(534e) Directed Evolution of Phenylalanine Ammonia-Lyase (PAL), a Key Enzyme for the Treatment of Phenylketonuria (PKU)

Phenylalanine ammonia lyase (PAL) enzymes are widely found associated with secondary metabolism in plants, bacteria, and fungi. Biocatalytic applications for natural product and fine chemical synthesis has driven the discovery, expression, characterization, and engineering of PAL. More recently, development of PALs for phenylketonuria (PKU) management and cancer therapy has further increase interest in engineering this class of enzymes. Comparative structure and enzyme analysis have informed numerous efforts to improve enzyme stability and alter substrate specificity through rational design. While site-specific mutagenesis has been employed for improving PAL, a more comprehensive mutational landscape has yet to be explored for this class of enzymes. Here, we report development of a directed evolution strategy to engineer PAL enzymes. Central to this approach is a high-throughput enrichment that couples E. coli growth to PAL activity. Applying this method to the PAL from Anabaena variabilis, used in the FDA approved drug Pegvaliase, for PKU treatment, we identified mutations at residues previously unknown as relevant for function that increase higher turnover frequency almost twofold after only a single round of engineering. This work demonstrates the power our technique for ammonia lyase enzyme engineering and is the first example of improving the specific activity of a PAL toward its native substrate.

Mays, Mohan et al. (2020) ChemComm