Biotransformation of lignin: Mechanisms,applications and future work

As one of the most abundant polymers in biosphere, lignin has attracted extensive attention as a kind of promising feedstock for biofuel and bio-based products. However, the utilization of lignin presents various challenges in that its complex composition and structure and high resistance to degradation. Lignin conversion through biological platform harnesses the catalytic power of microorganisms to decompose complex lignin molecules and obtain value-added products through biosynthesis. Given the heterogeneity of lignin, various microbial metabolic pathways are involved in lignin bioconversion processes, which has been characterized in extensive research work. With different types of lignin substrates (e.g., model compounds, technical lignin, and lignocellulosic biomass), several bacterial and fungal species have been proved to own lignin-degrading abilities and accumulate microbial products (e.g., lipid and polyhydroxyalkanoates), while the lignin conversion efficiencies are still relatively low. Genetic and metabolic strategies have been developed to enhance lignin biodegradation by reprogramming microbial metabolism, and diverse products, such as vanillin and dicarboxylic acids were also produced from lignin. This article aims at presenting a comprehensive review on lignin bioconversion including lignin degradation mechanisms, metabolic pathways, and applications for the production of value-added bioproducts. Advanced techniques on genetic and metabolic engineering are also covered in the recent development of biological platforms for lignin utilization. To conclude this article, the existing challenges for efficient lignin bioprocessing are analyzed and possible directions for future work are proposed.     

Microbial production of lactic acid: the latest development

Lactic acid is an important platform chemical for producing polylactic acid (PLA) and other value-added products. It is naturally produced by a wide spectrum of microbes including bacteria, yeast and filamentous fungi. In general, bacteria ferment C5 and C6 sugars to lactic acid by either homo- or hetero-fermentative mode. Xylose isomerase, phosphoketolase, transaldolase, l- and d-lactate dehydrogenases are the key enzymes that affect the ways of lactic acid production. Metabolic engineering of microbial strains are usually needed to produce lactic acid from unconventional carbon sources. Production of d-LA has attracted much attention due to the demand for producing thermostable PLA, but large scale production of d-LA has not yet been commercialized. Thermophilic Bacillus coagulans strains are able to produce l-lactic acid from lignocellulose sugars homo-fermentatively under non-sterilized conditions, but the lack of genetic tools for metabolically engineering them severely affects their development for industrial applications. Pre-treatment of agriculture biomass to obtain fermentable sugars is a pre-requisite for utilization of the huge amounts of agricultural biomass to produce lactic acid. The major challenge is to obtain quality sugars of high concentrations in a cost effective-way. To avoid or minimize the use of neutralizing agents during fermentation, genetically engineering the strains to make them resist acidic environment and produce lactic acid at low pH would be very helpful for reducing the production cost of lactic acid.     

Recent advances on production of 2, 3-butanediol using engineered microbes

As a significant platform chemical, 2, 3-butanediol (2, 3-BD) has found wide applications in industry. The success of microbial 2, 3-BD production was limited by the use of pathogenic microorganisms and low titer in engineered hosts. The utilization of cheaply available feedstock such as lignocellulose was another major challenge to achieve economic production of 2, 3-BD. To address those issues, engineering strategies including both genetic modifications and process optimization have been employed. In this review, we summarized the state-of-the-art progress in the biotechnological production of 2, 3-BD. Metabolic engineering and process engineering strategies were discussed.     

Direct production of commodity chemicals from lignocellulose using Myceliophthora thermophila

The production of fuels and chemicals from renewable plant biomass has been proposed as a feasible strategy for global sustainable development. However, the economic efficiency of biorefineries is low. Here, through metabolic engineering, Myceliophthora thermophila, a cellulolytic thermophilic fungus, was constructed into a platform that can efficiently convert lignocellulose into important bulk chemicals—four carbon 1, 4-diacids (malic and succinic acid), building blocks for biopolymers—without the need for extra hydrolytic enzymes. Titers of >200 g/L from crystalline cellulose and 110 g/L from plant biomass (corncob) were achieved during fed-batch fermentation. Our study represents a milestone in consolidated bioprocessing technology and offers a new and promising system for the cost-effective production of chemicals and fuels from biomass.     

Strain engineering for microbial production of value-added chemicals and fuels from glycerol

While the widespread reliance on fossil fuels is driven by their low cost and relative abundance, this fossil-based economy has been deemed unsustainable and, therefore, the adoption of sustainable and environmentally compatible energy sources is on the horizon. Biorefinery is an emerging approach that integrates metabolic engineering, synthetic biology, and systems biology principles for the development of whole-cell catalytic platforms for biomanufacturing. Due to the high degree of reduction and low cost, glycerol, either refined or crude, has been recognized as an ideal feedstock for the production of value-added biologicals, though microbial dissimilation of glycerol sometimes can be difficult particularly under anaerobic conditions. While strain development for glycerol biorefinery is widely reported in the literature, few, if any, commercialized bioprocesses have been developed as a result, such that engineering of glycerol metabolism in microbial hosts remains an untapped opportunity in biomanufacturing. Here we review the recent progress made in engineering microbial hosts for the production of biofuels, diols, organic acids, biopolymers, and specialty chemicals from glycerol. We begin with a broad outline of the major pathways for fermentative and respiratory glycerol dissimilation and key end metabolites, and then focus our analysis on four key genera of bacteria known to naturally dissimilate glycerol, i.e. Klebsiella, Citrobacter, Clostridium, and Lactobacillus, in addition to Escherichia coli, and systematically review the progress made toward engineering these microorganisms for glycerol biorefinery. We also identify the major biotechnological and bioprocessing advantages and disadvantages of each genus, and bottlenecks limiting the production of target metabolites from glycerol in engineered strains. Our analysis culminates in the development of potential strategies to overcome the current technical limitations identified for commonly employed strains, with an outlook on the suitability of different hosts for the production of key metabolites and avenues for their future development into biomanufacturing platforms.     

Efficient,environmentally-friendly and specific valorization of lignin: promising role of non-radical lignolytic enzymes

Lignin is the second most abundant bio-resource in nature. It is increasingly important to convert lignin into high value-added chemicals to accelerate the development of the lignocellulose biorefinery. Over the past several decades, physical and chemical methods have been widely explored to degrade lignin and convert it into valuable chemicals. Unfortunately, these developments have lagged because of several difficulties, of which high energy consumption and non-specific cleavage of chemical bonds in lignin remain the greatest challenges. A large number of enzymes have been discovered for lignin degradation and these are classified as radical lignolytic enzymes and non-radical lignolytic enzymes. Radical lignolytic enzymes, including laccases, lignin peroxidases, manganese peroxidases and versatile peroxidases, are radical-based bio-catalysts, which degrade lignins through non-specific cleavage of chemical bonds but can also catalyze the radical-based re-polymerization of lignin fragments. In contrast, non-radical lignolytic enzymes selectively cleave chemical bonds in lignin and lignin model compounds and, thus, show promise for use in the preparation of high value-added chemicals. In this mini-review, recent developments on non-radical lignolytic enzymes are discussed. These include recently discovered non-radical lignolytic enzymes, their metabolic pathways for lignin conversion, their recent application in the lignin biorefinery, and the combination of bio-catalysts with physical/chemical methods for industrial development of the lignin refinery.     

Plant genetic engineering to improve biomass characteristics for biofuels

Currently, most ethanol produced in the United States is derived from maize kernel, at levels in excess of four billion gallons per year. Plant lignocellulosic biomass is renewable, cheap and globally available at 10-50 billion tons per year. At present, plant biomass is converted to fermentable sugars for the production of biofuels using pretreatment processes that disrupt the lignocellulose and remove the lignin, thus allowing the access of microbial enzymes for cellulose deconstruction. Both the pretreatments and the production of enzymes in microbial tanks are expensive. Recent advances in plant genetic engineering could reduce biomass conversion costs by developing crop varieties with less lignin, crops that self-produce cellulase enzymes for cellulose degradation and ligninase enzymes for lignin degradation, or plants that have increased cellulose or an overall biomass yield.     

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.     

Thermophilic xylanases: from bench to bottle

Lignocellulosic biomass is a valuable raw material. As technology has evolved, industrial interest in new ways to take advantage of this raw material has grown. Biomass is treated with different microbial cells or enzymes under ideal industrial conditions to produce the desired products. Xylanases are the key enzymes that degrade the xylosidic linkages in the xylan backbone of the biomass, and commercial enzymes are categorized into different glycoside hydrolase families. Thermophilic microorganisms are excellent sources of industrially relevant thermostable enzymes that can withstand the harsh conditions of industrial processing. Thermostable xylanases display high-specific activity at elevated temperatures and distinguish themselves in biochemical properties, structures, and modes of action from their mesophilic counterparts. Natural xylanases can be further improved through genetic engineering. Rapid progress with genome editing, writing, and synthetic biological techniques have provided unlimited potential to produce thermophilic xylanases in their natural hosts or cell factories including bacteria, yeasts, and filamentous fungi. This review will discuss the biotechnological potential of xylanases from thermophilic microorganisms and the ways they are being optimized and produced for various industrial applications.     

Engineering Escherichia coli BL21 (DE3) for high-yield production of germacrene A,a precursor of β-elemene via combinatorial metabolic engineering strategies

β-elemene is one of the most commonly used antineoplastic drugs in cancer treatment. As a plant-derived natural chemical, biologically engineering microorganisms to produce germacrene A to be converted to β-elemene harbors great expectations since chemical synthesis and plant isolation methods come with their production deficiencies. In this study, we report the design of an Escherichia coli cell factory for the de novo production of germacrene A to be converted to β-elemene from a simple carbon source. A series of systematic approaches of engineering the isoprenoid and central carbon pathways, translational and protein engineering of the sesquiterpene synthase, and exporter engineering yielded high-efficient β-elemene production. Specifically, deleting competing pathways in the central carbon pathway ensured the availability of acetyl-coA, pyruvate, and glyceraldehyde-3-phosphate for the isoprenoid pathways. Adopting lycopene color as a high throughput screening method, an optimized NS^Y305N was obtained via error-prone polymerase chain reaction mutagenesis. Further overexpression of key pathway enzymes, exporter genes, and translational engineering produced 1161.09 mg/L of β-elemene in a shake flask. Finally, we detected the highest reported titer of 3.52 g/L of β-elemene and 2.13 g/L germacrene A produced by an E. coli cell factory in a 4-L fed-batch fermentation. The systematic engineering reported here generally applies to microbial production of a broader range of chemicals. This illustrates that rewiring E. coli central metabolism is viable for producing acetyl-coA-derived and pyruvate-derived molecules cost-effectively.     

微生物木糖发酵产乙醇的代谢工程

利用木质纤维素发酵生产乙醇具有广泛的应用前景。而自然界中缺少有效转化木糖为乙醇的微生物是充分利用纤维素水解产物、提高乙醇产率、降低生产成本的关键因素。多年来研究者利用分子生物学技术对微生物菌株进行了代谢工程改造,使其能更有效地利用木糖生产乙醇。以下主要对运动发酵单胞菌、大肠杆菌和酵母等候选产乙醇微生物的木糖代谢工程研究进展进行了概述。     

Sugar transporters in efficient utilization of mixed sugar substrates: current knowledge and outlook

There is increasing interest in production of transportation fuels and commodity chemicals from lignocellulosic biomass, most desirably through biological fermentation. Considerable effort has been expended to develop efficient biocatalysts that convert sugars derived from lignocellulose directly to value-added products. Glucose, the building block of cellulose, is the most suitable fermentation substrate for industrial microorganisms such as Escherichia coli, Corynebacterium glutamicum, and Saccharomyces cerevisiae. Other sugars including xylose, arabinose, mannose, and galactose that comprise hemicellulose are generally less efficient substrates in terms of productivity and yield. Although metabolic engineering including introduction of functional pentose-metabolizing pathways into pentose-incompetent microorganisms has provided steady progress in pentose utilization, further improvements in sugar mixture utilization by microorganisms is necessary. Among a variety of issues on utilization of sugar mixtures by the microorganisms, recent studies have started to reveal the importance of sugar transporters in microbial fermentation performance. In this article, we review current knowledge on diversity and functions of sugar transporters, especially those associated with pentose uptake in microorganisms. Subsequently, we review and discuss recent studies on engineering of sugar transport as a driving force for efficient bioconversion of sugar mixtures derived from lignocellulose.     

Methods for Facilitating Microbial Growth on Pulp Mill Waste Streams and Characterization of the Biodegradation Potential of Cultured Microbes

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high quality paper. Black liquor is a pulping waste generated by the kraft process that has potential for downstream bioconversion. However, the recalcitrant nature of the lignocellulose resources, its chemical derivatives that constitute the majority of available organic carbon within black liquor, and its basic pH present challenges to microbial biodegradation of this waste material. Methods for the collection and modification of black liquor for microbial growth are aimed at utilization of this pulp waste to convert the lignin, organic acids, and polysaccharide degradation byproducts into valuable chemicals. The lignocellulose extraction techniques presented provide a reproducible method for preparation of lignocellulose growth substrates for understanding metabolic capacities of cultured microorganisms. Use of gas chromatography-mass spectrometry enables the identification and quantification of the fermentation products resulting from the growth of microorganisms on pulping waste. These methods when used together can facilitate the determination of the metabolic activity of microorganisms with potential to produce fermentation products that would provide greater value to the pulping system and reduce effluent waste, thereby increasing potential paper milling profits and offering additional uses for black liquor.     

Systems biology based metabolic engineering for non-natural chemicals

Production of chemicals in microorganisms is no longer restricted to products arising from native metabolic potential. In this review, we highlight the evolution of metabolic engineering studies, from the production of natural chemicals fermented from biomass hydrolysates, to the engineering of microorganisms for the production of non-natural chemicals. Advances in synthetic biology are accelerating the successful development of microbial cell factories to directly produce value-added chemicals. Here we outline the emergence of novel computational tools for the creation of synthetic pathways, for designing artificial enzymes for non-natural reactions and for re-wiring host metabolism to increase the metabolic flux to products. We also highlight exciting opportunities for applying directed evolution of enzymes, dynamic control of growth and production, growth-coupling strategies as well as decoupled strategies based on orthogonal pathways in the context of non-natural chemicals.     

生物质多糖的高效降解与降解酶（系）的精确定制

自然界中多糖类生物质资源十分丰富,然而其复杂的抗降解屏障限制了生物转化的进程.近年来,随着生物质多糖结构的快速解析以及大量多糖降解酶的鉴定研究,针对不同底物结构或产物需求,仿制高效微生物多糖代谢途径,精确定制多糖降解酶系,促进生物质高效转化已成为可能.本文分析中性多糖(纤维素和木聚糖)、碱性多糖(几丁质和壳聚糖)以及酸性多糖(褐藻胶)的精细结构组成与基团性质,总结3类多糖主要降解酶的活性架构特征及其底物精确结合模式.文章还阐述蛋白质工程设计与定制策略,针对酶分子不同功能区的分析,可为酶分子的功能快速设计与改造提供靶点,以获得适宜于工业应用的高效酶分子,此外,根据微生物胞外降解酶系的降解次序与协同关系,可基于应用需求精确定制复杂多糖降解酶系,实现生物质的高效与高值降解转化.     

Microbial production of lactate-containing polyesters

Due to our increasing concerns on environmental problems and limited fossil resources, biobased production of chemicals and materials through biorefinery has been attracting much attention. Optimization of the metabolic performance of microorganisms, the key biocatalysts for the efficient production of the desired target bioproducts, has been achieved by metabolic engineering. Metabolic engineering allowed more efficient production of polyhydroxyalkanoates, a family of microbial polyesters. More recently, non-natural polyesters containing lactate as a monomer have also been produced by one-step fermentation of engineered bacteria. Systems metabolic engineering integrating traditional metabolic engineering with systems biology, synthetic biology, protein/enzyme engineering through directed evolution and structural design, and evolutionary engineering, enabled microorganisms to efficiently produce natural and non-natural products. Here, we review the strategies for the metabolic engineering of microorganisms for the in vivo biosynthesis of lactate-containing polyesters and for the optimization of whole cell metabolism to efficiently produce lactate-containing polyesters. Also, major problems to be solved to further enhance the production of lactate-containing polyesters are discussed.     

Screening of carbon sources for beta-glucosidase production by Aspergillus saccharolyticus

The fungus Aspergillus saccharolyticus was found to produce a culture broth rich in beta-glucosidase activity, an enzyme which plays an essential role for efficient and complete hydrolysis of lignocellulosic biomass. Direct application of fungal fermentation broths produced on-site in a biorefinery may be an integral part of a biorefinery for lowering the cost associated with the use of commercial enzymes for saccharification of biomass. Utilization of low value slip streams from the biorefinery as substrates for such an on-site enzyme production would be ideal to reduce costs. In order to understand which carbon sources that support growth and trigger A. saccharolyticus to produce beta-glucosidases, carbon sources, ranging from monomer sugars to complex lignocellulosic biomasses, including pretreated and hydrolyzed corn stover fractions, were investigated as substrates and inducers of enzyme production. A convenient micro titer plate experimental setup was developed that facilitated a fast screening for beta-glucosidase activity on the different carbon sources. The greatest beta-glucosidase activity was found when A. saccharolyticus was cultivated on media containing xylose, xylan, wheat bran, and pretreated corn stover. In a refinery, beta-glucosidase production by A. saccharolyticus could with success be based on the biomass hemicelluloses and their degradation products which cannot be converted by conventional yeast.     

Bioconversion of lignocellulosic biomass: biochemical and molecular perspectives

In view of rising prices of crude oil due to increasing fuel demands, the need for alternative sources of bioenergy is expected to increase sharply in the coming years. Among potential alternative bioenergy resources, lignocellulosics have been identified as the prime source of biofuels and other value-added products. Lignocelluloses as agricultural, industrial and forest residuals account for the majority of the total biomass present in the world. To initiate the production of industrially important products from cellulosic biomass, bioconversion of the cellulosic components into fermentable sugars is necessary. A variety of microorganisms including bacteria and fungi may have the ability to degrade the cellulosic biomass to glucose monomers. Bacterial cellulases exist as discrete multi-enzyme complexes, called cellulosomes that consist of multiple subunits. Cellulolytic enzyme systems from the filamentous fungi, especially Trichoderma reesei, contain two exoglucanases or cellobiohydrolases (CBH1 and CBH2), at least four endoglucanases (EG1, EG2, EG3, EG5), and one β-glucosidase. These enzymes act synergistically to catalyse the hydrolysis of cellulose. Different physical parameters such as pH, temperature, adsorption, chemical factors like nitrogen, phosphorus, presence of phenolic compounds and other inhibitors can critically influence the bioconversion of lignocellulose. The production of cellulases by microbial cells is governed by genetic and biochemical controls including induction, catabolite repression, or end product inhibition. Several efforts have been made to increase the production of cellulases through strain improvement by mutagenesis. Various physical and chemical methods have been used to develop bacterial and fungal strains producing higher amounts of cellulase, all with limited success. Cellulosic bioconversion is a complex process and requires the synergistic action of the three enzymatic components consisting of endoglucanases, exoglucanases and β-glucosidases. The co-cultivation of microbes in fermentation can increase the quantity of the desirable components of the cellulase complex. An understanding of the molecular mechanism leading to biodegradation of lignocelluloses and the development of the bioprocessing potential of cellulolytic microorganisms might effectively be accomplished with recombinant DNA technology. For instance, cloning and sequencing of the various cellulolytic genes could economize the cellulase production process. Apart from that, metabolic engineering and genomics approaches have great potential for enhancing our understanding of the molecular mechanism of bioconversion of lignocelluloses to value added economically significant products in the future. JIMB 2008: BioEnergy - Special issue.     

微生物木糖代谢途径改造制备生物基化学品

当前,全球经济的高速发展与日益减少的石油资源储备进一步加剧了能源供需矛盾。人类对开发利用可再生的纤维素生物质资源寄予厚望。木糖是木质纤维素水解产物中含量仅次于葡萄糖的一种单糖,因此对木糖高效率生物转化的研究成为影响其工业化前景的关键因素之一。针对近几年的研究,文中综述了生物转化木糖方面的进展,包括木糖代谢途径的鉴定和设计、木糖运输途径的改造、生物基化学品制备。为了解决当前全球面临的能源危机与环境问题,运用合成生物学技术发展新一代生物燃料技术,特别是开发能够代谢木糖高产乙醇的微生物工程菌株是实现可持续发展的重要方式。