Culture by Three Isolatesof Claviceps Purpurea

Home , Claviceps purpurea, Ergocristine, Ergocryptine

APPLIED MICROBIOLOGY, Sept. 1969, p. 464-468 Vol. 18, No. 3 Copyright © 1969 American Society for Microbiology Printed in U.S.A. Production of Peptide Ergot Alkaloids in Submerged Culture by Three Isolates of Claviceps purpurea

A. M. AMICI, A. MINGHEITI, T. SCO1TI, C. SPALLA, AND L. TOGNOLI Istituto Ricerche, Farmitalia, Milano, Italy Received for publication 7 April 1969

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.

High yields of lysergic acid alkaloids in sub- NaOH; sterilization was achieved by heating at 110 C merged culture were first obtained in 1961 by for 20 min. Arcamone et al. (6), who described the produc- Medium Pep3 (peptone-sucrose) contained (g per liter): sucrose, 300; peptone, 10; KH2PO4, 0.5; tion of a-hydroxyethyl-lysergamide by a strain of MgSO4-7H20, 0.5; FeSO4 7H20, 0.007; ZnSO4 Claviceps paspali (Fr.) Tul. Subsequently, other 7H20, 0.006; agar, 18; and tap water to a volume of authors (8, 13) obtained the same compound 1,000 ml. The pH was not adjusted and sterilization with different strains of C. paspali. Later, Kobel was achieved by heating at 110 C for 20 min. et al. (11) described the production of 6-methyl- Medium TS contained (g per liter): sucrose, 100; ergol-8-ene-8-carboxylic acid by different strains asparagine, 10; KH2PO4, 0.5; MgSO4 7H20, 0.3; of the same species. In 1966, Amici et al. (2) FeSO4 7H20, 0.007; ZnSO4 7H20, 0.006; yeast ex- described the production in high yield of ergota- tract, 0.1; and distilled water to a volume of 1,000 mine by a submerged culture of a strain of C. ml. The pH was adjusted to 5.2 with NaOH, and sterilization was achieved by heating at 110 C for purpurea. Studies on the physiology, genetics, 20 min. and metabolism of the same strain were also Medium TG contained (g per liter): glucose, 100; published (3-5). citric acid, 10; KH2PO4, 0.5; MgSO4- 7H20, 0.3; In 1967, we isolated three strains of C. pur- FeSO4 7H20, 0.007; ZnSO4 7H20, 0.006; yeast ex- purea that produce large amounts of ergocryptine tract, 0.1; and distilled water to a volume of 1,000 ml. and ergotamine, ergocornine and ergosine, and The pH was adjusted to 5.2 with aqueous ammonia; ergocristine in submerged culture. This paper sterilization was achieved by heating at 120 C for deals with a description of these strains and of 20 min. their production processes. Medium TV contained (g per liter): sucrose, 100; Vegedor, 10; KH2PO4, 0.5; MgSO4 7H2O, 0.3; FeSO4 7H20, 0.007; ZnSO4 7H20, 0.006; and dis- MATERIAL AND METHODS tilled water to a volume of 1,000 ml. The pH was not Culture media. Medium CZ 4M contained (g per adjusted; sterilization was achieved by heating at 120 liter): glucose, 40; Vegedor (a vegetable extract pro- C for 20 min. duced by Liebig, Italy), 1; (NH4)2HP04, 5; K2HPO4, Medium T25 contained (g per liter): sucrose, 300; 1; MgSO4 7H20, 2.5; KCl, 0.5; FeSO4 7H20, 0.01; citric acid, 15; KH2PO4, 0.5; MgSO4 7H20, 0.5; KCl, ZnSO4- 7H20, 0.01; agar, 18; and distilled water to a 0.12; FeSO4 7H20, 0.007; ZnSO4 7H20, 0.006; yeast volume of 1,000 ml. The pH was not adjusted and extract, 0.1; and tap water to a volume of 1,000 ml. sterilization was achieved by heating at 110 C for The pH was adjusted to 5.2 with aqueous ammonia; 20 min. sterilization was achieved by heating at 120 C for Medium TS5 contained (g per liter): sucrose, 100; 20 min. asparagine, 5; KH2PO4, 0.25; MgSO4 7H20, 0.15; Methods of growth. A portion (1 cm2) of the my- yeast extract, 0.05; agar, 18; and distilled water to a celial mat of each strain, grown on slants of a solid volume of 1,000 ml. The pH was adjusted to 5.2 with medium at 28 C for 8 days, was mashed with a sterile 464 VOL. 18, 1969 PEPTIDE ERGOT ALKALOIDS 465

TABLE 1. Products of alkaline and acid a field near Brunico, South Tyrol, Italy. It some- hydrolysis of ergot alkaloids times produces conidia on certain solid-culture Alkaloids Alkaline hydrolysis Acid media. Normally, it is maintained on medium hydrolysis TS5 on which it never produces conidia. For alkaloid production with this strain, medium TG Ergotamine Lysergic acid + pyru- Proline + phenylala- vic acid nine is employed for the inoculum phase and medium Ergosine Lysergic acid + pyru- Proline + leucine T25 is used for the production phase. Alkaloid vic acid production reaches about 1,800 j4g/ml. The Ergocryptine Lysergic acid + di- Proline + leucine alkaloids consist of a mixture of practically methylpyruvic acid Ergocornine Lysergic acid + di- Proline + valine equal amounts of ergocryptine and ergotamine. methylpyruvic acid The course of a typical fermentation is shown in Ergocristine Lysergic acid + di- Proline + phenylala- Fig. 1. methylpyruvic acid nine Strain FI 43/14 of C. purpurea was isolated from a sclerotium collected from an ear of rye spatula and transferred to a 300-ml Erlenmeyer flask grown near Usseglio, Piedmont, Italy, and was containing 50 ml of an inoculum medium. The flasks maintained on medium CZ4M. It does not nor- were incubated for 6 days at 24 C on a rotary shaker mally form conidia on the usual solid-culture operating at 220 rev/min, and describing a circle media. For alkaloid production, medium TS is 8 cm in diameter. Samples (5 ml) of the cultures employed for the inoculum phase and medium thus obtained were employed as inocula for 300-ml T25 is used for the production phase. The alka- flasks, each containing 50 ml of a production medium. loids reach a level of about 1,000 to 1,100 pig/ml These were incubated for 10 to 14 days, according to within 12 to 14 days and consist of almost equal the strain, as described for the inoculum cultures. and Methods of analyses. Alkaloids, sterols, sugars, dry quantities of ergocornine ergosine. The weight, lipids, protein nitrogen, and citric acid were course of a typical fermentation is shown in Fig. determined by the methods of Amici et al. (3). All of 2. the strains studied were found to retain more than half Strain Fl S40 of C. purpurea was isolated from of the alkaloids produced in their mycelia. a sclerotium collected from a ear of rye grown in Extractions of the alkaloids were carried out by adding to the culture broths the equivalent volume of an aqueous solution of 4% tartaric acid and two volumes of acetone. After a careful homogenization, the mixture was filtered and its pH was adjusted to 8.5 with saturated Na2CO3 solution; alkaloids were then extracted with chloroform. The solvent was removed under vacuum at 30 C and the crude chloroform extract was passed through a silica-gel column. Elution was then carried out with increasing amounts of methanol to separate the alkaloids, which were later purified and crystallized in the proper solvent (10). The alkaloids were identified by thin-layer chromatography by using silica-gel plates with the following solvents: ethyl acetate-N, N-dimethylformamide-ethyl alcohol [13:1.9:0.1 (12)] or chloroform-methanol- concentrated ammonia [80:20:0.2 (1)]. Identification was also made by paper chromatography by using Whatman No. 1 paper soaked with formamide in benzene-petroleum ether [6:4 (9)]. The identity of the alkaloids was further confirmed by comparing their rotatory powers (10), melting points (10), and mass spectrograms with those of authentic samples. They were also subjected to acid and alkaline hydrol- yses, and the degradation products obtained were identified and compared with the data reported in the literature (10). The results are summarized in Table 1. RESULTS Strain F1 32/17 of C. purpurea was isolated FIG. 1. Course of a fermentation on medium T25 in from a sclerotium collected from an ear of rye in submerged culture of strain Fl 32/17 of C. purpurea. 466 AMICI ET AL. APPL. MICROBIOL. terized by the fact that, under our conditions, they do not form conidia or, at best, that they produce very few of them. This behavior was E~~~~~~~~~ investigated and discussed in previous studies (4, 14) in which it was also established that alka- /E°cm-9 stEl X E loid production is correlated with the hetero- karyotic condition of the producing strains. This condition is not normally compatible with the -0105 presence of mononucleate forms, such as conidia, since segregation of the single nuclei and conse- quent disjunction of the heterokaryon occurs j.5 during the sporulation. cm~~~~~~~~~~~~2815 I ~ .. / / 5....0200251 Of the four strains mentioned in Table 2, three produce alkaloids in medium T25; only one needs medium TS. All of the four strains 24085400EE13V~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~112lt00 \ - H lipids || elaborate high concentrations of lipids and 200 401000 ' c*itri 481020j 11l sterols. In a previous study with ergotamine-producing strains, a positive correlation was found between the two characteristics described above 20 alkaloids 60 .0302 i5 and the alkaloid production (3). The problem was studied further by determining the lipid and I0 acidtIklod sterol content of five strains of C. purpurea, freshly isolated from sclerotia and grown on 125600* inorganic P / .. prten medium T25 under submerged conditions, which failed to synthetize the alkaloids. The lipid content of the mycelium of these strains varied 0 2 4 6 8 10 12 14 days between 12.1 and 19.3%. (In the four alkaloid- producing strains, it ranged from 39 to 69%7.) FIG. 2. Course of a fermentation on medium T25 in The sterol production varied between 48 and 166 submerged culture of strain Fl 43/14 of C. purpurea. Ag/ml. (The four alkaloid-producing strains syn- thesized them in concentrations of 316 to 510 the Goms district of the Rhone Valley, Switzer- ,4g/ml). The correlation between alkaloid bio- land and was maintained on Pep3 medium. It synthesis and the accumulation of lipids and does not generally form conidia on the common solid-culture media. For alkaloid production by this strain, medium TV is employed for the inoculum phase and medium TS is used for the production phase. About 1,000 to 1,100 ,ug of ergocristine per ml of culture is produced within 8 to 10 days. The course of a typical fermentation is shown in Fig. 3. DISCUSSION Of the peptide ergot alkaloids, only ergotamine (2-5) had been produced in large amounts under submerged conditions. Our data demonstrate that ergosine, ergocryptine, ergocornine, and ergocristine can also be formed under the same submerged conditions. It is possible, therefore, to produce large amounts of the alkaloids belong- ing to the two important groups of peptide alkaloids, the ergotamine group and the ergotoxine group, with C. purpurea. A comparison between the main characteristics of the strains described in this paper and those of strain 275 Fl, the producer of ergotamine described in preceding papers (2-5), is sum- FIG. 3. Course of a fermentation on medium TS in marized in Table 2. All of the strains are charac- submerged culture of strain FI S40 of C. purpurea. VOL. 18, 1969 PEPTIDE ERGOT ALKALOIDS 467 TABLE 2. Comparison among the main characteristics of the strains of Claviceps purpurea producing peptide alkaloids in shaken cultures

Strains Characteristic 275 FI (2) FI 32/17 FI 43/14 FI S40

Conidia ...... Absent.. Sometimes pres- Generally absent Generally ab- ent sent Alkaloids produced ...... Ergotamine Ergocryptine and Ergocornine and Ergocristine ergotamine ergosine Alkaloids (Iug/ml) ...... 1,150 1,800 1,100 1,000 Production media ...... T25 T25 T25 TS Dry weight (mg/ml) ...... 38.5 40 68 33 Lipids (mg/ml) ...... 15 24 47 18.5 Lipids (per cent of dry weight). 39 60 69 54 Protein nitrogen (mg/100 ml) .. 92 89 149 88 Sterols (sg/ml) ...... 316 444 393 510 Sucrose utilized (g/liter) ...... 160 75 145 100 Citric acid utilized (g/liter) 14 15 6 Ammonium nitrogen utilized (mg/100 ml)...... 246 227 203 sterols in C. purpurea seems, therefore, to be a the exhaustion of one of the substances present general rule. in the medium (7). This seems plausible because For a more detailed examination of the course it is known that P or Mg exhaustion causes an of the fermentations, strains F1 32/17 and FI accumulation of lipids (7), and the importance 43/14 were considered apart from strain F1 of the initial P concentration in the medium for S40. The fermentation process in the first two alkaloid production has been demonstrated by strains could be separated into two phases. The Taber and Vining (15) and by Arcamone et al. first one is characterized by the rapid utilization (5). The latter authors demonstrated that alka- and exhaustion of the phosphate contained in loid production is favored by growth-limiting the medium, by the rapid uptake of ammonia phosphate concentrations and concluded that nitrogen, citric acid, and (in strain Fl 32/17) "during the second phase of growth the reduc- sucrose. In this phase, there is rapid growth and tion of protein synthesis should make available low production of alkaloids. The second phase, the simple nitrogenous precursors (i.e. amino which begins between 4 and 6 days, is character- acids) for LAD (lysergic acid derivatives) synthe- ized by slower growth (particularly evident if the sis." We believe this conclusion to be valid also protein nitrogen is taken as an index of growth) for the strains described in this paper. and by a marked tendency to accumulate lipids, sterols, and alkaloids. An analogous pattern has ACKNOWLEDGMENTS been reported for strain 275 F1 incubated in We gratefully acknowledge the support and interest of B. flasks (3) and in fermentors (5). In the latter case, Camerino throughout these investigations. We also thank A. a third phase, characterized by the absence of Giangualano and A. Pavesi for technical assistance. ammonia nitrogen in the medium, was also described during which there is only a slight LITERATURE CITED increase in the dry weight and little utilization of 1. Agurell, S. 1965. Thin-layer chromatographic and thin-layer carbohydrates; there is, however, further accumu- electrophoretic analysis of ergot alkaloids. Relations between structures, RM value and electrophoretic mobility lation of alkaloids. This phase has not been in the clavine series. Acta Pharm. Suec. 2:357-374. taken into consideration in fermentations carried 2. Amici, A. M., A. Minghetti, T. Scotti, C. Spalla, and L. out in flasks because in these cases there is not a Tognoli. 1966. Production of ergotamine by a strain of significant increase in alkaloids. Although the Claviceps purpurea (Fr.) Tul. Experientia 22:415-416. a 3. Amici, A. M., A. Minghetti, T. Scotti, C. Spalla, and L. data for strain FL S40 are not as complete, Tognoli. 1967. Ergotamine production in submerged cul- growth phase and an alkaloid production phase ture and physiology of Claviceps purpurea. Appl. Micro- can be distinguished. The first phase is character- biol. 15:597-602. ized by the rapid utilization and exhaustion of the 4. Amici, A. M., T. Scotti, C. Spalla, and L. Tognoli. 1967. Heterokaryosis and alkaloid production in Claviceps pur- phosphate contained in the medium. purea. Appl. Microbiol. 15:611-615. In all of the strains, the passage from the first 5. Arcamone, F., G. Cassinelli, G. Ferni, S. Penco, P. Pennella, to the second phase is probably determined by and C. Pol. 1968. Ergotamine production and metabolism 468 AMICI ET AL. APPL. MICROBIOL.

of Claviceps purpurea strain in stirred fermenters. Third In- 11. Kobel, H., E. Schreier, and J. Rutschmann. 1964. 6-Methyl- ternational Fermentation Symposium, New Brunswick, N.J. A8.9-ergolen-8-carbonsaure, ein neues Ergolinderivat aus 6. Arcamone, F., E B. Chain, A. Ferretti, A. Minghetti, P. Kulturen eines Stammes von Claviceps paspali Stevens et Pennella, A. Tonolo, and L. Vero. 1961. Production of a Hall. Helv. Chim. Acta 47:1052-1064. new lysergic acid derivative in submerged culture by a 12. McLaughlin, J. L., J. E. Goyan, and A. G. Paul. 1964. Thin- strain of Claviceps paspali Stevens and Hall. Proc. Roy. layer chromatography of ergot alkaloids. J. Pharm. Sci. Soc. Ser. B Biol. Sci. 155:26-54. 53:306-310. 7. Borrow, A., E. G. Jefferys, R. H. J. Kessell, E. C. Lloyd, 13. Pacifici, L. R., W. J. Kelleher, and A. E. Schwarting. 1962. P. B. Lloyd, and I. S. Nixon. 1961. The metabolism of Production of lysergic acid derivatives in submerged cul- Gibberella fujikuroi in stirred culture. Can. J. Microbiol. ture. L. Fermentation studies. Lloydia 25:37-45. 7:227-276. 14. Spalla, C., A. M. Amici, T. Scotti, and L. Tognoli. 1968. 8. Groger, D., and V. E. Tyler, Jr. 1963. Alkaloid production Heterokaryosis of alkaloid producing strains of Claviceps by Claviceps paspali in submerged culture. Lloydia 26: purpurea in saprophytic and parasitic conditions. Third 174-191. International Fermentation Symposium, New Brunswick, 9. Hais, J. M., and K. Macek. 1963. Paper chromatography, N.J. p. 591, Academic Press Inc., New York. 15. Taber, W. A., and L. C. Vining. 1958. The influence of cer- 10. Hofmann, A. 1964. Die Mutterkornalkaloide, p. 19-27, 75. tain factors on the in vitro production of ergot alkaloids by F. Enke Verlag, Stuttgart. Claviceps purpurea (Fr.) Tul. Can. J. Microbiol. 4:611-626