Genomic and proteomic approaches to explore the diversity of cellwall-degrading
enzymes
Rendez-Vous Protéomique 2009
April 16, 2009Adrian TsangAdrian Tsang
Concordia University
The futureThe future
Graphics obtained from U.S. Department of Energy Genomics program, http://genomics.energy.gov
Back to the present Back to the present
Fungi and bacteria
Graphics obtained from Genomics:GTL Roadmap, U.S. Department of Energy Office of Science, August 2005, http://genomicsgtl.energy.gov/roadmap/
Some of the general challenges to be considered for technology developmentSome of the general challenges to be considered for technology development
Feedstock – food/feed vs fuel Feedstock food/feed vs fuel Greenhouse gas emissions Energy balance Energy balance Water consumption P bli t Public acceptance The list goes on …
This presentationThis presentation
1. The lignocellulose (woody biomass, plant cell wall) promise p
2. The lignocellulose challenge Bioconversion technology y
3. The role for genomic science4. Examples of cellulosic bioprocesses
Reduction in greenhouse gas (GHG) emissions of biofuels as compared to gasoline
100
Corn ethanol78
Sugarcane ethanol44
Oilseed biodiesel
9
32
Lignocellulosic ethanol*9
0 20 40 60 80 100
GHG emissions compared to gasoline (%)
Source: “Green dreams”, October 2007, National Geographic; *projection
Energy balance of fuel ethanol and biodiesel
1.3Corn ethanol
2.5 Oilseed biodiesel
8 Sugarcane ethanol
36Lignocellulosic ethanol*
2
0 10 20 30 40
Units of energy output from
g
Units of energy output from one unit of input
Source: “Green dreams”, October 2007, National Geographic; *projection
Feedstock heterogeneityFeedstock heterogeneity
Cellulose48%
Pectin 1%
Extractives5% Pectin
4 5%
Ash 10%48%
Hemicellulose21%
Cellulose26%
4.5%Extractives
8%
Lignin 22%
Hemicellulose22.5%
Lignin 18%
Hardwood stemCorn stover
Cellulose
Pectin 3%
Extractives
Ash 10%
Corn stover
Cellulose33%
H i ll l Li i
Extractives13%
Wheat strawHemicellulose
23%Lignin 17%
Conversion of lignocellulose to fuels and products
Feedstock
Pretreatment
Enzyme hydrolysis
Fermentation
Fuels and products
Enzyme cost
Corn starch
Fuel ethanol
l 1 litreglucose
Straw
Source: US DOE National Renewable Energy Laboratory ; *economic viability, $0.04
Lignocellulose-active proteinsLignocellulose-active proteins
Cellulose
Cellulose• Endoglucanase, exoglucanase, beta-glucosidase
• Sowellenin, expansin
Lignin Laccaselignin peroxidasemanganese peroxidase Lignocellulose
HemicelluloseXylan –endoxylanase, xylosidase, acetyl-
Pectin y y y
xylanesterase, feruloyl esterase, alpha-glucuronidase,Mannan – mannanase, mannosidaseArabinan – arabinofuranosidase, arabinase arabinase,
Source of biomass-degrading enzymes –microbial extracellular proteins
Aerobic environment (fungi)
Soluble, secreted proteins
Anaerobic environment (bacteria & fungi)( g )
Cellulosome
Graphics obtained from U.S. Department of Energy Genomics program, http://genomics.energy.gov
Fungal genomes to industrial applications –a vertical approach
Sequence analysis (~30 fungal genomes)
Transcriptomeanalysis
Secreted proteins (computational & analytical)(computational & analytical)
R bi t t iRecombinant proteins
Biochemical characterization
Applications testing
Computational prediction of fungal secreted proteins
Protein models of fungal genomes
Si lP 3Phobius
SignalP v3
TMHMM
Signal peptide
M b
Signal/membrane
GH i O h bi
TargetP
Membrane
Mitochondria
ER/Golgi19%
GH proteins12%
Other biomass-degrading enzymes
14%
xKDEL
Endoplasmic reticulum
U k s
Others1%
Computational secretome
(3-11% of genome)
l l f d
Manual annotation of predicted secreted proteins from 15 fungi
Unknowns54%
Analysis pipeline for prediction of secreted proteins
secreted proteins from 15 fungi
Verified Aspergillus niger secretomeVerified Aspergillus niger secretome2D-LC-MS/MS
Trypsindigestion
Secreted proteins
Analytical secretome
Verified Verified secretome
Computational secretome
ER/Golgi19%
GH proteins12%
Other biomass-degrading enzymes
14%GH proteins
40%
Unknowns15%
Others
Others%
15%
Verified secretome of l f
Unknowns54%
1% Other biomass-degrading enzymes
30%
Verified secretome of Aspergillus niger cultured on complex carbohydrates
Manual annotation of computational secretome
A. niger cellulases induced by feedstockA. niger cellulases induced by feedstock
l d ll l
related to extracellular endo‐…
endoglucanase
8
10
12
14
cted
enzym
es detectedundetected
candidate endoglucanase
related to endoglucanase
putative cellulase
related to extracellular …Barley
triticale
hemp
flax
0
2
4
6
umbe
r of predic
0 1 2 3 4
endoglucanase A
endoglucanase B
endoglucanase Cflax
canola
alfalfa
n
0 1 2 3 4
cellobiohydrolase cbhA
exoglucanase
Barleyhypothetical beta‐glucosidase
hypothetical beta‐glucosidase
beta‐glucosidase
Barley
related to cellobiohydrolase
candidate cellobiohydrolase
cellobiohydrolase cbhBtriticale
hemp
flax
canolahypothetical beta‐glucosidase
beta‐glucosidase bglA
related to beta‐glucosidase
related to beta‐glucosidase triticale
hemp
flax
canola
0 2 4 6 8 10 12 alfalfa
0 2 4 6 8
hypothetical beta‐glucosidase alfalfa
A. niger pectinases induced by feedstockA. niger pectinases induced by feedstockdetected undetected
6
8
ed enzym
es
detected undetected
exopolygalacturonase X
exo‐polygalacturonase C
0
2
4
mbe
r of predicte
endo‐polygalacturonase C
endo‐polygalacturonase I
exo‐polygalacturonase B
p yg
Barley
triticale
num
pectin lyase C
hypothetical exo‐rhamnogalacturonase
xylogalacturonase A hemp
flax
l
related to pectin methyl esterase
pectinesterase A
hypothetical pectinesterasecanola
alfalfa
pectin lyase A
candidate rhamnogalcturonan lyase
rhamnogalacturonan lyase
0 2 4 6 8 10 12 14 16
Conclusions from mass spectrometric analysis
Only a fraction of biomass degrading enzymes are induced 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
Plans for producing recombinant biomass-degrading enzymes
Express all genes predicted to encode biomass
Aspergillus niger49 GH families
to encode biomass-degrading enzymes
239 GH proteins
Characterization Guide the development of unknowns of enzyme cocktails
2D-LC-MS/MS
Genome annotation( d l ti )(gene model correction)
Clone and express lignocellulolytic enzymes – ~4,200 genes and ~2,000 enzymes g y
96-well cultures
A. niger transformants
• Characterization Expression screening
Activity screening
• Application• Hybrid enzymes
p g
Activity screening
Characterization of unknown extracellular proteins - ~1,400 unknown genes and 500 enzymes
Recombinant hypothetical, secreted proteins are screened f ti it n 36 n t l nd s nth ti s bst t s
larabinase
2
for activity on 36 natural and synthetic substrates.
protease5
esterase-lipase3exoglucanase
11
chitinase5
2
endoglucanaseendoglucanase
20
phosphatase2
5
xylanase4
pectinase15
Application – transgenic crops and fermentation organisms to further reduce
Application – transgenic crops and fermentation organisms to further reduce
Novel fungal
enzymes
enzyme costenzyme cost
Transformation Transformation into tobacco
Transformation
Transformation into alfalfa
f minto fermentation
microbes
Remove chemicals in bleaching and reduce energy in pulpinggy p p g
Canadian pulp & paper industry:• World’s largest exporter
d l h d lbl h d
• World s largest exporter• $32B revenue• 90,000 jobs
Wood 25-30% lignin
Bleached pulp<0.5% lignin
Unbleached pulp
• 30 M tonnes / year
Five-stage pulp bleaching
ClO2 NaOH + O2 ClO2 NaOH + H2O2 ClO2
Data source: Natural Resources Canada (2007) Canada’s Forests- annual report 2007 http://foretscanada.rncan.gc.ca/rpt
Improve lignocellulose digestibility in cattle feed
Cattle industry in Canada – $8B revenue
10%2.3 M
tonnes3.0 M
tonnes
Lignocellulosedegradation in = +
forages
Grain ti
Solid manure
D t f th U it d St t H tfi ld R D R l h J d G bb J H (1999) C ll ll t t l f d ti
consumption
Data for the United States: Hatfield, R.D., Ralph, J., and Grabber, J.H. (1999) Cell wall structural foundations: Molecular basis for improving forage digestibilities. Crop Sci. 39:27-37
Concluding remarks
Development of lignocellulosic fuels and products does 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
Partners and collaborators
• Manoj Kuma• Rutger van Rooijen• Rob Meima
• Serge Laberge• Tim McAllister• Margie Gruber
d é h
• Greg Butler• Justin Powlowski
R i ld St • Anja Reimens• Lisette Mohrmann• Alard van Dijk• Ilja Westerlaken
• André Larouche• Dan Brown• Rima Menassa
• Reginald Storms•Tricia John•Annie Bellemare•Tieling Zhang
N d I h lj
• Diana Gutker• Nadeeza Ishmael•Kimberly Bull• Nick O’Toole
• Scott Baker• Ellen Panisko• Jon Magnuson
• Sylvie LaBoissiere• Ken Dewar• Marcos Di Falco• Line Roy• Gary Levequeg
• Michael Paice • Robert Bourbonnais • Sylvie Renaud • Vineet Dua
Gary Leveque
• Theresa White• John Tomashek
• Xiao Zhang • David Nguyen