A bimodular PKS platform that expands the biological design space

Abstract

Traditionally engineered to produce novel bioactive molecules, Type I modular polyketide synthases (PKSs) could be engineered as a new biosynthetic platform for the production of de novo fuels, commodity chemicals, and specialty chemicals. Previously, our investigations manipulated the first module of the lipomycin PKS to produce short chain ketones, 3-hydroxy acids, and saturated, branched carboxylic acids. Building upon this work, we have expanded to multi-modular systems by engineering the first two modules of lipomycin to generate unnatural polyketides as potential biofuels and specialty chemicals in Streptomyces albus. First, we produce 20.6 mg/L of the ethyl ketone, 4,6 dimethylheptanone through a reductive loop exchange in LipPKS1 and a ketoreductase knockouts in LipPKS2. We then show that an AT swap in LipPKS1 and a reductive loop exchange in LipPKS2 can produce the potential fragrance 3-isopropyl-6-methyltetrahydropyranone. Highlighting the challenge of maintaining product fidelity, in both bimodular systems we observed side products from premature hydrolysis in the engineered first module and stalled dehydration in reductive loop exchanges.Collectively, our work expands the biological design space and moves the field closer to the production of "designer" biomolecules. Highlights• Engineered lipomycin module 1 and module 2 to produce unnatural polyketides as valuable bio-based chemicals • A reductive loop swap and ketoreductase knockout used to produce 20 mg/mL of a novel ethyl ketone, a gasoline replacement • An acyltransferase swap and reductive loop swap successfully produced δ-lactone, a potential fragrant compound • Incomplete reduction and premature hydrolysis observed in engineered modules

Biofuels for a sustainable future

“…Owing to its multi-modular nature, PKS provides a versatile synthetic platform where multiple modules can be assembled with complete fidelity toward rational design and synthesis of novel chemicals. Such multi-modular system has been successfully implemented to produce the pre-designed molecules (Kalkreuter et al, 2019), including the production of ethyl ketones, a potential biofuel with favorable properties (Zargar et al, 2020b). However, side products are typically observed due to gatekeeping domains and other mismatched enzymatic considerations.…”