title: A method for gene amplification and simultaneous deletion in Corynebacterium glutamicum genome without any genetic markers.
abstract: A method for the simultaneous replacement of a given gene by a target gene, leaving no genetic markers, has been developed. The method is based on insertional inactivation and double-crossover homologous recombination. With this method, the lysC(T311I), fbp and ddh genes were inserted into Corynebacterium glutamicum genome, and the pck, alaT and avtA genes were deleted. Mobilizable plasmids with lysC(T311I), fbp and ddh cassettes and two homologous arms on the ends of pck, alaT and avtA were constructed, and then transformed into C. glutamicum. The target-expression cassettes were inserted in the genome via the first homologous recombination, and the genetic markers were removed via the second recombination. The target-transformants were sequentially screened from kanamycin-resistance and sucrose-resistance plates. The enzyme activities of transformants were stably maintained for 30 generations under non-selective culture conditions, suggesting that the integrated cassettes in host were successfully expressed and maintained as stable chromosomal insertions in C. glutamicum. The target-transformants were used to optimize the l-lysine production, showing that the productions were strongly increased because the selected genes were closely linked to l-lysine production. In short, this method can be used to construct amino acid high-producing strains with unmarked gene amplification and simultaneous deletion in genome.
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800(#) Lihu Road, WuXi 214122, People's Republic of China.
authors.forename: Jianzhong
authors.initials: J
authors.lastname: Xu
authors.affiliation: Wuxi Institute of Commerce, 809(#) Qianhu Road, WuXi 214122, People's Republic of China.
authors.forename: Xiuhua
authors.initials: X
authors.lastname: Xia
authors.affiliation: OriGene Biotechnology Co., Ltd., 88(#) Meiliang Road, WuXi 214122, People's Republic of China.
authors.forename: Junlan
authors.initials: J
authors.lastname: Zhang
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800(#) Lihu Road, WuXi 214122, People's Republic of China.
authors.forename: Yanfeng
authors.initials: Y
authors.lastname: Guo
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800(#) Lihu Road, WuXi 214122, People's Republic of China.
authors.forename: He
authors.initials: H
authors.lastname: Qian
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800(#) Lihu Road, WuXi 214122, People's Republic of China. Electronic address: xujz126@126.com.
authors.forename: Weiguo
authors.initials: W
authors.lastname: Zhang
mesh_terms: D003001:Cloning, Molecular; D048230:Corynebacterium glutamicum / Q000235:genetics* / Q000254:growth & development; D005784:Gene Amplification; D017353:Gene Deletion; D005798:Genes, Bacterial; D005818:Genetic Engineering; D005819:Genetic Markers; D016680:Genome, Bacterial; D059765:Homologous Recombination; D008239:Lysine / Q000096:biosynthesis; D010957:Plasmids / Q000235:genetics; D014169:Transformation, Bacterial
chemical_list: D005819:Genetic Markers; D008239:Lysine
keywords: Corynebacterium glutamicum; Double-crossover recombination; Gene amplification; Gene deletion; Integration; Marker free