Enhanced Supply Of Aspartic Acid For Promoting The Production Of Bacitracin In Bacillus Licheniformis DW2

Bacitracin is a polypeptide antibiotic which is formed by 11 amino acids.Bacitracin can repress gram-positive bacteria,and can also inhibit partial gram-negative bacteria.Bacitracin owns the features of strong antibacterial activity,high safety and good stability,thus,it has the broad application prospects in many fields,such as poultry and animal husbandry.At present,the production of bacitracin mainly depends on microbial fermentation.Some studies have proven that the insufficient supply of precursor amino acids might play a key role on account of restricting of the production of bacitracin,and it is beneficial to break-through the striction of bacitracin under natural conditions by strengthening the supply of precursor amino acids to achieve the purpose of high-level production of bacitracin.In this study,Bacillus licheniformis DW2,a strain with high-level producton of bacitracin,was used as an original strain.The strain was stored by our group.Firstly,different concentrations of aspartic acid were added at different times(12 h,24 h,36 h)during bacitracin fermentation,and our results confirmed that exogenous addition of Asp benefited bacitracin production of Bacillus licheniformis DW2.The bacitracin fermentation results showed that when Asp was added at a final concentration of 60 mg/L at 24 h,the yield was the highest,and it is 19.2%higher than that of control(0 mg/L).After that,a series of genes in Asp metabolic pathways(including plasmid overexpression and replacing the promoter by the proven strong promoter P_{bac A})were strengthened via plasmid overexpression or promoter replacement.Based on our results,several genes have shown the significant effects on the improvement of bacitracin yield.Among them,the bacitracin yield of aspartate aminotransferase Asp B over-expressing strain DW2/p HY-asp B was increased by 5.5%compared to the strain DW2/p HY-300,while the bacitracin yield of promoter replacement strain DW2-P_{bac A}(aps B)was increased by 2.3%.Pyruvate carboxylase Pyc A was strengthened by via promoter replacement,and yield of the strain reached 844.5 U/m L,increased by 8.8%compared to DW2.Meanwhile,the bacitracin yields of aspartate aminolyase gene asp A and malate dehydrogenase gene mal S deletion strains were 891.3 U/m L and 833.5 U/m L,which were 14.8%and 7.3%higher than that of DW2,respectively.Furthermore,there are variety of amino acid transporters own the capability for Asp transportation in bacteria,based on the previous research,among which Yve A is the less reported one,meanwhile,the direction of transportation is not the same in many bacteria.In this study,yve A deletion strain and overexpression strain were both constructed,named as DW2?yve A and DW2/p HY-yve A,respectively.It was found that the yield of bacitracin on DW2?yve A strain was 887.3 U/m L,which was 14.3%higher than DW2,and the yield of Yve A overexpression strain DW2/p HY-yve A reached 642.4U/m L,decreased by 4.2%compared to DW2/p HY-300 strain.In addition,the intracellular concentration of Lys in DW2?yve A increased by 4.8%,while it was decreased by 14.1%in Yve A overexpression strain DW2/p HY-yve A compared to DW2/p HY-300,which were consistent with the trend of bacitracin yields.In the results,the intracellular level of Asp in DW2?yve A increased by 4.8%with DW2,this might due to that Asp has been rapidly consumed by other metabolic pathways,thus,it is difficult to accumulate during bacitracin fermentation by genetic modification on only a single gene.This experiment also preliminarily confirmed that aspartate transporter Yve A in Bacillus licheniformis DW2 acts as an exporter.At last,the genes with efficiency results,which have been confirmed in the previous results,were overlaid in DW2,and the highest bacitracin yield was attained by the final strain ASP4.The bacitracin yield of ASP4 reached 932.7 U/m L,increased by 20.1%compared to the original strain DW2,and the intracellular Asp concentrations were increased by 137.1%.Taken together,our reuslts indicate that enhancing precursor Asp supply was an effective strategy to increase bacitracin production in Bacillus licheniformis.