Abstract:
Fungal secondary metabolites are known for their incredible diversity and potential benefits to society, such as the renowned secondary metabolite penicillin. Loline alkaloids from the Epichloë fungi have unique insecticidal secondary metabolites, produced as part of a well-studied, mutualistic relationship with grasses. Penexpandine, a novel secondary metabolite, has been identified in the fruit-rotting fungus Penicillium expansum and contains a proposed loline moiety. In this study, a gene cluster in P. expansum containing six known loline genes (lolC, lolF, lolD, lolT, lolO, lolE) and two novel genes, lolB and lolG, has been proposed for penexpandine biosynthesis. Sequence analysis of these two novel genes indicated that they encode a putative non-ribosomal peptide synthetase (NRPS)-like enzyme (lolB) and a methyltransferase (lolG), which are consistent with the structure of penexpandine. In addition, an orthologue of another loline gene (lolN) was also identified in P. expansum. Taken together, a pathway for penexpandine biosynthesis has been proposed. The proposed pathway involves an undescribed capability for an NRPS and initial investigations of this have been performed at the sequence level. Split-marker deletion was used for the first time in this fungus to delete the two novel genes lolB and lolG. Expression analysis using qPCR was used to identify the promising conditions for loline gene expression in P. expansum, and under these conditions chemical detection of loline intermediates was achieved for the first time. Synteny analysis performed around the proposed penexpandine biosynthesis gene cluster identified elements of interest to the function of lolB and a putative transposable element with implications for the evolution of the loline biosynthesis gene cluster. In conclusion, a role for the proposed penexpandine gene cluster in loline synthesis was established.
