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