Genetic, molecular, and biochemical basis of fungal tropolone biosynthesis

Abstract

A gene cluster encoding the biosynthesis of the fungal tropolone stipitatic acid was discovered in Talaromyces stipitatus ( Penicillium stipitatum ) and investigated by targeted gene knockout. A minimum of three genes are required to form the tropolone nucleus: tropA encodes a nonreducing polyketide synthase which releases 3-methylorcinaldehyde; tropB encodes a FAD-dependent monooxygenase which dearomatizes 3-methylorcinaldehyde via hydroxylation at C-3; and tropC encodes a non-heme Fe(II)-dependent dioxygenase which catalyzes the oxidative ring expansion to the tropolone nucleus via hydroxylation of the 3-methyl group. The tropA gene was characterized by heterologous expression in Aspergillus oryzae , whereas tropB and tropC were successfully expressed in Escherichia coli and the purified TropB and TropC proteins converted 3-methylorcinaldehyde to a tropolone in vitro. Finally, knockout of the tropD gene, encoding a cytochrome P450 monooxygenase, indicated its place as the next gene in the pathway, probably responsible for hydroxylation of the 6-methyl group. Comparison of the T. stipitatus tropolone biosynthetic cluster with other known gene clusters allows clarification of important steps during the biosynthesis of other fungal compounds including the xenovulenes, citrinin, sepedonin, sclerotiorin, and asperfuranone.

Heterologous Production of Fungal Maleidrides Reveals the Cryptic Cyclization Involved in their Biosynthesis

“…It is interesting to note that fungi have evolved at least two distinct routes to maleic anhydride moieties. Other organisms oxidize vicinal aromatic methyl groups to form this biologically important motif 24, 25…”

Pictet–Spengler reaction-based biosynthetic machinery in fungi

“…Some fungi biosynthesize aryl aldehydes through the nonreducing polyketide synthase (23,24), which may be hypothetically condensable with β-arylethylamine derivatives under the FPS catalysis according to the logic of PS reaction (13). Accordingly, our endeavor began with the screening for the activator of the cryptic FPS gene and a suitable β-arylethylamine substrate that may undergo the PS reaction with the fungal aldehyde upon the microbial cultivation.…”

“…To bypass the obstacle, we have introduced the GIEI approach that was expected to facilitate the identification of precursors or intermediates suggestive of the fungal-derived substrate. The C. globosum genome was scrutinized to carry as many as 90 cytochrome P450 (CYP450) and 2 flavin-containing monooxygenases genes (SI Appendix, Table S6), which might have expressed enzymes capable of diversifying the PS reaction products (23,26). The inhibition of monooxygenases was hypothesized to stabilize the intact or less-oxidized PS reaction product(s).…”

Crystal structure of 3,5,7-tris(morpholinomethyl)tropolone·0.67 hydrate, C22H33N3O5·0.67H2O

“…Tropolone and its derivatives have been reported as antimicrobial agents. It also possess antiviral, antitumor, antioxidant and enzyme inhibiting properties [3][4][5][6][7]. The title compound exhibit antiviral activities [7], while the isopropyl derivatives of tropolone (β-thujaplicin and γ-thujaplicin) were reported to have antifungal and antibacterial properties [6].…”