Regulation of Ansamitocin P-3 Biosynthesis by the Response Regulator CrsB in Actinosynnema pretiosum.

Ansamitocin P-3 (AP-3) is an ansamycin antibiotic produced by Actinosynnema pretiosum with high clinical and commercial value. Its biosynthesis is tightly controlled, yet the underlying transcriptional network is not fully understood. Here, we identified the two-component system CrsAB and characterized the response regulator CrsB. Deletion of crsB resulted in a 57% reduction in AP-3 yield without impairing morphological development or biomass accumulation. RNA‑Seq showed that CrsB acts as a global regulator, influencing energy metabolism, cell integrity, and biosynthetic capacity. At the fermention at 48 h, genes asm23 and asm43-47 within the biosynthetic gene cluster, which are primarily responsible for the synthesis of AHBH from UDP-glucose, were significantly downregulated upon crsB deletion. Conversely, genes of the pentose phosphate pathway, zwf, opcA and pgl, were markedly upregulated, whereas their expression levels showed no significant differences between the two strains at 72 h. These temporal transcriptional changes were confirmed by qRT‑PCR. Collectively, our data suggest that CrsB positively coordinates primary and secondary metabolism primarily during the early stage to promote AP-3 production, offering new targets for metabolic engineering.