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Teth137, a Conserved Factor of Unknown Function from Thermoanaerobacter ethanolicus JW200, Represses the Transcription of the adhE Gene In Vitro.

ABSTRACT: Teth137, a 13.7 kD protein of unknown function from Thermoanaerobacter ethanolicus JW200, is encoded by 360 nucleotides and has been obtained by DNA-coupled column previously. However, no function study of Teth137 has been published. Homologous modeling of Teth137 shows the protein is comprised of a helix-turn-helix motif which is a typical DNA-binding domain. Therefore, it is speculated Teth137 is a DNA-binding protein and involved in transcription of the adhE gene (encodes alcohol dehydrogenase E). To investigate the function of Teth137, recombinant Teth137 is overexpressed in Escherichia coli JM109 and purified by DEAE column. Purified Teth137 exhibits the affinity with the adhE promoter region in gel electrophoresis mobility shift assay (GEMSA). Teth137 at the concentration of 48 ?M retards the migration of 5 nM of probe in the presence of the competitor DNA. Mutant analysis indicates that S69, T70, P71 and T72 are critical to protein-DNA interface; Gly substitutions at these residues results in the loss of the binding ability with the adhE promoter region. Moreover, T. ethanolicus JW200 RNA polymerase, ? subunit and template plasmid are prepared for in vitro transcription assay to detect the regulation function of Teth137. The results of the in vitro transcription show that the transcription of 5 nM of the template plasmid is inhibited by 48 ?M of Teth137.

SUBMITTER: Jing Q

PROVIDER: S-EPMC3626947 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Teth137, a Conserved Factor of Unknown Function from Thermoanaerobacter ethanolicus JW200, Represses the Transcription of the adhE Gene In Vitro.

Jing Qingqing Q Wang Jingkai J Wu Guogan G

Indian journal of microbiology 20121209 2

Teth137, a 13.7 kD protein of unknown function from Thermoanaerobacter ethanolicus JW200, is encoded by 360 nucleotides and has been obtained by DNA-coupled column previously. However, no function study of Teth137 has been published. Homologous modeling of Teth137 shows the protein is comprised of a helix-turn-helix motif which is a typical DNA-binding domain. Therefore, it is speculated Teth137 is a DNA-binding protein and involved in transcription of the adhE gene (encodes alcohol dehydrogenas ...[more]

PMID: 24426101

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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.

Dataset Information

Teth137, a Conserved Factor of Unknown Function from Thermoanaerobacter ethanolicus JW200, Represses the Transcription of the adhE Gene In Vitro.

Publications

Teth137, a Conserved Factor of Unknown Function from Thermoanaerobacter ethanolicus JW200, Represses the Transcription of the adhE Gene In Vitro.

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