Tolerant Yeast Research Articles

Analysis of the chloroplast crotonylome of wheat seedling leaves reveals the roles of crotonylated proteins involved in salt-stress responses.

Lysine crotonylation (Kcr) is a novel post-translational modification and its function in plant salt-stress responses remains unclear. In this study, we performed the first comprehensive chloroplast crotonylome analysis of wheat seedling leaves to examine the potential functions of Kcr proteins in salt-stress responses. In a total of 471 chloroplast proteins, 1290 Kcr sites were identified as significantly regulated by salt stress, and the Kcr proteins were mainly involved in photosynthesis, protein folding, and ATP synthesis. The identified Kcr sites that responded to salt stress were concentrated within KcrK and KcrF motifs, with the conserved KcrF motif being identified in the Kcr proteins of wheat chloroplasts for the first time. Notably, 10 Kcr sites were identified in fructose-1,6-bisphosphate aldolase (TaFBA6), a key chloroplast metabolic enzyme involved in the Calvin-Benson cycle. Site-directed mutagenesis of TaFBA6 showed that the Kcr at K367 is critical in maintaining its enzymatic activity and in conferring salt tolerance in yeast. Further molecular dynamic simulations and analyses of surface electrostatic potential indicated that the Kcr at K367 could improve the structural stability of TaFBA6 by decreasing the distribution of positive charges on the protein surface to resist alkaline environments, thereby promoting both the activity of TaFBA6 and salt tolerance.

Molecular identification of yeasts from Turkish traditional cheeses: Extracellular enzyme activities and physiological properties important for dairy industry

Abstract. Gunay M, Genc TT. 2023. Molecular identification of yeasts from Turkish traditional cheeses: Extracellular enzyme activities and physiological properties important for dairy industry. Nusantara Bioscience 15: 1-11. The determination of yeast microbiota in cheeses and the physiological properties of yeasts are very important for the dairy industry. In addition, the physiological features, proteolytic and lipolytic activities, and stress tolerance of yeasts have a significant role in the selection of starter yeast species for cheese ripening. This study aimed to determine industrially important yeasts isolated from cheese samples. Molecular techniques identified the isolated yeast strains. The yeast strains’ extracellular enzyme activities, fermentation capacities, and thermotolerance and osmotolerance properties were also evaluated. A total of 81 yeast strains were isolated and characterized from three types of cheese samples. PCR-RFLP determined the isolated yeast strains and sequence analysis of ITS1-5.8S-ITS2 and 26S rDNA regions. A maximum parsimony tree was constructed by MEGA X software to evaluate the phylogenetic relationship of identified yeast strains. Candida intermedia, Candida parapsilosis, Clavispora lusitaniae, Debaryomyces hansenii, Kluyveromyces marxianus, Pichia kudriavzevii, and Wickerhamomyces anomalus yeast species were identified on cheese samples. The distribution of identified yeast species on cheese samples was determined as 48.1% for W. anomalus, 17.3% for K. marxianus, 14.8% for C. parapsilosis, 8.6% for D. hansenii, 4.9% for Cl. lusitaniae, 3.7% for C. intermedia and 2.5% for P. kudriavzevii. The W. anomalus yeast species was common in three cheese types. All strains of W. anomalus and P. kudriavzevii yeast species, three C. parapsilosis, and two Cl. lusitaniae yeast strains have important physiological properties for industrial applications. These yeast strains have the potential to be used in combination as starter cultures to improve cheese maturation in the future. This comprehensive study identifies yeast species by ITS1-5.8S-ITS2 and 26S rDNA regions and determines industrially important yeast species using multiple criteria (extracellular enzyme activity, stress tolerance, and fermentation capacity).

Sumoylation is Largely Dispensable for Normal Growth but Facilitates Heat Tolerance in Yeast.

Numerous proteins are sumoylated in normally growing yeast and SUMO conjugation levels rise upon exposure to several stress conditions. We observe high levels of sumoylation also during early exponential growth and when nutrient-rich medium is used. However, we find that reduced sumoylation (∼75% less than normal) is remarkably well-tolerated, with no apparent growth defects under nonstress conditions or under osmotic, oxidative, or ethanol stresses. In contrast, strains with reduced activity of Ubc9, the sole SUMO conjugase, are temperature-sensitive, implicating sumoylation in the heat stress response, specifically. Aligned with this, a mild heat shock triggers increased sumoylation which requires functional levels of Ubc9, but likely also depends on decreased desumoylation, since heat shock reduces protein levels of Ulp1, the major SUMO protease. Furthermore, we find that a ubc9 mutant strain with only ∼5% of normal sumoylation levels shows a modest growth defect, has abnormal genomic distribution of RNA polymerase II (RNAPII), and displays a greatly expanded redistribution of RNAPII after heat shock. Together, our data implies that SUMO conjugations are largely dispensable under normal conditions, but a threshold level of Ubc9 activity is needed to maintain transcriptional control and to modulate the redistribution of RNAPII and promote survival when temperatures rise.

Evolutionary and reverse engineering in Saccharomyces cerevisiae reveals a Pdr1p mutation-dependent mechanism for 2-phenylethanol tolerance

Background2-Phenylethanol (2-PE), a higher alcohol with a rose-like odor, inhibits growth of the producer strains. However, the limited knowledge regarding 2-PE tolerance mechanisms renders our current knowledge base insufficient to inform rational design.ResultsTo improve the growth phenotype of Saccharomyces cerevisiae under a high 2-PE concentration, adaptive laboratory evolution (ALE) was used to generate an evolved 19–2 strain. Under 2-PE stress, its OD600 and growth rate increased by 86% and 22% than that of the parental strain, respectively. Through whole genome sequencing and reverse engineering, transcription factor Pdr1p mutation (C862R) was revealed as one of the main causes for increased 2-PE tolerance. Under 2-PE stress condition, Pdr1p mutation increased unsaturated fatty acid/saturated fatty acid ratio by 42%, and decreased cell membrane damage by 81%. Using STRING website, we identified Pdr1p interacted with some proteins, which were associated with intracellular ergosterol content, reactive oxygen species (ROS), and the ATP-binding cassette transporter. Also, the results of transcriptional analysis of genes encoded these proteins confirmed that Pdr1p mutation induced the expression of these genes. Compared with those of the reference strain, the ergosterol content of the PDR1_862 strain increased by 72%–101%, and the intracellular ROS concentration decreased by 38% under 2-PE stress. Furthermore, the Pdr1p mutation also increased the production of 2-PE (11% higher).ConclusionsIn the present work, we have demonstrated the use of ALE as a powerful tool to improve yeast tolerance to 2-PE. Based on the reverse engineering, transcriptional and physiological analysis, we concluded that Pdr1p mutation significantly enhanced the 2-PE tolerance of yeast by regulating the fatty acid proportion, intracellular ergosterol and ROS. It provides new insights on Pdr1p mediated 2-PE tolerance, which could help in the design of more robust yeasts for natural 2-PE synthesis.

The potential of multistress tolerant yeast, Saccharomycodes ludwigii, for second-generation bioethanol production

Ethanol production at high temperatures using lignocellulosic biomass as feedstock requires a highly efficient thermo and lignocellulosic inhibitor-tolerant ethanologenic yeast. In this study, sixty-three yeast isolates were obtained from tropical acidic fruits using a selective acidified medium containing 80 mM glacial acetic acid. Twenty-nine of the yeast isolates exhibited significant thermo and acetic acid-tolerant fermentative abilities. All these isolates were classified into three major yeast species, namely Saccharomycodes ludwigii, Pichia kudriavzevii, and P. manshurica, based on molecular identification. Saccharomycodes ludwigii APRE2 displayed an ability to grow at high temperatures of up to 43 °C and exhibited significant multistress tolerance toward acetic acid, furfural, 5-hydroxymethyl furfural (5-HMF), and ethanol among the isolated yeast species. It can produce a maximum ethanol concentration of 63.07 g/L and productivity of 1.31 g/L.h in yeast extract malt extract (YM) medium containing 160 g/L glucose and supplemented with 80 mM acetic acid and 15 mM furfural as a cocktail inhibitor. When an acid-pretreated pineapple waste hydrolysate (PWH) containing approximately 106 g/L total sugars, 131 mM acetic acid, and 3.95 mM furfural was used as a feedstock, 38.02 g/L and 1.58 g/L.h of ethanol concentration and productivity, respectively, were achieved. Based on the results of the current study, the new thermo and acetic acid-tolerant yeast S. ludwigii APRE2 exhibited excellent potential for second-generation bioethanol production at high temperatures.

Genome-Wide Analysis of Wheat GATA Transcription Factor Genes Reveals Their Molecular Evolutionary Characteristics and Involvement in Salt and Drought Tolerance.

GATA transcription factor genes participate in plant growth, development, morphogenesis, and stress response. In this study, we carried out a comprehensive genome-wide analysis of wheat GATA transcription factor genes to reveal their molecular evolutionary characteristics and involvement in salt and drought tolerance. In total, 79 TaGATA genes containing a conserved GATA domain were identified in the wheat genome, which were classified into four subfamilies. Collinear analysis indicated that fragment duplication plays an important role in the amplification of the wheat GATA gene family. Functional disproportionation analysis between subfamilies found that both type I and type II functional divergence simultaneously occurs in wheat GATA genes, which might result in functional differentiation of the TaGATA gene family. Transcriptional expression analysis showed that TaGATA genes generally have a high expression level in leaves and in response to drought and salt stresses. Overexpression of TaGATA62 and TaGATA73 genes significantly enhanced the drought and salt tolerance of yeast and Arabidopsis. Protein-protein docking indicated that TaGATAs can enhance drought and salt tolerance by interacting between the DNA-binding motif of GATA transcription factors and photomorphogenesis-related protein TaCOP9-5A. Our results provided a base for further understanding the molecular evolution and functional characterization of the plant GATA gene family in response to abiotic stresses.

Genome-Wide Characterization and Function Analysis of ZmERD15 Genes’ Response to Saline Stress in Zea mays L.

Early responsive dehydration (ERD) genes can be rapidly induced by dehydration. ERD15 genes have been confirmed to regulate various stress responses in plants. However, the maize ERD15 members have not been characterized. In the present study, a total of five ZmERD15 genes were identified from the maize genome and named ZmERD15a, ZmERD15b, ZmERD15c, ZmERD15d, and ZmERD15e. Subsequently, their protein properties, gene structure and duplication, chromosomal location, cis-acting elements, subcellular localization, expression pattern, and over-expression in yeast were analyzed. The results showed that the ZmERD15 proteins were characterized by a similar size (113-159 aa) and contained a common domain structure, with PAM2 and adjacent PAE1 motifs followed by an acidic region. The ZmERD15 proteins exhibited a close phylogenetic relationship with OsERD15s from rice. Five ZmERD15 genes were distributed on maize chromosomes 2, 6, 7, and 9 and showed a different exon-intron organization and were expanded by duplication. Besides, the promoter region of the ZmERD15s contained abundant cis-acting elements that are known to be responsive to stress and hormones. Subcellular localization showed that ZmERD15b and ZmERD15c were localized in the nucleus. ZmERD15a and ZmERD15e were localized in the nucleus and cytoplasm. ZmERD15d was localized in the nucleus and cell membrane. The results of the quantitative real-time PCR (qRT-PCR) showed that the expression of the ZmERD15 genes was regulated by PEG, salinity, and ABA. The heterologous expression of ZmERD15a, ZmERD15b, ZmERD15c, and ZmERD15d significantly enhanced salt tolerance in yeast. In summary, a comprehensive analysis of ZmERD15s was conducted in the study. The results will provide insights into further dissecting the biological function and molecular mechanism of ZmERD15s regulating of the stress response in maize.

Isolation of Three Metallothionein Genes and Their Roles in Mediating Cadmium Resistance

Isolating the genes responsible for cadmium (Cd) accumulation and tolerance in oilseed rape and uncovering their functional mechanism is of great significance for guiding genetic improvement to cope with heavy metal pollution. In this study, we screened the cDNA library of Brassica napus cv. Westar using a yeast genetic complementation system and isolated BnMT2-22a, BnMT2-22b and BnMT3b, which can mediate Cd tolerance in yeast. They all have two cysteine-rich domains in their sequence. Ectopic expression of these MTs demonstrated that all of them enhanced Cd and Cu tolerance in yeast, but had no effect on Mn and Zn tolerance. The fusion of the red fluorescent protein mRFP did not affect their function in mediating Cd tolerance, and using these functional fusion proteins we observed that they were all localized in cytosol. Meanwhile, their expression in yeast did not affect the accumulation of Cd in the yeast transformants. Gene expression analyses found that BnMT2-22a, BnMT2-22b and BnMT3b were all induced by Cd in roots, and BnMT3b was also significantly induced in shoots. These results indicate that the genes BnMT2-22a, BnMT2-22b and BnMT3b isolated with cDNA library screening can mediate Cd tolerance, and they may detoxify Cd via cytosolic chelation.

Impact of ergosterol content on acetic and lactic acids toxicity to Saccharomyces cerevisiae.

Organic acid stress often represents a major hurdle in industrial bio-based microbial processes. Organic acids can be released from lignocellulosic feedstocks pretreatment and can also be desirable products obtained by microbial fermentation with applications in different industrial sectors. Yeasts are prominent cell factories. However, the presence of organic acids can compromise yeast metabolism, impairing fermentation performances and limiting the economic feasibility of the processes. Plasma membrane remodeling is deeply involved in yeast tolerance to organic acids, but the detailed mechanisms and potentials of this phenomenon remain largely to be studied and exploited. We investigated the impact of ergosterol on Saccharomyces cerevisiae tolerance against organic acid stress by coupling in vitro and in vivo assays. In the in vitro assay, synthetic lipid vesicles were prepared containing different concentrations of ergosterol. We observed changes in organic acids diffusion through the membrane as a function of ergosterol content. Then, we extended our approach in vivo, engineering S. cerevisiae with the aim of changing the ergosterol content of cells. We focused on ECM22, an important transcription factor, involved in the regulation of ergosterol biosynthesis. The overexpression of ECM22 was sufficient to increase ergosterol levels in S. cerevisiae, resulting in an enhanced tolerance toward lactic acid stress. In this work we propose an in vitro approach, using synthetic lipid vesicles, as a complementary method to be used when studying the impact of the plasma membrane lipid composition on the diffusion of organic acids.

A Na+/H+ antiporter localized on the Golgi-to-vacuole transport system from Camellia sinensis, CsNHX6, plays a positive role in salt tolerance

Tea plant (Camellia sinensis) is an important traditional horticultural plant known for the tea products processed from its leaves, which often faces many different adverse conditions, including saline environments. Na+/H+ antiporters (NHXs) are extensively involved in the process of plant response to salt stress and resistance acquisition, but studies in tea plant are less common. In this study, a novel NHX gene named CsNHX6 was cloned from tea plant, which encodes 528 amino acids with 12 typical transmembrane domains. Our results showed that CsNHX6 had both Na+ and K+ dual transport function, and the transport activity depended on an appropriate H+ concentration. In addition, three conserved acidic residues, D164, E188 and D193 in CsNHX6, are essential for Na+ and K+ transport. Further, we found that CsNHX6 was significantly induced by salt stress, and its overexpression enhanced the tolerance of yeast and Arabidopsis to salt stress, this was closely related to the improvement of Na+ storage capacity of cells. Furthermore, subcellular localization assay revealed that CsNHX6 was localized in a Golgi-to-vacuole transport system, including Golgi, TGN, PVC and vacuole, and this localized distribution could be enhanced by salt stress. Taken together, these findings suggest that a potential Na+ transport network is dominated by CsNHX6 under salt stress, which directly or indirectly achieves the regionalization of excessive Na+, thus endowing organisms with salt tolerance.

Identification of acetic acid sensitive strains through biosensor-based screening of a Saccharomyces cerevisiae CRISPRi library

BackgroundAcetic acid tolerance is crucial for the development of robust cell factories for conversion of lignocellulosic hydrolysates that typically contain high levels of acetic acid. Screening mutants for growth in medium with acetic acid is an attractive way to identify sensitive variants and can provide novel insights into the complex mechanisms regulating the acetic acid stress response.ResultsAn acetic acid biosensor based on the Saccharomyces cerevisiae transcription factor Haa1, was used to screen a CRISPRi yeast strain library where dCas9-Mxi was set to individually repress each essential or respiratory growth essential gene. Fluorescence-activated cell sorting led to the enrichment of a population of cells with higher acetic acid retention. These cells with higher biosensor signal were demonstrated to be more sensitive to acetic acid. Biosensor-based screening of the CRISPRi library strains enabled identification of strains with increased acetic acid sensitivity: strains with gRNAs targeting TIF34, MSN5, PAP1, COX10 or TRA1.ConclusionsThis study demonstrated that biosensors are valuable tools for screening and monitoring acetic acid tolerance in yeast. Fine-tuning the expression of essential genes can lead to altered acetic acid tolerance.

The characterisation of Wickerhamomyces anomalus M15, a highly tolerant yeast for bioethanol production using seaweed derived medium.

Advanced generation biofuels have potential for replacing fossil fuels as society moves forward into a net-zero carbon future. Marine biomass is a promising source of fermentable sugars for fermentative bioethanol production; however the medium derived from seaweed hydrolysis contains various inhibitors, such as salts that affected ethanol fermentation efficiency. In this study the stress tolerance of a marine yeast, Wickerhamomyces anomalus M15 was characterised. Specific growth rate analysis results showed that Wickerhamomyces anomalus M15 could tolerate up to 600 g/L glucose, 150 g/L xylose and 250 g/L ethanol, respectively. Using simulated concentrated seaweed hydrolysates, W. anomalus M15’s bioethanol production potential using macroalgae derived feedstocks was assessed, in which 5.8, 45.0, and 19.9 g/L ethanol was produced from brown (Laminaria digitata), green (Ulva linza) and red seaweed (Porphyra umbilicalis) based media. The fermentation of actual Ulva spp. hydrolysate harvested from United Kingdom shores resulted in a relatively low ethanol concentration (15.5 g/L) due to challenges that arose from concentrating the seaweed hydrolysate. However, fed-batch fermentation using simulated concentrated green seaweed hydrolysate achieved a concentration of 73 g/L ethanol in fermentations using both seawater and reverse osmosis water. Further fermentations conducted with an adaptive strain W. anomalus M15-500A showed improved bioethanol production of 92.7 g/L ethanol from 200 g/L glucose and reduced lag time from 93 h to 24 h in fermentation with an initial glucose concentration of 500 g/L. The results indicated that strains W. anomalus M15 and W. anomalus M15-500A have great potential for industrial bioethanol production using marine biomass derived feedstocks. It also suggested that if a concentrated high sugar content seaweed hydrolysate could be obtained, the bioethanol concentration could achieve 90 g/L or above, exceeding the minimum industrial production threshold.

The eisosomes contribute to acid tolerance of yeast by maintaining cell membrane integrity

Microbes have evolved multiple mechanisms to resist environmental stresses, which are regulated in complex and delicate ways. Though the role of cell membranes in acid resistance from the perspective of physicochemical properties and membrane proteins has been deeply studied, the function of eisosomes is still in its infancy. In this study, we firstly reported the dynamic changes of eisosomes under acid stress and the decreased acid tolerance of yeasts caused by eisosome disruption. Physiological indicators and non-targeted lipid profiling revealed that eisosome disruption caused changes in multiple lipids and imbalances in lipid homeostasis, which are responsible for membrane integrity damage. Thus the increased infiltration of carboxylic acids and the raised ROS levels were detected in strains with disrupted eisosome assembly, resulting in decreased cellular tolerance. The results here provide novel insights into the acid-resistant mechanism of yeasts from the perspective of the cell membrane subdomain, which has practical impacts on green biological manufacturing and food preservation.

Protective effects of peptides on the cell wall structure of yeast under osmotic stress.

Three peptides (LL, LML, and LLL) were used to examine their influences on the osmotic stress tolerance and cell wall properties of brewer's yeast. Results suggested that peptide supplementation improved the osmotic stress tolerance of yeast through enhancing the integrity and stability of the cell wall. Transmission electron micrographs showed that the thickness of yeast cell wall was increased by peptide addition under osmotic stress. Additionally, quantitative analysis of cell wall polysaccharide components in the LL and LLL groups revealed that they had 27.34% and 24.41% higher chitin levels, 25.73% and 22.59% higher mannan levels, and 17.86% and 21.35% higher β-1,3-glucan levels, respectively, than the control. Furthermore, peptide supplementation could positively modulate the cell wall integrity pathway and up-regulate the expressions of cell wall remodeling-related genes, including FKS1, FKS2, KRE6, MNN9, and CRH1. Thus, these results demonstrated that peptides improved the osmotic stress tolerance of yeast via remodeling the yeast cell wall and reinforcing the structure of the cell wall. KEY POINTS: • Peptide supplementation improved yeast osmotic stress tolerance via cell wall remodeling. • Peptide supplementation enhanced cell wall thickness and stability under osmotic stress. • Peptide supplementation positively modulated the CWI pathway under osmotic stress.

Intracellular trehalose accumulation via the Agt1 transporter promotes freeze-thaw tolerance in Saccharomyces cerevisiae.

This study is to investigate the use of a constitutively expressed trehalose transport protein to directly control intracellular trehalose levels and protect baker's yeast (Saccharomyces cerevisiae) cells against freeze-thaw stress in vivo. We used a constitutively overexpressed Agt1 transporter to investigate the role of trehalose in the freeze-thaw tolerance of yeast cells by regulating intracellular trehalose concentrations independently of intracellular biosynthesis. Using this method, we found that increasing intracellular trehalose in yeast cells improved cell survival rate after 8 days of freezing at -80 and -20°C. We also observed that freeze-thaw tolerance promoted by intracellular trehalose only occurs in highly concentrated cell pellets rather than cells in liquid suspension. Trehalose is sufficient to provide freeze-thaw tolerance using our Agt1 overexpression system. Freeze-thaw tolerance can be further enhanced by deletion of genes encoding intracellular trehalose degradation enzymes. These findings are relevant to improving the freeze-thaw tolerance of baker's yeast in the frozen baked goods industry through engineering strains that can accumulate intracellular trehalose via a constitutively expressed trehalose transporter and inclusion of trehalose into the growth medium.

Genome-wide identification of plasma-membrane intrinsic proteins in pumpkin and functional characterization of CmoPIP1-4 under salinity stress

Plasma membrane intrinsic proteins ( PIPs ) play a crucial role in the salinity and drought tolerance of plants. PIPs operate as vital water conduits for regulating water homeostasis during salt stress. Pumpkin is an important Cucurbitaceae crop, and no research on PIPs in pumpkin has been carried out. The current study, presents the first genome-wide characterization of the PIP gene family and functional characterization of CmoPIP1–4 with knockout mutants in pumpkin. According to their phylogeny, 21 CmoPIP genes have been identified and divided into 2 subgroups: CmoPIP1s and CmoPIP2s . Functional studies on their NPA motifs, ar/R filter, and Froger's positions predicted that these CmoPIP proteins would be substrate specific. The CmoPIP1s expression was found to be responsive to salt stress in leaves and roots. CmoPIP1–4 plays an important role in the salt stress tolerance of pumpkin, and the overexpression of CmoPIP1–4 confers salinity tolerance to yeast. However, hyper sensitivity to salt was observed in CmoPIP1–4 CRISPR plants phenotypically (weaker health, lower relative water contents and reduced photosynthetic activities), physiologically (higher Na + /K + ratios, malondialdehyde, ROS and membrane stability index, lower the contents of proline, glycine betaine and also decreased antioxidative enzyme activities), and molecularly (lower expression levels of salt-associated genes CmoSOS1 , CmoHKT1;1 , CmoNHX4 , and a lower expression levels antioxidative genes CmoSOD1/2, CmoPOD1/2 and CmoCAT1/2 ). This study highlights that CmoPIP1–4 is a key player in the stress-signaling pathway in pumpkin plants and could be valuable for developing stress-resistant cucurbit crops in the future. • Twenty-one plasma membrane intrinsic proteins ( PIPs ) were genome-wide characterized in Cucurbita moschata . • Salinity stress decreases the expression of most CmoPIPs . • Overexpression of CmoPIP1-4 conferred salt tolerance in yeast. • A salt-sensitive phenotype was observed after the CmoPIP1-4 knockout in pumpkin roots.

Full-length transcriptional analysis reveals the complex relationship of leaves and roots in responses to cold-drought combined stress in common vetch.

Plant responses to single or combined abiotic stresses between aboveground and underground parts are complex and require crosstalk signaling pathways. In this study, we explored the transcriptome data of common vetch (Vicia sativa L.) subjected to cold and drought stress between leaves and roots via meta-analysis to identify the hub abiotic stress-responsive genes. A total of 4,836 and 3,103 differentially expressed genes (DEGs) were identified in the leaves and roots, respectively. Transcriptome analysis results showed that the set of stress-responsive DEGs to concurrent stress is distinct from single stress, indicating a specialized and unique response to combined stresses in common vetch. Gene Ontology (GO) enrichment analyses identified that “Photosystem II,” “Defence response,” and “Sucrose synthase/metabolic activity” were the most significantly enriched categories in leaves, roots, and both tissues, respectively. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis results indicated that “ABC transporters” are the most enriched pathway and that all of the genes were upregulated in roots. Furthermore, 29 co-induced DEGs were identified as hub genes based on the consensus expression profile module of single and co-occurrence stress analysis. In transgenic yeast, the overexpression of three cross-stress tolerance candidate genes increased yeast tolerance to cold-drought combined stress. The elucidation of the combined stress-responsive network in common vetch to better parse the complex regulation of abiotic responses in plants facilitates more adequate legume forage breeding for combined stress tolerance.

Metabolic characteristics of intracellular trehalose enrichment in salt-tolerant Zygosaccharomyces rouxii.

The salt-tolerant flavor yeast Zygosaccharomyces rouxii is an important food flavor microorganism, but its intracellular stress-resistant trehalose synthesis efficiency has been shown to be low, resulting in its weak high-temperature resistance. The intracellular and extracellular levels of carbohydrates, organic acids, and amino acids of Z. rouxii in a 20-L mechanically stirred ventilated fermenter were analyzed using metabolomics research methods. Our results showed that glucose supplementation could promote the growth of yeast cells, but high temperatures (> 35°C) significantly prevented cell growth. Under three different growth strategies, extracellular glucose was continuously utilized and intracellular glucose was continuously metabolized, but glucose overflow metabolism was inhibited by high temperature, which showed that the level of intracellular/extracellular ethanol was stable. High temperature stimulated significant intracellular trehalose accumulation (c20.5h = 80.78 mg/g Dry Cell Weight (DCW)) but not efflux, as well as xylitol accumulation (c20.5h = 185.97 mg/g DCW) but with efflux (c20.5h = 29.78 g/L). Moreover, heat resistance evaluation showed that xylitol and trehalose had heat-protective effects on Z. rouxii. In addition, a large amount of propionic acid and butyric acid accumulated inside and outside these cells, showing that the conversion of glucose to acid in yeast cells becomes the main pathway of glucose overflow metabolism in high temperatures. In addition, the increased demand of yeast cells for phenylalanine, threonine, and glycine at high temperatures suggested that these metabolites participated in the temperature adaptation of Z. rouxii in different ways. Valine and leucine/isoleucine [branched-chain amino acids (BCAAs)] were mainly affected by the addition of glucose, while glucose, sucrose, aspartic acid/asparagine, and glutamate/glutamine were not affected by this temperature regulation as a whole. This study could help deepen our understanding of the high-temperature adaptation mechanism of salt-tolerant Z. rouxii, and has theoretical significance for the application of highly tolerant yeast to food brewing.

The cell wall and the response and tolerance to stresses of biotechnological relevance in yeasts.

In industrial settings and processes, yeasts may face multiple adverse environmental conditions. These include exposure to non-optimal temperatures or pH, osmotic stress, and deleterious concentrations of diverse inhibitory compounds. These toxic chemicals may result from the desired accumulation of added-value bio-products, yeast metabolism, or be present or derive from the pre-treatment of feedstocks, as in lignocellulosic biomass hydrolysates. Adaptation and tolerance to industrially relevant stress factors involve highly complex and coordinated molecular mechanisms occurring in the yeast cell with repercussions on the performance and economy of bioprocesses, or on the microbiological stability and conservation of foods, beverages, and other goods. To sense, survive, and adapt to different stresses, yeasts rely on a network of signaling pathways to modulate the global transcriptional response and elicit coordinated changes in the cell. These pathways cooperate and tightly regulate the composition, organization and biophysical properties of the cell wall. The intricacy of the underlying regulatory networks reflects the major role of the cell wall as the first line of defense against a wide range of environmental stresses. However, the involvement of cell wall in the adaptation and tolerance of yeasts to multiple stresses of biotechnological relevance has not received the deserved attention. This article provides an overview of the molecular mechanisms involved in fine-tuning cell wall physicochemical properties during the stress response of Saccharomyces cerevisiae and their implication in stress tolerance. The available information for non-conventional yeast species is also included. These non-Saccharomyces species have recently been on the focus of very active research to better explore or control their biotechnological potential envisaging the transition to a sustainable circular bioeconomy.

Recent advances in yeast genome evolution with stress tolerance for green biological manufacturing.

Green biological manufacturing is a revolutionary industrial model utilizing yeast as a significant microbial cell factory to produce biofuels and other biochemicals. However, biotransformation efficiency is often limited owing to several stress factors resulting from environmental changes or metabolic imbalance, leading to the slow growth of cells, compromised yield, and enhanced energy consumption. These factors make biological manufacturing competitively less economical. In this regard, minimizing the stress impact on microbial cell factories and strong robust performance have been an interesting area of interest in the last few decades. In this review, we focused on revealing the stress factors and their associated mechanisms for yeast in biological manufacturing. To improve yeast tolerance, rational and irrational strategies were introduced, and the molecular basis of genome evolution in yeast was also summarized. Furthermore, strategies of genome-directed evolution such as homology directed repair and nonhomologous end-joining, and the synthetic chromosome recombination and modification by LoxP-mediated evolution and their association with stress tolerance was highlighted. We hope that genome evolution provides new insights for solving the limitations of the natural phenotypes of microorganisms in industrial fermentation for the production of valuable compounds.