Establishment Of An Efficient Gene Editing Method Based On CRISPR/Cas9 System For Bacillus Coagulans

The unique biological characteristics of Bacillus coagulans have great potential in the industrial fermentation of lactic acid.Its efficiency of gene editing has been very low,which greatly increases the difficulty of genetic engineering of B.coagulans.CRISPR-Cas9 system has the characteristics of high efficiency and accurate editing,which has been favored by scientists.In this study,the effective method of gene editing for B.coagulans based on CRISPR-Cas9 system was estabolished.Firstly,the CRISPR-Cas9 system suitable for B.coagulans ATCC7050 was constructed,and the result of Western-Blot showed that the BcCas9n protein modified by codon preference according to Cas9 protein was successfully expressed in B.coagulans ATCC7050,then the ldhL1 gene in B.coagulans ATCC7050 was mutated and knocked out by CRISPR-Cas9 system,and ldhD gene was replaced by ldhLl gene.Studies on the mutant strains showed that the ldhLl-mutated strain had obviously lower lactic acid productivity;the ldhLl knocked-out strain nearly did not produce lactic acid;the over-expressed strain had enhanced production and productivity of lactic acid,and the optical purity of lactic acid also increased from 99.43±0.08%to 99.68±0.06%.The above results showed that the ldhL1 gene was the most important lactic acid dehydrogenase gene in the biosynthesis of lactic acid,and laid an important foundation for the efficient genetic manipulation of B.coagulans.