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1.
Elife ; 112022 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-35638899

RESUMO

The lives of microbes unfold at the micron scale, and their molecular machineries operate at the nanoscale. Their study at these resolutions is key toward achieving a better understanding of their ecology. We focus on cellulose degradation of the canonical Clostridium thermocellum system to comprehend how microbes build and use their cellulosomal machinery at these nanometer scales. Degradation of cellulose, the most abundant organic polymer on Earth, is instrumental to the global carbon cycle. We reveal that bacterial cells form 'cellulosome capsules' driven by catalytic product-dependent dynamics, which can increase the rate of hydrolysis. Biosynthesis of this energetically costly machinery and cell growth are decoupled at the single-cell level, hinting at a division-of-labor strategy through phenotypic heterogeneity. This novel observation highlights intrapopulation interactions as key to understanding rates of fiber degradation.


Assuntos
Celulossomas , Clostridium thermocellum , Proteínas de Bactérias/metabolismo , Metabolismo dos Carboidratos , Celulose/metabolismo , Celulossomas/metabolismo , Fibras na Dieta/metabolismo , Hidrólise
2.
Int J Biol Macromol ; 207: 784-790, 2022 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-35351552

RESUMO

Polysaccharides derived from lignocellulose are promising sustainable carbon sources. Cellulosome is a supramolecular machine integrating multi-function enzymes for effective lignocellulose bio-saccharification. However, how various non-cellulose components of lignocellulose affect the cellulosomal saccharification is hitherto unclear. This study first investigated the stability and oxygen sensitivity of the cellulosome from Clostridium thermocellum during long-term saccharification process. Then, the differential inhibitory effects of non-cellulose components, including lignin, xylan, and arabinoxylan, on the cellulosome-based saccharification were determined. The results showed that lignin played inhibitory roles by non-productively adsorbing extracellular proteins of C. thermocellum. Differently, arabinoxylan preferred to bind with the cellulosomal components. Almost no adsorption of cellulosomal proteins on solid xylan was detected. Instead, xylan in water-dissolved form interacted with the cellulosomal proteins, especially the key exoglucanase Cel48S, leading to the xylan inhibitory effect. Compared to xylan, xylooligosaccharides influenced the cellulosome activity slightly. Hence, this work demonstrates that the timely hydrolysis or removal of dissolved xylan is important for cellulosome-based lignocellulose saccharification.


Assuntos
Celulossomas , Clostridium thermocellum , Proteínas de Bactérias/metabolismo , Hidrólise , Lignina/metabolismo , Xilanos/metabolismo
3.
J Hazard Mater ; 431: 128596, 2022 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-35248959

RESUMO

Widespread application of synthetic dyes could generate colored wastewaters causing a range of serious environmental problems. Due to the complex nature of effluents from textile industries, it is difficult to obtain satisfactory treatment of dyes-contaminated wastewater using one single method. Biohybrids coupling of photocatalysts and biocatalysts have great potential in environmental purification. However, how to select suitable organisms and enhance the hybrid's catalytic activities remain challenging. Here, a novel biohybrid system (Clostridium thermocellum-CdS), created for light-driven biodecolorization under thermophilic treatment by using non-photosynthetic microorganism C. thermocellum self-photosensitized with CdS nanoparticles was established. The biohybrids exhibited remarkable decolorization effects on triphenylmethane dyes. The highest decolorization rate was 0.206 min-1. More importantly, enhanced catalytic activities of cadmium sulfide (CdS)-based biohybrids by controlling the particle sizes of semiconductors were demonstrated. Biohybrids systems (Clostridium thermocellum-CdS) through the self-precipitation of CdS with different particle sizes not only showed dramatic changes in the optical properties but also exhibited a very different decolorization rate. This work can not only further broaden targeted applications of CdS-based biohybrids but also demonstrate a promising route for improving biohybrids corresponding photocatalytic capabilities through in situ precipitation CdS with different particle sizes.


Assuntos
Compostos de Cádmio , Clostridium thermocellum , Corantes , Sulfetos , Compostos de Tritil , Águas Residuárias
4.
Appl Microbiol Biotechnol ; 106(5-6): 2133-2145, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35157106

RESUMO

An anaerobic thermophilic bacterial strain, A9 (NITE P-03545), that secretes ß-glucosidase was newly isolated from wastewater sediments by screening using esculin. The 16S rRNA gene sequence of strain A9 had 100% identity with that of Thermobrachium celere type strain JW/YL-NZ35. The complete genome sequence of strain A9 showed 98.4% average nucleotide identity with strain JW/YL-NZ35. However, strain A9 had different physiological properties from strain JW/YL-NZ35, which cannot secrete ß-glucosidases or grow on cellobiose as the sole carbon source. The key ß-glucosidase gene (TcBG1) of strain A9, which belongs to glycoside hydrolase family 1, was characterized. Recombinant ß-glucosidase (rTcBG1) hydrolyzed cellooligosaccharides to glucose effectively. Furthermore, rTcBG1 showed high thermostability (at 60°C for 2 days) and high glucose tolerance (IC50 = 0.75 M glucose), suggesting that rTcBG1 could be used for biological cellulose saccharification in cocultures with Clostridium thermocellum. High cellulose degradation was observed when strain A9 was cocultured with C. thermocellum in a medium containing 50 g/l crystalline cellulose, and glucose accumulation in the culture supernatant reached 35.2 g/l. In contrast, neither a monoculture of C. thermocellum nor coculture of C. thermocellum with strain JW/YL-NZ35 realized efficient cellulose degradation or high glucose accumulation. These results show that the ß-glucosidase secreted by strain A9 degrades cellulose effectively in combination with C. thermocellum cellulosomes and has the potential to be used in a new biological cellulose saccharification process that does not require supplementation with ß-glucosidases. KEY POINTS: • Strain A9 can secrete a thermostable ß-glucosidase that has high glucose tolerance • A coculture of strain A9 and C. thermocellum showed high cellulose degradation • Strain A9 achieves biological saccharification without addition of ß-glucosidase.


Assuntos
Clostridium thermocellum , Celulose/metabolismo , Clostridiaceae , Clostridium thermocellum/genética , Clostridium thermocellum/metabolismo , Técnicas de Cocultura , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , beta-Glucosidase/metabolismo
5.
Enzyme Microb Technol ; 156: 110002, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35168167

RESUMO

The composition of cellulosomal carbohydrate-active enzymes (CAZymes) secreted from a cellulolytic bacterium Clostridium thermocellum varies depending on the cellulosic substrate used during cultivation. C. thermocellum detects the polysaccharides in cellulosic material via anti-sigma factors and expresses the appropriate CAZyme gene via alternative sigma factors, SigIs. Previous studies on the regulation of CAZyme gene expression via SigIs in C. thermocellum have been conducted in vitro or in a heterologous host, because of the limited genetic tools available for C. thermocellum. To characterize the in vivo function of SigIs, in the present study, we established a sigI7 gene expression strain of C. thermocellum. Transcriptome analysis of this strain revealed that SigI7 induced the expression of cellulosomal CAZyme genes and cellulosomal scaffold genes. However, there was a decrease in the degradation ability of the exoproteome from the sigI7 expression strain; the product of the downregulated gene, Clo1313_1002, rescued the activity of the C. thermocellum exoproteome from the sigI7 expression strain. In this study, we demonstrate the in vivo function of SigI7 and discuss the CAZymes that are important for cellulosic biomass degradation by C. thermocellum.


Assuntos
Proteínas de Bactérias , Clostridium thermocellum , Fator sigma , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biomassa , Clostridium thermocellum/genética , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Fator sigma/genética , Fator sigma/metabolismo
6.
Proteins ; 90(7): 1457-1467, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35194841

RESUMO

Clostridium thermocellum is actively being developed as a microbial platform to produce biofuels and chemicals from renewable plant biomass. An attractive feature of this bacterium is its ability to efficiently degrade lignocellulose using surface-displayed cellulosomes, large multi-protein complexes that house different types of cellulase enzymes. Clostridium thermocellum tailors the enzyme composition of its cellulosome using nine membrane-embedded anti-σ factors (RsgI1-9), which are thought to sense different types of extracellular carbohydrates and then elicit distinct gene expression programs via cytoplasmic σ factors. Here we show that the RsgI9 anti-σ factor interacts with cellulose via a C-terminal bi-domain unit. A 2.0 Å crystal structure reveals that the unit is constructed from S1C peptidase and NTF2-like protein domains that contain a potential binding site for cellulose. Small-angle X-ray scattering experiments of the intact ectodomain indicate that it adopts a bi-lobed, elongated conformation. In the structure, a conserved RsgI extracellular (CRE) domain is connected to the bi-domain via a proline-rich linker, which is expected to project the carbohydrate-binding unit ~160 Å from the cell surface. The CRE and proline-rich elements are conserved in several other C. thermocellum anti-σ factors, suggesting that they will also form extended structures that sense carbohydrates.


Assuntos
Celulossomas , Clostridium thermocellum , Proteínas de Bactérias/química , Biomassa , Celulose/metabolismo , Celulossomas/química , Clostridium thermocellum/metabolismo , Prolina/metabolismo , Fator sigma/química
7.
Metab Eng ; 69: 286-301, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34982997

RESUMO

Clostridium thermocellum is a promising candidate for consolidated bioprocessing because it can directly ferment cellulose to ethanol. Despite significant efforts, achieved yields and titers fall below industrially relevant targets. This implies that there still exist unknown enzymatic, regulatory, and/or possibly thermodynamic bottlenecks that can throttle back metabolic flow. By (i) elucidating internal metabolic fluxes in wild-type C. thermocellum grown on cellobiose via 13C-metabolic flux analysis (13C-MFA), (ii) parameterizing a core kinetic model, and (iii) subsequently deploying an ensemble-docking workflow for discovering substrate-level regulations, this paper aims to reveal some of these factors and expand our knowledgebase governing C. thermocellum metabolism. Generated 13C labeling data were used with 13C-MFA to generate a wild-type flux distribution for the metabolic network. Notably, flux elucidation through MFA alluded to serine generation via the mercaptopyruvate pathway. Using the elucidated flux distributions in conjunction with batch fermentation process yield data for various mutant strains, we constructed a kinetic model of C. thermocellum core metabolism (i.e. k-ctherm138). Subsequently, we used the parameterized kinetic model to explore the effect of removing substrate-level regulations on ethanol yield and titer. Upon exploring all possible simultaneous (up to four) regulation removals we identified combinations that lead to many-fold model predicted improvement in ethanol titer. In addition, by coupling a systematic method for identifying putative competitive inhibitory mechanisms using K-FIT kinetic parameterization with the ensemble-docking workflow, we flagged 67 putative substrate-level inhibition mechanisms across central carbon metabolism supported by both kinetic formalism and docking analysis.


Assuntos
Clostridium thermocellum , Celobiose/metabolismo , Clostridium thermocellum/genética , Clostridium thermocellum/metabolismo , Etanol/metabolismo , Fermentação , Cinética
8.
Appl Environ Microbiol ; 88(1): e0153121, 2022 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-35015978

RESUMO

Clostridium thermocellum is a thermophilic, anaerobic bacterium that natively ferments cellulose to ethanol and is a candidate for cellulosic biofuel production. Recently, we identified a hypermutator strain of C. thermocellum with a C669Y mutation in the polC gene, which encodes a DNA polymerase III enzyme. Here, we reintroduced this mutation using recently developed CRISPR tools to demonstrate that this mutation is sufficient to recreate the hypermutator phenotype. The resulting strain shows an approximately 30-fold increase in the mutation rate. This mutation is hypothesized to function by interfering with metal ion coordination in the PHP (polymerase and histidinol phosphatase) domain, which is responsible for proofreading. The ability to selectively increase the mutation rate in C. thermocellum is a useful tool for future directed evolution experiments. IMPORTANCE Cellulosic biofuels are a promising approach to decarbonize the heavy-duty-transportation sector. A longstanding barrier to cost-effective cellulosic biofuel production is the recalcitrance of cellulose to solubilization. Native cellulose-consuming organisms, such as Clostridium thermocellum, are promising candidates for cellulosic biofuel production; however, they often need to be genetically modified to improve product formation. One approach is adaptive laboratory evolution. Our findings demonstrate a way to increase the mutation rate in this industrially relevant organism, which can reduce the time needed for adaptive evolution experiments.


Assuntos
Clostridium thermocellum , Composição de Bases , Clostridium thermocellum/genética , DNA Polimerase III , Nucleotídeos , Fenótipo , Filogenia , RNA Ribossômico 16S , Análise de Sequência de DNA
9.
Protein Sci ; 31(2): 498-512, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34865273

RESUMO

The export of antimicrobial peptides is mediated by diverse mechanisms in bacterial quorum sensing pathways. One such binary system employed by gram-positive bacteria is the PCAT1 ABC transporter coupled to a cysteine protease. The focus of this study is the N-terminal C39 peptidase (PEP) domain from Clostridium thermocellum PCAT1 that processes its natural substrate CtA by cleaving a conserved -GG- motif to separate the cargo from the leader peptide prior to secretion. In this study, we are primarily interested in elucidating the dynamic and structural determinants of CtA binding and how it is coupled to cleavage efficiency in the PCAT1 PEP domain. To this end, we have characterized CtA interactions with PEP domain and PCAT1 transporter in detergent micelles using solution nuclear magnetic resonance spectroscopy. The bound CtA structure revealed the disordered C-terminal cargo peptide is linked by a sterically hindered cleavage site to a helix docked within a hydrophobic cavity in the PEP domain. The wide range of internal motions detected by amide nitrogen (N15 ) relaxation measurements in the free enzyme and substrate-bound complex suggests the binding site is relatively floppy. This flexibility plays a key role in the structural rearrangement necessary to relax steric inhibition in the bound substrate. In conjunction with previously reported PCAT1 structures, we offer fresh insight into the ATP-mediated association between PEP and transmembrane domains as a putative mechanism to optimize peptide cleavage by regulating the width and flexibility of the enzyme active site.


Assuntos
Transportadores de Cassetes de Ligação de ATP , Proteínas de Bactérias , Clostridium thermocellum , Domínios Proteicos , Transportadores de Cassetes de Ligação de ATP/química , Proteínas de Bactérias/química , Clostridium thermocellum/química , Peptídeo Hidrolases/química , Sinais Direcionadores de Proteínas
10.
Appl Environ Microbiol ; 88(4): e0185721, 2022 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-34936842

RESUMO

The atypical glycolysis of Clostridium thermocellum is characterized by the use of pyrophosphate (PPi) as a phosphoryl donor for phosphofructokinase (Pfk) and pyruvate phosphate dikinase (Ppdk) reactions. Previously, biosynthetic PPi was calculated to be stoichiometrically insufficient to drive glycolysis. This study investigates the role of a H+-pumping membrane-bound pyrophosphatase, glycogen cycling, a predicted Ppdk-malate shunt cycle, and acetate cycling in generating PPi. Knockout studies and enzyme assays confirmed that clo1313_0823 encodes a membrane-bound pyrophosphatase. Additionally, clo1313_0717-0718 was confirmed to encode ADP-glucose synthase by knockouts, glycogen measurements in C. thermocellum, and heterologous expression in Escherichia coli. Unexpectedly, individually targeted gene deletions of the four putative PPi sources did not have a significant phenotypic effect. Although combinatorial deletion of all four putative PPi sources reduced the growth rate by 22% (0.30 ± 0.01 h-1) and the biomass yield by 38% (0.18 ± 0.00 gbiomass gsubstrate-1), this change was much smaller than what would be expected for stoichiometrically essential PPi-supplying mechanisms. Growth-arrested cells of the quadruple knockout readily fermented cellobiose, indicating that the unknown PPi-supplying mechanisms are independent of biosynthesis. An alternative hypothesis that ATP-dependent Pfk activity circumvents a need for PPi altogether was falsified by enzyme assays, heterologous expression of candidate genes, and whole-genome sequencing. As a secondary outcome, enzymatic assays confirmed functional annotation of clo1313_1832 as ATP- and GTP-dependent fructokinase. These results indicate that the four investigated PPi sources individually and combined play no significant PPi-supplying role, and the true source(s) of PPi, or alternative phosphorylating mechanisms, that drive(s) glycolysis in C. thermocellum remain(s) elusive. IMPORTANCE Increased understanding of the central metabolism of C. thermocellum is important from a fundamental as well as from a sustainability and industrial perspective. In addition to showing that H+-pumping membrane-bound PPase, glycogen cycling, a Ppdk-malate shunt cycle, and acetate cycling are not significant sources of PPi supply, this study adds functional annotation of four genes and availability of an updated PPi stoichiometry from biosynthesis to the scientific domain. Together, this aids future metabolic engineering attempts aimed to improve C. thermocellum as a cell factory for sustainable and efficient production of ethanol from lignocellulosic material through consolidated bioprocessing with minimal pretreatment. Getting closer to elucidating the elusive source of PPi, or alternative phosphorylating mechanisms, for the atypical glycolysis is itself of fundamental importance. Additionally, the findings of this study directly contribute to investigations into trade-offs between thermodynamic driving force versus energy yield of PPi- and ATP-dependent glycolysis.


Assuntos
Clostridium thermocellum , Clostridium thermocellum/metabolismo , Difosfatos/metabolismo , Glucose-1-Fosfato Adenililtransferase/metabolismo , Pirofosfatase Inorgânica/metabolismo , Fosfatos/metabolismo , Piruvato Ortofosfato Diquinase/genética , Piruvato Ortofosfato Diquinase/metabolismo , Ácido Pirúvico/metabolismo
11.
N Biotechnol ; 67: 12-22, 2022 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-34915174

RESUMO

Lactic acid (LA) has several applications in the food, cosmetics and pharmaceutical industries, as well as in the production of biodegradable plastic polymers, namely polylactides. Industrial production of LA is essentially based on microbial fermentation. Recent reports have shown the potential of the cellulolytic bacterium Clostridium thermocellum for direct LA production from inexpensive lignocellulosic biomass. However, C. thermocellum is highly sensitive to acids and does not grow at pH < 6.0. Improvement of LA tolerance of this microorganism is pivotal for its application in cost-efficient production of LA. In the present study, the LA tolerance of C. thermocellum strains LL345 (wild-type fermentation profile) and LL1111 (high LA yield) was increased by adaptive laboratory evolution. At large inoculum size (10 %), the maximum tolerated LA concentration of strain LL1111 was more than doubled, from 15 g/L to 35 g/L, while subcultures evolved from LL345 showed 50-85 % faster growth in medium containing 45 g/L LA. Gene mutations (pyruvate phosphate dikinase, histidine protein kinase/phosphorylase) possibly affecting carbohydrate and/or phosphate metabolism have been detected in most LA-adapted populations. Although improvement of LA tolerance may sometimes also enable higher LA production in microorganisms, C. thermocellum LA-adapted cultures showed a yield of LA, and generally of other organic acids, similar to or lower than parental strains. Based on its improved LA tolerance and LA titer similar to its parent strain (LL1111), mixed adapted culture LL1630 showed the highest performing phenotype and could serve as a framework for improving LA production by further metabolic engineering.


Assuntos
Clostridium thermocellum , Clostridium thermocellum/genética , Clostridium thermocellum/metabolismo , Etanol/metabolismo , Fermentação , Ácido Láctico , Engenharia Metabólica
12.
Enzyme Microb Technol ; 151: 109918, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34649693

RESUMO

As the only glycoside hydrolase family 48 member in Clostridium thermocellum, the exoglucanase Cel48S plays a crucial role in the extremely high activity of the cellulosome against crystalline cellulose. Although the importance of Cel48S in the hydrolysis of crystalline cellulose has been widely accepted, an efficient production system has not yet been established because Cel48S is usually expressed in Escherichia coli within inactive inclusion bodies. For unstable proteins like Cel48S, translocation across the inner membrane can be more advantageous than cytoplasmic production due to the presence of folding modulators in the periplasm and the absence of cytoplasmic proteases. In this study, we evaluated whether the production of Cel48S in the periplasmic space of E. coli could enhance its functional expression. To do so, we attached the PelB signal peptide, which mediates post-translational secretion, to the N-terminal end of Cel48S (P-Cel48S). The PelB signal peptide allowed catalytically active Cel48S to be successfully produced in the culture medium. In addition, we investigated the role of an alternative co-translational pathway on the extracellular production of Cel48S, finding that co-translational secretion yielded a specific activity of recombinant Cel48S of 135.1 ± 10.0 U/mg cell in the culture medium, which was 2.2 times higher than that associated with P-Cel48S expression. Therefore, we believe that our approach has potential applications for the cost-effective conversion of lignocellulosic biomass and the industrial production of other unstable proteins.


Assuntos
Celulase , Clostridium thermocellum , Celulase/genética , Celulase/metabolismo , Clostridium thermocellum/genética , Clostridium thermocellum/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Periplasma/metabolismo , Partícula de Reconhecimento de Sinal
13.
Biomater Sci ; 9(22): 7444-7455, 2021 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-34647546

RESUMO

The development of protein and microorganism engineering have led to rising expectations of biotechnology in the design of emerging biomaterials, putatively of high interest to reduce our dependence on fossil carbon resources. In this way, cellulose, a renewable carbon based polysaccharide and derived products, displays unique properties used in many industrial applications. Although the functionalization of cellulose is common, it is however limited in terms of number and type of functions. In this work, a Carbohydrate-Binding Module (CBM) was used as a central core to provide a versatile strategy to bring a large diversity of functions to cellulose surfaces. CBM3a from Clostridium thermocellum, which has a high affinity for crystalline cellulose, was flanked through linkers with a streptavidin domain and an azide group introduced through a non-canonical amino acid. Each of these two extra domains was effectively produced and functionalized with a variety of biological and chemical molecules. Structural properties of the resulting tripartite chimeric protein were investigated using molecular modelling approaches, and its potential for the multi-functionalization of cellulose was confirmed experimentally. As a proof of concept, we show that cellulose can be labelled with a fluorescent version of the tripartite protein grafted to magnetic beads and captured using a magnet.


Assuntos
Clostridium thermocellum , Nanopartículas , Sítios de Ligação , Celulose , Polissacarídeos
14.
Enzyme Microb Technol ; 150: 109887, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34489040

RESUMO

Lignocellulose is a prominent source of carbohydrates to be used in biorefineries. One of the main challenges associated with its use is the low yields obtained during enzymatic hydrolysis, as well as the high cost associate with enzyme acquisition. Despite the great attention in using the fraction composed by hexoses, nowadays, there is a growing interest in enzymatic blends to deconstruct the pentose-rich fraction. Among the organisms studied as a source of enzymes to lignocellulose deconstruction, the anaerobic bacterium Clostridium thermocellum stands out. Most of the remarkable performance of C. thermocellum in degrading cellulose is related to its capacity to assemble enzymes into well-organized enzymatic complexes, cellulosomes. A mini-version of a cellulosome was designed in the present study, using the xylanase XynA and the N-terminus portion of scaffolding protein, mCipA, harboring one CBM3 and two cohesin I domains. The formed mini-xylanosome displayed maximum activity between 60 and 70 °C in a pH range from 6 to 8. Although biochemical properties of complexed/non-complexed enzymes were similar, the formed xylanosome displayed higher hydrolysis at 60 and 70 °C for alkali-treated sugarcane bagasse. Lignocellulose deconstruction using fungal secretome and the mini-xylanosome resulted in higher d-glucose yield, and the addition of the mCipA scaffolding protein enhanced cellulose deconstruction when coupled with fungal enzymes. Results obtained in this study demonstrated that the assembling of xylanases into mini-xylanosomes could improve sugarcane deconstruction, and the mCipA protein can work as a cellulose degradation enhancer.


Assuntos
Celulossomas , Clostridium thermocellum , Composição de Bases , Clostridium thermocellum/genética , Lignina , Filogenia , RNA Ribossômico 16S , Análise de Sequência de DNA
15.
Bioresour Technol ; 341: 125837, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34461408

RESUMO

In present study, the water-soluble extractives removal prior to alkali pretreatment of sugarcane tops (SCT) was carried out. The solid alkali pretreated SCT (apSCT) recovered on Field-emission scanning electron microscopy (FE-SEM) analysis showed exposure of cellulosic fibres as compared with raw SCT. The analyses of apSCT by Fourier Transform Infrared (FT-IR) Spectroscopy, X-ray diffraction (XRD) and High performance liquid chromatography (HPLC) analysis also confirmed the enhanced cellulose content in apSCT. Optimum conditions for response surface methodology based saccharification of apSCT at 40 °C, 150 rpm were 2.14% (w/v) apSCT loading in citrate-phosphate buffer (50 mM, pH 6.0), recombinant hydrolytic enzymes (from Clostridium/Hungateiclostridium thermocellum) loading for endo-1,4-ß-glucanase (CtCel8A) = 213.2 U/g, cellobiohydrolase (CtCBH5A) = 272.5 U/g and ß-glucosidase (HtBg1) = 299.8 U/g for 49.2 h. Under optimized saccharification conditions, the total reducing sugar yield was 265 mg/g (glucose 214 mg/g) of apSCT. Fermentation of produced glucose by S. cerevisiae gave 0.19 g/g glucose of bioethanol.


Assuntos
Clostridium thermocellum , Saccharum , Fermentação , Hidrólise , Saccharomyces cerevisiae , Espectroscopia de Infravermelho com Transformada de Fourier
16.
Chemistry ; 27(63): 15688-15698, 2021 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-34436794

RESUMO

ß-1→4-Glucan polysaccharides like cellulose, derivatives and analogues, are attracting attention due to their unique physicochemical properties, as ideal candidates for many different applications in biotechnology. Access to these polysaccharides with a high level of purity at scale is still challenging, and eco-friendly alternatives by using enzymes in vitro are highly desirable. One prominent candidate enzyme is cellodextrin phosphorylase (CDP) from Ruminiclostridium thermocellum, which is able to yield cellulose oligomers from short cellodextrins and α-d-glucose 1-phosphate (Glc-1-P) as substrates. Remarkably, its broad specificity towards donors and acceptors allows the generation of highly diverse cellulose-based structures to produce novel materials. However, to fully exploit this CDP broad specificity, a detailed understanding of the molecular recognition of substrates by this enzyme in solution is needed. Herein, we provide a detailed investigation of the molecular recognition of ligands by CDP in solution by saturation transfer difference (STD) NMR spectroscopy, tr-NOESY and protein-ligand docking. Our results, discussed in the context of previous reaction kinetics data in the literature, allow a better understanding of the structural basis of the broad binding specificity of this biotechnologically relevant enzyme.


Assuntos
Clostridium thermocellum , Glucosiltransferases , Espectroscopia de Ressonância Magnética , Polissacarídeos
17.
Bioresour Technol ; 337: 125441, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34182347

RESUMO

Consolidated bio-saccharification (CBS) technology employs cellulosome-producing bacterial cells, rather than fungal cellulases, as biocatalysts for cost-effective production of lignocellulosic sugars. Extracellular ß-glucosidase (BGL) expression in the whole-cell arsenal is indispensable, due to severe cellobiose inhibition of the cellulosome. However, high-level BGL expression in Clostridium thermocellum is challenging, and the optimal BGL production level for efficient cellulose saccharification is currently unknown. Herein, we obtained new CBS biocatalysts by transforming BGL-expressing plasmids into C. thermocellum, which produced abundant BGL proteins and hydrolyzed cellulose effectively. The optimal ratio of extracellular BGL-to-cellulosome activity was determined to be in a range of 5.5 to 21.6. Despite the critical impact of BGL, both excessive BGL expression and its assembly on the cellulosome via type I cohesin-dockerin interaction led to reduced cellulosomal activity, which further confirmed the importance of coordinated BGL expression with the cellulosome. This study will further promote industrial CBS application in lignocellulose conversion.


Assuntos
Celulossomas , Clostridium thermocellum , Proteínas de Bactérias , Lignina , beta-Glucosidase
18.
Biomol NMR Assign ; 15(2): 329-334, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-33876380

RESUMO

The cellulosome is a highly efficient cellulolytic complex containing cellulolytic enzymes and non-catalytic subunits, i.e. scaffoldins, which are assembled by the interactions between the dockerin modules of the enzymes and the cohesin modules of the primary scaffoldins. The cellulosome attaches to the cell surface via the S-layer homology (SLH) modules of the anchoring scaffoldins. Clostridium thermocellum DSM1313 is a thermophilic cellulosome-producing bacterium with great potential in lignocellulose bioconversion and biofuel production. The bacterium contains four anchoring scaffoldins ScaB, ScaC, ScaD and ScaF, among which ScaF is the only one that contains an additional module of unknown function (ScaF-X) between the cohesin and SLH modules. The gene of ScaF is located outside the scaffoldin gene cluster of scaA, scaB, scaC and scaD. Previous studies showed unique regulation properties and function of ScaF compared to other anchoring scaffoldins, which might be related to the additional ScaF-X module. Here we report the NMR chemical shift assignments of ScaF-X from C. thermocellum DSM1313. The well-dispersed NMR spectrum and the secondary structure prediction based on the chemical shifts of ScaF-X indicated that ScaF-X is a well-folded protein module. The chemical shift assignments provide the basis for future studies on the structure of this module and its function in cellulosomes.


Assuntos
Clostridium thermocellum
19.
Acta Crystallogr F Struct Biol Commun ; 77(Pt 4): 95-104, 2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33830074

RESUMO

A novel member of the family 3 carbohydrate-binding modules (CBM3s) is encoded by a gene (Cthe_0271) in Clostridium thermocellum which is the most highly expressed gene in the bacterium during its growth on several types of biomass substrates. Surprisingly, CtCBM3-0271 binds to at least two different types of xylan, instead of the common binding of CBM3s to cellulosic substrates. CtCBM3-0271 was crystallized and its three-dimensional structure was solved and refined to a resolution of 1.8 Å. In order to learn more about the role of this type of CBM3, a comparative study with its orthologue from Clostridium clariflavum (encoded by the Clocl_1192 gene) was performed, and the three-dimensional structure of CcCBM3-1192 was determined to 1.6 Šresolution. Carbohydrate binding by CcCBM3-1192 was found to be similar to that by CtCBM3-0271; both exhibited binding to xylan rather than to cellulose. Comparative structural analysis of the two CBM3s provided a clear functional correlation of structure and binding, in which the two CBM3s lack the required number of binding residues in their cellulose-binding strips and thus lack cellulose-binding capabilities. This is an enigma, as CtCBM3-0271 was reported to be a highly expressed protein when the bacterium was grown on cellulose. An additional unexpected finding was that CcCBM3-1192 does not contain the calcium ion that was considered to play a structural stabilizing role in the CBM3 family. Despite the lack of calcium, the five residues that form the calcium-binding site are conserved. The absence of calcium results in conformational changes in two loops of the CcCBM3-1192 structure. In this context, superposition of the non-calcium-binding CcCBM3-1192 with CtCBM3-0271 and other calcium-binding CBM3s reveals a much broader two-loop region in the former compared with CtCBM3-0271.


Assuntos
Clostridiales/metabolismo , Clostridium thermocellum/metabolismo , Proteínas de Membrana/metabolismo , Polissacarídeos/metabolismo , Sequência de Aminoácidos , Clostridiales/química , Clostridiales/genética , Clostridium thermocellum/química , Clostridium thermocellum/genética , Cristalização , Proteínas de Membrana/química , Proteínas de Membrana/genética , Polissacarídeos/química , Polissacarídeos/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
20.
Sci Rep ; 11(1): 7880, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33846482

RESUMO

Since the pandemic outbreak of Covid-19 in December 2019, several lateral flow assay (LFA) devices were developed to enable the constant monitoring of regional and global infection processes. Additionally, innumerable lateral flow test devices are frequently used for determination of different clinical parameters, food safety, and environmental factors. Since common LFAs rely on non-biodegradable nitrocellulose membranes, we focused on their replacement by cellulose-composed, biodegradable papers. We report the development of cellulose paper-based lateral flow immunoassays using a carbohydrate-binding module-fused to detection antibodies. Studies regarding the protein binding capacity and potential protein wash-off effects on cellulose paper demonstrated a 2.7-fold protein binding capacity of CBM-fused antibody fragments compared to the sole antibody fragment. Furthermore, this strategy improved the spatial retention of CBM-fused detection antibodies to the test area, which resulted in an enhanced sensitivity and improved overall LFA-performance compared to the naked detection antibody. CBM-assisted antibodies were validated by implementation into two model lateral flow test devices (pregnancy detection and the detection of SARS-CoV-2 specific antibodies). The CBM-assisted pregnancy LFA demonstrated sensitive detection of human gonadotropin (hCG) in synthetic urine and the CBM-assisted Covid-19 antibody LFA was able to detect SARS-CoV-2 specific antibodies present in serum. Our findings pave the way to the more frequent use of cellulose-based papers instead of nitrocellulose in LFA devices and thus potentially improve the sustainability in the field of POC diagnostics.


Assuntos
Anticorpos Antivirais/análise , Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , Carboidratos/química , Colódio/química , Imunoensaio/métodos , Materiais Biocompatíveis , Gonadotropina Coriônica/química , Clostridium thermocellum/imunologia , Humanos , Fragmentos de Imunoglobulinas/química , Imunoglobulina G/química , Sistemas Automatizados de Assistência Junto ao Leito , Ligação Proteica , SARS-CoV-2/imunologia , Urinálise
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