Metabolic control analysis of glycerol synthesis in Saccharomyces cerevisiae

Glycerol, a major by-product of ethanol fermentation by Saccharomyces cerevisiae, is of significant importance to the wine, beer, and ethanol production industries. To gain a clearer understanding of and to quantify the extent to which parameters of the pathway affect glycerol flux in S. cerevisiae, a kinetic model of the glycerol synthesis pathway has been constructed. Kinetic parameters were collected from published values. Maximal enzyme activities and intracellular effector concentrations were determined experimentally. The model was validated by comparing experimental results on the rate of glycerol production to the rate calculated by the model. Values calculated by the model agreed well with those measured in independent experiments. The model also mimics the changes in the rate of glycerol synthesis at different phases of growth. Metabolic control analysis values calculated by the model indicate that the NAD(+)-dependent glycerol 3-phosphate dehydrogenase-catalyzed reaction has a flux control coefficient (C(J)v1) of approximately 0.85 and exercises the majority of the control of flux through the pathway. Response coefficients of parameter metabolites indicate that flux through the pathway is most responsive to dihydroxyacetone phosphate concentration (R(J)DHAP= 0.48 to 0.69), followed by ATP concentration (R(J)ATP = -0.21 to -0.50). Interestingly, the pathway responds weakly to NADH concentration (R(J)NADH = 0.03 to 0.08). The model indicates that the best strategy to increase flux through the pathway is not to increase enzyme activity, substrate concentration, or coenzyme concentration alone but to increase all of these parameters in conjunction with each other.     

Metabolic Engineering of Saccharomyces cerevisiae

Comprehensive knowledge regarding Saccharomyces cerevisiae has accumulated over time, and today S. cerevisiae serves as a widley used biotechnological production organism as well as a eukaryotic model system. The high transformation efficiency, in addition to the availability of the complete yeast genome sequence, has facilitated genetic manipulation of this microorganism, and new approaches are constantly being taken to metabolicially engineer this organism in order to suit specific needs. In this paper, strategies and concepts for metabolic engineering are discussed and several examples based upon selected studies involving S. cerevisiae are reviewed. The many different studies of metabolic engineering using this organism illustrate all the categories of this multidisciplinary field: extension of substrate range, improvements of producitivity and yield, elimination of byproduct formation, improvement of process performance, improvements of cellular properties, and extension of product range including heterologous protein production.     

Metabolic engineering of Saccharomyces cerevisiae.

Comprehensive knowledge regarding Saccharomyces cerevisiae has accumulated over time, and today S. cerevisiae serves as a widley used biotechnological production organism as well as a eukaryotic model system. The high transformation efficiency, in addition to the availability of the complete yeast genome sequence, has facilitated genetic manipulation of this microorganism, and new approaches are constantly being taken to metabolicially engineer this organism in order to suit specific needs. In this paper, strategies and concepts for metabolic engineering are discussed and several examples based upon selected studies involving S. cerevisiae are reviewed. The many different studies of metabolic engineering using this organism illustrate all the categories of this multidisciplinary field: extension of substrate range, improvements of producitivity and yield, elimination of byproduct formation, improvement of process performance, improvements of cellular properties, and extension of product range including heterologous protein production.     

A cybernetic view of microbial growth: modeling of cells as optimal strategists

A cybernetic framework is presented which views microbial response in multiple substrate environments as a judicious investment of cellular resources in synthesizing different key proteins according to an optimal regulatory strategy. A mathematical model is developed within the cybernetic framework for the diauxic growth of Klebsiella pneumoniae on a mixture of D-glucose and D-xylose. The "bang-bang" optimal policy describes well the experimental observations obtained using a fermenter coupled to an Apple II microcomputer. Striking variations in respiratory levels are observed experimentally during the switching of the cell's adaptive machinery for the utilization of the less preferred substrate.     

Schemes of flux control in a model of Saccharomyces cerevisiae glycolysis.

We used parameter scanning to emulate changes to the limiting rate for steps in a fitted model of glucose-derepressed yeast glycolysis. Three flux-control regimes were observed, two of which were under the dominant control of hexose transport, in accordance with various experimental studies and other model predictions. A third control regime in which phosphofructokinase exerted dominant glycolytic flux control was also found, but it appeared to be physiologically unreachable by this model, and all realistically obtainable flux control regimes featured hexose transport as a step involving high flux control.     

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.     

Filamentation of Metabolic Enzymes in Saccharomyces cerevisiae

Compartmentation via filamentation has recently emerged as a novel mechanism for metabolic regulation. In order to identify filamentforming metabolic enzymes systematically, we performed a genome-wide screening of all strains available from an open reading frameGFP collection in Saccharomyces cerevisiae. We discovered nine novel filament-forming proteins and also confirmed those identified previously. From the 4159 strains, we found 23 proteins, mostly metabolic enzymes, which are capable of forming filaments in vivo. In silico protein-protein interaction analysis suggests that these filament-forming proteins can be clustered into several groups, including translational initiation machinery and glucose and nitrogen metabolic pathways. Using glutamine-utilising enzymes as examples, we found that the culture conditions affect the occurrence and length of the metabolic filaments. Furthermore, we found that two CTP synthases(Ura7p and Ura8p) and two asparagine synthetases(Asn1p and Asn2p) form filaments both in the cytoplasm and in the nucleus.Live imaging analyses suggest that metabolic filaments undergo sub-diffusion. Taken together, our genome-wide screening identifies additional filament-forming proteins in S. cerevisiae and suggests that filamentation of metabolic enzymes is more general than currently appreciated.     

Metabolic engineering of the phenylpropanoid pathway in Saccharomyces cerevisiae

Flavonoids are valuable natural products derived from the phenylpropanoid pathway. The objective of this study was to create a host for the biosynthesis of naringenin, the central precursor of many flavonoids. This was accomplished by introducing the phenylpropanoid pathway with the genes for phenylalanine ammonia lyase (PAL) from Rhodosporidium toruloides, 4-coumarate:coenzyme A (CoA) ligase (4CL) from Arabidopsis thaliana, and chalcone synthase (CHS) from Hypericum androsaemum into two Saccharomyces cerevisiae strains, namely, AH22 and a pad1 knockout mutant. Each gene was cloned and inserted into an expression vector under the control of a separate individual GAL10 promoter. Besides its PAL activity, the recombinant PAL enzyme showed tyrosine ammonia lyase activity, which enabled the biosynthesis of naringenin without introducing cinnamate 4-hydroxylase (C4H). 4CL catalyzed the conversion of both trans-cinnamic acid and p-coumaric acid to their corresponding CoA products, which were further converted to pinocembrin chalcone and naringenin chalcone by CHS. These chalcones were cyclized to pinocembrin and naringenin. The yeast AH22 strain coexpressing PAL, 4CL, and CHS produced approximately 7 mg liter(-1) of naringenin and 0.8 mg liter(-1) of pinocembrin. Several by-products, such as 2',4',6'-trihydroxydihydrochalcone and phloretin, were also identified. Precursor feeding studies indicated that metabolic flux to the engineered flavonoid pathway was limited by the flux to the precursor l-tyrosine.     

Saccharomyces cerevisiae as a model organism: a comparative study

Background

Model organisms are used for research because they provide a framework on which to develop and optimize methods that facilitate and standardize analysis. Such organisms should be representative of the living beings for which they are to serve as proxy. However, in practice, a model organism is often selected ad hoc, and without considering its representativeness, because a systematic and rational method to include this consideration in the selection process is still lacking.

Methodology/Principal Findings

In this work we propose such a method and apply it in a pilot study of strengths and limitations of Saccharomyces cerevisiae as a model organism. The method relies on the functional classification of proteins into different biological pathways and processes and on full proteome comparisons between the putative model organism and other organisms for which we would like to extrapolate results. Here we compare S. cerevisiae to 704 other organisms from various phyla. For each organism, our results identify the pathways and processes for which S. cerevisiae is predicted to be a good model to extrapolate from. We find that animals in general and Homo sapiens in particular are some of the non-fungal organisms for which S. cerevisiae is likely to be a good model in which to study a significant fraction of common biological processes. We validate our approach by correctly predicting which organisms are phenotypically more distant from S. cerevisiae with respect to several different biological processes.

Conclusions/Significance

The method we propose could be used to choose appropriate substitute model organisms for the study of biological processes in other species that are harder to study. For example, one could identify appropriate models to study either pathologies in humans or specific biological processes in species with a long development time, such as plants.     

Metabolic Engineering of Glycerol Production in Saccharomyces cerevisiae

Inactivation of TPI1, the Saccharomyces cerevisiae structural gene encoding triose phosphate isomerase, completely eliminates growth on glucose as the sole carbon source. In tpi1-null mutants, intracellular accumulation of dihydroxyacetone phosphate might be prevented if the cytosolic NADH generated in glycolysis by glyceraldehyde-3-phosphate dehydrogenase were quantitatively used to reduce dihydroxyacetone phosphate to glycerol. We hypothesize that the growth defect of tpi1-null mutants is caused by mitochondrial reoxidation of cytosolic NADH, thus rendering it unavailable for dihydroxyacetone-phosphate reduction. To test this hypothesis, a tpi1Δ nde1Δ nde2Δ gut2Δ quadruple mutant was constructed. NDE1 and NDE2 encode isoenzymes of mitochondrial external NADH dehydrogenase; GUT2 encodes a key enzyme of the glycerol-3-phosphate shuttle. It has recently been demonstrated that these two systems are primarily responsible for mitochondrial oxidation of cytosolic NADH in S. cerevisiae. Consistent with the hypothesis, the quadruple mutant grew on glucose as the sole carbon source. The growth on glucose, which was accompanied by glycerol production, was inhibited at high-glucose concentrations. This inhibition was attributed to glucose repression of respiratory enzymes as, in the quadruple mutant, respiratory pyruvate dissimilation is essential for ATP synthesis and growth. Serial transfer of the quadruple mutant on high-glucose media yielded a spontaneous mutant with much higher specific growth rates in high-glucose media (up to 0.10 h⁻¹ at 100 g of glucose·liter⁻¹). In aerated batch cultures grown on 400 g of glucose·liter⁻¹, this engineered S. cerevisiae strain produced over 200 g of glycerol·liter⁻¹, corresponding to a molar yield of glycerol on glucose close to unity.     

Metabolic engineering of glycerol production in Saccharomyces cerevisiae

Inactivation of TPI1, the Saccharomyces cerevisiae structural gene encoding triose phosphate isomerase, completely eliminates growth on glucose as the sole carbon source. In tpi1-null mutants, intracellular accumulation of dihydroxyacetone phosphate might be prevented if the cytosolic NADH generated in glycolysis by glyceraldehyde-3-phosphate dehydrogenase were quantitatively used to reduce dihydroxyacetone phosphate to glycerol. We hypothesize that the growth defect of tpi1-null mutants is caused by mitochondrial reoxidation of cytosolic NADH, thus rendering it unavailable for dihydroxyacetone-phosphate reduction. To test this hypothesis, a tpi1delta nde1delta nde2delta gut2delta quadruple mutant was constructed. NDE1 and NDE2 encode isoenzymes of mitochondrial external NADH dehydrogenase; GUT2 encodes a key enzyme of the glycerol-3-phosphate shuttle. It has recently been demonstrated that these two systems are primarily responsible for mitochondrial oxidation of cytosolic NADH in S. cerevisiae. Consistent with the hypothesis, the quadruple mutant grew on glucose as the sole carbon source. The growth on glucose, which was accompanied by glycerol production, was inhibited at high-glucose concentrations. This inhibition was attributed to glucose repression of respiratory enzymes as, in the quadruple mutant, respiratory pyruvate dissimilation is essential for ATP synthesis and growth. Serial transfer of the quadruple mutant on high-glucose media yielded a spontaneous mutant with much higher specific growth rates in high-glucose media (up to 0.10 h(-1) at 100 g of glucose. liter(-1)). In aerated batch cultures grown on 400 g of glucose. liter(-1), this engineered S. cerevisiae strain produced over 200 g of glycerol. liter(-1), corresponding to a molar yield of glycerol on glucose close to unity.     

代谢工程改造酿酒酵母合成肌醇

【目的】肌醇别名环己六醇,是一种具有生物活性的糖醇,在医药、食品和饲料等领域具有重要的应用价值。为获得生产肌醇的微生物细胞工厂,通过代谢工程改造,构建生产肌醇的酿酒酵母工程菌株。【方法】对酿酒酵母肌醇合成途径的正负调控同时改造,过表达肌醇-3-磷酸合成酶基因ino1,敲除肌醇生物合成的转录抑制子基因opi1和抗性基因kan MX,获得重组菌。利用气相色谱法检测重组菌发酵液中肌醇含量。【结果】构建了生物安全性的产肌醇基因工程菌株,摇瓶培养产量为1.021 g/L。【结论】通过过表达ino1和敲除opi1来改造酿酒酵母,能够有效提高重组菌的肌醇产量,为下一步的微生物发酵法产肌醇的工业应用奠定基础。     

Metabolic surprises in Saccharomyces cerevisiae during adaptation to saline conditions: questions, some answers and a model

This review describes the metabolic alterations and adaptations of yeast cells in response to osmotic stress. The basic theme of the cellular response is known to be exclusion of the extracellular stress agent salt and intracellular accumulation of the compatible solute glycerol. Molecular details of these basic processes are currently rather well known. However, analysis of expression changes during adaptation to salt has revealed a number of metabolic surprises. These include the induced expression of genes involved in glycerol dissimilation as well as trehalose turnover. The physiological rationale for these responses to osmotic stress is discussed. A model is presented in which it is hypothesised that the two pathways function as glycolytic safety valves during adaptation to stress.     

Metabolic flux analysis of xylose metabolism in recombinant Saccharomyces cerevisiae using continuous culture

This study focused on elucidating metabolism of xylose in a Saccharomyces cerevisiae strain that overexpresses xylose reductase and xylitol dehydrogenase from Pichia stipitis, as well as the endogenous xylulokinase. The influence of xylose on overall metabolism was examined supplemented with low glucose levels with emphasis on two potential bottlenecks; cofactor requirements and xylose uptake. Results of metabolic flux analysis in continuous cultivations show changes in central metabolism due to the cofactor imbalance imposed by the two-step oxidoreductase reaction of xylose to xylulose. A comparison between cultivations on 27:3g/L xylose-glucose mixture and 10g/L glucose revealed that the NADPH-generating flux from glucose-6-phosphate to ribulose-5-phosphate was almost tenfold higher on xylose-glucose mixture and due to the loss of carbon in that pathway the total flux to pyruvate was only around 60% of that on glucose. As a consequence also the fluxes in the citric acid cycle were reduced to around 60%. As the glucose level was decreased to 0.1g/L the fluxes to pyruvate and in the citric acid cycle were further reduced to 30% and 20%, respectively. The results from in vitro and in vivo xylose uptake measurements showed that the specific xylose uptake rate was highest at the lowest glucose level, 0.1g/L.     

Metabolic engineering of Saccharomyces cerevisiae for the production of n-butanol

Background

The thermotolerant methylotrophic yeast Hansenula polymorpha is capable of alcoholic fermentation of xylose at elevated temperatures (45 – 48°C). Such property of this yeast defines it as a good candidate for the development of an efficient process for simultaneous saccharification and fermentation. However, to be economically viable, the main characteristics of xylose fermentation of H. polymorpha have to be improved.

Results

Site-specific mutagenesis of H. polymorpha XYL1 gene encoding xylose reductase was carried out to decrease affinity of this enzyme toward NADPH. The modified version of XYL1 gene under control of the strong constitutive HpGAP promoter was overexpressed on a Δxyl1 background. This resulted in significant increase in the K_M for NADPH in the mutated xylose reductase (K341 → R N343 → D), while K_M for NADH remained nearly unchanged. The recombinant H. polymorpha strain overexpressing the mutated enzyme together with native xylitol dehydrogenase and xylulokinase on Δxyl1 background was constructed. Xylose consumption, ethanol and xylitol production by the constructed strain were determined for high-temperature xylose fermentation at 48°C. A significant increase in ethanol productivity (up to 7.3 times) was shown in this recombinant strain as compared with the wild type strain. Moreover, the xylitol production by the recombinant strain was reduced considerably to 0.9 mg × (L × h)^-1 as compared to 4.2 mg × (L × h)^-1 for the wild type strain.

Conclusion

Recombinant strains of H. polymorpha engineered for improved xylose utilization are described in the present work. These strains show a significant increase in ethanol productivity with simultaneous reduction in the production of xylitol during high-temperature xylose fermentation.