A novel multifunctional C-23 Oxidase, CYP714E19, is involved in asiaticoside biosynthesis

Ok Tae Kim, Yurry Um, Mei Lan Jin, Jang Uk Kim, Daniela Hegebarth, Lucas Busta, Radu C. Racovita, Reinhard Jetter

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

Centella asiatica is widely used as a medicinal plant due to accumulation of the ursane-type triterpene saponins asiaticoside and madecassoside. The molecular structure of both compounds suggests that they are biosynthesized from α-amyrin via three hydroxylations, and the respective Cyt P450-dependent monooxygenases (P450 enzymes) oxidizing the C-28 and C-2α positions have been reported. However, a third enzyme hydroxylating C-23 remained elusive. We previously identified 40,064 unique sequences in the transcriptome of C. asiatica elicited by methyl jasmonate, and among them we have now found 149 unigenes encoding putative P450 enzymes. In this set, 23 full-length cDNAs were recognized, 13 of which belonged to P450 subfamilies previously implicated in secondary metabolism. Four of these genes were highly expressed in response to jasmonate treatment, especially in leaves, in accordance with the accumulation patterns of asiaticoside. The functions of these candidate genes were tested using heterologous expression in yeast cells. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that yeast expressing only the oxidosqualene synthase CaDDS produced the asiaticoside precursor α-amyrin (along with its isomer β-amyrin), while yeast co-expressing CaDDS and CYP716A83 also contained ursolic acid along with oleanolic acid. This P450 enzyme thus acts as a multifunctional triterpenoid C-28 oxidase converting amyrins into corresponding triterpenoid acids. Finally, yeast strains co-expressing CaDDS, CYP716A83 and CYP714E19 produced hederagenin and 23-hydroxyursolic acid, showing that CYP714E19 is a multifunctional triterpenoid oxidase catalyzing the C-23 hydroxylation of oleanolic acid and ursolic acid. Overall, our results demonstrate that CaDDS, CYP716A83 and CYP714E19 are C. asiatica enzymes catalyzing consecutive steps in asiaticoside biosynthesis.

Original languageEnglish (US)
Pages (from-to)1200-1213
Number of pages14
JournalPlant and Cell Physiology
Volume59
Issue number6
DOIs
StatePublished - Jun 1 2018
Externally publishedYes

Bibliographical note

Funding Information:
This work was supported by the Cooperative Research Program for Agricultural Science & Technology Development [Project No. PJ00849903]; the Rural Development Administation, Republic of Korea; the Natural Sciences and Engineering Research Council of Canada; and the Canada Foundation for Innovation.

Keywords

  • Asiaticoside
  • Centella asiatica
  • Cytochrome P450
  • Methyl jasmonate
  • Oxidosqualene cyclases

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