Increasing The Bacitracin Yield By Modifying The Synthesis And Transport Pathway Of The Branch Chain Amino Acids

Bacitracin is a type of antimicrobial peptide which is constituted by 12 amino acids residues.Previous study has demonstrated bacitracin is a broad-spectrum antibiotic,which is biosynthesized through non-ribosomal peptide synthetases by Bacillus subtilis and Bacillus licheniformis.Bacitracin is typically used for adding feed because it can repress gram positive bacteria and partial gram negative bacteria.Precursor amino acids supply might be the limit factor during bacitracin biosynthesis.Previous studies have found that increasing Ile and Leu supplies can improve the production of bacitracin,and the effect of Ile supply is more remarkable.In this study,we increased the supply of branch chain amino acids(BCAA)by strengthening the biosynthesis pathway of BCAA,weakening the branch pathway of BCAA and modifying the transporter system of BCAA.We found that the integration overexpression of gene ilvB and deletion of transporter YhdG can increase the production of bacitracin.Finally,high bacitracin production strain was achieved by integration overexpression of accetohydroxy acid synthase gene ilvB combined with deletion of transporter YhdG.According to the information in KEGG and NCBI databases,there are four key enzymes relevant to the biosynthesis of Ile in Escherichia coli and Corynebacterium glutamicum,which are aspartokinase,homoserine kinase,threonine deaminase,accetohydroxy acid synthase,and accetohydroxy acid synthase is the common key enzyme of BCAA.The synthesis of Met,the branch pathway of BCAA is limited by homoserine transsucciyllase which is encoded by gene met A.Meanwhile,previous study have demonstrated BrnFE,BrnQ and YhdG(BcaP)are BCAA transporters,and YhdG is the most efficient transporter in B.subtilis.In this study,we constructed plasmid overexpression strains for overexpressing the five key genes relevant to the biosynthesis of BCAA,gene deletion strains for weakening the Met biosynthesis pathway and modifying the transporter system.Fermentation results showed that the ilvB overexpression strain's bacitracin yield increased 11.01%compared to the original strains B.licheniformis DW2.Then,we integrated the gene ilvB to the B.licheniformis DW2 genome to eliminate the negative effect of plasmid and the bacitracin yield increased 14.06%compared to DW2.Meanwhile,the transporter YhdG deletion strain's bacitracin yield reached 917.35 U/mL,which increased 11.21%compared to B.licheniformis DW2.Finally we knockouted the transporter gene yhdG of the ilvB integration overexpression strain and the bacitracin yield reached 995.25 U/mL,which was 21.11%higher than B.licheniformis DW2.To study the effect of ilvB integration overexpression on the metabolism in B.licheniformis DW2,we detected the intracellular and extracellular content of relative amino acids.The results showed that the intracellular BCAA increase compared to B.licheniformis DW2 during the synthesis of bacitracin,but the intracellular Lys and Met were almost the same as B.licheniformis DW2.We also researched the effect of the expression level of YhdG on bacitracin by overexpressing and deleting gene yhdG separately.We demonstrated that higher the expression level of YhdG,lower the bacitracin yield.Then,we detected the the intracellular and extracellular BCAA content during fermentation at yhdG deletion strain.The results supported the fact that the transporter YhdG act as an exporter for BCAA in B.licheniformis DW2.What's more,we also demonstrated YhdG is a non-specific transporter for BCAA in B.licheniformis DW2 by detecting the other 17 category amino acids.Our work constucted bacitracin high-production strain DW2::ilvB AyhdG,the bacitracin titer of which reached 1060.03U/mL after optimizing the content of soybean from 10%to 8%.Meanwhile,we also reduced the cost of fermentation,which laid foundation for the industrial production of bacitracin.