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

<p>Abstract</p> <p>Background</p> <p>The yeast <it>Saccharomyces cerevisiae </it>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 <it>S. cerevisiae </it>but understanding of the oxygen dependence of intracellular flux distributions is still scarce.</p> <p>Results</p> <p>Metabolic flux distributions of <it>S. cerevisiae </it>CEN.PK113-1A growing in glucose-limited chemostat cultures at a dilution rate of 0.1 h^-1with 20.9%, 2.8%, 1.0%, 0.5% or 0.0% O₂in 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 <it>S. cerevisiae</it>.</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

Evidence that the gene YLR070c of Saccharomyces cerevisiae encodes a xylitol dehydrogenase

The open reading frame YLR070c of Saccharomyces cerevisiae has high sequence similarity to S. cerevisiae sorbitol dehydrogenase and to xylitol dehydrogenase of Pichia stipitis. Overexpression of this open reading frame in S. cerevisiae resulted in xylitol dehydrogenase activity. The enzyme is specific for NADH. The following Michaelis constants were estimated: D-xylulose, 1.1 mM; NADH, 240 μM (at pH 7.0); xylitol, 25 mM; NAD, 100 μM (at pH 9.0). Xylitol dehydrogenase activity with the same kinetic properties can also be induced by xylose in wild type S. cerevisiae cells. Copyright (C) 1999 Federation of European Biochemical Societies.</p

Endogenous xylose pathway in Saccharomyces cerevisiae

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

Metabolic engineering of Saccharomyces cerevisiae for conversion of D-glucose to xylitol and other five-carbon sugars and sugar alcohols

Recombinant Saccharomyces cerevisiae strains that produce the sugar alcohols xylitol and ribitol and the pentose sugar d-ribose from d-glucose in a single fermentation step are described. A transketolase-deficient S. cerevisiae strain accumulated d-xylulose 5-phosphate intracellularly and released ribitol and pentose sugars (d-ribose, d-ribulose, and d-xylulose) into the growth medium. Expression of the xylitol dehydrogenase-encoding gene XYL2 of Pichia stipitis in the transketolase-deficient strain resulted in an 8.5-fold enhancement of the total amount of the excreted sugar alcohols ribitol and xylitol. The additional introduction of the 2-deoxy-glucose 6-phosphate phosphatase-encoding gene DOG1 into the transketolase-deficient strain expressing the XYL2 gene resulted in a further 1.6-fold increase in ribitol production. Finally, deletion of the endogenous xylulokinase-encoding gene XKS1 was necessary to increase the amount of xylitol to 50% of the 5-carbon sugar alcohols excreted

Microbial d-xylonate production

d-Xylonic acid is a versatile platform chemical with reported applications as complexing agent or chelator, in dispersal of concrete, and as a precursor for compounds such as co-polyamides, polyesters, hydrogels and 1,2,4-butanetriol. With increasing glucose prices, d-xylonic acid may provide a cheap, non-food derived alternative for gluconic acid, which is widely used (about 80 kton/year) in pharmaceuticals, food products, solvents, adhesives, dyes, paints and polishes. Large-scale production has not been developed, reflecting the current limited market for d-xylonate. d-Xylonic acid occurs naturally, being formed in the first step of oxidative metabolism of d-xylose by some archaea and bacteria via the action of d-xylose or d-glucose dehydrogenases. High extracellular concentrations of d-xylonate have been reported for various bacteria, in particular Gluconobacter oxydans and Pseudomonas putida. High yields of d-xylonate from d-xylose make G. oxydans an attractive choice for biotechnical production. G. oxydans is able to produce d-xylonate directly from plant biomass hydrolysates, but rates and yields are reduced because of sensitivity to hydrolysate inhibitors. Recently, d-xylonate has been produced by the genetically modified bacterium Escherichia coli and yeast Saccharomyces cerevisiae and Kluyveromyces lactis. Expression of NAD(+)-dependent d-xylose dehydrogenase of Caulobacter crescentus in either E. coli or in a robust, hydrolysate-tolerant, industrial Saccharomyces cerevisiae strain has resulted in d-xylonate titres, which are comparable to those seen with G. oxydans, at a volumetric rate approximately 30 % of that observed with G. oxydans. With further development, genetically modified microbes may soon provide an alternative for production of d-xylonate at industrial scale