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Boisset et al, unpublished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