Pathway reprogramming and catalytic network engineering for the production of bioactive aspertetranones from deep-sea Aspergillus versicolor ADS-F20

Abstract

Aspertetranones are a unique class of marine fungal meroditerpenoids characterized by a highly oxygenated, linear 6/6/6/6 tetracyclic core fused to an 𝛼-pyrone scaffold. Although the pathway of aspertetranone biosynthesis in Aspergillus ochraceopetaliformis has been partially elucidated, the full potential of these compounds remains untapped. The structural diversity and enzyme promiscuity of tailoring reactions offer unexplored opportunities for the generation of bioactive derivatives through combinatorial biosynthesis. In this study, we identified the atn biosynthetic gene cluster responsible for aspertetranone production in deep-sea-derived Aspergillus versicolor ADS-F20. Through the systematic heterologous expression of 12 key genes in Aspergillus oryzae, the full pathway reconstitution and targeted biosynthesis of 17 metabolites were achieved, thus expanding the known chemical space of meroterpenoids. Notably, bioactivity screening identified compound 6 as having potent antibacterial and antifungal activities against Vibrio vulnificus ATCC 27562 (MIC = 4.50 𝜇g/mL) and Phytophthora nicotianae (MIC = 9.01 𝜇g/mL). Compound 11 demonstrated broad-spectrum anticancer and cytotoxic effects against the K-562, MCF7, and PATU8988T cell lines. This study underscores the power of pathway reprogramming and catalytic network engineering as versatile strategies for expanding the structural and functional diversity of biosynthetic pathway components.

Keywords

Fungal meroterpenoids / Aspertetranones / Combinatorial biosynthesis / Pathway reprogramming / Bioactivity

Data availability statement

The data that support the findings of this study are available in the supplementary material of this article.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

CRediT authorship contribution statement

Peiyuan Feng: Writing – original draft, Project administration, Methodology, Investigation. Moli Sang: Investigation, Data curation, Conceptualization. Wei Zhang: Writing – review & editing, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

References

[1] R. Geris, T.J. Simpson, Meroterpenoids produced by fungi, Nat. Prod. Rep. 26 (2009) 1063-1094, doi: 10.1039/b820413f.

[2] Y. Matsuda, I. Abe, Biosynthesis of fungal meroterpenoids, Nat. Prod. Rep. 33 (2016) 26-53, doi: 10.1039/c5np00090d.

[3] M. Jiang, Z. Wu, L. Liu, S. Chen, The chemistry and biology of fungal meroterpenoids (2009—2019), Org. Biomol. Chem. 19 (2021) 1644-1704, doi: 10.1039/d0ob02162h.

[4] T. Zeng, J. Li, R. Wu, Natural product databases for drug discovery: features and applications, Pharm. Sci. Adv. 2 (2024) 100050, doi: 10.1016/j.pscia.2024.100050.

[5] Y. Wang, S. Qi, Y. Zhan, N. Zhang, A.A. Wu, F. Gui, K. Guo, Y. Yang, S. Cao, Z. Hu, Z. Zheng, S. Song, Q. Xu, Y. Shen, X. Deng, Aspertetranones A—D, putative meroterpenoids from the marine algal—associated fungus Aspergillus sp. ZL0—1b14 , J. Nat. Prod. 78 (2015) 2405-2410, doi: 10.1021/acs.jnatprod.5b00487.

[6] J. Wang, X. Wei, X. Qin, X. Tian, L. Liao, K. Li, X. Zhou, X. Yang, F. Wang, T. Zhang, Z. Tu, B. Chen, Y. Liu, Antiviral merosesquiterpenoids produced by the antarctic fungus Aspergillus ochraceopetaliformis SCSIO 05702 , J. Nat. Prod. 79 (2016) 59-65, doi: 10.1021/acs.jnatprod.5b00650.

[7] R.A. Hill, A. Sutherland, Hot off the press, Nat. Prod. Rep. 41 (2024) 1214-1218, doi: 10.1039/d4np90034k.

[8] Y. Li, M. Cong, W. Wang, X. Zhang, Y. Zhu, Y. Song, W. Zhang, H. Xiao, Y. Liu, C. Zhang, J. Wang, Y. Yan, An enzymatic carbon—carbon bond cleavage and aldol reaction cascade converts an angular scaffold into the linear tetracyclic core of ochraceopones, Angew. Chem. Int. Ed. Engl. 63 (2024) e202403365, doi: 10.1002/anie.202403365.

[9] K. Ma, J. Liu, Z. Huang, M. Wu, D. Liu, J. Ren, A. Fan, W. Lin, Three—dimensional structural alignment based discovery and molecular basis of AtoB, catalyzing linear tetracyclic formation, Chem. Sci. 15 (2024) 18490-18496, doi: 10.1039/d4sc05590j.

[10] F.J. Jin, J. Maruyama, P.R. Juvvadi, M. Arioka, K. Kitamoto, Development of a novel quadruple auxotrophic host transformation system by argB gene disruption using adeA gene and exploiting adenine auxotrophy in Aspergillus oryzae , FEMS Microbiol. Lett. 239 (2004) 79-85, doi: 10.1016/j.femsle.2004.08.025.

[11] K. Blin, S. Shaw, H.E. Augustijn, Z.L. Reitz, F. Biermann, M. Alanjary, A. Fetter, B.R. Terlouw, W.W. Metcalf, E.J.N. Helfrich, G.P. van Wezel, M.H. Medema, T. Weber, antiSMASH 7.0: new and improved predictions for detection, regulation, chemical structures and visualisation, Nucleic Acids Res. 51 (2023) W46-W50, doi: 10.1093/nar/gkad344.

[12] T. Fujii, H. Yamaoka, K. Gomi, K. Kitamoto, C. Kumagai, Cloning and nucleotide sequence of the ribonuclease T1 gene (rntA) from Aspergillus oryzae and its expression in Saccharomyces cerevisiae and Aspergillus oryzae , Biosci. Biotechnol. Biochem. 59 (1995) 1869-1874, doi: 10.1271/bbb.59.1869.

[13] O. Yamada, S. Na Nan, T. Akao, M. Tominaga, H. Watanabe, T. Satoh, H. Enei, O. Akita, dffA gene from Aspergillus oryzae encodes l—ornithine N5—oxygenase and is indispensable for deferriferrichrysin biosynthesis , J. Biosci. Bioeng. 95 (2003) 82-88, doi: 10.1016/S1389—1723(03)80153—6.

[14] F.J. Jin, J.—i. Maruyama, P.R. Juvvadi, M. Arioka, K. Kitamoto, Adenine auxotrophic mutants of Aspergillus oryzae: development of a novel transformation system with triple auxotrophic hosts , Biosci. Biotechnol. Biochem. 68 (2004) 656-662, doi: 10.1271/bbb.68.656.

[15] M. Heneghan, A. Yakasai, L. Halo, Z. Song, A. Bailey, T. Simpson, R. Cox, C. Lazarus, First heterologous reconstruction of a complete functional fungal biosynthetic multigene cluster, Chembiochem 11 (2010) 1508-1512, doi: 10.1002/cbic.201000259.

[16] P.—H. Xue, N. Zhang, D. Liu, Q.—R. Zhang, J.—S. Duan, Y.—Q. Yu, J.—Y. Li, S.—J. Cao, F. Zhao, N. Kang, F. Qiu, Cytotoxic and anti—inflammatory sesquiterpenes from the whole plants of Centipeda minima , J. Nat. Prod. 84 (2021) 247-258, doi: 10.1021/acs.jnatprod.0c00884.

[17] L. Zhao, J.—Y. Tao, S.—L. Zhang, R. Pang, F. Jin, J.—H. Dong, Y.—J. Guo, Inner anti—inflammatory mechanisms of petroleum ether extract from melilotus suaveolens Ledeb , Inflammation 30 (2007) 213-223, doi: 10.1007/s10753—007—9039—x.

[18] H. Tominaga, M. Ishiyama, F. Ohseto, K. Sasamoto, T. Hamamoto, K. Suzuki, M. Watanabe, A water—soluble tetrazolium salt useful for colorimetric cell viability assay, Anal. Commun. 36 (1999) 47-50, doi: 10.1039/A809656B.

[19] M.—Y. Xu, S.Y. Lee, S.S. Kang, Y.S. Kim, Antitumor activity of jujuboside B and the underlying mechanism via induction of apoptosis and autophagy, J. Nat. Prod. 77 (2014) 370-376, doi: 10.1021/np401022g.

[20] I. Wiegand, K. Hilpert, R.E.W. Hancock, Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances, Nat. Protoc. 3 (2008) 163-175, doi: 10.1038/nprot.2007.521.

[21] Y.K. Kim, H. Tomoda, H. Nishida, T. Sunazuka, R. Obata, S. Omura, Pyripyropenes, novel inhibitors of acyl—CoA: cholesterol acyltransferase produced by Aspergillus fumigatus. II. Structure elucidation of pyripyropenes A, B, C and D , J. Antibiot. 47 (1994) 154-162 (Tokyo), doi: 10.7164/antibiotics.47.154.

[22] T. Itoh, K. Tokunaga, Y. Matsuda, I. Fujii, I. Abe, Y. Ebizuka, T. Kushiro, Reconstitution of a fungal meroterpenoid biosynthesis reveals the involvement of a novel family of terpene cyclases, Nat. Chem. 2 (2010) 858-864, doi: 10.1038/nchem.764.

[23] X. Wei, T. Matsuyama, H. Sato, D. Yan, P.M. Chan, K. Miyamoto, M. Uchiyama, Y. Matsuda, Molecular and computational bases for spirofuranone formation in setosusin biosynthesis, J. Am. Chem. Soc. 143 (2021) 17708-17715, doi: 10.1021/jacs.1c08336.

[24] T.X. Bai, Z.Y. Quan, R. Zhai, T. Awakawa, Y. Matsuda, I. Abe, Elucidation and heterologous reconstitution of chrodrimanin B biosynthesis, Org. Lett. 20 (2018) 7504-7508, doi: 10.1021/acs.orglett.8b03268.

[25] Y. Matsuda, T. Wakimoto, T. Mori, T. Awakawa, I. Abe, Complete biosynthetic pathway of anditomin: nature’s sophisticated synthetic route to a complex fungal meroterpenoid, J. Am. Chem. Soc. 136 (2014) 15326-15336, doi: 10.1021/ja508127q.

[26] Z. Quan, T. Awakawa, Recent developments in the engineered biosynthesis of fungal meroterpenoids, Beilstein. J. Org. Chem. 20 (2024) 578-588, doi: 10.3762/bjoc.20.50.

[27] X. Zhang, J. Guo, F. Cheng, S. Li, Cytochrome P450 enzymes in fungal natural product biosynthesis, Nat. Prod. Rep. 38 (2021) 1072-1099, doi: 10.1039/d1np00004g.