Energy coupling in Saccharomyces cerevisiae: selected opportunities for metabolic engineering

Free-energy (ATP) conservation during product formation is crucial for the maximum product yield that can be obtained, but often overlooked in metabolic engineering strategies. Product pathways that do not yield ATP or even demand input of free energy (ATP) require an additional pathway to supply the ATP needed for product formation, cellular maintenance, and/or growth. On the other hand, product pathways with a high ATP yield may result in excess biomass formation at the expense of the product yield. This mini-review discusses the importance of the ATP yield for product formation and presents several opportunities for engineering free-energy (ATP) conservation, with a focus on sugar-based product formation by Saccharomyces cerevisiae. These engineering opportunities are not limited to the metabolic flexibility within S.?cerevisiae itself, but also expression of heterologous reactions will be taken into account. As such, the diversity in microbial sugar uptake and phosphorylation mechanisms, carboxylation reactions, product export, and the flexibility of oxidative phosphorylation via the respiratory chain and H(+) -ATP synthase can be used to increase or decrease free-energy (ATP) conservation. For product pathways with a negative, zero or too high ATP yield, analysis and metabolic engineering of the ATP yield of product formation will provide a promising strategy to increase the product yield and simplify process conditions.     

Engineering cytosolic acetyl-coenzyme A supply in Saccharomyces cerevisiae: Pathway stoichiometry,free-energy conservation and redox-cofactor balancing

Saccharomyces cerevisiae is an important industrial cell factory and an attractive experimental model for evaluating novel metabolic engineering strategies. Many current and potential products of this yeast require acetyl coenzyme A (acetyl-CoA) as a precursor and pathways towards these products are generally expressed in its cytosol. The native S. cerevisiae pathway for production of cytosolic acetyl-CoA consumes 2 ATP equivalents in the acetyl-CoA synthetase reaction. Catabolism of additional sugar substrate, which may be required to generate this ATP, negatively affects product yields. Here, we review alternative pathways that can be engineered into yeast to optimize supply of cytosolic acetyl-CoA as a precursor for product formation. Particular attention is paid to reaction stoichiometry, free-energy conservation and redox-cofactor balancing of alternative pathways for acetyl-CoA synthesis from glucose. A theoretical analysis of maximally attainable yields on glucose of four compounds (n-butanol, citric acid, palmitic acid and farnesene) showed a strong product dependency of the optimal pathway configuration for acetyl-CoA synthesis. Moreover, this analysis showed that combination of different acetyl-CoA production pathways may be required to achieve optimal product yields. This review underlines that an integral analysis of energy coupling and redox-cofactor balancing in precursor-supply and product-formation pathways is crucial for the design of efficient cell factories.     

Evolutionary optimization of metabolic pathways. Theoretical reconstruction of the stoichiometry of ATP and NADH producing systems

The structural design of ATP and NADH producing systems, such as glycolysis and the citric acid cycle (TCA), is analysed using optimization principles. It is assumed that these pathways combined with oxidative phosphorylation have reached, during their evolution, a high efficiency with respect to ATP production rates. On the basis of kinetic and thermodynamic principles, conclusions are derived concerning the optimal stoichiometry of such pathways. Extending previous investigations, both the concentrations of adenine nucleotides as well as nicotinamide adenine dinucleotides are considered variable quantities. This implies the consideration of the interaction of an ATP and NADH producing system, an ATP consuming system, a system coupling NADH consumption with ATP production and a system consuming NADH decoupled from ATP production. It is examined in what respect real metabolic pathways can be considered optimal by studying a large number of alternative pathways. The kinetics of the individual reactions are described by linear or bilinear functions of reactant concentrations. In this manner, the steady-state ATP production rate can be calculated for any possible ATP and NADH producing pathway. It is shown that most of the possible pathways result in a very low ATP production rate and that the very efficient pathways share common structural properties. Optimization with respect to the ATP production rate is performed by an evolutionary algorithm. The following results of our analysis are in close correspondence to the real design of glycolysis and the TCA cycle. (1) In all efficient pathways the ATP consuming reactions are located near the beginning. (2) In all efficient pathways NADH producing reactions as well as ATP producing reactions are located near the end. (3) The number of NADH molecules produced by the consumption of one energy-rich molecule (glucose) amounts to four in all efficient pathways. A distance measure and a measure for the internal ordering of reactions are introduced to study differences and similarities in the stoichiometries of metabolic pathways.     

Kinetic and thermodynamic principles determining the structural design of ATP-producing systems

It is theoretically analysed whether the structural design of ATP-producing pathways, in particular the design of glycolysis, may be explained by optimization principles. On the basis of kinetic and thermodynamic principles conclusions are derived concerning the stoichiometry of these pathways in states of high ATP production rates. One of the extensions to previous investigations is that the concentrations of the adenine nucleotides are taken into account as variable quantities. This necessitates the consideration of an interaction of the ATP-producing system I with an external ATP-consuming system II. A great variety of pathways is studied which differ in the number and location of ATP-consuming reactions, ATP-producing reactions and reactions involving inorganic phosphate. The corresponding number of possible pathways may be calculated in an explicit manner as a function of the number of those reactions which do not couple to ATP or inorganic phosphate. The kinetics of the individual reactions are described by linear or bilinear functions of reactant concentrations and all rate equations are expressed in terms of equilibrium constants and characteristic times. A thermodynamical analysis of the two coupled systems yields upper and lower limits for the concentration of ATP and an explicit expression for the maximal difference between the number of ATP-producing and ATP-consuming reactions of system I. The following results of the optimization are obtained. (i) The ATP production rate always increases if the ATP-producing reactions as well as those reactions characterized by an uptake of inorganic phosphate are shifted as far as possible towards the end of system I. (ii) Explicit conditions for the optimal location of the ATP-consuming reactions are presented. The results are discussed in the context of characteristic times as well as in terms of enzyme kinetic parameters. (iii) For two sets of characteristic times the resulting stoichiometries and their corresponding steady-state fluxes are investigated in detail. One of these stoichiometries shows a close correspondence to contemporary standard glycolysis. (iv) It is shown that most possible pathways result in a very low steady-state flux, that is, the optimal stoichiometry is characterized by a significant selective advantage. (v) The standard free energy profile of a pathway with an optimal stoichiometry is discussed. It differs significantly from the free energy profiles of nonoptimized pathways.     

What Process Is Glycolytic Stoichiometry Optimal For?

It has been proposed that the glycolytic stoichiometry of 2 ATP per glucose is the result of an optimization that maximizes the rate of ATP production. However, using a nonequilibrium thermodynamic approach, we show here that glycolysis operates under optimal output power and not at optimal flow of ATP production. Furthermore, it can be proved that the same maximal output power can be achieved with different stoichiometries. However, changes in the glycolytic stoichiometry would dramatically affect the efficiency of all those cellular processes powered by ATP. Our results suggest that the stoichiometric coefficient, as found in most contemporary cells, may be the outcome of an evolutionary process leading to yield an operative quantum energy for the hydrolysis of ATP. [Reviewing Editor: Dr. Antony Dean]     

Affinity labeling of the (Na+ + Mg2+)-ATPase from A choleplasma laidlawii B membranes by the 2′,3′-dialdehyde derivative of adenosine 5′-triphosphate

The (Na⁺ + Mg²⁺)-ATPase of the Acholeplasma laidlawii B plasma membrane was inactivated by the 2′,3′-dialdehyde derivative of ATP (oATP). oATP behaved as a reversible competitive inhibitor of this ATPase and was slowly hydrolyzed by the enzyme. In addition, oATP induced an irreversible inactivation of the enzyme. A 62% inactivation of the enzyme correlated with the binding of 16 moles of oATP per mole of the enzyme. In the presence of 5′-adenylyl imidodiphosphate, a non-hydrolyzable substrate analogue, the stoichiometry was 8 moles oATP per mole of ATPase. By SDS-polyacrylamide gel electrophoresis, [U-¹⁴C]oATP was found to bind covalently to four of the five subunits of the enzyme, but specific labeling was highest for the γ-subunit of the ATPase.     

The stoichiometry of the chloroplast ATP synthase oligomer III in Chlamydomonas reinhardtii is not affected by the metabolic state

The chloroplast H⁺-ATP synthase is a key component for the energy supply of higher plants and green algae. An oligomer of identical protein subunits III is responsible for the conversion of an electrochemical proton gradient into rotational motion. It is highly controversial if the oligomer III stoichiometry is affected by the metabolic state of any organism. Here, the intact oligomer III of the ATP synthase from Chlamydomonas reinhardtii has been isolated for the first time. Due to the importance of the subunit III stoichiometry for energy conversion, a gradient gel system was established to distinguish oligomers with different stoichiometries. With this methodology, a possible alterability of the stoichiometry in respect to the metabolic state of the cells was examined. Several growth parameters, i.e., light intensity, pH value, carbon source, and CO₂ concentration, were varied to determine their effects on the stoichiometry. Contrary to previous suggestions for E. coli, the oligomer III of the chloroplast H⁺-ATP synthase always consists of a constant number of monomers over a wide range of metabolic states. Furthermore, mass spectrometry indicates that subunit III from C. reinhardtii is not modified posttranslationally. Data suggest a subunit III stoichiometry of the algae ATP synthase divergent from higher plants.     

The ATP paradox is the expression of an economizing fuel mechanism

The strong negative correlation between glycolytic flux and intracellular ATP concentration observed in yeast has long been an intriguing and counterintuitive phenomenon, which has been referred to as the ATP paradox. Herein, using principles of irreversible thermodynamics it was shown that if the ATP-consuming pathways are more sensitive to extracellular glucose than glycolysis, then upon glucose addition glycolysis performance can switch from an efficient working regime to a dissipative regime, and vice versa, depending on glucose availability. The efficient regime represents a good compromise between high output power and low dissipation, whereas the dissipative working regime offers a higher output power although at a high glucose cost. The physiological and evolutionary implications of this switch strategy are discussed.     

Effects of dinitrophenol and oligomycin on the coupling between anaerobic metabolism and anaerobic sodium transport by the isolated turtle bladder

Dinitrophenol (1 x 10^-5 M) has been found to inhibit anaerobic sodium transport by the isolated urinary bladder of the fresh water turtle. Concurrently, anaerobic glycolysis was stimulated markedly. However, tissue ATP levels diminished only modestly, remaining at approximately 75% of values observed under anaerobic conditions without DNP. The utilization of glucose (from endogenous glycogen) corresponded closely to that predicted from the molar quantities of lactate formed. Thus the glycolytic pathway was completed in the presence of DNP and if ATP were synthesized normally during glycolysis, synthesis should have been increased. On the other hand, the decrease in Na transport should have decreased ATP utilization. Oligomycin did not block sodium transport either aerobically or anaerobically, but ATP concentrations did decrease. When anaerobic glycolysis was blocked by iodoacetate, pyruvate did not sustain sodium transport thus suggesting that no electron acceptors were available in the system. Two explanations are entertained for the anaerobic effect of DNP: (a) Stimulation by DNP of plasma membrane as well as mitochondrial ATPase activity; (b) inhibition of a high energy intermediate derived from glycolytic ATP or from glycolysis per se. The arguments relevant to each possibility are presented in the text. Although definitive resolution is not possible, we believe that the data favor the hypothesis that there was a high energy intermediate in the anaerobic system and that this intermediate, rather than ATP, served as the immediate source of energy for the sodium pump.     

Radiation induces senescence and a bystander effect through metabolic alterations

Cellular senescence is a state of irreversible growth arrest; however, the metabolic processes of senescent cells remain active. Our previous studies have shown that radiation induces senescence of human breast cancer cells that display low expression of securin, a protein involved in control of the metaphase–anaphase transition and anaphase onset. In this study, the protein expression profile of senescent cells was resolved by two-dimensional gel electrophoresis to investigate associated metabolic alterations. We found that radiation induced the expression and activation of glyceraldehyde-3-phosphate dehydrogenase that has an important role in glycolysis. The activity of lactate dehydrogenase A, which is involved in the conversion of pyruvate to lactate, the release of lactate and the acidification of the extracellular environment, was also induced. Inhibition of glycolysis by dichloroacetate attenuated radiation-induced senescence. In addition, radiation also induced activation of the 5′-adenosine monophosphate-activated protein kinase (AMPK) and nuclear factor kappa B (NF-κB) pathways to promote senescence. We also found that radiation increased the expression of monocarboxylate transporter 1 (MCT1) that facilitates the export of lactate into the extracellular environment. Inhibition of glycolysis or the AMPK/NF-κB signalling pathways reduced MCT1 expression and rescued the acidification of the extracellular environment. Interestingly, these metabolic-altering signalling pathways were also involved in radiation-induced invasion of the surrounding, non-irradiated breast cancer and normal endothelial cells. Taken together, radiation can induce the senescence of human breast cancer cells through metabolic alterations.     

Bonded Cumomer Analysis of Human Melanoma Metabolism Monitored by 13C NMR Spectroscopy of Perfused Tumor Cells

A network model for the determination of tumor metabolic fluxes from ¹³C NMR kinetic isotopomer data has been developed and validated with perfused human DB-1 melanoma cells carrying the BRAF V600E mutation, which promotes oxidative metabolism. The model generated in the bonded cumomer formalism describes key pathways of tumor intermediary metabolism and yields dynamic curves for positional isotopic enrichment and spin-spin multiplets. Cells attached to microcarrier beads were perfused with 26 mm [1,6-¹³C₂]glucose under normoxic conditions at 37 °C and monitored by ¹³C NMR spectroscopy. Excellent agreement between model-predicted and experimentally measured values of the rates of oxygen and glucose consumption, lactate production, and glutamate pool size validated the model. ATP production by glycolytic and oxidative metabolism were compared under hyperglycemic normoxic conditions; 51% of the energy came from oxidative phosphorylation and 49% came from glycolysis. Even though the rate of glutamine uptake was ∼50% of the tricarboxylic acid cycle flux, the rate of ATP production from glutamine was essentially zero (no glutaminolysis). De novo fatty acid production was ∼6% of the tricarboxylic acid cycle flux. The oxidative pentose phosphate pathway flux was 3.6% of glycolysis, and three non-oxidative pentose phosphate pathway exchange fluxes were calculated. Mass spectrometry was then used to compare fluxes through various pathways under hyperglycemic (26 mm) and euglycemic (5 mm) conditions. Under euglycemic conditions glutamine uptake doubled, but ATP production from glutamine did not significantly change. A new parameter measuring the Warburg effect (the ratio of lactate production flux to pyruvate influx through the mitochondrial pyruvate carrier) was calculated to be 21, close to upper limit of oxidative metabolism.     

Biochemical thresholds for pathological presentation of ATP synthase deficiencies

Mitochondrial ATP synthase is responsible for production of the majority of cellular ATP. Disorders of ATP synthase in humans can be caused by numerous mutations in both structural subunits and specific assembly factors. They are associated with variable pathogenicity and clinical phenotypes ranging from mild to the most severe mitochondrial diseases. To shed light on primary/pivotal functional consequences of ATP synthase deficiency, we explored human HEK 293 cells with a varying content of fully assembled ATP synthase, selectively downregulated to 15–80% of controls by the knockdown of F₁ subunits γ, δ and ε. Examination of cellular respiration and glycolytic flux revealed that enhanced glycolysis compensates for insufficient mitochondrial ATP production while reduced dissipation of mitochondrial membrane potential leads to elevated ROS production. Both insufficient energy provision and increased oxidative stress contribute to the resulting pathological phenotype. The threshold for manifestation of the ATP synthase defect and subsequent metabolic remodelling equals to 10–30% of residual ATP synthase activity. The metabolic adaptations are not able to sustain proliferation in a galactose medium, although sufficient under glucose-rich conditions. As metabolic alterations occur when the content of ATP synthase drops below 30%, some milder ATP synthase defects may not necessarily manifest with a mitochondrial disease phenotype, as long as the threshold level is not exceeded.     

13C-metabolic flux analysis of Clostridium ljungdahlii illuminates its core metabolism under mixotrophic culture conditions

Carbon dioxide-fixing acetogenic bacteria (acetogens) utilizing the Wood-Ljungdahl Pathway (WLP) play an important role in CO₂ fixation in the biosphere and in the development of biological processes – alone or in cocultures, under both autotrophic and mixotrophic conditions – for production of chemicals and fuels. To date, limited work has been reported in experimentally validating and quantifying reaction fluxes of their core metabolic pathways. Here, the core metabolic model of the acetogen Clostridium ljungdahlii was interrogated using ¹³C-metabolic flux analysis (¹³C-MFA), which required the development of a new defined culture medium. Autotrophic, heterotrophic, and mixotrophic growth in defined medium was possible by adding 1 mM methionine to replace yeast extract. Our ¹³C-MFA found an incomplete TCA cycle and inactive core pathways/reactions, notably those of the oxidative pentose phosphate pathway, Entner-Doudoroff pathway, and malate dehydrogenase. ¹³C-MFA during mixotrophic growth using the parallel tracers [1–¹³C]fructose, [1,2–¹³C]fructose, [1,2,3–¹³C]fructose, and [U–¹³C]asparagine found that externally supplied CO₂ contributed the majority of carbon consumed. All internally-produced CO₂ from the catabolism of asparagine and fructose was consumed by the WLP. While glycolysis of fructose was active, it was not a major contributor to overall production of ATP, NADH, and acetyl-CoA. Gluconeogenic reactions were active despite the availability of organic carbon. Asparagine was catabolized equally via conversion to threonine and subsequent cleavage to produce acetaldehyde and glycine, and via deamination to fumarate and then the anaplerotic conversion of malate to pyruvate. Both pathways for asparagine catabolism produced acetyl-CoA, either directly via pyruvate or indirectly via the WLP. Cofactor stoichiometry based on our data predicted an essentially zero flux through the ferredoxin-dependent transhydrogenase (Nfn) reaction. Instead, nearly all of NADPH generated from the hydrogenase reaction was consumed by the WLP. Reduced ferredoxin produced by the hydrogenase reaction and glycolysis was mostly used for ATP generation via the RNF/ATPase system, with the remainder consumed by the WLP. NADH produced by RNF/ATPase was entirely consumed via the WLP.     

Channeling in native microbial pathways: Implications and challenges for metabolic engineering

Intracellular enzymes can be organized into a variety of assemblies, shuttling intermediates from one active site to the next. Eukaryotic compartmentalization within mitochondria and peroxisomes and substrate tunneling within multi-enzyme complexes have been well recognized. Intriguingly, the central pathways in prokaryotes may also form extensive channels, including the heavily branched glycolysis pathway. In vivo channeling through cascade enzymes is difficult to directly measure, but can be inferred from in vitro tests, reaction thermodynamics, transport/reaction modeling, analysis of molecular diffusion and protein interactions, or steady state/dynamic isotopic labeling. Channeling presents challenges but also opportunities for metabolic engineering applications. It rigidifies fluxes in native pathways by trapping or excluding metabolites for bioconversions, causing substrate catabolite repressions or inferior efficiencies in engineered pathways. Channeling is an overlooked regulatory mechanism used to control flux responses under environmental/genetic perturbations. The heterogeneous distribution of intracellular enzymes also confounds kinetic modeling and multiple-omics analyses. Understanding the scope and mechanisms of channeling in central pathways may improve our interpretation of robust fluxomic topology throughout metabolic networks and lead to better design and engineering of heterologous pathways.     

Reconstruction of the Saccharopolyspora erythraea genome-scale model and its use for enhancing erythromycin production

Genome-scale metabolic reconstructions are routinely used for the analysis and design of metabolic engineering strategies for production of primary metabolites. The use of such reconstructions for metabolic engineering of antibiotic production is not common due to the lack of simple design algorithms in the absence of a cellular growth objective function. Here, we present the metabolic network reconstruction for the erythromycin producer Saccharopolyspora erythraea NRRL23338. The model was manually curated for primary and secondary metabolism pathways and consists of 1,482 reactions (2,075 genes) and 1,646 metabolites. As part of the model validation, we explored the potential benefits of supplying amino acids and identified five amino acids “compatible” with erythromycin production, whereby if glucose is supplemented with this amino acid on a carbon mole basis, the in silico model predicts that high erythromycin yield is possible without lowering biomass yield. Increased erythromycin titre was confirmed for four of the five amino acids, namely valine, isoleucine, threonine and proline. In bioreactor experiments, supplementation with 2.5?% carbon mole of valine increased the growth rate by 20?% and simultaneously the erythromycin yield on biomass by 50?%. The model presented here can be used as a framework for the future integration of high-throughput biological data sets in S. erythraea and ultimately to realise strain designs capable of increasing erythromycin production closer to the theoretical yield.     

Adenine and adenosine salvage pathways in erythrocytes and the role of S-adenosylhomocysteine hydrolase. A theoretical study using elementary flux modes

This article is devoted to the study of redundancy and yield of salvage pathways in human erythrocytes. These cells are not able to synthesize ATP de novo. However, the salvage (recycling) of certain nucleosides or bases to give nucleotide triphosphates is operative. As the salvage pathways use enzymes consuming ATP as well as enzymes producing ATP, it is not easy to see whether a net synthesis of ATP is possible. As for pathways using adenosine, a straightforward assumption is that these pathways start with adenosine kinase. However, a pathway bypassing this enzyme and using S-adenosylhomocysteine hydrolase instead was reported. So far, this route has not been analysed in detail. Using the concept of elementary flux modes, we investigate theoretically which salvage pathways exist in erythrocytes, which enzymes belong to each of these and what relative fluxes these enzymes carry. Here, we compute the net overall stoichiometry of ATP build-up from the recycled substrates and show that the network has considerable redundancy. For example, four different pathways of adenine salvage and 12 different pathways of adenosine salvage are obtained. They give different ATP/glucose yields, the highest being 3:10 for adenine salvage and 2:3 for adenosine salvage provided that adenosine is not used as an energy source. Implications for enzyme deficiencies are discussed.