RESUMO
To understand the role of the X25 domains of the amylopullulanase enzyme from Thermoanaerobacter brockii brockii (T. brockii brockii), four truncated variants that are TbbApuΔX25-1-SH3 (S130-A1484), TbbApuΔX25-2-SH3 (T235-A1484), TbbApuΔX25-1-CBM20 (S130-P1254), and TbbApuΔX25-2-CBM20 (T235-P1254) were constructed, expressed and characterized together with the SH3 and CBM20 domain truncated variants (TbbApuΔSH3 (V1-A1484) and TbbApuΔCBM20 (V1-P1254). TbbApuΔSH3 showed improved affinity and specificity for both pullulan and soluble starch than full-length TbbApu with lower Km and higher kcat/Km values. It indicates that SH3 is a disposable domain without any effect on the activity and stability of the enzyme. However, TbbApuΔX25-1-SH3, TbbApuΔX25-2-SH3, TbbApuΔX25-1-CBM20, TbbApuΔX25-2-CBM20 (T235-P1254) and TbbApuΔCBM20 showed higher Km and lower kcat/Km values than TbbApuΔSH3 to both soluble starch and pullulan. It specifies that the X25 domains and CBM20 play an important role in both α-amylase and pullulanase activity. Also, it is revealed that while truncation of the CBM20 domain as starch binding domain (SBD) did not affect on raw starch binding ability of the enzyme, truncation of both X25 domains caused almost complete loss of the raw starch binding ability of the enzyme. All these results enlightened the function of the X25 domains that play a more crucial role than CBM20 in the enzyme's binding to raw starch and also play a crucial role in its activity.
Assuntos
Glicosídeo Hidrolases , Domínios Proteicos , Thermoanaerobacter , Thermoanaerobacter/enzimologia , Thermoanaerobacter/genética , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Amido/metabolismo , Especificidade por Substrato , Cinética , Estabilidade Enzimática , Glucanos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismoRESUMO
To search for a novel thermostable esterase for optimized industrial applications, esterase from a thermophilic eubacterium species, Thermoanaerobacter tengcongensis MB4, was purified and characterized in this work. Sequence analysis of T. tengcongensis esterase with other homologous esterases of the same family revealed an apparent tail at the C-terminal that is not conserved across the esterase family. Hence, it was hypothesized that the tail is unlikely to have an essential structural or catalytic role. However, there is no documented report of any role for this tail region. We probed the role of the C-terminal domain on the catalytic activity and substrate preference of T. tengcongensis esterase EstA3 with a view to see how it could be engineered for enhanced properties. To achieve this, we cloned, expressed, and purified the wild-type and the truncated versions of the enzyme. In addition, a naturally occurring member of the family (from Brevibacillus brevis) that lacks the C-terminal tail was also made. In vitro characterization of the purified enzymes showed that the C-terminal domain contributes significantly to the catalytic activity and distinct substrate preference of T. tengcongensis esterase EstA3. All three recombinant enzymes showed the highest preference for paranitrophenyl butyrate (pNPC4), which suggests they are true esterases, not lipases. Kinetic data revealed that truncation had a slight effect on the substrate-binding affinity. Thus, the drop in preference towards long-chain substrates might not be a result of substrate binding affinity alone. The findings from this work could form the basis for future protein engineering allowing the modification of esterase catalytic properties through domain swapping or by attaching a modular protein domain.
Assuntos
Proteínas de Bactérias , Esterases , Firmicutes , Esterases/metabolismo , Sequência de Aminoácidos , Hidrólise , Proteínas de Bactérias/metabolismo , Thermoanaerobacter/genética , Thermoanaerobacter/química , Estabilidade Enzimática , Especificidade por Substrato , Clonagem MolecularRESUMO
Aldehyde:ferredoxin oxidoreductases (AORs) have been isolated and biochemically-characterized from a handful of anaerobic or facultative aerobic archaea and bacteria. They catalyze the ferredoxin (Fd)-dependent oxidation of aldehydes to acids. Recently, the involvement of AOR in the reduction of organic acids to alcohols with electrons derived from sugar or synthesis gas was demonstrated, with alcohol dehydrogenases (ADHs) carrying out the reduction of the aldehyde to the alcohol (AOR-ADH pathway). Here, we describe the biochemical characterization of an AOR of the thermophilic fermentative bacterium Thermoanaerobacter sp. strain X514 (AORX514). The putative aor gene (Teth514_1380) including a 6x-His-tag was introduced into the genome of the genetically-accessible, related species Thermoanaerobacter kivui. The protein was purified to apparent homogeneity, and indeed revealed AOR activity, as measured by acetaldehyde-dependent ferredoxin reduction. AORX514 was active over a wide temperature (10 to 95 °C) and pH (5.5 to 11.5) range, utilized a wide variety of aldehydes (short and branched-chained, aliphatic, aromatic) and resembles archaeal sensu stricto AORs, as the protein is active in a homodimeric form. The successful, recombinant production of AORX514 in a related, well-characterized and likewise strict anaerobe paves the road towards structure-function analyses of this enzyme and possibly similar oxygen-sensitive or W/Mo-dependent proteins in the future.
Assuntos
Aldeídos , Ferredoxinas , Ferredoxinas/genética , Thermoanaerobacter/genética , Acetaldeído , Álcool Desidrogenase , Archaea , DNA Topoisomerases Tipo IRESUMO
A major electron carrier involved in energy and carbon metabolism in the acetogenic model organism Thermoanaerobacter kivui is ferredoxin, an iron-sulfur-containing, electron-transferring protein. Here, we show that the genome of T. kivui encodes four putative ferredoxin-like proteins (TKV_c09620, TKV_c16450, TKV_c10420 and TKV_c19530). All four genes were cloned, a His-tag encoding sequence was added and the proteins were produced from a plasmid in T. kivui. The purified proteins had an absorption peak at 430 nm typical for ferredoxins. The determined iron-sulfur content is consistent with the presence of two predicted [4Fe4S] clusters in TKV_c09620 and TKV_c19530 or one predicted [4Fe4S] cluster in TKV_c16450 and TKV_c10420 respectively. The reduction potential (Em ) for TKV_c09620, TKV_c16450, TKV_c10420 and TKV_c19530 was determined to be -386 ± 4 mV, -386 ± 2 mV, -559 ± 10 mV and -557 ± 3 mV, respectively. TKV_c09620 and TKV_c16450 served as electron carriers for different oxidoreductases from T. kivui. Deletion of the ferredoxin genes led to only a slight reduction of growth on pyruvate or autotrophically on H2 + CO2 . Transcriptional analysis revealed that TKV_c09620 was upregulated in a ΔTKV_c16450 mutant and vice versa TKV_c16450 in a ΔTKV_c09620 mutant, indicating that TKV_c09620 and TKV_c16450 can replace each other. In sum, our data are consistent with the hypothesis that TKV_c09620 and TKV_c16450 are ferredoxins involved in autotrophic and heterotrophic metabolism of T. kivui.
Assuntos
Ferredoxinas , Thermoanaerobacter , Thermoanaerobacter/química , Thermoanaerobacter/genética , Thermoanaerobacter/metabolismo , Ferredoxinas/química , Ferredoxinas/genética , Ferredoxinas/metabolismo , Genoma Bacteriano/genética , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Espectroscopia FotoeletrônicaRESUMO
Thermoanaerobacter thermocopriae-derived thermostable cycloisomaltooligosaccharide (CI)-forming enzymes catalyze the production of CIs from dextran. The primary structure of the enzyme is comprised of CI glucanotransferase (TtCITase) at the N-terminal region and long isomaltooligosaccharide-forming enzyme (TtTGase) at the C-terminal region connected by carbohydrate-binding module family 35 (CBM, TtCBM). Three truncated mutants of CI-forming enzymes were successfully produced in Corynebacterium glutamicum, a food-grade host system, and their biochemical properties were characterized. The enzymes had optimum at pH 6.0 and pH-stability (5.0-12.0). Three enzymes had optimum temperature over 55 °C and they maintained 80% activity at 55 °C for 2 h, 12 h, and 18 h, respectively. Enzymes without CBM showed weaker allosteric behavior than those of other enzymes, which suggests the important role of CBM in allosteric behavior. However, CBM bearing enzymes showed high production of CIs with various degree of polymerization. These enzymes have potential application as the encapsulating material for insoluble pharmaceutical biomaterials.
Assuntos
Glucosiltransferases , Thermoanaerobacter , Carboidratos , Clostridium , Glucosiltransferases/química , Glucosiltransferases/genética , Thermoanaerobacter/genéticaRESUMO
This study uses differential scanning calorimetry, X-ray crystallography, and molecular dynamics simulations to investigate the structural basis for the high thermal stability (melting temperature 97.5°C) of a FN3-like protein domain from thermophilic bacteria Thermoanaerobacter tengcongensis (FN3tt). FN3tt adopts a typical FN3 fold with a three-stranded beta sheet packing against a four-stranded beta sheet. We identified three solvent exposed arginine residues (R23, R25, and R72), which stabilize the protein through salt bridge interactions with glutamic acid residues on adjacent strands. Alanine mutation of the three arginine residues reduced melting temperature by up to 22°C. Crystal structures of the wild type (WT) and a thermally destabilized (∆Tm -19.7°C) triple mutant (R23L/R25T/R72I) were found to be nearly identical, suggesting that the destabilization is due to interactions of the arginine residues. Molecular dynamics simulations showed that the salt bridge interactions in the WT were stable and provided a dynamical explanation for the cooperativity observed between R23 and R25 based on calorimetry measurements. In addition, folding free energy changes computed using free energy perturbation molecular dynamics simulations showed high correlation with melting temperature changes. This work is another example of surface salt bridges contributing to the enhanced thermal stability of thermophilic proteins. The molecular dynamics simulation methods employed in this study may be broadly useful for in silico surface charge engineering of proteins.
Assuntos
Proteínas de Bactérias/química , Domínio de Fibronectina Tipo III , Cloreto de Sódio/química , Thermoanaerobacter/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Temperatura Alta , Simulação de Dinâmica Molecular , Domínios Proteicos , Estabilidade Proteica , Thermoanaerobacter/genéticaRESUMO
Glucose isomerase (GI) is a key enzyme in the preparation of high fructose corn syrup (HFCS). In this study, a mutant TEGI-M-L38 M/V137 L (TEGI-M2) of glucose isomerase (TEGI-M) originated from Thermoanaerobacter ethanalicus CCSD1 was obtained by site-directed mutagenesis. The TEGI-M2 showed an optimal activity at 85 â and pH 6.5 with the divalent cations Co2+ and Mg2+. The structural differences between TEGI-M and TEGI-M2 were investigated based on the homology modeling and molecular docking, to elucidate the mechanism of improvement in the enzymatic properties. Compared with the original enzyme, the TEGI-M2 showed a 2.0-fold increased enzyme activity and a decreased Km from 234.2 mM to 85.9 mM. Finally, the application of mutant TEGI-M2 in HFCS one-step biosynthesis was attempted, resulting in a d-fructose yield of 67.3 %, which was 14.3 % higher than that of TEGI-M. This improved catalytic performance of TEGI-M2 was of great importance for the industrial preparation of d-fructose in one-step process.
Assuntos
Aldose-Cetose Isomerases , Thermoanaerobacter , Aldose-Cetose Isomerases/genética , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Thermoanaerobacter/genéticaRESUMO
Isomaltooligosaccharide (IMO), considered to be a prebiotic, reportedly has health effects, particularly in terms of digestion; however, the prebiotic effects of IMOs depend largely on the degree of polymerization. Currently, IMOs are commercially produced using transglucosidase (TG) derived from Aspergillus niger. Here, we report a novel Thermoanaerobacter thermocopriae-derived TG (TtTG) that can produce long-chain IMOs (L-IMOs) using maltodextrin as the main substrate. A putative carbohydrate-binding gene comprising carbohydrate-binding module 35 and glycoside hydrolase family 15 domain was cloned and successfully overexpressed in Escherichia coli BL21 (DE3) cells. The resulting purified recombinant enzyme (TtTG) had a molecular mass of 94 kDa. TtTG displayed an optimal pH of 4.0 (higher than that of commercial TG) and an optimal temperature of 60 °C (same as that of commercial TG). TtTG also enabled the synthesis of oligosaccharides using various saccharides, such as palatinose, kojibiose, sophorose, maltose, cellobiose, isomaltose, gentiobiose, and trehalose, which acted as specific acceptors. TtTG could also produce a medium-sized L-IMO, different from that by dextran-dextrinase and TG, from maltodextrin, as the sole substrate. Thus, the novel combination of maltodextrin and TtTG shows potential as an effective method for commercially producing L-IMOs with improved prebiotic effects.
Assuntos
Glucosiltransferases , Thermoanaerobacter , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Oligossacarídeos , Polissacarídeos , Especificidade por Substrato , Thermoanaerobacter/genéticaRESUMO
Thermoanaerobacter ethanolicus can produce acetate, lactate, hydrogen, and ethanol from sugars resulting from plant carbohydrate polymer degradation at temperatures above 65°C. T. ethanolicus is a promising candidate for thermophilic ethanol fermentations due to the utilization of both pentose and hexose. Although an ethanol balance model in T. ethanolicus has been developed, only a few physiological or biochemical experiments regarding the function of important enzymes in ethanol formation have been carried out. To address this issue, we developed a thermostable Cas9-based system for genome editing of T. ethanolicus As a proof of principle, three genes, including the thymidine kinase gene (tdk), acetaldehyde-alcohol dehydrogenase gene (adhE), and redox sensing protein gene (rsp), were chosen as editing targets, and these genes were edited successfully. As a genetic tool, we tested the gene knockout and a small DNA fragment knock-in. After optimization of the transformation strategies, 77% genome-editing efficiency was observed. Furthermore, our in vivo results revealed that redox sensing protein (RSP) plays a more important role in regulation of energy metabolism, including hydrogen production and ethanol formation. The genetic system provides us with an effective strategy to identify genes involved in biosynthesis and energy metabolism.IMPORTANCE Interest in thermophilic microorganisms as emerging metabolic engineering platforms to produce biofuels and chemicals has surged. Thermophilic microbes for biofuels have attracted great attention, due to their tolerance of high temperature and wide range of substrate utilization. On the basis of the biochemical experiments of previous investigation, the formation of ethanol was controlled via transcriptional regulation and influenced by the relevant properties of specific enzymes in T. ethanolicus Thus, there is an urgent need to understand the physiological function of these key enzymes, which requires genetic manipulations such as deletion or overexpression of genes encoding putative key enzymes. Here, we developed a thermostable Cas9-based engineering tool for gene editing in T. ethanolicus The thermostable Cas9-based genome-editing tool may further be applied to metabolically engineer T. ethanolicus to produce biofuels. This genetic system represents an important expansion of the genetic tool box of anaerobic thermophile T. ethanolicus strains.
Assuntos
Proteínas de Bactérias/genética , Sistemas CRISPR-Cas , Edição de Genes , Thermoanaerobacter/genética , Anaerobiose , Proteínas de Bactérias/metabolismo , Etanol/metabolismo , Fermentação , Thermoanaerobacter/enzimologiaRESUMO
Riboswitches are structured RNA motifs that recognize metabolites to alter the conformations of downstream sequences, leading to gene regulation. To investigate this molecular framework, we determined crystal structures of a preQ1-I riboswitch in effector-free and bound states at 2.00 Å and 2.65 Å-resolution. Both pseudoknots exhibited the elusive L2 loop, which displayed distinct conformations. Conversely, the Shine-Dalgarno sequence (SDS) in the S2 helix of each structure remained unbroken. The expectation that the effector-free state should expose the SDS prompted us to conduct solution experiments to delineate environmental changes to specific nucleobases in response to preQ1. We then used nudged elastic band computational methods to derive conformational-change pathways linking the crystallographically-determined effector-free and bound-state structures. Pathways featured: (i) unstacking and unpairing of L2 and S2 nucleobases without preQ1-exposing the SDS for translation and (ii) stacking and pairing L2 and S2 nucleobases with preQ1-sequestering the SDS. Our results reveal how preQ1 binding reorganizes L2 into a nucleobase-stacking spine that sequesters the SDS, linking effector recognition to biological function. The generality of stacking spines as conduits for effector-dependent, interdomain communication is discussed in light of their existence in adenine riboswitches, as well as the turnip yellow mosaic virus ribosome sensor.
Assuntos
Simulação de Dinâmica Molecular , Riboswitch , Pareamento de Bases , Regulação Bacteriana da Expressão Gênica , Guanina/análogos & derivados , Dodecilsulfato de Sódio/química , Thermoanaerobacter/genéticaRESUMO
Glycerol-rich waste streams produced by the biodiesel, bioethanol and oleochemical industries can be treated and valorized by anaerobic microbial communities to produce methane. As current knowledge of the microorganisms involved in thermophilic glycerol conversion to methane is scarce, thermophilic glycerol-degrading methanogenic communities were enriched. A co-culture of Thermoanaerobacter and Methanothermobacter species was obtained, pointing to a non-obligately syntrophic glycerol degradation. This hypothesis was further studied by incubating Thermoanaerobacter brockii subsp. finnii and T. wiegelii with glycerol (10 mM) in pure culture and with different hydrogenotrophic methanogens. The presence of the methanogen accelerated glycerol fermentation by the two Thermoanaerobacter strains up to 3.3 mM day-1 , corresponding to 12 times higher volumetric glycerol depletion rates in the methanogenic co-cultures than in the pure bacterial cultures. The catabolic pathways of glycerol conversion were identified by genome analysis of the two Thermoanaerobacter strains. NADH and reduced ferredoxin formed in the pathway are linked to proton reduction, which becomes thermodynamically favourable when the hydrogen partial pressure is kept low by the hydrogenotrophic methanogenic partner.
Assuntos
Glicerol , Thermoanaerobacter , Anaerobiose , Metano , Methanobacteriaceae/genética , Thermoanaerobacter/genéticaRESUMO
Isomaltooligosaccharides (IMOs) have good prebiotic effects, and long IMOs (LIMOs) with a degree of polymerization (DP) of 7 or above show improved effects. However, they are not yet commercially available, and require costly enzymes and processes for production. The Nterminal region of the thermostable Thermoanaerobacter thermocopriae cycloisomaltooligosaccharide glucanotransferase (TtCITase) shows cyclic isomaltooligosaccharide (CI)-producing activity owing to a catalytic domain of glycoside hydrolase (GH) family 66 and carbohydrate-binding module (CBM) 35. In the present study, we elucidated the activity of the C-terminal region of TtCITase (TtCITase-C; Met740-Phe1,559), including a CBM35-like region and the GH family 15 domain. The domain was successfully cloned, expressed, and purified as a single protein with a molecular mass of 115 kDa. TtCITase-C exhibited optimal activity at 40°C and pH 5.5, and retained 100% activity at pH 5.5 after 18-h incubation. TtCITase-C synthesized α-1,6 glucosyl products with over seven degrees of polymerization (DP) by an α-1,6 glucosyl transfer reaction from maltopentaose, isomaltopentaose, or commercialized maltodextrins as substrates. These results indicate that TtCITase-C could be used for the production of α-1,6 glucosyl oligosaccharides with over DP7 (LIMOs) in a more cost-effective manner, without requiring cyclodextran.
Assuntos
Glucosiltransferases/química , Glucosiltransferases/metabolismo , Oligossacarídeos/metabolismo , Thermoanaerobacter/enzimologia , Domínio Catalítico , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Glucosiltransferases/genética , Glicosídeo Hidrolases , Concentração de Íons de Hidrogênio , Peso Molecular , Polimerização , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Análise de Sequência de Proteína , Temperatura , Thermoanaerobacter/genéticaRESUMO
Acetogenic bacteria recently attracted attention because they reduce carbon dioxide (CO2 ) with hydrogen (H2 ) to acetate or to other products such as ethanol. Besides gases, acetogens use a broad range of substrates, but conversion of the sugar alcohol mannitol has rarely been reported. We found that the thermophilic acetogenic bacterium Thermoanaerobacter kivui grew on mannitol with a specific growth rate of 0.33 h-1 to a final optical density (OD600 ) of 2.2. Acetate was the major product formed. A lag phase was observed only in cultures pre-grown on glucose, not in those pre-grown on mannitol, indicating that mannitol metabolism is regulated. Mannitol-1-phosphate dehydrogenase (MtlD) activity was observed in cell-free extracts of cells grown on mannitol only. A gene cluster (TKV_c02830-TKV_c02860) for mannitol uptake and conversion was identified in the T. kivui genome, and its involvement was confirmed by deleting the mtlD gene (TKV_c02860) encoding the key enzyme MtlD. Finally, we overexpressed mtlD, and the recombinant MtlD carried out the reduction of fructose-6-phosphate with NADH, at a high VMAX of 1235 U mg-1 at 65°C. The enzyme was thermostable for 40 min at 75°C, thereby representing the first characterized MtlD from a thermophile.
Assuntos
Manitol/metabolismo , Desidrogenase do Álcool de Açúcar/metabolismo , Thermoanaerobacter/enzimologia , Estabilidade Enzimática , Genes Bacterianos , Família Multigênica , Thermoanaerobacter/genética , Thermoanaerobacter/crescimento & desenvolvimentoRESUMO
The ancient reductive acetyl-CoA pathway is employed by acetogenic bacteria to form acetate from inorganic energy sources. Since the central pathway does not gain net ATP by substrate-level phosphorylation, chemolithoautotrophic growth relies on the additional formation of ATP via a chemiosmotic mechanism. Genome analyses indicated that some acetogens only have an energy-converting, ion-translocating hydrogenase (Ech) as a potential respiratory enzyme. Although the Ech-encoding genes are widely distributed in nature, the proposed function of Ech as an ion-translocating chemiosmotic coupling site has neither been demonstrated in bacteria nor has it been demonstrated that it can be the only energetic coupling sites in microorganisms that depend on a chemiosmotic mechanism for energy conservation. Here, we show that the Ech complex of the thermophilic acetogenic bacterium Thermoanaerobacter kivui is indeed a respiratory enzyme. Experiments with resting cells prepared from T. kivui cultures grown on carbon monoxide (CO) revealed CO oxidation coupled to H2 formation and the generation of a transmembrane electrochemical ion gradient ([Formula: see text]). Inverted membrane vesicles (IMVs) prepared from CO-grown cells also produced H2 and ATP from CO (via a loosely attached CO dehydrogenase) or a chemical reductant. Finally, we show that Ech activity led to the translocation of both H+ and Na+ across the membrane of the IMVs. The H+ gradient was then used by the ATP synthase for energy conservation. These data demonstrate that the energy-converting hydrogenase in concert with an ATP synthase may be the simplest form of respiration; it combines carbon dioxide fixation with the synthesis of ATP in an ancient pathway.
Assuntos
Fenômenos Bioquímicos , Redes e Vias Metabólicas , Oxirredutases/metabolismo , Força Próton-Motriz/fisiologia , Thermoanaerobacter/metabolismo , Trifosfato de Adenosina/metabolismo , Aldeído Oxirredutases/metabolismo , Ciclo do Carbono , Monóxido de Carbono/metabolismo , Membrana Celular/metabolismo , Hidrogênio/metabolismo , Complexos Multienzimáticos/metabolismo , Família Multigênica , Oxirredução , Vesículas Secretórias/metabolismo , Sódio/metabolismo , Thermoanaerobacter/enzimologia , Thermoanaerobacter/genéticaRESUMO
Site-directed mutagenesis was employed to generate five different triple point mutations in the double mutant (C295A/I86A) of Thermoanaerobacter ethanolicus alcohol dehydrogenase (TeSADH) by computer-aided modeling with the aim of widening the small alkyl-binding pocket. TeSADH engineering enables the enzyme to accept sterically hindered substrates that could not be accepted by the wild-type enzyme. The underline in the mutations highlights the additional point mutation on the double mutant TeSADH introduced in this work. The catalytic efficiency (kcat/KM) of the M151A/C295A/I86A triple TeSADH mutant for acetophenone increased about 4.8-fold higher than that of the double mutant. A 2.4-fold increase in conversion of 3'-methylacetophenone to (R)-1-(3-methylphenyl)-ethanol with a yield of 87% was obtained by using V115A/C295A/I86A mutant in asymmetric reduction. The A85G/C295A/I86A mutant also produced (R)-1-(3-methylphenyl)-ethanol (1.7-fold) from 3'-methylacetophenone and (R)-1-(3-methoxyphenyl)-ethanol (1.2-fold) from 3'- methoxyacetophenone, with improved yield. In terms of thermal stability, the M151A/ C295A/I86A and V115A/C295A/I86A mutants significantly increased ΔT1/2 by +6.8°C and +2.4°C, respectively, with thermal deactivation constant (kd) close to the wild-type enzyme. The M151A/C295A/I86A mutant reacts optimally at 70 °C with almost 4 times more residual activity than the wild type. Considering broad substrate tolerance and thermal stability together, it would be promising to produce (R)-1-(3-methylphenyl)-ethanol from 3'- methylacetophenone by V115A/C295A/I86A, and (R)-1-phenylethanol from acetophenone by M151A/C295A/I86A mutant, in large-scale bioreduction processes.
Assuntos
Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Mutação Puntual , Thermoanaerobacter/enzimologia , Thermoanaerobacter/genética , Acetofenonas/química , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Álcoois/química , Domínio Catalítico/genética , Ensaios Enzimáticos , Estabilidade Enzimática , Engenharia Genética , Cetonas/química , Cinética , Modelos Moleculares , Conformação Molecular , Mutagênese Sítio-Dirigida , Análise de Sequência de DNA , Especificidade por SubstratoRESUMO
In past years, lots of research has been focused on the indigenous bacteria and their mechanisms, which help in enhanced oil recovery. Most of the oil wells in Indian subcontinent have temperature higher than 60⯰C. Also, the role of methanogenic consortia from high temperature petroleum reservoir for enhanced oil recovery (EOR) has not been explored much. Hence, in the present study methanogens isolated from thermophilic oil wells (70⯰C) were evaluated for enhanced oil recovery. Methane gas is produced by methanogens, which helps in oil recovery from depleted oil wells through reservoir re-pressurization and also can be recovered from reservoir along with crude oil as alternative energy source. Therefore, in this study indigenous methanogenic consortium (TERIL146) was enriched from high temperature oil reservoir showing (12â¯mmol/l) gas production along with other metabolites. Sequencing analysis revealed the presence of Methanothermobacter sp., Thermoanaerobacter sp., Gelria sp. and Thermotoga sp. in the consortium. Furthermore, the developed indigenous consortium TERIL146 showed 8.3% incremental oil recovery in sandpack assay. The present study demonstrates successful recovery of both oil and energy (gas) by the developed indigenous methanogenic consortium TERIL146 for potential application in thermophilic depleted oil wells of Indian subcontinent.
Assuntos
Bactérias/isolamento & purificação , Methanobacteriaceae/isolamento & purificação , Consórcios Microbianos , Campos de Petróleo e Gás/microbiologia , Bactérias/classificação , Bactérias/genética , Temperatura Alta , Microbiologia Industrial , Metano/metabolismo , Methanobacteriaceae/classificação , Methanobacteriaceae/genética , Filogenia , Análise de Sequência de DNA , Thermoanaerobacter/classificação , Thermoanaerobacter/genética , Thermoanaerobacter/isolamento & purificação , Thermotoga maritima/classificação , Thermotoga maritima/genética , Thermotoga maritima/isolamento & purificaçãoRESUMO
Cellulose and hemicellulose are the most abundant components in plant biomass. A preferred Consolidated Bioprocessing (CBP) system is one which can directly convert both cellulose and hemicellulose into target products without adding the costly hydrolytic enzyme cocktail. In this work, the thermophilic, cellulolytic, and anaerobic bacterium, Clostridium thermocellum DSM 1313, was engineered to grow on xylose in addition to cellulose. Both xylA (encoding for xylose isomerase) and xylB (encoding for xylulokinase) genes from the thermophilic anaerobic bacterium Thermoanaerobacter ethanolicus were introduced to enable xylose utilization while still retaining its inherent ability to grow on 6-carbon substrates. Targeted integration of xylAB into C. thermocellum genome realized simultaneous fermentation of xylose with glucose, with cellobiose (glucose dimer), and with cellulose, respectively, without carbon catabolite repression. We also showed that the respective H2 and ethanol production were twice as much when both xylose and cellulose were consumed simultaneously than when consuming cellulose alone. Moreover, the engineered xylose consumer can also utilize xylo-oligomers (with degree of polymerization of 2-7) in the presence of xylose. Isotopic tracer studies also revealed that the engineered xylose catabolism contributed to the production of ethanol from xylan which is a model hemicellulose in mixed sugar fermentation, demonstrating immense potential of this enhanced CBP strain in co-utilizing both cellulose and hemicellulose for the production of fuels and chemicals.
Assuntos
Celulose/metabolismo , Clostridium thermocellum/genética , Clostridium thermocellum/metabolismo , Fermentação , Engenharia Metabólica/métodos , Polissacarídeos/metabolismo , Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/metabolismo , Anaerobiose , Celobiose/metabolismo , Clonagem Molecular , Clostridium thermocellum/crescimento & desenvolvimento , Glucose/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool) , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Thermoanaerobacter/enzimologia , Thermoanaerobacter/genética , Xilose/metabolismoRESUMO
Thermoanaerobacter kivui is one of the very few thermophilic acetogenic microorganisms. It grows optimally at 66°C on sugars but also lithotrophically with H2 + CO2 or with CO, producing acetate as the major product. While a genome-derived model of acetogenesis has been developed, only a few physiological or biochemical experiments regarding the function of important enzymes in carbon and energy metabolism have been carried out. To address this issue, we developed a method for targeted markerless gene deletions and for integration of genes into the genome of T. kivui The strain naturally took up plasmid DNA in the exponential growth phase, with a transformation frequency of up to 3.9 × 10-6 A nonreplicating plasmid and selection with 5-fluoroorotate was used to delete the gene encoding the orotate phosphoribosyltransferase (pyrE), resulting in a ΔpyrE uracil-auxotrophic strain, TKV002. Reintroduction of pyrE on a plasmid or insertion of pyrE into different loci within the genome restored growth without uracil. We subsequently studied fructose metabolism in T. kivui The gene fruK (TKV_c23150) encoding 1-phosphofructosekinase (1-PFK) was deleted, using pyrE as a selective marker via two single homologous recombination events. The resulting ΔfruK strain, TKV003, did not grow on fructose; however, growth on glucose (or on mannose) was unaffected. The combination of pyrE as a selective marker and the natural competence of the strain for DNA uptake will be the basis for future studies on CO2 reduction and energy conservation and their regulation in this thermophilic acetogenic bacterium.IMPORTANCE Acetogenic bacteria are currently the focus of research toward biotechnological applications due to their potential for de novo synthesis of carbon compounds such as acetate, butyrate, or ethanol from H2 + CO2 or from synthesis gas. Based on available genome sequences and on biochemical experiments, acetogens differ in their energy metabolism. Thus, there is an urgent need to understand the carbon and electron flows through the Wood-Ljungdahl pathway and their links to energy conservation, which requires genetic manipulations such as deletion or overexpression of genes encoding putative key enzymes. Unfortunately, genetic systems have been reported for only a few acetogenic bacteria. Here, we demonstrate proof of concept for the genetic modification of the thermophilic acetogenic species Thermoanaerobacter kivui The genetic system will be used to study genes involved in biosynthesis and energy metabolism, and may further be applied to metabolically engineer T. kivui to produce fuels and chemicals.
Assuntos
Frutose/metabolismo , Genoma Bacteriano , Thermoanaerobacter/genética , Ciclo do Carbono , Metabolismo Energético/genética , Frutose/farmacologia , Deleção de Genes , Glucose/farmacologia , Recombinação Homóloga , Manose/farmacologia , Ácido Orótico/análogos & derivados , Ácido Orótico/farmacologia , Fosfofrutoquinases/deficiência , Fosfofrutoquinases/genética , Thermoanaerobacter/efeitos dos fármacos , Thermoanaerobacter/enzimologia , Thermoanaerobacter/crescimento & desenvolvimentoRESUMO
Secondary alcohol dehydrogenase (SADH) from Thermoanaerobacter ethanolicus reduces ketones to chiral alcohols, and generally obeys Prelog's Rule, with binding pockets for large and small alkyl substituents, giving (S)-alcohols. We have previously shown that mutations in both the large and small pockets can alter both substrate specificity and stereoselectivity. In the present work, Met-151 and Thr-153, residues located in the small pocket, were mutated to alanine. The M151A mutant SADH shows significantly lower activity and lower stereoselectivity for reduction of aliphatic ketones than wild-type SADH. Furthermore, M151A showed non-linear kinetics for reduction of acetone. T153A SADH shows lower activity but similar stereoselectivity for ketone reduction compared to wild-type SADH. The I86A/M151A/C295A and I86A/T153A/C295A triple mutant SADH show altered specificity for reduction of substituted acetophenones. These results confirm that these mutations are useful to combine with I86A/C295A SADH to expand the small pocket of SADH and broaden the substrate specificity.
Assuntos
Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Thermoanaerobacter/enzimologia , Thermoanaerobacter/genética , Oxirredutases do Álcool/química , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico/genética , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Especificidade por SubstratoRESUMO
The use of microbial cells to convert plant biomass directly to fuels and chemicals is referred to as consolidated bioprocessing (CBP). Members of the bacterial genus, Caldicellulosiruptor (Gram-positive, anaerobic hyperthermophiles) are capable of deconstructing plant biomass without enzymatic or chemical pretreatment. This is accomplished by the production and secretion of free, multi-domain enzymes that outperform commercial enzyme cocktails on some substrates. Here, we show that the exoproteome of Caldicellulosiruptor bescii may be enhanced by the heterologous expression of enzymes from Acidothermus cellulolyticus that act synergistically to improve sugar release from complex substrates; as well as improve cell growth. In this work, co-expression of the A. cellulolyticus Acel_0615 ß-glucanase (GH6 and GH12) and E1 endoglucanase (GH5) enzymes resulted in an increase in the activity of the exoproteome on Avicel; as well as an increase in growth of C. bescii on Avicel compared to the parental strain or the strain expressing the ß-glucanase alone. Our ability to engineer the composition and effectiveness of the exoproteome of these bacteria provides insight into the natural mechanism of plant cell wall deconstruction, as well as future directions for improving CBP. Biotechnol. Bioeng. 2017;114: 2474-2480. © 2017 Wiley Periodicals, Inc.