Thermoanaerobacter ethanolicus CCSD1
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PROVIDER: PRJNA33633 | ENA |
REPOSITORIES: ENA
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Project description:BackgroundThermoanaerobacter ethanolicus produces a considerable amount of ethanol from a range of carbohydrates and is an attractive candidate for applications in bioconversion processes. A genetic system with reusable selective markers would be useful for deleting acid production pathways as well as other genetic modifications.ResultsThe thymidine kinase (tdk) gene was deleted from T. ethanolicus JW200 to allow it to be used as a selectable marker, resulting in strain X20. Deletion of the tdk gene reduced growth rate by 20 %; however, this could be reversed by reintroducing the tdk gene (strain X20C). The tdk and high-temperature kanamycin (htk) markers were tested by using them to delete lactate dehydrogenase (ldh). During positive selection of ldh knockouts in strain X20 on kanamycin agar plates, six out of seven picked colonies were verified transformants. Deletion of ldh reduced lactic acid production by 90 %. The tdk and 5-fluoro-2'-deoxyuridine (FUDR) combination worked reliably as demonstrated by successful tdk removal in all 21 colonies tested.ConclusionA gene deletion and integration system with reusable markers has been developed for Thermoanaerobacter ethanolicus JW200 with positive selection on kanamycin and negative selection on FUDR. Gene deletion was demonstrated by ldh gene deletion and gene integration was demonstrated by re-integration of the tdk gene. Transformation via a natural competence protocol could use DNA PCR products amplified directly from Gibson Assembly mixture for efficient genetic modification.
| S-EPMC4857275 | biostudies-literature
Project description:Thermophilic ethanol-producing bacterium
| PRJNA18603 | ENA
Project description: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.
| S-EPMC7755235 | biostudies-literature
Project description:This study was conducted to identify the genes involved in the synthesis of membrane-spanning lipids in Thermoanaerobacter ethanolicus JW 200.
2022-10-26 | GSE211546 | GEO
Project description:The genes encoding xylose isomerase (xylA) and xylulose kinase (xylB) from the thermophilic anaerobe Thermoanaerobacter ethanolicus were found to constitute an operon with the transcription initiation site 169 nucleotides upstream from the previously assigned (K. Dekker, H. Yamagata, K. Sakaguchi, and S. Udaka, Agric. Biol. Chem. 55:221-227, 1991) promoter region. The bicistronic xylAB mRNA was processed by cleavage within the 5'-terminal portion of the XylB-coding sequence. Transcription of xylAB was induced in the presence of xylose, and, unlike in all other xylose-utilizing bacteria studied, was not repressed by glucose. The existence of putative xyl operator sequences suggested that xylose utilization is controlled by a repressor-operator mechanism. The T. ethanolicus xylB gene coded for a 500-amino-acid-residue protein with a deduced amino acid sequence highly homologous to those of other XylBs. This is the first report of an xylB nucleotide sequence and an xyLAB operon from a thermophilic anaerobic bacterium.
| S-EPMC106996 | biostudies-literature
Project description:Thermoanaerobacter ethanolicus JW 200 has been identified as a potential sustainable biofuel producer due to its ability to readily ferment carbohydrates to ethanol. A hybrid sequencing approach, combining Oxford Nanopore and Illumina DNA sequence reads, was applied to produce a single contiguous genome sequence of 2,911,280?bp.
| S-EPMC6346170 | biostudies-literature
Project description:This SuperSeries is composed of the SubSeries listed below.
2022-10-26 | GSE211548 | GEO
Project description:The Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase I86A mutant is stereospecific for (R)-alcohols instead of (S)-alcohols. Pyramidal crystals grown in the presence of (R)-phenylethanol via the hanging-drop vapour-diffusion method diffracted to 3.2 A resolution at the Canadian Light Source. The crystal belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 80.23, b = 124.90, c = 164.80 A. The structure was solved by molecular replacement using the structure of T. brockii SADH (PDB entry 1ykf).
| S-EPMC2898473 | biostudies-literature
Project description:Thermoanaerobacter ethanolicus LL1204 resequencing genome sequencing
| PRJNA333453 | ENA
Project description:Thermoanaerobacter ethanolicus LL1205 resequencing genome sequencing
| PRJNA333454 | ENA