Continuous Engineering of Phenylalanine Ammonia Lyase from Lettuce (<i>Lactuca sativa</i> L.) for Efficient Synthesis of 3,4-Substituted Phenylalanine

Phenylalanine ammonia lyase (PAL) is a promising catalyst for synthesizing non-natural amino acids. <i>Ls</i>PAL3 from lettuce is a potential candidate for protein engineering. Using alanine scanning and CASTing mutation strategies, we developed a highly effective triple mutant, L126C/F129I/L130C (named <i>Ls</i>M3), which exhibited superior catalytic activity toward 3,4-dimethoxy-substituted substrates. Further enhancements of thermal stability resulted in a robust combined mutant, <i>Ls</i>MC6 (which integrates <i>Ls</i>M3 with additional mutations G62A/S516A/V705A). Its activity was 4.6 times that of <i>Ls</i>M3 in the ammonia addition reaction of 3,4-dimethoxy-substituted cinnamic acid, and its half-life of thermal inactivation at 60 °C was 3.5 times that of <i>Ls</i>M3. <i>Ls</i>MC6 demonstrated significantly improved activity over previously described PALs in the ammonia addition reactions of seven 3,4-substituted cinnamic acid derivatives. Docking and molecular dynamics (MD) simulations revealed that L126C/F129I/L130C mutations reshaped the catalytic pocket, while the incorporation of G62A, S516A, and V705A mutations significantly reduced atomic displacements, thereby enhancing the activity and stability of <i>Ls</i>MC6.