Metabolic engineering of the tryptophan and phenylalanine biosynthetic pathways in rice

Aromatic amino acids function as building blocks of proteins and as precursors for secondary metabolism. To obtain plants that accumulate tryptophan (Trp) and phenylalanine (Phe), we modified the biosynthetic pathways for these amino acids in rice and dicot species. By introducing a gene encoding a feedback-insensitive anthranilate synthase (AS) alpha subunit, we successfully obtained transgenic plants that over-accumulated Trp. In addition, we found mutant calli that accumulated Phe and Trp at high concentrations. The causal gene (mtr1-D) encoded an arogenate dehydratase (ADT)/prephenate dehydratase (PDT) that catalyzes the final reaction in Phe biosynthesis. The wild-type enzyme was sensitive to feedback inhibition by Phe, but the mutant enzyme encoded by mtr1-D was relatively insensitive. Further, detailed analysis of downstream secondary metabolism from Trp in rice revealed that the Trp pathway, by producing serotonin, is involved in the defense response against pathogenic infection. Based on these findings we propose that the reactions catalyzed by AS and ADT are critical regulatory points in the biosynthesis of Trp and Phe, respectively. In addition, detailed characterization of transgenic lines that accumulate these aromatic amino acids provided new insights into the regulation of downstream secondary metabolism, translocation of aromatic amino acids, and effects of accumulation of aromatic amino acids on various agronomic traits.