scholarly journals Model Parameterization with Quantitative Proteomics: Case Study with Trehalose Metabolism in Saccharomyces cerevisiae

Processes ◽  
2021 ◽  
Vol 9 (1) ◽  
pp. 139
Author(s):  
Chuan Fu Yap ◽  
Manuel Garcia-Albornoz ◽  
Andrew F. Jarnuczak ◽  
Simon J. Hubbard ◽  
Jean-Marc Schwartz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Stress ◽  
Quantitative Proteomics ◽  
Metabolic Flux ◽  
Glycolytic Flux ◽  
Trehalose Metabolism ◽  
Regulatory Effects ◽  
Flux Increase ◽  
Using Data ◽  
Fructose 1,6 Bisphosphate

When Saccharomyces cerevisiae undergoes heat stress it stimulates several changes that are necessary for its survival, notably in carbon metabolism. Notable changes include increase in trehalose production and glycolytic flux. The increase in glycolytic flux has been postulated to be due to the regulatory effects in upper glycolysis, but this has not been confirmed. Additionally, trehalose is a useful industrial compound for its protective properties. A model of trehalose metabolism in S. cerevisiae was constructed using Convenient Modeller, a software that uses a combination of convenience kinetics and a genetic algorithm. The model was parameterized with quantitative omics under standard conditions and validated using data collected under heat stress conditions. The completed model was used to show that feedforward activation of pyruvate kinase by fructose 1,6-bisphosphate during heat stress contributes to the increase in metabolic flux. We were also able to demonstrate in silico that overexpression of enzymes involved in production and degradation of trehalose can lead to higher trehalose yield in the cell. By integrating quantitative proteomics with metabolic modelling, we were able to confirm that the flux increase in trehalose metabolic pathways during heat stress is due to regulatory effects and not purely changes in enzyme expression. The overexpression of enzymes involved in trehalose metabolism is a potential approach to be exploited for trehalose production without need for increasing temperature.

scholarly journals Measuring glycolytic flux in single yeast cells with an orthogonal synthetic biosensor

2019 ◽  
Author(s):  
Francisca Monteiro ◽  
Georg Hubmann ◽  
Justin Norder ◽  
Johan Hekelaar ◽  
Joana Saldida ◽  
...  
Keyword(s):  
Protein Design ◽  
Metabolic Flux ◽  
Single Cells ◽  
Yeast Cells ◽  
Glycolytic Flux ◽  
Cell Level ◽  
Yeast Cultures ◽  
Intrinsic Correlation ◽  
Metabolic Heterogeneity ◽  
Fructose 1,6 Bisphosphate

AbstractMetabolic heterogeneity between individual cells of a population harbors offers significant challenges for fundamental and applied research. Identifying metabolic heterogeneity and investigating its emergence requires tools to zoom into metabolism of individual cells. While methods exist to measure metabolite levels in single cells, we lack capability to measure metabolic flux, i.e. the ultimate functional output of metabolic activity, on the single-cell level. Here, combining promoter engineering, computational protein design, biochemical methods, proteomics and metabolomics, we developed a biosensor to measure glycolytic flux in single yeast cells, by drawing on the robust cell-intrinsic correlation between glycolytic flux and levels of fructose-1,6-bisphosphate (FBP), and by transplanting the B. subtilis FBP-binding transcription factor CggR into yeast. As proof of principle, using fluorescence microscopy, we applied the sensor to identify metabolic subpopulations in yeast cultures. We anticipate that our biosensor will become a valuable tool to identify and study metabolic heterogeneity in cell populations.

scholarly journals Fine tuning the glycolytic flux ratio of EP-bifido pathway for mevalonate production by enhancing glucose-6-phosphate dehydrogenase (Zwf) and CRISPRi suppressing 6-phosphofructose kinase (PfkA) in Escherichia coli

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Ying Li ◽  
He Xian ◽  
Ya Xu ◽  
Yuan Zhu ◽  
Zhijie Sun ◽  
...  
Keyword(s):  
Pentose Phosphate Pathway ◽  
Conversion Rate ◽  
Metabolic Flux ◽  
Flux Ratio ◽  
Reducing Power ◽  
Pentose Phosphate ◽  
Fine Tuning ◽  
High Yield ◽  
Glycolytic Flux ◽  
Acetyl Coa

Abstract Background Natural glycolysis encounters the decarboxylation of glucose partial oxidation product pyruvate into acetyl-CoA, where one-third of the carbon is lost at CO2. We previously constructed a carbon saving pathway, EP-bifido pathway by combining Embden-Meyerhof-Parnas Pathway, Pentose Phosphate Pathway and “bifid shunt”, to generate high yield acetyl-CoA from glucose. However, the carbon conversion rate and reducing power of this pathway was not optimal, the flux ratio of EMP pathway and pentose phosphate pathway (PPP) needs to be precisely and dynamically adjusted to improve the production of mevalonate (MVA). Result Here, we finely tuned the glycolytic flux ratio in two ways. First, we enhanced PPP flux for NADPH supply by replacing the promoter of zwf on the genome with a set of different strength promoters. Compared with the previous EP-bifido strains, the zwf-modified strains showed obvious differences in NADPH, NADH, and ATP synthesis levels. Among them, strain BP10BF accumulated 11.2 g/L of MVA after 72 h of fermentation and the molar conversion rate from glucose reached 62.2%. Second, pfkA was finely down-regulated by the clustered regularly interspaced short palindromic repeats interference (CRISPRi) system. The MVA yield of the regulated strain BiB1F was 8.53 g/L, and the conversion rate from glucose reached 68.7%. Conclusion This is the highest MVA conversion rate reported in shaken flask fermentation. The CRISPRi and promoter fine-tuning provided an effective strategy for metabolic flux redistribution in many metabolic pathways and promotes the chemicals production.

scholarly journals A synthetic RNA-based biosensor for fructose-1,6-bisphosphate that reports glycolytic flux

2021 ◽  
Author(s):  
Alvaro Darío Ortega ◽  
Vakil Takhaveev ◽  
Silke Roelie Vedelaar ◽  
Yi Long ◽  
Neus Mestre-Farràs ◽  
...  
Keyword(s):  
Glycolytic Flux ◽  
Fructose 1,6 Bisphosphate

scholarly journals Molecular Basis of Fructose Utilization by the Wine Yeast Saccharomyces cerevisiae: a Mutated HXT3 Allele Enhances Fructose Fermentation

2007 ◽  
Vol 73 (8) ◽  
pp. 2432-2439 ◽  
Author(s):  
Carole Guillaume ◽  
Pierre Delobel ◽  
Jean-Marie Sablayrolles ◽  
Bruno Blondin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Basis ◽  
Alcoholic Fermentation ◽  
Wine Yeast ◽  
Glycolytic Flux ◽  
Hexose Transporter ◽  
Wine Yeasts ◽  
Yeast Saccharomyces Cerevisiae ◽  
High Fructose ◽  
Fructose Fermentation

ABSTRACT Fructose utilization by wine yeasts is critically important for the maintenance of a high fermentation rate at the end of alcoholic fermentation. A Saccharomyces cerevisiae wine yeast able to ferment grape must sugars to dryness was found to have a high fructose utilization capacity. We investigated the molecular basis of this enhanced fructose utilization capacity by studying the properties of several hexose transporter (HXT) genes. We found that this wine yeast harbored a mutated HXT3 allele. A functional analysis of this mutated allele was performed by examining expression in an hxt1-7Δ strain. Expression of the mutated allele alone was found to be sufficient for producing an increase in fructose utilization during fermentation similar to that observed in the commercial wine yeast. This work provides the first demonstration that the pattern of fructose utilization during wine fermentation can be altered by expression of a mutated hexose transporter in a wine yeast. We also found that the glycolytic flux could be increased by overexpression of the mutant transporter gene, with no effect on fructose utilization. Our data demonstrate that the Hxt3 hexose transporter plays a key role in determining the glucose/fructose utilization ratio during fermentation.

Studies with GIP/Ins cells indicate secretion by gut K cells is KATPchannel independent

2003 ◽  
Vol 284 (5) ◽  
pp. E988-E1000 ◽  
Author(s):  
Song Yan Wang ◽  
Maggie M.-Y. Chi ◽  
Lin Li ◽  
Kelle H. Moley ◽  
Burton M. Wice
Keyword(s):  
Tricarboxylic Acid Cycle ◽  
Calcium Mobilization ◽  
Enteroendocrine Cells ◽  
Glycolytic Flux ◽  
K Cells ◽  
Antibody Staining ◽  
Methyl Pyruvate ◽  
Fructose 1,6 Bisphosphate ◽  
Insulinoma Cells

K cells are a subpopulation of enteroendocrine cells that secrete glucose-dependent insulinotropic polypeptide (GIP), a hormone that promotes glucose homeostasis and obesity. Therefore, it is important to understand how GIP secretion is regulated. GIP-producing (GIP/Ins) cell lines secreted hormones in response to many GIP secretagogues except glucose. In contrast, glyceraldehyde and methyl pyruvate stimulated hormone release. Measurements of intracellular glucose 6-phosphate, fructose 1,6-bisphosphate, and pyruvate levels, as well as glycolytic flux, in glucose-stimulated GIP/Ins cells indicated that glycolysis was not impaired. Analogous results were obtained using glucose-responsive MIN6 insulinoma cells. Citrate levels increased similarly in glucose-treated MIN6 and GIP/Ins cells. Thus pyruvate entered the tricarboxylic acid cycle. Glucose and methyl pyruvate stimulated 1.4- and 1.6-fold increases, respectively, in the ATP-to-ADP ratio in GIP/Ins cells. Glyceraldehyde profoundly reduced, rather than increased, ATP/ADP. Thus nutrient-regulated secretion is independent of the ATP-dependent potassium (KATP) channel. Antibody staining of mouse intestine demonstrated that enteroendocrine cells producing GIP, glucagon-like peptide-1, CCK, or somatostatin do not express detectable levels of inwardly rectifying potassium (Kir) 6.1 or Kir 6.2, indicating that release of these hormones in vivo may also be KATPchannel independent. Conversely, nearly all cells expressing chromogranin A or substance P and ∼50% of the cells expressing secretin or serotonin exhibited Kir 6.2 staining. Compounds that activate calcium mobilization were potent secretagogues for GIP/Ins cells. Secretion was only partially inhibited by verapamil, suggesting that calcium mobilization from intracellular and extracellular sources, independent from KATPchannels, regulates secretion from some, but not all, subpopulations of enteroendocrine cells.

scholarly journals Combined Biosynthetic Pathway Engineering and Storage Pool Expansion for High-Level Production of Ergosterol in Industrial Saccharomyces cerevisiae

2021 ◽  
Vol 9 ◽  
Author(s):  
Zhi-Jiao Sun ◽  
Jia-Zhang Lian ◽  
Li Zhu ◽  
Yi-Qi Jiang ◽  
Guo-Si Li ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Biosynthetic Pathway ◽  
Metabolic Flux ◽  
Feeding Strategy ◽  
Pathway Engineering ◽  
Ergosterol Biosynthesis ◽  
Sterol Esters ◽  
Storage Pool ◽  
Ergosterol Content ◽  
Dry Cell Weight

Ergosterol, a terpenoid compound produced by fungi, is an economically important metabolite serving as the direct precursor of steroid drugs. Herein, ergsosterol biosynthetic pathway modification combined with storage capacity enhancement was proposed to synergistically improve the production of ergosterol in Saccharomyces cerevisiae. S. cerevisiae strain S1 accumulated the highest amount of ergosterol [7.8 mg/g dry cell weight (DCW)] among the wild-type yeast strains tested and was first selected as the host for subsequent metabolic engineering studies. Then, the push and pull of ergosterol biosynthesis were engineered to increase the metabolic flux, overexpression of the sterol acyltransferase gene ARE2 increased ergosterol content to 10 mg/g DCW and additional overexpression of a global regulatory factor allele (UPC2-1) increased the ergosterol content to 16.7 mg/g DCW. Furthermore, considering the hydrophobicity sterol esters and accumulation in lipid droplets, the fatty acid biosynthetic pathway was enhanced to expand the storage pool for ergosterol. Overexpression of ACC1 coding for the acetyl-CoA carboxylase increased ergosterol content from 16.7 to 20.7 mg/g DCW. To address growth inhibition resulted from premature accumulation of ergosterol, auto-inducible promoters were employed to dynamically control the expression of ARE2, UPC2-1, and ACC1. Consequently, better cell growth led to an increase of ergosterol content to 40.6 mg/g DCW, which is 4.2-fold higher than that of the starting strain. Finally, a two-stage feeding strategy was employed for high-density cell fermentation, with an ergosterol yield of 2986.7 mg/L and content of 29.5 mg/g DCW. This study provided an effective approach for the production of ergosterol and other related terpenoid molecules.

scholarly journals Metabolic Fluxes in Corynebacterium glutamicum during Lysine Production with Sucrose as Carbon Source

2004 ◽  
Vol 70 (12) ◽  
pp. 7277-7287 ◽  
Author(s):  
Christoph Wittmann ◽  
Patrick Kiefer ◽  
Oskar Zelder
Keyword(s):  
Carbon Source ◽  
Corynebacterium Glutamicum ◽  
Metabolic Flux ◽  
Tca Cycle ◽  
Pentose Phosphate ◽  
Flux Analysis ◽  
Phosphotransferase System ◽  
Lysine Production ◽  
Metabolic Fluxes ◽  
Fructose 1,6 Bisphosphate

ABSTRACT Metabolic fluxes in the central metabolism were determined for lysine-producing Corynebacterium glutamicum ATCC 21526 with sucrose as a carbon source, providing an insight into molasses-based industrial production processes with this organism. For this purpose, 13C metabolic flux analysis with parallel studies on [1-13CFru]sucrose, [1-13CGlc]sucrose, and [13C6 Fru]sucrose was carried out. C. glutamicum directed 27.4% of sucrose toward extracellular lysine. The strain exhibited a relatively high flux of 55.7% (normalized to an uptake flux of hexose units of 100%) through the pentose phosphate pathway (PPP). The glucose monomer of sucrose was completely channeled into the PPP. After transient efflux, the fructose residue was mainly taken up by the fructose-specific phosphotransferase system (PTS) and entered glycolysis at the level of fructose-1,6-bisphosphate. Glucose-6-phosphate isomerase operated in the gluconeogenetic direction from fructose-6-phosphate to glucose-6-phosphate and supplied additional carbon (7.2%) from the fructose part of the substrate toward the PPP. This involved supply of fructose-6-phosphate from the fructose part of sucrose either by PTSMan or by fructose-1,6-bisphosphatase. C. glutamicum further exhibited a high tricarboxylic acid (TCA) cycle flux of 78.2%. Isocitrate dehydrogenase therefore significantly contributed to the total NADPH supply of 190%. The demands for lysine (110%) and anabolism (32%) were lower than the supply, resulting in an apparent NADPH excess. The high TCA cycle flux and the significant secretion of dihydroxyacetone and glycerol display interesting targets to be approached by genetic engineers for optimization of the strain investigated.

scholarly journals Molecular Basis for Anaerobic Growth of Saccharomyces cerevisiae on Xylose, Investigated by Global Gene Expression and Metabolic Flux Analysis

2004 ◽  
Vol 70 (4) ◽  
pp. 2307-2317 ◽  
Author(s):  
Marco Sonderegger ◽  
Marie Jeppsson ◽  
Bärbel Hahn-Hägerdal ◽  
Uwe Sauer
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Metabolic Flux Analysis ◽  
Metabolic Flux ◽  
Global Gene Expression ◽  
Anaerobic Growth ◽  
Flux Analysis ◽  
Atp Production ◽  
Redox Imbalance ◽  
Chemostat Cultures

ABSTRACT Yeast xylose metabolism is generally considered to be restricted to respirative conditions because the two-step oxidoreductase reactions from xylose to xylulose impose an anaerobic redox imbalance. We have recently developed, however, a Saccharomyces cerevisiae strain that is at present the only known yeast capable of anaerobic growth on xylose alone. Using transcriptome analysis of aerobic chemostat cultures grown on xylose-glucose mixtures and xylose alone, as well as a combination of global gene expression and metabolic flux analysis of anaerobic chemostat cultures grown on xylose-glucose mixtures, we identified the distinguishing characteristics of this unique phenotype. First, the transcript levels and metabolic fluxes throughout central carbon metabolism were significantly higher than those in the parent strain, and they were most pronounced in the xylose-specific, pentose phosphate, and glycerol pathways. Second, differential expression of many genes involved in redox metabolism indicates that increased cytosolic NADPH formation and NADH consumption enable a higher flux through the two-step oxidoreductase reaction of xylose to xylulose in the mutant. Redox balancing is apparently still a problem in this strain, since anaerobic growth on xylose could be improved further by providing acetoin as an external NADH sink. This improved growth was accompanied by an increased ATP production rate and was not accompanied by higher rates of xylose uptake or cytosolic NADPH production. We concluded that anaerobic growth of the yeast on xylose is ultimately limited by the rate of ATP production and not by the redox balance per se, although the redox imbalance, in turn, limits ATP production.

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