Applied Microbiology and Biotechnology

, Volume 103, Issue 17, pp 7029–7039 | Cite as

Identification of RoCYP01 (CYP716A155) enables construction of engineered yeast for high-yield production of betulinic acid

  • Jiajian Huang
  • Wenlong Zha
  • Tianyue An
  • Hua Dong
  • Ying Huang
  • Dong Wang
  • Rongmin Yu
  • Lixin Duan
  • Xueli Zhang
  • Reuben J. PetersEmail author
  • Zhubo DaiEmail author
  • Jiachen ZiEmail author
Biotechnological products and process engineering


Betulinic acid (BA) and its derivatives possess potent pharmacological activity against cancer and HIV. As with many phytochemicals, access to BA is limited by the requirement for laborious extraction from plant biomass where it is found in low amounts. This might be alleviated by metabolically engineering production of BA into an industrially relevant microbe such as Saccharomyces cerevisiae (yeast), which requires complete elucidation of the corresponding biosynthetic pathway. However, while cytochrome P450 enzymes (CYPs) that can oxidize lupeol into BA have been previously identified from the CYP716A subfamily, these generally do not seem to be specific to such biosynthesis and, in any case, have not been shown to enable high-yielding metabolic engineering. Here RoCYP01 (CYP716A155) was identified from the BA-producing plant Rosmarinus officinalis (rosemary) and demonstrated to effectively convert lupeol into BA, with strong correlation of its expression and BA accumulation. This was further utilized to construct a yeast strain that yields > 1 g/L of BA, providing a viable route for biotechnological production of this valuable triterpenoid.


Betulinic acid Cytochrome P450 Synthetic biology Yeast 



We thank Prof. David R. Nelson for annotation of CYP716A155.

Funding information

This work was supported by grants from the National Natural Science Foundation of China (NSFC, No. 81673530), China Postdoctoral Science Foundation (2018 M633289), and Open Fund of Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research (No. 2016B 030301004).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2019_10004_MOESM1_ESM.pdf (456 kb)
ESM 1 (PDF 455 kb)


  1. Ajikumar PK, Xiao WH, Tyo KE, Wang Y, Simeon F, Leonard E, Mucha O, Phon TH, Pfeifer B, Stephanopoulos G (2010) Isoprenoid pathway optimization for taxol precursor overproduction in Escherichia coli. Science 330:70–74. CrossRefGoogle Scholar
  2. Bian G, Deng Z, Liu T (2017) Strategies for terpenoid overproduction and new terpenoid discovery. Curr Opin Biotechnol 48:234–241. CrossRefGoogle Scholar
  3. Boachon B, Buell CR, Crisovan E, Dudareva N, Garcia N, Godden G, Henry L, Kamileen MO, Kates HR, Kilgore MB, Lichman BR, Mavrodiev EV, Newton L, Rodriguez-Lopez C, O’Connor SE, Soltis D, Soltis P, Vaillancourt B, Wiegert-Rininger K, Zhao D (2018) Phylogenomic mining of the mints reveals multiple mechanisms contributing to the evolution of chemical diversity in Lamiaceae. Mol Plant 11:1084–1096. CrossRefGoogle Scholar
  4. Boutanaev AM, Moses T, Zi J, Nelson DR, Mugford ST, Peters RJ, Osbourn A (2015) Investigation of terpene diversification across multiple sequenced plant genomes. Proc Natl Acad Sci U S A 112:E81–E88. CrossRefGoogle Scholar
  5. Carelli M, Biazzi E, Panara F, Tava A, Scaramelli L, Porceddu A, Graham N, Odoardi M, Piano E, Arcioni S, May S, Scotti C, Calderini O (2011) Medicago truncatula CYP716A12 is a multifunctional oxidase invoved in the biosynthesis of hemolytic saponins. Plant Cell 23:3070–3081. CrossRefGoogle Scholar
  6. Chintharlapalli S, Papineni S, Ramaiah SK, Safe S (2007) Betulinic acid inhibits prostate cancer growth through inhibition of specificity protein transcription factors. Cancer Res 67:2816–2823. CrossRefGoogle Scholar
  7. Csuk R, Schmuck K, Schäfer R (2006) A practical synthesis of betulinic acid. Tetrahedron Lett 47:8769–8770. CrossRefGoogle Scholar
  8. Dai Z, Liu Y, Zhang X, Shi M, Wang B, Wang D, Huang L, Zhang X (2013) Metabolic engineering of Saccharomyces cerevisiae for production of ginsenosides. Metab Eng 20:146–156. CrossRefGoogle Scholar
  9. Dai Z, Wang B, Liu Y, Shi M, Wang D, Zhang X, Liu T, Huang L, Zhang X (2014) Producing aglycons of ginsenosides in bakers’ yeast. Sci Rep 4:3698. CrossRefGoogle Scholar
  10. Dang Z, Ho P, Zhu L, Qian K, Lee KH, Huang L, Chen CH (2013) New betulinic acid derivatives for bevirimat-resistant human immunodeficiency virus type-1. J Med Chem 56:2029–2037. CrossRefGoogle Scholar
  11. Dewick PM (2002) Medicinal natural products: a biosynthetic approach, 2nd edn. John Wiley & Sons, New York, pp 167–290Google Scholar
  12. Donald KAG, Hampton RY, Fritz IB (1997) Effects of overproduction of the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme a reductase on squalene synthesis in Saccharomyces cerevisiae. Appl Environ Microbiol 63:3341–3344Google Scholar
  13. Engels B, Dahm P, Jennewein S (2008) Metabolic engineering of taxadiene biosynthesis in yeast as a first step towards Taxol (paclitaxel) production. Metab Eng 10:201–206. CrossRefGoogle Scholar
  14. Fukushima EO, Seki H, Ohyama K, Ono E, Umemoto N, Mizutani M, Saito K, Muranaka T (2011) CYP716A subfamily members are multifunctional oxidases in triterpenoid biosynthesis. Plant Cell Physiol 52:2050–2061. CrossRefGoogle Scholar
  15. Ghosh S (2017) Triterpene structural diversification by plant cytochrome P450 enzymes. Front Plant Sci 8:1886. CrossRefGoogle Scholar
  16. Guo J, Zhou YJ, Hillwig ML, Shen Y, Yang L, Wang Y, Zhang X, Liu W, Peters RJ, Chen X, Zhao ZK, Huang L (2013) CYP76AH1 catalyzes turnover of miltiradiene in tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts. Proc Natl Acad Sci U S A 110:12108–12113. CrossRefGoogle Scholar
  17. Huang L, Li J, Ye H, Li C, Wang H, Liu B, Zhang Y (2012) Molecular characterization of the pentacyclic triterpenoid biosynthetic pathway in Catharanthus roseus. Planta 236:1571–1581. CrossRefGoogle Scholar
  18. Ignea C, Athanasakoglou A, Ioannou E, Georgantea P, Trikka FA, Loupassaki S, Roussis V, Makris AM, Kampranis SC (2016) Carnosic acid biosynthesis elucidated by a synthetic biology platform. Proc Natl Acad Sci U S A 113:3681–3686. CrossRefGoogle Scholar
  19. Jones DT, Taylor WR, Thornton JM (1992) The rapid generation of mutation data matrices from protein sequences. Comput Appl Biosci 8:275–282. Google Scholar
  20. Khakimov B, Kuzina V, Erthmann PØ, Fukushima EO, Augustin JM, Olsen CE, Scholtalbers J, Volpin H, Andersen SB, Hauser TP, Muranaka T, Bak S (2015) Identification and genome organization of saponin pathway genes from a wild crucifer, and their use for transient production of saponins in Nicotiana benthamiana. Plant J 84:478–490. CrossRefGoogle Scholar
  21. Krasutsky PA, Carlson RM, Nesterenko VV, Kolomitsyn IM, Edwardson CF (2003) Birch bark processing and the isolation of natural products from birch bark. US patent No US 6,634,575 B2. 2003-10-21Google Scholar
  22. Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874. CrossRefGoogle Scholar
  23. Lenihan JR, Tsuruta H, Diola D, Renninger NS, Regentin R (2008) Developing an industrial artemisinic acid fermentation process to support the cost-effective production of antimalarial artemisinin-based combination therapies. Biotechnol Prog 24:1026–1032. CrossRefGoogle Scholar
  24. Li Y, Pfeifer BA (2014) Heterologous production of plant-derived isoprenoid products in microbes and the application of metabolic engineering and synthetic biology. Curr Opin Plant Biol 19:8–13. CrossRefGoogle Scholar
  25. Li F, Goila-Gaur R, Salzwedel K, Kilgore NR, Reddick M, Matallana C, Castillo A, Zoumplis D, Martin DE, Orenstein JM, Allaway GP, Freed EO, Wild CT (2003) PA-457: a potent HIV inhibitor that disrupts core condensation by targeting a late step in gag processing. Proc Natl Acad Sci U S A 100:13555–13560. CrossRefGoogle Scholar
  26. Li M, Kildegaard KR, Chen Y, Rodriguez A, Borodina I, Nielsen J (2015) De novo production of resveratrol from glucose or ethanol by engineered Saccharomyces cerevisiae. Metab Eng 32:1–11. CrossRefGoogle Scholar
  27. Liu X, Jutooru I, Lei P, Kim K, Lee S, Brents LK, Prather PL, Safe S (2012) Betulinic acid targets YY1 and ErbB2 through cannabinoid receptor-dependent disruption of microRNA-27a:ZBTB10 in breast cancer. Mol Cancer Ther 11:1421–1431. CrossRefGoogle Scholar
  28. Majeed R, Hamid A, Sangwan PL, Chinthakindi PK, Koul S, Rayees S, Singh G, Mondhe DM, Mintoo MJ, Singh SK, Rath SK, Saxena AK (2014) Inhibition of phosphotidylinositol-3 kinase pathway by a novel naphthol derivative of betulinic acid induces cell cycle arrest and apoptosis in cancer cells of different origin. Cell Death Dis 5:e1459. CrossRefGoogle Scholar
  29. Mertens-Talcott SU, Noratto GD, Li X, Angel-Morales G, Bertoldi MC, Safe S (2013) Betulinic acid decreases ER-negative breast cancer cell growth in vitro and in vivo: role of Sp transcription factors and microRNA-27a:ZBTB10. Mol Carcinog 52:591–602. CrossRefGoogle Scholar
  30. Paddon CJ, Westfall PJ, Pitera DJ, Benjamin K, Fisher K, McPhee D, Leavell MD, Tai A, Main A, Eng D, Polichuk DR, Teoh KH, Reed DW, Treynor T, Lenihan J, Fleck M, Bajad S, Dang G, Dengrove D, Diola D, Dorin G, Ellens KW, Fickes S, Galazzo J, Gaucher SP, Geistlinger T, Henry R, Hepp M, Horning T, Iqbal T, Jiang H, Kizer L, Lieu B, Melis D, Moss N, Regentin R, Secrest S, Tsuruta H, Vazquez R, Westblade LF, Xu L, Yu M, Zhang Y, Zhao L, Lievense J, Covello PS, Keasling JD, Reiling KK, Renninger NS, Newman JD (2013) High-level semi-synthetic production of the potent antimalarial artemisinin. Nature 496:528–532. CrossRefGoogle Scholar
  31. Pai SR, Joshi RK (2014) Distribution of betulinic acid in plant kingdom. Plant Sci Today 1:103–107. CrossRefGoogle Scholar
  32. Pal A, Ganguly A, Chowdhuri S, Yousuf M, Ghosh A, Barui AK, Kotcherlakota R, Adhikari S, Banerjee R (2015) Bis-arylidene oxindole−betulinic acid conjugate: a fluorescent cancer cell detector with potent anticancer activity. ACS Med Chem Lett 6:612–616. CrossRefGoogle Scholar
  33. Pezzuto JM, Kim DSHL (1998) Improved methods of manufacturing betulinic acid. PCT patent no. WO 98/43936. 1998-10-08Google Scholar
  34. Pisha E, Chai H, Lee IS, Chagwedera TE, Farnsworth NR, Cordell GA, Beecher CW, Fong HH, Kinghorn AD, Brown DM, Wani MC, Wall ME, Hieken TJ, Das Gupta TK, Pezzuto JM (1995) Discovery of betulinic acid as a selective inhibitor of human melanoma that functions by induction of apoptosis. Nat Med 1:1046–1051CrossRefGoogle Scholar
  35. Qian K, Kuo RY, Chen CH, Huang L, Morris-Natschke SL, Lee KH (2010) Anti-AIDS agents 81. Design, synthesis, and structure-activity relationship study of betulinic acid and moronic acid derivatives as potent HIV maturation inhibitors. J Med Chem 53:3133–3141. CrossRefGoogle Scholar
  36. Razboršek MI, Vončina DB, Doleček V, Vončina E (2007) Determination of major phenolic acids, phenolic diterpenes and triterpenes in rosemary (Rosmarinus officinalis L.) by gas chromatography and mass spectrometry. Acta Chim Slov 54:60–67Google Scholar
  37. Ressmann A, Kremsmayr T, Gaertner P, Zirbs R, Bica K (2017) Toward a benign strategy for the manufacturing of betulinic acid. Green Chem 19:1014–1022. CrossRefGoogle Scholar
  38. Ryu SY, Lee CK, Lee CO, Kim HS, Zee OP (1992) Antiviral triterpenes from Prunella vulgaris. Arch Pharm Res 15:242–245CrossRefGoogle Scholar
  39. Siddiqui N, Aeri V (2016) Optimization of betulinic acid extraction from Tecomella undulata bark using a box-Behnken design and its densitometric validation. Molecules 21:393–404. CrossRefGoogle Scholar
  40. Šiman P, Filipová A, Tichá A, Niang M, Bezrouk A, Havelek R (2016) Effective method of purification of betulin from birch bark: the importance of its purity for scientific and medicinal use. PLoS One 11:e0154933. CrossRefGoogle Scholar
  41. Tamura K, Seki H, Suzuki H, Kojoma M, Saito K, Muranaka T (2017) CYP716A179 functions as a triterpene C-28 oxidase in tissue-cultured stolons of Glycyrrhiza uralensis. Plant Cell Rep 36:437–445. CrossRefGoogle Scholar
  42. Thimmappa R, Geisler K, Louveau T, O’Maille P, Osbourn A (2014) Triterpene biosynthesis in plants. Annu Rev Plant Biol 65:225–257. CrossRefGoogle Scholar
  43. Tulisalo J, Pirttimaa M, Alakurtti S, Ylikauhaluoma J, Koskimies S (2013) Method for preparation of betulinic acid. PCT patent No. WO 2013/038314 A1. 2013-03-21Google Scholar
  44. Wang D, Liu Y, Xu J, Wang J, Dai Z, Zhang X, Huang L (2018) Construction of efficient yeast cell factories for production of ginsenosides precursor dammarenediol-II. Acta Pharm Sin 53:1233–1241. Google Scholar
  45. Wong J, de Rond T, d’Espaux L, van der Horst C, Dev I, Rios-Solis L, Kirby J, Scheller H, Keasling J (2018) High-titer production of lathyrane diterpenoids from sugar by engineered Saccharomyces cerevisiae. Metab Eng 45:142–148. CrossRefGoogle Scholar
  46. Ye Y, Zhang T, Yuan H, Li D, Lou H, Fan P (2017) Mitochondria-targeted lupane triterpenoid derivatives and their selective apoptosis-inducing anticancer mechanisms. J Med Chem 60:6353–6363. CrossRefGoogle Scholar
  47. Zhao Y, Gu Q, Morris-Natschke SL, Chen CH, Lee KH (2016) Incorporation of privileged structures into bevirimat can improve activity against wild-type and bevirimat-resistant HIV-1. J Med Chem 59:9262–9268. CrossRefGoogle Scholar
  48. Zhou YJ, Gao W, Rong Q, Jin G, Chu H, Liu W, Yang W, Zhu Z, Li G, Zhu G, Huang L, Zhao ZK (2012) Modular pathway engineering of diterpenoid synthases and the mevalonic acid pathway for miltiradiene production. J Am Chem Soc 134:3234–3241. CrossRefGoogle Scholar
  49. Zhou C, Li J, Li C, Zhang Y (2016) Improvement of betulinic acid biosynthesis in yeast employing multiple strategies. BMC Biotechnol 16:59. CrossRefGoogle Scholar
  50. Zi J, Peters RJ (2013) Characterization of CYP76AH4 clarifies phenolic diterpenoid biosynthesis in the Lamiaceae. Org Biomol Chem 11:7650–7652. CrossRefGoogle Scholar
  51. Zi J, Matsuba Y, Hong YJ, Jackson AJ, Tantillo DJ, Pichersky E, Peters RJ (2014) Biosynthesis of lycosantalonol, a cis-prenyl derived diterpenoid. J Am Chem Soc 136:16951–16953. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Biotechnological Institute of Chinese Materia MedicJinan UniversityGuangzhouChina
  2. 2.Tianjin Institute of Industrial BiotechnologyChinese Academy of SciencesTianjinChina
  3. 3.Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine and New Drugs ResearchJinan UniversityGuangzhouChina
  4. 4.Joint Laboratory for Translational Cancer Research of Chinese Medicine of the Ministry of Education of the People’s Republic of China, International Institute for Translational Chinese MedicineGuangzhou University of Chinese MedicineGuangzhouChina
  5. 5.Roy J. Carver Department of Biochemistry, Biophysics, Molecular BiologyIowa State UniversityAmesUSA

Personalised recommendations