Biosynthesis of High-Molecular-Weight Poly(d-lactate)-Containing Block Copolyesters Using Evolved Sequence-Regulating Polyhydroxyalkanoate Synthase PhaCAR
ACS SUSTAINABLE CHEMISTRY & ENGINEERING(2023)
Abstract
Bacterial polyhydroxyalkanoate (PHA) synthase PhaC(AR) is a unique enzyme that can synthesize block copolymers.In thisstudy, poly(d-lactate) (PDLA)-containing block copolymerswere synthesized using PhaC(AR) and its mutated variants.Recombinant Escherichia coli harboring phaC (AR) and relevant genes were cultivated withsupplementation of the corresponding monomer precursors. Consequently,PhaC(AR) synthesized poly(3-hydroxybutyrate)-b-2 mol % PDLA [P(3HB)-b-PDLA]. The incorporationof the d-lactate (LA) enantiomer was confirmed by chiralgas chromatography. Previously identified beneficial mutations inPhaC(AR), N149D (ND), and F314H (FH), which increased activitytoward a medium-chain-length substrate 3-hydroxyhexanoyl (3HHx)-CoA,improved the incorporation of LA units. The combined pairwise mutationNDFH 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 yieldedP(3HHx)-b-PDLA. The block copolymerization of 3HHxand D-LA units was confirmed by NMR analyses and solvent fractionationof polymers. The PDLA crystal in P(3HHx)-b-PDLA wasdetected using differential scanning calorimetry and wide-angle X-raydiffraction. Its mass-average molecular weight was 8.6 x 10(5). Thus, block copolymerization utilized high-molecular-weightPDLA as a component of PHAs. Newsynthetic method of block copolyester with isotacticPDLA segment from renewable resources using engineered polyester synthase.
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Key words
PHA synthase,in vitro evolution,sequenceregulation,PDLA-like copolymers,block copolymer
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