RESUMO
Clostridium kluyveri is an anaerobic microorganism that is well-known for producing butyrate and hexanoate using ethanol and acetate. It is also an important bacterium in the production of Chinese strong flavour baijiu (SFB). To obtain a comprehensive understanding of its metabolism, a curated genome-scale metabolic model (GSMM) of C. kluyveri, including 708 genes, 994 reactions, and 804 metabolites, was constructed and named iCKL708. This model was used to simulate the growth of C. kluyveri on different carbon substrates and the results agreed well with the experimental data. The butyrate, pentanoate, and hexanoate biosynthesis pathways were also elucidated. Flux balance analysis indicated that the ratio of ethanol to acetate, as well as the uptake rate of carbon dioxide, affected hexanoate production. The GSMM iCKL708 described here provides a platform to further our understanding and exploration of the metabolic potential of C. kluyveri.
Assuntos
Clostridium kluyveri/genética , Genoma Bacteriano/genética , Redes e Vias Metabólicas/genética , Acetatos/metabolismo , Butiratos/metabolismo , Caproatos/metabolismo , Clostridium kluyveri/metabolismo , Etanol/metabolismoRESUMO
Chinese strong-flavor liquor (CSFL), accounting for more than 70% of both Chinese liquor production and sales, was produced by complex fermentation with pit mud. Clostridium kluyveri, an important species coexisted with other microorganisms in fermentation pit mud (FPM), could produce caproic acid, which was subsequently converted to the key CSFL flavor substance ethyl caproate. In this study, we present the first complete genome sequence of C. kluyveri isolated from FPM. Clostridium kluyveri JZZ contains one circular chromosome and one circular plasmid with length of 4,454,353 and 58,581 bp, respectively. 4158 protein-coding genes were predicted and 2792 genes could be assigned with COG categories. It possesses the pathway predicted for biosynthesis of caproic acid with ethanol. Compared to other two C. kluyveri genomes, JZZ consists of longer chromosome with multiple gene rearrangements, and contains more genes involved in defense mechanisms, as well as DNA replication, recombination, and repair. Meanwhile, JZZ contains fewer genes involved in secondary metabolites biosynthesis, transport, and catabolism, including genes encoding Polyketide Synthases/Non-ribosomal Peptide Synthetases. Additionally, JZZ possesses 960 unique genes with relatively aggregating in defense mechanisms and transcription. Our study will be available for further research about C. kluyveri isolated from FPM, and will also facilitate the genetic engineering to increase biofuel production and improve fragrance flavor of CSFL.
Assuntos
Clostridium kluyveri/genética , Genoma Bacteriano , Vinho/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Caproatos/metabolismo , China , Clostridium kluyveri/isolamento & purificação , Clostridium kluyveri/metabolismo , Etanol/metabolismo , Fermentação , Aromatizantes/metabolismoRESUMO
A strain of Clostridium kluyveri was isolated from the bovine rumen in a medium containing ethanol as an electron donor and acetate and succinate (common products of rumen fermentation) as electron acceptors. The isolate displayed a narrow substrate range but wide temperature and pH ranges atypical of ruminal bacteria and a maximum specific growth rate near the typical liquid dilution rate of the rumen. Quantitative real-time PCR revealed that C. kluyveri was widespread among bovine ruminal samples but was present at only very low levels (0.00002% to 0.0002% of bacterial 16S rRNA gene copy number). However, the species was present in much higher levels (0.26% of bacterial 16S rRNA gene copy number) in lucerne silage (but not maize silage) that comprised much of the cows' diet. While C. kluyveri may account for several observations regarding ethanol utilization and volatile fatty acid production in the rumen, its population size and growth characteristics suggest that it is not a significant contributor to ruminal metabolism in typical dairy cattle, although it may be a significant contributor to silage fermentation. The ability of unadapted cultures to produce substantial levels (12.8 g L(-1)) of caproic (hexanoic) acid in vitro suggests that this strain may have potential for industrial production of caproic acid.
Assuntos
Carga Bacteriana , Clostridium kluyveri/isolamento & purificação , Rúmen/microbiologia , Acetatos/metabolismo , Animais , Bovinos , Clostridium kluyveri/classificação , Clostridium kluyveri/genética , Meios de Cultura/química , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Dieta , Etanol/metabolismo , Concentração de Íons de Hidrogênio , Medicago sativa/microbiologia , Dados de Sequência Molecular , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Ácido Succínico/metabolismo , Temperatura , Zea mays/microbiologiaRESUMO
Clostridium kluyveri is unique among the clostridia; it grows anaerobically on ethanol and acetate as sole energy sources. Fermentation products are butyrate, caproate, and H2. We report here the genome sequence of C. kluyveri, which revealed new insights into the metabolic capabilities of this well studied organism. A membrane-bound energy-converting NADH:ferredoxin oxidoreductase (RnfCDGEAB) and a cytoplasmic butyryl-CoA dehydrogenase complex (Bcd/EtfAB) coupling the reduction of crotonyl-CoA to butyryl-CoA with the reduction of ferredoxin represent a new energy-conserving module in anaerobes. The genes for NAD-dependent ethanol dehydrogenase and NAD(P)-dependent acetaldehyde dehydrogenase are located next to genes for microcompartment proteins, suggesting that the two enzymes, which are isolated together in a macromolecular complex, form a carboxysome-like structure. Unique for a strict anaerobe, C. kluyveri harbors three sets of genes predicted to encode for polyketide/nonribosomal peptide synthetase hybrides and one set for a nonribosomal peptide synthetase. The latter is predicted to catalyze the synthesis of a new siderophore, which is formed under iron-deficient growth conditions.
Assuntos
Clostridium kluyveri/genética , Genoma Bacteriano , Acetatos/metabolismo , Álcool Desidrogenase/metabolismo , Aldeído Oxirredutases/metabolismo , Clostridium kluyveri/enzimologia , Clostridium kluyveri/metabolismo , Etanol/metabolismo , Fermentação , Glicerol/metabolismo , Dados de Sequência Molecular , Fenóis/metabolismo , Ácido Succínico/metabolismo , Tiazóis/metabolismoRESUMO
It was recently found that the cytoplasmic butyryl-coenzyme A (butyryl-CoA) dehydrogenase-EtfAB complex from Clostridium kluyveri couples the exergonic reduction of crotonyl-CoA to butyryl-CoA with NADH and the endergonic reduction of ferredoxin with NADH via flavin-based electron bifurcation. We report here on a second cytoplasmic enzyme complex in C. kluyveri capable of energetic coupling via this novel mechanism. It was found that the purified iron-sulfur flavoprotein complex NfnAB couples the exergonic reduction of NADP+ with reduced ferredoxin (Fdred) and the endergonic reduction of NADP+ with NADH in a reversible reaction: Fdred2-+NADH+2 NADP++H+=Fdox+NAD++2 NADPH. The role of this energy-converting enzyme complex in the ethanol-acetate fermentation of C. kluyveri is discussed.
Assuntos
Clostridium kluyveri/enzimologia , Ferredoxinas/metabolismo , NADP/metabolismo , NAD/metabolismo , Acil Coenzima A/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clostridium kluyveri/genética , OxirreduçãoRESUMO
A metabolically engineered Escherichia coli has been constructed for the production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] from unrelated carbon sources. Genes involved in succinate degradation in Clostridium kluyveri and P(3HB) accumulation pathway of Ralstonia eutropha were co-expressed for the synthesis of the above copolyester. E. coli native succinate semialdehyde dehydrogenase genes sad and gabD were both deleted for eliminating succinate formation from succinate semialdehyde, which functioned to enhance the carbon flux to 4HB biosynthesis. The metabolically engineered E. coli produced 9.4 gl(-1) cell dry weight containing 65.5% P(3HB-co-11.1 mol% 4HB) using glucose as carbon source in a 48 h shake flask growth. The presence of 1.5-2 gl(-1) alpha-ketoglutarate or 1.0 gl(-1) citrate enhanced the 4HB monomer content from 11.1% to more than 20%. In a 6l fermentor study, a 23.5 gl(-1) cell dry weight containing 62.7% P(3HB-co-12.5 mol% 4HB) was obtained after 29 h of cultivation. To the best of our knowledge, this study reports the highest 4HB monomer content in P(3HB-co-4HB) produced from unrelated carbon sources.
Assuntos
Carbono/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Hidroxibutiratos/metabolismo , Redes e Vias Metabólicas/genética , Poliésteres/metabolismo , Ácido Cítrico/metabolismo , Clostridium kluyveri/enzimologia , Clostridium kluyveri/genética , Cupriavidus necator/enzimologia , Cupriavidus necator/genética , Fermentação , Engenharia Genética , Glucose/metabolismo , Ácidos Cetoglutáricos/metabolismo , Deleção de Sequência , Succinato-Semialdeído Desidrogenase/genética , Ácido Succínico/metabolismoRESUMO
It has been suggested that ethanol metabolism in the strict anaerobe Clostridium kluyveri occurs within a metabolosome, a subcellular proteinaceous bacterial microcompartment. Two bacterial microcompartment shell proteins [EtuA (ethanol utilization shell protein A) and EtuB] are found encoded on the genome clustered with the genes for ethanol utilization. The function of the bacterial microcompartment is to facilitate fermentation by sequestering the enzymes, substrates and intermediates. Recent structural studies of bacterial microcompartment proteins have revealed both hexamers and pentamers that assemble to generate the pseudo-icosahedral bacterial microcompartment shell. Some of these shell proteins have pores on their symmetry axes. Here we report the structure of the trimeric bacterial microcompartment protein EtuB, which has a tandem structural repeat within the subunit and pseudo-hexagonal symmetry. The pores in the EtuB trimer are within the subunits rather than between symmetry related subunits. We suggest that the evolutionary advantage of this is that it releases the pore from the rotational symmetry constraint allowing more precise control of the fluxes of asymmetric molecules, such as ethanol, across the pore. We also model EtuA and demonstrate that the two proteins have the potential to interact to generate the casing for a metabolosome.
Assuntos
Proteínas de Bactérias/química , Clostridium kluyveri , Etanol/metabolismo , Multimerização Proteica , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Clostridium kluyveri/química , Clostridium kluyveri/genética , Clostridium kluyveri/metabolismo , Clostridium kluyveri/ultraestrutura , Cristalografia por Raios X , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Porinas/química , Porinas/genética , Porinas/isolamento & purificação , Porinas/metabolismo , Multimerização Proteica/fisiologia , Homologia de Sequência de AminoácidosRESUMO
A small enzyme showing diaphorase activity was purified from culture supernatant of Clostridium kluyveri and its N-terminal amino acid sequence was determined. This sequence identified a gene (diaA) encoding a protein (DiaA) of 229 amino acids with a predicted molecular weight of 24,981 in the genomic DNA sequence database of C. kluyveri constructed by the Research Institute of Innovative Technology for the Earth. The predicted protein was composed of a flavin reductase-like domain and a rubredoxin-like domain from its N-terminus. The diaA gene was cloned into an expression vector and expressed in an Escherichia coli recombinant. Recombinant enzyme rDiaA showed NADH/NADPH diaphorase activity with 2,6-dichlorophenolindophenol and nitro blue tetrazolium. The enzyme was most active at pH 8.0 at 40 degrees C. The UV-visible absorption spectrum and thin layer chromatography (TLC) analyses indicated that one rDiaA molecule contained a tightly bound FMN molecule as a prosthetic group. An iron molecule was also detected in an enzyme molecule.
Assuntos
Clostridium kluyveri/genética , NADH Desidrogenase/genética , Sequência de Aminoácidos , Proteínas de Bactérias , Clonagem Molecular/métodos , Clostridium kluyveri/enzimologia , Mononucleotídeo de Flavina , Expressão Gênica , Genes Bacterianos , Ferro , Dados de Sequência Molecular , NADH Desidrogenase/isolamento & purificaçãoRESUMO
The Clostridium kluyveri bfmBC gene encoding a putative dihydrolipoyl dehydrogenase (DLD; EC 1.8.1.4) was expressed in Escherichia coli, and the recombinant enzyme rBfmBC was characterized. UV-visible absorption spectrum and thin layer chromatography analysis of rBfmBC indicated that the enzyme contained a noncovalently but tightly attached FAD molecule. rBfmBC catalyzed the oxidation of dihydrolipoamide (DLA) with NAD(+) as a specific electron acceptor, and the apparent K(m) values for DLA and NAD(+) were 0.3 and 0.5 mM respectively. In the reverse reaction, the apparent K(m) values for lipoamide and NADH were 0.42 and 0.038 mM respectively. Like other DLDs, this enzyme showed NADH dehydrogenase (diaphorase) activity with some synthetic dyes, such as 2,6-dichlorophenolindophenol and nitro blue tetrazolium. rBfmBC was optimally active at 40 degrees C at pH 7.0, and the enzyme maintained some activity after a 30-min incubation at 60 degrees C.
Assuntos
Clostridium kluyveri/enzimologia , Di-Hidrolipoamida Desidrogenase/genética , Di-Hidrolipoamida Desidrogenase/metabolismo , NADH Desidrogenase/metabolismo , Clostridium kluyveri/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Cinética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência , Espectrofotometria UltravioletaRESUMO
Fluorescence in-situ hybridization (FISH) is a common and popular method used to investigate microbial communities in natural and engineered environments. In this study, two specific 16S rRNA-targeted oligonucleotide probes, CLZ and KCLZ, were designed and verified to quantify the genus Clostridium and the species Clostridium kluyveri. The optimal concentration of hybridization buffer solution for both probes was 30% (w/v). The specificity of the designed probes was high due to the use of pellets from pure reference strains. Feasibility was tested using samples of Chinese liquor from the famed Luzhou manufacturing cellar. The effectiveness of detecting target cells appears to vary widely in different environments. In pit mud, the detection effectiveness of the target cell by probes CLZ and KCLZ was 49.11% and 32.14%, respectively. Quantitative analysis by FISH technique of microbes in pit mud and fermented grains showed consistency with the results detected by qPCR and PCR-DGGE techniques, which showed that the probes CLZ and KCLZ were suitable to analyze the biomass of Clostridium spp. and C. kluyveri during liquor fermentation. Therefore, this study provides a method for quantitative analysis of Clostridium spp. and C. kluyveri and monitoring their community dynamics in microecosystems.
Assuntos
Bebidas Alcoólicas/microbiologia , Clostridium/isolamento & purificação , Hibridização in Situ Fluorescente , RNA Ribossômico 16S/genética , Biomassa , Clostridium/genética , Clostridium kluyveri/genética , Clostridium kluyveri/isolamento & purificação , Estudos de Viabilidade , Fermentação , Corantes Fluorescentes/química , Sondas de Oligonucleotídeos/química , Reação em Cadeia da Polimerase em Tempo Real , Especificidade da EspécieRESUMO
Clostridium kluyveri is unique in fermenting ethanol and acetate to butyrate, caproate, and H2. The genes encoding butyrate-producing enzymes, including electron-bifurcating butyryl-CoA dehydrogenase/electron transfer flavoprotein complex and NADH-dependent reduced ferredoxin:NADP(+) oxidoreductase, form a cluster, which is preceded by a gene annotated as the transcriptional regulator Rex. Northern blotting and RT-PCR experiments indicated that the gene cluster forms a large transcriptional unit that possibly includes several small transcriptional units. The deduced Rex protein contains a winged helix DNA-binding domain and a Rossmann fold potentially interacting with NAD(H). Bioinformatics analysis revealed that Rex can bind the promoter regions of numerous genes, which are involved in carbon and energy metabolism, including NADH oxidation, hydrogen production, ATP synthesis, butyrate formation, and succinate metabolism. Rex may regulate the transcription of genes encoding certain transcriptional regulators and transporters. Electrophoretic mobility shift and isothermal titration calorimetry assays revealed that Rex specifically formed protein-DNA complexes with the promoter regions of target genes, which could be inhibited by NADH but restored by an excess amount of NAD(+). These results suggest that Rex plays a key role in the carbon and energy metabolism of C. kluyveri as a global transcriptional regulator in response to the cellular NADH/NAD(+) ratio.
Assuntos
Clostridium kluyveri/genética , Clostridium kluyveri/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Genes Bacterianos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Butiratos/metabolismo , OxirreduçãoRESUMO
Chinese strong-aroma type liquor (CSAL) is a popular distilled alcoholic beverage in China. It is produced by a complex fermentation process that is conducted in pits in the ground. Ethyl caproate is a key flavor compound in CSAL and is thought to originate from caproic acid produced by Clostridia inhabiting the fermentation pit mud. However, the particular species of Clostridium associated with this production are poorly understood and problematic to quantify by culturing. In this study, a total of 28 closest relatives including 15 Clostridia and 8 Bacilli species in pit muds from three CSAL distilleries, were detected by culture-dependent and -independent methods. Among them, Clostridium kluyveri was identified as the main producer of caproic acid. One representative strain C. kluyveri N6 could produce caproic, butyric and octanoic acids and their corresponding ethyl esters, contributing significantly to CSAL flavor. A real time quantitative PCR assay of C. kluyveri in pit muds developed showed that a concentration of 1.79×10(7) 16S rRNA gene copies/g pit mud in LZ-old pit was approximately six times higher than that in HLM and YH pits and sixty times higher than that in LZ-new pit respectively. This method can be used to improve the management of pit mud microbiology and its impact on CSAL quality.
Assuntos
Bebidas Alcoólicas , Caproatos/metabolismo , Clostridium kluyveri/isolamento & purificação , Fermentação , Bacillus/genética , Bacillus/isolamento & purificação , China , Clostridium kluyveri/classificação , Clostridium kluyveri/genética , RNA Ribossômico 16S/genética , Olfato , Microbiologia do SoloRESUMO
The synthesis of citrate from acetyl-coenzyme A and oxaloacetate is catalyzed in most organisms by a Si-citrate synthase, which is Si-face stereospecific with respect to C-2 of oxaloacetate. However, in Clostridium kluyveri and some other strictly anaerobic bacteria, the reaction is catalyzed by a Re-citrate synthase, whose primary structure has remained elusive. We report here that Re-citrate synthase from C. kluyveri is the product of a gene predicted to encode isopropylmalate synthase. C. kluyveri is also shown to contain a gene for Si-citrate synthase, which explains why cell extracts of the organism always exhibit some Si-citrate synthase activity.