Biosynthesis of polyhydroxyalkanoate copolymers consisting of α-methylated monomer units from glucose and propionate: thermal properties and chiral configuration

Expression of the Ascaris suum ketothiolase (Acat3) in recombinant Escherichia coli enables the production of α-methylated monomers such as 3-hydroxy-2-methylvalerate (3H2MV) and 3-hydroxy-2-methylbutyrate (3H2MB) for polyhydroxyalkanoate (PHA) biosynthesis from glucose and propionate as carbon sources. However, the chiral configurations and thermal properties of biosynthesized PHAs remain poorly understood.

In this study, 3-hydroxybutyrate (3HB)-based PHA copolymers containing 3H2MV and 3H2MB units were synthesized from glucose and propionate using Acat3-expressing Escherichia coli LSBJ. The 3H2MV fraction of the synthesized PHA reached 15.7 mol%, while the 3H2MB fraction remained at approximately 0.2 mol%. Chirality analysis revealed that (2S,3R)- and (2R,3R)-3H2MV units were both detected; however, (2R,3R)-3H2MV units were dominant in the PHA copolymer produced by the strain expressing the (R)-specific enoyl-CoA hydratase (PhaJ_Ac). To evaluate the effect of α-methylated monomers on the crystallization behavior of PHA copolymers, cold crystallization was compared for PHA polymers with different mol% 3-hydroxyvalerate (3HV) monomer units. The cold crystallization of the copolymer containing 11 mol% 3H2MV and 30 mol% 3HV was detected at 68 °C, while the non-α-methylated copolymer containing 24 mol% 3HV did not exhibit cold crystallization, indicating that α-methylated PHA had a greater tendency to crystallize.

This study conclusively demonstrated that (2S,3R)- and (2R,3R)-3H2MV units were both incorporated into PHA by expressing Acat3; however, the (2R,3R)-isomer became the dominant 3H2MV unit in the PHA copolymers by additionally expressing PhaJ_Ac. The 3H2MV repeating unit facilitated the crystallization of PHA copolymers despite the high fraction of the 3HV unit.