Control of the flux in the arginine pathway of Neurospora crassa. Modulations of enzyme activity and concentration.

The influence of particular enzyme activities on the flux of metabolites in a pathway can be estimated by 'modulating' enzymes (i.e. changing turnover or concentration) and measuring the response in various parts of the system. By controlling the nuclear ration of two genetically different nuclear types in heterokaryons, the enzyme concentrations at four different steps in the arginine pathway were decreased over a range. This range was extended by the use of bradytrophs, mutant strains specifying enzymes with greatly diminished enzyme activities. Strains altered simultaneously at more than one step were also constructed by genetic recombination. By measuring the outputs of the pathway and the steady-state concentrations of intermediate pools, the fluxes in different parts of the pathway were calculated. This allowed the construction of flux/enzyme relationships, the slope of which is a measure of the sensitivity of a flux to the change in enzyme activity at that step. All fluxes were found to be considerably buffered for quite substantial decreases in the activities of all enzymes. Mass action plays an important part in this phenomenon, as do inhibition and repression. Because of the existence of expansion fluxes in growing systems, we find quantitatively different fluxes in different parts of the single pathway. For the same reason some enzyme modulations given decreased fluxes in one part and increased fluxes in another. The understanding of control in the pathway thus involves consideration of many mechanisms operating simultaneously and the estimation of changes in the whole system. The concept of a 'rate-limiting step' is found to be inadequate and is replaced by a quantitative measure, the Sensitivity Coefficient, which takes account of all the interactions. It is shown that control of the flux is shared among all the enzymes of the pathway. The results are discussed in terms of the theory of flux control.     

Metabolic modeling of polyhydroxybutyrate biosynthesis

A mathematical model describing intracellular polyhydroxybutyrate (PHB) synthesis in Alcaligenes eutrophus has been constructed. The model allows investigation of issues such as the existence of rate-limiting enzymatic steps, possible regulatory mechanisms in PHB synthesis, and the effects different types of rate expressions have on model behavior. Simulations with the model indicate that activities of all PHB pathway enzymes influence overall PHB flux and that no single enzymatic step can easily be identified as rate limiting. Simulations also support regulatory roles for both thiolase and reductase, mediated through AcCoA/CoASH and NADPH/NADP+ ratios, respectively. To make the model more realistic, complex rate expressions for enzyme-catalyzed reactions were used which reflect both the reversibility of the reactions and the reaction mechanisms. Use of the complex kinetic expressions dramatically changed the behavior of the system compared to a simple model containing only Michaelis-Menten kinetic expressions; the more complicated model displayed different responses to changes in enzyme activities as well as inhibition of flux by the reaction products CoASH and NADP+. These effects can be attributed to reversible rate expressions, which allow prediction of reaction rates under conditions both near and far from equilibrium.     

Intermediate-sensor assisted push–pull strategy and its application in heterologous deoxyviolacein production in Escherichia coli

Because high-throughput screening tools are typically unavailable when using the pathway-engineering approach, we developed a new strategy, named intermediate sensor-assisted push–pull strategy, which enables sequential pathway optimization by incorporating a biosensor targeting a key pathway intermediate. As proof of concept, we constructed an l-Trp biosensor and used it to optimize the deoxyviolacein biosynthetic pathway, which we divided into two modules with l-Trp being the product of the upstream and the substrate of the downstream module for deoxyviolacein synthesis. Using the biosensor and fluorescence-activated cell sorting, the activities of the two modules were sequentially and independently optimized in Escherichia coli to achieve the desired phenotypes. By this means, we increased the deoxyviolacein titer 4.4-fold (1.92 g/L), which represents the greatest deoxyviolacein production reported. This work suggests that a biosynthetic pathway can be enhanced to produce a value-added secondary metabolite(s) without available end-product screening method by using a central metabolic junction molecule biosensor(s).     

Toward predicting metabolic fluxes in metabolically engineered strains

Predicting metabolic fluxes of a genetically engineered organism is an important step toward rational pathway design. However, because of various regulatory mechanisms, which are complex, often ill-characterized, and sometimes undiscovered, predicting metabolic fluxes using kinetic simulation is difficult. We propose to incorporate regulatory constraints in flux calculation to allow prediction of the steady-state fluxes without complete kinetics. The regulatory constraint, in its linear form, is derived from the dynamic metabolic control theory and involves the flux control coefficients. It is shown that with these constraints, the responses to metabolic perturbation can be predicted. Conversely, the regulatory constraints and the control coefficients can be determined by comparing the experimental data with the prediction. Therefore, this approach may offer a practical direction toward prediction of fluxes for metabolically engineered organisms.     

Sensitivity of pathway rate to activities of substrate-cycle enzymes: application to gluconeogenesis and glycolysis

In a study of metabolic regulation, it is frequently useful to consider the degree to which an enzyme can influence the rate of its pathway. The most productive expression of rate-controlling influence is the fractional change in pathway rate per fractional change in enzyme activity (called control strength or sensitivity coefficient). We have developed a system for considering how a substrate-cycle enzyme's control strength depends on its flux and reaction order and on related features of other enzymes of its pathway. We have applied this system to the gluconeogenic pathway of rat liver and the glycolytic pathway of bovine sperm, where enough fluxes and reaction orders have been published to allow valid estimates of several control strengths. In normal fed animals where gluconeogenesis is slow and unidirectional substrate-to-product and product-to-substrate fluxes are comparable, all substrate-cycle limbs have very high and similar control strengths regardless of their flux rates and positions in the pathway. The activity of a step affects all substrate-cycle control strengths similarly as it affects unidirectional end-to-end fluxes relative to net rate. Control strengths of non-substrate-cycle enzymes are negligible compared to those of substrate cycles. In fasting animals, on the other hand, where unidirectional Pyr----Glc flux is much greater than Glc----Pyr flux, upstream enzymes (near Pyr) have a regulatory advantage over downstream enzymes (near Glc). In this circumstance, control strength of each substrate-cycle enzyme is inversely related to rate limitingness between its substrate and the pathway substrate. Because the Pyr/PEP cycle is significantly rate limiting, the control strength of the Pyr----PEP limb is much greater than that of pyruvate kinase and all downstream enzymes. In the glycolytic pathway of bovine sperm, strong product inhibition of hexokinase detracts greatly from its rate limitingness and control strength, which are very small despite its position at the beginning of the pathway and its large free energy. Because the glucose-transport-hexokinase segment is not rate limiting, phosphofructo 1-kinase has almost as much control strength as it would have as the first enzyme of the pathway, and because the F6P/FDP cycle is only moderately rate limiting, Fru-1,6-P2ase and enzymes further downstream have substantial control strengths. When glycolysis is accelerated by stimulation of phosphofructo 1-kinase, control strength shifts from phosphofructo-1-kinase and all downstream enzymes to the transporthesokinase segment.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulatory role of acetylation on enzyme activity and fluxes of energy metabolism pathways

BackgroundMost of the enzymes involved in the central carbon metabolism are acetylated in Lys residues. It has been claimed that this covalent modification represents a novel regulatory mechanism by which both enzyme/transporter activities and pathway fluxes can be modulated.MethodsTo establish which enzymes are regulated by acetylation, a systematic experimental analysis of activities and acetylation profile for several energy metabolism enzymes and pathway fluxes was undertaken in cells and mitochondria.ResultsThe majority of the glycolytic and neighbor enzymes as well as mitochondrial enzymes indeed showed Lys-acetylation, with GLUT1, HPI, CS, ATP synthase displaying comparatively lower acetylation patterns. The incubation of cytosolic and mitochondrial fractions with recombinant Sirt-3 produced lower acetylation signals, whereas incubation with acetyl-CoA promoted protein acetylation. Significant changes in acetylation levels of MDH and IDH-2 from rat liver mitochondria revealed no change in their activities. Similar observations were attained for the cytosolic enzymes from AS-30D and HeLa cells. A minor but significant (23%) increase in the AAT-MDH complex activity induced by acetylation was observed. To examine this question further, AS-30D and HeLa cells were treated with nicotinamide and valproic acid. These compounds promoted changes in the acetylation patterns of glycolytic proteins, although their activities and the glycolytic flux (as well as the OxPhos flux) revealed no clear correlation with acetylation.ConclusionAcetylation seems to play no predominant role in the control of energy metabolism enzyme activities and pathway fluxes.General significanceThe physiological function of protein acetylation on energy metabolism pathways remains to be elucidated.     

The regulatory design of an allosteric feedback loop: the effect of saturation by pathway substrate

Aspects of metabolic regulation can be fruitfully studied with a combination of generic modelling, control analysis and graphical analysis using rate characteristics. This paper analyses a prototypical supply-demand system consisting of a biosynthetic subsystem subject to allosteric inhibition by its product and a demand process that consumes this product. The effect of changes in affinity of the committing supply enzyme for the pathway substrate on the regulatory properties of the supply subsystem is compared for the Monod-Wyman-Changeux and the reversible Hill allosteric enzyme models. We found that the Hill model has a distinct advantage in that the steady-state concentration at which it maintains the product is set by the half-saturating product concentration and is independent of changes in the degree of saturation for substrate. In contrast, with the Monod-Wyman-Changeux model this set point varies with affinity for substrate. Explicitly incorporating reversibility in all rate equations made it possible to distinguish between kinetic and thermodynamic aspects of regulation. Combining the supply and demand rate characteristics allows us to explore both the control distribution at steady state and the regulatory performance of the system over a wide range of demand activities.     

Feedfoward inhibition in biosynthetic pathways: inhibition of the aminoacyl-tRNA synthetase by intermediates of the pathway.

The stability of an amino acid biosynthetic pathway controlled by end-product inhibition is significantly improved if, in addition, the corresponding aminoacyl-tRNA synthetase is inhibited by an intermediate in the pathway. The more proximal the feedforward modifier is to the initial substrate, the more stable is the system. The temporal responsiveness of a system having both feedback and feedforward inhibition also is improved by having the feedforward modifier located at the beginning of the pathway. According to all other criteria that have been used previously to determine the functional effectiveness of biosynthetic pathways, the behavior of such a system essentially is unaffected by the position of the feedforward modifier in the pathway.     

Experimental determination of group flux control coefficients in metabolic networks

Grouping of reactions around key metabolite branch points can facilitate the study of metabolic control of complex metabolic networks. This top-down Metabolic Control Analysis is exemplified through the introduction of group (flux, as well as concentration) control coefficients whose magnitudes provide a measure of the relative impact of each reaction group on the overall network flux, as well as on the overall network stability, following enzymatic amplification. In this article, we demonstrate the application of previously developed theory to the determination of group flux control coefficients. Experimental data for the changes in metabolic fluxes obtained in response to the introduction of six different environmental perturbations are used to determine the group flux control coefficients for three reaction groups formed around the phosphoenolpyruvate/pyruvate branch point. The consistency of the obtained group flux control coefficient estimates is systematically analyzed to ensure that all necessary conditions are satisfied. The magnitudes of the determined control coefficients suggest that the control of lysine production flux in Corynebacterium glutamicum cells at a growth base state resides within the lysine biosynthetic pathway that begins with the PEP/PYR carboxylation anaplorotic pathway. Copyright 1998 John Wiley & Sons, Inc.     

A typical N-terminal extensions confer novel regulatory properties on GTP cyclohydrolase isoforms in Drosophila melanogaster

The cofactor tetrahydrobiopterin plays critical roles in the modulation of the signaling molecules dopamine, serotonin, and nitric oxide. Deficits in cofactor synthesis have been associated with several human hereditary diseases. Responsibility for the regulation of cofactor pools resides with the first enzyme in its biosynthetic pathway, GTP cyclohydrolase I. Because organisms must be able to rapidly respond to environmental and developmental cues to adjust output of these signaling molecules, complex regulatory mechanisms are vital for signal modulation. Mammalian GTP cyclohydrolase is subject to end-product inhibition via an associated regulatory protein and to positive regulation via phosphorylation, although target residues are unknown. GTP cyclohydrolase is composed of a highly conserved homodecameric catalytic core and non-conserved N-terminal domains proposed to be regulatory sites. We demonstrate for the first time in any organism that the N-terminal arms of the protein serve regulatory functions. We identify two different modes of regulation of the enzyme mediated through the N-terminal domains. The first is end-product feedback inhibition, catalytically similar to that of the mammalian enzyme, except that feedback inhibition by the cofactor requires sequences in the N-terminal arms rather than a separate regulatory protein. The second is a novel inhibitory interaction between the N-terminal arms and the active sites, which can be alleviated through the phosphorylation of serine residues within the N termini. Both mechanisms allow for acute and highly responsive regulation of cofactor production as required by downstream signaling pathways.     

Analysis of metabolic fluxes in batch and continuous cultures of Bacillus subtilis

It is well recognized that metabolic fluxes are the key variables that must be determined in order to understand metabolic regulation and patterns. However, owing to difficulties in measuring the flux values, evaluation of metabolic fluxes has not been an integral part of the most metabolic studies. Flux values for metabolites of glycolysis, tricarboxylic acid (TCA) cycle, and hexose monophosphate (HMP) pathway were obtained for batch and glucose-limited continuous cultures of Bacillus subtilis by combining the information from the stoichiometry of key biosynthetic reactions with the experimental data on concentrations of glucose and metabolic by-products, CO(2) evolution, and oxygen uptake rates. The results indicate that (1) the metabolic fluxes and energetic yield as well as the extent of flux mismatch in metabolic activity of glycolysis and the TCA cycle reactions can be accurately quantified; (2) the flux through the TCA cycle in continuous culture is much in excess of cell energetic and biosynthetic demands for precursors; (3) for the range of growth rates examined the TCA cycle flux increases almost in proportion to growth rate and is significantly repressed only at very high growth rates of batch cultures; and (4) for continuous cultures the isocitrate dehydrogenase catalyzed reaction of the TCA cycle is the major source of the reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH) used in biosynthesis. (c) 1993 John Wiley & Sons, Inc.     

Pathway analysis, engineering, and physiological considerations for redirecting central metabolism

The rate and yield of producing a metabolite is ultimately limited by the ability to channel metabolic fluxes from central metabolism to the desired biosynthesis pathway. Redirection of central metabolism thus is essential to high-efficiency production of biochemicals. This task begins with pathway analysis, which considers only the stoichiometry of the reaction networks but not the regulatory mechanisms. An approach extended from convex analysis is used to determine the basic reaction modes, which allows the determination of optimal and suboptimal flux distributions, yield, and the dispensable sets of reactions. Genes responsible for reactions in the same dispensable set can be deleted simultaneously. This analysis serves as an initial guideline for pathway engineering. Using this analysis, we successfully constructed an Escherichia coli strain that can channel the metabolic flow from carbohydrate to the aromatic pathway with theoretical yield. This analysis also predicts a novel cycle involving phosphoenolpyruvate (PEP) carboxykinase (Pck) and the glyoxylate shunt, which can substitute the tricarboxylic acid cycle with only slightly less efficiency. However, the full cycle could not be confirmed in vivo, possibly because of the regulatory mechanism not considered in the pathway analysis.In addition to the kinetic regulation, we have obtained evidence suggesting that central metabolites are involved in specific regulons in E. coli. Overexpression of PEP-forming enzymes (phosphoenolpyruvate synthase [Pps] and Pck) stimulates the glucose consumption rate, represses the heat shock response, and negatively regulates the Ntr regulon. These results suggest that some glycolytic intermediates may serve as a signal in the regulation of the phosphotransferase system, heat shock response, and nitrogen regulation. However, the role of central metabolites in these regulations has not been determined conclusively. (c) 1996 John Wiley & Sons, Inc.     

Improving key enzyme activity in phenylpropanoid pathway with a designed biosensor

Overexpressing key enzymes of biosynthetic pathways for overproduction of value-added products usually imposes metabolic burdens on cells, which can be circumvented by improving the key enzyme activities. p-Coumarate: CoA ligase (4CL) is a critical enzyme in the phenylpropanoid pathway that synthesizes various natural products. To screen for 4CL with improved activity, a biosensor of resveratrol whose biosynthetic pathway involves 4CL was designed by engineering the TtgR regulatory protein. The biosensor exhibited good specificity and robustness, allowing rapid and sensitive selection of resveratrol hyper-producers. A 4CL variant with improved activity was selected from a 4CL mutagenesis library constructed in the resveratrol biosynthetic pathway in Escherichia coli. This mutant led to increased production of not only resveratrol but also the flavonoid naringenin, when introduced in their corresponding biosynthetic pathways. These findings demonstrate the feasibility of improving key enzyme activities in important biosynthetic pathways with the aid of designed biosensors of pathway products.     

Development of bifunctional biosensors for sensing and dynamic control of glycolysis flux in metabolic engineering

Glycolysis is the primary metabolic pathway in all living organisms. Maintaining the balance of glycolysis flux and biosynthetic pathways is the crucial matter involved in the microbial cell factory. Few regulation systems can address the issue of metabolic flux imbalance in glycolysis. Here, we designed and constructed a bifunctional glycolysis flux biosensor that can dynamically regulate glycolysis flux for overproduction of desired biochemicals. A series of positive-and negative-response biosensors were created and modified for varied thresholds and dynamic ranges. These engineered glycolysis flux biosensors were verified to be able to characterize in vivo fructose-1,6-diphosphate concentration. Subsequently, the biosensors were applied for fine-tuning glycolysis flux to effectively balance the biosynthesis of two chemicals: mevalonate and N-acetylglucosamine. A glycolysis flux-dynamically controlled Escherichia coli strain achieved a 111.3 g/L mevalonate titer in a 1L fermenter.     

Pathway analysis and metabolic engineering in Corynebacterium glutamicum

The gram-positive bacterium Corynebacterium glutamicum is used for the industrial production of amino acids, e.g. of L-glutamate and L-lysine. During the last 15 years, genetic engineering and amplification of genes have become fascinating methods for studying metabolic pathways in greater detail and for the construction of strains with the desired genotypes. In order to obtain a better understanding of the central metabolism and to quantify the in vivo fluxes in C. glutamicum, the [13C]-labelling technique was combined with metabolite balancing to achieve a unifying comprehensive pathway analysis. These methods can determine the flux distribution at the branch point between glycolysis and the pentose phosphate pathway. The in vivo fluxes in the oxidative part of the pentose phosphate pathway calculated on the basis of intracellular metabolite concentrations and the kinetic constants of the purified glucose-6-phosphate and 6-phosphogluconate dehydrogenases determined in vitro were in full accordance with the fluxes measured by the [13C]-labelling technique. These data indicate that the oxidative pentose phosphate pathway in C. glutamicum is mainly regulated by the ratio of NADPH/NADP concentrations and the specific activity of glucose-6-phosphate dehydrogenase. The carbon flux via the oxidative pentose phosphate pathway correlated with the NADPH demand for L-lysine synthesis. Although it has generally been accepted that phosphoenolpyruvate carboxylase fulfills a main anaplerotic function in C. glutamicum, we recently detected that a biotin-dependent pyruvate carboxylase exists as a further anaplerotic enzyme in this bacterium. In addition to the activities of these two carboxylases three enzymes catalysing the decarboxylation of the C4 metabolites oxaloacetate or malate are also present in this bacterium. The individual flux rates at this complex anaplerotic node were investigated by using [13C]-labelled substrates. The results indicate that both carboxylation and decarboxylation occur simultaneously in C. glutamicum so that a high cyclic flux of oxaloacetate via phosphoenolpyruvate to pyruvate was found. Furthermore, we detected that in C. glutamicum two biosynthetic pathways exist for the synthesis of DL-diaminopimelate and L-lysine. As shown by NMR spectroscopy the relative use of both pathways in vivo is dependent on the ammonium concentration in the culture medium. Mutants defective in one pathway are still able to synthesise enough L-lysine for growth, but the L-lysine yields with overproducers were reduced. The luxury of having these two pathways gives C. glutamicum an increased flexibility in response to changing environmental conditions and is also related to the essential need for DL-diaminopimelate as a building block for the synthesis of the murein sacculus.     

Carbon metabolism and the sign of control coefficients in metabolic adaptations underlying K-ras transformation

Metabolic adaptations are associated with changes in enzyme activities. These adaptations are characterized by patterns of positive and negative changes in metabolic fluxes and concentrations of intermediate metabolites. Knowledge of the mechanism and parameters governing enzyme kinetics is rarely available. However, the signs-increases or decreases-of many of these changes can be predicted using the signs of metabolic control coefficients. These signs require the only knowledge of the structure of the metabolic network and a limited qualitative knowledge of the regulatory dependences, which is widely available for carbon metabolism. Here, as a case study, we identified control coefficients with fixed signs in order to predict the pattern of changes in key enzyme activities which can explain the observed changes in fluxes and concentrations underlying the metabolic adaptations in oncogenic K-ras transformation in NIH-3T3 cells. The fixed signs of control coefficients indicate that metabolic changes following the oncogenic transformation-increased glycolysis and oxidative branch of the pentose-phosphate pathway, and decreased concentration in sugar-phosphates-could be associated with increases in activity for glucose-6-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase, and decrease for transketolase. These predictions were validated experimentally by measuring specific activities. We conclude that predictions based on fixed signs of control coefficients are a very robust tool for the identification of changes in enzyme activities that can explain observed metabolic adaptations in carbon metabolism.     

Discovery of parallel pathways of kanamycin biosynthesis allows antibiotic manipulation

Kanamycin is one of the most widely used antibiotics, yet its biosynthetic pathway remains unclear. Current proposals suggest that the kanamycin biosynthetic products are linearly related via single enzymatic transformations. To explore this system, we have reconstructed the entire biosynthetic pathway through the heterologous expression of combinations of putative biosynthetic genes from Streptomyces kanamyceticus in the non-aminoglycoside-producing Streptomyces venezuelae. Unexpectedly, we discovered that the biosynthetic pathway contains an early branch point, governed by the substrate promiscuity of a glycosyltransferase, that leads to the formation of two parallel pathways in which early intermediates are further modified. Glycosyltransferase exchange can alter flux through these two parallel pathways, and the addition of other biosynthetic enzymes can be used to synthesize known and new highly active antibiotics. These results complete our understanding of kanamycin biosynthesis and demonstrate the potential of pathway engineering for direct in vivo production of clinically useful antibiotics and more robust aminoglycosides.     

Sequential Metabolic Phases as a Means to Optimize Cellular Output in a Constant Environment

Temporal changes of gene expression are a well-known regulatory feature of all cells, which is commonly perceived as a strategy to adapt the proteome to varying external conditions. However, temporal (rhythmic and non-rhythmic) changes of gene expression are also observed under virtually constant external conditions. Here we hypothesize that such changes are a means to render the synthesis of the metabolic output more efficient than under conditions of constant gene activities. In order to substantiate this hypothesis, we used a flux-balance model of the cellular metabolism. The total time span spent on the production of a given set of target metabolites was split into a series of shorter time intervals (metabolic phases) during which only selected groups of metabolic genes are active. The related flux distributions were calculated under the constraint that genes can be either active or inactive whereby the amount of protein related to an active gene is only controlled by the number of active genes: the lower the number of active genes the more protein can be allocated to the enzymes carrying non-zero fluxes. This concept of a predominantly protein-limited efficiency of gene expression clearly differs from other concepts resting on the assumption of an optimal gene regulation capable of allocating to all enzymes and transporters just that fraction of protein necessary to prevent rate limitation. Applying this concept to a simplified metabolic network of the central carbon metabolism with glucose or lactate as alternative substrates, we demonstrate that switching between optimally chosen stationary flux modes comprising different sets of active genes allows producing a demanded amount of target metabolites in a significantly shorter time than by a single optimal flux mode at fixed gene activities. Our model-based findings suggest that temporal expression of metabolic genes can be advantageous even under conditions of constant external substrate supply.     

The oxidative pentose phosphate pathway: structure and organisation

The oxidative pentose phosphate pathway is a major source of reducing power and metabolic intermediates for biosynthetic processes. Some, if not all, of the enzymes of the pathway are found in both the cytosol and plastids, although the precise distribution of their activities varies. The apparent absence of sections of the pathway from the cytosol potentially complicates metabolism. These complications are partly offset, however, by exchange of intermediates between the cytosol and the plastids through the activities of a family of plastid phosphate translocators. Molecular analysis is confirming the widespread presence of multiple genes encoding each of the enzymes of the oxidative pentose phosphate pathway. Differential expression of these isozymes may ensure that the kinetic properties of the activity that catalyses a specific reaction match the metabolic requirements of a particular tissue. This hypothesis can be tested thanks to recent developments in the application of 13C-steady-state labelling strategies. These strategies make it possible to quantify flux through metabolic networks and to discriminate between pathways of carbohydrate oxidation in the cytosol and plastids.