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

• Published in: Fungal genetics and biology 2018-07, Vol.116, p.14-23
• Main Authors: Lebar, Matthew D., Cary, Jeffrey W., Majumdar, Rajtilak, Carter-Wientjes, Carol H., Mack, Brian M., Wei, Qijian, Uka, Valdet, De Saeger, Sarah, Diana Di Mavungu, José
• Format: Article
• Language: English
• Subjects: Aspergillus flavus; Aspergillus flavus - genetics; Aspergillus flavus - metabolism; Gene cluster; Gene Knockdown Techniques; Genes, Fungal; Multigene Family; Natural products; Pyrazines - metabolism; Pyrazinones; Siderophores
• ISSN: 1087-1845; 1096-0937
• DOI: 10.1016/j.fgb.2018.04.009

•Aspergillus flavus gene cluster 11 is responsible for production of aspergillic acid.•Aspergillic acid production is abolished in the cluster 11 NRPS-like protein KO.•The cluster 11 P450 oxidoreductase installs hydroxamic acid functionality.•An Ankyrin domain protein is required for aspergillic acid production in A. flavus. Aspergillus flavus can colonize important food staples and produce aflatoxins, a group of toxic and carcinogenic secondary metabolites. Previous in silico analysis of the A. flavus genome revealed 56 gene clusters predicted to be involved in the biosynthesis of secondary metabolites. A. flavus secondary metabolites produced during infection of maize seed are of particular interest, especially with respect to their roles in the biology of the fungus. A predicted nonribosomal peptide synthetase-like (NRPS-like) gene, designated asaC (AFLA_023020), present in the uncharacterized A. flavus secondary metabolite gene cluster 11 was previously shown to be expressed during the earliest stages of maize kernel infection. Cluster 11 is composed of six additional genes encoding a number of putative decorating enzymes as well as a transporter and transcription factor. We generated knock-out mutants of the seven predicted cluster 11 genes. 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 asaC mutant showed no production of aspergillic acid or its precursors. Knockout of the cluster 11 P450 oxidoreductase afforded a pyrazinone metabolite, the aspergillic acid precursor deoxyaspergillic acid. The formation of hydroxyaspergillic acid was abolished in a desaturase/hydroxylase mutant. The hydroxamic acid functional group in aspergillic acid allows the molecule to bind to iron resulting in the production of a red pigment in A. flavus identified previously as ferriaspergillin. A reduction of aflatoxin B1 and cyclopiazonic acid that correlated with reduced fungal growth was observed in maize kernel infection assays when aspergillic acid biosynthesis in A. flavus is halted.