Pichia stipitis genes for alcohol dehydrogenase with fermentative and respiratory functions

Two genes coding for isozymes of alcohol dehydrogenase (ADH); designated PsADH1 and PsADH2, have been identified and isolated from Pichia stipitis CBS 6054 genomic DNA by Southern hybridization to Saccharomyces cerevisiae ADH genes, and their physiological roles have been characterized through disruption. The amino acid sequences of the PsADH1 and PsADH2 isozymes are 80.5% identical to one another and are 71.9 and 74.7% identical to the S. cerevisiae ADH1 protein. They also show a high level identity with the group I ADH proteins from Kluyveromyces lactis. The PsADH isozymes are presumably localized in the cytoplasm, as they do not possess the amino-terminal extension of mitochondrion-targeted ADHs. Gene disruption studies suggest that PsADH1 plays a major role in xylose fermentation because PsADH1 disruption results in a lower growth rate and profoundly greater accumulation of xylitol. Disruption of PsADH2 does not significantly affect ethanol production or aerobic growth on ethanol as long as PsADH1 is present. The PsADH1 and PsADH2 isozymes appear to be equivalent in the ability to convert ethanol to acetaldehyde, and either is sufficient to allow cell growth on ethanol. However, disruption of both genes blocks growth on ethanol. P. stipitis strains disrupted in either PsADH1 or PsADH2 still accumulate ethanol, although in different amounts, when grown on xylose under oxygen-limited conditions. The PsADH double disruptant, which is unable to grow on ethanol, still produces ethanol from xylose at about 13% of the rate seen in the parental strain. Thus, deletion of both PsADH1 and PsADH2 blocks ethanol respiration but not production, implying a separate path for fermentation.     

Development of ascending colon in human fetal period

Study of glucose and xylose utilization by Pichia stipitis in a limited oxygen supply condition revealed that the rate of glucose utilization decreased rapidly while that of xylose decreased slowly until the time that glucose and xylose were shown to level out, at which point the rate of xylose utilization increased rapidly. Based on the results, ethanol fermentation technology in continuous connective tower fermenters was advanced, e.g., fermentation by P. stipitis in an airlift loop tower focusing on xylose utilization and then residue glucose utilization by Saccharomyces cerevisiae in an overflow tower. When the fed hydrolyzate of bagasse was concentrated in five folds and the dilution rate was 0.1 h-1, the total utilization ratio of reducing sugar was 97.2%; the concentration of ethanol was 46.4 g/L.h.     

Modifications to the ADH1 promoter of Saccharomyces cerevisiae for efficient production of heterologous proteins

The promoter of alcohol dehydrogenase I of the yeast Saccharomyces cerevisiae was studied using Bacillus amyloliquefaciens alpha-amylase as a marker protein. On glucose, activity of the original ADH1 promoter decreases during late exponential, ethanol production growth phase. When 1100 bp (from -414 bp to -1500 bp) of the upstream sequence are deleted, activity increases into the late ethanol consumption phase but the promoter becomes active only after ethanol production growth phase (Ruohonen et al. (1991) Yeast 7, 337-346). We have now restored 300 bp (from -414 bp to -700 bp) upstream of the deletion site and obtained expression from the ADH1 promoter throughout the yeast growth cycle. The restored sequence allowed alpha-amylase expression to start during early exponential growth phase indicating that it is required for activation of the ADH1 promoter during ethanol production growth phase, possibly through glucose induction. On ethanol, all the promoters were active, but the short promoter was temporally activated first, suggesting that the restored sequence is not required for promoter activity during early oxidative growth.     

Age of initiating selected health-risk behaviors among high school students in the United States

The XYL1 and XYL2 genes from Pichia stipitis encoding xylose reductase (XR) and xylilitol dehydrogenase (XDH), respectively, were transformed into Saccharomyces cerevisiae. These two genes were placed in different directions under the control of the alcohol dehydrogenase I (ADHI) and phosphoglycerate kinase (PGK) promoters and inserted into the E. coli-yeast shuttle plasmid YEp24. Different recombinant S. cerevisiae strains were constructed with different specific activities of XR and XDH. The highest XR or XDH activities were obtained when the expressed gene was controlled by the PGK promoter and located downstream after the ADHI promoter-gene-terminator sequence. The XR/XDH ratio (ratio of specific enzyme activities of XR and XDH) in these recombinant S. cerevisiae strains varied from 17.5 to 0.06. In order to enhance xylose utilization, in the XYL1, XYL2 containing S. cerevisiae strains, the native TKL1 gene encoding transketolase and the TALI gene encoding transaldolase were also overexpressed, which showed considerably good growth on the xylose plate. Fermentation of the recombinant S. cerevisiae strains containing XYL1, XYL2, TKL1, and TAL1 were studied with mixtures of glucose and xylose. The strain with XR/XDH ratio of 0.06 consumed 3.25 g/L xylose and formed no xylitol and less glycerol and acetic acid, but more ethanol compared with the strains with a higher XR/XDH ratio.     

Heterologous HIS3 marker and GFP reporter modules for PCR-targeting in Saccharomyces cerevisiae

We have fused the open reading frames of his3-complementing genes from Saccharomyces kluyveri and Schizosac-charomyces pombe to the strong TEF gene promotor of the filamentous fungus Ashbya gossypii. Both chimeric modules and the cognate S. kluyveri HIS3 gene were tested in transformations of his3 S. cerevisiae strains using PCR fragments flanked by 40 bp target guide sequences. The 1.4 kb chimeric Sz. pombe module (HIS3MX6) performed best. With less than 5% incorrectly targeted transformants, it functions as reliably as the widely used geniticin resistance marker kanMX. The rare false-positive His+ transformants seem to be due to non-homologous recombination rather than to gene conversion of the mutated endogenous his3 allele. We also cloned the green fluorescent protein gene from Aequorea victoria into our pFA-plasmids with HIS3MX6 and kanMX markers. The 0.9 kb GFP reporters consist of wild-type GFP or GFP-S65T coding sequences, lacking the ATG, fused to the S. cerevisiae ADH1 terminator. PCR-synthesized 2.4 kb-long double modules flanked by 40-45 bp-long guide sequences were successfully targeted to the carboxy-terminus of a number of S. cerevisiae genes. We could estimate that only about 10% of the transformants carried inactivating mutations in the GFP reporter.     

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

Xylitol production from xylose was studied using recombinant Saccharomyces cerevisiae 2805 containing xylose reductase genes (XYL1) of Pichia stipitis at chromosomal delta-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 neor 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.     

Caspase-3-induced gelsolin fragmentation contributes to actin cytoskeletal collapse, nucleolysis, and apoptosis of vascular smooth muscle cells exposed to proinflammatory cytokines

It has been postulated that ethanol-induced pancreatic injury may be mediated by the oxidation of ethanol within the pancreas with secondary toxic metabolic changes, but there is little evidence of pancreatic ethanol oxidation. The aims of this study were to determine whether pancreatic acinar cells metabolize significant amounts of ethanol and, if so, to compare their rate of ethanol oxidation to that of hepatocytes. Cultured rat pancreatic acinar cells and hepatocytes were incubated with 5 to 50 mmol/L carbon 14-labeled ethanol (25 dpm/nmol). Ethanol oxidation was calculated from the production of 14C-labeled acetate that was isolated by Dowex ion-exchange chromatography. Ethanol oxidation by pancreatic acinar cells was demonstrable at all ethanol concentrations tested. At an intoxicating ethanol concentration (50 mmol/L), 14C-labeled acetate production (227+/-20 nmol/10(6) cells/h) approached that of hepatocytes (337+/-61 nmol/10(6) cells/h). Phenanthroline (an inhibitor of classes I through III isoenzymes of alcohol dehydrogenase (ADH)) inhibited pancreatic ethanol oxidation by 90%, but 4-methylpyrazole (a class I and II ADH inhibitor), carbon monoxide (a cytochrome P450 inhibitor), and sodium azide (a catalase inhibitor) had no effect. This study has shown that pancreatic acinar cells oxidize significant amounts of ethanol. At intoxicating concentrations of ethanol, pancreatic acinar cell ethanol oxidation may have the potential to contribute to pancreatic cellular injury. The mechanism appears to involve the class III isoenzyme of ADH.     

Hemiacetal dehydrogenation activity of alcohol dehydrogenases in Saccharomyces cerevisiae

Some methylotrophic yeasts produce methyl formate from methanol and formaldehyde via hemiacetal formation. We investigated Saccharomyces cerevisiae to find whether this yeast has a carboxylate ester producing pathway that proceeds via hemiacetal dehydrogenation. We confirmed that the purified alcohol dehydrogenase (Adh) protein from S. cerevisiae can catalyze the production of esters. High specific activities were observed toward the hemiacetals corresponding to the primary alcohols when ether groups were substituted for methylene groups, resulting in the formation of formate esters. Both ADH and methyl formate synthesizing activities were sharply reduced in the delta adh1 delta adh2 mutant. The ADH1 and ADH2 genes encode the major Adh proteins in S. cerevisiae. Thus, it was concluded that the S. cerevisiae Adh protein catalyzes activities for the production of certain carboxylate esters.     

Modulation of glycerol and ethanol yields during alcoholic fermentation in Saccharomyces cerevisiae strains overexpressed or disrupted for GPD1 encoding glycerol 3-phosphate dehydrogenase

The possibility of the diversion of carbon flux from ethanol towards glycerol in Saccharomyces cerevisiae during alcoholic fermentation was investigated. Variations in the glycerol 3-phosphate dehydrogenase (GPDH) level and similar trends for alcohol dehydrogenase (ADH), pyruvate decarboxylase and glycerol-3-phosphatase were found when low and high glycerol-forming wine yeast strains were compared. GPDH is thus a limiting enzyme for glycerol production. Wine yeast strains with modulated GPD1 (encoding one of the two GPDH isoenzymes) expression were constructed and characterized during fermentation on glucose-rich medium. Engineered strains fermented glucose with a strongly modified [glycerol] : [ethanol] ratio. gpd1delta mutants exhibited a 50% decrease in glycerol production and increased ethanol yield. Overexpression of GPD1 on synthetic must (200 g/l glucose) resulted in a substantial increase in glycerol production ( x 4) at the expense of ethanol. Acetaldehyde accumulated through the competitive regeneration of NADH via GPDH. Accumulation of by-products such as pyruvate, acetate, acetoin, 2,3 butane-diol and succinate was observed, with a marked increase in acetoin production.     

Interaction between the Adh and Odh loci in response to ethanol in Drosophila melanogaster

Six Drosophila melanogaster strains were constructed from two isofemale lines. The strains had four allele combinations at the alcohol dehydrogenase (Adh) and octanol dehydrogenase (Odh) loci, while all alpha-glycerophosphate dehydrogenase (alpha Gpdh), malate dehydrogenase (Mdh), and aldehyde oxidase (Aldox) alleles were identical. Second-instar and early and late third-instar larvae were exposed to different concentrations of ethanol (0, 5, and 7.5%) and 3 days later fresh weights and the activities of ADH, ODH, alpha GPDH, and MDH were measured. Activity differences were observed between the two Adh genotypes: ADHF allozyme had considerably higher activity than ADHS. Exogenous ethanol resulted in the highest increase in ADH activity in the second- and early third-instar stages. This ADH induction depended on the allele combination at the Adh and Odh loci; e.g., in the strain having the AdhS-OdhS allele combination, increased ADH activity was observed only after exposure to 7.5% ethanol. ODH activities differed according to the Odh genotypes, in that the ODHS allozyme had a higher activity than ODHF. ODH activities did not appreciably respond to different ethanol treatments. All six strains had identical alleles at the Mdh and alpha Gpdh loci, but nevertheless, the responses of these enzymes to ethanol depended on the allele combinations at the Adh and Odh loci. alpha GPDH activity followed that of ADH in all experiments. MDH activities were not influenced by exogenous ethanol in the strains homozygous for the AdhS allele. In AdhF strains, however, exposure to 7.5% ethanol resulted in a considerable decrease in MDH activity in the second-instar larvae. Correlations among the response variables showed that ODH activities were strongly associated with fresh weight and the activities of all other enzymes, except for ADH. ADH activity, however, showed a significant correlation only with alpha GPDH activity throughout the larval life. Both MDH and ODH activities were found to be in strong negative correlation with ADH activity in the second-instar larvae. At this most sensitive life stage, the metabolic response to ethanol is highly correlated.     

The osmotic hypersensitivity of the yeast Saccharomyces cerevisiae is strain and growth media dependent: quantitative aspects of the phenomenon

Osmotic hypersensitivity is manifested as cellular death at magnitudes of osmotic stress that can support growth. Cellular capacity for survival when plated onto high NaCl media was examined for a number of laboratory and industrial strains of Saccharomyces cerevisiae. During respiro-fermentative growth in rich medium with glucose as energy and carbon source, the hypersensitivity phenomenon was fairly strain invariant with a threshold value of about 1 M-NaCl; most strains fell within a 300 mM range in LD10 values (lethal dose yielding 10% survival). Furthermore, all but one of the strains displayed similar differential death responses above the threshold value, i.e. ten-fold decreased viability for every 250 mM increase in salinity. Addition of small amounts of salt to the growth medium drastically improved tolerance and shifted the hypersensitivity threshold to higher NaCl concentrations. This salt-instigated tolerance could partly be reversed by washing in water. The washing procedure depleted cells of the glycerol that they had accumulated under saline growth, and the contribution from glycerol to the improved tolerance was about 50% in the two strains examined. Growth on derepressing carbon sources like galactose, ethanol or glycerol gave strain-dependent responses. The laboratory strain X2180-1A drastically improved tolerance while the bakers' yeast strain Y41 did so only marginally. It was concluded that all strains of S. cerevisiae display the osmotic hypersensitivity phenomenon in qualitative terms while the quantitative values differ. It was also proposed that growth rate does not dictate the level of osmotic hypersensitivity of S. cerevisiae.