1 / 27

Study of the adaptation of

Study of the adaptation of S. cerevisiae strains to winemaking conditions by means of directed evolution and competition experiments with bar-coded YKO strains. Long-term objective.

kipling
Download Presentation

Study of the adaptation of

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Study of the adaptation of S. cerevisiae strains to winemaking conditions by means of directed evolution and competition experiments with bar-coded YKO strains

  2. Long-term objective Comprehensive identification of genes involved in the adaptation of Saccharomyces cerevisiae to the winemaking environment.

  3. Some limitations of transcriptomic approaches • Genes relevant for many biological processes are not subject to transcriptional regulation in response to environmental conditions that influence these processes (Birrell et al. 2002; PNAS). • Not all genes showing a transcriptional change in response to a given culture condition are required for fitness under these conditions (Tai et al., 2007; Microbiology SGM).

  4. Some alternative genome-wide approaches (wine) Proteomics • Complementary information • Usually no direct correlation with transcription data Comparative genomics by hybridization (aCGH or low coverage sequencing) • Strains showing different fermentation phenotypes • Wine vs. non-wine strains Whole genome sequencing • Horizontal transfer • ¿New mobile elements?

  5. HaploInsuficiency Profiling/HOmozygous Profiling HIP/HOP BY4743 BY4742 BY4741 Construction of YKO S. cerevisiae collections X Heterozygous strains x6000 Homozygous strains x4500

  6. HaploInsuficiency Profiling/HOmozygous Profiling HIP/HOP

  7. Some considerations about HIP/HOP analysis of wine fermentation • Environmental conditions experiment dramatic changes • Low number of generations in similar conditions glucose O.D. CO2·t-1 ethanol Alternative approach Continuous culture

  8. Simulation of wine fermentation in continuous culture

  9. Simulation of first step of wine fermentation in continuous culture • 10 generation times for homozygous competition (SM) • 20 generation times for heterozygous competition (SM) • Controls for 10 and 20 generation times in YPD • 3 biological replicates for each of the above

  10. HIP-HOP results for the first step of alcoholic fermentation At least 150 heterozygous deleted strains showed deficient growth in synthetic must after 20 generations (>2-fold reduced fitness as compared to fitness in YPD) At least 126 homozygous deleted strains showed deficient growth in synthetic must after 10 generations (>2-fold reduced fitness as compared to fitness in YPD)

  11. Individual phenotypic characterization of selected strains.

  12. Relevant functions from HIP analysis • Vacuolar functions, including autophagy • Different functions in the “DNA-to-protein” pathway • mRNA processing and stability • Protein synthesis • Secretion (ER functions) Relevant functions from HOP analysis • Adenine and lysine biosynthesis • Inositol biosynthesis • Biosynthesis of phospholipids

  13. Apparent limitations of the HIP/HOP approach • Limited to loss-of-function phenotypes • Difficulty to estimate wine-related phenotypes in a BY4743 background Complementary approaches • Directed evolution of laboratory strains • QTL mapping by high throughput methods

  14. Directed evolution of laboratory strains • Haploid laboratory strain (BY4741) • Continuous culture in conditions emulating the first step of alcoholic fermentation • Working volume 40-50 ml • 150-250 generations (three biological replicates) • Verification of the “evolved” phenotype • Whole genome sequence analysis of the evolved strains

  15. Phenotype of evolved strains. Batch culture Adaptation to first steps does not involve improved overall fermentation performance, rather the opposite

  16. Phenotype of evolved strains. Continuous culture

  17. Summary of mutations already identified • SNPs in non-coding regions • Nonsense mutations • Missense mutations • 50% mutations Mutations requiring further confirmation • Deletions/insertions • Changes in copy-number • Chromosomal rearrangements

  18. RSP5: E3 Ubiquitinligase BY4741 E18 BV19 AV16

  19. Ubiquitin-proteasome pathway mutants

  20. Pilar Morales Maite Novo Manuel Quirós Zoel Salvadó Martijn Wapenaar Ana Mangado www.icvv.es/winehiphop

  21. Coincidences with previous studies 28 overlapping HOP genes 19 overlapping HIP genes 3 overlapping HIP genes

  22. Previous reports of HIP/HOP analysis of wine fermentation Delneri et al. 2008 • Commercial grape must (100 g/L sugar) (among several other media) • Chemostat • Not supplemented with uridine. Aerobic. • Only HIP analysis • No unstressed contrast • Concluded all nutritional requirements were provided by must

  23. Previous reports of HIP/HOP analysis of wine fermentation Piggott et al. 2011 • Synthetic must (200 g/L sugar) • Single biological replicate each (HIP and HOP analyses) • Time-course • YPD amplification of samples • No unstressed contrast • Autophagy and ubiquitin-proteasome functions required • Proficient deleted strains also identified (ribosomal and peroxisomal functions) • FUR4

  24. Some genes to watch From the HIP analysis SAM1 and SAM2; URE2; DUR1,2; MAL12, OCA6; CDC19; genes involved in Gap1p sorting; genes involved in chromatin remodeling and histone modification From the HOP analysis NPR2, NPR3 and RTC1; CAR1 and CAN1; GPD1 y GPD2; UBR1; STB5; BCK1; BUL2; ADH3; AQR1; genes coding for ribosomal proteins; genes involved in protein folding in the ER

  25. HIP

  26. HOP

More Related