Changes in intracellular metabolism underlying the adaptation of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains to ethanol stress

Saccharomyces cerevisiae is often stressed by the ethanol which accumulates during the production of bioethanol by the fermentation process. The study of ethanol-adapted S. cerevisiae strains provide an opportunity to clarify the molecular mechanism underlying the adaptation or tolerance of S. cerevisiae to ethanol stress. The aim of this study was to clarify this molecular mechanism by investigating the ethanol adaptation-associated intracellular metabolic changes in S. cerevisiae using a gas chromatography–mass spectrometry-based metabolomics strategy. A partial least-squares-discriminant analysis between the parental strain and ethanol-adapted strains identified 12 differential metabolites of variable importance with a projection value of >1. The ethanol-adapted strains had a more activated glycolysis pathway and higher energy production than the parental strain, suggesting the possibility that an increased energy production and energy requirement might be partly responsible for an increased ethanol tolerance. An increased glycine content also partly contributed to the higher ethanol tolerance of the ethanol-adapted strains. The decreased oleic acid content may be a self-protection mechanism of ethanol-adapted strains to maintain membrane integrity through decreasing membrane fluidity. We suggest that while being exposed to ethanol stress, ethanol-adapted S. cerevisiae cells may remodel their metabolic phenotype and the composition of their cell membrane to adapt to ethanol stress and acquire higher ethanol tolerance.     

Stress Tolerance Variations in Saccharomyces cerevisiae Strains from Diverse Ecological Sources and Geographical Locations

The budding yeast Saccharomyces cerevisiae is a platform organism for bioethanol production from various feedstocks and robust strains are desirable for efficient fermentation because yeast cells inevitably encounter stressors during the process. Recently, diverse S. cerevisiae lineages were identified, which provided novel resources for understanding stress tolerance variations and related shaping factors in the yeast. This study characterized the tolerance of diverse S. cerevisiae strains to the stressors of high ethanol concentrations, temperature shocks, and osmotic stress. The results showed that the isolates from human-associated environments overall presented a higher level of stress tolerance compared with those from forests spared anthropogenic influences. Statistical analyses indicated that the variations of stress tolerance were significantly correlated with both ecological sources and geographical locations of the strains. This study provides guidelines for selection of robust S. cerevisiae strains for bioethanol production from nature.     

Physiological and molecular characterisation of Saccharomyces cerevisiae cachaça strains isolated from different geographic regions in Brazil

In this work, 74 Saccharomyces cerevisiae strains isolated from cachaça fermentation of six different geographic regions in Brazil were characterized by mitochondrial DNA restriction fragment length polymorphism (mtDNA-RFLP) and by their ability to grow on stress conditions occurring during the cachaça fermentation process. Cachaça S. cerevisiae strains showed high mtDNA-RFLP polymorphism with the occurrence of 32 different molecular patterns. The S. cerevisiae strains presenting prevalent mtDNA were able to grow better in the stress conditions than strains represented by infrequent patterns. The principal coordinate analysis on 17 stress conditions revealed that the major source of growth variation were high ethanol concentrations and low temperatures. These results indicate that the stress conditions occurring in the fermentation process influence the prevalence of the most adapted S. cerevisiae strains in each distillery. The physiological tests used in our study can be used as a criterion for rapidly selecting autochthonous yeast strains for further purposes such as the selection of fermentative starters of S. cerevisiae strains.     

Purification and biochemical characterization of Msi3, an essential Hsp110 molecular chaperone in Candida albicans

Hsp110s are unique and essential molecular chaperones in the eukaryotic cytosol. They play important roles in maintaining cellular protein homeostasis. Candida albicans is the most prevalent yeast opportunistic pathogen that causes fungal infections in humans. As the only Hsp110 in Candida albicans, Msi3 is essential for the growth and infection of Candida albicans. In this study, we have expressed and purified Msi3 in nucleotide-free state and carried out biochemical analyses. Sse1 is the major Hsp110 in budding yeast S. cerevisiae and the best characterized Hsp110. Msi3 can substitute Sse1 in complementing the temperature-sensitive phenotype of S. cerevisiae carrying a deletion of SSE1 gene although Msi3 shares only 63.4% sequence identity with Sse1. Consistent with this functional similarity, the purified Msi3 protein shares many similar biochemical activities with Sse1 including binding ATP with high affinity, changing conformation upon ATP binding, stimulating the nucleotide-exchange for Hsp70, preventing protein aggregation, and assisting Hsp70 in refolding denatured luciferase. These biochemical characterizations suggested that Msi3 can be used as a model for studying the molecular mechanisms of Hsp110s.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12192-021-01213-5.     

松萝内生酿酒酵母菌株耐酸生理特性与分子机制探究

【目的】对我国西藏地区来源的不同酵母菌株进行有机酸发酵性能测试,此外,对具有良好产酸性能的分离自松萝内部的酿酒酵母菌株Saccharomy cescerevisiae 2-2进行耐酸性能分析,并探究其耐酸较强的分子机制。【方法】比较不同糖浓度培养基液体发酵培养过程中pH的变化,并比较低pH胁迫条件下菌株的生长,检测酿酒酵母菌株的产酸潜力和耐酸特性;对菌株2-2和模式酵母菌株S288C进行比较基因组分析,并利用实时荧光定量聚合酶链式反应(real-time fluorescence quantitative polymerase chain reaction,RT-qPCR)分析关键基因的转录,探究菌株2-2耐酸分子机制。【结果】松萝内生酿酒酵母2-2在所有检测的菌株中产酸潜力较大,耐酸性能较好。在菌株2-2中与胁迫耐受性相关的基因PDR15、PDR12和SUR1在低pH胁迫条件下存在显著的上调或下调,但这些基因转录变化趋势与菌株S288C相反。【结论】松萝内生酿酒酵母2-2是一株产酸耐酸性能较好的菌株,对其独特的调节机制进行深入分析,有希望选育性能更好的产酸酵母菌株。     

Oxidative stress and aging: Learning from yeast lessons

The yeast Saccharomyces cerevisiae has played a vital role in the understanding of the molecular basis of aging and the relationship of aging process with oxidative stress (non-homeostatic accumulation of Reactive Oxygen Species, ROS). The mammalian and yeast antioxidant responses are similar and over 25 % of human-degenerative disease related genes have close homologues in yeast. The reduced genetic redundancy of yeast facilitates visualization of the effect of a deleted or mutated gene. By manipulating growth conditions, yeast cells can survive only fermenting (low ROS levels) or respiring (increased ROS levels), which facilitates the elucidation of the mechanisms involved with acquisition of tolerance to oxidative stress. Furthermore, the yeast databases are the most complete of all eukaryotic models. In this work, we highlight the value of S. cerevisiae as a model to investigate the oxidative stress response and its potential impact on aging and age-related diseases.     

Activity of the α-glucoside transporter Agt1 in Saccharomyces cerevisiae cells during dehydration-rehydration events

Microbial cells can enter a state of anhydrobiosis under desiccating conditions. One of the main determinants of viability during dehydration-rehydration cycles is structural integrity of the plasma membrane. Whereas much is known about phase transitions of the lipid bilayer, there is a paucity of information on changes in activity of plasma membrane proteins during dehydration-rehydration events. We selected the α-glucoside transporter Agt1 to gain insights into stress mechanisms/responses and ecophysiology during anhydrobiosis. As intracellular water content of S. cerevisiae strain 14 (a strain with moderate tolerance to dehydration-rehydration) was reduced to 1.5 g water/g dry weight, the activity of the Agt1 transporter decreased by 10–15 %. This indicates that functionality of this trans-membrane and relatively hydrophobic protein depends on water. Notably, however, levels of cell viability were retained. Prior incubation in the stress protectant xylitol increased stability of the plasma membrane but not Agt1. Studies were carried out using a comparator yeast which was highly resistant to dehydration-rehydration (S. cerevisiae strain 77). By contrast to S. cerevisiae strain 14, there was no significant reduction of Agt1 activity in S. cerevisiae strain 77 cells. These findings have implications for the ecophysiology of S. cerevisiae strains in natural and industrial systems.     

The antioxidant system in Suaeda salsa under salt stress

ABSTRACTSuaeda salsaL. is a typical euhalophyte and is widely distributed throughout the world. Suaeda plants are important halophyte resources, and the physiological and biochemical characteristics of their various organsand their response to salt stress have been intensively studied. Leaf succulence, intracellular ion localization, increased osmotic regulation and enhanced antioxidant capacities are important responses for Suaeda plants to adapt to salt stress. Among these responses, scavenging of reactive oxygen species (ROS) is an important mechanism for plants to withstand oxidative stress and improve salt tolerance. The generation and scavenging pathways of ROS, as well as the expression of scavenging enzymes change under salt stress. This article reviews the antioxidant system constitute of S. salsa, and the mechanisms by which S. salsaantioxidant capacity is improved for salt tolerance. In addition, the differences between types of antioxidant mechanisms in S. salsaare reviewed, thereby revealing the adaptation mechanisms of Suaeda to different habitats. The review provides important clues for the comprehensive understanding of the salt tolerance mechanisms of halophytes.KEYWORDS: Suaeda salsa, halophyte, salt-tolerance mechanism, oxidative stress, antioxidant system     

Photosynthesis and antioxidative defense mechanisms in deciphering drought stress tolerance of crop plants

Crop plants are regularly exposed to an array of abiotic and biotic stresses, among them drought stress is a major environmental factor that shows adverse effects on plant growth and productivity. Because of this these factors are considered as hazardous for crop production. Drought stress elicits a plethora of responses in plants resulting in strict amendments in physiological, biochemical, and molecular processes. Photosynthesis is the most fundamental physiological process affected by drought due to a reduction in the CO₂ assimilation rate and disruption of primary photosynthetic reactions and pigments. Drought also expedites the generation of reactive oxygen species (ROS), triggering a cascade of antioxidative defense mechanisms, and affects many other metabolic processes as well as affecting gene expression. Details of the drought stress-induced changes, particularly in crop plants, are discussed in this review, with the major points: 1) leaf water potentials and water use efficiency in plants under drought stress; 2) increased production of ROS under drought leading to oxidative stress in plants and the role of ROS as signaling molecules; 3) molecular responses that lead to the enhanced expression of stress-inducible genes; 4) the decrease in photosynthesis leading to the decreased amount of assimilates, growth, and yield; 5) the antioxidant defense mechanisms comprising of enzymatic and non-enzymatic antioxidants and the other protective mechanisms; 6) progress made in identifying the drought stress tolerance mechanisms; 7) the production of transgenic crop plants with enhanced tolerance to drought stress.     

酿酒酵母中基于CRISPR/Cas9工具的基因编辑与代谢调控研究进展

CRISPR/Cas9系统已广泛用于各种生物体的基因编辑和代谢工程。本文综述了CRISPR/Cas9在酿酒酵母中的基本原理和实际应用。首先总结了CRISPR/Cas9技术的发展历史、酿酒酵母基因组中基因缺失和多DNA片段插入的成功案例。这一先进的系统减少了劳动力,增强了对分子遗传学的理解,加速了微生物工程的发展。其次总结了基于CRISPR/Cas9的系统在生产高附加值化学品和提高酿酒酵母耐应激性方面的研究进展。该综述对酿酒酵母的遗传和合成生物学研究具有重要的参考价值。     

In Vivo Assessment of Cold Tolerance through Chlorophyll-a Fluorescence in Transgenic Zoysiagrass Expressing Mutant Phytochrome A

Chlorophyll-a fluorescence analysis provides relevant information about the physiology of plants growing under abiotic stress. In this study, we evaluated the influence of cold stress on the photosynthetic machinery of transgenic turfgrass, Zoysia japonica, expressing oat phytochrome A (PhyA) or a hyperactive mutant phytochrome A (S599A) with post-translational phosphorylation blocked. Biochemical analysis of zoysiagrass subjected to cold stress revealed reduced levels of hydrogen peroxide, increased proline accumulation, and enhanced specific activities of antioxidant enzymes compared to those of control plants. Detailed analyses of the chlorophyll-a fluorescence data through the so-called OJIP test exhibited a marked difference in the physiological status among transgenic and control plants. Overall, these findings suggest an enhanced level of cold tolerance in S599A zoysiagrass cultivars as reflected in the biochemical and physiological analyses. Further, we propose that chlorophyll-a fluorescence analysis using OJIP test is an efficient tool in determining the physiological status of plants under cold stress conditions.     

The ethanol stress response and ethanol tolerance of Saccharomyces cerevisiae

Saccharomyces cerevisiae is traditionally used for alcoholic beverage and bioethanol production; however, its performance during fermentation is compromised by the impact of ethanol accumulation on cell vitality. This article reviews studies into the molecular basis of the ethanol stress response and ethanol tolerance of S. cerevisiae; such knowledge can facilitate the development of genetic engineering strategies for improving cell performance during ethanol stress. Previous studies have used a variety of strains and conditions, which is problematic, because the impact of ethanol stress on gene expression is influenced by the environment. There is however some commonality in Gene Ontology categories affected by ethanol assault that suggests that the ethanol stress response of S. cerevisiae is compromised by constraints on energy production, leading to increased expression of genes associated with glycolysis and mitochondrial function, and decreased gene expression in energy‐demanding growth‐related processes. Studies using genome‐wide screens suggest that the maintenance of vacuole function is important for ethanol tolerance, possibly because of the roles of this organelle in protein turnover and maintaining ion homoeostasis. Accumulation of Asr1 and Rat8 in the nucleus specifically during ethanol stress suggests S. cerevisiae has a specific response to ethanol stress although this supposition remains controversial.     

Improve carbon metabolic flux in Saccharomyces cerevisiae at high temperature by overexpressed TSL1 gene

This study describes a novel strategy to improve the glycolysis flux of Saccharomyces cerevisiae at high temperature. The TSL1 gene-encoding regulatory subunit of the trehalose synthase complex was overexpressed in S. cerevisiae Z-06, which increased levels of trehalose synthase activity in extracts, enhanced stress tolerance and glucose consuming rate of the yeast cells. As a consequence, the final ethanol concentration of 185.5 g/L was obtained at 38 °C for 36 h (with productivity up to 5.2 g/L/h) in 7-L fermentor, and the ethanol productivity was 92.7 % higher than that of the parent strain. The results presented here provide a novel way to enhance the carbon metabolic flux at high temperature, which will be available for the purposes of producing other primary metabolites of commercial interest using S. cerevisiae as a host.     

Characterization of the Viable but Nonculturable (VBNC) State in Saccharomyces cerevisiae

The Viable But Non Culturable (VBNC) state has been thoroughly studied in bacteria. In contrast, it has received much less attention in other microorganisms. However, it has been suggested that various yeast species occurring in wine may enter in VBNC following sulfite stress.In order to provide conclusive evidences for the existence of a VBNC state in yeast, the ability of Saccharomyces cerevisiae to enter into a VBNC state by applying sulfite stress was investigated. Viable populations were monitored by flow cytometry while culturable populations were followed by plating on culture medium. Twenty-four hours after the application of the stress, the comparison between the culturable population and the viable population demonstrated the presence of viable cells that were non culturable. In addition, removal of the stress by increasing the pH of the medium at different time intervals into the VBNC state allowed the VBNC S. cerevisiae cells to “resuscitate”. The similarity between the cell cycle profiles of VBNC cells and cells exiting the VBNC state together with the generation rate of cells exiting VBNC state demonstrated the absence of cellular multiplication during the exit from the VBNC state. This provides evidence of a true VBNC state. To get further insight into the molecular mechanism pertaining to the VBNC state, we studied the involvement of the SSU1 gene, encoding a sulfite pump in S. cerevisiae. The physiological behavior of wild-type S. cerevisiae was compared to those of a recombinant strain overexpressing SSU1 and null Δssu1 mutant. Our results demonstrated that the SSU1 gene is only implicated in the first stages of sulfite resistance but not per se in the VBNC phenotype. Our study clearly demonstrated the existence of an SO₂-induced VBNC state in S. cerevisiae and that the stress removal allows the “resuscitation” of VBNC cells during the VBNC state.