1. Evolutionary Biology The Crabtree effect and its influence on fitness of yeast populations from natural isolates Presented by Emelyne Cunnington Co-supervised by Thomas Pfeiffer and Austen Ganley

2. Energy metabolism The Crabtree effect Research questions Methodology Pilot study Conclusion ATP ~ An energetic compound

3. Energy metabolism The Crabtree effect Research questions Methodology Pilot study Conclusion ATP ~ As a component of yeast fitness Glucose Glycolysis ATP RESPIRATION FERMENTATION Pyruvate Pdh Pdc TCA Cycle OXPHOS Adh Ethanol CO2 Acetaldehyde Acetyl-CoA ATP

4. Energy metabolism The Crabtree effect Research questions Methodology Pilot study Conclusion Crabtree effect ~Aerobic alcoholic fermentation~ • Certain yeasts produce ethanol in aerobic condition • Crabtree-positive yeasts • Ferment sugar in addition to respiration • In presence of oxygen • Above a critical [sugar] • By opposition: Crabtree-negative yeasts • Use solely the respiration pathway Crabtree, Biochem. J. (1929)

5. Energy metabolism The Crabtree effect Research questions Methodology Pilot study Conclusion Crabtree effect ~Aerobic alcoholic fermentation~ • Crabtree-positive yeasts • Typical growth • On batch culture • Diauxic shift • Two time growth Monje-Casas, Biochem, J. (2004)

6. Energy metabolism The Crabtree effect Research questions Methodology Pilot study Conclusion Crabtree effect ~Aerobic alcoholic fermentation~ • Crabtree-positive yeasts • Typical growth • On chemostat • Dilution rate • Drop in growth yield Dijken, Antonie Van Leeuwenhoek (1993)

7. Energy metabolism The Crabtree effect Research questions Methodology Pilot study Conclusion Costs and Benefits • Why do yeasts use the ‘wasteful’ fermentation pathway in presence of oxygen? • Rate of ATP production • Yield in ATP production Neijsel, Mol. Microbiol. (1994)

8. Energy metabolism The Crabtree effect Research questions Methodology Pilot study Conclusion Costs and Benefits • Why do yeasts use the ‘wasteful’ fermentation pathway in presence of oxygen? • Aerobic fermentation = higher rate in ATP production • Competitive advantage • Come at the cost of lower yield of ATP production Trade-off between yield and rate Pfeiffer, Nature (2001)

9. Energy metabolism The Crabtree effect Research questions Methodology Pilot study Conclusion Costs and Benefits • Yield/Rate Trade-off theory: • Growth rate at the cost of growth yield Selection for growth yield Coupled with decrease in growth rate Jasmin, Proc. R. Soc. B (2012)

10. Energy metabolism The Crabtree effect Research questions Methodology Pilot study Conclusion Investigation of the Crabtree effect • Selection for growth yield Such as Environment dependant • Unstructured environment  Competitive trait Crabtree-positive: advantage of higher rate • Structured environment  Co-operative trait Crabtree-negative: advantage of higher yield MacLean, Nature (2006)

11. Energy metabolism The Crabtree effect Research questions Methodology Pilot study Conclusion Experimental evolution ~Selection for growth yield~ Bachemann, PNAS (2013)

12. Energy metabolism The Crabtree effect Research questions Methodology Pilot study Conclusion Distinction between natural isolates without engineered populations 72h growth

13. Acknowledgement • Thomas Pfeiffer • Austen Ganley • Rainey’s group • Bas Teusink

14. Thank you for your attention

15. Energy metabolism The Crabtree effect Research questions Methodology Pilot study Conclusion PERSPECTIVES On the investigation of the Crabtree effect • Relevance of the Yield/Rate Trade-off theory • ‘Typical’ yeast metabolic behavior in nature • Influence of the Crabtree effect on population fitness • Influence of the population structures & interactions

16. Yield in ATP production NADH = 3 ATP - FADH2 = 2ATP - GTP = ATP

17. Aerobic Chemostat Culture Controlled culture in Bioreactor

18. Ethanol production as a function of sugar concentration F R verduyn, Appl. Microbiol. Biotechnol. (1984)

19. Energy metabolism The Crabtree effect Research questions Methodology Pilot study Conclusion Costs and Benefits • Why do yeasts use the ‘wasteful’ fermentation pathway in presence of oxygen? M.A.C Theory Glucose Glycolysis ATP RESPIRATION FERMENTATION Pyruvate Pdh Pdc TCA Cycle OXPHOS Adh Ald, Acs Ethanol CO2 Acetaldehyde Acetyl-CoA ATP ATP

20. Yield/Rate trade-off in ATP production Pfeiffer, Trend in Biochemical Sciences (2005)

21. Fitness as a function of sugar concentration Population frequency: Population growth rate: Monod’s equation: while Monod, Herman et Cie (1946)

22. Population frequency to Population fitness Population frequency: Population growth rate: In competition:and

23. Day 1 Experimental design ~ Competition Strain A Strain B Overnight culture 1ml 1ml Day 3 0h 0h 4h 4h 8h 8h 12h 12h

24. Camera setting AxB T12 Strain A AxB T8 AxB T0 AxB T4 Strain B

25. Energy metabolism The Crabtree effect Research questions Methodology Pilot study Conclusion Distinction between natural isolates without engineered populations • Using time lapse photography to record colony growth on agar plates  Collection of picture • Subtraction of light background  Isolation of pixel’s colonies 72h growth 0h growth

26. Camera setting

27. Camera setting