title: Engineering the aveC gene to enhance the ratio of doramectin to its CHC-B2 analogue produced in Streptomyces avermitilis.
abstract: Avermectin and its analogues are produced by the actinomycete Streptomyces avermitilis and are major commercial products for parasite control in the fields of animal health, agriculture, and human infections. Historically, the avermectin analogue doramectin (CHC-B1), which is sold commercially as Dectomax is co-produced during fermentation with the undesired analogue CHC-B2 at a CHC-B2:CHC-B1 ratio of 1.6:1. Although the identification of the avermectin gene cluster has allowed for characterization of most of the biosynthetic pathway, the mechanism for determining the avermectin B2:B1 ratio remains unclear. The aveC gene, which has an essential role in avermectin biosynthesis, was inactivated by insertional inactivation and mutated by site-specific mutagenesis and error-prone PCR. Several unrelated mutations were identified that resulted in improved ratios of the desirable avermectin analogue CHC-B1, produced relative to the undesired CHC-B2 fermentation component. High-throughput (HTP) screening of cultures grown on solid-phase fermentation plates and analysis using electrospray mass spectrometry was implemented to significantly increase screening capability. An aveC gene with mutations that result in a 4-fold improvement in the ratio of doramectin to CHC-B2 was identified. Subsequent integration of the enhanced aveC gene into the chromosome of the S. avermitilis production strain demonstrates the successful engineering of a specific biosynthetic pathway gene to significantly improve fermentation productivity of a commercially important product.
authors.affiliation: Pfizer Global Research and Development, Eastern Pt Rd, Groton, Connecticut 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: Frank
authors.initials: F
authors.lastname: Kaczmarek
authors.forename: Hamish
authors.initials: H
authors.lastname: McArthur
authors.forename: Anke
authors.initials: A
authors.lastname: Krebber
authors.forename: Yan
authors.initials: Y
authors.lastname: Chen
authors.forename: Jeremy
authors.initials: J
authors.lastname: Minshull
authors.forename: Sun Ai
authors.initials: SA
authors.lastname: Raillard
authors.forename: Claes
authors.initials: C
authors.lastname: Gustafsson
mesh_terms: D001483:Base Sequence; D018118:Chloride Channels; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D020869:Gene Expression Profiling / Q000379:methods; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D024861:Genetic Enhancement / Q000379:methods; D014644:Genetic Variation / Q000235:genetics; D016680:Genome, Bacterial; D007559:Ivermectin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering / Q000379:methods*; D011786:Quality Control; D011995:Recombination, Genetic; D013045:Species Specificity; D013302:Streptomyces / Q000145:classification / Q000235:genetics* / Q000378:metabolism*
chemical_list: D018118:Chloride Channels; D007559:Ivermectin; C019264:avermectin; C084101:doramectin