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

K Stutzman-Engwall, S Conlon, R Fedechko… - Metabolic …, 2005 - Elsevier
K Stutzman-Engwall, S Conlon, R Fedechko, H McArthur, K Pekrun, Y Chen, S Jenne, C La…
Metabolic engineering, 2005Elsevier
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 …
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.
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