Metabolomic Analysis Reveals Key Metabolites Related to the Rapid Adaptation of Saccharomyce cerevisiae to Multiple Inhibitors of Furfural, Acetic Acid, and Phenol

Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University , Tianjin, People's Republic of China .
Omics: a journal of integrative biology (Impact Factor: 2.36). 02/2013; 17(3). DOI: 10.1089/omi.2012.0093
Source: PubMed


Abstract During hydrolysis of lignocellulosic biomass, a broad range of inhibitors are generated, which interfere with yeast growth and bioethanol production. In order to improve the strain tolerance to multiple inhibitors-acetic acid, furfural, and phenol (three representative lignocellulose-derived inhibitors) and uncover the underlying tolerant mechanism, an adaptation experiment was performed in which the industrial Saccharomyces cerevisiae was cultivated repeatedly in a medium containing multiple inhibitors. The adaptation occurred quickly, accompanied with distinct increase in growth rate, glucose utilization rate, furfural metabolism rate, and ethanol yield, only after the first transfer. A similar rapid adaptation was also observed for the lab strains of BY4742 and BY4743. The metabolomic analysis was employed to investigate the responses of the industrial S. cereviaise to three inhibitors during the adaptation. The results showed that higher levels of 2-furoic acid, 2, 3-butanediol, intermediates in glycolytic pathway, and amino acids derived from glycolysis, were discovered in the adapted strains, suggesting that enhanced metabolic activity in these pathways may relate to resistance against inhibitors. Additionally, through single-gene knockouts, several genes related to alanine metabolism, GABA shunt, and glycerol metabolism were verified to be crucial for the resistance to multiple inhibitors. This study provides new insights into the tolerance mechanism against multiple inhibitors, and guides for the improvement of tolerant ethanologenic yeast strains for lignocellulose-bioethanol fermentation.

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Available from: Bing-Zhi Li, Jul 26, 2014
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    • "In glutamate degradation, glutamate is degraded into gammaaminobutyrate (GABA) by glutamate decarboxylase and then degraded into succinate by succinate semialdehyde dehydrogenases Uga1p and Uga2p (Coleman et al. 2001). It has been revealed that the cellular GABA level is lower in yeast cells which have a tolerance towards multiple inhibitors (Wang et al. 2013), and yeast cells are more sensitive to oxidative stress when GABA degradation are blocked (Coleman et al. 2001). In this study, Gdh1p and Uga1p were differently expressed in the yeast cells under AFP stress as shown in Fig. 8. "
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    ABSTRACT: Toxic compounds including acids, furans, and phenols (AFP) were generated from the pretreatment of lignocellulose. We cultivated Saccharomyces cerevisiae cells in a batch mode, besides the cell culture of original yeast strain in AFP-free medium which was referred as C0, three independent subcultures were cultivated under multiple inhibitors AFP and were referred as C1, C2, and C3 in time sequence. Comparing to C0, the cell density was lowered while the ethanol yield was maintained stably in the three yeast cultures under AFP stress, and the lag phase of C1 was extended while the lag phases of C2 and C3 were not extended. In proteomic analysis, 194 and 215 unique proteins were identified as differently expressed proteins at lag phase and exponential phase, respectively. Specifically, the yeast cells co-regulated protein folding and protein synthesis process to prevent the generation of misfolded proteins and to save cellular energy, they increased the activity of glycolysis, redirected metabolic flux towards phosphate pentose pathway and the biosynthesis of ethanol instead of the biosynthesis of glycerol and acetic acid, and they upregulated several oxidoreductases especially at lag phase and induced programmed cell death at exponential phase. When the yeast cells were cultivated under AFP stress, the new metabolism homeostasis in favor of cellular energy and redox homeostasis was generated in C1, then it was inherited and optimized in C2 and C3, enabling the yeast cells in C2 and C3 to enter the exponential phase in a short period after inoculation, which thus significantly shortened the fermentation time.
    Applied Microbiology and Biotechnology 01/2014; 98(5). DOI:10.1007/s00253-014-5519-8 · 3.34 Impact Factor
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    • "Quenching and metabolite extraction were performed according to the methods described previously with minor modifications (Wang et al., 2013). Briefly, cells were quickly sprayed into 60% (v/v) methanol solution (À40 C) to arrest metabolism instantaneously, cooled at À40 C for 30 s and then collected by centrifugation at 4,000g and À20 C for 5 min. "
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    ABSTRACT: During lignocellulosic ethanol fermentation, yeasts are exposed to various lignocellulose-derived inhibitors, which disrupt the efficiency of hexose and pentose co-fermentation. To understand the metabolic response of fermentation microbes to these inhibitors, a comparative metabolomic investigation was performed on a xylose-fermenting Saccharomyces cerevisiae 424A (LNH-ST) and its parental strain 4124 with and without three typical inhibitors (furfural, acetic acid, and phenol). Three traits were uncovered according to fermentation results. First, the growth of strain 424A (LNH-ST) was more sensitive to inhibitors than strain 4124. Through metabolomic analysis, the variance of trehalose, cadaverine, glutamate and g-aminobutyric acid (GABA) suggested that strain 424A (LNH-ST) had a lower capability to buffer redox changes caused by inhibitors. Second, lower ethanol yield in glucose and xylose co-fermentation than glucose fermentation was observed in strain 424A (LNH-ST), which was considered to be correlated with the generation of xylitol, as well as the reduced levels of lysine, glutamate, glycine and isoleucine in strain 424A (LNH-ST). Accumulation of glycerol, galactinol and mannitol was also observed in strain 424A (LNH-ST) during xylose fermentation. Third, xylose utilization of strain 424A (LNH-ST) was more significantly disturbed by inhibitors than glucose utilization. Through the analysis of fermentation and metabolomic results, it was suggested that xylose catabolism and energy supply, rather than xylose uptake, were the limiting steps in xylose utilization in the presence of inhibitors.
    Biotechnology and Bioengineering 01/2014; 111(1):152-64. DOI:10.1002/bit.24992 · 4.13 Impact Factor
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    • "Furfural, which is a by-product of the dehydration of xylose, has been considered one of the main inhibitors in the following fermentation (Li and Yuan 2010). The main inhibitors produced in pretreatment are furans and acids (Ding et al. 2012; Wang et al. 2013), and the yields of these inhibitors were much lower in alkaline pretreatment than in acidic pretreatment (Chundawat et al. 2010). "
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    ABSTRACT: Acids and alkalis are considered important catalysts in biomass pretreatment, which is essential to overcome the recalcitrance of lignocellulose for sugar release. In this study, the effects of various chemicals and temperatures on the pretreatment and subsequent enzymatic hydrolysis of wheat straw were investigated. The conversions of glucan and xylan during pretreatment and enzymatic hydrolysis were examined. The temperature and different ions in pretreatment govern the dissociation constant and hydrogen ion concentration. Due to higher dissociation at higher temperature, weak acids and weak alkalis can produce high glucose yields, similar to strong acids and alkalis. The concept of modified combined severity for weak acid pretreatment was explored. The pH value and real combined severity of weak acids at reaction temperatures were estimated according to xylan recovery during pretreatment. Glucose yield in enzymatic hydrolysis is mainly decided by xylan recovery for acidic pretreatment and by total content of xylan and acid-insoluble-lignin in solids for alkaline pretreatment.
    Bioresources 11/2013; 9(1). DOI:10.15376/biores.9.1.24-38 · 1.43 Impact Factor
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