Poly[(R)-3-hydroxybutyrate] formation in Escherichia coli from glucose through an enoyl-CoA hydratase-mediated pathway.
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 beta-oxidation multienzyme FadB(Ec). When grown on glucose as the sole carbon source, the cells accumulated P(3HB) up to an amount 6.5 wt% 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.
Sato
Shun
S
Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan.
Nomura
Christopher T
CT
Abe
Hideki
H
Doi
Yoshiharu
Y
Tsuge
Takeharu
T
D000333:Aeromonas / Q000201:enzymology* / Q000235:genetics; D020118:Cupriavidus necator / Q000201:enzymology* / Q000235:genetics; D004746:Enoyl-CoA Hydratase / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D024861:Genetic Enhancement; D005947:Glucose / Q000378:metabolism*; D006885:Hydroxybutyrates / Q000378:metabolism*; D011091:Polyesters / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D000094122:Polyhydroxybutyrates
D004746:Enoyl-CoA Hydratase; D005947:Glucose; D006885:Hydroxybutyrates; D011091:Polyesters; D011994:Recombinant Proteins; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates