Transcript Metabolic Engineering of Saccharomyces cerevesiae

Bioethanol from
Lignocellulose
Group 10:
Alessandro Fazio
Fen Yang
Marcelo Bertalan
Vijaya Krishna
Woril Dudley
International collobaration
for the production
of Bioethanol
ECONOMICAL
Biomass Sources
Corn
Starch
Corn
Fiber
Sugar Cane
Switch
Grass
Cottonwoods
Paper
Wood Chips
Stover
ABUNDANT & AVAILABLE
The Products
Ethanol:
• Fuel (crops and residues) 68%
– Anhydrous Ethanol, gasoline aditive
– Hydroethanol destined for Biofuels
• Beverages (crops) 11%
• Perfumes & Pharmacology (crops) 21%
Alternative Products:
• Sugar Powder (crops)
• Biodegradable Plastic (crops)
– Polyhydroxybutyrate-PHB
*In Sugarcane the bagasse and stillage can be used for the production
of energy (ethanol and biogas) as well as component sugars (glucose,
xylose, xylitol)
The World Ethanol Market
•Total World Ethanol production in 2004: 40.92 billion Litres.
•Global ethanol market will be worth over US$16 billion by 2005
•The largest consuming regions are South America and Asia.
•In Brazil the sugar-ethanol market trade reaches about $7.5 billion/yr.
World Ethanol Production
40
35
30
%
25
20
15
10
5
0
Brazil
U.S.
China
India
Others
The Brazilian Ethanol Experience
•Oil price: 1973: $2.50/barrel. 1979: $20.00. 1981: $34.40/barrel
•In 1973 Brazil development of the first car fueled by hydrated
ethanol in the world.
•Today there are 9 million vehicles with hydrated ethanol.
•Anhydrous ethanol is utilized in 25% blend with gasoline.
•The production of ethanol reduces petroleum importation. In the
last 22 yr, an economy of US$1.8 billion/yr.
% Ethanol in
Gasoline
(gasohol)
1977: 4.5%
1979: 15%
1981: 20%
1985: 22%
1998: 24%
1999: 20%
2002: 22%
2005: 25%
Ethanol cost x Oil cost
•The direct cost of 1 l of gasoline in the USA
was US$0.21 and the cost of 1 l of ethanol
was US$0.34.
•The average cost of sugarcane production in
Brazil was US$180/t of sugar or US$0.20/L of
ethanol.
•However, the energy originating from 1 L of
ethanol corresponds to 20.5 MJ, and from 1 L
of gasoline, 30.5 MJ.
Criteria for microorganisms
•
Broad substrate utilization
•
Converting hexose and pentose to ethanol efficiently.
•
High ethanol yield (>90% of theoretical) and
productivity
•
High tolerance to acids, ethanol, inhibitors and
process hardiness.
•
Can be robust to simple growth medium
• However, no natural microorganism
displays all of the features.
• Metabolic engineering of
microorganism is a very efficient tool
for increasing bioethanol yield.
Bacteria
Yeast
Escherichia coli
Klebsiella oxytoca
Zymomonas mobilis
Bacillus stearothermophilus
Saccharomyces cerevisiae
Pachysolen tannophilus
Candida shehatae
Pichia stipitis
Escherichia coli
• An important vehicle for the cloning and
modification of genes
• Ferment hexose and pentose as well with
high ethanol yield by recombinant strains
• High glycolytic fluxes
• Reasonable ethanol tolerance
Klebsiella.oxytoca
•
•
•
Wide sugar utilization
Form ethanol through the PFL pathway
after being modified
High ethanol yield
Zymomonas mobilis
•
A gram-negative, natural fermentative bacteria in
ethanol production
•
The only bacteria which can use Entner-Doudoroff
pathway anaerobically
•
Unable to ferment pentose but hexose
•
Limitation of using lignocellulose
•
•
Relatively easier to receive and maintain foreign
genes
High ethanol yield
Bacillus stearothermophilus
• Thermophilic organisms fermenting
hexose and pentose after being modified
• Avoid the limitation of high concentration
of ethanol harmful to fermentaion
Saccharomyces cerevisiae
• The most common and natural fermentative
yeast for ethanol
• Only convert glucose to ethanol for wild-type
• Limitation of using lignocellulose
• Relative high ethanol yield
• Can be easily modified by metabolic
engineering to ferment pentose
Other yeasts
Pachysolen tannophilus, Candida
shehatae, and Pichia stipitis
• Ferment xylose
• Low ethanol yields
• High sensitivity to inhibitors, low PH
and high concentration of ethanol
BioEthanol from Bacteria: Klebsiella
oxytoca
The most promising ethanologenic
bacteria are:
•Escherichia coli
•Zymomonas mobilis
•Klebsiella oxytoca
BioEthanol from Bacteria: Klebsiella oxytoca
Main features:
• Enteric Bacterium (Gram negative)
• EtOH is formed through the PFL (Pyruvate Formate Lyase) pathway, like in
E. coli
• It produces its own β-GLUCOSIDASE and therefore it is able to metabolize
dimeric (cellobiose) and trimeric (cellotriose) sugars, besides monomeric
(hexoses and pentoses) sugars
• Less enzymes are required for the pre-treatment of cellulose: economic
advantage for the solubilization of cellulose
SSF conditions:
35-37 C
pH 5.0-5.4
Dien et al.
(2003)
Klebsiella oxytoca: casAB operon
Ingram et al.
(1999)
casA and casB genes allow K. oxytoca to transport and metabolize cellobiose
Klebsiella oxytoca: EtOH production
EtOH is naturally produced
through the Pyruvate Formate
Lyase (PFL) pathway (similarly
to E. coli)
Dien et al. (2003)
Klebsiella oxytoca: metabolic engineering for
EtOH production
Strategy: redirection of metabolism
towards EtOH production through the
insertion of pet operon
PDC
ADH
Pet operon: Pyruvate
decarboxylase (PDC) and
Alcohol dehydrogenase (ADH)
Two main strains were produced:
K. Oxytoca M5A1 + plasmids with pet operon = K. Oxytoca M5A1 (pLOI555)
K. Oxytoca M5A1 + chromosomal integration of pdc and adhB from Z. mobilis = K. Oxytoca
P2
Klebsiella oxytoca: metabolic engineering for
cellulose hydrolysis
K. Oxytoca P2
+ two extracellular endoglucanase genes (CelZ
and CelY) from Erwinia chrysanthemi.
+ out gene for secretion from Erwinia
chrysanthemi
= K. oxytoca SZ21 (pCPP2006)
However, the strain fermented poorly
cellulose without addition of commercial
cellulose
Zhou and Ingram (2000)
K. oxytoca, E. coli, Z. mobilis
Dien et al. (2003)
Possible strategy for the future
•
Since casAB operon insertion has been
attempted in E.coliKO11, a possible strategy
could be the integration of casAB operon and
endoglucanase genes in S.cerevisiae genome
in order to allow this yeast to solubilize
cellulose and, therefore, to reduce the cost of
the process
Bottlenecks in using bacteria for industrial
production of EtOH
•Production of EtOH in large reactors
•Contamination
•GRAS status
•Relevant economic advantages respect to yeasts (e.g.
reduced need for enzymes)
Moreover, industrial acceptance of recombinant bacteria will
depend upon the relative success of yeast microbiologists in
developing industrially relevant pentose-fermenting
Saccharomyces strains.
Metabolic Engineering
of
Saccharomyces cerevesiae
• Saccharomyces cerevesiae is unable to ferment
pentoses. Metabolic engineering can be used to make
S.cerevesiae able to ferment xylose, the main
component of pentoses.
• The efficiency of the constructed strain depends on its
substrate utilization range, to use all the sugars of
lignocellulose substrate
• Xylose metabolism involves conversion of xylose to
xylulose, whcih after phosphorylation, is metabolized
through pentose phosphate pathway
Strategies Employed
Strategy
Result
Insertion of pentose utilization genes XYL1(xylose
reductase) and XYL2 (xylitol dehydrogenase) from
P.Stipitis
a) Over expression of XYL1+XYL2
b) Chromosomal integration of XYL1 and XYL2
c) Expressing different ratios of XR and XDH and
over expression of TKL1 and TAL1
a & b) Could grow on xylose but the ethanol yield was
less
Improvement of xylulose consumption
a)
Capable of growing on xylose alone. xylitol yield
was still high. xylose was fermented with 66% of
the theoretical yield. In a mixture of sugars, 90% of
the yield was achieved but arabinose was not
metabolised.
b) Expressing the genes XKS1, XYL1 and XYL2 in a
multi-copy vector.
b)
Unstable in non-selective media.
c) Chromosomal integration of the above strain.
c)
Chromosomal integration solved the problem
resulting in a stable strain. 70% of the theoretical
yield attained in a glucose-xylose mixture.
d)
25% of the theoretical yield was attained in a
minimal medium containing glucose and xylose.
c) Could produce more ethanol but still was not
economically viable. A ratio of 0.06 had higher
xylose consumption and lower xylitol formation.
a) Expressing the gene XKS1 (xylulo kianase) XYL1
and XYL2
d) Expressing the genes XKS1, XYL1 and XYL2 in a
single-copy vector.
• Now S.cerevesiae can ferment xylose
efficiently through genetic modifications
• But the expected ethanol cannot be
obtained in any case and resulted in a
low rate of xylose consumption and
substantial xylitol secretion.
• The problem of xylitol excretion is
attributed to the cofactor imbalance
(NAD+ and NADPH)
First Strategy
• The metabolic strategy applied was to delete the zwf1 gene encoding the
glucose-6-phosphate dehydrogenase in the strain with the genes XKS1,
XYL1 and XYL2 expressed in a multi-copy vector.
• As it can be seen the main source of NADPH originating form the
oxidative part of the pentose phosphate pathway has there by been
reduced
The strategy of redox metabolism to improve
the strain for the conversion of xylose to
ethanol
• Xylitol + NADP+ <=XR=> D-xylose + NADPH + H+ ……… (1)
• Xylitol + NAD+ <=XDH=> D-xylulose + NADH + H+....…… (2)
•
As it can be seen from the reaction (1) that xylose reductase
is NADPH dependent and reaction (2) that xylitol
dehydrogenase is NAD+ dependent.
• The imbalance leads to more of the first reaction and less
second reaction, thus forming a lot of xylitol and less
converted to xylulose.
Results of first strategy:
• Significant improvement of ethanol yield.
• Reduction of xylitol yield.
Explanation:
• The possible explanation for this is that with the less
availability of NADPH, it is using NADH to convert xylose
to xylitol releasing NAD+. Inorder to reconvert the NAD+
it is utilising it to form xylulose from xylitol.
Second Strategy
The strategy applied was to modulating the redox metabolism to favour xylose
metabolism through metabolic engineering of ammonium assimilation in the strain
with the genes XKS1, XYL1 and XYL2 expressed in a multi-copy vector.
a) Deletion of GDH1
Reaction 1 is encoded by GDH1 and reaction 2 is encoded by GDH2
L-Glutamate + NAD+ + H2O <=> 2-Oxoglutarate + NH3 +NADH + H+ …….…… (1)
L-Glutamate + NADP+ + H2O <=> 2-Oxoglutarate + NH3+ NADPH + H+……..…(2)
b) Over expression of GDH2 or GS-GOGAT system (GLT1+GLN1)
Reaction 1 is encoded by GLT1 and reaction 2 is encoded by GLN1 (Alternate pathway)
2 L-Glutamate + NAD+ <=> L-Glutamine + 2-Oxoglutarate+ NADH ………… (1)
ATP + L-Glutamate + NH3 <=> ADP + Orthophosphate +L-Glutamine ………... (2)
Results
a) Results:
• Increased Ethanol yield.
• Decreased Glycerol yield.
• The specific growth rate reduced dramatically.
b) Results:
• Specific growth rate could now be recovered.
Experimental results:
• Glycerol decreased in both the cases
• The specific growth rate could be recovered in the second
case
• But deletion of gdh1 alone reduced the ethanol yield
substantially.
Possible strategies for the future
A future possibility is to find a mutant strain that can
ferment both xylose and arabinose, thus utilizing all the
pentoses of lignocellulose.
• Insertion of genes for arabinose metabolism and xylose
transport will increase the pentose utilization. Genes for
arabinose metabolism can be obtained form yeasts such
as Candida aurigiensis and for xylose transport from
P.stipitis.
• Expression of the genes araA (L-arabinose isomerase),
araB (L-ribulokinase), araD (L-ribulose-5phosphate-4epimerase) from E.coli into the mutant strain of
S.cerevesiae for arabinose metabolism
Bioethanol efficiency production
Sugar cane yields the best energy balance in production of ethanol.
Raw Material
Energy Output / Energy Input
Wheat
1.2
Corn
1.3 – 1.8
Sugar Beet
1.9
Sugar Cane
(under Brazilian production conditions)
8.3
Macedo, I. et alii, F.O. Lichts 2004
Raw Material
Energy Output / Energy Input
Wood
0.47
Switchgrass
0.50
Corn
0.71
David Pimentel D. And Tad W. Patzek 2005
Fermentation efficiency production
Alternatives approach in Bioethanol production
?%
20% - ?%
10%
Source
Pre-treatment
Fermentation
Plant improvement:
Sucrose content
Pathogen response
Photoreceptors
Aluminum tolerance
Ethanol
Microbial improvement:
Fixing nitrogen to the plant
Phytohormones: Auxin, giberillin
and cytokinin.
Antagonism against pathogens.
Pre-treatment of Lignocellulose for
bioethanol fermentation
• It was considered necessary to give a brief overview of this pretreatment step, since the method employed can have implications
for fermentation conditions and the choice of microbe.
• The hydrolysis is usually carried out by the use of enzymes or by
chemical treatment.
• Enzymatic Hydrolysis
• This is carried out by cellulose enzymes which are highly specific.
• Novozymes is launching three new enzymes which make the
production of ethanol from wheat, rye and barley up to 20%
• The new enzymes break down components of the grain which would
otherwise result in a thick consistency. This saves producers the
amount of water and energy that would otherwise be required to
dilute and handle the mash. A thinner mash also makes life easier
for the enzymes in the next stage of the process, which break the
material down into sugars for fermentation into ethanol (alcohol).
Ethical and Conclusion
•
Lands used for lignocellulose production for
ethanol production, could be used for edible
crops, in helping to alleviate current food
shortage
million hectares
Brazil´s Territory 850.00
Total Arable Land 320.00
Cultivated - all crops 60.40
- with Sugar Cane 5.34
-for ethanol 2.66
Denmark´s Territory 4.3
Total Arable Land 2.679
• From the present statistics, about 57% more
energy is required to produce a litre of
ethanol than the energy harvested from
ethanol using lignocellulose. The poor
tropical countries of the world are best
suited for the growth of sugar cane, and
most of these countries have vast unused
lands that could be utilized for this purpose.
• It would therefore be an advantage to all
parties to used the vast resources being
spent on trying to make something work
which might not be economically viable, to
helping these countries cultivate sugar cane
on a large scale, and then either locating
ethanol plants there, or having the
harvested cane shipped to the developed
countries for the fermentation process. It
would provide much needed cash flow for
some of these countries.
• Ethanol from sugar cane although
more efficient, still consumes more
energy than is produced. It therefore
means that a lot of the energies being
channelled into metabolic engineering
for lignocellulose bioethanol production
could be used for finding means of
improving this process, which
represents greater economic viability.
Blend gasoline - urban pollution
• Studies have found (Australia) that the use of
E10:
– Decreased CO emission by 32%;
– Decreased HC emission by 12% ;
– Decreased toxic emissions of 1-3 butadiene
(19%), benzene (27%), toluene (30%) and
xylene (27%);
– Decreased carcinogenic risk by 24%.
• In the USA, wintertime CO emissions have been
reduced by 25% to 30%.
• Conclusion:
• For bioethanol from lignocellulose to be a
viable alternative to fossil fuel, then the
cost of production will have to be reduced.
• The perfect microbe that provides broad
substrate utilization, give high ethanol
yields and is tolerant to the harsh
conditions after chemical pretreatment will
have to be engineered
• Reduction in process costs, by integrating
process engineering tools with metabolic
engineering