A ligand-mediated eGFP-expression system (LiMEx) was developed as a novel flow cytometry based screening platform that relies on a competitive conversion/binding of arginine between arginine deiminase and arginine repressor. Unlike product-driven detection systems, the competitive screening platform allows to evolve enzymes toward efficient operation at low substrate concentrations under physiological conditions. The principle of LiMEx was validated by evolving arginine deiminase (ADI, an anticancer therapeutic) for stronger inhibition of tumor growth. After screening of ∼8.2 × 10(6) clones in three iterative rounds of epPCR libraries, PpADI (ADI from Pseudomonas plecoglossicida) variant M31 with reduced S0.5 value (0.17 mM compared to 1.23 mM (WT)) and, importantly, increased activity at physiological arginine concentration (M31:6.14 s(-1); WT: not detectable) was identified. Moreover, M31 showed a significant inhibitory effect against SK-MEL-28 and G361 melanoma cell lines. (IC50 = 0.02 μg/mL for SK-MEL-28 and G361).
Keywords: LiMEx; arginine deiminase; competitive screening system; directed evolution; protein engineering; synthetic biology.