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

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 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.

Aspergillus korhogoensis, a Novel Aflatoxin Producing Species from the Côte d’Ivoire

“…Aflatrem and paspaline were also present in the sclerotium extracts, as well as another related compound that appears to be an uncharacterized hydroxylated form of aflatrem ([M + H] + /z = 518.28992; deviation = −0.347 ppm) previously evoked by Nicholson et al [ 33 ]. Leporin B and its precursor were previously detected in culture extracts [ 25 ].…”

Applications of Fourier Transform Ion Cyclotron Resonance (FT-ICR) and Orbitrap Based High Resolution Mass Spectrometry in Metabolomics and Lipidomics

“…LC-MS analysis has been extensively used in metabolomics and lipidomics studies over the last decade [ 93 , 94 , 95 , 96 , 97 , 98 , 99 , 100 , 101 , 102 , 103 , 104 , 105 , 106 , 107 , 108 , 109 , 110 , 111 , 112 , 113 , 114 , 115 , 116 , 117 , 118 ]. It offers high sensitivity, high resolution and covers wide polarity and molecular weight range of analytes.…”

“…Analytical advantages of HRMS make it broadly applicable to other fields beyond metabolomics and lipidomics. Numerous studies have been published employing HRMS based techniques in many research areas, such as glycomics [ 68 , 69 , 70 ], food science [ 71 , 72 , 73 ], forensics [ 157 ], toxicology [ 157 , 158 ], natural products discovery [ 74 , 75 ], agriculture [ 37 , 96 , 159 , 160 , 161 , 162 , 163 , 164 , 165 , 166 , 167 ], environmental [ 76 , 77 ] and pharmaceutical [ 78 , 79 ] studies. In recent years, food authenticity and safety have become a global concern prompting the development of novel analytical techniques to address food safety issues.…”

Foodomics as a promising tool to investigate the mycobolome

“…Therefore, only some representative studies are detailed here. In regard to genomics related to biosynthesis of mycotoxins, LC-Orbitrap TM 20 has been applied to unravel the genes responsible for generating aflatoxins [49]. For most of the fungi affecting food almost no efficient strategies for control have been found.…”