Development of arbuscular mycorrhizal biotechnology and industry: current achievements and bottlenecks

Advanced scientific knowledge on arbuscular mycorrhizal symbioses recently enhanced potential for implementation of mycorrhizal biotechnology in horticulture and agriculture plant production, landscaping, phytoremediation and other segments of the plant market. The advances consist in significant findings regarding:—new molecular detection tools for tracing inoculated fungi in the field;—the coexistence mechanisms of various fungi in the single root system;—new knowledge on in vitro physiology of the AM fungi grown in root organ cultures;—mechanisms of synergistic interactions with other microbes like PGPR or saprotrophic fungi; discovery of mycorrhiza supportive compounds such as strigolactones. Scientific knowledge has been followed by technological developments like novel formulations for liquid applications or seed coating, mycorrhiza stimulating compounds or new application modes. Still the missing components of biotechnology are appropriate, cheap, highly reproducible and effective methods for inocula purity testing and quality control. Also there is a weak traceability of the origin of the mycorrhizal fungi strains used in commercial inocula. Numerous poor quality products can still be found on the markets claiming effective formation mycorrhiza which have very low capacity to do so. These products usually rely in their effects on plant growth not on support of host plants via formation of effective mycorrhizal symbiosis but on fertilizing compounds added to products. There is growing number of enterprises producing mycorrhiza based inocula recently not only in developed world but increasingly in emerging markets. Also collaboration between private sector and scientific community has an improving trend as the development of private sector can fuel further research activities. Last but not least there is apparent growing pull of the market and increasing tendency of reduction of agrochemical inputs and employment of alternative strategies in planting and plant production. These circumstances support further developments of mycorrhizal inocula production and applications and maturation of the industry.     

丝状真菌纤维素酶合成诱导及转录调控

利用廉价可再生木质纤维素资源水解产生的可发酵糖生产生物能源和生物基化学品是近年来国内外研究的热点。纤维素酶酶解是木质纤维素原料生物降解的重要手段,但目前纤维素酶生产成本过高,限制了纤维素生物转化和生物炼制的工业化应用。对丝状真菌纤维素酶基因表达和调控进行研究,有利于进一步选育纤维素酶高产菌株,降低纤维素酶生产成本。随着高通量测序及丝状真菌遗传操作等技术的进步,对丝状真菌纤维素酶诱导和基因表达调控机理有了更深入的认识。本文综述了近年来丝状真菌纤维素酶诱导和纤维素酶基因表达调控的最新进展,重点论述糖转运蛋白、转录因子和染色质重塑对纤维素酶表达调控的影响,并对利用人工锌指蛋白进行丝状真菌纤维素酶诱导调控研究进行了展望。     

Plant biotechnology for lignocellulosic biofuel production

Lignocelluloses from plant cell walls are attractive resources for sustainable biofuel production. However, conversion of lignocellulose to biofuel is more expensive than other current technologies, due to the costs of chemical pretreatment and enzyme hydrolysis for cell wall deconstruction. Recalcitrance of cell walls to deconstruction has been reduced in many plant species by modifying plant cell walls through biotechnology. These results have been achieved by reducing lignin content and altering its composition and structure. Reduction of recalcitrance has also been achieved by manipulating hemicellulose biosynthesis and by overexpression of bacterial enzymes in plants to disrupt linkages in the lignin–carbohydrate complexes. These modified plants often have improved saccharification yield and higher ethanol production. Cell wall‐degrading (CWD) enzymes from bacteria and fungi have been expressed at high levels in plants to increase the efficiency of saccharification compared with exogenous addition of cellulolytic enzymes. In planta expression of heat‐stable CWD enzymes from bacterial thermophiles has made autohydrolysis possible. Transgenic plants can be engineered to reduce recalcitrance without any yield penalty, indicating that successful cell wall modification can be achieved without impacting cell wall integrity or plant development. A more complete understanding of cell wall formation and structure should greatly improve lignocellulosic feedstocks and reduce the cost of biofuel production.     

A critical review of the application of white rot fungus to environmental pollution control

Research on white rot fungi for environmental biotechnology has been conducted for more than 20 years. In this article, we have reviewed processes for cell growth and enzyme production including the factors influencing enzyme productivity and the methods for enhancement of enzyme production. Significant progress has been achieved in molecular biology related to white rot fungi, especially related to the extraction of genetic material (RNA and DNA), gene cloning and the construction of genetically engineered microorganisms. The development of biotechnologies using white rot fungi for environmental pollution control has been implemented to treat various refractory wastes and to bioremediate contaminated soils. The current status and future research needs for fundamentals and application are addressed in this review.     

Tailoring lignin biosynthesis for efficient and sustainable biofuel production

Increased global interest in a bio‐based economy has reinvigorated the research on the cell wall structure and composition in plants. In particular, the study of plant lignification has become a central focus, with respect to its intractability and negative impact on the utilization of the cell wall biomass for producing biofuels and bio‐based chemicals. Striking progress has been achieved in the last few years both on our fundamental understanding of lignin biosynthesis, deposition and assembly, and on the interplay of lignin synthesis with the plant growth and development. With the knowledge gleaned from basic studies, researchers are now able to invent and develop elegant biotechnological strategies to sophisticatedly manipulate the quantity and structure of lignin and thus to create economically viable bioenergy feedstocks. These concerted efforts open an avenue for the commercial production of cost‐competitive biofuel to meet our energy needs.     

几种丝状真菌原生质体的形成与再生*

本文报道从黑曲霉、米曲霉、酱油曲霉、康宁木霉、拟青零等工业上有重要用途的7种丝状真菌菌丝制备原生质体,以及对这些真菌原生质再生过程中形态学变化进行观察的结果。实验表明用商品纤维素酶、蜗牛酶、溶菌酶配成的混合酶液,可成功地对上述丝状真菌制备出原生质体。在所有供试菌株中均观察到由原生质体直接长出菌丝,及原生质体先形成酵母状物再长出菌丝这两类再生方式。     

几种丝状真菌原生质体的形成与再生

本文报道从黑曲霉、米曲霉、酱油曲霉、康宁木霉、拟青零等工业上有重要用途的7种丝状真菌菌丝制备原生质体,以及对这些真菌原生质再生过程中形态学变化进行观察的结果。实验表明用商品纤维素酶、蜗牛酶、溶菌酶配成的混合酶液,可成功地对上述丝状真菌制备出原生质体。在所有供试菌株中均观察到由原生质体直接长出菌丝,及原生质体先形成酵母状物再长出菌丝这两类再生方式。     

Transfection and expression of plasmid DNA in plant cells by an arginine-rich intracellular delivery peptide without protoplast preparation

The delivery and expression of exogenous genes in plant cells have been of particular interest for plant research and biotechnology. Here, we present results demonstrating a simple DNA transfection system in plants. Short arginine-rich intracellular delivery peptide, a protein transduction domain, was capable of delivering plasmid DNA into living plant cells non-covalently. This peptide-mediated DNA delivery conferred several advantages, such as nuclear targeting, non-toxic effect, and ease of preparation without protoplast formulation. Thus, this novel technology shall provide a powerful tool to investigate gene function in vivo, and lay the foundation for the production of transgenic plants in future.     

Seaweed protoplasts: status,biotechnological perspectives and needs

Protoplasts are living plant cells without cell walls which offer a unique uniform single cell system that facilitates several aspects of modern biotechnology, including genetic transformation and metabolic engineering. Extraction of cell wall lytic enzymes from different phycophages and microbial sources has greatly improved protoplast isolation and their yield from a number of anatomically more complex species of brown and red seaweeds which earlier remained recalcitrant. Recently, recombinant cell wall lytic enzymes were also produced and evaluated with native ones for their potential abilities in producing viable protoplasts from Laminaria. Reliable procedures are now available to isolate and culture protoplasts from diverse groups of seaweeds. To date, there are 89 species belonging to 36 genera of green, red and brown seaweeds from which successful protoplast isolation and regeneration has been reported. Of the total species studied for protoplasts, most belonged to Rhodophyta with 41 species (13 genera) followed by Chlorophyta and Phaeophyta with 24 species each belonging to 5 and 18 genera, respectively. Regeneration of protoplast-to-plant system is available for a large number of species, with extensive literature relating to their culture methods and morphogenesis. In the context of plant genetic manipulation, somatic hybridization by protoplast fusion has been accomplished in a number of economically important species with various levels of success. Protoplasts have also been used for studying foreign gene expression in Porphyra and Ulva. Isolated protoplasts are also exploited in numerous miscellaneous studies involving membrane function, cell structure, bio-chemical synthesis of cell walls etc. This article briefly reviews the status of various developments in seaweed protoplasts research and their potentials in genetic improvement of seaweeds, along with needs that must to be fulfilled for effective realization of the objectives envisaged for protoplast research.     

Recent trends in nanomaterials immobilised enzymes for biofuel production

Application of nanomaterials as novel supporting materials for enzyme immobilisation has generated incredible interest in the biotechnology community. These robust nanostructured forms, such as nanoparticles, nanofibres, nanotubes, nanoporous, nanosheets, and nanocomposites, possess a high surface area to volume ratios that can cause a high enzyme loading and facilitate reaction kinetics, thus improving biocatalytic efficiency for industrial applications. In this article, we discuss research opportunities of nanoscale materials in enzyme biotechnology and highlight recent developments in biofuel production using advanced material supports for enzyme immobilisation and stabilisation. Synthesis and functionalisation of nanomaterial forms using different methods are highlighted. Various simple and effective strategies designed to result in a stable, as well as functional protein-nanomaterial conjugates are also discussed. Analytical techniques confirming enzyme loading on nanomaterials and assessing post-immobilisation changes are discussed. The current status of versatile nanomaterial support for biofuel production employing cellulases and lipases is described in details. This report concludes with a discussion on the likely outcome that nanomaterials will become an integral part of sustainable bioenergy production.     

Marginal lands for bioenergy in China; an outlook in status,potential and management

Energy consumption and CO₂ emissions have been increasing continuously over the past few decades in China and there is a pressing need to replace the fossil fuel‐based economy with an efficient low‐carbon system, tailor‐made to future requirements. China is starting an energy transition with the aim of building an energy system for the future. China has made tremendous progress in increasing the amount of renewable energy and reducing the cost of renewable energy over the last 20 years. According to the 14th 5 year plan, China aims to incorporate 20% of renewable energy to the primary energy mix and attain 27% reduction in CO₂ emissions. Bioenergy crops constitute a significant proportion of biomass‐based bioenergy and have recently been promoted by the Chinese Government to help overcome food and fuel conflict. Steps are being taken to promote bioenergy crops on marginal lands in China, and various regions across the country with soil marginality have been evaluated for bioenergy crop cultivation. The present paper reviews the status of bioenergy in China and the potential status of marginal lands from different regions of China. It also elaborates on some of the policies, subsidies and incentives allocated by the Chinese Government for the promotion of biomass‐based energy. Land management and plant improvement strategies were discussed, which are effective in making marginal lands suitable for bioenergy crop cultivation. Managing planting strategies, intercropping and crop rotation are effective management practices used in China for the utilization of marginal lands. A national investigation is desirable for creating an inventory of technical and economic potential of biomass feedstocks that could be planted on marginal lands. This would assist with highlighting the pros and cons of using marginal lands for bioenergy production and effective policy making.     

Production, partial purification and characterization of feruloyl esterase by Aspergillus awamori in submerged fermentation

Microbial feruloyl esterases acting on plant cell wall polymers represent key tools for the degradation of plant cell wall. In this paper, we describe in detail the microbial production, partial purification and characterization of feruloyl esterase from a culture medium of Aspergillus awamori strain IFO4033 obtained from a crude hemicellulose preparation of wheat straw, corncobs and wheat germ. Feruloyl esterase was extracted using centrifugation and dialysis, and then purified by ion exchange chromatography and microfiltration to homogeneity, which was checked by SDSPAGE and isoelectric focusing-PAGE. Protein content and activity of the enzyme were measured in each step of extraction and purification. Biomass was determined by the dry weight method. pH and temperature optima of feruloyl esterase enzyme were also determined. The effects of culturing time, and carbon and nitrogen sources on enzyme production were systematically investigated. Finally, enzyme activities under different storage conditions were examined.     

The realm of cellulases in biorefinery development

Geopolitical concerns (unstable supply of gasoline, environmental pollution, and regular price hikes), economic, and employment concerns have been prompting researchers, entrepreneurs, and policy makers to focus on harnessing the potential of lignocellulosic feedstock for fuel ethanol production and its commercialization. The carbohydrate skeleton of plant cell walls needs to be depolymerised into simpler sugars for their application in fermentation reactions as a chief carbon source of suitable ethnologic strains for ethanol production. The role of cellulolytic enzymes in the degradation of structural carbohydrates of the plant cell wall into ready-to-fermentable sugar stream is inevitable. Cellulase synergistically acts upon plant cell wall polysaccharides to release glucose into the liquid media. Cellulase predominantly dominates all the plant cell wall degrading enzymes due to their vast and diverse range of applications. Apart from the major applications of cellulases such as in detergent formulations, textile desizing, and development of monogastric feed for ruminants, their role in biorefinery is truly remarkable. This is a major area where new research tools based upon fermentation based formulations, biochemistry, and system biology to expedite the structure-function relationships of cellulases including cellulosomes and new designer enzymatic cocktails are required. In the last two decades, a considerable amount of research work has been performed on cellulases and their application in biomass saccharification. However, there are still technical and economic impediments to the development of an inexpensive commercial cellulase production process. Advancements in biotechnology such as screening of microorganisms, manipulation of novel cellulase encoding traits, site-specific mutagenesis, and modifications to the fermentation process could enhance the production of cellulases. Commercially, cheaper sources of carbohydrates and modified fermentation conditions could lead to more cost-effective production of cellulases with the goal to reduce the cost of ethanol production from lignocellulosics. Implementation of integrated steps like cellulase production and cellulase mediated saccharification of biomass in conjunction with the fermentation of released sugars in ethanol in a single step so called consolidated bio-processing (CBP) is very important to reduce the cost of bioethanol. This paper aims to explore and review the important findings in cellulase biotechnology and the forward path for new cutting edge opportunities in the success of biorefineries.     

The realm of cellulases in biorefinery development

Geopolitical concerns (unstable supply of gasoline, environmental pollution, and regular price hikes), economic, and employment concerns have been prompting researchers, entrepreneurs, and policy makers to focus on harnessing the potential of lignocellulosic feedstock for fuel ethanol production and its commercialization. The carbohydrate skeleton of plant cell walls needs to be depolymerised into simpler sugars for their application in fermentation reactions as a chief carbon source of suitable ethnologic strains for ethanol production. The role of cellulolytic enzymes in the degradation of structural carbodydrates of the plant cell wall into ready-to-fermentable sugar stream is inevitable. Cellulase synergistically acts upon plant cell wall polysaccharides to release glucose into the liquid media. Cellulase predominantly dominates all the plant cell wall degrading enzymes due to their vast and diverse range of applications. Apart from the major applications of cellulases such as in detergent formulations, textile desizing, and development of monogastric feed for ruminants, their role in biorefinery is truly remarkable. This is a major area where new research tools based upon fermentation based formulations, biochemistry, and system biology to expedite the structure–function relationships of cellulases including cellulosomes and new designer enzymatic cocktails are required. In the last two decades, a considerable amount of research work has been performed on cellulases and their application in biomass saccharification. However, there are still technical and economic impediments to the development of an inexpensive commercial cellulase production process. Advancements in biotechnology such as screening of microorganisms, manipulation of novel cellulase encoding traits, site-specific mutagenesis, and modifications to the fermentation process could enhance the production of cellulases. Commercially, cheaper sources of carbohydrates and modified fermentation conditions could lead to more cost-effective production of cellulases with the goal to reduce the cost of ethanol production from lignocellulosics. Implementation of integrated steps like cellulase production and cellulase mediated saccharification of biomass in conjunction with the fermentation of released sugars in ethanol in a single step so called consolidated bio-processing (CBP) is very important to reduce the cost of bioethanol. This paper aims to explore and review the important findings in cellulase biotechnology and the forward path for new cutting edge opportunities in the success of biorefineries.     

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。     

Hypocrea jecorina CEL6A protein engineering

The complex technology of converting lignocellulose to fuels such as ethanol has advanced rapidly over the past few years, and enzymes are a critical component of this technology. The production of effective enzyme systems at cost structures that facilitate commercial processes has been the focus of research for many years. Towards this end, the H. jecorina cellobiohydrolases, CEL7A and CEL6A, have been the subject of protein engineering at Genencor. Our first rounds of cellobiohydrolase engineering were directed towards improving the thermostability of both of these enzymes and produced variants of CEL7A and CEL6A with apparent melting temperatures above 70°C, placing their stability on par with that of H. jecorina CEL5A (EG2) and CEL3A (BGL1). We have now moved towards improving CEL6A- and CEL7A-specific performance in the context of a complete enzyme system under industrially relevant conditions. Achievement of these goals required development of new screening strategies and tools. We discuss these advances along with some results, focusing mainly on engineering of CEL6A.     

Fungal and other β-d-glucosidases — Their properties and applications

β-d-Glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) has been described in a variety of fungi and bacteria. Its function — to catalyse the hydrolysis of cellobiose, and aryl and alkyl β-d-glucosides — depends upon the nature of its source. Recent interest in this enzyme centres on its role in the enzymatic hydrolysis of cellulose. The rate and extent of cellulose hydrolysis can be increased by supplementing commercial cellulases with immobilized β-d-glucosidase, which has high stability and can be recovered and reused. The current state of β-d-glucosidase biotechnology is described.     

Plants to power: bioenergy to fuel the future

Bioenergy should play an essential part in reaching targets to replace petroleum-based transportation fuels with a viable alternative, and in reducing long-term carbon dioxide emissions, if environmental and economic sustainability are considered carefully. Here, we review different platforms, crops, and biotechnology-based improvements for sustainable bioenergy. Among the different platforms, there are two obvious advantages to using lignocellulosic biomass for ethanol production: higher net energy gain and lower production costs. However, the use of lignocellulosic ethanol as a viable alternative to petroleum-based transportation fuels largely depends on plant biotechnology breakthroughs. We examine how biotechnology, such as lignin modification, abiotic stress resistance, nutrition usage, in planta expression of cell wall digestion enzymes, biomass production, feedstock establishment, biocontainment of transgenes, metabolic engineering, and basic research, can be used to address the challenges faced by bioenergy crop production.     

Simple and reliable system for transient gene expression for the characteristic signal sequences and the estimation of the localization of target protein in plant cell

The efficiency of transient gene expression in plants credibly demonstrated characteristics of gene functions in numerous studies. Two key strategies of transient expression became favorites among researchers: protoplast transfection and agroinfiltration. Each of them, alongside the advantages, has its own constraints. In this work, an easy, rapid, and reliable system for characterization of the signal sequences and determinations of target protein localization in a plant cell is proposed and tested. This system—called the AgI–PrI—implies production of protoplasts from plant tissues after agroinfiltration. Reliability of the proposed system for transient gene expression has been proved using characterized signal sequences in Nicotiana benthamiana cells. The corresponding protocol is less expensive and depends to a lesser degree on the professional skills in the area of protoplast isolation and transfection; furthermore, it may be applicable to other plant species with either available efficient methods of agroinfiltration and protoplast isolation or with the potential for one of the protocols to be supplemented. Thus, the AgI–PrI technique makes it possible to combine the advantages of two widely used methods for the transient gene expression in plants—agroinfiltration and protoplast isolation and transfection—and concurrently avoids their critical points.     

Development of a sample preparation method for fungal proteomics

Since filamentous fungi including basidiomycetous fungi possess an exceptionally robust cell wall as in microorganisms, effective extraction of intracellular proteins is a key step for fungal proteomic studies. To overcome the experimental obstacle caused by cell walls, we utilized fungal protoplasts, prepared from the brown-rot basidiomycete, Tyromyces palustris. The amount and quality of proteins extracted from the protoplast cells were much higher than that from the mycelial cells. Quantitative comparisons of proteome maps prepared from mycelial and protoplast cells indicated protein spots with a wider range of molecular weights and pIs in the protoplast sample. Furthermore, no streaking or tailing was observed in the protoplasts, suggesting that effective extraction of intracellular proteins from protoplasts might help suppress degradation of proteins during this process. In addition to the efficiency of protein extraction, simple and efficient subcellular fractionation was also achieved using protoplast cells.