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Biochemistry. 2017 Oct 10;56(40):5260-5268. doi: 10.1021/acs.biochem.7b00602. Epub 2017 Sep 21.

Fluorescence Polarization Assay for Small Molecule Screening of FK506 Biosynthesized in 96-Well Microtiter Plates.

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Department of Chemistry, Columbia University in the City of New York , 550 West 120th Street, Northwest Corner Building 1206, New York, New York 10027, United States.
Integrated Program in Cellular, Molecular and Biomedical Studies, Columbia University in the City of New York , New York, New York 10032, United States.
Department of Systems Biology, Irving Cancer Research Center, Columbia University in the City of New York , 1130 St. Nicholas Avenue, New York, New York 10032, United States.


The fluorescence polarization (FP) assay has been widely used to study enzyme kinetics, antibody-antigen interactions, and other biological interactions. We propose that the FP assay can be adapted as a high-throughput and potentially widely applicable screen for small molecules. This is useful in metabolic engineering, which is a promising approach to synthesizing compounds of pharmaceutical, agricultural, and industrial importance using bioengineered strains. There, the development of high-yield strains is often a costly and time-consuming process. This problem can be addressed by generating and testing large mutant strain libraries. However, a current key bottleneck is the lack of high-throughput screens to detect the small molecule products. The FP assay is quantitative, sensitive, fast, and cheap. As a proof of principle, we established the FP assay to screen for FK506 (tacrolimus) produced by Streptomyces tsukubaensis, which was cultivated in 96-well plates. An ultraviolet mutagenized library of 160 colonies was screened to identify strains showing higher FK506 productivities. The FP assay has the potential to be generalized to detect a wide range of other small molecules.

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