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Novel Enzyme Paradigms for Biomass Conversion toBiofuels
Citation preview
Ed BayerDepartment of Biological
ChemistryThe Weizmann Institute of Science
Rehovot, Israel
Novel Enzyme Paradigms for Biomass Conversion toBiofuels
The Plant Cell Wall
Cellulose Other stuff
Carpita NC, Gibeaut DM (1993)
Glucose Cellobiose
Intra- and Interchain Hydrogen Bonding
Crystalline Structure of Cellulose
Catalyticmodule
CBMDockerinmodule
Cellulases (and friends)are Multi-modular Enymes
Cellulases are not “normal” enzymes
Catalyticmodule
CBMDockerinmodule
Cellulases are sophisticated enzymes
Cellulases (and friends)are Multi-modular Enymes
Cellulose
Catalytic module
CBM
Free Enzyme Paradigm
Synergism
22 22 10 3 3 3 43 6
Cellulose
Hemicellulose 1Hemicellulose 2
Catalytic modules
CBMs CBMCBMs
Multi-functional Enzyme Paradigm
22 22 10 3 3 3 43 6
Cellmembrane
Peptido-glycan
Cell surfacepolysaccharides
CellInterior
SLH
ExtracellularMatrix
Cell-Surface Enzyme Paradigm
22 10 9 9
CellulosomeDiscovery
1983
Bayer & Lamed (1983)
Cell
Cellulose
Enzymatic subunits
IIII
Anchoring protein
TheCellulosome Paradigm
Cellulosome
CBMScaffoldin subunit
Dockerin
Cohesin
DockerinCohesin
Type IType II
A Molecular Lego !
C. thermocellum Genome — Cellulosome Components
Some Advantages of the Cellulosome
• More efficient synergism due to enzyme proximity.
• The enzymes are kept attached to the cell.
• Common targeting of enzymes to the substrate.
• The whole cell is attached to the substrate.
20% cellulose
0
20
40
60
80
100
0 40 80 120 160 200
Time (h)
%So
lubi
lizat
ion
+Cellulosome
Can the Cellulosome Do the Job?
Avicel
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0 2 4 6 8 10 12 14 16 18
C. thermocellum (cellulosome)
Trichoderma reesei (free enzyme system)
Incubation time (hours)
Glu
cose
rele
ased
(mg/
ml)
C. thermocellum Cellulosome vs. Fungal Enzymes
Boisset et al, unpublished
USA
DOE Roadmap: Biomass to Biofuels
•Purpose: To define barriers and challenges to a rapid expansion of cellulosic-ethanol production.
•Key goals:
• Conclusion: The core barrier is cellulosic-biomass recalcitrance to processing to ethanol.
•To understand plant cell-wall chemical and physical structures — how are they synthesized and how can they be deconstructed?
•To design and produce improved enzymes.
•To improve enzyme production and make it cost effective.
•To design and produce more appropriate plants.
375 Million Dollars !
12
3
Enzymes
Plant Fiber
Pretreatment
Microbes Yeast
Sugars
Biomass to Ethanol
Cellulose Degraders – Bacteria and Fungi
Major cellulose-degrading systems:
• Free cellulases - common in fungi and aerobic bacteria- large amounts of enzymes produced
• Cellulosomes - particularly efficient cellulose-degrading systems- common in anaerobic bacteria- meager amounts of enzymes produced
Designer Cellulosomes
1994
Designer Cellulosomes
Native Cellulosome Designer Cellulosome
Random incorporation Controlled incorporation
BA CNative dockerin-containing enzymes
BA C
Chimaeric dockerin-containing enzymes
CBM 1 32Native scaffoldin
CBM 1 32Chimaeric scaffoldin
Catalyticmodule CBM
Free Cellulases
Production of Chimaeric Cellulases
Dockerin
Cellulosomal Cellulases
CatalyticmoduleDockerinCatalytic
module
ChimaericCellulases
DockerinCatalyticmodule
Chimaeric Cellulases
Assembly of Designer Cellulosomes
1 2 3 4 5 6
CBM
2
CBM
1
Chimaeric Scaffoldin
Fierobe et al (2002) JBC 277, 49621Fierobe et al (2001) JBC 276, 21257
Henri-Pierre Fierobe
Adva Mechaly
Fierobe et al (2002) JBC 277, 49621
C B M1 3
8
8Cc
48
48Ft
C B M 1x C B M2
+
8Cc 48Ft
848
Enhanced Synergism of Bi-functional Designer Cellulosomes
Stimulation factor = 4.1
TargetingEffect
Proximity Effect
Fierobe et al (2002) JBC 277, 49621
Substrate: Cellulose
FreeEnzymes
Enz
ymes
Scaffoldins
Libraries of Designer Cellulosome Components
5A
6A
6B
48A
9A
9B
T. fusca
5G
8A
9R
48S
C. thermocellum
9K
8A-c8 8A-t8 8A-f88
5G-t5 5G-f5
48S-t48 48
5 f-5A5 b-5A5v-5A5
4848A-t48
6
Dockerins
CBM
CBM
CBM
CBM
CBM
CBM
CBM
CBM
HatchedWheat Straw
[Moraïs et al, mBio 2010]
Cellulase/xylanaseproximity effect
5 10
11
XBM
48
Wild-type enzymes
10 548
Divalentdesigner cellulosomes
Tetravalentdesigner cellulosome
10 548
XBM
11
XBM
11
Exo-glucanase
Endo-glucanase
Xylanases
HatchedWheat StrawHatched
Wheat Straw
[Moraïs et al, mBio 2010]
Cellulase/xylanaseproximity effect
5
10
11
XBM
48 Wild-type enzymes
10
11
XBM 548Divalent
designer cellulosomes
Tetravalentdesigner cellulosome
10 5
11XBM48
Europe
WP1Enzymatichydrolysis
WP2Ethanol
production
WP3Process
technology
WP4Socio-economic and environmental
impacts & development strategy
WP5Evaluation of lignocellulosic
ethanolfor automotive applications
WP7: Project management
WP6Disseminationand training
Major cellulases of Major cellulases of Trichoderma reeseiTrichoderma reesei
77 77
Cellobiohydrolase ICellobiohydrolase I((Cel7ACel7A))
Endoglucanase IEndoglucanase I((Cel7BCel7B))
66
Cellobiohydrolase IICellobiohydrolase II((Cel6ACel6A))
55
Endoglucanase IIEndoglucanase II((Cel5ACel5A))
II II IIII
CBM Catalytic Module
Nile: Weizmann + CNRS
CNRSCNRS WeizmannWeizmann
4545 II 6161 II 7474 II1212 II55
Endo’ase VEndo’ase V Endo’ase IVEndo’ase IV Endo’ase IIIEndo’ase III -Mannanase-MannanaseXyloglucanase )Egl6(Xyloglucanase )Egl6(
Nile: Weizmann + CNRS
7 I -H
7 -H
7 IIIa -H
7 I -H
7 IIIa -H
CBHI, WT
CBH3a
catCBH-Dt
CBHI-Dt
CBH3a-Dt
7 I -H
7 IIIa -H
7 -H
7 I -H
-H7 IIIa
EGL1, WT
EGL3a
catEGl-coht
EGl1-coht
EGl3a-coht
5I --HH
EglII, WTEglII, WT
5I --HHEglII-DfEglII-Df
5 --HHEglII-DfEglII-Df
--HH6
Dc-CatCBHIIDc-CatCBHII
--HHI 6
CBHII-DcCBHII-Dc
--HHI 6
CBHII-Cohf-DcCBHII-Cohf-Dc
6 --HH
CatCBHII-Cohf-DcCatCBHII-Cohf-Dc
6I --HH
CBHII, WTCBHII, WT
CNRS Weizmann
Genetic constructions
√ √ √
Nile: Weizmann + CNRS
7 I -H
7 -H
7 IIIa -H
CBHI, WT
CBH3a
catCBH-Dt
7 I -H
7 IIIa -H
7 -H
EGL1, WT
EGL3a
catEGl-coht
5I --HH
EglII, WTEglII, WT
5I --HHEglII-DfEglII-Df
5 --HHEglII-DfEglII-Df
CNRS Weizmann
Production/secretion in T. reesei
Grafting bact
erial ce
llulosomal modules
failed
!
Nile: Weizmann + CNRS:
Joint meeting in Israel )May 29th-June 3rd 2008(
-Decided to abandon the nanosomeapproach
-Suggestedapproach for the time left: Bifunctional exo/endo enzymes( exclusivelycomposed of T. reesei modules)
7
Cellobiohydrolase I(Cel7A)
I 7
Endoglucanase I(Cel7B)
I 6
Cellobiohydrolase II(Cel6A)
I5
Endoglucanase II(Cel5A)
I
57 I 67 I
Nile: Weizmann + CNRS:
CNRS Weizmann
New genetic contructs performed over the past year
Fusions: CBH1/Egl2
57 I -H
57 -H
CBH1-catEGl2
catCBH1-catEGl2
7
67 I
Fusions: Egl1/CBH2
-H
6 -H
Egl1-catCBH2
catEgl1-catCBH2
Provided to Partner INRA Provided to Partner VTT
Weizmann Fusions: Egl1/CBH2Enzymatic assays
6
7 I
7 I 6+
67 I
7 6
New Chapter in Cellulase Research
Bacillussubtilis
Saccharomycescerevisiae
Aspergillusniger
Clostridium acetobutylicum
in vitroassembly
Engineering of Cellulolytic Bioreactors
DesignerCellulosomes
4 65
D E F67 I
67I
Multifunctional complexes
Sugars ButanolAcetone Ethanol
Engineering of Cellulolytic Bioreactors
CellulosicBiomass
Bacillussubtilis
Saccharomycescerevisiae
Aspergillusniger
Clostridium acetobutylicum
in vitroassembly
With thanks to …
Group and CollaboratorsIsrael
Jean-Pierre BelaichAnne BelaichHenri-Pierre FierobeFlorence MingardonFrédéric MonotAntoine MargeotEric RecordChantal TardiffSandrine PagesBernard HenrissatPedro CoutinhoVeronique Receveur-BrechotMichal Hammel
France
Yoav BarakRachel HaimovitzAlonKarpolJonathan CaspiMichael AnbarBareketDassaIlitNoachOrlyAlberMichal SlutzkiSarah MoraisYael BarkanHadarGilaryShacharYoavGiladGefenAlikDemishteinAdvaMechalyDavid Nakar
Raphael LamedEly MoragYuval ShohamFelix FrolowSvetlana PetkunLinda ShimonIlyaBorovokSadanariJindouYitzhak HadarOra Furman
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