Interplay of Aro80 and GATA activators in regulation of genes for catabolism of aromatic amino acids in Saccharomyces cerevisiae

Aro80, a member of the Zn₂Cys₆ family proteins, activates expression of the ARO9 and ARO10 genes involved in catabolism of aromatic amino acids in response to aromatic amino acids that act as inducers. ARO9 and ARO10 are also under the control of nitrogen catabolite repression, but the direct roles for GATA factors, Gat1 and Gln3, in this regulation have not yet been elucidated. Here, we demonstrate that Aro80 is constitutively bound to its target promoters and activated by inducers at the level of transactivation. Although Aro80 also binds to its own promoter, ARO80 expression is induced only by rapamycin, but not by tryptophan. We show that Aro80 is absolutely required for Gat1 binding to the ARO9, ARO10 and ARO80 promoters upon rapamycin treatment. Gln3 binding to these promoters shows a partial requirement for Aro80. Rapamycin‐dependent Gat1 and Gln3 binding to the Aro80 target promoters is not affected by tryptophan availability, suggesting that transactivation activity of Aro80 is not necessary for the recruitment of GATA factors. Rapamycin‐dependent induction of Aro80 target genes also requires PP2A phosphatase complex, but not Sit4 phosphatase, acting downstream of TORC1.     

Characterisation of Saccharomyces cerevisiae ARO8 and ARO9 genes encoding aromatic aminotransferases I and II reveals a new aminotransferase subfamily

The ARO8 and ARO9 genes of Saccharomyces cerevisiae were isolated by complementation of the phenylalanine/tyrosine auxotrophy of an aro8 aro9 double-mutant strain that is defective in aromatic aminotransferases I (aro8) and II (aro9). The genes were sequenced, and deletion mutants were constructed and analysed. The expression of ARO8 and ARO9 was studied. The deduced amino acid sequences of Aro8p and Aro9p suggest that the former is a 500-residue, 56168-Da polypeptide and the latter a 513-residue, 58516-Da polypeptide. They correspond, respectively, to Ygl202p and Yhr137p, two putative proteins of unknown function revealed by systematic sequencing of the yeast genome. We show that aromatic aminotransferases I and II are homologous proteins, members of aminotransferase subgroup I, and, together with three other proteins, they constitute within the subgroup a new subfamily of enzymes specialised for aromatic amino acid and α-aminoadipate transamination. ARO8 expression is subject to the general control of amino acid biosynthesis. ARO9 expression is induced when aromatic amino acids are present in the growth medium and also in aro8 mutants grown on minimal ammonia medium. An autonomously replicating sequence (ARS) element is located between the ARO8 gene and YGL201c which encodes a protein of the minichromosome maintenance family. Received: 18 June 1997 / Accepted: 23 September 1997     

Enhanced Enzymatic Activity of Glycerol-3-Phosphate Dehydrogenase from the Cryophilic Saccharomyces kudriavzevii

During the evolution of the different species classified within the Saccharomyces genus, each one has adapted to live in different environments. One of the most important parameters that have influenced the evolution of Saccharomyces species is the temperature. Here we have focused on the study of the ability of certain species as Saccharomyces kudriavzevii to grow at low temperatures, in contrast to Saccharomyces cerevisiae. We observed that S. kudriavzevii strains isolated from several regions are able to synthesize higher amounts of glycerol, a molecule that has been shown to accumulate in response to freeze and cold stress. To explain this observation at the molecular level we studied the expression of glycerol biosynthetic pathway genes and we observed a higher expression of GPD1 gene in S. kudriavzevii compared to S. cerevisiae in micro-vinification conditions. We observed higher enzymatic activity of Gpd1p in S. kudriavzevii in response to osmotic and cold stress. Also, we determined that S. kudriavzevii Gpd1p enzyme presents increased catalytic properties that will contribute to increase glycerol production. Finally, we evaluated the glycerol production with S. cerevisiae, S. kudriavzevii or a recombinant Gpd1p variant in the same background and observed that the S. kudriavzevii enzyme produced increased glycerol levels at 12 or 28°C. This suggests that glycerol is increased in S. kudriavzevii mainly due to increased V _max of the Gpd1p enzyme. All these differences indicate that S. kudriavzevii has changed the metabolism to promote the branch of the glycolytic pathway involved in glycerol production to adapt to low temperature environments and maintain the NAD⁺/NADH ratio in alcoholic fermentations. This knowledge is industrially relevant due to the potential use, for example, of S. cerevisiae-S. kudriavzevii hybrids in the wine industry where glycerol content is an important quality parameter.     

Alleviation of feedback inhibition in Saccharomyces cerevisiae aromatic amino acid biosynthesis: quantification of metabolic impact

A quantitative analysis of the impact of feedback inhibition on aromatic amino acid biosynthesis was performed in chemostat cultures of Saccharomyces cerevisiae. Introduction of a tyrosine-insensitive allele of ARO4 (encoding 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase) caused a three-fold increase of intracellular phenylalanine and tyrosine concentrations. These amino acids were not detected extracellularly. However, an over 100-fold increase of the extracellular levels of phenylacetate, phenylethanol and their para-hydroxyl analogues was observed. The total increase of the flux through the aromatic pathway was estimated to be over four-fold. Individual overexpression of either the wild-type or feedback insensitive allele of ARO7 (encoding chorismate mutase had no significant impact. However when they were combined with the Tyr-insensitive ARO4 allele in combination with the Tyr-insensitive ARO4 allele, extracellular concentrations of aromatic compounds were increased by over 200-fold relative to the reference strain, corresponding to a 4.5-fold increase of the flux through the aromatic amino acid biosynthesis pathway. Elimination of allosteric control on these two key reactions in aromatic amino acid metabolism significantly affected intracellular concentrations of several non-aromatic amino acids. This broader impact of amino acid biosynthesis presents a challenge in rational optimization of the production of specific amino acids and derived flavour compounds.     

Characterisation of Saccharomyces cerevisiae ARO8 and ARO9 genes encoding aromatic aminotransferases I and II reveals a new aminotransferase subfamily

The ARO8 and ARO9 genes of Saccharomyces cerevisiae were isolated by complementation of the phenylalanine/tyrosine auxotrophy of an aro8 aro9 double-mutant strain that is defective in aromatic aminotransferases I (aro8) and II (aro9). The genes were sequenced, and deletion mutants were constructed and analysed. The expression of ARO8 and ARO9 was studied. The deduced amino acid sequences of Aro8p and Aro9p suggest that the former is a 500-residue, 56168-Da polypeptide and the latter a 513-residue, 58516-Da polypeptide. They correspond, respectively, to Ygl202p and Yhr137p, two putative proteins of unknown function revealed by systematic sequencing of the yeast genome. We show that aromatic aminotransferases I and II are homologous proteins, members of aminotransferase subgroup I, and, together with three other proteins, they constitute within the subgroup a new subfamily of enzymes specialised for aromatic amino acid and α-aminoadipate transamination. ARO8 expression is subject to the general control of amino acid biosynthesis. ARO9 expression is induced when aromatic amino acids are present in the growth medium and also in aro8 mutants grown on minimal ammonia medium. An autonomously replicating sequence (ARS) element is located between the ARO8 gene and YGL201c which encodes a protein of the minichromosome maintenance family.     

Genetic identification of new biological species <Emphasis Type="Italic">Saccharomyces arboricolus</Emphasis> Wang et Bai

Direct genetic testing for hybrid sterility unambiguously showed that the newly described yeast Saccharomyces arboricolus Wang et Bai is reproductively isolated from Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces cariocanus, Saccharomyces kudriavzevii, Saccharomyces mikatae and Saccharomyces paradoxus and, therefore, represents a new biological species of the genus Saccharomyces. Combined phylogenetic analysis of the rDNA repeat sequences (18S, 26S, ITS), nuclear ACT1 and mitochondrial ATP9 genes revealed that S. arboricolus, along with S. kudriavzevii and S. bayanus, is distantly related to the other four biological species.     

Molecular genetic diversity of the Saccharomyces yeasts in Taiwan: Saccharomyces arboricola, Saccharomyces cerevisiae and Saccharomyces kudriavzevii

Genetic hybridization, sequence and karyotypic analyses of natural Saccharomyces yeasts isolated in different regions of Taiwan revealed three biological species: Saccharomyces arboricola, Saccharomyces cerevisiae and Saccharomyces kudriavzevii. Intraspecies variability of the D1/D2 and ITS1 rDNA sequences was detected among S. cerevisiae and S. kudriavzevii isolates. According to molecular and genetic analyses, the cosmopolitan species S. cerevisiae and S. kudriavzevii contain local divergent populations in Taiwan, Malaysia and Japan. Six of the seven known Saccharomyces species are documented in East Asia: S. arboricola, S. bayanus, S. cerevisiae, S. kudriavzevii, S. mikatae, and S. paradoxus.     

Environmental systems biology of cold‐tolerant phenotype in Saccharomyces species adapted to grow at different temperatures

Temperature is one of the leading factors that drive adaptation of organisms and ecosystems. Remarkably, many closely related species share the same habitat because of their different temporal or micro‐spatial thermal adaptation. In this study, we seek to find the underlying molecular mechanisms of the cold‐tolerant phenotype of closely related yeast species adapted to grow at different temperatures, namely S. kudriavzevii CA111 (cryo‐tolerant) and S. cerevisiae 96.2 (thermo‐tolerant). Using two different systems approaches, i. thermodynamic‐based analysis of a genome‐scale metabolic model of S. cerevisiae and ii. large‐scale competition experiment of the yeast heterozygote mutant collection, genes and pathways important for the growth at low temperature were identified. In particular, defects in lipid metabolism, oxidoreductase and vitamin pathways affected yeast fitness at cold. Combining the data from both studies, a list of candidate genes was generated and mutants for two predicted cold‐favouring genes, GUT2 and ADH3, were created in two natural isolates. Compared with the parental strains, these mutants showed lower fitness at cold temperatures, with S. kudriavzevii displaying the strongest defect. Strikingly, in S. kudriavzevii, these mutations also significantly improve the growth at warm temperatures. In addition, overexpression of ADH3 in S. cerevisiae increased its fitness at cold. These results suggest that temperature‐induced redox imbalances could be compensated by increased glycerol accumulation or production of cytosolic acetaldehyde through the deletion of GUT2 or ADH3, respectively.     

Metabolome segregation of four strains of Saccharomyces cerevisiae,Saccharomyces uvarum and Saccharomyces kudriavzevii conducted under low temperature oenological conditions

The monitoring of fermentation at low temperatures (12–15°C) is a current practice in the winery for retention and enhancement of the flavour volatile content of wines. Among Saccharomyces species, Saccharomyces uvarum and Saccharomyces kudriavzevii have revealed interesting industrial properties, including better adaptation at low temperatures. To gather deeper knowledge of the fermentative metabolism at a low temperature of these species together with S. cerevisiae, we performed a comparative metabolomic analysis using four representative strains. We used batch cultures to obtain an exhaustive and dynamic image of the metabolome of strains passing through the sequential stresses related to the winemaking environment. A great variety of intra- and extracellular metabolites (>500 compounds) were quantified across fermentation using distinct chromatographic methods. Besides a global decrease in the lipid composition of the four strains when they entered into the stationary phase, we reported some strain-specific high magnitude changes. Examples of these differences included divergent patterns of production of short-chain fatty acids and erythritol in the S. uvarum strain. Strains also differed in expression for aromatic amino acid biosynthesis and sulphur metabolism, including the glutathione pathway. These data will allow us to refine and obtain the most value of fermentations with this alternative Saccharomyces species.     

Stuck at work? Quantitative proteomics of environmental wine yeast strains reveals the natural mechanism of overcoming stuck fermentation

During fermentation oenological yeast cells are subjected to a number of different stress conditions and must respond rapidly to the continuously changing environment of this harsh ecological niche. In this study we gained more insights into the cell adaptation mechanisms by linking proteome monitoring with knowledge on physiological behaviour of different strains during fermentation under model winemaking conditions. We used 2D‐DIGE technology to monitor the proteome evolution of two newly discovered environmental yeast strains Saccharomyces bayanus and triple hybrid Saccharomyces cerevisiae × Saccharomyces kudriavzevii × S. bayanus and compared them to data obtained for the commercially available S. cerevisiae strain. All strains examined showed (i) different fermentative behaviour, (ii) stress resistance as well as (iii) susceptibility to stuck fermentation which was reflected in significant differences in protein expression levels. During our research we identified differentially expressed proteins in 155 gel spots which correspond to 70 different protein functions. Differences of expression between strains were observed mainly among proteins involved in stress response, proteins degradation pathways, cell redox homeostasis and amino acids biosynthesis. Interestingly, the newly discovered triple hybrid S. cerevisiae × S. kudriavzevii × S. bayanus strain which has the ability to naturally restart stuck fermentation showed a very strong induction of expression of two proteolytic enzymes: Pep4 and Prc1 that appear as numerous isoforms on the gel image and which may be the key to its unique properties. This study is an important step towards the better understanding of wine fermentations at a molecular level.     

Differentiation of six sibling species in the Saccharomyces sensu stricto complex by multilocus enzyme electrophoresis and UP-PCR analysis

UP-PCR analysis and multilocus enzyme electrophoresis were used to characterize 37 strains of the sibling species Saccharomyces cerevisiae, S. bayanus, S. cariocanus, S. kudriavzevii, S. mikatae and S. paradoxus. The results demonstrate that both molecular approaches are useful for discriminating between these phenotypically indistinguishable Saccharomyces species. The data obtained are in excellent agreement with previously reported genetic analyses, sequencing of the 18S rRNA and ITS regions, and DNA-DNA reassociation data. This revised version was published online in June 2006 with corrections to the Cover Date.     

Natural populations of Saccharomyces kudriavzevii in Portugal are associated with oak bark and are sympatric with S. cerevisiae and S. paradoxus

Here we report the isolation of four Saccharomyces species (former Saccharomyces sensu stricto group) from tree bark. The employment of two temperatures (10 degrees C in addition to the more commonly used 30 degrees C) resulted in the isolation of S. kudriavzevii and S. uvarum, two species that grow at low temperatures, in addition to S. cerevisiae and S. paradoxus. A clear bias was found toward the bark of certain trees, particularly certain oak species. Very often, more than one Saccharomyces species was found in one locality and occasionally even in the same bark sample. Our evidence strongly suggests that (markedly) different growth temperature preferences play a fundamental role in the sympatric associations of Saccharomyces species uncovered in this survey. S. kudriavzevii was isolated at most of the sites sampled in Portugal, indicating that the geographic distribution of this species is wider than the distribution assumed thus far. However, the Portuguese S. kudriavzevii population exhibited important genetic differences compared to the type strain of the species that represents a Japanese population. In this study, S. kudriavzevii stands out as the species that copes better with low temperatures.     

Substrate Specificity of Thiamine Pyrophosphate-Dependent 2-Oxo-Acid Decarboxylases in Saccharomyces cerevisiae

Fusel alcohols are precursors and contributors to flavor and aroma compounds in fermented beverages, and some are under investigation as biofuels. The decarboxylation of 2-oxo acids is a key step in the Ehrlich pathway for fusel alcohol production. In Saccharomyces cerevisiae, five genes share sequence similarity with genes encoding thiamine pyrophosphate-dependent 2-oxo-acid decarboxylases (2ODCs). PDC1, PDC5, and PDC6 encode differentially regulated pyruvate decarboxylase isoenzymes; ARO10 encodes a 2-oxo-acid decarboxylase with broad substrate specificity, and THI3 has not yet been shown to encode an active decarboxylase. Despite the importance of fusel alcohol production in S. cerevisiae, the substrate specificities of these five 2ODCs have not been systematically compared. When the five 2ODCs were individually overexpressed in a pdc1Δ pdc5Δ pdc6Δ aro10Δ thi3Δ strain, only Pdc1, Pdc5, and Pdc6 catalyzed the decarboxylation of the linear-chain 2-oxo acids pyruvate, 2-oxo-butanoate, and 2-oxo-pentanoate in cell extracts. The presence of a Pdc isoenzyme was also required for the production of n-propanol and n-butanol in cultures grown on threonine and norvaline, respectively, as nitrogen sources. These results demonstrate the importance of pyruvate decarboxylases in the natural production of n-propanol and n-butanol by S. cerevisiae. No decarboxylation activity was found for Thi3 with any of the substrates tested. Only Aro10 and Pdc5 catalyzed the decarboxylation of the aromatic substrate phenylpyruvate, with Aro10 showing superior kinetic properties. Aro10, Pdc1, Pdc5, and Pdc6 exhibited activity with all branched-chain and sulfur-containing 2-oxo acids tested but with markedly different decarboxylation kinetics. The high affinity of Aro10 identified it as a key contributor to the production of branched-chain and sulfur-containing fusel alcohols.