Inhibition and Inactivation of Glucose-phosphorylating Enzymes from Saccharomyces cerevisiae by D-Xylose (original) (raw)

Regulation of phosphotransferases in glucose- and xylose-fermenting yeasts

Applied Biochemistry and Biotechnology, 1997

This research examined the titers of hexokinase (HK), phosphofructokinase (PFK), and xylulokinase (XUK) in Saccharomyces cerevisiae and two xylose fermenting yeasts, Pachysolen tannophilus and Candida shehatae, following shifts in carbon source and aeration. Xylose-grown C. shehatae, glucose-grown P. tannophilus, and glucose-grown S. cerevisiae, had the highest specific activities of XUK, HK, and PFK, respectively. XUK was induced by xylose to moderate levels in both P. tannophilus and C. shehatae, but was present only in trace levels in S. cerevisiae. HK activities in P. tannophilus were two to three fold higher when cells were grown on glucose than when grown on xylose, but HK levels were less inducible in C. shehatae. The PFK activities in S. cerevisiae were 1.5 to 2 times higher than in the two xylose-fermenting yeasts. Transfer from glucose to xylose rapidly inactivated HK in P. tannophilus, and transfer from xylose to glucose inactivated XUK in C. shehatae. The patterns of induction and inactivation indicate that the basic regulatory mechanisms differ in the two xylose fermenting yeasts.

Used but not Sensed - The Paradox of D-xylose Metabolism in Saccharomyces cerevisiae

2019

The realization that the extraction and combustion of fossil fuels is having serious effects on the environment and the climate, together with the ever-growing need for fuels, has led to the development of the concept of the biorefinery. Biorefineries are refineries in which fossil resources, such as oil, are replaced by renewable biomaterials to produce biofuels and biochemicals. Non-edible biomass is used in a lignocellulose-based refinery, which avoids the conflict between fuel and food production, but a number of inherent technical challenges must be overcome. The robust and genetic engineering-friendly yeast Saccharomyces cerevisiae (baker's yeast) is a promising platform organism for biomass fermentation, but it lacks functional assimilatory pathways to utilise Dxylose, the second most abundant sugar in a wide range of lignocellulosic materials. During the past two decades, recombinant forms of S. cerevisiae have been developed able to efficiently convert D-xylose to ethanol. However, the rate of conversion is slow, and D-xylose appears not to be recognised by S. cerevisiae as a fermentable sugar. This thesis is focused on investigating the role of the sugar sensing and signalling routes in the unusual behaviour of S. cerevisiae on D-xylose. A panel of in vivo biosensors coupled to D-glucose signalling routes was used under different physiological conditions and in the presence of different genetic modifications. The green fluorescent protein gene (yEGFP3) was coupled to different endogenous yeast promoters known to be regulated by at least one of the three main sugar pathways: Snf3p/Rgt2p, cAMP/PKA and SNF1/Mig1p. The signallome investigation revealed that a recombinant strain of S. cerevisiae able to assimilate D-xylose could sense high concentrations of D-xylose, but the signal was similar to that observed with low levels of D-glucose: inducing SUC2p (SNF1/Mig1p pathway) and HXT2p (Snf3p/Rgt2p pathway) but repressing HXT1p (Snf3p/Rgt2p and cAMP/PKA pathway). Strains unable to metabolise D-xylose provided no clear signal in the presence of Dxylose due to heterogeneity in the population of the biosensor strains. However, in strains that were able to assimilate D-xylose, the signalling induction pattern was completely opposite to the signal obtained when protein kinase A (PKA) was activated by high levels of D-glucose. It was therefore hypothesized that the signal triggered by a high D-xylose level similar to a low D-glucose signal was due to a low PKA activity. Further validation of the role of sugar signalling was obtained by using targeted deletants known to improve the Dxylose consumption rate without being directly associated with D-xylose catabolic routes. Notably, it was found that the signalling response on D-xylose changed from a low D-glucose signal in the background strain, to simultaneous signalling of high and low D-glucose in the best strain (ira2∆isu1∆). Since IRA2 is a repressor of PKA activity, this finding supported the hypothesis of the malfunction of PKA activity on D-xylose due to poor sensing through this route. This study also focused on understanding whether the sensing signal observed in the presence of high concentrations of D-xylose could be linked to a metabolite acting as a pathway regulator. Using strains in which PGI1, which encodes an isomerase enabling the reversible conversion of glucose-6-phosphate and fructose-6phosphate, had been deleted, it was possible to link changes in signalling to disturbances in the levels of glycolytic intermediates. The findings presented in this thesis support the hypothesis of a dysfunctional sugar signalling mechanism on D-xylose, and show that the phenotype is a result of the lack of membrane sensing in connection with alterations in intracellular signalling.

Repression of xylose utilization by glucose in xylose-fermenting yeasts

Canadian Journal of Microbiology, 1988

xylose utilization by glucose in xylose-fermenting yeasts. Can. J. Microbiol. 34: 1316-1320. The xylose-fermenting yeasts Pichia stipitis, Candida steatolytica, and Candida shehatae were subjected to fermentations in synthetic media containing mixtures of glucose and xylose. In all cases, repression of xylose uptake by glucose was observed, although the extent of repression was different with each yeast. While Candida shehatae was found to utilize xylose effectively in the presence of approximately 5% (wlv) glucose, Candida steatolytica could only utilize xylose when the glucose concentration was below 3 % (wlv), and Pichia stipitis required the glucose concentration in the medium to be below 2% (wlv) before significant xylose utilization occurred.

The binding of glucose and nucleotides to hexokinase from Saccharomyces cerevisiae

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1988

The binding of glucose, ADP and AdoPPINH]P, to the native PII dimer and PII monomer and the proteolytically-modified SII monomer of hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) from Saccharomyces cerevisiae was monitored at pH 6.7 by the concomitant quenching of protein fluorescence. The data were analysed in terms of Qm~,, the maximal quenching of fluorescence at saturating concentrations of ligand, and [L]0.s, the concentration of ligand at half-maximal quenching. No changes in fluorescence were observed with free enzyme and nucleotide alone. In the presence of saturating levels of glucose, Qmax induced by nucleotide was between 2 and 7%, and [L]0. 5 was between 0.12 and 0.56 mM, depending on the nucleotide and enzyme species. Omax induced by glucose alone was between 22 and 25%, while [L] 0.5 was approx. 0.4 mM for either of the monomeric hexokinase forms and 3.4 for PII dimer. In the presence of 6 mM ADP or 2 mM AdoPP[NH]P, Qm~ for glucose was increased by up to 4% and [L]0. s was diminished 3-fold for hexokinase PII monomer, 6-fold for SII monomer, and 15-fold for PII dimer. The results are interpreted in terms of nucleotide-induced conformational change of bexokinase in the presence of glucose and synergistic binding interactions between glucose and nucleotide.

Regulation of xylose metabolism in recombinant Saccharomyces cerevisiae

Microbial Cell Factories, 2008

Background: Considerable interest in the bioconversion of lignocellulosic biomass into ethanol has led to metabolic engineering of Saccharomyces cerevisiae for fermentation of xylose. In the present study, the transcriptome and proteome of recombinant, xylose-utilising S. cerevisiae grown in aerobic batch cultures on xylose were compared with those of glucose-grown cells both in glucose repressed and derepressed states. The aim was to study at the genome-wide level how signalling and carbon catabolite repression differ in cells grown on either glucose or xylose. The more detailed knowledge whether xylose is sensed as a fermentable carbon source, capable of catabolite repression like glucose, or is rather recognised as a non-fermentable carbon source is important for further engineering this yeast for more efficient anaerobic fermentation of xylose.

Physiological Properties of Saccharomyces cerevisiae from Which Hexokinase II Has Been Deleted

Applied and Environmental Microbiology, 2001

Hexokinase II is an enzyme central to glucose metabolism and glucose repression in the yeast Saccharomyces cerevisiae. Deletion of HXK2, the gene which encodes hexokinase II, dramatically changed the physiology of S. cerevisiae. The hxk2-null mutant strain displayed fully oxidative growth at high glucose concentrations in early exponential batch cultures, resulting in an initial absence of fermentative products such as ethanol, a postponed and shortened diauxic shift, and higher biomass yields. Several intracellular changes were associated with the deletion of hexokinase II. The hxk2 mutant had a higher mitochondrial H ؉ -ATPase activity and a lower pyruvate decarboxylase activity, which coincided with an intracellular accumulation of pyruvate in the hxk2 mutant. The concentrations of adenine nucleotides, glucose-6-phosphate, and fructose-6-phosphate are comparable in the wild type and the hxk2 mutant. In contrast, the concentration of fructose-1,6-bisphosphate, an allosteric activator of pyruvate kinase, is clearly lower in the hxk2 mutant than in the wild type. The results suggest a redirection of carbon flux in the hxk2 mutant to the production of biomass as a consequence of reduced glucose repression.

Enzymatic and physiological study of d-xylose metabolism by Candida shehatae

Applied Microbiology and Biotechnology, 1989

Candida shehatae carbon metabolic pathways were correlated with fermentative activity under different growth conditions. Reduced nicotine adenine dinucleotide (NADPH) is the coenzyme preferred for xylose reductase by C. shehatae under in vitro anoxic cell culture conditions. To prevent a redox imbalance derived from intracellular accumulation of NADH in the second enzymatic step of xylose metabolism, the operation of phosphoketolase via in addition the classic pentose phosphate pathway essential for NADH dissimilation is suggested. Variation in cultivation conditions showed a different N A D H / N A D P H ratio coupled to xylose reductase activity. The existence of two xylose reductases is discussed. Like ethanol, xylitol accumulates only under oxygen-limited or anaerobic conditions. Xylitol accumulation under anaerobic conditions was higher when using respiring cells than respirofermentative cells. This fact suggests that cells pregrown under oxygen limitation are better adapted to starting alcoholic fermentation than cells previously grown under aerobic conditions.

Endogenous Xylose Pathway in Saccharomyces cerevisiae

Applied and Environmental Microbiology, 2004

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Enhancement of xylose uptake in 2-deoxyglucose tolerant mutant of Saccharomyces cerevisiae

Journal of Bioscience and Bioengineering, 2011

Chemical mutation of Saccharomyces cerevisiae using ethyl methane sulfonate was performed to enhance its ability of xylose uptake for ethanol production from lignocellulose under microaerobic condition. Among the appeared mutants, the mutant no. 2 (M2) strain screened using inhibitory effects of 2-deoxyglucose (DOG) showed more than 4-fold high ability in xylose uptake compared with the wild type strain, under the presence of glucose. The catabolite repression by glucose was sufficiently reduced in M2 strain due to its tolerance to the high concentration of DOG (0.5%, wt./vol.). Metabolomic analyses of various sugars in the cell revealed that some of xylose was reduced to xylitol in M2 cell, providing the concentration gradient of xylose and more uptake of xylose. Xylulose-5-phosphate was significantly detected in the crude cell extract from M2 strain, indicating higher metabolic activity in pentose phosphate pathway. This was also confirmed by in vitro analyses of key enzymes involved in glucose and xylose metabolism, such as hexokinase, glucose-6-phosphate dehydrogenase and xylose reductase. Glucose uptake was moderately suppressed in the presence of trehalose-6-phosphate inhibiting the activation of hexokinase, resulting in more uptake of xylose through hexose transport system. To our knowledge, this study is the first report verifying that the mutation technique successfully enhances the xylose uptake by S. cerevisiae, particularly under the presence of glucose.