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

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 <i>Streptomyces tsukubaensis</i>, 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

HbIDI, SlIDI and EcIDI: A comparative study of isopentenyl diphosphate isomerase activity and structure

In this study, we cloned, expressed and purified the isopentenyl diphosphate isomerases (IDIs) from two plants, Hevea brasiliensis and Solanum lycopersicum, and compared them to the already well characterized Escherichia coli IDI. Phylogenetic analysis showed high homology between the three enzymes. Their catalytic activity was investigated in vitro with recombinant purified enzymes and in vivo by complementation colorimetric tests. The three enzymes displayed consistent activities both in vitro and in vivo. In term of structure, studied by ATR-FTIR and molecular modeling, it is clear that both plant enzymes are more related to their human homologue than to E. coli IDI. But it is assumed that EcIDI represent the minimalistic part of the catalytic core, as both plant enzymes present a supplementary sequence forming an extra alpha-helice surrounding the catalytic site that could facilitate the biocatalysis. New potential biotechnological applications may be envisaged. (C) 2016 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved