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 Evolutionary Biology

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

Energy metabolism The Crabtree effect Research questions Methodology Pilot study Conclusion ATP ~ As a component of yeast fitness Glucose Pyruvate Acetaldehyde CO 2 ATP Ethanol Acetyl-CoA RESPIRATIONFERMENTATION TCA Cycle OXPHOS Adh Glycolysis PdhPdc

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 Energy metabolism The Crabtree effect Research questions Methodology Pilot study Conclusion Crabtree effect ~Aerobic alcoholic fermentation~ Crabtree, Biochem. J. (1929)

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

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

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

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 Costs and Benefits Energy metabolism The Crabtree effect Research questions Methodology Pilot study Conclusion Pfeiffer, Nature (2001)

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

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

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

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

Thomas Pfeiffer Austen Ganley Rainey’s group Bas Teusink Acknowledgement

Thank you for your attention

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

Yield in ATP production RespirationFermentation Glycolysis Pyruvate oxidation (x2) Glycolysis Ethanol production ATP2--2 NADH612-- FADH2--2 GTP--1 Total815 (x2)20 Pathway382 NADH = 3 ATP-FADH2 = 2ATP-GTP = ATP

Aerobic Chemostat Culture Controlled culture in Bioreactor

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

Energy metabolism The Crabtree effect Research questions Methodology Pilot study Conclusion Glucose Pyruvate Acetaldehyde CO 2 ATP Ethanol Acetyl-CoA ATP RESPIRATIONFERMENTATION TCA Cycle OXPHOS Adh Glycolysis PdhPdc Ald, Acs Why do yeasts use the ‘wasteful’ fermentation pathway in presence of oxygen? M.A.C Theory Costs and Benefits

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

Fitness as a function of sugar concentration Monod, Herman et Cie (1946)

Population frequency to Population fitness

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

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

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

Camera setting