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.
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 pathway-specific 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.
Lorenz
Nicole
N
Institut für Botanik, Westfalische Wilhelms-Universität Münster, Schlossgarten 3, D-48149 Münster, Germany.
Olsovská
Jana
J
Sulc
Miroslav
M
Tudzynski
Paul
P
D053898:Biosynthetic Pathways; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D002967:Claviceps / Q000201:enzymology* / Q000235:genetics; D003067:Coenzymes / Q000032:analysis; D004873:Ergolines / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D005816:Genetic Complementation Test; D013058:Mass Spectrometry; D008954:Models, Biological; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000031:analogs & derivatives*
D003067:Coenzymes; D004873:Ergolines; D005656:Fungal Proteins; D005182:Flavin-Adenine Dinucleotide; C006959:chanoclavine; D014364:Tryptophan; D010088:Oxidoreductases