Paper Title
Targeted Biochemical Genomics of Plant P450 Genes Through in Vivo Product Analysis in Transgenic Yeasts

Each plant species harbours a few hundred cytochrome P450 genes, which are responsible for a wide range of common or species-specific natural products. However, the identification of P450 genes has been slow in part due to the lack of a genomics approach. Here, we developed a relatively simple genomics method to screen plant P450 genes by generating transgenic yeasts that exhibit gain-of-function biochemical mutants. To achieve this goal, we generated 141 independent yeast transgenic strains that expressed rice full-length P450 cDNAs. Thereafter, the yeast strains were incubated and induced independently in the presence of various substrates, followed by HPLC detection of expected products. Among the 141 yeast transgenic strains, we found only one yeast strain that produced 4-coumaric acid when challenged with cinnamic acid, proving that the transgenic yeast strain expressed P450 gene coding for cinnamate 4-hydroxylase (C4H). To further confirm the efficacy of this system, the yeast transgenic strains were induced independently in the presence of ferulic acid and tryptamine to clone ferulate 5-hydroxylase (F5H) and tryptamine 5-hydroxylase (T5H), respectively. The yeast transgenic strains 11 and 29 produced exclusively the synthesis of 5-hydroxyferulic acid and serotonin, respectively. The in vivo syntheses of products were further verified by measuring F5H and T5H enzyme activity in vitro. These yeast mutants provide a rapid and sensitive fishing system for identifying the function of P450 genes. Keywords - Caffeic acid, P450, Rice, Serotonin, ,Tryptamine