Cloning, targeted integration and heterologous expression of Fusarium metabolite pathway genes

The Fusarium genus is capable of producing many secondary metabolites of which some have interesting bioactivities or relevant pharmaceutical properties. Fungal genes involved in biosynthesis of a specific secondary metabolite are clustered together in areas of tight regulation and epigenetic control. Many gene clusters are activated by specific stimuli and are therefore inactive under laboratory conditions.

Heterologous expression of pathway genes in a model host is a popular option for seeking elucidation of uncharacterized fungal biosynthesis pathways. The aim of our project is to capture entire biosynthetic gene clusters in a vector system usable for fungal transformation. To meet this aim we prepared a multipurpose shuttle vector (Y-GOTL) for selection and propagation in Saccharomyces cerevisiae (2µ ori and URA3) and Escherichia coli and Agrobacterium tumefaciens (oriV, TrfA, Kan^R). The vector contains a T-DNA cassette comprising a multiple cloning site (MCS), an NptII resistance marker and two regions for targeted integration in F. graminearum near the active tubulin locus.

Here we present the successful transfer of the recently identified cytokinin biosynthetic cluster from Fusarium pseudograminearum into the non-producer Fusarium graminearum. The 8kbp gene cluster consisting of four genes was amplified by PCR as three overlapping fragments. The outermost fragments were amplified with primers containing tails with 30 bp homology to the Y-GOTL MCS. Linearized vector and PCR fragments containing the cytokinin cluster were assembled by in vivo homologous recombination in S. cerevisiae. A validated construct was purified and electroporated into Agrobacterium tumefaciens and subsequently transformed into spores of F. graminearum by Agrobacterium-mediated transformation. The resulting F. graminearum mutants were able to produce cytokinins, showing that the gene cluster has functionally been transferred and integrated. In the future, we will use this approach on some of the many uncharacterized secondary metabolite gene clusters in Fusarium.