The Response Regulator CrsR Positively Regulates Ansamitocin P-3 Biosynthesis in Actinosynnema pretiosum

Peipei Zhang^1,2Gongli Zong^1,2Tengfei Wang²Shuangying Zhao²Ruoyi Sun²Jiafang Fu^1,2Meng Liu^3*Guangxiang Cao^1,2*

• ¹Shandong Academy of Medical Sciences (SDAMS), Jinan, China
• ²Shandong First Medical University, Jinan, China
• ³Shandong Jianzhu University, Jinan, China

Ansamitocin P-3 (AP-3), a maytansinoid antibiotic produced by Actinosynnema pretiosum, exhibits potent anticancer activity. However, its biosynthetic regulation in A. pretiosum remains largely unknown. Two-component systems (TCSs) are ubiquitous in actinomycetes and primarily regulate the biosynthesis of secondary metabolites. In this work, we identified a novel TCS, designated CrsRK, in A. pretiosum X47 through sequence analysis. Deletion of the response regulator gene crsR 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, deletion of crsR also significantly downregulated transcription of the AP-3 biosynthetic genes, including asm7, asm10-15, asm21, asm23-24, asmAB, and asm43-47, which encode enzymes for multiple steps in AP-3 biosynthesis. Electrophoretic mobility shift assays confirmed direct binding of CrsR to promoters of asm21, asm43-44, and asm45-47 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 as well as TCS functions in A. pretiosum.