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.

Scheme 1.

Isotopic labeling patterns observed in stipitatic acid biosynthesis.

Fig. 1.

Involvement of tspks1 (tropA) in the biosynthesis of methylorcinaldehyde and tropolones in T. stipitatus. PKS domains: SAT, starter unit acyl transferase; KAS, ketosynthase; AT, acyl transferase; PT, product template; ACP, acyl carrier protein; CMeT, C-methyl transferase; R, acyl CoA thiolester reductase. HPLC analysis of tspks1 KO: (A) UV chromatogram at 260 nm for WT T. stipitatus; (B) UV chromatogram at 260 nm for T. stipitatus tspks1 KO. HPLC analysis of tspks1 expression in A. oryzae: (C) UV chromatogram at 293 nm for untransformed A. oryzae; (D) UV chromatogram at 293 nm for A. oryzae expressing aspks1; (E) UV chromatogram at 293 nm for A. oryzae expressing tspks1.

Scheme 2.

Overview of the results from KO experiments. Compounds in square brackets were not observed directly. Note that the absolute configurations of 11, 13, and 14 are unknown.

Scheme 3.

In vitro assay of TsL1 (TropB) and TsR5 (TropC) using purified proteins: (A) HPLC trace (300 nm) of boiled TropB incubated with 3 (2 mM), potassium phosphate (20 mM, pH 7.6), EDTA (1 mM), and NADPH (3 mM); (B) TropB incubated with 3 (2 mM) under the same conditions (ESMS, electrospray mass spectrometry); (C) HPLC trace (375 nm) of boiled TropC incubated with 11 (2.5 mM), Tris (50 mM, pH 7.5), ascorbate (4 mM), α-ketoglutarate (2.5 mM), and FeSO₄ (0.1 mM); (D) TropC incubated with 11 (2.5 mM) under the same conditions. See SI Appendix for experimental details and further controls.

Scheme 4.

Proposed mechanisms for the production of 15 and 16 by TropC.

Scheme 5.

Biosynthetic relationships between tropolones and azaphilones: Bold lines indicate likely PKS starter units; black circles denote carbons derived from S-adenosyl methionine.