Studies on the Chemical Diversities of Secondary Metabolites Produced by Neosartorya fischeri via the OSMAC Method

The One Strain Many Compounds (OSMAC) method was applied to explore the chemical diversities of secondary metabolites produced by Neosartorya fischeri NRRL 181. Four pyripyropenes 1–4, eight steroids 5–11, and four prenylated indole alkaloids 12–15, were obtained from the fungus cultured in petri dishes containing potato dextrose agar (PDA). 1,7,11-trideacetylpyripyropene A (1) and 1,11-dideacetyl pyripyropene A (2) were obtained and spectroscopically characterized (1D, 2D NMR, and HR-ESI-MS) from a natural source for the first time. It offered a sustainable source of these two compounds, which were usually used as starting materials in preparing pyripyropene derivatives. In addition, as compared with all the other naturally occurring pyripyropenes, 1 and 2 possessed unique acetylation patterns that did not follow the established late-step biosynthetic rules of pyripyropenes. The natural occurrence of 1 and 2 in the fungus implied that the timing and order of hydroxylation and acetylation in the late-step biosynthetic pathway of pyripyropenes remained to be revealed. The isolation and identification of 1–15 indicated that the OSMAC method could remarkably alter the metabolic profile and enrich the chemical diversities of fungal metabolites. Compounds 1–4 exhibited no obvious cytotoxicity against the triple-negative breast cancer cell line MDA-MB-231 as compared with taxol.


Introduction
Filamentous fungi are prolific producers of bioactive natural products [1], as exemplified by the antibiotic penicillin [2] and the anti-hypercholesterolemia drug lovastatin [3]. In recent years, data from genome sequencing have revealed an inconsistency between the number of biosynthetic gene clusters (BGCs) identified as potentially encoding secondary metabolites and the actual number of chemically characterized secondary metabolites from any given fungi [4]. The reason underlying the phenomenon is that many BGCs are not expressed under laboratory conditions and are generally considered as silent or cryptic. Inducing the expression of these silent BGCs could unlock the chemical diversity they control, allowing the discovery of novel molecules of both medical and biotechnological interest. Therefore, both genetic and cultivation-based techniques have been developed aimed at stimulating expression of these silent BGCs [4]. The principles behind the cultivation based approaches have been conceptualized in the "one strain many compounds" (OSMAC) framework, which underlines how a single strain can produce different molecules when grown under different environmental conditions [5]. Unlike genetic manipulations, the OSMAC approach is not targeted to the activation of a specific cryptic gene cluster, but to the systematic alteration of readily accessible culturing parameters including medium components (salts, amino acids, and carbon source), pH, culture aeration (including the type of culture vessel used), and temperature of growth [6]. This makes the OSMAC approach an accessible, versatile, inexpensive, and relatively simple tool for regulating the metabolism of fungi [6].
Neosartorya fischeri is a thermotolerance fungus belonging to the Aspergillus subgenus Fumigati subgroup Fumigati, and shares several common physical traits with Aspergillus fumigatus [7]. As compared with A. fumigatus, the secondary metabolites of N. fischeri have not been extensively studied and only resulted in the discovery of limited types of compounds [8]. Nevertheless, bioinformatics prediction by antiSMASH [9] revealed that the genome of N. fischeri NRRL181 harbored 39 BGCs, with 25 of them not related to any previously reported natural products (https://fungismash.secondarymetabolites.org/upload/fungi-d6eb54a9-0d96-4fa0-bf97ba8be5065608/index.html), indicating great potential for mining the fungus for novel secondary metabolites. In our previous endeavor at discovering novel structures from fungi, we focused on two N. fischeri strains, i.e., N. fischeri CGMCC 3.5378 and N. fischeri NRRL181. In-depth studies on the secondary metabolites of N. fischeri CGMCC 3.5378 led to the isolation of five new prenylated indole alkaloids cultured on wheat bran [10,11] and moist corn [12], while the preliminary studies on N. fischeri NRRL181 furnished two new fumiquinazolines and a known compound pyripyropene A [13]. The promising bioactivities and rare natural occurrence of pyripyropenes [14,15] encouraged us to explore the chemical diversities of this kind of natural products. As guided by the OSMAC strategy, N. fischeri NRRL181 was cultured in 20 different conditions (Table S1) and analyzed for the production of pyripyropenes by HPLC. Herein, we report the isolation, structure characterization, and bioassay of the induced secondary metabolites from the fungus cultured in petri dishes containing PDA agar.
Compound 2 was finally identified as 1,11-dideacetylpyripyropene A on the basis of comprehensive elucidation of the 2D-NMR data ( Figure 2). As part of our ongoing screening of natural products against triple-negative breast cancer, compounds 1−4 were subjected to cytotoxicity assay against MDA-MB-231 cell line by the method we reported previously [30,31]. Unfortunately, none of them showed obvious activity as compared with the positive control, taxol.

Discussion
In the present work, OSMAC method was used to study the metabolic potential of the fungus N. fischeri NRRL181 and the chemical diversity of its secondary metabolites. Along with the targeted isolation of four pyripyropenes 1−4, eight steroids 5−11 and four prenylated indole alkaloids 12−15 were also identified from the fungus cultured on PDA agar in petri dishes. Compounds 1−3 and 5−10 have never been obtained in our previous chemical investigations on this fungus and were regarded as the induced metabolites. Compounds 5−10 are oxygenated steroids biogenetically related to ergosterol (11), with compound 5 featuring a rearranged 6/6/5/6 ring system. Their occurrence herein indicated that silent genes encoding the enzymes for rearrangement and oxidation may be activated in the present culturing condition and lead to the production of this series of oxygenated steroids.

Discussion
In the present work, OSMAC method was used to study the metabolic potential of the fungus N. fischeri NRRL181 and the chemical diversity of its secondary metabolites. Along with the targeted isolation of four pyripyropenes 1-4, eight steroids 5-11 and four prenylated indole alkaloids 12-15 were also identified from the fungus cultured on PDA agar in petri dishes. Compounds 1-3 and 5-10 have never been obtained in our previous chemical investigations on this fungus and were regarded as the induced metabolites. Compounds 5-10 are oxygenated steroids biogenetically related to ergosterol (11), with compound 5 featuring a rearranged 6/6/5/6 ring system. Their occurrence herein indicated that silent genes encoding the enzymes for rearrangement and oxidation may be activated in the present culturing condition and lead to the production of this series of oxygenated steroids.
Pyripyropenes are meroterpenoids first isolated from Aspergillus fumigatus by theŌmura group [17]. To date, only 25 pyripyropenes bearing different substitution (hydroxyl, acetoxyl, or propionyloxy groups) at C-1, C-7, C-11 and C-13 of the terpenoid moiety have been obtained from natural sources, with pyripyropenes A-R from A. fumigatus FO-1289 and its mutant [17,32,33], pyripyropenes S-T from A. similanensis [34,35], 1-deacetylpyripyropene A, 11-deacetylpyripyropene O and 13-dehydroxy-1,11-deacetylpyripyropene A from Fusarium lateritium [36], 7-deacetylpyripyropene A (3) and 13-dehydroxylpyripyropene A from N. pseudofischeri [16]. Pyripyropenes, in particular pyripyropene A (4), were discovered as a highly potent selective inhibitor of the ACAT-2 isoform, which was considered a new therapeutic target for the treatment and prevention of hypercholesterolemia and atherosclerosis [14]. The in vivo efficacy of pyripyropene A (4) has also been proved [37]. Recently, pyripyropene A (4) has also gained attention as a promising lead for developing insecticides since it exhibited excellent insecticidal activity against aphids by both foliar application and soil drenching without serious toxicological issues [15,38,39]. Compounds 1 and 2 were previously prepared by chemical deacetylation of pyripyropene A (4) [40,41] and used as starting materials in preparing pyripyropene derivatives. This is the first report on the isolation of the two compounds from a natural source, and the spectroscopic data were also completely reported and precisely assigned for the first time. The acquisition of them enriched the limited structure diversity of naturally occurring pyripyropenes and offered a sustainable way of preparing the two compounds. The BGC of pyripyropenes (pyr cluster) was identified in A. fumigatus Af293 by Itoh et al. [42], containing nine genes encoding one CoA ligase (pyr1), one polyketide synthase (pyr2), one terpene cyclase (pyr4), one flavin adenine dinucleotide dependent monooxygenase (pyr5), one prenyltransferase (pyr6), two cytochrome P450s (pyr3 and pyr9), and two acetyltransferases (pyr7 and pyr8). They also precisely proved the early steps in pyripyropenes biosynthesis, in which pyr1, pyr2, pyr6, pyr5, and pyr4 worked sequentially to construct the meroterpenoid core of pyripyropenes (deacetyl-pyripyropene E). However, the hydroxylation and acetylation mechanisms for the late steps were left unresolved. Hu et al. [43,44] identified the second BGC of pyripyropenes (ppb cluster) in another pyripyropene A producing strain Penicillium coprobium PF1169, where four tailoring genes ppb3, ppb4, ppb8, and ppb9 were highly homologous to pyr3, pyr9, pyr7, and pyr8, respectively. By introducing the four tailoring genes individually into the model fungus A. oryzae and feeding the transformants with certain predicted intermediates, they reported the functions of ppb3 (P450-1), ppb4 (P450-2), ppb8 (AT-1), and ppb9 (AT-2), and proposed the mechanism for the late steps of pyripyropene biosynthesis [43,44]. According to the pathway they proposed, compounds 1 and 2 obtained in the present study were not on the pathway and should be considered as shunt products. However, given that the conclusion has been drawn based on the bioconversion of some predicted intermediates, it is reasonable to infer that one might get different results and depict different pathways if different intermediates were used. The natural occurrence of 1 and 2 in N. fischeri NRRL181 offered us with clues that the timing and order of hydroxylation and acetylation in the late-step biosynthetic pathway of pyripyropenes may be different from those reported. Hence, the mechanisms underlying these biosynthetic steps are still open and warrant further in vitro and in vivo studies.

Fungus and Culture Conditions
The fungus was purchased from DSMZ (DE-Braunschweig). The cultivation was carried out on static potato dextrose agar (PDA) medium at 30 • C for 25 days.

Extraction and Isolation
The cultivated PDA medium along with the N. fischeri NRRL 181 mycelium was successively extracted with methanol (seven days each). The solvent was evaporated under reduced pressure to give a crude extract (13.8

Bioassay
All isolates were evaluated for the cytotoxic activities against MDA-MB-231 cell line according to protocols we previously reported [30,31], employing taxol as the positive control.

Conclusions
By applying the OSMAC method to N. fischeri NRRL 181, four pyripyropenes, eight steroids, and four prenylated indole alkaloids, were obtained from the fungus cultured in petri dishes containing PDA medium. The results validated the effectiveness of the OSMAC method in diversifying the fungal secondary metabolites. The acquisition of the two new naturally-occurring pyripyropene derivatives may help to understand the late-step biosynthetic mechanism of pyripyropenes.