Genomic and proteomic approaches to explore the diversity of cellwall-degrading enzymes

Transcription

1 Genomic and proteomic approaches to explore the diversity of cellwall-degrading enzymes Rendez-Vous Protéomique 2009 April 16, 2009 Adrian Tsang Concordia University

2 The future Graphics obtained from U.S. Department of Energy Genomics program,

3 Back to the present Fungi and bacteria Graphics obtained from Genomics:GTL Roadmap, U.S. Department of Energy Office of Science, August 2005,

4 Some of the general challenges to be considered for technology development Feedstock food/feed vs fuel Greenhouse gas emissions Energy balance Water consumption Public acceptance The list goes on

5 This presentation 1. The lignocellulose (woody biomass, plant cell wall) promise 2. The lignocellulose challenge Bioconversion technology 3. The role for genomic science 4. Examples of cellulosic bioprocesses

6 Reduction in greenhouse gas (GHG) emissions of biofuels as compared to gasoline Corn ethanol 44 Sugarcane ethanol 32 Oilseed biodiesel Lignocellulosic ethanol* GHG emissions compared to gasoline (%) Source: Green dreams, October 2007, National Geographic; *projection

7 Energy balance of fuel ethanol and biodiesel 1.3 Corn ethanol 2.5 Oilseed biodiesel 8 Sugarcane ethanol 2 36 Lignocellulosic ethanol* Units of energy output from one unit of input Source: Green dreams, October 2007, National Geographic; *projection

8 Feedstock heterogeneity Hemicellulose 21% Pectin 1% Extractives Cellulose 5% Pectin 48% 45% 4.5% Extractives 8% Ash 10% Cellulose 26% Hardwood stem Lignin 22% Hemicellulose 22.5% Corn stover Lignin 18% Pectin 3% Extractives 13% Ash 10% Cellulose 33% Wheat straw Hemicellulose l Lignin i 23% 17%

9 Conversion of lignocellulose to fuels and products Feedstock Pretreatment Enzyme hydrolysis Fermentation Fuels and products

10 Enzyme cost Corn starch Fuel ethanol glucose 1 litre Straw Source: US DOE National Renewable Energy Laboratory ; *economic viability, $0.04

11 Lignocellulose-active proteins Cellulose Endoglucanase, exoglucanase, beta-glucosidase Sowellenin, expansin Lignin Laccase lignin peroxidase manganese peroxidase Lignocellulose Pectin Hemicellulose Xylan endoxylanase, xylosidase, acetyl- xylanesterase, feruloyl esterase, alpha-glucuronidase, Mannan mannanase, mannosidase Arabinan arabinofuranosidase, arabinase,

12 Source of biomass-degrading enzymes microbial extracellular proteins Aerobic environment (fungi) Soluble, secreted proteins Anaerobic environment (bacteria & fungi) Cellulosome Graphics obtained from U.S. Department of Energy Genomics program,

13 Fungal genomes to industrial applications a vertical approach Sequence analysis (~30 fungal genomes) Transcriptome analysis Secreted proteins (computational & analytical) Recombinant proteins Biochemical characterization Applications testing

14 Computational prediction of fungal secreted proteins Protein models of fungal genomes Phobius SignalP v3 Signal peptide Signal/membrane TMHMM TargetP Mitochondria Membrane ER/Golgi 19% GH proteins Oh Other biomass- 12% degrading enzymes 14% xkdel Endoplasmic reticulum Computational secretome (3-11% of genome) Analysis pipeline for prediction of secreted proteins Unknowns 54% Others 1% Manual annotation of predicted secreted proteins from 15 fungi

15 Verified Aspergillus niger secretome 2D-LC-MS/MS Secreted proteins Trypsin digestion Analytical secretome Computational secretome Verified secretome ER/Golgi 19% GH proteins 12% Other biomassdegrading enzymes 14% Others 15% Unknowns 15% GH proteins 40% Unknowns 54% Others 1% Other biomassdegrading enzymes 30% Manual annotation of computational secretome Verified secretome of Aspergillus niger cultured on complex carbohydrates

16 A. niger cellulases induced by feedstock umber of predic cted enzymes n detected undetected related to extracellular endo related to extracellular l putative cellulase related to endoglucanase candidate endoglucanase endoglucanase C endoglucanase B endoglucanase A endoglucanase Barley triticale hemp flax canola alfalfa hypothetical beta glucosidase beta glucosidase exoglucanase hypothetical beta glucosidase Barley cellobiohydrolase cbha Barley related to beta glucosidase related to beta glucosidase beta glucosidase bgla triticale hemp flax cellobiohydrolase cbhb candidate cellobiohydrolase triticale hemp flax hypothetical beta glucosidase canola related to cellobiohydrolase canola hypothetical beta glucosidase alfalfa alfalfa

17 A. niger pectinases induced by feedstock detected undetected exo polygalacturonase C exopolygalacturonase X exo polygalacturonase B endo polygalacturonase I endo polygalacturonase C xylogalacturonase A Barley triticale hemp num mber of predicte ed enzymes hypothetical exo rhamnogalacturonase pectin lyase C hypothetical pectinesterase pectinesterase A flax canola alfalfa related to pectin methyl esterase rhamnogalacturonan lyase candidate rhamnogalcturonan lyase pectin lyase A

18 Conclusions from mass spectrometric analysis Only a fraction of biomass-degrading enzymes are induced by agricultural feedstocks Similar set of enzymes induced by different feedstocks Esterases, enzymes that cleave the linkages between polymers, appear to be selectively induced It i l if th i d d fl t (1) th i It is unclear if the enzymes induced reflect (1) their effectiveness in hydrolyzing biomass or (2) gene regulation in response to feedstock

19 Plans for producing recombinant biomassdegrading enzymes Aspergillus niger 49 GH families 239 GH proteins Characterization of unknowns Express all genes predicted to encode biomassdegrading enzymes Guide the development of enzyme cocktails 2D-LC-MS/MS Genome annotation (gene model correction)

20 Clone and express lignocellulolytic enzymes ~4,200 genes and ~2,000 enzymes 96-well cultures A. niger transformants Characterization Application Hybrid enzymes Expression screening Activity screening

21 Characterization of unknown extracellular proteins - ~1,400 unknown genes and 500 enzymes Recombinant hypothetical, secreted proteins are screened for activity it on 36 natural and synthetic ti substrates. t s endoglucanase 20 exoglucanase 11 endoglucanase xylanase 4 esterase-lipase 3 arabinase 2 protease 5 chitinase 5 pectinase 15 phosphatase 2

22 Application transgenic crops and fermentation organisms to further reduce enzyme cost Novel fungal enzymes Transformation into tobacco Transformation into alfalfa Transformation into fermentation microbes

23 Remove chemicals in bleaching and reduce energy in pulping p Canadian pulp & paper industry: World s largest exporter $32B revenue 90,000 jobs 30 M tonnes / year Wood Unbleached Bleached pulp 25-30% lignin pulp <0.5% lignin Five-stage pulp bleaching ClO 2 NaOH + O 2 ClO 2 NaOH + H 2 O 2 ClO 2 Data source: Natural Resources Canada (2007) Canada s Forests- annual report

24 Improve lignocellulose digestibility in cattle feed Cattle industry in Canada $8B revenue 10% 2.3 M tonnes 3.0 M tonnes Lignocellulose degradation in = + forages Solid manure Grain consumption Data for the United States: t Hatfield, R.D., Ralph, J., and Grabber, J.H. (1999) Cell wall structural t foundations: Molecular basis for improving forage digestibilities. Crop Sci. 39:27-37

25 Concluding remarks Development of lignocellulosic fuels and products does not require breakthrough technology, but needs efficient processes Ethanol and other fuels are simply a few of the products that can be generated from lignocellulose Genomic science plays an important role in many Genomic science plays an important role in many aspects of products development

26 Partners and collaborators Greg Butler Justin Powlowski Reginald Storms Tricia John Annie Bellemare Tieling Zhang Nadeeza Ishmael Kimberly Bull Nick O Toole Manoj Kuma Rutger van Rooijen Rob Meima Anja Reimens Lisette Mohrmann Alard van Dijk Ilja Westerlaken Diana Gutker Serge Laberge Tim McAllister Margie Gruber André Larouche Dan Brown Rima Menassa Michael Paice Robert Bourbonnais Sylvie Renaud Vineet Dua Xiao Zhang David Nguyen Scott Baker Ellen Panisko Jon Magnuson Sylvie LaBoissiere Ken Dewar Marcos Di Falco Line Roy Gary Leveque Theresa White John Tomashek