A Novel Multifunctional C-23 Oxidase, CYP714E19, is Involved in Asiaticoside Biosynthesis

Area of Science:

• Plant biochemistry
• Metabolic engineering
• Medicinal plant research

Background:

• Centella asiatica accumulates valuable triterpene saponins like asiaticoside and madecassoside.
• Biosynthesis is thought to involve α-amyrin hydroxylation by cytochrome P450 enzymes, but key enzymes remained unidentified.

Purpose of the Study:

• Identify and characterize novel cytochrome P450 enzymes involved in asiaticoside and madecassoside biosynthesis in Centella asiatica.
• Elucidate the specific roles of these enzymes in the metabolic pathway.

Main Methods:

• Transcriptome analysis of methyl jasmonate-elicited Centella asiatica.
• Identification and isolation of candidate P450 genes.
• Heterologous expression of genes in yeast.
• Biochemical characterization using Gas Chromatography-Mass Spectrometry (GC-MS).

Main Results:

• Identified 149 unigenes encoding putative P450 enzymes, with four highly expressed under jasmonate treatment.
• Yeast expressing CaDDS and CYP716A83 produced ursolic and oleanolic acids, indicating CYP716A83's C-28 oxidase activity.
• Yeast co-expressing CaDDS, CYP716A83, and CYP714E19 produced hederagenin and 23-hydroxyursolic acid, confirming CYP714E19's C-23 hydroxylase activity.

Conclusions:

• CaDDS, CYP716A83, and CYP714E19 are key enzymes in Centella asiatica for consecutive steps in asiaticoside biosynthesis.
• These enzymes catalyze crucial hydroxylations and oxidations in the triterpenoid saponin pathway.
• Findings provide insights for metabolic engineering of medicinal plant compounds.