Identification and characterization of a novel light-induced promoter for recombinant protein production in Pleurotus ostreatus

  • Chaomin YinEmail author
  • Xiuzhi Fan
  • Kun Ma
  • Zheya Chen
  • Defang Shi
  • Fen Yao
  • Hong Gao
  • Aimin MaEmail author


A lectin gene (plectin) with a high level of expression was previously identified by comparative transcriptome analysis of Pleurotus ostreatus. In this study, we cloned a 733-bp DNA fragment from the start codon of the plectin gene. Sequence analysis showed that the plectin promoter (Plp) region contained several eukaryotic transcription factor binding motifs, such as the TATA-box, four possible CAAT-box, light responsiveness motifs and MeJA-responsiveness motifs. To determine whether the Plp promoter was a light-regulated promoter, we constructed an expression vector with the fused egfp-hph fragment under the control of the Plp promoter and transformed P. ostreatus mycelia via Agrobacterium tume-faciens. PCR and Southern blot analyses confirmed the Plp-egfp-hph fragment was integrated into the chromosomal DNA of transformants. qRT-PCR, egfp visualization, and intra-cellular egfp determination experiments showed the Plp promoter could be a light-induced promoter that may be suitable for P. ostreatus genetic engineering. This study lays the foundation for gene homologous expression in P. ostreatus.


Pleurotus ostreatus lectin, promoter Agrobacte-rium tumefaciens light-induction 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.



This work was supported by grants from the National Natural Science Foundation of China (No. 31801921, 31772375 & 31601806), the Natural Science Foundation of Hubei Province (No. 2018CFB188), and the Hubei Academy of Agricultural Sciences Foundation for Young Researchers (No. 2017nkyjj04).


  1. Amore, A., Honda, Y., and Faraco, V. 2012a. Copper induction of enhanced green fluorescent protein expression in Pleurotus os-treatus driven by laccase poxa1b promoter. FEMS Microbiol. Lett. 337, 155–163.CrossRefGoogle Scholar
  2. Amore, A., Honda, Y., and Faraco, V. 2012b. Enhanced green fluorescent protein expression in Pleurotus ostreatus for in vivo analysis of fungal laccase promoters. Appl. Biochem. Biotechnol. 168, 761–769.CrossRefGoogle Scholar
  3. Blumhoff, M., Steiger, M.G., Marx, H., Mattanovich, D., and Sauer, M. 2013. Six novel constitutive promoters for metabolic engineering of Aspergillus niger. Appl. Microbiol. Biotechnol. 97, 259–267.CrossRefGoogle Scholar
  4. Bradford, M.M. 1976. A rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.CrossRefGoogle Scholar
  5. Burns, C., Gregory, K.E., Kirby, M., Cheung, M.K., Riquelme, M., Elliott, T.J., Challen, M.P., Bailey, A., and Foster, G.D. 2005. Efficient GFP expression in the mushrooms Agaricus bisporus and Coprinus cinereus requires introns. Fungal Genet. Biol. 42, 191–199.CrossRefGoogle Scholar
  6. Collins, C.M., Heneghan, M.N., Kilaru, S., Bailey, A.M., and Foster, G.D. 2010. Improvement of the Coprinopsis cinerea molecular toolkit using new construct design and additional marker genes. J. Microbiol. Methods 82, 156–162.CrossRefGoogle Scholar
  7. Cong, W.R., Liu, Y., Li, Q.Z., and Zhou, X.W. 2014. Cloning and analysis of a functional promoter of fungal immunomodulatory protein from Flammulina velutipes. Mol Biol. Rep. 41, 4381–4387.CrossRefGoogle Scholar
  8. Ding, Y., Liang, S., Lei, J., Chen, L, Kothe, E., and Ma, A. 2011. Agro-bacterium tumefaciens mediated fused egfp-hph gene expression under the control of gpd promoter in Pleurotus ostreatus. Microbiol. Res. 166, 314–322.CrossRefGoogle Scholar
  9. Fischer, S., Engstler, C., Procopio, S., and Becker, T. 2016. Induced gene expression in industrial Saccharomyces pastorianus var. carls-bergensis TUM 34/70: evaluation of temperature and ethanol inducible native promoters. FEMS Yeast Res. 16, pii: fow014Google Scholar
  10. Fuller, K.K., Dunlap, J.C., and Loros, J.J. 2018. Light-regulated promoters for tunable, temporal, and affordable control of fungal gene expression. Appl. Microbiol. Biotechnol. 102, 3849–3863.CrossRefGoogle Scholar
  11. Ganzlin, M. and Rinas, U. 2008. In-depth analysis of the Aspergillus niger glucoamylase (glaA) promoter performance using high-throughput screening and controlled bioreactor cultivation techniques. J. Biotechnol. 135, 266–271.CrossRefGoogle Scholar
  12. Hernandez-Garcia, C.M. and Finer, J.J. 2014. Identification and validation of promoters and cis-acting regulatory elements. Plant Sci. 218, 109–119.CrossRefGoogle Scholar
  13. Huang, X., Lu, X., and Li, J.J. 2014. Cloning, characterization and application of a glyceraldehyde-3-phosphate dehydrogenase promoter from Aspergillus terreus. J. Ind. Microbiol. Biotechnol. 41, 585–592.CrossRefGoogle Scholar
  14. Joshi, C.P. 1987. An inspection of the domain between putative TATA box and translation start site in 79 plant genes. Nucleic Acids Res. 15, 6643–6653.CrossRefGoogle Scholar
  15. Kilaru, S., Hoegger, P.J., Majcherczyk, A., Burns, C, Shishido, K., Bailey, A., Foster, G.D., and Kües, U. 2006. Expression of laccase gene lcc1 in Coprinopsis cinerea under control of various basi-diomycetous promoters. Appl. Microbiol. Biotechnol. 71, 200–210.CrossRefGoogle Scholar
  16. Kluge, J., Terfehr, D., and Kück, U. 2018. Inducible promoters and functional genomic approaches for the genetic engineering of filamentous fungi. Appl. Microbiol. Biotechnol. 102, 6357–6372.CrossRefGoogle Scholar
  17. Li, J., Liu, X.B., Zhao, Z.W., and Yang, Z.L 2019. Genetic diversity, core collection and breeding history of Pleurotus ostreatus in China. Mycoscience 60, 14–24.CrossRefGoogle Scholar
  18. Li, J., Xue, L.X, Yan, H.X., Liu, H.T., and Liang, J.Y. 2008. Inducible EGFP expression under the control of the nitrate reductase gene promoter in transgenic Dunaliella salina. J. Appl. Phycol. 20, 137–145.CrossRefGoogle Scholar
  19. Lin, Y.J., Huang, L.H., and Huang, C.T. 2013. Enhancement of heterologous gene expression in Flammulina velutipes using poly-cistronic vectors containing a viral 2A cleavage sequence. PLoS One 8, e59099.CrossRefGoogle Scholar
  20. Livak, K.J. and Schmittgen, T.D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2 method. Methods 25, 402–408.CrossRefGoogle Scholar
  21. Luo, Q.L, Li, Y.G., Gu, H.Q., Zhao, L., Gu, XP., and Li, W.B. 2013. The promoter of soybean photoreceptor GmPLP1 gene enhances gene expression under plant growth regulator and light stresses. Plant Cell Tiss. Org. 114, 109–119.CrossRefGoogle Scholar
  22. Ma, B., Mayfield, M.B., and Gold, M.H. 2001. The green fluorescent protein gene functions as a reporter of gene expression in Pha-nerochaete chrysosporium. Appl. Environ. Microbiol. 67, 948–955.CrossRefGoogle Scholar
  23. Mahnaz, M., Jamshid, F., Aliakbar, H.M., and Mohammadreza, H. 2019. Molecular cloning, expression and characterization of poxa1b gene from Pleurotus ostreatus. Mol. Biol. Rep. 46, 981–990.CrossRefGoogle Scholar
  24. Matsuzawa, T., Tohda, H., and Takegawa, K. 2013. Ethanol-inducible gene expression using gld1 + promoter in the fission yeast Schizo-saccharomyces pombe. Appl. Microbiol. Biotechnol. 97, 6835–6843.CrossRefGoogle Scholar
  25. Meyer, V., Wanka, F., van Gent, J., Arentshorst, M., van den Hondel, C.A., and Ram, A.F. 2011. Fungal gene expression on demand: an inducible, tunable, and metabolism-independent expression system for Aspergillus niger. Appl. Environ. Microbiol. 77, 2975–2983.CrossRefGoogle Scholar
  26. Müller, T., Benjdia, M., Avolio, M., Voigt, B., Menzel, D., Pardo, A., Frommer, W.B., and Wipf, D. 2006. Functional expression of the green fluorescent protein in the ectomycorrhizal model fungus Hebeloma cylindrosporum. Mycorrhiza 16, 437–442.CrossRefGoogle Scholar
  27. Nitta, Y., Miyazaki, Y., Nakamura, M., Iimura, Y., Shishido, K., Kajita, S., and Morohoshi, N. 2004. Molecular cloning of the promoter region of the glyceraldehyde-3-phosphate dehydrogenase gene that contributes to the construction of a new transformation system in Coriolus versicolo. Mycoscience 45, 131–136.CrossRefGoogle Scholar
  28. Salinas, F., Rojas, V., Delgado, V., Agosin, E., and Larrondo, L.F. 2017. Optogenetic switches for light-controlled gene expression in yeast. Appl. Microbiol. Biotechnol. 101, 2629–2640.CrossRefGoogle Scholar
  29. Sato, M., Kurahashi, A., Nishibori, K., and Fujimori, F. 2015. Development of a transformation system for the edible mushroom Grifola frondosa: Demonstrating heterologous gene expression and RNAi-mediated gene silencing. Mycoscience 56, 364–372.CrossRefGoogle Scholar
  30. Shi, L., Fang, X., Li, M., Mu, D., Ren, A., Tan, Q., and Zhao, M. 2012. Development of a simple and efficient transformation system for the basidiomycetous medicinal fungus Ganoderma lucidum. World J. Microbiol. Biotechnol. 28, 283–291.CrossRefGoogle Scholar
  31. Sun, S.J., Chen, D.X., Xie, B.G., Hu, F.P., and Zheng, J.G. 2009. Isolation of GPD promoter from Tremella fuciformis and driving expression of EGFP gene. DNA Cell Biol. 28, 65–70.CrossRefGoogle Scholar
  32. Wang, L., Zhao, S., Chen, X.X., Deng, Q.P., Li, C.X., and Feng, J.X. 2018. Secretory overproduction of a raw starch-degrading glu-coamylase in Penicillium oxalicum using strong promoter and signal peptide. Appl. Microbiol. Biotechnol. 102, 9291–9301.CrossRefGoogle Scholar
  33. Yin, Y., Liu, Y., Jin, H., Wang, S., Zhao, S., Geng, X., Li, M., and Xu, F. 2012. Polyethylene glycol-mediated transformation of fused egfp-hph gene under the control of gpd promoter in Pleurotus eryngii. Biotechnol. Lett. 34, 1895–1900.CrossRefGoogle Scholar
  34. Yin, C., Zheng, L., Zhu, J., Chen, L., and Ma, A. 2015a. Characterization of the highly active fragment of glyceraldehyde-3-phos-phate dehydrogenase gene promoter for recombinant protein expression in Pleurotus ostreatus. FEMS Microbiol. Lett. 362, pii: fnv010.Google Scholar
  35. Yin, C., Zheng, L., Zhu, J., Chen, L., and Ma, A. 2015b. Enhancing stress tolerance by overexpression of a methionine sulfoxide re-ductase A (MsrA) gene in Pleurotus ostreatus. Appl. Microbiol. Biotechnol. 99, 3115–3126.CrossRefGoogle Scholar
  36. Zhang, G., Liu, P., Wei, W., Wang, X., Wei, D., and Wang, W. 2016. A light-switchable bidirectional expression system in filamentous fungus Trichoderma reesei. J. Biotechnol. 240, 85–93.CrossRefGoogle Scholar

Copyright information

© The Microbiological Society of Korea 2019

Authors and Affiliations

  1. 1.Institute of Agro-Products Processing and Nuclear-Agricultural TechnologyHubei Academy of Agricultural SciencesWuhanP. R. China
  2. 2.National Research and Development Center for Edible Fungi Processing (Wuhan)WuhanP. R. China
  3. 3.College of Food Science and TechnologyHuazhong Agricultural UniversityWuhanP. R. China

Personalised recommendations