Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10234682 | Metabolic Engineering | 2005 | 11 Pages |
Abstract
The avermectin analog doramectin (CHC-B1), sold commercially as DectomaxTM, 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.
Keywords
Related Topics
Physical Sciences and Engineering
Chemical Engineering
Bioengineering
Authors
Kim Stutzman-Engwall, Steve Conlon, Ronald Fedechko, Hamish McArthur, Katja Pekrun, Yan Chen, Stephane Jenne, Charlene La, Na Trinh, Seran Kim, Ying-Xin Zhang, Richard Fox, Claes Gustafsson, Anke Krebber,