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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.

  • glycosylation of hyperthermostable designer Cellulosome components yields enhanced stability and cellulose hydrolysis
    FEBS Journal, 2020
    Co-Authors: Amaranta Kahn, Sarah Morais, Michael E Himmel, Daehwan Chung, Nicholas S. Sarai, Neal Hengge, Edward A. Bayer, Audrey Kahn, Yannick J. Bomble
    Abstract:

    : Biomass deconstruction remains integral for enabling second-generation biofuel production at scale. However, several steps necessary to achieve significant solubilization of biomass, notably harsh pretreatment conditions, impose economic barriers to commercialization. By employing hyperthermostable cellulase machinery, biomass deconstruction can be made more efficient, leading to milder pretreatment conditions and ultimately lower production costs. The hyperthermophilic bacterium Caldicellulosiruptor bescii produces extremely active hyperthermostable cellulases, including the hyperactive multifunctional cellulase CbCel9A/Cel48A. Recombinant CbCel9A/Cel48A components have been previously produced in Escherichia coli and integrated into synthetic hyperthermophilic designer Cellulosome complexes. Since then, glycosylation has been shown to be vital for the high activity and stability of CbCel9A/Cel48A. Here, we studied the impact of glycosylation on a hyperthermostable designer Cellulosome system in which two of the cellulosomal components, the scaffoldin and the GH9 domain of CbCel9A/Cel48A, were glycosylated as a consequence of employing Ca. bescii as an expression host. Inclusion of the glycosylated components yielded an active Cellulosome system that exhibited long-term stability at 75°C. The resulting glycosylated designer Cellulosomes showed significantly greater synergistic activity compared to the enzymatic components alone, as well as higher thermostability than the analogous nonglycosylated designer Cellulosomes. These results indicate that glycosylation can be used as an essential engineering tool to improve the properties of designer Cellulosomes. Additionally, Ca. bescii was shown to be an attractive candidate for production of glycosylated designer Cellulosome components, which may further promote the viability of this bacterium both as a cellulase expression host and as a potential consolidated bioprocessing platform organism.

  • 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.

  • dynamic interactions of type i cohesin modules fine tune the structure of the Cellulosome of clostridium thermocellum
    Proceedings of the National Academy of Sciences of the United States of America, 2018
    Co-Authors: Anders Barth, Yoav Barak, Jelle Hendrix, Daniel B Fried, Edward A. Bayer, Don C Lamb
    Abstract:

    Efficient degradation of plant cell walls by selected anaerobic bacteria is performed by large extracellular multienzyme complexes termed Cellulosomes. The spatial arrangement within the Cellulosome is organized by a protein called scaffoldin, which recruits the cellulolytic subunits through interactions between cohesin modules on the scaffoldin and dockerin modules on the enzymes. Although many structural studies of the individual components of cellulosomal scaffoldins have been performed, the role of interactions between individual cohesin modules and the flexible linker regions between them are still not entirely understood. Here, we report single-molecule measurements using FRET to study the conformational dynamics of a bimodular cohesin segment of the scaffoldin protein CipA of Clostridium thermocellum . We observe compacted structures in solution that persist on the timescale of milliseconds. The compacted conformation is found to be in dynamic equilibrium with an extended state that shows distance fluctuations on the microsecond timescale. Shortening of the intercohesin linker does not destabilize the interactions but reduces the rate of contact formation. Upon addition of dockerin-containing enzymes, an extension of the flexible state is observed, but the cohesin–cohesin interactions persist. Using all-atom molecular-dynamics simulations of the system, we further identify possible intercohesin binding modes. Beyond the view of scaffoldin as “beads on a string,” we propose that cohesin–cohesin interactions are an important factor for the precise spatial arrangement of the enzymatic subunits in the Cellulosome that leads to the high catalytic synergy in these assemblies and should be considered when designing Cellulosomes for industrial applications.

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

  • modular organization of the thermobifida fusca exoglucanase cel6b impacts cellulose hydrolysis and designer Cellulosome efficiency
    Biotechnology Journal, 2017
    Co-Authors: Eva Setterlamed, Sarah Morais, Raphael Lamed, Johanna Stern, Edward A. Bayer
    Abstract:

    : Cellulose deconstruction can be achieved by three distinct enzymatic paradigms: free enzymes, multifunctional enzymes, and self-assembled, multi-enzyme complexes (Cellulosomes). To study their comparative efficiency, the simple and efficient cellulolytic system of the aerobic bacterium, Thermobifida fusca, is developed as an enzymatic model. In previous studies, most of its cellulases are successfully converted to the cellulosomal mode and exhibited high cellulolytic activities, except for Cel6B, a key exoglucanase of the T. fusca enzymatic system. Here, the impact of the modular organization of Cel6B on enzymatic activity is investigated. The position of the cellulose-binding module (CBM), its family and linker segment are shown to affect activity. Surprisingly, exchange of the native family-2 CBM to family-3 generates an increase in Cel6B activity on cellulosic substrates. Conversion of Cel6B to the cellulosomal mode by fusing a cohesin to the catalytic module enables formation of divalent enzyme complexes with dockerin-bearing enzymes. The resultant pseudo-Cellulosomes, containing Cel6B combined with endoglucanase Cel5A, exhibits enhanced enzymatic activity, compared to mixtures of wild-type enzymes or bifunctional enzymes, unlike similar pseudo-Cellulosomes containing endoglucanase Cel6A or proccessive endoglucanase Cel9A. Insight into the different enzymatic paradigms benefits ongoing development of efficient cellulolytic systems for conversion of plant-derived biomass into valuable sugars. NOVELTY STATEMENT: The protein engineering of the modular arrangement of a key exoglucanase from a highly cellulolytic bacterium, Thermobifida fusca, served to explore and compare three major enzymatic paradigms for cellulose degradation. This approach revealed highly active chimaeric forms of the exoglucanase that act in synergy together with a potent endoglucanase in bifunctional enzymes or divalent pseudo-Cellulosome-like complexes. Such engineered enzymes could be further integrated into larger enzymatic complexes, thereby providing a significant step forward towards conversion of the entire T. fusca free cellulolytic system into the cellulosomal modex and the enhanced conversion of cellulosic biomass into soluble sugars.

  • how does Cellulosome composition influence deconstruction of lignocellulosic substrates in clostridium ruminiclostridium thermocellum dsm 1313
    Biotechnology for Biofuels, 2017
    Co-Authors: Shahar Yoav, Bareket Dassa, Raphael Lamed, Ely Morag, Yoav Barak, Yitzhak Hadar, Ilya Borovok, Melina Shamshoum, Edward A. Bayer
    Abstract:

    Bioethanol production processes involve enzymatic hydrolysis of pretreated lignocellulosic biomass into fermentable sugars. Due to the relatively high cost of enzyme production, the development of potent and cost-effective cellulolytic cocktails is critical for increasing the cost-effectiveness of bioethanol production. In this context, the multi-protein cellulolytic complex of Clostridium (Ruminiclostridium) thermocellum, the Cellulosome, was studied here. C. thermocellum is known to assemble Cellulosomes of various subunit (enzyme) compositions, in response to the available carbon source. In the current study, different carbon sources were used, and their influence on both cellulosomal composition and the resultant activity was investigated. Glucose, cellobiose, microcrystalline cellulose, alkaline-pretreated switchgrass, alkaline-pretreated corn stover, and dilute acid-pretreated corn stover were used as sole carbon sources in the growth media of C. thermocellum strain DSM 1313. The purified Cellulosomes were compared for their activity on selected cellulosic substrates. Interestingly, Cellulosomes derived from cells grown on lignocellulosic biomass showed no advantage in hydrolyzing the original carbon source used for their production. Instead, microcrystalline cellulose- and glucose-derived Cellulosomes were equal or superior in their capacity to deconstruct lignocellulosic biomass. Mass spectrometry analysis revealed differential composition of catalytic and structural subunits (scaffoldins) in the different Cellulosome samples. The most abundant catalytic subunits in all Cellulosome types include Cel48S, Cel9K, Cel9Q, Cel9R, and Cel5G. Microcrystalline cellulose- and glucose-derived Cellulosome samples showed higher endoglucanase-to-exoglucanase ratios and higher catalytic subunit-per-scaffoldin ratios compared to lignocellulose-derived Cellulosome types. The results reported here highlight the finding that Cellulosomes derived from cells grown on glucose and microcrystalline cellulose are more efficient in their action on cellulosic substrates than other Cellulosome preparations. These results should be considered in the future development of C. thermocellum-based cellulolytic cocktails, designer Cellulosomes, or engineering of improved strains for deconstruction of lignocellulosic biomass.

  • 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.

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.

  • glycosylation of hyperthermostable designer Cellulosome components yields enhanced stability and cellulose hydrolysis
    FEBS Journal, 2020
    Co-Authors: Amaranta Kahn, Sarah Morais, Michael E Himmel, Daehwan Chung, Nicholas S. Sarai, Neal Hengge, Edward A. Bayer, Audrey Kahn, Yannick J. Bomble
    Abstract:

    : Biomass deconstruction remains integral for enabling second-generation biofuel production at scale. However, several steps necessary to achieve significant solubilization of biomass, notably harsh pretreatment conditions, impose economic barriers to commercialization. By employing hyperthermostable cellulase machinery, biomass deconstruction can be made more efficient, leading to milder pretreatment conditions and ultimately lower production costs. The hyperthermophilic bacterium Caldicellulosiruptor bescii produces extremely active hyperthermostable cellulases, including the hyperactive multifunctional cellulase CbCel9A/Cel48A. Recombinant CbCel9A/Cel48A components have been previously produced in Escherichia coli and integrated into synthetic hyperthermophilic designer Cellulosome complexes. Since then, glycosylation has been shown to be vital for the high activity and stability of CbCel9A/Cel48A. Here, we studied the impact of glycosylation on a hyperthermostable designer Cellulosome system in which two of the cellulosomal components, the scaffoldin and the GH9 domain of CbCel9A/Cel48A, were glycosylated as a consequence of employing Ca. bescii as an expression host. Inclusion of the glycosylated components yielded an active Cellulosome system that exhibited long-term stability at 75°C. The resulting glycosylated designer Cellulosomes showed significantly greater synergistic activity compared to the enzymatic components alone, as well as higher thermostability than the analogous nonglycosylated designer Cellulosomes. These results indicate that glycosylation can be used as an essential engineering tool to improve the properties of designer Cellulosomes. Additionally, Ca. bescii was shown to be an attractive candidate for production of glycosylated designer Cellulosome components, which may further promote the viability of this bacterium both as a cellulase expression host and as a potential consolidated bioprocessing platform organism.

  • 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, Yael Vazana, Edward A. Bayer, 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.

Yoav Barak - One of the best experts on this subject based on the ideXlab platform.

  • dynamic interactions of type i cohesin modules fine tune the structure of the Cellulosome of clostridium thermocellum
    Proceedings of the National Academy of Sciences of the United States of America, 2018
    Co-Authors: Anders Barth, Yoav Barak, Jelle Hendrix, Daniel B Fried, Edward A. Bayer, Don C Lamb
    Abstract:

    Efficient degradation of plant cell walls by selected anaerobic bacteria is performed by large extracellular multienzyme complexes termed Cellulosomes. The spatial arrangement within the Cellulosome is organized by a protein called scaffoldin, which recruits the cellulolytic subunits through interactions between cohesin modules on the scaffoldin and dockerin modules on the enzymes. Although many structural studies of the individual components of cellulosomal scaffoldins have been performed, the role of interactions between individual cohesin modules and the flexible linker regions between them are still not entirely understood. Here, we report single-molecule measurements using FRET to study the conformational dynamics of a bimodular cohesin segment of the scaffoldin protein CipA of Clostridium thermocellum . We observe compacted structures in solution that persist on the timescale of milliseconds. The compacted conformation is found to be in dynamic equilibrium with an extended state that shows distance fluctuations on the microsecond timescale. Shortening of the intercohesin linker does not destabilize the interactions but reduces the rate of contact formation. Upon addition of dockerin-containing enzymes, an extension of the flexible state is observed, but the cohesin–cohesin interactions persist. Using all-atom molecular-dynamics simulations of the system, we further identify possible intercohesin binding modes. Beyond the view of scaffoldin as “beads on a string,” we propose that cohesin–cohesin interactions are an important factor for the precise spatial arrangement of the enzymatic subunits in the Cellulosome that leads to the high catalytic synergy in these assemblies and should be considered when designing Cellulosomes for industrial applications.

  • dynamic interactions of type i cohesin modules fine tune the structure of the Cellulosome of clostridium thermocellum
    bioRxiv, 2018
    Co-Authors: Anders Barth, Yoav Barak, Jelle Hendrix, Daniel B Fried, Edward A. Bayer, Don C Lamb
    Abstract:

    Efficient degradation of plant cell walls by selected anaerobic bacteria is performed by large extracellular multienzyme complexes termed Cellulosomes. The spatial arrangement within the Cellulosome is organized by a protein called scaffoldin, which recruits the cellulolytic subunits through interactions between cohesin modules on the scaffoldin and dockerin modules on the enzymes. Although many structural studies of the individual components of cellulosomal scaffoldins have been performed, the role of interactions between individual cohesin modules and the flexible linker regions between them are still not entirely understood. Here, we report single-molecule measurements using Forster resonance energy transfer to study the conformational dynamics of a bimodular tandem cohesin segment of the scaffoldin protein CipA of Clostridium thermocellum. Our data reveal the existence of compacted structures in solution that persist on the timescale of milliseconds. The compacted conformation is found to be in dynamic equilibrium with an extended state that shows distance fluctuations on the microsecond timescale. Shortening of the inter-cohesin linker does not significantly alter the structural dynamics. Upon addition of dockerin-containing enzymes, an extension of the flexible state is observed but the cohesin-cohesin interactions persist. This suggests that the dockerin-binding interfaces are not involved in cohesin-cohesin interactions. The formation of cohesin-cohesin interactions is also observed in all-atom molecular dynamics simulations of the system. From the simulations, we identify possible inter-cohesin binding modes, none of which show obstruction of the cohesin-dockerin binding interfaces. Our results go beyond the view of scaffoldin as 'beads on a string'. We propose that both the flexibility and cohesin-cohesin interactions are important factors for the precise spatial arrangement of the enzymatic subunits in the Cellulosome that leads to the high catalytic synergy in these assemblies. Hence, the flexibility of the linker region and cohesin-cohesin interactions should be considered when designing Cellulosomes for industrial applications.

  • how does Cellulosome composition influence deconstruction of lignocellulosic substrates in clostridium ruminiclostridium thermocellum dsm 1313
    Biotechnology for Biofuels, 2017
    Co-Authors: Shahar Yoav, Bareket Dassa, Raphael Lamed, Ely Morag, Yoav Barak, Yitzhak Hadar, Ilya Borovok, Melina Shamshoum, Edward A. Bayer
    Abstract:

    Bioethanol production processes involve enzymatic hydrolysis of pretreated lignocellulosic biomass into fermentable sugars. Due to the relatively high cost of enzyme production, the development of potent and cost-effective cellulolytic cocktails is critical for increasing the cost-effectiveness of bioethanol production. In this context, the multi-protein cellulolytic complex of Clostridium (Ruminiclostridium) thermocellum, the Cellulosome, was studied here. C. thermocellum is known to assemble Cellulosomes of various subunit (enzyme) compositions, in response to the available carbon source. In the current study, different carbon sources were used, and their influence on both cellulosomal composition and the resultant activity was investigated. Glucose, cellobiose, microcrystalline cellulose, alkaline-pretreated switchgrass, alkaline-pretreated corn stover, and dilute acid-pretreated corn stover were used as sole carbon sources in the growth media of C. thermocellum strain DSM 1313. The purified Cellulosomes were compared for their activity on selected cellulosic substrates. Interestingly, Cellulosomes derived from cells grown on lignocellulosic biomass showed no advantage in hydrolyzing the original carbon source used for their production. Instead, microcrystalline cellulose- and glucose-derived Cellulosomes were equal or superior in their capacity to deconstruct lignocellulosic biomass. Mass spectrometry analysis revealed differential composition of catalytic and structural subunits (scaffoldins) in the different Cellulosome samples. The most abundant catalytic subunits in all Cellulosome types include Cel48S, Cel9K, Cel9Q, Cel9R, and Cel5G. Microcrystalline cellulose- and glucose-derived Cellulosome samples showed higher endoglucanase-to-exoglucanase ratios and higher catalytic subunit-per-scaffoldin ratios compared to lignocellulose-derived Cellulosome types. The results reported here highlight the finding that Cellulosomes derived from cells grown on glucose and microcrystalline cellulose are more efficient in their action on cellulosic substrates than other Cellulosome preparations. These results should be considered in the future development of C. thermocellum-based cellulolytic cocktails, designer Cellulosomes, or engineering of improved strains for deconstruction of lignocellulosic biomass.

  • complexity of the ruminococcus flavefaciens fd 1 Cellulosome reflects an expansion of family related protein protein interactions
    Scientific Reports, 2017
    Co-Authors: Vered Israeliruimy, Bareket Dassa, Sarah Morais, Pedro Bule, Yoav Barak, Ilya Borovok, Yuval Hamberg, Sadanari Jindou, Michal Slutzki, Vânia Cardoso
    Abstract:

    Protein-protein interactions play a vital role in cellular processes as exemplified by assembly of the intricate multi-enzyme Cellulosome complex. Cellulosomes are assembled by selective high-affinity binding of enzyme-borne dockerin modules to repeated cohesin modules of structural proteins termed scaffoldins. Recent sequencing of the fiber-degrading Ruminococcus flavefaciens FD-1 genome revealed a particularly elaborate Cellulosome system. In total, 223 dockerin-bearing ORFs potentially involved in Cellulosome assembly and a variety of multi-modular scaffoldins were identified, and the dockerins were classified into six major groups. Here, extensive screening employing three complementary medium- to high-throughput platforms was used to characterize the different cohesin-dockerin specificities. The platforms included (i) cellulose-coated microarray assay, (ii) enzyme-linked immunosorbent assay (ELISA) and (iii) in-vivo co-expression and screening in Escherichia coli. The data revealed a collection of unique cohesin-dockerin interactions and support the functional relevance of dockerin classification into groups. In contrast to observations reported previously, a dual-binding mode is involved in Cellulosome cell-surface attachment, whereas single-binding interactions operate for Cellulosome integration of enzymes. This sui generis Cellulosome model enhances our understanding of the mechanisms governing the remarkable ability of R. flavefaciens to degrade carbohydrates in the bovine rumen and provides a basis for constructing efficient nano-machines applied to biological processes.

  • 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.

Carlos M. G. A. Fontes - One of the best experts on this subject based on the ideXlab platform.

  • Cellulosome assembly: paradigms are meant to be broken!
    Current Opinion in Structural Biology, 2018
    Co-Authors: Pedro Bule, Carlos M. G. A. Fontes, Virgínia M. R. Pires, Victor D. Alves
    Abstract:

    Cohesin–Dockerin interactions are at the core of cellulosomal assembly and organization. They are highly specific and form stable complexes, allowing Cellulosomes to adopt distinct conformations. Each cellulosomal system seems to have a particular organizational strategy that can vary in complexity according to the nature of its Cohesin–Dockerin interactions. Hence, several efforts have been undertaken to reveal the mechanisms that govern the specificity, affinity and flexibility of these protein–protein interactions. Here we review the most recent studies that have focused on the structural aspects of Cohesin–Dockerin recognition. They reveal an ever-increasing number of subtle intricacies suggesting that Cellulosome assembly is more complex than was initially thought.

  • Assembly of Ruminococcus flavefaciens Cellulosome revealed by structures of two cohesin-dockerin complexes.
    Scientific Reports, 2017
    Co-Authors: Pedro Bule, Harry J Gilbert, Victor D. Alves, Vered Israeli-ruimy, Luís M. A. Ferreira, Steven P. Smith, Shabir Najmudin, Edward A. Bayer, Ana Carvalho, Carlos M. G. A. Fontes
    Abstract:

    Cellulosomes are sophisticated multi-enzymatic nanomachines produced by anaerobes to effectively deconstruct plant structural carbohydrates. Cellulosome assembly involves the binding of enzyme-borne dockerins (Doc) to repeated cohesin (Coh) modules located in a non-catalytic scaffoldin. Docs appended to cellulosomal enzymes generally present two similar Coh-binding interfaces supporting a dual-binding mode, which may confer increased positional adjustment of the different complex components. Ruminococcus flavefaciens’ Cellulosome is assembled from a repertoire of 223 Doc-containing proteins classified into 6 groups. Recent studies revealed that Docs of groups 3 and 6 are recruited to the Cellulosome via a single-binding mode mechanism with an adaptor scaffoldin. To investigate the extent to which the single-binding mode contributes to the assembly of R. flavefaciens Cellulosome, the structures of two group 1 Docs bound to Cohs of primary (ScaA) and adaptor (ScaB) scaffoldins were solved. The data revealed that group 1 Docs display a conserved mechanism of Coh recognition involving a single-binding mode. Therefore, in contrast to all Cellulosomes described to date, the assembly of R. flavefaciens Cellulosome involves single but not dual-binding mode Docs. Thus, this work reveals a novel mechanism of Cellulosome assembly and challenges the ubiquitous implication of the dual-binding mode in the acquisition of Cellulosome flexibility.

  • single binding mode integration of hemicellulose degrading enzymes via adaptor scaffoldins in ruminococcus flavefaciens Cellulosome
    Journal of Biological Chemistry, 2016
    Co-Authors: Pedro Bule, Harry J Gilbert, Victor D. Alves, Luís M. A. Ferreira, Steven P. Smith, Shabir Najmudin, Edward A. Bayer, Andre Leitao, Carlos M. G. A. Fontes
    Abstract:

    : The assembly of one of Nature's most elaborate multienzyme complexes, the Cellulosome, results from the binding of enzyme-borne dockerins to reiterated cohesin domains located in a non-catalytic primary scaffoldin. Generally, dockerins present two similar cohesin-binding interfaces that support a dual binding mode. The dynamic integration of enzymes in Cellulosomes, afforded by the dual binding mode, is believed to incorporate additional flexibility in highly populated multienzyme complexes. Ruminococcus flavefaciens, the primary degrader of plant structural carbohydrates in the rumen of mammals, uses a portfolio of more than 220 different dockerins to assemble the most intricate Cellulosome known to date. A sequence-based analysis organized R. flavefaciens dockerins into six groups. Strikingly, a subset of R. flavefaciens cellulosomal enzymes, comprising dockerins of groups 3 and 6, were shown to be indirectly incorporated into primary scaffoldins via an adaptor scaffoldin termed ScaC. Here, we report the crystal structure of a group 3 R. flavefaciens dockerin, Doc3, in complex with ScaC cohesin. Doc3 is unusual as it presents a large cohesin-interacting surface that lacks the structural symmetry required to support a dual binding mode. In addition, dockerins of groups 3 and 6, which bind exclusively to ScaC cohesin, display a conserved mechanism of protein recognition that is similar to Doc3. Groups 3 and 6 dockerins are predominantly appended to hemicellulose-degrading enzymes. Thus, single binding mode dockerins interacting with adaptor scaffoldins exemplify an evolutionary pathway developed by R. flavefaciens to recruit hemicellulases to the sophisticated Cellulosomes acting in the gastrointestinal tract of mammals.

  • elaborate Cellulosome architecture of acetivibrio cellulolyticus revealed by selective screening of cohesin dockerin interactions
    PeerJ, 2014
    Co-Authors: Yuval Hamberg, Bareket Dassa, Raphael Lamed, Carlos M. G. A. Fontes, Yoav Barak, Kate Cameron, Edward A. Bayer, Vered Ruimyisraeli, Daniel B Fried
    Abstract:

    Cellulosic waste represents a significant and underutilized carbon source for the biofuel industry. Owing to the recalcitrance of crystalline cellulose to enzymatic degradation, it is necessary to design economical methods of liberating the fermentable sugars required for bioethanol production. One route towards unlocking the potential of cellulosic waste lies in a highly complex class of molecular machines, the Cellulosomes. Secreted mainly by anaerobic bacteria, Cellulosomes are structurally diverse, cell surface-bound protein assemblies that can contain dozens of catalytic components. The key feature of the Cellulosome is its modularity, facilitated by the ultra-high affinity cohesin–dockerin interaction. Due to the enormous number of cohesin and dockerin modules found in a typical cellulolytic organism, a major bottleneck in understanding the biology of cellulosomics is the purification of each cohesin- and dockerin-containing component, prior to analyses of their interaction. As opposed to previous approaches, the present study utilized proteins contained in unpurified whole-cell extracts. This strategy was made possible due to an experimental design that allowed for the relevant proteins to be “purified” via targeted affinity interactions as a function of the binding assay. The approach thus represents a new strategy, appropriate for future medium- to high-throughput screening of whole genomes, to determine the interactions between cohesins and dockerins. We have selected the Cellulosome of Acetivibrio cellulolyticus for this work due to its exceptionally complex Cellulosome systems and intriguing diversity of its cellulosomal modular components. Containing 41 cohesins and 143 dockerins, A. cellulolyticus has one of the largest number of potential cohesin–dockerin interactions of any organism, and contains unusual and novel cellulosomal features. We have surveyed a representative library of cohesin and dockerin modules spanning the Cellulosome’s total cohesin and dockerin sequence diversity, emphasizing the testing of unusual and previously-unknown protein modules. The screen revealed several novel cell-bound Cellulosome architectures, thus expanding on those previously known, as well as soluble cellulose systems that are not bound to the bacterial cell surface. This study sets the stage for screening the entire complement of cellulosomal components from A. cellulolyticus and other organisms with large Cellulosome systems. The knowledge gained by such efforts brings us closer to understanding the exceptional catalytic abilities of Cellulosomes and will allow the use of novel cellulosomal components in artificial assemblies and in enzyme cocktails for sustainable energy-related research programs.

  • construction of gh16 β glucanase mini Cellulosomes to improve the nutritive value of barley based diets for broilers
    Journal of Agricultural and Food Chemistry, 2014
    Co-Authors: Monica Costa, Luís M. A. Ferreira, Vania O Fernandes, T Ribeiro, Vânia Cardoso, Helena Santos, Madalena Lordelo, Luis Serrano, Carlos M. G. A. Fontes
    Abstract:

    Anaerobic cellulolytic bacteria organize a comprehensive range of cellulases and hemicellulases in high molecular weight multienzyme complexes termed Cellulosomes. Integration of cellulosomal components occurs via highly ordered protein–protein interactions between cohesins and dockerins. This paper reports the production of mini-Cellulosomes containing one (GH16-1C) or three (GH16-3C) copies of Clostridium thermocellum glucanase 16A (CtGlc16A). Barley β-1,3–1,4-glucans are known to be antinutritive for monogastric animals, particularly for poultry. GH16-1C and GH16-3C were used to supplement barley-based diets for broilers. The data revealed that the two mini-Cellulosomes effectively improved the nutritive value of barley-based diets for broilers. Analysis of mini-Cellulosome molecular integrity revealed that linker sequences separating protein domains in scaffoldins and cellulosomal catalytic units are highly susceptible to proteolytic attack in vivo. The data suggest that linker protection could result...