DC Field | Value | Language |
---|---|---|
dc.contributor.author | Kwon, Kil Koang | ko |
dc.contributor.author | Lee, Dae-Hee | ko |
dc.contributor.author | Kim, Su Jin | ko |
dc.contributor.author | Choi, Su-Lim | ko |
dc.contributor.author | Rha, Eugene | ko |
dc.contributor.author | Yeom, Soo-Jin | ko |
dc.contributor.author | Subhadra, Bindu | ko |
dc.contributor.author | Lee, Jinhyuk | ko |
dc.contributor.author | Jeong, Ki Jun | ko |
dc.contributor.author | Lee, Seung-Goo | ko |
dc.date.accessioned | 2018-03-21T02:05:58Z | - |
dc.date.available | 2018-03-21T02:05:58Z | - |
dc.date.created | 2018-02-27 | - |
dc.date.created | 2018-02-27 | - |
dc.date.created | 2018-02-27 | - |
dc.date.created | 2018-02-27 | - |
dc.date.issued | 2018-02 | - |
dc.identifier.citation | SCIENTIFIC REPORTS, v.8 | - |
dc.identifier.issn | 2045-2322 | - |
dc.identifier.uri | http://hdl.handle.net/10203/240559 | - |
dc.description.abstract | Genetic circuit-based biosensors are useful in detecting target metabolites or in vivo enzymes using transcription factors (Tx) as a molecular switch to express reporter signals, such as cellular fluorescence and antibiotic resistance. Herein, a phenol-detecting Tx (DmpR) was employed as a critical tool for enzyme engineering, specifically for the rapid analysis of numerous mutants with multiple mutations at the active site of tryptophan-indole lyase (TIL, EC 4.1.99.1). Cellular fluorescence was monitored cell-by-cell using flow cytometry to detect the creation of phenolic compounds by a new tyrosine-phenollyase (TPL, EC 4.1.99.2). In the TIL scaffold, target amino acids near the indole ring (Asp(137), Phe(304), Val(394), Ile(396) and His(463)) were mutated randomly to construct a large diversity of specificity variations. Collection of candidate positives by cell sorting using flow cytometry and subsequent shuffling of beneficial mutations identified a critical hit with four mutations (D137P, F304D, V394L, and I396R) in the TIL sequence. The variant displayed one-thirteenth the level of TPL activity, compared with native TPLs, and completely lost the original TIL activity. The findings demonstrate that hypersensitive, Tx-based biosensors could be useful critically to generate new activity from a related template, which would alleviate the current burden to high-throughput screening. | - |
dc.language | English | - |
dc.publisher | NATURE PUBLISHING GROUP | - |
dc.title | Evolution of enzymes with new specificity by high-throughput screening using DmpR-based genetic circuits and multiple flow cytometry rounds | - |
dc.type | Article | - |
dc.identifier.wosid | 000424449100037 | - |
dc.identifier.scopusid | 2-s2.0-85052863670 | - |
dc.type.rims | ART | - |
dc.citation.volume | 8 | - |
dc.citation.publicationname | SCIENTIFIC REPORTS | - |
dc.identifier.doi | 10.1038/s41598-018-20943-8 | - |
dc.contributor.localauthor | Jeong, Ki Jun | - |
dc.contributor.nonIdAuthor | Kwon, Kil Koang | - |
dc.contributor.nonIdAuthor | Lee, Dae-Hee | - |
dc.contributor.nonIdAuthor | Kim, Su Jin | - |
dc.contributor.nonIdAuthor | Choi, Su-Lim | - |
dc.contributor.nonIdAuthor | Rha, Eugene | - |
dc.contributor.nonIdAuthor | Yeom, Soo-Jin | - |
dc.contributor.nonIdAuthor | Subhadra, Bindu | - |
dc.contributor.nonIdAuthor | Lee, Jinhyuk | - |
dc.contributor.nonIdAuthor | Lee, Seung-Goo | - |
dc.description.isOpenAccess | Y | - |
dc.type.journalArticle | Article | - |
dc.subject.keywordPlus | TYROSINE PHENOL-LYASE | - |
dc.subject.keywordPlus | TRYPTOPHAN INDOLE-LYASE | - |
dc.subject.keywordPlus | ESCHERICHIA-COLI | - |
dc.subject.keywordPlus | DIRECTED EVOLUTION | - |
dc.subject.keywordPlus | PROTEIN DESIGN | - |
dc.subject.keywordPlus | ACTIVE-SITE | - |
dc.subject.keywordPlus | MECHANISM | - |
dc.subject.keywordPlus | INACTIVATION | - |
dc.subject.keywordPlus | PERSPECTIVE | - |
dc.subject.keywordPlus | BIOLOGY | - |
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