Prof. Arkady P.Sinitsyn

Transcription

1 Enzyme complex for highly efficient saccharification of lignocellulosic feedstocks Prof. Arkady P.Sinitsyn A.N.Bach Institute of Biochemistry, Russian Academy of Sciences German-Russian Forum Biotechnology At the BIOTECHNICA 2011, Hannover

2 Concept of Pilot Plant for complex utilization of lignocellulosic feed-stocks Lignocellulosic feed-stocks Corporation of Biotechnology Pretreatment (milling) Enzymatic hydrolysis Fermentation Fermentation products BEA Separation of BEA Butanol Scaling up. Nanomembranes. Apparatus design Chemistry of mechanoactivation Phytosterin Extractives Design and optimization of multienzymatic compositions. Apparatus design (continuous saccharification) Biodiversity & screening. Genomic sequence. Metabolic engineering. Apparatus design Tolerancy to solvents Thermostability Improvement of products yield DDG Aceton EtOH Chemical industry Lignin СО 2, Н 2 Pharma Wood chemistry Enzyme preparations Yeast Energy Energy

3 Selection of enzymes for saccharification of different lignocellulosic feed-stocks DDG Grain hulls Corn stalks & stower Straw Woody materials Pretreatment Bagasse Grass Selection of enzymes for hydrolysis of polysaccharides Eg-s CBH-s β-g Xylanases Mannanases Accessory enzymes Fermentable sugars (C6 and C5)

4 Pretreatment leads to destruction of a crystalline structure of cellulose and/or to lignin degradation

5 Different dry mills Impeller mill IM450 Ball mill-activator AGO-2

6 Efficiency of milling on the Impeller mill IM450 Initial aspen chips Aspen after milling Aspen particles after milling (light microscopy)

7 Comparison of reaction ability of different pretreated lignocellulosic feed-stocks Relative glucose yield, % Before Prt After Prt Bagasse / steam explosion 2. Wheat straw / steam explosion 3. Oat hulls / steam explosion 4. Rice hulls / steam explosion 5. Hard wood / dry milling 6. Soft wood / steam explosion 7. Soft wood / organosolv 8. High kappa cellulosic wood pulp Saccharification by cellulases, 10 FPU/g + 20 bgu/g, ph 5, 50ºC, 24 h

8 Novel strains producers of cellulases, hemicellulases and related enzymes available in INBI-MSU-Fermtech Mutants of Penicillium sp.: Strain B Strain B221-6 Mutants of Trichoderma sp.: Strain TW-1 Strain TW-307

9 Selection of unique cellulolytic enzymes: hydrolysis of steam-exploded Douglas Fir by different cellulases Penicillium enzymes 70.0 Conversion, % Trichoderma enzymes TR2 UBC1 UBC2 TR Time (hrs) 10 FPU per 1 g of substrate, [S] = 5%, рн 5, 50 о С Sinitsyn et al, Appl.Biochem.Biotechnol., 2005, v , p.219

10 Glucose yield by action of individual enzymes Pretreated Corn Cobs, ph5, 50ºС, 72 hours. Total protein concentration 0.2 mg/ml, cellobiase 0.1 U/ml Controls: Spezyme CP, Multifect XL, CP + XL T r C B H I 6 6 T r C B H I 5 7 T r C B H II 6 0 T r C B H II 5 0 T r E G I h m T r E G I lm T r E G II h m T r E G II lm T r X y l II 2 1 A.j. B G L P v C B H I 6 6 (A ) P v C B H I 5 5 P v C B H II 6 0 P v E G I 7 0 P v E G I 5 2 P v E G II 3 9 P v E G III 2 5 P v B G L P v X y l 2 3 S p e z y m e 0,1 g /l M u ltife c t 0,1 g /l S 0,0 7 g /l + M 0,0 2 5 g /l B a c k g ro u n d s u b s tra te Trichoderma individual enzymes Penicillium individual enzymes G lu c o s e ( g /L

11 Penicillium sp. strain producer of cellulase complex with remarkably better saccharification ability than Trichoderma sp. Possible driving forces of Penicillium product: High specific activity of different cellulases Diverse and efficient enzymatic cocktail Production of β-glucosidase (cellobiase) Less inhibition by lignin and lignin residue Less unproductive adsorption of cellulose

12 Results of classical mutagenesis of Penicillium verruculosum strain (х5) wild type strain

13 Strategy for creation of recombinant strain producers of enzymes for saccharification of lignocellulosic feedstocks. GENETIC ENGINEERING APPROACH Penicillium 537 NiaD - (host strain) CBH-promoter Hist-promoter Target enzyme(s) producer Basal cellulase complex + target enzyme(s) producer

14 Example of high level expression of single target enzyme by recombinant Penicillium strain using cbh1 promoter (heterologous β-glucosidase) Penicillium 537 host strain Penicillium strain producer of β-glucosidase (CBH-promoter) CBH 1 35% CBH 2 25% Eg-s 1% CBH 2 4% Xyl 1% CBH 1 14% Other proteins 19% bgluc 1% Xyl 3% Eg-s 15% bgluc 80%

15 Results of saccharification of milled softwood [S] = 10%, рн 5, 50 о С Penicillium Penicillium β-glucosidase β-glucosidase Penicillium 537 [RS], g/l 10 0 β-glucosidase Hours

16 Cloning & expression of different genes using cbh1 promoter Strain Gene Enzyme T.reesei and P.verruculosum T.reesei and P.verruculosum cbh1 cbh2 egi1 egi2 Cellobiohydrolase 1 Cellobiohydrolase 2 Endoglucanase 1 Endoglucanase 2 T. reesei egi4 Endoglucanase 4 P. canescens xyla Xylanase А T. reesei xyliii Xylanase 3 T. reesei manb Mannanase В

17 g/l Dosage dependence (in mg of protein per 1 g of substrate) on reducing sugars yield in the course of saccharification of dry milled hardwood by recombinant enzyme preparations ([S]= 100 g/l, ph5, 50 C) g/l 2 mg/g 5 mg/g Time, hours Time, hours g/l 10 mg/g Time, hours

18 Results of saccharification of dry milled bagasse by recombinant enzyme preparations (cbh1 promoter) RS release in the course of saccharification of milled bagasse by XylА-4, EgIV-2 and reference enzyme preparations RS, g/l [E]= 5 mg/g, [S]= 100 g/l, ph 5, 50 С Time, hours

19 Composition of the most efficient recombinant enzyme preparations obtained using cbh1 promoter КсилА-4 XylА-4 КсилА рек; 18% другие белки; 19% Other-19% XylArec-18% CBH1-35% ЦБГ I; 35% Ксил; 6% КГ; 3% bглю; 4% Other-13% Egs-10% ЭГ; EgIV-2 ЦБГ I; CBH1-32% Ксил; 2% КГ; 2% bглю; 2% CBH1-12% ЦБГ II; 12% ЭГ; Egs-10% Eg4rec-14% ЭГIV рек; 15% ЦБГ II; CBH1-28% Ксил; 4% КГ; 2% bглю; 4% ЭГ; Egs-16% Other-10% CBH1-32% ЦБГ I; 35% Control CBH1-34% ЦБГ II;

20 Cloning of different genes using hist4 promoter Strain Gene Enzyme A.niger bgl (AN: AF302657) β-glucosidase P.canescens xyla (AN: AY756109) Xylanase А P.verruculosum egi2 Endoglucanase 2

21 Results of screening of recombinant enzyme preparations obtained by hist4 promoter by saccharification ability, dry milled hardwood and bagasse [E]= 5 мg/g, [S]= 100 g/l, ph 5, 50 0 С, 24 hours ВС Глюкоза ВС Глюкоза 0 P.verruculosum CV12 F10 P.verruculosum Ксибетен-Целл Spezyme CP Celluclast 1.5L Accellerase1000 CellicCtec1 P.verruculosum CV12 F10 P.verruculosum Ксибетен-Целл Spezyme CP Celluclast 1.5L Accellerase1000 CellicCtec1 Концентрация сахаров, г/л Концентрация сахаров, г/л g/l RS Gluc g/l RS Gluc Hist-βGl Dry milled hardwood Dry milled bagasse Hist-βGl

22 Composition of the most efficient recombinant enzyme preparation obtained using hist4 promoter (heterologous β-glucosidase) Penicillium 537 host strain Penicillium strain producer of cellulase complex enriched by β-glucosidase (hist-promoter) CBH 1 35% CBH 2 25% CBH 1 34% CBH 2 26% Other proteins 19% bgluc 1% Xyl 3% Eg-s 15% Other proteins 7% bgluc 13% Xyl 5% Eg-s 15%

23 Creation of recombinant strain producers of efficient enzyme complex for saccharification of lignocellulosic feedstocks by simultaneous expression of several target genes Penicillium 537 NiaD - (host strain) CBH-promoter Hist-promoter Basal cellulase complex + number of expressed target enzymes (e.g. EG-s + CBH-s + β-glucosidase, etc.)

24 Results of hydrolysis of dry milled bagasse by enzyme preparations obtained by recombinant Penicillium strains after simultaneous expression of different target enzymes (EG-s, CBH-s, bg) Control, Penicillium 537 Different transformants, obtained after simultaneous expression С, g/l С(RS) C(Glc) 10 0 # # # # # # # # # # # # mg/g of protein loading, [S] = 10%, рн 5, 50 о С

25 Thank you! +7 (495) (916)