Plant pathogens as a source of diverse enzymes for lignocellulose digestion

The plant cell wall is a major barrier that many plant pathogens must surmount for successful invasion of their plant hosts. Full genome sequencing of a number of plant pathogens has revealed often large, complex, and redundant enzyme systems for degradation of plant cell walls. Recent surveys have noted that plant pathogenic fungi are highly competent producers of lignocellulolytic enzymes, and their enzyme activity patterns reflect host specificity. We propose that plant pathogens may contribute to biofuel production as diverse sources of accessory enzymes for more efficient conversion of lignocellulose into fermentable sugars.     

Fungal Bioconversion of Lignocellulosic Residues; Opportunities & Perspectives

The development of alternative energy technology is critically important because of the rising prices of crude oil, security issues regarding the oil supply, and environmental issues such as global warming and air pollution. Bioconversion of biomass has significant advantages over other alternative energy strategies because biomass is the most abundant and also the most renewable biomaterial on our planet. Bioconversion of lignocellulosic residues is initiated primarily by microorganisms such as fungi and bacteria which are capable of degrading lignocellulolytic materials. Fungi such as Trichoderma reesei and Aspergillus niger produce large amounts of extracellular cellulolytic enzymes, whereas bacterial and a few anaerobic fungal strains mostly produce cellulolytic enzymes in a complex called cellulosome, which is associated with the cell wall. In filamentous fungi, cellulolytic enzymes including endoglucanases, cellobiohydrolases (exoglucanases) and β-glucosidases work efficiently on cellulolytic residues in a synergistic manner. In addition to cellulolytic/hemicellulolytic activities, higher fungi such as basidiomycetes (e.g. Phanerochaete chrysosporium) have unique oxidative systems which together with ligninolytic enzymes are responsible for lignocellulose degradation. This review gives an overview of different fungal lignocellulolytic enzymatic systems including extracellular and cellulosome-associated in aerobic and anaerobic fungi, respectively. In addition, oxidative lignocellulose-degradation mechanisms of higher fungi are discussed. Moreover, this paper reviews the current status of the technology for bioconversion of biomass by fungi, with focus on mutagenesis, co-culturing and heterologous gene expression attempts to improve fungal lignocellulolytic activities to create robust fungal strains.     

Lentinus edodes and Pleurotus species lignocellulolytic enzymes activity in submerged and solid-state fermentation of lignocellulosic wastes of different composition

Lentinus edodes and Pleurotus species from various origins were compared for the first time for their ability to produce lignocellulolytic enzyme in solid-state (SSF) and submerged (SF) fermentation of various plant raw material. Fungi cultivation in identical culture conditions revealed wide differences among both species and strains of the same species. The yields of CMCase (62.3Uml(-1)), xylanase (84.1 U ml(-1)), FPA (5.9 U ml(-1)), and laccase (4103 Ul(-1)) are the best so far obtained with the strains of oyster mushrooms. The study pointed out that the nature of lignocellulosic material and the method of fungi cultivation are factors determining the expression of lignocellulolytic potential of fungi as well as the ratio of individual enzymes in enzyme complex. SSF of tree leaves is favorable for laccase and MnP secretion by the majority L. edodes and Pleurotus strains, whereas SF provides better production of hydrolytic enzymes.     

The major cellulases CBH‐1 and CBH‐2 of Neurospora crassa rely on distinct ER cargo adaptors for efficient ER‐exit

Filamentous fungi are native secretors of lignocellulolytic enzymes and are used as protein‐producing factories in the industrial biotechnology sector. Despite the importance of these organisms in industry, relatively little is known about the filamentous fungal secretory pathway or how it might be manipulated for improved protein production. Here, we use Neurospora crassa as a model filamentous fungus to interrogate the requirements for trafficking of cellulase enzymes from the endoplasmic reticulum to the Golgi. We characterized the localization and interaction properties of the p24 and ERV‐29 cargo adaptors, as well as their role in cellulase enzyme trafficking. We find that the two most abundantly secreted cellulases, CBH‐1 and CBH‐2, depend on distinct ER cargo adaptors for efficient exit from the ER. CBH‐1 depends on the p24 proteins, whereas CBH‐2 depends on the N. crassa homolog of yeast Erv29p. This study provides a first step in characterizing distinct trafficking pathways of lignocellulolytic enzymes in filamentous fungi.     

白蚁及共生微生物木质纤维素水解酶的种类

白蚁是热带生态系统重要的木质纤维素降解者。白蚁种类丰富,可分成高等白蚁和低等白蚁,食性也具有各自特点。白蚁自身可以产生纤维素酶,主要是GHF9的内切葡聚糖酶(EG),也有β-葡萄糖苷酶(GB)。低等白蚁共生的原虫中已发现丰富的纤维素酶基因,属于GHF5,7和45。同时还有其他相关功能基因,如木聚糖酶和果胶类物质水解酶。高等白蚁肠道中没有共生原虫。高等培菌白蚁可以利用共生蚁巢伞属真菌促进木质纤维素降解,真菌可以产生纤维素酶,果胶质水解酶类、木聚糖酶,同时还产生可能与木质素分解相关的一种漆酶,但是从分子水平,关于共生真菌纤维素水解酶的研究还较少。白蚁肠道已分离出许多具有木质纤维素降解能力的菌株,最近的研究也发现了大量细菌纤维素酶基因。白蚁-共生系统丰富的木质纤维素水解酶类为发展生物方法开发纤维素乙醇这一思路提供有价值的资源。     

贝壳状革耳菌和黄孢平革菌固体培养酶系比较

白腐菌黄孢平革菌(Phanerochaete chrysosporium) 与贝壳状革耳菌(Panus conchatus)在类似自然状态的固体培养条件下酶的分泌情况有 较大差异。P.conchatus和P.chrysosporium的主要木素降解酶分别是漆酶和锰过氧化物酶 ;两种菌均产生较高水平的木聚糖酶;P.conchatus在整个培养过程中所产生的内切葡 聚糖酶、微晶纤维素酶和纤维二糖酶活力均比P.chrysosporium相应酶的活力低得多, 尤其是内切葡聚糖酶。研究结果初步揭示了P.conchaus降解木素的主要酶系及选择性降 解木素的原因。     

Biological pretreatment and bioconversion of agricultural wastes,using ligninolytic and cellulolytic fungal consortia

Agricultural wastes have attractive potential as alternate energy sources. However, a major bottleneck is to identify eco-friendly treatment methodologies to utilize them. The large diversity of unexplored, novel, and potential microorganisms hold great promise and require periodic isolation and characterization of microorganisms for bioprospection. In this study, approximately 100 fungal isolates were tested for their lignocellulolytic enzyme activities, based on plate assay, followed by quantification of enzyme activity. From this, M2E (Inonotus tropicalis) and 2a (Cerrena unicolor) showed good growth and proficient ligninolytic activity; isolates GK1 (Chaetomium globosum) and GK2 (Chaetomium brasiliense) exhibited exceptional cellulolytic activity on lignocellulosic substrates such as rice straw and sugarcane bagasse. Consortia of the potential ligninolytic and cellulolytic isolates were set up to determine their ability to biodegrade the lignocellulosic substrates such as rice straw and sugarcane bagasse. The efficiency of the consortia was determined on the basis of the increase in enzyme activity; it was also evident through scanning electron microscopy, x-ray diffraction analysis of the degraded substrates, and the sugar yield. Experiments were also carried out to compare the biological with the physical pretreatment methods. The consortium of ligninolytic and cellulolytic marine-derived fungi developed in this study prove to have the potential for application in the effective utilization of agricultural wastes.     

Microbial characterization of organic carrier colonization during a model biofiltration experiment

AIMS: Dynamic microbial characterization of the colonization of organic carrier during a model biofiltration experiment using methanol as air pollutant. METHODS AND RESULTS: A model biofilter was used in order to characterize the micro-organisms involved in the colonization of a model organic carrier. The model system consisted of closed vial as biofilter, peanut shells as lignocellulosic carrier and methanol as air pollutant. The micro-organisms involved in biofiltration were identified and characterized for their lignocellulolytic and methylotrophic activities. Fungi presented a higher lignocellulolytic activity than bacteria. A steady-state was reached after 15 to 20 days. CONCLUSIONS: The consortium naturally associated to peanut shells is limited to few aerobic bacteria and lignocellulolytic fungi. This consortium was able to degrade methanol without external nutrient supply. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first paper that focuses on carrier degradation processes and the micro-organisms involved during the start-up period of a biofiltration process.     

Exploring the bioprospecting and biotechnological potential of white-rot and anaerobic Neocallimastigomycota fungi: peptidases,esterases, and lignocellulolytic enzymes

Fungi constitute an invaluable natural resource for scientific research, owing to their diversity; they offer a promising alternative for bioprospecting, thus contributing to biotechnological advances. For a long time, extensive information has been exploited and fungal products have been tested as a source of natural compounds. In this context, enzyme production remains a field of interest, since it offers an efficient alternative to the hazardous processes of chemical transformations. Owing to their vast biodiversity and peculiar biochemical characteristics, two fungal categories, white-rot and anaerobic Neocallimastigomycota, have gathered considerable attention for biotechnological applications. These fungi are known for their ability to depolymerize complex molecular structures and are used in degradation of lignocellulosic biomass, improvement of animal feed digestibility, biogas and bioethanol production, and various other applications. However, there are only limited reports that describe proteolytic enzymes and esterases in these fungi and their synergistic action with lignocellulolytic enzymes on degradation of complex polymers. Thus, in this minireview, we focus on the importance of these organisms in enzyme technology, their bioprospecting, possibility of integration of their enzyme repertoire, and their prospects for future biotechnological innovation.

     

全基因组水平扫描鉴定粗糙脉孢菌糖转运蛋白及其在酿酒酵母己糖发酵中的评价

木质纤维素降解真菌粗糙脉孢菌天然具有吸收利用多种单糖和寡糖的能力,但是目前基因组中注释的预测糖转运蛋白仍然有过半功能未知。本研究从全基因组水平系统分析了粗糙脉孢菌预测糖转运蛋白的转运底物。研究发现两个转运蛋白(NCU01868和NCU08152)具有转运多种己糖底物的功能,因此分别命名为NcHXT-1和NcHXT-2。利用荧光共振能量转移技术(FRET)确认了NcHXT-1/-2具有葡萄糖转运功能。在己糖转运蛋白全缺酿酒酵母EBY.VW4000中分别过表达NcHXT-1/-2,能恢复其在葡萄糖、半乳糖或甘露糖的液体培养基中生长并生成乙醇的能力。NcHXT-1/-2在很多纤维素降解真菌中均具有保守的同源蛋白。本研究通过全基因组扫描鉴定,发现了两个保守的丝状真菌己糖转运蛋白,为真菌降解利用木质纤维素及酵母利用单糖发酵提供了新的改造靶点。     

High-throughput enzymatic hydrolysis of lignocellulosic biomass via in-situ regeneration

The high cost of lignocellulolytic enzymes is one of the main barriers towards the development of economically competitive biorefineries. Enzyme engineering can be used to significantly increase the production rate as well as specific activity of enzymes. However, the success of enzyme optimization efforts is currently limited by a lack of robust high-throughput (HTP) cellulase screening platforms for insoluble pretreated lignocellulosic substrates. We have developed a cost-effective microplate based HTP enzyme-screening platform for ionic liquid (IL) pretreated lignocellulose. By performing in-situ biomass regeneration in micro-volumes, we can volumetrically meter biomass (sub-mg loading) and also precisely control the amount of residual IL for engineering novel IL-tolerant cellulases. Our platform only requires straightforward liquid-handling steps and allows the integration of biomass regeneration, washing, saccharification, and imaging steps in a single microtiter plate. The proposed method can be used to screen individual cellulases as well as to develop novel cellulase cocktails.     

Lignocellulose-degrading marine fungi

Evidence for lignocellulose-degrading ability among marine fungi is reviewed. Enzyme production, mass loss and micromorphological data suggest that most strains capable of decay activity are likely to be soft-rot fungi, with relatively few capable of white-rot decay. This probably reflects the relatively high number of ascomycete genera compared to basidiomycetes described to date. The ecological and biotechnological importance of marine fungal lignocellulolytic enzymes is discussed.     

Lignocellulolytic enzyme production from submerged fermentation of paddy straw

Five strains of cellulolytic bacteria and four strains of Phanerochaete chrysosporium were evaluated for the lignocellulolytic enzyme production during submerged fermentation (SmF) of paddy straw. Extra-cellular enzyme assay for CMCase, FPase, Cellobiase, Xylanase, Lignin peroxidase and Laccase enzymes was performed after 7 and 15 days of submerged fermentation. Cellulomonas cellulans MTCC 23, Cytophaga hutchinsonii NCIM 2338 and Phanerochaete chrysosporium MTCC 787 were found to produce higher lignocellulolytic enzyme activities than rest of the cultures after 15 days of fermentation.     

Expression of lignocellulolytic enzymes in Pichia pastoris

ABSTRACT: BACKGROUND: Sustainable utilization of plant biomass as renewable source for fuels and chemical building blocks requires a complex mixture of diverse enzymes, including hydrolases which comprise the largest class of lignocellulolytic enzymes. These enzymes need to be available in large amounts at a low price to allow sustainable and economic biotechnological processes. Over the past years Pichia pastoris has become an attractive host for the cost-efficient production and engineering of heterologous (eukaryotic) proteins due to several advantages. RESULTS: In this paper codon optimized genes and synthetic alcohol oxidase 1 promoter variants were used to generate Pichia pastoris strains which individually expressed cellobiohydrolase 1, cellobiohydrolase 2 and beta-mannanase from Trichoderma reesei and xylanase A from Thermomyces lanuginosus. For three of these enzymes even gram quantities of enzyme per liter were obtained by fed-batch cultivation. Additionally, we compared our achieved yields of secreted enzymes and the corresponding activities to literature data. CONCLUSION: In our experiments we could clearly see the importance of gene optimization and strain characterization for successfully improving secretion levels. We also give a basic guideline for understanding the interplay of promoter strength and gene dosage for a successful improvement of the secretory production of lignocellulolytic enzymes in Pichia pastoris.