Characterization and Functional Analyses of R -Specific Enoyl Coenzyme A Hydratases in Polyhydroxyalkanoate-Producing Ralstonia eutropha

A genome survey of polyhydroxyalkanoate (PHA)-producingRalstonia eutropha H16 detected the presence of 16 orthologs ofR -specific enoyl coenzyme A (enoyl-CoA) hydratase, among which three proteins shared high homologies with the enzyme specific to enoyl-CoAs of medium chain length encoded byphaJ4 fromPseudomonas aeruginosa (phaJ4Pa ). The recombinant forms of the three proteins, termed PhaJ4aRe to PhaJ4cRe , actually showed enoyl-CoA hydratase activity withR specificity, and the catalytic efficiencies were elevated as the substrate chain length increased from C4 to C8 . PhaJ4aRe and PhaJ4bRe showed >10-fold-higher catalytic efficiency than PhaJ4cRe . The functions of the new PhaJ4 proteins were investigated using previously engineeredR. eutropha strains as host strains; these strains are capable of synthesizing poly((R )-3-hydroxybutyrate-co -(R )-3-hydroxyhexanoate) [P(3HB-co -3HHx)] from soybean oil. Deletion ofphaJ4aRe from the chromosome resulted in significant decrease of 3HHx composition in the accumulated copolyester, whereas no change was observed with deletion ofphaJ4bRe orphaJ4cRe , indicating that only PhaJ4aRe was one of the major enzymes supplying the (R )-3HHx-CoA monomer through β-oxidation. Introduction ofphaJ4aRe orphaJ4bRe into theR. eutropha strains using a broad-host-range vector enhanced the 3HHx composition of the copolyesters, but the introduction ofphaJ4cRe did not. The two genes were then inserted into thepha operon on chromosome 1 of the engineeredR. eutropha by homologous recombination. These modifications enabled the biosynthesis of P(3HB-co -3HHx) composed of a larger 3HHx fraction without a negative impact on cell growth and PHA production on soybean oil, especially whenphaJ4aRe orphaJ4bRe was tandemly introduced withphaJAc fromAeromonas caviae .