Tolerant Yeast Research Articles

Membrane Fluidity of Saccharomyces cerevisiae from Huangjiu (Chinese Rice Wine) Is Variably Regulated by OLE1 To Offset the Disruptive Effect of Ethanol.

An evolution and resequencing strategy was used to research the genetic basis of Saccharomyces cerevisiae BR20 (with 18 vol% ethanol tolerance) and the evolved strain F23 (with 25 vol% ethanol tolerance). Whole-genome sequencing and RNA sequencing (RNA-seq) indicated that the enhanced ethanol tolerance under 10 vol% ethanol could be attributed to amino acid metabolism, whereas 18 vol% ethanol tolerance was due to fatty acid metabolism. Ultrastructural analysis indicated that F23 exhibited better membrane integrity than did BR20 under ethanol stress. At low concentrations (<5 vol%), the partition of ethanol into the membrane increased the membrane fluidity, which had little effect on cell growth. However, the toxic effects of medium and high ethanol concentrations (5 to 20 vol%) tended to decrease the membrane fluidity. Under high ethanol stress (>10 vol%), the highly tolerant strain was able to maintain a relatively constant fluidity by increasing the content of unsaturated fatty acid (UFA), whereas less-tolerant strains show a continuous decrease in fluidity and UFA content. OLE1, which was identified as the only gene with a differential single-nucleotide polymorphism (SNP) mutation site related to fatty acid metabolism, was significantly changed in response to ethanol. The role of OLE1 in membrane fluidity was positively validated in its overexpressed transformants. Therefore, OLE1 lowered the rate of decline in membrane fluidity and thus enabled the yeast to better fight the deleterious effects of ethanol.IMPORTANCE Yeasts with superior ethanol tolerance are desirable for winemakers and wine industries. In our previous work, strain F23 was evolved with superior ethanol tolerance and fermentation activity to improve the flavor profiles of Chinese rice wine. Therefore, exploring the genomic variations and ethanol tolerance mechanism of strain F23 could contribute to an understanding of its effect on the flavor characteristics in the resulting Chinese rice wine. The cellular membrane plays a vital role in the ethanol tolerance of yeasts; however, how the membrane is regulated to fight the toxic effect of ethanol remains to be elucidated. This study suggests that the membrane fluidity is variably regulated by OLE1 to offset the disruptive effect of ethanol. Current work will help develop more ethanol-tolerant yeast strains for wine industries and contribute to a deep understanding of its high flavor-producing ability.

Critical Roles of the Pentose Phosphate Pathway and GLN3 in Isobutanol-Specific Tolerance in Yeast.

Branched-chain alcohols are attractive advanced biofuels; however, their cellular toxicity is an obstacle to engineering microbes to produce them at high titers. We performed genome-wide screens on the Saccharomyces cerevisiae gene deletion library to identify cell systems involved in isobutanol-specific tolerance. Deletion of pentose phosphate pathway genes GND1 or ZWF1 causes hypersensitivity to isobutanol but not to ethanol. By contrast, deletion of GLN3 increases yeast tolerance specifically to branched-chain alcohols. Transcriptomic analyses revealed that isobutanol induces a nitrogen starvation response via GLN3 and GCN4, upregulating amino acid biosynthesis and nitrogen scavenging while downregulating glycolysis, cell wall biogenesis, and membrane lipid biosynthesis. Disruption of this response by deleting GLN3 is enough to enhance tolerance and boost isobutanol production 4.9-fold in engineered strains. This study illustrates how adaptive mechanisms to tolerate stress can lead to toxicity in microbial fermentations for chemical production and how genetic interventions can boost production by evading such mechanisms.

Lipid and carotenoid production from wheat straw hydrolysates by different oleaginous yeasts

This work aimed to support the development of a new process for the production of lipids and carotenoids by oleaginous yeasts using both cellulosic and hemicellulosic fractions of biomass as feedstock for fermentation. The hypothesis behind this idea is that this strategy could lead to a sustainable pathway to produce these valuable components with potential to reduce the production costs. For the experiments, six oleaginous yeasts were initially cultivated in synthetic media containing glucose or xylose as carbon sources and different carbon to nitrogen ratios in order to identify the top producers. Rhodosporidium toruloides NRRL Y-1091 and Lipomyces starkeyi NRRL Y-1389 produced the highest amount of carotenoids and lipids, respectively. Then, the performance of these two strains when cultivated in wheat straw cellulosic and hemicellulosic hydrolysates was assessed. Detoxification of the cellulosic and hemicellulosic hydrolysates with activated charcoal removed 87.80% and 77.17% of inhibitors, respectively, and significantly improved the performance of the two yeasts. The highest concentration of lipids, 3.99 ± 0.35 g/L, was obtained by cultivating L. starkeyi in washed cellulosic hydrolysate; while the highest production of carotenoids, 24.58 ± 1.88 mg/L, was obtained by cultivating R. toruloides in decolorized cellulosic hydrolysate. Increasing the tolerance of oleaginous yeasts to inhibitors resulting from biomass pretreatment and/or using cost-effective detoxification methods were identified as key parameters for further development of this process at commercial scale.

Autophagy is required for sulfur dioxide tolerance in Saccharomyces cerevisiae.

SummarySulfiting agents are among the most widely used preservatives in the food and beverages industries, including winemaking, and one of their main functions is inhibition of spoilage microorganisms. We have used a whole genome quantitative fitness analysis in order to improve our knowledge on yeast tolerance to sulfites. Apart from the contribution of sulfite efflux to tolerance, results point to vesicle‐mediated transport, autophagy and vacuolar activity as the main cellular functions required to survive sulfite challenges. The involvement of autophagic and vacuolar functions in sulfite tolerance was further confirmed by pairwise competition using a newly constructed atg2‐defective strain, as well as by showing induction of ATG8 expression by sulfite. Autophagy is required for the turnover of proteins and subcellular structures damaged by sulfite. In addition, the requirement for vacuolar functions might be related to its role in intracellular pH homeostasis. Finally, the involvement of the sulfite pump Ssu1 and the transcription factor Fzf1 in sulfite tolerance by Saccharomyces cerevisiae was confirmed; a result that validates the experimental approach used in this work. These findings have relevance for understanding sulfite toxicity and tolerance, as well as for the eventual design of strategies aiming to control yeast spoilage.

Isolation and Partial Characterization of 2-Deoxy-D-glucose Resistant Saccharomyces cerevisiae Strain from Fruits Harvested in Yamagata, Japan

Aims: The study aimed to isolate and characterize 2-deoxy-D-glucose (2-DG) resistant Saccharomyces cerevisiae from fruits to establish distinctive bread making technology using wild-type yeasts in the future.&#x0D; Study Design: The research was conducted experimentally.&#x0D; Place and Duration of Study: Yamagata University, Yamagata, Japan, from April 2015 to March 2019.&#x0D; Methodology: Wild-type yeasts with 2-DG resistance were isolated using the following experiments: 1. Separation by yeast nitrogen base-maltose plate medium, 2. Carbon dioxide (CO2) and ethanol production tests, 3. Leavening ability tests using bread doughs, 4. Sequence analysis. The identified yeast strain was used for freezing and drying torelance tests. Moreover, it tried to improve drying tolerance of yeasts.&#x0D; Results: Yeasts were separated from twenty varieties of five fruits species. Among them, a yeast strain (YTPR1) isolated from pear Redbartllet fruits was identified as S. cerevisiae. YTPR1 possessed high fermentation ability and freezing tolerance, however, CO2 and ethanol production decreased after lyophilization of yeasts. In contrast, the cultivation with trehalose, glycerol, and L-glutamic acid at low concentration enhanced the fermentation ability of YTPR1.&#x0D; Conclusion: Yeast YTPR1 isolated from pear Redbartllet fruits utilized maltose as well as glucose, fructose, and sucrose. To improve drying tolerance of yeast YTPR1, it was useful to incubate with 6% trehalose, 0.1-2.0% glycerol and 1.0% L-glutamic acid.

Characterization of tolerance and multi-enzyme activities in non-Saccharomyces yeasts isolated from Vidal blanc icewine fermentation.

This work aim to study the tolerance and aroma-related enzymes activities of the non-Saccharomyces yeasts in Vidal blanc icewine from the Huanren region of China. The strains were identified by sequencing internal transcribed spacer (ITS) region and the 26S rDNA D1/D2 domain genes and all representative non-Saccharomyces yeasts were belonged to genera Metschnikowia, Hanseniaspora, Torulaspora, Candida, and Debaryomyces. A total of 28 strains were carried out for tolerance experiments and results suggested that most of them could tolerate 500g/L glucose, 4% ethanol, 20g/L tartaric acid, and 350mg/L SO2 . Finally, a total of 17 strains with better tolerance were carried out for the β-glucosidase, β-xylosidase, and pectinase activities experiments. The results showed that Candida railenensis HC08 and the strains of Hanseniaspora genus have satisfactory multi-enzyme activities, which can be used to design mixed fermentation to produce characteristic icewine. PRACTICAL APPLICATIONS: Non-Saccharomyces yeasts produces a series of hydrolytic enzymes that are thought to have a significant contribution to the aroma complexity of wines, however, are poorly explored in icewine. In this work, most of the non-Saccharomyces yeasts screened from Chinese icewine can adapt well to the high-sugar and high-acid environment of icewine, and can secrete hydrolase. The application of these strains in mixed fermentation could provide a prospect for the production of characteristic icewine.

Proteasome subunit \u03b11 overexpression preferentially drives canonical proteasome biogenesis and enhances stress tolerance in yeast

The 26S proteasome conducts the majority of regulated protein catabolism in eukaryotes. At the heart of the proteasome is the barrel-shaped 20S core particle (CP), which contains two β-rings sandwiched between two α-rings. Whereas canonical CPs contain α-rings with seven subunits arranged α1-α7, a non-canonical CP in which a second copy of the α4 subunit replaces the α3 subunit occurs in both yeast and humans. The mechanisms that control canonical versus non-canonical CP biogenesis remain poorly understood. Here, we have repurposed a split-protein reporter to identify genes that can enhance canonical proteasome assembly in mutant yeast producing non-canonical α4-α4 CPs. We identified the proteasome subunit α1 as an enhancer of α3 incorporation, and find that elevating α1 protein levels preferentially drives canonical CP assembly under conditions that normally favor α4-α4 CP formation. Further, we demonstrate that α1 is stoichiometrically limiting for α-ring assembly, and that enhancing α1 levels is sufficient to increase proteasome abundance and enhance stress tolerance in yeast. Together, our data indicate that the abundance of α1 exerts multiple impacts on proteasome assembly and composition, and we propose that the limited α1 levels observed in yeast may prime cells for alternative proteasome assembly following environmental stimuli.

The zinc-regulated protein (ZIP) family genes and glutathione s-transferase (GST) family genes play roles in Cd resistance and accumulation of pak choi (Brassica campestris ssp. chinensis)

The molecular mechanisms of the differences among the Cd tolerance and accumulation of different pak choi cultivars are essential to further breed Cd-safe genotypes pak choi. In our research, via morphological comparison, qRT-PCR and yeast function complementary approaches, we explored the differences of Cd tolerance and capacity for Cd uptake in nine various pak choi varieties. Results showed that higher expressions of BcZIPs involved in Cd uptake in ‘Kang Re605’ may lead to its higher capacity for Cd accumulation. The lowest expressions of transporter gene in ‘Wu Yueman’ were consistent with its fewest ability to uptake Cd. Beyond that, the difference of resistance was very great among varieties. Meanwhile, the expressions of the BcGSTUs were differentially induced by Cd exposure in different pak choi varieties, and ‘Kang Re605’ performed the highest BcGSTUs expression overall. To verify the role of GSTUs played in Cd resistance of pak choi, four BcGSTUs, BcGSTU4, BcGSTU11, BcGSTU12 and BcGSTU22 in a high-Cd accumulation and tolerance variety ‘Kang Re605’ were cloned, quantitated and transferred to Cd-sensitive yeast mutant strain. And finally found that BcGSTU11 increased the Cd tolerance of yeast, which may associate with a high Cd resistance of ‘Kang Re605’. Simultaneously, less BcGSTUs abundance in ‘Shang Haiqing’ may result in its weak tolerance to Cd. These findings will help us to comprehend the roles of BcZIPs and BcGSTUs in Cd absorption and detoxification as well as promote our understanding of the Cd-resistant and Cd-accumulated mechanisms in pak choi.

Highlight on the expression and the function of a novel MnSOD from diploid wheat (T. monococcum) in response to abiotic stress and heavy metal toxicity

Superoxide dismutases (SODs) play a pivotal role in improving abiotic stress tolerance in plant cells. A novel manganese superoxide dismutase gene, denoted as TmMnSOD, was identified from Triticum monococcum. The encoded protein displayed high sequence identity with MnSOD family members and was highly homologous to TdMnSOD from durum wheat. Furthermore, the 3D structure analysis revealed that TmMnSOD displayed homotetramer subunit organization, incorporating four Mn2+ ions. Notably, TmMnSOD structure contains predominantly alpha helices with three beta sheets. On the other hand, under stress conditions, TmMnSOD transcript level was significantly up-regulated by salt, oxidative and heavy metal stresses. At the functional level, TmMnSOD imparts tolerance of yeast and E. coli cells under diverse stresses. Promoter analysis of TmMnSOD gene showed the presence of a great number of salt and pathogen-responsive cis-regulatory elements, highlighting the interest of this gene in breeding programs towards improved tolerance to salt stress in wheat.

Coordination of the Cell Wall Integrity and High-Osmolarity Glycerol Pathways in Response to Ethanol Stress in Saccharomyces cerevisiae.

During fermentation, a high ethanol concentration is a major stress that influences the vitality and viability of yeast cells, which in turn leads to a termination of the fermentation process. In this study, we show that the BCK1 and SLT2 genes encoding mitogen-activated protein kinase kinase kinase (MAPKKK) and mitogen-activated protein kinase (MAPK) of the cell wall integrity (CWI) pathway, respectively, are essential for ethanol tolerance, suggesting that the CWI pathway is involved in the response to ethanol-induced cell wall stress. Upon ethanol exposure, the CWI pathway induces the expression of specific cell wall-remodeling genes, including FKS2, CRH1, and PIR3 (encoding β-1,3-glucan synthase, chitin transglycosylase, and O-glycosylated cell wall protein, respectively), which eventually leads to the remodeling of the cell wall structure. Our results revealed that in response to ethanol stress, the high-osmolarity glycerol (HOG) pathway plays a collaborative role with the CWI pathway in inducing cell wall remodeling via the upregulation of specific cell wall biosynthesis genes such as the CRH1 gene. Furthermore, the substantial expression of CWI-responsive genes is also triggered by external hyperosmolarity, suggesting that the adaptive changes in the cell wall are crucial for protecting yeast cells against not only cell wall stress but also osmotic stress. On the other hand, the cell wall stress-inducing agent calcofluor white has no effect on promoting the expression of GPD1, a major target gene of the HOG pathway. Collectively, these findings suggest that during ethanol stress, the CWI and HOG pathways collaboratively regulate the transcription of specific cell wall biosynthesis genes, thereby leading to adaptive changes in the cell wall.IMPORTANCE The budding yeast Saccharomyces cerevisiae has been widely used in industrial fermentations, including the production of alcoholic beverages and bioethanol. During fermentation, an increased ethanol concentration is the main stress that affects yeast metabolism and inhibits ethanol production. This work presents evidence that in response to ethanol stress, both CWI and HOG pathways cooperate to control the expression of cell wall-remodeling genes in order to build the adaptive strength of the cell wall. These findings will contribute to a better understanding of the molecular mechanisms underlying adaptive responses and tolerance of yeast to ethanol stress, which is essential for successful engineering of yeast strains for improved ethanol tolerance.

Protein and transcript profiling analysis of the response of Yarrowia lipolytica Y‐2 in the degradation of ochratoxin A

AbstractThe occurrence of ochratoxin A (OTA) has been reported in cereals, feeds and food. Biological control of OTA has been considered as a promising strategy. Our previous study has shown that Yarrowia lipolytica Y‐2 could degrade OTA. In this study, proteome and transcriptome analysis were performed to investigate the possible mechanisms involved in OTA degradation by Y. lipolytica Y‐2 and the responses of this antagonistic yeast to OTA. The results showed that many proteins and genes involved in removal of reactive O2 species were up‐regulated in Y. lipolytica Y‐2 to respond to the oxidative stress caused by OTA. Also, some proteins and genes involved in the removal of generated toxicants under stress condition were up‐regulated to improve tolerance of this antagonistic yeast to OTA stress. Furthermore, some genes encoding transport proteins and multidrug resistance proteins were up‐regulated significantly to transport and expel OTA from cells and alleviate the toxicity to Y. lipolytica Y‐2. Notably, the gene ECM14 (encoding putative metallocarboxypeptidase ECM14) directly involved in OTA degradation was up‐regulated in Y. lipolytica Y‐2 treated with this mycotoxin. To defend against OTA stress, some genes related to tricarboxylic acid cycle were induced by OTA to generate abundant energy for Y. lipolytica Y‐2. Besides, the down‐regulation of some genes involved in DNA replication and repair decreased the ability of Y. lipolytica Y‐2 to repair DNA damage caused by OTA. These results may imply that OTA degradation by this antagonistic yeast was the synergetic effect of multifarious defence responses to OTA stress.

Enhanced acetic acid stress tolerance and ethanol production in Saccharomyces cerevisiae by modulating expression of the de novo purine biosynthesis genes

BackgroundYeast strains that are tolerant to multiple environmental stresses are highly desired for various industrial applications. Despite great efforts in identifying key genes involved in stress tolerance of budding yeast Saccharomyces cerevisiae, the effects of de novo purine biosynthesis genes on yeast stress tolerance are still not well explored. Our previous studies showed that zinc sulfate addition improved yeast acetic acid tolerance, and key genes involved in yeast stress tolerance were further investigated in this study.ResultsThree genes involved in de novo purine biosynthesis, namely, ADE1, ADE13, and ADE17, showed significantly increased transcription levels by zinc sulfate supplementation under acetic acid stress, and overexpression of these genes in S. cerevisiae BY4741 enhanced cell growth under various stress conditions. Meanwhile, ethanol productivity was also improved by overexpression of the three ADE genes under stress conditions, among which the highest improvement attained 158.39% by ADE17 overexpression in the presence of inhibitor mixtures derived from lignocellulosic biomass. Elevated levels of adenine-nucleotide pool “AXP” ([ATP] + [ADP] + [AMP]) and ATP content were observed by overexpression of ADE17, both under control condition and under acetic acid stress, and is consistent with the better growth of the recombinant yeast strain. The global intracellular amino acid profiles were also changed by overexpression of the ADE genes. Among the changed amino acids, significant increase of the stress protectant γ-aminobutyric acid (GABA) was revealed by overexpression of the ADE genes under acetic acid stress, suggesting that overexpression of the ADE genes exerts control on both purine biosynthesis and amino acid biosynthesis to protect yeast cells against the stress.ConclusionWe proved that the de novo purine biosynthesis genes are useful targets for metabolic engineering of yeast stress tolerance. The engineered strains developed in this study with improved tolerance against multiple inhibitors can be employed for efficient lignocellulosic biorefinery to produce biofuels and biochemicals.

Functional characterization of metallothionein-like genes from Physcomitrella patens: expression profiling, yeast heterologous expression, and disruption of PpMT1.2a gene.

Physcomitrella patens contains four metallothionein-like genes. Three were shown to confer metal tolerance in yeast. Transcript profiling suggests their roles in senescence and reproductive development or cadmium and oxidative stress. Metallothioneins (MTs) have been suggested to play various roles including metal detoxification, nutrient remobilization, ROS scavenging, stress tolerance, and plant development. However, little is known about the forms and functions of MTs in bryophytes. The moss Physcomitrella patens genome was found to contain four MT-like genes. Amino acid sequence composition showed that the P. patens MTs (PpMTs) were clustered with Type 1 plant MTs, and could be further classified into two sub-types, herein referred to as sub-type 1: PpMT1.1a and PpMT1.1b and sub-type 2: PpMT1.2a and PpMT1.2b. Transcript abundance of PpMT1.1b and PpMT1.2b was upregulated in the gametophore compared to protonema, and all, except PpMT1.2a, were highly induced in senescing gametophytes. PpMT1.1a and PpMT1.1b transcripts were upregulated in protonema treated with cadmium and hydrogen peroxide. Unlike many higher plant MTs, the PpMT transcript abundance was not strongly induced in response to copper and zinc. These results suggest that PpMTs may play a role in protecting P. patens from cadmium and oxidative stress and may be involved in tissues senescence and reproductive development. The PpMTs, except PpMT1.2b, were also able to confer metal tolerance and accumulation when heterologously expressed in the ∆cup1 yeast. A P. patens mutant lacking PpMT1.2a through targeted gene disruption was generated. However, it did not show any alteration in growth phenotypes under senescence-induced conditions or hypersensitivity to cadmium, copper, zinc, H2O2, and NaCl stresses. Further characterization of additional P. patens mutants lacking single or multiple PpMTs may provide insight into the physiological roles of bryophytic MTs.

Evolutionary engineering in Saccharomyces cerevisiae reveals a TRK1-dependent potassium influx mechanism for propionic acid tolerance

BackgroundPropionic acid (PA), a key platform chemical produced as a by-product during petroleum refining, has been widely used as a food preservative and an important chemical intermediate in many industries. Microbial PA production through engineering yeast as a cell factory is a potentially sustainable alternative to replace petroleum refining. However, PA inhibits yeast growth at concentrations well below the titers typically required for a commercial bioprocess.ResultsAdaptive laboratory evolution (ALE) with PA concentrations ranging from 15 to 45 mM enabled the isolation of yeast strains with more than threefold improved tolerance to PA. Through whole genome sequencing and CRISPR–Cas9-mediated reverse engineering, unique mutations in TRK1, which encodes a high-affinity potassium transporter, were revealed as the cause of increased propionic acid tolerance. Potassium supplementation growth assays showed that mutated TRK1 alleles and extracellular potassium supplementation not only conferred tolerance to PA stress but also to multiple organic acids.ConclusionOur study has demonstrated the use of ALE as a powerful tool to improve yeast tolerance to PA. Potassium transport and maintenance is not only critical in yeast tolerance to PA but also boosts tolerance to multiple organic acids. These results demonstrate high-affinity potassium transport as a new principle for improving organic acid tolerance in strain engineering.

The Goldilocks effect of respiration on canavanine tolerance in Saccharomyces cerevisiae.

When glucose is available, Saccharomyces cerevisiae prefers fermentation to respiration. In fact, it can live without respiration at all. Here, we study the role of respiration in stress tolerance in yeast. We found that colony growth of respiratory-deficient yeast (petite) is greatly inhibited by canavanine, the toxic analog of arginine that causes proteotoxic stress. We found lower amounts of the amino acids involved in arginine biosynthesis in petites compared with WT. This finding may be explained by the fact that petite cells exposed to canavanine show reduction in the efficiency of targeting of proteins required for arginine biosynthesis. The retrograde (RTG) pathway signals mitochondrial stress. It positively controls production of arginine precursors. We show that canavanine abrogates RTG signaling especially in petite cells, and mutants in the RTG pathway are extremely sensitive to canavanine. We suggest that petite cells are naturally ineffective in production of some amino acids; combination of this fact with the effect of canavanine on the RTG pathway is the simplest explanation why petite cells are inhibited by canavanine. Surprisingly, we found that canavanine greatly inhibits colony formation when WT cells are forced to respire. Our research proposes a novel connection between respiration and proteotoxic stress.

Identification and expression analysis of the GDSL esterase/lipase family genes, and the characterization of SaGLIP8 in Sedum alfredii Hance under cadmium stress.

BackgroundThe herb Sedum alfredii (S. alfredii) Hance is a hyperaccumulator of heavy metals (cadmium (Cd), zinc (Zn) and lead (Pb)); therefore, it could be a candidate plant for efficient phytoremediation. The GDSL esterase/lipase protein (GELP) family plays important roles in plant defense and growth. Although the GELP family members in a variety of plants have been cloned and analyzed, there are limited studies on the family’s responses to heavy metal-stress conditions.MethodsMultiple sequence alignments and phylogenetic analyses were performed according to the criteria described. A WGCNA was used to construct co-expression regulatory networks. The roots of S. alfredii seedlings were treated with 100 µM CdCl2 for qRT-PCR to analyze expression levels in different tissues. SaGLIP8 was transformed into the Cd sensitive mutant strain yeast Δycf1 to investigate its role in resistance and accumulation to Cd.ResultsWe analyzed GELP family members from genomic data of S. alfredii. A phylogenetic tree divided the 80 identified family members into three clades. The promoters of the 80 genes contained certain elements related to abiotic stress, such as TC-rich repeats (defense and stress responsiveness), heat shock elements (heat stress) and MYB-binding sites (drought-inducibility). In addition, 66 members had tissue-specific expression patterns and significant responses to Cd stress. In total, 13 hub genes were obtained, based on an existing S. alfredii transcriptome database, that control 459 edge genes, which were classified into five classes of functions in a co-expression subnetwork: cell wall and defense function, lipid and esterase, stress and tolerance, transport and transcription factor activity. Among the hub genes, Sa13F.102 (SaGLIP8), with a high expression level in all tissues, could increase Cd tolerance and accumulation in yeast when overexpressed.ConclusionBased on genomic data of S. alfredii, we conducted phylogenetic analyses, as well as conserved domain, motif and expression profiling of the GELP family under Cd-stress conditions. SaGLIP8 could increase Cd tolerance and accumulation in yeast. These results indicated the roles of GELPs in plant responses to heavy metal exposure and provides a theoretical basis for further studies of the SaGELP family’s functions.

Study on the Soil Fertilizing Activities of Salt Tolerant Yeast Isolates

Study on the Soil Fertilizing Activities of Salt Tolerant Yeast Isolates

SCRaMbLE generates evolved yeasts with increased alkali tolerance

BackgroundStrains with increased alkali tolerance have a broad application in industrial, especially for bioremediation, biodegradation, biocontrol and production of bio-based chemicals. A novel synthetic chromosome recombination and modification by LoxP-mediated evolution (SCRaMbLE) system has been introduced in the synthetic yeast genome (Sc 2.0), which enables generation of a yeast library with massive structural variations and potentially drives phenotypic evolution. The structural variations including deletion, inversion and duplication have been detected within synthetic yeast chromosomes.ResultsHaploid yeast strains harboring either one (synV) or two (synV and synX) synthetic chromosomes were subjected to SCRaMbLE. Seven of evolved strains with increased alkali tolerance at pH 8.0 were generated through multiple independent SCRaMbLE experiments. Various of structural variations were detected in evolved yeast strains by PCRTag analysis and whole genome sequencing including two complex structural variations. One possessed an inversion of 20,743 base pairs within which YEL060C (PRB1) was deleted simultaneously, while another contained a duplication region of 9091 base pairs in length with a deletion aside. Moreover, a common deletion region with length of 11,448 base pairs was mapped in four of the alkali-tolerant strains. We further validated that the deletion of YER161C (SPT2) within the deleted region could increase alkali tolerance in Saccharomyces cerevisiae.ConclusionsSCRaMbLE system provides a simple and efficient way to generate evolved yeast strains with enhanced alkali tolerance. Deletion of YER161C (SPT2) mapped by SCRaMbLE can improve alkali tolerance in S. cerevisiae. This study enriches our understanding of alkali tolerance in yeast and provides a standard workflow for the application of SCRaMbLE system to generate various phenotypes that may be interesting for industry and extend understanding of phenotype-genotype relationship.

DEVELOPMENT OF SOME TOLERANT YEAST (SACCHAROMYCES CEREVISIAE) STRAINS TO HEAT AND SALT STRESSES

All living organisms are subjected to changing in conservational conditions, to which they must adapt to. Stress is defined as a threat refers to the physiological balance of systems critical to survival organisms. Five yeast strains (Saccharomyces cerevisiae) were subjected to different adverse environmental situations, such as thermal, osmotic and oxidative (salt) stresses. The objective of this work was to detect the most tolerant yeast strains under salt or heat stresses. Five yeast strains were exposed in a first experiment to heat stress at 20°C or 40°C beside to the control at 30°C to detect the more tolerant strain. The same yeast strains were subjected in a second experiment to two different concentrations of salt stress (NaCl); 0.5 or 1.0 M, separately, for two days (at 30°C as normal temperature for growth), other strains were exposed to 0.5 M concentration of NaCl for 24 hours, then 1 M for another 24 hours. For the heat stress results, strain S4 was more tolerant at 40oCwith insignificant difference compared to the control (30oC), while it showed significant difference at 20°C. Strain S5 also was more tolerant at 20oC with insignificant difference compared to the control. For salt treatment, the only insignificant value was for strain S3at 0.5M NaCl compared with the control.

A repeat region from the Brassica juncea HMA4 gene BjHMA4R is specifically involved in Cd2+ binding in the cytosol under low heavy metal concentrations

BackgroundHMA4 transporters are involved in the transport and binding of divalent heavy metals (Cd, Zn, Pb [lead] and Co [cobalt]). In general, as efflux pumps, HMA4 transporters can increase the heavy metal tolerance of yeast and Escherichia coli. Additional research has shown that the C-terminus of HMA4 contains a heavy metal-binding domain and that heterologous expression of a portion of peptides from this C-terminal domain in yeast provides a high level of Cd tolerance and Cd hyperaccumulation.ResultsWe cloned BjHMA4 from Brassica juncea, and quantitative real-time PCR analysis revealed that BjHMA4 was upregulated by Zn and Cd in the roots, stems and leaves. Overexpression of BjHMA4 dramatically affects Zn/Cd distribution in rice and wheat seedlings. Interestingly, BjHMA4 contains a repeat region named BjHMA4R within the C-terminal region; this repeat region is not far from the last transmembrane domain. We further characterized the detailed function of BjHMA4R via yeast and E. coli experiments. Notably, BjHMA4R greatly and specifically improved Cd tolerance, and BjHMA4R transformants both grew on solid media that contained 500 μM CdCl2 and presented improved Cd accumulation (approximately twice that of wild-type [WT] strains). Additionally, visualization via fluorescence microscopy indicated that BjHMA4R clearly localizes in the cytosol of yeast. Overall, these findings suggest that BjHMA4R specifically improves Cd tolerance and Cd accumulation in yeast by specifically binding Cd2+ in the cytosol under low heavy metal concentrations. Moreover, similar results in E. coli experiments corroborate this postulation.ConclusionBjHMA4R can specifically bind Cd2+ in the cytosol, thereby substantially and specifically improving Cd tolerance and accumulation under low heavy metal concentrations.