Metabolic modeling of Saccharomyces cerevisiae using the optimal control of homeostasis: a cybernetic model definition

A model is presented to describe the observed behavior of microorganisms that aim at metabolic homeostasis while growing and adapting to their environment in an optimal way. The cellular metabolism is seen as a network with a multiple controller system with both feedback and feedforward control, i.e., a model based on a dynamic optimal metabolic control. The dynamic network consists of aggregated pathways, each having a control setpoint for the metabolic states at a given growth rate. This set of strategies of the cell forms a true cybernetic model with a minimal number of assumptions. The cellular strategies and constraints were derived from metabolic flux analysis using an identified, biochemically relevant, stoichiometry matrix derived from experimental data on the cellular composition of continuous cultures of Saccharomyces cerevisiae. Based on these data a cybernetic model was developed to study its dynamic behavior. The growth rate of the cell is determined by the structural compounds and fluxes of compounds related to central metabolism. In contrast to many other cybernetic models, the minimal model does not consist of any assumed internal kinetic parameters or interactions. This necessitates the use of a stepwise integration with an optimization of the fluxes at every time interval. Some examples of the behavior of this model are given with respect to steady states and pulse responses. This model is very suitable for describing semiquantitatively dynamics of global cellular metabolism and may form a useful framework for including structured and more detailed kinetic models.     

The glutamate synthase (GOGAT) of Saccharomyces cerevisiae plays an important role in central nitrogen metabolism

Central nitrogen metabolism contains two pathways for glutamate biosynthesis, glutaminases and glutamate synthase (GOGAT), using glutamine as the sole nitrogen source. GOGAT's importance for cellular metabolism is still unclear. For a further physiological characterisation of the GOGAT function in central nitrogen metabolism, a GOGAT-negative (Deltaglt1) mutant strain (VWk274 LEU(+)) was studied in glutamine-limited continuous cultures. As reference, we did the same experiments with a wild-type strain (VWk43). Intracellular and extracellular metabolites were analysed during different steady states in both strains. The redox state of the cell was taken into account and the NAD(H) and NADP(H) concentrations were determined as well as the reduced and oxidised forms of glutathione (GSH and GSSG, respectively). The results of this study confirm an earlier suggestion, based on a metabolic network model, that GOGAT may be a link between the carbon catabolic reactions (energy production) and nitrogen anabolic reactions (biomass production) by working as a shuttle between cytosol and mitochondria.     

Rewiring central carbon metabolism for tyrosol and salidroside production in Saccharomyces cerevisiae

Metabolic engineering of Saccharomyces cerevisiae for high-level production of aromatic chemicals has received increasing attention in recent years. Tyrosol production from glucose by S. cerevisiae is considered an environmentally sustainable and safe approach. However, the production of tyrosol and salidroside by engineered S. cerevisiae has been reported to be lower than 2 g/L to date. In this study, S. cerevisiae was engineered with a push-pull-restrain strategy to efficiently produce tyrosol and salidroside from glucose. The biosynthetic pathways of ethanol, phenylalanine, and tryptophan were restrained by disrupting PDC1, PHA2, and TRP3. Subsequently, tyrosol biosynthesis was enhanced with a metabolic pull strategy of introducing PcAAS and EcTyrA^M53I/A354V. Moreover, a metabolic push strategy was implemented with the heterologous expression of phosphoketolase (Xfpk), and then erythrose 4-phosphate was synthesized simultaneously by two pathways, the Xfpk-based pathway and the pentose phosphate pathway, in S. cerevisiae. Furthermore, the heterologous expression of Xfpk alone in S. cerevisiae efficiently improved tyrosol production compared with the coexpression of Xfpk and phosphotransacetylase. Finally, the tyrosol yield increased by approximately 135-folds, compared with that of parent strain. The total amount of tyrosol and salidroside with glucose fed-batch fermentation was over 10 g/L and reached levels suitable for large-scale production.     

Tendency modeling: a new approach to obtain simplified kinetic models of metabolism applied to Saccharomyces cerevisiae

A novel approach to construct kinetic models of metabolic pathways, to be used in metabolic engineering, is presented: the tendency modeling approach. This approach greatly facilitates the construction of these models and can easily be applied to complex metabolic networks. The resulting models contain a minimal number of parameters; identification of their values is straightforward. Use of in vitro obtained information in the identification of the kinetic equations is minimized. The tendency modeling approach has been used to derive a dynamic model of primary metabolism for aerobic growth of Saccharomyces cerevisiae on glucose, in which compartmentation is included. Simulation results obtained with the derived model are satisfying for most of the carbon metabolites that have been measured. Compared to a more detailed model, the simulations of our model are less accurate, but taking into account the much smaller number of kinetic parameters (35 instead of 84), the tendency the modeling approach is considered promising.     

Neuromechanics: an integrative approach for understanding motor control

Neuromechanics seeks to understand how muscles, sense organs,motor pattern generators, and brain interact to produce coordinatedmovement, not only in complex terrain but also when confrontedwith unexpected perturbations. Applications of neuromechanicsinclude ameliorating human health problems (including prosthesisdesign and restoration of movement following brain or spinalcord injury), as well as the design, actuation and control ofmobile robots. In animals, coordinated movement emerges fromthe interplay among descending output from the central nervoussystem, sensory input from body and environment, muscle dynamics,and the emergent dynamics of the whole animal. The inevitablecoupling between neural information processing and the emergentmechanical behavior of animals is a central theme of neuromechanics.Fundamentally, motor control involves a series of transformationsof information, from brain and spinal cord to muscles to body,and back to brain. The control problem revolves around the specifictransfer functions that describe each transformation. The transferfunctions depend on the rules of organization and operationthat determine the dynamic behavior of each subsystem (i.e.,central processing, force generation, emergent dynamics, andsensory processing). In this review, we (1) consider the contributionsof muscles, (2) sensory processing, and (3) central networksto motor control, (4) provide examples to illustrate the interplayamong brain, muscles, sense organs and the environment in thecontrol of movement, and (5) describe advances in both roboticsand neuromechanics that have emerged from application of biologicalprinciples in robotic design. Taken together, these studiesdemonstrate that (1) intrinsic properties of muscle contributeto dynamic stability and control of movement, particularly immediatelyafter perturbations; (2) proprioceptive feedback reinforcesthese intrinsic self-stabilizing properties of muscle; (3) controlsystems must contend with inevitable time delays that can simplifyor complicate control; and (4) like most animals under a varietyof circumstances, some robots use a trial and error processto tune central feedforward control to emergent body dynamics.     

The concentration of ammonia regulates nitrogen metabolism in Saccharomyces cerevisiae.

Saccharomyces cerevisiae was grown in a continuous culture at a single dilution rate with input ammonia concentrations whose effects ranged from nitrogen limitation to nitrogen excess and glucose limitation. The rate of ammonia assimilation (in millimoles per gram of cells per hour) was approximately constant. Increased extracellular ammonia concentrations are correlated with increased intracellular glutamate and glutamine concentrations, increases in levels of NAD-dependent glutamate dehydrogenase activity and its mRNA (gene GDH2), and decreases in levels of NADPH-dependent glutamate dehydrogenase activity and its mRNA (gene GDH1), as well as decreases in the levels of mRNA for the amino acid permease-encoding genes GAP1 and PUT4. The governing factor of nitrogen metabolism might be the concentration of ammonia rather than its flux.     

An integrated pathway system modeling of Saccharomyces cerevisiae HOG pathway: a Petri net based approach

Biochemical networks comprise many diverse components and interactions between them. It has intracellular signaling, metabolic and gene regulatory pathways which are highly integrated and whose responses are elicited by extracellular actions. Previous modeling techniques mostly consider each pathway independently without focusing on the interrelation of these which actually functions as a single system. In this paper, we propose an approach of modeling an integrated pathway using an event-driven modeling tool, i.e., Petri nets (PNs). PNs have the ability to simulate the dynamics of the system with high levels of accuracy. The integrated set of signaling, regulatory and metabolic reactions involved in Saccharomyces cerevisiae’s HOG pathway has been collected from the literature. The kinetic parameter values have been used for transition firings. The dynamics of the system has been simulated and the concentrations of major biological species over time have been observed. The phenotypic characteristics of the integrated system have been investigated under two conditions, viz., under the absence and presence of osmotic pressure. The results have been validated favorably with the existing experimental results. We have also compared our study with the study of idFBA (Lee et al., PLoS Comput Biol 4:e1000–e1086, 2008) and pointed out the differences between both studies. We have simulated and monitored concentrations of multiple biological entities over time and also incorporated feedback inhibition by Ptp2 which has not been included in the idFBA study. We have concluded that our study is the first to the best of our knowledge to model signaling, metabolic and regulatory events in an integrated form through PN model framework. This study is useful in computational simulation of system dynamics for integrated pathways as there are growing evidences that the malfunctioning of the interplay among these pathways is associated with disease.     

Role of NAD-linked glutamate dehydrogenase in nitrogen metabolism in Saccharomyces cerevisiae.

We cloned GDH2, the gene that encodes the NAD-linked glutamate dehydrogenase in the yeast Saccharomyces cerevisiae, by purifying the enzyme, making polyclonal antibodies to it, and using the antibodies to screen a lambda gt11 yeast genomic library. A yeast strain with a deletion-disruption allele of GDH2 which replaced the wild-type gene grew very poorly with glutamate as a nitrogen source, but growth improved significantly when the strain was also provided with adenine or other nitrogenous compounds whose biosynthesis requires glutamine. Our results indicate that the NAD-linked glutamate dehydrogenase catalyzes the major, but not sole, pathway for generation of ammonia from glutamate. We also isolated yeast mutants that lacked glutamate synthase activity and present evidence which shows that normally NAD-linked glutamate dehydrogenase is not involved in glutamate biosynthesis, but that if the enzyme is overexpressed, it may function reversibly in intact cells.     

A multisite integrative cassette for the yeast Saccharomyces cerevisiae.

We have developed a cassette for the integration of cloned DNA sequences at multiple sites in the Saccharomyces cerevisiae genome, taking advantage of the naturally repeated sigma sequences. This cassette contains one engineered sigma element which allows the targeting of an embedded gene at different genomic sigma elements by gene replacement. Two yeast genes, ARG4 and URA3, were thus integrated in the absence of any bacterial sequences, individually or sequentially on twelve chromosomes. Consequently, these studies led to the genetical tagging of individual members of the sigma family.     

Sterol homeostasis in the budding yeast, Saccharomyces cerevisiae

Lipid related diseases, such as obesity, type 2 diabetes, and atherosclerosis are epidemics in developed civilizations. A common underlying factor among these syndromes is excessive subcellular accumulation of lipids such as cholesterol and triglyceride. The homeostatic events that govern these metabolites are understood to varying degrees of sophistication. We describe here the utilization of a genetically powerful model organism, budding yeast, to identify and characterize novel aspects of sterol and lipid homeostasis.     

Dynamic modeling of the central carbon metabolism of Escherichia coli

Application of metabolic engineering principles to the rational design of microbial production processes crucially depends on the ability to describe quantitatively the systemic behavior of the central carbon metabolism to redirect carbon fluxes to the product-forming pathways. Despite the importance for several production processes, development of an essential dynamic model for central carbon metabolism of Escherichia coli has been severely hampered by the current lack of kinetic information on the dynamics of the metabolic reactions. Here we present the design and experimental validation of such a dynamic model, which, for the first time, links the sugar transport system (i.e., phosphotransferase system [PTS]) with the reactions of glycolysis and the pentose-phosphate pathway. Experimental observations of intracellular concentrations of metabolites and cometabolites at transient conditions are used to validate the structure of the model and to estimate the kinetic parameters. Further analysis of the detailed characteristics of the system offers the possibility of studying important questions regarding the stability and control of metabolic fluxes.     

Dynamic genome-scale modeling of Saccharomyces cerevisiae unravels mechanisms for ester formation during alcoholic fermentation

Fermentation employing Saccharomyces cerevisiae has produced alcoholic beverages and bread for millennia. More recently, S. cerevisiae has been used to manufacture specific metabolites for the food, pharmaceutical, and cosmetic industries. Among the most important of these metabolites are compounds associated with desirable aromas and flavors, including higher alcohols and esters. Although the physiology of yeast has been well-studied, its metabolic modulation leading to aroma production in relevant industrial scenarios such as winemaking is still unclear. Here we ask what are the underlying metabolic mechanisms that explain the conserved and varying behavior of different yeasts regarding aroma formation under enological conditions? We employed dynamic flux balance analysis (dFBA) to answer this key question using the latest genome-scale metabolic model (GEM) of S. cerevisiae. The model revealed several conserved mechanisms among wine yeasts, for example, acetate ester formation is dependent on intracellular metabolic acetyl-CoA/CoA levels, and the formation of ethyl esters facilitates the removal of toxic fatty acids from cells using CoA. Species-specific mechanisms were also found, such as a preference for the shikimate pathway leading to more 2-phenylethanol production in the Opale strain as well as strain behavior varying notably during the carbohydrate accumulation phase and carbohydrate accumulation inducing redox restrictions during a later cell growth phase for strain Uvaferm. In conclusion, our new metabolic model of yeast under enological conditions revealed key metabolic mechanisms in wine yeasts, which will aid future research strategies to optimize their behavior in industrial settings.     

Oscillatory metabolism of Saccharomyces cerevisiae: an overview of mechanisms and models

The budding yeast Saccharomyces cerevisiae displays steady oscillations in continuous cultures under certain conditions. Oscillatory responses are important both metabolically and in process applications. Although much information has become available, a definitive theory to explain and model these oscillations is yet to be formulated. Models of oscillatory cultivation have focussed primarily either on intracellular reactions or on transport processes coupled to substantially lumped intracellular kinetics. This review discusses the development of the models and the directions they provide for a comprehensive model of oscillatory metabolism.     

A steady-state modeling approach to validate an in vivo mechanism of the GAL regulatory network in Saccharomyces cerevisiae.

Cellular regulation is a result of complex interactions arising from DNA-protein and protein-protein binding, autoregulation, and compartmentalization and shuttling of regulatory proteins. Experiments in molecular biology have identified these mechanisms recruited by a regulatory network. Mathematical models may be used to complement the knowledge-base provided by in vitro experimental methods. Interactions identified by in vitro experiments can lead to the hypothesis of multiple candidate models explaining the in vivo mechanism. The equilibrium dissociation constants for the various interactions and the total component concentration constitute constraints on the candidate models. In this work, we identify the most plausible in vivo network by comparing the output response to the experimental data. We demonstrate the methodology using the GAL system of Saccharomyces cerevisiae for which the steady-state analysis reveals that Gal3p neither dimerizes nor shuttles between the cytoplasm and the nucleus.     

Gis4, a new component of the ion homeostasis system in the yeast Saccharomyces cerevisiae

Gis4 is a new component of the system required for acquisition of salt tolerance in Saccharomyces cerevisiae. The gis4Delta mutant is sensitive to Na(+) and Li(+) ions but not to osmotic stress. Genetic evidence suggests that Gis4 mediates its function in salt tolerance, at least partly, together with the Snf1 protein kinase and in parallel with the calcineurin protein phosphatase. When exposed to salt stress, mutants lacking gis4Delta display a defect in maintaining low intracellular levels of Na(+) and Li(+) ions and exporting those ions from the cell. This defect is due to diminished expression of the ENA1 gene, which encodes the Na(+) and Li(+) export pump. The protein sequence of Gis4 is poorly conserved and does not reveal any hints to its molecular function. Gis4 is enriched at the cell surface, probably due to C-terminal farnesylation. The CAAX box at the C terminus is required for cell surface localization but does not seem to be strictly essential for the function of Gis4 in salt tolerance. Gis4 and Snf1 seem to share functions in the control of ion homeostasis and ENA1 expression but not in glucose derepression, the best known role of Snf1. Together with additional evidence that links Gis4 genetically and physically to Snf1, it appears that Gis4 may function in a pathway in which Snf1 plays a specific role in controlling ion homeostasis. Hence, it appears that the conserved Snf1 kinase plays roles in different pathways controlling nutrient as well as stress response.