Osmotic stress response: quantification of cell maintenance and metabolic fluxes in a lysine-overproducing strain of Corynebacterium glutamicum

Osmotic stress diminishes cell productivity and may cause cell inactivation in industrial fermentations. The quantification of metabolic changes under such conditions is fundamental for understanding and describing microbial behavior during bioprocesses. We quantified the gradual changes that take place when a lysine-overproducing strain of Corynebacterium glutamicum is grown in continuous culture with saline gradients at different dilution rates. The use of compatible solutes depended on environmental conditions; certain osmolites predominated at different dilution rates and extracellular osmolalities. A metabolic flux analysis showed that at high dilution rates C. glutamicum redistributed its metabolic fluxes, favoring energy formation over growth. At low dilution rates, cell metabolism accelerated as the osmolality was steadily increased. Flexibility in the oxaloacetate node proved to be key for the energetic redistribution that occurred when cells were grown at high dilution rates. Substrate and ATP maintenance coefficients increased 30- and 5-fold, respectively, when the osmolality increased, which demonstrates that energy pool management is fundamental for sustaining viability.     

Impact of transport processes in the osmotic response of Corynebacterium glutamicum

Osmoregulation, the adaptation of cells to changes in the external osmolarity, is an important aspect of the bacterial stress response, in particular for a soil bacterium like Corynebacterium glutamicum. Consequently, this organism is equipped with several redundant systems for coping with both hyper- and hypoosmotic stress. For the adaptation to hypoosmotic stress C. glutamicum possesses at least three different mechanosensitive (MS) channels. To overcome hyperosmotic stress C. glutamicum accumulates so-called compatible solutes either by means of biosynthesis or by uptake. Uptake of compatible solutes is in general preferred to de novo synthesis because of lower energy costs. Noticeable, only secondary transporters belonging to the MHS (ProP) or the BCCT-family (BetP, EctP and LcoP) are involved in the uptake of proline, betaine and ectoine. In contrast to Escherichia coli or Bacillus subtilis no ABC-transporters were found catalyzing uptake of compatible solutes. BetP was one of the first examples of the growing group of osmosensory proteins to be analyzed in detail. This transporter is characterized, besides the catalytic activity of betaine uptake, by the ability to sense osmotic changes (osmosensing) and to respond to the extent of osmotic stress by adaptation of transport activity (osmoregulation). BetP detects hyperosmotic stress via an increase in the internal K(+) concentration following a hyperosmotic shift, and thus acts as a chemosensor.     

Modelling and analysis of central metabolism operating regulatory interactions in salt stress conditions in a L-carnitine overproducing E. coli strain

Based on experimental data from E. coli cultures, we have devised a mathematical model in the GMA-power law formalism that describes the central and L-carnitine metabolism in and between two steady states, non-osmotic and hyperosmotic (0.3 M NaCl). A key feature of this model is the introduction of type of kinetic order, the osmotic stress kinetic orders (g(OSn)), derived from the power law general formalism, which represent the effect of osmotic stress in each metabolic process of the model.By considering the values of the g(OSn) linked to each metabolic process we found that osmotic stress has a positive and determinant influence on the increase in flux in energetic metabolism (glycolysis); L-carnitine biosynthesis production; the transformation/excretion of Acetyl-CoA into acetate and ethanol; the input flux of peptone into the cell; the anabolic use of pyruvate and biomass decomposition. In contrast, we found that although the osmotic stress has an inhibitory effect on the transformation flux from the glycolytic end products (pyruvate) to Acetyl-CoA, this inhibition is counteracted by other effects (the increase in pyruvate concentration) to the extent that the whole flux increases. In the same vein, the down regulation exerted by osmotic stress on fumarate uptake and its oxidation and the production and export of lactate and pyruvate are reversed by other factors up to the point that the first increased and the second remained unchanged.The model analysis shows that in osmotic conditions the energy and fermentation pathways undergo substantial rearrangement. This is illustrated by the observation that the increase in the fermentation fluxes is not connected with fluxes towards the tricaboxylic acid intermediates and the synthesis of biomass. The osmotic stress associated with these fluxes reflects these changes. All these observations support that the responses to salt stress observed in E. coli might be conserved in halophiles.Flux evolution during osmotic adaptations showed a hyperbolic (increasing or decreasing) pattern except in the case of peptone and fumarate uptake by the cell, which initially decreased. Finally, the model also throws light on the role of L-carnitine as osmoprotectant.     

Engineering metabolism and product formation in Corynebacterium glutamicum by coordinated gene overexpression

Single gene overexpression in product pathways such as lysine synthesis has often been employed in metabolic engineering efforts aiming at pathway flux amplification and metabolite overproduction. This approach is limited due to metabolic flux imbalances that often lead to unpredictable physiological responses and suboptimal metabolite productivity. This deficiency can be overcome by the coordinated overexpression of more than one flux controlling genes in a production pathway selected by considering their individual contributions on the cell physiology This concept is demonstrated by the simultaneous overexpression of pyruvate carboxylase and aspartate kinase, two key enzymes in central carbon metabolism and the lysine production pathway in Corynebacterium glutamicum. Contrary to expectations based on the importance of each of these two genes in lysine production, the monocistronic overexpression of either gene results in marginal changes in the overall lysine productivity due to either reduced cell growth or reduced lysine specific productivity. In contrast, the simultaneous amplification of the activities of the two enzymes yielded more than 250% increase of the lysine specific productivity in lactate minimal medium without affecting the growth rate or final cell density of the culture. These results demonstrate that significant flux amplification in complex pathways involving central carbon metabolism is possible through coordinated overexpression of more than one gene in the pathway. This can be achieved either by external, gene expression inducing, controls or controls responding to the physiological cellular state.     

Metabolic pathway analysis for rational design of L-methionine production by Escherichia coli and Corynebacterium glutamicum

Metabolic pathway analysis was carried out to predict the metabolic potential of Corynebacterium glutamicum and Escherichia coli for the production of L-methionine. Based on detailed stoichiometric models for these organisms, this allowed the calculation of the theoretically optimal methionine yield and related metabolic fluxes for various scenarios involving different mutants and process conditions. The theoretical optimal methionine yield on the substrates glucose, sulfate and ammonia for the wildtype of C. glutamicum is 0.49 (C-mol) (C-mol)(-1), whereas the E. coli wildtype exhibits an even higher potential of 0.52 (C-mol) (C-mol)(-1). Both strains showed completely different optimal flux distributions. C. glutamicum has a high flux through the pentose phosphate pathway (PPP), whereas the TCA cycle flux is very low. Additionally, it recruits a metabolic cycle, which involves 2-oxoglutarate and glutamate. In contrast, E. coli does minimize the flux through the PPP, and the flux through the TCA cycle is high. The improved potential of the E. coli wildtype is due to its membrane-bound transhydrogenase and its glycine cleavage system as shown by additional simulations with theoretical mutants. A key point for maximizing methionine yield is the choice of the sulfur source. Replacing sulfate by thiosulfate or sulfide increased the maximal theoretical yield in C. glutamicum up to 0.68 (C-mol) (C-mol)(-1). A further increase is possible by the application of additional C1 sources. The highest theoretical potential was obtained for C. glutamicum applying methanethiol as combined source for C1 carbon and sulfur (0.91 (C-mol) (C-mol)(-1)). Substrate requirement for maintenance purposes reduces theoretical methionine yields. In the case of sulfide used as sulfur source a maintenance requirement of 9.2 mmol ATP g(-1) h(-1), as was observed under stress conditions, would reduce the maximum theoretical yield from 67.8% to 47% at a methionine production rate of 0.65 mmol g(-1) h(-1). The enormous capability of both organisms encourages the development of biotechnological methionine production, whereby the use of metabolic pathway analysis, as shown, provides valuable advice for future strategies in strain and process improvement.     

Cell Death Induced by Mild Physical Perturbations Could Be Related to Transient Plasma Membrane Modifications

An understanding of membrane destabilization induced by osmotic treatments is important to better control cell survival during biotechnological processes. The effects on the membranes of the yeast Saccharomyces cerevisiae of perturbations similar in intensity (same amount of energy) but differing in the source type (heat, compression and osmotic gradient) were investigated. The anisotropy of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene was measured before and after each treatment to assess the reversibility of the membrane changes related to each treatment. Except for heat shock at 75°C, changes in membrane fluidity were reversible after the return to initial conditions, showing that two kinds of physical stress can be distinguished regarding the reversibility of membrane changes: high and mild energy stresses. With the application of osmotic gradients, anisotropy was assessed during treatment with five osmotic pressure levels from 30.7 to 95.4 MPa with two different yeast strains and related to the rate of cell death caused by each stress. The exposure of cells to increasing osmotic pressures involved a progressive lowering of membrane anisotropy during lethal perturbations. Osmotic stresses associated with reversible fluidity changes of increasing intensity in the membrane led to proportional death rates and time-dependant cell death of increasing rapidity during the application of the stress. Finally, a hypothesis relating the extent of membrane structural changes to the kinetic of cell death is proposed.     

Metabolic phenotype of phosphoglucose isomerase mutants of Corynebacterium glutamicum

A series of experiments reported in the literature using fluxomics as an efficient functional genomics tool revealed that the L-lysine production of the Corynebacterium glutamicum strain MH20-22B correlates with the extent of intracellular NADPH supply. Some alternative metabolic engineering strategies to increase intracellular NADPH supply in the C. glutamicum strain DSM5715 were considered and finally the redirection of carbon flux through the pentose phosphate pathway with two NADPH generating enzymatic reactions was favored. Elsewhere, the construction of a phosphoglucose isomerase (Pgi) null mutant of the C. glutamicum strain DSM5715 has been described by utilizing genetic engineering as well as some aspects of its metabolic phenotype. Most interestingly, it was shown that not only could the L-lysine formation be increased by 1.7-fold but the by-product concentration for the null mutant strain was also able to be drastically reduced. In this publication we discuss this metabolic phenotype in detail and present additional data on by-product formation as well as yield considerations. Results from isotope based metabolic flux analysis in combination with considerations on NADPH metabolism clearly exclude the existence of Pgi isoenzymes in C. glutamicum strain DSM5715. The genome region containing the pgi gene was analyzed. It cannot be excluded that polar effects might have been caused by the disruption of the pgi gene and might have contributed to the observed metabolic phenotype of C. glutamicum Pgi mutants. We illustrate growth characteristics of a Pgi mutant of an industrial L-lysine production strain. A reduced growth rate and a biphasic growth behavior was observed. The importance of NADPH reoxidation for well balanced growth in Pgi mutants is discussed. Another phosphoglucose isomerase mutant of C. glutamicum has been described in literature with which an increase in L-lysine yield from 42 to 52% was observed. This finding highlights the general potential of metabolic flux redirection towards the pentose phosphate pathway, which could be used for metabolic engineering of the biotechnological synthesis of (1) aromatic amino acids and (2) chemicals whose synthesis depends on intracellular NADPH supply.     

Der Einfluss verschiedener Salze und verschiedener Inhibitoren auf den Wurzeldruck von Zea mays

The effect of different salts and inhibitors on the root pressure of Zea mays.— The influence of various salt solutions and inhibitors on the exudation rate has been investigated with young excised primary roots of Zea mays. The following results were obtained. — The effect of chlorides on the exudation rate was higher than the effect of sulphates K⁺ and Na⁺ effected higher flux rates than Ca²⁺ and Mg²⁺ The highest exudation rate was obtained with KCl. — In comparison to an isotonic Lutrol-solution (a liquid condensed polyethylenoxid) a 0.5525 molar KCl-solution, applicated on the root stump, increased the exudation rate considerably. — Metabolic inhibitors and anaerobic conditions decreased the exudation rate. — Experiments, concerning the influence of metabolic inhibitors on the exudation and on the Rb-uptake showed a highly significant positive correlation (r =+0.72***) between the exudation rates and the Rb-concentrations in the exudates. The Rb-accumulation in the root tissue was not correlated to the exudation rate. — The experimental data agree with the concept of a transversal water transport in the root tissue, effected by osmotic forces. The root pressure is based on the osmotic gradient between the xylem sap and the outer solution. This gradient is built up by the metabolic secretion of ions into the xylem sap. It is supposed that the transversal water transport in the roots mainly goes through the free space of the cortex.     

Effects of the changes in enzyme activities on metabolic flux redistribution around the 2-oxoglutarate branch in glutamate production by<Emphasis Type="Italic"> Corynebacterium glutamicum</Emphasis>

An experimental method for metabolic control analysis (MCA) was applied to the investigation of a metabolic network of glutamate production by Corynebacterium glutamicum. A metabolic reaction (MR) model was constructed and used for flux distribution analysis (MFA). The flux distribution at a key branch point, 2-oxoglutarate, was investigated in detail. Activities of isocitrate dehydrogenase (ICDH), glutamate dehydrogenase (GDH), and 2-oxoglutarate dehydrogenase complex (ODHC) around this the branch point were changed, using two genetically engineered strains (one with enhanced ICDH activity and the other with enhanced GDH activity) and by controlling environmental conditions (i.e. biotin-deficient conditions). The mole flux distribution was determined by an MR model, and the effects of the changes in the enzyme activities on the mole flux distribution were compared. Even though both GDH and ICDH activities were enhanced, the mole flux distribution was not significantly changed. When the ODHC activity was attenuated, the flux through ODHC decreased, and glutamate production was markedly increased. The flux control coefficients of the above three enzymes for glutamate production were determined based on changes in enzyme activities and the mole flux distributions. It was found that the factor with greatest impact on glutamate production in the metabolic network was obtained by attenuation of ODHC activity.     

Metabolic network analysis during fed-batch cultivation of Corynebacterium glutamicum for pantothenic acid production: first quantitative data and analysis of by-product formation

A first generation genetically modified strain of Corynebacterium glutamicum has been assessed for its potential to synthesise and accumulate the vitamin pantothenic acid in the medium using fed-batch cultivation technology, with biomass concentration controlled by isoleucine limitation. Kinetic analysis of specific rates throughout the process has been used to model carbon flux through both central metabolism and the specific pathways involved in product formation. Flux towards pantothenic acid is potentially high but much of this flux is dissipated as by-products within associated pathways, notably linked to amino acid synthesis. The major limitation of vitamin production in this strain is linked to the tenfold higher flux of keto-isovalerate towards valine rather than pantothenic acid. Attempts to modify this ratio by imposing nitrogen limitation provoked carbon overflow as unidentified non-nitrogenous compounds. The observed accumulation of glycine suggests that the flux towards pantothenate production may by limited by the rate of the pathway intermediate (5,10-methylene-tetrahydrofolate) regeneration.     

Responding phospholipid membranes--interplay between hydration and permeability.

Osmotic forces are important in regulating a number of physiological membrane processes. The effect of osmotic pressure on lipid phase behavior is of utmost importance for the extracellular lipids in stratum corneum (the outer part of human skin), due to the large gradient in water chemical potential between the water-rich tissue on the inside, and the relative dry environment on the outside of the body. We present a theoretical model for molecular diffusional transport over an oriented stack of two-component lipid bilayers in the presence of a gradient in osmotic pressure. This gradient serves as the driving force for diffusional motion of water. It also causes a gradient in swelling and phase transformations, which profoundly affect the molecular environment and thus the local diffusion properties. This feedback mechanism generates a nonlinear transport behavior, which we illustrate by calculations of the flux of water and solute (nicotine) through the bilayer stack. The calculated water flux shows qualitative agreement with experimental findings for water flux through stratum corneum. We also present a physical basis for the occlusion effect. Phase behavior of binary phospholipid mixtures at varying osmotic pressures is modeled from the known interlamellar forces and the regular solution theory. A first-order phase transformation from a gel to a liquid--crystalline phase can be induced by an increase in the osmotic pressure. In the bilayer stack, a transition can be induced along the gradient. The boundary conditions in water chemical potential can thus act as a switch for the membrane permeability.     

Glycerolization of Chinese hamster ovary cells in monolayer culture

CHO monolayer cultures were used as a model system to examine the kinetics of cell glycerolization and deglycerolization. Both influx and efflux of [³H]glycerol appear to be first-order processes, the rate of flux being proportional to glycerol concentration. When net flux ceases, the intracellular and extracellular concentrations of glycerol appear to be identical. Influx can be described by a single first-order curve, whereas efflux requires at least two such functions. Flux rates are sensitive to changes in osmolality, the fast efflux component accelerating and the slow component decelerating with increased application of osmotic stress. The rate of influx slows as the temperature drops and as the viscosity of the medium increases. Cells can tolerate a much steeper deglycerolizing gradient at 37 °C than at 0 °C.     

Glycine betaine uptake after hyperosmotic shift in Corynebacterium glutamicum.

Osmoregulatory uptake of glycine betaine in whole cells of Corynebacterium glutamicum ATCC 13032 (wild type) was studied. The cells actively take up glycine betaine when they are osmotically shocked. The total accumulation and uptake rate were dependent on the osmotic strength of the medium. Kinetic analysis revealed a high-affinity transport system (Km, 8.6 +/- 0.4 microM) with high maximum velocity (110 nmol.min-1.mg [dry weight]-1). Glycine betaine functioned as a compatible solute when added to the medium and allowed growth at an otherwise inhibitory osmotic strength of 1.5 M NaCl. Proline and ectoine could also be used as osmoprotectants. Glycine betaine is neither synthesized nor metabolized by C. glutamicum. The glycine betaine transport system is constitutively expressed at a basal level of activity. It can be induced up to eightfold by osmotic stress and is strongly regulated at the level of activity. The transport system is highly specific and has its pH optimum in the slightly alkaline range at about pH 8. The uptake of the zwitterionic glycine betaine is mediated by a secondary symport system coupled to cotransport of at least two Na+ ions. It is thus driven both by the membrane potential and the Na+ gradient. An extremely high accumulation (internal/external) ratio of up to 4 x 10(6) was measured, which represents the highest accumulation ratio observed for any transport system.     

Comparative study of flux redistribution of metabolic pathway in glutamate production by two coryneform bacteria

In amino acid production by coryneform bacteria, study on relationship between change in enzyme activities and production of a target amino acid is important. In glutamate production, Kawahara et al. discovered that the effect of decrease in 2-oxoglutamate dehydrogenase complex (ODHC) on glutamate production is essential (Kawahara et al., Biosci. Biotechnol. Biochem. 61(7) (1997) 1109). Significant reduction of the ODHC activity was observed in the cells under the several glutamate-productive conditions in Corynebacterium glutamicum. Recent progress in metabolic engineering enables us to quantitatively compare the flux redistribution of the different strains after change in enzyme activity precisely. In this paper, relationship between flux redistribution and change in enzyme activities after biotin deletion and addition of detergent (Tween 40) was studied in two coryneform bacteria, C. glutamicum and a newly isolated strain, Corynebacterium efficiens (Fudou et al., Int. J. Syst. Evol. Microbiol. 52(Part 4) 1127), based on metabolic flux analysis (MFA). It was observed that in both species the specific activities of isocitrate dehydrogenase (ICDH) and glutamate dehydrogenase (GDH) did not significantly change throughout the fermentation, while that of the ODHC significantly decreased after biotin depletion and Tween 40 addition. Flux redistribution clearly occurred after the decrease in ODHC specific activity. The difference in glutamate production between C. glutamicum and C. efficiens was caused by the difference in the degree of decrease in ODHC specific activity. The difference in Michaelis-Menten constants or K(m) value between ICDH, GDH, and ODHC explained the mechanism of flux redistribution at the branch point of 2-oxoglutarate. It was found that the K(m) values of ICDH and ODHC were much lower than that of GDH for both strains. It was quantitatively proved that the ODHC plays the most important role in controlling flux distribution at the key branch point of 2-oxoglutarate in both coryneform bacteria. Flux redistribution mechanism was well simulated by a Michaelis-Menten-based model with kinetic parameters. The knowledge of the mechanism of flux redistribution will contribute to improvement of glutamate production in coryneform bacteria.     

Genealogy profiling through strain improvement by using metabolic network analysis: metabolic flux genealogy of several generations of lysine-producing corynebacteria

A comprehensive approach of metabolite balancing, (13)C tracer studies, gas chromatography-mass spectrometry, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and isotopomer modeling was applied for comparative metabolic network analysis of a genealogy of five successive generations of lysine-producing Corynebacterium glutamicum. The five strains examined (C. glutamicum ATCC 13032, 13287, 21253, 21526, and 21543) were previously obtained by random mutagenesis and selection. Throughout the genealogy, the lysine yield in batch cultures increased markedly from 1.2 to 24.9% relative to the glucose uptake flux. Strain optimization was accompanied by significant changes in intracellular flux distributions. The relative pentose phosphate pathway (PPP) flux successively increased, clearly corresponding to the product yield. Moreover, the anaplerotic net flux increased almost twofold as a consequence of concerted regulation of C(3) carboxylation and C(4) decarboxylation fluxes to cover the increased demand for lysine formation; thus, the overall increase was a consequence of concerted regulation of C(3) carboxylation and C(4) decarboxylation fluxes. The relative flux through isocitrate dehydrogenase dropped from 82.7% in the wild type to 59.9% in the lysine-producing mutants. In contrast to the NADPH demand, which increased from 109 to 172% due to the increasing lysine yield, the overall NADPH supply remained constant between 185 and 196%, resulting in a decrease in the apparent NADPH excess through strain optimization. Extrapolated to industrial lysine producers, the NADPH supply might become a limiting factor. The relative contributions of PPP and the tricarboxylic acid cycle to NADPH generation changed markedly, indicating that C. glutamicum is able to maintain a constant supply of NADPH under completely different flux conditions. Statistical analysis by a Monte Carlo approach revealed high precision for the estimated fluxes, underlining the fact that the observed differences were clearly strain specific.     

Impact of osmotic stress on volume regulation,cytoplasmic solute composition and lysine production in Corynebacterium glutamicum MH20-22B

The response of the L-lysine producing Corynebacterium glutamicum strain MH20-22B to osmotic stress was studied in batch cultures. To mimic the conditions during a fermentation process the long term adaptation of cells subjected to a constant osmotic stress between 1.0 and 2.5 osM was investigated. Cytoplasmic water content and volume of C. glutamicum cells were found to depend on growth phase, extent of osmotic stress and availability of betaine. The maximal cytoplasmic volumes, which were highest at maximal growth rate, were linearily related to osmotic stress, whereas in stationary cells no active volume regulation was observed. Under severe osmotic stress proline was the prominent compatible solute in growing cells. Uptake of betaine, if available in the medium, reduced the concentration of proline from 750 to 300 mM, indicating that uptake of compatible solutes is preferred to synthesis. Furthermore, betaine was shown to have a higher efficiency to counteract osmotic stress, since the overall concentration of compatible solutes was lower in the presence of betaine. Under severe osmotic stress, the addition of betaine shifted L-lysine production in MH20-22B to earlier fermentation times and increased both product concentration and yield in these phases, but did not improve the final L-lysine yield.     

Analysis of Growth and Water Relations of Tomato Fruits in Relation to Air Vapor Pressure Deficit and Plant Fruit Load

The influence of air vapor pressure deficit (VPD) and plant fruit load on the expansion and water relations of young tomato fruits grown in a glasshouse were evaluated under summer Mediterranean conditions. The contributions of phloem, xylem and transpiration fluxes to the fruit volume increase were estimated at an hourly scale from the growth curves of intact, heat-girdled and detached fruits, measured using displacement transducers. High VPD conditions reduced the xylem influx and increased the fruit transpiration, but hardly affected the phloem influx. Net water accumulation and growth rate were reduced, and a xylem efflux even occurred during the warmest and driest hours of the day. Changes in xylem flux could be explained by variations in the gradient of water potential between stem and fruit, due to changes in stem water potential. Misting reduced air VPD and alleviated the reduction in fruit volume increase through an increase in xylem influx and a decrease in fruit transpiration. Under low fruit load, the competition for assimilates being likely reduced, the phloem flux to fruits increased, similarly to the xylem and transpiration fluxes, without any changes in the fruit water potential. However, different diurnal dynamics among treatments assume variable contributions of turgor and osmotic pressure in F3 and F6 fruits, and hypothetical short-term variations in the water potential gradient between stem and fruit, preventing xylem efflux in F3 fruits.     

The rate of metabolism in marine animals: environmental constraints, ecological demands and energetic opportunities

The rates of metabolism in animals vary tremendously throughout the biosphere. The origins of this variation are a matter of active debate with some scientists highlighting the importance of anatomical or environmental constraints, while others emphasize the diversity of ecological roles that organisms play and the associated energy demands. Here, we analyse metabolic rates in diverse marine taxa, with special emphasis on patterns of metabolic rate across a depth gradient, in an effort to understand the extent and underlying causes of variation. The conclusion from this analysis is that low rates of metabolism, in the deep sea and elsewhere, do not result from resource (e.g. food or oxygen) limitation or from temperature or pressure constraint. While metabolic rates do decline strongly with depth in several important animal groups, for others metabolism in abyssal species proceeds as fast as in ecologically similar shallow-water species at equivalent temperatures. Rather, high metabolic demand follows strong selection for locomotory capacity among visual predators inhabiting well-lit oceanic waters. Relaxation of this selection where visual predation is limited provides an opportunity for reduced energy expenditure. Large-scale metabolic variation in the ocean results from interspecific differences in ecological energy demand.