EFFECT OF VITREOSCILLA HEMOGLOBIN DOSAGE ON MICROAEROBIC ESCHERICHIA-COLI CARBON AND ENERGY-METABOLISM

The amount of Vitreoscilla hemoglobin (VHb) expression was modulated o ver a broad range with an isopropyl-beta-D-thiogalactopyranoside- (IPT G-) inducible plasmid, and the consequences on microaerobic Escherichi a coli physiology were examined in glucose fed-batch cultivations. The effect of IPTG induction on growth under oxygen-limited conditions wa s most visible during late fed-batch phase where the final cell densit y increased initially linearly with increasing VHb concentrations, ult imately saturating at a 2.7-fold increase over the VHb-negative (VHb(- )) control. During the same growth phase, the specific excretions of f ermentation by-products, acetate, ethanol, formate, lactate, and succi nate from the culture expressing the highest amount of VHb were reduce d 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. Var ying the amount of VHb from 0 to 3.8 mu mol/g dry cell weight (DCW) in creased the specific growth rate, the growth yield, and the oxygen con sumption rate by 33%, 35%, and 60%, respectively. Increasing VHb conce ntration to 3.8 mu mol/g DCW suppressed the rate of carbon dioxide evo lution 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 phosp hate pathway and a smaller fraction of carbon through the tricarboxyli c acid cycle from acetyl coenzyme A. The overall nicotinamide adenine dinucleotide [NAD(P)H] flux balance indicates that VHb-expressing cell s generate a net NADH flux by the NADH/NADPH transhydrogenase while th e VHb(-) cells yield a net NADPH flux under the same growth conditions . Flux distribution analysis also reveals that VHb(+) cells have a sma ller adenosine triphosphate (ATP) synthesis rate from substrate-level phosphorylation but a larger overall ATP production rate under microae robic conditions. The thermodynamic efficiency of growth, based on red ucing equivalents generated per unit of biomass produced, is greater f or VHb(+) cells. (C) 1996 John Wiley & Sons, Inc.