Evolution of Efficient Modular Polyketide Synthases by Homologous Recombination
Citations Over TimeTop 10% of 2015 papers
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.
Related Papers
- → Probing the interactions of an acyl carrier protein domain from the 6‐deoxyerythronolide B synthase(2011)60 cited
- → A dual transacylation mechanism for polyketide synthase chain release in enacyloxin antibiotic biosynthesis(2019)40 cited
- → De Novo Design and Implementation of a Tandem Acyl Carrier Protein Domain in a Type I Modular Polyketide Synthase(2018)14 cited
- → Synthesis and Characterisation of Acyl Carrier Protein Bound Polyketide Analogues(2002)18 cited
- → The Substrate‐Protein and Protein‐Protein Interactions of Fatty Acid and Polyketide Mega‐Synthases(2016)