Abstract
Meroterpenoids are hybrid natural products of both terpenoid and polyketide origin. We identified a biosynthetic gene cluster that is responsible for the production of the meroterpenoid pyripyropene in the fungus Aspergillus fumigatus through reconstituted biosynthesis of up to five steps in a heterologous fungal expression system. The cluster revealed a previously unknown terpene cyclase with an unusual sequence and protein primary structure. The wide occurrence of this sequence in other meroterpenoid and indole–diterpene biosynthetic gene clusters indicates the involvement of these enzymes in the biosynthesis of various terpenoid-bearing metabolites produced by fungi and bacteria. In addition, a novel polyketide synthase that incorporated nicotinyl-CoA as the starter unit and a prenyltransferase, similar to that in ubiquinone biosynthesis, was found to be involved in the pyripyropene biosynthesis. The successful production of a pyripyropene analogue illustrates the catalytic versatility of these enzymes for the production of novel analogues with useful biological activities.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Christianson, D. W. Structural biology and chemistry of the terpenoid cyclases. Chem. Rev. 106, 3412–3442 (2006).
Fischbach, M. A. & Walsh, C. T. Assembly-line enzymology for polyketide and nonribosomal peptide antibiotics: logic, machinery, and mechanisms. Chem. Rev. 106, 3468–3496 (2006).
Geris, R. & Simpson, T. J. Meroterpenoids produced by fungi. Nat. Prod. Rep. 26, 1063–1097 (2009).
Ōmura, S., Tomoda, H., Kim, Y. K. & Nishida, H. Pyripyropenes, highly potent inhibitors of acyl-CoA : cholesterol acyltransferase produced by Aspergillus fumigatus. J. Antibiot. 46, 1168–1169 (1993).
Tomoda, H., Kim, Y. K., Nishida, H., Masuma, R. & Ōmura, S. Pyripyropenes, novel inhibitors of acyl-CoA : cholesterol acyltransferase produced by Aspergillus fumigatus. J. Antibiot. 47, 148–153 (1994).
Das, A., Davis, M. A., Tomoda, H., Ōmura, S. & Rudel, L. L. Identification of the interaction site within acyl-CoA:cholesterol acyltransferase 2 for the isoform-specific inhibitor pyripyropene A. J. Biol. Chem. 283, 10453–10460 (2008).
Lee, S. S., Peng, F. C., Chiou, C. M. & Ling, K. H. NMR assignments of territrems A, B, and C and the structure of MB2, the major metabolite of territrem B by rat liver microsomal fraction. J. Nat. Prod. 55, 251–255 (1992).
Kuno, F., Otoguro, K., Shiomi, K., Iwai, Y. & Ōmura, S. Arisugacins A and B, novel and selective acetylcholinesterase inhibitors from Penicillium sp. FO-4259. J. Antibiot. 49, 742–747 (1996).
Chen, J. W., Luo, Y. L., Hwang, M. J., Peng, F. C. & Ling, K. H. Territrem B, a tremorgenic mycotoxin that inhibits acetylcholinesterase with a noncovalent yet irreversible binding mechanism. J. Biol. Chem. 274, 34916–34923 (1999).
Ridley, C. P. & Khosla, C. Synthesis and biological activity of novel pyranopyrones derived from engineered aromatic polyketides. ACS Chem. Biol. 2, 104–108 (2007).
Kuzuyama, T., Noel, J. P. & Richard, S. B. Structural basis for the promiscuous biosynthetic prenylation of aromatic natural products. Nature 435, 983–987 (2005).
Kawasaki, T. et al. Biosynthesis of a natural polyketide–isoprenoid hybrid compound, furaquinocin A: identification and heterologous expression of the gene cluster. J. Bacteriol. 188, 1236–1244 (2006).
Haagen, Y. et al. A gene cluster for prenylated naphthoquinone and prenylated phenazine biosynthesis in Streptomyces cinnamonensis DSM 1042. ChemBioChem 7, 2016–2027 (2006).
Winter, J. M. et al. Molecular basis for chloronium-mediated meroterpene cyclization. J. Biol. Chem. 282, 16362–16368 (2007).
Tomoda, H. et al. Biosynthesis of pyripyropene A. J. Org. Chem. 61, 882–886 (1996).
Nierman, W. C. et al. Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus. Nature 438, 1151–1156 (2005).
Fujii, I. et al. Cloning of the polyketide synthase gene atX from Aspergillus terreus and its identification as the 6-methylsalicylic acid synthase gene by heterologous expression. Mol. Gen. Genet. 253, 1–10 (1996).
Watanabe, A. et al. Product identification of polyketide synthase coded by Aspergillus nidulans wA gene. Tetrahedron Lett. 39, 7733–7736 (1998).
Watanabe, A. et al. Re-identification of Aspergillus nidulans wA gene to code for a polyketide synthase of naphthopyrone. Tetrahedron Lett. 40, 91–94 (1999).
Fujii, I. et al. Heterologous expression and product identification of Colletotrichum lagenarium polyketide synthase encoded by the PKS1 gene involved in melanin biosynthesis. Biosci. Biotechnol. Biochem. 63, 1445–1452 (1999).
Fujii, I. et al. Enzymatic synthesis of 1,3,6,8-tetrahydroxynaphthalene solely from malonyl coenzyme A by a fungal iterative type I polyketide synthase PKS1. Biochemistry 39, 8853–8858 (2000).
Watanabe, A. et al. Aspergillus fumigatus alb1 encodes naphthopyrone synthase when expressed in Aspergillus oryzae. FEMS Microbiol. Lett. 192, 39–44 (2000).
Fujii, I., Watanabe, A., Sankawa, U. & Ebizuka, Y. Identification of Claisen cyclase domain in fungal polyketide synthase WA, a naphthopyrone synthase of Aspergillus nidulans. Chem. Biol. 8, 189–197 (2001).
Fujii, I., Yoshida, N., Shimomaki, S., Oikawa, H. & Ebizuka, Y. An iterative type I polyketide synthase PKSN catalyzes synthesis of the decaketide alternapyrone with regio-specific octa-methylation. Chem. Biol. 12, 1301–1309 (2005).
Kasahara, K., Fujii, I., Oikawa, H. & Ebizuka, Y. Expression of Alternaria solani PKSF generates a set of complex reduced-type polyketides with different carbon-lengths and cyclization. ChemBioChem 7, 920–924 (2006).
Jeong, T. S. et al. GERI-BP001 compounds, new inhibitors of acyl-CoA : cholesterol acyltransferase from Aspergillus fumigatus F37. J. Antibiot. 48, 751–756 (1995).
Heide, L. Prenyl transfer to aromatic substrates: genetics and enzymology. Curr. Opin. Chem. Biol. 13, 171–179 (2009).
Zhao, Y. J., Chng, S. S. & Loh, T. P. Lewis acid-promoted intermolecular acetal-initiated cationic polyene cyclizations. J. Am. Chem. Soc. 129, 492–493 (2007).
Saikia, S., Parker, E. J., Koulman, A. & Scott, B. Four gene products are required for the fungal synthesis of the indole–diterpene, paspaline. FEBS Lett. 580, 1625–1630 (2006).
Young, C., McMillan, L., Telfer, E. & Scott, B. Molecular cloning and genetic analysis of an indole–diterpene gene cluster from Penicillium paxilli. Mol. Microbiol. 39, 754–764 (2001).
Zhang, S., Monahan, B. J., Tkacz, J. S. & Scott, B. Indole–diterpene gene cluster from Aspergillus flavus. Appl. Environ. Microbiol. 70, 6875–6883 (2004).
Dairi, T. Studies on biosynthetic genes and enzymes of isoprenoids produced by actinomycetes. J. Antibiot. 58, 227–243 (2005).
Smith, A. B. III, Kinsho, T., Sunazuka, T. & Ōmura, S. Biomimetic total synthesis of the ACAT inhibitor (+)-pyripyropene E. Tetrahedron Lett. 37, 6461–6464 (1996).
Thoma, R. et al. Insight into steroid scaffold formation from the structure of human oxidosqualene cyclase. Nature 432, 118–122 (2004).
Erkel, G., Rether, J., Anke, T. & Sterner, O. S14-95, a novel inhibitor of the JAK/STAT pathway from a Penicillium species. J. Antibiot. 56, 337–343 (2003).
Acknowledgements
We thank T.S. Jeong for providing the pyripyropene-producing strain, and K. Gomi and K. Kitamoto for their support in fungal transformation and expression. A part of this work was supported financially by a Grant-in-Aid for Young Scientists (B) (No. 21710222) from the Japan Society for the Promotion of Science (JSPS) and The Mochida Memorial Foundation for Medical and Pharmaceutical Research to T.K., a Grant-in-Aid for Scientific Research (A) (No. 20241049) to Y.E. from JSPS, a Grant-in-Aid for JSPS Fellows to T.I. from JSPS and a Grant-in-Aid for Scientific Research on Priority Areas ‘Applied Genomics’ to I.F. from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Author information
Authors and Affiliations
Contributions
T.I., Y.E. and T.K. conceived and designed the experiments, T.I., K.T. and Y.M. performed the experiments, T.I., K.T., Y.M., I.A., Y.E. and T.K. analysed the data, I.F. contributed the fungal expression system and T.I., Y.E. and T.K. co-wrote the paper.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary information
Supplementary information (PDF 2428 kb)
Rights and permissions
About this article
Cite this article
Itoh, T., Tokunaga, K., Matsuda, Y. et al. Reconstitution of a fungal meroterpenoid biosynthesis reveals the involvement of a novel family of terpene cyclases. Nature Chem 2, 858–864 (2010). https://doi.org/10.1038/nchem.764
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nchem.764