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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