The understanding of OTA biosynthesis: new insight from Aspergillus carbonarius

The increasing availability of fungal genomes and bioinformatic tools have led to the identification of clusters of known metabolites and to the prediction of novel cryptic clusters for still unknown 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 (1). The genome sequencing of Aspergillus carbonarius has advanced the knowledge of the molecular mechanism of biosynthesis of ochratoxin A (OTA), one of the most important mycotoxin contaminating several commodities. Differently from other mycotoxins, the elucidation of the genetic background of OTA biosynthesis has remained uncompleted for a long time. Aspergillus carbonarius is the major responsible of OTA contamination of wine and other grape products in the Mediterranean area, constituting a great health risk and cause of important economic losses (2). The analysis of A. carbonarius genome has revealed the presence of a great number of PKSs and NRPSs, enzymes having an essential role in the synthesis of fungal secondary metabolites. Subsequently, the identification of the PKS putatively involved in the biosynthesis of OTA has led to an extensive study of the adjacent genomic region, in the attempt to identify other genes involved and to define the OTA biosynthesis cluster. The roles of three key genes -AcOTApks, AcOTAnrps and AcOTAhal - have been demonstrated by gene knock-out approach and the order of the fundamental enzymatic steps in the biosynthesis pathway of OTA has been clarified. These studies demonstrated that the enzymatic step involving the addition of phenilalanine to the polyketide ring takes place before the chlorination step. Moreover, it was demonstrated that OT? is not a precursor of OTA but rather a product of OTA hydrolysis (3, 4). Other predicted genes in the cluster need to be further investigated to fully clarify the structural and regulatory mechanisms of toxin production, among which the genes coding a p450 monooxygenase, a transcription factor, a transporter protein and an aspartyl protease. Transcriptomic analyses are in progress to study and clarify at a deeper level the complex genetic picture of the fungus during OTA biosynthesis. References 1. Brakhage A.A., 2013. Nature Reviews Microbiology 11.1: 21-32. 2. Perrone G. et al., 2008. Aspergillus in the genomic era, Academic Publishers, Wageningen, 2008, 179-212. 3. Gallo A. et al., 2012. Appl. Environ. Microbiol., 78 (23), 8208-8218. 4. Ferrara M. et al., 2016. Appl. Environ. Microbiol., 82 (18), 5631-5641.