title: Toward a generalized and high-throughput enzyme screening system based on artificial genetic circuits.
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.
authors.affiliation: Biochemicals and Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology , 125 Gwahak-ro, Yuseong-gu, Daejeon, South Korea.
authors.forename: Su-Lim
authors.initials: SL
authors.lastname: Choi
authors.forename: Eugene
authors.initials: E
authors.lastname: Rha
authors.forename: Sang Jun
authors.initials: SJ
authors.lastname: Lee
authors.forename: Haseong
authors.initials: H
authors.lastname: Kim
authors.forename: Kilkoang
authors.initials: K
authors.lastname: Kwon
authors.forename: Young-Su
authors.initials: YS
authors.lastname: Jeong
authors.forename: Young Ha
authors.initials: YH
authors.lastname: Rhee
authors.forename: Jae Jun
authors.initials: JJ
authors.lastname: Song
authors.forename: Hak-Sung
authors.initials: HS
authors.lastname: Kim
authors.forename: Seung-Goo
authors.initials: SG
authors.lastname: Lee
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D004798:Enzymes / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000379:methods*; D054892:Metagenome; D059010:Single-Cell Analysis; D058615:Synthetic Biology / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D004798:Enzymes