Molecular characterization and properties of (R)-specific enoyl-CoA hydratases from Pseudomonas aeruginosa: metabolic tools for synthesis of polyhydroxyalkanoates via fatty acid beta-oxidation

The use of (R)-specific enoyl-coenzyme A (CoA) hydratase (PhaJ) provides a powerful tool for polyhydroxyalkanoate (PHA) synthesis from fatty acids or plant oils in recombinant bacteria. PhaJ provides monomer units for PHA synthesis from the fatty acid ß-oxidation cycle. Previously, two phaJ genes (phaJ1_Pa and phaJ2_Pa) were identified in Pseudomonas aeruginosa. This report identifies two new phaJ genes (phaJ3_Pa and phaJ4_Pa) in P. aeruginosa through a genomic database search. The abilities of the four PhaJ_Pa proteins and the (R)-3-hydroxyacyl-acyl carrier protein (R)-3HA-ACP] dehydrases, FabA_Pa and FabZ_Pa, to supply monomers from enoyl-CoA substrates for PHA synthesis were determined. The presence of either PhaJ1_Pa or PhaJ4_Pa in recombinant Escherichia coli led to the high levels of PHA accumulation (as high as 36–41 wt.% in dry cells) consisting of mainly short- (C4–C6) and medium-chain-length (C6–C10) 3HA units, respectively. Furthermore, detailed characterizations of PhaJ1_Pa and PhaJ4_Pa were performed using purified samples. Kinetic analysis revealed that only PhaJ4_Pa exhibits almost constant maximum reaction rates (V_max) irrespective of the chain length of the substrates. The assay for stereospecific hydration revealed that, unlike PhaJ1_Pa, PhaJ4_Pa has relatively low (R)-specificity. These hydratases may be very useful as monomer-suppliers for the synthesis of designed PHAs in recombinant bacteria.