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Title: Use of UHPLC high-resolution Orbitrap mass spectrometry to investigate the genes involved in the production of secondary metabolites in Aspergillus flavus

Author
item ARROYO-MANZANARES, NATALIA - Ghent University
item DIANA DI MAVUNGU, JOSE - Ghent University
item UKA, VALDET - Ghent University
item MALYSHEVA, SVETLANA - Ghent University
item Cary, Jeffrey
item EHRLICH, KENNETH - Retired ARS Employee
item VANHAECKE, LYNN - Ghent University
item Bhatnagar, Deepak
item DE SAEGER, SARAH - Ghent University

Submitted to: Food Additives & Contaminants
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/9/2015
Publication Date: 8/17/2015
Citation: Arroyo-Manzanares, N., Diana Di Mavungu, J., Uka, V., Malysheva, S.V., Cary, J.W., Ehrlich, K.C., Vanhaecke, L., Bhatnagar, D., De Saeger, S. 2015. Use of UHPLC high-resolution Orbitrap mass spectrometry to investigate the genes involved in the production of secondary metabolites in Aspergillus flavus. Food Additives & Contaminants. Part A, 32(10):1656-1673. doi:10.1080/19440049.2015.1071499.

Interpretive Summary: Filamentous fungi produce a number of secondary metabolic compounds that have been shown to be both of great value (i.e. antibiotics and anti-hypercholesterolemics) and great harm (i.e. aflatoxins and trichothecenes). The fungus Aspergillus flavus is known for its ability to produce the toxic and carcinogenic aflatoxins in food and feed. While aflatoxins are of most concern, A. flavus is predicted to be capable of producing many more secondary metabolites based on a study of its complete genome sequence. Some of these metabolites could be of great importance in food and feed safety. We describe an analytical methodology based on Orbitrap High Resolution Mass Spectrometry technology that allows the determination of fungal metabolites, in support of studies on the function of genes involved in secondary metabolism in fungi. This technology has been used to assist in the identification of a number of A. flavus secondary metabolites including one responsible for the production of a class of compounds termed leporins. Besides identification of the known compounds, leporin B and leporin C, we also identified for the first time 5 other compounds including dimers and trimers belonging to this class of fungal secondary metabolites. The trimer was found to be capable of binding iron. This suggests that leporins may function in acquisition of iron from the environment.

Technical Abstract: The fungus Aspergillus flavus is known for its ability to produce the toxic and carcinogenic aflatoxins in food and feed. While aflatoxins are of most concern, A. flavus is predicted to be capable of producing many more metabolites based on a study of its complete genome sequence. Some of these metabolites could be of great importance in food and feed safety. Here, we describe an analytical methodology based on Orbitrap HRMS technology that allows the untargeted determination of fungal metabolites, in support of the study of the function of genes involved in secondary metabolism in fungi. The applied strategy implies the detection and identification of differentially expressed metabolites in extracts of wild-type and mutant fungal strains, using Orbitrap HRMS accurate mass data. The suitability of this approach was demonstrated by the confirmation of previously characterized genes involved in the aflatoxin biosynthetic pathway, namely a polyketide synthase (pksA), an oxidoreductase (ordA) and a methyltransferase (omtA) gene. Subsequently, the proposed methodology was applied for the detection and identification of metabolites produced by a yet uncharacterized gene cluster in A. favus, cluster 23. Comparative Orbitrap HRMS analysis of extracts of A. flavus wild-type strain and an overexpression mutant for the transcription factor of gene cluster 23 (lepE) demonstrated that this gene cluster is responsible for the production a set of 2-pyridone derivatives, the leporins. Besides the known derivatives leporin B and leporin B precursor that could be identified by automatic dereplication of the accurate mass data, 5 other compounds belonging to this class of fungal secondary metabolites were detected and identified for the first time, combining MS and multiple stage MS data.