The production of L-tryptophan through che- mical synthesis, direct fermentation, bioconversion and enzymatic conversion has been reported. However, the role of the transport system for the aromatic amino acids in L- ...The production of L-tryptophan through che- mical synthesis, direct fermentation, bioconversion and enzymatic conversion has been reported. However, the role of the transport system for the aromatic amino acids in L- tryptophan producing strains has not been fully explored. In this study, the atop gene of the L-tryptophan producing Escherichia coli TRTH strain was disrupted using Red recombination technology and an atoP mutant E. coli TRTH AaroP was constructed. Fed-batch fermentation of E. coli TRTH △aroP was carried out in 30-L fermentor to investigate the L-tryptophan production. Compared with E. coli TRTH, the atoP mutant was able to maintain a higher growth rate during the exponential phase of the fermentation and the L-tryptophan production increased by 13.3%.展开更多
We applied a resistance split-fusion strategy to increase the in vivo direct cloning efficiency mediated by Red recombination. The cat cassette was divided into two parts： cma （which has a homologous sequence with ...We applied a resistance split-fusion strategy to increase the in vivo direct cloning efficiency mediated by Red recombination. The cat cassette was divided into two parts： cma （which has a homologous sequence with cmb） and cmb, each of which has no resistance separately unless the two parts are fused together. The crab sequence was integrated into one flank of a target clon- ing region in the chromosome, and a linear vector containing the cma sequence was electroporated into the cells to directly capture the target region. Based on this strategy, we successfully cloned an approximately 48 kb DNA fragment from the E. coli DH1-Z chromosome with a positive frequency of approximately 80%. Combined with double-strand breakage-stimulated homologous recombination, we applied this strategy to successfully replace the corresponding region of the E. coli DH36 chromosome and knock out four non-essential genomic regions in one step. This strategy could provide a powerful tool for the heterologous expression of microbial natural product biosynthetic pathways for genome assembly and for the functional study of DNA sequences dozens of kilobases in length.展开更多
文摘The production of L-tryptophan through che- mical synthesis, direct fermentation, bioconversion and enzymatic conversion has been reported. However, the role of the transport system for the aromatic amino acids in L- tryptophan producing strains has not been fully explored. In this study, the atop gene of the L-tryptophan producing Escherichia coli TRTH strain was disrupted using Red recombination technology and an atoP mutant E. coli TRTH AaroP was constructed. Fed-batch fermentation of E. coli TRTH △aroP was carried out in 30-L fermentor to investigate the L-tryptophan production. Compared with E. coli TRTH, the atoP mutant was able to maintain a higher growth rate during the exponential phase of the fermentation and the L-tryptophan production increased by 13.3%.
基金supported by the National Natural Science Foundation of China(81373286)National Basic Research Program of China(2011CBA00800)
文摘We applied a resistance split-fusion strategy to increase the in vivo direct cloning efficiency mediated by Red recombination. The cat cassette was divided into two parts： cma （which has a homologous sequence with cmb） and cmb, each of which has no resistance separately unless the two parts are fused together. The crab sequence was integrated into one flank of a target clon- ing region in the chromosome, and a linear vector containing the cma sequence was electroporated into the cells to directly capture the target region. Based on this strategy, we successfully cloned an approximately 48 kb DNA fragment from the E. coli DH1-Z chromosome with a positive frequency of approximately 80%. Combined with double-strand breakage-stimulated homologous recombination, we applied this strategy to successfully replace the corresponding region of the E. coli DH36 chromosome and knock out four non-essential genomic regions in one step. This strategy could provide a powerful tool for the heterologous expression of microbial natural product biosynthetic pathways for genome assembly and for the functional study of DNA sequences dozens of kilobases in length.
