Dissection of central carbon metabolism of hemoglobin-expressing Escherichia coli by 13C nuclear magnetic resonance flux distribution analysis in microaerobic bioprocesses.
Escherichia coli MG1655 cells expressing Vitreoscilla hemoglobin (VHb), Alcaligenes eutrophus flavohemoprotein (FHP), the N-terminal hemoglobin domain of FHP (FHPg), and a fusion protein which comprises VHb and the A. eutrophus C-terminal reductase domain (VHb-Red) were grown in a microaerobic bioreactor to study the effects of low oxygen concentrations on the central carbon metabolism, using fractional (13)C-labeling of the proteinogenic amino acids and two-dimensional [(13)C, (1)H]-correlation nuclear magnetic resonance (NMR) spectroscopy. The NMR data revealed differences in the intracellular 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 with a branched tricarboxylic acid (TCA) cycle. Furthermore, cellular demands for ATP and reduction equivalents in VHb- and VHb-Red-expressing cells were met by an increased flux through glycolysis. In contrast, in E. coli cells expressing A. eutrophus hemeproteins, the TCA cycle is running cyclically, indicating a shift towards a more aerobic regulation. Consistently, E. coli cells displaying FHP and FHPg activity showed lower production of the typical anaerobic by-products formate, acetate, and D-lactate. The implications of these observations for biotechnological applications are discussed.
Frey
A D
AD
Institute of Biotechnology, ETH Zürich, CH-8093 Zürich, Switzerland.
Fiaux
J
J
Szyperski
T
T
Wüthrich
K
K
Bailey
J E
JE
Kallio
P T
PT
D000693:Anaerobiosis; D019149:Bioreactors; D001709:Biotechnology / Q000379:methods; D002244:Carbon / Q000378:metabolism*; D002247:Carbon Isotopes / Q000378:metabolism; D004926:Escherichia coli / Q000254:growth & development* / Q000378:metabolism; D006454:Hemoglobins / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy
D002247:Carbon Isotopes; D006454:Hemoglobins; D002244:Carbon