Genome Mining and Post-PKS Tailoring Mechanism of a Disaccharide-containing Polyene NPP in Pseudonocardia autotrophica

A novel polyene compound NPP identified in a rare actinomycestes, Pseudonocardia autotrophica KCTC9441, was proved to contain an aglycone identical to nystatin but to harbor a unique di-sugar moiety, mycosaminyl-(a1-4)-N-acetyl-glucosamine, which led to higher solubility and reduced hemolytic activity. Although the nppDI was proved to be responsible for the transfer of first polyene sugar, mycosamine in NPP biosynthesis, the gene responsible for the second sugar extending glycosyltransferase (GT) as well as NPP post-PKS mechanism remained unknown. Through P. autotrophica genome mining, here we identified a novel NPP-specific second sugar extending GT gene named nppY. Targeted nppY gene deletion and its complementation proved that nppY is indeed responsible for the transfer of second sugar, N-acetyl-glucosamine in NPP biosynthesis. Site-directed mutagenesis on nppY also revealed some amino acid residues critical for NppY GT function. Moreover, a combination of deletions and complementations of two GT genes (nppDI and nppY) and one P450 hydroxylase gene (nppL) involved in the NPP post-PKS biosynthesis revealed that NPP aglycone is sequentially modified by the two different GTs encoded by nppDI and nppY, respectively, followed by the nppL-driven regio-specific hydroxylation at the NPP C10 position. These results set the stage for the biotechnological application of sugar diversification for the biosynthesis of novel polyene compounds in actinomycetes. This work is supported by grants PJ009522 from RDA.