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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Boisset et al, unpublished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


Ed Bayer