Genetic engineering, high resolution mass spectrometry and nuclear magnetic resonance spectroscopy elucidate the bikaverin biosynthetic pathway in Fusarium fujikuroi

• A new bikaverin intermediate, oxo-pre-bikaverin, was isolated and its structure elucidated.
• High resolution mass spectrometric data provided a putative bikaverin biosynthesis.
• Bik2 and Bik3 presumably catalyze alternating reaction steps in bikaverin biosynthesis.

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.

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