Yeast Biodiversity and Strategies for Industrial Applications
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A brief summary of my research career working with Saccharomyces yeast hybrids and their applications to the industry
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Yeast Biodiversity and Strategies for Industrial Applications
- 1. Yeast Biodiversity and Strategies for Industrial Applications David Peris, Postdoctoral Marie Curie Fellow Biotechnology Department, SBYBI Group 17th November 2017 @djperis
- 2. Yeasts vs Eubacteria Image provided by: ceiba.cc.ntu.edu.twImage provided by: http://faculty.ccbcmd.edu/courses/bio141/lecguide/unit4/fungi/u1fig35.html
- 3. The symbiogenesis (hybrid nature) of the Eukaryote cell Pittis et al 2016 Nature
- 4. Yeasts in the Network of Life (NoL) context www.tolweb.org
- 5. Yeasts in the Network of Life (NoL) context www.tolweb.org
- 6. Neolithic revolution Hornsey 2012 RSCPublishing
- 7. An opened opportunity for fermentation Hornsey 2012 RSCPublishing
- 8. Raw material for alcoholic fermentation Carbon sources Glucose Fructose Glu Glu Maltose Glu Glu Glu Maltotriose Barley/Wheat Apple Grapes Rice
- 9. Stefanini et al 2012 PNAS Raw material is essential for the fermentation conducted by yeasts
- 10. Wine S. cerevisiae close relatives are found in Mediterranean oaks Almeida et al 2015 Mol Ecol
- 11. Yeast infection from Mediterranean oaks (wine case) Stefanini et al 2012 PNAS
- 12. Yeast reproduction and accumulation of new mutations Hornsey 2012 RSCPublishing
- 13. Bottlenecks can fix disgusting yeast products Hornsey 2012 RSCPublishing
- 14. Bottlenecks can fix disgusting yeast products Hornsey 2012 RSCPublishing X
- 15. Bottlenecks can fix non-adapted yeasts Hornsey 2012 RSCPublishing
- 16. Bottlenecks can fix non-adapted yeasts Hornsey 2012 RSCPublishing X
- 17. Bottlenecks can fix the best variants Hornsey 2012 RSCPublishing
- 18. Unconscious domestication of S. cerevisiae strains Carbon sources Glucose Fructose Glu Glu Maltose Glu Glu Glu Maltotriose Barley/Wheat Apple Grapes Rice Gallone et al 2016 Cell Gallone et al 2018 Cur Op Biotechnol
- 19. Unconscious domestication of S. cerevisiae strains? Carbon sources Glucose Fructose Glu Glu Maltose Glu Glu Glu Maltotriose glycolysis Carbon products Ethanol Other compounds fermentation Flavours Glycerol CO2
- 21. The World is changing... Nicholas 2015, Scientific American
- 22. Climatic change is accelerating maturation Low acidity (high pH) Altered phenolic maturation Altered tannin content Higher sugar levels
- 23. New yeasts must be domesticated Low acidity (high pH) Altered phenolic maturation Altered tannin content Higher sugar levels High glycerol production Increase the acidity
- 24. ...the consumer preference is changing as well
- 25. ...an opened window for yeast innovation Low ethanol production Low temperature profile Improve flavours
- 26. Based on the Biological Species Concept S. paradoxus S. mikatae S. arboricola S. kudriavzevii S. uvarum S. cerevisiae S. eubayanus S. jureii
- 27. How do we define a species? Life cycle MAT MATa MAT/MATa Haploid (n) Haploid (n) Diploid (2n) 2 Sexual types (MAT locus)
- 28. In rich conditions, yeast divides by mitosis MAT MATa MAT/MATa Haploid (n) Haploid (n) Diploid (2n) Clonal divisions (mitosis)
- 29. Sexual competent cells can mate MAT MATa MAT/MATa Haploid (n) Haploid (n) Diploid (2n) MAT MATa X MAT/MATa schmoo MATa MATMATa MAT
- 30. Sporulation is promoted under starvation or stressful conditions MAT/MATa Diploid (2n) aa Tetrad Spore Sporulation
- 31. A diploid gets sexual competent by sporulating MAT MAT/MATa Haploid (n) Diploid (2n) MAT MATa X MAT/MATa aa
- 32. Rare mating mechanisms allows diploid to mate MAT MAT/MATa Haploid (n) Diploid (2n) MAT MATa/MATa X MAT/MATa/MATa Homing endonuclease (HO) MATa MAT HO MATa MATa 1)
- 33. Rare mating mechanisms allows diploid to mate MAT MAT/MATa Haploid (n) Diploid (2n) MAT MATa/MATa X Homing endonuclease (HO) MATa MAT HO MATa MATa 1) MAT inactivation MATa MAT 2) MAT/MATa/MATa
- 34. Rare mating mechanisms allows diploids to mate MAT MAT/MATa Haploid (n) Diploid (2n) MAT MATa/MATa X Homing endonuclease (HO) MATa MAT HO MATa MATa 1) MAT inactivation MATa MAT 2) Loss of 1 copy of Chr III MATa3) MAT/MATa/MATa
- 35. Interspecific crosses are possible (no prezygotic barrier) MAT Haploid (n) – Species A MAT X MAT/MATa MATa Haploid (n) – Species B MATa aa
- 36. MAT Haploid (n) – Species A MAT X MAT/MATa MATa Haploid (n) – Species B MATa aa x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x xx x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x: No growth 4/128 = 3.1% If spore viability below 5% suggests different species (postzygotic barrier)
- 38. Huge diversity America C 0.05 n = 980 strains S. cerevisiae S. paradoxus S. mikatae S. kudriavzevii S. arboricola S. uvarum S. eubayanus EU & America A Far East America B Holarctic & Patagonia B
- 39. The closest species are different as human and macaques America C 0.05 n = 980 strains S. cerevisiae S. paradoxus S. mikatae S. kudriavzevii S. arboricola S. uvarum S. eubayanus EU & America A Far East America B Holarctic & Patagonia B Dujon 2006 TIG
- 40. Saccharomyces yeasts are different as human and chicken America C 0.05 n = 980 strains S. cerevisiae S. paradoxus S. mikatae S. kudriavzevii S. arboricola S. uvarum S. eubayanus EU & America A Far East America B Holarctic & Patagonia B Dujon 2006 TIG
- 41. Closely related S. kudriavzevii strains Peris et al 2016 Food Microbiology VRB CECT1939 IFO1815 CA111 CR89 CR90 CR85 CR91 ZP591 IFO1802 CBS7001 0.05 100/1 100/1 100/0.99 100/1 100/1 93/1 S. uvarum S. kudriavzevii S. mikatae S. paradoxus S. cerevisiae
- 42. Closely related strains show different fermentative profiles Peris et al 2016 Food Microbiology PC2(26.4%) PC1 (43.0%) CR90 CR91 CA111 CR89 CR85 IFO 1802 VRB Ethyl acetate VRB CECT1939 IFO1815 CA111 CR89 CR90 CR85 CR91 ZP591 IFO1802 CBS7001 0.05 100/1 100/1 100/0.99 100/1 100/1 93/1 S. uvarum S. kudriavzevii S. mikatae S. paradoxus S. cerevisiae
- 43. We can try to isolate the best performers under industrial conditions n = 141 strains x 28 conditions x 3 replicates Total = 11844 Growth curves
- 44. Ideal growth curve n = 141 strains x 28 conditions x 3 replicates Total = 11844 Growth curves
- 45. Diversity in the consumption rate and growth curves n = 141 strains x 28 conditions x 3 replicates Total = 11844 Growth curves No growth: 0 Late or low growth: 1 Small growth: 2 Double curve: 4 First curve: 3 Early and high growth: 7 Late and high growth: 5 Middle and high growth: 6 Flocculation: 8 Growth Capacity & Growth Curve Type
- 46. Diversity in the consumption rate and growth curves n = 141 strains x 28 conditions x 3 replicates Total = 11844 Growth curves No growth: 0 Late or low growth: 1 Small growth: 2 Double curve: 4 First curve: 3 Early and high growth: 7 Late and high growth: 5 Middle and high growth: 6 Flocculation: 8 Growth Capacity & Growth Curve Type
- 47. Diversity in the consumption rate and growth curves n = 141 strains x 28 conditions x 3 replicates Total = 11844 Growth curves No growth: 0 Late or low growth: 1 Small growth: 2 Double curve: 4 First curve: 3 Early and high growth: 7 Late and high growth: 5 Middle and high growth: 6 Flocculation: 8 Growth Capacity & Growth Curve Type
- 48. Diversity in the consumption rate and growth curves n = 141 strains x 28 conditions x 3 replicates Total = 11844 Growth curves No growth: 0 Late or low growth: 1 Small growth: 2 Double curve: 4 First curve: 3 Early and high growth: 7 Late and high growth: 5 Middle and high growth: 6 Flocculation: 8 Growth Capacity & Growth Curve Type
- 49. Diversity in the consumption rate and growth curves n = 141 strains x 28 conditions x 3 replicates Total = 11844 Growth curves No growth: 0 Late or low growth: 1 Small growth: 2 Double curve: 4 First curve: 3 Early and high growth: 7 Late and high growth: 5 Middle and high growth: 6 Flocculation: 8 Growth Capacity & Growth Curve Type
- 50. Diversity in the consumption rate and growth curves n = 141 strains x 28 conditions x 3 replicates Total = 11844 Growth curves No growth: 0 Late or low growth: 1 Small growth: 2 Double curve: 4 First curve: 3 Early and high growth: 7 Late and high growth: 5 Middle and high growth: 6 Flocculation: 8 Growth Capacity & Growth Curve Type
- 51. Diversity in the consumption rate and growth curves n = 141 strains x 28 conditions x 3 replicates Total = 11844 Growth curves No growth: 0 Late or low growth: 1 Small growth: 2 Double curve: 4 First curve: 3 Early and high growth: 7 Late and high growth: 5 Middle and high growth: 6 Flocculation: 8 Growth Capacity & Growth Curve Type
- 52. Diversity in the consumption rate and growth curves n = 141 strains x 28 conditions x 3 replicates Total = 11844 Growth curves No growth: 0 Late or low growth: 1 Small growth: 2 Double curve: 4 First curve: 3 Early and high growth: 7 Late and high growth: 5 Middle and high growth: 6 Flocculation: 8 Growth Capacity & Growth Curve Type
- 53. Nucleotide diversity is translated in different phenotypic traits ConditionsStrains Growth Capacity & Growth Curve Type
- 54. Some interesting industrial traits ConditionsStrains Growth Capacity & Growth Curve Type Maltose Maltose Glycerol Glycerol 10⁰C 10⁰C 10⁰C Maltotriose Maltotriose Maltotriose
- 55. Non-cerevisiae strains do not tolerate industrial conditions ConditionsStrains Growth Capacity & Growth Curve Type Maltose Maltose Glycerol Glycerol 10⁰C 10⁰C 10⁰C Maltotriose Maltotriose Maltotriose
- 56. Alternatives to non-tolerant species: Hybridization S. paradoxus S. mikatae S. arboricola S. kudriavzevii S. uvarum S. cerevisiae S. eubayanus S. jureii S. cer x S. kud Triple Hybrids S. cer x S. kud x S. uva
- 57. Scer x Skud hybrids are diverse and might be generated multiple times Peris et al 2012 Yeast Peris et al 2012 BMC Genomics Peris et al 2012 PloS One Peris et al 2017 Yeast
- 58. Alternatives to non-tolerant species: Hybridization S. pastorianus S. bayanus 95% Saaz (Group 1) Frohberg (Group 2) S. paradoxus S. mikatae S. arboricola S. kudriavzevii S. uvarum S. cerevisiae S. eubayanus S. jureii
- 59. Wild S. eubayanus strain was found in Patagonia in 2011 Libkind et al 2011
- 60. Now, we have multitude of S. eubayanus strains around the world Peris et al 2014 Mol Ecol Peris et al 2016 PloS Genetics A. saccharumF. grandifoliaNothofagus trees Pinus taedaCedrus spp. Quercus rubra Araucaria araucana Wild Hybrid
- 61. Is the Tibetan S. eubayanus strain the close relative? Saaz Frohberg Peris et al 2016 PloS Genetics
- 62. Can we see different ancestries in genomic regions? Peris et al 2016 PloS Genetics
- 63. Peris et al 2016 PloS Genetics None of the wild S. eubayanus is the close relative of parental donor
- 64. Potentially two different hybridization events Peris et al 2016 PloS Genetics
- 65. What do hybrids have in common? Fermentations at low temperature
- 66. Hybridization combines the best of parentals Saccharomyces uvarum Saccharomyces eubayanus Saccharomyces cerevisiae Saccharomyces kudriavzevii
- 67. What is going on with the mitochondrial genome? MAT MATa X S. cerevisiae S. kudriavzevii
- 68. Uniparental inheritance MAT MATa X S. cerevisiae S. kudriavzevii rhoScer
- 69. Uniparental inheritance MAT MATa X S. cerevisiae S. kudriavzevii rhoScer rhoSkud
- 70. Uniparental inheritance MAT MATa X S. cerevisiae S. kudriavzevii rhoScer rhoScerxSkud rhoSkud
- 71. Network explanation: haplotype CATTATGCCGTAT Hap 1 or 1
- 72. Network explanation: haplotype frequency CATTATGCCGTAT CATTATGCCGTAT Hap 1 or 1 Hap 1 or 1
- 73. Network explanation: haplotypes connected CATTATGCCGTAT CGTTACGCCATAC Hap 1 or 1 Hap 2 or 2
- 74. Network explanation: reticulate events CATTATGCCGTAT CGTTACGCCATAC CGTTACGCCGTAT Hap 1 or 1 Hap 2 or 2 Hap 3 or 3
- 75. Most industrial hybrids (96.4%) have a non-S. cerevisiae mtDNA Total strains =798 Hybrids = 139
- 76. Mitochondrial genome influences in temperature tolerance Scer-Ale ρ+ x Seub ρ⁰ Scer-Ale ρ⁰ x Seub ρ+ Scer-Ale ρ+ Scer-Ale ρ⁰ Seub ρ+ Seub ρ⁰ 10⁰C
- 77. Mitochondrial genome influences in temperature tolerance Scer-Ale ρ+ x Seub ρ⁰ Scer-Ale ρ⁰ x Seub ρ+ Scer-Ale ρ+ Scer-Ale ρ⁰ Seub ρ+ Seub ρ⁰ Scer-Ale ρ+ x Seub ρ⁰ Scer-Ale ρ⁰ x Seub ρ+ Scer-Ale ρ+ Scer-Ale ρ⁰ Seub ρ+ Seub ρ⁰ 10⁰C 37⁰C
- 78. Generating yeast diversity by mitochondrial introgression for wine innovation MITOGRESSION
- 79. Biodiversity is also translated in different aromatic interspecies profile Saccharomyces uvarum Saccharomyces eubayanus Saccharomyces cerevisiae Saccharomyces kudriavzevii
- 80. 80 In diploid hybrids we lose genomic diversity Peris et al 2017 Yeast
- 81. 81Peris et al 2017 Yeast Classical rare-mating and protoplast fusion are tedious
- 82. HyPr promotes gene conversion in the MAT locus NATMX HYGMX NATMX HYGMX HO expression MAT/MATa MATa/MATa MAT/MATa MAT/MAT Homing endonuclease (HO) MATa MAT HO MATa MATa 1) Alexander, Peris et al 2016 Fungal Gen & Biol
- 83. Convert the rare-mating to frequent-mating with HyPr X NATMX HYGMX NATMX HYGMX NATMX HYGMX HO expression MATa/MATa/MAT/MAT MAT/MATa MATa/MATa MAT/MATa MAT/MAT Alexander, Peris et al 2016 Fungal Gen & Biol
- 84. Marker-free hybrids and fully exploiting all the genomic diversity of parental genomes X NATMX HYGMX NATMX HYGMX NATMX HYGMX HO expression Remove selection pressure Alexander, Peris et al 2016 Fungal Gen & Biol MATa/MATa/MAT/MAT MAT/MATa MATa/MATa MAT/MATa MAT/MAT MATa/MATa/MAT/MAT
- 85. S. cer rhoScer S. kud rhoSkud Cybrids (2n) MATa/MATa MAT/MAT HyPr (Hybrid Production) technology to generate cybrids
- 86. HyPr (Hybrid Production) technology to generate cybrids & hybrids S. cer rhoScer S. kud rhoSkud x Hybrids (4n) Scer x Skud ρSkud Scer x Skud ρScer MATa/MATa MAT/MAT S. cer rhoScer S. kud rhoSkud MATa/MATa MAT/MAT Cybrids (2n)
- 87. Combined the best yeasts to generate new wine products Synthetic Must HPLC GC Mass Loss
- 88. Summary There is a huge diversity in yeasts in general, and Saccharomyces in particular waiting to be discovered and exploited
- 89. Summary There is a huge diversity in yeasts in general, and Saccharomyces in particular waiting to be discovered and exploited Hybridization has occurred multiple times indicating an important domestication mechanism for industrial processes
- 90. Summary There is a huge diversity in yeasts in general, and Saccharomyces in particular waiting to be discovered and exploited Hybridization has occurred multiple times indicating an important domestication mechanism for industrial processes Hybridization is a short-term solution to combine different strains with interesting industrial traits
- 91. Summary There is a huge diversity in yeasts in general, and Saccharomyces in particular waiting to be discovered and exploited Hybridization has occurred multiple times indicating an important domestication mechanism for industrial processes Hybridization is a short-term solution to combine different strains with interesting industrial traits Hybridization can offer a solution to generate new strains to solve the industrial challenges derived from climatic change and consumer demands.
- 92. Amparo Querol Laura Pérez David Lázaro Querol Lab Members Eladio Barrio Barrio Lab Members Carmela Belloch José Guillamón Guillamón Lab Members Sergi Puig Sergi Lab Members Diego Libkind Juan Eizaguirre Jose Paulo Sampaio Paula Gonçalves Christian Landry Jean-Baptiste Leducq Guillaume Charron Justin Fay Katie Hyma Li Xueying Fengyan Bai Qi Ming Wang Chris T. Hittinger’s lab Quinn Langdon Emily Baker Ryan Moryarty Kayla Sylvester Christina Kuang Hittinger Lab Members William G Alexander Thank you UW & GLBRC CollaborationSBYBI