High Concentrations Of Sorbitol Research Articles

Development and Regulation of the Extreme Biofilm Formation of Deinococcus radiodurans R1 under Extreme Environmental Conditions.

To grow in various harsh environments, extremophiles have developed extraordinary strategies such as biofilm formation, which is an extremely complex and progressive process. However, the genetic elements and exact mechanisms underlying extreme biofilm formation remain enigmatic. Here, we characterized the biofilm-forming ability of Deinococcus radiodurans in vitro under extreme environmental conditions and found that extremely high concentrations of NaCl or sorbitol could induce biofilm formation. Meantime, the survival ability of biofilm cells was superior to that of planktonic cells in different extreme conditions, such as hydrogen peroxide stress, sorbitol stress, and high UV radiation. Transcriptome profiles of D. radiodurans in four different biofilm development stages further revealed that only 13 matched genes, which are involved in environmental information processing, carbohydrate metabolism, or stress responses, share sequence homology with genes related to the biofilm formation of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Overall, 64% of the differentially expressed genes are functionally unknown, indicating the specificity of the regulatory network of D. radiodurans. The mutation of the drRRA gene encoding a response regulator strongly impaired biofilm formation ability, implying that DrRRA is an essential component of the biofilm formation of D. radiodurans. Furthermore, transcripts from both the wild type and the drRRA mutant were compared, showing that the expression of drBON1 (Deinococcus radioduransBON domain-containing protein 1) significantly decreased in the drRRA mutant during biofilm development. Further analysis revealed that the drBON1 mutant lacked the ability to form biofilm and DrRRA, and as a facilitator of biofilm formation, could directly stimulate the transcription of the biofilm-related gene drBON1. Overall, our work highlights a molecular mechanism mediated by the response regulator DrRRA for controlling extreme biofilm formation and thus provides guidance for future studies to investigate novel mechanisms that are used by D. radiodurans to adapt to extreme environments.

Plasticizer concentration effect on films and coatings based on poly(vinyl alcohol) and cationic starch blends

Films based on poly(vinyl alcohol) (PVA) and cationic starch (CS) were combined with different percentages of sorbitol (S; 15.0, 22.5, and 30.0% w v-1) to assess the effect of plasticizer on the films. Spectroscopic analyses confirmed the interaction between them. However, micrographs indicated the formation of sorbitol crystals on the surface of the films, especially at higher sorbitol concentrations. The blends presented low water vapor transmission rate values, reaching (7.703 ± 0.000) g h−1 m−2 (PVA75CS25S15), and low solubility values for the films containing higher CS amounts. The lack of statistical differences in most parameters suggests that no significant gain comes from increasing the amount of sorbitol at percentages higher than 15%. As a coating, the blend PVA75CS25S15 successfully decreased the loss of moisture content in acerolas by 1.15 times (compared to the control), confirming the suitability of this matrix as a fruit coating.

Physical properties of the cytoplasm modulate the rates of microtubule polymerization and depolymerization.

The cytoplasm is a crowded, visco-elastic environment whose physical properties change according to physiological or developmental states. How the physical properties of the cytoplasm impact cellular functions invivo remains poorly understood. Here, we probe the effects of cytoplasmic concentration on microtubules by applying osmotic shifts to fission yeast, moss, and mammalian cells. We show that the rates of both microtubule polymerization and depolymerization scale linearly and inversely with cytoplasmic concentration; an increase in cytoplasmic concentration decreases the rates of microtubule polymerization and depolymerization proportionally, whereas a decrease in cytoplasmic concentration leads to the opposite. Numerous lines of evidence indicate that these effects are due to changes in cytoplasmic viscosity rather than cellular stress responses or macromolecular crowding per se. We reconstituted these effects on microtubules invitro by tuning viscosity. Our findings indicate that, even in normal conditions, the viscosity of the cytoplasm modulates the reactions that underlie microtubule dynamic behaviors.

Effect of glycerol and sorbitol on the mechanical and barrier properties of films based on pea protein isolate produced by high‐moisture extrusion processing

AbstractDisposable food packaging accounts for a large proportion of the plastic waste in the environment. Bio‐based and biodegradable alternatives could help to reduce plastic pollution but must perform similarly to current synthetic materials. Here, we investigated the effect of two plasticizers (glycerol and sorbitol) on the mechanical and barrier properties of films composed of extruded pea protein isolate. We evaluated physical properties such as tensile strength, elongation at break, moisture content, surface energy, residence time distribution, and gas barrier properties as a function of plasticizer concentration. The flexibility and workability of the films generally increased with higher concentrations of the plasticizer. The residence time distribution (RTD) showed that sorbitol mixtures remained in the extruder longer than glycerol mixtures, exacerbating the loss of quality. Glycerol films generally retained more moisture than sorbitol films and the tensile strength declined with increasing glycerol content, but the elongation at break increased. The polar fraction of the surface energy increased with higher concentrations of sorbitol, indicating that sorbitol is more polar than the pea protein isolate. In terms of barrier properties, both the water vapor transmission rate (WVTR) and oxygen transmission rate (OTR) were higher for films plasticized with sorbitol. Specifically, the OTR for sorbitol‐plasticized films at 50% (vol/vol) was 519 cm3*100 μm/(m2*d*bar) compared to 300 cm3*100 μm/(m2*d*bar) for the glycerol‐plasticized films, and the WVTR for sorbitol‐plasticized films at 50% (vol/vol) was 2633 g*100 μm/(m2*d) compared to 895 g*100 μm/(m2*d) for the glycerol‐plasticized films. For both plasticizers, the WVTR increased with increasing plasticizer concentration. In contrast, the OTR declined with increasing glycerol concentrations, whereas no trend was observed for sorbitol. Our study shows that pea protein isolate is suitable for the manufacture of bio‐based films and confirms that plasticizers influence the resulting mechanical and barrier properties. Further research is necessary to optimize the properties of the films.

Seven Years at High Salinity-Experimental Evolution of the Extremely Halotolerant Black Yeast Hortaea werneckii.

The experimental evolution of microorganisms exposed to extreme conditions can provide insight into cellular adaptation to stress. Typically, stress-sensitive species are exposed to stress over many generations and then examined for improvements in their stress tolerance. In contrast, when starting with an already stress-tolerant progenitor there may be less room for further improvement, it may still be able to tweak its cellular machinery to increase extremotolerance, perhaps at the cost of poorer performance under non-extreme conditions. To investigate these possibilities, a strain of extremely halotolerant black yeast Hortaea werneckii was grown for over seven years through at least 800 generations in a medium containing 4.3 M NaCl. Although this salinity is well above the optimum (0.8–1.7 M) for the species, the growth rate of the evolved H. werneckii did not change in the absence of salt or at high concentrations of NaCl, KCl, sorbitol, or glycerol. Other phenotypic traits did change during the course of the experimental evolution, including fewer multicellular chains in the evolved strains, significantly narrower cells, increased resistance to caspofungin, and altered melanisation. Whole-genome sequencing revealed the occurrence of multiple aneuploidies during the experimental evolution of the otherwise diploid H. werneckii. A significant overrepresentation of several gene groups was observed in aneuploid regions. Taken together, these changes suggest that long-term growth at extreme salinity led to alterations in cell wall and morphology, signalling pathways, and the pentose phosphate cycle. Although there is currently limited evidence for the adaptive value of these changes, they offer promising starting points for future studies of fungal halotolerance.

The effect of bulking agent and type of chocolate on the physicochemical characteristics of sucrose-free chocolate using stevia as a sweetener

The aim of this research is to find out the effect addition of bulking agents on the physicochemical properties of sucrose-free chocolate products using stevia as a sweetener. Sucrose-free chocolate using stevia as a sweetener needs various bulking agents to make the same physical characteristics as the commercial chocolate. The alternative bulking agent that could be used for sucrose-free chocolate is the combination of inulin and erythritol or inulin and sorbitol. The combination of inulin and sorbitol as a bulking agent on dark chocolate can improve the protein and fat content and lower the moisture content than the reference chocolate. Milk chocolate with inulin and erythritol as a bulking agent has a higher protein, fat content but lower carbohydrate content than commercial chocolate. A high concentration of inulin will increase the level of carbohydrate content both in dark and milk chocolate. Higher concentrations of sorbitol can decrease the fat content on sucrose-free dark chocolate. Erythritol with a high concentration will increase the color brightness and reduce moisture. Higher concentrations of inulin will decrease the fat content on sucrose-free milk chocolate.

Mixed Brazilian Cerrado fruits preserves without added sugar: the effect of bodying agents

PurposeThe purpose of this paper is to evaluate the effect of the bodying agents (erythritol, sorbitol, xylitol and polydextrose) and their mixtures on the preparation of mixed Brazilian Cerrado fruit preserves (marolo, soursop and sweet passion fruit).Design/methodology/approachThe simplex mixture design was used for product optimization and the preserves evaluated by physical, physicochemical and sensory properties. The research data were analyzed using regression equations on SAS University and exploratory analysis by principal component analysis and parallel factors techniques on Sensomaker software.FindingsThe results show that erythritol and polydextrose bodying agents should not be used as pure components because they provoked changes in the properties of the final product and negatively influenced the sensory attributes. While the high concentrations of xylitol and sorbitol provided better sensorial acceptance, being considered, therefore, good substitutes for sucrose.Practical implicationsThis research has shown it is feasible to use bodying agents in the preparation of mixed Brazilian Cerrado fruit preserves without added sugar.Social implicationsThe development of mixed preserves could increase population access to the Brazilian Cerrado fruits. In addition, sugar-free preserves are a viable alternative for adding value to the product and satisfy all the consumers.Originality/valueThe combination of properties of two or more fruits and use of bodying agents can improve sensory, physical and physicochemical characteristics of the final product. However, there are technological challenges to evaluate in the preparation of mixed Brazilian Cerrado fruit preserves without added sugar.

Combined impacts of prolonged drought and warming on plant size and foliar chemistry.

Future shifts in precipitation regimes and temperature are expected to affect plant traits dramatically. To date, many studies have explored the effects of acute stresses, but few have investigated the consequences of prolonged shifts in climatic conditions on plant growth and chemistry. Plant size and metabolite profiles were assessed on naturally occurring Plantago lanceolata plants growing under different precipitation (ambient, 50 % less than ambient = drought) and temperature (ambient, +0.8, +2.4 and +4.0 °C above ambient) treatments at the Boston Area Climate Experiment (constructed in 2007). The analysis of primary and secondary metabolites revealed pronounced effects of drought, and a precipitation × temperature interaction. Strikingly, the effects of precipitation were minimal at the two lower temperatures but marked at the two higher temperatures. Compared with the ambient condition, plants in the drought plots had lower concentrations of foliar nitrogen, amino acids and most sugars, and higher concentrations of sorbitol, citrate and malate, common stress-induced metabolites. This pattern was especially evident at high temperatures. Moreover, drought-exposed plants showed lower concentrations of catalpol, an iridoid glycoside. While the effect of warming on the metabolite profiles was less pronounced, differences were marked when combined with drought. Given the interactive effect of environmental variables on leaf chemistry, and the fact that woody and herbaceous plants seem to differ in their responses to temperature and precipitation, future studies should account for the direct and indirect effects of the community response to multifactorial field conditions.

Hyper-Osmotic Stress Elicits Membrane Depolarization and Decreased Permeability in Halotolerant Marine Debaryomyces hansenii Strains and in Saccharomyces cerevisiae.

The use of seawater and marine microorganisms can represent a sustainable alternative to avoid large consumption of freshwater performing industrial bioprocesses. Debaryomyces hansenii, which is a known halotolerant yeast, possess metabolic traits appealing for developing such processes. For this purpose, we studied salt stress exposure of two D. hansenii strains isolated from marine fauna. We found that the presence of sea salts during the cultivation results in a slight decrease of biomass yields. Nevertheless, higher concentration of NaCl (2 M) negatively affects other growth parameters, like growth rate and glucose consumption rate. To maintain an isosmotic condition, the cells accumulate glycerol as compatible solute. Flow cytometry analysis revealed that the osmotic adaptation causes a reduced cellular permeability to cell-permeant dye SYBR Green I. We demonstrate that this fast and reversible phenomenon is correlated to the induction of membrane depolarization, and occurred even in presence of high concentration of sorbitol. The decrease of membrane permeability induced by osmotic stress confers to D. hansenii resistance to cationic drugs like Hygromycin B. In addition, we describe that also in Saccharomyces cerevisiae the exposure to hyper-osmotic conditions induced membrane depolarization and reduced the membrane permeability. These aspects are very relevant for the optimization of industrial bioprocesses, as in the case of fermentations and bioconversions carried out by using media/buffers containing high nutrients/salts concentrations. Indeed, an efficient transport of molecules (nutrients, substrates, and products) is the prerequisite for an efficient cellular performance, and ultimately for the efficiency of the industrial process.

Postharvest dehydration induces variable changes in the primary metabolism of grape berries

Postharvest dehydration causes changes in texture, color, taste and nutritional value of food due to the high temperatures and long drying times required. In grape berries, a gradual dehydration process is normally utilized for raisin production and for making special wines. Here we applied a raisin industry-mimicking dehydration process for eleven days at 50°C to intact berry clusters from cv. Sémillon plants, and a set of molecular, cellular and biochemical analyses were performed to study the impact of postharvest dehydration in the primary metabolism. Transcriptional analyses by real time qPCR showed that several aquaporins (VvTIP1;2 and VvSIP1) and sugar transporters (VvHT1, VvSWEET11, VvSWEET15, VvTMT1, VvSUC12) genes were strongly upregulated. Moreover, the study of key enzymes of osmolytes metabolism, including mannitol dehydrogenase (VvMTD) and sorbitol dehydrogenase (VvSDH), at gene expression and protein activity level, together with the transcriptional analysis of the polyol transporter gene VvPLT1, showed an enhanced polyol biosynthesis capacity, which was supported by the detection of sorbitol in dehydrated grapes only. The metabolism of organic acids was also modulated, by the induction of transcriptional and biochemical activity modifications in malate dehydrogenases and malic enzymes that led to organic acid degradation, as demonstrated by HPLC analysis. Taken together, this study showed that primary metabolism of harvested berries was severely influenced in response to dehydration treatments towards lower organic acid and higher sorbitol concentrations, while sugar transporter and aquaporin genes were significantly upregulated.

Yeast Kch1 and Kch2 membrane proteins play a pleiotropic role in membrane potential establishment and monovalent cation homeostasis regulation.

The Kch1 and Kch2 plasma-membrane proteins were identified in Saccharomyces cerevisiae as being essential for the activation of a high-affinity Ca2+ influx system. We searched for Kch proteins roles in the maintenance of cation homeostasis and tested the effect of kch1 and/or kch2 deletions on various physiological parameters. Compared to wild-type, kch1 kch2 mutant cells were smaller, relatively hyperpolarised, grew better under limited K+ conditions and exhibited altered growth in the presence of monovalent cations. The absence of Kch1 and Kch2 did not change the intracellular pH in cells growing at low potassium or the tolerance of cells to divalent cations, high concentration of sorbitol or extreme external pH. The overexpression of KCH1 only increased the intracellular pH in the presence of elevated K+ in media. None of the phenotypes associated with the deletion of KCH1 and KCH2 in wild type were observed in a strain lacking KCH genes and main K+ uptake systems Trk1 and Trk2. The role of the Kch homologue in cation homeostasis was also tested in Candida albicans cells. Our data demonstrate that Kch proteins significantly contribute to the maintenance of optimal cation homeostasis and membrane potential in S. cerevisiae but not in C. albicans.

Ionotropic Receptor 76b Is Required for Gustatory Aversion to Excessive Na+ in Drosophila.

Avoiding ingestion of excessively salty food is essential for cation homeostasis that underlies various physiological processes in organisms. The molecular and cellular basis of the aversive salt taste, however, remains elusive. Through a behavioral reverse genetic screening, we discover that feeding suppression by Na+-rich food requires Ionotropic Receptor 76b (Ir76b) in Drosophila labellar gustatory receptor neurons (GRNs). Concentrated sodium solutions with various anions caused feeding suppression dependent on Ir76b. Feeding aversion to caffeine and high concentrations of divalent cations and sorbitol was unimpaired in Ir76b-deficient animals, indicating sensory specificity of Ir76b-dependent Na+ detection and the irrelevance of hyperosmolarity-driven mechanosensation to Ir76b-mediated feeding aversion. Ir76b-dependent Na+-sensing GRNs in both L− and s-bristles are required for repulsion as opposed to the previous report where the L-bristle GRNs direct only low-Na+ attraction. Our work extends the physiological implications of Ir76b from low-Na+ attraction to high-Na+ aversion, prompting further investigation of the physiological mechanisms that modulate two competing components of Na+-evoked gustation coded in heterogeneous Ir76b-positive GRNs.

Mitogen activated protein kinases SakA(HOG1) and MpkC collaborate for Aspergillus fumigatus virulence.

Here, we investigated which stress responses were influenced by the MpkC and SakA mitogen-activated protein kinases of the high-osmolarity glycerol (HOG) pathway in the fungal pathogen Aspergillus fumigatus. The ΔsakA and the double ΔmpkC ΔsakA mutants were more sensitive to osmotic and oxidative stresses, and to cell wall damaging agents. Both MpkC::GFP and SakA::GFP translocated to the nucleus upon osmotic stress and cell wall damage, with SakA::GFP showing a quicker response. The phosphorylation state of MpkA was determined post exposure to high concentrations of congo red and Sorbitol. In the wild-type strain, MpkA phosphorylation levels progressively increased in both treatments. In contrast, the ΔsakA mutant had reduced MpkA phosphorylation, and surprisingly, the double ΔmpkC ΔsakA had no detectable MpkA phosphorylation. A. fumigatus ΔsakA and ΔmpkC were virulent in mouse survival experiments, but they had a 40% reduction in fungal burden. In contrast, the ΔmpkC ΔsakA double mutant showed highly attenuated virulence, with approximately 50% mice surviving and a 75% reduction in fungal burden. We propose that both cell wall integrity (CWI) and HOG pathways collaborate, and that MpkC could act by modulating SakA activity upon exposure to several types of stresses and during CW biosynthesis.

Response of enzyme activities and microbial communities to soil amendment with sugar alcohols.

SummaryChanges in microbial community structure are widely known to occur after soil amendment with low‐molecular‐weight organic compounds; however, there is little information on concurrent changes in soil microbial functional diversity and enzyme activities, especially following sorbitol and mannitol amendment. Soil microbial functional diversity and enzyme activities can be impacted by sorbitol and mannitol, which in turn can alter soil fertility and quality. The objective of this study was to investigate the effects of sorbitol and mannitol addition on microbial functional diversity and enzyme activities. The results demonstrated that sorbitol and mannitol addition altered the soil microbial community structure and improved enzyme activities. Specifically, the addition of sorbitol enhanced the community‐level physiological profile (CLPP) compared with the control, whereas the CLPP was significantly inhibited by the addition of mannitol. The results of a varimax rotated component matrix demonstrated that carbohydrates, polymers, and carboxylic acids affected the soil microbial functional structure. Additionally, we found that enzyme activities were affected by both the concentration and type of inputs. In the presence of high concentrations of sorbitol, the urease, catalase, alkaline phosphatase, β‐glucosidase, and N‐acetyl‐β‐d‐glucosaminidase activities were significantly increased, while invertase activity was decreased. Similarly, this increase in invertase, catalase, and alkaline phosphatase and N‐acetyl‐β‐d‐glucosaminidase activities was especially evident after mannitol addition, and urease activity was only slightly affected. In contrast, β‐glucosidase activity was suppressed at the highest concentration. These results indicate that microbial community diversity and enzyme activities are significantly affected by soil amendment with sorbitol and mannitol.

Study of model systems to test the potential function of Artemia group 1 late embryogenesis abundant (LEA) proteins.

Embryos of the brine shrimp, Artemia franciscana, are genetically programmed to develop either ovoviparously or oviparously depending on environmental conditions. Shortly upon their release from the female, oviparous embryos enter diapause during which time they undergo major metabolic rate depression while simultaneously synthesize proteins that permit them to tolerate a wide range of stressful environmental events including prolonged periods of desiccation, freezing, and anoxia. Among the known stress-related proteins that accumulate in embryos entering diapause are the late embryogenesis abundant (LEA) proteins. This large group of intrinsically disordered proteins has been proposed to act as molecular shields or chaperones of macromolecules which are otherwise intolerant to harsh conditions associated with diapause. In this research, we used two model systems to study the potential function of the group 1 LEA proteins from Artemia. Expression of the Artemia group 1 gene (AfrLEA-1) in Escherichia coli inhibited growth in proportion to the number of 20-mer amino acid motifs expressed. As well, clones of E. coli, transformed with the AfrLEA-1 gene, expressed multiple bands of LEA proteins, either intrinsically or upon induction with isopropyl-β-thiogalactoside (IPTG), in a vector-specific manner. Expression of AfrLEA-1 in E. coli did not overcome the inhibitory effects of high concentrations of NaCl and KCl but modulated growth inhibition resulting from high concentrations of sorbitol in the growth medium. In contrast, expression of the AfrLEA-1 gene in Saccharomyces cerevisiae did not alter the growth kinetics or permit yeast to tolerate high concentrations of NaCl, KCl, or sorbitol. However, expression of AfrLEA-1 in yeast improved its tolerance to drying (desiccation) and freezing. Under our experimental conditions, both E. coli and S. cerevisiae appear to be potentially suitable hosts to study the function of Artemia group 1 LEA proteins under environmentally stressful conditions.

Anoxia-induced changes in optimal substrate for peripheral nerve

Hyperglycemia accentuates the injury produced by anoxia both in the central and peripheral nervous system. To understand whether this is a consequence of changes in metabolic pathways produced by anoxia, the effect of the metabolic substrate used by the rat peripheral nerve on the nerve action potential (NAP) was studied in the presence and absence of anoxia. In the continuously oxygenated state, the NAP was well preserved with glucose, lactate, as well as with high concentrations of sorbitol and fructose but not β-hydroxybutyrate, acetate or galactose. With intermittent anoxia, the pattern of substrate effects on the NAP changed markedly so that low concentrations of fructose became able to support neurophysiologic activity but not high concentrations of glucose. These alterations occurred gradually with repeated episodes of anoxia as reflected by the progressive increase in the time needed for the NAP to disappear during anoxia when using glucose as substrate. This “preconditioning” effect was not seen with other substrates and an opposite effect was seen with lactate. In fact, the rate at which the NAP disappeared during anoxia was not simply related to degree of recovery after anoxia. These are distinct phenomena. For example, the NAP persisted longest during anoxia in the setting of hyperglycemia but this was the state in which the anoxic damage was most severe.Correlating the results with existing literature on the metabolic functions of Schwann cells and axons generates testable hypotheses for the mechanism of hyperglycemic damage during anoxia and lead to discussions of the role for a metabolic shuttle between Schwann cells and axons as well as a potential important role of glycogen.

Photosystem I shows a higher tolerance to sorbitol‐induced osmotic stress than photosystem II in the intertidal macro‐algae Ulva prolifera (Chlorophyta)

The photosynthetic performance of the desiccation-tolerant, intertidal macro-algae Ulva prolifera was significantly affected by sorbitol-induced osmotic stress. Our results showed that photosynthetic activity decreased significantly with increases in sorbitol concentration. Although the partial activity of both photosystem I (PS I) and photosystem II (PS II) was able to recover after 30 min of rehydration, the activity of PS II decreased more rapidly than PS I. At 4 M sorbitol concentration, the activity of PS II was almost 0 while that of PS I was still at about one third of normal levels. Following prolonged treatment with 1 and 2 M sorbitol, the activity of PS I and PS II decreased slowly, suggesting that the effects of moderate concentrations of sorbitol on PS I and PS II were gradual. Interestingly, an increase in non-photochemical quenching occurred under these conditions in response to moderate osmotic stress, whereas it declined significantly under severe osmotic stress. These results suggest that photoprotection in U. prolifera could also be induced by moderate osmotic stress. In addition, the oxidation of PS I was significantly affected by osmotic stress. P700(+) in the thalli treated with high concentrations of sorbitol could still be reduced, as PS II was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), but it could not be fully oxidized. This observation may be caused by the higher quantum yield of non-photochemical energy dissipation in PS I due to acceptor-side limitation (Y(NA)) during rehydration in seawater containing DCMU.

Pain relief, functional recovery and associated medical treatments reduction in large-scale population with osteoarthritis receiving injections of viscosupplement incorporating high concentration of sorbitol

Pain relief, functional recovery and associated medical treatments reduction in large-scale population with osteoarthritis receiving injections of viscosupplement incorporating high concentration of sorbitol

Mutations of the TATA-binding protein confer enhanced tolerance to hyperosmotic stress in Saccharomyces cerevisiae

Previously, it was shown that overexpression of either of two SPT15 mutant alleles, SPT15-M2 and SPT15-M3, which encode mutant TATA-binding proteins, confer enhanced ethanol tolerance in Saccharomyces cerevisiae. In this study, we demonstrated that strains overexpressing SPT15-M2 or SPT15-M3 were tolerant to hyperosmotic stress caused by high concentrations of glucose, salt, and sorbitol. The enhanced tolerance to high glucose concentrations in particular improved ethanol production from very high gravity (VHG) ethanol fermentations. The strains displayed constitutive and sustained activation of Hog1, a central kinase in the high osmolarity glycerol (HOG) signal transduction pathway of S. cerevisiae. However, the cell growth defect known to be caused by constitutive and sustained activation of Hog1 was not observed. We also found that reactive oxygen species (ROS) were accumulated to a less extent upon exposure to high glucose concentration in our osmotolerant strains. We identified six new genes (GPH1, HSP12, AIM17, SSA4, USV1, and IGD1), the individual deletion of which renders cells sensitive to 50% glucose. In spite of the presence of multiple copies of stress response element in their promoters, it was apparent that those genes were not controlled at the transcriptional level by the HOG pathway under the high glucose conditions. Combined with previously published results, overexpression of SPT15-M2 or SPT15-M3 clearly provides a basis for improved tolerance to ethanol and osmotic stress, which enables construction of strains of any genetic background that need enhanced tolerance to high concentrations of ethanol and glucose, promoting the feasibility for VHG ethanol fermentation.

Overexpression of TsGOLS2, a galactinol synthase, in Arabidopsis thaliana enhances tolerance to high salinity and osmotic stresses

Galactinol synthase (GOLS, EC 2.4.1.123), a key enzyme in the synthesis of raffinose family oligosaccharides (RFOs), catalyzes the condensation of UDP-galactose with myo-inositol to produce galactinol as the sole donor for the synthesis of RFOs. RFOs have been implicated in mitigating effects of environmental stresses on plants. TsGOLS2, was cloned from Thellungiella salsuginea with high homology to AtGOLS2. TsGOLS2 was up-regulated by several abiotic stresses. We overexpressed TsGOLS2 in Arabidopsis thaliana. The contents of galactinol, raffinose, and α-ketoglutaric acid were significantly increased in transgenic plants. Compared to wild type plants, salt-stressed transgenic A.thaliana exhibited higher germination rate, photosynthesis ability, and seedling growth. After being treated with osmotic stress by high concentration of sorbitol, transgenic plants retained high germination rates and grew well during early development. These results indicated that overexpression of TsGOLS2 in A.thaliana improved the tolerance of transgenic plants to high salinity and osmotic stress.