GIÁO TRÌNH CÔNG NGHỆ enzyme

Trichoderma reesei genome model and research of « helper » enzymes to improve efficiency and to reduce the cost for enzymatic hydrolysis of lignocelluloses Anthony Levasseur, Isabelle H

Trichoderma reesei genome model and research of

« helper » enzymes to improve efficiency and to reduce the cost for enzymatic hydrolysis of

lignocelluloses

Anthony Levasseur, Isabelle Herpoël-Gimbert, Eva Uzan, Sacha Grisel, Anne Lomascolo, Eric Record, Marcel Asther,

Jean-Claude Sigoillot.

UMR INRA/Université de Provence de Biotechnologie des

champignons filamenteux (UMR-BCF) –ESIL- Case 925 -

163, Av de Luminy - 13288 Marseille Cedex 09

Hanọ march 2009

L’UMR 1163 de Biotechnologie des Champignons Filamenteux

Campus de Luminy

umrbcf@esil.univmed-mrs.fr

04 91 82 86 00 http://umrbcf.esil.univ-mrs.fr

Le campus de Luminy

un environnement scientifique dense (3 000 personnes impliquées dans des activités de recherche )

2 facultés, 6 écoles et instituts (9 000 étudiants)

une infrastructure pour la vie étudiante: restaurants et

résidences universitaires, complexes sportifs …

un campus dans un site préservé, le massif des Calanques

« Cultiver votre passion pour les études, la recherche

et la nature »

Vue sur le campus

Massif des Calanques : Sugiton

Hanọ march 2009

DESTRUCTURATION ENZYMES OF LIGNOCELLULOSES

HELPER ENZYMES

Cinnamoyl ester

hydrolases

Cellulases

Hémicellulases

LIGNOLYTIC ENZYMES

FOLymes

Wood

or

Annual

plants

Bioraffinery : total valorisation of biomasse using

fungal enzymes

Déstructuration

lisation

Agromaterials Solvents Polyaromatic resins

Speciality papers

Bioéthanol

INRA

Modified fibres

Hanọ march 2009

II

I

III Heterologous expression

Centre Français de Ressources Fongiques

Phylogenomic studies

Functionnal high throuput screening

Genome modeling

Gene isolation

Nanosome conception

Basical research

Exploration

of biodiversity

Bioraffinery

« Reticulation » Agromaterials

« Biobleaching » Special papers

« Hydrolysis »

Bioéthanol

INRA

Green Chemistry

Integrated research strategy UMR Biotechnology of Filamentous Fungi

Cost effective production of clean bioethanol from lignocellulosic biomass for use as transport fuel

Principaux partenaires:

•IFP, SAF-ISIS, INRA, CNRS

•ENITecnologie, Roal Oy,Svensk Etanolkemi

•DIREVO Biotech, Granit,Centro Ricerche FIAT,

•VTT, ETHZ, ULUND, Weizmann Institute

•European Renewable Energy Centres Agency

Lignocellulose degradation studies in biofuel field

Use of pentoses and lactose

for cellulase production by

par T reesei

Use of helper enzymes (FAE, laccase ) associated

to cellulases

Hanọ march 2009

Bioethanol production

Use of wheat-straw Three step production:

- « flash » hydrolysis by steam explosion ( generate inhibitors such as furfurals)

- hydrolysis of cellulose by cellulases

from Trichoderma reesei ( Lack of cellobiohydrolases)

- Use of dextrose by S cerevisiae

(Alcoholic fermentation)(What about pentoses?)

Ethanol production from lignocellulosic biomass :

typical flow sheet

LCB Pretreatment Cellulose + Lignin Enzymatic

hydrolysis Glucose

Fermentation

Ethanol

Cellulase production

Hemicellulose

hydrolyzate

Pentoses

Hanọ march 2009

Production de cellulases en mode FEDBATCH par

T.reesei CL847

Growth medium:

Initial lactose concentration : 35 g/l

Lactose 28 g

4 N Medium 400 ml (final 2N)

Antifoam (BASF) 0,4 ml

Production medium

Sucres : 250 g/l

4 N Medium 250 ml (final 2N)

Cellulase production by T.reesei CL847 (IFP conditions)

-5

0

5

10

15

20

25

30

35

40

Culture time (hours)

glucose lactose xylose proteines poids sec

Hanọ march 2009

dilution µg/cuve gL

100 34,99 17,50

100 33,16 16,58 17,04

100 34,99 17,50

100 37,66 18,83 18,16

suivi des paramêtres de croissance culture CL847.7 et CL847.8

-5

0

5

10

15

20

25

30

35

40

45

17 41 65 89 116.15 145.45 160.15 184,30 210 234

Culture time (hours)

lact xylo galac lact galac prot prot

PS

Ps

Cellulase production by T.reesei CL847 (Modified conditions)

0 5 10 15 20 25 30 35 40 45 50

0 50 100 150 200 250 300

Temps (h)

lactose glucose xylose galactose protéines

Control of fed-batch cellulases production

fed-batch T.reesei CL847

Mainly xylanases, Very efficient cocktail

50 g/L

T reesei

Pilote 75 L

Hanọ march 2009

Surproductives strains (home made)

- CBHI et EGLI (T reesei) : mainly cellulases

- Nano-tools : in progress: Expression of chimerical enzymes

Expression system : Trichoderma reesei

Rut-C30

Sequenced genome

CL 847

Industrial strain

cellulase 50 g/L

Tolypocladium geodes LysC

Lab strain (cellulase less)

QM 9414

Lab strain

ura-Auxotrophy markers

Antibiotic resistance

markers

• Same transformation method than A niger (protoplasts)

• Promoters gpd et cbhI

• auxotrophic markers or antibiotics (phléomycin and hygromycin)

Bifunctional enzymes

TtrpC

PgpdA ssgla

A

glaA (514aa)

Kex 2 site

I

cbm3a + Histag +stop

Multifunctional enzymes

Two ways :

- Gene fusion with linker

- Cellulosome-like system with

dockerin and cohesin

Possible addition of a carbohydrate

binding domain (CBM)

Hanọ march 2009

faeA linker xynB linker cbm

Bifunctional enzymes

complementary functions

Bifunctional enzyme expression : hyperglycosylated linker strategy

FAEA

(A niger) XynB(A niger)

Chimerical enzymes

chimerical cellulosome

Cc :Clostridium cellulolyticum Cth : Clostridium thermocellum

Fungal Enzyme -bacterial dockerin

Hanọ march 2009

Development of tools for high throughput

screening

Automate GENESIS

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