Better yeast for better wine Saccharomyces cerevisiae strains for winemaking

Transcrição

1 Better yeast for better wine Saccharomyces cerevisiae strains for winemaking Dorit-Elisabeth Schuller Centro de Biologia Molecular e Ambiental, Universidade do Minho, Braga

2 Identification of polymorphisms among individuals within a species To study the genetic basis of phenotypic differences elucidate To evaluate the evolutionary history of the species 30,097 SNPs per strain Liti et al., Nature, 009 Schacherer et al., Nature, 009

3 Fermentation technologies are deeply rooted in the history of most societies Saccharomyces cerevisiae strains from winemaking environments o o o o o o o o high ethanol production and tolerance growth at high sugar concentrations high fermentative activity (complete sugar consumption) growth at low temperatures glycerol production low sulphur dioxide production hydrogen sulphide production production of flavouring compounds Today: Winemakers use commercial winemaking strains

4 Fermentation technologies are deeply rooted in the history of most societies Adapted from Swiegers et al, Australian Journal of Grape and Wine Research, 005

5 Main topics o Biogeography and populational analysis of indigenous S. cerevisiae strains from winemaking environments o Constitution of a S. cerevisiae bio-databank comprising ca. 700 strains from distinct winemaking regions o Selection of novel S. cerevisiae winemaking strains o Selection of novel S. cerevisiae winemaking strains - Linking genetic profiles and phenotypic information o Genomic approaches to unravel the genetic characteristics of S. cerevisiae winemaking strains

6 Biogeography and populational analysis of indigenous S. cerevisiae strains from winemaking environments 3 - sampling sites Arinto Aragonês Avesso Alvarinho Baga Castelão Terrantez M. Gomes 6 5 Verdelho Loureiro 3 Touriga Nacional Bical

7 - Summary of S. cerevisiae strains from Portuguese winemaking regions- Nºof grape samples Spontaneous fermentations Nº of isolates Nº of different Saccharomyces cerevisiae strains Vinhos Verdes Bairrada (*) Estremadura Alentejo (*) Palmela Ribatejo 7 0 (*) Douro 6 80 (*) Dão (*) Açores (*) Total (*) underway

8 - molecular typing methods - Mitochondrial DNA restriction analysis (mtdna RFLP) Interdelta sequence amplification Preliminary screening In depth characterization Microsatelite analysis AAT AAT5 AAT6 AAT3 AAT AAT Microsatellite Chrom. Position/ORF Repeat Ref. ScAAT XIII ATT ScAAT II YBL08c ATT ScAAT 3 IV YDR60w ATT ScAAT VII ATT ScAAT 5 XVI TAA ScAAT 6 IX TAA YPL009 XV YOR56c TAA ScYOR67C XV YOR67c TGT C XV TAA+TAG C5 VI GT Pérez and Gallego, 00 Legras et al., 005

9 Allele frequency Allele frequency - Populational analysis of S. cerevisiae populations from distinct winemaking regions and grape varieties - Vinho Verde 5 3,5 3,0,5,0,5,0 Touriga Nacional - vineyard ( strains) Loureiro - vineyard (6 strains) Arinto - vineyard 5 (7 strains) Avesso - vineyard 7 (3 strains) Alvarinho - vineryard (0 strains) 7 0,5 0, Microsatellite AAT AAT AAT3 Allele (Nº of repeats) AAT AAT5 AAT6 C C5 YPL009 YOR67C Bairrada 3,0,5,0,5 Touriga Nacional - vineyard 3 (8 strains) Maria Gomes - vineyard (0 strains) Baga - vineyard 5 ( strains) Aragonez - vineyard 5 (3 strains) 3 5,0 0,5 0, Microsatellite Allele (Nº of repeats) AAT AAT AAT3 AAT AAT5 AAT6 C C5 YPL009 YOR67C

10 Bairrada Populational analysis of S. cerevisiae populations from distinct winemaking regions and grape varieties Vinho Verde 0.5 Coefficient Year R = 0, Coefficient _Al_Pro_3 7 7_Ar_Lou_7 3 7_TN_Mea_ 7_Ba_QU_8 7_Ba_Ca_9 7_MG_Ant_0 7_TN_QU_9 7_Ar_EV_ 7_TN_EV_ 3 7_Al_So_6 8 7_Av_Co_3 9 7_Av_Ca_5 7_Lou_EV_8 7_Lou_Am_ Coefficient Alvarinho Loureiro Avesso Arinto Touriga Nacional Coefficient Year R = 0,78 Cluster analysis (UPGMA) based on a Euclidean distance dissimilarity matrix of allelic frequencies. Genetic proximity is correlated with geographic distance between wine regions Within wine regions, S. cerevisiae populations are associated with the grape variety _Al_Pro_7 6_TN_Mea_ 6_Ara_Ou_3 6_Ba_QU_ 6_Ba_Ca_8 6_Al_So_5 6_Ar_Lou_0 6_Lou_Am_3 6_TN_So_5 6_Av_Co_3 Bical Baga Maria Gomes Aragonês Schuller et al., 005 Schuller et al., 007

11 Constitution of a S. cerevisiae bio-databank comprising ca. 600 strains from distinct winemaking regions Queries Isolate Grape varieties Vineyard Wine Region Sampling year Allelic combinations for each strain Schuller et al., 005 Schuller et al., 007 Valero et al., 007 Schuller and Casal, 007

12 Linking genetic profiles and phenotypic information by computational approaches Phenotypic tests Glucose Ribose Arabinose Sucrose Galactose Raffinose Maltose Glycerol Potassium acetate Peptone Ammonium sulphate Imidazole Urea Ethanol tolerance Temperature Osmotic stress H S production Cerulenin resistance Strains TFL resistance Cells in the heatmap represent values of O.D.

13 Phenotypic tests A 60 =0. A 60 =0. A 60 =. A 60 <.0 A 60.0 A A 60 = 0. A 60 = 0. A 60 =. A 60 = 0.8 A A A 60 =.0 A 60 =.3 A 60 =. A 60 =. A classes, and 3 (sub-groups) ºC ºC 8 o C 30 o C 30 o C 5 o C 5 o C YNB + galactose YNB + galactose YNB + maltose YNB + maltose YNB + raffinose YNB + raffinose YNB + ammonium sulphate YNB + urea YNB + urea ethanol 6% H S production Modelling the relation between individual phenotypes and genotype information Modelling technique linear linear knn tree knn linear linear linear linear linear tree knn linear linear linear linear linear tree AUC Percentage (%) ,3 00. AUC = area under receiver operation score Franco-Duarte et al., 009 COMPUTATIONAL APPROACHES FOR THE GENETIC AND PHENOTYPIC CHARACTERIZATION OF A SACCHAROMYCES CEREVISIAE WINE YEAST COLLECTION

14 Comparative genomics of yeast strains isolated from diverse ecological niches unveils important genome diversity - unravel intraspecific natural genome diversity - S. cerevisiae winemaking strains S. cerevisiae clinical strains Nacional Facility for DNA Microarrays Variability is associated with the sub-telomeric regions of some chromosomes Carreto et al., 008 Wine strains : reduced number of Ty elements and the flanking genes

15 Differential expression of co-regulated genes distinguishes Saccharomyces cerevisiae strains during fermentation Winemaking strains Nacional Facility for DNA Microarrays Clinical strain S88c Carreto et al., in prep.

16 Differential expression of co-regulated genes distinguishes Saccharomyces cerevisiae strains during fermentation Nacional Facility for DNA Microarrays M M M M T T Carreto et al., in prep. M Amino acid metabolism T Amino acid transport

17 Ongoing projects INNOYEAST - Innovation and improvement of European wine industry competitiveness by the research and development of native microencapsulated wine yeasts to produce quality wines

18 PhenoMet - Integrative pheno-metabolomic and genomic approaches for Saccharomyces cerevisiae winemaking yeasts PTDC/AGR-ALI/0339/008 Project diagram CBMA Centro de Biologia Molecular e Ambiental CBQF Centro de Biotecnologia e Química Fina Strain collection Phenotypic screen Genetic characterization Saccharomyces cerevisiae biodiversity database 3 Fermentations Functional genomics Data analysis IBB Institute for Biotechnology and Bioengineering INETI Instituto Nacional de Engenharia Tecnologia e Inovação CESAM Centre for Environmental and Marine Studies Fiber optics spectroscopy Hyperspectral microscopy, Image analysis 5 Data quality / signal processing Phenotypes Microarrays Image analysis Fiber optics spectroscopy HPLC/GC-MS data warehousing relational databases 6 Fermentations GC-MS LC-MS HPLC Metabolic network reconstruction Network topologies and complexity Critical pathway analysis Network hub compounds In-Silico S. cerevisiae experiments

19 Acknowledgements Ana Magalhães Eugénia Vieira João Drumonde Nuno Fonseca Raquel Pereira Ricardo Duarte Sylvie Dequin INRA Montpellier, France Jean-Luc Legras INRA Colmar, France Universidade de Aveiro Manuel Santos, Laura Carreto BIOCANT Catarina Gomes Susana Sousa EVN Filomena Duarte Rui Cunha Anselmo Mendes Euclides Rodrigues José Domingues João Melícias Frederico Gomes Leonor Novais Magda Silva Graça Sofia Machado Barbara Dellinger Adega Cooperativa de Cantanhede Comissão de Viticultura da Região dos Vinhos Verdes Companhia das Quintas Estação Vitivinícola Amândio Galhano PROVAM Produtores de Vinhos Alvarinho de Monção Quinta de Ameal Quinta da Cancela Quinta de Covela Quinta de Lourosa Quinta da Pedra Quinta da Soalheira Sociedade Agrícola Gabriel Francisco Dias & Irmãs Solar de Bouças

20 Phenotypic characterization of a S. cerevisiae strain collection Current research and future perspectives Neural networks (SOM) Enzymatic activities (API) Selection of a genetically most diverse set of strains Fermentative and aromatic profiling Genetic profiles Molecular markers that correlate with specific phenotypes / traits

21 dco /dt (g/lh) Abordagens a desenvolver. Genética Comportamento fermentativo AAT AAT6 AAT5 AAT AAT3,75,50 Perfil fermentativo,5,00 38 Integração de dados,75genéticos e fisiológicos 39,50 30,5,00 0,75 Bioinformática 0,50 0,5 Métodos computacionais 0,00 horas hours QA3 VL 7B Previsão de características desejáveis a partir de dados genéticos Analítica

22 Conclusions The finding of 50 S. cerevisiae strains reveals a fascinating genetic diversity in vineyard environments. Microsatellite analysis permitted a high resolution populational screen, showing that genetic differences among S. cerevisiae populations derived from both diagnostic vineyard-, specific alleles and the accumulation of small allele-frequency differences across ten microsatellite loci; each vineyard contains differentiated S. cerevisiae populations, hypothesizing the occurrence of specific native strains that can be associated with a terroir.

23 Acknowledgements Magda Silva Graça Hugo Alves Sofia Machado Barbara Dellinger Adega Cooperativa de Cantanhede Comissão de Viticultura da Região dos Vinhos Verdes Companhia das Quintas Estação Vitivinícola Amândio Galhano PROVAM Produtores de Vinhos Alvarinho de Monção Quinta de Ameal Quinta da Cancela Quinta de Covela Quinta de Lourosa Quinta da Pedra Quinta da Soalheira Sociedade Agrícola Gabriel Francisco Dias & Irmãs Solar de Bouças Jean-Luc Legras INRA Colmar, France Rui Cunha Anselmo Mendes Euclides Rodrigues José Domingues João Melícias Frederico Gomes Leonor Novais

24 Santa Maria São Miguel Terceira 3 Baía do Tagarete Baía de S. Lourenço S. Vicente 0 Biscoitos Sul Baía da Maia 5 P. Delgada 6 Caloura 7 8 Vila Ponta Franca Garça 9 Faial da Terra 3 Pesqueiro Porto Martins Salga Graciosa Pico 5 Vitória 6 Santa Cruz da Graciosa 8 Lagoa Outeiro Lagidos 9 Canada das Almas Included in appellations of origin Not included in appellations of or Candelária 3 7 Luz S. Mateus 5 Terra do Pão 0 Manhenha Faial S. Jorge Flores Norte Pequeno Capelinhos 6 Varadouro Praia do Norte 35 Urzelina Fajã do Ouvidor 3 3 Fajã da Ribeira da Areia 33 Fajã dos Vimes Fajã de S. João 3 30 Ponta do Topo 36 Santa Cruz das Flores

25 bioytec Molecular Biotechnology Fermentation technology: Ongoing projects: o Integrative Pheno-Metabolomic and genomic approaches for Saccharomyces cerevisiae winemaking yeasts o The indigenous microbiome of Portuguese wine fermentations o Implementation of a National Facility for DNA Microarrays

26 T. Nacional Avesso Arinto Alvarinho Loureiro Vinhos Verdes Spontaneous Fermentations No fermentation ongoing Same vineyard S. cerevisiae strains involved in spontaneous fermentations

27 Spontaneous Fermentations S. cerevisiae strains involved in spontaneous fermentations Baga Bical M. Gomes Aragonês T. Nacional Bairrada No fermentation ongoinfg Same vineyard Non-Saccharomyces

28 Douro Estremadura Estremadura Alentejo Alentejo Alentejo Alentejo Estremadura Estremadura Estremadura Estremadura Palmela Ribatejo Alentejo Estremadura Alentejo Alentejo Dão Douro Estremadura Estremadura Spontaneous Fermentations S. cerevisiae strains involved in spontaneous fermentations Aragonês Arinto Castelão Trincadeira T. Nacional Ribatejo No fermentation ongoinfg Same vineyard Non-Saccharomyces

29 Graciosa Pico S. Jorge Terceira Graciosa Pico S. Jorge Terceira Pico S. Jorge Terceira Faial Flores Graciosa Pico S. Jorge S. Miguel S. Maria Terceira Spontaneous Fermentations Fermentações Espontâneas S. cerevisiae strains involved in spontaneous fermentations Açores Arinto Verdelho Terrantez Híbridos Produtores directos No fermentation ongoinfg Same vineyard Non-Saccharomyces

30 Traditional 3 grape varieties Hybrid varieties from non-abandoned vineyards Hybrid varieties from abandoned vineyards Number 6 os samples Number of fermentations Day of the beggining of the fermentation (average),0,,3 Duration of the fermentations (average) Finished fermentations (%) 33,3 7, 83,3 Fermentations completed by S. cerevisiae (%) 75,0 56,3 56,0 Fermentations 5,0 3,7,0 completed by non-saccharomyces species (%) Total number f S. cerevisiae strains Minimum number of S. cerevisiae strains / sample Maximum number of S. cerevisiae strains / sample 3

31 The BIONUMERICS software for databasing and cluster analysis Entries from multiple fingerprint and character types Approaches with single/multiple (polyphasic) fingerprint and character types Binary/numerical value experiment file(s) Porto, 5/6 Junho 007

32 - distribution of the most frequent microsatellite alleles - Locus Nº of alleles Allele (nº of repeats) Most frequent alleles AAT5 AAT3 AAT AAT6 AAT ScAAT ScAAT ScAAT ScAAT ScAAT ScAAT C C YPL ScYOR Total: 9 alleles

33 bioytec Molecular Biotechnology Fermentation technology: wine Main topics o Biogeography and populational analysis of indigenous S. cerevisiae strains from winemaking environments o Constitution of a S. cerevisiae bio-databank comprising ca. 700 strains from distinct winemaking regions o Selection of novel S. cerevisiae winemaking strains o Linking genetic profiles and phenotypic information by computational approaches o Genomic approaches to unravel the genetic characteristics that are responsible for distinct fermentative behavior (S. cerevisiae winemaking strains versus other S. cerevisiae strains)