Biosynthetic Pathway for Furanosteroid Demethoxyviridin and Identification

ARTICLE DOI: 10.1038/s41467-018-04298-2 OPEN Biosynthetic pathway for furanosteroid demethoxyviridin and identification of an unusual pregnane side-chain cleavage Gao-Qian Wang1, Guo-Dong Chen1, Sheng-Ying Qin2, Dan Hu 1, Takayoshi Awakawa3, Shao-Yang Li1, Jian-Ming Lv1, Chuan-Xi Wang 1, Xin-Sheng Yao1, Ikuro Abe 3 & Hao Gao 1 1234567890():,; Furanosteroids, represented by wortmannin, viridin, and demethoxyviridin, are a special group of fungal-derived, highly oxygenated steroids featured by an extra furan ring. They are well-known nanomolar-potency inhibitors of phosphatidylinositol 3-kinase and widely used in biological studies. Despite their importance, the biosyntheses of these molecules are poorly understood. Here, we report the identification of the biosynthetic gene cluster for demethoxyviridin, consisting of 19 genes, and among them 15 biosynthetic genes, including six cytochrome P450 monooxygenase genes, are deleted. As a result, 14 biosynthetic intermediates are isolated, and the biosynthetic pathway for demethoxyviridin is elucidated. Notably, the pregnane side-chain cleavage requires three enzymes: flavin-dependent Baeyer-Villiger monooxygenase, esterase, and dehydrogenase, in sharp contrast to the single cytochrome P450-mediated process in mammalian cells. Structure–activity analyses of these obtained biosynthetic intermediates reveal that the 3-keto group, the C1β–OH, and the aromatic ring C are important for the inhibition of phosphatidylinositol 3-kinase. 1 Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy/Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan University, 510632 Guangzhou, People’s Republic of China. 2 Clinical Experimental Center, First Affiliated Hospital of Jinan University, 510630 Guangzhou, People’s Republic of China. 3 Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 HongoBunkyo-kuTokyo 113-0033, Japan. These authors contributed equally: Gao-Qian Wang, Guo-Dong Chen, Sheng-Ying Qin. Correspondence and requests for materials should be addressed to D.H. (email: [email protected]) or to I.A. (email: [email protected]) or to H.G. (email: [email protected]) NATURE COMMUNICATIONS | (2018) 9:1838 | DOI: 10.1038/s41467-018-04298-2 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04298-2 teroids are modified triterpenoids containing the tetracyclic further insight into the biosynthesis of demethoxyviridin from system of lanosterol, but lacking the three methyl groups at lanosterol, Hanson et al. identified a group of ergosterol side- S fi C4 and C14. Further modi cations in the side chain lead to chain-derived C6 and C7 alcohols sharing a common origin with – different sub-classes of steroids bearing C18 C29 skeletons. They demethoxyviridin. This suggested that the removal of the sterol are some of the most widely distributed small molecules in nature side chain during demethoxyviridin biosynthesis occurs between and serve a myriad of biological functions. Sterols are the most C20 and C22, in a pathway analogous to the sterol side-chain important form of steroids, with a hydroxyl group at C3 and a cleavage during the biosynthesis of steroid hormones in mam- skeleton derived from cholestane, among which cholesterol in malian cells18, 19. Despite these advances, the genetic basis for the animals, sitosterol in plants, and ergosterol in fungi are well- biosynthesis of furanosteroids has remained elusive. Recently, a known molecules, as they are the essential components of the transcriptional comparison of Trichoderma virens IMI 304061, a cellular membranes in these eukaryotic organisms1, 2. In addition, high producer of viridin, and its mutant strain deficient in sec- sterols are important precursors for many biologically important ondary metabolite production identified a four-gene cluster pre- molecules, such as the steroid hormones from animals and the dicted to be responsible for the biosynthesis of viridin;20 however, cardenolides from plants, by extensive carbon degradation3, 4.In it was soon realized that this gene cluster is involved in the fungi, the oxidative removal of carbons from sterol precursors biosynthesis of volatile terpene compounds, rather than viridin21. also produces active molecules, such as wortmannin, viridin, and During our manuscript submission, Bansal et al. reported a demethoxyviridin (1), which are called furanosteroids because all biosynthetic gene cluster for viridin, but they did not provide of these molecules contain an extra furan ring fused between C4 substantial evidence for the biosynthetic pathway of viridin22. and C6 of the steroidal framework (Fig. 1a)5. Since viridin was In our previous explorations for bioactive secondary first discovered in 19456, extensive biological studies of this class metabolites from fungi23, 24, we identified the endolichenic fungus of compounds have been performed, which revealed that Nodulisporium sp. (no. 65-12-7-1), which can produce large furanosteroids possess a variety of important biological proper- amounts of demethoxyviridin (1) and several analogs25, 26. These ties, including antifungal, anti-inflammatory, and antibacterial findings provided a good chance to elucidate its biosynthesis. activities7, 8. Especially, furanosteroids are nanomolar-potency Here, we report the identification of the gene cluster and the inhibitors of phosphatidylinositol 3-kinase (PI3K), among which biosynthetic pathway for 1, by the combinational use of a wortmannin has been developed as a commercial PI3K inhibitor transcriptome comparison analysis, CRISPR-Cas9-based gene widely used in various biological studies9, 10. Notably, a semi- disruption, an Aspergillus oryzae NSAR1 heterologous gene synthetic analog of wortmannin, PX-866, was tested in a phase II expression system, and an in vitro enzymatic assay. Our study clinical trial for treating cancers11. The intriguing structures and sets the stage to uncover the biosyntheses of other furanosteroids excellent biological activities of furanosteroids have thus led to and expands the chemical diversity of pharmaceutically impor- extensive efforts toward their total chemical synthesis over the tant furanosteroids by engineered biosyntheses. past 20 years, and the stereoselective synthesis of wortmannin and (–)-viridin was finally achieved in 201712, 13. However, as compared with the progress in chemical synthesis, the bio- Results synthesis of these important molecules in fungi is poorly Identification of the gene cluster for demethoxyviridin. understood. Although in most cases, terpene cyclase is often clustered with its Early isotope labeling experiments with 13C-labeled mevalo- downstream modification enzymes in fungal genomes27, the nate or acetate revealed that viridin, demethoxyvridin, and lanosterol-derived triterpenes/steroids, including ergosterol and wortmannin are formed from two farnesyl units in a steroid-like ganoderic acid, do not conform to this rule28, 29. This could be the – manner, and do not originate from a diterpenoid14 16.A reason why Kenerley et al. failed to find the biosynthetic gene subsequent study using 14C-labeled lanosterol confirmed that it is cluster of viridin, when using terpene cyclase as the starting converted to viridin via a normal steroidal pathway17. To gain point21. Therefore, a different strategy based on other features of a O O O MeO AcO OH OH C D MeO O A B H O 4 E 6 O O O O O O O O Wortmannin Viridin Demethoxyviridin (1) b vidA vidB vidC vidD vidE vidF vidG vidH vidI vidJ vidK vidL vidM vidN vidO vidP vidQ vidR vidS 42.7 kb CYP BVFMO Unknown Transporter Dehydrogenase Esterase Bet v1-like protein Oxidoreductase Glyoxalase I Demethoxyviridin biosynthetic gene cluster from Nodulisporium sp. (No. 65-12-7-1) Fig. 1 Representative furanosteroids and biosynthetic gene cluster of demethoxyviridin (1). a Structures of wortmannin, viridin, and demethoxyviridin (1). b Gene map of the demethoxyviridin biosynthetic gene cluster from Nodulisporium sp. (no. 65-12-7-1), consisting of 19 genes from vidA (g3266)tovidS (g3284). The arrow indicates the direction from the start to the stop codon. Different types of genes are indicated by different colors and among them red arrows indicate six CYP genes 2 NATURE COMMUNICATIONS | (2018) 9:1838 | DOI: 10.1038/s41467-018-04298-2 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04298-2 ARTICLE ELSD 12 O O O a q 11 13 1 OH 17 2 19 OH JN1001(wt) 1 14 10 8 15 3 O 7 O b 4 3 4 6 HO O O O ΔvidK 29 O O O 132 c 6 7 5 ΔvidA d 8 6 O O O ΔvidD 7 e 8 Δ H H vidH HO O HO O HO O O O O 9 f ΔvidP 4 56 g 11 10 12 ΔvidF OH OH O O h 13 Δ vidS H H H O O HO O HO O 14 O O O i ΔvidQ 7 89 j 2 ΔvidJ O O O 1 20 k ΔvidM O H H HO H O O HO O HO l ΔvidE O O 10 11 12 m ΔvidG O O n ΔvidN H H o ΔvidO HO O HO OH O OH O OH 13 14 p ΔvidR New compounds 10.015.0 20.0 25.0 30.0 35.0 40.0 Retention time (min) Fig. 2 Metabolite analysis of the vid gene deletion mutants. a–p HPLC profiles of extracts from the vid gene deletion mutants. q Structures of the compounds isolated from the vid gene deletion mutants. The chromatograms were monitored with an evaporative light scattering detector (ELSD). The new compounds are indicated by red numbers furanosteroids should be adopted. Since demethoxyviridin pos- three clusters, and found that only in cluster V, all of the genes sesses a highly oxygenated structure, we inferred that there should including six CYP genes were upregulated under the demethox- be multiple cytochrome P450 monooxygenase (CYP) genes in its yviridin productive conditions (Supplementary Fig. 4). This result biosynthetic gene cluster. Thus, the CYP gene clusters in the was further confirmed by RNA sequencing (RNA-Seq), and the genome could be potential targets. To identify the biosynthetic genes from g3262 to g3284 were found to be upregulated, except gene cluster of demethoxyviridin, we sequenced the whole gen- for the non-expressed gene, g3263 (Supplementary Fig.

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