1 Kluyver Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands
2 DSM Life Sciences, Bakery Ingredients Cluster, PO Box 1, 2600 MA Delft, The Netherlands
Correspondence
Pascale Daran-Lapujade
P.Lapujade{at}tnw.tudelft.nl
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ABSTRACT |
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Supplementary expression profiles and transcriptome analysis data are available with the online version of this paper.
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INTRODUCTION |
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Prolonged chemostat cultivation under defined conditions offers an excellent approach to study evolution, and has been applied to various micro-organisms and nutrient limitations (Francis & Hansche, 1972; Jansen et al., 2004
; Rosenzweig et al., 1994
; Wick et al., 2002
). Under nutrient-limited conditions, selection is primarily for an improved affinity for the growth-limiting nutrient. Any adaptation that results in a higher specific growth rate (µ) at the ambient (often vanishingly low) residual substrate concentration will result in an improved competitiveness in comparison with non-adapted cells. A general trend observed during prolonged chemostat cultivation is a progressive, generally hyperbolic, decrease of the residual concentration of the growth-limiting nutrient (Kovarova-Kovar & Egli, 1998
; Senn et al., 1994
). This decrease reflects a selection for cells with a higher affinity [µmax·Ks1, in which Ks is the substrate-saturation constant for the growth-limiting nutrient (Monod, 1942
), and µmax is the maximum specific growth rate]. In addition, studies on mutative adaptation of micro-organisms in chemostat cultures have demonstrated changes in cellular activity (Novick & Szilard, 1950a
; van Schie et al., 1989
; Weikert et al., 1997
) and morphology (Adams et al., 1985
; Brown & Hough, 1965
). The main challenge now resides in the identification of the molecular basis for the adaptation. Some pioneering studies with Saccharomyces cerevisiae exploited the availability of the complete yeast genome and of genomics tools such as DNA microarrays. Genome-wide transcriptome analysis, performed during prolonged chemostat cultivation of S. cerevisiae on glucose (Ferea et al., 1999
), revealed changes in expression of many genes, including several genes encoding proteins involved in central carbon metabolism. Furthermore, the strong selection pressure in these cultures resulted in the enrichment of mutants with one or more duplications of particular HXT genes, encoding high-affinity hexose transporters (Brown et al., 1998
). Although these studies yielded important insights into the dynamics of the yeast genome under selective conditions, the changes that were observed at the transcriptome level were not correlated to enzyme levels or metabolic capacities.
A crucial feature of bakers' yeast is its capacity to produce CO2, referred to as fermentative capacity (van Hoek et al., 1998). After prolonged glucose-limited cultivation of S. cerevisiae, in addition to an increased affinity for glucose, we observed a dramatic decrease in fermentative capacity. Consequently, the aim of the present study was to perform an integral analysis of the long-term adaptation of S. cerevisiae during prolonged glucose-limited, aerobic cultivation in chemostat cultures, with special emphasis on the regulation of glucose transport and glycolytic capacity. To this end, we applied an integrated approach that combined transcriptome analysis, measurement of fermentative capacity and activities of glucose transport and glycolytic enzymes, and characterization of cellular morphology.
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METHODS |
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Media.
Synthetic medium containing mineral salts and vitamins was prepared and sterilized as described by Verduyn et al. (1992). For chemostat cultivation, the glucose concentration in reservoir media was 7·5 g l1 (0·25 mol C l1). This medium composition has previously been demonstrated to sustain glucose-limited cultivation of S. cerevisiae CEN.PK113-7D (Lange & Heijnen, 2001
; Verduyn et al., 1992
). For batch cultivation, the initial glucose concentration was 20 g l1.
Chemostat cultivation.
Aerobic chemostat cultivation was performed at a dilution rate of 0·10 h1 at 30 °C in 1·5 l laboratory fermenters (Applikon) at a stirrer speed of 800 r.p.m. The working volume of the cultures was kept at 1·0 l by a peristaltic effluent pump coupled to an electrical level sensor. This set-up ensured that under all growth conditions, biomass concentrations in samples taken directly from the culture differed by <1 % from those in samples taken from the effluent line (Noorman et al., 1991). The exact working volume was measured after each experiment. The pH was kept at 5·0±0·1 by an ADI 1030 biocontroller (Applikon), via the automatic addition of 2 mol KOH l1. The fermenter was flushed with air at a flow rate of 0·5 l min1 using a Brooks 5876 mass-flow controller. The dissolved-oxygen concentration was continuously monitored with an oxygen electrode (model 34 100 3002; Ingold), and it remained above 60 % of air saturation. Chemostat cultures were routinely checked for potential bacterial and fungal infection by phase-contrast microscopy.
Batch cultivation in fermenters.
For batch cultivation in fermenters, the same set-up as for chemostat cultivation was used, except that no medium feed rate, and consequently no effluent removal rate, was applied. The starting volume of these fermentations was 1·0 l. Samples were withdrawn at appropriate intervals for determination of dry weight and metabolite concentrations.
Off-gas analysis.
The exhaust gas was cooled in a condenser (2 °C), and dried with a Perma Pure dryer type PD-625-12P. O2 and CO2 concentrations were determined with a Rosemount NGA2000 analyser. Determination of the exhaust gas flow rate and calculation of specific rates of CO2 production and O2 consumption were performed as described previously (van Urk et al., 1988; Weusthuis et al., 1994
).
Determination of culture dry weight.
Culture samples (10 ml) were filtered through preweighed nitrocellulose filters (pore size, 0·45 µm; Gelman Sciences). After removal of medium, the filters were washed with demineralized water, dried in a Whirlpool Easytronic M591 microwave oven for 20 min at 360 W output, and weighed. Duplicate determinations varied by <1 %.
Extracellular metabolite analysis.
Glucose, ethanol, glycerol, acetate and pyruvate present in the supernatant of chemostat cultures were determined by HPLC analysis using an HPX-87H Aminex ion-exchange column (300x7·8 mm, Bio-Rad) at 60 °C. The column was eluted with 5 mM sulfuric acid at a flow rate of 0·6 ml min1. Pyruvate and acetate were detected by a Waters 441 UV-meter at 214 nm, coupled to a Waters 741 data module. Glucose, ethanol and glycerol were detected by an ERMA type ERC-7515A refractive-index detector coupled to a Hewlett Packard type 3390A integrator. Glucose concentrations in reservoir media were also analysed by HPLC.
Residual glucose measurements of continuous cultures.
Samples of cells (5 ml) were rapidly (within 3 s) transferred from the chemostat culture into a syringe containing 62·0 g cold steel beads (diameter, 4 mm; temperature, 20 °C) (Mashego et al., 2003). After withdrawal from the fermenter, the sample was directly filtered (0·2 µm pore-size filter; Schleicher & Schuell). The supernatant was analysed for glucose by a commercial kit (catalogue no. 716251; Diffchamb Biocontrol), as described previously (Jansen et al., 2002
).
Fermentative capacity assays.
Samples containing exactly 200 mg dry weight of biomass were harvested from a steady-state chemostat culture by centrifugation (5000 g, 3 min), and resuspended in 10 ml fivefold-concentrated synthetic medium (pH 5·6). Subsequently, these cell suspensions were introduced into a thermostat-controlled (30 °C) vessel. The volume was adjusted to 40 ml with demineralized water. After 10 min incubation, 10 ml glucose solution (100 g l1) was added, and samples (1 ml) were taken at appropriate time intervals for 30 min. The 10 ml headspace was continuously flushed with water-saturated CO2 at a flow rate of approximately 30 ml min1. The ethanol concentration in the supernatant was analysed using a colorimetric assay (Verduyn et al., 1984). Fermentative capacity can be calculated from the linear increase in ethanol concentration and is expressed as mmol ethanol produced (g dry yeast biomass)1 h1 (van Hoek et al., 1998
). Growth during these assays can be neglected, as no significant change in biomass concentration was observed.
Preparation of cell extracts.
For preparation of cell extracts, culture samples were harvested by centrifugation, washed twice with phosphate buffer pH 7·5 (10 mM potassium phosphate, 2 mM EDTA), concentrated fourfold, and stored at 20 °C. Before assaying, the cells were thawed, washed and resuspended in phosphate buffer pH 7·5 (100 mM potassium phosphate, 2 mM MgCl2, 1 mM DTT). Intracellular proteins were released by sonication at 0 °C using glass beads (0·7 mm diameter) in an MSE Soniprep 150 sonicator (150 W output, 8 µm peak-to-peak amplitude) for 3 min at 0·5 min intervals. Unbroken cells and cell debris were removed by centrifugation (4 °C, 20 min at 36 000 g), and the supernatant was used as the cell extract for enzyme assays. In all cell extracts, this method released 53±4 % of the total cellular proteins.
Enzyme assays.
Enzyme assays were performed with a Hitachi model 100-60 spectrophotometer at 30 °C and 340 nm (340 of reduced pyridine-dinucleotide cofactors 6·3 mM1) with freshly prepared cell extracts. All enzyme activities are expressed as µmol substrate converted per min per mg protein [U (mg protein)1]. When necessary, extracts were diluted in sonication buffer. All assays were performed with two concentrations of cell extract. Specific activities of these duplicate experiments differed by <10 %.
Hexokinase (HXK; EC 2.7.1.1) was assayed according to Postma et al. (1989). Phosphoglucose isomerase (PGI; EC 5.3.1.9) was assayed according to Bergmeyer (1974)
, with minor modifications. The assay mixture contained: Tris/HCl buffer (pH 8·0) 50 mM, MgCl2 5 mM, NADP+ 0·4 mM, glucose-6-phosphate dehydrogenase (Roche) 1·8 U ml1 and cell extract. The reaction was started with 2 mM fructose 6-phosphate. Phosphofructokinase (PFK; EC 2.7.1.11) was assayed according to de Jong-Gubbels et al. (1995)
, with minor modifications. The assay mixture contained: imidazole/HCl (pH 7·0) 50 mM, MgCl2 5 mM, NADH 0·15 mM, fructose 2,6-diphosphate 0·10 mM, fructose-1,6-diphosphate aldolase (FBA; Roche) 0·45 U ml1, glycerol-3-phosphate dehydrogenase (Roche) 0·6 U ml1, triosephosphate isomerase (TPI) 1·8 U ml1 (Roche) and cell extract. The endogenous activity was measured after adding 0·25 mM fructose 6-phosphate. The reaction was started with 0·5 mM ATP. FBA (EC 4.1.2.13) was assayed according to van Dijken et al. (1978)
. TPI (EC 5.3.1.1) was assayed according to van Hoek (2000)
. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) was assayed according to van Hoek (2000)
, with minor modifications. The assay mixture contained the following: triethanolamine/HCl buffer (pH 7·6) 100 mM, ATP 1 mM, EDTA 1 mM, MgSO4 1·5 mM, NADH 0·15 mM, phosphoglycerate kinase (PGK) 25·0 U ml1 (Sigma) and cell extract. The reaction was started with 5 mM 3-phosphoglycerate (trihexylammonium salt). PGK (EC 2.7.2.3) was assayed using the same method as for GAPDH, except that PGK was replaced by glyceraldehyde-3-phosphate dehydrogenase, 8·0 U ml1 (Roche). Phosphoglycerate mutase (PGM; EC 5.4.2.1) was assayed according to Bergmeyer (1974)
. Enolase (ENO; EC 4.2.1.11) was assayed according to van Hoek (2000)
. Pyruvate kinase (PYK; EC 2.7.1.40) was assayed according to de Jong-Gubbels et al. (1995)
, with minor modifications. The assay mixture contained the following: cacodylic acid/KOH (pH 6·2) 100 mM, KCl 100 mM, ADP 10 mM, fructose 1,6-diphosphate 1 mM, MgCl2 25 mM, NADH 0·15 mM, lactate dehydrogenase (Roche) 11·25 U ml1 and cell extract. The reaction was started with 2 mM phosphoenolpyruvate. Pyruvate decarboxylase (PDC; EC 4.1.1.1) and alcohol dehydrogenase (ADH; EC 1.1.1.1) were assayed according to Postma et al. (1989)
.
Total RNA isolation.
Cells were rapidly (within 3 s) transferred from the chemostat culture into liquid nitrogen to immediately quench the metabolism. The frozen cell suspension (about 40 g cell broth) was thawed gently on ice. After complete thawing, the cell suspension was centrifuged at 0 °C, 5000 g, for 5 min. Total RNA extraction from the pellets was performed using the hot-phenol method (Schmitt et al., 1990).
Microarray analysis.
The results for each growth condition were derived from three independently cultured replicates. Sampling of cells from chemostats, probe preparation, and hybridization to Affymetrix GeneChip microarrays, as well as data acquisition and analysis, were performed as previously described (Daran-Lapujade et al., 2004; Piper et al., 2002
). Statistical analysis was performed using Microsoft Excel running the significance analysis of microarrays add-in (SAM; version 1.12; Tusher et al., 2001
). Genes were considered as being changed in expression if they were identified as being significantly changed by at least twofold using SAM (expected median false-discovery rate of 1 %). Promoter analysis was performed using web-based software Regulatory Sequence Analysis Tools (http://rsat.ulb.ac.be/rsat/; van Helden et al., 2000
), as previously described by Daran-Lapujade et al. (2004)
. The complete dataset can be found at http://www.bt.tudelft.nl/glucose-selection, and the genes with significant change in expression are listed in Supplementary Figure S1 with the online version of this paper.
Image analysis.
Microscopic images were taken using an Olympus IMT-2 reverse microscope, and analysed using an Olympus camera adaptor, a CCD camera, Olympus MTV-3, and the image analyser software Leica Qwin, version pro 2.2.
Restart of chemostat cultivation.
A stored glycerol stock (80 °C) of a prolonged glucose-limited chemostat cultivation culture (containing 30 %, v/v, sterile glycerol) was streaked out once for purity on a synthetic medium plate containing 0·8 % (w/v) glucose. Single colonies were used for inoculation of a shake-flask containing 0·8 % (w/v) glucose, which, after growth to stationary phase, was used as the inoculum for the chemostat culture.
Glucose-pulse experiments.
After a steady-state had been established at a dilution rate of 0·10 h1, the medium-supply and effluent-removal pumps were switched off. Immediately afterwards, 18 ml sterile 50 % (w/v) glucose solution was aseptically added to the culture. At appropriate time intervals, samples were taken for measurement of metabolite concentrations, OD660 and dry weight. Sugar concentrations and metabolite levels in the supernatants were determined by HPLC analysis.
Intracellular metabolite analysis.
Samples (525 µl) for intracellular metabolite analysis were taken rapidly (within 3 s) from the fermenter, and collected on ice in perchloric acid (5 %, v/v, final concentration). Samples were neutralized after 15 min by addition of 150 µl 2 M K2CO3 (0·35 M final concentration), and stored at 20 °C. Before analysis, samples were centrifuged for 1 min at 16 000 g. Intracellular metabolites were measured on a COBAS-FARA automatic analyser (Roche). Intracellular concentrations were calculated assuming that 1 mg protein corresponds to 3·75 µl intracellular volume (Richard et al., 1996; Teusink et al., 1998a
). Furthermore, it was assumed that cells of the reference and the evolved strains had the same cell volume.
Hexose transport assays.
Cells from chemostat cultures were harvested by centrifugation at 4 °C (5 min, 5000 g), washed once in 0·1 M potassium phosphate buffer (pH 6·5), and resuspended in the same buffer to a concentration of approximately 4 g protein l1. Cells were kept on ice until further use. Zero-trans influx of glucose was determined at 30 °C, according to Walsh et al. (1994). All data fitted well to one-component kinetics.
Protein determinations.
Protein concentrations in cell extracts used for enzyme analysis, and in cell suspensions used for hexose transport studies, were estimated by the Lowry method. Dried bovine serum albumin (fatty-acid free, obtained from Sigma) was used as a standard.
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RESULTS |
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Prolonged glucose-limited chemostat cultivation results in altered glucose uptake kinetics
The evolved strain exhibited a lower Km (0·54±0·07 mM) and a higher Vmax [865±39 nmol min1 (mg protein)1] for glucose uptake than the reference strain [Km 1·54±0·23 mM, and Vmax 551±4·5 nmol min1 (mg protein)1]. These kinetic data for glucose transport were consistent with the higher affinity for glucose of the evolved strain as reflected by the lower residual glucose concentration in the chemostat cultures. However, the increased capacity of glucose transport in the evolved strain indicates that glucose transport does not control its strongly reduced fermentative capacity.
Transcript levels of glycolytic genes, but not HXT genes, correlated with activity assays
To assess whether the major changes in glycolytic enzyme levels and glucose-uptake kinetics were controlled at the level of transcription, transcript levels of relevant structural genes were analysed with DNA microarrays. A Student's t-test was used to assess the statistical significance of the observed changes (Tables 1 and 2). For most glycolytic enzymes, the changes in enzyme levels observed in cell extracts were qualitatively consistent with changes at the mRNA level. Notable exceptions were PFK, FBA, TPI and ADH. Transcript levels of PFK and ADH were lower in the evolved strain, whereas in vitro enzyme activities remained constant. On the other hand, FBA and TPI showed a downregulation of in vitro enzyme activities, but no significant change at the mRNA level. Quantitatively, the correlation between changes in transcript level and in vitro enzyme activity did not always match. Changes in transcript levels for GAPDH, PGK, PGM, ENO and PDC were less strong than the changes in in vitro enzyme activity. These observations suggest that the levels of some glycolytic enzymes are partially controlled at a post-transcriptional level.
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Unbiased transcriptome analysis
To investigate whether, in addition to the transcription of glycolytic genes, transcription of other genes was affected in the evolved strain, a genome-wide transcript analysis was performed. Statistical analysis identified 249 transcripts (4·1 % of the genome) whose levels significantly differed in the evolved and parental strains. Among the 186 genes that yielded a higher transcript level in the evolved strain, several were involved in cell cycle and DNA processing (34 genes, 18 %). Many of these are crucial for cell cycle progression or are involved in cell morphology (see Supplementary Fig. S1 with the online version of this paper). Upregulation of these genes may contribute to the decreased maximum specific growth rate of the evolved strain in batch cultures (Table 4, see following section) and its elongated morphology. Among the 63 genes that showed a reduced transcript level in the evolved strain, the most interesting encode proteins involved in metabolism (20 genes, 32 %), including the glycolytic enzyme genes ENO1, ENO2, TDH1, PYK1 and PDC1. Remarkably, eight additional genes were involved in stress response (HSP30, YRO2, etc.; for complete list see Supplementary Fig. S1).
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DISCUSSION |
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A possible explanation for the decreased glycolytic enzyme levels in the evolved strain is that glucose-limited cultivation provides a selective pressure to economize on protein synthesis, which is an energetically expensive process (Forrest & Walker, 1971; Oura, 1972
; Stouthamer, 1973
). In S. cerevisiae, glycolytic enzymes represent around 1015 % of the total cellular protein during aerobic, sugar-limited growth (van Hoek, 2000
). The estimated change in total cellular protein represented by glycolysis in the evolved strain is
7 % relative to the parental strain. Although this decrease is too small to be clearly reflected in the biomass yield on glucose (Fig. 1b
), it may well be significant during long-term selection. The observation that levels of enzymes in the upper half of glycolysis, such as HXK, were not affected may reflect their involvement in maintaining a low intracellular glucose concentration and, consequently, a high affinity of in vivo glucose uptake (Bisson & Fraenkel, 1983
; Teusink et al., 1998a
).
An interesting implication of the present study is that the evolved strain was capable of growing on glucose at the same specific growth rate in chemostat and with the same biomass yield on glucose as the parental strain, even though levels of key glycolytic enzymes were drastically different. The most probable explanation is that levels of important glycolytic intermediates and/or low-molecular-weight effectors of glycolytic enzymes were different in the two strains. In addition to being of fundamental interest, this might provide an attractive means of challenging kinetic models of glycolysis. This option has been investigated in a separate study (Mashego et al., 2005).
Transcriptome analysis as a tool for studying selected strains
DNA-microarray analysis provides quantitative, reproducible and genome-wide data on mRNA levels. Such analyses can, in principle, be used to investigate the molecular basis for phenotypic differences between different microbial strains, and to study the selective pressures to which micro-organisms are exposed in nature, in the laboratory or in industry. However, our results underline some inherent limitations of this approach.
Although a major change in glucose-uptake kinetics was observed in the evolved strain, this could not be clearly attributed to a different transcript level of any of the known HXT-encoded glucose transporters, nor did the observed Km coincide with that of any of the known hexose transporters in S. cerevisiae (Boles & Hollenberg, 1997; Özcan & Johnston, 1999
; Reifenberger et al., 1995
). Interestingly, a similar high-affinity glucose-transport system has been found in hxk2 mutants of this yeast (Petit et al., 2000
). Several mechanisms may be responsible for the observed reduction of the Km, including point mutations in the structural genes, involvement of other proteins, and changes in membrane composition.
A recent study in which glycolytic fluxes and transcript levels of glycolytic genes were compared in chemostat cultures grown on different carbon sources demonstrated that glycolytic mRNA levels are poor indicators for glycolytic flux (Daran-Lapujade et al., 2004). The present study shows that this conclusion also holds for a comparison between different S. cerevisiae strains grown under identical conditions. Indeed, the reduced glycolytic enzyme activities could not be fully correlated to a decrease in transcription, indicating that modifications in post-transcriptional processes were also involved in the selection process.
A non-biased transcriptome analysis yielded a large number of genes that showed a significantly different transcript level in the parental and evolved strains. Most of these changes in expression could not be linked to phenotype, and the transcriptome analysis clearly failed to identify the molecular basis of the evolution. In this work, we isolated three single-cell lines from a single prolonged chemostat culture, resulting in highly similar evolved strains. Ideally, statistical analysis of a large number of independent selection experiments would reveal whether selection ultimately converges to the same or similar genotypes, or whether different genotypes may become dominant. In the latter case, transcriptome analysis would probably be more helpful in identifying the mutations responsible for the evolved phenotype. Ferea et al. (1999) used independently selected aerobic glucose-limited cultures of S. cerevisiae for transcriptome analysis, and could indeed identify a somewhat smaller set of 88 transcripts with significantly changed expression between short- and long-term cultivations. They also observed a decreased expression of genes encoding enzymes in the lower part of glycolysis, indicating that this feature represents a significant competitive advantage for S. cerevisiae grown under aerobic glucose limitation. However, most of the other genes with changed expression do not overlap between this study and that of Ferea et al. (1999)
. This is probably due to the use of slightly different culture set-up between the two studies (different dilution rate, different strain, different metabolism).
Implications for biotechnological application and evolutionary engineering of S. cerevisiae
Chemostats are perfectly suited tools for strain improvement via evolutionary engineering, thus providing an appealing alternative to empirical strain improvement, and a valuable addition to metabolic engineering approaches. The principle of evolutionary engineering is to confront a micro-organism with a certain environment, and let natural selection engineer its genome until mutants with the desired phenotype (novel catabolic activity, improved stress resistance, etc.; for review see Sauer, 2001) take over the culture. Clearly, the strongly reduced fermentative capacity that is obtained after long-term selection in glucose-limited chemostat cultures disqualifies this procedure as a means of obtaining improved bakers' yeast strains. This study highlights the necessity to rationally design the chemostat for selection pressure and culture condition in order to direct evolution towards the desired phenotype.
Evolutionary engineering has been successfully applied to improve industrially relevant physiological properties (Flores et al., 1996; Hall & Hauer, 1993
; Sauer, 2001
). However, during long-term glucose-limited cultivation, we observed that an improved affinity was accompanied by a strongly delayed response to glucose excess. In a recent study on evolution of S. cerevisiae in maltose-limited chemostat cultures, we observed a similar apparent trade-off between affinity in nutrient-limited chemostat cultures and the ability to cope with a sudden exposure to sugar excess (Jansen et al., 2004
). These observations underline that selection, under steady-state nutrient-limited conditions, of spontaneous or induced mutants with desirable traits may come at the expense of their ability to cope with changes in the nutrient concentration. This is relevant when chemostat cultures are used for the directed selection of strains with industrially relevant properties (Flores et al., 1996
; Kuyper et al., 2004
; Sauer, 2001
). Furthermore, evolutionary engineering will become a valuable tool for rationally designed metabolic engineering approaches only if the molecular basis of the desired phenotype can be identified and used to genetically engineer micro-organisms. The present work exemplifies the difficulty of discovering the mutated gene(s) responsible for adaptation, and underlines the current limitation and challenges of evolutionary engineering.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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---|
Bergmeyer, H. U. (1974). Methods of Enzymatic Analysis, 2nd edn. New York & London: Academic Press.
Bisson, L. F. (1988). High-affinity glucose transport in Saccharomyces cerevisiae is under general glucose repression control. J Bacteriol 170, 48384845.[Medline]
Bisson, L. F. & Fraenkel, D. G. (1983). Involvement of kinases in glucose and fructose uptake by Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 80, 17301734.
Boer, V. M., de Winde, J. H., Pronk, J. T. & Piper, M. D. (2003). The genome-wide transcriptional responses of Saccharomyces cerevisiae grown on glucose in aerobic chemostat cultures limited for carbon, nitrogen, phosphorus, or sulfur. J Biol Chem 278, 32653274.
Boles, E. & Hollenberg, C. P. (1997). The molecular genetics of hexose transport in yeasts. FEMS Microbiol Rev 21, 85111.[CrossRef][Medline]
Brown, C. M. & Hough, J. S. (1965). Elongation of yeast cells in continuous culture. Nature 206, 676678.[Medline]
Brown, C. J., Todd, K. M. & Rosenzweig, R. F. (1998). Multiple duplications of yeast hexose transport genes in response to selection in a glucose-limited environment. Mol Biol Evol 15, 931942.[Abstract]
Chambers, A., Packham, E. A. & Graham, I. R. (1995). Control of glycolytic gene expression in the budding yeast (Saccharomyces cerevisiae). Curr Genet 29, 19.[CrossRef][Medline]
de Jong-Gubbels, P., Vanrolleghem, P., van Dijken, J. P. & Pronk, J. T. (1995). Regulation of carbon metabolism in chemostat cultures of Saccharomyces cerevisiae grown on mixtures of glucose and ethanol. Yeast 11, 407418.[Medline]
Daran-Lapujade, P., Jansen, M. L., Daran, J. M., Van Gulik, W., de Winde, J. H. & Pronk, J. T. (2004). Role of transcriptional regulation in controlling fluxes in central carbon metabolism of Saccharomyces cerevisiae: a chemostat culture study. J Biol Chem 279, 91259138.
Diderich, J. A., Schuurmans, J. M., van Gaalen, M. C., Kruckeberg, A. L. & van Dam, K. (2001). Functional analysis of the hexose transporter homologue HXT5 in Saccharomyces cerevisiae. Yeast 18, 15151524.[CrossRef][Medline]
Dunham, M. J., Badrane, H., Ferea, T. L., Adams, J., Brown, P. O. & Rosenzweig, R. F. (2002). Characteristic genome rearrangements in experimental evolution of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 99, 1614416149.
Ferea, T. L., Botstein, D., Brown, P. O. & Rosenzweig, R. F. (1999). Systematic changes in gene expression patterns following adaptive evolution in yeast. Proc Natl Acad Sci U S A 96, 97219726.
Flikweert, M. T., Kuyper, M., van Maris, A. J. A., Kotter, P., van Dijken, J. P. & Pronk, J. T. (1999). Steady-state and transient-state analysis of growth and metabolite production in a Saccharomyces cerevisiae strain with reduced pyruvate-decarboxylase activity. Biotechnol Bioeng 66, 4250.[CrossRef][Medline]
Flores, N., Xiao, J., Berry, A., Bolivar, F. & Valle, F. (1996). Pathway engineering for the production of aromatic compounds in Escherichia coli. Nat Biotechnol 14, 620623.[CrossRef][Medline]
Forrest, W. W. & Walker, D. J. (1971). The generation and utilization of energy during growth. Adv Microb Physiol 5, 213274.[Medline]
Francis, J. C. & Hansche, P. E. (1972). Directed evolution of metabolic pathways in microbial populations. I. Modification of the acid phosphatase pH optimum in S. cerevisiae. Genetics 70, 5973.
Hall, B. G. & Hauer, B. (1993). Acquisition of new metabolic activities by microbial populations. Methods Enzymol 224, 603613.[Medline]
Iyer, V. R., Horak, C. E., Scafe, C. S., Botstein, D., Snyder, M. & Brown, P. O. (2001). Genomic binding sites of the yeast cell-cycle transcription factors SBF and MBF. Nature 409, 533538.[CrossRef][Medline]
Jansen, M. L. A., de Winde, J. H. & Pronk, J. T. (2002). Hxt-carrier-mediated glucose efflux upon exposure of Saccharomyces cerevisiae to excess maltose. Appl Environ Microbiol 68, 42594265.
Jansen, M. L., Daran-Lapujade, P., de Winde, J. H., Piper, M. D. & Pronk, J. T. (2004). Prolonged maltose-limited cultivation of Saccharomyces cerevisiae selects for cells with improved maltose affinity and hypersensitivity. Appl Environ Microbiol 70, 19561963.
Kovarova-Kovar, K. & Egli, T. (1998). Growth kinetics of suspended microbial cells: from single-substrate-controlled growth to mixed-substrate kinetics. Microbiol Mol Biol Rev 62, 646666.
Kubitschek, H. E. (1970). Introduction to Research with Continuous Cultures. Englewood Cliffs, NJ: Prentice Hall.
Kuyper, M., Winkler, A. A., van Dijken, J. P. & Pronk, J. T. (2004). Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principle. FEMS Yeast Res 4, 655664.[CrossRef][Medline]
Lange, H. C. & Heijnen, J. J. (2001). Statistical reconciliation of the elemental and polymeric biomass composition of Saccharomyces cerevisiae. Biotechnol Bioeng 75, 334344.[CrossRef][Medline]
Mäenpää, P. H., Raivio, K. O. & Kekomäki, M. P. (1968). Liver adenine nucleotides: fructose-induced depletion and its effect on protein synthesis. Science 161, 12531254.[Medline]
Martinez-Pastor, M. T., Marchler, G., Schuller, C., Marchler-Bauer, A., Ruis, H. & Estruch, F. (1996). The Saccharomyces cerevisiae zinc finger proteins Msn2p and Msn4p are required for transcriptional induction through the stress response element (STRE). EMBO J 15, 22272235.[Abstract]
Mashego, M. R., van Gulik, W. M., Vinke, J. L. & Heijnen, J. J. (2003). Critical evaluation of sampling techniques for residual glucose determination in carbon-limited chemostat culture of Saccharomyces cerevisiae. Biotechnol Bioeng 83, 395399.[CrossRef][Medline]
Mashego, M. R., Jansen, M. L., Vinke, J. L., van Gulik, W. M. & Heijnen, J. J. (2005). Changes in the metabolome of Saccharomyces cerevisiae associated with evolution in aerobic glucose-limited chemostats. FEMS Yeast Res 5, 419430.[CrossRef][Medline]
Monod, J. (1942). Recherche sur la Croissance des Cultures Bactériennes. Paris: Hermann et Cie.
Nishi, K., Park, C. S., Pepper, A. E., Eichinger, G., Innis, M. A. & Holland, M. J. (1995). The GCR1 requirement for yeast glycolytic gene expression is suppressed by dominant mutations in the SGC1 gene, which encodes a novel basic-helix-loop-helix protein. Mol Cell Biol 15, 26462653.[Abstract]
Noorman, H. J., Baksteen, J., Heijnen, J. J., Luyben, K. & Ch, A. M. (1991). The bioreactor overflow device: an undesired selective separator in continuous cultures? J Gen Microbiol 13, 21712177.
Novick, A. & Szilard, L. (1950a). Experiments with the chemostat on spontaneous mutations of bacteria. Proc Natl Acad Sci U S A 36, 708719.[Medline]
Novick, A. & Szilard, L. (1950b). Description of the chemostat. Science 112, 715716.[Medline]
Oura, E. (1972). Reactions leading to the formation of yeast cell material from glucose and ethanol. PhD thesis, University of Helsinki.
Özcan, S. & Johnston, M. (1999). Function and regulation of yeast hexose transporters. Microbiol Mol Biol Rev 63, 554569.
Petit, T., Diderich, J. A., Kruckeberg, A. L., Gancedo, C. & van Dam, K. (2000). Hexokinase regulates kinetics of glucose transport and expression of genes encoding hexose transporters in Saccharomyces cerevisiae. J Bacteriol 182, 68156818.
Piper, M. D., Daran-Lapujade, P., Bro, C., Regenberg, B., Knudsen, S., Nielsen, J. & Pronk, J. T. (2002). Reproducibility of oligonucleotide microarray transcriptome analyses. An interlaboratory comparison using chemostat cultures of Saccharomyces cerevisiae. J Biol Chem 277, 3700137008.
Postma, E., Scheffers, W. A. & van Dijken, J. P. (1989). Kinetics of growth and glucose transport in glucose-limited chemostat cultures of Saccharomyces cerevisiae CBS 8066. Yeast 5, 159165.[Medline]
Ramos, J., Szkutnicka, K. & Cirillo, V. P. (1988). Relationship between low- and high-affinity glucose transport systems of Saccharomyces cerevisiae. J Bacteriol 170, 53755377.[Medline]
Reifenberger, E., Freidel, K. & Ciriacy, M. (1995). Identification of novel HXT genes in Saccharomyces cerevisiae reveals the impact of individual hexose transporters on glycolytic flux. Mol Microbiol 16, 157167.[Medline]
Richard, P., Teusink, B., Hemker, M. B., van Dam, K. & Westerhoff, H. V. (1996). Sustained oscillations in free-energy state and hexose phosphates in yeast. Yeast 12, 731740.[CrossRef][Medline]
Rieger, M., Käppeli, O. & Fiechter, A. (1983). The role of limited respiration in the complete oxidation of glucose by Saccharomyces cerevisiae. J Gen Microbiol 129, 653661.
Rosenzweig, R. F., Sharp, R. R., Treves, D. S. & Adams, J. (1994). Microbial evolution in a simple unstructured environment: genetic differentiation in Escherichia coli. Genetics 137, 903917.
Rutherford, J. C., Jaron, S. & Winge, D. R. (2003). Aft1p and Aft2p mediate iron-responsive gene expression in yeast through related promoter elements. J Biol Chem 278, 2763627643.
Sauer, U. (2001). Evolutionary engineering of industrially important microbial phenotypes. Adv Biochem Eng Biotechnol 73, 129169.[Medline]
Schmitt, M. E., Brown, T. A. & Trumpower, T. L. (1990). A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. Nucleic Acids Res 18, 30913092.[Medline]
Senn, H., Lendenmann, U., Snozzi, M., Hamer, G. & Egli, T. (1994). The growth of Escherichia coli in glucose-limited chemostat cultures: a re-examination of the kinetics. Biochim Biophys Acta 1201, 424436.[Medline]
Stouthamer, A. H. (1973). A theoretical study on the amount of ATP required for synthesis of microbial cell material. Antonie van Leeuwenhoek 39, 545565.[Medline]
Teusink, B., Diderich, J. A., Westerhoff, H. V., van Dam, K. & Walsh, M. C. (1998a). Intracellular glucose concentration in derepressed yeast cells consuming glucose is high enough to reduce the glucose transport rate by 50 %. J Bacteriol 180, 556562.
Teusink, B., Walsh, M. C., van Dam, K. & Westerhoff, H. V. (1998b). The danger of metabolic pathways with turbo design. Trends Biochem Sci 23, 162169.[CrossRef][Medline]
Tusher, V. G., Tibshirani, R. & Chu, G. (2001). Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 98, 51165121.
van Dijken, J. P., Harder, W., Beardsmore, A. J. & Quayle, J. R. (1978). Dihydroxyacetone: an intermediate in the assimilation of methanol by yeasts? FEMS Microbiol Lett 4, 97102.[CrossRef]
van Helden, J., Andre, B. & Collado-Vides, J. (2000). A web site for the computational analysis of yeast regulatory sequences. Yeast 16, 177187.[CrossRef][Medline]
van Hoek, P. (2000). Fermentative capacity in aerobic cultures of bakers' yeast. PhD thesis, Technical University of Delft.
van Hoek, P., van Dijken, J. P. & Pronk, J. T. (1998). Effect of specific growth rate on fermentative capacity of baker's yeast. Appl Environ Microbiol 64, 42264233.
van Maris, A. J. A., Bakker, B. M., Brandt, M., Boorsma, A., Teixeira de Mattos, M. J., Grivell, L. A., Pronk, J. T. & Blom, J. (2001). Modulating the distribution of fluxes among respiration and fermentation by overexpression of HAP4 in Saccharomyces cerevisiae. FEMS Yeast Res 1, 139149.[CrossRef][Medline]
van Schie, B. J., Rouwenhorst, R. J., van Dijken, J. P. & Kuenen, J. G. (1989). Selection of glucose-assimilating variants of Acinetobacter calcoaceticus LMD 79.41 in chemostat culture. Antonie van Leeuwenhoek 55, 3952.[Medline]
van Urk, H., Mak, P. R., Scheffers, W. A. & van Dijken, J. P. (1988). Metabolic responses of Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621 upon transition from glucose limitation to glucose excess. Yeast 4, 283291.[Medline]
van Urk, H., Voll, W. S. L., Scheffers, W. A. & van Dijken, J. P. (1990). Transient-state analysis of metabolic fluxes in Crabtree-positive and Crabtree-negative yeasts. Appl Environ Microbiol 56, 282286.
Verduyn, C., van Dijken, J. P. & Scheffers, W. A. (1984). Colorimetric alcohol assays with alcohol oxidase. J Microbiol Methods 2, 1525.[CrossRef]
Verduyn, C., Postma, E., Scheffers, W. A. & van Dijken, J. P. (1992). Effect of benzoic acid on metabolic fluxes in yeasts: a continuous study on regulation of respiration and alcoholic fermentation. Yeast 8, 501517.[Medline]
Walsh, M. C., Smits, H. P., Scholte, M. & van Dam, K. (1994). Affinity of glucose transport in Saccharomyces cerevisiae is modulated during growth on glucose. J Bacteriol 176, 953958.[Abstract]
Weikert, C., Sauer, U. & Bailey, J. E. (1997). Use of a glycerol-limited, long-term chemostat for isolation of Escherichia coli mutants with improved physiological properties. Microbiology 143, 15671574.[Medline]
Weusthuis, R. A., Luttik, M. A. H., Scheffers, W. A., van Dijken, J. P. & Pronk, J. T. (1994). Is the Kluyver effect in yeast caused by product inhibition? Microbiology 140, 17231729.[Medline]
Wick, L. M., Weilenmann, H. & Egli, T. (2002). The apparent clock-like evolution of Escherichia coli in glucose-limited chemostats is reproducible at large but not at small population sizes and can be explained with Monod kinetics. Microbiology 148, 28892902.[Medline]
Received 20 August 2004;
revised 1 February 2005;
accepted 9 February 2005.
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