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Extensive Exometabolome Analysis Reveals Extended Overflow Paczia et al. Microbial Cell Factories 2012, 11:122 http://www.microbialcellfactories.com/content/11/1/122 RESEARCH Open Access Extensive exometabolome analysis reveals extended overflow metabolism in various microorganisms Nicole Paczia, Anke Nilgen, Tobias Lehmann, Jochem Gätgens, Wolfgang Wiechert and Stephan Noack* Abstract Overflow metabolism is well known for yeast, bacteria and mammalian cells. It typically occurs under glucose excess conditions and is characterized by excretions of by-products such as ethanol, acetate or lactate. This phenomenon, also denoted the short-term Crabtree effect, has been extensively studied over the past few decades, however, its basic regulatory mechanism and functional role in metabolism is still unknown. Here we present a comprehensive quantitative and time-dependent analysis of the exometabolome of Escherichia coli, Corynebacterium glutamicum, Bacillus licheniformis, and Saccharomyces cerevisiae during well-controlled bioreactor cultivations. Most surprisingly, in all cases a great diversity of central metabolic intermediates and amino acids is found in the culture medium with extracellular concentrations varying in the micromolar range. Different hypotheses for these observations are formulated and experimentally tested. As a result, the intermediates in the culture medium during batch growth must originate from passive or active transportation due to a new phenomenon termed “extended” overflow metabolism. Moreover, we provide broad evidence that this could be a common feature of all microorganism species when cultivated under conditions of carbon excess and non-inhibited carbon uptake. In turn, this finding has consequences for metabolite balancing and, particularly, for intracellular metabolite quantification and 13C-metabolic flux analysis. Keywords: Overflow metabolism, Crabtree effect, Exometabolome, Mass spectrometry, Intracellular metabolite quantification Background Escherichia coli and is also termed the bacterial Crabtree Overflow metabolism, i.e. producing ethanol under aer- effect [7,8]. Acetate overflow predominantly occurs obic and glucose excess conditions, is one of the out- under conditions of high glucose concentrations, i.e. in standing characteristics of a Crabtree-positive yeast such the exponential growth phase of batch cultures or in as Saccharomyces cerevisiae [1,2]. The Crabtree effect chemostat cultures operated at high dilution rates. In involves glucose-induced inhibition of respiration and contrast, no extracellular acetate accumulation was oxidative phosphorylation accompanied by a parallel up- found under glucose-limiting conditions, i.e. the station- regulation of glucose uptake and glycolysis. However, ary phase of batch cultures or chemostat cultures oper- the precise mechanism is still unresolved [3-5]. More re- ated at low dilution rates [9]. Similar to the Crabtree cently, it has been discussed that this phenomenon is a effect in yeast, its regulatory mechanism in bacteria is general survival strategy of Saccharomyces yeast in order still under investigation [10-12]. to outcompete other microorganisms in fast sugar con- Interestingly, recent experimental data from shake- sumption [6]. flask cultivations of several bacterial species including E. In the same way as ethanol in yeast, acetate overflow coli indicated that metabolites originating from central is well-known for prokaryotic organisms such as carbon metabolism are present in the culture media [13]. A similar observation was made for S. cerevisiae * Correspondence: [email protected] and E. coli in glucose-limited chemostat cultures [14,15]. Institute of Bio- and Geosciences, Biotechnology, Systems Biotechnology, The fact that this has not been observed before is due to Forschungszentrum Jülich GmbH, Jülich, Germany © 2012 Paczia 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. Paczia et al. Microbial Cell Factories 2012, 11:122 Page 2 of 14 http://www.microbialcellfactories.com/content/11/1/122 the improved mass spectrometry capabilities allowing Corynebacterium glutamicum, were rigorously and concentration measurements down to the nanomolar broadly analyzed over the time course of batch cultiva- range. However, except for singular measurements, no tions under well-controlled bioreactor conditions. In ac- systematic investigation on the microorganism’s exome- cordance with these results, several hypotheses to explain tabolome including time-dependent effects of metabolite the sometimes considerable amounts of metabolites excretion and uptake has been carried out so far. detected in the broth were formulated and tested. In this paper, we address the conundrum related to the presence of metabolites originating from central carbon Results and discussion metabolism in the culture medium during typical bioreac- Organism-wide exometabolome analysis tor cultivations. The exometabolomes of four biotechnolo- The growth of E. coli proceeded in a typical batch mode gically relevant model organisms, i.e. the wild types of manner (Figure 1A). During the exponential phase the Escherichia coli, Bacillus licheniformis and Saccharomyces substrate glucose was converted to biomass and the cerevisiae as well as an L-lysine producing strain of main by-products carbon dioxide, pyruvate and acetate. (A) (B) Escherichia coli Bacillus licheniformis Figure 1 Exometabolome analysis of Escherichia coli and Bacillus licheniformis. (A) Batch cultivation of E. coli WT on defined media with 20 g l-1 glucose. (B) Batch cultivation of B. licheniformis WT on defined media with 16 g l-1 glucose. Abbreviations: OD, optical density; Glc, glucose; CO2, offgas carbon dioxide; pO2, dissolved oxygen; G6P, glucose-6-phosphate; PYR, pyruvate; ACT, acetate; HOM, homoserine; TYR, tyrosine; VAL, valine; F6P, fructose-6-phosphate; GLY, glycine; ASN, asparagine; LYS, lysine. Paczia et al. Microbial Cell Factories 2012, 11:122 Page 3 of 14 http://www.microbialcellfactories.com/content/11/1/122 Table 1 Extracellular metabolite concentrations in the culture broth of batch cultures of E. coli, B. licheniformis, S. cerevisiae and C. glutamicum Central metabolic intermediates E. coli B. licheniformis S. cerevisiae C. glutamicum (K12 W3110) (DSM13D102) (CEN.PK 113-7D) (DM1800) Glucose-6-phosphate 17.65 ± 0.53 (t = 11) 88.53 ± 7.64 (t = 26) 1.25 ± 0.01 (t = 26) 10.16 ± 0.74 (t = 182) Fructose-6-phosphate 29.29 ± 0.60 (t = 10) 19.25 ± 0.96 (t = 15) 0.87 ± 0.01 (t = 17) 3.84 ± 0.22 (t = 182) Fructose-1,6-bisphosphate 34.21 ± 0.72 (t = 11) 9.41 ± 0.60 (t = 14) 11.43 ± 0.29 (t = 7) 3.36 ± 0.07 (t = 110) Dihydroxyacetone phosphate 50.40 ± 1.74 (t = 12) 19.75 ± 0.14 (t = 9) 0.41 ± 0.01 (t = 26) 8.67 ± 0.10 (t = 19) Glyceraldehyde-3-phosphate 15.04 ± 0.24 (t = 12) 2.67 ± 0.05 (t = 8) < LOD 0.56 ± 0.01 (t = 27) 2/3-phosphoglycerate 19.45 ± 1.55 (t = 16) 66.26 ± 2.01 (t = 15) 2.19 ± 0.01 (t = 26) 12.17 ± 1.07 (t = 182) Phosphoenolpyruvate 20.43 ± 0.28 (t = 9) 21.76 ± 0.48 (t = 12) 1.08 ± 0.02 (t = 26) < LOD Pyruvate 4924 ± 509 (t = 8) 2553 ± 25 (t = 8) 734.5 ± 33.2 (t = 6) < LOD 6-Phosphogluconate < LOD < LOD < LOD < LOD Ribose-5-phosphate 10.29 ± 0.22 (t = 16) 23.03 ± 0.73 (t = 9) < LOD 0.61 ± 0.03 (t = 0.03) Ribu-/Xylulose-5-phosphate 3.69 ± 0.04 (t = 8) 12.27 ± 0.38 (t = 11) 0.02 ± 0.01 (t = 17) 0.58 ± 0.05 (t = 182) Erythrose-4-phosphate 1.31 ± 0.21 (t = 15) 2.04 ± 0.01 (t = 17) < LOD 2.47 ± 1.14 (t = 18) Sedoheptulose-7-phosphate < LOD < LOD < LOD < LOD Acetyl-CoA < LOD < LOD < LOD < LOD Citrate 139.8 ± 6.2 (t = 12) 104.1 ± 3.6 (t = 9) 26.44 ± 0.11 (t = 26) n.d. Cis-aconitate 14.13 ± 0.40 (t = 12) 12.27 ± 0.15 (t = 9) 1.65 ± 0.01 (t = 26) < LOD Isocitrate 23.13 ± 0.54 (t = 12) 18.28 ± 0.38 (t = 9) 6.12 ± 0.12 (t = 17) n.d. α-Ketoglutarate 200.0 ± 17.7 (t = 16) 75.73 ± 1.62 (t = 8) 50.13 ± 0.92 (t = 26) < LOD Fumarate 12.45 ± 2.31 (t = 11) 120.8 ± 2.6 (t = 8) 12.35 ± 0.38 (t = 16) < LOD Malate < LOD < LOD < LOD < LOD Glyoxylate < LOD < LOD < LOD < LOD Cometabolites AXP < LOD < LOD < LOD < LOD NAD(P) < LOD < LOD < LOD < LOD NAD(P)H < LOD < LOD < LOD < LOD Amino acids Alanine 1.69 ± 0.19 (t = 9) 138.0 ± 2.4 (t = 8) 5.47 ± 1.18 (t = 26) 64.80 ± 4.21 (t = 182) Leucine/Isoleucine 20.17 ± 0.21 (t = 26) 11.23 ± 0.4 (t = 13) 8.07 ± 0.40 (t = 26) < LOD Valine 88.50 ± 2.18 (t = 26) 31.67 ± 1.30 (t = 13) 7.69 ± 0.61 (t = 26) 56.00 ± 6.66 (t = 92) Aspartate 20.87 ± 0.55 (t = 26) 4.01 ± 0.32 (t = 16) 4.20 ± 0.25 (t = 26) 28.73 ± 0.32 (t = 48) Homoserine 2.43 ± 0.48 (t = 26) 9.15 ± 0.86 (t = 9) < LOD 46.61 ± 0.8 (t = 182) Threonine 2.59 ± 0.30 (t = 14) < LOD < LOD < LOD Methionine 0.55 ± 0.01 (t = 26) 123.7 ± 3.3 (t = 26) 0.15 ± 0.01 (t = 26) 26.67 ± 0.75 (t = 182) Lysine < LOD 158.8 ± 2.8 (t = 17) 3.99 ± 1.52 (t = 9) 5957 ± 80 (t = 182) Tryptophane 0.25 ± 0.01 (t = 12) 9.15 ± 0.17 (t = 26) < LOD < LOD Tyrosine 29.80 ± 0.90 (t = 16) 67.20 ± 2.80 (t = 26) 1.41 ± 0.08 (t = 26) 8.21 ± 0.36 (t = 182) Phenylalanine 28.00 ± 0.56 (t = 26) 206.4 ± 4.5 (t = 26) 4.48 ± 0.22 (t = 26) < LOD Glutamate < LOD 55.20 ± 1.39 (t = 8) 28.32 ± 0.56 (t = 26) 180.1 ± 0.9 (t = 17) Glutamine < LOD < LOD 2.71 ± 0.15 (t = 9) < LOD Proline 0.54 ± 0.13 (t = 13) 20.71 ± 0.82 (t = 11) 7.40 ± 0.28 (t = 5) 34.69 ± 1.75 (t = 17) Serine 0.87 ± 0.52 (t = 13) 1.73 ± 0.19 (t = 16) 1.60 ± 0.17 (t = 6) 24.27 ± 1.22 (t = 98) Glycine 1.23 ± 0.52 (t = 13) 6.84 ± 1.11 (t = 4) < LOD 698.4 ± 60.2 (t = 182) Arginine 9.61 ± 0.91 (t = 7) < LOD 76.80 ± 0.01 (t = 10) 35.47 ± 1.43 (t = 182) Paczia et al.
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