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Applied and Environmental Microbiology, August 2005, p. 4503-4509, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4503-4509.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Chemobiosynthesis of Novel 6-Deoxyerythronolide B Analogues by Mutation of the Loading Module of 6-Deoxyerythronolide B Synthase 1

Sumati Murli, Karen S. MacMillan,{dagger} Zhihao Hu, Gary W. Ashley, Steven D. Dong, James T. Kealey, Christopher D. Reeves, and Jonathan Kennedy*

Kosan Biosciences Inc., 3832 Bay Center Place, Hayward, California 94545

Received 6 January 2005/ Accepted 2 March 2005

Chemobiosynthesis (J. R. Jacobsen, C. R. Hutchinson, D. E. Cane, and C. Khosla, Science 277:367-369, 1997) is an important route for the production of polyketide analogues and has been used extensively for the production of analogues of 6-deoxyerythronolide B (6-dEB). Here we describe a new route for chemobiosynthesis using a version of 6-deoxyerythronolide B synthase (DEBS) that lacks the loading module. When the engineered DEBS was expressed in both Escherichia coli and Streptomyces coelicolor and fed a variety of acyl-thioesters, several novel 15-R-6-dEB analogues were produced. The simpler "monoketide" acyl-thioester substrates required for this route of 15-R-6-dEB chemobiosynthesis allow greater flexibility and provide a cost-effective alternative to diketide-thioester feeding to DEBS KS1o for the production of 15-R-6-dEB analogues. Moreover, the facile synthesis of the monoketide acyl-thioesters allowed investigation of alternative thioester carriers. Several alternatives to N-acetyl cysteamine were found to work efficiently, and one of these, methyl thioglycolate, was verified as a productive thioester carrier for mono- and diketide feeding in both E. coli and S. coelicolor.

* Corresponding author. Mailing address: Kosan Biosciences Inc., 3832 Bay Center Place, Hayward, CA 94545. Phone: (510) 731-5236. Fax: (510) 732-8401. E-mail: kennedy{at}kosan.com.

{dagger} Present address: Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037.

Applied and Environmental Microbiology, August 2005, p. 4503-4509, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4503-4509.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.





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