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Title 

Toward a generalized and high-throughput enzyme screening system based on artificial genetic circuits

 

인공 유전자회로를 이용한 초고속 효소탐색시스템 개발

Authors 

Su-Lim ChoiEugene RhaSang Jun LeeHaseong KimKilkoang KwonY S JeongY H RheeJae Jun SongH S KimSeung Goo Lee

Publisher 

American Chemical Society

Issue Date 

2014

Citation 

ACS Synthetic Biology, vol. 3, no. 0, pp. 163-171

Keywords 

DmpRenzyme screeninggenetic circuithigh-throughput screeningmetagenomephenolic compounds

Abstract 

Large-scale screening of enzyme libraries is essential for the development of cost-effective biological processes, which will be indispensable for the production of sustainable biobased chemicals. Here, we introduce a genetic circuit termed the Genetic Enzyme Screening System that is highly useful for high-throughput enzyme screening from diverse microbial metagenomes. The circuit consists of two AND logics. The first AND logic, the two inputs of which are the target enzyme and its substrate, is responsible for the accumulation of a phenol compound in cell. Then, the phenol compound and its inducible transcription factor, whose activation turns on the expression of a reporter gene, interact in the other logic gate. We confirmed that an individual cell harboring this genetic circuit can present approximately a 100-fold higher cellular fluorescence than the negative control and can be easily quantified by flow cytometry depending on the amounts of phenolic derivatives. The high sensitivity of the genetic circuit enables the rapid discovery of novel enzymes from metagenomic libraries, even for genes that show marginal activities in a host system. The crucial feature of this approach is that this single system can be used to screen a variety of enzymes that produce a phenol compound from respective synthetic phenyl-substrates, including cellulase, lipase, alkaline phosphatase, tyrosine phenol-lyase, and methyl parathion hydrolase. Consequently, the highly sensitive and quantitative nature of this genetic circuit along with flow cytometry techniques could provide a widely applicable toolkit for discovering and engineering novel enzymes at a single cell level.

ISSN 

2161-5063

Link 

http://dx.doi.org/10.1021/sb400112u

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2019-05-02


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