Metabolic-Flux Profiling of the Yeasts Saccharomyces cerevisiae and Pichia stipitis

Institute of Molecular Biology and Biophysics, ETH Zürich, CH-8093 Zürich, Switzerland.
Eukaryotic Cell (Impact Factor: 3.18). 03/2003; 2(1):170-80. DOI: 10.1128/EC.2.1.170-180.2003
Source: PubMed

ABSTRACT The so far largely uncharacterized central carbon metabolism of the yeast Pichia stipitis was explored in batch and glucose-limited chemostat cultures using metabolic-flux ratio analysis by nuclear magnetic resonance.
The concomitantly characterized network of active metabolic pathways was compared to those identified in Saccharomyces cerevisiae, which led to the following conclusions. (i) There is a remarkably low use of the non-oxidative pentose phosphate (PP) pathway
for glucose catabolism in S. cerevisiae when compared to P. stipitis batch cultures. (ii) Metabolism of P. stipitis batch cultures is fully respirative, which contrasts with the predominantly respiro-fermentative metabolic state of S. cerevisiae. (iii) Glucose catabolism in chemostat cultures of both yeasts is primarily oxidative. (iv) In both yeasts there is significant
in vivo malic enzyme activity during growth on glucose. (v) The amino acid biosynthesis pathways are identical in both yeasts.
The present investigation thus demonstrates the power of metabolic-flux ratio analysis for comparative profiling of central
carbon metabolism in lower eukaryotes. Although not used for glucose catabolism in batch culture, we demonstrate that the
PP pathway in S. cerevisiae has a generally high catabolic capacity by overexpressing the Escherichia coli transhydrogenase UdhA in phosphoglucose isomerase-deficient S. cerevisiae.

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Available from: Z. Petek Cakar, Sep 25, 2015
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    • "In addition to problems associated with xylose transport, xylose utilization in S. cerevisiae is hampered by bottlenecks in the metabolism of xylose through the PPP (Fiaux et al., 2003). Several studies have been conducted to improve the metabolic fluxes through the PPP, including the overexpression of genes encoding key enzymes in the pathway such as transaldolase (TAL1), transketolase (TKL1), ribose-5-phosphate isomerase (RK11), and ribulose 5-phosphate epimerase (RPE1) (Karhumaa et al., 2005). "
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    ABSTRACT: Lignocellulosic biomass is a viable source of renewable energy for bioethanol production. For the efficient conversion of biomass into bioethanol, it is essential that sugars from both the cellulose and hemicellulose fractions of lignocellulose be utilised. We describe the development of a recombinant yeast system for the fermentation of cellulose and xylose, the most abundant pentose sugar in the hemicellulose fraction of biomass. The brewer's yeast Saccharomyces pastorianus was chosen as a host as significantly higher recombinant enzyme activities are achieved, when compared to the more commonly used S. cerevisiae. When expressed in S. pastorianus, the Trichoderma reesei xylose oxidoreductase pathway was more efficient at alcohol production from xylose than the xylose isomerase pathway. The alcohol yield was influenced by the concentration of xylose in the medium and was significantly improved by the additional expression of a gene encoding for xylulose kinase. The xylose reductase, xylitol dehydrogenase and xylulose kinase genes were co-expressed with genes encoding for the three classes of T. reesei cellulases, namely endoglucanase (EG2), cellobiohydrolysase (CBH2) and β-glucosidase (BGL1). The initial metabolism of xylose by the engineered strains facilitated production of cellulases at fermentation temperatures. The sequential metabolism of xylose and cellulose generated an alcohol yield of 82% from the available sugars. Several different types of biomass, such as the energy crop Miscanthus sinensis and the industrial waste, brewer's spent grains, were examined as biomass sources for fermentation using the developed yeast strains. Xylose metabolism and cell growth were inhibited in fermentations carried out with acid-treated spent grain liquor, resulting in a 30% reduction in alcohol yield compared to fermentations carried out with mixed sugar substrates. Reconstitution of complete enzymatic pathways for cellulose hydrolysis and xylose utilisation in S. pastorianus facilitates the co-fermentation of cellulose and xylose without the need for added exogenous cellulases and provides a basis for the development of a consolidated process for co-utilisation of hemicellulose and cellulose sugars.
    Microbial Cell Factories 04/2015; 14(1):61. DOI:10.1186/s12934-015-0242-4 · 4.22 Impact Factor
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    • "c o m / l o c a t e / f m as " Crabtree effect " (Fiaux et al., 2003; Gancedo, 1998). This results in repression of the tricarboxylic acid (TCA) cycle and respiration, even in the presence of oxygen (Blank and Sauer, 2004; Fiaux et al., 2003). Several studies show that such actively growing cells show reduced stress resistance, including reduced tolerance to heatshock , starvation and osmotic stress conditions (Verstrepen et al., 2004; Werner-Washburne et al., 1993). "
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    ABSTRACT: Fermentation of sugars into CO2, ethanol and secondary metabolites by baker's yeast (Saccharomyces cerevisiae) during bread making leads to leavening of dough and changes in dough rheology. The aim of this study was to increase our understanding of the impact of yeast on dough related aspects by investigating the effect of harvesting yeast at seven different points of the growth profile on its fermentation performance, metabolite production, and the effect on critical dough fermentation parameters, such as gas retention potential. The yeast cells harvested during the diauxic shift and post-diauxic growth phase showed a higher fermentation rate and, consequently, higher maximum dough height than yeast cells harvested in the exponential or stationary growth phase. The results further demonstrate that the onset of CO2 loss from fermenting dough is correlated with the fermentation rate of yeast, but not with the amount of CO2 that accumulated up to the onset point. Analysis of the yeast metabolites produced in dough yielded a possible explanation for this observation, as they are produced in different levels depending on physiological phase and in concentrations that can influence dough matrix properties. Together, our results demonstrate a strong effect of yeast physiology at the time of harvest on subsequent dough fermentation performance, and hint at an important role of yeast metabolites on the subsequent gas holding capacity.
    Food Microbiology 05/2014; 39:108–115. DOI:10.1016/ · 3.33 Impact Factor
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    • "S. stipitis might start fermentation when glucose concentration or oxygen concentration decreased by S. cerevisiae activity in ICR. It was reported that, even at high glucose concentrations, crabtree negative S. stipitis exhibits predominantly respirative metabolism (Fiaux et al., 2003) and the onset of the fermentation in S. stipitis is not dependent on sugar concentration and this yeast needs oxygen limiting conditions for fermentation (Papini et al., 2012). Although remarkable amount of glucose and xylose were utilized by S. stipitis, very low amount of ethanol was produced in ICR filled only with S. stipitis cells (Figs. 3 and 4). "
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    ABSTRACT: In this research, Scheffersomyces stipitis and Saccharomyces cerevisiae in immobilized and suspended state were used to convert pentose and hexose sugars to ethanol. In batch and continuous systems, S. stipitis and S. cerevisiae co-culture performance was better than S. cerevisiae. Continuous ethanol production was performed in packed bed immobilized cell reactor (ICR). In ICR, S. stipitis cells were found to be more sensitive to oxygen concentration and other possible mass transfer limitations as compared to S. cerevisiae. Use of co-immobilized S. stipitis and S. cerevisiae resulted in maximum xylose consumption (73.92%) and 41.68g/Lday ethanol was produced at HRT (hydraulic retention time) of 6h with wheat straw hydrolysate. At HRT of 0.75h, the highest amount of ethanol with the values of 356.21 and 235.43g/Lday was produced when synthetic medium and wheat straw hydrolysate were used as feeding medium in ICR, respectively.
    Bioresource Technology 02/2014; 158C:286-293. DOI:10.1016/j.biortech.2014.02.022 · 4.49 Impact Factor
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