Novel Enzyme Paradigms for Biomass Conversion toBiofuels Ed Bayer Department of Biological Chemistry The Weizmann Institute of Science Rehovot, Israel
The Plant Cell Wall
Cellulose
Other stuff
(Carpita NC, Gibeaut DM (1993
Glucose
Cellobiose
Intra- and Interchain Hydrogen Bonding
Crystalline Structure of Cellulose
Cellulases (and friends) are Multi-modular Enymes
Catalytic module
CBM
Dockerin module
Cellulases are not “normal� enzymes
Cellulases (and friends) are Multi-modular Enymes
Catalytic module
CBM
Dockerin module
Cellulases are sophisticated enzymes
Free Enzyme Paradigm
Catalytic module CBM
Cellulose
Synergism
Multi-functional Enzyme Paradigm
Catalytic modules CBMs
CBMs
CBM
22
22
10
3
3
3
43
6
22
22
10
3
3
3
43
6
Hemicellulose 2
Hemicellulose 1 Cellulose
Cell-Surface Enzyme Paradigm Cell surface polysaccharides
Peptidoglycan
Extracellular Matrix
22
Cell membrane
Cell Interior
10
9
9 SLH
Cellulosome Discovery 1983
(Bayer & Lamed (1983
TheCellulosome Paradigm
Cellulosome Cell
Enzymatic subunits
Type II
Type I
Dockerin Cohesin
Dockerin
II
Cohesin
II
Scaffoldin subunit CBM
Anchoring protein
Cellulose
A Molecular Lego !
C. thermocellum Genome — Cellulosome Components
Some Advantages of the Cellulosome • More efficient synergism due to enzyme proximity.
• Common targeting of enzymes to the substrate.
• The enzymes are kept attached to the cell.
• The whole cell is attached to the substrate.
?Can the Cellulosome Do the Job
Solubilization%
Avicel
cellulose 20%
100 80
Cellulosome+
60 40 20 0 0
40
80
120
(Time (h
160
200
(Glucose released (mg/ml
C. thermocellum Cellulosome vs. Fungal Enzymes
0,8
(C. thermocellum (cellulosome
0,7 0,6 0,5 0,4 0,3
Trichoderma reesei (free enzyme system(
0,2 0,1 0 0
2
4
6
8
10
12
14
16
18
(Incubation time (hours
Boisset et al, unpublished
USA
DOE Roadmap: Biomass to Biofuels Purpose: To define barriers and challenges to a rapid• .expansion of cellulosic-ethanol production Conclusion: The core barrier is cellulosic-biomass • .recalcitrance to processing to ethanol :Key goals• 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 •
! Million Dollars 375
1 2 3
Biomass to Ethanol Plant Fiber
Microbes
Enzymes
Sugars
Pretreatment
Yeast
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 Native dockerincontaining enzymes
Native scaffoldin
A
CBM
C
B
1
Designer Cellulosome
2
3
Random incorporation
A
CBM
C
B
1
2
3
Controlled incorporation
Chimaeric dockerincontaining enzymes
Chimaeric scaffoldin
Production of Chimaeric Cellulases
Catalytic module
CBM
Free Cellulases
Catalytic Catalytic module module
Dockerin Dockerin
ChimaericCellulases Chimaeric Cellulases
Catalytic module
Dockerin
Cellulosomal Cellulases
Assembly of Designer Cellulosomes
1
2
3
CBM
4 5
6
Adva Mechaly
Henri-Pierre Fierobe Chimaeric Scaffoldin
1
2
CBM
Fierobe et al (2001( JBC 276, 21257 Fierobe et al (2002( JBC 277, 49621
Fierobe et al (2002( JBC 277, 49621
Enhanced Synergism of Bi-functional Designer Cellulosomes Substrate: Cellulose
8Cc
48Ft
8
48
Stimulation factor = 4.1 1
Proximity Effect
CB M x
1
3
48Ft
8Cc 8
Targeting Effect
CB M
+
48
2
CB M
Free Enzymes
Fierobe et al (2002( JBC 277, 49621
Libraries of Designer Cellulosome Components C. thermocellum
Scaffoldins
5G 8A
CBM
5 5G-t 8
8
8A-c
8 8A-t
5 5G-f 8 8A-f
9K CBM
Enzymes
9R CBM
CBM
CBM
48S T. fusca 5A v-5A 5
48 48S-t
5
48
5 f-5A
5 b-5A
6A 6B
CBM
6
9A CBM
CBM
9B 48A
48 48A-t
Dockerins
48
Cellulase/xylanaseproximity effect
-Exo glucanase
-Endo glucanase
48
5
48
10
5
XBM
10
10
XBM
48
11
11
11
XBM
5
Hatched Wheat Straw
Xylanases Wild-type enzymes
Divalent designer cellulosomes
Tetravalent designer cellulosome
[Mora誰s et al, mBio 2010]
Hatched Hatched Wheat Straw Wheat Straw
Cellulase/xylanaseproximity effect
11 10
5
XBM
48
Tetravalent designer cellulosome
11 XBM
10
5 48
11
Divalent designer cellulosomes
Wild-type enzymes
XBM
10
5
48
[Mora誰s et al, mBio 2010]
Europe
WP7: Project management
WP1 Enzymatic hydrolysis
WP4 Socio-economic and environmental impacts & development strategy
WP3 Process technology
WP2 Ethanol production
WP6 Dissemination and training
WP5 Evaluation of lignocellulosic ethanol for automotive applications
Nile: Weizmann + CNRS
Major cellulases of Trichoderma reesei CNRS
Weizmann
I
7
I
Cellobiohydrolase I (Cel7A(
45
I
Endo’ase V
61
6
Cellobiohydrolase II (Cel6A(
12
I
Endo’ase IV
Endo’ase III
CBM
I
7
Endoglucanase I (Cel7B(
74
5
I
Endoglucanase II (Cel5A(
I
(Xyloglucanase (Egl6
Catalytic Module
5
I
β-Mannanase
Nile: Weizmann + CNRS Genetic constructions Weizmann
CNRS
7
H-
I
IIIa
H-
I 5
I
H-
6
I
5
EglII-Df EglII-Df
H-
CBH3a-Dt
7
H-
H-
IIIa
H-
EGL3a
7
HcatEGl-coht
H-
7
H-
CatCBHII-Cohf-Dc CatCBHII-Cohf-Dc
I
H-
EGl1-coht
CBHII-Cohf-Dc CBHII-Cohf-Dc 6
IIIa
H-
6
I
CBHI-Dt
7
H-
Dc-CatCBHII Dc-CatCBHII
H-
I
EGL1, WT
6
HcatCBH-Dt
7
CBHII-Dc CBHII-Dc
EglII-Df EglII-Df 7
H-
CBHII, CBHII, WT WT
H-
CBH3a
7
6
I
EglII, EglII, WT WT
CBHI, WT 7
5
I
7
IIIa EGl3a-coht
H-
Nile: Weizmann + CNRS
d e l Weizmann ai f s e l u d o Hm l a om
Production/secretion in T. reesei CNRS
7
H-
I
CBHI, WT
7
IIIa
√
H-
7
HcatCBH-Dt
!G
n i t f a r
EglII, EglII, WT WT
5
I
l e c
EglII-Df EglII-Df
CBH3a
b g
H-
5
I
l a i r e t ac
√ s
Ho l u l
5
EglII-Df EglII-Df
H-
√
I
7
EGL1, WT
7
IIIa
H-
EGL3a
7
HcatEGl-coht
:Nile: Weizmann + CNRS
(Joint meeting in Israel (May 29th-June 3rd 2008 Decided to abandon the nanosomeapproachSuggestedapproach for the time left: Bifunctional exo/endo enzymes (exclusivelycomposed of T. reesei modules (
7
I
I
Cellobiohydrolase I (Cel7A(
7
5
Endoglucanase II (Cel5A(
I
5
7
I
I
Endoglucanase I (Cel7B(
7
6
Cellobiohydrolase II (Cel6A(
I
6
:Nile: Weizmann + CNRS New genetic contructs performed over the past year CNRS
Weizmann
Fusions: CBH1/Egl2
Fusions: Egl1/CBH2
7
5
I
H-
7
CBH1-catEGl2
7
5
H-
catCBH1-catEGl2
Provided to Partner INRA
I
6
H-
Egl1-catCBH2
7
6
H-
catEgl1-catCBH2
Provided to Partner VTT
Weizmann
Fusions: Egl1/CBH2
Enzymatic assays
+
I
7
I
7
6
7
6
7
I 6
New Chapter in Cellulase Research
6
Engineering of Cellulolytic Bioreactors Designer Cellulosomes
Bacillus subtilis
D
E
F
4
5
6
Aspergillus niger
Multifunctional complexes
in vitro assembly
7
I
6
I
Clostridium acetobutylicum
7
6
Saccharomyces cerevisiae
Engineering of Cellulolytic Bioreactors Cellulosic Biomass
Bacillus subtilis
Aspergillus niger
Sugars
in vitro assembly
Clostridium acetobutylicum
Butanol Acetone
Saccharomyces cerevisiae
Ethanol
With thanks to ‌
Group and Collaborators France
Israel Yoav Barak Rachel Haimovitz AlonKarpol Jonathan Caspi Michael Anbar BareketDassa IlitNoach OrlyAlber Michal Slutzki Sarah Morais Yael Barkan HadarGilary ShacharYoav GiladGefen AlikDemishtein AdvaMechaly David Nakar
Raphael Lamed Ely Morag Yuval Shoham Felix Frolow Svetlana Petkun Linda Shimon IlyaBorovok SadanariJindou Yitzhak Hadar Ora Furman
Jean-Pierre Belaich Anne Belaich Henri-Pierre Fierobe Florence Mingardon FrĂŠdĂŠric Monot Antoine Margeot Eric Record Chantal Tardiff Sandrine Pages Bernard Henrissat Pedro Coutinho Veronique Receveur-Brechot Michal Hammel