title: Semi-synthetic DNA shuffling of aveC leads to improved industrial scale production of doramectin by Streptomyces avermitilis.
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.
authors.affiliation: Pfizer Global Research & Development, Gorton Labortories, Eastern Pt. Rd. MS 4123, Groton, CT 06340, USA. stutzmanengw@groton.pfizer.com
authors.forename: Kim
authors.initials: K
authors.lastname: Stutzman-Engwall
authors.forename: Steve
authors.initials: S
authors.lastname: Conlon
authors.forename: Ronald
authors.initials: R
authors.lastname: Fedechko
authors.forename: Hamish
authors.initials: H
authors.lastname: McArthur
authors.forename: Katja
authors.initials: K
authors.lastname: Pekrun
authors.forename: Yan
authors.initials: Y
authors.lastname: Chen
authors.forename: Stephane
authors.initials: S
authors.lastname: Jenne
authors.forename: Charlene
authors.initials: C
authors.lastname: La
authors.forename: Na
authors.initials: N
authors.lastname: Trinh
authors.forename: Seran
authors.initials: S
authors.lastname: Kim
authors.forename: Ying-Xin
authors.initials: YX
authors.lastname: Zhang
authors.forename: Richard
authors.initials: R
authors.lastname: Fox
authors.forename: Claes
authors.initials: C
authors.lastname: Gustafsson
authors.forename: Anke
authors.initials: A
authors.lastname: Krebber
mesh_terms: D032621:DNA Shuffling / Q000379:methods*; D019020:Directed Molecular Evolution / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D024861:Genetic Enhancement / Q000379:methods*; D014644:Genetic Variation / Q000235:genetics; D007218:Industrial Microbiology / Q000379:methods*; D007559:Ivermectin / Q000031:analogs & derivatives* / Q000378:metabolism*; D009154:Mutation; D010865:Pilot Projects; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism*
chemical_list: D007559:Ivermectin; C084101:doramectin