Use of UHPLC high-resolution Orbitrap mass spectrometry to investigate the genes involved in the production of secondary metabolites in Aspergillus flavus.
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 high-resolution mass spectrometry (HRMS) accurate mass data. The suitability of this approach was demonstrated by the confirmation of previously characterised 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 uncharacterised gene cluster in A. favus, cluster 23. Comparative Orbitrap HRMS analysis of extracts of A. flavus wild-type strain and an over-expression 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 de-replication of the accurate mass data, five 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.
a Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences , Ghent University , Ghent , Belgium.|b Department of Analytical Chemistry, Faculty of Sciences , University of Granada , Granada , Spain.
Natalia
N
Arroyo-Manzanares
a Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences , Ghent University , Ghent , Belgium.
José Diana
JD
Di Mavungu
a Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences , Ghent University , Ghent , Belgium.
Valdet
V
Uka
a Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences , Ghent University , Ghent , Belgium.
Svetlana V
SV
Malysheva
c Southern Regional Research Center, Agricultural Research Service , US Department of Agriculture , New Orleans , LA , USA.
Jeffrey W
JW
Cary
c Southern Regional Research Center, Agricultural Research Service , US Department of Agriculture , New Orleans , LA , USA.
Kenneth C
KC
Ehrlich
d Laboratory of Chemical Analysis, Department of Veterinary Public Health and Food Safety, Faculty of Veterinary Medicine , Ghent University , Merelbeke , Belgium.
Lynn
L
Vanhaecke
c Southern Regional Research Center, Agricultural Research Service , US Department of Agriculture , New Orleans , LA , USA.
Deepak
D
Bhatnagar
a Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences , Ghent University , Ghent , Belgium.
Sarah
S
De Saeger
D000348:Aflatoxins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D001231:Aspergillus flavus / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001578:Benzopyrans / Q000302:isolation & purification / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D059022:Food Safety; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D006801:Humans; D013058:Mass Spectrometry; D008780:Methyltransferases / Q000235:genetics* / Q000378:metabolism; D005810:Multigene Family; D009154:Mutation; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D048630:Polyketide Synthases / Q000235:genetics* / Q000378:metabolism; D011728:Pyridones / Q000302:isolation & purification / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
D000348:Aflatoxins; D001578:Benzopyrans; D005656:Fungal Proteins; D011728:Pyridones; D014157:Transcription Factors; C479226:leporin B; D048630:Polyketide Synthases; D010088:Oxidoreductases; D008780:Methyltransferases; C096006:omtA protein, Aspergillus
Aspergillus spp.; comparative metabolomics; gene function; high-resolution mass spectrometry