Post-PKS Tailoring Mechanism and Molecular Dissection of a Glycosyltransferase Involved in a Second Sugar Attachment to the Disaccharide-containing Polyene

Previously, a novel polyene compound named NPP, which exhibited more than 300-fold higher solubility and 10-fold reduced hemolytic activity than nystatin, was proved to contain an aglycone identical to nystatin and harbors a unique di-sugar moiety, mycosaminyl-(a1-4)-N-acetyl-glucosamine in Pseudonocardia autotrophica KCTC9441. Although the nppDI was proved to be responsible for the first sugar mycosamine transfer in NPP biosynthesis, the gene responsible for the second sugar, N-acetyl-glucosamine transfer and its mechanism in NPP biosynthesis remained unknown. Through P. autotrophica whole genome mining, here we identified a NPP-specific glycosyltransferase (GT) gene named nppY, which was located upstream of the NPP biosynthetic gene cluster. PCR-targeted nppY disruption and its complementation proved that nppY is responsible for the second sugar transfer in NPP biosynthesis. Domain swapping and site-directed mutagenesis of nppY revealed some motifs critical for the polyene GT substrate specificity. Moreover, serial deletions and expressions of two GT genes (nppDI and nppY) and one P450 hydroxylase gene (nppL) involved in the post-PKS NPP biosynthesis revealed that NPP aglycon is sequentially modified with two sugars by nppDI and nppY, followed by the nppL-driven 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.