Molecular Biosynthetic Process Development for Polyketide-family Cryptic Polyene via Genome Reconstr

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Summary

Polyene antibiotics such as nystatin are a large family of very valuable antifungal polyketide compounds typically produced by soil actinomycetes. Previously, using a polyene cytochrome P450 hydroxylase-specific genome screening strategy, Pseudonocardia autotrophica KCTC9441 was determined to contain an approximately 125.7 kb region of contiguous DNA with a total of 23 open reading frames, which are involved in the biosynthesis and regulation of a structurally unique polyene natural product named NPP. Here, we report the complete structure of NPP, which contains an aglycone identical to nystatin and harbors a unique di-sugar moiety, mycosaminyl- (1-4)-N-acetyl-glucosamine. A mutant generated by inactivation of a sole glycosyltransferase gene (nppDI) within the npp gene cluster can be complemented in trans either by nppDI-encoded protein, or by its nystatin counterpart, NysDI, suggesting that the two sugars might be attached by two different glycosyltransferases. Compared with nystatin (which bears a single sugar moiety), the di-sugar containing NPP exhibits approximately 300-fold higher water solubility and 10-fold reduced hemolytic activity, while retaining about 50% antifungal activity against Candida albicans. These characteristics reveal NPP as a promising candidate for further development into a pharmacokinetically improved, less-cytotoxic polyene antifungal antibiotic.

Remarkable

we report the complete NPP structure bearing an aglycone identical to nystatin with a unique di-sugar moiety, mycosamine (1-4)-N-acetyl-glucosamine. Another new molecule was generated following in-frame deletion of the NPP PKS enolyreductase (ER) domain in module 5, which converted the conjugated tetraene NPP to its corresponding heptaene analogue. In addition, we inactivated nppDI, the sole glycosyltransferase encoding gene within the npp gene cluster, and found that this mutant can be functionally complemented in trans either by nppDI or its nystatin counterpart, nysDI. This result indicates that two sugars might be attached to the aglycone by two independent glycosyltransferases. Remarkably, when the disaccharide containing NPP was compared with a mycosamine-containing nystatin, the former exhibited approximately 300-fold higher water solubility and 10-fold reduced hemolytic activity, while retaining about 50% of its antifungal activity. These results, suggests that NPP is a promising lead compound for further development into a pharmacokinetically improved, and less cytotoxic polyene antifungal agent.

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