A method for simultaneous gene overexpression and inactivation in the Corynebacterium glutamicum genome

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• 作者：Jianzhong Xu ; Junlan Zhang ; Mei Han…
• 关键词：Corynebacterium glutamicum ; Mobilizable plasmid ; Homologous double crossover ; Integration ; Marker free
• 刊名：Journal of Industrial Microbiology and Biotechnology
• 出版年：2016
• 出版时间：October 2016
• 年：2016
• 卷：43
• 期：10
• 页码：1417-1427
• 全文大小：1,351 KB
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Chemistry
  Biotechnology
  Genetic Engineering
  Biochemistry
  Bioinformatics
  Microbiology
  Microbial Genetics and Genomics
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1476-5535
• 卷排序：43

文摘

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 lysCT311I 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.