GFP reporter screens for the engineering of amino acid degrading enzymes from libraries expressed in bacteria

Abstract

There is significant interest in engineering human amino acid degrading enzymes as non-immunogenic chemotherapeutic agents. We describe a high-throughput fluorescence activated cell sorting (FACS) assay for detecting the catalytic activity of amino acid degrading enzymes in bacteria, at the single cell level. This assay relies on coupling the synthesis of the GFP reporter to the catalytic activity of the desired amino acid degrading enzyme in an appropriate E. coli genetic background. The method described here allows facile screening of much larger libraries (10(6)-10(7)) than was previously possible. We demonstrate the application of this technique in the screening of libraries of bacterial and human asparaginases and also for the catalytic optimization of an engineered human methionine gamma lyase.

Fig. 1

(1) Engineer E coli strain that can no longer make a metabolic intermediate needed for growth. (2) Create mutagenized libraries of enzymes that have the potential to make the missing metabolic intermediate. (3) Co-transform auxotrophic strain with library and GFP plasmids that have mutually exclusive promoters. (4) Culture under nonselective conditions and induce expression of enzyme plasmids. (5) Shift culture to selective conditions and induce expression of GFP. If an enzyme variant can rescue the auxotrophy, then the cell will express greater levels of GFP. (6) Use rounds of FACS sorting to select cells with high GFP fluorescence. (7) Use secondary screen/assay to rank order clones and if necessary repeat mutagenesis on variants with increased activity.

Fig. 2

Summary of genes deleted in E coli to create l-aspartic acid auxotroph strain that can be rescued by recombinant asparaginase activity.

Fig. 3

Flow cytometric analysis of E coli D-aux strain expressing an error-prone library of hASRGL-1 variants. Representative fluorescence histograms are shown from round 1 (R1) of sorting to round 7 (R7), along with a positive control containing l-Asp (see Note 4). Mean of the fluorescence histograms is reported in FACS diagram.

Fig. 4

(a) Summary of the pathway and gene deletions in E coli needed to create an α-ketobutyrate auxotrophic strain that can be rescued by recombinant methionine-γ-lyase activity. (b) Summary of the pathway and gene deletions needed to create an l-methionine auxotroph to prevent parental cystathionine-γ-lyase activity from rescuing α-ketobutyrate auxotrophy.

Fig. 5

Histogram showing enrichment of positive enzyme control out of a mixed pool of 10,000:1 hCGL to pMGL over the course of four rounds as well as the signals of a positive enzyme control alone (pMGL-Ile). (Inset) A histogram of a positive and negative control experiment using hCGL in the presence or absence of l-Ile (+/− Ile).