A High-Throughput Screening System Based on Droplet Microfluidics for Glucose Oxidase Gene Libraries

Abstract

Glucose oxidase (GOx) is an important industrial enzyme that can be optimized for specific applications by mutagenesis and activity-based screening. To increase the efficiency of this approach, we have developed a new ultrahigh-throughput screening platform based on a microfluidic lab-on-chip device that allows the sorting of GOx mutants from a saturation mutagenesis library expressed on the surface of yeast cells. GOx activity was measured by monitoring the fluorescence of water microdroplets dispersed in perfluorinated oil. The signal was generated via a series of coupled enzyme reactions leading to the formation of fluorescein. Using this new method, we were able to enrich the yeast cell population by more than 35-fold for GOx mutants with higher than wild-type activity after two rounds of sorting, almost double the efficiency of our previously described flow cytometry platform. We identified and characterized novel GOx mutants, the most promising of which (M6) contained a combination of six point mutations that increased the catalytic constant k_cat by 2.1-fold compared to wild-type GOx and by 1.4-fold compared to a parental GOx variant. The new microfluidic platform for GOx was therefore more sensitive than flow cytometry and supports comprehensive screens of gene libraries containing multiple mutations per gene.

Keywords: enzyme optimization; fluorescent label; protein engineering; sorting.

Figure 1

A schematic showing the reaction pathway for the vanadium bromoperoxide-coupled fluorescence assay for the detection of glucose oxidase activity within aqueous microdroplets.

Figure 2

The 16 positions in glucose oxidase that were changed by site-directed mutagenesis.

Figure 3

Analysis of glucose oxidase (GOx) library L1 using the new microfluidics system (upper row) and on standard agar plates (lower row). The library was analyzed before sorting (BS), after the first (S1), second (S2) and third (S3) rounds of sorting (S3). Red fluorescence (showing the number of GOx molecules) reflects the detection of a C-terminal Myc epitope using a mouse primary anti-c-Myc antibody and a secondary goat anti-mouse IgG antibody labeled with DyLight 550. The sorting gate in the upper panels is between the green and pink lines.

Figure 4

Proportion of glucose oxidase variants in the L1 library based on activity compared to the wild-type enzyme. The charts show the proportion of variants with no activity, less than wild-type activity, and more than wild-type activity before sorting (BS), and after the first (S1), second (S2) and third (S3) rounds of sorting.

Figure 5

The M6 mutant of glucose oxidase features amino acid substitutions at six positions.