Rimocidin

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 a target, and the kanamycin resistance gene (neo) under the control of PrimA was chosen as the 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. 79 mutants were obtained in total, of which 67 mutants showed a 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 with M527-pAN. Notably, 3 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 with 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 with M527-pAN. These results confirmed that the RGMS approach was successful in improving the rimocidin production in S. rimosus M527.

The polyene macrolide rimocidin, produced by Streptomyces rimosus M527, is highly effective against a broad range of fungal plant pathogens, but at low yields. Elicitation is an effective method of stimulating the yield of bioactive secondary metabolites. In this study, the biomass and filtrate of a culture broth of Escherichia coli JM109, Bacillus subtilis WB600, Saccharomyces cerevisiae, and Fusarium oxysporum f. sp. cucumerinum were employed as elicitors to promote rimocidin production in S. rimosus M527. Adding culture broth and biomass of S. cerevisiae (A3) and F. oxysporum f. sp. cucumerinum (B4) resulted in an increase of rimocidin production by 51.2% and 68.3% respectively compared with the production under normal conditions in 5-l fermentor. In addition, quantitative RT-PCR analysis revealed that the transcriptions of ten genes (rimA to rimK) located in the gene cluster involved in rimocidin biosynthesis in A3 or B4 elicitation experimental group were all higher than those of a control group. Using a β-glucuronidase (GUS) reporter system, GUS enzyme activity assay, and Western blot analysis, we discovered that elicitation of A3 or B4 increased protein synthesis in S. rimosus M527. These results demonstrate that the addition of elicitors is a useful approach to improve rimocidin production.Key Points · An effective strategy for enhancing rimocidin production in S. rimosus M527 is demonstrated. · Overproduction of rimocidin is a result of higher expressed structural genes followed by an increase in protein synthesis.

Precursor engineering is an effective strategy for the overproduction of secondary metabolites. The polyene macrolide rimocidin, which is produced by Streptomyces rimosus M527, exhibits a potent activity against a broad range of phytopathogenic fungi. It has been predicted that malonyl-CoA is used as extender units for rimocidin biosynthesis. Based on a systematic analysis of three sets of time-series transcriptome microarray data of S. rimosus M527 fermented in different conditions, the differentially expressed accsr gene that encodes acetyl-CoA carboxylase (ACC) was found. To understand how the formation of rimocidin is being influenced by the expression of the accsr gene and by the concentration of malonyl-CoA, the accsr gene was cloned and over-expressed in the wild-type strain S. rimosus M527 in this study. The recombinant strain S. rimosus M527-ACC harboring the over-expressed accsr gene exhibited better performances based on the enzymatic activity of ACC, intracellular malonyl-CoA concentrations, and rimocidin production compared to S. rimosus M527 throughout the fermentation process. The enzymatic activity of ACC and intracellular concentration of malonyl-CoA of S. rimosus M527-ACC were 1.0- and 1.5-fold higher than those of S. rimosus M527, respectively. Finally, the yield of rimocidin produced by S. rimosus M527-ACC reached 320.7 mg/L, which was 34.0% higher than that of S. rimosus M527. These results confirmed that malonyl-CoA is an important precursor for rimocidin biosynthesis and suggested that an adequate supply of malonyl-CoA caused by accsr gene over-expression led to the improvement in rimocidin production.