Poly[(R)-3-Hydroxybutyrate] Formation in Escherichia coli from Glucose through an Enoyl-CoA Hydratase-Mediated Pathway(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)

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In this study, a new metabolic pathway for the synthesis of poly [(R)-3-hydroxybutyrate] [P(3HB)] was constructed in a recombinant Escherichia coli strain that utilized forward and reverse reactions catalyzed by two substrate-specific enoyl-CoA hydratases, R-hydratase (PhaJ) and S-hydratase (FadB), to epimerize (S)-3HB-CoA to (R)-3HB-CoA via a crotonyl-CoA intermediate. The R-hydratase gene (phaJ_<Ac>) from Aeromonas caviae was coexpressed with the PHA synthase gene (phaC_<Re>) and 3-ketothiolase gene (phaA_<Re>) from Ralstonia eutropha in fadR mutant E. coli strains (CAG18497 and LS5218), which had constitutive levels of the β-oxidation multienzyme FadB_<Ec>. When grown on glucose as the sole carbon source, the cells accumulated P(3HB) up to an amount 6.5wt% of the dry cell weight, whereas the control cells without phaJ_<Ac> or fadR mutation accumulated significantly smaller amounts of P(3HB). These results suggest that PhaJ_<Ac> and FadB_<Ec> played an important role in supplying monomers for P(3HB) synthesis in the pathway. Furthermore, by using this pathway, a P(3HB)-concentration-dependent fluorescent staining screening technique was developed to rapidly identify cells that possess active R-hydratase.
公益社団法人日本生物工学会の論文
2007-01-25