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

A. D. Frey, J. Fiaux, T. Szyperski, K. Wüthrich, J. E. Bailey, P. T. Kallio*

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

26 Citations (Scopus)

Abstract

Escherichia coli MG1655 cells expressing Vitreoscilla hemoglobin (VHb), Alcaligenes eutrophus flavohemoprorein (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 13C-labeling of the proteinogenic amino acids and two-dimensional [13C, 1H]-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.

Original languageEnglish
Pages (from-to)680-687
Number of pages8
JournalApplied and Environmental Microbiology
Volume67
Issue number2
DOIs
Publication statusPublished - Feb 2001
MoE publication typeA1 Journal article-refereed

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