title: Antisense RNA-based High-Throughput Screen System for Directed Evolution of Quorum Quenching Enzymes.
abstract: Quorum quenching (QQ) enzymes, which disrupt the quorum sensing signaling process, have attracted considerable attention as new antimicrobial agents. However, their low catalytic efficiency for quorum sensing molecules remains a challenge. Herein, we present an antisense RNA-based high-throughput screen system for directed evolution of a quorum quenching enzyme. The screening system was constructed by incorporating an antisense RNA (RyhB) into a synthetic module to quantitatively regulate the expression of a reporter gene fused with a sense RNA (sodB). To control the expression of a reporter gene in response to the catalytic activity of a quorum quenching enzyme, the region of interaction and mode between a pair of antisense (RyhB) and sense (sodB) RNAs was designed and optimized through the prediction of the secondary structure of the RNA pair. The screening system constructed was shown to lead to a significant reduction in the false-positive rate (average 42%) in the screening of N-acyl-homoserine lactonase (AiiA) with increased catalytic activity, resulting in a true-positive frequency of up to 76%. The utility and efficiency of the screening system were demonstrated by selecting an AiiA with 31-fold higher catalytic efficiency than the wild-type in three rounds of directed evolution. The present approach can be widely used for the screening of quorum quenching enzymes with the desired catalytic property, as well as for a synthetic network for a stringent regulation of the gene expression.
authors.affiliation: Department of Biological Sciences, Korea Advanced Institute of Science and Technology , 373-1, Gusung-dong, Yusung-gu, Daejon, 305-701, Korea.
authors.forename: Sang-Soo
authors.initials: SS
authors.lastname: Han
authors.affiliation: Department of Biological Sciences, College of Natural Sciences, Chonnam National University , Gwangju 500-757, Korea.|Department of Molecular Medicine (BK21plus), Chonnam National University Graduate School, Chonnam National University , Gwangju 500-757, Korea.
authors.forename: Won-Ji
authors.initials: WJ
authors.lastname: Park
authors.affiliation: Department of Biological Sciences, Korea Advanced Institute of Science and Technology , 373-1, Gusung-dong, Yusung-gu, Daejon, 305-701, Korea.
authors.forename: Hak-Sung
authors.initials: HS
authors.lastname: Kim
authors.affiliation: Department of Biological Sciences, College of Natural Sciences, Chonnam National University , Gwangju 500-757, Korea.
authors.forename: Geun-Joong
authors.initials: GJ
authors.lastname: Kim
mesh_terms: D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D001419:Bacteria / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005453:Fluorescence; D053038:Quorum Sensing / Q000235:genetics; D016372:RNA, Antisense / Q000235:genetics / Q000378:metabolism*; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; C104908:N-hexanoyl-L-homoserine lactone; D016372:RNA, Antisense; D054852:Small Molecule Libraries; D015107:4-Butyrolactone