Emerging strategies of lignin engineering and degradation for cellulosic biofuel production

Ethanol and other biofuels produced from lignocellulosic biomass represent a renewable, more carbon-balanced alternative to both fossil fuels and corn-derived or sugarcane-derived ethanol. Unfortunately, the presence of lignin in plant cell walls impedes the breakdown of cell wall polysaccharides to simple sugars and the subsequent conversion of these sugars to usable fuel. Recent advances in the understanding of lignin composition, polymerization, and regulation have revealed new opportunities for the rational manipulation of lignin in future bioenergy crops, augmenting the previous successful approach of manipulating lignin monomer biosynthesis. Furthermore, recent studies on lignin degradation in nature may provide novel resources for the delignification of dedicated bioenergy crops and other sources of lignocellulosic biomass.     

Genetic engineering of grass cell wall polysaccharides for biorefining

Grasses represent an abundant and widespread source of lignocellulosic biomass, which has yet to fulfil its potential as a feedstock for biorefining into renewable and sustainable biofuels and commodity chemicals. The inherent recalcitrance of lignocellulosic materials to deconstruction is the most crucial limitation for the commercial viability and economic feasibility of biomass biorefining. Over the last decade, the targeted genetic engineering of grasses has become more proficient, enabling rational approaches to modify lignocellulose with the aim of making it more amenable to bioconversion. In this review, we provide an overview of transgenic strategies and targets to tailor grass cell wall polysaccharides for biorefining applications. The bioengineering efforts and opportunities summarized here rely primarily on (A) reprogramming gene regulatory networks responsible for the biosynthesis of lignocellulose, (B) remodelling the chemical structure and substitution patterns of cell wall polysaccharides and (C) expressing lignocellulose degrading and/or modifying enzymes in planta. It is anticipated that outputs from the rational engineering of grass cell wall polysaccharides by such strategies could help in realizing an economically sustainable, grass‐derived lignocellulose processing industry.     

微生物法降解木质素的研究进展

生物质是代替石化资源生产能源和化学品的关键资源,木质素作为植物细胞壁的主要成分已经在很多行业中得到了广泛的应用。然而,由于木质素结构复杂且难以降解,成为生物质资源利用的最大障碍,因此,去除或者降解木质素是利用细胞壁中其他成分的关键步骤。许多行业使用有害化学物质降解木质素,严重危害了生态环境,自然界中木质素经常被包括真菌和细菌在内的微生物降解,因此,研究微生物降解木质素的机制为解决这一问题提供了可能性。本文讨论了木质素的化学组成成分,重点讨论了自然界降解木质素的微生物种类及其降解机制,包括各种真菌和细菌的木质素降解活性,描述了由各种微生物特别是白腐真菌、褐腐真菌和细菌产生的木质素降解酶,并展望了今后木质素生物降解的研究和应用的可能方向。     

Overexpression of GA20‐OXIDASE1 impacts plant height,biomass allocation and saccharification efficiency in maize

Increased biomass yield and quality are of great importance for the improvement of feedstock for the biorefinery. For the production of bioethanol, both stem biomass yield and the conversion efficiency of the polysaccharides in the cell wall to fermentable sugars are of relevance. Increasing the endogenous levels of gibberellic acid (GA) by ectopic expression of GA20‐OXIDASE1 (GA20‐OX1), the rate‐limiting step in GA biosynthesis, is known to affect cell division and cell expansion, resulting in larger plants and organs in several plant species. In this study, we examined biomass yield and quality traits of maize plants overexpressing GA20‐OX1 (GA20‐OX1). GA20‐OX1 plants accumulated more vegetative biomass than control plants in greenhouse experiments, but not consistently over two years of field trials. The stems of these plants were longer but also more slender. Investigation of GA20‐OX1 biomass quality using biochemical analyses showed the presence of more cellulose, lignin and cell wall residue. Cell wall analysis as well as expression analysis of lignin biosynthetic genes in developing stems revealed that cellulose and lignin were deposited earlier in development. Pretreatment of GA20‐OX1 biomass with NaOH resulted in a higher saccharification efficiency per unit of dry weight, in agreement with the higher cellulose content. On the other hand, the cellulose‐to‐glucose conversion was slower upon HCl or hot‐water pretreatment, presumably due to the higher lignin content. This study showed that biomass yield and quality traits can be interconnected, which is important for the development of future breeding strategies to improve lignocellulosic feedstock for bioethanol production.     

Ferulic acid: a key component in grass lignocellulose recalcitrance to hydrolysis

In the near future, grasses must provide most of the biomass for the production of renewable fuels. However, grass cell walls are characterized by a large quantity of hydroxycinnamic acids such as ferulic and p‐coumaric acids, which are thought to reduce the biomass saccharification. Ferulic acid (FA) binds to lignin, polysaccharides and structural proteins of grass cell walls cross‐linking these components. A controlled reduction of FA level or of FA cross‐linkages in plants of industrial interest can improve the production of cellulosic ethanol. Here, we review the biosynthesis and roles of FA in cell wall architecture and in grass biomass recalcitrance to enzyme hydrolysis.     

Maize and sorghum: genetic resources for bioenergy grasses

The highly photosynthetic-efficient C4 grasses, such as switchgrass (Panicum virgatum), Miscanthus (Miscanthusxgiganteus), sorghum (Sorghum bicolor) and maize (Zea mays), are expected to provide abundant and sustainable resources of lignocellulosic biomass for the production of biofuels. A deeper understanding of the synthesis, deposition and hydrolysis of the distinctive cell walls of grasses is crucial to gain genetic control of traits that contribute to biomass yield and quality. With a century of genetic investigations and breeding success, recently completed genome sequences, well-characterized cell wall compositions, and a close evolutionary relationship with future bioenergy perennial grasses, we propose that maize and sorghum are key model systems for gene discovery relating to biomass yield and quality in the bioenergy grasses.     

Polymorphisms in monolignol biosynthetic genes are associated with biomass yield and agronomic traits in European maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

Background  

Reduced lignin content leads to higher cell wall digestibility and, therefore, better forage quality and increased conversion of lignocellulosic biomass into ethanol. However, reduced lignin content might lead to weaker stalks, lodging, and reduced biomass yield. Genes encoding enzymes involved in cell wall lignification have been shown to influence both cell wall digestibility and yield traits.     

Cell wall remodeling under salt stress: Insights into changes in polysaccharides,feruloylation, lignification,and phenolic metabolism in maize

Although cell wall polymers play important roles in the tolerance of plants to abiotic stress, the effects of salinity on cell wall composition and metabolism in grasses remain largely unexplored. Here, we conducted an in-depth study of changes in cell wall composition and phenolic metabolism induced upon salinity in maize seedlings and plants. Cell wall characterization revealed that salt stress modulated the deposition of cellulose, matrix polysaccharides and lignin in seedling roots, plant roots and stems. The extraction and analysis of arabinoxylans by size-exclusion chromatography, 2D-NMR spectroscopy and carbohydrate gel electrophoresis showed a reduction of arabinoxylan content in salt-stressed roots. Saponification and mild acid hydrolysis revealed that salinity also reduced the feruloylation of arabinoxylans in roots of seedlings and plants. Determination of lignin content and composition by nitrobenzene oxidation and 2D-NMR confirmed the increased incorporation of syringyl units in lignin of maize roots. Salt stress also induced the expression of genes and the activity of enzymes enrolled in phenylpropanoid biosynthesis. The UHPLC–MS-based metabolite profiling confirmed the modulation of phenolic profiling by salinity and the accumulation of ferulate and its derivatives 3- and 4-O-feruloyl quinate. In conclusion, we present a model for explaining cell wall remodeling in response to salinity.     

Biochemical control of xylan biosynthesis - which end is up?

Xylans are major components of land plant secondary cell walls and are required for normal plant growth and development. Secondary walls also account for the bulk of lignocellulosic biomass, a potential feedstock for large-scale production of biofuels. Glucuronoxylan and arabinoxylan affect the conversion of lignocellulosic biomass to fermentable sugar, a crucial and expensive step in biofuel production. Thus, knowledge of xylan biosynthesis may provide tools to modify secondary cell wall structure and thereby improve the bioprocessing characteristics of biomass. Recent studies have shown that glucuronoxylan structure and biosynthesis are far more complex than previously appreciated and the number of glycosyltransferases implicated in this process continues to increase. New hypotheses regarding the mechanisms of glucuronoxylan biosynthesis challenge some widely held views.     

Enzymatic processes involved in the incorporation of hydroxycinnamates into grass cell walls

Many plant species have one or more types of acylation of cell wall polymers. Grasses (Poaceae family) are unique with abundant acylation of specific cell wall polymers by hydroxycinnamates. The most common hydroxycinnamates found in a wide range of grasses are ferulates (trans-4-hydroxy-3-methoxycinnamate) and p-coumarates (trans-4-hydroxycinnamate). These two hydroxycinnamates are synthesized by the phenylpropanoid pathway. Though structurally related, they seem to have different functional roles within the cell wall. Ferulates have been shown to have a critical role in cross-linking cell wall components; forming links between structural polysaccharides and links between structural polysaccharides and lignin. They are incorporated into the cell wall by distinctly different mechanisms. Ferulic acid is incorporated into cell walls as ester linked substituents on arabinoxylans. The exact role p-coumarates play in plant cell walls is unknown, but it has been shown that p-coumaric acid is ester-linked to monolignols and shuttled out to the wall to become incorporated into newly forming lignin polymers. Both processes require the activity of specific hydroxycinnamoyl transferases utilizing CoA derivatives to drive the transferase reactions.     

High throughput screening of hydrolytic enzymes from termites using a natural substrate derived from sugarcane bagasse

Background

Short rotation coppice willow is a potential lignocellulosic feedstock in the United Kingdom and elsewhere; however, research on optimising willow specifically for bioethanol production has started developing only recently. We have used the feedstock Salix viminalis × Salix schwerinii cultivar 'Olof' in a three-month pot experiment with the aim of modifying cell wall composition and structure within the stem to the benefit of bioethanol production. Trees were treated for 26 or 43 days with tension wood induction and/or with an application of the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile that is specific to secondary cell walls. Reaction wood (tension and opposite wood) was isolated from material that had received the 43-day tension wood induction treatment.

Results

Glucan content, lignin content and enzymatically released glucose were assayed. All measured parameters were altered without loss of total stem biomass yield, indicating that enzymatic saccharification yield can be enhanced by both alterations to cell wall structure and alterations to absolute contents of either glucan or lignin.

Conclusions

Final glucose yields can be improved by the induction of tension wood without a detrimental impact on biomass yield. The increase in glucan accessibility to cell wall degrading enzymes could help contribute to reducing the energy and environmental impacts of the lignocellulosic bioethanol production process.     

Heterogeneity and Glycan Masking of Cell Wall Microstructures in the Stems of Miscanthus x giganteus,and Its Parents M. sinensis and M. sacchariflorus

Plant cell walls, being repositories of fixed carbon, are important sources of biomass and renewable energy. Miscanthus species are fast growing grasses with a high biomass yield and they have been identified as potential bioenergy crops. Miscanthus x giganteus is the sterile hybrid between M. sinensis and M. sacchariflorus, with a faster and taller growth than its parents. In this study, the occurrence of cell wall polysaccharides in stems of Miscanthus species has been determined using fluorescence imaging with sets of cell wall directed monoclonal antibodies. Heteroxylan and mixed linkage-glucan (MLG) epitopes are abundant in stem cell walls of Miscanthus species, but their distributions are different in relation to the interfascicular parenchyma and these epitopes also display different developmental dynamics. Detection of pectic homogalacturonan (HG) epitopes was often restricted to intercellular spaces of parenchyma regions and, notably, the high methyl ester LM20 HG epitope was specifically abundant in the pith parenchyma cell walls of M. x giganteus. Some cell wall probes cannot access their target glycan epitopes because of masking by other polysaccharides. In the case of Miscanthus stems, masking of xyloglucan by heteroxylan and masking of pectic galactan by heteroxylan and MLG was detected in certain cell wall regions. Knowledge of tissue level heterogeneity of polysaccharide distributions and molecular architectures in Miscanthus cell wall structures will be important for both understanding growth mechanisms and also for the development of potential strategies for the efficient deconstruction of Miscanthus biomass.     

Phylogeny in Defining Model Plants for Lignocellulosic Ethanol Production: A Comparative Study of Brachypodium distachyon,Wheat, Maize,and Miscanthus x giganteus Leaf and Stem Biomass

The production of ethanol from pretreated plant biomass during fermentation is a strategy to mitigate climate change by substituting fossil fuels. However, biomass conversion is mainly limited by the recalcitrant nature of the plant cell wall. To overcome recalcitrance, the optimization of the plant cell wall for subsequent processing is a promising approach. Based on their phylogenetic proximity to existing and emerging energy crops, model plants have been proposed to study bioenergy-related cell wall biochemistry. One example is Brachypodium distachyon, which has been considered as a general model plant for cell wall analysis in grasses. To test whether relative phylogenetic proximity would be sufficient to qualify as a model plant not only for cell wall composition but also for the complete process leading to bioethanol production, we compared the processing of leaf and stem biomass from the C₃ grasses B. distachyon and Triticum aestivum (wheat) with the C₄ grasses Zea mays (maize) and Miscanthus x giganteus, a perennial energy crop. Lambda scanning with a confocal laser-scanning microscope allowed a rapid qualitative analysis of biomass saccharification. A maximum of 108–117 mg ethanol·g⁻¹ dry biomass was yielded from thermo-chemically and enzymatically pretreated stem biomass of the tested plant species. Principal component analysis revealed that a relatively strong correlation between similarities in lignocellulosic ethanol production and phylogenetic relation was only given for stem and leaf biomass of the two tested C₄ grasses. Our results suggest that suitability of B. distachyon as a model plant for biomass conversion of energy crops has to be specifically tested based on applied processing parameters and biomass tissue type.     

Epigallocatechin gallate incorporation into lignin enhances the alkaline delignification and enzymatic saccharification of cell walls

ABSTRACT: BACKGROUND: Lignin is an integral component of the plant cell wall matrix but impedes the conversion of biomass into biofuels. The plasticity of lignin biosynthesis should permit the inclusion of new compatible phenolic monomers such as flavonoids into cell wall lignins that are consequently less recalcitrant to biomass processing. In the present study, epigallocatechin gallate (EGCG) was evaluated as a potential lignin bioengineering target for rendering biomass more amenable to processing for biofuel production. RESULTS: In vitro peroxidase-catalyzed polymerization experiments revealed that both gallate and pyrogallyl (B-ring) moieties in EGCG underwent radical cross-coupling with monolignols mainly by beta--O--4-type cross-coupling, producing benzodioxane units following rearomatization reactions. Biomimetic lignification of maize cell walls with a 3:1 molar ratio of monolignols and EGCG permitted extensive alkaline delignification of cell walls (72 to 92 %) that far exceeded that for lignified controls (44 to 62 %). Alkali-insoluble residues from EGCG-lignified walls yielded up to 34 % more glucose and total sugars following enzymatic saccharification than lignified controls. CONCLUSIONS: It was found that EGCG readily copolymerized with monolignols to become integrally cross-coupled into cell wall lignins, where it greatly enhanced alkaline delignification and subsequent enzymatic saccharification. Improved delignification may be attributed to internal trapping of quinone-methide intermediates to prevent benzyl ether cross-linking of lignin to structural polysaccharides during lignification, and to the cleavage of ester intra-unit linkages within EGCG during pretreatment. Overall, our results suggest that apoplastic deposition of EGCG for incorporation into lignin would be a promising plant genetic engineering target for improving the delignification and saccharification of biomass crops.     

Over-expression of F5H in COMT-deficient Arabidopsis leads to enrichment of an unusual lignin and disruption of pollen wall formation

The presence of the phenylpropanoid polymer lignin in plant cell walls impedes breakdown of polysaccharides to the fermentable sugars that are used in biofuel production. Genetically modified plants with altered lignin properties hold great promise to improve biomass degradability. Here, we describe the generation of a new type of lignin enriched in 5-hydroxy-guaiacyl units by over-expressing ferulate 5-hydroxylase in a line of Arabidopsis lacking caffeic acid O-methyltransferase. The lignin modification strategy had a profound impact on plant growth and development and cell-wall properties, and resulted in male sterility due to complete disruption of formation of the pollen wall. The modified plants showed significantly improved cell-wall enzymatic saccharification efficiency without a reduction in post-harvest biomass yield despite the alterations in the overall growth morphology. This study demonstrated the plasticity of lignin polymerization in terms of incorporation of unusual monomers that chemically resemble conventional monomers, and also revealed the link between the biosynthetic pathways of lignin and the pollen wall-forming sporopollenin.