Evolution of Efficient Modular Polyketide Synthases by Homologous Recombination

Area of Science:

• Biochemistry
• Synthetic Biology
• Natural Product Synthesis

Background:

• Type I polyketide synthases (PKS) are large, multifunctional enzymes responsible for producing complex natural products.
• The modular structure of PKS allows for engineering custom biocatalysts by modifying enzyme domains.
• Previous PKS engineering efforts often resulted in reduced enzyme activity and limited product yields.

Purpose of the Study:

• To develop a versatile method for generating and identifying functional chimeric PKS enzymes.
• To synthesize custom macrolactones and macrolides using engineered PKS.
• To explore the potential of PKS engineering for creating novel antibiotic-like molecules.

Main Methods:

• Hybridization of PKS genes from pikromycin and erythromycin pathways in Saccharomyces cerevisiae to create hybrid libraries.
• Utilized a 96-well plate format for high-throughput plasmid purification, transformation, sequencing, protein expression, in vitro reactions, and metabolite analysis.
• Generated and screened chimeric PKS enzymes for novel functionality and metabolite production.

Main Results:

• Successfully generated and identified active chimeric PKS enzymes with new functionalities.
• Streptomyces venezuelae strains expressing these chimeric PKS produced engineered macrolactones.
• A novel macrolide analogue was synthesized by further functionalizing a macrolactone produced by selected PKS chimeras.

Conclusions:

• The developed method provides a robust platform for engineering PKS pathways as modular components.
• This approach facilitates the creation of custom macrolactones and macrolides with potential therapeutic applications.
• The modular assembly of PKS offers a promising strategy for discovering and producing new antibiotic-like compounds.