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Cyclic peptides with cyclophane linkers are an attractive compound type owing to the fine‐tuned rigid three‐dimensional structures and unusual biophysical features. Cytochrome P450 enzymes are capable of catalyzing not only the C−C and C−O oxidative coupling reactions found in vancomycin and other nonribosomal peptides (NRPs), but they also exhibit novel catalytic activities to generate cyclic ribosomally synthesized and post‐translationally modified peptides (RiPPs) through cyclophane linkage. To discover more P450‐modified multicyclic RiPPs, we set out to find cryptic and unknown P450‐modified RiPP biosynthetic gene clusters (BGCs) through genome mining. Synergized bioinformatic analysis reveals that P450‐modified RiPP BGCs are broadly distributed in bacteria and can be classified into 11 classes. Focusing on two classes of P450‐modified RiPP BGCs where precursor peptides contain multiple conserved aromatic amino acid residues, we characterized 11 novel P450‐modified multicyclic RiPPs with different cyclophane linkers through heterologous expression. Further mutation of the key ring‐forming residues and combinatorial biosynthesis study revealed the order of bond formation and the specificity of P450s. This study reveals the functional diversity of P450 enzymes involved in the cyclophane‐containing RiPPs and indicates that P450 enzymes are promising tools for rapidly obtaining structurally diverse cyclic peptide derivatives. [ABSTRACT FROM AUTHOR]

Flavoprotein monooxygenases (FPMOs) play important roles in generating structural complexity and diversity in natural products biosynthesized by type II polyketide synthases (PKSs). In this study, we used genome mining to discover novel mutaxanthene analogues and investigated the biosynthesis of these aromatic polyketides and their unusual xanthene framework. We determined the complete biosynthetic pathway of mutaxathene through in vivo gene deletion and in vitro biochemical experiments. We show that a multifunctional FPMO, MtxO4, catalyzes ring rearrangement and generates the required xanthene ring through a multistep transformation. In addition, we successfully obtained all necessary enzymes for in vitro reconstitution and completed the total biosynthesis of mutaxanthene in a stepwise manner. Our results revealed the formation of a rare xanthene ring in type II polyketide biosynthesis, and demonstrate the potential of using total biosynthesis for the discovery of natural products synthesized by type II PKSs. [ABSTRACT FROM AUTHOR]

Pyranoanthocyanins have been reported to possess better chemical stability and bioactivities than monomeric anthocyanins in some aspects. The hypocholesterolemic activity of pyranoanthocyanins is unclear. In view of this, this study was conducted to compare the cholesterol-lowering activities of Vitisin A with the anthocyanin counterpart Cyanidin-3-O-glucoside(C3G) in HepG2 cells and to investigate the interaction of Vitisin A with the expression of genes and proteins associated with cholesterol metabolism. HepG2 cells were incubated with 40 μM cholesterol and 4 μM 25-hydroxycholeterol with various concentrations of Vitisin A or C3G for 24 h. It was found that Vitisin A decreased the cholesterol levels at the concentrations of 100 μM and 200 μM with a dose–response relationship, while C3G exhibited no significant effect on cellular cholesterol. Furthermore, Vitisin A could down-regulate 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR) to inhibit cholesterol biosynthesis through a sterol regulatory element-binding protein 2 (SREBP2)-dependent mechanism, and up-regulate low-density lipoprotein receptor (LDLR) and blunt the secretion of proprotein convertase subtilisin/kexin type 9 (PCSK9) protein to promote intracellular LDL uptake without LDLR degradation. In conclusion, Vitisin A demonstrated hypocholesterolemic activity, by inhibiting cholesterol biosynthesis and enhancing LDL uptake in HepG2 cells. [ABSTRACT FROM AUTHOR]

Lorneic acid and related natural products are characterized by a trialkyl‐substituted benzene ring. The formation of the aromatic core in the middle of the polyketide chain is unusual. We characterized a cytochrome P450 enzyme that can catalyze the hallmark benzene ring formation from an acyclic polyene substrate through genetic and biochemical analysis. Using this P450 as a beacon for genome mining, we obtained 12 homologous type I polyketide synthase (PKS) gene clusters, among which two gene clusters are activated and able to produce trialkyl‐substituted aromatic polyketides. Quantum chemical calculations were performed to elucidate the plausible mechanism for P450‐catalyzed benzene ring formation. Our work expands our knowledge of the catalytic diversity of cytochrome P450. [ABSTRACT FROM AUTHOR]

Sordarin (1) is a fungal diterpene glycoside that displays potent antifungal bioactivity through inhibition of elongation factor 2. The structures of sordarin and related compounds feature a highly rearranged tetracyclic diterpene core. In this study, we identified a concise pathway in the biosynthesis of sordarin. A diterpene cyclase (SdnA) generates the 5/8/5 cycloaraneosene framework, which is decorated by a set of P450s that catalyze a series of oxidation reactions, including hydroxylation, desaturation, and C−C bond oxidative cleavage, to give a carboxylate intermediate with a terminal alkene and a cyclopentadiene moiety. A novel Diels–Alderase SdnG catalyzes an intramolecular Diels–Alder (IMDA) reaction on this intermediate to forge the sordarin core structure. Subsequent methyl hydroxylation and glycosylation complete the biosynthesis of sordarin. Our work discloses a new strategy used by nature for the formation of the rearranged diterpene skeleton. [ABSTRACT FROM AUTHOR]

Macrocyclization is an important process that affords morphed scaffold in biosynthesis of bioactive natural products. Nature has adapted diverse biosynthetic strategies to form macrocycles. In this work, we report the identification and characterization of a small enzyme AvmM that can catalyze the construction of a 16-membered macrocyclic ring in the biosynthesis of alchivemycin A (1). We show through in vivo gene deletion, in vitro biochemical assay and isotope labelling experiments that AvmM catalyzes tandem dehydration and Michael-type addition to generate the core scaffold of 1. Mechanistic studies by crystallography, DFT calculations and MD simulations of AvmM reveal that the reactions are achieved with assistance from the special tenuazonic acid like moiety of substrate. Our results thus uncover an uncharacterized macrocyclization strategy in natural product biosynthesis. Macrocyclization is an important process in bioactive natural product synthesis. Here, the authors report on the study of a macrocyclic ring constructing enzyme in the biosynthesis of alchivemycin A and using gene deletion, biochemical assays and isotope labelling show the enzyme catalyses tandem dehydration and Michael-type addition. [ABSTRACT FROM AUTHOR]

Redox tailoring enzymes play key roles in generating structural complexity and diversity in type II polyketides. In chartreusin biosynthesis, the early 13C‐labeling experiments and bioinformatic analysis suggest the unusual aglycone is originated from a tetracyclic anthracyclic polyketide. Here, we demonstrated that the carbon skeleton rearrangement from a linear anthracyclic polyketide to an angular pentacyclic biosynthetic intermediate requires two redox enzymes. The flavin‐dependent monooxygenase ChaZ catalyses a Baeyer–Villiger oxidation on resomycin C to form a seven‐membered lactone. Subsequently, a ketoreductase ChaE rearranges the carbon skeleton and affords the α‐pyrone containing pentacyclic intermediate in an NADPH‐dependent manner via tandem reactions including the reduction of the lactone carbonyl group, Aldol‐type reaction, followed by a spontaneous γ‐lactone ring formation, oxidation and aromatization. Our work reveals an unprecedented function of a ketoreductase that contributes to generate structural complexity of aromatic polyketide. [ABSTRACT FROM AUTHOR]

[Display omitted] Azaspirene derivatives are widespread natural products in fungi. Here we reported five new azaspirenes A-E (1 – 5) from the marine mud-derived endophytic fungus Aspergillus micronesiensis NF666. Their structures were unambiguously determined by an extensive analysis of HRESIMS and NMR data. All compounds were evaluated on antimicrobial activities against five pathogens by the agar diffusion method. Moreover, a plausible biosynthetic pathway of azaspirenes A (1) is proposed in this work. [ABSTRACT FROM AUTHOR]

[Display omitted] Salinopyridins A and B, two novel polyethers containing a rare pyridine moiety, were isolated from an earwig symbiotic Streptomyces sp. NA4227. Their structures were determined by extensive spectroscopic analysis. Based on the structure, bioinformatics analysis and gene mutation experiments, we proposed that salinopyridins share the same biosynthetic pathway with salinomycin, and the formation of pyridine moiety is assembled through a nonenzymatic process. Salinopyridins A and B exhibited potent activity against several Gram-positive bacteria. [ABSTRACT FROM AUTHOR]