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PLuginbuhl-SIM-2008

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PLuginbuhl-SIM-2008

  1. 1. From Bugs to Biofuels The Termite Hindgut Metagenome Peter Luginbühl, Ph.D. Verenium Corporation
  2. 2. 2 Biomass to Ethanol Conversion ETHANOL Corn STARCH CELLULOSIC BIOMASS Corn StoverWood Chips Bagasse Switchgrass Enzymes enable the economic conversion of starch and pretreated cellulosic biomass into mixed sugars for production of biofuels a-amylase glucoamylase pretreatment LC enzyme cocktail
  3. 3. 3 Cellulosic Biomass Structure Plant cell wall structure Cellulose (glucose) 38 - 50% Hemicellulose (C5 and C6) 23 - 32% Lignin (phenolics) 15 - 25% Compositional variability
  4. 4. 4 Biomass Degradation Programs Integrated Corn Biorefinery New Zealand Biofuels Bagasse Conversion Cellulosic Enzymes from Termite Guts Different wood pulps digested with Verenium’s LC enzyme cocktail
  5. 5. 5 Cellulosic Ethanol Process 1.4 MGY Demonstration Plant Jennings, Louisiana
  6. 6. 6 Cellulose Degradation Endo-cellulase (endoglucanase) Attacks amorphous, non-crystalline regions of the chain producing oligosaccharides Exo-cellulase (cellobiohydrolase) Attacks chain ends producing cellobiose β-glucosidase Attacks oligosaccharides and cellobiose producing glucose
  7. 7. 7 Cellulase Sources • Trichoderma reesei – Discovered in the 1940’s by Elwyn Reese – “Jungle Rot” – Six decades of strain development – Culture broth containing complex mixture of many proteins – Not optimized to feedstock • Verenium – Unrivaled access to environments that contain biomass degrading enzymes – bioprospecting, biotraps – HT functional screens and metagenomics to identify key enzymes – No limitation to enzyme source (fungal or bacterial) – Large collection of active plant cell wall degrading enzymes – Optimize enzymes to high performance – GSSM™, Gene Reassembly™ – Ability to tailor enzyme cocktails to feedstock
  8. 8. 8 Xylanases from Insect Guts • Unusual microbial xylanases from insect guts – Brennan et al., Appl. Environ. Microbiol. 70, 3609-3617 (2004) Termite Moth Caterpillar Beechwood Xylan Wheat Arabinoxylan
  9. 9. 9 Termite Hindgut Metagenome • Termites – an extremely successful group of wood-degrading organisms • Metabolic capabilities of microbes living symbiotically in their hindguts • Important for their roles in carbon turnover in the environment – 2% of global CO2 emissions and 4% of global CH4 emissions (Sugimoto et al., 2000) • Potential source of biocatalysts for cellulosic biomass degradation • Metagenomic analysis of the microbial community in the hindgut of a ‘higher’ Nasutitermes species – Warneke et al., Nature 450, 560-565 (2007)
  10. 10. 10 Workflow and Strategy Sampling Hindgut section P3 DNA extraction - Verenium 3, 8 and 40 kb libraries - Verenium Sequencing (100 Mbp) - JGI Assembly - JGI Metabolic reconstruction Dissection COII phylogeny JGI Activity screening Verenium 16S rRNA survey (30 Mbp) JGI Microbial community structure Host IDHost morphology Transfer arrayed clones Enzyme activity testing Verenium JGI, Caltech, Verenium
  11. 11. 11 Termite Collection Morning of 24 May 2005 Secondary forest of Bosque Lluvioso (INBio private reserve) near the town of Guápiles, Costa Rica
  12. 12. 12 Host Identification • Mitochondrial cytochrome oxidase II gene – A nucleotide-based dendrogram using 661 aligned and filtered positions – B amino acid-based dendrogram using 228 aligned and filtered positions • Termite specimens were assigned to Nasutitermes sp.
  13. 13. 13 1 mm P2“Mixed Segment” 3rd Proctodeal Segment DNA Extraction
  14. 14. 14 Microbial Community Structure Leptospiraceae Phylum Spirochaetes [109] other Spirochaetaceae Phylum Fibrobacteres [49] Termite Treponema group 1 [23]  TTG-2 [8]  TTG-3 [1] TTG-4 [7]  TTG-5 [6] TTG-6 [2] TTG-7 [9]  TTG-8 [2] TTG-9 [5] TTG-10 [39]  T. primitia termite group [1] Spirochaeta (Treponema) caldaria, M71240.1 T. pallidum & amino acid fermentors T. bryantii & saccharolytic strains Spirochaeta coccoides SPN1, AJ698092.1 TSG-1 [1] Spirochaeta aurantia, AY599019.1 TSG-2 [3] TSG-3 [1] TLG-1 [1] Leptospira Fibrobacter Candidate order TFG-1 [14]  Candidate order TG3-1 [32]  Candidate order TG3-2 [3] Bacteroidetes [14]  Chlorobi & gut clones [1] Cyanobacteria & gut clones [2] Candidate phylum ZB3 [3] Proteobacteria [9]  Acidobacteria [10]  Candidate phylum Endomicrobia [1] Firmicutes [16] Deferribacteres [1] Aminanaerobia [1] 0.10 Treponema • Phylogenetic diversity of the P3 luminal microbiota • 1,703 16S rRNA sequences from a PCR-based inventory and from the metagenome • 216 phylotypes (99% sequence identity threshold) were identified – Treponema: 103 – Fibrobacteres: 49 • Red dot: at least one of these phylotypes was represented in the metagenome • White shading: reference groups not represented in termite gut derived inventories
  15. 15. 15 Termite Hindgut Metagenome • Two shotgun libraries from the P3 genomic DNA were prepared • 2–4 kb small insert library – 92,160 reads comprising 68.47 Mb • 32 kb insert fosmid library – 13,824 reads comprising 2.91 Mb • Assembled metagenome – 41,765 contigs covering 44.4 Mb – Longest contig was 14.7 kb and contained 72 reads – 25% of the reads remained as singlets – 72,926 ORFs – Over 700 glycoside hydrolase (GH) catalytic domains from 45 different sequence families – Rich diversity of putative cellulases and hemicellulases
  16. 16. 16 Glycoside Hydrolase Inventory termite gut community human gut community soil metagenome cellulolytic bacteria Family Known Acitvities (CAZy) TGC HGC-7 HGC-8 SM Fs Sd Ch Tf Cs Ct Ac Bl Glycoside Hydrolases GH1 b-glucosidase, b-galactosidase, b-mannosidase, others 22 20 23 9 0 2 1 2 1 2 1 0 GH2 b-galactosidase, b-mannosidase, others 23 13 17 7 1 6 0 0 3 1 0 1 GH3 b-1,4-glucosidase, b-1,4-xylosidase, b-1,3-glucosidase, a-L- arabinofuranosidase, others 69 20 23 44 2 6 6 2 2 2 3 2 GH4 a-glucosidase, a-galactosidase, a-glucuronidase, others 14 4 5 7 0 0 0 1 2 0 0 0 GH5 cellulase, b-1,4-endoglucanase, b-1,3-glucosidase, b-1,4- endoxylanase, b-1,4-endomannanase, others 56 1 1 2 11 17 4 3 5 10 2 0 GH6 endoglucanase, cellobiohydrolase 0 0 0 3 0 1 0 2 0 0 2 0 GH8 cellulase, b-1,3-glucosidase, b-1,4-endoxylanase, b-1,4- endomannanase, others 5 0 1 1 6 0 5 0 0 1 0 0 GH9 endoglucanase, cellobiohydrolase, b-glucosidase 9 0 0 2 8 3 6 2 2 16 2 0 GH10 xylanase, b-1,3-endoxylanase 46 0 2 11 7 5 3 2 5 5 2 0 GH11 xylanase 14 0 0 0 4 2 1 1 0 1 0 0 GH12 endoglucanase, b-1,3-1,4-glucanase, xyloglucan hydrolase 0 0 0 1 0 0 0 0 0 0 2 0 GH13 a-amylase, catalytic domain, and related enzymes 48 27 45 42 3 10 4 6 4 2 7 8 GH15 glucoamylase, glucodextranase 0 0 0 17 0 1 1 2 1 1 1 0 GH16 b-1,3(4)-endoglucanase, others 1 0 1 8 4 8 2 0 1 2 0 0 GH26 b-1,3-xylanase, mannanase 15 0 1 0 4 1 1 0 1 2 0 0 GH31 a-glucosidase, a-xylosidase, others 26 9 14 11 0 1 1 1 1 0 0 2 GH38 a-mannosidase 11 2 2 6 0 0 0 0 0 0 0 3 GH43 xylanase, b-xylosidase, a-L-arabinofuranosidase, arabinanase, others 16 4 4 5 13 13 2 1 4 4 0 7 GH45 endoglucanase (mainly eukaryotic, 2 bacterial) 4 0 0 0 4 0 0 0 0 0 0 0 GH48 endoglucanase, cellobiohydrolase 0 0 0 0 0 0 0 1 1 2 1 0 GH57 a-amylase, 4-a-glucanotransferase, a-galactosidase, others 17 0 0 3 3 0 1 0 0 0 0 0 Carbohydrate-binding modules CBM2 cellulose-binding domain 0 0 2 3 0 19 0 14 0 0 9 0 CBM3 cellulose-binding domain 0 0 0 1 0 0 0 2 10 19 9 0 CBM4 amorphous cellulose-, xylan- and glucan-binding domain 5 0 4 0 4 5 2 1 9 12 4 2 CBM6 amorphous cellulose- and xylan-binding domain 13 0 1 2 32 45 2 1 1 17 1 1 CBM32 galactose- and lactose-binding domain 4 10 23 12 1 26 1 1 1 1 0 6
  17. 17. 17 Proteomics and Activity Family Known Activities (CAZy) Termite Gut Community Source Organisms Proteomics Activity GH1 b-glucosidase, b-galactosidase, b-mannosidase, others 22 5 treponema 1 GH3 b-1,4-glucosidase, b-1,4-xylosidase, b-1,3-glucosidase, a-L- arabinofuranosidase, others 69 14 treponema 1 GH4 a-glucosidase, a-galactosidase, a-glucuronidase, others 14 3 treponema 2 GH5 cellulase, b-1,4-endoglucanase, b-1,3-glucosidase, b-1,4- endoxylanase, b-1,4-endomannanase, others 56 6 treponema 2 26 of 30 GH9 endoglucanase, cellobiohydrolase, b-glucosidase 9 2 fibrobacter 7 of 12 GH10 xylanase, b-1,3-endoxylanase 46 11 treponema 1 GH13 a-amylase, catalytic domain, and related enzymes 48 6 treponema 2 GH42 b-galactosidase 24 4 treponema 1 GH45 endoglucanase (mainly eukaryotic, 2 bacterial) 4 1 fibrobacter 2 of 2 • Source organisms – Fragments larger than 1kb were binned using PhyloPhytia, a phylogenetic classifier for the composition-based assignment of fragments at different taxonomic ranks • Proteomics – 3D LC-MS/MS data were searched against the termite hindgut metagenome database using SEQUEST • Activity – Genes were retrieved in full length, then subcloned and expressed in E.coli – Activity assays were conducted with phosphoric acid swollen cellulose (PASC) and microcrystalline cellulose (Avicel)
  18. 18. 18 Novel Cellulases GH5 GH9
  19. 19. 19 Novel Hemicellulases GH10 GH11
  20. 20. 20 Conclusions • Nasutitermes hindguts are dominated by spirochetes (treponema) and fibrobacters • Nasutitermes hindgut symbionts encode a rich diversity of wood- degrading enzymes – Novel cellulases and hemicellulases in metagenome – Glycoside hydrolases are expressed and secreted as monitored by proteomics experiments – Recombinantly expressed cellulases are active on model substrates and perform well in combinatorial enzyme cocktails on various feedstocks • Gut symbionts for DOE’s Bio Energy Science Center (BESC)
  21. 21. 21 Acknowledgements

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