A multi-omics approach to lignocellulolytic enzyme discovery reveals a new ligninase activity from Parascedosporium putredinis NO1.

Lignocellulose, the structural component of plant cells, is a major agricultural byproduct and the most abundant terrestrial source of biopolymers on Earth. The complex and insoluble nature of lignocellulose limits its conversion into value-added commodities, and currently, efficient transformation requires expensive pretreatments and high loadings of enzymes. Here, we report on a fungus from the Parascedosporium genus, isolated from a wheat-straw composting community, that secretes a large and diverse array of carbohydrate-active enzymes (CAZymes) when grown on lignocellulosic substrates. We describe an oxidase activity that cleaves the major ß-ether units in lignin, thereby releasing the flavonoid tricin from monocot lignin and enhancing the digestion of lignocellulose by polysaccharidase mixtures. We show that the enzyme, which holds potential for the biorefining industry, is widely distributed among lignocellulose-degrading fungi from the Sordariomycetes phylum.

Rerouting of the lignin biosynthetic pathway by inhibition of cytosolic shikimate recycling in transgenic hybrid aspen.

Lignin is a phenolic polymer deposited in the plant cell wall, and is mainly polymerized from three canonical monomers (monolignols), i.e. p-coumaryl, coniferyl and sinapyl alcohols. After polymerization, these alcohols form different lignin substructures. In dicotyledons, monolignols are biosynthesized from phenylalanine, an aromatic amino acid. Shikimate acts at two positions in the route to the lignin building blocks. It is part of the shikimate pathway that provides the precursor for the biosynthesis of phenylalanine, and is involved in the transesterification of p-coumaroyl-CoA to p-coumaroyl shikimate, one of the key steps in the biosynthesis of coniferyl and sinapyl alcohols. The shikimate residue in p-coumaroyl shikimate is released in later steps, and the resulting shikimate becomes available again for the biosynthesis of new p-coumaroyl shikimate molecules. In this study, we inhibited cytosolic shikimate recycling in transgenic hybrid aspen by accelerated phosphorylation of shikimate in the cytosol through expression of a bacterial shikimate kinase (SK). This expression elicited an increase in p-hydroxyphenyl units of lignin and, by contrast, a decrease in guaiacyl and syringyl units. Transgenic plants with high SK activity produced a lignin content comparable to that in wild-type plants, and had an increased processability via enzymatic saccharification. Although expression of many genes was altered in the transgenic plants, elevated SK activity did not exert a significant effect on the expression of the majority of genes responsible for lignin biosynthesis. The present results indicate that cytosolic shikimate recycling is crucial to the monomeric composition of lignin rather than for lignin content.

Degradation of lignin ß-aryl ether units in Arabidopsis thaliana expressing LigD, LigF and LigG from Sphingomonas paucimobilis SYK-6.

Lignin is a major polymer in the secondary plant cell wall and composed of hydrophobic interlinked hydroxyphenylpropanoid units. The presence of lignin hampers conversion of plant biomass into biofuels; plants with modified lignin are therefore being investigated for increased digestibility. The bacterium Sphingomonas paucimobilis produces lignin-degrading enzymes including LigD, LigF and LigG involved in cleaving the most abundant lignin interunit linkage, the ß-aryl ether bond. In this study, we expressed the LigD, LigF and LigG (LigDFG) genes in Arabidopsis thaliana to introduce postlignification modifications into the lignin structure. The three enzymes were targeted to the secretory pathway. Phenolic metabolite profiling and 2D HSQC NMR of the transgenic lines showed an increase in oxidized guaiacyl and syringyl units without concomitant increase in oxidized ß-aryl ether units, showing lignin bond cleavage. Saccharification yield increased significantly in transgenic lines expressing LigDFG, showing the applicability of our approach. Additional new information on substrate specificity of the LigDFG enzymes is also provided.

p-Coumaroyl-CoA:monolignol transferase (PMT) acts specifically in the lignin biosynthetic pathway in Brachypodium distachyon.

Grass lignins contain substantial amounts of p-coumarate (pCA) that acylate the side-chains of the phenylpropanoid polymer backbone. An acyltransferase, named p-coumaroyl-CoA:monolignol transferase (OsPMT), that could acylate monolignols with pCA in vitro was recently identified from rice. In planta, such monolignol-pCA conjugates become incorporated into lignin via oxidative radical coupling, thereby generating the observed pCA appendages; however p-coumarates also acylate arabinoxylans in grasses. To test the authenticity of PMT as a lignin biosynthetic pathway enzyme, we examined Brachypodium distachyon plants with altered BdPMT gene function. Using newly developed cell wall analytical methods, we determined that the transferase was involved specifically in monolignol acylation. A sodium azide-generated Bdpmt-1 missense mutant had no (<0.5%) residual pCA on lignin, and BdPMT RNAi plants had levels as low as 10% of wild-type, whereas the amounts of pCA acylating arabinosyl units on arabinoxylans in these PMT mutant plants remained unchanged. pCA acylation of lignin from BdPMT-overexpressing plants was found to be more than three-fold higher than that of wild-type, but again the level on arabinosyl units remained unchanged. Taken together, these data are consistent with a defined role for grass PMT genes in encoding BAHD (BEAT, AHCT, HCBT, and DAT) acyltransferases that specifically acylate monolignols with pCA and produce monolignol p-coumarate conjugates that are used for lignification in planta.

Estimation of Syringyl Units in Wood Lignins by FT-Raman Spectroscopy.

Syringyl (S) lignin content and the syringyl-to-guaiacyl (S/G) lignin ratio are important characteristics of wood and lignocellulosic biomass. Although numerous methods are available for estimating S lignin units and the S/G ratio, in this work, a new method based on Raman spectroscopy that uses the 370 cm-1 Raman band-area intensity (370-area) was developed. The reliability of the Raman approach for determining S content was first tested by the quantitative analysis of three syringyl lignin models by sampling them, separately, in dioxane and in Avicel. Good linear correlations between the 370 cm-1 intensity and model concentrations were obtained. Next, the percent syringyl (%S) lignin units in various woods were measured by correlating the 370 cm-1 Raman intensity data with values of S units in lignin determined by three regularly used methods, namely, thioacidolysis, DFRC, and 2D-HSQC NMR. The former two methods take into account only the monomers cleaved from ß-O-4-linked lignin units, whereas the NMR method reports S content on the whole cell wall lignin. When the 370-area intensities and %S values from the regularly used methods were correlated, good linear correlations were obtained ( R2 = 0.767, 0.731, and 0.804, respectively, for the three methods). The correlation with the highest R2, i.e., with the 2D NMR method, is proposed for estimating S units in wood lignins by Raman spectroscopy as, in principle, both represent the whole cell wall lignin and not just the portion of lignin that gets cleaved to release monomers. The Raman analysis method is quick, uses minimal harmful chemicals, is carried out nondestructively, and is insensitive to the wet or dry state of the sample. The only limitations are that the sample of wood contains at least 30% S and not be significantly fluorescent, although the latter can be mitigated in some cases.

Self-esteem change and diurnal cortisol secretion in older adulthood.

OBJECTIVE: Research suggests that self-esteem can decline in older adulthood. This process could remove a buffer that normally protects individuals against distress-related changes in cortisol secretion. We examined this possibility by testing whether change in self-esteem would predict alterations in cortisol secretion, particularly among older adults who reported high levels of depressive symptoms or perceived stress. METHODS: 147 older adults (aged 60+) completed three days of diurnal cortisol measurements at three different time points, namely every two years over a total period of four years. Measures of self-esteem, depressive symptoms, and perceived stress were assessed at T1 and T2. Potential demographic and health-related confounds were measured at baseline (partnership status, SES, mortality risk index, and medication). RESULTS: Linear regression models indicated that a decline in self-esteem from T1 to T2 predicted elevated cortisol output (AUCG) from T2 to T3, F (1, 137)=8.09, ß=-.25, R(2)=.05, p=.005. Interaction analyses revealed that this association was particularly strong among participants who experienced higher T1 or T2 levels of depressive symptoms or perceived stress, +1SD: ßs=-.34 to -.51, ps<.001, but not significant among their counterparts who reported relatively lower levels of depressive symptoms or perceived stress, -1SD: ßs=.03 to 11, ps>.43. CONCLUSIONS: Declines in self-esteem represent a mechanism that contributes to higher levels of diurnal cortisol secretion if older adults experience psychological distress. Increases in self-esteem, by contrast, can ameliorate older adults' cortisol regulation in stressful circumstances.