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ARS Home » Southeast Area » New Orleans, Louisiana » Southern Regional Research Center » Food and Feed Safety Research » Research » Publications at this Location » Publication #345206

Research Project: Genetic and Environmental Factors Controlling Aflatoxin Biosynthesis

Location: Food and Feed Safety Research

Title: Identification and functional analysis of the aspergillic acid gene cluster in Aspergillus flavus

Author
item Lebar, Matthew
item Cary, Jeffrey
item Majumdar, Raj
item Carter-wientjes, Carol
item Mack, Brian
item Wei, Qijian - Mei Mei
item Uka, Valdet - Ghent University
item De Saeger, Sarah - Ghent University
item Diana Di Mavungu, Jose - Ghent University

Submitted to: Fungal Genetics and Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/12/2018
Publication Date: 4/16/2018
Citation: Lebar, M.D., Cary, J.W., Majumdar, R., Carter-Wientjes, C.H., Mack, B.M., Wei, Q., Uka, V., De Saeger, S., Diana Di Mavungu, J. 2018. Identification and functional analysis of the aspergillic acid gene cluster in Aspergillus flavus. Fungal Genetics and Biology. 116:14-23.

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 genes responsible for producing a particular secondary metabolite are often clustered together on the chromosome. We have identified a secondary metabolic cluster in the fungus Aspergillus flavus and shown it is responsible for the production of compound termed aspergillic acid. We determined the enzymatic steps and chemical intermediates involved in aspergillic acid biosynthesis. Aspergillic acid can form a trimer with iron, producing a red pigment called ferriaspergillin. Fungi use iron-binding compounds for uptake, transport, and storage of iron. Aspergillic acid is also antimicrobial and could be used by the fungus against microbial threats or, possibly, as a virulence aid.

Technical Abstract: Aspergillus flavus can colonize important food staples and produces aflatoxins, toxic and carcinogenic secondary metabolites. In silico analysis of the A. flavus genome revealed 56 gene clusters encoding for secondary metabolites. How these many of these metabolites affect fungal development, survival, and virulence is not known. A. flavus metabolites produced during infection of maize seed are of particular interest. RNA-Seq analysis of all predicted A. flavus secondary metabolic gene cluster ‘backbone’ genes during maize kernel infection showed that in addition to the aflatoxin cluster polyketide synthase (PKS) gene, aflC, one of the earliest genes expressed was the uncharacterized Cluster 11 nonribosomal peptide synthetase (NRPS) gene, asaC (AFLA_023020). We focused on seven genes in Cluster 11, which encode the putative NRPS as well as Ankyrin domain protein (AFLA_023000), desaturase/hydroxylase (AFLA_023010), P450 oxidoreductase (AFLA_023030), MFS transporter (AFLA_023050), hypothetical protein (AFLA_023060) and C6 transcription factor (AFLA_023040). LC-MS analysis of extracts from knockout mutants of these genes showed that they were responsible for the synthesis of the previously characterized antimicrobial mycotoxin aspergillic acid. Extracts of the NRPS knockout showed no production of aspergillic acid or its precursors. Knockout of the P450 oxidoreductase afforded a pyrazinone metabolite, the aspergillic acid precursor deoxyaspergillic acid. The formation of hydroxyaspergillic acid was abolished in the desaturase/hydroxylase knockout. The bioactive properties of aspergillic acid are attributed to its ability to chelate iron as defense against competing organisms. Iron chelation, which generates the red pigment ferriaspergillin, could also be used offensively, as a virulence factor aiding the fungus in colonizing maize kernels. We observe a reduction of aflatoxin B1 and cyclopiazonic acid in corn kernel infection assays when aspergillic acid biosynthesis in A. flavus is halted suggesting that interfering with the fungus’s ability to modulate iron can lessen toxin production in infected crops.