The kinetics of batch and fed-batch cultures of recombinant Escherichia coli producing human-like collagen was investigated. In the batch culture, a kinetic model of a simple growth-association system was concluded wi...The kinetics of batch and fed-batch cultures of recombinant Escherichia coli producing human-like collagen was investigated. In the batch culture, a kinetic model of a simple growth-association system was concluded without consideration of cell endogeneous metabolism. The cell lag time, the maximum specific growth rate and Yx/s were determined as 1.75h, 0.65h^-1 and 0.51g·g^-1, respectively. In the fed-batch culture, different specific growth rates were set at （0.15, 0.2, 0.25h^-1） by the method of pseudo-exponential feeding, and the expressions for the specific rate of substrate consumption, the growth kinetics and the product formation kinetics of each phase were obtained. The result shows that the concentrations of cell and product can reach 77.5g·L^-1 and 10.2g·L^-1 respectively. The modal predictions are in good agreement with the experimental data.展开更多
Disulfide bond formation protein A （DsbA） is one of the important helper proteins for folding in protein synthesis in vivo. In this study, purification of recombinant DsbA was investigated by examining four importan...Disulfide bond formation protein A （DsbA） is one of the important helper proteins for folding in protein synthesis in vivo. In this study, purification of recombinant DsbA was investigated by examining four important factors with Box-Behnken design method, a statistic-based design of experiments. The optimal operation conditions were obtained by adopting the effectiveness coefficient method on the multi-objective problem, which takes the protein recovery, purification efficiency and throughput of ion-exchange chromatography into account. After the optimization, protein recovery of 96.8% and purity higher than 95% DsbA was achieved, and the productivity was （377.9±1.7） mg soluble DsbA per liter broth. The purified protein was identified by peptide mass fingerprinting matching the record of gil2624856, a mutant of DsbA. The DsbA was preliminarily applied to the refolding of denatured lysozyme in vitro.展开更多
Promoters are the most important tools to control and regulate the gene expression in synthetic biology and metabolic engineering. The expression of target genes in Escherichia coli is usually controlled by the high-s...Promoters are the most important tools to control and regulate the gene expression in synthetic biology and metabolic engineering. The expression of target genes in Escherichia coli is usually controlled by the high-strength inducible promoter with the result that the abnormally high transcription of these genes creates excessive metabolic load on the host, which decreases product formation. The constitutive expression systems are capable of avoiding these defects. In this study, to enrich the application of constitutive promoters in metabolic engineering, four promoters from the glycolytic pathway of E. coli were cloned and characterized using the enhanced green fluorescent protein as reporter. Among these promoters, Pgap Awas determined as the strongest one, the strength of which was about 8.92% of that of the widely used inducible promoter PT7. This promoter was used to control the expression of heterologous xylose reductase in E. coli for xylitol synthesis so as to verify its function in pathway engineering. The maximum xylitol titer(40.6 g·L-1) produced by engineered E. coli under the control of the constitutive promoter Pgap Awas obviously higher than that under the control of the inducible promoter PT7,indicating the feasibility and superiority of promoter Pgap Ain the metabolic engineering of E. coli.展开更多
5-Aminolevulinic acid （ALA） is a common precursor for tetrapyrrole compounds in all kinds of organ isms and has wide applications in agriculture and medicines. In this study, a new strategy, i.e. short-term dissolve...5-Aminolevulinic acid （ALA） is a common precursor for tetrapyrrole compounds in all kinds of organ isms and has wide applications in agriculture and medicines. In this study, a new strategy, i.e. short-term dissolved oxygen （DO） shock during aerobic fermentation, was introduced to produce 5-aminolevulinic acid with a recombi-nant E. coli. Effects of duration time of DO shock operation on plasmid concentration, intracellular ALA synthase （ALAS） activity and ALA production were investigated in Erlenmeyer shake flasks. The results indicated that both ALAS activity and ALA yield were enhanced in an anaerobic operation of 45 rain in the early exponential phase during fermentation, while they decreased when the anaerobic operation time was further increased to 60 rain. The DO shock protocol was confirmed with the fed-batch fermentation in a 15 L fermenter and the ALA production achieved 9.4 g.L-1 （72 mmol.L-1）, which is the highest yield in the fermentation broth reported up to now.展开更多
The discovery of new, highly active, biomass-degrading enzymes is important to the development of a sustainable biofuels industry. Dictyoglomus turgidum, a thermophilic, anaerobic eubacterium that ferments cellulose a...The discovery of new, highly active, biomass-degrading enzymes is important to the development of a sustainable biofuels industry. Dictyoglomus turgidum, a thermophilic, anaerobic eubacterium that ferments cellulose and produces ethanol and hydrogen, was chosen as a candidate to screen for novel enzymes. A novel thermostable endoglucanase, CelA, was identified and purified during screening of a shotgun library of Dic（yoglomus turgidum and subsequently subcloned and expressed in E. coli. The celA gene coding for a 312 amino acid protein showed low homology to proteins outside the genus Dictoglomi and lacked an apparent signal peptide. CelA had a broad substrate range, possessing both endo and exo activity on soluble and insoluble β-（1,4）-Iinked glucose-containing substrates as well as endo activity on soluble and insoluble β-（1,4）-linked mannose containing substrates. The specific activity of CelA was 226 U/rag using β-glucan, 66 U/mg using glucomannan, and 63 U/mg using CMC as substrates. The high temperature optimum of 70 ℃ to 80 ℃ and wide substrate range of the enzyme might make it an excellent tool for biomass degradation at high temperature.展开更多
基金Supported by the National Science and Technology Key Funds (2003DA901A32) and the National Natural Science Foundationof China (No.20476085).
文摘The kinetics of batch and fed-batch cultures of recombinant Escherichia coli producing human-like collagen was investigated. In the batch culture, a kinetic model of a simple growth-association system was concluded without consideration of cell endogeneous metabolism. The cell lag time, the maximum specific growth rate and Yx/s were determined as 1.75h, 0.65h^-1 and 0.51g·g^-1, respectively. In the fed-batch culture, different specific growth rates were set at （0.15, 0.2, 0.25h^-1） by the method of pseudo-exponential feeding, and the expressions for the specific rate of substrate consumption, the growth kinetics and the product formation kinetics of each phase were obtained. The result shows that the concentrations of cell and product can reach 77.5g·L^-1 and 10.2g·L^-1 respectively. The modal predictions are in good agreement with the experimental data.
基金Supported by the National Natural Science Foundation of China （21036005）.
文摘Disulfide bond formation protein A （DsbA） is one of the important helper proteins for folding in protein synthesis in vivo. In this study, purification of recombinant DsbA was investigated by examining four important factors with Box-Behnken design method, a statistic-based design of experiments. The optimal operation conditions were obtained by adopting the effectiveness coefficient method on the multi-objective problem, which takes the protein recovery, purification efficiency and throughput of ion-exchange chromatography into account. After the optimization, protein recovery of 96.8% and purity higher than 95% DsbA was achieved, and the productivity was （377.9±1.7） mg soluble DsbA per liter broth. The purified protein was identified by peptide mass fingerprinting matching the record of gil2624856, a mutant of DsbA. The DsbA was preliminarily applied to the refolding of denatured lysozyme in vitro.
基金Supported by the National High Technology Research and Development Program of China(863 Program)(2012AA02A704)the Major State Basic Research Development Program of China(973 Program)(2013CB733900)+1 种基金the National Natural Science Foundation of China(21176028,21376028)the National Research Foundation for the Doctoral Program of Higher Education of China(20121101110050)
文摘Promoters are the most important tools to control and regulate the gene expression in synthetic biology and metabolic engineering. The expression of target genes in Escherichia coli is usually controlled by the high-strength inducible promoter with the result that the abnormally high transcription of these genes creates excessive metabolic load on the host, which decreases product formation. The constitutive expression systems are capable of avoiding these defects. In this study, to enrich the application of constitutive promoters in metabolic engineering, four promoters from the glycolytic pathway of E. coli were cloned and characterized using the enhanced green fluorescent protein as reporter. Among these promoters, Pgap Awas determined as the strongest one, the strength of which was about 8.92% of that of the widely used inducible promoter PT7. This promoter was used to control the expression of heterologous xylose reductase in E. coli for xylitol synthesis so as to verify its function in pathway engineering. The maximum xylitol titer(40.6 g·L-1) produced by engineered E. coli under the control of the constitutive promoter Pgap Awas obviously higher than that under the control of the inducible promoter PT7,indicating the feasibility and superiority of promoter Pgap Ain the metabolic engineering of E. coli.
基金Supported by the National Natural Science Foundation of China （20306026 and 20876141） and the National Basic Research program of China （2007CB707805）.
文摘5-Aminolevulinic acid （ALA） is a common precursor for tetrapyrrole compounds in all kinds of organ isms and has wide applications in agriculture and medicines. In this study, a new strategy, i.e. short-term dissolved oxygen （DO） shock during aerobic fermentation, was introduced to produce 5-aminolevulinic acid with a recombi-nant E. coli. Effects of duration time of DO shock operation on plasmid concentration, intracellular ALA synthase （ALAS） activity and ALA production were investigated in Erlenmeyer shake flasks. The results indicated that both ALAS activity and ALA yield were enhanced in an anaerobic operation of 45 rain in the early exponential phase during fermentation, while they decreased when the anaerobic operation time was further increased to 60 rain. The DO shock protocol was confirmed with the fed-batch fermentation in a 15 L fermenter and the ALA production achieved 9.4 g.L-1 （72 mmol.L-1）, which is the highest yield in the fermentation broth reported up to now.
文摘The discovery of new, highly active, biomass-degrading enzymes is important to the development of a sustainable biofuels industry. Dictyoglomus turgidum, a thermophilic, anaerobic eubacterium that ferments cellulose and produces ethanol and hydrogen, was chosen as a candidate to screen for novel enzymes. A novel thermostable endoglucanase, CelA, was identified and purified during screening of a shotgun library of Dic（yoglomus turgidum and subsequently subcloned and expressed in E. coli. The celA gene coding for a 312 amino acid protein showed low homology to proteins outside the genus Dictoglomi and lacked an apparent signal peptide. CelA had a broad substrate range, possessing both endo and exo activity on soluble and insoluble β-（1,4）-Iinked glucose-containing substrates as well as endo activity on soluble and insoluble β-（1,4）-linked mannose containing substrates. The specific activity of CelA was 226 U/rag using β-glucan, 66 U/mg using glucomannan, and 63 U/mg using CMC as substrates. The high temperature optimum of 70 ℃ to 80 ℃ and wide substrate range of the enzyme might make it an excellent tool for biomass degradation at high temperature.
