Evidence of the Involvement of a Cyclase Gene in the Biosynthesis of Ochratoxin A in Aspergillus carbonarius.

Giancarlo Perrone,Massimo Ferrara,Carla Cervini,Antonia Gallo,Lucia Gambacorta,Michele Solfrizzo,Scott E. Baker

doi:10.3390/toxins13120892

Abstract

Ochratoxin A (OTA) is a well-known mycotoxin with wide distribution in food and feed. Fungal genome sequencing has great utility for identifying secondary metabolites gene clusters for known and novel compounds. A comparative analysis of the OTA-biosynthetic cluster in A. steynii, A. westerdijkiae, A. niger, A. carbonarius, and P. nordicum has revealed a high synteny in OTA cluster organization in five structural genes (otaA, otaB, ota, otaR1, and otaD). Moreover, a recent detailed comparative genome analysis of Aspergilli OTA producers led to the identification of a cyclase gene, otaY, located in the OTA cluster between the otaA and otaB genes, encoding for a predicted protein with high similarity to SnoaLs domain. These proteins have been shown to catalyze ring closure steps in the biosynthesis of polyketide antibiotics produced in Streptomyces. In the present study, we 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 and their role in OTA biosynthesis by complete gene deletion. Our results pointed out the involvement of a cyclase gene in OTA biosynthetic pathway for the first time. They represent a step forward in the understanding of the molecular basis of OTA biosynthesis in A. carbonarius.

Highlights

Ochratoxin A (OTA) is a well-known mycotoxin with wide distribution in food and feed, including cereal products, grapes and by-products, coffee, beverages, cocoa, nuts, dried fruits, and cured meat [1,2]
We demonstrate the role of the otaY gene by complete gene deletion using the CRISPR/Cas9 approach
The production of OTA by UPLC-FLD in mycelium collected after growth on 0.99 and 0.93 aw Grape Juice Medium (GJM) at 18/31 ◦C under 10 h/14 h dark/light photoperiod accounted for the OTA permissive condition (0.99 aw GJM) in 472.23 and 452.82 ng/g at 4 and 7 dpi, respectively

Summary

Introduction

Ochratoxin A (OTA) is a well-known mycotoxin with wide distribution in food and feed, including cereal products, grapes and by-products, coffee, beverages, cocoa, nuts, dried fruits, and cured meat [1,2]. A detailed comparative analysis of Aspergilli genomes has recently led us to identify a gene whose sequence encoded a predicted protein similar to bacterial polyketide cyclases This gene is located in the OTA cluster between the otaA and otaB genes; it is present in the genome sequences of all currently sequenced OTA-producing fungi, and it was hypothesized to be involved in OTA production [12]. The RT-PCR analysis of transcription of OTA cluster genes in strains ITEM 5010 and AC2021 after 4 dpi on MM agar medium (see oligonucleotides used in Table 2) revealed the expression of the complete set of genes in wild-type strain ITEM 5010 and the absence of the otaY transcript only in the mutant strain AC2021 (data not shown).

OTA Analysis

Fungal Strains and Growth Conditions

Cyclase Gene Expression Analysis

Cyclase Gene Deletion: sgRNAs Selection and Fungal Transformation

Genome Sequencing and Assembly

Findings