Breeding of a higher producer enables identification of a negative transcription factor controlling the biosynthesis of xiamenmycin in Streptomyces xiamenensis

Xiamenmycin, a prenylated benzopyran compound produced by Streptomyces xiamenensis 318, exhibits promising anti-fibrotic activity but suffers from low productivity, limiting its pharmaceutical development. In this study, we obtained a mutant strain N-8 producing 226.3 mg/L xiamenmycin B (28-fold increase relative to the parent strain MT-XN) via N -methyl- N ′-nitro- N -nitrosoguanidine mutagenesis. Further iterative ARTP treatments yielded mutant strain A5-8 with a titer of 429.4 mg/L. This screening was achieved using a fused antibiotic marker ( neo ) downstream of the xiamenmycin biosynthetic genes, which enabled kanamycin resistance-based selection. Furthermore, intracellular precursors of xiamenmycin were increased, as indicated by transcriptome analysis to be due to upregulation of biosynthetic pathway genes. Genome re-sequencing revealed deletions of ximA and a 138 bp upstream region of ximB promoter in strain N-8. In situ complementation of the 138 bp region significantly reduced xiamenmycin B production. Mechanistically, we identified a TetR-family transcription factor, XimN1, that specifically binds to the upstream of the ximB promoter and exerts a negative regulatory effect. Deletion of this region alleviated XimN1-mediated repression, upregulates ximBCDE transcription, and enhances xiamenmycin B production. The upstream region also contains the binding sites for AdpA. EMSA analysis further revealed that both proteins bind within the 138 bp region. In summary, we successfully constructed high-yield xiamenmycin mutants and elucidated a novel trans -acting transcription factor, XimN1 control xiamenmycin biosynthesis. This study presents practical strategies for improving the production efficiency of metabolites and offers novel insights into the transcriptional regulatory underlying secondary metabolism in Streptomyces .