20-nor-Isopimarane and isopimarane diterpenoids produced by Aspergillus sp. WT03.

Five new uncommon 20-nor-isopimarane diterpenoids, aspewentins N-R (1-5), and three related known congeners (6-8), along with an isopimarane diterpenoid, sphaeropsidin C (9), were isolated from a Coptis chinensis Franch. rhizosphere soil-derived fungal strain, Aspergillus sp. WT03. The structures of compounds 1-9 were characterized based on the comprehensive analysis of the spectroscopic data, and their absolute configurations were elucidated by single crystal X-ray diffraction and time-dependent density functional theory (TDDFT)-ECD calculations. Compounds 1-5 represent rare examples of 20-nor-isopimarane analogues featuring a 1,2,3,4-tetrahydronaphthalene and 3,4-dihydronaphthalen-1(2H)-one moiety. The biosynthetic pathway of these diterpenoids was also proposed. Additionally, compounds 1, 3 and 4 showed moderate cytotoxic activity against MCF-7, A549 and HT-29 cell lines.

Characterization of Multifunctional and Non-stereoselective Oxidoreductase RubE7/IstO, Expanding the Functional Diversity of the Flavoenzyme Superfamily.

Flavin-dependent enzymes enable a broad range of redox transformations and generally act as monofunctional and stereoselective catalysts. Herein, we report the investigation of a multifunctional and non-stereoselective FMN-dependent oxidoreductase RubE7 from the rubrolone biosynthetic pathway. Our study outlines a single RubE7-catalysed sequential reduction of three spatially distinct bonds in a tropolone ring and a reversible double-bond reduction and dehydrogenation. The crystal structure of IstO (a RubE7 homologue) with 2.0 Šresolution reveals the location of the active site at the interface of two monomers, and the size of active site is large enough to permit both flipping and free rotation of the substrate, resulting in multiple nonselective reduction reactions. Molecular docking and site mutation studies demonstrate that His106 is oriented towards the substrate and is important for the reverse dehydrogenation reaction.

Discovery and Characterization of a New Family of Diterpene Cyclases in Bacteria and Fungi.

Diterpene cyclases from bacteria and basidiomycete fungi are seldom studied. Here, we presented the identification and verification of EriG, a member of the UbiA superfamily, as the enzyme responsible for the cyclization of the cyathane skeleton in the mushroom Hericium erinaceum. Genome mining using the EriG protein sequence as a probe led to the discovery of a new family of ubiquitous UbiA-related diterpene cyclases in bacteria and fungi. We successfully characterized seven new diterpene cyclases from bacteria or basidiomycete fungi with the help of an engineered Escherichia coli strain and determined the structures of their corresponding products. A new diterpene with an unusual skeleton was generated during this process. The discovery of this new family of diterpene cyclases provides new insight into the UbiA superfamily.

Bioactive Sesquiterpenes from the Edible Mushroom Flammulina velutipes and Their Biosynthetic Pathway Confirmed by Genome Analysis and Chemical Evidence.

Twelve putative sesquiterpene synthases genes were found in clades along with enzymes with 1,6-, 1,10-, and 1,11-cyclase activities in the genome of Flammulina velutipes. Chemistry investigation of F. velutipes led to the identification of two seco-cuparane sesquiterpenes, flammufuranone A (1) and B (2); 13 new sesquiterpenes with nor-eudesmane, spiroaxane, cadinane, and cuparane skeletons (3-14, 16); as well as two new ergosterol derivatives (17 and 18). Sesquiterpenes (3-14) derived from 1,10-cyclizing enzyme were first reported from this mushroom. The absolute configurations in 1 (3R,7S) and 2 (3R,7R) were assigned by electronic circular dichroism (ECD) calculation. The absolute configuration in 3 was confirmed by X-ray diffraction analysis. The absolute configurations in the 1,2-diol moiety of 13, and in the 1,3-diol moiety of 17 and 18 were determined using Snatzke's method. Among these compounds, 3, 5, 13, and 14 were found to inhibit the HMG-CoA reductase with IC50 of 114.7, 77.6, 55.5, and 87.1 µM, respectively. Compounds 5, 6, 7, 10, 13, and 14 showed DPP-4 inhibitory activity with IC50 of 75.9, 83.7, 70.9, 79.7, 80.5, and 74.8 µM, respectively. The biosynthesis for sesquiterpenes in F. velutipes was also discussed.

Sesquiterpenoids with PTP1B Inhibitory Activity and Cytotoxicity from the Edible Mushroom Pleurotus citrinopileatus.

One new perhydrobenzannulated 5,5-spiroketal sesquiterpene, pleurospiroketal F (1), as well as six new modified bisabolene sesquiterpenes pleurotins A-F (2-7) were isolated from solid-state fermentation of Pleurotus citrinopileatus. The structures of compounds 1-7 were determined by NMR and MS spectroscopic analysis. The absolute configuration of 1 was determined by X-ray diffraction analysis, while the absolute configurations of 3-7 were assigned using the in situ dimolybdenum circular dichroism method and circular dichroism data comparison. Protein tyrosine phosphatase 1B plays a crucial role as a negative regulator of the insulin-dependent signal cascades. Therefore, the protein tyrosine phosphatase 1B inhibitor can be used for treating type 2 diabetes mellitus and obesity. Compounds 2 and 6 showed moderate inhibitory effects on protein tyrosine phosphatase 1B with IC50 s of 32.1 µM and 30.5 µM, respectively. The kinetic study confirmed compound 2 to be a noncompetitive inhibitor. Compounds 1-7 did not show cytotoxic activity against cancer cell lines (IC50 > 50 µM).

Characterization and Biosynthesis of a Rare Fungal Hopane-Type Triterpenoid Glycoside Involved in the Antistress Property of Aspergillus fumigatus.

A rare fungal hopane-type triterpenoid glycoside fumihopaside A was identified by genome mining in combination with HPLC-MS/MS in Aspergillus fumigatus. Combining genetic deletions in A. fumigatus with heterologous reconstitutions in Aspergillus nidulans of the afum gene cluster, we identified one fungal squalene hopane cyclase AfumA charging the cyclization of the hopene skeleton, one cytochrome P450, and one UDP-glycosyltransferase. Bioassays indicated that fumihopaside A plays important roles in protecting A. fumigatus against heat or ultraviolet stress.

Diketopiperazines and 2H-pyran-2-ones with antioxidant activity from the rice fermented with Aspergillus luchuensis.

Aspergillus luchuensis is widely used as a starter of saccharification in the koji industry, but no secondary metabolites have been reported from this fungus. Herein, we report the isolation and identification of four new diketopiperazine derivatives (1-4), one new methyl 4-(3-acetyl-2, 6-dihydroxyphenyl)-2-methoxybutanoate (5), and six known compounds (6-11) from the rice koji of A. luchuensis. The structures of 1-5 were determined by extensive spectral analysis including 1D and 2D NMR, HRESIMS, and CD, and ECD calculation. In antioxidant assays, compound 10 displayed moderate DPPH scavenging activity with an EC50 value of 60.8µM; compounds 1-4, 10 and 11 showed reducing ability with EC50 values ranging from 8.73 to 176.39µM. Compounds 1-11 showed no cytotoxicity against cell lines A549, K562, ASPC, and H460 at 200µM. Our current reports support the safety of A. luchuensis in food chemistry and confirm this fungus to be a new source of natural antioxidants.

New sesquiterpenoids from the edible mushroom Pleurotus cystidiosus and their inhibitory activity against α-glucosidase and PTP1B.

Nine new sesquiterpenoids, clitocybulol derivatives, clitocybulols G-O (1-9) and three known sesquiterpenoids, clitocybulols C-E (10-12), were isolated from the solid culture of the edible fungus Pleurotus cystidiosus. The structures of compounds 1-12 were determined by spectroscopic methods. The absolute configurations of compounds 1-9 were assigned via the circular dichroism (CD) data analysis. Compounds 1, 6 and 10 showed moderate inhibitory activity against protein tyrosine phosphatase-1B (PTP1B) with IC50 values of 49.5, 38.1 and 36.0µM, respectively.

Gloeophyllins A-J, Cytotoxic Ergosteroids with Various Skeletons from a Chinese Tibet Fungus Gloeophyllum abietinum.

Ten new ergosteroids, gloeophyllins A-J (1-10), have been isolated from the solid cultures of Gloeophyllum abietinum. The absolute configurations of 1, 2, and 9 were determined by X-ray crystallographic analysis. Compound 1 has a rare C-nor-D-homosteroid skeleton. Compound 9 possesses an unusual ergostane skeleton having a 10-oxabicyclo [4.3.1] decane moiety replacing 6/5 fused C/D rings. Compound 10 represents the first ergosteroid featuring the cleavage of a C8-C14 bond. The cytotoxicity of 1-10 was tested against the human cancer cell lines K562 and HCT116. The biosynthetic pathway for 1-10 is postulated.