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1.
Bioresour Technol ; 308: 123260, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32251860

RESUMO

Feedstock availability and its pretreatment, high process economics and insufficient ethanol (HEt) titres necessitated the bioprocesses that are sustainable. The advanced consolidated bioprocessing (CBPSeq) strategy presently considered for improved HEt production involves, sequential coupling of CBP thermophile, Clostridium thermocellum ATCC-27405 with mesophilic microaerobe, Pichia stipitis NCIM-3498. Biogenic municipal solid waste (BMSW) pretreated with 0.5% NaOH (CSPBMSW) served as the sole carbon source. CBPSeq (23.99 g/L) fared better than CBP standalone (18.10 g/L) wherein 1.32-folds improvement in HEt titre was recorded. Considering insufficient xylanase titre in cellulosome complex of C. thermocellum, CBPSeq was performed employing exogenous xylanases (CBPSeqE) to improve xylan digestibility and HEt yield. CBPSeqE-II biosystem at pH 5 showed maximum HEt titre of 36.90 g/L which corresponds to yield of 0.26 g HEt/ g CSPBMSW. This study substantiates efficacy of CBPSeqE-II biosystem in sustainable bioethanol production from BMSW in a single reactor without laborious steps.


Assuntos
Celulossomas , Clostridium thermocellum , Etanol , Fermentação , Resíduos Sólidos
2.
Proc Natl Acad Sci U S A ; 117(5): 2385-2394, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31953261

RESUMO

Cellulosomes, which are multienzyme complexes from anaerobic bacteria, are considered nature's finest cellulolytic machinery. Thus, constructing a cellulosome in an industrial yeast has long been a goal pursued by scientists. However, it remains highly challenging due to the size and complexity of cellulosomal genes. Here, we overcame the difficulties by synthesizing the Clostridium thermocellum scaffoldin gene (CipA) and the anchoring protein gene (OlpB) using advanced synthetic biology techniques. The engineered Kluyveromyces marxianus, a probiotic yeast, secreted a mixture of dockerin-fused fungal cellulases, including an endoglucanase (TrEgIII), exoglucanase (CBHII), ß-glucosidase (NpaBGS), and cellulase boosters (TaLPMO and MtCDH). The confocal microscopy results confirmed the cell-surface display of OlpB-ScGPI and fluorescence-activated cell sorting analysis results revealed that almost 81% of yeast cells displayed OlpB-ScGPI. We have also demonstrated the cellulosome complex formation using purified and crude cellulosomal proteins. Native polyacrylamide gel electrophoresis and mass spectrometric analysis further confirmed the cellulosome complex formation. Our engineered cellulosome can accommodate up to 63 enzymes, whereas the largest engineered cellulosome reported thus far could accommodate only 12 enzymes and was expressed by a plasmid instead of chromosomal integration. Interestingly, CipA 2B9C (with two cellulose binding modules, CBM) released significantly higher quantities of reducing sugars compared with other CipA variants, thus confirming the importance of cohesin numbers and CBM domain on cellulosome complex. The engineered yeast host efficiently degraded cellulosic substrates and released 3.09 g/L and 8.61 g/L of ethanol from avicel and phosphoric acid-swollen cellulose, respectively, which is higher than any previously constructed yeast cellulosome.


Assuntos
Membrana Celular/metabolismo , Celulossomas/metabolismo , Kluyveromyces/genética , Kluyveromyces/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Celulase/genética , Celulase/metabolismo , Celulose/metabolismo , Celulossomas/enzimologia , Celulossomas/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/genética , Clostridium thermocellum/genética , Etanol/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Kluyveromyces/enzimologia , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , beta-Glucosidase/genética , beta-Glucosidase/metabolismo
3.
Mol Biotechnol ; 61(11): 826-835, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31435842

RESUMO

The cellulosomal enzyme, RfGH51/2, of Ruminococcus flavefaciens contains an N-terminal module, a family 5 glycoside hydrolase GH5_4 with a putative endoglucanase activity, while C-terminal domain is a putative endo-mannanase (GH5_7). The two putative catalytic modules are separated by family 80 carbohydrate binding module (CBM80) having wide ligand specificity. The putative endo-mannanase module, GH5_7 (RfGH5_7), was cloned, expressed in Escherichia coli BL-21(DE3) cells and purified. SDS-PAGE analysis of purified RfGH5_7 showed molecular size ~ 35 kDa. Substrate specificity analysis of RfGH5_7 showed maximum activity against locust bean galactomannan (298.5 U/mg) followed by konjac glucomannan (256.2 U/mg) and carob galactomannan (177.2 U/mg). RfGH5_7 showed maximum activity at optimum pH 6.0 and temperature 60 °C. RfGH5_7 displayed stability in between pH 6.0 and 9.0 and thermostability till 50 °C. 10 mM Ca2+ ions increased the enzyme activity by 33%. The melting temperature of RfGH5_7 was 84 °C that was not affected by Ca2+ ions or chelating agents. RfGH5_7 showed, Vmax, 389 U/mg and Km, 0.92 mg/mL for locust bean galactomannan. TLC analysis revealed that RfGH5_7 hydrolysed locust bean galactomannan predominantly to mannose, mannobiose, mannotriose and higher degree of polymerization of manno-oligosaccharides indicating an endo-acting catalytic mechanism. This study revealed a highly active and thermostable endo-mannanase with considerable biotechnological potential.


Assuntos
Celulase/metabolismo , Ruminococcus/enzimologia , beta-Manosidase/metabolismo , Sequência de Aminoácidos/genética , Celulase/biossíntese , Celulase/química , Celulase/genética , Celulossomas/enzimologia , Quelantes , Cromatografia em Camada Delgada , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Galactanos/química , Galactanos/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Mananas/química , Mananas/metabolismo , Oligossacarídeos/química , Gomas Vegetais/química , Gomas Vegetais/metabolismo , Ruminococcus/genética , Especificidade por Substrato , Temperatura , beta-Manosidase/química , beta-Manosidase/genética
4.
Biotechnol Appl Biochem ; 66(5): 720-730, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31408226

RESUMO

Lignocellulose, one of the most abundant renewable sources of sugar, can be converted into bioenergy through hydrolysis of cellulose and hemicellulose. Due to its renewability and availability in large quantities, bioenergy is considered as a possible alternative to fossil energy and attracts the attention of the world with increased concerns about environmental protection and energy crisis. The depolymerization of cellulosic substrate to monomer is the rate-limiting step in the bioconversion of lignocellulose by cellulolytic microbes. Cellulosome, a multienzyme complex from anaerobic cellulolytic bacteria, can efficiently degrade the cellulosic substrates. Previous studies have shown that the reconstitution of cellulosome in vitro and its heterologous expression or display on the cell surface can help to solve the low yield problem of cellulosome in cellulolytic bacteria. This paper reviews the research progress in the reconstitution of cellulosome as well as its application in biorefinery, including the construction of cellulosome as well as different methods for cellulosome reconstitution and its surface display. This review will promote the understanding of cellulosome and its reconstitution.


Assuntos
Celulossomas/metabolismo , Celulossomas/química
5.
Int J Mol Sci ; 20(13)2019 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-31288425

RESUMO

Cellulosomes are an extracellular supramolecular multienzyme complex that can efficiently degrade cellulose and hemicelluloses in plant cell walls. The structural and unique subunit arrangement of cellulosomes can promote its adhesion to the insoluble substrates, thus providing individual microbial cells with a direct competence in the utilization of cellulosic biomass. Significant progress has been achieved in revealing the structures and functions of cellulosomes, but a knowledge gap still exists in understanding the interaction between cellulosome and lignocellulosic substrate for those derived from biorefinery pretreatment of agricultural crops. The cellulosomic saccharification of lignocellulose is affected by various substrate-related physical and chemical factors, including native (untreated) wood lignin content, the extent of lignin and xylan removal by pretreatment, lignin structure, substrate size, and of course substrate pore surface area or substrate accessibility to cellulose. Herein, we summarize the cellulosome structure, substrate-related factors, and regulatory mechanisms in the host cells. We discuss the latest advances in specific strategies of cellulosome-induced hydrolysis, which can function in the reaction kinetics and the overall progress of biorefineries based on lignocellulosic feedstocks.


Assuntos
Celulossomas/química , Lignina/química , Bactérias/classificação , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Celulossomas/metabolismo , Hidrólise , Filogenia , Ligação Proteica , RNA Ribossômico 16S , Especificidade por Substrato
6.
Appl Microbiol Biotechnol ; 103(17): 6885-6902, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31309267

RESUMO

The secretome, the complement of extracellular proteins, is a reflection of the interaction of an organism with its host or substrate, thus a determining factor for the organism's fitness and competitiveness. Hence, the secretome impacts speciation and organismal evolution. The zoosporic Chytridiomycota, Blastocladiomycota, Neocallimastigomycota, and Cryptomycota represent the earliest diverging lineages of the Fungal Kingdom. The review describes the enzyme compositions of these zoosporic fungi, underscoring the enzymes involved in biomass degradation. The review connects the lifestyle and substrate affinities of the zoosporic fungi to the secretome composition by examining both classical phenotypic investigations and molecular/genomic-based studies. The carbohydrate-active enzyme profiles of 19 genome-sequenced species are summarized. Emphasis is given to recent advances in understanding the functional role of rumen fungi, the basis for the devastating chytridiomycosis, and the structure of fungal cellulosome. The approach taken by the review enables comparison of the secretome enzyme composition of anaerobic versus aerobic early-diverging fungi and comparison of enzyme portfolio of specialized parasites, pathogens, and saprotrophs. Early-diverging fungi digest most major types of biopolymers: cellulose, hemicellulose, pectin, chitin, and keratin. It is thus to be expected that early-diverging fungi in its entirety represents a rich and diverse pool of secreted, metabolic enzymes. The review presents the methods used for enzyme discovery, the diversity of enzymes found, the status and outlook for recombinant production, and the potential for applications. Comparative studies on the composition of secretome enzymes of early-diverging fungi would contribute to unraveling the basal lineages of fungi.


Assuntos
Celulossomas/enzimologia , Proteínas Fúngicas/metabolismo , Fungos/classificação , Fungos/enzimologia , Animais , Evolução Biológica , Biopolímeros/metabolismo , Celulossomas/genética , Celulossomas/metabolismo , Proteínas Fúngicas/genética , Fungos/genética , Fungos/metabolismo , Genoma Fúngico/genética , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rúmen/microbiologia
7.
Chem Commun (Camb) ; 55(57): 8219-8222, 2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-31210215

RESUMO

Here we reported a new strategy to construct synthetic metabolons using dCas9-guided assembly. Three orthogonal dCas9 proteins were exploited to guide the independent and site-specific assembly of their fusion partners onto a single DNA scaffold. This new platform was applied towards the construction of a two-component cellulosome. Because of the superior binding affinity, the resulting structures exhibited both improved assembly and reducing sugar production. Conditional enzyme assembly was made possible by utilizing toehold-gated sgRNA (thgRNA), which blocks cellulosome formation until the spacer region is unblocked by a RNA trigger. This platform is highly modular owing to the ease of target synthesis, combinations of possible Cas9-fusion arrangements, and expansion to other metabolic pathways.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , RNA Guia/metabolismo , Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/genética , Celulase/química , Celulase/genética , Celulase/metabolismo , Celulossomas/química , Celulossomas/metabolismo , DNA/química , DNA/metabolismo , Ligação Proteica , Domínios Proteicos , RNA Guia/genética
8.
Nucleic Acids Res ; 47(11): 5988-5997, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31106374

RESUMO

The σ70 family alternative σI factors and their cognate anti-σI factors are widespread in Clostridia and Bacilli and play a role in heat stress response, virulence, and polysaccharide sensing. Multiple σI/anti-σI factors exist in some lignocellulolytic clostridial species, specifically for regulation of components of a multienzyme complex, termed the cellulosome. The σI and anti-σI factors are unique, because the C-terminal domain of σI (SigIC) and the N-terminal inhibitory domain of anti-σI (RsgIN) lack homology to known proteins. Here, we report structure and interaction studies of a pair of σI and anti-σI factors, SigI1 and RsgI1, from the cellulosome-producing bacterium, Clostridium thermocellum. In contrast to other known anti-σ factors that have N-terminal helical structures, RsgIN has a ß-barrel structure. Unlike other anti-σ factors that bind both σ2 and σ4 domains of the σ factors, RsgIN binds SigIC specifically. Structural analysis showed that SigIC contains a positively charged surface region that recognizes the promoter -35 region, and the synergistic interactions among multiple interfacial residues result in the specificity displayed by different σI/anti-σI pairs. We suggest that the σI/anti-σI factors represent a distinctive mode of σ/anti-σ complex formation, which provides the structural basis for understanding the molecular mechanism of the intricate σI/anti-σI system.


Assuntos
Proteínas de Bactérias/metabolismo , Celulossomas/metabolismo , Clostridium thermocellum/genética , Clostridium thermocellum/metabolismo , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Fator sigma/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/química , RNA Polimerases Dirigidas por DNA/química , Espectroscopia de Ressonância Magnética , Mutagênese , Plasmídeos/metabolismo , Conformação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Ressonância de Plasmônio de Superfície
9.
Methods Enzymol ; 617: 241-263, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30784404

RESUMO

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.


Assuntos
Celulossomas/genética , Lactobacillus plantarum/genética , Proteínas de Bactérias/genética , Eletroporação/métodos , Expressão Gênica , Lactobacillus plantarum/crescimento & desenvolvimento , Engenharia Metabólica/métodos , Plasmídeos/genética
10.
Methods Enzymol ; 617: 363-383, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30784409

RESUMO

Proteins are not designed to be standalone entities and must coordinate their collective action for optimum performance. Nature has developed through evolution the ability to colocalize the functional partners of a cascade enzymatic reaction in order to ensure efficient exchange of intermediates. Inspired by these natural designs, synthetic scaffolds have been created to enhance the overall biological pathway performance. In this chapter, we describe several DNA- and protein-based scaffold approaches to assemble artificial enzyme cascades for a wide range of applications. We highlight the key benefits and drawbacks of these approaches to provide insights on how to choose the appropriate scaffold for different cascade systems.


Assuntos
Bactérias/enzimologia , DNA/química , Enzimas Imobilizadas/química , Bactérias/química , Biocatálise , Materiais Biocompatíveis/química , Celulossomas/química , Celulossomas/enzimologia , Modelos Moleculares
11.
Chembiochem ; 20(11): 1394-1399, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-30697892

RESUMO

Polymer-protein core-shell nanoparticles have been explored for enzyme immobilization. This work reports on the development of functional polymeric micelles for immobilizing His6 -tagged cellulases with controlled spatial orientation of enzymes, resulting in "artificial cellulosomes" for effective cellulose hydrolysis. Poly(styrene)-b-poly(styrene-alt-maleic anhydride) was prepared through one-pot reversible addition-fragmentation chain-transfer polymerization and modified with nitrilotriacetic acid (NTA) to afford an amphiphilic block copolymer. The self-assembled polymer was mixed with a solution of NiSO4 to form Ni-NTA-functionalized micelles, which could successfully capture His6 -tagged cellulases and form hierarchically structured core-shell nanoparticles with cellulases as the corona. Because the anchored enzymes are site-specifically oriented and in close proximity, synergistic catalysis that results in over twofold activity enhancement has been achieved.


Assuntos
Celulases/química , Celulossomas/química , Proteínas de Fluorescência Verde/química , Proteínas Luminescentes/química , Nanopartículas/química , Polímeros , Enzimas Imobilizadas , Micelas , Polimerização , Polímeros/síntese química , Polímeros/química
12.
Curr Microbiol ; 76(3): 355-360, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30684027

RESUMO

The secretomes of the strain Cellulosimicrobium cellulans F16 grown on different carbon sources were analyzed by zymography, and the subcellular surface structures were extensively studied by electron microscope. The exo-cellulase and xylanase systems were sparse when cells were grown on soluble oligosaccharides, but were significantly increased when grown on complex and water-insoluble polysaccharides, such as Avicel, corn cob, and birchwood xylan. The cellulosome-like protuberant structures were clearly observed on the cell surfaces of strain F16 grown on cellulose, with diameters of 15-20 nm. Fibrous structures that connected the adjacent cells can be seen under microscope. Moreover, protuberances that adsorbed the cell to cellulose were also observed. As the adhesion of Cellulosimicrobium cellulans cells onto cellulose surfaces occurs via thick bacterial curdlan-type exopolysaccharides (EPS), such surface layer is potentially important in the digestion of insoluble substrates such as cellulose or hemicellulose, and the previously reported xylanosomes are part of such complex glycocalyx layer on the surface of the bacterial cell.


Assuntos
Actinobacteria/enzimologia , Actinobacteria/ultraestrutura , Carbono/metabolismo , Actinobacteria/metabolismo , Aderência Bacteriana , Celulose/metabolismo , Celulossomas/ultraestrutura , Glicocálix/ultraestrutura , Polissacarídeos Bacterianos/metabolismo , Polissacarídeos Bacterianos/ultraestrutura , Xilosidases/metabolismo , beta-Glucanas/metabolismo , beta-Glucosidase/metabolismo
13.
Appl Biochem Biotechnol ; 187(3): 994-1010, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30136170

RESUMO

The cellulosome is a supramolecular multienzyme complex formed via species-specific interactions between the cohesin modules of scaffoldin proteins and the dockerin modules of a wide variety of polysaccharide-degrading enzymes. Here, we report a comparative analysis of cellulosomes prepared from the thermophilic anaerobic bacteria Clostridium (Ruminiclostridium) clariflavum DSM 19732 and Clostridium (Ruminiclostridium) thermocellum ATCC 27405 grown on delignified rice straw. The results indicate that the isolated C. clariflavum cellulosome exhibits lower activity for insoluble cellulosic substrates and higher activity for hemicellulosic substrates, especially for xylan, compared to the isolated C. thermocellum cellulosome. The C. clariflavum cellulosome was separated into large and small complexes by size exclusion chromatography, and the high xylanase activity of the intact complex is mainly attributed to the small complex. Furthermore, both C. clariflavum and C. thermocellum cellulosomes efficiently converted delignified rice straw into soluble sugars with different compositions, whereas a mixture of these cellulosomes exhibited essentially no synergy for the saccharification of delignified rice straw. This is the first study to report that isolated C. clariflavum cellulosomes exhibit greater xylanase activity than isolated C. thermocellum cellulosomes. We also report the effect of a combination of intact cellulosome complexes isolated from different species on the saccharification of plant biomass.


Assuntos
Biomassa , Celulossomas/metabolismo , Clostridium thermocellum/citologia , Oryza/química , Proliferação de Células
14.
Biomol NMR Assign ; 13(1): 97-101, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30377946

RESUMO

Cellulosomes are highly efficient multienzyme complexes for lignocellulose degradation secreted by some lignocellulolytic bacteria. Cellulosomes are assembled through protein modules named cohesin and dockerin, and multiple cohesin modules in the scaffold protein generally determine the complexity of the cellulosomes. Some cellulosomal proteins contain multiple dockerin modules, which may generate more complex cellulosomal architectures. Genome mining revealed that cellulosomal proteins containing double dockerin modules and a protease module exist in many cellulosome-producing bacteria, and these proteins together with cellulosomal protease inhibitors were proposed to have regulatory roles. However, the structures and functions of these multiple-dockerin proteins in cellulosome have not been reported before. In this paper, we present the NMR chemical shift assignments of the double-dockerin of a cellulosomal protease from Clostridium thermocellum DSM1313. The secondary structures predicted from the chemical shifts agree with the structural arrangement of the tandem dockerin modules. The chemical shift assignments here provide the basis for the structural and functional studies of multiple-dockerin proteins in future.


Assuntos
Proteínas de Bactérias/química , Celulossomas/química , Clostridium thermocellum/química , Ressonância Magnética Nuclear Biomolecular , Isótopos de Nitrogênio , Estrutura Secundária de Proteína , Prótons
15.
Proc Natl Acad Sci U S A ; 115(48): E11274-E11283, 2018 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-30429330

RESUMO

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.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Celulossomas/química , Celulossomas/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Clostridium thermocellum/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Celulossomas/genética , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Clostridium thermocellum/química , Clostridium thermocellum/genética , Transferência Ressonante de Energia de Fluorescência , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Ligação Proteica
16.
World J Microbiol Biotechnol ; 34(10): 155, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30276481

RESUMO

Anaerobic fungi (phylum Neocallimastigomycota), an early branching family of fungi, are commonly encountered in the digestive tract of mammalian herbivores. To date, isolates from ten described genera have been reported, and several novel taxonomic groupings are detected using culture-independent molecular methods. Anaerobic fungi are recognized as playing key roles in the decomposition of lignocellulose (up to 50% of the ingested and untreated lignocellulose), with their physical penetration and extracellular enzymatical secretion of an unbiased diverse repertoire of cell-wall-degrading enzymes. The secreted cell-wall-degrading enzymes of anaerobic fungi include both free enzymes and extracellular multi-enzyme complexes called cellulosomes, both of which have potential as fiber degraders in industries. In addition, anaerobic fungi can provide large amounts of substrates such as hydrogen, formate, and acetate for their co-cultured methanogens. Consequently, large amounts of methane can be produced. And thus, it is promising to use the co-culture of anaerobic fungi and methanogens in the biogas process to intensify the biogas yield owing to the efficient and robust degradation of recalcitrant biomass by anaerobic fungi and improved methane production from co-cultures of anaerobic fungi and methanogens.


Assuntos
Biodegradação Ambiental , Biotecnologia , Fermentação , Fungos/metabolismo , Metano/metabolismo , Neocallimastigomycota/metabolismo , Ácido Acético/metabolismo , Anaerobiose/fisiologia , Biocombustíveis , Biomassa , Celulase/genética , Celulase/metabolismo , Celulossomas/enzimologia , Técnicas de Cocultura , Fibra de Algodão , Euryarchaeota/metabolismo , Formiatos/metabolismo , Fungos/classificação , Fungos/enzimologia , Fungos/genética , Hidrogênio/metabolismo , Lignina/metabolismo , Neocallimastigomycota/classificação , Neocallimastigomycota/enzimologia , Neocallimastigomycota/genética , Polissacarídeos/metabolismo , Especificidade por Substrato
17.
Microb Cell Fact ; 17(1): 122, 2018 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-30086751

RESUMO

BACKGROUND: The self-assembly of cellulosomes on the surface of yeast is a promising strategy for consolidated bioprocessing to convert cellulose into ethanol in one step. RESULTS: In this study, we developed a novel synthetic cellulosome that anchors to the endogenous yeast cell wall protein a-agglutinin through disulfide bonds. A synthetic scaffoldin ScafAGA3 was constructed using the repeated N-terminus of Aga1p and displayed on the yeast cell surface. Secreted cellulases were then fused with Aga2p to assemble the cellulosome. The display efficiency of the synthetic scaffoldin and the assembly efficiency of each enzyme were much higher than those of the most frequently constructed cellulosome using scaffoldin ScafCipA3 from Clostridium thermocellum. A complex cellulosome with two scaffoldins was also constructed using interactions between the displayed anchoring scaffoldin ScafAGA3 and scaffoldin I ScafCipA3 through disulfide bonds, and the assembly of secreted cellulases to ScafCipA3. The newly designed cellulosomes enabled yeast to directly ferment cellulose into ethanol. CONCLUSIONS: This is the first report on the development of complex multiple-component assembly system through disulfide bonds. This strategy could facilitate the construction of yeast cell factories to express synergistic enzymes for use in biotechnology.


Assuntos
Celulossomas/metabolismo , Celulases/metabolismo , Celulose/metabolismo , Clostridium thermocellum/efeitos dos fármacos , Clostridium thermocellum/metabolismo , Etanol/farmacologia
18.
Bioresour Technol ; 250: 860-867, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30001594

RESUMO

The production of bioethanol was studied by the cultivation of Clostridium thermocellum ATCC 31924 in MTC medium including crystalline cellulose as the sole substrate. The effects of key operational parameters that affect bioethanol production from microcrystalline cellulose were optimized. Under optimum conditions (pH 8.0, temperature 55 °C, inoculum size 4% (v/v) and 0.5% (w/v) substrate concentration), a maximum ethanol yield of 0.30 g ethanol/g cellulose consumed and 95.32% cellulose conversion was obtained. An inclusion of modest acetate concentration in the medium showed that carbon flux shifted away from lactate accompanied by 20% increase in ethanol production. It suggests that strain ATCC 31924 differed in its cellulose conversion efficacy and optimum pH requirements compared to the other reported strains of Clostridium thermocellum. The purified cellulosome of strain ATCC 31924 found to be rich in both cellulase and xylanase enzymes emphasizing the importance of this strain for the degradation of lignocellulosic biomass.


Assuntos
Clostridium thermocellum/metabolismo , Etanol/metabolismo , Celulase , Celulose , Celulossomas , Fermentação
19.
Methods Mol Biol ; 1796: 67-84, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29856047

RESUMO

Cell wall degradation by cellulases is extensively explored owing to its potential contribution to biofuel production. The cellulosome is an extracellular multienzyme complex that can degrade the plant cell wall very efficiently, and cellulosomal enzymes are therefore of great interest. The cellulosomal cellulases are defined as enzymes that contain a dockerin module, which can interact with a cohesin module contained in multiple copies in a noncatalytic protein, termed scaffoldin. The assembly of the cellulosomal cellulases into the cellulosomal complex occurs via specific protein-protein interactions. Cellulosome systems have been described initially only in several anaerobic cellulolytic bacteria. However, owing to ongoing genome sequencing and metagenomic projects, the discovery of novel cellulosome-producing bacteria and the description of their cellulosomal genes have dramatically increased in the recent years. In this chapter, methods for discovery of novel cellulosomal cellulases from a DNA sequence by bioinformatics and biochemical tools are described. Their biochemical characterization is also described, including both the enzymatic activity of the putative cellulases and their assembly into mature designer cellulosomes.


Assuntos
Bioquímica/métodos , Celulases/metabolismo , Celulossomas/metabolismo , Genômica/métodos , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Ciclo Celular/genética , Celulases/química , Celulose/metabolismo , Proteínas Cromossômicas não Histona/genética , Biologia Computacional , Sequência Conservada , Genoma Bacteriano , Filogenia , Ruminococcus/enzimologia , Ruminococcus/genética
20.
Methods Mol Biol ; 1796: 135-151, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29856052

RESUMO

Cellulose deconstruction is achieved in nature through two main enzymatic paradigms, i.e., free enzymes and enzymatic complexes (called cellulosomes). Gaining insights into the mechanism of action and synergy among the different cellulases is of high interest, notably in the field of renewable energy, and specifically, for the conversion of cellulosic biomass to soluble sugars, en route to biofuels. In this context, designer cellulosomes are artificially assembled, chimaeric protein complexes that are used as a tool to comparatively study cellulose degradation by different enzymatic paradigms, and could also serve to improve cellulose deconstruction. Various molecular biology techniques are employed in order to design and engineer the various components of designer cellulosomes. In this chapter, we describe the cloning processes through which the appropriate modules are selected and assembled at the molecular level.


Assuntos
Celulossomas/enzimologia , Clonagem Molecular/métodos , Sequência de Aminoácidos , Biocatálise , Proteínas de Ciclo Celular/metabolismo , Celulases/química , Celulases/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Ligação Proteica , Proteínas Recombinantes/química
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