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Molecular characterization of the pentacyclic triterpenoid biosynthetic pathway in Catharanthus roseus

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Abstract

Catharanthus roseus is an important medicinal plant and the sole commercial source of monoterpenoid indole alkaloids (MIA), anticancer compounds. Recently, triterpenoids like ursolic acid and oleanolic acid have also been found in considerable amounts in C. roseus leaf cuticular wax layer. These simple pentacyclic triterpenoids exhibit various pharmacological activities such as anti-inflammatory, anti-tumor and anti-microbial properties. Using the EST collection from C. roseus leaf epidermome (http://www.ncbi.nlm.nih.gov/dbEST), we have successfully isolated a cDNA (CrAS) encoding 2,3-oxidosqualene cyclase (OSC) and a cDNA (CrAO) encoding amyrin C-28 oxidase from the leaves of C. roseus. The functions of CrAS and CrAO were analyzed in yeast (Saccharomyces cerevisiae) systems. CrAS was characterized as a novel multifunctional OSC producing α- and β-amyrin in a ratio of 2.5:1, whereas CrAO was a multifunctional C-28 oxidase converting α-amyrin, β-amyrin and lupeol to ursolic-, oleanolic- and betulinic acids, respectively, via a successive oxidation at the C-28 position of the substrates. In yeast co-expressing CrAO and CrAS, ursolic- and oleanolic acids were detected in the yeast cell extracts, while the yeast cells co-expressing CrAO and AtLUP1 from Arabidopsis thaliana produced betulinic acid. Both CrAS and CrAO genes show a high expression level in the leaf, which was consistent with the accumulation patterns of ursolic- and oleanolic acids in C. roseus. These results suggest that CrAS and CrAO are involved in the pentacyclic triterpene biosynthesis in C. roseus.

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Abbreviations

CrAS:

Catharanthus roseus mixed amyrin synthase

CrAO:

Catharanthus roseus amyrin oxidase

EST:

Expressed sequence tag

ORF:

Open reading frame

LC–MS:

Liquid chromatogram mass spectrum

P450:

Cytochrome P450 monoxygenase

QRT-PCR:

Quantitative reverse transcription polymerase chain reaction

RACE:

Rapid amplification of cDNA ends

TAIL-PCR:

Thermal asymmetric interlaced polymerase chain reaction

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Acknowledgments

This project was partially supported by the Grant for One Hundred Talents Program of the Chinese Academy of Sciences, China (Project No. Y129441R01), the National Science and Technology Program of China during the Twelveth Five-Year Plan period and the National Natural Science Foundation of China (Project No. 91733A1001).

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Authors and Affiliations

  1. CAS Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, The Chinese Academy of Science, 430074, Wuhan, China

    Lili Huang, Jia Li, Changfu Li & Yansheng Zhang

  2. Key Laboratory of Plant Molecular Physiology, Institute of Botany, The Chinese Academy of Science, 100093, Beijing, China

    Lili Huang, Hechun Ye & Benye Liu

  3. Graduate University of the Chinese Academy of Science, 100049, Beijing, China

    Lili Huang & Hong Wang

Authors
  1. Lili Huang
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  2. Jia Li
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  3. Hechun Ye
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  4. Changfu Li
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  5. Hong Wang
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  6. Benye Liu
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  7. Yansheng Zhang
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Correspondence to Yansheng Zhang.

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Huang, L., Li, J., Ye, H. et al. Molecular characterization of the pentacyclic triterpenoid biosynthetic pathway in Catharanthus roseus . Planta 236, 1571–1581 (2012). https://doi.org/10.1007/s00425-012-1712-0

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