Down-regulation of the caffeic acid O-methyltransferase gene in switchgrass reveals a novel monolignol analog

Abstract

BackgroundDown-regulation of the caffeic acid 3-O-methyltransferase EC 2.1.1.68 (COMT) gene in the lignin biosynthetic pathway of switchgrass (Panicum virgatum) resulted in cell walls of transgenic plants releasing more constituent sugars after pretreatment by dilute acid and treatment with glycosyl hydrolases from an added enzyme preparation and from Clostridium thermocellum. Fermentation of both wild-type and transgenic switchgrass after milder hot water pretreatment with no water washing showed that only the transgenic switchgrass inhibited C. thermocellum. Gas chromatography–mass spectrometry (GCMS)-based metabolomics were undertaken on cell wall aqueous extracts to determine the nature of the microbial inhibitors.ResultsGCMS confirmed the increased concentration of a number of phenolic acids and aldehydes that are known inhibitors of microbial fermentation. Metabolomic analyses of the transgenic biomass additionally revealed the presence of a novel monolignol-like metabolite, identified as trans-3, 4-dimethoxy-5-hydroxycinnamyl alcohol (iso-sinapyl alcohol) in both non-pretreated, as well as hot water pretreated samples. iso-Sinapyl alcohol and its glucoside were subsequently generated by organic synthesis and the identity of natural and synthetic materials were confirmed by mass spectrometric and NMR analyses. The additional novel presence of iso-sinapic acid, iso-sinapyl aldehyde, and iso-syringin suggest the increased activity of a para-methyltransferase, concomitant with the reduced COMT activity, a strict meta-methyltransferase. Quantum chemical calculations were used to predict the most likely homodimeric lignans generated from dehydration reactions, but these products were not evident in plant samples.ConclusionsDown-regulation of COMT activity in switchgrass resulted in the accumulation of previously undetected metabolites resembling sinapyl alcohol and its related metabolites, but that are derived from para-methylation of 5-hydroxyconiferyl alcohol, and related precursors and products; the accumulation of which suggests altered metabolism of 5-hydroxyconiferyl alcohol in switchgrass. Given that there was no indication that iso-sinapyl alcohol was integrated in cell walls, it is considered a monolignol analog. Diversion of substrates from sinapyl alcohol to free iso-sinapyl alcohol, its glucoside, and associated upstream lignin pathway changes, including increased phenolic aldehydes and acids, are together associated with more facile cell wall deconstruction, and to the observed inhibitory effect on microbial growth. However, iso-sinapyl alcohol and iso-sinapic acid, added separately to media, were not inhibitory to C. thermocellum cultures.

Paving the Way for Lignin Valorisation: Recent Advances in Bioengineering, Biorefining and Catalysis

“…Foracomprehensive discussion, the reader is referred to arecent review article. [11] Them ajority of phenylpropanoid genetic strategies have been directed towards ad ecrease in lignin content across plant species,w ith research relating to biomass conversion being targeted at hardwoods, [42][43][44] softwoods, [45] monocots (grasses), [46][47][48][49][50][51][52][53] and dicots (including Arabidopsis and alfalfa/ truncatula). [43, However,a ni ncrease in saccharification yield will not boost the economics of abiorefinery operation if the lignin fraction, at 15-30 wt %o fd ry biomass,b ecomes amore recalcitrant material.…”

Structural Characterization of Lignin in Wild-Type versus COMT Down-Regulated Switchgrass

“…One of the promising approaches for reducing biomass recalcitrance is the development of genetically engineered plants involving down-regulation/overexpression of key enzymes involved in lignin biosynthesis that can achieve an improved sugar release performance with reduced pretreatment severities (Li et al, 2003;Chen and Dixon, 2007;Weng et al, 2008;Pu et al, 2011b;Shen et al, 2012;Tschaplinski et al, 2012). A recent report by Shen et al (2012) has demonstrated that overexpression of PvMYB4 genes in switchgrass resulted in an approximately threefold increase in sugar release efficiency from transgenic cell wall residues.…”

Tricin, a Flavonoid Monomer in Monocot Lignification

“…Several novel monomers, all deriving from the monolignol biosynthetic pathway, have been found to incorporate into lignin in wild-type and transgenic plants. For example, monolignol acetate, p-hydroxybenzoate, and p-coumarate ester conjugates have all been shown to incorporate into lignin polymers and are the source of naturally acylated lignins (Ralph et al, 2004;Lu and Ralph, 2008); lignins derived solely from caffeyl alcohol were found in the seed coats of both monocot and dicot plants (Chen et al, 2012a(Chen et al, , 2012b; lignins derived solely from 5-hydroxyconiferyl alcohol were found in a cactus (for example, in a member of the genera Astrophytum) seed coat (Chen et al, 2012a); a Medicago truncatula transgenic deficient in cinnamyl alcohol dehydrogenase exhibited a lignin that was overwhelmingly derived from hydroxycinnamaldehydes (instead of their usual hydroxycinnamyl alcohol analogs; Zhao et al, 2013); and iso-sinapyl alcohol was implicated as a monomer in caffeic acid O-methyltransferase downregulated switchgrass (Panicum virgatum; Tschaplinski et al, 2012). These findings imply that plants are quite flexible in being able to use a variety of monomers during lignification to form the heterogenous lignin polymer.…”