Biosynthesis of High-Molecular-Weight Poly(d‑lactate)-Containing Block Copolyesters Using Evolved Sequence-Regulating Polyhydroxyalkanoate Synthase PhaC_AR

Bacterial polyhydroxyalkanoate (PHA) synthase PhaCAR is a unique enzyme that can synthesize block copolymers. In this study, poly(d-lactate) (PDLA)-containing block copolymers were synthesized using PhaCAR and its mutated variants. Recombinant Escherichia coli harboring phaCAR and relevant genes were cultivated with supplementation of the corresponding monomer precursors. Consequently, PhaCAR synthesized poly(3-hydroxybutyrate)-b-2 mol % PDLA [P(3HB)-b-PDLA]. The incorporation of the d-lactate (LA) enantiomer was confirmed by chiral gas chromatography. Previously identified beneficial mutations in PhaCAR, N149D (ND), and F314H (FH), which increased activity toward a medium-chain-length substrate 3-hydroxyhexanoyl (3HHx)-CoA, improved the incorporation of LA units. The combined pairwise mutation NDFH synergistically increased the LA fraction to 21 mol % in P(3HB)-b-PDLA. Interestingly, a large amount of LA units (51 mol %) was incorporated by copolymerization with 3HHx units, which yielded P(3HHx)-b-PDLA. The block copolymerization of 3HHx and D-LA units was confirmed by NMR analyses and solvent fractionation of polymers. The PDLA crystal in P(3HHx)-b-PDLA was detected using differential scanning calorimetry and wide-angle X-ray diffraction. Its mass-average molecular weight was 8.6 × 105. Thus, block copolymerization utilized high-molecular-weight PDLA as a component of PHAs