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ISSY33 - 2017 06 26

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Mining diversity in the Saccharomyces genus and biotechnological applications by making higher order hybrids

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ISSY33 - 2017 06 26

  1. 1. Mining Saccharomyces diversity and experimental evolution for cellulosic biofuel and beer applications David Peris, Postdoctoral Research Associate Department of Genetics, University of Wisconsin-Madison 27th June 2017 @djperis
  2. 2. Domesticated Saccharomyces cerevisiae for alcoholic beverage production Carbon sources Glucose Fructose Glu Glu Maltose Glu Glu Glu Maltotriose glycolysis Carbon products Ethanol Other compounds fermentation
  3. 3. Most S. cerevisiae can not consume xylose Proteins, Oils, Ash (0-2%) Hemicellulose (19-34%) Lignin (21-32%) Cellulose (33-51%) Glucose Xylose Sugars (C6/C5) Piotrowski et al 2014 Front Microbiol
  4. 4. Toxins inhibits the ethanol production Proteins, Oils, Ash (0-2%) Hemicellulose (19-34%) Lignin (21-32%) Cellulose (33-51%) Glucose Xylose HMF Ferulic acid p-coumaric acid Feruloyl amide Sodium acetate Acetamide Sugars (C6/C5) Hydrolysate Toxins Piotrowski et al 2014 Front Microbiol Ethanol
  5. 5. Engineering chassis S. cerevisiae for xylose consumption The most tolerant of a panel of S. cerevisiae Engineered with xylose utilization genes Wohlbach et al. 2009 PNAS Sato et al. 2013 AEM Y732n CHASSIS Xylose
  6. 6. S. cerevisiae is still not happy with the toxins Wohlbach et al. 2009 PNAS Sato et al. 2013 AEM Y732n CHASSIS Xylose Hydrolysate toxins The most tolerant of a panel of S. cerevisiae Engineered with xylose utilization genes
  7. 7. The diversity in S. cerevisiae is low Liti et al 2009 Nature S. cerevisiae 0.8% nucleotide diversity
  8. 8. America C Surveying the diversity of Saccharomyces genus Phylogenomic tree Strains from 23 available lineages 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 Peris et al 2017 BFB
  9. 9. America C The Saccharomyces diversity is huge 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 Phylogenomic tree Strains from 23 available lineages Dujon 2006 TIG
  10. 10. America C S. mikatae & S. paradoxus are more tolerant to ACSH conditions 0.05 S. cerevisiae S. paradoxus S. mikatae S. kudriavzevii S. arboricola S. uvarum S. eubayanus EU & America A Far East America B Peris et al 2017 BFB
  11. 11. S. uvarum can consume xylose 0.05 S. cerevisiae S. paradoxus S. mikatae S. kudriavzevii S. arboricola S. uvarum S. eubayanus Peris et al 2017 BFB
  12. 12. Improvement of S. cerevisiae chassis strain The most tolerant of a panel of S. cerevisiae Engineered with xylose utilization genes Engineered with Hydrolysate tolerance traits Y732n CHASSIS v2.0 Xylose Hydrolysate toxins
  13. 13. Industrial Saccharomyces hybrids Gonzalez et al. 2008 Dunn et al. 2008 Peris et al. 2012a,b,c,2014,2016,2017b Libkind et al. 2011 Almeida et al 2014 S. pastorianus S. paradoxus S. mikatae S. arboricola S. kudriavzevii S. uvarum S. cerevisiae S. eubayanus S. bayanus
  14. 14. Generation of hybrids to combine genetic traits and generate diversity S.mikatae XHaploid (n) S. cerevisiae CHASSIS Peris et al 2017 BFB Haploid (n) MATa MAT MATa/MAT
  15. 15. 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 2 3 4 5 0 1 2 3 4 5 RC/MRC I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI sppIDer (species IDentifier) 1:1 chromosome of each parent Chromosome Peris et al 2017 BFB Quinn et al In preparation Chromosome Sequencing coverage S. cerevisiae S. mikatae
  16. 16. Adaptive evolution of the unstable hybrid: new mutations Peris et al 2017 BFB R1 30ºC 14days
  17. 17. Bottlenecks and new passages in fresh media R1 … 30ºC 14days Peris et al 2017 BFB
  18. 18. The best variants will survive R1 R9… 50 Generation 30ºC 14days ACSH Peris et al 2017 BFB
  19. 19. One of the evolved hybrids retain the genetic traits of the parents Peris et al 2017 BFB The evolved hybrids consume similar levels of xylose The growth rate of the evolved hybrid is similar to the parents More generation might generate a better strain than the parents.
  20. 20. One of the evolved hybrids retain the genetic traits of the parents Peris et al 2017 BFB The evolved hybrids consume similar levels of xylose The growth rate of the evolved hybrid is similar to the parents More generation might generate a better strain than the parents. Chromosome Chromosome Sequencing coverage S. cerevisiae S. mikatae
  21. 21. HyPr: Hybrid Production a/ a/ NATMX HYGMX Alexander et al 2016 FGB
  22. 22. HyPr promotes gene conversion in the MAT locus a/ a/ a/a / NATMX HYGMX NATMX HYGMX HO expression Alexander et al 2016 FGB
  23. 23. Convert the rare-mating to frequent-mating with HyPr a/ a/ a/a / X NATMX HYGMX NATMX HYGMX NATMX HYGMX HO expression a/a// Alexander et al 2016 FGB
  24. 24. Marker-free hybrids a/ a/ a/a / X NATMX HYGMX NATMX HYGMX NATMX HYGMX HO expression a/a// a/a// Remove selection pressure Alexander et al 2016 FGB
  25. 25. Ryan Moriarty X X X S. paradoxus S. mikatae S. arboricola S. kudriavzevii S. uvarum S. cerevisiae Proof of concept: higher order hybrids using HyPr?
  26. 26. Double hybrids were done! X X X S. paradoxus S. mikatae S. arboricola S. kudriavzevii S. uvarum S. cerevisiae
  27. 27. A four species hybrid was done! X X X X S. paradoxus S. mikatae S. arboricola S. kudriavzevii S. uvarum S. cerevisiae
  28. 28. sppIDer for the four species hybrid Spar Sarb Suva Scer
  29. 29. mitoSppIDer for the four species hybrid RF2 RPM1COBCOX1 COX1 ATP8FSparIII21S ATP6 COB COBCOX1 COX1 VAR1 15S COX2 COB COBCOX1 ATP9 COX321S ATP6VAR1 COBCOBCOX1COX1 15SRPM1 COX2 21SCOBCOBCOX1COX1ATP8 ATP9 FSuvaIIICOBCOX1COX1COX1 COX3 VAR1 RPM1 COBCOX1 COX2ATP8 21SCOBCOX1 15S ATP9 ATP6 FSuvaIIICOBCOX1 COX3 15SCOX1COX1 COBATP6 RPM1COX1COX1 COBVAR1 COX2 21SCOX1COX1 COB COX3ATP9 COX1 COX1COB ATP8VAR1 RF3ATP9 Spar_EUSuva_HolEUSuva_SAASeub_HolSeub_PB 0 2 4 6 0 2 4 6 0 2 4 6 0 2 4 6 4 6 log2(AverageDepth) Species Spar_ Suva_ Suva_ Seub_ Seub_ yHRVM495_mitoPop Avg depth of coverage RF2 RPM1COBCOX1 COX1 ATP8FSparIII21S ATP6 COB COBCOX1 COX1 VAR1 15S COX2 COB COBCOX1 ATP9 COX321S ATP6VAR1 COBCOBCOX1COX1 15SRPM1 COX2 21SCOBCOBCOX1COX1ATP8 ATP9 FSuvaIIICOBCOX1COX1COX1 COX3 VAR1 RPM1 COBCOX1 COX2ATP8 21SCOBCOX1 15S ATP9 ATP6 FSuvaIIICOBCOX1 COX3 15SCOX1COX1 COBATP6 RPM1COX1COX1 COBVAR1 COX2 21SCOX1COX1 COB COX3ATP9 COX1 COX1COBCOX221S COX1 COX1COX3 ATP6RPM115S COX1 COX1COB ATP8VAR1 RF3ATP9 0 20000 40000 60000 0 2 4 6 0 2 4 6 0 2 4 6 0 2 4 6 0 2 4 6 Genome Position log2(AverageDepth) yHRVM495_mitoPop Avg depth of coverage COBCOB 15S1 COX2 21SCOBCOB FSuvaIIICOB COX3 B COX2 21S 15S FSuvaIIICOX3 15SCOB RPM1 COB COX2 21SCOB COX3 COX1 COX1COBCOX2 COX1 COX1COX3 ATP6RPM1 COX1 COX1COB ATP8RF3 Suva_HolEUSuva_SAASeub_HolSeub_PB 40000 60000 Genome Position Species Spar_EU Suva_HolEU Suva_SAA Seub_Hol Seub_PB S. paradoxus S. uvarum
  30. 30. We have evidence for a six species hybrid! X X X X X S. paradoxus S. mikatae S. arboricola S. kudriavzevii S. uvarum S. cerevisiae
  31. 31. Restriction fragment Length Polymorphism give some clues M349-Sc M456–Sc M495–ScxSpxSaxSu M359-Sa M356-Su M461–Sax Su BRE5 S.cerevisiae S.paradoxus S.mikatae S.kudriavzevii S.uvarum S.arboricola Hybrid(6x->12n) X X
  32. 32. Complex hybrids generate diversity for industrial applications X X X X X S. paradoxus S. mikatae S. arboricola S. kudriavzevii S. uvarum S. cerevisiae
  33. 33. Conclusions There is a huge diversity in yeasts in general, and Saccharomyces in particular waiting to be discovered and exploited
  34. 34. Conclusions There is a huge diversity in yeasts in general, and Saccharomyces in particular waiting to be discovered and exploited Hybridization is a short-term solution to combine interesting strain with interesting industrial traits and a method for characterizing those traits
  35. 35. Conclusions There is a huge diversity in yeasts in general, and Saccharomyces in particular waiting to be discovered and exploited Hybridization is a short-term solution to combine interesting strain with interesting industrial traits and a method for characterizing those traits With HyPr we are able to generate higher order hybrids and diversity to address important genetic questions and fix industrial problems
  36. 36. Thank you Chris T. Hittinger Ryan V. Moriarty Quinn Langdon William Alexander Meihua Kuang Kayla Sylvester Emily Baker Hittinger Lab Members Wild YEAST program Trey Sato Li Hinchman Lucas Parreiras Jeff Piotrowski Diego Libkind Jose Paulo Sampaio Paula Gonçalves Christian Landry Jean-Baptiste Leducq Guillaume Charron Justin Fay Katie Hyma Fengyan Bai Qi Ming Wang Yaoping Zhang Alex Reau Haibo Li David Benton Yury Bukhman HPLC Service Mick McGee Audrey Gasch Maria Sardi UW & GLBRC Collaboration

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