Nielsen, Morten Thrane6; Nielsen, Jakob Blæsbjerg7; Anyaogu, Dianna Chinyere8; Holm, Dorte Koefoed1; Nielsen, Kristian Fog9; Larsen, Thomas Ostenfeld10; Mortensen, Uffe Hasbro7
1 Department of Systems Biology, Technical University of Denmark2 Eucaryotic Molecular Cell Biology, Department of Systems Biology, Technical University of Denmark3 Metabolomics Platform, Department of Systems Biology, Technical University of Denmark4 Metabolic Signaling and Regulation, Department of Systems Biology, Technical University of Denmark5 Natural Product Chemistry, Department of Systems Biology, Technical University of Denmark6 Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark7 Eukaryotic Molecular Cell Biology, Department of Biotechnology and Biomedicine, Technical University of Denmark8 Technical University of Denmark9 DTU Metabolomics Core, Department of Biotechnology and Biomedicine, Technical University of Denmark10 Natural Product Discovery, Department of Biotechnology and Biomedicine, Technical University of Denmark
Fungal natural products are a rich resource for bioactive molecules. To fully exploit this potential it is necessary to link genes to metabolites. Genetic information for numerous putative biosynthetic pathways has become available in recent years through genome sequencing. However, the lack of solid methodology for genetic manipulation of most species severely hampers pathway haracterization. Here we present a simple PCR based approach for heterologous reconstitution of intact gene clusters. Specifically, the putative gene cluster responsible for geodin production from Aspergillus terreus was transferred in a two step procedure to an expression platform in A. nidulans. The individual cluster fragments were generated by PCR and assembled via efficient USER fusion prior to ransformation and integration via re-iterative gene targeting. A total of 13 open reading frames contained in 25 kb of DNA were successfully transferred between the two species enabling geodin synthesis in A. nidulans. Subsequently, functions of three genes in the cluster were validated by genetic and chemical analyses. Specifically, ATEG_08451 (gedC) encodes a polyketide synthase, ATEG_08453 (gedR) encodes a transcription factor responsible for activation of the geodin gene cluster and ATEG_08460 (gedL) encodes a halogenase that catalyzes conversion of sulochrin to dihydrogeodin. We expect that our approach for transferring intact biosynthetic pathways to a fungus with a well developed genetic toolbox will be instrumental in characterizing the many exciting pathways for secondary metabolite production that are currently being uncovered by the fungal genome sequencing projects.