Construction Of A Markerless Genetic Manipulation System In Corynebacterium Glutamicum

Corynebacterium glutamicum is an important microorganism for the industrial production of various amino acids. It has been widly used in medicine, food and feed industry. It possesses rapid growth and good adaptability, which confered it broad prospect and great potential to serve as a novel cellular factory. However, the availability of its gene manipulation technology has generally lagged behind traditional genetic models such as Escherichia coli and Saccharomyces cerevisiae. In this research, a double crosssover recombination system for direct genetic manipulation of the C. glutamicum was developed. This system was based on a novel counter-selectable marker combined with the intramolecular homologous recombination stimulated by the double strand break(DSB). On this basis, a PCR-generated fragment was successfully used for the markerless genetic modification with the aid of recombinase from E. coli.Genetic manipulation system based on the plasmid pK18 mobsacB has now been widly used for markerless gene manipulation in C. glutamicum. But the counter-seletable marker sacB tended to result in many false-positive colonies in the selection process. In this study, we firstly verified the feasibility of upp as a counter-selectable marker by comparing the 5-FU tolerance of wild-type(WT) and upp deletion mutant(WT-?upp). It was also confiermed that upp showed about 10-fold lower false positive rate than sacB when the two gene were both expressed from promter Ptrc. Furthermore, we determined the concentration of 5-FU for the counter-selection was 100 ?M.To construct the double crossover recombination system, universal vector pCupp and helper plasmid pEC-XK99E-Sce I-recET were constructed. pCupp contained upp-cassette and pEC-XK99E-Sce I-recET could express endonuclease I-SceI with IPTG induction. Based on the two plasmids, upp was combined with double strand break(DSB) repair caused by exogenous endonuclease I-SceI. In-frame deletion of ldhA was performed with this system. The dosage of I-SceI was critical for efficient pop-out of upp-cassette.Thus, the optimal concentration of inducer IPTG and the suitable induction time for the pop-out frequency was explored. The results showed that the pop-out frequency reached to 3.68×10-5, which was a maximum value, when we use 1.5 mM IPTG to induce I-SceI expression for 24 h.With the arabinose induced expression of RecE/RecT from the helper plasmid pEC-XK99E-Sce I-recET, dsDNA PCR product for the deletion of ldhA was transformed into WT-?upp. And it was successfully inserted into the chromosome of WT-?upp. This was the first report of dsDNA PCR product realizing recombination in C. glutamicum. At last, the helper plasmid pEC-XK99E-SceI-recET could be eliminated by serial sub-cultivation. This meant that the realization of markerless mutation with this system was practicable.