Engineering Novel Polyhydroxyalkanoates Using Polyketide Synthases in Pseudomonas putida.

Polyhydroxyalkanoates (PHAs) are bio-based polyesters with the potential to replace petroleum-based products. However, their thermal and mechanical properties are limited by the composition of the monomers used to make them. Current approaches to expanding monomer diversity require individualized pathway engineering for each targeted monomer, limiting the scalability of PHA design. In this work, we establish polyketide synthases (PKSs) as a modular and programmable platform for expanding PHA monomer diversity with variable chain length and branching. Using Pseudomonas putidaas a host, we integrate four engineered PKSs and their variants with heterologous CoA-activating enzymes and short-chain-length PHA polymerases to produce diverse (R)-3-hydroxyacid monomers with defined substitution and branching at the ɑ- and β-positions and enable their in vivo polymerization. We also apply active learning-assisted directed evolution to improve the heterodimeric PHA polymerase CapPhaEC, achieving a 3.6-fold increase in incorporation of α-branched monomers. Leveraging the modularity of PKSs, we demonstrate the biosynthesis and polymerization of linear, branched, and aromatic monomers by exchanging acyltransferase (AT) and ketoreductase (KR) domains to control chain length, branching, and stereochemistry. This study illustrates the utility of using a flexible, programmable PKS platform for rapidly designing and testing new-to-nature PHAs.