Engineering Functionally Specialized Monolignol 4-O-Methyltransferases to Alter Lignin Structure in Energy Crop

Lignin is an irregularly cross‐linked biopolymer and primarily composed of p-hydroxyphenyl, guaiacyl, and syringyl subunits. Lignin composition dictates the degree of lignin condensation, reactivity, and thus the degradability of plant cell walls. Over the years we have been pursuing a strategy of etherifying the para-hydroxyl of the monolignols to disturb lignin biosynthesis. We reason that preferentially modifying the para-hydroxyl of particular lignin precursor to deprive its dehydrogenation propensity would prevent the formation of particular lignin subunits therefore may change lignin structure. Combining crystal structure determination with combinatorial active site saturation mutagenesis, we engineered a set of monolignol 4-O-methyltransferases that preferentially methylate the 4-hydroxyl of coniferyl alcohol, 5-hydroxylconiferyl alcohol, sinapyl alcohol, or ferulic acid, respectively. Crystal structure determination revealed that the engineered enzyme variants showed substantial alteration in their substrate-binding pocket that governs their substrate preferences. Expressing one of the engineered variants with preference for the phenolic moiety of syringyl lignin precursor in hybrid aspens, we found that the action of this variant resulted in a significant alteration of lignin composition. The syringyl-lignin subunits and the "wall-bound" phenolics were reduced by more than 50% and, concomitantly, the cellulose fiber content was increased by up to 12%, compared to the control plants. These changes rendered a 62% increase in the simple sugar release upon saccharification of transgenic wood and up to 49% increase in ethanol yield, when the pre-treated and untreated woody biomass was subjected to the simultaneous saccharification and fermentation, compared to corresponding controls. Moreover, the greenhouse-grown transgenic plants showed no obvious biomass yield penalty. These data suggest that our strategy of etherifying lignin precursors by MOMT represents a useful biotechnological solution for effectively tailoring the digestibility of Populus woody biomass; the created aspens have promising biomass potential as feedstocks for biofuel production.