Targeting hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase for lignin modification in Brachypodium distachyon.

BACKGROUND: Hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) is a central enzyme of the so-called "esters" pathway to monolignols. As originally envisioned, HCT functions twice in this pathway, to form coumaroyl shikimate and then, in the "reverse" direction, to convert caffeoyl shikimate to caffeoyl CoA. The discovery of a caffeoyl shikimate esterase (CSE) that forms caffeic acid directly from caffeoyl shikimate calls into question the need for the reverse HCT reaction in lignin biosynthesis. Loss of function of HCT gives severe growth phenotypes in several dicot plants, but less so in some monocots, questioning whether this enzyme, and therefore the shikimate shunt, plays the same role in both monocots and dicots. The model grass Brachypodium distachyon has two HCT genes, but lacks a classical CSE gene. This study was therefore conducted to evaluate the utility of HCT as a target for lignin modification in a species with an "incomplete" shikimate shunt. RESULTS: The kinetic properties of recombinant B. distachyon HCTs were compared with those from Arabidopsis thaliana, Medicago truncatula, and Panicum virgatum (switchgrass) for both the forward and reverse reactions. Along with two M. truncatula HCTs, B. distachyon HCT2 had the least kinetically unfavorable reverse HCT reaction, and this enzyme is induced when HCT1 is down-regulated. Down regulation of B. distachyon HCT1, or co-down-regulation of HCT1 and HCT2, by RNA interference led to reduced lignin levels, with only modest changes in lignin composition and molecular weight. CONCLUSIONS: Down-regulation of HCT1, or co-down-regulation of both HCT genes, in B. distachyon results in less extensive changes in lignin content/composition and cell wall structure than observed following HCT down-regulation in dicots, with little negative impact on biomass yield. Nevertheless, HCT down-regulation leads to significant improvements in biomass saccharification efficiency, making this gene a preferred target for biotechnological improvement of grasses for bioprocessing.

Allelopathic effects of exogenous phenylalanine: a comparison of four monocot species.

MAIN CONCLUSION: Exogenous phenylalanine stunted annual ryegrass but not switchgrass or winter grain rye, with deuterium incorporation up to 3% from phenyalanine-d 8 . Toxicity to duckweed varied with illumination intensity and glucose uptake. Isotopic labeling of biomolecules through biosynthesis from deuterated precursors has successfully been employed for both structural studies and metabolic analysis. Phenylalanine is the precursor of many products synthesized by plants, including the monolignols used for synthesis of lignin. Possible allelochemical effects of phenylalanine have not been reported, although its deamination product cinnamic acid is known to have deleterious effects on root elongation and growth of several plant species. The effects of phenylalanine and its deuterated analog phenylalanine-d 8 added to growth media were studied for annual ryegrass (Lolium multiflorum), winter grain rye (Secale cereale), and switchgrass (Panicum virgatum) cultivated under hydroponic conditions. Growth of annual ryegrass was inhibited by phenylalanine while switchgrass and rye were not significantly affected. Growth was less affected by deuterated phenylalanine-d 8 than by its protiated counterpart, which may be a typical deuterium kinetic isotope effect resulting in slower enzymatic reaction rates. Deuterium incorporation levels of 2-3% were achieved in biomass of switchgrass and annual ryegrass. Both protiated and deuterated phenylalanine were moderately toxic (IC25 values 0.6 and 0.8 mM, respectively) to duckweed (Lemna minor) grown using a 12 h diurnal cycle under photoautotrophic conditions. A significant increase in toxicity, greater for the deuterated form, was noted when duckweed was grown under higher intensity, full spectrum illumination with a metal halide lamp compared to fluorescent plant growth lamps emitting in the blue and red spectral regions. Supplementation with glucose increased toxicity of phenylalanine consistent with synergy between hexose and amino acid uptake that has been reported for duckweed.

High shear homogenization of lignin to nanolignin and thermal stability of nanolignin-polyvinyl alcohol blends.

A new method to prepare nanolignin using a simple high shear homogenizer is presented. The kraft lignin particles with a broad distribution ranging from large micron- to nano-sized particles were completely homogenized to nanolignin particles with sizes less than 100 nm after 4 h of mechanical shearing. The (13) C nuclear magnetic resonance (NMR) and (31) P NMR analysis showed that there were no major changes in the chemical composition between the starting kraft lignin particles and the nanolignin obtained after 4 h of mechanical treatment. The nanolignin particles did not show any change in molecular weight distribution and polydispersity compared to the original lignin particles. The nanolignin particles when used with polyvinyl alcohol (PVA) increased the thermal stability of nanolignin/PVA blends more effectively compared to the original lignin/PVA blends.

Effect of lignin content on changes occurring in poplar cellulose ultrastructure during dilute acid pretreatment.

BACKGROUND: Obtaining a better understanding of the complex mechanisms occurring during lignocellulosic deconstruction is critical to the continued growth of renewable biofuel production. A key step in bioethanol production is thermochemical pretreatment to reduce plant cell wall recalcitrance for downstream processes. Previous studies of dilute acid pretreatment (DAP) have shown significant changes in cellulose ultrastructure that occur during pretreatment, but there is still a substantial knowledge gap with respect to the influence of lignin on these cellulose ultrastructural changes. This study was designed to assess how the presence of lignin influences DAP-induced changes in cellulose ultrastructure, which might ultimately have large implications with respect to enzymatic deconstruction efforts. RESULTS: Native, untreated hybrid poplar (Populus trichocarpa x Populus deltoids) samples and a partially delignified poplar sample (facilitated by acidic sodium chlorite pulping) were separately pretreated with dilute sulfuric acid (0.10 M) at 160°C for 15 minutes and 35 minutes, respectively . Following extensive characterization, the partially delignified biomass displayed more significant changes in cellulose ultrastructure following DAP than the native untreated biomass. With respect to the native untreated poplar, delignified poplar after DAP (in which approximately 40% lignin removal occurred) experienced: increased cellulose accessibility indicated by increased Simons' stain (orange dye) adsorption from 21.8 to 72.5 mg/g, decreased cellulose weight-average degree of polymerization (DPw) from 3087 to 294 units, and increased cellulose crystallite size from 2.9 to 4.2 nm. These changes following DAP ultimately increased enzymatic sugar yield from 10 to 80%. CONCLUSIONS: Overall, the results indicate a strong influence of lignin content on cellulose ultrastructural changes occurring during DAP. With the reduction of lignin content during DAP, the enlargement of cellulose microfibril dimensions and crystallite size becomes more apparent. Further, this enlargement of cellulose microfibril dimensions is attributed to specific processes, including the co-crystallization of crystalline cellulose driven by irreversible inter-chain hydrogen bonding (similar to hornification) and/or cellulose annealing that converts amorphous cellulose to paracrystalline and crystalline cellulose. Essentially, lignin acts as a barrier to prevent cellulose crystallinity increase and cellulose fibril coalescence during DAP.

Cellulose Isolation Methodology for NMR Analysis of Cellulose Ultrastructure.

In order to obtain accurate information about the ultrastructure of cellulose from native biomass by 13C cross polarization magic angle spinning (CP/MAS) NMR spectroscopy the cellulose component must be isolated due to overlapping resonances from both lignin and hemicellulose. Typically, cellulose isolation has been achieved via holocellulose pulping to remove lignin followed by an acid hydrolysis procedure to remove the hemicellulose components. Using 13C CP/MAS NMR and non-linear line-fitting of the cellulose C4 region, it was observed that the standard acid hydrolysis procedure caused an apparent increase in crystallinity of ~10% or less on the cellulose isolated from Populus holocellulose. We have examined the effect of the cellulose isolation method, particularly the acid treatment time for hemicellulose removal, on cellulose ultrastructural characteristics by studying these effects on cotton, microcrystalline cellulose (MCC) and holocellulose pulped Populus. 13C CP/MAS NMR of MCC indicated that holocellulose pulping and acid hydrolysis has little effect on the crystalline ultrastructural components of cellulose. Although any chemical method to isolate cellulose from native biomass will invariably alter substrate characteristics, especially those related to regions accessible to solvents, we found those changes to be minimal and consistent in samples of typical crystallinity and lignin/hemicellulose content. Based on the rate of the hemicellulose removal, as determined by HPLC-carbohydrate analysis and magnitude of cellulose ultrastructural alteration, the most suitable cellulose isolation methodology utilizes a treatment of 2.5 M HCl at 100 °C for a standard residence time between 1.5 and 4 h. However, for the most accurate crystallinity results this residence time should be determined empirically for a particular sample.

Increase in 4-coumaryl alcohol units during lignification in alfalfa (Medicago sativa) alters the extractability and molecular weight of lignin.

The lignin content of biomass can impact the ease and cost of biomass processing. Lignin reduction through breeding and genetic modification therefore has potential to reduce costs in biomass-processing industries (e.g. pulp and paper, forage, and lignocellulosic ethanol). We investigated compositional changes in two low-lignin alfalfa (Medicago sativa) lines with antisense down-regulation of p-coumarate 3-hydroxylase (C3H) or hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (HCT). We investigated whether the difference in reactivity during lignification of 4-coumaryl alcohol (H) monomers versus the naturally dominant sinapyl alcohol and coniferyl alcohol lignin monomers alters the lignin structure. Sequential base extraction readily reduced the H monomer content of the transgenic lines, leaving a residual lignin greatly enriched in H subunits; the extraction profile highlighted the difference between the control and transgenic lines. Gel permeation chromatography of isolated ball-milled lignin indicated significant changes in the weight average molecular weight distribution of the control versus transgenic lines (CTR1a, 6000; C3H4a, 5500; C3H9a, 4000; and HCT30a, 4000).

Enhanced enzymatic hydrolysis of spruce by alkaline pretreatment at low temperature.

Alkaline pretreatment of spruce at low temperature in both presence and absence of urea was studied. It was found that the enzymatic hydrolysis rate and efficiency can be significantly improved by the pretreatment. At low temperature, the pretreatment chemicals, either NaOH alone or NaOH-urea mixture solution, can slightly remove lignin, hemicelluloses, and cellulose in the lignocellulosic materials, disrupt the connections between hemicelluloses, cellulose, and lignin, and alter the structure of treated biomass to make cellulose more accessible to hydrolysis enzymes. Moreover, the wood fiber bundles could be broken down to small and loose lignocellulosic particles by the chemical treatment. Therefore, the enzymatic hydrolysis efficiency of untreated mechanical fibers can also be remarkably enhanced by NaOH or NaOH/urea solution treatment. The results indicated that, for spruce, up to 70% glucose yield could be obtained for the cold temperature pretreatment (-15 degrees C) using 7% NaOH/12% urea solution, but only 20% and 24% glucose yields were obtained at temperatures of 23 degrees C and 60 degrees C, respectively, when other conditions remained the same. The best condition for the chemical pretreatment regarding this study was 3% NaOH/12% urea, and -15 degrees C. Over 60% glucose conversion was achieved upon this condition.