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Applied and Environmental Microbiology, November 2007, p. 7400-7407, Vol. 73, No. 22
0099-2240/07/$08.00+0     doi:10.1128/AEM.01122-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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

Aina Nedal,1 Håvard Sletta,2 Trygve Brautaset,2 Sven E. F. Borgos,2 Olga N. Sekurova,1 Trond E. Ellingsen,2 and Sergey B. Zotchev1*

Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway,1 SINTEF Materials and Chemistry, Department of Biotechnology, SINTEF, N-7465 Trondheim, Norway2

Received 20 May 2007/ Accepted 19 September 2007

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 β-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.

* Corresponding author. Mailing address: Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway. Phone: 47 73 59 86 79. Fax: 47 73 59 12 83. E-mail: sergey.zotchev{at}nt.ntnu.no

{triangledown} Published ahead of print on 28 September 2007.

Applied and Environmental Microbiology, November 2007, p. 7400-7407, Vol. 73, No. 22
0099-2240/07/$08.00+0     doi:10.1128/AEM.01122-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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