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. 2007 Nov;73(22):7400-7.
doi: 10.1128/AEM.01122-07. Epub 2007 Sep 28.

Analysis of the mycosamine biosynthesis and attachment genes in the nystatin biosynthetic gene cluster of Streptomyces noursei ATCC 11455

Aina Nedal  1 , Håvard Sletta, Trygve Brautaset, Sven E F Borgos, Olga N Sekurova, Trond E Ellingsen, Sergey B Zotchev
Affiliations

Analysis of the mycosamine biosynthesis and attachment genes in the nystatin biosynthetic gene cluster of Streptomyces noursei ATCC 11455

Aina Nedal et al. Appl Environ Microbiol. 2007 Nov.

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 beta-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.

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Figures

FIG. 1.
FIG. 1.
Proposed biosynthetic pathway for mycosamine, whereby the mycosamine (in the open rectangle) is shown to be attached at the C-19 position of the nystatin aglycone.
FIG. 2.
FIG. 2.
(A) Western blot demonstrating the expression of NysDIII (38 kDa) expressed in Flag-CTC vector and NysDII (37 kDa) expressed in Flag-MAC vector (anti-Flag). Lane 1, NysDIII expressed in DIII-CTC; lane 2, NysDII expressed in DII-MAC; lane 3, molecular mass marker. (B) HPLC chromatograms confirming the GDP-d-mannose 4,6-dehydratase activity of NysDIII. The top panel shows the substrate GDP-mannose. The bottom panel shows the conversion of GDP-mannose to GDP-4-keto-6-deoxy-d-mannose after incubation with recombinant NysDIII.
FIG. 3.
FIG. 3.
HPLC-diode array detector-MS analysis of nystatin and its analogue production in S. noursei wild type (top), NDD1 (middle), and NDD1 complemented with nysDI (bottom). Nystatin, Mw 925.50; nystatinolide, Mw 780.43; 10-deoxynystatinolide, Mw 764.43; and 10-deoxynystatin, Mw 909.50.
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