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Characterisation of intermediates involved in the biosynthesis of ochratoxin A by Penicillium verrucosum
Ochratoxin A (OA) is a nephrotoxin and possible carcinogen that has been detected in a variety of foods, including cereals and cereal products, pork, grapes and grape products, coffee and spices. Once it is present in food or animal feed, it is difficult to remove, because it is not completely degraded during processing or cooking. People in northern Europe often have detectable levels of OA in their blood serum. The main dietary source of this toxin in northern Europe is cereal products, and the meat of animals (in particular pigs) that have eaten contaminated grain. Infection occurs when grain is stored in cool, damp conditions, and is infected with the mould, Penicillium verrucosum. The EU limits for OA in foods are 5,0 µg/kg in raw cereals, 3,0 µg/kg in all cereal products, and 0,5 µg/kg in baby foods and processed cereal-based food for infants and young children.
Penicillium verrucosum is just one of many moulds that produce OA in foods. Four genes involved in OA synthesis and excretion have been characterised in Penicillium nordicum, a species that is closely related to P. verrucosum. Disruption of the four putative biosynthesis genes in P. nordicum by insertional mutagenesis causes OA production to decrease markedly. Primers to similar genes in P. verrucosum have been developed. But the involvement of these genes in biosynthesis of OA by P. verrucosum has not yet been demonstrated.
The molecular component of this project will focus on disrupting these genes in P. verrucosum by insertional mutagenesis, as has been demonstrated for P. nordicum. Techniques include generating protoplasts, and screening and characterising mutants. We will collaborate with Prof. Rolf Geisen, (Federal Research Centre for Nutrition and Food Molecular Food Mycology, Karlsruhe, Germany), who led the original studies on P. nordicum.
If the biosynthesis pathway has been blocked at a particular stage by gene inactivation, then molecules that are ‘intermediates’ to OA are likely to accumulate. The structure of such intermediates will be elucidated by LC-MS and NMR in collaboration with docent Anders Broberg, natural products chemist. Intermediates will also be characterised in wild type strains, naturally non- or weakly-toxigenic strains, and non-toxigenic strains produced by random mutagenesis. We also hope to find these intermediates in unrelated moulds that also produce OA, e.g. Aspergillus ochraceus and A. carbonarius. It is possible that these other moulds may synthesise OA by a different pathway; thus different intermediates may be involved.
• General mycology, including preparation of fungal culture media
• Extraction and HPLC analysis of ochratoxin A
• Insertional mutagenesis in fungi
• Preparation of samples for advanced chemical analyses (LC-MS, NMR)
We are looking for a student with broad skills in general microbiology and molecular biology. Experience and interest in chemical structures and analysis would be an advantage.
This project is supervised by Su-lin Leong, a postdoctoral fellow from Australia. Project discussions will be conducted in English.
Other collaborators on this project
• Anders Broberg, Chemistry, SLU
• Rolf Geisen, Federal Research Centre for Nutrition and Food Molecular Food Mycology, Karlsruhe, Germany
For information on the Department of Microbiology, SLU, visit our web-site: http://www.mikrob.slu.se
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