title: Evolution of Efficient Modular Polyketide Synthases by Homologous Recombination.
abstract: The structural scaffolds of many complex natural products are produced by multifunctional type I polyketide synthase (PKS) enzymes that operate as biosynthetic assembly lines. The modular nature of these mega-enzymes presents an opportunity to construct custom biocatalysts built in a lego-like fashion by inserting, deleting, or exchanging native or foreign domains to produce targeted variants of natural polyketides. However, previously engineered PKS enzymes are often impaired resulting in limited production compared to native systems. Here, we show a versatile method for generating and identifying functional chimeric PKS enzymes for synthesizing custom macrolactones and macrolides. PKS genes from the pikromycin and erythromycin pathways were hybridized in Saccharomyces cerevisiae to generate hybrid libraries. We used a 96-well plate format for plasmid purification, transformations, sequencing, protein expression, in vitro reactions and analysis of metabolite formation. Active chimeric enzymes were identified with new functionality. Streptomyces venezuelae strains that expressed these PKS chimeras were capable of producing engineered macrolactones. Furthermore, a macrolactone generated from selected PKS chimeras was fully functionalized into a novel macrolide analogue. This method permits the engineering of PKS pathways as modular building blocks for the production of new antibiotic-like molecules.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Chemler
authors.forename: Ashootosh
authors.initials: A
authors.lastname: Tripathi
authors.forename: Douglas A
authors.initials: DA
authors.lastname: Hansen
authors.affiliation: Sequoia Sciences, Inc. , 1912 Innerbelt Business Center Drive, Saint Louis, Missouri 63114, United States.
authors.forename: Mark
authors.initials: M
authors.lastname: O'Neil-Johnson
authors.affiliation: Sequoia Sciences, Inc. , 1912 Innerbelt Business Center Drive, Saint Louis, Missouri 63114, United States.
authors.forename: Russell B
authors.initials: RB
authors.lastname: Williams
authors.affiliation: Sequoia Sciences, Inc. , 1912 Innerbelt Business Center Drive, Saint Louis, Missouri 63114, United States.
authors.forename: Courtney
authors.initials: C
authors.lastname: Starks
authors.forename: Sung Ryeol
authors.initials: SR
authors.lastname: Park
authors.forename: David H
authors.initials: DH
authors.lastname: Sherman
mesh_terms: D004917:Erythromycin / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D019143:Evolution, Molecular; D059765:Homologous Recombination; D018942:Macrolides / Q000378:metabolism; D048630:Polyketide Synthases / Q000235:genetics* / Q000378:metabolism*; D015202:Protein Engineering; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013302:Streptomyces / Q000378:metabolism
chemical_list: D018942:Macrolides; D004917:Erythromycin; D048630:Polyketide Synthases; C003145:picromycin