Anaerobic Xylose Fermentation by Recombinant Saccharomyces cerevisiae Carrying XYL1, XYL2, and XKS1 in Mineral Medium Chemostat Cultures

For ethanol production from lignocellulose, the fermentation of xylose is an economic necessity. Saccharomyces cerevisiae has been metabolically engineered with a xylose-utilizing pathway. However, the high ethanol yield and productivity seen with glucose have not yet been achieved. To quantitatively analyze metabolic fluxes in recombinant S. cerevisiae during metabolism of xylose-glucose mixtures, we constructed a stable xylose-utilizing recombinant strain, TMB 3001. The XYL1 and XYL2 genes from Pichia stipitis, encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, and the endogenous XKS1 gene, encoding xylulokinase (XK), under control of the PGK1 promoter were integrated into the chromosomal HIS3 locus of S. cerevisiae CEN.PK 113-7A. The strain expressed XR, XDH, and XK activities of 0.4 to 0.5, 2.7 to 3.4, and 1.5 to 1.7 U/mg, respectively, and was stable for more than 40 generations in continuous fermentations. Anaerobic ethanol formation from xylose by recombinant S. cerevisiae was demonstrated for the first time. However, the strain grew on xylose only in the presence of oxygen. Ethanol yields of 0.45 to 0.50 mmol of C/mmol of C (0.35 to 0.38 g/g) and productivities of 9.7 to 13.2 mmol of C h⁻¹ g (dry weight) of cells⁻¹ (0.24 to 0.30 g h⁻¹ g [dry weight] of cells⁻¹) were obtained from xylose-glucose mixtures in anaerobic chemostat cultures, with a dilution rate of 0.06 h⁻¹. The anaerobic ethanol yield on xylose was estimated at 0.27 mol of C/(mol of C of xylose) (0.21 g/g), assuming a constant ethanol yield on glucose. The xylose uptake rate increased with increasing xylose concentration in the feed, from 3.3 mmol of C h⁻¹ g (dry weight) of cells⁻¹ when the xylose-to-glucose ratio in the feed was 1:3 to 6.8 mmol of C h⁻¹ g (dry weight) of cells⁻¹ when the feed ratio was 3:1. With a feed content of 15 g of xylose/liter and 5 g of glucose/liter, the xylose flux was 2.2 times lower than the glucose flux, indicating that transport limits the xylose flux.     

Starvation response of Saccharomyces cerevisiae grown in anaerobic nitrogen- or carbon-limited chemostat cultures

Anaerobic starvation conditions are frequent in industrial fermentation and can affect the performance of the cells. In this study, the anaerobic carbon or nitrogen starvation response of Saccharomyces cerevisiae was investigated for cells grown in anaerobic carbon or nitrogen-limited chemostat cultures at a dilution rate of 0.1 h(-1) at pH 3.25 or 5. Lactic or benzoic acid was present in the growth medium at different concentrations, resulting in 16 different growth conditions. At steady state, cells were harvested and then starved for either carbon or nitrogen for 24 h under anaerobic conditions. We measured fermentative capacity, glucose uptake capacity, intracellular ATP content, and reserve carbohydrates and found that the carbon, but not the nitrogen, starvation response was dependent upon the previous growth conditions. All cells subjected to nitrogen starvation retained a large portion of their initial fermentative capacity, independently of previous growth conditions. However, nitrogen-limited cells that were starved for carbon lost almost all their fermentative capacity, while carbon-limited cells managed to preserve a larger portion of their fermentative capacity during carbon starvation. There was a positive correlation between the amount of glycogen before carbon starvation and the fermentative capacity and ATP content of the cells after carbon starvation. Fermentative capacity and glucose uptake capacity were not correlated under any of the conditions tested. Thus, the successful adaptation to sudden carbon starvation requires energy and, under anaerobic conditions, fermentable endogenous resources. In an industrial setting, carbon starvation in anaerobic fermentations should be avoided to maintain a productive yeast population.     

Induction of Stress Genes in Saccharomyces cerevisiae during Starvation

In order to analyze the response of Saccharomyces cerevisiae to starvation on a gene expression level, microarray experiments were performed using a yeast whole genome array. It is well known that under stress conditions like heat, high salt concentrations, pressure or the presence of toxins, special stress response genes are induced in Saccharomyces cerevisiae. This includes the genes encoding the typical heat shock proteins as well as numerous genes concerning cell membrane composition, central carbon metabolism or cell cycle. In this contribution, the Saccharomyces cerevisiae starvation‐stress response is analyzed. Starvation is a living condition often experienced by yeast in natural surroundings. As Saccharomyces cerevisiae is an eukaryote, many results from the gene expression analysis are valid for mammalians as well. The understanding of response of the yeast to the absence of a nutrient is also important for the development of feeding strategies in cultivations. Therefore, knowledge about the gene expression during starvation is important for both research and industrial applications. The regulation of 233 genes, which are involved in the stress response according to the literature, was examined via microarray experiments. In addition, a screening was carried out identifying 115 genes, which are hitherto not known to be comprised in the stress response, but which were significantly up‐regulated during starvation.     

Ribonucleic Acid Regulation in Amino Acid-Limited Cultures of Escherichia coli Grown in a Chemostat

The regulation of ribonucleic acid (RNA) synthesis was examined in cultures of bacteria whose growth was limited in the chemostat by the supply of a required amino acid. Strains possessing the relaxed (relA) mutation accumulated excess RNA (relative to protein) at low growth rates when growth was limited by arginine, histidine, or cysteine but not when limited by methionine. In contrast, stringent (relA(+)) strains maintained a constant RNA/protein ratio with decreasing growth rate regardless of the amino acid used to limit growth. The presence of excess RNA in relaxed strains was accompanied by an absence of increase in RNA production upon addition of chloramphenicol, a lag upon shift-up in growth by addition of excess of the limiting amino acid, and a decreased rate of production of beta-galactosidase upon induction. Analysis of the RNA accumulated in relaxed strains indicated it was present as transfer RNA as well as 50S and 30S ribosomal subunits. Microscope examination of the relaxed strains during histidine-, arginine-, or cysteine-limited growth in the chemostat showed them to be 10 to 20 times longer in size than the stringent strains. Also, cell density was reduced to one-tenth when the increased size was observed. An analysis of the amount of ppGpp present in all slow-growing amino acid-limited cultures (relaxed and stringent) demonstrated that only basal levels of ppGpp were made. These data are consistent with the hypothesis that when growth is limited in the chemostat by an initiation event in protein synthesis, i.e., limited methionine, RNA regulation occurs in relaxed as well as stringent strains. Also, when other amino acids are limiting in concentration during translation, errors occur in relaxed strains, resulting in misread proteins.     

Regulation of the Expression of the Photosynthetic Apparatus of Rhodobacter capsulatus Grown in Nitrogen-Limited Chemostat Cultures

Rhodobacter capsulatus was grown in chemostat cultures under different dilution rates and with ammonium ions as the limiting nutrient. The maximal growth rate (μmax) and the Monod cell growth saturation coefficient (K_s), were calculated from batch cultures grown at different concentrations of NH₄ ⁺. The experiments in chemostat were carried out at 0.25 mM (NH₄)₂SO₄, and the dilution rates were varied between 38% and 75% of μmax. The results indicated that under continuous culture conditions the cell yield coefficient (Y) (mg dry weight × μmol consumed ammonium sulfate⁻¹) decreased with increasing dilution rate (D). On the contrary, the cell yield was constant when expressed as mg cellular protein ×μmol consumed ammonium sulfate⁻¹. This occurred as a consequence of both an increase in the consumed ammonium sulfate and a simultaneous decrease in the cell biomass production at increasing growth rates. The cells produced at higher growth rates had a higher protein content per cell. The specific content of bacteriochlorophyll (Bchl) decreased (between 3 and 4 times) with increasing growth rates measured in either cells or chromatophores. However, the absorption spectra of the cells indicated that the ratio LHI (light-harvesting complex I) to LHII (light-harvesting complex II) Bchl complexes did not change. The reaction center (RC) complex content varied in parallel with the total Bchl content, yielding a constant photosynthetic unit of 65 mol Bchl × mol RC⁻¹ at different Ds. On the other hand, the uncoupled ATPase-specific activity measured in chromatophores was usually between 30% and 40% higher at the highest growth rates reached in these experiments. Received: 22 January 1996 / Accepted: 9 March 1996     

Improved Anaerobic Use of Arginine by Saccharomyces cerevisiae

Anaerobic arginine catabolism in Saccharomyces cerevisiae was genetically modified to allow assimilation of all four rather than just three of the nitrogen atoms in arginine. This was accomplished by bypassing normal formation of proline, an unusable nitrogen source in the absence of oxygen, and causing formation of glutamate instead. A pro3 ure2 strain expressing a PGK1 promoter-driven PUT2 allele encoding Δ¹-pyrroline-5-carboxylate dehydrogenase lacking a mitochondrial targeting sequence produced significant cytoplasmic activity, accumulated twice as much intracellular glutamate, and produced twice as much cell mass as the parent when grown anaerobically on limiting arginine as sole nitrogen source.     

Sporulation Synchrony of Saccharomyces cerevisiae Grown in Various Carbon Sources

Sporulation of several strains of Saccharomyces cerevisiae grown in a variety of carbon sources that do not repress the tricarboxylic acid cycle enzymes was more synchronous than the sporulation of cells grown in medium containing dextrose which does repress those enzymes. Dextrose-grown cells showed optimal sporulation synchrony when inoculated into sporulation medium from early stationary phase when the dextrose in the medium is exhausted. Logarithmic-phase cells grown in either non-fermentable carbon sources (acetate and glycerol) or a fermentable carbon source that does not repress tricarboxylic acid cycle enzymes (galactose) sporulated more synchronously than the early stationary-phase dextrose cells. Attempts were made to sporulate cells taken from both complex and semidefined media. The semidefined acetate medium failed to support the growth of a number of strains. However, cells grown in the complex acetate medium, as well as both complex and semidefined glycerol and galactose media, sporulated with better synchrony than did the dextrose-grown cells.     

Respiratory Development in Saccharomyces cerevisiae Grown at Controlled Oxygen Tension

Saccharomyces cerevisiae was grown in batch culture over a wide range of oxygen concentrations, varying from the anaerobic condition to a maximal dissolved oxygen concentration of 3.5 muM. The development of cells was assayed by measuring amounts of the aerobic cytochromes aa(3), b, c, and c(1), the cellular content of unsaturated fatty acids and ergosterol, and the activity of respiratory enzyme complexes. The half-maximal levels of membrane-bound cytochromes aa(3), b, and c(1), were reached in cells grown in O(2) concentrations around 0.1 muM; this was similar to the oxygen concentration required for half-maximal levels of unsaturated fatty acid and sterol. However, the synthesis of ubiquinone and cytochrome c and the increase in fumarase activity were essentially linear functions of the dissolved oxygen concentration up to 3.5 muM oxygen. The synthesis of the succinate dehydrogenase, succinate cytochrome c reductase, and cytochrome c oxidase complexes showed different responses to changes in O(2) concentration in the growth medium. Cyanide-insensitive respiration and P(450) cytochrome content were maximal at 0.25 muM oxygen and declined in both more anaerobic and aerobic conditions. Cytochrome c peroxidase and catalase activities in cell-free homogenates were high in all but the most strictly anaerobic cells.     

Oxygen Response of the Wine Yeast Saccharomyces cerevisiae EC1118 Grown under Carbon-Sufficient,Nitrogen-Limited Enological Conditions

Discrete additions of oxygen play a critical role in alcoholic fermentation. However, few studies have quantitated the fate of dissolved oxygen and its impact on wine yeast cell physiology under enological conditions. We simulated the range of dissolved oxygen concentrations that occur after a pump-over during the winemaking process by sparging nitrogen-limited continuous cultures with oxygen-nitrogen gaseous mixtures. When the dissolved oxygen concentration increased from 1.2 to 2.7 μM, yeast cells changed from a fully fermentative to a mixed respirofermentative metabolism. This transition is characterized by a switch in the operation of the tricarboxylic acid cycle (TCA) and an activation of NADH shuttling from the cytosol to mitochondria. Nevertheless, fermentative ethanol production remained the major cytosolic NADH sink under all oxygen conditions, suggesting that the limitation of mitochondrial NADH reoxidation is the major cause of the Crabtree effect. This is reinforced by the induction of several key respiratory genes by oxygen, despite the high sugar concentration, indicating that oxygen overrides glucose repression. Genes associated with other processes, such as proline uptake, cell wall remodeling, and oxidative stress, were also significantly affected by oxygen. The results of this study indicate that respiration is responsible for a substantial part of the oxygen response in yeast cells during alcoholic fermentation. This information will facilitate the development of temporal oxygen addition strategies to optimize yeast performance in industrial fermentations.     

Long-Term Changes in Chemostat Cultures of Cytophaga johnsonae

Long-term studies with a gliding, heterotrophic bacterium, Cytophaga johnsonae, were conducted in a glucose-limited chemostat at a high and a low dilution rate. To test the stability of the steady state during long-term experiments the following parameters were monitored: optical density, glucose concentration, glucose uptake potential, ATP content of the cells, and plate counts on two different agar media. Biomass remained relatively constant, although the observed changes could have been possible in both directions. During all steady states, glucose uptake showed a stepwise increase and the glucose concentration showed a corresponding decrease. Glucose uptake potential and glucose concentration in the chemostat were inversely proportional. The ATP content of the cells varied up to 33% during the steady state, but did not show a general trend. After long cultivation in all chemostats, plate counts on both agars dropped to values less than 20% of the original steady-state level. These decreases were due to an inability of the cells to grow on agar plates, not to a lack of vitality of the cells in the chemostat. This study showed that even during shorter chemostat runs, e.g., 1 week, changes in important parameters with the steady state must be expected, especially in the uptake potential and the concentration of the limiting substrate.     

Evolutionary Engineering of Saccharomyces cerevisiae for Anaerobic Growth on Xylose

Xylose utilization is of commercial interest for efficient conversion of abundant plant material to ethanol. Perhaps the most important ethanol-producing organism, Saccharomyces cerevisiae, however, is incapable of xylose utilization. While S. cerevisiae strains have been metabolically engineered to utilize xylose, none of the recombinant strains or any other naturally occurring yeast has been able to grow anaerobically on xylose. Starting with the recombinant S. cerevisiae strain TMB3001 that overexpresses the xylose utilization pathway from Pichia stipitis, in this study we developed a selection procedure for the evolution of strains that are capable of anaerobic growth on xylose alone. Selection was successful only when organisms were first selected for efficient aerobic growth on xylose alone and then slowly adapted to microaerobic conditions and finally anaerobic conditions, which indicated that multiple mutations were necessary. After a total of 460 generations or 266 days of selection, the culture reproduced stably under anaerobic conditions on xylose and consisted primarily of two subpopulations with distinct phenotypes. Clones in the larger subpopulation grew anaerobically on xylose and utilized both xylose and glucose simultaneously in batch culture, but they exhibited impaired growth on glucose. Surprisingly, clones in the smaller subpopulation were incapable of anaerobic growth on xylose. However, as a consequence of their improved xylose catabolism, these clones produced up to 19% more ethanol than the parental TMB3001 strain produced under process-like conditions from a mixture of glucose and xylose.     

Carbon Starvation Can Induce Energy Deprivation and Loss of Fermentative Capacity in Saccharomyces cerevisiae

Seven different strains of Saccharomyces cerevisiae were tested for the ability to maintain their fermentative capacity during 24 h of carbon or nitrogen starvation. Starvation was imposed by transferring cells, exponentially growing in anaerobic batch cultures, to a defined growth medium lacking either a carbon or a nitrogen source. After 24 h of starvation, fermentative capacity was determined by addition of glucose and measurement of the resulting ethanol production rate. The results showed that 24 h of nitrogen starvation reduced the fermentative capacity by 70 to 95%, depending on the strain. Carbon starvation, on the other hand, provoked an almost complete loss of fermentative capacity in all of the strains tested. The absence of ethanol production following carbon starvation occurred even though the cells possessed a substantial glucose transport capacity. In fact, similar uptake capacities were recorded irrespective of whether the cells had been subjected to carbon or nitrogen starvation. Instead, the loss of fermentative capacity observed in carbon-starved cells was almost surely a result of energy deprivation. Carbon starvation drastically reduced the ATP content of the cells to values well below 0.1 μmol/g, while nitrogen-starved cells still contained approximately 6 μmol/g after 24 h of treatment. Addition of a small amount of glucose (0.1 g/liter at a cell density of 1.0 g/liter) at the initiation of starvation or use of stationary-phase instead of log-phase cells enabled the cells to preserve their fermentative capacity also during carbon starvation. The prerequisites for successful adaptation to starvation conditions are probably gradual nutrient depletion and access to energy during the adaptation period.     

Osmotic Properties of Spheroplasts from Saccharomyces cerevisiae Grown at Different Temperatures

Spheroplasts were prepared from cells of Saccharomyces cerevisiae NCYC 366, grown at 30 or 15 C, by incubating cells with snail-gut juice after pretreatment with 2-mercaptoethanol. Walls of cells grown batchwise or in continuous culture at 15 C were more resistant to digestion with snail juice than walls on cells grown under the same conditions as 30 C. Spheroplasts lysed when suspended in hypotonic solutions of mannitol. The resistance of spheroplasts to osmotic lysis tended to increase when the test temperature was lowered below 30 C. The increased resistance was greater with spheroplasts from cells grown at 15 C. Cations, especially Ca²⁺, protected spheroplasts against osmotic lysis. In general, the protective effects, measured at 30 C, were smaller with spheroplasts from cells grown at 15 C compared with 30 C. Citrate and ethylenediaminetetraacetate (EDTA) decreased the resistance of spheroplasts to osmotic lysis. On the whole, the decrease was greater with spheroplasts from cells grown at 30 C rather than 15 C. In the presence of EDTA, spheroplasts from cells grown at 30 C were less resistant to osmotic lysis at 5 C than at 30 C; when spheroplasts from cells grown at 15 C were similarly examined, they were more resistant to lysis at 5 C than at 30 C. Spheroplast membranes from cells grown at 15 C had slightly but significantly greater contents of Mg²⁺, Ca²⁺, K⁺, and Na⁺ compared with spheroplast membranes from cells grown at 15 C. Mg²⁺ and Ca²⁺ were more easily extracted with EDTA from membranes of 30 C-grown cells than from 15 C-grown cells.     

Metabolic Regulation in Glucose-Limited Chemostat Cultures of Escherichia coli

Glucose-limited chemostat cultures of Escherichia coli, growing at dilution rates above 0.3/hr, continue to grow at the restricted rate after removal of glucose restriction. In a glycogenless strain, the specific rates of increase of mass, protein, and ribonucleic acid (RNA) were equal before and after supplementation with 0.05% glucose and did not increase detectably until after 30 to 60 min. The unrestricted specific growth rate was reached after two to three doublings of cell mass. Supplementation with glucose plus 20 amino acids, but not with glucose plus vitamins or ribosides, produced an immediate increase in the specific rates of mass and RNA synthesis followed by an increase in the specific rate of protein synthesis. In a wild-type strain, synthesis of protein and RNA continued at the restricted rate after glucose supplementation, but the specific rate of increase of mass immediately increased due to rapid synthesis of glycogen. At dilution rates less than 0.3/hr, the specific rates of increase of mass, protein, and RNA increased immediately after supplementation with glucose, but did not immediately attain the unrestricted growth. The results at dilution rates greater than 0.3/hr are interpreted to mean that the regulation of a number of enzymatic reactions is entirely through control of enzyme synthesis, without modulation of enzyme function. The levels of such enzymes are controlled so that operation with zero-order kinetics precisely meets the demands for balanced growth. It was shown that glutamic dehydrogenase and glutamic-oxalacetic transaminase are regulated in this manner.