Thermobifida fusca YX strain:YX
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REPOSITORIES: ENA
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| GCA_000012405.1_sequence_report.txt | Txt |
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Project description:Thermobifida fusca transcriptome
| PRJNA281773 | ENA
Project description:Thermobifida fusca is a moderately thermophilic soil bacterium that belongs to Actinobacteria. It is a major degrader of plant cell walls and has been used as a model organism for the study of secreted, thermostable cellulases. The complete genome sequence showed that T. fusca has a single circular chromosome of 3,642,249 bp predicted to encode 3,117 proteins and 65 RNA species with a coding density of 85%. Genome analysis revealed the existence of 29 putative glycoside hydrolases in addition to the previously identified cellulases and xylanases. The glycosyl hydrolases include enzymes predicted to exhibit mainly dextran/starch- and xylan-degrading functions. T. fusca possesses two protein secretion systems: the sec general secretion system and the twin-arginine translocation system. Several of the secreted cellulases have sequence signatures indicating their secretion may be mediated by the twin-arginine translocation system. T. fusca has extensive transport systems for import of carbohydrates coupled to transcriptional regulators controlling the expression of the transporters and glycosylhydrolases. In addition to providing an overview of the physiology of a soil actinomycete, this study presents insights on the transcriptional regulation and secretion of cellulases which may facilitate the industrial exploitation of these systems.
| S-EPMC1899369 | biostudies-literature
Project description:Allylic alcohols are valuable precursors in the synthesis of pharmaceutical intermediates, agrochemicals and natural products. Regioselective oxidation of parental alkenes is a challenging task for chemical catalysts and requires several steps including protection and deprotection. Many cytochrome P450 enzymes are known to catalyse selective allylic hydroxylation under mild conditions. Here, we describe CYP154E1 from Thermobifida fusca YX that enables this type of oxidation. Several acyclic terpenoids were tested as possible substrates for CYP154E1, and the regio- and chemoselectivity of their oxidation was investigated. Using a previously established bioinformatics approach we identified position 286 in the active site of CYP154E1 which is putatively involved in substrate binding and thereby might have an effect on enzyme selectivity. To tune regio- and chemoselectivity of the enzyme three mutants at position 286 were constructed and used for substrate oxidation. All formed products were analysed with GC-MS and identified using chemically synthesised authentic samples and known compounds as references. Best regioselectivity towards geraniol and nerol was observed with the wild type enzyme mainly leading to 8-hydroxy derivatives (8-hydroxygeraniol or 8-hydroxynerol) with high selectivity (100% and 96% respectively). Highest selectivities during the oxidation of geranylacetone and nerylacetone were observed with the following variants: V286F led mainly to 7-hydroxygeranylacetone (60% of the total product) and V286A produced predominantly 12-hydroxynerylacetone (75% of total product). Thus, CYP154E1 and its mutants expand the tool-box for allylic hydroxylation in synthetic chemistry.
| S-EPMC4077532 | biostudies-literature
Project description:Thermostability and specific activity of enzymes are two of the most important properties for industrial biocatalysts. Here, we developed a petri dish-based double-layer high-throughput screening (HTS) strategy for rapid identification of desired mutants of polyphosphate glucokinase (PPGK) from a thermophilic actinobacterium, Thermobifida fusca YX, with both enhanced thermostability and activity. Escherichia coli colonies representing a PPGK mutant library were grown on the first-layer Phytagel-based plates, which can remain solid for 1 h, even at heat treatment temperatures of more than 100°C. The second layer that was poured on the first layer contained agarose, substrates, glucose 6-phosphate dehydrogenase (G6PDH), the redox dye tetranitroblue tetrazolium (TNBT), and phenazine methosulfate. G6PDH was able to oxidize the product from the PPGK-catalyzed reaction and generate NADH, which can be easily examined by a TNBT-based colorimetric assay. The best mutant obtained after four rounds of directed evolution had a 7,200-fold longer half-life at 55°C, 19.8°C higher midpoint of unfolding temperature (Tm ), and a nearly 3-fold enhancement in specific activities compared to those of the wild-type PPGK. The best mutant was used to produce 9.98 g/liter myo-inositol from 10 g/liter glucose, with a theoretical yield of 99.8%, along with two other hyperthermophilic enzymes at 70°C. This PPGK mutant featuring both great thermostability and high activity would be useful for ATP-free production of glucose 6-phosphate or its derived products.IMPORTANCE Polyphosphate glucokinase (PPGK) is an enzyme that transfers a terminal phosphate group from polyphosphate to glucose, producing glucose 6-phosphate. A petri dish-based double-layer high-throughput screening strategy was developed by using ultrathermostable Phytagel as the first layer instead of agar or agarose, followed by a redox dye-based assay for rapid identification of ultrathermostable PPGK mutants. The best mutant featuring both great thermostability and high activity could produce glucose 6-phosphate from glucose and polyphosphate without in vitro ATP regeneration.
| S-EPMC6070756 | biostudies-literature
Project description:Acyl-CoA carboxylases (AcCCase) are biotin-dependent enzymes that are capable of carboxylating more than one short chain acyl-CoA substrate. We have conducted structural and kinetic analyses of such an AcCCase from Thermobifida fusca YX, which exhibits promiscuity in carboxylating acetyl-CoA, propionyl-CoA, and butyryl-CoA. The enzyme consists of two catalytic subunits (TfAcCCA and TfAcCCB) and a non-catalytic subunit, TfAcCCE, and is organized in quaternary structure with a A6B6E6 stoichiometry. Moreover, this holoenzyme structure appears to be primarily assembled from two A3 and a B6E6 subcomplexes. The role of the TfAcCCE subunit is to facilitate the assembly of the holoenzyme complex, and thereby activate catalysis. Based on prior studies of an AcCCase from Streptomyces coelicolor, we explored whether a conserved Asp residue in the TfAcCCB subunit may have a role in determining the substrate selectivity of these types of enzymes. Mutating this D427 residue resulted in alterations in the substrate specificity of the TfAcCCase, increasing proficiency for carboxylating acetyl-CoA, while decreasing carboxylation proficiency with propionyl-CoA and butyryl-CoA. Collectively these results suggest that residue D427 of AcCCB subunits is an important, but not sole determinant of the substrate specificity of AcCCase enzymes.
| S-EPMC7835884 | biostudies-literature
Project description:A thermophilic Thermobifida fusca strain UPMC 901, harboring highly thermostable cellulolytic activity, was successfully isolated from oil palm empty fruit bunch compost. Its endoglucanase had the highest activity at 24?hours of incubation in carboxymethyl-cellulose (CMC) and filter paper. A maximum endoglucanase activity of 0.9?U/mL was achieved at pH 5 and 60?°C using CMC as a carbon source. The endoglucanase properties were further characterized using crude enzyme preparations from the culture supernatant. Thermal stability indicated that the endoglucanase activity was highly stable at 70?°C for 24?hours. Furthermore, the activity was found to be completely maintained without any loss at 50?°C and 60?°C for 144?hours, making it the most stable than other endoglucanases reported in the literature. The high stability of the endoglucanase at an elevated temperature for a prolonged period of time makes it a suitable candidate for the biorefinery application.
| S-EPMC6753106 | biostudies-literature
Project description:Thermobifida fusca is of biotechnological interest due to its ability to produce an array of plant cell wall hydrolytic enzymes. Nonetheless, only one T. fusca bacteriophage with genome information has been reported to date. This study was aimed at discovering more relevant bacteriophages to expand the existing knowledge of phage diversity for this host species. With this end in view, a thermostable T. fusca bacteriophage P318, which belongs to the Siphoviridae family, was isolated and characterized. P318 has a double-stranded DNA genome of 48,045 base pairs with 3'-extended COS ends, on which 52 putative ORFs are organized into clusters responsible for the order of genome replication, virion morphogenesis, and the regulation of the lytic/lysogenic cycle. In comparison with T. fusca and the previously discovered bacteriophage P1312, P318 has a much lower G+C content in its genome except at the region encompassing ORF42, which produced a protein with unknown function. P1312 and P318 share very few similarities in their genomes except for the regions encompassing ORF42 of P318 and ORF51 of P1312 that are homologous. Thus, acquisition of ORF42 by lateral gene transfer might be an important step in the evolution of P318.
| S-EPMC6893835 | biostudies-literature
Project description:The proteomics study of thermophilic microorganisms in the sludge heat-treated at 75 degrees Celsius, to investigate the heat-resistant enzymes related to the hydrolysis of sludge organic matter.
2023-05-05 | PXD042029 |
Project description:Actinomycetes have a long history of being the source of numerous valuable natural products and medicinals. To expedite product discovery and optimization of biochemical production, high-throughput technologies can now be used to screen the library of compounds present (or produced) at a given time in an organism. This not only facilitates chemical product screening, but also provides a comprehensive methodology to the study cellular metabolic networks to inform cellular engineering. Here, we present some of the first metabolomic data of the industrial cellulolytic actinomycete Thermobifida fusca generated using LC-MS/MS. The underlying objective of conducting global metabolite profiling was to gain better insight on the innate capabilities of T. fusca, with a long-term goal of facilitating T. fusca-based bioprocesses. The T. fusca metabolome was characterized for growth on two cellulose-relevant carbon sources, cellobiose and Avicel. Furthermore, the comprehensive list of measured metabolites was computationally integrated into a metabolic model of T. fusca, to study metabolic shifts in the network flux associated with carbohydrate and amino acid metabolism.
| S-EPMC5746737 | biostudies-literature
Project description:A relationship between processivity and synergism has not been reported for cellulases, although both characteristics are very important for hydrolysis of insoluble substrates. Mutation of two residues located in the active site tunnel of Thermobifida fusca exocellulase Cel6B increased processivity on filter paper. Surprisingly, mixtures of the Cel6B mutant enzymes and T. fusca endocellulase Cel5A did not show increased synergism or processivity, and the mutant enzyme which had the highest processivity gave the poorest synergism. This study suggests that improving exocellulase processivity might be not an effective strategy for producing improved cellulase mixtures for biomass conversion. The inverse relationship between the activities of many of the mutant enzymes with bacterial microcrystalline cellulose and their activities with carboxymethyl cellulose indicated that there are differences in the mechanisms of hydrolysis for these substrates, supporting the possibility of engineering Cel6B to target selected substrates.
| S-EPMC2772456 | biostudies-literature