FI86082B - Process for the preparation of antibiotic complex A- 21978C by culturing Streptomyces roseosporus NRRL 15998 - Google Patents

Abstract

The invention relates to a process for the preparation of the antibiotic A-21978C complex or factors thereof by culturing Streptomyces roseosporus NRRL 15998 or A-21978C- producing mutants, variants or recombinants thereof. The micro-organism is cultured on a culture medium containing sources of assimilable carbon, nitrogen and inorganic salts, under aerobic, submerged fermentation conditions. If desired the obtained A-21978C-complex is separated and the individual A-21978C- factors are separated from the complex.

Description

1 86082

A method for preparing the antibiotic complex A-21978C by culturing Streptomyces roseosporus NRRL 15998

Divided separated from application 853910.

This invention relates to a process for the preparation of antibiotics A-21978C of formula (I) ϊ r io / \ / V \

u f II

X t \ / * / \ / \ / \, o ° = .; 'ϊ Y h / C0NHa. — 7 · A 3 «J lv J« 15 ^> WyY z \ "\ h h! H ~ -» ϊ y / yv 2o ° = \ y h

x — 7 * ίΗ »o V» X

“K Y λ / w” ϊ Ϊ j *

HOAc

Wherein R is 8-methyldecanoyl, 10-methylundecanoyl, 10-methyldodecanoyl, 9-methylundecanoyl, n-dodecanoyl or n-decanoyl. The method of the invention is characterized in that Streptomyces roseosporus 30 NRRL 15998 or a progeny thereof producing A-21978C is cultured in a medium containing sources of assimilable carbon, nitrogen and inorganic salts under aerobic submerged culture conditions until A-21978C is formed. A-21978C antibiotics are separated from the medium.

2 86082 Antibiotics belonging to the family A-21978C are excellent antibacterial agents. A particularly significant group of A-21978C derivatives are those of formula I (see also: Berbard J. Abbott, Davis S. Fukuda and Manuel 5 Debono, U.S. Patent No. 4,537,717). The preparation of these derivatives previously required a multi-step process which was time consuming, yield reducing and expensive. An improved method can now be used to prepare these A-21978-C derivatives directly. The previous method for preparing a derivative of formula I, such as the n-decanoyl derivative of A-21978C, requires the following steps: 1. Fermentation of a culture producing A-21978C.

a. Initiation of fermentation by means of an ampoule containing liquid nitrogen 15.

b. Primary transfer phase (48 hours).

c. Secondary transfer phase (24 hours).

d. Tertiary transfer phase (24 hours).

e. Fermentation (140 hours).

20 2. Filtration, adsorption on resin and elution as well as concentration.

3. Preparation of t-Boc- (t-butoxycarbonyl-protected) complex.

4. Concentration of the protected complex 5. Fermentation of the deacylated culture using e.g. Actinoplanes utahensis 25 a. Initiation of fermentation by means of an ampoule containing liquid nitrogen.

b. Primary transfer phase (72 hours).

c. Fermentation (67 hours).

6. Deacylation of the complex by deacylated culture.

30 7. Filtration, adsorption on resin and elution as well as concentration.

. . 8. Reacylation.

9. Hydrolysis of the protecting group.

”10. Final cleaning.

The new method comprises the addition of a C2-C14 alkanoic acid (ROH- * 35 compound in which R is as defined above) or 3,86082 of its ester or salt to the A-21978C-producing culture during the production step (step le) of the corresponding formula I to obtain the compound. This method eliminates steps 3, 4, 5, 6, 7, 8 and 9 of the previous method. In addition, the new method increases the yields considerably higher than those obtained using the previous method.

Streptomyces roseosporus strain NRRL 15998 (A-21978,65), a mutant strain of NRRL 11379, is a useful A-21978C-producing culture. Said crops are part of the basic crop collection maintained by the Northern Regional Research Center, U.S. Pat. Department of Agriculture, Agricultural Research Service, Peoria, Illinois 61604, from which they are available to the public under accession numbers 15 NRRL 11379 and NRRL 15998. A culture of S. roseosporus NRRL 11379 and the conditions for its use in the production of A-21978C antibiotics are described by Robert L. Hamil and Marvin M. Hoehn in U.S. Patent 4,331,594, which is incorporated herein by reference.

The naturally occurring A-21978C factors described in U.S. Patent 4,331,594 are factors and C5. In factors A and C5, R in formula I is specific

Ci0-C12 alkanoyl group. A-21978C factor C0, previously thought to have a particularly branched C10-alkan-25 noyl side chain, has been found to be a mixture of the two components in an approximately 2: 1 ratio. The main component has a branched -C10 side chain and the smaller component has a straight C10 side chain.

When a compound of formula I is prepared by the method described herein, it is also referred to as A-21978C for convenience. With the exception of naturally occurring factors, side chain length is used to refer to the factor. Thus, for example, a compound of formula I in which R = octanoyl is called, if prepared by this method, the factor A-21978Ce.

Sii.

86082

The alkyl moiety of the C2-C14 alkanoic acid, ester or salt (substrate) used in the process described may be straight or branched chain. For the preparation of naturally occurring A-21978C factors Cx, C2 or C3, for example, 8-methyldecanoic, 10-methyldodecane or 10-methylundecanoic acid, ester or salt could be used. C2-C14 straight chain acids, esters and salts are recommended for use in the process due to their availability and cheapness. A particularly preferred substrate is n-decanoic acid, its esters or salts.

When a C2-C14 alkanoic acid ester is used, C1-C4 alkyl esters are preferred. In such an ester, the C 1 -C 4 alkyl group may also be straight or branched chain.

Typical suitable salts of C2-C14 alkanoic acids which can be used in the process are those formed using alkali metals and alkaline earth metals such as sodium, potassium, lithium, cesium, rubidium, barium, calcium or magnesium. Suitable amine salts include ammonium, primary, secondary and tertiary C1-C4 alkyl ammonium and hydroxy C2-C4 alkyl ammonium salts.

The substrate is preferably added to the fermentation medium in the form of a sterile solution. A particularly useful solvent for this purpose is methyl oleate, although other solvents such as ethanol, ethyl acetate and C 1 -C 4 esters of unsaturated fatty acids may be used. If the substrate is suitably liquid at fermentation temperature, it will add directly.

The rate of addition of the substrate should be low enough to avoid the formation of a fermentation toxic effect, but high enough to increase the yield of the desired compound of formula I. For addition rates of about 0.05 to about 0.5 ml per liter, 35 fermentation broths per hour are recommended. As an addition rate of about 0.1 to about 1.2 ml per liter of fermentation broth per hour is preferred.

5,86082

The substrate is added to the growing A-21978C-producing culture during the fermentation production step from about 15 to about 32 hours and until the fermentation is complete. The substrate can be added by various methods, but it is preferable to add it by a method that best achieves a uniform flow.

After fermentation, the resulting compound of formula I can be recovered, if desired, using methods known in the art (see, e.g., U.S. Patent 10,433,594).

The method according to the invention uses a new microorganism, Streptomyces roseosporus NRRL 15998. This microorganism also produces A-21978C antibiotics. In particular, this invention relates to an improved method for producing novel A-21978C antibiotics in culture

Streptomyces roseosporus, NRRL 15998 strain under aerobic submerged culture conditions until a substantial amount of A-21978C antibiotics is formed. The A-21978C anti-biotic complex can be extracted from the fermentation broth and felts with polar organic solvents. The individual elements of A-21978-C can also be separated and further purified by methods known in the art, such as column chromatography.

A culture isolated from the wild ("wild species") 25 produces an antibiotic in low yields. The formation of an antibiotic is often capricious. Strains with improved potency and strains that continuously produce antibiotics are therefore very valuable.

This aspect of the invention provides a greatly improved method for preparing A-21978C antibiotics. . by culturing Streptomyces roseosporus NRRL 15998 * or its A-21978C-producing progeny in a culture medium containing assimilable sources of carbon, nitrogen and inorganic salts under aerobic submerged culture conditions until A-21978C is formed. The A-21978C antibiotic complex or individual factors can be recovered using a variety of isolation and purification methods known in the art.

The microorganism used in the method according to the invention has been designated A-21978.65. This strain was developed by selecting and modifying the strain from a culture designated A-21978.6 (NRRL 11379). Strain A-21978.6, studied and characterized by Frederick P. 10 Mertz and Ralph E. Kastner (Lilly Research Laboratories), in turn was developed from a native strain obtained from the soil of Mount Ararat in Turkey. Strain A-21978.6 is classified as a new strain named Streptomyces roseosporus, Falcäo de Morias and Daliä Maia 1961. This classification was made after comparison with published reports [R.E. Buchanan and N. E. Gibbons, "Bergey's Manual of Determinative Bacteriology," The Williams and Wilkins Company, 9th Edition, 1974; and E.B. Shirling and D. Cottlieb, "Cooperative Description of Type 20 Strains of Streptomyces," Intern. Journal of Systematic Bacteriol., 808-809 (1972)].

The classification was based on the methods recommended for the International Streptomyces Project [E.B. Shirling and D. Cottlieb, "Methods of Characterization of 25 Streptomyces Species," Intern. Journal of Systematic Bac- ·: * ·: teriol. 16, 313-340 (1966)] in conjunction with certain supplementary experiments. Carbon utilization was determined in ISP No. 9 base medium to which carbon sources were added so much that the final concentration became 1.0%. 30 Carbon sources were sterilized by filtration; the medium was sterilized by heating in an autoclave. The plates were examined after incubation for 14 days at 30 ° C.

• Cell wall sugars were determined using the method of Lechevalier (M.P. Lechevalier, "Chemical Methods as 35 Criteria for the Separation of Actinomycetes into Genera", 7 86082

Seminar sponsored by the Actinomycetes subcommittee of the American Society of Microbiology, chaired by Dr. Thomas G. Pridham, a seminar held at the Institute of Microbiology (Rutgers University, The State University of New Jersey, New Brunswick, New Jersey, 1971). The isomer of diaminopimelic acid was determined using the method of Becker et al. [B. Becker, et al., "Rapid Differentiation Between Norcardia and Streptomyces by Paper Chromato-10 Graph of Whole Cell Hydrolysates," Appl. Microbiol. 11, 421-423)]. Amino acid analysis was performed by washing the cell wall fragments. Melanoid pigments were determined using ISP No. 1 (tryptone yeast extract broth), ISP No. 6 (peptone-yeast extract iron agar), ISP No. 7-15 (tyrosine agar), ISP No. 7 modified (ISP No. 7 without tyrosine) and the tyrosine assay [Yuzuru Mikami, et al., "Modified Arai and Mikani Melanin Formation Test of Streptomyces", Intern. Journal of Systematic Bac-teriol. 27 (3), 290 (1977)]. The hydrolysis of the starch was performed by testing the starch present with iodine.

The temperature range, NaCl tolerance, pH range, and antibiotic sensitivity were examined using ISP No. 2 agar medium. The temperature range was as follows; 25, 28, 30, 34, 37, 40, 45, 50 and 55 ° C. The tolerance of NaCl was measured by adding NaCl to the agar to such an extent that the concentrations became; 0, 1, 2, 3, 4, 5, 6, 8, 10 and 12%. These were incubated at 30 ° C. The pH range was measured by adjusting the pH of the agar from 3.0 to 11.0 L with increments of pH units just prior to casting. Antibiotic Sensitive:. The assay was determined using sensitivity discs plated on inoculated agar plates.

. . Color names were labeled according to the ISCC-NBS method (K.L. Kelly and D.B. Judd, "The ISCC-NBS Methods of Designing Colors and a Dictionary of Color Names," U.S. De-35 partment of Commerce Cire. 553 Washington, D.C., 1955).

8 86082

The numbers in parentheses refer to the Tresner and Backus color series [H.D. Tresner and E.J. Backus, "System of Color Heels for Streptomycete Taxonomy," Appl. Microbiol. 11, 335-338 (1956)]. Color stripe markings are under-5 referenced. Merz and Paul color plate markings are enclosed in square brackets (A. Maerz and M.R. Paul, "Dictionary of Color," McGraw-Hill Book Company, Inc., New York, N.Y. 1950).

Morphology 10 The morphology of culture A-21978.6 consists of sporophores classified as Rectus-Flexibilis (RF). Spore chains have> 10 spores per chain. The surface of the spore is smooth.

Culture A-21978.6 is characterized in that it produces predominantly red aerial spore mass color 15 reverse color is reddish. A light brown water-soluble pigment is also present. These characteristics are present in three of the fourteen agar plate culture media (ISP No. 2, ISP No. 7, TPO). These three substrates are the only ones that support 20 abundant aerial and vegetative growth.

Two agar plating media, ISP # 4 and glukoo-Si-asparagine agar, produced a color between white and gray aerial spore mass reverse color of yellow. No water-soluble pigment was removed. These two substrates supported good, though not abundant, aerial and vegetative growth.

Nine other agar plate media were used, but these produced poor or non-existent growth and spore formation. Color of air mass, if any. 30 masses were present, albeit faintly, in the color range between white and gray.

. . Melanoid pigments are absent. The main constituents of the cell wall are: LL-DAP, glycine, glucose and ribose. This indicates a type I cell wall and a type C sugar structure. (R.E. Buchanan and N.E.

9 86082

Gibbons, Eds., Bergeys's Manual of Determinative Bacteriology, The Williams & Wilkins Company, 8th ed., 1974, p. 685).

The following five cultures were compared in laboratory-5 experiments to A-21978.6:

Streptomyces albovinaceous ISP 5136; ATCC 15833 Streptomyces candidus ISP 5141; ATCC 19891 Streptomyces moderatus ISP 5529, ATCC 23443 Streptomyces roseosporus ISP 5122, ATCC 23958 Streptomyces setonii ISP 5395; ATCC 25497 These cultures belong to the white and red of VA comonomer in a series, they have a RF type sporophore morphology, morfolo-ogy, smooth spore surface ornamentation, and the ISP descriptions that they are melanin negative and do not have an account to the list of reverse color or water-soluble pigments.

These characteristics, together with the carbon utilization model and other secondary features, are consistent with the characteristics of culture A-21978.6.

When these cultures were compared to A-21978.6 under 20 laboratory conditions, four of them were discarded. S. candidus and S. setonii produced yellow aerial mass on many media, thus separating from culture A-21978.6. Albivinaceuos S. and S. moderatus showed dark distinctive reverse color, water-soluble pigments, and produced 25 Melanoid pigments, all of which showed differences in culture A21978.6 relation. S. Modera-tion-cultures were ISP description shows a strong reddish or brown reverse color, but does not show such a characteristic of S. albovinaceous culture. Neither of the 30 cultures are classified as melanin-positive.

Culture A-21978.6 was therefore classified as . known as Streptomyces roseosporus,

Falcäo de Morias and Dalian Maia 1961. This classification was based on a comparison with published descriptions and direct 35 laboratory comparisons. The following 10 e 'neo C i_> U u L · type data on cultivation mainly present direct comparative studies.

Culture characteristics Morphology 5 A21978.6 S.roseosporus

Sporophores straight or tortuous (RF), no hooks, loops, or coils were observed. Spore chains> 10. Spore surface smoothness determined by scanning electron microscopy.

10 Spores: oblong - oblong - cylindrical oval

Average size: 0.85 x 1.78 pm 1.01 x 2.47 pm Range: 0.65-0.97 x 0.97-2.6 pm 0.97-1.3 x 1.63-3, 25 pm, 86082

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15 86082

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GM Φ · Η> i O + · fl) <0 · ... · <(D ^ 4r-IG CL CO P> ie 86082 The specified characteristics of strain A-21978.6 differ from the published characteristics of S. roseosporus strain. 21978.6 differs from the published strain in spore size, carrot and potato plug growth, NaCl tolerance, and nitrate 5 reduction.

The A-21978.65 strain used in the invention has the identification characteristics of the A-21978.6 strain, but differs in the amount of A-21978C antibiotics produced therefrom. At best, the previous strain did not produce more than 10 pg of A-21978C antibiotics per ml of fermentation broth. The improved strain A-21978.65 of this invention produces at least 2 1/2 times this amount in tank fermentations and has produced as much as 18 times this amount when performing fermentation in a shake flask. With this improved production property of A-21978C, the new strain provides a greatly improved method for obtaining these antibiotics.

As with other organisms, the characteristics of the new A-21978C-producing culture used in this invention, Streptomyces roseosporus NRRL 15998, are susceptible to variation. Recombinants, variants and mutants of strain NRRL 15998 can be obtained by treatment with various known mutagens, such as ultraviolet rays, X-rays, high frequency waves, radioactive rays and chemicals. Progeny of Streptomyces roseosporus NRRL 15998 that retain their ability to produce A-21978C antibiotics in high yields can also be used in this invention.

The culture medium used to grow the culture of Streptomyces roseosporus NRRL 15998 can be one of a number of media: Due to the economics of production, the optimal yield and the ease of isolation of the product, some culture media are recommended. Thus, for example, a preferred source of carbon for large scale fermentation is tapioca dextrin, although glucose, fructose, galactose, maltose, mannose, cottonseed oil, methyl oleate, glycerol, refined soybean oil and their soybean oil may also be used. A preferred source of nitrogen is enzyme hydrolyzed casein, although soluble meat peptone, soybean meal, soybean hydrolyzate, soybean meal, yeast, amino acids such as L-asparagine and DL-leucine and the like are also useful. Suitable inorganic salts as nutrients that can be included in the culture media include soluble salts capable of producing potassium, ammonium, chloride, sulfate, nitrate ions and the like. Among these, K2SO4 is particularly useful for antibiotic production. Molasses ash, ash dialysate and a synthetic mineral mixture are also useful.

For the preparation of A-21978C antibiotics, it is preferred to use distilled or deionized water in the fermentation broth. Some minerals in tap water, such as calcium and carbonate, appear to be detrimental to the production of antibiotics.

Essential trace elements essential for the growth and development of the organism should also be included in the culture medium. Such trace elements are generally present in other media in sufficient amounts to meet the growth requirements of the 25 organisms.

The addition of small amounts (e.g. 0.2 ml / liter) of an antifoam, such as polypropylene glycol, to large scale fermentation media may be necessary if foaming is a problem.

To prepare substantial amounts of A-21978C antibiotics, it is preferred to perform aerobic, submerged culture in tanks. However, small amounts of A-21978C antibiotics can be obtained by shake flask culture. Due to the slowdown that usually occurs in the production of antibiotics, which is usually associated with spore-shaped inoculation of the organism in large containers, it is preferred to use vegetative inoculation. The vegetative inoculum is prepared by inoculating a small volume of culture medium with the spore form of the organism or mycelial fragments of the organism for 5 min to obtain a fresh, actively growing culture. The vegetative inoculum is then transferred to a larger container.

The novel A-21978C-producing organism can be grown at temperatures between about 20 ° C and about 40 ° C. Production of A-21978C by Opti-10 appears to occur at temperatures between about 30 and 32 ° C.

As with conventional aerobic submerged culture methods, sterile air is dispersed through the culture medium. For efficient production of A-21978C antibiotics, the percentage of air saturation should be greater than 20%, preferably greater than 30% (at 30 ° C and one atmospheric pressure) for tank production.

For fermentation in a tank, it is preferred to keep the pH of the fermentation medium between about 6.5 and 7.0. This can be done by adding appropriate amounts of, for example, sodium hydroxide (in the early stages) and hydrochloric acid (in the later stages).

The production of A-21978C antibiotics can be monitored during fermentation by testing samples taken from broth or extracts of mycelium solids for their antibiotic activity against organisms known to be sensitive to antibiotics. One test organism useful for testing these antibiotics is Micrococcus luteus. The bioassay is preferably performed by paper disc assay on agar plates.

Alternatively, culture solids, including medium and mycelium, can be used without extraction or separation, but preferably after dehydration, as a source of A-21978C antibiotics. Example 35 After producing the A-21978C antibiotic activity, the culture medium can be dried by lyophilization and mixed directly into the premix to be fed.

The compounds of formula I are excellent antibacterial agents.

The following examples are provided to more fully illustrate the present invention.

Example 1 Production of A-21978C complex

The stock culture is prepared and maintained in the vapor phase of liquid nitrogen. Streptomyces roseosporus NRRL 15998, previously stored in the vapor phase of liquid nitrogen, was used to inoculate a 50 ml medium of the following composition:

Ingredient Amount (%) 15 Trypticase Soy Broth * 3.0 dextrin 2.5 water (deionized) 94.5 ♦ Baltimore Biological Laboratories, Cockeysville MD.

The inoculated medium was incubated in a 250 ml Erlenmeyer flask at 30 ° C for 48 hours on a shaker rotating a 5.08 cm arc at 250 rpm. The finished growth culture was transferred to several wells (0.5 ml / well) and stored in the liquid nitrogen vapor phase.

To obtain a larger uniform batch of stored material, a 1 ml culture stored in liquid nitrogen was used to inoculate an 80 ml batch of the medium described above. The inoculated medium 30 was incubated in a 250 ml Erlenmeyer flask at 30 ° C for 48 hours on a shaker rotating a 5.08 cm diameter arc at 250 rpm.

10 ml of such a culture was used to inoculate a 450 ml second stage vegetative medium having the same composition as the primary vegetative medium described above. The second stage medium was incubated in a 2-liter Erlenmeyer flask for 24 hours at 30 ° C on a shaker with a 5.08 cm diameter arc at 250 rpm for 5 minutes.

1 liter of second stage vegetative culture was used to inoculate a sterile tertiary inoculum of 39 liters with the following composition: 10 Ingredient Amount (%) of soybean meal 0.5 yeast extract * 0.5 calcium gluconate 1.0 KClb 0.02 15 MgSO 4 · 7H 2 O 2 0.02

FeSO 4 · 7H 2 Ob 0.0004

Sag 471 (antifoam) c 0.03 water 97.9296

20 “Difco Laboratories, Detroit MI

bMinivals were prepared as follows:

FeSO 4 · 7H 2 O (7.8 g) was dissolved in concentrated HCl (76 mL). MgSO 4 * 7H 2 O (380 g), KCl (380 g) and deionized water were added to a total volume of 3800 25 mL. To obtain the specified minerals, 80 ml of solution per 39 liters per tertiary inoculum development step is used.

cUnion Carbide, Danbuty CT.

The inoculated medium was incubated for 24 hours in a stainless steel vessel at 30 ° C. The vessel was aerated with sterile air at a rate of 0.85 v / v / min and agitated with conventional stirrers at 350-450 rpm. The pressure in the vessel was maintained at 0.35 kg / cm 2 overpressure (5 PSIG).

23 8 6 0 8 2 1 liter of incubated tertiary inoculum was used to inoculate 119 liters of sterile production medium with the following composition: Ingredient Amount (%) 5 soybean meal 2.2

Fe (NH4) 2SO4 ♦ 6H2O 0, 066 dextrose 0.825

Sag 471 0.022 potato dextrin 3.3 10 molasses (sugar cane molasses) 0.275 tap water 93.312 The pH was adjusted to 7.0 after the addition of the first two ingredients and again after all ingredients were added and immediately before sterilization.

The inoculated production medium was incubated for 6 days in a stainless steel vessel at 30 ° C and aerated with sterile air at a rate of 0.5 v / v / min. The medium was agitated with conventional agitators at 250 rpm for the first 15 hours and at no-20 at 350 rpm after these 15 hours. The pH was maintained at or above 6.5 by the addition of ammonium hydroxide solution. The yield of A-21978C complex was 0.282 g per liter of broth at the end of fermentation. The distribution of factors is shown in Table 1.

Example 2 Elevated production of A-21978CH

The primary, secondary and tertiary growth steps were performed as described in Example 1. The production step was started as described in Example 1, except that from 28 hours onwards was fed. . a sterile solution containing 50% v / v in a fermentation vessel. caprylic acid (octanoic acid) and methyl oleate, at a rate of 0.13 ml per liter of fermentation broth per hour and this rate was maintained until the end of fermentation at 144 to 35 hours. The yield of the A-21978C complex was 1.255 g per liter of 24,86082 broth, which represents a 445% increase in yield compared to the yield of Example 1. Factor A-21978Ce (a compound of formula I wherein R is octanoyl) represented 9% of the total A-21978C complex prepared by this method; No A-21978Ce was detected in the A-21978C complex prepared by the method of Example 1.

Example 3 Elevated production of A-21978Cq

The primary, secondary and tertiary graft development steps were performed in the same manner as in Example 1. The production step was started as described in Example 1, except that from 28 hours a sterile solution containing 25% v / v was added to the fermentor. nonanoic acid and 75% methyl oleate, at a rate of 0.13 ml per liter of fermentation broth per hour and this rate of addition was maintained for 144 hours until the end of fermentation. The yield of A-21978C complex was 0.821 g per liter of broth, which represents a 293% increase over that obtained in Example 1. Factor A-21978C, 20 (Formula I: R = nonanyl) represented 10% of the total A-21978C complex prepared by this method; none of A-21978C9 was detected in the A-21978C complex prepared by the method of Example 1.

Example 4 Increased production of factor A-21978Cin (formula I: R = n-decanoyl

The vegetative growth stages of the primary and secondary stages were cultured as described in Example 1. In the tertiary step, 800 ml of a second-30 inoculum culture of 950 liters of sterile was used. . for the transplantation of a tertiary transplant, a development platform of the following size: 25 86082

Ingredient Amount (%) dextrose 2.0 calcium carbonate 0.2 soybean meal 2.0 5 yeast extract 0.1 KCl * 0.02

MgSO 4 · 7H 2 O “0.02

FeSO 4 · 7H 2 Oa 0.0004

Sag 471 (antifoam) 0.02 10 water 95.6396 "A trace mineral solution was prepared as described in Example 1.

The inoculated medium was incubated for 24 hours in a stainless steel vessel at 30 ° C. The vessel was aerated with sterile air at a rate of 0.8 v / v / min and agitated using conventional agitators. One liter of this tertiary stage inoculum was used to inoculate 119 liters of the 20 production stage medium described in Example 1. The production step was also started in the same manner as in Example 1, except that from 28 hours a sterile solution containing 50% v / v was fed to the fermentation medium. capric acid (decanoic acid) and 50% methyl oleate, at a rate of 0.26 ml per liter of fermentation broth per hour, and this rate of addition was maintained for 223 hours until the end of fermentation. The yield of the A-21978C complex was 1.94 g per liter of broth, which represents a 687% increase compared to that obtained in Example 1. The concentration of A-30 21978C10 factor (formula I: R = n-decanoyl) was 1.63 g per liter, i.e. 84% of the total A-21978C complex. This was 13583% higher than the amount of A-21978C10 produced using the method of Example 1.

26 8 6 0 8 2

Example 5 Alternative method for elevated production of A-217978C10

The developmental stages of primary, secondary and tertiary grafting were performed as described in Example 1. The production step was started as described in Example 1, except that from 28 hours a sterile solution containing 25% v / v was added to the fermentation chamber. capric acid ethyl ester (ethyl captate) 10 and 75% methyl oleate, at a rate of 0.13 ml per liter of fermentation broth per hour and this rate of addition was maintained until the end of fermentation at 144 hours. The yield of A-21978C complex was 1.022 g per liter, which represents a 362% increase over that obtained in Example 1. The concentration of A-21978C10 in Teki-15 was 0.202 g per liter, or 20% of the total A-21978C complex. This was 1683% higher than the concentration of A-21978C10- obtained using the method of Example 1.

Example 6 20 An alternative method for the elevated production of A-21978Cin

The stages of primary and secondary phase vegetative growth were cultured as described in Example 1. The tertiary phase transfer was cultured as described in Example 4, except that the volume of the medium was 1900 liters and the duration of the phase was extended to 48 hours. The aeration rate was 0.3 v / v / min in hours 0.24, 0.45 v / v / min in hours 24 to 40 and 0.90 v / v / min in hours 40 to 48. The production phase was started in advance. -30 as described in character 1. At 23 hours, 0.004% yeast extract was added as a sterile slurry in one portion to the fermentation vessel. From 36 hours onwards, a solution of glycerol and ammonium decanoate was added at a rate of 0.84 ml per liter of fermentation broth per hour. The feed solution contained glycerol: 35 (3600 g), deionized water (9000 mL), capric acid (1 27 86 0S2 liters) and concentrated ammonium hydroxide solution (620 mL). The feed was maintained at this rate until fermentation was stopped at 43 hours. The yield of A-21978C complex was 1.772 g per liter, which represents a 628% increase compared to that obtained in Example 1. The concentration of A-21978C10 was determined to be 0.739 g per liter, or 42% of the total A-21978C complex. This was 6158% higher than the concentration of A-21978C10 produced by the method of Example 1.

10 Example 7 Elevated production of A-217978C11

The primary, secondary and tertiary inoculation steps were performed as described in Example 1. The production step was started as described in Example 1, except that from 28 hours a sterile solution containing 25% v / v was added to the fermentation vessel. undecanoic acid and 75% methyl oleate, at a rate of 0.13 ml per liter of fermentation broth per hour until fermentation was stopped at 144 hours. The yield of A-21978C complex 20 was 1.62 g per liter, which represents a 574% increase compared to that obtained in Example 1. The concentration of factor A-21978Cn (formula I: R = undecanoyl) was determined to be 0.70 g per liter, i.e. 43% of the total A-21978C-: · complex. Factor A-21978Cn could not be detected in the A-21978C complex prepared by the example method:.

Example 8 Elevated production of A-21978CU

The primary, secondary and tertiary inoculation steps were performed as described in Example 1. The production step was started as described in Example 1, except that from 28 hours a sterile solution containing 25% v / v was added to the fermentation vessel. lauric acid and 75% methyl oleate, at a rate of 0.13 ml per liter of fermentation broth per hour therefore until fermentation. 35 were terminated in 144 hours. The yield of A-21978C complex 28,80082 was 1.12 g per liter, which represents a 400% increase compared to that obtained in Example 1. The concentration of factor A-21978C5 (formula I: R = dodecanoyl) was determined to be 0.372 g per liter, i.e. 33% of the total A-5 21978C complex. This was 5314% higher than the concentration of A-21978C5 found in the A-21978C complex prepared by the method of Example 1.

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Example 9 Alternative Production of A-21978C Complex A-21978C complex is produced using the method of Example 1 but using a culture of Streptomyces roseosporus NRRL 5 11379.

Example 10

An alternative method for increasing the production of A-217978C10

A-21978C10 is produced using the method of Example 6 but using Streptomyces roseosporus NRRL

11379 culture.

Example 11 Production of A-21978C Complex in a Shake Flask The general procedure described in Example 1 is used, but the shake flask conditions and the following fermentation medium are used:

Ingredient Amount (g / liter) glucose 7.5 dextrin® 30 20 enzyme hydrolysed caseiniab 5 peptoniac 5 molasses 2.5 deionized water q.s. up to one liter

--- ·· 25 * Statex 11, A.E. Staley Co., Decatur IL

The yield of bNZ Amine A, Humko Sheffield Chemical, Lyndhurst NJ cBiosate, Baltimore Biological Laboratories, Cockeysville MD A-21978C complex from this fermentation was 1800 pg per ml of broth.

Claims (3)

3i 86082 A process for the preparation of A-21978C antibiotics of the formula 5 s Γ / \ / Hs / \ / \ HOsc / l f 'I I 10. f. N / Y il H «T <ri H = C ^ JAA · 'V> / V”> - <H 1 π! where R is 8-methyldecanoyl, 10-methylundecanoyl, 10-methyldodecanoyl, 9-methylundecanoyl, n-dodecanoyl or n-decanoyl , characterized in that Streptomyces roseosporus NRRL 15998 or its A-. . An offspring producing 21978C in a medium containing sources of assimilable carbon, nitrogen and inorganic salts under aerobic submerged culture conditions until A-21978C antibiotics have formed and, if desired, the A-21978C antibiotics are separated from the medium. 32 86082 A method according to claim 1, characterized in that the A-21978C antibiotics are separated from the medium. Method according to Claim 1 or 2, characterized in that the individual A-21978C antibiotic factors C0, Cx, C2, C3, CA or C5 are produced. 33 86082