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Tropolone sesquiterpenoids (TS) are meroterpenoid natural products that share the conserved structural feature of a polyketide-derived tropolone nucleus connected to a humulene-derived macrocycle via a bridging dihydropyran ring. Here, the biosynthesis of the TS xenovulene A [1] was investigated using a combination of heterologous gene expression in the fungal host Aspergillus oryzae NSAR1 and in E. coli BL21. Heterologous expression experiments validated a minimal xenovulene A [1] biosynthetic gene set encoding eight dedicated enzymes involved in tropolone formation, humulene formation and DIELS-ALDER chemistry. Reconstitution of key enzymatic steps in vitro identified a new type of class I terpene cyclase (AsR6), that catalyzes the stereoselective formation of ɑ-humulene [2] from farnesyl pyrophosphate [3] or either enantiomer of nerolidyl pyrophosphate [4]. The biosynthesis of the structurally related bistropolones eupenifeldin [5] and noreupenifeldin B [6] in Phaeosphaeriaceae sp. CF-150626 was also investigated. Isotopic labelling studies identified an unusual oxidative ring contraction that putatively converts [5] into [6]. Through a combined genetic and chemical approach, a candidate biosynthetic gene cluster for [5]-biosynthesis was identified (the eup2 BGC). With EupR3 a homologue of AsR6 was characterized that stereoselectively produces 2Z-humulene [7], a geometric isomer of [2]. In cooperation with the Helmholtz Institute for Infection Research (Braunschweig, Germany) the crystal structure of AsR6 in the unliganded state and in complex with thiolo-S-diphosphate [8] and an in crystallo cyclized reaction product was obtained. A new pyrophosphate binding site was identified that consists of a binuclear magnesium cluster and a conserved lysine residue. Site- directed mutagenesis validated the motif and identified a key amino acid residue, L/M285, that drives the stereoselective formation of either [2] or [7]. New-to-nature TS natural products were produced through heterologous expression of different combinations of biosynthetic enzymes from the xenovulene A [1], eupenifeldin [5] and pycnidione [9] pathways. The rational design of expression experiments resulted in the formation and characterization of seven new derivatives. The obtained non-natural products differ in the nature of the polyketide moiety, the substitution pattern of the humulene macrocycle and the degree of hydroxylation.

Xenovulene A is a complex fungal meroterpenoid, produced by the organism hitherto known as Acremonium strictum IMI 501407, for which limited biosynthetic evidence exists. Here, we generate a draft genome and show that the producing organism is previously unknown and should be renamed as Sarocladium schorii. A biosynthetic gene cluster is discovered which bears resemblance to those involved in the biosynthesis of fungal tropolones, with additional genes of unknown function. Heterologous reconstruction of the entire pathway in Aspergillus oryzae allows the chemical steps of biosynthesis to be dissected. The pathway shows very limited similarity to the biosynthesis of other fungal meroterpenoids. The pathway features: the initial formation of tropolone intermediates; the likely involvement of a hetero Diels–Alder enzyme; a terpene cyclase with no significant sequence homology to any known terpene cyclase and two enzymes catalysing oxidative-ring contractions., Xenovulene A is a fungal compound that has the potential to be used as an antidepressant. Here, the authors unravel the pathway leading to its formation in fungi and discover a new class of enzymes, which accounts for some unusual chemistry in the synthesis of xenovulene.