Discovery of fernane-type triterpenoids from Diaporthe discoidispora using genome mining and HSQC-based SMART technology

Abstract

In this study, we employed a combination of genome mining and heteronuclear single quantum coherence (HSQC)-based small molecule accurate recognition technology (SMART) technology to search for fernane-type triterpenoids. Initially, potential endophytic fungi were identified through genome mining. Subsequently, fine fractions containing various fernane-type triterpenoids were selected using HSQC data collection and SMART prediction. These triterpenoids were then obtained through targeted isolation and identification. Finally, their antifungal activity was evaluated. As a result, three fernane-type triterpenoids, including two novel compounds, along with two new sesquiterpenes and four known compounds were isolated from one potential strain, Diaporthe discoidispora. Their structures were elucidated through analysis of high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and nuclear magnetic resonance (NMR) spectroscopic data. The absolute configurations were determined using single-crystal X-ray diffraction analysis and electron capture detector (ECD) analysis. Compound 3 exhibited moderate antifungal activity against Candida albicans CMCC 98001 and Aspergillus niger.

Keywords

Fernane / Triterpenoid / Genome mining / SMART technology / Endophytic fungi / Diaporthe discoidispora

References

[1] Zhang HW, Song YC, Tan RX.Biology and chemistry of endophytes. Nat Prod Rep. 2006; 23(5):753-771. https://doi.org/10.1039/b609472b.

[2] Singh A, Singh DK, Kharwar RN, et al. Fungal endophytes as efficient sources of plant-derived bioactive compounds and their prospective applications in natural product drug discovery: insights, avenues, and challenges. Microorganisms. 2021; 9(1):197. https://doi.org/10.3390/microorganisms9010197.

[3] Liu ZG, Sun Y, Tang MY, et al. Trichodestruxins A-D: cytotoxic cyclodepsipeptides from the endophytic fungus Trichoderma harzianum. J Nat Prod. 2020; 83(12):3635-3641. https://doi.org/10.1021/acs.jnatprod.0c00808.

[4] Gakuubi MM, Munusamy M, Liang ZX, et al. Fungal endophytes: a promising frontier for discovery of novel bioactive compounds. J Fungi. 2021; 7(10):786. https://doi.org/10.3390/jof7100786.

[5] Cheng JT, Wang HM, Yu JH, et al. Discovery of a potential liver fibrosis inhibitor from a mushroom endophytic fungus by genome mining of a silent biosynthetic gene cluster. J Agric Food Chem. 2021; 69(38):11303-11310. https://doi.org/10.1021/acs.jafc.1c03639.

[6] Morishita Y, Tsukada K, Murakami K, et al. Synthetic biology-based discovery of diterpenoid pyrones from the genome of Eupenicillium shearii. J Nat Prod. 2022; 85(2):384-390. https://doi.org/10.1021/acs.jnatprod.1c00973.

[7] Shen QY, Dai GZ, Li AY, et al. Genome-guided discovery of highly oxygenated aromatic polyketides, saccharothrixins D-M, from the rare marine Actinomycete Saccharothrix sp. D09. J Nat Prod. 2021; 84(11):2875-2884. https://doi.org/10.1021/acs.jnatprod.1c00617.

[8] Schwartz RE, Smith SK, Onishi JC, et al. The isolation and structure determination of enfumafungin, a triterpene glycoside antifungal agent from the fermenation of a Hormonema sp. J Am Chem Soc. 2000;122:4882-4886.

[9] Kuhnert E, Li Y, Lan N, et al. Enfumafungin synthase represents a novel lineage of fungal triterpene cyclases. Environ Microbiol. 2018; 20(9):3325-3342. https://doi.org/10.1111/1462-2920.14333.

[10] Li XY, Lv JM, Cao ZQ, et al. Biosynthetic characterization of the antifungal fernane-type triterpenoid polytolypin for generation of new analogues via combinatorial biosynthesis. Org Biomol Chem. 2023; 21(4):851-857. https://doi.org/10.1039/d2ob02158g.

[11] Li SF, Yu XQ, Li YL, et al. Vibsane-type diterpenoids from Viburnum odoratissimum and their cytotoxic activities. J Bioorg Chem. 2021;106:104498. https://doi.org/10.1016/j.bioorg.2020.104498.

[12] Bai M, Xu W, Zhang X, et al. HSQC-based small molecule accurate recognition technology discovery of diverse cytotoxic sesquiterpenoids from Elephantopus tomentosus L. and structural revision of molephantins A and B. Phytochemistry. 2023;206:113562. https://doi.org/10.1016/j.phytochem.2022.113562.

[13] Shigematsu N, Tsujii E, Kayakiri N, et al. WF11605, an antagonist of leukotriene B4 produced by a fungus II. Structure determination. J Antibiot. 1992; 45(5):704-708. https://doi.org/10.7164/antibiotics.45.704.

[14] Kaur R, Macleod J, Foley W, et al. Gluconic acid: and antifungal agent produced by Pseudomonas species in biological control of take-all. Phytochemistry. 2006; 67(6):595-604. https://doi.org/10.1016/j.phytochem.2005.12.011.

[15] Tan X, Zhang X, Yu M, et al. Sesquiterpenoids and mycotoxin swainsonine from the locoweed endophytic fungus Alternaria oxytropis. Phytochemistry. 2019; 164:154-161. https://doi.org/10.1016/j.phytochem.2019.05.012.

[16] Brauers G, Edrada RA, Ebel R, et al. Anthraquinones and betaenone derivatives from the sponge-associated fungus Microsphaeropsis species: novel inhibitors of protein kinases. J Nat Prod. 2000; 63(6):739-745. https://doi.org/10.3168/jds.S0022-0302(01)74580-8.

[17] Schlingmann G, Milne L, Carter GT.Isolation and identification of antifungal polyesters from the marine fungus Hypoxylon oceanicum LL-15G256. Tetrahedron. 2002; 58(34):6825-6835. https://doi.org/10.1016/S0040-4020(02)00746-9.

[18] Xiao Y, Liang W, Zhang Z, et al. Polyketide derivatives from the endophytic fungus Phaeosphaeria sp. LF5 isolated from Huperzia serrata and their acetylcholinesterase inhibitory activities. J Fungi. 2022; 8(3):232. https://doi.org/10.3390/jof8030232.

[19] Gao J, Li XM, Li X, et al.New lactone and isocoumarin derivatives from the marine mangrove-derived endophytic fungus Penicillium coffeae MA-314. Phytochem Lett. 2019; 32:1-5. https://doi.org/10.1016/j.phytol.2019.04.018.

[20] Dolomanov OV, Bourhis LJ, Gildea RJ, et al. OLEX2: a complete structure solution, refinement and analysis program. J Appl Cryst. 2009; 42(2):339-341. https://doi.org/10.1515/ncrs-2023-0461.

[21] Sheldrick GM.SHELXT-Integrated space-group and crystal-structure determination. Acta Cryst. 2015; A71:3-8. https://doi.org/10.1107/S2053273314026370.

[22] Sheldrick GM.Crystal structure refinement with SHELXL. Acta Cryst. 2015; C71:3-8. https://doi.org/10.1107/S2053229614024218.