Enzymatic basis for C-lignin monomer biosynthesis in the seed coat of Cleome hassleriana.

C-lignin is a linear polymer of caffeyl alcohol, found in the seed coats of several exotic plant species, with promising properties for generation of carbon fibers and high value chemicals. In the ornamental plant Cleome hassleriana, guaiacyl (G) lignin is deposited in the seed coat for the first 6-12 days after pollination, after which G-lignin deposition ceases and C-lignin accumulates, providing an excellent model system to study C-lignin biosynthesis. We performed RNA sequencing of seed coats harvested at 2-day intervals throughout development. Bioinformatic analysis identified a complete set of lignin biosynthesis genes for Cleome. Transcript analysis coupled with kinetic analysis of recombinant enzymes in Escherichia coli revealed that the switch to C-lignin formation was accompanied by down-regulation of transcripts encoding functional caffeoyl CoA- and caffeic acid 3-O-methyltransferases (CCoAOMT and COMT) and a form of cinnamyl alcohol dehydrogenase (ChCAD4) with preference for coniferaldehyde as substrate, and up-regulation of a form of CAD (ChCAD5) with preference for caffealdehyde. Based on these analyses, blockage of lignin monomer methylation by down-regulation of both O-methyltransferases (OMTs) and methionine synthase (for provision of C1 units) appears to be the major factor in diversion of flux to C-lignin in the Cleome seed coat, although the change in CAD specificity also contributes based on the reduction of C-lignin levels in transgenic Cleome with down-regulation of ChCAD5. Structure modeling and mutational analysis identified amino acid residues important for the preference of ChCAD5 for caffealdehyde.

Systematic approaches to C-lignin engineering in Medicago truncatula.

BACKGROUND: C-lignin is a homopolymer of caffeyl alcohol present in the seed coats of a variety of plant species including vanilla orchid, various cacti, and the ornamental plant Cleome hassleriana. Because of its unique chemical and physical properties, there is considerable interest in engineering C-lignin into the cell walls of bioenergy crops as a high-value co-product of bioprocessing. We have used information from a transcriptomic analysis of developing C. hassleriana seed coats to suggest strategies for engineering C-lignin in a heterologous system, using hairy roots of the model legume Medicago truncatula. RESULTS: We systematically tested strategies for C-lignin engineering using a combination of gene overexpression and RNAi-mediated knockdown in the caffeic acid/5-hydroxy coniferaldehyde 3/5-O-methyltransferase (comt) mutant background, monitoring the outcomes by analysis of lignin composition and profiling of monolignol pathway metabolites. In all cases, C-lignin accumulation required strong down-regulation of caffeoyl CoA 3-O-methyltransferase (CCoAOMT) paired with loss of function of COMT. Overexpression of the Selaginella moellendorffii ferulate 5-hydroxylase (SmF5H) gene in comt mutant hairy roots resulted in lines that unexpectedly accumulated high levels of S-lignin. CONCLUSION: C-Lignin accumulation of up to 15% of total lignin in lines with the greatest reduction in CCoAOMT expression required the strong down-regulation of both COMT and CCoAOMT, but did not require expression of a heterologous laccase, cinnamyl alcohol dehydrogenase (CAD) or cinnamoyl CoA reductase (CCR) with preference for 3,4-dihydroxy-substituted substrates in M. truncatula hairy roots. Cell wall fractionation studies suggested that the engineered C-units are not present in a heteropolymer with the bulk of the G-lignin.

Discovery, disassembly, depolymerization and derivatization of catechyl lignin in Chinese tallow seed coats.

The search for feedstock of catechyl lignin (C-lignin) is great interest and importance, as C-lignin featuring homogeneity and linearity is considered as an "ideal lignin" archetype for valorization and exits in only a few plant seed coats. In this study, naturally occurring C-lignin is first discovered in the seed coats of Chinese tallow, which has the highest content of C-lignin (15.4 wt%) as compared with other known feedstocks. An optimized extraction procedure by ternary deep eutectic solvents (DESs) enables the complete disassembly of C-lignin and G/S-lignin coexisted in Chinese tallow seed coats, and characterizations revealed that the as-separated C-lignin sample is abundant in benzodioxane units with no observation of ß-O-4 structures from G/S-lignin. Catalytic depolymerization of C-lignin results in a simplex catechol product in 129 mg per gram seed coats, being higher than other reported feedstocks. Derivatizing the "black" C-lignin via the nucleophilic isocyanation of benzodioxane γ-OH leads to a "whitened C-lignin" with uniform laminar structure and excellent crystallization ability, being conducive to fabricating functional materials. Overall, this contribution showed that Chinses tallow seed coats are suitable feedstock for acquiring C-lignin biopolymer.

Release of Small Phenolic Metabolites from Isotopically Labeled 13C Lignin in the Pig Cecum Model.

A diet with a high dietary fiber content is often recommended in today's nutrition due to several beneficial health effects related to its intake. Lignin as a part of dietary fiber is the second most abundant natural polymer and considered to be stable during digestion. However, some studies indicate a partial degradation during the intestinal metabolism. To further elucidate this hypothesis, the aim of this study was to investigate whether lignin is metabolized by the gut microbiota using the ex vivo pig cecum model. As potential lignin-derived metabolites might already naturally occur in the pig cecal matrix, an approach using isotopically labeled 13C lignin was chosen for this study. Ten small phenolic lignin degradation products and their time-dependent metabolism were identified via an untargeted HPLC-HRMS approach, and the quantity of the metabolites was estimated. From the results, we conclude that lignin is partially degraded releasing small phenolic metabolites.