Two Cytochrome P450 Enzymes Form the Tricyclic Nested Skeleton of Meroterpenoids by Sequential Oxidative Reactions.

Meroterpenoid clavilactones feature a unique benzo-fused ten-membered carbocyclic ring unit with an α,ß-epoxy-γ-lactone moiety, forming an intriguing 10/5/3 tricyclic nested skeleton. These compounds are good inhibitors of the tyrosine kinase, attracting a lot of chemical synthesis studies. However, the natural enzymes involved in the formation of the 10/5/3 tricyclic nested skeleton remain unexplored. Here, we identified a gene cluster responsible for the biosynthesis of clavilactone A in the basidiomycetous fungus Clitocybe clavipes. We showed that a key cytochrome P450 monooxygenase ClaR catalyzes the diradical coupling reaction between the intramolecular hydroquinone and allyl moieties to form the benzo-fused ten-membered carbocyclic ring unit, followed by the P450 ClaT that exquisitely and stereoselectively assembles the α,ß-epoxy-γ-lactone moiety in clavilactone biosynthesis. ClaR unprecedentedly acts as a macrocyclase to catalyze the oxidative cyclization of the isopentenyl to the nonterpenoid moieties to form the benzo-fused macrocycle, and a multifunctional P450 ClaT catalyzes a ten-electron oxidation to accomplish the biosynthesis of the 10/5/3 tricyclic nested skeleton in clavilactones. Our findings establish the foundation for the efficient production of clavilactones using synthetic biology approaches and provide the mechanistic insights into the macrocycle formation in the biosynthesis of fungal meroterpenoids.

Mining an O-methyltransferase for de novo biosynthesis of physcion in Aspergillus nidulans.

Physcion is one of natural anthraquinones, registered as a novel plant-derived fungicide due to its excellent prevention of plant disease. However, the current production of physcion via plant extraction limits its yield promotion and application. Here, a pair of polyketide synthases (PKS) in emodin biosynthesis were used as probes to mining the potential O-methyltransferase (OMT) responsible for physcion biosynthesis. Further refinement using the phylogenetic analysis of the mined OMTs revealed a distinct OMT (AcOMT) with the ability of transferring a methyl group to C-6 hydroxyl of emodin to form physcion. Through introducing AcOMT, we successfully obtained the de novo production of physcion in Aspergillus nidulans. The physcion biosynthetic pathway was further rationally engineered by expressing the decarboxylase genes from different fungi. Finally, the titer of physcion reached to 64.6 mg/L in shake-flask fermentation through enhancing S-adenosylmethionine supply. Our work provides a native O-methyltransferase for physcion biosynthesis and lays the foundation for further improving the production of physcion via a sustainable route. KEY POINTS: • Genome mining of the native O-methyltransferase responsible for physcion biosynthesis • De novo biosynthesis of physcion in the engineered Aspergillus nidulans • Providing an alternative way to produce plant-derived fungicide physcion.

Cadinane-type sesquiterpenoid dimeric diastereomers hibisceusones A-C from infected stems of Hibiscus tiliaceus with cytotoxic activity against triple-negative breast cancer cells.

Three new cadinane-type sesquiterpenoid dimeric diastereomers (1-3) named hibisceusones A-C were obtained from the infected stems of Hibiscus tiliaceus. The structures were determined by NMR spectroscopy and MS techniques, and the absolute configurations were assigned by ECD and single-crystal X-ray diffraction techniques. Compounds 1-3 are diastereomers, and contain a 1,4-dioxane ring linearly fused to different cadinane-type polycyclic skeletons. This is the first time that such a structure has been identified in natural products. Compounds 1-3 exhibited cytotoxic activities, and 2 showed a significantly high anti-triple-negative breast cancer (TNBC) effect. The anti-cancer effect of compound 2 was 3-4 fold higher than that of 1 and 3. The anti-cancer effect was generated via the induction of the apoptosis of the MDA-MB-231 cells by inhibiting the PI3Kα pathway.

Synthesis, Purification, and Selective ß2-AR Agonist and Bronchodilatory Effects of Catecholic Tetrahydroisoquinolines from Portulaca oleracea.

A green, biomimetic, phosphate-mediated Pictet-Spengler reaction was used in the synthesis of three catecholic tetrahydroisoquinolines, 1, 2, and 12, present in the medicinal plant Portulaca oleracea, as well as their analogues 3-11, 13, and 14, with dopamine hydrochloride and aldehydes as the substrates. AB-8 macroporous resin column chromatography was applied for purification of the products from the one-step high-efficacy synthesis. It eliminated the difficulties in the isolation of catecholic tetrahydroisoquinolines from the aqueous reaction system and unreacted dopamine hydrochloride. Activity screening in CHO-K1/Gα15 cell models consistently expressing α1B-, ß1-, or ß2-adrenergic receptors indicated that 12 and 2, compounds that are present in P. oleracea, possessed the most potent ß2-adrenergic receptor agonist activity and 2 was a selective ß2-adrenergic receptor agonist at the concentration of 100 µM. Both 12 and 2 exhibited dose-dependent bronchodilator effects on the histamine-induced contraction of isolated guinea-pig tracheal smooth muscle, with EC50 values of 0.8 and 2.8 µM, respectively. These findings explain the scientific rationale of P. oleracea use as an antiasthmatic herb in folk medicine and provide the basis for the discovery of novel antiasthma drugs.

Abietane-Type Diterpenoids From Nepeta bracteata Benth. and Their Anti-Inflammatory Activity.

Terpenes possess a wide range of structural features and pharmaceutical activities and are promising for drug candidates. With the aim to find bioactive terpene molecules, eight new compounds were isolated from the medicinal plant Nepeta bracteata Benth., including seven new abietane-type diterpenoids (1-7), along with a new ursane-type triterpenoid (8). The structures of compounds 1-8 were elucidated through the detailed spectroscopic analyses of their 1D and 2D NMR and MS data, and the absolute configurations of compounds 1-7 were determined by comparing their experimental and calculated ECD spectra. Compound 1 was a novel degraded carbon diterpene with the disappearing of methyl signal at C-19, while compound 7 possessed a new norabietane-type diterpenoid carbon skeleton with the presence of five-membered lactone arising from ring rearrangement. The anti-inflammatory of all obtained isolates were evaluated on lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and the results of anti-inflammatory activity screening showed that compared with the LPS model group, all compounds were significantly down-regulation the TNF-α inflammatory factor at the specific concentration, except for compound 6.

Catecholic alkaloid sulfonates and aromatic nitro compounds from Portulaca oleracea and screening of their anti-inflammatory and anti-microbial activities.

Acidic compounds were enriched from a water decoction of Portulaca oleracea using 717 anion exchange resin column chromatography. A total of 22 compounds including 9 catecholamine derivatives, of which six were rare sulfonic acid derivatives, and 9 nitro derivatives, were further isolated through various column chromatographic methods, and their structures were elucidated by interpreting their spectroscopic data and ECD calculations. Among them, 16 compounds were isolated from P. oleracea for the first time, 8 of which were undescribed compounds and four compounds were natural products. Pharmacological screening indicated that cis-3-(3-nitro-4-hydroxyphenyl)-methyl acrylate exhibited anti-inflammatory activity, measured as inhibition of nitric oxide production in LPS-stimulated RAW264.7 macrophage cells, with an EC50 value of 18.0 µM, The compounds showed only weak anti-microbial activity with (2R)-(+)-2-chloro-3-(3-nitro-4-hydroxyphenyl)-propionic acid methyl ester inhibiting Candida albicans with a MIC of 256 µg/mL, and 3-methoxy-4,5-dinitrophenol inhibiting Shigella sonnei with a MIC of 512 µg/mL.