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Identification of a cyclohexylcarbonyl CoA biosynthetic gene cluster and application in the production of doramectin

Nature Biotechnology volume 18, pages 980–983 (2000)Cite this article

Abstract

The side chain of the antifungal antibiotic ansatrienin A from Streptomyces collinus contains a cyclohexanecarboxylic acid (CHC)-derived moiety. This moiety is also observed in trace amounts of ω-cyclohexyl fatty acids (typically less than 1% of total fatty acids) produced by S. collinus. Coenzyme A-activated CHC (CHC-CoA) is derived from shikimic acid through a reductive pathway involving a minimum of nine catalytic steps. Five putative CHC-CoA biosynthetic genes in the ansatrienin biosynthetic gene cluster of S. collinus have been identified. Plasmid-based heterologous expression of these five genes in Streptomyces avermitilis or Streptomyces lividans allows for production of significant amounts of ω-cyclohexyl fatty acids (as high as 49% of total fatty acids). In the absence of the plasmid these organisms are dependent on exogenously supplied CHC for ω-cyclohexyl fatty acid production. Doramectin is a commercial antiparasitic avermectin analog produced by fermenting a bkd mutant of S. avermitilis in the presence of CHC. Introduction of the S. collinus CHC-CoA biosynthetic gene cassette into this organism resulted in an engineered strain able to produce doramectin without CHC supplementation. The CHC-CoA biosynthetic gene cluster represents an important genetic tool for precursor-directed biosynthesis of doramectin and has potential for directed biosynthesis in other important polyketide-producing organisms.

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Figure 1: The CHC-CoA biosynthetic pathway and its role in ansatrienin biosynthesis.
Figure 2: The role of precursors in the biosynthesis of avermectin and doramectin in S. avermitilis.
Figure 3: The CHC-CoA biosynthetic genes of the ansatrienin biosynthetic gene cluster (A) cloned into a streptomycetes expression plasmid, pAC12 (B).
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Figure 5

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Acknowledgements

We thank Ron Fedechcko and Hamish McArthur at Pfizer, for providing doramectin and for carrying out LC-MS analyses of the S. avermitilis/pAC12 fermentations. We also thank Claudio Denoya (Pfizer) for advice for working with the bkd mutant of S. avermitilis. We thank Stephanie Patton for carrying out 2-cyclohexenylcarbonyl CoA isomerase assays. This work was supported in part by a grant from the National Science Foundation (MCB 9418581).

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  1. Department of Medicinal Chemistry and Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, 800 E. Leigh St. Suite 212, Richmond, 23219, VA

    T. Ashton Cropp, Dennis J. Wilson & K.A. Reynolds

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  1. T. Ashton Cropp
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  2. Dennis J. Wilson
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  3. K.A. Reynolds
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Correspondence to K.A. Reynolds.

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Cropp, T., Wilson, D. & Reynolds, K. Identification of a cyclohexylcarbonyl CoA biosynthetic gene cluster and application in the production of doramectin. Nat Biotechnol 18, 980–983 (2000). https://doi.org/10.1038/79479

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