4Net 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,
5Major 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.
6Renewable Energy Biomass Program Next generation:Renewable Energy Biomass ProgramThe 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.
7To be a viable alternative, a biofuel program should: Provide a net energy gainHave environmental benefitsBe economically competitiveBe producible in large quantities without reducing food supplies
8Current 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
10DEGREE OF DIFFICULTY in PRODUCING ETHANOL EASIEST AND MOST ECONOMICAL WAY TO MAKE ETHANOL TODAYONLY COMMERCIAL ROUTE TODAYGLUCOSESingle six carbon sugar“Free” Six carbon sugarYeastSUCROSESix carbon sugar dimerEthanolSTARCHPolymer of glucoseCELLULOSEPolymer of glucose; intertwined with ligninand hemicelluloseHEMICELLULOSEPolymer of six and five carbonsugars (PENTOSES); intertwinedwith ligninNOT COMMERCIALLY VIABLE TODAYFive carbon sugarGMO YeastEColiOtherOrganismsMOST DIFFICULT AND LEASTECONOMICAL WAY TO MAKEETHANOL TODAY?Ethanol
11Challenges in Biofuels Production Stephanopoulos, G. (2007). Science 315,
12A combination of 3 enzymes is required to degrade Cellulose: endoglucanases (endo--1,4-glucanases, EG)-GlucosidasesCellobiohydrolases (exo-b-1,4-glucanases, CBHs)
15FIG. 3. Hypothesis for the role of oligomers during microbially and enzymatically mediated cellulose hydrolysis.
16The 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.
17Two major approaches for bioethanol production: A separate step to produce cellulasesCombining cellulase production, hydrolysis, and fermentation in a single organism.SHF -- separate hydrolysis & fermentationSSF -- simultaneous saccharification & fermentationSSCF -- simultaneous saccharification & combined fermentationCPB -- consolidated bioprocession
23C. thermocellum both cellulolytic and ethanogenic Highly efficient cellulosomeLow ethanol producing capabilityLow ethanol tolerannceSlow growingNot accessible to genetic manipulation
24P. chrysosporium lignin degradation cellulases and xylanse producing No genetic toolNon-ethanol producing
25S. cerevisiae, Zymomonas mobilis, E S. cerevisiae, Zymomonas mobilis, E. coli , and Klebsiella oxytoca are ethanol-tolerant.S. cerevisiae and Zymomonas mobilis are also ethanolic.
26Anaerobic Glucose Respiration (Fermentation to Ethanol) Most Important Bug:Saccharomyces cerevisiaePossible Contender:Zymomonas mobilisC6H12O6 → 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 moreEthanol tolerance at 12-15% (v/v); butanol much less
27Zymomonas mobilisa metabolically engineered bacteria used for fermenting bothglucose and xylose to ethanol.Science, vol 315, pp , 2007.
28Zymomonas mobilisIts 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.
29Zymomonas mobilis haslow biomass yield, biomass competing with ethanol for the available carbon source(s),high speed of substrate conversion to metabolic products, andcomparatively simple glycolytic pathways
30S. cerevisiae as a CBP host -- additional advantages Robust growth under industrial production conditionsinhibitor tolerancehigh ethanol productivityExcellent genetic system
32Construction of Xylose utilizing yeast S. cerevisiae does not naturally ferment xylose, but other fungi and many bacteria do.
33Figure 1. Metabolic pathways for xylose utilization. Xylose reductaseXylose isomeraseXylitol dehydrogenaseXylulose kinaseFigure 1Metabolic pathways for xylose utilization. A. The XR-XDH pathway. B. The XI pathway.fungalbacterialFigure 1. Metabolic pathways for xylose utilization.
34Anaerobic 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).
35Additional 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.
37The 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.
38Take 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.
39Figure 1. Metabolic pathways for xylose utilization. Xylose reductaseXylose isomeraseXylitol dehydrogenaseXylulose kinaseFigure 1Metabolic pathways for xylose utilization. A. The XR-XDH pathway. B. The XI pathway.fungalbacterialFigure 1. Metabolic pathways for xylose utilization.
40Figure 2. Aerobic growth of TMB 3057 (XR-XDH) (■) and TMB 3066 (XI) (▲) in mineral medium with xylose (50 g/l) as the sole carbon sourceKarhumaa et al. (2007). Microb Cell Fact. 2007; 6: 5.
41Symbols: 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;× acetateKarhumaa et al. (2007). Microb Cell Fact. 2007; 6: 5.
42Figure 4Fermentation 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.
46Expression of cellulases in S. cerevisiae Ref: van Zyl et al. (2007). Adv. Biochem. Engin/Biotechnol. 108:
47A combination of 3 enzymes is required to degrade Cellulose: endoglucanases (endo--1,4-glucanases, EG)-GlucosidasesCellobiohydrolases (exo-b-1,4-glucanases, CBHs)
48For 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?
50General conclusions:A relative low titer of secreted CBH is found, with a variable range between 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.
51In 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
54A combination of 3 enzymes is required to degrade Cellulose: endoglucanases (endo--1,4-glucanases, EG)-GlucosidasesCellobiohydrolases (exo-b-1,4-glucanases, CBHs)
55Rationale: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).
56The action of the endoglucanase encoded by Trichoderma reesei EGI(cel7B) yields mainly cellobiose and glucose from PASC as substrate.
57Terms:EGI: an endoglucanase of Trichoderma reeseiBGL1: the -glucosidase of Saccharomycopsis fibuligeraPASC: phosphoricacid swollen cellulose
58Plasmid constructs: pCEL5 -- pEGI -- Pro sec EGI Pro sec BGL1 Pro sec
59Haan 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
60Extracellular endoglucanase activity β -Glucosidase activity,Extracellular endoglucanase activityY294[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
61Growth curve ethanol production Y294[CEL5] (●, ○)Growth curveY294[CEL5] glucose preculture (●, ○)ethanol productionFig. 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
62Haan 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