Systematic promoter design for plasmid-encoded <i>S</i>-adenosylmethionine sensing systems
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- Watanabe Taro
- Department of Applied Chemistry, Faculty of Science, and Engineering, Waseda University Kirin Central Research Institute, Kirin Holdings Company, Limited
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- Kimura Yuki
- Department of Applied Chemistry, Faculty of Science, and Engineering, Waseda University
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- Umeno Daisuke
- Department of Applied Chemistry, Faculty of Science, and Engineering, Waseda University
抄録
<p>S-adenosylmethionine (SAM) is an important biomolecule that mainly acts as a methyl donor and plays many roles in a variety of biological functions. SAM is also required for the biosynthesis of valuable methylated compounds, but its supply is a bottleneck for these biosynthetic pathways. To overcome this bottleneck and to reconfigure SAM homeostasis, a high-throughput sensing system for changes in intracellular SAM availability is required. We constructed a plasmid that can detect the factors that can alter SAM availability using minimal components. It does so by placing a fluorescent protein under a promoter controlled by endogenous MetJ, a transcription factor that represses its own regulons upon binding with SAM. Next, to validate SAM-responsive behavior, we systematically reconstructed 10 synthetic promoters with different positions and with different number of metbox sites, sequences of MetJ binding. We found that a position between the −35 box and the −10 box was the most effective for repression and that this setup was suitable for detecting the genetic or environmental factors that can deplete and recover the intracellular SAM availability. Overall, the response patterns of the synthetic MetJ-regulated promoters characterized in this study may be useful for the development of further SAM biosensing systems.</p>
収録刊行物
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- The Journal of General and Applied Microbiology
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The Journal of General and Applied Microbiology advpub (0), 2024
公益財団法人 応用微生物学・分子細胞生物学研究奨励会