Designer organisms: From cellulosics to ethanol production

1. Designer organisms: From cellulosics to ethanol production Ming-Che Shih施明哲 Agricultural Biotechnology Research Center Academia Sinica

2. Current Ethanol Production Methods Adopted from US DOE

3. Main feedstocks for current generation biofuels • Biodiesel --- Soybean • Ethanol -- Corn (U.S.) • Sugarcane (Brazil)

4. Net energy balance (NEB) for corn grain ethanol and soybean biodiesel production. Hill et al. (2006). PNAS 103, 11206-11210.

5. Major problems: • Not energy efficient & not enough feed stock supply • If all the U.S. corn and soybean harvested in 2005 were used for biofuel production, it would provide: Only a net energy gain equivalent to 2.4% and 2.9% of U.S. gasoline and diesel consumption.

6. Next generation: Renewable Energy Biomass Program • The vast bulk of plant material is cell wall, which consists of cellulose (40-50%), hemicellulose (20-30%), and lignin (20-30%), depending on plant species. • The race now is to develop technology to use cellulose and hemicellulose for bioethanol production.

7. To be a viable alternative, a biofuel program should: • Provide a net energy gain • Have environmental benefits • Be economically competitive • Be producible in large quantities without reducing food supplies

8. Current efforts focus on three areas • Identify feedstcoks that can grow on marginal lands and have good biomass production. Such feedstocks can be further improved through genetic engineering. • Develop technology to break cellulose and hemicellulose down to their component sugars. • Biorefinery will then be used to convert these sugars into fuel ethanol or other building block chemicals. -- saccharification step -- fermentation step

10. DEGREE OF DIFFICULTY in PRODUCING ETHANOL EASIEST AND MOST ECONOMICAL WAY TO MAKE ETHANOL TODAY ONLY COMMERCIAL ROUTE TODAY GLUCOSE Single six carbon sugar “Free” Six carbon sugar Yeast SUCROSE Six carbon sugar dimer Ethanol STARCH Polymer of glucose CELLULOSE Polymer of glucose; intertwined with lignin and hemicellulose HEMICELLULOSE Polymer of six and five carbon sugars (PENTOSES); intertwined with lignin NOT COMMERCIALLY VIABLE TODAY Five carbon sugar GMO Yeast EColi Other Organisms MOST DIFFICULT AND LEAST ECONOMICAL WAY TO MAKE ETHANOL TODAY ? Ethanol

11. Challenges in Biofuels Production Stephanopoulos, G. (2007). Science 315, 801 - 804.

12. A combination of 3 enzymes is required to degrade Cellulose: endoglucanases (endo--1,4-glucanases, EG) -Glucosidases Cellobiohydrolases (exo-b-1,4-glucanases, CBHs)

16. The key step is to breakdown cellulose into glucose and hemicellulose into xylose. • Two main obstacles in cellulose breakdown: • Lignins prevent access of cellulose to enzyme attack. • Cellulose in crystalline form cannot be degraded efficiently by cellulases.

17. Two major approaches for bioethanol production: A separate step to produce cellulases Combining cellulase production, hydrolysis, and fermentation in a single organism. • SHF -- separate hydrolysis & fermentation • SSF -- simultaneous saccharification & fermentation • SSCF -- simultaneous saccharification & combined fermentation • CPB -- consolidated bioprocession

19. Current status: SSF Source: US DOE

20. Future goal: CBP Source: US DOE

21. An ideal CBP host should be: • Cellulotic -- able to produce efficient cellulases • Ethanolic -- • ethanol tolerant & &

22. CBP host candidates: Clostridium thermocellum Phanerochaete chrysosporium Saccharomyces cerevisiae Zymomonas mobilis E. coli Klebsiella oxytoca

23. C. thermocellum • both cellulolytic and ethanogenic • Highly efficient cellulosome • Low ethanol producing capability • Low ethanol tolerannce • Slow growing • Not accessible to genetic manipulation

24. P. chrysosporium • lignin degradation • cellulases and xylanse producing • No genetic tool • Non-ethanol producing

25. S. cerevisiae,Zymomonas mobilis,E. coli, and Klebsiella oxytoca are ethanol-tolerant. • S. cerevisiae andZymomonas mobilis are also ethanolic.

26. Anaerobic Glucose Respiration(Fermentation to Ethanol) C6H12O6 → 2 C2H5OH + 2 CO2 + 2ATP (MW = 180) (MW = 92) (MW = 88) Most Important Bug: Saccharomyces cerevisiae Possible Contender: Zymomonas mobilis • Factoids: • Theoretical maximum yield (w/w) = 51% • Energy content of EtOH/Gas = 2/3; butanol more • Ethanol tolerance at 12-15% (v/v); butanol much less

27. Zymomonas mobilis a metabolically engineered bacteria used for fermenting both glucose and xylose to ethanol. Science, vol 315, pp 802-803, 2007.

28. Zymomonas mobilis • Its ethanol yield reaches 98% of the theoretical maximum compared to ~90% of S. cerevisiae. • It is the only to-date identified bacterium that is toxicologically tolerant to high ethanol concentrations.

29. Zymomonas mobilis has low biomass yield, biomass competing with ethanol for the available carbon source(s), high speed of substrate conversion to metabolic products, and comparatively simple glycolytic pathways

30. S. cerevisiae as a CBP host -- additional advantages • Robust growth under industrial production conditions • inhibitor tolerance • high ethanol productivity • Excellent genetic system

32. Construction of Xylose utilizing yeast S. cerevisiae does not naturally ferment xylose, but other fungi and many bacteria do.

33. Xylose reductase Xylose isomerase Xylitol dehydrogenase Xylulose kinase fungal bacterial Figure 1. Metabolic pathways for xylose utilization.

34. Anaerobic xylose fermentation by S. cerevisiae was first demonstrated by heterologous expression of xylose reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis together with overexpression of the endogenous xylulokinase (XK).

35. Additional findings from studies of Xylose utilizing yeast: • Genetic modifications other than the sole introduction of initial xylose utilization pathway are needed for efficient xylose metabolism. • The combination of overexpressed XK, overexpressed non-oxidative pentose phosphate pathway (PPP) and deletion of the endogenous aldose reductase gene GRE3 have been shown to enhance both aerobic and anaerobic xylose utilization in XR-XDH- as well as XI- carrying strains.

37. The overexpression of XK is necessary to overcome the naturally low expression level of this enzyme. • The overexpression of the PPP enzymes enables efficient incorporation of xylulose-5-phosphate into the central metabolism. • The gene GRE3 codes for an unspecific reductase that functions as an NADPH-dependent xylose reductase, and contributes to xylitol formation with concomitant inhibition of XI activity.

38. Take home message: • It is possible to improve efficiencies in production of specific metabolites through metabolic engineering by changing the levels of transoprters or key enzymes in the relevant pathways. • However, an deep understanding of metabolic network is needed, since it is likely that changes in the level of one enzyme or cofactors will affect the entire pathway.

39. Xylose reductase Xylose isomerase Xylitol dehydrogenase Xylulose kinase fungal bacterial Figure 1. Metabolic pathways for xylose utilization.

40. Figure 2. Aerobic growth of TMB 3057 (XR-XDH) (■) and TMB 3066 (XI) (▲) in mineral medium with xylose (50 g/l) as the sole carbon source Karhumaa et al. (2007). Microb Cell Fact. 2007; 6: 5.

41. Symbols: xylose; * xylitol; ■ glycerol; ▲ethanol; × acetate Karhumaa et al. (2007). Microb Cell Fact. 2007; 6: 5.

42. Symbols: mannose; □glucose; galactose; xylose; *xylitol; ■ glycerol; ▲ethanol; × acetate. Karhumaa et al. (2007). Microb Cell Fact. 2007; 6: 5.

44. Anaerobic batch fermentation of 50 of xylose by different sttrains

46. Expression of cellulases in S. cerevisiae Ref: van Zyl et al. (2007). Adv. Biochem.Engin/Biotechnol. 108:205-235.

47. A combination of 3 enzymes is required to degrade Cellulose: endoglucanases (endo--1,4-glucanases, EG) -Glucosidases Cellobiohydrolases (exo-b-1,4-glucanases, CBHs)

48. For S. cerevisiae as a CBP microbe, two questions need to be answered. How much saccharolytic enzymes, particularly cellulase expression, is enough to enable CBP conversion of plant material to ethanol, and is that amount feasible in S. cerevisiae? How do we accomplish those levels of expression?

50. General conclusions: • A relative low titer of secreted CBH is found, with a variable range between 0.002 to 1.5% of total cellular proteins. • This observation, coupled with the low specific activity of CBHs, suggests that CBH expression is a limiting factor for CBP using yeast.