Expression of dehydrin gene from Arctic Cerastium arcticum increases abiotic stress tolerance and enhances the fermentation capacity of a genetically engineered Saccharomyces cerevisiae laboratory strain

We investigated Arctic plants to determine if they have a specific mechanism enabling them to adapt to extreme environments because they are subject to such conditions throughout their life cycles. Among the cell defense systems of the Arctic mouse-ear chickweed Cerastium arcticum, we identified a stress-responsive dehydrin gene CaDHN that belongs to the SK₅ subclass and contains conserved regions with one S segment at the N-terminus and five K segments from the N-terminus to the C-terminus. To investigate the molecular properties of CaDHN, the yeast Saccharomyces was transformed with CaDHN. CaDHN-expressing transgenic yeast (TG) cells recovered more rapidly from challenge with exogenous stimuli, including oxidants (hydrogen peroxide, menadione, and tert-butyl hydroperoxide), high salinity, freezing and thawing, and metal (Zn²⁺), than wild-type (WT) cells. TG cells were sensitive to copper, cobalt, and sodium dodecyl sulfate. In addition, the cell survival of TG cells was higher than that of WT cells when cells at the mid-log and stationary stages were exposed to increased ethanol concentrations. There was a significant difference in cultures that have an ethanol content >16 %. During glucose-based batch fermentation at generally used (30 °C) and low (18 °C) temperatures, TG cells produced a higher alcohol concentration through improved cell survival. Specifically, the final alcohol concentrations were 13.3 and 13.2 % in TG cells during fermentation at 30 and 18 °C, respectively, whereas they were 10.2 and 9.4 %, respectively, in WT cells under the same fermentation conditions. An in vitro assay revealed that purified CaDHN acted as a reactive oxygen species scavenger by neutralizing H₂O₂ and a chaperone by preventing high temperature-mediated catalase inactivation. Taken together, our results show that CaDHN expression in transgenic yeast confers tolerance to various abiotic stresses by improving redox homeostasis and enhances fermentation capacity, especially at low temperatures (18 °C).     

Ectopic expression of sweet potato MuS1 increases acquired stress tolerance and fermentation yield in Saccharomyces cerevisiae

The MuS1 gene is highly homologous to many stress-related proteins in plants. Here, we characterized whether a new candidate gene, MuS1, is related to multiple stress tolerance in yeast as it is in plants. Transgenic yeast strain expressing MuS1 were more resistant to hydrogen peroxide, menadione, high salinity, metals (i.e., cadmium, copper, iron, and zinc), ethanol, and lactic acid than wild-type strain transformed with a vector alone. In addition, the alcohol yield of the transgenic yeast strain was higher than that of the wild-type strain during the batch fermentation process. These results show that MuS1-expressing transgenic yeast strain exhibits enhanced alcohol yield as well as tolerance to abiotic stresses, especially metal stress.     

Exploration of a natural reservoir of flocculating genes from various Saccharomyces cerevisiae strains and improved ethanol fermentation using stable genetically engineered flocculating yeast strains

Flocculating yeast strains with good fermentation ability are desirable for brewing industry as well as for fuel ethanol production, however, the genetic diversity of the flocculating genes from natural yeast strains is largely unexplored. In this study, FLO1, FLO5, FLO9, FLO10 and FLO11 PCR products were obtained from 16 yeast strains from various sources, and the PCR product amplified from FLO1 of the self-flocculating yeast strain SPSC01 was used for the construction of expression cassette flanked by homologous fragments of the endonuclease gene HO for chromosome integration. A genetically engineered flocculating yeast BHL01 with good fermentation performance was obtained by transforming an industrial strain Saccharomyces cerevisiae 4126 with the expression cassette. The fermentation performances of SPSC01 and BHL01 in flask fermentation were compared using 208 g/L glucose. BHL01 completed the fermentation 8 h earlier than SPSC01, while no significant difference between BHL01 and S. cerevisiae 4126 was observed. In very high gravity repeated batch ethanol fermentation using 255 g/L glucose, BHL01 maintained stable flocculation for at least over 24 batches, while SPSC01 displayed severe deflocculation under the same conditions. The natural reservoir of flocculating genes from yeast strains may represent an unexplored gene source for the construction of new flocculating yeast strains for improved ethanol production.     

Mitochondrial and cytosolic expression of human peroxiredoxin 5 in Saccharomyces cerevisiae protect yeast cells from oxidative stress induced by paraquat

Human peroxiredoxin 5 is a recently discovered mitochondrial, peroxisomal and cytosolic thioredoxin peroxidase able to reduce hydrogen peroxide and alkyl hydroperoxides. To gain insight into peroxiredoxin 5 antioxidant role in cell protection, we investigated the resistance of yeast cells expressing human peroxiredoxin 5 in mitochondria or in the cytosol against oxidative stress induced by paraquat. The herbicide paraquat is a redox active drug known to generate superoxide anions in mitochondria and the cytosol of yeast and mammalian cells leading to the formation of several reactive oxygen species. Here, we report that mitochondrial and cytosolic human peroxiredoxin 5 protect yeast cells from cytotoxicity and lipid peroxidation induced by paraquat.     

Monitoring of Saccharomyces cerevisiae in commercial bakers' yeast fermentation

In the highly competitive market of commercial bakers' yeast, fermentations are operated for maximum efficiency and minimum production cost. In order to maintain competitiveness, the fermentations must be highly consistent with minimum variation in yeast performance, maximum yield on raw materials, and minimum production of undesirable side products. The use of advanced instrumentation is of critical importance to achieving these goals by the production engineer. An in situ optical density probe was used to determine the yeast cell density in full-scale commercial bakers' yeast fermentations. The optical density probe results were compared with oxygen uptake rate analyses, packed cell volume, and off-line measured cell dry weights. The most accurate measurement of cell density was found to be the optical density probe. This instrument allowed the on-line determination of cell density with highly consistent results from fermentation batch to batch and with out the need for intermittent recalibration. (c) 1995 John Wiley & Sons, Inc.     

GPX2, encoding a phospholipid hydroperoxide glutathione peroxidase homologue, codes for an atypical 2-Cys peroxiredoxin in Saccharomyces cerevisiae

We have previously reported that Saccharomyces cerevisiae has three glutathione peroxidase homologues (GPX1, GPX2, and GPX3) (Inoue, Y., Matsuda, T., Sugiyama, K., Izawa, S., and Kimura, A. (1999) J. Biol. Chem. 274, 27002-27009). Of these, the GPX2 gene product (Gpx2) shows the greatest similarity to phospholipid hydroperoxide glutathione peroxidase. Here we show that GPX2 encodes an atypical 2-Cys peroxiredoxin which uses thioredoxin as an electron donor. Gpx2 was essentially in a reduced form even in mutants defective in glutathione reductase or glutaredoxin under oxidative stressed conditions. On the other hand, Gpx2 was partially oxidized in a mutant defective in cytosolic thioredoxin (trx1Deltatrx2Delta) under non-stressed conditions and completely oxidized in tert-butyl hydroperoxide-treated cells of trx1Deltatrx2Delta and thioredoxin reductase-deficient mutant cells. Alanine scanning of cysteine residues of Gpx2 revealed that an intramolecular disulfide bond was formed between Cys37 and Cys83 in vivo. Gpx2 was purified to determine whether it functions as a peroxidase that uses thioredoxin as an electron donor in vitro. Gpx2 reduced H2O2 and tert-butyl hydroperoxide in the presence of thioredoxin, thioredoxin reductase, and NADPH (for H2O2, Km= 20 microm, kcat = 9.57 x 10(2) s(-1); for tert-butyl hydroperoxide, Km= 62.5 microm, kcat = 3.68 x 10(2) s(-1)); however, it showed remarkably less activity toward these peroxides in the presence of glutathione, glutathione reductase, and NADPH. The sensitivity of yeast cells to tert-butyl hydroperoxide was found to be exacerbated by the co-existence of Ca2+, a tendency that was most obvious in gpx2Delta cells. Although the redox state of Gpx2 was not affected by Ca2+, the Gpx2 level was markedly increased in the presence of both tert-butyl hydroperoxide and Ca2+. Gpx2 is likely to play an important role in the protection of cells from oxidative stress in the presence of Ca2+.     

Cloning and characterization of a 2-Cys peroxiredoxin from Pisum sativum

A cDNA sequence coding for a pea (Pisum sativum L.) 2-Cys peroxiredoxin (2-Cys Prx) has been cloned. The deduced amino acid sequence showed a high sequence homology to the 2-Cys Prx enzymes of Phaseolus vulgaris (86%), Arabidopsis thaliana (75%), and Spinacia oleracea (75%), and contained a chloroplast target sequence at its N-terminus. The mature enzyme, without the transit peptide, has a molecular mass of 22 kDa as well as two cysteine residues (Cys-53 and Cys-175) which are well conserved among proteins of this group. The protein was expressed in a heterologous system using the expression vector pET3d, and was purified to homogeneity by three sequential chromatographic steps. The enzyme exhibits peroxidase activity on hydrogen peroxide (H(2)O(2)) and t-butyl hydroperoxide (TBHP) with DTT as reducing agent. Although both pea Trxs f and m reduce oxidized 2-Cys Prx, Trx m is more efficient. The precise conditions for oligomerization of 2-Cys Prx through extensive gel filtration studies are also reported. The transition dimer-decamer produced in vitro between pH 7.5 and 8.0 and the influence of DTT suggest that a great change in the enzyme quaternary structure of 2-Cys Prx may take place in the chloroplast during the dark-light transition. In addition, the cyclophilin-dependent reduction of chloroplast 2-Cys Prx is shown.     

Deletion of PHO13 improves aerobic l-arabinose fermentation in engineered Saccharomyces cerevisiae

Journal of Industrial Microbiology & Biotechnology - Pentose sugars are increasingly being used in industrial applications of Saccharomyces cerevisiae. Although l-arabinose is a highlighted...     

Molecular characterization of a 2-Cys peroxiredoxin induced by abiotic stress in mungbean

A mungbean low temperature-inducible VrPrx1 encoding 2-Cys peroxiredoxin (2-Cys Prx) was cloned by subtractive suppression hybridization. The deduced VrPrx1 amino acid sequence showed highest sequence homology to 2-Cys Prxs of Phaseolus vulgaris (95%), Pisum sativum (89%), and Arabidopsis thaliana (87%). VrPrx1 RNA and protein levels were increased by low temperature, hydrogen peroxide (H₂O₂), and wounding but decreased by high salinity, drought, and exogenous abscisic acid. Recombinant His-tagged VrPrx1 recombinant protein protected DNA and glutamine synthetase activity from degradation via the thiol/Fe(III) oxygen mixed-function oxidation system, and exhibited peroxidase activity to H₂O₂ in the presence of the reducing agent dithiothreitol (DTT) in vitro. The oxidized dimers and oligomers of the VrPrx1 recombinant protein were reduced to monomers by DTT or thioredoxin. Subcellular localization studies confirmed that VrPrx1-GFP was targeted to the plastid. To evaluate the function of VrPrx1 in planta, the antioxidant activities and photosynthetic efficiency were investigated in VrPrx1-overexpressing Arabidopsis plants. VrPrx1 ectopic expression conferred improved photosynthetic efficiency under oxidative stress conditions. Hence, mungbean VrPrx1 may play an important role in protecting the photosynthetic apparatus against oxidative and abiotic stress conditions.     

Production of S-lactoylglutathione by glycerol-adapted Saccharomyces cerevisiae and genetically engineered Escherichia coli cells

Summary The enzymatic production of S-lactoylglutathione was studied by applying glyoxalase I to glycerol-grown cells of Saccharomyces cerevisiae and Escherichia coli cells dosed with Pseudomonas putida glyoxalase I gene. The glyoxalase I in S. cerevisiae cells was markedly induced when the cells were grown on glycerol. The activity of the enzyme in glycerol-grown cells was more than 20-fold higher compared with that of the glucose-grown cells. By using extracts of glycerol-grown yeast cells, about 5 mmol/1 (2 g/l) of S-lactoylglutathione was produced from 10 mM methylglyoxal and 50 mM glutathione within 1 h. The extracts of E. coli cells carrying a hybrid plasmid pGI423, which contains P. putida glyoxalase I gene, showed approximately 170-fold higher glyoxalase I activity than that of E. coli cells without pGI423. The extracts were used for production of S-lactoylglutathione and, under optimal conditions, about 40 mmol/l (15 g/l) of S-lactoylglutathione was produced from 50 mM methylglyoxal and 100mM glutathione within 1 h.     

酵母海藻糖酶缺失突变株的构建及其耐性

[目的]构建酵母海藻糖酶缺失突变株,并进行耐性分析,进一步研究海藻糖与酵母耐性之间的关系,为商业生产打下一定的基础.[方法]利用同源重组的方法,敲除了编码酸性海藻糖酶的ATH1基因和中性海藻糖酶的NTH1基因,构建了酸性海藻糖酶缺失突变株(△ath1)、中性海藻糖酶缺失突变株(△nth1)和双缺失突变株(△ath1△nth1),并进行了耐性分析.[结果]结合PCR和Southernblot的结果,验证了突变株构建的正确.所有突变株的海藻糖积累量和细胞密度均高于亲本,冷冻、高温、高糖和酒精耐性提高了.[结论]说明海藻糖含量与酵母耐性有一定的相关性.突变株耐性的改善,表明它们在酿造和烘焙产业中具有潜在的商业价值.     

Over-expression of Isu1p and Jac1p increases the ethanol tolerance and yield by superoxide and iron homeostasis mechanism in an engineered Saccharomyces cerevisiae yeast

Journal of Industrial Microbiology & Biotechnology - The ethanol stress response in ethanologenic yeast during fermentation involves the swishing of several adaptation mechanisms. In...     

The STF2p hydrophilin from Saccharomyces cerevisiae is required for dehydration stress tolerance

The yeast Saccharomyces cerevisiae is able to overcome cell dehydration; cell metabolic activity is arrested during this period but restarts after rehydration. The yeast genes encoding hydrophilin proteins were characterised to determine their roles in the dehydration-resistant phenotype, and STF2p was found to be a hydrophilin that is essential for survival after the desiccation-rehydration process. Deletion of STF2 promotes the production of reactive oxygen species and apoptotic cell death during stress conditions, whereas the overexpression of STF2, whose gene product localises to the cytoplasm, results in a reduction in ROS production upon oxidative stress as the result of the antioxidant capacity of the STF2p protein.     

The role of peroxisomes in xylose alcoholic fermentation in the engineered Saccharomyces cerevisiae

Xylose is a second‐most abounded sugar after glucose in lignocellulosic hydrolysates and should be efficiently fermented for economically viable second‐generation ethanol production. Despite significant progress in metabolic and evolutionary engineering, xylose fermentation rate of recombinant Saccharomyces cerevisiae remains lower than that for glucose. Our recent study demonstrated that peroxisome‐deficient cells of yeast Ogataea polymorpha showed a decrease in ethanol production from xylose. In this work, we have studied the role of peroxisomes in xylose alcoholic fermentation in the engineered xylose‐utilizing strain of S. cerevisiae. It was shown that peroxisome‐less pex3Δ mutant possessed 1.5‐fold decrease of ethanol production from xylose. We hypothesized that peroxisomal catalase Cta1 may have importance for hydrogen peroxide, the important component of reactive oxygen species, detoxification during xylose alcoholic fermentation. It was clearly shown that CTA1 deletion impaired ethanol production from xylose. It was found that enhancing the peroxisome population by modulation the peroxisomal biogenesis by overexpression of PEX34 activates xylose alcoholic fermentation.     

Oligomerization and chaperone activity of a plant 2-Cys peroxiredoxin in response to oxidative stress

Plant 2-Cys peroxiredoxins (2-Cys Prxs) have been reported to localize to chloroplasts and perform antioxidative roles during plant development and photosynthesis. In this study, we identified that, in addition to the well-known function of thioredoxin (Trx)-dependent peroxidase, the plant 2-Cys Prx in Chinese cabbage 2-Cys Prx1, designated C2C-Prx1, also behaves as a molecular chaperone under oxidative stress conditions, like the yeast and mammalian 2-Cys Prxs. By the chaperone function of C2C-Prx1, the protein efficiently prevented the denaturation of citrate synthase and insulin from heat shock and dithiothreitol (DTT)-induced chemical stresses. Also, the protein structure of C2C-Prx1 was shown to have discretely sized multiple structures, whose molecular sizes were in the diverse ranges of low molecular weight (LMW) proteins to high molecular weight (HMW) protein complexes. The dual functions of C2C-Prx1 acting as a peroxidase and as a molecular chaperone are alternatively switched by heat shock and oxidative stresses, accompanying with its structural changes. The peroxidase function predominates in the lower MW forms, but the chaperone function predominates in the higher MW complexes. The precise regulation of C2C-Prx1 structures and functions may play a pivotal role in the protection of plant chloroplasts from photo-oxidative stress.     

Genetic dissection of ethanol tolerance in the budding yeast Saccharomyces cerevisiae

Uncovering genetic control of variation in ethanol tolerance in natural populations of yeast Saccharomyces cerevisiae is essential for understanding the evolution of fermentation, the dominant lifestyle of the species, and for improving efficiency of selection for strains with high ethanol tolerance, a character of great economic value for the brewing and biofuel industries. To date, as many as 251 genes have been predicted to be involved in influencing this character. Candidacy of these genes was determined from a tested phenotypic effect following gene knockout, from an induced change in gene function under an ethanol stress condition, or by mutagenesis. This article represents the first genomics approach for dissecting genetic variation in ethanol tolerance between two yeast strains with a highly divergent trait phenotype. We developed a simple but reliable experimental protocol for scoring the phenotype and a set of STR/SNP markers evenly covering the whole genome. We created a mapping population comprising 319 segregants from crossing the parental strains. On the basis of the data sets, we find that the tolerance trait has a high heritability and that additive genetic variance dominates genetic variation of the trait. Segregation at five QTL detected has explained approximately 50% of phenotypic variation; in particular, the major QTL mapped on yeast chromosome 9 has accounted for a quarter of the phenotypic variation. We integrated the QTL analysis with the predicted candidacy of ethanol resistance genes and found that only a few of these candidates fall in the QTL regions.     

Response of wine yeast (Saccharomyces cerevisiae) aldehyde dehydrogenases to acetaldehyde stress during Icewine fermentation

AIMS: We previously reported that the aldehyde dehydrogenase encoded by ALD3 but not ALD6 was responsible, in part, for the increased acetic acid found in Icewines based on the expression profile of these genes during fermentation. We have now completed the expression profile of the remaining yeast aldehyde dehydrogenase genes ALD2, ALD4 and ALD5 during these fermentations to determine their contribution to acetic acid production. The contribution of acetaldehyde stress as a signal to stimulate ALD expression during these fermentations was investigated for all ALD genes. The expression of glycerol-3-phosphate encoded by GPD2 was also followed during these fermentations to determine its role in addition to the role we already identified for GPD1 in the elevated glycerol produced during Icewine fermentation. METHODS AND RESULTS: Icewine juice (38.5 degrees Brix, 398 +/- 5 g l(-1) sugar), diluted Icewine juice (20.8 degrees Brix, 196 +/- 4 g l(-1) sugar) and the diluted juice with sugar levels equal to the original Icewine juice (36.6 degrees Brix, 395 +/- 6 g l(-1) sugar) were fermented in duplicate using the commercial wine yeast K1-V1116. Acetic acid and glycerol production increased 8.4- and 2.7-fold in the Icewine vs the diluted juice fermentation, respectively, accompanied by a fourfold transient increase in acetaldehyde in the Icewine condition during the first week. Both mitochondrial aldehyde dehydrogenases encoded by ALD4 and ALD5 were expressed, with ALD5 expression highest at the start of all fermentations and ALD4 expression increasing during the first week of each condition. ALD2, ALD4, ALD5 and GPD2 showed no differential expression between the three fermentation conditions indicating their lack of involvement in elevating acetic acid and glycerol in Icewine. When yeast fermenting the diluted fermentation was exposed to exogenous acetaldehyde, the transient spike in acetaldehyde increased the expression of ALD3 but this response alone was not sufficient to cause an increase in acetic acid. Expression of the other aldehyde dehydrogenases was unaffected by the acetaldehyde addition. CONCLUSIONS: The aldehyde dehydrogenases encoded by ALD2, ALD4 and ALD5 do not contribute to the elevated acetic acid production during Icewine fermentation. Expression of GPD2 was not upregulated in high sugar fermentations and does not reflect the elevated levels of glycerol found in these wines. Acetaldehyde at a concentration produced during Icewine fermentation stimulates the expression of ALD3, but has no impact on the expression of ALD2, -4, -5 and -6. Upregulation of ALD3 alone in the dilute fermentation is not sufficient to increase acetic acid in wine and requires additional responses found in cells under hyperosmotic stress. SIGNIFICANCE AND IMPACT OF THE STUDY: This work confirms that increased acetic acid and glycerol production during Icewine fermentation follows upregulation of ALD3 and GPD1 respectively, but upregulation of ALD3 alone is not sufficient to increase acetic acid production. Additional responses of cells under osmotic stress are required to increase acetic acid in Icewine.