Construction and application of an efficient multiple-gene-deletion system in Corynebacterium glutamicum

Gene deletion techniques are important for modifying Corynebacterium glutamicum, the bacterium for industrial production of amino acids. In this study, a novel multiple-gene-deletion system for C glutamicum was developed. The system is composed of three plasmids pDTW109, pDTW201 and pDTW202. pDTW109 is a temperature-sensitive vector which harbors a cat gene under the tacM promoter, a cre gene under the tac promoter, an origin oriE for replicating in Escherichia coli, and another origin rep(TS) for replicating in C glutamicum only at low temperatures; it has high transformation efficiency in C glutamicum and can be easily eliminated by growing at 37 C. pDTW201 and pDTW202 carry loxp-flanked or mutant lox-flanked kan, respectively. This deletion system combines homologous recombination and Cre/lox site-specific recombination, could efficiently delete the aceE gene from the chromosome of C glutamicum ATCC13032, ATCC13869 or ATCC14067, respectively, and could also delete both genes of aceE and ilvA from the chromosome of C glutamicum ATCC14067. The system is simple and efficient, and can be easily implemented for multiple-gene-deletion in C glutamicum. (C) 2013 Elsevier Inc. All rights reserved.