1. From Bugs to Biofuels
The Termite Hindgut Metagenome
Peter Luginbühl, Ph.D.
Verenium Corporation

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
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
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
Cellulosic Ethanol Process
1.4 MGY Demonstration Plant
Jennings, Louisiana

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
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
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
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
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
Termite Collection
Morning of 24 May 2005
Secondary forest of Bosque Lluvioso
(INBio private reserve) near the town
of Guápiles, Costa Rica

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
1 mm
P2“Mixed Segment”
3rd Proctodeal Segment
DNA Extraction

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
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
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
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
Novel Cellulases
GH5 GH9

19. 19
Novel Hemicellulases
GH10 GH11

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
Acknowledgements