Dissecting enzyme function with microfluidic-based deep mutational scanning

Abstract

Natural enzymes are incredibly proficient catalysts, but engineering them to have new or improved functions is challenging due to the complexity of how an enzyme's sequence relates to its biochemical properties. Here, we present an ultrahigh-throughput method for mapping enzyme sequence-function relationships that combines droplet microfluidic screening with next-generation DNA sequencing. We apply our method to map the activity of millions of glycosidase sequence variants. Microfluidic-based deep mutational scanning provides a comprehensive and unbiased view of the enzyme function landscape. The mapping displays expected patterns of mutational tolerance and a strong correspondence to sequence variation within the enzyme family, but also reveals previously unreported sites that are crucial for glycosidase function. We modified the screening protocol to include a hightemperature incubation step, and the resulting thermotolerance landscape allowed the discovery of mutations that enhance enzyme thermostability. Droplet microfluidics provides a general platform for enzyme screening that, when combined with DNAsequencing technologies, enables high-throughput mapping of enzyme sequence space.protein engineering | droplet-based microfluidics | high-throughput DNA sequencing E nzymes are powerful biological catalysts capable of remarkably accelerating the rates of chemical transformations (1). The molecular bases of these rate accelerations are often complex, using multiple steps, multiple catalytic mechanisms, and relying on numerous molecular interactions, in addition to those provided by the main catalytic groups. This complexity imposes a significant barrier to understanding how an enzyme's sequence impacts its function and, thus, on our ability to rationally design biocatalysts with new or enhanced functions (2-4).Comprehensive mappings of sequence-function relationships can be used to dissect the molecular basis of protein function in an unbiased manner (5). Growth selections or in vitro binding screens can be combined with next-generation DNA sequencing to generate detailed mappings between a protein's sequence and its biochemical properties, such as binding affinity, enzymatic activity, and stability (6-9). This deep mutational scanning approach has been used to study the structure of the protein fitness landscape, discover new functional sites, improve molecular energy functions, and identify beneficial combinations of mutations for protein engineering. However, these methods rely on functional assays coupled to cell growth or protein binding, severely limiting the types of proteins that can be analyzed. For example, most enzymes of biological or industrial relevance cannot be analyzed using existing methods because they do not catalyze a reaction that can be directly coupled to cell growth. Experimental advances are needed to broaden the applicability of deep mutational scanning to the diverse palette of functions performed by enzymes.In this paper, we present a general method for mapping protein sequence-func...

Robotic automation of droplet microfluidics

“…This workflow, and its variants, are valuable for applications of droplet microfluidics, including sequence-function mapping, in vitro enzyme evolution, and single-cell PCR, among others. [20][21][22][23] The in vitro evolution process consists of iterative cycles of diversity generation (mutagenesis) and screening (droplet sorting). For mutagenesis, error-prone PCR can amplify a wild type sequence to generate a diverse library of mutants.…”

Droplet microfluidics for synthetic biology

“…Continuous methods employ flow-focusing, in which an outer flow stream, often an oil phase with a droplet-stabilizing surfactant, encapsulates an inner aqueous phase to generate droplets. 6,7 Continuous methods can rapidly generate monodisperse droplets (up to 1-10 kHz), and are especially useful for high-throughput screening applications. 8,9 However, they allow limited control over addition or subtraction of reagents once droplets are formed.…”

“…Examples include the measurement of protein amount by expressing a protein with a fluorescent tag (e.g., GFP) and the measurement of enzyme activity by using fluorogenic substrates. 7,49,63 Fluorescent-activated droplet sorting (FADS, Fig. 3a recently been used for high-throughput screening.…”

Enzyme engineering in biomimetic compartments

“…A workflow in which droplet assays were combined with next generation sequencing was recently published [83]. …”