scholarly journals Effect of Linker Length and Dockerin Position on Conversion of a Thermobifida fusca Endoglucanase to the Cellulosomal Mode

2009 ◽  
Vol 75 (23) ◽  
pp. 7335-7342 ◽  
Author(s):  
Jonathan Caspi ◽  
Yoav Barak ◽  
Rachel Haimovitz ◽  
Diana Irwin ◽  
Raphael Lamed ◽  
...  
Keyword(s):  
Cell Wall ◽  
Proximity Effect ◽  
Plant Cell Wall ◽  
Thermobifida Fusca ◽  
Scaffolding Proteins ◽  
Wild Type ◽  
Catalytic Module ◽  
Synergistic Response ◽  
Cellulose Binding ◽  
Designer Cellulosome

ABSTRACT We have been developing the cellulases of Thermobifida fusca as a model to explore the conversion from a free cellulase system to the cellulosomal mode. Three of the six T. fusca cellulases (endoglucanase Cel6A and exoglucanases Cel6B and Cel48A) have been converted in previous work by replacing their cellulose-binding modules (CBMs) with a dockerin, and the resultant recombinant “cellulosomized” enzymes were incorporated into chimeric scaffolding proteins that contained cohesin(s) together with a CBM. The activities of the resultant designer cellulosomes were compared with an equivalent mixture of wild-type enzymes. In the present work, a fourth T. fusca cellulase, Cel5A, was equipped with a dockerin and intervening linker segments of different lengths to assess their contribution to the overall activity of simple one- and two-enzyme designer cellulosome complexes. The results demonstrated that cellulose binding played a major role in the degradation of crystalline cellulosic substrates. The combination of the converted Cel5A endoglucanase with the converted Cel48A exoglucanase also exhibited a measurable proximity effect for the most recalcitrant cellulosic substrate (Avicel). The length of the linker between the catalytic module and the dockerin had little, if any, effect on the activity. However, positioning of the dockerin on the opposite (C-terminal) side of the enzyme, consistent with the usual position of dockerins on most cellulosomal enzymes, resulted in an enhanced synergistic response. These results promote the development of more complex multienzyme designer cellulosomes, which may eventually be applied for improved degradation of plant cell wall biomass.

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 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 Synergistic Effect of Different Plant Cell Wall–Degrading Enzymes Is Important for Virulence of Fusarium graminearum

2017 ◽  
Vol 30 (11) ◽  
pp. 886-895 ◽  
Author(s):  
Maria Chiara Paccanaro ◽  
Luca Sella ◽  
Carla Castiglioni ◽  
Francesca Giacomello ◽  
Ana Lilia Martínez-Rocha ◽  
...  
Keyword(s):  
Cell Wall ◽  
Fusarium Graminearum ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Xylanase Activity ◽  
Cellulase Activity ◽  
Wild Type ◽  
Cell Wall Degrading Enzymes ◽  
Xylanase Production ◽  
Significant Difference

Endo-polygalacturonases (PGs) and xylanases have been shown to play an important role during pathogenesis of some fungal pathogens of dicot plants, while their role in monocot pathogens is less defined. Pg1 and xyr1 genes of the wheat pathogen Fusarium graminearum encode the main PG and the major regulator of xylanase production, respectively. Single- and double-disrupted mutants for these genes were obtained to assess their contribution to fungal infection. Compared with wild-type strain, the ∆pg mutant showed a nearly abolished PG activity, slight reduced virulence on soybean seedlings, but no significant difference in disease symptoms on wheat spikes; the ∆xyr mutant was strongly reduced in xylanase activity and moderately reduced in cellulase activity but was as virulent as wild type on both soybean and wheat plants. Consequently, the ΔpgΔxyr double mutant was impaired in xylanase, PG, and cellulase activities but, differently from single mutants, was significantly reduced in virulence on both plants. These findings demonstrate that the concurrent presence of PG, xylanase, and cellulase activities is necessary for full virulence. The observation that the uronides released from wheat cell wall after a F. graminearum PG treatment were largely increased by the fungal xylanases suggests that these enzymes act synergistically in deconstructing the plant cell wall.

scholarly journals Extracellular Glycanases of Rhizobium leguminosarum Are Activated on the Cell Surface by an Exopolysaccharide-Related Component

2000 ◽  
Vol 182 (5) ◽  
pp. 1304-1312 ◽  
Author(s):  
Angeles Zorreguieta ◽  
Christine Finnie ◽  
J. Allan Downie
Keyword(s):  
Cell Wall ◽  
Agrobacterium Tumefaciens ◽  
Cell Surface ◽  
Rhizobium Leguminosarum ◽  
Plant Cell Wall ◽  
Agar Medium ◽  
Wild Type ◽  
Close Proximity ◽  
Mutant Strains ◽  
Colony Surface

ABSTRACT Rhizobium leguminosarum secretes two extracellular glycanases, PlyA and PlyB, that can degrade exopolysaccharide (EPS) and carboxymethyl cellulose (CMC), which is used as a model substrate of plant cell wall cellulose polymers. When grown on agar medium, CMC degradation occurred only directly below colonies of R. leguminosarum, suggesting that the enzymes remain attached to the bacteria. Unexpectedly, when a PlyA-PlyB-secreting colony was grown in close proximity to mutants unable to produce or secrete PlyA and PlyB, CMC degradation occurred below that part of the mutant colonies closest to the wild type. There was no CMC degradation in the region between the colonies. By growing PlyB-secreting colonies on a lawn of CMC-nondegrading mutants, we could observe a halo of CMC degradation around the colony. Using various mutant strains, we demonstrate that PlyB diffuses beyond the edge of the colony but does not degrade CMC unless it is in contact with the appropriate colony surface. PlyA appears to remain attached to the cells since no such diffusion of PlyA activity was observed. EPS defective mutants could secrete both PlyA and PlyB, but these enzymes were inactive unless they came into contact with an EPS+ strain, indicating that EPS is required for activation of PlyA and PlyB. However, we were unable to activate CMC degradation with a crude EPS fraction, indicating that activation of CMC degradation may require an intermediate in EPS biosynthesis. Transfer of PlyB to Agrobacterium tumefaciens enabled it to degrade CMC, but this was only observed if it was grown on a lawn ofR. leguminosarum. This indicates that the surface ofA. tumefaciens is inappropriate to activate CMC degradation by PlyB. Analysis of CMC degradation by other rhizobia suggests that activation of secreted glycanases by surface components may occur in other species.

scholarly journals The Xylanase Inhibitor TAXI-I Increases Plant Resistance to Botrytis cinerea by Inhibiting the BcXyn11a Xylanase Necrotizing Activity

Plants ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 601
Author(s):  
Silvio Tundo ◽  
Maria Chiara Paccanaro ◽  
Ibrahim Elmaghraby ◽  
Ilaria Moscetti ◽  
Renato D’Ovidio ◽  
...  
Keyword(s):  
Cell Wall ◽  
Botrytis Cinerea ◽  
Plant Cell Wall ◽  
Wild Type ◽  
Cell Wall Degrading Enzymes ◽  
Targeted Deletion ◽  
Plant Infection ◽  
Transgenic Lines ◽  
Xylanase Inhibitor ◽  
Main Component

During host plant infection, pathogens produce a wide array of cell wall degrading enzymes (CWDEs) to break the plant cell wall. Among CWDEs, xylanases are key enzymes in the degradation of xylan, the main component of hemicellulose. Targeted deletion experiments support the direct involvement of the xylanase BcXyn11a in the pathogenesis of Botrytis cinerea. Since the Triticum aestivum xylanase inhibitor-I (TAXI-I) has been shown to inhibit BcXyn11a, we verified if TAXI-I could be exploited to counteract B. cinerea infections. With this aim, we first produced Nicotiana tabacum plants transiently expressing TAXI-I, observing increased resistance to B. cinerea. Subsequently, we transformed Arabidopsis thaliana to express TAXI-I constitutively, and we obtained three transgenic lines exhibiting a variable amount of TAXI-I. The line with the higher level of TAXI-I showed increased resistance to B. cinerea and the absence of necrotic lesions when infiltrated with BcXyn11a. Finally, in a droplet application experiment on wild-type Arabidopsis leaves, TAXI-I prevented the necrotizing activity of BcXyn11a. These results would confirm that the contribution of BcXyn11a to virulence is due to its necrotizing rather than enzymatic activity. In conclusion, our experiments highlight the ability of the TAXI-I xylanase inhibitor to counteract B. cinerea infection presumably by preventing the necrotizing activity of BcXyn11a.

scholarly journals Pyramiding Unmarked Deletions in Ralstonia solanacearum Shows That Secreted Proteins in Addition to Plant Cell-Wall-Degrading Enzymes Contribute to Virulence

2005 ◽  
Vol 18 (12) ◽  
pp. 1296-1305 ◽  
Author(s):  
Huanli Liu ◽  
Shuping Zhang ◽  
Mark A. Schell ◽  
Timothy P. Denny
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Ralstonia Solanacearum ◽  
Plant Cell Wall ◽  
Secreted Proteins ◽  
Cellulolytic Enzymes ◽  
Wild Type ◽  
Cell Wall Degrading Enzymes ◽  
Tomato Plants ◽  
Degrading Enzymes

Ralstonia solanacearum, like many phytopathogenic bacteria, makes multiple extracellular plant cell-wall-degrading enzymes (CWDE), some of which contribute to its ability to cause wilt disease. CWDE and many other proteins are secreted to the milieu via the highly conserved type II protein secretion system (T2SS). R. solanacearum with a defective T2SS is weakly virulent, but it is not known whether this is due to absence of all the CWDE or the loss of other secreted proteins that contribute to disease. These alternatives were investigated by creating mutants of wild-type strain GMI1000 lacking either the T2SS or up to six CWDE and comparing them for virulence on tomato plants. To create unmarked deletions, genomic regions flanking the target gene were polymerase chain reaction (PCR)-amplified, were fused using splice overlap extension PCR, were cloned into a suicide plasmid harboring the sacB counter-selectable marker, and then, were site-specifically introduced into the genome. Various combinations of five deletions (δpehA, δpehB, δpehC, δpme, and δegl) and one inactivated allele (cbhA::aphA-3) resulted in 15 mutants missing one to six CWDE. In soil-drench inoculation assays, virulence of mutants lacking only pectic enzymes (PehA, PehB, PehC, and Pme) was not statistically different from GMI1000, but all the mutants lacking one or both cellulolytic enzymes (Egl or CbhA) wilted plants significantly more slowly than did the wild type. The GMI-6 mutant that lacks all six CWDE was more virulent than the mutant lacking only its two cellulolytic enzymes, and both were significantly more virulent than the T2SS mutant (GMI-D). Very similar results were observed in wounded-petiole inoculation assays, so GMI-6 and GMI-D appear to be less capable of colonizing tomato tissues after invasion. Because the T2SS mutant was much less virulent than the sixfold CWDE mutant, we conclude that other secreted proteins contribute substantially to the ability of R. solanacearum GMI1000 to systemically colonize tomato plants.

scholarly journals Extensin arabinosylation is involved in root response to elicitors and limits oomycete colonization

2019 ◽  
Vol 125 (5) ◽  
pp. 751-763 ◽  
Author(s):  
Romain Castilleux ◽  
Barbara Plancot ◽  
Bruno Gügi ◽  
Agnès Attard ◽  
Corinne Loutelier-Bourhis ◽  
...  
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Root Colonization ◽  
Wild Type ◽  
Differential Distribution ◽  
Root Cells ◽  
Structural Conformation ◽  
Microbe Interactions ◽  
Cross Links

Abstract Background and Aims Extensins are hydroxyproline-rich glycoproteins thought to strengthen the plant cell wall, one of the first barriers against pathogens, through intra- and intermolecular cross-links. The glycan moiety of extensins is believed to confer the correct structural conformation to the glycoprotein, leading to self-assembly within the cell wall that helps limit microbial adherence and invasion. However, this role is not clearly established. Methods We used Arabidopsis thaliana mutants impaired in extensin arabinosylation to investigate the role of extensin arabinosylation in root–microbe interactions. Mutant and wild-type roots were stimulated to elicit an immune response with flagellin 22 and immunolabelled with a set of anti-extensin antibodies. Roots were also inoculated with a soilborne oomycete, Phytophthora parasitica, to assess the effect of extensin arabinosylation on root colonization. Key Results A differential distribution of extensin epitopes was observed in wild-type plants in response to elicitation. Elicitation also triggers altered epitope expression in mutant roots compared with wild-type and non-elicited roots. Inoculation with the pathogen P. parasitica resulted in enhanced root colonization for two mutants, specifically xeg113 and rra2. Conclusions We provide evidence for a link between extensin arabinosylation and root defence, and propose a model to explain the importance of glycosylation in limiting invasion of root cells by pathogenic oomycetes.

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 Proteomic and Transcriptomic Analysis of Extracellular Proteins and mRNA Levels in Thermobifida fusca Grown on Cellobiose and Glucose

2007 ◽  
Vol 189 (17) ◽  
pp. 6260-6265 ◽  
Author(s):  
Shaolin Chen ◽  
David B. Wilson
Keyword(s):  
Cell Wall ◽  
Plant Cell Wall ◽  
Extracellular Proteins ◽  
Transcriptional Analysis ◽  
Thermobifida Fusca ◽  
Transcriptional Level ◽  
Mrna Levels ◽  
Egg White Lysozyme ◽  
Extracellular Proteome ◽  
Genes Encoding

ABSTRACT Thermobifida fusca secretes proteins that carry out plant cell wall degradation. Using two-dimensional electrophoresis, the extracellular proteome of T. fusca grown on cellobiose was compared to that of cells grown on glucose. Extracellular proteins, the expression of which is induced by cellobiose, mainly are cellulases and cellulose-binding proteins. Other major extracellular proteins induced by cellobiose include a xylanase (Xyl10A) and two unknown proteins, the C-terminal regions of which are homologous to a lytic transglycosylase goose egg white lysozyme domain and an NLPC_P60 domain (which defines a family of cell wall peptidases), respectively. Transcriptional analysis of genes encoding cellobiose-induced proteins suggests that their expression is controlled at the transcriptional level and that their expression also is induced by cellulose. Some other major extracellular proteins produced by T. fusca grown on both cellobiose and glucose include Lam81A and three unknown proteins that are homologous to aminopeptidases and xylanases or that contain a putative NLPC_P60 domain.

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