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Diels-Alder reactions are among the most powerful synthetic transformations to construct complex natural products. Despite that increasing of enzymatic intramolecular Diels-Alder reactions have been discovered, natural intermolecular Diels-Alderases are rarely described. Here, we report an intermolecular hetero-Diels-Alder reaction in the biosynthesis of tropolonic sesquiterpenes and functionally characterize EupfF as the first fungal intermolecular hetero-Diels-Alderase. We demonstrate that EupfF catalyzed the dehydration of a hydroxymethyl-containing tropolone (5) to generate a reactive tropolone o-quinone methide (6) and might further stereoselectively control the subsequent intermolecular hetero-Diels-Alder reaction with (1E,4E,8Z)-humulenol (8) to produce enantiomerically pure neosetophomone B (1). Our results reveal the biosynthetic pathway of 1 and expand the repertoire of activities of Diels-Alder cyclases.

Duclauxins are dimeric and heptacyclic fungal polyketides with notable bioactivities. We characterized the cascade of redox transformations in the biosynthetic pathway of duclauxin from Talaromyces stipitatus. The redox reaction sequence is initiated by a cupin family dioxygenase DuxM that performs an oxidative cleavage of the peri-fused tricyclic phenalenone and affords a transient hemiketal-oxaphenalenone intermediate. Additional redox enzymes then morph the oxaphenoalenone into either an anhydride or a dihydrocoumarin-containing monomeric building block that is found in dimeric duxlauxins. Oxidative coupling between the monomers to form the initial C-C bond was shown to be catalyzed by a P450 monooxygenase, although the enzyme responsible for the second C-C bond formation was not found in the pathway. Collectively, the number and variety of redox enzymes used in the duclauxin pathway showcase Nature's strategy to generate structural complexity during natural product biosynthesis.

The small-molecule biosynthetic potential of most filamentous fungi has remained largely unexplored and represents an attractive source for the discovery of new compounds. Genome sequencing of Calcarisporium arbuscula, a mushroom-endophytic fungus, revealed 68 core genes that are involved in natural product biosynthesis. This is in sharp contrast to the predominant production of the ATPase inhibitors aurovertin B and D in the wild-type fungus. Inactivation of a histone H3 deacetylase led to pleiotropic activation and overexpression of more than 75 % of the biosynthetic genes. Sampling of the overproduced compounds led to the isolation of ten compounds of which four contained new structures, including the cyclic peptides arbumycin and arbumelin, the diterpenoid arbuscullic acid A, and the meroterpenoid arbuscullic acid B. Such epigenetic modifications therefore provide a rapid and global approach to mine the chemical diversity of endophytic fungi.

Despite the remarkable versatility displayed by flavin-dependent monooxygenases (FMOs) in natural product biosynthesis, one notably missing activity is the oxidative generation of carbonate functional groups. We describe a multifunctional Baeyer-Villiger monooxygenase, CcsB, which catalyzes the formation of an in-line carbonate in the macrocyclic portion of cytochalasin E. This study expands the repertoire of activities of FMOs and provides a possible synthetic strategy for transformation of ketones into carbonates.

A challenge for biomedical research is the development of pharmaceuticals that appropriately target disease mechanisms. Natural products can be a rich source of bioactive chemicals for medicinal applications but can act through unknown mechanisms and can be difficult to produce or obtain. To address these challenges, we developed a new marine-derived, renewable natural products resource and a method for linking bioactive derivatives of this library to the proteins and biological processes that they target in cells. We used cell-based screening and computational analysis to match gene expression signatures produced by natural products to those produced by small interfering RNA (siRNA) and synthetic microRNA (miRNA) libraries. With this strategy, we matched proteins and miRNAs with diverse biological processes and also identified putative protein targets and mechanisms of action for several previously undescribed marine-derived natural products. We confirmed mechanistic relationships for selected siRNAs, miRNAs, and compounds with functional roles in autophagy, chemotaxis mediated by discoidin domain receptor 2, or activation of the kinase AKT. Thus, this approach may be an effective method for screening new drugs while simultaneously identifying their targets.

Aurovertins are the structurally diverse polyketides that distribute widely in different fungal species. They feature a 2,6-dioxabicyclo[3.2.1]-octane ring in structure and exhibit the potential antitumor activity against breast cancer as F1-ATPase β subunit inhibitor. In this study, we constructed the biosynthetic pathway of aurovertin in an Aspergillus nidulans host and obtained seven aurovertin-type compounds. Surprisingly, three new aurovertin geometric isomers were characterized. By introducing an inducible promoter xylP(p) in the pathway gene acyltransferase aurG, we can control the product ratios among different aurovertin compounds by adding glucose and/or inducer xylose. The yields of aurovertins could be increased up to about 20 times by adding a constitutive promoter gpdA(p) to transcription factor AurF, which indicates AurF's positive role in the biosynthesis of aurovertin. Taken together, our results provided not only an efficient way to generate bioactive fungal natural products but also realized the rational controlling their yields with designed promoters. [ABSTRACT FROM AUTHOR]