Ng Yao Zong, Baldera-Aguayo Pedro A, Cornish Virginia W
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.
Biochemistry. 2017 Oct 10;56(40):5260-5268. doi: 10.1021/acs.biochem.7b00602. Epub 2017 Sep 21.
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.
荧光偏振（FP）分析已被广泛用于研究酶动力学、抗体-抗原相互作用及其他生物相互作用。我们提出，FP分析可被改编为一种用于小分子的高通量且可能广泛适用的筛选方法。这在代谢工程中很有用，代谢工程是一种利用生物工程菌株合成具有药物、农业和工业重要性的化合物的有前景的方法。在那里，高产菌株的开发通常是一个成本高昂且耗时的过程。这个问题可以通过生成和测试大型突变菌株文库来解决。然而，当前一个关键瓶颈是缺乏用于检测小分子产物的高通量筛选方法。FP分析具有定量、灵敏、快速且廉价的特点。作为原理验证，我们建立了FP分析方法来筛选筑波链霉菌产生的FK506（他克莫司），该菌在96孔板中培养。对一个由160个菌落组成的紫外线诱变文库进行筛选，以鉴定出显示出更高FK506生产力的菌株。FP分析有潜力被推广用于检测广泛的其他小分子。
