Lignin Modification via Expression of a Tyrosine Rich Cell Wall Peptide in Hybrid Poplar. The Pennsylvania State University, USA

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1 Lignin Modification via Expression of a Tyrosine Rich Cell Wall Peptide in Hybrid Poplar John Carlson 1, Yajun Ya 2, Xinli Xia 2, Weilun Yin 2, Haiying Liang 3, Chris Frost 1, icole Brown 1, Ming Tien 1 1 The Pennsylvania State University, USA 2 Beijing Forestry University, PRC 3 Clemson University, USA International Poplar Commission 23 rd Session

2 Importance of lignin Approximately 20% of all carbon is fixed in lignin Lignin provides plants with structural rigidity Lignin is removed from wood for paper production and interferes with ethanol production C Representative Structure of Lignin Adapted from Adler H 3 C H 3 C 2 H 16 2 H H 3 C 3 etc. 2 H 15 1 H 3 C H 3 2 H H 14 H HH 2 C H 3 C 2 H 3 4 C C Carbohydrate H 3 C 3 H 3 C 3 5 H H 13 2 H 3 2 H C 6 H HH 2 C 3 Cβ Cα H 7 H 9 3 H 3 C 3 H 3 C H H 2 H etc. 2 H 2 H H 2 C 10 H

3 Lignin Degradation is of Central Importance in Biomass Utilization Mosier et al Pretreatment (acid and/heat treatment) is the most expensive part of lignocellulose utilization for biofuels. Pretreatment disrupts the lignin encasement around the cellulose and disrupts the crystallinity of cellulose (increase access of hydrolytic enzymes.

4 Impact of Lignin on Polysaccharide digestibility Polysacchridase Digestibility (%) Lignin content (%)

5 Strategies for dealing with the lignin barrier Post Harvest Treat lignocellulosic material with fungal enzymes or whole fungi (area of on-going research) Feedstock Modification Much effort has been expended in decreasing lignin content And in modifying lignin composition, i.e. increase in the syringyl to guaiacyl monomer ratio

6 A ovel Strategy for Lignin Modification in Poplar ur Hypothesis: Free radical coupling between lignin subunits and TYR will result in a lignin structure that can be partially hydrolyzed with protease pretreatment. This could permit more efficient extraction of lignin and enzymatic conversion of wood to ethanol. 2 H H Model for free radical coupling between the lignol precursors and the phenolic TYR. HH 2 C H 3 C H R H R 3 3 H R 2 H H R R H 2 H 2 H H 3 C HH 2 C 3 H 3 C R R H 3 R 3 H 3 HH 2 C H 3 C H 3 H R 2 H H HH 2 C H tyrosine R 3

7 ur Approach: Transform hybrid Poplar with a TYR-rich gene construct. Design TYR-rich peptides differing in length and sequence Express peptide transgenes in lignifying tissue in poplar Populus x Euramericana cv. eva at Beijing Forestry University Populus x gy hybrid at Penn State University Characterize transgenic plants Plant fitness Lignin structure Ethanol production Populus euramericana cv. eva Populus x gy

8 Glycine-rich protein (from pea) MATIHRLPSL VFLVLLALGV CSARRALLTL DAGYGLGHGT GGGYGGAAGS 50 YGGGGGGGSG GGGGYAGEHG VVGYGGGSGG GQGGGVGYGG DQGAGYGGGG 100 GSGGGGGVAY GGGGERGGYG GGQGGGAGGG YGAGGEHGIG YGGGGGSGAG 150 GGGGYAGGA 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 GGVP Has elevated Tyrosine level of 7%. Second generation peptides have over 40% Tyrosine

9 Binary Vector Construction RB S-pro PTII S-ter PAL2 pro Gly-rich gene S-ter LB Leader sequence Poplar phenylalanine ammonia-lyase (PAL2) promoter Leader sequences Pine cell wall Glucosidase signal peptide - Poplar Laccase signal peptide

10 PCR and of genomic Southern to show transformation transgenic gy: transgenic eva: ck-ck+m ck-ck+m HRGP gene PTⅡ gene

11 Transgenic poplar plants transgenic eva: transgenic gy plants in the green house

12 Detection of transcriptional expression of Gly-rich protein construct by Real-time PCR

13 Histochemical staining for lignin o visible differences for lignin content between Wild-type and Transformed plants

14 Lignin content not compromised

15 Digestibility assay with protease K Reducing sugar concentrations in stem tissue extracts of hybrid poplar gy 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.

16 Protease tests with eva transgenics A275nm Untransformed poplar Transformed poplar Time(h) UV absorbance at the wavelength of 275nm over time

17 Dynamic mechanical analysis Averages of 5 measurements on each of 2 samples from plants of the same transgenic line.

18 Populus x gy at Penn State U John Carlson, Prof Ming Tien, Prof Haiying Liang, Assist Prof icole Brown, Assist Prof Fang Cong, PhD student Chris Frost, Post-doc Acknowledgements Populus x Euramericana at Beijing Forestry U Yajun Ya, PhD student Xinli Xia, Assioc Prof Weilun Yin, Prof Thank you for attention! Funding: DE Energy Bioscience Huck Institute Discovery Grant

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