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Sample records for heterologous expression analyses

  1. Differential gene expression in recombinant Pichia pastoris analysed by heterologous DNA microarray hybridisation

    PubMed Central

    Sauer, Michael; Branduardi, Paola; Gasser, Brigitte; Valli, Minoska; Maurer, Michael; Porro, Danilo; Mattanovich, Diethard

    2004-01-01

    Background Pichia pastoris is a well established yeast host for heterologous protein expression, however, the physiological and genetic information about this yeast remains scanty. The lack of a published genome sequence renders DNA arrays unavailable, thereby hampering more global investigations of P. pastoris from the beginning. Here, we examine the suitability of Saccharomyces cerevisiae DNA microarrays for heterologous hybridisation with P. pastoris cDNA. Results We could show that it is possible to obtain new and valuable information about transcriptomic regulation in P. pastoris by probing S. cerevisiae DNA microarrays. The number of positive signals was about 66 % as compared to homologous S. cerevisiae hybridisation, and both the signal intensities and gene regulations correlated with high significance between data obtained from P. pastoris and S. cerevisiae samples. The differential gene expression patterns upon shift from glycerol to methanol as carbon source were investigated in more detail. Downregulation of TCA cycle genes and a decrease of genes related to ribonucleotide and ribosome synthesis were among the major effects identified. Conclusions We could successfully demonstrate that heterologous microarray hybridisations allow deep insights into the transcriptomic regulation processes of P. pastoris. The observed downregulation of TCA cycle and ribosomal synthesis genes correlates to a significantly lower specific growth rate during the methanol feed phase. PMID:15610561

  2. Heterologous Expression of Peroxidases

    NASA Astrophysics Data System (ADS)

    de Weert, Sandra; Lokman, B. Christien

    The industrial importance of peroxidases has led to much research in the past two decades on the development of a cost effective and efficient production process for peroxidases. Unfortunately, even today, no clear answers can be given to questions such as (1) should the peroxidase be expressed in bacteria, yeast, or fungi? (2) which is the optimal production strain (e.g., protease deficient, heme overproducing)? (3) which expression vector should be chosen? and (4) what purification method should be used? Strategies that have proven successful for one peroxidase can fail for another one; for each individual peroxidase, a new strategy has to be developed. This chapter gives an overview of the heterologous production of heme containing peroxidases in various systems. It focuses on the heterologous production of fungal peroxidases as they have been subject of considerable research for their industrial and environmental applications. An earlier study has also been performed by Conesa et al. [1] and is extended with recent proceedings.

  3. Heterologous and cell free protein expression systems.

    PubMed

    Farrokhi, Naser; Hrmova, Maria; Burton, Rachel A; Fincher, Geoffrey B

    2009-01-01

    In recognition of the fact that a relatively small percentage of 'named' genes in databases have any experimental proof for their annotation, attention is shifting towards the more accurate assignment of functions to individual genes in a genome. The central objective will be to reduce our reliance on nucleotide or amino acid sequence similarities as a means to define the functions of genes and to annotate genome sequences. There are many unsolved technical difficulties associated with the purification of specific proteins from extracts of biological material, especially where the protein is present in low abundance, has multiple isoforms or is found in multiple post-translationally modified forms. The relative ease with which cDNAs can be cloned has led to the development of methods through which cDNAs from essentially any source can be expressed in a limited range of suitable host organisms, so that sufficient levels of the encoded proteins can be generated for functional analysis. Recently, these heterologous expression systems have been supplemented by more robust prokaryotic and eukaryotic cell-free protein synthesis systems. In this chapter, common host systems for heterologous expression are reviewed and the current status of cell-free expression systems will be presented. New approaches to overcoming the special problems encountered during the expression of membrane-associated proteins will also be addressed. Methodological considerations, including the characteristics of codon usage in the expressed DNA, peptide tags that facilitate subsequent purification of the expressed proteins and the role of post-translational modifications, are examined.

  4. Heterologous gene expression in filamentous fungi.

    PubMed

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

    2012-01-01

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

  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.

  6. Utility of Aspergillus niger citrate synthase promoter for heterologous expression.

    PubMed

    Dave, Kashyap; Punekar, Narayan S

    2011-09-10

    Citrate synthase is a central player in the acidogenic metabolism of Aspergillus niger. The 5' upstream sequence (0.9kb DNA) of citrate synthase gene (citA) from A. niger NCIM 565 was analyzed and its promoter function demonstrated through the heterologous expression of two proteins. The cloned citrate synthase promoter (PcitA) sequence was able to express bar coding sequence thereby conferring phosphinothricin resistance. This sequence was further analyzed by systematic deletions to define an effective but compact functional promoter. The PcitA driven egfp expression showed that PcitA was active in all differentiation cell-stages of A. niger. EGFP expression was highest on non-repressible carbon sources like acetate and glycerol. Mycelial EGFP levels increased during acidogenic growth suggesting that PcitA is functional throughout this cultivation. A. niger PcitA is the first Krebs cycle gene promoter used to express heterologous proteins in filamentous fungi.

  7. Homologous versus heterologous gene expression in the yeast, Saccharomyces cerevisiae.

    PubMed Central

    Chen, C Y; Oppermann, H; Hitzeman, R A

    1984-01-01

    DNA sequences normally flanking the highly expressed yeast 3-phosphoglycerate kinase (PGK) gene have been placed adjacent to heterologous mammalian genes on high copy number plasmid vectors and used for expression experiments in yeast. For many genes thus far expressed with this system, expression has been 15-50 times lower than the expression of the natural homologous PGK gene on the same plasmid. We have extensively investigated this dramatic difference and have found that in most cases it is directly proportional to the steady-state levels of mRNAs. We demonstrate this phenomenon and suggest possible causes for this effect on mRNA levels. Images PMID:6096814

  8. 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.

  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 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.

  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. Combinatorial engineering for heterologous gene expression.

    PubMed

    Zwick, Friederike; Lale, Rahmi; Valla, Svein

    2013-01-01

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

  13. Combinatorial engineering for heterologous gene expression

    PubMed Central

    Zwick, Friederike; Lale, Rahmi; Valla, Svein

    2013-01-01

    Tools for strain engineering with predictable outcome are of crucial importance for the nascent field of synthetic biology. The success of combining different DNA biological parts is often restricted by poorly understood factors deriving from the complexity of the systems. We have previously identified variants for different regulatory elements of the expression cassette XylS/Pm. When such elements are combined they act in a manner consistent with their individual behavior, as long as they affect different functions, such as transcription and translation. Interestingly, sequence context does not seem to influence the final outcome significantly. Expression of reporter gene bla could be increased up to 75 times at the protein level by combining three variants in one cassette. For other tested reporter genes similar results were obtained, except that the stimulatory effect was quantitatively less. Combination of individually characterized DNA parts thus stands as suitable method to achieve a desired phenotype. PMID:23644416

  14. Heterologous expression of antigenic peptides in Bacillus subtilis biofilms.

    PubMed

    Vogt, Cédric M; Schraner, Elisabeth M; Aguilar, Claudio; Eichwald, Catherine

    2016-08-11

    Numerous strategies have been developed for the display of heterologous proteins in the surface of live bacterial carriers, which can be used as vaccines, immune-modulators, cancer therapy or bioremediation. Bacterial biofilms have emerged as an interesting approach for the expression of proteins of interest. Bacillus subtilis is a well-described, endospore-forming organism that is able to form biofilms and also used as a probiotic, thus making it a suitable candidate for the display of heterologous proteins within the biofilm. Here, we describe the use of TasA, an important structural component of the biofilms formed by B. subtilis, as a genetic tool for the display of heterologous proteins. We first engineered the fusion protein TasA-mCherry and showed that was widely deployed within the B. subtilis biofilms. A significant enhancement of the expression of TasA-mCherry within the biofilm was obtained when depleting both tasA and sinR genes. We subsequently engineered fusion proteins of TasA to antigenic peptides of the E. granulosus parasite, paramyosin and tropomyosin. Our results show that the antigens were well expressed within the biofilm as denoted by macrostructure complementation and by the detection of the fusion protein in both immunoblot and immunohistochemistry. In addition, we show that the recombinant endospores of B. subtilis preserve their biophysical and morphological properties. In this work we provide strong evidence pointing that TasA is a suitable candidate for the display of heterologous peptides, such as antigens, cytokines, enzymes or antibodies, in the B. subtilis biofilms. Finally, our data portray that the recombinant endospores preserve their morphological and biophysical properties and could be an excellent tool to facilitate the transport and the administration.

  15. 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.

  16. Heterologous gene expression in Hansenula polymorpha: Efficient secretion of glucoamylase

    SciTech Connect

    Gellissen, G.; Janowicz, Z.A.; Merckelbach, A.; Keup, P.; Weydemann, U.; Strasser, A.W.M. ); Piontek, M.; Hollenberg, C.P. )

    1991-03-01

    The authors have introduced the glucoamylase gene (GAM1) from Schwanniomyces occidentalis into the genome of the methylotrophic yeast Hansenula polymorpha to study the potential of this organism as a host for high-level expression of a heterologous gene encoding a secretory protein. Transformants of H. polymorpha containing GAM1 under control of the formate dehydrogenase (FMD) promoter are stable and efficiently secrete an active glucoamylase that is faithfully processed and modified. Yields of up to 1.4 g/l of active enzyme were obtained at cell densities of 100-130 grams dry weight per liter.

  17. Heterologous expression of human H1 histones in yeast.

    PubMed

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

    1998-09-18

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

    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

  20. Heterologous Expression of Novobiocin and Clorobiocin Biosynthetic Gene Clusters

    PubMed Central

    Eustáquio, Alessandra S.; Gust, Bertolt; Galm, Ute; Li, Shu-Ming; Chater, Keith F.; Heide, Lutz

    2005-01-01

    A method was developed for the heterologous expression of biosynthetic gene clusters in different Streptomyces strains and for the modification of these clusters by single or multiple gene replacements or gene deletions with unprecedented speed and versatility. λ-Red-mediated homologous recombination was used for genetic modification of the gene clusters, and the attachment site and integrase of phage φC31 were employed for the integration of these clusters into the heterologous hosts. This method was used to express the gene clusters of the aminocoumarin antibiotics novobiocin and clorobiocin in the well-studied strains Streptomyces coelicolor and Streptomyces lividans, which, in contrast to the natural producers, can be easily genetically manipulated. S. coelicolor M512 derivatives produced the respective antibiotic in yields comparable to those of natural producer strains, whereas S. lividans TK24 derivatives were at least five times less productive. This method could also be used to carry out functional investigations. Shortening of the cosmids' inserts showed which genes are essential for antibiotic production. PMID:15870333

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

    PubMed

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

    2017-01-01

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

  2. Noncytopathic Sindbis virus RNA vectors for heterologous gene expression

    PubMed Central

    Agapov, Eugene V.; Frolov, Ilya; Lindenbach, Brett D.; Prágai, Béla M.; Schlesinger, Sondra; Rice, Charles M.

    1998-01-01

    Infection of vertebrate cells with alphaviruses normally leads to prodigious expression of virus-encoded genes and a dramatic inhibition of host protein synthesis. Recombinant Sindbis viruses and replicons have been useful as vectors for high level foreign gene expression, but the cytopathic effects of viral replication have limited their use to transient studies. We recently selected Sindbis replicons capable of persistent, noncytopathic growth in BHK cells and describe here a new generation of Sindbis vectors useful for long-term foreign gene expression based on such replicons. Foreign genes of interest as well as the dominant selectable marker puromycin N-acteyltransferase, which confers resistance to the drug puromycin, were expressed as subgenomic transcripts of noncytopathic replicons or defective-interfering genomes complemented in trans by a replicon. Based on these strategies, we developed vectors that can be initiated via either RNA or DNA transfection and analyzed them for their level and stability of foreign gene expression. Noncytopathic Sindbis vectors express reasonably high levels of protein in nearly every cell. These vectors should prove to be flexible tools for the rapid expression of heterologous genes under conditions in which cellular metabolism is not perturbed, and we illustrate their utility with a number of foreign proteins. PMID:9789028

  3. 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

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

    PubMed

    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 rules for

  5. 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

  6. Induction of photosensitivity by heterologous expression of melanopsin.

    PubMed

    Qiu, Xudong; Kumbalasiri, Tida; Carlson, Stephanie M; Wong, Kwoon Y; Krishna, Vanitha; Provencio, Ignacio; Berson, David M

    2005-02-17

    Melanopsin has been proposed to be the photopigment of the intrinsically photosensitive retinal ganglion cells (ipRGCs); these photoreceptors of the mammalian eye drive circadian and pupillary adjustments through direct projections to the brain. Their action spectrum (lambda(max) approximately 480 nm) implicates an opsin and melanopsin is the only opsin known to exist in these cells. Melanopsin is required for ipRGC photosensitivity and for behavioural photoresponses that survive disrupted rod and cone function. Heterologously expressed melanopsin apparently binds retinaldehyde and mediates photic activation of G proteins. However, its amino-acid sequence differs from vertebrate photosensory opsins and some have suggested that melanopsin may be a photoisomerase, providing retinoid chromophore to an unidentified opsin. To determine whether melanopsin is a functional sensory photopigment, here we transiently expressed it in HEK293 cells that stably expressed TRPC3 channels. Light triggered a membrane depolarization in these cells and increased intracellular calcium. The light response resembled that of ipRGCs, with almost identical spectral sensitivity (lambda(max) approximately 479 nm). The phototransduction pathway included Gq or a related G protein, phospholipase C and TRPC3 channels. We conclude that mammalian melanopsin is a functional sensory photopigment, that it is the photopigment of ganglion-cell photoreceptors, and that these photoreceptors may use an invertebrate-like phototransduction cascade.

  7. Heterologous expression of functional Ptr ToxA.

    PubMed

    Tuori, R P; Wolpert, T J; Ciuffetti, L M

    2000-04-01

    Ptr ToxA, a proteinaceous host-selective toxin (HST) produced by the fungus Pyrenophora tritici-repentis, was expressed in Escherichia coli and purified as a polyhistidine-tagged, fusion protein (NC-FP). NC-FP, consisting of both the N and C domains of the ToxA open reading frame (ORF), is produced as an insoluble protein in E. coli at approximately 10 to 16 mg per liter of culture. Following in vitro refolding, NC-FP elicits cultivar-specific necrosis in wheat, with a specific activity similar to that of native Ptr ToxA. A fusion protein consisting of only the C domain has approximately 10 to 20% of the activity of native Ptr ToxA. These data suggest that (i) the N domain is important for maximal activity of Ptr ToxA, (ii) the N domain does not function to eliminate activity of the protoxin, and (iii) post-translational modifications of Ptr ToxA are not essential for activity. A C domain construct with a cysteine residue mutated to glycine is inactive. This, plus the observation that toxin activity is sensitive to reducing agents, provides evidence that the two cysteine residues in Ptr ToxA are involved in a disulfide bond that is essential for activity. The heterologous expression of Ptr ToxA provides a valuable tool for addressing a number of issues such as receptor binding studies, structure/function studies, and screening wheat cultivars for disease resistance.

  8. 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

  9. Heterologous expression of an active chitin synthase from Rhizopus oryzae.

    PubMed

    Salgado-Lugo, Holjes; Sánchez-Arreguín, Alejandro; Ruiz-Herrera, José

    2016-12-01

    Chitin synthases are highly important enzymes in nature, where they synthesize structural components in species belonging to different eukaryotic kingdoms, including kingdom Fungi. Unfortunately, their structure and the molecular mechanism of synthesis of their microfibrilar product remain largely unknown, probably because no fungal active chitin synthases have been isolated, possibly due to their extreme hydrophobicity. In this study we have turned to the heterologous expression of the transcript from a small chitin synthase of Rhizopus oryzae (RO3G_00942, Chs1) in Escherichia coli. The enzyme was active, but accumulated mostly in inclusion bodies. High concentrations of arginine or urea solubilized the enzyme, but their dilution led to its denaturation and precipitation. Nevertheless, use of urea permitted the purification of small amounts of the enzyme. The properties of Chs1 (Km, optimum temperature and pH, effect of GlcNAc) were abnormal, probably because it lacks the hydrophobic transmembrane regions characteristic of chitin synthases. The product of the enzyme showed that, contrasting with chitin made by membrane-bound Chs's and chitosomes, was only partially in the form of short microfibrils of low crystallinity. This approach may lead to future developments to obtain active chitin synthases that permit understanding their molecular mechanism of activity, and microfibril assembly.

  10. Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host

    PubMed Central

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

    2011-01-01

    Background Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. Methodology/Principal Findings The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. Conclusions/Significance Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein. PMID:22216205

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

    PubMed

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

    2011-01-01

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

  12. 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.

  13. Modulation of heterologous expression from PBAD promoter in Escherichia coli production strains.

    PubMed

    Széliová, Diana; Krahulec, Ján; Šafránek, Martin; Lišková, Veronika; Turňa, Ján

    2016-10-20

    Promoter PBAD is frequently used for heterologous gene expression due to several advantages, such as moderately high expression levels, induction by an inexpensive and non-toxic monosaccharide L-arabinose and tight regulation of transcription, which is particularly important for expression of toxic proteins. A drawback of this promoter is all-or-none induction that occurs at subsaturating inducer concentrations. Although the overall expression level of the cell culture seems to correlate with increasing arabinose concentrations, the population is a mixture of induced and uninduced cells and with increasing arabinose concentrations, only the fraction of induced cells increases. This phenomenon is caused by autocatalytic gene expression - the expression of the arabinose transporter AraE is induced by the transported molecule. In this work the promoter PE, controlling the expression of araE, was exchanged for the stronger PBAD promoter in two Escherichia coli strains commonly used for heterologous protein production. This modification should increase a basal number of arabinose transporters in the cell wall and reduce the threshold concentration required for induction and thus reduce heterogeneity of cell population. Heterogeneity and level of expression in individual cells were analysed by flow cytometry using gfp as a reporter gene. In the strain BL21ai, the promoter exchange increased the number of induced cells at subsaturating arabinose concentrations as well as a yield of protein at saturating inducer concentration. In contrast, the modification did not improve these characteristics in RV308ai. In both strains it was possible to modulate the expression level in induced cells 3-6-fold even at subsaturating arabinose concentrations. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Heterologously expressed family 51 alpha-L-arabinofuranosidases from Oenococcus oeni and Lactobacillus brevis.

    PubMed

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

    2011-02-01

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

  15. Barriers to heterologous expression of a selenoprotein gene in bacteria.

    PubMed Central

    Tormay, P; Böck, A

    1997-01-01

    The specificity parameters counteracting the heterologous expression in Escherichia coli of the Desulfomicrobium baculatum gene (hydV) coding for the large subunit of the periplasmic hydrogenase which is a selenoprotein have been studied. hydV'-'lacZ fusions were constructed, and it was shown that they do not direct the incorporation of selenocysteine in E. coli. Rather, the UGA codon is efficiently suppressed by some other aminoacyl-tRNA in an E. coli strain possessing a ribosomal ambiguity mutation. The suppression is decreased by the strA1 allele, indicating that the hydV selenocysteine UGA codon has the properties of a "normal" and suppressible nonsense codon. The SelB protein from D. baculatum was purified; in gel shift experiments, D. baculatum SelB displayed a lower affinity for the E. coli fdhF selenoprotein mRNA than E. coli SelB did and vice versa. Coexpression of the hydV'-'lacZ fusion and of the selB and tRNA(Sec) genes from D. baculatum, however, did not lead to selenocysteine insertion into the protein, although the formation of the quaternary complex between SelB, selenocysteyl-tRNA(Sec), and the hydV mRNA recognition sequence took place. The results demonstrate (i) that the selenocysteine-specific UGA codon is readily suppressed under conditions where the homologous SelB protein is absent and (ii) that apart from the specificity of the SelB-mRNA interaction, a structural compatibility of the quaternary complex with the ribosome is required. PMID:9006007

  16. 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

  17. Heterologous expression and manipulation of three tetracycline biosynthetic pathways.

    PubMed

    Wang, Peng; Kim, Woncheol; Pickens, Lauren B; Gao, Xue; Tang, Yi

    2012-10-29

    A very accommodating host: Three tetracycline biosynthetic pathways were overexpressed and manipulated in the heterologous host Streptomyces lividans K4-114. Through the inactivation of various genes and characterization of the resulting biosynthetic intermediates, new tetracycline-modifying enzymes were identified (see scheme). Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. [Heterologous genes expression on Escherichia coli chromosome lac operon using Red recombination].

    PubMed

    Li, Shanhu; Shi, Qingguo; Huang, Cuifen; Zhou, Jianguang

    2008-04-01

    To achieve efficient and stable expression of heterologous exogenetic protein or antigen in E. coli chromosome, the luciferase report gene was knocked in lacZ site of chromosome lac operon by using Red recombination system and selection-counterselection kan/sacB technology. The quantitative analysis of exogenous gene expression indicated that the target gene could be efficiently expressed at lacZ site of lac operon. The results confirmed the efficient screening and stable expression of heterologous protein or antigen on chromosome by using the recombinant engineering technique. This study demonstrated that the chromosome could be used as a vector for heterologous protein or antigen and the stable expression of exogenous gene on E. coli chromosome had no side effect on the bacterial growth and propagation.

  19. Comparison of three expression systems for heterologous xylanase production by S. cerevisiae in defined medium.

    PubMed

    Görgens, Johann F; Planas, Jordi; van Zyl, Willem H; Knoetze, Johannes H; Hahn-Hägerdal, Bärbel

    2004-10-30

    The influence of the auxotrophic deficiencies of the host strain and expression vector selection on the production of a heterologous protein was investigated. Heterologous xylanase production by two prototrophic S. cerevisiae transformants, containing either a plasmid-based, YEp-type expression system or an integrative, YIp-type expression system, were compared with production by an auxotrophic transformant, containing an identical YEp-type expression system, in batch and continuous cultivation, using a chemically defined medium. Heterologous xylanase production by the auxotrophic strains in defined medium was critically dependent on the availability of amino acids, as extracellular xylanase production increased dramatically when amino acids were over-consumed from the medium to the point of saturating the cell. Saturation with amino acids, indicated by an increased leakage of amino acids from the cell, was thus a prerequisite for high level of heterologous protein production by the auxotrophic strain. Maximal xylanase production levels by the auxotrophic strain corresponded to the levels obtained with a similar prototrophic strain during cultivation in defined medium without amino acids. Superfluous auxotrophic markers thus had a strong deleterious effect on heterologous protein production by recombinant yeasts, and the use of such strains should be limited to initial exploratory investigations. The increased copy number and foreign gene dosage of the YEp-based expression vector, stabilized by the ura3 fur1 autoselection system, significantly improved production levels of heterologous xylanase, compared to the YIp system, which is based on a single integration into the yeast genome. No evidence was found of the possible saturation of the host secretory capacity by multicopy overexpression. Stable production of heterologous xylanase at high levels by the prototrophic YEp-based recombinant strain, compared to the YIp system, was demonstrated.

  20. [Progress in heterologous expression of fungal natural products-A review].

    PubMed

    Ma, Zihui; Li, Wei; Yin, Wenbing

    2016-03-04

    Fungal products are important resources for natural drugs. Availabilities of many fungal genome sequences indicate that fungi have the huge potential to produce secondary metabolites. However, many valuable fungal natural products are hard to obtain under regular lab conditions because fungi are slow to grow, difficult to genetically modify, or have low yield of desired metabolites. Therefore, developing a heterologous expression system has been an efficient approach to discover novel natural product and elucidate the complicated biosynthetic pathways. This review describes several heterologous expression systems that have been successfully used for the expression of fungal biosynthetic gene clusters. Also, we address the application perspective based on the emerging DNA assembly technologies.

  1. Recent advances in discovery, heterologous expression, and molecular engineering of cyclodextrin glycosyltransferase for versatile applications.

    PubMed

    Han, Ruizhi; Li, Jianghua; Shin, Hyun-Dong; Chen, Rachel R; Du, Guocheng; Liu, Long; Chen, Jian

    2014-01-01

    Cyclodextrin glycosyltransferase (CGTase) is an important enzyme with multiple functions, in particular the production of cyclodextrins. It is also widely applied in baking and carbohydrate glycosylation because it participates in various types of catalytic reactions. New applications are being found with novel CGTases being isolated from various organisms. Heterologous expression is performed for the overproduction of CGTases to meet the requirements of these applications. In addition, various directed evolution techniques have been applied to modify the molecular structure of CGTase for improved performance in industrial applications. In recent years, substantial progress has been made in the heterologous expression and molecular engineering of CGTases. In this review, we systematically summarize the heterologous expression strategies used for enhancing the production of CGTases. We also outline and discuss the molecular engineering approaches used to improve the production, secretion, and properties (e.g., product and substrate specificity, catalytic efficiency, and thermal stability) of CGTase. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Optimizing heterologous protein production in the periplasm of E. coli by regulating gene expression levels

    PubMed Central

    2013-01-01

    Background In Escherichia coli many heterologous proteins are produced in the periplasm. To direct these proteins to the periplasm, they are equipped with an N-terminal signal sequence so that they can traverse the cytoplasmic membrane via the protein-conducting Sec-translocon. For poorly understood reasons, the production of heterologous secretory proteins is often toxic to the cell thereby limiting yields. To gain insight into the mechanism(s) that underlie this toxicity we produced two secretory heterologous proteins, super folder green fluorescent protein and a single-chain variable antibody fragment, in the Lemo21(DE3) strain. In this strain, the expression intensity of the gene encoding the target protein can be precisely controlled. Results Both SFGFP and the single-chain variable antibody fragment were equipped with a DsbA-derived signal sequence. Producing these proteins following different gene expression levels in Lemo21(DE3) allowed us to identify the optimal expression level for each target gene. Too high gene expression levels resulted in saturation of the Sec-translocon capacity as shown by hampered translocation of endogenous secretory proteins and a protein misfolding/aggregation problem in the cytoplasm. At the optimal gene expression levels, the negative effects of the production of the heterologous secretory proteins were minimized and yields in the periplasm were optimized. Conclusions Saturating the Sec-translocon capacity can be a major bottleneck hampering heterologous protein production in the periplasm. This bottleneck can be alleviated by harmonizing expression levels of the genes encoding the heterologous secretory proteins with the Sec-translocon capacity. Mechanistic insight into the production of proteins in the periplasm is key to optimizing yields in this compartment. PMID:23497240

  3. 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.

  4. Modulation of the Immune Response by Nematode Secreted Acetylcholinesterase Revealed by Heterologous Expression in Trypanosoma musculi

    PubMed Central

    Vaux, Rachel; Schnoeller, Corinna; Berkachy, Rita; Roberts, Luke B.; Hagen, Jana; Gounaris, Kleoniki

    2016-01-01

    Nematode parasites secrete molecules which regulate the mammalian immune system, but their genetic intractability is a major impediment to identifying and characterising the biological effects of these molecules. We describe here a novel system for heterologous expression of helminth secreted proteins in the natural parasite of mice, Trypanosoma musculi, which can be used to analyse putative immunomodulatory functions. Trypanosomes were engineered to express a secreted acetylcholinesterase from Nippostrongylus brasiliensis. Infection of mice with transgenic parasites expressing acetylcholinesterase resulted in truncated infection, with trypanosomes cleared early from the circulation. Analysis of cellular phenotypes indicated that exposure to acetylcholinesterase in vivo promoted classical activation of macrophages (M1), with elevated production of nitric oxide and lowered arginase activity. This most likely occurred due to the altered cytokine environment, as splenocytes from mice infected with T. musculi expressing acetylcholinesterase showed enhanced production of IFNγ and TNFα, with diminished IL-4, IL-13 and IL-5. These results suggest that one of the functions of nematode secreted acetylcholinesterase may be to alter the cytokine environment in order to inhibit development of M2 macrophages which are deleterious to parasite survival. Transgenic T. musculi represents a valuable new vehicle to screen for novel immunoregulatory proteins by extracellular delivery in vivo to the murine host. PMID:27802350

  5. New tools for reconstruction and heterologous expression of natural product biosynthetic gene clusters

    PubMed Central

    Luo, Yunzi; Enghiad, Behnam; Zhao, Huimin

    2015-01-01

    Natural product scaffolds remain a major source and inspiration for human therapeutics. However, generation of a natural product in the post-genomic era often requires reconstruction of the corresponding biosynthetic gene cluster in a heterologous host. In the burgeoning fields of synthetic biology and metabolic engineering, a significant amount of efforts has been devoted to develop DNA assembly techniques with higher efficiency, fidelity, and modularity, and heterologous expression systems with higher productivity and yield. Here we describe recent advances in DNA assembly and host engineering and highlight their applications in natural product discovery and engineering. PMID:26647833

  6. New tools for reconstruction and heterologous expression of natural product biosynthetic gene clusters.

    PubMed

    Luo, Yunzi; Enghiad, Behnam; Zhao, Huimin

    2016-02-01

    Natural product scaffolds remain a major source and inspiration for human therapeutics. However, generation of a natural product in the post-genomic era often requires reconstruction of the corresponding biosynthetic gene cluster in a heterologous host. In the burgeoning fields of synthetic biology and metabolic engineering, a significant amount of efforts has been devoted to develop DNA assembly techniques with higher efficiency, fidelity, and modularity, and heterologous expression systems with higher productivity and yield. Here we describe recent advances in DNA assembly and host engineering and highlight their applications in natural product discovery and engineering.

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

    PubMed Central

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

    2011-01-01

    Putative α-l-arabinofuranosidases of Oenococcus oeni and Lactobacillus brevis were heterologously expressed and characterized. We report the basic functional properties of the recombinant enzymes in comparison to those of a commercial family 51 arabinosidase of Aspergillus niger. PMID:21169445

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

    PubMed Central

    Phulara, Suresh Chandra; Chaturvedi, Preeti

    2016-01-01

    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

  9. The yeast Kluyveromyces lactis as an efficient host for heterologous gene expression.

    PubMed

    Swinkels, B W; van Ooyen, A J; Bonekamp, F J

    Several different yeast species have been developed into systems for efficient heterologous gene expression. In this paper we review foreign gene expression in the dairy yeast Kluyveromyces lactis. This yeast presents several advantageous properties in comparison to other yeast species. These include its impressive secretory capacities, its excellent fermentation characteristics on large scale, its food grade status and the availability of both episomal and integrative expression vectors. Moreover, in contrast to the methylotrophic yeasts that are frequently used for the expression of foreign genes, K. lactis does not require explosion-proof fermentation equipment. Here, we present an overview of the available tools for heterologous gene expression in K. lactis (available promoters, vector systems, etc). Also, the production of prochymosin, human serum albumin and pancreatic phospholipase by K. lactis is discussed in more detail, and used to rate the achievements of K. lactis with respect to other micro-organisms in which these proteins have been produced.

  10. 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.

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

    PubMed

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

    2017-01-01

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

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

    PubMed

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

    2016-01-04

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

  13. 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.

  14. Chlorella species as hosts for genetic engineering and expression of heterologous proteins: Progress, challenge and perspective.

    PubMed

    Yang, Bo; Liu, Jin; Jiang, Yue; Chen, Feng

    2016-10-01

    The species of Chlorella represent a highly specialized group of green microalgae that can produce high levels of protein. Many Chlorella strains can grow rapidly and achieve high cell density under controlled conditions and are thus considered to be promising protein sources. Many advances in the genetic engineering of Chlorella have occurred in recent years, with significant developments in successful expression of heterologous proteins for various applications. Nevertheless, a lot of obstacles remain to be addressed, and a sophisticated and stable Chlorella expression system has yet to emerge. This review provides a brief summary of current knowledge on Chlorella and an overview of recent progress in the genetic engineering of Chlorella, and highlights the advances in the development of a genetic toolbox of Chlorella for heterologous protein expression. Research directions to further exploit the Chlorella expression system with respect to both challenges and perspectives are also discussed. This paper serves as a comprehensive literature review for the Chlorella community and will provide valuable insights into future exploration of Chlorella as a promising host for heterologous protein expression.

  15. A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro

    PubMed Central

    Purushotham, Pallinti; Cho, Sung Hyun; Díaz-Moreno, Sara M.; Kumar, Manish; Nixon, B. Tracy; Bulone, Vincent; Zimmer, Jochen

    2016-01-01

    Plant cell walls are a composite material of polysaccharides, proteins, and other noncarbohydrate polymers. In the majority of plant tissues, the most abundant polysaccharide is cellulose, a linear polymer of glucose molecules. As the load-bearing component of the cell wall, individual cellulose chains are frequently bundled into micro and macrofibrils and are wrapped around the cell. Cellulose is synthesized by membrane-integrated and processive glycosyltransferases that polymerize UDP-activated glucose and secrete the nascent polymer through a channel formed by their own transmembrane regions. Plants express several different cellulose synthase isoforms during primary and secondary cell wall formation; however, so far, none has been functionally reconstituted in vitro for detailed biochemical analyses. Here we report the heterologous expression, purification, and functional reconstitution of Populus tremula x tremuloides CesA8 (PttCesA8), implicated in secondary cell wall formation. The recombinant enzyme polymerizes UDP-activated glucose to cellulose, as determined by enzyme degradation, permethylation glycosyl linkage analysis, electron microscopy, and mutagenesis studies. Catalytic activity is dependent on the presence of a lipid bilayer environment and divalent manganese cations. Further, electron microscopy analyses reveal that PttCesA8 produces cellulose fibers several micrometers long that occasionally are capped by globular particles, likely representing PttCesA8 complexes. Deletion of the enzyme’s N-terminal RING-finger domain almost completely abolishes fiber formation but not cellulose biosynthetic activity. Our results demonstrate that reconstituted PttCesA8 is not only sufficient for cellulose biosynthesis in vitro but also suffices to bundle individual glucan chains into cellulose microfibrils. PMID:27647898

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

    DOE PAGES

    Wang, Wei; Wei, Hui; Alahuhta, Markus; ...

    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

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

    PubMed

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

    2016-01-01

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

  18. Streptomyces and Saccharopolyspora hosts for heterologous expression of secondary metabolite gene clusters.

    PubMed

    Baltz, Richard H

    2010-08-01

    Natural products discovery from actinomycetes has been on the decline in recent years, and has suffered from a lack of innovative ways to discover new secondary metabolites within a background of the thousands of known compounds. Recent advances in whole genome sequencing have revealed that actinomycetes with large genomes encode multiple secondary metabolite pathways, most of which remain cryptic. One approach to address the expression of cryptic pathways is to first identify novel pathways by bioinformatics, then clone and express them in well-characterized hosts with known secondary metabolomes. This process should eliminate the tedious dereplication process that has hampered natural products discovery. Several laboratory and industrial production strains have been used for heterologous production of secondary metabolite pathways. This review discusses the results of these studies, and the pros and cons of using various Streptomyces and one Saccharopolyspora strain for heterologous expression. This information should provide an experimental basis to help researchers choose hosts for current application and future development to express heterologous secondary metabolite pathways in yields sufficient for rapid scale-up, biological testing, and commercial production.

  19. A protocol for heterologous expression and functional assay for mouse pheromone receptors.

    PubMed

    Dey, Sandeepa; Zhan, Senmiao; Matsunami, Hiroaki

    2013-01-01

    Innate social behaviors like intermale aggression, fear, and mating rituals are important for survival and propagation of a species. In mice, these behaviors have been implicated to be mediated by peptide pheromones that are sensed by a class of G protein-coupled receptors, vomeronasal receptor type 2 (V2Rs), expressed in the pheromone-detecting vomeronasal organ (VNO) (Chamero et al., Nature 450:899-902, 2007; Haga et al., Nature 466:118-122, 2010; Kimoto et al., Curr Biol 17:1879-1884, 2007; Leinders-Zufall et al., Nat Neurosci 12:1551-1558, 2009; Papes et al., Cell 141:692-703, 2010). Matching V2Rs with their cognate ligands is required to understand what receptors the biologically relevant pheromones are acting on. However, this goal has been greatly limited by the unavailability of appropriate heterologous tools commonly used to carry out receptor deorphanization, due to the fact that this family of receptors fails to traffic to the surface of heterologous cells. We have demonstrated that calreticulin, a housekeeping chaperone commonly expressed in most eukaryotic cells, is sparsely expressed in the vomeronasal sensory neurons (VSNs). Stable knock down of calreticulin in a HEK293T derived cell line (R24 cells) allows us to functionally express V2Rs on the surface of heterologous cells. In this chapter we describe protocols for maintenance and expansion of the R24 cell line and functional assays for V2Rs using these cells.

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

    PubMed

    Mardones, Wladimir; Callegari, Eduardo; Eyzaguirre, Jaime

    2015-12-01

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

  1. 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

  2. The expression of canine cardiac phospholamban in heterologous systems.

    PubMed

    Cook, E A; Huggins, J P; Sathe, G; England, P J; Piggott, J R

    1989-12-01

    A synthetic phospholamban gene has been cloned and expressed in Escherichia coli, producing both native phospholamban and a fusion protein with 81 amino acids of the influenza virus NS1 protein. Both the native phospholamban and fusion proteins produced extensive cell lysis upon induction of gene expression, but only the native protein underwent spontaneous pentamer formation in E. coli. Translation in vitro of mRNA produced by transcription in vitro of phospholamban cDNA was used to demonstrate the spontaneous aggregation of phospholamban to form pentamers in this system also, both in the presence and absence of exogenous microsomes from canine pancreas or heart. Phospholamban produced by translation in vitro was apparently susceptible to proteolysis by enzymes present in the particulate fractions in these experiments.

  3. Heterologous expression of pikromycin biosynthetic gene cluster using Streptomyces artificial chromosome system.

    PubMed

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

    2017-05-31

    Heterologous expression of biosynthetic gene clusters of natural microbial products has become an essential strategy for titer improvement and pathway engineering of various potentially-valuable natural products. A Streptomyces artificial chromosomal conjugation vector, pSBAC, was previously successfully applied for precise cloning and tandem integration of a large polyketide tautomycetin (TMC) biosynthetic gene cluster (Nah et al. in Microb Cell Fact 14(1):1, 2015), implying that this strategy could be employed to develop a custom overexpression scheme of natural product pathway clusters present in actinomycetes. To validate the pSBAC system as a generally-applicable heterologous overexpression system for a large-sized polyketide biosynthetic gene cluster in Streptomyces, another model polyketide compound, the pikromycin biosynthetic gene cluster, was preciously cloned and heterologously expressed using the pSBAC system. A unique HindIII restriction site was precisely inserted at one of the border regions of the pikromycin biosynthetic gene cluster within the chromosome of Streptomyces venezuelae, followed by site-specific recombination of pSBAC into the flanking region of the pikromycin gene cluster. Unlike the previous cloning process, one HindIII site integration step was skipped through pSBAC modification. pPik001, a pSBAC containing the pikromycin biosynthetic gene cluster, was directly introduced into two heterologous hosts, Streptomyces lividans and Streptomyces coelicolor, resulting in the production of 10-deoxymethynolide, a major pikromycin derivative. When two entire pikromycin biosynthetic gene clusters were tandemly introduced into the S. lividans chromosome, overproduction of 10-deoxymethynolide and the presence of pikromycin, which was previously not detected, were both confirmed. Moreover, comparative qRT-PCR results confirmed that the transcription of pikromycin biosynthetic genes was significantly upregulated in S. lividans containing tandem

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

    PubMed

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

    2015-06-01

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

  5. Heterologous Expression and Purification Systems for Structural Proteomics of Mammalian Membrane Proteins

    PubMed Central

    2002-01-01

    Membrane proteins (MPs) are responsible for the interface between the exterior and the interior of the cell. These proteins are implicated in numerous diseases, such as cancer, cystic fibrosis, epilepsy, hyperinsulinism, heart failure, hypertension and Alzheimer's disease. However, studies on these disorders are hampered by a lack of structural information about the proteins involved. Structural analysis requires large quantities of pure and active proteins. The majority of medically and pharmaceutically relevant MPs are present in tissues at very low concentration, which makes heterologous expression in large-scale production-adapted cells a prerequisite for structural studies. Obtaining mammalian MP structural data depends on the development of methods that allow the production of large quantities of MPs. This review focuses on the different heterologous expression systems, and the purification strategies, used to produce large amounts of pure mammalian MPs for structural proteomics. PMID:18629259

  6. Heterologous expression and purification of membrane-bound pyrophosphatases.

    PubMed

    Kellosalo, J; Kajander, T; Palmgren, M G; Lopéz-Marqués, R L; Goldman, A

    2011-09-01

    Membrane-bound pyrophosphatases (M-PPases) are enzymes that couple the hydrolysis of inorganic pyrophosphate to pumping of protons or sodium ions. In plants and bacteria they are important for relieving stress caused by low energy levels during anoxia, drought, nutrient deficiency, cold and low light intensity. While they are completely absent in mammalians, they are key players in the survival of disease-causing protozoans making these proteins attractive pharmacological targets. In this work, we aimed at the purification of M-PPases in amounts suitable for crystallization as a first step to obtain structural information for drug design. We have tested the expression of eight integral membrane pyrophosphatases in Saccharomyces cerevisiae, six from bacterial and archaeal sources and two from protozoa. Two proteins originating from hyperthermophilic organisms were purified in dimeric and monodisperse active states. To generate M-PPases with an increased hydrophilic surface area, which potentially should facilitate formation of crystal contacts, phage T4 lysozyme was inserted into different extramembraneous loops of one of these M-PPases. Two of these fusion proteins were active and expressed at levels that would allow their purification for crystallization purposes.

  7. Heterologous expression of tylosin polyketide synthase and production of a hybrid bioactive macrolide in Streptomyces venezuelae.

    PubMed

    Jung, Won Seok; Lee, Sang Kil; Hong, Jay Sung Joong; Park, Sung Ryeol; Jeong, Soon Jeong; Han, Ah Reum; Sohng, Jae Kyung; Kim, Byung Gee; Choi, Cha Yong; Sherman, David H; Yoon, Yeo Joon

    2006-10-01

    Tylosin polyketide synthase (Tyl PKS) was heterologously expressed in an engineered strain of Streptomyces venezuelae bearing a deletion of pikromycin PKS gene cluster using two compatible low-copy plasmids, each under the control of a pikAI promoter. The mutant strain produced 0.5 mg/l of the 16-membered ring macrolactone, tylactone, after a 4-day culture, which is a considerably reduced culture period to reach the maximum production level compared to other Streptomyces hosts. To improve the production level of tylactone, several precursors for ethylmalonyl-CoA were fed to the growing medium, leading to a 2.8-fold improvement (1.4 mg/ml); however, switching the pikAI promoter to an actI promoter had no observable effect. In addition, a small amount of desosamine-glycosylated tylactone was detected from the extract of the mutant strain, revealing that the native glycosyltransferase DesVII displayed relaxed substrate specificity in accepting the 16-membered ring macrolactone to produce the glycosylated tylactone. These results demonstrate a successful attempt for a heterologous expression of Tyl PKS in S. venezuelae and introduce S. venezuelae as a rapid heterologous expression system for the production of secondary metabolites.

  8. 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.

  9. 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.

  10. 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.

  11. Comparative Effects of Heterologous TRPV1 and TRPM8 Expression in Rat Hippocampal Neurons

    PubMed Central

    Crawford, Devon C.; Moulder, Krista L.; Gereau, Robert W.; Story, Gina M.; Mennerick, Steven

    2009-01-01

    Heterologous channel expression can be used to control activity in select neuronal populations, thus expanding the tools available to modern neuroscience. However, the secondary effects of exogenous channel expression are often left unexplored. We expressed two transient receptor potential (TRP) channel family members, TRPV1 and TRPM8, in cultured hippocampal neurons. We compared functional expression levels and secondary effects of channel expression and activation on neuronal survival and signaling. We found that activation of both channels with appropriate agonist caused large depolarizing currents in voltage-clamped hippocampal neurons, exceeding the amplitude responses to a calibrating 30 mM KCl stimulation. Both TRPV1 and TRPM8 currents were reduced but not eliminated by 4 hr incubation in saturating agonist concentration. In the case of TRPV1, but not TRPM8, prolonged agonist exposure caused strong calcium-dependent toxicity. In addition, TRPV1 expression depressed synaptic transmission dramatically without overt signs of toxicity, possibly due to low-level TRPV1 activation in the absence of exogenous agonist application. Despite evidence of expression at presynaptic sites, in addition to somatodendritic sites, TRPM8 expression alone exhibited no effects on synaptic transmission. Therefore, by a number of criteria, TRPM8 proved the superior choice for control over neuronal membrane potential. This study also highlights the need to explore potential secondary effects of long-term expression and activation of heterologously introduced channels. PMID:19997638

  12. Porcine purple acid phosphatase: heterologous expression, characterization, and proteolytic analysis.

    PubMed

    Naseri, Joseph Itor; Truong, Ngoc Thanh; Hörentrup, Jens; Kuballa, Petric; Vogel, Andreas; Rompel, Annette; Spener, Friedrich; Krebs, Bernt

    2004-12-01

    Uteroferrin is an iron-binding glycoprotein, which is abundantly synthesized in porcine uterine glandular endometrium and believed to be involved in maternal/fetal iron transport. In the present study, uteroferrin has been cloned and functionally expressed using baculovirus-infected insect host cells Spodoptera frugiperda. The work also addresses the possible role of proteolytic cleavage to facilitate the release of uteroferrin-bound iron. The enzyme secreted in culture medium exhibits a molecular mass and catalytic properties similar to native porcine uteroferrin. The specific activity was estimated at 233 U/mg using p-nitrophenyl phosphate as substrate. Partial cleavage of the enzyme with trypsin resulted in a 1.7-fold enhancement in specific activity and a two-subunit polypeptide as observed in preparations of most mammalian purple acid phosphatases. Digestion with the aspartic protease pepsin resulted in a 2.5-fold enzyme inactivation correlated with the appearance of low molecular weight polypeptide fragments and the release of enzyme-bound iron.

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

    PubMed Central

    Gomez‐Escribano, Juan Pablo; Bibb, Mervyn J.

    2011-01-01

    Summary 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. PMID:21342466

  14. Heterologous Expression of Glycosyl Hydrolases in planta: A New Departure for Biofuels

    SciTech Connect

    Taylor II, L. E.; Dai, Z.; Decker, S. R.; Brunecky, R.; Adney, W. S.; Ding, S. Y.; Himmel M. E.

    2008-01-01

    The concept of expressing non-plant glycosyl hydrolase genes in plant tissue is nearly two decades old, yet relatively little work in this field has been reported. However, resurgent interest in technologies aimed at enabling processes that convert biomass to sugars and fuels has turned attention toward this intuitive solution. There are several challenges facing researchers in this field, including the development of better and more specifically targeted delivery systems for hydrolytic genes, the successful folding and post-translational modification of heterologous proteins and the development of cost-effective process strategies utilizing these transformed plants. The integration of these concepts, from the improvement of biomass production and conversion characteristics to the heterologous production of glycosyl hydrolases in a high yielding bioenergy crop, holds considerable promise for improving the lignocellulosic conversion of biomass to ethanol and subsequently to fuels.

  15. Heterologous Expression of Glycosyl Hydrolases in planta: A New Departure for Biofuels

    SciTech Connect

    Taylor, Larry E.; Dai, Ziyu; Decker, Stephen R.; Brunecky, Roman; Adney, William S.; Ding, Shi-You; Himmel, Michael E.

    2008-08-01

    The concept of expressing non-plant glycosyl hydrolase genes in plant tissue is less than two decades old, and yet little work in this field has been reported. However, the resurgent interest in technologies aimed at enabling biomass to sugars/fuels processes have again pointed to this intuitive solution. Research challenges in this regard include developing better and more specifically targeted delivery systems for hydrolytic genes, achieving successful folding and post-translational modification of heterologous proteins, and developing cost effective process strategies utilizing these transformed plants. Integration of these concepts, from the improvement of biomass production and conversion characteristics to heterologous production of glycosyl hydrolases in a high yielding bioenergy crop, holds considerable promise for improving the lignocellulosic biomass to ethanol/fuels process.

  16. [The heterologous expression and purification of membrane protein from Mycobacterium tuberculosis].

    PubMed

    Liao, Dan; Xie, Jian-Ping; Wang, Hong-Hai

    2007-10-01

    Membrane proteins fulfill a wide range of central functions in the cell, but their structure determination remains one of the great challenges in structural biology. The heterologous overexpression is a demanding task. Here, we provide an overview of recent advance to heterologous expression and purification of membrane protein from Mycobacterium tuberculosis, whose membrane proteins represent the majority of the new potential drug targets in this bacillus, which is ranked as the number1 cause of infectious disease mortality in the world. A detailed structural and functional understanding of the membranes protein of Mycobacterium tuberculosis will be critical both for an understanding of the biology of infection and for the rational development of novel therapeutics. The procedures for functional expression followed by purification of membranes protein are reviewed here together with nonfunctional expression in inclusion bodies and subsequent refolding to produce functional proteins. The new expression systems, new approaches to soluble expression of recombinant proteins, new methods for membrane protein folding in vitro and new purification technology will provide a basis for choosing the best expression and purification protocol for a given membrane protein. The goal of this review is to aid researchers in the choice of a suitable expression system for their favourite proteins and make overproduction of functional membrane proteins becomes easier.

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

    PubMed Central

    Laxa, Miriam

    2017-01-01

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

  18. Engineering low-temperature expression systems for heterologous production of cold-adapted enzymes.

    PubMed

    Bjerga, Gro Elin Kjæreng; Lale, Rahmi; Williamson, Adele Kim

    2016-01-01

    Production of psychrophilic enzymes in the commonly used mesophilic expression systems is hampered by low intrinsic stability of the recombinant enzymes at the optimal host growth temperatures. Unless strategies for low-temperature expression are advanced, research on psychrophilic enzymes may end up being biased toward those that can be stably produced in commonly used mesophilic host systems. Two main strategies are currently being explored for the development of low-temperature expression in bacterial hosts: (i) low-temperature adaption of existing mesophilic expression systems, and (ii) development of new psychrophilic hosts. These developments include genetic engineering of the expression cassettes to optimize the promoter/operator systems that regulate heterologous expression. In this addendum we present our efforts in the development of such low-temperature expression systems, and speculate about future advancements in the field and potential applications.

  19. Engineering low-temperature expression systems for heterologous production of cold-adapted enzymes

    PubMed Central

    Bjerga, Gro Elin Kjæreng; Lale, Rahmi; Williamson, Adele Kim

    2016-01-01

    ABSTRACT Production of psychrophilic enzymes in the commonly used mesophilic expression systems is hampered by low intrinsic stability of the recombinant enzymes at the optimal host growth temperatures. Unless strategies for low-temperature expression are advanced, research on psychrophilic enzymes may end up being biased toward those that can be stably produced in commonly used mesophilic host systems. Two main strategies are currently being explored for the development of low-temperature expression in bacterial hosts: (i) low-temperature adaption of existing mesophilic expression systems, and (ii) development of new psychrophilic hosts. These developments include genetic engineering of the expression cassettes to optimize the promoter/operator systems that regulate heterologous expression. In this addendum we present our efforts in the development of such low-temperature expression systems, and speculate about future advancements in the field and potential applications. PMID:26710170

  20. 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.

  1. Genome-minimized Streptomyces host for the heterologous expression of secondary metabolism

    PubMed Central

    Komatsu, Mamoru; Uchiyama, Takuma; Ōmura, Satoshi; Cane, David E.; Ikeda, Haruo

    2010-01-01

    To construct a versatile model host for heterologous expression of genes encoding secondary metabolite biosynthesis, the genome of the industrial microorganism Streptomyces avermitilis was systematically deleted to remove nonessential genes. A region of more than 1.4 Mb was deleted stepwise from the 9.02-Mb S. avermitilis linear chromosome to generate a series of defined deletion mutants, corresponding to 83.12–81.46% of the wild-type chromosome, that did not produce any of the major endogenous secondary metabolites found in the parent strain. The suitability of the mutants as hosts for efficient production of foreign metabolites was shown by heterologous expression of three different exogenous biosynthetic gene clusters encoding the biosynthesis of streptomycin (from S. griseus Institute for Fermentation, Osaka [IFO] 13350), cephamycin C (from S. clavuligerus American type culture collection (ATCC) 27064), and pladienolide (from S. platensis Mer-11107). Both streptomycin and cephamycin C were efficiently produced by individual transformants at levels higher than those of the native-producing species. Although pladienolide was not produced by a deletion mutant transformed with the corresponding intact biosynthetic gene cluster, production of the macrolide was enabled by introduction of an extra copy of the regulatory gene pldR expressed under control of an alternative promoter. Another mutant optimized for terpenoid production efficiently produced the plant terpenoid intermediate, amorpha-4,11-diene, by introduction of a synthetic gene optimized for Streptomyces codon usage. These findings highlight the strength and flexibility of engineered S. avermitilis as a model host for heterologous gene expression, resulting in the production of exogenous natural and unnatural metabolites. PMID:20133795

  2. A series of promoters for constitutive expression of heterologous genes in fission yeast.

    PubMed

    Matsuyama, Akihisa; Shirai, Atsuko; Yoshida, Minoru

    2008-05-01

    Inducible/repressible promoters are useful for the maintenance of toxic genes or timely expression. For ectopic expression of cloned genes in the fission yeast Schizosaccharomyces pombe, the thiamine-regulatable nmt1 promoter has been widely used, since the transcriptional activity of this promoter can be controlled by thiamine. However, this property sometimes limits a certain type of research, since the expression inevitably requires cells to be cultivated under the conditions that induce promoter activation. To allow constitutive expression of heterologous genes, we cloned three promoters of cam1+, tif51+ and ef1a-c+. Construction of a series of vectors comprising these promoters and their introduction into the fission yeast cells demonstrated that the activity was different among these promoters but was not affected by cultured media commonly used in fission yeast. Therefore, a promoter with appropriate strength would be selectable from these promoters, depending on the genes to be expressed.

  3. Pigeons: A Novel GUI Software for Analysing and Parsing High Density Heterologous Oligonucleotide Microarray Probe Level Data

    PubMed Central

    Lai, Hung-Ming; May, Sean T.; Mayes, Sean

    2014-01-01

    Genomic DNA-based probe selection by using high density oligonucleotide arrays has recently been applied to heterologous species (Xspecies). With the advent of this new approach, researchers are able to study the genome and transcriptome of a non-model or an underutilised crop species through current state-of-the-art microarray platforms. However, a software package with a graphical user interface (GUI) to analyse and parse the oligonucleotide probe pair level data is still lacking when an experiment is designed on the basis of this cross species approach. A novel computer program called Pigeons has been developed for customised array data analysis to allow the user to import and analyse Affymetrix GeneChip® probe level data through XSpecies. One can determine empirical boundaries for removing poor probes based on genomic hybridisation of the test species to the Xspecies array, followed by making a species-specific Chip Description File (CDF) file for transcriptomics in the heterologous species, or Pigeons can be used to examine an experimental design to identify potential Single-Feature Polymorphisms (SFPs) at the DNA or RNA level. Pigeons is also focused around visualization and interactive analysis of the datasets. The software with its manual (the current release number version 1.2.1) is freely available at the website of the Nottingham Arabidopsis Stock Centre (NASC). PMID:27605027

  4. Heterologous Expression and Characterization of a Thermostable Exo-β-D-Glucosaminidase from Aspergillus oryzae.

    PubMed

    Wu, Dingxin; Wang, Linchun; Li, Yuwei; Zhao, Shumiao; Peng, Nan; Liang, Yunxiang

    2016-02-01

    An exo-β-D-glucosaminidase (AorCsxA) from Aspergillus oryzae FL402 was heterologously expressed and purified. The deduced amino acid sequence indicated that AorCsxA belonged to glycoside hydrolase family 2. AorCsxA digested colloid chitosan into glucosamine but not into chitosan oligosaccharides, demonstrating exo-β-D-glucosaminidase (CsxA) activity. AorCsxA exhibited optimal activity at pH 5.5 and 50°C; however, the enzyme expressed in Pichia pastoris (PpAorCsxA) showed much stronger thermostability at 50°C than that expressed in Escherichia coli (EcAorCsxA), which may be related to glycosylation. AorCsxA activity was inhibited by EDTA and most of the tested metal ions. A single amino acid mutation (F769W) in AorCsxA significantly enhanced the specific activity and hydrolysis velocity as revealed by comparison of Vmax and kcat values with those of the wild-type enzyme. The three-dimensional structure suggested the tightened pocket at the active site of F769W enabled efficient substrate binding. The AorCsxA gene was heterologously expressed in P. pastoris, and one transformant was found to produce 222 U/ml activity during the high-cell-density fermentation. This AorCsxA-overexpressing P. pastoris strain is feasible for large-scale production of AorCsxA.

  5. Cloning and heterologous expression of chlorophyll a synthase in Rhodobacter sphaeroides.

    PubMed

    Ipekoğlu, Emre M; Göçmen, Koray; Öz, Mehmet T; Gürgan, Muazzez; Yücel, Meral

    2017-03-01

    Rhodobacter sphaeroides is a purple non-sulfur bacterium which photoheterotrophically produces hydrogen from organic acids under anaerobic conditions. A gene coding for putative chlorophyll a synthase (chlG) from cyanobacterium Prochlorococcus marinus was amplified by nested polymerase chain reaction and cloned into an inducible-expression plasmid which was subsequently transferred to R. sphaeroides for heterologous expression. Induced expression of chlG in R. sphaeroides led to changes in light absorption spectrum within 400-700 nm. The hydrogen production capacity of the mutant strain was evaluated on hydrogen production medium with 15 mM malate and 2 mM glutamate. Hydrogen yield and productivity were increased by 13.6 and 22.6%, respectively, compared to the wild type strain. The results demonstrated the feasibility of genetic engineering to combine chlorophyll and bacteriochlorophyll biosynthetic pathways which utilize common intermediates. Heterologous expression of key enzymes from biosynthetic pathways of various pigments is proposed here as a general strategy to improve absorption spectra and yield of photosynthesis and hydrogen gas production in bacteria. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    PubMed

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

    2015-01-01

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

  7. 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

  8. Heterologous expression of the adenosine A1 receptor in transgenic mouse retina.

    PubMed

    Li, Ning; Salom, David; Zhang, Li; Harris, Tim; Ballesteros, Juan A; Golczak, Marcin; Jastrzebska, Beata; Palczewski, Krzysztof; Kurahara, Carole; Juan, Todd; Jordan, Steven; Salon, John A

    2007-07-17

    Traditional cell-based systems used to express integral membrane receptors have yet to produce protein samples of sufficient quality for structural study. Herein we report an in vivo method that harnesses the photoreceptor system of the retina to heterologously express G protein-coupled receptors in a biochemically homogeneous and pharmacologically functional conformation. As an example we show that the adenosine A1 receptor, when placed under the influence of the mouse opsin promoter and rhodopsin rod outer segment targeting sequence, localized to the photoreceptor cells of transgenic retina. The resulting receptor protein was uniformly glycosylated and pharmacologically well behaved. By comparison, we demonstrated in a control experiment that opsin, when expressed in the liver, had a complex pattern of glycosylation. Upon solubilization, the retinal adenosine A1 receptor retained binding characteristics similar to its starting material. This expression method may prove generally useful for generating high-quality G protein-coupled receptors for structural studies.

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

    PubMed Central

    Round, Ekaterina; Shevchenko, Vitaly; Gushchin, Ivan; Polovinkin, Vitaly; Borshchevskiy, Valentin; Gordeliy, Valentin

    2015-01-01

    Heterologous overexpression of functional membrane proteins is a major bottleneck of structural biology. Bacteriorhodopsin from Halobium salinarum (bR) is a striking example of the difficulties in membrane protein overexpression. We suggest a general approach with a finite number of steps which allows one to localize the underlying problem of poor expression of a membrane protein using bR as an example. Our approach is based on constructing chimeric proteins comprising parts of a protein of interest and complementary parts of a homologous protein demonstrating advantageous expression. This complementary protein approach allowed us to increase bR expression by two orders of magnitude through the introduction of two silent mutations into bR coding DNA. For the first time the high quality crystals of bR expressed in E. Coli were obtained using the produced protein. The crystals obtained with in meso nanovolume crystallization diffracted to 1.67 Å. PMID:26046789

  10. Expression of heterologous proteins flanked by NS3-4A cleavage sites within the hepatitis C virus polyprotein.

    PubMed

    Horwitz, Joshua A; Dorner, Marcus; Friling, Tamar; Donovan, Bridget M; Vogt, Alexander; Loureiro, Joana; Oh, Thomas; Rice, Charles M; Ploss, Alexander

    2013-04-25

    Hepatitis C virus (HCV) contributes substantially to human morbidity and mortality world-wide. The development of HCV genomes expressing heterologous proteins has enhanced the ability to study viral infection, but existing systems have drawbacks. Recombinant viruses often require adaptive mutations to compensate for reduced viral titers, or rely on an artificial genomic organization that uncouples viral protein expression from recombinant gene expression. Here, we sought to exploit the viral polyprotein processing machinery to express heterologous proteins within the context of the HCV polyprotein. We show that HCV genotypes 2a and 1b permit insertion of reporter proteins between NS5A and NS5B with minimal impact on viral fitness. Using this strategy we constructed reporter genomes exhibiting a wide dynamic range, simplifying analysis of HCV infection in primary hepatocytes. Expression of heterologous proteins within the HCV genome offers new opportunities to analyze HCV infection in experimental systems without perturbing functions of individual viral proteins.

  11. Expression of heterologous proteins flanked by NS3-4A cleavage sites within the hepatitis C virus polyprotein

    PubMed Central

    Horwitz, Joshua A.; Dorner, Marcus; Friling, Tamar; Donovan, Bridget M.; Vogt, Alexander; Loureiro, Joana; Oh, Thomas; Rice, Charles M.; Ploss, Alexander

    2013-01-01

    Hepatitis C virus (HCV) contributes substantially to human morbidity and mortality world-wide. The development of HCV genomes expressing heterologous proteins has enhanced the ability to study viral infection, but existing systems have drawbacks. Recombinant viruses often require adaptive mutations to compensate for reduced viral titers, or rely on an artificial genomic organization that uncouples viral protein expression from recombinant gene expression. Here, we sought to exploit the viral polyprotein processing machinery to express heterologous proteins within the context of the HCV polyprotein. We show that HCV genotypes 2a and 1b permit insertion of reporter proteins between NS5A and NS5B with minimal impact on viral fitness. Using this strategy we constructed reporter genomes exhibiting a wide dynamic range, simplifying analysis of HCV infection in primary hepatocytes. Expression of heterologous proteins within the HCV genome offers new opportunities to analyze HCV infection in experimental systems without perturbing functions of individual viral proteins. PMID:23485372

  12. Functional characterization of a sex pheromone receptor in the pest moth Spodoptera littoralis by heterologous expression in Drosophila.

    PubMed

    Montagné, Nicolas; Chertemps, Thomas; Brigaud, Isabelle; François, Adrien; François, Marie-Christine; de Fouchier, Arthur; Lucas, Philippe; Larsson, Mattias C; Jacquin-Joly, Emmanuelle

    2012-09-01

    Moth sex pheromone communication is recognised as a long-standing model for insect olfaction studies, and a widespread knowledge has been accumulated on this subject thanks to numerous chemical, electrophysiological and behavioural studies. A key step has been the identification of candidate sex pheromone receptors, opening new routes to understanding the specificity and sensitivity of this communication system, but only few of these receptors have as yet been functionally characterised. In this context, we aim at unravelling the molecular bases of pheromone reception in the noctuid moth Spodoptera littoralis. Taking advantage of a collection of antennal-expressed sequence tags, we previously identified three fragments of candidate pheromone receptors in this species. Here, we report full-length cloning of one of these receptors, named SlitOR6. Both sequence and expression pattern analyses were consistent with its annotation as a pheromone receptor, which we further confirmed by functional characterization. Using Drosophila antennae as a heterologous expression system, we identified a single component of the pheromone blend of S. littoralis, (Z,E)-9,12-tetradecadienyl acetate, as the ligand of SlitOR6. Two strategies were employed: (i) expressing SlitOR6 in the majority of Drosophila olfactory neurons, in addition to endogenous receptors, and monitoring the responses to pheromone stimuli by electroantennography; (ii) replacing the Drosophila pheromone receptor OR67d with SlitOR6 and monitoring the response by single sensillum recordings. Results were fully congruent and responses to (Z,E)-9,12-tetradecadienyl acetate were highly specific in both heterologous systems. This approach appears to be efficient and reliable for studying moth pheromone receptors in an in vivo context.

  13. Effect of heterologous xylose transporter expression in Candida tropicalis on xylitol production rate.

    PubMed

    Jeon, Woo Young; Shim, Woo Yong; Lee, Sung Hyeon; Choi, Joon Ho; Kim, Jung Hoe

    2013-06-01

    Xylose utilization is inhibited by glucose uptake in xylose-assimilating yeasts, including Candida tropicalis, resulting in limitation of xylose uptake during the fermentation of glucose/xylose mixtures. In this study, a heterologous xylose transporter gene (At5g17010) from Arabidopsis thaliana was selected because of its high affinity for xylose and was codon-optimized for functional expression in C. tropicalis. The codon-optimized gene was placed under the control of the GAPDH promoter and was integrated into the genome of C. tropicalis strain LXU1 which is xyl2-disrupted and NXRG (codon-optimized Neurospora crassa xylose reductase) introduced. The xylose uptake rate was increased by 37-73 % in the transporter expression-enhanced strains depending on the glucose/xylose mixture ratio. The recombinant strain LXT2 in 500-mL flask culture using glucose/xylose mixtures showed a xylose uptake rate that was 29 % higher and a xylitol volumetric productivity (1.14 g/L/h) that was 25 % higher than the corresponding rates for control strain LXU1. Membrane protein extraction and Western blot analysis confirmed the successful heterologous expression and membrane localization of the xylose transporter in C. tropicalis.

  14. 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

  15. 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.

  16. 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

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

    PubMed Central

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

    2009-01-01

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

  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. 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.

  20. Developing a xylanase XYNZG from Plectosphaerella cucumerina for baking by heterologously expressed in Kluyveromyces lactis.

    PubMed

    Zhan, Fei Xiang; Wang, Qin Hong; Jiang, Si Jing; Zhou, Yu Ling; Zhang, Gui Min; Ma, Yan He

    2014-12-16

    Xylanase can replace chemical additives to improve the volume and sensory properties of bread in the baking. Suitable baking xylanase with improved yield will promote the application of xylanase in baking industry. The xylanase XYNZG from the Plectosphaerella cucumerina has been previously characterized by heterologous expression in Pichia pastoris. However, P. pastoris is not a suitable host for xylanase to be used in the baking process since P. pastoris does not have GRAS (Generally Regarded As Safe) status and requires large methanol supplement during the fermentation in most conditions, which is not allowed to be used in the food industry. Kluyveromyces lactis, as another yeast expression host, has a GRAS status, which has been successfully used in food and feed applications. No previous work has been reported concerning the heterologous expression of xylanase gene xynZG in K. lactis with an aim for application in baking. The xylanase gene xynZG from the P. cucumerina was heterologously expressed in K. lactis. The recombinant protein XYNZG in K. lactis presented an approximately 19 kDa band on SDS-PAGE and zymograms analysis. Transformant with the highest halo on the plate containing the RBB-xylan (Remazol Brilliant Blue-xylan) was selected for the flask fermentation in different media. The results indicated that the highest activity of 115 U/ml at 72 h was obtained with the YLPU medium. The mass spectrometry analysis suggested that the hydrolytic products of xylan by XYNZG were mainly xylobiose and xylotriose. The results of baking trials indicated that the addition of XYNZG could reduce the kneading time of dough, increase the volume of bread, improve the texture, and have more positive effects on the sensory properties of bread. Xylanase XYNZG is successfully expressed in K. lactis, which exhibits the highest activity among the published reports of the xylanase expression in K. lactis. The recombinant XYNZG can be used to improve the volume and sensory

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

    PubMed Central

    Birkholtz, Lyn-Marie; Blatch, Gregory; Coetzer, Theresa L; Hoppe, Heinrich C; Human, Esmaré; Morris, Elizabeth J; Ngcete, Zoleka; Oldfield, Lyndon; Roth, Robyn; Shonhai, Addmore; Stephens, Linda; Louw, Abraham I

    2008-01-01

    Malaria remains the world's most devastating tropical infectious disease with as many as 40% of the world population living in risk areas. The widespread resistance of Plasmodium parasites to the cost-effective chloroquine and antifolates has forced the introduction of more costly drug combinations, such as Coartem®. In the absence of a vaccine in the foreseeable future, one strategy to address the growing malaria problem is to identify and characterize new and durable antimalarial drug targets, the majority of which are parasite proteins. Biochemical and structure-activity analysis of these proteins is ultimately essential in the characterization of such targets but requires large amounts of functional protein. Even though heterologous protein production has now become a relatively routine endeavour for most proteins of diverse origins, the functional expression of soluble plasmodial proteins is highly problematic and slows the progress of antimalarial drug target discovery. Here the status quo of heterologous production of plasmodial proteins is presented, constraints are highlighted and alternative strategies and hosts for functional expression and annotation of plasmodial proteins are reviewed. PMID:18828893

  2. Expression of sea anemone equistatin in potato. Effects of plant proteases on heterologous protein production.

    PubMed

    Outchkourov, Nikolay S; Rogelj, Boris; Strukelj, Borut; Jongsma, Maarten A

    2003-09-01

    Plants are increasingly used as production platforms of various heterologous proteins, but rapid protein turnover can seriously limit the steady-state expression level. Little is known about specific plant proteases involved in this process. In an attempt to obtain potato (Solanum tuberosum cv Desirée) plants resistant to Colorado potato beetle (Leptinotarsa decemlineata Say) larvae, the protease inhibitor equistatin was expressed under the control of strong, light-inducible and constitutive promoters and was targeted to the secretory pathway with and without endoplasmic reticulum retention signal. All constructs yielded similar stepwise protein degradation patterns, which considerably reduced the amount of active inhibitor in planta and resulted in insufficient levels for resistance against Colorado potato beetle larvae. Affinity purification of the degradation products and N-terminal sequencing allowed the identification of the amino acid P(1)-positions (asparagine [Asn]-13, lysine-56, Asn-82, and arginine-151) that were cleaved in planta. The proteases involved in the equistatin degradation were characterized with synthetic substrates and inhibitors. Kininogen domain 3 completely inhibited equistatin degradation in vitro. The results indicate that arginine/lysine-specific and legumain-type Asn-specific cysteine proteases seriously impede the functional accumulation of recombinant equistatin in planta. General strategies to improve the resistance to proteases of heterologous proteins in plants are proposed.

  3. Cloning, sequencing, analysis, and heterologous expression of the fredericamycin biosynthetic gene cluster from Streptomyces griseus.

    PubMed

    Wendt-Pienkowski, Evelyn; Huang, Yong; Zhang, Jian; Li, Bensheng; Jiang, Hao; Kwon, Hyungjin; Hutchinson, C Richard; Shen, Ben

    2005-11-30

    Fredericamycin (FDM) A, a pentadecaketide featuring two sets of peri-hydroxy tricyclic aromatic moieties connected through a unique chiral spiro carbon center, exhibits potent cytotoxicity and has been studied as a new type of anticancer drug lead because of its novel molecular architecture. The fdm gene cluster was localized to 33-kb DNA segment of Streptomyces griseus ATCC 49344, and its involvement in FDM A biosynthesis was proven by gene inactivation, complementation, and heterologous expression experiments. The fdm cluster consists of 28 open reading frames (ORFs), encoding a type II polyketide synthase (PKS) and tailoring enzymes as well as several regulatory and resistance proteins. The FDM PKS features a KSalpha subunit with heretofore unseen tandem cysteines at its active site, a KSbeta subunit that is distinct phylogenetically from KSbeta of hexa-, octa-, or decaketide PKSs, and a dedicated phosphopantetheinyl transferase. Further study of the FDM PKS could provide new insight into how a type II PKS controls chain length in aromatic polyketide biosynthesis. The availability of the fdm genes, in vivo characterization of the fdm cluster in S. griseus, and heterologous expression of the fdm cluster in Streptomyces albus set the stage to investigate FDM A biosynthesis and engineer the FDM biosynthetic machinery for the production of novel FDM A analogues.

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

    PubMed Central

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

    2011-01-01

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

  5. [Construction of a new bacterial artificial chromosome (BAC) vector for cloning of large DNA fragments and heterologous expression in Streptomyces].

    PubMed

    Huang, Sheng; Li, Na; Zhou, Jun; He, Jing

    2012-01-01

    Many natural product biosynthetic gene clusters are too large to be entirely cloned into one cosmid for heterologous expression. Because bacterial artificial chromosome (BAC) vectors are well known for their capacity of cloning large DNA fragments, we constructed a new BAC vector for cloning and heterologous expression of natural product biosynthesis gene clusters in Streptomyces. The chloramphenicol resistance gene on the original BAC vector pCUGIBAC1 was substituted with a streptomycin resistance gene via lambda RED-mediated PCR-targeting technique. The streptomycin resistance gene was then excised by digestion with NheI and the left gap was filled with the origin of transfer (oriT), the phiC31 integrase gene, the integrating attP site, and an apramycin resistance gene. We achieved the final BAC vector pMSBBACs. To test the newly established vector, pMSBBACs was used to build up a genomic BAC library of Streptomyces U27. The average size of inserts in the library is about 100kb. A 140 kb BAC plasmid as a representative was successfully introduced into heterologous hosts, S. lividans and S. albus, by either conjugation or protoplast transformation. It demonstrated that the BAC plasmids constructed by pMSBBACs could be integrated into chromosomes via site-specific recombination for heterologous expression. The newly constructed pMSBBACs was verified to be a good BAC vector for cloning of large DNA fragments and heterologous expression in Streptomyces.

  6. Polysaccharide-degrading thermophiles generated by heterologous gene expression in Geobacillus kaustophilus HTA426.

    PubMed

    Suzuki, Hirokazu; Yoshida, Ken-ichi; Ohshima, Toshihisa

    2013-09-01

    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.

  7. 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

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

    PubMed Central

    Law, Wenjing; Wuescher, Leah M.; Ortega, Amanda; Hapiak, Vera M.; Komuniecki, Patricia R.; Komuniecki, Richard

    2015-01-01

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

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

    PubMed

    Law, Wenjing; Wuescher, Leah M; Ortega, Amanda; Hapiak, Vera M; Komuniecki, Patricia R; Komuniecki, Richard

    2015-04-01

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

  10. Expression of Heterologous Sigma Factor Expands the Searchable Space for Biofuel Tolerance Mechanisms.

    PubMed

    Tomko, Timothy A; Dunlop, Mary J

    2017-04-03

    Microorganisms can produce hydrocarbons that can serve as replacements or additions to conventional liquid fuels for use in the transportation sector. However, a common problem in the microbial synthesis of biofuels is that these compounds often have toxic effects on the cell. In this study, we focused on mitigating the toxicity of the biojet fuel precursor pinene on Escherichia coli. We used genomic DNA from Pseudomonas putida KT2440, which has innate solvent-tolerance properties, to create transgenic libraries in an E. coli host. We exposed cells containing the library to pinene, selecting for genes that improved tolerance. Importantly, we found that expressing the sigma factor RpoD from P. putida greatly expanded the diversity of tolerance genes recovered. With low expression of rpoDP.putida, we isolated a single pinene tolerance gene; with increased expression of the sigma factor our selection experiments returned multiple distinct tolerance mechanisms, including some that have been previously documented and also new mechanisms. Interestingly, high levels of rpoDP.putida induction resulted in decreased diversity. We found that the tolerance levels provided by some genes are highly sensitive to the level of induction of rpoDP.putida, while others provide tolerance across a wide range of rpoDP.putida levels. This method for unlocking diversity in tolerance screening using heterologous sigma factor expression was applicable to both plasmid and fosmid-based transgenic libraries. These results suggest that by controlling the expression of appropriate heterologous sigma factors, we can greatly increase the searchable genomic space within transgenic libraries.

  11. Heterologous expression and extracellular secretion of cellulolytic enzymes by Zymomonas mobilis.

    PubMed

    Linger, Jeffrey G; Adney, William S; Darzins, Al

    2010-10-01

    Development of the strategy known as consolidated bioprocessing (CBP) involves the use of a single microorganism to convert pretreated lignocellulosic biomass to ethanol through the simultaneous production of saccharolytic enzymes and fermentation of the liberated monomeric sugars. In this report, the initial steps toward achieving this goal in the fermentation host Zymomonas mobilis were investigated by expressing heterologous cellulases and subsequently examining the potential to secrete these cellulases extracellularly. Numerous strains of Z. mobilis were found to possess endogenous extracellular activities against carboxymethyl cellulose, suggesting that this microorganism may harbor a favorable environment for the production of additional cellulolytic enzymes. The heterologous expression of two cellulolytic enzymes, E1 and GH12 from Acidothermus cellulolyticus, was examined. Both proteins were successfully expressed as soluble, active enzymes in Z. mobilis although to different levels. While the E1 enzyme was less abundantly expressed, the GH12 enzyme comprised as much as 4.6% of the total cell protein. Additionally, fusing predicted secretion signals native to Z. mobilis to the N termini of E1 and GH12 was found to direct the extracellular secretion of significant levels of active E1 and GH12 enzymes. The subcellular localization of the intracellular pools of cellulases revealed that a significant portion of both the E1 and GH12 secretion constructs resided in the periplasmic space. Our results strongly suggest that Z. mobilis is capable of supporting the expression and secretion of high levels of cellulases relevant to biofuel production, thereby serving as a foundation for developing Z. mobilis into a CBP platform organism.

  12. Heterologous expression of Streptococcus mutans Cnm in Lactococcus lactis promotes intracellular invasion, adhesion to human cardiac tissues and virulence.

    PubMed

    Freires, Irlan A; Avilés-Reyes, Alejandro; Kitten, Todd; Simpson-Haidaris, P J; Swartz, Michael; Knight, Peter A; Rosalen, Pedro L; Lemos, José A; Abranches, Jacqueline

    2017-01-02

    In S. mutans, the expression of the surface glycoprotein Cnm mediates binding to extracellular matrix proteins, endothelial cell invasion and virulence in the Galleria mellonella invertebrate model. To further characterize Cnm as a virulence factor, the cnm gene from S. mutans strain OMZ175 was expressed in the non-pathogenic Lactococcus lactis NZ9800 using a nisin-inducible system. Despite the absence of the machinery necessary for Cnm glycosylation, Western blot and immunofluorescence microscopy analyses demonstrated that Cnm was effectively expressed and translocated to the cell wall of L. lactis. Similar to S. mutans, expression of Cnm in L. lactis enabled robust binding to collagen and laminin, invasion of human coronary artery endothelial cells and increased virulence in G. mellonella. Using an ex vivo human heart tissue colonization model, we showed that Cnm-positive strains of either S. mutans or L. lactis outcompete their Cnm-negative counterparts for tissue colonization. Finally, Cnm expression facilitated L. lactis adhesion and colonization in a rabbit model of infective endocarditis. Collectively, our results provide unequivocal evidence that binding to extracellular matrices mediated by Cnm is an important virulence attribute of S. mutans and confirm the usefulness of the L. lactis heterologous system for further characterization of bacterial virulence factors.

  13. Generation of Arabidopsis mutants by heterologous expression of a full length cDNA library from tomato fruits

    USDA-ARS?s Scientific Manuscript database

    Heterologous expression of cDNA libraries in Arabidopsis and other plants has been used for gene identifications. To identify functions of tomato genes, we expressed a tomato full-length cDNA library in Arabidopsis thaliana and generated over 7,000 mutants. We constructed a tomato cDNA library with ...

  14. 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. © 2015 German Botanical Society and The Royal Botanical Society of the Netherlands.

  15. Heterologous expression and purification of catalytic domain of CESA1 from Arabidopsis thaliana

    DOE PAGES

    Vandavasi, Venu Gopal; O'Neill, Hugh Michael

    2016-10-20

    Here, heterologous expression of plant cellulose synthase (CESA) and its purification has remained a challenge for decades impeding detailed biophysical, biochemical and structural characterization of this key enzyme. An in-depth knowledge of structure and function of CESA proteins would enable us to better understand the hierarchical structure of the plant cell wall. Here, we report a detailed, and reproducible method of purification of catalytic domain of CESA1 from Arabidopsis thaliana that was recombinantly expressed in Escherichia coli. The method relies on a two stage purification procedure to obtain the catalytic domain in monomer and trimer forms. The biochemical and biophysicalmore » data including low resolution structures of the protein have been published. Currently the crystallization studies of this protein are underway.« less

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

    PubMed

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

    2017-12-01

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

  17. Expression of Heterologous Antigens in Commensal Neisseria spp.: Preservation of Conformational Epitopes with Vaccine Potential

    PubMed Central

    O'Dwyer, Clíona A.; Reddin, Karen; Martin, Denis; Taylor, Stephen C.; Gorringe, Andrew R.; Hudson, Michael J.; Brodeur, Bernard R.; Langford, Paul R.; Kroll, J. Simon

    2004-01-01

    Commensal neisseriae share with Neisseria meningitidis (meningococcus) a tendency towards overproduction of the bacterial outer envelope, leading to the formation and release during growth of outer membrane vesicles (OMVs). OMVs from both meningococci and commensal neisseriae have shown promise as vaccines to protect against meningococcal disease. We report here the successful expression at high levels of heterologous proteins in commensal neisseriae and the display, in its native conformation, of one meningococcal outer membrane protein vaccine candidate, NspA, in OMVs prepared from such a recombinant Neisseria flavescens strain. These NspA-containing OMVs conferred protection against otherwise lethal intraperitoneal challenge of mice with N. meningitidis serogroup B, and sera raised against them mediated opsonophagocytosis of meningococcal strains expressing this antigen. This development promises to facilitate the design of novel vaccines containing membrane protein antigens that are otherwise difficult to present in native conformation that provide cross-protective efficacy in the prevention of meningococcal disease. PMID:15501782

  18. The Zygosaccharomyces bailii antifungal virus toxin zygocin: cloning and expression in a heterologous fungal host.

    PubMed

    Weiler, Frank; Rehfeldt, Klaus; Bautz, Frank; Schmitt, Manfred J

    2002-11-01

    Zygocin, a monomeric protein toxin secreted by a virus-infected killer strain of the osmotolerant spoilage yeast Zygosaccharomyces bailii, kills a broad spectrum of human and phytopathogenic yeasts and filamentous fungi by disrupting cytoplasmic membrane function. The toxin is encoded by a double-stranded (ds)RNA killer virus (ZbV-M, for Z. bailii virus M) that stably persists within the yeast cell cytosol. In this study, the protein toxin was purified, its N-terminal amino acid sequence was determined, and a full-length cDNA copy of the 2.1 kb viral dsRNA genome was cloned and successfully expressed in a heterologous fungal system. Sequence analysis as well as zygocin expression in Schizosaccharomyces pombe indicated that the toxin is in vivo expressed as a 238-amino-acid preprotoxin precursor (pptox) consisting of a hydrophobic N-terminal secretion signal, followed by a potentially N-glycosylated pro-region and terminating in a classical Kex2p endopeptidase cleavage site that generates the N-terminus of the mature and biologically active protein toxin in a late Golgi compartment. Matrix-assisted laser desorption mass spectrometry further indicated that the secreted toxin is a monomeric 10.4 kDa protein lacking detectable post-translational modifications. Furthermore, we present additional evidence that in contrast with other viral antifungal toxins, zygocin immunity is not mediated by the toxin precursor itself and, therefore, heterologous pptox expression in a zygocin-sensitive host results in a suicidal phenotype. Final sequence comparisons emphasize the conserved pattern of functional elements present in dsRNA killer viruses that naturally infect phylogenetically distant hosts (Saccharomyces cerevisiae and Z. bailii) and reinforce models for the sequence elements that are in vivo required for viral RNA packaging and replication.

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

    NASA Astrophysics Data System (ADS)

    Yurong, Chai; Yumin, Lu; Tianyun, Wang; Weihong, Hou; Lexun, Xue

    2006-12-01

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

  20. 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.

  1. Synthesis of Novel Lipids in Saccharomyces cerevisiae by Heterologous Expression of an Unspecific Bacterial Acyltransferase

    PubMed Central

    Kalscheuer, Rainer; Luftmann, Heinrich; Steinbüchel, Alexander

    2004-01-01

    The bifunctional wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT) is the key enzyme in storage lipid accumulation in the gram-negative bacterium Acinetobacter calcoaceticus ADP1, mediating wax ester, and to a lesser extent, triacylglycerol (TAG) biosynthesis. Saccharomyces cerevisiae accumulates TAGs and steryl esters as storage lipids. Four genes encoding a DGAT (Dga1p), a phospholipid:diacylglycerol acyltransferase (Lro1p) and two acyl-coenzyme A:sterol acyltransferases (ASATs) (Are1p and Are2p) are involved in the final esterification steps in TAG and steryl ester biosynthesis in this yeast. In the quadruple mutant strain S. cerevisiae H1246, the disruption of DGA1, LRO1, ARE1, and ARE2 leads to an inability to synthesize storage lipids. Heterologous expression of WS/DGAT from A. calcoaceticus ADP1 in S. cerevisiae H1246 restored TAG but not steryl ester biosynthesis, although high levels of ASAT activity could be demonstrated for WS/DGAT expressed in Escherichia coli XL1-Blue in radiometric in vitro assays with cholesterol and ergosterol as substrates. In addition to TAG synthesis, heterologous expression of WS/DGAT in S. cerevisiae H1246 resulted also in the accumulation of fatty acid ethyl esters as well as fatty acid isoamyl esters. In vitro studies confirmed that WS/DGAT is capable of utilizing a broad range of alcohols as substrates comprising long-chain fatty alcohols like hexadecanol as well as short-chain alcohols like ethanol or isoamyl alcohol. This study demonstrated the highly unspecific acyltransferase activity of WS/DGAT from A. calcoaceticus ADP1, indicating the broad biocatalytic potential of this enzyme for biotechnological production of a large variety of lipids in vivo in prokaryotic as well as eukaryotic expression hosts. PMID:15574908

  2. Heterologous expression and characterization of the manganese-oxidizing protein from Erythrobacter sp. strain SD21.

    PubMed

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

    2014-11-01

    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. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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

    PubMed Central

    KOMATSU, MAMORU; KOMATSU, KYOKO; KOIWAI, HANAE; YAMADA, YUUKI; KOZONE, IKUKO; IZUMIKAWA, MIHO; HASHIMOTO, JUNKO; TAKAGI, MOTOKI; OMURA, SATOSHI; SHIN-YA, KAZUO; CANE, DAVID E.; IKEDA, HARUO

    2014-01-01

    An industrial microorganism Streptomyces avermitilis, which is a producer of anthelmintic macrocyclic lactones, avermectins, has been constructed as a versatile model host for heterologous expression of genes encoding secondary metabolite biosynthesis. Twenty of the entire biosynthetic gene clusters for secondary metabolites were successively cloned and introduced into a versatile model host S. avermitilis SUKA17 or 22. Almost all S. avermitilis transformants carrying the entire gene cluster produced metabolites as a result of the expression of biosynthetic gene clusters introduced. A few transformants were unable to produce metabolites but their production was restored by the expression of biosynthetic genes using an alternative promoter or the expression of a regulatory gene in the gene cluster that controls the expression of biosynthetic genes in the cluster using an alternative promoter. Production of metabolites in some transformants of the versatile host was higher than that of the original producers and cryptic biosynthetic gene clusters in the original producer were also expressed in a versatile host. PMID:23654282

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

    PubMed

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

    2013-02-01

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

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

    SciTech Connect

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

    2013-02-15

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

  6. In Vitro Characterization of a Heterologously Expressed Nonribosomal Peptide Synthetase Involved in Phosphinothricin Tripeptide Biosynthesis†

    PubMed Central

    2009-01-01

    The late stages of biosynthesis of phosphinothricin tripeptide (PTT) involve peptide formation and methylation on phosphorus. The exact timing of these transformations is not known. To provide insight into this question, we developed a heterologous expression system for PhsA, one of three NRPS proteins in PTT biosynthesis. The apparent kcat/Km value for ATP−pyrophosphate exchange activity for d,l-N-acetylphosphinothricin was 3.5 μM−1 min−1, whereas the kcat/Km,app for l-N-acetyldemethylphosphinothricin was 0.5 μM−1 min−1, suggesting the former might be the physiological substrate. Each substrate could be loaded onto the phosphopantetheine arm of the thiolation domain as observed by Fourier transform mass spectrometry (FTMS). PMID:19432442

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

    PubMed

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

    2017-01-01

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

  8. Substrates and inhibitors display different sensitivity to expression level of the dopamine transporter in heterologously expressing cells.

    PubMed

    Chen, Nianhang; Reith, Maarten E A

    2007-04-01

    The use of heterologous expression systems for studying dopamine (DA) transporter (DAT) function has provided important information corroborating and complementing in situ obtained knowledge. Preliminary experiments with human embryonic kidney cells (HEK293) heterologously expressing varying amounts of DAT suggested fluctuations in the potency of cocaine in inhibiting DA uptake and led to the present systematic assessment of the impact of the density of DAT on its function. Transiently expressing intact HEK293 cells, transfected with increasing amounts of DAT cDNA, displayed increasing levels of surface DAT, binding of the cocaine analog [(3)H]2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane ([(3)H]CFT), and uptake of [(3)H]DA, [(3)H]N-methyl-4-phenylpyridinium ([(3)H]MPP(+)), [(3)H]norepinephrine, and [(3)H]serotonin. However, the amount of DAT cDNA and the DAT expression level required to produce 50% of maximal activity was threefold higher for CFT binding than for DA uptake. Increased DAT expression was accompanied by weakened potency in inhibiting [(3)H]DA uptake for cocaine, CFT, benztropine, and its analog JHW025, GBR 12909 and mazindol; their potency in inhibiting [(3)H]CFT binding was unaffected. Inhibition of uptake by the substrates DA, m-tyramine, d-amphetamine, or MPP(+) was also unaffected. Increasing DAT in stably expressing HEK293 cells by stimulation of gene expression with sodium butyrate also decreased the uptake inhibitory potency of a number of the above blockers without affecting the interaction between substrates and DAT. The present results prompt discussion of models explaining how factors regulating DAT expression at the plasma membrane can regulate DAT function and pharmacology.

  9. Heterologous protein expression in Trichoderma reesei using the cbhII promoter.

    PubMed

    Meng, Fanju; Wei, Dongzhi; Wang, Wei

    2013-09-01

    To express homologous or heterologous proteins in fungi, a protein expression system using the promoter of cellobiohydrolase II gene (cbhII) was constructed by generating an expression vector called pWEIIF00. The obtained vector possesses the left and right borders, a hygromycin phosphotransferase B selective marker and a strong promoter and terminator of cbhII from Trichoderma reesei. It can easily undergo random recombination. The applicability of the vector was tested by red fluorescent protein gene (DsRed2) expression detection in T. reesei Rut C30. Using this system, a recombinant Cel5A variant, N342R (Qin et al., 2008), was then selected to express in Rut-C30. Compared to that of the parent strain, integration of the N342R gene resulted in 31.09% increased carboxymethyl-cellulose-degrading (CMCase) activity at pH 5.0 and 56.06% increased activity at pH 6.0. The increased CMCase activity of the recombinant strains would be beneficial for its application uses in multiple industries. The vector constructed in this study can used in fungi to produce industrial proteins.

  10. [Alkaline-adapted beta-mannanase of Bacillus pumilus: gene heterologous expression and enzyme characterization].

    PubMed

    Tang, Jiajie; Guo, Su; Wang, Wei; Wei, Wei; Wei, Dongzhi

    2015-11-04

    We expressed a novel alkaline-adapted beta-mannanase gene and characterized the enzyme for potential industrial applications. We obtained a mannanase gene (named man(B)) from Bacillus pumilus Nsic2 and expressed the gene man(B) in Escherichia coli and Bacillus subtilis. Furthermore, we characterized the enzyme. The gene man(B) had an open reading frame of 1104 bp that encoded a polypeptide of 367-amino-acid beta-mannanase (Man(B)). The protein sequence showed the highest identity with the beta-mannanase from B. pumilus CCAM080065. We expressed the gene man(B) in E. coli BL21 (DE3) with the enzyme activity of 11021.3 U/mL. Compared with other mannanases, Man(B) showed higher stability under alkaline conditions and was stable at pH6.0 -9.0. The specific activity of purified Man(B) was 4191 ± 107 U/mg. The K(m) and V(max) values of purified Man(B) were 35.7 mg/mL and 14.9 μmol/(mL x min), respectively. Meanwhile, we achieved recombinant protein secretion expression in B. subtilis WB800N. We achieved heterologous expression of the gene man(B) and characterized its enzyme. The alkaline-adapted Man(B) showed potential value in industrial applications due to its pH stability.

  11. Optimizing heterologous expression in Dictyostelium: importance of 5′ codon adaptation

    PubMed Central

    Vervoort, Elisa B.; van Ravestein, Arno; van Peij, Noël N. M. E.; Heikoop, Judith C.; van Haastert, Peter J. M.; Verheijden, Gijs F.; Linskens, Maarten H. K.

    2000-01-01

    Expression of heterologous proteins in Dictyostelium discoideum presents unique research opportunities, such as the functional analysis of complex human glycoproteins after random mutagenesis. In one study, human chorionic gonadotropin (hCG) and human follicle stimulating hormone were expressed in Dictyostelium. During the course of these experiments, we also investigated the role of codon usage and of the DNA sequence upstream of the ATG start codon. The Dictyostelium genome has a higher AT content than the human, resulting in a different codon preference. The hCG-β gene contains three clusters with infrequently used codons that were changed to codons that are preferred by Dictyostelium. The results reported here show that optimizing the first 5–17 codons of the hCG gene contributes to 4- to 5-fold increased expression levels, but that further optimization has no significant effect. These observations suggest that optimal codon usage contributes to ribosome stabilization, but does not play an important role during the elongation phase of translation. Furthermore, adapting the 5′-sequence of the hCG gene to the Dictyostelium ‘Kozak’-like sequence increased expression levels ~1.5-fold. Thus, using both codon optimization and ‘Kozak’ adaptation, a 6- to 8-fold increase in expression levels could be obtained for hCG. PMID:10773074

  12. Development of a Site-Directed Integration Plasmid for Heterologous Gene Expression in Mycoplasma gallisepticum

    PubMed Central

    Indikova, Ivana; Szostak, Michael P.

    2013-01-01

    Deciphering the molecular basis of the interactions between the parasite Mycoplasma gallisepticum and its avian hosts suffers from the lack of genetic tools available for the pathogen. In the absence of well established methods for targeted disruption of relevant M. gallisepticum genes, we started to develop suicide vectors and equipped them with a short fragment of M. gallisepticum origin or replication (oriCMG). We failed to create a disruption vector, although by adding a further short fragment of the M. gallisepticum tufB upstream region we created a “Trojan horse” plasmid. This is fully integrated into the genomic DNA of M. gallisepticum, always at the same site, oriCMG, and is able to carry and express any gene of interest in the genetic background of M. gallisepticum. Successful expression of a heterologous gene was shown with the lacZ gene of E. coli. When used for gene complementation or expression of hybrid genes in M. gallisepticum, a site-specific combined integration/expression vector constitutes an improvement on randomly integrating transposons, which might have unexpected effects on the expression of chromosomal genes. PMID:24278444

  13. 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

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

    DOE PAGES

    Javidpour, Pouya; Deutsch, Samuel; Mutalik, Vivek K.; ...

    2016-03-14

    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 studymore » 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

  15. The Role of Heterologous Chloroplast Sequence Elements in Transgene Integration and Expression1[W][OA

    PubMed Central

    Ruhlman, Tracey; Verma, Dheeraj; Samson, Nalapalli; Daniell, Henry

    2010-01-01

    Heterologous regulatory elements and flanking sequences have been used in chloroplast transformation of several crop species, but their roles and mechanisms have not yet been investigated. Nucleotide sequence identity in the photosystem II protein D1 (psbA) upstream region is 59% across all taxa; similar variation was consistent across all genes and taxa examined. Secondary structure and predicted Gibbs free energy values of the psbA 5′ untranslated region (UTR) among different families reflected this variation. Therefore, chloroplast transformation vectors were made for tobacco (Nicotiana tabacum) and lettuce (Lactuca sativa), with endogenous (Nt-Nt, Ls-Ls) or heterologous (Nt-Ls, Ls-Nt) psbA promoter, 5′ UTR and 3′ UTR, regulating expression of the anthrax protective antigen (PA) or human proinsulin (Pins) fused with the cholera toxin B-subunit (CTB). Unique lettuce flanking sequences were completely eliminated during homologous recombination in the transplastomic tobacco genomes but not unique tobacco sequences. Nt-Ls or Ls-Nt transplastomic lines showed reduction of 80% PA and 97% CTB-Pins expression when compared with endogenous psbA regulatory elements, which accumulated up to 29.6% total soluble protein PA and 72.0% total leaf protein CTB-Pins, 2-fold higher than Rubisco. Transgene transcripts were reduced by 84% in Ls-Nt-CTB-Pins and by 72% in Nt-Ls-PA lines. Transcripts containing endogenous 5′ UTR were stabilized in nonpolysomal fractions. Stromal RNA-binding proteins were preferentially associated with endogenous psbA 5′ UTR. A rapid and reproducible regeneration system was developed for lettuce commercial cultivars by optimizing plant growth regulators. These findings underscore the need for sequencing complete crop chloroplast genomes, utilization of endogenous regulatory elements and flanking sequences, as well as optimization of plant growth regulators for efficient chloroplast transformation. PMID:20130101

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

    PubMed

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

    2016-02-15

    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.

  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

    2016-08-04

    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.

  18. 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.

  19. 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.

  20. Heterologous expression of a bacterial homospermidine synthase gene in transgenic tobacco: effects on the polyamine pathway.

    PubMed

    Kaiser, Annette; Sell, Simone; Hehl, Reinhard

    2002-04-01

    Homospermidine synthase (HSS) is a branch-point enzyme that links the secondary pathway (pyrrolizidine alkaloids) to primary metabolism (polyamines). Since the diamine putrescine is a precursor of homospermidine and nicotine in tobacco, we performed heterologous expression of a bacterial homospermidine synthase gene (hss)in Nicotiana tabacum and determined the effect on free and conjugated polyamine levels. The hss gene from Rhodopseudomonas viridis was placed under the control of the cauliflower mosaic virus (CaMV) 35S promoter in Agrobacterium tumefaciens Ti-plasmid in sense and antisense orientation and both hss constructs were transformed into tobacco plants. Expression of the hss gene was verified by "Northern" and "Southern Blot" analysis. 2 transgenic sense lines were generated from 1000 calli which showed weak expression of homospermidine synthase, i.e. 50 pktal/mg protein and 45 pktal/mg protein. These transgenic sense plants showed a significantly decreased content of free spermidine while the pool of conjugated spermidine was not affected. The 2 sense plants exhibited a range of abnormal phenotypes such as dwarfness and stunted growth. Homospermidine was sporadically detectable in wild type tobacco. To our knowledge, this is the first biotechnological approach to express a prokaryotic homospermidine synthase gene in tobacco plants.

  1. 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

  2. Biologically meaningful expression profiling across species using heterologous hybridization to a cDNA microarray

    PubMed Central

    Renn, Susan CP; Aubin-Horth, Nadia; Hofmann, Hans A

    2004-01-01

    Background Unravelling the path from genotype to phenotype, as it is influenced by an organism's environment, is one of the central goals in biology. Gene expression profiling by means of microarrays has become very prominent in this endeavour, although resources exist only for relatively few model systems. As genomics has matured into a comparative research program, expression profiling now also provides a powerful tool for non-traditional model systems to elucidate the molecular basis of complex traits. Results Here we present a microarray constructed with ~4500 features, derived from a brain-specific cDNA library for the African cichlid fish Astatotilapia burtoni (Perciformes). Heterologous hybridization, targeting RNA to an array constructed for a different species, is used for eight different fish species. We quantified the concordance in gene expression profiles across these species (number of genes and fold-changes). Although most robust when target RNA is derived from closely related species (<10 MA divergence time), our results showed consistent profiles for other closely related taxa (~65 MA divergence time) and, to a lesser extent, even very distantly related species (>200 MA divergence time). Conclusion This strategy overcomes some of the restrictions imposed on model systems that are of importance for evolutionary and ecological studies, but for which only limited sequence information is available. Our work validates the use of expression profiling for functional genomics within a comparative framework and provides a foundation for the molecular and cellular analysis of complex traits in a wide range of organisms. PMID:15238158

  3. mRNA Distribution and Heterologous Expression of Orphan Cytochrome P450 20A1

    PubMed Central

    Stark, Katarina; Wu, Zhong-Liu; Bartleson, Cheryl J.; Guengerich, F. Peter

    2015-01-01

    Cytochrome P450 (P450) 20A1 is one of the so-called “orphan” P450s without assigned biological function. mRNA expression was detected in human liver and extrahepatic expression was noted in several human brain regions, including substantia nigra, hippocampus, and amygdala, using conventional polymerase chain reaction and RNA dot blot analysis. Adult human liver contained 3-fold higher overall mRNA levels than whole brain, although specific regions (i.e., hippocampus and substantia nigra) exhibited higher mRNA expression levels than liver. Orthologous full-length and truncated transcripts of P450 20A1 were transcribed and sequenced from rat liver, heart, and brain. In rat, the concentrations of full-length transcripts were 3–4 fold higher in brain and heart than liver. In situ hybridization of rat whole brain sections showed a similar mRNA expression pattern as observed for human P450 20A1, indicating expression in substantia nigra, hippocampus, and amygdala. A number of N-terminal modifications of the codon-optimized human P450 20A1 sequence were prepared and expressed in Escherichia coli, and two of the truncated derivatives showed characteristic P450 spectra (200–280 nmol P450/l). Although the recombinant enzyme system oxidized NADPH, no catalytic activity was observed with the heterologously expressed protein when a number of potential steroids and biogenic amines were surveyed as potential substrates. The function of P450 20A1 remains unknown; however, the sites of mRNA expression in human brain and the conservation among species may suggest possible neurophysiological function. PMID:18541694

  4. Heterologous expression of rice calnexin (OsCNX) confers drought tolerance in Nicotiana tabacum.

    PubMed

    Sarwat, Maryam; Naqvi, Afsar Raza

    2013-09-01

    Calnexin (CNX) is an integral membrane protein of endoplasmic reticulum (ER) and is a critical component of ER quality control machinery. It acts as a chaperone and ensures proper folding of newly synthesised glycoproteins. CNX shares a considerable homology with its luminal counterpart calreticulin (CRT). Together, they constitute CNX/CRT cycle which is imperative for proper folding of nascent proteins. CNX deficient organisms develop severe complications because of improper folding of proteins and consequently ER stress. CNX maintains calcium homeostasis by binding to the Ca(2+) which is a central node in various signaling pathways. Phosphorylation of cytoplasmic tail of CNX controls the sarco endoplasmic reticulum calcium ATPase and thus the movement of Ca(2+) in and out of its store-house, i.e. ER. Our studies on Oryza sativa CNX (OsCNX) reveal constitutive expression at various developmental stages and various tissues, thereby proving its requirement throughout the plant development. Further, its expression under various stress conditions gives an insight of the crosstalk existing between ER stress and abiotic stress signaling. This was confirmed by heterologous expression of OsCNX (OsCNX-HE) in tobacco and the OsCNX-HE lines were observed to exhibit better germination under mannitol stress and survival under dehydration stress conditions. The dehydration tolerance conferred by OsCNX appears to be ABA-dependent pathway.

  5. A novel Escherichia coli solubility enhancer protein for fusion expression of aggregation-prone heterologous proteins.

    PubMed

    Song, Jong-Am; Lee, Dae-Sung; Park, Jin-Seung; Han, Kyung-Yeon; Lee, Jeewon

    2011-07-10

    Through the proteome analysis of Escherichia coli BL21(DE3), we previously identified the stress-responsive protein, arsenate reductase (ArsC), that showed a high cytoplasmic solubility and a folding capacity even in the presence of stress-inducing reagents. In this study, we used ArsC as an N-terminal fusion partner to synthesize nine aggregation-prone proteins as water-soluble forms. As a result, solubility of the aggregation-prone proteins increased dramatically by the fusion of ArsC, due presumably to its tendency to facilitate the folding of target proteins. Also, we evaluated and confirmed the efficacy of ArsC-fusion expression in making the fusion-expressed target proteins have their own native function or structure. That is, the self-assembly function of human ferritin light chain, l-arginine-degrading function of arginine deiminase, and the correct secondary structure of human granulocyte colony stimulating factor were clearly observed through transmission electron microscope analysis, colorimetric enzyme activity assay, and circular dichroism, respectively. It is strongly suggested that ArsC can be in general an efficient fusion expression partner for the production of soluble and active heterologous proteins in E. coli. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. A new high-performance heterologous fungal expression system based on regulatory elements from the Aspergillus terreus terrein gene cluster.

    PubMed

    Gressler, Markus; Hortschansky, Peter; Geib, Elena; Brock, Matthias

    2015-01-01

    Recently, the Aspergillus terreus terrein gene cluster was identified and selected for development of a new heterologous expression system. The cluster encodes the specific transcription factor TerR that is indispensable for terrein cluster induction. To identify TerR binding sites, different recombinant versions of the TerR DNA-binding domain were analyzed for specific motif recognition. The high affinity consensus motif TCGGHHWYHCGGH was identified from genes required for terrein production and binding site mutations confirmed their essential contribution to gene expression in A. terreus. A combination of TerR with its terA target promoter was tested as recombinant expression system in the heterologous host Aspergillus niger. TerR mediated target promoter activation was directly dependent on its transcription level. Therefore, terR was expressed under control of the regulatable amylase promoter PamyB and the resulting activation of the terA target promoter was compared with activation levels obtained from direct expression of reporters from the strong gpdA control promoter. Here, the coupled system outcompeted the direct expression system. When the coupled system was used for heterologous polyketide synthase expression high metabolite levels were produced. Additionally, expression of the Aspergillus nidulans polyketide synthase gene orsA revealed lecanoric acid rather than orsellinic acid as major polyketide synthase product. Domain swapping experiments assigned this depside formation from orsellinic acid to the OrsA thioesterase domain. These experiments confirm the suitability of the expression system especially for high-level metabolite production in heterologous hosts.

  7. A new high-performance heterologous fungal expression system based on regulatory elements from the Aspergillus terreus terrein gene cluster

    PubMed Central

    Gressler, Markus; Hortschansky, Peter; Geib, Elena; Brock, Matthias

    2015-01-01

    Recently, the Aspergillus terreus terrein gene cluster was identified and selected for development of a new heterologous expression system. The cluster encodes the specific transcription factor TerR that is indispensable for terrein cluster induction. To identify TerR binding sites, different recombinant versions of the TerR DNA-binding domain were analyzed for specific motif recognition. The high affinity consensus motif TCGGHHWYHCGGH was identified from genes required for terrein production and binding site mutations confirmed their essential contribution to gene expression in A. terreus. A combination of TerR with its terA target promoter was tested as recombinant expression system in the heterologous host Aspergillus niger. TerR mediated target promoter activation was directly dependent on its transcription level. Therefore, terR was expressed under control of the regulatable amylase promoter PamyB and the resulting activation of the terA target promoter was compared with activation levels obtained from direct expression of reporters from the strong gpdA control promoter. Here, the coupled system outcompeted the direct expression system. When the coupled system was used for heterologous polyketide synthase expression high metabolite levels were produced. Additionally, expression of the Aspergillus nidulans polyketide synthase gene orsA revealed lecanoric acid rather than orsellinic acid as major polyketide synthase product. Domain swapping experiments assigned this depside formation from orsellinic acid to the OrsA thioesterase domain. These experiments confirm the suitability of the expression system especially for high-level metabolite production in heterologous hosts. PMID:25852654

  8. [Characteristics of extracellular invertase of Saccharomyces cerevisiae in Heterologous expression of the suc2 gene in Solarium Tuberosum plants].

    PubMed

    Deriabin, A N; Berdichevets, I N; Burakhanova, E A; Trunova, T I

    2014-01-01

    Some properties and activity of extracellular invertase in the Saccharomyces cerevisiae yeasts encoded by the suc2 gene in heterologous expression were described. It was shown that the target suc2 gene is actively expressed in the genome of the transformed potato plants and S. cerevisiae invertase synthesized by this gene is transported into the apoplast due to the signal peptide of the proteinase II inhibitor. This enzyme is present in the apoplast in a soluble form and absorbed into the cell wall.

  9. Effectiveness and stability of heterologous proteins expressed in plants by Turnip mosaic virus vector at five different insertion sites.

    PubMed

    Chen, Chin-Chih; Chen, Tsung-Chi; Raja, Joseph A J; Chang, Chin-An; Chen, Li-Wen; Lin, Shih-Shun; Yeh, Shyi-Dong

    2007-12-01

    The N-terminal (NT) regions of particular protein-coding sequences are generally used for in-frame insertion of heterologous open reading frames (ORFs) in potyviral vectors for protein expression in plants. An infectious cDNA clone of Turnip mosaic virus (TuMV) isolate YC5 was engineered at the generally used NT regions of HC-Pro and CP, and other possibly permissive sites to investigate their effectiveness to express the GFP (jellyfish green fluorescent protein) and Der p 5 (allergen from the dust mite, Dermatophagoides pteronyssinus) ORFs. The results demonstrated the permissiveness of the NT regions of P3, CIP and NIb to carry the ORFs and express the translates as part of the viral polyprotein, the processing of which released free-form proteins in the host cell milieu. However, these sites varied in their permissiveness to retain the ORFs intact and hence affect the heterologous protein expression. Moreover, strong influence of the inserted ORF and host plants in determining the permissiveness of a viral genomic context to stably carry the alien ORFs and hence to support their prolonged expression was also noticed. In general, the engineered sites were relatively more permissive to the GFP ORF than to the Der p 5 ORF. Among the hosts, the local lesion host, Chenopodium quinoa Willd. showed the highest extent of support to TuMV to stably carry the heterologous ORFs at the engineered sites and the protein expression therefrom. Among the systemic hosts, Nicotiana benthamiana Domin proved more supportive to TuMV to carry and express the heterologous ORFs than the Brassica hosts, whereas the protein expression levels were significantly higher and more stable in the plants of Brassica campestris L. var. chinensis and B. campestris L. var. ching-geeng than those in the plants of B. juncea L. and B. campestris L. var. pekinensis.

  10. Investigation of the Function of Candida albicans Als3 by Heterologous Expression in Candida glabrata

    PubMed Central

    Fu, Yue; Phan, Quynh T.; Luo, Guanpingsheng; Solis, Norma V.; Liu, Yaoping; Cormack, Brendan P.; Edwards, John E.; Ibrahim, Ashraf S.

    2013-01-01

    During hematogenously disseminated infection, blood-borne Candida albicans invades the endothelial cell lining of the vasculature to invade the deep tissues. Although the C. albicans Als3 invasin is critical for invasion and damage of endothelial cells in vitro, a C. albicans als3Δ/Δ mutant has normal virulence in the mouse model of disseminated infection. We hypothesized that the contribution of Als3 to virulence is obscured by the presence of additional C. albicans invasins. To elucidate the in vivo function of Als3, we heterologously expressed C. albicans ALS3 in Candida glabrata, a yeast that lacks a close ALS3 ortholog and has low virulence in mice. We found that following intravenous inoculation into mice, the ALS3-expressing strain preferentially trafficked to the brain, where it induced significantly elevated levels of myeloperoxidase, tumor necrosis factor, monocyte chemoattractant protein 1, and gamma interferon. Also, the ALS3-expressing strain had enhanced adherence to and invasion of human brain microvascular endothelial cells in vitro, demonstrating a potential mechanism for ALS3-mediated neurotropism. In addition, upon initiation of infection, the ALS3-expressing strain had increased trafficking to the cortex of the kidneys. With prolonged infection, this strain persisted in the kidneys at significantly higher levels than the control strain but did not induce an elevated inflammatory response. Finally, the ALS3-expressing strain had increased resistance to neutrophil killing in vitro. These results indicate that during disseminated infection, Als3 mediates initial trafficking to the brain and renal cortex and contributes to fungal persistence in the kidneys. PMID:23630968

  11. 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.

  12. Cloning, Heterologous Expression, and Distinct Substrate Specificity of Protein Farnesyltransferase from Trypanosoma brucei*

    PubMed Central

    Buckner, Frederick S.; Yokoyama, Kohei; Nguyen, Lisa; Grewal, Anita; Erdjument-Bromage, Hediye; Tempst, Paul; Strickland, Corey L.; Xiao, Li; Van Voorhis, Wesley C.; Gelb, Michael H.

    2010-01-01

    Protein prenylation occurs in the protozoan that causes African sleeping sickness (Trypanosoma brucei), and the protein farnesyltransferase appears to be a good target for developing drugs. We have cloned the α- and β-subunits of T. brucei protein farnesyltransferase (TB-PFT) using nucleic acid probes designed from partial amino acid sequences obtained from the enzyme purified from insect stage parasites. TB-PFT is expressed in both bloodstream and insect stage parasites. Enzymatically active TB-PFT was produced by heterologous expression in Escherichia coli. Compared with mammalian protein farnesyltransferases, TB-PFT contains a number of inserts of >25 residues in both subunits that reside on the surface of the enzyme in turns linking adjacent α-helices. Substrate specificity studies with a series of 20 peptides SSCALX (where X indicates a naturally occurring amino acid) show that the recombinant enzyme behaves identically to the native enzyme and displays distinct specificity compared with mammalian protein farnesyltransferase. TB-PFT prefers Gln and Met at the X position but not Ser, Thr, or Cys, which are good substrates for mammalian protein farnesyltransferase. A structural homology model of the active site of TB-PFT provides a basis for understanding structure-activity relations among substrates and CAAX mimetic inhibitors. PMID:10749864

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

    PubMed

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

    2013-06-15

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

  14. Heterologous expression and characterization of recombinant purple acid phosphatase from red kidney bean.

    PubMed

    Vogel, Andreas; Börchers, Torsten; Marcus, Katrin; Meyer, Helmut E; Krebs, Bernt; Spener, Friedrich

    2002-05-15

    Purple acid phosphatases (PAPs) are dinuclear metallohydrolases of widespread occurrence. In a first step to understand structure-function relationship of PAP from red kidney bean (kbPAP), we cloned its cDNA and functionally expressed the enzyme in insect cells. kbPAP cDNA encodes a protein of 459 amino acids with 99% identity to the published primary structure (T. Klabunde et al., Eur. J. Biochem. 226 (1994) 369-375). N-terminally the cDNA encodes 27 amino acids with characteristics for a signal directing the nascent protein to the endoplasmic reticulum. A baculovirus vector was constructed containing cDNAs of kbPAP and green fluorescent protein, the latter to serve as transfection and infection marker. Heterologous expression in High Five insect cells afforded a dimeric, disulfide-linked phosphatase of 110 kDa, identical to the mass of native kbPAP. Purification in three steps yielded 1.5 mg recombinant protein per liter of culture medium with a specific activity of 266 units/mg, slightly exceeding that of native kbPAP. The recombinant protein was functionally indistinguishable from native kbPAP, despite differences in glycosylation and sensitivity to redox reagents. (c) 2002 Elsevier Science (USA).

  15. 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.

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

    PubMed Central

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

    2012-01-01

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

  17. 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.

  18. Heterologous expression of a Nelumbo nucifera phytochelatin synthase gene enhances cadmium tolerance in Arabidopsis thaliana.

    PubMed

    Liu, Zhaolei; Gu, Chunsun; Chen, Fadi; Yang, Dongyin; Wu, Kunwei; Chen, Sumei; Jiang, Jiafu; Zhang, Zhen

    2012-02-01

    Phytochelatin synthase (PCS) is a key enzyme involved in the synthesis of phytochelatins, which are thought to play important roles in heavy metal detoxification. The sacred lotus (Nelumbo nucifera), one of the most popular ornamental species, has been shown to be a potential phytoremediator of heavy metal polluted water. However, the phytochelatin synthase gene in N. nucifera has not been identified yet. Here, we report the isolation and function characterization of a N. nucifera homologue of phytochelatin synthase. The sequence obtained shares a high degree of similarity with PCSs from other plant species and was named as Nelumbo nucifera phytochelatin synthase1 (NnPCS1). By using quantitative RT-PCR, we found that the expression of NnPCS1 in leaves of N. nucifera was dramatically increased in response to Cadmium (Cd) treatment. We further showed that, when exposed to Cd stress, Arabidopsis transgenic plants heterologous expressing NnPCS1 accumulated more Cd when compared with wild type. These results suggest that NnPCS1 involved in the response of N. nucifera to Cd stress and may represent a useful target gene for the phytoremediation of Cd-polluted water.

  19. 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

  20. Heterologous expression of the plant cysteine protease bromelain and its inhibitor in Pichia pastoris.

    PubMed

    Luniak, Nora; Meiser, Peter; Burkart, Sonja; Müller, Rolf

    2017-01-01

    Expression of proteases in heterologous hosts remains an ambitious challenge due to severe problems associated with digestion of host proteins. On the other hand, proteases are broadly used in industrial applications and resemble promising drug candidates. Bromelain is an herbal drug that is medicinally used for treatment of oedematous swellings and inflammatory conditions and consists in large part of proteolytic enzymes. Even though various experiments underline the requirement of active cysteine proteases for biological activity, so far no investigation succeeded to clearly clarify the pharmacological mode of action of bromelain. The potential role of proteases themselves and other molecules of this multi-component extract currently remain largely unknown or ill defined. Here, we set out to express several bromelain cysteine proteases as well as a bromelain inhibitor molecule in order to gain defined molecular entities for subsequent studies. After cloning the genes from its natural source Ananas comosus (pineapple plant) into Pichia pastoris and subsequent fermentation and purification, we obtained active protease and inhibitor molecules which were subsequently biochemically characterized. Employing purified bromelain fractions paves the way for further elucidation of pharmacological activities of this natural product. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:54-65, 2017. © 2016 American Institute of Chemical Engineers.

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

    PubMed

    Bey, Mathieu; Berrin, Jean-Guy; Poidevin, Laetitia; Sigoillot, Jean-Claude

    2011-12-28

    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. 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. We demonstrate that CDH offers an attractive tool for saccharification process enhancement due to gluconic acid production from raw lignocellulosic material.

  2. Lasso peptides from proteobacteria: Genome mining employing heterologous expression and mass spectrometry.

    PubMed

    Hegemann, Julian D; Zimmermann, Marcel; Zhu, Shaozhou; Klug, Dennis; Marahiel, Mohamed A

    2013-09-01

    Lasso peptides are natural products with a unique three dimensional structure resembling a lariat knot. They are from ribosomal origin and are post-translationally modified by two enzymes (B and C), one of which shares little similarity to enzymes outside of lasso peptide biosynthetic gene clusters and as such is a useful target for genome mining. In this study, we demonstrate a B protein-centric genome mining approach through which we were able to identify 102 putative lasso peptide biosynthetic gene clusters from a total of 87 different proteobacterial strains. Ten of these clusters were cloned into the pET41a expression vector, optimized through incorporation of a ribosomal binding site and heterologously expressed in Escherichia coli BL21(DE3). All 12 predicted lasso peptides (namely burhizin, caulonodin I, caulonodin II, caulonodin III, rhodanodin, rubrivinodin, sphingonodin I, sphingonodin II, syanodin I, sphingopyxin I, sphingopyxin II, and zucinodin) were detected by high-resolution Fourier transform mass spectrometry and their proposed primary structure was confirmed through tandem mass spectrometry. High yields (ranging from 0.4 to 5.2 mg/L) were observable for eight of these compounds, while thermostability assays revealed five new representatives of heat labile lasso peptides. Copyright © 2013 Wiley Periodicals, Inc.

  3. 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. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  4. Effects of unsaturated fatty acids on the kinetics of voltage‐gated proton channels heterologously expressed in cultured cells

    PubMed Central

    Kawanabe, Akira

    2016-01-01

    Key points Arachidonic acid (AA) greatly enhances the activity of the voltage‐gated proton (Hv) channel, although its mechanism of action and physiological function remain unclear.In the present study, we analysed the effects of AA on proton currents through Hv channels heterologously expressed in HEK293T cells.The dramatic increase in proton current amplitude elicited by AA was accompanied by accelerated activation kinetics and a leftward shift in the voltage‐dependence of activation.Mutagenesis studies suggest the two aforementioned effects of AA reflect two distinct structural mechanisms.Application of phospholipase A2, which liberates AA from phospholipids in the membrane, also enhances Hv channel activity, supporting the idea that AA modulates Hv channel activity within physiological contexts. Abstract Unsaturated fatty acids are key components of the biological membranes of all cells, and precursors of mediators for cell signalling. Arachidonic acid (AA) is an unsaturated fatty acid known to modulate the activities of various ion channels, including the voltage‐gated proton (Hv) channel, which supports the rapid production of reactive oxygen species (ROS) in phagocytes through regulation of pH and membrane potential. However, the molecular mechanisms and physiological functions of the effects of AA on Hv channels remain unclear. In the present study, we report an electrophysiological analysis of the effects of AA on the mouse Hv channel (mHv1) heterologously expressed in HEK293T cells. Application of AA to excised inside‐out patch membranes rapidly induced a robust increase in the amplitude of the proton current through mHv1. The current increase was accompanied by accelerated activation kinetics and a small leftward shift of the current–voltage relationship. In monomeric channels lacking the coiled‐coil region of the channel protein, the shift in the current–voltage relationship was diminished but activation and deactivation remained

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

    PubMed Central

    Gerile; Sogawa, Chiharu; Ohyama, Kazumi; Masuko, Takashi; Kusama, Tadashi; Morita, Katsuya; Sogawa, Norio; Kitayama, Shigeo

    2012-01-01

    Betaine/γ-aminobutyric acid (GABA) transporter (BGT1, SLC6A12) is a member of the Na+- and Cl−-dependent neurotransmitter transporter gene family with a homology to the GABA transporters (GATs), GAT1 (SLC6A1), GAT2 (SLC6A13) and GAT3 (SLC6A11) (HUGO nomenclature). Since antidepressants have been reported to inhibit GABA uptake, we examined those effects on mouse BGT1 (mBGT1) in comparison with other mouse GAT (mGAT) subtypes in the heterologously expressed cell cultures. All antidepressants tested here inhibited the [3H]GABA uptake through mBGT1 and mGATs in a rank order of potency with mBGT1 > mGAT1-3. Kinetic analyses for maprotilline, mianserine and trimipramine revealed that they inhibited mBGT1 and mGAT1 noncompetitively, except that mianserine competitively inhibited mBGT1. These results provided a clue to investigate the structure-function relationship of mBGT1 using antidepressants as a tool, leading to the identification of potential candidates for selective and specific inhibitors of mBGT1. PMID:22489112

  6. Housing and Husbandry of Xenopus laevis Affect the Quality of Oocytes for Heterologous Expression Studies

    PubMed Central

    Delpire, Eric; Gagnon, Kenneth B; Ledford, Jonathan J; Wallace, Jeanne M

    2011-01-01

    To assess the effect of Xenopus husbandry on oocyte quality for membrane transport physiology experiments, we compared a recirculating-water housing system with a static-water system in a 23-mo study. Two groups of frogs (n = 8) were maintained separately for the entire study: one group was housed in a multiinvestigator centrally managed Xenopus facility, which consists of 33 tanks placed on a shared and recirculating water system; the other group was housed in a satellite facility used by a single investigator and consisting of static tanks placed in a dedicated cold-room. The activity of a heterologously expressed membrane transporter was assessed every 4 to 5 wk for a total of 23 mo. Activity of the mouse cotransporter NKCC1 was assessed through isotopic 86Rb influx measurements under 2 experimental conditions: stimulation of cotransporter by coinjection of regulatory kinases and by exposure to a hypertonic solution. The results showed a significant difference in the level of ion fluxes under these 2 experimental conditions between the 2 groups of oocytes. During the entire period, oocytes isolated from frogs maintained in the static facility demonstrated consistently robust NKCC1 function, whereas oocytes isolated from frogs maintained in the recirculating facility showed inconsistent and weaker cotransporter function. Furthermore, the oocytes isolated from frogs maintained in the recirculating facility showed significant deterioration during the summer months (April to August), a seasonal variation that was muted in frog oocytes maintained in the static facility. PMID:21333163

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

    PubMed

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

    2011-04-15

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

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

    PubMed

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

    2011-08-01

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

  9. 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

  10. Heterologous expression, refolding and functional characterization of two antifreeze proteins from Fragilariopsis cylindrus (Bacillariophyceae).

    PubMed

    Uhlig, Christiane; Kabisch, Johannes; Palm, Gottfried J; Valentin, Klaus; Schweder, Thomas; Krell, Andreas

    2011-12-01

    Antifreeze proteins (AFPs) provide protection for organisms subjected to the presence of ice crystals. The psychrophilic diatom Fragilariopsis cylindrus which is frequently found in polar sea ice carries a multitude of AFP isoforms. In this study we report the heterologous expression of two antifreeze protein isoforms from F. cylindrus in Escherichia coli. Refolding from inclusion bodies produced proteins functionally active with respect to crystal deformation, recrystallization inhibition and thermal hysteresis. We observed a reduction of activity in the presence of the pelB leader peptide in comparison with the GS-linked SUMO-tag. Activity was positively correlated to protein concentration and buffer salinity. Thermal hysteresis and crystal deformation habit suggest the affiliation of the proteins to the hyperactive group of AFPs. One isoform, carrying a signal peptide for secretion, produced a thermal hysteresis up to 1.53°C±0.53°C and ice crystals of hexagonal bipyramidal shape. The second isoform, which has a long preceding N-terminal sequence of unknown function, produced thermal hysteresis of up to 2.34°C±0.25°C. Ice crystals grew in form of a hexagonal column in presence of this protein. The different sequences preceding the ice binding domain point to distinct localizations of the proteins inside or outside the cell. We thus propose that AFPs have different functions in vivo, also reflected in their specific TH capability. Copyright © 2011 Elsevier Inc. All rights reserved.

  11. Housing and husbandry of Xenopus laevis affect the quality of oocytes for heterologous expression studies.

    PubMed

    Delpire, Eric; Gagnon, Kenneth B; Ledford, Jonathan J; Wallace, Jeanne M

    2011-01-01

    To assess the effect of Xenopus husbandry on oocyte quality for membrane transport physiology experiments, we compared a recirculating-water housing system with a static-water system in a 23-mo study. Two groups of frogs (n = 8) were maintained separately for the entire study: one group was housed in a multiinvestigator centrally managed Xenopus facility, which consists of 33 tanks placed on a shared and recirculating water system; the other group was housed in a satellite facility used by a single investigator and consisting of static tanks placed in a dedicated cold-room. The activity of a heterologously expressed membrane transporter was assessed every 4 to 5 wk for a total of 23 mo. Activity of the mouse cotransporter NKCC1 was assessed through isotopic (86) Rb influx measurements under 2 experimental conditions: stimulation of cotransporter by coinjection of regulatory kinases and by exposure to a hypertonic solution. The results showed a significant difference in the level of ion fluxes under these 2 experimental conditions between the 2 groups of oocytes. During the entire period, oocytes isolated from frogs maintained in the static facility demonstrated consistently robust NKCC1 function, whereas oocytes isolated from frogs maintained in the recirculating facility showed inconsistent and weaker cotransporter function. Furthermore, the oocytes isolated from frogs maintained in the recirculating facility showed significant deterioration during the summer months (April to August), a seasonal variation that was muted in frog oocytes maintained in the static facility.

  12. Fermentation strategies for improved heterologous expression of laccase in Pichia pastoris.

    PubMed

    Hong, Feng; Meinander, Nina Q; Jönsson, Leif J

    2002-08-20

    Improved expression of recombinant laccase by Pichia pastoris carrying the lcc1 cDNA isolated from Trametes versicolor was achieved by optimization of the cultivation conditions in a fermentor equipped with a methanol sensor system. The results indicated that the activity obtained in fermentor cultivations was at least 7 times higher than in shake-flask cultures. Three different strategies for fermentor cultivations were compared: A (30 degrees C, 1.0% methanol), B (20 degrees C, 1.0% methanol), and C (20 degrees C, 0.5% methanol). The laccase activity, particularly the specific activity, could be improved by decreasing the cultivation temperature. The mechanisms behind the temperature effect on the laccase activity may be ascribed to poor stability, release of more proteases from dead cells, and folding problems at higher temperature. The results showed that the methanol concentration had a marked effect on the production of active heterologous laccase. A fivefold higher volumetric laccase activity was obtained when the methanol concentration was kept at 0.5% instead of 1.0%. The detrimental effect of methanol on the production of recombinant laccase may be attributed to lower laccase stability, a higher proteolytic activity, and folding problems due to higher growth rate at 1.0% methanol.

  13. Heterologous Expression of an Alkali and Thermotolerant Lipase from Talaromyces thermophilus in Trichoderma reesei.

    PubMed

    Zhang, Xu; Li, Xueqi; Xia, Liming

    2015-07-01

    To heterologously express a Talaromyces thermophilus lipase gene in Trichoderma reesei, an efficient binary vector pChph-pCBH1sigpro-ttl which includes a newly designed cbh1 promoter and hygromycin-resistant marker was constructed. This plasmid was then transformed into T. reesei via improved Agrobacterium EHA 105-mediated transformation. After modification of co-culture conditions and enzymolysis treatment of conidia, 258 transformants were produced. A two-step screening method based on antibiotic resistance and capacity to utilize lactose and tributyrin was introduced to further select promising candidates, which would be additionally verified by PCR analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and lipase activity assay. Lipase production was carried out in shaking flasks, and the activity reached 241 IU/mL (7415.4 IU/mg) after 84-h fermentation. It was found that this lipase performed high alkali and thermostable tolerance with the optimal pH 9.5 and temperature 60 °C, and it could retain more than 70 % activity after being disposed in pH 11 or 70 °C for 1 h. This study herein would benefit the genetic engineering of T. reesei and the industrial application of this important fungal lipase.

  14. The mammalian beta-tubulin repertoire: hematopoietic expression of a novel, heterologous beta-tubulin isotype

    PubMed Central

    1986-01-01

    We describe the structure of a novel and unusually heterologous beta- tubulin isotype (M beta 1) isolated from a mouse bone marrow cDNA library, and a second isotype (M beta 3) isolated from a mouse testis cDNA library. Comparison of M beta 1 and M beta 3 with the completed (M beta 4, M beta 5) or extended (M beta 2) sequence of three previously described beta-tubulin isotypes shows that each includes a distinctive carboxy-terminal region, in addition to multiple amino acid substitutions throughout the polypeptide chain. In every case where a mammalian interspecies comparison can be made, both the carboxy- terminal and internal amino acid substitutions that distinguish one isotype from another are absolutely conserved. We conclude that these characteristic differences are important in determining functional distinctions between different kinds of microtubule. The amino acid homologies between M beta 2, M beta 3, M beta 4, and M beta 5 are in the range of 95-97%; however the homology between M beta 1 and all the other isotypes is very much less (78%). The dramatic divergence in M beta 1 is due to multiple changes that occur throughout the polypeptide chain. The overall level of expression of M beta 1 is low, and is restricted to those tissues (bone marrow, spleen, developing liver and lung) that are active in hematopoiesis in the mouse. We predict that the M beta 1 isotype is functionally specialized for assembly into the mammalian marginal band. PMID:3782288

  15. Heterologous expression of a thermophilic diacylglycerol acyltransferase triggers triglyceride accumulation in Escherichia coli

    PubMed Central

    Santín, Omar; Cabezas, Matilde; Milagre, Cintia D. F.; de la Cruz, Fernando

    2017-01-01

    Triglycerides (TAGs), the major storage molecules of metabolic energy and source of fatty acids, are produced as single cell oil by some oleogenic microorganisms. However, these microorganisms require strict culture conditions, show low carbon source flexibilities, lack efficient genetic modification tools and in some cases pose safety concerns. TAGs have essential applications such as behaving as a source for added-value fatty acids or giving rise to the production of biodiesel. Hence, new alternative methods are urgently required for obtaining these oils. In this work we describe TAG accumulation in the industrially appropriate microorganism Escherichia coli expressing the heterologous enzyme tDGAT, a wax ester synthase/triacylglycerol:acylCoA acyltranferase (WS/DGAT). With this purpose, we introduce a codon-optimized gene from the thermophilic actinomycete Thermomonospora curvata coding for a WS/DGAT into different E. coli strains, describe the metabolic effects associated to the expression of this protein and evaluate neutral lipid accumulation. We observe a direct relation between the expression of this WS/DGAT and TAG production within a wide range of culture conditions. More than 30% TAGs were detected within the bacterial neutral lipids in 90 minutes after induction. TAGs were observed to be associated with the hydrophobic enzyme while forming round intracytoplasmic bodies, which could represent a bottleneck for lipid accumulation in E. coli. We detected an increase of almost 3-fold in the monounsaturated fatty acids (MUFA) occurring in the recombinant strains. These MUFA were predominant in the accumulated TAGs achieving 46% of the TAG fatty acids. These results set the basis for further research on the achievement of a suitable method towards the sustainable production of these neutral lipids. PMID:28448543

  16. 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.

  17. 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

  18. Heterologous expression and purification of a biologically active legume lectin from Cratylia mollis seeds (CRAMOLL 1).

    PubMed

    Varejão, Nathalia; Almeida, Marcius da Silva; De Cicco, Nuccia N T; Atella, Geórgia C; Coelho, Luana C B B; Correia, M A Tereza S; Foguel, Debora

    2010-09-01

    CRAMOLL 1 is a mannose/glucose isolectin isolated from Cratylia mollis seeds. This lectin has 82% sequence identity with Con A and essentially the same quaternary structure. As with Con A, CRAMOLL 1 seems to undergo complex post-translational processing which makes it difficult to the use of traditional molecular cloning for heterologous expression. Here we report the expression and purification of functional recombinant CRAMOLL 1 (rCRAMOLL 1) in Escherichia coli. This was accomplished by introducing a chemically synthesized DNA encoding the mature CRAMOLL 1 amino acid sequence into a bacterial expression vector under T7 promoter control. Most of the recombinant lectin was found in insoluble aggregates (inclusion bodies), but we were able to recover reasonable amounts of soluble lectin in the active form by decreasing the protein induction temperature. The recombinant lectin was purified to homogeneity with one-step affinity chromatography. The plant CRAMOLL 1 (pCRAMOLL 1) and rCRAMOLL 1 share several physicochemical properties such as molecular mass, charge density and secondary and tertiary structures. However, pCRAMOLL 1 has a lower thermodynamic stability than rCRAMOLL 1 when probed by acidification, high temperature or high hydrostatic pressure, and this is probably caused by the presence of tetramers composed of fragmented monomers, which are formed in the plant cotyledon but absent from the recombinant protein. rCRAMOLL 1 behaves identically to its plant counterpart with respect to its specificity for monosaccharides, and to its agglutinating activities against rabbit erythrocytes and Trypanosoma cruzi epimastigote cells. Copyright © 2010 Elsevier B.V. All rights reserved.

  19. Heterologous expression of the Crassostrea gigas (Pacific oyster) alternative oxidase in the yeast Saccharomyces cerevisiae.

    PubMed

    Robertson, Aaron; Schaltz, Kyle; Neimanis, Karina; Staples, James F; McDonald, Allison E

    2016-10-01

    Alternative oxidase (AOX) is a terminal oxidase within the inner mitochondrial membrane (IMM) present in many organisms where it functions in the electron transport system (ETS). AOX directly accepts electrons from ubiquinol and is therefore capable of bypassing ETS Complexes III and IV. The human genome does not contain a gene coding for AOX, so AOX expression has been suggested as a gene therapy for a range of human mitochondrial diseases caused by genetic mutations that render Complex III and/or IV dysfunctional. An effective means of screening mutations amenable to AOX treatment remains to be devised. We have generated such a tool by heterologously expressing AOX from the Pacific oyster (Crassostrea gigas) in the yeast Saccharomyces cerevisiae under the control of a galactose promoter. Our results show that this animal AOX is monomeric and is correctly targeted to yeast mitochondria. Moreover, when expressed in yeast, Pacific oyster AOX is a functional quinol oxidase, conferring cyanide-resistant growth and myxothiazol-resistant oxygen consumption to yeast cells and isolated mitochondria. This system represents a high-throughput screening tool for determining which Complex III and IV genetic mutations in yeast will be amenable to AOX gene therapy. As many human genes are orthologous to those found in yeast, our invention represents an efficient and cost-effective way to evaluate viable research avenues. In addition, this system provides the opportunity to learn more about the localization, structure, and regulation of AOXs from animals that are not easily reared or manipulated in the lab.

  20. Chromosomal position effect influences the heterologous expression of genes and biosynthetic gene clusters in Streptomyces albus J1074.

    PubMed

    Bilyk, Bohdan; Horbal, Liliya; Luzhetskyy, Andriy

    2017-01-04

    Efforts to construct the Streptomyces host strain with enhanced yields of heterologous product have focussed mostly on engineering of primary metabolism and/or the deletion of endogenous biosynthetic gene clusters. However, other factors, such as chromosome compactization, have been shown to have a significant influence on gene expression levels in bacteria and fungi. The expression of genes and biosynthetic gene clusters may vary significantly depending on their location within the chromosome. Little is known about the position effect in actinomycetes, which are important producers of various industrially relevant bioactive molecules. To demonstrate an impact of the chromosomal position effect on the heterologous expression of genes and gene clusters in Streptomyces albus J1074, a transposon mutant library with randomly distributed transposon that includes a β-glucuronidase reporter gene was generated. Reporter gene expression levels have been shown to depend on the position on the chromosome. Using a combination of the transposon system and a φC31-based vector, the aranciamycin biosynthetic cluster was introduced randomly into the S. albus genome. The production levels of aranciamycin varied up to eightfold depending on the location of the gene cluster within the chromosome of S. albus J1074. One of the isolated mutant strains with an artificially introduced attachment site produced approximately 50% more aranciamycin than strains with endogenous attBs. In this study, we demonstrate that expression of the reporter gene and aranciamycin biosynthetic cluster in Streptomyces albus J1074 varies up to eightfold depending on its position on the chromosome. The integration of the heterologous cluster into different locations on the chromosome may significantly influence the titre of the produced substance. This knowledge can be used for the more efficient engineering of Actinobacteria via the relocation of the biosynthetic gene clusters and insertion of additional

  1. Development of plasmid cloning vectors for Thermus thermophilus HB8: Expression of a heterologous, plasmid-borne kanamycin nucleotidyltransferase gene

    SciTech Connect

    Mather, M.W.; Fee, J.A. )

    1992-01-01

    While several thermus genes have been cloned and T. thermophilus has been shown to be transformable, molecular genetic studies of these thermophiles have been hampered by the absence of selectable cloning vectors. The authors have constructed a selectable plasmid by random insertion of a heterologous gene encoding a thermostable kanamycin nucleotidyltransferase activity into a cryptic, multicopy plasmid from T. thermophilus HB8. This plasmid should serve as a suitable starting point for the development of a gene expression system for T. thermophilus.

  2. 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.

  3. Genetic characterisation and heterologous expression of leucocin C, a class IIa bacteriocin from Leuconostoc carnosum 4010.

    PubMed

    Wan, Xing; Li, Ruiqing; Saris, Per E J; Takala, Timo M

    2013-04-01

    Leuconostoc carnosum 4010 is a protective culture for meat products. It kills the foodborne pathogen Listeria monocytogenes by producing two class IIa (pediocin-like) bacteriocins, leucocin A and leucocin C. The genes for leucocin A production have previously been characterised from Leuconostoc gelidum UAL 187, whereas no genetic studies about leucocin C has been published. Here, we characterised the genes for the production of leucocins A and C in L. carnosum 4010. In this strain, leucocin A and leucocin C operons were localised in different plasmids. Unlike in L. gelidum, leucocin A operon in L. carnosum 4010 only contained the structural and the immunity genes lcaAB without transporter genes lcaECD. On the contrary, leucocin C cluster included two intact operons. Novel genes lecCI encode the leucocin C precursor and the 97-aa immunity protein LecI, respectively. LecI shares 48 % homology with the immunity proteins of sakacin P and listeriocin. Another leucocin C operon lecXTS, encoding an ABC transporter and an accessory protein, was 97 % identical with the leucocin A transporter operon lcaECD of L. gelidum. For heterologous expression of leucocin C in Lactococcus lactis, the mature part of the lecC gene was fused with the signal sequence of usp45 in the secretion vector pLEB690. L. lactis secreted leucocin C efficiently, as shown by large halos on lawns of L. monocytogenes and Leuconostoc mesenteroides indicators. The function of LecI was then demonstrated by expressing the gene lecI in L. monocytogenes. LecI-producing Listeria was less sensitive to leucocin C than the vector strain, thus corroborating the immunity function of LecI.

  4. 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

  5. Identification of Xanthomonas citri ssp. citri host specificity genes in a heterologous expression host.

    PubMed

    Rybak, Myrian; Minsavage, Gerald V; Stall, Robert E; Jones, Jeffrey B

    2009-03-01

    We provide the first conclusive evidence that Xanthomonas axonopodis pv. citri Asiatic strain (Xac-A) and, in particular, Xac-A(w), a unique citrus canker A strain isolated from Key lime in Wellington, Florida, induces a hypersensitive reaction (HR) in grapefruit leaves. Using the heterologous tomato pathogen X. perforans, as a recipient of the Xac-A(w) genomic library, we identified a 1599-bp open reading frame responsible for HR in grapefruit, but not Key lime, and designated it avrGf1. Xac-A(w)DeltaavrGf1 produced typical, although visibly reduced, citrus canker symptoms (i.e. raised pustules) in grapefruit and typical canker symptoms in Key lime. We also determined that the X. perforans transconjugant carrying an Xac-A(w) hrpG elicited HR in grapefruit and Key lime leaves, and that xopA in X. perforans was partly responsible for HR. Xac-A transconjugants carrying the X. perforans xopA were reduced in ability to grow in grapefruit leaves relative to wild-type Xac-A. The X. perforans xopA appears to be a host-limiting factor. An avrBs3 homologue, which contained 18.5 repeats and induced HR in tomato, was designated avrTaw. This gene, when expressed in a pustule-minus Xac-A(w), did not complement pustule formation; however, pthA(w), a functional pthA homologue, complemented the mutant strain to produce typical pustules in Key lime, but markedly reduced pustules in grapefruit. Both avrBs3 homologues, when expressed in a typical Xac-A strain, resulted in typical citrus canker pustules in grapefruit, indicating that neither homologue suppressed pustule size in grapefruit. Xac-A(w) contains other unidentified factors that suppress development in grapefruit.

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

    PubMed

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

    2017-02-01

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

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

    PubMed

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

    1996-05-15

    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).

  8. 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

  9. Withania coagulans tryptophan decarboxylase gene cloning, heterologous expression, and catalytic characteristics of the recombinant enzyme.

    PubMed

    Jadaun, Jyoti Singh; Sangwan, Neelam Singh; Narnoliya, Lokesh Kumar; Tripathi, Sandhya; Sangwan, Rajender Singh

    2017-01-01

    Tryptophan decarboxylase (EC 4.1.1.28) catalyzes pyridoxal 5'-phosphate (PLP)-dependent decarboxylation of tryptophan to produce tryptamine for recruitment in a myriad of biosynthetic pathways of metabolites possessing indolyl moiety. A recent report of certain indolyl metabolites in Withania species calls for a possible predominant functional role of tryptophan decarboxylase (TDC) in the genome of Withania species to facilitate production of the indolyl progenitor molecule, tryptamine. Therefore, with this metabolic prospection, we have identified and cloned a full-length cDNA sequence of TDC from aerial tissues of Withania coagulans. The functional WcTDC gene comprises of 1506 bp open reading frame (ORF) encoding a 502 amino acid protein with calculated molecular mass and pI value of 56.38 kDa and 8.35, respectively. The gene was expressed in Escherichia coli, and the recombinant enzyme was affinity-purified to homogeneity to discern its kinetics of catalysis. The enzyme (WcTDC) exhibited much higher Km value for tryptophan than for pyridoxal 5'-phosphate and was dedicated to catalyze decarboxylation of only tryptophan or, to a limited extent, of its analogue (like 5-hydroxy tryptophan). The observed optimal catalytic functionality of the enzyme on the slightly basic side of the pH scale and at slightly higher temperatures reflected adaptability of the plant to hot and arid regions, the predominant natural habitat of the herb. This pertains to be the first report on cloning and characterization of heterologously expressed recombinant enzyme from W. coagulans and forms a starting point to further understanding of withanamide biosynthesis.

  10. 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.

  11. 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.

  12. Heterologous Expression, Purification and Immunoreactivity of the Antigen 5 from Polybia paulista Wasp Venom.

    PubMed

    Bazon, Murilo Luiz; Perez-Riverol, Amilcar; Dos Santos-Pinto, José Roberto Aparecido; Fernandes, Luis Gustavo Romani; Lasa, Alexis Musacchio; Justo-Jacomini, Débora Laís; Palma, Mario Sergio; Zollner, Ricardo de Lima; Brochetto-Braga, Márcia Regina

    2017-08-24

    Polybia paulista (Hymenoptera: Vespidae) is responsible for a high number of sting accidents and anaphylaxis events in Southeast Brazil, Argentina and Paraguay. The specific detection of allergy to the venom of this wasp is often hampered by the lack of recombinant allergens currently available for molecular diagnosis. Antigen 5 (~23 kDa) from P. paulista venom (Poly p 5) is a highly abundant and glycosylated allergenic protein that could be used for development of component-resolved diagnosis (CRD). Here, we describe the cloning and heterologous expression of the antigen 5 (rPoly p 5) from P. paulista venom using the eukaryotic system Pichia pastoris. The expression as a secreted protein yielded high levels of soluble rPoly p 5. The recombinant allergen was further purified to homogeneity (99%) using a two-step chromatographic procedure. Simultaneously, the native form of the allergen (nPoly p 5) was purified from the wasp venom by Ion exchange chromatography. The rPoly p 5 and nPoly p 5 were then submitted to a comparative analysis of IgE-mediated immunodetection using sera from patients previously diagnosed with sensitization to wasp venoms. Both rPoly p 5 and nPoly p 5 were recognized by specific IgE (sIgE) in the sera of the allergic individuals. The high levels of identity found between nPoly p 5 and rPoly p 5 by the alignment of its primary sequences as well as by 3-D models support the results obtained in the immunoblot. Overall, we showed that P. pastoris is a suitable system for production of soluble rPoly p 5 and that the recombinant allergen represents a potential candidate for molecular diagnosis of P.paulista venom allergy.

  13. Overproduction of Trehalose: Heterologous Expression of Escherichia coli Trehalose-6-Phosphate Synthase and Trehalose-6-Phosphate Phosphatase in Corynebacterium glutamicum

    PubMed Central

    Padilla, Leandro; Krämer, Reinhard; Stephanopoulos, Gregory; Agosin, Eduardo

    2004-01-01

    Trehalose is a disaccharide with potential applications in the biotechnology and food industries. We propose a method for industrial production of trehalose, based on improved strains of Corynebacterium glutamicum. This paper describes the heterologous expression of Escherichia coli trehalose-synthesizing enzymes trehalose-6-phosphate synthase (OtsA) and trehalose-6-phosphate phosphatase (OtsB) in C. glutamicum, as well as its impact on the trehalose biosynthetic rate and metabolic-flux distributions, during growth in a defined culture medium. The new recombinant strain showed a five- to sixfold increase in the activity of OtsAB pathway enzymes, compared to a control strain, as well as an almost fourfold increase in the trehalose excretion rate during the exponential growth phase and a twofold increase in the final titer of trehalose. The heterologous expression described resulted in a reduced specific glucose uptake rate and Krebs cycle flux, as well as reduced pentose pathway flux, a consequence of downregulated glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The results proved the suitability of using the heterologous expression of Ots proteins in C. glutamicum to increase the trehalose biosynthetic rate and yield and suggest critical points for further improvement of trehalose overproduction in C. glutamicum. PMID:14711665

  14. Heterologous expression of a tannic acid-inducible laccase3 of Cryphonectria parasitica in Saccharomyces cerevisiae

    PubMed Central

    2010-01-01

    Background A tannic acid-inducible and mycoviral-regulated laccase3 (lac3) from the chestnut blight fungus Cryphonectria parasitica has recently been identified, but further characterization was hampered because of the precipitation of protein products by tannic acid supplementation. The present study investigated the heterologous expression of the functional laccase3 using a yeast Saccharomyces cerevisiae. Results Laccase activity in the culture broth of transformants measured using a laccase-specific substrate suggested that the lac3 gene was successfully expressed and the corresponding protein product secreted into the culture media. In addition, activity staining and Western blot analysis of a native gel revealed that the enzyme activity co-existed with the protein product specific to anti-laccase3 antibody, confirming that the cloned lac3 gene is responsible for the laccase activity. When transformants were grown on plates containing tannic acid-supplemented media, brown coloration was observed around transformed cells, indicating the oxidation of tannic acid. However, the enzymatic activity was measurable only in the selective ura- media and was negligible in nonselective nutrient-rich culture conditions. This was in part because of the increased plasmid instability in the nonselective media. Moreover, the protein product of lac3 appears to be sensitive to the cultured nonselective nutrient-rich broth, because a rapid decline in enzymatic activity was observed when the cultured broth of ura- media was mixed with that of nonselective nutrient-rich broth. In addition, constitutive expression of the lac3 gene resulted in a reduced cell number of the lac3 transformants compared to that of vector-only transformed control. However, the presence of recombinant vector without lac3 induction did not affect the growth of transformants. Conclusions The results suggest that expression of the lac3 gene has an inhibitory effect on the growth of transformed S. cerevisiae

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

    PubMed

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

    2012-11-01

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

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

    PubMed

    Cox, Robert H; Fromme, Samantha

    2016-12-01

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

  17. Identification, heterologous expression and characterization of a dye-decolorizing peroxidase of Pleurotus sapidus.

    PubMed

    Lauber, Christiane; Schwarz, Tatiana; Nguyen, Quoc Khanh; Lorenz, Patrick; Lochnit, Guenter; Zorn, Holger

    2017-08-23

    The coding sequence of a peroxidase from the secretome of Pleurotus sapidus was cloned from a cDNA library. Bioinformatic analyses revealed an open reading frame of 1551 bp corresponding to a primary translation product of 516 amino acids. The DyP-type peroxidase was heterologously produced in Trichoderma reesei with an activity of 55,000 U L(-1). The enzyme was purified from the culture supernatant, biochemically characterized and the kinetic parameters were determined. The enzyme has an N-terminal signal peptide composed of 62 amino acids. Analysis by Blue Native PAGE and activity staining with ABTS, as well as gel filtration chromatography showed the native dimeric state of the enzyme (115 kDa). Analysis of the substrate range revealed that the recombinant enzyme catalyzes, in addition to the conversion of some classic peroxidase substrates such as 2,2'-azino-bis(3-ethylthiazoline-6-sulfonate) and substituted phenols like 2,6-dimethoxyphenol, also the decolorization of the anthraquinonic dye Reactive Blue 5. The enzyme also catalyzes bleaching of natural colorants such as β-carotene and annatto. Surprisingly, β-carotene was transformed in the presence and absence of H2O2 by rPsaDyP, however enzyme activity was increased by the addition of H2O2. This indicates that the rPsaDyP has an oxidase function in addition to a peroxidase activity. As a consequence of the high affinity to the characteristic substrate Reactive Blue 5 the rPsaDyP belongs functionally to the dyp-type peroxidase family.

  18. Heterologous expression of glycopeptide resistance vanHAX gene clusters from soil bacteria in Enterococcus faecalis.

    PubMed

    Hasman, Henrik; Aarestrup, Frank M; Dalsgaard, Anders; Guardabassi, Luca

    2006-04-01

    The aim of the study was to determine whether glycopeptide resistance gene clusters from soil bacteria could be heterologously expressed in Enterococcus faecalis and adapt to the new host following exposure to vancomycin. The vanHAX clusters from Paenibacillus thiaminolyticus PT-2B1, Paenibacillus apiarius PA-B2B and Amycolatopsis coloradensis DSM 44225 were separately cloned in an appropriately constructed shuttle vector containing the two-component regulatory system (vanRS) of Tn1546. The complete vanA(PT) operon (vanRSHAXY) from P. thiaminolyticus PT-2B1 was cloned in the same shuttle vector lacking enterococcal vanRS. All plasmid constructs were electroporated into E. faecalis JH2-2 and the MICs of vancomycin and teicoplanin were determined for each recombinant strain before and following exposure to sublethal concentrations of vancomycin. The vanHAX clusters from P. thiaminolyticus and P. apiarius conferred high-level vancomycin resistance (MIC > or = 125 mg/L) in E. faecalis JH2-2. In contrast, cloning of the vanHAX cluster from A. coloradensis did not result in a significant increase of vancomycin resistance (MIC = 0.7 mg/L). Resistance to vancomycin was not observed after cloning the complete vanA(PT) operon from P. thiaminolyticus (MIC = 2 mg/L), but this recombinant rapidly adapted to high concentrations of vancomycin (MIC = 500 mg/L) following exposure to sub-lethal concentrations of this antibiotic. The results showed that vanA(PT) in P. thiaminolyticus is a possible ancestor of vanA-mediated glycopeptide resistance in enterococci. Experimental evidence supported the hypothesis that enterococci did not acquire glycopeptide resistance directly from glycopeptide-producing organisms such as A. coloradensis.

  19. Malolactic enzyme from Oenococcus oeni: heterologous expression in Escherichia coli and biochemical characterization.

    PubMed

    Schümann, Christina; Michlmayr, Herbert; Del Hierro, Andrés M; Kulbe, Klaus D; Jiranek, Vladimir; Eder, Reinhard; Nguyen, Thu-Ha

    2013-01-01

    Malolactic enzymes (MLE) are known to directly convert L-malic acid into L-lactic acid with a catalytical requirement of nicotinamide adenine dinucleotide (NAD (+) ) and Mn ( 2+) ; however, the reaction mechanism is still unclear. To study a MLE, the structural gene from Oenococcus oeni strain DSM 20255 was heterologously expressed in Escherichia coli, yielding 22.9 kU l (-1) fermentation broth. After affinity chromatography and removal of apparently inactive protein by precipitation, purified recombinant MLE had a specific activity of 280 U mg (-1) protein with a recovery of approximately 61%. The enzyme appears to be a homodimer with a molecular mass of 128 kDa consisting of two 64 kDa subunits. Characterization of the recombinant enzyme showed optimum activity at pH 6.0 and 45°C, and Km, Vmax and kcat values of 4.9 mM, 427 U mg (-1) and 456 sec (-1) for L-malic acid, 91.4 µM, 295 U mg (-1) and 315 sec (-1) for NAD (+) and 4.6 µM, 229 U mg (-1) and 244 sec (-1) for Mn ( 2+) , respectively. The recombinant MLE retained 95% of its activity after 3 mo at room temperature and 7 mo at 4°C. When using pyruvic acid as substrate, the enzyme showed the conversion of pyruvic acid with detectable L-lactate dehydrogenase (L-LDH) activity and oxidation of NADH. This interesting observation might explain that MLE catalyzes a redox reaction and hence, the requirements for NAD (+) and Mn ( 2+) during the conversion of L-malic to L-lactic acid.

  20. Sulfide Consumption in Sulfurimonas denitrificans and Heterologous Expression of Its Three Sulfide-Quinone Reductase Homologs.

    PubMed

    Han, Yuchen; Perner, Mirjam

    2016-04-01

    Sulfurimonas denitrificans is a sulfur-oxidizing epsilonproteobacterium. It has been reported to grow with sulfide and to harbor genes that encode sulfide-quinone reductases (SQRs) (catalyze sulfide oxidation). However, the actual sulfide concentrations at which S. denitrificans grows and whether its SQRs are functional remain enigmatic. Here, we illustrate the sulfide concentrations at which S. denitrificans exhibits good growth, namely, 0.18 mM to roughly 1.7 mM. Around 2.23 mM, sulfide appears to inhibit growth. S. denitrificans harbors three SQR homolog genes on its genome (Suden_2082 for type II SQR, Suden_1879 for type III SQR, and Suden_619 for type IV SQR). They are all transcribed in S. denitrificans. According to our experiments, they appear to be loosely bound to the membrane. Each individual S. denitrificans SQR was heterologously expressed in the Rhodobacter capsulatus SB1003 sqr deletion mutant, and all exhibited SQR activities individually. This suggests that all of these three genes encode functional SQRs. This study also provides the first experimental evidence of a functional bacterial type III SQR. Although the epsilonproteobacterium Sulfurimonas denitrificans has been described as using many reduced sulfur compounds as electron donors, there is little knowledge about its growth with sulfide. In many bacteria, the sulfide-quinone reductase (SQR) is responsible for catalyzing sulfide oxidation. S. denitrificans has an array of different types of sqr genes on its genome and so do several other sulfur-oxidizing Epsilonproteobacteria. However, whether these SQRs are functional has remained unknown. Here, we shed light on sulfide metabolism in S. denitrificans. Our study provides the first experimental evidence of active epsilonproteobacterial SQRs and also gives the first report of a functional bacterial type III SQR. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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

    PubMed

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

    2016-11-01

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

  2. Fenamate-induced enhancement of heterologously expressed HERG currents in Xenopus oocytes.

    PubMed

    Malykhina, Anna P; Shoeb, Fouzia; Akbarali, Hamid I

    2002-10-11

    The human ether-a-go-go related gene (HERG) product encodes for the pore-forming subunit of the rapid component of the delayed rectifier K(+) channel that mediates repolarization of cardiac action potential. HERG channels are also potential targets of a large variety of pharmacological agents most of which tend to block HERG currents. In this study, we examined the effects of the non-steroidal anti-inflammatory agents, flufenamic acid and niflumic acid, on heterologously expressed HERG channels in oocytes. The cRNA of HERG (30 ng) was injected into Xenopus oocytes and currents were recorded using two-electrode voltage clamp technique in a low Cl(-) solution. Flufenamic and niflumic acids (10(-4)-5 x 10 (-4) M) enhanced the amplitude of outward currents evoked by depolarizing pulses. At potentials positive to 0 mV, an initial transient component was also evident in the presence of fenamates. Fenamates accelerated the activation rate of HERG channels and decelerated their deactivation. Flufenamic acid (5 x 10 (-4) M) shifted the I(tail)-V relationship from -26.7+/-0.1 to -31.4+/-0.2 mV. Neither flufenamic acid or niflumic acid affected the kinetics of HERG channel inactivation. Using a voltage protocol that mimicked the cardiac action potential, both fenamates increased the outward current during the plateau and during the phase 3 repolarization of action potential. The effects of the fenamates were blocked by the HERG channel blocker, E-4031 and were also not observed in water-injected oocytes. Our data suggest that fenamates enhance HERG currents and affect the action potential duration in the heart.

  3. 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).

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

    PubMed Central

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

    2017-01-01

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

  5. 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.

  6. 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.

  7. Molecular cloning and heterologous expression analysis of JrVTE1 gene from walnut (Juglans regia).

    PubMed

    Wang, Cancan; Li, Chuanrong; Leslie, Charles A; Sun, Qingrong; Guo, Xianfeng; Yang, Keqiang

    Tocopherol cyclase (VTE1) plays a key role in promoting the production of γ-tocopherol and improving total tocopherol content in photosynthetic organisms. Walnut is an important source of tocopherols in the human diet, and γ-tocopherol is the major tocopherol compound in walnut kernels. In this study, a full-length cDNA of the VTE1 gene was isolated from walnut using RT-PCR and RACE, and designated as JrVTE1. The full-length cDNA of the JrVTE1 gene contained a 1353-bp open-reading frame encoding a 451-amino-acid protein with a calculated molecular weight of 49.5 kDa. The deduced JrVTE1 protein had a considerable homology with other plant VTE1s and belonged to the tocopherol cyclase family. Functional characterization of JrVTE1 by heterologous expression was carried out in E. coli BL21 (DE3) and microshoot lines of the fruit trees jujube (Zizyphus jujuba var. spinosa) and pear (Pyrus communis) cultivar 'Old Home'. JrVTE1 in E. coli expressed as a 50 kDa protein, as expected. One or two copies of the transferred JrVTE1 gene were detected in the genomes of representative transgenic lines (from the initial transgenic plants) of jujube and pear by gel blots analysis. Over-expression of JrVTE1 in jujube and pear resulted in an accumulation of tocopherol and a shift in tocopherol composition in leaf, root and stem tissues. In the transgenic jujube, the total tocopherol content increased by 29.8 μg/g in the stems of line J3, 43.7 and 22.5 μg/g in the roots and leaves of line J1, respectively, whereas in the transgenic pear it increased by 47.3 μg/g in the leaf of line P3, and 16.7 and 10.4 μg/g in roots and stems of line P9, respectively. In the examined tissues of transgenic plants, the highest accumulation rate was the γ-tocopherol. These results indicate that JrVTE1 is one of the rate-limiting enzymes for tocopherol production and could be used to improve the tocopherol content of tree crops through genetic engineering.

  8. [Molecular cloning and heterologous expression of a new xylanase gene from Verticillium dahliae].

    PubMed

    Zhang, Guimin; Rao, Ben; Ye, Jian; Ma, Lixin; Zhang, Xian'en

    2008-06-01

    Fungus Verticillium dahliae caused greensickness of cotton and xylanase is necessary in this pathogenesis. Cloning xylanase gene from V. dahliae and heterologous expression might obtain new xylanase. By comparing the amino acid sequences of over 10 xylanases in 11 families from fungi through BLAST, we found 2 highly conserved regions, with a fragment of about 150 amino acids coding sequence in between. Degenerate primers complementary to the ends of these two conserved regions were designed to amplify the in-between sequence from V. dahliae. The whole xylanse gene containing intron was achieved by Genome-walking PCR method. A 63 bp intron was found through BLAST, the whole cDNA xynG was cloned by Dpn I -mediated PCR to delete intron. The cDNA was inserted into pHBM905 and expressed in Pichia pastoris GS115, xylanase-secreting transformants were selected on plate containing RBB-xylan. The transformant with the largest halos was selected for study the character of xylanase. The deduced amino acid sequence showed 72% identity with endo-beta-1, 4-xylanase from Cochliobolus carbonum and C. sativus in the GenBank, which means xynG is a new xylanase gene. The optimal pH of the purified recombinant enzyme was pH6. It remains over 50% relative activity at pH5-9. The optimal temperature was 45 degrees C. The most favorable substrate for the xylanase (XYNG) is Beechwood xylan. Mg2+ and Ca2+ improve the enzyme activity by 33.7% and 16.6%, respectively. EDTA, beta-Mercaptoethanol and NaN3 don't affect the enzyme activity. Tween-80 and DMSO activated enzyme activity by 28.4% and 12.8%. Hg+, in concentration of 5 mmol/L, also inhibited the enzyme activity. The xylanase gene xynG was firstly cloned from the fungi that caused greensickness of cotton. The xylanase genes containing one intron can be efficiently cloned from plant pathogens and white-rot fungi using strategy in this research. It is unnecessary to explore enzyme expression condition and measure enzyme activity of the

  9. 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.

  10. CXCR4 Is Required by a Nonprimate Lentivirus: Heterologous Expression of Feline Immunodeficiency Virus in Human, Rodent, and Feline Cells

    PubMed Central

    Poeschla, Eric M.; Looney, David J.

    1998-01-01

    A heterologous feline immunodeficiency virus (FIV) expression system permitted high-level expression of FIV proteins and efficient production of infectious FIV in human cells. These results identify the FIV U3 element as the sole restriction to the productive phase of replication in nonfeline cells. Heterologous FIV expression in a variety of human cell lines resulted in profuse syncytial lysis that was FIV env specific, CD4 independent, and restricted to cells that express CXCR4, the coreceptor for T-cell-line-adapted strains of human immunodeficiency virus. Stable expression of human CXCR4 in CXCR4-negative human and rodent cell lines resulted in extensive FIV Env-mediated, CXCR4-dependent cell fusion and infection. In feline cells, stable overexpression of human CXCR4 resulted in increased FIV infectivity and marked syncytium formation during FIV replication or after infection with FIV Env-expressing vectors. The use of CXCR4 is a fundamental feature of lentivirus biology independent of CD4 and a shared cellular link to infection and cytopathicity for distantly related lentiviruses that cause AIDS. Their conserved use implicates chemokine receptors as primordial lentivirus receptors. PMID:9658135

  11. Heterologous expression and N-terminal His-tagging processes affect the catalytic properties of staphylococcal lipases: a monolayer study.

    PubMed

    Horchani, Habib; Sabrina, Lignon; Régine, Lebrun; Sayari, Adel; Gargouri, Youssef; Verger, Robert

    2010-10-15

    The interfacial and kinetic properties of wild type, untagged recombinant and tagged recombinant forms of three staphylococcal lipases (SSL, SXL and SAL3) were compared using the monomolecular film technique. A kinetic study on the dependence of the stereoselectivity of these nine lipase forms on the surface pressure was performed using the three dicaprin isomers spread in the form of monomolecular films at the air-water interface. New parameters, termed Recombinant expression Effects on Catalysis (REC), N-Tag Effects on Catalysis (TEC), and N-Tag and Recombinant expression Effects on Catalysis (TREC), were introduced. The findings obtained showed that with all the lipases tested, the recombinant expression process and the N-terminal His-tag slightly affect the sn-1 preference for dicaprin enantiomers as well as the penetration capacity into monomolecular films of phosphatidylcholine but significantly decrease the catalytic rate of hydrolysis of three dicaprin isomers. This rate reduction is more pronounced at high surface pressures, i.e. at low interfacial energies. In conclusion, the effects of the heterologous expression process on the catalytic properties of the staphylococcal lipases are three times more deleterious than the presence of an N-terminal tag extension. In the case of the situation most commonly encountered in the literature, i.e. the heterologous expression of a tagged lipase, the rate of catalysis can be decreased by these processes by 42-83% on average in comparison with the values measured with the corresponding wild type form.

  12. Heterologous expression of bacterial phosphoenol pyruvate carboxylase and Entner-Doudoroff pathway in Saccharomyces cerevisiae for improvement of isobutanol production.

    PubMed

    Morita, Keisuke; Nomura, Yuta; Ishii, Jun; Matsuda, Fumio; Kondo, Akihiko; Shimizu, Hiroshi

    2017-09-01

    Bacterial phosphoenol pyruvate carboxylase (PPC) and enzymes in the Entner-Doudoroff (ED) pathway were heterologously expressed in Saccharomyces cerevisiae to improve the NADPH supply required for the bio-production of chemicals such as isobutanol. The heterologous expression of PPC from Synechocystis sp. PCC6803 increased in the isobutabol titer 1.45-fold (93.2±1.6 mg/L) in metabolically engineered S. cerevisiae strains producing isobutanol. This result suggested that the pyruvate and NADPH supply for isobutanol biosynthesis was activated by PPC overexpression. On the other hand, the expression of two enzymes organizing the ED pathway (6-phosphogluconate dehydratase [6PGD] and 2-dehydro-3-deoxy-phosphogluconate aldolase [KDPGA]) had no effect to isobutabol bio-production. Further analysis, however, revealed that additional expression of 6PGD and KDPGA improved the growth rate of S. cerevisiae strain BY4742 gnd1Δ. A (13)C-labeling experiment using [1-(13)C] glucose also suggested that metabolic flow levels in the ED pathway increased slightly with the additional expression. These results showed that the ED pathway was successfully constructed in S. cerevisiae, even though activity of the pathway was too weak to improve isobutanol biosynthesis. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  13. Increasing the metabolic capacity of Escherichia coli for hydrogen production through heterologous expression of the Ralstonia eutropha SH operon.

    PubMed

    Ghosh, Dipankar; Bisaillon, Ariane; Hallenbeck, Patrick C

    2013-08-26

    Fermentative hydrogen production is an attractive means for the sustainable production of this future energy carrier but is hampered by low yields. One possible solution is to create, using metabolic engineering, strains which can bypass the normal metabolic limits to substrate conversion to hydrogen. Escherichia coli can degrade a variety of sugars to hydrogen but can only convert electrons available at the pyruvate node to hydrogen, and is unable to use the electrons available in NADH generated during glycolysis. Here, the heterologous expression of the soluble [NiFe] hydrogenase from Ralstonia eutropha H16 (the SH hydrogenase) was used to demonstrate the introduction of a pathway capable of deriving substantial hydrogen from the NADH generated by fermentation. Successful expression was demonstrated by in vitro assay of enzyme activity. Moreover, expression of SH restored anaerobic growth on glucose to adhE strains, normally blocked for growth due to the inability to re-oxidize NADH. Measurement of in vivo hydrogen production showed that several metabolically engineered strains were capable of using the SH hydrogenase to derive 2 mol H2 per mol of glucose consumed, close to the theoretical maximum. Previous introduction of heterologous [NiFe] hydrogenase in E. coli led to NAD(P)H dependent activity, but hydrogen production levels were very low. Here we have shown for the first time substantial in vivo hydrogen production by a heterologously expressed [NiFe] hydrogenase, the soluble NAD-dependent H2ase of R. eutropha (SH hydrogenase). This hydrogenase was able to couple metabolically generated NADH to hydrogen production, thus rescuing an alcohol dehydrogenase (adhE) mutant. This enlarges the range of metabolism available for hydrogen production, thus potentially opening the door to the creation of greatly improved hydrogen production. Strategies for further increasing yields should revolve around making additional NADH available.

  14. Heterologous expression of bacteriocins using the mesentericin Y105 dedicated transport system by Leuconostoc mesenteroides.

    PubMed

    Morisset, Dany; Frère, Jacques

    2002-01-01

    Mesentericin Y105 (MesY105) is a class IIa anti-Listeria bacteriocin, produced by Leuconostoc (Ln.) mesenteroides Y105 and with potential food grade application. This bacterium produces a second bacteriocin, mesentericin B105 (MesB105), that does not belong to the same class. To study secretion of bacteriocins by the use of the MesY105 dedicated transport system (DTS), plasmids were constructed for heterologous expression by Ln. mesenteroides. pFBYC04 (Microbiology 144 (1998) 2845) harbours two divergent operons required for MesY105 secretion, i.e. the mesYI operon, encoding pre-MesY105 and immunity, respectively, and the mesCDE operon for secretion. A pFBYC04 derivative, pDMJF01 was constructed by divergent PCR to remove the mesY gene. Ln. mesenteroides DSM20484(pDMJF01) was unable to produce MesY105. The mesYI operon and mesB, mesH and mesF genes, encoding pre-MesB105, MesB105 immunity and a putative protein with unknown function, respectively, were cloned independently into a compatible pDMJF01 plasmid to produce, respectively, pDMJF:YI and pDMJF:BHF. DSM20484 transformed independently with these plasmids was unable to secrete any bacteriocin. MesY105 and MesB105 secretion was observed for DSM20484(pDMJF01) harbouring both pDMJF:YI and pDMJF:BHF. This indicates that the MesY105 DTS permits the transport of MesB105. MesY105 secretion machinery was used to secrete pediocin PA-1 (PedPA-1) by DSM20484 by an in-frame gene fusion strategy where the gene portions corresponding to the MesY105 leader peptide and the mature PedPA-1 were ligated. Thus, MesY105 secretion machinery appears to be a useful tool for secretion of class II bacteriocins by Leuconostoc.

  15. 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.

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

    PubMed

    Watanabe, Kenji; Oguri, Hiroki; Oikawa, Hideaki

    2009-04-01

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

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

    PubMed

    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.

  18. 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

  19. Heterologous expression of the kanamycin biosynthetic gene cluster (pSKC2) in Streptomyces venezuelae YJ003.

    PubMed

    Thapa, Laxmi Prasad; Oh, Tae-Jin; Lee, Hei Chan; Liou, Kwangkyoung; Park, Je Won; Yoon, Yeo Joon; Sohng, Jae Kyung

    2007-10-01

    The pSKC2 cosmid, which has 32 kb and 28 open-reading frames, was isolated from Streptomyces kanamyceticus ATCC12853 as the gene cluster of kanamycin. This gene cluster includes the minimal biosynthetic genes of kanamycin with the resistance and regulatory genes. It was heterologously expressed in Streptomyces venezuelae YJ003, which has the advantage of fast growth, good efficiency of the transformation host, and rapid production of the aminoglycosides antibiotic. The isolated compound was analyzed by electrospray ionization-mass spectrometry, liquid chromatography-mass spectrometry, high-performance liquid chromatography, and tandem mass spectrometry and shows a molecular weight of 485 as kanamycin A.

  20. Functional heterologous expression of an engineered full length CipA from Clostridium thermocellum in Thermoanaerobacterium saccharolyticum

    PubMed Central

    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 ΔcipA 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. PMID:23448319

  1. 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.

  2. Rapid cloning and heterologous expression of the meridamycin biosynthetic gene cluster using a versatile Escherichia coli-streptomyces artificial chromosome vector, pSBAC.

    PubMed

    Liu, Hongbo; Jiang, Hao; Haltli, Bradley; Kulowski, Kerry; Muszynska, Elwira; Feng, Xidong; Summers, Mia; Young, Mairead; Graziani, Edmund; Koehn, Frank; Carter, Guy T; He, Min

    2009-03-27

    Expression of biosynthetic pathways in heterologous hosts is an emerging approach to expedite production improvement and biosynthetic modification of natural products derived from microbial secondary metabolites. Herein we describe the development of a versatile Escherichia coli-Streptomyces shuttle Bacterial Artificial Chromosomal (BAC) conjugation vector, pSBAC, to facilitate the cloning, genetic manipulation, and heterologous expression of actinomycetes secondary metabolite biosynthetic gene clusters. The utility of pSBAC was demonstrated through the rapid cloning and heterologous expression of one of the largest polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) biosynthetic pathways: the meridamycin biosynthesis gene cluster (mer). The entire mer gene cluster ( approximately 90 kb) was captured in a single pSBAC clone through a straightforward restriction enzyme digestion and cloning approach and transferred into Streptomyces lividans. The production of meridamycin (1) in the heterologous host was achieved after replacement of the original promoter with an ermE* promoter and was enhanced by feeding with a biosynthetic precursor. The success of heterologous expression of such a giant gene cluster demonstrates the versatility of BAC cloning technology and paves the road for future exploration of expression of the meridamycin biosynthetic pathway in various hosts, including strains that have been optimized for polyketide production.

  3. Heterologous expression of BetL, a betaine uptake system, enhances the stress tolerance of Lactobacillus salivarius UCC118.

    PubMed

    Sheehan, Vivien M; Sleator, Roy D; Fitzgerald, Gerald F; Hill, Colin

    2006-03-01

    Given the increasing commercial and clinical relevance of probiotic cultures, improving the technological robustness of what are often process-sensitive cultures is an important biological goal. The nisin-controlled expression system was used to direct the heterologous expression of the listerial betaine uptake system BetL in the probiotic strain Lactobacillus salivarius UCC118. Following nisin induction, strains expressing betL exhibited a significant increase in resistance to several stresses, including elevated osmo-, cryo-, baro-, and chill tolerance, as well as increased resistance to spray- and freeze-drying. The ability to confer additional stress tolerance on a probiotic culture may be an important step in delivering viable cultures for maximal efficacy.

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

    PubMed

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

    2017-09-01

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

  5. Heterologous Expression of the Thiopeptide Antibiotic GE2270 from Planobispora rosea ATCC 53733 in Streptomyces coelicolor Requires Deletion of Ribosomal Genes from the Expression Construct

    PubMed Central

    Flinspach, Katrin; Kapitzke, Claudia; Tocchetti, Arianna; Sosio, Margherita; Apel, Alexander K.

    2014-01-01

    GE2270 is a thiopeptide antibiotic generated by extensive posttranslational modifications of a ribosomally generated precursor peptide. Thiopeptides are especially active against Gram-positive bacteria, including methicillin resistant Staphylococcus aureus (MRSA). In this study the GE2270 biosynthetic gene cluster (pbt) from Planobispora rosea ATCC 53733 was successfully expressed in the heterologous host strain Streptomyces coelicolor M1146. Notably, exconjugants containing the pbt gene cluster could only be obtained after deletion of the major part of the ribosomal genes flanking the gene cluster. This is a striking example that genes belonging to primary metabolism can prevent the successful conjugative transfer of DNA from phylogenetic distant species and thus complicate heterologous expression of secondary metabolite gene clusters. GE2270 production in the heterologous producer strain increased after introduction of the constitutive ermE* promoter upstream of the GE2270 resistance gene tuf from P. rosea. Insertion of the inducible tcp830 promoter resulted in inducible GE2270 production. When the regulatory gene pbtR was deleted, the resulting strain ceased to produce GE2270, suggesting an essential role of PbtR as a putative transcriptional activator of GE2270 expression. PMID:24598591

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

    PubMed

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

    2012-08-01

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

  7. Production of conjugated linoleic acid by heterologous expression of linoleic acid isomerase in oleaginous fungus Mortierella alpina.

    PubMed

    Hao, Danhui; Chen, Haiqin; Hao, Guangfei; Yang, Bo; Zhang, Baixi; Zhang, Hao; Chen, Wei; Chen, Yong Q

    2015-10-01

    To increase the commercial value of oleaginous fungus Mortierella alpina by incorporation of trans-10,cis-12 conjugated linoleic acid (CLA) into the polyunsaturated fatty acids (PUFAs) of M. alpina via Propionibacterium acnes isomerase (PAI) conversion. The PAI gene and the codon optimization version were heterologously expressed in M. alpina via Agrobacterium tumefaciens-mediated transformation (ATMT). Coding usage modification significantly improved the translation of PAI transcripts and trans-10,cis-12 CLA was produced up to 1.2 mg l(-1), which corresponds to approx. 0.05% of the total fatty acid (TFA). Since PAI prefers free linoleic acid as a substrate rather than any other forms, 5 μM long-chain acyl CoA synthetase inhibitor was added and the trans-10,cis-12 CLA content increased approx. 24-fold to 29 mg l(-1), reaching up to 1.2% (w/w) of the TFA in M. alpina. Heterologous expression of PAI in M. alpina by ATMT methods is a practicable way in biosynthesis of CLA and this system may be a feasible platform for industrial production of CLA.

  8. Heterologous expression of the yeast arsenite efflux system ACR3 improves Arabidopsis thaliana tolerance to arsenic stress.

    PubMed

    Ali, Waqar; Isner, Jean-Charles; Isayenkov, Stanislav V; Liu, Wenju; Zhao, Fang-Jie; Maathuis, Frans J M

    2012-05-01

    • Arsenic contamination has a negative impact on crop cultivation and on human health. As yet, no proteins have been identified in plants that mediate the extrusion of arsenic. Here, we heterologously expressed the yeast (Saccharomyces cerevisiae) arsenite efflux transporter ACR3 into Arabidopsis to evaluate how this affects plant tolerance and tissue arsenic contents. • ACR3 was cloned from yeast and transformed into wild-type and nip7;1 Arabidopsis. Arsenic tolerance was determined at the cellular level using vitality stains in protoplasts, in intact seedlings grown on agar plates and in mature plants grown hydroponically. Arsenic efflux was measured from protoplasts and from intact plants, and arsenic levels were measured in roots and shoots of plants exposed to arsenate. • At the cellular level, all transgenic lines showed increased tolerance to arsenite and arsenate and a greater capacity for arsenate efflux. With intact plants, three of four stably transformed lines showed improved growth, whereas only transgenic lines in the wild-type background showed increased efflux of arsenite into the external medium. The presence of ACR3 hardly affected tissue arsenic levels, but increased arsenic translocation to the shoot. • Heterologous expression of yeast ACR3 endows plants with greater arsenic resistance, but does not lower significantly arsenic tissue levels. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

  9. A novel salt-inducible vector for efficient expression and secretion of heterologous proteins in Bacillus subtilis.

    PubMed

    Promchai, Ruangurai; Promdonkoy, Boonhiang; Tanapongpipat, Sutipa; Visessanguan, Wonnop; Eurwilaichitr, Lily; Luxananil, Plearnpis

    2016-03-20

    Bacillus subtilis is commonly used as a host for heterologous protein production via plasmid-based expression system. In order to improve product safety, avoid carbon catabolite repression and lower production cost, a novel salt-inducible vector, pSaltExSePR5, was developed based on a natural plasmid of Lactobacillus plantarum BCC9546. Salt-inducible promoter opuAA and a DNA fragment encoding a signal peptide of subtilisin E (SubE) were sequentially added to the core shuttle vector to facilitate expression and secretion of a target protein in B. subtilis. To evaluate the effectiveness of this system under salt induction, a protease gene from Halobacillus sp. without its native signal sequence was inserted in the pSaltExSePR5 plasmid downstream of SubE signal sequence and transformed into B. subtilis WB800. Protease activities from cell-free supernatants of the recombinant bacteria cultures induced with 0.5-6% NaCl were analyzed. The highest protease activity of 9.1 U/ml was obtained after induction with 4% NaCl, while the non-induced culture exhibited activity of 0.128 U/ml. The results demonstrated that pSaltExSePR5 provides an alternative vector for efficient and simple production of heterologous proteins in B. subtilis with a safer and more economic inducer.

  10. Heterology Expression of the Arabidopsis C-Repeat/Dehydration Response Element Binding Factor 1 Gene Confers Elevated Tolerance to Chilling and Oxidative Stresses in Transgenic Tomato1

    PubMed Central

    Hsieh, Tsai-Hung; Lee, Jent-Turn; Yang, Pei-Tzu; Chiu, Li-Hui; Charng, Yee-yung; Wang, Yu-Chie; Chan, Ming-Tsair

    2002-01-01

    In an attempt to improve stress tolerance of tomato (Lycopersicon esculentum) plants, an expression vector containing an Arabidopsis C-repeat/dehydration responsive element binding factor 1 (CBF1) cDNA driven by a cauliflower mosaic virus 35S promoter was transferred into tomato plants. Transgenic expression of CBF1 was proved by northern- and western-blot analyses. The degree of chilling tolerance of transgenic T1 and T2 plants was found to be significantly greater than that of wild-type tomato plants as measured by survival rate, chlorophyll fluorescence value, and radical elongation. The transgenic tomato plants exhibited patterns of growth retardation; however, they resumed normal growth after GA3 (gibberellic acid) treatment. More importantly, GA3-treated transgenic plants still exhibited a greater degree of chilling tolerance compared with wild-type plants. Subtractive hybridization was performed to isolate the responsive genes of heterologous Arabidopsis CBF1 in transgenic tomato plants. CATALASE1 (CAT1) was obtained and showed activation in transgenic tomato plants. The CAT1 gene and catalase activity were also highly induced in the transgenic tomato plants. The level of H2O2 in the transgenic plants was lower than that in the wild-type plants under either normal or cold conditions. The transgenic plants also exhibited considerable tolerance against oxidative damage induced by methyl viologen. Results from the current study suggest that heterologous CBF1 expression in transgenic tomato plants may induce several oxidative-stress responsive genes to protect from chilling stress. PMID:12114563

  11. 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.

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

    PubMed

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

    2017-09-05

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

  13. 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

  14. Cell surface engineering of Bacillus subtilis improves production yields of heterologously expressed α-amylases.

    PubMed

    Cao, Haojie; van Heel, Auke J; Ahmed, Hifza; Mols, Maarten; Kuipers, Oscar P

    2017-04-04

    Bacillus subtilis is widely used as a cell factory for numerous heterologous proteins of commercial value and medical interest. To explore the possibility of further enhancing the secretion potential of this model bacterium, a library of engineered strains with modified cell surface components was constructed, and the corresponding influences on protein secretion were investigated by analyzing the secretion of α-amylase variants with either low-, neutral- or high- isoelectric points (pI). Relative to the wild-type strain, the presence of overall anionic membrane phospholipids (phosphatidylglycerol and cardiolipin) increased dramatically in the PssA-, ClsA- and double KO mutants, which resulted in an up to 47% higher secretion of α-amylase. Additionally, we demonstrated that the appropriate net charge of secreted targets (AmyTS-23, AmyBs and AmyBm) was beneficial for secretion efficiency as well. In B. subtilis, the characteristics of cell membrane phospholipid bilayer and the pIs of heterologous α-amylases appear to be important for their secretion efficiency. These two factors can be engineered to reduce the electrostatic interaction between each other during the secretion process, which finally leads to a better secretion yield of α-amylases.

  15. Heterologous expression and purification of catalytic domain of CESA1 from Arabidopsis thaliana

    SciTech Connect

    Vandavasi, Venu Gopal; O'Neill, Hugh Michael

    2016-10-20

    Here, heterologous expression of plant cellulose synthase (CESA) and its purification has remained a challenge for decades impeding detailed biophysical, biochemical and structural characterization of this key enzyme. An in-depth knowledge of structure and function of CESA proteins would enable us to better understand the hierarchical structure of the plant cell wall. Here, we report a detailed, and reproducible method of purification of catalytic domain of CESA1 from Arabidopsis thaliana that was recombinantly expressed in Escherichia coli. The method relies on a two stage purification procedure to obtain the catalytic domain in monomer and trimer forms. The biochemical and biophysical data including low resolution structures of the protein have been published. Currently the crystallization studies of this protein are underway.

  16. Functional Interactions between Heterologously Expressed Starch-Branching Enzymes of Maize and the Glycogen Synthases of Brewer's Yeast1

    PubMed Central

    Seo, Beom-seok; Kim, Seungtaek; Scott, M. Paul; Singletary, George W.; Wong, Kit-sum; James, Martha G.; Myers, Alan M.

    2002-01-01

    Starch-branching enzymes (SBEs) catalyze the formation of α(1→6) glycoside bonds in glucan polymers, thus, affecting the structure of amylopectin and starch granules. Two distinct classes of SBE are generally conserved in higher plants, although the specific role(s) of each isoform in determination of starch structure is not clearly understood. This study used a heterologous in vivo system to isolate the function of each of the three known SBE isoforms of maize (Zea mays) away from the other plant enzymes involved in starch biosynthesis. The ascomycete Brewer's yeast (Saccharomyces cerevisiae) was employed as the host species. All possible combinations of maize SBEs were expressed in the absence of the endogenous glucan-branching enzyme. Each maize SBE was functional in yeast cells, although SBEI had a significant effect only if SBEIIa and SBEIIb also were present. SBEI by itself did not support glucan accumulation, whereas SBEIIa and SBEIIb both functioned along with the native glycogen synthases (GSs) to produce significant quantities of α-glucan polymers. SBEIIa was phenotypically dominant to SBEIIb in terms of glucan structure. The specific branching enzyme present had a significant effect on the molecular weight of the product. From these data we suggest that SBEs and GSs work in a cyclically interdependent fashion, such that SBE action is needed for optimal GS activity; and GS, in turn, influences the further effects of SBE. Also, SBEIIa and SBEIIb appear to act before SBEI during polymer assembly in this heterologous system. PMID:11950968

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

    PubMed

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

    2007-01-01

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

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

    PubMed

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

    2010-10-25

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

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

    PubMed Central

    2010-01-01

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

  20. Heterologous expression of a rice metallothionein isoform (OsMTI-1b) in Saccharomyces cerevisiae enhances cadmium, hydrogen peroxide and ethanol tolerance.

    PubMed

    Ansarypour, Zahra; Shahpiri, Azar

    2017-02-04

    Metallothioneins are a superfamily of low-molecular-weight, cysteine (Cys)-rich proteins that are believed to play important roles in protection against metal toxicity and oxidative stress. The main purpose of this study was to investigate the effect of heterologous expression of a rice metallothionein isoform (OsMTI-1b) on the tolerance of Saccharomyces cerevisiae to Cd(2+), H2O2 and ethanol stress. The gene encoding OsMTI-1b was cloned into p426GPD as a yeast expression vector. The new construct was transformed to competent cells of S. cerevisiae. After verification of heterologous expression of OsMTI-1b, the new strain and control were grown under stress conditions. In comparison to control strain, the transformed S. cerevisiae cells expressing OsMTI-1b showed more tolerance to Cd(2+) and accumulated more Cd(2+) ions when they were grown in the medium containing CdCl2. In addition, the heterologous expression of GST-OsMTI-1b conferred H2O2 and ethanol tolerance to S. cerevisiae cells. The results indicate that heterologous expression of plant MT isoforms can enhance the tolerance of S. cerevisiae to multiple stresses.

  1. 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

  2. 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.

  3. Ubiquitin-like prokaryotic MoaD as a fusion tag for expression of heterologous proteins in Escherichia coli

    PubMed Central

    2014-01-01

    Background Eukaryotic ubiquitin and SUMO are frequently used as tags to enhance the fusion protein expression in microbial host. They increase the solubility and stability, and protect the peptides from proteolytic degradation due to their stable and highly conserved structures. Few of prokaryotic ubiquitin-like proteins was used as fusion tags except ThiS, which enhances the fusion expression, however, reduces the solubility and stability of the expressed peptides in E. coli. Hence, we investigated if MoaD, a conserved small sulfur carrier in prokaryotes with the similar structure of ubiquitin, could also be used as fusion tag in heterologous expression in E. coli. Results Fusion of MoaD to either end of EGFP enhanced the expression yield of EGFP with a similar efficacy of ThiS. However, the major parts of the fusion proteins were expressed in the aggregated form, which was associated with the retarded folding of EGFP, similar to ThiS fusions. Fusion of MoaD to insulin chain A or B did not boost their expression as efficiently as ThiS tag did, probably due to a less efficient aggregation of products. Interestingly, fusion of MoaD to the murine ribonuclease inhibitor enhanced protein expression by completely protecting the protein from intracellular degradation in contrast to ThiS fusion, which enhanced degradation of this unstable protein when expressed in E. coli. Conclusions Prokaryotic ubiquitin-like protein MoaD can act as a fusion tag to promote the fusion expression with varying mechanisms, which enriches the arsenal of fusion tags in the category of insoluble expression. PMID:24444081

  4. Heterologous expression of porcine reproductive and respiratory syndrome virus glycoprotein 5 in Bordetella bronchisepticaaroA mutant

    PubMed Central

    PARK, Sang Ik; SEO, Ja Young; KIM, Tae Jung

    2016-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important disease around the globe. Protection against this virus remains problematic. Here, we evaluated antibody (IgG & IgA) inducibility of a heterologous PRRSV glycoprotein 5 (GP5) expressed in a live attenuated Bordetella bronchisepticaaroA mutant strain (BBS-GP5). Mice and pigs were primed with recombinant GP5 (rGP5) subcutaneously followed by boosting with live BBS-GP5. As a result, anti-GP5 IgG was induced in both mice (P<0.001) and pigs (P<0.1). Pigs were challenged with live PRRSV (VR2332). Viral RNA was found to be significantly (P<0.01) removed in the vaccinated pig group. Overall, BBS-GP5 is a good candidate as a live attenuated vaccine against PRRSV infection. PMID:27349762

  5. 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.

  6. Impact of Heterologous Expression of Escherichia coli UDP-Glucose Pyrophosphorylase on Trehalose and Glycogen Synthesis in Corynebacterium glutamicum

    PubMed Central

    Padilla, Leandro; Morbach, Susanne; Krämer, Reinhard; Agosin, Eduardo

    2004-01-01

    Trehalose is a disaccharide with a wide range of applications in the food industry. We recently proposed a strategy for trehalose production based on improved strains of the gram-positive bacterium Corynebacterium glutamicum. This microorganism synthesizes trehalose through two major pathways, OtsBA and TreYZ, by using UDP-glucose and ADP-glucose, respectively, as the glucosyl donors. In this paper we describe improvement of the UDP-glucose supply through heterologous expression in C. glutamicum of the UDP-glucose pyrophosphorylase gene from Escherichia coli, either expressed alone or coexpressed with the E. coli ots genes (galU otsBA synthetic operon). The impact of such expression on trehalose accumulation and excretion, glycogen accumulation, and the growth pattern of new recombinant strains is described. Expression of the galU otsBA synthetic operon resulted in a sixfold increase in the accumulated and excreted trehalose relative to that in a wild-type strain. Surprisingly, single expression of galU also resulted in an increase in the accumulated trehalose. This increase in trehalose synthesis was abolished upon deletion of the TreYZ pathway. These results proved that UDP-glucose has an important role not only in the OtsBA pathway but also in the TreYZ pathway. PMID:15240254

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

    PubMed

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

    2016-01-01

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

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

    SciTech Connect

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

    2016-03-14

    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

  9. Collagenolytic Serine-Carboxyl Proteinase from Alicyclobacillus sendaiensis Strain NTAP-1: Purification, Characterization, Gene Cloning, and Heterologous Expression

    PubMed Central

    Tsuruoka, Naoki; Nakayama, Toru; Ashida, Masako; Hemmi, Hisashi; Nakao, Masahiro; Minakata, Hiroyuki; Oyama, Hiroshi; Oda, Kohei; Nishino, Tokuzo

    2003-01-01

    Enzymatic degradation of collagen produces peptides, the collagen peptides, which show a variety of bioactivities of industrial interest. Alicyclobacillus sendaiensis strain NTAP-1, a slightly thermophilic, acidophilic bacterium, extracellularly produces a novel thermostable collagenolytic activity, which exhibits its optimum at the acidic region (pH 3.9) and is potentially applicable to the efficient production of such peptides. Here, we describe the purification to homogeneity, characterization, gene cloning, and heterologous expression of this enzyme, which we call ScpA. Purified ScpA is a monomeric, pepstatin-insensitive carboxyl proteinase with a molecular mass of 37 kDa which exhibited the highest reactivity toward collagen (type I, from a bovine Achilles tendon) among the macromolecular substrates examined. On the basis of the sequences of the peptides obtained by digestion of collagen with ScpA, the following synthetic peptides were designed as substrates for ScpA and kinetically analyzed: Phe-Gly-Pro-Ala*Gly-Pro-Ile-Gly (kcat, 5.41 s−1; Km, 32 μM) and Met-Gly-Pro-Arg*Gly-Phe-Pro-Gly-Ser (kcat, 351 s−1; Km, 214 μM), where the asterisks denote the scissile bonds. The cloned scpA gene encoded a protein of 553 amino acids with a calculated molecular mass of 57,167 Da. Heterologous expression of the scpA gene in the Escherichia coli cells yielded a mature 37-kDa species after a two-step proteolytic cleavage of the precursor protein. Sequencing of the scpA gene revealed that ScpA was a collagenolytic member of the serine-carboxyl proteinase family (the S53 family according to the MEROPS database), which is a recently identified proteinase family on the basis of crystallography results. Unexpectedly, ScpA was highly similar to a member of this family, kumamolysin, whose specificity toward macromolecular substrates has not been defined. PMID:12513991

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

    PubMed

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

    2017-01-10

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

  11. Partial Reconstruction of the Ergot Alkaloid Pathway by Heterologous Gene Expression in Aspergillus nidulans

    PubMed Central

    Ryan, Katy L.; Moore, Christopher T.; Panaccione, Daniel G.

    2013-01-01

    Ergot alkaloids are pharmaceutically and agriculturally important secondary metabolites produced by several species of fungi. Ergot alkaloid pathways vary among different fungal lineages, but the pathway intermediate chanoclavine-I is evolutionarily conserved among ergot alkaloid producers. At least four genes, dmaW, easF, easE, and easC, are necessary for pathway steps prior to chanoclavine-I; however, the sufficiency of these genes for chanoclavine-I synthesis has not been established. A fragment of genomic DNA containing dmaW, easF, easE, and easC was amplified from the human-pathogenic, ergot alkaloid-producing fungus Aspergillus fumigatus and transformed into Aspergillus nidulans, a model fungus that does not contain any of the ergot alkaloid synthesis genes. HPLC and LC-MS analyses demonstrated that transformed A. nidulans strains produced chanoclavine-I and an earlier pathway intermediate. Aspergillus nidulans transformants containing dmaW, easF, and either easE or easC did not produce chanoclavine-I but did produce an early pathway intermediate and, in the case of the easC transformant, an additional ergot alkaloid-like compound. We conclude that dmaW, easF, easE, and easC are sufficient for the synthesis of chanoclavine-I in A. nidulans and expressing ergot alkaloid pathway genes in A. nidulans provides a novel approach to understanding the early steps in ergot alkaloid synthesis. PMID:23435153

  12. Heterologous expression and characterization of a novel branching enzyme from the thermoalkaliphilic anaerobic bacterium Anaerobranca gottschalkii.

    PubMed

    Thiemann, Volker; Saake, Bodo; Vollstedt, Angela; Schäfer, Thomas; Puls, Jürgen; Bertoldo, Costanzo; Freudl, Roland; Antranikian, Garabed

    2006-08-01

    The gene encoding the branching enzyme (BE) from the thermoalkaliphilic, anaerobic bacterium Anaerobranca gottschalkii was fused with a twin arginine translocation protein secretory-pathway-dependent signal sequence from Escherichia coli and expressed in Staphylococcus carnosus. The secreted BE was purified using hydrophobic interaction and gel filtration chromatography. The monomeric enzyme (72 kDa) shows maximal activity at 50 degrees C and pH 7.0. With amylose the BE displays high transglycosylation and extremely low hydrolytic activity. The conversion of amylose and linear dextrins was analysed by applying high-performance anion exchange chromatography and quantitative size-exclusion chromatography. Amylose (10(4)-4 x 10(7) g/mol) was converted to a major extent to products displaying molecular masses of 10(4)-4 x 10(5) g/mol, indicating that the enzyme could be applicable for the production of starch or dextrins with narrow molecular mass distributions. The majority of the transferred oligosaccharides, determined after enzymatic hydrolysis of the newly synthesized alpha-1,6 linkages, ranged between 10(3) and 10(4) g/mol, which corresponds to a degree of polymerisation (DP) of 6-60. The minimal donor chain length is DP 16. Furthermore, the obtained results support the hypotheses of a random endocleavage mechanism of BE and the occurrence of interchain branching.

  13. Escherichia coli EDA is a novel fusion expression partner to improve solubility of aggregation-prone heterologous proteins.

    PubMed

    Kang, Yoon-Sik; Song, Jong-Am; Han, Kyung-Yeon; Lee, Jeewon

    2015-01-20

    Since the use of solubility enhancer proteins is one of the effective methods to produce active recombinant proteins within Escherichia coli, the development of a novel fusion expression partner that can be applied to various aggregation-prone proteins is of crucial importance. In our previous work, two-dimensional electrophoresis (2-DE) was employed to systematically analyze the E. coli BL21 (DE3) proteome profile in response to heat treatment, and KDPG aldolase (EDA) was identified as a heat-responsive and aggregation-resistant protein. When used as fusion expression partner, EDA significantly increased the solubility of seven aggregation-prone heterologous proteins in the E. coli cytoplasm. The efficacy of EDA as a fusion expression partner was evaluated through the analysis of bioactivity or secondary structure of several target proteins: EDA-fusion expression resulted in the synthesis of bioactive human ferritin light chain and bacterial arginine deiminase and the formation of correct secondary structure of human granulocyte colony stimulation factor. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Heterologous expression of rab4 reduces glucose transport and GLUT4 abundance at the cell surface in oocytes.

    PubMed Central

    Mora, S; Monden, I; Zorzano, A; Keller, K

    1997-01-01

    To evaluate the role of the small rab GTP-binding proteins in glucose transporter trafficking, we have heterologously co-expressed rab4 or rab5 and GLUT4 or GLUT1 glucose transporters in Xenopus oocytes. Co-injection of rab4 and GLUT4 cRNAs resulted in a dose-dependent decrease in glucose transport; this effect was specific for rab4, since co-injection of an inactive rab4 mutant or rab5 cRNA did not have any effect on glucose transport. The effect of rab4 was selective for GLUT4, since no effect was detected in GLUT1-expressing oocytes. The inhibitory effect of rab4 on GLUT4-induced glucose transport was not the result of a change in overall cellular levels of GLUT4 glucose transporters. However, rab4 expression caused a marked decrease in the abundance of GLUT4 transporters present at the cell surface. Finally, rab4 and inhibitors of PtdIns 3-kinase showed additive effects in decreasing glucose transport in GLUT4-expressing oocytes. We conclude that rab4 plays an important role in the regulation of the intracellular GLUT4 trafficking pathway, by contributing to the intracellular retention of GLUT4 through a PtdIns 3-kinase-independent mechanism. PMID:9182703

  15. 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.

  16. A Simple, Highly Efficient Method for Heterologous Expression in Mammalian Primary Neurons Using Cationic Lipid-mediated mRNA Transfection

    PubMed Central

    Williams, Damian J.; Puhl, Henry L.; Ikeda, Stephen R.

    2010-01-01

    Expression of heterologous proteins in adult mammalian neurons is a valuable technique for the study of neuronal function. The post-mitotic nature of mature neurons prevents effective DNA transfection using simple, cationic lipid-based methods. Adequate heterologous protein expression is often only achievable using complex techniques that, in many cases, are associated with substantial toxicity. Here, a simple method for high efficiency transfection of mammalian primary neurons using in vitro transcribed mRNA and the cationic lipid transfection reagent Lipofectamine™ 2000 is described. Optimal transfection conditions were established in adult mouse dissociated dorsal root ganglion (DRG) neurons using a 96-well based luciferase activity assay. Using these conditions, a transfection efficiency of 25% was achieved in DRG neurons transfected with EGFP mRNA. High transfection efficiencies were also obtained in dissociated rat superior cervical ganglion (SCG) neurons and mouse cortical and hippocampal cultures. Endogenous Ca2+ currents in EGFP mRNA-transfected SCG neurons were not significantly different from untransfected neurons, which suggested that this technique is well suited for heterologous expression in patch clamp recording experiments. Functional expression of a cannabinoid receptor (CB1R), a G protein inwardly rectifying K+ channel (GIRK4) and a dominant-negative G protein α-subunit mutant (GoA G203T) indicate that the levels of heterologous protein expression attainable using mRNA transfection are suitable for most functional protein studies. This study demonstrates that mRNA transfection is a straightforward and effective method for heterologous expression in neurons and is likely to have many applications in neuroscience research. PMID:21267423

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

    PubMed

    Ni-Komatsu, Li; Orlow, Seth J

    2006-03-01

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

  18. Heterologous expression and characterization of choline oxidase from the soil bacterium Arthrobacter nicotianae.

    PubMed

    Ribitsch, D; Karl, W; Wehrschütz-Sigl, E; Tutz, S; Remler, P; Weber, H J; Gruber, K; Stehr, R; Bessler, C; Hoven, N; Sauter, K; Maurer, K H; Schwab, H

    2009-01-01

    In the course of a microbial screening of soil samples for new oxidases, different enrichment strategies were carried out. With choline as the only carbon source, a microorganism was isolated and identified as Arthrobacter nicotianae. From this strain, a gene coding for a choline oxidase was isolated from chromosomal DNA. This gene named codA was cloned in Escherichia coli BL21-Gold and the protein (An_CodA) heterologously overexpressed as a soluble intracellular protein of 59.1 kDa. Basic biochemical characterization of purified protein revealed a pH optimum of 7.4 and activity over a broad temperature range (15-70 degrees C). Specific activities were determined toward choline chloride (4.70 +/- 0.12 U/mg) and the synthetic analogs bis(2-hydroxyethyl)-dimethylammonium chloride (0.05 +/- 0.45 x 10(-2) U/mg) and tris-(2-hydroxyethyl)-methylammonium methylsulfate (0.01 +/- 0.12 x 10(-2) U/mg). With increasing number of oxidizable groups, a significant decrease in activity was noted. Determination of kinetic parameters in atmorspheric oxygen resulted in K (M) = 1.51 +/- 0.09 mM and V (max) = 42.73 +/- 0.42 mU/min for choline chloride and K (M) = 4.77 +/- 0.76 mM and V (max) = 48.40 +/- 2.88 mU/min for the reaction intermediate betaine aldehyde respectively. Nuclear magnetic resonance spectroscopic analysis of the products formed during the enzyme reaction with choline chloride showed that in vitro the intermediate betaine aldehyde exists also free in solution.

  19. Chemical and Steady-State Kinetic Analyses of a Heterologously Expressed Heme Dependent Chlorite Dismutase†

    PubMed Central

    Streit, Bennett R.; DuBois, Jennifer L.

    2013-01-01

    Chlorite dismutase carries out the heme-catalyzed decomposition of ClO2– to Cl– and O2, an unusual transformation with biotechnological and bioremediative applications. The enzyme has been successfully overexpressed for the first time in highly functional form in Escherichia coli and its steady state kinetics studied. The purified enzyme is abundant (55 mg/L cell culture), highly active (~4.7 × 103 μmol of ClO2– min–1 mg–1 subunit) and nearly stoichiometric in heme; further, it shares spectroscopic and physicochemical features with chlorite dismutases previously isolated from three organisms. A careful study of the enzyme's steady state kinetics has been carried out. ClO2– consumption and O2 release rates were measured, yielding comparable values of kcat (4.5 × 105 min–1), Km (~215 μM), and kcat/Km (3.5 × 107 M–1 s–1) via either method (4 °C, pH 6.8; all values referenced per heme-containing subunit). ClO2–:O2 stoichiometry exhibited a 1:1 relationship under all conditions measured. Though the value of kcat/Km indicates near diffusion control of the reaction, viscosogens had no effect on kcat/Km or Vmax. The product O2 did not inhibit the reaction at saturating [O2], but Cl– is a mixed inhibitor with relatively high values of KI (225 mM for enzyme and 95.6 mM for the enzyme–substrate complex), indicating a relatively low affinity of the heme iron for halogen ions. Chlorite irreversibly inactivates the enzyme after ~1.7 × 104 turnovers (per heme) and with a half-life of 0.39 min, resulting in bleaching of the heme chromophore. The inactivation KI (Kinact) of 166 μM is similar in magnitude to Km, consistent with a common Michaelis complex on the pathway to both reaction and inactivation. The one-electron peroxidase substrate guaiacol offers incomplete protection of the enzyme from inactivation. Mechanisms in keeping with the available data and the properties of other well-described heme enzymes are proposed. PMID:18422344

  20. Heterologous protein-DNA interactions lead to biased allelic expression of circadian clock genes in interspecific hybrids

    PubMed Central

    Ng, Danny W-K.; Chen, Helen H. Y.; Chen, Z. Jeffrey

    2017-01-01

    Genomic interactions in allopolyploids create expression variation of homoeologous alleles through protein-protein and protein-DNA interactions. However, the molecular basis for this is largely unknown. Here we investigated the protein-protein and protein-DNA interactions among homoeologous transcription factors in the circadian-clock feedback loop, consisting of CCA1 HIKING EXPEDITION (CHE), CIRCADIAN CLOCK ASSOCIATED1 (CCA1), and TIMING OF CAB EXPRESSION1 (TOC1), plus the interaction with a chromatin factor, HISTONE DEACETYLASE1 (HD1). In the allotetraploids formed between A. thaliana (At) and Arabidopsis arenosa (Aa), AtCCA1 is expressed at lower levels than AaCCA1, which could alter clock output traits. The reduced AtCCA1 expressions in the allotetraploids are consistent with the biochemical data that AaCHE showed preferential binding to the AtCCA1 promoter, in which AaCHE interacts with a higher affinity to AtHD1 than AtCHE. AaCHE also showed a higher affinity to TOC1 than AtCHE, consistent with the effect of TOC1 on repressing CCA1. Thus, stronger AaCHE-TOC1 and AaCHE-AtHD1 interactions reduce AtCC1 allelic expression. Our current data suggest a biochemical basis for protein interactions in trans with a preference to the cis-acting elements in heterologous combinations to reduce AtCCA1 expression, while altered CCA1 expression has been shown to affect metabolic and biomass heterosis in interspecific hybrids or allotetraploids. PMID:28345627

  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. Heterologous protein-DNA interactions lead to biased allelic expression of circadian clock genes in interspecific hybrids.

    PubMed

    Ng, Danny W-K; Chen, Helen H Y; Chen, Z Jeffrey

    2017-03-27

    Genomic interactions in allopolyploids create expression variation of homoeologous alleles through protein-protein and protein-DNA interactions. However, the molecular basis for this is largely unknown. Here we investigated the protein-protein and protein-DNA interactions among homoeologous transcription factors in the circadian-clock feedback loop, consisting of CCA1 HIKING EXPEDITION (CHE), CIRCADIAN CLOCK ASSOCIATED1 (CCA1), and TIMING OF CAB EXPRESSION1 (TOC1), plus the interaction with a chromatin factor, HISTONE DEACETYLASE1 (HD1). In the allotetraploids formed between A. thaliana (At) and Arabidopsis arenosa (Aa), AtCCA1 is expressed at lower levels than AaCCA1, which could alter clock output traits. The reduced AtCCA1 expressions in the allotetraploids are consistent with the biochemical data that AaCHE showed preferential binding to the AtCCA1 promoter, in which AaCHE interacts with a higher affinity to AtHD1 than AtCHE. AaCHE also showed a higher affinity to TOC1 than AtCHE, consistent with the effect of TOC1 on repressing CCA1. Thus, stronger AaCHE-TOC1 and AaCHE-AtHD1 interactions reduce AtCC1 allelic expression. Our current data suggest a biochemical basis for protein interactions in trans with a preference to the cis-acting elements in heterologous combinations to reduce AtCCA1 expression, while altered CCA1 expression has been shown to affect metabolic and biomass heterosis in interspecific hybrids or allotetraploids.

  3. Cytochrome c550 from Thiobacillus versutus: cloning, expression in Escherichia coli, and purification of the heterologous holoprotein.

    PubMed Central

    Ubbink, M; Van Beeumen, J; Canters, G W

    1992-01-01

    The gene coding for cytochrome c550 from Thiobacillus versutus, cycA, has been cloned and sequenced. It codes for a protein of 134 amino acids plus a 19-amino-acid-long signal peptide. Both coding and noncoding DNA sequences of the clone are homologous to the Paracoccus denitrificans DNA sequence. An expression vector was constructed by cloning the cycA gene directly behind the lac promoter of pUC. The cycA gene was expressed in Escherichia coli under semianaerobic conditions, and mature holo-cytochrome c550 was isolated with the periplasmic soluble protein fraction. Under both aerobic and anaerobic conditions, significantly less cytochrome c550 was produced. The heterologously expressed cytochrome c550 was isolated and purified to better than 95% purity and was compared with cytochrome c550 isolated and purified from T. versutus. No structural differences could be detected by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis UV-visible light spectroscopy, and 1H nuclear magnetic resonance spectroscopy, indicating that E. coli produces the cytochrome and attaches the heme correctly. Images PMID:1339423

  4. Do voltage-dependent K+ channels require Ca2+? A critical test employing a heterologous expression system.

    PubMed Central

    Armstrong, C M; Miller, C

    1990-01-01

    Removal of Ca2+ from the solution bathing neurons is known in many cases to alter the gating properties of voltage-dependent K+ channels and to induce a large, nonselective "leak" conductance. We used a heterologous expression system to test whether the leak conductance observed in neurons is mediated by voltage-dependent K+ channels in an altered, debased conformation. Voltage-dependent K+ channels were expressed in an insect cell line infected with a recombinant baculovirus carrying the cDNA for Drosophila Shaker "A-type" K+ channels. These expressed channels respond to low Ca2+ identically to voltage-dependent K+ channels in native neuronal membranes; upon removal of external Ca2+, Shaker K+ currents disappear and are replaced by a steady, nonselective leak conductance. However, control cells devoid of Shaker channels were free of any voltage-dependent conductances and did not generate a leak when external Ca2+ was removed. These results show that Ca2+ is essential for proper function of voltage-dependent K+ channels and is required to stabilize the native conformations of these membrane proteins. PMID:2217187

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

    PubMed

    Lemtiri-Chlieh, Fouad; Ali, Rashid

    2013-01-01

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

  6. Heterologous expression of stress-responsive DUF538 domain containing protein and its morpho-biochemical consequences.

    PubMed

    Gholizadeh, Ashraf

    2011-06-01

    As a usual response, plants induce/activate various proteins which are thought to be involved in defense mechanisms against the biotic and abiotic stresses they may be confronted with. The novel DUF538 domain containing proteins with unknown functions have been found to be induced/activated in response to different environmental stress stimuli in plants. In order to perform biochemical studies with these new plant stress-responsive proteins, a cDNA containing DUF538 domain was amplified from Celosia cristata full-length leaf expression library using a specific primer set. The isolated cDNA was subsequently expressed in Escherichia coli as a part of maltose-binding fusion protein (MBP-DUF538 construct) and purified at the yield of about 32 mg per liter of cell culture by affinity chromatography without affecting the recombinant bacterial cell growth. The purified fusion product was exogenously applied (10 μg per 4 cm(2)) on the leaves of Nicotiana tobaccum L. The results revealed that fused DUF538 protein does not induce morphological reposes, but elevates redox enzyme activities including catalase, peroxidase, polyphenol oxidase and phenyalanine ammonia lyase. This is the first time ever time report with respect to the heterologous expression of a plant stress-responsive DUF538 domain that may provide a basis to study its physiological roles and biochemical activities in vitro and in vivo.

  7. 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.

  8. Heterologous expression of GmSIP1;3 from soybean in tobacco showed and growth retardation and tolerance to hydrogen peroxide.

    PubMed

    Zhang, Dayong; Huang, Yihong; Kumar, Manoj; Wan, Qun; Xu, Zhaolong; Shao, Hongbo; Pandey, Girdhar K

    2017-10-01

    Aquaporins (AQPs) are transmembrane protein channels that are members of Major Intrinsic Proteins (MIP) superfamily. AQPs play important roles in plant reproduction, cell elongation, osmoregulation, influence leaf physiology and are responsive to drought and salt tolerance. Small intrinsic proteins (SIPs)belongs to one of the groups of AQPs, which are mainly localized to endoplasmic reticulum(ER). While this group of aquaporin is being well studied in Arabidopsis, grape and other plant species, not much is known about the molecular regulatory mechanisms driven by ER-type AQPs in Glycine Max. In this study, the function of GmSIP1;3 is studied in detail by using both yeast and plant systems. GmSIP1;3 showed a ubiquitous expression pattern in all different tissues in Glycine Max. Heterologous expression of GmSIP1;3 in Nicotiana tabacum conferred a short root phenotype,growth retardation at seedling stage and significant tolerance to oxidative stress (H2O2) both in yeast and plant systems. Auxin (IAA) content significantly increased in transgenic plants compared with that of wild type, however, the abscisic acid (ABA) content was significantly reduced. Subcellular localization and colocalization analyses showed GmSIP1;3 localized to ER plasma membrane. On the basis of these observations, we postulate that GmSIP1;3 is involved in oxidative stress pathways. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2016-05-01

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

  10. [Characterization of β-1, 4-mannanase from Bacillus pumilus and heterologous expression in Lactobacillus casei].

    PubMed

    Zou, Yexia; Lin, Jinzhong; Bu, Xuemei; Jiang, Lulu; Chen, Zhengjun; Ge, Xiangyang

    2015-12-04

    Lactobacillus casei is widely used in food production and feed industry. The aim of this study was to construct the recombinant expression mannanase Lb. casei. The mature peptide gene of β-1,4-mannanase from Bacillus pumilus was cloned into expression vectors pELX1 and pELSH, then electroporated into Lb. casei, establishing an intracellular and a secretion expression mannanase Lb. casei respectively. After incubation, the specific activity of β-1,4-mannanase was 23 U/mg whole cell protein for intracellular expression and 8.8 U/mL for secretion expression in supernatant. Mannanase gene expression in Lb. casei provides application prospect and deserves further study.

  11. Cold adaptation of a psychrophilic chaperonin from Psychrobacter sp. and its application for heterologous protein expression.

    PubMed

    Kim, Han-Woo; Wi, Ah Ram; Jeon, Byoung Wook; Lee, Jun Hyuck; Shin, Seung Chul; Park, Hyun; Jeon, Sung-Jong

    2015-09-01

    A chaperonin, PsyGroELS, from the Antarctic psychrophilic bacterium Psychrobacter sp. PAMC21119, was examined for its role in cold adaptation when expressed in a mesophilic Escherichia coli strain. Growth of E. coli harboring PsyGroELS at 10 °C was increased compared to the control strain. A co-expression system using PsyGroELS was developed to increase productivity of the psychrophilic enzyme PsyEst9. PsyEst9 was cloned and expressed using three E. coli variants that co-expressed GroELS from PAMC21119, E. coli, or Oleispira antarctica RB8(T). Co-expression with PsyGroELS was more effective for the production of PsyEst9 compared tothe other chaperonins. PsyGroELS confers cold tolerance to E. coli, and shows potential as an effective co-expression system for the stable production of psychrophilic proteins.

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

    PubMed

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

    2016-12-01

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

  13. 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.

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

    PubMed

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

    2017-01-20

    Welan gum is a microbial polysaccharide produced by Sphingomonas sp. Its production is limited by the dissolved oxygen levels in the highly viscous fermentation. A strategy of heterologous expression of the Vitreoscilla hemoglobin gene in Sphingomonas sp. HT-1 was investigated to alleviate oxygen limitation and improve the yield of welan gum. Ultimately, the welan gum production increased from 25.3g/L to 34.6g/L, whereas the rheological behavior of welan gum solutions remained virtually unchanged. The transcriptional levels of the key genes in the electron transfer chain, TCA cycle and welan gum synthesis pathway, as well as ATP level revealed that the VHb expression in Sphingomonas sp. HT-1 enhanced welan gum biosynthesis by improving respiration and ATP supply. This study would pave the genetic manipulation way for enhancing welan gum yield, and it's also of great importance for the industrial applications of welan gum under harsh conditions. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. 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

  16. 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

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

    PubMed Central

    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

  18. Co-expression of multiple target proteins in plants from a tobacco mosaic virus vector using a combination of homologous and heterologous subgenomic promoters.

    PubMed

    Roy, Gourgopal; Weisburg, Sangeetha; Foy, Kelly; Rabindran, Shailaja; Mett, Vadim; Yusibov, Vidadi

    2011-11-01

    To co-express multiple target proteins, we engineered a single-component chimeric tobacco mosaic virus (TMV)-based vector containing homologous and heterologous capsid protein subgenomic RNA promoters. Delivery of this vector into Nicotiana benthamiana plants via agroinfiltration resulted in co-expression of two reporter genes within a single cell. Furthermore, co-expression of a host-specific antisense RNA or a silencing suppressor protein from this vector augmented the accumulation of green fluorescent protein or a vaccine antigen, hemagglutinin from avian influenza virus A/Vietnam/1194/04. These findings suggest that this chimeric vector utilizing the homologous and heterologous subgenomic TMV promoters has a potential for high-level production of multiple therapeutic proteins including monoclonal antibodies.

  19. 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.

  20. 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

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

    PubMed

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

    2015-02-01

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

  2. Heterologous expression of Candida albicans Pma1p in Saccharomyces cerevisiae

    PubMed Central

    Keniya, Mikhail V.; Cannon, Richard D.; Nguyễn, ẤnBình; Tyndall, Joel D. A.; Monk, Brian C.

    2015-01-01

    Candida albicans is a major cause of opportunistic and life-threatening systemic fungal infections, especially in the immunocompromised. The plasma membrane proton pumping ATPase (Pma1p) is an essential enzyme that generates the electrochemical gradient required for cell growth. We expressed C. albicans Pma1p (CaPma1p) in Saccharomyces cerevisiae to facilitate screening for inhibitors. Replacement of S. cerevisiae PMA1 with C. albicans PMA1 gave clones expressing CaPma1p that grew slowly at low pH. CaPma1p was expressed at significantly lower levels and had lower specific activity than the native Pma1p. It also conferred pH sensitivity, hygromycin B resistance and low levels of glucose-dependent proton pumping. Recombination between CaPMA1 and the homologous non-essential ScPMA2 resulted in chimeric suppressor mutants that expressed functional CaPma1p with improved H+-ATPase activity and growth rates at low pH. Molecular models of suppressor mutants identified specific amino acids (between 531-595 in CaPma1p) that may affect regulation of the activity of Pma1p oligomers in S. cerevisiae. A modified CaPma1p chimeric construct containing only 5 amino acids from ScPma2p enabled the expression of a fully functional enzyme for drug screens and structural resolution. PMID:23374681

  3. Characterization of the human artemis promoter by heterologous gene expression in vitro and in vivo.

    PubMed

    Multhaup, Megan M; Gurram, Sweta; Podetz-Pedersen, Kelly M; Karlen, Andrea D; Swanson, Debra L; Somia, Nikunj V; Hackett, Perry B; Cowan, Morton J; McIvor, R Scott

    2011-10-01

    Artemis is an endonucleolytic enzyme involved in nonhomologous double-strand break repair and V(D)J recombination. Deficiency of Artemis results in a B- T- radiosensitive severe combined immunodeficiency, which may potentially be treatable by Artemis gene transfer into hematopoietic stem cells. However, we recently found that overexpression of Artemis after lentiviral transduction resulted in global DNA damage and increased apoptosis. These results imply the necessity of effecting natural levels of Artemis expression, so we isolated a 1 kilobase DNA sequence upstream of the human Artemis gene to recover and characterize the Artemis promoter (APro). The sequence includes numerous potential transcription factor-binding sites, and several transcriptional start sites were mapped by 5' rapid amplification of cDNA ends. APro and deletion constructs conferred significant reporter gene expression in vitro that was markedly reduced in comparison to expression regulated by the human elongation factor 1-α promoter. Ex vivo lentiviral transduction of an APro-regulated green fluorescent protein (GFP) construct in mouse marrow supported GFP expression throughout hematopoeitic lineages in primary transplant recipients and was sustained in secondary recipients. The human Artemis promoter thus provides sustained and moderate levels of gene expression that will be of significant utility for therapeutic gene transfer into hematopoeitic stem cells.

  4. Characterization of the Human Artemis Promoter by Heterologous Gene Expression In Vitro and In Vivo

    PubMed Central

    Multhaup, Megan M.; Gurram, Sweta; Podetz-Pedersen, Kelly M.; Karlen, Andrea D.; Swanson, Debra L.; Somia, Nikunj V.; Hackett, Perry B.; Cowan, Morton J.

    2011-01-01

    Artemis is an endonucleolytic enzyme involved in nonhomologous double-strand break repair and V(D)J recombination. Deficiency of Artemis results in a B− T− radiosensitive severe combined immunodeficiency, which may potentially be treatable by Artemis gene transfer into hematopoietic stem cells. However, we recently found that overexpression of Artemis after lentiviral transduction resulted in global DNA damage and increased apoptosis. These results imply the necessity of effecting natural levels of Artemis expression, so we isolated a 1 kilobase DNA sequence upstream of the human Artemis gene to recover and characterize the Artemis promoter (APro). The sequence includes numerous potential transcription factor-binding sites, and several transcriptional start sites were mapped by 5′ rapid amplification of cDNA ends. APro and deletion constructs conferred significant reporter gene expression in vitro that was markedly reduced in comparison to expression regulated by the human elongation factor 1-α promoter. Ex vivo lentiviral transduction of an APro-regulated green fluorescent protein (GFP) construct in mouse marrow supported GFP expression throughout hematopoeitic lineages in primary transplant recipients and was sustained in secondary recipients. The human Artemis promoter thus provides sustained and moderate levels of gene expression that will be of significant utility for therapeutic gene transfer into hematopoeitic stem cells. PMID:21663454

  5. 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.

  6. 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.

  7. 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.

  8. Heterologous expression of the bacteriocin mesentericin Y105 using the dedicated transport system and the general secretion pathway.

    PubMed

    Biet, F; Berjeaud, J M; Worobo, R W; Cenatiempo, Y; Fremaux, C

    1998-10-01

    Two different N-terminal extensions have been identified within class II bacteriocin precursors. The first one is a two-glycine-type leader peptide associated with a dedicated ATP-binding cassette transporter. The second is a signal peptide which directs the bacteriocin precursor to the general secretion machinery. Mesentericin Y105 is a class II anti-Listeria bacteriocin produced by Leuconostoc mesenteroides Y105 via a dedicated transport system (DTS). To investigate heterologous expression systems capable of producing mesentericin Y105 in various hosts, two different secretion vectors were constructed. One of them, containing the mesentericin Y105 structural gene fused to the segment encoding the divergicin A signal peptide, was introduced into Escherichia coli, Leuconostoc subsp. and Lactococcus subsp. In E. coli, mesentericin Y105 production was linked to a putative periplasmic toxicity. To take advantage of this secretion system, the mesentericin Y105 precursor was also produced in E. coli. It was demonstrated that this pre-bacteriocin exhibited some antagonistic activity against Listeria. To allow for a comparison between the two different transport systems, mesentericin Y105 production using the vector containing the mesentericin Y105 structural gene and its DTS transporter operon was examined. The production of mesentericin Y105 was monitored by a new fast purification method followed by MS analysis. It was shown that, in Leuconostoc, the production of mesentericin Y105 is enhanced via the DTS compared to the general secretion pathway.

  9. Mining Xanthomonas and Streptomyces genomes for new pectinase-encoding sequences and their heterologous expression in Escherichia coli.

    PubMed

    Xiao, Zhizhuang; Boyd, Jason; Grosse, Stephan; Beauchemin, Manon; Coupe, Elizabeth; Lau, Peter C K

    2008-04-01

    Microbial genome sequencing has left a legacy of annotated yet uncharacterized genes or open reading frames, activities that may have useful applications in health and/or the environment. We are interested in the discovery and characterization of potentially new pectinolytic activities for the enzymatic retting of natural bast fibers such as hemp and flax. A highlight in this study is the discovery of a cold-active pectate lyase among five pectate-lyase-encoding sequences and two polygalacturonase-encoding sequences that we have cloned from the genomes of Xanthomonas campestris pv. campestris and Streptomyces coelicolor A3(2). Heterologous expression of these sequences as active pectate lyases and polygalacturonases required their subcloning in Escherichia coli Rosetta cells. The most active recombinant pectate lyase (XcPL NP_638163), a cold-active pectate lyase (XcPL NP_636037), and a polygalacturonase (XcPG NP_638805) were purified to near homogeneity and their kinetic parameters were determined. A significant amount of pectin degradation products was shown to be released by the two pectate lyases but not the polygalacturonase when hemp fiber pectin was used as substrate. Results of this study showed that genome data mining, besides an economical approach to new gene acquisition, may uncover new findings such as the discovery of a cold-active pectate-lyase-encoding sequence from X. campestris, a mesophilic microorganism.

  10. 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.

  11. 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.

  12. Heterologous expression and nonsense suppression provide insights into agonist behavior at α6β2 nicotinic acetylcholine receptors.

    PubMed

    Post, Michael R; Limapichat, Walrati; Lester, Henry A; Dougherty, Dennis A

    2015-10-01

    The α6-containing subtypes of the nicotinic acetylcholine receptor (nAChR) are localized to presynaptic terminals of the dopaminergic pathways of the central nervous system. Selective ligands for these nAChRs are potentially useful in both Parkinson's disease and addiction. For these and other goals, it is important to distinguish the binding behavior of agonists at the α6-β2 binding site versus other subtypes. To study this problem, we apply nonsense suppression-based non-canonical amino acid mutagenesis. We report a combination of four mutations in α6β2 that yield high-level heterologous expression in Xenopus oocytes. By varying mRNA injection ratios, two populations were observed with unique characteristics, likely due to differing stoichiometries. Responses to nine known nAChR agonists were analyzed at the receptor, and their corresponding EC50 values and efficacies are reported. The system is compatible with nonsense suppression, allowing structure-function studies between Trp149 - a conserved residue on loop B found to make a cation-π interaction at several nAChR subtypes - and several agonists. These studies reveal that acetylcholine forms a strong cation-π interaction with the conserved tryptophan, while nicotine and TC299423 do not, suggesting a unique pharmacology for the α6β2 nAChR.

  13. Heterologous expression of AtClo1, a plant oil body protein, induces lipid accumulation in yeast.

    PubMed

    Froissard, Marine; D'andréa, Sabine; Boulard, Céline; Chardot, Thierry

    2009-05-01

    Proteomic approaches on lipid bodies have led to the identification of proteins associated with this compartment, showing that, rather than the inert fat depot, lipid droplets appear as complex dynamic organelles with roles in metabolism control and cell signaling. We focused our investigations on caleosin [Arabidopsis thaliana caleosin 1 (AtClo1)], a minor protein of the Arabidopsis thaliana seed lipid body. AtClo1 shares an original triblock structure, which confers to the protein the capacity to insert at the lipid body surface. In addition, AtClo1 possesses a calcium-binding domain. The study of plants deficient in caleosin revealed its involvement in storage lipid degradation during seed germination. Using Saccharomyces cerevisiae as a heterologous expression system, we investigated the potential role of AtClo1 in lipid body biogenesis and filling. The green fluorescent protein-tagged protein was correctly targeted to lipid bodies. We observed an increase in the number and size of lipid bodies. Moreover, transformed yeasts accumulated more fatty acids (+46.6%). We confirmed that this excess of fatty acids was due to overaccumulation of lipid body neutral lipids, triacylglycerols and steryl esters. We showed that the original intrinsic properties of AtClo1 protein were sufficient to generate a functional lipid body membrane and to promote overaccumulation of storage lipids in yeast oil bodies.

  14. Biosynthesis of a natural polyketide-isoprenoid hybrid compound, furaquinocin A: identification and heterologous expression of the gene cluster.

    PubMed

    Kawasaki, Takashi; Hayashi, Yutaka; Kuzuyama, Tomohisa; Furihata, Kazuo; Itoh, Nobuya; Seto, Haruo; Dairi, Tohru

    2006-02-01

    Furaquinocin (FQ) A, produced by Streptomyces sp. strain KO-3988, is a natural polyketide-isoprenoid hybrid compound that exhibits a potent antitumor activity. As a first step toward understanding the biosynthetic machinery of this unique and pharmaceutically useful compound, we have cloned an FQ A biosynthetic gene cluster by taking advantage of the fact that an isoprenoid biosynthetic gene cluster generally exists in flanking regions of the mevalonate (MV) pathway gene cluster in actinomycetes. Interestingly, Streptomyces sp. strain KO-3988 was the first example of a microorganism equipped with two distinct mevalonate pathway gene clusters. We were able to localize a 25-kb DNA region that harbored FQ A biosynthetic genes (fur genes) in both the upstream and downstream regions of one of the MV pathway gene clusters (MV2) by using heterologous expression in Streptomyces lividans TK23. This was the first example of a gene cluster responsible for the biosynthesis of a polyketide-isoprenoid hybrid compound. We have also confirmed that four genes responsible for viguiepinol [3-hydroxypimara-9(11),15-diene] biosynthesis exist in the upstream region of the other MV pathway gene cluster (MV1), which had previously been cloned from strain KO-3988. This was the first example of prokaryotic enzymes with these biosynthetic functions. By phylogenetic analysis, these two MV pathway clusters were identified as probably being independently distributed in strain KO-3988 (orthologs), rather than one cluster being generated by the duplication of the other cluster (paralogs).

  15. Heterologous expression of glycoside hydrolase family 2 and 42 β-galactosidases of lactic acid bacteria in Lactococcus lactis.

    PubMed

    Schwab, Clarissa; Sørensen, Kim I; Gänzle, Michael G

    2010-10-01

    This study characterized a glycoside hydrolase family 42 (GH42) β-galactosidase of Lactobacillus acidophilus (LacA) and compared lactose hydrolysis, hydrolysis of oNPG, pNPG and pNPG-analogues and galactooligosaccharides (GOSs) formation to GH2 β-galactosidases of Streptococcus thermophilus (LacZ type), Lactobacillus plantarum and Leuconostoc mesenteroides subsp. cremoris (both LacLM type). Beta-galactosidases were heterologously expressed in Lactococcus lactis using a p170 derived promoter; experiments were performed with L. lactis crude cell extract (CCE). The novel GH42 β-galactosidase of Lb. acidophilus had lower activity on lactose, oNPG and pNPG but higher relative activity on pNP analogues compared to GH2 β-galactosidases, and did not transgalactosylate at high lactose concentrations. Temperature and pH optima for lactose hydrolysis varied between GH2 β-galactosidases. oNPG and pNPG were the preferred substrates for hydrolysis; in comparison, activity on pNPG-analogues was less than 1.5%. GH2 β-galactosidases formed structurally similar GOS with varying preferences. The diversity of lactic acid bacteria β-galactosidase activity in L. lactis CCE can be exploited in future nutritional or therapeutic applications. Copyright © 2010 Elsevier GmbH. All rights reserved.

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

    PubMed

    Srichaisupakit, Akkaraphol; Ohashi, Takao; Fujiyama, Kazuhito

    2014-09-01

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

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

    PubMed

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

    2013-08-01

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

  18. Dynamin-like protein-dependent formation of Woronin bodies in Saccharomyces cerevisiae upon heterologous expression of a single protein.

    PubMed

    Würtz, Christian; Schliebs, Wolfgang; Erdmann, Ralf; Rottensteiner, Hanspeter

    2008-06-01

    Filamentous ascomycetes harbor Woronin bodies and glyoxysomes, two types of microbodies, within one cell at the same time. The dominant protein of the Neurospora crassa Woronin body, HEX1, forms a hexagonal core crystal via oligomerization and evidence has accumulated that Woronin bodies bud off from glyoxysomes. We analyzed whether HEX1 is sufficient to induce Woronin body formation upon heterologous expression in Saccharomyces cerevisiae, an organism devoid of this specialized organelle. In wild-type strain BY4742, initial import of HEX1 into existing peroxisomes enabled the formation of organelles with a hexagonal crystal. The observed structures mimicked the shape of genuine Woronin bodies, but exhibited a lower density and were significantly larger. Double-immunofluorescence analysis revealed that hexagonal HEX1 structures only occasionally co-localized with peroxisomal marker proteins, indicating that the Woronin-body-like structures are well separated from peroxisomes. In cells lacking Vps1p and Dnm1p, dynamin-like proteins required for the division of peroxisomes, the Woronin-body-like organelles remained attached to peroxisomes. The data indicate that Woronin bodies emerge after the formation of a HEX1 core crystal within peroxisomes followed by Vps1p- and Dnm1p-mediated fission.

  19. Effect of heterologous expression of molecular chaperone DnaK from Tetragenococcus halophilus on salinity adaptation of Escherichia coli.

    PubMed

    Sugimoto, Shinya; Nakayama, Jiro; Fukuda, Daisuke; Sonezaki, Shino; Watanabe, Maki; Tosukhowong, Amonlaya; Sonomoto, Kenji

    2003-01-01

    Molecular chaperone DnaK of halophilic Tetragenococcus halophilus JCM5888 was characterized under salinity conditions both in vitro and in vivo. The dnaK gene was cloned into an expression vector and transformed into Escherichia coli. The DnaK protein obtained from the recombinant E. coli showed a significantly higher refolding activity of denatured lactate dehydrogenase than that from non-halophilic Lactococcus lactis under NaCl concentrations higher than 1 M. E. coli without the overexpression of DnaK exhibited a growth profile with a prolonged lag phase and suppressed maximum cell density in Luria-Bertani medium containing 5% (0.86 M) NaCl. On the contrary, the overexpression of T. halophilus DnaK greatly shortened this prolonged lag phase with no effect on maximum growth, while that of L. lactis DnaK decreased maximum growth. The amount of protein aggregates was increased by salt stress in the E. coli cells, while this aggregation was greatly suppressed by the overexpression of T, halophilus DnaK. These results suggest that heterologous overexpression of T. halophilus DnaK, via its chaperone activity, promotes salinity adaptation of E. coli.

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

    PubMed

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

    2017-12-01

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

  2. Erysipelothrix rhusiopathiae YS-1 as a live vaccine vehicle for heterologous protein expression and intranasal immunization of pigs.

    PubMed

    Shimoji, Yoshihiro; Oishi, Eiji; Kitajima, Takashi; Muneta, Yoshihiro; Shimizu, Shinya; Mori, Yasuyuki

    2002-01-01

    We have developed a system in which a foreign antigen is delivered and expressed on the surface of an attenuated strain of Erysipelothrix rhusiopathiae YS-1 and have examined the ability of a such recombinant E. rhusiopathiae strain to function as a mucosal vaccine vector. The C-terminal portion, including two repeat regions, R1 and R2, of the P97 adhesin of Mycoplasma hyopneumoniae strain E-1 was successfully translocated and expressed on the E. rhusiopathiae YS-1 cell surface after it was fused to SpaA.1, a cell surface protective antigen of E. rhusiopathiae. BALB/c mice subcutaneously immunized with the E. rhusiopathiae recombinant strains developed specific antibodies against SpaA.1 protein and were protected from lethal challenge with the highly virulent homologous E. rhusiopathiae Fujisawa-SmR strain, showing the efficacy of this heterologous-antigen expression system as a vaccine against E. rhusiopathiae infection. To determine whether protective immune responses are induced in target species, newborn, specific-pathogen-free piglets were immunized intranasally with a recombinant strain designated YS-19. The immunized piglets developed specific anti-SpaA.1 immunoglobulin G (IgG) antibodies in their serum and were protected from death by erysipelas, showing that mucosal vaccination of piglets with YS-19 induces systemic immune responses. Furthermore, YS-19-immunized piglets showed higher levels of P97-specific IgA antibodies in the respiratory tract than did YS-1-immunized piglets. Thus, E. rhusiopathiae YS-1 appears to be a promising vaccine vector for mucosal delivery that can induce local and systemic immune responses.

  3. A new system for fast and quantitative analysis of heterologous gene expression in plants.

    PubMed

    Thévenin, J; Dubos, C; Xu, W; Le Gourrierec, J; Kelemen, Z; Charlot, F; Nogué, F; Lepiniec, L; Dubreucq, B

    2012-01-01

    • Large-scale analysis of transcription factor-cis-acting element interactions in plants, or the dissection of complex transcriptional regulatory mechanisms, requires rapid, robust and reliable systems for the quantification of gene expression. • Here, we describe a new system for transient expression analysis of transcription factors, which takes advantage of the fast and easy production and transfection of Physcomitrella patens protoplasts, coupled to flow cytometry quantification of a fluorescent protein (green fluorescent protein). Two small-sized and high-copy Gateway® vectors were specifically designed, although standard binary vectors can also be employed. • As a proof of concept, the regulation of BANYULS (BAN), a key structural gene involved in proanthocyanidin biosynthesis in Arabidopsis thaliana seeds, was used. In P. patens, BAN expression is activated by a complex composed of three proteins (TT2/AtMYB123, TT8/bHLH042 and TTG1), and is inhibited by MYBL2, a transcriptional repressor, as in Arabidopsis. Using this approach, two new regulatory sequences that are necessary and sufficient for specific BAN expression in proanthocyanidin-accumulating cells were identified. • This one hybrid-like plant system was successfully employed to quantitatively assess the transcriptional activity of four regulatory proteins, and to identify their target recognition sites on the BAN promoter.

  4. 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.

  5. 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.

  6. 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.

  7. Heterologous Expression of Gene of Interest Using the Marine Protozoan Perkinsus marinus

    NASA Astrophysics Data System (ADS)

    Cold, E. R.

    2016-02-01

    Perkinsus marinus is a marine protozoan parasite that causes "Dermo" disease in eastern oysters (Crassostrea virginica). P. marinus is closely related to Plasmodium falciparum which causes malaria. A recent study has showed that P. marinus causes no pathology damage but an immune response in humanized mouse, providing the bases for a genetically modified P. marinus expressing Plasmodium genes to be used as a vaccination delivery system for malaria and other pathogenic diseases. A modified plasmid vector (pMOE-GFP) based on highly expressed gene tagged with green fluorescence protein and targeted to P. marinus cell wall was used to clone MSP8 and HAP2. MSP8 encodes for merozoite surface in P. falciparum and HAP2 is essential for fusion of male and female gametes; genetic disruption of the HAP2 locus revealed that parasite fertilization is prevented. Using electroporation, MSP8 and HAP2 plasmid were introduced into the P. marinus trophozoites. As controls pMOE-GFP was transfected into P. mediterraneus, P. atlanticus and P. chesapeaki. Transfection conditions included 5x107 Perkinsus trophozoites and 10 µg of plasmid using Nucleofector® technology (D-023 program). The cells were recovered in 3 mL of Perkinsus culture media and transfected trophozoites were examined for green fluorescence. To facilitate subcloning of cells expressing GFP, we optimized a DME: HAM's F12 -5% FBS -containing agar solid medium for plating Perkinsus. Examination of all transfected cells indicates expression of both MSP8 and HAP2. This is the first time that genes of a protozoan parasite have been expressed in a marine protozoan. It was also concluded that P. mediterraneus, P. atlanticus and P. chesapeaki were stable mutation and can be isolated for further research.

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

    PubMed

    Hudson, Lauren E; Fasken, Milo B; McDermott, Courtney D; McBride, Shonna M; Kuiper, Emily G; Guiliano, David B; Corbett, Anita H; Lamb, Tracey J

    2014-01-01

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

  9. Functional Heterologous Protein Expression by Genetically Engineered Probiotic Yeast Saccharomyces boulardii

    PubMed Central

    Hudson, Lauren E.; Fasken, Milo B.; McDermott, Courtney D.; McBride, Shonna M.; Kuiper, Emily G.; Guiliano, David B.; Corbett, Anita H.; Lamb, Tracey J.

    2014-01-01

    Recent studies have suggested the potential of probiotic organisms to be adapted for the synthesis and delivery of oral therapeutics. The probiotic yeast Saccharomyces boulardii would be especially well suited for this purpose due to its ability, in contrast to probiotic prokaryotes, to perform eukaryotic post translational modifications. This probiotic yeast thus has the potential to express a broad array of therapeutic proteins. Currently, however, use of wild type (WT) S. boulardii relies on antibiotic resistance for the selection of transformed yeast. Here we report the creation of auxotrophic mutant strains of S. boulardii that can be selected without antibiotics and demonstrate that these yeast can express functional recombinant protein even when recovered from gastrointestinal immune tissues in mice. A UV mutagenesis approach was employed to generate three uracil auxotrophic S. boulardii mutants that show a low rate of reversion to wild type growth. These mutants can express recombinant protein and are resistant in vitro to low pH, bile acid salts, and anaerobic conditions. Critically, oral gavage experiments using C57BL/6 mice demonstrate that mutant S. boulardii survive and are taken up into gastrointestinal immune tissues on a similar level as WT S. boulardii. Mutant yeast recovered from gastrointestinal immune tissues furthermore retain expression of functional recombinant protein. These data show that auxotrophic mutant S. boulardii can safely express recombinant protein without antibiotic selection and can deliver recombinant protein to gastrointestinal immune tissues. These auxotrophic mutants of S. boulardii pave the way for future experiments to test the ability of S. boulardii to deliver therapeutics and mediate protection against gastrointestinal disorders. PMID:25391025

  10. Heterologous expression of gentian MYB1R transcription factors suppresses anthocyanin pigmentation in tobacco flowers.

    PubMed

    Nakatsuka, Takashi; Yamada, Eri; Saito, Misa; Fujita, Kohei; Nishihara, Masahiro

    2013-12-01

    Single-repeat MYB transcription factors, GtMYB1R1 and GtMYB1R9 , were isolated from gentian. Overexpression of these genes reduced anthocyanin accumulation in tobacco flowers, demonstrating their applicability to modification of flower color. RNA interference (RNAi) has recently been used to successfully modify flower color intensity in several plant species. In most floricultural plants, this technique requires prior isolation of target flavonoid biosynthetic genes from the same or closely related species. To overcome this limitation, we developed a simple and efficient method for reducing floral anthocyanin accumulation based on genetic engineering using novel transcription factor genes isolated from Japanese gentians. We identified two single-repeat MYB genes--GtMYB1R and GtMYB1R9--predominantly expressed in gentian petals. Transgenic tobacco plants expressing these genes were produced, and their flowers were analyzed for flavonoid components and expression of flavonoid biosynthetic genes. Transgenic tobacco plants expressing GtMYB1R1 or GtMYB1R9 exhibited significant reductions in floral anthocyanin accumulation, resulting in white-flowered phenotypes. Expression levels of chalcone isomerase (CHI), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS) genes were preferentially suppressed in these transgenic tobacco flowers. A yeast two-hybrid assay demonstrated that both GtMYB1R1 and GtMYB1R9 proteins interacted with the GtbHLH1 protein, previously identified as an anthocyanin biosynthesis regulator in gentian flowers. In addition, a transient expression assay indicated that activation of the gentian GtDFR promoter by the GtMYB3-GtbHLH1 complex was partly canceled by addition of GtMYB1R1 or GtMYB1R9. These results suggest that GtMYB1R1 and GtMYB1R9 act as antagonistic transcription factors of anthocyanin biosynthesis in gentian flowers. These genes should consequently be useful for manipulating anthocyanin accumulation via genetic engineering in

  11. 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.

  12. Seed-Specific Heterologous Expression of a Nasturtium FAE Gene in Arabidopsis Results in a Dramatic Increase in the Proportion of Erucic Acid1

    PubMed Central

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

    2004-01-01

    The fatty acid elongase [often designated FAE or β-(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 β-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. PMID:15333757

  13. 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.

  14. Heterologous expression and purification of active human phosphoribosylglycinamide formyltransferase as a single domain.

    PubMed

    Kan, C C; Gehring, M R; Nodes, B R; Janson, C A; Almassy, R J; Hostomska, Z

    1992-10-01

    We report here for the first time that the GART domain of the human trifunctional enzyme possessing GARS, AIRS, and GART activities can be expressed independently in Escherichia coli at high levels as a stable protein with enzymatic characteristics comparable to those of native trifunctional protein. Human trifunctional enzyme is involved in de novo purine biosynthesis, and has long been recognized as a target for antineoplastic intervention. The GART domain was expressed in E. coli under the control of bacteriophage T7 promotor and isolated by a three-step chromatographic procedure. Two residues, Asp 951 and His 915, were shown to be catalytically crucial by site-directed mutagenesis and subsequent characterization of purified mutant proteins. The active monofunctional GART protein produced in E. coli can serve as a valuable substitute of trifunctional enzyme for structural and functional studies which have been until now hindered because of insufficient quantity, instability, and size of the trifunctional GART protein.

  15. 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

  16. Heterologous expression in Saccharopolyspora erythraea of a pentaketide synthase derived from the spinosyn polyketide synthase.

    PubMed

    Martin, Christine J; Timoney, Máire C; Sheridan, Rose M; Kendrew, Steven G; Wilkinson, Barrie; Staunton, James C; Leadlay, Peter F

    2003-12-07

    A truncated version of the spinosyn polyketide synthase comprising the loading module and the first four extension modules fused to the erythromycin thioesterase domain was expressed in Saccharopolyspora erythraea. A novel pentaketide lactone product was isolated, identifying cryptic steps of spinosyn biosynthesis and indicating the potential of this approach for the biosynthetic engineering of spinosyn analogues. A pathway for the formation of the tetracyclic spinosyn aglycone is proposed.

  17. Cloning, sequencing and heterologous expression of the monoamine oxidase gene from Aspergillus niger.

    PubMed

    Schilling, B; Lerch, K

    1995-05-20

    The gene encoding the flavin-containing monoamine oxidase (MAO-N) of the filamentous fungus Aspergillus niger was cloned. MAO-N is the first nonvertebrate monoamine oxidase described to date. Three partial cDNA clones, isolated from an expression library, were used to identify and clone the structural gene (maoN) from an A. niger genomic DNA library. The maoN gene was sequenced, and analysis revealed an open reading frame that codes for a protein of 495 amino acids with a calculated molecular mass of 55.6 kDa. Sequencing of an internal proteolytic fragment of the purified enzyme confirmed the derived amino acid sequence. Analysis of the deduced amino acid sequence indicates that MAO-N is structurally related to the human monoamine oxidases MAO-A and MAO-B. In particular, the regions known to be involved in the binding of the FAD cofactor show a high degree of homology; however, the conserved cysteine residue to which the flavin cofactor is covalently bound in the mammalian forms is absent in the fungal enzyme. MAO-N has the C-terminal tripeptide Ala-Arg-Leu, which corresponds to the consensus targeting sequence found in many peroxisomal enzymes. The full-length cDNA for MAO-N was expressed in Escherichia coli from the T7 promoter of the expression vector pET3a, yielding a soluble and fully active enzyme form.

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

    PubMed

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

    2013-06-01

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

  19. Heterologous expression and characterization of Indian Sahiwal cattle (Bos indicus) alpha inhibin.

    PubMed

    Bhardwaj, Anuradha; Nayan, Varij; Yadav, Paras; De, Sachinandan; Datta, Tirtha K; Goswami, Surender Lal

    2012-04-01

    Inhibin is a non-steroidal glycoprotein hormone of gonadal origin with major action as negative feedback control of the production of FSH by the anterior pituitary gland. The physiological role of inhibin has led to the development of inhibin immunogens for fertility enhancement in farm animals. It is envisaged that a reduction of endogenous inhibin secretion would increase FSH concentrations and thus offers a potential for increasing the number of ovulatory follicles in the ovary. The present work was carried out to produce recombinant bovine (Indian Sahiwal Cattle; Bos indicus) alpha inhibin (bINH-α) in E. coli by optimizing its expression and purification in biologically active form and to study its immunological characterization. A bacterial protein expression vector system based on the phage T(5) promoter was used. The bINH-α encoding gene was successfully cloned and expressed in E. coli and the purified recombinant bINH-α was characterized. Recombinant bINH-α (25 µg mL(-1)) immunized guinea pigs had a significant increase in litter size compared to the control group. These results indicate a role for recombinant bINH-α as a fecundity vaccine to enhance the ovulation rate and litter size in animals.

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

    PubMed

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

    2010-05-30

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

  1. Heterologous expression of Oenococcus oeni malolactic enzyme in Lactobacillus plantarum for improved malolactic fermentation

    PubMed Central

    2012-01-01

    Lactobacillus plantarum is involved in a multitude of food related industrial fermentation processes including the malolactic fermentation (MLF) of wine. This work is the first report on a recombinant L. plantarum strain successfully conducting MLF. The malolactic enzyme (MLE) from Oenococcus oeni was cloned into the lactobacillal expression vector pSIP409 which is based on the sakacin P operon of Lactobacillus sakei and expressed in the host strain L. plantarum WCFS1. Both recombinant and wild-type L. plantarum strains were tested for MLF using a buffered malic acid solution in absence of glucose. Under the conditions with L-malic acid as the only energy source and in presence of Mn2+ and NAD+, the recombinant L. plantarum and the wild-type strain converted 85% (2.5 g/l) and 51% (1.5 g/l), respectively, of L-malic acid in 3.5 days. Furthermore, the recombinant L. plantarum cells converted in a modified wine 15% (0.4 g/l) of initial L-malic acid concentration in 2 days. In conclusion, recombinant L. plantarum cells expressing MLE accelerate the malolactic fermentation. PMID:22452826

  2. Heterologous expression of Oenococcus oeni malolactic enzyme in Lactobacillus plantarum for improved malolactic fermentation.

    PubMed

    Schümann, Christina; Michlmayr, Herbert; Eder, Reinhard; Del Hierro, Andrés M; Kulbe, Klaus D; Mathiesen, Geir; Nguyen, Thu-Ha

    2012-03-27

    Lactobacillus plantarum is involved in a multitude of food related industrial fermentation processes including the malolactic fermentation (MLF) of wine. This work is the first report on a recombinant L. plantarum strain successfully conducting MLF. The malolactic enzyme (MLE) from Oenococcus oeni was cloned into the lactobacillal expression vector pSIP409 which is based on the sakacin P operon of Lactobacillus sakei and expressed in the host strain L. plantarum WCFS1. Both recombinant and wild-type L. plantarum strains were tested for MLF using a buffered malic acid solution in absence of glucose. Under the conditions with L-malic acid as the only energy source and in presence of Mn2+ and NAD+, the recombinant L. plantarum and the wild-type strain converted 85% (2.5 g/l) and 51% (1.5 g/l), respectively, of L-malic acid in 3.5 days. Furthermore, the recombinant L. plantarum cells converted in a modified wine 15% (0.4 g/l) of initial L-malic acid concentration in 2 days. In conclusion, recombinant L. plantarum cells expressing MLE accelerate the malolactic fermentation.

  3. 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. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. Characterization of Trichoderma reesei endoglucanase II expressed heterologously in Pichia pastoris for better biofinishing and biostoning.

    PubMed

    Samanta, Sutanu; Basu, Asitava; Halder, Umesh Chandra; Sen, Soumitra Kumar

    2012-06-01

    The endoglucanase II of Trichoderma reesei is considered the most effective enzyme for biofinishing cotton fabrics and biostoning denim garments. However, the commercially available preparation of endoglucanase II is usually mixed with other cellulase components, especially endoglucanase I, resulting in hydrolysis and weight loss of garments during biofinishing and biostoning. We thus isolated the endoglucanase II gene from T. reesei to express this in Pichia pastoris, under the control of a methanol-inducible AOX1 promoter, to avoid the presence of other cellulase components. A highly expressible Mut(+) transformant was selected and its expression in BMMH medium was found most suitable for the production of large amounts of the recombinant protein. Recombinant endoglucanase II was purified to electrophoretic homogeneity, and functionally characterized by activity staining. The specific activity of recombinant endoglucanase II was found to be 220.57 EU/mg of protein. Purified recombinant endoglucanase II was estimated to have a molecular mass of 52.8 kDa. The increase in molecular mass was likely due to hyperglycosylation. Hyperglycosylation of recombinant endoglucanase II secreted by P. pastoris did not change the temperature or pH optima as compared to the native protein, but did result in increased thermostability. Kinetic analysis showed that recombinant endoglucanase was most active against amorphous cellulose, such as carboxymethyl cellulose, for which it also had a high affinity.

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

    PubMed Central

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

    2015-01-01

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

  6. [Heterologous expression and characterization of Yarrowia lipolytica lipase 4 and lipase 5 in Pichia pastoris].

    PubMed

    Zhao, Heyun; Xiao, Xiao; Xu, Li; Liu, Yun; Yan, Yunjun

    2011-10-01

    To clone cDNA sequences of lipase 4 (LIP4) and lipase 5 (LIPS), analyze gene structures and express them in Pichia pastoris so as to investigate their enzymatic characteristics. We first cloned cDNA sequences of LIP4 and LIP5 by reverse transcription PCR and analyzed their gene structures by SignalP 3.0. Then, intracellular expression vectors pPIC3. 5K-Lip4, pPIC3. 5K-Lip5 and inducible secretion vectors pPIC9K-Lip4, pPIC9K-Lip5 were constructed. All vectors were transformed into Pichia pastoris GS115 by electroporation, resulting in a series of engineered strains. After fermentation and NTA-Ni resin purification, the enzymatic properties of LIP4 and LIP5 were examined. The cloned cDNA sequences revealed that there was no intron in both of Lip4 and Lip5. The two lipases both contained catalytic triads and conserved GHSLG motifs. Their optimal substrate, pH, temperature were respectively pNP-caprylate (C8), 7.0 and 40 degrees C. The activities of LIP4 and LIPS were 10.16 U/mg and 5.1 U/mg, respectively. It was found that LIP4 was more sensitive to the variations of pH and temperature than LIP5. LIP4 and LIP5 could both be stimulated by Ca2+, besides LIPS could also be activated by Mg2+. They were both strongly inhibited by Hg2+, Phenylmethanesulfonyl fluoride (PMSF) and Dithiothreitol (DTT). The cloning of Lip4 and Lip5, expression in P. pastoris and characterization of their properties would offer a solid basis for their large-scale production and future application. In addition, the results also enriched the data for a systematic research on the lipase gene family of Y. lipolytica.

  7. 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

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

    PubMed

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

    2014-07-01

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

  9. Selective dopaminergic neurotoxicity of isoquinoline derivatives related to Parkinson's disease: studies using heterologous expression systems of the dopamine transporter.

    PubMed

    Storch, Alexander; Ott, Stefanie; Hwang, Yu I; Ortmann, Rainer; Hein, Andreas; Frenzel, Stefan; Matsubara, Kazuo; Ohta, Shigeru; Wolf, Hans Uwe; Schwarz, Johannes

    2002-03-01

    Endogenous isoquinoline (IQ) derivatives structurally related to the selective dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite 1-methyl-4-phenylpyridine (MPP(+)) may contribute to dopaminergic neurodegeneration in Parkinson's disease. We addressed the importance of the DAT molecule for selective dopaminergic toxicity by testing the differential cytotoxicity of 22 neutral and quaternary compounds from three classes of isoquinoline derivatives (3, IQs; 4,3,4-dihydroisoquinolines and 15, 1,2,3,4-tetrahydroisoquinolines) as well as MPP(+) in non-neuronal and neuronal heterologous expression systems of the DAT gene (human embryonic kidney HEK-293 and mouse neuroblastoma Neuro-2A cells, respectively). Cell death was estimated using the MTT assay and the Trypan blue exclusion method. Nine isoquinolines and MPP(+) showed general cytotoxicity in both parental cell lines after 72hr with half-maximal toxic concentrations (TC(50) values) in the micromolar range. The rank order of toxic potency was: papaverine>salsolinol=tetrahydropapaveroline=1-benzyl-TIQ=norsalsolinol>tetrahydropapaverine>2[N]-methyl-salsolinol>2[N]-methyl-norsalsolinol>2[N]-Me-IQ(+)=MPP(+). Besides MPP(+), only the 2[N]-methylated compounds 2[N]-methyl-IQ(+), 2[N]-methyl-norsalsolinol and 2[N]-methyl-salsolinol showed enhanced cytotoxicity in both DAT expressing cell lines with 2- to 14-fold reduction of TC(50) values compared to parental cell lines. The rank order of selectivity in both cell systems was: MPP(+)>2[N]-Me-IQ(+)>2[N]-methyl-norsalsolinol=2[N]-methyl-salsolinol. Our results suggest that 2[N]-methylated isoquinoline derivatives structurally related to MPTP/MPP(+) are selectively toxic to dopaminergic cells via uptake by the DAT, and therefore may play a role in the pathogenesis of Parkinson's disease.

  10. Molecular cloning and heterologous expression of laccase from Aeromonas hydrophila NIU01 in Escherichia coli with parameters optimization in production.

    PubMed

    Ng, I-Son; Zhang, Xia; Zhang, Yu; Lu, Yinghua

    2013-04-01

    Prior studies disclosed that Aeromonas hydrophila NIU01 was a biodecolorization and bioelectricity bacterium which was isolated from a cross-strait of Taiwan. However, enzymatic function, laccase, involved in this strain had never been reported. This first attempt is to explore its laccase activity, the molecular cloning and heterologous recombinant expression in Escherichia coli. A full-length novel gene of 1,647 bp, LacA, encoding of 549 amino acids was successfully cloned by polymerase chain reaction. The recombinant pET-15b(+)-NIU-LacA expression was compared in different E. coli strains. By applying Taguchi's L9 in culture optimization, the soluble laccase increased to 22.7 %, in which the conditions were obtained at 22 °C with initial shaking speed at 200 rpm, addition of lactose of 0.2 mM and CuSO4 of 0.5 mM to the medium, and shaking off while cell mass reached to OD(600nm) of 1.5. NIU-LacA was strongly inhibited by chloride ion. The optimal temperature was 60 °C and the optimum pH for ABTS (2,2'-azino-bis (3-ethylbenzthiazolinesulfonic acid) and 2,6-DMP (2,6-dimethoxyphenol) were pH 2.1 and pH 7.5 which enzymatic activity was 274.6 and 44.8 U/L, respectively. Further study in structural modeling of NIU-LacA showed the C terminal domain was the major variance in the three most closely A. hydrophila strains.

  11. 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.

  12. Heterologous expression of the flavonoid 3',5'-hydroxylase gene of Vinca major alters flower color in transgenic Petunia hybrida.

    PubMed

    Mori, S; Kobayashi, H; Hoshi, Y; Kondo, M; Nakano, M

    2004-01-01

    Flavonoid 3',5'-hydroxylase (F3'5'H) is the key enzyme for the expression of blue or purple flower color. A full-length cDNA for the F3'5'H gene was cloned from petals of Vinca major, and its genomic clone, designated VmFH1 (accession number AB078781 in the GenBank/EMBL/DDBJ databases), was isolated from leaves by a PCR-based strategy. Nucleotide sequence analysis revealed that VmFH1 contains one intron and an open reading frame encoding a polypeptide of 506 amino acid residues. The deduced amino acid sequence shows between 51% and 83% identity with those of previously reported F3'5'H genes. Southern blot analysis showed that there are 3-4 copies of the F3'5'H gene in the genome of V. major. Transcripts of the F3'5'H gene were detected in young flower petals but not in leaves as revealed by RT-PCR analysis. When VmFH1 was expressed in transgenic Petunia hybrida under the control of the cauliflower mosaic virus 35S promoter, some transgenic plants showed drastic flower color alteration from red to deep red with deep purple sectors. These transgenic plants accumulated 3',5'-hydroxylated anthocyanins in their petals, which were never detected in non-transgenic plants by high-performance liquid chromatography analysis. These results indicate that VmFH1 isolated from V. major encodes F3'5'H and is active in a heterologous plant species.

  13. Heterologous expression, and biochemical and cellular characterization of CaPLA1 encoding a hot pepper phospholipase A1 homolog.

    PubMed

    Seo, Young Sam; Kim, Eun Yu; Mang, Hyung Gon; Kim, Woo Taek

    2008-03-01

    Phospholipid signaling has been recently implicated in diverse cellular processes in higher plants. We identified a cDNA encoding the phospholipase A1 homolog (CaPLA1) from 5-day-old early roots of hot pepper. The deduced amino acid sequence showed that the lipase-specific catalytic triad is well conserved in CaPLA1. In vitro lipase assays and site-directed mutagenesis revealed that CaPLA1 possesses PLA1 activity, which catalyzes the hydrolysis of phospholipids at the sn-1 position. CaPLA1 was selectively expressed in young roots, at days 4-5 after germination, and rapidly declined thereafter, suggesting that the expression of CaPLA1 is subject to control by a development-specific mechanism in roots. Because transgenic work was extremely difficult in hot peppers, in this study we overexpressed CaPLA1 in Arabidopsis so as to provide cellular information on the function of this gene. CaPLA1 overexpressors had significantly longer roots, leaves and petioles, and grew more rapidly than the wild-type plants, leading to an early bolting phenotype with prolonged inflorescence. Microscopic analysis showed that the vegetative tissues of 35S:CaPLA1 plants contained an increased number of small-sized cells, which resulted in highly populated cell layers. In addition, mRNAs for cell cycle-controlled proteins and fatty acid catabolizing enzymes were coordinately upregulated in CaPLA1-overexpressing plants. These results suggest that CaPLA1 is functionally relevant in heterologous Arabidopsis cells, and hence might participate in a subset of positive control mechanisms of cell and tissue growth in transgenic lines. We discuss possible biochemical and cellular functions of CaPLA1 in relation to the phospholipid signaling pathway in hot pepper and transgenic Arabidopsis plants.

  14. 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.

  15. Heterologous expression and characterisation of the Aspergillus aspartic protease involved in the hydrolysis and decolorisation of red-pigmented proteins.

    PubMed

    Takenaka, Shinji; Umeda, Mayo; Senba, Hisanori; Koyama, Dai; Tanaka, Kosei; Yoshida, Ken-Ichi; Doi, Mikiharu

    2017-01-01

    Aspergillus repens strain MK82 produces an aspartic protease (PepA_MK82) that efficiently decolorises red-pigmented proteins during dried bonito fermentation. However, further expansion of the industrial applications of PepA_MK82 requires the high-level production and efficient preparation of the recombinant enzyme. The genomic DNA and cDNA fragments encoding the protease were cloned from strain MK82 and sequenced. Phylogenetic analysis of PepA_MK82 and comparisons with previously reported fungal aspartic proteases showed that PepA_MK 82 clusters with different groups of these enzymes. Heterologous expression of PepA_MK82 in Pichia pastoris yielded preparations of higher purity than obtained with an Escherichia coli expression system. Total protease activity in a 100-mL culture of the P. pastoris transformant was 14 times higher than that from an equivalent culture of A. repense MK82. The recombinant PepA_MK82 was easily obtained via acetone precipitation; the final recovery was 83%. PepA_MK82 and its recombinant had similar characteristics in terms of their optimal pH, thermostability, and decolorisation activity. The recombinant was also able to decolorise flaked, dried bonito and to bleach a blood-stained cloth. Given its ability to hydrolyse and decolorise red-pigmented proteins, recombinant PepA_MK8 can be exploited in the food industry and as a stain-removal agent in laundry applications. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  16. Cloning, characterization, and heterologous expression of a novel glucosyltransferase gene from sophorolipid-producing Candida bombicola.

    PubMed

    Solaiman, Daniel K Y; Liu, Yanhong; Moreau, Robert A; Zerkowski, Jonathan A

    2014-04-25

    Candida bombicola is well-studied for the production of a biosurfactant, the sophorolipids. In this paper, the cloning of a glucosyltransferase gene using polymerase-chain-reaction (PCR) technique is described. Degenerative primer-pairs were first designed based on the highly conserved amino-acid sequences of several selected yeast glucosyltransferases. Using these primers, an amplified sequence (amplicon) of 700 base-pair from C. bombicola was obtained and subsequently sequenced. Based on the sequence of this amplicon, additional target-specific PCR primers were designed for use in subsequent rounds of 3'- and 5'-extension using DNA walking technique to eventually obtain a C. bombicola genomic sequence containing an open-reading-frame putatively identified as a glucosyltransferase (gtf-1). The gene was subcloned in Saccharomyces cerevisiae for expression and functional characterization. Quantitative RT-PCR confirmed the expression of gtf-1 in the recombinant S. cerevisiae. In vitro assay with the sonicated cells of the recombinant yeast confirms the presence of glucosylation activity on sterol and hydroxy fatty acid substrates. This study reports for the first time the cloning and characterization of a broad-specificity lipid glucosylation gene from C. bombicola, and the functional activity of its gene product. Published by Elsevier B.V.

  17. 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.

  18. Stable transfection and continuous expression of heterologous genes in Entamoeba invadens.

    PubMed

    Singh, Nishant; Ojha, Sandeep; Bhattacharya, Alok; Bhattacharya, Sudha

    2012-07-01

    Amoebiasis is spread by the ingestion of dormant Entamoeba histolytica cysts. Intervention of encystation could break the transmission cycle, thereby reducing disease burden. The model system used to study trophozoite to cyst differentiation is Entamoeba invadens. Here we describe an electroporation-based method for stable transfection of E. invadens with a plasmid pEiNEO-LUC containing the neomycin phosphotransferase gene under the control of E. invadens ribosomal protein gene S10 promoter. The plasmid also contains luciferase reporter gene expressed from the promoter of ribosomal protein gene L3. After electroporation, cells receiving the plasmid were selected by growth in 10μgml(-1) G418 and stable lines were obtained in four to five weeks. The plasmid was replicated episomally to ∼10 copies per haploid genome. In the absence of drug selection 50% of the plasmid copies were lost in 9-10 days. In cells growing under drug selection the reporter gene was continuously expressed throughout the differentiation process from trophozoite to cyst and back, making this system suitable for analysis of genes involved in differentiation. Copyright © 2012. Published by Elsevier B.V.

  19. Molecular cloning and heterologous expression of progesterone 5beta-reductase from Digitalis lanata Ehrh.

    PubMed

    Herl, Vanessa; Fischer, Gabriele; Müller-Uri, Frieder; Kreis, Wolfgang

    2006-02-01

    A full-length cDNA clone that encodes progesterone 5beta-reductase (5beta-POR) was isolated from Digitalis lanata leaves. The reading frame of the 5beta-POR gene is 1170 nucleotides corresponding to 389 amino acids. For expression, a Sph1/Sal1 5beta-POR fragment was cloned into the pQE vector and was transformed into Escherichia coli strain M15[pREP4]. The recombinant gene was functionally expressed and the recombinant enzyme was characterized. The K(m) and v(max) values for the putative natural substrate progesterone were calculated to be 0.120 mM and 45 nkat mg(-1) protein, respectively. Only 5beta-pregnane-3,20-dione but not its alpha-isomer was formed when progesterone was used as the substrate. Kinetic constants for cortisol, cortexone, 4-androstene-3,17-dione and NADPH were also determined. The molecular organization of the 5beta-POR gene in D. lanata was determined by Southern blot analysis. The 5beta-POR is highly conserved within the genus Digitalis and the respective genes and proteins share considerable homology to putative progesterone reductases from other plant species.

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

    PubMed

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

    2008-08-01

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

  1. Isoform characterisation, heterologous expression and functional analysis of two lectins from Vatairea macrocarpa.

    PubMed

    Alves Filho, João Garcia; do Nascimento, Antonia Sâmia Fernandes; Gondim, Ana Cláudia Silva; Pereira, Ronniery Hilario; da Cunha, Rodrigo Maranguape Silva; Nagano, Celso Shiniti; Teixeira, Edson Holanda; Nascimento, Kyria Santiago; Cavada, Benildo Sousa

    2013-11-01

    VML is a lectin from Vatairea macrocarpa seeds that has various biological activities. Here, we describe three new lectin isoforms from V. macrocarpa identified through genomic DNA analysis. One of these isoforms has high similarity to VML, while another that has noteworthy differences. We have denoted the new isoforms as VML-2, VML-3 and VML-4. Recombinant VML (rVML) and VML-2 (rVML-2) were expressed in Escherichia coli and were anticipated to have similar biological activity compared to native VML. Recombinant lectins were produced using a synthetic gene strategy to improve the expression levels. We obtained two active recombinant lectin isoforms from V. macrocarpa, and there was no significant difference between their biological activities. The conservation between carbohydrate-binding sites of recombinant and native proteins was demonstrated by specific inhibition of hemagglutin activity by D-galactose and lactose. However, no inhibition was observed in the presence of glucose and mannose. Our data show that the recombinant lectins VML and VML-2 are active and capable of recognising D-galactose and lactose. Moreover, the absence of glycosylation does not interfere with their biological activity.

  2. 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.

  3. 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

  4. The adpA-like regulatory gene from Actinoplanes teichomyceticus: in silico analysis and heterologous expression.

    PubMed

    Ostash, Bohdan; Yushchuk, Oleksandr; Tistechok, Stepan; Mutenko, Halyna; Horbal, Lilia; Muryn, Andriy; Dacyuk, Yuriy; Kalinowski, Jorn; Luzhetskyy, Andriy; Fedorenko, Victor

    2015-08-01

    Analysis of the draft sequence of the genome of teicoplanin producer Actinoplanes teichomyceticus (NRRL-B16726) led to identification of several genes encoding AraC-family regulators that resemble AdpA, master regulator of transcription in Streptomyces. We elucidated possible regulatory functions of one of the identified genes, adpA19(at), most similar to archetypal adpA from model Streptomyces species, in a series of expression experiments. Introduction of adpA19 at under control of its own promoter on moderate copy number vector pKC1139 into NRRL-B16726 had no influence on antibiotic production and sporulation. Introduction of adpA19 at into Streptomyces coelicolor M145 and several S. ghanaensis strains had major influence on antibiotic production by these bacteria. Finally, adpA19 at expression in a set of soil actinomycete isolates led to induction of synthesis of antibiotic compounds. Our data point to pleiotropic regulatory role of adpA19(at), warranting its use as a tool to manipulate secondary metabolome of actinomycetes.

  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. Fungal His-Tagged Nitrilase from Gibberella intermedia: Gene Cloning, Heterologous Expression and Biochemical Properties

    PubMed Central

    Gong, Jin-Song; Li, Heng; Zhu, Xiao-Yan; Lu, Zhen-Ming; Wu, Yan; Shi, Jing-Song; Xu, Zheng-Hong

    2012-01-01

    Background Nitrilase is an important member of the nitrilase superfamiliy. It has attracted substantial interest from academia and industry for its function of converting nitriles directly into the corresponding carboxylic acids in recent years. Thus nitrilase has played a crucial role in production of commercial carboxylic acids in chemical industry and detoxification of nitrile-contaminated wastes. However, conventional studies mainly focused on the bacterial nitrilase and the potential of fungal nitrilase has been far from being fully explored. Research on fungal nitrilase gene expression will advance our understanding for its biological function of fungal nitrilase in nitrile hydrolysis. Methodology/Principal Findings A fungal nitrilase gene from Gibberella intermedia was cloned through reverse transcription-PCR. The open reading frame consisted of 963 bp and potentially encoded a protein of 320 amino acid residues with a theoretical molecular mass of 35.94 kDa. Furthermore, the catalytic triad (Glu-45, Lys-127, and Cys-162) was proposed and confirmed by site-directed mutagenesis. The encoding gene was expressed in Escherichia coli Rosetta-gami (DE3) and the recombinant protein with His6-tag was purified to electrophoretic homogeneity. The purified enzyme exhibited optimal activity at 45°C and pH 7.8. This nitrilase was specific towards aliphatic and aromatic nitriles. The kinetic parameters Vmax and Km for 3-cyanopyridine were determined to be 0.81 µmol/min·mg and 12.11 mM through Hanes-Woolf plot, respectively. 3-Cyanopyridine (100 mM) could be thoroughly hydrolyzed into nicotinic acid within 10 min using the recombinant strain with the release of about 3% nicotinamide and no substrate was detected. Conclusions/Significance In the present study, a fungal nitrilase was cloned from the cDNA sequence of G. intermedia and successfully expressed in E. coli Rosetta-gami (DE3). The recombinant strain displayed good 3-cyanopyridine degradation efficiency and wide

  7. Heterologous expression and characterization of a malathion-hydrolyzing carboxylesterase from a thermophilic bacterium, Alicyclobacillus tengchongensis.

    PubMed

    Xie, Zhenrong; Xu, Bo; Ding, Junmei; Liu, Lingyun; Zhang, Xuelin; Li, Junjun; Huang, Zunxi

    2013-08-01

    A carboxylesterase gene from thermophilic bacterium, Alicyclobacillus tengchongensis, was cloned and expressed in Escherichia coli BL21 (DE3). The gene coded for a 513 amino acid protein with a calculated molecular mass of 57.82 kDa. The deduced amino acid sequence had structural features highly conserved among serine hydrolases, including Ser204, Glu325, and His415 as a catalytic triad, as well as type-B carboxylesterase serine active site (FGGDPENITIGGQSAG) and type-B carboxylesterase signature 2 (EDCLYLNIWTP). The purified enzyme exhibited optimum activity with β-naphthyl acetate at 60 °C and pH 7 as well as stability at 25 °C and pH 7. One unit of the enzyme hydrolyzed 5 mg malathion l(-1) by 50 % within 25 min and 89 % within 100 min. The enzyme strongly degraded malathion and has a potential use for the detoxification of malathion residues.

  8. 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.

  9. Building Triketide α-Pyrone-Producing Yeast Platform Using Heterologous Expression of Sporopollenin Biosynthetic Genes.

    PubMed

    Kim, Sung Soo

    2015-11-01

    Sporopollenin is a poorly characterized mixed aliphatic and aromatic polymer with ester and ether linkages. Recent studies have reported that α-pyrone polyketide compounds generated by Arabidopsis thaliana, polyketide synthase A (PKSA) and tetraketide α-pyrone reductase 1 (TKPR1), are previously unknown sporopollenin precursors. Here, the yeast Saccharomyces cerevisiae was introduced to test potential sporopollenin biosynthetic pathways in vivo. A PKSA/TKPR1 dual expressor was generated and various chain-length alkyl α-pyrones were identified by GC-MS. The growth rate of the strain containing PKSA/TKPR1 appeared normal. These results indicate that PKSA/TKPR1-expressing yeast would be a starting platform to investigate in vivo sporopollenin metabolism.

  10. Characterization of lysosomal acid lipase by site-directed mutagenesis and heterologous expression.

    PubMed

    Sheriff, S; Du, H; Grabowski, G A

    1995-11-17

    Lysosomal acid lipase (LAL) is essential for the hydrolysis of cholesterol esters and triglycerides that are delivered to the lysosomes via the low density lipoprotein receptor system. The deficiency of LAL is associated with cholesteryl ester storage disease (CESD) and Wolman's disease (WD). We cloned the human LAL cDNA and expressed the active enzyme in the baculovirus system. Two molecular forms (M(r) approximately 41,000 and approximately 46,000) with different glycosylation were found intracellularly, and approximately 24% of the M(r) approximately 46,000 form was secreted into the medium. Tunicamycin treatment produced only an inactive M(r) approximately 41,000 form. This result implicates glycosylation occupancy in the proper folding for active-site function. Catalytic activity was greater toward cis- than trans-unsaturated fatty acid esters of 4-methylumbelliferone and toward esters with 7-carbon length acyl chains. LAL cleaved cholesterol esters and mono-, tri-, and diglycerides. Heparin had a biphasic effect on enzymatic activity with initial activation followed by inhibition. Inhibition of LAL activity by tetrahydrolipstatin and diethyl p-nitrophenyl phosphate suggested the presence of active serines in binding/catalytic domain(s) of the protein. Site-directed mutagenesis at two putative active centers, GXSXG, showed that Ser153 was important to catalytic activity, whereas Ser99 was not and neither was the catalytic nucleophile. Three reported mutations (L179P, L336P, and delta AG302 deletion) from CESD patients were created and expressed in the Sf9 cell system. None cleaved cholesterol esters, and L179P and L336P cleaved only triolein at approximately 4% of wild-type levels. These results suggest that mechanisms, in addition to LAL defects, may operate in the selective accumulation of cholesterol esters or triglycerides in CESD and WD patients.

  11. Heterologous high yield expression and purification of neurotensin and its functional fragment in Escherichia coli.

    PubMed

    Tapaneeyakorn, Satita; Ross, Simon; Attrill, Helen; Watts, Anthony

    2010-11-01

    Peptide synthesis is widely used for the production of small proteins and peptides, but producing uniformly isotopically labelled peptides for NMR and other biophysical studies could be limited for economic reasons. Here, we propose a use of a modified pGEV-1 plasmid to express neurotensin (NT(1-13)), pGlu(1)-Leu(2)-Tyr(3)-Glu(4)-Asn(5)-Lys(6)-Pro(7)-Arg(8)-Arg(9)-Pro(10)-Tyr(11)-Ile(12)-Leu(13)-OH, as a C-terminal fusion protein with the GB1 domain of streptococcal protein G. The free carboxyl-terminus is important for the function of several peptide hormones, including neurotensin. Therefore, for the pGEV-NT(1-13) construct, the C-terminal pGEV-encoded 6xHis tag was removed and an N-terminal 8xHis tag was introduced for affinity purification. To facilitate removal of tags using CNBr cleavage, a methionine was introduced at the N-terminal of the peptide. Furthermore, this pGEV-NT(1-13) plasmid was used as a template to include a Pro-7 to Met mutation for CNBr cleavage, giving NT(8-13), the sub-fragment crucial for the biological activity of this peptide. These two constructs are being used to produce uniformly labelled NT(1-13) and NT(8-13) in high yield and in a cost effective way, using cheap (15)N and/or (13)C source. The modification proposed here using the pGEV-1 plasmid could be an alternative option for the high expression of other isotopically labelled and unlabelled short peptides, including hormones and hydrophobic membrane peptides.

  12. Honeybee blue- and ultraviolet-sensitive opsins: cloning, heterologous expression in Drosophila, and physiological characterization.

    PubMed

    Townson, S M; Chang, B S; Salcedo, E; Chadwell, L V; Pierce, N E; Britt, S G

    1998-04-01

    The honeybee (Apis mellifera) visual system contains three classes of retinal photoreceptor cells that are maximally sensitive to light at 440 nm (blue), 350 nm (ultraviolet), and 540 nm (green). We performed a PCR-based screen to identify the genes encoding the Apis blue- and ultraviolet (UV)-sensitive opsins. We obtained cDNAs that encode proteins having a high degree of sequence and structural similarity to other invertebrate and vertebrate visual pigments. The Apis blue opsin cDNA encodes a protein of 377 amino acids that is most closely related to other invertebrate visual pigments that are thought to be blue-sensitive. The UV opsin cDNA encodes a protein of 371 amino acids that is most closely related to the UV-sensitive Drosophila Rh3 and Rh4 opsins. To test whether these novel Apis opsin genes encode functional visual pigments and to determine their spectral properties, we expressed them in the R1-6 photoreceptor cells of blind ninaE mutant Drosophila, which lack the major opsin of the fly compound eye. We found that the expression of either the Apis blue- or UV-sensitive opsin in transgenic flies rescued the visual defect of ninaE mutants, indicating that both genes encode functional visual pigments. Spectral sensitivity measurements of these flies demonstrated that the blue and UV visual pigments are maximally sensitive to light at 439 and 353 nm, respectively. These maxima are in excellent agreement with those determined previously by single-cell recordings from Apis photoreceptor cells and provide definitive evidence that the genes described here encode visual pigments having blue and UV sensitivity.

  13. Heterologous Expression of Aspergillus Niger --beta--D-Xylosidase (XInD): Characterization on Lignocellulosic Substrates

    SciTech Connect

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

    2008-01-01

    The gene encoding a glycosyl hydrolase family 3 xylan 1,4-beta-xylosidase, xlnD, was successfully cloned from Aspergillus niger strain ATCC 10864. The recombinant product was expressed in Aspergillus awamori, purified by column chromatography, and verified by matrix-assisted laser desorption ionization, tandem time of flight (MALDI-TOF/TOF) mass spectroscopy of tryptic digests. The T{sub max} was determined using differential scanning microcalorimetry (DSC) to be 78.2 C; the K{sub m} and k{sub cat} were found to be 255 {micro}M and 13.7 s{sup -1}, respectively, using {rho}NP-{Beta}-d-xylopyranoside as substrate. End-product inhibition by d-xylose was also verified and shown to be competitive; the K{sub i} for this inhibition was estimated to be 3.3 mM. XlnD was shown to efficiently hydrolyze small xylo-oligomers to monomeric xylose, making it a critical hydrolytic activity in cases where xylose is to be recovered from biomass conversion processes. In addition, the presence of the XlnD was shown to synergistically enhance the ability of an endoxylanase, XynA from Thermomyces lanuginosus, to convert xylan present in selected pretreated lignocellulosic substrates. Furthermore, the addition of the XynA/XlnD complex was effective in enhancing the ability of a simplified cellulase complex to convert glucan present in the substrates.

  14. 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.

  15. Heterologous expression of Aspergillus niger beta-D-xylosidase (XlnD): characterization on lignocellulosic substrates.

    PubMed

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

    2008-03-01

    The gene encoding a glycosyl hydrolase family 3 xylan 1,4-beta-xylosidase, xlnD, was successfully cloned from Aspergillus niger strain ATCC 10864. The recombinant product was expressed in Aspergillus awamori, purified by column chromatography, and verified by matrix-assisted laser desorption ionization, tandem time of flight (MALDI-TOF/TOF) mass spectroscopy of tryptic digests. The T (max) was determined using differential scanning microcalorimetry (DSC) to be 78.2 degrees C; the K (m) and k (cat) were found to be 255 microM and 13.7 s(-1), respectively, using pNP-beta-D-xylopyranoside as substrate. End-product inhibition by D-xylose was also verified and shown to be competitive; the K (i) for this inhibition was estimated to be 3.3 mM. XlnD was shown to efficiently hydrolyze small xylo-oligomers to monomeric xylose, making it a critical hydrolytic activity in cases where xylose is to be recovered from biomass conversion processes. In addition, the presence of the XlnD was shown to synergistically enhance the ability of an endoxylanase, XynA from Thermomyces lanuginosus, to convert xylan present in selected pretreated lignocellulosic substrates. Furthermore, the addition of the XynA/XlnD complex was effective in enhancing the ability of a simplified cellulase complex to convert glucan present in the substrates.

  16. Heterologous expression of specific K+ channels in T lymphocytes: functional consequences for volume regulation.

    PubMed Central

    Deutsch, C; Chen, L Q

    1993-01-01

    It has been postulated that the K+ channel isoform Kv1.3 plays a role in regulatory volume decrease (RVD) in response to hypotonic shock. We show that a mouse cytotoxic T-lymphocyte line, CTLL-2, is devoid of voltage-dependent K+ channels and is unable to volume regulate. Transient transfection of these cells with Kv1.3 reconstitutes their ability to volume regulate. As predicted by our model, this ability depends critically on volume-induced changes in membrane potential and the isoform of the K+ channel used. When the cells were transfected with Kv3.1, an isoform believed to be expressed in a specific subclass of mouse thymocytes, the CTLL-2 cells did not show RVD. The difference in the ability of the two isoforms to confer the capacity for RVD is expected from differences in the voltage dependence of activation of the channels, according to our proposed model for RVD. The experimental approach that we use, transient transfection and panning to select positive transfectants, is highly effective; it has a > 95% efficiency. This method, and this cell line, may be important tools in studying lymphocyte K+ channels and their function in situ. Images Fig. 3 PMID:8234253

  17. Cloning, post-translational modifications, heterologous expression and ligand-binding of boar salivary lipocalin.

    PubMed

    Loebel, D; Scaloni, A; Paolini, S; Fini, C; Ferrara, L; Breer, H; Pelosi, P

    2000-09-01

    Boar submaxillary glands produce the sex-specific salivary lipocalin (SAL), which binds steroidal sex pheromones as endogenous ligands. The cDNA encoding SAL was cloned and sequenced. From a single individual, two protein isoforms, differing in three amino acid residues, were purified and structurally characterized by a combined Edman degradation/MS approach. These experiments ascertained that the mature polypeptide is composed of 168 amino acid residues, that one of the three putative glycosylation sites is post-translationally modified and the structure of the bound glycosidic moieties. Two of the cysteine residues are paired together in a disulphide bridge, whereas the remaining two occur as free thiols. SAL bears sequence similarity to other lipocalins; on this basis, a three-dimensional model of the protein has been built. A SAL isoform was expressed in Escherichia coli in good yields. Protein chemistry and CD experiments verified that the recombinant product shows the same redox state at the cysteine residues and that the same conformation is observed as in the natural protein, thus suggesting similar folding. Binding experiments on natural and recombinant SAL were performed with the fluorescent probe 1-aminoanthracene, which was efficiently displaced by the steroidal sex pheromone, as well as by several odorants.

  18. Ani s 10, a new Anisakis simplex allergen: cloning and heterologous expression.

    PubMed

    Caballero, Maria Luisa; Umpierrez, Ana; Moneo, Ignacio; Rodriguez-Perez, Rosa

    2011-06-01

    Anisakiasis is a human disease caused by accidental ingestion of larval nematodes belonging to the Anisakidae family. Anisakiasis is often associated with a strong allergic response. Diagnosis of A. simplex allergy is currently carried out by test based on the IgE reactivity to a complete extract of L3 Anisakis larvae although the specificity of these diagnostic tests is poor. Improving the specificity of the diagnostic test is possible using purified recombinant allergens. A new Anisakis allergen, named Ani s 10, was detected by immunoscreening an expression cDNA library constructed from L3 Anisakis simplex larvae. The new allergen was overproduced in Escherichia coli; it is a protein of 212 amino acids and it was localized as a 22 kDa protein band in an ethanol fractionated extract from the parasite. Ani s 10 has no homology with any other described protein, and its sequence is composed by seven almost identical repetitions of 29 amino acids each. A total of 30 of 77 Anisakis allergic patients (39%) were positive both to rAni s 10 and natural Ani s 10 by immunoblotting. The new allergen could be useful in a component-resolved diagnosis system for Anisakis allergy. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  19. 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

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

    PubMed

    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.

  1. 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

  2. Cloning, post-translational modifications, heterologous expression and ligand-binding of boar salivary lipocalin.

    PubMed Central

    Loebel, D; Scaloni, A; Paolini, S; Fini, C; Ferrara, L; Breer, H; Pelosi, P

    2000-01-01

    Boar submaxillary glands produce the sex-specific salivary lipocalin (SAL), which binds steroidal sex pheromones as endogenous ligands. The cDNA encoding SAL was cloned and sequenced. From a single individual, two protein isoforms, differing in three amino acid residues, were purified and structurally characterized by a combined Edman degradation/MS approach. These experiments ascertained that the mature polypeptide is composed of 168 amino acid residues, that one of the three putative glycosylation sites is post-translationally modified and the structure of the bound glycosidic moieties. Two of the cysteine residues are paired together in a disulphide bridge, whereas the remaining two occur as free thiols. SAL bears sequence similarity to other lipocalins; on this basis, a three-dimensional model of the protein has been built. A SAL isoform was expressed in Escherichia coli in good yields. Protein chemistry and CD experiments verified that the recombinant product shows the same redox state at the cysteine residues and that the same conformation is observed as in the natural protein, thus suggesting similar folding. Binding experiments on natural and recombinant SAL were performed with the fluorescent probe 1-aminoanthracene, which was efficiently displaced by the steroidal sex pheromone, as well as by several odorants. PMID:10947950

  3. Heterologous expression of transaldolase gene Tal from Saccharomyces cerevisiae in Fusarium oxysporum for enhanced bioethanol production.

    PubMed

    Fan, Jin-Xia; Yang, Xiao-Xue; Song, Jin-Zhu; Huang, Xiao-Mei; Cheng, Zhong-Xiang; Yao, Lin; Juba, Olivia S; Liang, Qing; Yang, Qian; Odeph, Margaret; Sun, Yan; Wang, Yun

    2011-08-01

    The filamentous fungus Fusarium oxysporum is known for its ability to ferment xylose-producing ethanol. However, efficiency of xylose utilization and ethanol yield was low. In this study, the transaldolase gene from Saccharomyces cerevisiae has been successfully expressed in F. oxysporum by an Agrobacterium tumefaciens-mediated transformation method. The enzymatic activity of the recombinant fungus (cs28pCAM-Sctal4) was 0.195 times higher than that of the wild-type strain (cs28). The recombinant strain also exhibited a 28.83% increase in ethanol yield on xylose media compared to the parental strain. Enhanced ethanol production and a reduction in the biomass were observed during xylose fermentation. Ethanol yield from rice straw by simultaneous saccharification and fermentation with cs28pCAM-Sctal4 was 0.25 g g⁻¹ of rice straw. The transgenic strain of F. oxysporum cs28pCAM-Sctal4 might therefore have potential applications in industrial bioenergy production.

  4. Heterologous expression of pneumococcal virulence factor PspC on the surface of Lactococcus lactis confers adhesive properties.

    PubMed

    Asmat, Tauseef M; Klingbeil, Katharina; Jensch, Inga; Burchhardt, Gerhard; Hammerschmidt, Sven

    2012-03-01

    Lactococcus lactis is a non-pathogenic bacterium that is used in the food industry but is also used as a heterologous host to reveal protein functions of pathogenic bacteria. The adhesin PspC from Streptococcus pneumoniae is a choline-binding protein that is non-covalently anchored to the bacterial cell wall. To assess the exclusive impact of pneumococcal surface protein C (PspC) on the interplay with its host we generated recombinant L. lactis producing a nisin-inducible and covalently anchored variant of PspC on the lactococcal cell surface. A translational fusion of the 5'-end of pspC3.4 with the 3'-end of hic (pspC11.4) was designed to decorate the surface of L. lactis with a chimeric PspC. The PspC3.4 part comprises the first 281 aa residues of PspC3.4, while the Hic sequence consists of the proline-rich and sortase-anchored domain. The results demonstrated that PspC is sufficient for adhesion and subsequent invasion of host epithelial cells expressing the human polymeric Ig receptor (hpIgR). Moreover, invasion via hpIgR was even more pronounced when the chimeric PspC was produced by lactococci compared with pneumococci. This study shows also for the first time that PspC plays no significant role during phagocytosis by macrophages. In contrast, recruitment of Factor H via the PspC chimer has a dramatic effect on phagocytosis of recombinant but not wild-type lactococci, as Factor H interacts specifically with the amino-terminal part of PspC and mediates the contact with phagocytes. Furthermore, L. lactis expressing PspC increased intracellular calcium levels in pIgR-expressing epithelial cells, thus resembling the effect of pneumococci, which induced release of Ca(2+) from intracellular stores via the PspC-pIgR mechanism. In conclusion, expression of the chimeric PspC confers adhesive properties to L. lactis and indicates the potential of L. lactis as a suitable host to study the impact of individual bacterial factors on their capacity to interfere with the

  5. Heterologous expression of an uncharacterized universal stress protein gene (SbUSP) from the extreme halophyte, Salicornia brachiata, which confers salt and osmotic tolerance to E. coli.

    PubMed

    Udawat, Pushpika; Mishra, Avinash; Jha, Bhavanath

    2014-02-15

    Salicornia brachiata is an extreme halophyte considered to be a rich source of stress responsive genes and an EST database revealed that 30% of its genes are uncharacterized. In order to ascertain its function, a gene (Sal-E-56) of unknown function was made full length using RACE, cloned and characterized. The full length gene (873 bp; accession no. KF164282) contained an open reading frame (ORF) of 486 bp encoding for a protein that belongs to the universal stress protein (USP) family that was named SbUSP. The SbUSP interacted with adenosine monophosphate and exhibited characteristic motifs, phosphorylation, glycosylation and ATP binding sites. Further, in-silico analyses suggested a probable role in metabolic process of phosphate-containing compounds including signal transduction. In planta transcript profiling exhibited a significant expression response (7.8-fold) to salt stress, additionally abundant of SbUSP transcripts were observed during drought, heat and cold stress, reaching a maximum increase of 3.66-, 2.64- and 2.14-fold, respectively, at 12 or 24h. The heterologous expression of this gene in Escherichia coli provided enhanced stress tolerance and recombinant cells have higher growth rate compared to vector alone and showed growth at up to a 10(-5) dilution in the spot assay. It was predicted that SbUSP may be directly involved in tolerance mechanisms or function as a molecular switch (signaling molecule) to activate the stress adaptive mechanisms. However, further investigation will be required to determine its role as a molecular switch and mode of action during stress.

  6. Exploring Three PIPs and Three TIPs of Grapevine for Transport of Water and Atypical Substrates through Heterologous Expression in aqy-null Yeast

    PubMed Central

    Sabir, Farzana; Leandro, Maria José; Martins, Ana Paula; Loureiro-Dias, Maria C.; Moura, Teresa F.; Soveral, Graça; Prista, Catarina

    2014-01-01

    Aquaporins are membrane channels that facilitate the transport of water and other small molecules across the cellular membranes. We examined the role of six aquaporins of Vitis vinifera (cv. Touriga nacional) in the transport of water and atypical substrates (other than water) in an aqy-null strain of Saccharomyces cerevisiae. Their functional characterization for water transport was performed by stopped-flow fluorescence spectroscopy. The evaluation of permeability coefficients (Pf) and activation energies (Ea) revealed that three aquaporins (VvTnPIP2;1, VvTnTIP1;1 and VvTnTIP2;2) are functional for water transport, while the other three (VvTnPIP1;4, VvTnPIP2;3 and VvTnTIP4;1) are non-functional. TIPs (VvTnTIP1;1 and VvTnTIP2;2) exhibited higher water permeability than VvTnPIP2;1. All functional aquaporins were found to be sensitive to HgCl2, since their water conductivity was reduced (24–38%) by the addition of 0.5 mM HgCl2. Expression of Vitis aquaporins caused different sensitive phenotypes to yeast strains when grown under hyperosmotic stress generated by KCl or sorbitol. Our results also indicate that Vitis aquaporins are putative transporters of other small molecules of physiological importance. Their sequence analyses revealed the presence of signature sequences for transport of ammonia, boron, CO2, H2O2 and urea. The phenotypic growth variations of yeast cells showed that heterologous expression of Vitis aquaporins increased susceptibility to externally applied boron and H2O2, suggesting the contribution of Vitis aquaporins in the transport of these species. PMID:25111598

  7. 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.

  8. Alcohol dehydrogenases from Kluyveromyces marxianus: heterologous expression in Escherichia coli and biochemical characterization

    PubMed Central

    2014-01-01

    Background Kluyveromyces marxianus has recently become a species of interest for ethanol production since it can produce ethanol at high temperature and on a wide variety of substrates. However, the reason why this yeast can produce ethanol at high temperature is largely unknown. Results The ethanol fermentation capability of K. marxianus GX-UN120 at 40°С was found to be the same as that of Saccharomyces cerevisiae at 34°С. Zymogram analysis showed that alcohol dehydrogenase 1 (KmAdh1) was largely induced during ethanol production, KmAdh4 was constitutively expressed at a lower level and KmAdh2 and KmAdh3 were almost undetectable. The genes encoding the four alcohol dehydrogenases (ADHs) were cloned from strain GX-UN120. Each KmADH was expressed in Escherichia coli and each recombinant protein was digested with enterokinase to remove the fusion protein. The optimum pH of the purified recombinant KmAdh1 was 8.0 and that of KmAdh2, KmAdh3 and KmAdh4 was 7.0. The optimum temperatures of KmAdh1, KmAdh2, KmAdh3 and KmAdh4 were 50, 45, 55 and 45°C, respectively. The Km values of the recombinant KmAdh1 and KmAdh2 were 4.0 and 1.2 mM for acetaldehyde and 39.7 and 49.5 mM for ethanol, respectively. The Vmax values of the recombinant KmAdh1 and KmAdh2 were 114.9 and 21.6 μmol min-1 mg-1 for acetaldehyde and 57.5 and 1.8 μmol min-1 mg-1 for ethanol, respectively. KmAdh3 and KmAdh4 catalyze the oxidation reaction of ethanol to acetaldehyde but not the reduction reaction of acetaldehyde to ethanol, and the K m values of the recombinant KmAdh3 and KmAdh4 were 26.0 and 17.0 mM for ethanol, respectively. The Vmax values of the recombinant KmAdh3 and KmAdh4 were 12.8 and 56.2 μmol min-1 mg-1 for ethanol, respectively. Conclusion These data in this study collectively indicate that KmAdh1 is the primary ADH responsible for the production of ethanol from the reduction of acetaldehyde in K. marxianus. The relatively high optimum temperature of KmAdh1 may partially

  9. 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

  10. Tryparedoxin peroxidase of Leishmania donovani: molecular cloning, heterologous expression, specificity, and catalytic mechanism.

    PubMed

    Flohé, Leopold; Budde, Heike; Bruns, Karsten; Castro, Helena; Clos, Joachim; Hofmann, Birgit; Kansal-Kalavar, Sonia; Krumme, Dirk; Menge, Ulrich; Plank-Schumacher, Karin; Sztajer, Helena; Wissing, Joseph; Wylegalla, Claudia; Hecht, Hans-Jürgen

    2002-01-15

    Tryparedoxin peroxidase (TXNPx) of Trypanosomatidae is the terminal peroxidase of a complex redox cascade that detoxifies hydroperoxides by NADPH (Nogoceke et al., Biol. Chem. 378, 827-836, 1997). A gene putatively coding for a peroxiredoxin-type TXNPx was identified in L. donovani and expressed in Escherichia coli to yield an N-terminally His-tagged protein (LdH6TXNPx). LdH6TXNPx proved to be an active peroxidase with tryparedoxin (TXN) 1 and 2 of Crithidia fasciculata as cosubstrates. LdH6TXNPx efficiently reduces H2O2, is moderately active with t-butyl and cumene hydroperoxide, but only marginally with linoleic acid hydroperoxide and phosphatidyl choline hydroperoxide. The enzyme displays ping-pong kinetics with a k(cat) of 11.2 s(-1) and limiting K(m) values for t-butyl hydroperoxide and CfTXN1 of 50 and 3.6 microM, respectively. Site-directed mutagenesis confirmed that C52 and C173, as in related peroxiredoxins, are involved in catalysis. Exchanges of R128 against D and T49 against S and V, supported by molecular modelling, further disclose that the SH group of C52 builds the center of a novel catalytic triad. By hydrogen bonding with the OH of T49 and by the positive charge of R128 the solvent-exposed thiol of C52 becomes deprotonated to react with ROOH. Molecular models of oxidized TXNPx show C52 disulfide-bridged with C173' that can be attacked by C41 of TXN2. By homology, the deduced mechanism may apply to most peroxiredoxins and complements current views of peroxiredoxin catalysis. (c)2002 Elsevier Science.

  11. 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

  12. 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.; ...

    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

  13. Heterologous Expression and Characterization of Two 1-Hydroxy-2-Naphthoic Acid Dioxygenases from Arthrobacter phenanthrenivorans

    PubMed Central

    Kavakiotis, Konstantinos; Kallimanis, Aristeidis; Kyrpides, Nikos C.; Drainas, Constantin; Koukkou, Anna-Irini

    2012-01-01

    A protein fraction exhibiting 1-hydroxy-2-naphthoic acid (1-H2NA) dioxygenase activity was purified via ion exchange, hydrophobic interactions, and gel filtration chromatography from Arthrobacter phenanthrenivorans sp. nov. strain Sphe3 isolated from a Greek creosote-oil-polluted site. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and tandem MS (MS-MS) analysis revealed that the amino acid sequences of oligopeptides of the major 45-kDa protein species, as analyzed by SDS-PAGE and silver staining, comprising 29% of the whole sequence, exhibited strong homology with 1-H2NA dioxygenase of Nocardioides sp. strain KP7. A BLAST search of the recently sequenced Sphe3 genome revealed two putative open reading frames, named diox1 and diox2, showing 90% nucleotide identity to each other and 85% identity at the amino acid level with the Nocardia sp. homologue. diox1 was found on an indigenous Sphe3 plasmid, whereas diox2 was located on the chromosome. Both genes were induced by the presence of phenanthrene used as a sole carbon and energy source, and as expected, both were subject to carbon catabolite repression. The relative RNA transcription level of the chromosomal (diox2) gene was significantly higher than that of its plasmid (diox1) homologue. Both diox1 and diox2 putative genes were PCR amplified, cloned, and overexpressed in Escherichia coli. Recombinant E. coli cells expressed 1-H2NA dioxygenase activity. Recombinant enzymes exhibited Michaelis-Menten kinetics with an apparent Km of 35 μM for Diox1 and 29 μM for Diox2, whereas they showed similar kinetic turnover characteristics with Kcat/Km values of 11 × 106 M−1 s−1 and 12 × 106 M−1 s−1, respectively. Occurrence of two diox1 and diox2 homologues in the Sphe3 genome implies that a replicative transposition event has contributed to the evolution of 1-H2NA dioxygenase in A. phenanthrenivorans. PMID:22101055

  14. Heterologous Expression of Der Homologs in an Escherichia coli der Mutant and Their Functional Complementation

    PubMed Central

    Choi, Eunsil; Kang, Nalae; Jeon, Young; Pai, Hyun-Sook

    2016-01-01

    ABSTRACT The unique Escherichia coli GTPase Der (double Era-like GTPase), which contains tandemly repeated GTP-binding domains, has been shown to play an essential role in 50S ribosomal subunit biogenesis. The depletion of Der results in the accumulation of precursors of 50S ribosomal subunits that are structurally unstable at low Mg2+ concentrations. Der homologs are ubiquitously found in eubacteria. Conversely, very few are conserved in eukaryotes, and none is conserved in archaea. In the present study, to verify their conserved role in bacterial 50S ribosomal subunit biogenesis, we cloned Der homologs from two gammaproteobacteria, Klebsiella pneumoniae and Salmonella enterica serovar Typhimurium; two pathogenic bacteria, Staphylococcus aureus and Neisseria gonorrhoeae; and the extremophile Deinococcus radiodurans and then evaluated whether they could functionally complement the E. coli der-null phenotype. Only K. pneumoniae and S. Typhimurium Der proteins enabled the E. coli der-null strain to grow under nonpermissive conditions. Sucrose density gradient experiments revealed that the expression of K. pneumoniae and S. Typhimurium Der proteins rescued the structural instability of 50S ribosomal subunits, which was caused by E. coli Der depletion. To determine what allows their complementation, we constructed Der chimeras. We found that only Der chimeras harboring both the linker and long C-terminal regions could reverse the growth defects of the der-null strain. Our findings suggest that ubiquitously conserved essential GTPase Der is involved in 50S ribosomal subunit biosynthesis in various bacteria and that the linker and C-terminal regions may participate in species-specific recognition or interaction with the 50S ribosomal subunit. IMPORTANCE In Escherichia coli, Der (double Era-like GTPase) is an essential GTPase that is important for the production of mature 50S ribosomal subunits. However, to date, its precise role in ribosome biogenesis has not been

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

    PubMed

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

    2013-05-25

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

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

    PubMed Central

    2013-01-01

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

  17. Impact of HIV-1 Backbone on Neutralization Sensitivity: Neutralization Profiles of Heterologous Envelope Glycoproteins Expressed in Native Subtype C and CRF01_AE Backbone

    PubMed Central

    Sanders-Buell, Eric; Wesberry, Maggie; Towle, Teresa; Pillis, Devin M.; Molnar, Sebastian; McLinden, Robert; Edmonds, Tara; Hirsch, Ivan; O’Connell, Robert; McCutchan, Francine E.; Montefiori, David C.; Ochsenbauer, Christina; Kappes, John C.; Kim, Jerome H.; Polonis, Victoria R.; Tovanabutra, Sodsai

    2013-01-01

    Standardized assays to assess vaccine and antiviral drug efficacy are critical for the development of protective HIV-1 vaccines and drugs. These immune assays will be advanced by the development of standardized viral stocks, such as HIV-1 infectious molecular clones (IMC), that i) express a reporter gene, ii) are representative of globally diverse subtypes and iii) are engineered to easily exchange envelope (env) genes for expression of sequences of interest. Thus far, a subtype B IMC backbone expressing Renilla luciferase (LucR), and into which the ectodomain of heterologous env coding sequences can be expressed has been successfully developed but as execution of HIV-1 vaccine efficacy trials shifts increasingly to non-subtype B epidemics (Southern African and Southeast Asia), non-subtype B HIV-1 reagents are needed to support vaccine development. Here we describe two IMCs derived from subtypes C and CRF01_AE HIV-1 primary isolates expressing LucR (IMC.LucR) that were engineered to express heterologous gp160 Envs. 18 constructs expressing various subtypes C and CRF01_AE Envs, mostly acute, in subtype-matched and –unmatched HIV backbones were tested for functionality and neutralization sensitivity. Our results suggest a possible effect of non-env HIV-1 genes on the interaction of Env and neutralizing antibodies and highlight the need to generate a library of IMCs representative of the HIV-1 subtype spectrum to be used as standardized neutralization assay reagents for assessing HIV-1 vaccine efficacy. PMID:24312165

  18. Heterologous expression of a gene of Magnaporthe oryzae chrysovirus 1 strain A disrupts growth of the human pathogenic fungus Cryptococcus neoformans.

    PubMed

    Urayama, Syun-Ichi; Fukuhara, Toshiyuki; Moriyama, Hiromitsu; Toh-E, Akio; Kawamoto, Susumu

    2014-05-01

    Magnaporthe oryzae chrysovirus 1 strain A (MoCV1-A) is the causal agent of growth repression and attenuated virulence (hypovirulence) of the rice blast fungus, M. oryzae. We have previously reported that heterologous expression of MoCV1-A ORF4 in Saccharomyces cerevisiae results in growth defects, a large central vacuole and other cytological changes. In this study, the effects of open reading frame (ORF) 4 expression in Cryptococcus neoformans, a human pathogenic fungus responsible for severe opportunistic infection, were investigated. Cells expressing the ORF4 gene in C. neoformans showed remarkably enlarged vacuoles, nuclear diffusion and a reduced growth rate. In addition, expression of ORF4 apparently suppressed formation of the capsule that surrounds the entire cell wall, which is one of the most important components of expression of virulence. After 5-fluoroorotic acid treatment of ORF4-expressing cells to remove the plasmid carrying the ORF4 gene, the resultant plasmid-free cells recovered normal morphology and growth, indicating that heterologous expression of the MoCV1-A ORF4 gene induces negative effects in C. neoformans. These data suggest that the ORF4 product is a candidate for a pharmaceutical protein to control disease caused by C. neoformans.

  19. Heterologous expression of Paranosema (Antonospora) locustae hexokinase in lepidopteran, Sf9, cells is followed by accumulation of the microsporidian protein in insect cell nuclei.

    PubMed

    Timofeev, Sergey A; Senderskiy, Igor V; Tsarev, Alexander A; Tokarev, Yuri S; Dolgikh, Viacheslav V

    2017-02-01

    Paranosema (Nosema, Antonospora) locustae is the only microsporidium produced as a commercial product for biological control. Molecular mechanisms of the effects of this pathogen and other invertebrate microsporidia on host cells remain uncharacterized. Previously, we immunolocalized P. locustae hexokinase in nuclei of Locusta migratoria infected adipocytes. Here, the microsporidian protein was expressed in the yeast Pichia pastoris and in lepidopteran Sf9 cells. During heterologous expression, P. locustae hexokinase was accumulated in the nuclei of insect cells but not in yeast cell nuclei. This confirms nuclear localization of hexokinase secreted by microsporidia into infected host cells and suggests convenient model for its further study.

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

    SciTech Connect

    Kirimura, Kohtaro Watanabe, Shotaro; Kobayashi, Keiichi

    2016-05-13

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

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

    PubMed Central

    2011-01-01

    Background Alkaline α-amylases have potential applications for hydrolyzing starch under high pH conditions in the starch and textile industries and as ingredients in detergents for automatic dishwashers and laundries. While the alkaline α-amylase gains increased industrial interest, the yield of alkaline α-amylases from wild-type microbes is low, and the combination of genetic engineering and process optimization is necessary to achieve the overproduction of alkaline α-amylase. Results The alkaline α-amylase gene from Bacillus alcalophilus JN21 (CCTCC NO. M 2011229) was cloned and expressed in Bacillus subtilis strain WB600 with vector pMA5. The recombinant alkaline α-amylase was stable at pH from 7.0 to 11.0 and temperature below 40°C. The optimum pH and temperature of alkaline α-amylase was 9.0 and 50°C, respectively. Using soluble starch as the substrate, the Km and Vmax of alkaline α-amylase were 9.64 g/L and 0.80 g/(L·min), respectively. The effects of medium compositions (starch, peptone, and soybean meal) and temperature on the recombinant production of alkaline α-amylase in B. subtilis were investigated. Under the optimal conditions (starch concentration 0.6% (w/v), peptone concentration 1.45% (w/v), soybean meal concentration 1.3% (w/v), and temperature 37°C), the highest yield of alkaline α-amylase reached 415 U/mL. The yield of alkaline α-amylase in a 3-L fermentor reached 441 U/mL, which was 79 times that of native alkaline α-amylase from B. alcalophilus JN21. Conclusions This is the first report concerning the heterologous expression of alkaline α-amylase in B. subtilis, and the obtained results make it feasible to achieve the industrial production of alkaline α-amylase with the recombinant B. subtilis. PMID:21978209

  2. Effects of co-expression of molecular chaperones on heterologous soluble expression of the cold-active lipase Lip-948.

    PubMed

    Shuo-shuo, Cui; Xue-zheng, Lin; Ji-hong, Shen

    2011-06-01

    The cold-active lipase gene Lip-948, cloned from Antarctic psychrotrophic bacterium Psychrobacter sp. G, was ligated into plasmid pColdI. The recombinant plasmid pColdI+Lip-948 was then transformed into Escherichia coli BL21. SDS-PAGE analysis showed that there was substantive expression of lipase LIP-948 in E. coli with a yield of about 39% of total protein, most of which was present in the inclusion body. The soluble protein LIP-948 only consisted of 1.7% of total LIP-948 with a specific activity of 66.51U/mg. Co-expression of molecular chaperones with the pColdI+Lip-948 were also carried out. The results showed that co-expression of different chaperones led to an increase or decrease in the formation of soluble LIP-948 in varying degrees. Co-expression of pColdI+Lip-948 with chaperone pTf16 and pGro7 decreased the amount of soluble LIP-948, while the soluble expression was enhanced when pColdI+Lip-948 was co-expressed with "chaperone team" plasmids (pKJE7, pG-Tf2, pG-KJE8), respectively. LIP-948 was most efficiently expressed in soluble form when it was co-expressed with pG-KJE8, which was up to 19.8% of intracellular soluble proteins and with a specific activity of 108.77U/mg. The soluble LIP-948 was purified with amylase affinity chromatography and its enzymatic characters were studied. The optimal temperature and pH of LIP-948 was 35°C and 8, respectively. The activity of LIP-948 dropped dramatically after incubation at 50°C for 15min and was enhanced by Sr(2+), Ca(2+). It preferentially hydrolyzed 4-nitrophenyl esters with the shorter carbon chain. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Heterologous inducible expression of Enterococcus faecalis pCF10 aggregation substance asc10 in Lactococcus lactis and Streptococcus gordonii contributes to cell hydrophobicity and adhesion to fibrin.

    PubMed

    Hirt, H; Erlandsen, S L; Dunny, G M

    2000-04-01

    Aggregation substance proteins encoded by the sex pheromone plasmid family of Enterococcus faecalis have been shown previously to contribute to the formation of a stable mating complex between donor and recipient cells and have been implicated in the virulence of this increasingly important nosocomial pathogen. In an effort to characterize the protein further, prgB, the gene encoding the aggregation substance Asc10 on pCF10, was cloned in a vector containing the nisin-inducible nisA promoter and its two-component regulatory system. Expression of aggregation substance after nisin addition to cultures of E. faecalis and the heterologous bacteria Lactococcus lactis and Streptococcus gordonii was demonstrated. Electron microscopy revealed that Asc10 was presented on the cell surfaces of E. faecalis and L. lactis but not on that of S. gordonii. The protein was also found in the cell culture supernatants of all three species. Characterization of Asc10 on the cell surfaces of E. faecalis and L. lactis revealed a significant increase in cell surface hydrophobicity upon expression of the protein. Heterologous expression of Asc10 on L. lactis also allowed the recognition of its binding ligand (EBS) on the enterococcal cell surface, as indicated by increased transfer of a conjugative transposon. We also found that adhesion of Asc10-expressing bacterial cells to fibrin was elevated, consistent with a role for the protein in the pathogenesis of enterococcal endocarditis. The data demonstrate that Asc10 expressed under the control of the nisA promoter in heterologous species will be an useful tool in the detailed characterization of this important enterococcal conjugation protein and virulence factor.

  4. Pulchellin, a highly toxic type 2 ribosome-inactivating protein from Abrus pulchellus. Cloning heterologous expression of A-chain and structural studies.

    PubMed

    Silva, André L C; Goto, Leandro S; Dinarte, Anemari R; Hansen, Daiane; Moreira, Renato A; Beltramini, Leila M; Araújo, Ana P U

    2005-03-01

    Pulchellin is a type 2 ribosome-inactivating protein isolated from seeds of the Abrus pulchellus tenuiflorus plant. This study aims to obtain active and homogeneous protein for structural and biological studies that will clarify the functional aspects of this toxin. The DNA fragment encoding pulchellin A-chain was cloned and inserted into pGEX-5X to express the recombinant pulchellin A-chain (rPAC) as a fusion protein in Escherichia coli. The deduced amino acid sequence analyses of the rPAC presented a high sequential identity (> 86%) with the A-chain of abrin-c. The ability of the rPAC to depurinate rRNA in yeast ribosome was also demonstrated in vitro. In order to validate the toxic activity we promoted the in vitro association of the rPAC with the recombinant pulchellin binding chain (rPBC). Both chains were incubated in the presence of a reduced/oxidized system, yielding an active heterodimer (rPAB). The rPAB showed an apparent molecular mass of approximately 60 kDa, similar to the native pulchellin. The toxic activities of the rPAB and native pulchellin were compared by intraperitoneal injection of different dilutions into mice. The rPAB was able to kill 50% of the tested mice with doses of 45 microg x kg(-1). Our results indicated that the heterodimer showed toxic activity and a conformational pattern similar to pulchellin. In addition, rPAC produced in this heterologous system might be useful for the preparation of immunoconjugates with potential as a therapeutic agent.

  5. Molecular cloning and heterologous expression of a 10-deacetylbaccatin III-10-O-acetyl transferase cDNA from Taxus x media.

    PubMed

    Guo, Binhui; Kai, Guoyin; Gong, Yifu; Jin, Hongbin; Wang, Yechun; Miao, Zhiqi; Sun, Xiaofen; Tang, Kexuan

    2007-06-01

    A full-length cDNA encoding 10-deacetylbaccatin III-10-O-acetyl transferase (designated as TmDBAT), which catalyzes the acetylation of the C-10 hydroxyl group of the advanced metabolite 10-deacetylbaccatin III (10-DAB) to yield baccatin III, the immediate diterpenoid precursor of Taxol, was isolated from Taxus x media. Heterologous expression of TmDBAT in E. coli demonstrated that TmDBAT was a functional gene. Tissue expression pattern analysis revealed that TmDBAT expressed strongly in leaves, weak in stems and no expression could be detected in fruits, implying that TmDBAT was tissue-specific. Expression profiling analysis of TmDBAT under different elicitor treatments including silver nitrate, ammonium ceric sulphate and methyl jasmonate indicated that TmDBAT was an elicitor-responsive gene. Southern blot analysis suggested that TmDBAT belonged to a small multigene family.

  6. Molecular characterization, expression analysis and heterologous expression of two translationally controlled tumor protein genes from Cucumis sativus

    PubMed Central

    Meng, Xiang nan; Chen, Qiu min; Song, Tie feng; Cui, Na; Zhao, Ju yong; Jia, Shu min; Meng, Ke xin

    2017-01-01

    The translationally controlled tumor protein (TCTP) is a family of abundant and ubiquitous proteins involved in several important primary functions. Cucumbers harbor two TCTP genes, CsTCTP1 and CsTCTP2; however, their functional roles remain unclear. In this study, we isolated CsTCTP1 and CsTCTP2 (XP-004134215 and XP-004135602, respectively) promoters, full-length cDNA and genomic sequences from Cucumis sativus. Bioinformatics analysis, containing cis-acting elements, structural domains, phylogenetic tree and conserved motifs, suggested the conservation and divergence of CsTCTP1 and CsTCTP2, thus providing knowledge regarding their functions. Expression analysis indicated that CsTCTP1 and CsTCTP2 exhibited tissue-specific expression and were regulated by biotic or abiotic stresses in C. sativus. Furthermore, CsTCTP1 and CsTCTP2 were regulated by ABA and may be associated with the TOR (target of rapamycin) signaling pathway. In a prokaryotic expression analysis, CsTCTP1 and CsTCTP2 showed positive responses to salt and heat stresses and a negative response to drought and HgCl2 stresses. TCTP may exert multiple functions in various cellular processes. PMID:28926624

  7. Short communication: Improving the activity of bile salt hydrolases in Lactobacillus casei based on in silico molecular docking and heterologous expression.

    PubMed

    Xiong, Zhi-Qiang; Wang, Qiao-Hui; Kong, Ling-Hui; Song, Xin; Wang, Guang-Qiang; Xia, Yong-Jun; Zhang, Hui; Sun, Yong; Ai, Lian-Zhong

    2017-02-01

    Bile salt hydrolase (BSH) plays an essential role in the cholesterol-removing effect of lactic acid bacteria, which hydrolyze conjugated bile salts to amino acid and deconjugated bile salts. However, Lactobacillus casei lacks the bsh gene, which may make it highly sensitive to bile salt stress. We wanted to improve the BSH activity of L. casei for various food-industry applications (e.g., milk fermentation). Plate assay testing indicated that Lactobacillus plantarum AR113 has the highest BSH activity. We cloned and sequenced 4 bsh genes from the genome of L. plantarum AR113. Structure modeling and molecular docking of BSH indicated that BSH1 and BSH3 could react efficiently with bile salts, so we selected BSH1 and BSH3 for heterologous expression in L. casei. Compared with single expression of BSH1 or BSH3, co-expression of both protein sequences showed the highest hydrolysis activity by HPLC analysis. Our results suggested that heterologous expression of BSH in L. casei can significantly improve host activity against bile salts, and in silico molecular docking could be an efficient method of rapid screening for BSH with high activity.

  8. The Heterologous Expression of the Chrysanthemum R2R3-MYB Transcription Factor CmMYB1 Alters Lignin Composition and Represses Flavonoid Synthesis in Arabidopsis thaliana

    PubMed Central

    Chen, Sumei; Jiang, Jiafu; Gu, Chunsun; Zhou, Guoqin; Chen, Yu; Song, Aiping; Chen, Fadi

    2013-01-01

    Plant R2R3-MYB transcription factor genes are widely distributed in higher plants and play important roles in the regulation of many secondary metabolites at the transcriptional level. In this study, a chrysanthemum subgroup 4 R2R3-MYB transcription factor gene, designated CmMYB1, was isolated through screening chrysanthemum EST (expressed sequence tag) libraries and using rapid application of cDNA ends (RACE) methods and functionally characterized. CmMYB1 is expressed in the root, stem, leaf and flowers, but most strongly in the stem and most weakly in the root. Its heterologous expression in Arabidopsis thaliana reduced the lignin content and altered the lignin composition. The heterologous expression also repressed the flavonoids content in A. thaliana. Together, these results suggested that CmMYB1 is a negative regulator of genes involved in the lignin pathway and flavonoid pathway, it may be a promising gene for controlling lignin and flavonoids profiles in plants. PMID:23840353

  9. Heterologous production of kasugamycin, an aminoglycoside antibiotic from Streptomyces kasugaensis, in Streptomyces lividans and Rhodococcus erythropolis L-88 by constitutive expression of the biosynthetic gene cluster.

    PubMed

    Kasuga, Kano; Sasaki, Akira; Matsuo, Takashi; Yamamoto, Chika; Minato, Yuiko; Kuwahara, Naoya; Fujii, Chikako; Kobayashi, Masayuki; Agematu, Hitosi; Tamura, Tomohiro; Komatsu, Mamoru; Ishikawa, Jun; Ikeda, Haruo; Kojima, Ikuo

    2017-02-27

    Kasugamycin (KSM), an aminoglycoside antibiotic isolated from Streptomyces kasugaensis cultures, has been used against rice blast disease for more than 50 years. We cloned the KSM biosynthetic gene (KBG) cluster from S. kasugaensis MB273-C4 and constructed three KBG cassettes (i.e., cassettes I-III) to enable heterologous production of KSM in many actinomycetes by constitutive expression of KBGs. Cassette I comprised all putative transcriptional units in the cluster, but it was placed under the control of the P neo promoter from Tn5. It was not maintained stably in Streptomyces lividans and did not transform Rhodococcus erythropolis. Cassette II retained the original arrangement of KBGs, except that the promoter of kasT, the specific activator gene for KBG, was replaced with P rpsJ , the constitutive promoter of rpsJ from Streptomyces avermitilis. To enhance the intracellular concentration of myo-inositol, an expression cassette of ino1 encoding the inositol-1-phosphate synthase from S. avermitilis was inserted into cassette II to generate cassette III. These two cassettes showed stable maintenance in S. lividans and R. erythropolis to produce KSM. Particularly, the transformants of S. lividans induced KSM production up to the same levels as those produced by S. kasugaensis. Furthermore, cassette III induced more KSM accumulation than cassette II in R. erythropolis, suggesting an exogenous supply of myo-inositol by the ino1 expression in the host. Cassettes II and III appear to be useful for heterologous KSM production in actinomycetes. Rhodococcus exhibiting a spherical form in liquid cultivation is also a promising heterologous host for antibiotic fermentation.

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

    PubMed

    An, Jing; Hu, Zhenmin; Che, Benning; Chen, Haiying; Yu, Bingjun; Cai, Weiming

    2017-01-01

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

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

    PubMed Central

    An, Jing; Hu, Zhenmin; Che, Benning; Chen, Haiying; Yu, Bingjun; Cai, Weiming

    2017-01-01

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

  12. Heterologous protein production by filamentous fungi.

    PubMed

    Jeenes, D J; Mackenzie, D A; Roberts, I N; Archer, D B

    1991-01-01

    There are clearly many facets to successful production of heterologous proteins from filamentous fungi. The objectives are to exploit the natural ability of some species to secrete high levels of protein. The heterologous target proteins produced in a fungal host must be acceptable to the public and be economic to produce, i.e. the targets must be authentic (in structure and activity) and be produced in high yield to necessary levels of purity. The appearance of heterologous products from fungi on the market is testament to some success but, equally, there are considerable limitations in our ability to produce desired yields of many target proteins. We endorse the view of van den Hondel, Punt and van Gorcom (1991) that for the commercial production of heterologous proteins from filamentous fungi more information is required on transcriptional control, introns, mRNA stability and processing, translational efficiency, protein secretion, glycosylation and proteolysis. In addition, there is scope for yield improvement based on a better understanding of the physiology of growth/product secretion coupled to appropriate bioreactor operation. The authenticity of product is an aspect which will assume increasing importance, particularly for therapeutic proteins. The level at which the structures and functional activity of heterologous proteins are assessed will ultimately be determined by legislation. The analytical methods currently available are not always sufficient, for example, to reveal folded structures, and most proteins are not amenable to analysis by two-dimensional NMR. The authenticity of target heterologous proteins will also need to be assessed in relation to the glycosylation level and pattern. This is not easily done and explains the paucity of detailed information published to date on glycosylation of fungal proteins. Novel engineered proteins are already being produced from filamentous fungi where expression is an aid to investigation of structure

  13. Boosting isoprene production via heterologous expression of the Kudzu isoprene synthase gene (kIspS) into Bacillus spp. cell factory.

    PubMed

    Gomaa, Lamis; Loscar, Michael E; Zein, Haggag S; Abdel-Ghaffar, Nahed; Abdelhadi, Abdelhadi A; Abdelaal, Ali S; Abdallah, Naglaa A

    2017-08-08

    Isoprene represents a key building block for the production of valuable materials such as latex, synthetic rubber or pharmaceutical precursors and serves as basis for advanced biofuel production. To enhance the production of the volatile natural hydrocarbon isoprene, released by plants, animals and bacteria, the Kudzu isoprene synthase (kIspS) gene has been heterologously expressed in Bacillus subtilis DSM 402 and Bacillus licheniformis DSM 13 using the pHT01 vector. As control, the heterologous expression of KIspS in E. coli BL21 (DE3) with the pET28b vector was used. Isoprene production was analyzed using Gas Chromatography Flame Ionization Detector. The highest isoprene production was observed by recombinant B. subtilis harboring the pHT01-kIspS plasmid which produced 1434.3 μg/L (1275 µg/L/OD) isoprene. This is threefold higher than the wild type which produced 388 μg/L (370 μg/L/OD) isoprene, when both incubated at 30 °C for 48 h and induced with 0.1 mM IPTG. Additionally, recombinant B. subtilis produced fivefold higher than the recombinant B. licheniformis, which produced 437.2 μg/L (249 μg/L/OD) isoprene when incubated at 37 °C for 48 h induced with 0.1 mM IPTG. This is the first report of optimized isoprene production in B. licheniformis. However, recombinant B. licheniformis showed less isoprene production. Therefore, recombinant B. subtilis is considered as a versatile host for heterologous production of isoprene.

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

    PubMed

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

    2017-05-01

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

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

    PubMed

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

    2006-01-01

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

  16. [Heterologous expression of functionally active human cytochrome P-450s. Cytochrome P-450IIIA4 catalyzes the biotransformation of the anabolic steroid hormone methandrostenolone].

    PubMed

    Krynetskiĭ, E Iu; Kovaleva, I E; Luzikov, V N

    1994-02-01

    The expression of the cytochrome P450IIIA4 gene in the Saccharomyces cerevisiae yeast using the shuttle vector pYeDP1-8/2 has been carried out. The microsomal fraction isolated from the transformed yeast cells was used for biotransformation of the anabolic steroid hormone-methandrostenolone (MA). The microsomal oxidation products were analyzed by HPLC and two-dimensional TLC. It was shown that microsomes of the yeasts expressing human cytochrome P450IIIA4 catalyze the MA conversion into its 6 beta-hydroxy derivative. An identical product is formed via a reaction catalyzed by human liver microsomes. The use of the heterological system of cytochrome P450IIIA4 expression has made it possible to establish its role in MA metabolism. The experimental system simulates the first phase of the drug biotransformation in liver cells.

  17. Heterologous expression of metK1-sp and afsR-sp in Streptomyces venezuelae for the production of pikromycin.

    PubMed

    Maharjan, Sushila; Oh, Tae-Jin; Lee, Hei Chan; Sohng, Jae Kyung

    2008-09-01

    Two regulator genes, metK1-sp and afsR-sp, from Streptomyces peucetius ATCC 27952 were heterologously expressed in S. venezuelae ATCC 15439, to produce 14-membered pikromycin antibiotics. The production of pikromycin was increased by 1.6-fold and 2.6-fold by the expression of metK1-sp and afsR-sp, respectively. The overexpression of metK1-sp and afsR-sp in S. venezuelae stimulated the expression of the pathway-specific regulatory genes, pikD and ketosynthase, as demonstrated by RT-PCR. The elevated transcripts of the pikD and ketosynthase genes were consistent with the enhanced production of pikromycin.

  18. High-level intracellular expression of heterologous proteins in Brevibacillus choshinensis SP3 under the control of a xylose inducible promoter

    PubMed Central

    2013-01-01

    Background In past years research has focused on the development of alternative Gram positive bacterial expression systems to produce industrially relevant proteins. Brevibacillus choshinensis is an easy to handle non-sporulating bacterium, lacking extracellular proteases, that has been already shown to provide a high level of recombinant protein expression. One major drawback, limiting the applicability of the Brevibacillus expression system, is the absence of expression vectors based on inducible promoters. Here we used the PxylA inducible promoter, commonly employed in other Bacillae expression systems, in Brevibacillus. Results Using GFP, α-amylase and TcdA-GT as model proteins, high level of intracellular protein expression (up to 250 mg/L for the GFP) was achieved in Brevibacillus, using the pHis1522 vector carrying the B. megaterium xylose-inducible promoter (PxylA). The GFP expression yields were more than 25 fold higher than those reported for B. megaterium carrying the same vector. All the tested proteins show significant increment in their expression levels (2-10 folds) than those obtained using the available plasmids based on the P2 constitutive promoter. Conclusion Combining the components of two different commercially available Gram positive expression systems, such as Brevibacillus (from Takara Bio) and B. megaterium (from Mobitec), we demonstrate that vectors based on the B. megaterium PxylA xylose inducible promoter can be successfully used to induce high level of intracellular expression of heterologous proteins in Brevibacillus. PMID:23374160

  19. Translational Arrest Due to Cytoplasmic Redox Stress Delays Adaptation to Growth on Methanol and Heterologous Protein Expression in a Typical Fed-Batch Culture of Pichia pastoris

    PubMed Central

    Edwards-Jones, Bryn; Aw, Rochelle; Barton, Geraint R.; Tredwell, Gregory D.; Bundy, Jacob G.; Leak, David J.

    2015-01-01

    Results We have followed a typical fed-batch induction regime for heterologous protein production under the control of the AOX1 promoter using both microarray and metabolomic analysis. The genetic constructs involved 1 and 3 copies of the TRY1 gene, encoding human trypsinogen. In small-scale laboratory cultures, expression of the 3 copy-number construct induced the unfolded protein response (UPR) sufficiently that titres of extracellular trypsinogen were lower in the 3-copy construct than with the 1-copy construct. In the fed-batch-culture, a similar pattern was observed, with higher expression from the 1-copy construct, but in this case there was no significant induction of UPR with the 3-copy strain. Analysis of the microarray and metabolomic information indicates that the 3-copy strain was undergoing cytoplasmic redox stress at the point of induction with methanol. In this Crabtree-negative yeast, this redox stress appeared to delay the adaptation to growth on methanol and supressed heterologous protein production, probably due to a block in translation. Conclusion Although redox imbalance as a result of artificially imposed hypoxia has previously been described, this is the first time that it has been characterised as a result of a transient metabolic imbalance and shown to involve a stress response which can lead to translational arrest. Without detailed analysis of the underlying processes it could easily have been mis-interpreted as secretion stress, transmitted through the UPR. PMID:25785713

  20. 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

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

    PubMed

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

    2014-02-01

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

  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

    2015-10-13

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

  3. Heterologous expression of yeast Hxt2 in Arabidopsis thaliana alters sugar uptake, carbon metabolism and gene expression leading to glucose tolerance of germinating seedlings.

    PubMed

    Padilla-Chacón, Daniel; Cordoba, Elizabeth; Olivera, Teresa; Sánchez, Sobeida; Coello, Patricia; León, Patricia; Tiessen, Axel; Martínez-Barajas, Eleazar

    2010-04-01

    The hexose transporter 2 gene (Hxt2) from Saccharomyces cerevisiae was expressed in Arabidopsis thaliana under control of the 35S promoter. Several independent transgenic lines were selected after confirming single gene insertion by southern blot analysis in the T4 generation. Northern blots revealed the presence of heterologous transcript. Radiolabeling experiments revealed an increased rate of incorporation of the non-metabolizable analog 3-O-methyl-[U-14C]-glucose. This confirmed that the yeast Hxt2 transporter was functional in Arabidopsis. No phenotypic changes at the vegetative and reproductive stages could be detected in the transgenic lines when compared to wild type plants. Shortly after germination some differences in development and glucose signaling were observed. Transgenic seedlings cultivated in liquid medium or on solid agar plates were able to grow with 3% glucose (producing bigger plants and longer roots), while development of wild type plants was delayed under those conditions. Metabolite analysis revealed that the Hxt2 transgenic lines had higher rates of sugar utilization. Transcriptional profiling showed that particular genes were significantly up- or down-regulated. Some transcription factors like At1g27000 were repressed, while others, such as At3g58780, were induced. The mRNA from classical sugar signaling genes such as STP1, Hxk1, and ApL3 behaved similarly in transgenic lines and wild type lines. Results suggest that the Hxt2 transgene altered some developmental processes related to the perception of high carbon availability after the germination stage. We conclude that the developmental arrest of wild type plants at 3% glucose not only depends on Hxk1 as the only sugar sensor but might also be influenced by the route of hexose transport across the plasma membrane.

  4. RecET direct cloning and Redαβ recombineering of biosynthetic gene clusters, large operons or single genes for heterologous expression.

    PubMed

    Wang, Hailong; Li, Zhen; Jia, Ruonan; Hou, Yu; Yin, Jia; Bian, Xiaoying; Li, Aiying; Müller, Rolf; Stewart, A Francis; Fu, Jun; Zhang, Youming

    2016-07-01

    Full-length RecE and RecT from Rac prophage mediate highly efficient linear-linear homologous recombination that can be used to clone large DNA regions directly from genomic DNA into expression vectors, bypassing library construction and screening. Homologous recombination mediated by Redαβ from lambda phage has been widely used for recombinant DNA engineering. Here we present a protocol for direct cloning and engineering of biosynthetic gene clusters, large operons or single genes from genomic DNA using one Escherichia coli host that harbors both RecET and Redαβ systems. The pipeline uses standardized cassettes for horizontal gene transfer options, as well as vectors with different replication origins configured to minimize recombineering background through the use of selectively replicating templates or CcdB counterselection. These optimized reagents and protocols facilitate fast acquisition of transgenes from genomic DNA preparations, which are ready for heterologous expression within 1 week.

  5. Heterologous Expression of Equine CYP3A94 and Investigation of a Tunable System to Regulate Co-Expressed NADPH P450 Oxidoreductase Levels

    PubMed Central

    Dettwiler, Ramona; Schmitz, Andrea L.; Plattet, Philippe; Zielinski, Jana; Mevissen, Meike

    2014-01-01

    The activity of cytochrome P450 enzymes depends on the enzyme NADPH P450 oxidoreductase (POR). The aim of this study was to investigate the activity of the equine CYP3A94 using a system that allows to regulate the POR protein levels in mammalian cells. CYP3A94 and the equine POR were heterologously expressed in V79 cells. In the system used, the POR protein regulation is based on a destabilizing domain (DD) that transfers its instability to a fused protein. The resulting fusion protein is therefore degraded by the ubiquitin-proteasome system (UPS). Addition of “Shield-1” prevents the DD fusion protein from degradation. The change of POR levels at different Shield-1 concentrations was demonstrated by cytochrome c reduction, Western immunoblot analysis, and immunocytochemistry. The alteration of CYP3A94 activity was investigated using a substrate (BFC) known to detect CYP3A4 activity. Equine CYP3A94 was demonstrated to be metabolically active and its activity could be significantly elevated by co-expression of POR. Cytochrome c reduction was significantly increased in V79-CYP3A94/DD-POR cells compared to V79-CYP3A94 cells. Surprisingly, incubation with different Shield-1 concentrations resulted in a decrease in POR protein shown by Western immunoblot analysis. Cytochrome c reduction did not change significantly, but the CYP3A94 activity decreased more than 4-fold after incubation with 500 nM and 1 µM Shield-1 for 24 hours. No differences were obtained when V79-CYP3A94 POR cells with and without Shield-1 were compared. The basal activity levels of V79-CYP3A94/DD-POR cells were unexpectedly high, indicating that DD/POR is not degraded without Shield-1. Shield-1 decreased POR protein levels and CYP3A94 activity suggesting that Shield-1 might impair POR activity by an unknown mechanism. Although regulation of POR with the pPTuner system could not be obtained, the cell line V79-CYP3A94/DD-POR system can be used for further experiments to characterize the equine CYP3A

  6. Heterologous prime-boost vaccination with DNA and MVA vaccines, expressing HIV-1 subtype C mosaic Gag virus-like particles, is highly immunogenic in mice

    PubMed Central

    Douglass, Nicola; Chege, Gerald; Williamson, Anna-Lise

    2017-01-01

    In an effort to make affordable vaccines suitable for the regions most affected by HIV-1, we have constructed stable vaccines that express an HIV-1 subtype C mosaic Gag immunogen (BCG-GagM, MVA-GagM and DNA-GagM). Mosaic immunogens have been designed to address the tremendous diversity of this virus. Here we have shown that GagM buds from cells infected and transfected with MVA-GagM and DNA-GagM respectively and forms virus-like particles. Previously we showed that a BCG-GagM prime MVA-GagM boost generated strong cellular immune responses in mice. In this study immune responses to the DNA-GagM and MVA-GagM vaccines were evaluated in homologous and heterologous prime-boost vaccinations. The DNA homologous prime boost vaccination elicited predominantly CD8+ T cells while the homologous MVA vaccination induced predominantly CD4+ T cells. A heterologous DNA-GagM prime MVA-GagM boost induced strong, more balanced Gag CD8+ and CD4+ T cell responses and that were predominantly of an effector memory phenotype. The immunogenicity of the mosaic Gag (GagM) was compared to a naturally occurring subtype C Gag (GagN) using a DNA homologous vaccination regimen. DNA-GagN expresses a natural Gag with a sequence that was closest to the consensus sequence of subtype C viruses sampled in South Africa. DNA-GagM homologous vaccination induced cumulative HIV-1 Gag-specific IFN-γ ELISPOT responses that were 6.5-fold higher than those induced by the DNA-GagN vaccination. Similarly, DNA-GagM vaccination generated 7-fold higher levels of cytokine-positive CD8+ T cells than DNA-GagN, indicating that this subtype C mosaic Gag elicits far more potent immune responses than a consensus-type Gag. Cells transfected and infected with DNA-GagM and MVA-GagM respectively, expressed high levels of GagM and produced budding virus-like particles. Our data indicates that a heterologous prime boost regimen using DNA and MVA vaccines expressing HIV-1 subtype C mosaic Gag is highly immunogenic in mice and

  7. Heterologous prime-boost vaccination with DNA and MVA vaccines, expressing HIV-1 subtype C mosaic Gag virus-like particles, is highly immunogenic in mice.

    PubMed

    Chapman, Ros; Jongwe, Tsungai Ivai; Douglass, Nicola; Chege, Gerald; Williamson, Anna-Lise

    2017-01-01

    In an effort to make affordable vaccines suitable for the regions most affected by HIV-1, we have constructed stable vaccines that express an HIV-1 subtype C mosaic Gag immunogen (BCG-GagM, MVA-GagM and DNA-GagM). Mosaic immunogens have been designed to address the tremendous diversity of this virus. Here we have shown that GagM buds from cells infected and transfected with MVA-GagM and DNA-GagM respectively and forms virus-like particles. Previously we showed that a BCG-GagM prime MVA-GagM boost generated strong cellular immune responses in mice. In this study immune responses to the DNA-GagM and MVA-GagM vaccines were evaluated in homologous and heterologous prime-boost vaccinations. The DNA homologous prime boost vaccination elicited predominantly CD8+ T cells while the homologous MVA vaccination induced predominantly CD4+ T cells. A heterologous DNA-GagM prime MVA-GagM boost induced strong, more balanced Gag CD8+ and CD4+ T cell responses and that were predominantly of an effector memory phenotype. The immunogenicity of the mosaic Gag (GagM) was compared to a naturally occurring subtype C Gag (GagN) using a DNA homologous vaccination regimen. DNA-GagN expresses a natural Gag with a sequence that was closest to the consensus sequence of subtype C viruses sampled in South Africa. DNA-GagM homologous vaccination induced cumulative HIV-1 Gag-specific IFN-γ ELISPOT responses that were 6.5-fold higher than those induced by the DNA-GagN vaccination. Similarly, DNA-GagM vaccination generated 7-fold higher levels of cytokine-positive CD8+ T cells than DNA-GagN, indicating that this subtype C mosaic Gag elicits far more potent immune responses than a consensus-type Gag. Cells transfected and infected with DNA-GagM and MVA-GagM respectively, expressed high levels of GagM and produced budding virus-like particles. Our data indicates that a heterologous prime boost regimen using DNA and MVA vaccines expressing HIV-1 subtype C mosaic Gag is highly immunogenic in mice and

  8. Solubility enhancement of aggregation-prone heterologous proteins by fusion expression using stress-responsive Escherichia coli protein, RpoS.

    PubMed

    Park, Jin-Seung; Han, Kyung-Yeon; Lee, Jong-Ho; Song, Jong-Am; Ahn, Keum-Young; Seo, Hyuk-Seong; Sim, Sang-Jun Jun; Kim, Seung-Wook; Lee, Jeewon

    2008-02-19

    The most efficient method for enhancing solubility of recombinant proteins appears to use the fusion expression partners. Although commercial fusion partners including maltose binding protein and glutathione-S-transferase have shown good performance in enhancing the solubility, they cannot be used for the proprietory production of commercially value-added proteins and likely cannot serve as universal helpers to solve all protein solubility and folding issues. Thus, novel fusion partners will continue to be developed through systematic investigations including proteome mining presented in this study. We analyzed the Escherichia coli proteome response to the exogenous stress of guanidine hydrochloride using 2-dimensional gel electrophoresis and found that RpoS (RNA polymerase sigma factor) was significantly stress responsive. While under the stress condition the total number of soluble proteins decreased by about 7 %, but a 6-fold increase in the level of RpoS was observed, indicating that RpoS is a stress-induced protein. As an N-terminus fusion expression partner, RpoS increased significantly the solubility of many aggregation-prone heterologous proteins in E. coli cytoplasm, indicating that RpoS is a very effective solubility enhancer for the synthesis of many recombinant proteins. RpoS was also well suited for the production of a biologically active fusion mutant of Pseudomonas putida cutinase. RpoS is highly effective as a strong solubility enhancer for aggregation-prone heterologous proteins when it is used as a fusion expression partner in an E. coli expression system. The results of these findings may, therefore, be useful in the production of other biologically active industrial enzymes, as successfully demonstrated by cutinase.

  9. Functional Genome Mining for Metabolites Encoded by Large Gene Clusters through Heterologous Expression of a Whole-Genome Bacterial Artificial Chromosome Library in Streptomyces spp.

    PubMed Central

    Xu, Min; Wang, Yemin; Zhao, Zhilong; Gao, Guixi; Huang, Sheng-Xiong; Kang, Qianjin; He, Xinyi; Lin, Shuangjun; Pang, Xiuhua; Deng, Zixin

    2016-01-01

    ABSTRACT Genome sequencing projects in the last decade revealed numerous cryptic biosynthetic pathways for unknown secondary metabolites in microbes, revitalizing drug discovery from microbial metabolites by approaches called genome mining. In this work, we developed a heterologous expression and functional screening approach for genome mining from genomic bacterial artificial chromosome (BAC) libraries in Streptomyces spp. We demonstrate mining from a strain of Streptomyces rochei, which is known to produce streptothricins and borrelidin, by expressing its BAC library in the surrogate host Streptomyces lividans SBT5, and screening for antimicrobial activity. In addition to the successful capture of the streptothricin and borrelidin biosynthetic gene clusters, we discovered two novel linear lipopeptides and their corresponding biosynthetic gene cluster, as well as a novel cryptic gene cluster for an unknown antibiotic from S. rochei. This high-throughput functional genome mining approach can be easily applied to other streptomycetes, and it is very suitable for the large-scale screening of genomic BAC libraries for bioactive natural products and the corresponding biosynthetic pathways. IMPORTANCE Microbial genomes encode numerous cryptic biosynthetic gene clusters for unknown small metabolites with potential biological activities. Several genome mining approaches have been developed to activate and bring these cryptic metabolites to biological tests for future drug discovery. Previous sequence-guided procedures relied on bioinformatic analysis to predict potentially interesting biosynthetic gene clusters. In this study, we describe an efficient approach based on heterologous expression and functional screening of a whole-genome library for the mining of bioactive metabolites from Streptomyces. The usefulness of this function-driven approach was demonstrated by the capture of four large biosynthetic gene clusters for metabolites of various chemical types, including

  10. Development of a gene cloning system in a fast-growing and moderately thermophilic Streptomyces species and heterologous expression of Streptomyces antibiotic biosynthetic gene clusters

    PubMed Central

    2011-01-01

    Background Streptomyces species are a major source of antibiotics. They usually grow slowly at their optimal temperature and fermentation of industrial strains in a large scale often takes a long time, consuming more energy and materials than some other bacterial industrial strains (e.g., E. coli and Bacillus). Most thermophilic Streptomyces species grow fast, but no gene cloning systems have been developed in such strains. Results We report here the isolation of 41 fast-growing (about twice the rate of S. coelicolor), moderately thermophilic (growing at both 30°C and 50°C) Streptomyces strains, detection of one linear and three circular plasmids in them, and sequencing of a 6996-bp plasmid, pTSC1, from one of them. pTSC1-derived pCWH1 could replicate in both thermophilic and mesophilic Streptomyces strains. On the other hand, several Streptomyces replicons function in thermophilic Streptomyces species. By examining ten well-sporulating strains, we found two promising cloning hosts, 2C and 4F. A gene cloning system was established by using the two strains. The actinorhodin and anthramycin biosynthetic gene clusters from mesophilic S. coelicolor A3(2) and thermophilic S. refuineus were heterologously expressed in one of the hosts. Conclusions We have developed a gene cloning and expression system in a fast-growing and moderately thermophilic Streptomyces species. Although just a few plasmids and one antibiotic biosynthetic gene cluster from mesophilic Streptomyces were successfully expressed in thermophilic Streptomyces species, we expect that by utilizing thermophilic Streptomyces-specific promoters, more genes and especially antibiotic genes clusters of mesophilic Streptomyces should be heterologously expressed. PMID:22032628

  11. Endogenous PttHb1 and PttTrHb, and heterologous Vitreoscilla vhb haemoglobin gene expression in hybrid aspen roots with ectomycorrhizal interaction

    PubMed Central

    Jokipii, Soile; Häggman, Hely; Brader, Günter; Kallio, Pauli T.; Niemi, Karoliina

    2008-01-01

    Present knowledge on plant non-symbiotic class-1 (Hb1) and truncated (TrHb) haemoglobin genes is almost entirely based on herbaceous species while the corresponding tree haemoglobin genes are not well known. The function of these genes has recently been linked with endosymbioses between plants and microbes. In this work, the coding sequences of hybrid aspen (Populus tremula×tremuloides) PttHb1 and PttTrHb were characterized, indicating that the key residues of haem and ligand binding of both genes were conserved in the deduced amino acid sequences. The expression of PttHb1 and PttTrHb was examined in parallel with that of the heterologous Vitreoscilla haemoglobin gene (vhb) during ectomycorrhiza/ectomycorrhizal (ECM) interaction. Both ECM fungi studied, Leccinum populinum and Xerocomus subtomentosus, enhanced root formation and subsequent growth of roots of all hybrid aspen lines, but only L. populinum was able to form mycorrhizas. Real-time PCR results show that the dual culture with the ECM fungus, with or without emergence of symbiotic structures, increased the expression of both PttHb1 and PttTrHb in the roots of non-transgenic hybrid aspens. PttHb1 and PttTrHb had expression peaks 5 h and 2 d after inoculation, respectively, pointing to different functions for these genes during interaction with root growth-improving fungi. In contrast, ECM fungi were not able to enhance the expression of hybrid aspen endogenous haemoglobin genes in the VHb lines, which may be a consequence of the compensating action of heterologous haemoglobin. PMID:18544611

  12. Green targeting predictor and ambiguous targeting predictor 2: the pitfalls of plant protein targeting prediction and of transient protein expression in heterologous systems.

    PubMed

    Fuss, Janina; Liegmann, Oliver; Krause, Kirsten; Rensing, Stefan A

    2013-12-01

    The challenges of plant protein targeting prediction are the existence of dual subcellular targets and the bias of experimentally confirmed data towards few and mostly nonplant model species. To assess whether training with proteins from evolutionarily distant species has a negative impact on prediction accuracy, we developed the Green Targeting Predictor tool, which was trained with a species-specific data set for Physcomitrella patens. Its performance was compared with that of the same tool trained with a mixed data set. In addition, we updated the Ambiguous Targeting Predictor. We found that predictions deviated from in vivo observations predominantly for proteins diverging within the green lineage, as well as for dual targeted proteins. To evaluate the usefulness of heterologous expression systems, selected proteins were subjected to localization studies in P. patens, Arabidopsis thaliana and Nicotiana tabacum. Four out of six proteins that show dual targeting in the original plant system were located only in a single compartment in one or both heterologous systems. We conclude that targeting signals of divergent plant species exhibit differences, calling for custom in silico and in vivo approaches when aiming to unravel the actual distribution patterns of proteins within a plant cell. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

  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.

  14. 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

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

    PubMed Central

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

    2011-01-01

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

  16. 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

  17. The alkaline pectate lyase PEL168 of Bacillus subtilis heterologously expressed in Pichia pastoris is more stable and efficient for degumming ramie fiber

    PubMed Central

    2013-01-01

    Background The conventional degumming process of ramie with alkaline treatment at high temperature causes severe environmental pollution. Pectate lyases can be used to remove pectin from ramie in a degumming process with reduced environmental pollution and energy consumption. Pectate lyase PEL168 from Bacillus subtilis has been previously characterized and the protein structure was resolved. However, Bacillus is not a suitable host for pectate lyases during the degumming process since most Bacillus produce cellulases endogenously with a detrimental effect to the fiber. Pichia pastoris, which does not express endogenous cellulases and has high secretion capability, will be an ideal host for the expression. No previous work was reported concerning the heterologous expression of pectate lyase PEL168 in P. pastoris with an aim for industrial application in ramie bio-degumming. Results The gene pel168 was expressed in P. pastoris in this study. The recombinant protein PEL168 in P. pastoris (PEL168P) showed two bands of 48.6 kDa and 51.4 kDa on SDS-PAGE whereas the enzyme expressed in E. coli (PEL168E) was the same as predicted with a band of 46 kDa. Deglycosylation digestion suggested that PEL168P was glycosylated. The optimum reaction temperature of the two PEL168s was 50°C, and the optimum pH 9.5. After preincubation at 60°C for 20 min, PEL168E completely lost its activity, whereas PEL168P kept 26% of the residual activity. PEL168P had a specific activity of 1320 U/mg with a Km of 0.09 mg/ml and a Vmax of 18.13 μmol/min. K+, Li+, Ni2+ and Sr2+ showed little or no inhibitory effect on PEL168P activity, and Ca2+ enhanced enzyme activity by 38%. PEL168P can remove the pectin from ramie effectively in a degumming process. A 1.5 fold increase of PEL168 enzyme expression in P. pastoris was achieved by further codon optimization. Conclusions Pectate lyase PEL168 with an available protein structure can be heterologously expressed in P. pastoris. The characterized

  18. 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.

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

    PubMed Central

    Rohlfs, Marko; Anyaogu, Diana Chinyere; Nielsen, Jakob Blæsbjerg; Gotfredsen, Charlotte Held; Andersen, Mikael Rørdam; Hansen, Bjarne Gram; Mortensen, Uffe Hasbro; Larsen, Thomas Ostenfeld

    2013-01-01

    Secondary metabolites are known to serve a wide range of specialized functions including communication, developmental control and defense. Genome sequencing of several fungal model species revealed that the majority of predicted secondary metabolite related genes are silent in laboratory strains, indicating that fungal secondary metabolites remain an underexplored resource of bioactive molecules. In this study, we combine heterologous expression of regulatory proteins in Aspergillus nidulans with systematic variation of growth conditions and observe induced synthesis of insect juvenile hormone-III and methyl farnesoate. Both compounds are sesquiterpenes belonging to the juvenile hormone class. Juvenile hormones regulate developmental and metabolic processes in insects and crustaceans, but have not previously been reported as fungal metabolites. We found that feeding by Drosophila melanogaster larvae induced synthesis of juvenile hormone in A. nidulans indicating a possible role of juvenile hormone biosynthesis in affecting fungal-insect antagonisms. PMID:23991191

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

    PubMed

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

    2017-08-19

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

  1. Genetically modified bacterial strains and novel bacterial artificial chromosome shuttle vectors for constructing environmental libraries and detecting heterologous natural products in multiple expression hosts.

    PubMed

    Martinez, Asuncion; Kolvek, Steven J; Yip, Choi Lai Tiong; Hopke, Joern; Brown, Kara A; MacNeil, Ian A; Osburne, Marcia S

    2004-04-01

    The enormous diversity of uncultured microorganisms in soil and other environments provides a potentially rich source of novel natural products, which is critically important for drug discovery efforts. Our investigators reported previously on the creation and screening of an Escherichia coli library containing soil DNA cloned and expressed in a bacterial artificial chromosome (BAC) vector. In that initial study, our group identified novel enzyme activities and a family of antibacterial small molecules encoded by soil DNA cloned and expressed in E. coli. To continue our pilot study of the utility and feasibility of this approach to natural product drug discovery, we have expanded our technology to include Streptomyces lividans and Pseudomonas putida as additional hosts with different expression capabilities, and herein we describe the tools we developed for transferring environmental libraries into all three expression hosts and screening for novel activities. These tools include derivatives of S. lividans that contain complete and unmarked deletions of the act and red endogenous pigment gene clusters, a derivative of P. putida that can accept environmental DNA vectors and integrate the heterologous DNA into the chromosome, and new BAC shuttle vectors for transferring large fragments of environmental DNA from E. coli to both S. lividans and P. putida by high-throughput conjugation. Finally, we used these tools to confirm that the three hosts have different expression capabilities for some known gene clusters.

  2. Heterologous expression of Zygosaccharomyces rouxii glycerol 3-phosphate dehydrogenase gene (ZrGPD1) and glycerol dehydrogenase gene (ZrGCY1) in Saccharomyces cerevisiae.

    PubMed

    Watanabe, Yasuo; Tsuchimoto, Syoko; Tamai, Youichi

    2004-01-01

    We examined the effects of heterologous expression of the open reading frames (ORF) of two genes on salt tolerance and glycerol production in a Saccharomyces cerevisiae strain deficient in glycerol synthesis (gpd1Deltagpd2Delta). When the ORF of the Zygosaccharomyces rouxii glycerol 3-phosphate dehydrogenase gene (ZrGPD1) was expressed under the control of the GAL10 promoter, salt tolerance and glycerol production increased; when the ORF of the glycerol dehydrogenase gene (ZrGCY1) was expressed under the control of the GAL1 promoter, no such changes were observed. Zrgcy1p had a weak effect on glycerol production. These results suggest that Zrgpd1p is the primary enzyme involved in Z. rouxii glycerol production, following a mechanism similar to that of S. cerevisiae (Gpd1p). When the ORFs of the S. cerevisiae glycerol 3-phosphatase gene (GPP2) and ZrGPD1 were simultaneously expressed, glycerol production increased, compared with that in yeast expressing only ZrGPD1.

  3. Molecular cloning and heterologous expression of novel glucosyltransferases from tobacco cultured cells that have broad substrate specificity and are induced by salicylic acid and auxin.

    PubMed

    Taguchi, G; Yazawa, T; Hayashida, N; Okazaki, M

    2001-07-01

    Scopoletin is one of the phytoalexins in tobacco. Cells of the T-13 cell line (Nicotiana tabacum L. Bright Yellow) accumulate a large amount of scopoletin, also known as 7-hydroxy-6-methoxycoumarin, as a glucoconjugate, scopolin, in vacuoles. We report here the molecular cloning of glucosyltransferases that can catalyze the glucosylation of many kinds of secondary metabolites including scopoletin. Two cDNAs encoding glucosyltransferase (NtGT1a and NtGT1b) were isolated from a cDNA library derived from the tobacco T-13 cell line by screening with heterologous cDNAs as a probe. The deduced amino-acid sequences of NtGT1a and NtGT1b exhibited 92% identity with each other, approximately 20-50% identities with other reported glucosyltransferases. Heterologous expression of these genes in Escherichia coli showed that the recombinant enzymes had glucosylation activity against both flavonoids and coumarins. They also strongly reacted with 2-naphthol as a substrate. These recombinant enzymes can utilize UDP-glucose as the sugar donor, but they can also utilize UDP-xylose as a weak donor. RNA blot analysis showed that these genes are induced by salicylic acid and auxin, but the time course of the expression was different. This result is similar to the changes in scopoletin glucosylation activity in these tobacco cells after addition of these plant growth regulators. These results might suggest that one of the roles of the products of these genes is scopoletin glucosylation, in response to salicylic acid and/or auxin, together with the other glucosyltransferases in tobacco cells.

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

    PubMed Central

    Joseph, Joan; Fernández-Lloris, Raquel; Pezzat, Elías; Saubi, Narcís; Cardona, Pere-Joan; Mothe, Beatriz; Gatell, Josep Maria

    2010-01-01

    Mycobacterium bovis Bacillus Calmette-Guérin (BCG) as a live vector of recombinant bacterial vaccine is a promising system to be used. In this study, we evaluate the disrupted expression of heterologous HIV-1gp120 gene in BCG Pasteur host strain using replicative vectors pMV261 and pJH222. pJH222 carries a lysine complementing gene in BCG lysine auxotrophs. The HIV-1 gp120 gene expression was regulated by BCG hsp60 promoter (in plasmid pMV261) and Mycobacteria spp. α-antigen promoter (in plasmid pJH222). Among 14 rBCG:HIV-1gp120 (pMV261) colonies screened, 12 showed a partial deletion and two showed a complete deletion. However, deletion was not observed in all 10 rBCG:HIV-1gp120 (pJH222) colonies screened. In this study, we demonstrated that E. coli/Mycobacterial expression vectors bearing a weak promoter and lysine complementing gene in a recombinant lysine auxotroph of BCG could prevent genetic rearrangements and disruption of HIV 1gp120 gene expression, a key issue for engineering Mycobacterial based vaccine vectors. PMID:20617151

  5. 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

  6. 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

  7. Heterologous expression of mycobacterial Esx complexes in Escherichia coli for structural studies is facilitated by the use of maltose binding protein fusions.

    PubMed

    Arbing, Mark A; Chan, Sum; Harris, Liam; Kuo, Emmeline; Zhou, Tina T; Ahn, Christine J; Nguyen, Lin; He, Qixin; Lu, Jamie; Menchavez, Phuong T; Shin, Annie; Holton, Thomas; Sawaya, Michael R; Cascio, Duilio; Eisenberg, David

    2013-01-01

    The expression of heteroligomeric protein complexes for structural studies often requires a special coexpression strategy. The reason is that the solubility and proper folding of each subunit of the complex requires physical association with other subunits of the complex. The genomes of pathogenic mycobacteria encode many small protein complexes, implicated in bacterial fitness and pathogenicity, whose characterization may be further complicated by insolubility upon expression in Escherichia coli, the most common heterologous protein expression host. As protein fusions have been shown to dramatically affect the solubility of the proteins to which they are fused, we evaluated the ability of maltose binding protein fusions to produce mycobacterial Esx protein complexes. A single plasmid expression strategy using an N-terminal maltose binding protein fusion to the CFP-10 homolog proved effective in producing soluble Esx protein complexes, as determined by a small-scale expression and affinity purification screen, and coupled with intracellular proteolytic cleavage of the maltose binding protein moiety produced protein complexes of sufficient purity for structural studies. In comparison, the expression of complexes with hexahistidine affinity tags alone on the CFP-10 subunits failed to express in amounts sufficient for biochemical characterization. Using this strategy, six mycobacterial Esx complexes were expressed, purified to homogeneity, and subjected to crystallization screening and the crystal structures of the Mycobacterium abscessus EsxEF, M. smegmatis EsxGH, and M. tuberculosis EsxOP complexes were determined. Maltose binding protein fusions are thus an effective method for production of Esx complexes and this strategy may be applicable for production of other protein complexes.

  8. 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

    2014-10-05

    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. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Biochemical and EPR-spectroscopic investigation into heterologously expressed vinyl chloride reductive dehalogenase (VcrA) from Dehalococcoides mccartyi strain VS.

    PubMed

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

    2015-03-18

    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, Fe(3+), 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.

  10. Isolation, Characterization, and Heterologous Expression of the Novel Lantibiotic Epicidin 280 and Analysis of Its Biosynthetic Gene Cluster

    PubMed Central

    Heidrich, Christoph; Pag, Ulrike; Josten, Michaele; Metzger, Jörg; Jack, Ralph W.; Bierbaum, Gabriele; Jung, Günther; Sahl, Hans-Georg

    1998-01-01

    Epicidin 280 is a novel type A lantibiotic produced by Staphylococcus epidermidis BN 280. During C18 reverse-phase high-performance liquid chromatography two epicidin 280 peaks were obtained; the two compounds had molecular masses of 3,133 ± 1.5 and 3,136 ± 1.5 Da, comparable antibiotic activities, and identical amino acid compositions. Amino acid sequence analysis revealed that epicidin 280 exhibits 75% similarity to Pep5. The strains that produce epicidin 280 and Pep5 exhibit cross-immunity, indicating that the immunity peptides cross-function in antagonization of both lantibiotics. The complete epicidin 280 gene cluster was cloned and was found to comprise at least five open reading frames (eciI, eciA, eciP, eciB, and eciC, in that order). The proteins encoded by these open reading frames exhibit significant sequence similarity to the biosynthetic proteins of the Pep5 operon of Staphylococcus epidermidis 5. A gene for an ABC transporter, which is present in the Pep5 gene cluster but is necessary only for high yields (G. Bierbaum, M. Reis, C. Szekat, and H.-G. Sahl, Appl. Environ. Microbiol. 60:4332–4338, 1994), was not detected. Instead, upstream of the immunity gene eciI we found an open reading frame, eciO, which could code for a novel lantibiotic modification enzyme involved in reduction of an N-terminally located oxopropionyl residue. Epicidin 280 produced by the heterologous host Staphylococcus carnosus TM 300 after introduction of eciIAPBC (i.e., no eciO was present) behaved homogeneously during reverse-phase chromatography. PMID:9726851

  11. Progress toward heterologous expression of active G-protein-coupled receptors in Saccharomyces cerevisiae: Linking cellular stress response with translocation and trafficking

    PubMed Central

    O'Malley, Michelle A; Mancini, J Dominic; Young, Carissa L; McCusker, Emily C; Raden, David; Robinson, Anne S

    2009-01-01

    High-level expression of mammalian G-protein-coupled receptors (GPCRs) is a necessary step toward biophysical characterization and high-resolution structure determination. Even though many heterologous expression systems have been used to express mammalian GPCRs at high levels, many receptors are improperly trafficked or are inactive in these systems. En route to engineering a robust microbial host for GPCR expression, we have investigated the expression of 12 GPCRs in the yeast Saccharomyces cerevisiae, where all receptors are expressed at the mg/L scale. However, only the human adenosine A2a (hA2aR) receptor is active for ligand-binding and located primarily at the plasma membrane, whereas other tested GPCRs are mainly retained within the cell. Selective receptors associate with BiP, an ER-resident chaperone, and activated the unfolded protein response (UPR) pathway, which suggests that a pool of receptors may be folded incorrectly. Leader sequence cleavage of the expressed receptors was complete for the hA2aR, as expected, and partially cleaved for hA2bR, hCCR5R, and hD2LR. Ligand-binding assays conducted on the adenosine family (hA1R, hA2aR, hA2bR, and hA3R) of receptors show that hA2aR and hA2bR, the only adenosine receptors that demonstrate leader sequence processing, display activity. Taken together, these studies point to translocation as a critical limiting step in the production of active mammalian GPCRs in S. cerevisiae. PMID:19760666

  12. Improved production of fatty acid ethyl esters in Saccharomyces cerevisiae through up-regulation of the ethanol degradation pathway and expression of the heterologous phosphoketolase pathway.

    PubMed

    de Jong, Bouke Wim; Shi, Shuobo; Siewers, Verena; Nielsen, Jens

    2014-03-12

    Due to an increasing demand of transportation fuels, a lower availability of cheap crude oil and a lack of sustainability of fossil fuels, a gradual shift from petroleum based fuels towards alternative and renewable fuel resources will be required in the near future. Fatty acid ethyl esters (FAEEs) have properties similar to current crude diesel and could therefore form an important contribution to the development of sustainable transportation fuels in future. It is important to develop novel cell factories for efficient production of FAEEs and their precursors. Here, a Saccharomyces cerevisiae cell factory expressing a heterologous wax ester synthase (ws2) from Marinobacter hydrocarbonoclasticus was used to produce FAEEs from ethanol and acyl-coenzyme A (acyl-CoA). The production of acyl-CoA requires large amounts of NADPH and acetyl-CoA. Therefore, two metabolic engineering strategies for improved provision of NADPH and acetyl-CoA were evaluated. First, the ethanol degradation pathway was employed to re-channel carbon flow towards the synthesis of acetyl-CoA. Therefore, ADH2 and ALD6 encoding, respectively, alcohol dehydrogenase and acetaldehyde dehydrogenase were overexpressed together with the heterologous gene acsSEL641P encoding acetyl-CoA synthetase. The co-overexpression of ADH2, ALD6 and acsSEL641P with ws2 resulted in 408 ± 270 μg FAEE gCDW-1, a 3-fold improvement. Secondly, for the expression of the PHK pathway two genes, xpkA and ack, both descending from Aspergillus nidulans, were co-expressed together with ws2 to catalyze, respectively, the conversion of xylulose-5-phosphate to acetyl phosphate and glyceraldehyde-3-phosphate and acetyl phosphate to acetate. Alternatively, ack was substituted with pta from Bacillus subtilis, encoding phosphotransacetylase for the conversion of acetyl phosphate to acetyl-CoA. Both PHK pathways were additionally expressed in a strain with multiple chromosomally integrated ws2 gene, which resulted in respectively

  13. Improved production of fatty acid ethyl esters in Saccharomyces cerevisiae through up-regulation of the ethanol degradation pathway and expression of the heterologous phosphoketolase pathway

    PubMed Central

    2014-01-01

    Background Due to an increasing demand of transportation fuels, a lower availability of cheap crude oil and a lack of sustainability of fossil fuels, a gradual shift from petroleum based fuels towards alternative and renewable fuel resources will be required in the near future. Fatty acid ethyl esters (FAEEs) have properties similar to current crude diesel and could therefore form an important contribution to the development of sustainable transportation fuels in future. It is important to develop novel cell factories for efficient production of FAEEs and their precursors. Results Here, a Saccharomyces cerevisiae cell factory expressing a heterologous wax ester synthase (ws2) from Marinobacter hydrocarbonoclasticus was used to produce FAEEs from ethanol and acyl-coenzyme A (acyl-CoA). The production of acyl-CoA requires large amounts of NADPH and acetyl-CoA. Therefore, two metabolic engineering strategies for improved provision of NADPH and acetyl-CoA were evaluated. First, the ethanol degradation pathway was employed to re-channel carbon flow towards the synthesis of acetyl-CoA. Therefore, ADH2 and ALD6 encoding, respectively, alcohol dehydrogenase and acetaldehyde dehydrogenase were overexpressed together with the heterologous gene acsSEL641P encoding acetyl-CoA synthetase. The co-overexpression of ADH2, ALD6 and acsSEL641P with ws2 resulted in 408 ± 270 μg FAEE gCDW−1, a 3-fold improvement. Secondly, for the expression of the PHK pathway two genes, xpkA and ack, both descending from Aspergillus nidulans, were co-expressed together with ws2 to catalyze, respectively, the conversion of xylulose-5-phosphate to acetyl phosphate and glyceraldehyde-3-phosphate and acetyl phosphate to acetate. Alternatively, ack was substituted with pta from Bacillus subtilis, encoding phosphotransacetylase for the conversion of acetyl phosphate to acetyl-CoA. Both PHK pathways were additionally expressed in a strain with multiple chromosomally integrated ws2 gene, which

  14. Targeted Capture and Heterologous Expression of the Pseudoalteromonas Alterochromide Gene Cluster in Escherichia coli Represents a Promising Natural Product Exploratory Platform

    PubMed Central

    2015-01-01

    Marine pseudoalteromonads represent a very promising source of biologically important natural product molecules. To access and exploit the full chemical capacity of these cosmopolitan Gram-(−) bacteria, we sought to apply universal synthetic biology tools to capture, refactor, and express biosynthetic gene clusters for the production of complex organic compounds in reliable host organisms. Here, we report a platform for the capture of proteobacterial gene clusters using a transformation-associated recombination (TAR) strategy coupled with direct pathway manipulation and expression in Escherichia coli. The ∼34 kb pathway for production of alterochromide lipopeptides by Pseudoalteromonas piscicida JCM 20779 was captured and heterologously expressed in E. coli utilizing native and E. coli-based T7 promoter sequences. Our approach enabled both facile production of the alterochromides and in vivo interrogation of gene function associated with alterochromide’s unusual brominated lipid side chain. This platform represents a simple but effective strategy for the discovery and biosynthetic characterization of natural products from marine proteobacteria. PMID:25140825

  15. Heterologous expression and characterization of a glycoside hydrolase family 45 endo-β-1,4-glucanase from a symbiotic protist of the lower termite, Reticulitermes speratus.

    PubMed

    Otagiri, Masato; Lopez, Crisanto M; Kitamoto, Katsuhiko; Arioka, Manabu; Kudo, Toshiaki; Moriya, Shigeharu

    2013-03-01

    The termite symbiotic system is one of the efficient lignocellulose degradation systems. We tried to express and characterize a novel cellulolytic enzyme from this system. Here, we report the isolation of an endo-β-1,4-glucanase gene homolog of glycoside hydrolase family 45 from a symbiotic protistan community of Reticulitermes speratus. Heterologous expression of this gene was performed using the expression system of Aspergillus oryzae. Analysis of enzymatic properties revealed 786 μmol/min/mg protein in specific activity, a V max of 833.0 units/mg protein, and a K m value of 2.58 mg/ml with carboxymethyl cellulose as the substrate. Thin-layer chromatography analysis showed that RsSymEG2 produces cellobiose from cellodextrins larger than cellohexaose. This enzyme showed high specific activity like other endo-β-1,4-glucanases from the symbiotic system of termites. It means that the termite symbiotic system is a good resource for highly active endo-β-1,4-glucanases.

  16. Cloning a novel endo-1,4-β-D-glucanase gene from Trichoderma virens and heterologous expression in E. coli.

    PubMed

    Zeng, Rong; Hu, Qiao; Yin, Xiao-Yan; Huang, Hao; Yan, Jia-Bao; Gong, Zhi-Wei; Yang, Zhong-Hua

    2016-12-01

    Endo-1,4-β-D-glucanase (EG), as a key constituent of cellulase taking the responsibility of cutting β-1,4 glycosidic bonds, plays the essential role in the process of degrading cellulose by cellulase. Cloning and expressing the EG gene is important to the cellulase research and application. In this work, a novel EG gene was cloned from Trichoderma virens ZY-01, which was a cellulase secreting microbe isolated by our laboratory. The DNA sequence showed that the length of the cloned EG is 1069 bp, which had 95.2% similarity to the EG IV from T. viride AS 3.3711. Further, the expression vector pET-32a-EG was constructed and was successfully heterologously expressed in Escherichia coli. The expression product was purified with Ni(2+) affinity chromatography and its enzymatic properties were investigated. The SDS-PAGE showed the target protein is 39 kDa, which is consistent with the translated result from the DNA sequence. The kinetic parameter for the expression product was Km = 13.71 mg/mL and Vmax=0.51 μmol/min·mL. The optimal reaction pH and temperature was pH = 7.0 and T = 40 °C, which is similar to the native EG produced by Trichoderma virens ZY-01. It provides the foundation for the endo-1,4-β-D-glucanase further evolution and application.

  17. 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.

  18. Heterologous expression in Pichia pastoris and characterization of a β-glucosidase from the xylophagous cockroach Panesthia angustipennis spadica displaying high specific activity for cellobiose.

    PubMed

    Li, Yihai; Arakawa, Gaku; Tokuda, Gaku; Watanabe, Hirofumi; Arioka, Manabu

    2017-02-01

    A β-glucosidase (BG), PaBG1b, from the xylophagous cockroach Panesthia angustipennis spadica was heterologously expressed in the methylotrophic yeast Pichia pastoris, purified, and biochemically characterized. Post-translational modification and N-terminal sequencing analysis demonstrated that the expression product was comprised of two polypeptides with different N-terminal sequences, presumably due to the presence of lysine-arginine (KR) sequence in the putative mature region. Substrate specificity analysis showed that PaBG1b hydrolyzed a broad range of substrates including cellohexaose, with the preference for aryl β-d-fucosyl linkage and laminaribiose. Although the glucose tolerance of PaBG1b was moderate (Ki=200.3±1.1mM), PaBG1b demonstrated high specific activity and catalytic efficiency towards cellobiose with Vmax and kcat/Km values of 436.7±6.3U/mg and 109.8mM(-1)s(-1), respectively. In addition, PaBG1b was not inhibited by cellobiose up to the highest concentration tested (100mM). Collectively, our work demonstrates that PaBG1b is a potentially valuable BG for commercial bioethanol production from cellulose. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Cloning and heterologous expression of the entire gene clusters for PD 116740 from Streptomyces strain WP 4669 and tetrangulol and tetrangomycin from Streptomyces rimosus NRRL 3016.

    PubMed Central

    Hong, S T; Carney, J R; Gould, S J

    1997-01-01

    The genes for the complete pathways for two polycyclic aromatic polyketides of the angucyclinone class have been cloned and heterologously expressed. Genomic DNAs of Streptomyces rimosus NRRL 3016 and Streptomyces strain WP 4669 were partially digested with MboI, and libraries (ca. 40-kb fragments) in Escherichia coli XL1-Blue MR were prepared with the cosmid vector pOJ446. Hybridization with the actI probe from the actinorhodin polyketide synthase genes identified two clusters of polyketide genes from each organism. After transfer of the four clusters to Streptomyces lividans TK24, expression of one cluster from each organism was established through the identification of pathway-specific products by high-performance liquid chromatography with photodiode array detection. Peaks were identified from the S. rimosus cluster (pksRIM-1) for tetrangulol, tetrangomycin, and fridamycin E. Peaks were identified from the WP 4669 cluster (pksWP-2) for tetrangulol, 19-hydroxytetrangulol, 8-O-methyltetrangulol, 19-hydroxy-8-O-methyltetrangulol, and PD 116740. Structures were confirmed by 1H nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. PMID:8990300

  20. Heterologous expression and characterization of wild-type and mutant forms of a 26 kDa endochitinase from barley (Hordeum vulgare L.).

    PubMed Central

    Andersen, M D; Jensen, A; Robertus, J D; Leah, R; Skriver, K

    1997-01-01

    To investigate structure-function relationships in plant chitinases, we have developed a heterologous expression system for the 26 kDa endochitinase from Hordeum vulgare L. (barley). Escherichia coli cells harbouring the gene in a T7 RNA polymerase-based expression vector synthesized completely insoluble recombinant protein under standard induction conditions at 37 degrees C. However, a concentration of soluble recombinant protein of approx. 15 mg/l was achieved by inducing bacteria at low temperature (15 degrees C). Recombinant endochitinase was purified to homogeneity and shown to be structurally and functionally identical to the seed protein. An average of three disulphide bonds are present in the recombinant enzyme, consistent with the number found in the natural form. The seed and recombinant proteins showed the same specific activity towards a high-molecular-mass substrate and exhibited similar anti-fungal activity towards Tricoderma reesei. Site-directed mutagenesis was used to replace residues that are likely to be involved in the catalytic event, based on structural similarities with lysozyme and on sequence alignments with related chitinases. The Glu67-->Gln mutation resulted in a protein with undetectable activity, while the Glu89-->Gln mutation yielded an enzyme with 0. 25% of wild-type specific activity. This suggests that two acidic residues are essential for catalytic activity, similar to the situation with many other glycosyl hydrolases. Examination of conserved residues stretching into the proposed substrate binding cleft suggests that Asn124 also plays an important functional role. PMID:9148754

  1. Cloning and expression of the ponB gene, encoding penicillin-binding protein 1B of Escherichia coli, in heterologous systems.

    PubMed Central

    Plá, J; Rojo, F; de Pedro, M A; Ayala, J A

    1990-01-01

    A fragment from the ponB region of the Escherichia coli chromosome comprising the promoterless sequence encoding penicillin-binding protein 1B (PBP 1B) has been cloned in a broad-host-range expression vector under the control of the kanamycin resistance gene promoter present in the vector. The hybrid plasmid (pJP3) was used to transform appropriate strains of Salmonella typhimurium, Pseudomonas putida, and Pseudomonas aeruginosa. In all instances, the coding sequence was expressed in the heterologous hosts, yielding a product with electrophoretic mobility, protease accessibility, membrane location, and beta-lactam-binding properties identical to those of native PBP 1B in E. coli. These results indicated that PBP 1B of E. coli is compatible with the cytoplasmic membrane environment of unrelated bacterial species and support the idea that interspecific transfer of mutated alleles of genes coding for PBPs could potentially be an efficient spreading mechanism for intrinsic resistance to beta-lactams. Images PMID:2198260

  2. Heterologous viral RNA export elements improve expression of severe acute respiratory syndrome (SARS) coronavirus spike protein and protective efficacy of DNA vaccines against SARS.

    PubMed

    Callendret, Benoît; Lorin, Valérie; Charneau, Pierre; Marianneau, Philippe; Contamin, Hugues; Betton, Jean-Michel; van der Werf, Sylvie; Escriou, Nicolas

    2007-07-05

    The SARS-CoV spike glycoprotein (S) is the main target of the protective immune response in humans and animal models of SARS. Here, we demonstrated that efficient expression of S from the wild-type spike gene in cultured cells required the use of improved plasmid vectors containing donor and acceptor splice sites, as well as heterologous viral RNA export elements, such as the CTE of Mazon-Pfizer monkey virus or the PRE of Woodchuck hepatitis virus (WPRE). The presence of both splice sites and WPRE markedly improved the immunogenicity of S-based DNA vaccines against SARS. Upon immunization of mice with low doses (2 microg) of naked DNA, only intron and WPRE-containing vectors could induce neutralizing anti-S antibodies and provide protection against challenge with SARS-CoV. Our observations are likely to be useful for the construction of plasmid and viral vectors designed for optimal expression of intronless genes derived from cytoplasmic RNA viruses.

  3. Heterologous expression in Escherichia coli of native and mutant forms of the major intrinsic protein of rat eye lens (MIP26).

    PubMed Central

    Dilsiz, N; Crabbe, M J

    1995-01-01

    The complete cDNA of rat eye lens major intrinsic protein (MIP26) was sequenced using the dideoxy chain termination method. The sequence displayed 89% nucleotide identity and 95% identity at the amino acid level with bovine MIP26 [Gorin, Yancey, Cline, Revel and Horwitz (1984) Cell, 39, 49-54]. Both native and mutant cDNAs coding for rat MIP26 were amplified by PCR and subcloned into the pPOW expression vector for expression of Escherichia coli. A membrane signal peptide (PelB) was used for secretion of MIP26 into the cytoplasmic membrane. A hydrophilic octapeptide tail (FLAG) was fused to either the N- or C-terminus of MIP26 to aid monoclonal antibody-mediated identification and purification. Heterologously expressed MIP26 was identified by using a monoclonal antibody corresponding to the FLAG peptide located at the termini of MIP26. Immunofluorescently labelled monoclonal antibody was used to determine the localization of MIP26 in the cytoplasmic membrane. The majority of the protein was integrated into cell plasma membrane. MIP26 was extracted with n-octyl beta-D-glucopyranoside and then purified on an affinity gel column. Rat MIP26 cDNA contains an -Asn-Gly- sequence at the C-terminus, which has been shown in other proteins to be particularly susceptible to spontaneous deamidation [Takemoto and Emmons (1991) Curr. Eye Res. 10, 863-869]. We therefore modified the MIP26 molecule using a site-directed mutagenesis method to generate a mutant MIP26 at the appropriate asparagine residue (Asn244-->Asp) near the C-terminus. The mutation was confirmed by DNA sequencing. The mutant MIP26 protein was also expressed in E. coli and incorporated predominantly into the cytoplasmic membrane. Images Figure 5 Figure 6 Figure 7 PMID:7848273

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

    PubMed

    Sato, Toshitsugu; Irie, Toshikazu; Yoshino, Fumihiko

    2017-08-01

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