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

2020 ◽  
Author(s):  
Alvaro D. Ortega ◽  
Vakil Takhaveev ◽  
Silke Bonsing-Vedelaar ◽  
Yi Long ◽  
Neus Mestre-Farràs ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Hammerhead Ribozyme ◽  
Single Cells ◽  
Glycolytic Flux ◽  
Metabolic Heterogeneity ◽  
Fructose 1,6 Bisphosphate ◽  
Glycolytic Rate ◽  
Selection Of

ABSTRACTMetabolic heterogeneity, the occurrence of different metabolic phenotypes among cells, represents a key challenge in health and biotechnology. To unravel its molecular basis, tools probing metabolism of single cells are needed. While RNA devices harbor huge potential for the development of such tools, until today, it is challenging to create in vivo-functional sensors for any given metabolite. Here, we developed from scratch an RNA-based sensor for fructose-1,6-bisphosphate (FBP), a doubly phosphorylated intermediate of glycolysis. Starting from in vitro selection of an RNA aptamer and its structural analyses, we developed libraries of RNA-based regulatory devices with this aptamer and the hammerhead ribozyme as an actuator. Through FACS-seq-based high-throughput screening in yeast, we identified in vivo-functional FBP-sensing devices that generate fluorescent readout dependent on intracellular FBP concentration. As FBP reports the flux through glycolysis, the developed RNA device can be used to sense the glycolytic rate in single cells, offering unprecedented possibilities to investigate the causes of metabolic heterogeneity.

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 SUGAR-seq enables simultaneous detection of glycans, epitopes, and the transcriptome in single cells

2021 ◽  
Vol 7 (8) ◽  
pp. eabe3610
Author(s):  
Conor J. Kearney ◽  
Stephin J. Vervoort ◽  
Kelly M. Ramsbottom ◽  
Izabela Todorovski ◽  
Emily J. Lelliott ◽  
...  
Keyword(s):  
Single Cell ◽  
Posttranslational Modification ◽  
Cellular Differentiation ◽  
Single Cells ◽  
Simultaneous Detection ◽  
Surface Protein ◽  
Rna Seq ◽  
Cell Level ◽  
Simultaneous Integration ◽  
Unique Surface

Multimodal single-cell RNA sequencing enables the precise mapping of transcriptional and phenotypic features of cellular differentiation states but does not allow for simultaneous integration of critical posttranslational modification data. Here, we describe SUrface-protein Glycan And RNA-seq (SUGAR-seq), a method that enables detection and analysis of N-linked glycosylation, extracellular epitopes, and the transcriptome at the single-cell level. Integrated SUGAR-seq and glycoproteome analysis identified tumor-infiltrating T cells with unique surface glycan properties that report their epigenetic and functional state.

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

A CONVENIENT METHOD OF OBTAINING ASCOSPORES FROM BAKERS' YEAST

1949 ◽  
Vol 27c (4) ◽  
pp. 179-189 ◽  
Author(s):  
A. M. Adams
Keyword(s):  
Convenient Method ◽  
Sodium Acetate ◽  
Solid Medium ◽  
Genetic Research ◽  
Yeast Cells ◽  
Sporulation Medium ◽  
Direct Transfer ◽  
Yeast Cultures ◽  
High Yields ◽  
Solid Nutrient Medium

The superiority of methods involving the use of sporulation media containing acetate, first introduced by Stantial and Elder, over several commonly employed methods is established. A new method for obtaining ascospores from bakers' yeast cultures is recommended involving the direct transfer of vegetative cells from a solid nutrient medium to a solid medium containing acetate. High yields of ascospores are consistently produced after seven days' incubation. This method should lend itself particularly to use in the preparation of ascospores for instructional work, and for genetic research in yeast, and may also find application in yeast taxonomy. The technique recommended is as follows: vegetative yeast cells are multiplied on tomato juice agar or on dextrose nutrient agar, and are then transferred to a solid sporulation medium containing 0.04% dextrose, 0.14% anhydrous sodium acetate, and 2% agar.

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.

Differentiation of Yeast Cultures in Ruminant Feedingstuffs Using a Rapid DNA Fingerprinting Technique

1994 ◽  
Vol 1994 ◽  
pp. 101-101
Author(s):  
Elizabeth Moore ◽  
Denis R. Headon
Keyword(s):  
Sodium Dodecyl ◽  
Yeast Cells ◽  
Microbial Populations ◽  
Yeast Culture ◽  
Yeast Cultures ◽  
Yeast Strains ◽  
Genetic Sequences ◽  
Polymerase Chain ◽  
Different Strains ◽  
Triton X

Research indicates that certain yeast strains are beneficial in their capacity to stimulate key microbial populations. This stimulation is strain specific with similar yeast strains exerting their effect on totally different microbial populations. Future yeast culture supplements may contain mixtures of different strains designed to suit specific diets. This, therefore, requires the development of a rapid sensitive technique to differentiate among taxonomically similar yeast strains in animal diets. This technique, termed the Randomly Amplified Polymorphic DNA (RAPD) assay, is based upon the use of randomly designed short polynucleotide primers to amplify genetic sequences from the DNA of the desired yeast strain. Our objective involves the development of this technique to distinguish between closely related yeast strains present in feed. The feed sample investigated was a standard cattle ration containing three strains of Saccharomyces cerevisiae (1026, 2045 and 2020) and Candida utilis 3001 at a concentration of 106 CFU/g respectively. Isolation of single colonies of yeast strains present was achieved by feed extraction in dilution buffer followed by plating a series of dilutions on rose-bengal agar. Thirty randomly selected colonies were cultured in YPD (1% yeast extract, 2% peptone, 2% glucose) broth for 24 - 30 hours at 30°C. Genomic DNA was isolated from yeast cells by standard methods based on subjection of the cells to vortex mixing in the presence of glass beads, triton X-100, sodium dodecyl sulphate, phenol and chloroform. Isolated DNA from randomly selected colonies was amplified by Polymerase Chain Reaction (PCR) for 45 cycles of 1 min at 94°C, 1 min at 36°C and 1 min at 72°C using randomly designed 10 bp primers.

scholarly journals Glucose Signaling Pathway and Growth Conditions Regulate Gene Expression in Retrotransposon Ty2

2009 ◽  
Vol 64 (7-8) ◽  
pp. 526-532 ◽  
Author(s):  
Sezai Türkel ◽  
Özgür Bayram ◽  
Elif Arık
Keyword(s):  
Gene Expression ◽  
Growth Conditions ◽  
Glucose Sensors ◽  
Yeast Cells ◽  
Growth Stages ◽  
Regulate Gene Expression ◽  
Glucose Signaling ◽  
Yeast Cultures ◽  
Membrane Bound ◽  
High Level

Gene expression in the yeast retrotransposon Ty2 is regulated at transcriptional and translational levels. In this study, we have shown that the transcription of Ty2 is partially dependent on the membrane-bound glucose sensors Gpr1p and Mth1p in Saccharomyces cerevisiae. Transcription of Ty2 decreased approx. 3-fold in the gpr1, mth1 yeast mutant. Moreover, our results revealed that the transcription of Ty2 fluctuates during the growth stages of S. cerevisae. Both transcription and the frameshift rate of Ty2 rapidly dropped when the stationary stage yeast cells were inoculated into fresh medium. There was an instant activation of Ty2 transcription and a high level expression during the entire logarithmic stage of yeast growth. However, the transcription of Ty2 decreased 2-fold when the yeast cultures entered the stationary stage. The frameshift rate in Ty2 also varied depending on the growth conditions. The highest frameshift level was observed during the mid-logarithmic stage. It decreased up to 2-fold during the stationary stage. Furthermore, we have found that the frameshift rate of Ty2 diminished at least 5-fold in slowly growing yeasts. These results indicate that the transcription and the frameshift efficiency are coordinately regulated in the retrotransposon Ty2 depending on the growth conditions of S. cerevisiae.

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