Claudio Martínez

Purification and characterization of a p-coumarate decarboxylase and a vinylphenol reductase from Brettanomyces bruxellensis

The presence of Brettanomyces bruxellensis has been correlated with an increase of phenolic aromas in wine. The production of these aromas results from the metabolization of cinnamic acids, present in the wine, to their ethyl derivatives. Hence, the participation of two enzymes has been proposed: a p-coumarate decarboxylase (CD) and a vinylphenol reductase (VR). Both enzymes were purified and characterized from B. bruxellensis. In denaturing conditions, the CD enzyme had a molecular mass of 21 kDa, while in native conditions its mass was 41 kDa. The optimal activity was obtained at a temperature of 40 degrees C and a pH of 6.0. For p-coumaric acid, the Km value and Vmax were 1.22+/-0.08 mM and 98+/-0.15 micromol/min mg, respectively. The VR enzyme had a molecular mass of 37 kDa in SDS-PAGE, while in natural conditions its mass was 118 kDa. The Km value was > 3.37+/-2.05 mM and its Vmax was 107.62+/-50.38 micromol/min mg for NADPH used as a cofactor. Both enzymatic activities were stable at pH 3.4, but in the presence of ethanol the CD activity decreased drastically while the VR activity was more stable. This is the first report that shows the presence of a CD and a VR enzyme in B. bruxellensis.

Yeasts associated to Malbec grape berries from Mendoza, Argentina.

Aims:  The aims of this work were to evaluate different pre‐isolation treatments applied to complete yeast extraction from grapes and to identify the yeast microflora associated to Malbec grapes from two vineyards located in Mendoza, Argentina.

Genomic organization of the structural genes controlling the astaxanthin biosynthesis pathway of Xanthophyllomyces dendrorhous

The cloning and nucleotide sequence of the genes (idi, crtE, crtYB, crtl and crtS) controlling the astaxanthin biosynthesis pathway of the wild-type ATCC 24230 strain of Xanthophyllomyces dendrorhous in their genomic and cDNA version were obtained. The idi, crtE, crtYB, crtl and crtS genes were cloned, as fragments of 10.9, 11.5, 15.8, 5.9 and 4 kb respectively. The nucleotide sequence data analysis indicates that the idi, crtE, crtYB, crtl and crtS genes have 4, 8,4, 11, and 17 introns and 5, 9, 5, 12 and 18 exons respectively. In addition, a highly efficient site-directed mutagenesis system was developed by transformation by integration, followed by mitotic recombination (the double recombinant method). Heterozygote idi (idi+/idi-::hph), crtE (crtE+/crtE-::hph), crtYB (crtYB+/crtYB-::hph), crtI (crtI+/crtI-::hph) and crtS (crtS+/crtS-::hph) and homozygote mutants crtYB (crtYB-::hph/crtYB-::hph), crtI (crtI-::hph/crtI-::hph) and crtS (crtS-::hph/crtS-::hph) were constructed. All the heterozygote mutants have a pale phenotype and produce less carotenoids than the wild-type strain. The genetic analysis of the crtYB, crtl and crtS loci in the wild-type, heterozygote, and homozygote give evidence of the diploid constitution of ATCC 24230 strains. In addition, the cloning of a truncated form of the crtYB that lacks 153 amino acids of the N-terminal region derived from alternatively spliced mRNA was obtained. Their heterologous expression in Escherichia coli carrying the carotenogenic cluster of Erwinia uredovora result in trans-complementation and give evidence of its functionality in this bacterium, maintaining its phytoene synthase activity but not the lycopene cyclase activity.

The Genetic Basis of Natural Variation in Oenological Traits in Saccharomyces cerevisiae

Saccharomyces cerevisiae is the main microorganism responsible for wine alcoholic fermentation. The oenological phenotypes resulting from fermentation, such as the production of acetic acid, glycerol, and residual sugar concentration are regulated by multiple genes and vary quantitatively between different strain backgrounds. With the aim of identifying the quantitative trait loci (QTLs) that regulate oenological phenotypes, we performed linkage analysis using three crosses between highly diverged S. cerevisiae strains. Segregants from each cross were used as starter cultures for 20-day fermentations, in synthetic wine must, to simulate actual winemaking conditions. Linkage analysis on phenotypes of primary industrial importance resulted in the mapping of 18 QTLs. We tested 18 candidate genes, by reciprocal hemizygosity, for their contribution to the observed phenotypic variation, and validated five genes and the chromosome II right subtelomeric region. We observed that genes involved in mitochondrial metabolism, sugar transport, nitrogen metabolism, and the uncharacterized ORF YJR030W explained most of the phenotypic variation in oenological traits. Furthermore, we experimentally validated an exceptionally strong epistatic interaction resulting in high level of succinic acid between the Sake FLX1 allele and the Wine/European MDH2 allele. Overall, our work demonstrates the complex genetic basis underlying wine traits, including natural allelic variation, antagonistic linked QTLs and complex epistatic interactions between alleles from strains with different evolutionary histories.

Taqman real-time PCR for the detection and enumeration of Saccharomyces cerevisiae in wine

Saccharomyces cerevisiae is the main yeast species responsible for wine fermentation; however, its presence during maturing or barrel-ageing can sometimes result in a reduction in the quality of wine by refermentation. In this work, we developed a quantitative real-time PCR (QPCR) for the rapid detection and quantification of S. cerevisiae in wine. The primers and the hydrolysis probe (TaqMan) were designed from the sequence of a DNA fragment present only in S. cerevisiae and absent in other wine yeasts obtained from an RAPD-PCR analysis. The QPCR developed was highly reproducible, allowing the specific detection and quantification of this yeast in artificially contaminated wines, with a detection limit of 78 CFU/mL. Furthermore, the usefulness of the QPCR developed was evaluated through the quantification of the yeast in wine samples obtained from vineyards, confirming the quantitative capacity of the method. The methodology developed was specific, fast and a sensitive tool for the detection and enumeration of S. cerevisiae cells in wine.

High degree of correlation between molecular polymorphism and geographic origin of wine yeast strains.

Aims: To guarantee the endemic genetic background of the isolates obtained in yeast isolation programs, it is necessary to differentiate between endemic and commercial strains because the progressive use of commercial yeast in wine areas around the world would affect the autochthonous yeast populations.

Study of the coumarate decarboxylase and vinylphenol reductase activities of Dekkera bruxellensis (anamorph Brettanomyces bruxellensis) isolates

Aim:  To evaluate the coumarate descarboxylase (CD) and vinylphenol reductase (VR) activities in Dekkera bruxellensis isolates and study their relationship to the growth rate, protein profile and random amplified polymorphic DNA (RAPD) molecular pattern.

Mapping Genetic Variants Underlying Differences in the Central Nitrogen Metabolism in Fermenter Yeasts

Different populations within a species represent a rich reservoir of allelic variants, corresponding to an evolutionary signature of withstood environmental constraints. Saccharomyces cerevisiae strains are widely utilised in the fermentation of different kinds of alcoholic beverages, such as, wine and sake, each of them derived from must with distinct nutrient composition. Importantly, adequate nitrogen levels in the medium are essential for the fermentation process, however, a comprehensive understanding of the genetic variants determining variation in nitrogen consumption is lacking. Here, we assessed the genetic factors underlying variation in nitrogen consumption in a segregating population derived from a cross between two main fermenter yeasts, a Wine/European and a Sake isolate. By linkage analysis we identified 18 main effect QTLs for ammonium and amino acids sources. Interestingly, majority of QTLs were involved in more than a single trait, grouped based on amino acid structure and indicating high levels of pleiotropy across nitrogen sources, in agreement with the observed patterns of phenotypic co-variation. Accordingly, we performed reciprocal hemizygosity analysis validating an effect for three genes, GLT1, ASI1 and AGP1. Furthermore, we detected a widespread pleiotropic effect on these genes, with AGP1 affecting seven amino acids and nine in the case of GLT1 and ASI1. Based on sequence and comparative analysis, candidate causative mutations within these genes were also predicted. Altogether, the identification of these variants demonstrate how Sake and Wine/European genetic backgrounds differentially consume nitrogen sources, in part explaining independently evolved preferences for nitrogen assimilation and representing a niche of genetic diversity for the implementation of practical approaches towards more efficient strains for nitrogen metabolism.

Self-fertilization is the main sexual reproduction mechanism in native wine yeast populations

Saccharomyces cerevisiae is a model eukaryotic organism for classical genetics and genomics, and yet its ecology is still largely unknown. In this work, a population genetic analysis was performed on five yeast populations isolated from wine-making areas with different enological practices using simple sequence repeats and restriction fragment length polymorphism of mitochondrial DNA as molecular markers on 292 strains. In accordance with other studies, genome size estimation suggests that native S. cerevisiae strains are mainly homothallic and diploids. Analysis of mtDNA data showed that yeast populations from nonindustrial areas have 40% higher genetic diversity than populations isolated from industrial areas, demonstrating that industrial enological practices are likely to affect native yeast populations negatively by reducing its biodiversity. On the other hand, genetic differentiation analysis based on their microsatellite showed no correlation between genetic and geographic distance and a nonsignificant value when a Mantel test was applied. Finally, in the five populations studied, positive inbreeding (F(is)) values from 0.4 to 0.75, a low but significant level of linkage disequilibrium and a high number of multilocus genotypes were detected. These results strongly advocate that sexual reproduction is frequent enough to erase clonal signature in natural populations and that self-fertilization is the main mating system.

Cultivable psychrotolerant yeasts associated with Antarctic marine sponges

Unlike filamentous fungi and bacteria, very little is known about cultivable yeasts associated with marine sponges, especially those from Antarctic seas. During an expedition to King George Island, in the Antarctica, samples of 11 marine sponges were collected by scuba-diving. From these sponges, 20 psychrotolerant yeast isolates were obtained. Phylogenetic analyses of D1/D2 and ITS rRNA gene sequences revealed that the marine ascomycetous yeast Metschnikowia australis is the predominant organism associated with these invertebrates. Other species found belonged to the Basidiomycota phylum: Cystofilobasidium infirmominiatum, Rhodotorula pinicola, Leucosporidiella creatinivora and a new yeast from the Leucosporidiella genus. None of these yeasts have been previously associated with marine sponges. A screening to estimate the ability of these yeasts as producers of extracellular enzymatic activities at several pH and temperature conditions was performed. Several yeast isolates demonstrated amylolytic, proteolytic, lipolytic or cellulolytic activity, but none of them showed xylanolytic activity under the conditions assayed. To our knowledge, this work is the first description of cultivable yeasts associated with marine sponges from the Antarctic sea.