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Recombinant E. Coli (recombinant + e._coli)
Selected AbstractsEffects of feeding and induction strategy on the production of BmR1 antigen in recombinant E. coliLETTERS IN APPLIED MICROBIOLOGY, Issue 5 2009A. Norsyahida Abstract Aim:, To investigate the effects of feeding and induction strategies on the production of BmR1 recombinant antigen. Methods and Results:, Fed-batch fermentation was studied with respect to the specific growth rate and mode of induction to assess the growth potential of the bacteria in a bioreactor and to produce high yield of BmR1 recombinant antigen. Cells were grown at a controlled specific growth rate (,set) during pre-induction, followed by constant feeding postinduction. The highest biomass (24·3 g l,1) was obtained during fed-batch process operated at ,set of 0·15 h,1, whereby lower ,set (0·075 h,1) gave the highest protein production (9·82 mg l,1). The yield of BmR1 was increased by 1·2-fold upon induction with 1 mmol l,1 IPTG (isopropyl-,- d -thiogalactoside) compared to using 5 mmol l,1 and showed a further 3·5-fold increase when the culture was induced twice at the late log phase. Conclusions:, Combination of feeding at a lower ,set and twice induction with 1 mmol l,1 IPTG yielded the best result of all variables tested, promising an improved method for BmR1 production. Significance and Impact of the Study:, This method can be used to increase the production scale of the BmR1 recombinant antigen to meet the increasing demand for Brugia RapidÔ, a commercial diagnostic test for detection of brugian filariasis. [source] Simple enzymatic procedure for l -carnosine synthesis: whole-cell biocatalysis and efficient biocatalyst recyclingMICROBIAL BIOTECHNOLOGY, Issue 1 2010Jan Heyland Summary , -Peptides and their derivates are usually stable to proteolysis and have an increased half-life compared with , -peptides. Recently, , -aminopeptidases were described as a new enzyme class that enabled the enzymatic degradation and formation of , -peptides. As an alternative to the existing chemical synthesis routes, the aim of the present work was to develop a whole-cell biocatalyst for the synthesis and production of , -peptides using this enzymatic activity. For the optimization of the reaction system we chose the commercially relevant ,,, -dipeptide l -carnosine (, -alanine- l -histidine) as model product. We were able to show that different recombinant yeast and bacteria strains, which overexpress a , -peptidase, could be used directly as whole-cell biocatalysts for the synthesis of l -carnosine. By optimizing relevant reaction conditions for the best-performing recombinant Escherichia coli strain, such as pH and substrate concentrations, we obtained high l -carnosine yields of up to 71%. Long-time as well as biocatalyst recycling experiments indicated a high stability of the developed biocatalyst for at least five repeated batches. Application of the recombinant E. coli in a fed-batch process enabled the accumulation of l -carnosine to a concentration of 3.7 g l,1. [source] Farnesol production from Escherichia coli by harnessing the exogenous mevalonate pathwayBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2010Chonglong Wang Abstract Farnesol (FOH) production has been carried out in metabolically engineered Escherichia coli. FOH is formed through the depyrophosphorylation of farnesyl pyrophosphate (FPP), which is synthesized from isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) by FPP synthase. In order to increase FPP synthesis, E. coli was metabolically engineered to overexpress ispA and to utilize the foreign mevalonate (MVA) pathway for the efficient synthesis of IPP and DMAPP. Two-phase culture using a decane overlay of the culture broth was applied to reduce volatile loss of FOH produced during culture and to extract FOH from the culture broth. A FOH production of 135.5,mg/L was obtained from the recombinant E. coli harboring the pTispA and pSNA plasmids for ispA overexpression and MVA pathway utilization, respectively. It is interesting to observe that a large amount of FOH could be produced from E. coli without FOH synthase by the augmentation of FPP synthesis. Introduction of the exogenous MVA pathway enabled the dramatic production of FOH by E. coli while no detectable FOH production was observed in the endogenous MEP pathway-only control. Biotechnol. Bioeng. 2010;107: 421,429. © 2010 Wiley Periodicals, Inc. [source] Microbial bio-production of a recombinant stimuli-responsive biosurfactantBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2009W. Kaar Abstract Biosurfactants have been the subject of recent interest as sustainable alternatives to petroleum-derived compounds in areas ranging from soil remediation to personal and health care. The production of naturally occurring biosurfactants depends on the presence of complex feed sources during microbial growth and requires multicomponent enzymes for synthesis within the cells. Conversely, designed peptide surfactants can be produced recombinantly in microbial systems, enabling the generation of improved variants by simple genetic manipulation. However, inefficient downstream processing is still an obstacle for the biological production of small peptides. We present the production of the peptide biosurfactant GAM1 in recombinant E. coli. Expression was performed in fusion to maltose binding protein using chemically defined minimal medium, followed by a single-step affinity capture and enzymatic cleavage using tobacco etch virus protease. Different approaches to the isolation of peptide after cleavage were investigated, with special emphasis on rapid and simple procedures. Solvent-, acid-, and heat-mediated precipitation of impurities were successfully applied as alternatives to post-cleavage chromatographic peptide purification, and gave peptide purities exceeding 90%. Acid precipitation was the method of choice, due to its simplicity and the high purification factor and recovery rate achieved here. The functionality of the bio-produced peptide was tested to ensure that the resulting peptide biosurfactant was both surface active and able to be triggered to switch between foam-stabilizing and foam-destabilizing states. Biotechnol. Bioeng. 2009;102: 176,187. © 2008 Wiley Periodicals, Inc. [source] Directing vanillin production from ferulic acid by increased acetyl-CoA consumption in recombinant Escherichia coli,BIOTECHNOLOGY & BIOENGINEERING, Issue 1 2009Eun-Gyeong Lee Abstract The amplification of gltA gene encoding citrate synthase of TCA cycle was required for the efficient conversion of acetyl-CoA, generated during vanillin production from ferulic acid, to CoA, which is essential for vanillin production. Vanillin of 1.98 g/L was produced from the E. coli DH5, (pTAHEF-gltA) with gltA amplification in 48 h of culture at 3.0 g/L of ferulic acid, which was about twofold higher than the vanillin production of 0.91 g/L obtained by the E. coli DH5, (pTAHEF) without gltA amplification. The icdA gene encoding isocitrate dehydrogenase of TCA cycle was deleted to make the vanillin producing E. coli utilize glyoxylate bypass which enables more efficient conversion of acetyl-CoA to CoA in comparison with TCA cycle. The production of vanillin by the icdA null mutant of E. coli BW25113 harboring pTAHEF was enhanced by 2.6 times. The gltA amplification of the glyoxylate bypass in the icdA null mutant remarkably increased the production rate of vanillin with a little increase in the amount of vanillin production. The real synergistic effect of gltA amplification and icdA deletion was observed with use of XAD-2 resin reducing the toxicity of vanillin produced during culture. Vanillin of 5.14 g/L was produced in 24 h of the culture with molar conversion yield of 86.6%, which is the highest so far in vanillin production from ferulic acid using recombinant E. coli. Biotechnol. Bioeng. 2009;102: 200,208. © 2008 Wiley Periodicals, Inc. [source] Carbon metabolism and product inhibition determine the epoxidation efficiency of solvent-tolerant Pseudomonas sp. strain VLB120,CBIOTECHNOLOGY & BIOENGINEERING, Issue 6 2007Jin-Byung Park Abstract Utilization of solvent tolerant bacteria as biocatalysts has been suggested to enable or improve bioprocesses for the production of toxic compounds. Here, we studied the relevance of solvent (product) tolerance and inhibition, carbon metabolism, and the stability of biocatalytic activity in such a bioprocess. Styrene degrading Pseudomonas sp. strain VLB120 is shown to be solvent tolerant and was engineered to produce enantiopure (S)-styrene oxide from styrene. Whereas glucose as sole source for carbon and energy allowed efficient styrene epoxidation at rates up to 97 µmol/min/(g cell dry weight), citrate was found to repress epoxidation by the engineered Pseudomonas sp. strain VLB120,C emphasizing that carbon source selection and control is critical. In comparison to recombinant Escherichia coli, the VLB120,C-strain tolerated higher toxic product levels but showed less stable activities during fed-batch cultivation in a two-liquid phase system. Epoxidation activities of the VLB120,C-strain decreased at product concentrations above 130 mM in the organic phase. During continuous two-liquid phase cultivations at organic-phase product concentrations of up to 85 mM, the VLB120,C-strain showed stable activities and, as compared to recombinant E. coli, a more efficient glucose metabolism resulting in a 22% higher volumetric productivity. Kinetic analyses indicated that activities were limited by the styrene concentration and not by other factors such as NADH availability or catabolite repression. In conclusion, the stability of activity of the solvent tolerant VLB120,C-strain can be considered critical at elevated toxic product levels, whereas the efficient carbon and energy metabolism of this Pseudomonas strain augurs well for productive continuous processing. Biotechnol. Bioeng. 2007;98: 1219,1229. © 2007 Wiley Periodicals, Inc. [source] Augmented biosynthesis of cadmium sulfide nanoparticles by genetically engineered Escherichia coliBIOTECHNOLOGY PROGRESS, Issue 5 2009Yen-Lin Chen Abstract Microorganisms can complex and sequester heavy metals, rendering them promising living factories for nanoparticle production. Glutathione (GSH) is pivotal in cadmium sulfide (CdS) nanoparticle formation in yeasts and its synthesis necessitates two enzymes: ,-glutamylcysteine synthetase (,-GCS) and glutathione synthetase (GS). Hereby, we constructed two recombinant E. coli ABLE C strains to over-express either ,-GCS or GS and found that ,-GCS over-expression resulted in inclusion body formation and impaired cell physiology, whereas GS over-expression yielded abundant soluble proteins and barely impeded cell growth. Upon exposure of the recombinant cells to cadmium chloride and sodium sulfide, GS over-expression augmented GSH synthesis and ameliorated CdS nanoparticles formation. The resultant CdS nanoparticles resembled those from the wild-type cells in size (2,5 nm) and wurtzite structures, yet differed in dispersibility and elemental composition. The maximum particle yield attained in the recombinant E. coli was ,2.5 times that attained in the wild-type cells and considerably exceeded that achieved in yeasts. These data implicated the potential of genetic engineering approach to enhancing CdS nanoparticle biosynthesis in bacteria. Additionally, E. coli -based biosynthesis offers a more energy-efficient and eco-friendly method as opposed to chemical processes requiring high temperature and toxic solvents. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] | ||||||||||