Effects Of Extracellaluar Proteases And CodY Of Bacillus Licheniformis On The Bacitracin Biosynthesis

Bacitracin is an antimicrobial peptide composed of 12 amino acid residues.Due to its broad-spectrum antibacterial effects,bacitracin has been widely used as feed additives.Amino acids are the precursors for the biosynthesis of bacitracin,hence,the intake and biosynthesis of amino acids strongly affect the bacitracin biosynthesis.Protein in culture medium are degraded into oligopeptides and free amino acids by extracellaluar proteases,CodY,a global transcription factor,has an inhibiting effect on the biosynthesis of branched chain amino acids.In order to construct highly bacitracin producing strain,a series of Bacillus licheniformis DW2 mutants were constructed,effects of enhanced expression of extracellaluar proteases(Epr,AprE,BprA and Vpr)on the bacitracin biosynthesis were investigated,as well as the effect of deletion codY gene.Enhanced expression of Epr,AprE,BprA and Vpr were achieved by construction of genetically engineered strains DW2/pHY-P43epr,DW2/pHY-P43aprE,DW2/pHY-P43bprA,DW2/pHY-P43vpr,DW2aprE,DW2bprA.The extracellular proteinase activity of these mutants were determined at the end of bacitracin fermentation.Compared with the control group,the extracellular proteinase activity of DW2/pHY-P43vpr and DW2/pHY-P43epr showed no significant difference,and the extracellular proteinase activities of DW2/pHY-P43aprE,DW2/pHY-P43bprA,DW2aprE and DW2bprA increased by 101.3%,28.1%,28.6%and 8.6%,respectively.In addition,the enhancement of Epr,Vpr and AprE activity showed no effect to improve the total bacitracin yield.The total bacitracin yield of DW2/pHY-P43bprA was 20.5%higher than that of the control group.Moreover,the bacitracin A component yield of DW2/pHY-P43aprE was 52.6%higher than the control group.The results indicated that the episomal expression of AprE could significantly enhanced the yield of bacitracin A component.In order to improve the yield of branched chain amino acids,the AcodY mutant was constructed.The valine,leucine and isoleucine yield of mutant strain were 8.6-fold,2.8-fold and 2.4-fold higher than that of control group,respectively.The bacitracin yield,however,was reduced by 41.7%.By RT-qPCR analysis,the transcriptional levels of genes related to the synthesis of branched chain amino acid,ilvB,leuA and bcd were increased by 4.7-fold,4.9-fold and 5.3-fold,respectively compared with the control group.Nonetheless,the transcriptional levels of bacA,bacT and bcrB,genes related to bacitracin synthesiswere decreased by 79%,76%and 52%,respectively.Although the biosysthesis of bacitracin precursor(valine,leucine and isoleucine)was enhanced by the deletion of codY,the genes responsible for bacitracin synthesis were suppressed.