Open AccessAlkaloid 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
Author(s) -
Nicole Lorenz,
Jana Olšovská,
Miroslav Šulc,
Paul Tudzynski
Publication year - 2010
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.00737-09
Subject(s) - biochemistry , biosynthesis , biology , gene cluster , claviceps purpurea , oxidoreductase , stereochemistry , chemistry , gene , enzyme
Ergot alkaloids are indole-derived secondary metabolites synthesized by the phytopathogenic ascomyceteClaviceps 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 geneccsA , originally namedeasE and preliminarily annotated as coding for a flavin adenine dinucleotide-containing oxidoreductase, was deleted in theC. 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 ofN -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 ofccsA led to a block in alkaloid biosynthesis beyond Me-DMAT formation. Complementation with accsA /gfp fusion construct restored alkaloid biosynthesis. These data indicate thatccsA encodes the chanoclavine I synthase or a component thereof catalyzing the conversion ofN -methyl-dimethylallyltryptophan to chanoclavine I.