Identifying new lignin bioengineering targets: 1. Monolignol-substitute impacts on lignin formation and cell wall fermentability

BACKGROUND: Recent discoveries highlighting the metabolic malleability of plant lignification indicate that lignin can be engineered to dramatically alter its composition and properties. Current plant biotechnology efforts are primarily aimed at manipulating the biosynthesis of normal monolignols, but in the future apoplastic targeting of phenolics from other metabolic pathways may provide new approaches for designing lignins that are less inhibitory toward the enzymatic hydrolysis of structural polysaccharides, both with and without biomass pretreatment. To identify promising new avenues for lignin bioengineering, 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 of fibrous crops used for ruminant livestock or biofuel production. RESULTS: In the absence of anatomical constraints to digestion, lignification with normal monolignols hindered both the rate and extent of cell wall hydrolysis by rumen microflora. Inclusion of methyl caffeate, caffeoylquinic acid, or feruloylquinic acid with monolignols considerably depressed lignin formation and strikingly improved the degradability of cell walls. In contrast, dihydroconiferyl alcohol, guaiacyl glycerol, epicatechin, epigallocatechin, and epigallocatechin gallate readily formed copolymer-lignins with normal monolignols; cell wall degradability was moderately enhanced by greater hydroxylation or 1,2,3-triol functionality. Mono- or diferuloyl esters with various aliphatic or polyol groups readily copolymerized with monolignols, but in some cases they accelerated inactivation of wall-bound peroxidase and reduced lignification; cell wall degradability was influenced by lignin content and the degree of ester group hydroxylation. CONCLUSION: Overall, monolignol substitutes improved the inherent degradability of non-pretreated cell walls by restricting lignification or possibly by reducing lignin hydrophobicity or cross-linking to structural polysaccharides. Furthermore some monolignol substitutes, chiefly readily cleaved bi-phenolic conjugates like epigallocatechin gallate or diferuloyl polyol esters, are expected to greatly boost the enzymatic degradability of cell walls following chemical pretreatment. 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.