Engineered Zea mays phenylalanine ammonia-lyase for improve the catalytic efficiency of biosynthesis trans-cinnamic acid and p-coumaric acid

Abstract

Phenylalanine ammonia-lyase (PAL) plays a pivotal role in the biosynthesis of phenylalanine. PAL from Zea mays (ZmPAL2) exhibits a bi-function of direct deamination of L-phenylalanine (L-Phe) or L-tyrosine(-L-Tyr) to form trans-cinnamic acid or p-coumaric acid. trans-Cinnamic acid and p-coumaric acid are mainly used in flavors and fragrances, food additives, pharmaceutical and other fields. Here, the Activity of ZmPAL2 toward L-Phe or L-Tyr was improved by using semi-rational and rational designs. The catalytic efficiency (k_cat/K_m) of mutant PT10 (V258I/I459V/Q484N) against L-Phe was 30.8 μM⁻¹ s⁻¹, a 4.5-fold increase compared to the parent, and the catalytic efficiency of mutant PA1 (F135H/I459L) to L-tyrosine exhibited 8.6 μM⁻¹ s⁻¹, which was 1.6-fold of the parent. The yield of trans-cinnamic acid in PT10 reached 30.75 g/L with a conversion rate of 98%. Meanwhile, PA1 converted L-Tyr to yield 3.12 g/L of p-coumaric acid with a conversion rate of 95%. Suggesting these two engineered ZmPAL2 to be valuable biocatalysts for the synthesis of trans-cinnamic acid and p-coumaric acid. In addition, MD simulations revealed that the underlying mechanisms of the increased catalytic efficiency of both mutant PT10 and PA1 are attributed to the substrate remaining stable within the pocket and closer to the catalytically active site. This also provides a new perspective on engineered PAL.