The Experts below are selected from a list of 2874 Experts worldwide ranked by ideXlab platform

Edward A Bayer - One of the best experts on this subject based on the ideXlab platform.

  • Minimalistic Cellulosome of the Butanologenic Bacterium Clostridium saccharoperbutylacetonicum.
    Mbio, 2020
    Co-Authors: Bosmat Levi Hevroni, Sarah Morais, Ely Morag, Yonit Ben-david, Edward A Bayer
    Abstract:

    ABSTRACT Clostridium saccharoperbutylacetonicum is a mesophilic, anaerobic, butanol-producing bacterium, originally isolated from soil. It was recently reported that C. saccharoperbutylacetonicum possesses multiple cellulosomal elements and would potentially form the smallest cellulosome known in nature. Its genome contains only eight dockerin-bearing enzymes, and its unique scaffoldin bears two cohesins (Cohs), three X2 modules, and two carbohydrate-binding modules (CBMs). In this study, all of the cellulosome-related modules were cloned, expressed, and purified. The recombinant cohesins, dockerins, and CBMs were tested for binding activity using enzyme-linked immunosorbent assay (ELISA)-based techniques. All the enzymes were tested for their comparative enzymatic activity on seven different cellulosic and hemicellulosic substrates, thus revealing four cellulases, a xylanase, a mannanase, a xyloglucanase, and a lichenase. All dockerin-containing enzymes interacted similarly with the second cohesin (Coh2) module, whereas Coh1 was more restricted in its interaction pattern. In addition, the polysaccharide-binding properties of the CBMs within the scaffoldin were examined by two complementary assays, affinity electrophoresis and affinity pulldown. The scaffoldin of C. saccharoperbutylacetonicum exhibited high affinity for cellulosic and hemicellulosic substrates, specifically to microcrystalline cellulose and xyloglucan. Evidence that supports substrate-dependent in vivo secretion of Cellulosomes is presented. The results of our analyses contribute to a better understanding of simple cellulosome systems by identifying the key players in this minimalistic system and the binding pattern of its cohesin-dockerin interaction. The knowledge gained by our study will assist further exploration of similar minimalistic Cellulosomes and will contribute to the significance of specific sets of defined cellulosomal enzymes in the degradation of cellulosic biomass. IMPORTANCE Cellulosome-producing bacteria are considered among the most important bacteria in both mesophilic and thermophilic environments, owing to their capacity to deconstruct recalcitrant plant-derived polysaccharides (and notably cellulose) into soluble saccharides for subsequent processing. In many ecosystems, the cellulosome-producing bacteria are particularly effective “first responders.” The massive amounts of sugars produced are potentially amenable in industrial settings to further fermentation by appropriate microbes to biofuels, notably ethanol and butanol. Among the solvent-producing bacteria, Clostridium saccharoperbutylacetonicum has the smallest cellulosome system known thus far. The importance of investigating the building blocks of such a small, multifunctional nanomachine is crucial to understanding the fundamental activities of this efficient enzymatic complex.

  • Creation of a functional hyperthermostable designer cellulosome
    Biotechnology for Biofuels, 2019
    Co-Authors: Amaranta Kahn, Sarah Morais, Michael E Himmel, Yannick J. Bomble, Anastasia P. Galanopoulou, Daehwan Chung, Nicholas S. Sarai, Neal Hengge, Dimitris G. Hatzinikolaou, Edward A Bayer
    Abstract:

    Renewable energy has become a field of high interest over the past decade, and production of biofuels from cellulosic substrates has a particularly high potential as an alternative source of energy. Industrial deconstruction of biomass, however, is an onerous, exothermic process, the cost of which could be decreased significantly by use of hyperthermophilic enzymes. An efficient way of breaking down cellulosic substrates can also be achieved by highly efficient enzymatic complexes called Cellulosomes. The modular architecture of these multi-enzyme complexes results in substrate targeting and proximity-based synergy among the resident enzymes. However, Cellulosomes have not been observed in hyperthermophilic bacteria. Here, we report the design and function of a novel hyperthermostable “designer cellulosome” system, which is stable and active at 75 °C. Enzymes from Caldicellulosiruptor bescii, a highly cellulolytic hyperthermophilic anaerobic bacterium, were selected and successfully converted to the cellulosomal mode by grafting onto them divergent dockerin modules that can be inserted in a precise manner into a thermostable chimaeric scaffoldin by virtue of their matching cohesins. Three pairs of cohesins and dockerins, selected from thermophilic microbes, were examined for their stability at extreme temperatures and were determined stable at 75 °C for at least 72 h. The resultant hyperthermostable cellulosome complex exhibited the highest levels of enzymatic activity on microcrystalline cellulose at 75 °C, compared to those of previously reported designer cellulosome systems and the native cellulosome from Clostridium thermocellum. The functional hyperthermophilic platform fulfills the appropriate physico-chemical properties required for exothermic processes. This system can thus be adapted for other types of thermostable enzyme systems and could serve as a basis for a variety of cellulolytic and non-cellulolytic industrial objectives at high temperatures.

  • cell surface display of designer Cellulosomes by lactobacillus plantarum
    Methods in Enzymology, 2019
    Co-Authors: Yonit Bendavid, Sarah Morais, Johanna Stern, Itzhak Mizrahi, Edward A Bayer
    Abstract:

    Abstract Cell-surface display of designer Cellulosomes complexes has attracted increased interest in recent years. These engineered microorganisms can efficiently degrade lignocellulosic biomass that represents an abundant resource for conversion into fermentable sugars, suitable for production of biofuels. The designer cellulosome is an artificial enzymatic complex that mimics the architecture of the natural cellulosome and allows the control of the positions, type, and copy number of the cellulosomal enzymes within the complex. Lactobacillus plantarum is an attractive candidate for metabolic engineering of lignocellulosic biomass to biofuels, as its natural characteristics include high ethanol and acid tolerance and the ability to metabolize hexose sugars. In recent years, successful expression of a variety of designer Cellulosomes on the cell surface of this bacterium has been demonstrated using the cell-consortium approach. This strategy minimized genomic interference on each strain upon genetic engineering, thereby maximizing the ability of each strain to grow, express, and secrete each enzyme. In addition, this strategy allows stoichiometric control of the cellulosome elements and facile exchange of the secreted proteins. A detailed procedure for display of designer Cellulosomes on the cell surface of L. plantarum is described in this chapter.

  • The cohesin module is a major determinant of cellulosome mechanical stability
    Journal of Biological Chemistry, 2018
    Co-Authors: Albert Galera-prat, Sarah Morais, Edward A Bayer, Yael Vazana, Mariano Carrión-vázquez
    Abstract:

    : Cellulosomes are bacterial protein complexes that bind and efficiently degrade lignocellulosic substrates. These are formed by multimodular scaffolding proteins known as scaffoldins, which comprise cohesin modules capable of binding dockerin-bearing enzymes and usually a carbohydrate-binding module that anchors the system to a substrate. It has been suggested that Cellulosomes bound to the bacterial cell surface might be exposed to significant mechanical forces. Accordingly, the mechanical properties of these anchored Cellulosomes may be important to understand and improve cellulosome function. Here we used single-molecule force spectroscopy to study the mechanical properties of selected cohesin modules from scaffoldins of different Cellulosomes. We found that cohesins located in the region connecting the cell and the substrate are more robust than those located outside these two anchoring points. This observation applies to cohesins from primary scaffoldins (i.e. those that directly bind dockerin-bearing enzymes) from different Cellulosomes despite their sequence differences. Furthermore, we also found that cohesin nanomechanics (specifically, mechanostability and the position of the mechanical clamp of cohesin) are not significantly affected by other cellulosomal components, including linkers between cohesins, multiple cohesin repeats, and dockerin binding. Finally, we also found that cohesins (from both the connecting and external regions) have poor refolding efficiency but similar refolding rates, suggesting that the high mechanostability of connecting cohesins may be an evolutionarily conserved trait selected to minimize the occurrence of cohesin unfolding, which could irreversibly damage the cellulosome. We conclude that cohesin mechanostability is a major determinant of the overall mechanical stability of the cellulosome.

  • colocalization and disposition of Cellulosomes in clostridium clariflavum as revealed by correlative superresolution imaging
    Mbio, 2018
    Co-Authors: Lior Artzi, Sarah Morais, Tali Dadosh, Elad Milrot, Smadar Levinzaidman, Ely Morag, Edward A Bayer
    Abstract:

    ABSTRACT Cellulosomes are multienzyme complexes produced by anaerobic, cellulolytic bacteria for highly efficient breakdown of plant cell wall polysaccharides. Clostridium clariflavum is 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 in C. clariflavum and 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. IMPORTANCE Conversion 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.

Raphael Lamed - One of the best experts on this subject based on the ideXlab platform.

  • Unique organization and unprecedented diversity of the Bacteroides (Pseudobacteroides) cellulosolvens cellulosome system
    Biotechnology for Biofuels, 2017
    Co-Authors: Olga Zhivin, Bareket Dassa, Sagar M. Utturkar, Sarah Morais, Raphael Lamed, Steven D Brown, Bernard Henrissat, Edward A Bayer
    Abstract:

    Background(Pseudo) Bacteroides cellulosolvens is an anaerobic, mesophilic, cellulolytic, cellulosome-producing clostridial bacterium capable of utilizing cellulose and cellobiose as carbon sources. Recently, we sequenced the B. cellulosolvens genome, and subsequent comprehensive bioinformatic analysis, herein reported, revealed an unprecedented number of cellulosome-related components, including 78 cohesin modules scattered among 31 scaffoldins and more than 200 dockerin-bearing ORFs. In terms of numbers, the B. cellulosolvens cellulosome system represents the most intricate, compositionally diverse cellulosome system yet known in nature.ResultsThe organization of the B. cellulosolvens cellulosome is unique compared to previously described cellulosome systems. In contrast to all other known Cellulosomes, the cohesin types are reversed for all scaffoldins i.e., the type II cohesins are located on the enzyme-integrating primary scaffoldin, whereas the type I cohesins are located on the anchoring scaffoldins. Many of the type II dockerin-bearing ORFs include X60 modules, which are known to stabilize type II cohesin–dockerin interactions. In the present work, we focused on revealing the architectural arrangement of cellulosome structure in this bacterium by examining numerous interactions between the various cohesin and dockerin modules. In total, we cloned and expressed 43 representative cohesins and 27 dockerins. The results revealed various possible architectures of cell-anchored and cell-free Cellulosomes, which serve to assemble distinctive cellulosome types via three distinct cohesin–dockerin specificities: type I, type II, and a novel-type designated R (distinct from type III interactions, predominant in ruminococcal Cellulosomes).ConclusionsThe results of this study provide novel insight into the architecture and function of the most intricate and extensive cellulosomal system known today, thereby extending significantly our overall knowledge base of cellulosome systems and their components. The robust cellulosome system of B. cellulosolvens, with its unique binding specificities and reversal of cohesin–dockerin types, has served to amend our view of the cellulosome paradigm. Revealing new cellulosomal interactions and arrangements is critical for designing high-efficiency artificial Cellulosomes for conversion of plant-derived cellulosic biomass towards improved production of biofuels.

  • Unique organization and unprecedented diversity of the Bacteroides (Pseudobacteroides) cellulosolvens cellulosome system
    Biotechnology for Biofuels, 2017
    Co-Authors: Olga Zhivin, Bareket Dassa, Sagar M. Utturkar, Sarah Morais, Raphael Lamed, Steven D Brown, Bernard Henrissat, Edward A Bayer
    Abstract:

    (Pseudo) Bacteroides cellulosolvens is an anaerobic, mesophilic, cellulolytic, cellulosome-producing clostridial bacterium capable of utilizing cellulose and cellobiose as carbon sources. Recently, we sequenced the B. cellulosolvens genome, and subsequent comprehensive bioinformatic analysis, herein reported, revealed an unprecedented number of cellulosome-related components, including 78 cohesin modules scattered among 31 scaffoldins and more than 200 dockerin-bearing ORFs. In terms of numbers, the B. cellulosolvens cellulosome system represents the most intricate, compositionally diverse cellulosome system yet known in nature. The organization of the B. cellulosolvens cellulosome is unique compared to previously described cellulosome systems. In contrast to all other known Cellulosomes, the cohesin types are reversed for all scaffoldins i.e., the type II cohesins are located on the enzyme-integrating primary scaffoldin, whereas the type I cohesins are located on the anchoring scaffoldins. Many of the type II dockerin-bearing ORFs include X60 modules, which are known to stabilize type II cohesin–dockerin interactions. In the present work, we focused on revealing the architectural arrangement of cellulosome structure in this bacterium by examining numerous interactions between the various cohesin and dockerin modules. In total, we cloned and expressed 43 representative cohesins and 27 dockerins. The results revealed various possible architectures of cell-anchored and cell-free Cellulosomes, which serve to assemble distinctive cellulosome types via three distinct cohesin–dockerin specificities: type I, type II, and a novel-type designated R (distinct from type III interactions, predominant in ruminococcal Cellulosomes). The results of this study provide novel insight into the architecture and function of the most intricate and extensive cellulosomal system known today, thereby extending significantly our overall knowledge base of cellulosome systems and their components. The robust cellulosome system of B. cellulosolvens, with its unique binding specificities and reversal of cohesin–dockerin types, has served to amend our view of the cellulosome paradigm. Revealing new cellulosomal interactions and arrangements is critical for designing high-efficiency artificial Cellulosomes for conversion of plant-derived cellulosic biomass towards improved production of biofuels.

  • adaptor scaffoldins an original strategy for extended designer Cellulosomes inspired from nature
    Mbio, 2016
    Co-Authors: Johanna Stern, Sarah Morais, Raphael Lamed, Edward A Bayer
    Abstract:

    ABSTRACT Designer 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. IMPORTANCE Plant 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.

  • Clostridium clariflavum: Key Cellulosome Players Are Revealed by Proteomic Analysis
    Mbio, 2015
    Co-Authors: Lior Artzi, Raphael Lamed, Ely Morag, Yoav Barak, Edward A Bayer
    Abstract:

    Clostridium clariflavum is an anaerobic, cellulosome-forming thermophile, containing in its genome genes for a large number of cellulosomal enzyme and a complex scaffoldin system. Previously, we described the major cohesin-dockerin interactions of the cellulosome components, and on this basis a model of diverse cellulosome assemblies was derived. In this work, we cultivated C. clariflavum on cellobiose-, microcrystalline cellulose-, and switchgrass-containing media and isolated cell-free cellulosome complexes from each culture. Gelfiltration separation of the cellulosome samples revealed two major frac- tions, which were analyzed by label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) in order to identify the key players of the cellulosome assemblies therein. From the 13 scaffoldins present in the C. clariflavum genome, 11 were identi- fied, and a variety of enzymes from different glycoside hydrolase and carbohydrate esterase families were identified, including the glycoside hydrolase families GH48, GH9, GH5, GH30, GH11, and GH10. The expression level of the cellulosomal proteins varied as a function of the carbon source used for cultivation of the bacterium. In addition, the catalytic activity of each cellulo- some was examined on different cellulosic substrates, xylan and switchgrass. The cellulosome isolated from the microcrystalline cellulose-containing medium was the most active of all the Cellulosomes that were tested. The results suggest that the expression of the cellulosome proteins is regulated by the type of substrate in the growth medium. Moreover, both cell-free and cell-bound cellulosome complexes were produced which together may degrade the substrate in a synergistic manner. These observations are compatible with our previously published model of cellulosome assemblies in this bacterium. IMPORTANCE Because the reservoir of unsustainable fossil fuels, such as coal, petroleum, and natural gas, is overutilized and continues to contribute to environmental pollution and CO2emission, the need for appropriate alternative energy sources be- comes more crucial. Bioethanol produced from dedicated crops and cellulosic waste can provide a partial answer, yet a cost- effective production method must be developed. The cellulosome system of the anaerobic thermophile C. clariflavum comprises a large number of cellulolytic and hemicellulolytic enzymes, which self-assemble in a number of different cellulosome architec- tures for enhanced cellulosic biomass degradation. Identification of the major cellulosomal components expressed during growth of the bacterium and their influence on its catalytic capabilities provide insight into the performance of the remarkable cellulosome of this intriguing bacterium. Thefindings, together with the thermophilic characteristics of the proteins, render C. clariflavum of great interest for future use in industrial cellulose conversion processes.

  • Cellulosome domains for novel biotechnological application
    Progress in Biotechnology, 2014
    Co-Authors: Edward A Bayer, Raphael Lamed, Ely Morag, Meir Wilchek, Sima Yaron, Yuval Shoham
    Abstract:

    Abstract The functional domains of Cellulosomes from cellulolytic bacteria can be exploited for an astonishing variety of both conventional and nonconventional applications. Using a combination of molecular biological technique and chemical probes, the various domains can be mixed and matched, shuffled and scrambled to create new species of functionally altered complexes. For example, biotinylated cellulose-binding domains, attached to cellulose, serves as a basis for a new, simple and inexpensive type of avidin column for use in avidin-biotin technology. In addition, the domains responsible for integrating the catalytic subunits into the cellulosome can be reorganized for incorporation of foreign types of enzyme into heteroCellulosomes and/or chimeric complexes. Using this strategy, improved types of "supercellulosome" may be produced which will degrade cellulosic materials more efficiently. In the future, hybrid biomolecules, which comprise selected cellulosomal domains in conjunction with other affinity or enzymatic components, should find broad application in research, medicine and industry.

Sarah Morais - One of the best experts on this subject based on the ideXlab platform.

  • Minimalistic Cellulosome of the Butanologenic Bacterium Clostridium saccharoperbutylacetonicum.
    Mbio, 2020
    Co-Authors: Bosmat Levi Hevroni, Sarah Morais, Ely Morag, Yonit Ben-david, Edward A Bayer
    Abstract:

    ABSTRACT Clostridium saccharoperbutylacetonicum is a mesophilic, anaerobic, butanol-producing bacterium, originally isolated from soil. It was recently reported that C. saccharoperbutylacetonicum possesses multiple cellulosomal elements and would potentially form the smallest cellulosome known in nature. Its genome contains only eight dockerin-bearing enzymes, and its unique scaffoldin bears two cohesins (Cohs), three X2 modules, and two carbohydrate-binding modules (CBMs). In this study, all of the cellulosome-related modules were cloned, expressed, and purified. The recombinant cohesins, dockerins, and CBMs were tested for binding activity using enzyme-linked immunosorbent assay (ELISA)-based techniques. All the enzymes were tested for their comparative enzymatic activity on seven different cellulosic and hemicellulosic substrates, thus revealing four cellulases, a xylanase, a mannanase, a xyloglucanase, and a lichenase. All dockerin-containing enzymes interacted similarly with the second cohesin (Coh2) module, whereas Coh1 was more restricted in its interaction pattern. In addition, the polysaccharide-binding properties of the CBMs within the scaffoldin were examined by two complementary assays, affinity electrophoresis and affinity pulldown. The scaffoldin of C. saccharoperbutylacetonicum exhibited high affinity for cellulosic and hemicellulosic substrates, specifically to microcrystalline cellulose and xyloglucan. Evidence that supports substrate-dependent in vivo secretion of Cellulosomes is presented. The results of our analyses contribute to a better understanding of simple cellulosome systems by identifying the key players in this minimalistic system and the binding pattern of its cohesin-dockerin interaction. The knowledge gained by our study will assist further exploration of similar minimalistic Cellulosomes and will contribute to the significance of specific sets of defined cellulosomal enzymes in the degradation of cellulosic biomass. IMPORTANCE Cellulosome-producing bacteria are considered among the most important bacteria in both mesophilic and thermophilic environments, owing to their capacity to deconstruct recalcitrant plant-derived polysaccharides (and notably cellulose) into soluble saccharides for subsequent processing. In many ecosystems, the cellulosome-producing bacteria are particularly effective “first responders.” The massive amounts of sugars produced are potentially amenable in industrial settings to further fermentation by appropriate microbes to biofuels, notably ethanol and butanol. Among the solvent-producing bacteria, Clostridium saccharoperbutylacetonicum has the smallest cellulosome system known thus far. The importance of investigating the building blocks of such a small, multifunctional nanomachine is crucial to understanding the fundamental activities of this efficient enzymatic complex.

  • Creation of a functional hyperthermostable designer cellulosome
    Biotechnology for Biofuels, 2019
    Co-Authors: Amaranta Kahn, Sarah Morais, Michael E Himmel, Yannick J. Bomble, Anastasia P. Galanopoulou, Daehwan Chung, Nicholas S. Sarai, Neal Hengge, Dimitris G. Hatzinikolaou, Edward A Bayer
    Abstract:

    Renewable energy has become a field of high interest over the past decade, and production of biofuels from cellulosic substrates has a particularly high potential as an alternative source of energy. Industrial deconstruction of biomass, however, is an onerous, exothermic process, the cost of which could be decreased significantly by use of hyperthermophilic enzymes. An efficient way of breaking down cellulosic substrates can also be achieved by highly efficient enzymatic complexes called Cellulosomes. The modular architecture of these multi-enzyme complexes results in substrate targeting and proximity-based synergy among the resident enzymes. However, Cellulosomes have not been observed in hyperthermophilic bacteria. Here, we report the design and function of a novel hyperthermostable “designer cellulosome” system, which is stable and active at 75 °C. Enzymes from Caldicellulosiruptor bescii, a highly cellulolytic hyperthermophilic anaerobic bacterium, were selected and successfully converted to the cellulosomal mode by grafting onto them divergent dockerin modules that can be inserted in a precise manner into a thermostable chimaeric scaffoldin by virtue of their matching cohesins. Three pairs of cohesins and dockerins, selected from thermophilic microbes, were examined for their stability at extreme temperatures and were determined stable at 75 °C for at least 72 h. The resultant hyperthermostable cellulosome complex exhibited the highest levels of enzymatic activity on microcrystalline cellulose at 75 °C, compared to those of previously reported designer cellulosome systems and the native cellulosome from Clostridium thermocellum. The functional hyperthermophilic platform fulfills the appropriate physico-chemical properties required for exothermic processes. This system can thus be adapted for other types of thermostable enzyme systems and could serve as a basis for a variety of cellulolytic and non-cellulolytic industrial objectives at high temperatures.

  • cell surface display of designer Cellulosomes by lactobacillus plantarum
    Methods in Enzymology, 2019
    Co-Authors: Yonit Bendavid, Sarah Morais, Johanna Stern, Itzhak Mizrahi, Edward A Bayer
    Abstract:

    Abstract Cell-surface display of designer Cellulosomes complexes has attracted increased interest in recent years. These engineered microorganisms can efficiently degrade lignocellulosic biomass that represents an abundant resource for conversion into fermentable sugars, suitable for production of biofuels. The designer cellulosome is an artificial enzymatic complex that mimics the architecture of the natural cellulosome and allows the control of the positions, type, and copy number of the cellulosomal enzymes within the complex. Lactobacillus plantarum is an attractive candidate for metabolic engineering of lignocellulosic biomass to biofuels, as its natural characteristics include high ethanol and acid tolerance and the ability to metabolize hexose sugars. In recent years, successful expression of a variety of designer Cellulosomes on the cell surface of this bacterium has been demonstrated using the cell-consortium approach. This strategy minimized genomic interference on each strain upon genetic engineering, thereby maximizing the ability of each strain to grow, express, and secrete each enzyme. In addition, this strategy allows stoichiometric control of the cellulosome elements and facile exchange of the secreted proteins. A detailed procedure for display of designer Cellulosomes on the cell surface of L. plantarum is described in this chapter.

  • The cohesin module is a major determinant of cellulosome mechanical stability
    Journal of Biological Chemistry, 2018
    Co-Authors: Albert Galera-prat, Sarah Morais, Edward A Bayer, Yael Vazana, Mariano Carrión-vázquez
    Abstract:

    : Cellulosomes are bacterial protein complexes that bind and efficiently degrade lignocellulosic substrates. These are formed by multimodular scaffolding proteins known as scaffoldins, which comprise cohesin modules capable of binding dockerin-bearing enzymes and usually a carbohydrate-binding module that anchors the system to a substrate. It has been suggested that Cellulosomes bound to the bacterial cell surface might be exposed to significant mechanical forces. Accordingly, the mechanical properties of these anchored Cellulosomes may be important to understand and improve cellulosome function. Here we used single-molecule force spectroscopy to study the mechanical properties of selected cohesin modules from scaffoldins of different Cellulosomes. We found that cohesins located in the region connecting the cell and the substrate are more robust than those located outside these two anchoring points. This observation applies to cohesins from primary scaffoldins (i.e. those that directly bind dockerin-bearing enzymes) from different Cellulosomes despite their sequence differences. Furthermore, we also found that cohesin nanomechanics (specifically, mechanostability and the position of the mechanical clamp of cohesin) are not significantly affected by other cellulosomal components, including linkers between cohesins, multiple cohesin repeats, and dockerin binding. Finally, we also found that cohesins (from both the connecting and external regions) have poor refolding efficiency but similar refolding rates, suggesting that the high mechanostability of connecting cohesins may be an evolutionarily conserved trait selected to minimize the occurrence of cohesin unfolding, which could irreversibly damage the cellulosome. We conclude that cohesin mechanostability is a major determinant of the overall mechanical stability of the cellulosome.

  • colocalization and disposition of Cellulosomes in clostridium clariflavum as revealed by correlative superresolution imaging
    Mbio, 2018
    Co-Authors: Lior Artzi, Sarah Morais, Tali Dadosh, Elad Milrot, Smadar Levinzaidman, Ely Morag, Edward A Bayer
    Abstract:

    ABSTRACT Cellulosomes are multienzyme complexes produced by anaerobic, cellulolytic bacteria for highly efficient breakdown of plant cell wall polysaccharides. Clostridium clariflavum is 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 in C. clariflavum and 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. IMPORTANCE Conversion 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.

Michael E Himmel - One of the best experts on this subject based on the ideXlab platform.

  • Modeling the Self-assembly of the Cellulosome Enzyme
    2020
    Co-Authors: Yannick J. Bomble, Michael E Himmel, Gregg T. Beckham, James F. Matthews, Mark R. Nimlos, Michael F. Crowley
    Abstract:

    Most bacteria use free enzymes to degrade plant cell walls in nature. However, some bacteria have adopted a different strategy wherein enzymes can either be free or tethered on a protein scaffold forming a complex called a cellulosome. The study of the structure and mechanism of these large macromolecular complexes is an active and ongoing research topic, with the goal of finding ways to improve biomass conversion using Cellulosomes. Several mechanisms involved in cellulosome formation remain unknown, including how cellulosomal enzymes assemble on the scaffoldin and what governs the population of Cellulosomes created during self-assembly. Here, we present a coarse-grained model to study the self-assembly of Cellulosomes. The model captures most of the physical characteristics of three cellulosomal enzymes (Cel5B, CelS, and CbhA) and the scaffoldin (CipA) from Clostridium thermocellum. The protein structures are represented by beads connected by restraints to mimic the flexibility and shapes of these proteins. From a large simulation set, the assembly of cellulosomal enzyme complexes is shown to be dominated by their shape and modularity. The multimodular enzyme, CbhA, binds statistically more frequently to the scaffoldin than CelS or Cel5B. The enhanced binding is attributed to the flexible nature and multimodularity of this enzyme, providing a longer residence time around the scaffoldin. The characterization of the factors influencing the cellulosome assembly process may enable new strategies to create designers Cellulosomes.

  • Creation of a functional hyperthermostable designer cellulosome
    Biotechnology for Biofuels, 2019
    Co-Authors: Amaranta Kahn, Sarah Morais, Michael E Himmel, Yannick J. Bomble, Anastasia P. Galanopoulou, Daehwan Chung, Nicholas S. Sarai, Neal Hengge, Dimitris G. Hatzinikolaou, Edward A Bayer
    Abstract:

    Renewable energy has become a field of high interest over the past decade, and production of biofuels from cellulosic substrates has a particularly high potential as an alternative source of energy. Industrial deconstruction of biomass, however, is an onerous, exothermic process, the cost of which could be decreased significantly by use of hyperthermophilic enzymes. An efficient way of breaking down cellulosic substrates can also be achieved by highly efficient enzymatic complexes called Cellulosomes. The modular architecture of these multi-enzyme complexes results in substrate targeting and proximity-based synergy among the resident enzymes. However, Cellulosomes have not been observed in hyperthermophilic bacteria. Here, we report the design and function of a novel hyperthermostable “designer cellulosome” system, which is stable and active at 75 °C. Enzymes from Caldicellulosiruptor bescii, a highly cellulolytic hyperthermophilic anaerobic bacterium, were selected and successfully converted to the cellulosomal mode by grafting onto them divergent dockerin modules that can be inserted in a precise manner into a thermostable chimaeric scaffoldin by virtue of their matching cohesins. Three pairs of cohesins and dockerins, selected from thermophilic microbes, were examined for their stability at extreme temperatures and were determined stable at 75 °C for at least 72 h. The resultant hyperthermostable cellulosome complex exhibited the highest levels of enzymatic activity on microcrystalline cellulose at 75 °C, compared to those of previously reported designer cellulosome systems and the native cellulosome from Clostridium thermocellum. The functional hyperthermophilic platform fulfills the appropriate physico-chemical properties required for exothermic processes. This system can thus be adapted for other types of thermostable enzyme systems and could serve as a basis for a variety of cellulolytic and non-cellulolytic industrial objectives at high temperatures.

  • Modeling the Self-assembly of the Cellulosome Enzyme Complex
    Journal of Biological Chemistry, 2010
    Co-Authors: Yannick J. Bomble, Michael E Himmel, Gregg T. Beckham, James F. Matthews, Mark R. Nimlos, Michael F. Crowley
    Abstract:

    Abstract Most bacteria use free enzymes to degrade plant cell walls in nature. However, some bacteria have adopted a different strategy wherein enzymes can either be free or tethered on a protein scaffold forming a complex called a cellulosome. The study of the structure and mechanism of these large macromolecular complexes is an active and ongoing research topic, with the goal of finding ways to improve biomass conversion using Cellulosomes. Several mechanisms involved in cellulosome formation remain unknown, including how cellulosomal enzymes assemble on the scaffoldin and what governs the population of Cellulosomes created during self assembly. Here, we present a coarse grained model to study the self assembly of Cellulosomes. The model captures most of the physical characteristics of three cellulosomal enzymes (Cel5B, CelS, and CbhA) and the scaffoldin (CipA) from Clostridium thermocellum. The protein structures are represented by beads connected by restraints to mimic the flexibility and shapes of these proteins. From a large simulation set, the assembly of cellulosomal enzyme complexes is shown to be dominated by their shape and modularity. The multi-modular enzyme, CbhA, binds statistically more frequently to the scaffoldin than CelS or Cel5B. The enhanced binding is attributed to the flexible nature and multi-modularity of this enzyme, providing a longer residence time around the scaffoldin. The characterization of the factors influencing the cellulosome assembly process may enable new strategies to create designers Cellulosomes.

  • the potential of cellulases and Cellulosomes for cellulosic waste management
    Current Opinion in Biotechnology, 2007
    Co-Authors: Edward A Bayer, Raphael Lamed, Michael E Himmel
    Abstract:

    Lignocellulose is the most abundant plant cell wall component of the biosphere and the most voluminous waste produced by our society. Fortunately, it is not toxic or directly harmful, but our major waste disposal facilities — the landfills — are rapidly filling up with few realistic alternatives. Because cellulose is pure glucose, its conversion to fine products or fuels has remained a romantic and popular notion; however, the heterogeneous and recalcitrant nature of cellulosic waste presents a major obstacle for conventional conversion processes. One paradigm for the conversion of biomass to products in nature relies on a multienzyme complex, the cellulosome. Microbes that produce Cellulosomes convert lignocelluose to microbial cell mass and products (e.g. ethanol) simultaneously. The combination of designer Cellulosomes with novel production concepts could in the future provide the breakthroughs necessary for economical conversion of cellulosic biomass to biofuels.

  • The potential of cellulases and Cellulosomes for cellulosic waste management
    Current Opinion in Biotechnology, 2007
    Co-Authors: Edward A Bayer, Raphael Lamed, Michael E Himmel
    Abstract:

    Lignocellulose is the most abundant plant cell wall component of the biosphere and the most voluminous waste produced by our society. Fortunately, it is not toxic or directly harmful, but our major waste disposal facilities - the landfills - are rapidly filling up with few realistic alternatives. Because cellulose is pure glucose, its conversion to fine products or fuels has remained a romantic and popular notion; however, the heterogeneous and recalcitrant nature of cellulosic waste presents a major obstacle for conventional conversion processes. One paradigm for the conversion of biomass to products in nature relies on a multienzyme complex, the cellulosome. Microbes that produce Cellulosomes convert lignocelluose to microbial cell mass and products (e.g. ethanol) simultaneously. The combination of designer Cellulosomes with novel production concepts could in the future provide the breakthroughs necessary for economical conversion of cellulosic biomass to biofuels. © 2007 Elsevier Ltd. All rights reserved.

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  • catalytic subunit exchanges in the Cellulosomes produced by ruminiclostridium cellulolyticum suggest unexpected dynamics and adaptability of their enzymatic composition
    FEBS Journal, 2020
    Co-Authors: Romain Borne, Henripierre Fierobe, Chantal Tardif, Minhuyen Dao Ti, Nicolas Vita, Sandrine Pages
    Abstract:

    Cellulosomes are complex nanomachines produced by cellulolytic anaerobic bacteria such as Ruminiclostridium cellulolyticum (formerly known as Clostridium cellulolyticum). Cellulosomes are composed of a scaffoldin protein displaying several cohesin modules on which enzymatic components can bind to through their dockerin module. Although Cellulosomes have been studied for decades, very little is known about the dynamics of complex assembly. We have investigated the ability of some dockerin-bearing enzymes to chase the catalytic subunits already bound onto a miniscaffoldin displaying a single cohesin. The stability of the preassembled enzyme-scaffoldin complex appears to depend on the nature of the dockerin, and we have identified a key position in the dockerin sequence that is involved in the stability of the complex with the cohesin. Depending on the residue occupying this position, the dockerin can establish with the cohesin partner either a nearly irreversible or a reversible interaction, independently of the catalytic domain associated with the dockerin. Site-directed mutagenesis of this residue can convert a dockerin able to form a highly stable complex with the miniscaffoldin into a reversible complex forming one and vice versa. We also show that refunctionalization can occur with natural purified Cellulosomes. Altogether, our results shed light on the dynamics of Cellulosomes, especially their capacity to be remodeled even after their assembly is 'achieved', suggesting an unforeseen adaptability of their enzymatic composition over time.

  • the Cellulosomes from clostridium cellulolyticum
    FEBS Journal, 2009
    Co-Authors: Imen Fendri, Henripierre Fierobe, Sandrine Pages, Chantal Tardif, Stephanie Perret, Odile Valette, Sabrina Lignon
    Abstract:

    Cellulosomes produced by Clostridium cellulolyticum grown on cellulose were purified and separated using anion-exchange chromatography. SDS/PAGE analysis of six fractions showed variations in their cellulosomal protein composition. Hydrolytic activity on carboxymethyl cellulose, xylan, crystalline cellulose and hatched straw differed from one fraction to another. Fraction F1 showed a high level of activity on xylan, whereas fractions F5 and F6 were most active on crystalline cellulose and carboxymethyl cellulose, respectively. Several cellulosomal components specific to fractions F1, F5 and F6 were investigated using MS analysis. Several hemicellulases were identified, including three xylanases in F1, and several cellulases belonging to glycoside hydrolase families 9 and 5 and, a cystein protease inhibitor were identified in F5 and F6. Synergies were observed when two or three fractions were combined. A mixture containing fractions F1, F3 and F6 showed the most divergent cellulosomal composition, the most synergistic effects and the highest level of activity on straw (the most heterogeneous substrate tested). These findings show that on complex substrates such as straw, synergies occur between differently composed Cellulosomes and the degradation efficiency of the Cellulosomes is correlated with their enzyme diversity.

  • Exploration of new geometries in cellulosome-like chimeras.
    Applied and Environmental Microbiology, 2007
    Co-Authors: Florence Mingardon, Edward A Bayer, Chantal Tardif, Angélique Chanal, Henripierre Fierobe
    Abstract:

    In this study, novel cellulosome chimeras exhibiting atypical geometries and binding modes, wherein the targeting and proximity functions were directly incorporated as integral parts of the enzyme components, were designed. Two pivotal cellulosomal enzymes (family 48 and 9 cellulases) were thus appended with an efficient cellulose-binding module (CBM) and an optional cohesin and/or dockerin. Compared to the parental enzymes, the chimeric cellulases exhibited improved activity on crystalline cellulose as opposed to their reduced activity on amorphous cellulose. Nevertheless, the various complexes assembled using these engineered enzymes were somewhat less active on crystalline cellulose than the conventional designer Cellulosomes containing the parental enzymes. The diminished activity appeared to reflect the number of protein-protein interactions within a given complex, which presumably impeded the mobility of their catalytic modules. The presence of numerous CBMs in a given complex, however, also reduced their performance. Furthermore, a “covalent cellulosome” that combines in a single polypeptide chain a CBM, together with family 48 and family 9 catalytic modules, also exhibited reduced activity. This study also revealed that the cohesin-dockerin interaction may be reversible under specific conditions. Taken together, the data demonstrate that cellulosome components can be used to generate higher-order functional composites and suggest that enzyme mobility is a critical parameter for cellulosome efficiency.

  • action of designer Cellulosomes on homogeneous versus complex substrates controlled incorporation of three distinct enzymes into a defined trifunctional scaffoldin
    Journal of Biological Chemistry, 2005
    Co-Authors: Henripierre Fierobe, Raphael Lamed, Florence Mingardon, Adva Mechaly, Anne Belaich, Marco T Rincon, Sandrine Pages, Chantal Tardif, J P Belaich, Edward A Bayer
    Abstract:

    Abstract In recent work (Fierobe, H.-P., Bayer, E. A., Tardif, C., Czjzek, M., Mechaly, A., Belaich, A., Lamed, R., Shoham, Y., and Belaich, J.-P. (2002) J. Biol. Chem. 277, 49621–49630), we reported the self-assembly of a comprehensive set of defined “bifunctional” chimeric Cellulosomes. Each complex contained the following: (i) a chimeric scaffoldin possessing a cellulose-binding module and two cohesins of divergent specificity and (ii) two cellulases, each bearing a dockerin complementary to one of the divergent cohesins. This approach allowed the controlled integration of desired enzymes into a multiprotein complex of predetermined stoichiometry and topology. The observed enhanced synergy on recalcitrant substrates by the bifunctional designer Cellulosomes was ascribed to two major factors: substrate targeting and proximity of the two catalytic components. In the present work, the capacity of the previously described chimeric Cellulosomes was amplified by developing a third divergent cohesin-dockerin device. The resultant trifunctional designer Cellulosomes were assayed on homogeneous and complex substrates (microcrystalline cellulose and straw, respectively) and found to be considerably more active than the corresponding free enzyme or bifunctional systems. The results indicate that the synergy between two prominent cellulosomal enzymes (from the family-48 and -9 glycoside hydrolases) plays a crucial role during the degradation of cellulose by Cellulosomes and that one dominant family-48 processive endoglucanase per complex is sufficient to achieve optimal levels of synergistic activity. Furthermore cooperation within a cellulosome chimera between cellulases and a hemicellulase from different microorganisms was achieved, leading to a trifunctional complex with enhanced activity on a complex substrate.

  • use of antisense rna to modify the composition of Cellulosomes produced by clostridium cellulolyticum
    Molecular Microbiology, 2004
    Co-Authors: Chantal Tardif, J P Belaich, Stephanie Perret, Hedia Maamar
    Abstract:

    Summary The enzymatic composition of the Cellulosomes produced by Clostridium cellulolyticum was modified by inhibiting the synthesis of Cel48F that is the major cellulase of the Cellulosomes. The strain ATCC 35319 (pSOSasrF) was developed to over-produce a 469 nucleotide-long antisense-RNA (asRNA) directed against the ribosome-binding site region and the beginning of the coding region of the cel48F mRNAs. The cellulolytic system secreted by the asRNA-producing strain showed a markedly lower amount of Cel48F, compared to the control strain transformed with the empty plasmid (pSOSzero). This was correlated with a 30% decrease of the specific activity of the cellulolytic system on Avicel cellulose, indicating that Cel48F plays an important role in the recalcitrant cellulose degradation. However, only minor effects were observed on the growth parameters on cellulose. In both transformant strains, cellulosome production was found to be reduced and two unknown proteins (P105 and P98) appeared as major components of their cellulolytic systems. These proteins did not contain any dockerin domain and were shown to be not included into the Cellulosomes; they are expected to participate to the non-cellulosomal cellulolytic system of C. cellulolyticum.