Effect of Vitreoscilla hemoglobin dosage on microaerobic Escherichia coli carbon and energy metabolism

Abstract

The amount of Vitreoscilla hemoglobin (VHb) expression was modulated over a broad range with an isopropyl-P-D-thiogalactopyranoside-(IPTG-) inducible plasmid, and the consequences on microaerobic Escherichia coli physiology were examined in glucose fed-batch cultivations. The effect of IPTG induction on growth under oxygenlimited conditions was most visible during late fed-batch phase where the final cell density increased initially linearly with increasing VHb concentrations, ultimately saturating at a 2.7-fold increase over the VHb-negative (VHb-) control. During the same growth phase, the specific excretions of fermentation by-products, acetate, ethanol, formate, lactate, and succinate from the culture expressing the highest amount of VHb were reduced by 25%, 49%, 68%, 72%, and 50%, respectively, relative to the VHb-control. During the exponential growth phase, VHb exerted a positive but smaller control on growth rate, growth yield, and respiration. Varying the amount of VHb from 0 to 3.8 pmol/g dry cell weight (DCW) increased the specific growth rate, the growth yield, and the oxygen consumption rate by 33%, 35%, and 6O%, respectively. Increasing VHb concentration to 3.8 kmolig DCW suppressed the rate of carbon dioxide evolution in the exponential phase by 30%. A metabolic flux distribution analysis incorporating data from these cultivations discloses that VHb+ cells direct a larger fraction of glucose toward the pentose phosphate pathway and a smaller fraction of carbon through the tricarboxylic acid cycle from acetyl coenzyme A. The overall nicotinamide adenine dinucleotide [NAD(P)H] flux balance indicates that VHb-expressing cells generate a net NADH flux by the NADHiNADPH transhydrogenase while the VHbcells yield a net NADPH flux under the same growth conditions. Flux distribution analysis also reveals that VHb+ cells have a smaller adenosine triphosphate (ATP) synthesis rate from substrate-level phosphorylation but a larger overall ATP production rate under microaerobic conditions. The thermodynamic efficiency of growth, based on reducing equivalents generated per unit of biomass produced, is greater for VHb' cells. 0 1996 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

“…148261) was purchased from Roche Diagnostics. Quantifications of D-lactate, succinate, and formate concentrations were performed using enzymatic assays adapted for the Beckman autoanalyzer system (5,49).…”

“…Direction of additional glucose through PPP generates an excess amount of NADPH and results in a transhydrogenation reaction, which creates an H ϩ flux from NADPH to NAD ϩ . VHbexpressing cells also displayed strongly reduced formate and D-lactate excretion levels relative to those of controls (49).…”

“…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). Direction of additional glucose through PPP generates an excess amount of NADPH and results in a transhydrogenation reaction, which creates an H ϩ flux from NADPH to NAD ϩ .…”

Plant Hemoglobins

“…Sequence comparisons indicate that Vitreoscilla Hb is more homologous to plant than to animal Hbs (Arredondo-Peter and Escamilla, 1991), suggesting that early plant Hbs could have had functions similar to Vitreoscilla Hb. For example, overproduction of Vitreoscilla Hb in E. coli enhances cell growth by changing flux patterns to the pentose phosphate, TCA, NADH, and ATP pathways, prolonging cell growth during oxygen-limited conditions (Tsai et al, 1996).…”

Expression of Vitreoscilla hemoglobin in Gordonia amarae enhances biosurfactant production

“…These figures are within the ranges seen in previous work. In some cases the presence of VHb in recombinant bacteria has resulted in no growth advantage or even a slight growth disadvantage compared to VHb-free cells [4,22], and sometimes to advantages as high as 2-3X on a dry cell mass basis [38] and as high as about 40X on a viable cell basis [39].…”