Ferricyanide reduction by Escherichia coli: Kinetics, mechanism, and application to the optimization of recombinant fermentations

Ferricyanide reduction was studied by now injection analysis (FIA) and chro noamperometry (CA) using two host strains and one recombinant strain of E. coli. Samples taken from batch cultures off, coli JM105 and HB101 showed ma ximal specific ferricyanide reduction rates in the late exponential phase o f growth, with values (mu mol/min.g) of 24 (FIA) and 17 (CA) for JM105, and 36 (FIA) for HB101, when shake-flask cultures were sampled, and 70 for HB1 01, when a chemostat was used to control pH and dissolved oxygen concentrat ion throughout the cultivation. Remarkably higher ferricyanide reduction ra tes were obtained with HB101 cells; cultivated continuously at very slow gr owth rate, when chilled, resuspended cell samples were incubated for 5 min in solutions containing 10 mM succinate or formate. These compounds are sub strates for primary, membrane-bound dehydrogenases that transfer electrons via ubiquinone to the cytochrome oxidase complexes. Apparent Michaelis-Ment en kinetics were observed with respect to ferricyanide concentration when 1 0 mM succinate was included in the assay buffer; apparent K-m values of 10. 1 +/- 0.6 mRI and 14.4 +/- 1.2 mM ferricyanide were obtained for exponentia l- and stationary-phase E. coli JM105, respectively. Cyanide inhibition stu dies show that ferricyanide is reduced mainly by cytochrome o oxidase in ex ponentially growing cells. The large difference in ferricyanide reduction r ates observed in the absence and presence of succinate and formate were use d to signal stationary-phase entry 5 h after induction of recombinant human Cu/Zn superoxide dismutase expression in a batch fermentation off. coli HM S174(DE3)(pET3ahSOD). This new method can be used as an adjunct to the quan titation of medium components for the optimization of recombinant fermentat ions.