Biosynthesis and incorporation of side‐chain‐truncated lignin monomers to reduce lignin polymerization and enhance saccharification

Abstract

SummaryLignocellulosic biomass is utilized as a renewable feedstock in various agro-industrial activities. Lignin is an aromatic, hydrophobic and mildly branched polymer integrally associated with polysaccharides within the biomass, which negatively affects their extraction and hydrolysis during industrial processing. Engineering the monomer composition of lignins offers an attractive option towards new lignins with reduced recalcitrance. The presented work describes a new strategy developed in Arabidopsis for the overproduction of rare lignin monomers to reduce lignin polymerization degree (DP). Biosynthesis of these 'DP reducers' is achieved by expressing a bacterial hydroxycinnamoyl-CoA hydratase-lyase (HCHL) in lignifying tissues of Arabidopsis inflorescence stems. HCHL cleaves the propanoid side-chain of hydroxycinnamoylCoA lignin precursors to produce the corresponding hydroxybenzaldehydes so that plant stems expressing HCHL accumulate in their cell wall higher amounts of hydroxybenzaldehyde and hydroxybenzoate derivatives. Engineered plants with intermediate HCHL activity levels show no reduction in total lignin, sugar content or biomass yield compared with wild-type plants. However, cell wall characterization of extract-free stems by thioacidolysis and by 2D-NMR revealed an increased amount of unusual C 6 C 1 lignin monomers most likely linked with lignin as end-groups. Moreover the analysis of lignin isolated from these plants using size-exclusion chromatography revealed a reduced molecular weight. Furthermore, these engineered lines show saccharification improvement of pretreated stem cell walls. Therefore, we conclude that enhancing the biosynthesis and incorporation of C 6 C 1 monomers ('DP reducers') into lignin polymers represents a promising strategy to reduce lignin DP and to decrease cell wall recalcitrance to enzymatic hydrolysis.

Silencing CHALCONE SYNTHASE in Maize Impedes the Incorporation of Tricin into Lignin and Increases Lignin Content

“…Therefore, at least part of the lower Glc release is due to the reduced cellulose content in the leaves of C2-Idf plants. It has been established that saccharification efficiency is negatively correlated with lignin amount in both monocot and dicot species (Chen and Dixon, 2007;Sattler et al, 2010;Xu et al, 2011;Bouvier d'Yvoire et al, 2013;Jung et al, 2013;Van Acker et al, 2013;Vanholme et al, 2013a) and that a lower degree of polymerization of lignin eases its extraction from the biomass (Eudes et al, 2012;Vanholme et al, 2012;Shen et al, 2013). The alkaline pretreatment hydrolyzes ester bonds, thereby releasing (di)ferulates that acylate the hemicellulose (and of which a part is also coupled to lignin) and p-coumarates that additionally acylate the lignin backbone (Ralph, 2010).…”

Development of a new high-throughput method to determine the composition of ten monosaccharides including 4-<i>O</i>-methyl glucuronic acid from plant cell walls using ultra-performance liquid chromatography

“…Rha content in A. thaliana cell wall in this study (20.5±1.5 µg mg −1 AIR) was ca. 1.5 to 3 times higher than the previous studies, but relatively close to the data (14.4 and 17.3 µg mg −1 AIR) determined by the combination of the two step-hydrolysis with sulfuric acids and the quantification with GC (Lee et al 2011b(Lee et al , 2012, while the data determined with HPEAC-PAD system (9.6 and 10.6 µg mg −1 AIR, Eudes et al 2012;Iwase et al 2009) and the data determined by the combination of one-step hydrolysis and GC (6.72 µg mg −1 AIR, Persson et al 2007) were low. Therefore higher content of Rha in A. thaliana in this study might be caused by the difference of peak resolution of quantification apparatus (HPAEC or GC) and hydrolysis condition between our study (two-step) and the previous studies.…”

Impact of Altered Cell Wall Composition on Saccharification Efficiency in Stem Tissue of Arabidopsis RABA GTPase-Deficient Knockout Mutants

“…The first of these focuses on efficiency of pre-treatment, and the second focuses on exploration of genetic modification to increase susceptibility to pre-treatment and/or enzymatic hydrolysis. Currently, in the literature, various types of pretreatment for Arabidopsis have been described, which tend to use treatment with acids, alkalis and hot water [5][6][7][8], and a number of protocols for high-throughput analysis have also been described [9,10]. However, a consensus throughout the community has yet to be established.…”

“…The second line of research has been the exploration of gene knockouts which could increase the susceptibility to enzyme hydrolysis. Most of the work in this field has focused on genes affecting lignin and xylan composition [6][7][8][11][12][13]. However, direct interference with cell wall biosynthesis machinery could have an adverse effect on the agronomic properties of the crop.…”