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 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.