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

Abstract

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.