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

Main feedstocks for current generation biofuels

• Biodiesel --- Soybean

• Ethanol -- Corn (U.S.) Sugarcane (Brazil)

Hill et al. (2006). PNAS 103, 11206-11210.

Net energy balance (NEB) for corn grain ethanol and soybean biodiesel production.

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.

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

• 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.

Current efforts focus on three areas

-- saccharification step

-- fermentation step

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DEGREE OF DIFFICULTY in PRODUCING ETHANOL

GLUCOSE Single six carbon sugar

SUCROSE Six carbon sugar dimer

STARCH Polymer of glucose

CELLULOSE Polymer of glucose; intertwined with lignin and hemicellulose

MOST DIFFICULT AND LEAST ECONOMICAL WAY TO MAKE

ETHANOL TODAY

HEMICELLULOSE Polymer of six and five carbon sugars (PENTOSES); intertwined with lignin

“Free” Six carbon sugar

Ethanol

Yeast

EASIEST AND MOST ECONOMICAL WAY TO MAKE

ETHANOL TODAYONLY COMMERCIAL ROUTE TODAY

Five carbon sugar

GMO YeastEColiOtherOrganisms

NOT COMMERCIALLY VIABLE TODAY

Ethanol

?

Challenges in Biofuels Production

Stephanopoulos, G. (2007). Science 315, 801 - 804.

A combination of 3 enzymes is required to degrade Cellulose:

Cellobiohydrolases (exo--1,4-glucanases, CBHs)

endoglucanases (endo--1,4-glucanases, EG)

-Glucosidases

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:

1. A separate step to produce cellulases

2. 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

Source: US DOE

Future goal: CBP

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. 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:

1. Theoretical maximum yield (w/w) = 51%

2. Energy content of EtOH/Gas = 2/3; butanol more

3. Ethanol tolerance at 12-15% (v/v); butanol much less

Zymomonas mobilis

a metabolically engineered bacteria used for fermenting bothglucose and xylose to ethanol.

Science, vol 315, pp 802-803, 2007.

• 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

1. low biomass yield, biomass competing with ethanol for the available carbon source(s),

2. high speed of substrate conversion to metabolic products, and

3. comparatively simple glycolytic pathways

Zymomonas mobilis has

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.

Xylose reductase

Xylitol dehydrogenase

Xylulose kinase

Xylose isomerase

Figure 1. Metabolic pathways for xylose utilization.

fungal bacterial

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).

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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.

Xylose reductase

Xylitol dehydrogenase

Xylulose kinase

Xylose isomerase

Figure 1. Metabolic pathways for xylose utilization.

fungal bacterial

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

Karhumaa et al. (2007). Microb Cell Fact. 2007; 6: 5.

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:

Cellobiohydrolases (exo--1,4-glucanases, CBHs)

endoglucanases (endo--1,4-glucanases, EG)

-Glucosidases

For S. cerevisiae as a CBP microbe, two questions need to be answered.

1.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?

2.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:

Cellobiohydrolases (exo--1,4-glucanases, CBHs)

endoglucanases (endo--1,4-glucanases, EG)

-Glucosidases

• 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).

Rationale:

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:

pCEL--

pEGI --

sec EGI BGL1Pro sec

sec EGIPro

Pro

Haan et al. (2007). Meta Engin. 9: 87-94

Haan et al. (2007). Meta Engin. 9: 87-94

Y294[REF] (▾, ▿); Y294[SFI] (▴, ▵); Y294[EGI] (, ﾗ ); Y294[CEL5] (●, ○)

β -Glucosidase activity,

Extracellular endoglucanase activity

Haan et al. (2007). Meta Engin. 9: 87-94

Growth curve

ethanol production

Y294[CEL5] (●, ○)

Y294[CEL5] glucose preculture (●, ○)

Haan et al. (2007). Meta Engin. 9: 87-94

Science, vol 315, pp1488-1450, 2007.