Studies on the synthesis and biosynthesis of PR-Toxin

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The unknown biosynthesis of the terpenoid PR-Toxin from the fungus Penicillium roqueforti will be elucidated. For this purpose a total synthesis of PR-Toxin via a route that also makes diverse hypothetical intermediates accessible will be developed. The prepared compounds will be used as analytical standards to identify potential intermediates in culture extracts by GC/MS and HPLC/MS. The potential intermediates will subsequently also be prepared carrying a 13C-labelling, and used in feeding experiments to distinguish intermediates from non-convertable biosynthetic shunt products. The question whether an isomerisation along the PR-Toxin biosynthesis is spontaneous or enzyme-catalysed will also be addressed in a suitable isotopic labelling experiment. The genes of the biosynthetic gene cluster for PR-Toxin will be cloned and heterologously expressed. The expressed proteins will be purified and used in incubation experiments with synthetic potential substrates. The insights obtained by the different strategies will be used to establish a robust model of the biosynthesis of PR-Toxin.