VOL. XXXIX NO. 7 THE JOURNAL OF 1011

PRODUCTION OF times. The extract was concentrated and passed (PHOSPHONOMYCIN) through a carbon column with water. The BY PSEUDOMONAS SYRINGAE effluent was concentrated and applied to a Biogel P-2 column with water. The active eluate JUN'ICHI SHOJI*, TOSHIYUKI KATO, from the column was freeze-dried to give a glassy HIROSHI HINOO, TERUO HATTORI, residue (3 g). It was chromatographed on a KEIICHIRO HIROOKA, KOICHI MATSUMOTO, DEAE-Sephadex column in linear gradient man- TATSUO TANIMOTOand EIJI KONDO ner with water and 0.4 M NH4HCO3. The active eluate was once freeze-dried and then passed Shionogi Research Laboratories, through an Amberlite CG-50 (Na+) column with Shionogi & Co., Ltd., Fukushima-ku, Osaka 553, Japan water. Freeze-dry of the effluent gave a crude powder (230 mg) of the as a sodium (Received for publication March 7, 1986) salt. It was then purified by chromatographies on an Avicel cellulose column with 75 % propanol In recent years, it has become to be recognized followed by 75% acetonitrile. Concentration that can produce antibiotics of wide and freeze-dry of the active eluate gave a colorless structural diversity, some of which are known powder (21 mg) of the antibiotic as a sodium also as the products of Actinomycetales1~5) salt. Here we report the isolation of fosfomycin The antibiotic obtained as above is a water- (phosphonomycin)6~8) from a bacterial culture, soluble acidic substance. No UV absorption which was noticed in our screening work by was observed with an aqueous solution of the causing spheroplast formation of sodium salt. The IR spectrum (KBr) and the LS-1 (a supersensitive mutant to 0-lactam anti- 1H NMR spectrum (D 2O) were substantially biotics) in a hypertonic medium. identical with those of a commercially available The producing organism, PB-5,123, was iso- fosfomycin sodium salt (Meiji Seika Kaisha, lated from pond water in Kyoto city, and iden- Ltd.). The identity with fosfomycin was also tified as Pseudomonas syringae9~10)by the follow- shown by color reaction and TLC experiments. ing characteristics. The organism was aerobic, The same color tones were observed by reaction Gram-negative, non-sporulating rods (1.0 x with molybdate reagent. Identical mobilities 1.8 - 2.0 Elm) with rounded ends. Motility was on TLC were shown as follows: Silica gel plate observed with polar multi-trichous flagellation. (Merck); CHCl3 - MeOH - 28% ammoniacal Colonies on a nutrient agar were circular, convex, water (1:3:2), Rf 0.52; 70% propanol, Rf 0.30; smooth and shining with orange color. Glucose cellulose plate (Avicel) : butanol - acetic acid - was metabolized oxidatively. The following H2O (3: 1: 1), Rf 0.30; 70% propanol, Rf 0.40; tests gave positive results; catalase, gelatin butanol - pyridine - acetic acid - H2O (10: 6: 1: liquefaction, citrate utilization, arginine dihy- 4), Rf 0.27; 75 % acetonitrile, Rf 0.20. drolase, and formation of fluorescent pigments. Methanolysis products (2) were prepared ac- But, the following were negative; oxidase, starch cording to the method of CHRISTENSENet al.') hydrolysis, nitrate reduction, and accumulation from both our specimen and authentic fosfo- of poly-8-hydroxybutyrate. Growth was ob- mycin (1) and their optical activities were com- served at 28°C and 37°C, but not at 5°C or pared. 42°C. Product from our specimen: [a]D -5.1 _0.5°, The producing organism was cultivated by jar [a]3°, -17.3=0.6° (c 0.00909, H2O). fermentation using a medium consisting of Product from fosfomycin: [a]D -7.9±0.3°, glycerol 3.0%, glucose 0.1%, Polypeptone [a]3°, -25.3±0.4° (c 0.0157, H2O). 0.5%, beef extract 0.5%, NaCl 0.5%, under aerobic conditions for 24 hours. The antibiotic Fig. 1. in the culture filtrate (ca. 160 liters) was adsorbed H H H3CO H on a Dowex-1X2 (Cl-) column and eluted with H •PO3H2 5 yo NaCl. The active eluate was freeze-dried H3C O PO3H2 H3C H to give a powder (120 g). Extraction with methanol from the powder was repeated several I 2 1012 THE JOURNAL OF ANTIBIOTICS JULY 1986

From the above, the antibiotic produced by 1545, 1984 Pseudomonas syringae was concluded to be 6) STAPLEY,E. O.; D. HENDLIN, J. M. MATA, M. identical with fosfomycin including its stereo- JACKSON, H. WALLICK, S. HERNANDEZ, S. chemistry. MOCHALES,S. A. CURRIE & R. M. MILLER: Phosphonomycin. I. Discovery and in vitro

References biological characterization. Antimicrob. Agents Chemother. -1969, 284-290, 1970 1) LECHEVALIER,H. A.: Production of the same 7) CHRISTENSEN,B.G. ; W.J. LEANZA,T. R. BEATTIE, antibiotics by members of different genera of A. A. PATCHETT,B. H. ARISON,R. E. ORMOND, microorganisms. Adv. Appl. Microbiol. 19: F. A. KLEHL, Jr., G. ALBERS-SCHONBERG& O. 25-45, 1975 JARDETZKY: Phosphonomycin: Structure and 2) BRIGITTE,K.; H. REICHENBACH,H. AUGUSTINIAK synthesis. Science 166: 123-125, 1969 & G. HoFLE: Isolation and identification of 8) OKUIIARA, M.; Y. KURODA, K. UMEHARA,M. althiomycin from Cystobacter filscus (Myxo- KOSAKA, H. AOKI & H. IMANAKA (Fujisawa bacteriales). J. Antibiotics 35: 635636, 1982 Pharm.): Preparation of fosfomycin. Jpn. 3) SINGH, P. D.; P. C. WARD, J. S. WELLS, C. M. Kokai 54989 ('76), May 14, 1976 RICCA, W. H. TREJO, P. A. PRINCIPE & R. B. 9) BUCHANAN, R. E. & N. E. GIBBONS (Ed.): SYKES: Bacterial production of deacetoxyce- Part 7. Gram-negative aerobic rods and cocci. phalosporin C. J. Antibiotics 35: 13971399, Family 1. Pseudomonadaceae Winslow, Broad- 1982 hurst, Buchanan, Krumwiede, Rogers and Smith 4) TYMIAK,A. A.; C. A. CULVER,J. F. GOODMAN, 1917, 555. In BERGEY'SManual of Determina- V. S. SEINER & R. B. SYKES: Oxazinomycin tive Bacteriology. 8th Ed., pp. 217253, Wil- produced by a Pseudommnas species. J. Anti- liams & Wilkins Co., Baltimore, 1974 biotics 37: 416~418, 1984 10) PALLERONI,N. J.; Genus I. Pseudomonas. In 5) HARADA, S.; S. TSUBOTANI, H. ONO & H. BERGEY'S Manual of Systematic Bacteriology. OKAZAKI: Cephabacins, new cephem anti- Vol. 1, Eds., N. R. KRIEG & J. G. HOLT, pp. biotics of bacterial origin. II. Isolation and 141 - 199, Williams & Wilkins Co., Baltimore, characterization. J. Antibiotics 37: 1536~ 1984