Functional Analysis of the Fusarielin Biosynthetic Gene Cluster.

Fusarielins are polyketides with a decalin core produced by various species of Aspergillus and Fusarium. Although the responsible gene cluster has been identified, the biosynthetic pathway remains to be elucidated. In the present study, members of the gene cluster were deleted individually in a Fusarium graminearum strain overexpressing the local transcription factor. The results suggest that a trans-acting enoyl reductase (FSL5) assists the polyketide synthase FSL1 in biosynthesis of a polyketide product, which is released by hydrolysis by a trans-acting thioesterase (FSL2). Deletion of the epimerase (FSL3) resulted in accumulation of an unstable compound, which could be the released product. A novel compound, named prefusarielin, accumulated in the deletion mutant of the cytochrome P450 monooxygenase FSL4. Unlike the known fusarielins from Fusarium, this compound does not contain oxygenized decalin rings, suggesting that FSL4 is responsible for the oxygenation.

Fusarium graminearum PKS14 is involved in orsellinic acid and orcinol synthesis.

The available genome sequences show that the number of secondary metabolite genes in filamentous fungi vastly exceeds the number of known products. This is also true for the global plant pathogenic fungus Fusarium graminearum, which contains 15 polyketide synthase (PKS) genes, of which only 6 have been linked to products. To help remedy this, we focused on PKS14, which has only been shown to be expressed during plant infections or when cultivated on rice or corn meal (RM) based media. To enhance the production of the resulting product we introduced a constitutive promoter in front of PKS14 and cultivated two of the resulting mutants on RM medium. This led to the production of two compounds, which were only detected in the PKS14 overexpressing mutants and not in the wild type or PKS14 deletion mutants. The two compounds were tentatively identified as orsellinic acid and orcinol by comparing spectroscopic data (mass spectroscopy and chromatography) to authentic standards. NMR analysis of putative orcinol isolated from the PKS14 overexpressing mutant supported our identification. Orcinol and orsellinic acid, not previously detected in Fusarium, have primarily been detected in lichen fungi. Orsellinic acid is hypothesized to be the PKS release product which is transformed to orcinol through decarboxylation. Phylogenetic analyses of PKSs placed PKS14 in a subclade of known OA synthases. Expression analysis by microarray of 55 experiments identified seven genes near PKS14 that were expressed in a similar manner. One of the seven genes encodes a predicted carboxylase, which could be responsible for transforming orsellinic acid to orcinol.