title: A Cell-Cell Communication-Based Screening System for Novel Microbes with Target Enzyme Activities.
abstract: The development of synthetic biological devices has increased rapidly in recent years and the practical benefits of such biological devices are becoming increasingly clear. Here, we further improved the design of a previously reported high-throughput genetic enzyme screening system by investigating device-compatible biological components and phenol-mediated cell-cell communication, both of which increased the efficiency and practicality of the screening device without requiring the use of flow cytometry analysis. A sensor cell was designed to detect novel microbes with target enzyme activities on solid media by forming clear, circular colonies with fluorescence around the unknown microbes producing target enzymes. This mechanism of detection was enabled by the combination of pre-effector phenolic substrate treatment in the presence of target enzyme-producing microbes and control of the growth and fluorescence of remote sensor cells via phenol-mediated cell-cell communication. The sensor cells were applied to screen soil bacteria with phosphatase activity using phenyl phosphate as phenolic substrates. The sensor cells facilitated successful visualization of phosphatase activity in unknown microbes, which were identified by 16S rRNA analysis. Enzyme activity assays confirmed that the proposed screening technique was able to find 23 positive clones out of 33 selected colonies. Since many natural enzymatic reactions produce phenolic compounds from phenol-derived substrates, we anticipate that the proposed technique may have broad applications in the assessment and screening of novel microbes with target enzymes of interest. This method also can provide insights into the identification of novel enzymes for which screening assays are not yet available.
authors.affiliation: Synthetic Biology & Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology , 125 Gwahak-ro, Yuseong-gu, Daejeon, South Korea.
authors.forename: Haseong
authors.initials: H
authors.lastname: Kim
authors.affiliation: Synthetic Biology & Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology , 125 Gwahak-ro, Yuseong-gu, Daejeon, South Korea.
authors.forename: Eugene
authors.initials: E
authors.lastname: Rha
authors.affiliation: Synthetic Biology & Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology , 125 Gwahak-ro, Yuseong-gu, Daejeon, South Korea.|Biosystems and Bioengineering Program, University of Science and Technology , 217 Gajung-ro, Yuseong-gu, Daejeon, South Korea.
authors.forename: Wonjae
authors.initials: W
authors.lastname: Seong
authors.affiliation: Synthetic Biology & Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology , 125 Gwahak-ro, Yuseong-gu, Daejeon, South Korea.
authors.forename: Soo-Jin
authors.initials: SJ
authors.lastname: Yeom
authors.affiliation: Synthetic Biology & Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology , 125 Gwahak-ro, Yuseong-gu, Daejeon, South Korea.|Biosystems and Bioengineering Program, University of Science and Technology , 217 Gajung-ro, Yuseong-gu, Daejeon, South Korea.
authors.forename: Dae-Hee
authors.initials: DH
authors.lastname: Lee
authors.affiliation: Synthetic Biology & Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology , 125 Gwahak-ro, Yuseong-gu, Daejeon, South Korea.|Biosystems and Bioengineering Program, University of Science and Technology , 217 Gajung-ro, Yuseong-gu, Daejeon, South Korea.
authors.forename: Seung-Goo
authors.initials: SG
authors.lastname: Lee
mesh_terms: D000333:Aeromonas / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002450:Cell Communication; D002841:Chromatiaceae / Q000201:enzymology / Q000235:genetics; D004269:DNA, Bacterial / Q000302:isolation & purification; D004925:Escherichia / Q000201:enzymology / Q000235:genetics; D005434:Flow Cytometry; D005798:Genes, Bacterial; D057166:High-Throughput Screening Assays; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000302:isolation & purification; D056910:Republic of Korea; D017422:Sequence Analysis, DNA; D012705:Serratia / Q000201:enzymology / Q000235:genetics; D012763:Shigella flexneri / Q000201:enzymology / Q000235:genetics; D012988:Soil Microbiology; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; C080133:DmpR protein, Pseudomonas; D012336:RNA, Ribosomal, 16S; D015534:Trans-Activators; D010744:Phosphoric Monoester Hydrolases
keywords: cell−cell communication; enzyme; genetic circuit; high-throughput screening; metagenome; microbiome; phenol