Halotolerant Yeast Research Articles

Physiological Basis for the Tolerance of Yeast Zygosaccharomyces bisporus to Salt Stress

Zygosaccharomyces bisporus is a moderately halotolerant yeast isolated from highly sugary and salty foods. We performed various evident biochemical and in vivo experiments as first of its kind to sketch out the possible overlay of salt tolerance mechanism in this model organism. The growth and survival curve analysis revealed that 1.0 M NaCl concentration (sublethal) enacts growth inhibitory effects with prompting immediate delay in cell division cycle; however, yeast cells adopted modified stress physiologically with further stretched stress spans which was accompanied by an upsurge in the level of cellular metabolites such as trehalose (reserve carbohydrate) and chiefly glycerol (polyols) as major compatible osmolytes, suggesting their role in defense mechanism against osmotic stress. To further elucidate the relation of osmotic stress cell physiology to salinity, thiobarbituric acid reactive substances, protein carbonyl, and reduced glutathione content were measured in salt-stressed cells demonstrating positive correlation of reactive oxygen species generation in Z. bisporus with an elevated concentration of lipid and protein oxidation, thereby damaging cell membrane and eventually causing cell death. We assessed NaCl exposure sourcing increased intracellular reactive oxygen species concentration, by an electron transfer-based colorimetric cupric-reducing antioxidant capacity assay justifying that cellular total antioxidant capacity which uses all the combined antioxidant activities present within vitamins, proteins, lipids, and glutathione reverses these deleterious stress effects. Henceforth, performance of Z. bisporus MTCC 4801 mounted because of stress regime seems to be multifactorial.

Debaryomyces hansenii up regulates superoxide dismutase gene expression and enhances the immune response and survival in Pacific red snapper (Lutjanus peru) leukocytes after Vibrio parahaemolyticus infection

Application of yeast is increasing to improve welfare and promotes growth in aquaculture. The halotolerant yeast Debaryomyces hansenii is normally a non-pathogenic yeast with probiotic properties and potential source of antioxidant enzymes as superoxide dismutase. Here, first, we characterized the sequence features of MnSOD and icCu/ZnSOD from Pacific red snapper, and second, we evaluated the potential antioxidant immune responses of the marine yeast Debaryomyces hansenii strain CBS004 in leukocytes which were then subjected to Vibrio parahaemolyticus infection. In silico analysis revealed that LpMnSOD consisted of 1186 bp, with an ORF of 678 bp encoding a 225 amino acid protein and LpicCu/ZnSOD consisted of 1090 bp in length with an ORF of 465 bp encoding a 154 amino acid protein. Multiple alignment analyzes revealed many conserved regions and active sites among its orthologs. In vitro assays using head-kidney and spleen leukocytes immunostimulated with D. hansenii and zymosan in response to V. parahaemolyticus infection reveled that D. hansenii strain CBS004 significantly increased transcriptions of MnSOD and icCu/ZnSOD genes. Flow cytometry assay showed that D. hansenii was able to inhibit apoptosis caused by V. parahaemolyticus in the Pacific red snapper leukocytes and enhanced the phagocytic capacity in head-kidney leukocytes. Immunological assays reveled an increased in superoxide dismutase and peroxidase activities, as well as, in nitric oxide production and reactive oxygen species production (respiratory burst) in fish stimulated with D. hansenii. Finally, our results. These results strongly support the idea that marine yeast Debaryomyces hansenii strain CBS004 can stimulate the antioxidant immune mechanism in head-kidney and spleen leukocytes.

The serine/threonine phosphatase DhSIT4 modulates cell cycle, salt tolerance and cell wall integrity in halo tolerant yeast Debaryomyces hansenii

The highly conserved family of Phosphoprotein phosphatases (PPP) regulates several major physiological processes in yeast. However, very little is known about the PPP orthologs from the yeast species inhabiting extreme environmental niches. In the present study we have identified DhSIT4, a member of PPP6 class of serine threonine phosphatases from the halotolerant yeast Debaryomyces hansenii. Deletion of DhSIT4 in D. hansenii was not lethal but the mutant exhibited reduced growth due to its effect on the cell cycle. The knock out mutant Dhsit4Δ showed sensitivity towards Li+, Na+ and cell wall damaging agents. The expression of DhSit4p rescued salt, caffeine and calcofluor white sensitivity of Dhmpk1Δ strain and thereby indicating a genetic interaction of this phosphatase with the cell wall integrity pathway in this species. Our study also demonstrated the antagonistic roles of DhSit4p and DhPpz1p in maintaining the cell cycle and ion homeostasis in D. hansenii.

The halotolerant Debaryomyces hansenii, the Cinderella of non-conventional yeasts.

Debaryomyces hansenii is a halotolerant yeast with a high biotechnological potential, particularly in the food industry. However, research in this yeast is limited by its molecular peculiarities. In this review we summarize the state of the art of research in this microorganisms, describing both pros and cons. We discuss (i) its halotolerance, (ii) the molecular factors involved in saline and osmotic stress, (iii) its high gene density and ambiguous CUG decoding, and (iv) its biotechnological and medical interests. We trust that all the bottlenecks in its study will soon be overcome and D. hansenii will become a fundamental organism for food biotechnological processes. Copyright © 2016 John Wiley & Sons, Ltd.

Differential hypersaline stress response in Zygosaccharomyces rouxii complex yeasts: a physiological and transcriptional study.

The Zygosaccharomyces rouxii complex comprises three distinct lineages of halotolerant yeasts relevant in food processing and spoilage, such as Z. sapae, Z. rouxii and a mosaic group of allodiploid strains. They manifest plastic genome architecture (variation in karyotype, ploidy level and Na(+)/H(+) antiporter-encoding gene copy number), and exhibit diverse tolerances to salt concentrations. Here, we investigated accumulation of compatible osmolytes and transcriptional regulation of Na(+)/H(+) antiporter-encoding ZrSOD genes during salt exposure in strains representative for the lineages, namely Z. sapae ABT301(T) (low salt tolerant), Z. rouxii CBS 732(T) (middle salt tolerant) and allodiploid strain ATCC 42981 (high salt tolerant). Growth curve modelling in 2 M NaCl-containing media supplemented with or without yeast extract as nitrogen source indicates that moderate salt tolerance of CBS 732(T) mainly depends on nitrogen availability rather than intrinsic inhibitory effects of salt. All the strains produce glycerol and not mannitol under salt stress and use two different glycerol balance strategies. ATCC 42981 produces comparatively more glycerol than Z. sapae and Z. rouxii under standard growth conditions and better retains it intracellularly under salt injuries. Conversely, Z. sapae and Z. rouxii enhance glycerol production under salt stress and intracellularly retain glycerol less efficiently than ATCC 42981. Expression analysis shows that, in diploid Z. sapae and allodiploid ATCC 42981, transcription of gene variants ZrSOD2-22/ZrSOD2 and ZrSOD22 is constitutive and salt unresponsive.

Both BAT1 and ARO8 are responsible for unpleasant odor generation in halo-tolerant yeast Zygosaccharomyces rouxii.

Soy sauce yeast Zygosaccharomyces rouxii plays a central role in the production of flavor compounds in soy sauce, while the flor-forming strain spoils its quality by producing 2-methylpropanoic acid, 2-methylbutanoic acid, and 3-methylbutanoic acid, which have an unpleasant odor. To investigate the relationship between flor formation and unpleasant odor, we measured the volatile compounds that accumulated under various growth conditions. As a result, marked amounts of 2-methylpropanoic acid, 2-methylbutanoic acid, or 3-methylbutanoic acid accumulated in synthetic medium containing valine, isoleucine, or leucine, respectively, under aerobic growth conditions. These results implied that the unpleasant compounds were produced from their corresponding branched chain amino acid (BCAA) when the cell was placed under aerobic condition through flor formation. The first step in BCAA catabolism and the last step in BCAA anabolism are both catalyzed by a BCAA transaminase. A mutant lacking the BCAA transaminase gene, BAT1, resulted in valine and isoleucine auxotrophy, while a mutant lacking both BAT1 and the α-aminoadipate aminotransferase gene, ARO8, resulted in valine, isoleucine, and leucine auxotrophy. Although the bat1∆ aro8∆ double mutant formed flor similarly to the wild-type strain, the mutant exhibited less unpleasant odor generation. These results suggest that the interconversion between 4-methyl-2-oxopentanoate and leucine is catalyzed by both Bat1p and Aro8p in Z. rouxii. Taken together, these results indicate that flor formation is not seemed to be directly linked to unpleasant odor generation. These findings encourage us to breed flor-forming yeasts without an unpleasant odor.

Isolation and characterization of malachite green-removing yeast from a traditional fermented fishery product

The screening of malachite green (MG)-degrading microorganisms was carried out using various sources, namely, fish farms and a traditional fermented fishery product in Myanmar and Thailand. The enrichment culture method was performed using MG-containing broth media, and colonies that showed the decolorization of MG on plate media were isolated as MG-degrading candidates. From the results of the sequencing of the D1/D2 domain of the 26S rRNA gene, strain M3, a representative strain of MG-degrading candidates was identified as a halotolerant yeast, Debaryomycesnepalensis. Liquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis indicated that during the incubation of this strain, the MG concentration gradually decreased and eventually reached undetectable levels. Conversely, the concentration of leucomalachite green (LMG) increased, and the final amount of LMG in the broth culture was estimated to be approximately 40 % of the initial MG amount. In addition, results of proton nuclear magnetic resonance spectroscopy (1H-NMR) analysis also showed that MG and the tautomer of MG or other aromatic decomposition products of MG were not detected as a major component at the end of incubation. These results suggest that strain M3 removed MG and changed approximately 40 % to LMG and 60 % to some metabolites other than LMG.

Non-targeted metabolomic reveals the effect of salt stress on global metabolite of halotolerant yeast Candida versatilis and principal component analysis.

As one of the major microbes in the soy sauce fermentation, Candida versatilis enriches the flavor and improves the quality of soy sauce. In this study, a combination of five different GC-MS and LC-MS-based metabolome analytical approaches was used to analyze the intracellular, extracellular and whole metabolites of C. versatilis. Our results found out that a total of 132, 244 and 267 different metabolites were detectable from the intracellular, extracellular and whole part, respectively. When exposed to 0. 9 and 18% salt, respectively, 114, 123 and 129 different intracellular metabolites, 184, 200 and 178 extracellular metabolites and 177, 188 and 186 whole metabolites were detected, respectively. Our data showed that salt enhances the metabolic capacity of C. versatilis, especially its amino acid and enhances the synthesis and secretion of some metabolites of C. versatilis, especially the aldehydes and phenols, such as vanillin, guaiacol and 5-hydroxymethylfurfural. Our data also showed that special attention has to be paid to the generation of biogenic amines when C. versatilis was treated with salt.

Adaptation to high salt concentrations in halotolerant/halophilic fungi: a molecular perspective.

Molecular studies of salt tolerance of eukaryotic microorganisms have until recently been limited to the baker's yeast Saccharomyces cerevisiae and a few other moderately halotolerant yeast. Discovery of the extremely halotolerant and adaptable fungus Hortaea werneckii and the obligate halophile Wallemia ichthyophaga introduced two new model organisms into studies on the mechanisms of salt tolerance in eukaryotes. H. werneckii is unique in its adaptability to fluctuations in salt concentrations, as it can grow without NaCl as well as in the presence of up to 5 M NaCl. On the other hand, W. ichthyophaga requires at least 1.5 M NaCl for growth, but also grows in up to 5 M NaCl. Our studies have revealed the novel and intricate molecular mechanisms used by these fungi to combat high salt concentrations, which differ in many aspects between the extremely halotolerant H. werneckii and the halophilic W. ichthyophaga. Specifically, the high osmolarity glycerol signaling pathway that is important for sensing and responding to increased salt concentrations is here compared between H. werneckii and W. ichthyophaga. In both of these fungi, the key signaling components are conserved, but there are structural and regulation differences between these pathways in H. werneckii and W. ichthyophaga. We also address differences that have been revealed from analysis of their newly sequenced genomes. The most striking characteristics associated with H. werneckii are the large genetic redundancy, the expansion of genes encoding metal cation transporters, and a relatively recent whole genome duplication. In contrast, the genome of W. ichthyophaga is very compact, as only 4884 protein-coding genes are predicted, which cover almost three quarters of the sequence. Importantly, there has been a significant increase in their hydrophobins, cell-wall proteins that have multiple cellular functions.

Characterization of New Polyol/H+ Symporters in Debaryomyces hansenii

Debaryomyces hansenii is a halotolerant yeast that produces and assimilates a wide variety of polyols. In this work we evaluate polyol transport in D. hansenii CBS 767, detecting the occurrence of polyol/H+ (and sugar/H+) symporter activity, through the transient extracellular alkalinization of unbuffered starved cell suspensions. From the D. hansenii genome database, we selected nine ORFs encoding putative transporter proteins to clone in a centromeric plasmid with C-terminal GFP tagging and screened for polyol/H+ symporters by heterologous expression in Saccharomyces cerevisiae. Five distinct D. hansenii polyol/H+ symporters were identified and characterized, with different specificities and affinities for polyols, namely one glycerol-specific (DhStl1), one D-galactitol-specific (DhSgl1, Symporter galactitol/H+ 1), one D-(+)-chiro-inositol-specific (DhSyi1, Symporter D-(+)-chiro-inositol/H+ 1), one for D-sorbitol/D-mannitol/ribitol/D-arabitol/D-galactitol (DhSyl1, Symporter Polyols 1) and another for D-sorbitol/D-mannitol/ribitol/D-arabitol (DhSyl2, Symporter Polyols 2). This work contributed to the annotation of new yeast polyol transporters, including two specific for uncommon substrates as galactitol and D-(+)-chiro-inositol.

Insertion of a Specific Fungal 3′-phosphoadenosine-5′-phosphatase Motif into a Plant Homologue Improves Halotolerance and Drought Tolerance of Plants

Soil salinity and drought are among the most serious agricultural and environmental problems of today. Therefore, investigations of plant resistance to abiotic stress have received a lot of attention in recent years. In this study, we identified the complete coding sequence of a 3′-phosphoadenosine-5′-phosphatase protein, ApHal2, from the halotolerant yeast Aureobasidium pullulans. Expression of the ApHAL2 gene in a Saccharomyces cerevisiae hal2 mutant complemented the mutant auxotrophy for methionine, and rescued the growth of the hal2 mutant in media with high NaCl concentrations. A 21-amino-acids-long region of the ApHal2 enzyme was inserted into the Arabidopsis thaliana homologue of Hal2, the SAL1 phosphatase. The inserted sequence included the META motif, which has previously been implicated in increased sodium tolerance of the Hal2 homologue from a related fungal species. Transgenic Arabidopsis plants overexpressing this modified SAL1 (mSAL1) showed improved halotolerance and drought tolerance. In a medium with an elevated salt concentration, mSAL1-expressing plants were twice as likely to have roots in a higher length category in comparison with the wild-type Arabidopsis and with plants overexpressing the native SAL1, and had 5% to 10% larger leaf surface area under moderate and severe salt stress, respectively. Similarly, after moderate drought exposure, the mSAL1-expressing plants showed 14% increased dry weight after revitalisation, with no increase in dry weight of the wild-type plants. With severe drought, plants overexpressing native SAL1 had the worst rehydration success, consistent with the recently proposed role of SAL1 in severe drought. This was not observed for plants expressing mSAL1. Therefore, the presence of this fungal META motif sequence is beneficial under conditions of increased salinity and moderate drought, and shows no drawbacks for plant survival under severe drought. This demonstrates that adaptations of extremotolerant fungi should be considered as a valuable resource for improving stress-tolerance in plant breeding in the future.

The branched mitochondrial respiratory chain from Debaryomyces hansenii: Components and supramolecular organization

The branched respiratory chain in mitochondria from the halotolerant yeast Debaryomyces hansenii contains the classical complexes I, II, III and IV plus a cyanide-insensitive, AMP-activated, alternative-oxidase (AOX). Two additional alternative oxidoreductases were found in this organism: an alternative NADH dehydrogenase (NDH2e) and a mitochondrial isoform of glycerol-phosphate dehydrogenase (MitGPDH). These monomeric enzymes lack proton pump activity. They are located on the outer face of the inner mitochondrial membrane. NDH2e oxidizes exogenous NADH in a rotenone-insensitive, flavone-sensitive, process. AOX seems to be constitutive; nonetheless, most electrons are transferred to the cytochromic pathway. Respiratory supercomplexes containing complexes I, III and IV in different stoichiometries were detected. Dimeric complex V was also detected. In-gel activity of NADH dehydrogenase, mass spectrometry, and cytochrome c oxidase and ATPase activities led to determine the composition of the putative supercomplexes. Molecular weights were estimated by comparison with those from the yeast Y. lipolytica and they were IV2, I–IV, III2–IV4, V2, I–III2, I–III2–IV, I–III2–IV2, I–III2–IV3 and I–III2–IV4. Binding of the alternative enzymes to supercomplexes was not detected. This is the first report on the structure and organization of the mitochondrial respiratory chain from D. hansenii.

16 strains of Debaryomyces hansenii tested against Penicillium digitatum, a post harvest fungus, affecting citrus crops in Puerto Rico

Citrus crops contribute 11 million dollars to the Puerto Rican economy. Unfortunately, Penicillium spp. infects these crops causing a great economic loss. Debaryomyces hansenii, a marine halotolerant yeast, has the ability to produce a killer toxin which seems to be homologous to the KHR (killer toxin) gene of Saccharomyces cerevisiae whose activity in the reduction of the decay of citrus fruit caused by Penicillium spp. has been reported. Microbiology and molecular biology techniques have been used to characterize sixteen D. hansenii strains and isolate their killer toxin. Different assays of the killer toxin activity in vivo were realized to determine its potential as a biological control. Two strains, D. hansenii BCS 006 and YB‐17914, were more efficient against Penicillium digitatum.

Proteomic changes in response to potassium starvation in the extremophilic yeast Debaryomyces hansenii

In this work, we performed for the first time a proteomic approach to the processes induced by long-term potassium starvation in the halotolerant yeast Debaryomyces hansenii. The proteomic profile under this ionic stress conditions shows that important changes in gene expression take place as an adaptive response. We found a significant protein expression repression as well as metabolic changes such as the inhibition of the upper part of the glycolysis, the amino acid synthesis, and the Krebs cycle. On the other hand, genes related to stress responses, protein degradation, and sterols synthesis were upregulated in response to potassium deprivation. The findings in this study provide important information about how this particular yeast copes with ionic stress at molecular levels, which might further enrich the global understanding of salt tolerance processes in eukaryal systems and moreover highlighting the importance of the 'omics' approaches as a complement to the classical physiological studies.

Elucidation of the function of 16 newly identified genes of D. hansenii and their role in osmoadaptation

Debaromyces hansenii, a halotolerant yeast, is a model system for the study of osmoadaptation, tolerating up to 4M NaCl. This osmotic stress has been monitored with microarrays, where 109 genes displayed a significant change in expression. Among them, 16 genes have not had their function annotated. The characterization of these genes or their protein products by various bioinformatics analysis tools is key for the determination of their role in the halotolerant properties and preparedness of D. hansenii as an extremophilic organism. These results will assist in the determination of the appropriate experimental conditions for the complete elucidation of these genes, and the analysis of the potential application of D. hansenii in environmental and agricultural biotechnology currently underway in our laboratory.

Repeated capture of a cytoplasmic linear plasmid by the host nucleus in Debaryomyces hansenii

Debaryomyces hansenii is a halotolerant yeast species that has been shown to carry various nuclear genes of plasmid or viral origin (NUPAVs). However, a recent ancestor of such NUPAVs has not been identified. Here we determined for the first time the molecular structure of an entire cytoplasmic linear plasmid, pDH1A, indigenous to this species. The element is related to non-autonomous killer plasmids from Kluyveromyces lactis and Pichia acaciae and carries a B-type DNA polymerase as well as remnants of a killer toxin system, a secreted chitin-binding protein. Other essential toxin subunits or an immunity function, however, appear to be lost, while two additional small open reading frames are present. Transcripts for all four genes located on pDH1A could be verified by RT-PCR. Interestingly, all genes from pDH1A could be identified as ancestors of NUPAVs located at different chromosomes within the nucleus of D. hansenii, suggesting repeated nuclear capture of fragments originating from pDH1A.

Screening and identification of halotolerant yeast for hydrocarbon degrading and its properties studies

A strain of hydrocarbon degrading yeast named YH-41 was isolated and purified from the soil polluted by crude oil from Shengli oil field, China. The strain was identified by morphological, biochemical characteristics and 18S rDNA sequence analysis, the results show that the strain was similar to the haploid nucleus of Pichia ohmen, which belong toCandida., The GC analysis showed that the yeasts degradated diesel oil. Through bioremediation experiments, we can study the feasibility of bioremediation of oil-polluted soil by yeasts. The results show that the haploid nucleus of P. ohmen has a certain role on accelerating oil degradation in soil early in the test. The degradation rate of residual oil in the same period of time increased about 20% compared with other treatment without adding the yeasts. Key words: 18S rDNA, Candida, Pichia ohmen, haploid, GC analysis and bioremediation

手延べ乾麺の空隙形成に関与する耐塩性酵母

手延べ乾麺の空隙形成に関与する耐塩性酵母

A Statistical Approach to Optimize Xylitol Production by <i>Debaryomyces nepalensis</i> NCYC 3413 <i>in Vitro</i>

Debaryomyces nepalensis NCYC 3413, halotolerant yeast isolated from rotten apple, was capable of utilizing components of hemicellulose hydrolysate such as glucose, galactose, mannose, xylose and arabinose. The organism utilizes xylose as a sole carbon source and produces xylitol. The Plackett-Burman design was applied to determine the specific medium components affecting xylitol production and found that xylose, K2HPO4, and ZnSO4 were critical in augmenting xylitol production. These significant parameters were further optimized using response surface methodology. The optimum concentrations of xylose, K2HPO4, and ZnSO4 were found to be 100 g/l, 10.6 g/l and 8.9 mg/l respectively. Under these optimal conditions the xylitol production increased from 27 g/l to 36 g/l with a yield of 0.44 g/g (57% increase in total yield). In addition, formation of the by product (glycerol) was decreased under optimal conditions.

Purification and biochemical characterization of a moderately halotolerant NADPH dependent xylose reductase from Debaryomyces nepalensis NCYC 3413

A Xylose reductase (XR) from the halotolerant yeast, Debaryomyces nepalensis NCYC 3413 was purified to apparent homogeneity. The enzyme has a molecular mass of 74kDa with monomeric subunit of 36.4kDa (MALDI-TOF/MS) and pI of 6.0. The enzyme exhibited its maximum activity at pH 7.0 and 45°C (21.2U/mg). In situ gel digestion and peptide mass fingerprinting analysis showed 12–22% sequence homology with XR from other yeasts. Inhibition of the enzyme by DEPC (diethylpyrocarbonate) confirmed the presence of histidine residue in its active site. The enzyme exhibited high preference for pentoses over hexoses with greater catalytic efficiency for arabinose than xylose. The enzyme also showed absolute specificity with NADPH over NADH. The enzyme retained 90% activity with 100mM of NaCl or KCl and 40% activity with 1M KCl which suggest that the enzyme is moderately halotolerant and can be utilized for commercial production of xylitol under conditions where salts are present.