Molecular characterization of the PR-toxin gene cluster in Penicillium roqueforti and Penicillium chrysogenum: cross talk of secondary metabolite pathways.

The PR-toxin is a potent mycotoxin produced by Penicillium roqueforti in moulded grains and grass silages and may contaminate blue-veined cheese. The PR-toxin derives from the 15 carbon atoms sesquiterpene aristolochene formed by the aristolochene synthase (encoded by ari1). We have cloned and sequenced a four gene cluster that includes the ari1 gene from P. roqueforti. Gene silencing of each of the four genes (named prx1 to prx4) resulted in a reduction of 65-75% in the production of PR-toxin indicating that the four genes encode enzymes involved in PR-toxin biosynthesis. Interestingly the four silenced mutants overproduce large amounts of mycophenolic acid, an antitumor compound formed by an unrelated pathway suggesting a cross-talk of PR-toxin and mycophenolic acid production. An eleven gene cluster that includes the above mentioned four prx genes and a 14-TMS drug/H(+) antiporter was found in the genome of Penicillium chrysogenum. This eleven gene cluster has been reported to be very poorly expressed in a transcriptomic study of P. chrysogenum genes under conditions of penicillin production (strongly aerated cultures). We found that this apparently silent gene cluster is able to produce PR-toxin in P. chrysogenum under static culture conditions on hydrated rice medium. Noteworthily, the production of PR-toxin was 2.6-fold higher in P. chrysogenum npe10, a strain deleted in the 56.8kb amplifiable region containing the pen gene cluster, than in the parental strain Wisconsin 54-1255 providing another example of cross-talk between secondary metabolite pathways in this fungus. A detailed PR-toxin biosynthesis pathway is proposed based on all available evidence.

A single cluster of coregulated genes encodes the biosynthesis of the mycotoxins roquefortine C and meleagrin in Penicillium chrysogenum.

A single gene cluster of Penicillium chrysogenum contains genes involved in the biosynthesis and secretion of the mycotoxins roquefortine C and meleagrin. Five of these genes have been silenced by RNAi. Pc21g15480 (rds) encodes a nonribosomal cyclodipeptide synthetase for the biosynthesis of both roquefortine C and meleagrin. Pc21g15430 (rpt) encodes a prenyltransferase also required for the biosynthesis of both mycotoxins. Silencing of Pc21g15460 or Pc21g15470 led to a decrease in roquefortine C and meleagrin, whereas silencing of the methyltransferase gene (Pc21g15440; gmt) resulted in accumulation of glandicolin B, indicating that this enzyme catalyzes the conversion of glandicolin B to meleagrin. All these genes are transcriptionally coregulated. Our results prove that roquefortine C and meleagrin derive from a single pathway.

The heterotrimeric Galpha protein pga1 regulates biosynthesis of penicillin, chrysogenin and roquefortine in Penicillium chrysogenum.

We have studied the role of the pga1 gene of Penicillium chrysogenum, encoding the alpha subunit of a heterotrimeric G protein, in secondary metabolite production. The dominant activating pga1(G42R) mutation caused an increase in the production of the three secondary metabolites penicillin, the yellow pigment chrysogenin and the mycotoxin roquefortine, whereas the dominant inactivating pga1(G203R) allele and the deletion of the pga1 gene resulted in a decrease of the amount of produced penicillin and roquefortine. Chrysogenin is produced in solid medium as a yellow pigment, and its biosynthesis is clearly enhanced by the presence of the dominant activating pga1(G42R) allele. Roquefortine is produced associated with mycelium during the first 3 days in submerged cultures, and is released to the medium afterwards; dominant activating and inactivating pga1 mutations result in upregulation and downregulation of roquefortine biosynthesis recpectively. Pga1 regulates penicillin biosynthesis by controlling expression of the penicillin biosynthetic genes; the three genes pcbAB, pcbC and penDE showed elevated transcript levels in transformants expressing the pga1(G42R) allele, whereas in transformants with the inactivating pga1(G203R) allele and in the pga1-deleted mutant their transcript levels were lower than those in the parental strains. Increase of intracellular cAMP levels had no effect on penicillin production. In summary, the dominant activating pga1(G42R) allele upregulates the biosynthesis of three secondary metabolites in Penicillium chrysogenum to a different extent.