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

Abstract:

[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, trpE1 and trpE2 genes which are key genes in chorismate-competing pathway were knocked out in the lomofungin high-production mutant, and the global regulatory gene afsR 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 trpE1 and trpE2 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 afsR 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.