Directed evolution of Anabaena variabilis phenylalanine ammonia-lyase (PAL) identifies mutants with enhanced activities

Abstract

There is broad interest in engineering phenylalanine ammonia-lyase (PAL) for its biocatalytic applications in industry (fine-chemicals and natural product synthesis) and medicine (phenylketoruria/PKU and cancer treatment). While site-specific mutagenesis has been employed to improve PAL stability or substrate specificity, a more comprehensive mutational landscape has yet to be explored for this class of enzymes. Here, we report development of a directed evolution technique to engineer PAL enzymes. Central to this approach is a high-throughput enrichment that couples E. coli growth to PAL activity. Using the clinically-relevant PAL from Anabaena variabilis, which is used on the formulation of pegvaliase for PKU therapy, we identified mutations at residues previously unknown as relevant for function that increase turnover frequency almost twofold after only a single round of engineering. This work demonstrates the power our technique for ammonia-lyase enzyme engineering.