Abstract
Triterpenoids, a group of specialized plant metabolites with substantial structural diversity, are promising for healthcare applications. Ursolic acid, a pentacyclic triterpenoid with therapeutic potential, is also important as a precursor of corosolic acid, which is known as a "phyto-insulin" for its insulin-like properties. Ursolic acid is synthesized from a linear 30-carbon precursor 2,3-oxidosqualene via cyclization to produce triterpene scaffold α-amyrin, followed by a series of oxidation steps at the C-28 position mediated by cytochrome P450 monooxygenases (CYPs) in the CYP716A subfamily. The Tsukuba system was developed for the high-level transient expression of foreign proteins in plant cells based on the use of a binary vector equipped with geminiviral replication system and a double terminator. In this study, we used the Tsukuba system to produce ursolic acid in Nicotiana benthamiana leaves via transient pathway reconstruction. We used an oxidosqualene cyclase identified from the medicinal legume Bauhinia forficata, exhibiting a preponderant α-amyrin-producing activity. Wild-type Medicago truncatula CYP716A12 and its mutants were assessed in terms of ursolic acid production. We improved the performance of MtCYP716A12 by co-expressing it with the appropriate cytochrome P450 reductase (CPR) isozyme as an electron-transfer partner and tested different Agrobacterium infiltration ratios to optimize the CPR : CYP ratio to maximize ursolic acid production. We also achieved high yield of corosolic acid by co-expressing Avicennia marina CYP716C53 with ursolic acid biosynthetic enzymes. Moreover, engineering of AmCYP716C53 significantly improved corosolic acid yield, resulting in a yield exceeding the content found in banaba leaves, a well-known rich source of corosolic acid.