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

摘要

A genome survey of polyhydroxyalkanoate (PHA)-producing Ralstonia eutropha H16 detected the presence of 16 orthologs of R -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 by phaJ4 from Pseudomonas aeruginosa ( phaJ4_(Pa) ). The recombinant forms of the three proteins, termed PhaJ4a_( Re ) to PhaJ4c_( Re ), actually showed enoyl-CoA hydratase activity with R specificity, and the catalytic efficiencies were elevated as the substrate chain length increased from C_(4) to C_(8). PhaJ4a_( Re ) and PhaJ4b_( Re ) showed >10-fold-higher catalytic efficiency than PhaJ4c_( Re ). The functions of the new PhaJ4 proteins were investigated using previously engineered R. 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 of phaJ4a_(Re) from the chromosome resulted in significant decrease of 3HHx composition in the accumulated copolyester, whereas no change was observed with deletion of phaJ4b_(Re) or phaJ4c_(Re) , indicating that only PhaJ4a_( Re ) was one of the major enzymes supplying the ( R )-3HHx-CoA monomer through β-oxidation. Introduction of phaJ4a_(Re) or phaJ4b_(Re) into the R. eutropha strains using a broad-host-range vector enhanced the 3HHx composition of the copolyesters, but the introduction of phaJ4c_(Re) did not. The two genes were then inserted into the pha operon on chromosome 1 of the engineered R. 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 when phaJ4a_(Re) or phaJ4b_(Re) was tandemly introduced with phaJ_(Ac) from Aeromonas caviae .