Applied Microbiology and Biotechnology

, Volume 86, Issue 5, pp 1367–1374 | Cite as

Enhanced biosynthetic gene expressions and production of ganoderic acids in static liquid culture of Ganoderma lucidum under phenobarbital induction

  • Cui-Xia Liang
  • Ying-Bo Li
  • Jun-Wei Xu
  • Jia-Le Wang
  • Xiao-Ling Miao
  • Ya-Jie Tang
  • Tingyue Gu
  • Jian-Jiang ZhongEmail author
Biotechnological Products and Process Engineering


Static liquid culture of Ganoderma lucidum, a traditional Chinese medicinal mushroom, is a proven technology for producing ganoderic acids, which are secondary metabolites that possess antitumor properties. In this work, the addition of phenobarbital, a P450 inducer, was used to enhance the production of total and individual ganoderic acids in a two-stage cultivation involving a period of initial shake flask culture followed by static liquid culture of G. lucidum. The dosage and time of phenobarbital induction were critical for the enhanced production of ganoderic acids. The addition of 100 μM (final concentration) phenobarbital on day 5 after the shake flask culture was converted to the static liquid culture was found to be optimal, resulting in a maximal amount of total ganoderic acids of 41.4 ± 0.6 mg/g cell dry weight and increases in the levels of ganoderic acid-Mk, -T, -S, and -Me in the treated cells by 47%, 28%, 36%, and 64%, respectively. Meanwhile, the accumulation of lanosterol, a key intermediate, was found to decrease and transcriptions of three key genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase, squalene synthase, and lanosterol synthase in the triterpene biosynthetic pathway were up-regulated under phenobarbital induction. This work demonstrated a useful strategy for the enhanced production of ganoderic acids by G. lucidum.


Ganoderma lucidum Ganoderic acid Phenobarbital induction Gene expression Mushroom fermentation technology 



Financial support from the National Natural Science Foundation of China (NSFC Project No. 20776084, No. 30821005), the National High Technology R&D Program (863 Project No. 2007AA021506), Shanghai Science & Technology Commission (Project No. 08DZ1971900), and the Shanghai Leading Academic Discipline Project (Project Nos. B203 and B505) is gratefully acknowledged. We also thank the reviewers for their time and helpful comments.


  1. Abe I, Rohmer M, Prestwich GD (1993) Enzymatic cyclization of squalene and oxidosqualene to sterols and triterpenes. Chem Rev 93:2189–2206CrossRefGoogle Scholar
  2. Blättler SM, Rencurel F, Kaufmann MR, Meyer UA (2007) In the regulation of cytochrome P450 genes, phenobarbital targets LKB1 for necessary activation of AMP-activated protein kinase. Proc Natl Acad Sci USA 104:1045–1050CrossRefGoogle Scholar
  3. Chen NH, Liu JW, Zhong JJ (2008) Ganoderic Acid Me inhibits tumor invasion through down-regulating matrix metalloproteinases 2/9 gene expression. J Pharmacol Sci 108:212–216CrossRefGoogle Scholar
  4. Contin A, Collu G, Van Der Heijden R, Verpoorte R (1999) The effects of phenobarbital and ketoconazole on the alkaloid biosynthesis in Catharanthus roseus cell suspension cultures. Plant Physiol Biochem 37:139–144CrossRefGoogle Scholar
  5. El-Mekkawy S, Meselhy MR, Nakamura N, Tezuka Y, Hattori M, Kakiuchi N, Shimotohno K, Kawahata T, Otake T (1998) Anti-HIV-1 and anti-HIV-protease substances from Ganoderma lucidum. Phytochemistry 49:1651–1657CrossRefGoogle Scholar
  6. Fang QH, Zhong JJ (2002) Two-stage culture process for improved production of ganoderic acid by liquid fermentation of higher fungus Ganoderma lucidum. Biotechnol Prog 18:51–54CrossRefGoogle Scholar
  7. Hirotani M, Asaka I, Furuya T (1990) Investigation of the biosynthesis of 3-hydroxy triterpenoids, ganoderic acids T and S by application of a feeding experiment using [1, 2-13C2]acetate. J Chem Soc Perkin Trans 1:2751–2754CrossRefGoogle Scholar
  8. Hsieh C, Tseng MH, Liu CJ (2006) Production of polysaccharides from Ganoderma lucidum (CCRC 36041) under limitation of nutrients. Enzyme Microb Technol 38:109–117CrossRefGoogle Scholar
  9. Hu FX, Zhong JJ (2008) Jasmonic acid mediates gene transcription of ginsenoside biosynthesis in cell cultures of Panax notoginseng treated with chemically synthesized 2-hydroxyethyl jasmonate. Process Biochem 43:113–118CrossRefGoogle Scholar
  10. Jennewein S, Rithner CD, Williams RM, Croteau RB (2001) Taxol biosynthesis: taxane 13 α-hydroxylase is a cytochrome P450-dependent monooxygenase. Proc Natl Acad Sci USA 98:13595–13600CrossRefGoogle Scholar
  11. Lambert CB, Spire C, Claude N, Guillouzo A (2009) Dose- and time-dependent effects of phenobarbital on gene expression profiling in human hepatoma HepaRG cells. Toxicol Appl Pharmacol 234:345–360CrossRefGoogle Scholar
  12. Lee JH, Yoon YH, Kim HY, Shin DH, Kim DU, Lee IJ, Kim KU (2002) Cloning and expression of squalene synthase cDNA from hot pepper (Capsicum annuum L.). Mol Cells 13:436–443Google Scholar
  13. Mitsuguchi H, Seshime Y, Fujii I, Shibuya M, Ebizuka Y, Kushiro T (2009) Biosynthesis of steroidal antibiotic fusidanes: functional analysis of oxidosqualene cyclase and subsequent tailoring enzymes from Aspergillus fumigatus. J Am Chem Soc 131:6402–6411CrossRefGoogle Scholar
  14. Qian ZG, Zhao ZJ, Xu YF, Qian XH, Zhong JJ (2004) Novel chemically synthesized hydroxyl-containing jasmonates as powerful inducing signals for plant secondary metabolism. Biotechnol Bioeng 86:809–816CrossRefGoogle Scholar
  15. Rijhwani S, Shanks JV (1998) Effect of elicitor dosage and exposure time on biosynthesis of indole alkaloids by Catharanthus roseus hairy root cultures. Biotechnol Prog 14:442–449CrossRefGoogle Scholar
  16. Robinson GW, Tsay YH, Kienzle BK, Smith-Monroy CA, Bishop RW (1993) Conservation between human and fungal squalene synthetases: similarities in structure, function, and regulation. Mol Cell Biol 13:2706–2717Google Scholar
  17. Shang CH, Zhu F, Li N, Yang X, Shi L, Zhao MW, Li YX (2008) Cloning and characterization of a gene encoding HMG-CoA reductase from Ganoderma lucidum and its functional identification in yeast. Biosci Biotechnol Biochem 72:1333–1339CrossRefGoogle Scholar
  18. Shiao MS (1992) Triterpenoid natural products in the fungus Ganoderma lucidum. J Chin Chem Soc 39:669–674Google Scholar
  19. Shibuya M, Hoshino M, Katsube Y, Hayashi H, Kushiro T, Ebizuka Y (2006) Identification of β-amyrin and sophoradiol 24-hydroxylase by expressed sequence tag mining and functional expression assay. FEBS J 273:948–959CrossRefGoogle Scholar
  20. Tang W, Liu JW, Zhao WM, Wei DZ, Zhong JJ (2006) Ganoderic acid T from Ganoderma lucidum mycelia induces mitochondria mediated apoptosis in lung cancer cells. Life Sci 80:205–211CrossRefGoogle Scholar
  21. Wagner R, Mitchell DA, Sassaki GL, De Almeida Amazonas MAL, Berovič M (2003) Current techniques for the cultivation of Ganoderma lucidum for the production of biomass, ganoderic acid and polysaccharides. Food Technol Biotechnol 41:371–382Google Scholar
  22. Wang W, Zhao ZJ, Xu Y, Qian X, Zhong JJ (2006) Efficient induction of ginsenoside biosynthesis and alteration of ginsenoside heterogeneity in cell cultures of Panax notoginseng by using chemically synthesized 2-hydroxyethyl jasmonate. Appl Microbiol Biotechnol 70:298–307CrossRefGoogle Scholar
  23. Wang G, Zhao J, Liu J, Huang Y, Zhong JJ, Tang W (2007) Enhancement of IL-2 and IFN-γ expression and NK cells activity involved in the anti-tumor effect of ganoderic acid Me in vivo. Int Immunopharmacol 7:864–870CrossRefGoogle Scholar
  24. Xu JW, Xu YN, Zhong JJ (2010) Production of individual ganoderic acids and expression of biosynthetic genes in liquid static and shaking cultures of Ganoderma lucidum. Appl Microbiol Biotechnol. doi: 10.1007/s00253-009-2106-5
  25. Yuan JP, Wang JH, Liu X, Kuang HC, Huang XN (2006) Determination of ergosterol in Ganoderma spore lipid from the germinating spores of Ganoderma lucidum by high-performance liquid chromatography. J Agric Food Chem 54:6172–6176CrossRefGoogle Scholar
  26. Yeh SF, Chou CS, Lin LJ, Shiao MS (1989) Biosynthesis of oxygenated triterpenoids in Ganoderma lucidum. Proc Natl Sci Council B ROC 13:119–127Google Scholar
  27. Yue CJ, Zhong JJ (2008) Manipulation of ginsenoside heterogeneity of Panax notoginseng cells in flask and bioreactor cultivations with addition of phenobarbital. Bioprocess Biosyst Eng 31:95–100CrossRefGoogle Scholar
  28. Zhao MW, Zhong JY, Liang WQ, Chen MJ, Zhang DB, Pan YJ, Jong SC (2007) Cloning and characterization of squalene synthase (SQS) gene from Ganoderma lucidum. J Microbiol Biotechnol 17:1106–1112Google Scholar
  29. Zhong JJ, Xiao JH (2009) Secondary metabolites from higher fungi: discovery, bioactivity, and bioproduction. Adv Biochem Eng Biotechnol 113:79–150Google Scholar
  30. Zhu LW, Zhong JJ, Tang YJ (2008) Significance of fungal elicitors on the production of ganoderic acid and Ganoderma polysaccharides by the submerged culture of medicinal mushroom Ganoderma lucidum. Process Biochem 43:1359–1370CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Cui-Xia Liang
    • 1
  • Ying-Bo Li
    • 2
  • Jun-Wei Xu
    • 1
  • Jia-Le Wang
    • 1
  • Xiao-Ling Miao
    • 1
  • Ya-Jie Tang
    • 3
  • Tingyue Gu
    • 4
  • Jian-Jiang Zhong
    • 1
    Email author
  1. 1.Molecular Biochemical Engineering Group, Key Laboratory of Microbial Metabolism (Ministry of Education), School of Life Sciences and BiotechnologyShanghai Jiao Tong UniversityShanghaiChina
  2. 2.State Key Laboratory of Bioreactor Engineering, School of BiotechnologyEast China University of Science and TechnologyShanghaiChina
  3. 3.Key Laboratory of Fermentation Engineering (Ministry of Education) and Hubei Provincial Key Laboratory of Industrial Microbiology, College of BioengineeringHubei University of TechnologyWuhanChina
  4. 4.Department of Chemical and Biomolecular EngineeringOhio UniversityAthensUSA

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