthetic operons that allows for changes to regulatory element sequences, as well as modular assembly of multiple operons for simultaneous heterologous expression in E. coli (or potentially other microbial hosts). We also describe in vivo functional tests of putative anammox homologs of the phytoene desaturase CrtI, which plays an important role in the hypothesized ladderane pathway, and a method for soluble purification of one of these enzymes. This study is, to our knowledge, the first experimental effort focusing on the role of specific anammox genes in the production of ladderanes, and lays the foundation for future efforts toward determination of the ladderane biosynthetic pathway. Our substantial, but far from comprehensive, efforts at elucidating the ladderane biosynthetic pathway were not successful. We invite the scientific community to take advantage of the considerable synthetic biology resources and

  10. Investigation of Proposed Ladderane Biosynthetic Genes from Anammox Bacteria by Heterologous Expression in E. coli

    PubMed Central

    Javidpour, Pouya; Deutsch, Samuel; Mutalik, Vivek K.; Hillson, Nathan J.; Petzold, Christopher J.; Keasling, Jay D.; Beller, Harry R.

    2016-01-01

    Ladderanes are hydrocarbon chains with three or five linearly concatenated cyclobutane rings that are uniquely produced as membrane lipid components by anammox (anaerobic ammonia-oxidizing) bacteria. By virtue of their angle and torsional strain, ladderanes are unusually energetic compounds, and if produced biochemically by engineered microbes, could serve as renewable, high-energy-density jet fuel components. The biochemistry and genetics underlying the ladderane biosynthetic pathway are unknown, however, previous studies have identified a pool of 34 candidate genes from the anammox bacterium, Kuenenia stuttgartiensis, some or all of which may be involved with ladderane fatty acid biosynthesis. The goal of the present study was to establish a systematic means of testing the candidate genes from K. stuttgartiensis for involvement in ladderane biosynthesis through heterologous expression in E. coli under anaerobic conditions. This study describes an efficient means of assembly of synthesized, codon-optimized candidate ladderane biosynthesis genes in synthetic operons that allows for changes to regulatory element sequences, as well as modular assembly of multiple operons for simultaneous heterologous expression in E. coli (or potentially other microbial hosts). We also describe in vivo functional tests of putative anammox homologs of the phytoene desaturase CrtI, which plays an important role in the hypothesized ladderane pathway, and a method for soluble purification of one of these enzymes. This study is, to our knowledge, the first experimental effort focusing on the role of specific anammox genes in the production of ladderanes, and lays the foundation for future efforts toward determination of the ladderane biosynthetic pathway. Our substantial, but far from comprehensive, efforts at elucidating the ladderane biosynthetic pathway were not successful. We invite the scientific community to take advantage of the considerable synthetic biology resources and

  11. Heterologous expression of natural product biosynthetic gene clusters in Streptomyces coelicolor: from genome mining to manipulation of biosynthetic pathways.

    PubMed

    Gomez-Escribano, Juan Pablo; Bibb, Mervyn J

    2014-02-01

    Heterologous gene expression is one of the main strategies used to access the full biosynthetic potential of actinomycetes, as well as to study the metabolic pathways of natural product biosynthesis and to create unnatural pathways. Streptomyces coelicolor A3(2) is the most studied member of the actinomycetes, bacteria renowned for their prolific capacity to synthesize a wide range of biologically active specialized metabolites. We review here the use of strains of this species for the heterologous production of structurally diverse actinomycete natural products.

  12. A Chimeric Affinity Tag for Efficient Expression and Chromatographic Purification of Heterologous Proteins from Plants.

    PubMed

    Sainsbury, Frank; Jutras, Philippe V; Vorster, Juan; Goulet, Marie-Claire; Michaud, Dominique

    2016-01-01

    The use of plants as expression hosts for recombinant proteins is an increasingly attractive option for the production of complex and challenging biopharmaceuticals. Tools are needed at present to marry recent developments in high-yielding gene vectors for heterologous expression with routine protein purification techniques. In this study, we designed the Cysta-tag, a new purification tag for immobilized metal affinity chromatography (IMAC) of plant-made proteins based on the protein-stabilizing fusion partner SlCYS8. We show that the Cysta-tag may be used to readily purify proteins under native conditions, and then be removed enzymatically to isolate the protein of interest. We also show that commonly used protease recognition sites for linking purification tags are differentially stable in leaves of the commonly used expression host Nicotiana benthamiana, with those linkers susceptible to cysteine proteases being less stable then serine protease-cleavable linkers. As an example, we describe a Cysta-tag experimental scheme for the one-step purification of a clinically useful protein, human α1-antitrypsin, transiently expressed in N. benthamiana. With potential applicability to the variety of chromatography formats commercially available for IMAC-based protein purification, the Cysta-tag provides a convenient means for the efficient and cost-effective purification of recombinant proteins from plant tissues. PMID:26913045

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

    PubMed

    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.

  14. A glucose-derepressed promoter for expression of heterologous products in the filamentous fungus Aspergillus nidulans.

    PubMed

    Hintz, W E; Lagosky, P A

    1993-07-01

    We describe a putative binding sequence (GCGGGGC) for the glucose-responsive repressor protein CreA at two positions upstream of the transcription start site of the alcohol dehydrogenase I (alcA) gene of Aspergillus nidulans. To positively identify the putative binding sites as CreA-specific, the GCGGGGC blocks were mutated at five internal nucleotide positions to GTACTAC and reintroduced into the wild type alcA promoter driving expression of the endogenous alcohol dehydrogenase I gene. This CreA-binding site variant was then transformed into an AlcR constitutive A. nidulans host strain (T2625) and growth was monitored in the presence of the non-metabolized glucose analogue, 2-deoxyglucose. Positive transformants were selected by their ability to grow using ethanol as a carbon source in the presence of 2-deoxyglucose. Similar CreA binding site variant alcA promoters should permit the alcA-driven expression of heterologous genes in A. nidulans in the presence of glucose, the preferred carbon source for biomass accumulation and provides a model for controlling carbon-catabolite regulated expression in other expression systems.

  15. Genetic engineering and heterologous expression of the disorazol biosynthetic gene cluster via Red/ET recombineering

    PubMed Central

    Tu, Qiang; Herrmann, Jennifer; Hu, Shengbiao; Raju, Ritesh; Bian, Xiaoying; Zhang, Youming; Müller, Rolf

    2016-01-01

    Disorazol, a macrocyclic polykitide produced by the myxobacterium Sorangium cellulosum So ce12 and it is reported to have potential cytotoxic activity towards several cancer cell lines, including multi-drug resistant cells. The disorazol biosynthetic gene cluster (dis) from Sorangium cellulosum (So ce12) was identified by transposon mutagenesis and cloned in a bacterial artificial chromosome (BAC) library. The 58-kb dis core gene cluster was reconstituted from BACs via Red/ET recombineering and expressed in Myxococcus xanthus DK1622. For the first time ever, a myxobacterial trans-AT polyketide synthase has been expressed heterologously in this study. Expression in M. xanthus allowed us to optimize the yield of several biosynthetic products using promoter engineering. The insertion of an artificial synthetic promoter upstream of the disD gene encoding a discrete acyl transferase (AT), together with an oxidoreductase (Or), resulted in 7-fold increase in disorazol production. The successful reconstitution and expression of the genetic sequences encoding for these promising cytotoxic compounds will allow combinatorial biosynthesis to generate novel disorazol derivatives for further bioactivity evaluation. PMID:26875499

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

    PubMed

    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. PMID:23108028

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

    PubMed

    Yang, Jie; Xu, Xinqi; Ng, Tzi Bun; Lin, Juan; Ye, Xiuyun

    2016-01-01

    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. PMID:27527131

  18. A Chimeric Affinity Tag for Efficient Expression and Chromatographic Purification of Heterologous Proteins from Plants

    PubMed Central

    Sainsbury, Frank; Jutras, Philippe V.; Vorster, Juan; Goulet, Marie-Claire; Michaud, Dominique

    2016-01-01

    The use of plants as expression hosts for recombinant proteins is an increasingly attractive option for the production of complex and challenging biopharmaceuticals. Tools are needed at present to marry recent developments in high-yielding gene vectors for heterologous expression with routine protein purification techniques. In this study, we designed the Cysta-tag, a new purification tag for immobilized metal affinity chromatography (IMAC) of plant-made proteins based on the protein-stabilizing fusion partner SlCYS8. We show that the Cysta-tag may be used to readily purify proteins under native conditions, and then be removed enzymatically to isolate the protein of interest. We also show that commonly used protease recognition sites for linking purification tags are differentially stable in leaves of the commonly used expression host Nicotiana benthamiana, with those linkers susceptible to cysteine proteases being less stable then serine protease-cleavable linkers. As an example, we describe a Cysta-tag experimental scheme for the one-step purification of a clinically useful protein, human α1-antitrypsin, transiently expressed in N. benthamiana. With potential applicability to the variety of chromatography formats commercially available for IMAC-based protein purification, the Cysta-tag provides a convenient means for the efficient and cost-effective purification of recombinant proteins from plant tissues. PMID:26913045

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

    PubMed

    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. PMID:25681576

  20. Multicopy Integration and Expression of Heterologous Genes in Methylobacterium extorquens ATCC 55366†

    PubMed Central

    Choi, Young J.; Bourque, Denis; Morel, Lyne; Groleau, Denis; Míguez, Carlos B.

    2006-01-01

    High-level expression of chromosomally integrated genes in Methylobacterium extorquens ATCC 55366 was achieved under the control of the strong M. extorquens AM1 methanol dehydrogenase promoter (PmxaF) using the mini-Tn7 transposon system. Stable maintenance and expression of the integrated genes were obtained in the absence of antibiotic selective pressure. Furthermore, using this technology, a multicopy integration protocol for M. extorquens was also developed. Chromosomal integration of one to five copies of the gene encoding the green fluorescent protein (gfp) was achieved. The multicopy-based expression system permitted expression of a preset number of gene copies. A unique specific Tn7 integration locus in the chromosome of M. extorquens, known as the Tn7 attachment site (attTn7 site), was identified. This single attTn7 site was identified in an intergenic region between glmS, which encodes the essential enzyme glucosamine-6-phosphate synthetase, and dhaT, which encodes 1,3-propanediol dehydrogenase. The fact that the integration event is site specific and the fact that the attTn7 site is a noncoding region of the chromosome make the mini-Tn7 transposon system very useful for insertion of target genes and subsequent expression. In all transformants tested, expression and segregation of the transforming gene were stable without generation of secondary mutations in the host. In this paper, we describe single and multicopy chromosome integration and stable expression of heterologous genes (bgl [β-galactosidase], est [esterase], and gfp [green fluorescent protein]) in M. extorquens. PMID:16391115

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

    PubMed

    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.

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

    PubMed

    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. PMID:27138199

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

    PubMed

    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.

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

    PubMed

    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. PMID:25939548

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

    PubMed

    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.

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

    PubMed

    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. PMID:23516605

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

    PubMed

    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.

  8. Expression level tuning for optimal heterologous protein secretion in Saccharomyces cerevisiae.

    PubMed

    Parekh, R N; Wittrup, K D

    1997-01-01

    The relationship between expression level and secretion of bovine pancreatic trypsin inhibitor (BPTI) was determined in Saccharomyces cerevisiae using a tunable amplifiable delta integration vector. Optimal secretory productivity of 15 mg of BPTI/g cell dry weight yields 180 mg/L secreted active BPTI in test-tube cultures, an order of magnitude increase over 2 mu plasmid-directed secretion. Maximum productivity is determined by the protein folding capacity of the endoplasmic reticulum (ER). Unfolded protein accumulates in the ER as synthesis increases, until a physiological instability is reached and secretion decreases precipitously despite high BPTI mRNA levels. Optimal specific productivity of a standard laboratory strain of S. cerevisiae is double that reported for secretion of BPTI by Pichia pastoris, indicating that efficient utilization of S. cerevisiae's available secretory capacity can eliminate apparent differences among yeast species in their capacity for heterologous protein secretion. Although not generally recognized, the existence of an optimum synthesis level for secretion is apparently a general feature of eucaryotic expression systems and could be of substantial significance for maximization of protein secretion in mammalian and insect cell culture. PMID:9104035

  9. Bioethanol production by heterologous expression of Pdc and AdhII in Streptomyces lividans.

    PubMed

    Lee, Jae Sun; Chi, Won-Jae; Hong, Soon-Kwang; Yang, Ji-Won; Chang, Yong Keun

    2013-07-01

    Two genes from Zymomonas mobilis that are responsible for ethanol production, pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhII), were heterologously expressed in the Gram-positive bacterium Streptomyces lividans TK24. An examination of carbon distribution revealed that a significant portion of carbon metabolism was switched from biomass and organic acid biosynthesis to ethanol production upon the expression of pdc and adhII. The recombinant S. lividans TK24 produced ethanol from glucose with a yield of 23.7% based on the carbohydrate consumed. The recombinant was able to produce ethanol from xylose, L-arabinose, mannose, L-rhamnose, galactose, ribose, and cellobiose with yields of 16.0, 25.6, 21.5, 33.6, 30.6, 14.6, and 33.3%, respectively. Polymeric substances such as starch and xylan were directly converted to ethanol by the recombinant with ethanol yields of 18.9 and 8.8%, respectively. The recombinant S. lividans TK24/Tpet developed in this study is potentially a useful microbial resource for ethanol production from various sources of biomasses, especially microalgae.

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

    PubMed

    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.

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

    PubMed Central

    2011-01-01

    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. PMID:22204630

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

    PubMed

    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. PMID:26031839

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

    PubMed Central

    Melo, Sônia C.; Santos, Regineide X.; Melgaço, Ana C.; Pereira, Alanna C. F.; Pungartnik, Cristina; Brendel, Martin

    2015-01-01

    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. PMID:26039235

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

    PubMed

    Melo, Sônia C; Santos, Regineide X; Melgaço, Ana C; Pereira, Alanna C F; Pungartnik, Cristina; Brendel, Martin

    2015-06-01

    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.

  15. Heterologous expression and characterization of laccase 2 from Coprinopsis cinerea capable of decolourizing different recalcitrant dyes

    PubMed Central

    Tian, Yong-Sheng; Xu, Hu; Peng, Ri-He; Yao, Quan-Hong; Wang, Rong-Tan

    2014-01-01

    The gene (CcLcc2) encoding laccase from the basidiomycete Coprinopsis cinerea Okayama-7 #130 was synthesized by polymerase chain reaction-based two-step DNA synthesis, and heterologously expressed in Pichia pastoris. The recombinant protein was purified by ammonium sulphate precipitation and nickel nitrilotriacetic acid chromatography. The molecular mass of CcLcc2 was estimated to be 54 kDa by denaturing polyacrylamide gel electrophoresis. The optimum pH and temperature for laccase catalysis for the oxidation of 2,2ʹ-azino-bis(3-ethylbenzothiazoline-6-sulphonate) (ABTS) were 2.6 and 45 °C, respectively. The Km values of the enzyme towards the substrates ABTS, 2,6-dimethoxyphenol (2,6-DMP) and guaiacol were 0.93, 1.02 and 28.07 mmol·L−1, respectively. The decolourization of methyl orange, crystal violet and malachite green, commonly used in the textile industry, was assessed. The decolourization percentage of crystal violet and malachite green was 80% after 4 h of reaction, and that of methyl orange was 50% at 4 h. These results show that the CcLcc2 has enormous potential for the decolourization of highly stable triphenylmethane dyes. PMID:26019510

  16. Cloning, heterologous expression and properties of a recombinant active turnip peroxidase.

    PubMed

    Rodriguez-Cabrera, Norma A; Regalado, C; Garcia-Almendarez, Blanca E

    2011-07-13

    Turnip (Brassica napus) roots peroxidase isoforms have been used in diagnostic kits and can also efficiently polymerize phenolic compounds from wastewaters. Heterologous expression of a turnip acidic peroxidase (BnPA) was investigated to increase availability of this widely used enzyme. The mature BnPA was ligated into the pET28a(+) vector and used to transform Escherichia coli Rosetta 2. Recombinant BnPA peroxidase was overexpressed and accumulated in inclusion bodies from which it was purified to homogeneity by immobilized metal affinity chromatography under denaturing conditions. Peroxidase activity was observed after a refolding process under oxidative conditions. The yield of pure recombinant BnPA was 29 mg L(-1) of culture with a specific activity of 981 ± 20 ABTS units mg(-1) at optimal conditions (pH 6, 45 °C). Recombinant BnPA showed similar kinetic properties compared to native turnip peroxidase, and its secondary structure evaluated by circular dichroism comprised 20% α-helix, 32% β-sheet and 48% random structure. Recombinant BnPA showed high yield and good kinetic properties which are key steps for future structure-function studies and biotechnological applications. PMID:21591783

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

    PubMed

    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.

  18. Phage p1-derived artificial chromosomes facilitate heterologous expression of the FK506 gene cluster.

    PubMed

    Jones, Adam C; Gust, Bertolt; Kulik, Andreas; Heide, Lutz; Buttner, Mark J; Bibb, Mervyn J

    2013-01-01

    We describe a procedure for the conjugative transfer of phage P1-derived Artificial Chromosome (PAC) library clones containing large natural product gene clusters (≥70 kilobases) to Streptomyces coelicolor strains that have been engineered for improved heterologous production of natural products. This approach is demonstrated using the gene cluster for FK506 (tacrolimus), a clinically important immunosuppressant of high commercial value. The entire 83.5 kb FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488 present in one 130 kb PAC clone was introduced into four different S. coelicolor derivatives and all produced FK506 and smaller amounts of the related compound FK520. FK506 yields were increased by approximately five-fold (from 1.2 mg L(-1) to 5.5 mg L(-1)) in S. coelicolor M1146 containing the FK506 PAC upon over-expression of the FK506 LuxR regulatory gene fkbN. The PAC-based gene cluster conjugation methodology described here provides a tractable means to evaluate and manipulate FK506 biosynthesis and is readily applicable to other large gene clusters encoding natural products of interest to medicine, agriculture and biotechnology.

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

    PubMed

    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.

  20. Heterologous expression, purification and characterization of rat class theta glutathione transferase T2-2.

    PubMed Central

    Jemth, P; Stenberg, G; Chaga, G; Mannervik, B

    1996-01-01

    Rat glutathione transferase (GST) T2-2 of class Theta (rGST T2-2), previously known as GST 12-12 and GST Yrs-Yrs, has been heterologously expressed in Escherichia coli XLI-Blue. The corresponding cDNA was isolated from a rat hepatoma cDNA library, ligated into and expressed from the plasmid pKK-D. The sequence is the same as that of the previously reported cDNA of GST Yrs-Yrs. The enzyme was purified using ion-exchange chromatography followed by affinity chromatography with immobilized ferric ions, and the yield was approx. 200 mg from a 1 litre bacterial culture. The availability of a stable recombinant rGST T2-2 has paved the way for a more accurate characterization of the enzyme. The functional properties of the recombinant rGST T2-2 differ significantly from those reported earlier for the enzyme isolated from rat tissues. These differences probably reflect the difficulties in obtaining fully active enzyme from sources where it occurs in relatively low concentrations, which has been the case in previous studies. 1-Chloro-2,4-dinitrobenzene, a substrate often used with GSTs of classes Alpha, Mu and Pi, is a substrate also for rGST T2-2, but the specific activity is relatively low. The Km value for glutathione was determined with four different electrophiles and was found to be in the range 0.3 mM-0.8 mM. The Km values for some electrophilic substrates were found to be in the micromolar range, which is low compared with those determined for GSTs of other classes. The highest catalytic efficiency was obtained with menaphthyl sulphate, which gave a Kcat/Km value of 2.3 x 10(6) s-1.M-1 and a rate enhancement over the uncatalysed reaction of 3 x 10(10). PMID:8645195

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

    PubMed

    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. PMID:26792119

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

    PubMed

    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. PMID:27101009

  3. Heterologous expression and characterization of CYP61A1 from dandruff-causing Malassezia globosa.

    PubMed

    Ohk, Seul-Ong; Park, Hyoung-Goo; Lee, Hwayoun; Kwon, Yeo-Jung; Kim, Beom Joon; Kim, Donghak; Chun, Young-Jin

    2015-10-01

    Malassezia globosa is pathogenic fungus that causes skin disorders including dandruff in humans. Many yeast cytochrome CYP enzymes are involved in the biosynthesis of sterols and are considered major targets of azole antifungal agents. Here, we report on the expression and characterization of the MGL_0310 gene product (CYP61A1), a sterol C-22 desaturase in M. globosa. The open reading frame of the CYP61A1 gene was amplified by PCR from M. globosa CBS 7966 genomic DNA and cloned into a pCW vector. The CYP61A1 gene was heterologously expressed in Escherichia coli and purified using a Ni(2+)-NTA affinity column. The purified CYP61A1 protein exhibited a CO-difference spectrum typical of CYPs with a maximum absorption at 452nm. Binding spectral titration with β-sitosterol and campesterol demonstrated the type I binding mode with an increase at 411nm and a decrease at 432nm. The calculated Kd values are 5.4±0.6μM and 6.1±1.0μM for β-sitosterol and campesterol, respectively. No metabolic product, however, was observed in the CYP61A1-supported enzyme reaction with these sterols. The purified CYP61A1 protein exhibited tight binding to azole agents, suggesting that this enzyme may be a target for the pathogenic M. globosa fungus. Moreover, several fatty acids were found to bind to CYP61A1, indicating that the architecture of the enzyme includes a relatively large active site space. This study provides new insight into the biosynthesis of fungal sterols in M. globosa and a basis for the development of antifungal as potential therapeutic agents to treat dandruff. PMID:26160660

  4. Heterologous expression and enzymatic characterization of fructosyltransferase from Aspergillus niger in Pichia pastoris.

    PubMed

    Yang, Hailin; Wang, Yitian; Zhang, Ling; Shen, Wei

    2016-01-25

    In this work, the cDNA encoding fructosyltransferase (FTase) from Aspergillus niger YZ59 (CICIM F0901) was obtained and expressed in the methylotrophic yeast Pichia pastoris strain GS115. The yield of recombinant FTase in a 5-L fermentor reached 1020.0 U/mL after 96 h of induction, which was 1160.4 times higher that of native FTase from A. niger YZ59. The specific activity of recombinant FTase was 6.8×10(4) U/mg. The optimum temperature and pH of the recombinant FTase were 55 °C and 5.5, respectively. The recombinant FTase was stable below 40 °C and at pH from 3.0 to 10.0. Using sucrose as the substrate, the Km and Vmax values of recombinant FTase were 159.8 g/L and 0.66 g/(L min), respectively. The turnover number (kcat) and catalytic efficiency (kcat/Km) of recombinant FTase was 1.1×10(4) min(-1) and 68.8 L/(g min), respectively. The recombinant FTase was slightly activated by 5mM Ni(2+), Mg(2+), K(+), Fe(3+), or Mn(2+), but inhibited by all other metal ions (Na(+), Li(+), Ba(2+), Ca(2+), Zn(2+), and Cu(2+)). The highest yield of fructooligosaccharides for purified FTase reached approximately 343.3 g/L (w/v). This is the first study reporting the heterologous expression of FTases from A. niger in P. pastoris. This study plays an important role in the fructooligosaccharide synthesis industry by recombinant FTases.

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

    PubMed Central

    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. PMID:27101009

  6. Heterologous expression and functional characterization of a plant alkaline phytase in Pichia pastoris.

    PubMed

    Johnson, Steven C; Yang, Mimi; Murthy, Pushpalatha P N

    2010-12-01

    Phytases catalyze the sequential hydrolysis of phytic acid (myo-insositol hexakisphosphate), the most abundant inositol phosphate in cells. Phytic acid constitutes 3-5% of the dry weight of cereal grains and legumes such as corn and soybean. The high concentration of phytates in animal feed and the inability of non-ruminant animals such as swine and poultry to digest phytates leads to phosphate contamination of soil and water bodies. The supplementation of animal feed with phytases results in increased bioavailability to animals and decreased environmental contamination. Therefore, phytases are of great commercial importance. Phytases with a range of properties are needed to address the specific digestive needs of different animals. Alkaline phytase (LlALP1 and LlALP2) which possess unique catalytic properties that have the potential to be useful as feed and food supplement has been identified in lily pollen. Substantial quantities of alkaline phytase are needed for animal feed studies. In this paper, we report the heterologous expression of LlALP2 from lily pollen in Pichia pastoris. The expression of recombinant LlALP2 (rLlALP2) was optimized by varying the cDNA coding for LlALP2, host strain and growth conditions. The catalytic properties of recombinant LlALP2 were investigated extensively (substrate specificity, pH- and temperature dependence, and the effect of Ca(2+), EDTA and inhibitors) and found to be very similar to that of the native LlALP2 indicating that rLlALP2 from P. pastoris can serve as a potential source for structural and animal feed studies. PMID:20655385

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

    SciTech Connect

    Nakamura, Tomofumi; Ichinose, Hirofumi; Wariishi, Hiroyuki

    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.

  8. Heterologous expression and characterization of Bacillus coagulans L-arabinose isomerase.

    PubMed

    Zhou, Xingding; Wu, Jin Chuan

    2012-05-01

    Bacillus coagulans has been of great commercial interest over the past decade owing to its strong ability of producing optical pure L: -lactic acid from both hexose and pentose sugars including L: -arabinose with high yield, titer and productivity under thermophilic conditions. The L: -arabinose isomerase (L-AI) from Bacillus coagulans was heterologously over-expressed in Escherichia coli. The open reading frame of the L-AI has 1,422 nucleotides encoding a protein with 474 amino acid residues. The recombinant L-AI was purified to homogeneity by one-step His-tag affinity chromatography. The molecular mass of the enzyme was estimated to be 56 kDa by SDS-PAGE. The enzyme was most active at 70°C and pH 7.0. The metal ion Mn(2+) was shown to be the best activator for enzymatic activity and thermostability. The enzyme showed higher activity at acidic pH than at alkaline pH. The kinetic studies showed that the K (m), V (max) and k (cat)/K (m) for the conversion of L: -arabinose were 106 mM, 84 U/mg and 34.5 mM(-1)min(-1), respectively. The equilibrium ratio of L: -arabinose to L: -ribulose was 78:22 under optimal conditions. L: -ribulose (97 g/L) was obtained from 500 g/l of L: -arabinose catalyzed by the enzyme (8.3 U/mL) under the optimal conditions within 1.5 h, giving at a substrate conversion of 19.4% and a production rate of 65 g L(-1) h(-1).

  9. Effects of heterologous expression of phosphoenolpyruvate carboxykinase and phosphoenolpyruvate carboxylase on organic acid production in Aspergillus carbonarius.

    PubMed

    Yang, Lei; Lübeck, Mette; Lübeck, Peter S

    2015-11-01

    Aspergillus carbonarius has a potential as a cell factory for production of various organic acids. In this study, the organic acid profile of A. carbonarius was investigated under different cultivation conditions. Moreover, two heterologous genes, pepck and ppc, which encode phosphoenolpyruvate carboxykinase in Actinobacillus succinogenes and phosphoenolpyruvate carboxylase in Escherichia coli, were inserted individually and in combination in A. carbonarius to enhance the carbon flux toward the reductive TCA branch. Results of transcription analysis and measurement of enzyme activities of phosphoenolpyruvate carboxykinase and phosphoenolpyruvate carboxylase in the corresponding single and double transformants demonstrated that the two heterologous genes were successfully expressed in A. carbonarius. The production of citric acid increased in all the transformants in both glucose- and xylose-based media at pH higher than 3 but did not increase in the pH non-buffered cultivation compared with the wild type.

  10. Structural Diversification of Lyngbyatoxin A by Host-Dependent Heterologous Expression of the tleABC Biosynthetic Gene Cluster.

    PubMed

    Zhang, Lihan; Hoshino, Shotaro; Awakawa, Takayoshi; Wakimoto, Toshiyuki; Abe, Ikuro

    2016-08-01

    Natural products have enormous structural diversity, yet little is known about how such diversity is achieved in nature. Here we report the structural diversification of a cyanotoxin-lyngbyatoxin A-and its biosynthetic intermediates by heterologous expression of the Streptomyces-derived tleABC biosynthetic gene cluster in three different Streptomyces hosts: S. lividans, S. albus, and S. avermitilis. Notably, the isolated lyngbyatoxin derivatives, including four new natural products, were biosynthesized by crosstalk between the heterologous tleABC gene cluster and the endogenous host enzymes. The simple strategy described here has expanded the structural diversity of lyngbyatoxin A and its biosynthetic intermediates, and provides opportunities for investigation of the currently underestimated hidden biosynthetic crosstalk.

  11. Cloning and Heterologous Expression of the Thioviridamide Biosynthesis Gene Cluster from Streptomyces olivoviridis

    PubMed Central

    Izawa, Masumi; Kawasaki, Takashi

    2013-01-01

    Thioviridamide is a unique peptide antibiotic containing five thioamide bonds from Streptomyces olivoviridis. Draft genome sequencing revealed a gene (the tvaA gene) encoding the thioviridamide precursor peptide. The thioviridamide biosynthesis gene cluster was identified by heterologous production of thioviridamide in Streptomyces lividans. PMID:23995943

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

    PubMed

    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. PMID:23933077

  13. Comparison of seven different heterologous protein expression systems for the production of the serotonin transporter.

    PubMed

    Tate, Christopher G; Haase, Jana; Baker, Cara; Boorsma, Marco; Magnani, Francesca; Vallis, Yvonne; Williams, D Clive

    2003-02-17

    The rat serotonin transporter (rSERT) is an N-glycosylated integral membrane protein with 12 transmembrane regions; the N-glycans improve the ability of the SERT polypeptide chain to fold into a functional transporter, but they are not required for the transmembrane transport of serotonin per se. In order to define the best system for the expression, purification and structural analysis of serotonin transporter (SERT), we expressed SERT in Escherichia coli, Pichia pastoris, the baculovirus expression system and in four different stable mammalian cell lines. Two stable cell lines that constitutively expressed SERT (Imi270 and Coca270) were constructed using episomal plasmids in HEK293 cells expressing the EBNA-1 antigen. SERT expression in the three different inducible stable mammalian cell lines was induced either by a decrease in temperature (cell line pCytTS-SERT), the addition of tetracycline to the growth medium (cell line T-REx-SERT) or by adding DMSO which caused the cells to differentiate (cell line MEL-SERT). All the mammalian cell lines expressed functional SERT, but SERT expressed in E. coli or P. pastoris was nonfunctional as assessed by 5-hydroxytryptamine uptake and inhibitor binding assays. Expression of functional SERT in the mammalian cell lines was assessed by an inhibitor binding assay; the cell lines pCytTS-SERT, Imi270 and Coca270 contained levels of functional SERT similar to that of the standard baculovirus expression system (250,000 copies per cell). The expression of SERT in induced T-REx-SERT cells was 400,000 copies per cell, but in MEL-SERT it was only 80,000 copies per cell. All the mammalian stable cell lines expressed SERT at the plasma membrane as assessed by [3H]-5-hydroxytryptamine uptake into whole cells, but the V(max) for the T-Rex-SERT cell line was 10-fold higher than any of the other cell lines. It was noticeable that the cell lines that constitutively expressed SERT grew extremely poorly, compared to the inducible cell lines

  14. Bupropion-induced inhibition of α7 nicotinic acetylcholine receptors expressed in heterologous cells and neurons from dorsal raphe nucleus and hippocampus.

    PubMed

    Vázquez-Gómez, Elizabeth; Arias, Hugo R; Feuerbach, Dominik; Miranda-Morales, Marcela; Mihailescu, Stefan; Targowska-Duda, Katarzyna M; Jozwiak, Krzysztof; García-Colunga, Jesús

    2014-10-01

    The pharmacological activity of bupropion was compared between α7 nicotinic acetylcholine receptors expressed in heterologous cells and hippocampal and dorsal raphe nucleus neurons. The inhibitory activity of bupropion was studied on GH3-α7 cells by Ca2+ influx, as well as on neurons from the dorsal raphe nucleus and interneurons from the stratum radiatum of the hippocampal CA1 region by using a whole-cell voltage-clamp technique. In addition, the interaction of bupropion with the α7 nicotinic acetylcholine receptor was determined by [3H]imipramine competition binding assays and molecular docking. The fast component of acetylcholine- and choline-induced currents from both brain regions was inhibited by methyllycaconitine, indicating the participation of α7-containing nicotinic acetylcholine receptors. Choline-induced currents in hippocampal interneurons were partially inhibited by 10 µM bupropion, a concentration that could be reached in the brain during clinical administration. Additionally, both agonist-induced currents were reversibly inhibited by bupropion at concentrations that coincide with its inhibitory potency (IC50=54 µM) and binding affinity (Ki=63 µM) for α7 nicotinic acetylcholine receptors from heterologous cells. The [3H]imipramine competition binding and molecular docking results support a luminal location for the bupropion binding site(s). This study may help to understand the mechanisms of actions of bupropion at neuronal and molecular levels related with its therapeutic actions on depression and for smoking cessation.

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

    PubMed Central

    Vuoristo, Kiira S.; Mars, Astrid E.; van Loon, Stijn; Orsi, Enrico; Eggink, Gerrit; Sanders, Johan P. M.; Weusthuis, Ruud A.

    2015-01-01

    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 catalyzed 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 Escherichia coli with production titres up to 560 mg/L. PMID:26347730

  16. Heterologous expression of glycerol 3-phosphate dehydrogenase gene [DhGPD1] from the osmotolerant yeast Debaryomyces hansenii in Saccharomyces cerevisiae.

    PubMed

    Thomé, Patricia E

    2005-08-01

    The role for the gene encoding glycerol 3-phosphate dehydrogenase (DhGPD1) from the osmotolerant yeast Debaryomyces hansenii, in glycerol production and halotolerance, was studied through its heterologous expression in a Saccharomyces cerevisiae strain deficient in glycerol synthesis (gpd1Delta). The expression of the DhGPD1 gene in the gpd1Delta background restored glycerol production and halotolerance to wild type levels, corroborating its role in the salt-induced production of glycerol. Although the gene was functional in S. cerevisiae, its heterologous expression was not efficient, suggesting that the regulatory mechanism may not be shared by these two yeasts.

  17. Heterologous expression of antifreeze protein gene AnAFP from Ammopiptanthus nanus enhances cold tolerance in Escherichia coli and tobacco.

    PubMed

    Deng, Long-Qun; Yu, Hao-Qiang; Liu, Yan-Ping; Jiao, Pei-Pei; Zhou, Shu-Feng; Zhang, Su-Zhi; Li, Wan-Chen; Fu, Feng-Ling

    2014-04-10

    Antifreeze proteins are a class of polypeptides produced by certain animals, plants, fungi and bacteria that permit their survival under the subzero environments. Ammopiptanthus nanus is the unique evergreen broadleaf bush endemic to the Mid-Asia deserts. It survives at the west edge of the Tarim Basin from the disappearance of the ancient Mediterranean in the Tertiary Period. Its distribution region is characterized by the arid climate and extreme temperatures, where the extreme temperatures range from -30 °C to 40 °C. In the present study, the antifreeze protein gene AnAFP of A. nanus was used to transform Escherichia coli and tobacco, after bioinformatics analysis for its possible function. The transformed E. coli strain expressed the heterologous AnAFP gene under the induction of isopropyl β-D-thiogalactopyranoside, and demonstrated significant enhancement of cold tolerance. The transformed tobacco lines expressed the heterologous AnAFP gene in response to cold stress, and showed a less change of relative electrical conductivity under cold stress, and a less wilting phenotype after 16 h of -3 °C cold stress and thawing for 1h than the untransformed wild-type plants. All these results imply the potential value of the AnAFP gene to be used in genetic modification of commercially important crops for improvement of cold tolerance. PMID:24502990

  18. Heterologous expression of antifreeze protein gene AnAFP from Ammopiptanthus nanus enhances cold tolerance in Escherichia coli and tobacco.

    PubMed

    Deng, Long-Qun; Yu, Hao-Qiang; Liu, Yan-Ping; Jiao, Pei-Pei; Zhou, Shu-Feng; Zhang, Su-Zhi; Li, Wan-Chen; Fu, Feng-Ling

    2014-04-10

    Antifreeze proteins are a class of polypeptides produced by certain animals, plants, fungi and bacteria that permit their survival under the subzero environments. Ammopiptanthus nanus is the unique evergreen broadleaf bush endemic to the Mid-Asia deserts. It survives at the west edge of the Tarim Basin from the disappearance of the ancient Mediterranean in the Tertiary Period. Its distribution region is characterized by the arid climate and extreme temperatures, where the extreme temperatures range from -30 °C to 40 °C. In the present study, the antifreeze protein gene AnAFP of A. nanus was used to transform Escherichia coli and tobacco, after bioinformatics analysis for its possible function. The transformed E. coli strain expressed the heterologous AnAFP gene under the induction of isopropyl β-D-thiogalactopyranoside, and demonstrated significant enhancement of cold tolerance. The transformed tobacco lines expressed the heterologous AnAFP gene in response to cold stress, and showed a less change of relative electrical conductivity under cold stress, and a less wilting phenotype after 16 h of -3 °C cold stress and thawing for 1h than the untransformed wild-type plants. All these results imply the potential value of the AnAFP gene to be used in genetic modification of commercially important crops for improvement of cold tolerance.

  19. Heterologous expression of plant vacuolar pyrophosphatase in yeast demonstrates sufficiency of the substrate-binding subunit for proton transport

    SciTech Connect

    Kim, E.J.; Zhen, R.G.; Rea, P.A. )

    1994-06-21

    The membrane bounding the vacuole of plant cells contains two electrogenic proton pumps. These are the vacuolar H[sup +]-ATPase (EC 3.6.1.3), an enzyme common to all eukaryotes, and a vacuolar H[sup +]-translocating pyrophosphatase (EC 3.6.1.1), which is ubiquitous in plants but otherwise known in only a few phototrophic bacteria. Although the substrate-binding subunit of the vacuolar H[sup +]-pyrophosphatase has been identified and purified and cDNAs encoding it have been isolated and characterized, the minimal unit competent in pyrophosphate (PPi)-energized H[sup +] translocation is not known. Here the authors address this question and show that heterologous expression of the cDNA (AVP) encoding the substrate-binding subunit of the vacuolar H[sup +]-pyrophosphatase from the vascular plant Arabidopsis thaliana in the yeast Saccharomyces cerevisiae results in the production of vacuolarly localized functional enzyme active in PPi-dependent H[sup +] translocation. Since the heterologously expressed pump is indistinguishable from the native plant enzyme with respect to PPi hydrolysis, H[sub +] translocation, activation by potassium, and selective inhibition by calcium and 1,1-diphosphonates, it is concluded that all of the known catalytic functions of the enzyme map to the one subunit encoded by AVP.

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

    PubMed

    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.

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

    PubMed

    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

    2015-09-18

    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.

  2. Simple and rapid direct cloning and heterologous expression of natural product biosynthetic gene cluster in Bacillus subtilis via Red/ET recombineering

    PubMed Central

    Liu, Qingshu; Shen, Qiyao; Bian, Xiaoying; Chen, Hanna; Fu, Jun; Wang, Hailong; Lei, Ping; Guo, Zhaohui; Chen, Wu; Li, Dingjun; Zhang, Youming

    2016-01-01

    Heterologous expression of biosynthetic pathways is an important way to research and discover microbial natural products. Bacillus subtilis is a suitable host for the heterologous production of natural products from bacilli and related Firmicutes. Existing technologies for heterologous expression of large biosynthetic gene clusters in B. subtilis are complicated. Herein, we present a simple and rapid strategy for direct cloning based heterologous expression of biosynthetic pathways in B. subtilis via Red/ET recombineering, using a 5.2 kb specific direct cloning vector carrying homologous sequences to the amyE gene in B. subtilis and CcdB counterselection marker. Using a two-step procedure, two large biosynthetic pathways for edeine (48.3 kb) and bacillomycin (37.2 kb) from Brevibacillus brevis X23 and B. amyloliquefaciens FZB42, respectively, were directly cloned and subsequently integrated into the chromosome of B. subtilis within one week. The gene cluster for bacillomycin was successfully expressed in the heterologous host, although edeine production was not detectable. Compared with similar technologies, this method offers a simpler and more feasible system for the discovery of natural products from bacilli and related genera. PMID:27687863

  3. Functional identification of the glycerol permease activity of Arabidopsis thaliana NLM1 and NLM2 proteins by heterologous expression in Saccharomyces cerevisiae.

    PubMed

    Weig, A R; Jakob, C

    2000-09-22

    NLM proteins (NOD26-like major intrinsic proteins) from plants contain amino acid sequence signatures which can be found in aquaporins including plant plasma membrane intrinsic proteins and tonoplast intrinsic proteins and glycerol permeases such as the Escherichia coli GlpF and the yeast FPS1 proteins. Heterologous expression of two members of the NLM subgroup from Arabidopsis thaliana (AtNLM1 and AtNLM2) in baker's yeast demonstrated the glycerol permease activity in addition to the previously described aquaporin activity of AtNLM1. The transport was non-saturable up to 100 mM extracellular glycerol concentration. Longer-chain sugar alcohols did not compete with the transport of radiolabelled glycerol and hexoses were also not transported through the pore.

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

    PubMed

    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

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

    PubMed

    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

  6. Molecular Cloning and Heterologous Expression of the Dehydrophos Biosynthetic Gene Cluster

    PubMed Central

    Circello, Benjamin T.; Eliot, Andrew C.; Lee, Jin-Hee; van der Donk, Wilfred A.; Metcalf, William W.

    2010-01-01

    Summary Dehydrophos is a vinyl phosphonate tripeptide produced by Streptomyces luridus with demonstrated broad spectrum antibiotic activity. To identify genes necessary for biosynthesis of this unusual compound we screened a fosmid library of S. luridus for the presence of the phosphoenolpyruvate mutase gene, which is required for biosynthesis of most phosphonates. Integration of one such fosmid clone into the chromosome of Streptomyces lividans led to heterologous production of dehydrophos. Deletion analysis of this clone allowed identification of the minimal contiguous dehydrophos cluster, which contained 17 open reading frames (ORFs). Bioinformatic analyses of these ORFs are consistent with a proposed biosynthetic pathway that generates dehydrophos from phosphoenolpyruvate. The early steps of this pathway are supported by analysis of intermediates accumulated by blocked mutants and in vitro biochemical experiments. PMID:20416511

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

    PubMed Central

    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. PMID:26716454

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

    PubMed

    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. PMID:26716454

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

    PubMed

    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. PMID:26904091

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

    PubMed Central

    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. PMID:26904091

  11. Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production.

    PubMed

    Daly, Rachel; Hearn, Milton T W

    2005-01-01

    The use of the methylotrophic yeast, Pichia pastoris, as a cellular host for the expression of recombinant proteins has become increasing popular in recent times. P. pastoris is easier to genetically manipulate and culture than mammalian cells and can be grown to high cell densities. Equally important, P. pastoris is also a eukaryote, and thereby provides the potential for producing soluble, correctly folded recombinant proteins that have undergone all the post-translational modifications required for functionality. Additionally, linearized foreign DNA can be inserted in high efficiency via homologous recombination procedures to generate stable cell lines whilst expression vectors can be readily prepared that allow multiple copies of the target protein, multimeric proteins with different subunit structures, or alternatively the target protein and its cognate binding partners, to be expressed. A further benefit of the P. pastoris system is that strong promoters are available to drive the expression of a foreign gene(s) of interest, thus enabling production of large amounts of the target protein(s) with relative technical ease and at a lower cost than most other eukaryotic systems. The purpose of this review is to summarize important developments and features of this expression system and, in particular, to examine from an experimental perspective the genetic engineering, protein chemical and molecular design considerations that have to be taken into account for the successful expression of the target recombinant protein. Included in these considerations are the influences of P. pastoris strain selection; the choice of expression vectors and promoters; procedures for the transformation and integration of the vectors into the P. pastoris genome; the consequences of rare codon usage and truncated transcripts; and techniques employed to achieve multi-copy integration numbers. The impact of the alcohol oxidase (AOX) pathways in terms of the mut+ and mut(s) phenotypes

  12. Molecular basis for chloronium-mediated meroterpene cyclization: cloning, sequencing, and heterologous expression of the napyradiomycin biosynthetic gene cluster.

    PubMed

    Winter, Jaclyn M; Moffitt, Michelle C; Zazopoulos, Emmanuel; McAlpine, James B; Dorrestein, Pieter C; Moore, Bradley S

    2007-06-01

    Structural inspection of the bacterial meroterpenoid antibiotics belonging to the napyradiomycin family of chlorinated dihydroquinones suggests that the biosynthetic cyclization of their terpenoid subunits is initiated via a chloronium ion. The vanadium-dependent haloperoxidases that catalyze such reactions are distributed in fungi and marine algae and have yet to be characterized from bacteria. The cloning and sequence analysis of the 43-kb napyradiomycin biosynthetic cluster (nap) from Streptomyces aculeolatus NRRL 18422 and from the undescribed marine sediment-derived Streptomyces sp. CNQ-525 revealed 33 open reading frames, three of which putatively encode vanadium-dependent chloroperoxidases. Heterologous expression of the CNQ-525-based nap biosynthetic cluster in Streptomyces albus produced at least seven napyradiomycins, including the new analog 2-deschloro-2-hydroxy-A80915C. These data not only revealed the molecular basis behind the biosynthesis of these novel meroterpenoid natural products but also resulted in the first in vivo verification of vanadium-dependent haloperoxidases.

  13. Luminmycins A-C, cryptic natural products from Photorhabdus luminescens identified by heterologous expression in Escherichia coli.

    PubMed

    Bian, Xiaoying; Plaza, Alberto; Zhang, Youming; Müller, Rolf

    2012-09-28

    The 18 kb "silent" luminmycin biosynthetic pathway from Photorhabdus luminescens was cloned into a vector by using the newly established linear-linear homologous recombination and successfully expressed in Escherichia coli. Luminmycins A-C (1-3) were isolated from the heterologous host, and their structures were elucidated using 2D NMR spectroscopy and HRESIMS. Luminmycin A is a deoxy derivative of the previously reported glidobactin A, while luminmycins B and C most likely represent its acyclic biosynthetic intermediates. Compound 1 showed cytotoxicity against the human colon carcinoma HCT-116 cell line with an IC(50) value of 91.8 nM, while acyclic 2 was inactive at concentrations as high as 100 μg/mL. PMID:22909174

  14. [Heterologous expression, purification, and properties of a chymotrypsin inhibitor isolated from potatoes].

    PubMed

    2013-01-01

    The PKPIJ-B gene encoding a chymotrypsin inhibitor from a subfamily of potato Kunitz-type proteinase inhibitors (PKPI) in potatoes (Solanum tuberosum L. cv. Yubilei Zhukova) was cloned into a pET23a vector and then expressed in Escherichia coli. The recombinant PKPIJ-B protein obtained in the inclusion bodies was denatured, purified by high-performance liquid chromatography (HPLC) on Mono Q under denaturing conditions, and renaturated. The renaturated protein was additionally purified using HPLC on DEAE-ToyoPearl. The PKPIJ-B protein efficiently suppressed chymotrypsin activity, had a weaker effect on trypsin, and inhibited the growth and development of phytopathogenic microorganisms affecting potato plants. PMID:23662448

  15. Heterologous expression of Arabidopsis phytochrome B in transgenic potato influences photosynthetic performance and tuber development

    SciTech Connect

    Thiele, A.; Herold, M.; Lenk, I.; Gatz, C. . Albrecht von Haller Inst. fuer Pflanzenwissenschaften); Quail, P.H. )

    1999-05-01

    Transgenic potato (Solanum tuberosum) plants expressing Arabidopsis phytochrome B were characterized morphologically and physiologically under white light in a greenhouse to explore their potential for improved photosynthesis and higher tuber yields. As expected, overexpression of functional phytochrome B caused pleiotropic effects such as semidwarfism, decreased apical dominance, a higher number of smaller but thicker leaves, and increased pigmentation. Because of increased numbers of chloroplasts in elongated palisade cells, photosynthesis per leaf area and in each individual plant increased. In addition, photosynthesis was less sensitive to photoinactivation under prolonged light stress. The beginning of senescence was not delayed, but deceleration of chlorophyll degradation extended the lifetime of photosynthetically active plants. Both the higher photosynthetic performance and the longer lifespan of the transgenic plants allowed greater biomass production, resulting in extended underground organs with increased tuber yields.

  16. Biochemical and EPR-Spectroscopic Investigation into Heterologously Expressed Vinyl Chloride Reductive Dehalogenase (VcrA) from Dehalococcoides mccartyi Strain VS

    PubMed Central

    Parthasarathy, Anutthaman; Stich, Troy A.; Lohner, Svenja T.; Lesnefsky, Ann; Britt, R. David; Spormann, Alfred M.

    2015-01-01

    Reductive dehalogenases play a critical role in the microbial detoxification of aquifers contaminated with chloroethenes and chlorethanes by catalyzing the reductive elimination of a halogen. We report here the first heterologous production of vinyl chloride reductase VcrA from Dehalococcoides mccartyi strain VS. Heterologously expressed VcrA was reconstituted to its active form by addition of hydroxocobalamin/adenosylcobalamin, Fe3+, and sulfide in the presence of mercaptoethanol. The kinetic properties of reconstituted VcrA catalyzing vinyl chloride reduction with Ti(III)-citrate as reductant and methyl viologen as mediator were similar to those obtained previously for VcrA as isolated from D. mccartyi strain VS. VcrA was also found to catalyze a novel reaction, the environmentally important dihaloelimination of 1,2-dichloroethane to ethene. Electron paramagnetic resonance (EPR) spectroscopic studies with reconstituted VcrA in the presence of mercaptoethanol revealed the presence of Cob(II)alamin. Addition of Ti(III)-citrate resulted in the appearance of a new signal characteristic of a reduced [4Fe–4S] cluster and the disappearance of the Cob(II)alamin signal. UV–vis absorption spectroscopy of Ti(III)citrate-treated samples revealed the formation of two new absorption maxima characteristic of Cob(I)alamin. No evidence for the presence of a [3Fe–4S] cluster was found. We postulate that during the reaction cycle of VcrA, a reduced [4Fe–4S] cluster reduces Co(II) to Co(I) of the enzyme-bound cobalamin. Vinyl chloride reduction to ethene would be initiated when Cob(I)alamin transfers an electron to the substrate, generating a vinyl radical as a potential reaction intermediate. PMID:25686300

  17. [Heterologous expression and enzymatic analysis of Streptomyces griseus trypsin in Streptomyces lividans].

    PubMed

    Ma, Tengbo; Ling, Zhenmin; Kang, Zhen; Li, Jianghua; Du, Guocheng; Chen, Jian

    2013-04-01

    Trypsin as an important serine protease has been widely used in food, pharmaceutical and tanning industries. In this study, we successfully expressed trypsin (cloning from Streptomyces griseus ATCC10137) in Streptomyces lividans TK24 and comparatively investigated its enzymatic properties. Specifically, applying S. griseus ATCC 10137 genome as template, we obtained the sprT gene and sub-cloned it into the expression plasmid pIJ86, generating the recombinant strain S. lividans TK24/pIJ86-sprT. When cultivated in R2YE and SELF, the activity of rSGT reached 9.21 U/mL and 8.61 U/mL, respectively. Meanwhile, the results of the enzymatic analysis showed that rSGT exhibited a higher acid tolerance and a higher specificity to hydrolyze amide bonds compared with bovine trypsin (BT). In addition, Zn2+ and organic solvents up-regulated esterase and amidase of rSGT. Taken together, the results obtained herein provide meaningful information for further modification of rSGT and its industrial application.

  18. Functional Analysis of the Chloroplast Division Complex Using Schizosaccharomyces pombe as a Heterologous Expression System.

    PubMed

    TerBush, Allan D; Porzondek, Chris A; Osteryoung, Katherine W

    2016-04-01

    Chloroplast division is driven by a macromolecular complex that assembles at the midplastid. The FtsZ ring (Z ring) is the central structure in this complex, and is composed of the functionally distinct cytoskeletal proteins FtsZ1 and FtsZ2. Recent studies in the heterologous Schizosaccharomyces pombe system showed that Arabidopsis FtsZ1 and FtsZ2 filaments have distinct assembly and turnover characteristics. To further analyze these FtsZs, we employed this system to compare the assembly and dynamic properties of FtsZ1 and FtsZ2 lacking their N- and/or C-termini with those of their full-length counterparts. Our data provide evidence that the N-terminus of FtsZ2 is critical for its structural dominance over FtsZ1, and that the N- and C-termini promote polymer bundling and turnover of both FtsZs and contribute to their distinct behaviors. We also assessed how ARC6 affects FtsZ2 filament dynamics, and found that it interacts with and stabilizes FtsZ2 filaments in S. pombe independent of its presumed Z-ring tethering function in planta. Finally, we generated FtsZ1-FtsZ2 coexpression constructs to facilitate reconstitution of more complex interaction networks. Our experiments yield new insight into factors influencing FtsZ behavior and highlight the utility of S. pombe for analyzing chloroplast FtsZs and their assembly regulators.

  19. Heterologous expression of VHb can improve the yield and quality of biocontrol fungus Paecilomyces lilacinus, during submerged fermentation.

    PubMed

    Zhang, Shumeng; Wang, Jieping; Wei, Yale; Tang, Qing; Ali, Maria Kanwal; He, Jin

    2014-10-10

    Paecilomyces lilacinus is an egg-parasitic fungus which is effective against plant-parasitic nematodes and it has been successfully commercialized for the control of many plant-parasitic nematodes. However, during the large-scale industrial fermentation process of the filamentous fungus, the dissolved oxygen supply is a limiting factor, which influences yield, product quality and production cost. To solve this problem, we intended to heterologously express VHb in P. lilacinus ACSS. After optimizing the vgb gene, we fused it with a selection marker gene nptII, a promoter PgpdA and a terminator TtrpC. The complete expression cassette PgpdA-nptII-vgb-TtrpC was transferred into P. lilacinus ACSS by Agrobacterium tumefaciens-mediated transformation. Consequently, we successfully screened an applicable fungus strain PNVT8 which efficiently expressed VHb. The submerged fermentation experiments demonstrated that the expression of VHb not only increased the production traits of P. lilacinus such as biomass and spore production, but also improved the beneficial product quality and application value, due to the secretion of more protease and chitinase. It can be speculated that the recombinant strain harboring vgb gene will have a growth advantage over the original strain under anaerobic conditions in soil and therefore will possess higher biocontrol efficiency against plant-parasitic nematodes.

  20. Two novel heat shock genes encoding proteins produced in response to heterologous protein expression in Escherichia coli.

    PubMed

    Allen, S P; Polazzi, J O; Gierse, J K; Easton, A M

    1992-11-01

    In Escherichia coli high-level production of some heterologous proteins (specifically, human prorenin, renin, and bovine insulin-like growth factor 2) resulted in the induction of two new E. coli heat shock proteins, both of which have molecular masses of 16 kDa and are tightly associated with inclusion bodies formed during heterologous protein production. We named these inclusion body-associated proteins IbpA and IbpB. The coding sequences for IbpA and IbpB were identified and isolated from the Kohara E. coli gene bank. The genes for these proteins (ibpA and ibpB) are located at 82.5 min on the chromosome. Nucleotide sequencing of the two genes revealed that they are transcribed in the same direction and are separated by 110 bp. Putative Shine-Dalgarno sequences are located upstream from the initiation codons of both genes. A putative heat shock promoter is located upstream from ibpA, and a putative transcription terminator is located downstream from ibpB. A temperature upshift experiment in which we used a wild-type E. coli strain and an isogenic rpoH mutant strain indicated that a sigma 32-containing RNA polymerase is involved in the regulation of expression of these genes. There is 57.5% identity between the genes at the nucleotide level and 52.2% identity at the amino acid level. A search of the protein data bases showed that both of these 16-kDa proteins exhibit low levels of homology to low-molecular-weight heat shock proteins from eukaryotic species.

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

    PubMed Central

    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

  2. Sensitivities of Two Zebrafish TRPA1 Paralogs to Chemical and Thermal Stimuli Analyzed in Heterologous Expression Systems.

    PubMed

    Oda, Mai; Kurogi, Mako; Kubo, Yoshihiro; Saitoh, Osamu

    2016-03-01

    Transient receptor potential A1 (TRPA1) is the only member of the mouse, chick, and frog TRPA family, whereas 2 paralogs (zTRPA1a and zTRPA1b) are present in zebrafish. We herein investigated functional differences in the 2 zebrafish TRPA1s. HEK293T cells were used as heterologous expression systems, and the sensitivities of these cells to 4 chemical irritants (allyl isothiocyanate [AITC], caffeine, auto-oxidized epigallocatechin gallate [EGCG], and hydrogen peroxide [H2O2]) were compared with Ca(2+) imaging techniques. Sensitivities to the activators for AITC, oxidized EGCG, and H2O2 were higher in cells expressing zTRPA1a than in those expressing zTRPA1b, whereas caffeine appeared to activate both cells equally. We also characterized the thermal sensitivity of Xenopus oocytes expressing each TRPA1 electrophysiologically using a 2-electrode voltage clamp. Although endogenous currents induced by a cold stimulation were observed in control oocytes in some batches, oocytes expressing zTRPA1b showed significantly stronger cold- and heat-induced responses. However, significant thermal activation was not observed in oocytes expressing zTRPA1a. The results obtained using in vitro expression systems suggest that zTRPA1a is specialized for chemical sensing, whereas zTRPA1b responds to thermal stimuli. Furthermore, characterization of the chimeric molecule of TRPA1a and 1b revealed the importance of the N-terminal region in chemical and thermal sensing by zTRPA1s.

  3. Heterologous expression and biochemical and functional characterization of a recombinant alpha-type myotoxin inhibitor from Bothrops alternatus snake.

    PubMed

    Santos-Filho, Norival A; Boldrini-França, Johara; Santos-Silva, Ludier K; Menaldo, Danilo L; Henrique-Silva, Flávio; Sousa, Tiago S; Cintra, Adélia C O; Mamede, Carla C N; Oliveira, Fábio; Arantes, Eliane C; Antunes, Lusânia M Greggi; Cilli, Eduardo M; Sampaio, Suely V

    2014-10-01

    Venomous and non-venomous snakes possess phospholipase A2 (PLA2) inhibitory proteins (PLIs) in their blood serum. This study shows the expression and biochemical and functional characterization of a recombinant alpha inhibitor from Bothrops alternatus snake, named rBaltMIP. Its expression was performed in Pichia pastoris heterologous system, resulting in an active recombinant protein. The expressed inhibitor was tested regarding its ability to inhibit the phospholipase activity of different PLA2s, showing slight inhibitions especially at the molar ratios of 1:1 and 1:3 (PLA2:PLI). rBaltMIP was also effective in decreasing the myotoxic activity of the tested toxins at molar ratios greater than 1:0.4 (myotoxin:PLI). The inhibition of the myotoxic activity of different Asp49 (BthTX-II and PrTX-III) and Lys49 (BthTX-I and PrTX-I) myotoxins was also performed without the prior incubation of myotoxins/inhibitor in order to analyze the real possibility of using snake plasma inhibitors or recombinant inhibitors as therapeutic agents for treating envenomations. As a result, rBaltMIP was able to significantly inhibit the myotoxicity of Lys49 myotoxins. Histopathological analysis of the gastrocnemius muscles of mice showed that the myotoxins are able to induce severe damage to the muscle fibers of experimental animals by recruiting a large number of leukocyte infiltrates, besides forming an intense accumulation of intercellular fluid, leading to local edema. When those myotoxins were incubated with rBaltMIP, a reduction of the damage site could be observed. Furthermore, the cytotoxic activity of Asp49 PLA2s and Lys49 PLA2-like enzymes on C2C12 cell lines was decreased, as shown by the higher cell viabilities after preincubation with rBaltMIP. Heterologous expression would enable large-scale obtainment of rBaltMIP, thus allowing further investigations for the elucidation of possible mechanisms of inhibition of snake PLA2s, which have not yet been fully clarified. PMID:25047442

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

    PubMed

    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.

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

    PubMed

    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. PMID:27190148

  6. Development of an Escherichia coli-Lactobacillus casei shuttle vector for heterologous protein expression in Lactobacillus casei.

    PubMed

    Suebwongsa, Namfon; Lulitanond, Viraphong; Mayo, Baltasar; Yotpanya, Panjamaporn; Panya, Marutpong

    2016-01-01

    There is an increasing interest to develop various lactic acid bacteria (LAB) species as mucosal delivery vehicles, for which the development of a variety of cloning and expression systems for these bacteria is of primary importance. This study reports the complete nucleotide sequence of the cryptic plasmid pRCEID7.6 derived from the chicken probiotic LAB strain Lactobacillus casei TISTR1341. Sequence analysis and comparison showed that pRCEID7.6 is composed of nine putative open reading frames. The replicon origin of pRCEID7.6 consisted of untranslated origin of replication and translated replication protein B sequences. This region was used to construct Escherichia coli/L. casei shuttle vectors carrying erythromycin and chloramphenicol resistance genes as selective markers. Segregation and structural stability of the vectors in L. casei was sufficient for most genetic applications. The feasibility of this vector for heterologous protein expression in L. casei was determined by cloning in pRCEID-LC7.6, the gene encoding the nucleocapsid protein (NP), from the influenza A virus under the control of the homologous promoter from the lactate dehydrogenase gene. L. casei carrying this recombinant plasmid was shown to successfully express the NP protein. Therefore, this shuttle vector can be used for further study in the development of mucosal delivery vehicles. PMID:27026866

  7. Heterologous expression of rat epitope-tagged histamine H2 receptors in insect Sf9 cells

    PubMed Central

    Beukers, M W; Klaassen, C H W; De Grip, W J; Verzijl, D; Timmerman, H; Leurs, R

    1997-01-01

    Rat histamine H2 receptors were epitope-tagged with six histidine residues at the C-terminus to allow immunological detection of the receptor. Recombinant baculoviruses containing the epitope-tagged H2 receptor were prepared and were used to infect insect Sf9 cells. The His-tagged H2 receptors expressed in insect Sf9 cells showed typical H2 receptor characteristics as determined with [125I]-aminopotentidine (APT) binding studies. In Sf9 cells expressing the His-tagged H2 receptor histamine was able to stimulate cyclic AMP production 9 fold (EC50=2.1±0.1 μM) by use of the endogenous signalling pathway. The classical antagonists cimetidine, ranitidine and tiotidine inhibited histamine induced cyclic AMP production with Ki values of 0.60±0.43 μM, 0.25±0.15 μM and 28±7 nM, respectively (mean±s.e.mean, n=3). The expression of the His-tagged H2 receptors in infected Sf9 cells reached functional levels of 6.6±0.6 pmol mg−1 protein (mean±s.e.mean, n=3) after 3 days of infection. This represents about 2×106 copies of receptor/cell. Preincubation of the cells with 0.03 mM cholesterol-β-cyclodextrin complex resulted in an increase of [125I]-APT binding up to 169±5% (mean±s.e.mean, n=3). The addition of 0.03 mM cholesterol-β-cyclodextrin complex did not affect histamine-induced cyclic AMP production. The EC50 value of histamine was 3.1±1.7 μM in the absence of cholesterol-β-cyclodextrin complex and 11.1±5.5 μM in the presence of cholesterol-β-cyclodextrin complex (mean±s.e.mean, n=3). Also, the amount of cyclic AMP produced in the presence of 100 μM histamine was identical, 85±18 pmol/106 cells in the absence and 81±11 pmol/106 cells in the presence of 0.03 mM cholesterol-β-cyclodextrin complex (mean±s.e.mean, n=3). Immunofluorescence studies with an antibody against the His-tag revealed that the majority of the His-tagged H2 receptors was localized inside the insect Sf9 cells, although plasma membrane labelling could be

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

    PubMed

    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.

  9. Optimization of the production and purification processes of carnobacteriocins Cbn BM1 and Cbn B2 from Carnobacterium maltaromaticum CP5 by heterologous expression in Escherichia coli.

    PubMed

    Jasniewski, Jordane; Cailliez-Grimal, Catherine; Gelhaye, Eric; Revol-Junelles, Anne-Marie

    2008-04-01

    An optimization of the production and purification processes of carnobacteriocins Cbn BM1 and Cbn B2 from Carnobacterium maltaromaticum CP5, by heterologous expression in Escherichia coli is described. The genes encoding mature bacteriocin were cloned into an E. coli expression system and expressed as a fusion protein with a thermostable thioredoxin. Recombinant E. coli were cultivated following a fed-batch fermentation process with pH, temperature and oxygenation regulation. The overexpression of the fusion proteins was improved by replacing IPTG by lactose. The fusion proteins were purified by thermal coagulation followed by affinity chromatography. The thioredoxin fusion protein was removed by using CNBr instead of enterokinase and the carnobacteriocins were recovered by reverse-phase chromatography. These optimizations led us to produce up to 320 mg of pure protein per liter of culture, which is four to ten fold higher than what is described for other heterologous expression systems.

  10. Heterologously-expressed and Liposome-reconstituted Human Transient Receptor Potential Melastatin 4 Channel (TRPM4) is a Functional Tetramer

    PubMed Central

    Constantine, Maryrose; Liew, Chu Kong; Lo, Victor; Macmillan, Alex; Cranfield, Charles G.; Sunde, Margaret; Whan, Renee; Graham, Robert M.; Martinac, Boris

    2016-01-01

    Mutation, irregular expression and sustained activation of the Transient Receptor Potential Channel, type Melastatin 4 (TRPM4), have been linked to various cardiovascular diseases. However, much remains unknown about the structure of this important ion channel. Here, we have purified a heterologously expressed TRPM4-eGFP fusion protein and investigated the oligomeric state of TRPM4-eGFP in detergent micelles using crosslinking, native gel electrophoresis, multi-angle laser light scattering and electron microscopy. Our data indicate that TRPM4 is tetrameric, like other TRP channels studied to date. Furthermore, the functionality of liposome reconstituted TRPM4-eGFP was examined using electrophysiology. Single-channel recordings from TRPM4-eGFP proteoliposomes showed inhibition of the channel using Flufenamic acid, a well-established inhibitor of TRPM4, suggesting that the channels are functional upon reconstitution. Our characterisation of the oligomeric structure of TRPM4 and the ability to reconstitute functional channels in liposomes should facilitate future studies into the structure, function and pharmacology of this therapeutically relevant channel. PMID:26785754

  11. Heterologously-expressed and Liposome-reconstituted Human Transient Receptor Potential Melastatin 4 Channel (TRPM4) is a Functional Tetramer.

    PubMed

    Constantine, Maryrose; Liew, Chu Kong; Lo, Victor; Macmillan, Alex; Cranfield, Charles G; Sunde, Margaret; Whan, Renee; Graham, Robert M; Martinac, Boris

    2016-01-01

    Mutation, irregular expression and sustained activation of the Transient Receptor Potential Channel, type Melastatin 4 (TRPM4), have been linked to various cardiovascular diseases. However, much remains unknown about the structure of this important ion channel. Here, we have purified a heterologously expressed TRPM4-eGFP fusion protein and investigated the oligomeric state of TRPM4-eGFP in detergent micelles using crosslinking, native gel electrophoresis, multi-angle laser light scattering and electron microscopy. Our data indicate that TRPM4 is tetrameric, like other TRP channels studied to date. Furthermore, the functionality of liposome reconstituted TRPM4-eGFP was examined using electrophysiology. Single-channel recordings from TRPM4-eGFP proteoliposomes showed inhibition of the channel using Flufenamic acid, a well-established inhibitor of TRPM4, suggesting that the channels are functional upon reconstitution. Our characterisation of the oligomeric structure of TRPM4 and the ability to reconstitute functional channels in liposomes should facilitate future studies into the structure, function and pharmacology of this therapeutically relevant channel. PMID:26785754

  12. Cross Protection against Influenza A Virus by Yeast-Expressed Heterologous Tandem Repeat M2 Extracellular Proteins.

    PubMed

    Lee, Yu-Na; Kim, Min-Chul; Lee, Young-Tae; Hwang, Hye Suk; Lee, Jongsang; Kim, Cheol; Kang, Sang-Moo

    2015-01-01

    The influenza M2 ectodomain (M2e) is well conserved across human influenza A subtypes, but there are few residue changes among avian and swine origin influenza A viruses. We expressed a tandem repeat construct of heterologous M2e sequences (M2e5x) derived from human, swine, and avian origin influenza A viruses using the yeast expression system. Intramuscular immunization of mice with AS04-adjuvanted M2e5x protein vaccines was effective in inducing M2e-specific antibodies reactive to M2e peptide and native M2 proteins on the infected cells with human, swine, or avian influenza virus, mucosal and systemic memory cellular immune responses, and cross-protection against H3N2 virus. Importantly, M2e5x immune sera were found to confer protection against different subtypes of H1N1 and H5N1 influenza A viruses in naïve mice. Also, M2e5x-immune complexes of virus-infected cells stimulated macrophages to secrete cytokines via Fc receptors, indicating a possible mechanism of protection. The present study provides evidence that M2e5x proteins produced in yeast cells could be developed as a potential universal influenza vaccine. PMID:26366729

  13. Characterization of Heterologously Expressed Acetyl Xylan Esterase1 Isolated from the Anaerobic Rumen Fungus Neocallimastix frontalis PMA02

    PubMed Central

    Kwon, Mi; Song, Jaeyong; Park, Hong-Seog; Park, Hyunjin; Chang, Jongsoo

    2016-01-01

    Acetyl xylan esterase (AXE), which hydrolyzes the ester linkages of the naturally acetylated xylan and thus known to have an important role for hemicellulose degradation, was isolated from the anaerobic rumen fungus Neocallimastix frontatlis PMA02, heterologously expressed in Escherichi coli (E.coli) and characterized. The full-length cDNA encoding NfAXE1 was 1,494 bp, of which 978 bp constituted an open reading frame. The estimated molecular weight of NfAXE1 was 36.5 kDa with 326 amino acid residues, and the calculated isoelectric point was 4.54. The secondary protein structure was predicted to consist of nine α-helixes and 12 β-strands. The enzyme expressed in E.coli had the highest activity at 40°C and pH 8. The purified recombinant NfAXE1 had a specific activity of 100.1 U/mg when p-nitrophenyl acetate (p-NA) was used as a substrate at 40°C, optimum temperature. The amount of liberated acetic acids were the highest and the lowest when p-NA and acetylated birchwood xylan were used as substrates, respectively. The amount of xylose released from acetylated birchwod xylan was increased by 1.4 fold when NfAXE1 was mixed with xylanase in a reaction cocktail, implying a synergistic effect of NfAXE1 with xylanase on hemicellulose degradation. PMID:27383808

  14. The effects of a stimulating intron on the expression of heterologous genes in Arabidopsis thaliana.

    PubMed

    Emami, Shahram; Arumainayagam, Dinah; Korf, Ian; Rose, Alan B

    2013-06-01

    Introns are often added to transgenes to increase expression, although the mechanism through which introns stimulate gene expression in plants and other eukaryotes remains mysterious. While introns vary in their effect on expression, it is unknown whether different genes respond similarly to the same stimulatory intron. Furthermore, the degree to which gene regulation is preserved when expression is increased by an intron has not been thoroughly investigated. To test the effects of the same intron on the expression of a range of genes, GUS translational fusions were constructed using the promoters of eight Arabidopsis genes whose expression was reported to be constitutive (GAE1, CNGC2 and ROP10), tissue specific (ADL1A, YAB3 and AtAMT2) or regulated by light (ULI3 and MSBP1). For each gene, a fusion containing the first intron from the UBQ10 gene was compared to fusions containing the gene's endogenous first intron (if the gene has one) or no intron. In every case, the UBQ10 intron increased expression relative to the intronless control, although the magnitude of the change and the level of expression varied. The UBQ10 intron also changed the expression patterns of the CNGC2 and YAB3 fusions to include strong activity in roots, indicating that tissue specificity was disrupted by this intron. In contrast, the regulation of the ULI3 and MSBP1 genes by light was preserved when their expression was stimulated by the intron. These findings have important implications for biotechnology applications in which a high level of transgene expression in only certain tissues is desired.

  15. Secretory expression of a heterologous protein, Aiio-AIO6BS, in Bacillus subtilis via a non-classical secretion pathway.

    PubMed

    Pan, Xingliang; Yang, Yalin; Liu, Xuewei; Li, Dong; Li, Juan; Guo, Xiaoze; Zhou, Zhigang

    2016-09-16

    The quenching enzyme AIO6 (AiiO-AIO6) has been reported as a feed additive preparation for application in aquaculture and biological control of pathogenic Aeromonas hydrophila. We developed an economical strategy to express AIO6BS (AiiO-AIO6BS, codon optimized AIO6 in Bacillus subtilis) in Bacillus subtilis for facilitating its widespread application. Promoter p43 without the signal peptide was used for secretory expression of AIO6BS in B. subtilis. Western blotting analysis demonstrated that AIO6BS was successfully expressed and secreted into the cell culture. Expression analysis of AIO6BS in the single or double mutant of the lytC and lytD genes for cell autolysis in B. subtilis 1A751 and cell autolysis-resistant engineered strain LM2531 derived from the wild type 168 indicated that the release of the heterologous protein AIO6BS was not simply mediated by cell lysis. Expression level of AIO6BS did not change in the mutants of B. subtilis that harbored mutations in the secA, tatAC, or ecsA genes compared with that in the parent wild type strain. These results suggested the AIO6BS protein was likely secreted via a non-classical secretion pathway. The expression analysis of the various N- or C-terminal truncated gene products indicated that AIO6BS probably acts as an export signal to direct its self-secretion across the cell membrane. PMID:27514447

  16. Construction of a novel twin-arginine translocation (Tat)-dependent type expression vector for secretory production of heterologous proteins in Corynebacterium glutamicum.

    PubMed

    Zhang, Lirong; Jia, Huimin; Xu, Daqing

    2015-11-01

    Corynebacterium glutamicum is recognized as a favorable host for the secretory production of heterologous proteins. However, there are few secretion-type expression vectors available for protein production in C. glutamicum. In this study, we constructed a shuttle expression vector pAU3, which harbors the strong promoter tac-M for constitutive gene transcription, the consensus RBS sequence for protein translation, and the strong cgR_0949 signal sequence for protein secretion via the Tat pathway in C. glutamicum. The applicability of pAU3 was confirmed by the highly efficient expression and secretion of the CAT protein in C. glutamicum. The vector pAU3 is highly useful for secretory production of heterologous proteins in C. glutamicum.

  17. A ubiquitous splice variant and a common polymorphism affect heterologous expression of recombinant human SCN5A heart sodium channels.

    PubMed

    Makielski, Jonathan C; Ye, Bin; Valdivia, Carmen R; Pagel, Matthew D; Pu, Jielin; Tester, David J; Ackerman, Michael J

    2003-10-31

    Amino acid sequence variations in SCN5A are known to affect function of wild-type channels and also those with coexisting mutations; therefore, it is important to know the exact sequence and function of channels most commonly present in human myocardium. SCN5A was analyzed in control panels of human alleles, demonstrating that the existing clones (hH1, hH1a, hH1b) each contained a rare variant and thus none represented the common sequence. Confirming prior work, the H558R polymorphism was present in approximately 30% of subjects. Quantitative mRNA analysis from human hearts showed that a shorter 2015 amino acid splice variant lacking glutamine at position 1077 (Q1077del) made up 65% of the transcript in every heart examined. Age, sex, race, or structural heart disease did not affect this proportion of Q1077del. Estimated population frequencies for the four common variants were 25% SCN5A, 10% [H558R], 45% [Q1077del], and 20% [H558R;Q1077del], where the reference sequence SCN5A is GenBank AC137587. When expressed in HEK-293 cells, these common variants had a more positive mid-point of the voltage dependence of inactivation than the standard clone hH1. Also, channels containing Q1077 expressed smaller currents. When H558R was present with Q1077 ([H558R]), current expression was profoundly reduced despite normal trafficking to the cell surface. Thus, four variant sequences for SCN5A are commonly present in human myocardium and they exhibit functional differences among themselves and with the previous standard clone. These results have implications for the choice of background sequence for experiments with heterologous expression systems, and possibly implications for electrophysiological function in vivo. PMID:14500339

  18. Isolated yeast promoter sequence and a method of regulated heterologous expression

    DOEpatents

    Gao, Johnway; Skeen, Rodney S.; Hooker, Brian S.; Anderson, Daniel B.

    2005-05-31

    The present invention provides the promoter clone discovery of a glucoamylase gene of a starch utilizing yeast strain Schwanniomyces castellii. The isolated glucoamylase promoter is an inducible promoter, which can regulate strong gene expression in starch culture medium.

  19. Isotopic labeling of mammalian G protein-coupled receptors (GPCRs) heterologously expressed in Caenorhabditis elegans*

    PubMed Central

    Salom, David; Cao, Pengxiu; Yuan, Yiyuan; Miyagi, Masaru; Feng, Zhaoyang; Palczewski, Krzysztof

    2015-01-01

    High-resolution structural determination and dynamic characterization of membrane proteins by nuclear magnetic resonance (NMR) require their isotopic labeling. Although a number of labeled eukaryotic membrane proteins have been successfully expressed in bacteria, they lack posttranslational modifications and usually need to be refolded from inclusion bodies. This shortcoming of bacterial expression systems is particularly detrimental for the functional expression of G protein-coupled receptors (GPCRs), the largest family of drug targets, due to their inherent instability. In this work we show that proteins expressed by a eukaryotic organism can be isotopically labeled and produced with a quality and quantity suitable for NMR characterization. Using our previously described expression system in Caenorhabditis elegans, we showed the feasibility of labeling proteins produced by these worms with 15N,13C by providing them with isotopically labeled bacteria. 2H labeling also was achieved by growing C. elegans in presence of 70% heavy water. Bovine rhodopsin, simultaneously expressed in muscular and neuronal worm tissues, was employed as the ‘test’ GPCR to demonstrate the viability of this approach. Although the worms’ cell cycle was slightly affected by the presence of heavy isotopes, the final protein yield and quality was appropriate for NMR structural characterization. PMID:25461480

  20. Expression of an Engineered Heterologous Antimicrobial Peptide in Potato Alters Plant Development and Mitigates Normal Abiotic and Biotic Responses

    PubMed Central

    Goyal, Ravinder K.; Hancock, Robert E. W.; Mattoo, Autar K.; Misra, Santosh

    2013-01-01

    Antimicrobial cationic peptides (AMPs) are ubiquitous small proteins used by living cells to defend against a wide spectrum of pathogens. Their amphipathic property helps their interaction with negatively charged cellular membrane of the pathogen causing cell lysis and death. AMPs also modulate signaling pathway(s) and cellular processes in animal models; however, little is known of cellular processes other than the pathogen-lysis phenomenon modulated by AMPs in plants. An engineered heterologous AMP, msrA3, expressed in potato was previously shown to cause resistance of the transgenic plants against selected fungal and bacterial pathogens. These lines together with the wild type were studied for growth habits, and for inducible defense responses during challenge with biotic (necrotroph Fusarium solani) and abiotic stressors (dark-induced senescence, wounding and temperature stress). msrA3-expression not only conferred protection against F. solani but also delayed development of floral buds and prolonged vegetative phase. Analysis of select gene transcript profiles showed that the transgenic potato plants were suppressed in the hypersensitive (HR) and reactive oxygen species (ROS) responses to both biotic and abiotic stressors. Also, the transgenic leaves accumulated lesser amounts of the defense hormone jasmonic acid upon wounding with only a slight change in salicylic acid as compared to the wild type. Thus, normal host defense responses to the pathogen and abiotic stressors were mitigated by msrA3 expression suggesting MSRA3 regulates a common step(s) of these response pathways. The stemming of the pathogen growth and mitigating stress response pathways likely contributes to resource reallocation for higher tuber yield. PMID:24147012

  1. Expression of an engineered heterologous antimicrobial peptide in potato alters plant development and mitigates normal abiotic and biotic responses.

    PubMed

    Goyal, Ravinder K; Hancock, Robert E W; Mattoo, Autar K; Misra, Santosh

    2013-01-01

    Antimicrobial cationic peptides (AMPs) are ubiquitous small proteins used by living cells to defend against a wide spectrum of pathogens. Their amphipathic property helps their interaction with negatively charged cellular membrane of the pathogen causing cell lysis and death. AMPs also modulate signaling pathway(s) and cellular processes in animal models; however, little is known of cellular processes other than the pathogen-lysis phenomenon modulated by AMPs in plants. An engineered heterologous AMP, msrA3, expressed in potato was previously shown to cause resistance of the transgenic plants against selected fungal and bacterial pathogens. These lines together with the wild type were studied for growth habits, and for inducible defense responses during challenge with biotic (necrotroph Fusarium solani) and abiotic stressors (dark-induced senescence, wounding and temperature stress). msrA3-expression not only conferred protection against F. solani but also delayed development of floral buds and prolonged vegetative phase. Analysis of select gene transcript profiles showed that the transgenic potato plants were suppressed in the hypersensitive (HR) and reactive oxygen species (ROS) responses to both biotic and abiotic stressors. Also, the transgenic leaves accumulated lesser amounts of the defense hormone jasmonic acid upon wounding with only a slight change in salicylic acid as compared to the wild type. Thus, normal host defense responses to the pathogen and abiotic stressors were mitigated by msrA3 expression suggesting MSRA3 regulates a common step(s) of these response pathways. The stemming of the pathogen growth and mitigating stress response pathways likely contributes to resource reallocation for higher tuber yield.

  2. Heterologous expression of FMDV immunodominant epitopes and HSP70 in P. pastoris and the subsequent immune response in mice.

    PubMed

    Su, Chunxia; Duan, Xiangguo; Wang, Xiuqing; Wang, Chen; Cao, Rubing; Zhou, Bin; Chen, Puyan

    2007-10-01

    Mycobacterium tuberculosis heat shock protein70 (HSP70) is a major antigen with both chaperone and cytokine functions. It has been used as an adjuvant to induce or potentiate humoral and cellular immunity, both in the form of a mixture with peptide antigens, and as a fusion protein. We have evaluated the effects of HSP70 on foot and mouth virus (FMDV) subunit vaccines. FMDV VP1, and a synthetic multi-epitope FMDV (EG), and VP1-HSP70 and EG-HSP70 fusion proteins were all heterologously expressed in the yeast Pichia pastoris, and used as antigen in mice. The recombinant VP1 and EG alone was able to induce both humoral and marginal cell-mediated immune responses, while the HSP70 fusions markedly enhanced both the humoral and cell-mediated immune responses. The most prominent immune responses arose from vaccination with the EG-HSP70 fusion product. Both fusion protein-induced Th1-like cytokine (IFN-gamma) and Th2-like cytokine (IL-4) were identified.

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

    PubMed

    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.

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

    PubMed

    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.

  5. Identification of the thiazolyl peptide GE37468 gene cluster from Streptomyces ATCC 55365 and heterologous expression in Streptomyces lividans.

    PubMed

    Young, Travis S; Walsh, Christopher T

    2011-08-01

    Thiazolyl peptides are bacterial secondary metabolites that potently inhibit protein synthesis in Gram-positive bacteria and malarial parasites. Recently, our laboratory and others reported that this class of trithiazolyl pyridine-containing natural products is derived from ribosomally synthesized preproteins that undergo a cascade of posttranslational modifications to produce architecturally complex macrocyclic scaffolds. Here, we report the gene cluster responsible for production of the elongation factor Tu (EF-Tu)-targeting 29-member thiazolyl peptide GE37468 from Streptomyces ATCC 55365 and its heterologous expression in the model host Streptomyces lividans. GE37468 harbors an unusual β-methyl-δ-hydroxy-proline residue that may increase conformational rigidity of the macrocycle and impart reduced entropic costs of target binding. Isotope feeding and gene knockout were employed in the engineered S. lividans strain to identify the P450 monooxygenase GetJ as the enzyme involved in posttranslational transformation of isoleucine 8 to β-methyl-δ-hydroxy-proline through a predicted tandem double hydroxylation/cyclization mechanism. Loss of Ile8 oxygenative cyclization or mutation of Ile8 to alanine via preprotein gene replacement resulted in a 4-fold and 2-fold drop in antibiotic activity, respectively. This report of genetic manipulation of a 29-member thiazolyl peptide sets the stage for further genetic examination of structure activity relationships in the EF-Tu targeting class of thiazolyl peptides.

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

    PubMed

    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.

  7. Heterologous expression of xylanase enzymes in lipogenic yeast Yarrowia lipolytica

    SciTech Connect

    Wang, Wei; Wei, Hui; Alahuhta, Markus; Chen, Xiaowen; Hyman, Deborah; Johnson, David K.; Zhang, Min; Himmel, Michael E.

    2014-12-02

    In order to develop a direct microbial sugar conversion platform for the production of lipids, drop-in fuels and chemicals from cellulosic biomass substrate, we chose Yarrowia lipolytica as a viable demonstration strain. Y. lipolytica is known to accumulate lipids intracellularly and is capable of metabolizing sugars to produce lipids; however, it lacks the lignocellulose-degrading enzymes needed to break down biomass directly. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica. The XynII and XlnD expressing Yarrowia strains exhibited an ability to grow on xylan mineral plates. This was shown by Congo Red staining of halo zones on xylan mineral plates. Enzymatic activity tests further demonstrated active expression of XynII and XlnD in Y. lipolytica. Furthermore, synergistic action in converting xylan to xylose was observed when XlnD acted in concert with XynII. Finally, the successful expression of these xylanases in Yarrowia further advances us toward our goal to develop a direct microbial conversion process using this organism.

  8. Heterologous expression of codon optimized Trichoderma reesei Cel6A in Pichia pastoris.

    PubMed

    Sun, Fubao Fuelbiol; Bai, Renhui; Yang, Huimin; Wang, Fei; He, Jing; Wang, Chundi; Tu, Maobing

    2016-10-01

    The Cel6A deficiency has become one of the limiting factors for cellulose saccharification in biochemical conversion of cellulosic biomass to fuels and chemicals. The work attempted to use codon optimization to enhance Trichoderma reesei Cel6A expression in Pichia pastoris. Two recombinants P. pastoris GS115 containing AOX1 and GAP promotors were successfully constructed, respectively. The optimal temperatures and pHs of the expressed Cel6A from two recombinants were consistent with each other, were also in the extremely similar range to that reported on the native Cel6A from T. reesei. Based on the shake flask fermentation, AOX1 promotor enabled the recombinant to produce 265U/L and 300mg/L of the Cel6A enzyme, and the GAP promotor resulted in 145U/L and 200mg/L. High cell density fed batch (HCDFB) fermentation significantly improved the enzyme titer (1100U/L) and protein yield (2.0g/L) for the recombinant with AOX1 promotor. Results have showed that the AOX1 promotor is more suitable than the GAP for the Cel6A expression in P. pastoris. And the HCDFB cultivation is a favorable way to express the Cel6A highly in the methanol inducible yeast. PMID:27542751

  9. Heterologous expression of codon optimized Trichoderma reesei Cel6A in Pichia pastoris.

    PubMed

    Sun, Fubao Fuelbiol; Bai, Renhui; Yang, Huimin; Wang, Fei; He, Jing; Wang, Chundi; Tu, Maobing

    2016-10-01

    The Cel6A deficiency has become one of the limiting factors for cellulose saccharification in biochemical conversion of cellulosic biomass to fuels and chemicals. The work attempted to use codon optimization to enhance Trichoderma reesei Cel6A expression in Pichia pastoris. Two recombinants P. pastoris GS115 containing AOX1 and GAP promotors were successfully constructed, respectively. The optimal temperatures and pHs of the expressed Cel6A from two recombinants were consistent with each other, were also in the extremely similar range to that reported on the native Cel6A from T. reesei. Based on the shake flask fermentation, AOX1 promotor enabled the recombinant to produce 265U/L and 300mg/L of the Cel6A enzyme, and the GAP promotor resulted in 145U/L and 200mg/L. High cell density fed batch (HCDFB) fermentation significantly improved the enzyme titer (1100U/L) and protein yield (2.0g/L) for the recombinant with AOX1 promotor. Results have showed that the AOX1 promotor is more suitable than the GAP for the Cel6A expression in P. pastoris. And the HCDFB cultivation is a favorable way to express the Cel6A highly in the methanol inducible yeast.

  10. Heterologous expression and functional characterization of matrix metalloproteinase-11 from canine mammary tumor.

    PubMed

    Sunil Kumar, B V; Kumar, K Aswani; Padmanath, K; Sharma, Bhaskar; Kataria, Meena

    2013-01-01

    Matrix metalloproteinases (MMPs) are reported to be involved in tumor growth, apoptosis, angiogenesis, invasion, and development of metastases. These are zinc containing metalloproteases, known for their role in extracellular matrix degradation. MMP-11 (stromelysin3) is reported to be highly expressed in breast cancer, therefore it may act as marker enzyme for breast cancer progression. The present work was carried out to produce recombinant canine (Canis lupus familiaris) MMP-11 lacking the signal and propeptide in E. coli by optimizing its expression and purification in biologically active form and to functionally characterize it. A bacterial protein expression vector pPROEX HTc was used. The MMP-11 mature peptide encoding gene was successfully cloned and expressed in E. coli and the purified recombinant enzyme was found to be functionally active. The recombinant enzyme exhibited caseinolytic activity and could be activated by Trypsin and 4-Amino phenyl mercuric acetate (APMA). However Ethylene diamine tertra acetate (EDTA) inhibited the enzyme's caseinolytic activity. The recombinant enzyme degraded extracellular matrix constituents and facilitated migration of MDCK (Madin-Darby canine kidney) cells through BD Biocoat Matrigel invasion chambers. These results suggest that in vivo MMP-11 could play a significant role in the turnover of extracellular matrix constituents. PMID:23394368

  11. Cloning of a yeast alpha-amylase promoter and its regulated heterologous expression

    DOEpatents

    Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR; Hooker, Brian S [Kennewick, WA; Anderson, Daniel B [Pasco, WA

    2003-04-01

    The present invention provides the promoter clone discovery of an alpha-amylase gene of a starch utilizing yeast strain Schwanniomyces castellii. The isolated alpha-amylase promoter is an inducible promoter, which can regulate strong gene expression in starch culture medium.

  12. Properties of wild-type and fluorescent protein-tagged mouse tetrodotoxin-resistant sodium channel (Na V 1.8) heterologously expressed in rat sympathetic neurons.

    PubMed

    Schofield, Geoffrey G; Puhl, Henry L; Ikeda, Stephen R

    2008-04-01

    The tetrodotoxin (TTX)-resistant Na(+) current arising from Na(V)1.8-containing channels participates in nociceptive pathways but is difficult to functionally express in traditional heterologous systems. Here, we show that injection of cDNA encoding mouse Na(V)1.8 into the nuclei of rat superior cervical ganglion (SCG) neurons results in TTX-resistant Na(+) currents with amplitudes equal to or exceeding the currents arising from natively expressing channels of mouse dorsal root ganglion (DRG) neurons. The activation and inactivation properties of the heterologously expressed Na(V)1.8 Na(+) channels were similar but not identical to native TTX-resistant channels. Most notably, the half-activation potential of the heterologously expressed Na(V)1.8 channels was shifted about 10 mV toward more depolarized potentials. Fusion of fluorescent proteins to the N- or C-termini of Na(V)1.8 did not substantially affect functional expression in SCG neurons. Unexpectedly, fluorescence was not concentrated at the plasma membrane but found throughout the interior of the neuron in a granular pattern. A similar expression pattern was observed in nodose ganglion neurons expressing the tagged channels. In contrast, expression of tagged Na(V)1.8 in HeLa cells revealed a fluorescence pattern consistent with sequestration in the endoplasmic reticulum, thus providing a basis for poor functional expression in clonal cell lines. Our results establish SCG neurons as a favorable surrogate for the expression and study of molecularly defined Na(V)1.8-containing channels. The data also indicate that unidentified factors may be required for the efficient functional expression of Na(V)1.8 with a biophysical phenotype identical to that found in sensory neurons. PMID:18272876

  13. Successful expression of heterologous egfp gene in the mitochondria of a photosynthetic eukaryote Chlamydomonas reinhardtii.

    PubMed

    Hu, Zhangli; Zhao, Zhonglin; Wu, Zhihua; Fan, Zhun; Chen, Jun; Wu, Jinxia; Li, Jiancheng

    2011-09-01

    The efficient expression of exogenous gene in mitochondria of photosynthetic organism has been an insurmountable problem. In this study, the pBsLPNCG was constructed by inserting the egfp gene into a site between TERMINVREP-Left repeats and the cob gene in a fragment of mitochondrial DNA of Chlamydomonas reinhardtii CC-124 and introduced into the mitochondria of respiratory deficient dum-1 mutation of C. reinhardtii CC-2654. Sequencing and DNA Southern analyses revealed that egfp gene had been integrated into the mitochondrial genome of transgenic algae as expected and no other copy of egfp existed in their nucleic genome. Both the fluorescence detection and Western blot analysis confirmed the presence of eGFP protein in the transgenic algae; it indicated that the egfp gene was successfully expressed in the mitochondria of C. reinhardtii. PMID:21664493

  14. Cloning and heterologous expression of Plasmodium ovale dihydrofolate reductase-thymidylate synthase gene

    PubMed Central

    Tirakarn, Srisuda; Riangrungroj, Pinpunya; Kongsaeree, Palangpon; Imwong, Mallika; Yuthavong, Yongyuth; Leartsakulpanich, Ubolsree

    2012-01-01

    Plasmodial bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a validated antimalarial drug target. In this study, expression of the putative dhfr-ts of Plasmodium ovale rescued the DHFR chemical knockout and a TS null bacterial strain, demonstrating its DHFR and TS catalytic functions. PoDHFR-TS was expressed in Escherichia coli BL21 (DE3) and affinity purified by Methotrexate Sepharose column. Biochemical and enzyme kinetics characterizations indicated that PoDHFR-TS is similar to other plasmodial enzymes, albeit with lower catalytic activity but better tolerance of acidic pH. Importantly, the PoDHFR from Thai isolate EU266602 remains sensitive to the antimalarials pyrimethamine and cycloguanil, in contrast to P. falciparum and P. vivax isolates where resistance to these drugs is widespread. PMID:22234170

  15. Heterologous expression of AtPAP2 in transgenic potato influences carbon metabolism and tuber development.

    PubMed

    Zhang, Youjun; Sun, Feng; Fettke, Joerg; Schöttler, Mark Aurel; Ramsden, Lawrence; Fernie, Alisdair R; Lim, Boon Leong

    2014-10-16

    Changes in carbon flow and sink/source activities can affect floral, architectural, and reproductive traits of plants. In potato, overexpression (OE) of the purple acid phosphatase 2 of Arabidopsis (AtPAP2) resulted in earlier flowering, faster growth rate, increased tubers and tuber starch content, and higher photosynthesis rate. There was a significant change in sucrose, glucose and fructose levels in leaves, phloem and sink biomass of the OE lines, consistent with an increased expression of sucrose transporter 1 (StSUT1). Furthermore, the expression levels and enzyme activity of sucrose-phosphate synthase (SPS) were also significantly increased in the OE lines. These findings strongly suggest that higher carbon supply from the source and improved sink strength can improve potato tuber yield.

  16. Heterologous Expression of Hepatitis C Virus Core Protein in Oil Seeds of Brassica napus L.

    PubMed Central

    Mohammadzadeh, Sara; Roohvand, Farzin; Ajdary, Soheila; Ehsani, Parastoo; Hatef Salmanian, Ali

    2015-01-01

    Background: Hepatitis c virus (HCV), prevalent among 3% of the world population, is a major worldwide public health concern and an effective vaccination could help to overcome this problem. Plant seeds as low-cost vaccine expression platforms are highly desirable to produce antigens. Objectives: The present study was aimed at investigating the possible expression of recombinant HCV core protein, as a leading HCV vaccine candidate, in canola (Brassica napus) plant seeds in order to be used as an effective immunogen for vaccine researches. Materials and Methods: A codon-optimized gene harboring the Kozak sequence, 6 × His-tag, HCVcp (1 - 122 residues) and KDEL (Lys-Asp-Glu-Leu) peptide in tandem was designed and expressed under the control of the seed specific promoter, fatty acid elongase 1 (FAE1), to accumulate the recombinant protein in canola (B. napus L.) seeds. Transgenic lines were screened and the presence of the transgene was confirmed in the T0 plants by polymerase chain reaction (PCR). The quantity and quality of the HCV core protein (HCVcp) in transgenic seeds were evaluated by enzyme-linked immunosorbent assay (ELISA) and western blot, respectively. Results: Western blot analysis using anti-His antibody confirmed the presence of a 15 kDa protein in the seeds of T1 transgenic lines. The amount of antigenic protein accumulated in the seeds of these transgenic lines was up to 0.05% of the total soluble protein (TSP). Conclusions: The canola oilseeds could provide a useful expression system to produce HCV core protein as a vaccine candidate. PMID:26855744

  17. Heterologous expression of betaine aldehyde dehydrogenase gene from Ammopiptanthus nanus confers high salt and heat tolerance to Escherichia coli.

    PubMed

    Yu, Hao-Qiang; Wang, Ying-Ge; Yong, Tai-Ming; She, Yue-Hui; Fu, Feng-Ling; Li, Wan-Chen

    2014-10-01

    Betaine aldehyde dehydrogenase (BADH) catalyzes the synthesis of glycine betaine, a regulator of osmosis, and therefore BADH is considered to play a significant role in response of plants to abiotic stresses. Here, based on the conserved residues of the deduced amino acid sequences of the homologous BADH genes, we cloned the AnBADH gene from the xerophytic leguminous plant Ammopiptanthus nanus by using reverse transcription PCR and rapid amplification of cDNA ends. The full-length cDNA is 1,868 bp long without intron, and contains an open reading frame of 1512 bp, and 3'- and 5'-untranslated regions of 294 and 62 bp. It encodes a 54.71 kDa protein of 503 amino acids. The deduced amino acid sequence shares high homology, conserved amino acid residues and sequence motifs crucial for the function with the BADHs in other leguminous species. The sequence of the open reading frame was used to construct a prokaryotic expression vector pET32a-AnBADH, and transform Escherichia coli. The transformants expressed the heterologous AnBADH gene under the induction of isopropyl β-D-thiogalactopyranoside, and demonstrated significant enhancement of salt and heat tolerance under the stress conditions of 700 mmol L(-1) NaCl and 55°C high temperature. This result suggests that the AnBADH gene might play a crucial role in adaption of A. nanus to the abiotic stresses, and have the potential to be applied to transgenic operations of commercially important crops for improvement of abiotic tolerance. PMID:25046139

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

    PubMed

    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. PMID:26615395

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

    PubMed

    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.

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

    PubMed

    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.

  1. Heterologous Expression of Lysergic Acid and Novel Ergot Alkaloids in Aspergillus fumigatus

    PubMed Central

    Robinson, Sarah L.

    2014-01-01

    Different lineages of fungi produce distinct classes of ergot alkaloids. Lysergic acid-derived ergot alkaloids produced by fungi in the Clavicipitaceae are particularly important in agriculture and medicine. The pathway to lysergic acid is partly elucidated, but the gene encoding the enzyme that oxidizes the intermediate agroclavine is unknown. We investigated two candidate agroclavine oxidase genes from the fungus Epichloë festucae var. lolii × Epichloë typhina isolate Lp1 (henceforth referred to as Epichloë sp. Lp1), which produces lysergic acid-derived ergot alkaloids. Candidate genes easH and cloA were expressed in a mutant strain of the mold Aspergillus fumigatus, which typically produces a subclass of ergot alkaloids not derived from agroclavine or lysergic acid. Candidate genes were coexpressed with the Epichloë sp. Lp1 allele of easA, which encodes an enzyme that catalyzed the synthesis of agroclavine from an A. fumigatus intermediate; the agroclavine then served as the substrate for the candidate agroclavine oxidases. Strains expressing easA and cloA from Epichloë sp. Lp1 produced lysergic acid from agroclavine, a process requiring a cumulative six-electron oxidation and a double-bond isomerization. Strains that accumulated excess agroclavine (as a result of Epichloë sp. Lp1 easA expression in the absence of cloA) metabolized it into two novel ergot alkaloids for which provisional structures were proposed on the basis of mass spectra and precursor feeding studies. Our data indicate that CloA catalyzes multiple reactions to produce lysergic acid from agroclavine and that combining genes from different ergot alkaloid pathways provides an effective strategy to engineer important pathway molecules and novel ergot alkaloids. PMID:25107976

  2. Heterologous expression of mammalian Plk1 in Drosophila reveals divergence from Polo during late mitosis

    SciTech Connect

    Pearson, John . E-mail: jrobpea@upo.es; Godinho, Susana A.; Tavares, Alvaro; Glover, David M.

    2006-04-01

    Drosophila Polo kinase is the founder member of a conserved kinase family required for multiple stages of mitosis. We assessed the ability of mouse Polo-like kinase 1 (Plk1) to perform the multiple mitotic functions of Polo kinase, by expressing a Plk1-GFP fusion in Drosophila. Consistent with the previously reported localization of Polo kinase, Plk1-GFP was strongly localized to centrosomes and recruited to the centromeric regions of condensing chromosomes during early mitosis. However, in contrast to a functional Polo-GFP fusion, Plk1-GFP failed to localize to the central spindle midzone in both syncytial embryo mitosis and the conventional mitoses of cellularized embryos and S2 cells. Moreover, unlike endogenous Polo kinase and Polo-GFP, Plk1-GFP failed to associate with the contractile ring. Expression of Plk1-GFP enhanced the lethality of hypomorphic polo mutants and disrupted the organization of the actinomyosin cytoskeleton in a dominant-negative manner. Taken together, our results suggest that endogenous Polo kinase has specific roles in regulating actinomyosin rearrangements during Drosophila mitoses that its mammalian counterpart, Plk1, cannot fulfill. Consistent with this hypothesis, we observed defects in the cortical recruitment of myosin and myosin regulatory light chain in Polo deficient cells.

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

    PubMed

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

    2016-02-01

    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. PMID:26907343

  4. Development of a novel thermostable Newcastle disease virus vaccine vector for expression of a heterologous gene.

    PubMed

    Wen, Guoyuan; Chen, Chen; Guo, Jing; Zhang, Zhenyu; Shang, Yu; Shao, Huabin; Luo, Qingping; Yang, Jun; Wang, Hongling; Wang, Hongcai; Zhang, Tengfei; Zhang, Rongrong; Cheng, Guofu; Yu, Qingzhong

    2015-06-01

    Thermostable Newcastle disease virus (NDV) vaccines have been used widely to control Newcastle disease for village poultry flocks, due to their independence of cold chains for delivery and storage. To explore the potential use of thermostable NDV as a vaccine vector, an infectious clone of thermostable avirulent NDV strain TS09-C was developed using reverse genetics technology. The GFP gene, along with the self-cleaving 2A gene of foot-and-mouth disease virus and ubiquitin monomer (2AUbi), were inserted immediately upstream of the NP (nucleocapsid protein), M (matrix protein) or L (large polymerase protein) gene translation start codon in the TS09-C infectious clone. Detection of GFP expression in the recombinant virus-infected cells showed that the recombinant virus, rTS-GFP/M, with the GFP gene inserted into the M gene expressed the highest level of GFP. The rTS-GFP/M virus retained the same thermostability, growth dynamics and pathogenicity as its parental rTS09-C virus. Vaccination of specific-pathogen-free chickens with the rTS-GFP/M virus conferred complete protection against virulent NDV challenge. Taken together, the data suggested that the rTS09-C virus could be used as a vaccine vector to develop bivalent thermostable vaccines against Newcastle disease and the target avian diseases for village chickens, especially in the developing and least-developed countries. PMID:25626679

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

    PubMed

    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.

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

    PubMed Central

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

    2016-01-01

    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. PMID:26907343

  7. Heterologous expression and localization of gentisate transporter Ncg12922 from Corynebacterium glutamicum ATCC 13032

    SciTech Connect

    Xu Ying; Yan Dazhong; Zhou Ningyi . E-mail: n.zhou@pentium.whiov.ac.cn

    2006-07-28

    Ralstonia sp. strain U2 metabolizes naphthalene via gentisate (2,5-dihydroxybenzoate) to central metabolites, but it was found unable to utilize gentisate as growth substrate. A putative gentisate transporter encoded by ncg12922 from Corynebacterium glutamicum ATCC 13032 was functionally expressed in Ralstonia sp. strain U2, converting strain U2 to a gentisate utilizer. After ncg12922 was inserted into plasmid pGFPe with green fluorescence protein gene gfp, the expressed fusion protein Ncg12922-GFP could be visualized in the periphery of Escherichia coli cells under confocal microscope, consistent with a cytoplasmic membrane location. In contrast, GFP was ubiquitous in the cytoplasm of E. coli cells carrying pGFPe only. Gentisate 1,2-dioxygenase activity was present in the cell extract from strain U2 induced with gentisate but at a much lower level (one-fifth) than that obtained with salicylate. However, it exhibited a similar level in strain U2 containing Ncg12922 induced either by salicylate or gentisate.

  8. Cloning and heterologous expression of a thermostable pectate lyase from Penicillium occitanis in Escherichia coli.

    PubMed

    Damak, Naourez; Abdeljalil, Salma; Koubaa, Aida; Trigui, Sameh; Ayadi, Malika; Trigui-Lahiani, Hèla; Kallel, Emna; Turki, Nadia; Djemal, Lamia; Belghith, Hafeth; Taieb, Noomen Hadj; Gargouri, Ali

    2013-11-01

    The entire pectate lyase cDNA (Pel1) of Penicillium occitanis was cloned from a cDNA bank and sequenced. The ORF exhibited a great homology to Penicillium marneffei and conservation of all features of fungal pectate lyases such as the barrel structure with "eight right-handed parallel β-helix" architecture. The structure modeling also showed the interesting resemblance with thermostable pectate lyases since several specific residues were also shared by Pel1 and these thermostable enzymes. Having shown that the enzyme retains its activity after endoH-mediated deglycosylation, we investigated its expression in Escherichia coli BL21 using the pET28-a vector. This expression was shown to be optimum when cells were induced at room temperature in 2YT medium rather than at 37 °C and LB medium. In such conditions, the recombinant protein was apparently produced more in soluble form than as inclusion bodies. The effect of NaCl concentration was investigated during the binding and elution steps of recombinant His-tagged enzyme on MagneHis Ni-particles. The purified enzyme was shown to retain its thermo-activity as well as a great tolerance to high concentration of NaCl and imidazole.

  9. Heterologous expression and functional characterization of avian mu-class glutathione S-transferases.

    PubMed

    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

  10. Purification, characterization, and heterologous expression of an antifungal protein from the endophytic Bacillus subtilis strain Em7 and its activity against Sclerotinia sclerotiorum.

    PubMed

    Wang, N N; Gao, X N; Yan, X; Li, Z P; Kang, Z S; Huang, L L; Han, Q M

    2015-01-01

    An antifungal protein exhibiting a high activity against Sclerotinia sclerotiorum in vivo was purified by ammonium sulfate precipitation, hydrophobic chromatography, and gel filtration chromatography from the culture filtrate of the endophytic Bacillus subtilis strain Em7. The protein was characterized as a β-1,3-1,4-glucanase according to amino acid analysis, and showed excellent properties in thermal stability and acid resistance. At the same time, the antifungal protein was cloned and heterologously expressed in Escherichia coli BL21. The recombinant protein was purified and showed similar enzymatic properties to the native protein, exhibiting strong inhibitory activity against S. sclerotiorum. This shows that the β-1,3-1,4-glucanase may play a very important role in B. subtilis Em7 biocontrol function. In addition, many physiochemical properties of the native and purified recombinant protein were compared, including the effect of pH, temperature, metal cations, substrate specificity, and kinetic parameters. All parameters were similar between the native and recombinant purified protein, indicating that the purified recombinant protein has potential for industrial applications. PMID:26634515

  11. Directed Evolution of a Secretory Leader for the Improved Expression of Heterologous Proteins and Full-Length Antibodies in S. cerevisiae

    PubMed Central

    Rakestraw, J. Andy; Sazinsky, Stephen L.; Piatesi, Andrea; Antipov, Eugene; Wittrup, K. Dane

    2010-01-01

    Because of its eukaryotic nature, simple fermentation requirements, and pliable genetics, there have been many attempts at improving recombinant protein production in S. cerevisiae. These strategies typically involve altering the expression of a native protein thought to be involved in heterologous protein trafficking. Usually, these approaches yield three to ten-fold improvements over wild-type strains and are almost always specific to one type of protein. In this study, a library of mutant alpha mating factor 1 leader peptides (MFα1pp) is screened for the enhanced secretion of a single-chain antibody. One of the isolated mutants is shown to enhance the secretion of the scFv up to sixteen-fold over wild-type. These leaders also confer a secretory improvement to two other scFvs as well as two additional, structurally unrelated proteins. Moreover, the improved leader sequences, combined with strain engineering, allow for a one-hundred eighty fold improvement over previous reports in the secretion of full length, functional, glycosylated human IgG1. The production of full-length IgG1 at milligram per liter titers in a simple, laboratory-scale system will significantly expedite drug discovery and reagent synthesis while reducing antibody cloning, production, and characterization costs. PMID:19459139

  12. Heterologous Expression, Purification, and Biochemical Characterization of α-Humulene Synthase from Zingiber zerumbet Smith.

    PubMed

    Alemdar, Semra; Hartwig, Steffen; Frister, Thore; König, Jan Christoph; Scheper, Thomas; Beutel, Sascha

    2016-02-01

    The α-humulene synthase from Zingiber zerumbet Smith was expressed as a polyhistidine-tagged protein in an E. coli BL21(DE3) strain. Induction time and inductor (isopropyl-β-D-thiogalactopyranoside) concentration were optimized. The enzyme was successfully purified directly from cell lysate by NTA affinity column chromatography and careful selection of coordinated metal ion and imidazole elution conditions. Bioactivity assays were conducted with the natural substrate farnesyl diphosphate (FDP) in a two-phase system with in situ extraction of products. The conversion of FDP to α-humulene (~94.5%) and β-caryophyllene (~5.5%) could be monitored by gas chromatography-flame ionization detection (GC-FID). Optimal pH and temperature as well as kinetic parameters K M and k cat were determined using a discontinuous kinetic assay. PMID:26463657

  13. Effect of culture temperature on the heterologous expression of Pleurotus eryngii versatile peroxidase in Aspergillus hosts.

    PubMed

    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.

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

    PubMed

    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.

  15. Heterologous, Expression, and Characterization of Thermostable Glucoamylase Derived from Aspergillus flavus NSH9 in Pichia pastoris

    PubMed Central

    Karim, Kazi Muhammad Rezaul; Hossain, Md. Anowar; Sing, Ngieng Ngui; Mohd Sinang, Fazia; Hussain, Mohd Hasnain Md.; Roslan, Hairul Azman

    2016-01-01

    A novel thermostable glucoamylase cDNA without starch binding domain (SBD) of Aspergillus flavus NSH9 was successfully identified, isolated, and overexpressed in Pichia pastoris GS115. The complete open reading frame of glucoamylase from Aspergillus flavus NSH9 was identified by employing PCR that encodes 493 amino acids lacking in the SBD. The first 17 amino acids were presumed to be a signal peptide. The cDNA was cloned into Pichia pastoris and the highest expression of recombinant glucoamylase (rGA) was observed after 8 days of incubation period with 1% methanol. The molecular weight of the purified rGA was about 78 kDa and exhibited optimum catalytic activity at pH 5.0 and temperature of 70°C. The enzyme was stable at higher temperature with 50% of residual activity observed after 20 min at 90°C and 100°C. Low concentration of metal (Mg++, Fe++, Zn++, Cu++, and Pb++) had positive effect on rGA activity. This rGA has the potential for use and application in the saccharification steps, due to its thermostability, in the starch processing industries. PMID:27504454

  16. Heterologous, Expression, and Characterization of Thermostable Glucoamylase Derived from Aspergillus flavus NSH9 in Pichia pastoris.

    PubMed

    Karim, Kazi Muhammad Rezaul; Husaini, Ahmad; Hossain, Md Anowar; Sing, Ngieng Ngui; Mohd Sinang, Fazia; Hussain, Mohd Hasnain Md; Roslan, Hairul Azman

    2016-01-01

    A novel thermostable glucoamylase cDNA without starch binding domain (SBD) of Aspergillus flavus NSH9 was successfully identified, isolated, and overexpressed in Pichia pastoris GS115. The complete open reading frame of glucoamylase from Aspergillus flavus NSH9 was identified by employing PCR that encodes 493 amino acids lacking in the SBD. The first 17 amino acids were presumed to be a signal peptide. The cDNA was cloned into Pichia pastoris and the highest expression of recombinant glucoamylase (rGA) was observed after 8 days of incubation period with 1% methanol. The molecular weight of the purified rGA was about 78 kDa and exhibited optimum catalytic activity at pH 5.0 and temperature of 70°C. The enzyme was stable at higher temperature with 50% of residual activity observed after 20 min at 90°C and 100°C. Low concentration of metal (Mg(++), Fe(++), Zn(++), Cu(++), and Pb(++)) had positive effect on rGA activity. This rGA has the potential for use and application in the saccharification steps, due to its thermostability, in the starch processing industries. PMID:27504454

  17. Heterologous expression and kinetic characterisation of Neurospora crassa β-xylosidase in Pichia pastoris.

    PubMed

    Kirikyali, N; Connerton, I F

    2014-04-10

    To degrade plant hemicelluloses fungi employ β-xylosidases to hydrolyse xylooligosaccharides, released by endo-xylanases, into xylose. We have expressed the β-xylosidase from Neurospora crassa in Pichia pastoris under the control of alcohol oxidase 1 (AOX1) promoter. The recombinant enzyme is optimally active at 50 °C and pH 5.0 with Km and Vmax values of 8.9 mM and 1052 μmol min⁻¹ mg⁻¹ respectively against 4-nitrophenyl β-xylopyranoside. Xylose is a non-competitive inhibitor with a K(i) of 1.72 mM. The enzyme is characterised to be an exo-cutting enzyme releasing xylose from the non-reducing ends of β-1,4 linked xylooligosaccharides (X₂, X₃ and X₄) but also capable of transxylosilation. Catalytic conversion of X₂, X₃ and X4 decreases (V(max) and k(cat)) with increasing chain length. PMID:24629269

  18. Enhanced degradation of haloacid by heterologous expression in related Burkholderia species.

    PubMed

    Su, Xianbin; Deng, Liyu; Kong, Ka Fai; Tsang, Jimmy S H

    2013-10-01

    Haloacids are environmental pollutant and can be transformed to non-toxic alkanoic acids by microbial dehalogenase. Bacterium Burkholderia species MBA4 was enriched from soil for its ability to bioremediate haloacids such as mono-chloroacetate (MCA), mono-bromoacetate (MBA), 2-mono-chloropropionate, and 2-mono-bromopropionate. MBA4 produces an inducible dehalogenase Deh4a that catalyzes the dehalogenation process. The growth of MBA4 on haloacid also relies on the presence of a haloacid-uptake system. Similar dehalogenase genes can be found in the genome of many related species. However, wildtype Burkholderia caribensis MWAP64, Burkholderia phymatum STM815, and Burkholderia xenovorans LB400 were not able to grow on MCA. When a plasmid containing the regulatory and structural gene of Deh4a was transformed to these species, they were able to grow on haloacid. The specific enzyme activities in these recombinants ranges from 2- to 30-fold that of MBA4 in similar condition. Reverse transcription-quantitative real-time PCR showed that the relative transcript levels in these recombinant strains ranges from 9 to over 1,600 times that of MBA4 in similar condition. A recombinant has produced nearly five times of dehalogenase that MBA4 could ever achieve. While the expressions of Deh4a were more relaxed in these phylogenetically related species, an MCA-uptake activity was found to be inducible. These metabolically engineered strains are better degraders than the haloacid-enriched MBA4.

  19. Expression of peach sucrose transporters in heterologous systems points out their different physiological role.

    PubMed

    Zanon, Laura; Falchi, Rachele; Hackel, Aleksandra; Kühn, Christina; Vizzotto, Giannina

    2015-09-01

    Sucrose is the major phloem-translocated component in a number of economically important plant species. The comprehension of the mechanisms involved in sucrose transport in peach fruit appears particularly relevant, since the accumulation of this sugar, during ripening, is crucial for the growth and quality of the fruit. Here, we report the functional characterisation and subcellular localisation of three sucrose transporters (PpSUT1, PpSUT2, PpSUT4) in peach, and we formulate novel hypotheses about their role in accumulation of sugar. We provide evidence, about the capability of both PpSUT1 and PpSUT4, expressed in mutant yeast strains to transport sucrose. The functionality of PpSUT1 at the plasma membrane, and of PpSUT4 at the tonoplast, has been demonstrated. On the other hand, the functionality of PpSUT2 was not confirmed: this protein is unable to complement two sucrose uptake-deficient mutant yeast strains. Our results corroborate the hypotheses that PpSUT1 partakes in phloem loading in leaves, and PpSUT4 sustains cell metabolism by regulating sucrose efflux from the vacuole.

  20. Heterologous, Expression, and Characterization of Thermostable Glucoamylase Derived from Aspergillus flavus NSH9 in Pichia pastoris.

    PubMed

    Karim, Kazi Muhammad Rezaul; Husaini, Ahmad; Hossain, Md Anowar; Sing, Ngieng Ngui; Mohd Sinang, Fazia; Hussain, Mohd Hasnain Md; Roslan, Hairul Azman

    2016-01-01

    A novel thermostable glucoamylase cDNA without starch binding domain (SBD) of Aspergillus flavus NSH9 was successfully identified, isolated, and overexpressed in Pichia pastoris GS115. The complete open reading frame of glucoamylase from Aspergillus flavus NSH9 was identified by employing PCR that encodes 493 amino acids lacking in the SBD. The first 17 amino acids were presumed to be a signal peptide. The cDNA was cloned into Pichia pastoris and the highest expression of recombinant glucoamylase (rGA) was observed after 8 days of incubation period with 1% methanol. The molecular weight of the purified rGA was about 78 kDa and exhibited optimum catalytic activity at pH 5.0 and temperature of 70°C. The enzyme was stable at higher temperature with 50% of residual activity observed after 20 min at 90°C and 100°C. Low concentration of metal (Mg(++), Fe(++), Zn(++), Cu(++), and Pb(++)) had positive effect on rGA activity. This rGA has the potential for use and application in the saccharification steps, due to its thermostability, in the starch processing industries.

  1. Cloning, sequencing, and heterologous expression of a gene coding for Arthromyces ramosus peroxidase.

    PubMed

    Sawai-Hatanaka, H; Ashikari, T; Tanaka, Y; Asada, Y; Nakayama, T; Minakata, H; Kunishima, N; Fukuyama, K; Yamada, H; Shibano, Y

    1995-07-01

    To understand the relationship between the structure and functions of the peroxidase of Arthromyces ramosus, a novel taxon of hyphomycete, and the evolutionary relationship of the A.ramosus peroxidase (ARP) with the other peroxidases, we isolated complementary and genomic DNA clones encoding ARP and characterized them. The sequence analyses of the ARP and cDNA coding for ARP showed that a mature ARP consists of 344 amino acids with a N-terminal pyroglutamic acid preceded by a signal peptide of 20 amino acid residues. The amino acid sequence of ARP was 99% identical to that of the peroxidase of Coprinus cinereus, a basidiomycete, and also had very high similarities (41-43% identity) to those of basidiomycetous lignin peroxidases, although we could find no lignin peroxidase activities for ARP when assayed with lignin model compounds. We could identified His184 and His56 as proximal and distal ligands to heme, respectively, and Arg52 as an essential Arg. Comparison of the sequences of complementary and genomic DNAs found that protein-encoding DNA is interrupted by 14 intervening sequences. The ARP cDNA was expressed in the yeast Saccharomyces cerevisiae under the promoter of the glyceraldehyde 3-phosphate dehydrogenase gene, yielding 0.02 units/ml of a secreted active peroxidase.

  2. Heterologous expression and characterisation of a laccase from Colletotrichum lagenarium and decolourisation of different synthetic dyes.

    PubMed

    Wang, Bo; Yan, Ying; Tian, Yongsheng; Zhao, Wei; Li, Zhengjun; Gao, Jianjie; Peng, Rihe; Yao, Quanhong

    2016-03-01

    Laccases have received considerable attention in recent decades because of their ability to oxidise a large spectrum of phenolic and non-phenolic organic substrates and highly recalcitrant environmental pollutants. In this research, a laccase gene from Colletotrichum lagenarium was chemically synthesised using yeast bias codons and expressed in Pichia pastoris. The molecular mass of the recombinant laccase was estimated to be 64.6 kDa by SDS-PAGE, and the enzyme exhibited maximum activity at pH 3.6-4.0 but more stability in buffer with higher pH (>pH 3.6). The optimal reaction temperature of the enzyme was 40 °C, beyond which stability significantly decreased. By using 2,2'-azino-bis-(3-ethylbenzothiazoline)-6-sulphonate (ABTS) as a substrate, K m and V max values of 0.34 mM and 7.11 mM min(-1) mg(-1), respectively, were obtained. Using ABTS as a mediator, the laccase could oxidise hydroquinone to p-benzoquinone and decolourise the synthetic dyes malachite green, crystal violet and orange G. These results indicated that the laccase could be used to treat industrial effluents containing artificial dyes. PMID:26867601

  3. Heterologous expression and biochemical characterization of glucose isomerase from Thermobifida fusca.

    PubMed

    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.

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

    PubMed Central

    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

  5. Expression of peach sucrose transporters in heterologous systems points out their different physiological role.

    PubMed

    Zanon, Laura; Falchi, Rachele; Hackel, Aleksandra; Kühn, Christina; Vizzotto, Giannina

    2015-09-01

    Sucrose is the major phloem-translocated component in a number of economically important plant species. The comprehension of the mechanisms involved in sucrose transport in peach fruit appears particularly relevant, since the accumulation of this sugar, during ripening, is crucial for the growth and quality of the fruit. Here, we report the functional characterisation and subcellular localisation of three sucrose transporters (PpSUT1, PpSUT2, PpSUT4) in peach, and we formulate novel hypotheses about their role in accumulation of sugar. We provide evidence, about the capability of both PpSUT1 and PpSUT4, expressed in mutant yeast strains to transport sucrose. The functionality of PpSUT1 at the plasma membrane, and of PpSUT4 at the tonoplast, has been demonstrated. On the other hand, the functionality of PpSUT2 was not confirmed: this protein is unable to complement two sucrose uptake-deficient mutant yeast strains. Our results corroborate the hypotheses that PpSUT1 partakes in phloem loading in leaves, and PpSUT4 sustains cell metabolism by regulating sucrose efflux from the vacuole. PMID:26259193

  6. Heterologous expression of a rice miR395 gene in Nicotiana tabacum impairs sulfate homeostasis.

    PubMed

    Yuan, Ning; Yuan, Shuangrong; Li, Zhigang; Li, Dayong; Hu, Qian; Luo, Hong

    2016-01-01

    Sulfur participates in many important mechanisms and pathways of plant development. The most common source of sulfur in soil -SO4(2-)- is absorbed into root tissue and distributed into aerial part through vasculature system, where it is reduced into sulfite and finally sulfide within the subcellular organs such as chloroplasts and mitochondria and used for cysteine and methionine biosynthesis. MicroRNAs are involved in many regulation pathways by repressing the expression of their target genes. MiR395 family in Arabidopsis thaliana has been reported to be an important regulator involved in sulfate transport and assimilation, and a high-affinity sulphate transporter and three ATP sulfurylases (ATPS) were the target genes of AthmiR395 (Arabidopsis thaliana miR395). We have cloned a miR395 gene from rice (Oryza sativa) and studied its function in plant nutritional response. Our results indicated that in rice, transcript level of OsamiR395 (Oryza sativa miR395) increased under sulfate deficiency conditions, and the two predicted target genes of miR395 were down-regulated under the same conditions. Overexpression of OsamiR395h in tobacco impaired its sulfate homeostasis, and sulfate distribution was also slightly impacted among leaves of different ages. One sulfate transporter (SULTR) gene NtaSULTR2 was identified to be the target of miR395 in Nicotiana tobacum, which belongs to low affinity sulfate transporter group. Both miR395 and NtaSULTR2 respond to sulfate starvation in tobacco. PMID:27350219

  7. Heterologous expression of a rice miR395 gene in Nicotiana tabacum impairs sulfate homeostasis

    PubMed Central

    Yuan, Ning; Yuan, Shuangrong; Li, Zhigang; Li, Dayong; Hu, Qian; Luo, Hong

    2016-01-01

    Sulfur participates in many important mechanisms and pathways of plant development. The most common source of sulfur in soil –SO42−– is absorbed into root tissue and distributed into aerial part through vasculature system, where it is reduced into sulfite and finally sulfide within the subcellular organs such as chloroplasts and mitochondria and used for cysteine and methionine biosynthesis. MicroRNAs are involved in many regulation pathways by repressing the expression of their target genes. MiR395 family in Arabidopsis thaliana has been reported to be an important regulator involved in sulfate transport and assimilation, and a high-affinity sulphate transporter and three ATP sulfurylases (ATPS) were the target genes of AthmiR395 (Arabidopsis thaliana miR395). We have cloned a miR395 gene from rice (Oryza sativa) and studied its function in plant nutritional response. Our results indicated that in rice, transcript level of OsamiR395 (Oryza sativa miR395) increased under sulfate deficiency conditions, and the two predicted target genes of miR395 were down-regulated under the same conditions. Overexpression of OsamiR395h in tobacco impaired its sulfate homeostasis, and sulfate distribution was also slightly impacted among leaves of different ages. One sulfate transporter (SULTR) gene NtaSULTR2 was identified to be the target of miR395 in Nicotiana tobacum, which belongs to low affinity sulfate transporter group. Both miR395 and NtaSULTR2 respond to sulfate starvation in tobacco. PMID:27350219

  8. Heterologous Expression and Biochemical Characterisation of Fourteen Esterases from Helicoverpa armigera

    PubMed Central

    Li, Yongqiang; Coppin, Chris W.; Devonshire, Alan L.; Scott, Colin; East, Peter; Russell, Robyn J.; Oakeshott, John G.

    2013-01-01

    Esterases have recurrently been implicated in insecticide resistance in Helicoverpa armigera but little is known about the underlying molecular mechanisms. We used a baculovirus system to express 14 of 30 full-length esterase genes so far identified from midgut cDNA libraries of this species. All 14 produced esterase isozymes after native PAGE and the isozymes for seven of them migrated to two regions of the gel previously associated with both organophosphate and pyrethroid resistance in various strains. Thirteen of the enzymes obtained in sufficient yield for further analysis all showed tight binding to organophosphates and low but measurable organophosphate hydrolase activity. However there was no clear difference in activity between the isozymes from regions associated with resistance and those from elsewhere in the zymogram, or between eight of the isozymes from a phylogenetic clade previously associated with resistance in proteomic and quantitative rtPCR experiments and five others not so associated. By contrast, the enzymes differed markedly in their activities against nine pyrethroid isomers and the enzymes with highest activity for the most insecticidal isomers were from regions of the gel and, in some cases, the phylogeny that had previously been associated with pyrethroid resistance. Phospholipase treatment confirmed predictions from sequence analysis that three of the isozymes were GPI anchored. This unusual feature among carboxylesterases has previously been suggested to underpin an association that some authors have noted between esterases and resistance to the Cry1Ac toxin from Bacillus thuringiensis. However these three isozymes did not migrate to the zymogram region previously associated with Cry1Ac resistance. PMID:23799064

  9. Heterologous expression of family 10 xylanases from Acidothermus cellulolyticus enhances the exoproteome of Caldicellulosiruptor bescii and growth on xylan substrates

    DOE PAGES

    Kim, Sun -Ki; Chung, Daehwan; Himmel, Michael E.; Bomble, Yannick J.; Westpheling, Janet

    2016-08-22

    The ability to deconstruct plant biomass without conventional pretreatment has made members of the genus Caldicellulosiruptor the target of investigation for the consolidated processing of lignocellulosic biomass to biofuels and bioproducts. These Gram-positive bacteria are hyperthermophilic anaerobes and the most thermophilic cellulolytic organisms so far described. They use both C5 and C6 sugars simultaneously and have the ability to grow well on xylan, a major component of plant cell walls. This is an important advantage for their use to efficiently convert biomass at yields sufficient for an industrial process. For commodity chemicals, yield from substrate is perhaps the most importantmore » economic factor. In an attempt to improve even further the ability of C. bescii to use xylan, we introduced two xylanases from Acidothermus cellulolyticus. Acel_0180 includes tandem carbohydrate-binding modules (CBM2 and CBM3) located at the C-terminus, one of which, CBM2, is not present in C. bescii. Also, the sequences of Xyn10A and Acel_0180 have very little homology with the GH10 domains present in C. bescii. For these reasons, we selected these xylanases as potential candidates for synergistic interaction with those in the C. bescii exoproteome. As a result, heterologous expression of two xylanases from Acidothermus cellulolyticus in Caldicellulosiruptor bescii resulted in a modest, but significant increase in the activity of the exoproteome of C. bescii on xylan substrates. Even though the increase in extracellular activity was modest, the ability of C. bescii to grow on these substrates was dramatically improved suggesting that the xylan substrate/microbe interaction substantially increased deconstruction over the secreted free enzymes alone. In conclusion, we anticipate that the ability to efficiently use xylan, a major component of plant cell walls for conversion of plant biomass to products of interest, will allow the conversion of renewable, sustainable, and

  10. Cloning of ε-poly-L-lysine (ε-PL) synthetase gene from a newly isolated ε-PL-producing Streptomyces albulus NK660 and its heterologous expression in Streptomyces lividans.

    PubMed

    Geng, Weitao; Yang, Chao; Gu, Yanyan; Liu, Ruihua; Guo, Wenbin; Wang, Xiaomeng; Song, Cunjiang; Wang, Shufang

    2014-03-01

    ε-Poly-L-lysine (ε-PL), showing a wide range of antimicrobial activity, is now industrially produced as a food additive by a fermentation process. A new strain capable of producing ε-PL was isolated from a soil sample collected from Gutian, Fujian Province, China. Based on its morphological and biochemical features and phylogenetic similarity with 16S rRNA gene, the strain was identified as Streptomyces albulus and named NK660. The yield of ε-PL in 30 l fed-batch fermentation with pH control was 4.2 g l(-1) when using glycerol as the carbon source. The structure of ε-PL was determined by nuclear magnetic resonance (NMR) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Previous studies have shown that the antimicrobial activity of ε-PL is dependent on its molecular size. In this study, the polymerization degree of the ε-PL produced by strain NK660 ranged from 19 to 33 L-lysine monomers, with the main component consisting of 24-30 L-lysine monomers, which implied that the ε-PL might have higher antimicrobial activity. Furthermore, the ε-PL synthetase gene (pls) was cloned from strain NK660 by genome walking. The pls gene with its native promoter was heterologously expressed in Streptomyces lividans ZX7, and the recombinant strain was capable of synthesizing ε-PL. Here, we demonstrated for the first time heterologous expression of the pls gene in S. lividans. The heterologous expression of pls gene in S. lividans will open new avenues for elucidating the molecular mechanisms of ε-PL synthesis.

  11. Cloning of ε-poly-L-lysine (ε-PL) synthetase gene from a newly isolated ε-PL-producing Streptomyces albulus NK660 and its heterologous expression in Streptomyces lividans

    PubMed Central

    Geng, Weitao; Yang, Chao; Gu, Yanyan; Liu, Ruihua; Guo, Wenbin; Wang, Xiaomeng; Song, Cunjiang; Wang, Shufang

    2014-01-01

    ε-Poly-L-lysine (ε-PL), showing a wide range of antimicrobial activity, is now industrially produced as a food additive by a fermentation process. A new strain capable of producing ε-PL was isolated from a soil sample collected from Gutian, Fujian Province, China. Based on its morphological and biochemical features and phylogenetic similarity with 16S rRNA gene, the strain was identified as Streptomyces albulus and named NK660. The yield of ε-PL in 30 l fed-batch fermentation with pH control was 4.2 g l−1 when using glycerol as the carbon source. The structure of ε-PL was determined by nuclear magnetic resonance (NMR) and matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI-TOF MS). Previous studies have shown that the antimicrobial activity of ε-PL is dependent on its molecular size. In this study, the polymerization degree of the ε-PL produced by strain NK660 ranged from 19 to 33 L-lysine monomers, with the main component consisting of 24–30 L-lysine monomers, which implied that the ε-PL might have higher antimicrobial activity. Furthermore, the ε-PL synthetase gene (pls) was cloned from strain NK660 by genome walking. The pls gene with its native promoter was heterologously expressed in Streptomyces lividans ZX7, and the recombinant strain was capable of synthesizing ε-PL. Here, we demonstrated for the first time heterologous expression of the pls gene in S. lividans. The heterologous expression of pls gene in S. lividans will open new avenues for elucidating the molecular mechanisms of ε-PL synthesis. PMID:24423427

  12. Heterologous expression, secretion and characterization of the Geobacillus thermoleovorans US105 type I pullulanase.

    PubMed

    Zouari Ayadi, Dorra; Ben Ali, Mamdouh; Jemli, Sonia; Ben Mabrouk, Sameh; Mezghani, Monia; Ben Messaoud, Ezzedine; Bejar, Samir

    2008-03-01

    Pullulanase type I of Geobacillus thermoleovorans US105 strain (PUL US105) was produced and secreted efficiently in the E. coli periplasmic or extracellular fraction using two different signal peptides. Hence, the open reading frame was connected downstream of the lipase A signal peptide of Bacillus subtilis strain leading to an efficient secretion of an active form enzyme on the periplasmic fraction. In addition, pul US105 was fused to the alpha-amylase signal sequence of the Bacillus stearothermophilus US100 strain. The monitoring of the pullulanase activity and Western blot analysis for this last construction showed that the most activity was found in the supernatant culture, proving the efficient secretion of this natively cytoplasmic enzyme as an active form. The PUL US105 was purified to homogeneity from the periplasmic fraction, using heat treatment, size exclusion, and anion-exchange chromatography. The native pullulanase has a molecular mass of 160 kDa and is composed of two identical subunits of 80 kDa each. It was independent for metallic ions for its activity, while its thermostability was obviously improved in presence of only 0.1 mM CaCl2. PMID:18183386

  13. Fumarate Production by Torulopsis glabrata: Engineering Heterologous Fumarase Expression and Improving Acid Tolerance

    PubMed Central

    Chen, Xiulai; Song, Wei; Gao, Cong; Qin, Wen; Luo, Qiuling; Liu, Jia; Liu, Liming

    2016-01-01

    Fumarate is a well-known biomass building block compound. However, the poor catalytic efficiency of fumarase is one of the major factors preventing its widespread production. To address this issue, we selected residues 159HPND162 of fumarase from Rhizopus oryzae as targets for site-directed mutagenesis based on molecular docking analysis. Twelve mutants were generated and characterized in detail. Kinetic studies showed that the Km values of the P160A, P160T, P160H, N161E, and D162W mutants were decreased, whereas Km values of H159Y, H159V, H159S, N161R, N161F, D162K, and D162M mutants were increased. In addition, all mutants displayed decreased catalytic efficiency except for the P160A mutant, whose kcat/Km was increased by 33.2%. Moreover, by overexpressing the P160A mutant, the engineered strain T.G-PMS-P160A was able to produce 5.2 g/L fumarate. To further enhance fumarate production, the acid tolerance of T.G-PMS-P160A was improved by deleting ade12, a component of the purine nucleotide cycle, and the resulting strain T.G(△ade12)-PMS-P160A produced 9.2 g/L fumarate. The strategy generated in this study opens up new avenues for pathway optimization and efficient production of natural products. PMID:27711153

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To gain insight into the molecular process regulating ripening in apple, and to compare to tomato, 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. T...

  15. Evaluation of Streptomyces coelicolor A3(2) as a heterologous expression host for the cyanobacterial protein kinase C activator lyngbyatoxin A.

    PubMed

    Jones, Adam C; Ottilie, Sabine; Eustáquio, Alessandra S; Edwards, Daniel J; Gerwick, Lena; Moore, Bradley S; Gerwick, William H

    2012-04-01

    Filamentous marine cyanobacteria are extremely rich sources of bioactive natural products and often employ highly unusual biosynthetic enzymes in their assembly. However, the current lack of techniques for stable DNA transfer into these filamentous organisms, combined with the absence of heterologous expression strategies for nonribosomal cyanobacterial gene clusters, prohibit the creation of mutant strains or the heterologous production of these cyanobacterial compounds in other bacteria. In this study, we evaluated the capability of a derivative of the model actinomycete Streptomyces coelicolor A3(2) to express enzymes involved in the biosynthesis of the protein kinase C activator lyngbyatoxin A from a Hawaiian strain of Moorea producta (previously classified as Lyngbya majuscula). Despite large differences in GC content between these two bacteria and the presence of rare TTA/UUA leucine codons in lyngbyatoxin ORFs we were able to achieve expression of the cytochrome P450 monooxygenase LtxB and reverse prenyltransferase LtxC in S. coelicolor M512 and confirmed the in vitro functionality of S. coelicolor overexpressed LtxC. Attempts to express the entire lyngbyatoxin A gene cluster in S. coelicolor M512 were not successful because of transcript termination observed for the ltxA gene, which encodes a large nonribosomal peptide synthetase. However, these attempts did show a detectable level of cyanobacterial promoter recognition in Streptomyces. Successful expression of lyngbyatoxin A proteins in Streptomyces provides a new platform for biochemical investigation of natural product enzymes from Moorea strains.

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

    PubMed

    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. PMID:26976138

  17. Isolation of dihydroflavonol 4-reductase cDNA clones from Angelonia x angustifolia and heterologous expression as GST fusion protein in Escherichia coli.

    PubMed

    Gosch, Christian; Nagesh, Karthik Mudigere; Thill, Jana; Miosic, Silvija; Plaschil, Sylvia; Milosevic, Malvina; Olbricht, Klaus; Ejaz, Shaghef; Rompel, Annette; Stich, Karl; Halbwirth, Heidi

    2014-01-01

    Blue Angelonia × angustifolia flowers can show spontaneous mutations resulting in white/blue and white flower colourations. In such a white line, a loss of dihydroflavonol 4-reductase (DFR) activity was observed whereas chalcone synthase and flavanone 3-hydroxylase activity remained unchanged. Thus, cloning and characterization of a DFR of Angelonia flowers was carried out for the first time. Two full length DFR cDNA clones, Ang.DFR1 and Ang.DFR2, were obtained from a diploid chimeral white/blue Angelonia × angustifolia which demonstrated a 99% identity in their translated amino acid sequence. In comparison to Ang.DFR2, Ang.DFR1 was shown to contain an extra proline in a proline-rich region at the N-terminus along with two exchanges at the amino acids 12 and 26 in the translated amino acid sequence. The recombinant Ang.DFR2 obtained by heterologous expression in yeast was functionally active catalyzing the NADPH dependent reduction of dihydroquercetin (DHQ) and dihydromyricetin (DHM) to leucocyanidin and leucomyricetin, respectively. Dihydrokaempferol (DHK) in contrast was not accepted as a substrate despite the presence of asparagine in a position assumed to determine DHK acceptance. We show that substrate acceptance testing of DFRs provides biased results for DHM conversion if products are extracted with ethyl acetate. Recombinant Ang.DFR1 was inactive and functional activity could only be restored via exchanges of the amino acids in position 12 and 26 as well as the deletion of the extra proline. E. coli transformation of the pGEX-6P-1 vector harbouring the Ang.DFR2 and heterologous expression in E. coli resulted in functionally active enzymes before and after GST tag removal. Both the GST fusion protein and purified DFR minus the GST tag could be stored at -80°C for several months without loss of enzyme activity and demonstrated identical substrate specificity as the recombinant enzyme obtained from heterologous expression in yeast.

  18. Increasing the stearate content in seed oil of Brassica juncea by heterologous expression of MlFatB affects lipid content and germination frequency of transgenic seeds.

    PubMed

    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. PMID:26351151

  19. Increasing the stearate content in seed oil of Brassica juncea by heterologous expression of MlFatB affects lipid content and germination frequency of transgenic seeds.

    PubMed

    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.

  20. Heterologous Expression and Functional Characterization of the Exogenously Acquired Aminoglycoside Resistance Methyltransferases RmtD, RmtD2, and RmtG

    PubMed Central

    Corrêa, Laís L.; Witek, Marta A.; Zelinskaya, Natalia; Picão, Renata C.

    2015-01-01

    The exogenously acquired 16S rRNA methyltransferases RmtD, RmtD2, and RmtG were cloned and heterologously expressed in Escherichia coli, and the recombinant proteins were purified to near homogeneity. Each methyltransferase conferred an aminoglycoside resistance profile consistent with m7G1405 modification, and this activity was confirmed by in vitro 30S methylation assays. Analyses of protein structure and interaction with S-adenosyl-l-methionine suggest that the molecular mechanisms of substrate recognition and catalysis are conserved across the 16S rRNA (m7G1405) methyltransferase family. PMID:26552988

  1. Heterologous expression and purification of plantaricin NC8, a two-peptide bacteriocin against Salmonella spp. from Lactobacillus plantarum ZJ316.

    PubMed

    Jiang, Han; Li, Ping; Gu, Qing

    2016-11-01

    Bacteriocin, which is produced by lactic acid bacteria (LAB), has the potential to act as natural preservatives in the food industry. To develop strategies to overproduce such peptides, plantaricin NC8, a class IIb LAB bacteriocin that consists of two peptides, PLNC8α and PLNC8β, was successfully heterologously expressed in Escherichia coli BL21 (DE3). PLNC8α and PLNC8β peptides were expressed as His6-tag fusion proteins and were separated by Ni(2+) chelating affinity chromatography. To get the PLNC8α and PLNC8β peptides without extra amino acids in the N-terminus, the fusion proteins were cleaved by enterokinase and further purified using the Ni-NTA Sefinose™ Resin Kit. The molecular masses of peptides were checked using Tricine-SDS-PAGE and MALDI-TOF-MS. The yield of purified PLNC8α was around 2-2.5 mg/L, and the yield of PLNC8β was around 1.5-2 mg/L. The antimicrobial spectrum of cleaved peptides was detected and the synergistic action of PLNC8α and PLNC8β was preliminarily confirmed. It was found that E. coli was a suitable host for heterologous expression of plantaricin NC8 with a significant yield. Importantly, the bacteriocin appeared to be very active for controlling and inhibiting the food-borne pathogenic Gram-negative bacteria Salmonella spp., and might be useful as a natural preservative candidate. PMID:27373940

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

    PubMed

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

    2015-01-01

    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. PMID:26459865

  3. Short communication: Naturally sensitive Bacillus thuringiensis EG10368 produces thurincin H and acquires immunity after heterologous expression of the one-step-amplified thurincin H gene cluster.

    PubMed

    Wang, G; Manns, D C; Churey, J J; Worobo, R W

    2014-07-01

    Heterologous expression of bacteriocin genetic determinants (or operons) has long been a research interest for the functional analysis of genes involved in bacteriocin biosynthesis, regulation, modification, and immunity. Previously, construction of genomic libraries of the bacteriocin producer strains was usually required to identify new bacteriocin operons, a method that is tedious and time consuming. For the first time, we directly amplified an 8.14-kb bioinformatically identified thurincin H gene cluster using a one-step PCR method with 100% accuracy. This amplified gene cluster was cloned into plasmid pHT315, resulting in plasmid pGW139, and subsequently transformed to Bacillus thuringiensis EG10368, a strain naturally sensitive to thurincin H. Heterologous expression of the gene cluster makes the sensitive B. thuringiensis EG10368 produce thurincin H at a higher level compared with the wild-type producer, B. thuringiensis SF361. Moreover, B. thuringiensis EG10368pGW139 acquired complete immunity to thurincin H. The results indicated that one-step PCR is a promising tool to accurately amplify long bacteriocin gene clusters used in bacteriocin functional analysis studies and it is an effective way to produce bacteriocins at a higher level, without the need to clone large chromosomal fragments.

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

    PubMed

    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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26459865

  6. Heterologous expression and characterization of tyrosine decarboxylase from Enterococcus faecalis R612Z1 and Enterococcus faecium R615Z1.

    PubMed

    Liu, Fang; Xu, Wenjuan; Du, Lihui; Wang, Daoying; Zhu, Yongzhi; Geng, Zhiming; Zhang, Muhan; Xu, Weimin

    2014-04-01

    Tyrosine decarboxylase (TDC) is responsible for tyramine production and can catalyze phenylalanine to produce β-phenylethylamine. Enterococcus strains are a group of bacteria predominantly producing tyramine and β-phenylethylamine in water-boiled salted duck. In this study, the heterologous expression and characterization of two TDCs from Enterococcus faecalis R612Z1 (612TDC) and Enterococcus faecium R615Z1 (615TDC) were studied. The recombinant putative proteins of 612TDC and 615TDC were heterologously expressed in Escherichia coli. 612TDC is a 620-amino-acid protein with a molecular mass of 70.0 kDa, whereas 615TDC is a 625-amino-acid protein with a molecular mass of 70.3 kDa. Both 612TDC and 615TDC showed an optimum temperature of 25 °C for the tyrosine and phenylalanine substrates. However, 612TDC revealed maximal activity at pH 5.5, whereas 615TDC revealed maximal activity at pH 6.0. Kinetic studies showed that 612TDC and 615TDC exhibited higher specificity for tyrosine than for phenylalanine. The catalysis abilities of both 612TDC and 615TDC for phenylalanine were restrained significantly with the increase in NaCl concentration, but this was not the case for tyrosine. This study revealed that the enzyme properties of the purified recombinant 612TDC and 615TDC were similar, although their amino acid sequences had 84% identity. PMID:24680070

  7. Characterisation of Cyanobacterial Bicarbonate Transporters in E. coli Shows that SbtA Homologs Are Functional in This Heterologous Expression System

    PubMed Central

    Du, Jiahui; Förster, Britta; Rourke, Loraine; Howitt, Susan M.; Price, G. Dean

    2014-01-01

    Cyanobacterial HCO3- transporters BCT1, SbtA and BicA are important components of cyanobacterial CO2-concentration mechanisms. They also show potential in applications aimed at improving photosynthetic rates and yield when expressed in the chloroplasts of C3 crop species. The present study investigated the feasibility of using Escherichia coli to assess function of a range of SbtA and BicA transporters in a heterologous expression system, ultimately for selection of transporters suitable for chloroplast expression. Here, we demonstrate that six β-forms of SbtA are active in E. coli, although other tested bicarbonate transporters were inactive. The sbtA clones were derived from Synechococcus sp. WH5701, Cyanobium sp. PCC7001, Cyanobium sp. PCC6307, Synechococcus elongatus PCC7942, Synechocystis sp. PCC6803, and Synechococcus sp. PCC7002. The six SbtA homologs varied in bicarbonate uptake kinetics and sodium requirements in E. coli. In particular, SbtA from PCC7001 showed the lowest uptake affinity and highest flux rate and was capable of increasing the internal inorganic carbon pool by more than 8 mM relative to controls lacking transporters. Importantly, we were able to show that the SbtB protein (encoded by a companion gene near sbtA) binds to SbtA and suppresses bicarbonate uptake function of SbtA in E. coli, suggesting a role in post-translational regulation of SbtA, possibly as an inhibitor in the dark. This study established E. coli as a heterologous expression and analysis system for HCO3- transporters from cyanobacteria, and identified several SbtA transporters as useful for expression in the chloroplast inner envelope membranes of higher plants. PMID:25536191

  8. Functional properties of homomeric, human alpha 7-nicotinic acetylcholine receptors heterologously expressed in the SH-EP1 human epithelial cell line.

    PubMed

    Zhao, Lingke; Kuo, Yen-Ping; George, Andrew A; Peng, Jian-Hong; Purandare, Madhuri Singh; Schroeder, Katherine M; Lukas, Ronald J; Wu, Jie

    2003-06-01

    alpha 7-Nicotinic acetylcholine receptors (alpha 7-nAChRs) are broadly distributed in the central nervous system, where they play important roles in chemical and electrical signaling, and perhaps in neurite outgrowth, synaptic plasticity, and neuronal death/survival. To help elucidate their normal and pathophysiological roles, we have heterologously expressed human alpha 7-nAChR in transfected SH-EP1 human epithelial cells. Reverse transcription-polymerase chain reaction and mRNA fluorescence in situ hybridization analyses demonstrate expression of human alpha 7 subunits as messenger RNA. Patch-clamp recordings exploiting a novel strategy to prevent functional rundown of whole-cell peak current responses to repeated acute challenges with nicotinic agonists show successful expression of functional alpha 7-nAChR that mediate inward currents characterized by rapid phases of activation and inactivation. Concentration-response curves show that nicotine, acetylcholine, and choline are efficacious agonists at human alpha 7-nAChRs. Current-voltage relationships show inward rectification for agonist-induced currents. Human alpha 7-nAChRs exhibit some sensitivity to alpha 7-nAChR antagonists alpha-bungarotoxin (Bgt) or methyllycaconitine (MLA) when applied coincidentally with agonist, but much higher affinity block occurs when cells and alpha 7-nAChRs are pre-exposed to antagonists for 2 min before challenge with agonist. Both Bgt and MLA are competitive inhibitors of alpha 7-nAChR function. Whole-cell current peak amplitudes and half-times for inactivation of alpha 7-nAChR functional responses to nicotine are dramatically reduced in the absence of extracellular Ca2+, suggestive of high Ca2+ permeability of the alpha 7-nAChR channel. Thus, heterologously expressed human alpha 7-nAChR in mammalian cells have properties of native alpha 7-nAChR or of alpha 7-nAChR heterologously expressed in other systems and serve as excellent models for studies of molecular bases of alpha 7-n

  9. Α-L-arabinofuranosidase 3 from Penicillium purpurogenum (ABF3): potential application in the enhancement of wine flavour and heterologous expression of the enzyme.

    PubMed

    Ravanal, Maria Cristina; Rosa, Lorena; Eyzaguirre, Jaime

    2012-09-15

    An α-l-arabinofuranosidase (ABF3) from Penicillium purpurogenum was purified and its possible biotechnological application in the enhancement of wine flavour combined with P. purpurogenum β-glucosidase was studied. A must from Muscat of Alexandria was used to isolate the glycosides. The total monosaccharide (glucose, arabinose and xylose) levels in the glycosides were determined after acid hydrolysis, and were compared with the result of enzymatic hydrolysis. These results were analogous to those obtained in similar experiments using a commercial preparation, thus suggesting that the enzyme from P. purpurogenum may prove useful in this particular application. This prompted us to express the enzyme heterologously. The abf3 gene was thus expressed in Pichia pastoris. The recombinant enzyme was purified and it shows the same properties of the native ABF3 (substrate specificity, kinetic constants, pH and temperature optima and antibody cross-reactivity).

  10. Production of a new hybrid anthracycline 4-O-methylepelmycin by heterologous expression of dnrK in epelmycin-producing Streptomyces violaceus.

    PubMed

    Miyamoto, Y; Ohta, S; Johdo, O; Nagamatsu, Y; Yoshimoto, A

    2000-08-01

    A new hybrid anthracycline antibiotic was produced by heterologous expression of dnrK encoding carminomycin 4-O-metyltransferase in an epelmycin-producing Streptomyces violaceus. pMK100 was constructed by insertion of Steptomyces peucetius dnrK gene in Steptomyces-expression vector pIJ6021 and introduced to the epelmycin producer. The transformant produced a hybrid anthracycline antibiotic together with host epelmycins when cultured in antibiotic production medium in the presence of thiostrepton. The hybrid anthracycline was determined to be 7-O-L-rhodosaminyl-4-O-methyl-epsilon-rhodomycinone (4-O-methylepelmycin D). However, the attempts on production of hybrid 4-O-methylaclarubicin and 4-O-methyl-1-deoxyobelmycin by the transformants of aclarubicin and 1-deoxyobelmycin producers with pMK 100 were unsuccessful. PMID:11079805

  11. Ethanol production from acid- and alkali-pretreated corncob by endoglucanase and β-glucosidase co-expressing Saccharomyces cerevisiae subject to the expression of heterologous genes and nutrition added.

    PubMed

    Feng, Chunying; Zou, Shaolan; Liu, Cheng; Yang, Huajun; Zhang, Kun; Ma, Yuanyuan; Hong, Jiefang; Zhang, Minhua

    2016-05-01

    Low-cost technologies to overcome the recalcitrance of cellulose are the key to widespread utilization of lignocellulosic biomass for ethanol production. Efficient enzymatic hydrolysis of cellulose requires the synergism of various cellulases, and the ratios of each cellulase are required to be regulated to achieve the maximum hydrolysis. On the other hand, engineering of cellulolytic Saccharomyces cerevisiae strains is a promising strategy for lignocellulosic ethanol production. The expression of cellulase-encoding genes in yeast would affect the synergism of cellulases and thus the fermentation ability of strains with exogenous enzyme addition. However, such researches are rarely reported. In this study, ten endoglucanase and β-glucosidase co-expressing S. cerevisiae strains were constructed and evaluated by enzyme assay and fermentation performance measurement. The results showed that: (1) maximum ethanol titers of recombinant strains exhibited high variability in YPSC medium (20 g/l peptone, 10 g/l yeast extract, 100 g/l acid- and alkali-pretreated corncob) within 10 days. However, they had relatively little difference in USC medium (100 g/l acid- and alkali-pretreated corncob, 0.33 g/l urea, pH 5.0). (2) Strains 17# and 19#, with ratio (CMCase to β-glucosidase) of 7.04 ± 0.61 and 7.40 ± 0.71 respectively, had the highest fermentation performance in YPSC. However, strains 11# and 3# with the highest titers in USC medium had a higher ratio of CMCase to β-glucosidase, and CMCase activities. These results indicated that nutrition, enzyme activities and the ratio of heterologous enzymes had notable influence on the fermentation ability of cellulase-expressing yeast.

  12. Isolation of Dihydroflavonol 4-Reductase cDNA Clones from Angelonia x angustifolia and Heterologous Expression as GST Fusion Protein in Escherichia coli

    PubMed Central

    Gosch, Christian; Nagesh, Karthik Mudigere; Thill, Jana; Miosic, Silvija; Plaschil, Sylvia; Milosevic, Malvina; Olbricht, Klaus; Ejaz, Shaghef; Rompel, Annette; Stich, Karl; Halbwirth, Heidi

    2014-01-01

    Blue Angelonia × angustifolia flowers can show spontaneous mutations resulting in white/blue and white flower colourations. In such a white line, a loss of dihydroflavonol 4-reductase (DFR) activity was observed whereas chalcone synthase and flavanone 3-hydroxylase activity remained unchanged. Thus, cloning and characterization of a DFR of Angelonia flowers was carried out for the first time. Two full length DFR cDNA clones, Ang.DFR1 and Ang.DFR2, were obtained from a diploid chimeral white/blue Angelonia × angustifolia which demonstrated a 99% identity in their translated amino acid sequence. In comparison to Ang.DFR2, Ang.DFR1 was shown to contain an extra proline in a proline-rich region at the N-terminus along with two exchanges at the amino acids 12 and 26 in the translated amino acid sequence. The recombinant Ang.DFR2 obtained by heterologous expression in yeast was functionally active catalyzing the NADPH dependent reduction of dihydroquercetin (DHQ) and dihydromyricetin (DHM) to leucocyanidin and leucomyricetin, respectively. Dihydrokaempferol (DHK) in contrast was not accepted as a substrate despite the presence of asparagine in a position assumed to determine DHK acceptance. We show that substrate acceptance testing of DFRs provides biased results for DHM conversion if products are extracted with ethyl acetate. Recombinant Ang.DFR1 was inactive and functional activity could only be restored via exchanges of the amino acids in position 12 and 26 as well as the deletion of the extra proline. E. coli transformation of the pGEX-6P-1 vector harbouring the Ang.DFR2 and heterologous expression in E. coli resulted in functionally active enzymes before and after GST tag removal. Both the GST fusion protein and purified DFR minus the GST tag could be stored at −80°C for several months without loss of enzyme activity and demonstrated identical substrate specificity as the recombinant enzyme obtained from heterologous expression in yeast. PMID:25238248

  13. Heterologous, PKC-Mediated Desensitization of Human Histamine H3 Receptors Expressed in CHO-K1 Cells.

    PubMed

    Montejo-López, Wilber; Rivera-Ramírez, Nayeli; Escamilla-Sánchez, Juan; García-Hernández, Ubaldo; Arias-Montaño, José-Antonio

    2016-09-01

    Desensitization is a major mechanism to regulate the functional response of G protein-coupled receptors. In this work we studied whether the human histamine H3 receptor of 445 amino acids (hH3R445) experiences heterologous desensitization mediated by PKC activation. Bioinformatic analysis indicated the presence of Serine and Threonine residues susceptible of PKC-mediated phosphorylation on the third intracellular loop and the carboxyl terminus of the hH3R445. In CHO-K1 cells stably transfected with the hH3R445 direct PKC activation by phorbol 12-myristate 13-acetate (TPA, 200 nM) abolished H3R-mediated inhibition of forskolin-stimulated cAMP accumulation. Activation of endogenous purinergic receptors by ATP (adenosine 5'-triphosphate, 10 μM) increased the free calcium intracellular concentration ([Ca(2+)]i) confirming their coupling to phospholipase C stimulation. Incubation with ATP also abolished H3R-mediated inhibition of forskolin-induced cAMP accumulation, and this effect was prevented by the PKC inhibitors Ro-31-8220 and Gö-6976. Pre-incubation with TPA or ATP reduced H3R-mediated stimulation of [(35)S]-GTPγS binding to membranes from CHO-K1-hH3R445 cells by 39.7 and 54.2 %, respectively, with no change in the agonist potency, and the effect was prevented by either Ro-31-8220 or Gö-6976. Exposure to ATP or TPA also resulted in the loss of cell surface H3Rs (-30.4 and -45.1 %) as evaluated by [(3)H]-NMHA binding to intact cells. These results indicate that the hH3R445 undergoes heterologous desensitization upon activation of receptors coupled to PKC stimulation. PMID:27350581

  14. Heterologous production of fungal secondary metabolites in Aspergilli

    PubMed Central

    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. PMID:25713568

  15. Heterologous expression, purification, crystallization and preliminary X-ray analysis of raucaffricine glucosidase, a plant enzyme specifically involved in Rauvolfia alkaloid biosynthesis

    SciTech Connect

    Ruppert, Martin; Panjikar, Santosh; Barleben, Leif; Stöckigt, Joachim

    2006-03-01

    Raucaffricine glucosidase, an enzyme involved in the biosynthesis of monoterpenoid indole alkaloids in the plant Rauvolfia serpentina, was crystallized by the hanging-drop vapour-diffusion method using PEG4000 as precipitant. The crystals diffract to 2.3 Å resolution and belong to space group I222. Raucaffricine glucosidase (RG) is an enzyme that is specifically involved in the biosynthesis of indole alkaloids from the plant Rauvolfia serpentina. After heterologous expression in Escherichia coli cells, crystals of RG were obtained by the hanging-drop vapour-diffusion technique at 293 K with 0.3 M ammonium sulfate, 0.1 M sodium acetate pH 4.6 buffer and 11% PEG 4000 as precipitant. Crystals belong to space group I222 and diffract to 2.30 Å, with unit-cell parameters a = 102.8, b = 127.3, c = 215.8 Å.

  16. A Systematic Analysis of the Structures of Heterologously Expressed Proteins and Those from Their Native Hosts in the RCSB PDB Archive

    PubMed Central

    Zhou, Ren-Bin; Lu, Hui-Meng; Liu, Jie; Shi, Jian-Yu; Zhu, Jing; Lu, Qin-Qin; Yin, Da-Chuan

    2016-01-01

    Recombinant expression of proteins has become an indispensable tool in modern day research. The large yields of recombinantly expressed proteins accelerate the structural and functional characterization of proteins. Nevertheless, there are literature reported that the recombinant proteins show some differences in structure and function as compared with the native ones. Now there have been more than 100,000 structures (from both recombinant and native sources) publicly available in the Protein Data Bank (PDB) archive, which makes it possible to investigate if there exist any proteins in the RCSB PDB archive that have identical sequence but have some difference in structures. In this paper, we present the results of a systematic comparative study of the 3D structures of identical naturally purified versus recombinantly expressed proteins. The structural data and sequence information of the proteins were mined from the RCSB PDB archive. The combinatorial extension (CE), FATCAT-flexible and TM-Align methods were employed to align the protein structures. The root-mean-square distance (RMSD), TM-score, P-value, Z-score, secondary structural elements and hydrogen bonds were used to assess the structure similarity. A thorough analysis of the PDB archive generated five-hundred-seventeen pairs of native and recombinant proteins that have identical sequence. There were no pairs of proteins that had the same sequence and significantly different structural fold, which support the hypothesis that expression in a heterologous host usually could fold correctly into their native forms. PMID:27517583

  17. A Systematic Analysis of the Structures of Heterologously Expressed Proteins and Those from Their Native Hosts in the RCSB PDB Archive.

    PubMed

    Zhou, Ren-Bin; Lu, Hui-Meng; Liu, Jie; Shi, Jian-Yu; Zhu, Jing; Lu, Qin-Qin; Yin, Da-Chuan

    2016-01-01

    Recombinant expression of proteins has become an indispensable tool in modern day research. The large yields of recombinantly expressed proteins accelerate the structural and functional characterization of proteins. Nevertheless, there are literature reported that the recombinant proteins show some differences in structure and function as compared with the native ones. Now there have been more than 100,000 structures (from both recombinant and native sources) publicly available in the Protein Data Bank (PDB) archive, which makes it possible to investigate if there exist any proteins in the RCSB PDB archive that have identical sequence but have some difference in structures. In this paper, we present the results of a systematic comparative study of the 3D structures of identical naturally purified versus recombinantly expressed proteins. The structural data and sequence information of the proteins were mined from the RCSB PDB archive. The combinatorial extension (CE), FATCAT-flexible and TM-Align methods were employed to align the protein structures. The root-mean-square distance (RMSD), TM-score, P-value, Z-score, secondary structural elements and hydrogen bonds were used to assess the structure similarity. A thorough analysis of the PDB archive generated five-hundred-seventeen pairs of native and recombinant proteins that have identical sequence. There were no pairs of proteins that had the same sequence and significantly different structural fold, which support the hypothesis that expression in a heterologous host usually could fold correctly into their native forms.

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

    PubMed Central

    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

  19. Heterologous expression of polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase genes from a novel pyrene-degrading betaproteobacterium.

    PubMed

    Singleton, David R; Hu, Jing; Aitken, Michael D

    2012-05-01

    A betaproteobacterium within the family Rhodocyclaceae previously identified as a pyrene degrader via stable-isotope probing (SIP) of contaminated soil (designated pyrene group 1 or PG1) was cultivated as the dominant member of a mixed bacterial culture. A metagenomic library was constructed, and the largest contigs were analyzed for genes associated with polycyclic aromatic hydrocarbon (PAH) metabolism. Eight pairs of genes with similarity to the α- and β-subunits of ring-hydroxylating dioxygenases (RHDs) associated with aerobic bacterial PAH degradation were identified and linked to PG1 through PCR analyses of a simplified enrichment culture. In tandem with a ferredoxin and reductase found in close proximity to one pair of RHD genes, six of the RHDs were cloned and expressed in Escherichia coli. Each cloned RHD was tested for activity against nine PAHs ranging in size from two to five rings. Despite differences in their predicted protein sequences, each of the six RHDs was capable of transforming phenanthrene and pyrene. Three RHDs could additionally transform naphthalene and fluorene, and these genotypes were also associated with the ability of the E. coli constructs to convert indole to indigo. Only one of the six cloned RHDs was capable of transforming anthracene and benz[a]anthracene. None of the tested RHDs were capable of significantly transforming fluoranthene, chrysene, or benzo[a]pyrene.

  20. Heterologous Expression of Polycyclic Aromatic Hydrocarbon Ring-Hydroxylating Dioxygenase Genes from a Novel Pyrene-Degrading Betaproteobacterium

    PubMed Central

    Hu, Jing; Aitken, Michael D.

    2012-01-01

    A betaproteobacterium within the family Rhodocyclaceae previously identified as a pyrene degrader via stable-isotope probing (SIP) of contaminated soil (designated pyrene group 1 or PG1) was cultivated as the dominant member of a mixed bacterial culture. A metagenomic library was constructed, and the largest contigs were analyzed for genes associated with polycyclic aromatic hydrocarbon (PAH) metabolism. Eight pairs of genes with similarity to the α- and β-subunits of ring-hydroxylating dioxygenases (RHDs) associated with aerobic bacterial PAH degradation were identified and linked to PG1 through PCR analyses of a simplified enrichment culture. In tandem with a ferredoxin and reductase found in close proximity to one pair of RHD genes, six of the RHDs were cloned and expressed in Escherichia coli. Each cloned RHD was tested for activity against nine PAHs ranging in size from two to five rings. Despite differences in their predicted protein sequences, each of the six RHDs was capable of transforming phenanthrene and pyrene. Three RHDs could additionally transform naphthalene and fluorene, and these genotypes were also associated with the ability of the E. coli constructs to convert indole to indigo. Only one of the six cloned RHDs was capable of transforming anthracene and benz[a]anthracene. None of the tested RHDs were capable of significantly transforming fluoranthene, chrysene, or benzo[a]pyrene. PMID:22427500

  1. Heterologous expression of proteorhodopsin enhances H2 production in Escherichia coli when endogenous Hyd-4 is overexpressed.

    PubMed

    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.

  2. Molecular cloning and heterologous expression in Pichia pastoris of X-prolyl-dipeptidyl aminopeptidase from basidiomycete Ustilago maydis.

    PubMed

    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.

  3. d-2,3-Butanediol Production Due to Heterologous Expression of an Acetoin Reductase in Clostridium acetobutylicum ▿ †

    PubMed Central

    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

  4. 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.

    PubMed

    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.

  5. Heterologous expression of pyrroloquinoline quinone (pqq) gene cluster confers mineral phosphate solubilization ability to Herbaspirillum seropedicae Z67.

    PubMed

    Wagh, Jitendra; Shah, Sonal; Bhandari, Praveena; Archana, G; Kumar, G Naresh

    2014-06-01

    Gluconic acid secretion mediated by the direct oxidation of glucose by pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (GDH) is responsible for mineral phosphate solubilization in Gram-negative bacteria. Herbaspirillum seropedicae Z67 (ATCC 35892) genome encodes GDH apoprotein but lacks genes for the biosynthesis of its cofactor PQQ. In this study, pqqE of Erwinia herbicola (in plasmid pJNK1) and pqq gene clusters of Pseudomonas fluorescens B16 (pOK53) and Acinetobacter calcoaceticus (pSS2) were over-expressed in H. seropedicae Z67. Transformants Hs (pSS2) and Hs (pOK53) secreted micromolar levels of PQQ and attained high GDH activity leading to secretion of 33.46 mM gluconic acid when grown on 50 mM glucose while Hs (pJNK1) was ineffective. Hs (pJNK1) failed to solubilize rock phosphate, while Hs (pSS2) and Hs (pOK53) liberated 125.47 μM and 168.07 μM P, respectively, in minimal medium containing 50 mM glucose under aerobic conditions. Moreover, under N-free minimal medium, Hs (pSS2) and Hs (pOK53) not only released significant P but also showed enhanced growth, biofilm formation, and exopolysaccharide (EPS) secretion. However, indole acetic acid (IAA) production was suppressed. Thus, the addition of the pqq gene cluster, but not pqqE alone, is sufficient for engineering phosphate solubilization in H. seropedicae Z67 without compromising growth under nitrogen-fixing conditions.

  6. cDNA sequence and heterologous expression of monomeric spinach pullulanase: multiple isomeric forms arise from the same polypeptide.

    PubMed Central

    Renz, A; Schikora, S; Schmid, R; Kossmann, J; Beck, E

    1998-01-01

    The spinach pullulanase gene was cloned and sequenced using peptide sequences of the purified enzyme as a starting point and employing PCR techniques and cDNA library screening. Its open reading frame codes for a protein of 964 amino acids which represents a precursor of the pullulanase. The N-terminal transit peptide consists of 65 amino acids, and the mature protein, comprising 899 amino acids, has a calculated molecular mass of 99kDa. Pullulanase is a member of the alpha-amylase family. In addition to a characteristic catalytic (beta/alpha)8-barrel domain, it contains a domain, F, that is specific for branching and debranching enzymes. Pullulanase cDNA was expressed in Escherichia coli, and the purified protein was compared with the enzyme from spinach leaves. Identity of the two proteins was confirmed in terms of catalytic properties, N-terminal amino acid sequences and molecular masses. The pullulanase produced by E. coli showed the same microheterogeneity as the spinach leaf enzyme: it could be resolved into two substrate-induced forms by electrophoresis in amylopectin-containing polyacrylamide gels, and, in the absence of substrate, into several free forms (charge isomers) by isoelectric focusing or chromatofocusing. Rechromatofocusing of single free forms resulted in the originally observed pattern of molecular forms. However, heterogeneity of the protein disappeared on isoelectric focusing under completely denaturing conditions when only one protein band was observed. Post-translational modifications such as glycosylation and phosphorylation could be excluded as potential explanations for the protein heterogeneity. Therefore the microheterogeneity of spinach leaf pullulanase results from neither genetic variation nor post-translational modifications, but is a property of the single unmodified gene product. The different interconvertible forms of the pullulanase represent protein populations of different tertiary structure of the same polypeptide. PMID

  7. A More Desirable Balanced Polyunsaturated Fatty Acid Composition Achieved by Heterologous Expression of Δ15/Δ4 Desaturases in Mammalian Cells

    PubMed Central

    Zhu, Guiming; Ou, Qin; Zhang, Tao; Jiang, Xudong; Sun, Guozhi; Zhang, Ning; Wang, Kunfu; Fang, Heng; Wang, Mingfu; Sun, Jie; Ge, Tangdong

    2013-01-01

    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. PMID:24391980

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

    SciTech Connect

    Narayan, Om Prakash; Kumari, Nidhi; Rai, Lal Chand

    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.

  9. Heterologous expression of ACC deaminase from Trichoderma asperellum improves the growth performance of Arabidopsis thaliana under normal and salt stress conditions.

    PubMed

    Zhang, Fuli; Zhang, Ju; Chen, Long; Shi, Xiaoying; Lui, Zhihua; Li, Chengwei

    2015-09-01

    Transgenic Arabidopsis thaliana plants expressing the 1-aminocyclopropane-1-carboxylate deaminase gene (ACCD) of Trichoderma asperellum ACCC30536 (TaACCD) were created and their growth performance was assessed under normal and salt stress conditions. In order to characterize their growth, root length, root number, fresh weight (FW), relative water content (RWC), seed production, and seed number were measured. Under normal growing condition, all growth parameters except for dry weight (DW) of the transgenic plants increased significantly compared to WT plants. Furthermore, the transgenic line also exhibited higher tolerance and faster growth than WT plants in the presence of 150 mM NaCl. The increased salt stress tolerance of the transgenic plants is attributed to a greater RWC, root weight, root length, root number and FW under salt stress, and to reduced reactive oxygen species (ROS) level, cell death and electrolyte leakage compared to WT plants. The reduction in ROS levels could be explained by increased activity of several antioxidant enzymes, including peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT). Thus, we propose that heterologous expression of TaACCD could be used to improve salt stress tolerance in plants. PMID:26004912

  10. Heterologous expression of ACC deaminase from Trichoderma asperellum improves the growth performance of Arabidopsis thaliana under normal and salt stress conditions.

    PubMed

    Zhang, Fuli; Zhang, Ju; Chen, Long; Shi, Xiaoying; Lui, Zhihua; Li, Chengwei

    2015-09-01

    Transgenic Arabidopsis thaliana plants expressing the 1-aminocyclopropane-1-carboxylate deaminase gene (ACCD) of Trichoderma asperellum ACCC30536 (TaACCD) were created and their growth performance was assessed under normal and salt stress conditions. In order to characterize their growth, root length, root number, fresh weight (FW), relative water content (RWC), seed production, and seed number were measured. Under normal growing condition, all growth parameters except for dry weight (DW) of the transgenic plants increased significantly compared to WT plants. Furthermore, the transgenic line also exhibited higher tolerance and faster growth than WT plants in the presence of 150 mM NaCl. The increased salt stress tolerance of the transgenic plants is attributed to a greater RWC, root weight, root length, root number and FW under salt stress, and to reduced reactive oxygen species (ROS) level, cell death and electrolyte leakage compared to WT plants. The reduction in ROS levels could be explained by increased activity of several antioxidant enzymes, including peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT). Thus, we propose that heterologous expression of TaACCD could be used to improve salt stress tolerance in plants.

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

    PubMed

    An, Lei; Pang, Yun-Wei; Gao, Hong-Mei; Tao, Li; Miao, Kai; Wu, Zhong-Hong; Tian, Jian-Hui

    2012-11-23

    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. PMID:23103373

  12. Cloning of dfdA genes from Terrabacter sp. strain DBF63 encoding dibenzofuran 4,4a-dioxygenase and heterologous expression in Streptomyces lividans.

    PubMed

    Kasuga, Kano; Nitta, Atsushi; Kobayashi, Masayuki; Habe, Hiroshi; Nojiri, Hideaki; Yamane, Hisakazu; Omori, Toshio; Kojima, Ikuo

    2013-05-01

    A dibenzofuran (DF)-degrader Terrabacter sp. strain DBF63 harbors the dbfA and dbfBC genes for DF degradation and the fln-dbfA, pht, and pca gene clusters for the utilization of fluorene (FN) as a sole carbon source. From this strain, dfdA1, the gene encoding the second DF dioxygenase was detected using degenerate polymerase chain reaction (PCR) and the dfdA1A2A3A4 genes were cloned from a cosmid library of the DBF63 genome. Nucleotide sequencing revealed that the dfdA genes showed considerably high identities with those of other actinobacteria, such as Terrabacter sp. strain YK3 and Rhodococcus sp. strain HA01. In the neighboring region of the dfdA genes, as many as 11 homologs for transposase and integrase genes and the putative extradiol dioxygenase gene disrupted by an insertion sequence (dfdB::ISTesp2) were found, suggesting that repeated gene rearrangement had occurred. Quantitative reverse transcription-PCR analysis revealed that dfdA1 was expressed primarily in the DF-fed strain, whereas dbfA1 was expressed in the FN-cultured strain, apparently indicating that the dfdA genes are induced by DF for the initial hydroxylation of DF in strain DBF63. Furthermore, two polycistronic gene cassettes consisting of either dfdA or dbfA together with the dbfBC gene were constructed and expressed heterologously in Streptomyces lividans, degrading DF to salicylate. Furthermore, the expressed DfdA dioxygenase degraded dibenzo-p-dioxin, carbazole, dibenzothiophene, anthracene, phenanthrene, and biphenyl, thereby exhibiting a broader substrate range than that of the DbfA dioxygenase.

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

    PubMed

    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.

  14. Highly efficient strategy for the heterologous expression and purification of soluble Cowpea chlorotic mottle virus capsid protein and in vitro pH-dependent assembly of virus-like particles.

    PubMed

    Díaz-Valle, Armando; García-Salcedo, Yardena M; Chávez-Calvillo, Gabriela; Silva-Rosales, Laura; Carrillo-Tripp, Mauricio

    2015-12-01

    Obtaining pure and soluble viral capsid proteins (CPs) has been a major challenge in the fields of science and technology in recent decades. In many cases, the CPs can self-assemble in the absence of a viral genome, resulting in non-infectious, empty virus-like particles (VLPs) which can be safely handled. The use of VLPs has found great potential in biotechnology and health purposes. In addition, VLPs are a good model system to study protein-protein interactions at the molecular level. In this work, an optimized strategy for the heterologous expression of the Cowpea chlorotic mottle virus (CCMV) CP based in Escherichia coli is described. The method is efficient, inexpensive and it consistently produces higher yields and greater purity levels than those reported so far. Additionally, one of the main advantages of this method is the prevention of the formation of inclusion bodies, thus allowing to directly obtain high amounts of the CP in a soluble and functionally active state with the capacity to readily form VLPs in vitro. The CCMV CP self-assembly pH dependence was also investigated, providing guidelines to easily modulate the process.

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

    SciTech Connect

    An, Lei; Pang, Yun-Wei; Gao, Hong-Mei; Tao, Li; Miao, Kai; Wu, Zhong-Hong; 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.

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

    PubMed

    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.

  17. Seed-specific heterologous expression of a nasturtium FAE gene in Arabidopsis results in a dramatic increase in the proportion of erucic acid.

    PubMed

    Mietkiewska, Elzbieta; Giblin, E Michael; Wang, Song; Barton, Dennis L; Dirpaul, Joan; Brost, Jennifer M; Katavic, Vesna; Taylor, David C

    2004-09-01

    The fatty acid elongase [often designated FAE or beta-(or 3-) ketoacyl-CoA synthase] is a condensing enzyme and is the first component of the elongation complex involved in synthesis of erucic acid (22:1) in seeds of garden nasturtium (Tropaeolum majus). Using a degenerate primers approach, a cDNA of a putative embryo FAE was obtained showing high homology to known plant elongases. This cDNA contains a 1,512-bp open reading frame that encodes a protein of 504 amino acids. A genomic clone of the nasturtium FAE was isolated and sequence analyses indicated the absence of introns. Northern hybridization showed the expression of this nasturtium FAE gene to be restricted to the embryo. Southern hybridization revealed the nasturtium beta-ketoacyl-CoA synthase to be encoded by a small multigene family. To establish the function of the elongase homolog, the cDNA was introduced into two different heterologous chromosomal backgrounds (Arabidopsis and tobacco [Nicotiana tabacum]) under the control of a seed-specific (napin) promoter and the tandem 35S promoter, respectively. Seed-specific expression resulted in up to an 8-fold increase in erucic acid proportions in Arabidopsis seed oil, while constitutive expression in transgenic tobacco tissue resulted in increased proportions of very long chain saturated fatty acids. These results indicate that the nasturtium FAE gene encodes a condensing enzyme involved in the biosynthesis of very long chain fatty acids, utilizing monounsaturated and saturated acyl substrates. Given its strong and unique preference for elongating 20:1-CoA, the utility of the FAE gene product for directing or engineering increased synthesis of erucic acid is discussed.

  18. Molecular cloning, heterologous expression and functional characterization of a novel translationally-controlled tumor protein (TCTP) family member from Loxosceles intermedia (brown spider) venom.

    PubMed

    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. Synthesis of FAEEs from glycerol in engineered Saccharomyces cerevisiae using endogenously produced ethanol by heterologous expression of an unspecific bacterial acyltransferase.

    PubMed

    Yu, Kyung Ok; Jung, Ju; Kim, Seung Wook; Park, Chul Hwan; Han, Sung Ok

    2012-01-01

    The high price of petroleum-based diesel fuel has led to the development of alternative fuels, such as ethanol. Saccharomyces cerevisiae was metabolically engineered to utilize glycerol as a substrate for ethanol production. For the synthesis of fatty acid ethyl esters (FAEEs) by engineered S. cerevisiae that utilize glycerol as substrate, heterologous expression of an unspecific acyltransferase from Acinetobacter baylyi with glycerol utilizing genes was established. As a result, the engineered YPH499 (pGcyaDak, pGupWs-DgaTCas) strain produced 0.24 g/L FAEEs using endogenous ethanol produced from glycerol. And this study also demonstrated the possibility of increasing FAEE production by enhancing ethanol production by minimizing the synthesis of glycerol. The overall FAEE production in strain YPH499 fps1Δ gpd2Δ (pGcyaDak, pGupWs-DgaTCas) was 2.1-fold more than in YPH499 (pGcyaDak, pGupWs-DgaTCas), with approximately 0.52 g/L FAEEs produced, while nearly 17 g/L of glycerol was consumed. These results clearly indicated that FAEEs were synthesized in engineered S. cerevisiae by esterifying exogenous fatty acids with endogenously produced ethanol from glycerol. This microbial system acts as a platform in applying metabolic engineering that allows the production of FAEEs from cheap and abundant substrates specifically glycerol through the use of endogenous bioethanol.

  20. Type I Diacylglycerol Acyltransferase (MtDGAT1) from Macadamia tetraphylla: Cloning, Characterization, and Impact of Its Heterologous Expression on Triacylglycerol Composition in Yeast.

    PubMed

    Arroyo-Caro, José María; Mañas-Fernández, Aurora; Alonso, Diego López; García-Maroto, Federico

    2016-01-13

    Acyltransferase enzymes have been reported as useful biotechnological tools in order to increase oil yield and modify fatty acid composition. Macadamia species are able to accumulate unusually high levels of palmitoleic acid that besides oleic acid amounts to over 80% of monounsaturated fatty acids in the seed oil. In this work, a gene encoding a type 1 acyl-CoA:diacylglycerol acyltransferase (DGAT1) was cloned from M. tetraphylla. DGAT activity of the protein encoded by MtDGAT1 was confirmed by heterologous expression in a yeast mutant. Fatty acid composition of triacylglycerols synthesized by MtDGAT1 was compared to that of DGAT1 enzymes from Arabidopsis and Echium, with the results suggesting a substrate preference for monounsaturated over polyunsaturated fatty acids. Characteristics of MtDGAT1 may contribute to biochemical mechanisms determining the particular fatty acid composition of Macadamia oil and also indicate the possibility of using this enzyme in biotechnological approaches where a reduction of polyunsaturated fatty acids in the oil is desired.

  1. Molecular cloning, heterologous expression and functional characterization of a novel translationally-controlled tumor protein (TCTP) family member from Loxosceles intermedia (brown spider) venom.

    PubMed

    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. PMID:22051631

  2. Effects of volatile and intravenous anesthetics on the uptake of GABA, glutamate and dopamine by their transporters heterologously expressed in COS cells and in rat brain synaptosomes.

    PubMed

    Sugimura, M; Kitayama, S; Morita, K; Irifune, M; Takarada, T; Kawahara, M; Dohi, T

    2001-08-01

    Although the neurotransmitter uptake system is considered a possible target for the presynaptic action of anesthetic agents, observations are inconsistent concerning effects on the transporter and their clinical relevance. The present study examined the effects of volatile and intravenous anesthetics on the uptake of GABA, glutamate and dopamine in COS cells heterologously expressing the transporters for these neurotransmitters and in the rat brain synaptosomes. Halothane and isoflurane, but not thiamylal or thiopental, significantly inhibited uptake by COS cell systems of GABA, dopamine and glutamic acid in a concentration-dependent manner within clinically relevant ranges for anesthesia induced by these agents. Similarly, in synaptosomes halothane and isoflurane but not thiopental significantly suppressed the uptake of GABA and glutamic acid, respectively. These results do not support the hypothesis that volatile and intravenous anesthetics exert their action via specific inhibition of GABA uptake to enhance inhibitory GABAergic neuronal activity. Rather, they suggest that presynaptic uptake systems for various neurotransmitters including GABA may be the molecular targets for volatile anesthetic agents.

  3. D-ribulose-5-phosphate 3-epimerase: Cloning and heterologous expression of the spinach gene, and purification and characterization of the recombinant enzyme

    SciTech Connect

    Chen, Y.R.; Hartman, F.C.; Lu, T.Y.S.; Larimer, F.W.

    1998-09-01

    The authors have achieved, to their knowledge, the first high-level heterologous expression of the gene encoding D-ribulose-5-phosphate 3-epimerase from any source, thereby permitting isolation and characterization of the epimerase as found in photosynthetic organisms. The extremely labile recombinant spinach (Spinacia oleracea L.) enzyme was stabilized by DL-{alpha}-glycerophosphate or ethanol and destabilized by D-ribulose-5-phosphate or 2-mercaptoethanol. Despite this lability, the unprecedentedly high specific activity of the purified material indicates that the structural integrity of the enzyme is maintained throughout isolation. Ethylenediaminetetraacetate and divalent metal cations did not affect epimerase activity, thereby excluding a requirement for the latter in catalysis. As deduced from the sequence of the cloned spinach gene and the electrophoretic mobility under denaturing conditions of the purified recombinant enzyme, its 25-kD subunit size was about the same as that of the corresponding epimerases of yeast and mammals. However, in contrast to these other species, the recombinant spinach enzyme was octameric rather than dimeric, as assessed by gel filtration and polyacrylamide gel electrophoresis under nondenaturing conditions. Western-blot analyses with antibodies to the purified recombinant enzyme confirmed that the epimerase extracted from spinach leaves is also octameric.

  4. Nucleotide sequences and heterologous expression of tcmG and tcmP, biosynthetic genes for tetracenomycin C in Streptomyces glaucescens.

    PubMed Central

    Decker, H; Motamedi, H; Hutchinson, C R

    1993-01-01

    The nucleotide sequence of the tcmIII, tcmIc, and tcmVII region of the tetracenomycin (TCM) C gene cluster of Streptomyces glaucescens ETH 22794 (GLA.0) revealed the presence of two genes, tcmP and tcmG. The deduced product of tcmG resembles flavoprotein hydroxylases found in several other bacteria, whereas the predicted amino acid sequence of tcmP is not significantly similar to those of any known proteins in the available data bases. Southern blot hybridization revealed an approximately 180-bp deletion in a tcmIII (tcmG) mutant and a 1,800-bp insertion in a tcmVII (tcmP) mutant. Heterologous expression of tcmG and tcmP in Streptomyces lividans and tcmP in Escherichia coli established that tcmP encodes an O-methyltransferase, catalyzing the methylation of the C-9 carboxy group of TCM E to yield TCM A2, and that tcmG is responsible for the hydroxylation of TCM A2 at positions C-4, C-4a, and C-12a to give TCM C. These are the final two steps of TCM C biosynthesis. Images PMID:8509339

  5. Type I Diacylglycerol Acyltransferase (MtDGAT1) from Macadamia tetraphylla: Cloning, Characterization, and Impact of Its Heterologous Expression on Triacylglycerol Composition in Yeast.

    PubMed

    Arroyo-Caro, José María; Mañas-Fernández, Aurora; Alonso, Diego López; García-Maroto, Federico

    2016-01-13

    Acyltransferase enzymes have been reported as useful biotechnological tools in order to increase oil yield and modify fatty acid composition. Macadamia species are able to accumulate unusually high levels of palmitoleic acid that besides oleic acid amounts to over 80% of monounsaturated fatty acids in the seed oil. In this work, a gene encoding a type 1 acyl-CoA:diacylglycerol acyltransferase (DGAT1) was cloned from M. tetraphylla. DGAT activity of the protein encoded by MtDGAT1 was confirmed by heterologous expression in a yeast mutant. Fatty acid composition of triacylglycerols synthesized by MtDGAT1 was compared to that of DGAT1 enzymes from Arabidopsis and Echium, with the results suggesting a substrate preference for monounsaturated over polyunsaturated fatty acids. Characteristics of MtDGAT1 may contribute to biochemical mechanisms determining the particular fatty acid composition of Macadamia oil and also indicate the possibility of using this enzyme in biotechnological approaches where a reduction of polyunsaturated fatty acids in the oil is desired. PMID:26666454

  6. Selective Toxicity of the Anthelmintic Emodepside Revealed by Heterologous Expression of Human KCNMA1 in Caenorhabditis elegansS⃞

    PubMed Central

    Crisford, Anna; Murray, Caitriona; O'Connor, Vincent; Edwards, Richard J.; Kruger, Nina; Welz, Claudia; von Samson-Himmelstjerna, Georg; Harder, Achim; Walker, Robert J.

    2011-01-01

    Emodepside is a resistance-breaking anthelmintic of a new chemical class, the cyclooctadepsipeptides. A major determinant of its anthelmintic effect is the calcium-activated potassium channel SLO-1. SLO-1 belongs to a family of channels that are highly conserved across the animal phyla and regulate neurosecretion, hormone release, muscle contraction, and neuronal network excitability. To investigate the selective toxicity of emodepside, we performed transgenic experiments in which the nematode SLO-1 channel was swapped for a mammalian ortholog, human KCNMA1. Expression of either the human channel or Caenorhabditis elegans slo-1 from the native slo-1 promoter in a C. elegans slo-1 functional null mutant rescued behavioral deficits that otherwise resulted from loss of slo-1 signaling. However, worms expressing the human channel were 10- to 100-fold less sensitive to emodepside than those expressing the nematode channel. Strains expressing the human KCNMA1 channel were preferentially sensitive to the mammalian channel agonists NS1619 and rottlerin. In the C. elegans pharyngeal nervous system, slo-1 is expressed in neurons, not muscle, and cell-specific rescue experiments have previously shown that emodepside inhibits serotonin-stimulated feeding by interfering with SLO-1 signaling in the nervous system. Here we show that ectopic overexpression of slo-1 in pharyngeal muscle confers sensitivity of the muscle to emodepside, consistent with a direct interaction of emodepside with the channel. Taken together, these data predict an emodepside-selective pharmacophore harbored by SLO-1. This has implications for the development of this drug/target interface for the treatment of helminth infections. PMID:21415309

  7. Heterologous Expression of Mannanase and Developing a New Reporter Gene System in Lactobacillus casei and Escherichia coli.

    PubMed

    Lin, Jinzhong; Zou, Yexia; Ma, Chengjie; She, Qunxin; Liang, Yunxiang; Chen, Zhengjun; Ge, Xiangyang

    2015-01-01

    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 of ManB during growth, GusA activity was not; western blot analysis indicated that while the amount of GusA protein increased during later growth stages, GusA activity gradually decreased, indicating that the enzyme was inactive during cell growth. A similar trend was observed in E. coli JM109. We chose to use the more stable mannanase gene as the reporter to test secretion expression in L. casei. Two pELX1-based secretion vectors were constructed: one carried the signal peptide of the unknown secretion protein Usp45 from Lactococcus lactis (pELSH), and the other contained the full-length SlpA protein 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. casei and E.coli. PMID:26562012

  8. [Heterologous extracellular expression and initial characterization of the peroxisomal catalase from the methylotrophic yeast Hansenula polymorpha in Pichia pastoris].

    PubMed

    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+.

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

    PubMed

    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. PMID:26603122

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

    PubMed

    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.

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

    PubMed

    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.

  12. Heterologous expression of human cytochrome P450 2E1 in HepG2 cell line

    PubMed Central

    Zhuge, Jian; Luo, Ye; Yu, Ying-Nian

    2003-01-01

    AIM: Human cytochrome P-450 2E1 (CYP2E1) takes part in the biotransformation of ethanol, acetone, many small-molecule 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 ± 0.025 nmol·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. PMID:14669323

  13. A ripening associated peroxidase from papaya having a role in defense and lignification: heterologous expression and in-silico and in-vitro experimental validation.

    PubMed

    Pandey, Veda P; Dwivedi, Upendra N

    2015-01-25

    Fruit ripening associated full length cDNA of a peroxidase from papaya was cloned and heterologously expressed. The expressed peroxidase was activated by in-vitro re-folding in the presence of hemin and calcium. The purified recombinant peroxidase exhibited broad substrate affinity in the order of o-dianisidine>pyrogallol>guaiacol and was found to be a homotetramer of 155kDa with each subunit having a size of 38kDa. The basis of the distinctive preferences for various substrates was investigated through in-silico molecular modeling approaches. Thus, when the modeled papaya peroxidase-heme complex was docked with these substrates, the in-silico binding efficiency was found to be in agreement with those of wet lab results with the involvement of Arg37, Phe40, His41, Pro137, Asn138, His139, His167, and Phe239 as the common interacting residues in all the cases. However, the binding of the different substrates were found to be associated with conformational changes in the peroxidase. Thus, in the case of o-dianisidine (the most efficient substrate), the protein was folded in the most compact fashion when compared to guaiacol (the least efficient substrate). Protein function annotation analyses revealed that the papaya peroxidase may have biological roles in oxidation-reduction processes, stresses, defense responses etc. In order to further validate its role in lignifications, the papaya peroxidase was compared with a lignin biosynthetic peroxidase from Leucaena leucocephala, a tree legume. Thus, based on 3D structure superimposition and docking, both peroxidases exhibited a great extent of similarity suggesting the papaya peroxidase having a role in lignification (defense response) too. The predicted functions of papaya peroxidase in defense response and lignification were further validated experimentally using qRT-PCR analyses and measurement of oxidation of coniferyl alcohol.

  14. A phase 1 study of a heterologous prime-boost vaccination involving a truncated HER2 sequence in patients with HER2-expressing breast cancer

    PubMed Central

    Kim, Sung-Bae; Ahn, Jin-Hee; Kim, Jeongeun; Jung, Kyung Hae

    2015-01-01

    A phase 1 clinical trial was conducted to assess the safety, tolerability, and preliminary efficacy of a heterologous prime-boost strategy involving plasmid DNA (pHM-GM-CSF, expressing truncated human epidermal growth factor receptor 2 (HER2) and granulocyte macrophage colony-stimulation factor (GM-CSF) as a bicistronic message) and an adenoviral vector (Ad-HM, containing the same modified HER2 sequence only), in patients with stage III–IV metastatic breast cancer expressing HER2. Nine eligible subjects were divided into three cohorts based on the dosages (2, 4, and 8 mg/patient/visit) of pHM-GM-CSF used as the primer, which was intramuscularly injected three times at weeks 0, 2, and 4. It was followed by a single injection of Ad-HM (3 × 109 virus particles), used as a booster, at week 6. During the 6-month follow-up period, adverse events (AEs), pharmacokinetics and pharmacodynamics, and HER2-specific cellular and humoral immune responses were evaluated. Seven cases of minor grade 1 toxicities in four of nine subjects and no serious drug-related AEs were reported. HER2-specific cell-mediated or humoral immunity was produced in all (100%) or three subjects (33%), respectively. One subject showed a partial response, and seven subjects had stable diseases. However, there were no differences in clinical tumor response and HER2-specific immune responses among the cohorts. These results showed that intramuscular injections of pHM-GM-CSF and Ad-HM were well tolerated and safe. PMID:26445724

  15. A phase 1 study of a heterologous prime-boost vaccination involving a truncated HER2 sequence in patients with HER2-expressing breast cancer.

    PubMed

    Kim, Sung-Bae; Ahn, Jin-Hee; Kim, Jeongeun; Jung, Kyung Hae

    2015-01-01

    A phase 1 clinical trial was conducted to assess the safety, tolerability, and preliminary efficacy of a heterologous prime-boost strategy involving plasmid DNA (pHM-GM-CSF, expressing truncated human epidermal growth factor receptor 2 (HER2) and granulocyte macrophage colony-stimulation factor (GM-CSF) as a bicistronic message) and an adenoviral vector (Ad-HM, containing the same modified HER2 sequence only), in patients with stage III-IV metastatic breast cancer expressing HER2. Nine eligible subjects were divided into three cohorts based on the dosages (2, 4, and 8 mg/patient/visit) of pHM-GM-CSF used as the primer, which was intramuscularly injected three times at weeks 0, 2, and 4. It was followed by a single injection of Ad-HM (3 × 10(9) virus particles), used as a booster, at week 6. During the 6-month follow-up period, adverse events (AEs), pharmacokinetics and pharmacodynamics, and HER2-specific cellular and humoral immune responses were evaluated. Seven cases of minor grade 1 toxicities in four of nine subjects and no serious drug-related AEs were reported. HER2-specific cell-mediated or humoral immunity was produced in all (100%) or three subjects (33%), respectively. One subject showed a partial response, and seven subjects had stable diseases. However, there were no differences in clinical tumor response and HER2-specific immune responses among the cohorts. These results showed that intramuscular injections of pHM-GM-CSF and Ad-HM were well tolerated and safe. PMID:26445724

  16. Single-step co-integration of multiple expressible heterologous genes into the ribosomal DNA of the methylotrophic yeast Hansenula polymorpha.

    PubMed

    Klabunde, J; Diesel, A; Waschk, D; Gellissen, G; Hollenberg, C P; Suckow, M

    2002-05-01

    We have investigated the methylotrophic yeast Hansenula polymorpha as a host for the co-integration and expression of multiple heterologous genes using an rDNA integration approach. The ribosomal DNA (rDNA) of H. polymorpha was found to consist of a single rDNA cluster of about 50-60 repeats of an 8-kb unit located on chromosome II. A 2.4-kb segment of H. polymorpha rDNA encompassing parts of the 25S, the complete 5S and the non-transcribed spacer region between 25S and 18S rDNA was isolated and inserted into conventional integrative H. polymorpha plasmids harboring the Saccharomyces- cerevisiae-derived URA3 gene for selection. These rDNA plasmids integrated homologously into the rDNA repeats of a H. polymorpha (odc1) host as several independent clusters. Anticipating that this mode of multiple-cluster integration could be used for the simultaneous integration of several distinct rDNA plasmids, the host strain was co-transformed with a mixture of up to three different plasmids, all bearing the same URA3 selection marker. Transformations indeed resulted in mitotically stable strains harboring one, two, or all three plasmids integrated into the rDNA. The overall copy number of the plasmids integrated did not exceed the number of rDNA repeats present in the untransformed host strain, irrespective of the number of different plasmids involved. Strains harboring different plasmids co-expressed the introduced genes, resulting in functional proteins. Thus, this approach provides a new and attractive tool for the rapid generation of recombinant strains that simultaneously co-produce several proteins in desired stoichiometric ratios. PMID:12021801

  17. A ripening associated peroxidase from papaya having a role in defense and lignification: heterologous expression and in-silico and in-vitro experimental validation.

    PubMed

    Pandey, Veda P; Dwivedi, Upendra N

    2015-01-25

    Fruit ripening associated full length cDNA of a peroxidase from papaya was cloned and heterologously expressed. The expressed peroxidase was activated by in-vitro re-folding in the presence of hemin and calcium. The purified recombinant peroxidase exhibited broad substrate affinity in the order of o-dianisidine>pyrogallol>guaiacol and was found to be a homotetramer of 155kDa with each subunit having a size of 38kDa. The basis of the distinctive preferences for various substrates was investigated through in-silico molecular modeling approaches. Thus, when the modeled papaya peroxidase-heme complex was docked with these substrates, the in-silico binding efficiency was found to be in agreement with those of wet lab results with the involvement of Arg37, Phe40, His41, Pro137, Asn138, His139, His167, and Phe239 as the common interacting residues in all the cases. However, the binding of the different substrates were found to be associated with conformational changes in the peroxidase. Thus, in the case of o-dianisidine (the most efficient substrate), the protein was folded in the most compact fashion when compared to guaiacol (the least efficient substrate). Protein function annotation analyses revealed that the papaya peroxidase may have biological roles in oxidation-reduction processes, stresses, defense responses etc. In order to further validate its role in lignifications, the papaya peroxidase was compared with a lignin biosynthetic peroxidase from Leucaena leucocephala, a tree legume. Thus, based on 3D structure superimposition and docking, both peroxidases exhibited a great extent of similarity suggesting the papaya peroxidase having a role in lignification (defense response) too. The predicted functions of papaya peroxidase in defense response and lignification were further validated experimentally using qRT-PCR analyses and measurement of oxidation of coniferyl alcohol. PMID:25447898

  18. Natural and Heterologous Production of Bacteriocins

    NASA Astrophysics Data System (ADS)

    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

  19. Discovery of [NiFe] hydrogenase genes in metagenomic DNA: cloning and heterologous expression in Thiocapsa roseopersicina.

    PubMed

    Maróti, Gergely; Tong, Yingkai; Yooseph, Shibu; Baden-Tillson, Holly; Smith, Hamilton O; Kovács, Kornél L; Frazier, Marvin; Venter, J Craig; Xu, Qing

    2009-09-01

    Using a metagenomics approach, we have cloned a piece of environmental DNA from the Sargasso Sea that encodes an [NiFe] hydrogenase showing 60% identity to the large subunit and 64% to the small subunit of a Thiocapsa roseopersicina O2-tolerant [NiFe] hydrogenase. The DNA sequence of the hydrogenase identified by the metagenomic approach was subsequently found to be 99% identical to the hyaA and hyaB genes of an Alteromonas macleodii hydrogenase, indicating that it belongs to the Alteromonas clade. We were able to express our new Alteromonas hydrogenase in T. roseopersicina. Expression was accomplished by coexpressing only two accessory genes, hyaD and hupH, without the need to express any of the hyp accessory genes (hypABCDEF). These results suggest that the native accessory proteins in T. roseopersicina could substitute for the Alteromonas counterparts that are absent in the host to facilitate the assembly of a functional Alteromonas hydrogenase. To further compare the complex assembly machineries of these two [NiFe] hydrogenases, we performed complementation experiments by introducing the new Alteromonas hyaD gene into the T. roseopersicina hynD mutant. Interestingly, Alteromonas endopeptidase HyaD could complement T. roseopersicina HynD to cleave endoproteolytically the C-terminal end of the T. roseopersicina HynL hydrogenase large subunit and activate the enzyme. This study refines our knowledge on the selectivity and pleiotropy of the elements of the [NiFe] hydrogenase assembly machineries. It also provides a model for functionally analyzing novel enzymes from environmental microbes in a culture-independent manner. PMID:19633107

  20. Heterologous expression and characterization of bacterial 2-C-methyl-D-erythritol-4-phosphate pathway in Saccharomyces cerevisiae.

    PubMed

    Carlsen, Simon; Ajikumar, Parayil Kumaran; Formenti, Luca Riccardo; Zhou, Kang; Phon, Too Heng; Nielsen, Michael Lynge; Lantz, Anna Eliasson; Kielland-Brandt, Morten C; Stephanopoulos, Gregory

    2013-07-01

    Transfer of a biosynthetic pathway between evolutionary distant organisms can create a metabolic shunt capable of bypassing the native regulation of the host organism, hereby improving the production of secondary metabolite precursor molecules for important natural products. Here, we report 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 machinery was unable to functionalize ispG and ispH. However, we have found that leuC and leuD, encoding the heterodimeric iron-sulfur cluster protein, isopropylmalate isomerase, can complement the S. cerevisiae leu1 auxotrophy. To our knowledge, this is the first time a bacterial iron-sulfur cluster protein has been functionally expressed in the cytosol of S. cerevisiae under aerobic conditions and shows that S. cerevisiae has the capability to functionally express at least some bacterial iron-sulfur cluster proteins in its cytosol.

  1. Identification, cDNA cloning and heterologous expression of a hyaluronidase from the tarantula Brachypelma vagans venom.

    PubMed

    Clement, Herlinda; Olvera, Alejandro; Rodríguez, Mabel; Zamudio, Fernando; Palomares, Laura A; Possani, Lourival D; Odell, George V; Alagón, Alejandro; Sánchez-López, Rosana

    2012-12-01

    Hyaluronidases (Hyal) present in the venom of poisonous animals have been considered as "spreading factors" that facilitate a fast penetration of the venom in the prey. We have found that hyaluronidase from the tarantula Brachypelma vagans venom (BvHyal) displays a substrate-specific Hyal activity against hyaluronan. By using a combined strategy based on peptide sequencing and RT-PCR, we have cloned a BvHyal cDNA. Active recombinant BvHyal was efficiently expressed in a baculovirus system in insect cell. PMID:22982117

  2. Heterologous expression of the halophyte Zoysia matrella H⁺-pyrophosphatase gene improved salt tolerance in Arabidopsis thaliana.

    PubMed

    Chen, Yu; Li, Lanlan; Zong, Junqin; Chen, Jingbo; Guo, Hailin; Guo, Aigui; Liu, Jianxiu

    2015-06-01

    A number of vacuolar H(+)-pyrophosphatase (VP) family genes play important roles in plant growth under salt stress condition. Despite their biological importance in plant salt-stress regulation, there is no report about VP in the halophytic turfgrass Zoysia matrella. Here, we isolated ZmVP1, a type I VP homologues gene encoding 768 amino acids by using the degenerated PCR and RACE PCR methods from Zoysia matrella. The expression level of ZmVP1 was significantly induced by salinity, drought and cold, but not by heat. ZmVP1 can restore the salt-tolerant ability of a salt-sensitive yeast strain. Overexpression of ZmVP1 in Arabidopsis thaliana resulted in more vigorous growth under salt stress. Moreover, the transgenic Arabidopsis accumulated more Na(+) and K(+) in the leaves compared to that of wild type plants under salt stress, had higher activities of V-ATPase and V-PPase, and showed higher relative gene expression levels of 5 stress-related genes (AtNHX1, AtLEA, AtP5CS, AtMn-SOD, AtAPX1). These results demonstrated that ZmVP1 from Z. matrella was a functional tonoplast H(+)-pyrophosphatase contributing to salt tolerance potentially through regulating the Na(+) compartment in vacuole, K(+) assimilation, osmotic regulation and antioxidant response. PMID:25874657

  3. Heterologous expression of Ceratophyllum demersum phytochelatin synthase, CdPCS1, in rice leads to lower arsenic accumulation in grain.

    PubMed

    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.

  4. Molecular cloning of amphioxus uncoupling protein and assessment of its uncoupling activity using a yeast heterologous expression system

    SciTech Connect

    Chen, Kun; Sun, Guoxun; Lv, Zhiyuan; Wang, Chen; Jiang, Xueyuan; Li, Donghai; Zhang, Chenyu

    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.

  5. Heterologous expression of Ceratophyllum demersum phytochelatin synthase, CdPCS1, in rice leads to lower arsenic accumulation in grain.

    PubMed

    Shri, Manju; Dave, Richa; Diwedi, Sanjay; Shukla, Devesh; Kesari, Ravi; Tripathi, Rudra Deo; Trivedi, Prabodh Kumar; Chakrabarty, Debasis

    2014-01-01

    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. PMID:25048298

  6. Heterologous expression of Ceratophyllum demersum phytochelatin synthase, CdPCS1, in rice leads to lower arsenic accumulation in grain

    PubMed Central

    Shri, Manju; Dave, Richa; Diwedi, Sanjay; Shukla, Devesh; Kesari, Ravi; Tripathi, Rudra Deo; Trivedi, Prabodh Kumar; Chakrabarty, Debasis

    2014-01-01

    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. PMID:25048298

  7. Increasing free-energy (ATP) conservation in maltose-grown Saccharomyces cerevisiae by expression of a heterologous maltose phosphorylase.

    PubMed

    de Kok, Stefan; Yilmaz, Duygu; Suir, Erwin; Pronk, Jack T; Daran, Jean-Marc; van Maris, Antonius J A

    2011-09-01

    Increasing free-energy conservation from the conversion of substrate into product is crucial for further development of many biotechnological processes. In theory, replacing the hydrolysis of disaccharides by a phosphorolytic cleavage reaction provides an opportunity to increase the ATP yield on the disaccharide. To test this concept, we first deleted the native maltose metabolism genes in Saccharomyces cerevisiae. The knockout strain showed no maltose-transport activity and a very low residual maltase activity (0.03 μmol mg protein(-1)min(-1)). Expression of a maltose phosphorylase gene from Lactobacillus sanfranciscensis and the MAL11 maltose-transporter gene resulted in relatively slow growth (μ(aerobic) 0.09 ± 0.03 h(-1)). Co-expression of Lactococcus lactis β-phosphoglucomutase accelerated maltose utilization via this route (μ(aerobic) 0.21 ± 0.01 h(-1), μ(anaerobic) 0.10 ± 0.00 h(-1)). Replacing maltose hydrolysis with phosphorolysis increased the anaerobic biomass yield on maltose in anaerobic maltose-limited chemostat cultures by 26%, thus demonstrating the potential of phosphorolysis to improve the free-energy conservation of disaccharide metabolism in industrial microorganisms.

  8. Identification, cloning and heterologous expression of active [NiFe]-hydrogenase 2 from Citrobacter sp. SG in Escherichia coli.

    PubMed

    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.

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

    PubMed

    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 <4) in a human serum bactericidal assay (hSBA) using anti-rNMB1612 sera, although another strain (MC168) expressing the same 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

  10. Heterologous expression and purification of a multiheme cytochrome from a Gram-positive bacterium capable of performing extracellular respiration.

    PubMed

    Costa, N L; Carlson, H K; Coates, J D; Louro, R O; Paquete, C M

    2015-07-01

    Microbial electrochemical technologies are emerging as environmentally friendly biotechnological processes. Recently, a thermophilic Gram-positive bacterium capable of electricity production in a microbial fuel cell was isolated. Thermincola potens JR contains several multiheme c-type cytochromes that were implicated in the process of electricity production. In order to understand the molecular basis by which Gram-positive bacteria perform extracellular electron transfer, the relevant proteins need to be characterized in detail. Towards this end, a chimeric gene containing the signal peptide from Shewanella oneidensis MR-1 small tetraheme cytochrome c (STC) and the gene sequence of the target protein TherJR_0333 was constructed. This manuscript reports the successful expression of this chimeric gene in the Gram-negative bacterium Escherichia coli and its subsequent purification and characterization. This methodology opens the possibility to study other multiheme cytochromes from Gram-positive bacteria, allowing the extracellular electron transfer mechanisms of this class of organisms to be unraveled.

  11. Heterologous expression, purification, and enzymatic characterization of the acyclic carotenoid 1,2-hydratase from Rubrivivax gelatinosus.

    PubMed

    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.

  12. Heterologous expression of ATG8c from soybean confers tolerance to nitrogen deficiency and increases yield in Arabidopsis.

    PubMed

    Xia, Tongmei; Xiao, Dong; Liu, Dong; Chai, Wenting; Gong, Qingqiu; Wang, Ning Ning

    2012-01-01

    Nitrogen is an essential element for plant growth and yield. Improving Nitrogen Use Efficiency (NUE) of crops could potentially reduce the application of chemical fertilizer and alleviate environmental damage. To identify new NUE genes is therefore an important task in molecular breeding. Macroautophagy (autophagy) is an intracellular process in which damaged or obsolete cytoplasmic components are encapsulated in double membraned vesicles termed autophagosomes, then delivered to the vacuole for degradation and nutrient recycling. One of the core components of autophagosome formation, ATG8, has been shown to directly mediate autophagosome expansion, and the transcript of which is highly inducible upon starvation. Therefore, we postulated that certain homologs of Saccharomyces cerevisiae ATG8 (ScATG8) from crop species could have potential for NUE crop breeding. A soybean (Glycine max, cv. Zhonghuang-13) ATG8, GmATG8c, was selected from the 11 family members based on transcript analysis upon nitrogen deprivation. GmATG8c could partially complement the yeast atg8 mutant. Constitutive expression of GmATG8c in soybean callus cells not only enhanced nitrogen starvation tolerance of the cells but accelerated the growth of the calli. Transgenic Arabidopsis over-expressing GmATG8c performed better under extended nitrogen and carbon starvation conditions. Meanwhile, under optimum growth conditions, the transgenic plants grew faster, bolted earlier, produced larger primary and axillary inflorescences, eventually produced more seeds than the wild-type. In average, the yield was improved by 12.9%. We conclude that GmATG8c may serve as an excellent candidate for breeding crops with enhanced NUE and better yield. PMID:22629371

  13. Efficient Production of 2,5-Diketo-d-Gluconate via Heterologous Expression of 2-Ketogluconate Dehydrogenase in Gluconobacter japonicus

    PubMed Central

    Kataoka, Naoya; Matsutani, Minenosuke; Matsushita, Kazunobu

    2015-01-01

    2,5-Diketo-d-gluconate (2,5DKG) is a compound that can be the intermediate for d-tartrate and also vitamin C production. Although Gluconobacter oxydans NBRC3293 produces 2,5DKG from d-glucose via d-gluconate and 2-keto-d-gluconate (2KG), with accumulation of the product in the culture medium, the efficiency of 2,5DKG production is unsatisfactory because there is a large amount of residual d-gluconate at the end of the biotransformation process. Oxidation of 2KG to 2,5DKG is catalyzed by a membrane-bound flavoprotein-cytochrome c complex: 2-keto-gluconate dehydrogenase (2KGDH). Here, we studied the kgdSLC genes encoding 2KGDH in G. oxydans NBRC3293 to improve 2,5DKG production by Gluconobacter spp. The kgdS, kgdL, and kgdC genes correspond to the small, large, and cytochrome subunits of 2KGDH, respectively. The kgdSLC genes were cloned into a broad-host-range vector carrying a DNA fragment of the putative promoter region of the membrane-bound alcohol dehydrogenase gene of G. oxydans for expression in Gluconobacter spp. According to our results, 2KGDH that was purified from the recombinant Gluconobacter cells showed characteristics nearly the same as those reported previously. We also expressed the kgdSLC genes in a mutant strain of Gluconobacter japonicus NBRC3271 (formerly Gluconobacter dioxyacetonicus IFO3271) engineered to produce 2KG efficiently from a mixture of d-glucose and d-gluconate. This mutant strain consumed almost all of the starting materials (d-glucose and d-gluconate) to produce 2,5DKG quantitatively as a seemingly unique metabolite. To our knowledge, this is the first report of a Gluconobacter strain that produces 2,5DKG efficiently and homogeneously. PMID:25769838

  14. Molecular Characterization of Laccase Genes from the Basidiomycete Coprinus cinereus and Heterologous Expression of the Laccase Lcc1

    PubMed Central

    Yaver, Debbie S.; Overjero, Maria Del Carmen; Xu, Feng; Nelson, Beth A.; Brown, Kim M.; Halkier, Torben; Bernauer, Sheryl; Brown, Stephen H.; Kauppinen, Sakari

    1999-01-01

    A laccase from Coprinus cinereus is active at alkaline pH, an essential property for some potential applications. We cloned and sequenced three laccase genes (lcc1, lcc2, and lcc3) from the ink cap basidiomycete C. cinereus. The lcc1 gene contained 7 introns, while both lcc2 and lcc3 contained 13 introns. The predicted mature proteins (Lcc1 to Lcc3) are 58 to 80% identical at the amino acid level. The predicted Lcc1 contains a 23-amino-acid C-terminal extension rich in arginine and lysine, suggesting that C-terminal processing may occur during its biosynthesis. We expressed the Lcc1 protein in Aspergillus oryzae and purified it. The Lcc1 protein as expressed in A. oryzae has an apparent molecular mass of 66 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and absorption maxima at 278 and 614 nm. Based on the N-terminal protein sequence of the laccase, a 4-residue propeptide was processed during the maturation of the enzyme. The dioxygen specificity of the laccase showed an apparent Km of 21 ± 2 μM and a catalytic constant of 200 ± 10 min−1 for O2 with 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) as the reducing substrate at pH 5.5. Lcc1 from A. oryzae may be useful in industrial applications. This is the first report of a basidiomycete laccase whose biosynthesis involves both N-terminal and C-terminal processing. PMID:10543807

  15. Detection of infectious myonecrosis virus using monoclonal antibody specific to N and C fragments of the capsid protein expressed heterologously.

    PubMed

    Kunanopparat, Areerat; Chaivisuthangkura, Parin; Senapin, Saengchan; Longyant, Siwaporn; Rukpratanporn, Sombat; Flegel, Timothy W; Sithigorngul, Paisarn

    2011-01-01

    The gene encoding the capsid protein in ORF1 of the genome of infectious myonecrosis virus (IMNV) (GenBank AY570982) was amplified into three parts named CP-N (nucleotides 2248-3045), CP-I (nucleotides 3046-3954) and CP-C (nucleotides 3955-4953). The CP-N fragment was inserted into expression vector pTYB1 while CP-I and CP-C were each inserted into expression vector pGEX-6P-1 for transformation of BL21 E. coli strain. After induction, intein-CP-N (84 kDa), glutathione-S-transferase (GST)-CP-I (60 kDa) and GST-CP-C (62 kDa) fusion proteins were produced. They were separated by SDS-PAGE and electroeluted before immunization of Swiss mice for monoclonal antibody (MAb) production. Two MAbs specific to CP-N and one MAb specific to CP-C were selected for use for detection of natural IMNV infections in Penaeus vannamei by dot blotting, Western blotting and immunohistochemistry. There was no cross-reaction with shrimp tissues or common shrimp viruses including white spot syndrome virus (WSSV), yellow head virus (YHV), Taura syndrome virus (TSV), Penaeus monodon nucleopolyhedrovirus (PemoNPV), Penaeus stylirostris densovirus (PstDNV) and Penaeus monodon densovirus (PmDNV). The detection sensitivities of the MAbs were approximately 6 fmol/spot of purified recombinant intein-CP-N protein and 8 fmol/spot of GST-CP-C as determined by dot blotting. A combination of all three MAbs resulted in a twofold increase in sensitivity over use of any single MAb. However, this sensitivity was approximately 10 times lower than that of one-step RT-PCR using the same sample. Immunohistochemical analysis using MAbs specific to CP-N and CP-C in IMNV-infected shrimp revealed intense staining patterns in muscles, the lymphoid organ, gills, the heart, hemocytes and connective tissue.

  16. Characterization of Four Type IV Pilin Homologues in Stigmatella aurantiaca DSM17044 by Heterologous Expression in Myxococcus xanthus

    PubMed Central

    Pan, Hongwei; Zhou, Xiuwen; Liu, Xin; Luo, Ningning; Hu, Wei; Li, Yuezhong

    2013-01-01

    As prokaryotic models for multicellular development, Stigmatellaaurantiaca and Myxococcus xanthus share many similarities in terms of social behaviors, such as gliding motility. Our current understanding of myxobacterial grouped-cell motilities comes mainly from the research on M. xanthus, which shows that filamentous type IV pili (TFP), composed of type IV pilin (also called PilA protein) subunits, are the key apparatus for social motility (S-motility). However, little is known about the pilin protein in S. aurantiaca. We cloned and sequenced four genes (pilASa1~4) from S. aurantiaca DSM17044 that are homologous to pilAMx (pilA gene in M. xanthus DK1622). The homology and similarities among PilASa proteins and other myxobacterial homologues were systematically analyzed. To determine their potential biological functions, the four pilASa genes were expressed in M. xanthus DK10410 (ΔpilAMx), which did not restore S-motility on soft agar or EPS production to host cells. After further analysis of the motile behaviors in a methylcellulose solution, the M. xanthus strains were categorized into three types. YL6101, carrying pilASa1, and YL6104, carrying pilASa4, produced stable but unretractable surface pili; YL6102, carrying pilASa2, produced stable surface pili and exhibited reduced TFP-dependent motility in methylcellulose; YL6103, carrying pilASa3, produced unstable surface pili. Based on these findings, we propose that pilASa2 might be responsible for the type IV pilin production involved in group motility in S. aurantiaca DSM17044. After examining the developmental processes, it was suggested that the expression of PilASa4 protein might have positive effects on the fruiting body formation of M. xanthus DK10410 cells. Moreover, the formation of fruiting body in M. xanthus cells with stable exogenous TFPSa were compensated by mixing them with S. aurantiaca DSM17044 cells. Our results shed some light on the features and functions of type IV pilin homologues in S

  17. Structure-function relationships of purple acid phosphatase from red kidney beans based on heterologously expressed mutants.

    PubMed

    Truong, Ngoc Thanh; Naseri, Joseph Itor; Vogel, Andreas; Rompel, Annette; Krebs, B

    2005-08-01

    Purple acid phosphatases are binuclear metalloenzymes, which catalyze the conversion of orthophosphoric monoesters to alcohol and orthophosphate. The enzyme from red kidney beans is characterized with a Fe(III)-Zn(II) active center. So far, the reaction mechanisms postulated for PAPs assume the essentiality of two amino acids, residing near the bimetallic active site. Based on the amino acid sequence of kidney bean PAP (kbPAP), residues H296 and H202 are believed to be essential for catalytic function of the enzyme. In the present study, the role of residue H202 has been elucidated. Mutants H202A and H202R were prepared by site-directed mutagenesis and expressed in baculovirus-infected insect cells. Based on kinetic studies, residue H202 is assumed to play a role in stabilizing the transition state, particularly in charge compensation, steric positioning of the substrate, and facilitating the release of the product by protonating the substrate leaving groups. The study confirmed the essentiality and elucidates the functional role of H202 in the catalytic mechanism of kbPAP.

  18. Crystallization and preliminary characterization of three different crystal forms of human saposin C heterologously expressed in Pichia pastoris

    SciTech Connect

    Schultz-Heienbrok, Robert; Rossocha, Maksim; Saenger, Wolfram

    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.

  19. Structure-function relationships of purple acid phosphatase from red kidney beans based on heterologously expressed mutants.

    PubMed

    Truong, Ngoc Thanh; Naseri, Joseph Itor; Vogel, Andreas; Rompel, Annette; Krebs, B

    2005-08-01

    Purple acid phosphatases are binuclear metalloenzymes, which catalyze the conversion of orthophosphoric monoesters to alcohol and orthophosphate. The enzyme from red kidney beans is characterized with a Fe(III)-Zn(II) active center. So far, the reaction mechanisms postulated for PAPs assume the essentiality of two amino acids, residing near the bimetallic active site. Based on the amino acid sequence of kidney bean PAP (kbPAP), residues H296 and H202 are believed to be essential for catalytic function of the enzyme. In the present study, the role of residue H202 has been elucidated. Mutants H202A and H202R were prepared by site-directed mutagenesis and expressed in baculovirus-infected insect cells. Based on kinetic studies, residue H202 is assumed to play a role in stabilizing the transition state, particularly in charge compensation, steric positioning of the substrate, and facilitating the release of the product by protonating the substrate leaving groups. The study confirmed the essentiality and elucidates the functional role of H202 in the catalytic mechanism of kbPAP. PMID:16009331

  20. Thermostability and Ca2+ binding properties of wild type and heterologously expressed PsbO protein from cyanobacterial photosystem II.

    PubMed

    Loll, Bernhard; Gerold, Gisa; Slowik, Daria; Voelter, Wolfgang; Jung, Christiane; Saenger, Wolfram; Irrgang, Klaus-Dieter

    2005-03-29

    Oxygenic photosynthesis takes place in the thylakoid membrane of cyanobacteria, algae, and higher plants. Initially light is absorbed by an oligomeric pigment-protein complex designated as photosystem II (PSII), which catalyzes light-induced water cleavage under release of molecular oxygen for the biosphere on our planet. The membrane-extrinsic manganese stabilizing protein (PsbO) is associated on the lumenal side of the thylakoids close to the redox-active (Mn)(4)Ca cluster at the catalytically active site of PSII. Recombinant PsbO from the thermophilic cyanobacterium Thermosynechococcus elongatus was expressed in Escherichia coli and spectroscopically characterized. The secondary structure of recombinant PsbO (recPsbO) was analyzed in the absence and presence of Ca(2+) using Fourier transform infrared spectroscopy (FTIR) and circular dichroism spectropolarimetry (CD). No significant structural changes could be observed when the PSII subunit was titrated with Ca(2+) in vitro. These findings are compared with data for spinach PsbO. Our results are discussed in the light of the recent 3D-structural analysis of the oxygen-evolving PSII and structural/thermodynamic differences between the two homologous proteins from thermophilic cyanobacteria and plants.

  1. A xylanase from Streptomyces sp. FA1: heterologous expression, characterization, and its application in Chinese steamed bread.

    PubMed

    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. PMID:26803505

  2. Construction of chromosomally located T7 expression system for production of heterologous secreted proteins in Bacillus subtilis.

    PubMed

    Chen, Po Ting; Shaw, Jei-Fu; Chao, Yun-Peng; David Ho, Tuan-Hua; Yu, Su-May

    2010-05-12

    Bacillus subtilis is most commonly employed for secretion of recombinant proteins. To circumvent the problems caused by using plasmids, the T7 expression system known for its high efficiency was rebuilt in B. subtilis. Accordingly, a markerless and replicon-free method was developed for genomic insertion of DNAs. By the act of homologous recombination via the guide DNA, a suicidal vector carrying the gene of interest was integrated into genomic loci of bacteria. Removal of the inserted selection marker and replicon flanked by FRT sites was mediated by the FLP recombinase. By using the mentioned system, B. subtilis strain PT5 was constructed to harbor a genomic copy of the spac promoter-regulated T7 gene 1 located at wprA (encoding the cell wall-associated protease). Similarly, the T7 promoter-driven nattokinase or endoglucanase E1 of Thermomonospora fusca genes were also integrated into mpr (encoding an extracellular protease) of strain PT5. Consequently, the integrant PT5/Mmp-T7N or PT5/MT1-E1 resulted in a "clean" producer strain deprived of six proteases. After 24 h, the strain receiving induction was able to secret nattokinase and endoglucanase E1 with the volumetric activity reaching 10860 CU/mL and 8.4 U/mL, respectively. This result clearly indicates the great promise of the proposed approach for high secretion of recombinant proteins in B. subtilis.

  3. Characterization and heterologous expression of a new matrix attachment region binding protein from the unicellular green alga Dunaliella salina.

    PubMed

    Wang, Tianyun; Hou, Guiqin; Wang, Yafeng; Xue, Lexun

    2010-12-01

    Although interactions between the nuclear matrix and special regions of chromosomal DNA called matrix attachment regions (MARs) are implicated in various nuclear functions, the understanding of the regulatory mechanism of MARs is still poor. A few MAR-binding proteins (MARBP) have been isolated from some plants and animals, but not from the unicellular algae. Here, we identify a novel MAR-binding protein, namely DMBP-1, from the halotolerant alga Dunaliella salina. The cDNA of DMBP-1 is 2322-bp long and contains a 1626 bp of an open reading frame encoding a polypeptide of 542 amino acids (59 kDa). The DMBP-1 expressed in Escherichia coli specifically binds A/T-rich MAR DNA. The DMBP-1 fused to green fluorescent protein appears only inside the nuclei of Chinese hamster ovarian cells transfected with the pEGFP-MBP, indicating that the protein is located in the nuclei. The findings mentioned above may contribute to better understanding of the nuclear matrix-MAR interactions.

  4. Heterologous expression and biochemical characterization of an endo-β-1,4-glucanase from Thermobifida fusca.

    PubMed

    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.

  5. Heterologous Expression and Characterization of a GH3 β-Glucosidase from Thermophilic Fungi Myceliophthora thermophila in Pichia pastoris.

    PubMed

    Zhao, Junqi; Guo, Chao; Tian, Chaoguang; Ma, Yanhe

    2015-09-01

    A novel β-glucosidase of glycoside hydrolase (GH) family 3 from Myceliophthora thermophila (mtbgl3b) was successfully expressed in Pichia pastoris. The full-length gene consists of 2613 bp nucleotides encoding a protein of 870 amino acids. MtBgl3b showed maximum activity at pH 5.0 and remained more than 70 % relative activity at 3.5-6.0. The enzyme displayed the highest activity at 60 °C and kept about 90 % relative activity for 50-65 °C; besides, the enzyme showed psychrophilic trait and remains 51 % relative activity at 40 °C. MtBgl3b exhibited good stability over a wide pH range of 3.0-10.0 and was thermostable at 60 and 65 °C. The enzyme displayed highest activity towards p-nitrophenyl-β-D-glucopyranoside (pNPG), followed by p-nitrophenyl-D-cellobioside (pNPC), cellotetraose, cellotriose, cellobiose, and gentiobiose. When using 10 % cellobiose (w/v) as the substrate, the enzyme showed transglycosylation activity to produce the cellotriose. The kinetic parametric of K m and V max were 2.78 mM and 927.9 μM mg(-1) min(-1), respectively. Finally, the reaction mode of the enzyme and the substrates were analyzed by molecular docking approach.

  6. Crystallization and preliminary characterization of three different crystal forms of human saposin C heterologously expressed in Pichia pastoris

    PubMed Central

    Schultz-Heienbrok, Robert; Remmel, Natascha; Klingenstein, R.; Rossocha, Maksim; Sandhoff, Konrad; Saenger, Wolfram; Maier, Timm

    2006-01-01

    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. PMID:16511279

  7. Human GPR42 is a transcribed multisite variant that exhibits copy number polymorphism and is functional when heterologously expressed

    PubMed Central

    Puhl III, Henry L.; Won, Yu-Jin; Lu, Van B.; Ikeda, Stephen R.

    2015-01-01

    FFAR3 (GPR41) is a G-protein coupled receptor for which short-chain fatty acids serve as endogenous ligands. The receptor is found on gut enteroendocrine L-cells, pancreatic β-cells, and sympathetic neurons, and is implicated in obesity, diabetes, allergic airway disease, and altered immune function. In primates, FFAR3 is segmentally duplicated resulting in GPR42, a gene currently classified as a suspected pseudogene. In this study, we sequenced FFAR3 and GPR42 open reading frames from 56 individuals and found an unexpectedly high frequency of polymorphisms contributing to several complex haplotypes. We also identified a frequent (18.8%) structural variation that results in GPR42 copy number polymorphism. Finally, sequencing revealed that 50.6% of GPR42 haplotypes differed from FFAR3 by only a single non-synonymous substitution and that the GPR42 reference sequence matched only 4.4% of the alleles. Sequencing of cDNA from human sympathetic ganglia and colon revealed processed transcripts matching the GPR42 genotype. Expression of several GPR42 haplotypes in rat sympathetic neurons revealed diverse pharmacological phenotypes that differed in potency and efficacy. Our data suggest that GPR42 be reclassified as a functioning gene and that recognition of sequence and copy number polymorphism of the FFAR3/GPR42 complex be considered during genetic and pharmacological investigation of these receptors. PMID:26260360

  8. Heterologous Expression of Two Jatropha Aquaporins Imparts Drought and Salt Tolerance and Improves Seed Viability in Transgenic Arabidopsis thaliana.

    PubMed

    Khan, Kasim; Agarwal, Pallavi; Shanware, Arti; Sane, Vidhu Aniruddha

    2015-01-01

    Drought and high salinity are environmental conditions that cause adverse effects on the growth and productivity of crops. Aquaporins are small integral membrane proteins that belong to the family of the major intrinsic proteins (MIPs), with members in animals, plants and microbes, where they facilitate the transport of water and/or small neutral solutes thereby affecting water balance. In this study we characterized two aquaporin genes namely, plasma membrane intrinsic protein (PIP2;7) and tonoplast intrinsic protein TIP1;3 from Jatropha curcas that are localised to the plasma membrane and vacuole respectively. Transgenic Arabidopsis thaliana lines over-expressing JcPIP2;7 and JcTIP1;3 under a constitutive promoter show improved germination under high salt and mannitol compared to control seeds. These transgenic plants also show increased root length under abiotic stress conditions compared to wild type Col-0 plants. Transgenic lines exposed to drought conditions by withholding water for 20 days, were able to withstand water stress and attained normal growth after re-watering unlike control plants which could not survive. Transgenic lines also had better seed yield than control under salt stress. Importantly, seed viability of transgenic plants grown under high salt concentration was 35%-45% compared to less than 5% for control seeds obtained from plants growing under salt stress. The effect of JcPIP2;7 and JcTIP1;3 on improving germination and seed viability in drought and salinity make these important candidates for genetic manipulation of plants for growth in saline soils. PMID:26067295

  9. A unique mono- and diacylglycerol lipase from Penicillium cyclopium: heterologous expression, biochemical characterization and molecular basis for its substrate selectivity.

    PubMed

    Tan, Zhong-Biao; Li, Jian-Fang; Li, Xue-Ting; Gu, Ying; Wu, Min-Chen; Wu, Jing; Wang, Jun-Qing

    2014-01-01

    A cDNA gene encoding a mature peptide of the mono- and diacylglycerol lipase (abbreviated to PcMdl) from Penicillium cyclopium PG37 was cloned and expressed in Pichia pastoris GS115. The recombinant PcMdl (rePcMdl) with an apparent molecular weight of 39 kDa showed the highest activity (40.5 U/mL of culture supernatant) on 1,2-dibutyrin substrate at temperature 35°C and pH 7.5. The rePcMdl was stable at a pH range of 6.5-9.5 and temperatures below 35°C. The activity of rePcMdl was inhibited by Hg2+ and Fe3+, but not significantly affected by EDTA or the other metal ions such as Na+, K+, Li+, Mg2+, Zn2+, Ca2+, Mn2+, Cu2+, and Fe2+. PcMdl was identified to be strictly specific to mono- and diacylglycerol, but not triacylglycerol. Stereographic view of PcMdl docked with substrate (tri- or diacylglycerol) analogue indicated that the residue Phe256 plays an important role in conferring the substrate selectivity. Phe256 projects its side chain towards the substrate binding groove and makes the sn-1 moiety difficult to insert in. Furthermore, sn-1 moiety prevents the phosphorus atom (substitution of carboxyl carbon) from getting to the Oγ of Ser145, which results in the failure of triacylglycerol hydrolysis. These results should provide a basis for molecular engineering of PcMdl and expand its applications in industries. PMID:25051359

  10. Heterologous Expression of Two Jatropha Aquaporins Imparts Drought and Salt Tolerance and Improves Seed Viability in Transgenic Arabidopsis thaliana

    PubMed Central

    Khan, Kasim; Agarwal, Pallavi; Shanware, Arti; Sane, Vidhu Aniruddha

    2015-01-01

    Drought and high salinity are environmental conditions that cause adverse effects on the growth and productivity of crops. Aquaporins are small integral membrane proteins that belong to the family of the major intrinsic proteins (MIPs), with members in animals, plants and microbes, where they facilitate the transport of water and/or small neutral solutes thereby affecting water balance. In this study we characterized two aquaporin genes namely, plasma membrane intrinsic protein (PIP2;7) and tonoplast intrinsic protein TIP1;3 from Jatropha curcas that are localised to the plasma membrane and vacuole respectively. Transgenic Arabidopsis thaliana lines over-expressing JcPIP2;7 and JcTIP1;3 under a constitutive promoter show improved germination under high salt and mannitol compared to control seeds. These transgenic plants also show increased root length under abiotic stress conditions compared to wild type Col-0 plants. Transgenic lines exposed to drought conditions by withholding water for 20 days, were able to withstand water stress and attained normal growth after re-watering unlike control plants which could not survive. Transgenic lines also had better seed yield than control under salt stress. Importantly, seed viability of transgenic plants grown under high salt concentration was 35%-45% compared to less than 5% for control seeds obtained from plants growing under salt stress. The effect of JcPIP2;7 and JcTIP1;3 on improving germination and seed viability in drought and salinity make these important candidates for genetic manipulation of plants for growth in saline soils. PMID:26067295

  11. Heterologous Expression of Two Jatropha Aquaporins Imparts Drought and Salt Tolerance and Improves Seed Viability in Transgenic Arabidopsis thaliana.

    PubMed

    Khan, Kasim; Agarwal, Pallavi; Shanware, Arti; Sane, Vidhu Aniruddha

    2015-01-01

    Drought and high salinity are environmental conditions that cause adverse effects on the growth and productivity of crops. Aquaporins are small integral membrane proteins that belong to the family of the major intrinsic proteins (MIPs), with members in animals, plants and microbes, where they facilitate the transport of water and/or small neutral solutes thereby affecting water balance. In this study we characterized two aquaporin genes namely, plasma membrane intrinsic protein (PIP2;7) and tonoplast intrinsic protein TIP1;3 from Jatropha curcas that are localised to the plasma membrane and vacuole respectively. Transgenic Arabidopsis thaliana lines over-expressing JcPIP2;7 and JcTIP1;3 under a constitutive promoter show improved germination under high salt and mannitol compared to control seeds. These transgenic plants also show increased root length under abiotic stress conditions compared to wild type Col-0 plants. Transgenic lines exposed to drought conditions by withholding water for 20 days, were able to withstand water stress and attained normal growth after re-watering unlike control plants which could not survive. Transgenic lines also had better seed yield than control under salt stress. Importantly, seed viability of transgenic plants grown under high salt concentration was 35%-45% compared to less than 5% for control seeds obtained from plants growing under salt stress. The effect of JcPIP2;7 and JcTIP1;3 on improving germination and seed viability in drought and salinity make these important candidates for genetic manipulation of plants for growth in saline soils.

  12. A Recombinant Chimeric Ad5/3 Vector Expressing a Multistage Plasmodium Antigen Induces Protective Immunity in Mice Using Heterologous Prime-Boost Immunization Regimens.

    PubMed

    Cabrera-Mora, Monica; Fonseca, Jairo Andres; Singh, Balwan; Zhao, Chunxia; Makarova, Natalia; Dmitriev, Igor; Curiel, David T; Blackwell, Jerry; Moreno, Alberto

    2016-10-01

    An ideal malaria vaccine should target several stages of the parasite life cycle and induce antiparasite and antidisease immunity. We have reported a Plasmodium yoelii chimeric multistage recombinant protein (P. yoelii linear peptide chimera/recombinant modular chimera), engineered to express several autologous T cell epitopes and sequences derived from the circumsporozoite protein and the merozoite surface protein 1. This chimeric protein elicits protective immunity, mediated by CD4(+) T cells and neutralizing Abs. However, experimental evidence, from pre-erythrocytic vaccine candidates and irradiated sporozoites, has shown that CD8(+) T cells play a significant role in protection. Recombinant viral vectors have been used as a vaccine platform to elicit effective CD8(+) T cell responses. The human adenovirus (Ad) serotype 5 has been tested in malaria vaccine clinical trials with excellent safety profile. Nevertheless, a major concern for the use of Ad5 is the high prevalence of anti-vector neutralizing Abs in humans, hampering its immunogenicity. To minimize the impact of anti-vector pre-existing immunity, we developed a chimeric Ad5/3 vector in which the knob region of Ad5 was replaced with that of Ad3, conferring partial resistance to anti-Ad5 neutralizing Abs. Furthermore, we implemented heterologous Ad/protein immunization regimens that include a single immunization with recombinant Ad vectors. Our data show that immunization with the recombinant Ad5/3 vector induces protective efficacy indistinguishable from that elicited by Ad5. Our study also demonstrates that the dose of the Ad vectors has an impact on the memory profile and protective efficacy. The results support further studies with Ad5/3 for malaria vaccine development. PMID:27574299

  13. Increased mannosylphosphorylation of N-glycans by heterologous expression of YlMPO1 in glyco-engineered Saccharomyces cerevisiae for mannose-6-phosphate modification.

    PubMed

    Gil, Jin Young; Park, Jeong-Nam; Lee, Kyung Jin; Kang, Ji-Yeon; Kim, Yeong Hun; Kim, Seonghun; Kim, Sang-Yoon; Kwon, Ohsuk; Lim, Yong Taik; Kang, Hyun Ah; Oh, Doo-Byoung

    2015-07-20

    Mannosylphosphorylated N-glycans found in yeasts can be converted to those containing mannose-6-phosphate, which is a key factor for lysosomal targeting. In the traditional yeast Saccharomyces cerevisiae, both ScMNN4 and ScMNN6 genes are required for efficient mannosylphosphorylation. ScMnn4 protein has been known to be a positive regulator of ScMnn6p, a real enzyme for mannosylphosphorylation. On the other hand, YlMpo1p, a ScMnn4p homologue, mediates mannosylphosphorylation in Yarrowia lypolytica without the involvement of ScMnn6p homologues. In this study, we show that heterologous expression of YlMpo1p can perform and enhance mannosylphosphorylation in S. cerevisiae in the absence of ScMnn4p and ScMnn6p. Moreover, the level of mannosylphosphorylation of N-glycans enhanced by YlMpo1p overexpression is much higher than that with ScMnn4p overexpression, and this is highlighted further in Scmnn4- and Scmnn6-disrupted mutants. When YlMpo1p overexpression is applied to glyco-engineered S. cerevisiae in which the synthesis of immunogenic glycans is abolished, a great increase of bi-mannosylphosphorylated glycan is observed. Through an in vitro process involving the uncapping of the outer mannose residue, this bi-mannosylphosphorylated structure is changed to a bi-phosphorylated structure with high affinity for mannose-6-phosphate receptor. The superior ability of YlMpo1p to increase bi-mannosylphosphorylated glycan in yeast shows promise for the production of therapeutic enzymes with improved lysosomal targeting capability.

  14. Heterologous expression and characterization of a novel thermo-halotolerant endoglucanase Cel5H from Dictyoglomus thermophilum.

    PubMed

    Shi, Runrun; Li, Zhimin; Ye, Qin; Xu, Jianhe; Liu, Yan

    2013-08-01

    A novel β-1,4-endoglucanase gene was cloned from Dictyoglomus thermophilum, designated as Cel5H for being a member of glycoside hydrolase family 5. The purified recombinant endoglucanase showed high hydrolytic activities on carboxylmethyl cellulose with a broad optimal temperature of 50-85°C and an optimal pH of 5.0. Furthermore, this enzyme was highly thermostable with a half-life of 336 h at 70°C and retained more than 80% of the initial activity after 135 days incubation at 50°C. To enhance the performance of the thermophilic endoglucanase, chimeric enzymes containing Cel5H and syncretic cellulose binding module (CBM) were constructed. The results showed that all the CBMs were effective. In addition, Cel5H was highly tolerant against high salt concentration and distinguished from salt-tolerant bacteria since it was independent of high salt concentration. Three-dimensional structure of Cel5H was developed by homology modeling methods and surface electrostatic analysis was performed. PMID:23747445

  15. pZMO7-Derived shuttle vectors for heterologous protein expression and proteomic applications in the ethanol-producing bacterium Zymomonas mobilis

    PubMed Central

    2014-01-01

    Background The ethanol-producing bacterium Zymomonas mobilis has attracted considerable scientific and commercial interest due to its exceptional physiological properties. Shuttle vectors derived from native plasmids have previously been successfully used for heterologous gene expression in this bacterium for a variety of purposes, most notably for metabolic engineering applications. Results A quantitative PCR (qPCR) approach was used to determine the copy numbers of two endogenous double stranded DNA plasmids: pZMO1A (1,647 bp) and pZMO7 (pZA1003; 4,551 bp) within the NCIMB 11163 strain of Z. mobilis. Data indicated pZMO1A and pZMO7 were present at ca. 3-5 and ca. 1-2 copies per cell, respectively. A ca. 1,900 bp fragment from plasmid pZMO7 was used to construct two Escherichia coli – Z. mobilis shuttle vectors (pZ7C and pZ7-184). The intracellular stabilities and copy numbers of pZ7C and pZ7-184 were characterized within the NCIMB 11163, ATCC 29191 and (ATCC 10988-derived) CU1 Rif2 strains of Z. mobilis. Both shuttle vectors could be stably maintained within the ATCC 29191 strain (ca. 20-40 copies per cell), and the CU1 Rif2 strain (ca. 2-3 copies per cell), for more than 50 generations in the absence of an antibiotic selectable marker. A selectable marker was required for shuttle vector maintenance in the parental NCIMB 11163 strain; most probably due to competition for replication with the endogenous pZMO7 plasmid molecules. N-terminal glutathione S-transferase (GST)-fusions of four endogenous proteins, namely the acyl-carrier protein (AcpP); 2-dehydro-3-deoxyphosphooctonate aldolase (KdsA); DNA polymerase III chi subunit (HolC); and the RNA chaperone protein Hfq; were successfully expressed from pZ7C-derived shuttle vectors, and their protein-protein binding interactions were analyzed in Z. mobilis ATCC 29191. Using this approach, proteins that co-purified with AcpP and KdsA were identified. Conclusions We show that a shuttle vector-based protein affinity

  16. Comparative analysis of heterologous expression, biochemical characterization optimal production of an alkaline α-amylase from alkaliphilic Alkalimonas amylolytica in Escherichia coli and Pichia pastoris.

    PubMed

    Yang, Haiquan; Liu, Long; Shin, Hyun-dong; Chen, Rachel R; Li, Jianghua; Du, Guocheng; Chen, Jian

    2013-01-01

    An alkaline α-amylase gene from alkaliphilic Alkalimonas amylolytica was synthesized based on the preferred codon usage of Escherichia coli and Pichia pastoris, respectively, and then was expressed in the according heterologous host, E. coli BL21 (DE3) and P. pastoris GS115. The alkaline α-amylase expressed in E. coli was designated AmyA, whereas that produced by P. pastoris was designated AmyB. The specific activity of AmyA and AmyB was 16.0 and 16.6 U/mg at pH 9.5 and 50°C, respectively. The optimal pH and pH stability of AmyA and AmyB were similar, whereas the optimum temperature and thermal stability of AmyB were slightly enhanced compared with those of AmyA. The AmyA and AmyB had a similar melting temperature of 64°C and the same catalytic efficiency (k(cat) /K(m) ) of 2.0 × 10(6) L/(mol min). AmyA and AmyB were slightly activated by 1 mM Co(2+) , Ca(2+) , or Na(+) , but inhibited by all other metal ions (K(+) , Mg(2+) , Fe(3+) , Fe(2+) , Zn(2+) , Mn(2+) , and Cu(2+) ). Tween 80 or Tween 60 (10% (w/v)) had little influence on the stability of AmyA and AmyB, while the 10% (w/v) sodium dodecyl sulfate caused the complete loss of AmyA and AmyB activities. The AmyA and AmyB were stable in the presence of solid detergents (washing powder), while were less stable in liquid detergents. Under the optimal conditions in 3-L bioreactor, the extracellular AmyB activity reached 600 U/mL, which was about 10 times as that of AmyA. These results indicated that P. pastoris was a preferable host for alkaline α-amylase expression and the produced alkaline α-amylase had a certain application potential in solid detergents.

  17. Molecular Cloning, Heterologous Expression, and Functional Characterization of an NADPH-Cytochrome P450 Reductase Gene from Camptotheca acuminata, a Camptothecin-Producing Plant

    PubMed Central

    Chen, Fei; Yang, Yun; Yang, Lixia; Zhang, Guolin; Luo, Yinggang

    2015-01-01

    Camptothecin (CAM), a complex pentacyclic pyrroloqinoline alkaloid, is the starting material for CAM-type drugs that are well-known antitumor plant drugs. Although many chemical and biological research efforts have been performed to produce CAM, a few attempts have been made to uncover the enzymatic mechanism involved in the biosynthesis of CAM. Enzyme-catalyzed oxidoreduction reactions are ubiquitously presented in living organisms, especially in the biosynthetic pathway of most secondary metabolites such as CAM. Due to a lack of its reduction partner, most catalytic oxidation steps involved in the biosynthesis of CAM have not been established. In the present study, an NADPH-cytochrome P450 reductase (CPR) encoding gene CamCPR was cloned from Camptotheca acuminata, a CAM-producing plant. The full length of CamCPR cDNA contained an open reading frame of 2127-bp nucleotides, corresponding to 708-amino acid residues. CamCPR showed 70 ~ 85% identities to other characterized plant CPRs and it was categorized to the group II of CPRs on the basis of the results of multiple sequence alignment of the N-terminal hydrophobic regions. The intact and truncate CamCPRs with N- or C-terminal His6-tag were heterologously overexpressed in Escherichia coli. The recombinant enzymes showed NADPH-dependent reductase activity toward a chemical substrate ferricyanide and a protein substrate cytochrome c. The N-terminal His6-tagged CamCPR showed 18- ~ 30-fold reduction activity higher than the C-terminal His6-tagged CamCPR, which supported a reported conclusion, i.e., the last C-terminal tryptophan of CPRs plays an important role in the discrimination between NADPH and NADH. Co-expression of CamCPR and a P450 monooxygenase, CYP73A25, a cinnamate 4-hydroxylase from cotton, and the following catalytic formation of p-coumaric acid suggested that CamCPR transforms electrons from NADPH to the heme center of P450 to support its oxidation reaction. Quantitative real-time PCR analysis showed that

  18. Characterization of Endogenous Sodium Channels in the ND7-23 Neuroblastoma Cell Line: Implications for Use as a Heterologous Ion Channel Expression System Suitable for Automated Patch Clamp Screening

    PubMed Central

    Zidar, Nace; Kikelj, Danijel; Kirby, Robert W.

    2016-01-01

    Abstract The rodent neuroblastoma cell line, ND7-23, is used to express voltage-dependent sodium (Nav) and other neuronal ion channels resistant to heterologous expression in Chinese hamster ovary (CHO) or human embryonic kidney (HEK) cells. Their advantage is that they provide endogenous factors and signaling pathways to promote ion channel peptide folding, expression, and function at the cell surface and are also amenable to automated patch clamping. However, ND7-23 cells exhibit endogenous tetrodotoxin (TTX)-sensitive Nav currents, and molecular profiling has revealed the presence of Nav1.2, Nav1.3, Nav1.6, and Nav1.7 transcripts, but no study has determined which subtypes contribute to functional channels at the cell surface. We profiled the repertoire of functional Nav channels endogenously expressed in ND7-23 cells using the QPatch automated patch clamp platform and selective toxins and small molecules. The potency and subtype selectivity of the ligands (Icagen compound 68 from patent US-20060025415-A1-20060202, 4,9 anhydro TTX, and Protoxin-II) were established in human Nav1.3, Nav1.6, and Nav1.7 channel cell lines before application of selective concentrations to ND7-23 cells. Our data confirm previous studies that >97% of macroscopic Nav current in ND7-23 cells is carried by TTX-sensitive channels (300 nM TTX) and that Nav1.7 is the predominant channel contributing to this response (65% of peak inward current), followed by Nav1.6 (∼20%) and negligible Nav1.3 currents (∼2%). In addition, our data are the first to assess the Nav1.6 potency (50% inhibitory concentration [IC50] of 33 nM) and selectivity (50-fold over Nav1.7) of 4,9 anhydro TTX in human Nav channels expressed in mammalian cells, confirming previous studies of rodent Nav channels expressed in oocytes and HEK cells. PMID:26991361

  19. Harnessing the beneficial heterologous effects of vaccination.

    PubMed

    Goodridge, Helen S; Ahmed, S Sohail; Curtis, Nigel; Kollmann, Tobias R; Levy, Ofer; Netea, Mihai G; Pollard, Andrew J; van Crevel, Reinout; Wilson, Christopher B

    2016-06-01

    Clinical evidence strongly suggests that certain live vaccines, in particular bacille Calmette-Guérin (BCG) and measles vaccines, can reduce all-cause mortality, most probably through protection against non-targeted pathogens in addition to the targeted pathogen. The underlying mechanisms are currently unknown. We discuss how heterologous lymphocyte activation and innate immune memory could promote protection beyond the intended target pathogen and consider how vaccinologists could leverage heterologous immunity to improve outcomes in vulnerable populations, in particular the very young and the elderly. PMID:27157064

  20. The influence of cis-acting P1 protein and translational elements on the expression of Potato virus Y helper-component proteinase (HCPro) in heterologous systems and its suppression of silencing activity.

    PubMed

    Tena Fernández, Fátima; González, Inmaculada; Doblas, Paula; Rodríguez, César; Sahana, Nandita; Kaur, Harpreet; Tenllado, Francisco; Praveen, Shelly; Canto, Tomas

    2013-06-01

    In the Potyvirus genus, the P1 protein is the first N-terminal product processed from the viral polyprotein, followed by the helper-component proteinase (HCPro). In silencing suppression patch assays, we found that Potato virus Y (PVY) HCPro expressed from a P1-HCPro sequence increased the accumulation of a reporter gene, whereas protein expressed from an HCPro sequence did not, even with P1 supplied in trans. This enhancing effect of P1 has been noted in other potyviruses, but has remained unexplained. We analysed the accumulation of PVY HCPro in infiltrated tissues and found that it was higher when expressed from P1-HCPro than from HCPro sequences. Co-expression of heterologous suppressors increased the steady-state level of mRNA expressed from the HCPro sequence, but not that of protein. This suggests that, in the absence of P1 upstream, either HCPro acquires a conformation that affects negatively its activity or stability, or that its translation is reduced. To test these options, we purified HCPro expressed in the presence or absence of upstream P1, and found no difference in purification pattern and final soluble state. By contrast, alteration of the Kozak context in the HCPro mRNA sequence to favour translation increased partially suppressor accumulation and activity. Furthermore, protein activity was not lower than in protein expressed from P1-HCPro sequences. Thus, a direct role for P1 on HCPro suppressor activity or stability, by influencing its conformation during translation, can be excluded. However, P1 could still have an indirect effect favouring HCPro accumulation. Our data highlight the relevance of cis-acting translational elements in the heterologous expression of HCPro.

  1. Construction and expression of a heterologous protein in Lactococcus lactis by using the nisin-controlled gene expression system: the case of the PRRSV ORF6 gene.

    PubMed

    Wang, Z H; Wang, Y L; Zeng, X Y

    2014-01-01

    The porcine reproductive and respiratory syndrome virus (PRRSV) continues to be a threat, exerting significant economic effects on the swine industry worldwide. However, none of the current commercially available vaccines can completely prevent respiratory infection, trans-placental transmission, pig-to-pig transmission of the virus, or maintain immune protection in sows. This study provides information on PRRSV and a review of available options for PRRS control strategies based on its pathogenic characteristics, immune properties, and biological characteristics. In this study, the nisin-controlled expression system of Lactococcus lactis was selected as a vector to express the ORF6 gene of PRRSV. Food-grade recombinant, L. lactis PNZ8149/NZ3900-M/PRRS, which contained the lactose operon, was successfully constructed. The molecular weight of the expressed recombinant protein was approximately 19 kDa. Furthermore, the recombinant protein was located on the surface of L. lactis and showed reactogenicity with the antibody against PRRSV. Results of this study are expected to lay a theoretical foundation for development of genetically engineered L. lactis mucosal vaccines and to provide information related to its immune activity and adjuvant effects. PMID:24634130

  2. Additive Expression of Consolidated Memory through Drosophila Mushroom Body Subsets.

    PubMed

    Yang, Chu-Huai; Shih, Meng-Fu Maxwell; Chang, Ching-Ching; Chiang, Meng-Hsuan; Shih, Hsiang-Wen; Tsai, Ya-Lun; Chiang, Ann-Shyn; Fu, Tsai-Feng; Wu, Chia-Lin

    2016-05-01

    Associative olfactory memory in Drosophila has two components called labile anesthesia-sensitive memory and consolidated anesthesia-resistant memory (ARM). Mushroom body (MB) is a brain region critical for the olfactory memory and comprised of 2000 neurons that can be classified into αβ, α'β', and γ neurons. Previously we demonstrated that two parallel pathways mediated ARM consolidation: the serotonergic dorsal paired medial (DPM)-αβ neurons and the octopaminergic anterior paired lateral (APL)-α'β' neurons. This finding prompted us to ask how this composite ARM is retrieved. Here, we showed that blocking the output of αβ neurons and that of α'β' neurons each impaired ARM retrieval, and blocking both simultaneously had an additive effect. Knockdown of radish and octβ2R in αβ and α'β' neurons, respectively, impaired ARM. A combinatorial assay of radish mutant background rsh1 and neurotransmission blockade confirmed that ARM retrieved from α'β' neuron output is independent of radish. We identified MBON-β2β'2a and MBON-β'2mp as the MB output neurons downstream of αβ and α'β' neurons, respectively, whose glutamatergic transmissions also additively contribute to ARM retrieval. Finally, we showed that α'β' neurons could be functionally subdivided into α'β'm neurons required for ARM retrieval, and α'β'ap neurons required for ARM consolidation. Our work demonstrated that two parallel neural pathways mediating ARM consolidation in Drosophila MB additively contribute to ARM expression during retrieval.

  3. Using fluorometry and ion-sensitive microelectrodes to study the functional expression of heterologously-expressed ion channels and transporters in Xenopus oocytes

    PubMed Central

    Musa-Aziz, Raif; Boron, Walter F.; Parker, Mark D.

    2010-01-01

    The Xenopus laevis oocyte is a model system for the electrophysiological study of exogenous ion transporters. Three main reasons make the oocyte suitable for this purpose: (a) it has a large cell size (~1 mm diameter), (b) it has an established capacity to produce—from microinjected mRNAs or cRNAs—exogenous ion transporters with close-to-physiological post-translational modifications and actions, and (c) its membranes contain endogenous ion-transport activities which are usually smaller in magnitude than the activities of exogenously-expressed ion transporters. The expression of ion-transporters as green-fluorescent-protein fusions allows the fluorometric assay of transporter yield in living oocytes. Monitoring of transporter-mediated movement of ions such as Cl−, H+ (and hence base equivalents like OH−1 and HCO3−), K+, and Na+ is achieved by positioning the tips of ion-sensitive microelectrodes inside the oocyte and/or at the surface of the oocyte plasma membrane. The use of ion-sensitive electrodes is critical for studying net ion-movements mediated by electroneutral transporters. The combined use of fluorometry and electrophysiology expedites transporter study by allowing measurement of transporter yield prior to electrophysiological study and correlation of relative transporter yield with transport rates. PMID:20051266

  4. Heterologous Production of Curcuminoids

    PubMed Central

    Rodrigues, J. L.; Prather, K. L. J.

    2015-01-01

    SUMMARY Curcuminoids, components of the rhizome of turmeric, show several beneficial biological activities, including anticarcinogenic, antioxidant, anti-inflammatory, and antitumor activities. Despite their numerous pharmaceutically important properties, the low natural abundance of curcuminoids represents a major drawback for their use as therapeutic agents. Therefore, they represent attractive targets for heterologous production and metabolic engineering. The understanding of biosynthesis of curcuminoids in turmeric made remarkable advances in the last decade, and as a result, several efforts to produce them in heterologous organisms have been reported. The artificial biosynthetic pathway (e.g., in Escherichia coli) can start with the supplementation of the amino acid tyrosine or phenylalanine or of carboxylic acids and lead to the production of several natural curcuminoids. Unnatural carboxylic acids can also be supplemented as precursors and lead to the production of unnatural compounds with possibly novel therapeutic properties. In this paper, we review the natural conversion of curcuminoids in turmeric and their production by E. coli using an artificial biosynthetic pathway. We also explore the potential of other enzymes discovered recently or already used in other similar biosynthetic pathways, such as flavonoids and stilbenoids, to increase curcuminoid yield and activity. PMID:25631288

  5. Heterologous Expression of MeLEA3: A 10 kDa Late Embryogenesis Abundant Protein of Cassava, Confers Tolerance to Abiotic Stress in Escherichia coli with Recombinant Protein Showing In Vitro Chaperone Activity.

    PubMed

    Barros, Nicolle L F; da Silva, Diehgo T; Marques, Deyvid N; de Brito, Fabiano M; dos Reis, Savio P; de Souza, Claudia R B

    2015-01-01

    Late embryogenesis abundant (LEA) proteins are small molecular weight proteins involved in acquisition of tolerance to drought, salinity, high temperature, cold, and freezing stress in many plants. Previous studies revealed a cDNA sequence coding for a 10 kDa atypical LEA protein, named MeLEA3, predicted to be located into mitochondria with potential role in salt stress response of cassava (Manihot esculenta Crantz). Here we aimed to produce the recombinant MeLEA3 protein by heterologous expression in Escherichia coli and evaluate the tolerance of bacteria expressing this protein under abiotic stress. Our result revealed that the recombinant MeLEA3 protein conferred a protective function against heat and salt stress in bacterial cells. Also, the recombinant MeLEA3 protein showed in vitro chaperone activity by protection of NdeI restriction enzyme activity under heat stress. PMID:25990084

  6. Heterologous production of the lipopeptide biosurfactant serrawettin W1 in Escherichia coli.

    PubMed

    Thies, Stephan; Santiago-Schübel, Beatrix; Kovačić, Filip; Rosenau, Frank; Hausmann, Rudolf; Jaeger, Karl-Erich

    2014-07-10

    The non-ionic biosurfactant serrawettin W1 is a lipopeptide produced by red-pigmented strains of Serratia marcescens which shows antimicrobial, antitumor and plant protecting properties. Here, we report a simple method for heterologous production of this biosurfactant. S. marcescens strain DSM12481 was identified as a novel serrawettin W1 producer and the respective nonribosomal peptide synthetase gene swrW was cloned and expressed in Escherichia coli BL21 Gold. Chemical analysis of heterologous serrawettin W1 revealed that E. coli mainly produced serrawettin with C10 fatty acids as does S. marcescens. Additionally, serrawettin species with longer fatty acids (C13, C14) were detected in S. marcescens which were absent in E. coli. The expression system described here paves the way for the large scale production of this biotechnologically important biosurfactant.

  7. Yeast systems for the commercial production of heterologous proteins.

    PubMed

    Buckholz, R G; Gleeson, M A

    1991-11-01

    Yeasts are attractive hosts for the production of heterologous proteins. Unlike prokaryotic systems, their eukaryotic subcellular organization enables them to carry out many of the post-translational folding, processing and modification events required to produce "authentic" and bioactive mammalian proteins. In addition, they retain the advantages of a unicellular microorganism, with respect to rapid growth and ease of genetic manipulation. The vast majority of yeast expression work has focused on the well-characterized baker's yeast Saccharomyces cerevisiae. However, with the development of DNA transformation technologies, a growing number of non-Saccharomyces yeasts are becoming available as hosts for recombinant polypeptide production. These include Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, Schizosaccharomyces pombe, Schwanniomyces occidentalis and Yarrowia lipolytica. The performance of these alternative yeast expression systems is reviewed here relative to S. cerevisiae, and the advantages and limitations of these systems are discussed.

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

    PubMed Central

    Chen, Fengping; Lin, Dong; Wang, Jingyuan; Li, Botao; Duan, Hongxia; Liu, Junli; Liu, Xili

    2015-01-01

    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. PMID:26042103

  9. Regulated delayed expression of rfc enhances the immunogenicity and protective efficacy of a heterologous antigen delivered by live attenuated Salmonella enterica vaccines.

    PubMed

    Kong, Qingke; Liu, Qing; Jansen, Angela M; Curtiss, Roy

    2010-08-23

    The Salmonella rfc gene encodes the O-antigen polymerase. We constructed three strains in which we replaced the native rfc promoter with the arabinose-dependent araC P(BAD) promoter so that rfc expression was dependent on exogenously supplied arabinose provided during in vitro growth. The three mutant strains were designed to synthesize different amounts of Rfc by altering the ribosome-binding sequence and start codon. We examined these strains for a number of in vitro characteristics compared to an isogenic Deltarfc mutant and the wild-type parent strain. One promoter-replacement mutation, DeltaP(rfc174), yielded an optimal profile, exhibiting wild-type characteristics when grown with arabinose, and Deltarfc characteristics when grown without arabinose. In addition, when administered orally, the DeltaP(rfc174) strain was completely attenuated in for virulence in mice. The DeltaP(rfc174) mutation was introduced into attenuated Salmonella vaccine strain chi9241 (DeltapabA DeltapabB DeltaasdA) followed by introduction of an Asd(+) balanced-lethal plasmid to designed for expression of the pneumococcal surface protein PspA. Mice immunized with either chi9241 or its DeltaP(rfc174) derivative expressing pspA were protected against S. pneumoniae challenge.

  10. Heterologous Expression Implicates a GATA Factor in Regulation of Nitrogen Metabolic Genes and Ion Homeostasis in the Halotolerant Yeast Debaryomyces hansenii†

    PubMed Central

    García-Salcedo, Raúl; Casamayor, Antonio; Ruiz, Amparo; González, Asier; Prista, Catarina; Loureiro-Dias, Maria C.; Ramos, José; Ariño, Joaquín

    2006-01-01

    The yeast Debaryomyces hansenii has a remarkable capacity to proliferate in salty and alkaline environments such as seawater. A screen for D. hansenii genes able to confer increased tolerance to high pH when overexpressed in Saccharomyces cerevisiae yielded a single gene, named here DhGZF3, encoding a putative negative GATA transcription factor related to S. cerevisiae Dal80 and Gzf3. Overexpression of this gene in wild-type S. cerevisiae increased caffeine and rapamycin tolerance, blocked growth in low glucose concentrations and nonfermentable carbon sources, and resulted in lithium- and sodium-sensitive cells. Sensitivity to salt could be attributed to a reduced cation efflux, most likely because of a decrease in expression of the ENA1 Na+-ATPase gene. Overexpression of DhGZF3 did not affect cell growth in a gat1 mutant but was lethal in the absence of Gln3. These are positive factors that oppose both Gzf3 and Dal80. Genome-wide transcriptional profiling of wild-type cells overexpressing DhGZF3 shows decreased expression of a number of genes that are usually induced in poor nitrogen sources. In addition, the entire pathway leading to Lys biosynthesis was repressed, probably as a result of a decrease in the expression of the specific Lys14 transcription factor. In conclusion, our results demonstrate that DhGzf3 can play a role as a negative GATA transcription factor when expressed in S. cerevisiae and that it most probably represents the only member of this family in D. hansenii. These findings also point to the GATA transcription factors as relevant elements for alkaline-pH tolerance. PMID:16896222

  11. Heterologous expression of Lactobacillus casei RecO improved the multiple-stress tolerance and lactic acid production in Lactococcus lactis NZ9000 during salt stress.

    PubMed

    Wu, Chongde; Zhang, Juan; Du, Guocheng; Chen, Jian

    2013-09-01

    The aim of this study was to investigate the effect of nisin-inducible RecO expression on the stress tolerance of Lactococcus lactis NZ9000. RecO protein from Lactobacillus casei Zhang was introduced into Lactococcus lactis NZ9000 by using a nisin-inducible expression system. The recombinant strain (NZ-RecO) exhibited higher growth performances and survival rate compared with the control strain (NZ-Vector) under stress conditions. In addition, the NZ-RecO strain exhibited 1.37-, 1.41-, and 1.42-fold higher biomass, lactate production, lactate productivity, compared with the corresponding values for NZ-Vector during NaCl-stressed condition. Analysis of lactate dehydrogenase (LDH) activity showed that the production of RecO maintained the stability of LDH during salt stress. These results suggest that overproduction of RecO improved the multiple-stress tolerance and lactic acid production in Lactococcus lactis NZ9000 during salt stress. Results presented in this study may help to enhance the industrial utility of lactic acid bacteria. PMID:23796607

  12. Heterologous vaccine effects.

    PubMed

    Saadatian-Elahi, Mitra; Aaby, Peter; Shann, Frank; Netea, Mihai G; Levy, Ofer; Louis, Jacques; Picot, Valentina; Greenberg, Michael; Warren, William

    2016-07-25

    The heterologous or non-specific effects (NSEs) of vaccines, at times defined as "off-target effects" suggest that they can affect the immune response to organisms other than their pathogen-specific intended purpose. These NSEs have been the subject of clinical, immunological and epidemiological studies and are increasingly recognized as an important biological process by a growing group of immunologists and epidemiologists. Much remain to be learned about the extent and underlying mechanisms for these effects. The conference "Off-target effects of vaccination" held in Annecy-France (June 8-10 2015) intended to take a holistic approach drawing from the fields of immunology, systems biology, epidemiology, bioinformatics, public health and regulatory science to address fundamental questions of immunological mechanisms, as well as translational questions about vaccines NSEs. NSE observations were examined using case-studies on live attenuated vaccines and non-live vaccines followed by discussion of studies of possible biological mechanisms. Some possible pathways forward in the study of vaccines NSE were identified and discussed by the expert group. PMID:27312214

  13. Heterologously expressed Aspergillus aculeatus β-glucosidase in Saccharomyces cerevisiae is a cost-effective alternative to commercial supplementation of β-glucosidase in industrial ethanol production using Trichoderma reesei cellulases.

    PubMed

    Treebupachatsakul, Treesukon; Nakazawa, Hikaru; Shinbo, Hideaki; Fujikawa, Hiroki; Nagaiwa, Asami; Ochiai, Nobuhiro; Kawaguchi, Takashi; Nikaido, Mitsuru; Totani, Kazuhide; Shioya, Koki; Shida, Yosuke; Morikawa, Yasushi; Ogasawara, Wataru; Okada, Hirofumi

    2016-01-01

    Trichoderma reesei is a filamentous organism that secretes enzymes capable of degrading cellulose to cellobiose. The culture supernatant of T. reesei, however, lacks sufficient activity to convert cellobiose to glucose using β-glucosidase (BGL1). In this study, we identified a BGL (Cel3B) from T. reesei (TrCel3B) and compared it with the active β-glucosidases from Aspergillus aculeatus (AaBGL1). AaBGL1 showed higher stability and conversion of sugars to ethanol compared to TrCel3B, and therefore we chose to express this recombinant protein for use in fermentation processes. We expressed the recombinant protein in the yeast Saccharomyces cerevisiae, combined it with the superb T. reesei cellulase machinery and used the combination in a simultaneous saccharification and fermentation (SSF) process, with the hope that the recombinant would supplement the BGL activity. As the sugars were processed, the yeast immediately converted them to ethanol, thereby eliminating the problem posed by end product inhibition. Recombinant AaBGL1 activity was compared with Novozyme 188, a commercially available supplement for BGL activity. Our results show that the recombinant protein is as effective as the commercial supplement and can process sugars with equal efficiency. Expression of AaBGL1 in S. cerevisiae increased ethanol production effectively. Thus, heterologous expression of AaBGL1 in S. cerevisiae is a cost-effective and efficient process for the bioconversion of ethanol from lignocellulosic biomass.

  14. Characterization and heterologous expression of hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/benzoyltransferase from elicited cell cultures of carnation, Dianthus caryophyllus L.

    PubMed

    Yang, Q; Reinhard, K; Schiltz, E; Matern, U

    1997-12-01

    Benzoyl-CoA:anthranilate N-benzoyltransferase catalyzes the first committed reaction of phytoalexin biosynthesis in carnation (Dianthus caryophyllus L.), and the product N-benzoylanthranilate is the precursor of several sets of dianthramides. The transferase activity is constitutively expressed in suspension-cultured carnation cells and can be rapidly induced by the addition of yeast extract. The enzyme was purified to homogeneity from yeast-induced carnation cells and shown to consist of a single polypeptide chain of 53 kDa. Roughly 20% of the sequence was identified by micro-sequencing of tryptic peptides, and some of these sequences differed in a few amino acid residues only suggesting the presence of isoenzymes. A specific 0.8 kb cDNA probe was generated by RT-PCR, employing degenerated oligonucleotide primers complementary to two of the tryptic peptides and using poly(A)+ RNA from elicited carnation cells. Five distinct benzoyltransferase clones were isolated from a cDNA library, and three cDNAs, pchcbt1-3, were sequenced and shown to encode full-size N-benzoyltransferases. The translated peptide sequences revealed more than 95% identity among these three clones. The additional two clones harbored insert sequences mostly homologous with pchcbt 1 but differing in the 3'-flanking regions due to variable usage of poly(A) addition sites. The identity of the clones was confirmed by matching the translated polypeptides with the tryptic enzyme sequences as well as by the activity of the benzoyltransferase expressed in Escherichia coli. Therefore, carnation encodes a small family of anthranilate N-benzoyltransferase genes. In vitro, the benzoyltransferases exhibited narrow substrate specificity for anthranilate but accepted a variety of aromatic acyl-CoAs. Catalytic rates with cinnamoyl- or 4-coumaroyl-CoA exceeded those observed with benzoyl-CoA, although the corresponding dianthramides did not accumulate in vivo. Thus the cDNAs described represent also the first

  15. Additives

    NASA Technical Reports Server (NTRS)

    Smalheer, C. V.

    1973-01-01

    The chemistry of lubricant additives is discussed to show what the additives are chemically and what functions they perform in the lubrication of various kinds of equipment. Current theories regarding the mode of action of lubricant additives are presented. The additive groups discussed include the following: (1) detergents and dispersants, (2) corrosion inhibitors, (3) antioxidants, (4) viscosity index improvers, (5) pour point depressants, and (6) antifouling agents.

  16. Effect of homologous serotonin receptor loop substitutions on the heterologous expression in Pichia of a chimeric acetylcholine-binding protein with alpha-bungarotoxin-binding activity.

    PubMed

    Paulo, Joao A; Hawrot, Edward

    2009-10-01

    The molluscan acetylcholine-binding protein (AChBP) is a soluble homopentameric homolog of the extracellular domain of various ligand-gated ion channels. Previous studies have reported that AChBP, when fused to the ion pore domain of the serotonin receptor (5HT(3A)R), can form a functional ligand-gated chimeric channel only if the AChBP loop regions between beta-strands beta1 and beta2 (beta1-beta2), beta6 and beta7 (beta6-beta7), and beta8 and beta9 (beta8-beta9) are replaced with those of the 5HT(3A)R. To investigate further the potential interactions among these three important loop regions in a membrane- and detergent-free system, we designed AChBP constructs in which loops beta1-beta2, beta6-beta7, and beta8-beta9 of the AChBP were individually and combinatorially substituted in all permutations with the analogous loops of the 5HT(3A)R. These chimeras were expressed as secreted proteins using the Pichia pastoris yeast expression system. [(125)I]-alpha-Bungarotoxin-binding was detected in the culture media obtained from homologous recombinant clones expressing the wild-type AChBP, the beta1-beta2 loop-only chimera, and the chimera containing all three 5HT(3A)R loop substitutions. The remaining chimeras failed to show [(125)I]-alpha-bungarotoxin binding, and further analysis of cellular extracts allowed us to determine that these binding-negative chimeric constructs accumulated intracellularly and were not secreted into the culture medium. Our results demonstrate that coordinated interactions among loops beta1-beta2, beta6-beta7, and beta8-beta9 are essential for the formation of a functional ligand-binding site, as evidenced by [(125)I]-alpha-bungarotoxin-binding, and for efficient protein secretion. In addition, the constructs described here demonstrate the feasibility of utilizing soluble scaffolds to explore functionally important interactions within the extracellular domain of membrane-bound proteins. PMID:19427904

  17. Heterologous Production of Hyaluronic Acid in an ε-Poly-l-Lysine Producer, Streptomyces albulus

    PubMed Central

    Yoshimura, Tomohiro; Shibata, Nobuyuki; Hamano, Yoshimitsu

    2015-01-01

    Hyaluronic acid (HA) is used in a wide range of medical applications, where its performance and therapeutic efficacy are highly dependent on its molecular weight. In the microbial production of HA, it has been suggested that a high level of intracellular ATP enhances the productivity and molecular weight of HA. Here, we report on heterologous HA production in an ε-poly-l-lysine producer, Streptomyces albulus, which has the potential to generate ATP at high level. The hasA gene from Streptococcus zooepidemicus, which encodes HA synthase, was refactored and expressed under the control of a late-log growth phase-operating promoter. The expression of the refactored hasA gene, along with genes coding for UDP-glucose dehydrogenase, UDP-N-acetylglucosamine pyrophosphorylase, and UDP-glucose pyrophosphorylase, which are involved in HA precursor sugar biosynthesis, resulted in efficient production of HA in the 2.0 MDa range, which is greater than typical bacterial HA, demonstrating that a sufficient amount of ATP was provided to support the biosynthesis of the precursor sugars, which in turn promoted HA production. In addition, unlike in the case of streptococcal HA, S. albulus-derived HA was not cell associated. Based on these findings, our heterologous production system appears to have several advantages for practical HA production. We propose that the present system could be applicable to the heterologous production of a wide variety of molecules other than HA in the case their biosynthesis pathways require ATP in vivo. PMID:25795665

  18. Biochemical Characterization of Paracoccidioides brasiliensis α-1,3-Glucanase Agn1p, and Its Functionality by Heterologous Expression in Schizosaccharomyces pombe

    PubMed Central

    Villalobos-Duno, Héctor; San-Blas, Gioconda; Paulinkevicius, Maryan; Sánchez-Martín, Yolanda; Nino-Vega, Gustavo

    2013-01-01

    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 GH-71 into at

  19. Heterologous expression of human carnitine palmitoyltransferase (CPT) II in yeast: A model for the molecular analysis of mitochondrial fatty acid oxidation defects

    SciTech Connect

    Cavadini, P.; Invernizzi, F.; Baratta, S.

    1994-09-01

    The CPT enzyme system, which is composed of two distinct mitochondrial membrane-bound proteins (CPT I and CPT II), provides the mechanism whereby long-chain fatty acids are transferred from the cytosol to the mitochondrial matrix to undergo {beta}-oxidation. Here, we report the development of an expression system for investigating genotype/phenotype correlations in CPT II deficiency and, potentially, other mitochondrial fatty acid oxidation defects. To explore yeast as an expression system, we introduced a cDNA encoding the entire human CPT II precursor into Saccharomyces cerevisiae. Expression was programmed by using an inducible galactose operon promoter (GAL1). Following induction, human CPT II was expressed at high levels, with activity 4- to 16-fold greater than in human fibroblasts. Levels of expression paralleled those of respiration, being higher in cells grown on a nonfermentable carbon source than in those grown on glucose. Immunoprecipitation of pulse-labeled transformed cells demonstrated that human CPT II expressed in yeast was targeted to mitochondria with correct proteolytic processing of its 25-residue mitochondrial leader sequence. Preliminary results on the expression of a number of mutant CPT II alleles associated with different clinical phenotypes demonstrated the value of this system for examining the functional consequences of disease-causing mutations and investigating genotype/phenotype correlations in patients with CPT II deficiency.

  20. Structural requisites of 2-(p-chlorophenoxy)propionic acid analogues for activity on native rat skeletal muscle chloride conductance and on heterologously expressed CLC-1

    PubMed Central

    Liantonio, Antonella; De Luca, Annamaria; Pierno, Sabata; Didonna, Maria Paola; Loiodice, Fulvio; Fracchiolla, Giuseppe; Tortorella, Paolo; Laghezza, Antonio; Bonerba, Elisabetta; Traverso, Sonia; Elia, Laura; Picollo, Alessandra; Pusch, Michael; Camerino, Diana Conte

    2003-01-01

    The 2-(p-chlorophenoxy)propionic acid (CPP) modulates in a stereoselective manner the macroscopic chloride conductance (gCl), the electrical parameter sustained by the CLC-1 channel, of skeletal muscle. In order to determine the structural requirements for modulating native gCl and to identify high-affinity ligands, the effects of newly synthesised CPP analogues have been evaluated on gCl of rat EDL muscle fibres by means of the two-microelectrode current-clamp technique. Each type of the following independent modification of CPP structure led to a three- to 10-fold decrease or to a complete lack of gCl-blocking activity: replacement of the electron-attractive chlorine atom of the aromatic ring, substitution of the oxygen atom of the phenoxy group, modification at the chiral centre and substitution of the carboxylic function with a phosphonate one. The analogues bearing a second chlorophenoxy group on the asymmetric carbon atom showed a significant gCl-blocking activity. Similar to racemate CPP, the analogue with this group, spaced by an alkyl chain formed by three methylenic groups, blocked gCl by 45% at 100 μM. These latter derivatives were tested on heterelogously expressed CLC-1 performing inside-out patch-clamp recordings to further define how interaction between drug and channel protein could take place. Depending on the exact chemical nature of modification, these derivatives strongly blocked CLC-1 with KD values at −140 mV ranging from about 4 to 180 μM. In conclusion, we identified four molecular determinants pivotal for the interaction with the binding site on muscle CLC-1 channels: (a) the carboxylic group that confers the optimal acidity and the negative charge; (b) the chlorophenoxy moiety that might interact with a hydrophobic pocket; (c) the chiral centre that allows the proper spatial disposition of the molecule; (d) an additional phenoxy group that remarkably stabilises the binding by interacting with a second hydrophobic pocket. PMID:12890704

  1. Heterologously expressed Aspergillus aculeatus β-glucosidase in Saccharomyces cerevisiae is a cost-effective alternative to commercial supplementation of β-glucosidase in industrial ethanol production using Trichoderma reesei cellulases.

    PubMed

    Treebupachatsakul, Treesukon; Nakazawa, Hikaru; Shinbo, Hideaki; Fujikawa, Hiroki; Nagaiwa, Asami; Ochiai, Nobuhiro; Kawaguchi, Takashi; Nikaido, Mitsuru; Totani, Kazuhide; Shioya, Koki; Shida, Yosuke; Morikawa, Yasushi; Ogasawara, Wataru; Okada, Hirofumi

    2016-01-01

    Trichoderma reesei is a filamentous organism that secretes enzymes capable of degrading cellulose to cellobiose. The culture supernatant of T. reesei, however, lacks sufficient activity to convert cellobiose to glucose using β-glucosidase (BGL1). In this study, we identified a BGL (Cel3B) from T. reesei (TrCel3B) and compared it with the active β-glucosidases from Aspergillus aculeatus (AaBGL1). AaBGL1 showed higher stability and conversion of sugars to ethanol compared to TrCel3B, and therefore we chose to express this recombinant protein for use in fermentation processes. We expressed the recombinant protein in the yeast Saccharomyces cerevisiae, combined it with the superb T. reesei cellulase machinery and used the combination in a simultaneous saccharification and fermentation (SSF) process, with the hope that the recombinant would supplement the BGL activity. As the sugars were processed, the yeast immediately converted them to ethanol, thereby eliminating the problem posed by end product inhibition. Recombinant AaBGL1 activity was compared with Novozyme 188, a commercially available supplement for BGL activity. Our results show that the recombinant protein is as effective as the commercial supplement and can process sugars with equal efficiency. Expression of AaBGL1 in S. cerevisiae increased ethanol production effectively. Thus, heterologous expression of AaBGL1 in S. cerevisiae is a cost-effective and efficient process for the bioconversion of ethanol from lignocellulosic biomass. PMID:26073313

  2. Heterologous expression of Fusarium oxysporum tomatinase in Saccharomyces cerevisiae increases its resistance to saponins and improves ethanol production during the fermentation of Agave tequilana Weber var. azul and Agave salmiana must.

    PubMed

    Cira, Luis Alberto; González, Gloria Angélica; Torres, Juan Carlos; Pelayo, Carlos; Gutiérrez, Melesio; Ramírez, Jesús

    2008-03-01

    This paper describes the effect of the heterologous expression of tomatinase from Fusarium oxysporum f. sp lycopersici in Saccharomyces cerevisiae. The gene FoTom1 under the control of the S. cerevisiae phosphoglycerate kinase (PGK1) promoter was cloned into pYES2. S. cerevisiae strain Y45 was transformed with this vector and URA3 transformant strains were selected for resistance to alpha-tomatine. Two transformants were randomly selected for further study (designated Y45-1 and Y45-2). Control strain Y45 was inhibited at 50 muM alpha-tomatine, in contrast, transformants Y45-1 and Y45-2 did not show inhibition at 200 muM. Tomatinase activity was detected by HPLC monitoring tomatine disappearance and tomatidine appearance in the supernatants of culture medium. Maximum tomatinase activity was observed in the transformants after 6 h, remaining constant during the following 24 h. No tomatinase activity was detected in the parental strain. Moreover, the transformants were able to grow and produce ethanol in a mix of Agave tequilana Weber var. azul and Agave salmiana must, contrary to the Y45 strain which was unable to grow and ferment under these conditions.

  3. Expression of a Heterologous Xylose Transporter in a Saccharomyces cerevisiae Strain Engineered to Utilize Xylose Improves Aerobic Xylose Co-consumption

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Strains of Saccharomyces cerevisiae have been engineered to utilize xylose by expression of the genes for xylose reductase and xylitol dehydrogenase, or xylose isomerase. These strains are still limited in their ability to efficiently use xylose. Unlike native xylose assimilating yeasts such as Pi...

  4. Expression of a heterologous xylose transporter in a Saccharomyces cerevisiae strain engineered to utilize xylose increases xylose uptake and improves xylose/glucose co-consumption

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Strains of Saccharomyces cerevisiae have been engineered to utilize xylose by expressing either the genes for xylose reductase and xylitol dehydrogenase, or for xylose isomerase. These strains still use xylose at sub-optimal rates for industrial fermentation. Unlike natural xylose fermenting yeast...

  5. Molecular Characterization of the Elaeis guineensis Medium-Chain Fatty Acid Diacylglycerol Acyltransferase DGAT1-1 by Heterologous Expression in Yarrowia lipolytica

    PubMed Central

    Aymé, Laure; Jolivet, Pascale; Nicaud, Jean-Marc; Chardot, Thierry

    2015-01-01

    Diacylglycerol acyltransferases (DGAT) are involved in the acylation of sn-1,2-diacylglycerol. Palm kernel oil, extracted from Elaeis guineensis (oil palm) seeds, has a high content of medium-chain fatty acids mainly lauric acid (C12:0). A putative E. guineensis diacylglycerol acyltransferase gene (EgDGAT1-1) is expressed at the onset of lauric acid accumulation in the seed endosperm suggesting that it is a determinant of medium-chain triacylglycerol storage. To test this hypothesis, we thoroughly characterized EgDGAT1-1 activity through functional complementation of a Yarrowia lipolytica mutant strain devoid of neutral lipids. EgDGAT1-1 expression is sufficient to restore triacylglycerol accumulation in neosynthesized lipid droplets. A comparative functional study with Arabidopsis thaliana DGAT1 highlighted contrasting substrate specificities when the recombinant yeast was cultured in lauric acid supplemented medium. The EgDGAT1-1 expressing strain preferentially accumulated medium-chain triacylglycerols whereas AtDGAT1 expression induced long-chain triacylglycerol storage in Y. lipolytica. EgDGAT1-1 localized to the endoplasmic reticulum where TAG biosynthesis takes place. Reestablishing neutral lipid accumulation in the Y. lipolytica mutant strain did not induce major reorganization of the yeast microsomal proteome. Overall, our findings demonstrate that EgDGAT1-1 is an endoplasmic reticulum DGAT with preference for medium-chain fatty acid substrates, in line with its physiological role in palm kernel. The characterized EgDGAT1-1 could be used to promote medium-chain triacylglycerol accumulation in microbial-produced oil for industrial chemicals and cosmetics. PMID:26581109

  6. Regulated delayed expression of rfaH in an attenuated Salmonella enterica serovar typhimurium vaccine enhances immunogenicity of outer membrane proteins and a heterologous antigen.

    PubMed

    Kong, Qingke; Liu, Qing; Roland, Kenneth L; Curtiss, Roy

    2009-12-01

    RfaH is a transcriptional antiterminator that reduces the polarity of long operons encoding secreted and surface-associated cell components of Salmonella enterica serovar Typhimurium, including O antigen and lipopolysaccharide core sugars. A DeltarfaH mutant strain is attenuated in mice (50% lethal dose [LD(50)], >10(8) CFU). To examine the potential for using rfaH in conjunction with other attenuating mutations, we designed a series of strains in which we replaced the native rfaH promoter with the tightly regulated arabinose-dependent araC P(BAD) promoter so that rfaH expression was dependent on exogenously supplied arabinose provided during in vitro growth. Following colonization of host lymphoid tissues, where arabinose was not available, the P(BAD) promoter was no longer active and rfaH was not expressed. In the absence of RfaH, O antigen and core sugars were not synthesized. We constructed three mutant strains that expressed different levels of RfaH by altering the ribosome-binding sequence and start codon. One mutation, DeltaP(rfaH178), was introduced into the attenuated vaccine strain chi9241 (DeltapabA DeltapabB DeltaasdA) expressing the pneumococcal surface protein PspA from an Asd(+) balanced-lethal plasmid. Mice immunized with this strain and boosted 4 weeks later induced higher levels of serum immunoglobulin G specific for PspA and for outer membrane proteins from other enteric bacteria than either an isogenic DeltarfaH derivative or the isogenic RfaH(+) parent. Eight weeks after primary oral immunization, mice were challenged with 200 LD(50) of virulent Streptococcus pneumoniae WU2. Immunization with DeltaP(rfaH178) mutant strains led to increased levels of protection compared to that of the parent chi9241 and of a DeltarfaH derivative of chi9241.

  7. Molecular Characterization of the Elaeis guineensis Medium-Chain Fatty Acid Diacylglycerol Acyltransferase DGAT1-1 by Heterologous Expression in Yarrowia lipolytica.

    PubMed

    Aymé, Laure; Jolivet, Pascale; Nicaud, Jean-Marc; Chardot, Thierry

    2015-01-01

    Diacylglycerol acyltransferases (DGAT) are involved in the acylation of sn-1,2-diacylglycerol. Palm kernel oil, extracted from Elaeis guineensis (oil palm) seeds, has a high content of medium-chain fatty acids mainly lauric acid (C12:0). A putative E. guineensis diacylglycerol acyltransferase gene (EgDGAT1-1) is expressed at the onset of lauric acid accumulation in the seed endosperm suggesting that it is a determinant of medium-chain triacylglycerol storage. To test this hypothesis, we thoroughly characterized EgDGAT1-1 activity through functional complementation of a Yarrowia lipolytica mutant strain devoid of neutral lipids. EgDGAT1-1 expression is sufficient to restore triacylglycerol accumulation in neosynthesized lipid droplets. A comparative functional study with Arabidopsis thaliana DGAT1 highlighted contrasting substrate specificities when the recombinant yeast was cultured in lauric acid supplemented medium. The EgDGAT1-1 expressing strain preferentially accumulated medium-chain triacylglycerols whereas AtDGAT1 expression induced long-chain triacylglycerol storage in Y. lipolytica. EgDGAT1-1 localized to the endoplasmic reticulum where TAG biosynthesis takes place. Reestablishing neutral lipid accumulation in the Y. lipolytica mutant strain did not induce major reorganization of the yeast microsomal proteome. Overall, our findings demonstrate that EgDGAT1-1 is an endoplasmic reticulum DGAT with preference for medium-chain fatty acid substrates, in line with its physiological role in palm kernel. The characterized EgDGAT1-1 could be used to promote medium-chain triacylglycerol accumulation in microbial-produced oil for industrial chemicals and cosmetics.

  8. Molecular Characterization of the Elaeis guineensis Medium-Chain Fatty Acid Diacylglycerol Acyltransferase DGAT1-1 by Heterologous Expression in Yarrowia lipolytica.

    PubMed

    Aymé, Laure; Jolivet, Pascale; Nicaud, Jean-Marc; Chardot, Thierry

    2015-01-01

    Diacylglycerol acyltransferases (DGAT) are involved in the acylation of sn-1,2-diacylglycerol. Palm kernel oil, extracted from Elaeis guineensis (oil palm) seeds, has a high content of medium-chain fatty acids mainly lauric acid (C12:0). A putative E. guineensis diacylglycerol acyltransferase gene (EgDGAT1-1) is expressed at the onset of lauric acid accumulation in the seed endosperm suggesting that it is a determinant of medium-chain triacylglycerol storage. To test this hypothesis, we thoroughly characterized EgDGAT1-1 activity through functional complementation of a Yarrowia lipolytica mutant strain devoid of neutral lipids. EgDGAT1-1 expression is sufficient to restore triacylglycerol accumulation in neosynthesized lipid droplets. A comparative functional study with Arabidopsis thaliana DGAT1 highlighted contrasting substrate specificities when the recombinant yeast was cultured in lauric acid supplemented medium. The EgDGAT1-1 expressing strain preferentially accumulated medium-chain triacylglycerols whereas AtDGAT1 expression induced long-chain triacylglycerol storage in Y. lipolytica. EgDGAT1-1 localized to the endoplasmic reticulum where TAG biosynthesis takes place. Reestablishing neutral lipid accumulation in the Y. lipolytica mutant strain did not induce major reorganization of the yeast microsomal proteome. Overall, our findings demonstrate that EgDGAT1-1 is an endoplasmic reticulum DGAT with preference for medium-chain fatty acid substrates, in line with its physiological role in palm kernel. The characterized EgDGAT1-1 could be used to promote medium-chain triacylglycerol accumulation in microbial-produced oil for industrial chemicals and cosmetics. PMID:26581109

  9. Heterologous expression and functional analysis of rat Nav1.8 (SNS) voltage-gated sodium channels in the dorsal root ganglion neuroblastoma cell line ND7-23.

    PubMed

    John, Victoria H; Main, Martin J; Powell, Andrew J; Gladwell, Zoe M; Hick, Caroline; Sidhu, Harjeet S; Clare, Jeff J; Tate, Simon; Trezise, Derek J

    2004-03-01

    The voltage-gated sodium channel NaV1.8 (SNS, PN3) is thought to be a molecular correlate of the dorsal root ganglion (DRG) tetrodotoxin resistant (TTX-R) Na+ current. TTX-R/NaV1.8 is an attractive therapeutic drug target for inflammatory and neuropathic pain on the basis of its specific distribution in sensory neurones and its modulation by inflammatory mediators. However, detailed analysis of recombinant NaV1.8 has been hampered by difficulties in stably expressing the functional protein in mammalian cells. Here, we show stable expression and functional analysis of rat NaV1.8 (rNaV1.8) in the rat DRG/mouse N18Tg2 neuroblastoma hybridoma cell line ND7-23. Rat NaV1.8 Na+ currents were recorded (789 +/- 89 pA, n=62, over 20-cell passages) that qualitatively resembled DRG TTX-R in terms of gating kinetics and voltage-dependence of activation and inactivation. The local anaesthetic drug tetracaine produced tonic inhibition of rNaV1.8 (mean IC50 value 12.5 microM) and in repeated gating paradigms (2-10 Hz) also showed frequency-dependent block. There was a correlation between the ability of several analogues of the anticonvulsant/analgesic compound lamotrigine to inhibit TTX-R and rNaV1.8 (r=0.72, P<0.001). RT-PCR analysis of wild type ND7-23 cells revealed endogenous expression of the beta1 and beta3 accessory Na+ channel subunits-the possibility that the presence of these subunits assists and stabilises expression of rNaV1.8 is discussed. We conclude that the neuroblastoma ND7-23 cell line is a suitable heterologous expression system for rNaV1.8 Na+ channels in that it allows stable expression of a channel with biophysical properties that closely resemble the native TTX-R currents in DRG neurones. This reagent will prove useful in the search for pharmacological inhibitors of rNaV1.8 as novel analgesics. PMID:14975698

  10. Brucella abortus strain RB51 as a vector for heterologous protein expression and induction of specific Th1 type immune responses.

    PubMed

    Vemulapalli, R; He, Y; Boyle, S M; Sriranganathan, N; Schurig, G G

    2000-06-01

    Brucella abortus strain RB51 is a stable, rough, attenuated mutant widely used as a live vaccine for bovine brucellosis. Our ultimate goal is to develop strain RB51 as a preferential vector for the delivery of protective antigens of other intracellular pathogens to which the induction of a strong Th1 type of immune response is needed for effective protection. As a first step in that direction, we studied the expression of a foreign reporter protein, beta-galactosidase of Escherichia coli, and the 65-kDa heat shock protein (HSP65) of Mycobacterium bovis in strain RB51. We cloned the promoter sequences of Brucella sodC and groE genes in pBBR1MCS to generate plasmids pBBSODpro and pBBgroE, respectively. The genes for beta-galactosidase (lacZ) and HSP65 were cloned in these plasmids and used to transform strain RB51. An enzyme assay in the recombinant RB51 strains indicated that the level of beta-galactosidase expression is higher under the groE promoter than under the sodC promoter. In strain RB51 containing pBBgroE/lacZ, but not pBBSODpro/lacZ, increased levels of beta-galactosidase expression were observed after subjecting the bacteria to heat shock or following internalization into macrophage-like J774A.1 cells. Mice vaccinated with either of the beta-galactosidase-expressing recombinant RB51 strains developed specific antibodies of predominantly the immunoglobulin G2a (IgG2a) isotype, and in vitro stimulation of their splenocytes with beta-galactosidase induced the secretion of gamma interferon (IFN-gamma), but not interleukin-4 (IL-4). A Th1 type of immune response to HSP65, as indicated by the presence of specific serum IgG2a, but not IgG1, antibodies, and IFN-gamma, but not IL-4, secretion by the specific-antigen-stimulated splenocytes, was also detected in mice vaccinated with strain RB51 containing pBBgroE/hsp65. Studies with mice indicated that expression of beta-galactosidase or HSP65 did not alter either the attenuation characteristics of strain RB51 or

  11. Heterologous expression of fungal cytochromes P450 (CYP5136A1 and CYP5136A3) from the white-rot basidiomycete Phanerochaete chrysosporium: Functionalization with cytochrome b5 in Escherichia coli.

    PubMed

    Hatakeyama, Mayumi; Kitaoka, Takuya; Ichinose, Hirofumi

    2016-07-01

    Cytochromes P450 from the white-rot basidiomycete Phanerochaete chrysosporium, CYP5136A1 and CYP5136A3, are capable of catalyzing oxygenation reactions of a wide variety of exogenous compounds, implying their significant roles in the metabolism of xenobiotics by the fungus. It is therefore interesting to explore their biochemistry to better understand fungal biology and to enable the use of fungal enzymes in the biotechnology sector. In the present study, we developed heterologous expression systems for CYP5136A1 and CYP5136A3 using the T7 RNA polymerase/promoter system in Escherichia coli. Expression levels of recombinant P450s were dramatically improved by modifications and optimization of their N-terminal amino acid sequences. A CYP5136A1 reaction system was reconstructed in E. coli whole cells by coexpression of CYP5136A1 and a redox partner, NADPH-dependent P450 reductase (CPR). The catalytic activity of CYP5136A1 was significantly increased when cytochrome b5 (Cyt-b5) was further coexpressed with CPR, indicating that Cyt-b5 supports electron transfer reactions from NAD(P)H to CYP5136A1. Notably, P450 reaction occurred in E. coli cells that harbored CYP5136A1 and Cyt-b5 but not CPR, implying that the reducing equivalents required for the P450 catalytic cycle were transferred via a CPR-independent pathway. Such an "alternative" electron transfer system in CYP5136A1 reaction was also demonstrated using purified enzymes in vitro. The fungal P450 reaction system may be associated with sophisticated electron transfer pathways.

  12. 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.

    PubMed

    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.

  13. Biosynthetic pathway for γ-cyclic sarcinaxanthin in Micrococcus luteus: heterologous expression and evidence for diverse and multiple catalytic functions of C(50) carotenoid cyclases.

    PubMed

    Netzer, Roman; Stafsnes, Marit H; Andreassen, Trygve; Goksøyr, Audun; Bruheim, Per; Brautaset, Trygve

    2010-11-01

    We report the cloning and characterization of the biosynthetic gene cluster (crtE, crtB, crtI, crtE2, crtYg, crtYh, and crtX) of the γ-cyclic C(50) carotenoid sarcinaxanthin in Micrococcus luteus NCTC2665. Expression of the complete and partial gene cluster in Escherichia coli hosts revealed that sarcinaxanthin biosynthesis from the precursor molecule farnesyl pyrophosphate (FPP) proceeds via C(40) lycopene, C(45) nonaflavuxanthin, C(50) flavuxanthin, and C(50) sarcinaxanthin. Glucosylation of sarcinaxanthin was accomplished by the crtX gene product. This is the first report describing the biosynthetic pathway of a γ-cyclic C(50) carotenoid. Expression of the corresponding genes from the marine M. luteus isolate Otnes7 in a lycopene-producing E. coli host resulted in the production of up to 2.5 mg/g cell dry weight sarcinaxanthin in shake flasks. In an attempt to experimentally understand the specific difference between the biosynthetic pathways of sarcinaxanthin and the structurally related ε-cyclic decaprenoxanthin, we constructed a hybrid gene cluster with the γ-cyclic C(50) carotenoid cyclase genes crtYg and crtYh from M. luteus replaced with the analogous ε-cyclic C(50) carotenoid cyclase genes crtYe and crtYf from the natural decaprenoxanthin producer Corynebacterium glutamicum. Surprisingly, expression of this hybrid gene cluster in an E. coli host resulted in accumulation of not only decaprenoxanthin, but also sarcinaxanthin and the asymmetric ε- and γ-cyclic C(50) carotenoid sarprenoxanthin, described for the first time in this work. Together, these data contributed to new insight into the diverse and multiple functions of bacterial C(50) carotenoid cyclases as key catalysts for the synthesis of structurally different carotenoids. PMID:20802040

  14. Biosynthetic Pathway for γ-Cyclic Sarcinaxanthin in Micrococcus luteus: Heterologous Expression and Evidence for Diverse and Multiple Catalytic Functions of C50 Carotenoid Cyclases▿ †

    PubMed Central

    Netzer, Roman; Stafsnes, Marit H.; Andreassen, Trygve; Goksøyr, Audun; Bruheim, Per; Brautaset, Trygve

    2010-01-01

    We report the cloning and characterization of the biosynthetic gene cluster (crtE, crtB, crtI, crtE2, crtYg, crtYh, and crtX) of the γ-cyclic C50 carotenoid sarcinaxanthin in Micrococcus luteus NCTC2665. Expression of the complete and partial gene cluster in Escherichia coli hosts revealed that sarcinaxanthin biosynthesis from the precursor molecule farnesyl pyrophosphate (FPP) proceeds via C40 lycopene, C45 nonaflavuxanthin, C50 flavuxanthin, and C50 sarcinaxanthin. Glucosylation of sarcinaxanthin was accomplished by the crtX gene product. This is the first report describing the biosynthetic pathway of a γ-cyclic C50 carotenoid. Expression of the corresponding genes from the marine M. luteus isolate Otnes7 in a lycopene-producing E. coli host resulted in the production of up to 2.5 mg/g cell dry weight sarcinaxanthin in shake flasks. In an attempt to experimentally understand the specific difference between the biosynthetic pathways of sarcinaxanthin and the structurally related ɛ-cyclic decaprenoxanthin, we constructed a hybrid gene cluster with the γ-cyclic C50 carotenoid cyclase genes crtYg and crtYh from M. luteus replaced with the analogous ɛ-cyclic C50 carotenoid cyclase genes crtYe and crtYf from the natural decaprenoxanthin producer Corynebacterium glutamicum. Surprisingly, expression of this hybrid gene cluster in an E. coli host resulted in accumulation of not only decaprenoxanthin, but also sarcinaxanthin and the asymmetric ɛ- and γ-cyclic C50 carotenoid sarprenoxanthin, described for the first time in this work. Together, these data contributed to new insight into the diverse and multiple functions of bacterial C50 carotenoid cyclases as key catalysts for the synthesis of structurally different carotenoids. PMID:20802040

  15. Heterologous Expression of Phanerochaete chrysoporium Glyoxal Oxidase and its Application for the Coupled Reaction with Manganese Peroxidase to Decolorize Malachite Green

    PubMed Central

    Son, Yu-Lim; Kim, Hyoun-Young; Thiyagarajan, Saravanakumar; Xu, Jing Jing

    2012-01-01

    cDNA of the glx1 gene encoding glyoxal oxidase (GLX) from Phanerochaete chrysosporium was isolated and expressed in Pichia pastoris. The recombinant GLX (rGLX) produces H2O2 over 7.0 nmol/min/mL using methyl glyoxal as a substrate. Use of rGLX as a generator of H2O2 improved the coupled reaction with recombinant manganese peroxidase resulting in decolorization of malachite green up to 150 µM within 90 min. PMID:23323052

  16. Engineered Biosynthesis of Natural Products in Heterologous Hosts

    PubMed Central

    Luo, Yunzi; Li, Bing-Zhi; Liu, Duo; Zhang, Lu; Chen, Yan; Jia, Bin; Zeng, Bo-Xuan; Zhao, Huimin; Yuan, Ying-Jin

    2015-01-01

    Natural products produced by microorganisms and plants are a major resource of antibacterial and anticancer drugs as well as industrially useful compounds. However, the native producers often suffer from low productivity and titers. Here we summarize the recent applications of heterologous biosynthesis for the production of several important classes of natural products such as terpenoids, flavonoids, alkaloids, and polyketides. In addition, we will discuss the new tools and strategies at multi-scale levels including gene, pathway, genome and community levels for highly efficient heterologous biosynthesis of natural products. PMID:25960127

  17. Heterologous expression of Cenchritis muricatus protease inhibitor II (CmPI-II) in Pichia pastoris system: Purification, isotopic labeling and preliminary characterization.

    PubMed

    Cabrera-Muñoz, Aymara; Rojas, Laritza; Gil, Dayrom F; González-González, Yamile; Mansur, Manuel; Camejo, Ayamey; Pires, José R; Alonso-Del-Rivero Antigua, Maday

    2016-10-01

    Cenchritis muricatus protease inhibitor II (CmPI-II) is a tight-binding serine protease inhibitor of the Kazal family with an atypical broad specificity, being active against several proteases such as bovine pancreatic trypsin, human neutrophil elastase and subtilisin A. CmPI-II 3D structures are necessary for understanding the molecular basis of its activity. In the present work, we describe an efficient and straightforward recombinant expression strategy, as well as a cost-effective procedure for isotope labeling for NMR structure determination purposes. The vector pCM101 containing the CmPI-II gene, under the control of Pichia pastoris AOX1 promoter was constructed. Methylotrophic Pichia pastoris strain KM71H was then transformed with the plasmid and the recombinant protein (rCmPI-II) was expressed in benchtop fermenter in unlabeled or (15)N-labeled forms using ammonium chloride ((15)N, 99%) as the sole nitrogen source. Protein purification was accomplished by sequential cation exchange chromatography in STREAMLINE DirectHST, anion exchange chromatography on Hitrap Q-Sepharose FF and gel filtration on Superdex 75 10/30, yielding high quantities of pure rCmPI-II and (15)N rCmPI-II. Recombinant proteins displayed similar functional features as compared to the natural inhibitor and NMR spectra indicated folded and homogeneously labeled samples, suitable for further studies of structure and protease-inhibitor interactions. PMID:27353494

  18. Heterologous expression of Cenchritis muricatus protease inhibitor II (CmPI-II) in Pichia pastoris system: Purification, isotopic labeling and preliminary characterization.

    PubMed

    Cabrera-Muñoz, Aymara; Rojas, Laritza; Gil, Dayrom F; González-González, Yamile; Mansur, Manuel; Camejo, Ayamey; Pires, José R; Alonso-Del-Rivero Antigua, Maday

    2016-10-01

    Cenchritis muricatus protease inhibitor II (CmPI-II) is a tight-binding serine protease inhibitor of the Kazal family with an atypical broad specificity, being active against several proteases such as bovine pancreatic trypsin, human neutrophil elastase and subtilisin A. CmPI-II 3D structures are necessary for understanding the molecular basis of its activity. In the present work, we describe an efficient and straightforward recombinant expression strategy, as well as a cost-effective procedure for isotope labeling for NMR structure determination purposes. The vector pCM101 containing the CmPI-II gene, under the control of Pichia pastoris AOX1 promoter was constructed. Methylotrophic Pichia pastoris strain KM71H was then transformed with the plasmid and the recombinant protein (rCmPI-II) was expressed in benchtop fermenter in unlabeled or (15)N-labeled forms using ammonium chloride ((15)N, 99%) as the sole nitrogen source. Protein purification was accomplished by sequential cation exchange chromatography in STREAMLINE DirectHST, anion exchange chromatography on Hitrap Q-Sepharose FF and gel filtration on Superdex 75 10/30, yielding high quantities of pure rCmPI-II and (15)N rCmPI-II. Recombinant proteins displayed similar functional features as compared to the natural inhibitor and NMR spectra indicated folded and homogeneously labeled samples, suitable for further studies of structure and protease-inhibitor interactions.

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

    PubMed

    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.

  20. Heterologous protein expression by transimmortalized differentiated liver cell lines derived from transgenic mice (hepatomas/alpha 1 antitrypsin/ONC mouse).

    PubMed

    Dalemans, W; Perraud, F; Le Meur, M; Gerlinger, P; Courtney, M; Pavirani, A

    1990-07-01

    A number of therapeutic plasma proteins are synthesized by human hepatocytes. Since many of these proteins undergo liver-specific post-translational modifications which are required for full biological activity, it may therefore be necessary to develop hepatocyte-based expression systems for their production. Using transgenic mice we have developed a transimmortalisation technique for the isolation of differentiated hepatic cell lines, already engineered to secrete human alpha 1 antitrypsin (alpha 1 AT), a plasma protein which is produced mainly in liver cells. This was achieved by co-expression of the mouse c-myc proto-oncogene and a genomic copy of the human alpha 1 AT gene, both under the control of the human alpha 1 AT promoter. Transgenic mice carrying this construct developed hepatomas producing human alpha 1 AT. Under defined culture conditions, cell lines secreting active alpha 1 AT were derived from these tumours. These cells maintain a differentiated hepatic phenotype and continue to secrete human alpha 1 AT for at least 40 generations. PMID:2257132

  1. Heterologous expression of type I antifreeze peptide GS-5 in baker's yeast increases freeze tolerance and provides enhanced gas production in frozen dough.

    PubMed

    Panadero, Joaquin; Randez-Gil, Francisca; Prieto, Jose Antonio

    2005-12-28

    The demand for frozen-dough products has increased notably in the baking industry. Nowadays, no appropriate industrial baker's yeast with optimal gassing capacity in frozen dough is, however, available, and it is unlikely that classical breeding programs could provide significant improvements of this trait. Antifreeze proteins, found in diverse organisms, display the ability to inhibit the growth of ice, allowing them to survive at temperatures below 0 degrees C. In this study a recombinant antifreeze peptide GS-5 was expressed from the polar fish grubby sculpin (Myoxocephalus aenaeus) in laboratory and industrial baker's yeast strains of Saccharomyces cerevisiae. Production of the recombinant protein increased freezing tolerance in both strains tested. Furthermore, expression of the GS-5 encoding gene enhanced notably the gassing rate and total gas production in frozen and frozen sweet doughs. These effects are unlikely to be due to reduced osmotic damage during freezing/thawing, because recombinant cells showed growth behavior similar to that of the parent under hypermosmotic stress conditions. PMID:16366681

  2. Silencing and heterologous expression of ppo-2 indicate a specific function of a single polyphenol oxidase isoform in resistance of dandelion (Taraxacum officinale) against Pseudomonas syringae pv. tomato.

    PubMed

    Richter, Carolin; Dirks, Mareike E; Gronover, Christian Schulze; Prüfer, Dirk; Moerschbacher, Bruno M

    2012-02-01

    Dandelion (Taraxacum officinale) possesses an unusually high degree of disease resistance. As this plant exhibits high polyphenol oxidase (PPO) activity and PPO have been implicated in resistance against pests and pathogens, we analyzed the potential involvement of five PPO isoenzymes in the resistance of dandelion against Botrytis cinerea and Pseudomonas syringae pv. tomato. Only one PPO (ppo-2) was induced during infection, and ppo-2 promoter and β-glucuronidase marker gene fusions revealed strong induction of the gene surrounding lesions induced by B. cinerea. Specific RNAi silencing reduced ppo-2 expression only, and concomitantly increased plant susceptibility to P. syringae pv. tomato. At 4 days postinoculation, P. syringae pv. tomato populations were strongly increased in the ppo-2 RNAi lines compared with wild-type plants. When the dandelion ppo-2 gene was expressed in Arabidopsis thaliana, a plant having no PPO gene, active protein was formed and protein extracts of the transgenic plants exhibited substrate-dependent antimicrobial activity against P. syringae pv. tomato. These results clearly indicate a strong contribution of a specific, single PPO isoform to disease resistance. Therefore, we propose that specific PPO isoenzymes be included in a new family of pathogenesis-related (PR) proteins.

  3. Identification of a Δ12 fatty acid desaturase from oil palm (Elaeis guineensis Jacq.) involved in the biosynthesis of linoleic acid by heterologous expression in Saccharomyces cerevisiae.

    PubMed

    Sun, Ruhao; Gao, Lingchao; Yu, Xiaoping; Zheng, Yusheng; Li, Dongdong; Wang, Xinguang

    2016-10-10

    Oil palm (Elaeis guineensis Jacq.) is one of the highest oil-yield crops in the world. A Δ12-desaturases associated with the primary steps of long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis were successfully cloned from oil palm and their functions identified. The open reading frames (ORFs) of egFAD2 (GenBank accession: KT023602) consisted of 1176bp and code for 391 amino acids. Their deduced polypeptides showed 75-93% identity to microsomal Δ12-desaturases from other higher plants, and each contained the three histidine clusters typical of the catalytic domains of such enzymes. RT-PCR experiment indicated that the egFAD2 gene exhibited the highest accumulation in the mesocarp of fruits at 120-140 DAP (i.e. the fourth period of fruit development) and, despite having different expression levels, the other four stages were at significantly lower levels compared with the fourth stage. Plasmid pYES2-egFAD2 was transformed into Saccharomyces cerevisiae strain INVSc1 using lithium acetate method for expression under the induction of galactose. Yeast cells transformed with plasmid constructs containing egFAD12 produced an appreciable amount of linoleic acids (18:2(Δ9,)(12)), not normally present in wild-type yeast cells, indicating that the genes encoded functional Δ12-desaturase enzymes. PMID:27370696

  4. Heterologous expression of newly identified galectin-8 from sea urchin embryos produces recombinant protein with lactose binding specificity and anti-adhesive activity

    PubMed Central

    Karakostis, Kostantinos; Costa, Caterina; Zito, Francesca; Matranga, Valeria

    2015-01-01

    Galectin family members specifically bind beta-galactoside derivatives and are involved in different cellular events, including cell communication, signalling, apoptosis, and immune responses. Here, we report a tandem-repeat type galectin from the Paracentrotus lividus sea urchin embryo, referred to as Pl-GAL-8. The 933nt sequence encodes a protein of 34.73 kDa, containing the conserved HFNPRF and WGxExR motifs in the two highly similar carbohydrate-recognition domains (CRD). The three-dimensional protein structure model of the N-CRD confirms the high evolutionary conservation of carbohydrate binding sites. The temporal gene expression is regulated during development and transcripts localize at the tip of the archenteron at gastrula stage, in a subset of the secondary mesenchyme cells that differentiate into blastocoelar (immune) cells. Functional studies using a recombinant Pl-GAL-8 expressed in bacteria demonstrate its hemo-agglutinating activity on human red blood cells through the binding to lactose, as well as its ability in inhibiting the adhesion of human Hep-G2 cells to the substrate. The recent implications in autoimmune diseases and inflammatory disorders make Gal-8 an attractive candidate for therapeutic purposes. Our results offer a solid basis for addressing the use of the new Pl-GAL-8 in functional and applicative studies, respectively in the developmental and biomedical fields. PMID:26640155

  5. Identification of a Δ12 fatty acid desaturase from oil palm (Elaeis guineensis Jacq.) involved in the biosynthesis of linoleic acid by heterologous expression in Saccharomyces cerevisiae.

    PubMed

    Sun, Ruhao; Gao, Lingchao; Yu, Xiaoping; Zheng, Yusheng; Li, Dongdong; Wang, Xinguang

    2016-10-10

    Oil palm (Elaeis guineensis Jacq.) is one of the highest oil-yield crops in the world. A Δ12-desaturases associated with the primary steps of long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis were successfully cloned from oil palm and their functions identified. The open reading frames (ORFs) of egFAD2 (GenBank accession: KT023602) consisted of 1176bp and code for 391 amino acids. Their deduced polypeptides showed 75-93% identity to microsomal Δ12-desaturases from other higher plants, and each contained the three histidine clusters typical of the catalytic domains of such enzymes. RT-PCR experiment indicated that the egFAD2 gene exhibited the highest accumulation in the mesocarp of fruits at 120-140 DAP (i.e. the fourth period of fruit development) and, despite having different expression levels, the other four stages were at significantly lower levels compared with the fourth stage. Plasmid pYES2-egFAD2 was transformed into Saccharomyces cerevisiae strain INVSc1 using lithium acetate method for expression under the induction of galactose. Yeast cells transformed with plasmid constructs containing egFAD12 produced an appreciable amount of linoleic acids (18:2(Δ9,)(12)), not normally present in wild-type yeast cells, indicating that the genes encoded functional Δ12-desaturase enzymes.

  6. Heterologous Infection During Chagas' Disease

    NASA Astrophysics Data System (ADS)

    Sibona, G. J.; Condat, C. A.; Cossi Isasi, S.

    2007-05-01

    Human populations are often infected with more than one parasite strain. This is frequently the case with ChagasŠ disease, which is endemic to large regions of Latin America. In the present work we study the dynamics of the heterologous infection for this disease, using a model for the interaction between the trypanosoma cruzi parasite and the immune system. We find the dependence of the nature of the post-acute stage on the parameters characterizing the inoculated infectious strains.

  7. Heterologous expression of a plastid EF-Tu reduces protein thermal aggregation and enhances CO2 fixation in wheat (Triticum aestivum) following heat stress.

    PubMed

    Fu, Jianming; Momcilović, Ivana; Clemente, Thomas E; Nersesian, Natalya; Trick, Harold N; Ristic, Zoran

    2008-10-01

    Heat stress is a major constraint to wheat production and negatively impacts grain quality, causing tremendous economic losses, and may become a more troublesome factor due to global warming. At the cellular level, heat stress causes denaturation and aggregation of proteins and injury to membranes leading to alterations in metabolic fluxes. Protein aggregation is irreversible, and protection of proteins from thermal aggregation is a strategy a cell uses to tolerate heat stress. Here we report on the development of transgenic wheat (Triticum aestivum) events, expressing a maize gene coding for plastidal protein synthesis elongation factor (EF-Tu), which, compared to non-transgenic plants, display reduced thermal aggregation of leaf proteins, reduced heat injury to photosynthetic membranes (thylakoids), and enhanced rate of CO(2) fixation after exposure to heat stress. The results support the concept that EF-Tu ameliorates negative effects of heat stress by acting as a molecular chaperone. This is the first demonstration of the introduction of a plastidal EF-Tu in plants that leads to protection against heat injury and enhanced photosynthesis after heat stress. This is also the first demonstration that a gene other than HSP gene can be used for improvement of heat tolerance and that the improvement is possible in a species that has a complex genome, hexaploid wheat. The results strongly suggest that heat tolerance of wheat, and possibly other crop plants, can be improved by modulating expression of plastidal EF-Tu and/or by selection of genotypes with increased endogenous levels of this protein.

  8. A novel ICK peptide from the Loxosceles intermedia (brown spider) venom gland: cloning, heterologous expression and immunological cross-reactivity approaches.

    PubMed

    Matsubara, Fernando Hitomi; Gremski, Luiza Helena; Meissner, Gabriel Otto; Constantino Lopes, Eduardo Soares; Gremski, Waldemiro; Senff-Ribeiro, Andrea; Chaim, Olga Meiri; Veiga, Silvio Sanches

    2013-09-01

    The venom of a Loxosceles spider is composed of a complex mixture of biologically active components, consisting predominantly of low molecular mass molecules (3-45 kDa). Transcriptome analysis of the Loxosceles intermedia venom gland revealed ESTs with similarity to the previously described LiTx peptides. Sequences similar to the LiTx3 isoform were the most abundant, representing approximately 13.9% of all ESTs and 32% of the toxin-encoding messengers. These peptides are grouped in the ICK (Inhibitor Cystine Knot) family, which contains single chain molecules with low molecular mass (3-10 kDa). Due to their high number of cysteine residues, ICK peptides form intramolecular disulfide bridges. The aims of this study were to clone and express a novel ICK peptide isoform, as well as produce specific hyperimmune serum for immunoassays. The corresponding cDNA was amplified by PCR using specific primers containing restriction sites for the XhoI and BamHI enzymes; this PCR product was then ligated in the pET-14b vector and transformed into E. coli AD494 (DE3) cells. The peptide was expressed by IPTG induction for 4 h at 30 °C and purified by affinity chromatography with Ni-NTA resin. Hyperimmune serum to the recombinant peptide was produced in rabbits and was able to specifically recognize both the purified recombinant peptide and the native form present in the venom. Furthermore, the recombinant peptide was recognized by antisera raised against L. intermedia, L. gaucho and L. laeta whole venoms. The recombinant peptide obtained will enable future studies to characterize its biological activity, as well as investigations regarding possible biotechnological applications.

  9. Human Cytomegalovirus Nuclear Egress Proteins Ectopically Expressed in the Heterologous Environment of Plant Cells are Strictly Targeted to the Nuclear Envelope

    PubMed Central

    Lamm, Christian E.; Link, Katrin; Wagner, Sabrina; Milbradt, Jens; Marschall, Manfred; Sonnewald, Uwe

    2016-01-01

    In all eukaryotic cells, the nucleus forms a prominent cellular compartment containing the cell’s nuclear genome. Although structurally similar, animal and plant nuclei differ substantially in details of their architecture. One example is the nuclear lamina, a layer of tightly interconnected filament proteins (lamins) underlying the nuclear envelope of metazoans. So far no orthologous lamin genes could be detected in plant genomes and putative lamin-like proteins are only poorly described in plants. To probe for potentially conserved features of metazoan and plant nuclear envelopes, we ectopically expressed the core nuclear egress proteins of human cytomegalovirus pUL50 and pUL53 in plant cells. pUL50 localizes to the inner envelope of metazoan nuclei and recruits the nuclear localized pUL53 to it, forming heterodimers. Upon expression in plant cells, a very similar localization pattern of both proteins could be determined. Notably, pUL50 is specifically targeted to the plant nuclear envelope in a rim-like fashion, a location to which coexpressed pUL53 becomes strictly corecruited from its initial nucleoplasmic distribution. Using pUL50 as bait in a yeast two-hybrid screening, the cytoplasmic re-initiation supporting protein RISP could be identified. Interaction of pUL50 and RISP could be confirmed by coexpression and coimmunoprecipitation in mammalian cells and by confocal laser scanning microscopy in plant cells, demonstrating partial pUL50-RISP colocalization in areas of the nuclear rim and other intracellular compartments. Thus, our study provides strong evidence for conserved structural features of plant and metazoan nuclear envelops and identifies RISP as a potential pUL50-interacting plant protein. PMID:26978388

  10. Exon 3 splicing and mutagenesis identify residues influencing cell surface density of heterologously expressed silkworm (Bombyx mori) glutamate-gated chloride channels.

    PubMed

    Furutani, Shogo; Ihara, Makoto; Nishino, Yuri; Akamatsu, Miki; Jones, Andrew K; Sattelle, David B; Matsuda, Kazuhiko

    2014-12-01

    Glutamate-gated chloride channels (GluCls) mediate fast inhibitory neurotransmission in invertebrate nervous systems. Insect GluCls show alternative splicing, and, to determine its impact on channel function and pharmacology, we isolated GluCl cDNAs from larvae of the silkworm (Bombyx mori). We show that six B. mori glutamate-gated chloride channel variants are generated by splicing in exons 3 and 9 and that exons 3b and 3c are common in the brain and third thoracic ganglion. When expressed in Xenopus laevis oocytes, the three functional exon 3 variants (3a, b, c) all had similar EC50 values for l-glutamate and ivermectin (IVM); however, Imax (the maximum l-glutamate- and IVM-induced response of the channels at saturating concentrations) differed strikingly between variants, with the 3c variant showing the largest l-glutamate- and IVM-induced responses. By contrast, a partial deletion detected in exon 9 had a much smaller impact on l-glutamate and IVM actions. Binding assays using [(3)H]IVM indicate that diversity in IVM responses among the GluCl variants is mainly due to the impact on channel assembly, altering receptor cell surface numbers. GluCl variants expressed in HEK293 cells show that structural differences influenced Bmax but not Kd values of [(3)H]IVM. Domain swapping and site-directed mutagenesis identified four amino acids in exon 3c as hot spots determining the highest amplitude of the l-glutamate and IVM responses. Modeling the GluCl 3a and 3c variants suggested that three of the four amino acids contribute to intersubunit contacts, whereas the other interacts with the TM2-TM3 linker, influencing the receptor response. PMID:25261427

  11. Human Cytomegalovirus Nuclear Egress Proteins Ectopically Expressed in the Heterologous Environment of Plant Cells are Strictly Targeted to the Nuclear Envelope.

    PubMed

    Lamm, Christian E; Link, Katrin; Wagner, Sabrina; Milbradt, Jens; Marschall, Manfred; Sonnewald, Uwe

    2016-03-10

    In all eukaryotic cells, the nucleus forms a prominent cellular compartment containing the cell's nuclear genome. Although structurally similar, animal and plant nuclei differ substantially in details of their architecture. One example is the nuclear lamina, a layer of tightly interconnected filament proteins (lamins) underlying the nuclear envelope of metazoans. So far no orthologous lamin genes could be detected in plant genomes and putative lamin-like proteins are only poorly described in plants. To probe for potentially conserved features of metazoan and plant nuclear envelopes, we ectopically expressed the core nuclear egress proteins of human cytomegalovirus pUL50 and pUL53 in plant cells. pUL50 localizes to the inner envelope of metazoan nuclei and recruits the nuclear localized pUL53 to it, forming heterodimers. Upon expression in plant cells, a very similar localization pattern of both proteins could be determined. Notably, pUL50 is specifically targeted to the plant nuclear envelope in a rim-like fashion, a location to which coexpressed pUL53 becomes strictly corecruited from its initial nucleoplasmic distribution. Using pUL50 as bait in a yeast two-hybrid screening, the cytoplasmic re-initiation supporting protein RISP could be identified. Interaction of pUL50 and RISP could be confirmed by coexpression and coimmunoprecipitation in mammalian cells and by confocal laser scanning microscopy in plant cells, demonstrating partial pUL50-RISP colocalization in areas of the nuclear rim and other intracellular compartments. Thus, our study provides strong evidence for conserved structural features of plant and metazoan nuclear envelops and identifies RISP as a potential pUL50-interacting plant protein.

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

    PubMed

    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)

  13. Heterologous expression of two FAD-dependent oxidases with (S)-tetrahydroprotoberberine oxidase activity from Arge mone mexicana and Berberis wilsoniae in insect cells.

    PubMed

    Gesell, Andreas; Chávez, Maria Luisa Díaz; Kramell, Robert; Piotrowski, Markus; Macheroux, Peter; Kutchan, Toni M

    2011-06-01

    Berberine, palmatine and dehydrocoreximine are end products of protoberberine biosynthesis. These quaternary protoberberines are elicitor inducible and, like other phytoalexins, are highly oxidized. The oxidative potential of these compounds is derived from a diverse array of biosynthetic steps involving hydroxylation, intra-molecular C-C coupling, methylenedioxy bridge formation and a dehydrogenation reaction as the final step in the biosynthesis. For the berberine biosynthetic pathway, the identification of the dehydrogenase gene is the last remaining uncharacterized step in the elucidation of the biosynthesis at the gene level. An enzyme able to catalyze these reactions, (S)-tetrahydroprotoberberine oxidase (STOX, EC 1.3.3.8), was originally purified in the 1980s from suspension cells of Berberis wilsoniae and identified as a flavoprotein (Amann et al. 1984). We report enzymatic activity from recombinant STOX expressed in Spodoptera frugiperda Sf9 insect cells. The coding sequence was derived successively from peptide sequences of purified STOX protein. Furthermore, a recombinant oxidase with protoberberine dehydrogenase activity was obtained from a cDNA library of Argemone mexicana, a traditional medicinal plant that contains protoberberine alkaloids. The relationship of the two enzymes is discussed regarding their enzymatic activity, phylogeny and the alkaloid occurrence in the plants. Potential substrate binding and STOX-specific amino acid residues were identified based on sequence analysis and homology modeling. PMID:21327819

  14. Heterologous expression of the Na+,K+-ATPase γ subunit in Xenopus oocytes induces an endogenous, voltage-gated large diameter pore

    PubMed Central

    Sha, Qun; Lansbery, Kristan L; Distefano, Darcy; Mercer, Robert W; Nichols, Colin G

    2001-01-01

    The γ subunit is a specific component of the plasmalemmal Na+,K+-ATPase. Like structurally related single-spanning membrane proteins such as cardiac phospholemman, Mat-8 and renal CHIF, large ion conductances are activated when γ subunits are expressed in Xenopus oocytes. Here we report critical properties of the γ-activated conductance. The γ-activated conductance showed non-selective cationic and anionic permeation, and extremely slow kinetics, with an activation time constant > 1 s following steps to -100 mV. The γ-activated conductance was inhibited by extracellular divalent ions including Ba2+ (Ki= 0.7 mm) and Ca2+ (Ki= 0.4 mm). 2-Deoxyglucose (MW ∼180), inulin (MW ∼5000) and spermidine (MW ∼148) efflux could occur through the γ-activated conductance pathway, indicating a large pore diameter. In contrast, dextran-70 (MW ∼70 000) did not pass through the γ-activated channel, indicating an upper limit to the pore size of ∼50 Å (5 nm). Similar conductances that are permeable to large molecules were activated by extreme hyperpolarization (> -150 mV) of uninjected oocytes. We conclude that the Na+,K+-ATPase γ subunits activate Ca2+- and voltage-gated, non-selective, large diameter pores that are intrinsically present within the oocyte membrane. PMID:11533133

  15. Aspergillus niger lipase: Heterologous expression in Pichia pastoris, molecular modeling prediction and the importance of the hinge domains at both sides of the lid domain to interfacial activation.

    PubMed

    Shu, Zhengyu; Duan, Mojie; Yang, Jiangke; Xu, Li; Yan, Yunjun

    2009-01-01

    Aspergillus niger lipase (ANL) is an important biocatalyst in the food processing industry. However, there is no report of its detailed three-dimensional structure because of difficulties in crystallization. In this article, based on experimental data and bioinformational analysis results, the structural features of ANL were simulated. Firstly, two recombinant ANLs expressed in Pichia pastoris were purified to homogeneity and their corresponding secondary structure compositions were determined by circular dichroism spectra. Secondly, the primary structure, the secondary structure and the three-dimensional structure of ANL were modeled by comparison with homologous lipases with known three-dimensional structures using the BioEdit software, lipase engineering database (http://www.led.uni-stuttgart.de/), PSIPRED server and SwissModel server. The predicted molecular structure of ANL presented typical features of the alpha/beta hydrolase fold including positioning of the putative catalytic triad residues and the GXSXG signature motif. Comparison of the predicted three-dimensional structure of ANL with the X-ray three-dimensional structure of A. niger feruloyl esterase showed that the functional difference of interfacial activation between lipase and esterase was concerned with the difference in position of the lid. Our three-dimensional model of ANL helps to modify lipase structure by protein engineering, which will further expand the scope of application of ANL. PMID:19248178

  16. Heterologous expression of two FAD-dependent oxidases with (S)-tetrahydroprotoberberine oxidase activity from Arge mone mexicana and Berberis wilsoniae in insect cells.

    PubMed

    Gesell, Andreas; Chávez, Maria Luisa Díaz; Kramell, Robert; Piotrowski, Markus; Macheroux, Peter; Kutchan, Toni M

    2011-06-01

    Berberine, palmatine and dehydrocoreximine are end products of protoberberine biosynthesis. These quaternary protoberberines are elicitor inducible and, like other phytoalexins, are highly oxidized. The oxidative potential of these compounds is derived from a diverse array of biosynthetic steps involving hydroxylation, intra-molecular C-C coupling, methylenedioxy bridge formation and a dehydrogenation reaction as the final step in the biosynthesis. For the berberine biosynthetic pathway, the identification of the dehydrogenase gene is the last remaining uncharacterized step in the elucidation of the biosynthesis at the gene level. An enzyme able to catalyze these reactions, (S)-tetrahydroprotoberberine oxidase (STOX, EC 1.3.3.8), was originally purified in the 1980s from suspension cells of Berberis wilsoniae and identified as a flavoprotein (Amann et al. 1984). We report enzymatic activity from recombinant STOX expressed in Spodoptera frugiperda Sf9 insect cells. The coding sequence was derived successively from peptide sequences of purified STOX protein. Furthermore, a recombinant oxidase with protoberberine dehydrogenase activity was obtained from a cDNA library of Argemone mexicana, a traditional medicinal plant that contains protoberberine alkaloids. The relationship of the two enzymes is discussed regarding their enzymatic activity, phylogeny and the alkaloid occurrence in the plants. Potential substrate binding and STOX-specific amino acid residues were identified based on sequence analysis and homology modeling.

  17. Heterologous production of glidobactins/luminmycins in Escherichia coli Nissle containing the glidobactin biosynthetic gene cluster from Burkholderia DSM7029.

    PubMed

    Bian, Xiaoying; Huang, Fan; Wang, Hailong; Klefisch, Thorsten; Müller, Rolf; Zhang, Youming

    2014-10-13

    Natural product peptide-based proteasome inhibitors show great potential as anticancer drugs. Here we have cloned the biosynthetic gene cluster of a potent proteasome inhibitor-glidobactin from Burkholderia DSM7029-and successfully detected glidobactins/luminmycins in E. coli Nissle. We have also improved the yield of glidobactin A tenfold by promoter change in a heterologous host. In addition, two new biosynthetic intermediates were identified by comparative MS/MS fragmentation analysis. Identification of acyclic luminmycin E implies substrate specificity of the TE domain for cyclization. The establishment of a heterologous expression system for syrbactins provided the basis for the generation of new syrbactins as proteasome inhibitors by molecular engineering, but the TE domain's specificity cannot be ignored. PMID:25147087

  18. Priming with two DNA vaccines expressing hepatitis C virus NS3 protein targeting dendritic cells elicits superior heterologous protective potential in mice.

    PubMed

    Guan, Jie; Deng, Yao; Chen, Hong; Yin, Xiao; Yang, Yang; Tan, Wenjie

    2015-10-01

    Development an effective vaccine may offer an alternative preventive and therapeutic strategy against HCV infection. DNA vaccination has been shown to induce robust humoral and cellular immunity and overcome many problems associated with conventional vaccines. In this study, mice were primed with either conventional pVRC-based or suicidal pSC-based DNA vaccines carrying DEC-205-targeted NS3 antigen (DEC-NS3) and boosted with type 5 adenoviral vectors encoding the partial NS3 and core antigens (C44P). The prime boost regimen induced a marked increase in antigen-specific humoral and T-cell responses in comparison with either rAd5-based vaccines or DEC-205-targeted DNA immunization in isolation. The protective effect against heterogeneous challenge was correlated with high levels of anti-NS3 IgG and T-cell-mediated immunity against NS3 peptides. Moreover, priming with a suicidal DNA vaccine (pSC-DEC-NS3), which elicited increased TNF-α-producing CD4+ and CD8+ T-cells against NS3-2 peptides (aa 1245-1461), after boosting, showed increased heterogeneous protective potential compared with priming with a conventional DNA vaccine (pVRC-DEC-NS3). In conclusion, a suicidal DNA vector (pSC-DEC-NS3) expressing DEC-205-targeted NS3 combined with boosting using an rAd5-based HCV vaccine (rAd5-C44P) is a good candidate for a safe and effective vaccine against HCV infection.

  19. Heterologous expression and biochemical characterization of two calcium-dependent protein kinase isoforms CaCPK1 and CaCPK2 from chickpea.

    PubMed

    Syam Prakash, S R; Jayabaskaran, Chelliah

    2006-11-01

    In plants, calcium-dependent protein kinases (CPKs) constitute a unique family of enzymes consisting of a protein kinase catalytic domain fused to carboxy-terminal autoregulatory and calmodulin-like domains. We isolated two cDNAs encoding calcium-dependent protein kinase isoforms (CaCPK1 and CaCPK2) from chickpea. Both isoforms were expressed as fusion proteins in Escherichia coli. Biochemical analyses have identified CaCPK1 and CaCPK2 as Ca(2+)-dependent protein kinases since both enzymes phosphorylated themselves and histone III-S as substrate only in the presence of Ca(2+). The kinase activity of the recombinant enzymes was calmodulin independent and sensitive to CaM antagonists W7 [N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide] and calmidazoilum. Phosphoamino acid analysis revealed that the isoforms transferred the gamma-phosphate of ATP only to serine residues of histone III-S and their autophosphorylation occurred on serine and threonine residues. These two isoforms showed considerable variations with respect to their biochemical and kinetic properties including Ca(2+) sensitivities. The recombinant CaCPK1 has a pH and temperature optimum of pH 6.8-8.6 and 35-42 degrees C, respectively, whereas CaCPK2 has a pH and temperature optimum of pH 7.2-9 and 35-42 degrees C, respectively. Taken together, our results suggest that CaCPK1 and CaCPK2 are functional serine/threonine kinases and may play different roles in Ca(2+)-mediated signaling in chickpea plants.

  20. Cumulative effect of heterologous AtWRI1 gene expression and endogenous BjAGPase gene silencing increases seed lipid content in Indian mustard Brassica juncea.

    PubMed

    Bhattacharya, Surajit; Das, Natasha; Maiti, Mrinal K

    2016-10-01

    The production of vegetable oil in many countries of the world, including India has not been able to keep pace with the increasing requirement, leading to a very large gap in the demand-supply chain. Thus, there is an urgent need to increase the yield potential of the oilseed crops so as to enhance the storage lipid productivity. The present study describes a novel metabolic engineering ploy involving the constitutive down-regulation of endogenous ADP-glucose pyrophosphorylase (BjAGPase) enzyme and the seed-specific expression of WRINKLED1 transcription factor (AtWRI1) from Arabidopsis thaliana in Indian mustard (Brassica juncea) with an aim to divert the photosynthetically fixed carbon pool from starch to lipid synthesis in the seeds for the enhanced production of storage lipids in the seeds of transgenic mustard plants. The starch content, in both the vegetative leaf and developing seed tissues of the transgenic B. juncea lines exhibited a reduction by about 45-53% compared to the untransformed control, whereas the soluble sugar content was increased by 2.4 and 1.3-fold in the leaf and developing seed tissues, respectively. Consequently, the transgenic lines showed a significant enhancement in total seed lipid content ranging between 7.5 and 16.9%. The results indicate that the adopted metabolic engineering strategy was successful in significantly increasing the seed oil content. Therefore, findings of our research suggest that the metabolic engineering strategy adopted in this study for shifting the anabolic carbon flux from starch synthesis to lipid biosynthesis can be employed for increasing the storage lipid content of seeds in other plant species. PMID:27314514

  1. Characterization of a Functional Soluble Form of a Brassica napus Membrane-Anchored Endo-1,4-β-Glucanase Heterologously Expressed in Pichia pastoris1

    PubMed Central

    Mølhøj, Michael; Ulvskov, Peter; Dal Degan, Florence

    2001-01-01

    The Brassica napus gene, Cel16, encodes a membrane-anchored endo-1,4-β-glucanase with a deduced molecular mass of 69 kD. As for other membrane-anchored endo-1,4-β-glucanases, Cel16 consists of a predicted intracellular, charged N terminus (methionine1-lysine70), a hydrophobic transmembrane domain (isoleucine71-valine93), and a periplasmic catalytic core (lysine94-proline621). Here, we report the functional analysis of Δ1-90Cel16, the N terminally truncated Cel16, missing residues 1 through 90 and comprising the catalytic domain of Cel16 expressed recombinantly in the methylotrophic yeast Pichia pastoris as a soluble protein. A two-step purification protocol yielded Δ1-90Cel16 in a pure form. The molecular mass of Δ1-90Cel16, when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was about 130 kD and about 60 kD after enzymatic removal of N-glycans, fitting the expected molecular mass of 59 kD. Δ1-90Cel16 was highly N glycosylated as compared with the native B. napus Cel16 protein. Δ1-90Cel16 had a pH optimum of 6.0. The activity of Δ1-90Cel16 was inhibited by EDTA and exhibited a strong dependence on calcium. Δ1-90Cel16 showed substrate specificity for low substituted carboxymethyl-cellulose and amorphous cellulose. It did not hydrolyze crystalline cellulose, xyloglycan, xylan, (1→3),(1→4)-β-d-glucan, the highly substituted hydroxyethylcellulose, or the oligosaccharides cellotriose, cellotetraose, cellopentaose, or xylopentaose. Size exclusion analysis of Δ1-90Cel16-hydrolyzed carboxymethylcellulose showed that Δ1-90Cel16 is a true endo-acting glucanase. PMID:11598241

  2. A novel cold-active and salt-tolerant α-amylase from marine bacterium Zunongwangia profunda: molecular cloning, heterologous expression and biochemical characterization.

    PubMed

    Qin, Yongjun; Huang, Zongqing; Liu, Ziduo

    2014-03-01

    A novel gene (amyZ) encoding a cold-active and salt-tolerant α-amylase (AmyZ) was cloned from marine bacterium Zunongwangia profunda (MCCC 1A01486) and the protein was expressed in Escherichia coli. The gene has a length of 1785 bp and encodes an α-amylase of 594 amino acids with an estimated molecular mass of 66 kDa by SDS-PAGE. The enzyme belongs to glycoside hydrolase family 13 and shows the highest identity (25%) to the characterized α-amylase TVA II from thermoactinomyces vulgaris R-47. The recombinant α-amylase showed the maximum activity at 35 °C and pH 7.0, and retained about 39% activity at 0 °C. AmyZ displayed extreme salt tolerance, with the highest activity at 1.5 M NaCl and 93% activity even at 4 M NaCl. The catalytic efficiency (k cat/K m) of AmyZ increased from 115.51 (with 0 M NaCl) to 143.30 ml mg(-1) s(-1) (with 1.5 M NaCl) at 35 °C and pH 7.0, using soluble starch as substrate. Besides, the thermostability of the enzyme was significantly improved in the presence of 1.5 M NaCl or 1 mM CaCl2. AmyZ is one of the very few α-amylases that tolerate both high salinity and low temperatures, making it a potential candidate for research in basic and applied biology.

  3. Comparison of Steroid Modulation of Spontaneous Inhibitory Postsynaptic Currents in Cultured Hippocampal Neurons and Steady-State Single-Channel Currents from Heterologously Expressed α1β2γ2L GABA(A) Receptors.

    PubMed

    Chakrabarti, Sampurna; Qian, Mingxing; Krishnan, Kathiresan; Covey, Douglas F; Mennerick, Steven; Akk, Gustav

    2016-04-01

    Neuroactive steroids are efficacious modulators of γ-aminobutyric acid type A receptor (GABA(A)) receptor function. The effects of steroids on the GABA(A) receptor are typically determined by comparing steady-state single-channel open probability or macroscopic peak responses elicited by GABA in the absence and presence of a steroid. Due to differences in activation conditions (exposure duration, concentration of agonist), it is not obvious whether modulation measured using typical experimental protocols can be used to accurately predict the effect of a modulator on native receptors under physiologic conditions. In the present study, we examined the effects of 14 neuroactive steroids and analogs on the properties of spontaneous inhibitory postsynaptic currents (sIPSCs) in cultured rat hippocampal neurons. The goal was to determine whether the magnitude of modulation of the decay time course of sIPSCs correlates with the extent of modulation and kinetic properties of potentiation as determined in previous single-channel studies. The steroids were selected to cover a wide range of efficacy on heterologously expressed rat α1β2γ2L GABA(A) receptors, ranging from essentially inert to highly efficacious (strong potentiators of single-channel and macroscopic peak responses). The data indicate a strong correlation between prolongation of the decay time course of sIPSCs and potentiation of single-channel open probability. Furthermore, changes in intracluster closed time distributions were the single best predictor of prolongation of sIPSCs. We infer that the information obtained in steady-state single-channel recordings can be used to forecast modulation of synaptic currents.

  4. cDNA cloning, heterologous expressions, and functional characterization of malonyl-coenzyme a:anthocyanidin 3-o-glucoside-6"-o-malonyltransferase from dahlia flowers.

    PubMed

    Suzuki, Hirokazu; Nakayama, Toru; Yonekura-Sakakibara, Keiko; Fukui, Yuko; Nakamura, Noriko; Yamaguchi, Masa-Atsu; Tanaka, Yoshikazu; Kusumi, Takaaki; Nishino, Tokuzo

    2002-12-01

    In the flowers of important ornamental Compositae plants, anthocyanins generally carry malonyl group(s) at their 3-glucosyl moiety. In this study, for the first time to our knowledge, we have identified a cDNA coding for this 3-glucoside-specific malonyltransferase for anthocyanins, i.e. malonyl-coenzyme A:anthocyanidin 3-O-glucoside-6"-O-malonyltransferase, from dahlia (Dahlia variabilis) flowers. We isolated a full-length cDNA (Dv3MaT) on the basis of amino acid sequences specifically conserved among anthocyanin acyltransferases of the versatile plant acyltransferase family. Dv3MaT coded for a protein of 460 amino acids. Quantitative real-time PCR analyses of Dv3MaT showed that the transcript was present in accordance with the distribution of 3MaT activities and the anthocyanin accumulation pattern in the dahlia plant. The Dv3MaT cDNA was expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity and characterized. The recombinant Dv3MaT catalyzed the regiospecific transfer of the malonyl group from malonyl-coenzyme A (K(m), 18.8 microM) to pelargonidin 3-O-glucoside (K(m), 46.7 microM) to produce pelargonidin 3-O-6"-O-malonylglucoside with a k(cat) value of 7.3 s(-1). The other enzymatic profiles of the recombinant Dv3MaT were closely related to those of native anthocyanin malonyltransferase activity in the extracts of dahlia flowers. Dv3MaT cDNA was introduced into petunia (Petunia hybrida) plants whose red floral color is exclusively provided by cyanidin 3-O-glucoside and 3,5-O-diglucoside. Thirteen transgenic lines of petunia were found to produce malonylated products of these anthocyanins (11-63 mol % of total anthocyanins in the flower). The spectral stability of cyanidin 3-O-6"-O-malonylglucoside at the pHs of intracellular milieus of flowers was significantly higher than that of cyanidin 3-O-glucoside. Moreover, 6"-O-malonylation of cyanidin 3-O-glucoside effectively prevented the anthocyanin from attack of beta

  5. Application of image cytometry to characterize heterologous lipid flippases in yeast.

    PubMed

    Jensen, Maria S; Costa, Sara R; Theorin, Lisa; Christensen, Jan Pravsgaard; Pomorski, Thomas Günther; López-Marqués, Rosa L

    2016-07-01

    Lipid flippases are integral membrane proteins that play a central role in moving lipids across cellular membranes. Some of these transporters are ATPases that couple lipid translocation to ATP hydrolysis, whereas others function without any discernible metabolic energy input. A growing number of lipid flippases has been identified but key features of their activity remain to be elucidated. A well-established method to characterize ATP-driven flippases is based on their heterologous expression in yeast, followed by incubation of the cells with fluorescent lipids. Internalization of these probes is typically monitored by flow cytometry, a costly and maintenance-intensive method. Here, we have optimized a protocol to use an automated image-based cell counter to accurately measure lipid uptake by heterologous lipid flippases expressed in yeast. The method was validated by comparison with the classical flow cytometric evaluation of lipid-labeled cells. In addition, we demonstrated that expression of fluorescently tagged flippase complexes can be directly co-related with fluorescent lipid uptake using the image-based cell counter system. The method extends the number of techniques available for characterization of lipid flippase activity, and should be readily adaptable to analyze a variety of other transport systems in yeast, parasites, and mammalian cells. © 2016 International Society for Advancement of Cytometry. PMID:27272389

  6. μ-Opioid receptor desensitization: homologous or heterologous?

    PubMed

    Llorente, Javier; Lowe, Janet D; Sanderson, Helen S; Tsisanova, Elena; Kelly, Eamonn; Henderson, Graeme; Bailey, Chris P

    2012-12-01

    There is considerable controversy over whether μ-opioid receptor (MOPr) desensitization is homologous or heterologous and over the mechanisms underlying such desensitization. In different cell types MOPr desensitization has been reported to involve receptor phosphorylation by various kinases, including G-protein-coupled receptor kinases (GRKs), second messenger and other kinases as well as perturbation of the MOPr effector pathway by GRK sequestration of G protein βγ subunits or ion channel modulation. Here we report that in brainstem locus coeruleus (LC) neurons prepared from relatively mature rats (5-8 weeks old) rapid MOPr desensitization induced by the high-efficacy opioid peptides methionine enkephalin and DAMGO was homologous and not heterologous to α(2)-adrenoceptors and somatostatin SST(2) receptors. Given that these receptors all couple through G proteins to the same set of G-protein inwardly rectifying (GIRK) channels it is unlikely therefore that in mature neurons MOPr desensitization involves G protein βγ subunit sequestration or ion channel modulation. In contrast, in slices from immature animals (less than postnatal day 20), MOPr desensitization was observed to be heterologous and could be downstream of the receptor. Heterologous MOPr desensitization was not dependent on protein kinase C or c-Jun N-terminal kinase activity, but the change from heterologous to homologous desensitization with age was correlated with a decrease in the expression levels of GRK2 in the LC and other brain regions. The observation that the mechanisms underlying MOPr desensitization change with neuronal development is important when extrapolating to the mature brain results obtained from experiments on expression systems, cell lines and immature neuronal preparations.

  7. Heterologous xylose isomerase pathway and evolutionary engineering improve xylose utilization in Saccharomyces cerevisiae

    PubMed Central

    Qi, Xin; Zha, Jian; Liu, Gao-Gang; Zhang, Weiwen; Li, Bing-Zhi; Yuan, Ying-Jin

    2015-01-01

    Xylose utilization is one key issue for the bioconversion of lignocelluloses. It is a promising approach to engineering heterologous pathway for xylose utilization in Saccharomyces cerevisiae. Here, we constructed a xylose-fermenting yeast SyBE001 through combinatorial fine-tuning the expression of XylA and endogenous XKS1. Additional overexpression of genes RKI1, RPE1, TKL1, and TAL1 in the non-oxidative pentose phosphate pathway (PPP) in SyBE001 increased the xylose consumption rate by 1.19-fold. By repetitive adaptation, the xylose utilization rate was further increased by ∼10-fold in the resultant strain SyBE003. Gene expression analysis identified a variety of genes with significantly changed expression in the PPP, glycolysis and the tricarboxylic acid cycle in SyBE003. PMID:26539187

  8. Heterologous laccase production and its role in industrial applications

    PubMed Central

    Pezzella, Cinzia; Giardina, Paola; Faraco, Vincenza; Sannia, Giovanni

    2010-01-01

    Laccases are blue multicopper oxidases, catalyzing the oxidation of an array of aromatic substrates concomitantly with the reduction of molecular oxygen to water. These enzymes are implicated in a variety of biological activities. Most of the laccases studied thus far are of fungal origin. The large range of substrates oxidized by laccases has raised interest in using them within different industrial fields, such as pulp delignification, textile dye bleaching and bioremediation. Laccases secreted from native sources are usually not suitable for large-scale purposes, mainly due to low production yields and high cost of preparation/purification procedures. Heterologous expression may provide higher enzyme yields and may permit to produce laccases with desired properties (such as different substrate specificities, or improved stabilities) for industrial applications. This review surveys researches on heterologous laccase expression focusing on the pivotal role played by recombinant systems towards the development of robust tools for greening modern industry. PMID:21327057

  9. Heterologous laccase production and its role in industrial applications.

    PubMed

    Piscitelli, Alessandra; Pezzella, Cinzia; Giardina, Paola; Faraco, Vincenza; Giovanni, Sannia

    2010-01-01

    Laccases are blue multicopper oxidases, catalyzing the oxidation of an array of aromatic substrates concomitantly with the reduction of molecular oxygen to water. These enzymes are implicated in a variety of biological activities. Most of the laccases studied thus far are of fungal origin. The large range of substrates oxidized by laccases has raised interest in using them within different industrial fields, such as pulp delignification, textile dye bleaching, and bioremediation. Laccases secreted from native sources are usually not suitable for large-scale purposes, mainly due to low production yields and high cost of preparation/purification procedures. Heterologous expression may provide higher enzyme yields and may permit to produce laccases with desired properties (such as different substrate specificities, or improved stabilities) for industrial applications. This review surveys researches on heterologous laccase expression focusing on the pivotal role played by recombinant systems towards the development of robust tools for greening modern industry. PMID:21327057

  10. Improving heterologous polyketide production in Escherichia coli by transporter engineering.

    PubMed

    Yang, Jingya; Xiong, Zhi-Qiang; Song, Shu-Jie; Wang, Jian-Feng; Lv, Hua-Jun; Wang, Yong

    2015-10-01

    Expelling heterologous compounds out of hosts by transporters is a potential strategy to enhance product titers in microbial cell factories. In this work, to increase heterologous polyketide 6-deoxyerythronolide B (6dEB, erythromycin precursor) production, tripartite multidrug efflux pumps MacAB-TolC, AcrAB-TolC, MdtEF-TolC, and MexAB-OprM were modulated in a 6dEB production strain. Compared with the control, overexpression of a single component of efflux pumps (except oprM) repressed 6dEB production, but modulation of two components MacA and MacB or the complete pumps MacAB-TolC and MdtEF-TolC significantly improved 6dEB titer by 100 ± 11, 118 ± 54, and 98 ± 12 %, respectively. In addition, to avoid the challenging fine-tuning components of pumps, the transcriptional regulators of efflux pumps were modulated to improve the 6dEB production. Overexpression of RpoH (activator of MdtEF-TolC) and EvgA (activator of EmrKY-TolC and AcrAD-TolC) strongly increased 6dEB titer by 152 ± 54 and 142 ± 85 %, respectively. This is the first report of transporter engineering for improving heterologous polyketide production in Escherichia coli. Our results provide an effective strategy for improving the yield of the heterologous products in chassis cell. PMID:26062534

  11. Recombinant measles virus incorporating heterologous viral membrane proteins for use as vaccines.

    PubMed

    Swett-Tapia, Cindy; Bogaert, Lies; de Jong, Pascal; van Hoek, Vladimir; Schouten, Theo; Damen, Irma; Spek, Dirk; Wanningen, Patrick; Radošević, Katarina; Widjojoatmodjo, Myra N; Zahn, Roland; Custers, Jerome; Roy, Soumitra

    2016-09-01

    Recombinant measles virus (rMV) vectors expressing heterologous viral membrane protein antigens are potentially useful as vaccines. Genes encoding the mumps virus haemagglutinin-neuraminidase (MuV-HN), the influenza virus haemagglutinin (Flu-HA) or the respiratory syncytial virus fusion (RSV-F) proteins were inserted into the genome of a live attenuated vaccine strain of measles virus. Additionally, in this case rMV with the MuV-HN or the influenza HA inserts, chimeric constructs were created that harboured the measles virus native haemagglutinin or fusion protein cytoplasmic domains. In all three cases, sucrose-gradient purified preparations of rMV were found to have incorporated the heterologous viral membrane protein on the viral membrane. The possible utility of rMV expressing RSV-F (rMV.RSV-F) as a vaccine was tested in a cotton rat challenge model. Vaccination with rMV.RSV-F efficiently induced neutralizing antibodies against RSV and protected animals from infection with RSV in the lungs. PMID:27311834

  12. Heterologous carotenoid-biosynthetic enzymes: functional complementation and effects on carotenoid profiles in Escherichia coli.

    PubMed

    Song, Gyu Hyeon; Kim, Se Hyeuk; Choi, Bo Hyun; Han, Se Jong; Lee, Pyung Cheon

    2013-01-01

    A limited number of carotenoid pathway genes from microbial sources have been studied for analyzing the pathway complementation in the heterologous host Escherichia coli. In order to systematically investigate the functionality of carotenoid pathway enzymes in E. coli, the pathway genes of carotenogenic microorganisms (Brevibacterium linens, Corynebacterium glutamicum, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodopirellula baltica, and Pantoea ananatis) were modified to form synthetic expression modules and then were complemented with Pantoea agglomerans pathway enzymes (CrtE, CrtB, CrtI, CrtY, and CrtZ). The carotenogenic pathway enzymes in the synthetic modules showed unusual activities when complemented with E. coli. For example, the expression of heterologous CrtEs of B. linens, C. glutamicum, and R. baltica influenced P. agglomerans CrtI to convert its substrate phytoene into a rare product-3,4,3',4'-tetradehydrolycopene-along with lycopene, which was an expected product, indicating that CrtE, the first enzyme in the carotenoid biosynthesis pathway, can influence carotenoid profiles. In addition, CrtIs of R. sphaeroides and R. capsulatus converted phytoene into an unusual lycopene as well as into neurosporene. Thus, this study shows that the functional complementation of pathway enzymes from different sources is a useful methodology for diversifying biosynthesis as nature does. PMID:23144136

  13. Heterologous protein production using the twin arginine translocation pathway

    DOEpatents

    Pohlschroder, Mechtild; Kissinger, Jessica C; Rose, R. Wesley; Brueser, Thomas; Dilks, Kieran

    2008-11-04

    Provided are means for evaluating and identifying putative substrates of the twin arginine translocation (Tat) secretory pathway in Streptomyces and other bacterial species. Also provided, therefore, are simple ways to express, secrete and purify correctly folded heterologous proteins on a large scale using host microorganisms, such as, Streptomyces and the Tat pathway therein. Many of the thus-produced proteins are of significant therapeutic value in the pharmaceutical and biochemical industries, particularly when they can be secreted from the host in fully-folded active form. Accordingly, there are further provided the heterologous proteins produced by the Tat secretion pathway using the foregoing methods, and the computer algorithm used to identify the Tat signal sequence and putative substrates.

  14. Engineering the Expression and Characterization of Two Novel Laccase Isoenzymes from Coprinus comatus in Pichia pastoris by Fusing an Additional Ten Amino Acids Tag at N-Terminus

    PubMed Central

    Gu, Chunjuan; Zheng, Fei; Long, Liangkun; Wang, Jing; Ding, Shaojun

    2014-01-01

    The detail understanding of physiological/biochemical characteristics of individual laccase isoenzymes in fungi is necessary for fundamental and application purposes, but our knowledge is still limited for most of fungi due to difficult to express laccases heterologously. In this study, two novel laccase genes, named lac3 and lac4, encoding proteins of 547 and 532-amino acids preceded by 28 and 16-residue signal peptides, respectively, were cloned from the edible basidiomycete Coprinus comatus. They showed 70% identity but much lower homology with other fungal laccases at protein level (less than 58%). Two novel laccase isoenzymes were successfully expressed in Pichia pastoris by fusing an additional 10 amino acids (Thr-Pro-Phe-Pro-Pro-Phe-Asn-Thr-Asn-Ser) tag at N-terminus, and the volumetric activities could be dramatically enhanced from undetectable level to 689 and 1465 IU/l for Lac3 and Lac4, respectively. Both laccases possessed the lowest Km and highest kcat/Km value towards syringaldazine, followed by ABTS, guaiacol and 2,6-dimethylphenol similar as the low redox potential laccases from other microorganisms. Lac3 and Lac4 showed resistant to SDS, and retained 31.86% and 43.08% activity in the presence of 100 mM SDS, respectively. Lac3 exhibited higher decolorization efficiency than Lac4 for eleven out of thirteen different dyes, which may attribute to the relatively higher catalytic efficiency of Lac3 than Lac4 (in terms of kcat/Km) towards syringaldazine and ABTS. The mild synergistic decolorization by two laccases was observed for triphenylmethane dyes but not for anthraquinone and azo dyes. PMID:24710109

  15. Functional phytohemagglutinin (PHA) and Galanthus nivalis agglutinin (GNA) expressed in Pichia pastoris correct N-terminal processing and secretion of heterologous proteins expressed using the PHA-E signal peptide.

    PubMed

    Raemaekers, R J; de Muro, L; Gatehouse, J A; Fordham-Skelton, A P

    1999-10-01

    Phytohemagglutinin (Phaseolus vulgaris agglutinin; PHA; E- and L-forms) and snowdrop lectin (Galanthus nivalis agglutinin; GNA) were expressed in Pichia pastoris using native signal peptides, or the Saccharomyces alpha-factor preprosequence, to direct proteins into the secretory pathway. PHA and GNA were present as soluble, functional proteins in culture supernatants when expressed from constructs containing the alpha-factor preprosequence. The recombinant lectins, purified by affinity chromatography, agglutinated rabbit erythrocytes at concentrations similar to the respective native lectins. However, incomplete processing of the signal sequence resulted in PHA-E, PHA-L and GNA with heterogenous N-termini, with the majority of the protein containing N-terminal extensions derived from the alpha-factor prosequence. Polypeptides in which most of the alpha-factor prosequence was present were also glycosylated. Inclusion of Glu-Ala repeats at the C-terminal end of the alpha-factor preprosequence led to efficient processing N-terminal to the Glu-Ala sequence, but inefficient removal of the repeats themselves, resulting in polypeptides with heterogenous N-termini still containing N-terminal extensions. In contrast, PHA expressed with the native signal peptide was secreted, correctly processed, and also fully functional. No expression of GNA from a construct containing the native GNA signal peptide was observed. The PHA-E signal peptide directed correct processing and secretion of both GNA and green fluorescent protein (GFP) when used in expression constructs, and is suggested to have general utility for synthesis of correctly processed proteins in Pichia.

  16. Kluyveromyces marxianus as a host for heterologous protein synthesis.

    PubMed

    Gombert, Andreas K; Madeira, José Valdo; Cerdán, María-Esperanza; González-Siso, María-Isabel

    2016-07-01

    The preferentially respiring and thermotolerant yeast Kluyveromyces marxianus is an emerging host for heterologous protein synthesis, surpassing the traditional preferentially fermenting yeast Saccharomyces cerevisiae in some important aspects: K . marxianus can grow at temperatures 10 °C higher than S. cerevisiae, which may result in decreased costs for cooling bioreactors and reduced contamination risk; has ability to metabolize a wider variety of sugars, such as lactose and xylose; is the fastest growing eukaryote described so far; and does not require special cultivation techniques (such as fed-batch) to avoid fermentative metabolism. All these advantages exist together with a high secretory capacity, performance of eukaryotic post-translational modifications, and with a generally regarded as safe (GRAS) status. In the last years, replication origins from several Kluyveromyces spp. have been used for the construction of episomal vectors, and also integrative strategies have been developed based on the tendency for non-homologous recombination displayed by K. marxianus. The recessive URA3 auxotrophic marker and the dominant Kan(R) are mostly used for selection of transformed cells, but other markers have been made available. Homologous and heterologous promoters and secretion signals have been characterized, with the K. marxianus INU1 expression and secretion system being of remarkable functionality. The efficient synthesis of roughly 50 heterologous proteins has been demonstrated, including one thermophilic enzyme. In this mini-review, we summarize the physiological characteristics of K. marxianus relevant for its use in the efficient synthesis of heterologous proteins, the efforts performed hitherto in the development of a molecular toolbox for this purpose, and some successful examples. PMID:27260286

  17. Additional disulfide bonds in insulin: Prediction, recombinant expression, receptor binding affinity, and stability

    PubMed Central

    Vinther, Tine N; Pettersson, Ingrid; Huus, Kasper; Schlein, Morten; Steensgaard, Dorte B; Sørensen, Anders; Jensen, Knud J; Kjeldsen, Thomas; Hubalek, František

    2015-01-01

    The structure of insulin, a glucose homeostasis-controlling hormone, is highly conserved in all vertebrates and stabilized by three disulfide bonds. Recently, we designed a novel insulin analogue containing a fourth disulfide bond located between positions A10-B4. The N-terminus of insulin's B-chain is flexible and can adapt multiple conformations. We examined how well disulfide bond predictions algorithms could identify disulfide bonds in this region of insulin. In order to identify stable insulin analogues with additional disulfide bonds, which could be expressed, the Cβ cut-off distance had to be increased in many instances and single X-ray structures as well as structures from MD simulations had to be used. The analogues that were identified by the algorithm without extensive adjustments of the prediction parameters were more thermally stable as assessed by DSC and CD and expressed in higher yields in comparison to analogues with additional disulfide bonds that were more difficult to predict. In contrast, addition of the fourth disulfide bond rendered all analogues resistant to fibrillation under stress conditions and all stable analogues bound to the insulin receptor with picomolar affinities. Thus activity and fibrillation propensity did not correlate with the results from the prediction algorithm. PMID:25627966

  18. Heterologous Expression of Moss Light-harvesting Complex Stress-related 1 (LHCSR1), the Chlorophyll a-Xanthophyll Pigment-protein Complex Catalyzing Non-photochemical Quenching, in Nicotiana sp.*

    PubMed Central

    Pinnola, Alberta; Ghin, Leonardo; Gecchele, Elisa; Merlin, Matilde; Alboresi, Alessandro; Avesani, Linda; Pezzotti, Mario; Capaldi, Stefano; Cazzaniga, Stefano; Bassi, Roberto

    2015-01-01

    Oxygenic photosynthetic organisms evolved mechanisms for thermal dissipation of energy absorbed in excess to prevent formation of reactive oxygen species. The major and fastest component, called non-photochemical quenching, occurs within the photosystem II antenna system by the action of two essential light-harvesting complex (LHC)-like proteins, photosystem II subunit S (PSBS) in plants and light-harvesting complex stress-related (LHCSR) in green algae and diatoms. In the evolutionary intermediate Physcomitrella patens, a moss, both gene products are active. These proteins, which are present in low amounts, are difficult to purify, preventing structural and functional analysis. Here, we report on the overexpression of the LHCSR1 protein from P. patens in the heterologous systems Nicotiana benthamiana and Nicotiana tabacum using transient and stable nuclear transformation. We show that the protein accumulated in both heterologous systems is in its mature form, localizes in the chloroplast thylakoid membranes, and is correctly folded with chlorophyll a and xanthophylls but without chlorophyll b, an essential chromophore for plants and algal LHC proteins. Finally, we show that recombinant LHCSR1 is active in quenching in vivo, implying that the recombinant protein obtained is a good material for future structural and functional studies. PMID:26260788

  19. Heterologous expression of moss light-harvesting complex stress-related 1 (LHCSR1), the chlorophyll a-xanthophyll pigment-protein complex catalyzing non-photochemical quenching, in Nicotiana sp.

    PubMed

    Pinnola, Alberta; Ghin, Leonardo; Gecchele, Elisa; Merlin, Matilde; Alboresi, Alessandro; Avesani, Linda; Pezzotti, Mario; Capaldi, Stefano; Cazzaniga, Stefano; Bassi, Roberto

    2015-10-01

    Oxygenic photosynthetic organisms evolved mechanisms for thermal dissipation of energy absorbed in excess to prevent formation of reactive oxygen species. The major and fastest component, called non-photochemical quenching, occurs within the photosystem II antenna system by the action of two essential light-harvesting complex (LHC)-like proteins, photosystem II subunit S (PSBS) in plants and light-harvesting complex stress-related (LHCSR) in green algae and diatoms. In the evolutionary intermediate Physcomitrella patens, a moss, both gene products are active. These proteins, which are present in low amounts, are difficult to purify, preventing structural and functional analysis. Here, we report on the overexpression of the LHCSR1 protein from P. patens in the heterologous systems Nicotiana benthamiana and Nicotiana tabacum using transient and stable nuclear transformation. We show that the protein accumulated in both heterologous systems is in its mature form, localizes in the chloroplast thylakoid membranes, and is correctly folded with chlorophyll a and xanthophylls but without chlorophyll b, an essential chromophore for plants and algal LHC proteins. Finally, we show that recombinant LHCSR1 is active in quenching in vivo, implying that the recombinant protein obtained is a good material for future structural and functional studies. PMID:26260788

  20. Heterologous expression of moss light-harvesting complex stress-related 1 (LHCSR1), the chlorophyll a-xanthophyll pigment-protein complex catalyzing non-photochemical quenching, in Nicotiana sp.

    PubMed

    Pinnola, Alberta; Ghin, Leonardo; Gecchele, Elisa; Merlin, Matilde; Alboresi, Alessandro; Avesani, Linda; Pezzotti, Mario; Capaldi, Stefano; Cazzaniga, Stefano; Bassi, Roberto

    2015-10-01

    Oxygenic photosynthetic organisms evolved mechanisms for thermal dissipation of energy absorbed in excess to prevent formation of reactive oxygen species. The major and fastest component, called non-photochemical quenching, occurs within the photosystem II antenna system by the action of two essential light-harvesting complex (LHC)-like proteins, photosystem II subunit S (PSBS) in plants and light-harvesting complex stress-related (LHCSR) in green algae and diatoms. In the evolutionary intermediate Physcomitrella patens, a moss, both gene products are active. These proteins, which are present in low amounts, are difficult to purify, preventing structural and functional analysis. Here, we report on the overexpression of the LHCSR1 protein from P. patens in the heterologous systems Nicotiana benthamiana and Nicotiana tabacum using transient and stable nuclear transformation. We show that the protein accumulated in both heterologous systems is in its mature form, localizes in the chloroplast thylakoid membranes, and is correctly folded with chlorophyll a and xanthophylls but without chlorophyll b, an essential chromophore for plants and algal LHC proteins. Finally, we show that recombinant LHCSR1 is active in quenching in vivo, implying that the recombinant protein obtained is a good material for future structural and functional studies.

  1. Emotional expression in music: contribution, linearity, and additivity of primary musical cues.

    PubMed

    Eerola, Tuomas; Friberg, Anders; Bresin, Roberto

    2013-01-01

    The aim of this study is to manipulate musical cues systematically to determine the aspects of music that contribute to emotional expression, and whether these cues operate in additive or interactive fashion, and whether the cue levels can be characterized as linear or non-linear. An optimized factorial design was used with six primary musical cues (mode, tempo, dynamics, articulation, timbre, and register) across four different music examples. Listeners rated 200 musical examples according to four perceived emotional characters (happy, sad, peaceful, and scary). The results exhibited robust effects for all cues and the ranked importance of these was established by multiple regression. The most important cue was mode followed by tempo, register, dynamics, articulation, and timbre, although the ranking varied across the emotions. The second main result suggested that most cue levels contributed to the emotions in a linear fashion, explaining 77-89% of variance in ratings. Quadratic encoding of cues did lead to minor but significant increases of the models (0-8%). Finally, the interactions between the cues were non-existent suggesting that the cues operate mostly in an additive fashion, corroborating recent findings on emotional expression in music (Juslin and Lindström, 2010). PMID:23908642

  2. Using Transcriptional Control To Increase Titers of Secreted Heterologous Proteins by the Type III Secretion System

    PubMed Central

    Metcalf, Kevin J.; Finnerty, Casey; Azam, Anum; Valdivia, Elias

    2014-01-01

    The type III secretion system (T3SS) encoded at the Salmonella pathogenicity island 1 (SPI-1) locus secretes protein directly from the cytosol to the culture media in a concerted, one-step process, bypassing the periplasm. While this approach is attractive for heterologous protein production, product titers are too low for many applications. In addition, the expression of the SPI-1 gene cluster is subject to native regulation, which requires culturing conditions that are not ideal for high-density growth. We used transcriptional control to increase the amount of protein that is secreted into the extracellular space by the T3SS of Salmonella enterica. The controlled expression of the gene encoding SPI-1 transcription factor HilA circumvents the requirement of endogenous induction conditions and allows for synthetic induction of the secretion system. This strategy increases the number of cells that express SPI-1 genes, as measured by promoter activity. In addition, protein secretion titer is sensitive to the time of addition and the concentration of inducer for the protein to be secreted and SPI-1 gene cluster. Overexpression of hilA increases secreted protein titer by >10-fold and enables recovery of up to 28 ± 9 mg/liter of secreted protein from an 8-h culture. We also demonstrate that the protein beta-lactamase is able to adopt an active conformation after secretion, and the increase in secreted titer from hilA overexpression also correlates to increased enzyme activity in the culture supernatant. PMID:25038096

  3. Identification of maize embryo-preferred promoters suitable for high-level heterologous protein production.

    PubMed

    Streatfield, Stephen J; Bray, Jeffrey; Love, Robert T; Horn, Michael E; Lane, Jeffrey R; Drees, Carol F; Egelkrout, Erin M; Howard, John A

    2010-01-01

    The production of heterologous proteins in plants at levels consistent with commercialization of protein products requires molecular tools to ensure high-level transgene expression. The identification of strong promoters, preferably specific to the target expression tissue, is a focus for improving foreign protein yields using transgenic cereals as a production system. Thus, there is a requirement for strong embryo preferred monocot promoters. We obtained the sequences of 500 randomly selected maize cDNA clones to determine gene expression profiles in embryo tissues at multiple stages during development. Promoters corresponding to the most abundant clones were identified and isolated. These promoters were fused to the b-glucuronidase reporter and their tissue specificity and developmental expression characteristics assessed in transgenic maize. All of the isolated promoters tested drove transgene expression predominantly in the embryo and were most active late in embryogenesis during storage protein deposition. One of the most active promoters assessed by transgene expression was associated with the globulin-1 protein. Sequence identified here extended approximately 1.6 kb distal to the previously identified extent of the globulin-1 promoter, and this additional sequence boosted expression over two-fold. The extended globulin-1 promoter sequence isolated in this study has the potential for driving transgene expression at higher levels than those previously reported for cereals. Also, other highly active embryo promoters identified here offer opportunities to express multiple foreign proteins simultaneously at high levels in embryo tissues, while avoiding concerns over gene silencing due to the repeated use of a single promoter. PMID:21844671

  4. Heterologous production of ribostamycin derivatives in engineered Escherichia coli.

    PubMed

    Kurumbang, Nagendra Prasad; Park, Je Won; Yoon, Yeo Joon; Liou, Kwangkyoung; Sohng, Jae Kyung

    2010-09-01

    Aminoglycosides are a class of important antibiotic compounds used for various therapeutic indications. In recent times, their efficacy has been curtailed due to the rapid development of bacterial resistance. There is a need to develop novel derivatives with an improved spectrum of activity and higher sensitivity against pathogenic bacteria. Although efforts have been focused on the development of newer therapeutic agents by chemical synthesis, to our knowledge, there has been no attempt to harness the potential of microorganisms for this purpose. Escherichia coli affords a widely studied cellular system that could be utilized not only for understanding but also for attempting to engineer the biosynthetic pathway of secondary metabolites. The primary metabolic pathway of E. coli can be engineered to divert the precursor pool required for the biosynthesis of secondary metabolites. Utilizing this approach previously, we engineered E. coli host and generated E. coli M1. Here, we produced a ribostamycin derivative in the engineered host by heterologous expression of the recombinants constructed from the genes encoding the biosynthetic pathway in aminoglycoside-producing strains. The products obtained from the transformants were isolated, analyzed and verified to be ribostamycin derivatives. The study further demonstrated the importance of E. coli as surrogate antibiotic producer and also offered future possibility for the production of other aminoglycoside derivatives through genetic engineering and expression in a heterologous background.

  5. Heterologous expressed toxic and non-toxic peptide variants of toxin CssII are capable to produce neutralizing antibodies against the venom of the scorpion Centruroides suffusus suffusus.

    PubMed

    Hernández-Salgado, Kenya; Estrada, Georgina; Olvera, Alejandro; Coronas, Fredy I; Possani, Lourival D; Corzo, Gerardo

    2009-08-15

    Two toxic and one non-toxic recombinant peptide variants of the mammalian neurotoxin CssII was cloned into the expression vector pQE30 containing a 6His-tag and a Factor Xa proteolytic cleavage site. The toxic recombinant peptides rCssII, HisrCssII and the non-toxic rCssIIE15R were expressed under induction with isopropyl thiogalactoside (IPTG), isolated using chromatographic techniques and folded correctly in vitro. The three recombinant variants showed similar secondary structures as the native CssII, but only the rCssIIE15R was not toxic to mice at concentrations up to 30microg/20g mouse body weight when injected intraperitoneally. All three recombinant peptides were capable of displacing the native CssII from their receptor sites in rat brain synaptosomes, suggesting that they had similar structural and functional characteristics of the native peptides. The three recombinant variants of CssII and the native one were used as antigens for immunization of New Zealand rabbits. The antibodies present in the rabbit antisera were able to recognize the native CssII. Additionally and more importantly, the sera of the immunized rabbits were able to neutralize both the native toxin CssII and the whole soluble venom of the scorpion Centruroides suffusus suffusus. These results indicate that the recombinant peptides can be used to produce antidotes against the venom of this species of scorpion.

  6. Treatment of Prion Disease with Heterologous Prion Proteins

    PubMed Central

    Skinner, Pamela J.; Kim, Hyeon O.; Bryant, Damani; Kinzel, Nikilyn J.; Reilly, Cavan; Priola, Suzette A.; Ward, Anne E.; Goodman, Patricia A.; Olson, Katherine; Seelig, Davis M.

    2015-01-01

    Prion diseases such as Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy in cattle, and scrapie in sheep are fatal neurodegenerative diseases for which there is no effective treatment. The pathology of these diseases involves the conversion of a protease sensitive form of the cellular prion protein (PrPC) into a protease resistant infectious form (PrPsc or PrPres). Both in vitro (cell culture and cell free conversion assays) and in vivo (animal) studies have demonstrated the strong dependence of this conversion process on protein sequence homology between the initial prion inoculum and the host’s own cellular prion protein. The presence of non-homologous (heterologous) proteins is often inhibitory to this conversion process. We hypothesize that the presence of heterologous prion proteins from one species might therefore constitute an effective treatment for prion disease in another species. To test this hypothesis, we infected mice intracerebrally with murine adapted RML-Chandler scrapie and treated them with heterologous prion protein (purified bacterially expressed recombinant hamster prion protein) or vehicle alone. Treated animals demonstrated reduced disease associated pathology, decreased accumulation of protease-resistant disease-associated prion protein, with delayed onset of clinical symptoms and motor deficits. This was concomitant with significantly increased survival times relative to mock-treated animals. These results provide proof of principle that recombinant hamster prion proteins can effectively and safely inhibit prion disease in mice, and suggest that hamster or other non-human prion proteins may be a viable treatment for prion diseases in humans. PMID:26134409

  7. Treatment of Prion Disease with Heterologous Prion Proteins.

    PubMed

    Skinner, Pamela J; Kim, Hyeon O; Bryant, Damani; Kinzel, Nikilyn J; Reilly, Cavan; Priola, Suzette A; Ward, Anne E; Goodman, Patricia A; Olson, Katherine; Seelig, Davis M

    2015-01-01

    Prion diseases such as Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy in cattle, and scrapie in sheep are fatal neurodegenerative diseases for which there is no effective treatment. The pathology of these diseases involves the conversion of a protease sensitive form of the cellular prion protein (PrPC) into a protease resistant infectious form (PrPsc or PrPres). Both in vitro (cell culture and cell free conversion assays) and in vivo (animal) studies have demonstrated the strong dependence of this conversion process on protein sequence homology between the initial prion inoculum and the host's own cellular prion protein. The presence of non-homologous (heterologous) proteins is often inhibitory to this conversion process. We hypothesize that the presence of heterologous prion proteins from one species might therefore constitute an effective treatment for prion disease in another species. To test this hypothesis, we infected mice intracerebrally with murine adapted RML-Chandler scrapie and treated them with heterologous prion protein (purified bacterially expressed recombinant hamster prion protein) or vehicle alone. Treated animals demonstrated reduced disease associated pathology, decreased accumulation of protease-resistant disease-associated prion protein, with delayed onset of clinical symptoms and motor deficits. This was concomitant with significantly increased survival times relative to mock-treated animals. These results provide proof of principle that recombinant hamster prion proteins can effectively and safely inhibit prion disease in mice, and suggest that hamster or other non-human prion proteins may be a viable treatment for prion diseases in humans. PMID:26134409

  8. Controlled Gene Expression in the Plant Pathogen Cryphonectria parasitica by Use of a Copper-Responsive Element▿

    PubMed Central

    Willyerd, Karyn L.; Kemp, Amanda M.; Dawe, Angus L.

    2009-01-01

    We have developed a tool for controlled expression of heterologous or ectopic genes in the chestnut pathogen Cryphonectria parasitica using the promoter region from a putative copper-regulated transporter gene. In addition, we have found that expression control via this system is not affected by the virulence-attenuating hypovirus CHV1-EP713. PMID:19542340

  9. Shades of Emotion: What the Addition of Sunglasses or Masks to Faces Reveals about the Development of Facial Expression Processing

    ERIC Educational Resources Information Center

    Roberson, Debi; Kikutani, Mariko; Doge, Paula; Whitaker, Lydia; Majid, Asifa

    2012-01-01

    Three studies investigated developmental changes in facial expression processing, between 3 years-of-age and adulthood. For adults and older children, the addition of sunglasses to upright faces caused an equivalent decrement in performance to face inversion. However, younger children showed "better" classification of expressions of faces wearing…

  10. Thinking-for-Speaking of Chinese EFL Learners in the Use of Additional Expressions of Manner in the Expression of Motion Events

    ERIC Educational Resources Information Center

    Ziyan, Xu

    2013-01-01

    This study adopts Talmy's (1985, 1991, 2000) theory of linguistic typology and Slobin's (2003, 2004) hypothesis of Thinking-for-Speaking to investigate the use of additional expressions of Manner in the expression of Motion events by Chinese EFL learners in comparison with French EFL learners. The aim is to find out the Thinking-for-Speaking…

  11. Scaling Up the Production of Recombinant Antimicrobial Plantaricin E from a Heterologous Host, Escherichia coli.

    PubMed

    Pal, Gargi; Srivastava, Sheela

    2015-09-01

    Enhanced production of heterologously expressed plantaricin (plnE) from Escherichia coli BL21 (DE3) was achieved from a small- to large-scale batch culture. Starting from a 15-ml shake-flask culture grown in Luria-Bertani (LB) broth, the protein expression could be scaled up using 50 ml, 100 ml, 1 l, and 2 l batch culture. Using similar condition, plantaricin E (PlnE) was successfully expressed in a 30-l stirred fermenter. The protein was expressed as TRX-(His)6-fusion protein and separated by Ni(2+) affinity chromatography. Growth in two complex media, LB and Terrific broth (TB), was optimized and compared for the production of PlnE, which was higher in LB in comparison with that of TB. In the fermenter, 140 and 180 mg of PlnE could be produced from 12 l of culture volume at 30 and 25 °C, respectively. The yield of heterologously purified PlnE was found to be 1.2-1.5%, which was much higher in comparison with the plantaricins produced from the native strain of Lactobacillus plantarum (0.3-0.7%). Overproduction of PlnE with the help of heterologous expression can overcome the constraint of the low yield from producer strain and provides an easy and low-cost strategy for large-scale production.

  12. Insights into the Biosynthesis of 12-Membered Resorcylic Acid Lactones from Heterologous Production in Saccharomyces cerevisiae

    PubMed Central

    2015-01-01

    The phytotoxic fungal polyketides lasiodiplodin and resorcylide inhibit human blood coagulation factor XIIIa, mineralocorticoid receptors, and prostaglandin biosynthesis. These secondary metabolites belong to the 12-membered resorcylic acid lactone (RAL12) subclass of the benzenediol lactone (BDL) family. Identification of genomic loci for the biosynthesis of lasiodiplodin from Lasiodiplodia theobromae and resorcylide from Acremonium zeae revealed collaborating iterative polyketide synthase (iPKS) pairs whose efficient heterologous expression in Saccharomyces cerevisiae provided a convenient access to the RAL12 scaffolds desmethyl-lasiodiplodin and trans-resorcylide, respectively. Lasiodiplodin production was reconstituted in the heterologous host by co-expressing an O-methyltransferase also encoded in the lasiodiplodin cluster, while a glutathione-S-transferase was found not to be necessary for heterologous production. Clarification of the biogenesis of known resorcylide congeners in the heterologous host helped to disentangle the roles that biosynthetic irregularities and chemical interconversions play in generating chemical diversity. Observation of 14-membered RAL homologues during in vivo heterologous biosynthesis of RAL12 metabolites revealed “stuttering” by fungal iPKSs. The close global and domain-level sequence similarities of the orthologous BDL synthases across different structural subclasses implicate repeated horizontal gene transfers and/or cluster losses in different fungal lineages. The absence of straightforward correlations between enzyme sequences and product structural features (the size of the macrocycle, the conformation of the exocyclic methyl group, or the extent of reduction by the hrPKS) suggest that BDL structural variety is the result of a select few mutations in key active site cavity positions. PMID:24597618

  13. Insights into the biosynthesis of 12-membered resorcylic acid lactones from heterologous production in Saccharomyces cerevisiae.

    PubMed

    Xu, Yuquan; Zhou, Tong; Espinosa-Artiles, Patricia; Tang, Ying; Zhan, Jixun; Molnár, István

    2014-05-16

    The phytotoxic fungal polyketides lasiodiplodin and resorcylide inhibit human blood coagulation factor XIIIa, mineralocorticoid receptors, and prostaglandin biosynthesis. These secondary metabolites belong to the 12-membered resorcylic acid lactone (RAL12) subclass of the benzenediol lactone (BDL) family. Identification of genomic loci for the biosynthesis of lasiodiplodin from Lasiodiplodia theobromae and resorcylide from Acremonium zeae revealed collaborating iterative polyketide synthase (iPKS) pairs whose efficient heterologous expression in Saccharomyces cerevisiae provided a convenient access to the RAL12 scaffolds desmethyl-lasiodiplodin and trans-resorcylide, respectively. Lasiodiplodin production was reconstituted in the heterologous host by co-expressing an O-methyltransferase also encoded in the lasiodiplodin cluster, while a glutathione-S-transferase was found not to be necessary for heterologous production. Clarification of the biogenesis of known resorcylide congeners in the heterologous host helped to disentangle the roles that biosynthetic irregularities and chemical interconversions play in generating chemical diversity. Observation of 14-membered RAL homologues during in vivo heterologous biosynthesis of RAL12 metabolites revealed "stuttering" by fungal iPKSs. The close global and domain-level sequence similarities of the orthologous BDL synthases across different structural subclasses implicate repeated horizontal gene transfers and/or cluster losses in different fungal lineages. The absence of straightforward correlations between enzyme sequences and product structural features (the size of the macrocycle, the conformation of the exocyclic methyl group, or the extent of reduction by the hrPKS) suggest that BDL structural variety is the result of a select few mutations in key active site cavity positions. PMID:24597618

  14. Biology of Trypanosoma (Trypanozoon) evansi in experimental heterologous mammalian hosts.

    PubMed

    Misra, K K; Roy, S; Choudhury, A

    2016-09-01

    Trypanosoma (Trypanozoon) evansi is a causative agent of the dreadful mammalian disease trypanosomiasis or 'Surra' and carried as a latent parasite in domestic cattle but occasionally proves fatal when transmitted to horses and camel. Sporadic outbreak of 'Surra' to different animals (beside their natural hosts) reminds that T. evansi may be zoonotic, as their close relative cause sleeping sickness to human being. This haemoflagellate is mechanically transmitted by horse fly and its effect on different host varies depending on certain factors including the effectiveness of transmission by mechanical vector, the suitability and susceptibility of the host as well as most importantly the ability of the disease establishment of parasite to adapt itself to the host's resistance, etc. The course of the disease caused by T. evansi is similar to that of human sleeping sickness caused by T. (T.) brucei gambiense. The target organs and symptoms show close similarity. T. evansi can successfully be transmitted among unnatural hosts i.e., other classes of vertebrates, like chicken. In transmission experiments, the unnatural hosts may sometimes induce profound changes in the biology of trypanosomes. Hence, in present study the observations are the biology of different morphological changes of T. evansi as well as its ability of disease formation within some heterologous mammal viz., albino rat, guineapig, bandicoot, mongoose, domestic cat and common monkey. Blood smears of infected albino rats, bandicoot, and mongoose revealed only monomorphic form. Interestingly, blood smears of infected cat and monkey, T. evansi shows slender trypomastigote form and short intermediate form whereas organ smears shows other two forms of haemoflagellate viz., sphaeromastigote and amastigote form. The haemoflagellate maintains a common reproductive cycle in all the experimental heterologous hosts whereas disease symptoms differ. T. evansi infected cat and monkey shows nervous symptoms. Infected

  15. Molecular cloning and characterization of a KCS gene from Cardamine graeca and its heterologous expression in Brassica oilseeds to engineer high nervonic acid oils for potential medical and industrial use.

    PubMed

    Taylor, David C; Francis, Tammy; Guo, Yiming; Brost, Jennifer M; Katavic, Vesna; Mietkiewska, Elzbieta; Michael Giblin, E; Lozinsky, Sharla; Hoffman, Travis

    2009-12-01

    Nervonic acid 24:1 Delta15 (cis-tetracos-15-enoic acid) is a very long-chain monounsaturated fatty acid and exists in nature as an elongation product of oleic acid. There is an increasing interest in production of high nervonic acid oils for pharmaceutical, nutraceutical and industrial applications. Using a polymerase chain reaction approach, we have isolated a gene from Cardamine graeca L., which encodes a 3-ketoacyl-CoA synthase (KCS), the first component of the elongation complex involved in synthesis of nervonic acid. Expression of the Cardamine KCS in yeast resulted in biosynthesis of nervonic acid, which is not normally present in yeast cells. We transformed Arabidopsis and Brassica carinata with the Cardamine KCS under the control of the seed-specific promoter, napin. The T(3) generations of transgenic Arabidopsis and B. carinata plants expressing the Cardamine KCS showed that seed-specific expression resulted in relatively large comparative increases in nervonic acid proportions in Arabidopsis seed oil, and 15-fold increase in nervonic acid proportions in B. carinata seed oil. The highest nervonic acid level in transgenic B. carinata lines reached 44%, with only 6% of residual erucic acid. In contrast, similar transgenic expression of the Cardamine KCS in high erucic B. napus resulted in 30% nervonic acid but with 20% residual erucic acid. Experiments using the Lunaria KCS gene gave results similar to the latter. In both cases, the erucic acid content is too high for human or animal consumption. Thus, the Cardamine KCS: B. carinata high nervonic/highly reduced erucic transgenic seed oils will be the most suitable for testing in pharmaceutical/nutraceutical applications to improve human and animal health.

  16. 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.

    PubMed

    Fernandes, Sara; Tuohy, Maria G; Murray, Patrick G

    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 TeXR K271R+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.

  17. Heterologous overexpression of Glomerella cingulata FAD-dependent glucose dehydrogenase in Escherichia coli and Pichia pastoris

    PubMed Central

    2011-01-01

    Background FAD dependent glucose dehydrogenase (GDH) currently raises enormous interest in the field of glucose biosensors. Due to its superior properties such as high turnover rate, substrate specificity and oxygen independence, GDH makes its way into glucose biosensing. The recently discovered GDH from the ascomycete Glomerella cingulata is a novel candidate for such an electrochemical application, but also of interest to study the plant-pathogen interaction of a family of wide-spread, crop destroying fungi. Heterologous expression is a necessity to facilitate the production of GDH for biotechnological applications and to study its physiological role in the outbreak of anthracnose caused by Glomerella (anamorph Colletotrichum) spp. Results Heterologous expression of active G. cingulata GDH has been achieved in both Escherichia coli and Pichia pastoris, however, the expressed volumetric activity was about 4800-fold higher in P. pastoris. Expression in E. coli resulted mainly in the formation of inclusion bodies and only after co-expression with molecular chaperones enzymatic activity was detected. The fed-batch cultivation of a P. pastoris transformant resulted in an expression of 48,000 U L-1 of GDH activity (57 mg L-1). Recombinant GDH was purified by a two-step purification procedure with a yield of 71%. Comparative characterization of molecular and catalytic properties shows identical features for the GDH expressed in P. pastoris and the wild-type enzyme from its natural fungal source. Conclusions The heterologous expression of active GDH was greatly favoured in the eukaryotic host. The efficient expression in P. pastoris facilitates the production of genetically engineered GDH variants for electrochemical-, physiological- and structural studies. PMID:22151971

  18. The GH26 β-mannanase RsMan26H from a symbiotic protist of the termite Reticulitermes speratus is an endo-processive mannobiohydrolase: heterologous expression and characterization.

    PubMed

    Tsukagoshi, Hikaru; Nakamura, Akihiko; Ishida, Takuya; Otagiri, Masato; Moriya, Shigeharu; Samejima, Masahiro; Igarashi, Kiyohiko; Kitamoto, Katsuhiko; Arioka, Manabu

    2014-09-26

    Symbiotic protists in the gut of termites are prominent natural resources for enzymes involved in lignocellulose degradation. Here we report expression, purification, and biochemical characterization of a glycoside hydrolase family 26 mannanase RsMan26H from the symbiotic protist of the lower termite, Reticulitermes speratus. Biochemical analysis of RsMan26H demonstrates that this enzyme is an endo-processive mannobiohydrolase producing mannobiose from oligo- and polysaccharides, followed by a minor accumulation of oligosaccharides larger than mannobiose. To our knowledge, this is the first report describing the unique mannobiohydrolase enzyme from the eukaryotic origin.

  19. Differential Expression of Functional Fc-Receptors and Additional Immune Complex Receptors on Mouse Kidney Cells

    PubMed Central

    Suwanichkul, Adisak; Wenderfer, Scott E.

    2013-01-01

    The precise mechanisms by which circulating immune complexes accumulate in the kidney to form deposits in glomerulonephritis are not well understood. In particular, the role of resident cells within glomeruli of the kidney has been widely debated. Immune complexes have been shown to bind one glomerular cell type (mesangial cells) leading to functional responses such as pro-inflammatory cytokine production. To further assess the presence of functional immunoreceptors on resident glomerular cells, cultured mouse renal epithelial, endothelial, and mesangial cells were treated with heat-aggregated mouse IgG or preformed murine immune complexes. Mesangial and renal endothelial cells were found to bind IgG complexes, whereas glomerular epithelial cell binding was minimal. A blocking antibody for Fc-gamma receptors reduced binding to mesangial cells but not renal endothelial cells, suggesting differential immunoreceptor utilization. RT-PCR and immunostaining based screening of cultured renal endothelial cells showed limited low-level expression of known Fc-receptors and Igbinding proteins. The interaction between mesangial cells and renal endothelial cells and immune complexes resulted in distinct, cell-specific patterns of chemokine and cytokine production. This novel pathway involving renal endothelial cells likely contributes to the predilection of circulating immune complex accumulation within the kidney and to the inflammatory responses that drive kidney injury. PMID:23911392

  20. Molecular cloning and expression of an additional epidermal growth factor receptor-related gene.

    PubMed Central

    Plowman, G D; Whitney, G S; Neubauer, M G; Green, J M; McDonald, V L; Todaro, G J; Shoyab, M

    1990-01-01

    Epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), and amphiregulin are structurally and functionally related growth regulatory proteins. These secreted polypeptides all bind to the 170-kDa cell-surface EGF receptor, activating its intrinsic kinase activity. However, amphiregulin exhibits different activities than EGF and TGF-alpha in a number of biological assays. Amphiregulin only partially competes with EGF for binding EGF receptor, and amphiregulin does not induce anchorage-independent growth of normal rat kidney cells (NRK) in the presence of TGF-beta. Amphiregulin also appears to abrogate the stimulatory effect of TGF-alpha on the growth of several aggressive epithelial carcinomas that overexpress EGF receptor. These findings suggest that amphiregulin may interact with a separate receptor in certain cell types. Here we report the cloning of another member of the human EGF receptor (HER) family of receptor tyrosine kinases, which we have named "HER3/ERRB3." The cDNA was isolated from a human carcinoma cell line, and its 6-kilobase transcript was identified in various human tissues. We have generated peptide-specific antisera that recognizes the 160-kDa HER3 protein when transiently expressed in COS cells. These reagents will allow us to determine whether HER3 binds amphiregulin or other growth regulatory proteins and what role HER3 protein plays in the regulation of cell growth. Images PMID:2164210

  1. Heterologous recombinant protein with decapacitating activity prevents and reverts cryodamage in ram sperm: An emerging biotechnological tool for cryobiology.

    PubMed

    Zalazar, L; Ledesma, A; Hozbor, F; Cesari, A

    2016-01-01

    During the last decades fundamental and applied aspects of mammalian ram sperm cryopreservation have been increasingly explored by scientists and biotechnologists. Many works report modifications in the composition of the freezing extenders and explore the beneficial and detrimental effects of seminal plasma or seminal plasma components in cryopreservation. Seminal plasma is known to contain stabilizing proteins, thereby this is a good start point to study the maintenance of membrane stability based on the basic knowledge of sperm physiology. However, seminal plasma composition is variable among rams and also the introduction of exogenous seminal plasma or its fractions to commercial semen can be associated with the transmission of viral diseases. Our work shows that a mouse protein, called SPINK3 (Serine Protease Inhibitor Kazal type 3) with decapacitating activity interacts with heterologous ram sperm when it is produced as a recombinant molecule. By immunocytochemistry assays we demonstrate that this protein (naturally expressed by mouse seminal vesicle under androgenic control) binds to the apical portion of both fresh and frozen ram sperm, the same localization described in mouse homologous sperm. Furthermore, it significantly improves sperm progressive motility compared to non-treated samples when it is added to freezing extenders and to dilution media after thawing. On the contrary, addition of SPINK3 does not modify sperm viability. The percentage of sperm with intact acrosome after ionophore induction was also significantly higher in sperm frozen in the presence of SPINK3 compared to control samples and the addition of SPINK3 after thawing significantly reduced both induced and non induced acrosomal loss, indicating that heterologous SPINK3 might act as a calcium inhibitor transport as described in mouse. Based on our results SPINK3 may find a place as a desirable biotechnological tool to achieve a higher proportion of competent sperm to fertilize. PMID

  2. Heterologous recombinant protein with decapacitating activity prevents and reverts cryodamage in ram sperm: An emerging biotechnological tool for cryobiology.

    PubMed

    Zalazar, L; Ledesma, A; Hozbor, F; Cesari, A

    2016-01-01

    During the last decades fundamental and applied aspects of mammalian ram sperm cryopreservation have been increasingly explored by scientists and biotechnologists. Many works report modifications in the composition of the freezing extenders and explore the beneficial and detrimental effects of seminal plasma or seminal plasma components in cryopreservation. Seminal plasma is known to contain stabilizing proteins, thereby this is a good start point to study the maintenance of membrane stability based on the basic knowledge of sperm physiology. However, seminal plasma composition is variable among rams and also the introduction of exogenous seminal plasma or its fractions to commercial semen can be associated with the transmission of viral diseases. Our work shows that a mouse protein, called SPINK3 (Serine Protease Inhibitor Kazal type 3) with decapacitating activity interacts with heterologous ram sperm when it is produced as a recombinant molecule. By immunocytochemistry assays we demonstrate that this protein (naturally expressed by mouse seminal vesicle under androgenic control) binds to the apical portion of both fresh and frozen ram sperm, the same localization described in mouse homologous sperm. Furthermore, it significantly improves sperm progressive motility compared to non-treated samples when it is added to freezing extenders and to dilution media after thawing. On the contrary, addition of SPINK3 does not modify sperm viability. The percentage of sperm with intact acrosome after ionophore induction was also significantly higher in sperm frozen in the presence of SPINK3 compared to control samples and the addition of SPINK3 after thawing significantly reduced both induced and non induced acrosomal loss, indicating that heterologous SPINK3 might act as a calcium inhibitor transport as described in mouse. Based on our results SPINK3 may find a place as a desirable biotechnological tool to achieve a higher proportion of competent sperm to fertilize.

  3. Expressed Peptide Tags: An additional layer of data for genome annotation

    SciTech Connect

    Savidor, Alon; Donahoo, Ryan S; Hurtado-Gonzales, Oscar; Verberkmoes, Nathan C; Shah, Manesh B; Lamour, Kurt H; McDonald, W Hayes

    2006-01-01

    While genome sequencing is becoming ever more routine, genome annotation remains a challenging process. Identification of the coding sequences within the genomic milieu presents a tremendous challenge, especially for eukaryotes with their complex gene architectures. Here we present a method to assist the annotation process through the use of proteomic data and bioinformatics. Mass spectra of digested protein preparations of the organism of interest were acquired and searched against a protein database created by a six frame translation of the genome. The identified peptides were mapped back to the genome, compared to the current annotation, and then categorized as supporting or extending the current genome annotation. We named the classified peptides Expressed Peptide Tags (EPTs). The well annotated bacterium Rhodopseudomonas palustris was used as a control for the method and showed high degree of correlation between EPT mapping and the current annotation, with 86% of the EPTs confirming existing gene calls and less than 1% of the EPTs expanding on the current annotation. The eukaryotic plant pathogens Phytophthora ramorum and Phytophthora sojae, whose genomes have been recently sequenced and are much less well annotated, were also subjected to this method. A series of algorithmic steps were taken to increase the confidence of EPT identification for these organisms, including generation of smaller sub-databases to be searched against, and definition of EPT criteria that accommodates the more complex eukaryotic gene architecture. As expected, the analysis of the Phytophthora species showed less correlation between EPT mapping and their current annotation. While ~77% of Phytophthora EPTs supported the current annotation, a portion of them (7.2% and 12.6% for P. ramorum and P. sojae, respectively) suggested modification to current gene calls or identified novel genes that were missed by the current genome annotation of these organisms.

  4. Biosynthesis and Heterologous Production of Epothilones

    NASA Astrophysics Data System (ADS)

    Müller, Rolf

    Although a variety of chemical syntheses for the epothilones and various derivatives have been described, modifying the backbone of those natural products remains a major challenge. One alternative to chemical alteration is the elucidation and subsequent manipulation of the biosynthetic pathway via genetic engineering in the producing organism. This type of approach is known as “combinatorial biosynthesis” and holds great promise, especially in conjunction with semi-synthesis methods to alter the structure of the natural product. In parallel, production can be optimized in the natural producer if the regulatory mechanisms governing the biosynthesis are understood. Alternatively, the entire gene cluster can be transferred into a heterologous host, more amenable both to genetic alteration and overexpression.

  5. Heterologous over-expression of ACC SYNTHASE8 (ACS8) in Populus tremula x P. alba clone 717-1B4 results in elevated levels of ethylene and induces stem dwarfism and reduced leaf size through separate genetic pathways.

    PubMed

    Plett, Jonathan M; Williams, Martin; LeClair, Gaetan; Regan, Sharon; Beardmore, Tannis

    2014-01-01

    Plant height is an important agronomic and horticultural trait that impacts plant productivity, durability and esthetic appeal. A number of the plant hormones such as gibberellic acid (GA), auxin and ethylene have been linked to control of plant architecture and size. Reduction in GA synthesis and auxin transport result in dwarfism while ethylene may have a permissive or repressive effect on tissue growth depending upon the age of plant tissues or the environmental conditions considered. We describe here an activation-tagged mutant of Populus tremula x P. alba clone 717-1B4 identified from 2000 independent transgenic lines due to its significantly reduced growth rate and smaller leaf size. Named dwarfy, the phenotype is due to increased expression of PtaACC SYNTHASE8, which codes for an enzyme in the first committed step in the biosynthesis of ethylene. Stems of dwarfy contain fiber and vessel elements that are reduced in length while leaves contain fewer cells. These morphological differences are linked to PtaACS8 inducing different transcriptomic programs in the stem and leaf, with genes related to auxin diffusion and sensing being repressed in the stem and genes related to cell division found to be repressed in the leaves. Altogether, our study gives mechanistic insight into the genetics underpinning ethylene-induced dwarfism in a perennial model organism. PMID:25414707

  6. Heterologous over-expression of ACC SYNTHASE8 (ACS8) in Populus tremula x P. alba clone 717-1B4 results in elevated levels of ethylene and induces stem dwarfism and reduced leaf size through separate genetic pathways

    PubMed Central

    Plett, Jonathan M.; Williams, Martin; LeClair, Gaetan; Regan, Sharon; Beardmore, Tannis

    2014-01-01

    Plant height is an important agronomic and horticultural trait that impacts plant productivity, durability and esthetic appeal. A number of the plant hormones such as gibberellic acid (GA), auxin and ethylene have been linked to control of plant architecture and size. Reduction in GA synthesis and auxin transport result in dwarfism while ethylene may have a permissive or repressive effect on tissue growth depending upon the age of plant tissues or the environmental conditions considered. We describe here an activation-tagged mutant of Populus tremula x P. alba clone 717-1B4 identified from 2000 independent transgenic lines due to its significantly reduced growth rate and smaller leaf size. Named dwarfy, the phenotype is due to increased expression of PtaACC SYNTHASE8, which codes for an enzyme in the first committed step in the biosynthesis of ethylene. Stems of dwarfy contain fiber and vessel elements that are reduced in length while leaves contain fewer cells. These morphological differences are linked to PtaACS8 inducing different transcriptomic programs in the stem and leaf, with genes related to auxin diffusion and sensing being repressed in the stem and genes related to cell division found to be repressed in the leaves. Altogether, our study gives mechanistic insight into the genetics underpinning ethylene-induced dwarfism in a perennial model organism. PMID:25414707

  7. Harnessing heterologous and endogenous CRISPR-Cas machineries for efficient markerless genome editing in Clostridium

    PubMed Central

    Pyne, Michael E.; Bruder, Mark R.; Moo-Young, Murray; Chung, Duane A.; Chou, C. Perry

    2016-01-01

    Application of CRISPR-Cas9 systems has revolutionized genome editing across all domains of life. Here we report implementation of the heterologous Type II CRISPR-Cas9 system in Clostridium pasteurianum for markerless genome editing. Since 74% of species harbor CRISPR-Cas loci in Clostridium, we also explored the prospect of co-opting host-encoded CRISPR-Cas machinery for genome editing. Motivation for this work was bolstered from the observation that plasmids expressing heterologous cas9 result in poor transformation of Clostridium. To address this barrier and establish proof-of-concept, we focus on characterization and exploitation of the C. pasteurianum Type I-B CRISPR-Cas system. In silico spacer analysis and in vivo interference assays revealed three protospacer adjacent motif (PAM) sequences required for site-specific nucleolytic attack. Introduction of a synthetic CRISPR array and cpaAIR gene deletion template yielded an editing efficiency of 100%. In contrast, the heterologous Type II CRISPR-Cas9 system generated only 25% of the total yield of edited cells, suggesting that native machinery provides a superior foundation for genome editing by precluding expression of cas9 in trans. To broaden our approach, we also identified putative PAM sequences in three key species of Clostridium. This is the first report of genome editing through harnessing native CRISPR-Cas machinery in Clostridium. PMID:27157668

  8. Effects of maternally exposed coloring food additives on receptor expressions related to learning and memory in rats.

    PubMed

    Ceyhan, Betul Mermi; Gultekin, Fatih; Doguc, Duygu Kumbul; Kulac, Esin

    2013-06-01

    Exposure to artificial food colors and additives (AFCAs) has been implicated in the induction and severity of some childhood behavioral and learning disabilities. N-methyl-D-aspartate receptors (NMDARs) and nicotinic acetylcholine receptors (nACHRs) are thought to be effective in the learning and memory-generating process. In this study, we investigated the effects of intrauterine exposure to AFCAs on subunit concentrations of NMDARs and nAChRs isoforms in rats. We administered a mixture of AFCAs (Eritrosin, Ponceau 4R, Allura Red AC, Sunset Yellow FCF, Tartrazin, Amaranth, Brilliant Blue, Azorubin and Indigotin) to female rats before and during gestation. The concentration of NR2A and NR2B subunits and nAChR α7, α4β2 isoforms in their offspring's hippocampi were measured by Western Blotting. Expressions of NR2B and nAChR β2 were significantly increased (17% and 6.70%, respectively), whereas expression of nAChR α4 was significantly decreased (5.67%) in male experimental group compared to the male control group (p<0.05). In the female experimental group, AFCAs caused a 14% decrease in NR2B expression when compared to the female control group (p<0.05). Our results indicate that exposure to AFCAs during the fetal period may lead to alterations in expressions of NMDARs and nAChRs in adulthood. These alterations were different between male and female genders. PMID:23429044

  9. The F130S point mutation in the Arabidopsis high-affinity K+ transporter AtHAK5 increases K+ over Na+ and Cs+ selectivity and confers Na+ and Cs+ tolerance to yeast under heterologous expression

    PubMed Central

    Alemán, Fernando; Caballero, Fernando; Ródenas, Reyes; Rivero, Rosa M.; Martínez, Vicente; Rubio, Francisco

    2014-01-01

    Potassium (K+) is an essential macronutrient required for plant growth, development and high yield production of crops. Members of group I of the KT/HAK/KUP family of transporters, such as HAK5, are key components for K+ acquisition by plant roots at low external K+ concentrations. Certain abiotic stress conditions such as salinity or Cs+-polluted soils may jeopardize plant K+ nutrition because HAK5-mediated K+ transport is inhibited by Na+ and Cs+. Here, by screening in yeast a randomly-mutated collection of AtHAK5 transporters, a new mutation in AtHAK5 sequence is identified that greatly increases Na+ tolerance. The single point mutation F130S, affecting an amino acid residue conserved in HAK5 transporters from several species, confers high salt tolerance, as well as Cs+ tolerance. This mutation increases more than 100-fold the affinity of AtHAK5 for K+ and reduces the Ki values for Na+ and Cs+, suggesting that the F130 residue may contribute to the structure of the pore region involved in K+ binding. In addition, this mutation increases the Vmax for K+. All this changes occur without increasing the amount of the AtHAK5 protein in yeast and support the idea that this residue is contributing to shape the selectivity filter of the AtHAK5 transporter. PMID:25228905

  10. Self-made phage libraries with heterologous inserts in the Mtd of Bordetella bronchiseptica.

    PubMed

    Overstreet, Cathie M; Yuan, Tom Z; Levin, Aron M; Kong, Calvin; Coroneus, John G; Weiss, Gregory A

    2012-04-01

    Phage display libraries are widely used as tools for identifying, dissecting and optimizing ligands. Development of a simple method to access greater library diversities could expedite and expand the technique. This paper reports progress toward harnessing the naturally occurring diversity generating retroelement used by Bordetella bronchiseptica bacteriophage to alter its tail-fiber protein. Mutagenesis and testing identified four sites amenable to the insertion of <19-residue heterologous peptides within the variable region. Such sites allow auto-generation of peptide libraries surrounded by a scaffold with additional variations. The resultant self-made phage libraries were used successfully for selections targeting anti-FLAG antibody, immobilized metal affinity chromatography microtiter plates and HIV-1 gp41. The reported experiments demonstrate the utility of the major tropism determinant protein of B.bronchiseptica as a natural scaffold for diverse, phage-constructed libraries with heterologous self-made phage libraries.

  11. Combinatorial therapeutic activation with heparin and AICAR stimulates additive effects on utrophin A expression in dystrophic muscles.

    PubMed

    Péladeau, Christine; Ahmed, Aatika; Amirouche, Adel; Crawford Parks, Tara E; Bronicki, Lucas M; Ljubicic, Vladimir; Renaud, Jean-Marc; Jasmin, Bernard J

    2016-01-01

    Upregulation of utrophin A is an attractive therapeutic strategy for treating Duchenne muscular dystrophy (DMD). Over the years, several studies revealed that utrophin A is regulated by multiple transcriptional and post-transcriptional mechanisms, and that pharmacological modulation of these pathways stimulates utrophin A expression in dystrophic muscle. In particular, we recently showed that activation of p38 signaling causes an increase in the levels of utrophin A mRNAs and protein by decreasing the functional availability of the destabilizing RNA-binding protein called K-homology splicing regulatory protein, thereby resulting in increases in the stability of existing mRNAs. Here, we treated 6-week-old mdx mice for 4 weeks with the clinically used anticoagulant drug heparin known to activate p38 mitogen-activated protein kinase, and determined the impact of this pharmacological intervention on the dystrophic phenotype. Our results show that heparin treatment of mdx mice caused a significant ∼1.5- to 3-fold increase in utrophin A expression in diaphragm, extensor digitorum longus and tibialis anterior (TA) muscles. In agreement with these findings, heparin-treated diaphragm and TA muscle fibers showed an accumulation of utrophin A and β-dystroglycan along their sarcolemma and displayed improved morphology and structural integrity. Moreover, combinatorial drug treatment using both heparin and 5-amino-4-imidazolecarboxamide riboside (AICAR), the latter targeting 5' adenosine monophosphate-activated protein kinase and the transcriptional activation of utrophin A, caused an additive effect on utrophin A expression in dystrophic muscle. These findings establish that heparin is a relevant therapeutic agent for treating DMD, and illustrate that combinatorial treatment of heparin with AICAR may serve as an effective strategy to further increase utrophin A expression in dystrophic muscle via activation of distinct signaling pathways.

  12. Genome-wide meta-analysis of maize heterosis reveals the potential role of additive gene expression at pericentromeric loci

    PubMed Central

    2014-01-01

    QTL, and also suggests a model for the potential role of additive expression in the formation and conservation of heterosis for GY via dominant, multigenic quantitative trait loci. Our findings contribute to a deeper understanding of the multifactorial phenomenon of heterosis, and thus to the breeding of new high yielding varieties. PMID:24693880

  13. Boosting heterologous protein production in transgenic dicotyledonous seeds using Phaseolus vulgaris regulatory sequences.

    PubMed

    De Jaeger, Geert; Scheffer, Stanley; Jacobs, Anni; Zambre, Mukund; Zobell, Oliver; Goossens, Alain; Depicker, Ann; Angenon, Geert

    2002-12-01

    Over the past decade, several high value proteins have been produced in different transgenic plant tissues such as leaves, tubers, and seeds. Despite recent advances, many heterologous proteins accumulate to low concentrations, and the optimization of expression cassettes to make in planta production and purification economically feasible remains critical. Here, the regulatory sequences of the seed storage protein gene arcelin 5-I (arc5-I) of common bean (Phaseolus vulgaris) were evaluated for producing heterologous proteins in dicotyledonous seeds. The murine single chain variable fragment (scFv) G4 (ref. 4) was chosen as model protein because of the current industrial interest in producing antibodies and derived fragments in crops. In transgenic Arabidopsis thaliana seed stocks, the scFv under control of the 35S promoter of the cauliflower mosaic virus (CaMV) accumulated to approximately 1% of total soluble protein (TSP). However, a set of seed storage promoter constructs boosted the scFv accumulation to exceptionally high concentrations, reaching no less than 36.5% of TSP in homozygous seeds. Even at these high concentrations, the scFv proteins had antigen-binding activity and affinity similar to those produced in Escherichia coli. The feasibility of heterologous protein production under control of arc5-I regulatory sequences was also demonstrated in Phaseolus acutifolius, a promising crop for large scale production.

  14. Structural responses of amoebae to the injection of heterologous cytoplasm.

    PubMed

    Flickinger, C J; Read, G A; Kabana, E M

    1980-10-01

    Responses to the introduction of heterologous cytoplasm and the fate of foreign organelles were investigated in amoebae. Heterologous cytoplasm was transferred by microinjection from Pelomyxa carolinensis to Amoeba discoides. In control experiments, homologous cytoplasm was transferred from one A. discoides to another. Recipient cells were observed by light microscopy, and samples were prepared for ultrastructural study at intervals between 15 min nad 3 days after operation. Recipients of heterologous cytoplasm showed two main responses. First, about 40% recipients expelled small amounts of cytoplasm by a blebbing process within 30 min after injection. Second, organelles were segregated and broken down in membrane-bounded cytoplasmic vacuoles between 6 h and 2 days after operation. Acid phosphatase reaction product was observed in these vacuoles along with altered organelles. Use of electron-dense thoria particles to mark donor cells demonstrated the presence of injected cytoplasm in the vacuoles. In contrast, when amoebae were injected with homologous cytoplasm, none was expelled and vacuoles containing degenerating organelles were uncommon. The survival rate and general appearance of recipients of heterologous cytoplasm were much poorer than those of homologous recipients, and most of the former died by I week after operation. It is postulated that amoeba are capable of recognizing heterologous organelles introduced into the cytoplasm and that they respond by expulsion and/or destruction of the foreign cellular components. The previously described lethal effect of heterologous cytoplasm was confirmed. PMID:7462341

  15. Modulation of GABA receptors expressed in Xenopus oocytes by 13-L-hydroxylinoleic acid and food additives.

    PubMed

    Aoshima, H; Tenpaku, Y

    1997-12-01

    To study the effects of 13-L-hydroxylinoleic acid (LOH) and food additives on gamma-aminobutyric acid (GABA) receptors, ionotropic GABA receptors were expressed in Xenopus oocytes by injecting mRNAs prepared from rat whole brain. LOH, which was prepared by reduction of 13-L-hydroperoxylinoleic acid (LOOH), inhibited the response of GABA receptors in the presence of high concentrations of GABA. LOH also inhibited nicotinic acetylcholine, glycine, and kainate receptors, while it had little effect on NMDA receptors expressed in Xenopus oocytes. However, LOH potentiated the response of GABA receptors as well as LOOH in the presence of low concentrations of GABA, possibly increasing the affinity of GABA for the receptors, while linoleic acid did not. Since some modification of the compounds seemed to change their effects on GABA receptors, the responses of GABA receptors elicited by 10 microM GABA were measured in the presence of compounds with various kinds of functional groups or the structural isomers of pentanol. Potentiation of GABA receptors depended strongly on the species of functional groups and also depended on the structure of the isomers. Then effects of various kinds of food additives on GABA receptors were also examined; perfumes such as alcohols or esters potentiated the responses strongly, while hexylamine, nicotinamide, or caffeine inhibited the responses, mainly in a competitive manner, and vanillin inhibited the responses noncompetitively. These results suggest the possibility that production of LOOH and LOH, or intake of much of some food additives, modulates the neural transmission in the brain, especially through ionotropic GABA receptors and changes the frame of the human mind, as alcohol or tobacco does.

  16. Additive Regulation of Adiponectin Expression by the Mediterranean Diet Olive Oil Components Oleic Acid and Hydroxytyrosol in Human Adipocytes

    PubMed Central

    Scoditti, Egeria; Massaro, Marika; Carluccio, Maria Annunziata; Pellegrino, Mariangela; Wabitsch, Martin; Calabriso, Nadia; Storelli, Carlo; De Caterina, Raffaele

    2015-01-01

    Adiponectin, an adipocyte-derived insulin-sensitizing and anti-inflammatory hormone, is suppressed in obesity through mechanisms involving chronic inflammation and oxidative stress. Olive oil consumption is associated with beneficial cardiometabolic actions, with possible contributions from the antioxidant phenol hydroxytyrosol (HT) and the monounsaturated fatty acid oleic acid (OA, 18:1n-9 cis), both possessing anti-inflammatory and vasculo-protective properties. We determined the effects of HT and OA, alone and in combination, on adiponectin expression in human and murine adipocytes under pro-inflammatory conditions induced by the cytokine tumor necrosis factor(TNF)-α. We used human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes and murine 3T3-L1 adipocytes as cell model systems, and pretreated them with 1-100 μmol/L OA, 0.1-20 μmol/L HT or OA plus HT combination before stimulation with 10 ng/mL TNF-α. OA or HT significantly (P<0.05) prevented TNF-α-induced suppression of total adiponectin secretion (by 42% compared with TNF-α alone) as well as mRNA levels (by 30% compared with TNF-α alone). HT and OA also prevented—by 35%—TNF-α-induced downregulation of peroxisome proliferator-activated receptor PPARγ. Co-treatment with HT and OA restored adiponectin and PPARγ expression in an additive manner compared with single treatments. Exploring the activation of JNK, which is crucial for both adiponectin and PPARγ suppression by TNF-α, we found that HT and OA additively attenuated TNF-α-stimulated JNK phosphorylation (up to 55% inhibition). In conclusion, the virgin olive oil components OA and HT, at nutritionally relevant concentrations, have additive effects in preventing adiponectin downregulation in inflamed adipocytes through an attenuation of JNK-mediated PPARγ suppression. PMID:26030149

  17. Low-Affinity Memory CD8+ T Cells Mediate Robust Heterologous Immunity.

    PubMed

    Krummey, Scott M; Martinez, Ryan J; Andargachew, Rakieb; Liu, Danya; Wagener, Maylene; Kohlmeier, Jacob E; Evavold, Brian D; Larsen, Christian P; Ford, Mandy L

    2016-03-15

    Heterologous immunity is recognized as a significant barrier to transplant tolerance. Whereas it has been established that pathogen-elicited memory T cells can have high or low affinity for cross-reactive allogeneic peptide-MHC, the role of TCR affinity during heterologous immunity has not been explored. We established a model with which to investigate the impact of TCR-priming affinity on memory T cell populations following a graft rechallenge. In contrast to high-affinity priming, low-affinity priming elicited fully differentiated memory T cells with a CD45RB(hi) status. High CD45RB status enabled robust secondary responses in vivo, as demonstrated by faster graft rejection kinetics and greater proliferative responses. CD45RB blockade prolonged graft survival in low affinity-primed mice, but not in high affinity-primed mice. Mechanistically, low affinity-primed memory CD8(+) T cells produced more IL-2 and significantly upregulated IL-2Rα expression during rechallenge. We found that CD45RB(hi) status was also a stable marker of priming affinity within polyclonal CD8(+) T cell populations. Following high-affinity rechallenge, low affinity-primed CD45RB(hi) cells became CD45RB(lo), demonstrating that CD45RB status acts as an affinity-based differentiation switch on CD8(+) T cells. Thus, these data establish a novel mechanism by which CD45 isoforms tune low affinity-primed memory CD8(+) T cells to become potent secondary effectors following heterologous rechallenge. These findings have direct implications for allogeneic heterologous immunity by demonstrating that despite a lower precursor frequency, low-affinity priming is sufficient to generate memory cells that mediate potent secondary responses against a cross-reactive graft challenge. PMID:26864034

  18. Heterologous DNA Uptake in Cultured Symbiodinium spp. Aided by Agrobacterium tumefaciens.

    PubMed

    Ortiz-Matamoros, Mario Fernando; Islas-Flores, Tania; Voigt, Boris; Menzel, Diedrik; Baluška, František; Villanueva, Marco A

    2015-01-01

    Plant-targeted pCB302 plasmids containing sequences encoding gfp fusions with a microtubule-binding domain; gfp with the fimbrin actin-binding domain 2; and gfp with AtRACK1C from Arabidopsis thaliana, all harbored in Agrobacterium tumefaciens, were used to assay heterologous expression on three different clades of the photosynthetic dinoflagellate, Symbiodinium. Accessibility to the resistant cell wall and through the plasma membrane of these dinoflagellates was gained after brief but vigorous shaking in the presence of glass beads and polyethylene glycol. A resistance gene to the herbicide Basta allowed appropriate selection of the cells expressing the hybrid proteins, which showed a characteristic green fluorescence, although they appeared to lose their photosynthetic pigments and did not further divide. Cell GFP expression frequency measured as green fluorescence emission yielded 839 per every 106 cells for Symbiodinium kawagutii, followed by 640 and 460 per every 106 cells for Symbiodinium microadriaticum and Symbiodinium sp. Mf11, respectively. Genomic PCR with specific primers amplified the AtRACK1C and gfp sequences after selection in all clades, thus revealing their presence in the cells. RT-PCR from RNA of S. kawagutii co-incubated with A. tumefaciens harboring each of the three vectors with their respective constructs, amplified products corresponding to the heterologous gfp sequence while no products were obtained from three distinct negative controls. The reported procedure shows that mild abrasion followed by co-incubation with A. tumefaciens harboring heterologous plasmids with CaMV35S and nos promoters can lead to expression of the encoded proteins into the Symbiodinium cells in culture. Despite the obvious drawbacks of the procedure, this is an important first step towards a stable transformation of Symbiodinium.

  19. Heterologous DNA Uptake in Cultured Symbiodinium spp. Aided by Agrobacterium tumefaciens

    PubMed Central

    Voigt, Boris; Menzel, Diedrik; Baluška, František; Villanueva, Marco A.

    2015-01-01

    Plant-targeted pCB302 plasmids containing sequences encoding gfp fusions with a microtubule-binding domain; gfp with the fimbrin actin-binding domain 2; and gfp with AtRACK1C from Arabidopsis thaliana, all harbored in Agrobacterium tumefaciens, were used to assay heterologous expression on three different clades of the photosynthetic dinoflagellate, Symbiodinium. Accessibility to the resistant cell wall and through the plasma membrane of these dinoflagellates was gained after brief but vigorous shaking in the presence of glass beads and polyethylene glycol. A resistance gene to the herbicide Basta allowed appropriate selection of the cells expressing the hybrid proteins, which showed a characteristic green fluorescence, although they appeared to lose their photosynthetic pigments and did not further divide. Cell GFP expression frequency measured as green fluorescence emission yielded 839 per every 106 cells for Symbiodinium kawagutii, followed by 640 and 460 per every 106 cells for Symbiodinium microadriaticum and Symbiodinium sp. Mf11, respectively. Genomic PCR with specific primers amplified the AtRACK1C and gfp sequences after selection in all clades, thus revealing their presence in the cells. RT-PCR from RNA of S. kawagutii co-incubated with A. tumefaciens harboring each of the three vectors with their respective constructs, amplified products corresponding to the heterologous gfp sequence while no products were obtained from three distinct negative controls. The reported procedure shows that mild abrasion followed by co-incubation with A. tumefaciens harboring heterologous plasmids with CaMV35S and nos promoters can lead to expression of the encoded proteins into the Symbiodinium cells in culture. Despite the obvious drawbacks of the procedure, this is an important first step towards a stable transformation of Symbiodinium. PMID:26167858

  20. Heterologous fermentation of a diterpene from Alternaria brassisicola

    PubMed Central

    Arens, Julia; Bergs, Dominik; Mewes, Mirja; Merz, Juliane; Schembecker, Gerhard; Schulz, Frank

    2014-01-01

    A variety of different applications render terpenes and terpenoids attractive research targets. A promising but so far insufficiently explored family of terpenoids are the fusicoccanes that comprise a characteristic 5-8-5 fused tricyclic ring system. Besides herbicidal effects, these compounds also show apoptotic and anti-tumour effects on mammalian cells. The access to fusicoccanes from natural sources is scarce. Recently, we introduced a metabolically engineered Saccharomyces cerevisiae strain to enable the heterologous fermentation of the shared fusicoccane–diterpenoid precursor, fusicocca-2,10(14)-diene. Here, we show experiments towards the identification of bottlenecks in this process. The suppression of biosynthetic by-products via medium optimisation was found to be an important aspect. In addition, the fermentation process seems to be improved under oxygen limitation conditions. Under fed-batch conditions, the fermentation yield was reproducibly increased to approximately 20 mg/L. Furthermore, the impact of the properties of the terpene synthase on the fermentation yield is discussed, and the preliminary studies on the engineering of this key enzyme are presented. PMID:25379342

  1. High Titer Heterologous Production of Lyngbyatoxin in E. coli, a Protein Kinase C Activator from an Uncultured Marine Cyanobacterium

    PubMed Central

    Ongley, Sarah E.; Bian, Xiaoying; Zhang, Youming; Chau, Rocky; Gerwick, William H.; Müller, Rolf; Neilan, Brett A.

    2013-01-01

    Many chemically-complex cyanobacterial polyketides and nonribosomal peptides are of great pharmaceutical interest, but the levels required for exploitation are difficult to achieve from native sources. Here we develop a framework for the expression of these multifunctional cyanobacterial assembly lines in Escherichia coli using the lyngbyatoxin biosynthetic pathway, derived from a marine microbial assemblage dominated by the cyanobacterium Moorea producens. Heterologous expression of this pathway afforded high titers of both lyngbyatoxin A (25.6 mg L-1) and its precursor indolactam-V (150 mg L-1). Production, isolation and identification of all expected chemical intermediates of lyngbyatoxin biosynthesis in E. coli also confirmed the previously proposed biosynthetic route setting a solid chemical foundation for future pathway engineering. The successful production of the nonribosomal peptide lyngbyatoxin A in E. coli also opens the possibility for future heterologous expression, characterization and exploitation of other cyanobacterial natural product pathways. PMID:23751865

  2. Heterologous induction of Ty1 retrotransposition: Reverse transcriptase plays a key role in initiation of the retrotransposition cycle

    SciTech Connect

    Reznik, N.L.; Kidgotko, O.V.; Zolotova, L.I.

    1995-12-01

    A new method was developed to study the mechanism of initiation of the retrotransposition cycle: retrotransposons of Drosophila melanogaster, gypsy, copia, and 17.6 were expressed in yeast under the control of strong yeast promoters. Expression of retrotransposons induced formation of viruslike particles (VLPs) associated with full-length Ty1 RNA and DNA sequences. This phenomenon was termed heterologous induction. When the gene for reverse transcriptase of human immunodeficiency virus (HIV) was expressed in yeast, the same results were obtained. These data allowed us to assume that the excess of active reverse transcriptase plays the key role in induction of transposition. Possible mechanisms of induction of Ty1 transposition by homologous and heterologous elements are discussed. 34 refs., 5 figs.

  3. Modulation of chicken intestinal immune gene expression by small cationic peptides as feed additives during the first week posthatch.

    PubMed

    Kogut, Michael H; Genovese, Kenneth J; He, Haiqi; Swaggerty, Christina L; Jiang, Yiwei

    2013-09-01

    We have been investigating modulation strategies tailored around the selective stimulation of the host's immune system as an alternative to direct targeting of microbial pathogens by antibiotics. One such approach is the use of a group of small cationic peptides (BT) produced by a Gram-positive soil bacterium, Brevibacillus texasporus. These peptides have immune modulatory properties that enhance both leukocyte functional efficiency and leukocyte proinflammatory cytokine and chemokine mRNA transcription activities in vitro. In addition, when provided as a feed additive for just 4 days posthatch, BT peptides significantly induce a concentration-dependent protection against cecal and extraintestinal colonization by Salmonella enterica serovar Enteritidis. In the present studies, we assessed the effects of feeding BT peptides on transcriptional changes on proinflammatory cytokines, inflammatory chemokines, and Toll-like receptors (TLR) in the ceca of broiler chickens with and without S. Enteritidis infection. After feeding a BT peptide-supplemented diet for the first 4 days posthatch, chickens were then challenged with S. Enteritidis, and intestinal gene expression was measured at 1 or 7 days postinfection (p.i.) (5 or 11 days of age). Intestinal expression of innate immune mRNA transcripts was analyzed by quantitative real-time PCR (qRT-PCR). Analysis of relative mRNA expression showed that a BT peptide-supplemented diet did not directly induce the transcription of proinflammatory cytokine, inflammatory chemokine, type I/II interferon (IFN), or TLR mRNA in chicken cecum. However, feeding the BT peptide-supplemented diet primed cecal tissue for increased (P ≤ 0.05) transcription of TLR4, TLR15, and TLR21 upon infection with S. Enteritidis on days 1 and 7 p.i. Likewise, feeding the BT peptides primed the cecal tissue for increased transcription of proinflammatory cytokines (interleukin 1β [IL-1β], IL-6, IL-18, type I and II IFNs) and inflammatory chemokine (CxCLi2

  4. Mutations in the coat protein gene of plum pox virus suppress particle assembly, heterologous encapsidation and complementation in transgenic plants of Nicotiana benthamiana.

    PubMed

    Varrelmann, M; Maiss, E

    2000-03-01

    Two different motifs in the coat protein (CP) of Plum pox virus (PPV) (R(3015)Q(3016), D(3059)) were mutated by replacing the respective amino acids with others possessing different chemical properties. The mutated CP genes were introduced into an infectious full-length clone of PPV (p35PPV-NAT) to investigate their influence on systemic infection of transgenic wild-type PPV CP-expressing and non-transgenic plants of Nicotiana benthamiana. All mutants failed to establish systemic infections in non-transgenic N. benthamiana plants, but were complemented by intact CP in transgenic plants. Moreover, the CP-RQ-D mutant (carrying mutations in both the RQ and D motifs) was introduced into p35PPV-NAT engineered to express beta-glucuronidase (GUS) for direct observation of systemic movement and particle assembly in N. benthamiana leaves. GUS-staining revealed that the CP mutant (RQ-D) was restricted to initially infected cells without forming virions. Systemic movement and particle assembly were restored in CP-transgenic N. benthamiana plants. Finally, transgenic N. benthamiana plants were generated that expressed each of the three mutated CP genes. Homozygous T(2) lines were selected and tested for resistance to PPV. Immunogold labelling and electron microscopy revealed that heterologous encapsidation with challenging Chilli veinal mottle virus and Potato virus Y was suppressed in these lines. In addition, assembly mutants did not complement CP-defective p35PPV-NAT. The possible use of modified viral CP genes for the production of virus-resistant transgenic plants, thereby reducing the putative risks of heterologous encapsidation and complementation, is discussed. PMID:10675394

  5. Additive transgene expression and genetic introgression in multiple green-fluorescent protein transgenic crop x weed hybrid generations.

    PubMed

    Halfhill, M D; Millwood, R J; Weissinger, A K; Warwick, S I; Stewart, C N

    2003-11-01

    The level of transgene expression in crop x weed hybrids and the degree to which crop-specific genes are integrated into hybrid populations are important factors in assessing the potential ecological and agricultural risks of gene flow associated with genetic engineering. The average transgene zygosity and genetic structure of transgenic hybrid populations change with the progression of generations, and the green fluorescent protein (GFP) transgene is an ideal marker to quantify transgene expression in advancing populations. The homozygous T(1) single-locus insert GFP/ Bacillus thuringiensis (Bt) transgenic canola ( Brassica napus, cv Westar) with two copies of the transgene fluoresced twice as much as hemizygous individuals with only one copy of the transgene. These data indicate that the expression of the GFP gene was additive, and fluorescence could be used to determine zygosity status. Several hybrid generations (BC(1)F(1), BC(2)F(1)) were produced by backcrossing various GFP/Bt transgenic canola ( B. napus, cv Westar) and birdseed rape ( Brassica rapa) hybrid generations onto B. rapa. Intercrossed generations (BC(2)F(2) Bulk) were generated by crossing BC(2)F(1) individuals in the presence of a pollinating insect ( Musca domestica L.). The ploidy of plants in the BC(2)F(2) Bulk hybrid generation was identical to the weedy parental species, B. rapa. AFLP analysis was used to quantify the degree of B. napus introgression into multiple backcross hybrid generations with B. rapa. The F(1) hybrid generations contained 95-97% of the B. napus-specific AFLP markers, and each successive backcross generation demonstrated a reduction of markers resulting in the 15-29% presence in the BC(2)F(2) Bulk population. Average fluorescence of each successive hybrid generation was analyzed, and homozygous canola lines and hybrid populations that contained individuals homozygous for GFP (BC(2)F(2) Bulk) demonstrated significantly higher fluorescence than hemizygous hybrid

  6. Additive transgene expression and genetic introgression in multiple green-fluorescent protein transgenic crop x weed hybrid generations.

    PubMed

    Halfhill, M D; Millwood, R J; Weissinger, A K; Warwick, S I; Stewart, C N

    2003-11-01

    The level of transgene expression in crop x weed hybrids and the degree to which crop-specific genes are integrated into hybrid populations are important factors in assessing the potential ecological and agricultural risks of gene flow associated with genetic engineering. The average transgene zygosity and genetic structure of transgenic hybrid populations change with the progression of generations, and the green fluorescent protein (GFP) transgene is an ideal marker to quantify transgene expression in advancing populations. The homozygous T(1) single-locus insert GFP/ Bacillus thuringiensis (Bt) transgenic canola ( Brassica napus, cv Westar) with two copies of the transgene fluoresced twice as much as hemizygous individuals with only one copy of the transgene. These data indicate that the expression of the GFP gene was additive, and fluorescence could be used to determine zygosity status. Several hybrid generations (BC(1)F(1), BC(2)F(1)) were produced by backcrossing various GFP/Bt transgenic canola ( B. napus, cv Westar) and birdseed rape ( Brassica rapa) hybrid generations onto B. rapa. Intercrossed generations (BC(2)F(2) Bulk) were generated by crossing BC(2)F(1) individuals in the presence of a pollinating insect ( Musca domestica L.). The ploidy of plants in the BC(2)F(2) Bulk hybrid generation was identical to the weedy parental species, B. rapa. AFLP analysis was used to quantify the degree of B. napus introgression into multiple backcross hybrid generations with B. rapa. The F(1) hybrid generations contained 95-97% of the B. napus-specific AFLP markers, and each successive backcross generation demonstrated a reduction of markers resulting in the 15-29% presence in the BC(2)F(2) Bulk population. Average fluorescence of each successive hybrid generation was analyzed, and homozygous canola lines and hybrid populations that contained individuals homozygous for GFP (BC(2)F(2) Bulk) demonstrated significantly higher fluorescence than hemizygous hybrid