An NADH/NAD-favored aldo-keto reductase facilitates avilamycin A biosynthesis by primarily catalyzing oxidation of avilamycin C.

Avilamycins, which possess potent inhibitory activity against Gram-positive bacteria, are a group of oligosaccharide antibiotics produced by . Among these structurally related oligosaccharide antibiotics, avilamycin serves as the main bioactive component in veterinary drugs and animal feed additives, which differs from avilamycin only in the redox state of the two-carbon branched-chain of the terminal octose moiety. However, the mechanisms underlying assembly and modification of the oligosaccharide chain to diversify individual avilamycins remain poorly understood. Here, we report that AviZ1, an aldo-keto reductase in the avilamycin pathway, can catalyze the redox conversion between avilamycins and . Remarkably, the ratio of these two components produced by AviZ1 depends on the utilization of specific redox cofactors, namely NADH/NAD or NADPH/NADP. These findings are inspired by gene disruption and complementation experiments and are further supported by enzymatic activity assays, kinetic analyses, and cofactor affinity studies on AviZ1-catalyzed redox reactions. Additionally, the results from sequence analysis, structure prediction, and site-directed mutagenesis of AviZ1 validate it as an NADH/NAD-favored aldo-keto reductase that primarily oxidizes avilamycin to form avilamycin by utilizing abundant NAD . Building upon the biological function and catalytic activity of AviZ1, overexpressing AviZ1 in is thus effective to improve the yield and proportion of avilamycin in the fermentation profile of avilamycins. This study represents, to our knowledge, the first characterization of biochemical reactions involved in avilamycin biosynthesis and contributes to the construction of high-performance strains with industrial value.IMPORTANCEAvilamycins are a group of oligosaccharide antibiotics produced by , which can be used as veterinary drugs and animal feed additives. Avilamycin is the most bioactive component, differing from avilamycin only in the redox state of the two-carbon branched-chain of the terminal octose moiety. Currently, the biosynthetic pathway of avilamycins is not clear. Here, we report that AviZ1, an aldo-keto reductase in the avilamycin pathway, can catalyze the redox conversion between avilamycins and . More importantly, AviZ1 exhibits a unique NADH/NAD preference, allowing it to efficiently catalyze the oxidation of avilamycin to form avilamycin using abundant NAD in cells. Thus, overexpressing AviZ1 in is effective to improve the yield and proportion of avilamycin in the fermentation profile of avilamycins. This study serves as an enzymological guide for rational strain design, and the resulting high-performance strains have significant industrial value.