Identification of the decumbenone biosynthetic gene cluster in Penicillium decumbens and the importance for production of calbistrin.

Background: Filamentous fungi are important producers of secondary metabolites, low molecular weight molecules that often have bioactive properties. Calbistrin A is a secondary metabolite with an interesting structure that was recently found to have bioactivity against leukemia cells. It consists of two polyketides linked by an ester bond: a bicyclic decalin containing polyketide with structural similarities to lovastatin, and a linear 12 carbon dioic acid structure. Calbistrin A is known to be produced by several uniseriate black Aspergilli, Aspergillus versicolor -related species, and Penicillia. Penicillium decumbens produces calbistrin A and B as well as several putative intermediates of the calbistrin pathway, such as decumbenone A-B and versiol.

Results: A comparative genomics study focused on the polyketide synthase (PKS) sets found in three full genome sequence calbistrin producing fungal species, P. decumbens, A. aculeatus and A. versicolor , resulted in the identification of a novel, putative 13-membered calbistrin producing gene cluster ( calA to calM ). Implementation of the CRISPR/Cas9 technology in P. decumbens allowed the targeted deletion of genes encoding a polyketide synthase ( calA ), a major facilitator pump ( calB ) and a binuclear zinc cluster transcription factor ( calC ). Detailed metabolic profiling, using UHPLC-MS, of the ∆ calA (PKS) and ∆ calC (TF) strains confirmed the suspected involvement in calbistrin productions as neither strains produced calbistrin nor any of the putative intermediates in the pathway. Similarly analysis of the excreted metabolites in the ∆ calB (MFC-pump) strain showed that the encoded pump was required for efficient export of calbistrin A and B.

Conclusion: Here we report the discovery of a gene cluster ( calA - M ) involved in the biosynthesis of the polyketide calbistrin in P. decumbens . Targeted gene deletions proved the involvement of CalA (polyketide synthase) in the biosynthesis of calbistrin, CalB (major facilitator pump) for the export of calbistrin A and B and CalC for the transcriptional regulation of the cal -cluster. This study lays the foundation for further characterization of the calbistrin biosynthetic pathway in multiple species and the development of an efficient calbistrin producing cell factory.