Exploring Class I polyhydroxyalkanoate synthases with broad substrate specificity for polymerization of structurally...

Area of Science:

• Biotechnology
• Polymer Science
• Microbial Biochemistry

Background:

• Polyhydroxyalkanoates (PHAs) are biodegradable thermoplastics produced by bacteria.
• Class I PHA synthases (PhaCs) are crucial for synthesizing 3-hydroxybutyrate (3HB)-based copolymers.
• A scarcity of Class I PhaCs with broad substrate specificities limits the diversity of industrially relevant PHAs.

Purpose of the Study:

• To identify and characterize novel Class I PhaCs with broad substrate specificities.
• To explore the potential of these novel PhaCs for producing diverse PHA copolymers.
• To engineer a promising PhaC for enhanced polymerization and specificity.

Main Methods:

• Homology search in GenBank using a known broad-specificity Class I PhaC (PhaC_Ac) as a template.
• Expression and characterization of four novel PhaCs from marine bacteria (Ferrimonas marina, Plesiomonas shigelloides, Shewanella pealeana, Vibrio metschnikovii) in Escherichia coli.
• Evaluation of polymerization ability and substrate specificity using various hydroxyalkanoate monomers.
• Site-directed mutagenesis of the identified broad-specificity PhaC (PhaC_Ps).

Main Results:

• All four novel PhaCs synthesized poly(3-hydroxybutyrate) (P(3HB)) with high molecular weight in E. coli, exceeding that of PhaC_Ac.
• PhaC from Plesiomonas shigelloides (PhaC_Ps) demonstrated notable broad substrate specificity.
• Engineered variants of PhaC_Ps exhibited improved polymerization ability and substrate specificity.

Conclusions:

• Novel Class I PhaCs from marine bacteria possess significant potential for P(3HB) synthesis.
• PhaC_Ps is a promising candidate for producing diverse PHA copolymers due to its broad substrate specificity.
• Enzyme engineering can further enhance the utility of PhaC_Ps for tailored PHA production.