An earlier stoichiometric analysis of oxygen‐limited metabolism of Escherichia coli expressing cloned Vitreoscilla hemoglobin (VHb) suggested improved efficiency of ATP production relative to wild‐type controls [Khosla, C., Curtis, J. E., DeModena, J., Rinas, J. & Bailey, J. E. (1990) Bio‐Technol. 8, 849–853]. This hypothesis has been further examined by determining several energetic parameters of different VHb‐expressing E. coli (VHb+) strains relative to controls not expressing VHb (VHb–). The H+/O ratio, the transmembrane ΔpH, and the ATP content of VHb+ constructs are 1.5, 1.6 and 2 times, respectively, corresponding values in VHb– controls. VHb was expressed using a low‐copy‐number vector in E. coli mutant strains lacking cytochrome o, cytochrome d, or both terminal oxidases; significant growth enhancement due to VHb expression was observed only in the strain having functional cytochrome o and lacking cytochrome d. All of these data obtained using different E. coli strains are consistent with a model of VHb action that hypothesizes enhancement by VHb of activity of the lower oxygen‐affinity, higher proton‐pumping‐efficiency cytochrome o terminal oxidase under oxygen‐limited growth conditions.

The classical method of metabolic engineering, identifying a rate-determining step in a pathway and alleviating the bottleneck by enzyme overexpression, has motivated much research but has enjoyed only limited practical success. Intervention of other limiting steps, of counterbalancing regulation, and of unknown coupled pathways often confounds this direct approach. Here the concept of inverse metabolic engineering is codified and its application is illustrated with several examples. Inverse metabolic engineering means the elucidation of a metabolic engineering strategy by: first, identifying, constructing, or calculating a desired phenotype; second, determining the genetic or the particular environmental factors conferring that phenotype; and third, endowing that phenotype on another strain or organism by directed genetic or environmental manipulation. This paradigm has been successfully applied in several contexts, including elimination of growth factor requirements in mammalian cell culture and increasing the energetic efficiency of microaerobic bacterial respiration. 0 1996 John Wiley & Sons, Inc.

The classical method of metabolic engineering, identifying a rate-determining step in a pathway and alleviating the bottleneck by enzyme overexpression, has motivated much research but has enjoyed only limited practical success. Intervention of other limiting steps, of counter-balancing regulation, and of unknown coupled pathways often confounds this direct approach. Here the concept of inverse metabolic engineering is codified and its application is illustrated with several examples. Inverse metabolic engineering means the elucidation of a metabolic engineering strategy by: first, identifying, constructing, or calculating a desired phenotype; second, determining the genetic or the particular environmental factors conferring that phenotype; and third, endowing that phenotype on another strain or organism by directed genetic or environmental manipulation. This paradigm has been successfully applied in several contexts, including elimination of growth factor requirements in mammalian cell culture and increasing the energetic efficiency of microaerobic bacterial respiration.

No 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.