Identification, biosynthesis, and characterization of polyhydroxyalkanoate copolymer consisting of 3-hydroxybutyrate and 3-hydroxy-4-methylvalerate

Abstract

In this study, we found that Ralstonia eutropha strain PHB(-)4 expressing the polyhydroxyalkanoate (PHA) synthase 1 (PhaC1(Ps)) from Pseudomonas sp. 61-3 synthesized a PHA copolymer containing a 3-hydroxybutyrate (3HB) and small amounts of 3-hydroxy-4-methylvalerate (3H4MV) and 3-hydroxyvalerate (3HV) from fructose as a sole carbon source. 3H4MV is a monomer unit that has hitherto not been reported as a naturally occurring component of PHAs. To increase the 3H4MV fraction in PHA copolymers, the culture medium was supplemented with four structural analogs that served as 3H4MV precursors. Of these, 4-methylvalerate (4MV) was the most efficient in increasing the 3H4MV content of PHA. The R. eutropha strain PHB(-)4 expressing Aeromonas cavaie PHA synthase (PhaC(Ac)) and its mutant (PhaC(Ac) NSDG mutant) were also able to synthesize 3H4MV-containing PHA from 4MV, increasing 3H4MV molar fraction in PHA copolymer up to 38 mol %. The structure and physical properties of P(3HB-co-3H4MV) copolymers were characterized by gel permeation chromatography, NMR spectroscopy, differential scanning calorimetry, and tensile testing. It can be shown that P(3HB-co-3H4MV) samples synthesized by PhaC(Ac) and its mutant NSDG were of higher molecular weights than those by PhaC1(Ps) and were a flexible and ductile material with moderate toughness. Additionally, the ductility can be kept for at least 180 days without significant deterioration caused by secondary crystallization when 3H4MV molar fraction was higher than 14 mol %. The newly identified 3H4MV unit is a promising monomer for improving material property and stability of 3HB-based copolymers.