The plasmid-expression system is routinely plagued by potential plasmid instability. Chromosomal integration is one powerful approach to overcome the problem. Herein we report a plasmid-free hyper-producer E.coli stra...The plasmid-expression system is routinely plagued by potential plasmid instability. Chromosomal integration is one powerful approach to overcome the problem. Herein we report a plasmid-free hyper-producer E.coli strain for coenzyme Q10 production. A series of integration expression vectors, pxKC3T5b and pxKT5b, were constructed for chemically inducible chromosomal evolution(multiple copy integration) and replicon-free and markerless chromosomal integration(single copy integration), respectively. A coenzyme Q10 hyper-producer Escherichia coli TBW20134 was constructed by applying chemically inducible chromosomal evolution,replicon-free and markerless chromosomal integration as well as deletion of menaquinone biosynthetic pathway.The engineered E. coli TBW20134 produced 10.7 mg per gram of dry cell mass(DCM) of coenzyme Q10 when supplemented with 0.075 g·L-1of 4-hydroxy benzoic acid; this yield is unprecedented in E. coli and close to that of the commercial producer Agrobacterium tumefaciens. With this strain, the coenzyme Q10 production capacity was very stable after 30 sequential transfers and no antibiotics were required during the fermentation process. The strategy presented may be useful as a general approach for construction of stable production strains synthesizing natural products where various copy numbers for different genes are concerned.展开更多
Recently, engineered minichromosomes have been produced using a telomere-mediated truncation technique in some plants. However, the study on transferring genes to minichromosomes is very limited.Here, telomere-mediate...Recently, engineered minichromosomes have been produced using a telomere-mediated truncation technique in some plants. However, the study on transferring genes to minichromosomes is very limited.Here, telomere-mediated truncation was successfully performed in common wheat（Triticum aestivum）to generate stable truncated chromosomes accompanied by a relatively high frequency of chromosomal rearrangements. After the cross between transgenic parents, a promoter-less DsRed gene in a chromosome from one parent was transferred to another chromosome from the other parent at the site behind a maize ubiquitin promoter via the Cre/lox system. DsRed transcripts and red fluorescent proteins were detected in the recombinant plants. In one such seedling, transgenic signals were detected at the centric terminus of chromosome 4D and the distal terminus of chromosome 3A. Clear translocations could be detected at the transgenic loci of these two chromosomes. Intriguingly, signals of centric-specific sequences were co-localized with the translocated D-group chromosomal segment in the terminal region of chromosome 3A. Our results indicate that the Cre/lox system induces the gene swapping to the target chromosome and non-homologous chromosomal recombination simultaneously. These approaches could offer a platform to transfer large DNA fragments or even terminal chromosomal segments to other chromosomes of the natural genome.展开更多
Precise chromosome engineering has traditionally relied on the Cre-Lox recombination system-an approach in which the enzyme Cre functions like molecular scissors,cutting and rejoining DNA at specific“Lox”sites to ad...Precise chromosome engineering has traditionally relied on the Cre-Lox recombination system-an approach in which the enzyme Cre functions like molecular scissors,cutting and rejoining DNA at specific“Lox”sites to add,remove,or flip genomic DNA segments inside living cells.展开更多
EST-PCR based molecular markers specific for alien chromosomes are not only useful for the detection of the introgressed alien chromatin in the wheat background, but also provide evidence of the syntenic relationship ...EST-PCR based molecular markers specific for alien chromosomes are not only useful for the detection of the introgressed alien chromatin in the wheat background, but also provide evidence of the syntenic relationship between homoeologous chromosomes. In the present study, in order to develop high density and evenly distributed molecular markers on chromosome 4V of Haynaldia villosa, a total of 607 primer pairs were designed according to the EST sequences, which were previously located in 23 different bins of wheat chromosomes 4A, 4B and 4D. By using the Triticum durum-H, villosa amphiploid and T. aestivum-H, villosa alien chromosome lines involving chromosome 4V, it was found that 9.23% of the tested primers could amplify specific bands for chromosome 4V. Thirty and twenty-six specific markers could be assigned to chromosome arms 4VS and 4VL, respectively. These 4V specific markers provided efficient tools for the characterization of structural variation involving the chromosome 4V as well as for the selection of useful genes located on chromosome 4V in breeding programs.展开更多
基金Supported by the National Natural Science Foundation of China(30970089,20876181,21276289)the Natural Science Foundation of Guangdong Province(9351027501000003,S2011010001396)
文摘The plasmid-expression system is routinely plagued by potential plasmid instability. Chromosomal integration is one powerful approach to overcome the problem. Herein we report a plasmid-free hyper-producer E.coli strain for coenzyme Q10 production. A series of integration expression vectors, pxKC3T5b and pxKT5b, were constructed for chemically inducible chromosomal evolution(multiple copy integration) and replicon-free and markerless chromosomal integration(single copy integration), respectively. A coenzyme Q10 hyper-producer Escherichia coli TBW20134 was constructed by applying chemically inducible chromosomal evolution,replicon-free and markerless chromosomal integration as well as deletion of menaquinone biosynthetic pathway.The engineered E. coli TBW20134 produced 10.7 mg per gram of dry cell mass(DCM) of coenzyme Q10 when supplemented with 0.075 g·L-1of 4-hydroxy benzoic acid; this yield is unprecedented in E. coli and close to that of the commercial producer Agrobacterium tumefaciens. With this strain, the coenzyme Q10 production capacity was very stable after 30 sequential transfers and no antibiotics were required during the fermentation process. The strategy presented may be useful as a general approach for construction of stable production strains synthesizing natural products where various copy numbers for different genes are concerned.
基金supported by the National Key Research and Development Program of China(2016YFD0102003)the Ministry of Science and Technology(MOST)Project(2016ZX08010002 and 2014ZX0801006B)
文摘Recently, engineered minichromosomes have been produced using a telomere-mediated truncation technique in some plants. However, the study on transferring genes to minichromosomes is very limited.Here, telomere-mediated truncation was successfully performed in common wheat（Triticum aestivum）to generate stable truncated chromosomes accompanied by a relatively high frequency of chromosomal rearrangements. After the cross between transgenic parents, a promoter-less DsRed gene in a chromosome from one parent was transferred to another chromosome from the other parent at the site behind a maize ubiquitin promoter via the Cre/lox system. DsRed transcripts and red fluorescent proteins were detected in the recombinant plants. In one such seedling, transgenic signals were detected at the centric terminus of chromosome 4D and the distal terminus of chromosome 3A. Clear translocations could be detected at the transgenic loci of these two chromosomes. Intriguingly, signals of centric-specific sequences were co-localized with the translocated D-group chromosomal segment in the terminal region of chromosome 3A. Our results indicate that the Cre/lox system induces the gene swapping to the target chromosome and non-homologous chromosomal recombination simultaneously. These approaches could offer a platform to transfer large DNA fragments or even terminal chromosomal segments to other chromosomes of the natural genome.
文摘Precise chromosome engineering has traditionally relied on the Cre-Lox recombination system-an approach in which the enzyme Cre functions like molecular scissors,cutting and rejoining DNA at specific“Lox”sites to add,remove,or flip genomic DNA segments inside living cells.
基金supported by the National High-Tech R & D Program of China(2011AA10010103,2011AA10010201)the National Natural Science Foundation of China (31201204)+1 种基金the Natural Science Foundation of Jiangsu Province,China (BK2010448)the Technology Support Program of Jiangsu Province,Chian (BE2012306)
文摘EST-PCR based molecular markers specific for alien chromosomes are not only useful for the detection of the introgressed alien chromatin in the wheat background, but also provide evidence of the syntenic relationship between homoeologous chromosomes. In the present study, in order to develop high density and evenly distributed molecular markers on chromosome 4V of Haynaldia villosa, a total of 607 primer pairs were designed according to the EST sequences, which were previously located in 23 different bins of wheat chromosomes 4A, 4B and 4D. By using the Triticum durum-H, villosa amphiploid and T. aestivum-H, villosa alien chromosome lines involving chromosome 4V, it was found that 9.23% of the tested primers could amplify specific bands for chromosome 4V. Thirty and twenty-six specific markers could be assigned to chromosome arms 4VS and 4VL, respectively. These 4V specific markers provided efficient tools for the characterization of structural variation involving the chromosome 4V as well as for the selection of useful genes located on chromosome 4V in breeding programs.
