Improving degradation of
biomass by improving cell wall
digestibility
Felice Cervone
Dip. Biologia Vegetale – Università di Roma “Sapienza”
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SACCHARIFICATION
Soource: EPOBIO
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• Enzymatic hydrolysis is considered the most
promising, environmentally friendly technology
available for biorefining
A bottleneck for the industrial scale-up of this process is
recalcitrance of cell walls to enzymatic hydrolysis due to:
- heterogeneity and complexity of cell wall
structural components
- presence of inhibitors of microbial enzymes
- degree of lignification
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Cell wall degradability may be improved by:
- lowering lignin composition (though lignin is required for
mechanical strength)
- increasing levels of hexose- compared to pentosecontaining polymers
- weakening cross-linkages between wall components such
as hemicelluloses, lignin and cellulose
- altering the levels of polymer modifications, such as
esterification, to promote enzyme accessibility and
digestibility
-altering those components that act as a “glue” of cell
walls, i.e. pectin and hemicellulose in middle lamellae
and primary cell walls
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WALL-DECO: Deconstructing cell walls to
improve the processing of biomass from crops
• identification of crop natural accessions with high cell
wall degradability
• isolation of genetic loci involved in cell wall degradability
• isolation of novel CWDEs from plant, bacterial and
fungal sources for improved cell wall degradation
• generation of tailor-made CWDEs and inhibitors for
improved cell wall degradation
• generation and characterization of “self-deconstructing
plants”
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Expression of cell wall-degrading enzymes (CWDEs) to
improve degradability of plant biomass
Selection of
microbial strains
Characterization
Of CWDEs
Expression of
CWDEs in plants
Increased
biomass degradation
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Mechanisms to control activity of CWDEs in planta
• Inducible promoters (e.g. heat, alcohol-activated
promoters)
• Targeted mutagenesis to alter enzyme activity
. Use of specific inhibitors
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Plant Cell Structure
middle
lamella
Pectin (10-35%)
primary
cell wall
Cellulose (9-25%)
plasma
membrane
Hemicellulose (25-50%)
50 nm
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Pectin
Pectins are a group of polysaccharides in the primary cell
walls and intercellular regions of higher plants
The key feature for the major pectic polysaccharides is
the presence of linear chain regions comprised of (1→4)linked-α-D-galactopyranosyluronic acid units.
COOCH3
O H
OH H
COOH
O
OH
O H
H
H
H
OH
O
H
H
OH
COOH
O
O H
OH H
H
H
OH
O
H
COOH
O H
OH H
H
OH
O
COOCH3
O H
OH H
O
H
H
OH
COOCH3
O H
OH H
H
H
OH
COOH
O
O H
OH H
H
H
O
OH
Homogalacturonan
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Polygalacturases (PG)
-PG is the first enzyme to be secreted by most
phytopathogenic microorganisms and is an important
pathogenicity factor.
-Its action on homogalacturonan of the plant cell wall
is a pre-requisite for the accessibility of substrate to
other CWDEs.
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The crystal structure of PG
from Fusarium moniliforme (1.7Å)
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H188
D212
D213
R267
D191
K269
PG
Federici et al. Proc Natl Acad Sci U S A 2001, 98:13425-30 .
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PG plants have dwarf phenotype
wt
#5
#7
#16
PG#16 x
PvPGIP2
PvPGIP2
Tabacco
#1
#5
Col-0
Arabidopsis
PG201
#4
#1
Ws-0
(Capodicasa et al. Plant Physiol. 2004)
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Defence genes are constitutively
expressed
Ws
PG plants
Pdf1.2
PR1
0,040
0,8
0,030
0,6
0,20
0,020
0,4
0,10
0,010
0,2
0,00
0,000
0,0
0,40
Arbitrary Units
AtPGIP1
0,30
Real-Time RT-PCR
.
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Polygalacturonase-inhibiting protein (PGIP),
a plant recognition protein for
non-self polygalacturonases, is a leucine-rich
repeat
(LRR) protein
Federici et al. 2003, PNAS 98: 13425-13430
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Predicted PGIP area of interaction
L89
V181
S207
Q253
H300
Q320
A326
A340
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Molecular docking of the PG-PGIP complex
PGIP
PG
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MOLECULAR DOCKING
PvPGIP2 and
A. niger
PG
F. moniliforme
PG
B. cinerea
PG
Sicilia et al, Plant Phys 2005; Federici et al.
Trends in Plant Science 2006
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Pectin MethylEsterases (PMEs)
•Plant cell wall proteins involved in remodeling
of plant cell wall during plant growth and
development
•Plant PMEs typically occur in multigene families
(∼60 PME-related genes in Arabidopsis).
•PMEs are also produced by phytopathogenic
fungi and bacteria
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Demethylation of pectins by PMEs
PMEs remove the methyl ester groups from
homogalacturonan producing methanol and stretches
of acidic residues
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PME
PMEI
• “Four Helix Bundle” of the
inhibitor
• 2 disulphide bridges necessary
to maintain fold
• 1:1 stoichiometry
• Inibitors bind active site of
PME preventing substrate
binding
Di Matteo et al., Plant Cell
2005
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Overexpression of AtPMEI-1 in A. thaliana
mRNA
WT
1-1
protein
1-5 1-43
Mw
Std
WT 1-1 1-5 1-43
AtPMEI
AtPMEI
21 kDa
18 kDa
14 kDa
UBQ5
PME actvity decreases
Degree of methylation increases
100
83,8%
80
52,3%
46,7%
60
40
20
0
WT
1-43
1-1
1-5
Degree of
Methylesterification (%)
P M E a ctiv ity (% )
120
70
60
a
a
WT
1-43
b
b
1-1
1-5
50
40
30
20
10
0
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Increased root length
WT
AtPMEI
Ca2+
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XIP
GH10 xylanase
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GH10 xylanase /XIP complex
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Acknowledgements
Università di Roma “La Sapienza”
Dip. Biologia Vegetale
Roberta Galletti
Vincenzo Lionetti
Fedra Francocci
Flavio Scaloni
Manuel Benedetti
S. Ferrari
D. Bellincampi
G. De Lorenzo
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