Construction Of A Bacillus Subtilis Mutant Deficient In LipA And RecA

Bacillus subtilis can secrete one kind of lipase named LipA directly into the culture medium. LipA shows good activity when it hydrolyzes substrates with short or middle chain fatty acids. Construction of LipA over-expression strain by B.Subtilis and its lipA gene is an efficient method to produce LipA. B.Subtilis DB104 is a mutant deficient in a neutral metalloprotease and an alkaline serine protease which is suitable to be the host strain for expressing lipase. But lipA gene on DB104's chorosome would act with the same gene on the recombined vector and influence the screening of the expression strain. The recombination function involved with recA gene would lead to the unstable of recombined plasmid in the strain. What's more, the increased production of acidic glycolysis products would lead to the decreasing of pH in glucose containing media. LipA would misfold after secretion across the cell wall under low pH condition which results in low enzymatical activity. So we have to modify the gene of DB104.We constructed the lipA deficient mutant TZ10. By using TZ10 and the fat-media both, the expression strain of LipA can be identified easily. CcpA encoded by ccpA gene is the key regulator which mediates the CCR effect, so the knock-out of CcpA could eliminate the CCR effect driven by glucose and slow down the rate of pH decreasing. So TZ10 deficient in CcpA could be a host strain to identify the effect of CcpA to the production of LipA. Based on TZ10 and TZ20, the recA gene was further knocked out result in TZ10K, TZ20K and TZ76. Compared with the strain with intact recA gene, the three strains growed at a very low rate.