Lignin serves a vital role as an inter- and intra-molecular glue strengthening plant cell walls, but it hinders numerous agro-industrial processes such as the chemical pulping of woody crops, forage digestion by livestock, and the enzymatic saccharification and fermentation of lignocellulosic biomass into liquid biofuels. As a result, considerable effort has been directed towards reducing or altering the biosynthesis of lignin in plants to permit more efficient utilization of plant cell walls [1
]. In angiosperms, lignin is normally formed by the oxidative copolymerization of monolignols, principally coniferyl alcohol (CA) and sinapyl alcohol (SA), Figure . Perturbing single or multiple genes in the monolignol pathway can lead to massive structural changes in the polymer due to dramatic shifts in the deposition of normal monolignols [8
], and/or incorporation of pathway intermediates and other phenolic compounds [12
]. The inherent malleability of plant lignification is further illustrated by the natural incorporation of various γ-acylated monolignols [13
] and ferulate arabinoxylan esters [19
] into lignin and the recent discovery of a seed-coat lignin surprisingly formed solely from caffeyl alcohol [21
]. These findings support the notion that plants could be genetically engineered to make use of precursors from alternate phenolic pathways to form lignins that are more amenable to processing [20
Structures of conventional monolignols, coniferyl alcohol (CA) and sinapyl alcohol (SA), and flavonols and gallate derivatives used in this study, epigallocatechin gallate (EGCG), epigallocatechin (EGC), and ethyl gallate (EG).
In order to identify the most promising genetic engineering targets for modifying lignin, we have used a biomimetic cell wall model system to test a variety of plant-derived phenolics as alternative precursors for lignification [31
]. These studies have demonstrated that copolymerization of hydroxycinnamate conjugates such as coniferyl ferulate [34
] and rosmarinic acid [31
] with normal monolignols dramatically improves the alkali extractability of lignin and the subsequent enzymatic hydrolysis of fiber. Accompanying in vitro
lignification studies demonstrated that these conjugates readily participate in peroxidase-catalyzed copolymerization reactions with normal monolignols. The resulting lignin contains readily cleaved ester linkages in the backbone of the polymer which permit lignin depolymerization under mild alkaline conditions [31
]. Subsequent cell wall studies revealed that several flavonoid and gallate derivatives hold promise as monolignol substitutes for modulating the adverse effects of lignin to enhance the inherent fermentability of cell walls [32
]. Among these, epigallocatechin gallate (EGCG, Figure ) was particularly attractive because it readily formed wall-bound polymers with normal monolignols and enhanced the fermentability of non-pretreated cell walls by 25% [32
]. Similarly to the aforementioned hydroxycinnamate conjugates, incorporation of EGCG could introduce easily cleaved ester linkages into the lignin backbone via oxidative coupling of its epigallocatechin and gallate moieties with monolignols. However, the involvement of these EGCG moieties in coupling reactions with monolignols is not known. It is also not known whether EGCG incorporation into lignin could enhance the delignification of cell walls by chemical pretreatment and/or their saccharification by hydrolytic enzymes.
Therefore in the present study, we examined the copolymerization of EGCG and CA into dehydrogenation polymers (synthetic lignins, DHPs), utilizing an in vitro
horseradish peroxidase (HRP)-catalyzed polymerization system that models lignin polymerization in vivo
]. Two-dimensional nuclear magnetic resonance (NMR) experiments with the DHPs revealed one major cross-coupling mode between CA and both epigallocatechin and gallate moieties of EGCG. We then subjected cell wall dehydrogenation polymers (CWDHPs) formed by artificially lignifying maize cell walls with CA, SA, and EGCG [32
] to alkaline pretreatment and enzymatic hydrolysis. Wet chemical and NMR analyses of CWDHPs, alkali-insoluble residues, and enzyme hydrolysates revealed that EGCG incorporation into lignin dramatically enhanced the delignification and enzymatic saccharification of cell walls.