Alkaloid Cluster Gene ccsA of the Ergot Fungus Claviceps purpurea Encodes Chanoclavine I Synthase, a Flavin Adenine Dinucleotide-Containing Oxidoreductase Mediating the Transformation of N -Methyl-Dimethylallyltryptophan to Chanoclavine I

Abstract

Ergot alkaloids are indole-derived secondary metabolites synthesized by the phytopathogenic ascomycete Claviceps purpurea. In wild-type strains, they are exclusively produced in the sclerotium, a hibernation structure; for biotechnological applications, submerse production strains have been generated by mutagenesis. It was shown previously that the enzymes specific for alkaloid biosynthesis are encoded by a gene cluster of 68.5 kb. This ergot alkaloid cluster consists of 14 genes coregulated and expressed under alkaloid-producing conditions. Although the role of some of the cluster genes in alkaloid biosynthesis could be confirmed by a targeted knockout approach, further functional analyses are needed, especially concerning the early pathwayspecific steps up to the production of clavine alkaloids. Therefore, the gene ccsA, originally named easE and preliminarily annotated as coding for a flavin adenine dinucleotide-containing oxidoreductase, was deleted in the C. purpurea strain P1, which is able to synthesize ergot alkaloids in axenic culture. Five independent knockout mutants were analyzed with regard to alkaloid-producing capability. Thin-layer chromatography (TLC), ultrapressure liquid chromatography (UPLC), and mass spectrometry (MS) analyses revealed accumulation of N-methyl-dimethylallyltryptophan (Me-DMAT) and traces of dimethylallyltryptophan (DMAT), the first pathway-specific intermediate. Since other alkaloid intermediates could not be detected, we conclude that deletion of ccsA led to a block in alkaloid biosynthesis beyond Me-DMAT formation. Complementation with a ccsA/gfp fusion construct restored alkaloid biosynthesis. These data indicate that ccsA encodes the chanoclavine I synthase or a component thereof catalyzing the conversion of N-methyl-dimethylallyltryptophan to chanoclavine I.

Periglandula, a new fungal genus within the Clavicipitaceae and its association with Convolvulaceae

“…The order and orientation of the genes ccsA (encoding a component of chanoclavine cyclase; Lorenz et al 2010), easF (encoding an N-methyltransferase; Rigbers and Li 2008), easG (encoding a reductase/dehydrogenase) and dmaW within the cluster parallels the arrangement of genes in C. purpurea and C. fusiformis, and differs from their arrangement in Aspergillus fumigatus (Schardl et al 2006, Wallwey et al 2010, a member of the only distantly related Trichocomaceae (Eurotiales).…”

Ergot Alkaloids of the Family Clavicipitaceae

“…2) (Gerhards et al 2014; Robinson and Panaccione 2015; Schardl et al 2006; Young et al 2015) begins with the formation of 4-(γ,γ)-dimethylallyltryptophan (DMAT) from dimethylallyl diphosphate (DMAPP) and L-tryptophan, catalyzed by the enzyme, DMAT synthase (Heinstein et al 1971), which is encoded by the gene dmaW (Tsai et al 1995). The next biosynthetic step is catalyzed by DMAT N -methyltransferase encoded by easF (Otsuka et al 1980), and subsequent steps by a catalase encoded by easC (Goetz et al 2011), and chanoclavine-I synthase, a flavine-dependent oxidoreductase encoded by easE (= ccsA ) (Lorenz et al 2010). Chanoclavine I is the simplest clavine known to be a pathway end-product, since some fungi such as Epichloë elymi E56 possess dmaW, easF, easC, and easE as their only four functional ergot-alkaloid biosynthesis genes (Schardl et al 2013c).…”

Partial Reconstruction of the Ergot Alkaloid Pathway by Heterologous Gene Expression in Aspergillus nidulans

“…[5], the synthesis of chanoclavine-I occurs in at least five steps. Gene knockout and heterologous expression studies have demonstrated that at least four genes, dmaW , easF , easE , and easC , are required prior to chanoclavine-I [6,7,8,9]. However, it is currently unknown whether these genes are sufficient to synthesize chanoclavine-I.…”