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.
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