Modification And Expression Of LipA Promoter In Bacillus Subtilis

At present, the application of lipase that produced by Microbe is prevailing. This pathway of producing lipase has a lot of advantage. The Bacillus subtilis lipase has the smallest molecule mass known so far. It exhibits a broad substrate range, which shows its latent application value. To construct lipase overexpression strain through genetic engineering and put it into real production is the very need for detergent, chiral compounds synthesizing, paper making and biomedicine technology exploration. Construction of LipA over-expression strain by B.subtilis and its lipA gene is an efficient method to produce LipA. Bacillus Subtilis DB104 is a mutant deficient in a neutral metallo protease and an alkaline serine protease which is suitable to be the host strain for expressing lipase. In order to obtain good screen marker we had to modify the lipA gene of DB104. The method we adopt is modify the -35 section and -10 section of promoter of lipA gene to accord with the consensus sequence.Take Bacillus Subtilis DB104 as origination we constructed the lipA deficient mutant SH-1 which has erm resistance. Homologous recombination fragment including the promoter of lipA gene was obtained by overlap PCR, and was then ligated with plasmid pUC18, then transformed to B. subtilis SH-1, where homologous recombination happened in the site of lipA, the erm fragment was replaced by lipA fragment, the lipA mutation recovered strain B. subtilis SH-2 which had no erm resistance. Compared with B. subtilis SH-1, B. subtilis SH-2 can grow in minimum plate of only using olive oil as carbon source. Fermentation data showed that the maximum extracellular enzyme activity of and SH-2 was 90.80 U/L using olive oil as substrate, whereas DB104 only 21.62U/L.