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Title: Identification and characterization of the polyketide synthase involved in ochratoxin A biosynthesis in Aspergillus carbonarius

Abstract

Ochratoxin A (OTA) is a potent mycotoxin produced by Aspergillus and Penicillium species and is a common contaminant of a wide variety of food commodities, with Aspergillus carbonarius being the main producer of OTA contamination in grapes and wine. The molecular structure of OTA is composed of a dihydroisocoumarin ring linked to phenylalanine and, as shown in different producing fungal species, a polyketide synthase (PKS) is a component of the OTA biosynthetic pathway. Similar to observations in other filamentous ascomycetes, the genome sequence of A. carbonarius contains a large number of genes predicted to encode PKSs. In this work a pks gene identified within the putative OTA cluster of A. carbonarius, designated as AcOTApks, was inactivated and the resulting mutant strain was unable to produce OTA, confirming the role of AcOTApks in this biosynthetic pathway. AcOTApks protein is characteristic of the highly reduced (HR)-PKS family, and also contains a putative methyltransferase domain likely responsible for the addition of the methyl group to the OTA polyketide structure. AcOTApks is different from the ACpks protein that we previously described which showed an expression profile compatible with OTA production. We performed phylogenetic analyses of the β-ketosynthase and acyl-transferase domains of the OTA PKSsmore » which had been identified and characterized in different OTA producing fungal species. The phylogenetic results were similar for both the two domains analyzed and showed that OTA PKS of A. carbonarius, Aspergillus niger, and Aspergillus ochraceus clustered in a monophyletic group with 100% bootstrap support suggesting a common origin, while the other OTA PKSs analyzed were phylogenetically distant. A qRT-PCR assay monitored AcOTApks expression during fungal growth and concomitant production of OTA by A. carbonarius in synthetic grape medium. A clear correlation between the expression profile of AcOTApks and kinetics of OTA production was observed with AcOTApks which reached its maximum level of transcription before OTA accumulation in mycelium reached its highest level, confirming the fact that gene transcription always precedes phenotypic production.« less

Authors:
; ; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1140098
Report Number(s):
PNNL-SA-101669
BM0101020
DOE Contract Number:  
AC05-76RL01830
Resource Type:
Journal Article
Journal Name:
International Journal of Food Microbiology, 179:10-17
Additional Journal Information:
Journal Name: International Journal of Food Microbiology, 179:10-17
Country of Publication:
United States
Language:
English
Subject:
fungi, Aspergillus, mycotoxin, ochratoxin, carbonarious,

Citation Formats

Gallo, Antonia, Knox, Benjamin P., Bruno, Kenneth S., Solfrizzo, Michele, Baker, Scott E., and Perrone, Giancarlo. Identification and characterization of the polyketide synthase involved in ochratoxin A biosynthesis in Aspergillus carbonarius. United States: N. p., 2014. Web. doi:10.1016/j.ijfoodmicro.2014.03.013.
Gallo, Antonia, Knox, Benjamin P., Bruno, Kenneth S., Solfrizzo, Michele, Baker, Scott E., & Perrone, Giancarlo. Identification and characterization of the polyketide synthase involved in ochratoxin A biosynthesis in Aspergillus carbonarius. United States. https://doi.org/10.1016/j.ijfoodmicro.2014.03.013
Gallo, Antonia, Knox, Benjamin P., Bruno, Kenneth S., Solfrizzo, Michele, Baker, Scott E., and Perrone, Giancarlo. 2014. "Identification and characterization of the polyketide synthase involved in ochratoxin A biosynthesis in Aspergillus carbonarius". United States. https://doi.org/10.1016/j.ijfoodmicro.2014.03.013.
@article{osti_1140098,
title = {Identification and characterization of the polyketide synthase involved in ochratoxin A biosynthesis in Aspergillus carbonarius},
author = {Gallo, Antonia and Knox, Benjamin P. and Bruno, Kenneth S. and Solfrizzo, Michele and Baker, Scott E. and Perrone, Giancarlo},
abstractNote = {Ochratoxin A (OTA) is a potent mycotoxin produced by Aspergillus and Penicillium species and is a common contaminant of a wide variety of food commodities, with Aspergillus carbonarius being the main producer of OTA contamination in grapes and wine. The molecular structure of OTA is composed of a dihydroisocoumarin ring linked to phenylalanine and, as shown in different producing fungal species, a polyketide synthase (PKS) is a component of the OTA biosynthetic pathway. Similar to observations in other filamentous ascomycetes, the genome sequence of A. carbonarius contains a large number of genes predicted to encode PKSs. In this work a pks gene identified within the putative OTA cluster of A. carbonarius, designated as AcOTApks, was inactivated and the resulting mutant strain was unable to produce OTA, confirming the role of AcOTApks in this biosynthetic pathway. AcOTApks protein is characteristic of the highly reduced (HR)-PKS family, and also contains a putative methyltransferase domain likely responsible for the addition of the methyl group to the OTA polyketide structure. AcOTApks is different from the ACpks protein that we previously described which showed an expression profile compatible with OTA production. We performed phylogenetic analyses of the β-ketosynthase and acyl-transferase domains of the OTA PKSs which had been identified and characterized in different OTA producing fungal species. The phylogenetic results were similar for both the two domains analyzed and showed that OTA PKS of A. carbonarius, Aspergillus niger, and Aspergillus ochraceus clustered in a monophyletic group with 100% bootstrap support suggesting a common origin, while the other OTA PKSs analyzed were phylogenetically distant. A qRT-PCR assay monitored AcOTApks expression during fungal growth and concomitant production of OTA by A. carbonarius in synthetic grape medium. A clear correlation between the expression profile of AcOTApks and kinetics of OTA production was observed with AcOTApks which reached its maximum level of transcription before OTA accumulation in mycelium reached its highest level, confirming the fact that gene transcription always precedes phenotypic production.},
doi = {10.1016/j.ijfoodmicro.2014.03.013},
url = {https://www.osti.gov/biblio/1140098}, journal = {International Journal of Food Microbiology, 179:10-17},
number = ,
volume = ,
place = {United States},
year = {Mon Jun 02 00:00:00 EDT 2014},
month = {Mon Jun 02 00:00:00 EDT 2014}
}