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
The stability and high productivity of heterogeneous terpenoid production in Escherichia coli expression system is one of the most key issues for its large scale industrialization.In the current study on taking lycope...The stability and high productivity of heterogeneous terpenoid production in Escherichia coli expression system is one of the most key issues for its large scale industrialization.In the current study on taking lycopene biosynthesis as an example,an integrated Escherichia coli system has been generated successfully,which resulted into stable and high lycopene production.In this process,two modules of mevalonate(MVA)pathway and one module of lycopene expression pathway were completely integrated in the chromosome.Firstly,the copy number and integrated position of three modules of heterologous pathways were rationally optimized.Later,a strain DH416 equipped with heterogeneous expression pathways through chromosomal integration was efficiently derived from parental strain DH411.The evolving DH416 strain efficiently produced the lycopene level of 1.22 g/L(49.9 mg/g DCW)in a 5 L fermenter with mean productivity of 61.0 mg/L/h.Additionally,the integrated strain showed more genetic stability than the plasmid systems after successive 21st passage.展开更多
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.
基金supported by grants from the National Key Research and Development Program of China(2018YFA0900300 and 2019YFA090141)the National Science and Technology Major Project(2017ZX07402003).
文摘The stability and high productivity of heterogeneous terpenoid production in Escherichia coli expression system is one of the most key issues for its large scale industrialization.In the current study on taking lycopene biosynthesis as an example,an integrated Escherichia coli system has been generated successfully,which resulted into stable and high lycopene production.In this process,two modules of mevalonate(MVA)pathway and one module of lycopene expression pathway were completely integrated in the chromosome.Firstly,the copy number and integrated position of three modules of heterologous pathways were rationally optimized.Later,a strain DH416 equipped with heterogeneous expression pathways through chromosomal integration was efficiently derived from parental strain DH411.The evolving DH416 strain efficiently produced the lycopene level of 1.22 g/L(49.9 mg/g DCW)in a 5 L fermenter with mean productivity of 61.0 mg/L/h.Additionally,the integrated strain showed more genetic stability than the plasmid systems after successive 21st passage.
