Dynamic metabolomics differentiates between carbon and energy starvation in recombinant Saccharomyces cerevisiae fermenting xylose

Abstract

BackgroundThe 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.ResultsUsing 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.ConclusionsThe 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.

Physiological effects of over-expressing compartment-specific components of the protein folding machinery in xylose-fermenting Saccharomyces cerevisiae

“…One explanation could be that the previously observed accumulation of fumarate [31] does not occur in the cytosol, but is restricted to the mitochondria. To investigate this possibility we over-expressed the OSM1 gene, alone and in combination with ERO1 , and evaluated the fermentation performance in both defined and complex media with 20 g/L glucose and 50 g/L xylose.…”

Leveraging transcription factors to speed cellobiose fermentation by

“…For example, central carbon metabolism is thought to be highly regulated at the post-transcriptional level [81]. Additional methods such as ribosome profiling [82] and metabolomics [83] could be used to identify additional bottlenecks in central carbon metabolism that could be addressed in the future. These methods to address post-transcriptional regulation could also be used to identify a small number of targets that could be engineered to improve cofermentation of plant cell wall-derived sugars (such as cellobiose and xylose, and to improve the production of "drop-in" fuels and other renewable chemicals using yeast.…”

Metabolomics applied in bioenergy

“…The results suggest that xylose can be primarily used as an energy source, as both strains maintained a high energy charge during the transition to xylose fermentation. However, it seems that xylose fermentation uncouples energy and carbon metabolism [22]. These findings were uncovered solely through metabolomics analysis.…”

“…However, a more holistic approach could also provide the metabolic reconstruction of most biologically relevant and predictive models to improve the fermentative ability of S. cerevisiae [93]. Recently, dynamic metabolomics studies of two recombinant strains of S. cerevisiae during anaerobic batch fermentation of a glucose/xylose mixture were conducted using LC-MS [22]. The results suggest that xylose can be primarily used as an energy source, as both strains maintained a high energy charge during the transition to xylose fermentation.…”

“…Therefore, it is considered a powerful tool for the unbiased characterization of genotypes and phenotypes with application in multiple areas, such as evaluation of genetically modified organisms [10][11][12], functional genomics [13][14][15][16][17], responses to environmental factors [18][19][20][21][22][23], metabolic engineering [24,25], and quantitative genetics [26][27][28][29][30]. Furthermore, metabolomics, together with multivariate and correlation analyses, is an excellent tool for the study of systems biology, being widely recognized as the cornerstone of this emerging area [22,[31][32][33][34]. In this review, we focus on how metabolomics approaches have been used to identify novel metabolic routes for microbial biomass conversion and also highlight biochemical pathways important for plant biomass production.…”