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

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)
Net energy balance (NEB) for corn grain ethanol and
soybean biodiesel production.
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:
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 (4050%), 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. -- saccharification step
• Biorefinery will then be used to convert
these sugars into fuel ethanol or other
building block chemicals. -- fermentation step
DEGREE OF DIFFICULTY in PRODUCING
ETHANOL
EASIEST AND MOST
ECONOMICAL WAY TO MAKE
ETHANOL TODAY
ONLY COMMERCIAL ROUTE TODAY
GLUCOSE
“Free” Six carbon sugar
Single six carbon sugar
SUCROSE
Yeast
Ethanol
Six carbon sugar dimer
STARCH
Polymer of glucose
CELLULOSE
Polymer of glucose; intertwined with lignin
and hemicellulose
HEMICELLULOSE
NOT COMMERCIALLY VIABLE TODAY
Five carbon sugar
?
Ethanol
GMO Yeast
EColi
Other
Organisms
Polymer of six and five carbon
sugars (PENTOSES); intertwined
with lignin
MOST DIFFICULT AND LEAST
ECONOMICAL WAY TO MAKE
ETHANOL TODAY
10
Challenges in Biofuels Production
Stephanopoulos, G. (2007). Science 315, 801 - 804.
A combination of 3 enzymes is required to degrade
Cellulose:
b-Glucosidases
endoglucanases
(endo-b-1,4glucanases, EG)
Cellobiohydrolases
(exo-b-1,4glucanases, CBHs)
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
• SHF -- separate hydrolysis & fermentation
• SSF -- simultaneous saccharification &
fermentation
• SSCF -- simultaneous saccharification &
combined fermentation
2. Combining cellulase production, hydrolysis,
and fermentation in a single organism.
• 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.
coli , and Klebsiella oxytoca are ethanoltolerant.
• 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 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
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
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
Xylose isomerase
Xylitol dehydrogenase
Xylulose kinase
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.
35
• 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-5phosphate into the central metabolism.
• The gene GRE3 codes for an unspecific
reductase that functions as an NADPHdependent 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
Xylose isomerase
Xylitol dehydrogenase
Xylulose kinase
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
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:
b-Glucosidases
endoglucanases
(endo-b-1,4glucanases, EG)
Cellobiohydrolases
(exo-b-1,4glucanases, CBHs)
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:
b-Glucosidases
endoglucanases
(endo-b-1,4glucanases, EG)
Cellobiohydrolases
(exo-b-1,4glucanases, CBHs)
Rationale:
• Endoglucanases are active on the
amorphousregions of cellulose and yield
cellobiose and cellooligosaccharidesas
hydrolysis products.
• b-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 b-glucosidase of Saccharomycopsis
fibuligera
PASC: phosphoricacid swollen cellulose
Plasmid constructs:
pCEL5 -Pro sec
EGI
pEGI -Pro sec
EGI
Pro sec
BGL1
Haan et al. (2007). Meta Engin. 9: 87-94
β -Glucosidase activity,
Extracellular
endoglucanase
activity
Y294[REF] (▾, ▿);
Y294[SFI] (▴, ▵);
Y294[EGI] (, ﾗ);
Y294[CEL5] (●, ○)
Haan et al. (2007). Meta Engin. 9: 87-94
Y294[CEL5] (●, ○)
Growth curve
Y294[CEL5] glucose preculture (●, ○)
ethanol production
Haan et al. (2007). Meta Engin. 9: 87-94
Haan et al. (2007). Meta Engin. 9: 87-94
Science, vol 315, pp1488-1450, 2007.