Biomass conversion: opportunities in lignin management. Clint Chapple Department of Biochemistry Purdue University

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1 Biomass conversion: opportunities in lignin management Clint Chapple Department of Biochemistry Purdue University

2 Targets for biomass improvement Yield Water and nutrient use efficiency Pest and pathogen resistance Biomass quality

3 Targets for biomass improvement Yield Water and nutrient use efficiency Pest and pathogen resistance Biomass quality

4 Lignin is critical for plant survival structural support water transport important impact on bioenergy and bioprocessing large commitment of fixed carbon high energy density

5 Lignin is a biosynthetically plastic polymer Bonawitz and Chapple, Ann Rev Genetics, 2010

6 Lignin is a biosynthetically plastic polymer Nucleic acid synthesis Template-dependent Protein synthesis Template-dependent H H Me H H H Me Me H H Me Me H Me H Me H Me Polysaccharide synthesis Me Enzyme specificity-directed H Me H H H Lignin synthesis Random radical coupling dependent on precursor supply

7 pportunities for lignin engineering Block lignin synthesis Change lignin composition Redirect lignin monomers Alter lignin localization

8 Mutant screens can identify genes important to processes of interest Normal plant mutagenesis Mutant with trait of interest gene identification Understanding of gene function

9 Lignin is a biosynthetically plastic polymer Bonawitz and Chapple, Ann Rev Genetics, 2010

10 Lignin modification-induced dwarfing (LMID) Schilmiller et al., Plant J. 2009

11 Lignin is a biosynthetically plastic polymer Bonawitz and Chapple, Ann Rev Genetics, 2010

12 Lack of C3'H reduces lignin content and leads to novel lignins Franke et al., Plant J, 2002

13 Lignin is a biosynthetically plastic polymer Bonawitz and Chapple, Ann Rev Genetics, 2010

14 ref4 and rfr1 are required for lignin regulation

15 ref4 and rfr1 are required for lignin regulation

16 REF4 and RFR1 are components of the Mediator complex P Cramer Lab, Gene Center, University of Munich

17 Disruption of MED5 rescues the dwarf phenotype of ref8 wild type Bonawitz et al., Nature, 2014 med5a/b ref8-1 med5a/b ref8-1 ref8-2 med5a/b ref8-2

18 ref8-1 mutants show widespread MED5-dependent transcriptional reprogramming med5a/b ref med5a/b ref med5a/b ref med5a/b ref8-1 up-regulated down-regulated (DeSeq, false discovery rate <0.05) Bonawitz et al., Nature, 2014

19 med5a/b ref8-1 mutants synthesize predominantly H lignin wild type med5a/b med5a/b ref8-1 syringyl (S) guaiacyl (G) p-hydroxyphenyl (H) Yuki Tobimatsu, John Ralph, Wisconsin

20 med5a/b ref8 mutants show facilitated saccharification 100 no pretreatment 100 liquid hot water pretreated glucose release (%) wild type med5a/b med5a/b ref time (hrs) Eduardo Ximenes, Mike Ladisch, Purdue

21 Suppressor screens can identify genes important to processes of interest Normal plant mutagenesis Mutant with trait of interest mutagenesis Mutant plant in which trait is abolished

22 Suppressors of ref4-3 may identify other proteins required for phenylpropanoid regulation Dolan et al., unpublished

23 Whole genome sequencing identified intragenic suppressor mutations and Mediator subunit suppressors Dolan et al., unpublished

24 REF4 and RFR1 are components of the Mediator complex P Cramer Lab, Gene Center, University of Munich

25 Maybe lignin isn t all bad Significant proportion of plant biomass More highly reduced than sugars and polysaccharides Amenable to catalytic degradation and conversion H H Me H Me H H Me Me H H Me Me H H Me H H Me H Me H H Me

26 Parsell et al., Green Chemistry 2015 Maybe lignin isn t all bad

27 Shuai et al., Science 2016 Maybe lignin isn t all bad

28 Summary We are developing an understanding of the catalysts and regulators that provide us with control over lignin The study of Arabidopsis has revealed a previously unknown regulatory circuit for lignin biosynthesis that involves Mediator The dwarfing seen in some lignin-deficient plants can be suppressed genetically, indicating that it is an active proteinmediated process Plants with high H-lignin are viable and could provide novel inputs for the biorefinery Catalytic conversion of lignin into fuels may make more efficient use of biomass

29 Acknowledgements Purdue and C3Bio Nick Bonawitz Whitney Dolan Jake Stout Candy Mao Bryon Donohoe Peter Ciesielski Eduardo Ximenes Mike Ladisch GCEP Sirius Li Yuki Tobimatsu John Ralph Chris McClellan Claire Halpin Wout Boerjan Brian Dilkes Charles Addo-Quaye

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