Optimizing Nystatin Biosynthesis Through Targeted Manipulation of nysL and nysGtf in Streptomyces noursei

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Nystatin, a polyene macrolide antifungal agent produced by Streptomyces noursei, exists as three major derivatives (nystatin A1, A3, polyfungin B) with distinct C-10 and C-35 modifications. Although the core catalytic functions of NysL and NysGtf have been established, the metabolic crosstalk between the C-10 hydroxylation and C-35 digitosylation branches, together with the in vivo substrate promiscuity of NysGtf in industrial S. noursei, remains poorly understood. In this study, we constructed gene-disruption, complementation and overexpression mutants of S. noursei 72-22-1 to characterize nysL and nysGtf. While NysL has previously been established as the C-10 hydroxylase, we characterized its role in the industrial derivative strain 72-22-1. nysL disruption abolished C-10 hydroxylation, eliminated nystatin A1/A3 and caused accumulation of 10-deoxynystatin and polyfungin B, while complementation and/or overexpression restored A1/A3 formation. nysGtf inactivation blocked C-35 glycosylation, raising nystatin A1 titer to 0.587 mg/mL and decreasing polyfungin B to 0.027 mg/mL. Its overexpression enhanced nystatin A3 production to 1.195 mg/mL. Our findings define the physiological roles of NysL and NysGtf in nystatin biosynthesis, identify polyfungin B as the key antifungal component, and provide a feasible strategy for metabolic engineering of S. noursei to generate high-activity nystatin derivatives.