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

cell wall types

Acid phloroglucinol Coniferyl lignin

Chlorine – sulfite Syringyl lignin

Diazonium cds Phenolic acids

Stain Substrate

Akin, 2006

Acid Phloroglucinol-Stained Grass Stem

AP+ = Epidermis, Sclerenchyma ring, Vascular bundles,

(Coniferyl lignin) Akin, 2006

Grass StemAcid Phloroglucinol-Stained Biodegradation

AP+ and non-degradable cell types

= Epidermis, Sclerenchyma ring, Vascular bundles

(Coniferyl lignin stained) Akin, 2006

Strategies for dealing with the lignin barrier

• Back end (post harvest)– Treat lignocellulosic material with fungal

enzymes or whole fungi (area of on-going research)

• Front end – During past decade, mainly for the pulp and

paper industry, much effort expended toward decreasing lignin content (these strategies can be directly transferred to biofuel application).

– Lignin composition has also been altered, syringylto guaiacyl ratio increased to 3-fold

Back end treatment: How does nature degrade lignin?

Brown rot

White rot

•Predominant degraders of lignin are filamentous fungi•Two major modes of decay studied in my lab

•Brown rot•White rot

•Both processes are oxidative involving free radicals

TimeR

elat

ive

amou

nt

Time

Rel

ativ

e am

ount

CarbohydratesLignin

Brown versus white rot fungiWhite rot Brown rot

Carbohydrate-relatedEndoglucanaseCellobiosehydrolaseEndoxylanases

YesYesYes

YesYesYes

Lignin-relatedPeroxidasesLaccasesQuinone reductases

YesYesYes

NoNoYes

Small moleculesOxalateQuinones

YesNo

YesYes

Digestibility of grasses impacted by lignin

Extent of crosslinking of phenolic acids in cell wall

Extent of lignification of cell walls and vascular system

Rates of digestion controlled by

Leaves R2 = 0.25

60

70

80

90

4 5 6 7Klason lignin% Dry Matter

IVD

MD

Leaves

total monomers and dimers mg /g dry wt

60

70

80

90

0 2 4 6

IVD

MD

R = 0.842 Stem R2= 0.97

Klason lignin% Dry Matter

60

70

80

90

4 5 6 7

IVD

MD

Xyl----------------Xyl-------------Xyl------------------------Xyl----------------------Xyl-

OH

OCH3

C

O

O

C

OH

OCH

C

O

O

C O

H3O

OH

3 3H

C

O

O

CH3

C O

O

Ara AraAra

O

OC

LIGNIN

Xyl-------------Xyl-----------Xyl------------Xyl--------------Xyl----------Xyl-------

Ara

C

OLIGNIN

O

Ara

Coniferyl alcohol

O

OC

OCH3O

C

C CH2OHH

H2

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

Glycine-rich protein (from pea)MATIHRLPSL VFLVLLALGV CSARRALLTL DAGYGLGHGT GGGYGGAAGS 50 YGGGGGGGSG GGGGYAGEHG VVGYGGGSGG GQGGGVGYGG DQGAGYGGGG 100 GSGGGGGVAY GGGGERGGYG GGQGGGAGGG YGAGGEHGIG YGGGGGSGAG 150 GGGGYNAGGA QGGGYGTGGG AGGGGGGGGD HGGGYGGGQG AGGGAGGGYG 200 GGGEHGGGGG GGQGGGAGGG YGAGGEHGGG AGGGQGGGAG GGYGAGGEHG 250 GGAGGGQGGG AGGGYGAGGE HGGGAGGGQG GGAGGGYGAG GEHGGGAGGG 300 QGGGAGGGYG AGGEHGGGGG GGQGGGAGGG YAAVGEHGGG YGGGQGGGDG 350 GGYGTGGEHG GGYGGGQGGG AGGGYGTGGE HGGGYGGGQG GGGGYGAGGD 400 HGAAGYGGGE GGGGGSGGGY GDGGAHGGGY GGGAGGGGGY GAGGAHGGGY 450 GGGGGIGGGH GGNVP

•Also has elevated Tyr (7%).•Second generation has over 40% Tyr

Vector Construction

NOS-pro NPTII NOS-ter PAL2 pro Gly-rich gene NOS-terRB LB

Leader sequence

•Poplar phenylalanine ammonia-lyase (PAL2) promoter•Poplar leader sequence (GLac90 laccase gene putative signal peptide)

Transgenic poplar

Expression of Gly-rich proteinreal-time PCR

Histochemical staining for lignin

• 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