RESUMO
Sequence-regulating polyhydroxyalkanoate synthase PhaCAR is a chimeric enzyme comprising PhaCs from Aeromonas caviae and Ralstonia eutropha (Cupriavidus necator). It spontaneously synthesizes a short-chain-length (SCL, ≤C5) 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 PhaCAR was performed to increase its activity toward a medium-chain-length (MCL, C6-12) 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 PhaCAR 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.