Potential application of yeasts from Ecuadorian chichas in controlled beer and chicha production.

The potential of yeasts isolated from traditional chichas as starter cultures, either for controlled production of the native beverage or for industrial beer production, has been investigated. Three S. cerevisiae strains and one T. delbrueckii strain isolated from four different Ecuadorian chichas were compared to ale and lager beer strains with respect to fermentation performance, sugar utilisation, phenolic off-flavour production, flocculation and growth at low temperature. Fermentations were performed in 15 °P all-malt wort and in a model chicha substrate at 12 °C and 20 °C. Tall-tube fermentations (1.5 L) were also performed with both substrates to assess yeast performance and beer quality. Among the strains tested, only one Ecuadorian S. cerevisiae strain was able to ferment the wort sugars maltose and maltotriose. Fermentations with all Ecuadorian strains were poor in wort at 12 °C relative to 20 °C, but were similar in model chicha substrate at both temperatures. The aromatic profile was different between species and strains. These results indicate the potential of yeasts derived from traditional Andean fermented beverages for commercial applications. One of the chicha strains demonstrated traits typical of domesticated brewery strains and could be suitable for ale fermentation, while the other strains may have potential for low-alcohol beer or chicha production.

Biotechnological impact of stress response on wine yeast.

Wine yeast deals with many stress conditions during its biotechnological use. Biomass production and its dehydration produce major oxidative stress, while hyperosmotic shock, ethanol toxicity and starvation are relevant during grape juice fermentation. Most stress response mechanisms described in laboratory strains of Saccharomyces cerevisiae are useful for understanding the molecular machinery devoted to deal with harsh conditions during industrial wine yeast uses. However, the particularities of these strains themselves, and the media and conditions employed, need to be specifically looked at when studying protection mechanisms.

Mitochondria inheritance is a key factor for tolerance to dehydration in wine yeast production.

UNLABELLED: Mitochondria are the cell's powerhouse when organisms are grown in the presence of oxygen. They are also the source of reactive oxygen species that cause damage to the biochemical components of the cell and lead to cellular ageing and death. Under winemaking conditions, Saccharomyces yeasts exclusively have a fermentative metabolism due to the high sugar content of grape must. However, their production as an active dry yeast (ADY) form required aerobic propagation and a dehydration process. In these industrial steps, oxidative stress is particularly harmful for the cell. In this work, we analysed the impact of the mitochondrial genome on oxidative stress response, longevity and dehydration tolerance using the synthetic interspecific hybrids obtained between two S. cerevisiae and S. uvarum strains. The isogenic nature of nuclear DNA of such hybrids allowed us to analyse the impact of mitochondrial DNA for fermentative and oxidative stress conditions. Under grape must conditions, the inheritance of mitochondrial DNA poorly impacted the fermentative performance of interspecific hybrids, unlike the hybrids with S. cerevisiae mitochondrial inheritance, which displayed increased tolerance to oxidative stress and dehydration, and showed an extended chronological longevity when cells were grown with aeration. SIGNIFICANCE AND IMPACT OF THE STUDY: In modern oenology, yeast starters are employed to inoculate grape juice, usually in the form of active dry yeast (ADY). The dehydration process implies stressful conditions that lead to oxidative damage. Other yeast species and interspecific hybrids other than Saccharomyces cerevisiae may be used to confer novel properties to the final product. However, these yeasts are usually more sensitive to drying. Understanding the causes of oxidative stress tolerance is therefore necessary for developing the use of these organisms in industry. This study indicates the impact of mitochondrial DNA inheritance for oxidative stress resistance in an interspecific context using isogenic Saccharomyces cerevisiae × Saccharomyces uvarum hybrids.

Wine yeast strains engineered for glycogen overproduction display enhanced viability under glucose deprivation conditions.

We used metabolic engineering to produce wine yeasts with enhanced resistance to glucose deprivation conditions. Glycogen metabolism was genetically modified to overproduce glycogen by increasing the glycogen synthase activity and eliminating glycogen phosphorylase activity. All of the modified strains had a higher glycogen content at the stationary phase, but accumulation was still regulated during growth. Strains lacking GPH1, which encodes glycogen phosphorylase, are unable to mobilize glycogen. Enhanced viability under glucose deprivation conditions occurs when glycogen accumulates in the strain that overexpresses GSY2, which encodes glycogen synthase and maintains normal glycogen phosphorylase activity. This enhanced viability is observed under laboratory growth conditions and under vinification conditions in synthetic and natural musts. Wines obtained from this modified strain and from the parental wild-type strain don't differ significantly in the analyzed enological parameters. The engineered strain might better resist some stages of nutrient depletion during industrial use.

Performance of industrial strains of Saccharomyces cerevisae during wine fermentation is affected by manipulation strategies based on sporulation.

Genetic manipulation of industrial wine yeast strains has become an essential tool for both the study of the molecular mechanisms underlaying their physiology and the improvement of their fermentative properties. The construction of null mutants for any gene in these usually diploid strains, by using a procedure based on sporulation of a heterozygote lacking one copy of the gene of interest, has been tested as an alternative to the tedious work of sequential disruption of the complete set of copies. Our results indicate that most of the homozygotes resulting from sporulation of wine yeast strains are defective in glucose consumption under microvinification conditions in synthetic must and produce stuck fermentations. These kinds of defects are observed even for strains derived from sporulation of wild type. Alteration of genomic features of wine strains by sporulation is responsible for these defects.

Transcriptional and structural study of a region of two convergent overlapping yeast genes.

The exceptionally close packing of many yeast genes and other chromosomal elements raises the question of how those elements are functionally insulated. All published work shows that natural insulators are very effective, but transcriptional interference (TI) occurs if they are mutated or if their natural context is altered. Mechanisms to avoid TI are poorly understood, but are thought to involve an interplay of cis sequences and trans factors in a chromatin context. We have studied the case of two convergent closely packed ORFs (56 bp of separation) in chromosome IX of Saccharomyces cerevisiae. mRNAs from POT1 and YIL161w overlap by up to 115 nt. Convergent transcription causes a small but noticeable negative effect on the level of POT1 mRNA and nucleosome displacement in the intergenic region. This suggests for the first time that some TI could occur in convergently transcribed yeast genes, even in a natural chromosomal context.

Stochastic nucleosome positioning in a yeast chromatin region is not dependent on histone H1.

To gain a better understanding of the function of the yeast histone H1, its role in nucleosome positioning was studied. With this objective in mind, we analyzed a chromatin region of the yeast Chromosome (Chr) IX, in which there are two closely packed open reading frames (ORFs), POT1 and YIL161w. This locus shows a regular ladder of 13 stochastically positioned nucleosomes, which is unaffected by the absence of the HHO1 gene. This suggests that histone H1 has no effect on nucleosome positioning in yeast.

Chromatin structure of the yeast SUC2 promoter in regulatory mutants.

We have previously suggested that two positioned nucleosomes are removed from the promoter of the Saccharomyces cerevisiae SUC2 gene upon depression by glucose starvation. To gain further insight into the changes accompanying derepression at the chromatin level we have studied the chromatin structure of the SUC2 promoter in several mutants affecting SUC2 expression. The non-derepressible mutants snf1, snf2 and snf5 present a chromatin structure characteristic of the repressed state, irrespective of the presence or absence of glucose. The non-repressible mutants, mig1 and ssn6, as well as the double mutant snfs sn6 exhibit an opened chromatin structure even in the presence of glucose. These results suggest that the DNA-binding protein encoded by MIG1 is necessary to produce the characteristic pattern of repressed chromatin and that the SNF1 protein kinase is sufficient to produce the derepressed chromatin pattern. A model is presented for the transitions that result in opening up of the chromatin structure.

Genome mapping with anchored clones: theoretical aspects.

As part of our effort to construct a physical map of the genome of Arabidopsis thaliana we have made a mathematical analysis of our experimental approach of anchoring yeast artificial chromosome clones with genetically mapped RFLPs and RAPDs. The details of this analysis are presented and their implications for mapping the Arabidopsis genome are discussed.

A new glucose-repressible gene identified from the analysis of chromatin structure in deletion mutants of yeast SUC2 locus.

We have previously shown that some changes occur in the chromatin structure of the 3' flank of the yeast SUC2 gene in going from a repressed to an active state. In an attempt to find out the causes of these changes, we have carried out experiments in which mutant copies of SUC2 locus lacking either 5' or 3' flanks have been analysed for their transcriptional activity and chromatin structure. These experiments allowed us to discard any relationship between SUC2 transcription and chromatin changes within its 3'flank. Sequencing of this flank and mRNA analysis, however, resulted in the location of a putative peroxisomal 3-oxoacyl-CoA thiolase gene (POT1), which is repressible by glucose. The disruption of the gene produced a yeast strain unable to use oleic acid as a carbon source. This is the first time that chromatin structure analysis has permitted the identification of a new gene.

Chromatin structure of transposon Tn903 cloned into a yeast plasmid.

Transposon Tn903 contains the APH gene for kanamycin resistance, which is active in yeast [A. Jiménez and J. Davies (1980) Nature (London) 287, 869-871] and is flanked by two inverted repeats (IR) 1057 bp long. When plasmid pAJ50, carrying Tn903 and the 2-microns circle origin of replication, is cloned into Saccharomyces cerevisiae, nucleosomes are assembled in vivo on the prokaryotic DNA of the transposon. Indirect end labeling revealed that three nucleosomes are preferentially positioned on symmetrical sequences from both IRs. DNase I digestion also confirmed that the chromatin structure is symmetrical in both IRs. This suggests that sequence determinants are decisive for chromatin structure in these regions. We have calculated the rotational and translational fits [H. R. Drew and C. R. Calladine (1987) J. Mol. Biol. 195, 143-173] for the Tn903 sequence and the results indicate that the nucleosome positioning on the IRs is sequence-directed. Nucleosome deposition on the APH gene also occurs, but no clear positioning exists. Some sequence preference for positioning nucleosomes on the promoter can be predicted, especially from the translational fit. Experimental data indicate, however, that nucleosomes are absent from the promoter. Therefore, chromatin can be organized on prokaryotic DNA in a manner that resembles the typical eukaryotic chromatin structure.

In vivo assembly of chromatin on pBR322 sequences cloned into yeast plasmids.

In order to study the in vivo assembly of chromatin on prokaryotic DNA templates, we have transformed yeast cells with plasmids pAJ50 and pRB58, which contain pBR322 sequences. In both cases nucleosomes are assembled in vivo on pBR322 DNA, although the nucleosomes are not homogeneous in size. To explore whether there is any preference for nucleosome assembly along pBR322 sequences, we have used an indirect end labeling method. The results indicate that most nucleosomes are placed at random on pBR322, although the probability for histone octamers to interact with some short regions is somewhat reduced. These regions coincide with sequences in which the frequency distribution of nucleosomes reconstituted in vitro (E. Caffarelli et al. (1988) Eur. J. Biochem. 171, 497-501) is low. Sequence determinants that direct chromatin assembly in vitro seem thereby to act to some extent in vivo.

Fine analysis of the chromatin structure of the yeast SUC2 gene and of its changes upon derepression. Comparison between the chromosomal and plasmid-inserted genes.

Micrococcal nuclease digestion has been used to investigate some fine details of the chromatin structure of the yeast SUC2 gene for invertase. Precisely positioned nucleosomes have been found on a 2 kb sequence from the 3' non-coding region, and four nucleosomes also seem to occupy fixed positions on the 5' flank. Eleven nucleosomes lie on the coding region, although their positioning is not as precise as in the flanks. When the gene is derepressed, these latter nucleosomes adopt a more open conformation and so do two of the nucleosomes positioned on the 5' flank. A dramatic change occurs in the 3' flank, whose involvement in the structural transitions of chromatin upon gene activation is postulated. All the observed features are conserved when the gene is inserted in either a single copy centromeric plasmid or in a multicopy, 2 micron circle-based plasmid.

DNase I sensitivity of the chromatin of the yeast SUC2 gene for invertase.

The DNase I sensitivity of chromatin of the yeast SUC2 gene, which encodes two forms of invertase, has been studied both in the genome and in a multicopy plasmid carrying the gene and its flaking sequences. Whereas little if any difference in the DNase I sensitivity of the flanking regions was found between the repressed and the derepressed states, derepression of the gene was accompanied by a large increase in the sensitivity of the transcribed region. A well-defined DNase I hypersensitive site was found centered at approximately 120 bp downstream from the end of the coding region. This site seems to be flanked in the 3' non-coding region by strictly positioned nucleosomes, and the structure of this region changes upon derepression. In the 5' non-coding region two DNase I hypersensitive sites have been found flanking the TATA box and a set of three closely spaced hypersensitive sites occurs in an upstream regulatory sequence. The structure of these latter sites depends on the on-off state of transcription.

Sliding-end-labelling. A method to avoid artifacts in nucleosome positioning.

A method, termed 'sliding-end-labelling', has been devised to avoid a frequent artifact in nucleosome positioning by indirect end labelling, namely the appearing of DNA fragments originated by two nuclease cuts, one of them lying within the region covered by the probe. The method is applied to the nucleosome positioning in the yeast SUC2 gene for invertase.