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Metab Eng. 2005 Jan;7(1):27-37.

Semi-synthetic DNA shuffling of aveC leads to improved industrial scale production of doramectin by Streptomyces avermitilis.

Author information

1
Pfizer Global Research & Development, Gorton Labortories, Eastern Pt. Rd. MS 4123, Groton, CT 06340, USA. stutzmanengw@groton.pfizer.com

Abstract

The avermectin analog doramectin (CHC-B1), sold commercially as Dectomax, is biosynthesized by Streptomyces avermitilis. aveC, a gene encoding an unknown mechanistic function, plays an essential role in the production of doramectin (avermectin CHC-B1), modulating the production ratio of CHC-B1 to other avermectins, most notably the undesirable analog CHC-B2. To improve the production ratio for doramectin, the aveC gene was subjected to iterative rounds of semi-synthetic DNA shuffling. Libraries of shuffled aveC gene variants were transformed into S. avermitilis, screened using a miniaturized 96-well growth and production format, and analyzed by high throughput mass spectrometry to determine CHC-B2:CHC-B1 ratios. Several improved aveC variants were identified; the best shuffled gene encoded 10 amino acid mutations, and conferred a final CHC-B2:CHC-B1 ratio of 0.07:1, a 23-fold improvement over the starting gene (aveC wild type). Chromosomal insertion of an improved aveC shuffled gene into a high titer S. avermitilis strain yielded an improved doramectin production strain. This strain is under development to be used commercially, and is expected to provide considerable cost savings in large-scale manufacture, as well as significantly reducing by-product levels of CHC-B2 requiring disposal.

PMID:
15721808
DOI:
10.1016/j.ymben.2004.07.003
[Indexed for MEDLINE]

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