Modification of isoprene synthesis to enable production of curcurbitadienol synthesis in Saccharomyces cerevisiae

  • Jing Qiao
  • Zuliang Luo
  • Shengrong Cui
  • Huan Zhao
  • Qi Tang
  • Changming Mo
  • Xiaojun MaEmail author
  • Zimian DingEmail author
Fermentation, Cell Culture and Bioengineering - Original Paper


Cucurbitane-type triterpenoids such as mogrosides and cucurbitacins that are present in the plants of Cucurbitaceae are widely used in Asian traditional medicine. Cucurbitadienol is the skeleton of cucurbitane-type triterpenoids. As an alternative production strategy, we developed baker’s yeast Saccharomyces cerevisiae as a microbial host for the eventual transformation of cucurbitadienol. The synthetic pathway of cucurbitadienol was constructed in Saccharomyces cerevisiae by introducing the cucurbitadienol synthase gene from different plants, resulting in 7.80 mg cucurbitadienol from 1 L of fermentation broth. Improving supplies of isoprenoid precursors was then investigated for increasing cucurbitadienol production. Cucurbitadienol production increased to 21.47 mg/L through the overexpression of a global regulatory factor (UPC2) gene of triterpenoid synthase. In addition, knockout of the ERG7 gene increased cucurbitadienol production from 21.47 to 61.80 mg/L. Finally, fed-batch fermentation was performed, and 63.00 mg/L cucurbitadienol was produced. This work is an important step towards the total biosynthesis of valuable cucurbitane-type triterpenoids and demonstrates the potential for developing a sustainable and secure yeast biomanufacturing platform for triterpenoids.


Triterpenoids Cucurbitadienol Metabolic engineering Saccharomyces cerevisiae 



We thank Dr. Yuan Zhou (Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences) for providing the cucurbitadienol standard.


This work was supported by the National Natural Science Foundation of China (NO. 81573521), Beijing Municipal Natural Science Foundation (NO. 5172028) and CAMS Innovation Fund for Medical Sciences (NO. 2017-I2M-1-013).

Compliance with ethical standards

Conflict of interest

Authors declare that they have no conflicts of interest.

Supplementary material

10295_2018_2116_MOESM1_ESM.docx (134 kb)
Supplementary material 1 (DOCX 134 kb)


  1. 1.
    Ajikumar PK, Tyo K, Carlsen S, Mucha O, Phon TH, Stephanopoulos G (2008) Terpenoids: Opportunities for Biosynthesis of Natural Product Drugs Using Engineered Microorganisms. Mol Pharm 5(2):167–190. Google Scholar
  2. 2.
    Albertsen L, Chen Y, Bach LS, Rattleff S, Maury J, Brix S, Nielsen J, Mortensen UH (2011) Diversion of flux toward sesquiterpene production in Saccharomyces cerevisiae by fusion of host and heterologous enzymes. Appl Environ Microbiol 77(3):1033–1040. Google Scholar
  3. 3.
    Alper H, Fischer C, Nevoigt E, Stephanopoulos G (2005) Tuning genetic control through promoter engineering. Proc Natl Acad Sci USA 102(36):12678–12683. Google Scholar
  4. 4.
    Asadollahi MA, Maury J, Schalk M, Clark A, Nielsen J (2010) Enhancement of farnesyl diphosphate pool as direct precursor of sesquiterpenes through metabolic engineering of the mevalonate pathway in Saccharomyces cerevisiae. Biotechnol Bioeng 106(1):86–96. Google Scholar
  5. 5.
    Bai LH, Ma XJ, Mo CM, Shi L, Feng SX, Jiang XJ (2007) study on quantitative assessment of Siraitia grosvenorii germplasms by general index. China J Chin Mater Med 32:2482–2484. Google Scholar
  6. 6.
    Brennan TC, Turner CD, Krömer JO, Nielsen LK (2012) Alleviating monoterpene toxicity using a two-phase extractive fermentation for the bioproduction of jet fuel mixtures in Saccharomyces cerevisiae. Biotechnol Bioeng 109(10):2513–2522. Google Scholar
  7. 7.
    Carelli M, Calderini O (2011) Medicago truncatula CYP716A12 is a multifunctional oxidase involved in the biosynthesis of hemolytic saponins. Plant Cell 23(8):3070. Google Scholar
  8. 8.
    Chang MC, Keasling JD (2006) Production of isoprenoid pharmaceuticals by engineered microbes. Nat Chem Biol 2(12):674–681. Google Scholar
  9. 9.
    Corey EJ, Cheng H, Baker CH, Matsuda SPT, Li D, Song X (1997) Studies on the substrate binding segments and catalytic action of lanosterol synthase Affinity labeling with carbocations derived from mechanism-based analogs of 2,3-oxidosqualene and site-directed mutagenesis probes. J Am Chem Soc 119(6):1289–1296. Google Scholar
  10. 10.
    Dai L, Liu C, Zhu Y, Zhang J, Men Y, Zeng Y, Sun Y (2015) Functional characterization of cucurbitadienol synthase and triterpene glycosyltransferase involved in biosynthesis of mogrosides from Siraitia grosvenorii. Plant Cell Physiol 56(6):1172–1182. Google Scholar
  11. 11.
    Dai Z, Liu Y, Huang L, Zhang X (2012) Production of miltiradiene by metabolically engineered Saccharomyces cerevisiae. Biotechnol Bioeng 109(11):2845–2853. Google Scholar
  12. 12.
    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(5):146–156. Google Scholar
  13. 13.
    Dai ZB, Wang BB, Liu Y, Shi MY, Wang D, Zhang XN, Liu T, Huang LQ, Zhang XL (2014) Producing aglycons of ginsenosides in bakers’ yeast. Sci Rep 4(3698):3698. Google Scholar
  14. 14.
    Davidovich-Rikanati R, Shalev L, Baranes N, Meir A, Itkin M, Cohen S, Zimbler K, Portnoy V, Ebizuka Y, Shibuya M (2015) Recombinant yeast as a functional tool for understanding bitterness and cucurbitacin biosynthesis in watermelon (Citrullus spp.). Yeast 32(1):103–114. Google Scholar
  15. 15.
    Dejong JM, Liu Y, Bollon AP, Long RM, Jennewein S, Williams D, Croteau RB (2006) Genetic engineering of taxol biosynthetic genes in Saccharomyces cerevisiae. Biotechnol Bioeng 93(2):212–224. Google Scholar
  16. 16.
    Ding Y, Nguyen HT, Kim SI, Kim HW, Kim YH (2009) The regulation of inflammatory cytokine secretion in macrophage cell line by the chemical constituents of Rhus sylvestris. Bioorg Med Chem Lett 19(13):3607–3610. Google Scholar
  17. 17.
    Donald KA, 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(9):3341–3344Google Scholar
  18. 18.
    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(4):201–206. Google Scholar
  19. 19.
    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(12):2050–2061. Google Scholar
  20. 20.
    Gao LL, Wang QH, Liang HC, Gong T, Yang JL, Zhu P (2014) Construction of Saccharomyces cerevisiae haploid mutant deficient in lanosterol synthase gene. Acta Pharm Sin B 49(5):742–746Google Scholar
  21. 21.
    Geu-Flores F, Nour-Eldin HH, Nielsen MT, Halkier BA (2007) USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products. Nucleic Acids Res 35(7):e55. Google Scholar
  22. 22.
    Hoshino T (2017) β-Amyrin biosynthesis: catalytic mechanism and substrate recognition. Org Biomol Chem. Google Scholar
  23. 23.
    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(5):1571–1581. Google Scholar
  24. 24.
    Itkin M, Davidovich-Rikanati R, Cohen S, Portnoy V, Doron-Faigenboim A, Oren E, Freilich S, Tzuri G, Baranes N, Shen S (2016) The biosynthetic pathway of the nonsugar, high-intensity sweetener mogroside V from Siraitia grosvenorii. Proc Natl Acad Sci USA 113(47):E7619. Google Scholar
  25. 25.
    Kim YS, Cho JH, Park S, Han JY, Back K, Choi YE (2011) Gene regulation patterns in triterpene biosynthetic pathway driven by overexpression of squalene synthase and methyl jasmonate elicitation in Bupleurum falcatum. Planta 233(2):343–355. Google Scholar
  26. 26.
    Kürten C, Uhlén M, Syrén PO (2015) Overexpression of functional human oxidosqualene cyclase in Escherichia coli. Protein Expression Purif 115:46–53. Google Scholar
  27. 27.
    Leber R, Zenz R, Schröttner K, Fuchsbichler S, Pühringer B, Turnowsky F (2001) A novel sequence element is involved in the transcriptional regulation of expression of the ERG1 (squalene epoxidase) gene in Saccharomyces cerevisiae. FEBS J 268(4):914–924. Google Scholar
  28. 28.
    Lee MH, Jeong JH, Seo JW, Shin CG, Kim YS, In JG, Yang DC, Yi JS, Choi YE (2004) Enhanced triterpene and phytosterol biosynthesis in Panax ginseng overexpressing squalene synthase gene. Plant Cell Physiol 45(8):976–984. Google Scholar
  29. 29.
    Li D, Ikeda T, Huang Y, Liu J, Nohara T, Sakamoto T, Nonaka GI (2007) Seasonal variation of mogrosides in Lo Han Kuo ( Siraitia grosvenorii ) fruits. J Nat Med 61(3):307–312. Google Scholar
  30. 30.
    Lian J, Mishra S, Zhao H (2018) recent advances in metabolic engineering of Saccharomyces cerevisiae: new tools and their applications. Metab Eng. Google Scholar
  31. 31.
    Ma CL, Mitra A (2002) Expressing multiple genes in a single open reading frame with the 2A region of foot-and-mouth disease virus as a linker. Mol Breed 9(3):191–199. Google Scholar
  32. 32.
    Makapugay HC, Npd N, Soejarto DD, Kinghorn AD (1985) High-performance liquid chromatographic analysis of the major sweet principle of lo han kuo fruits. J Agric Food Chem 33(3):305–312. Google Scholar
  33. 33.
    Marienhagen J, Bott M (2013) Metabolic engineering of microorganisms for the synthesis of plant natural products. Bioengineered 163(2):166–178. Google Scholar
  34. 34.
    Minh CV, Nhiem NX, Yen HT, Kiem PV, Tai BH, Le TAH, Hien TT, Park S, Kim N, Kim SH (2015) Chemical constituents of Trichosanthes kirilowii and their cytotoxic activities. Arch Pharmacal Res 38(8):1443–1448. Google Scholar
  35. 35.
    Newman JD, Marshall J, Chang M, Nowroozi F, Paradise E, Pitera D, Newman KL, Keasling JD (2006) High-level production of amorpha-4,11-diene in a two-phase partitioning bioreactor of metabolically engineered Escherichia coli. Biotechnol Bioeng 95(4):684–691. Google Scholar
  36. 36.
    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(7446):528. Google Scholar
  37. 37.
    Qing ZX, Zhao H, Tang Q, Mo CM, Huang P, Cheng P, Yang P, Yang XY, Liu XB, Zheng YJ (2017) Systematic identification of flavonols, flavonol glycosides, triterpene and siraitic acid glycosides from Siraitia grosvenorii using high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry combined with a screening strategy. J Pharm Biomed Anal 138:240. Google Scholar
  38. 38.
    Ríos JL, Escandell JM, Recio MC (2005) New insights into the bioactivity of cucurbitacins. Stud Nat Prod Chem 32(05):429–469. Google Scholar
  39. 39.
    Ro DK, Paradise EM, Ouellet M, Fisher KJ, Newman KL, Ndungu JM, Ho KA, Eachus RA, Ham TS, Kirby J, Chang MC, Withers ST, Shiba Y, Sarpong R, Keasling JD (2006) Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440(7086):940–943. Google Scholar
  40. 40.
    Seo JW, Jeong JH, Shin CG, Lo SC, Han SS, Yu KW, Harada E, Han JY, Choi YE (2005) Overexpression of squalene synthase in Eleutherococcus senticosus increases phytosterol and triterpene accumulation. Phytochemistry 66(8):869–887. Google Scholar
  41. 41.
    Shang Y, Ma Y, Zhou Y, Zhang H, Duan L, Chen H, Zeng J, Zhou Q, Wang S, Gu W, Liu M, Ren J, Gu X, Zhang S, Wang Y, Yasukawa K, Bouwmeester HJ, Qi X, Zhang Z, Lucas WJ, Huang S (2014) Plant science. Biosynthesis, regulation, and domestication of bitterness in cucumber. Science 346(6213):1084–1088. Google Scholar
  42. 42.
    Shao Z, Zhao H, Zhao H (2009) DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways. Nucleic Acids Res 37(2):e16. Google Scholar
  43. 43.
    Shibuya M, Adachi S, Ebizuka Y (2004) Cucurbitadienol synthase, the first committed enzyme for cucurbitacin biosynthesis, is a distinct enzyme from cycloartenol synthase for phytosterol biosynthesis. Tetrahedron 35(45):6995–7003. Google Scholar
  44. 44.
    Sun J, Shao Z, Zhao H, Nair N, Wen F, Xu JH, Zhao H (2012) Cloning and characterization of a panel of constitutive promoters for applications in pathway engineering in Saccharomyces cerevisiae. Biotechnol Bioeng 109(8):2082–2092. Google Scholar
  45. 45.
    Swingle WT (1941) Momordica grosvenorii sp. nov. The source of the Chinese Lo Han Kuo. J Arnold Arbor 22(2):197–203Google Scholar
  46. 46.
    Tokuhiro K, Muramatsu M, Ohto C, Kawaguchi T, Obata S, Muramoto N, Hirai M, Takahashi H, Kondo A, Sakuradani E (2009) Overproduction of geranylgeraniol by metabolically engineered Saccharomyces cerevisiae. Appl Environ Microbiol 75(17):5536–5543. Google Scholar
  47. 47.
    Tu D, Ma X, Zhao H, Mo C, Tang Q, Wang L, Huang J, Pan L (2016) Cloning and expression of SgCYP450-4 from Siraitia grosvenorii. Acta Pharm. Sin. B 6(6):614–622. Google Scholar
  48. 48.
    Ukiya M, Akihisa T, Tokuda H, Toriumi M, Mukainaka T, Banno N, Kimura Y, Hasegawa J, Nishino H (2002) Inhibitory effects of cucurbitane glycosides and other triterpenoids from the fruit of Momordica grosvenorii on epstein-barr virus early antigen induced by tumor promoter 12-O-tetradecanoylphorbol-13-acetate. J Agric Food Chem 50(23):6710–6715. Google Scholar
  49. 49.
    Veen M, Stahl U, Lang C (2003) Combined overexpression of genes of the ergosterol biosynthetic pathway leads to accumulation of sterols in Saccharomyces cerevisiae. FEMS Yeast Res 4(1):87–95. Google Scholar
  50. 50.
    Verwaal R, Jiang Y, Wang J, Daran JM, Sandmann G, Ja VDB, van Ooyen AJ (2010) Heterologous carotenoid production in Saccharomyces cerevisiae induces the pleiotropic drug resistance stress response. Yeast 27(12):983–998. Google Scholar
  51. 51.
    Verwaal R, Wang J, Meijnen JP, Visser H, Sandmann G, Ja VDB, van Ooyen AJ (2007) High-level production of beta-carotene in Saccharomyces cerevisiae by successive transformation with carotenogenic genes from Xanthophyllomyces dendrorhous. Appl Environ Microbiol 73(13):4342. Google Scholar
  52. 52.
    Vickers CE, Bydder SF, Zhou Y, Nielsen LK (2013) Dual gene expression cassette vectors with antibiotic selection markers for engineering in Saccharomyces cerevisiae. Microb Cell Fact 12(1):96. Google Scholar
  53. 53.
    Westfall PJ, Pitera DJ, Lenihan JR, Eng D, Woolard FX, Regentin R, Horning T, Tsuruta H, Melis DJ, Owens A (2012) Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin. Proc Natl Acad Sci USA 109(3):111–118. Google Scholar
  54. 54.
    Xia Y, Riverohuguet ME, Hughes BH, Marshall WD (2008) Isolation of the sweet components from Siraitia grosvenorii. Food Chem 107(3):1022–1028. Google Scholar
  55. 55.
    Xie W, Ye L, Lv X, Xu H, Yu H (2015) Sequential control of biosynthetic pathways for balanced utilization of metabolic intermediates in Saccharomyces cerevisiae. Metab Eng 28:8–18. Google Scholar
  56. 56.
    Zhang J, Dai L, Yang J, Liu C, Men Y, Zeng Y, Cai Y, Zhu Y, Sun Y (2016) Oxidation of cucurbitadienol catalyzed by CYP87D18 in the biosynthesis of mogrosides from siraitia grosvenorii. Plant Cell Physiol 57(5):1000–1007. Google Scholar
  57. 57.
    Zhou YJ, Gao W, Rong Q, Jin G, Chu H, Liu W, Yang W, Zhu Z, Li G, Zhu G (2012) Modular pathway engineering of diterpenoid synthases and the mevalonic acid pathway for miltiradiene production. J Am Chem Soc 134(6):3234–3241. Google Scholar

Copyright information

© Society for Industrial Microbiology and Biotechnology 2018

Authors and Affiliations

  • Jing Qiao
    • 1
  • Zuliang Luo
    • 1
  • Shengrong Cui
    • 1
  • Huan Zhao
    • 2
  • Qi Tang
    • 3
  • Changming Mo
    • 4
  • Xiaojun Ma
    • 1
    Email author
  • Zimian Ding
    • 1
    Email author
  1. 1.Institute of Medicinal Plant DevelopmentChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijingChina
  2. 2.State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia MedicaAcademy of Chinese Medical SciencesBeijingChina
  3. 3.National and Local Union Engineering Research Center of Veterinary Herbal Medicine Resources and Initiative and Hunan Co-Innovation Center for Utilization of Botanical Functional IngredientsHunan Agricultural UniversityChangshaChina
  4. 4.Guangxi Botanical Garden of Medicinal PlantsNanningChina

Personalised recommendations