Saccharomyces cerevisiae CEN.PK113-1A was grown in glucose-limited chemostat culture with 0%, 0.5%, 1.0%, 2.8% or 20.9% O2 in the inlet gas (D=0.10 h(-1), pH 5, 30 degrees C) to determine the effects of oxygen on 17 metabolites and 69 genes related to central carbon metabolism. The concentrations of tricarboxylic acid cycle (TCA) metabolites and all glycolytic metabolites except 2-phosphoglycerate+3-phosphoglycerate and phosphoenolpyruvate were higher in anaerobic than in fully aerobic conditions. Provision of only 0.5-1% O2 reduced the concentrations of most metabolites, as compared with anaerobic conditions. Transcription of most genes analyzed was reduced in 0%, 0.5% or 1.0% O2 relative to cells grown in 2.8% or 20.9% O2. Ethanol production was observed with 2.8% or less O2. After steady-state analysis in defined oxygen concentrations, the conditions were switched from aerobic to anaerobic. Metabolite and transcript levels were monitored for up to 96 h after the transition, and this showed that more than 30 h was required for the cells to fully adapt to anaerobiosis. Levels of metabolites of upper glycolysis and the TCA cycle increased following the transition to anaerobic conditions, whereas those of metabolites of lower glycolysis generally decreased. Gene regulation was more complex, with some genes showing transient upregulation or downregulation during the adaptation to anaerobic conditions.

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.

The field of systems biology is often held back by difficulties in obtaining comprehensive, highquality, quantitative data sets. In this paper, we undertook an interlaboratory effort to generate such a data set for a very large number of cellular components in the yeast Saccharomyces cerevisiae, a widely used model organism that is also used in the production of fuels, chemicals, food ingredients and pharmaceuticals. With the current focus on biofuels and sustainability, there is much interest in harnessing this species as a general cell factory. In this study, we characterized two yeast strains, under two standard growth conditions. We ensured the high quality of the experimental data by evaluating a wide range of sampling and analytical techniques. Here we show significant differences in the maximum specific growth rate and biomass yield between the two strains. on the basis of the integrated analysis of the highthroughput data, we hypothesize that differences in phenotype are due to differences in protein metabolism.

The baker's yeast Saccharomyces cerevisiae is generally classified as a non-xylose-utilizing organism. We found that S. cerevisiae can grow on D-xylose when only the endogenous genes GRE3 (YHR104w), coding for a nonspecific aldose reductase, and XYL2 (YLR070c, ScXYL2), coding for a xylitol dehydrogenase (XDH), are overexpressed under endogenous promoters. In nontransformed S. cerevisiae strains, XDH activity was significantly higher in the presence of xylose, but xylose reductase (XR) activity was not affected by the choice of carbon source. The expression of SOR1, encoding a sorbitol dehydrogenase, was elevated in the presence of xylose as were the genes encoding transketolase and transaldolase. An S. cerevisiae strain carrying the XR and XDH enzymes from the xylose-utilizing yeast Pichia stipitis grew more quickly and accumulated less xylitol than did the strain overexpressing the endogenous enzymes. Overexpression of the GRE3 and ScXYL2 genes in the S. cerevisiae CEN.PK2 strain resulted in a growth rate of 0.01 g of cell dry mass liter ؊1 h ؊1 and a xylitol yield of 55% when xylose was the main carbon source.The pentose sugar xylose is a major constituent of lignocellulose. Saccharomyces cerevisiae cannot use xylose, instead converting it primarily to xylitol with only a small fraction going into biomass or ethanol (44,45). Recombinant xylose-metabolizing S. cerevisiae strains contain genes from the xylose-utilizing yeast Pichia stipitis coding enzymes for the first two steps in xylose conversion (23,38,46). However, the potential of S. cerevisiae's own enzymes, if they are overexpressed, has not been evaluated.In xylose-utilizing fungi, xylose reductase (XR) reduces xylose to xylitol, which is oxidized to xylulose by xylitol dehydrogenase (XDH). Xylulose is subsequently phosphorylated to xylulose 5-phosphate by xylulokinase and metabolized through the pentose phosphate pathway. S. cerevisiae cannot utilize xylose but can grow on xylulose (15, 43). Thus, the inability of S. cerevisiae to utilize xylose was attributed to its inability to convert xylose to xylulose (15), even though low XR and XDH activities are known in S. cerevisiae (4). A nonspecific aldose reductase, converting xylose to xylitol, was purified and characterized from S. cerevisiae (24); however, the genes coding for the putative XR and XDH enzymes remained unknown. The third enzyme in the xylose pathway, xylulokinase, is encoded by XKS1, a gene that has been cloned from, and probably is functional in, S. cerevisiae (19). Moderate increases in xylulokinase activity are beneficial in recombinant xylose-metabolizing S. cerevisiae strains (9,18,20,21,40).Based on the S. cerevisiae genome sequence (14), the Nterminal amino acid sequence of the previously purified aldoketo reductase corresponds to the open reading frame YHR104w (GRE3), which has 72% amino acid similarity to the XR enzyme of P. stipitis. This enzyme can reduce a wide variety of ketose substrates and requires a NADPH cofactor (24). The XR of P. stipitis can use either NADH or NADPH i...

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 27:3 g/L xylose-glucose mixture and 10 g/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 0.1 g/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, 0.1 g/L.