Identifying new lignin bioengineering targets: 1. Monolignol-substitute impacts on lignin formation and cell wall fermentability

Background  

Recent discoveries highlighting the metabolic malleability of plant lignification indicate that lignin can be engineered to dramatically alter its composition and properties. Current plant biotechnology efforts are primarily aimed at manipulating the biosynthesis of normal monolignols, but in the future apoplastic targeting of phenolics from other metabolic pathways may provide new approaches for designing lignins that are less inhibitory toward the enzymatic hydrolysis of structural polysaccharides, both with and without biomass pretreatment. To identify promising new avenues for lignin bioengineering, we artificially lignified cell walls from maize cell suspensions with various combinations of normal monolignols (coniferyl and sinapyl alcohols) plus a variety of phenolic monolignol substitutes. Cell walls were then incubated in vitro with anaerobic rumen microflora to assess the potential impact of lignin modifications on the enzymatic degradability of fibrous crops used for ruminant livestock or biofuel production.     

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.     

Peculiarities of brown-rot fungi and biochemical Fenton reaction with regard to their potential as a model for bioprocessing biomass

This work reviews the brown-rot fungal biochemical mechanism involved in the biodegradation of lignified plant cell walls. This mechanism has been acquired as an apparent alternative to the energetically expensive apparatus of lignocellulose breakdown employed by white-rot fungi. The mechanism relies, at least in the incipient stage of decay, on the oxidative cleavage of glycosidic bonds in cellulose and hemicellulose and the oxidative modification and arrangement of lignin upon attack by highly destructive oxygen reactive species such as the hydroxyl radical generated non-enzymatically via Fenton chemistry \( ({\text{F}}{{\text{e}}^{{{3} + }}} + {{\text{H}}_{{2}}}{{\text{O}}_{{2}}} \to {\text{F}}{{\text{e}}^{{{2} + }}} + \cdot {\text{OH}}{{ + }^{ - }}{\text{OH}}) \). Modifications in the lignocellulose macrocomponents associated with this non-enzymatic attack are believed to aid in the selective, near-complete removal of polysaccharides by an incomplete cellulase suite and without causing substantial lignin removal. Utilization of this process could provide the key to making the production of biofuel and renewable chemicals from lignocellulose biomass more cost-effective and energy efficient. This review highlights the unique features of the brown-rot fungal non-enzymatic, mediated Fenton reaction mechanism, the modifications to the major plant cell wall macrocomponents, and the implications and opportunities for biomass processing for biofuels and chemicals.     

Implications of chemical and physical factors on the biological processing of lignocellulose

It is hoped that recognition of the chemical and physical factors touched on above will lead to their assimilation early in the process development. Although the depolymerization of plant cell wall material and the subsequent metabolism of the fragments are entirely effected by biological processes in nature, practical considerations of rates, concentrations, selectivity and susceptibility will require chemical or physical processing as well for the production of chemicals and materials from wood. These processes will break the polymers down into small molecules or at the very least serve as pretreatments to confer accessibility to the polymers in lignified cell walls. Further conversion of the small molecules into useful chemicals and materials may then be carried out by either biological or chemical processing as desired.     

Microbial conversion of sugars from plant biomass to lactic acid or ethanol

Concerns for our environment and unease with our dependence on foreign oil have renewed interest in converting plant biomass into fuels and 'green' chemicals. The volume of plant matter available makes lignocellulose conversion desirable, although no single isolated organism has been shown to depolymerize lignocellulose and efficiently metabolize the resulting sugars into a specific product. This work reviews selected chemicals and fuels that can be produced from microbial fermentation of plant-derived cell-wall sugars and directed engineering for improvement of microbial biocatalysts. Lactic acid and ethanol production are highlighted, with a focus on engineered Escherichia coli .     

Recent advances in metabolic engineering of Corynebacterium glutamicum for bioproduction of value-added aromatic chemicals and natural products

Recent progress in synthetic and systems metabolic engineering technologies has explored the potential of microbial cell factories for the production of industrially relevant bulk and fine chemicals from renewable biomass resources in an eco-friendly manner. Corynebacterium glutamicum, a workhorse for industrial amino acid production, has currently evolved into a promising microbial platform for bioproduction of various natural and non-natural chemicals from renewable feedstocks. Notably, it has been recently demonstrated that metabolically engineered C. glutamicum can overproduce several commercially valuable aromatic and related chemicals such as shikimate, 4-hydroxybenzoate, and 4-aminobenzoate from sugars at remarkably high titer suitable to commercial application. On the other hand, overexpression and/or extension of its endogenous metabolic pathways by integrating heterologous metabolic pathways enabled production of structurally intricate and valuable natural chemicals like plant polyphenols, carotenoids, and fatty acids. In this review, we summarize recent advances in metabolic engineering of C. glutamicum for production of those value-added aromatics and other natural products, which highlights high potential and the versatility of this microbe for bioproduction of diverse chemicals.

     

A combined FT-IR microscopy and principal component analysis on softwood cell walls

A combined FT-IR microscopy and principle component analysis was used to investigate chemical variations between softwood species as well as types of wood cell walls; latewood tracheids, earlywood tracheids and earlywood ray parenchyma cells. The method allowed us to detect small spectral differences between cell types rather than species and to predict characteristic chemical components of each cell type. The method enabled information to be obtained which allowed a evaluation of the polysaccharide composition even in lignified woody plant cell walls.     

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。     

Development of biocatalysts for production of commodity chemicals from lignocellulosic biomass

Lignocellulosic biomass is recognized as potential sustainable source for production of power, biofuels and variety of commodity chemicals which would potentially add economic value to biomass. Recalcitrance nature of biomass is largely responsible for the high cost of its conversion. Therefore, it is necessary to introduce some cost effective pretreatment processes to make the biomass polysaccharides easily amenable to enzymatic attack to release mixed fermentable sugars. Advancement in systemic biology can provide new tools for the development of such biocatalysts for sustainable production of commodity chemicals from biomass. Integration of functional genomics and system biology approaches may generate efficient microbial systems with new metabolic routes for production of commodity chemicals. This paper provides an overview of the challenges that are faced by the processes converting lignocellulosic biomass to commodity chemicals. The critical factors involved in engineering new microbial biocatalysts are also discussed with more emphasis on commodity chemicals.     

Designing the deconstruction of plant cell walls

Cell wall architecture plays a key role in the regulation of plant cell growth and differentiation into specific cell types. Gaining genetic control of the amount, composition, and structure of cell walls in different cell types will impact both the quantity and yield of fermentable sugars from biomass for biofuels production. The recalcitrance of plant biomass to degradation is a function of how polymers crosslink and aggregate within walls. Novel imaging technologies provide an opportunity to probe these higher order structures in their native state. If cell walls are to be efficiently deconstructed enzymatically to release fermentable sugars, then we require a detailed understanding of their structural organization in future bioenergy crops.     

Metabolic engineering for the utilization of carbohydrate portions of lignocellulosic biomass

The petrochemical industry has grown to meet the need for massive production of energy and commodities along with an explosive population growth; however, serious side effects such as greenhouse gas emissions and global warming have negatively impacted the environment. Lignocellulosic biomass with myriad quantities on Earth is an attractive resource for the production of carbon-neutral fuels and chemicals through environmentally friendly processes of microbial fermentation. This review discusses metabolic engineering efforts to achieve economically feasible industrial production of fuels and chemicals from microbial cell factories using the carbohydrate portion of lignocellulosic biomass as substrates. The combined knowledge of systems biology and metabolic engineering has been applied to construct robust platform microorganisms with maximum conversion of monomeric sugars, such as glucose and xylose, derived from lignocellulosic biomass. By comprehensively revisiting carbon conversion pathways, we provide a rationale for engineering strategies, as well as their features, feasibility, and recent representative studies. In addition, we briefly discuss how tools in systems biology can be applied in the field of metabolic engineering to accelerate the development of microbial cell factories that convert lignocellulosic biomass into carbon-neutral fuels and chemicals with economic feasibility.     

Modifying plants for biofuel and biomaterial production

The productivity of plants as biofuel or biomaterial crops is established by both the yield of plant biomass per unit area of land and the efficiency of conversion of the biomass to biofuel. Higher yielding biofuel crops with increased conversion efficiencies allow production on a smaller land footprint minimizing competition with agriculture for food production and biodiversity conservation. Plants have traditionally been domesticated for food, fibre and feed applications. However, utilization for biofuels may require the breeding of novel phenotypes, or new species entirely. Genomics approaches support genetic selection strategies to deliver significant genetic improvement of plants as sources of biomass for biofuel manufacture. Genetic modification of plants provides a further range of options for improving the composition of biomass and for plant modifications to assist the fabrication of biofuels. The relative carbohydrate and lignin content influences the deconstruction of plant cell walls to biofuels. Key options for facilitating the deconstruction leading to higher monomeric sugar release from plants include increasing cellulose content, reducing cellulose crystallinity, and/or altering the amount or composition of noncellulosic polysaccharides or lignin. Modification of chemical linkages within and between these biomass components may improve the ease of deconstruction. Expression of enzymes in the plant may provide a cost‐effective option for biochemical conversion to biofuel.     

合成生物学在微生物细胞工厂构建中的应用

通过微生物发酵的方法生产大宗化学品和天然产物能够部分替代石油化工炼制和植物提取。合成生物学技术的发展极大地提高了构建微生物细胞工厂生产大宗化学品和天然产物的能力。一方面综述了合成生物学在构建细胞工厂时的关键技术,包括最优合成途径的设计、合成途径的创建与优化、细胞性能的优化;另一方面,介绍了应用这些技术构建细胞工厂生产燃料化学品、大宗化学品和天然产物的典型案例。     

Engineering biocatalysts for production of commodity chemicals

Lignocellulosic biomass is an attractive alternate to petroleum for production of both fuels and commodity chemicals. This conversion of biomass would require a new generation of microbial biocatalysts that can convert all the sugars present in the biomass to the desired compounds. In this review, the critical factors that need to be considered in engineering such microbial biocatalysts for cost-effective fermentation of sugars are discussed with specific emphasis on commodity chemicals such as lactic acid, succinic acid and acetic acid.     

Gravity: one of the driving forces for evolution

Mechanical load is 10(3) larger for land-living than for water-living organisms. As a consequence, antigravitational material in form of compound materials like lignified cell walls in plants and mineralised bones in animals occurs in land-living organisms preferentially. Besides cellulose, pectic substances of plant cell walls seem to function as antigravitational material in early phases of plant evolution and development. A testable hypothesis including vesicular recycling processes into the tensegrity concept is proposed for both sensing of gravitational force and responding by production of antigravitational material at the cellular level.     

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part I: Lignin

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation¹ and other products such as biocomposite materials⁷. Plant biomass remains one of the greatest untapped reserves on the planet⁴. It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses (matrix polysaccharides, and the polyphenol lignin⁶ and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls provide tensile strength to cells and the entire plants, ward off pathogens, and allow water to be transported throughout the plant; in the case of trees up to more the 100 m above ground level. Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant⁴. Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerization by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis. In this first part we focus on the analysis of the polyphenol lignin (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its lignin content by acetylbromide solubilization³, and its lignin composition in terms of its syringyl, guaiacyl- and p-hydroxyphenyl units⁵. The protocol for analyzing the carbohydrates in lignocellulosic biomass including cellulose content and matrix polysaccharide composition is discussed in Part II².     

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part II: Carbohydrates

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation² and other products such as biocomposite materials⁶. Plant biomass remains one of the greatest untapped reserves on the planet⁴. It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses, and the polyphenol lignin⁵ and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls contribute extensively to the strength and structural integrity of the entire plant. Despite its necessary rigidity, the cell wall is a highly dynamic entity that is metabolically active and plays crucial roles in numerous cell activities such as plant growth and differentiation⁵. Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant⁴. Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerisation by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its monosaccharide composition of the hemicelluloses and other matrix polysaccharides1, and its content of crystalline cellulose⁷. The protocol for analyzing the lignin components in lignocellulosic biomass is discussed in Part I³.     

毛竹茎细胞壁半纤维素多糖的免疫细胞化学定位研究

本文以毛竹(Phyllostachys pubescens)为材料,采用免疫细胞化学标记方法对两种细胞壁半纤维素多糖成分,即木聚糖(Xylan)和(1-3)(1-4)-β-葡聚糖［(1-3)(1-4)-β-glucan］在毛竹茎中的分布进行了观察。结果表明,应用免疫细胞化学方法可以准确、有效地观察这两种半纤维素多糖成分在细胞壁中的分布;木聚糖分布在已木质化的组织细胞的细胞壁中,与细胞壁木质化有密切关系;(1-3)(1-4)-β-葡聚糖在幼竹茎基本组织中分布于短薄壁细胞细胞壁中及长薄壁细胞胞间层,而在老龄竹茎基本组织中,仅分布于短薄壁细胞细胞壁中,而长薄壁细胞细胞壁却无此成分,反映出长、短薄壁细胞细胞壁组成上的差异。     

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.     

Endophytic Fungal Strains of Soybean for Lipid Production

Lipids created via microbial biosynthesis are a potential raw material to replace plant-based oil for biodiesel production. Oleaginous microbial species currently available are capable of accumulating high amount of lipids in their cell biomass, but rarely can directly utilize lignocellulosic biomass as substrates. Thus this research focused on the screening and selection of new fungal strains that generate both lipids and hydrolytic enzymes. To search for oleaginous fungal strains in the soybean plant, endophytic fungi and fungi close to the plant roots were studied as a microbial source. Among 33 endophytic fungal isolates screened from the soybean plant, 13 have high lipid content (>20 % dry biomass weight); among 38 fungal isolates screened from the soil surrounding the soybean roots, 14 have high lipid content. Also, five fungal isolates with both high lipid content and promising biomass production were selected for further studies on their cell growth, oil accumulation, lipid content and profile, utilization of various carbon sources, and cellulase production. The results indicate that most strains could utilize different types of carbon sources and some strains accumulated >40 % of the lipids based on the dry cell biomass weight. Among these promising strains, some Fusarium strains specifically showed considerable production of cellulase, which offers great potential for biodiesel production by directly utilizing inexpensive lignocellulosic material as feedstock.