Speaker
Description
Directed evolution (DE) is a process of iterative variant generation, screening, and selection to find enzyme variants with improved or specialized functional properties. Advances in droplet-based microfluidics have further expedited DE allowing for rapid screening of large enzyme libraries [1]. Enzyme-expressing genes can be compartmentalized with a fluorogenic substrate inside picoliter-scale water-in-oil emulsions for a high-throughput (~2 kHz) fluorescence-activated droplet sorting based on the enzyme performance. However, fluorogenic assays for enzymatic systems are limited.
To address this limitation, absorbance-based detection assays, which represent a larger spectrum have been reported [1, 2]. A range of different chromogenic substrates can be tested using these techniques, unlocking previously unaccessed sequences. Nevertheless, absorption is weak at the micron scale and droplet-interface-induced scattering decreases detection sensitivity. Measurements are therefore often performed at a cost of increasing droplet sizes or acquisition times, which limits throughput to <1 kHz. Moreover, studies to date have been mostly focused on absorbance-based identification and enrichment of enzymes with improved activity or thermostability (e.g., phenylalanine dehydrogenase [2]). However, redox potential, a key enzyme property that governs electron transfer (i.e., oxidation-reduction kinetics), remains largely unexplored via DE.
Here, we address the challenges associated with signal sensitivity using a custom confocal Absorbance-Activated Droplet Sorting (cAADS) system [3]. The illumination and detection optics of the confocal imaging platform are spatially conjugated and focused on the same diffraction-limited spot, allowing precise absorbance detection of droplets inside a microchannel and rejecting out-of-focus scattered light.
The increased sensitivity allows us to conduct absorbance measurements at ultrahigh throughput (5.4 kHz) from droplets as small as 10 pL, and sorting of 50 pL droplets at frequencies up to 2.6 kHz with 99% efficiency. The methodology is demonstrated by enrichment of active Bilirubin Oxidase (BOD) variants. Ultimately, we can screen libraries of ~1 million variants per day using chromogenic substrates as specific redox potential markers. As a proof of concept, a bacterial BOD with a low redox potential (+0.34V vs. Ag/AgCl at pH 7 [4]) will be used as a model with the objective to identify a BOD with a redox potential ~+0.5V vs. Ag/AgCl at pH 7 that can be efficiently used in biofuel cells.
References
[1] E. J. Medcalf, M. Gantz, T. S. Kaminski, and F. Hollfelder, "Ultra-high-throughput absorbance-activated droplet sorting for enzyme screening at kilohertz frequencies”, Anal. Chem., vol. 95, pp. 4597-4604, 2023.
[2] F. Gielen, R. Hours, S. Emond, M. Fischlechner, U. Schell, and F. Hollfelder, “Ultrahigh-throughput-directed enzyme evolution by absorbance-activated droplet sorting (AADS)”, Proc. Natl. Acad. Sci. U.S.A., vol. 113, pp. e7383-7389, 2016.
[3] A. M. Kaba, S. Gounel, T. Beneyton, L. Buisson, J-C Baret, N. Mano, “Confocal absorbance-activated droplet sorting (cAADS) for enzyme engineering”, Adv. Sci., vol. 12, e05324, 2025.
[4] S. Gounel, J. Rouhana, C. Stines-Chaumeil, M. Cadet, and N. Mano, "Increasing the catalytic activity of Bilirubin oxidase from Bacillus pumilus: importance of host strain and chaperones proteins", J. Biotech., vol. 20, pp. 19-25, 2016.