Evolution of Efficient Modular Polyketide Synthases by Homologous Recombination

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 fashi...

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Bibliographic Details
Published in:Journal of the American Chemical Society 2015-08, Vol.137 (33), p.10603-10609
Main Authors: Chemler, Joseph A, Tripathi, Ashootosh, Hansen, Douglas A, O’Neil-Johnson, Mark, Williams, Russell B, Starks, Courtney, Park, Sung Ryeol, Sherman, David H
Format: Article
Language:English
Subjects:
Erythromycin - metabolism
Escherichia coli - genetics
Evolution, Molecular
Homologous Recombination
Macrolides - metabolism
Polyketide Synthases - genetics
Polyketide Synthases - metabolism
Protein Engineering
Saccharomyces cerevisiae - genetics
Streptomyces - metabolism
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Summary: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.
ISSN:0002-7863
1520-5126
DOI:10.1021/jacs.5b04842