RESUMO
The anaerobe Clostridium acetobutylicum belongs to the most important industrially used bacteria. Whereas genome mining points to a high potential for secondary metabolism in C. acetobutylicum, the functions of most biosynthetic gene clusters are cryptic. We report that the addition of supra-physiological concentrations of cysteine triggered the formation of a novel natural product, clostrisulfone (1). Its structure was fully elucidated by NMR, MS and the chemical synthesis of a reference compound. Clostrisulfone is the first reported natural product with a diphenylsulfone scaffold. A biomimetic synthesis suggests that pentamethylchromanol-derived radicals capture sulfur dioxide to form 1. In a cell-based assay using murine macrophages a biphasic and dose-dependent regulation of the LPS-induced release of nitric oxide was observed in the presence of 1.
Assuntos
Clostridium acetobutylicum , Fatores Imunológicos , Sulfonas , Animais , Clostridium acetobutylicum/química , Clostridium acetobutylicum/efeitos dos fármacos , Clostridium acetobutylicum/genética , Clostridium acetobutylicum/metabolismo , Cisteína/farmacologia , Fatores Imunológicos/metabolismo , Fatores Imunológicos/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , Família Multigênica , Sulfonas/metabolismo , Sulfonas/farmacologiaRESUMO
Butanol fermentation comprises two successive and distinct stages, namely acidogenesis and solventogenesis. The current lack of clarity regarding the underlying metabolic regulation of fermentation impedes improvements in biobutanol production. Here, a proteomics study was performed in the acidogenesis phase, the lowest pH point (transition point), and the solventogenesis phase in the butanol-producing symbiotic system TSH06. Forty-two Clostridium acetobutylicum proteins demonstrated differential expression levels at different stages. The protein level of butanol dehydrogenase increased in the solventogenesis phase, which was in accordance with the trend of butanol concentration. Stress proteins were upregulated either at the transition point or in the solventogenesis phase. The cell division-related protein Maf was upregulated at the transition point. We disrupted the maf gene in C. acetobutylicum TSH1, and Bacillus cereus TSH2 was added to form a new symbiotic system. TSH06â³maf produced 13.9 ± 1.0 g/L butanol, which was higher than that of TSH06 (12.3 ± 0.9 g/L). Butanol was furtherly improved in fermentation at variable temperature with neutral red addition for both TSH06 and TSH06â³maf. The butanol titer of the maf deletion strain was higher than that of the wild type, although the exact mechanism remains to be determined.
Assuntos
Bacillus cereus/metabolismo , Butanóis/metabolismo , Clostridium acetobutylicum/metabolismo , Engenharia Metabólica , Oxirredutases do Álcool/metabolismo , Proteínas de Bactérias/metabolismo , Butanóis/análise , Clostridium acetobutylicum/efeitos dos fármacos , Técnicas de Cocultura , Fermentação , Concentração de Íons de Hidrogênio , Proteômica , SimbioseRESUMO
In recent years, increasing concerns over environment, energy and climate have renewed interest in biotechnological production of butanol. However, growth inhibition by fermentation products and inhibitory components from raw biomass has hindered the development of acetone-butanol-ethanol (ABE) fermentation. Improving the cellular robustness of Clostridium acetobutylicum is of great importance for efficient ABE production. In this study, we attempted to improve the robustness and butanol titers of C. acetobutylicum ATCC824 by overexpressing GroESL and DnaK from the extremely radioresistant bacterium Deinococcus wulumuqiensis R12 and from C. acetobutylicum ATCC824 itself. Three recombinant strains were obtained and designated 824(dnaK R12), 824(groESL R12) and 824(groESL824). These three recombinants were found to have significantly improved tolerances to stresses including butanol, furfural, oxidation and acid. Meanwhile, the butanol titers increased to 13.0g/L, 11.2g/L and 10.7g/L, which were 49.4%, 28.7% and 23.0% higher than that from the wild-type strain (8.7g/L), respectively. For 824(dnaK R12), the production of acetic and butyric acids decreased by 97.1% (1.4g/L vs. 0.04g/L) and 100% (0.3g/L vs. 0g/L), respectively, compared with the wild-type strain. Overexpressing GroESL and DnaK from D. wulumuqiensis R12 also resulted in better growth and ABE production than the wild-type strain on fermentation in the presence of 2.5g/L furfural. Strain 824(groESL R12) was superior to 824(groESL 824) in diverse types of stress-tolerance and butanol titer, indicating that GroESL from the extremophilic bacterium could perform its function more efficiently in the heterologous host than native GroESL. Our study provides evidence that extremophilic bacteria can be excellent resources for engineering C. acetobutylicum to improve its robustness and butanol titer.
Assuntos
Proteínas de Bactérias/genética , Clostridium acetobutylicum/genética , Proteínas de Choque Térmico/genética , 1-Butanol/farmacologia , Chaperoninas/genética , Clostridium acetobutylicum/efeitos dos fármacos , Clostridium acetobutylicum/crescimento & desenvolvimento , Escherichia coli/genética , Furaldeído/farmacologia , Peróxido de Hidrogênio/farmacologia , Concentração de Íons de Hidrogênio , Oxidantes/farmacologiaRESUMO
During the fermentation process, Clostridium acetobutylicum cells are often inhibited by the accumulated butanol. However, the mechanism underlying response of C. acetobutylicum to butanol stress remains poorly understood. This study was performed to clarify such mechanism through investigating the butanol stress-associated intracellular biochemical changes at acidogenesis phase (i.e., middle exponential phase) and solventogenesis phase (i.e., early stationary phase) by a gas chromatography-mass spectrometry-based metabolomics strategy. With the aid of partial least-squares-discriminant analysis, a pairwise discrimination between control group and butanol-treated groups was revealed, and 27 metabolites with variable importance in the projection value greater than 1 were identified. Under butanol stress, the glycolysis might be inhibited while TCA cycle might be promoted. Moreover, changes of lipids and fatty acids compositions, amino acid metabolism and osmoregulator concentrations might be the key factors involved in C. acetobutylicum metabolic response to butanol stress. It was suggested that C. acetobutylicum cells might change the levels of long acyl chain saturated fatty acids and branched-chain amino acids to maintain the integrity of cell membrane through adjusting membrane fluidity under butanol stress. The increased level of glycerol was considered to be correlated with osmoregulation and regulating redox balance. In addition, increased levels of some amino acids (i.e., threonine, glycine, alanine, phenylalanine, tyrosine, tryptophan, aspartate and glutamate) might also confer butanol tolerance to C. acetobutylicum. These results highlighted our knowledge about the response or adaptation of C. acetobutylicum to butanol stress, and would contribute to the construction of feasible butanologenic strains with higher butanol tolerance.
Assuntos
Biocombustíveis/microbiologia , Butanóis/metabolismo , Butanóis/farmacologia , Clostridium acetobutylicum/citologia , Clostridium acetobutylicum/metabolismo , Fermentação , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Clostridium acetobutylicum/efeitos dos fármacos , Clostridium acetobutylicum/fisiologia , Relação Dose-Resposta a Droga , Fermentação/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacosRESUMO
While extensively studied in several model organisms, the role of small, non-coding RNAs in the stress response remains largely unexplored in Clostridium organisms. About 100 years after the first industrial Acetone-Butanol-Ethanol fermentation process, based on the Weizmann Clostridium acetobutylicum strain, strain tolerance to butanol remains a crucial factor limiting the economics of the process. Several studies have examined the response of this organism to metabolite stress, and several genes have been engaged to impart enhanced tolerance, but no sRNAs have yet been directly engaged in this task. We show that the two stress-responsive sRNAs, 6S and tmRNA, upon overexpression impart tolerance to butanol as assessed by viability assays under process-relevant conditions. 6S overexpression enhances cell densities as well as butanol titres. We discuss the likely mechanisms that these two sRNAs might engage in this tolerance phenotype. Our data support the continued exploration of sRNAs as a basis for engineering enhanced tolerance and enhanced solvent production, especially because sRNA-based strategies impose a minimal metabolic burden on the cells.
Assuntos
Butanóis/metabolismo , Clostridium acetobutylicum/metabolismo , Tolerância a Medicamentos/genética , RNA Bacteriano/biossíntese , Pequeno RNA não Traduzido/biossíntese , RNA não Traduzido/biossíntese , Acetona/metabolismo , Clostridium acetobutylicum/efeitos dos fármacos , Clostridium acetobutylicum/genética , Etanol/metabolismo , Fermentação/fisiologia , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , RNA não Traduzido/genéticaRESUMO
Biobutanol outperforms bioethanol as an advanced biofuel, but is not economically competitive in terms of its titer, yield and productivity associated with feedstocks and energy cost. In this work, the synergistic effect of calcium and zinc was investigated in the acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum using glucose, xylose and glucose/xylose mixtures as carbon source(s). Significant improvements associated with enhanced glucose/xylose utilization, cell growth, acids re-assimilation and butanol biosynthesis were achieved. Especially, the maximum butanol and ABE production of 16.1 and 25.9 g L(-1) were achieved from 69.3 g L(-1) glucose with butanol/ABE productivities of 0.40 and 0.65 g L(-1) h(-1) compared to those of 11.7 and 19.4 g/L with 0.18 and 0.30 g L(-1) h(-1) obtained in the control respectively without any supplement. More importantly, zinc was significantly involved in the butanol tolerance based on the improved xylose utilization under various butanol-shock conditions (2, 4, 6, 8 and 10 g L(-1) butanol). Under the same conditions, calcium and zinc co-supplementation led to the best xylose utilization and butanol production. These results suggested that calcium and zinc could play synergistic roles improving ABE fermentation by C. acetobutylicum.
Assuntos
Butanóis/metabolismo , Cálcio/farmacologia , Clostridium acetobutylicum/metabolismo , Dissacarídeos/metabolismo , Zinco/farmacologia , Acetona/metabolismo , Biocombustíveis , Butanóis/farmacologia , Clostridium acetobutylicum/efeitos dos fármacos , Farmacorresistência Bacteriana , Sinergismo Farmacológico , Etanol/metabolismo , Fermentação/efeitos dos fármacosRESUMO
Paper mill sludge (PS), a solid waste from pulp and paper industry, was investigated as a feedstock for acetone-butanol-ethanol (ABE) production by simultaneous saccharification and fermentation (SSF). ABE fermentation of paper sludge by Clostridium acetobutylicum required partial removal of ash in PS to enhance its enzymatic digestibility. Enzymatic hydrolysis was found to be a rate-limiting step in the SSF. A total of 16.4-18.0g/L of ABE solvents were produced in the SSF of de-ashed PS with solid loading of 6.3-7.4% and enzyme loading of 10-15FPU/g-glucan, and the final solvent yield reached 0.27g/g sugars. No pretreatment and pH control were needed in ABE fermentation of paper sludge, which makes it an attractive feedstock for butanol production. The results suggested utilization of paper sludge should not only consider the benefits of buffering effect of CaCO3 in fermentation, but also take into account its inhibitory effect on enzymatic hydrolysis.
Assuntos
Acetona/metabolismo , Butanóis/metabolismo , Metabolismo dos Carboidratos , Etanol/metabolismo , Fermentação , Papel , Esgotos/química , Álcalis/farmacologia , Metabolismo dos Carboidratos/efeitos dos fármacos , Celulose/metabolismo , Clostridium acetobutylicum/efeitos dos fármacos , Clostridium acetobutylicum/metabolismo , Fermentação/efeitos dos fármacos , Glucose/metabolismo , Hidrólise , Panicum/efeitos dos fármacos , Xilose/metabolismoRESUMO
Lignocellulosic biomass and dedicated energy crops such as Jerusalem artichoke are promising alternatives for biobutanol production by solventogenic clostridia. However, fermentable sugars such as fructose or xylose released from the hydrolysis of these feedstocks were subjected to the incomplete utilization by the strains, leading to relatively low butanol production and productivity. When 0.001 g/L ZnSO4·7H2O was supplemented into the medium containing fructose as sole carbon source, 12.8 g/L of butanol was achieved with butanol productivity of 0.089 g/L/h compared to only 4.5 g/L of butanol produced with butanol productivity of 0.028 g/L/h in the control without zinc supplementation. Micronutrient zinc also led to the improved butanol production up to 8.3 g/L derived from 45.2 g/L xylose as sole carbon source with increasing butanol productivity by 31.7%. Moreover, the decreased acids production was observed under the zinc supplementation condition, resulting in the increased butanol yields of 0.202 g/g-fructose and 0.184 g/g-xylose, respectively. Similar improvements were also observed with increasing butanol production by 130.2 % and 8.5 %, butanol productivity by 203.4% and 18.4%, respectively, in acetone-butanol-ethanol fermentations from sugar mixtures of fructose/glucose (4:1) and xylose/glucose (1:2) simulating the hydrolysates of Jerusalem artichoke tubers and corn stover. The results obtained from transcriptional analysis revealed that zinc may have regulatory mechanisms for the sugar transport and metabolism of Clostridium acetobutylicum L7. Therefore, micronutrient zinc supplementation could be an effective way for economic development of butanol production derived from these low-cost agricultural feedstocks.
Assuntos
1-Butanol/metabolismo , Acetona/metabolismo , Etanol/metabolismo , Fermentação , Frutose/metabolismo , Xilose/metabolismo , Zinco/metabolismo , Biomassa , Metabolismo dos Carboidratos/efeitos dos fármacos , Clostridium acetobutylicum/efeitos dos fármacos , Clostridium acetobutylicum/metabolismo , Fermentação/efeitos dos fármacos , Glucose/metabolismo , Helianthus/química , Helianthus/metabolismo , Zea mays/química , Zea mays/metabolismo , Zinco/farmacologiaRESUMO
The micronutrient zinc plays vital roles in ABE fermentation by Clostridium acetobutylicum. In order to elucidate the zinc-associated response for enhanced glucose utilization and earlier solventogenesis, transcriptional analysis was performed on cells grown in glucose medium at the exponential growth phase of 16 h without/with supplementary zinc. Correspondingly, the gene glcG (CAC0570) encoding a glucose-specific PTS was significantly upregulated accompanied with the other two genes CAC1353 and CAC1354 for glucose transport in the presence of zinc. Additionally, genes involved in the metabolisms of six other carbohydrates (maltose, cellobiose, fructose, mannose, xylose and arabinose) were differentially expressed, indicating that the regulatory effect of micronutrient zinc is carbohydrate-specific with respects to the improved/inhibited carbohydrate utilization. More importantly, multiple genes responsible for glycolysis (glcK and pykA), acidogenesis (thlA, crt, etfA, etfB and bcd) and solventogenesis (ctfB and bdhA) of C. acetobutylicum prominently responded to the supplementary zinc at differential expression levels. Comparative analysis of intracellular metabolites revealed that the branch node intermediates such as acetyl-CoA, acetoacetyl-CoA, butyl-CoA, and reducing power NADH remained relatively lower whereas more ATP was generated due to enhanced glycolysis pathway and earlier initiation of solventogenesis, suggesting that the micronutrient zinc-associated response for the selected intracellular metabolisms is significantly pleiotropic.
Assuntos
Butanóis/metabolismo , Clostridium acetobutylicum/efeitos dos fármacos , Perfilação da Expressão Gênica/métodos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Zinco/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocombustíveis , Cromatografia Líquida , Clostridium acetobutylicum/genética , Clostridium acetobutylicum/metabolismo , Dissacarídeos/metabolismo , Fermentação/efeitos dos fármacos , Fermentação/genética , Glucose/metabolismo , Glicólise/genética , Monossacarídeos/metabolismo , Polissacarídeos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Solventes/metabolismo , Espectrometria de Massas em TandemRESUMO
This study aimed to quantitatively evaluate the inhibitor tolerance of butanol-producing bacterium Clostridium acetobutylicum. The inhibitory effect of the inhibitors generated by acid pretreatment of biomass feedstock on butanol fermentation decreased in the order of formic acid>oxalic acid>furfural>5-HMF>Na2SO4. C. acetobutylicum has a small tolerance range for furfural (1.06-2.6g/L) and 5-HMF (1.99-2.3g/L). However, the inhibitory effect of Na2SO4 appears to have a wide range, with a chronic toxicity for C. acetobutylicum. All the results could explain, in quantitative manner, the instability of butanol fermentation with C. acetobutylicum from various acid-pretreated feedstocks caused by the fermentation inhibitors.
Assuntos
Butanóis/metabolismo , Clostridium acetobutylicum/metabolismo , Microbiologia Industrial/métodos , Biomassa , Clostridium acetobutylicum/efeitos dos fármacos , Clostridium acetobutylicum/crescimento & desenvolvimento , Relação Dose-Resposta a Droga , Fermentação , Formiatos/metabolismo , Formiatos/farmacologia , Furaldeído/metabolismo , Furaldeído/farmacologia , Hidrogênio/metabolismo , Hidrólise , Ácido Oxálico/metabolismo , Ácido Oxálico/farmacologia , Sulfatos/farmacologiaRESUMO
This study conducted batch experiments to evaluate the potential of butanol production from microalgae biodiesel residues by Clostridium acetobutylicum. The results indicated that with 90 g/L of glucose as the sole substrate the highest butanol yield of 0.2 g/g-glucose was found, but the addition of butyrate significantly enhanced the butanol yield. The highest butanol yield of 0.4 g/g-glucose was found with 60 g/L of glucose and 18 g/L of butyrate. Using microalgae biodiesel residues as substrate, C. acetobutylicum produced 3.86 g/L of butanol and achieved butanol yield of 0.13 g/g-carbohydrate via ABE fermentation, but the results indicated that approximately one third of carbohydrate was not utilized by C. acetobutylicum. Biological butanol production from microalgae biodiesel residues can be possible, but further research on fermentation strategies are required to improve production yield.
Assuntos
Biocombustíveis , Butanóis/metabolismo , Clostridium acetobutylicum/metabolismo , Microalgas/metabolismo , Butiratos/farmacologia , Clostridium acetobutylicum/efeitos dos fármacos , Fermentação/efeitos dos fármacos , Glucose/farmacologia , Hidrólise , Microalgas/efeitos dos fármacos , ResíduosRESUMO
To obtain native strains resistant to butanol toxicity, a new isolating method and serial enrichment was used in this study. With this effort, mutant strain SE36 was obtained, which could withstand 35g/L (compared to 20g/L of the wild-type strain) butanol challenge. Based on 16s rDNA comparison, the mutant strain was identified as Clostridium acetobutylicum. Under the optimized condition, the phase shift was smoothly triggered and fermentation performances were consequently enhanced. The maximum total solvent and butanol concentration were 23.6% and 24.3%, respectively higher than that of the wild-type strain. Furthermore, the correlation between butanol produced and the butanol tolerance was investigated, suggesting that enhancing butanol tolerance could improve butanol production. These results indicate that the simple but effective isolation method and acclimatization process are a promising technique for isolation and improvement of butanol tolerance and production.
Assuntos
Acetona/metabolismo , Butanóis/metabolismo , Clostridium acetobutylicum/isolamento & purificação , Etanol/metabolismo , Manihot , Butanóis/toxicidade , Clostridium acetobutylicum/efeitos dos fármacos , Clostridium acetobutylicum/metabolismo , Compostos de Manganês , Sulfatos , Zea maysRESUMO
BACKGROUND: Clostridium acetobutylicum fermentations are promising for production of commodity chemicals from heterogeneous biomass due to the wide range of substrates the organism can metabolize. Much work has been done to elucidate the pathways for utilization of aldoses, but little is known about metabolism of more oxidized substrates. Two oxidized hexose derivatives, gluconate and galacturonate, are present in low cost feedstocks, and their metabolism will contribute to overall metabolic output of these substrates. RESULTS: A complete metabolic network for glucose, gluconate, and galacturonate utilization was generated using online databases, previous studies, genomic context, and experimental data. Gluconate appears to be metabolized via the Entner-Doudoroff pathway, and is likely dehydrated to 2-keto-3-deoxy-gluconate before phosphorylation to 2-keto-3-deoxy-6-P-gluconate. Galacturonate appears to be processed via the Ashwell pathway, converging on a common metabolite for gluconate and galacturonate metabolism, 2-keto-3-deoxygluconate. As expected, increasingly oxidized substrates resulted in increasingly oxidized products with galacturonate fermentations being nearly homoacetic. Calculations of expected ATP and reducing equivalent yields and experimental data suggested galacturonate fermentations were reductant limited. Galacturonate fermentation was incomplete, which was not due solely to product inhibition or the inability to utilize low concentrations of galacturonate. Removal of H2 and CO2 by agitation resulted in faster growth, higher cell densities, formation of relatively more oxidized products, and higher product yields for cultures grown on glucose or gluconate. In contrast, cells grown on galacturonate showed reduced growth rates upon agitation, which was likely due to loss in reductant in the form of H2. The growth advantage seen on agitated glucose or gluconate cultures could not be solely attributed to improved ATP economics, thereby indicating other factors are also important. CONCLUSIONS: The metabolic network presented in this work should facilitate similar reconstructions in other organisms, and provides a further understanding of the pathways involved in metabolism of oxidized feedstocks and carbohydrate mixtures. The nearly homoacetic fermentation during growth on galacturonate indicates further optimization of this and related organisms could provide a route to an effective biologically derived acetic acid production platform. Furthermore, the pathways could be targeted to decrease production of undesirable products during fermentations of heterogeneous biomass.
Assuntos
Clostridium acetobutylicum/metabolismo , Fermentação , Hexoses/metabolismo , Acetatos/metabolismo , Trifosfato de Adenosina/metabolismo , Reatores Biológicos/microbiologia , Carbono/farmacologia , Dióxido de Carbono/metabolismo , Cromatografia Líquida de Alta Pressão , Clostridium acetobutylicum/efeitos dos fármacos , Clostridium acetobutylicum/crescimento & desenvolvimento , Fermentação/efeitos dos fármacos , Ácidos Hexurônicos/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Oxirredução/efeitos dos fármacosRESUMO
Production of acetone-butanol-ethanol (ABE) from cassava was investigated with a fermentation-pervaporation (PV) coupled process. ABE products were in situ removed from fermentation broth to alleviate the toxicity of solvent to the Clostridium acetobutylicum DP217. Compared to the batch fermentation without PV, glucose consumption rate and solvent productivity increased by 15% and 21%, respectively, in batch fermentation-PV coupled process, while in continuous fermentation-PV coupled process running for 304 h, the substrate consumption rate, solvent productivity and yield increased by 58%, 81% and 15%, reaching 2.02 g/Lh, 0.76 g/Lh and 0.38 g/g, respectively. Silicalite-1 filled polydimethylsiloxane (PDMS)/polyacrylonitrile (PAN) membrane modules ensured media recycle without significant fouling, steadily generating a highly concentrated ABE solution containing 201.8 g/L ABE with 122.4 g/L butanol. After phase separation, a final product containing 574.3g/L ABE with 501.1g/L butanol was obtained. Therefore, the fermentation-PV coupled process has the potential to decrease the cost in ABE production.
Assuntos
Acetona/metabolismo , Biotecnologia/métodos , Butanóis/metabolismo , Etanol/metabolismo , Fermentação , Manihot/metabolismo , Amilases/metabolismo , Técnicas de Cultura Celular por Lotes , Carbono/farmacologia , Clostridium acetobutylicum/efeitos dos fármacos , Clostridium acetobutylicum/metabolismo , Fermentação/efeitos dos fármacos , Manihot/efeitos dos fármacos , Membranas ArtificiaisRESUMO
Clostridium acetobutylicum strain Rh8 is a butanol-tolerant mutant which can tolerate up to 19g/L butanol, 46% higher than that of its parent strain DSM 1731. We previously performed comparative cytoplasm- and membrane-proteomic analyses to understand the mechanism underlying the improved butanol tolerance of strain Rh8. In this work, we further extended this comparison to the genomic level. Compared with the genome of the parent strain DSM 1731, two insertion sites, four deletion sites, and 67 single nucleotide variations (SNVs) are distributed throughout the genome of strain Rh8. Among the 67 SNVs, 16 SNVs are located in the predicted promoters and intergenic regions; while 29 SNVs are located in the coding sequence, affecting a total of 21 proteins involved in transport, cell structure, DNA replication, and protein translation. The remaining 22 SNVs are located in the ribosomal genes, affecting a total of 12 rRNA genes in different operons. Analysis of previous comparative proteomic data indicated that none of the differentially expressed proteins have mutations in its corresponding genes. Rchange Algorithms analysis indicated that the mutations occurred in the ribosomal genes might change the ribosome RNA thermodynamic characteristics, thus affect the translation strength of these proteins. Take together, the improved butanol tolerance of C. acetobutylicum strain Rh8 might be acquired through regulating the translational process to achieve different expression strength of genes involved in butanol tolerance.
Assuntos
Butanóis/farmacologia , Clostridium acetobutylicum/metabolismo , Proteoma , Clostridium acetobutylicum/efeitos dos fármacos , Clostridium acetobutylicum/genética , Genes Bacterianos , MutaçãoRESUMO
Solvent screening for in situ liquid extraction of products from acetone-butanol-ethanol (ABE) fermentation was carried out, taking into account biological parameters (biocompatibility, bioavailability, and product yield) and extraction performance (partition coefficient and selectivity) determined in real fermentation broth. On the basis of different solvent characteristics obtained from literature, 16 compounds from different chemical families were selected and experimentally evaluated for their extraction capabilities in a real ABE fermentation broth system. From these compounds, nine potential solvents were also tested for their biocompatibility towards Clostridium acetobutylicum. Moreover, bioavailability and differences in substrate consumption and total n-butanol production with respect to solvent-free fermentations were quantified for each biocompatible solvent. Product yield was enhanced in the presence of organic solvents having higher affinity for butanol and butyric acid. Applying this methodology, it was found that the Guerbet alcohol 2-butyl-1-octanol presented the best extracting characteristics (the highest partition coefficient (6.76) and the third highest selectivity (644)), the highest butanol yield (27.4 %), and maintained biocompatibility with C. acetobutylicum.
Assuntos
Acetona/isolamento & purificação , Butanóis/isolamento & purificação , Clostridium acetobutylicum/crescimento & desenvolvimento , Clostridium acetobutylicum/metabolismo , Etanol/isolamento & purificação , Solventes/química , Acetona/metabolismo , Butanóis/metabolismo , Clostridium acetobutylicum/efeitos dos fármacos , Etanol/metabolismo , Fermentação , Programas de Rastreamento , Solventes/isolamento & purificação , Solventes/toxicidadeRESUMO
An unexpected promotion effect of Ginkgo leaf on the growth of Clostridium acetobutylicum ATCC 824 and acetone-butanol-ethanol (ABE) fermentation was investigated. Component analysis of Ginkgo leaf was carried out and flavonoids were determined as the potential key metabolites. Then the flavonoids feeding experiments were carried out. Results showed that addition of only 10 mg/L flavonoids to the fermentation broth can promote butanol and ABE titre up to 14.5 and 17.8 g/L after 5 days of fermentation, that is, 74 and 68% higher than the control. A 2.2-fold biomass also has been achieved. Furthermore, by employing such novel founding, we easily exploited flavonoids from soybean and some agriculture wastes as the wide-distributed and economic feasible ABE fermentation promoter. The mechanism of the above effects was investigated from the perspective of oxidation-reduction potential. This work opens a new way in the efforts to increase the titer of butanol.
Assuntos
Acetona/metabolismo , Butanóis/metabolismo , Clostridium acetobutylicum/efeitos dos fármacos , Clostridium acetobutylicum/metabolismo , Etanol/metabolismo , Flavonoides/farmacologia , Biomassa , Fermentação/efeitos dos fármacosRESUMO
Acetone-butanol-ethanol (ABE) was produced from rice straw using a process containing ethanol organosolv pretreatment, enzymatic hydrolysis, and fermentation by Clostridium acetobutylicum bacterium. Pretreatment of the straw with 75% (v/v) aqueous ethanol containing 1% w/w sulfuric acid at 150 °C for 60 min resulted in the highest total sugar concentration of 31 g/L in the enzymatic hydrolysis. However, the highest ABE concentration and productivity (10.5 g/L and 0.20 g/Lh, respectively) were obtained from the straw pretreated at 180 °C for 30 min. Enzymatic hydrolysis of the straw pretreated at 180 °C for 30 min with 5% solid loading resulted in glucose yield of 46.2%, which was then fermented to 80.3 g butanol, 21.1 g acetone, and 22.5 g ethanol, the highest overall yield of ABE production. Thus, the organosolv pretreatment can be applied for efficient production of the solvents from rice straw.
Assuntos
1-Butanol/metabolismo , Acetona/metabolismo , Biotecnologia/métodos , Etanol/metabolismo , Compostos Orgânicos/farmacologia , Oryza/efeitos dos fármacos , Solventes/farmacologia , Arabinose/metabolismo , Clostridium acetobutylicum/efeitos dos fármacos , Clostridium acetobutylicum/metabolismo , Fermentação/efeitos dos fármacos , Glucose/metabolismo , Hidrólise/efeitos dos fármacos , Oryza/metabolismo , Resíduos , Xilose/metabolismoRESUMO
Development of controllable hypermutable cells can greatly benefit understanding and harnessing microbial evolution. However, there have not been any similar systems developed for Clostridium, an important bacterial genus. Here we report a novel two-step strategy for developing controllable hypermutable cells of Clostridium acetobutylicum, an important and representative industrial strain. Firstly, the mutS/L operon essential for methyldirected mismatch repair (MMR) activity was inactivated from the genome of C. acetobutylicum to generate hypermutable cells with over 250-fold increased mutation rates. Secondly, a proofreading control system carrying an inducibly expressed mutS/L operon was constructed. The hypermutable cells and the proofreading control system were integrated to form a controllable hypermutable system SMBMutC, of which the mutation rates can be regulated by the concentration of anhydrotetracycline (aTc). Duplication of the miniPthl-tetR module of the proofreading control system further significantly expanded the regulatory space of the mutation rates, demonstrating hypermutable Clostridium cells with controllable mutation rates are generated. The developed C. acetobutylicum strain SMBMutC2 showed higher survival capacities than the control strain facing butanol-stress, indicating greatly increased evolvability and adaptability of the controllable hypermutable cells under environmental challenges.
Assuntos
Engenharia Celular/métodos , Clostridium acetobutylicum/citologia , Clostridium acetobutylicum/genética , Metilação de DNA/genética , Reparo de Erro de Pareamento de DNA/genética , Mutação , Butanóis/farmacologia , Clostridium acetobutylicum/efeitos dos fármacos , Clostridium acetobutylicum/fisiologia , Evolução Molecular , Genoma Bacteriano/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Óperon/genética , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genéticaRESUMO
BACKGROUND: Organisms of the genus Clostridium are Gram-positive endospore formers of great importance to the carbon cycle, human normo- and pathophysiology, but also in biofuel and biorefinery applications. Exposure of Clostridium organisms to chemical and in particular toxic metabolite stress is ubiquitous in both natural (such as in the human microbiome) and engineered environments, engaging both the general stress response as well as specialized programs. Yet, despite its fundamental and applied significance, it remains largely unexplored at the systems level. RESULTS: We generated a total of 96 individual sets of microarray data examining the transcriptional changes in C. acetobutylicum, a model Clostridium organism, in response to three levels of chemical stress from the native metabolites, butanol and butyrate. We identified 164 significantly differentially expressed transcriptional regulators and detailed the cellular programs associated with general and stressor-specific responses, many previously unexplored. Pattern-based, comparative genomic analyses enabled us, for the first time, to construct a detailed picture of the genetic circuitry underlying the stress response. Notably, a list of the regulons and DNA binding motifs of the stress-related transcription factors were identified: two heat-shock response regulators, HrcA and CtsR; the SOS response regulator LexA; the redox sensor Rex; and the peroxide sensor PerR. Moreover, several transcriptional regulators controlling stress-responsive amino acid and purine metabolism and their regulons were also identified, including ArgR (arginine biosynthesis and catabolism regulator), HisR (histidine biosynthesis regulator), CymR (cysteine metabolism repressor) and PurR (purine metabolism repressor). CONCLUSIONS: Using an exceptionally large set of temporal transcriptional data and regulon analyses, we successfully built a STRING-based stress response network model integrating important players for the general and specialized metabolite stress response in C. acetobutylicum. Since the majority of the transcription factors and their target genes are highly conserved in other organisms of the Clostridium genus, this network would be largely applicable to other Clostridium organisms. The network informs the molecular basis of Clostridium responses to toxic metabolites in natural ecosystems and the microbiome, and will facilitate the construction of genome-scale models with added regulatory-network dimensions to guide the development of tolerant strains.