| Bacitracin is an antimicrobial peptide of broad-spectrum antibacterial effect.The main production strain of bacitracin is Bacillus licheniformis.The more stable clathrate zinc bacitracin is widely used as in animal feed additive.However,strong odor pollution coming from the mass production of bacitracin seriously affects the physical and mental health of the residents around the factory.Branched chain carboxylic acids(BCCAs)are considered to be the main substances causing the odor.In this paper,we optimized the fermentation strain B.licheniformis DW2 to reduce the synthesis of the branched chain carboxylic acids and obtained the low odor strains.According to the BCCAs synthesis pathway,the mutant DW2△bkd R,DW2△bcd,DW2△ywa A,DW2△ybg E,DW2△ilv D,DW2△bkd AB,DW2△ptb were constructed by deletion of BKD operon transcription factor(Bkd R),leucine dehydrogenase(Bcd),two kinds of branched chain aminotransferase(Ywa A,Ybg E),dihydroxy acid dehydratase(Ilv D),alpha keto acid dehydrogenase complex E1 beta subunit(Bkd AB),phosphate butyryltransferase(Ptb)of B.licheniformis DW2.The concentration of BCCAs at fermentation end was detected by GC.The BCCAs concentration of DW2△bkd R was7.92 g/L,143.7% higher than DW2.The absence of branched chain amino transferase had no effect on the synthesis of branched chain short chain fatty acids.The BCCAs concentration of DW2△bcd,DW2△ilv D,DW2△bkd AB,DW2△ptb were 1.67,2.82,1.47,2.13 g/L,which were reduced by 48.6%,13.2%,54.5%,34.3% compared with DW2,respectively.The lack of Bcd,Bkd AB and Ptb significantly reduced the BCCAs synthesis.The total bacitracin yield in the supernatant at fermentation end was detected by HPLC,and DW2△bcd,DW2△bkd AB,DW2△ptb had the total bacitracin yield of 595.5,372.8and 631.9 U/m L and reduced by 18.1%,48.7%,13.1%.compared to DW2.The deletion of Bkd AB has a great influence on the ability of peptide synthesis,so it is not suitable to be used as the target enzyme to construct engineering bacteria.Bcd and Ptb null mutation effect bacitracin synthesis relatively small and can be used as low odor bacitracin producing engineering bacteria.Intracellular branched chain acyl coenzyme A and BCCAs could be used to synthetize branched chain fatty acid(BCFAs).Enhancing the utilization of intracellular branched chain acyl coenzyme A and BCCAs could reduce the concentration of extracellular BCCAs.Enhanced expression of beta ketoacyl ACP synthase Fab HA,Fab HB were achieved by construction of genetically engineered strains DW2/p HY-P43 fab HA and DW2/p HY-P43 fab HB.BCCAs concentration of DW2/p HY-P43 fab HA showed no significant difference with DW2,but the BCCAs concentration of DW2/p HY-P43 fab HB decreased by 28% compared with DW2.Enhanced expression of beta-keto ACP synthase Fab HB have obvious effect on the reduction of the peculiar smell and could be used as low odor bacitracin producing engineering bacteria. |
