A new polythioester (PTE), poly(3-mercapto-2-methylpropionate) [P(3M2MP)], and its copolymer with 3-hydroxybutyrate (3HB) were successfully biosynthesized from 3-mercapto-2-methylpropionic acid as a structurally-related precursor. This is the fourth PTE of biological origin and the first to be α-methylated. P(3M2MP) was biosynthesized using an engineered Escherichia coli LSBJ, which has a high molecular weight, amorphous structure, and elastomeric properties, reaching 2600% elongation at break. P(3HB-co-3M2MP) copolymers were synthesized by expressing 3HB-supplying enzymes. The copolymers were produced with high content in the cells and showed a high 3M2MP unit incorporation of up to 77.2 wt% and 54.8 mol%, respectively. As the 3M2MP fraction in the copolymer increased, the molecular weight decreased and the polymers became softer, more flexible, and less crystalline, with lower glass transition temperatures and higher elongations at break. The properties of this PTE were distinct from those of previously biosynthesized PTEs, indicating that the range of material properties can be further expanded by introducing α-methylated thioester monomers.

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Polyhydroxyalkanoate (PHA) synthases (PhaCs) are key enzymes in PHA polymerization. PhaCs with broad substrate specificity are attractive for synthesizing structurally diverse PHAs. In the PHA family, 3-hydroxybutyrate (3HB)-based copolymers are industrially produced using Class I PhaCs and can be used as practical biodegradable thermoplastics. However, Class I PhaCs with broad substrate specificities are scarce, prompting our search for novel PhaCs. In this study, four new PhaCs from the bacteria Ferrimonas marina, Plesiomonas shigelloides, Shewanella pealeana, and Vibrio metschnikovii were selected via a homology search against the GenBank database, using the amino acid sequence of Aeromonas caviae PHA synthase (PhaCAc), a Class I enzyme with a wide range of substrate specificities, as a template. The four PhaCs were characterized in terms of their polymerization ability and substrate specificity, using Escherichia coli as a host for PHA production. All the new PhaCs were able to synthesize P(3HB) in E. coli with a high molecular weight, surpassing PhaCAc. The substrate specificity of PhaCs was evaluated by synthesizing 3HB-based copolymers with 3-hydroxyhexanoate, 3-hydroxy-4-methylvalerate, 3-hydroxy-2-methylbutyrate, and 3-hydroxypivalate monomers. Interestingly, PhaC from P. shigelloides (PhaCPs) exhibited relatively broad substrate specificity. PhaCPs was further engineered through site-directed mutagenesis, and the variant resulted in an enzyme with improved polymerization ability and substrate specificity.