COMT, CRTZ, and F3′H regulate glycyrrhizic acid biosynthesis in Glycyrrhiza uralensis hairy roots

Glycyrrhiza uralensis Fisch. is prescribed as one of the original plants of licorice in Chinese Pharmacopoeia. This herbal medicine possesses numerous pharmacological activities and has been used in clinic in China since ancient times. Glycyrrhizic acid (GA) is a triterpenoid compound isolated from G. uralensis. It is often used as one of the marker components for the medicinal quality of the herb. Previously, we showed that the expression levels of three genes in G. uralensis were inversely correlated with the content of GA, including the caffeic acid 3-O-methyltransferase gene (COMT), the β-carotene 3-hydroxylase gene (CRTZ), and the flavonoid 3′-monooxygenase gene (F3′H). In this study, the main aim is to determine the roles of the three genes on GA production through gene knockout and overexpression in G. uralensis hairy roots. We observed that neither knockout nor overexpression of any of the genes affected the viability of the transgenic hairy roots, indicating that these genes are not essential for survival of hairy roots. Compared with the wild type and negative control hairy roots, GA content was significantly lower in hairy roots overexpressing COMT, CRTZ, or F3′H, but higher in those with anyone of the genes knocked out. Our findings demonstrate that the three genes, COMT, CRTZ, and F3′H, all negatively regulate the GA biosynthesis.