Improvement of Escherichia coli microaerobic oxygen metabolism by Vitreoscilla hemoglobin: New insights from NAD(P)H fluorescence and culture redox potential

Abstract

On-line NAD(P)H fluorescence and culture redox potential (CRP) measurements were utilized to investigate the role of Vitreoscilla hemoglobin (VHb) in perturbing oxygen metabolism of microaerobic Escherichia coli Batch cultures of a VHb-synthesizing E. coli strain and the iso-genic control under fully aerated conditions were subject to several high/low oxygen transitions, and the NAD(P)H fluorescence and CRP were monitored during these passages. The presence of VHb decreased the rate of net NAD(P)H generation by 2.4-fold under diminishing oxygen tension. In the absence of aeration, the strain producing VHb maintained a steady NAD(P)H level 1.8-fold less than that of the control, indicating that the presence of VHb keeps E. coli in a more oxidized state under oxygen-limited conditions. Estimated from CRP, the oxygen uptake rates near anoxia were 25% higher for cells with VHb than those without. These results suggest that VHb-expressing cells have a higher microaerobic electron transport chain turnover rate. To examine how NAD(P)H utilization of VHb-expressing cells responds to rapidly changing oxygen tension, which is common in large-scale fermentations, we pulsed air intermittently into a cell suspension and recorded the fluorescence response to the imposed dissolved oxygen (DO) fluctuation. Relative to the control, cells containing VHb had a sluggish fluorescence response to sudden changes of oxygen tension, suggesting that VHb buffers intracellular redox perturbations caused by extracellular DO fluctuations.(c) John Wiley & Sons, Inc.

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

“…The association equilibrium constants (K) determined for FHP and VHb attribute high oxygen affinity to FHP (K ϭ 250 M Ϫ1 ) and low oxygen affinity to VHb (K ϭ 0.014 M Ϫ1 ). Due to the very high values for k off , VHb-expressing cells have previously been proposed to scavenge oxygen and possibly provide O 2 rapidly to respiratory complexes in E. coli (20,(47)(48)(49). The mechanism of biochemical action of FHP globin on the cellular metabolism of E. coli is still unknown.…”

“…Furthermore, it has also been reported that the steady-state NAD(P)H level is 1.8-fold lower in a VHb-expressing strain than in control cells grown under nearly anoxic conditions (47), i.e., cells are in a more reduced state under oxygen-limited conditions (16). Anoxia is known to reduce electron flow through the respiratory chain so that NAD(P)H is consumed more slowly.…”

“…Therefore, one may hypothesize that, under low oxygen tension, the presence of VHb in E. coli increases the electron flux through the respiratory chain. Moreover, Tsai et al (47) postulated that this shift in the NAD ϩ /NADH concentration ratio might have implications on key steps of the central carbon metabolism in VHb-expressing E. coli. Therefore, metabolic flux analysis (MFA) was performed and a metabolic model suggested that VHb-positive cells direct a higher fraction of glucose through the pentose phosphate pathway (PPP) and channel less acetyl coenzyme A (AcCoA) through the tricarboxylic acid cycle (TCA) than wild-type E. coli (49).…”

Expression of Alcaligenes eutrophus Flavohemoprotein and Engineered Vitreoscilla Hemoglobin-Reductase Fusion Protein for Improved Hypoxic Growth of Escherichia coli

“…The expression of these two proteins resulted in 2.2-fold (FHP) and 2.5-fold (VHb-FAD-NAD) increases in final cell densities relative to the VHb-expressing strain. VHb expression has been shown to modulate cellular metabolism of E. coli (7,18,27,(38)(39)(40). VHb influences the energy level by interacting with the respiratory chain, thus leading to increased proton pumping, higher ATP production rates, and a less reduced contingent of pyridine nucleotide cofactors (7,18,38,39).…”

“…Although the exact mechanism by which VHb causes these effects is unknown, it has been hypothesized that due to its unusual kinetic parameters for oxygen binding and release (K D ϭ 72 M) (45), VHb is able to scavenge oxygen molecules from solution and provide them for cellular activities in heterologous organisms (18,45,49). More detailed observations of biochemical and physiological changes accompanying VHb expression in Escherichia coli indicate increased overall ATP production and turnover rates, increase in cytochrome o expression and specific activity, decreased levels of reduced pyridine nucleotides, and changes in central carbon metabolism (7,18,27,(38)(39)(40). Experiments were conducted with engineered E. coli to determine if globins besides VHb can be applied to enhance hypoxic growth and protein production levels.…”