Analysis of the Mycosamine Biosynthesis and Attachment Genes in the Nystatin Biosynthetic Gene Cluster of Streptomyces noursei ATCC 11455

Abstract

The polyene macrolide antibiotic nystatin produced by Streptomyces noursei contains a deoxyaminosugar mycosamine moiety attached to the C-19 carbon of the macrolactone ring through the ␤-glycosidic bond. The nystatin biosynthetic gene cluster contains three genes, nysDI, nysDII, and nysDIII, encoding enzymes with presumed roles in mycosamine biosynthesis and attachment as glycosyltransferase, aminotransferase, and GDP-mannose dehydratase, respectively. In the present study, the functions of these three genes were analyzed. The recombinant NysDIII protein was expressed in Escherichia coli and purified, and its in vitro GDP-mannose dehydratase activity was demonstrated. The nysDI and nysDII genes were inactivated individually in S. noursei, and analyses of the resulting mutants showed that both genes produced nystatinolide and 10-deoxynystatinolide as major products. Expression of the nysDI and nysDII genes in trans in the respective mutants partially restored nystatin biosynthesis in both cases, supporting the predicted roles of these two genes in mycosamine biosynthesis and attachment. Both antifungal and hemolytic activities of the purified nystatinolides were shown to be strongly reduced compared to those of nystatin, confirming the importance of the mycosamine moiety for the biological activity of nystatin.

New Nystatin-Related Antifungal Polyene Macrolides with Altered Polyol Region Generated via Biosynthetic Engineering of Streptomyces noursei

“…In vitro assays for determination of antifungal and hemolytic activities. Determination of in vitro antifungal activity was performed essentially as described previously (7,17). Indicator Candida albicans cells were grown in 96-well plates, and cell growth was monitored in the presence of 24 different concentrations of antibiotics.…”

Engineered biosynthesis of pimaricin derivatives with improved antifungal activity and reduced cytotoxicity

“…Assays of in vitro antifungal and hemolytic activities were performed as previously described (Borgos et al 2006;Nedal et al 2007). In vitro antifungal activity assays were done by cultivating Candida albicans ATCC 14053 and monitoring cell growth with antibiotic concentrations ranging from 0 to 8 μg/mL.…”

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

“…The mycosamine sugar of polyenes is synthesised from GDP--D-mannose (Nic Lochlainn and Caffrey, 2009;Nedal et al, 2007). GDP--D-mannose 4, 6 dehydratase catalyses formation of GDP-4-keto-6-deoxy--Dmannose, which is somehow isomerised to the 3-keto sugar (Fig.…”

“…While the 3, 4 ketoisomerisation can occur spontaneously, it is not known whether the reaction is enzyme-catalyzed in vivo (Hutchinson et al, 2010). The Zotchev group found that NysDIII GDP--Dmannose 4, 6 dehydratase and NysDII GDP-mycosamine synthase were not sufficient to reconstitute GDPmycosamine biosynthesis in vitro (Nedal et al, 2007). So far, bioinformatic analysis of genome sequences has not helped to resolve this issue.…”