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 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 Hanoï 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 Hanoï march 2009 Le campus de Luminy Vue sur le campus « Cultiver votre passion pour les études, la recherche et la nature » Massif des Calanques : Sugiton un environnement scientifique dense (3 000 personnes impliquées dans des activités de recherche ) facultés, 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 Hanoï march 2009 DESTRUCTURATION ENZYMES OF LIGNOCELLULOSES LIGNOLYTIC ENZYMES FOLymes HELPER ENZYMES Cinnamoyl ester hydrolases Hémicellulases Cellulases Hanoï march 2009 Bioraffinery : total valorisation of biomasse using fungal enzymes Délignification Wood or Annual plants Biobleaching Déstructuration Speciality papers Réticulation Functionna- Hydrolysis lisation INRA Bioéthanol Modified fibres Agromaterials Solvents Polyaromatic resins Hanoï march 2009 Integrated research strategy UMR Biotechnology of Filamentous Fungi Basical research Green Chemistry Nanosome conception Bioraffinery Heterologous expression I Gene isolation INRA « Hydrolysis » Bioéthanol Genome modeling III « Biobleaching » Special papers « Reticulation » Phylogenomic studies Agromaterials II Exploration of biodiversity Functionnal high throuput screening Centre Franỗais de Ressources Fongiques Hanoï march 2009 Hanoï march 2009 NILE :New Improvements for Ligno-cellulosic Ethanol 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 Hanoï march 2009 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 Hanoï 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?) Hanoï march 2009 Ethanol production from lignocellulosic biomass : typical flow sheet LCB Pretreatment Cellulose + Lignin Hemicellulose Enzymatic hydrolysis Glucose Cellulase production hydrolyzate Pentoses Fermentation Ethanol Hanoï march 2009 Production de cellulases en mode FEDBATCH par T.reesei CL847 Growth medium: Initial lactose concentration : 35 g/l Lactose 28 g N Medium 400 ml (final 2N) Antifoam (BASF) 0,4 ml Cornsteep g Production medium Sucres : 250 g/l Xylose Lactose N Medium BASF Water 50 g 75 g 250 ml (final 2N) ml q.s.p 250 ml Hanoï march 2009 Cellulase production by T.reesei CL847 (IFP conditions) 40 35 30 g/litre 25 glucose lactose 20 xylose 15 proteines poids sec 10 0 50 100 150 -5 Culture time (hours) Hanoï march 2009 200 250 300 Cellulase production by T.reesei CL847 (Modified conditions) suivi des paramêtres de croissance culture CL847.7 et CL847.8 45 40 35 30 lact xylo 25 galac g/l lact 20 galac prot prot 15 PS Ps 10 dilution 100 -5 100 100 100 17 µg/cuve 41 34,99 33,16 34,99 37,66 65 gL 17,50 16,58 17,50 18,83 89 116.15 145.45 17,04 Culture time (hours) 18,16 Hanoï march 2009 160.15 184,30 210 234 Control of fed-batch cellulases production fed-batch T.reesei CL847 50 g/L Mainly xylanases, Very efficient cocktail T reesei 50 45 Concentration (g/l) 40 35 lactose 30 glucose 25 xylose 20 galactose 15 protéines 10 0 50 100 150 200 250 300 Temps (h) Pilote 75 L 30 m3 Hanọ march 2009 Expression system : Trichoderma reesei • Same transformation method than A niger (protoplasts) • Promoters gpd et cbhI • auxotrophic markers or antibiotics (phléomycin and hygromycin) Rut-C30 QM 9414 Lab strain ura- Sequenced genome Antibiotic resistance markers Auxotrophy markers Tolypocladium geodes LysC Lab strain (cellulase less) CL 847 Industrial strain cellulase 50 g/L Surproductives strains (home made) - CBHI et EGLI (T reesei) : mainly cellulases - Nano-tools : in progress: Expression of chimerical enzymes Hanoï march 2009 Multifunctional enzymes NarI/NarI PgpdA ssgla A glaA (514aa) Two ways : -Gene fusion with linker - Cellulosome-like system with dockerin and cohesin Bifunctional enzymes Possible addition of a carbohydrate binding domain (CBM) Hanoï march 2009 BglII/BglI Kex I site cbm3a + Histag +stop TtrpC Bifunctional enzymes ™ Fusion of gene sequences coding for fungal enzymes with complementary functions Bifunctional enzyme expression : hyperglycosylated linker strategy FAEA (A niger) PgpdA PgpdA XynB (A niger) TTrpC faeA linker xynB faeA linker xynB linker cbm Hanoï march 2009 TTrpC Chimerical enzymes ™ FAEA (Aspergillus niger) ™ Dockerin (Clostridium thermocellum) Fungal Enzyme bacterial dockerin chimerical cellulosome Cc :Clostridium cellulolyticum Cth : Clostridium thermocellum Hanoï march 2009 Development of tools for high throughput screening Aspergillus niger cultures Coloured tests (+ / ) Automate GENESIS Pycnoporus Hanoïcinnabarinus march 2009 cultures Coloured tests (+ / )