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436 results on '"Gorwa-Grauslund, Marie"'

5. Evaluation of Pyrophosphate‐Driven Proton Pumps in Saccharomyces cerevisiae under Stress Conditions

Author

Sreenivas, Krishnan, Eisentraut, Leon, Brink, Daniel P., Persson, Viktor C., Carlquist, Magnus, Gorwa-Grauslund, Marie F., van Niel, Ed W. J., Sreenivas, Krishnan, Eisentraut, Leon, Brink, Daniel P., Persson, Viktor C., Carlquist, Magnus, Gorwa-Grauslund, Marie F., and van Niel, Ed W. J.

Abstract

In Saccharomyces cerevisiae, pH homeostasis is reliant on ATP due to the use of proton-translocating ATPase (H+-ATPase) which constitutes a major drain within cellular ATP supply. Here, an exogenous proton-translocating pyrophosphatase (H+-PPase) from Arabidopsis thaliana, which uses inorganic pyrophosphate (PPi) rather than ATP, was evaluated for its effect on reducing the ATP burden. The H+-Ppase was localized to the vacuolar membrane or to the cell membrane, and their impact was studied under acetate stress at a low pH. Biosensors (pHluorin and mQueen-2m) were used to observe changes in intracellular pH (pHi) and ATP levels during growth on either glucose or xylose. A significant improvement of 35% in the growth rate at a pH of 3.7 and 6 g·L−1 acetic acid stress was observed in the vacuolar membrane H+-PPase strain compared to the parent strain. ATP levels were elevated in the same strain during anaerobic glucose and xylose fermentations. During anaerobic xylose fermentations, co-expression of pHluorin and a vacuolar membrane H+-PPase improved the growth characteristics by means of an improved growth rate (11.4%) and elongated logarithmic growth duration. Our study identified a potential method for improving productivity in the use of S. cerevisiae as a cell factory under the harsh conditions present in industry.

Published
2024

43. Electrochemical probing of in vivo 5-hydroxymethyl furfural reduction in saccharomyces cerevisiae

Author

Kostesha, Natalie V., Almeida, Joao R.M., Heiskanen, Arto R., Gorwa-Grauslund, Marie F., Hahn-Hagerdal, Barbel, and Emneus, Jenny

Subjects
Genetically modified organisms -- Chemical properties, Brewer's yeast -- Chemical properties, Electrochemical analysis -- Methods, Chemistry
Abstract

In this work, mediated amperometry was used to evaluate whether differences in intracellular nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) level could be observed between a genetically modified Saccharomyces cerevisiae strain, engineered for NADPH dependent 5-hydroxymethyl-2-furaldehyde (HMF) reduction, and its control strain. Cells overexpressing the alcohol dehydrogenase 6 gene (ADH6 strain) and cells carrying the corresponding control plasmid (control strain) were each immobilized on Au-microelectrodes. The real-time dynamics of NAD(P)H availability in the two strains, preincubated with HMF, was probed using the menadione--ferricyanide double mediator system. A lower intracellular NADPH level as the consequence of more effective HMF reduction was observed for the ADH6 strain both with and without added glucose, which increases the overall cellular NADPH level. The mediated amperometric signal during real-time monitoring of the concentration dependent HMF reduction in living cells could be translated into the cellular enzyme kinetic parameters: [K.sup.app.sub.M,cell], [V.sub.MAX], [k.sub.cat,cell], and [k.sub.cat,cell]/ The results indicated that the overexpression of the ADH6 gene gave a 68% decrease in [K.sup.app.sub.M,cell] and 42% increase in [V.sub.MAX], resulting in a 4-fold increase in [k.sub.cat,cell]/[K.sup.app.sub.M,cell]. These results demonstrate that the mediated amperometric method is useful for monitoring the short-term dynamics of NAD(P)H variations and determining cellular enzyme kinetic parameters in S. cerevisiae cells. 10.1021/ac901402m

Published
2009

49. Amperometric response from the glycolytic versus the pentose phosphate pathway in Saccharomyces cerevisiae cells

Author

Spegel, Christer F., Heiskanen, Arto R., Kostesha, Natalie, Johanson, Ted H., Gorwa-Grauslund, Marie-F., Koudelka-Hep, Milena, Emneus, Jenny, and Ruzgas, Tautgirdas

Subjects
NAD (Coenzyme) -- Research, Brewer's yeast -- Psychological aspects, Glucose metabolism -- Research, Chemistry
Abstract

The two main metabolic pathways involved in sugar metabolism, i.e., the pentose phosphate pathway (PPP) and the glycolytic pathway (GP), were amperometrically monitored using a double-mediator system composed of menadione and ferricyanide. With the use of the Saccharomyces cerevisiae deletion mutant, EBY44, lacking the gene encoding for the branch point enzyme phosphoglucose isomerize, selective amperometric monitoring of the PPP, mainly producing NADPH, and the GP, mainly producing NADH, could be achieved. It was found that the bioelectrocatalytic current was primarily originating from NADPH. This conclusion was supported by metabolite flux analysis, confirming that, in the presence of menadione, the cells increase the rate of NADPH-producing reactions although these processes might be detrimental to cell survival. The higher rate of in vivo NADPHdependent menadione reduction can be ascribed to the fact that the intracellular NADPH/[NADP.sup.+] ratio is much higher than NADH/[NAD.sup.+] as well as that the former ratio is more tightly controlled. This tight control over the cofactor ratios is lost upon cell disintegration as observed from spectrophotometric assays using crude cell extract, and amperometric investigations of permeabilized cells indicate a higher rate of NADH- than NADPH-dependent menadione reduction. These in vitro experiments show a higher activity of NADH-dependent than NADPH-dependent menadione-reducing dehydrogenases in S. cerevisiae cells.

Published
2007

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