Xylitol production using recombinant Saccharomyces cerevisiae containing multiple xylose reductase genes at chromosomal δ-sequences

Xylitol production from xylose was studied using recombinant Saccharomyces cerevisiae 2805 containing xylose reductase genes (XYL1) of Pichia stipitis at chromosomal δ-sequences. S. cerevisiae 2805-39-40, which contains about 40 copies of the XYL1 gene on the chromosome, was obtained by a sequential transformation using a dominant selection marker neo^r and an auxotrophic marker URA3. The multiple XYL1 genes were stably maintained on the chromosome even after 21 and 10 days in the non-selective sequential batch and chemostat cultures, respectively, whereas S. cerevisiae 2805:pVTXR, which harbors the episomal plasmid pVTXR having the XYL1 gene, showed mitotic plasmid instability and more than 95% of the cells lost the plasmid under the same culture conditions. In the first batch (3 days) of the sequential batch culture, volumetric xylitol productivity was 0.18 g l⁻¹ h⁻¹ for S. cerevisiae 2805-39-40, as compared to 0.21 g l⁻¹ h⁻¹ for S. cerevisiae 2805:pVTXR. However, the xylitol productivity of the latter started to decrease rapidly in the third batch and dropped to 0.04 g l⁻¹ h⁻¹ in the seventh batch, whereas the former maintained the stable xylitol productivity at 0.18 g l⁻¹ h⁻¹ through the entire sequential batch culture. The xylitol production level in the chemostat culture was about 8 g l⁻¹ for S. cerevisiae 2805-39-40, as compared to 2.0 g l⁻¹ for S. cerevisiae 2805:pVTXR after 10 days of cultures even though the xylitol production level of the latter was higher than that of the former for the first 5 days. The results of this experiment indicate that S. cerevisiae containing the multiple XYL1 genes on the chromosome is much more efficient for the xylitol production in the long-term non-selective culture than S. cerevisiae harboring the episomal plasmid containing the XYL1 gene.     

Factors affecting conjugative transfer rates of plasmids in batch and continuous cultures

Factors affecting the rates of plasmid transfer were investigated using Escherichia coli LC102 bearing a conjugative plasmid R100-1 and E. coli DH1. The rate constant of transconjugant increase, k_ti, was used for presenting the degree of plasmid transmissibility instead of the plasmid transfer efficiency (pte). The rate constant was defined as the specific rate of transconjugant increase (srti, the number of transconjugants per donor per h) divided by the recipient cell concentration. The k_ti values ranged between 10⁻¹⁰ and 10⁻¹⁵ ml cells⁻¹ h⁻¹, when estimated under various conditions. Moderate liquid agitation had a favorable effect on k_tf but agitation rates higher than 33 s⁻¹ (intergrated shear force) greatly decreased the value of k_ti. The transconjugant-forming activity of the cells growing in continuous culture did not significantly change with the dilution rate, except those growing at dilution rates less than 0.1 h⁻¹. The rate constant k_ti at temperatures of 10–15°C was as low as the detection limit (10⁻¹⁵ ml cells⁻¹ h⁻¹).     

Plasmid stability during continuous culture in aSaccharomyces cerevisiae double mutant transformed by a plasmid carrying a eukaryotic gene

Summary An investigation of plasmid stability in aSaccharomyces cerevisiae double mutant has been performed. The host was a double recombinantura3 furl mutant containing a plasmid bearing the yeast URA3⁺ allele and an expression cassette for human ₁-antitrypsin. The mutant was grown in continuous culture employing a semi-defined medium containing added uracil to provide non selective growth conditions. After 150 generations of continuous growth, no cured cells had been detected: the specific expression level of ₁-antitrypsin remained constant throughout the experiment.     

Effects of Pyruvate Decarboxylase Overproduction on Flux Distribution at the Pyruvate Branch Point in Saccharomyces cerevisiae

A multicopy plasmid carrying the PDC1 gene (encoding pyruvate decarboxylase; Pdc) was introduced in Saccharomyces cerevisiae CEN.PK113-5D. The physiology of the resulting prototrophic strain was compared with that of the isogenic prototrophic strain CEN.PK113-7D and an empty-vector reference strain. In glucose-grown shake-flask cultures, the introduction of the PDC1 plasmid caused a threefold increase in the Pdc level. In aerobic glucose-limited chemostat cultures growing at a dilution rate of 0.10 h⁻¹, Pdc levels in the overproducing strain were 14-fold higher than those in the reference strains. Levels of glycolytic enzymes decreased by ca. 15%, probably due to dilution by the overproduced Pdc protein. In chemostat cultures, the extent of Pdc overproduction decreased with increasing dilution rate. The high degree of overproduction of Pdc at low dilution rates did not affect the biomass yield. The dilution rate at which aerobic fermentation set in decreased from 0.30 h⁻¹ in the reference strains to 0.23 h⁻¹ in the Pdc-overproducing strain. In the latter strain, the specific respiration rate reached a maximum above the dilution rate at which aerobic fermentation first occurred. This result indicates that a limited respiratory capacity was not responsible for the onset of aerobic fermentation in the Pdc-overproducing strain. Rather, the results indicate that Pdc overproduction affected flux distribution at the pyruvate branch point by influencing competition for pyruvate between Pdc and the mitochondrial pyruvate dehydrogenase complex. In respiratory cultures (dilution rate, <0.23 h⁻¹), Pdc overproduction did not affect the maximum glycolytic capacity, as determined in anaerobic glucose-pulse experiments.     

Prodigiosin formation by Serratia marcescens in a chemostat

In a study of the control of metabolite formation, prodigiosin production by Serratia marcescens was used as a model. Specific production rates of prodigiosin formation were determined using batch culture technique. Sucrose as carbon source and NH₄NO₃ as nitrogen source resulted in a specific production rate of 0.476 mg prodigiosin (g cell dry weight)⁻¹ h⁻¹. Prodigiosin formation and productivity was inversely correlated to growth rate when the bacterium was grown under carbon limitation on a defined medium in a chemostat culture. The maximum specific growth rate (μ_max) was 0.54 h⁻¹ and prodigiosin was formed in amounts over 1 mg l⁻¹ up to a growth rate (μ) of 0.3 h⁻¹ at steady state conditions. At a dilution rate of 0.1 h⁻¹ growth at steady state with carbon and phosphate limitation supported prodigiosin formation giving a similar specific yield [1.17 mg prodigiosin (g cell dry weight)⁻¹ and 0.94 mg g⁻¹, respectively], however, cells grown with nitrogen limitation [(NH₄)₂SO₄] did not form prodigiosin. Productivity in batch culture was 1.33 mg l⁻¹ h⁻¹ as compared to 0.57 mg l⁻¹ h⁻¹ in the chemostat.     

A system for temperature-controlled expression of a foreign gene with dual mode in Saccharomyces cerevisiae

A temperature-controlled expression system for a foreign gene in Saccharomyces cerevisiae was constructed. In this system, a MATa hmlα2-102 HMRa sir3–8^ts double mutant was used as host, and a DNA fragment bearing the promoter and pre- or pre-pro regions of the MFα1 gene encoding the α-factor of S. cerevisiae was used as a promoter for expression of a foreign gene cloned on a vector. When the host cells were incubated at a restrictive temperature for the sir3–8^ts mutation (30°C to 35°C) they showed the α mating type and a PHO5 DNA fragment of S. cerevisiae, encoding repressible acid phosphatase, connected downstream of the MFα1 promoter was expressed. But when they were incubated at permissive lower temperature (25°C), at which they have the a mating type, the PHO5 DNA was not expressed. Acid phosphatase activity was increased 30-fold by shifting the incubation temperature from 25°C to 30°C. In this system it may also be possible to express a foreign gene at lower temperature but shut off its expression at higher temperature by connecting the gene to a promoter DNA of an a-specific gene.     

Continuous high-ethanol fermentation from cane molasses by a flocculating yeast

Repeated-batch fermentation by a flocculating fusant, Saccharomyces cerevisiae HA 2, was done in a molasses medium that contained 20% (w/v) total sugar, at 30°C in an automatically controlled fermentor, and the effects of ethanol concentration on the specific growth rate and the specific production rate of ethanol were studied. Both the specific growth rate and the specific production rate of ethanol fell with increase of ethanol concentration, and there was a linear correlation between each rate and the concentration of thanol. The maximum specific growth rate (μ_max) and the maximum specific production rate of ethanol (q_max) were 0.12 h⁻¹ and 0.1 g ethanol/10⁹ cells·h, respectively. The specific growth rate and the specific production rate of ethanol fell to zero at ethanol concentration of 89 g/l and 95 g/l, respectively. The number of viable cells, calculated from the linear inhibition equation, was 1.3 × 10⁹ cells/ml for production of 85 g/l ethanol at a dilution rate (D₁) of 0.2 h⁻¹. Based on this estimation, a laboratory-scale continuous fermentation, using two fermentors in series, was done. In the second fermentor, 85 g/l ethanol was produced at a dilution rate (D₁) of 0.2 h⁻¹ by the active feedig of the fermented mash from the first fermentor into the second fermentor by pumping (hereafter called active feeding). To maintain the number of viable cells above 10⁹ cells/ml in the second fermentor, a active feeding ratio of more than 23% was required. Under these conditions, 81 g/l ethanol was produced in the second fermentor at a dilution rate (D_t) of 0.25 h⁻¹, and the high ethanol productivity of 20.3 g/l·h could be achieved. A bench-scale continuous fermentation, using two fermentors in series, with a active feeding ratio of 25% was done. An ethanol concentration of 84 g/l in the second fermentor at a dilution rate (D_t) of 0.25 h⁻¹ was achieved, just as it was in the laboratory-scale fermentation test.     

Stable continuous constitutive expression of a heterologous protein in Saccharomyces cerevisiae without selection pressure

The stability of heterologous protein expression in Saccharomyces cerevisiae during continuous culture without selection for plasmid-containing cells was investigated. The protein chosen was the leech thrombin inhibitor desulphato-hirudin, which is tolerated well by S. cerevisiae when over-expressed. Expression was from a 2- derived multicopy vector containing a synthetic hirudin gene under control of the constitutive glyceraldehyde-3-phosphate dehydrogenase derived GAPFL promoter. The behaviour of the system was studied at three dilution rates (D) corresponding to approximately 30% (0.06 h^–1), 60% (0.12 h^–1) and 90% (0.17 h^–1) of the estimated maximum D. The level of plasmid loss was low at all Ds, with only 5–10% plasmid-free cells observed at 75 generations. The plasmid was most stably maintained at the intermediate D of 0.12 h^–1, where the rate of loss was comparable to the loss of the native 2- plasmid. Hirudin expression was also highest at D=0.12 h^–1, possibly as a result of cell lysis at D=0.06 h^–1 and D=0.17 h^–1, leading to the release of vacuolar proteases and subsequent proteolysis of hirudin. Differences in expression levels were not a result of changes in plasmid copy number, which was in the range 40–60 throughout all three experiments. The high stability of this system at all Ds investigated shows that heterologous protein expression is not a burden to S. cerevisiae when the protein expressed is tolerated well. Correspondence to: M. Ibba     

Effect of dilution rates on the amount of wasted substrate during alcohol fermentation

Continuous ethanol production in a one-stage continuous stirred tank fermentor without recycle was carried out using a yeast strain Saccharomyces cerevisiae. Different dilution rates were used. Cell and ethanol concentrations in the culture medium decreased with increasing dilution rates, and the maximum value of 3.0 g l⁻¹h⁻¹was found at a dilution rate of 0.340 h⁻¹. Specific ethanol productivities increased as dilution rates were increased, and the highest value appeared at about the same dilution rate as that for the maximum fermentor productivity. A material balance equation, which relates total amount of spent medium to cell synsthesis, ethanol production, and overall maintenance, was introduced. The cellular yield and overall maintenance coefficients increased with increasing dilution rates. The fraction of limiting substrate utilized for overall maintenance, which includes the limiting substrate spent for purposes other than cell synthesis and ethanol production, decreased with increasing dilution rates. The non-product associated substrate utilization can be minimized if correct dilution rate is chosen.     

The effect of decreasing oxygen feed rates on growth and metabolism of Torulaspora delbrueckii

The effect of decreasing oxygen feed rates on the growth and metabolism of Torulaspora delbrueckii and Saccharomyces cerevisiae in chemostat cultures was investigated. The biosynthetic oxygen requirement, i.e. the minimum specific oxygen consumption rate required for steady-state growth at a dilution rate of 0.10 h^–1, of T. delbrueckii was quantified to be less than 0.1 mmol O₂ g^–1 h^–1. Under strict anaerobiosis, washout of T. delbrueckii occurred, whereas for S. cerevisiae it did not. Under oxygen-limited conditions, the increase in fermentative ability of T. delbrueckii with diminishing oxygen supply was less pronounced than that of S. cerevisiae. These results indicate that T. delbrueckii was more disturbed in its energy balance than S. cerevisiae under strict anaerobiosis, and they may explain why T. delbrueckii exhibits poorer growth than S. cerevisiae under this condition.     

Isolation of a cDNA from Saccharomyces cerevisiae that encodes a high affinity sulphate transporter at the plasma membrane

Resistance to selenate and chromate, toxic analogues of sulphate, was used to isolate a mutant of Saccharomyces cerevisiae deficient in the capacity to transport sulphate into the cells. A clone which complements this mutation was isolated from a cDNA library prepared from S. cerevisiae poly(A)⁺ RNA. This clone contains an insert which is 2775 by in length and has a single open reading frame that encodes a 859 amino acid polypeptide with a molecular mass of 96 kDa. Sequence motifs within the deduced amino acid sequence of this cDNA (SUL1) show homology with conserved areas of sulphate transport proteins from other organisms. Sequence analysis predicts the position of 12 putative membrane spanning domains in SUL1. When the cDNA for SUL1 was expressed in S. cerevisiae, a high affinity sulphate uptake activity (K_m = 7.5 ± 0.6 μM for SO ₄ ^2? ) was observed. A genomic mutant of S. cerevisiae in which 1096 by were deleted from the SUL1 coding region was constructed. This mutant was unable to grow on media containing less than 5 mM sulphate unless complemented with a plasmid containing the SUL1 cDNA. We conclude that the SUL1 cDNA encodes a S. cerevisiae high affinity sulphate transporter that is responsible for the transfer of sulphate across the plasma membrane from the external medium.     

Optimisation and scale-up of α-glucuronidase production by recombinant Saccharomyces cerevisiae in aerobic fed-batch culture with constant growth rate

α-Glucuronidase (EC 3.2.1.139) of family GH 115 from Scheffersomyces stipitis is a valuable enzyme for the modification of water-soluble xylan into insoluble biopolymers, due to its unique ability to act on polymeric xylans. The influence of growth rate on the production of α-glucuronidase by recombinant Saccharomyces cerevisiae MH1000pbk10D-glu in glucose-limited fed-batch culture was studied at 14 and 100 L scale. At and below the critical specific growth rate (μ_crit) of 0.12 h⁻¹ at 14 L scale, the biomass yield coefficient (Y_x/s) remained constant at 0.4 g g⁻¹ with no ethanol production, whereas ethanol yields relative to biomass (k_eth/x) of up to 0.54 g g⁻¹ and a steady decrease in Y_x/s were observed at μ > 0.12 h⁻¹. Production of α-glucuronidase was growth associated at a product yield (k_α-glu/x) of 0.45 mg g⁻¹, with the highest biomass (37.35 g/L) and α-glucuronidase (14.03 mg/L) concentrations, were recorded during fed-batch culture at or near to μ_crit. Scale-up with constant k_La from 14 to 100 L resulted in ethanol concentrations of up to 2.5 g/L at μ = 0.12 h⁻¹. At this scale, α-glucuronidase yield could be maximised at growth rates below μ_crit, to prevent localised high glucose concentration pockets at the feed entry zone that would induce oxido-reductive metabolism. This is the first report where recombinant production of α-glucuronidase (EC 3.2.1.139) by S. cerevisiae was optimised for application at pilot scale.     

Production of l-Methionine-enriched cells of a mutant derived from a methylotrophic yeast,Candida boidinii

l-Methionine-enriched cells production of an ethionine-resistant mutant of Candida boidinii no. 2201 was greatly improved by the control of pH and by feeding of methanol and other medium components during cultivation in a jar fermentor. Under the optimal conditions, 38.5 g (as dry weight)_of cells abd 282 mg of pool methionine (intracellular pool of free l-methionine) per l of culture broth were obtained after 11 d of cultivation.The culture conditions for production of l-methionine-enriched cells in continuous culture were investigated. With limited methanol in continuous cultivation, pool methionine productivity reached a maximum value of 1.14 mg·l⁻¹·h⁻¹ at a dilution rate of 0.05·h⁻¹. During methanol-limited growth in continuous cultivation, the pool methionine content of the mutant was about 20–35% higher than that in batch cultivation.     

Fish red blood cell carbon dioxide transport in vitro: A comparative study

Red blood cell (rbc) carbon dioxide transport was examined in vitro in three teleosts (Oncorhynchus mykiss, Anguilla anguilla, Scophthalmus maximus) and an elasmobranch (Scyliorhinus canicula) using a radioisotopic assay that measures the net conversion of plasma HCO₃⁻ to CO₂. The experiments were designed to compare the intrinsic rates of rbc CO₂ excretion and the impact of haemoglobin oxygenation/deoxygenation among the species.Under conditions simulating in vivo levels of plasma HCO₃⁻ and natural haematocrits, the rate of whole blood CO₂ excretion varied between 14.0 μmol ml⁻¹ h⁻¹ (S. canicula) and 17.6 μmol ml⁻¹ h⁻¹ (O. mykiss). The rate of CO₂ excretion in separated plasma was significantly greater in the dogfish, S. canicula. The contribution of the rbc to overall whole blood CO₂ excretion was low in the dogfish (46 ± 6%) compared to the teleosts (trout, 71 ± 4%; turbot, 64 ± 5%; eel, 55 ± 3%).To eliminate the naturally occurring differences in haematocrit and plasma [HCO₃⁻] as inter-specific variables, the rates of whole blood CO₂ excretion were determined in blood that had been resuspended to constant [HCO₃⁻] (5 mmol⁻¹) and haematocrit (20%) in appropriate teleost and elasmobranch Ringer solutions. Under such normalized conditions, the rate of whole blood CO₂ excretion was significantly higher in the turbot (22.4 ± 1.3 μmol ml⁻¹ h⁻¹) in comparison to the other species (16.4–18.4 μmol ml⁻¹ h⁻¹) and thus revealed a greater intrinsic rate of rbc CO₂ excretion in the turbot.To study the contribution of Bohr protons, the rates of whole blood CO₂ excretion were assessed in blood subjected to rapid oxygenation during the initial phase of the 3 min assay period. Rapid oxygenation significantly enhanced the rate of CO₂ excretion in the teleosts but not in the elasmobranch. The extent of the increase provided by the rapid oxygenation of haemoglobin was a linear function of the extent of the Haldane effect, as quantified in each species from in vitro CO₂ dissociation (combining) curves. Under steady-state conditions, deoxygenated blood exhibited greater rates of CO₂ excretion than oxygenated blood in the teleosts but not in the elasmobranch. As a consequence of the Haldane effect, rbc intracellular pH was increased in the teleosts by deoxygenation but was unaltered in the elasmobranch.The results, by extrapolation, suggest that the rates of CO₂ excretion in vivo are influenced by the magnitude of the Haldane effect and the extent of haemoglobin oxygenation during gill transit in addition to the intrinsic rate at which the rbc converts plasma HCO₃⁻ to CO₂.     

Cell mass production of Acinetobacter calcoaceticus in palm oil-limited chemostat culture

Chemostat culture of Acinetobacter calcoaceticus KB-2 was done under palm oil-limiting conditions for cell production, and variation of cell compositions and yield coefficients were investigated in connection with the specific growth rates. At the concentration of 0.6% palm oil, the productivity of cells and yield coefficient were 4.76 g cells/l/h and 1.18 g cells/g palm oil, respectively, at a practical dilution rate of 0.85 h⁻¹. About 80% of the palm oil was assimilated by the strain, and the maintenance coefficient was 0.035 g palm oil/g cells/h. Although the carbohydrate content remained essentially constant when the growth rate was varied, the lipid, protein, and nucleic acid contents were increased slightly at higher growth rates. Although the protein content increased only 3%, the protein yield coefficient (Y_p) increased about 1.5 times over the range of specific growth rates between 0.1 and 0.7 h⁻¹. The increase in Y_p was due to the higher protein content of the biomass and to higher values of the cell yield coefficient.     

Development of an industrial ethanol-producing yeast strain for efficient utilization of cellobiose

The BGL1 gene, encoding β-glucosidase in Saccharomycopsis fibuligera, was intracellular, secreted or cell-wall associated expressed in an industrial strain of Saccharomyces cerevisiae. The obtained recombinant strains were studied under aerobic and anaerobic conditions. The results indicated that both the wild type and recombinant strain expressing intracellular β-glucosidase cannot grow in medium using cellobiose as sole carbon source. As for the recombinant EB1 expressing secreted enzyme and WB1 expressing cell-wall associated enzyme, the maximum specific growth rates (μ_max) could reach 0.03 and 0.05 h⁻¹ under anaerobic conditions, respectively. Meanwhile, the surface-engineered S. cerevisiae utilized 5.2 g cellobiose L⁻¹ and produced 2.3 g ethanol L⁻¹ in 48 h, while S. cerevisiae secreting β-glucosidase into culture broth used 3.6 g cellobiose L⁻¹ and produced 1.5 g ethanol L⁻¹ over the same period, but no-full depletion of cellobiose were observed for both the used recombinant strains. The results suggest that S. cerevisiae used in industrial ethanol production is deficient in cellobiose transporter. However, when β-glucoside permease and β-glucosidase were co-expressed in this strain, it could uptake cellobiose and showed higher growth rate (0.11 h⁻¹) on cellobiose.     

ZIP4H (TEX11) deficiency in the mouse impairs meiotic double strand break repair and the regulation of crossing over

We have recently shown that hypomorphic Mre11 complex mouse mutants exhibit defects in the repair of meiotic double strand breaks (DSBs). This is associated with perturbation of synaptonemal complex morphogenesis, repair and regulation of crossover formation. To further assess the Mre11 complex's role in meiotic progression, we identified testis-specific NBS1-interacting proteins via two-hybrid screening in yeast. In this screen, Zip4h (Tex11), a male germ cell specific X-linked gene was isolated. Based on sequence and predicted structural similarity to the S. cerevisiae and A. thaliana Zip4 orthologs, ZIP4H appears to be the mammalian ortholog. In S. cerevisiae and A. thaliana, Zip4 is a meiosis-specific protein that regulates the level of meiotic crossovers, thus influencing homologous chromosome segregation in these organisms. As is true for hypomorphic Nbs1 (Nbs1^ΔB/ΔB) mice, Zip4h^−/Y mutant mice were fertile. Analysis of spermatocytes revealed a delay in meiotic double strand break repair and decreased crossover formation as inferred from DMC1 and MLH1 staining patterns, respectively. Achiasmate chromosomes at the first meiotic division were also observed in Zip4h^−/Y mutants, consistent with the observed reduction in MLH1 focus formation. These results indicate that meiotic functions of Zip4 family members are conserved and support the view that the Mre11 complex and ZIP4H interact functionally during the execution of the meiotic program in mammals.     

Continuous ethanol production by yeast immobilized on to channeled alumina beads

Vertical and near-horizontal (15° angle) packed-bed columns were compared for continuous ethanol fermentation using an alcohol- and glucose-tolerant Saccharomyces cerevisiae strain immobilized on to channeled alumina beads (5·0 × 10⁹ cells g⁻¹ beads). Spaces between beads (1·0–6·5 mm) and angle (15°) of near-horizontal reactor columns (with six ports in each) efficiently removed CO₂ and increased ethanol productivity. Malt-glucose-yeast-extract broth containing 16·7% glucose at 35°C fed at a dilution rate of 3· h⁻¹ to thw two horizontal columns (in series) yielded maximum ethanol productivity of 40·0 g liter⁻¹ h⁻¹. Feedstock flow rate and other factors (temperature, pH, nutrients, and glucose levels) affected productivities. The immobilized-cell system showed operational stability for >3 months without plugging, and could be stored for at least one year with no loss of bioreactor performance. Scanning electron micrographs of the beads revealed large numbers of yeast-cells attached on to internal and external surfaces of beads.     

Growth kinetics and Pho84 phosphate transporter activity of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> under phosphate-limited conditions

The effect of phosphate (P _i ) concentration on the growth behavior of Saccharomyces cerevisiae strain CEN.PK113-5D in phosphate-limited batch and chemostat cultures was studied. The range of dilution rates used in the present study was 0.08–0.45 h⁻¹. The batch growth of yeast cells followed Monod relationship, but growth of the cells in phosphate-limited chemostat showed change in growth kinetics with increasing dilution rates. The difference in growth kinetics of the yeast cells in phosphate-limited chemostat for dilution rates below and above approximately 0.2 h⁻¹ has been discussed in terms of the batch growth kinetic data and the change in the metabolic activity of the yeast cells. Immunological detection of a C-terminally myc epitope-tagged Pho84 fusion protein indicated derepressive expression of the Pho84 high-affinity P _i transporter in the entire range of dilution rates employed in this study. Phosphate transport activity mediated by Pho84 transporter was highest at very low dilution rates, i.e. 0.08–0.1 h⁻¹, corresponding to conditions in which the amount of synthesized Pho84 was at its maximum.