Study on the regulatory mechanism of NsdAsr on rimocidin biosynthesis in Streptomyces rimosus M527.

Background: We previously identified a regulator NsdA_sr, which negatively regulated rimocidin biosynthesis in Streptomyces rimosus M527. However, the exact regulatory mechanism of NsdA_sr on rimocidin production remains unknown.

Results: In this study, firstly, transcriptomic data demonstrated that the differentially expressed genes resulting from the over-expression of nsdA_sr were primarily associated with several key metabolic pathways, including glycolysis, oxidative phosphorylation, and ribosome-related genes, all of which were downregulated. This directly impacted the concentrations of CoA and NADH, as confirmed by concentration measurement assays. Subsequently, the results of the ChIP-seq experiments revealed that NsdA_sr directly binds to 49 target genes. Notably, these include RS18275 and RS18290 (both involved in fatty acid degradation) as well as rpoB (related to DNA transcription). The validity of the ChIP-seq assay for these three genes was further supported by in vitro electrophoretic mobility shift assays. Regarding RS18275 and RS18290, the results revealed that the binding of NsdA_sr to these elements led to the downregulation of gene expression. This, in turn, resulted in a decrease in the levels of butyryl-CoA and malonyl-CoA, which are known precursors for rimocidin biosynthesis. Consequently, this negatively impacted on the biosynthesis of rimocidin. In the case of rpoB, the results indicated that NsdA_sr binding led to a downregulation of overall protein levels. This was determined by enzymatic activity of report gene GUS and Western blot assay. Consequently, this resulted in a decrease in rimocidin yield.

Conclusion: This study reveals NsdA_sr's dual role in limiting rimocidin production by suppressing metabolic precursors and modulating protein expression. Integrated transcriptomic and ChIP-seq analyses provide critical insights into its regulatory mechanisms.