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. 2018 Sep 24;143(19):4747-4755.
doi: 10.1039/c8an00851e.

Ultra-high Throughput Functional Enrichment of Large Monoamine Oxidase (MAO-N) Libraries by Fluorescence Activated Cell Sorting

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Free PMC article

Ultra-high Throughput Functional Enrichment of Large Monoamine Oxidase (MAO-N) Libraries by Fluorescence Activated Cell Sorting

Joanna C Sadler et al. Analyst. .
Free PMC article

Abstract

Directed evolution enables the improvement and optimisation of enzymes for particular applications and is a valuable tool for biotechnology and synthetic biology. However, studies are often limited in their scope by the inability to screen very large numbers of variants to identify improved enzymes. One class of enzyme for which a universal, operationally simple ultra-high throughput (>106 variants per day) assay is not available is flavin adenine dinucleotide (FAD) dependent oxidases. The current high throughput assay involves a visual, colourimetric, colony-based screen, however this is not suitable for very large libraries and does not enable quantification of the relative fitness of variants. To address this, we describe an optimised method for the sensitive detection of oxidase activity within single Escherichia coli (E. coli) cells, using the monoamine oxidase from Aspergillus niger, MAO-N, as a model system. In contrast to other methods for the screening of oxidase activity in vivo, this method does not require cell surface expression, emulsion formation or the addition of an extracellular peroxidase. Furthermore, we show that fluorescence activated cell sorting (FACS) of large libraries derived from MAO-N under the assay conditions can enrich the library in functional variants at much higher rates than via the colony-based method. We demonstrate its use for directed evolution by identifying a new mutant of MAO-N with improved activity towards a novel secondary amine substrate. This work demonstrates, for the first time, an ultra-high throughput screening methodology widely applicable for the directed evolution of FAD dependent oxidases in E. coli.

Figures

Fig. 1
Fig. 1. Overview of the flow cytometric assay for detection of amine oxidase activity in E. coli with the three main steps comprising (1) expression of the amine oxidase variant library in E. coli; (2) staining of E. coli cells with a fluorogenic probe which is sensitive to oxidation by H2O2 in the presence of an intracellular, endogenous peroxidase and (3) fluorescence activated cell sorting (FACS) of active variants based on fluorescence of the oxidised probe.
Fig. 2
Fig. 2. (a) Coupling of MAO-N activity to fluorescence in vivo. (b) FCM analysis of induced and non-induced E. coli cells containing the MAO-N expression construct, treated with H2DCFDA and incubated with AMBA. (c) Comparison of fluorogenic dyes: FCM analysis of E. coli cells expressing MAO-N D5 stained with H2DHDCF, C-H2DHDCF or DHR and incubated with AMBA. (d) Optimisation of conditions for the expression of MAO-N D5 in E. coli: FCM analysis of E. coli cells incubated at 18 °C or 30 °C for 4 or 16 hours after inducing MAO-N D5 expression.
Fig. 3
Fig. 3. (a) Effect of substrate incubation time on fluorescence of E. coli cells expressing MAO-N D5 stained with 50 μM C-H2DCFDA and incubated with AMBA, compared to a non-induced control. (b) Effect of probe concentration on the maximum fluorescence of E. coli cells stained with C-H2DCFDA determined by the addition of 10 mM H2O2 (blue line) prior to analysis, in comparison to the fluorescence of C-H2DCFDA stained cells after incubation with AMBA for 5 (red line) and 20 (green line) minutes.
Fig. 4
Fig. 4. Fluorescence of C-H2DCFDA stained E. coli incubated with a library of amine substrates. Data were collected after 10 minutes incubation time in each case and was gated on FSC-A vs. SSC-A. n1%: Number of cells with fluorescence in the 99% percentile.
Fig. 5
Fig. 5. (a) Comparison between fluorescence of CASTing library and MAO-N D5 with MBEA under FCM assay conditions. (b) Functional enrichment of a library of MAO-N variants incubated with MBEA after two rounds of sorting. ROI: Region of interest.
Fig. 6
Fig. 6. (a) Flow cytometric analysis of MAO-N D5 T93GF466T activity with MBEA in comparison to MAO-N D5 and the variant library under assay conditions. (b) Position of T93 and F466 relative to the cofactor, FAD, in the active site of MAO-N D5 (PBD accession code: 2VVM).

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