improving degradation of biomass by improving cell wall ......5 • enzymatic hydrolysis is...
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Felice Cervone
Dip. Biologia Vegetale – Università di Roma “Sapienza”
Improving degradation of biomass by improving cell wall
digestibility
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SACCHARIFICATION
Soource: EPOBIO
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• Enzymatic hydrolysis is considered the most promising, environmentally friendly technology available for biorefiningA 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 pentose-containing 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) toimprove degradability of plant biomass
Selection of microbial strains
CharacterizationOf CWDEs
Expression of CWDEs in plants
Increasedbiomass 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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middlelamella
primarycell wall
plasmamembrane
Hemicellulose (25-50%)
Pectin (10-35%)
Cellulose (9-25%)
50 nm
Plant Cell Structure
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Pectin
Homogalacturonan
O HCOOCH3
H
O H
COOH
HO
O HCOOH
HO
O H
COOCH3
HO
O H
COOH
HO O
OH
H
H
OH
OH
H
H
OH
OH
H
H
OH
OH
H
H
OH
OH
H
H
OH
O
O HCOOH
H
O H
COOCH3
HO O
OH
H
H
OH
OH
H
H
OH
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.
Pectins are a group of polysaccharides in the primary cell walls and intercellular regions of higher plants
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Polygalacturases (PG)
-PG is the first enzyme to be secreted by mostphytopathogenic microorganisms and is an importantpathogenicity factor.
-Its action on homogalacturonan of the plant cell wallis a pre-requisite for the accessibility of substrate toother CWDEs.
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The crystal structure of PG from Fusarium moniliforme (1.7Å)
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H188D212
D213
D191
K269
R267
Federici et al. Proc Natl Acad Sci U S A 2001, 98:13425-30 .
PG
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PG plants have dwarf phenotype
(Capodicasa et al. Plant Physiol. 2004)
Tabacco
wt #5 #7 #16 PG#16 x PvPGIP2
PvPGIP2
Arabidopsis
PG201 #4
#1 Ws-0
Col-0#5#1
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Defence genes are constitutivelyexpressed
Arb
itrar
yU
nit s
0,0
0,2
0,4
0,6
0,8
PR1Pdf1.2 AtPGIP1
WsPG plants
0,00
0,10
0,20
0,30
0,40
0,000
0,010
0,020
0,030
0,040
.Real-Time RT-PCR
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Federici et al. 2003, PNAS 98: 13425-13430
Polygalacturonase-inhibiting protein (PGIP),a plant recognition protein for
non-self polygalacturonases, is a leucine-richrepeat
(LRR) protein
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S207
V181
Q253
H300
Q320
A326
L89
A340
Predicted PGIP area of interaction
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Molecular docking of the PG-PGIP complex
PGIP PG
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MOLECULAR DOCKING
PvPGIP2 and
F. moniliformePG
A. nigerPG
B. cinereaPG
Sicilia et al, Plant Phys 2005; Federici et al.Trends in Plant Science 2006
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•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 phytopathogenicfungi and bacteria
Pectin MethylEsterases (PMEs)
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PMEs remove the methyl ester groups from homogalacturonan producing methanol and stretches of acidic residues
Demethylation of pectins by PMEs
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Di Matteo et al., Plant Cell2005
PME PMEI
• “Four Helix Bundle” of the inhibitor
• 2 disulphide bridges necessaryto maintain fold
• 1:1 stoichiometry
• Inibitors bind active site of PME preventing substratebinding
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46,7%
83,8%
52,3%
0
20
40
60
80
100
120
WT 1-43 1-1 1-5
PM
E a
ctiv
ity (%
)
mRNA protein
PME actvity decreases Degree of methylation increases
a ab b
0
10
20
30
40
50
60
70
WT 1-43 1-1 1-5
Deg
ree
of
Met
hyle
ster
ifica
tion
(%)
Overexpression of AtPMEI-1 in A. thaliana
UBQ5
AtPMEI
1-1 1-5WT 1-43
AtPMEI 18 kDa21 kDa
14 kDa
1-1 1-5WT 1-43Mw Std
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Increased root length
AtPMEIWT
Ca2+
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XIP GH10 xylanase
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GH10 xylanase /XIP complex
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AcknowledgementsUniversità di Roma “La Sapienza”Dip. Biologia Vegetale
Roberta Galletti Vincenzo LionettiFedra FrancocciFlavio ScaloniManuel Benedetti
S. FerrariD. BellincampiG. De Lorenzo