Genome mining is a promising method to discover novel secondary metabolites in the postgenomic era. We applied the Aspergillus oryzae heterologous expression system to functionally characterize cryptic bifunctional terpene synthase genes found in fungal genomes and identified the sesterfisherol synthase gene (NfSS) from Neosartorya fischeri. Sesterfisherol contains a characteristic 5-6-8-5 tetracyclic ring system and is modified by cytochrome P450 monooxygenase (NfP450) to sesterfisheric acid. The cyclization mechanism was proposed on the basis of the analysis of in vivo and in vitro enzymatic reactions with isotopically labeled precursors. The mechanism involves C1 cation-olefin IV-olefin V cyclization followed by five hydride shifts, allowing us to propose a unified biogenesis for sesterterpenes branching from bicyclic (5-15), tricyclic (5-12-5), and tetracyclic (5-6-8-5) cation intermediates. Furthermore, the mechanism is distinct from that of a separate class of di/sesterterpenes including fusicoccins and ophiobolins. The difference between mechanisms is consistent with phylogenetic analysis of bifunctional terpene synthases, suggesting that the amino acid sequence reflects the initial cyclization mode, which is most likely related to the initial conformation of a linear prenyl diphosphate.

The [4+2] cycloaddition remains one of the most intriguing transformations in synthetic and natural products chemistry. In nature, however, there are remarkably few enzymes known to have this activity. We herein report an unprecedented enzymatic [4+2] cyclization cascade that has a central role in the biosynthesis of pyrroindomycins, which are pentacyclic spirotetramate natural products. Beginning with a linear intermediate that contains two pairs of 1,3-diene and alkene groups, the dedicated cyclases PyrE3 and PyrI4 act in tandem to catalyze the formation of two cyclohexene rings in the dialkyldecalin system and the tetramate spiro-conjugate of the molecules. The two cyclizations are completely enzyme dependent and proceed in a regio- and stereoselective manner to establish the enantiomerically pure pentacyclic core. Analysis of a related spirotetronate pathway confirms that homologs are functionally exchangeable, establishing the generality of these findings and explaining how nature creates diverse active molecules with similar rigid scaffolds.

Dendrodolides A-M (1-13), 13 new 12-membered macrolides, were isolated from Dendrodochium sp., a fungus associated with the sea cucumber Holothuria nobilis Selenka, which was collected from the South China Sea. The structures of the dendrodolides were elucidated by means of detailed spectroscopic analysis and X-ray single-crystal diffraction. The absolute configurations were assigned using the modified Mosher method, exciton-coupled circular dichroism (ECCD), electronic solution and solid-state circular dichroism (ECD) supported by time-dependent density functional theory (TDDFT) ECD calculations, and X-ray analysis. A detailed conformational analysis of the 13 derivatives indicated that the conformation of the flexible macrolide ring plays a decisive role in their chiroptical properties. Thus, it is highly recommended to apply advanced levels of theory and to avoid simple comparison of ECD spectra to determine the absolute configurations of these derivatives. In an in vitro bioassay, compounds 1-5, 7-9, 11, and 12 exhibited different levels of growth inhibitory activity against SMMC-7721 and HCT116 cells. This is the first report of 12-membered macrolides from the fungus of the genus Dendrodochium . The coisolation of four pairs of epimers is extremely interesting and indicates the complexity of β-ketoreductase stereospecificity in the biosynthesis of enigmatic iterative fungal polyketides.

OR, ECD and VCD are powerful methods to determine the absolute configuration of natural products either applied independently or in combination.

Four new indolo‐sesquiterpenes – dixiamycins A (1) and B (2), oxiamycin (3), and chloroxiamycin (4) – were isolated from a marine‐derived Actinomycete and characterized, together with the known compound xiamycin A (5). Dixiamycins A (1) and B (2) are the first examples of atropisomerism of naturally occurring N‐N‐coupled atropo‐diastereomers, with a dimeric indolo‐sesquiterpene skeleton and a stereogenic N‐N axis between sp3‐hybridized nitrogen atoms. Solution TDDFT ECD calculations were utilized to ascertain the axial chirality of 1 and 2, and rotational barriers and transitions states of the inversion were calculated. Oxiamycin (3) contains a seven‐membered 2,3,4,5‐tetrahydrooxepine ring. The two dimeric compounds 1 and 2 showed better antibacterial activities than the monomers 3–5.