The response regulator CrsR positively regulates ansamitocin P-3 biosynthesis in Actinosynnema pretiosum

Ansamitocin P-3 (AP-3), a maytansinoid antibiotic produced by <i>Actinosynnema pretiosum</i>, exhibits potent anticancer activity. However, its biosynthetic regulation in <i>A. pretiosum</i> remains largely unknown. Two-component systems (TCSs) are ubiquitous in <i>actinomycetes</i> and primarily regulate the biosynthesis of secondary metabolites. In this study, we identified a novel TCS, designated CrsRK, in <i>A. pretiosum</i> X47 through sequence analysis. Deletion of the response regulator gene <i>crsR</i> drastically decreased AP-3 production. RNA-seq revealed CrsR's global regulatory role, significantly altering transcription of primary metabolic genes, especially those in purine metabolism. Crucially, the deletion of <i>crsR</i> also significantly downregulated transcription of the AP-3 biosynthetic genes, including <i>asm7</i>, <i>asm10-15</i>, <i>asm21</i>, <i>asm23-24</i>, <i>asmAB</i>, and <i>asm43-47</i>, which encode enzymes for multiple steps in AP-3 biosynthesis. Electrophoretic mobility shift assays confirmed direct binding of CrsR to promoters of <i>asm21</i>, <i>asm43-44</i>, and <i>asm45-47</i> operons, indicating direct transcriptional control. Our results demonstrate that CrsR positively regulates AP-3 biosynthesis by directly and indirectly controlling transcription within the AP-3 biosynthetic gene cluster. In conclusion, this study elucidates the critical role of CrsR in AP-3 biosynthesis and expands our understanding of AP-3 regulatory mechanisms and TCS functions in <i>A. pretiosum.</i>