The Optimization Of SsDNA Homologous Recombination Efficiency In Corynebacterium Glutamicum

The mutant of Corynebacterium glutamicum is widely used in production of amino acid and other metabolites, driving the need for the rapid introduction of chromosomal mutations. Compared to traditional breeding techniques, synthetic ss DNA oligos recombine efficiently to generate point mutations, deletions, insertions and even multiple-sites with one manipulation. However, the low recombination efficiency limited its wide applicability. It is crucial to improve the efficiency of ss DNA recombination. Both the electro-transformation efficiency of ss DNA and expression of recombinase were optimized to improve the recombination efficiency of ss DNA in C. glutamicum ATCC 13032. In this paper, the main results are as follows:(1) The single-factor experiment was firstly carried out to demonstrate an optimal compentent medium. On this basis, single-factor experiments combined with response surface methodology was used to optimize four cell wall weakning agents supplemented in the compentent medium, obtained an optimal compentent medium, and improved electro-transformation efficiency of heterogenous DNA by 108-fold. Furthermore, several single-factor experiments were carried out to optimized the conditions of cell growth state, electroporation buffer, electroporation parameters, heat shock condition, the plasmid amount and post-culture time, improved the electro-transformation efficiency of heterogenous DNA by 11-fold, the electro-transformation efficiency of heterogenous DNA reached to 3.81×107 cfu/μg plasmid DNA, and recombination efficiency of ss DNA reached to 5.6×104 cfu/109 viable cells in C. glutamicum ATCC 13032.(2) An ss DNA targeted to modify rps L gene was used to optimize the ss DNA length, amount and phosphorothioate modification etc. Contrasting to rps L spontaneous mutation rate, the optimized result of ss DNA length of 90 nt base and amount of 10 μg/80 μL competent cells were chosen, with two phosphorothioates modification at 3’ end, the ss DNA homologous recombination efficiency reached to 9.8×104 cfu/109 viable cells in C. glutamicum ATCC 13032, which was 1.75-fold higher.(3) With C. glutamicum ATCC 13032 as a host system, 13032/p DXW-10-rec T strain was constructed, expressed recombinant protein Rec T in plasmid p DXW-10. With 13032/p DXW-10-rec T as the host strain, the ss DNA recombination effiency was improved by 1.7-fold, reached to 1.67×105 cfu/109 viable cells; the T7 promoter mediated inducible expression system 13032-DE3/p JC1-T7-rec T was constructed to further improve the quantity of protein expression, obtained recombination efficiency of 7.8×105 cfu/109 viable cells, which was 4.7-fold higher than 13032/p DXW-10-rec T; Furthermore, a genomic integration strain of 13032-DE3 Zif’::T7-rec T was constructed to get ride of using plasmids and antibiotics selection marker, a recombination efficiency of 6.7×105 cfu/109 viable cells was obtained, which was only slightly lower than 13032-DE3/p JC1-T7-rec T, but improved the convenience and operability of genetic modification in C. glutamicum ATCC 13032.(4) ss DNA with single point mutation was designed targeting to gene rpo B and gyr A respectively, to evaluate the optimized ss DNA homologous recombination protocal, obtained ss DNA recombination efficiency of 5.6×106 cfu/109 viable cells and 2.3×107 cfu/109 viable cells respectively. The results showed that the method was also applicable for rpo B and gyr A genes.