Process for producing nocardicin A

Abstract

A new species of Nocardiopsis, designated Nocardiopsis atra Huang sp. nov. ATCC 31511, when aerobically propagated in an aqueous nutrient medium, produces nocardicin A in good yield.

Description

BACKGROUND OF THE INVENTION

Among the best known and widely used class of antibacterial agents are the so-called .beta.-lactam antibiotics. These compounds are characterized in that they have a nucleus consisting of a 2-azetidinone (.beta.-lactam) ring. When the .beta.-lactam is fused to a thiazolidine ring, the compounds are usually referred to generically as penicillins. When the .beta.-lactam is fused to a dihydrothiazine ring, the compounds are referred to as cephalosporins.

The .beta.-lactam antibiotics are synthesized, for no apparent reason, by only a few microorganisms. All of the organisms recognized to produce .beta.-lactam antibiotics are filamentous microorganisms, but not all of these microorganisms are taxonomically related. Some are fungi (eukaryotes), whereas others are streptomycetes (prokaryotes). Thienamycin, U.S. Pat. No. 3,950,357, is an interesting new .beta.-lactam antibiotic produced by a species of Streptomyces.

Nocardicin A, U.S. Pat. No. 3,923,977, is a novel .beta.-lactam antibiotic produced by Nocardia uniformis var. tsuyamanensis ATCC 21806. Nocardicin A has an unusual .beta.-lactam moiety which is uniquely different from all other .beta.-lactam antibiotics in not being fused to another ring as shown in the following structure: ##STR1##

The unique structure of nocardicin A may account for its relative stability toward a variety of .beta.-lactamases as well as for its negligible immunological cross-reactivity with penicillins and cephalosporins.

Unfortunately, the outstanding in vitro and in vivo antimicrobial properties of the penicillins and caphalosporins are not shared by nocardicin A. However, the antibiotic's nucleus, 3-aminonocardicinic acid, of the following structure has been prepared, J. Am. Chem. Soc. 100, 3933 (1978): ##STR2##

The present invention is concerned with the discovery that nocardicin A is produced by a new species of Nocardiopsis designated Nocardiopsis atra Huang sp. nov. ATCC 31511. The microorganism with its potential for cultural and mutational development offers promise of the production of large amounts of nocardicin A, subsequent cleavage to 3-aminonocardicinic acid and the synthesis of a variety of clinically useful semisynthetic nocardicins paralleling those of the semisynthetic penicillins and cephalosporins.

SUMMARY OF THE INVENTION

Nocardicin A, previously reported .beta.-lactam antibiotic, is produced by the aerobic propagation in aqueous nutrient media of a new species of Nocardiopsis designated Nocardiopsis atra Huang sp. nov. ATCC 31511.

DETAILED DESCRIPTION OF THE INVENTION

The microorganism useful for the production of nocardicin A was isolated from a coil sample from Nomozaki, Japan. This culture, designated Nocardiopsis atra Huang sp. nov., has been deposited in The American Type Culture Collection, Rockville, Md. as the type culture under their accession number ATCC 31511. The permanency of the deposit of this culture at The American Type Culture Collection at Rockville, Md. and ready accessibility thereto by the public are afforded throughout the effective life of the patent in the event the patent is granted. Access to the culture is available during pendency of the application under 37 CFR 1.14 and 35 USC 112. All restrictions on the availability to the public of the culture deposited will be irrevocably removed upon granting of the patent.

The culture was planted from a slant into liquid ATCC No. 172 medium and grown for 4 days at 28.degree. C. on a shaker. It was then removed from the shaker, centrifuged for 20 minutes, washed three times with sterile distilled water and planted on media commonly used for identification of members of the Actinomycetales.

The inoculated plates were incubated at 28.degree. C. and records of results were made after suitable incubation time with most of the final results recorded at 14 days. The colors were described in common terminology, but exact color was determined by comparison with color chips from the Color Harmony Manual, fourth edition.

The methods of whole-cell and sugar analyses are those described by Becker, B. et al., Appl. Microbiol., 12, 421-423 (1964) and by Lechevalier, M.P., J. Lab. Clin. Med., 71, 934-944 (1968). Mycolate analyses were performed by the method of Lechevalier, M.P. et al., J. Bacteriol., 105, 313-318 (1971).

The results of chemical tests of whole-cell and cell wall indicated the new culture belonged in the genus Nocardiopsis. For comparison purposes, therefore, culture studies included Nocardia uniformis subsp. tsuyamanesis ATCC 21806 and Nocardiopsis dassonvillei ATCC 23218.

Identification media used for the characterization of the culture and references for their composition are as follows:

1. Tryptone Yeast Extract Broth (ISP #1 medium, Difco).

2. Yeast Extract-Malt Extract Agar (ISP #2 medium, Difco).

3. Oatmeal Agar (ISP #3 medium, Difco).

4. Inorganic Salts--Starch Agar (ISP #4 medium, Difco).

5. Glycerol--Asparagine Agar (ISP #5 medium, Difco).

6. Glycerol--Asparagine Agar (prepared in our lab).

7. Peptone--Yeast Extract Iron Agar (#6 medium, Difco).

8. Gelatin Agar--R. E. Gordon and J. M. Mihm, Jr. Bact. 73: 15-27, 1957.

9. Starch Agar--Ibid.

10. Organic Nitrate Broth--Ibid.

11. Dextrose Nitrate Broth--S. A. Waksman, The Actinomycetes, Vol. 2, medium no. 1. p. 328, 1961, and 3 g dextrose substituted for 30 g sucrose and agar omitted.

12. Potato Carrot Agar--M. P. Lechevalier, Jr. Lab. and Clinical Med. 71: 934-944, 1968 but use only 30 g potatoes, 2.5 g carrots and 20 g agar.

13. 2% Tap Water Agar

14. Czapek-Sucrose Agar--S. A. Waksman, The Actinomycetes, Vol. 2, medium no. 1, p. 328, 1961.

15. Glucose Asparagine Agar--Ibid, medium no. 2, p. 328.

16. Glucose-Yeast Extract Agar--Ibid, medium no. 29, p. 331

17. Emerson's Agar--Ibid, medium no. 28, p. 331.

18. Nutrient Agar--Ibid, medium no. 14, p. 330.

19. Bennet's Agar--Ibid, medium no. 30, p. 331.

20. Gordon and Smith' Tyrosine Agar R. E. Gordon and M. M. Smith, Jr. Bact. 69: 147-150, 1955.

21. Casein Agar--Ibid.

22. Calcium Malate Agar--S. A. Waksman, Bact. Rev. 21: 1-29, 1957.

23. Gauze's #1 Mineral Agar--G. F. Gauze et al. Problems in the Classification of Antagonistic Actinomycetes. English Ed., p. 13, 1957.

24. Gauze's #2 Organic Agar--Ibid.

25. Skim Milk--Difco

26. Cellulose utilization--

(a) H. L. Jensen, Proc. Linn. Soc. N.S.W. 55: 231-248, 1930. (b) M. Levine and H. W. Schoenlein, A Compilation of Culture Media, Medium 2511, 1930.

27. Utilization of Organic Acids--R. E. Gordon et al., Int. Jr. Syst. Bact. 24: 54-63, 1974.

28. Carbohydrate Utilization and Acid Production from Carbohydrates--Ibid.

29. Hydrolysis of Hippurate and Esculin--Ibid.

30. Decomposition of Adenine, Hypoxanthine, Xanthine, and Urea--Ibid.

31. Yeast Dextrose Agar for Studies of Temperature and Survival at 50.degree. C.--Ibid.

32. Resistance to Lysozyme--Ibid.

33. Glucose Broth--Ibid.

__________________________________________________________________________ Nocardia Nocardiopsis uniformis subsp. Nocardiopsis Cultural atra tsuyamanensis dassonvilleiAgar Character* ATCC 31511 ATCC 21806 ATCC 23218__________________________________________________________________________Yeast Extract- G Poor to moderate Good ExcellentMalt Extract SC Brown to greyish Orange White with paleAgar brown (4 ia to 4 lc) orange tint (4 lg to 4 li) (3 ea) T Thin, smooth to Thin to slightly Wrinkled with a slightly Sc raised, wrinkled fluffy edge, AM -- -- White RC Same as SC Same as SC Yellowish orange (3 ia to 3 lc) SP -- -- Pale yellowishOatmeal Agar G Moderate Moderate Moderate SC Dull white Pale brownish Colorless to (3 gc) white T Thin, smooth, Submerged, smooth Thin, smooth with with a few small with spreading spreading edge white dots edge AM None to scant -- White, sparse RC Same as SC Same as SC Same as SC SP Pale greenish -- --Inorganic G Moderate Moderate to good ModerateSalts-Starch SC Pale grey to Yellowish orange White to paleAgar greyish (near (3 ea, 3 ia to yellowish grey series, 3 la) (2 ga) 2 dc to 2 fe) with white tint T Thin, smooth Thin to slightly Thin to slightly raised raised smooth to slightly wrinkled AM White -- White RC Same as surface Same as surface Same as surface SP Pale greyish green -- -- (1 gc)Glycerol G Poor to moderate Poor to moderate PoorAsparagine SC Colorless to cream Colorless to pale Colorless toAgar yellowish brown pale cream(dehydrated (near 2 ea)powder from T Thin, appearing as Submerged, smooth Thin, smooth,Difco) confluent smear with a fluffy or very small edge isolated dots AM -- -- -- RC Same as surface Same as surface Same as surface SP -- -- --Glycerol G Good Moderate Moderate to goodAsparagine SC Grey, dark grey to Orange White to creamAgar black (near grey) (3 ga to 3 ia) (1 1/2 ca)(Prepared) series, 3 ih to 3 ml) T Raised, slightly Thin, smooth, with Raised, rough- wrinkled a spreading ened to edge wrinkled AM -- -- White RC Brown (4 pg to Same as surface Pale yellowish 4 pi) to black to pale yel- lowish green (1 1/2 ea to 1 1/2 gc) SP Greyish orange Very pale Pale yellowish (near 3 gc) to orange blackGelatin Agar G Good Good Good SC Grey to black Orange (3 ia) Cream to pale (near grey yellowish series, 3 fe) (2 ca to 2 ga) T Raised, wrinkled Thin, smooth but Thin, smooth but slightly wrin- slightly wrin- kled near the kled near the edge end of streak AM -- -- White RC Same as surface Same as surface Yellowish (2 ia) SP Greyish -- --Starch Agar G Good Good Good SC Greyish to black Orange (near 4 ia Yellowish brown (near grey or 4 la) (near 2 lc) series 2 ih to but white 7 ml near edge T Raised, roughened Slightly raised, Slightly raised, wrinkled wrinkled AM -- -- White RC Same as surface Same as surface Same as surface SP Greyish -- --Potato Carrot G Moderate Poor to moderate Poor to moderateAgar SC Cream (1 1/2 Colorless to pale Colorless, white ca) brown (2 ca) to pale brown (2 ca) T Thin, smooth, Submerged, thin, Thin, smooth, with some small smooth, with a with a fluffy, slightly raised spreading edge spreading edge dots AM None to sparse -- White RC Same as surface Same as surface Same as surface SP -- -- --Tap Water G Poor to moderate Poor PoorAgar SC Colorless to white Colorless Colorless T Thin, smooth, with Submerged, smooth, Thin, smooth a few white dots with a spread- ing edge AM None to sparse -- -- RC Same as surface Same as surface Same as surface SP -- -- --Czapek-Sucrose G Moderate Moderate ModerateAgar SC Pale greyish green Yellowish to Cream (1 1/2 ca) (1 1/2 ec) with yellowish with very pale some small black orange (1 1/2 ea greenish tint dots, greyish to 2 lc) near the end of streak (near grey series, 2 fe) T Thin, smooth Submerged, smooth Slightly raised with a spread- and roughened ing edge AM -- -- White, sparse RC Same as surface Same as surface Same as surface SP Pale greenish -- Pale greyish (1 1/2 gc)Glucose G Moderate Moderate ModerateAsparagine SC Pale brown (3 gc) Yellowish orange Cream (1 1/2 ca)Agar to black (3 ga to 3 la) T Slightly raised, Thin, smooth, with Thin to slightly smooth, with a few small raised appear- small raised raised dots ing as small black dots isolated dots, slightly wrinkled AM -- -- -- RC Brown to black Same as surface Same as surface SP Pale yellowish -- -- brownGlucose Yeast G Good Good GoodExtract Agar SC Black (near grey Orange White to cream series, 3 po) (near 4 ia) (2 ca) T Raised, wrinkled Slightly raised, Raised, strongly wrinkled wrinkled AM -- -- White RC Black Same as surface Yellowish brown (near 2 ic) SP Black -- Brown (3 ic)Emerson's G Good to excellent Good GoodAgar SC Black (near grey Orange White, cream to series, 3 ml) (4 ga to 4 ia) brown (2 ca to 3 le) T Raised, wrinkled Slightly raised, Slightly raised, wrinkled, but roughened to smooth near wrinkled, with the edge a fluffy, spreading edge AM -- -- White RC Black Same as surface Brown SP Black Yellowish brown Brown (4 ne) (3 lc)Nutrient Agar G Moderate to poor Moderate Moderate SC Pale grey to Pale orange Cream (2 ca) grey (near grey (between 3 ea series, 2 dc, 4 ea) 2 ih to 2 ml) T Thin, smooth, with Thin, smooth Thin, smooth or raised black appearing as dots small isolated dots, with a spreading edge AM -- -- White, sparse RC Same as surface Same as surface Cream to pale yellowish SP Pale greyish -- --Bennett's Agar G Good to excellent Moderate to good Good SC Black (near grey Yellowish orange Cream to yellow- series, 3 ml) (3 ga to 3 la) ish (2 ca to 2 ea) with a white edge T Raised, wrinkled, Thin, smooth to Moderately slightly wrin- raised, wrin- kled kled, with a fluffy edge AM -- -- White RC Black Same as surface Same as surface SP Black -- --Gordon and G Moderate Moderate to good ModerateSmith' SC Grey to dark grey Dark orange Dirty whiteTyrosine (near grey (near 4 lc)Agar series, 2 ih to 2 ml) T Thin to slightly Thin, smooth Thin, smooth or raised, slightly appearing as roughened to small isolated granular dots, with a spreading edge AM -- -- White RC Same as surface Same as surface Pale yellowish SP Greyish black Brown (near 3 ic) Brown (between 3 gc and 3 ic)Calcium G Moderate Poor to moderate ModerateMalate SC Black Yellowish to Dirty white toAgar yellowish brown cream (2 ga to 2 lc) (2 ca) T Thin, smooth, with Thin, smooth, with Appearing as some raised dots a spreading sub- isolated dots merged edge AM -- -- White RC Black Same as surface Yellowish brown (2 ic) SP Pale greenish -- --Gauze's #1 G Moderate Moderate to good Moderate to goodMineral Agar SC Pale greenish Yellowish orange White cream (3 ea to 3 ia) T Thin, with small Thin, smooth, with Slightly raised white to greyish a spreading edge and wrinkled dots AM White, sparse -- White RC Same as surface Same as surface Greyish brown (2 ie) SP Pale greenish (1 ca) -- Pale greyish (betwen 2 ec and 2 ge)Gauze's #2 G Moderate to good Good Moderate to goodOrganic Agar SC Dark brown to Orange (3 ga, Cream greyish (3 ni, 3 ia to 3 gc) near grey series 3 ih) T Raised, granular Slightly raised, Raised and to flaky wrinkled, with wrinkled a moderately spreading edge AM -- -- -- RC Dark brown to Same as surface Same as surface black SP Greyish brown Pale yellowish Pale yellowish orange__________________________________________________________________________ *G-growth, SCsurface color, Ttexture, AMaerial mycelium, RCreverse color, SPsoluble pigment, " means none. ______________________________________ NocardiaBiochemical and N. atra uniformis subsp. NocardiopsisPhysiological ATCC tsuyamanensis dassonvilleiProperties 31511 ATCC 21806 ATCC 23218______________________________________Gram stain + + +Acid-fastness - - -Decomposition of:Adenine - - +Calcium malate + + +Casein + + +Hypoxanthine - - +Tyrosine + - -Urea + + +Xanthine - - +Hydrolysis of:Esculin - + +Hippurate + - +Starch + + +Growth at:45.degree. C. - - -37.degree. C. very poor good good28.degree. C. good good good21.degree. C. very poor good goodSurvival at 50.degree. C. - + -8 hMelanin production - - -H.sub.2 S production - + +Gelatin liquefaction + + +Cellulosedecomposition - - -Growth incellulose broth:Jensen's poor good poorLevine &Schoenlein's - poor -Skim milk:Coagulation - + -peptonization - + -Acid from and(utilization of):Adonitol + (+) - (-) - (-)Arabinose + (+) + (.+-.) + (+)Cellobiose + (+) + (+) + (+)Dulcitol - (-) - (+) - (-)Erythritol - (+) - (.+-.) - (-)Fructose + (+) + (+) + (+)Galactose + (+) + (+) + (+)Glucose + (+) + (+) + (+)Glycerol + (+) + (+) + (+)Inositol + (+) - (-) - (-)Lactose + (+) + (+) - (.+-.)Maltose + (+) + (+) + (+ )Mannitol + (+) + (+) + (+)Mannose + (+) + (+) + (+)Melezitose - (.+-.) - (.+-.) - (.+-.)Melibiose + (+) + (+) - (-).alpha.-Methyl-d- + (+) - (-) - (.+-.)glucosideRaffinose + (+) + (+) - (.+-.)Rhamnose + (+) + (+) + (+)Ribose + (+) - (-) + (+)Salicin + (+) + (+) + (+)Sorbitol - (+) - (.+-.) - (-)Sorbose - (.+-.) - (.+-.) - (-)Starch + (+) + (+) + (+)Sucrose + (+) + (+) + (+)Trehalose + (+) + (+) + (+)Xylose + (+) + (+) + (+)Utilization of:Acetate + + +Benzoate - - -Citrate + - +Dextrin - - -Lactate + + +Malate + + +Mucate - - -Oxalate - - -Phenol - - -Propionate - - -Pyruvate + + +Succinate + + +Nitrite fromNitrate:Dextrose nitrate - + +brothOrganic nitrate - + +brothResistance to - + -lysozyme______________________________________ "+", positive; "-", negative; ".+-.", doubtfully utilized.

Morphological observations were made 1, 2, 3, 5, 7, 14 and 28 days after the inoculation of the new Nocardiopsis culture on Czapek sucrose agar. Substrate mycelium began to fragment into bacillary cells after 3 days of incubation; aerial mycelium appeared after 5 days of incubation; substrate and aerial mycelia fragmented after 14 days of incubation into rods of varying lengths which were smooth and measured 1.5-10 (or longer).times.0.6-0.9 .mu.m.

The cell wall contained meso-diaminopimelic acid but not diagnostic sugars. The cell wall contained no mycolates of any kind.

The new culture is characterized by gram-positive reaction, non-acid-fastness, black or greyish colonies, black or greyish colony reverse, black soluble pigment on some media and fragmentation of both aerial and substrate mycelia. These features plus the presence of mesodiaminopimelic acid in the cell wall but the absence of diagnostic sugars and mycolates place the new culture in the genus Nocardiopsis.

The biochemical and physiological properties distinguishing the new culture from Nocardia uniformis subsp. tsuyamanesis are the decomposition of tyrosine; hydrolysis of esculin and hippurate; growth at 21.degree. and 37.degree. C. survival at 50.degree. C. for 8 hours; H.sub.2 S production; growth in Jensen's cellulose broth; coagulation and peptonization of milk; nitrate reduction; resistance to lysozyme; acid production after utilization of adonitol, inositol, .alpha.-methyl-d-glucoside and ribose. The new culture differs from Nocardiopsis dassonvillei in hydrolysis of esculin; growth at 21.degree. and 37.degree. C.; H.sub.2 S production, nitrate reduction; decomposition of adenine, hypoxanthine tyrosine and xanthine; acid production from adonitol, inositol, lactose, melibiose, .alpha.-methyl-d-glucoside and raffinose; and utilization of adonitol, erythritol, inositol, melibiose and sorbitol.

The new culture was considered to be a new species of Nocardiopsis and was designated as Nocardiopsis atra Huang sp. nov.

Cultivation of Nocrdiopsis atra Huang sp. nov. ATCC 31511 preferably takes place in aqueous nutrient media at a temperature of 24-36.degree. C. and under submerged aerobic conditions with agitation. Nutrient media which are useful for such purposes include a source of assimilable carbon such as sugars, starches and glycerol; a source of organic nitrogen such as casein, enzymatic digest of casein, soybean meal, cottonseed meal, peanut meal, wheat gluten, soy flour, meat meal and fish meal. A source of growth substances such as grain solubles and yeast extract as well as salts such as sodium chloride and calcium carbonate and trace elements such as iron, zinc, cobalt and maganese may also be utilized with advantageous results. If excessive foaming is encountered during fermentation, antifoam agents such as vegetable oil or silicones may be added to the fermentation medium. Aeration of the medium in tanks for submerged growth is preferably maintained at the rate of about 1/2 to 2 volumes of free air per volume of broth per minute. Agitation may be maintained by means of agitators generally familiar to those in the fermentation industry. Aseptic conditions must, of course, be maintained through the transfer of the organism and throughout its growth.

Inoculum for the preparation of the antibiotic may be obtained by employing growth from a slant of the culture. The growth may be used to inoculate either shake flasks or inoculum tanks or the inoculum tanks may be seeded from the shake flasks. Growth in shaken flasks will generally have reached its maximum in 2 to 4 days whereas inoculum in submerged inoculum tanks will usually be at the most favorable period in 1.5-3 days. Substantial antibiotic activity is obtained in the final fermentor stage in approximately 2 to 5 days.

The process of antibiotic production is conveniently followed during fermentation by biological assay of the broth employing a sensitive strain of Comamonas terrigena ATTC 8461 or Micrococcus lutenus. Standard plate assay technique is employed in which the zone of inhibition surrounding a filter paper disc saturated with broth is used as a measure of antibiotic potency.

The antibiotic produced by Nocardiopsis atra Huang sp. nov. ATCC 31511 was recovered from the filtered fermentation broth and purified by a combination of carbon absorption and elution and column chromatography. The antibiotic was established to be identical with previously reported nocardicin A (U.S. Pat. No. 3,923,977) by comparison with the published physicochemical data and side by side comparisons with nocardicin A obtained from the fermentation broth of known producer Nocardia uniformis subsp. tsuyamanensis ATCC 21806.

EXAMPLE 1

A sterile aqueous medium having the following composition is prepared:

______________________________________Ingredient Grams/liter______________________________________Glucose 25Corn steep liquor 15Distillers' solubles 10Cottonseed meal 5Cobalt chloride 0.01Calcium carbonate 3______________________________________

Cells from a slant culture of Nocardiopsis atra ATCC 31511 are transferred to each of a number of 300 ml shake flasks each containing 40 ml of the above medium and shaken for 3 to 4 days at 28.degree. C.

Aliquots sufficient to provide a 5% v/v inoculum are transferred to fermentors each containing two liters of the above described sterile medium. The temperature is maintained at 30.degree. C. The broth is stirred at 200 r.p.m. and aerated at the rate of about one volume of air per volume of broth per minute. The fermentation is maintained until substantial antibiotic activity is obtained (3-4 days).

Nocardicin A may be recovered and separated by the methods described in U.S. Pat. No. 3,923,977.

EXAMPLE II

The method of Example I may be repeated with comparable results employing a fermentation medium of the following composition:

______________________________________Ingredient Grams/liter______________________________________Glucose 30Corn steep liquor 6Glycine 3Cottonseed meal 6DL-methionine 2Calcium carbonate 5______________________________________

EXAMPLE III

The method of Example I may be repeated with comparable results employing a fermentation medium of the following composition:

______________________________________Ingredient Grams/liter______________________________________Glucose 1Dextrin 24Yeast extract 5Beef extract 3Polypeptone 5Calcium carbonate 4______________________________________

Claims

1. A process for producing nocardicin A which comprises aerobically propagating Nocardiopsis atra Huang sp. nov. ATCC 31511 in an aqueous nutrient medium containing a source of assimilable carbon and a source of assimilable nitrogen until a substantial amount of nocardicin A is obtained.