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A Novel Approach to Facilitate Accessibility of Cellulose and Hemicellulose by Introducing a Tyrosine Rich Peptide Gene In Poplar Cell Walls. Haiying Liang 1 , Nicole Brown 2 , John Carlson 2 , Ming Tien 2 1 Clemson University, Clemson, SC 29634 - PowerPoint PPT Presentation
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Haiying Liang1, Nicole Brown2, John Carlson2, Ming Tien2 1 Clemson University, Clemson, SC 29634
2The Pennsylvania State University, University Park, PA 16802
A Novel Approach to Facilitate Accessibility of Cellulose and Hemicellulose by Introducing a Tyrosine Rich Peptide Gene In
Poplar Cell Walls
Energy Crisis
Don Tate III AMERICAN-STATESMAN ILLUSTRATION, from http://www.statesman.com/business/content/business/other/gas.html
Haiying Liang1, Nicole Brown2, John Carlson2, Ming Tien2 1 Clemson University, Clemson, SC 29634
2The Pennsylvania State University, University Park, PA 16802
A Novel Approach to Facilitate Accessibility of Cellulose and Hemicellulose by Introducing a Tyrosine Rich Peptide Gene In
Poplar Cell Walls
About Lignin
A major component of wood -raw material for pulp and paper production -a good renewable energy source
A component of lignocellulosic material -feedstock for livestock
2nd most abundant terrestrial biopolymer -approx. 30% of the organic carbon in the biosphere
Lignin Degradation is of Central Importance in Biomass Utilization
Undesired material in pulp and paper industries and
biomass utilization
Mosier et al. 2005
Difficult to degrade , no nutritional value
Approaches Being Taken
Schematic from Mosier et al. (2005) showing goals of pretreatment of lignocellulosic material
Expensive and environment-unfriendly
lignin network crystallinity of cellulose
1. Decrease lignin content2. Modify lignin monomer composition: G (guaiacyl) S (syringyl)
Biotechnology
PAL
C4H
CCR
CAD
4CL
COMT
monolignol synthesis
Essential Role of Lignin in Cell Wall
Essential component of cell wall:
- imparts rigidity to plants
- conducts water & solutes to different parts of plants
- provides physical barrier to invading pests
HypothesisFree radical coupling between lignol subunits and TYR will result in a lignin structure that can be partially hydrolyzed with proteases.
CHOHHC
HOCH2
H3COO
O
HCCH
CH2OH
HCHC
HOH2C
OCH3
OHOH3CO
OCH3
O
CHOHCH
CH2OH
OCH3
OCHOH
HCHOH2C
OCH3
CHOHCH
CH2OH
H3CO
HO
OCH3
O
N NO
O
R
RN N
O
O
R
RN N
O
O RN N
O
O
R
N NO
O
R
N NO
O R
OHOH
OCHOH
HC
HOH2C
OCH3
CHOHCH
CH2OH
H3CO
HO
OCH3
O
R
O
OCHOH
HC
HOH2C
OCH3R
Representative structure of peptide-cross-linked lignin via phenolic tyrosines.
1. Design TYR-rich peptide genes differing in length and sequence
2. Express transgene in lignifying tissue in poplar
3. Characterize transgenic plants:-Plant fitness -Lignin structure and lignin-tyrosine bonding in plants-Small scale pulping and ethanol production tests
Strategies
Strategy 1: Gene Design Binary Vector (Modified from pBI101)
Tyr rich gene (13%)
PAL promoter--CBG-leader
For secretion into the cell wall during lignification (Pinus contorta coniferin-
specific β-glucosidase)
Expression of PAL2-GUS gene fusion in poplar in the actively lignifying
phloem and xylem cells(PAL: Phenylalanine Ammonia-Lyase )
Transformation in Poplar
Ogy (P. deltoides Marsh. × P. nigra L.)
Transgenic lines1 Kb
Plasmid DNAWild type DNA
Example of PCR screening with transgene-specific primers
2W 2ZZ 2-II 2X M 4-2 Wt
1Kb
2Kb
Example of genomic southern hybridization
Transgenic Line
wt 2x 29 2-I 4c 4.2 2zz 10.1 2ee 2w 2-II
Ty
ros
ine
-ric
h t
ran
sg
en
e
ex
pre
ss
ion
(re
lati
ve
to
r1
8S
)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Strategy 2: Gene Expression Real-Time PCR
2-I 2X 2YY 2Z 2P
Strategy 3: CharacterizationHistochemical Staining of Lignin
Wt 2-5V 4-4 2EE
Phloroglucinol
Potassium permanganate
Phloroglucinol
Potassium permanganate
Strategy 3: CharacterizationKlason Lignin Content Analysis
wt
2yy
2-5
v
2I
2-5
2P
2II
2E
E
2A
2W
Lig
nin
Co
nte
nt
(% S
tem
Dry
We
igh
t)
05
101520253035
Further StudiesLocalization of TYR-rich peptide with antibodyPathogen susceptibility analysisTensile strength, lignin structure, etc.
Tyr
TyrLys
Glu
Lys
Glu
Lys
Glu
Tyr
Tyr
Tyr
Tyr
Tyr
Peptide showing salt bridging
Strategy 3: Characterizing Lignin-Protein Structure and Interactions
Isolated Lignin
MWL isolated from stemsHSQC and TOCSY (Hu et al.)
Compare to 13C-Tyrosine treated MWL fractions (from control plants)
Strategy 3: Characterizing Lignin-Protein Structure and Interactions
HSQC of MWL (Hu et al., Nature Biotechnol, 1999)
Solid samples
Method—CP/MAS NMR 15N Tyrosine tissues Plants supplied K15NO3
(Englesberger et al. 2006)
Cross polarization studies Basis: < 10 Å proximity Different 15N chemical shifts
should be detected for the various amino acids
Strategy 3: Characterizing Lignin-Protein Interactions at the Nanoscale
Morais et al. 1999, J. Braz. Chem. Soc.
Lignin-Protein Interactions
Solid state NMR (CP/MAS)
Variable Contact Time Cross Polarization Proton spin-lattice relaxation time in the rotating frame
(HT1)
Common HT1indicates nanoscale homogeneity, while different HT1 indicates nanoscale phase separation
Strategy 3: Characterizing Lignin-Protein Interactions at the Nanoscale
Strategy 3: Thermal Characterization
DMA/DSCLignin Glass Transition Region
Before and after plasticization (DMA) Control and modified plants Extracted MWL
Strategy 3: Bioenergy Studies
•Pulping efficiency and ethanol production from modified tissues
•Does protease pre-treatment of tyrosine-rich transgenic poplar plants impact these processes?
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.1
mg of weight loss /mg wood
chps
Wt T15 T17 T19 T28
Preliminary digestibility assay using protease K
Acknowledgments
• Alan Benesi, Penn State NMR Director• DOE & Huck Institutes for the Life Sciences at• Penn State for $$$