Differential importance of trehalose accumulation in Saccharomyces cerevisiae in response to various environmental stresses

Trehalose is believed to play an important role in stress tolerance in the yeast Saccharomyces cerevisiae. In this research, the responses to various environmental stresses, such as high ethanol concentration, heat, oxidative, and freezing stresses, were investigated in a strain with deletion of the NTH1, NTH2, and ATH1 genes encoding trehalases that are involved in trehalose degradation and the triple deletion strains overexpressing TPS1 or TPS2, both of which encode trehalose biosynthesis enzymes in S. cerevisiae. The contents of trehalose constitutively accumulated in the TPS1- and TPS2-overexpressing triple deletion strains were higher than that in the original triple deletion strain. High trehalose accumulation and growth activity were observed in the TPS2-overexpressing triple deletion strain after ethanol stress induction. The same was also observed in the triple deletion and the TPS1- and TPS2-overexpressing triple deletion strains after heat stress induction. In case of freezing stress, all the recombinant strains with high constitutive trehalose content showed high tolerance. However, in case of oxidative stress, trehalose accumulation could not make the yeast cells tolerant. Our results indicated that high trehalose accumulation can make yeast cells resistant to multiple stresses, but the importance of this accumulation before or after stress induction is varied depending on the type of stress.     

Proline accumulation in baker's yeast enhances high-sucrose stress tolerance and fermentation ability in sweet dough

During bread-making processes, yeast cells are exposed to various baking-associated stresses. High-sucrose concentrations exert severe osmotic stress that seriously damages cellular components by generation of reactive oxygen species (ROS). Previously, we found that the accumulation of proline conferred freeze-thaw stress tolerance and the baker's yeast strain that accumulated proline retained higher-level fermentation abilities in frozen doughs than the wild-type strain. In this study, we constructed self-cloning diploid baker's yeast strains that accumulate proline. These resultant strains showed higher cell viability and lower intracellular oxidation levels than that observed in the wild-type strain under high-sucrose stress condition. Proline accumulation also enhanced the fermentation ability in high-sucrose-containing dough. These results demonstrate the usefulness of proline-accumulating baker's yeast for sweet dough baking.     

Cellular mechanism for the improvement of multiple stress tolerance in brewer's yeast by potassium ion supplementation

The ethanol fermentation efficiency was affected by multiple stress tolerance of yeast during brewing and bioethanol industry. The effect of KCl on the multiple stress tolerance of yeast cells was examined. Results showed that KCl addition significantly enhanced the tolerance of yeast cells to osmotic and ethanol stress, which correlated with the decreased membrane permeability, the increased intracellular ergosterol and ATP content, and the improved activity of complex II and complex III in yeast cells. Biomass and viability of yeast cells under osmotic and ethanol stress were increased significantly by KCl addition. Supplementation of 4 and 10 g L⁻¹ KCl exhibited the best promotion activity for yeast cells present in medium with 500 g L⁻¹ sucrose and 10% (v v⁻¹) ethanol, respectively. These results suggested that exogenous potassium addition might be an effective strategy to improve yeast tolerance and fermentation efficiency during industrial very-high-gravity (VHG) fermentation.     

纤维乙醇用酵母菌的驯化

酵母菌菌种性能的优劣直接关系纤维乙醇发酵质量的好坏及设备的利用率等,进而影响企业的经济效益.该文通过对纤维乙醇生产用酵母菌株的驯化,获得一株驯化酵母菌株:并将该菌株与1308菌株、1300菌株和安琪酵母发酵、耐乙酸、耐糠醛、耐羟甲基糠醛能力进行了对比试验,结果表明该驯化酵母菌株发酵能力、耐受性均优于其他供试菌株;更适宜于在纤维乙醇生产中应用.     

酿酒酵母工业菌株胁迫条件耐受性分析

对酿酒酵母（Saccharomyces cerevisiae）工业菌株胁迫条件，包括高浓度酒精、高渗透压、高温、营养饥饿、氧化胁迫、糠醛毒性的耐受性进行了分析，同时测定了抗生素G418对这些菌株的最低抑菌浓度。结果表明，所测定的酵母菌株对这些逆境条件的耐受性有明显差别，表现出良好耐受性的是6508和安琪酵母菌株，同时多倍性的酿酒酵母工业菌株的耐受性均比单倍性实验室菌株高。     

Ethanol-tolerant Saccharomyces cerevisiae strains isolated under selective conditions by over-expression of a proofreading-deficient DNA polymerase δ

Ethanol damages the cell membrane and functional proteins, gradually reducing cell viability, and leading to cell death during fermentation which impairs effective bioethanol production by budding yeast Saccharomyces cerevisiae. To obtain more suitable strains for bioethanol production and to gain a better understanding of ethanol tolerance, ethanol-tolerant mutants were isolated using the novel mutagenesis technique based on the disparity theory of evolution. According to this theory evolution can be accelerated by affecting the lagging-strand synthesis in which DNA polymerase δ is involved. Expression of the pol3-01 gene, a proofreading-deficient of DNA polymerase δ, in S. cerevisiae W303-1A grown under conditions of increasing ethanol concentration resulted in three ethanol-tolerant mutants (YFY1, YFY2 and YFY3), which could grow in medium containing 13% ethanol. Ethanol productivity also increased in YFY strains compared to the wild-type strain in medium containing 25% glucose. Cell morphology of YFY strain cells was normal even in the presence of 8% ethanol, whereas W303-1A cells were expanded by a big vacuole. Furthermore, two of these mutants were also resistant to high-temperature, Calcofluor white and NaCl. Expression levels of TPS1 and TSL1, which are responsible for trehalose biosynthesis, were higher in YFY strains relative to W303-1A, resulting in high levels of intracellular trehalose in YFY strains. This contributed to the multiple-stress tolerance that makes YFY strains suitable for the production of bioethanol.     

Oleic acid and ergosterol supplementation mitigates oxidative stress in wine strains of Saccharomyces cerevisiae

During fermentation of high-sugar-containing medium lacking lipid nutrients, wine yeasts undergo oxidative stress and oxidative damage to cell membranes and proteins. Considering that cell membranes are important stress sensors, and that under hypoxic conditions wine yeasts modulate cell membranes composition by incorporating lipids available in the growth medium, in the present work, the effects of lipid nutrition on wine yeast oxidative stress response were evaluated on two strains of Saccharomyces cerevisiae. Biomarkers of oxidative stress, oxidative damage and antioxidant response were evaluated together with viability and acetic acid production during fermentation of a synthetic must lacking lipid nutrients as compared to added oleic acid and ergosterol. The results show that the availability of lipid nutrients causes a significant reduction in the intracellular content of reactive oxygen species and in the oxidative damage to membranes and proteins, as indicated by flow cytometry of cells stained with dihydroethidum (DHE) and propidium iodide (PI) and by Western blot of protein carbonyls. Accordingly, lipid nutrients feeding results in the increase in cell viability and superoxide activity, and the reduction in trehalose accumulation, proteinase A activity and production of acetic acid. In summary, these results are compatible with the hypothesis that the supplementation of lipid nutrients mitigates oxidative stress and oxidative damage in wine strains of S. cerevisiae during growth under unfavourable conditions.     

Saccharomyces cerevisiae engineered for xylose metabolism requires gluconeogenesis and the oxidative branch of the pentose phosphate pathway for aerobic xylose assimilation

Saccharomyces strains engineered to ferment xylose using Scheffersomyces stipitis xylose reductase (XR) and xylitol dehydrogenase (XDH) genes appear to be limited by metabolic imbalances, due to differing cofactor specificities of XR and XDH. The S. stipitis XR, which uses both NADH and NADPH, is hypothesized to reduce the cofactor imbalance, allowing xylose fermentation in this yeast. However, unadapted S. cerevisiae strains expressing this XR grow poorly on xylose, suggesting that metabolism is still imbalanced, even under aerobic conditions. In this study, we investigated the possible reasons for this imbalance by deleting genes required for NADPH production and gluconeogenesis in S. cerevisiae. S. cerevisiae cells expressing the XR-XDH, but not a xylose isomerase, pathway required the oxidative branch of the pentose phosphate pathway (PPP) and gluconeogenic production of glucose-6-P for xylose assimilation. The requirement for generating glucose-6-P from xylose was also shown for Kluyveromyces lactis. When grown in xylose medium, both K. lactis and S. stipitis showed increases in enzyme activity required for producing glucose-6-P. Thus, natural xylose-assimilating yeast respond to xylose, in part, by upregulating enzymes required for recycling xylose back to glucose-6-P for the production of NADPH via the oxidative branch of the PPP. Finally, we show that induction of these enzymes correlated with increased tolerance to the NADPH-depleting compound diamide and the fermentation inhibitors furfural and hydroxymethyl furfural; S. cerevisiae was not able to increase enzyme activity for glucose-6-P production when grown in xylose medium and was more sensitive to these inhibitors in xylose medium compared to glucose.     

Pretreatment of the yeast antagonist, Candida oleophila, with glycine betaine increases oxidative stress tolerance in the microenvironment of apple wounds

In response to wounding, harvested fruit tissues of apple and citrus exhibit the production of reactive oxygen species (ROS). ROS production is greater when yeast antagonists used as biocontrol agents are applied in the wounds. These phenomena result in an oxidative stress environment for the yeast antagonists. It has been demonstrated that pre-exposure of some of these yeast antagonists to sublethal abiotic stress (heat or hydrogen peroxide), or stress-ameliorating compounds such as glycine betaine (GB) can induce subsequent oxidative stress tolerance in the antagonistic yeast. The increased level of oxidative stress tolerance has been demonstrated in vitro and is characterized by higher levels of antioxidant gene expression, increased production of trehalose, and lower levels of ROS when yeast are exposed to a subsequent oxidative stress. The current study determined whether or not the effects of GB on yeast antagonists determined in vitro persist and are present in planta when yeast are applied to wounded apples. The effect of exogenous GB on the production of ROS in the yeast antagonist, Candida oleophila, was determined after the yeast was placed in apple wounds. Oxidative damage to yeast cells recovered from apple wounds was also monitored. Results indicated that GB treatment improved the adaptation of C. oleophila to apple fruit wounds. Compared to untreated control yeast cells, GB-treated cells recovered from the oxidative stress environment of apple wounds exhibited less accumulation of ROS and lower levels of oxidative damage to cellular proteins and lipids. Additionally, GB-treated yeast exhibited greater biocontrol activity against Penicillium expansum and Botrytis cinerea, and faster growth in wounds of apple fruits compared to untreated yeast. The expression of major antioxidant genes, including peroxisomal catalase, peroxiredoxin TSA1, and glutathione peroxidase was elevated in the yeast by GB treatment. This study supports the premise that activation of antioxidant response in biocontrol yeast can improve biocontrol efficacy.     

Fermentative capacity of Kluyveromyces marxianus and Saccharomyces cerevisiae after oxidative stress

Volatile compound production during alcoholic fermentation has been studied in the production of many beverages. Temperature, yeast strain, nutrients and pH have been identified as important factors in the production of volatile compounds. In addition, other factors could influence this production during the fermentation process as well. Oxidative stress could occur during yeast biomass production because oxygen is an essential nutrient that is added to the growth medium. The fermentation parameters and the volatile compound production of one Saccharomyces cerevisiae strain (MC4) and two Kluyveromyces marxianus strains (OFF1 and SLP1) were evaluated in relation to fermentation parameters after oxidative stress induced by hydrogen peroxide or menadione. These yeasts were compared with S. cerevisiae W303–1A and showed significant differences in ethanol production, ethanol yield and maximum ethanol production rate. K. marxianus (OFF1) showed better fermentative capacity after oxidative stress. The higher alcohol production decreased after oxidative stress by >35% after 72 h fermentation time, and the amyl alcohol decreased at a higher level (>60%); however, the isobutanol production increased after oxidative stress between 1.5 and 4 times. The yeasts produced significant concentrations of esters however ethyl lactate, ethyl caprylate and the ethyl caproate were not detected in the control fermentation, while in the stress fermentation they accounted for up to 3 mg/L. These results demonstrate that oxidative stress can play an important role in the final aroma profile; but it is necessary to guarantee adequate yeast growth to obtain the volatile compounds desired. Copyright © 2017 The Institute of Brewing & Distilling     

CAMBRIDGE PRIZE LECTURE IMPROVING YEAST FERMENTATION PERFORMANCE

A number of factors affecting yeast fermentation performance have been investigated. These include the mode of substrate feeding, nutrient supplementation, temperature, osmotic pressure, oxygen, intracellular ethanol accumulation, and yeast ethanol tolerance. Nutrient supplementation was observed to play a key role in yeast fermentations employing high gravity media and at high temperatures. Furthermore, complete attenuation of high gravity wort (25°P) could be achieved by optimizing the yeast pitching rate, fermentation temperature, and level of wort oxygenation. Genetic manipulation techniques, such as spheroplast fusion, were successfully employed to obtain ethanol and osmotolerant yeast strains. In addition, a number of stable 2-deoxy-D-glucose resistant mutants, isolated from brewing and non-brewing yeast strains, were observed to rapidly utilize maltose and maltotriose in the presence of high concentrations of glucose. Fermentation and ethanol production rates were increased in these strains. Therefore, employing strategies of optimized fermentation conditions and strain development have resulted in improvements in yeast fermentation performance.     

转录因子MSN2基因过表达对酿酒酵母耐受性的影响

cAMP信号通路在调控酵母细胞代谢、增殖、分化及压力抗性的获得过程中具有重要的作用。工业应用中对酵母的耐受性有很高的要求,在发酵过程中,胁迫环境如高温、高渗透压、营养饥饿和高浓度酒精毒性等不可避免,故而提高酵母菌种的耐受性,可以提高菌种的发酵性能,降低发酵过程中的能量消耗。本文构建的突变株在工业应用方面具有重要的意义。以实验室现有菌种AY12a为出发菌株,URA3基因作筛选标记,利用胞内重组,在MSN2基因的N端加上强启动子PGK1_p以实现基因的过表达,最终通过PCR验证,成功构建突变株AY12a-msn2。对酵母进行耐受性的测定,发现AY12a-msn2不具有一定的耐高温性能。同时将突变株与AY12a进行玉米高温浓醪发酵,并测定发酵完成后的酒度、残糖、48 h细胞存活率、CO_2失重及发酵时间。结果发现突变株AY12a-msn2酒度下降,残糖含量上升,48 h细胞存活率上升,发酵时间较长。     

Overexpression of vacuolar H+‐ATPase‐related genes in bottom‐fermenting yeast enhances ethanol tolerance and fermentation rates during high‐gravity fermentation

Vacuolar H⁺‐ATPase (V‐ATPase) is thought to play a role in stress tolerance. In this study it was found that bottom‐fermenting yeast strains, in which the V‐ATPase‐related genes DBF2, VMA41/CYS4/NHS5 and RAV2 were overexpressed, exhibited stronger ethanol tolerance than the parent strain and showed increased fermentation rates in a high‐sugar medium simulating high‐gravity fermentation. Among the strains examined, the DBF2‐overexpressing bottom‐fermenting yeast strain exhibited the highest ethanol tolerance and fermentation rate in YPM20 medium. Using this strain, high‐gravity fermentation was performed by adding sugar to the wort, which led to increased fermentation rates and yeast viability compared with the parent strain. These findings indicate that V‐ATPase is a stress target in high‐gravity fermentation and suggests that enhancing the V‐ATPase activity increases the ethanol tolerance of bottom‐fermenting yeast, thereby improving the fermentation rate and cell viability under high‐gravity conditions. Copyright © 2012 The Institute of Brewing & Distilling     

Respiration-deficient mutants of Zymomonas mobilis show improved growth and ethanol fermentation under aerobic and high temperature conditions

Respiration-deficient mutant (RDM) strains of Zymomonas mobilis were isolated from antibiotic-resistant mutants. These RDM strains showed various degrees of respiratory deficiency. All RDM strains exhibited much higher ethanol fermentation capacity than the wild-type strain under aerobic conditions. The strains also gained thermotolerance and exhibited greater ethanol production at high temperature (39°C), under both non-aerobic and aerobic conditions, compared with the wild-type strain. Microarray and subsequent quantitative PCR analyses suggest that enhanced gene expression involved in the metabolism of glucose to ethanol resulted in the high ethanol production of RDM strains under aerobic growth conditions. Reduction of intracellular oxidative stress may also result in improved ethanol fermentation by RDM strains at high temperatures.