Distinct Roles of Coexisting PAS-LuxR Regulators in Polyene Biosynthesis in Streptomyces ahygroscopicus: Insights from Gene Expression, Metabolomics, and In Silico Analysis

In Streptomyces ahygroscopicus, the homologous PAS-LuxR regulators NysRIV and TtmRIV govern nystatin and tetramycin biosynthesis, respectively. To confirm their functional relationship within the same strain, gene deletion, complementation, domain swapping, electrophoretic mobility shift assays, transcriptomics, qRT-PCR, and CoA-derived metabolite measurements were combined to characterize their regulatory roles. Deletion of nysRIV abolished nystatin production, and native nysRIV complementation restored nystatin partially (about 17% of the parental level) in the ΔttmS1ΔnysRIV background and not at all in ΔnysRIV, indicating that NysRIV is required for full nystatin production. TtmRIV and the two chimeric regulators failed to substitute for NysRIV, indicating limited regulatory interchangeability. Their DNA-binding preference regulators largely followed the origin of their HTH domains, supporting an important contribution of the C-terminal HTH region to differential promoter recognition in vitro. Their effects on polyene production also depended on genetic background. Transcriptomics of the nysRIV deletion mutant revealed changes in lipid, polyketide, pyruvate, and CoA-related metabolism, accompanied by altered acc-gene expression and intracellular CoA-derived metabolite pools. Together, these results indicate distinct but partially overlapping roles of NysRIV and TtmRIV in polyene biosynthesis and highlight the influence of regulator identity and host genetic background on PAS-LuxR regulatory outcomes.