earticle

영어

Molecular evolution has proved to be powerful for creation of enzymes with desired catalytic properties. However, this approach is often limited by the lack of high-throughput screening system. In particular, few HTS system is available for evolution of hydrolases with practical applications. In this study, we demonstrate the construction and use of a high-throughput screening system for directed evolution of organophosphate-degrading enzymes for degrading lethal nerve agents and toxic pesticides. This high -throughput screening system was combined with a fluorescence-activated cell sorting (FACS), full-HTS system and a genetic circuit that is composed of a transcription factor DmpR activated by phenolic compounds and EGFP. Accordingly, fluorescence intensities of host cells are dependent on the catalytic activity of the enzymes, and the cells expressing enzymes with high catalytic activities are screened by FACS and full-HTS. Methyl parathion hydrolase from Pseudomonas sp. WBC-3 and p-nitrophenyl diphenylphosphate were used as a model enzyme and ananalogue of G-type nerve agents, respectively. The utility of the genetic circuit-based HTS system was demonstrated by successfully selecting a triple mutant with a 100-fold higher kcat/Km than the wild-type enzyme after 3 rounds of directed evolution. Contribution of individual mutations to the catalytic efficiency was elucidated by mutational and structural analyses. Our results clearly demonstrate that the DmpR-based genetic circuit system can be effectively used for directed evolution of organophosphate-degrading enzymes.