C03026: An investigation into the potential use of DNA microarray technology for the identification of molecular mechanisms that control production of ochratoxin A in Penicillium verrucosum

Study Duration: January 2002 to March 2002

Contractor: Central Science Laboratory

Contamination of human and animal foodstuffs by fungal mycotoxins is a serious concern to the food industry and Government and there is legislation to set limits on mycotoxin levels.

Understanding the production of mycotoxin by fungi is attractive as this should lead to control over levels of production. However, this is complicated by the fact that within an individual fungal species it is possible to have strains that are producers (toxigenic strains), non-producers (non-toxigenic strains) and facultative producers.

In conjunction with bacteria, fungi are used for the fermentation of food. for example, penicillium species (such as Penicillium roqueforti, Penicillium camemberti) are involved in the fermentation of Roquefort and Camembert cheeses and also some meats. It is important that the fungi used in fermentation processes are non-toxigenic.

The only sure way to identify potentially toxigenic fungi is by the isolation and identification of the gene(s) involved in mycotoxin production. Using conventional techniques, this would require laboriously isolating, cloning and sequencing genes and identifying their function. Applying DNA microarray technology could be a method of more rapidly identifying the gene function.

P. verrucosum, is the main fungus responsible for ochratoxin A (OTA) production in the UK and northern European countries, and OTA is the subject of impending EU legislation. It is the aim of this research project to demonstrate whether DNA microarray technology can provide a short-cut to identification of those genes that are switched on in OTA producing strains of P. verrucosum.

All expressed genes in a toxigenic strain of P. verrucosum will be cloned to obtain a complimentary DNA (cDNA) library. Amongst other genes, the library will contain those genes that code for the enzymes involved in OTA production.

Individual clones from the cDNA library will be used to print microarray slides.

cDNA labelled with fluorescent dyes will be synthesised from both toxigenic and non-toxigenic strains of P. verrucosum, and used in probing the microarray slides. Any clones binding to the probes differentially will be marked as potential leads to OTA production pathways.

The cDNA clones identified on microarray slides will be sequenced to obtain information about the structure of each gene.

The gene sequences will be compared with other genes in Genebank database to establish a possible function for each gene in P. verrucosum.

The construction of a cDNA library from P. verrucosum, and initial screening of over 460 clones provided a successful initial assessment of the new technology. A useful cDNA library from a toxigenic strain of P. verrucosum was developed through DNA sequencing, generating a wealth of genetic information. Initial screening highlighted nine over-expressed genes in the OTA-producing strain PV11. These and another 179 randomly isolated clones from the cDNA library were sequenced to obtain information about the structure of each gene. This led to identification of the DNA sequence of a number of key genes, including polyketide synthase, which are likely to be involved in OTA synthesis in P. verrucosum.

This short study demonstrated the utility of DNA microarray technology for identification of genes that are differentially expressed in toxigenic and non-toxigenic strains of micro-organisms. It can be used to screen more genes, especially using those already identified in this study as potentially important in the production of OTA

The final report is available from the Agency's Information Centre.
To obtain a copy, please contact the Enquiry Desk, Information Services, Food Standards Agency (tel: 020 7276 8181/8182 or email: infocentre@foodstandards.gsi.gov.uk)

Contact: For any enquiries concerning this research project, please contact the relevant Programme contact or email: science@foodstandards.gsi.gov.uk