Continuous cultivation of the yeastSaccharomyces cerevisiae at different dilution rates and glucose concentrations in nutrient media

The influence of glucose concentration in nutrient media on the specific growth rate and biomass yield in the course of continuous fermentation ofSaccharomyces cerevisiae was investigated. An increase of glucose content in media decreased the specific growth rate and the biomass yield. Glucose concentration had significant effects on protein and phosphate contents of cells. However, an increased glucose concentration increased the fermentative power ofS. cerevisiae (SJA-method). An increase of the dilution rate decreased the cell concentration in the fermentor. Specific growth rate approached the values of the dilution rate. The best agreement has been obtained at a dilution rate of 0.20/h. This dilution rate proved to be most convenient for the investigated microorganism and cultivation conditions (media composition, pH, aeration intensity and temperature). Biomass yield proved to be decreased by an increase of the dilution rate.     

Growth and adaptation of Saccharomyces cerevisiae at different cadmium concentrations

The effect of 0, 5, 10 and 25 mg l-1 cadmium on the growth of Saccharomyces cerevisiae in defined medium has been investigated. It was found that the length of the lag phase increased with cadmium concentration and that metal uptake during the lag phase occurred only at a cadmium concentration of 25 mg l-1. However, metal uptake occurred at all cadmium concentrations during the exponential phase. The yeast was gradually adapted to cadmium by a series of subcultures which resulted in a decrease in the length of the lag phase. Adaptation also caused a reduction in the cadmium uptake during the lag phase at 25 mg l-1 cadmium but did not affect uptake during the exponential phase at any concentration. A single passage through cadmium-free medium partially reversed the adaptation process. Sulphide production was enhanced significantly when the yeast was grown in the presence of increasing cadmium concentrations. However, at 5 mg l-1 cadmium, adapted cells produced less sulphide than unadapted cells, whilst at 10 and 25 mg l-1 cadmium the production of sulphide was similar for adapted and unadapted cells.     

Transcriptional responses to glucose at different glycolytic rates in Saccharomyces cerevisiae.

The addition of glucose to Saccharomyces cerevisiae cells causes reprogramming of gene expression. Glucose is sensed by membrane receptors as well as (so far elusive) intracellular sensing mechanisms. The availability of four yeast strains that display different hexose uptake capacities allowed us to study glucose-induced effects at different glycolytic rates. Rapid glucose responses were observed in all strains able to take up glucose, consistent with intracellular sensing. The degree of long-term responses, however, clearly correlated with the glycolytic rate: glucose-stimulated expression of genes encoding enzymes of the lower part of glycolysis showed an almost linear correlation with the glycolytic rate, while expression levels of genes encoding gluconeogenic enzymes and invertase (SUC2) showed an inverse correlation. Glucose control of SUC2 expression is mediated by the Snf1-Mig1 pathway. Mig1 dephosphorylation upon glucose addition is known to lead to repression of target genes. Mig1 was initially dephosphorylated upon glucose addition in all strains able to take up glucose, but remained dephosphorylated only at high glycolytic rates. Remarkably, transient Mig1-dephosphorylation was accompanied by the repression of SUC2 expression at high glycolytic rates, but stimulated SUC2 expression at low glycolytic rates. This suggests that Mig1-mediated repression can be overruled by factors mediating induction via a low glucose signal. At low and moderate glycolytic rates, Mig1 was partly dephosphorylated both in the presence of phosphorylated, active Snf1, and unphosphorylated, inactive Snf1, indicating that Mig1 was actively phosphorylated and dephosphorylated simultaneously, suggesting independent control of both processes. Taken together, it appears that glucose addition affects the expression of SUC2 as well as Mig1 activity by both Snf1-dependent and -independent mechanisms that can now be dissected and resolved as early and late/sustained responses.     

Continuous cultivation of Saccharomyces cerevisiae at different biotin concentrations in nutrient media

An increase of biotin concentration in nutrient media increased the content of protein, phosphorus, total ribonucleic acids, activity of pyruvate carboxylase and isocitrate lyase in cells and decreased the content of trehalose, glycogen and respiratory quotient of yeast cells in the course of continuous cultivation of Saccharomyces cerevisiae.     

Increased ethanol productivity in xylose-utilizing Saccharomyces cerevisiae via a randomly mutagenized xylose reductase

Baker's yeast (Saccharomyces cerevisiae) has been genetically engineered to ferment the pentose sugar xylose present in lignocellulose biomass. One of the reactions controlling the rate of xylose utilization is catalyzed by xylose reductase (XR). In particular, the cofactor specificity of XR is not optimized with respect to the downstream pathway, and the reaction rate is insufficient for high xylose utilization in S. cerevisiae. The current study describes a novel approach to improve XR for ethanol production in S. cerevisiae. The cofactor binding region of XR was mutated by error-prone PCR, and the resulting library was expressed in S. cerevisiae. The S. cerevisiae library expressing the mutant XR was selected in sequential anaerobic batch cultivation. At the end of the selection process, a strain (TMB 3420) harboring the XR mutations N272D and P275Q was enriched from the library. The V(max) of the mutated enzyme was increased by an order of magnitude compared to that of the native enzyme, and the NADH/NADPH utilization ratio was increased significantly. The ethanol productivity from xylose in TMB 3420 was increased ～40 times compared to that of the parent strain (0.32 g/g [dry weight {DW}] × h versus 0.007 g/g [DW] × h), and the anaerobic growth rate was increased from ～0 h(-1) to 0.08 h(-1). The improved traits of TMB 3420 were readily transferred to the parent strain by reverse engineering of the mutated XR gene. Since integrative vectors were employed in the construction of the library, transfer of the improved phenotype does not require multicopy expression from episomal plasmids.     

Adaptation of a recombinant xylose-utilizing Saccharomyces cerevisiae strain to a sugarcane bagasse hydrolysate with high content of fermentation inhibitors

Adaptation of a xylose-utilizing genetically engineered strain of Saccharomyces cerevisiae to sugarcane bagasse hydrolysates by cultivation during 353h using medium with increasing concentrations of inhibitors, including phenolic compounds, furaldehydes and aliphatic acids, led to improved performance with respect to ethanol production. The remaining xylose concentration in the medium at the end of the cultivation was 5.2g l(-1), while it was 11gl(-1) in the feed, indicating that approximately half of the xylose was consumed. The performance of the adapted strain was compared with the parental strain with respect to its ability to ferment three bagasse hydrolysates with different inhibitor concentration. The ethanol yield after 24h of fermentation of the bagasse hydrolysate with lowest inhibitor concentration increased from 0.18gg(-1) of total sugar with the non-adapted strain to 0.38gg(-1) with the adapted strain. The specific ethanol productivity increased from 1.15g ethanol per g initial biomass per h with the non-adapted strain to 2.55gg(-1) h(-1) with the adapted strain. The adapted strain performed better than the non-adapted also in the two bagasse hydrolysates containing higher concentrations of inhibitors. The adapted strain converted the inhibitory furaldehydes 2-furaldehyde (furfural) and 5-hydroxymethyl-2-furaldehyde (HMF) at a faster rate than the non-adapted strain. The xylose-utilizing ability of the yeast strain did not seem to be affected by the adaptation and the results suggest that ethanol rather than xylitol was formed from the consumed xylose.     

Ethanol production with Saccharomyces cerevisiae under aerobic conditions at different potassium concentrations

The specific ethanol productivity with Saccharomyces cerevisiae grown aerobicly in a chemostat was found to be highly dependent on the ratio of intracellular to extracellular potassium concentration through variations in the energy consumption used for maintenance of the concentration gradient of potassium across the cell membrane. The specific ethanol productivity progressively rose from 0 to 20 mmol h(-1) g(-1) cell dry matter at a growth rate of 0.17 h(-1) when the ratio of intracellular to extracellular potassium concentration was increased from 10 to 80. The ethanol production under potassium limited growth conditions was caused neither by a reduction in the specific respiratory activity nor by variations in the potassium content in cell dry matter. Results which strongly that ethanol production under potassium limited growth conditions is brought about by changes in the ratio of pyruvate oxidase to pyruvate decarboxylase activity through changes in the intracellular pyruvate concentration are presented.     

Reduced oxidative pentose phosphate pathway flux in recombinant xylose-utilizing Saccharomyces cerevisiae strains improves the ethanol yield from xylose

In recombinant, xylose-fermenting Saccharomyces cerevisiae, about 30% of the consumed xylose is converted to xylitol. Xylitol production results from a cofactor imbalance, since xylose reductase uses both NADPH and NADH, while xylitol dehydrogenase uses only NAD(+). In this study we increased the ethanol yield and decreased the xylitol yield by lowering the flux through the NADPH-producing pentose phosphate pathway. The pentose phosphate pathway was blocked either by disruption of the GND1 gene, one of the isogenes of 6-phosphogluconate dehydrogenase, or by disruption of the ZWF1 gene, which encodes glucose 6-phosphate dehydrogenase. Decreasing the phosphoglucose isomerase activity by 90% also lowered the pentose phosphate pathway flux. These modifications all resulted in lower xylitol yield and higher ethanol yield than in the control strains. TMB3255, carrying a disruption of ZWF1, gave the highest ethanol yield (0.41 g g(-1)) and the lowest xylitol yield (0.05 g g(-1)) reported for a xylose-fermenting recombinant S. cerevisiae strain, but also an 84% lower xylose consumption rate. The low xylose fermentation rate is probably due to limited NADPH-mediated xylose reduction. Metabolic flux modeling of TMB3255 confirmed that the NADPH-producing pentose phosphate pathway was blocked and that xylose reduction was mediated only by NADH, leading to a lower rate of xylose consumption. These results indicate that xylitol production is strongly connected to the flux through the oxidative part of the pentose phosphate pathway.     

Biofilm build-up of Pseudomonas putida in a chemostat at different dilution rates

Summary A test system was set up where the build-up of a biofilm on a defined surface could be studied in a carbon source limited chemostat.The attachment of P. putida ATCC 11172 to glass when growing on L-asparagine was studied at different dilution rates (specific growth rates) from 0.1 to 1.5 h^–1 The number of attached colony forming units (cfu) increased with dilution rate from 1×10⁶ cfu/cm² at 0.1 h^–1 to 4×10⁷ cfu/cm² at 1.0 h^–1 and then the attachment decreased to about 6×10⁶ cfu/cm² at higher dilution rates (1.1–1.5 h^–1). The number of attached cfu was measured after 24 h exposure. The value of the maximum specific growth rate in batch culture was 0.6 h^–1.The total amount of attached cell-mass followed roughly the same pattern as the viable count.The viable count of the cells suspended in the growth medium showed its lowest value at the same dilution rate as resulted in maximum adhesion.It was shown that the effect of growth rate on the biofilm build-up of P. putida is significant, and ought to be borne in mind when continuous culture systems are set up and results evaluated.     

Generation of the improved recombinant xylose-utilizing Saccharomyces cerevisiae TMB 3400 by random mutagenesis and physiological comparison with Pichia stipitis CBS 6054

The recombinant xylose-utilizing Saccharomyces cerevisiae TMB 3399 was constructed by chromosomal integration of the genes encoding D-xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase (XK). S. cerevisiae TMB 3399 was subjected to chemical mutagenesis with ethyl methanesulfonate and, after enrichment, 33 mutants were selected for improved growth on D-xylose and carbon dioxide formation in Durham tubes. The best-performing mutant was called S. cerevisiae TMB 3400. The novel, recombinant S. cerevisiae strains were compared with Pichia stipitis CBS 6054 through cultivation under aerobic, oxygen-limited, and anaerobic conditions in a defined mineral medium using only D-xylose as carbon and energy source. The mutation led to a more than five-fold increase in maximum specific growth rate, from 0.0255 h(-1) for S. cerevisiae TMB 3399 to 0.14 h(-1) for S. cerevisiae TMB 3400, whereas P. stipitis grew at a maximum specific growth rate of 0.44 h(-1). All yeast strains formed ethanol only under oxygen-limited and anaerobic conditions. The ethanol yields and maximum specific ethanol productivities during oxygen limitation were 0.21, 0.25, and 0.30 g ethanol g xylose(-1) and 0.001, 0.10, and 0.16 g ethanol g biomass(-1) h(-1) for S. cerevisiae TMB 3399, TMB 3400, and P. stipitis CBS 6054, respectively. The xylitol yield under oxygen-limited and anaerobic conditions was two-fold higher for S. cerevisiae TMB 3399 than for TMB 3400, but the glycerol yield was higher for TMB 3400. The specific activity, in U mg protein(-1), was higher for XDH than for XR in both S. cerevisiae TMB 3399 and TMB 3400, while P. stipitis CBS 6054 showed the opposite relation. S. cerevisiae TMB 3400 displayed higher specific XR, XDH and XK activities than TMB 3399. Hence, we have demonstrated that a combination of metabolic engineering and random mutagenesis was successful to generate a superior, xylose-utilizing S. cerevisiae, and uncovered distinctive physiological properties of the mutant.     

Intracellular Signal Triggered by Cholera Toxin in Saccharomyces boulardii and Saccharomyces cerevisiae

As is the case for Saccharomyces boulardii, Saccharomyces cerevisiae W303 protects Fisher rats against cholera toxin (CT). The addition of glucose or dinitrophenol to cells of S. boulardii grown on a nonfermentable carbon source activated trehalase in a manner similar to that observed for S. cerevisiae. The addition of CT to the same cells also resulted in trehalase activation. Experiments performed separately on the A and B subunits of CT showed that both are necessary for activation. Similarly, the addition of CT but not of its separate subunits led to a cyclic AMP (cAMP) signal in both S. boulardii and S. cerevisiae. These data suggest that trehalase stimulation by CT probably occurred through the cAMP-mediated protein phosphorylation cascade. The requirement of CT subunit B for both the cAMP signal and trehalase activation indicates the presence of a specific receptor on the yeasts able to bind to the toxin, a situation similar to that observed for mammalian cells. This hypothesis was reinforced by experiments with ¹²⁵I-labeled CT showing specific binding of the toxin to yeast cells. The adhesion of CT to a receptor on the yeast surface through the B subunit and internalization of the A subunit (necessary for the cAMP signal and trehalase activation) could be one more mechanism explaining protection against the toxin observed for rats treated with yeasts.     

RNA and protein elongation rates in Saccharomyces cerevisiae

Summary The RNA elongation rate has been measured in yeast by the kinetics of appearance of radioactivity in the different molecular weight classes by the method first developed by Bremer and Yuan (1968). Despite the limitations caused by the breakdown of the 35s rRNA precursor, an estimate of 29 to 38 nucleotides/second at 30° has been obtained for the RNA elongation rate. The protein elongation rate has been calculated by the method of Maaløe and Kjeldgaard (1966) which consists of dividing the number of amino acids polymerized into protein per unit of time by the number of active ribosomes. This has given values of 7 to 9 amino acids/second at 30°.These numbers are of the same order as those found in Escherichia coli when corrected to 37°. Eucaryotic cells could thus have preserved part of the coupling found in bacteria between RNA and protein elongation rates.     

Nitrification rates in a saline medium at different dissolved oxygen concentrations

A kinetic study was carried out in a saline medium to assess the effect of O₂ on the two-step nitrification process: for the first nitritation step, 2–26 mg dissolved O₂ (DO) l^–1 was used and for the second nitratation step, 0.5–24 mg DO l^–1 was used. Nitritation rate was measured in the presence of sodium azide so as to inhibit nitratation. Ammonia-oxidizing (AOB) and nitrite-oxidizing (NOB) bacterial in the mixed culture were determined by 16 S rRNA hybridization. The affinity constants for oxygen O₂ of the AOB and the NOB were 1.66 mg O₂ l^–1 and 3 mg O₂ l^–1 respectively. The larger than the previously reported values of these constants might be due to the high salt content in the medium. High O₂ concentrations did inhibit the nitrification rate.     

Fine-Structure Analysis of Intercellular and Intracellular Mitochondrial Diversity in Saccharomyces cerevisiae

Crosses were made between haploid wild-type and suppressive petite strains of bakers' yeast to obtain zygotes for analysis of mitochondrial heterogeneity. Wild-type x petite zygotes contained about 40% noncristate mitochondria when immediate mating mixtures were examined. The frequency of defective mitochondria had decreased to an average of 9.2% in 1-week-old zygote isolate cultures, and to 4.4% in slant cultures 1.5 years after initial zygote isolation. The latter value was not significantly different from values obtained with wild x wild zygotes of either age. The noncristate mitochondria were of two types: one lacking inner membrane invaginations or elaborations and the other containing concentrically arranged loops of inner membrane. The significance of these two types of respiration-deficient mitochondria is unknown. The gradual decrease in frequency of noncristate mitochondria, perhaps due to selection pressures in mixed chondriomes, was discussed as a further indication of the semiautonomous nature of the yeast organelle.     

Production of hirudin by recombinant Saccharomyces cerevisiae in a membrane-recycle fermentor

Summary Recombinant Saccharomyces cerevisiae was employed to continuously produce hirudin in a membrane cell recycle fermentor. The gene cooing for the anticoagulant protein was combined with the GAL10 promoter for controlled expression and the MF 1 signal sequence for secretion to the fermentation broth. A dilution rate of 0.1h^–1 yielded a maximum hirudin concentration of 59mg / l with a specific hirudin concentration of 2.4 mg /g cell mass among dilution rates studied ranging from 0.05h^–1 to 0.3h^–1. Cell bleeding gave the same fermentation results as cell recycle fermentation without cell bleeding. The productivity of the cell recycle fermentation process was 6.0mg hirudin/l · hr, corresponding to a 1.7-fold increase compared with a conventional continuous culture.     

不同宿主来源的重组酿酒酵母混合糖代谢比较

【目的】研究不同工业酿酒酵母宿主背景对重组酵母木糖利用效率的影响。【方法】将木糖利用途径的木糖还原酶(XR)、木糖醇脱氢酶(XDH)和木酮糖激酶(XK)编码基因串联后分别转入3株不同的工业酿酒酵母中,得到重组酵母ZQ1、ZQ5和ZQ7。分别对3个木糖途径代谢基因的表达水平、酶活和重组菌株的木糖发酵效率进行比较。【结果】重组菌株在木糖代谢基因转录、酶活性和木糖利用性能方面有很大差异,其中ZQ5木糖代谢能力最强,ZQ7其次,ZQ1木糖利用能力最弱。ZQ7在初始木糖浓度为20 g/L时木糖利用速率快于ZQ5,表明木糖浓度对重组菌发酵性能评价具有影响。【结论】不同菌株的遗传背景和木糖浓度对重组菌木糖利用的影响很大,评价重组酵母的木糖利用需考虑宿主的遗传背景和底物浓度的影响。