Altered lignin biosynthesis using biotechnology to improve lignocellulosic biofuel feedstocks

Lignocellulosic feedstocks can be converted to biofuels, which can conceivably replace a large fraction of fossil fuels currently used for transformation. However, lignin, a prominent constituent of secondary cell walls, is an impediment to the conversion of cell walls to fuel: the recalcitrance problem. Biomass pretreatment for removing lignin is the most expensive step in the production of lignocellulosic biofuels. Even though we have learned a great deal about the biosynthesis of lignin, we do not fully understand its role in plant biology, which is needed for the rational design of engineered cell walls for lignocellulosic feedstocks. This review will recapitulate our knowledge of lignin biosynthesis and discuss how lignin has been modified and the consequences for the host plant.     

Lignocellulosic biomass: Biosynthesis,degradation, and industrial utilization

Lignocellulose biomass derived from plant cell walls is a rich source of biopolymers, chemicals, and sugars, besides being a sustainable alternative to petrochemicals. A natural armor protecting living protoplasts, the cell wall is currently the target of intense study because of its crucial importance in plant development, morphogenesis, and resistance to (a)biotic stresses. Beyond the intrinsic relevance related to the overall plant physiology, plant cell walls constitute an exquisite example of a natural composite material that is a constant source of inspiration for biotechnology, biofuel, and biomaterial industries. The aim of the present review is to provide the reader with an overview of the current knowledge concerning lignocellulosic biomass synthesis and degradation, by focusing on its three principal constituents, i.e. cellulose, hemicellulose (in particular xylan), and lignin. Furthermore, the current industrial exploitation of lignocellulose from fast growing fibre crops (such as hemp) is highlighted. We conclude this review by suggesting approaches for further research to fill gaps in our current knowledge and to highlight the potential of biotechnology and bioengineering in improving both biomass biosynthesis and degradation.     

Lignin Biosynthesis Studies in Plant Tissue Cultures

Lignin, a phenolic polymer abundant in cell walls of certain cell types, has given challenges to scientists studying its structure or biosynthesis. In plants lignified tissues are distributed between other, non-lignified tissues. Characterization of native lignin in the cell wall has been difficult due to the highly cross-linked nature of the wall components. Model systems, like plant tissue cultures with tracheary element differentiation or extracellular lignin formation, have provided useful information related to lignin structure and several aspects of lignin formation. For example, many enzyme activities in the phenylpropanoid pathway have been first identified in tissue cultures. This review focuses on studies where the use of plant tissue cultures has been advantageous in structural and biosynthesis studies of lignin, and discusses the validity of tissue cultures as models for lignin biosynthesis.     

Deciphering the enigma of lignification: precursor transport, oxidation, and the topochemistry of lignin assembly

Plant lignification is a tightly regulated complex cellular process that occurs via three sequential steps: the synthesis of monolignols within the cytosol; the transport of monomeric precursors across plasma membrane; and the oxidative polymerization of monolignols to form lignin macromolecules within the cell wall. Although we have a reasonable understanding of monolignol biosynthesis, many aspects of lignin assembly remain elusive. These include the precursors' transport and oxidation, and the initiation of lignin polymerization. This review describes our current knowledge of the molecular mechanisms underlying monolignol transport and oxidation, discusses the intriguing yet least-understood aspects of lignin assembly, and highlights the technologies potentially aiding in clarifying the enigma of plant lignification.     

Bacterial enzymes for lignin depolymerisation: new biocatalysts for generation of renewable chemicals from biomass

The conversion of polymeric lignin from plant biomass into renewable chemicals is an important unsolved problem in the biorefinery concept. This article summarises recent developments in the discovery of bacterial enzymes for lignin degradation, our current understanding of their molecular mechanism of action, and their use to convert lignin or lignocellulose into aromatic chemicals. The review also discusses the recent developments in screening of metagenomic libraries for new biocatalysts, and the use of protein engineering to enhance lignin degradation activity.     

Biodegradation of hardwood lignocellulosics by the western poplar clearwing borer, Paranthrene robiniae (Hy. Edwards)

Lignin in plant cell wall is a source of useful chemicals and also the major barrier for saccharification of lignocellulosic biomass for producing biofuel and bioproducts. Enzymatic lignin degradation/modification process could bypass the need for chemical pretreatment and thereby facilitate bioprocess consolidation. Herein, we reveal our new discovery in elucidating the process of hardwood lignin modification/degradation by clearwing borer, Paranthrene robiniae . The wood-boring clearwing borer, P. robiniae , effectively tunnels hardwood structures during the larval stage; its digestion products from wood components, however, has not yet been investigated. A series of analysis conducted in this study on tunnel walls and frass produced provided evidence of structural alterations and lignin degradation during such hardwood digestion process. The analysis included solid state (13)C cross-polarization magic angle spinning (CP/MAS) nuclear magnetic resonance (NMR) spectroscopy, attenuated total reflectance Fourier transform infrared (ATR-FTIR), pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS), and thermogravimetric (TG) analysis; the results strongly suggest that the structural alteration of lignin primarily involved a preferential degradation of syringyl units accompanied by oxidation on the side chains of lignin guaiacyl moieties. This study also further indicated that unlike the wood-feeding termite the clearwing borer does not target cellulose as an energy source, and thus its lignin degradation ability should provide potential information on how to disassemble and utilize hardwood lignin. Overall, this biological model with an efficient lignin disruption system will provide the new insight into novel enzyme system required for effective plant cell wall disintegration for enhanced cellulose accessibility by enzymes and production of value-added lignin derived products.     

Lignin: genetic engineering and impact on pulping

Lignin is a major component of wood, the most widely used raw material for the production of pulp and paper. Although the biochemistry and molecular biology underpinning lignin production are better understood than they are for the other wood components, recent work has prompted a number of re-evaluations of the lignin biosynthetic pathway. Some of the work on which these revisions have been based involved the investigation of transgenic plants with modified lignin biosynthesis. In addition to their value in elucidating the lignin biosynthetic pathway, such transgenic plants are also being produced with the aim of improving plant raw materials for pulp and paper production. This review describes how genetic engineering has yielded new insights into how the lignin biosynthetic pathway operates and demonstrates that lignin can be improved to facilitate pulping. The current technologies used to produce paper are presented in this review, followed by a discussion of the impact of lignin modification on pulp production. Fine-tuned modification of lignin content, composition, or both is now achievable and could have important economic and environmental benefits.     

Genetic modification of lignin biosynthesis for improved biofuel production

The energy in cellulosic biomass largely resides in plant cell walls. Cellulosic biomass is more difficult than starch to break down into sugars because of the presence of lignin and the complex structure of cell walls. Transgenic down-regulation of major lignin genes led to reduced lignin content, increased dry matter degradability, and improved accessibility of cellulases for cellulose degradation. This review provides background information on lignin biosynthesis and focuses on genetic manipulation of lignin genes in important monocot species as well as the dicot potential biofuel crop alfalfa. Reduction of lignin in biofuel crops by genetic engineering is likely one of the most effective ways of reducing costs associated with pretreatment and hydrolysis of cellulosic feedstocks, although some potential fitness issues should also be addressed.     

Lignin: Genetic Engineering and Impact on Pulping

Lignin is a major component of wood, the most widely used raw material for the production of pulp and paper. Although the biochemistry and molecular biology underpinning lignin production are better understood than they are for the other wood components, recent work has prompted a number of re-evaluations of the lignin biosynthetic pathway. Some of the work on which these revisions have been based involved the investigation of transgenic plants with modified lignin biosynthesis. In addition to their value in elucidating the lignin biosynthetic pathway, such transgenic plants are also being produced with the aim of improving plant raw materials for pulp and paper production. This review describes how genetic engineering has yielded new insights into how the lignin biosynthetic pathway operates and demonstrates that lignin can be improved to facilitate pulping. The current technologies used to produce paper are presented in this review, followed by a discussion of the impact of lignin modification on pulp production. Fine-tuned modification of lignin content, composition, or both is now achievable and could have important economic and environmental benefits.     

Lignin characteristics in soil profiles in different plant communities in a subtropical mixed forest

Aims Lignin is generally considered as an important indicator of soil organic carbon (SOC) storage and dynamics. To evaluate the effects of plant communities and soil depth on soil lignin is critical to better understand forest carbon cycling.Methods We compared lignin content and chemical signature in three soil depths of four major plant communities in a subtropical forest, which located in the north part of Wuling Mountains, China. Lignin was measured using CuO oxidation method.Important findings Both lignin content and its biochemical signature in plant litter varied among communities. However, these differences were mostly no longer exist in the upper soil layers. Lignin chemistry in soils inherited some of the biochemical signature of lignin in litter, but in a diminished magnitude. These results suggest that different plant communities had similar decomposition process with varying rates, caused diminished differences in lignin content and its biochemical signature. Lignin content decreased with soil depth, but the biochemical signature of lignin was not significantly different among soil layers for all communities, which suggests that vertical movement of lignin within the soil profile is very likely a key process causing this similar biochemical signature. These results emphasized the important roles of lignin inputs and soil eluviation in shaping lignin characteristics and distribution in forest soils, which pinpoint the urgent need to consider hydrological processes in studying forest soil carbon cycling.     

木质素生物合成途径及调控的研究进展

木质素是植物体中仅次于纤维素的一种重要大分子有机物质,具重要生物学功能。木质素填充于纤维素构架中增强植物体的机械强度,利于疏导组织的水分运输和抵抗不良外界环境的侵袭。陆生植物的木质素合成是适应陆地环境的重要进化特征之一。然而,制浆造纸的中心环节是用大量化学品将原料中的木质素与纤维素分离,纤维素用于造纸,分离的木质素等成为造纸工业的主要废弃物,对江河湖海的污染触目惊心。脱木质素的化学品投入及废液的碱回收处理需大量耗能并增加造纸成本。饲草的木质素还影响牲畜的消化与营养吸收,木质素含量的高低是饲草优劣…     

Identification of grass-specific enzyme that acylates monolignols with p-coumarate

Lignin is a major component of plant cell walls that is essential to their function. However, the strong bonds that bind the various subunits of lignin, and its cross-linking with other plant cell wall polymers, make it one of the most important factors in the recalcitrance of plant cell walls against polysaccharide utilization. Plants make lignin from a variety of monolignols including p-coumaryl, coniferyl, and sinapyl alcohols to produce the three primary lignin units: p-hydroxyphenyl, guaiacyl, and syringyl, respectively, when incorporated into the lignin polymer. In grasses, these monolignols can be enzymatically preacylated by p-coumarates prior to their incorporation into lignin, and these monolignol conjugates can also be "monomer" precursors of lignin. Although monolignol p-coumarate-derived units may comprise up to 40% of the lignin in some grass tissues, the p-coumarate moiety from such conjugates does not enter into the radical coupling (polymerization) reactions of lignification. With a greater understanding of monolignol p-coumarate conjugates, grass lignins could be engineered to contain fewer pendent p-coumarate groups and more monolignol conjugates that improve lignin cleavage. We have cloned and expressed an enzyme from rice that has p-coumarate monolignol transferase activity and determined its kinetic parameters.     

植物中SAM Mtases基因研究进展

SAM Mtases是从多种植物中分离到的一类S-腺苷-L-甲硫氨酸依赖性氧位甲基转移酶基因,该基因对植物体内木质素、类苯基丙烷、类黄酮类、生物碱和脂肪族化合物等许多次生代谢产物合成有直接的影响,并且在植物抗病、抗紫外线、杀虫、抗菌、植物激素生长和信号调节、植物共生、花粉管伸长和花粉生长等生理过程中起重要作用.该文总结了国内外已经克隆到的SAM Mtases同源基因的分离、分类及其功能,为进一步研究SAM Mtases基因在植物生理代谢调控中的地位及在植物抗性及药用成分育种上的应用提供参考.     

Degradation of xenobiotic compounds by lignin-degrading white-rot fungi: enzymology and mechanisms involved

White-rot fungi (WRF) are ubiquitous in nature with their natural ability to compete and survive. WRF are the only organisms known to have the ability to degrade and mineralize recalcitrant plant polymer lignin. Their potential to degrade second most abundant carbon reserve material lignin on the earth make them important link in global carbon cycle. WRF degrade lignin by its unique ligninolytic enzymatic machinery including lignin peroxidase, manganese peroxidase, laccase, cellobiose dehydrogenase, H2O2-generating enzymes, etc. The ligninolytic enzymes system is non-specific, extracellular and free radical based that allows them to degrade structurally diverse range of xenobiotic compounds. Lignin peroxidase and manganese peroxidase carry out direct and indirect oxidation as well as reduction of xenobiotic compounds. Indirect reactions involved redox mediators such as veratryl alcohol and Mn2+. Reduction reactions are carried out by carboxyl, superoxide and semiquinone radicals, etc. Methylation is used as detoxification mechanism by WRF. Highly oxidized chemicals are reduced by transmembrane redox potential. Degradation of a number of environmental pollutants by ligninolytic system of white rot fungi is described in the present review.     

Catalytic Profile of Arabidopsis Peroxidases,AtPrx-2, 25 and 71, Contributing to Stem Lignification

Lignins are aromatic heteropolymers that arise from oxidative coupling of lignin precursors, including lignin monomers (p-coumaryl, coniferyl, and sinapyl alcohols), oligomers, and polymers. Whereas plant peroxidases have been shown to catalyze oxidative coupling of monolignols, the oxidation activity of well-studied plant peroxidases, such as horseradish peroxidase C (HRP-C) and AtPrx53, are quite low for sinapyl alcohol. This characteristic difference has led to controversy regarding the oxidation mechanism of sinapyl alcohol and lignin oligomers and polymers by plant peroxidases. The present study explored the oxidation activities of three plant peroxidases, AtPrx2, AtPrx25, and AtPrx71, which have been already shown to be involved in lignification in the Arabidopsis stem. Recombinant proteins of these peroxidases (rAtPrxs) were produced in Escherichia coli as inclusion bodies and successfully refolded to yield their active forms. rAtPrx2, rAtPrx25, and rAtPrx71 were found to oxidize two syringyl compounds (2,6-dimethoxyphenol and syringaldazine), which were employed here as model monolignol compounds, with higher specific activities than HRP-C and rAtPrx53. Interestingly, rAtPrx2 and rAtPrx71 oxidized syringyl compounds more efficiently than guaiacol. Moreover, assays with ferrocytochrome c as a substrate showed that AtPrx2, AtPrx25, and AtPrx71 possessed the ability to oxidize large molecules. This characteristic may originate in a protein radical. These results suggest that the plant peroxidases responsible for lignin polymerization are able to directly oxidize all lignin precursors.     

Cinnamyl alcohol dehydrogenases-C and D, key enzymes in lignin biosynthesis, play an essential role in disease resistance in Arabidopsis

The deposition of lignin during plant–pathogen interactions is thought to play a role in plant defence. However, the function of lignification genes in plant disease resistance is poorly understood. In this article, we provide genetic evidence that the primary genes involved in lignin biosynthesis in Arabidopsis, CAD-C and CAD-D , act as essential components of defence to virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae pv. tomato , possibly through the salicylic acid defence pathway. Thus, in contrast with cellulose synthesis, whose alteration leads to an increase in disease resistance, alteration of the cell wall lignin content leads directly or indirectly to defects in some defence components.     

Glucuronoyl esterases: diversity,properties and biotechnological potential. A review

Glucuronoyl esterases (GEs) belonging to the carbohydrate esterase family 15 (CE15) are involved in microbial degradation of lignocellulosic plant materials. GEs are capable of degrading complex polymers of lignin and hemicellulose cleaving ester bonds between glucuronic acid residues in xylan and lignin alcohols. GEs promote separation of lignin, hemicellulose and cellulose which is crucial for efficient utilization of biomass as an energy source and feedstock for further processing into products or chemicals. Genes encoding GEs are found in both fungi and bacteria, but, so far, bacterial GEs are essentially unexplored, and despite being discovered >10?years ago, only a limited number of GEs have been characterized. The first laboratory scale example of improved xylose and glucuronic acid release by the synergistic action of GE with cellulolytic enzymes was only reported recently (improved C5 sugar and glucuronic acid yields) and, until now, not much is known about their biotechnology potential. In this review, we discuss the diversity, structure and properties of microbial GEs and consider the status of their action on natural substrates and in biological systems in relation to their future industrial use.