Four new N-acylated aminoalkanoic acids, namely clonoroseins E-H (1-4), together with three previously identified analogs, clonoroseins A, B, and D (5-7), were identified from the endophytic fungus Clonostachys rosea strain 15020 (CR15020), using Feature-based Molecular Networking (FBMN). The elucidation of their chemical structures, including their absolute configurations, was achieved through spectroscopic analysis combined with quantum chemical calculations. Bioinformatics analyses suggested that an iterative type I HR-PKS (CrsE) generates the polyketide side chain of these clonoroseins. Furthermore, a downstream adenylate-forming enzyme of the PKS (CrsD) was suspected to function as an amide synthetase. CrsD potentially facilitates the transformation of the polyketide moiety into an acyl-AMP intermediate, followed by nucleophilic substitution with either β-alanine or γ-aminobutyric acid to produce amide derivatives. These findings significantly expand our understanding of PKS-related products originating from C. rosea and also underscore the powerful application of FBMN analytical methods in characterization of new compounds.

Orange processing waste (OPW) generated by the processing of oranges, as well as other citrus fruits, is a major source of pectin in the market nowadays. The residues generated during the pectin extraction process may contain many phytochemicals, including flavonoids. We use state-of-the-art techniques such as liquid chromatography high-resolution mass spectrometry (LC-HRMS/MS) and feature-based molecular network (FBMN) to annotate the flavonoids in OPWs. In particular, four flavonoids, hesperidin, naringin, diosmin, and hesperetin were quantified in the samples by LC-TDQ-MS. In total, 32 flavonoids from different classes were annotated, of which 16 were polymethoxylated flavonoids, 13 were flavonoid glycosides and 3 were flavanone aglycones. The results showed that flavonoid glycosides remain in high concentrations in OPWs from pectin factories even after pectin extraction by harsh conditions. The results show an exciting opportunity to harness the untapped potential of pectin factory waste as a renewable source for the extraction of glycoside flavonoids.

Fungi have developed a wide array of defense strategies to overcome mechanical injuries and pathogen infections. Recently, photoactivity has been discovered by showing that pigments isolated from Cortinarius uliginosus produce singlet oxygen under irradiation. To test if this phenomenon is limited to dermocyboid Cortinarii, six colourful Cortinarius species belonging to different classical subgenera (i.e., Dermocybe, Leprocybe, Myxacium, Phlegmacium, and Telamonia) were investigated. Fungal extracts were explored by the combination of in vitro photobiological methods, UHPLC coupled to high-resolution tandem mass spectrometry (UHPLC-HRMS2), feature-based molecular networking (FBMN), and metabolite dereplication techniques. The fungi C. rubrophyllus (Dermocybe) and C. xanthophyllus (Phlegmacium) exhibited promising photobiological activity in a low concentration range (1-7 µg/mL). Using UHPLC-HRMS2-based metabolomic tools, the underlying photoactive principle was investigated. Several monomeric and dimeric anthraquinones were annotated as compounds responsible for the photoactivity. Furthermore, the results showed that light-induced activity is not restricted to a single subgenus, but rather is a trait of Cortinarius species of different phylogenetic lineages and is linked to the presence of fungal anthraquinones. This study highlights the genus Cortinarius as a promising source for novel photopharmaceuticals. Additionally, we showed that putative dereplication of natural photosensitizers can be done by FBMN.

Dark teas are prepared by a microbial fermentation process. Flavan-3-ol B-ring fission analogues (FBRFAs) are some of the key bioactive constituents that characterize dark teas. The precursors and the synthetic mechanism involved in the formation of FBRFAs are not known. Using a unique solid-state fermentation system with β-cyclodextrin inclusion complexation as well as targeted chromatographic isolation, spectroscopic identification, and Feature-based Molecular Networking on the Global Natural Products Social Molecular Networking web platform, we reveal that dihydromyricetin and the FBRFAs, including teadenol A and fuzhuanin A, are derived from epigallocatechin gallate upon exposure to fungal strains isolated from Fuzhuan brick tea. In particular, the strains from subphylum Pezizomycotina were key drivers for these B-/C-ring oxidation transformations. These are the same transformations seen during the fermentation process of dark teas. These discoveries set the stage to enrich dark teas and other food products for these health-promoting constituents.