Abstract
Terminators and introns are vital regulators of gene expression in many eukaryotes; however, the functional importance of these elements for controlling gene expression in Agaricomycetes remains unclear. In this study, the effects of Ceriporiopsis subvermispora terminators and introns on the expression of a recombinant hygromycin B phosphotransferase gene (hph) were characterized. Using a transient transformation system, we proved that a highly active terminator (e.g., the gpd terminator) is required for the efficient expression of the hph gene. Mutational analyses of the C. subvermispora gpd terminator revealed that hph expression was dictated by an A-rich region, which included a putative positioning element, and polyadenylation sites. In contrast, our results indicated that introns are not required for the expression of hph directed by the Csβ1-tub and Csgpd promoters in C. subvermispora. This study provides insights into the functions and cis-element requirements of transcriptional terminators in Agaricomycetes, which may be relevant for designing recombinant genes for this important fungal class.
Similar content being viewed by others
References
Burns, C., Gregory, K.E., Kirby, M., Cheung, M.K., Riqueime, 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.
Chan, S., Choi, E.A., and Shi, Y. 2011. Pre-mRNA 3′-end processing complex assembly and function. Wiley Interdiscrip. Rev. RNA 2, 321–335.
Chorev, M. and Carmel, L. 2012. The function of introns. Front. Genet. 3, 55.
Curran, K.A., Karim, A.S., Gupta, A., and Alper, H.S. 2013. Use of expression-enhancing terminators in Sacchromycetes cerevisiae to increase mRNA half-life and improve gene expression control for metabolic engineering applications. Metab. Eng. 19, 88–97.
de Mattos-Shipley, K.M.J., Ford, K.L., Alberti, F., Banks, A.M., Bailey, A.M., and Foster, G.D. 2016. The good, the bad and the tasty: The many role of mushrooms. Stud. Mycol. 85, 125–157.
Fernandez-Fueyo, E., Ruiz-Dueñas, F.J., Ferreira, P., Floudas, D., Hibbett, D.S., Canessa, P., Larrondo, L.F., James, T.Y., Seelenfreund, D., Lobos, S., et al. 2012. Comparative genomics of Ceriporiopsis subvermispora and Phanerochaete chrysosporium provide insight into selective ligninolysis. Proc. Natl. Acad. Sci. USA 109, 5458–5463.
Fitzgerald, M. and Shenk, T. 1981. The sequence 5′-AAUAAA-3′ forms part of the recognition site for polyadenylation of late SV40 mRNAs. Cell 24, 251–260.
Ford, K.L., Baumgarner, K., Henricot, B., Bailey, A.M., and Foster, G.D. 2016. A native promoter and inclusion of an intron is necessary for efficient expression of GFP or mRFP in Armillaria mellea. Sci. Rep. 6, 29226.
Gaunitz, F., Heise, H., and Gebhardt, R. 2004. A silencer element in the first intron of the glutamine synthetase gene represses induction by glucocorticoids. Mol. Endocrinol. 18, 63–69.
Gouka, R.J., Punt, P.J., Hessing, J.G., and van den Hondel, C.A. 1996. Analysis of heterologous protein production in defined recombinant Aspergillus awamori strains. Appl. Environ. Microbiol. 62, 1951–1957.
Guo, Z. and Sherman, F. 1996. 3′-end-forming signals of yeast mRNA. Trends Biochem. Sci. 21, 477–481.
Hibbett, D.S. and Donoghue, M.J. 2001. Analysis of character correlation among wood decay mechanisms, mating systems, and substrate ranges in homobasidiomycetes. Syst. Biol. 50, 215–242.
Honda, Y., Tanigawa, E., Tsukihara, T., Nguyen, X.D., Kawabe, H., Sakatoku, N., Watari, J., Sato, H., Yano, S., Tachiki, T., et al. 2019. Stable and transient transformation, and a promoter assay in the selective lignin-degrading fungus, Ceriporopsis subvermispora. AMB Express 9, 92.
Hoshida, H., Konda, M., Kobayashi, T., Yarimizu, T., and Akada, R. 2017. 5′-UTR introns enhance protein expression in the yeast Sacchoromyces cerevisiare. Appl. Microbiol. Biotechnol. 101, 241–251.
Huang, M.T. and Gorman, C.M. 1990. Intervening sequences increase efficiency of RNA 3′ processing and accumulation of cytoplasmic RNA. Nucleic Acids Res. 18, 937–947.
Ito, Y., Yamanishi, M., Ikeuchi, A., Imamura, C., Tokuhiro, K., Kitagawa, T., and Matsuyama, T. 2013. Characterization of five terminator region that increase the protein yield of transgene in Saccharomyces cerevisiae. J. Biotechnol. 168, 486–492.
Itoh, H., Wada, M., Honda, Y., Kuwahara, M., and Watanabe, T. 2003. Bioorganosolve pretreatments for simultaneous saccharification and fermentation of beech wood by ethanolysis and white rot fungi. J. Biotechnol. 103, 273–280.
Jung, K., Park, J., Choi, J., Park, B., Kim, S., Ahn, K., Choi, J., Choi, D., Kang, S., and Lee, Y.H. 2008. SNUGB: a versatile genome browser supporting comparative and functional fungal genomics. BMC Genomics 9, 586.
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 basidiomycetous promoters. Appl. Microbiol. Biotechnol. 71, 200–210.
Kuo, C.Y., Chou, S.Y., Hseu, R.S., and Huang, C.T. 2010. Heterologous expression of EGFP in enoki mushroom Flammulina velutipes. Bot. Stud. 51, 303–309.
Landowski, C.P., Huuskonen, A., Wahl, R., Westerholm-Parvinen, A., Kanerva, A., Hänninen, A.L., Salovuoni, N., Penttilä, M., Natunen, J., Ostermeiner, C., et al. 2015. Enabling low cost biopharmaceuticals: a systematic approach to delete proteases from a well-known protein production host Trichoderma reesei. PLoS ONE 10, e0134723.
Liu, K., Sun, B., You, H., Tu, J.L., Yu, X., Zhao, P., and Xu, J.W. 2020. Dual sgRNA-directed gene deletion in basidiomycetes Ganoderma lucidum using the CRISPR/Cas9 system. Microb. Biotechnol. 13, 386–386.
Lugones, L.G., Scholtmeijer, K., Klootwijk, R., and Wessels, J.G. 1999. Introns are necessary for mRNA accumulation in Schizophyllum commune. Mol. Microbiol. 32, 681–689.
Ma, B., Mayfield, M.B., and Gold, M.H. 2001. The green fluorescent protein gene functions as a reporter of gene expression in Phanerochaete chrysosporium. Appl. Environ. Microbiol. 67, 948–955.
Mandel, C.R., Bai, Y., and Tong, L. 2008. Protein factors in premRNA 3′-end processing. Cell. Mol. Life Sci. 65, 1099–1122.
Messner, K. and Srebotnik, E. 1994. Biopulping: An overview of developments in an environmentally safe paper-making technology. FEMS Microbiol. Rev. 13, 351–364.
Millevoi, S. and Vagner, S. 2010. Molecular mechanisms of eukaryotic pre-mRNA 3′ end processing regulation. Nucleic Acids Res. 38, 2757–2774.
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.
Nambu-Nishida, Y., Sakihama, Y., Ishii, J., Hasunuma, T., and Kondo, A. 2018. Selection of yeast Sacchromycetes cerevisiae promoters available for xylose cultivation and fermentation. J. Biosci. Bioeng. 125, 76–86.
Nayan, N., Sonnenberg, A.S.M., Hendriks, W.H., and Cone, J.W. 2018. Screening of white-rot fungi for bioprocessing of wheat straw into ruminant feed. J. Appl. Microbiol. 125, 468–479.
Neve, J., Patel, R., Wang, Z., Louey, A., and Furger, A.M. 2017. Cleavage and polyadenylation: ending the message expands gene regulation. RNA Biol. 14, 865–890.
Nguyen, D.X., Sakaguchi, T., Nakazawa, T., Sakamoto, M., and Honda, Y. 2020. A 14-bp stretch plays a critical role in regulating gene expression from ß1-tubulin promoters of basidiomycetes. Curr. Genet. 66, 217–228.
Nishida, H. 2015. Relationship between chromosomal GC content and isoelectric points of histones in fungi. J. Gen. Appl. Microbiol. 61, 24–26.
Peterson, R. and Nevalainen, H. 2012. Trichoderma reesei RUT-C30-thirty years of strain improvement. Microbiology 158, 59–68.
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.
Scholtmeijer, K., Wösten, H.A.B., Springer, J., and Wessels, J.G.H. 2001. Effect of introns and AT-rich sequences on expression of the bacterial hygromycin B resistance gene in the basidiomycete Schizophyllum commune. Appl. Environ. Microbiol. 67, 481–483.
Schuren, F.H.J. and Wessels, J.G.H. 1998. Expression of heterologous genes in Schizophyllum commune is often hampered by the formation of truncated transcripts. Curr. Genet. 33, 151–156.
Shaul, O. 2017. How intron enhance gene expression. Int. J. Biochem. Cell Biol. 91, 145–155.
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.
Tanaka, M., Sakai, Y., Yamada, O., Shintani, T., and Gomi, K. 2011. In silico analysis of 3′-end processing signal in Aspergillus oryzae using expressed sequence tags and genomic sequencing data. DNA Res. 18, 189–200.
Tian, B. and Graber, J.H. 2012. Signals for pre-mRNA cleavage and polyadenylation. Wiley Interdiscip. Rev. RNA 3, 385–396.
Todd, R.B., Zhou, M., Ohm, R.A., Leeggangers, H.A.C.F., Visser, L., and de Vries, R.P. 2014. Prevalence of transcription factor in ascomycetes and basidiomycetes fungi. BMC Genomics 15, 214.
Tokuoka, M., Tanaka, M., Ono, K., Takagi, S., Shintani, T., and Gomi, K. 2008. Codon optimization increases steady-state mRNA levels in Aspegillus oryzae heterologous gene expression. Appl. Environ. Microbiol. 74, 6538–6546.
Tourmente, S., Chapel, S., Dreau, D., Drake, M.E., Bruhat, A., Couders, J.L., and Dastugue, B. 1993. Enhancer and silencer elements within the first intron mediate the transcriptional regulation of the β3 tubulin gene by 20-hydroxyecdysone in Drosophila Kc cells. Insect Biochem. Mol. Biol. 23, 137–143.
Zhao, J., Hyman, L., and Moore, C. 1999. Formation of mRNA 3′ ends in Eukaryotes: mechanism, regulation and interrelationships with other steps in mRNA synthesis. Microbiol. Mol. Biol. Rev. 63, 405–445.
Acknowledgments
This work was supported by research grants from the Noda Institute for Scientific Research and the Sugiyama Institute of Industrial Chemistry as well as KAKENHI grants (25660137; 15K14771 and 19K22332 to YH).
Author information
Authors and Affiliations
Corresponding author
Additional information
Conflict of Interest
The authors declare no financial conflicts of interest.
Supplemental material for this article may be found at http://www.springerlink.com/content/120956.
Electronic Supplementary Material
Rights and permissions
About this article
Cite this article
Nguyen, D.X., Nishisaka, E., Kawauchi, M. et al. Characterization of the effects of terminators and introns on recombinant gene expression in the basidiomycete Ceriporiopsis subvermispora. J Microbiol. 58, 1037–1045 (2020). https://doi.org/10.1007/s12275-020-0213-2
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12275-020-0213-2