AUTHOR=Li Le , Lin Shumin , Chen Yuanyuan , Wang Yaqin , Xiao Luhua , Ye Xiaofang , Sun Lei , Zhan Ruoting , Xu Hui 

TITLE=Cytochrome P450 Monooxygenase/Cytochrome P450 Reductase Bi-Enzymatic System Isolated From Ilex asprella for Regio-Specific Oxidation of Pentacyclic Triterpenoids

JOURNAL=Frontiers in Plant Science

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2022.831401

DOI=10.3389/fpls.2022.831401

ISSN=1664-462X

ABSTRACT=<p><italic>Ilex asprella</italic> is a plant from Aquifoliaceae. Its root is commonly used as folk medicinal materials in southern China. The chemical compositions of <italic>I. asprella</italic> are rich in pentacyclic triterpenoids, which show various biological activities and demonstrate a good prospect for drug development. The elucidation of biosynthesis mechanism of triterpenoids in <italic>I. asprella</italic> could lay important foundations for the production of these precious plant secondary metabolites by metabolic engineering. Our previous studies have revealed IaAO1 (a CYP716A210 homolog) responsible for the C-28 oxidation of α- and β-amyrin. Herein, we reported the identification of three more cytochrome P450 monooxygenase genes <italic>IaAO2</italic> (a CYP716A212 homolog), <italic>IaAO4</italic> (CYP714E88), <italic>IaAO5</italic> (CYP93A220), and a cytochrome P450 reductase gene <italic>IaCPR</italic> by using <italic>Saccharomyces cerevisiae</italic> eukaryotic expression system and gas chromatography-mass spectrometry. Among them, the protein encoded by <italic>IaAO2</italic> can catalyze the C-28 oxidation of α-amyrin and β-amyrin, <italic>IaAO4</italic> can catalyze the C-23 oxidation of ursolic acid and oleanolic acid, while <italic>IaAO5</italic> is responsible for the C-24 oxidation of β-amyrin. By introducing three genes <italic>IaAO1</italic>, <italic>IaAO4</italic> and <italic>IaCPR</italic> into <italic>S. cerevisiae.</italic> We constructed an engineered yeast strain that can produce C-23 hydroxyl ursane-type triterpenoid derivatives. This study contributes to a thorough understanding of triterpenoid biosynthesis of medicinal plants and provides important tools for further metabolic engineering.</p>