Breeding And Fermentation Process Optimization Of Polymyxin B By Paenibacillus Polymyxa

Polymyxin B is a class of alkaline polypeptide antibiotics produced by Paenibacillus polymyxa.Polymyxin B consists of four main components,including B1,B2,B3 and B1-I.It has a significant effect on gram-negative bacteria.Clinically,it is mainly used to treat infection caused by drug-resistant bacteria.The production level of Polymyxin B is low in our country and the quality control of multi-component drug is difficult,so the Polymyxin B API mainly depends on imports now.In order to solve this problem,this paper focus on the breeding of polymyxin B-producing bacteria and optimization of fermentation process to improve the yield of polymyxin B and meet the requirements of the API,providing a foundation for the further industrial production.Firstly,P.polymyxa SIPI PB-1(837±50 mg/L)was used as the starter strain.Two screening methods,the inhibition zone screening and self-metabolite tolerance,were established.After serval rounds of natural isolation and mutagenesis breeding,the mutant strain SIPI PB-N14 was obtained.The yield of polymyxin B was 1297±60 mg/L,which was 55% higher than that of the original strain.The whole genome of the highyield strains was sequenced,and the biosynthetic gene cluster of polymyxin B was analyzed,which could provide reference for the further genetic modification of the strains.Secondly,SIPI PB-N14 fermentation medium and culture conditions were optimized.By exploring the fermentation medium 1,the optimal medium composition was determined as: glucose 15 g/L,wheat flour 80 g/L,hot cracked soybean powder 5 g/L,fish meal peptone 5 g/L,sodium glutamate 5 g/L and sodium chloride 3 g/L.The culture conditions were further optimized to obtain the seed culture time 20-24 h,the culture temperature 30℃,the pH before sterilization 8.5.After optimization,the yield of polymyxin B in flask culture reached 1362±70 mg/L.Then,the technology of polymyxin B on 5 L fermenter was carried out by feeding glucose and the p H control strategy in fermentation process.The result showed that the p H controlled at 6.0 was beneficial to the synthesis of polymyxin B.The fermentation production reached 1681±25mg/L,and the proportions of B1,B2,B3 and B1-I were75.2%,20.4%,1.3% and 3.0%,respectively,which met the proportion requirements of the API.Finally,the separation of polymyxin B was studied.D-152 cation exchange resin was used to adsorb the polymyxin B in the fermentation broth and eluted with 0.2 mol/L H2SO4 solution.Most of the impurities in the fermentation broth could be removed,and the purity of polymyxin B was 78.5% with recovery yield 69%.The proportions of components B1,B2,B3 and B1-I were 75.0%,19.7%,1.4% and 3.9%,which met the proportion requirements of each component.