Tremorgenic and neurotoxic paspaline-derived indole-diterpenes: biosynthetic diversity, threats and applications.

Indole-diterpenes (IDTs) such as the aflatrems, janthitrems, lolitrems, paspalitrems, penitrems, shearinines, sulpinines, and terpendoles are biogenetically related but structurally varied tremorgenic and neurotoxic mycotoxins produced by fungi. All these metabolites derive from the biosynthetic intermediate paspaline, a frequently occurring IDT on its own right. In this comprehensive review, we highlight the similarities and differences of the IDT biosynthetic pathways that lead to the generation of the main paspaline-derived IDT subgroups. We survey the taxonomic distribution and the regulation of IDT production in various fungi and compare the organization of the known IDT biosynthetic gene clusters. A detailed assessment of the highly diverse biological activities of these mycotoxins leads us to emphasize the significant losses that paspaline-derived IDTs cause in agriculture, and compels us to warn about the various hazards they represent towards human and livestock health. Conversely, we also describe the potential utility of these versatile molecules as lead compounds for pharmaceutical drug discovery, and examine the prospects for their industrial scale manufacture in genetically manipulated IDT producers or domesticated host microorganisms in synthetic biological production systems.

Inactivation of the indole-diterpene biosynthetic gene cluster of Claviceps paspali by Agrobacterium-mediated gene replacement.

The hypocrealean fungus Claviceps paspali is a parasite of wild grasses. This fungus is widely utilized in the pharmaceutical industry for the manufacture of ergot alkaloids, but also produces tremorgenic and neurotoxic indole-diterpene (IDT) secondary metabolites such as paspalitrems A and B. IDTs cause significant losses in agriculture and represent health hazards that threaten food security. Conversely, IDTs may also be utilized as lead compounds for pharmaceutical drug discovery. Current protoplast-mediated transformation protocols of C. paspali are inadequate as they suffer from inefficiencies in protoplast regeneration, a low frequency of DNA integration, and a low mitotic stability of the nascent transformants. We adapted and optimized Agrobacterium tumefaciens-mediated transformation (ATMT) for C. paspali and validated this method with the straightforward creation of a mutant strain of this fungus featuring a targeted replacement of key genes in the putative IDT biosynthetic gene cluster. Complete abrogation of IDT production in isolates of the mutant strain proved the predicted involvement of the target genes in the biosynthesis of IDTs. The mutant isolates continued to produce ergot alkaloids undisturbed, indicating that equivalent mutants generated in industrial ergot producers may have a better safety profile as they are devoid of IDT-type mycotoxins. Meanwhile, ATMT optimized for Claviceps spp. may open the door for the facile genetic engineering of these industrially and ecologically important organisms.

Functional characterization of the idtF and idtP genes in the Claviceps paspali indole diterpene biosynthetic gene cluster.

Claviceps paspali is used in the pharmaceutical industry for the production of ergot alkaloids. This fungus also biosynthesizes paspalitrems, indole diterpene (IDT) mycotoxins that cause significant economic losses in agriculture and represent safety concerns for ergot alkaloid manufacture. Here, we use Agrobacterium-mediated transformation to replace the idtP and the idtF genes in the IDT biosynthetic gene cluster of C. paspali with a selectable marker gene. We show that the ΔidtP knockout mutant produces paspaline, the first IDT intermediate of the pathway. The ΔidtF strain produces unprenylated IDTs such as paspalinine and paspaline. These experiments validate the function of idtP as the gene encoding the cytochrome P450 monooxygenase that oxidizes and demethylates paspaline to produce 13-desoxypaxilline, and that of idtF as the gene that encodes the α-prenyltransferase that prenylates paspalinine at the C20 or the C21 positions to yield paspalitrems A and C, respectively. In addition, we also show that axenic cultures of the wild type, the ΔidtP and the ΔidtF mutant C. paspali strains fail to produce an assembly of IDTs that are present in C. paspali-Paspalum spp. associations.