title: A method for simultaneous gene overexpression and inactivation in the Corynebacterium glutamicum genome.
abstract: The gene integration method is an important tool to stably express desirable genes in bacteria. To avoid heavy workload and cost, we constructed a rapid and efficient method for genome modification. This method depended on a mobilizable plasmid, which contains a P tac promoter, an introduced multiple cloning site (iMCS), and rrnBT1T2 terminator. Briefly, the mobilizable plasmid pK18-MBPMT with the P tac-iMCS-rrnBT1T2 cartridge derived from pK18mobsacB was prepared to directly integrate hetero-/homologous DNA into the Corynebacterium glutamicum genome. Like our previous method, this method was based on insertional inactivation and double-crossover homologous recombination, which simultaneously achieved gene overexpression and inactivation in the genome without the use of genetic markers. Compared to the previous method, this protocol omitted the construction of a recombinant expression plasmid and clone of the target gene(s) cassette, which significantly decreased the workload, cost, and operational time. Using this method, the heterologous gene amy and the homologous gene lysC (T311I) were successfully integrated into the C. glutamicum genome at alaT and avtA loci, respectively. Moreover, the operation time of this method was shorter than that of the previous method, especially for repeated integration. This method, which is based on the mobilizable plasmid pK18-MBPMT, thus represents a potentially attractive protocol for the integration of genes in the course of genetic modification of C. glutamicum.
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800# Lihu Road, 214122, Wuxi, People's Republic of China. xujz126@126.com.
authors.forename: Jianzhong
authors.initials: J
authors.lastname: Xu
authors.affiliation: OriGene Biotechnology Co., Ltd., 88# Meiliang Road, 214122, Wuxi, 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, 214122, Wuxi, People's Republic of China.|State Key Laboratory of Dairy Biotechnology, 1518# Jiangchang West Road, 200436, Shanghai, People's Republic of China.
authors.forename: Mei
authors.initials: M
authors.lastname: Han
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800# Lihu Road, 214122, Wuxi, People's Republic of China.
authors.forename: Weiguo
authors.initials: W
authors.lastname: Zhang
mesh_terms: D048230:Corynebacterium glutamicum / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D018014:Gene Transfer Techniques; D005798:Genes, Bacterial; D016680:Genome, Bacterial; D059765:Homologous Recombination; D016254:Mutagenesis, Insertional; D010957:Plasmids
keywords: Corynebacterium glutamicum; Homologous double crossover; Integration; Marker free; Mobilizable plasmid