Vol. 37, No. 3

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1979, p. 365-368

0099-2240/79/03-0365/04$02.00/0

Production of Mycophenolic Acid by Penicillium roqueforti Strains PHILIPPE LAFONT,'* JEAN-PAUL DEBEAUPUIS,' MICHEL GAILLARDIN,'

AND

JACQUES

PAYEN2

LN.S.E.R.M., Unite de Recherche de Toxicologie Alimentaire, 78110 Le Vesinet,' and E.N.S.A.IA., Laboratoire de Micro biologie, 54000 Nancy,2 France Received for publication 21 December 1978

Sixteen strains of Penicillium roqueforti Thom, isolated from blue-molded cheeses, were studied. In vitro, all of these strains produced mycophenolic acid, some on the order of 0.8 to 4 mg/g of dry culture. The greatest yields were obtained after 10 days of incubation of cultures at 15°C. However, under some experimental conditions, mycophenolic acid was not alone responsible for the toxicity of culture extracts to chicken embryos.

In a previous study of in vitro toxigenesis of Penicillium roqueforti isolated from bluemolded cheeses, we have shown that there is a great heterogeneity between strains in terms of toxin synthesis (7). The diversity of the data from three assay systems (chicken embryo developmental inhibition, cell culture toxicity, and mouse lethality) clearly showed that various fungal metabolites are involved. PR toxin, a sesquiterpene studied by Wei et al. (16, 17), and isofumigaclavine A and roquefortine, alkaloids isolated from P. roqueforti cultures (10, 14, 15), are not involved in this experimental toxicity.

Continuing our work, we have identified one of the metabolites responsible for the inhibition of chicken embryo and cell culture development as mycophenolic acid. Mycophenolic acid, although not previously described in extracts of P. roqueforti strains, has long been recognized as an antibiotic substance for bacteria and dermatophytic fungi; it interferes also with viral multiplication (11). For mammals, the toxicity of this product seems to be low. The 50% lethal doses per os are 2,500 mg/kg in rats and 700 mg/kg in mice. The intravenous 50% lethal doses are 550 and 450 mg/kg, respectively (18). Daily oral doses of 80 and 320 mg/kg for 1 year do not give apparent signs of toxicity in rabbits (1). However, in rats, oral administration of daily doses of the order of 30 mg/kg brings about anemia, and death follows several weeks later. Monkeys receiving 150 mg/kg develop abdominal pains, diarrhea with blood, and anemia after 2 weeks of feeding (18). MATERLALS AND METHODS Organisms. Sixteen strains of P. roqueforti Thom (13) were used. Two strains (221-30 and 280-67), originally isolated from French Roquefort cheese, were

obtained from the Centraalbureau voor Schimmelcultures, Baarn. We isolated the others from samples of Roquefort cheeses and of various French, Italian, or German blue cheeses (the origin of each strain is reported in Table 1). Stock cultures were prepared on modified Czapek medium (3) incubated at 25°C and stored at 4 to 50C. Conidia from 10-day-old subcultures, started from a stock culture and grown at 25°C, served as inocula for metabolite production. Mycophenolic acid production. Four media were tested for suitability as substrates: medium I was Czapek solution, with dextrose, modified according to Brian et al. (3), supplemented with 1% asparagine; medium II was a semisynthetic medium used for the production of a variety of mycotoxins (8), with Difco Casamino Acids as the nitrogen source; medium III was a yeast extract medium (2% yeast extract and 5% sucrose); and medium IV was curdled milk. Portions of medium (100 ml) in 500-ml flasks were inoculated with 2 x 106 to 2 x 107 spores, according to a method previously described by Lafont and Lafont (6). Surface cultures were incubated at 15 or 25°C without shaking, in the dark. Culture extracts. After incubation, cultures (mycelium and medium) were extracted three times with 1.5 parts of chloroform at 63°C, for 1 h. The chloroform extracts were decanted, filtered, pooled, and concentrated in vacuo. Isolation of mycophenolic acid. The chloroform crude extract was applied to a 5-cm-diameter chromatography column packed with 100 g of silica gel (Mallinckrodt; 100 mesh). The elution was performed on a linear gradient of chloroform-methanol (98:2, vol/vol, to 70:30, vol/vol), and 13-ml fractions were collected. The purification of one of the toxic metabolites, mycophenolic acid, required the application of fractions no. 25 to 35 to a second column (60 g of Mallinckrodt silica gel, 100 mesh, in a 2.2-cm-diameter column; elution with a linear gradient of n-hexaneacetone [9:1, vol/vol, to 6:4, vol/vol]). Mycophenolic acid was crystallized at 0°C from n-hexane-acetone. Mycophenolic acid assays by thin-layer chro-

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TABLE 1. Production of mycophenolic acid by 16 strains of P. roqueforti Mycophecd nolic acida Originnlc (mg/g of dry

Strain no. 221-30 280-67 549 553-2 603-A 616-A 596-A 596-D

CBSb

CBS Roquefort cheese Roquefort cheese Roquefort cheese Roquefort cheese Gorgonzola cheese French blue cheese (fourme d'Ambert) 596-E French blue cheese (fourme d'Ambert) 618-B French blue cheese (industrial) 618-A French blue cheese (industrial) 622 French blue cheese (industrial) 621 French blue cheese (industrial) 620 French blue cheese (industrial) French blue cheese (industrial) 619 596-F German blue cheese (industrial) a In 10-day cultures at 15°C, on medium I. b CBS, Centraalbureau voor Schimmelcultures,

culture) 0.090 0.870 1.725 0.0014 4.060 0.520 0.007 0.018 0.004 0.021 0.003 0.011 0.052 0.024 0.013 0.240

Baam.

matography. Successive dilutions of samples were chromatographed on glass plates precoated with silica gel (Merck, type 60, 0.25 mm thick). The developing system was water-saturated ethyl acetate-toluene (65: 35, vol/vol). Chromatograms were exposed to ammonia and observed under ultraviolet light (Hanovia Lamps, Slough, England). Quantitative estimation was performed by fluorodensitometric measurement (Photometer PHI 5, Vernon S.A., Paris; Hg source; excitation wavelength, 254 nm; emission wavelength, 410 to 470 nm). Standards were crystallized mycophenolic acid obtained from a Penicillium brevi-compactum strain. Assays on chicken embryos. According to the method of Lafont and Frayssinet (5), fertile eggs were inoculated, before incubation, with aqueous ethanol (50:50, vol/vol) solutions of: (i) crude extracts evaporated to dryness; (ii) chromatographic fractions; or (iii) crystallized mycophenolic acid. Quantitative estimations of toxicity were performed by the determination of 50% lethal dose (inoculation of successive dilutions in a series of 12 eggs; average + standard deviation for six assays by the probit transformation). The results were calculated in taking into account the hatchability of eggs receiving an equal volume (20 pl) of aqueous ethanol (for 630 eggs it was 87.22 ± 8.33%).

RESULTS Identification of a metabolite from P. roqueforti as mycophenolic acid. The toxic effects on chicken embryos of extracts from cultures not older than 10 days were directly linked with the presence of a single substance (see Fig. 2). The elution volume of this substance, in the n-hexane-acetone chromatographic system, was about 120 ml. On thin-layer chromatography it

was revealed as a blue spot with ferric chloride and as a pale-red spot with bisdiazoted benzidine. It gave colorless crystals with a melting point of 1410C. The mass spectrum showed a molecular ion peak at m/e 320. Its infrared spectrum had bands at 3,400 cm-' (free hydroxyl group), 3,200 to 2,900 cm-' (OH stretching), 1,720 and 1,685 cm-' (C=O stretching), 1,610 and 1,420 cm-' (ethylenic insaturation), and 1,200 to 950 cm-' (substitutions on aromatic ring). These characteristics are in agreement with the molecular structure of the mycophenolic acid (2). For chicken embryos, the 50% lethal dose was 1 ± 0.1 ,Ag (value calculated for 216 eggs). By the fluorometric method, the detection limit of mycophenolic acid was 0.03 ,ug; in assays, quantitative estimation was possible as far as 0.15 ,ug with an error lower than 6%. Mycophenolic acid production in surface culture. The level of mycophenolic acid in cultures of strain CBS 280-67 in medium I was determined daily during a 2-week incubation period at 150C. The production ofthe metabolite was noted at a very early stage of the culture. Its concentration reached a maximum at day 10 and decreased thereafter (Fig. 1). The same time scale was observed with three other strains

E0.75 0

0.2500

Days

FIG. 1. Production of mycophenolic acid by strain CBS 280-67 at 15°C on medium I.

VOL. 37, 1979

MYCOPHENOLIC ACID PRODUCTION BY P. ROQUEFORTI

(603A, 616B, and 549). In the case of strains 5532, 596A, and 596E, which exhibited a lower production, the maximum level was noted at 15 to 20 days of incubation at 15°C. The data in Table 1 show that all 16 strains studied produced mycophenolic acid, but only 4 produced it in relatively large quantities. Three of the latter strains were cultured concurrently on media I, II, III, and IV at 15°C. The levels of mycophenolic acid were similar in cultures grown on synthetic or semisynthetic media, but were much lower with curdled milk as a substrate (Table 2). Relation between mycophenolic acid level and toxicity of culture extract. Two or three flasks of culture of strain CBS 280-67 were extracted at 24-h intervals from 6 to 15 days. The extracts were assayed for mycophenolic acid TABLE 2. Production of mycophenolic acid by cultures on different media Strain

Mycophenolic acid'

Medium

603

(yg/g of medium)

81.0 60.7 74.8 IV 9.20 I 549 48.6 II 54.6 III 63.5 IV 1.62 280-67 I 26.7 II 25.2 III 21.7 IV 0.64 a Data obtained after incubation for 10 days at 15°C. I II III

367

level by fluorodensitometry and for toxicity by the chicken embryo test. The results (Fig. 2) were obtained from six separate analyses. For 6to 10-day-old cultures grown at 15°C, the toxicity of the crude extract, as revealed in the biological test, was that predicted on the basis of the mycophenolic acid content of the extract. On the other hand, with older cultures, the differences between the observed results of the biological and physical tests were evident, indicating that the observed toxic effects may be attributed only partially to the presence of mycophenolic acid. Similar observations were made in studying four other strains (221-30, 549, 603A, and 616A). For 15-day-old cultures grown at 15°C, irrespective of the strain and its capacity to produce mycophenolic acid, the ratio between the toxicity for chicken embryos of the crude extract and that of the mycophenolic acid alone, present in the extract, had an approximate value of 3 (Fig. 3). On the other hand, cultures developed at 25°C did not produce toxic metabolite other than mycophenolic acid. DISCUSSION Mycophenolic acid is a metabolite identified in extracts of various Penicillium species (or series): P. stoloniferum, P. brevi-compactum, P. bialowiezense, and P. viridicatum (18). According to Raper and Thom (12), the first three species (or series), as well as P. roqueforti, are classed in the P. asymmetrica-velutina section. The demonstration of mycophenolic acid production by the latter indicates metabolic resemblances, but we noted that the quantity of the

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XI'

a

E -av

C

I 0

.V

x

x 7

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Cutu incubdton time (days) FIG. 2. Production of mycophenolic acid and evolution of the toxicity of the culture crude extract to chicken

embryos.

APPL. ENVIRON. MICROBIOL.

LAFONT ET AL.

368

This work was supported in part by contract no. 77-12 from the Ministere de la Qualite de la Vie.

I I I

I

0t lai

It

I8

10d

15d.

15iC

1Od

15d (ae)

25-C

(tenperatur)

FIG. 3. Relation between toxicity of the crude extract and that due to the mycophenolic acidpresent in the extract. Data refer to strains CBS 280-67, CBS 221-30, 549, 603A, and 616A. Cultures at 15°C and 25°C; extractions after 10 and 15 days. Bars indicate standard deviation calculated for six assays with each strain.

metabolite obtained with the strains used in our experiments was lower than that synthesized by P. brevi-compactum (4, 9). On the other hand, some strains of P. brevi-compactum have been reported to produce no mycophenolic acid (4), whereas all of the P. roqueforti strains seem able to do so. According to the results reported here, the nature of the substrate on which the fungi grow plays a role in mycophenolic acid biosynthesis. In vitro the yield on curdled milk is poor. We have applied the fluorodensitometric assay system to analysis of marketed blue-molded cheeses (P. Lafont, M. G. Siriwardana, I. Combemale, and J. Lafont, Food Cosmet. Toxicol., in press); 38% of the studied samples were positive, but only 3% were at levels higher than 10 mg/kg. A correlation was noted between the intensity of in vitro mycophenolic acid production by the P. roqueforti strain used in ripening and the level of the fungal metabolite detected in the cheese. ACKNOWLEDGMENTS We are very grateful to J. M. Ziegler, Service de Spectrometrie de Masse, Faculte des Sciences Pharmaceutiques et Biologiques, Universite de Nancy I, Nancy, France, for taking the mass spectrum. We thank J. Sarfati for her help in preparing the fungal cultures.

LITERATURE CITED 1. Adams, E., G. Tood, and W. Gibson. 1975. Long-term toxicity study of mycophenolic acid in rabbits. Toxicol. Appl. Pharrnacol. 34:509-512. 2. Birkinshaw, J. H., H. Raistrick, and D. J. Ross. 1952. Studies in the biochemistry of micro-organisms. The molecular constitution of mycophenolic acid, a metabolic product of Penicillium brevi-compactum Dierckx. Part 3. Further observations on the structural formula for mycophenolic acid. Biochem. J. 50:630-634. 3. Brian, P. W., A. W. Dawkins, J. F. Grove, H. G. Hemming, D. Love, and G. L. Norris. 1961. Phytotoxic compounds produced by Fusarium equiseti. J. Exp. Bot. 12:1-12. 4. Clutterbuck, P. W., A. E. Oxford, H. Raistrick, and G. Smith. 1932. The metabolic products of the P. brevicompactum series. Biochem. J. 24:1441-1458. 5. Lafont, J., and C. Frayssinet. 1969. Mycotoxines elaborees par des Aspergillus. Leur activite sur l'embryon de poulet. C. R. Soc. Biol. 163:1362-1364. 6. Lafont, P., and J. Lafont. 1970. Production d'aflatoxine par AspergiUus flavus Link en culture statique. Ann. Inst. Pasteur 118:340-348. 7. Lafont, P., J. Lafont, J. Payen, E. Chany, G. Bertin, and C. Frayssinet. 1976. Toxin production by 50 strains of Penicillium used in the cheese industry. Food Cosmet. Toxicol. 14:137-139. 8. Lafont, P., M. G. Siriwardana, M. Gaillardin, and J. Lafont. 1977. Production d'hydroxy-aflatoxines par des cultures de Aspergillus du groupe flavus. Ann. Microbiol. 128:221-228. 9. Nulton, C. P., and I. M. Campbell. 1977. Mycophenolic acid is produced during balanced growth of Penicillium brevi-compactum. Can. J. Microbiol. 23:20-27. 10. Ohmomo, S., T. Sato, T. Utagawa, and M. Abe. 1975. Isolation of festuclavine and three new indole alkaloids, roquefortine A, B and C from the cultures of Penicillium roqueforti. XII. Production of alkaloids and related substances by fungi. J. Agric. Chem. Soc. Jpn. 49:615623. 11. Planterose, D. N. 1969. Antiviral and cytotoxic effects of mycophenolic acid. J. Gen. Virol. 4:629-630. 12. Raper, K. B., and C. Thom. 1968. A manual of Penicillia, p. 336418. Hafner Publishing Co., New York. 13. Samson, R. A., C. Eckardt, and R. Orth. 1977. The taxonomy of Penicillium species from fermented cheeses. Antonie van Leeuwenhoek J. Microbiol. Serol. 43:341-350. 14. Scott, P. M., B. P. C. Kennedy, J. Harwig, and B. J. Blanchfield. 1977. Study of conditions for production of roquefortine and other metabolites of Penicillium roqueforti. Appl. Environ. Microbiol. 33:249-253. 15. Scott, P. M., M. A. Merrien, and J. Polonsky. 1976. Roquefortine and isofumigaclavine A, metabolites from Penicillium roqueforti. Experientia 32:140-141. 16. Wei, R. D., H. K. Schnoes, P. A. Hart, and F. M. Strong. 1957. The structure of PR-toxin, a mycotoxin from PeniciUium roqueforti. Tetrahedron 31:109-114. 17. Wei, R. D., P. E. Still, E. B. Smalley, H. K. Schnoes, and F. M. Strong. 1973. Isolation and partial characterization of a mycotoxin from Penicillium roqueforti. Appl. Microbiol. 25:111-114. 18. Wilson, B. J. 1971. Miscellaneous PeniciUium toxins, p. 459-521. In A. Ciegler, S. Kadis, and S. J. Ajl (ed.), Mirwibial toxins, vol. VI. Academic Press Inc., New York.