Characterization and cloning of an (R)-specific trans-2,3-enoylacyl-CoA hydratase from Rhodospirillum rubrum and use of this enzyme for PHA production in Escherichia coli

An (R)-trans-2,3-enoylacyl-CoA hydratase was purified to near-homogeneity f rom Rhodospirillum rubrum. Protein sequencing of enriched protein fractions allowed the construction of a degenerate oligonucleotide. The gene encodin g the (R)-specific hydratase activity was cloned following three rounds of colony hybridization using the oligonucleotide, and overexpression of the g ene in E. coli led to the purification of the enzyme to homogeneity. The pu rified enzyme used crotonyl-CoA, trans-2,3-pentenoyl-CoA, and trans-2,3-hex enoyl-CoA with approximately equal specificity as substrates in the hydrati on reaction. However, no activity was observed using trans-2,3-octenoyl-CoA as a substrate, but this compound did partially inhibit crotonyl-CoA hydra tion. Based on the nucleotide sequence, the protein has a monomeric molecul ar weight of 15.4 kDa and is a homotetramer in its native form as determine d by gel filtration chromatography and native PAGE. The hydratase was expre ssed together with the PHA synthase from Thiocapsa pfennigii in E. coli str ain DH5 alpha. Growth of these strains on oleic acid resulted in the produc tion of the terpolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-h ydroxyhexanoate).