title: Genetic engineering, high resolution mass spectrometry and nuclear magnetic resonance spectroscopy elucidate the bikaverin biosynthetic pathway in Fusarium fujikuroi.
abstract: Secondary metabolites of filamentous fungi can be highly bioactive, ranging from antibiotic to cancerogenic properties. In this study we were able to identify a new, yet unknown metabolite produced by Fusarium fujikuroi, an ascomycetous rice pathogen. With the help of genomic engineering and high-performance liquid chromatography (HPLC) coupled to high resolution mass spectrometry (HRMS) followed by isolation and detailed structure elucidation, the new substance could be designated as an unknown bikaverin precursor, missing two methyl- and one hydroxy group, hence named oxo-pre-bikaverin. Though the bikaverin gene cluster has been extensively studied in the past, elucidation of the biosynthetic pathway remained elusive due to a negative feedback loop that regulates the genes within the cluster. To decipher the bikaverin biosynthetic pathway and to overcome these negative regulation circuits, the structural cluster genes BIK2 and BIK3 were overexpressed independently in the ΔΔBIK2/BIK3+OE::BIK1 mutant background by using strong constitutive promoters. Using the software tool MZmine 2, the metabolite profile of the generated mutants obtained by HPLC-HRMS was compared, revealing further intermediates.
authors.affiliation: Institute of Food Chemistry, Westfälische Wilhelms-Universität Münster, Corrensstraße 45, 48149 Münster, Germany; NRW Graduate School of Chemistry, Westfälische Wilhelms-Universität Münster, Wilhelm-Klemm-Str. 10, 48149 Münster, Germany.
authors.forename: Birgit
authors.initials: B
authors.lastname: Arndt
authors.affiliation: Institute of Molecular Biology and Biotechnology of Fungi, Westfälische Wilhelms-Universität Münster, Schlossplatz 8, 48149 Münster, Germany.
authors.forename: Lena
authors.initials: L
authors.lastname: Studt
authors.affiliation: Institute of Food Chemistry, Westfälische Wilhelms-Universität Münster, Corrensstraße 45, 48149 Münster, Germany; Institute of Molecular Biology and Biotechnology of Fungi, Westfälische Wilhelms-Universität Münster, Schlossplatz 8, 48149 Münster, Germany.
authors.forename: Philipp
authors.initials: P
authors.lastname: Wiemann
authors.affiliation: Institute of Food Chemistry, Westfälische Wilhelms-Universität Münster, Corrensstraße 45, 48149 Münster, Germany; Institute of Molecular Biology and Biotechnology of Fungi, Westfälische Wilhelms-Universität Münster, Schlossplatz 8, 48149 Münster, Germany.
authors.forename: Helena
authors.initials: H
authors.lastname: Osmanov
authors.affiliation: Institute of Food Chemistry, Westfälische Wilhelms-Universität Münster, Corrensstraße 45, 48149 Münster, Germany.
authors.forename: Karin
authors.initials: K
authors.lastname: Kleigrewe
authors.affiliation: Institute of Pharmaceutical and Medicinal Chemistry, Westfälische Wilhelms-Universität Münster, Corrensstraße 48, 48149 Münster, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Köhler
authors.affiliation: Institute of Food Chemistry, Westfälische Wilhelms-Universität Münster, Corrensstraße 45, 48149 Münster, Germany.
authors.forename: Isabel
authors.initials: I
authors.lastname: Krug
authors.affiliation: Institute of Molecular Biology and Biotechnology of Fungi, Westfälische Wilhelms-Universität Münster, Schlossplatz 8, 48149 Münster, Germany.
authors.forename: Bettina
authors.initials: B
authors.lastname: Tudzynski
authors.affiliation: Institute of Food Chemistry, Westfälische Wilhelms-Universität Münster, Corrensstraße 45, 48149 Münster, Germany; NRW Graduate School of Chemistry, Westfälische Wilhelms-Universität Münster, Wilhelm-Klemm-Str. 10, 48149 Münster, Germany. Electronic address: humpf@wwu.de.
authors.forename: Hans-Ulrich
authors.initials: HU
authors.lastname: Humpf
mesh_terms: D053898:Biosynthetic Pathways; D049109:Cell Proliferation / Q000187:drug effects; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005670:Fusarium / Q000235:genetics* / Q000378:metabolism*; D005800:Genes, Fungal; D005818:Genetic Engineering / Q000379:methods*; D056945:Hep G2 Cells; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D005810:Multigene Family; D009154:Mutation; D012275:Oryza / Q000382:microbiology; D044004:Xanthones / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D005656:Fungal Proteins; D044004:Xanthones; C000269:bikaverin
keywords: Bikaverin; Biosynthesis; Fusarium; Genomics; Mass spectrometry; NMR spectroscopy; Natural products