Tolerant Yeast Research Articles

Heavy Metals Induced Oxidative Stress in Multi-Metal Tolerant Yeast, Candida sp. PS33 and its Capability to Uptake Heavy Metals from Wastewater

Heavy Metals Induced Oxidative Stress in Multi-Metal Tolerant Yeast, Candida sp. PS33 and its Capability to Uptake Heavy Metals from Wastewater

Enhanced fermentative performance under stresses of multiple lignocellulose-derived inhibitors by overexpression of a typical 2-Cys peroxiredoxin from Kluyveromyces marxianus

BackgroundBioethanol from lignocellulosic materials is of great significance to the production of renewable fuels due to its wide sources. However, multiple inhibitors generated from pretreatments represent great challenges for its industrial-scale fermentation. Despite the complex toxicity mechanisms, lignocellulose-derived inhibitors have been reported to be related to the levels of intracellular reactive oxygen species (ROS), which makes oxidoreductase a potential target for the enhancement of the tolerance of yeasts to these inhibitors.ResultsA typical 2-Cys peroxiredoxin from Kluyveromyces marxianus Y179 (KmTPX1) was identified, and its overexpression was achieved in Saccharomyces cerevisiae 280. Strain TPX1 with overexpressed KmTPX1 gene showed an enhanced tolerance to oxidative stresses. Serial dilution assay indicated that KmTPX1 gene contributed to a better cellular growth behavior, when the cells were exposed to multiple lignocellulose-derived inhibitors, such as formic acid, acetic acid, furfural, ethanol, and salt. In particular, KmTPX1 expression also possessed enhanced tolerance to a mixture of formic acid, acetic acid, and furfural (FAF) with a shorter lag period. The maximum glucose consumption rate and ethanol generation rate in KmTPX1-expressing strain were significantly improved, compared with the control. The mechanism of improved tolerance to FAF depends on the lower level of intracellular ROS for cell survival under stress.ConclusionA new functional gene KmTPX1 from K. marxianus is firstly associated with the enhanced tolerance to multiple lignocellulose-derived inhibitors in S. cerevisiae. We provided a possible detoxification mechanism of the KmTPX1 for further theoretical research; meanwhile, we provided a powerful potential for application of the KmTPX1 overexpressing strain in ethanol production from lignocellulosic materials.

Phenol degradation and heavy metal tolerance of Antarctic yeasts.

In cold environments, biodegradation of organic pollutants and heavy metal bio-conversion requires the activity of cold-adapted or cold-tolerant microorganisms. In this work, the ability to utilize phenol, methanol and n-hexadecane as C source, the tolerance to different heavy metals and growth from 5 to 30 °C were evaluated in cold-adapted yeasts isolated from Antarctica. Fifty-nine percent of the yeasts were classified as psychrotolerant as they could grow in all the range of temperature tested, while the other 41% were classified as psychrophilic as they only grew below 25 °C. In the assimilation tests, 32, 78, and 13% of the yeasts could utilize phenol, n-hexadecane, and methanol as C source, respectively, but only 6% could assimilate the three C sources evaluated. In relation to heavy metals ions, 55, 68, and 80% were tolerant to 1mM of Cr(VI), Cd(II), and Cu(II), respectively. Approximately a half of the isolates tolerated all of them. Most of the selected yeasts belong to genera previously reported as common for Antarctic soils, but several other genera were also isolated, which contribute to the knowledge of this cold environment mycodiversity. The tolerance to heavy metals of the phenol-degrading cold-adapted yeasts illustrated that the strains could be valuable as inoculant for cold wastewater treatment in extremely cold environments.

Mitochondrial Superoxide Dismutase and Yap1p Act as a Signaling Module Contributing to Ethanol Tolerance of the Yeast Saccharomyces cerevisiae.

Baker's yeast harbors multiple systems that ensure tolerance to high concentrations of ethanol. Still, the role of mitochondria under severe ethanol stress in yeast is not completely clear. Our study revealed a signaling function of mitochondria which contributes significantly to the ethanol tolerance of yeast cells. We found that mitochondrial superoxide dismutase Sod2p and cytoplasmic hydrogen peroxide sensor Yap1p act together as a module of the mitochondrion-to-nucleus signaling pathway. We also report cross talk between this pathway and the conventional retrograde signaling cascade activated by dysfunctional mitochondria.

Improvement of yeast tolerance to acetic acid through Haa1 transcription factor engineering: towards the underlying mechanisms

BackgroundBesides being a major regulator of the response to acetic acid in Saccharomyces cerevisiae, the transcription factor Haa1 is an important determinant of the tolerance to this acid. The engineering of Haa1 either by overexpression or mutagenesis has therefore been considered to be a promising avenue towards the construction of more robust strains with improved acetic acid tolerance.ResultsBy applying the concept of global transcription machinery engineering to the regulon-specific transcription factor Haa1, a mutant allele containing two point mutations could be selected that resulted in a significantly higher acetic acid tolerance as compared to the wild-type allele. The level of improvement obtained was comparable to the level obtained by overexpression of HAA1, which was achieved by introduction of a second copy of the native HAA1 gene. Dissection of the contribution of the two point mutations to the phenotype showed that the major improvement was caused by an amino acid exchange at position 135 (serine to phenylalanine). In order to further study the mechanisms underlying the tolerance phenotype, Haa1 translocation and transcriptional activation of Haa1 target genes was compared between Haa1 mutant, overproduction and wild-type strains. While the rapid Haa1 translocation from the cytosol to the nucleus in response to acetic acid was not affected in the Haa1S135F mutant strain, the levels of transcriptional activation of four selected Haa1-target genes by acetic acid were significantly higher in cells of the mutant strain as compared to cells of the wild-type strain. Interestingly, the time-course of transcriptional activation in response to acetic acid was comparable for the mutant and wild-type strain whereas the maximum mRNA levels obtained correlate with each strain’s tolerance level.ConclusionOur data confirms that engineering of the regulon-specific transcription factor Haa1 allows the improvement of acetic acid tolerance in S. cerevisiae. It was also shown that the beneficial S135F mutation identified in the current work did not lead to an increase of HAA1 transcript level, suggesting that an altered protein structure of the Haa1S135F mutant protein led to an increased recruitment of the transcription machinery to Haa1 target genes.

Characteristics of antioxidant activity of soybean PM1 protein and enhancement of tolerance of recombinant yeast to copper stress

Characteristics of antioxidant activity of soybean PM1 protein and enhancement of tolerance of recombinant yeast to copper stress

Isolation and Characterization of Ethanol, Sugar and Thermo Tolerant Yeast Isolates in Ethiopia

Isolation and Characterization of Ethanol, Sugar and Thermo Tolerant Yeast Isolates in Ethiopia

Isolation and Characterization of Ethanol, Sugar and Thermo Tolerant Yeast Isolates in Ethiopia

Isolation and Characterization of Ethanol, Sugar and Thermo Tolerant Yeast Isolates in Ethiopia

Carbon metabolism and transcriptional variation in response to salt stress in the genome shuffled Candida versatilis and a wild-type salt tolerant yeast strain

The carbon metabolism and molecular mechanisms of adaptation response when exposed to conditions causing osmotic stress in strains of a wild-type of Candida versatilis (WT) and S3–5 were investigated.

Evolutionary engineering reveals divergent paths when yeast is adapted to different acidic environments

Tolerance of yeast to acid stress is important for many industrial processes including organic acid production. Therefore, elucidating the molecular basis of long term adaptation to acidic environments will be beneficial for engineering production strains to thrive under such harsh conditions. Previous studies using gene expression analysis have suggested that both organic and inorganic acids display similar responses during short term exposure to acidic conditions. However, biological mechanisms that will lead to long term adaptation of yeast to acidic conditions remains unknown and whether these mechanisms will be similar for tolerance to both organic and inorganic acids is yet to be explored. We therefore evolved Saccharomyces cerevisiae to acquire tolerance to HCl (inorganic acid) and to 0.3M L-lactic acid (organic acid) at pH 2.8 and then isolated several low pH tolerant strains. Whole genome sequencing and RNA-seq analysis of the evolved strains revealed different sets of genome alterations suggesting a divergence in adaptation to these two acids. An altered sterol composition and impaired iron uptake contributed to HCl tolerance whereas the formation of a multicellular morphology and rapid lactate degradation was crucial for tolerance to high concentrations of lactic acid. Our findings highlight the contribution of both the selection pressure and nature of the acid as a driver for directing the evolutionary path towards tolerance to low pH. The choice of carbon source was also an important factor in the evolutionary process since cells evolved on two different carbon sources (raffinose and glucose) generated a different set of mutations in response to the presence of lactic acid. Therefore, different strategies are required for a rational design of low pH tolerant strains depending on the acid of interest.

Fitness Trade-Offs Lead to Suppressor Tolerance in Yeast

Genetic variation among individuals within a population provides the raw material for phenotypic diversity upon which natural selection operates. Some given variants can act on multiple standing genomic variations simultaneously and release previously inaccessible phenotypes, leading to increased adaptive potential upon challenging environments. Previously, we identified such a variant related to a tRNA nonsense suppressor in yeast. When introduced into other genetic backgrounds, the suppressor led to an increased population phenotypic variance on various culture conditions, conferring background and environment specific selective advantages. Nonetheless, most isolates are intolerant to the suppressor on rich media due to a severe fitness cost. Here, we found that the tolerance to suppressor is related to a surprising level of fitness outburst, showing a trade-off effect to accommodate the cost of carrying the suppressor. To dissect the genetic basis of such trade-offs, we crossed strains with contrasting tolerance levels on rich media, and analyzed the fitness distribution patterns in the offspring. Combining quantitative tetrad analysis and bulk segregant analysis, we identified two genes, namely MKT1 and RGA1, involved in suppressor tolerance. We showed that alleles from the tolerant parent for both genes conferred a significant gain of fitness, which increased the suppressor tolerance. Our results present a detailed dissection of suppressor tolerance in yeast and provide insights into the molecular basis of trade-offs between fitness and evolutionary potential.

Identification of gene knockdown targets conferring enhanced isobutanol and 1-butanol tolerance to Saccharomyces cerevisiae using a tunable RNAi screening approach.

Improving yeast tolerance to 1-butanol and isobutanol is a step toward enabling high-titer production. To identify previously unknown genetic targets leading to increased tolerance, we establish a tunable RNA interference (RNAi) screening approach. Specifically, we optimized the efficiency and tunability of RNA interference library screening in yeast, ultimately enabling downregulation efficiencies from 0 to 94%. Using this system, we identified the Hsp70 family as a key regulator of isobutanol tolerance in a single round of screening, with downregulation of these genes conferring up to 64% increased growth in 12g/L isobutanol. For 1-butanol, we find through two rounds of iterative screening that the combined downregulation of alcohol dehydrogenase and enolase improves growth up to 3100% in 10g/L 1-butanol. Collectively, this work improves the tunability of RNAi in yeast as demonstrated by the discovery of novel effectors for these complex phenotypes.

Cloning and characterization of vacuolar H+-pyrophosphatase gene (AnVP1) from Ammopiptanthus nanus and its heterologous expression enhances osmotic tolerance in yeast and Arabidopsis thaliana

Vacuolar H+-pyrophosphatase (H+-PPase) is one of the proton pumps involved in the response of plants to osmotic stress. In the present study, the vacuolar H+-PPase gene (AnVP1) was cloned from an epibiotic super-xerophytic plant, Ammopiptanthus nanus, by using rapid amplification of cDNA ends. The full-length cDNA was 2684 bp long, containing an open reading frame of 2298 bp that encoded 765 amino acids with 13 transmembrane regions. The putative protein shared high homology, conserved domains and amino acid residues with the H+-PPase proteins from other plants. The result of quantitative real-time PCR showed that the expression of the AnVP1 gene was strongly induced by salt, dehydration and abscisic acid, and inhibited by cold and heat. Complementary of the AnVP1 gene in the yeast mutant ena1 improved its salt and osmotic tolerance. Heterologous expression of the AnVP1 gene in the Arabidopsis mutant significantly restored its salt and drought tolerance. Under salt and drought stress, the transgenic plants exhibited more vigorous growth, more developed root system, higher proline content and relative water content, and lower malondialdehyde content and relative electrolytic leakage, as well as more Na+ and K+ accumulation in leaf, than the untransformed mutant plants. All these results demonstrate that the AnVP1 gene encodes a functional vacuolar H+-pyrophosphatase that plays a crucial role in adaptation of A. nanus to abiotic stress. After further evaluation for its tolerant function, this gene is hopeful to be used to genetic engineering for improving abiotic tolerance of commercial crops.

A potential contribution of the less negatively charged cell wall to the high aluminum tolerance of Rhodotorula taiwanensis RS1.

Rhodotorula taiwanensis RS1 (Rt) is a high-aluminum (Al)-tolerant yeast that can survive Al at concentrations up to 200 mM. In this study, we compared Rt with an Al-sensitive congeneric strain, R. mucilaginosa AKU 4812 (Rm) and Al sensitive mutant 1 (alsm1) of Rt, to explore the Al tolerance mechanisms of Rt. The growth of Rm was completely inhibited by 1 mM Al, but that of Rt was not inhibited until Al concentration was more than 70 mM. The growth of alsm1 was inhibited much more by 70 mM and 100 mM Al than that of Rt. Compared with Rm cells, Rt cells accumulated less Al in the cell wall and cytoplasm. A time-course analysis showed that Al was absorbed by Rm cells much more rapidly than by Rt cells when exposed to the same Al concentration. Meanwhile, the Al content of alsm1 was higher than that of Rt. Although the cell wall of Rt was thicker than that of alsm1 and Rm under control and 0.1 mM Al, that of Rt was thinner than that of alsm1 under 70 mM Al despite that their cell walls were thickened. The alcian blue adsorption was lower and cell wall zeta-potential was higher in Rt and alsm1 than in Rm, indicating a less negative charge of cell wall of Rt and alsm1 than that of Rm. Taken together, the less negatively charged cell wall of Rt may restrict the adsorption of cationic Al in cells, potentially contributing to its high Al tolerance. Copyright © 2016 John Wiley & Sons, Ltd.

Transcription-coupled changes to chromatin underpin gene silencing by transcriptional interference

Long non-coding RNA (lncRNA) transcription into a downstream promoter frequently results in transcriptional interference. However, the mechanism of this repression is not fully understood. We recently showed that drug tolerance in fission yeast Schizosaccharomyces pombe is controlled by lncRNA transcription upstream of the tgp1+ permease gene. Here we demonstrate that transcriptional interference of tgp1+ involves several transcription-coupled chromatin changes mediated by conserved elongation factors Set2, Clr6CII, Spt6 and FACT. These factors are known to travel with RNAPII and establish repressive chromatin in order to limit aberrant transcription initiation from cryptic promoters present in gene bodies. We therefore conclude that conserved RNAPII-associated mechanisms exist to both suppress intragenic cryptic promoters during genic transcription and to repress gene promoters by transcriptional interference. Our analyses also demonstrate that key mechanistic features of transcriptional interference are shared between S. pombe and the highly divergent budding yeast Saccharomyces cerevisiae. Thus, transcriptional interference is an ancient, conserved mechanism for tightly controlling gene expression. Our mechanistic insights allowed us to predict and validate a second example of transcriptional interference involving the S. pombe pho1+ gene. Given that eukaryotic genomes are pervasively transcribed, transcriptional interference likely represents a more general feature of gene regulation than is currently appreciated.

Distribution of tannin-'tolerant yeasts isolated from Miang, a traditional fermented tea leaf (Camellia sinensis var. assamica) in northern Thailand

Miang is a fermented food product prepared from the tea leaves of Camellia sinensis var. assamica, and is traditionally produced in mountainous areas of northern Thailand. Although Miang has a long history and reveals deep-rooted cultural involvement with local people in northern Thailand, little is known regarding its microbial diversity. Yeasts were isolated from 47 Miang samples collected from 28 sampling sites, including eight provinces in upper northern Thailand. A hundred and seven yeast isolates were recovered and identified within 14 species based on the comparison of the D1/D2 sequence of the large subunit (LSU) rRNA gene. Candida ethanolica was determined to be the dominant species that was frequently found in Miang together with minor resident yeast species. All yeast isolates demonstrated their tannin-tolerant capability when cultivated on yeast malt agar (YMA) containing 50g/l tannin, but nine isolates displayed clear zones forming around their colonies, e.g., Debaryomyces hansenii, Cyberlindnera rhodanensis, and Sporidiobolus ruineniae. The results obtained from a visual reading method of tannase revealed that all yeast isolates were positive for methyl gallate, indicating that they possess tannase activity. It is assumed that a tannin-tolerant ability is one of the most important factors for developing a yeast community in Miang. This research study is the first report to describe tannin-tolerant yeasts and yeast communities in traditionally fermented tea leaves.

Implementing principles of traditional concentrated grape must fermentation to the production of new generation balsamic vinegars. Starter selection and effectiveness.

In an effort to implement principles of traditional concentrated grape must fermentation to the production of new generation balsamic vinegars (BVs), the specific goals of the study were the isolation and molecular identification of the predominant yeasts in concentrated grape must (cv. Xinomavro), their technological characterization and the evaluation of the fermentative aptitude of the selected strains. Tolerance against 5-hydroxymethyl-furfural (HMF) and furfural, acetic acid and glucose concentration was examined by appropriate methods and tests. The enological characteristics studied were acetic acid and H2S production, foaming and flocculation ability and key enzymatic activity. PCR-RFLP analysis revealed only the presence of Saccharomyces cerevisiae and Hanseniaspora uvarum among the 14 predominant osmophilic yeast isolates. Tolerance to both HMF and furfural was found strain- and dose-dependent and was suggested as a critical factor in the pre-selection of yeast starters. The most tolerant yeasts to these stress factors, a S. cerevisiae and a non-Saccharomyces strains, showed satisfactory growth in the presence of high glucose and acetic acid content (up to 600g/L and 2% w/w, respectively) and desirable enological characteristics. Results from the comparative evaluation of the fermentative aptitude of these strains with a commercial wine strain highlighted that the isolates had glucophilic behaviour and ability to produce desirable amounts of ethanol (100-120g/kg) in short time (~20d). The key volatiles useful for varietal discrimination and differentiation between the BVs and the traditional ones were also evaluated.

Simultaneous Saccharification and Fermentation of Low Strength Sodium Hydroxide Pre-treated Rice Straw for Higher Ethanol Yields: Batch and Fed Batch Hybrid Approach

Low strength sodium hydroxide pre-treated rice straw was hydrolysed at two different solid loadings i.e. 10 and 15 % (w/v) with in-house cellulases (IC) produced by Aspergillus terreus along with commercial cellulases (CC) by using batch and fed batch hybrid simultaneous saccharification and fermentation process. Optimization of process parameters enhanced the crude cellulase activities i.e. filter paper, β-glucosidase and endoglucanase by 1.6, 2.7 and 2.2 fold in 6 days as compared to activities obtained on a single substrate i.e. rice straw. Out of four fed batch approaches, approach III (A-III) at 10 % solid loading yielded higher ethanol concentrations of 30.55 and 28.66 g L−1 by using thermo tolerant in-house yeast strain Kluyveromyces marxianus at 42 °C. Combination of 9 FPU g−1 substrate and CC plus 30 CBU of commercial β-glucosidase (Cβ) yielded slightly higher theoretical ethanol yields of 92.24 % as compared to 86.54 % obtained from IC+Cβ. Dunnett’s Post Hoc Annova test also proved that CC+Cβ were found to be significant in batch as well fed batch experiments as compared to other combinations tried. Thus, fed batch A-III seems to be an optimistic one to overcome the problems associated with batch mode to achieve higher ethanol yields.

Identification of survival-promoting OSIP108 peptide variants and their internalization in human cells

The plant-derived decapeptide OSIP108 increases tolerance of yeast and human cells to apoptosis-inducing agents, such as copper and cisplatin. We performed a whole amino acid scan of OSIP108 and conducted structure-activity relationship studies on the induction of cisplatin tolerance (CT) in yeast. The use of cisplatin as apoptosis-inducing trigger in this study should be considered as a tool to better understand the survival-promoting nature of OSIP108 and not for purposes related to anti-cancer treatment. We found that charged residues (Arg, His, Lys, Glu or Asp) or a Pro on positions 4–7 improved OSIP108 activity by 10% or more. The variant OSIP108[G7P] induced the most pronounced tolerance to toxic concentrations of copper and cisplatin in yeast and/or HepG2 cells. Both OSIP108 and OSIP108[G7P] were shown to internalize equally into HeLa cells, but at a higher rate than the inactive OSIP108[E10A], suggesting that the peptides can internalize into cells and that OSIP108 activity is dependent on subsequent intracellular interactions. In conclusion, our studies demonstrated that tolerance/survival-promoting properties of OSIP108 can be significantly improved by single amino acid substitutions, and that these properties are dependent on (an) intracellular target(s), yet to be determined.

Protective Effects of Arginine on Saccharomyces cerevisiae Against Ethanol Stress.

Yeast cells are challenged by various environmental stresses in the process of industrial fermentation. As the currently main organism for bio-ethanol production, Saccharomyces cerevisiae suffers from ethanol stress. Some amino acids have been reported to be related to yeast tolerance to stresses. Here the relationship between arginine and yeast response to ethanol stress was investigated. Marked inhibitions of ethanol on cell growth, expression of genes involved in arginine biosynthesis and intracellular accumulation of arginine were observed. Furthermore, extracellular addition of arginine can abate the ethanol damage largely. To further confirm the protective effects of arginine on yeast cells, yeast strains with different levels of arginine content were constructed by overexpression of ARG4 involved in arginine biosynthesis or CAR1 encoding arginase. Intracellular arginine was increased by 18.9% or 13.1% respectively by overexpression of ARG4 or disruption of CAR1, which enhanced yeast tolerance to ethanol stress. Moreover, a 41.1% decrease of intracellular arginine was observed in CAR1 overexpressing strain, which made yeast cells keenly sensitive to ethanol. Further investigations indicated that arginine protected yeast cells from ethanol damage by maintaining the integrity of cell wall and cytoplasma membrane, stabilizing the morphology and function of organellae due to low ROS generation.