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

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 (σ(54))-dependent transcription factor (TF) can be a valuable component of these biose...

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Autores principales: Kwon, Kil Koang, Yeom, Soo-Jin, Choi, Su-Lim, Rha, Eugene, Lee, Hyewon, Kim, Haseong, Lee, Dae-Hee, Lee, Seung-Goo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7142073/
https://www.ncbi.nlm.nih.gov/pubmed/32269250
http://dx.doi.org/10.1038/s41598-020-62892-1
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author Kwon, Kil Koang
Yeom, Soo-Jin
Choi, Su-Lim
Rha, Eugene
Lee, Hyewon
Kim, Haseong
Lee, Dae-Hee
Lee, Seung-Goo
author_facet Kwon, Kil Koang
Yeom, Soo-Jin
Choi, Su-Lim
Rha, Eugene
Lee, Hyewon
Kim, Haseong
Lee, Dae-Hee
Lee, Seung-Goo
author_sort Kwon, Kil Koang
collection PubMed
description 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 (σ(54))-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 (σ(70)) TFs. Here, we show that these unique characteristics of σ(54)-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 σ(54) 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 σ(54)-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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spelling pubmed-71420732020-04-11 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 Sci Rep Article 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 (σ(54))-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 (σ(70)) TFs. Here, we show that these unique characteristics of σ(54)-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 σ(54) 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 σ(54)-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. Nature Publishing Group UK 2020-04-08 /pmc/articles/PMC7142073/ /pubmed/32269250 http://dx.doi.org/10.1038/s41598-020-62892-1 Text en © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Kwon, Kil Koang
Yeom, Soo-Jin
Choi, Su-Lim
Rha, Eugene
Lee, Hyewon
Kim, Haseong
Lee, Dae-Hee
Lee, Seung-Goo
Acclimation of bacterial cell state for high-throughput enzyme engineering using a DmpR-dependent transcriptional activation system
title Acclimation of bacterial cell state for high-throughput enzyme engineering using a DmpR-dependent transcriptional activation system
title_full Acclimation of bacterial cell state for high-throughput enzyme engineering using a DmpR-dependent transcriptional activation system
title_fullStr Acclimation of bacterial cell state for high-throughput enzyme engineering using a DmpR-dependent transcriptional activation system
title_full_unstemmed Acclimation of bacterial cell state for high-throughput enzyme engineering using a DmpR-dependent transcriptional activation system
title_short Acclimation of bacterial cell state for high-throughput enzyme engineering using a DmpR-dependent transcriptional activation system
title_sort acclimation of bacterial cell state for high-throughput enzyme engineering using a dmpr-dependent transcriptional activation system
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7142073/
https://www.ncbi.nlm.nih.gov/pubmed/32269250
http://dx.doi.org/10.1038/s41598-020-62892-1
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