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1.
J Biotechnol ; 353: 9-18, 2022 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-35659892

RESUMO

Acetogenic bacteria produce acetate following the fixation of CO2 via the Wood-Ljungdahl pathway. As such, they represent excellent process organisms for the production of novel chemicals and fuels from this waste greenhouse gas. Acetobacterium woodii is the model acetogen and numerous studies have been conducted investigating its biochemistry, gas consumption and use as a production chassis. However, there are a dearth of available tools for A. woodii gene modification which limits the research options available for genetic studies. Here, the previously proposed Clostridia Roadmap is implemented in A. woodii leading to the derivation of a knockout system for the generation of clean, in-frame deletions. The replicon of the Gram-positive plasmid pCD6 that originated in Clostridioides difficile was identified as being replication-defective in A. woodii, a property that was exploited to construct a pseudo-suicide knockout plasmid which was used to generate an auxotrophic, pyrE mutant. This allowed the subsequent use of a heterologous pyrE gene (from Clostridium acetobutylicum) as a counter selection marker and the deletion of a number of genes by allelic exchange. Specific mutants generated were affected in growth on glucose, fructose and ethanol as a consequence of deletion of fruA, pstG and adhE, respectively.


Assuntos
Acetobacterium , Clostridium acetobutylicum , Acetatos/metabolismo , Acetobacterium/genética , Acetobacterium/metabolismo , Dióxido de Carbono/metabolismo , Clostridium acetobutylicum/metabolismo , Deleção de Genes , Humanos
2.
Microb Cell Fact ; 21(1): 130, 2022 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-35761287

RESUMO

BACKGROUND: Lignocellulosic biomass is recognized as an effective potential substrate for biobutanol production. Though many pretreatment and detoxification methods have been set up, the fermentability of detoxicated lignocellulosic substrate is still far lower than that of starchy feedstocks. On the other hand, the number of recent efforts on rational metabolic engineering approaches to increase butanol production in Clostridium strains is also quite limited, demonstrating the physiological complexity of solventogenic clostridia. In fact, the strain performance is greatly impacted by process control. developing efficient process control strategies could be a feasible solution to this problem. RESULTS: In this study, oxidoreduction potential (ORP) controlling was applied to increase the fermentability of enzymatically hydrolyzed steam-exploded corn stover (SECS) for butanol production. When ORP of detoxicated SECS was controlled at - 350 mV, the period of fermentation was shortened by 6 h with an increase of 27.5% in the total solvent (to 18.1 g/L) and 34.2% in butanol (to 10.2 g/L) respectively. Silico modeling revealed that the fluxes of NADPH, NADH and ATP strongly differed between the different scenarios. Quantitative analysis showed that intracellular concentrations of ATP, NADPH/NADP+, and NADH/NAD+ were increased by 25.1%, 81.8%, and 62.5%. ORP controlling also resulted in a 2.1-fold increase in butyraldehyde dehydrogenase, a 1.2-fold increase in butanol dehydrogenase and 29% increase in the cell integrity. CONCLUSION: ORP control strategy effectively changed the intracellular metabolic spectrum and significantly improved Clostridium cell growth and butanol production. The working mechanism can be summarized into three aspects: First, Glycolysis and TCA circulation pathways were strengthened through key nodes such as pyruvate carboxylase [EC: 6.4.1.1], which provided sufficient NADH and NADPH for the cell. Second, sufficient ATP was provided to avoid "acid crash". Third, the key enzymes activities regulating butanol biosynthesis and cell membrane integrity were improved.


Assuntos
Butanóis , Clostridium acetobutylicum , 1-Butanol/metabolismo , Trifosfato de Adenosina/metabolismo , Butanóis/metabolismo , Clostridium/metabolismo , Clostridium acetobutylicum/metabolismo , Fermentação , NAD/metabolismo , NADP/metabolismo , Vapor , Zea mays/metabolismo
3.
mBio ; 13(3): e0076922, 2022 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-35638736

RESUMO

Anoxic microsites arising in fungal biofilms may foster the presence of obligate anaerobes. Here, we analyzed whether and to which degree hyphae of Coprinopsis cinerea thriving in oxic habitats enable the germination, growth, and dispersal of the obligate anaerobic soil bacterium Clostridium acetobutylicum. Time-resolved optical oxygen mapping, microscopy, and metabolite analysis revealed the formation and persistence of anoxic circum hyphal niches, allowing for spore germination, growth, and fermentative activity of the obligate anaerobe in an otherwise inhabitable environment. Hypoxic liquid films containing 80% ± 10% of atmospheric oxygen saturation around single air-exposed hyphae thereby allowed for efficient clostridial dispersal amid spatially separated (>0.5 cm) anoxic sites. Hyphae hence may serve as good networks for the activity and spatial organization of obligate anaerobic bacteria in oxygenated heterogeneous environments such as soil. IMPORTANCE Although a few studies have reported on the presence of anoxic microniches in fungal biofilms, knowledge of the effects of fungal oxygen consumption on bacterial-fungal interactions is limited. Here, we demonstrate the existence and persistence of oxygen-free zones in air-exposed mycelia enabling spore germination, growth, fermentative activity, and dispersal of the obligate anaerobe. Our study points out a previously overlooked role of aerobic fungi in creating and bridging anoxic microniches in ambient oxic habitats. Air-exposed hyphae hence may act as a scaffold for activity and dispersal of strictly anaerobic microbes. Given the short-term tolerance of strict anaerobes to oxygen and reduced oxygen content in the mycosphere, hyphae can promote spatial organization of both obligate anaerobic and aerobic bacteria. Such finding may be important for a better understanding of previously observed co-occurrences of aerobes and anaerobes in well-aerated habitats such as upland soils.


Assuntos
Bactérias Anaeróbias , Clostridium acetobutylicum , Ecossistema , Hifas , Solo
4.
Microbiol Spectr ; 10(2): e0228821, 2022 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-35412381

RESUMO

Transcription initiation is a tightly regulated process that is crucial for many aspects of prokaryotic physiology. High-throughput transcription start site (TSS) mapping can shed light on global and local regulation of transcription initiation, which in turn may help us understand and predict microbial behavior. In this study, we used Capp-Switch sequencing to determine the TSS positions in the genomes of three model solventogenic clostridia: Clostridium acetobutylicum ATCC 824, C. beijerinckii DSM 6423, and C. beijerinckii NCIMB 8052. We first refined the approach by implementing a normalization pipeline accounting for gene expression, yielding a total of 12,114 mapped TSSs across the species. We further compared the distributions of these sites in the three strains. Results indicated similar distribution patterns at the genome scale, but also some sharp differences, such as for the butyryl-CoA synthesis operon, particularly when comparing C. acetobutylicum to the C. beijerinckii strains. Lastly, we found that promoter structure is generally poorly conserved between C. acetobutylicum and C. beijerinckii. A few conserved promoters across species are discussed, showing interesting examples of how TSS determination and comparison can improve our understanding of gene expression regulation at the transcript level. IMPORTANCE Solventogenic clostridia have been employed in industry for more than a century, initially being used in the acetone-butanol-ethanol (ABE) fermentation process for acetone and butanol production. Interest in these bacteria has recently increased in the context of green chemistry and sustainable development. However, our current understanding of their genomes and physiology limits their optimal use as industrial solvent production platforms. The gene regulatory mechanisms of solventogenesis are still only partly understood, impeding efforts to increase rates and yields. Genome-wide mapping of transcription start sites (TSSs) for three model solventogenic Clostridium strains is an important step toward understanding mechanisms of gene regulation in these industrially important bacteria.


Assuntos
Acetona , Clostridium acetobutylicum , Acetona/metabolismo , Bactérias Anaeróbias , Butanóis/metabolismo , Clostridium/genética , Clostridium/metabolismo , Clostridium acetobutylicum/genética , Clostridium acetobutylicum/metabolismo , Fermentação
5.
Bioresour Technol ; 353: 127078, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35395367

RESUMO

The present study aimed to enzymatic deinking of waste papers and to valorize the effluent for biobutanol production. Application of fungal enzymatic cocktail (cellulase, amylase, xylanase, pectinase, lipase, and ligninase) on office used paper, newspaper, and ballpen written paper leading to improvement in brightness (84.91, 72.51, 76.69 % ISO), InKd (82.89, 68.95, 76.49%), κ-number (12.9, 13.6, and 13.1), opacity (27.91, 30.07, and 2.85%), tensile strength (49.24, 45.31, and 46.98 Nm/g), respectively and indices were consistent with chemical treated pulps. The quality of effluent generated during enzymatic deinking in respect to BOD and COD level was eco-friendlier than the chemical process. The enzyme-treated effluent was employed as supporting substrate for butanol (18.4 g/l) production by Clostridium acetobutylicum ATCC824. Material balance and life cycle assessment of the whole processes were evaluated to validate its industrial and environmental relevance.


Assuntos
Celulase , Clostridium acetobutylicum , 1-Butanol , Butanóis , Tinta , Papel
6.
Int J Biol Macromol ; 207: 324-332, 2022 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-35259435

RESUMO

The recently developed technologies for immobilization of cellulase may address the challenges in costly hydrolysis of cellulose for cellulosic butanol production. In this study, a "hybrid" hydrolysis was developed based on chemical hydrolysis of cellulose to its oligomers followed by enzymatic post-hydrolysis of the resulting "soluble oligomers" by cellulase immobilized on chitosan-coated Fe3O4 nanoparticles. This hybrid hydrolysis stage was utilized in the process of biobutanol production from a waste textile, jeans waste, leading to selective formation of glucose and high yield of butanol production by Clostridium acetobutylicum. After validating the immobilization process, the optimum immobilization parameters including enzyme concentration and time were achieved on 8 h and 15.0 mg/mL, respectively. The reusability of immobilized enzyme showed that immobilized cellulase could retain 51.5% of its initial activity after three times reuses. Dilute acid hydrolysis of regenerated cellulose at 120-180 °C for 60 min 0.5-1.0% phosphoric acid led to less than 10 g/L glucose production, and enzymatic post-hydrolysis of the oligomers resulted in up to 51.5 g/L glucose. Fermentation of the hydrolysate was accompanied by 5.3 g/L acetone-butanol-ethanol (ABE) production. The simultaneous co-saccharification and fermentation (SCSF) of soluble and insoluble oligomers of cellulose led to 17.4 g/L ABE production.


Assuntos
Celulase , Quitosana , Clostridium acetobutylicum , Nanopartículas de Magnetita , 1-Butanol , Acetona , Butanóis , Celulase/metabolismo , Celulose/metabolismo , Clostridium acetobutylicum/metabolismo , Etanol , Fermentação , Glucose , Hidrólise , Têxteis
7.
Biotechnol J ; 17(5): e2100515, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35077002

RESUMO

The capability of four genetically modified Acetobacterium woodii strains for improved production of acetone from CO2 and hydrogen was tested. The acetone biosynthesis pathway was constructed by combining genes from Clostridium acetobutylicum and Clostridium aceticum. Expression of acetone production genes was demonstrated in all strains. In bioreactors with continuous gas supply, all produced acetic acid, acetone, and, surprisingly, isopropanol. The production of isopropanol was caused by an endogenous secondary alcohol dehydrogenase (SADH) activity at low gas-feeding rate. Although high amounts of the natural end product acetic acid of A. woodii were formed,14.5 mM isopropanol and 7.6 mM acetone were also detected, showing that this is a promising approach for the production of new solvents from C1 gases. The highest acetic acid, acetone, and isopropanol production was detected in the recombinant A. woodii [pJIR750_ac1t1] strain, with final concentrations of 438 mM acetic acid, 7.6 mM acetone, and 14.5 mM isopropanol. The engineered strain A. woodii [pJIR750_ac1t1] was found to be the most promising strain for acetone production from a gas mixture of CO2 and H2 and the formation of isopropanol in A. woodii was shown for the first time.


Assuntos
Dióxido de Carbono , Clostridium acetobutylicum , 2-Propanol , Ácido Acético , Acetobacterium , Acetona , Dióxido de Carbono/metabolismo , Clostridium acetobutylicum/metabolismo , Hidrogênio/metabolismo
8.
ACS Synth Biol ; 11(2): 953-967, 2022 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-35081709

RESUMO

Anaerobic bacteria are promising biocatalysts to produce industrially relevant products from nonfood feedstocks. Several anaerobes are genetically accessible, and various molecular tools for metabolic engineering are available. Still, the use of bright fluorescent reporters, which are commonly used in molecular biological approaches is limited under anaerobic conditions. Therefore, the establishment of different anaerobic fluorescent reporter proteins is of great interest. Here, we present the establishment of the green- and red-fluorescent reporter proteins greenFAST and redFAST for use in different solventogenic and acetogenic bacteria. Green fluorescence of greenFAST was bright in Clostridium saccharoperbutylacetonicum, Clostridium acetobutylicum, Acetobacterium woodii, and Eubacterium limosum, while only C. saccharoperbutylacetonicum showed bright red fluorescence when producing redFAST. We used both reporter proteins in C. saccharoperbutylacetonicum for multicolor approaches. These include the investigation of the co-culture dynamics of metabolically engineered strains. Moreover, we established a tightly regulated inducible two-plasmid system and used greenFAST and redFAST to track the coexistence and interaction of both plasmids under anaerobic conditions in C. saccharoperbutylacetonicum. The establishment of greenFAST and redFAST as fluorescent reporters opens the door for further multicolor approaches to investigate cell dynamics, gene expression, or protein localization under anaerobic conditions.


Assuntos
Clostridium acetobutylicum , Bactérias Anaeróbias/genética , Clostridium acetobutylicum/genética , Engenharia Metabólica , Plasmídeos
9.
Bioresour Technol ; 346: 126405, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34826562

RESUMO

Microbial reduction of black strap molasses (BSM) by Clostridium acetobutylicum MTCC 11,274 was performed for the production of biobutanol. The optimum fermentation conditions were predicted using one factor at a time (OFAT) method. The identification of significant parameters was performed using Plackett-Burman Design (PBD). Furthermore the fermentation conditions were optimized using central composite design (CCD). The kinetics of substrate utilization and product formation were investigated. Initial pH, yeast extract concentration (g/L) and total reducing sugar concentration (g/L) were found as significant parameters affecting butanol production using C. acetobutylicum MTCC11274. The maximum butanol production under optimal condition was 10.27 + 0.82 g/L after 24 h. The waste black strap molasses obtained from sugar industry could be used as promising substrate for the production of next generation biofuel.


Assuntos
Clostridium acetobutylicum , Anaerobiose , Butanóis , Fermentação , Cinética , Melaço
10.
Biotechnol Adv ; 58: 107889, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-34929313

RESUMO

Solventogenic clostridia are not a strictly defined group within the genus Clostridium but its representatives share some common features, i.e. they are anaerobic, non-pathogenic, non-toxinogenic and endospore forming bacteria. Their main metabolite is typically 1-butanol but depending on species and culture conditions, they can form other metabolites such as acetone, isopropanol, ethanol, butyric, lactic and acetic acids, and hydrogen. Although these organisms were previously used for the industrial production of solvents, they later fell into disuse, being replaced by more efficient chemical production. A return to a more biological production of solvents therefore requires a thorough understanding of clostridial metabolism. Transcriptome analysis, which reflects the involvement of individual genes in all cellular processes within a population, at any given (sampling) moment, is a valuable tool for gaining a deeper insight into clostridial life. In this review, we describe techniques to study transcription, summarize the evolution of these techniques and compare methods for data processing and visualization of solventogenic clostridia, particularly the species Clostridium acetobutylicum and Clostridium beijerinckii. Individual approaches for evaluating transcriptomic data are compared and their contributions to advancements in the field are assessed. Moreover, utilization of transcriptomic data for reconstruction of computational clostridial metabolic models is considered and particular models are described. Transcriptional changes in glucose transport, central carbon metabolism, the sporulation cycle, butanol and butyrate stress responses, the influence of lignocellulose-derived inhibitors on growth and solvent production, and other respective topics, are addressed and common trends are highlighted.


Assuntos
Clostridium acetobutylicum , Clostridium beijerinckii , Butanóis/metabolismo , Clostridium/metabolismo , Clostridium acetobutylicum/genética , Clostridium acetobutylicum/metabolismo , Clostridium beijerinckii/genética , Clostridium beijerinckii/metabolismo , Fermentação , Solventes , Transcriptoma/genética
11.
Biotechnol Bioeng ; 119(1): 226-235, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34687217

RESUMO

Solventogenesis and sporulation of clostridia are the main responsive adaptations to the acidic environment during acetone-butanol-ethanol (ABE) fermentation. It was hypothesized that five orphan histidine kinases (HKs) including Cac3319, Cac0323, Cac0903, Cac2730, and Cac0437 determined the cell fates between sporulation and solventogenesis. In this study, the comparative genomic analysis revealed that a mutation in cac0437 appeared to contribute to the nonsporulating feature of ATCC 55025. Hence, the individual and interactive roles of five HKs in regulating cell growth, metabolism, and sporulation were investigated. The fermentation results of mutants with different HK expression levels suggested that cac3319 and cac0437 played critical roles in regulating sporulation and acids and butanol biosynthesis. Morphological analysis revealed that cac3319 knockout abolished sporulation (Stage 0) whereas cac3319 overexpression promoted spore development (Stage VII), and cac0437 knockout initiated but blocked sporulation before Stage II, indicating the progression of sporulation was altered through engineering HKs. By combinatorial HKs knockout, the interactive effects between two different HKs were investigated. This study elucidated the regulatory roles of HKs in clostridial differentiation and demonstrated that HK engineering can be effectively used to control sporulation and enhance butanol biosynthesis.


Assuntos
Proteínas de Bactérias , Butanóis/metabolismo , Clostridium acetobutylicum , Histidina Quinase , Esporos Bacterianos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clostridium acetobutylicum/enzimologia , Clostridium acetobutylicum/genética , Clostridium acetobutylicum/metabolismo , Clostridium acetobutylicum/fisiologia , Fermentação , Histidina Quinase/genética , Histidina Quinase/metabolismo , Engenharia Metabólica
12.
Bioresour Technol ; 343: 126093, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34624476

RESUMO

A multistep approach was undertaken for biobutanol production targeting valorization of agricultural waste. Optimum production of lignocellulolytic enzymes [CMCase (3822.93U/mg), FPase (3640.93U/mg), ß-glucosidase (3873.92U/mg), xylanase (3460.24U/mg), pectinase (3359.57U/mg), α-amylase (4136.54U/mg), and laccase (3863.16U/mg)] was accomplished through solid-substrate fermentation of pretreated mixed substrates (wheat bran, sugarcane bagasse and orange peel) by Aspergillus niger SKN1 and Trametes hirsuta SKH1. Partially purified enzyme cocktail was employed for saccharification of the said substrate mixture into fermentable sugar (69.23 g/L, product yield of 24% w/w). The recovered sugar with vegetable extract supplements was found as robust fermentable medium that supported 16.51 g/L biobutanol production by Clostridium acetobutylicum ATCC824. The sequential bioprocessing of low-priced substrates and exploitation of vegetable extract as growth factor for microbial butanol production will open a new vista in biofuel research.


Assuntos
Clostridium acetobutylicum , Biomassa , Butanóis , Fermentação , Hidrólise , Lignina , Polyporaceae , Trametes
13.
Biotechnol Bioeng ; 118(12): 4699-4707, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34491579

RESUMO

Glycolate is a bulk chemical with wide applications in the textile, food processing, and pharmaceutical industries. Glycolate can be produced from glucose via the glycolysis and glyoxylate shunt pathways, followed by reduction to glycolate. However, two problems limit the productivity and yield of glycolate when using glucose as the sole carbon source. The first is a cofactor imbalance in the production of glycolate from glucose via the glycolysis pathway, since NADPH is required for glycolate production, while glycolysis generates NADH. To rectify this imbalance, the NADP+ -dependent glyceraldehyde 3-phosphate dehydrogenase GapC from Clostridium acetobutylicum was introduced to generate NADPH instead of NADH in the oxidation of glyceraldehyde 3-phosphate during glycolysis. The soluble transhydrogenase SthA was further eliminated to conserve NADPH by blocking its conversion into NADH. The second problem is an unfavorable carbon flux distribution between the tricarboxylic acid cycle and the glyoxylate shunt. To solve this problem, isocitrate dehydrogenase (ICDH) was eliminated to increase the carbon flux of glyoxylate and thereby improve the glycolate titer. After engineering through the integration of gapC, combined with the inactivation of ICDH, SthA, and by-product pathways, as well as the upregulation of the two key enzymes isocitrate lyase (encoding by aceA), and glyoxylate reductase (encoding by ycdW), the glycolate titer increased to 5.3 g/L with a yield of 1.89 mol/mol glucose. Moreover, an optimized fed-batch fermentation reached a titer of 41 g/L with a yield of 1.87 mol/mol glucose after 60 h.


Assuntos
Escherichia coli , Glicolatos , Engenharia Metabólica/métodos , Proteínas de Bactérias/genética , Clostridium acetobutylicum/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Glucose/metabolismo , Glicolatos/análise , Glicolatos/metabolismo , Redes e Vias Metabólicas/genética
14.
J Microbiol Biotechnol ; 31(10): 1393-1400, 2021 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-34584034

RESUMO

Acetone-butanol-ethanol (ABE) fermentation by the anaerobic bacterium Clostridium acetobutylicum has been considered a promising process of industrial biofuel production. Phosphotransbutyrylase (phosphate butyryltransferase, PTB) plays a crucial role in butyrate metabolism by catalyzing the reversible conversion of butyryl-CoA into butyryl phosphate. Here, we report the crystal structure of PTB from the Clostridial host for ABE fermentation, C. acetobutylicum, (CaPTB) at a 2.9 Å resolution. The overall structure of the CaPTB monomer is quite similar to those of other acyltransferases, with some regional structural differences. The monomeric structure of CaPTB consists of two distinct domains, the N- and C-terminal domains. The active site cleft was formed at the interface between the two domains. Interestingly, the crystal structure of CaPTB contained eight molecules per asymmetric unit, forming an octamer, and the size-exclusion chromatography experiment also suggested that the enzyme exists as an octamer in solution. The structural analysis of CaPTB identifies the substrate binding mode of the enzyme and comparisons with other acyltransferase structures lead us to speculate that the enzyme undergoes a conformational change upon binding of its substrate.


Assuntos
Proteínas de Bactérias/química , Clostridium acetobutylicum/enzimologia , Fosfato Acetiltransferase/química , Acetona/metabolismo , Acil Coenzima A , Sequência de Aminoácidos , Butanóis/metabolismo , Domínio Catalítico , Etanol/metabolismo , Fermentação , Estrutura Quaternária de Proteína
15.
Nucleic Acids Res ; 49(19): e113, 2021 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-34417598

RESUMO

DNA methylation is widespread amongst eukaryotes and prokaryotes to modulate gene expression and confer viral resistance. 5-Methylcytosine (m5C) methylation has been described in genomes of a large fraction of bacterial species as part of restriction-modification systems, each composed of a methyltransferase and cognate restriction enzyme. Methylases are site-specific and target sequences vary across organisms. High-throughput methods, such as bisulfite-sequencing can identify m5C at base resolution but require specialized library preparations and single molecule, real-time (SMRT) sequencing usually misses m5C. Here, we present a new method called RIMS-seq (rapid identification of methylase specificity) to simultaneously sequence bacterial genomes and determine m5C methylase specificities using a simple experimental protocol that closely resembles the DNA-seq protocol for Illumina. Importantly, the resulting sequencing quality is identical to DNA-seq, enabling RIMS-seq to substitute standard sequencing of bacterial genomes. Applied to bacteria and synthetic mixed communities, RIMS-seq reveals new methylase specificities, supporting routine study of m5C methylation while sequencing new genomes.


Assuntos
5-Metilcitosina/metabolismo , Metilases de Modificação do DNA/metabolismo , Enzimas de Restrição do DNA/metabolismo , Escherichia coli K12/genética , Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Acinetobacter calcoaceticus/enzimologia , Acinetobacter calcoaceticus/genética , Aeromonas hydrophila/enzimologia , Aeromonas hydrophila/genética , Bacillus amyloliquefaciens/enzimologia , Bacillus amyloliquefaciens/genética , Sequência de Bases , Clostridium acetobutylicum/enzimologia , Clostridium acetobutylicum/genética , Metilação de DNA , Metilases de Modificação do DNA/genética , Enzimas de Restrição do DNA/genética , Escherichia coli K12/enzimologia , Regulação Bacteriana da Expressão Gênica , Haemophilus/enzimologia , Haemophilus/genética , Haemophilus influenzae/enzimologia , Haemophilus influenzae/genética , Humanos , Microbiota/genética , Análise de Sequência de DNA , Pele/microbiologia
16.
Dalton Trans ; 50(30): 10405-10422, 2021 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-34240096

RESUMO

The organometallic H-cluster of the [FeFe]-hydrogenase consists of a [4Fe-4S] cubane bridged via a cysteinyl thiolate to a 2Fe subcluster ([2Fe]H) containing CO, CN-, and dithiomethylamine (DTMA) ligands. The H-cluster is synthesized by three dedicated maturation proteins: the radical SAM enzymes HydE and HydG synthesize the non-protein ligands, while the GTPase HydF serves as a scaffold for assembly of [2Fe]H prior to its delivery to the [FeFe]-hydrogenase containing the [4Fe-4S] cubane. HydG uses l-tyrosine as a substrate, cleaving it to produce p-cresol as well as the CO and CN- ligands to the H-cluster, although there is some question as to whether these are formed as free diatomics or as part of a [Fe(CO)2(CN)] synthon. Here we show that Clostridium acetobutylicum (C.a.) HydG catalyzes formation of multiple equivalents of free CO at rates comparable to those for CN- formation. Free CN- is also formed in excess molar equivalents over protein. A g = 8.9 EPR signal is observed for C.a. HydG reconstituted to load the 5th "dangler" iron of the auxiliary [4Fe-4S][FeCys] cluster and is assigned to this "dangler-loaded" cluster state. Free CO and CN- formation and the degree of activation of [FeFe]-hydrogenase all occur regardless of dangler loading, but are increased 10-35% in the dangler-loaded HydG; this indicates the dangler iron is not essential to this process but may affect relevant catalysis. During HydG turnover in the presence of myoglobin, the g = 8.9 signal remains unchanged, indicating that a [Fe(CO)2(CN)(Cys)] synthon is not formed at the dangler iron. Mutation of the only protein ligand to the dangler iron, H272, to alanine nearly completely abolishes both free CO formation and hydrogenase activation, however results show this is not due solely to the loss of the dangler iron. In experiments with wild type and H272A HydG, and with different degrees of dangler loading, we observe a consistent correlation between free CO/CN- formation and hydrogenase activation. Taken in full, our results point to free CO/CN-, but not an [Fe(CO)2(CN)(Cys)] synthon, as essential species in hydrogenase maturation.


Assuntos
Hidrogenase , Clostridium acetobutylicum , Proteínas Ferro-Enxofre
17.
Environ Microbiol ; 23(8): 4112-4125, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34245087

RESUMO

Clostridia comprise bacteria of environmental, biotechnological and medical interest and many commensals of the gut microbiota. Because of their strictly anaerobic lifestyle, oxygen is a major stress for Clostridia. However, recent data showed that these bacteria can cope with O2 better than expected for obligate anaerobes through their ability to scavenge, detoxify and consume O2 . Upon O2 exposure, Clostridia redirect their central metabolism onto pathways less O2 -sensitive and induce the expression of genes encoding enzymes involved in O2 -reduction and in the repair of oxidized damaged molecules. While Faecalibacterium prausnitzii efficiently consumes O2 through a specific extracellular electron shuttling system requiring riboflavin, enzymes such as rubrerythrins and flavodiiron proteins with NAD(P)H-dependent O2 - and/or H2 O2 -reductase activities are usually encoded in other Clostridia. These two classes of enzymes play indeed a pivotal role in O2 tolerance in Clostridioides difficile and Clostridium acetobutylicum. Two main signalling pathways triggering O2 -induced responses have been described so far in Clostridia. PerR acts as a key regulator of the O2 - and/or reactive oxygen species-defence machinery while in C. difficile, σB , the sigma factor of the general stress response also plays a crucial role in O2 tolerance by controlling the expression of genes involved in O2 scavenging and repair systems.


Assuntos
Clostridioides difficile , Clostridium acetobutylicum , Clostridium/genética , Oxigênio , Fator sigma
18.
Microb Cell Fact ; 20(1): 149, 2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34325704

RESUMO

BACKGROUND: The intracellular ATP level is an indicator of cellular energy state and plays a critical role in regulating cellular metabolism. Depletion of intracellular ATP in (facultative) aerobes can enhance glycolysis, thereby promoting end product formation. In the present study, we examined this s trategy in anaerobic ABE (acetone-butanol-ethanol) fermentation using Clostridium acetobutylicum DSM 1731. RESULTS: Following overexpression of atpAGD encoding the subunits of water-soluble, ATP-hydrolyzing F1-ATPase, the intracellular ATP level of 1731(pITF1) was significantly reduced compared to control 1731(pIMP1) over the entire batch fermentation. The glucose uptake was markedly enhanced, achieving a 78.8% increase of volumetric glucose utilization rate during the first 18 h. In addition, an early onset of acid re-assimilation and solventogenesis in concomitant with the decreased intracellular ATP level was evident. Consequently, the total solvent production was significantly improved with remarkable increases in yield (14.5%), titer (9.9%) and productivity (5.3%). Further genome-scale metabolic modeling revealed that many metabolic fluxes in 1731(pITF1) were significantly elevated compared to 1731(pIMP1) in acidogenic phase, including those from glycolysis, tricarboxylic cycle, and pyruvate metabolism; this indicates significant metabolic changes in response to intracellular ATP depletion. CONCLUSIONS: In C. acetobutylicum DSM 1731, depletion of intracellular ATP significantly increased glycolytic rate, enhanced solvent production, and resulted in a wide range of metabolic changes. Our findings provide a novel strategy for engineering solvent-producing C. acetobutylicum, and many other anaerobic microbial cell factories.


Assuntos
Trifosfato de Adenosina/metabolismo , Clostridium acetobutylicum/metabolismo , Fermentação , Glicólise , Solventes/metabolismo , Acetona/metabolismo , Anaerobiose , Biocombustíveis , Butanóis/metabolismo , Clostridium acetobutylicum/genética , Etanol/metabolismo , Hidrólise
19.
Mol Microbiol ; 116(2): 648-662, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34028100

RESUMO

Group I biotin protein ligases (BPLs) catalyze the covalent attachment of biotin to its cognate acceptor proteins. In contrast, Group II BPLs have an additional N-terminal DNA-binding domain and function not only in biotinylation but also in transcriptional regulation of genes of biotin biosynthesis and transport. Most bacteria contain only a single biotin protein ligase, whereas Clostridium acetobutylicum contains two biotin protein ligase homologs: BplA and BirA'. Sequence alignments showed that BplA is a typical group I BPL, whereas BirA' lacked the C-terminal domain conserved throughout extant BPL proteins. This raised the questions of why two BPL homologs are needed and why the apparently defective BirA' has been retained. We have used in vivo and in vitro assays to show that BplA is a functional BPL whereas BirA' acts as a biotin sensor involved in transcriptional regulation of biotin transport. We also successfully converted BirA' into a functional biotin protein ligase with regulatory activity by fusing it to the C-terminal domain from BplA. Finally, we provide evidence that BplA and BirA' interact in vivo.


Assuntos
Biotina/metabolismo , Biotinilação/fisiologia , Carbono-Nitrogênio Ligases/metabolismo , Clostridium acetobutylicum/metabolismo , Transcrição Genética/genética , Biotina/biossíntese , Carbono-Nitrogênio Ligases/genética , Clostridium acetobutylicum/genética , Regulação Bacteriana da Expressão Gênica/genética , Domínios e Motivos de Interação entre Proteínas/fisiologia
20.
Biotechnol Bioeng ; 118(7): 2770-2780, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33871069

RESUMO

Microorganisms harbor bulks of functionally similar or undefined genes, which belong to paralogous gene family. There is a necessity of exploring combinatorial or interactive functions of these genes, but conventional loss-of-function strategy with one-by-one rounds suffers extremely low efficiency for generating mutant libraries with all gene permutations. Here, taking histidine kinases (HKs) in Clostridium acetobutylicum as a proof-of-concept, we developed a multi-plasmid cotransformation strategy for generating all theoretical HKs combinations in one round. For five HKs with 31 theoretical combinations, the library containing 22 mutants within all the possible HKs-inactivated combinations was constructed with 11 days compared to 242 days by conventional strategy, while the other 9 combinations cannot survive. Six mutants with the enhanced butanol production and tolerance were obtained with changes of cell development during fermentation, one of which could produce 54.2% more butanol (56.4% more solvents), while the butanol production of other mutants was unchanged or decreased. The cotransformation strategy demonstrated potentials for fast exploring pleiotropic function of paralogous family genes in cell survival, cell development, and target product metabolism.


Assuntos
Butanóis/metabolismo , Clostridium acetobutylicum , Histidina Quinase , Engenharia Metabólica , Mutação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clostridium acetobutylicum/genética , Clostridium acetobutylicum/metabolismo , Histidina Quinase/genética , Histidina Quinase/metabolismo
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