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Metabolic pathway engineering based on metabolomics confers acetic and formic acid tolerance to a recombinant xylose-fermenting strain of Saccharomyces cerevisiae.

ABSTRACT: The development of novel yeast strains with increased tolerance toward inhibitors in lignocellulosic hydrolysates is highly desirable for the production of bio-ethanol. Weak organic acids such as acetic and formic acids are necessarily released during the pretreatment (i.e. solubilization and hydrolysis) of lignocelluloses, which negatively affect microbial growth and ethanol production. However, since the mode of toxicity is complicated, genetic engineering strategies addressing yeast tolerance to weak organic acids have been rare. Thus, enhanced basic research is expected to identify target genes for improved weak acid tolerance.In this study, the effect of acetic acid on xylose fermentation was analyzed by examining metabolite profiles in a recombinant xylose-fermenting strain of Saccharomyces cerevisiae. Metabolome analysis revealed that metabolites involved in the non-oxidative pentose phosphate pathway (PPP) [e.g. sedoheptulose-7-phosphate, ribulose-5-phosphate, ribose-5-phosphate and erythrose-4-phosphate] were significantly accumulated by the addition of acetate, indicating the possibility that acetic acid slows down the flux of the pathway. Accordingly, a gene encoding a PPP-related enzyme, transaldolase or transketolase, was overexpressed in the xylose-fermenting yeast, which successfully conferred increased ethanol productivity in the presence of acetic and formic acid.Our metabolomic approach revealed one of the molecular events underlying the response to acetic acid and focuses attention on the non-oxidative PPP as a target for metabolic engineering. An important challenge for metabolic engineering is identification of gene targets that have material importance. This study has demonstrated that metabolomics is a powerful tool to develop rational strategies to confer tolerance to stress through genetic engineering.

SUBMITTER: Hasunuma T

PROVIDER: S-EPMC3025834 | biostudies-other | 2011 Jan

REPOSITORIES: biostudies-other

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Metabolic pathway engineering based on metabolomics confers acetic and formic acid tolerance to a recombinant xylose-fermenting strain of Saccharomyces cerevisiae.

Hasunuma Tomohisa T Sanda Tomoya T Yamada Ryosuke R Yoshimura Kazuya K Ishii Jun J Kondo Akihiko A

Microbial cell factories 20110110 1

<h4>Background</h4>The development of novel yeast strains with increased tolerance toward inhibitors in lignocellulosic hydrolysates is highly desirable for the production of bio-ethanol. Weak organic acids such as acetic and formic acids are necessarily released during the pretreatment (i.e. solubilization and hydrolysis) of lignocelluloses, which negatively affect microbial growth and ethanol production. However, since the mode of toxicity is complicated, genetic engineering strategies address ...[more]

PMID: 21219616

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Project description:BACKGROUND:The concerted effects of changes in gene expression due to changes in the environment are ultimately reflected in the metabolome. Dynamics of metabolite concentrations under a certain condition can therefore give a description of the cellular state with a high degree of functional information. We used this potential to evaluate the metabolic status of two recombinant strains of Saccharomyces cerevisiae during anaerobic batch fermentation of a glucose/xylose mixture. Two isogenic strains were studied, differing only in the pathways used for xylose assimilation: the oxidoreductive pathway with xylose reductase (XR) and xylitol dehydrogenase (XDH) or the isomerization pathway with xylose isomerase (XI). The isogenic relationship between the two strains ascertains that the observed responses are a result of the particular xylose pathway and not due to unknown changes in regulatory systems. An increased understanding of the physiological state of these strains is important for further development of efficient pentose-utilizing strains for bioethanol production. RESULTS:Using LC-MS/MS we determined the dynamics in the concentrations of intracellular metabolites in central carbon metabolism, nine amino acids, the purine nucleotides and redox cofactors. The general response to the transition from glucose to xylose was increased concentrations of amino acids and TCA-cycle intermediates, and decreased concentrations of sugar phosphates and redox cofactors. The two strains investigated had significantly different uptake rates of xylose which led to an enhanced response in the XI-strain. Despite the difference in xylose uptake rate, the adenylate energy charge remained high and stable around 0.8 in both strains. In contrast to the adenylate pool, large changes were observed in the guanylate pool. CONCLUSIONS:The low uptake of xylose by the XI-strain led to several distinguished responses: depletion of key metabolites in glycolysis and NADPH, a reduced GTP/GDP ratio and accumulation of PEP and aromatic amino acids. These changes are strong indicators of carbon starvation. The XR/XDH-strain displayed few such traits. The coexistence of these traits and a stable adenylate charge indicates that xylose supplies energy to the cells but does not suppress a response similar to carbon starvation. Particular signals may play a role in the latter, of which the GTP/GMP ratio could be a candidate as it decreased significantly in both strains.

Project description:BackgroundEfficient xylose fermentation still demands knowledge regarding xylose catabolism. In this study, metabolic flux analysis (MFA) and metabolomics were used to improve our understanding of xylose metabolism. Thus, a stoichiometric model was constructed to simulate the intracellular carbon flux and used to validate the metabolome data collected within xylose catabolic pathways of non-Saccharomyces xylose utilizing yeasts.ResultsA metabolic flux model was constructed using xylose fermentation data from yeasts Scheffersomyces stipitis, Spathaspora arborariae, and Spathaspora passalidarum. In total, 39 intracellular metabolic reactions rates were utilized validating the measurements of 11 intracellular metabolites, acquired by mass spectrometry. Among them, 80% of total metabolites were confirmed with a correlation above 90% when compared to the stoichiometric model. Among the intracellular metabolites, fructose-6-phosphate, glucose-6-phosphate, ribulose-5-phosphate, and malate are validated in the three studied yeasts. However, the metabolites phosphoenolpyruvate and pyruvate could not be confirmed in any yeast. Finally, the three yeasts had the metabolic fluxes from xylose to ethanol compared. Xylose catabolism occurs at twice-higher flux rates in S. stipitis than S. passalidarum and S. arborariae. Besides, S. passalidarum present 1.5 times high flux rate in the xylose reductase reaction NADH-dependent than other two yeasts.ConclusionsThis study demonstrated a novel strategy for metabolome data validation and brought insights about naturally xylose-fermenting yeasts. S. stipitis and S. passalidarum showed respectively three and twice higher flux rates of XR with NADH cofactor, reducing the xylitol production when compared to S. arborariae. Besides then, the higher flux rates directed to pentose phosphate pathway (PPP) and glycolysis pathways resulted in better ethanol production in S. stipitis and S. passalidarum when compared to S. arborariae.

Dataset Information

Metabolic pathway engineering based on metabolomics confers acetic and formic acid tolerance to a recombinant xylose-fermenting strain of Saccharomyces cerevisiae.

Publications

Metabolic pathway engineering based on metabolomics confers acetic and formic acid tolerance to a recombinant xylose-fermenting strain of Saccharomyces cerevisiae.

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