Dissection of central carbon metabolism of hemoglobin-expressing Escherichia coli by C-13 nuclear magnetic resonance flux distribution analysis in microaerobic bioprocesses

Escherichia coli MG1655 cells expressing Vitreoscilla hemoglobin (VHb), Alc aligenes eutrophus flavohemoprotein (FI IP), the N-terminal hemoglobin doma in of FHP (FHPg), and a fusion protein which comprises VHb and the A. eutro phics C-terminal reductase domain (VHb-Red) were grown in a microaerobic bi oreactor to study the effects of low oxygen concentrations on the central c arbon metabolism, using fractional C-13-labeling of the proteinogenic amino acids and two-dimensional [C-13, H-1]-correlation nuclear magnetic resonan ce (NMR) spectroscopy. The NMR data revealed differences in the intracellul ar carbon fluxes between E. coli cells expressing either VHb or VHb-Red and cells expressing A. eutrophus FHP or the truncated heme domain (FHPg), E. coli MG1655 cells expressing either VHb or VHb-Red were found to function w ith a branched tricarboxylic acid (TCA) cycle. Furthermore, cellular demand s for ATP and reduction equivalents in VHb- and VHb-Red-expressing cells we re met by an increased flux through glycolysis. In contrast, in E. coli cel ls expressing A. eutrophus hemeproteins, the TCA cycle is running cyclicall y, indicating a shift towards a more aerobic regulation. Consistently, E. c oli cells displaying FHP and FHPg activity showed lo,ver production of the typical anaerobic by-products formate, acetate, and D-lactate, The implicat ions of these observations for biotechnological applications are discussed.