Metabolic and kinetic analysis of poly(3-hydroxybutyrate) production by recombinant Escherichia coli

A quantitatively repeatable protocol was developed for poly(3-hydroxybutyra te) (PHB) production by Escherichia coli XL1-Blue (pSYL107). Two constant-g lucose fed-batch fermentations of duration 25 h were carried out in a 5-L b ioreactor, with the measured oxygen volumetric mass-transfer coefficient (k (L)a) held constant at 1.1 min(-1). All major consumption and production ra tes were quantified. The intracellular concentration profiles of acetyl-CoA (300 to 600 mug.g RCM-1) and 3-hydroxy-butyryl-CoA (20 to 40 mug.g RCM-1) were measured, which is the first time this has been performed for E. coli during PHB production. The kinetics of PHB production were examined and lik ely ranges were established for polyhydroxyalkanoate (PHA) enzyme activity and the concentration of pathway metabolites. These measured and estimated values are quite similar to the available literature estimates for the nati ve PHB producer Ralstonia eutropha. Metabolic control analysis performed on the PHB metabolic pathway showed that the PHB flux was highly sensitive to acetyl-CoA/CoA ratio (response coefficient 0.8), total acetyl-CoA + CoA co ncentration (response coefficient 0.7), and pH (response coefficient -1.25) . It was less sensitive (response coefficient 0.25) to NADPH/NADP ratio. NA DP(H) concentration (NADPH + NADP) had a negligible effect. No single enzym e had a dominant flux control coefficient under the experimental conditions examined (0.6, 0.25, and 0.15 for 3-ketoacyl-CoA reductase, PHA synthase, and 3-ketothiolase, respectively). In conjunction with metabolic flux analy sis, kinetic analysis was used to provide a metabolic explanation for the o bserved fermentation profile. In particular, the rapid onset of PHB product ion was shown to be caused by oxygen limitation, which initiated a cascade of secondary metabolic events, including cessation of TCA cycle flux and an increase in acetyl-CoA/CoA ratio. (C) 2001 John Wiley & Sons. Inc. Biotech nol Bioeng 74: 70-80, 2001.