Efficient Biomass Conversion: Delineating the Best Lignin Monomer-Substitutes

Transcription

1 Efficient Biomass Conversion: Delineating the Best Lignin Monomer-Substitutes Investigators John Ralph, Professor, Biochemistry; Xuejun Pan, Professor, Biological Systems Engineering; Sara Patterson, Professor, Horticulture; Dino Ress, Postdoctoral Researcher; Yuki Tobimatsu, Postdoctoral Researcher; Martina pietnik, Postdoctoral Researcher; Sasikumar Elumalai, Postdoctoral Researcher; Jenny Bolivar, Graduate Researcher; Christy Davidson, Technician, U. Wisconsin-Madison. Involved coworkers (unfunded): Hoon Kim and Fachuang Lu, Research Scientists, U. Wisconsin-Madison. Collaborators: John Grabber, Research Scientist, and Paul Schatz, Technician, US Dairy Forage Research Center, USDA-ARS. [This project has now completed its first full year] Abstract The three year plan is to delineate a set of approaches for successfully altering lignin structure, in a way that allows plant cell wall breakdown to produce biofuels in a more energy-efficient manner, by providing alternative plant-compatible monomers to the lignification process. The approach is to synthesize and test various classes of novel plant compatible monomer-substitutes for their abilities to incorporate into lignins, and then to determine how such incorporation affects biomass processing in biomimetic cell wall systems. The ability of a chosen monomer to incorporate into lignins (copolymerizing with the traditional monomers) is determined by in vitro biomimetic lignification involving the phenolic radical coupling reactions that typify the lignification process. Those that successfully make co-polymers are polymerized into a suspension-cultured cell wall system to further delineate their polymerization efficacy and to provide biomimetic cell wall material for preliminary testing of conversion efficiency following selected pretreatments and in a variety of processes. The most promising monomer-substitutes will be revealed to other GCEP researchers so that the process of understanding the pathways that produce the monomers and obtaining the required genes can proceed most expediently. Accomplishments this year include the following We have synthesized a set of monomer replacements for testing; further compounds have been obtained from natural sources or commercially. The classes of monomer-substitutes include: a) Difunctional monomers or monomer conjugates linked via cleavable ester or amide (and/or hydrophilic) functionality; b) Monomers that produce novel cleavable functionality in the polymer; c) Hydrophilic monomers; d) Monomers that minimize lignin-polysaccharide cross-linking; and e) Monomers that produce simpler lignins. Compounds have been sent

2 to another GCEP researcher, Boerjan, for identification and authentication of metabolites in Arabidopsis lines. Synthetic lignification studies have begun and are revealing the compatibility (or otherwise) of monolignol substitutes with the lignification process. Isolated primary cell walls have been ectopically lignified with several sets of monomersubstitutes to provide materials for testing the digestibility (saccharifiability) of ligninmodified cell walls, with and without pretreatment. In particular, introducing various catechins and gallates (from plant tannin pathways) into lignification produces quite remarkable improvements in degradability; the improvement is related quite strikingly to the phenolic-h content of the monomer-substitute; a provisional patent has been filed. In collaboration with Boerjan s group, we have tested a new multi-misregulation approach to produce the first transgenics that derive their lignins primarily from nontraditional monomers. The approach was aimed at improving digestibility by reducing the extent of cell-wall-polymer cross-linking, but the plants are not healthy, and not worthy of testing for digestibility implications. Nevertheless, the approach is informative in delineating just how far lignification can be perturbed away from normal monolignols and sheds light on how biosynthetic pathways interact. Finally, an approach toward providing gram-quantities of secondary wall elements in a culture system seems poised to also allow testing in relevant dicot systems. verall, a picture is emerging of the kinds of monolignol-substitutes that are compatible with lignification and allow for improved and/ or less energy-intensive saccharification of pretreated and non-pretreated cell walls. Introduction The objective of this work is to reduce the energy requirements for processing lignocellulosic materials by structurally altering lignin, by modifying its monomer complement, to allow the biomass resources to be more efficiently and sustainably utilized. It aims to identify lignin monomer-substitutes that are fully compatible with the polymerization processes inherent in plant lignification and that, additionally, can produce modified lignin polymers that render plant cell walls less recalcitrant toward processing to biofuels. The use of lignocellulosics for biofuels, and the improvements if feedstocks can be selected/engineered for easier processing, will contribute enormously to minimizing greenhouse gas production in the transportation fuels sector. The approach is to synthesize and test a strategic range of novel plant compatible monomer substitutes for their abilities to incorporate into lignins, and then to determine how such incorporation affects biomass processing in biomimetically lignified cell wall systems. The classes of monomer substitutes include: a) Difunctional monomers or monomer conjugates linked via cleavable ester or amide (and/or hydrophilic) functionality; b) Monomers that produce novel cleavable functionality in the polymer; c) Hydrophilic monomers; d) Monomers that minimize lignin-polysaccharide cross-linking; and e) Monomers that produce simpler lignins. The ability of a chosen monomer to incorporate into lignins (copolymerizing with the traditional monomers) is determined by in vitro biomimetic lignification involving

3 the phenolic radical coupling reactions that typify the lignification process. Those that successfully make co-polymers are next polymerized into a suspension-cultured cell wall system to further delineate their polymerization efficacy and to provide biomimetic cell wall material for preliminary testing of conversion efficiency following selected pretreatments and in a variety of processes. These materials also provide the cell-wall- NMR database to allow the success of plant transformations to be determined. Monomer-substitutes that are most promising are revealed to other GCEP researchers so that the process of understanding the pathways that produce the monomers, obtaining the required genes, and testing plant transformations, can proceed most expediently. Background ver the past decade it has become apparent that the metabolic malleability of lignification, the process of polymerization of phenolic monomers to produce lignin polymers, provides enormous potential for engineering the resistant polymer to be more amenable to processing, as reviewed. 1-5 Massive compositional changes can be realized by perturbing single genes in the monolignol pathway, particularly the hydroxylases. More strikingly, monomer substitution has been observed in the process of lignification, particularly in cases where a plant s ability to biosynthesize the usual complement of monolignols is compromised. These substitutions include products of incomplete monolignol biosynthesis such as 5-hydroxyconiferyl alcohol, 6 ferulic acid, 7 and coniferaldehyde and sinapaldehyde, 6 in some cases at quite high levels and without obvious pleiotropic effects. This suggests that lignin composition and structure can be altered, leading to plants with characteristics for improved processing to biofuels. Replacing the entire monomer component of lignification with a novel monomer is unlikely to be an effective strategy that is acceptable to the growing plant. Introducing strategic monomers into the normal monolignol pool is, however, a viable proposition. To date, incorporation of up to 30% novel monomer has produced plants with no pleiotropic effects or obvious growth phenotypes. A range of alternative monomers appears to be consistent with the GCEP RFP criteria of maintaining the plant s structural and functional integrity, but any approach will require empirical testing. The key here is to home in on the best strategies for plant-compatible monomer substitution that will produce lignins that substantially ease processing of the cell wall. bservations to date have allowed us to detail some ideal properties of monolignol substitutes. 2 When such compounds are introduced into lignins, even at significant levels, the plants show no obvious growth/development phenotype. Monomers that have accessible conjugation into the sidechain allowing for endwise β 4-coupling seem to fare the best. Examples are: 5-hydroxyconiferyl alcohol, the hydroxycinnamaldehydes, hydroxycinnamate esters, and acylated hydroxycinnamyl alcohols, Figure 1. Due to incompatibilities in radical coupling reactions, p-hydroxy phenyl moieties fare less well than guaiacyl or syringyl moieties, at least when incorporating into guaiacyl-syringyl lignins, but other phenolics have not been well studied.

4 a 5 H 1 B 4 Me hydroxycinnamyl alcohol monolignol β 5 A 4 1 Me H 2 H B Me Me A H + β 4-quinone methide intermediate H 2 H H B Me Me A H new β-ether end-unit on elongated polymer A B H PD H 2 2 C Me H 2 H H H H B C Me Me Me H A β-ether unit B in 2-unit elongated polymer A B C b R R B Me γ-acylated hydroxycinnamyl alcohol monomer γ A Me H 2 R A B Me Me H + β 4-quinone methide intermediate H 2 H R B Me Me A H new γ-acylated β-ether end-unit on elongated polymer A B H PD H 2 2 C Me H 2 H H H B C Me Me Me H A γ-acylated β-ether unit B in 2-unit elongated polymer A B C c H B Me hydroxycinnamaldehyde monomer A Me H H B Me Me A H + β 4-quinone methide intermediate H H C -H + A PD Me H 2 B Me H 2 2 Me H H H new cinnamaldehyde β-ether end-unit on elongated polymer A B H B C Me Me Me H A cimmamaldehyde β-ether unit B in 2-unit elongated polymer A B C d H B H Me 5-hydroxyconiferyl alcohol monomer A Me H 2 H A B Me H Me H + β 4-quinone methide intermediate H H H C H 2 H A H PD Me H + A B Me H 2 2 B Me H Me Me H new 5-hydroxyguaiacyl β-ether C end-unit on elongated polymer A B H H Me benzodioxane unit B in 2-unit elongated polymer A B C Figure 1: Cross-coupling and post-coupling reactions for various well-suited monomers incorporated into lignification. 2 Illustration is for the major β 4-coupling only. a) Normal hydroxycinnamyl alcohol radicals B cross-couple with the phenolic end of the growing polymer A, mainly by β 4-coupling, to produce an intermediate quinone methide which rearomatizes by nucleophilic water addition to produce the elongated lignin chain A B. The subsequent chain elongation via a further monolignol radical C etherifies the unit created by the prior monomer B addition, producing the

5 Figure 1: (ctd) C2-unit-elongated polymer unit A B C. b) Various γ-acylated monolignols (p-coumarate, p-hydroxybenzoate, and acetate) cross-couple equally well producing analogous products but with the β-ether unit B γ-acylated in the lignin polymer unit A B C. c) Hydroxycinnamaldehydes B may also cross-couple with the phenolic end of the growing polymer A, again mainly by β 4-coupling, to produce an intermediate quinone methide again, but one which rearomatizes by loss of the acidic β-proton, producing an unsaturated cinnamaldehyde-β 4-linked B end-unit. Incorporation further into the polymer by etherification is analogous to a). The unsaturated aldehyde units B give rise to unique thioacidolysis markers. d) 5-Hydroxy coniferyl alcohol monomer A also cross-couples with the phenolic end of the growing polymer A, mainly by β 4-coupling, to produce an intermediate quinone methide as usual which rearomatizes normally by nucleophilic water addition to produce the elongated lignin chain A B bearing a novel 5-hydroxyguaiacyl phenolic end-unit. The subsequent chain elongation via a further monolignol radical C coupling β 4 to the new phenolic end of A B, but this time the rearomatization of the quinone methide (not shown) is via internal attack of the 5-H producing novel benzodioxane units B C in the 2-unit-elongated polymer unit A B C. 5-Hydroxy coniferyl alcohol incorporation produces a lignin with a structure that deviates significantly from the normal lignin. The bolded bonds are the ones formed in the radical coupling steps. Without regard to plant biochemistry, it is easy to come up with a set of weird and wonderful monomers from simple chemical principles, from chemical catalogs, or by design. At this initial stage, however, the only monomers in contention are those that plants can biosynthesize; i.e., for which in planta biosynthetic pathways (and hence enzymes and genes) exist. All of the potential lignin monomers we intend to test have been isolated from various plant materials. The derivation of some is not entirely obvious but, if plants are truly making them, then enzymes and genes for the required biochemical pathways must be in place. The classes of monomers are considered the most fruitful to explore are as described above. Approach The project has now completed its first full year. The steps required to select and then test promising monomer substitutes are briefly described here. The actual progress is summarized in the next section (Progress). 1. Delineate monomer compatibility. Determining the compatibility of the chosen monomers with lignification via in vitro model coupling reactions is essential to test as any monomer that does not couple integrally into lignins is unlikely to be valuable. And, for as much as we know about radical coupling, coupling and cross-coupling propensities must be tested empirically! We have used such methods to define how ferulates couple into lignins, for example. 8 The models and model polymers also provide the NMR database required to identify the resulting products and pathways in the more complex

6 cell wall models and in transformed plants, and oligomers can be used for GC- or LC-MS (metabolomics) screening. 2. Biomimetically lignify the selected monomers into cell walls. Selected monomers, at varying levels relative to the normal monolignols, need to be incorporated into cell walls. Strategically 13 C-labeled monomers are used as appropriate. 3. Delineate the resultant cell wall lignin structure. Structural characterization of the walls reveals whether the monomers integrate into wall lignins as planned. Ectopic lignification of isolated cell walls also provides materials for conversion testing. Structure is examined by degradative methods and, most importantly, via our whole-cell-wall dissolution and NMR procedures (where strategic labeling can help reveal the bonding patterns) Test biomass processing impacts. Monomers are all selected for their potential to improve biomass processing efficiency. The walls from step 2 are tested under a variety of biomass conversion methods to delineate how much improvement might be expected in planta from utilization of the monomer substitutes are various levels. Progress 1. Synthesis of lignin monomer substitutes A major part of our activity during this year has been in the synthesis of various plant-compatible monomer substitutes that have the potential, when incorporated into lignification, to improve lignin degradability or access to cell wall polysaccharides. Round 1 monomer substitutes. Syntheses of large amounts of normal lignin monomers (coniferyl 1 and sinapyl 2 alcohols), as well as the first round of potential monomer-substitutes 3-16 (Figure 2), have been completed. These include examples from the above classes of: a) Difunctional monomers or monomer conjugates linked via cleavable ester and/or hydrophilic functionality and c) Hydrophilic monomers, along with some miscellaneous monomers of interest because they have been found at quite high levels in lignins (e.g., dihydroconiferyl alcohol 3). Caffeoyl quinic acid 6 has recently been implicated as an intermediate in the 3-hydroxylation step catalyzed by cinnamate 3-hydroxylase (C3H); 13 one of the GCEP targets in Boerjan s group is in the export of quinates to the wall. The analog, feruloyl quinic acid 8, was selected as a useful target having both the polar quinic acid moiety and a lignification-compatible ferulate unit. Caffeoyl quinic acid 6 (= chlorogenic acid) is readily available, but the ferulate analog is not; its non-trivial synthesis is provided in the Supplementary Material, Figure S1, along with the method for synthesizing the various feruloyl polyols Compounds are particularly attractive, having two ferulate moieties; their incorporation into lignins would potentially produce polymer chains with cleavable ester links in the backbone. Catechins, available from tannin pathways are also intriguing. Round 1 compounds include catechins 9-11 (Figure 2).

7 H H H H H H H Me H H 2 C H H H H Me Me Me H 1 2 H Me H 3 4 Me H H 5 6 Me H H 2 C H H H H Me H H H H H Me H H H 7 8 H 9 H H 10 H H 11 H H H H H H H Me H H Me H H H Me H Me Figure 2: Compounds synthesized (or obtained) H for Round 1 lignification studies: coniferyl alcohol Me 1, sinapyl alcohol 2, dihydroconiferyl alcohol 3, guaiacylglycerol 4, methyl caffeate 5, caffeoyl quinic acid 6, methyl ferulate 7, feruloyl quinic acid 8, epicatechin 9, epigallocatechin 10, epigallocatechin gallate 11, ethyl ferulate 12, feruloyl glycol 13, 1--feruloyl glycerol 14, 1,3-di--feruloyl glycerol 15, 2,3-di--feruloyl threitol H H H H H Me Me Round 2 monomer substitutes. Given the success in improving digestibility (see Section 3 below) using catechin derivatives, the vanillate 17 and ferulate 18 analogs, Figure 3, were of considerable interest. Again, incorporation of the various moieties into the lignin polymer would provide the opportunity to cleave the lignin via simple estercleavage reactions, and vanillate and ferulate are known to be highly compatible with lignification. Their syntheses, requiring regiospecific acylation of catechins by vanillate or ferulate, posed considerable difficulties, but have been accomplished using improved methods for selective protection of epicatechin as shown in Figure S2. ther so-called Round 2 compounds include: diferuloyl tartaric acid 19, a more polar bis-feruloyl component whose synthesis is shown in Figure S3; and the γ-glucosides of all three monolignols 21H/G/S these polar monolignol derivatives have been shown to produce higher-mw synthetic lignins using in vitro methods; 13 their syntheses have been completed as outlined in Figure S4. 14

8 H H H H H Me H H H H C 2 H H C 2 H H 17 Me H H 18 Me H H Me 19 H H 20 Figure 3: Compounds synthesized (or obtained) for Round 2 lignification studies: epicatechin vanillate 17, epicatechin ferulate 18, di-feruloyl tartaric acid 19, rosmarinic acid 20, and the monolignol γ-glucosides 21H/G/S. 2. Monomer compatibility with lignification. It is important that monomer substitutes are compatible with the radical coupling reactions of lignification. As we do not envision fully replacing normal monolignols, this means that monomers need to cross-couple with monolignols and with, primarily, guaiacyl and syringyl phenolic end-units in the growing polymer. We have already sufficiently documented the compatibility of ferulates with lignification, so reaffirming this for each of the ferulate-based monomers was not seen as crucial. [Incorporation into cell wall lignins, for feruloylquinic acid 8 and 1,4-di--feruloyl threitol 16, are provided below (Figure S7)]. That said, we shall be producing synthetic lignins, typically using 25% or less of the monomer-substitute in a synthetic lignin derived from 50:50 coniferyl:sinapyl alcohols to provide the necessary database for recognizing all of these components in lignins. More important has been to explore how well other phenolics, those for which we know less about their radical coupling behavior, incorporate into lignin polymers. For example, it was unknown whether the caffeates (such as in 5 and 6, Figure 2, and rosmarinic acid 20, Figure 3), rather commonly produced plant metabolites, will efficiently couple into lignins. Consequently, we produced synthetic lignins in which methyl caffeate 5 and rosmarinic acid 20 were used as low-level monomer-substitutes. As seen from the NMR evidence in Figures S5 and S6, caffeates incorporate integrally into guaiacyl lignins, even making the anticipated benzodioxane structures anticipated when the 3-H of a caffeoyl-derived moiety internally traps the intermediate quinone methide produced after a monolignol adds, at its β-position, to the catechol unit, at its 4 -position; analogous benzodioxanes have been well authenticated products of lignification with 5-hydroxyconiferyl alcohol in CMT-deficient angiosperms (see Section 5, below). In summary, although we were originally not convinced that caffeates and other o-diphenols were compatible with lignification, they appear to be so (although the full range of coupling and cross-coupling propensities remains to be determined). This means that the large number of plant metabolites in this class are open for consideration as possible lignin monomer substitutes. H H H H 21G/S/H R 1 21G: R 1 =Me, R 2 =H 21S: R 1 =R 2 =Me 21H: R 1 =R 2 =H H R 2

9 3. Cell Wall Lignification and Digestibility Testing. We have, via John Grabber s crucial collaboration, an excellent grass (corn) cell wall system for testing cell wall lignification and providing materials for digestibility testing (as well as for whole-cell-wall NMR characterization). This allows us to determine the likely efficacy of a monolignol-substitute strategy before undertaking a search for genes (involved in the substitute s biosynthesis) and plant engineering. As noted below, in Section 4, we are also seeking a dicot secondary wall system to improve the validity of testing beyond the grass system. Thus, we artificially lignified cell walls from maize cell suspensions with various combinations of normal monolignols (coniferyl and sinapyl alcohols) plus a variety of phenolic monolignol substitutes. Cell walls were then incubated in vitro with anaerobic rumen microflora to assess the potential impact of lignin modifications on the enzymatic degradability and fermentability of fibrous crops used for ruminant livestock or biofuel production. Round 1 monomer substitutes. Compounds from Round 1 have now all been incorporated into cell wall lignins; in ongoing work, we are characterizing the enzymatic saccharification of intact and chemically pretreated cell walls lignified by these and other monolignol substitutes to identify promising genetic engineering targets for improving plant fiber utilization. As alluded to above, in our search for monomer-substitutes that improve digestibility, one path explores reducing the hydrophobicity of lignin to permit greater penetration and hydrolysis of fiber by polysaccharidases. Lignin hydrophobicity could be modulated by the incorporation of phenolics with extensive sidechain or aromatic ring hydroxylation (e.g., guaiacyl glycerol 4 or epigallocatechin gallate 11) or substitution with hydrophilic groups (e.g., feruloylquinic acid 8 or 1--feruloyl glycerol 14). Another approach would be to incorporate phenolics with ortho-diol functionality (e.g., methyl caffeate 5, caffeoylquinic acid 6, epicatechin 9, epigallocatechin 10, and epigallocatechin gallate 11). The presence of such o-diphenols provides an intramolecular pathway to trap lignin quinone methide intermediates which form cross-links between lignin and structural polysaccharides; 2 such cross-links appear to limit the enzymatic hydrolysis of cell walls. 15 Another route, first illustrated by our work with coniferyl ferulate, 16,17 would be to incorporate readily cleaved bis-phenolic conjugates (e.g., epigallocatechin gallate 11,1,3-di--feruloyl glycerol 15, or 1,4-di--feruloyl threitol 16) to facilitate lignin depolymerization during pretreatment of biomass for saccharification. Evidence for the incorporation (or otherwise) of monomer-substitutes into the cell wall is provided in Figure S7. In this round, lignification with normal monolignols hindered the enzymatic hydrolysis and fermentation of cell walls at the molecular or cellular level. As this study is now published, 18 we summarize only the major results here. We artificially lignified maize cell walls with normal monolignols and a variety of phenolic monolignol substitutes normally associated with other plant metabolic pathways. Lignification with normal monolignols severely impeded structural polysaccharide hydrolysis and fermentation by rumen microflora at the molecular or cellular level in cell walls.

10 Inclusion of methyl caffeate 5, caffeoylquinic acid (chlorogenic acid) 6, or feruloylquinic acid 8 with monolignols considerably depressed lignin formation and strikingly improved the degradation and fermentation of non-pretreated cell walls. In contrast, dihydroconiferyl alcohol 3, guaiacyl glycerol 4, epicatechin 9, epigallocatechin 10, and epigallocatechin gallate 11 readily formed copolymer-lignins with normal monolignols, as did the phenylpropanoids dihydroconiferyl alcohol 3 and guaiacylglycerol 4 (although these can only become polymer end-groups). Cell wall fermentability was moderately enhanced by catechins with 1,2,3-triol functionality (in 10 and 11), while inclusion of phenylpropanoids with variously hydroxylated conjugate groups had little or no impact. Mono- or diferuloyl esters with various aliphatic or polyol groups readily formed copolymers with monolignols, but their tendency to accelerate peroxidase inactivation slightly diminished lignin formation in some cases. Relative to cell walls lignified with normal mono lignols, copolymerization of mono- or diferuloyl esters into lignin had negative Figure 5: Gas production (relatively to a normally-lignified control) for lignins augmented with various epicatechins having 5-8 phenolic hydroxyls. Gas production was markedly enhanced with increasing phenolic hydroxyl content of the monomer-substitute (in a virtually linear fashion). 19 Gas production (ml/g cell wall) Nonlignified CA+SA+FQA CA+SA CA+SA+MF Fermentation time (h) Figure 4. Blank-corrected in vitro gas production curves from nonlignified walls and from artificially lignified cell walls prepared with a 1:1 molar ratio of coniferyl alcohol 1 plus sinapyl alcohol 2 (CA+SA), or a 1:1:1 molar ratio of CA and SA plus feruloylquinic acid 8 (CA+SA+FQA) or methyl ferulate 7 (CA+SA+MF). 18 to weakly positive effects on cell wall hydrolysis and fermentability, which were in part dependent on the quantity of lignin formed and the degree of monomer hydroxylation. verall, monolignol substitutes improved the inherent fermentability of cell walls by depressing lignin formation or possibly by reducing lignin hydrophobicity or cross-linking to structural polysaccharides. For example, the improvement in gas production via rumen microbes (a good measure of cell wall fermentability) is shown in Figure 4. When epicatechin derivatives were fermented, gas production was markedly enhanced with increasing phenolic hydroxyl content of the monomersubstitute (in a virtually linear fashion), Figure 5.

11 The impacts on lignin formation, fermentability, and lignin extractablity for the control, epicatechin (5 phenols) and epigallocatechin gallate (8 phenols) are shown in Figure S8. Some monolignol substitutes, chiefly readily cleaved conjugates with two (or more) phenolic moieties separated by cleavable linkages, such as in epigallocatechin gallate 11 or diferuloyl polyol esters 15 and 16, are expected to greatly boost enzymatic saccharification of cell walls following chemical pretreatment. Such trials are still underway. Although producing cell walls that can be saccharified without requiring chemical pretreatments remains a goal, it is likely that pretreatment will be required to allow the CW polysaccharides to be fully utilized. Thus, monomer-substitutes that produce lignins that are essentially designed for simplified pretreatment, are particularly attractive targets. 4. Development of dicot cell wall culture system Although we have a cell wall system that allows us to produce ectopically lignified cell walls with lignins of our choice, the cell walls from the current maize system are in some ways too easy to delignify, even when lignified with traditional monolignols. Having a dicot system, and particularly a secondary cell wall system, would be advantageous. Although there are a number of culture systems that produce tracheary elements (secondary wall structures), the amounts required for lignification studies and the follow-up digestibility studies here are beyond the realms of many of these. The Patterson lab has primarily focused on optimizing a method of plant cell culture that will yield large amounts of purified tracheary elements to be used in biochemical experiments. We have initiated callus cultures, suspension lines and nodule cultures in Arabidopsis, poplar and tobacco and find that nodule cultures are an excellent system for tracheid enrichment. Nodules are spherical independent structures that can develop in tissue cultures from numerous plant species, both in liquid- and solid-based media. They have some internal organization, they contain several cell types, including numerous vascularized centers, from which more nodules can be easily regenerated; and nodules have been used successfully to regenerate Populus plants. 20 In Taxus, nodule cultures have been scaled up to produce large amounts of secondary metabolites (i.e., taxol). 21 In tobacco, we have established both callus culture and cell suspensions of the tobacco line Havana 425 in presence the cytokinin benzylaminopurine (BA) at 0.89 μm and the auxin naphtalenacetic acid (NAA) at 10 μm. From suspensions we started nodule cultures that were ready to be scaled up to large culture bottles after 4 weeks. We have experimented with cytokinin/auxin ratios to determine which concentrations are best to induce and promote sustained the growth of highly vascularized nodules. After five weeks of treatment with 8.88 μm BA, 10 µm NAA, nodules developed with a small internal void (Figures 6a and b) and numerous vascularized zones differentiated toward the internal zone of the nodule (Figure 6c). Nodules grown in medium with lower hormone concentrations (0.89 µm BA, 2.7 μm NAA) developed vascularized centers with rosette-

12 Figure 6: Tobacco nodules developed in presence of BA (8.88 μm) and NAA (10 μm). a. and b. External and internal view of the nodule. c. Vascularized zones in a cut section of the nodule stained with berberine hemisulfate. d. Detail of a rosette-like vascularized center observed in 2.7 μm of NAA and 0.89 µm of BA. e and f. Tracheary elements (TEs) present in the vascularized zones. e. stained with berberine hemisulfate. f. unstained TE. Figure 7: Percentage of occupied area by TEs, estimated by berberine hemisulfate staining of hand cut sections of nodules (as in Fig 6c) growing in three treatments with BA and 10 μm NAA, and one treatment with low NAA and 0.88 μm BA. like shapes (Figure 6d). Nearly 45% of the cut section of the nodules encompasses vascularized regions (Figure 7). Currently, we can generate approximately 60 g of nodules from a 5 g culture and roughly g of TE was obtained per bottle of culture. These results demonstrate the potential usefulness of nodule cultures to obtain grams of TEs, such as the ones showed in Fig. 6e and f, which then can be purified and be made available for biochemical experiments. We are now working on optimizing extraction and purification of TEs from the nodules. We are testing procedures involving maceration and filtration using sieves and mesh of different pore sizes (from 800 to 40 μm). In addition, we have established nodule cultures from poplar and Arabidopsis suspensions. Poplar represents a promising alternative to tobacco to produce large amounts of TEs. We will evaluate the poplar and Arabidopsis cultures as we have done with the tobacco system. These plants have the advantage of sequenced genomes and available mutants with limited lignin or other cell wall associated compounds. 5. Creation of a transgenic Arabidopsis in which a monolignol-substitute is the major monomer Creating a plant, even an unhealthy, growth-compromised one, in which the complement of normal monolignols is minor is simply an intriguing target, providing insight into just how far and in which directions lignin redesign might be contemplated. The goal here was to determine if we could develop plants in which the lignin approaches the state where none of its constitution derives from traditional monolignols! ur aim

13 Figure 8: Partial monolignol pathway showing the last steps toward the biosynthesis of the primary monolignols, coniferyl alcohol MG and sinapyl alcohol MS, and the resulting guaiacyl and syringyl units PG and PS resulting in the lignin polymer. CMTdownregulation has already been shown to result in the biosynthesis and transport to the wall of the novel monomer 5-hydroxyconiferyl alcohol M5H, and upregulation of F5H has demonstrated the effective shunting of the pathway away from coniferyl alcohol MG. Here were concomitantly upregulate F5H and downregulate CMT to provide 5-hydroxyconiferyl alcohol M5H as the major monomer for lignification. CCR H H Me H Me Me Me H H H coniferaldehyde 5-hydroxyconiferaldehyde sinapaldehyde H Me F5H H H H H Me CMT CAD CAD CAD MG coniferyl alcohol PG G unit (Guaiacyl) F5H P5H 5H unit (5-Hydroxyguaiacyl) CMT M5H 5-hydroxyconiferyl alcohol Me H H PS S unit (Syringyl) H Me MS sinapyl alcohol Peroxidase/H 2 2 Peroxidase/H 2 2 Peroxidase/H 2 2 L I G N I N here was not to simply test this concept, but arose from a desire to profoundly alter the lignin composition and structure for both academic and, potentially, practical purposes. Simple examination of the pathway, the relevant section of which is shown in Figure 8, suggests a strategy that might produce lignins that are derived almost entirely from non-traditional monolignols. Thus, if F5H is upregulated while CMT is concomitantly downregulated, it is anticipated that the cells will produce primarily 5-hydroxyconiferyl alcohol for lignification. The reasons are simple: F5H upregulation has already been shown to push the flux beyond coniferyl alcohol toward sinapyl alcohol, whereas CMT downregulation limits the cell s ability to produce sinapyl alcohol and results in the biosynthesis of 5-hydroxyconiferyl alcohol that, importantly, is exported to the wall and incorporated into lignins. If there is no feedback inhibition, therefore, such double and opposing misregulation might be expected to result in the production of little coniferyl alcohol, little sinapyl alcohol, and primarily 5-hydroxyconiferyl alcohol for lignification. Beyond just delineating how far it is possible to push the incorporation of a novel monomer into lignin, however, there is a potentially viable practical reason for pursuing such a course. Although substantial levels of 5-hydroxyconiferyl alcohol can apparently be tolerated in lignification without obvious adverse effects on plant growth and development, we anticipated that massive replacement of the traditional monolignols with 5-hydroxyconiferyl alcohol was likely to result in plant defects; after all, the structure, hydrophobicity, and mechanical properties of the resultant lignins would be significantly altered. But there is one feature of 5-hydroxyconiferyl alcohol incorporation

14 that might have considerable practical benefit lignin-polysaccharide cross-linking is likely to be significantly reduced, leading to potentially improved wall digestibility and saccharifiability. Thus, although the mechanism has only been tentatively proposed H γ H α β 5H 5 4 Me M5H. G Me PG. (e.g., G-end-unit) Me H 5H H Me G H PS H 2 QM5H/G intermediate Me 3 G 4 H β H ( PS) γ α H 5H H Me Me H 5H H P5H (New end-unit) α G γ β H Me MG. Me 5H H P5H. H G Me Me H γ 4 5 β α New benzodioxane unit in Lignin Figure 9: Mechanism by which the 5-hydroxyconiferyl alcohol monomer M5H incorporates into lignins forming novel benzodioxane units in the polymer. The scheme is shown using the example of incorporation of a 5-hydroxyconiferyl alcohol monomer into a guaiacyl lignin, but analogous coupling reactions occur to a PS unit in lignin (although the dotted arrow indicating the possibility of coupling to the 5-position is then not possible) and, in the bottom row, analogous coupling can occur from any of the monomers MG, MS, or M5H to continue chain extension; in the case of M5H, as is apparently prevalent in the lignin here, extended benzodioxane chains, see Figure 3, may result. An important aspect is the differing options for re-aromatizing the quinone methide intermediates QM. Normal β 4-guaiacyl or β 4-syringyl quinone methides, QMX/G or QMX/S, (X = G, S, or 5H) re-aromatize by external nucleophilic addition, usually by water, but also by polysaccharide hydroxyl attack. The latter produces benzylic -polysaccharide ethers that consitute a form of lignin-polysaccharide cross-linking. In contrast, the β 4-(5-hydroxyguaiacyl) quinone methide QMG/5H shown (or any quinone methide QMX/5H) will be rapidly trapped internally by the 5-H that is uniquely present in quinone methides derived from coupling of a monomer (at its β-position) with a 5-hydroxyguaiacyl end-unit. Importantly, polysaccharide trapping of quinone methide intermediates can not occur because the external nucleophilic attack cannot compete with the intramolecular process. As a consequence, lignin-polysaccharide cross-linking can not result when 5-hydroxyguaiacyl units are involved. Although crosslinking of the two polymers may be crucial to the plant s cell wall structural integrity, reduced cross-linking is expected to improve saccharifiability. H 5H H PS H 2 QMG/5H intermediate Me G H 5H Me

15 (by ourselves), CMT-downregulation in alfalfa does indeed produce plants which, despite having essentially the same lignin levels, are more digestible. Examination of the pathway by which 5-hydroxyconiferyl alcohol incorporates into lignin, Figure 9, reveals the basis for our contention. After 5-hydroxyconiferyl alcohol couples into the growing polymer, coupling of any monolignol, or 5-hydroxyconiferyl alcohol again, with the new 5-hydroxyguaiacyl end generates a quinone methide intermediate, as usual. However, whereas β 4-guaiacyl or β 4-syringyl quinone methides, QMX/G or QMX/S, (X = G, S, or 5H) re-aromatize by external nucleophilic addition, the β 4-(5- hydroxyguaiacyl) quinone methide QMG/5H shown (or any quinone methide QMX/5H) will be rapidly trapped internally by the 5-H that is uniquely present in the latter case. Quinone methide rearomatization is usually by water but can also be by polysaccharide hydroxyl attack, the latter resulting in lignin-polysaccharide cross-linking, Figure 9. As has been demonstrated previously for ferulate-mediated lignin-polysaccharide crosslinking in grasses, covalent cross-linking between the two polymer classes significantly reduces the degradability of plant cell wall polysaccharides by polysaccharidase enzymes and the microbes that produce them. Indeed, lignin-polysaccharide cross-linking is likely to be one of the major factors involved in recalcitrance to saccharification attributable to lignins. It is therefore logical that lignins produced primarily from 5-hydroxyconiferyl alcohol as a monomer will have markedly reduced cross-linking to polysaccharides, and that the cell walls in plants with such lignins may therefore be more readily degraded. a WT S : G : 5HG ~ 17.3 : 82.7 : b comt S : G : 5HG ~ 3.9 : 94.8 : c C4H:F5H1 S : G : 5HG ~ 100 : 0 : d comt C4H:F5H1 S : G : 5HG ~ 15.2 : 13.8 : e comt C4H:F5H1 chs S : G : 5HG ~ 46.4 : 33.5 : S2/6 5H6+S2/6 S2/6+5H H2 5H G G 2 5 Me R S 2 Me Me R H 125 Me H Figure 10: Partial short-range 13 C 1 H (HSQC) spectra (aromatic regions) of acetylated enzyme lignins. a: WT control lignin is a typically composed from S- and G-units. d: The lignin in the F5H-upregulated CMT-downregulated (comt C4H:F5H1) plants is largely derived from 5-hydroxyconiferyl alcohol, with only small amounts of traditional S and G units being detectable. ther reference lines are shown in b (CMT-deficient, with only low S-levels and detectable 5H-levels), c, F5H-upregulated, with very high levels of S-units, essentially no G-units), and e (CMT-downregulated, F5H-upregulated, and CHS-downregulated lines that restore some growth but lower the 5H-content).

16 It was to test these various hypotheses that we therefore sought to explore the ramifications of concomitant F5H-upregulation and CMT-downregulation, initially in Arabidopsis, in collaboration with the Belgian GCEP group lead by Boerjan. [Chapple s group, also in GCEP, has independently approached the same idea]. Boerjan s report will describe the project and findings in more detail. Strikingly, when Arabidopsis F5H1 is over-expressed in a comt mutant background, the lignin is derived from ~71% 5-hydroxyconiferyl alcohol, the monolignol-substitute, Figure 10. The resulting lignins contain an overwhelming proportion, ~92%, of novel benzodioxane units that are not detectable in control plants, Figure 11. Such a level of a single inter-unit type is unprecedented, in normal or transgenic plants, even for the predominant β-ether linkages in normal lignins. This is the first transgenic with a lignin that is comprised almost entirely from units that are undetectable in WT plants. As described in Boerjan s report, the plants are severely compromised and not worth testing for digestibility. Attempts to mitigate the plant growth defects by addressing the a) WT b) F5H, CMT A: 75.9%, B: 17.2%, C: 6.2%, D: 0.7%, J: 0.0% Bβ 50 A: 6.5%, B: 0.9%, C: 0.8%, D: 0.0%, J: 91.9% 50 Methoxyl Cβ Methoxyl Cβ Aγ + Dγ (+ PS) 60 Aγ + Jγ (+ PS) 60 Bγ Bγ Aα Cγ 70 Aα Cγ 70 Jβ Jα Aβ 80 Aβ 80 Dα Dβ Cα Bα H γ γ β H β H α γ β α α Me Cα 90 ppm c) F5H, CMT, CHS A: 59.1%, B: 1.4%, C: 1.5%, D: 0.0%, J: 38.0% Methoxyl 90 ppm 50 A β-aryl ether (β--4) B phenylcoumaran β-5 C resinol β-β Aγ + Jγ (+ PS) Bγ Me 70 Me Me α β H H 4 5 β α Methoxyl Aα Jα Aβ Jβ 80 D dibenzodioxocin 5-5/β--4 J benzodioxane (β--4) Unresolved, Unassigned, saccharides etc Figure 11: Partial short-range 13 C 1 H (HSQC) spectra (aliphatic regions) of acetylated enzyme lignins. a: WT control lignin. b: The lignin in the F5H-upregulated CMTdownregulated plants have huge amounts of new benzodioxane structures J and only traces of normal units A and C. c: Attempted rescue of growth defects by CHSdownregulation was also accompanied by lesser novel 5H monomers (and therefore produced fewer benzodioxane units J). Cα 90 ppm

17 overproduction of flavonols, using chalcone-synthase downregulation, 22 restored plant growth to near wild-type status, but the 5H level was not nearly as high. The resulting comt C4H:F5H1 chs plants, while resurrecting some of the growth defects, revealed tissue specific and genetic background-dependent interactions between phenylpropanoid and flavonoid biosynthesis that need to be taken into account in future cell wall engineering strategies. In summary, whereas this approach was successful in producing plants with lignin derived from primarily a novel monolignol-substitute, and whereas that lignin was quite simple and regular and, presumably, had reduced polysaccharide cross-linking, it has not resulted in plant materials that are agronomically sound. As the forthcoming publication will show (see Publication #4, from this work), the work has helped delineate how various pathways interact and, again, is the first report showing that lignin can be comprised almost entirely from units that are undetectable in WT plants. 6. Miscellaneous A number of other research endeavors accompany this work. We are collaborating with the Belgian GCEP group (Boerjan) on modeling synthetic lignification, and on identifying and authenticating plant phenolics that might be utilized as monolignolsubstitutes. We also continue to identify novel components in the lignins in natural, mutant, and transgenic plants, to provide further insight into the malleability of the lignification process and to provide new leads into potential modifications. Future Plans Although there is some logical deviation from the original proposal, based on the findings to date, the plan is to continue to test classes of compatible, plant-synthesized phenolics for their abilities to enter lignification, altering the lignin polymer in ways that allow simpler cell wall deconstruction and/or enhanced cell wall digestibility. Thus, further compounds, and further classes of compounds will continue to be tested. At the same time, cell walls now lignified with current round compounds need to be evaluated, particularly for their digestibility after simple chemical pretreatments. It is likely, again, that those monomer-substitutes that introduce readily cleavable linkages into the backbone of the polymer will respond particularly favorably to pretreatments that cleave those linkages, degrading the lignins to a far greater extent and/or under milder conditions, than is possible for native lignins. It is anticipated that, by the end of this project, we will have a very good idea of what types of targets are the best to pursue for producing plant cell walls that are ideally tailored toward industrial utilization. Conclusions The project described here, when coupled with collaborating studies from other labs,

18 will help delineate just how far lignification can be perturbed in various directions, and will develop new leads toward altered lignins with improved processing or utilization potential structurally altering lignin by altering its monomer complement will allow the biomass polysaccharides to be more efficiently and sustainably utilized. There is currently no way of knowing which of these lignin-alteration strategies might be tolerated by plants, but classes of targets that confer significant potential for improved biomass processing will be identified. Although it is impossible to detail the anticipated energy savings at this point, reducing the recalcitrance of plant cell walls to industrial processing, for biofuels and beyond, will have an enormous impact on biofuels production and, consequently, on mitigating net GHG emissions. As noted previously, we have been exploring one novel monomer system under prior auspices. 17 The preliminary results already attest to the potential of the GCEP approach for success. The prospective energy savings are indicated by the remarkable processing improvements on cell walls. For example, with 25% of the monomer-substitute coniferyl ferulate incorporated into lignins, alkaline pulping at 100 C resulted in the same degree of delignification (and produced 16% higher fiber yield) as from the normally-lignified material at 160 C. Introducing 60% of the monomer-substitute allowed the pulping to be carried out to the same level at 30 C and produced 67% higher fiber yield. Such gains portend enormous potential for sustainable local (and even small-scale) processing without massive facility costs; a conventional pulp mill digester facility currently costs ~$1 billion, for example. Similar energy savings, and consequent reductions in greenhouse gas emissions, are anticipated from reducing the energy requirements for processing biomass into liquid fuels. Along with the near carbon neutrality of utilizing plant biomass (instead of fossil fuel sources), these lignin-modified plant materials have the potential to significantly ameliorate greenhouse gas emission in the transportation fuel industry globally. Publications The following papers and presentations cite this GCEP grant. ne provisional patent has also been filed. Refereed Publications 1. R. Vanholme, K. Morreel, J. Ralph and W. Boerjan. Lignin biosynthesis and structure. Plant Physiology, 2010, in press. 2. F. van Parijs, K. Morreel, J. Ralph, W. Boerjan and R. M. H. Merks. Modeling lignin polymerization. Part 1: simulation model of dehydrogenation polymers. Plant Physiology, 2010, in press. 3. J. H. Grabber, P. F. Schatz, H. Kim, F. Lu and J. Ralph. Identifying new lignin bioengineering targets: 1. Monolignol substitute impacts on lignin formation and cell wall fermentability. BMC Plant Biology, 2010, in press. 4. R. Vanholme, J. Ralph, T. Akiyama, F. Lu, J. Rencoret Pazo, J. Christensen, A. Rohde, K. Morreel, R. DeRycke, H. Kim, B. Van Reusel and W. Boerjan. Engineering Traditional Monolignols out of Lignins. The Effects of Concomitant F5H1-up-regulation and CMT-down-regulation in Arabidopsis, 2010, in final preparation.

19 Presentations 1. J. H. Grabber, J. Ralph, D. K. Ress and X. Pan. Incorporation of epicatechin esters into lignin enhances cell wall fermentability. 32nd Symposium on Biotechnology for Fuels and Chemicals, Hilton Clearwater Beach, Clearwater, FL, 2010, Paper # J. H. Grabber, J. Ralph and X. Pan. Identifying new lignin bioengineering targets: Monolignol substitute impacts on lignin formation and cell wall utilization. 32nd Symposium on Biotechnology for Fuels and Chemicals, Hilton Clearwater Beach, Clearwater, FL, 2010, Paper # D. K. Ress, Y. Tobimatsu, H. Kim, F. Lu, J. Ralph, C. Davidson and J. H. Grabber. Development of a Systems Based Lignification Archetype Using Unconventional Lignin Monomer Substitutes. Global Climate & Energy Project, Stanford University, J. Ralph, H. Kim, F. Lu, D. K. Ress, Y. Tobimatsu, X. Pan, S. Patterson and J. H. Grabber. Efficient biomass conversion: delineating the best monolignol substitutes (introductory overview). Global Climate & Energy Project, Stanford University, J. Ralph, J. H. Grabber, F. Lu, H. Kim, J. M. Marita and R. D. Hatfield. Incorporation of monolignol conjugates into lignin for improved processing. in 2009 IUFR Tree Biotechnology Conference, Whistler, BC, Canada, J. Ralph, J. H. Grabber, F. Lu, H. Kim, J. M. Marita and R. D. Hatfield. Designing lignins for improved biomass processing. Global Climate and Energy Change (GCEP) Research Symposium, Frances C. Arrillaga Alumni Center, Stanford University, Stanford, CA, J. Ralph. A basis for... vercoming the lignin problem in biomass conversion Contemporary Biochemistry, Biofuels Seminar Series, Ebling Symposium Center, Microbial Sciences Building, U. Wisconsin-Madison, Ralph, J.; Hatfield, R. D.; Grabber, J. H.; Lu, F.; Kim, H.; Marita, J. M. Designing lignins for improved biomass processing, FuncFiber 2008 International Symposium on the biology and biotechnology of wood, Umeå, Sweden, March 10-12, 2008, 2008; Umeå, Sweden, 2008; p Ralph, J., Modification of cell wall structure to improve biomass processing. Scion Seminar Series, Scion, Rotorua, New Zealand, Patent Application 1. J. H. Grabber, J. Ralph. Incorporation of flavan-3-ols and gallic acid derivatives into lignin to improve biomass utilization. USA Provisional Patent, #61/325,695, April 19, References 1. J. Ralph. Perturbing Lignification. in The Compromised Wood Workshop 2007, eds. K. Entwistle, P. J. Harris and J. Walker, Wood Technology Research Centre, University of Canterbury, New Zealand, Canterbury, 2007, pp J. Ralph. What makes a good monolignol substitute? in The Science and Lore of the Plant Cell Wall Biosynthesis, Structure and Function, ed. T. Hayashi, Universal Publishers (BrownWalker Press), Boca Raton, FL, 2006, pp J. Ralph, K. Lundquist, G. Brunow, F. Lu, H. Kim, P. F. Schatz, J. M. Marita, R. D. Hatfield, S. A. Ralph, J. H. Christensen and W. Boerjan. Lignins: natural polymers from oxidative coupling of 4-hydroxyphenylpropanoids. Phytochemistry Reviews, 2004, 3,

20 4. W. Boerjan, J. Ralph and M. Baucher. Lignin biosynthesis. Annual Reviews in Plant Biology, 2003, 54, R. Vanholme, K. Morreel, J. Ralph and W. Boerjan. Lignin engineering. Current pinion in Plant Biology, 2008, 11, J. Ralph, C. Lapierre, J. Marita, H. Kim, F. Lu, R. D. Hatfield, S. A. Ralph, C. Chapple, R. Franke, M. R. Hemm, J. Van Doorsselaere, R. R. Sederoff, D. M. Malley, J. T. Scott, J. J. MacKay, N. Yahiaoui, A.-M. Boudet, M. Pean, G. Pilate, L. Jouanin and W. Boerjan. Elucidation of new structures in lignins of CAD- and CMT-deficient plants by NMR. Phytochemistry, 2001, 57, J. Ralph, H. Kim, F. Lu, J. H. Grabber, J.-C. Leplé, J. Berrio-Sierra, M. Mir Derikvand, L. Jouanin, W. Boerjan and C. Lapierre. Identification of the structure and origin of a thioacidolysis marker compound for ferulic acid incorporation into angiosperm lignins (and an indicator for cinnamoyl-coa reductase deficiency). The Plant Journal, 2008, 53, J. Ralph, R. F. Helm, S. Quideau and R. D. Hatfield. Lignin-feruloyl ester crosslinks in grasses. Part 1. Incorporation of feruloyl esters into coniferyl alcohol dehydrogenation polymers. Journal of the Chemical Society, Perkin Transactions 1, 1992, D. J. Yelle, J. Ralph and C. R. Frihart. Characterization of non-derivatized plant cell walls using high-resolution solution-state NMR spectroscopy. Magnetic Resonance in Chemistry, 2008, 46, H. Kim, J. Ralph and T. Akiyama. Solution-state 2D NMR of Ball-milled Plant Cell Wall Gels in DMS-d 6. BioEnergy Research, 2008, 1, J. Ralph and F. Lu. Cryoprobe 3D NMR of acetylated ball-milled pine cell walls. rganic and Biomolecular Chemistry, 2004, 2, F. Lu and J. Ralph. Non-degradative dissolution and acetylation of ball-milled plant cell walls; high-resolution solution-state NMR. The Plant Journal, 2003, 35, Y. Tobimatsu, T. Takano, H. Kamitakahara and F. Nakatsubo. Studies on the dehydrogenative polymerizations of monolignol beta-glycosides. Part 2: Horseradish peroxidase catalyzed dehydrogenative polymerization of isoconiferin. Holzforschung, 2006, 60, T. Takano, Y. Tobimatsu, T. Hosoya, T. Hattori, J. hnishi, M. Takano, H. Kamitakahara and F. Nakatsubo. Studies on the dehydrogenative polymerizations of monolignol beta-glycosides. Part 1. Syntheses of monolignol beta-glycosides, (E)-isconiferin, (E)-isosyringin, and (E)-triandrin. Journal of Wood Chemistry and Technology, 2006, 26, J. H. Grabber and R. D. Hatfield. Methyl esterification divergently affects the degradability of pectic uronosyls in nonlignified and lignified maize cell walls. Journal of Agricultural and Food Chemistry, 2005, 53, J. H. Grabber, R. D. Hatfield and J. Ralph. Apoplastic ph and Monolignol Addition Rate Effects on Lignin Formation and Cell Wall Degradability in Maize.

21 Journal of Agricultural and Food Chemistry, 2003, 51, J. H. Grabber, R. D. Hatfield, F. Lu and J. Ralph. Coniferyl ferulate incorporation into lignin enhances the alkaline delignification and enzymatic degradation of maize cell walls. Biomacromolecules, 2008, 9, J. H. Grabber, P. F. Schatz, H. Kim, F. Lu and J. Ralph. Identifying new lignin bioengineering targets: 1. Monolignol substitute impacts on lignin formation and cell wall fermentability. BMC Plant Biology, 2010, in press. 19. J. H. Grabber, J. Ralph, D. K. Ress and X. Pan. Incorporation of epicatechin esters into lignin enhances cell wall fermentability. in 32nd Symposium on Biotechnology for Fuels and Chemicals, Hilton Clearwater Beach, Clearwater, FL, Editon edn., 2010, p. Paper # B. H. McCown, E. L. Zeldin, H. A. Pinkalla and R. R. Dedolph. Nodule culture: a developmental pathway with high potential for regeneration, automated micropropagation and plant metabolite production from woody plants. in Genetic manipulation of woody plants, eds. J. W. Hanover, D. E. Keathley, C. M. Wilson and G. Kuny, Plenum, New York, 1988, pp D. D. Ellis, E. L. Zeldin, M. Brodhagen, W. A. Russin and B. H. McCown. Taxol Production in nodule cultures of Taxus. Journal of Natural Products, 1996, 59, S. Besseau, L. Hoffmann, P. Geoffroy, C. Lapierre, B. Pollet and M. Legrand. Flavonoid accumulation in Arabidopsis repressed in lignin synthesis affects auxin transport and plant growth. Plant Cell, 2007, 19, Contacts John Ralph: jralph@wisc.edu Xuejun Pan: xpan@wisc.edu Sara Patterson: spatters@wisc.edu

22 Supplementary Material Synthetic schemes for some of the monolignol-substittues, additional spectral evidence for lignificaiton reactions, and some additional data. N Me H 7 Me Me H Me Me a b c Si Si Si Me N H C 2H H C2H H H H d H e Si f Si - Na + H g H H 8 Me H Me Cl H h H Me H i Me H 13 H Me H H H H h H h H Me Me H j Me Me i H Me H H i Me H H 15 H 14 Me H H Me H H h H i,k H H H H Me Figure S1: Synthetic schemes for feruloyl quinic acid 8 and feruloyl polyols Reagents and conditions are: a. (CH 3 ) 3 SiCH 2 CH 2 CH 2 Cl, [(CH 3 ) 2 CH] 2 NH, CH 2 CL 2 ; b. 1M NaH; c. carbonyldiimidazole, DMF; d. cyclohexanone, DMF; e. 1M NaH, H 2, dioxane; f. NaH, DMF; g: 1M HCl; h. CH 2 Cl 2, pyridine, DMAP; i. pyrrolidine; j. 80% acetic acid; k 1M HCl. Me Me 16 Me

23 H H Cl Ac Ac Me H Ac 2 Pyridine (95%) Me Ac SCl 2 Toluene Reflux Me Ac Et 3 N CH 2 Cl 2 (90%) Ac Ac M + Pyrrolidine CH 2 Cl 2 (47%) H H A H H 9 H H H Ac 2 Pyridine Ac Ac Ac Ac H + Et 3 N H Ac H 18 Ac Ac M H H Ac 2 H SCl 2 Cl CH 2 Cl 2 (84%) Ac Me Me H Pyridine (97%) Me Ac Toluene Reflux Me Ac Pyrrolidine CH 2 Cl 2 (42%) H H Ac H H 17 Me H Figure S2: Synthesis of epicatechin vanillate 17, and epicatechin ferulate 18. Conditions and yields are provided on the figure.

24 H Bn H BnBr KH Bn Me H K 2 C 3 DMF (89%) Me H H Bn C 2 H C 2 H H 2, THF (90%) BnH Toluene Reflux (98%) Me H H Bn C 2 Bn C 2 Bn Figure S3: Synthesis of diferuloyl tartaric acid 19. Conditions and yields are provided on the figure. EDC. HCl CH 2 Cl 2 DMAP (70%) Me Bn H Me Pd(Ac) 2 Et 3 N, Et 3 SiH CH 2 Cl 2 (80%) C 2 Bn C 2 Bn Me C 2 H C 2 H H Me 19 H H H a b R 2 R 1 R 2 R 1 R 2 R 1 H Ac Ac 1G, 1S, 1H 2G, 2S, 2H 3G, 3S, 3H H H H c Piv Piv Piv Piv Piv H Piv Piv H Piv Piv Br d e Piv Piv Piv Piv f Piv Piv Piv H NH Piv 7 8 CCl 3 G: R1=Me, R2=H S: R1=R2=Me H: R1=R2=H 3G, 3S, or 3H 8 g Piv Piv Piv Piv R 1 R 1 Ac h H H H R 2 H H R 2 9G, 9S, 9H 21G/S/H Figure S4: Synthesis of monolignol glucosides 21H/G/S. 14 Note: the compounds numbered in this scheme do NT relate to those in the main section, except for the title product compounds 21. Reaction conditions: a. Ac 2 /Py; b. i: (CCl) 2 /DMF ii: NaBH 4 / DMF; c. PivCl/Py/CHCl 3 ; d. HBr/AcH/CH 2 Cl 2 ; e. Ag 2 C 3 /Acetone-H 2 (9:1, v/v); f. CCl 3 CN/DBU/CH 2 Cl 2 ; g. BF 3 -Et 2 /CH 2 Cl 2 ; h. NaMe/MeH.

25 Aγ Caff-Me X1γ Bγ Aα Cγ Cγ Eα 80 Eβ 80 Dα Cα Aβ Dβ Dα Cα Aβ Dβ 90 Bα 90 Bα Cβ Aγ (Eγ) Bγ Aα Bβ F2 [ppm] A: β--4; B: β-5; C: β-β; D: dibezodioxocin; E: 5.0 Benzodioxane 110 G2 YT2-8-CaffMeCADHP8 HSQC_ADIA_GEL.2.jr DMS D:\\ yt 17 DHP from CA&CaffMe (80/20, mol/mol) G2 Caffβ G5, G6 Cath2,Cath5, Cath6 (??) X2β 140 X1α X1β 140 X1α X1β 3.0 F2 [ppm] Me G5, G6 3.5 F1 [ppm] YT1-114-CA-DHP-114 HSQC_ADIA.jr DMS D:\\ yt 11 X2β 4.0 H G: Guaiacyl X1: coniferyl alcohol; X2: coniferyl aldehyde Cath: Cathecol; Caff: Caffeoate DHP from CA 4.5 F1 [ppm] F1 [ppm] DHP from CA&CaffMe (80/20, mol/mol) 60 X1γ YT2-8-CaffMeCADHP8 HSQC_ADIA_GEL.2.jr DMS D:\\ yt Cβ 120 Bβ DHP from CA F1 [ppm] YT1-114-CA-DHP-114 HSQC_ADIA.jr DMS D:\\ yt Caffα X2α X2α F2 [ppm] F2 [ppm] Figure S5: 2D HSQC NMR of sidechain (top) and aromatic (bottom) regions of synthetic lignins from solely coniferyl alcohol 1 (left) and 80:20 coniferyl alcohol:methyl caffeate 5 (right). New signals derived from incorporation of the caffeoyl moieties are seen in the aromatic region (bottom), and new benzodioxane structures in the lignin are noted by their diagnostic correlations (labeled E) in the sidechain regions; benzodioxanes are anticipated when a monolignol adds to an o-diphenol after β 4-radical coupling, the intermediate quinone methide produced is internally trapped by the 3-H.

26 F1 [ppm] F1 [ppm] YT1-114-CA-DHP-114 HSQC_ADIA.jr DMS D:\\ yt 11 DHP from CA YT2-22-RA-DHP22 HSQC_ADIA_GEL.2.jr DMS D:\\ yt 19 DHP from CA&RA (80/20, mol/mol) (RA7 ) Bβ Cβ Bβ Cβ Bα X1γ Aα Cγ Aβ Cα Bγ Aγ X1γ Bγ Aα Cγ (RA8 ) Eα Eβ Dα Cα Aβ Dβ Bα Aγ (Eγ) F2 [ppm] A: β--4; B: β-5; C: β-β; D: dibezodioxocin; E: Benzodioxane F2 [ppm] YT1-114-CA-DHP-114 HSQC_ADIA.jr DMS D:\\ yt 11 DHP from CA X2β G2 G5, G6 X1α X1β G: Guaiacyl; X1: coniferyl alcohol; X2: coniferyl aldehyde; RA: rosmarinic acid moiety F1 [ppm] YT2-22-RA-DHP22 HSQC_ADIA_GEL.2.jr DMS D:\\ yt 19 DHP from CA&RA RA2 (80/20, mol/mol)?? RA7 RA2 RA6 RA ?? H RA6 RA5 RA8 F1 [ppm] F2 [ppm] F2 [ppm] Figure S6: 2D HSQC NMR of sidechain (top) and aromatic (bottom) regions of synthetic lignins from solely coniferyl alcohol 1 (left) and 80:20 coniferyl alcohol:rosmarinic acid 20 (right). New signals derived from incorporation of the caffeoyl and o-diphenol moieties are seen in the aromatic region (bottom), and new benzodioxane structures in the lignin are noted by their diagnostic correlations (labeled E) in the sidechain regions; benzodioxanes are anticipated when a monolignol adds to an o-diphenol after β 4- radical coupling, the intermediate quinone methide produced is internally trapped by the 3-H. The apparent absence of rosmarinic acid s original 7 and 8 correlations suggest that other transformations have occurred during the lignification; products are still being elucidated.

27 a) b) S2/6 100 S2/ Me S 2 Me R α S 2/6 G2 } G5 + G6 Pyridine S 2/6 Pyridine G2 } G5 + G Sʹ Me Me R 6 G 2 5 Me R Pyridine Pyridine FA Me R c) EG6 EG8 S2/6 ppm 100 d) ppm 100 H 5 6 H 4 H EG 2 H H 1 3 H H H EG2 /6 G2 } G5 + G6 Pyridine FA2 G2 FA8 } G5 + G6 FA6 Pyridine Unassigned, polysaccharides etc Pyridine Pyridine FA ppm Figure S7. Delineating the incorporation of various monomers into cell walls. Aromatic regions from 13 C 1 H correlation gel-state 2D NMR spectra (HSQC) of whole cell walls in DMS-d 6 and pyridine-d 5. Maize cell walls were artificially lignified with a) coniferyl alcohol 1 and sinapyl alcohol 2 in a 1:1 molar ratio, b) coniferyl alcohol, sinapyl alcohol, and feruloylquinic acid 8 in a 1:1:1 molar ratio, c) coniferyl alcohol, sinapyl alcohol, and epigallocatechin gallate 11 in a 1:1:0.5 molar ratio, and d) coniferyl alcohol and 1,4-di-feruloyl threitol 16 in a 4:1 molar ratio. Note that in c and d, but not b, it is apparent that the monomer-substitutes have entered lignification in the cell wall. ppm

28 Epicatechin phenolic hydroxylation/o-diols/esters: Cell wall lignin content Epicatechin phenolic hydroxylation/o-diols/esters: Cell wall fermentability No impact on lignin content Not all epicatechin derivatives are readily incorporated into lignin Enhanced fermentability Related to less hydrophobic lignin & cross-linking? Normal monolignols Epicatechin Epigallocatechin gallate Normal monolignols Epicatechin Epigallocatechin gallate Epicatechin phenolic hydroxylation/o-diols/esters Epicatechin phenolic hydroxylation/o-diols/esters; Cell wall delignification Enhanced fermentability related to phenolic hydroxylation See poster for more information Enhanced lignin extractability Related to ionization of H groups in alkali and less crosslinking? Normal monolignols Epicatechin Epigallocatechin gallate Figure S8: Effects on lignification and on fermentability of incorporating epicatechin 9 and epigallocatechin gallate 11 into cell wall lignins. 19 The graph is the same as that in Figure 5 (in the main section -- see that caption).