GENOMIC COMPARATIVE ANALYSIS AND GENOME EDITING REVEALED THE INVOLVEMENT OF A CYCLASE GENE IN THE BIOSYNTHESIS OF OCHRATOXIN A

The widespread use of Next-Generation Sequencing (NGS) for fungal genome sequencing has led to identification of SM clusters for known metabolites as well as a significant number of novel predicted SM gene clusters. Fungal genome sequencing has great utility for identification of secondary metabolites gene clusters for known as well as novel compounds. Ochratoxin A (OTA) is a well-known mycotoxin with wide distribution on food and feed. A comparative analysis of gene cluster structure in 19 Aspergillus and 2 Penicillium OTA producers has revealed a high synteny across these species in OTA cluster, encoding five structural genes: otaA, otaB, otaC, otaR1, and otaD. Furthermore, we identify a previously undescribed additional gene. The new otaY gene is located between the otaA and otaB genes and encodes a protein with predicted SnoaL domain. These snoaL-domain containing proteins have been shown to catalyze ring closure steps in the biosynthesis of polyketide antibiotics produced in Streptomyces. Gene expression analysis has demonstrated an upregulation of the cyclase gene in A. carbonarius under OTA permissive conditions, consistent with the expression trends of the other OTA cluster genes. The role of in OTA biosynthesis has been demonstrated by CRISPR/Cas9 complete gene deletion. The presented results reveal for the first time the involvement of a cyclase gene in OTA biosynthetic pathway and redefine the structure of the OTA core cluster, consisting in six genes.