(424e) A Polyketide Synthase Platform to Produce Biofuels and Specialty Chemicals (Faculty Candidate)

Traditionally engineered to produce novel pharmaceuticals, 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. A significant challenge in PKS design is engineering a partially reductive module to produce a saturated β-carbon through a reductive loop exchange. In this presentation, we first establish that chemoinformatics, a field traditionally used in drug discovery, offers a viable strategy for reductive loop exchanges. We introduced a set of donor reductive loops of diverse genetic origin and chemical substrate structures into the first extension module of the lipomycin PKS (LipPKS1). Product titers of these engineered unimodular PKSs correlated with chemical similarity between the substrate of the donor reductive loops and recipient LipPKS1, reaching a titer of 165 mg/L of short chain fatty acids produced by Streptomyces albus J1074 harboring these engineered PKSs. Expanding this method to larger intermediates requiring bimodular communication, we introduced reductive loops of divergent chemosimilarity into LipPKS2 and determined triketide lactone production. We observed a statistically significant correlation between atom pair chemosimilarity and production, establishing a new chemoinformatic method that may aid in the engineering of PKSs to produce desired, unnatural products.

Building upon this work, we 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.