4. Conclusion

In summary, we report here the development of a sensitive, simple, and efficient fluorogenic assay that is suitable for high-throughput screening of ADHs. We demonstrated the potential of SDFA by performing directed evolution on PfODH through screening a combinatorial library. The selected enzyme variant exhibited a substantially higher kcat/km value toward (S )-2-octanol compared to its wild-type. In addition, the secretion-based nature of SDFA exempts the need for cell lysis, thus bypassing its disadvantages including the release of interfering cytosolic components, the additional cost of lysis reagent, the potential inconsistency of lysis efficiency, the extra effort of preserving intact culture for genome recovery. From this perspective, SDFA not only provides a sensitive and reliable way for determining ADHs activity, but also simplifies the screening procedure.
To the best of our knowledge, SDFA is the first secretion-based assay that can easily enable normalizing the catalytic activity of ADHs for high throughput screening. SDFA is in theory also applicable to other NAD(P)(H)-dependent enzymes besides ADHs and is suitable for high-throughput screening methods such as 96/384 well plate format. Although the method we have developed so far only works with the oxidative direction of the ADH-catalyzed reaction, SDFA should still be attractive to many protein engineers, because there are a large number of useful oxidative reactions catalyzed ADHs.