Biosynthesis of High-Molecular-Weight Poly(<scp>d</scp>-lactate)-Containing Block Copolyesters Using Evolved Sequence-Regulating Polyhydroxyalkanoate Synthase PhaC<sub>AR</sub>
Hien Phan, Shoko Furukawa, Koto Imai, Hiroya Tomita, Takuya Isono, Toshifumi Satoh, Ken’ichiro Matsumoto
Abstract
Bacterial polyhydroxyalkanoate (PHA) synthase PhaC AR is a unique enzyme that can synthesize block copolymers. In this study, poly( d -lactate) (PDLA)-containing block copolymers were synthesized using PhaC AR and its mutated variants. Recombinant Escherichia coli harboring phaC AR and relevant genes were cultivated with supplementation of the corresponding monomer precursors. Consequently, PhaC AR 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 PhaC AR, 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 × 10 5 . Thus, block copolymerization utilized high-molecular-weight PDLA as a component of PHAs.