高产洛蒙真菌素洛蒙德链霉菌高通量筛选方法的建立与复合育种

背景]洛蒙德链霉菌S015能生物合成具有广谱抗菌活性的吩嗪类化合物洛蒙真菌素。目的]因S015菌株的洛蒙真菌素产量较低,将S015菌株经复合诱变育种和基因工程改造,提高洛蒙真菌素产量。方法]建立洛蒙真菌素产生菌的高通量筛选方法,对出发菌株S0 15进行常压室温等离子体(atmospheric and room temperature plasma,ARTP)技术和紫外复合诱变,筛选得到高产菌株;并在高产菌株上敲除洛蒙真菌素的前体分支酸竟争途径中的关键基因trpE1、trpE2,再过表达全局调控基因afsR。结果]利用洛蒙真菌素在紫外波长375 nm处的特征吸收峰,以及洛蒙真菌素浓度和375 nm处吸光度值的正相关关系,建立了基于24孔深孔板发酵和酶标仪快速检测的高通量筛选方法。经过6轮ARTP和紫外复合诱变及高通量筛选,从4 320株突变株中筛选得到遗传稳定的高产菌株M6,其洛蒙真菌素的产量为61.33 mg/L,是S015菌株的7.35倍;M6菌株的分支途径基因trpE1、trpE2双敲株的洛蒙真菌素产量为81.89 mg/L,是S015菌株的9.82倍;在该基因工程菌株中过表达全局调控基因afsR,产量为109.53 mg/L,是S015菌株的13.13倍。结论]建立的高通量筛选方法可以有效筛选高产洛蒙真菌素的突变株,并且操作简单快速。通过ARTP和紫外复合诱变,结合高产株M6的基因工程改造,能进一步提升洛蒙真菌素的产量。

High throughput screening and combined breeding of Streptomyces lomondensis for high production of lomofungin

Background] Lomofungin, a phenazine compound with broad-spectrum antibacterial activity, can be biosynthesized by Streptomyces lomondensis S015. Objective] Due to the low production of lomofungin in S015, this study aims to enhance its production by combined mutation breeding and genetic engineering. Methods] A high throughput screening method was established to analyze the production of lomofungin of mutants, and the lomofungin high-production strains were screened after atmospheric and room temperature plasma(ARTP) technology and ultraviolet(UV) combined mutation breeding based on the starting strain S015. Then, trp E1 and trp E2 genes which are key genes in chorismate-competing pathway were knocked out in the lomofungin high-production mutant, and the global regulatory gene afs R was overexpressed. Results] According to lomofungin’s characterized UV absorption peak at 375 nm and the positive correlation between the concentration of lomofungin and the value of A375, a high throughput screening method, which is based on 24-deep-well plate culture and microplate reader, was built successfully. After 6 rounds of ARTP and UV combined mutation breeding, strain M6, a genetically stable and high lomofungin-production strain, was screened from 4 320 mutants. Its yield of lomofungin is 61.33 mg/L, which is 7.35 times the yield of starting strain S015. After two branch pathway genes trp E1 and trp E2 were knocked out from strain M6, the lomofungin production increased to 81.89 mg/L, and then it increased to 109.53 mg/L when afs R was overexpressed, which is 13.13 times the production of S015. Conclusion] The high throughput screening method developed in this study could efficiently analyze the production of lomofungin of mutants in a simple and fast way. Combined mutation breeding of ARTP and UV as well as genetic engineering is an effective mean to obtain high-yield strains of antibiotics.