Home About us Contact
|
Home About us Contact | ||
|
|
||
Heterologous Production (heterologous + production)
Selected AbstractsAn Approach for Enhancing Heterologous Production of Providencia Rettgeri Penicillin Acylase in Escherichia coliBIOTECHNOLOGY PROGRESS, Issue 3 2000C. Perry Chou Heterologous production of Providencia rettgeri penicillin acylase (PAC) was optimized inEscherichia coli. Several factors, including carbon, temperature, and host effects, were identified to be critical for the enzyme overproduction. The optimum culture conditions for the enzyme production vary for different host/vector systems. With the optimization, both volumetric and specific PAC activities could be significantly improved by more than 50-fold compared to the native expression in P. rettgeri. The heterologous production could be possibly limited by translation or posttranslational steps, depending on the culture temperature and host/vector system. To our knowledge, this is the first evidence demonstrating the limiting step for the production of P. rettgeri PAC and the existence of the P. rettgeri PAC precursor. [source] Heterologous production of the Piromyces equi cinnamoyl esterase in Trichoderma reesei for biotechnological applicationsLETTERS IN APPLIED MICROBIOLOGY, Issue 6 2009L. Poidevin Abstract Aims:, The objective of the study was to produce and characterize the cinnamoyl esterase EstA from the anaerobic fungus Piromyces equi for potential industrial applications. Methods and Results:, The catalytic domain EstA was produced in Trichoderma reesei. Because the two fungi displayed different genome features, including different codon usage and GC content, a synthetic gene was designed and expressed, leading to the production of the corresponding protein at around 33 mg per litre in the T. reesei culture medium. After the recombinant protein was purified, biochemical characterization showed that EstA presents peak activity at pH 6·5 and at 50,60°C. Furthermore, EstA remained stable at pH 6,8 and below 50°C. EstA was compared to cinnamoyl esterases FaeA and FaeB from Aspergillus niger in terms of ferulic acid (FA) release from wheat bran (WB), maize bran (MB) and sugar beet pulp (SBP). Conclusion:, The synthetic gene was successfully cloned and overexpressed in T. reesei. EstA from P. equi was demonstrated to efficiently release FA from various natural substrates. Significance and Impact of the Study:, Recombinant EstA produced in an industrial enzyme producer, T. reesei, was biochemically characterized, and its capacity to release an aromatic compound (FA) for biotechnological applications was demonstrated. [source] An Approach for Enhancing Heterologous Production of Providencia Rettgeri Penicillin Acylase in Escherichia coliBIOTECHNOLOGY PROGRESS, Issue 3 2000C. Perry Chou Heterologous production of Providencia rettgeri penicillin acylase (PAC) was optimized inEscherichia coli. Several factors, including carbon, temperature, and host effects, were identified to be critical for the enzyme overproduction. The optimum culture conditions for the enzyme production vary for different host/vector systems. With the optimization, both volumetric and specific PAC activities could be significantly improved by more than 50-fold compared to the native expression in P. rettgeri. The heterologous production could be possibly limited by translation or posttranslational steps, depending on the culture temperature and host/vector system. To our knowledge, this is the first evidence demonstrating the limiting step for the production of P. rettgeri PAC and the existence of the P. rettgeri PAC precursor. [source] Heterologous expression and characterization of the exopolysaccharide from Streptococcus thermophilus Sfi39FEBS JOURNAL, Issue 19 2001Jacques-Edouard Germond The genes responsible for exopolysaccharide (EPS) synthesis in Streptococcus thermophilus Sfi39 were identified on a 20-kb genomic fragment. The two genes, epsE and epsG, were shown to be involved in EPS synthesis as their disruption lead to the loss of the ropy phenotype. Several naturally selected nonropy mutants were isolated, one acquired an insertion sequence (IS)-element (IS905) in the middle of the eps gene cluster. The eps gene cluster was cloned and transferred into a nonEPS-producing heterologous host, Lactococcus lactis MG1363. The EPS produced was shown by chemical analysis and NMR spectroscopy to be identical to the EPS produced by S. thermophilus Sfi39. This demonstrated first that all genes needed for EPS production and export were present in the S. thermophilus Sfi39 eps gene cluster, and second that the heterologous production of an EPS was possible by transfer of the complete eps gene cluster alone, provided that the heterologous host possessed all necessary genetic information for precursor synthesis. [source] Biosynthesis of spectinomycin: heterologous production of spectinomycin and spectinamine in an aminoglycoside-deficient host, Streptomyces venezuelae YJ003JOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2008L.P. Thapa Abstract Aims:, To obtain spectinomycin and spectinamine by heterologous expression into the biosynthetic deoxysugar (desosamine) gene-deleted host Streptomyces venezuelae YJ003. Methods and Results:, The 17-kb spectinomycin biosynthetic gene cluster from Streptomyces spectabilis ATCC 27741 was heterologously expressed into Streptomyces venezuelae YJ003. Furthermore, the speA, speB and spcS2 encoded in the spectinomycin biosynthetic gene cluster of cosmid pSPC8 were also heterologously characterized to be responsible for the production of spectinamine. Conclusions:, The results of this study indicated that pSPC8 contains all the genes necessary for the biosynthesis of spectinomycin. We also concluded that SpeA, SpeB and SpcS2 are sufficient for the biosynthesis of spectinamine. We also verified that SpeB and SpcS2 show dual character in the biosynthetic pathway of spectinomycin in Streptomyces spectabilis. Significance and Impact of the Study:, This is the report regarding the expression of a biosynthetic gene cluster that gives rise to the production of aminoglycoside antibiotics in Streptomyces venezuelae YJ003. Therefore, this work may serve as a foundation for further research on spectinomycin biosynthesis and other aminoglycosides. [source] Construction and performance of heterologous polyketide-producing K-12- and B-derived Escherichia coliLETTERS IN APPLIED MICROBIOLOGY, Issue 2 2010J. Wu Abstract Aims:,Escherichia coli has emerged as a viable heterologous host for the production of complex, polyketide natural compounds. In this study, polyketide biosynthesis was compared between different E. coli strains for the purpose of better understanding and improving heterologous production. Methods and Results:, Both B and K-12 E. coli strains were genetically modified to support heterologous polyketide biosynthesis [specifically, 6-deoxyerythronolide B (6dEB)]. Polyketide production was analysed using a helper plasmid designed to overcome rare codon usage within E. coli. Each strain was analysed for recombinant protein production, precursor consumption, by-product production, and 6dEB biosynthesis. Of the strains tested for biosynthesis, 6dEB production was greatest for E. coli B strains. When comparing biosynthetic improvements as a function of mRNA stability vs codon bias, increased 6dEB titres were observed when additional rare codon tRNA molecules were provided. Conclusions:,Escherichia coli B strains and the use of tRNA supplementation led to improved 6dEB polyketide titres. Significance and Impact of the Study:, Given the medicinal potential and growing field of polyketide heterologous biosynthesis, the current study provides insight into host-specific genetic backgrounds and gene expression parameters aiding polyketide production through E. coli. [source] Production and characterization of an allergen panel for component-resolved diagnosis of celery allergyMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue S2 2008Merima Bublin Abstract In celery a relevant food allergen source, three allergens have been identified so far: Api g 1 and Api g 4, and one glycosylated protein, Api g 5. For component-resolved food allergy diagnosis high amounts of well-defined allergens are needed. Depending on the individual celery allergen, protocols for heterologous production and purification from natural source, respectively, were established to obtain homogenous protein batches. Afterwards the purified recombinant allergens, Api g 1, Api g 4 and natural Api g 5 were characterized regarding their structural integrity and immunological activity. Therefore, several methods were applied. Proteins were identified by partial N-terminal sequencing, protein mass was verified by MS and sequence integrity by MALDI-TOF and N-terminal sequencing after tryptic digestion. Presence of isoforms in natural allergen preparations was identified by 2-DE. Secondary and tertiary structures were evaluated by circular dichroism (CD) spectroscopy and NMR analysis. Finally, IgE binding capacity was verified using selected sera from celery allergic patients in IgE immunoblots and IgE ELISA. These well-defined celery allergens will be used to prove the concept of component-resolved diagnosis and will contribute to improve food allergy diagnosis in the future. [source] High-level expression and purification of Cys-loop ligand-gated ion channels in a tetracycline-inducible stable mammalian cell line: GABAA and serotonin receptorsPROTEIN SCIENCE, Issue 9 2010Zuzana Dostalova Abstract The human neuronal Cys-loop ligand-gated ion channel superfamily of ion channels are important determinants of human behavior and the target of many drugs. It is essential for their structural characterization to achieve high-level expression in a functional state. The aim of this work was to establish stable mammalian cell lines that enable high-level heterologous production of pure receptors in a state that supports agonist-induced allosteric conformational changes. In a tetracycline-inducible stable human embryonic kidney cells (HEK293S) cell line, GABAA receptors containing ,1 and ,3 subunits could be expressed with specific activities of 29,34 pmol/mg corresponding to 140,170 pmol/plate, the highest expression level reported so far. Comparable figures for serotonin (5-HT3A) receptors were 49,63 pmol/mg and 245,315 pmol/plate. The expression of 10 nmol of either receptor in suspension in a bioreactor required 0.3,3.0 L. Both receptor constructs had a FLAG epitope inserted at the N-terminus and could be purified in one step after solubilization using ANTI-FLAG affinity chromatography with yields of 30,40%. Purified receptors were functional. Binding of the agonist [3H]muscimol to the purified GABAAR was enhanced allosterically by the general anesthetic etomidate, and purified 5-hydroxytryptamine-3A receptor supported serotonin-stimulated cation flux when reconstituted into lipid vesicles. [source] A SELDI-TOF MS procedure for the detection, quantitation, and preliminary characterization of low-molecular-weight recombinant proteins expressed in transgenic plantsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 2 2009M. Amine Badri Abstract We describe a SELDI-TOF MS procedure for the rapid detection and quantitation of low-molecular-weight recombinant proteins expressed in plants. Transgenic lines of potato (Solanum tuberosum L.) expressing the clinically useful protein bovine aprotinin or the cysteine protease inhibitor corn cystatin II were generated by Agrobacterium tumefaciens -mediated transformation, and then used as test material for the analyses. Real-time RT-PCR amplifications and detection of the recombinant proteins by immunoblotting were first conducted for transformed potato lines accumulating the proteins in different cell compartments. Both proteins were found at varying levels in leaves, depending on their final cellular destination and transgene expression rate. These conclusions drawn from standard immunodetection assays were easily confirmed by SELDI-TOF MS comparative profiling, after immobilizing the leaf proteins of control and transformed lines on protein biochips for weak cationic exchange. This procedure, carried out in less than 2,h, allows for the rapid comparison of recombinant protein levels in transgenic plant lines. The molecular weight of immobilized proteins can also be determined directly from the MS spectra, thus providing a simple way to assess the structural integrity and homogeneity of recombinant proteins in planta, and to identify the most suitable cellular compartments for their heterologous production. [source] Production and characterization of a recombinant beta-1,4-endoglucanase (glycohydrolase family 9) from the termite Reticulitermes flavipesARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 3 2010Xuguo Zhou Abstract Cell-1 is a host-derived beta-1,4-endoglucanase (Glycohydrolase Family 9 [GHF9]) from the lower termite Reticulitermes flavipes. Here, we report on the heterologous production of Cell-1 using eukaryotic (Baculovirus Expression Vector System; BEVS) and prokaryotic (E. coli) expression systems. The BEVS-expressed enzyme was more readily obtained in solubilized form and more active than the E. coli,expressed enzyme. Km and Vmax values for BEVS-expressed Cell-1 against the model substrate CMC were 0.993% w/v and 1.056,µmol/min/mg. Additional characterization studies on the BEVS-expressed enzyme revealed that it possesses activity comparable to the native enzyme, is optimally active around pH 6.5,7.5 and 50,60°C, is inhibited by EDTA, and displays enhanced activity up to 70°C in the presence of CaCl2. These findings provide a foundation on which to begin subsequent investigations of collaborative digestion by coevolved host and symbiont digestive enzymes from R. flavipes that include GHF7 exoglucanases, GHF1 beta glucosidases, phenol-oxidizing laccases, and others. © 2010 Wiley Periodicals, Inc. [source] Heterologous expression of the biosynthetic gene clusters of coumermycin A1, clorobiocin and caprazamycins in genetically modified Streptomyces coelicolor strainsBIOPOLYMERS, Issue 9 2010Katrin Flinspach Abstract The biosynthetic gene clusters of the aminocoumarin antibiotics clorobiocin and coumermycin A1 and of the liponucleoside antibiotic caprazamycin were stably integrated into the genomes of different host strains derived from Streptomyces coelicolor A3(2). For the heterologous expression of clorobiocin derivatives in a chemically defined medium, inclusion of 0.6% of the siloxylated ethylene oxide/propylene oxide copolymer Q2-5247 into the growth medium proved to result in a 4.8-fold increase of productivity. Presumably, this copolymer acts as an oxygen carrier. The additional inclusion of cobalt chloride (0.2,2 mg l,1) dramatically increased the percentage of the desired compound clorobiocin within the total produced clorobiocin derivatives. This is very likely due to a stimulation of a cobalamin-dependent methylation reaction catalyzed by the enzyme CloN6 of clorobiocin biosynthesis. All three investigated host strains (S. coelicolor M512, M1146 and M1154) gave similar production rates of total clorobiocin derivatives (on average, 158 mg l,1 in the presence of 0.6% Q2-5247 and 0.2 mg l,1 CoCl2). In contrast, heterologous production of caprazamycin derivatives was optimal in strain M1154 (amounts of 152 mg l,1 on average). © 2010 Wiley Periodicals, Inc. Biopolymers 93: 823,832, 2010. [source] Efficient experimental design and micro-scale medium enhancement of 6-deoxyerythronolide B production through Escherichia coliBIOTECHNOLOGY PROGRESS, Issue 5 2009Michael Pistorino Abstract The recent use of heterologous hosts to produce natural products has shown significant potential, although limitations still exist regarding optimal production titers. In this study, we utilize micro-scale cultures and well-defined screening methods to identify key medium components that influence the heterologous production of the complex polyketide 6-deoxyerythronolide B (6dEB) through E. coli. It was determined that tryptone had a significant effect on 6dEB production and could supplement substrate requirements and improve recombinant protein levels of the essential deoxyerythronolide B synthase (DEBS) which catalyze 6dEB conversion. As a result, the study (1) demonstrates the feasibility of micro-scale cultures to study E. coli 6dEB production and effectively model larger-scale cultures; (2) identifies an enhanced medium which generates over 160 mg L,1 6dEB (a 22-fold improvement over current culture media); and (3) provides new insight and understanding related to the heterologous production of 6dEB from E. coli. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] | |||||||||||||