Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Uncovering hidden sesquiterpene biosynthetic pathway through expression boost area-mediated productivity enhancement in basidiomycete

The Journal of Antibiotics volume 73, pages 721–728 (2020)Cite this article

Subjects

Abstract

Basidiomycetes are known to biosynthesize many biologically interesting compounds, including terpenoids. However, they are notoriously difficult to manipulate. Previously, we identified the gene cluster encoding enzymes responsible for the biosynthesis of lagopodins, cuparene-type sesquiterpenoid quinone natural products in Coprinopsis cinerea. In this study, we attempted to increase the productivity of lagopodin B (1) and related pathway products by overexpressing the terpene cyclase gene cop6 in C. cinerea to determine the details of the complex lagopodin and hitoyol biosynthetic pathway. Random integration of the cop6 into the genome of the ku70-deficient C. cinerea strain resulted in an ~2.4-fold increase in the production of 1. However, integration of cop6 into a highly transcribed position within the chromosome we designated as an expression boost area (EBA) resulted in an ~14-fold greater production of 1. Furthermore, the EBA-integration strain allowed us to isolate a previously undetected product 2, which we determined to be the known compound, hydroxylagopodin B. This finding expanded our understanding of the lagopodin–hitoyol biosynthetic pathway and allowed us to hypothesize a possible mechanism for the biosynthesis of a related homodimeric compound, lagopodin C. Our results demonstrate the potential of targeting EBA to integrate key biosynthetic genes into the genome for enhancing the production of difficult-to-obtain compounds for studying the biosynthesis of complex secondary metabolites in basidiomycetes and other complex eukaryotic organisms.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  1. Stajich JE, Wilke SK, Ahrén D, Au CH, Birren BW, Borodovsky M, et al. Insights into evolution of multicellular fungi from the assembled chromosomes of the mushroom Coprinopsis cinerea (Coprinus cinereus). Proc Natl Acad Sci USA. 2010;107:11889–94.

    Article  CAS  Google Scholar 

  2. Masuya T, Tsunematsu Y, Hirayama Y, Sato M, Noguchi H, Nakazawa T, et al. Biosynthesis of lagopodins in mushroom involves a complex network of oxidation reactions. Org Biomol Chem. 2019;17:234–9.

    Article  CAS  Google Scholar 

  3. Liu YZ, Li YY, Sun YF, Zheng ZH, Song SY, Su WJ, et al. Highly acetylated lanostane-type triterpenoids from Coprinus cinereus 120. Helv Chim Acta. 2012;95:282–5.

    Article  CAS  Google Scholar 

  4. Lagoutte R, Winssinger N. Following the lead from nature with covalent inhibitors. Chimia. 2017;71:703–11.

    Article  CAS  Google Scholar 

  5. Bills GF, Gloer JB. Biologically active secondary metabolites from the fungi. Microbiol Spectr. 2016;4:1–32. FUNK-0009-2016.

    CAS  Google Scholar 

  6. Agger S, Lopez-Gallego F, Schmidt-Dannert C. Diversity of sesquiterpene synthases in the basidiomycete Coprinus cinereus. Mol Microbiol. 2009;72:1181–95.

    Article  CAS  Google Scholar 

  7. Otaka J, Hashizume D, Masumoto Y, Muranaka A, Uchiyama M, Koshino H, et al. Hitoyol A and B, two norsesquiterpenoids from the basidiomycete Coprinopsis cinerea. Org Lett. 2017;19:4030–3.

    Article  CAS  Google Scholar 

  8. Otaka J, Shimizu T, Futamura Y, Hashizume D, Osada H. Structures and synthesis of hitoyopodins: bioactive aromatic sesquiterpenoids produced by the mushroom Coprinopsis cinerea. Org Lett. 2018;20:6294–7.

    Article  CAS  Google Scholar 

  9. Muraguchi H, Umezawa K, Niikura M, Yoshida M, Kozaki T, Ishii K, et al. Strand-specific RNA-Seq analyses of fruiting body development in Coprinopsis cinerea. PLoS One. 2015;10:e0141586.

    Article  Google Scholar 

  10. Liu Y, Srivilai P, Loos S, Aebi M, Kues U. An essential gene for fruiting body initiation in the basidiomycete Coprinopsis cinerea is homologous to bacterial cyclopropane fatty acid synthase genes. Genetics. 2006;172:873–84.

    Article  CAS  Google Scholar 

  11. Nakazawa T, Ando Y, Kitaaki K, Nakahori K, Kamada T. Efficient gene targeting in ΔCc.ku70 or ΔCc.lig4 mutants of the agaricomycete Coprinopsis cinerea. Fungal Genet Biol. 2011;48:939–46.

    Article  CAS  Google Scholar 

  12. Nakazawa T, Tatsuta Y, Fujita T, Nakahori K, Kamada T. Mutations in the Cc.rmt1 gene encoding a putative protein arginine methyltransferase alter developmental programs in the basidiomycete Coprinopsis cinerea. Curr Genet. 2010;56:361–7.

    Article  CAS  Google Scholar 

  13. Nakazawa T, Honda Y. Absence of a gene encoding cytosine deaminase in the genome of the agaricomycete Coprinopsis cinerea enables simple marker recycling through 5-fluorocytosine counterselection. FEMS Microbiol Lett. 2015;362:fnv123.

    Article  Google Scholar 

  14. Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018;34:i884–90.

    Article  Google Scholar 

  15. Kim D, Paggi JM, Park C, Bennett C, Salzberg SL. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol. 2019;37:907–15.

    Article  CAS  Google Scholar 

  16. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The sequence alignment/map format and SAMtools. Bioinformatics. 2009;25:2078–9.

    Article  Google Scholar 

  17. Pertea M, Pertea GM, Antonescu CM, Chang TC, Mendell JT, Salzberg SL. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat Biotechnol. 2015;33:290–5.

    Article  CAS  Google Scholar 

  18. Nakazawa T, Ishiuchi K, Sato M, Tsunematsu Y, Sugimoto S, Gotanda Y, et al. Targeted disruption of transcriptional regulators in Chaetomium globosum activates biosynthetic pathways and reveals transcriptional regulator-like behavior of aureonitol. J Am Chem Soc. 2013;135:13446–55.

    Article  CAS  Google Scholar 

  19. Sugano SS, Suzuki H, Shimokita E, Chiba H, Noji S, Osakabe Y, et al. Genome editing in the mushroom-forming basidiomycete Coprinopsis cinerea, optimized by a high-throughput transformation system. Sci Rep. 2017;7:1260.

    Article  Google Scholar 

  20. Tsunematsu Y, Ishikawa N, Wakana D, Goda Y, Noguchi H, Moriya H, et al. Distinct mechanisms for spiro-carbon formation reveal biosynthetic pathway crosstalk. Nat Chem Biol. 2013;9:818–25.

    Article  CAS  Google Scholar 

  21. Winter JM, Cascio D, Dietrich D, Sato M, Watanabe K, Sawaya MR, et al. Biochemical and structural basis for controlling chemical modularity in fungal polyketide biosynthesis. J Am Chem Soc. 2015;137:9885–93.

    Article  CAS  Google Scholar 

  22. Gottlieb HE, Kotlyar V, Nudelman A. NMR chemical shifts of common laboratory solvents as trace impurities. J Org Chem. 1997;62:7512–5.

    Article  CAS  Google Scholar 

  23. Liu C, Minami A, Ozaki T, Wu J, Kawagishi H, Maruyama JI, et al. Efficient reconstitution of basidiomycota diterpene erinacine gene cluster in ascomycota host Aspergillus oryzae based on genomic DNA sequences. J Am Chem Soc. 2019;141:15519–23.

    Article  CAS  Google Scholar 

  24. Tsunematsu Y, Takanishi J, Asai S, Masuya T, Nakazawa T, Watanabe K. Genomic mushroom hunting decrypts coprinoferrin, a siderophore secondary metabolite vital to fungal cell development. Org Lett. 2019;21:7582–6.

    Article  CAS  Google Scholar 

  25. Bottom CB, Siehr DJ. Hydroxylagopodin B, a sesquiterpenoid quinone from a mutant strain of Coprinus macrorhizus var. Microsporus Phytochem. 1975;14:1433.

    Article  CAS  Google Scholar 

  26. Bollinger P. Ueber die konstitution und konfiguration der lagopodine A, B und C. Switzerland: Eidgenosichen Technischen Hockshule ZĂĽrich; 1965.

    Google Scholar 

  27. Wang P, Gao X, Tang Y. Complexity generation during natural product biosynthesis using redox enzymes. Curr Opin Chem Biol. 2012;16:362–9.

    Article  Google Scholar 

  28. Ishiuchi K, Nakazawa T, Yagishita F, Mino T, Noguchi H, Hotta K, et al. Combinatorial generation of complexity by redox enzymes in the chaetoglobosin A biosynthesis. J Am Chem Soc. 2013;135:7371–7.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We wish to thank the financial support from the Japan Society for the Promotion of Science (JSPS) (KW, 16H06449; 19H02898; 19K22291; 19KK0150; YT, 17K15265; 20K05866), the Takeda Science Foundation (KW), the Institution of Fermentation at Osaka (KW), the Japan Antibiotics Research Association (KW), the Uehara Memorial Foundation (KW), and the HOKUTO Bio-chemical Research Foundation (KW).

Author information

Author notes

  1. Junnosuke Otaka

    Present address: Biological Resources and Post-harvest Division, Japan International Research Center for Agricultural Sciences (JIRCAS), 1-1 Ohwashi, Tsukuba, Ibaraki, 305-8686, Japan

Authors and Affiliations

  1. Department of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, 422-8526, Japan

    Shihori Asai, Yuta Tsunematsu, Takahiro Masuya & Kenji Watanabe

  2. Chemical Biology Research Group, RIKEN Center for Sustainable Resource Science (CSRS), 2-1 Hirosawa, Wako-shi, Saitama, 351-0198, Japan

    Junnosuke Otaka & Hiroyuki Osada

Authors
  1. Shihori Asai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yuta Tsunematsu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Takahiro Masuya
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Junnosuke Otaka
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hiroyuki Osada
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kenji Watanabe
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kenji Watanabe.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Asai, S., Tsunematsu, Y., Masuya, T. et al. Uncovering hidden sesquiterpene biosynthetic pathway through expression boost area-mediated productivity enhancement in basidiomycete. J Antibiot 73, 721–728 (2020). https://doi.org/10.1038/s41429-020-0355-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41429-020-0355-9

This article is cited by

Search

Advanced search

Quick links