Abstract
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 (PhaC
Ac
), 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 PhaC
Ac
. 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
(PhaC
Ps
) exhibited relatively broad substrate specificity. PhaC
Ps
was further engineered through site-directed mutagenesis, and the variant resulted in an enzyme with improved polymerization ability and substrate specificity.