Directed Evolution of Sequence-Regulating Polyhydroxyalkanoate Synthase to Synthesize a Medium-Chain-Length–Short-Chain-Length (MCL–SCL) Block Copolymer

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Sequence-regulating polyhydroxyalkanoate synthase PhaC AR is a chimeric enzyme comprising PhaCs from Aeromonas caviae and Ralstonia eutropha ( Cupriavidus necator ). It spontaneously synthesizes a short-chain-length (SCL, ≤C 5 ) block copolymer poly(2-hydroxybutyrate)- b -poly(3-hydroxybutyrate) [P(2HB)- b -P(3HB)] from a mixture of monomer substrates. In this study, directed evolution of PhaC AR was performed to increase its activity toward a medium-chain-length (MCL, C 6–12 ) monomer, 3-hydroxyhexanoyl (3HHx)-coenzyme A (CoA). Random mutagenesis and selection based on P(3HB- co -3HHx) production in Escherichia coli found that beneficial mutations N149D and F314L increase the 3HHx fraction. The site-directed saturation mutagenesis at position 314, which is adjacent to the catalytic center C315, demonstrated that F314H synthesizes the P(3HHx) homopolymer. The F314H mutant exhibited increased activity toward 3HHx-CoA compared with the parent enzyme, whereas the activity toward 3HB-CoA decreased. The predicted tertiary structure of PhaC AR by AlphaFold2 provided insight into the mechanism of the beneficial mutations. In addition, this finding enabled the synthesis of a new PHA block copolymer, P(3HHx)- b -P(2HB). Solvent fractionation indicated the presence of a covalent linkage between the polymer segments. This novel MCL–SCL block copolymer considerably expands the range of the molecular design of PHA block copolymers.