Tolerant Yeast Research Articles

Stress Tolerance of Yeasts Dominating Reverse Osmosis Membranes for Whey Water Treatment.

Filamentous yeast species belonging to the closely related Saprochaete clavata and Magnusiomyces spicifer were recently found to dominate biofilm communities on the retentate and permeate surface of Reverse Osmosis (RO) membranes used in a whey water treatment system after CIP (Cleaning-In-Place). Microscopy revealed that the two filamentous yeast species can cover extensive areas due to their large cell size and long hyphae formation. Representative strains from these species were here further characterized and displayed similar physiological and biochemical characteristics. Both strains tested were able to grow in twice RO-filtrated permeate water and metabolize the urea present. Little is known about the survival characteristics of these strains. Here, their tolerance toward heat (60, 70, and 80°C) and Ultraviolet light (UV-C) treatment at 255 nm using UV-LED was assessed as well as their ability to form biofilm and withstand cleaning associated stress. According to the heat tolerance experiments, the D60°C of S. clavata and M. spicifer is 16.37 min and 7.24 min, respectively, while a reduction of 3.5 to >4.5 log (CFU/mL) was ensured within 5 min at 70°C. UV-C light at a dose level 10 mJ/cm2 had little effect, while doses of 40 mJ/cm2 and upward ensured a ≥4log reduction in a static laboratory scale set-up. The biofilm forming potential of one filamentous yeast and one budding yeast, Sporopachydermia lactativora, both isolated from the same biofilm, was compared in assays employing flat-bottomed polystyrene microwells and peg lids, respectively. In these systems, employing both nutrient rich as well as nutrient poor media, only the filamentous yeast was able to create biofilm. However, on RO membrane coupons in static systems, both the budding yeast and a filamentous yeast were capable of forming single strain biofilms and when these coupons were exposed to different simulations of CIP treatments both the filamentous and budding yeast survived these. The dominance of these yeasts in some filter systems tested, their capacity to adhere and their tolerance toward relevant stresses as demonstrated here, suggest that these slow growing yeasts are well suited to initiate microbial biofouling on surfaces in low nutrient environments.

Saltwater based fractionation and valorisation of macroalgae

AbstractBACKGROUNDMacroalgae are gaining increasing interest as an important biomass feedstock. Yet when valorising marine biomass, the presence of salt can pose a substantial obstacle to the effectiveness of downstream biological and chemical processes, as well as the engineering infrastructure required. Accordingly, dewatering, washing and drying are often considered the first and crucial primary steps in processing marine biomass such macroalgae. The high costs of these processes can make further marine biorefinery commercialisation prohibitive. This investigation assesses simple pre‐treatments for macroalgal biomass in saltwater, thereby reducing the freshwater footprint, and removing the need for an energy‐intensive washing and drying stage.RESULTSUsing acid and basic catalysts, the carbohydrate and soluble protein components were fractionated into a soluble aqueous phase, for further fermentation and a solid phase suitable for hydrothermal liquefaction. The presence of saltwater was found to aid the fractionation process, solubilising more of the biomass. The use of H2SO4 produced more monosaccharides, whereas NaOH solubilised higher levels of biomass at lower temperatures. The aqueous phase was demonstrated to be suitable for biological processing with the salt tolerant yeast Metschnikowia pulcherrima, and the residual solids suitable for processing via hydrothermal liquefaction.CONCLUSIONBy contrast with existing pre‐treatment strategies, we demonstrate that an entirely salt‐based biochemical conversion route is a potentially viable option. For the first time this work demonstrates that, rather than a hindrance, the presence of saltwater can be advantageous, and could provide an alternative, more cost‐effective pathway to achieving a successful macroalgal‐based biorefinery.

Ectopic expression of AmNAC1 from Avicennia marina (Forsk.) Vierh. confers multiple abiotic stress tolerance in yeast and tobacco

Abiotic factors like salinity, drought and cold affect agricultural productivity substantially worldwide. NAC (NAM, ATAF1/2 and CUC2) family transcription factors have been shown to play important roles in biotic and abiotic stress responses in many plant species. Our previous studies have shown that AmNAC1, a gene from mangrove Avicennia marina, is differentially regulated in response to salt, drought and cold stresses and salicylic acid treatment. To gain more insights into the function of AmNAC1 in abiotic stress, AmNAC1 was heterologously expressed in yeast and tobacco. Heterologous expression of AmNAC1 in yeast (Saccharomyces cerevisiae) resulted in improved tolerance to salinity (NaCl and LiCl), alkalinity (NaHCO3) stresses. In addition, transgenic yeast exhibited enhanced tolerance compared to control when subjected to high (50 °C) and freezing (− 20 °C) temperature. Similarly, overexpression of AmNAC1 in tobacco using the constitutive promoter (2xCaMV) demonstrated increased tolerance to abiotic stresses such as salinity, drought and cold. Specifically, compared to wild type (WT) and vector control (VC), AmNAC1 transgenic lines exhibited higher germination, increased root length and better survival rates at the whole plant level. Collectively, these results indicate a role for AmNAC1 in multiple abiotic stress tolerance. AmNAC1 is a stress responsive transcription factor from mangrove Avicennia marina and enhances better tolerance to NaCl, NaHCo3, LiCl, freezing and high temperature in transgenic yeast. Over expression of AmNAC1 in tobacco improved salt, cold and drought tolerance.

Heterologous expression of an Agropyron cristatum SnRK2 protein kinase gene (AcSnRK2.11) increases freezing tolerance in transgenic yeast and tobacco.

The sucrose non-fermenting-1 related protein kinase 2 (SnRK2) family plays an important role in the response to abiotic stress. To characterize the function of the SnRK2 gene from Agropyron cristatum in stress protection, we cloned the complete coding sequence of the AcSnRK2.11 gene from A. cristatum and generated AcSnRK2.11-overexpressing tobacco lines. The open reading frame of AcSnRK2.11 was 1083bp in length and encoded a polypeptide of 360 amino acid residues. The sequence analysis results showed that AcSnRK2.11 contained conserved domains typified in SnRK2 protein kinases. Subcellular localization analysis showed that AcSnRK2.11 was located in the nucleus. AcSnRK2.11 was constitutively expressed in all of the examined tissues, and its transcription was induced by cold, dehydration, and salt stress, but not by abscisic acid treatment. Overexpression of the AcSnRK2.11 gene conferred freezing tolerance in yeast. AcSnRK2.11-overexpressing tobacco lines showed higher tolerance to freezing stress than did wild-type (WT) based on higher survival rates, lower malondialdehyde content and increased relative water content retention, chlorophyll yields, superoxide dismutase activities, reactive oxygen species content, peroxidase levels, and soluble carbohydrates under low-temperature conditions. The transcripts of NtDREB1, NtDREB2, NtERD10A, NtERD10B, NtERD10C, NtERD10D, NtMnSOD, NtCDPK15, and NtMPK9 in AcSnRK2.11-overexpressing tobacco lines were more abundant than in WT plants under low-temperature stress. These results suggest that AcSnRK2.11 may function as a regulatory factor associated with a cold-response pathway and could be used in plant breeding for cold resistance.

The AlRabring7 E3-Ub-ligase mediates AlRab7 ubiquitination and improves ionic and oxidative stress tolerance in Saccharomyces cerevisiae

The maintenance of ROS homeostasis, membrane biogenesis and recycling of molecules are common stress responses involving specific and complex regulatory network. Ubiquitination is an important and common mechanism which facilitates environmental adaptation in eukaryotes. In the present study we have cloned the AlRabring7, an E3-Ub-ligase, previously identified as AlRab7 interacting partner. The role of AlRabring7 for ubiquitinating AlRab7 and facilitating stress tolerance is analysed. The AlRabring7, with an open-reading frame of 702 bp encodes a protein of 233 amino acids, with RING-HC domain of 40 amino acids. In silico analysis shows that AlRabring7 is a C3HC4-type RING E3 Ub ligase. The protein - protein docking show interaction dynamics between AlRab7-AlRabring7-Ubiquitin proteins. The AlRab7 and AlRabring7 transcript showed up-regulation in response to different salts i.e: NaCl, KCl, CaCl2, NaCl + KCl, NaCl + CaCl2, imposing ionic as well as hyperosmotic stress, and also with oxidative stress by H2O2 treatment. Interestingly, the AlRabring7 showed early transcript expression with maximum expression in shoots on combinatorial stresses. The AlRab7 showed delayed and maximum expression with NaCl + CaCl2 stress treatment. The AlRab7 complements yeast ypt7Δ mutants and restored the fragmented vacuole. The in vitro ubiquitination assay revealed that AlRabring7 function as E3 ubiquitin ligase and mediates AlRab7 ubiquitination. Overexpression of AlRab7 and AlRabring7 independently and when co-transformed enhanced the growth of yeast cells during stress conditions. Further, the bimolecular fluorescence complementation assay shows the in planta interaction of the two proteins. Our results suggest that AlRab7 and AlRabring7 confers enhanced stress tolerance in yeast.

Coordinated mechanisms of leaves and roots in response to drought stress underlying full-length transcriptome profiling in Vicia sativa L

BackgroundCommon vetch (Vicia sativa L.) is an important self-pollinating annual forage legume and is of interest for drought prone regions as a protein source to feed livestock and human consumption. However, the development and production of common vetch are negatively affected by drought stress. Plants have evolved common or distinct metabolic pathways between the aboveground and underground in response to drought stress. Little is known regarding the coordinated response of aboveground and underground tissues of common vetch to drought stress.ResultsOur results showed that a total of 30,427 full-length transcripts were identified in 12 samples, with an average length of 2278.89 bp. Global transcriptional profiles of the above 12 samples were then analysed via Illumina-Seq. A total of 3464 and 3062 differentially expressed genes (DEGs) were identified in the leaves and roots, respectively. Gene Ontology (GO) enrichment analyses identified that the dehydrin genes and Δ1-pyrroline-5-carboxylate synthase were induced for the biosynthesis of proline and water conservation. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis results indicated that the DEGs were significantly enriched in hormone signal transduction, starch and sucrose metabolism, and arginine and proline metabolism, and various drought response candidate genes were also identified. Abscisic acid (ABA; the AREB/ABF-SnRK2 pathway) regulates the activity of AMY3 and BAM1 to induce starch degradation in leaves and increase carbon export to roots, which may be associated with the drought stress responses in common vetch. Among the co-induced transcription factors (TFs), AREB/ABF, bHLH, MYB, WRKY, and AP2/ERF had divergent expression patterns and may be key in the crosstalk between leaves and roots during adaption to drought stress. In transgenic yeast, the overexpression of four TFs increased yeast tolerance to osmotic stresses.ConclusionThe multipronged approach identified in the leaves and roots broadens our understanding of the coordinated mechanisms of drought response in common vetch, and further provides targets to improve drought resistance through genetic engineering.

Selection and application of novel high temperature inducible promoters in the tolerant yeast Candida glycerinogenes

High temperature fermentation can substantially reduce the contaminations and condensation costs. Candida glycerinogenes was more resistant to high temperature than Saccharomyces cerevisiae. Quantitative real-time PCR results showed that 7 of 33 candidates served as potential high temperature inducible promoters in C.glycerinogenes. Fluorescence analysis indicated that PCgcwp1 showed the highest activity at 42°C. PCgaac and PCgpot1 showed medium-high expression, while PCghsp12, PCgatp1, PCgino1 and PCgscl were modest or weaker expression. Above seven promoters were further used to control the expression of xylose reductase gene from Neurospora crassa in C.glycerinogenes. Compared with 30°C, the xylitol yields of the seven recombinant strains were all improved at 42°C, and PCgcwp1 showed the highest xylitol production which was increased by 204% and reached 15.4g/L. These results showed a potential to high temperature fermentation on the stress tolerant C.glycerinogenes and some of novel high temperature inducible promoters.

Ionic Liquid Tolerance of Yeasts in Family Dipodascaceae and Genus Wickerhamomyces

In previous studies of ionic liquid (IL) tolerance of numerous species of ascomycetous yeasts, two strains of Wickerhamomyces ciferrii and Galactomyces candidus had unusually high tolerance in media containing up to 5% (w/v) of the 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]). The study aimed at investigating whether additional strains of these species, and additional species in the Dipodascaceae family, also possess IL tolerance, and to compare sensitivity to the acetate and chloride versions of the ionic liquid. Fifty five yeast strains in the family Dipodascaceae, which encompasses genera Galactomyces, Geotrichum, and Dipodascus, and seven yeast strains of species Wickerhamomyces ciferrii were tested for ability to grow in laboratory medium containing no IL, 242mM [C2C1Im][OAc], or 242mM [C2C1Im]Cl, and in IL-pretreated switchgrass hydrolysate. Many yeasts exhibited tolerance of one or both ILs, with higher tolerance of the chloride anion than of the acetate anion. Different strains of the same species exhibited varying degrees of IL tolerance. Galactomyces candidus, UCDFSTs 52-260, and 50-64, had exceptionally robust growth in [C2C1Im][OAc], and also grew well in the switchgrass hydrolysate. Identification of IL tolerant and IL resistant yeast strains will facilitate studies of the mechanism of IL tolerance, which could include superior efflux, metabolism or exclusion.

Arabidopsis Mitochondrial Voltage-Dependent Anion Channels Are Involved in Maintaining Reactive Oxygen Species Homeostasis, Oxidative and Salt Stress Tolerance in Yeast

Voltage-dependent anion channels (VDACs) are conserved proteins of the mitochondria. We have functionally compared Arabidopsis VDACs using Saccharomyces cerevisiae Δpor1 and M3 yeast system. VDAC (1, 2, and 4) were able to restore Δpor1 growth in elevated temperature, in oxidative and salt stresses, whereas VDAC3 only partially rescued Δpor1 in these conditions. The ectopic expression of VDAC (1, 2, 3, and 4) in mutant yeast recapitulated the mitochondrial membrane potential thus, enabled it to maintain reactive oxygen species homeostasis. Overexpression of these VDACs (AtVDACs) in M3 strain did not display any synergistic or antagonistic activity with the native yeast VDAC1 (ScVDAC1). Collectively, our data suggest that Arabidopsis VDACs are involved in regulating respiration, reactive oxygen species homeostasis, and stress tolerance in yeast.

SaHsfA4c From Sedum alfredii Hance Enhances Cadmium Tolerance by Regulating ROS-Scavenger Activities and Heat Shock Proteins Expression.

The heat shock transcription factor (Hsf) family, an important member in plant stress response, affects cadmium (Cd) tolerance in plants. In this study, we identified and functionally characterized a transcript of the Hsf A4 subgroup from Sedum alfredii. Designated as SaHsfA4c, the open reading frame was 1,302 bp long and encoded a putative protein of 433 amino acids containing a complete DNA-binding domain (DBD). Heterologous expression of SaHsfA4c in yeast enhanced Cd stress tolerance and accumulation, whereas expression of the alternatively spliced transcript InSaHsfA4c which contained an intron and harbored an incomplete DBD, resulted in relatively poor Cd stress tolerance and low Cd accumulation in transgenic yeast. The function of SaHsfA4c under Cd stress was characterized in transgenic Arabidopsis and non-hyperaccumulation ecotype S. alfredii. SaHsfA4c was able to rescue the Cd sensitivity of the Arabidopsis athsfa4c mutant. SaHsfA4c reduced reactive oxygen species (ROS) accumulation and increased the expression of ROS-scavenging enzyme genes and Hsps in transgenic lines. The present results suggest that SaHsfA4c increases plant resistance to stress by up-regulating the activities of ROS-scavenging enzyme and the expression of Hsps.

Pathway-based signature transcriptional profiles as tolerance phenotypes for the adapted industrial yeast Saccharomyces cerevisiae resistant to furfural and HMF.

The industrial yeast Saccharomyces cerevisiae has a plastic genome with a great flexibility in adaptation to varied conditions of nutrition, temperature, chemistry, osmolarity, and pH in diversified applications. A tolerant strain against 2-furaldehyde (furfural) and 5-hydroxymethyl-2-furaldehyde (HMF) was successfully obtained previously by adaptation through environmental engineering toward development of the next-generation biocatalyst. Using a time-course comparative transcriptome analysis in response to a synergistic challenge of furfural-HMF, here we report tolerance phenotypes of pathway-based transcriptional profiles as components of the adapted defensive system for the tolerant strain NRRL Y-50049. The newly identified tolerance phenotypes were involved in biosynthesis superpathway of sulfur amino acids, defensive reduction-oxidation reaction process, cell wall response, and endogenous and exogenous cellular detoxification. Key transcription factors closely related to these pathway-based components, such as Yap1, Met4, Met31/32, Msn2/4, and Pdr1/3, were also presented. Many important genes in Y-50049 acquired an enhanced transcription background and showed continued increased expressions during the entire lag phase against furfural-HMF. Such signature expressions distinguished tolerance phenotypes of Y-50049 from the innate stress response of its progenitor NRRL Y-12632, an industrial type strain. The acquired yeast tolerance is believed to be evolved in various mechanisms at the genomic level. Identification of legitimate tolerance phenotypes provides a basis for continued investigations on pathway interactions and dissection of mechanisms of yeast tolerance and adaptation at the genomic level.

Evaluation of Kuroda etal.: Insight into Yeast Isobutanol Tolerance with Advances Still Needed.

One snapshot of the peer review process for "Critical Roles of the Pentose Phosphate Pathway and GLN3 in Isobutanol-Specific Tolerance in Yeast" (Kuroda etal., 2019).

Machine Learning Approach for Candida albicans Fluconazole Resistance Detection Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

Candida albicans causes life-threatening systemic infections in immunosuppressed patients. These infections are commonly treated with fluconazole, an antifungal agent targeting the ergosterol biosynthesis pathway. Current Antifungal Susceptibility Testing (AFST) methods are time-consuming and are often subjective. Moreover, they cannot reliably detect the tolerance phenomenon, a breeding ground for the resistance. An alternative to the classical AFST methods could use Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass spectrometry (MS). This tool, already used in clinical microbiology for microbial species identification, has already offered promising results to detect antifungal resistance on non-azole tolerant yeasts. Here, we propose a machine-learning approach, adapted to MALDI-TOF MS data, to qualitatively detect fluconazole resistance in the azole tolerant species C. albicans. MALDI-TOF MS spectra were acquired from 33 C. albicans clinical strains isolated from 15 patients. Those strains were exposed for 3 h to 3 fluconazole concentrations (256, 16, 0 μg/mL) and with (5 μg/mL) or without cyclosporin A, an azole tolerance inhibitor, leading to six different experimental conditions. We then optimized a protein extraction protocol allowing the acquisition of high-quality spectra, which were further filtered through two quality controls. The first one consisted of discarding not identified spectra and the second one selected only the most similar spectra among replicates. Quality-controlled spectra were divided into six sets, following the sample preparation’s protocols. Each set was then processed through an R based script using pre-defined housekeeping peaks allowing peak spectra positioning. Finally, 32 machine-learning algorithms applied on the six sets of spectra were compared, leading to 192 different pipelines of analysis. We selected the most robust pipeline with the best accuracy. This LDA model applied to the samples prepared in presence of tolerance inhibitor but in absence of fluconazole reached a specificity of 88.89% and a sensitivity of 83.33%, leading to an overall accuracy of 85.71%. Overall, this work demonstrated that combining MALDI-TOF MS and machine-learning could represent an innovative mycology diagnostic tool.

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

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

The genetic basis of aneuploidy tolerance in wild yeast.

Aneuploidy is highly detrimental during development yet common in cancers and pathogenic fungi - what gives rise to differences in aneuploidy tolerance remains unclear. We previously showed that wild isolates of Saccharomyces cerevisiae tolerate chromosome amplification while laboratory strains used as a model for aneuploid syndromes do not. Here, we mapped the genetic basis to Ssd1, an RNA-binding translational regulator that is functional in wild aneuploids but defective in laboratory strain W303. Loss of SSD1 recapitulates myriad aneuploidy signatures previously taken as eukaryotic responses. We show that aneuploidy tolerance is enabled via a role for Ssd1 in mitochondrial physiology, including binding and regulating nuclear-encoded mitochondrial mRNAs, coupled with a role in mitigating proteostasis stress. Recapitulating ssd1Δ defects with combinatorial drug treatment selectively blocked proliferation of wild-type aneuploids compared to euploids. Our work adds to elegant studies in the sensitized laboratory strain to present a mechanistic understanding of eukaryotic aneuploidy tolerance.

Isolation of Indigenous Selenium Tolerant Yeast and Investigation of the Relationship Between Growth and Selenium Biotransformation.

Purpose: Organic selenium compound such as selenomethionine plays a significant function in the response to oxidative stress. Saccharomyces cerevisiae have the ability to accumulate selenium and selenium biotransformation. Selection of indigenous selenium tolerant yeast is our goals. The relationship between cell growth and selenium biotransformation was also investigated. Methods: The screening of the yeast cell was carried out at two steps in order to select yeast with high capacity for resistance and accumulation of selenium. The isolates were selected according to produced high biomass at different concentrations of selenium. Secondly, best yeast strains from previous step were grown in presence of 25 mg/L of sodium selenite and organic selenium content was measured. Results: The S17 isolate showed had maximum organic selenium accumulation (2515 ppm) and biomass production (2.73 g/L) compared to the other isolates. The biomass production and organic selenium accumulation of the S17 during 120 hours was shown a direct relationship between growth and biotransformation. Conclusion: This increase in organic selenium content was achieved with yeast screening. It is interesting to know that organic selenium has high bioavailability and low toxicity compared with inorganic selenium. Therefore, finding yeast strains which are resistant to selenium can be very helpful in cancer prevention.

A Role for COX20 in Tolerance to Oxidative Stress and Programmed Cell Death in Saccharomyces cerevisiae.

Industrial production of bioethanol from lignocellulosic materials (LCM′s) is reliant on a microorganism being tolerant to the stresses inherent to fermentation. Previous work has highlighted the importance of a cytochrome oxidase chaperone gene (COX20) in improving yeast tolerance to acetic acid, a common inhibitory compound produced during pre-treatment of LCM’s. The presence of acetic acid has been shown to induce oxidative stress and programmed cell death, so the role of COX20 in oxidative stress was determined. Analysis using flow cytometry revealed that COX20 expression was associated with reduced levels of reactive oxygen species (ROS) in hydrogen peroxide and metal-induced stress, and there was a reduction in apoptotic and necrotic cells when compared with a strain without COX20. Results on the functionality of COX20 have revealed that overexpression of COX20 induced respiratory growth in Δimp1 and Δcox18, two genes whose presence is essential for yeast respiratory growth. COX20 also has a role in protecting the yeast cell against programmed cell death.

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.