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

Current Ethanol Production Methods Adopted from US DOE

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

Net energy balance (NEB) for corn grain ethanol and soybean biodiesel production. Corn starch-based ethanol production has a net energy balance of only 1.25, which is very low. Hill et al. (2006). PNAS 103, 11206-11210.

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: The key is that starch or sucrose-based ethanol production will not be enough to produce sufficient ethanol needed. A second generation of technology is needed. Only a net energy gain equivalent to 2.4% and 2.9% of U.S. gasoline and diesel consumption.

Renewable Energy Biomass Program 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.

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

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

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

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

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

FIG. 3. Hypothesis for the role of oligomers during microbially and enzymatically mediated cellulose hydrolysis.

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.

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

Current status: SSF Source: US DOE

Future goal: CBP Source: US DOE

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

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

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

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

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

Anaerobic Glucose Respiration (Fermentation to Ethanol) Most Important Bug: Saccharomyces cerevisiae Possible Contender: Zymomonas mobilis C6H12O6 → 2 C2H5OH + 2 CO2 + 2ATP (MW = 180) (MW = 92) (MW = 88) 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

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

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.

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

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

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

Figure 1. Metabolic pathways for xylose utilization. Xylose reductase Xylose isomerase Xylitol dehydrogenase Xylulose kinase Figure 1 Metabolic pathways for xylose utilization. A. The XR-XDH pathway. B. The XI pathway. fungal bacterial Figure 1. Metabolic pathways for xylose utilization.

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).

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.

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.

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.

Figure 1. Metabolic pathways for xylose utilization. Xylose reductase Xylose isomerase Xylitol dehydrogenase Xylulose kinase Figure 1 Metabolic pathways for xylose utilization. A. The XR-XDH pathway. B. The XI pathway. fungal bacterial Figure 1. Metabolic pathways for xylose utilization.

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.

Symbols: xylose; * xylitol; ■ glycerol; ▲ethanol; × acetate Figure 3. Anaerobic fermentation profiles of strains TMB 3057 (P. stipitis XR and XDH on plasmid) (A), TMB 3066 (Piromyces XI on plasmid) (B) and TMB 3400 (industrial strain with chromosomally integrated P. stipitis XR and XDH) (C). Mineral medium with xylose (50 g/l) was used. The initial biomass concentration for all strains was 5 g/l. Symbols: xylose; * xylitol; ■ glycerol; ▲ethanol; × acetate. Symbols: xylose; * xylitol; ■ glycerol; ▲ethanol; × acetate Karhumaa et al. (2007). Microb Cell Fact. 2007; 6: 5.

Figure 4 Fermentation of lignocellulose hydrolysate by strains TMB 3057 (P. stipitis XR and XDH on plasmid) (A), TMB 3066 (Piromyces XI on plasmid) (B) and TMB 3400 (industrial strain with chromosomally integrated XR and XDH) (C). The initial cell concentration was 5–10 g/l for all the fermentation experiments shown. For clarity, the hydrolysate components and their consumption are shown on the left, and the accumulation of the products is shown on the right. Symbols: mannose; □glucose; galactose; xylose; *xylitol; ■ glycerol; ▲ethanol; × acetate. Symbols: mannose; □glucose; galactose; xylose; *xylitol; ■ glycerol; ▲ethanol; × acetate. Karhumaa et al. (2007). Microb Cell Fact. 2007; 6: 5.

Anaerobic batch fermentation of 50 of xylose by different sttrains

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

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

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?

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.

In a recent report, the amount of CBH1 required to enable growth on crystalline cellulose was found to be between 1 and 10% of total cellular proteins, which is within the capability of heterologous protein production in S. cerevisiae. Haan et al. (2007). Meta Engin. 9: 87-94

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

Rationale: Endoglucanases are active on the amorphousregions of cellulose and yield cellobiose and cellooligosaccharidesas hydrolysis products. -glucosidases convert cellobiose and some cello-oligosaccharides to glucose, combining these activities should enable degradation of an amorphous cellulosic substrate such asphosphoric acid swollen cellulose (PASC).

The action of the endoglucanase encoded by Trichoderma reesei EGI(cel7B) yields mainly cellobiose and glucose from PASC as substrate.

Terms: EGI: an endoglucanase of Trichoderma reesei BGL1: the -glucosidase of Saccharomycopsis fibuligera PASC: phosphoricacid swollen cellulose

Plasmid constructs: pCEL5 -- pEGI -- Pro sec EGI Pro sec BGL1 Pro sec

Haan et al. (2007). Meta Engin. 9: 87-94 ig. 1: Recombinant S. cerevisiae Y294 strains as plate cultures: (A) SC−URA medium with 20ﾊgﾊl−1 glucose. (B) YPC medium (10ﾊgﾊl−1 cellobiose) showing growth of BGL1 containing Y294 strains. (C) SC−URA medium (20ﾊgﾊl−1 glucose) supplemented with 0.1% CMC; after incubation colonies were washed off and the medium subsequently stained with Congo red. CMC degrading Y294 strains (containing EGI) showed clearing zones. (D) YP-PASC (10ﾊgﾊl−1 PASC) medium showing growth of the BGL1, EGI co-expressing strain Y294[CEL5]. (E) An enhanced top view of the YP-PASC plate in D to illustrate growth by strain Y294[CEL5]. The plates were photographed after 4 days of incubation at 30ﾊ｡C. Haan et al. (2007). Meta Engin. 9: 87-94

Extracellular endoglucanase activity β -Glucosidase activity, Extracellular endoglucanase activity Y294[REF] (▾, ▿); Y294[SFI] (▴, ▵); Y294[EGI] (, ﾗ); Y294[CEL5] (●, ○) Fig. 2: Time course of enzymatic activity of recombinant S. cerevisiae strains Y294[REF] (▾, ▿); Y294[SFI] (▴, ▵); Y294[EGI] (, ﾗ); Y294[CEL5] (●, ○) on YPD medium: (A) β -Glucosidase activity, indicated as total activity (supernatant and cell associatedﾑsolid symbols) and extracellular activity (supernatantﾑopen symbols) was measured on p -NPG. (B) Extracellular endoglucanase activity (solid symbols) was measured on CMC. Activities expressed as units per dry cell weight (DCW). Haan et al. (2007). Meta Engin. 9: 87-94

Growth curve ethanol production Y294[CEL5] (●, ○) Growth curve Y294[CEL5] glucose preculture (●, ○) ethanol production Fig. 3: (A) Growth curve (solid symbols) and (B) ethanol production (open symbols) time course of anaerobic cultures of recombinant S. cerevisiae strains Y294[REF] (▾, ▿); Y294[SFI] (▴, ▵); Y294[EGI] (, ﾗ); Y294[CEL5] glucose preculture (●, ○); Y294[CEL5] PASC preculture (■, □) on YP medium containing 10ﾊgﾊl−1 PASC as sole carbohydrate source. Haan et al. (2007). Meta Engin. 9: 87-94

Haan et al. (2007). Meta Engin. 9: 87-94 Fig. 4: Decreased viscosity of anaerobic YP-PASC cultures at the end of the 240ﾊh growth period. Viscosity measurements were done over 30 shear rates (2ﾐ200ﾊs−1) for the culture media after the growth period as well as for fresh YP-PASC (10ﾊgﾊl−1 PASC) medium. The average viscosities of the spent culture media were expressed as a percentage of the viscosity of fresh medium. Haan et al. (2007). Meta Engin. 9: 87-94

Science, vol 315, pp1488-1450, 2007.