AUTHOR=Sivashankari Ramamoorthi M , Mierzati Maierwufu , Miyahara Yuki , Mizuno Shoji , Nomura Christopher T. , Taguchi Seiichi , Abe Hideki , Tsuge Takeharu 

TITLE=Exploring Class I polyhydroxyalkanoate synthases with broad substrate specificity for polymerization of structurally diverse monomer units

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=11

YEAR=2023

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2023.1114946

DOI=10.3389/fbioe.2023.1114946

ISSN=2296-4185

ABSTRACT=<p>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 <italic>Ferrimonas marina</italic>, <italic>Plesiomonas shigelloides</italic>, <italic>Shewanella pealeana</italic>, and <italic>Vibrio metschnikovii</italic> were selected <italic>via</italic> a homology search against the GenBank database, using the amino acid sequence of <italic>Aeromonas caviae</italic> PHA synthase (PhaC<sub>Ac</sub>), 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 <italic>Escherichia coli</italic> as a host for PHA production. All the new PhaCs were able to synthesize P(3HB) in <italic>E. coli</italic> with a high molecular weight, surpassing PhaC<sub>Ac</sub>. 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 <italic>P. shigelloides</italic> (PhaC<sub>Ps</sub>) exhibited relatively broad substrate specificity. PhaC<sub>Ps</sub> was further engineered through site-directed mutagenesis, and the variant resulted in an enzyme with improved polymerization ability and substrate specificity.</p>