Abstract
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.
Highlights
Claviceps paspali is a hypocrealean fungus that has been used in the pharmaceutical industry for decades to produce ergot alkaloids
Genomic DNA from the wild-type strain and that transformed with the pAg-H3 vector did not yield PCR amplicons, as expected. These PCR experiments proved that the CPIDTF2, CPIDTF3, and CPIDTF7 isolates are homokaryotic for the ΔidtF allele, while the CPIDTP1, CPIDTP8, and CPIDTP11 isolates are homokaryotic for the ΔidtP allele
We identified the paspalitrem gene cluster of C. paspali by the disruption of the idtCBGF gene locus using A. tumefaciens-mediated gene disruption
Summary
Claviceps paspali is a hypocrealean fungus that has been used in the pharmaceutical industry for decades to produce ergot alkaloids. These alkaloids serve as precursors for the manufacture of drugs that treat Parkinson’s disease and migraine (Arcamone et al 1960; Tudzynski et al 2001). C. paspali forms an association with dallis grasses (Paspalum spp.) and produces ergot alkaloids and indole diterpenes (IDTs) such as paspalitrems (Cole et al 1977; Uhlig et al 2014). Paspalum stagger is rarely lethal for the intoxicated livestock, reduced body mass gain and culling due to accidents suffered by the animals as a result of uncoordinated movement lead to large losses in agriculture (Cole et al 1977; Moyano et al 2010; Cawdell-Smith et al 2010). Safety concerns and process economics both led to a demand in the pharmaceutical industries for paspalitrem nonproducing mutant C. paspali strains (Kozák et al 2018)
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