Engineered Phenylalanine Ammonia Lyases Enable Biocatalytic Synthesis of a Non-Canonical Phenylalanine Derivative at Scale.

Abstract

Phenylalanine ammonia lyases (PALs) are an enzyme class of high synthetic utility as they catalyze the synthesis of chiral non-canonical phenylalanine amino acids from readily available cinnamic acids using simple ammonia as the amine donor. However, the applications of PAL in chemical manufacturing have been rare due to the challenges in identifying PALs active under industrially relevant conditions. Herein, we report our efforts towards engineering a PAL enzyme from Planctomyces brasiliensis (PbPAL) for the synthesis of L-3-cyanophenylalanine as an intermediate in the synthesis of Enlicitide, the clinically approved macrocyclic peptide to lower cholesterol levels. Protein engineering identified several key mutations that resulted in an evolved PbPAL variant with >100-fold improved activity and enhanced recombinant expression in E. coli. The computational studies suggest the final variant has increased substrate accessibility to the active site, which enabled high conversion (up to 97%) to L-3-cyanophenylalanine with excellent enantioselectivity at low enzyme loading (3 w/w%), demonstrated at up to 250-gram scale. In addition, the final variant represents a broad substrate scope against a variety of heterocyclic cinnamic acids with high enantioselectivity. This work showcases how protein engineering can enable the biocatalytic synthesis of non-canonical amino acids at scale.