Coniferyl Ferulate Incorporation into Lignin Enhances the Alkaline Delignification and Enzymatic Degradation of Cell Walls John H. Grabber,* Ronald D. Hatfield, Fachuang Lu, and John Ralph United States Dairy Forage Research Center, USDA-ARS, 1925 Linden Drive West, Madison, Wisconsin 53706, and Department of Biochemistry, 433 Babcock Drive, University of Wisconsin, Madison, Wisconsin 53706 Received May 14, 2008; Revised Manuscript Received July 8, 2008 Incorporating ester interunit linkages into lignin could facilitate fiber delignification and utilization. In model studies with maize cell walls, we examined how partial substitution of coniferyl alcohol (a normal monolignol) with coniferyl ferulate (an ester conjugate from lignan biosynthesis) alters the formation and alkaline extractability of lignin and the enzymatic hydrolysis of structural polysaccharides. Coniferyl ferulate moderately reduced lignification and cell-wall ferulate copolymerization with monolignols. Incorporation of coniferyl ferulate increased lignin extractability by up to 2-fold in aqueous NaOH, providing an avenue for producing fiber with less noncellulosic and lignin contamination or of delignifying at lower temperatures. Cell walls lignified with coniferyl ferulate were more readily hydrolyzed with fibrolytic enzymes, both with and without alkaline pretreatment. Based on our results, bioengineering of plants to incorporate coniferyl ferulate into lignin should enhance lignocellulosic biomass saccharification and particularly pulping for paper production. Introduction Recent discoveries highlighting the metabolic malleability of plant lignification indicate that lignin can be engineered to dramatically diminish its adverse impact on fiber utilization for nutritional and industrial purposes. Perturbing single genes in the monolignol pathway can lead to dramatic shifts in the proportions of normal monolignols (1, 2, 3; Figure 1) polymerized into lignin or elevated incorporation of pathway intermediates into the polymer. 1 In normal plants, monolignols destined for lignin polymerization can also be extensively preacylated with acetate, p-hydroxybenzoate, or p-coumarate. 1 p-Coumarates acylate the γ-position of phenylpropanoid sidechains of mainly syringyl units in lignin. 2,3 Structural and enzymatic studies suggest that syringyl units are enzymatically preacylated with p-coumarate prior to their incorporation into lignin, 4 implicating sinapyl p-coumarate 5 as the logical precursor. Based on the analysis of isolated lignins and whole cell walls, sinapyl p-coumarate could comprise up to 40% of the lignin in some grass tissues. 2,5 p-Coumarate esters on lignin form few cross-linked structures mediated by radical coupling reactions and most remain as terminal units with an unsaturated side chain and a free phenolic group. 2 In contrast to p-coumarate, ferulates 4 esterified by simple alcohols, sugars, soluble pectins, or insoluble cell-wall xylans readily undergo diverse radical coupling reactions with each other and with lignin monomers and oligomers to form cross- linked networks. 6-10 Once polymerized into lignin, ferulate cannot be fully released by solvolytic methods. 8 Cleavage of ferulate ester linkages, however, contributes to the unusually high extractability of grass lignin and the dramatically improved enzymatic degradability of grass cell walls following mild alkaline treatments. 11 Ferulate-monolignol ester conjugates, such as coniferyl feru- late 6 or sinapyl ferulate 7 have not been identified in lignins, but they are naturally produced by some plants as secondary metabolites during, among other things, lignan biosynthesis. 12-15 This raises the exciting possibility that plants could be engi- neered to produce and transport coniferyl or sinapyl ferulate to the apoplastic space in a manner analogous to sinapyl p- coumarate, but with full incorporation of both the ferulate and the monolignol moieties of the conjugate into lignin (Figure 2). We anticipate that incorporation of these conjugates or other related diphenolics would improve lignin extraction during alkaline pulping 16 via one or more of the following mechanisms: (1) cleavage of ester interunit linkages to depolymerize lignin, (2) improved lignin solubility due to ionization of ferulic acid * To whom correspondence should be addressed. Tel.: 608-890-0059. Fax: 608-890-0076. E-mail: john.grabber@ars.usda.gov. Figure 1. Structures of p-coumaryl alcohol 1, coniferyl alcohol 2, sinapyl alcohol 3, ferulate 4, sinapyl p-coumarate 5, coniferyl ferulate 6, and sinapyl ferulate 7. Arrows indicate sites normally involved in radical coupling reactions during lignification. Biomacromolecules 2008, 9, 2510–2516 2510 10.1021/bm800528f CCC: $40.75  2008 American Chemical Society Published on Web 08/20/2008