A new understanding: Gene expression, cell characteristic and antioxidant enzymes of Zygosaccharomyces rouxii under the D-fructose regulation.

Zygosaccharomyces rouxii is a well-known salt-tolerant yeast. In our previous study, it was interesting that Z. rouxii could produce higher levels of 4-hydroxy-2, 5-dimethyl-3(2 H)-furanone in 120 g/L D-fructose and 180 g/L NaCl involved YPD medium at 5 d. In order to explore the resistance and furanone production mechanisms of Z. rouxii under D-fructose regulation, a comparative transcriptomics method in Z. rouxii was to set to find differentially expressed genes, the physiological and biochemical indexes (growth and cell morphology, lipid peroxidation and relative electrical conductivity, the antioxidant enzymes activity), and the expression of oxidoreductase activity genes. The results indicated that a larger number of different expressed genes at transcriptome analysis, such as the series antioxidant enzymes were related to the resistance characteristics. Research had confirmed that the living cell numbers and cell areas of D-fructose regulation group were significantly lower than the controls at the initial stage, while those higher than of the controls at the late stage. During the fermentation period, the lipid peroxidation and the relative electrical conductivity of the yeast cell membrane were increased. And also the D-fructose regulation group present lower inhibition superoxide anion ability. The activity of CAT in the D-fructose regulation group was always higher than that of the control group. Only the activity of GSH-Px was found to be significantly increased at 1 d except for other enzymes activities. Most of the oxidoreductase activity genes, such as especially the GSH-Px gene under D-fructose regulation conditions were expressed at higher levels than those of control groups. Combining the levels of transcription and enzymes activity data, those could understand that exogenous D-fructose had a stress effect on Z. rouxii at the early stage of culture. With the fermentation time progress, it was no longer a stressor substance for the Z. rouxii, and changed the nutrient to promote growth of Z. rouxii in the later stages. During the whole process, GSH-Px was the main defense enzyme and CAT was the sustained defense enzyme. Therefore, the experimental results might provide effective mechanisms in Z. rouxii for practical application of furanone production in the industry under exogenous D-fructose regulation.

Simultaneously saccharification and fermentation approach as a tool for enhanced fossil fuels biodesulfurization.

Biodesulfurization can be a complementary technology to the hydrodesulfurization, the commonly physical-chemical process used for sulfur removal from crude oil. The desulfurizing bacterium Gordonia alkanivorans strain 1B as a fructophilic microorganism requires fructose as C-source. In this context, the main goal of this work was the optimization of a simultaneous saccharification and fermentation (SSF) approach using the Zygosaccharomyces bailii strain Talf1 crude enzymes with invertase activity and sucrose as a cheaper fructose-rich commercial C-source (50% fructose) towards dibenzothiophene (DBT) desulfurization by strain 1B. The determination of optimal conditions, for both sucrose hydrolysis and DBT desulfurization was carried out through two sequential experimental uniform designs according to the Doehlert distribution for two factors: pH (5.5-7.5) and temperature (28-38 °C), with the enzyme load of 1.16 U/g/L; and enzyme load (0-4 U/g/L) and temperature (28-38 °C), with pH at 7.5. Based on 2-hydroxybiphenyl production, the analysis of the response surfaces obtained pointed out for pH 7.5, 32 °C and 1.8 U/g/L as optimal conditions. Further optimized SSF of sucrose during the DBT desulfurization process permitted to attain a 4-fold enhanced biodesulfurization. This study opens a new focus of research through the exploitation of sustainable low cost sucrose-rich feedstocks towards a more economical viable bioprocess scale-up.

Influence of culture conditions on production of phytase by Zygosaccharomyces baili var. balil.

Microbial phytases are phosphohydrolytic enzymes which are gaining attention for their commercial exploitation in feed and food industry. In the present study, ten yeasts were isolated from different soil samples and screened for their phytase producing capability. Among these isolates, the most promising yeast strain was Zygosaccharomyces bailii var. bailii which produced highest phytase yield (6.36 U ml(-1)) in malt yeast extract glucose peptone (MYGP) medium. In order to improve phytase production by Zygosaccharomyces bailii, different physio-chemical parameters were optimized. The optimal conditions for phytase production was found to be: incubation time-42 hr, temperature-30 degrees C, medium pH-6.0 and substrate (calcium phytate) concentration-0.1%. Glucose at 0.5% concentration supported higher phytase production (13.75 U ml(-1)) than other carbon sources tested. Metal ions (Ca+/+, Na+, K+, Mg++) and additives; ethylene diamine tetraacetate (EDTA), sodium dodecyl sulphate (SDS) and toluene did not affect enzyme production. However, Zn++, Ni++, Ba++, Pb++ and detergents like Triton X-100 and Tweens strongly inhibited (>90%) phytase production. An overall 2.21-fold enhancement in phytase activity (6.36-->14.03 U ml(-1)) was attained after optimization studies.

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.

Production and characterization of a novel yeast extracellular invertase activity towards improved dibenzothiophene biodesulfurization.

The main goal of this work was the production and characterization of a novel invertase activity from Zygosaccharomyces bailii strain Talf1 for further application to biodesulfurization (BDS) in order to expand the exploitable alternative carbon sources to renewable sucrose-rich feedstock. The maximum invertase activity (163 U ml(-1)) was achieved after 7 days of Z. bailii strain Talf1 cultivation at pH 5.5-6.0, 25 °C, and 150 rpm in Yeast Malt Broth with 25 % Jerusalem artichoke pulp as inducer substrate. The optimum pH and temperature for the crude enzyme activity were 5.5 and 50 °C, respectively, and moreover, high stability was observed at 30 °C for pH 5.5-6.5. The application of Talf1 crude invertase extract (1 %) to a BDS process by Gordonia alkanivorans strain 1B at 30 °C and pH 7.5 was carried out through a simultaneous saccharification and fermentation (SSF) approach in which 10 g l(-1) sucrose and 250 µM dibenzothiophene were used as sole carbon and sulfur sources, respectively. Growth and desulfurization profiles were evaluated and compared with those of BDS without invertase addition. Despite its lower stability at pH 7.5 (loss of activity within 24 h), Talf1 invertase was able to catalyze the full hydrolysis of 10 g l(-1) sucrose in culture medium into invert sugar, contributing to a faster uptake of the monosaccharides by strain 1B during BDS. In SSF approach, the desulfurizing bacterium increased its µmax from 0.035 to 0.070 h(-1) and attained a 2-hydroxybiphenyl productivity of 5.80 µM/h in about 3 days instead of 7 days, corresponding to an improvement of 2.6-fold in relation to the productivity obtained in BDS process without invertase addition.

Enhancement of dibenzothiophene desulfurization by Gordonia alkanivorans strain 1B using sugar beet molasses as alternative carbon source.

There are several problems limiting an industrial application of fossil fuel biodesulfurization, and one of them is the cost of culture media used to grow the microorganisms involved in the process. In this context, the utilization of alternative carbon sources resulting from agro-industrial by-products could be a strategy to reduce the investment in the operating expenses of a future industrial application. Recently, Gordonia alkanivorans 1B was described as a fructophilic desulfurizing bacterium, and this characteristic opens a new interest in alternative carbon sources rich in fructose. Thus, the goal of this study was to evaluate the utilization of sugar beet molasses (SBM) in the dibenzothiophene (DBT) desulfurization process using strain 1B. SBM firstly treated with 0.25% BaCl2 (w/v) was used after sucrose acidic hydrolysis or in a simultaneous saccharification and fermentation process with a Zygosaccharomyces bailii Talf1 invertase (1%), showing promising results. In optimal conditions, strain 1B presented a µ max of 0.0795 h(-1), and all DBT was converted to 2-hydroxybiphenyl (250 µM) within 48 h with a maximum production rate of 7.78 µM h(-1). Our results showed the high potential of SBM to be used in a future industrial fossil fuel biodesulfurization process using strain 1B.

Screening of novel yeast inulinases and further application to bioprocesses.

Inulin is a carbohydrate composed of linear chains of ß-2,1-linked D-fructofuranose molecules terminated by a glucose residue through a sucrose-type linkage at the reducing end. Jerusalem artichoke (JA) is one of the most interesting materials among unconventional and renewable raw materials, with levels of inulin reaching 50-80% of dry matter. Inulin or inulin-rich materials can be actively hydrolyzed by microbial inulinases to produce glucose and fructose syrups that can be used in bioprocesses. In this study, several microbial strains were isolated and their ability to inulinase biosynthesis was evaluated. The novel yeast strain Talf1, identified as Zygosaccharomyces bailii, was the best inulinase producer, attaining 8.67 U/ml of inulinase activity when JA juice was used as the inducer substrate. Z. bailii strain Talf1 and/or its enzymatic crude extract were further applied for bioethanol production and biodesulfurization (BDS) processes, using inulin and JA juice as carbon source. In a consolidated bioprocessing for ethanol production from 200 g/l inulin, Z. bailii strain Talf1 was able to produce 67 g/l of ethanol. This ethanol yield was improved in a simultaneous saccharification and fermentation (SSF) process, with the ethanologenic yeast Saccharomyces cerevisiae CCMI 885 and the Talf1 inulinases, achieving a production of 78 g/l ethanol. However, the highest ethanol yield (∼48%) was obtained in a SSF process from JA juice (∼130 g/l fermentable sugars), where the S. cerevisiae produced 63 g/l ethanol. Relatively to the dibenzothiophene BDS tests, the Gordonia alkanivorans strain 1B achieved a desulfurization rate of 4.8 µM/h within a SSF process using Talf1 inulinases and JA juice, highlighting the potential of JA as a less expensive alternative carbon source. These results showed the high potential of Z. bailii strain Talf1 inulinases as a versatile tool for bioprocesses using inulin-rich materials.

Production of glutaminase(E.C. 3.2.1.5) from Zygosaccharomyces rouxii in solid-state fermentation and modeling the growth of Z. rouxii therein.

Glutaminase production in Zygosacchromyces rouxii by solid-state fermentation (SSF) is detailed. Substrates screening showed best results with oatmeal (OM) and wheatbran (WB). Further, a 1:1 combination of OM: WB gave 0.614units/gds with artificial sea water (ASW) as moistening agent. Evaluation of additional carbon, nitrogen, aminoacids and minerals supplementation was done. A central composite design was employed to investigate effects of four variables, viz. moisture content, glucose, corn steep liquor and glutamine on production. A 4-fold increase in enzyme production was obtained. Studies were undertaken to analyze the time course model the microbial growth and nutrient utilization during SSF. Logistic equation (R2=0.8973), describing the growth model of Z.rouxii was obtained, with maximum values of micronm and Xm at 0.326h-1 and 7.35% of dry matter weight loss, respectively. A good-fit model to describe utilization of total carbohydrate (R2=0.9906) nitrogen concentration (R2=0.9869) with time was obtained. The model was used successfully to predict enzyme production (R2=0.7950).

Cloning of the Zygosaccharomyces bailii GAS1 homologue and effect of cell wall engineering on protein secretory phenotype.

BACKGROUND: Zygosaccharomyces bailii is a diploid budding yeast still poorly characterized, but widely recognised as tolerant to several stresses, most of which related to industrial processes of production. Because of that, it would be very interesting to develop its ability as a cell factory. Gas1p is a beta-1,3-glucanosyltransglycosylase which plays an important role in cell wall construction and in determining its permeability. Cell wall defective mutants of Saccharomyces cerevisiae and Pichia pastoris, deleted in the GAS1 gene, were reported as super-secretive. The aim of this study was the cloning and deletion of the GAS1 homologue of Z. bailii and the evaluation of its deletion on recombinant protein secretion. RESULTS: The GAS1 homologue of Z. bailii was cloned by PCR, and when expressed in a S. cerevisiae GAS1 null mutant was able to restore the parental phenotype. The respective Z. bailii Deltagas1 deleted strain was obtained by targeted deletion of both alleles of the ZbGAS1 gene with deletion cassettes having flanking regions of approximately 400 bp. The morphological and physiological characterization of the Z. bailii null mutant resulted very similar to that of the corresponding S. cerevisiae mutant. As for S. cerevisiae, in the Z. bailii Deltagas1 the total amount of protein released in the medium was significantly higher. Moreover, three different heterologous proteins were expressed and secreted in said mutant. The amount of enzymatic activity found in the medium was almost doubled in the case of the Candida rugosa lipase CRL1 and of the Yarrowia lipolytica protease XPR2, while for human IL-1beta secretion disruption had no relevant effect. CONCLUSIONS: The data presented confirm that the engineering of the cell wall is an effective way to improve protein secretion in yeast. They also confirmed that Z. bailii is an interesting candidate, despite the knowledge of its genome and the tools for its manipulation still need to be improved. However, as already widely reported in literature, our data confirmed that an "always working" solution to the problems related to recombinant protein production can be hardly, if never, found; instead, manipulations have to be finely tuned for each specific product and/or combination of host cell and product.

Production of glutaminase (E.C.3.2.1.5) from Zygosaccharomyces rouxii: statistical optimization using response surface methodology.

A face centered central composite design was employed to investigate the interactive effects of four variables, viz. concentrations of sucrose, yeast extract, sodium chloride, and glutamine, identified earlier by one-factor-at-a-time approach, on glutaminase production by Zygosaccharomyces rouxii. A significant influence of yeast extract on glutaminase production was noted. Response surface methodology (RSM) showed that a medium containing (g/l) sucrose, 17.8; yeast extract, 48.0; glutamine, 5.0 and sodium chloride, 55.6 to be optimum for the production of glutaminase. This medium was projected to produce, theoretically, an enzyme activity of 149.98 U/l and a specific activity of 0.488 U/mg protein. The applied methodology was validated using this optimized media and enzyme activity 155.89+/-1.68 U/l and specific activity of 0.468+/-0.088 U/mg protein was obtained. Further, this optimization strategy combined with an increase in inoculum enhanced the enzyme activity and specific activity by 2.94 and 3.58 fold, respectively, as compared to the unoptimized media.

Production of Yarrowia lipolytica Nha2 Na+/H+ antiporter improves the salt tolerance of Saccharomyces cerevisiae.

Yarrowia lipolytica plasma-membrane Na+/H+ antiporter, encoded by the YlNHA2 gene, is a very efficient exporter of surplus sodium from the cytosol. Its heterologous expression in Saccharomyces cerevisiae wild-type laboratory strains increased their sodium tolerance more efficiently than the expression of ZrSod2-22 antiporter from the osmotolerant yeast Zygosaccharomvces rouxii.

Isoenzyme patterns: a valuable molecular tool for the differentiation of Zygosaccharomyces species and detection of misidentified isolates.

Electrophoretic analysis of esterase, acid phosphatase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase and alcohol dehydrogenase isoenzymes was performed in 39 strains classified into six species of the yeast genus Zygosaccharomyces. The electrophoretic profiles obtained allowed the clear separation of Z. bailii, Z. bisporus, Z. florentinus, Z. lentus, Z. mellis and Z. rouxii, strains of the latter species clustering into two subgroups. Furthermore, this methodology enabled the detection of misidentified strains, as subsequently confirmed by DNA-DNA reassociation and sequencing of the D1/D2 domain of the 26S rRNA gene. Cluster analysis of the global electrophoretic data and those obtained using only two of the isoenzyme systems, esterase and lactate dehydrogenase, yielded similar grouping of the strains examined, indicating that these enzymes are good markers for the differentiation of Zygosaccharomyces species.

Yeast orthologues associated with glycerol transport and metabolism.

Glycerol is a key compound in the regulation of several metabolic pathways in Saccharomyces cerevisiae. From this yeast most of the genes involved in glycerol consumption, production and transport are now available. Some of the mechanisms involving glycerol metabolism and transport are common to other yeasts. This work presents a search for GPD1/2, GUT1, GUP1/2 and FPS1 orthologues in a series of hemiascomycetous yeasts. All the genes cloned were able to complement S. cerevisiae mutant phenotypes and presented a high degree of similarity to the corresponding genes in this yeast. A phylogenetic analysis is presented. The allocation of GUP genes in the membrane bound O-acyl transferases (MBOAT) family is suggested as more consistent than their inclusion in the TC-DB/glycerol uptake family.

Evolution of divergent DNA recognition specificities in VDE homing endonucleases from two yeast species.

Homing endonuclease genes (HEGs) are mobile DNA elements that are thought to confer no benefit to their host. They encode site-specific DNA endonucleases that perpetuate the element within a species population by homing and disseminate it between species by horizontal transfer. Several yeast species contain the VMA1 HEG that encodes the intein-associated VMA1-derived endonuclease (VDE). The evolutionary state of VDEs from 12 species was assessed by assaying their endonuclease activities. Only two enzymes are active, PI-ZbaI from Zygosaccharomyces bailii and PI-ScaI from Saccharomyces cariocanus. PI-ZbaI cleaves the Z.bailii recognition sequence significantly faster than the Saccharomyces cerevisiae site, which differs at six nucleotide positions. A mutational analysis indicates that PI-ZbaI cleaves the S.cerevisiae substrate poorly due to the absence of a contact that is analogous to one made in PI-SceI between Gln-55 and nucleotides +9/+10. PI-ZbaI cleaves the Z.bailii substrate primarily due to a single base-pair substitution (A/T+5 --> T/A+5). Structural modeling of the PI-ZbaI/DNA complex suggests that Arg-331, which is absent in PI-SceI, contacts T/A+5, and the reduced activity observed in a PI-ZbaI R331A mutant provides evidence for this interaction. These data illustrate that homing endonucleases evolve altered specificity as they adapt to recognize alternative target sites.

Isolation of an acetyl-CoA synthetase gene (ZbACS2) from Zygosaccharomyces bailii.

A gene homologous to Saccharomyces cerevisiae ACS genes, coding for acetyl-CoA synthetase, has been cloned from the yeast Zygosaccharomyces bailii ISA 1307, by using reverse genetic approaches. A probe obtained by PCR amplification from Z. bailii DNA, using primers derived from two conserved regions of yeast ACS proteins, RIGAIHSVVF (ScAcs1p; 210-219) and RVDDVVNVSG (ScAcs1p; 574-583), was used for screening a Z. bailii genomic library. Nine clones with partially overlapping inserts were isolated. The sequenced DNA fragment contains a complete ORF of 2027 bp (ZbACS2) and the deduced polypeptide shares significant homologies with the products of ACS2 genes from S. cerevisiae and Kluyveromyces lactis (81% and 82% identity and 84% and 89% similarity, respectively). Phylogenetic analysis shows that the sequence of Zbacs2 is more closely related to the sequences from Acs2 than to those from Acs1 proteins. Moreover, this analysis revealed that the gene duplication producing Acs1 and Acs2 proteins has occurred in the common ancestor of S. cerevisiae, K. lactis, Candida albicans, C. glabrata and Debaryomyces hansenii lineages. Additionally, the cloned gene allowed growth of S. cerevisiae Scacs2 null mutant, in medium containing glucose as the only carbon and energy source, indicating that it encodes a functional acetyl-CoA synthetase. Also, S. cerevisiae cells expressing ZbACS2 have a shorter lag time, in medium containing glucose (2%, w/v) plus acetic acid (0.1-0.35%, v/v). No differences in cell response to acetic acid stress were detected both by specific growth and death rates. The mode of regulation of ZbACS2 appears to be different from ScACS2 and KlACS2, being subject to repression by a glucose pulse in acetic acid-grown cells.

Heterologous expression of Zygosaccharomyces rouxii glycerol 3-phosphate dehydrogenase gene (ZrGPD1) and glycerol dehydrogenase gene (ZrGCY1) in Saccharomyces cerevisiae.

We examined the effects of heterologous expression of the open reading frames (ORF) of two genes on salt tolerance and glycerol production in a Saccharomyces cerevisiae strain deficient in glycerol synthesis (gpd1Deltagpd2Delta). When the ORF of the Zygosaccharomyces rouxii glycerol 3-phosphate dehydrogenase gene (ZrGPD1) was expressed under the control of the GAL10 promoter, salt tolerance and glycerol production increased; when the ORF of the glycerol dehydrogenase gene (ZrGCY1) was expressed under the control of the GAL1 promoter, no such changes were observed. Zrgcy1p had a weak effect on glycerol production. These results suggest that Zrgpd1p is the primary enzyme involved in Z. rouxii glycerol production, following a mechanism similar to that of S. cerevisiae (Gpd1p). When the ORFs of the S. cerevisiae glycerol 3-phosphatase gene (GPP2) and ZrGPD1 were simultaneously expressed, glycerol production increased, compared with that in yeast expressing only ZrGPD1.

Aerobic sugar metabolism in the spoilage yeast Zygosaccharomyces bailii.

Despite the importance of some Zygosaccharomyces species as agents causing spoilage of food, the carbon and energy metabolism of most of them is yet largely unknown. This is the case with Zygosaccharomyces bailii. In this study the occurrence of the Crabtree effect in the petite-negative yeast Z. bailii ATCC 36947 was investigated. In this yeast the aerobic ethanol production is strictly dependent on the carbon source utilised. In glucose-limited continuous cultures a very low level of ethanol was produced. In fructose-limited continuous cultures ethanol was produced at a higher level and its production increased with the dilution rate. As a consequence, on fructose the onset of respiro-fermentative metabolism caused a reduction in biomass yield. An immediate aerobic alcoholic fermentation in Z. bailii was observed during the transition from sugar limitation to sugar excess, both on glucose and on fructose. The analysis of some key enzymes of the fermentative metabolism showed a high level of acetyl-CoA synthetase in Z. bailii growing on fructose. At high dilution rates, the activities of glucose- and fructose-phosphorylating enzymes, as well as of pyruvate decarboxylase and alcohol dehydrogenase, were higher in cells during growth on fructose than on glucose.

Visualization of somatic deletions mediated by R/RS site-specific recombination and induction of germinal deletions caused by callus differentiation and regeneration in rice.

A transgenic rice plant expressing the recombinase of Zygosaccharomyces rouxii under the control of the CaMV 35S promoter was crossed with a transgenic plant carrying a cryptic (beta-glucuronidase) GUS reporter gene, which was activated by recombinase-mediated deletions between two specific recombination sites ( RSs). In F(1) plants, GUS activity was observed as blue spots and stripes in vascular bundles in several parts of the leaves. GUS expression was detected in all of the calli induced from F(1) seeds and throughout the regenerated plants. DNA analysis using the polymerase chain reaction and Southern blotting showed that R/ RS-mediated deletions occurred in all of the cells of the regenerated plants. Stable GUS expression was confirmed in the progeny resulting from self-pollination. Thus, the deletions obtained in the regenerated plants were genetically equivalent to the germinal deletions. These results indicate that the induction of callus differentiation and shoot regeneration is an effective manner to activate the R/ RS system and to produce plants with chromosomal deletions.

Cloning and characterization of genes encoding trehalose-6-phosphate synthase (TPS1) and trehalose-6-phosphate phosphatase (TPS2) from Zygosaccharomyces rouxii.

In many organisms, trehalose protects against several environmental stresses, such as heat, desiccation, and salt, probably by stabilizing protein structures and lipid membranes. Trehalose synthesis in yeast is mediated by a complex of trehalose-6-phosphate synthase (TPS1) and trehalose-6-phosphate phosphatase (TPS2). In this study, genes encoding TPS1 and TPS2 were isolated from Zygosaccharomyces rouxii (designated ZrTPS1 and ZrTPS2, respectively). They were functionally identified by their complementation of the tps1 and tps2 yeast deletion mutants, which are unable to grow on glucose medium and with heat, respectively. Full-length ZrTPS1 cDNA is composed of 1476 nucleotides encoding a protein of 492 amino acids with a molecular mass of 56 kDa. ZrTPS2 cDNA consists of 2843 nucleotides with an open reading frame of 2700 bp, which encodes a polypeptide of 900 amino acids with a molecular mass of 104 kDa. The amino acid sequence encoded by ZrTPS1 has relatively high homology with TPS1 of Saccharomyces cerevisiae and Schizosaccharomyces pombe, compared with TPS2. Western blot analysis showed that the antibody against S. cerevisiae TPS1 recognizes ZrTPS1. Under normal growth conditions, ZrTPS1 and ZrTPS2 were highly and constitutively expressed, unlike S. cerevisiae TPS1 and TPS2. Salt stress and heat stress reduced the expression of the ZrTPS1 and ZrTPS2 genes, respectively.

Molecular cloning and sequence analysis of the Zygosaccharomyces bailii HIS3 gene encoding the imidazole glycerolphosphate dehydratase.

Zygosaccharomyces bailii is a spoilage yeast belonging to the Zygosaccharomyces genus. In recent years these yeasts, due to their exceptional resistance to several stresses, have become more and more interesting as model organisms to study the molecular basis of the said resistance. A Z. bailii cDNA library has been built and the 672 bp nucleotide sequence coding for the HIS3 gene was cloned by complementation of a Saccharomyces cerevisiae his3 mutant strain. The deduced 223 amino acid sequence shares a high degree of homology with His3p homologues in other non-conventional yeast species. The GeneBank Accession No. is AY050224.