Cyclolization of D-lysergic acid alkaloid peptides.

The tripeptide chains of the ergopeptines, a class of pharmacologically important D-lysergic acid alkaloid peptides, are arranged in a unique bicyclic cyclol based on an amino-terminal α-hydroxyamino acid and a terminal orthostructure. D-lysergyl-tripeptides are assembled by the nonribosomal peptide synthetases LPS1 and LPS2 of the ergot fungus Claviceps purpurea and released as N-(D-lysergyl-aminoacyl)-lactams. We show total enzymatic synthesis of ergopeptines catalyzed by a Fe²âº/2-ketoglutarate-dependent dioxygenase (EasH) in conjunction with LPS1/LPS2. Analysis of the reaction indicated that EasH introduces a hydroxyl group into N-(D-lysergyl-aminoacyl)-lactam at α-C of the aminoacyl residue followed by spontaneous condensation with the terminal lactam carbonyl group. Sequence analysis revealed that EasH belongs to the wide and diverse family of the phytanoyl coenzyme A hydroxylases. We provide a high-resolution crystal structure of EasH that is most similar to that of phytanoyl coenzyme A hydroxylase, PhyH, from human.

Combinatorial assembly of simple and complex D-lysergic acid alkaloid peptide classes in the ergot fungus Claviceps purpurea.

The ergot fungus Claviceps purpurea produces both ergopeptines and simple d-lysergic acid alkylamides. In the ergopeptines, such as ergotamine, d-lysergic acid is linked to a bicyclic tripeptide in amide-like fashion, whereas in the d-lysergylalkanolamides it is linked to an amino alcohol derived from alanine. We show here that these compound classes are synthesized by a set of three non-ribosomal lysergyl peptide synthetases (LPSs), which interact in a combinatorial fashion for synthesis of the relevant product. The trimodular LPS1 assembles with LPS2, the d-lysergic acid recruiting module, to synthesize the d-lysergyltripeptide precursors of ergopeptines from d-lysergic acid and the three amino acids of the peptide chain. Alternatively, LPS2 can assemble with a distinct monomodular non-ribosomal peptide synthetase (NRPS) subunit (ergometrine synthetase) to synthesize the d-lysergic acid alkanolamide ergometrine from d-lysergic acid and alanine. The synthesis proceeds via covalently bound d-lysergyl alanine and release of dipeptide as alcohol with consumption of NADPH. Enzymatic and immunochemical analyses showed that ergometrine synthetase is most probably the enzyme LPS3 whose gene had been identified previously as part of the ergot alkaloid biosynthesis gene cluster in C. purpurea. Inspections of all LPS sequences showed no recognizable peptide linkers for their protein-protein interactions as in NRPS subunits of bacteria. Instead, they all carry conserved N-terminal domains (C0-domains) with similarity to the C-terminal halves of NRPS condensation domains pointing to an alternative mechanism of subunit-subunit interactions in fungal NRPS systems. Phylogenetic analysis of LPS modules and the C0-domains suggests that these enzyme systems most probably evolved by module duplications and rearrangements from a bimodular ancestor.

Use of a nonhomologous end joining deficient strain (Deltaku70) of the ergot fungus Claviceps purpurea for identification of a nonribosomal peptide synthetase gene involved in ergotamine biosynthesis.

The ergot fungus Claviceps purpurea uses mainly the nonhomologous-end-joining (NHEJ) system for integration of exogenous DNA, leading to a low frequency of homologous integration (1-2%). To improve gene targeting efficiency we deleted the C. purpurea ku70 gene in two different strains: the pathogenic strain 20.1 and the apathogenic, ergot alkaloid producing strain P1. The mutants were not impaired in vegetative and pathogenic development nor alkaloid production. Gene targeting efficiency was significantly increased (50-60%) in the Deltaku70 mutants. The P1 Deltaku70 strain (producing ergotamine and ergocryptine) was used for targeted deletion of lpsA1, one of the two trimodular NRPS genes present in the alkaloid gene cluster, encoding D-lysergyl peptide synthetases involved in formation of the tripeptide moiety of ergopeptines. Mutants lacking the lpsA1 gene were shown to be incapable of producing ergotamine but were still able to produce ergocryptine, proving that LpsA1 is involved in ergotamine biosynthesis.

Application of oxygen vectors to Claviceps purpurea cultivation.

The application of a two-phase fermentation system for the production of ergot peptide alkaloids by Claviceps purpurea is described. Perfluorocarbons (PFC) are used as oxygen vectors in Claviceps fermentation for the first time. In shake-flask cultivations, the inclusion of PFC in the medium brings about a five-fold increase in the total alkaloid production and a six-fold increase in the pharmaceutically important component, ergotamine. This rise cannot be correlated with the concentration of the added PFC and it is thought that the enhancement is due to a combination of factors, including the influence of PFC. Other oxygen vectors, such as several hydrocarbons, prove to be poor oxygen carriers in our study. Cultivations with PFC in a bioreactor are reproducible, the maximum total alkaloid and ergotamine production being attained on the 11th and 9th days, respectively. The relatively lower increase in the total alkaloid production in the bioreactor as compared to the shake-flasks is attributed to the unequal oxygen availability in the reactor. Processes with PFC offer the operational advantage of a five-fold reduction in aeration rate.

Biosynthesis of ergotamine by Claviceps purpurea (Fr.) Tul.

High-yielding strains of Claviceps purpurea (Fr.) Tul, grown on a defined medium, have been used for a study of the biosynthesis of the peptide ergot alkaloid, ergotamine. l-[U-(14)C]tryptophan, dl-[2-(14)C]mevalonic acid lactone, sodium [2-(14)C]acetate, sodium [(14)C]formate and the methyl group of l-[methyl-(14)C]methionine were efficiently incorporated into the peptide alkaloids and specifically labelled the ergoline moiety of ergotamine. These results are the same as previously found for the biosynthesis of other ergot alkaloids. Time-course incubation experiments demonstrated that l-[U-(14)C]phenylalanine, l-[U-(14)C]proline and l-[U-(14)C]alanine were incorporated into the peptide ergot alkaloids. Chemical degradation of the radioactive alkaloid derived from additional precursor incubation experiments showed that phenylalanine and proline function as the most efficient precursors, and specifically label the constitutive side-chain phenylalanyl and prolyl moieties of the alkaloid. The evidence obtained from l-[U-(14)C]alanine-incorporation experiments was inconclusive. However, degradation of ergotamine isolated after incubation with dl-[1-(14)C]alanine, showed that the carboxyl group of the labelled amino acid was specifically incorporated into the alpha-hydroxy-alpha-amino acid residue of the alkaloid. This, in conjunction with the l-[U-(14)C]alanine-incorporation results, showed conclusively that all three carbon atoms of alanine were incorporated as a biosynthetic unit into the alpha-hydroxy-alpha-amino acid moiety of ergotamine.

Production of peptide ergot alkaloids in submerged culture by three isolates of Claviceps purpurea.

Three strains of Claviceps purpurea (Fr.) Tul., isolated from sclerotia grown on rye, produce under submerged conditions ergocryptine and ergotamine, ergocornine and ergosine, and ergocristine, respectively. All of the strains either lacked the ability to produce conidia or formed them sparingly, but they accumulated large quantities of lipids and sterols. The fermentations are typically divided into two phases. The first is characterized by the rapid utilization and exhaustion of the phosphate contained in the medium, rapid uptake of ammonium nitrogen and of citric acid, rapid growth, and low alkaloid production; the second phase is characterized by slower growth and by a marked accumulation of lipids, sterols, and alkaloids.

Ergotamine production in submerged culture and physiology of Calviceps purpurea.

Strain 275 FI of Claviceps purpurea, which produces large amounts of peptide alkaloids in submerged culture, and strains V, C, and W, spontaneously obtained from 275 FI and practically unable to produce alkaloids, were compared. Strain 275 FI differs from the other strains in its capacity to accumulate lipids and sterols, as well as in its capacity to produce alkaloids. Strain 275 FI utilizes large quantities of sucrose and citric acid simultaneously; strain V utilizes large amounts of sucrose but little citric acid; strain C utilizes large quantities of citric acid but only small amounts of sucrose; strain W consumes only small amounts of both substances. We conclude that the production of large quantities of alkaloids, as well as the accumulation of lipids and sterols, is correlated with the simultaneous utilization of large amounts of sucrose and citric acid.

Heterokaryosis and alkaloid production in Claviceps purpurea.

Strain 275 FI of Claviceps purpurea, which produces large amounts of peptide alkaloids in submerged culture, is a heterokaryon; after several generations on agar media, it segregates the single components. These components (by us labeled V, C, and W) are stable and produce almost no alkaloids under described conditions of submerged culture. The mycelium of strain 275 FI consists of hyphae with multinucleate cells and does not produce conidia. Strains V, C, and W form numerous anastomoses when grown together on agar. By combining strains V and C, a heterokaryon similar to 275 FI in appearance has been obtained. This new strain produces amounts of alkaloids much larger than those produced by V and C separately or in associated submerged culture. We conclude, therefore, that in strain 275 FI the heterokaryotic condition is favorable to the production of alkaloids. Several conidiaproducing cultures of C. purpurea of various origins, as well as sclerotia of the same species, have been examined. The results demonstrated that the heterokaryotic condition is rare in the cultures, but it is frequent in the mycelium from sclerotia. Since it is known that the production of alkaloids is typical of the sclerotial phase in C. purpurea, it is suggested that this capacity is related to the heterokaryosis of the producing strains.