Study On Autolysis Gene Knockout Of Bacillus Licheniformis With Spore Deficiency

Bacillus licheniformis is one of the important industrial production strains in the enzyme preparation industry that can ferment and produce a variety of enzyme preparation products including alpha-amylase.Bacillus licheniformis is susceptible to the formation of spores and the formation of alkaline proteases during fermentation,especially in the middle and late stages of fermentation,affecting the stable accumulation of target enzymes.In addition,there is autolysis in the growth process of Bacillus licheniformis.In the spore-deleted bacteria,the autolysis phenomenon is more obvious,which is not conducive to the high-density fermentation of the cells,and ultimately affects the fermentation production of the cells and the target enzyme.Therefore,the gene knockout method is used to reduce the spore formation rate and the autolysis rate of Bacillus licheniformis,which is beneficial to the fermentation production of the target enzyme,and is a new industrial microbial breeding method.Spore formation and autolysis of bacteria were regulated by various genes.In this study,B.licheniformis DL-1 was used as the starting strain,and?-amylase was used as the target enzyme,and two genes spoIIQ,pcf directly related to B.licheniformis DL-1spore formation and autolysis of bacteria were selected as a target knock-out gene.The effects of the above two genes on the spore formation rate,bacterial autolysis rate and bacterial fermentation performance of Bacillus licheniformis were analyzed.The main results are as follows:1.Using the gene-free knockout technique and pTOPO-Cmr as a gene knockout vector,the recombinant vector pTOPO-Cmr-spoIIQ was constructed,and the mid-term regulatory gene spoIIQ of Bacillus licheniformis was knocked out to obtain the recombinant B.licheniformis DL-1?spoIIQ.The results showed that the spore formation rates of the starting bacteria and the recombinant bacteria were 79.82%and0.16%,respectively,this indicates that the deletion of the spoIIQ gene causes a significant decrease in the sporulation rate of B.licheniformis DL-1.However,the autolysis of B.licheniformis DL-1?spoIIQ was more obvious compared with the original strain B.licheniformis DL-1,the concentration of bacteria decreased by 39.7%,and the activities of?-amylase and alkaline protease decreased by 13.1%and 28.5%,respectively.Through the control of nutrient conditions,the autolysis rate of B.licheniformis DL-1?spoIIQ can be reduced by 52.6%,which can improve the autolysis of bacteria to some extent.Studies have shown that knocking out the spoIIQ gene can reduce the spore formation rate of Bacillus licheniformis,but the autolysis of the recombinant bacteria needs further solution.2.Based on the principle of homologous single exchange,based on the spore-deleted recombinant B.licheniformis DL-1?spoIIQ,the pcf fragment was ligated with the Cm~r fragment by overlapping PCR and electrotransformed into B.licheniformis DL-1?spoIIQ competent cells.Obtained the recombinant B.licheniformis DL-1?spoIIQ?pcf,the recombinant strain B.licheniformis DL-1?spoIIQ and the recombinant strain B.licheniformis DL-1?spoIIQ?pcf were cultured in Luria-Bertani medium.It was found that autolysis rate of the recombinant strain B.licheniformis DL-1?spoIIQ?pcf was reduced by 11.2%compared with the recombinant B.licheniformis DL-1?spoIIQ.The combination of nutrient conditions and autolysis gene pcf knockout reduced the autolysis rate of the bacteria by 71.2%.Due to the accumulation of bacterial biomass,the recombinant fermentation bacteria B.licheniformis DL-1?spoIIQ?pcf,?-amylase and alkaline protease activity were 434 U/mL and 94 U/mL,respectively,compared with the recombinant B.licheniformis DL-1?spoIIQ.The increase was 27.5%and 17.6%,and the?-amylase activity was 10.9%higher than the original strain B.licheniformis DL-1,and the alkaline protease was 16.1%lower than the starting bacteria B.licheniformis DL-1.The fermentation activity of 5 L fermenter showed that the maximum enzyme activities of the original bacteria B.licheniformis DL-1 and the recombinant B.licheniformis DL-1?spoIIQ?pcf?-amylase were 1247 U/mL and 1488U/mL,respectively,among them,the recombinant bacteria increased the activity of?-amylase by 19.3%,which was 3.2 times and 3.4 times higher than that of shake flask fermentation.The results showed that the deletion of spoIIQ gene can reduce the secretion of Bacillus alkaline protease.On the basis of spore deficiency,the pcf gene deletion can control the autolysis rate of the bacteria,increase the fermentation biomass,and increase the fermentation yield of the target enzyme.