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.The ergot fungus Claviceps purpurea is a phytopathogenic ascomycete which infects the ears of several grasses, replacing the ovary and producing a hibernation structure, the so-called sclerotium, in which the ergot alkaloids are formed. These substances show a high level of structural homology to some neurotransmitters like serotonin and dopamine and can therefore bind to the same receptors in the central nervous system (CNS), which is the basis for the application of ergot alkaloids in a variety of clinical conditions (15).The biochemistry of ergot alkaloid biosynthesis was first investigated by isolation of intermediates and postulation of a hypothetical pathway as well as enzymes needed for the successive biosynthetic steps of the production (Fig. ​(Fig.1).1). Most of the data were collected by pursuing the fate of radiolabeled precursors in feeding experiments (4). The first enzyme which could be assigned to alkaloid production was dimethylallyltryptophan synthetase (DMATS), which is the key enzyme of the pathway and is encoded by the gene dmaW (18). These analyses were performed with a Claviceps fusiformis strain, but a homolog of dmaW ({"type":"entrez-nucleotide","attrs":{"text":"AY259840","term_id":"32402653","term_text":"AY259840"}}AY259840) possessing a similar function could also be isolated in C. purpurea, as was confirmed by a knockout approach (N. Lorenz and P. Tudzynski, unpublished data). Using genome walking combined with cDNA screening, a 68.5-kb genomic region surrounding dmaW could be sequenced and revealed 14 open reading frames (ORFs) (putative genes) encoding, among others, nonribosomal peptide synthetases (NRPSs), a putative catalase, a CYP450-1 monooxygenase, a putative methyltransferase, and several oxidoreductases (6, 13, 19) (Fig. ​(Fig.2).2). Some of these genes were functionally and biochemically analyzed by a gene replacement approach which revealed their function within the pathway (2, 5, 7). However, there is still a deficit in functional analyses, especially with respect to the early steps within this pathway. The conversion from N-methyl-dimethylallyltryptophan (Me-DMAT) to agroclavine via chanoclavine I and chanoclavine I aldehyde includes successive oxidation and reduction steps mediated by a specific class of enzymes, the oxidoreductases (15) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Biosynthetic pathway of the ergot alkaloid biosynthesis of C. purpurea. Genes analyzed so far have been assigned to the corresponding enzyme at the corresponding position within the pathway. DMAPP, dimethylallyldiphosphate; DMAT, dimethylallyltryptophan; Me-DMAT, N-methyl-DMAT. (Adapted from reference 7 with permission of Wiley-VCH Verlag GmbH & Co. KGaA.)Open in a separate windowFIG. 2.Alkaloid biosynthesis gene cluster of C. purpurea. Highlighted in white is the gene of interest ccsA. (Adapted from reference 7 with permission of Wiley-VCH Verlag GmbH & Co. KGaA.)These enzymes are involved in the biosynthesis of many fungal secondary metabolites. A prominent example is the family of the cytochrome P450 monooxygenases (named after the characteristic peak of 450 nm when complexed with carbon monoxide). Cytochrome P450 (CYP450) monooxygenases catalyze the transfer of one oxygen atom from molecular oxygen to various substrates, mostly accomplished by the involvement of NAD(P)H as an electron donor. The eas cluster of C. purpurea also includes a gene encoding a CYP450 monooxygenase: cloA is involved in the oxidation of elymoclavine, leading to the formation of paspalic acid (7).No further monooxygenase-encoding genes seem to be present in the eas cluster, but several genes code for putative oxidoreductases (easA, easD, easE, easG, and easH). These oxidoreductases are most likely involved in the early steps within the pathway, but none of them has been functionally analyzed so far (15).We initiated a functional analysis of the putative oxidoreductase-encoding gene ccsA (formerly easE) (Fig. ​(Fig.2).2). The coding region of ccsA ({"type":"entrez-nucleotide","attrs":{"text":"AJ011965","term_id":"4499842","term_text":"AJ011965"}}AJ011965; 1,503 bp) is composed of two exons interrupted by an intron of 52 bp, yielding a coding capacity of 483 amino acids (aa). The gene product shows highest similarity to putative oxidoreductases of other ergot alkaloid-producing fungi: EasE of C. fusiformis (e⁻¹⁶⁰; {"type":"entrez-protein","attrs":{"text":"ABV57823","term_id":"157488556","term_text":"ABV57823"}}ABV57823), EasE of Neotyphodium lolii (e⁻¹¹⁸; {"type":"entrez-protein","attrs":{"text":"ABM91450","term_id":"124110194","term_text":"ABM91450"}}ABM91450) and CpoX1 of Aspergillus fumigatus (e⁻⁹⁶; {"type":"entrez-nucleotide","attrs":{"text":"XM_751049","term_id":"71002921","term_text":"XM_751049"}}XM_751049). Analyses of the protein sequence using the program PROSITE revealed a flavin adenine dinucleotide (FAD)-binding domain (pfam01565) spanning the region from amino acids 14 to 161 and a berberine bridge enzyme domain (BBE domain; pfam08031) from amino acids 412 to 457. The role of CcsA in the alkaloid biosynthesis pathway was investigated by knockout of the corresponding gene, followed by functional and biochemical analyses of the deletion mutants.