High-throughput droplet-based microfluidics for directed evolution of enzymes

Abstract

Natural enzymes have evolved over millions of years to allow for their effective operation within specific environments. However, it is significant to note that despite their wide structural and chemical diversity, relatively few natural enzymes have been successfully applied to industrial processes. To address this limitation, directed evolution (DE) (a method that mimics the process of natural selection to evolve proteins toward a user-defined goal) coupled with droplet-based microfluidics allows the detailed analysis of millions of enzyme variants on ultra-short timescales, and thus the design of novel enzymes with bespoke properties. In this review, we aim at presenting the development of DE over the last years and highlighting the most important advancements in droplet-based microfluidics, made in this context towards the high-throughput demands of enzyme optimization. Specifically, an overview of the range of microfluidic unit operations available for the construction of DE platforms is provided, focusing on their suitability and benefits for cell-based assays, as in the case of directed evolution experimentations.

Keywords: directed evolution; droplet-based microfluidics; high throughput screening; protein engineering; single-cell.

Figure 1

Schematic representation of a directed evolution campaign. The campaign comprises iterative rounds in which a parent gene, coding for an enzyme of interest, is subjected to random mutagenesis (or rational design) to yield a mutant library. Enzyme variants are subsequently expressed and screened for a desired property, such as activity. Improved variants are isolated, evaluated and then used to parent the next round of the evolution cycle. Reprinted and reproduced from 4, with the permission of SciELO Publishing.

Figure 2

Generic workflow of microfluidic sorting. Single cells expressing a library of recombinant enzymes together with cell lysis solution and substrate are encapsulated within pL‐volume droplets and their contents are mixed. Subsequently, droplets are incubated and then sorted according to the fluorescence signal. Reprinted and reproduced from 16, with the permission of Elsevier Publishing.

Figure 3

Conventional library screening methods for the directed evolution of enzymes. (A) (i) Selection screening and (ii) agar‐plate assays. (B) Microtiter‐plate (MTP) screening. (C) Cell‐based compartmentalization coupled with fluorescence‐activated cell sorting (FACS). This approach relies on either the substrate being able to freely diffuse across the cell membrane to reach cytoplasmically expressed enzyme, or enzyme being displayed on the cell surface to react with substrate. (D) (i) Whole‐cell and (ii) in vitro compartmentalization (IVC) coupled with FACS, where sorting is made possible by the production of w/o/w double emulsions. Reprinted and reproduced from 4, with the permission of SciELO Publishing.

Figure 4

(A)‐(E) Structures of the most commonly used flow‐based droplet generators. Aqueous flow and oil flow are labeled as “w” and “o” respectively, and arrows indicate flow directions. Reprinted and reproduced from 35, with the permission of MDPI Publishing.

Figure 5

Examples of droplet unit operations. (A) Pillar‐induced droplet merging. Reprinted and reproduced from 72, with the permission of Royal Society of Chemistry Publishing. (B) Self‐synchronising pairwise production of droplets by step emulsification. Reprinted and reproduced from 80, with the permission of American Institute of Physics Publishing. (i) Schematic of microfluidic droplet generator and (ii) time series of optical images displaying formation of two different droplet populations. (C) Reinjection and passive synchronization of droplets resulting in A‐B alternating pattern. Reprinted and reproduced from 83, with the permission of Royal Society of Chemistry Publishing. (D) Electric field‐triggered picoinjection. Reprinted and reproduced from 84, with the permission of National Academy of Sciences of the United States of America Publishing. (E) On‐chip droplet incubation using trap arrays. Reprinted and reproduced from 86, with the permission of Royal Society of Chemistry Publishing. (F) Fluorescence activated droplet sorting (FADS). Reprinted and reproduced from 87, with the permission of Royal Society of Chemistry Publishing. (G) On‐chip long‐term incubation in delay‐lines. Reprinted and reproduced from 88, with the permission of Nature Publishing Group.