Atypical phenylalanine ammonia-lyases that enhance phenylpropanoid production in plants

Plants synthesize diverse phenylpropanoids, which provide us essential medicine, nutrients, and biomaterials while also acting as a major global carbon sink. Phenylpropanoids are primarily derived from L-phenylalanine (Phe) via Phe ammonia-lyase (PAL), a highly regulated enzymatic activity that limits phenylpropanoid production. However, it remains poorly understood how different plant species regulate PAL enzymes. Phylogeny-guided biochemical screening uncovered complex and diverse feedback regulation of PAL enzymes from 14 different plant species. This screening found a previously uncharacterized PAL clade, namely PALnc (from noncanonical), with some members exhibiting feedback activation by p -coumaric acid, caffeic acid, and 4-hydroxyphenylpyruvic acid in vitro. Transient expression of two PALnc genes, from Elaeis guineensis ( EgPALnc ) and Piper methysticum ( PmPALnc2 ), in Nicotiana benthamiana leaves elevated phenylpropanoid content, supported by the high in planta stability of PALnc proteins compared to canonical PALs. Whereas overexpression of Arabidopsis thaliana PAL1 ( AtPAL1 ) in stable Arabidopsis transgenic lines resulted in very low accumulation of AtPAL1 protein and minor metabolic effects, introduction of EgPALnc rendered high content of EgPALnc protein and hyperaccumulation of hydroxycinnamates derivatives. Our findings uncover a previously overlooked diversity in the regulation of the entry step of phenylpropanoid biosynthesis across various plants and identify atypical PALs within the PALnc clade as valuable tools to debottleneck and enhance phenylpropanoid production in planta.