The Fermentation Optimizations For Bacitracin Biosynthesis By Bacillus Licheniformis

Bacitracin belongs to the non ribosomal peptide synthetase（NRPS）catalyticsynthesis of secondary metabolites with a 12-cyclic peptide structure.With the investigations on the relationship among cell growth,overflow metabolites and bacitracin synthesis,some regulation strategys was conducted,and an improved efficiency of the bacitracin synthesis was obtained.The results are significant for the guides on bacitracin industrial fermentation production,and the results are as follows:1.The single-factor experiments to determine the significant effects on bacitracin synthesis by metal ions,and the optimal results is ensured by orthogonal experiment:sodium molybdate 10 mg/L,5 mg/L ferric sulfate,manganese sulfate 20 mg/L,copper sulfate,0.5 mg/L.In 10 L fermentor process,the bacitracin production and synthetic rate of optimization addition between 12²8 h was increased by 20%⁵1%and 165%,respectively,compared to the control.Meanwhile,during the course of mid-late fermentation period,the concentration of acetic acid,propionic acid and isobutyric acid was only 46%,54%and 49%of the control,respectively,indacting that VFA shortage might affecte the bacitracin synthesis efficiency.Then,10 g/L glucose was added at 16 h,the cell autolysis time delay 8 h and the maximum cells numberwas increased by 18.1%than the control.The bacitracin production was 1004 U/mL that was 7.0%and 13.6%higher than control and that without additions of metal ions and glucose,respectively.2.Batch culture with H₂O₂ addition with 1 mL/min from 13 h to 29 h was conducted,the biomass in 40 h reached a maximum（592 ug/mL）,which was 5.1%higher than the control,and cell autolysis disappeared.Meanwhile,the contents of acetic acid,propionic acid,succinic acid and 2,3-butanediol were generally lower than control,contents of pyruvic acid and alpha ketoglutaric acid were higher than control.Results indicated that EMP carbin flux was higher and the carbon spilling effect was lower than control during the course of former-fermentation period,respectively.However,carbon flux in the oxidation branch of TCA cycle was higher than the control during the mid-later periods of the fermentation,which promoting the efficiency of the bacitracin synthesis.Thel bacitracin production was 1033 U/mL,9.6%higher than that of control.3.Othogonal experiments was done to optimize the content of CaCO₃,acetate and starch,and the results was acetate 1%,starch 2.4%and CaCO₃ 0.57%.However,Batch culture results showed bacitracin production was only 580 U/mL,61.8%of the control.4.Batch culture of base fermentation medium with acetate addition of 20 g at 30 h （Fermentation 1）was conducted,the biomass was nearly increased by 30%,and bacitracin production was 730 U/mL.Fermentation 1 with acetic acid addition to control pH（7.5-7.9）（Fermentation 2）was conducted,and the bacitracin production was783 U/mL at 28 h,7.3%higher than that of fermentation 2.