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.

Synthesis and Absolute Configuration of Natural 2‐Pyrones

“…[6] Nectriapyrone (2) was first isolated from Gyrostroma missouriense, [7] while gibepyrone A (3) was isolated from Fusarium fujikuroi. [10] Both enantiomers of compound 4 are known from nature: Phomapyrone C [(-)-4] was isolated from Leptosphaeria maculans [11] and Nocardiopsis spp., [12] while germicidin C [(+)-4] is known from Streptomyces coelicolor. [10] Both enantiomers of compound 4 are known from nature: Phomapyrone C [(-)-4] was isolated from Leptosphaeria maculans [11] and Nocardiopsis spp., [12] while germicidin C [(+)-4] is known from Streptomyces coelicolor.…”

“…[18] Gibepyrone A (3) shows a weak antibiotic activity [8] and a growth inhibition against morning glory and lettuce seedlings. [10] Extracts from L. maculans containing (-)-4 and 7 exhibited phytotoxic activity against Brassica spp., [11] its enantiomer (+)-4 had an inhibitory effect on the germination of its producer strain S. coelicolor, [13b] and for (-)-5 antibiotic activity was reported. [10] Extracts from L. maculans containing (-)-4 and 7 exhibited phytotoxic activity against Brassica spp., [11] its enantiomer (+)-4 had an inhibitory effect on the germination of its producer strain S. coelicolor, [13b] and for (-)-5 antibiotic activity was reported.…”

“…[12] The structural similarities of the discussed natural products point to a shared biosynthetic mechanism. [10] The production of the germicidins was assigned to a type III PKS in S. coelicolor. [20] Feeding experiments with 13 C-labelled acetate underpinned this biosynthetic model for 2, [21] and recently the iterative type I PKS Gpy1 was characterized that is responsible for production of 3 in F. fujikuroi ( Figure 1B).…”

“…All compounds show the characteristics of the polyketide synthase (PKS) assembly logic and can be derived from C 2 building blocks. [13a] In need for a synthetic standard during the investigation of the biosynthesis of 3, [10] different synthetic strategies to obtain small methylated 2-pyrones were evaluated in our laboratories. [10] The production of the germicidins was assigned to a type III PKS in S. coelicolor.…”

Induction of Nectriapyrone Biosynthesis in the Rice Blast Fungus Pyricularia oryzae by Disturbance of the Two‐Component Signal Transduction System

“…Nectriapyrone is similar to gibepyrones from Fusarium spp. ; the gebepyrones lack the methoxy group, and the O‐MT gene is absent from the gibepyrone biosynthetic gene cluster . Nectriapyrone is also similar to the germicidins from Streptomyces spp.…”