Improvement of rimocidin production in Streptomyces rimosus M527 by reporter-guided mutation selection.

In this study, we employed a reporter-guided mutation selection (RGMS) strategy to improve the rimocidin production of Streptomyces rimosus M527, which is based on a single-reporter plasmid pAN and atmospheric and room temperature plasma (ARTP). In plasmid pAN, PrimA, a native promoter of the loading module of rimocidin biosynthesis (RimA) was chosen as target, and the kanamycin resistance gene (neo) under the control of PrimA was chosen as reporter gene. The integrative plasmid pAN was introduced into the chromosome of S. rimosus M527 by conjugation to yield the initial strain S. rimosus M527-pAN. Subsequently, mutants of M527-pAN were generated by ARTP. Seventy-nine mutants were obtained in total, of which sixty-seven mutants showed higher level of kanamycin resistance (Kanr) than that of the initial strain M527-pAN. The majority of mutants exhibited a slight increase in rimocidin production compared to M527-pAN. Notably, three mutants, M527-pAN-S34, S38, and S52, which exhibited highest kanamycin resistance among all Kanr mutants, showed 34%, 52% and 45% increase in rimocidin production compared to M527-pAN, respectively. Quantitative RT-PCR analysis revealed that the transcriptional levels of neo and rim genes were increased in mutants M527-pAN-S34, S38, and S52 compared to M527-pAN. These results confirmed that the RGMS approach was successful in improving the rimocidin production in S. rimosus M527.