1. Enzyme Optimization for Cell-Free Ethanol Production Eric J. Allain Assistant Professor Dept. of Chemistry Appalachian State University

2. The Fuel Crisis

3. Ethanol as a fuel solution Renewable Greenhouse gas neutral Works with existing systems

4. Fuel Alcohol Production Process Flow Beer 190 Proof Ethanol Whole Stillage Three column distillation system Molecular sieves 5% Gasoline 200 proof ethanol 200 proof Denatured ethanol Evaporators Wet grain Thin stillage Drum dryer Centrifuge Syrup Condensate DDG Fermentation Whole corn Slurry tank Hammer mill Jet Cooker Liquef action Mash cooling Fresh water & 4 recycled water sources Yeast CO 2 Enzymes

5.  -amylase glucoamylase  -Amylase Breakdown of Gelatinized Starch Amylopectin Amylose Glucoamylase Hydrolysis of Dextrins to Glucose Glucose

6. Yeast Uses the Glucose for Energy and Makes Ethanol as a Byproduct

7. What about places that can’t grow corn? Wheat, barley, rye, etc. (starch containing plants) Sugar cane Cellulose – over half of the carbon in the biosphere!

8. Improving the economy of ethanol production Glucose BiomassYeast Ethanol Increase the rate of ethanol production The Cell-Free Ethanol Concept If yeast is the limiting factor then why not get rid of it and use only the enzymes needed to convert glucose to ethanol?

9. Cell-Free Ethanol Production An idea as old as biochemistry! Viability no longer a concern We can change enzyme levels to whatever we need them to be Ethanol toxicity not a problem Enzyme engineering to tailor enzymes for max ethanol production Greater process flexibility (ie higher temperature) Higher yield (no carbon used for yeast growth)

10. Modeling Ethanol Glycolysis Glc G6P F6P F16BP DHAPGAP BPG 3PG 2PG PEP Pyr AcAl EtOH HK PGI PFK Ald TPI GAPDH PGK PGM Eno PK PDC ADH ATP ADP ATP ADP NAD + NADH ADP ATP ADP ATP NADH NAD + PiPi 50 mM ATP ADP ATPase Teusink et al Eur. J. Biochem. 267, 5313-5329 (2000) V = k cat [Enz] [S] K M + [S]

11. Ethanol production rate = 2.2 mM/min

12. Model Predicted Steady State Ethanol Production Rate = 8.9 mM/min 1.7 mM/min Cellular enzyme level = 13035 mg /L of Cytoplasm 0.2 L cytoplasm / L Fermentation Adjusted Rate = 1.78 mM/min For consideration of Cell- Free Ethanol Production use total enzyme level of 2742 mg/L Can we adjust these enzyme levels to increase rate of ethanol production?

13. Optimization of Enzyme Levels for Cell Free Ethanol Production Glc G6P F6P F16BP DHAPGAP BPG 3PG 2PG PEP Pyr AcAl EtOH HK PGI PFK Ald TPI GAPDH PGK PGM Eno PK PDC ADH ATP ADP ATP ADP NAD + NADH ADP ATP ADP ATP NADH NAD + PiPi ATP ADP ATPase Use metabolic control analysis for optimization Find steady state flux Increase the level of one enzyme by a small amount Find new steady state flux = Control Coefficient  ln Flux  ln [Enz] Enzymes with high control coefficients are exerting a greater amount of control on the flux through the pathway

14. Optimization Algorithm Calculate control coefficients for each enzyme Raise the level of each enzyme by 10mg/L x the control coefficient for that enzyme Rescale so total enzyme level is still 2742 mg/L Calculate new rate of ethanol production Initial rate = 1.86 mM/min After 1 st iteration rate = 1.88 mM/min

15. Optimized Rate of Ethanol Production = 3.66 mM/min

17. Is the optimum a true optimum?

18. Recycling the enzyme ideas Immobilization Altering the process – high temperature reaction Other possibilities Enzyme engineering (PFK deregulation) Using the ATP Current Focus Validation of model predictions in the lab

19. Acknowledgements Collaborators:Dr. Eric Marland Dr. Rene Salinas Allain Lab:Kristi Bilotti Diana Dardugno Andrew Madison Brittany Overfield Laurin Robertson Benjamin Shepperd Russell Vegh Patrick Williams Funding:ASU URC

20. Funding…

21. 38% Cellulose 32% Hemicellulose 17% Lignin 13% Other Corn stover Yield = 72 gallons ethanol/dry ton = 30 lbs. corn stover/gallon = 30 lbs. corn stover/gallon 72% Starch 10% Cellulose/Hemicellulose 9% Protein 4.5% Oil 3.5% Other Corn Yield = 114 gallons ethanol/dry ton = 18 lbs. corn grain/gallon = 18 lbs. corn grain/gallon Acid pretreated corn stover 56% Cellulose 5% Hemicellulose 28% Lignin 13% Other Converting Biomass to Ethanol

22. 38% Cellulose 32% Hemicellulose 17% Lignin 13% Other Corn stover Yield = 72 gallons ethanol/dry ton = 30 lbs. corn stover/gallon = 30 lbs. corn stover/gallon Enzyme usage ~100 g protein/gallon 72% Starch 10% Cellulose/Hemicellulose 9% Protein 4.5% Oil 3.5% Other Corn Yield = 114 gallons ethanol/dry ton = 18 lbs. corn grain/gallon = 18 lbs. corn grain/gallon Enzyme usage ~ 1 g protein/gallon Acid pretreated corn stover 56% Cellulose 5% Hemicellulose 28% Lignin 13% Other Converting Biomass to Ethanol