Gibepyrone Biosynthesis in the Rice Pathogen Fusarium fujikuroi Is Facilitated by a Small Polyketide Synthase Gene Cluster

Abstract

The 2H-pyran-2-one gibepyrone A and its oxidized derivatives gibepyrones B-F have been isolated from the rice pathogenic fungus Fusarium fujikuroi already more than 20 years ago. However, these products have not been linked to the respective biosynthetic genes, and therefore, their biosynthesis has not yet been analyzed on a molecular level. Feeding experiments with isotopically labeled precursors clearly supported a polyketide origin for the formal monoterpenoid gibepyrone A, whereas the terpenoid pathway could be excluded. Targeted gene deletion verified that the F. fujikuroi polyketide synthase PKS13, designated Gpy1, is responsible for gibepyrone A biosynthesis. Next to Gpy1, the ATP-binding cassette transporter Gpy2 is encoded by the gibepyrone gene cluster. Gpy2 was shown to have only a minor impact on the actual efflux of gibepyrone A out of the cell. Instead, we obtained evidence that Gpy2 is involved in gene regulation as it represses GPY1 gene expression. Thus, GPY1 was up-regulated and gibepyrone A production was enhanced both extra- and intracellularly in Δgpy2 mutants. Furthermore, expression of GPY genes is strictly repressed by members of the fungus-specific velvet complex, Vel1, Vel2, and Lae1, whereas Sge1, a major regulator of secondary metabolism in F. fujikuroi, affects gibepyrone biosynthesis in a positive manner. The gibepyrone A derivatives gibepyrones B and D were shown to be produced by cluster-independent P450 monooxygenases, probably to protect the fungus from the toxic product. In contrast, the formation of gibepyrones E and F from gibepyrone A is a spontaneous process and independent of enzymatic activity.

Keywords: Fusarium fujikuroi; biosynthesis; fungi; gene regulation; gibepyrones; polyketide; secondary metabolism.

FIGURE 1.

Chemical structures of 2H-pyran-2-ones. Shown are structures of the parent compound 2H-pyran-2-one (A) and the fungal metabolites triacetic acid lactone (B); 6-pentyl-2H-pyran-2-one, its shorter and longer homologs, and derivatives with unsaturated side chain (C); gibepyrones A–F (D); fusalanipyrone (E); nectriapyrone and pestalopyrone (F).

FIGURE 2.

Synthesis of gibepyrones A, B, C, and E. NEt₃, triethyl amine; DCM, dichloromethane; m-CPBA, meta-chlorperbenzoic acid; MsOH, methanesulfonic acid; THF, tetrahydrofurane; PCC, pyridinium chlorochromate; SeO₂, selenium dioxide; t-BuOOH, tert-butyl hydroperoxide.

FIGURE 3.

Gibepyrone A production depends on the presence of Ppt1 (A) and the SM biosynthetic key enzyme PKS13, designated Gpy1 (B). A, HPLC-MS/MS analysis of culture fluids of the WT, Δppt1, and PPT1^C. The strains were grown in the presence of 6 mm glutamine for 7 days. The gibepyrone A peak was verified through comparison with the synthesized chemical standard. B, the WT, the Δgpy1 deletion mutant, and the complemented strain GPY1^C were grown and analyzed as described above.

FIGURE 4.

The gibepyrone biosynthetic gene cluster comprises a PKS-encoding gene and an ABC transporter-encoding gene, GPY1 and GPY2, respectively. A, schematic representation of GPY1 (FFUJ_12020), GPY2 (FFUJ_12021), and neighboring, non-cluster genes (gray). Arrows, direction of transcription; white bars, introns. B, HPLC-MS/MS analysis of culture fluids of the WT and indicated deletion mutants. The strains were grown in triplicate in the presence of 6 mm glutamine for 7 days with product formation of the WT being set to 100%. C, HPLC-MS/MS and Northern blot expressional analysis of the WT and the overexpression mutants of GPY1 and FFUJ_12023 (TF). The strains were grown and analyzed as described above for gibepyrone A quantification, whereas cell harvest was carried out after 3 days for expressional analysis. In the latter case, GPY1 and FFUJ_12023 were used as probes. Error bars, S.D.

FIGURE 5.

The ABC transporter Gpy2 represses expression of the PKS-encoding gene GPY1. A, HPLC-MS/MS analysis of extra- and intracellular gibepyrone A. The WT and indicated deletion and overexpression mutants were grown in the presence of 6 mm glutamine for 7 days. Culture fluids were directly measured for extracellular gibepyrone A content, whereas metabolite extraction from washed mycelium was applied before quantifying intracellular levels. The cultivation was carried out in triplicate, and product formation of the WT was set to 100%. B, qRT-PCR analysis of the relative expression (RE) of GPY1 and GPY2 using the ΔΔCt method. The WT, Δgpy2, and OE::GPY2 mutants were cultivated for 3 days, whereupon total RNA was isolated from the harvested mycelium. Error bars (S.D.) originate from a technical replicate, and expression of the WT was arbitrarily set to 1. C, HPLC-MS/MS analysis of extracellular gibepyrone A. The WT and Δgpy2 were grown in the presence of 6 mm glutamine for 1–3 days (d). The cultivation was carried out in triplicate, and product formation of the WT at 3 days was set to 100%. D, qRT-PCR analysis of the relative expression of GPY1 using the ΔΔCt method. The WT and Δgpy2 were cultivated for 1–3 days, whereupon total RNA was isolated from the harvested mycelium. Error bars (S.D.) originate from a technical replicate, and expression of the WT at 3 days was arbitrarily set to 1.

FIGURE 6.

Production of gibepyrone A derivatives, gibepyrones B–E. HPLC-HRMS analysis of culture fluids of the WT and the Δcpr mutant. The strains were grown in the presence of 6 mm glutamine for 7 days, and the supernatant was analyzed without further processing. Shown are extracted ion chromatograms for the calculated masses of gibepyrones A–E for the two strains as well as for the synthesized chemical standards. The gibepyrone E peak cannot be found in the cultures. However, in the presence of water, both the cultures and the standard show an identical double peak (Gibepyrone E derivatives).

FIGURE 7.

Regulation of gibepyrone A biosynthesis. A, HPLC-MS/MS analysis of culture fluids of the WT and indicated deletion mutants. The strains were grown in triplicate in the presence of 6 mm glutamine for 7 days with product formation of the WT being set to 100%. B, qRT-PCR analysis of the relative expression (RE) of GPY1 and GPY2 using the ΔΔCt method. The strains were cultivated for 3 days, whereupon total RNA was isolated from the harvested mycelium. Error bars (S.D.) originate from a technical replicate, and expression of the WT was arbitrarily set to 1. C, qRT-PCR analysis of the relative expression of the indicated genes using the ΔΔCt method. The WT and Δvel1 mutant were cultivated for 3 days, whereupon total RNA was isolated from the harvested mycelium. Error bars (S.D.) originate from a technical replicate, and expression of each gene in the WT was arbitrarily set to 1.

FIGURE 8.

Proposed pathway for gibepyrone A biosynthesis by the PKS Gpy1. As a first step, the acetyl-CoA starter unit is bound to the acyl carrier (ACP) domain of Gpy1 as thioester. It is condensed with three malonyl-CoA extender units under the release of carbon dioxide through the activities of the β-ketoacyl synthase (KS) and acyltransferase (AT) domains. We found experimental evidence that the methyltransferase (MT) domain of Gpy1 is responsible for the C-methylation of the polyketide backbone during two of the elongation steps using SAM as a precursor. Finally, gibepyrone A is released upon intramolecular cyclization. Heavy lines, C₂ units from acetate/malonate; black circles, methyl groups originating from SAM.

FIGURE 9.

Biosynthesis and regulation of gibepyrone A and its derivatives in F. fujikuroi. The PKS Gpy1 facilitates the condensation of the C₁₀ compound gibepyrone A. Beside the PKS, an ABC transporter-encoding gene, GPY2, belongs to the cluster. However, Gpy2 seems to have a minor impact on the efflux of gibepyrone A out of the cell, but it was shown to repress expression of the key gene GPY1. Furthermore, members of the velvet complex, Vel1, Vel2, and Lae1, negatively affect cluster gene expression and gibepyrone A product formation, whereas Sge1 represents a positive regulator of gibepyrone biosynthesis. The oxidized compounds gibepyrones B and D are derived from gibepyrone A through the activity of cluster-independent, CPR-dependent P450 monooxygenases, whereas gibepyrones E and F are formed in the absence of enzymatic activity.