Modifying Lignin Synthesis by Incorporation of Tyrosine- Containing Peptides Haiying Liang, John Carlson, Nicole Brown and Ming Tien Penn State Center for Nanocellulosics The Pennsylvania State University, University Park, PA 16802 2008 International Conference on Nanotechnology for the Forest Products Industry-Nanotechnology from Research to Applications St. Louis, Mo June 25, 2008
32
Embed
Modifying Lignin Synthesis by Incorporation of Tyrosine ... · C2 T9 T11 T4 T5 T2 T10 T3 T8 T1 T7 T6 T12 ... bm1(reduced CAD activity) Bm2 weak and inconsistent correlations between
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Modifying Lignin Synthesis by Incorporation of Tyrosine-
Containing PeptidesHaiying Liang, John Carlson, Nicole Brown
and Ming TienPenn State Center for Nanocellulosics
The Pennsylvania State University, University Park, PA 16802
2008 International Conference on Nanotechnology forthe Forest Products Industry-Nanotechnology from
Research to ApplicationsSt. Louis, Mo
June 25, 2008
What is lignin?
• Approximately 20% of all carbons fixed by photosynthesis is transformed into lignin– Accounts for a large percentage of the energy that the
earth captures from the sun• Plants adaptation to the more arduous terrestrial
lifestyle– Aquatic plants do not contain lignin (kelp uses gas-filled
bladders to be vertical)– Lignin provides plants with structural rigidity
• Lignin is removed from wood for paper making
Lignin: A statistical polymer of phenylpropanoidunits assembled by free radical coupling
OH
OH
R
R = H or OCH3
OH
R
e-
O.
OH
R
O
.OH
R
O
. OH
R
O
.
R R R R R
Free radical (& plasticity)
lignin synthesis
Note the plasticity of lignin biosynthesis: free radical coupling, in contrast to “enzyme active site” chemistry allows for incorporation of non-lignin components.
β
α
C
C
16
15
14
13
11
10
9
8
7
65
4
3
2
112
O
CH
CH
CH2OH
H3CO
O
COH
CH
CH2OH
H3CO
OH
CH
H3CO
CHOH
HC
HOCH2
H3COO
O
HC
CH
CH2OH
HC
HC
OCH3
HOH2C
OCH3
OO
H3CO
O
CH
CH2OH
OCH3
O
CHOH
CH
CH2OH
OCH3
OH
CH
HC
CH2OH
H3CO OCH3
O etc.
O
C
C
H3CO OCH3
Carbohydrate
OH
C
HC
CH2OH
OCH3
O
O
CHOH
HC
HOH2C
OCH3
CHOH
CH
CH2OH
O
HC
H3CO
HO
OCH3
O
HC
HC
H2C
OCH3
OCH2
O
OCH
CH
O
CH2OH
H3CO
HC
CH2OH
OHOCH3
CH CH2OH
O etc.
Adapted from Adler
CRepresentative Structure of Lignin
Recalcitrance:impact of Lignin on Polysaccharide digestibility
Pol
ysac
chrid
ase
Dig
estib
ility
(%) 100
80
60
40
20
0
Lignin content (%)0 5 10 15 20 25
Pol
ysac
chrid
ase
Dig
estib
ility
(%) 100
80
60
40
20
0
Lignin content (%)0 5 10 15 20 25
Switchgrass
Corn stover
Poplar
Spruce
Histochemical stains can identify sites and types of aromatic compounds within
Ester and ether linkages of ferulic acid in grass cell walls
Ether linkage
1,4,ß Xylans
LIGNIN
Ester linkage
Ester linkage
Ester linkage
Ester linkage
FAE
Expression of vacuolar targeted FAEA under the rice actin promoter in Festuca arundinacea
0
100
200
300
400
500
600
C2 T9 T11 T4 T5 T2 T10 T3 T8 T1 T7 T6 T12
FAE
act
ivity
un
its g
fres
h w
t¯¹
Control and Transgenics
In vitro dry matter digestibility [IVDMD]) of matureleaves of Lolium multiflorum FAEA expressing plants
IVD
MD
%
65
70
75
80
85C
ontro
l
8D7C
1
D18
C2
D19
C1
D8C
2
D7C
2
D18
C1
D19
C3
5D4C
1
D19
C2
D8C
4
D8C
3
D7C
1
D8C
5
Plant Number
Buanafina et al. (2006). Appl. Bioch. Biotech. 129-132: 416-426.
Front End Treatment: There are many targets
for modifying lignin biosynthesis
•While there are examples of lignin content being lowered without damaging fitness, many examples of compromised plant fitness…•Are there limits to this strategy? Is engineering trees without lignin similar to engineering boneless chicken?
Lignin and fitness of maizeSource of variation Effect on agricultural fitness Divergent selection for fiber
concentration
Insect resistant/susceptible hybrids
bm3(reduced COMT activity)
bm1(reduced CAD activity)
Bm2
weak and inconsistent correlations between lignin content and height, yield, lodging, and maturity
no difference in lignin content
reduced grain yield reduced dry matter yield reduced early season vigor 2- to 4-d-later maturity increased stalk breakage
decreased stalk strength decreased days to floweringno reduction in dry matter yield no effect on early season growth increased days to flowering
No information
Penn State Strategy
• Our strategy is to replace some lignin-lignin linkages with lignin-peptide linkages
• In doing so, different proteins can now be attached and thereby functionalize lignin
CHOHHC
HOCH2
H3COO
O
HCCH
CH2OH
HCHC
HOH2COCH3
OHOH3CO
OCH3
O
CHOHCH
CH2OH
OCH3
OCHOH
HCHOH2C
OCH3
CHOHCHCH2OH
H3CO
HO
OCH3
O
N NO
O
R
RN N
O
O
R
RN N
O
O RN N
O
O
RN N
O
O
RN N
O
O R
OHOH
OCHOH
HCHOH2C
OCH3
CHOHCHCH2OH
H3CO
HO
OCH3
O
R
O
OCHOH
HCHOH2C
OCH3R
Strategy cont’dFree radical coupling between lignol subunits and TYR will result in a lignin structure that can be partially hydrolyzed with proteases. This would permit more efficient extraction of lignin and enzymatic conversion of wood to ethanol.
Strategy cont’d• Design TYR-rich peptide genes differing in
length and sequence
• Express transgene in lignifying tissue in poplar
• Characterize transgenic plants– Plant fitness – Lignin structure and lignin-tyrosine bonding in
plants– Small scale pulping and ethanol production tests
• No visible differences between WT and transformed plants for lignin
Lignin content not compromised
Digestibility assay with protease K
Reducing sugar concentrations in stem tissue extracts of hybrid poplar “Ogy” wildtypes and transgenic lines. For each line, a portion of ground tissue was incubated with sequential incubations of protease K followed by cellulase and hemicellulase (shaded bars), while another portion of tissue was incubated only with cellulase and hemicellulase (open bars). Bars are means + SD of 2-3 replicates of individual samplings.
Dynamic mechanical analysis
• All samples are comprised of 5 measurements taken on each of 2 samples from the same sampling, except for T7 which had insufficient tissue to prepare a second sample and so is comprised of two samples from different samplings of the same transgenic line.
Functionalizing Lignin
• Attachment of proteins to lignin opens up the door to functionalizing lignin
• Generate reactive groups to crosslink between lignin and cellulose – Express CBDs with tyrosine content to crosslink
with lignin allows for stronger interaction between lignin and cellulose
Lignin
HN
YFPCBDStreptavidin Cellulose
Biotinetc
Functionalizing Lignin, cont’d
• Addition of polyhistidine for binding of metals – Infiltrate lignin with electron dense substituents for
subsequent spectroscopic visualization of lignin – Infiltrate lignin with heavy metals used for wood
preservative but slows their release.• Provide chelating sites for cations or provide cationic sites
for anions (arsenate) used as wood preservatives
N CHCCH2
O
NNH
HN CHC
CH2
O
NNH
HN CHCH2C
O
N
NH
HN
CHCH2C
O
N
NH
NHCH
CH2CO
N
NH
NHCH
CH2C
OH
ON
HN
Lignin Lignin
N CH C
CH2
O
CH2
CH2
CH2
NH3+
HN CH C
CH2
O
CH2
CH2
CH2
NH3+
HN CH C
H2C
O
H2C
H2C
H2C
+H3N
HN
CHC
H2C
O
H2C
H2C
H2C+H3N
NHCH
CH2C
OH
O
H2CH2C
H2C+H3N
Acknowledgements• People
– John Carlson
– Haiying Liang
– Nicole Brown
– Fang Cong
• Funding– DOE Energy Bioscience– Huck Institute Discovery Grant