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

• Developed a novel multiple-gene-deletion system for C. glutamicum.
• Constructed three plasmids pDTW109, pDTW201 and pDTW202 for the deletion system.
• Deleted the aceE gene in three different C. glutamicum strains by using the system.
• Deleted both aceE and ilvA genes in C. glutamicum ATCC14067 by using the system.

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 repTS 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.