Ethanol-inducible gene expression using gld1 + promoter in the fission yeast Schizosaccharomyces pombe

In the fission yeast Schizosaccharomyces pombe, the gld1 ⁺ gene encoding glycerol dehydrogenase is repressed by glucose and induced by ethanol and 1-propanol. The promoter region of gld1 ⁺ was cloned into a multicopy vector designated as pEG1 for evaluation as an ethanol-inducible expression vector using EGFP as a model heterologous protein. Expression of EGFP was repressed in the presence of high glucose and induced in the presence of ethanol, low-glucose, and 1-propanol in the absence of glucose. Addition of ethanol to cells harboring pEG1–EGFP was found to be the most effective means for inducing EGFP production. Protein yields were found to increase in proportion to ethanol concentration. As a further test of effectiveness, secreted recombinant human growth hormone was produced using the pEG1 expression vector in medium containing glycerol and ethanol. The pEG1 gene expression system is an effective tool for the production of heterologous proteins under glucose-limiting conditions, including medium containing glycerol as a carbon source.     

The gld1 + gene encoding glycerol dehydrogenase is required for glycerol metabolism in Schizosaccharomyces pombe

The budding yeast Saccharomyces cerevisiae is able to utilize glycerol as the sole carbon source via two pathways (glycerol 3-phosphate pathway and dihydroxyacetone [DHA] pathway). In contrast, the fission yeast Schizosaccharomyces pombe does not grow on media containing glycerol as the sole carbon source. However, in the presence of other carbon sources such as galactose and ethanol, S. pombe could assimilate glycerol and glycerol was preferentially utilized over ethanol and galactose. No equivalent of S. cerevisiae Gcy1/glycerol dehydrogenase has been identified in S. pombe. However, we identified a gene in S. pombe, SPAC13F5.03c (gld1 ⁺), that is homologous to bacterial glycerol dehydrogenase. Deletion of gld1 caused a reduction in glycerol dehydrogenase activity and prevented glycerol assimilation. The gld1Δ cells grew on 50 mM DHA as the sole carbon source, indicating that the glycerol dehydrogenase encoded by gld1 ⁺ is essential for glycerol assimilation in S. pombe. Strains of S. pombe deleted for dak1 ⁺ and dak2 ⁺ encoding DHA kinases could not grow on glycerol and showed sensitivity to a higher concentration of DHA. The dak1Δ strain showed a more severe reduction of growth on glycerol and DHA than the dak2Δ strain because the expression of dak1 ⁺ mRNA was higher than that of dak2 ⁺. In wild-type S. pombe, expression of the gld1 ⁺, dak1 ⁺, and dak2 ⁺ genes was repressed at a high concentration of glucose and was derepressed during glucose starvation. We found that gld1 ⁺ was regulated by glucose repression and that it was derepressed in scr1Δ and tup12Δ strains.     

Respiratory metabolism of a "petite negative"yeast Schizosaccharomyces pombe 972h-

The respiratory metabolism of Schizosaccharomyces pombe 972h(-), a fission, haplontic, "petite negative" yeast, was studied. Glucose and glycerol are good growth substrates and are oxidized under appropriate conditions. l-Lactate, ethanol, malate, and succinate are oxidized but are poor substrates for growth. d-Lactate and pyruvate are neither oxidized nor used for growth. Limited growth was observed under anaerobic conditions. The addition of 0.3% KNO(3) to a rich medium relieves the oxygen requirement. A continuous increase of cell respiration during growth on repressive concentration of glucose was observed, suggesting the presence of glucose repression of respiration. Reduced nicotinamide adenine dinucleotide (NADH), succinate, alpha-glycerophosphate, and ascorbate plus tetramethyl-p-phenylenediamine are oxidized by a mitochondrial fraction. NADH and succinate oxidations are inhibited by antimycin A and NaCN but not by rotenone, suggesting the absence of the phosphorylation site I and the presence of sites II and III. The effects of several mitochondrial inhibitors on growth and respiration indicate that the requirement of an oxidant for growth is related neither to the functioning of the respiratory electron transport chain nor to the formation of respiratory energy. The previously suggested correlations between the nonviability of vegetative "petites" mutants, the absence of repression of respiration by glucose, and the incapacity to grow under anaerobic conditions are thus not strictly valid for S. pombe.     

Role of growth phase and ethanol in freeze-thaw stress resistance of Saccharomyces cerevisiae.

The freeze-thaw tolerance of Saccharomyces cerevisiae was examined throughout growth in aerobic batch culture. Minimum tolerance to rapid freezing (immersion in liquid nitrogen; cooling rate, approximately 200 degrees C min-1) was associated with respirofermentative (exponential) growth on glucose. However, maximum tolerance occurred not during the stationary phase but during active respiratory growth on ethanol accumulated during respirofermentative growth on glucose. The peak in tolerance occurred several hours after entry into the respiratory growth phase and did not correspond to a transient accumulation of trehalose which occurred at the point of glucose exhaustion. Substitution of ethanol with other carbon sources which permit high levels of respiration (acetate and galactose) also induced high freeze-thaw tolerance, and the peak did not occur in cells shifted directly from fermentative growth to starvation conditions or in two respiratorily incompetent mutants. These results imply a direct link with respiration, rather than exhaustion of glucose. The role of ethanol as a cryoprotectant per se was also investigated, and under conditions of rapid freezing (cooling rate, approximately 200 degrees C min-1), ethanol demonstrated a significant cryoprotective effect. Under the same freezing conditions, glycerol had little effect at high concentrations and acted as a cryosensitizer at low concentrations. Conversely, under slow-freezing conditions (step freezing at -20, -70, and then -196 degrees C; initial cooling rate, approximately 3 degrees C min-1), glycerol acted as a cryoprotectant while ethanol lost this ability. Ethanol may thus have two effects on the cryotolerance of baker's yeast, as a respirable carbon source and as a cryoprotectant under rapid-freezing conditions.     

Fermentation of glycerol to 1,3-propanediol: use of cosubstrates

Three fermentable substances, glucose, 1,2-ethanediol and 1,2-propanediol were checked as cosubstrates for the fermentation of glycerol by Clostridium butyricum and Citrobacter freundii with the aim of achieving a complete conversion of glycerol to 1,3-propanediol. Glucose was fermented by C. butyricum mainly to acetate, CO₂ and reducing equivalents in the presence of glycerol and contributed markedly to the 1,3-propanediol yield. However, because of relatively slow growth on glucose, complete conversion was not achieved. If the two glycols were used as cosubstrates for glycerol fermentation, the 1,3-propanediol yield did not increase but dimished considerably, as they were converted to more reduced products, i.e. alcohols instead of acids. From 1,2-propanediol 2-propanol was formed in addition to 1-propanol. The ratio of the propanols was dependent on the culture conditions.     

Rhizomorph Formation in Fungi

The effect on growth and rhizomorph formation of 12 alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, iso-butyl alcohol, tert-butyl alcohol, 1-pentanol, iso-amyl alcohol, ethylene glycol and glycerol) at different concentrations has been examined for 2 isolates of Armillaria mellea (Vahl ex Fr.) Quél. and 1 of Clitocybe geotropa (Bull. ex Fr.) Quél. The fungi were cultivated for 28 days on a synthetic, liquid glucose medium with the alcohols as supplement. The following alcohols strongly stimulated growth and rhizomorph formation: ethanol, 1-propanol and 1-butanol. A great variation was demonstrated between the isolates in relation to rhizomorph production, morphology, and ability to be stimulated by different alcohols.     

Regulation of sugar transport in Neurospora crassa

Sugar uptake systems in Neurospora crassa are catabolically repressed by glucose. Synthesis of a low K(m) glucose uptake system (system II) in Neurospora is derepressed during starvation for an externally supplied source of carbon and energy. Fasting also results in the derepression of uptake systems for fructose, galactose, and lactose. In contrast to the repression observed when cells were grown on glucose, sucrose, or fructose, system II was not repressed by growth on tryptone and casein hydrolysate. System II was inactivated in the presence of 0.1 m glucose and glucose plus cycloheximide but not by cycloheximide alone. Inactivation followed first-order kinetics with a half-time of 40 min. The addition of glycerol to the uptake medium had no significant effect on the kinetics of 3-0-methyl glucose uptake, suggesting that the system was not feedback inhibitable by catabolites of glycerol metabolism.     

Effect of Nutrient Starvation on the Cellular Composition and Metabolic Capacity of Saccharomyces cerevisiae

This investigation addresses the following question: what are the important factors for maintenance of a high catabolic capacity under various starvation conditions? Saccharomyces cerevisiae was cultured in aerobic batch cultures, and during the diauxic shift cells were transferred and subjected to 24 h of starvation. The following conditions were used: carbon starvation, nitrogen starvation in the presence of glucose or ethanol, and both carbon starvation and nitrogen starvation. During the starvation period changes in biomass composition (including protein, carbohydrate, lipid, and nucleic acid contents), metabolic activity, sugar transport kinetics, and the levels of selected enzymes were recorded. Subsequent to the starvation period the remaining catabolic capacity was measured by addition of 50 mM glucose. The results showed that the glucose transport capacity is a key factor for maintenance of high metabolic capacity in many, but not all, cases. The results for cells starved of carbon, carbon and nitrogen, or nitrogen in the presence of glucose all indicated that the metabolic capacity was indeed controlled by the glucose transport ability, perhaps with some influence of hexokinase, phosphofructokinase, aldolase, and enolase levels. However, it was also demonstrated that there was no such correlation when nitrogen starvation occurred in the presence of ethanol instead of glucose.     

Effect of nutrient starvation on the cellular composition and metabolic capacity of Saccharomyces cerevisiae

This investigation addresses the following question: what are the important factors for maintenance of a high catabolic capacity under various starvation conditions? Saccharomyces cerevisiae was cultured in aerobic batch cultures, and during the diauxic shift cells were transferred and subjected to 24 h of starvation. The following conditions were used: carbon starvation, nitrogen starvation in the presence of glucose or ethanol, and both carbon starvation and nitrogen starvation. During the starvation period changes in biomass composition (including protein, carbohydrate, lipid, and nucleic acid contents), metabolic activity, sugar transport kinetics, and the levels of selected enzymes were recorded. Subsequent to the starvation period the remaining catabolic capacity was measured by addition of 50 mM glucose. The results showed that the glucose transport capacity is a key factor for maintenance of high metabolic capacity in many, but not all, cases. The results for cells starved of carbon, carbon and nitrogen, or nitrogen in the presence of glucose all indicated that the metabolic capacity was indeed controlled by the glucose transport ability, perhaps with some influence of hexokinase, phosphofructokinase, aldolase, and enolase levels. However, it was also demonstrated that there was no such correlation when nitrogen starvation occurred in the presence of ethanol instead of glucose.