Abstract
A novel polyene compound NPP identified in a rare actinomycetes, Pseudonocardia autotrophica KCTC9441, was shown to contain an aglycone identical to nystatin but to harbor a unique di-sugar moiety, mycosaminyl-(α1-4)-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 tailoring mechanism remained unknown. Here, we identified a NPP-specific second sugar extending GT gene named nppY, located at the edge of the NPP biosynthetic gene cluster. 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 several 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.
Highlights
Invasive fungal infections are becoming a serious problem in human health
The expression of the nppDI gene in the double knock-out mutant ESK621 generated 10-deoxyNPP. All these results indicate that NPP-specific glycosyltransferase (NppY) catalyzes the transfer of second sugar, N-acetylglucosamine first, and the NppL-driven regio-specific hydroxylation is the final post-polyketide synthases (PKSs) modification step in NPP biosynthesis (Fig 5)
The proposed post-PKS tailoring steps of NPP biosynthesis was re-confirmed by a series of complementation in a triple knockout mutant (ΔnppDIΔnppYΔnppL) (Table 2 and S4 Fig). Polyene macrolides such as amphotericin B and nystatin are very effective broad spectrum antifungal antibiotic that is active against human immunodeficiency virus and prion diseases
Summary
Invasive fungal infections are becoming a serious problem in human health. The mortality rates for fungal infections with three most common species of pathogens are Candida albicans of 20%-40%, Aspergillus fumigatus of 50%-90%, and Cryptococcus neoformans of 20%-70% [1,2,3]. These diseases usually occur in the setting of immunocompromised patients as a result of HIV infection, anticancer therapy, or immunosuppressive therapy after organ transplantation [1]. Azoles inhibit the fungal cytochrome P450 (CYP)-dependent enzyme involved in ergosterol biosynthesis, the emergence of Candida resistance to azoles has become an increasing.
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