Dissection of Central Carbon Metabolism of Hemoglobin-Expressing Escherichia coli by ¹³ C Nuclear Magnetic Resonance Flux Distribution Analysis in Microaerobic Bioprocesses

Abstract

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.Hemoglobins are a specific group of oxygen-binding proteins that can be found in mammals, plants, and microorganisms (15). The homodimeric hemoglobin of Vitreoscilla (VHb) is the best characterized bacterial hemoglobin. The expression of VHb is up-regulated by oxygen limitation (hypoxia) in Vitreoscilla (50), but its physiological functions have not yet been entirely elucidated. Nonetheless, heterologous expression of VHb has been used to alleviate physiologically unfavorable effects of oxygen limitation and to improve growth properties and productivity of various microorganisms, plants, and mammalian cells that yield industrially important metabolites (7, 18, 21, 28-30, 32, 35).Previous research on the effects arising from heterologous VHb expression has mainly focused on the operation of the respiratory chain. Expression of VHb in Escherichia coli cells lacking either cytochrome o (aerobic terminal oxidase) or cytochrome d complexes (microaerobic terminal oxidase) revealed a 5-fold increase in cytochrome o (in a cyd mutant, cyo ϩ strain) and a 1.5-fold increase in cytochrome d (in cyd ϩ , cyo mutant strain) complexes relative to wild-type cells (48). These results have led to the hypothesis that VHb is able to increase the effective intracellular oxygen concentration with concomitant increase of the amount of cytochrome o complexes (20): the proton translocation activity of cytochrome o complexes is characterized by a higher H/O ratio than cytochrome d complexes and is able to generate a larger proton gradient across the cell membrane (25,36,38). VHb-expressing cells are indeed able to generate a larger proton flux ...

Bacterial Hemoglobins and Flavohemoglobins for Alleviation of Nitrosative Stress in Escherichia coli

“…(20,21). FHP-or FHPg-expressing E. coli strains had higher oxygen uptake rates than strains producing VHb or VHb-Red under microaerobic conditions (9). These results correlate well with the observed higher NO ⅐ turnover rate of FHP and FHPg relative to VHb and VHb-Red.…”

Enhancement of ethanol production from potato-processing wastewater by engineering Escherichia coli using Vitreoscilla haemoglobin

“…Two other studies in E. coli found either substantially higher (66%; Frey et al . ) or lower (49%; Tsai et al . ) ethanol production correlated with VHb expression.…”

Flavohemoglobin Detoxifies Nitric Oxide in Aerobic, but Not Anaerobic, Escherichia coli

“…5) may limit the survival value of this NO detoxification system during anaerobic-to-aerobic transitions. Furthermore, growth stimulatory effects of flavoHbs and homologous single domain Hbs have been previously reported for microaerobic growth conditions and have been associated with altered glycolytic and citric acid cycle intermediates and activities (40). Thus, flavoHb may additionally benefit cells in which aconitase, the citric acid cycle, respiration, and energy production are threatened by NO inhibition.…”