Systematicly Engineering Of Branch Chain Amino Acids Supply For The Enhancement Of Bacitracin Yield In Bacillus Licheniformis DW2

Bacitracin is a type of cyclic peptide antibiotic which contains 11 kinds of amino acids.Bacitracin is widely used in feed industry because it owns the features of strong antibacterial activity,high safety and good stability.Previous studies have shown that the availability of precursor amino acids supply plays a key role during the biosynthesis of bacitracin,As important components of bacitracin,the ability of bacteria to synthesize branched-chain amino acids is beneficial to bacitracin production.In this study,the biosynthesis pathway of branch chain amino acids(BCAAs),BCAAs transporters and transcriptional regulators of Bacillus licheniformis DW2 were systematically modified,thus the intracellular BCAAs concentrations were greatly increased,and a novel strategy was provided for enhancing bacitracin production.In this study,Bacillus licheniformis DW2 was used as the original strain,and a series of genes in BCAAs biosynthesis pathway were episomal over-expressed,our results showed that the effects of episomal overexpression of acetolactate synthase gene ilv B,2-isopropylmalate synthase leu A and BCAAs aminotransferase gene ybg E were better than the control stain DW2/p HY300.To eliminate the adverse effects of free plasmids on the growth of bacteria,leu A,ilv B and ybg E were enhanced by gene integration or strong promoter replacement,and the bacitracin yields were increased by 11.67%,10.5% and 8.62% compared with DW2,respectively.Then,the intracellular and extracellular BCAAs concentrations of engineered strains during the rapid synthesis phase of bacitracin were determined,results showed that compared with DW2,the intracellular L-Leu concentration of DW2::leu A was increased by183.20%,while the concentrations of intracellular L-Ile and L-Val showed no significant differences;the intracellular BCAAs concentrations were significant higher in DW2-Pbac Ailv B and DW2-Pbac A-ybg E than those of DW2,the concentrations of intracellular L-Leu,LIle and L-Val were increased by 138.96%,28.07%,142.35% and 89.40%,25.81%,102.66%respectively.BraB,BrnQ and YvbW were annotated as the BCAA transporters in DW2 which have not been reported yet.In this study,the mutant strains with different expression levels of BraB,BrnQ and YvbW were constructed,fermentation results implied that the bacitracin yields of DW2-Pbac A-bra B,DW2-Pbac A-brn Q and DW2-Pbac A-yvb W were increased by 10.26%,7.10% and 5.39% respectively than those of DW2.Then,the intracellular and extracellular BCAAs concentrations of bra B,brn Q and yvb W over-expressed strains during the rapid synthesis phase of bacitracin were determined,results showed that compared with DW2,the intracellular BCAAs concentrations of three mutant strains were significantly increased,among which the intracellular BCAAs concentrations of DW2-Pbac A-brn Q showed the maximal promotion.Lrp is one type of transcriptional factors that widely presents in bacteria which regulates various physiological processes,including amino acid metabolism and antibiotic synthesis.In this study,it’s found that the bacitracin production increased by 12.60% after the deletion of lrp in DW2.Then,the intracellular and extracellular amino acid concentrations of DW2△lrp and DW2 during the rapid synthesis phase of bacitracin were determined,results showed that the concentrations of intracellular L-Ile,L-Val and L-Leu were increased by 148.05%,20.03%and 144.94% in DW2△lrp than those of DW2,while the intracellular concentrations of other precursor amino acids showed no significant differences between DW2 and DW2△lrp.In addition,the transcriptional levels of genes involved in the biosynthesis and transport of BCAAs in DW2Δlrp and DW2 during the early phase of bacitracin synthesis were determined,results showed that the transcriptional level of brn Q was enhanced by 2.82 fold in DW2△lrp than that of DW2,while the transcription levels of other genes showed no significant differences.Our results implied that the deletion of lrp increases the expression level of brn Q,thus allowed more BCAAs to be transported into the cell,thus promoting the synthesis of bacitracin.Finally,the genes with better effects above were deleted or over-expressed in DW2,and the bacitracin high-production strain DW08 were constructed.The bacitracin of DW08 was increased by 40.93% compared with DW2,and the intracellular BCAAs concentrations were also significantly increased.Overall,our results demonstrate that enhancing the supply of intracellular precursor amino acids in B.licheniformis was an effective strategy to increase the production of bacitracin.