Bergdahl et al. Biotechnology for Biofuels 2012, 5:34 http://www.biotechnologyforbiofuels.com/content/5/1/34 RESEARCH Open Access Dynamic metabolomics differentiates between carbon and energy starvation in recombinant Saccharomyces cerevisiae fermenting xylose Basti Bergdahl1*, Dominik Heer2, Uwe Sauer2, Bärbel Hahn-Hägerdal1 and Ed WJ van Niel1 Abstract 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. Keywords: Metabolomics, Yeast metabolism, Xylose fermentation, Metabolic status, Starvation, Bioethanol * Correspondence: [email protected] 1Applied Microbiology, Lund University, PO Box 124, SE-221 00 Lund, Sweden Full list of author information is available at the end of the article © 2012 Bergdahl et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Bergdahl et al. Biotechnology for Biofuels 2012, 5:34 Page 2 of 19 http://www.biotechnologyforbiofuels.com/content/5/1/34 Background enzymatic reactions through their thermodynamic proper- The yeast Saccharomyces cerevisiae has been the organ- ties [19]. The concerted effect of changes in gene expression ism of choice in the food and beverage industry due to in response to environmental variation is thus ultimately its excellent growth and fermentation capabilities under reflected in the metabolome. The dynamic changes in me- anaerobic conditions. These characteristics combined with tabolite concentrations under a certain perturbation can high tolerance to low pH and high sugar and ethanol con- therefore provide a description of the phenotype and the centrations yields a production organism, which is very cellular state with a higher degree of functional information robust in industrial processes. Thus, S. cerevisiae has also than a snap-shot of either the transcriptome or the prote- become the preferred organism for the production of bio- ome. We used this potential to evaluate the metabolic status fuels and fine chemicals [1]. An important step towards of two isogenic recombinant strains of S. cerevisiae harbour- the replacement of fossil raw materials is the efficient ing either the XR/XDH or the XI pathway during anaerobic utilization of renewable lignocellulose. This raw material batch fermentation of a glucose/xylose mixture. A recently is generated in the forest and agricultural sectors, and developed method for quantification of intracellular con- contains carbohydrates which can be converted into fuel- centrations of metabolites by LC-MS/MS [20] was used to grade ethanol by fermentation [2]. The pentose sugar xy- determine the dynamic response of targeted metabolites in lose constitutes a major fraction of the sugar monomers these recombinant strains when the carbon and energy obtained after hydrolysis of certain lignocellulose materials source changed from glucose to xylose. The difference in [3,4] and complete utilization of xylose is therefore neces- xylose uptake rate between the XI-strain and the XR/XDH- sary to obtain competitive process economics [5]. strain allows us to propose responses in metabolite concen- S. cerevisiae cannot naturally utilize xylose and has there- trations expected to occur during starvation for carbon and fore been extensively engineered to acquire this trait as energy. The results suggest that xylose is primarily used as summarized in several recent reviews [6-9]. To enable xy- an energy source as both strains maintained a high energy lose utilization and fermentation to ethanol by S. cerevisiae, charge during the transition to xylose fermentation while one of two heterologous pathways have been introduced: varying signs of carbon starvation were observed. the oxido-reductive pathway or the isomerisation pathway [10] (Figure 1A). The oxido-reductive pathway is found in Results fungi and consists of two enzymes, a NAD(P)H-dependent In this study we determined the dynamics in the concentra- xylose reductase (XR) and a NAD-dependent xylitol de- tions of metabolites in glycolysis, the pentose phosphate hydrogenase (XDH). Some XR enzymes can use both pathway (PPP), the TCA cycle, nine amino acids, the purine NADH and NADPH as cofactor although with a preference nucleotides and redox cofactors, in two recombinant strains for the latter [11]. This causes a cofactor imbalance between of Saccharomyces cerevisiae. The two strains have identical the XR and XDH reactions and leads to xylitol formation genetic background but employ two different pathways for and reduced ethanol yields [12-14]. This does not occur in xylose utilization; strain TMB 3057 [21] has the oxido- the isomerisation pathway, which only consists of a reductive fungal pathway consisting of XR and XDH from cofactor-independent xylose isomerase (XI). The XI with Pichia stipitis and strain TMB 3359 [22] has the isomerisa- highest activity when expressed in S. cerevisiae originates tion pathway consisting of the XI from Piromyces sp.Sam- from the fungus Piromyces sp. Even so, the activity of this ples for determination of metabolite concentrations were XI is too low to enable anaerobic growth on xylose without collected at specific time points during anaerobic batch fer- further evolution or adaptation of the recombinant strain mentation with 20 g/L glucose and 40 g/L xylose as sub- [10,15]. strates. Additionally, for the XR/XDH-strain measured The rational design of industrially applicable microorgan- intracellular metabolite concentrations were validated by isms requires an understanding of the cellular processes evaluating the simultaneous thermodynamic feasibility of that give rise to a certain phenotype. The functional infor- 237 metabolic and transport reactions at each sampling mation about a cellular phenotype is found in the biochem- point. ical processes that occur in response to environmental conditions and is commonly viewed as the connection be- Fermentation performance tween the three major ‘omes in the cell: transcriptome, Both the XR/XDH-strain and the XI-strain consumed all proteome and metabolome [16,17]. Both proteins and glucose within 20.5 hours (Figure 2A and B) and in the metabolites are directly involved in the cellular biochemis- same time the XR/XDH-strain co-consumed 5.7 ± 0.5 g/L try and thereby closely dictate the function of the organism xylose (Figure 2A), whereas the XI-strain only co- [18]. The functional role of intracellular metabolites is emi- consumed 0.6 ± 0.2 g/L xylose (Figure 2B). Until this point nent as they can influence the conversion rate by an en- the biomass yield per gram sugar consumed was similar zyme, either as substrate, product or allosteric effector, act for the two strains but higher yield of the by-products as signalling molecules and even influence the direction of glycerol and xylitol were obtained with the XR/XDH- Bergdahl et al. Biotechnology for Biofuels 2012, 5:34 Page 3 of 19 http://www.biotechnologyforbiofuels.com/content/5/1/34 strain (Additional file 1: Table S1). After glucose depletion, converted to xylitol and ethanol with a minor amount the XR/XDH-strain consumed an additional 10.8 ± 0.1 g/L used for biomass and glycerol (Figure 2A and C). The xylose (rxyl = 0.30 g/g CDW/h) most of which was XI-strainonlyconsumedanadditional0.6±0.1g/L Figure 1 (See legend on next page.) Bergdahl et al. Biotechnology for Biofuels 2012, 5:34 Page 4 of 19 http://www.biotechnologyforbiofuels.com/content/5/1/34 (See figure on previous page.) Figure 1 Overview of the main metabolic reactions in central carbon metabolism. A) Xylose can be assimilated through two pathways: the fungal oxidoreductive pathway consisting of XR and XDH or the bacterial isomerization pathway via an XI.