Evidence for an ergot alkaloid gene cluster in Claviceps purpurea

A gene (cpd1) coding for the dimethylallyltryptophan synthase (DMATS) that catalyzes the first specific step in the biosynthesis of ergot alkaloids, w as cloned from a strain of Claviceps purpurea that produces alkaloids in ax enic culture. The derived gene product (CPD1) shows only 70% similarity to the corresponding gene previously isolated from Claviceps strain ATCC 26245 , which is likely to be an isolate of C. fusiformis. Therefore, the related cpd1 most probably represents the first C. purpurea gene coding for an enz ymatic step of the alkaloid biosynthetic pathway to be cloned. Analysis of the 3'-flanking region of cpd1 revealed a second, closely linked ergot alka loid biosynthetic gene named cpps1, which codes for a 356-kDa polypeptide s howing significant similiarity to fungal modular peptide synthetases. The p rotein contains three amino acid-activating modules, and in the second modu le a sequence is found which matches that of an internal peptide (17 amino acids in length) obtained from a tryptic digest of lysergyl peptide synthet ase 1 (LPS1) of C. purpurea, thus confirming that cpps1 encodes LPS1. LPS1 activates the three amino acids of the peptide portion of ergot peptide alk aloids during D-lysergyl peptide assembly. Chromosome walking revealed the presence of additional genes upstream of cpd1 which are probably also invol ved in ergot alkaloid biosynthesis: cpox1 probably codes for an FAD-depende nt oxidoreductase (which could represent the chanoclavine cyclase), and a s econd putative oxidoreductase gene, cpox2, is closely linked to it in inver se orientation. RT-PCR experiments confirm that all four genes are expresse d under conditions of peptide alkaloid biosynthesis. These results strongly suggest that at least some genes of ergot alkaloid biosynthesis in C. purp urea are clustered, opening the way for a detailed molecular genetic analys is of the pathway.