Cloning of the cytochrome P450 enzyme from Perilla frutescens involved in nothoapiole biosynthesis.

Phenylpropanoid volatile components are found in various plants and are useful in medicines, health foods, and fragrances. While the pharmacological actions and toxicities of these compounds have been investigated, there are few reports of the cloning of genes that encode biosynthetic enzymes involved in substituent formation at diverse positions and numbers. Previously, using the expressed sequence tag (EST) libraries of pure perilla strains that have been maintained for over 30 years for their oil type, we characterized the P450 enzyme that produces an intermediate for dillapiole by adding a hydroxy group to myristicin. In this study, we selected a P450 enzyme involved in nothoapiole biosynthesis from the EST library. Heterologous expression of this enzyme in yeast showed that it is a hydroxylase that synthesizes an intermediate to produce nothoapiole from apiole and dillapiole. The enzyme has high amino acid sequence similarity with a previously cloned enzyme and is categorized into the CYP71D subfamily. Furthermore, we investigated the presence or absence of essential oil components and intermediates believed to be involved in nothoapiole biosynthesis by component analysis of perilla essential oil using GC-MS to help elucidate the biosynthetic pathway of nothoapiole. Only a small number of plant species contain nothoapiole as their principal component and thus few studies have reported the biosynthetic genes involved or the drug efficacy and toxicity of nothoapiole. The present study will aid in understanding the biosynthesis of phenylpropanoid volatile compounds, thereby contributing to further research on potentially useful compounds such as nothoapiole.