Nystatin Biosynthesis and Transport: nysH and nysG Genes Encoding a Putative ABC Transporter System in Streptomyces noursei ATCC 11455 Are Required for Efficient Conversion of 10-Deoxynystatin to Nystatin

Abstract

The genes nysH and nysG, encoding putative ABC-type transporter proteins, are located at the flank of the nystatin biosynthetic gene cluster in Streptomyces noursei ATCC 11455. To assess the possible roles of these genes in nystatin biosynthesis, they were inactivated by gene replacements leading to in-frame deletions. Metabolite profile analysis of the nysH and nysG deletion mutants revealed that both of them synthesized nystatin at a reduced level and produced considerable amounts of a putative nystatin analogue. Liquid chromatography-mass spectrometry and nuclear magnetic resonance structural analyses of the latter metabolite confirmed its identity as 10-deoxynystatin, a nystatin precursor lacking a hydroxyl group at C-10. Washing experiments demonstrated that both nystatin and 10-deoxynystatin are transported out of cells, suggesting the existence of an alternative efflux system(s) for the transport of nystatin-related metabolites. This notion was further corroborated in experiments with the ATPase inhibitor sodium o-vanadate, which affected the production of nystatin and 10-deoxynystatin in the wild-type strain and transporter mutants in a different manner. The data obtained in this study suggest that the efflux of nystatin-related polyene macrolides occurs through several transporters and that the NysH-NysG efflux system provides conditions favorable for C-10 hydroxylation.

The Final Step of Hygromycin A Biosynthesis, Oxidation of C-5″-Dihydrohygromycin A, Is Linked to a Putative Proton Gradient-Dependent Efflux

“…There are numerous precedents for the association of multiple efflux systems with antibiotic biosynthetic processes and, in particular, transporters that can excrete pathway intermediates. The 10-deoxynystatin intermediate, produced by deletion of the putative type III ABC transporter in S. noursei, is found in the fermentation broth, suggesting an additional efflux process (29). The premature efflux of landomycin D, an intermediate in landomycin A biosynthesis in S. cyanogenus, has been reported with either the overexpression of the proton gradient-dependent transporter gene lanJ or the disruption of the regulatory gene, lanK, that represses lanJ (19).…”

“…Furthermore, expression in the mutant strains of an additional copy of nysL, which presumably encodes a monooxygenase for C-10 hydroxylation, partially restored nystatin production with a concomitant decrease in 10-deoxynystatin. NysL has been expressed and has been shown to catalyze the hydroxylation of 10-deoxynystatin (29). The reaction has not been shown to be reversible (in contrast to the reaction observed for Hyg26), and there does not appear to be detectable inhibition of NysL by the product nystatin.…”

Characterization of the P450 Monooxygenase NysL, Responsible for C-10 Hydroxylation during Biosynthesis of the Polyene Macrolide Antibiotic Nystatin in Streptomyces noursei

“…The purification of 10-deoxynystatin was performed according to a previously described procedure (25). The antifungal activities of nystatin and 10-deoxynystatin were determined using Candida albicans as a test organism according to a method described previously (9).…”

“…Assessments of nystatin and 10-deoxynystatin production and accurate mass determinations were done by liquid chromatography-mass spectroscopy (LC-MS) of dimethyl sulfoxide extracts of 5-day-old cultures from well plate cultivations (9). Well plate cultivations were performed in semidefined 0.5ϫ SAO-23 medium according to a previously described protocol (25).…”

Polyene macrolide biosynthesis in streptomycetes and related bacteria: recent advances from genome sequencing and experimental studies

“…Introduction of NppY into S. noursei did not result in production of NPP, presumably because in this host 10-deoxynystatin is rapidly hydroxylated and exported before the second sugar can be added. In S. noursei, C-10 hydroxylation of nystatin is the last modification in the pathway and does not proceed to completion when export proteins are inactivated (Sletta et al, 2005). Possibly intracellular accumulation of nystatin shifts the reaction equilibrium towards the 10-deoxy form.…”