[논문]Acclimation of bacterial cell state for high-throughput enzyme engineering using a DmpR-dependent transcriptional activation system

Kwon, Kil Koang; Yeom, Soo-Jin; Choi, Su-Lim; Rha, Eugene; Lee, Hyewon; Kim, Haseong; Lee, Dae-Hee; Lee, Seung-Goo

doi:10.1038/s41598-020-62892-1

[해외논문] Acclimation of bacterial cell state for high-throughput enzyme engineering using a DmpR-dependent transcriptional activation system 원문보기

Scientific reports, v.10, 2020년, pp.6091 -

Kwon, Kil Koang (Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141 Republic of Korea) , Yeom, Soo-Jin (Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141 Republic of Korea) , Choi, Su-Lim (Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141 Republic of Korea) , Rha, Eugene (Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141 Republic of Korea) , Lee, Hyewon (Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141 Republic of Korea) , Kim, Haseong (Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141 Republic of Korea) , Lee, Dae-Hee (Synthetic Biology and Bioengineering Research Center, Korea Research I) , Lee, Seung-Goo

Abstract ▼ AI-Helper

Genetic circuit-based biosensors have emerged as an effective analytical tool in synthetic biology; these biosensors can be applied to high-throughput screening of new biocatalysts and metabolic pathways. Sigma 54 (σ54)-dependent transcription factor (TF) can be a valuable component of these biosensors owing to its intrinsic silent property compared to most of the housekeeping sigma 70 (σ70) TFs. Here, we show that these unique characteristics of σ54-dependent TFs can be used to control the host cell state to be more appropriate for high-throughput screening. The acclimation of cell state was achieved by using guanosine (penta)tetraphosphate ((p)ppGpp)-related genes (relA, spoT) and nutrient conditions, to link the σ54 TF-based reporter expression with the target enzyme activity. By controlling stringent programmed responses and optimizing assay conditions, catalytically improved tyrosine phenol lyase (TPL) enzymes were successfully obtained using a σ54-dependent DmpR as the TF component, demonstrating the practical feasibility of this biosensor. This combinatorial strategy of biosensors using σ factor-dependent TFs will allow for more effective high-throughput enzyme engineering with broad applicability.

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