Transcriptome of Saccharomyces cerevisiae during production of D-xylonate.

BACKGROUND: Production of D-xylonate by the yeast S. cerevisiae provides an example of bioprocess development for sustainable production of value-added chemicals from cheap raw materials or side streams. Production of D-xylonate may lead to considerable intracellular accumulation of D-xylonate and to loss of viability during the production process. In order to understand the physiological responses associated with D-xylonate production, we performed transcriptome analyses during D-xylonate production by a robust recombinant strain of S. cerevisiae which produces up to 50 g/L D-xylonate. RESULTS: Comparison of the transcriptomes of the D-xylonate producing and the control strain showed considerably higher expression of the genes controlled by the cell wall integrity (CWI) pathway and of some genes previously identified as up-regulated in response to other organic acids in the D-xylonate producing strain. Increased phosphorylation of Slt2 kinase in the D-xylonate producing strain also indicated that D-xylonate production caused stress to the cell wall. Surprisingly, genes encoding proteins involved in translation, ribosome structure and RNA metabolism, processes which are commonly down-regulated under conditions causing cellular stress, were up-regulated during D-xylonate production, compared to the control. The overall transcriptional responses were, therefore, very dissimilar to those previously reported as being associated with stress, including stress induced by organic acid treatment or production. Quantitative PCR analyses of selected genes supported the observations made in the transcriptomic analysis. In addition, consumption of ethanol was slower and the level of trehalose was lower in the D-xylonate producing strain, compared to the control. CONCLUSIONS: The production of organic acids has a major impact on the physiology of yeast cells, but the transcriptional responses to presence or production of different acids differs considerably, being much more diverse than responses to other stresses. D-Xylonate production apparently imposed considerable stress on the cell wall. Transcriptional data also indicated that activation of the PKA pathway occurred during D-xylonate production, leaving cells unable to adapt normally to stationary phase. This, together with intracellular acidification, probably contributes to cell death.

Integration of transcription and flux data reveals molecular paths associated with differences in oxygen-dependent phenotypes of Saccharomyces cerevisiae.

BACKGROUND: Saccharomyces cerevisiae is able to adapt to a wide range of external oxygen conditions. Previously, oxygen-dependent phenotypes have been studied individually at the transcriptional, metabolite, and flux level. However, the regulation of cell phenotype occurs across the different levels of cell function. Integrative analysis of data from multiple levels of cell function in the context of a network of several known biochemical interaction types could enable identification of active regulatory paths not limited to a single level of cell function. RESULTS: The graph theoretical method called Enriched Molecular Path detection (EMPath) was extended to enable integrative utilization of transcription and flux data. The utility of the method was demonstrated by detecting paths associated with phenotype differences of S. cerevisiae under three different conditions of oxygen provision: 20.9%, 2.8% and 0.5%. The detection of molecular paths was performed in an integrated genome-scale metabolic and protein-protein interaction network. CONCLUSIONS: The molecular paths associated with the phenotype differences of S. cerevisiae under conditions of different oxygen provisions revealed paths of molecular interactions that could potentially mediate information transfer between processes that respond to the particular oxygen availabilities.

Transcriptional responses of Saccharomyces cerevisiae to shift from respiratory and respirofermentative to fully fermentative metabolism.

In industrial fermentations of Saccharomyces cerevisiae, transient changes in oxygen concentration commonly occur and it is important to understand the behavior of cells during these changes. Glucose-limited chemostat cultivations were used to study the time-dependent effect of sudden oxygen depletion on the transcriptome of S. cerevisiae cells initially in fully aerobic or oxygen-limited conditions. The overall responses to anaerobic conditions of cells initially in different conditions were very similar. Independent of initial culture conditions, transient downregulation of genes related to growth and cell proliferation, mitochondrial translation and protein import, and sulphate assimilation was seen. In addition, transient or permanent upregulation of genes related to protein degradation, and phosphate and amino acid uptake was observed in all cultures. However, only in the initially oxygen-limited cultures was a transient upregulation of genes related to fatty acid oxidation, peroxisomal biogenesis, oxidative phosphorylation, TCA cycle, response to oxidative stress, and pentose phosphate pathway observed. Furthermore, from the initially oxygen-limited conditions, a rapid response around the metabolites of upper glycolysis and the pentose phosphate pathway was seen, while from the initially fully aerobic conditions, a slower response around the pathways for utilization of respiratory carbon sources was observed.

Low oxygen levels as a trigger for enhancement of respiratory metabolism in Saccharomyces cerevisiae.

BACKGROUND: The industrially important yeast Saccharomyces cerevisiae is able to grow both in the presence and absence of oxygen. However, the regulation of its metabolism in conditions of intermediate oxygen availability is not well characterised. We assessed the effect of oxygen provision on the transcriptome and proteome of S. cerevisiae in glucose-limited chemostat cultivations in anaerobic and aerobic conditions, and with three intermediate (0.5, 1.0 and 2.8% oxygen) levels of oxygen in the feed gas. RESULTS: The main differences in the transcriptome were observed in the comparison of fully aerobic, intermediate oxygen and anaerobic conditions, while the transcriptome was generally unchanged in conditions receiving different intermediate levels (0.5, 1.0 or 2.8% O2) of oxygen in the feed gas. Comparison of the transcriptome and proteome data suggested post-transcriptional regulation was important, especially in 0.5% oxygen. In the conditions of intermediate oxygen, the genes encoding enzymes of the respiratory pathway were more highly expressed than in either aerobic or anaerobic conditions. A similar trend was also seen in the proteome and in enzyme activities of the TCA cycle. Further, genes encoding proteins of the mitochondrial translation machinery were present at higher levels in all oxygen-limited and anaerobic conditions, compared to fully aerobic conditions. CONCLUSION: Global upregulation of genes encoding components of the respiratory pathway under conditions of intermediate oxygen suggested a regulatory mechanism to control these genes as a response to the need of more efficient energy production. Further, cells grown in three different intermediate oxygen levels were highly similar at the level of transcription, while they differed at the proteome level, suggesting post-transcriptional mechanisms leading to distinct physiological modes of respiro-fermentative metabolism.

Oxygen dependence of metabolic fluxes and energy generation of Saccharomyces cerevisiae CEN.PK113-1A.

BACKGROUND: The yeast Saccharomyces cerevisiae is able to adjust to external oxygen availability by utilizing both respirative and fermentative metabolic modes. Adjusting the metabolic mode involves alteration of the intracellular metabolic fluxes that are determined by the cell's multilevel regulatory network. Oxygen is a major determinant of the physiology of S. cerevisiae but understanding of the oxygen dependence of intracellular flux distributions is still scarce. RESULTS: Metabolic flux distributions of S. cerevisiae CEN.PK113-1A growing in glucose-limited chemostat cultures at a dilution rate of 0.1 h-1 with 20.9%, 2.8%, 1.0%, 0.5% or 0.0% O2 in the inlet gas were quantified by 13C-MFA. Metabolic flux ratios from fractional [U-13C]glucose labelling experiments were used to solve the underdetermined MFA system of central carbon metabolism of S. cerevisiae.While ethanol production was observed already in 2.8% oxygen, only minor differences in the flux distribution were observed, compared to fully aerobic conditions. However, in 1.0% and 0.5% oxygen the respiratory rate was severely restricted, resulting in progressively reduced fluxes through the TCA cycle and the direction of major fluxes to the fermentative pathway. A redistribution of fluxes was observed in all branching points of central carbon metabolism. Yet only when oxygen provision was reduced to 0.5%, was the biomass yield exceeded by the yields of ethanol and CO2. Respirative ATP generation provided 59% of the ATP demand in fully aerobic conditions and still a substantial 25% in 0.5% oxygenation. An extensive redistribution of fluxes was observed in anaerobic conditions compared to all the aerobic conditions. Positive correlation between the transcriptional levels of metabolic enzymes and the corresponding fluxes in the different oxygenation conditions was found only in the respirative pathway. CONCLUSION: 13C-constrained MFA enabled quantitative determination of intracellular fluxes in conditions of different redox challenges without including redox cofactors in metabolite mass balances. A redistribution of fluxes was observed not only for respirative, respiro-fermentative and fermentative metabolisms, but also for cells grown with 2.8%, 1.0% and 0.5% oxygen. Although the cellular metabolism was respiro-fermentative in each of these low oxygen conditions, the actual amount of oxygen available resulted in different contributions through respirative and fermentative pathways.

Transcription of hexose transporters of Saccharomyces cerevisiae is affected by change in oxygen provision.

BACKGROUND: The gene family of hexose transporters in Saccharomyces cerevisiae consists of 20 members; 18 genes encoding transporters (HXT1-HXT17, GAL2) and two genes encoding sensors (SNF3, RGT2). The effect of oxygen provision on the expression of these genes was studied in glucose-limited chemostat cultivations (D = 0.10 h(-1), pH 5, 30 degrees C). Transcript levels were measured from cells grown in five steady state oxygen levels (0, 0.5, 1, 2.8 and 20.9% O2), and from cells under conditions in which oxygen was introduced to anaerobic cultures or removed from cultures receiving oxygen. RESULTS: The expression pattern of the HXT gene family was distinct in cells grown under aerobic, hypoxic and anaerobic conditions. The transcription of HXT2, HXT4 and HXT5 was low when the oxygen concentration in the cultures was low, both under steady state and non-steady state conditions, whereas the expression of HXT6, HXT13 and HXT15/16 was higher in hypoxic than in fully aerobic or anaerobic conditions. None of the HXT genes showed higher transcript levels in strictly anaerobic conditions. Expression of HXT9, HXT14 and GAL2 was not detected under the culture conditions studied. CONCLUSION: When oxygen becomes limiting in a glucose-limited chemostat cultivation, the glucose uptake rate per cell increases. However, the expression of none of the hexose transporter encoding genes was increased in anaerobic conditions. It thus seems that the decrease in the moderately low affinity uptake and consequently the relative increase of high affinity uptake may itself allow the higher specific glucose consumption rate to occur in anaerobic compared to aerobic conditions.

Central carbon metabolism of Saccharomyces cerevisiae in anaerobic, oxygen-limited and fully aerobic steady-state conditions and following a shift to anaerobic conditions.

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.

Expression of xyloglucan endotransglycosylases of Gerbera hybrida and Betula pendula in Pichia pastoris.

The plant enzyme xyloglucan endotransglycosylase (XET; EC 2.4.1.207, xyloglucan:xyloglucosyl transferase) participates in selective modification of plant cell walls during cell growth. XETs are potential catalysts in various applications. Here, sequences encoding two XETs from Gerbera hybrida and Betula pendula are reported. The encoded proteins, which are 51% identical at the amino acid level, were expressed in the yeast Pichia pastoris in secreted form with the aid of mating factor alpha signal sequence. XET production in shake flask cultivations was better at 22 degrees C than at 30 degrees C. Both the yield of protein of expected molecular mass and the XET activity improved at the lower temperature. Under all cultivation conditions studied, higher amounts of XET from B. pendula (BXET) were expressed than XET from G. hybrida (GXET). Both XET enzymes were produced in 16l fed-batch bioreactor cultures. GXET was produced in methanol-limited fed-batch cultivation in minimal medium, and BXET in temperature-limited fed-batch (TLFB) in minimal or complex medium. Production was highest in TLFB in complex medium. BXET was purified from the culture filtrate and characterized. Based on the specific activity of the purified protein, 60-70 mg l(-1) BXET was produced in the TLFB in complex medium.

The ORF YNL274c (GOR1) codes for glyoxylate reductase in Saccharomyces cerevisiae.

The enzyme glyoxylate reductase reversibly reduces glyoxylate to glycolate, or alternatively hydroxypyruvate to D-glycerate, using either NADPH or NADH as a co-factor. The enzyme has multiple metabolic roles in different organisms. In this paper we show that GOR1 (ORF YNL274c) encodes a glyoxylate reductase and not a hydroxyisocaproate dehydrogenase in Saccharomyces cerevisiae, even though it also has minor activity on alpha-ketoisocaproate. In addition, we show that deletion of the glyoxylate reductase-encoding gene leads to higher biomass concentration after diauxic shift.

Xylose chemostat isolates of Saccharomyces cerevisiae show altered metabolite and enzyme levels compared with xylose, glucose, and ethanol metabolism of the original strain.

The efficient conversion of xylose-containing biomass hydrolysate by the ethanologenic yeast Saccharomyces cerevisiae to useful chemicals such as ethanol still remains elusive, despite significant efforts in both strain and process development. This study focused on the recovery and characterization of xylose chemostat isolates of a S. cerevisiae strain that overexpresses xylose reductase- and xylitol dehydrogenase-encoding genes from Pichia stipitis and the gene encoding the endogenous xylulokinase. The isolates were recovered from aerobic chemostat cultivations on xylose as the sole or main carbon source. Under aerobic conditions, on minimal medium with 30 g l(-1) xylose, the growth rate of the chemostat isolates was 3-fold higher than that of the original strain (0.15 h(-1) vs 0.05 h(-1)). In a detailed characterization comparing the metabolism of the isolates with the metabolism of xylose, glucose, and ethanol in the original strain, the isolates showed improved properties in the assumed bottlenecks of xylose metabolism. The xylose uptake rate was increased almost 2-fold. Activities of the key enzymes in the pentose phosphate pathway (transketolase, transaldolase) increased 2-fold while the concentrations of their substrates (pentose 5-phosphates, sedoheptulose 7-phosphate) decreased correspondingly. Under anaerobic conditions, on minimal medium with 45 g l(-1) xylose, the ethanol productivity (in terms of cell dry weight; CDW) of one of the isolates increased from 0.012 g g(-1) CDW h(-1) to 0.017 g g(-1) CDW h(-1) and the yield from 0.09 g g(-1) xylose to 0.14 g g(-1) xylose, respectively.