Journal of Microbiology

, Volume 57, Issue 2, pp 154–162 | Cite as

Gentic overexpression increases production of hypocrellin A in Shiraia bambusicola S4201

  • Dan Li
  • Ning Zhao
  • Bing-Jing Guo
  • Xi Lin
  • Shuang-Lin ChenEmail author
  • Shu-Zhen YanEmail author
Systems and Synthetic Microbiology and Bioinformatics


Hypocrellin A (HA) is a perylenequinone (PQ) isolated from Shiraia bambusicola that shows antiviral and antitumor activities, but its application is limited by the low production from wild fruiting body. A gene overexpressing method was expected to augment the production rate of HA in S. bambusicola. However, the application of this molecular biology technology in S. bambusicola was impeded by a low genetic transformation efficiency and little genomic information. To enhance the plasmid transformant ratio, the Polyethylene Glycol-mediated transformation system was established and optimized. The following green fluorescent protein (GFP) analysis showed that the gene fusion expression system we constructed with a GAPDH promoter Pgpd1 and a rapid 2A peptide was successfully expressed in the S. bambusicola S4201 strain. We successfully obtained the HA high-producing strains by overexpressing O-methyltransferase/FAD-dependent monooxygenase gene (mono) and the hydroxylase gene (hyd), which were the essential genes involved in our putative HA biosynthetic pathway. The overexpression of these two genes increased the production of HA by about 200% and 100%, respectively. In general, this study will provide a basis to identify the genes involved in the hypocrellin A biosynthesis. This improved transformation method can also be used in genetic transformation studies of other fungi.


Shiraia bambusicola genetic transformation hydroxylase gene O-methyltransferase/FAD-dependent monooxygenase gene 


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  1. Arai, R., Ueda, H., Kitayama, A., Kamiya, N., and Nagamune, T. 2001. Design of the linkers which effectively separate domains of a bifunctional fusion protein. Protein Eng. 14, 529–532.CrossRefGoogle Scholar
  2. Cai, Y.J., Liang, X.H., Liao, X.R., Ding, Y.R., Sun, J., and Li, X.H. 2010. High-yield hypocrellin A production in solid–state fermentation by Shiraia sp. SUPER-H168. Appl. Biochem. Biotechnol. 160, 2275–2286.CrossRefGoogle Scholar
  3. Cao, E.H., Xin, S.M., and Cheng, L.S. 1992. DNA damage induced by hypocrellin–A photosensitization. Int. J. Radiat. Biol. 61, 213–219.CrossRefGoogle Scholar
  4. Chen, H.Q., Lee, M.H., Daub, M.E., and Chung, K.R. 2007. Molecular analysis of the cercosporin biosynthetic gene cluster in Cercospora nicotianae. Mol. Microbiol. 64, 755–770.CrossRefGoogle Scholar
  5. Chi, M.H., Park, S.Y., Kim, S., and Lee, Y.H. 2009. A novel pathogenicity gene is required in the rice blast fungus to suppress the basal defenses of the host. PLoS Pathog. 5, e1000401.CrossRefGoogle Scholar
  6. Choi, S.Y., Baek, S.H., Chang, S.J., Song, Y., Rafique, R., Lee, K.T., and Park, T.J. 2016. Synthesis of upconversion nanoparticles conjugated with graphene oxide quantum dots and their use against cancer cell imaging and photodynamic therapy. Biosens. Bioelectron. 93, 267–273.CrossRefGoogle Scholar
  7. Daub, M.E., Herrero, S., and Chung, K.R. 2005. Photoactivated perylenequinone toxins in fungal pathogenesis of plants. FEMS Microbiol. Lett. 252, 197–206.CrossRefGoogle Scholar
  8. Dekkers, K.L., You, B.J., Gowda, V.S., Liao, H.L., Lee, M.H., Bau, H.J., Ueng, P.P., and Chung, K.R. 2007. The Cercospora nicotianae gene encoding dual O-methyltransferase and FAD-dependent monooxygenase domains mediates cercosporin toxin biosynthesis. Fungal Genet. Biol. 44, 444–454.CrossRefGoogle Scholar
  9. Deng, H.X., Gao, R.J., Chen, J.J., Liao, X.R., and Cai, Y.J. 2016. An efficient polyethylene glycol-mediated transformation system of lentiviral vector in Shiraia bambusicola. Process Biochem. 51, 1357–1362.CrossRefGoogle Scholar
  10. Gao, M.L., Liu, Y., Hai-Ning, Y.U., Hou, H.M., and Bao, Y.M. 2017. Study on preparation conditions of protoplast of Fusarium proliferatum. J. Anhui Agri. Sci. 45, 149–151.Google Scholar
  11. Guo, L.Y., Yan, S.Z., Li, Q., Xu, Q., Lin, X., Qi, S.S., Yu, S.Q., and Chen, S.L. 2017. Poly (lactic-co-glycolic) acid nanoparticles improve oral bioavailability of hypocrellin A in rat. RSC Adv. 7, 42073–42082.CrossRefGoogle Scholar
  12. Hess, R.A. and Nakai, M. 2000. Histopathology of the male reproductive system induced by the fungicide benomyl. Histol. Histopathol. 15, 207–224.Google Scholar
  13. Hu, M.M., Cai, Y.J., Liao, X., Liao, X.R., Hao, Z.K., and Liu, J.Y. 2012. Development of an HPLC method to analyze and prepare elsinochrome C and hypocrellin A in the submerged fermentation broth of Shiraia sp. SUPER-H168. Biomed. Chromatogr. 26, 737–742.CrossRefGoogle Scholar
  14. Jiang, H., Shen, Y., Liu, W., and Lu, L. 2013. Deletion of the putative stretch-activated ion channel Mid1 is hypervirulent in Aspergillus fumigatus. Fungal Genet. Biol. 62, 62–70.CrossRefGoogle Scholar
  15. Kishi, T., Tahara, S., Taniguchi, N., Tsuda, M., Tanaka, C., and Takahashi, S. 1991. New perylenenquiones from Shiraia bambusicola. Planta Med. 57, 376–379.CrossRefGoogle Scholar
  16. Liu, B., Bao, J.Y., Zhang, Z.B., Yan, R.M., Wang, Y., Yang, H.L., and Zhu, D. 2018. Enhanced production of perylenequinones in the endophytic fungus Shiraia sp. Slf14 by calcium/calmodulin signal transduction. Appl. Microbiol. Biotechnol. 102, 153–163.CrossRefGoogle Scholar
  17. Liu, X.Y., Shen, X.Y., Li, F., Gao, J., and Hou, C.L. 2016. High-efficiency biosynthesis of hypocrellin A in Shiraia sp. using gammaray mutagenesis. Appl. Microbiol. Biotechnol. 100, 4875–4883.CrossRefGoogle Scholar
  18. Okubo, A., Yamazak, S., and Fuwa, K. 1975. Biosynthesis of cercosporin. Agric. Biol. Chem. 39, 1173–1175.Google Scholar
  19. Pan, W.S., Ji, Y.Y., Yang, Z.Y., and Wang, J.W. 2012. Screening of high-yield hypocrellin A producing mutants from Shiraia sp. S8 by protoplast mutagenesis and ultraviolet irradiation. Chin. J. Bioprocess. Eng. 10, 18–23.Google Scholar
  20. Qi, S.S., Lin, X., Zhang, M.M., Yan S.Z., Yu, S.Q., and Chen, S.L. 2014. Preparation and evaluation of hypocrellin A loaded poly (lacticco-glycolic acid) nanoparticles for photodynamic therapy. RSC Adv. 4, 40085–40094.CrossRefGoogle Scholar
  21. Sun, Q., Wei, W., Zhao, J., Song, J., Peng, F., Zhang, S.P., Zheng, Y.L., Chen, P., and Zhu, W.J. 2015. An efficient PEG/CaCl2-mediated transformation approach for the medicinal fungus Wolfiporia cocos. J. Microbiol. Biotechnol. 25, 1528–1531.CrossRefGoogle Scholar
  22. Wang, J., Wu, Y.N., Gong, Y.F., Yu, S.W., and Liu, G. 2015. Enhancing xylanase production in the thermophilic fungus Myceliophthora thermophila by homologous overexpression of Mtxyr1. J. Ind. Microbiol. Biotechnol. 42, 1–9.CrossRefGoogle Scholar
  23. Yang, H.L., Wang, Y., Zhang, Z.B., Yan, R.M., and Zhu, D. 2014. Whole-genome shotgun assembly and analysis of the genome of Shiraia sp. strain Slf14, a novel endophytic fungus producing huperzine A and hypocrellin A. Genome Announc. 2, 343–349.Google Scholar
  24. Yang, H.L., Xiao, C.X., Ma, W.X., and He, G.Q. 2009. The production of hypocrellin colorants by submerged cultivation of the medicinal fungus Shiraia bambusicola. Dyes Pigments 82, 142–146.CrossRefGoogle Scholar
  25. Yu, M.Y. and Chang, S.T. 1987. Effects of osmotic stabilizers on the activities of mycolytic enzymes used in fungal protoplast liberation. MIRCEN J. Appl. Microbiol. Biotechnol. 3, 161–167.CrossRefGoogle Scholar
  26. Zhang, Y.H., Wei, D.S., Xing, L.J., and Li, M.C. 2008. A modified method for isolating DNA from fungus. Microbiology 35, 466–469.Google Scholar
  27. Zhao, N., Lin, X., Qi, S.S., Luo Z.M., Chen, S.L., and Yan, S.Z. 2016. De novo transcriptome assembly in Shiraia bambusicola to investigate putative genes involved in the biosynthesis of hypocrellin A. Int. J. Mol. Sci. 17, 311–324.CrossRefGoogle Scholar
  28. Zhou, X., Li, G., and Xu, J.R. 2011. Efficient approaches for generating GFP fusion and epitope–tagging constructs in filamentous fungi. Methods Mol. Biol. 722, 199–212.CrossRefGoogle Scholar
  29. Zhou, J.H., Liu, J.H., Xia, S.Q., Wang, X.S., and Zhang, B.W. 2005. Effect of chelation to lanthanum ions on the photodynamic properties of hypocrellin A. J. Phys. Chem. B. 109, 19529–19535.CrossRefGoogle Scholar

Copyright information

© The Microbiological Society of Korea and Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Jiangsu Key Laboratory for Microbes and Functional Genomics, College of Life SciencesNanjing Normal UniversityNanjingP. R. China

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