Genomic signatures of adaptation to wine biological ageing conditions in biofilm-forming flor yeasts.

The molecular and evolutionary processes underlying fungal domestication remain largely unknown despite the importance of fungi to bioindustry and for comparative adaptation genomics in eukaryotes. Wine fermentation and biological ageing are performed by strains of S. cerevisiae with, respectively, pelagic fermentative growth on glucose and biofilm aerobic growth utilizing ethanol. Here, we use environmental samples of wine and flor yeasts to investigate the genomic basis of yeast adaptation to contrasted anthropogenic environments. Phylogenetic inference and population structure analysis based on single nucleotide polymorphisms revealed a group of flor yeasts separated from wine yeasts. A combination of methods revealed several highly differentiated regions between wine and flor yeasts, and analyses using codon-substitution models for detecting molecular adaptation identified sites under positive selection in the high-affinity transporter gene ZRT1. The cross-population composite likelihood ratio revealed selective sweeps at three regions, including in the hexose transporter gene HXT7, the yapsin gene YPS6 and the membrane protein coding gene MTS27. Our analyses also revealed that the biological ageing environment has led to the accumulation of numerous mutations in proteins from several networks, including Flo11 regulation and divalent metal transport. Together, our findings suggest that the tuning of FLO11 expression and zinc transport networks are a distinctive feature of the genetic changes underlying the domestication of flor yeasts. Our study highlights the multiplicity of genomic changes underlying yeast adaptation to man-made habitats and reveals that flor/wine yeast lineage can serve as a useful model for studying the genomics of adaptive divergence.

Microbial diversity and biochemical characteristics of Borassus akeassii wine.

UNLABELLED: Palm wine produced traditionally and consumed by many people in the South-West of Burkina Faso is subject to alteration. In this study, we carried out a follow-up of two palm wines' fermentation during the 10 days in which palm wines are classically produced and consumed. We monitored biochemical characteristics of fermenting wines as well as followed the microflora kinetics using culture-dependent and culture-independent methods. The analysis of the acid content and the bacterial population revealed the correlation between the development of Lactic acid bacteria, acetic acid, and total acidity. Ribosomal intergenic spacer analysis and sequencing results revealed different yeast and bacterial populations for the two palm wines. Although Saccharomyces cerevisiae remained the sole yeast species in one fermentation, it was quickly replaced by Clavispora lusitaniae in the second fermentation, which had never been described until now in palm wine. When considering bacteria, the species Corynebacterium sp., Lactobacillus casei, Lactobacillus paracasei and Leuconostoc sp. were detected in both palm wines. But we also detected Acetobacter pasteurianus, Bacillus cereus and Bacillus thuringiensis in the second fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results highlight the evolution of palm wine during the 10 days separating palm tapping and consumption of the fermented wine. The fermentation step is performed within few hours and completed after 24 h. The next days, its acidity increases progressively with the production of lactic and acetic acids by bacteria. The high production of acetic acid is very likely one of the main cause of palm wine degradation during this period. This indicates that the solution to palm wine preservation might be protection against oxygen, as well as the limit of bacterial growth through the use of preservatives.

Ecological success of a group of Saccharomyces cerevisiae/Saccharomyces kudriavzevii hybrids in the northern european wine-making environment.

The hybrid nature of lager-brewing yeast strains has been known for 25 years; however, yeast hybrids have only recently been described in cider and wine fermentations. In this study, we characterized the hybrid genomes and the relatedness of the Eg8 industrial yeast strain and of 24 Saccharomyces cerevisiae/Saccharomyces kudriavzevii hybrid yeast strains used for wine making in France (Alsace), Germany, Hungary, and the United States. An array-based comparative genome hybridization (aCGH) profile of the Eg8 genome revealed a typical chimeric profile. Measurement of hybrids DNA content per cell by flow cytometry revealed multiple ploidy levels (2n, 3n, or 4n), and restriction fragment length polymorphism analysis of 22 genes indicated variable amounts of S. kudriavzevii genetic content in three representative strains. We developed microsatellite markers for S. kudriavzevii and used them to analyze the diversity of a population isolated from oaks in Ardèche (France). This analysis revealed new insights into the diversity of this species. We then analyzed the diversity of the wine hybrids for 12 S. cerevisiae and 7 S. kudriavzevii microsatellite loci and found that these strains are the products of multiple hybridization events between several S. cerevisiae wine yeast isolates and various S. kudriavzevii strains. The Eg8 lineage appeared remarkable, since it harbors strains found over a wide geographic area, and the interstrain divergence measured with a (δµ)(2) genetic distance indicates an ancient origin. These findings reflect the specific adaptations made by S. cerevisiae/S. kudriavzevii cryophilic hybrids to winery environments in cool climates.

Activation of two different resistance mechanisms in Saccharomyces cerevisiae upon exposure to octanoic and decanoic acids.

Medium-chain fatty acids (octanoic and decanoic acids) are well known as fermentation inhibitors. During must fermentation, the toxicity of these fatty acids is enhanced by ethanol and low pH, which favors their entrance in the cell, resulting in a decrease of internal pH. We present here the characterization of the mechanisms involved in the establishment of the resistance to these fatty acids. The analysis of the transcriptome response to the exposure to octanoic and decanoic acids revealed that two partially overlapping mechanisms are activated; both responses share many genes with an oxidative stress response, but some key genes were activated differentially. The transcriptome response to octanoic acid stress can be described mainly as a weak acid response, and it involves Pdr12p as the main transporter. The phenotypic analysis of knocked-out strains confirmed the role of the Pdr12p transporter under the control of WAR1 but also revealed the involvement of the Tpo1p major facilitator superfamily proteins (MFS) transporter in octanoic acid expulsion. In contrast, the resistance to decanoic acid is composite. It also involves the transporter Tpo1p and includes the activation of several genes of the beta-oxidation pathway and ethyl ester synthesis. Indeed, the induction of FAA1 and EEB1, coding for a long-chain fatty acyl coenzyme A synthetase and an alcohol acyltransferase, respectively, suggests a detoxification pathway through the production of decanoate ethyl ester. These results are confirmed by the sensitivity of strains bearing deletions for the transcription factors encoded by PDR1, STB5, OAF1, and PIP2 genes.

Genetic analysis of Saccharomyces cerevisiae strains isolated from palm wine in eastern Nigeria. Comparison with other African strains.

AIMS: To study the yeast diversity of Nigerian palm wines by comparison with other African strains. METHODS AND RESULTS: Twenty-three Saccharomyces cerevisiae strains were obtained from palm wine samples collected at four locations in eastern Nigeria, and characterized using different molecular techniques: internal transcribed spacer restriction fragment length polymorphism and sequence analysis, pulsed field gel electrophoresis, inter delta typing and microsatellite multilocus analysis. These techniques revealed that palm wine yeasts represent a group of closely related strains that includes other West African isolates (CBS400, NCYC110, DVPG6044). Population analysis revealed an excess of homozygote strains and an allelic richness similar to wine suggestive of local domestication. Several other African yeast strains were not connected to this group. Ghana sorghum beer strains and other African strains (DBVPG1853 and MUCL28071) displayed strikingly high relatedness with European bread, beer or wine strains, and the genome of strain MUCL30909 contained African and wine-type alleles, indicating its hybrid origin. CONCLUSIONS: Nigerian palm wine yeast represents a local specific yeast flora, whereas a European origin or hybrid was suspected for several other Africa isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents the first genetic characterization of an autochthonous African palm wine yeast population and confirms the idea that human intervention has favoured yeast migration.

Application of Multi Locus Sequence Typing to the analysis of the biodiversity of indigenous Saccharomyces cerevisiae wine yeasts from Lebanon.

AIMS: To assess suitability of Multi Locus Sequence Typing (MLST) for investigating the biodiversity of wine yeast strains. This method was compared with established ones like microsatellite analysis or amplification of genomic regions flanked by repeated (delta) elements. METHODS AND RESULTS: DNA fragments were amplified and sequenced for 26 loci representing housekeeping genes, open reading frames (ORFs) of unknown functions or intergenic regions. A set of seven loci was tested on 84 Saccharomyces cerevisiae strains, including 65 strains isolated from traditional wineries in Lebanon, commercial wine strains and Asian isolates. An overall sequence diversity of 2.05% was observed, consisting of single nucleotide polymorphisms, 60% of them occurring in a heterozygous state. The number of polymorphic sites per locus varied between 4 and 14. The same set of strains was analysed by microsatellite typing on six polymorphic loci and by interdelta amplification. CONCLUSIONS: Clustering of MLST profiles clearly differentiated the Asian group of strains from Lebanese and European commercial strains that appear closely related. The current MLST scheme appears less discriminatory (92.27%) on closely related wine yeasts than microsatellite or interdelta typing (>99%). SIGNIFICANCE AND IMPACT OF THE STUDY: MLST is a highly reliable method for relatedness inference and promising for wine yeast typing.

Predominance of Saccharomyces uvarum during spontaneous alcoholic fermentation, for three consecutive years, in an Alsatian winery.

AIMS: The purpose of this study was to determine the origin of the yeasts involved in the spontaneous alcoholic fermentation of an Alsatian wine. METHODS AND RESULTS: During three successive years, must was collected at different stages of the winemaking process and fermented in the laboratory or in the cellar. Saccharomyces yeasts were sampled at the beginning and at the end of the fermentations. Saccharomyces cerevisiae clones were genetically characterized by inter-delta PCR. Non-S. cerevisiae clones were identified as Saccharomyces uvarum by PCR-RFLP on MET2 gene and characterized at the strain level by karyotyping. The composition of the Saccharomyces population in the vineyard, after crushing and in the vat was analyzed. This led to three main results. First, the vineyard Saccharomyces population was rather homogeneous. Second, new non-resident strains had appeared in the must during the winemaking process. Finally, the yeast population in the vat only consisted in S. uvarum strains. CONCLUSION: This 3-year study has enabled us to show the involvement of indigenous S. uvarum in the alcoholic fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study gives a first insight into the polymorphism of S. uvarum strains involved in a spontaneous alcoholic fermentation.

Natural nitriles and their metabolism.

The present work reviews the numerous nitrile compounds that have been isolated from plants and animals. Two kinds of potentially toxic molecules are widespread, namely the cyanogenic glycosides and cyanollpids. Many other aromatic and allphatic nitriles are synthesized to a lesser extent. Different studies on the synthesis and degradation of these cyanogenic compounds are also reviewed to emphasize the potential use of different microorganisms for the detoxification of food and foodstuff.

Purification and properties of the beta-glucosidase from a nitrile hydratase-producing Brevibacterium sp. strain R312.

Besides its nitrile hydratase and wide spectrum amidase activities, the Brevibacterium sp. R312 strain also possesses a constitutive beta-glucosidase. Its optimum pH is 6. The enzyme was purified by fractionation precipitation with ammonium sulfate followed by chromatographic elutions on Q-Sepharose Fast Flow, Sephadex G-200 and Phenyl Superose. The resulting purification was 1960 folds for a 6% yield. The molecular weight of this enzyme was estimated at 180,000. It contains two identical sub-units. The pI is 5.5. This enzyme has a strong affinity for aryl-beta-glycosides:pNPG, prunassine; it could also degrade linamarine. It is inhibited by p-chloromercuribenzoate, delta-gluconolactone and glucose.