New insights in genetics of mycotoxin biosynthesis by genomic approach: the ochratoxin A story

In the nineties fungal secondary metabolites (SMs), such as antibiotics and mycotoxins, started to be genetically characterized. Then, the clustered arrangement of genes involved in the biosynthesis of a single SM was studied. In the pre-genomic era, gene cluster discovery in fungi was complex and time-consuming, involving cumbersome traditional molecular methods. Genomics has revolutionized the research on SM biosynthesis pathways, allowing the bypass of such approaches. The breakthrough of next-generation sequencing (NGS) technologies and the advent of Bioinformatics have opened a new era in the study of biological systems. NGS technologies contributed significantly to the increasing availability of fungal genomes and bioinformatic analysis lead to the identification of SM clusters of known metabolites and to the prediction of novel cryptic clusters for still unknown microbial metabolites. However, most of the clusters identified by genome analysis are still to be deeply examined to completely understand the pathway steps and the regulatory network behind the metabolite biosynthesis. Here, we present the example of how the genomic approach has led to the identification of biosynthetic genes and their role in ochratoxin A (OTA) production by Aspergillus carbonarius. From the genome sequencing and the subsequent prediction of OTA cluster, we demonstrated by gene knock-out approach the key role of three genes (AcOTApks, AcOTAnrps and AcOTAhal) in the OTA biosynthesis. Single gene knock-out mutant allowed us to elucidate the order of the enzymatic steps in the biosynthesis pathway. Other predicted genes in the cluster, such as a p450 monooxygenase and a transcription factor gene, need to be investigated for the full knowledge of the structural and regulatory mechanisms of toxin production. Furthermore, transcriptomic analyses are in progress to study and clarify at a deeper level the complex genetic picture of the fungus during OTA biosynthesis.