Development of efficient enzymes by directed evolution
The ultrahigh-throughput analytical capacity of droplet-based microfluidics allows to screen the activity of millions of enzyme variants in a single day and selecting those molecules with the highest catalytic rate. With this technology, we devise directed evolution procedures in which rounds of mutagenesis and ultrahigh-throughput screenings are performed to rapidly identify very efficient enzymes based on their turnover capacity. We demonstrated the efficiency of this selection scheme by selecting mutants of the X-motif ribozyme affording a 30-fold higher kcatwhile no longer suffering from product inhibition.
Typical microfluidic-assisted screening workflow used to selected rare improved catalysts contained in a mutant library.
- Ryckelynck M., Baudrey S., Rick C., Marin A., Coldren F., Westhof E., Griffiths A.D. (2015). Using droplet-based microfluidics to improve the catalytic properties of RNA under multiple-turnover conditions. RNA, 21, 458-469.
- Autour A., Ryckelynck M. (2017). Ultrahigh-Throughput Improvement and Discovery of Enzymes Using Droplet-Based Microfluidic Screening. Micromachines, 8(4), 128.