Jorge Rencoret

Monolignol Ferulate Transferase Introduces Chemically Labile Linkages into the Lignin Backbone

Constructed for Deconstruction Lignin provides strength to wood but also impedes efficient degradation when wood is used as biofuel. Wilkerson et al. (p. 90) engineered poplar to produce lignin that is more amenable to degradation. From a handful of plants that contain more digestible lignin monomers, Angelica sinensis was selected and its monolignol transferase activities analyzed. The enzyme involved, coniferyl ferulate feruloyl-CoA monolignol transferase, was then expressed in poplar. The resulting poplar trees showed no difference in growth habit under greenhouse conditions, but their lignin showed improved digestibility. Engineered poplar lignin with readily cleavable ester bonds in the polymer backbone improves wood degradability. Redesigning lignin, the aromatic polymer fortifying plant cell walls, to be more amenable to chemical depolymerization can lower the energy required for industrial processing. We have engineered poplar trees to introduce ester linkages into the lignin polymer backbone by augmenting the monomer pool with monolignol ferulate conjugates. Herein, we describe the isolation of a transferase gene capable of forming these conjugates and its xylem-specific introduction into poplar. Enzyme kinetics, in planta expression, lignin structural analysis, and improved cell wall digestibility after mild alkaline pretreatment demonstrate that these trees produce the monolignol ferulate conjugates, export them to the wall, and use them during lignification. Tailoring plants to use such conjugates during cell wall biosynthesis is a promising way to produce plants that are designed for deconstruction.

Lignin Composition and Structure in Young versus Adult Eucalyptus globulus Plants

Lignin changes during plant growth were investigated in a selected Eucalyptus globulus clone. The lignin composition and structure were studied in situ by a new procedure enabling the acquisition of two-dimensional nuclear magnetic resonance (2D-NMR) spectra on wood gels formed in the NMR tube as well as by analytical pyrolysis-gas chromatography-mass spectrometry. In addition, milled-wood lignins were isolated and analyzed by 2D-NMR, pyrolysis-gas chromatography-mass spectrometry, and thioacidolysis. The data indicated that p-hydroxyphenyl and guaiacyl units are deposited at the earlier stages, whereas the woods are enriched in syringyl (S) lignin during late lignification. Wood 2D-NMR showed that β-O-4′ and resinol linkages were predominant in the eucalypt lignin, whereas other substructures were present in much lower amounts. Interestingly, open β-1′ structures could be detected in the isolated lignins. Phenylcoumarans and cinnamyl end groups were depleted with age, spirodienone abundance increased, and the main substructures (β-O-4′ and resinols) were scarcely modified. Thioacidolysis revealed a higher predominance of S units in the ether-linked lignin than in the total lignin and, in agreement with NMR, also indicated that resinols are the most important nonether linkages. Dimer analysis showed that most of the resinol-type structures comprised two S units (syringaresinol), the crossed guaiacyl-S resinol appearing as a minor substructure and pinoresinol being totally absent. Changes in hemicelluloses were also shown by the 2D-NMR spectra of the wood gels without polysaccharide isolation. These include decreases of methyl galacturonosyl, arabinosyl, and galactosyl (anomeric) signals, assigned to pectin and related neutral polysaccharides, and increases of xylosyl (which are approximately 50% acetylated) and 4-O-methylglucuronosyl signals.

HSQC-NMR analysis of lignin in woody (Eucalyptus globulus and Picea abies) and non-woody (Agave sisalana) ball-milled plant materials at the gel state

Abstract In situ analysis of lignin by 2D NMR of whole plant material was carried out by swelling finely ball-milled samples in deuterated dimethylsulfoxide (DMSO-d6 ) and sonicated so that a gel was formed in the NMR analysis tube. Solution HSQC NMR spectra of different plant materials representative for hardwood (Eucalyptus globulus), softwood (Picea abies), and non-woody plants (Agave sisalana) are presented here. The spectra show signals corresponding to those of the main plant constituents, such as lignin and polysaccharides. The lignin signals were assigned by comparing the HSQC spectra of the whole plant materials with the HSQC spectra of their respective milled-wood lignins (MWLs). In general terms, the major lignin structural features, such as the relative abundances of the main lignin substructures, the syringyl/guaiacyl ratios and the extent of γ-acetylation of the lignin side-chain observed in the HSQC spectra of the whole plant materials, matched those obtained from the HSQC spectra of the isolated MWLs. Therefore, this technique, which needs only minor amounts of lignocellulosic material and minimal sample preparation, can be useful for the rapid screening of plant lignins without the need for tedious and time-consuming lignin isolation procedures.

Tricin, a Flavonoid Monomer in Monocot Lignification

Tricin, a flavonoid, is a lignification monomer in grasses where it functions as a nucleation site for lignin polymer formation. Tricin was recently discovered in lignin preparations from wheat (Triticum aestivum) straw and subsequently in all monocot samples examined. To provide proof that tricin is involved in lignification and establish the mechanism by which it incorporates into the lignin polymer, the 4′-O-β-coupling products of tricin with the monolignols (p-coumaryl, coniferyl, and sinapyl alcohols) were synthesized along with the trimer that would result from its 4′-O-β-coupling with sinapyl alcohol and then coniferyl alcohol. Tricin was also found to cross couple with monolignols to form tricin-(4′-O-β)-linked dimers in biomimetic oxidations using peroxidase/hydrogen peroxide or silver (I) oxide. Nuclear magnetic resonance characterization of gel permeation chromatography-fractionated acetylated maize (Zea mays) lignin revealed that the tricin moieties are found in even the highest molecular weight fractions, ether linked to lignin units, demonstrating that tricin is indeed incorporated into the lignin polymer. These findings suggest that tricin is fully compatible with lignification reactions, is an authentic lignin monomer, and, because it can only start a lignin chain, functions as a nucleation site for lignification in monocots. This initiation role helps resolve a long-standing dilemma that monocot lignin chains do not appear to be initiated by monolignol homodehydrodimerization as they are in dicots that have similar syringyl-guaiacyl compositions. The term flavonolignin is recommended for the racemic oligomers and polymers of monolignols that start from tricin (or incorporate other flavonoids) in the cell wall, in analogy with the existing term flavonolignan that is used for the low-molecular mass compounds composed of flavonoid and lignan moieties.

Pretreatment with laccase and a phenolic mediator degrades lignin and enhances saccharification of Eucalyptus feedstock

BackgroundBiofuel production from lignocellulosic material is hampered by biomass recalcitrance towards enzymatic hydrolysis due to the compact architecture of the plant cell wall and the presence of lignin. The purpose of this work is to study the ability of an industrially available laccase-mediator system to modify and remove lignin during pretreatment of wood (Eucalyptus globulus) feedstock, thus improving saccharification, and to analyze the chemical modifications produced in the whole material and especially in the recalcitrant lignin moiety.ResultsUp to 50% lignin removal from ground eucalypt wood was attained by pretreatment with recombinant Myceliophthora thermophila laccase and methyl syringate as mediator, followed by alkaline peroxide extraction in a multistage sequence. The lignin removal directly correlated with increases (approximately 40%) in glucose and xylose yields after enzymatic hydrolysis. The pretreatment using laccase alone (without mediator) removed up to 20% of lignin from eucalypt wood. Pyrolysis-gas chromatography/mass spectrometry of the pretreated wood revealed modifications of the lignin polymer, as shown by lignin markers with shortened side chains and increased syringyl-to-guaiacyl ratio. Additional information on the chemical modifications produced was obtained by two-dimensional nuclear magnetic resonance of the whole wood swollen in dimethylsulfoxide-d6. The spectra obtained revealed the removal of guaiacyl and syringyl lignin units, although with a preferential removal of the former, and the lower number of aliphatic side-chains per phenylpropane unit (involved in main β-O-4ʹ and β-βʹ inter-unit linkages), in agreement with the pyrolysis-gas chromatography/mass spectrometry results, without a substantial change in the wood polysaccharide signals. However, the most noticeable modification observed in the spectra was the formation of Cα-oxidized syringyl lignin units during the enzymatic treatment. Further insight into the modifications of lignin structure, affecting other inter-unit linkages and oxidized structures, was attained by nuclear magnetic resonance of the lignins isolated from the eucalypt feedstock after the enzymatic pretreatments.ConclusionsThis work shows the potential of an oxidative enzymatic pretreatment to delignify and improve cellulase saccharification of a hardwood feedstock (eucalypt wood) when applied directly on the ground lignocellulosic material, and reveals the main chemical changes in the pretreated material, and its recalcitrant lignin moiety, behind the above results.

HSQC-NMR analysis of lignin in woody (Eucalyptus globulus and Picea abies) and non-woody (Agave sisalana) ball-milled plant materials at the gel state 10th EWLP, Stockholm, Sweden, August 25–28, 2008

Abstract In situ analysis of lignin by 2D NMR of whole plant material was carried out by swelling finely ball-milled samples in deuterated dimethylsulfoxide (DMSO-d6 ) and sonicated so that a gel was formed in the NMR analysis tube. Solution HSQC NMR spectra of different plant materials representative for hardwood (Eucalyptus globulus), softwood (Picea abies), and non-woody plants (Agave sisalana) are presented here. The spectra show signals corresponding to those of the main plant constituents, such as lignin and polysaccharides. The lignin signals were assigned by comparing the HSQC spectra of the whole plant materials with the HSQC spectra of their respective milled-wood lignins (MWLs). In general terms, the major lignin structural features, such as the relative abundances of the main lignin substructures, the syringyl/guaiacyl ratios and the extent of γ-acetylation of the lignin side-chain observed in the HSQC spectra of the whole plant materials, matched those obtained from the HSQC spectra of the isolated MWLs. Therefore, this technique, which needs only minor amounts of lignocellulosic material and minimal sample preparation, can be useful for the rapid screening of plant lignins without the need for tedious and time-consuming lignin isolation procedures.

Lipid and lignin composition of woods from different eucalypt species

Abstract The lipid and lignin compositions of woods from the eucalypt species Eucalyptus globulus, E. nitens, E. maidenii, E. grandis, and E. dunnii have been characterized. The lipid composition was analyzed by GC and GC/MS using short- and medium-length high-temperature capillary columns, which allowed the detection of intact high-molecular-weight compounds. Similar lipid compositions were observed in all eucalypt woods, which were dominated by sitosterol, sitosterol esters and sitosteryl 3β-D-glucopyranoside. These substance classes are mainly responsible for pitch deposition during kraft pulping of eucalypt wood. However, some quantitative differences were found in the abundance of different lipid classes, with wood from E. globulus containing the lowest amounts of these pitch-forming compounds. The lignins of all eucalypt woods were analyzed in situ (without previous isolation) by pyrolysis-GC/MS. A predominance of syringyl (S) over guaiacyl (G) lignin units was observed and the S/G ratio was in the range from 2.7 to 4.1. E. globulus wood had the highest S/G ratio, a finding that, together with its low lignin content, explains its easy delignification under kraft cooking conditions.

Polymerization of lignosulfonates by the laccase-HBT (1-hydroxybenzotriazole) system improves dispersibility

The ability of laccases from Trametes villosa (TvL), Myceliophthora thermophila (MtL), Trametes hirsuta (ThL) and Bacillus subtilis (BsL) to improve the dispersion properties of calcium lignosulfonates 398 in the presence of HBT as a mediator was investigated. Size exclusion chromatography showed an extensive increase in molecular weight of the samples incubated with TvL and ThL by 107% and 572% from 28400 Da after 17h of incubation, respectively. Interestingly, FTIR spectroscopy, (13)C NMR and Py-GC/MS analysis of the treated samples suggested no substantial changes in the aromatic signal of the lignosulfonates, a good indication of the ability of TvL/ThL-HBT systems to limit their effect on functional groups without degrading the lignin backbone. Further, the enzymatic treatments led to a general increase in the dispersion properties, indeed a welcome development for its application in polymer blends.

Structural modification of eucalypt pulp lignin in a totally chlorine-free bleaching sequence including a laccase-mediator stage

Abstract Structural modification of eucalypt pulp lignin was investigated in a totally chlorine-free (TCF) bleaching sequence including a laccase-mediator stage. This stage was applied after two oxygen delignification stages, and was followed by an alkaline peroxide stage. After two oxygen delignification stages, two more stages with a laccase mediator and alkaline peroxide were applied. The residual lignins were enzymatically isolated from the different pulps and analyzed by spectroscopic techniques and analytical pyrolysis. The latter revealed high amounts of syringyl units (>70%) in the lignins. 13C-1H heteronuclear single quantum correlation (HSQC) NMR indicated high amounts of β-O-4′ inter-unit linkages (>75% side-chains). Changes in lignin composition and inter-unit linkages were demonstrated in the course of the bleaching sequence. Moreover, oxidative modification of the major syringyl units was shown by C2,6-H2,6 HSQC correlations and by the presence of oxidized pyrolysis markers in pyrograms. The existence of both Cα keto and carboxyl groups in the residual lignin, together with normal (Cα-hydroxylated) units, was revealed by heteronuclear multiple bond correlation (HMBC) between aromatic H2,6 and side-chain carbons. These Cα-oxidized structures represent nearly 60% of total units in the lignin isolated from the enzymatically treated pulp. Analysis of residual lignin after the final peroxide stage compared with a simple alkaline treatment revealed that most of the oxidatively altered lignin was removed by the alkali used in the peroxide stage. Thus, the kappa number decreased and the final residual lignin was more structurally related to that found before the oxidative stages, although it contained less resinols and more carboxyl group-bearing units. However, the action of peroxide is necessary to attain the high brightness required (>90% ISO).

Towards industrially-feasible delignification and pitch removal by treating paper pulp with Myceliophthora thermophila laccase and a phenolic mediator

The ability of two natural phenols to act as mediators of the recombinant Myceliophthora thermophila laccase (MtL) in eucalypt-pulp delignification was investigated. After alkaline peroxide extraction, the properties of the enzymatically-treated pulps improved with respect to the control. The pulp brightness increased (3.1 points) after the enzymatic treatment with MtL alone, but the highest improvements were obtained after the MtL treatment using syringaldehyde (4.7 points) and especially methyl syringate (8.3 points) as mediators. Likewise, a decrease in kappa number up to 2.7 points was obtained after the MtL-methyl syringate treatment, followed by decreases of 1.4 and 0.9 points after the treatments with MtL-syringaldehyde and MtL alone, respectively. On the other hand, removal of the main lipophilic extractives present in eucalypt pulp was observed after the above laccase-mediator treatments. Finally, the doses of both MtL and methyl syringate were reduced, and results compatible with industrial implementation were obtained.