High-throughput screening technologies for enzyme engineering

•Ultra-high-throughput technologies have emerged for enzyme engineering.•These technologies are enabling screens of millions of enzyme variants.•Cells or synthetic droplets can be used as compartments for enzyme reactions.•Microfluidic devices and microchamber arrays have been developed for screening.•Workflow, throughput, and assay format and flexibility varies for each technology.

Emerging technologies are enabling ultra-high-throughput screening of combinatorial enzyme libraries to identify variants with improved properties such as increased activity, altered substrate specificity, and increased stability. Each of these enzyme engineering platforms relies on compartmentalization of reaction components, similar to microtiter plate-based assays which have been commonly used for testing the activity of enzyme variants. The technologies can be broadly divided into three categories according to their spatial segregation strategy: (1) cells as reaction compartments, (2) in vitro compartmentalization via synthetic droplets, and (3) microchambers. Here, we discuss these emerging platforms, which in some cases enable the screening of greater than 10 million enzyme variants, and highlight benefits and limitations of each technology.

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