Aspergillus carbonarius proteases and their possible activity in ochratoxin A degradation.

Aspergillus carbonarius is the main responsible fungus of ochratoxin A (OTA) contamination of grapes and derived products. Recently, the biosynthetic mechanism of this mycotoxin has been mainly elucidated by experiments of knocking out of the key biosynthetic genes. The mutant strains of A. carbonarius, in which the AcOTAnrps gene had been disrupted, was unable to produce OTA but retained its ability to degrade OTA into OT? when it was grown in presence of exogenous OTA. Microbial degradation of OTA is due to the enzymatic cleavage of the amide bond between L-?-phenylalanine and OT? by proteolytic proteins. Then, an in silico screening has been made on the available genome sequence of A. carbonarius ITEM 5010 to identify genes encoding proteases and to investigate their involvement in the OTA degrading activity of A. carbonarius. Preliminary transcriptomic analysis allowed selecting eight protease encoding genes that were expressed at increased level during OTA production. From the analysis of functional domains of the deduced protein sequences, four identified genes encode for aspartic proteases, three of them encode for serine proteases and one for a metalloprotease. Wild type and three mutant strains of A. carbonarius ITEM 5010 (?AcOTAnrps, ?AcOTApks, ?AcOTAhal) previously obtained and resulted to be unable to produce OTA, have been incubated in presence of OTA under different conditions and time of growth. Expression levels during growth and activation rate of the selected protease genes are under investigation in order to establish their involvement in the degradation activity of A. carbonarius strains.