Transcriptomic Analysis of Kalopanax septemlobus and Characterization of KsBAS, CYP716A94 and CYP72A397 Genes Involved in Hederagenin Saponin Biosynthesis

Abstract

Kalopanax septemlobus, commonly named the castor aralia tree, is a highly valued woody medicinal tree belonging to the family Araliaceae. Kalopanax septemlobus contains approximately 15 triterpenoid saponins primarily constituted of hederagenin aglycones. Hederagenin is a representative precursor for hemolytic saponin in plants. In the present study, transcriptome analysis was performed to discover genes involved in hederagenin saponin biosynthesis in K. septemlobus. De novo assembly generated 82,698 unique sequences, including 17,747 contigs and 64,951 singletons, following 454 pyrosequencing. Oxidosqualene cyclases (OSCs) are enzymes that catalyze the formation of diverse triterpene skeletons from 2,3-oxidosqualene. Heterologous expression of an OSC sequence in yeast revealed that KsBAS is a β-amyrin synthase gene. Cytochrome P450 genes (CYPs) make up a supergene family in the plant genome and play a key role in the biosynthesis of sapogenin aglycones. In total, 95 contigs and 110 singletons annotated as CYPs were obtained by sequencing the K. septemlobus transcriptome. By heterologous expression in yeast, we found that CYP716A94 was β-amyrin 28-oxidase involved in oleanolic acid production from β-amyrin, and CYP72A397 was oleanolic acid 23-hydroxylase involved in hederagenin production from oleanolic acid. Engineered yeast co-expressing KsBAS, CYP716A94 and CYP72A397 produced hederagenin. Kalopanax septemlobus CYP72A397 is a novel CYP enzyme that synthesizes hederagenin aglycone from oleanolic acid as a single product. In conclusion, we characterized three genes participating in sequential steps for hederagenin biosynthesis from β-amyrin, which are likely to play a major role in hederagenin saponin biosynthesis in K. septemlobus.

The cytochrome P450 CYP72A552 is key to production of hederagenin‐based saponins that mediate plant defense against herbivores

“…Therefore, we concluded that CYP72A552 oxidises oleanolic acid at the C-23 position only to hederagenin, and that yeast and tobacco enzymes can further oxidise hederagenin to gypsogenic acid. In agreement with this conclusion, Han et al (2017) found that hederagenin was the only product of yeast microsomes harbouring Kalopanax septemlobus CYP72A397 when administrated with oleanolic acid. So, the gypsogenin-derived saponins found in B. vulgaris must be catalysed by another enzyme instead of CYP72A552.…”

“…In agreement with this conclusion, Han et al . () found that hederagenin was the only product of yeast microsomes harbouring Kalopanax septemlobus CYP72A397 when administrated with oleanolic acid. So, the gypsogenin‐derived saponins found in B. vulgaris must be catalysed by another enzyme instead of CYP72A552.…”

Identification and analysis of CYP450 and UGT supergene family members from the transcriptome of Aralia elata (Miq.) Seem reveal candidate genes for triterpenoid saponin biosynthesis

“…8). Members of the CYP72A subfamily have been shown to be involved in a variety of sapogenin biosynthesis reactions, with four uralensis CYP450 gene (CYP72A154), and one K. septemlobus gene (CYP72A397) having been shown to be involved in sapogenin biosynthesis [30,31,43,44]. The CYP72A68v2 enzyme in M. truncatula has been shown to catalyze the oleanolic acid to gypsogenic acid conversion via intermediate hederagenin formation, while CYP72A397 in K. septemlobus produces hederagenin as a single compound [30,31] (Fig.…”

Identification and analysis of CYP450 and UGT supergene family members from the transcriptome of Aralia elata (Miq.) Seem reveal candidate genes for triterpenoid saponin biosynthesis

“…8). Members of the CYP72A subfamily have been shown to be involved in a variety of sapogenin biosynthesis reactions, with four M. truncatula CYP450 genes (CYP72A63, CYP72A61v2, CYP72A67, and CYP72A68v2), one Glycyrrhiza uralensis CYP450 gene (CYP72A154), and one K. septemlobus gene (CYP72A397) having been shown to be involved in sapogenin biosynthesis [30,31,43,44]. The CYP72A68v2 enzyme in M. truncatula has been shown to catalyze the oleanolic acid to gypsogenic acid conversion via intermediate hederagenin formation, while CYP72A397 in K. septemlobus produces hederagenin as a single compound [30,31] (Fig.…”

“…Phylogenetic analyses revealed CYP716A295 and CYP716A296 to be grouped in the CYP716A subfamily and to be most closely related to Maesa lanceolata CYP716A75 and P. ginseng CYP716A52v2, respectively, both of which encode β-amyrin 28-oxidase enzymes involved in oleanolic acid production [29,32]. CYP72A763 and CYP72A776 clustered in the CYP72A group and was most closely related to M. truncatula CYP72A68v2 and Kalopanax septemlobus CYP72A397, respectively, both of which encode β-amyrin 23-hydroxylase enzymes [30,31] (Fig. 7).…”