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1.
Coenzyme A (CoA) and its thioester derivatives are important precursor molecules for many industrially useful compounds such as esters, PHBs, lycopene and polyketides. Previously, in our lab we could increase the intracellular levels of CoA and acetyl-Coenzyme A (acetyl-CoA) by overexpressing one of the upstream rate-controlling enzymes pantothenate kinase with a concomitant supplementation of the precursor pantothenic acid to the cell culture medium. In this study, we showed that the CoA/acetyl-CoA manipulation system could be used to increase the productivity of industrially useful compounds derived from acetyl-CoA. We chose the production of isoamyl acetate as a model system. Isoamyl acetate is an important flavor component of sake yeast and holds a great commercial value. Alcohol acetyl transferase (AAT) condenses isoamyl alcohol and acetyl-CoA to produce isoamyl acetate. The gene ATF2, coding for this AAT was cloned and expressed in Escherichia coli. This genetic engineered E. coli produces isoamyl acetate, an ester, from intracellular acetyl-CoA when isoamyl alcohol is added externally to the cell culture medium. In the current study, we showed that in a strain bearing ATF2 gene, an increase in intracellular CoA/acetyl-CoA by overexpressing panK leads to an increase in isoamyl acetate production. Additionally, the cofactor manipulation technique was combined with more traditional approach of competing pathway deletions to further increase isoamyl acetate production. The acetate production pathway competes with isoamyl acetate production for the common intracellular metabolite acetyl-CoA. Earlier we have shown that acetate pathway deletion (ackA-pta) increases isoamyl acetate production. The acetate production pathway was inactivated under elevated CoA/acetyl-CoA conditions, which lead to a further increase in isoamyl acetate production. 相似文献
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The possibility of using in situ addition of anion-exchange resin for the removal of acetate in the culture aimed at improving growth of E. coli and expression of periplasmic human interferon-α2b (PrIFN-α2b) was studied in shake flask culture and stirred tank bioreactor. Different types of anion-exchange resin were evaluated and the concentration of anion-exchange resin was optimized using response surface methodology. The addition of anion-exchange resins reduced acetate accumulation in the culture, which in turn, improved growth of E. coli and enhanced PrIFN-α2b expression. The presence of anion-exchange resins did not influence the physiology of the cells. The weak base anion-exchange resins, which have higher affinity towards acetate, yielded higher PrIFN-α2b expression as compared to strong anion-exchange resins. High concentrations of anion-exchange resin showed inhibitory effect towards growth of E. coli as well as the expression of PrIFN-α2b. The maximum yield of PrIFN-α2b in shake flask culture (501.8 μg/L) and stirred tank bioreactor (578.8 μg/L) was obtained at ion exchange resin (WA 30) concentration of 12.2 g/L. The production of PrIFN-α2b in stirred tank bioreactor with the addition of ion exchange resin was about 1.8-fold higher than that obtained in fermentation without ion exchange resin (318.4 μg/L). 相似文献
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C. Born M. Biselli C. Wandrey J. Thömmes M. -R. Kula 《Bioprocess and biosystems engineering》1996,15(1):21-29
Continuous culture may be an efficient way of producing proteins which are susceptible to secondary processing in the course of a fermentation process. Short residence times in these systems support the production of correctly assembled proteins by avoiding substrate limitations and product inhibitions and also minimize the contact of sensitive bioproducts with degrading enzymes. Thus products of increased stability and integrity are obtained from continuous processes. The downstream process following continuous culture has to be adapted to the specific conditions of continuous fermentations, e.g. large liquid volumes and diluted process solutions. In this paper an approach is shown how a fluidized bed adsorption as first recovery operation may be coupled directly to a continuous production. Immobilized hybridoma cells are cultivated in porous glass microcarriers in a continuous fluidized bed process, the cell containing harvest is purified by fluidized bed adsorption using an agarose based cation exchange matrix. By this coupled mode of operation the large biomass containing harvest volume resulting from the continuous cultivation may be applied directly to a fluidized chromatographic matrix without prior clarification, leading to a particle free and initially purified product solution of reduced volume. In an experimental setup a bench-scale fluidized bed bioreactor of 25 ml carrier volume was coupled to a fluidized bed adsorption column operated with 300 ml of adsorbent. This configuration yielded up to 20 mg of monoclonal antibody per day in a cell free solution at fourfold concentration and fivefold purification. The process was run for more than three weeks with consistent product output.The help of H. Schmitz, A. Bader, J. Gätgens and M. Halfar during the experiments is gratefully acknowledged. This work was partially funded by the ministry of science and research of the Federal Republic of Germany within the project Stoffumwandlung mit Biokatalysatoren. 相似文献
4.
Acetate excretion by Escherichia coli during aerobic growth on glucose is a major obstacle to enhanced recombinant protein production. We report here that the fraction of carbon flux through the anaplerotic pathways is one of the factors influencing acetate excretion. Flux analysis of E. coli central metabolic pathways predicts that increasing the fraction of carbon flux through the phosphoenolpyruvate carboxylase (PPC) pathway and the glyoxylate bypass reduces acetate production. We tested this prediction by overexpressing PPC and deregulating the glyoxylate bypass by using a fadR strain. Results show that the acetate yield by the fadR strain with PPC overexpression is decreased more than fourfold compared to the control, while the biomass yield is relatively unaffected. Apparently, the fraction of carbon flux through the anaplerotic pathways is one of the factors that influence acetate excretion. These results confirm the prediction of our flux analysis and further suggest that E. coli is not fully optimized for efficient utilization of glucose. 相似文献
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Jian Wang Li-Kun Cheng Jing Wang Qian Liu Tong Shen Ning Chen 《Applied microbiology and biotechnology》2013,97(17):7587-7596
Reducing the accumulation of acetate in Escherichia coli cultures can decrease carbon efflux as by-products and reduce acetate toxicity, and therefore enable high cell density cultivation. The concentration of intracellular amino acids can be decreased by genetic modifications of the corresponding amino acid transport systems. This can increase the levels of amino acids in the fermentation broth by decreasing the feedback inhibition on the corresponding biosynthetic pathways. Here, the effects of genetic manipulation of phosphate acetyltransferase (pta), high affinity tryptophan transporter (mtr) and aromatic amino acid exporter (yddG) on l-tryptophan production were investigated. The pta mutants accumulated less acetate and showed higher capacity for producing l-tryptophan as compared with the parental strain. The strains lacking mtr, or overexpressed yddG, or with the both mtr knockout and yddG overexpression, accumulated lower concentrations of intracellular tryptophan but higher production of extracellular l-tryptophan. In the l-tryptohan fed-batch fermentation of an E. coli derived from TRTH0709/pMEL03 having deletion of pta-mtr and overexpression of yddG in a 30-L fermentor, the maximum concentration of l-tryptophan (48.68 g/L) was obtained, which represented a 15.96 % increase as compared with the parental strain. Acetate accumulated to a concentration of 0.95 g/L. The intracellular concentration of l-tryptophan was low, and the glucose conversion rate reached a high level of 21.87 %, which was increased by 15.53 % as compared with the parent strain. 相似文献
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Covalently immobilized biotin was used as a biospecific adsorbant to investigate the application of streptavidin as an affinity domain for simultaneous purification and immobilization of recombinant proteins. A streptavidin-beta-galactosidase fusion protein was constructed and tested as a model system. The gene for streptavidin from Streptomyces avidinii was modified by polymerase chain reaction to mutate the stop codon and to facilitate cloning into an Escherichia coli expression vector yielding a versatile plasmid with 37 unique restriction enzyme sites at the 3' end. E. coli beta-galactosidase was cloned in-frame to the streptavidin gene. Analysis of lysates of induced recombinant E. coli cells by SDS-PAGE and Western blots indicated that the 133.6-kDa fusion protein was expressed. Sulfosuccinimidyl-6-(biotinamido) hexanoate was covalently immobilized on 3-aminopropyl-controled-pore glass beads. Exposure of recombinant cell lysates to this support indicated that streptavidin-beta-galactosidase was bioselectively adsorbed. The resulting biocatalyst contained 300 mg protein per gram of beads and exhibited a specific activity of 306 betamol/min per milligram protein with o-nitrophenyl-beta-D-galactopyranoside as substrate corresponding to approximately 50% of that observed for commercially pure E. coli beta-galactosidase. (c) 1994 John Wiley & Sons, Inc. 相似文献
11.
Xianglei Liu Jun Lin Haifeng Hu Bin Zhou Baoquan Zhu 《World journal of microbiology & biotechnology》2014,30(9):2543-2550
Shikimic acid (SA) is the key synthetic material of Oseltamivir, which is an effective drug for the prevention and treatment of influenza. In this study, to block the downstream metabolic pathway of SA, the shikimate kinase isoenzyme genes aroK and aroL were deleted by Red recombination. Moreover, the key enzyme genes aroG, aroB, tktA and aroE of SA pathway were co-expressed by constructing the recombinant vector pETDuet-GBAE. As a result, SA production of E. coli BW25113 (?aroL/aroK, DE3)/pETDuet-GBAE reached 1,077.6 mg/l when low amounts of sorbitol (5 g/l) were fed in shake flasks. The yield was 3.7 times that when glucose was used (P < 0.05). The results showed that sorbitol was an optimized carbon source for the high efficient accumulation of SA for the first time, which was applicable to use in the industry for high yields and low consumption. 相似文献
12.
Selection for signal sequence mutations that enhance production of secreted human proinsulin by Escherichia coli 总被引:1,自引:0,他引:1
This paper describes a method for the positive selection of signal sequence mutations that result in enhanced production of secreted human proinsulin by Escherichia coli. Coding sequences for the structural portion of beta-lactamase (EC 3.5.2.6) were substituted for those of the C terminus of proinsulin in a plasmid that normally directs the synthesis and secretion of proinsulin. The resulting plasmid directed the synthesis of a proinsulin/beta-lactamase fusion protein that was secreted into the periplasmic space and conferred resistance to low levels of ampicillin (Ap). Beneficial changes to the signal sequence were selected by the host's ability to grow on high levels of Ap. The beta-lactamase coding sequences were then replaced with those of human proinsulin, resulting in plasmids which directed enhanced production of secreted proinsulin. 相似文献
13.
Summary Aspartase containingEscherichia
coli cells were entrapped in cellulose acetate aggregated on cotton cloth. The enzyme activity of the cloth was stabilized by treatment with polyethylenimine and alkaline glutaraldehyde in the presence of 0.1 M sodium dithionite. A column packed with the cloth segments catalyzed 100% conversion of 1 M ammonium fumarate to aspartic acid at a space velocity of 7/h. When the same cloth segments were stirred at 100 rpm in the substrate solution, the productivity of the cloth increased 2.5 times. 相似文献
14.
Genetic manipulation of the host strain, by which cell physiology could be modulated, was exploited to enhance recombinant protein production in Escherichia coli. The effects of an inactivated stationary-phase gene (rmf or katF) on recombinant protein production in strains with two different expression systems (the pH-inducible and the lac promoters) were investigated. An improvement of recombinant protein production in the katF mutant at low growth rates was observed for both expression systems. A fourfold and a 30% increase in the volumetric recombinant protein activity were observed for the pH-inducible and the lac promoter system, respectively. The effect of the rmf mutation, on the other hand, depends on the expression system. A twofold increase in the volumetric recombinant protein activity was found for the pH-inducible promoter system, but there was no improvement for the lac promoter system. Improvement in culture performance for slow-growing cultures may have an impact on the design strategy of the host/vector system used in fed-batch cultures, where the specific growth rate is usually slow. The information may also be useful for developing optimal host/vector gene expression systems for recombinant protein production. (c) 1996 John Wiley & Sons, Inc. 相似文献
15.
Valente CA Prazeres DM Cabral JM Monteiro GA 《Applied and environmental microbiology》2004,70(8):5033-5036
The yield of human alpha 2b interferon in Escherichia coli was optimized by replacement of low-usage arginine codons located in the mRNA 5' end. The differences observed among the various gene variants suggest that codon usage, Shine-Dalgarno-like sequences, and mRNA secondary structure contribute to the performance of E. coli translation machinery. 相似文献
16.
Strain improvement to enhance the production of recombinant penicillin acylase in high-cell-density Escherichia coli cultures 总被引:1,自引:0,他引:1
Using fed-batch operation for high-cell-density cultivation, efforts are frequently made for optimization of culture parameters, particularly feeding strategy. The current study also emphasized the importance of selecting strains for the production of recombinant proteins in high-cell-density cultures. With Escherichia coli penicillin acylase (PAC) as a target protein, the host/vector system of MDdeltaP7 harboring pTrcKnPAC2902 and pKS12 was designed for optimization of fed-batch cultivation for recombinant protein production. The host, MDdeltaP7, potentially had a high translational and periplasmic processing efficiency for pac expression. On the other hand, the vector, pTrcKnPAC2902, was genetically constructed for pac overexpression. Coexistence of the other vector, pKS12, significantly enhanced PAC production by improving cell physiology and reducing the amount of inclusion body formation upon pac overexpression. An extremely high volumetric PAC activity at 37,500 U/L was obtained with the use of the developed host/vector system under optimum fed-batch culture conditions. 相似文献
17.
An integrated metabolic model for the production of acetate by growing Escherichia coli on glucose under aerobic conditions is presented. The model is based on parameters which are easily determined by experiments. Forming the basis for this integrated metabolic model are the 12 principal precursor metabolites for biosynthetic pathways, the Embden-Meyerhof-Parnas pathway, the pentose phosphate cycle, the tricarboxylic acid cycle and the anapleurotic reactions, the Crabtree effect, the Pasteur effect, and the details of bacterial respiration. The result can be used to explain phenomena often observed in industrial fermentations, i.e., increased acetate production which follows from high glucose uptake rate, a low oxygen concentration, a high specific growth rate, or a combination of these conditions. (c) 1993 John Wiley & Sons, Inc. 相似文献
18.
The culture of Escherichia coli for the commercial production of recombinant proteins has increased significantly in recent years. The production of acetate as a byproduct retards cell growth, inhibits protein formation, and diverts carbon from biomass to protein product. Our approach to reducing acetate accumulation was to disable the phosphoenolpyruvate:sugar phosphotransferase system (PEP-PTS) by deleting the ptsHI operon in the wild-type E. coli strain GJT001. The mutation caused a severe reduction in growth rate and glucose uptake rate in glucose-supplemented M9 minimal medium, which confirmed the mutation, and eliminated acetate accumulation. The mutant strain (TC110) apparently metabolized glucose by a non-PTS mechanism that we are currently investigating, followed by phosphorylation by glucokinase. In complex medium such as 2xLB broth with 2% glucose, TC110 was able to grow quickly and still retained the phenotype of significantly reduced acetate accumulation (9.1+/-6.6 vs. 90.4+/-1.6mM in GJT001, P<0.05). The reduced acetate accumulation resulted in a significant improvement in final OD (23.5+/-0.7 in TC110 vs. 8.0+/-0.1 in GJT001, P<0.05). We tested the strains for the production of model recombinant proteins such as green fluorescent protein (GFP) and beta-galactosidase. TC110 had a 385-fold improvement in final volumetric productivity of GFP over GJT001 in shake flasks with 2xLB broth with 2% glucose. The distribution of GFP fluorescence in the cell population, as determined by flow cytometry, was much broader in GJT001 (coefficient of variation=466+/-35%) than in TC110 (coefficient of variation=55+/-1%). In corn steep liquor medium with 2% glucose, we observed a 28.5-fold improvement in final volumetric production of GFP in TC110 over GJT001. TC110 had a 7.5-fold improvement in final volumetric productivity of beta-galactosidase over GJT001 in 2xLB broth with 2% glucose medium. When tested in a batch bioreactor cultures with 2xLB broth with 2% glucose medium, the volumetric production of GFP by TC110 was 25-fold higher than that of GJT001. In summary, the ptsHI mutant of GJT001 resulted in reduced acetate accumulation, which led to significant improvements in recombinant protein production in batch bioreactors. 相似文献
19.
Metabolic constraints during the production of recombinant protein in Escherichia coli impede the efficient utilization of resources by the cells, thus reducing their production potential. In order to minimize these adverse effects, we have proposed to segregate the cell population into two groups: the first one formed by non-induced cells, growing at a high specific growth rate and rapidly contributing cells to the system, and the second one formed by fully induced cells, growing slowly but using the cell machinery to express the target protein. An adequate balance between these two populations should maximize the protein expression in a given system. This segregation is accomplished experimentally by taking advantage of the "all or none" phenomenon, in which at subsaturated inducer conditions the cells are either fully induced or fully uninduced. Based on this two-population theory, a mathematical model was developed in which a parameter alpha was defined as the fraction of the fully induced cells in the total population. In this study three different induction strategies were investigated and their effect on the protein production was established. It was found that the linear increase of this fraction, achieving maximum induction (alpha = 1) only at the end of the fermentation and with a slope m = 0.15 gave the best results. Finally these results were validated experimentally with the finding that they closely match the mathematical simulation with a 26% increase in protein production with respect to the conventional induction approach described. 相似文献
20.
The uptake of acetate by Escherichia coli w 总被引:2,自引:0,他引:2
C Wagner R Odom W T Briggs 《Biochemical and biophysical research communications》1972,47(5):1036-1043