Tailoring lignin biosynthesis for emerging bioenergy and bioproduct applications. Scott Sattler

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1 Tailoring lignin biosynthesis for emerging bioenergy and bioproduct applications Scott Sattler Wheat, Sorghum and Forage Research Unit USDA-ARS Lincoln, Nebraska USA

2 Sorghum: a versatile bioenergy crop forage/energy sweet grain sugars & biomass biomass C4 photosynthesis/water Use Efficiency Wide Adaptation/High Yield Potential Drought Tolerance/Pest Resistance Existing Agricultural Infrastructure starch

3 genomics.energy.gov Biomass: Cell Walls Lignin Oak Ridge National Laboratory heterogeneous polymer composed of aromatic subunits polymerization catalyzed by freeradicals cross-links cell wall polysaccharides Lignin is a major factor reducing digestibility

4 brown midrib mutants LB neor/ kanr CaMV 35S transgenic plants CaMV E35S SbMyb60 Biological Conversion decreasing lignin RB Thermochemical Conversion increasing lignin livestock digestibility combustion Green chemistry cellulosic bioenergy pyrolysis

5 For conversion of biomass: Produce multiple biofuels Utilize all components of biomass as substrates Fuels High value green chemistry products Pharmaceuticals

6 Sorghum brown midrib (bmr) mutants Wild-type bmr6 bmr12 bmr6 + bmr12 Wild-type: outside bmr12: inside juicy stalk: left dry stalk: right Near-isogenic lines in forage and grain varieties bmr mutants reduce lignin content and alter lignin composition

7 Disrupting lignin synthesis using brown midrib (bmr) mutants Monolignol Pathway Bout and Vermerris (2003); Saballos et al. (2009, 2012); Sattler et al. (2009) Bmr6 encodes cinnamyl alcohol dehydrogenase (CAD) Bmr12 encodes caffeic acid O- methyltransferase (COMT) Bmr2 encodes a 4-coumarate: Coenzyme A ligase (4CL)

8 bmr mutants increase ethanol efficiency of biomass Ethanol Efficiency (% max, washed solids) R = bmr6, Lignin (mg/g) Reducing lignin is key for bioenergy crop improvement Dien, B, G. Sarath, J. Pedersen, S. Sattler, H. Chen, D. Funnell-Harris, N. Nichols and M. Cotta BioEnergy Research 2: WT

9 Exploring total energy content of sorghum biomass bomb calorimeter calories g WT bmr6 bmr12 stacked lower lignin Showed lignin content affects the available total energy

10 Increasing lignin synthesis through biotechnology In collaboration with Tom Clemente, University of Nebraska-Lincoln Monolignol pathway Identified/cloned 35S overexpressed Transformed Goal: increase flux through the pathway thereby increasing lignin

11 Increasing lignin through SbMyb60 transcription activation Positive regulators of lignin biosynthesis in Arabidopsis SbMyb60 Monolignol pathway proteins accumulate in Myb lines Scully E.D., T. Gries, G. Sarath, N.A. Palmer, L. Baird, M.J. Serapiglia, B.S. Dien, A.A. Boateng, Z. Ge, D.L. Funnell-Harris, P. Twigg, T.E. Clemente and S.E. Sattler Plant Journal. Scully, E. D., Gries, T., Palmer, N. A., Sarath, G., Funnell-Harris, D. L., Baird, L., Twigg, P., Seravalli, J., Clemente, T. E., Sattler, S. E New Phytologist.

12 SbMyb60 overexpression alters transcriptome Relative Expression of SbMyb60 via qrt-pcr Differentially expressed genes via RNAseq Relatively few genes differentially expressed in both tissues

13 Summary of the RNAseq and metabolite analyses Coexpression analysis Scully et al New Phytologist 35S::SbMyb60 activated expression of genes involved in aromatic amino acid, S- adenosyl methionine (SAM) and folate biosynthetic pathways. Genes involved in nitrogen (N) assimilation, carbon (C) metabolism, UDP-sugar biosynthesis and cellulose synthesis were also induced. SbMyb60 activates pathways that synthesize the substrates and cofactors required for lignin biosynthesis.

14 SbMyb60 alters lignin composition and content of biomass WT 10a 15a 2a ADL is 23% higher in 10a relative to WT Calories/g Bomb Calorimetry b a c bc WT 2a 10a 15a 2a had the highest energy values All three events have increased energy content Scully et al The Plant Journal

15 Hydroxycinnamic acids in grass cell walls Both p- coumaric and ferulic acid are involved in cross-linking within grass cell walls

16 Increased lignin staining and wall-bound monolignol pathway products Cell wall-bound hydroxycinnamates phloroglucinol staining WT 10a 15a 2a Scully et al New Phytologist Increased wall-bound phenolics in 2a Increased lignin staining in 10a and 15a and cortical expansion

17 SbMyb60 affects plant development Scully et al 2016 Plant Journal; Scully et al 2018 New Phytologist SbMyb60 overexpression affects flowering time and vegetative development

18 Caffeoyl-CoA LB leaves stalks neor/ kanr SAM CaMV 35S CCoAOMT overexpression Feruloyl-CoA CCoAOMT WT 9A 28B CaMV E35S RB actin SbCCoAOMT actin SbCCoAOMT Differentially Expressed Genes (DEGs) from RNAseq Up-regulated 28b leaf 20 9a leaf a stalk b stalk Down-regulated 28b leaf a leaf b stalk 7 6 9a stalk 84 CCoAOMT overexpression had a minimal effect on gene expression

19 calories/g % dry weight CCoAOMT overexpression altered energy Acid Detergent Lignin a,b a a Energy of Biomass b b a WT ZG A ZG B calories/g Energy of Detergent-Washed Biomass 4300 b 4250 a,b 4050 WT ZG A ZG B WT ZG A ZG B calories/g a Energy of Acid-Washed Biomass a a a Acid labile groups are responsible for increase in energy WT ZG A ZG B

20 peak area of major ion (x10 6 ) peak area of major ion (x10 3 ) 35S::CCoAOMT alters cell wall composition soluble wall bound WT 9a 28b soluble wall bound Ferulic Acid ** * * ** Sinapic Acid ** ** * * WT 9a 28b WT 9A 28B Increased autofluorescence and phloroglucinol staining are due to increased levels of hydroxycinnamoyl compounds in cell wall, and not lignin

21 dry stover yield (kg/ha) Field trial of 35S::SbMyb60 and CCoAOMT Stover Yield WT 35S::SbMyb60 2a 10a 15a 35S::SbCCoAOMT 9a 28b hybrid parent Hybrids affected performance based on transgenic event * Tool to increase feruloyl and sinapoyl groups in cell wall outside of lignin Females: AN32, AN4692, ATx3118 male: RTx430

22 ADL % Field Trial: cell wall composition Acid Detergent Lignin (ADL) * Females: AN32, AN4692, ATx3118 male: RTx430 calories/g WT Energy Concentrations 35S::SbCCoAOMT 9a 28b hybrid parent * 35S::SbMyb60 2a 10a 15a

23 Future directions: Combining approaches to alter monolignol biosynthesis brown midrib 2 bmr mutants X Monolignol Pathway Transgenic lines Goal: Sorghum plants with drastically altered phenylpropanoids -Address fundamental questions about cell walls and lignin -Test the impact of these changes on biotic interactions

24 Summary Demonstrated bmr6 and bmr12 improve saccharification Exploring ways to alter monolignol biosynthesis and affect biomass composition through biotechnology Shown SbMyb60 overexpression activates pathways leading to lignin synthesis Shown CCoAOMT overexpression increases hydroxycinnamates in the cell wall There may be greater potential to increase hydroxycinnamates in cell walls than lignin

25 Acknowledgements Wheat, Sorghum and Forage Research Unit University of San Diego USDA-ARS, Lincoln Lisa Baird Tammy Gries Hannah Tetreault USDA-ARS, Peoria, IL John Toy Bruce Dien Deanna Funnell-Harris Pat Dowd Gautam Sarath Mark Berhow Nathan Palmer Pat O Neill University of Nebraska-Kearney Jeff Pedersen Paul Twigg Sarah Finegan Lois Bernhardson University of Nebraska-Lincoln Tom Clemente Funding: USDA-ARS CRIS project # D Grant # USDA-ARS Manhattan, KS Erin Scully Washington State University ChulHee Kang USDA-ARS, Lubbock TX Zhanguo Xin University of Florida Wilfred Vermerris USDA-ARS, Wyndmoor, PA Michelle Serapiglia Kwasi Boateng

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