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Development of a Transformation System for the Medicinal Fungus Sanghuangporus baumii and Acquisition of High-Value Strain

  • Zengcai Liu (College of Forestry, Northeast Forestry University) ;
  • Ruipeng Liu (College of Forestry, Northeast Forestry University) ;
  • Li Zou (College of Forestry, Northeast Forestry University)
  • Received : 2023.03.22
  • Accepted : 2023.05.28
  • Published : 2023.06.30

Abstract

To further explore the molecular mechanism of triterpenoid biosynthesis and acquire high-value strain of Sanghuangporus baumii, the Agrobacterium tumefaciens-mediated transformation (ATMT) system was studied. The key triterpenoid biosynthesis-associated gene isopentenyl diphosphate isomerase (IDI) was transformed into S. baumii by ATMT system. Then, the qRT-PCR technique was used to analyze gene transcript level, and the widely targeted metabolomics was used to investigate individual triterpenoid content. Total triterpenoid content and anti-oxidant activity were determined by spectrophotometer. In this study, we for the first time established an efficient ATMT system and transferred the IDI gene into S. baumii. Relative to the wild-type (WT) strain, the IDI-transformant (IT) strain showed significantly higher transcript levels of IDI and total triterpenoid content. We then investigated individual triterpenoids in S. baumii, which led to the identification of 10 distinct triterpenoids. The contents of individual triterpenoids produced by the IT2 strain were 1.76-10.03 times higher than those produced by the WT strain. The triterpenoid production showed a significant positive correlation with the IDI gene expression. Besides, IT2 strain showed better anti-oxidant activity. The findings provide valuable information about the biosynthetic pathway of triterpenoids and provide a strategy for cultivating high-value S. baumii strains.

Keywords

Acknowledgement

This work was supported by the National Natural Science Foundation of China [32171792].

References

  1. Yang Y, He P, Li N. The antitumor potential of extract of the oak bracket medicinal mushroom Inonotus baumii in SMMC-7721 tumor cells. Evid Based Complement Alternat Med. 2019;2019:1242784.
  2. Cai CS, Ma JX, Han CR, et al. Extraction and antioxidant activity of total triterpenoids in the mycelium of a medicinal fungus, Sanghuangporus sanghuang. Sci Rep. 2019;9(1):7418.
  3. Liu MM, Zeng P, Li XT, et al. Antitumor and immunomodulation activities of polysaccharide from Phellinus baumii. Int J Biol Macromol. 2016;91:1199-1205. https://doi.org/10.1016/j.ijbiomac.2016.06.086
  4. Yang K, Zhang S, Ying YM, et al. Cultivated fruit body of Phellinus baumii: a potentially sustainable antidiabetic resource. ACS Omega. 2020;5(15):8596-8604. https://doi.org/10.1021/acsomega.9b04478
  5. Sun TT, Sun XY, Wang XT, et al. Construction of prokaryotic expression and overexpression vectors of squalene epoxidase gene from Sanghuangporus baumii. Chin Trad Herb Drugs. 2018;49:2632-2639.
  6. Li ZN, Li WJ, Wang JZ, et al. Defatted silkworm pupae hydrolysates as a nitrogen source to produce polysaccharides and flavonoids using Phellinus baumii. Biomass Conv. Bioref. 2021;11(2):527-537. https://doi.org/10.1007/s13399-020-00800-3
  7. Zhang DH, Jiang LX, Li N, et al. Overexpression of the squalene epoxidase gene alone and in combination with the 3-hydroxy-3-methylglutaryl coenzyme a gene increases ganoderic acid production in Ganoderma lingzhi. J Agric Food Chem. 2017;65(23):4683-4690. https://doi.org/10.1021/acs.jafc.7b00629
  8. Lee CH, Hsu KH, Wang SY, et al. Cloning and characterization of the lanosterol 14alpha-demethylase gene from Antrodia cinnamomea. J Agric Food Chem. 2010;58(8):4800-4807. https://doi.org/10.1021/jf904257h
  9. Sun TT, Zou L, Zhang LF, et al. Methyl jasmonate induces triterpenoid biosynthesis in Inonotus baumii. Biotechnol. Biotechnol. Equip. 2017;31(2):312-317. https://doi.org/10.1080/13102818.2017.1284023
  10. Xu YN, Xia XX, Zhong JJ. Induction of ganoderic acid biosynthesis by Mn2+ in static liquid cultivation of Ganoderma lucidum. Biotechnol Bioeng. 2014;111(11):2358-2365. https://doi.org/10.1002/bit.25288
  11. Xu YN, Xia XX, Zhong JJ. Induced effect of Na+ on ganoderic acid biosynthesis in static liquid culture of Ganoderma lucidum via calcineurin signal transduction. Biotechnol Bioeng. 2013;110(7):1913-1923. https://doi.org/10.1002/bit.24852
  12. Lu JMY, Fan WL, Wang WF, et al. Genomic and transcriptomic analyses of the medicinal fungus Antrodia cinnamomea for its metabolite biosynthesis and sexual development. Proc Natl Acad Sci USA. 2014;111(44):E4743-E4752. https://doi.org/10.1073/pnas.1417570111
  13. Zhou JS, Ji SL, Ren MF, et al. Enhanced accumulation of individual ganoderic acids in a submerged culture of Ganoderma lucidum by the overexpression of squalene synthase gene. Biochem. Eng. J. 2014;90:178-183. https://doi.org/10.1016/j.bej.2014.06.008
  14. Lin YL, Lee YR, Tsao NW, et al. Characterization of the 2,3-Oxidosqualene cyclase gene from Antrodia cinnamomea and enhancement of cytotoxic triterpenoid compound production. J Nat Prod. 2015;78(7):1556-1562. https://doi.org/10.1021/acs.jnatprod.5b00020
  15. Ajikumar PK, Xiao WH, Tyo KEJ, et al. Isoprenoid pathway optimization for taxol precursor overproduction in Escherichia coli. Science. 2010;330(6000):70-74. https://doi.org/10.1126/science.1191652
  16. Wu FL, Shi L, Yao J, et al. The cloning, characterization, and functional analysis of a gene encoding an isopentenyl diphosphate isomerase involved in triterpene biosynthesis in the lingzhi or reishi medicinal mushroom Ganoderma lucidum (higher basidiomycetes). Int J Med Mushrooms. 2013;15(3):223-232. https://doi.org/10.1615/IntJMedMushr.v15.i3.10
  17. Zhang DH, Li N, Yu X, et al. Overexpression of the homologous lanosterol synthase gene in ganoderic acid biosynthesis in Ganoderma lingzhi. Phytochemistry. 2017;134:46-53. https://doi.org/10.1016/j.phytochem.2016.11.006
  18. Wang YH, Wang GQ, Yi XM, et al. Hepatoprotective and antioxidant effects of total triterpenoids from Poria cocos. EJMP. 2017;21(2):1-9. https://doi.org/10.9734/EJMP/2017/37908
  19. Wang JR, Li YY, Liu DN. Cloning and characterization of farnesyl diphosphate synthase gene involved in triterpenoids biosynthesis from Poria cocos. Int J Mol Sci. 2014;15(12):22188-22202. https://doi.org/10.3390/ijms151222188
  20. Wu XD, Xie JZ, Qiu L, et al. The anti-inflammatory and analgesic activities of the ethyl acetate extract of Viburnum taitoense Hayata. J Ethnopharmacol. 2021;269:113742.
  21. Yang CS, Li WC, Li C, et al. Metabolism of ganoderic acids by a Ganoderma lucidum cytochrome P450 and the 3-keto sterol reductase ERG27 from yeast. Phytochemistry. 2018;155:83-92. https://doi.org/10.1016/j.phytochem.2018.07.009
  22. Wang XT, Wang SX, Xu XR, et al. Molecular cloning, characterization, and heterologous expression of an acetyl-CoA acetyl transferase gene from Sanghuangporus baumii. Protein Expr Purif. 2020;170:105592.
  23. Huang J, Wang K, Zuo S, et al. Unsaturated fatty acid promotes the production of triterpenoids in submerged fermentation of Sanghuangporus baumii. Food Biosci. 2020;37:100712.
  24. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2DDCT method. Methods. 2001;25(4):402-408. https://doi.org/10.1006/meth.2001.1262
  25. Liu ZC, Sun TT, Wang SX, et al. Cloning, molecular properties and differential expression analysis of the isopentenyl diphosphate isomerase gene in Sanghuangporus baumii. Biotechnol Biotechnol Equip. 2020;34(1):623-630. https://doi.org/10.1080/13102818.2020.1792342
  26. Maamoun AA, El-Akkad RH, Farag MA. Mapping metabolome changes in Luffa aegyptiaca Mill fruits at different maturation stages via MS-based metabolomics and chemometrics. J Adv Res. 2021;29:179-189. https://doi.org/10.1016/j.jare.2019.10.009
  27. Lv H, Zhang Y, Shi J, et al. Phytochemical profiles and antioxidant activities of Chinese dark teas obtained by different processing technologies. Food Res Int. 2017;100(Pt 3):486-493. https://doi.org/10.1016/j.foodres.2016.10.024
  28. Vain P. Thirty years of plant transformation technology development. Plant Biotechnol J. 2007;5(2):221-229. https://doi.org/10.1111/j.1467-7652.2006.00225.x
  29. Chen X, Stone M, Schlagnhaufer C, et al. A fruiting body tissue method for efficient Agrobacterium-mediated transformation of Agaricus bisporus. Appl Environ Microbiol. 2000;66(10):4510-4513. https://doi.org/10.1128/AEM.66.10.4510-4513.2000
  30. Michielse CB, Hooykaas PJJ, Hondel C, et al. Agrobacterium-mediated transformation of the filamentous fungus Aspergillus awamori. Nat Protoc. 2008;3(10):1671-1678. https://doi.org/10.1038/nprot.2008.154
  31. Shi L, Fang X, Li MJ, et al. Development of a simple and efficient transformation system for the basidiomycetous medicinal fungus Ganoderma lucidum. World J Microbiol Biotechnol. 2012;28(1):283-291. https://doi.org/10.1007/s11274-011-0818-z
  32. Wang S, Chen H, Wang Y, et al. Effects of Agrobacterium tumefaciens strain types on the Agrobacterium-mediated transformation efficiency of filamentous fungus Mortierella alpina. Lett Appl Microbiol. 2020;70(5):388-393. https://doi.org/10.1111/lam.13286
  33. Shi L, Qin L, Xu YJ, et al. Molecular cloning, characterization, and function analysis of a mevalonate pyrophosphate decarboxylase gene from Ganoderma lucidum. Mol Biol Rep. 2012;39(5):6149-6159. https://doi.org/10.1007/s11033-011-1431-9
  34. Liu RD, Kim WY, Paguirigan JA, et al. Establishment of Agrobacterium tumefaciens-mediated transformation of Cladonia macilenta, a model lichen-forming fungus. JOF. 2021;7(4):252.
  35. Liu N, Chen GQ, Ning GA, et al. Agrobacterium tumefaciens-mediated transformation: an efficient tool for insertional mutagenesis and targeted gene disruption in Harpophora oryzae. Microbiol Res. 2016;182:40-48. https://doi.org/10.1016/j.micres.2015.09.008
  36. Cardoza RE, Vizca ino JA, Hermosa MR, et al. Cloning and characterization of the erg1 gene of Trichoderma harzianum: effect of the erg1 silencing on ergosterol biosynthesis and resistance to terbinafine. Fungal Genet Biol. 2006;43(3):164-178. https://doi.org/10.1016/j.fgb.2005.11.002
  37. Sun J, Zhang YY, Liu H, et al. A novel cytoplasmic isopentenyl diphosphate isomerase gene from tomato (Solanum lycopersicum): cloning, expression, and color complementation. Plant Mol Biol Rep. 2010;28(3):473-480. https://doi.org/10.1007/s11105-009-0174-4
  38. Wang YC, Qiu CX, Zhang F, et al. Molecular cloning, expression profiling and functional analyses of a cDNA encoding isopentenyl diphosphate isomerase from Gossypium barbadense. Biosci Rep. 2009; 29(2):111-119. https://doi.org/10.1042/BSR20070052
  39. Miscioscia E, Shmalberg J, Scott KC. Measurement of 3-acetyl-11-keto-beta-boswellic acid and 11- keto-beta-boswellic acid in Boswellia serrata supplements administered to dogs. BMC Vet Res. 2019;15(1):1-7. https://doi.org/10.1186/s12917-018-1758-8
  40. Kim SH, Jung SH, Lee YJ, et al. Dammarenediol-II prevents VEGF-mediated microvascular permeability in diabetic mice. Phytother Res. 2015;29(12):1910-1916. https://doi.org/10.1002/ptr.5480
  41. Lu B, Liu L, Zhen X, et al. Anti tumor activity of triterpenoid rich extract from bamboo shavings Caulis bamfusae in Taeniam. Afr J Biotechnol. 2010;9:6430-6436.
  42. Supanimit T, Sasinun S, Chadarat A, et al. Pharmacokinetics of ganoderic acids a and f after oral administration of Ling Zhi preparation in healthy male volunteers. J. Evidence-Based Integr. Med. 2012;2012:780892.
  43. Jayaprakasha GK, Jadegoud Y, Nagana GGA, et al. Bioactive compounds from sour orange inhibit Colon cancer cell proliferation and induce cell cycle arrest. J Agric Food Chem. 2010;58(1):180-186. https://doi.org/10.1021/jf9027816
  44. Poulose SM, Harris ED, Patil BS. Citrus limonoids induce apoptosis in human neuroblastoma cells and have radical scavenging activity. J Nutr. 2005;135(4):870-877. https://doi.org/10.1093/jn/135.4.870
  45. Hajjaj H, Mace C, Roberts M, et al. Effect of 26-oxygenosterolsfrom Ganoderma lucidum and their activity as cholesterol synthesis inhibitors. Appl Environ Microbiol. 2005;71(7):3653-3658.
  46. Sawai S, Saito K. Triterpenoid biosynthesis and engineering in plants. Front Plant Sci. 2011;2:25.