Salt-tolerant Yeast Zygosaccharomyces Rouxii Research Articles

High Glucose Is a Stimulation Signal of the Salt-Tolerant Yeast Zygosaccharomyces rouxii on Thermoadaptive Growth.

The salt-tolerant yeast Zygosaccharomyces rouxii is a typical aroma-producing yeast used in food brewing, but its mechanism of high temperature tolerance is still unclear. In this study, the response mechanism of Z. rouxii to glucose under high temperature stress at 40 °C was explored, based on the total synthetic lowest-nutrient medium. The results of the growth curves and scanning electron microscopy showed that high glucose was necessary for Z. rouxii to restore growth under high temperature stress, with the biomass at 300 g/L of glucose (OD600, 120h = 2.44 ± 0.26) being 8.71 times higher than that at 20 g/L (OD600, 120h = 0.28 ± 0.08). The results of the transcriptome analysis, combined with RT-qPCR, showed that the KEGG analysis of differentially expressed genes was enriched in pathways related to glucose metabolism, and high glucose (300 g/L) could effectively stimulate the gene expression of glucose transporters, trehalose synthesis pathways, and xylitol synthesis pathways under a high temperature, especially the expression of the glucose receptor gene RGT2 (up-regulated 193.7 times at 12 h). The corresponding metabolic characteristics showed that the contents of intracellular metabolites, such as glucose (Cmax, 6h = 6.50 ± 0.12 mg/g DCW), trehalose (Cmax, 8h = 369.00 ± 17.82 μg/g DCW), xylitol (Cmax, 8h = 1.79 ± 0.27 mg/g DCW), and glycerol (Cmax, 8h = 268.10 ± 44.49 μg/g DCW), also increased with time. The accumulation of acetic acid, as the main product of overflow metabolism under high temperature stress (intracellular Cmax, 2h = 126.30 ± 10.96 μg/g DCW; extracellular Cmax, 12h = 499.63 ± 27.16 mg/L), indicated that the downstream glycolysis pathway was active. Compared with the normal physiological concentration of glucose, a high glucose concentration can effectively stimulate the gene expression and metabolism of salt-tolerant Z. rouxii under high-temperature conditions to restore growth. This study helps to deepen the current understanding of the thermoadaptive growth mechanism of salt-tolerant Z. rouxii.

Exponential feeding strategy of high-density cultivation of a salt-tolerant aroma-producing yeast Zygosaccharomyces rouxii in stirred fermenter

Salt-tolerant yeast Zygosaccharomyces rouxii is a key strain to produce flavor in soy sauce brewing. The aim of this research was to establish the optimal feeding strategy and parameter for high-density culture of Z. rouxii. An exponential feeding strategy was applied where the specific growth rate was fixed by limitation of the fed carbon source. The set point of specific growth rate of exponential feeding for fed-batch culture was chosen to be 0.03–0.07h−1. The optimal set point of specific growth rate (μset) of exponential feeding started after glucose depletion for fed-batch culture was found to be 0.05h−1. Furthermore, a relatively high-dense biomass (72.86g/L dry cell weight) was obtained in the culture of exponential feeding started after glucose was exhausted with μset=0.05h−1. Less than the level of Saccharomyces cerevisiae and Pichia pastoris, however the biomass concentration was much higher than data from other research about Z. rouxii culture.

Expression of key hydrolases for soy sauce fermentation in Zygosaccharomyces rouxii

Several key hydrolases in soy sauce fermentation such as proteases, peptidases, and glutaminases are supplied by Aspergillus sojae or Aspergillus oryzae. The genes encoding these hydrolases were successfully expressed in salt-tolerant yeast Zygosaccharomyces rouxii. These transformants are expected to supply extra hydrolases during soy sauce fermentation process.

The salt tolerant yeast Zygosaccharomyces rouxii possesses two plasma-membrane Na+/H+-antiporters (ZrNha1p and ZrSod2–22p) playing different roles in cation homeostasis and cell physiology

Antiporters exporting Na+ and K+ in exchange for protons are conserved among yeast species. The only exception so far has been Zygosaccharomyces rouxii, an osmotolerant species closely related to Saccharomyces cerevisiae. Z. rouxii was described as possessing one plasma-membrane antiporter transporting only Na+ (ZrSod2–22p in the CBS 732T type strain). We report the characterization of a second gene, ZrNHA1, encoding a new K+(Na+)/H+-antiporter capable of both K+ and Na+ export. Synteny analyses suggested that ZrSOD2–22 originated by single duplication of the ZrNHA1 gene. Substrate specificities and transport properties of ZrNha1p and ZrSod2–22p were compared upon heterologous expression in S. cerevisiae, and then directly in Z. rouxii. Deletion mutants and phenotype analyses revealed that ZrSod2–22 antiporter is important for Na+ detoxification, probably together with ZrEna1 ATPase; ZrNha1p is indispensable to maintain potassium homeostasis and ZrEna1p is not, in contrast to the situation in S. cerevisiae, involved in this function.

Co-expression of the Na/H-antiporter and H-ATPase genes of the salt-tolerant yeast in

We cloned two genes from the salt-tolerant yeast Zygosaccharomyces rouxii: ZrSOD2 for the cell membrane Na(+)/H(+)-antiporter and ZrPMA1 for the cell membrane H(+)-ATPase. The products of these genes play cooperative roles in the salt-tolerance of Z. rouxii, and the function of the ZrPMA1 product is regulated at the transcription level. We constructed a yeast expression vector that is able to co-express the ZrSOD2 and ZrPMA1 genes. Single expression of ZrSOD2 was effective in conferring salt-tolerance, and although a slight synergic effect was observed with co-expression of ZrSOD2 and ZrPMA1, the usefulness of this co-expression is likely to be minimal with regard to salt-tolerance.

Salt Shock Enhances the Expression of ZrATP2, the Gene for the Mitochondrial ATPase .BETA. Subunit of Zygosaccharomyces rouxii

In the course of a study of cell wall proteins from the salt-tolerant yeast Zygosaccharomyces rouxii , a protein that increased its expression as the NaCl concentration of the culture medium increased was identified. Several degenerate primers were constructed based on partial amino acid sequences of this protein and were used in PCR amplification of a gene termed ZrATP2 . The amino acid sequence deduced from nucleotide sequence of the gene revealed that ZrATP2 encodes the β subunit of mitochondrial F 1 ATPase. Northern blot analysis demonstrated that NaCl shock induced an elevation in ZrATP2 expression, which corresponded with the resumption of Z. rouxii cell growth after salt shock.

Salt shock enhances the expression of ZrATP2, the gene for the mitochondrial ATPase β subunit of Zygosaccharomyces rouxii

In the course of a study of cell wall proteins from the salt-tolerant yeast Zygosaccharomyces rouxii, a protein that increased its expression as the NaCl concentration of the culture medium increased was identified. Several degenerate primers were constructed based on partial amino acid sequences of this protein and were used in PCR amplification of a gene termed ZrATP2. The amino acid sequence deduced from nucleotide sequence of the gene revealed that ZrATP2 encodes the β subunit of mitochondrial F 1 ATPase. Northern blot analysis demonstrated that NaCl shock induced an elevation in ZrATP2 expression, which corresponded with the resumption of Z. rouxii cell growth after salt shock.

Role of the glutamic and aspartic residues in Na +-ATPase function in the ZrENA1 gene of Zygosaccharomyces rouxii

The effect of replacement of negatively charged amino acid residues on the function of Na + transport proteins of the salt-tolerant yeast Zygosaccharomyces rouxii was examined by heterologous expression of mutant proteins in a strain of Saccharomyces cerevisiae, RH16.6, that lacks native Na +-ATPase activity due to null mutations of ENA1, ENA2, ENA3, and ENA4. Mutants of Na +/H + antiporter gene ( ZrSOD2) and Na +-ATPase gene ( ZrENA1) of Z. rouxii were generated by site-directed mutagenesis. The significance of two aspartic residues arranged in tandem (D265 and D266) was demonstrated in Z. rouxii Na +/H + antiporter. Some Z. rouxii Na +-ATPase mutant genes, namely E778A, D852A, and E981A present in the transmembrane domains (TMDs) and D736A, D743A, D748A, D749A, D759A, and D760A present in the cytoplasmic space were constructed. A lower level of salt tolerance was bestowed by the mutant genes D852A and E981A present in TMDs and D748A and D749A present in cytoplasmic space, compared with the wild-type gene ( ZrENA1).

Concomitant extracellular accumulation of alpha-keto acids and higher alcohols by Zygosaccharomyces rouxii

Alpha-keto acids are key intermediates in the formation of higher alcohols, important flavor components in soy sauce, and produced by the salt-tolerant yeast Zygosaccharomyces rouxii. Unlike most of the higher alcohols, the alpha-keto acids are usually not extracellularly accumulated by Z. rouxii when it is cultivated with ammonium as the sole nitrogen source. To facilitate extracellular accumulation of the alpha-keto acids from aspartate-derived amino acid metabolism, the amino acids valine, leucine, threonine and methionine were exogenously supplied during batch and A-star cultivations of (routants of) Z. rouxii. It was shown that all alpha-keto acids from the aspartate-derived amino acid metabolism, except alpha-ketobutyrate, could be extracellularly accumulated. In addition, it appeared from the concomitant extracellular accumulation of alpha-keto acids and higher alcohols that in Z. rouxii, valine, leucine and methionine were converted via Ehrlich pathways similar to those in Saccharomyces cerevisiae. Unlike these amino acids, threonine was converted via both the Ehrlich and amino acid biosynthetic pathways in Z. rouxii.

Concomitant Extracellular Accumulation of Alpha-Keto Acids and Higher Alcohols by Zygosaccharomyces rouxii

Alpha-keto acids are key intermediates in the formation of higher alcohols, important flavor components in soy sauce, and produced by the salt-tolerant yeast Zygosaccharomyces rouxii. Unlike most of the higher alcohols, the alpha-keto acids are usually not extracellularly accumulated by Z. rouxii when it is cultivated with ammonium as the sole nitrogen source. To facilitate extracellular accumulation of the alpha-keto acids from aspartate-derived amino acid metabolism, the amino acids valine, leucine, threonine and methionine were exogenously supplied during batch and A-star cultivations of (routants of) Z. rouxii. It was shown that all alpha-keto acids from the aspartate-derived amino acid metabolism, except alpha-ketobutyrate, could be extracellularly accumulated. In addition, it appeared from the concomitant extracellular accumulation of alpha-keto acids and higher alcohols that in Z. rouxii, valine, leucine and methionine were converted via Ehrlich pathways similar to those in Saccharomyces cerevisiae. Unlike these amino acids, threonine was converted via both the Ehrlich and amino acid biosynthetic pathways in Z. rouxii.

Effect of nystatin on the release of glycerol from salt-stressed cells of the salt-tolerant yeast Zygosaccharomyces rouxii.

The polyene antibiotic nystatin, which affects fungal membrane permeability, inhibited the growth of Zygosaccharomyces rouxii grown in medium containing 15% (w/v) NaCl, whereas yeast grown in medium without NaCl were only slightly inhibited. Nystatin caused salt-stressed cells to release large amounts of glycerol and inhibited their growth, but amino acids and materials with an absorbance at 260 nm were not released from the cells. The leakage was increased by the addition of glucose, and more than 90% of the intracellular glycerol was released into the medium during a 2-h incubation with 0.11 microM nystatin and 2% (w/v) glucose. Glycerol was indispensable for the growth of Z. rouxii grown in culture medium containing 15% NaCl.

Two putative MAP kinase genes, ZrHOG1 and ZrHOG2, cloned from the salt-tolerant yeast Zygosaccharomyces rouxii are functionally homologous to the Saccharomyces cerevisiae HOG1 gene.

The salt-tolerant yeast Zygosaccharomyces rouxii can adjust its osmotic balance when responding to osmotic shock by accumulating glycerol as the compatible osmolyte. However, the mechanism of glycerol production in Z. rouxii cells and its genetic regulation remain to be elucidated. Two putative mitogen-activated protein (MAP) kinase genes, ZrHOG1 and ZrHOG2, were cloned from Z. rouxii by their homology with HOG1 from Saccharomyces cerevisiae. The deduced amino acid sequences of ZrHog1p and ZrHog2p indicated close homology to that of Hog1p and contained a TGY motif for phosphorylation by MAP kinase kinase. When ZrHOG1 or ZrHOG2 was expressed in an S. cerevisiae hog1delta null mutant, the salt tolerance and osmotic tolerance characteristics of wild-type S. cerevisiae were restored. In addition, the aberrant cell morphology and low glycerol content of the hog1delta null mutant were corrected, indicating that ZrHog1p and ZrHog2p have functions similar to Hog1p. While the transcription of the glycerol-3-phosphate dehydrogenase gene (GPD1) of the ZrHOG1-harbouring S. cerevisiae mutant was similar to that of wild-type S. cerevisiae, the ZrHOG2-harbouring strain showed prolonged GPD1 transcription. Both Zrhog1delta and Zrhog2delta Z. rouxii null mutants showed a decrease in salt tolerance compared to the wild-type strain. The present study suggested the presence of a high-osmolarity glycerol response (HOG) pathway in Z. rouxii similar to that elucidated in S. cerevisiae. Two putative MAP kinase genes in Z. rouxii appeared to be significant in either osmotic regulation or ion homeostasis.

Characterization of the Na +-ATPase gene ( ZENA1) from the salt-tolerant yeast Zygosaccharomyces rouxii

In order to clarify the relationship between the salt tolerance of Zygosaccharomyces rouxii and the function of Na +-ATPase, a gene which exhibited homology to the Na +-ATPase gene ( ZENA1) of Saccharomyces cerevisiae was isolated from Z. rouxii. This newly isolated gene ( ZENA1) encoded a product of 1048 amino acids. The predicted amino-acid sequence of Zena1p was highly homologous to that of S. cerevisiae Ena1p and Ena2p, and Schwanniomyces occidentalis Ena1p and Ena2p, but showed low homology to that of Zpma1p, which is the product of the Z. rouxii plasma membrane H +.ATPase gene ( ZENA1). Zena1p shares the peptide motifs which have been suggested to participate in the function of ATPase. Expression of ZENA1 was observed, but was independent of NaCl shock. When ZENA1 was expressed in salt-sensitive S. cerevisiae under the regulation of a GAL1 promoter by using the expression vector pYES2, salt tolerance of the transformants was observed. The growth characteristics of Zena1Δ-disruptants of Z. rouxii and the pH profiles of their plasma membrane ATPase activity were almost the same as those of the wild-type strain, indicating that the function of Zena1p is of little relevance to the salt tolerance property of Z. rouxii. By considering the close relationship between the salt tolerance of Z. rouxii and the function of its Na +/H +-antiporter, we concluded that the extrusion of Na + across the plasma membrane in Z. rouxii cells might be carried out mainly via the function of the Na +/H +-antiporter in a high salinity environment.

Regulation of intracellular osmotic pressure and some factors that influence the promotion of glycerol synthesis in a respiration-deficient mutant of the salt-tolerant yeast Zygosaccharomyces rouxii during salt stress.

The accumulation of glycerol and inorganic ions were examined in a respiration-deficient (RD) mutant isolated from the salt-tolerant yeast Zygosaccharomyces rouxii for 3 h after salt stress due to 1 M NaCl. After the start of salt stress, intracellular levels of glycerol continued to increase for up to 3 h, while the levels of Na + and Cl - ions in cells reached maximum values within 1 h and then decreased gradually. Increases in intracellular concentrations of solutes resulted in an osmotic pressure that was almost equivalent to the external osmotic pressure within 2 h after salt stress. The RD strain had the same ability to tolerate salt as the wild-type strain. Therefore, we used the RD strain to examine the mechanism in the glycolytic pathway that is responsible for the promotion of glycerol synthesis that is induced by NaCl. When exposed to medium with 1 M NaCl, RD cells diverted about one-sixteenth of the amount of ethanol that was produced in the medium without NaCl to the production of glycerol. This result suggests the presence of factors that mediate a change from the normal metabolism of glucose to the promotion of glycerol synthesis in response to external NaCl. The specific activities of glycerol-3-phosphate dehydrogenase (GPDH) in extracts of cells grown with and without 1 M NaCl were very low in reaction mixtures with NADH or NADPH, although the cellular activity of alcohol dehydrogenase (ADH) was high and was repressed by external Nacl. This result indicates that the pathway involving GPDH makes only a small contribution to the synthesis of glycerol and that an alternative pathway functions for the synthesis in Z. rouxii. The addition of sodium sulfite, which binds to acetaldehyde, and of glycidol, an inhibitor of triosephosphate isomerase (TPI), to the medium promoted the synthesis of glycerol in RD strain. These results suggest the possibility that the extra NADH resulted from the binding of sulfite to acetaldehyde, or the inhibition of ADH and/or TPI under the NaCl-stressed condition lead to the promotion of glycerol synthesis by Z. rouxii.

Efflux of sodium ions by a Na+/H+-antiporter during salt stress in the salt-tolerant yeast Zygosaccharomyces rouxii.

The flux of Na+, Cl- and H+ ions and the accumulation of glycerol in cells of a salt-tolerant yeast, Zygosaccharomyces rouxii, was examined during the initial stages of salt stress. Although the intracellular accumulation of glycerol that was required for osmoregulation began immediately after exposure of the cells to salt stress, rapid influx of Na+ and Cl- ions into cells also occurred. These ions were subsequently extruded from the cells and the intracellular levels of both ions were maintained much lower than those in the external medium. Furthermore, a rapid efflux of protons was induced by activation of the plasma membrane ATPase (PM- ATPase) immediately after the start of salt stress and then the influx (re-influx) of protons into cells occurred. The addition of a specific inhibitor of PM-ATPase, diethylstilbestrol (DES), and of inhibitors of the energy-generating system, namely sodium azide, antimycin A and dinitrophenol, inhibited the intracellular accumulation of glycerol and activation of PM-ATPase, and they retarded the rapid efflux of protons and the extrusion of Na+ and Cl- ions. The re-influx of protons into cells was inhibited by amphotericin B, probably because of the destruction of the structure of the plasma membrane. In mutants in which rapid activation of PM-ATPase was not observed immediately after the start of salt stress, the efflux of protons was markedly retarded and extrusion of Na+ and Cl- ions was also suppressed during salt stress. Amiloride, an inhibitor of the Na+/H+-antiporter, inhibited the re-influx of protons and the efflux of Na+ and Cl- ions, but this inhibitor did not inhibit the accumulation of glycerol, activation of PM-ATPase or the rapid efflux of protons. These results suggest that the Na+/H+-antiporter may play a significant role in extrusion of the ions that enter cells during the initial stages of salt stress.

Effect of salt stress on plasma membrane permeability and lipid saturation in the salt-tolerant yeast Zygosaccharomyces rouxii

Zygosaccharomyces rouxii mutants defective in NaCl tolerance were isolated. One of the mutants, 77-1S, could not grow in a medium containing 1% (w/v) NaCl at pH 6.0, but showed recovery of 15% NaCl tolerance at pH 4.0 in the same medium. The mutant 77-1S leaked intracellular glycerol through the plasma membrane (PM) under 10% NaCl at pH 6.0, but the glycerol leakage decreased under the 10% NaCl-tolerant condition at pH 4.0. The intracellular content of Na + in 77-1S cells grown in YPD medium containing 0.8% NaCl at pH 6.0 was about ninefold higher than that of the parent M7, whereas the Na + content was similar in the mutant and the parent at pH 4.0. The leakage of glycerol and the influx of Na + through the PM under the NaCl condition were considered to be caused by damage to the PM in the mutant 77-1S. Neither an increase of oleic acid nor a decrease of linoleic acid was observed under the 10% NaCl condition at pH 6.0 in the mutant 77-1S, although such changes were seen in the parent M7. The activity of PM H +-ATPase prepared from 77-1S grown at pH 6.0 was almost the same as that of the parent M7. The mutant 77-1S showed both an increase of oleic acid and a decrease of linoleic acid in the presence of 10% NaCl at pH 4.0 in the conditions under which the strain could grow. Therefore, lipid saturation seems to be important for salt tolerance in Z. rouxii cells under a high-NaCl condition.

Expression of plasma membrane proton-ATPase gene in salt-tolerant yeast Zygosaccharomyces rouxii is induced by sodium chloride

Expression of plasma membrane proton-ATPase gene in salt-tolerant yeast Zygosaccharomyces rouxii is induced by sodium chloride

Expression of plasma membrane proton-ATPase gene in salt-tolerant yeast Zygosaccharomyces rouxii is induced by sodium chloride

The amounts of mRNA and protein of plasma membrane proton-ATPase were measured in the salt-tolerant yeast Zygosaccharomyces rouxii by Northern and Western blot analyses. Although their amounts were independent of growth phase, their synthesis were induced when yeast cells were grown in the presence of NaCl or were subjected to NaCl shock. This finding was consistent with our previous result that plasma membrane proton-ATPase activity was elevated in Z. rouxii cells grown in medium containing high concentrations of NaCl.

Participation of cAMP in the induction of the synthesis of glycerol for the osmoregulatory response in the salt-tolerant yeast Zygosaccharomyces rouxii.

Participation of cAMP in the induction of the synthesis of glycerol for the osmoregulatory response in the salt-tolerant yeast Zygosaccharomyces rouxii.

A transient and rapid activation of plasma-membrane ATPase during the initial stages of osmoregulation in the salt-tolerant yeast Zygosaccharomyces rouxii

A transient and rapid activation of plasma-membrane ATPase during the initial stages of osmoregulation in the salt-tolerant yeast Zygosaccharomyces rouxii