scholarly journals Toward combined delignification and saccharification of wheat straw by a laccase-containing designer cellulosome

2016 ◽  
Vol 113 (39) ◽  
pp. 10854-10859 ◽  
Author(s):  
Lital Davidi ◽  
Sarah Moraïs ◽  
Lior Artzi ◽  
Doriv Knop ◽  
Yitzhak Hadar ◽  
...  
Keyword(s):  
Wheat Straw ◽  
Alternative Fuels ◽  
Plant Cell Wall ◽  
Plant Biomass ◽  
Soluble Sugars ◽  
Spatial Proximity ◽  
Aerobic Bacterium ◽  
Lignocellulose Conversion ◽  
Wide Range ◽  
Designer Cellulosome

Efficient breakdown of lignocellulose polymers into simple molecules is a key technological bottleneck limiting the production of plant-derived biofuels and chemicals. In nature, plant biomass degradation is achieved by the action of a wide range of microbial enzymes. In aerobic microorganisms, these enzymes are secreted as discrete elements in contrast to certain anaerobic bacteria, where they are assembled into large multienzyme complexes termed cellulosomes. These complexes allow for very efficient hydrolysis of cellulose and hemicellulose due to the spatial proximity of synergistically acting enzymes and to the limited diffusion of the enzymes and their products. Recently, designer cellulosomes have been developed to incorporate foreign enzymatic activities in cellulosomes so as to enhance lignocellulose hydrolysis further. In this study, we complemented a cellulosome active on cellulose and hemicellulose by addition of an enzyme active on lignin. To do so, we designed a dockerin-fused variant of a recently characterized laccase from the aerobic bacteriumThermobifida fusca. The resultant chimera exhibited activity levels similar to the wild-type enzyme and properly integrated into the designer cellulosome. The resulting complex yielded a twofold increase in the amount of reducing sugars released from wheat straw compared with the same system lacking the laccase. The unorthodox use of aerobic enzymes in designer cellulosome machinery effects simultaneous degradation of the three major components of the plant cell wall (cellulose, hemicellulose, and lignin), paving the way for more efficient lignocellulose conversion into soluble sugars en route to alternative fuels production.

scholarly journals Assembly of Xylanases into Designer Cellulosomes Promotes Efficient Hydrolysis of the Xylan Component of a Natural Recalcitrant Cellulosic Substrate

mBio ◽  
2011 ◽  
Vol 2 (6) ◽  
Author(s):  
Sarah Moraïs ◽  
Yoav Barak ◽  
Yitzhak Hadar ◽  
David B. Wilson ◽  
Yuval Shoham ◽  
...  
Keyword(s):  
Cell Wall ◽  
Wheat Straw ◽  
Enzymatic Degradation ◽  
Plant Cell Wall ◽  
Anaerobic Bacteria ◽  
Synergistic Action ◽  
Cellulosic Biomass ◽  
Thermobifida Fusca ◽  
Content Type ◽  
Designer Cellulosome

ABSTRACTIn nature, the complex composition and structure of the plant cell wall pose a barrier to enzymatic degradation. Nevertheless, some anaerobic bacteria have evolved for this purpose an intriguing, highly efficient multienzyme complex, the cellulosome, which contains numerous cellulases and hemicellulases. The rod-like cellulose component of the plant cell wall is embedded in a colloidal blend of hemicelluloses, a major component of which is xylan. In order to enhance enzymatic degradation of the xylan component of a natural complex substrate (wheat straw) and to study the synergistic action among different xylanases, we have employed a variation of the designer cellulosome approach by fabricating a tetravalent complex that includes the three endoxylanases ofThermobifida fusca(Xyn10A, Xyn10B, and Xyn11A) and an Xyl43A β-xylosidase from the same bacterium. Here, we describe the conversion of Xyn10A and Xyl43A to the cellulosomal mode. The incorporation of the Xyl43A enzyme together with the three endoxylanases into a common designer cellulosome served to enhance the level of reducing sugars produced during wheat straw degradation. The enhanced synergistic action of the four xylanases reflected their immediate juxtaposition in the complex, and these tetravalent xylanolytic designer cellulosomes succeeded in degrading significant (~25%) levels of the total xylan component of the wheat straw substrate. The results suggest that the incorporation of xylanases into cellulosome complexes is advantageous for efficient decomposition of recalcitrant cellulosic substrates—a distinction previously reserved for cellulose-degrading enzymes.IMPORTANCEXylanases are important enzymes for our society, due to their variety of industrial applications. Together with cellulases and other glycoside hydrolases, xylanases may also provide cost-effective conversion of plant-derived cellulosic biomass into soluble sugars en route to biofuels as an alternative to fossil fuels. Xylanases are commonly found in multienzyme cellulosome complexes, produced by anaerobic bacteria, which are considered to be among the most efficient systems for degradation of cellulosic biomass. Using a designer cellulosome approach, we have incorporated the entire xylanolytic system of the bacteriumThermobifida fuscainto defined artificial cellulosome complexes. The combined action of these designer cellulosomes versus that of the wild-type free xylanase system was then compared. Our data demonstrated that xylanolytic designer cellulosomes displayed enhanced synergistic activities on a natural recalcitrant wheat straw substrate and could thus serve in the development of advanced systems for improved degradation of lignocellulosic material.

scholarly journals Adaptor Scaffoldins: An Original Strategy for Extended Designer Cellulosomes, Inspired from Nature

mBio ◽  
2016 ◽  
Vol 7 (2) ◽  
Author(s):  
Johanna Stern ◽  
Sarah Moraïs ◽  
Raphael Lamed ◽  
Edward A. Bayer
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Soluble Sugars ◽  
Single Type ◽  
Wall Material ◽  
Type I ◽  
Type Ii ◽  
Recombinant Enzymes ◽  
Designer Cellulosome

ABSTRACTDesigner cellulosomes consist of chimeric cohesin-bearing scaffoldins for the controlled incorporation of recombinant dockerin-containing enzymes. The largest designer cellulosome reported to date is a chimeric scaffoldin that contains 6 cohesins. This scaffoldin represented a technical limit of sorts, since adding another cohesin proved problematic, owing to resultant low expression levels, instability (cleavage) of the scaffoldin polypeptide, and limited numbers of available cohesin-dockerin specificities—the hallmark of designer cellulosomes. Nevertheless, increasing the number of enzymes integrated into designer cellulosomes is critical, in order to further enhance degradation of plant cell wall material. Adaptor scaffoldins comprise an intermediate type of scaffoldin that can both incorporate various enzymes and attach to an additional scaffoldin. Using this strategy, we constructed an efficient form of adaptor scaffoldin that possesses three type I cohesins for enzyme integration, a single type II dockerin for interaction with an additional scaffoldin, and a carbohydrate-binding module for targeting to the cellulosic substrate. In parallel, we designed a hexavalent scaffoldin capable of connecting to the adaptor scaffoldin by the incorporation of an appropriate type II cohesin. The resultant extended designer cellulosome comprised 8 recombinant enzymes—4 xylanases and 4 cellulases—thereby representing a potent enzymatic cocktail for solubilization of natural lignocellulosic substrates. The contribution of the adaptor scaffoldin clearly demonstrated that proximity between the two scaffoldins and their composite set of enzymes is crucial for optimized degradation. After 72 h of incubation, the performance of the extended designer cellulosome was determined to be approximately 70% compared to that of native cellulosomes.IMPORTANCEPlant cell wall residues represent a major source of renewable biomass for the production of biofuels such as ethanol via breakdown to soluble sugars. The natural microbial degradation process, however, is inefficient for achieving cost-effective processes in the conversion of plant-derived biomass to biofuels, either from dedicated crops or human-generated cellulosic wastes. The accumulation of the latter is considered a major environmental pollutant. The development of designer cellulosome nanodevices for enhanced plant cell wall degradation thus has major impacts in the fields of environmental pollution, bioenergy production, and biotechnology in general. The findings reported in this article comprise a true breakthrough in our capacity to produce extended designer cellulosomes via synthetic biology means, thus enabling the assembly of higher-order complexes that can supersede the number of enzymes included in a single multienzyme complex.

scholarly journals Deconstruction of Lignocellulose into Soluble Sugars by Native and Designer Cellulosomes

mBio ◽  
2012 ◽  
Vol 3 (6) ◽  
Author(s):  
Sarah Moraïs ◽  
Ely Morag ◽  
Yoav Barak ◽  
Dan Goldman ◽  
Yitzhak Hadar ◽  
...  
Keyword(s):  
Wheat Straw ◽  
Plant Cell Wall ◽  
Soluble Sugar ◽  
Cellulosic Biomass ◽  
Thermobifida Fusca ◽  
Cellulolytic Bacterium ◽  
Artificial Enzyme ◽  
Wild Type ◽  
Content Type ◽  
Designer Cellulosome

ABSTRACTLignocellulosic biomass, the most abundant polymer on Earth, is typically composed of three major constituents: cellulose, hemicellulose, and lignin. The crystallinity of cellulose, hydrophobicity of lignin, and encapsulation of cellulose by the lignin-hemicellulose matrix are three major factors that contribute to the observed recalcitrance of lignocellulose. By means of designer cellulosome technology, we can overcome the recalcitrant properties of lignocellulosic substrates and thus increase the level of native enzymatic degradation. In this context, we have integrated six dockerin-bearing cellulases and xylanases from the highly cellulolytic bacterium,Thermobifida fusca, into a chimeric scaffoldin engineered to bear a cellulose-binding module and the appropriate matching cohesin modules. The resultant hexavalent designer cellulosome represents the most elaborate artificial enzyme composite yet constructed, and the fully functional complex achieved enhanced levels (up to 1.6-fold) of degradation of untreated wheat straw compared to those of the wild-type free enzymes. The action of these designer cellulosomes on wheat straw was 33 to 42% as efficient as the natural cellulosomes ofClostridium thermocellum. In contrast, the reduction of substrate complexity by chemical or biological pretreatment of the substrate removed the advantage of the designer cellulosomes, as the free enzymes displayed higher levels of activity, indicating that enzyme proximity between these selected enzymes was less significant on pretreated substrates. Pretreatment of the substrate caused an increase in activity for all the systems, and the native cellulosome completely converted the substrate into soluble saccharides.IMPORTANCECellulosic biomass is a potential alternative resource which could satisfy future demands of transportation fuel. However, overcoming the natural lignocellulose recalcitrance remains challenging. Current research and development efforts have concentrated on the efficient cellulose-degrading strategies of cellulosome-producing anaerobic bacteria. Cellulosomes are multienzyme complexes capable of converting the plant cell wall polysaccharides into soluble sugar products en route to biofuels as an alternative to fossil fuels. Using a designer cellulosome approach, we have constructed the largest form of homogeneous artificial cellulosomes reported to date, which bear a total of six different cellulases and xylanases from the highly cellulolytic bacteriumThermobifida fusca. These designer cellulosomes were comparable in size to natural cellulosomes and displayed enhanced synergistic activities compared to their free wild-type enzyme counterparts. Future efforts should be invested to improve these processes to approach or surpass the efficiency of natural cellulosomes for cost-effective production of biofuels.

scholarly journals Bioavailability of Carbohydrate Content in Natural and Transgenic Switchgrasses for the Extreme Thermophile Caldicellulosiruptor bescii

2017 ◽  
Vol 83 (17) ◽  
Author(s):  
Jeffrey V. Zurawski ◽  
Piyum A. Khatibi ◽  
Hannah O. Akinosho ◽  
Christopher T. Straub ◽  
Scott H. Compton ◽  
...  
Keyword(s):  
Cell Wall ◽  
Hydrothermal Treatment ◽  
Plant Cell Wall ◽  
Plant Biomass ◽  
Carbohydrate Content ◽  
Content Type ◽  
Caldicellulosiruptor Bescii ◽  
Physical Chemical ◽  
Lignocellulose Conversion ◽  
Natural Variant

ABSTRACT Improving access to the carbohydrate content of lignocellulose is key to reducing recalcitrance for microbial deconstruction and conversion to fuels and chemicals. Caldicellulosiruptor bescii completely solubilizes naked microcrystalline cellulose, yet this transformation is impeded within the context of the plant cell wall by a network of lignin and hemicellulose. Here, the bioavailability of carbohydrates to C. bescii at 70°C was examined for reduced lignin transgenic switchgrass lines COMT3(+) and MYB Trans, their corresponding parental lines (cultivar Alamo) COMT3(−) and MYB wild type (WT), and the natural variant cultivar Cave-in-Rock (CR). Transgenic modification improved carbohydrate solubilization by C. bescii to 15% (2.3-fold) for MYB and to 36% (1.5-fold) for COMT, comparable to the levels achieved for the natural variant, CR (36%). Carbohydrate solubilization was nearly doubled after two consecutive microbial fermentations compared to one microbial step, but it never exceeded 50% overall. Hydrothermal treatment (180°C) prior to microbial steps improved solubilization 3.7-fold for the most recalcitrant line (MYB WT) and increased carbohydrate recovery to nearly 50% for the least recalcitrant lines [COMT3(+) and CR]. Alternating microbial and hydrothermal steps (T→M→T→M) further increased bioavailability, achieving carbohydrate solubilization ranging from 50% for MYB WT to above 70% for COMT3(+) and CR. Incomplete carbohydrate solubilization suggests that cellulose in the highly lignified residue was inaccessible; indeed, residue from the T→M→T→M treatment was primarily glucan and inert materials (lignin and ash). While C. bescii could significantly solubilize the transgenic switchgrass lines and natural variant tested here, additional or alternative strategies (physical, chemical, enzymatic, and/or genetic) are needed to eliminate recalcitrance. IMPORTANCE Key to a microbial process for solubilization of plant biomass is the organism's access to the carbohydrate content of lignocellulose. Economically viable routes will characteristically minimize physical, chemical, and biological pretreatment such that microbial steps contribute to the greatest extent possible. Recently, transgenic versions of plants and trees have been developed with the intention of lowering the barrier to lignocellulose conversion, with particular focus on lignin content and composition. Here, the extremely thermophilic bacterium Caldicellulosiruptor bescii was used to solubilize natural and genetically modified switchgrass lines, with and without the aid of hydrothermal treatment. For lignocellulose conversion, it is clear that the microorganism, plant biomass substrate, and processing steps must all be considered simultaneously to achieve optimal results. Whether switchgrass lines engineered for low lignin or natural variants with desirable properties are used, conversion will depend on microbial access to crystalline cellulose in the plant cell wall.

scholarly journals Contribution of a Xylan-Binding Module to the Degradation of a Complex Cellulosic Substrate by Designer Cellulosomes

2010 ◽  
Vol 76 (12) ◽  
pp. 3787-3796 ◽  
Author(s):  
Sarah Moraïs ◽  
Yoav Barak ◽  
Jonathan Caspi ◽  
Yitzhak Hadar ◽  
Raphael Lamed ◽  
...  
Keyword(s):  
Wheat Straw ◽  
Plant Cell Wall ◽  
Proximity Effects ◽  
Synergistic Activity ◽  
Wild Type ◽  
Cellulosic Substrate ◽  
Specific Degradation ◽  
Designer Cellulosome ◽  
Insight Into ◽  
Enhance Activity

ABSTRACT Conversion of components of the Thermobifida fusca free-enzyme system to the cellulosomal mode using the designer cellulosome approach can be employed to discover the properties and inherent advantages of the cellulosome system. In this article, we describe the conversion of the T. fusca xylanases Xyn11A and Xyn10B and their synergistic interaction in the free state or within designer cellulosome complexes in order to enhance specific degradation of hatched wheat straw as a model for a complex cellulosic substrate. Endoglucanase Cel5A from the same bacterium and its recombinant dockerin-containing chimera were also studied for their combined effect, together with the xylanases, on straw degradation. Synergism was demonstrated when Xyn11A was combined with Xyn10B and/or Cel5A, and ∼1.5-fold activity enhancements were achieved by the designer cellulosome complexes compared to the free wild-type enzymes. These improvements in activity were due to both substrate-targeting and proximity effects among the enzymes contained in the designer cellulosome complexes. The intrinsic cellulose/xylan-binding module (XBM) of Xyn11A appeared to be essential for efficient substrate degradation. Indeed, only designer cellulosomes in which the XBM was maintained as a component of Xyn11A achieved marked enhancement in activity compared to the combination of wild-type enzymes. Moreover, integration of the XBM in designer cellulosomes via a dockerin module (separate from the Xyn11A catalytic module) failed to enhance activity, suggesting a role in orienting the parent xylanase toward its preferred polysaccharide component of the complex wheat straw substrate. The results provide novel mechanistic insight into the synergistic activity of designer cellulosome components on natural plant cell wall substrates.

scholarly journals Preliminary crystallographic analysis of a double mutant of the acetyl xylo-oligosaccharide esterase Axe2 in its dimeric form

2014 ◽  
Vol 70 (4) ◽  
pp. 476-481 ◽  
Author(s):  
Shifra Lansky ◽  
Onit Alalouf ◽  
Rachel Salama ◽  
Hay Dvir ◽  
Yuval Shoham ◽  
...  
Keyword(s):  
Plant Cell Wall ◽  
Plant Biomass ◽  
Thermophilic Bacterium ◽  
Crystallographic Analysis ◽  
Dimensional Structure ◽  
Orthorhombic Space Group ◽  
Data Set ◽  
Cell Parameters ◽  
Wide Range ◽  
Dimeric Form

Xylans are polymeric sugars constituting a significant part of the plant cell wall. They are usually substituted with acetyl side groups attached at positions 2 or 3 of the xylose backbone units. Acetylxylan esterases are part of the hemicellulolytic system of many microorganisms which utilize plant biomass for growth. These enzymes hydrolyze the ester linkages of the xylan acetyl groups and thus improve the accessibility of main-chain-hydrolyzing enzymes and their ability to break down the sugar backbone units. The acetylxylan esterases are therefore critically important for those microorganisms and as such could be used for a wide range of biotechnological applications. The structure of an acetylxylan esterase (Axe2) isolated from the thermophilic bacteriumGeobacillus stearothermophilusT6 has been determined, and it has been demonstrated that the wild-type enzyme is present as a unique torus-shaped octamer in the crystal and in solution. In order to understand the functional origin of this unique oligomeric structure, a series of rational noncatalytic, site-specific mutations have been made on Axe2. Some of these mutations led to a different dimeric form of the protein, which showed a significant reduction in catalytic activity. One of these double mutants, Axe2-Y184F-W190P, has recently been overexpressed, purified and crystallized. The best crystals obtained belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 71.1,b= 106.0,c= 378.6 Å. A full diffraction data set to 2.3 Å resolution has been collected from a flash-cooled crystal of this type at 100 K using synchrotron radiation. This data set is currently being used for the three-dimensional structure analysis of the Axe2-Y184F-W190P mutant in its dimeric form.

scholarly journals Proteome specialization of anaerobic fungi during ruminal degradation of recalcitrant plant fiber

2020 ◽  
Author(s):  
Live H. Hagen ◽  
Charles G. Brooke ◽  
Claire A. Shaw ◽  
Angela D. Norbeck ◽  
Hailan Piao ◽  
...  
Keyword(s):  
Plant Cell Wall ◽  
Plant Biomass ◽  
Soluble Sugars ◽  
Rumen Bacteria ◽  
Anaerobic Fungi ◽  
Host Animal ◽  
Bacterial Populations ◽  
Domain Specific ◽  
Gene Catalog ◽  
Ruminal Degradation

Abstract The rumen harbors a complex microbial mixture of archaea, bacteria, protozoa, and fungi that efficiently breakdown plant biomass and its complex dietary carbohydrates into soluble sugars that can be fermented and subsequently converted into metabolites and nutrients utilized by the host animal. While rumen bacterial populations have been well documented, only a fraction of the rumen eukarya are taxonomically and functionally characterized, despite the recognition that they contribute to the cellulolytic phenotype of the rumen microbiota. To investigate how anaerobic fungi actively engage in digestion of recalcitrant fiber that is resistant to degradation, we resolved genome-centric metaproteome and metatranscriptome datasets generated from switchgrass samples incubated for 48 h in nylon bags within the rumen of cannulated dairy cows. Across a gene catalog covering anaerobic rumen bacteria, fungi and viruses, a significant portion of the detected proteins originated from fungal populations. Intriguingly, the carbohydrate-active enzyme (CAZyme) profile suggested a domain-specific functional specialization, with bacterial populations primarily engaged in the degradation of hemicelluloses, whereas fungi were inferred to target recalcitrant cellulose structures via the detection of a number of endo- and exo-acting enzymes belonging to the glycoside hydrolase (GH) family 5, 6, 8, and 48. Notably, members of the GH48 family were amongst the highest abundant CAZymes and detected representatives from this family also included dockerin domains that are associated with fungal cellulosomes. A eukaryote-selected metatranscriptome further reinforced the contribution of uncultured fungi in the ruminal degradation of recalcitrant fibers. These findings elucidate the intricate networks of in situ recalcitrant fiber deconstruction, and importantly, suggest that the anaerobic rumen fungi contribute a specific set of CAZymes that complement the enzyme repertoire provided by the specialized plant cell wall degrading rumen bacteria.

scholarly journals The transcription factor Roc1 is a regulator of cellulose degradation in the wood-decaying mushroom Schizophyllum commune

2021 ◽  
Author(s):  
Ioana M Marian ◽  
Peter Jan Vonk ◽  
Ivan D Valdes ◽  
Kerrie Barry ◽  
Benedict Bostock ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Plant Cell Wall ◽  
Plant Biomass ◽  
Cellulose Degradation ◽  
Schizophyllum Commune ◽  
Building Blocks ◽  
Lignocellulose Degradation ◽  
Transcriptional Level ◽  
Wide Range

Wood-decaying fungi of the class Agaricomycetes (phylum Basidiomycota) are saprotrophs that break down lignocellulose and play an important role in the nutrient recycling. They secrete a wide range of extracellular plant cell wall degrading enzymes that break down cellulose, hemicellulose and lignin, the main building blocks of plant biomass. Although the production of these enzymes is regulated mainly at the transcriptional level, no activating regulators have been identified in any wood-decaying fungus in the class Agaricomycetes. We studied the regulation of cellulase expression in the wood-decaying fungus Schizophyllum commune. Comparative genomics and transcriptomics on two wild isolates revealed a Zn2Cys6-type transcription factor gene (roc1) that was highly up-regulated during growth on cellulose, when compared to glucose. It is only conserved in the class Agaricomycetes. A roc1 knockout strain showed an inability to grow on medium with cellulose as sole carbon source, and growth on cellobiose and xylan (other components of wood) was inhibited. Growth on non-wood-related carbon sources was not inhibited. Cellulase activity was reduced in the growth medium of the Δroc1 strain. ChIP-Seq identified 1474 binding sites of the Roc1 transcription factor. Promoters of genes involved in lignocellulose degradation were enriched with these binding sites, especially those of LPMO (lytic polysaccharide monooxygenase) CAZymes, indicating that Roc1 directly regulates these genes. A GC-rich motif was identified as the binding site of Roc1, which was confirmed by a functional promoter analysis. Together, Roc1 is a key regulator of cellulose degradation and the first identified in wood-decaying fungi in the phylum Basidiomycota.

scholarly journals Colocalization and Disposition of Cellulosomes in Clostridium clariflavum as Revealed by Correlative Superresolution Imaging

mBio ◽  
2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Lior Artzi ◽  
Tali Dadosh ◽  
Elad Milrot ◽  
Sarah Moraïs ◽  
Smadar Levin-Zaidman ◽  
...  
Keyword(s):  
Cell Surface ◽  
Bacterial Cell ◽  
Plant Cell Wall ◽  
Plant Biomass ◽  
Soluble Sugars ◽  
Attractive Alternative ◽  
Cellulolytic Bacteria ◽  
Extracellular Medium ◽  
Bacterial Cell Surface ◽  
Clostridium Clariflavum

ABSTRACTCellulosomes are multienzyme complexes produced by anaerobic, cellulolytic bacteria for highly efficient breakdown of plant cell wall polysaccharides.Clostridium clariflavumis an anaerobic, thermophilic bacterium that produces the largest assembled cellulosome complex in nature to date, comprising three types of scaffoldins: a primary scaffoldin, ScaA; an adaptor scaffoldin, ScaB; and a cell surface anchoring scaffoldin, ScaC. This complex can contain 160 polysaccharide-degrading enzymes. In previous studies, we proposed potential types of cellulosome assemblies inC. clariflavumand demonstrated that these complexes are released into the extracellular medium. In the present study, we explored the disposition of the highly structured, four-tiered cell-anchored cellulosome complex of this bacterium. Four separate, integral cellulosome components were subjected to immunolabeling: ScaA, ScaB, ScaC, and the cellulosome’s most prominent enzyme, GH48. Imaging of the cells by correlating scanning electron microscopy and three-dimensional (3D) superresolution fluorescence microscopy revealed that some of the protuberance-like structures on the cell surface represent cellulosomes and that the components are highly colocalized and organized by a defined hierarchy on the cell surface. The display of the cellulosome on the cell surface was found to differ between cells grown on soluble or insoluble substrates. Cell growth on microcrystalline cellulose and wheat straw exhibited dramatic enhancement in the amount of cellulosomes displayed on the bacterial cell surface.IMPORTANCEConversion of plant biomass into soluble sugars is of high interest for production of fermentable industrial materials, such as biofuels. Biofuels are a very attractive alternative to fossil fuels, both for recycling of agricultural wastes and as a source of sustainable energy. Cellulosomes are among the most efficient enzymatic degraders of biomass known to date, due to the incorporation of a multiplicity of enzymes into a potent, multifunctional nanomachine. The intimate association with the bacterial cell surface is inherent in its efficient action on lignocellulosic substrates, although this property has not been properly addressed experimentally. The dramatic increase in cellulosome performance on recalcitrant feedstocks is critical for the design of cost-effective processes for efficient biomass degradation.

scholarly journals Proteome specialization of anaerobic fungi during ruminal degradation of recalcitrant plant fiber

2020 ◽  
Author(s):  
Live H. Hagen ◽  
Charles G. Brooke ◽  
Claire Shaw ◽  
Angela D. Norbeck ◽  
Hailan Piao ◽  
...  
Keyword(s):  
Plant Cell Wall ◽  
Plant Biomass ◽  
Soluble Sugars ◽  
Rumen Bacteria ◽  
Anaerobic Fungi ◽  
Host Animal ◽  
Bacterial Populations ◽  
Domain Specific ◽  
Gene Catalogue ◽  
Ruminal Degradation

AbstractThe rumen harbors a complex microbial mixture of archaea, bacteria, protozoa and fungi that efficiently breakdown plant biomass and its complex dietary carbohydrates into soluble sugars that can be fermented and subsequently converted into metabolites and nutrients utilized by the host animal. While rumen bacterial populations have been well documented, only a fraction of the rumen eukarya are taxonomically and functionally characterized, despite the recognition that they contribute to the cellulolytic phenotype of the rumen microbiota. To investigate how anaerobic fungi actively engage in digestion of recalcitrant fiber that is resistant to degradation, we resolved genome-centric metaproteome and metatranscriptome datasets generated from switchgrass samples incubated for 48 hours in nylon bags within the rumen of cannulated dairy cows. Across a gene catalogue covering anaerobic rumen bacteria, fungi and viruses, a significant portion of the detected proteins originated from fungal populations. Intriguingly, the carbohydrate-active enzyme (CAZyme) profile suggested a domain-specific functional specialization, with bacterial populations primarily engaged in the degradation of polysaccharides such as hemicellulose, whereas fungi were inferred to target recalcitrant cellulose structures via the detection of a number of endo- and exo-acting enzymes belonging to the glycoside hydrolase (GH) family 5, 6, 8 and 48. Notably, members of the GH48 family were amongst the highest abundant CAZymes and detected representatives from this family also included dockerin domains that are associated with fungal cellulosomes. A eukaryote-selected metatranscriptome further reinforced the contribution of uncultured fungi in the ruminal degradation of recalcitrant fibers. These findings elucidate the intricate networks of in situ recalcitrant fiber deconstruction, and importantly, suggests that the anaerobic rumen fungi contribute a specific set of CAZymes that complement the enzyme repertoire provided by the specialized plant cell wall degrading rumen bacteria.

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