Molecular Characterization of the Cercosporin Biosynthetic Pathway in the Fungal Plant Pathogen Cercospora nicotianae

Abstract

Perylenequinones are a class of photoactivated polyketide mycotoxins produced by fungal plant pathogens that notably produce reactive oxygen species with visible light. The best-studied perylenequinone is cercosporin-a product of the Cercospora species. While the cercosporin biosynthetic gene cluster has been described in the tobacco pathogen Cercospora nicotianae, little is known of the metabolite's biosynthesis. Furthermore, in vitro investigations of the polyketide synthase central to cercosporin biosynthesis identified the naphthopyrone nor-toralactone as its direct product-an observation in conflict with published biosynthetic proposals. Here, we present an alternative biosynthetic pathway to cercosporin based on metabolites characterized from a series of biosynthetic gene knockouts. We show that nor-toralactone is the key polyketide intermediate and the substrate for the unusual didomain protein CTB3. We demonstrate the unique oxidative cleavage activity of the CTB3 monooxygenase domain in vitro. These data advance our understanding of perylenequinone biosynthesis and expand the biochemical repertoire of flavin-dependent monooxygenases.

Figure 1

The currently proposed cercosporin biosynthetic pathway. (a) The cercosporin toxin biosynthetic (CTB) gene cluster has been identified in C. nicotianae. (b) The proposed cercosporin biosynthesis hinges upon the formation of carboxylic acid 5 by the NR-PKS CTB1. The direct product of CTB1 is nor-toralactone (6), precluding the proposed biosynthetic scheme.

Figure 2

Metabolic profiles of CTB gene cluster mutant strains. Chromatograms at 250 nm of extracted metabolite profiles for (a) wild-type, (b) ΔCTB1, (c) ΔCTB2, (d) ΔCTB3c, (e) ΔCTB5, (f) ΔCTB6, and (g) ΔCTB7 strains displayed along with images of the mycelia for each strain. The metabolites were prepared at a concentration of 10 cm² colony surface area per mL in methanol. Identified cercosporin intermediate metabolites are displayed.

Figure 3

Phenotypic and genetic analysis of the CTB cluster. (a) Cercosporin biosynthesis was complemented at the colony-colony interface of the ΔCTB1/ΔCTB3c mutant pair (top/bottom, respectively). The numbers indicate the distance that colonies were inoculated apart from one another in cm. (b) Average colony diameters of CTB disruption mutants are displayed (n = 37). Error bars represent the 95% confidence interval. Stars indicate statistically significant difference from wild-type (p < 0.01). (c) Comparison of gene clusters similar to the CTB gene cluster of C. nicotianae (top).

Figure 4

Product profiles of in vitro reactions of CTB3. The 280 nm chromatograms of the following reactions are displayed: (a) CTB3-MT with nor-toralactone, (b) CTB3-MO with toralactone, (c) CTB3-MT and CTB3-MO with nor-toralactone, (d) CTB3-MT and CTB3-MO with nor-toralactone under reductive conditions, (e) CTB3-MO with toralactone under reductive conditions, and (f) CTB3 with nor-toralactone under reductive conditions. Peaks for nor-toralactone (6) and toralactone (7) are indicated along with peaks for products cercoquinone C (12) and cercoquinone D (11). A peak for DTT and other cosubstrates are observed, as applicable.

Figure 5

Proposed cercosporin biosynthetic pathway. On the strength of observed pathway intermediates, in vitro chemistry, phenotypic, genetic, and pairwise complementation a revised biosynthetic scheme for cercosporin is presented.

Figure 6

Structures of known fungal perylenequinone natural products. Common architectural features are observed in the family. The occurrence of methyoxy at 2, 2′ and 6, 6′ positions are invariable. As is the 2-oxypropyl derivatives at 7, 7′. The formation of the perylenequinone core also appears to be conserved.

Scheme 1

Proposed formation of cercoquinone A. Two mechanisms were considered. Addition of water followed by elimination of methanol (pictured) or enzymatic demethylation. Cercosporin is shown with a monomeric unit shown in red. CTB7 is postulated to form the dioxepine ring, a transformation that would require the elimination of a methyl group at the position of demethylation in cercoquinone A.

Scheme 2

Proposed mechanism for CTB3 flavin-dependent monooxygenase domain. (a) General mechanism of p-hydroxybenzoate (17) hydroxylase family members. (b) Proposed mechanism for CTB3 catalyzed oxidative aromatic ring cleavage of toralactone (7). (c) Mechanism of MHPCO and 5PAO oxidative aromatic ring cleavage.