Title of Invention

A PROCESS FOR THE PREPARATION OF PSEUDOMONIC ACID A BY MICROBIOLOGICAL METHOD

Abstract

A procedure for the preparation of pseudomonic acid A comprising submerged cultivation of a Pseudomonas bacterium strain capable of biosynthesis of the substantially pure pseudomonic acid A in aerated conditions via fermentation; and isolation of the desired compound is disclosed. In particular, the procedure of the present invention comprises cultivation of the Pseudomonas sp. bacterium strain No. 19/26 deposited under accession No. NCAIM(P)B 001235 in the National Collection of the Agricultural and Industrial Microorganisms, Budapest, Hungary, or its pseudomonic acid A-producing mutant or variant, on a medium at a temperature of between about 20°C and 30°C containing organic nitrogen and carbon sources and, optionally, mineral salts.

Full Text

FORM 2 THE PATENTS ACT 1970 [39 OF 1970] & THE PATENTS RULES, 2003 COMPLETE SPECIFICATION [See Section 10; rule 13] "A PROCESS FOR THE PREPARATION OF PSEUDOMONIC ACID A BY MICROBIOLOGICAL METHOD" TEVA GYOGYSZERGYAR RESZVENYTARSASAG (formerly known as BIOGAL GYOGYSZERGYAR RT), of Pallagi 13, H-4042 Debrecen, Hungary, The following specification particularly describes the invention and the manner in which it is to be performed: The present invention relates to a process for the preparation of pseudornonic acid A. The present invention relates to a microbiological method for the manufacture of the antibiotic pseudornonic acid A (mupirocin). BACKGROUND OF THE INVENTION Pseudornonic acid A, also known as mupirocin, is an antibiotic that has a growth inhibiting effect mainly against Gram positive bacteria (e.g. Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Klebsiella pneumoniae) and some Gram negative bacteria (e.g. Haemophilus influenzae, Neisseria gonorrhoeae) [A. Ward, D.M.Campoli-Richards, Drugs 32, 425-444 (1986)] and its minimal inhibiting concentration is in the range of 0.02-0.5 mg/dm3. Pseudornonic acid A, by inhibiting the isoleucine-tRNA synthase enzyme, affects the peptide synthesis of pathogen bacteria [J. Hughes and G. Mellows, Biochem. J. 191, 209-219 (1980)]. An advantageous feature of mis antibiotic is that it has very low toxicity both for humans and animals and it is negative in the Ames test. Pseudornonic acid A is presently used in human therapy, in various formulations, for the treatment of skin infections (e.g. impetigo, pyoderma), nose and external ear infections, acne, burns, eczema, psoriasis, in case of ulceration for treatment of secondary infections, and for prevention of hospital infections. The chemical structure of pseudornonic acid A was determined to be 9-{4[5S(2S,3S-epoxy-5S-hydroxy-4S-methylhexyl)-3R,4R-dihydroxy-tetrahydropyran-2S-yI]-3-methylbut-2(E)-enoyloxy}nonanoic acid [E. B. Chain and G. Mellows, J. C. S. Chem. Comm. 847-848 (1974); R..G. Alexander, J. P. Clayton, K. Luk, N.H. Rogers, T.J. King, J.C.S. Perkin 1. 561-565 (1978)], as depicted by formula (1): / It is known that Pseudomonasfluorescens is able to produce the pseudomonic acid A. According to the British Patent No. 1,395,907, the Pseudomonas fluorescein NCIB 10586 strain is able to biosynthesize the pseudomonic acid complex consisting of pseudomonic acid A and its isomer being a double bond in the cis position between the carbon atoms C2 and C3 and pseudomonic acid B. The ratio of the components is 4.5:4.5:1. According to the Japanese patent application No. 52-70083, however, the Pseudomonasfluorescens Y-I 1633 strain is able to biosynthesize the pseudomonic acid complex consisting of the pseudomonic acid A, pseudomonic acid B and further two components with unknown structures in the ratio of 9:0.5:0.5. SUMMARY OF THE INVENTION The present invention is directed to a procedure for the preparation of pseudomonic acid A comprising cultivating on a medium comprising at least one organic nitrogen or carbon source, in submerged aerated conditions, a Pseudomonas bacterium strain capable of the biosynthesis of pseudomonic acid A, and fermentation of the Pseudomonas culture such that pseudomonic acid A is formed. Preferably the Pseudomonas bacterium strain is Pseudomonas sp. bacterium strain No. 19/26 deposited under accession No. NCAIM(P)B 001235 in the National Collection of Agricultural and Industrial Microorganisms, Budapest, Hungary, or its pseudomonic acid A-producing mutant or variant. The present invention also is directed to a Pseudomonas culture capable of biosynthesizing pseudomonic acid A in submerged aerated conditions, consisting essentially of a novel Pseuaomonas sp bacterium strain No. 19/26. The present invention further is directed to a biologically pure culture of a novel Pseudomonas sp. bacterium strain No. 19/26. BRIEF DESCRIPTION OF THE DRAWING Figure 1 is an HPLC chromatogram of the pseudomonic acid complex produced by Pseudomonas sp. No. 19/26 [NCAIM(P)B 001235]. DETAILED DESCRIPTION OF THE INVENTION In the course of searching antimicrobial antibiotics produced by bacteria, 20,000 microorganisms were isolated on nutrient agar medium containing 10 Afg/ml candicin and 5µg/ml cycloheximide in order to prevent the growth of fungi. Antibacterial effectiveness of the shaken flask culture of the isolated bacterial strains was examined on different Gram positive and Gram negative test-microorganisms and their resistant variants for antibiotics occurring in the therapeutic practice. By the application of the antibiotic resistant test-microorganisms we wanted to promote the recognition of antibacterial antibiotics with new mode of action. In the course of the above described examinations a bacterium isolate, designated as "strain No. 19/26," was selected. Originally, this organism had been obtained from a soil sample collected in Argentina. Its culture broth had significant antibiotic effect against Bacillus subtilis ATCC 6633, Staphylococcus aureus SMITH, the penicillin-resistant Staphylococcus aureus 1110, an unnumbered Streptococcus pneumoniae strain, Streptococcus pyogenes A 115 ROBB, Alca/igenesfaecalis 140001, Bordetella hronchiseplica ATCC 4617, Klebsiella pneumoniae ATCC 10031, the meticillin- and aminoglucoside-resistam Staphylococcus aureus MRS A 1190R and two unnumbered Mycoplasma pneumoniae and Haemophilus injluenzae strains, respectively. Later the antibacterial metabolic product of strain No 19/26 was isolated from its culture broth and as a result of a chemical structure analysis it was found to be identical to the pseudomonic acid A. At the same time this selected pseudomonic acid A-producing bacterium strain was subjected to taxonomic studies First a preliminary comparative investigation was done with selected closely related bacterial strains using the Bio-Merieux ATB Expression equipment with ID 32 GN strip The latter is suitable for a relatively fast (e.g. generic level) taxonomic identification of a given unknown bacterial strain if its diagnostic propenies can show a desired hig!i degree of phenetic similarity to at least one of those of the authentic strains that were incorporated in the database of the equipment. Carbon-source utilization spectra of an unknown bacterial strain to be investigated can be determined with this equipment in 32 tests with 14 different sugars and 14 organic acids, respectively, added into a minimal culture medium completed with a complex.of growth factors. Evaluation of the utilization test results is done after the inoculation and passing 48 hours incubation time by means of automatic turbidity measurement on the developed microcultures designating the growth intensities (the reactions) as + or - or ?, respectively The results of the synchronously studied biochemical tests are evaluated by the software of the equipment. The obtained physiological-biochemical data of strain No. 19/26 were correlated with those of the bacterial strains that are listed in the database (the total number of such strains is in this case 114, among them 14 belonging to different Pseudomonas species. Pseudomonas aeruginosa I and //, Pseudomonas alcaligenes, Pseudomonas fluorescens I and //, Pseudomonas mendocina. Pseudomonas mesophi/ica, Pseudomonas pickettii, Pseudomonas pseudomallei, Pseudomonas vesicularis, Pseudomonas cepacia. Pseudomonas diminuta. Pseudomonas putida. and Pseudomonas slutzeri). According to the outcome of this correlation experiment, strain No 19/26 proved to be a member of the genus Pseudomonas. After successful identification at generic level of strains No. 19/26 we tried to identify it at species level by means of classical taxonomic investigation method. Cultures of strain No. 19/26 can multiply on suitable solid media in form of non-sporulating Gram negative rods, 0.6-1.1 to 1.3-4.0 microns. These are actively motile with polar flagella. These obligately aerobic chemoorganotrophic rods can attack glucose only oxidatively They carnot ferment and are unable to respire with nitrates as terminal electron acceptors, but show positive catalase reaction. They do not develop visible colonies on simple synthetic media and for their multiplication always require growth factors. Growing under optimal conditions their colonies are arginindihydrolase positive, produce fluorescent pigments but the presence and accumulation of poly-P-hydroxy-butyrate in the cells cannot be detected. Further features of strain No. 19/26 are the following: development of it; colonies was not observed at 41°C; it does not produce levan from sucrose, it does not hydrolyze gelatin and starch, it is oxidase positive, and it does not utilize glucose, 2-keto-gluconate, trehalose and meso-inositol as the sole carbon source. All of these diagnostic properties clearly separate the Pseudomonas strain No. 19/26 from the species P. aeruginosa, P fluorescens, P. chlororaphis, P. aureofaciens, P. syringae, P. viridiflava. P. dehorii, P. stutzeri, P. mendocina, P. a/caligenes, P. pseudoalcaligenes and P. ptitida species. Cultivation of strain No 19/26 can be carried out only on media prepared with (0.2-1.0%) yeast extracts, com steep liquor or other complex natural nutrients containing very different growth factors On the basis of these particular diagnostic properties strain No. 19/26 can be considered as an organism which belongs to the so called "non-typical Pseudomonas species." Such "non-typical Pseudomonas species" may be incorporated into a newly established bacterial taxon in the future. (N.J. Palleroni, "Psuedomonaceae," Bergey"s Manual of Systematic Bacteriology, 1,141-219, ed.: N.R. Kriegand J.G. Holt, Baltimore: Williams and Wilkins, 1984) Strain No 19/26 was deposited on July 16, 1996 under accession No NCA1M(P)B 00123.5 in the National Collection of Agricultural and Industrial Microorganisms, Budapest, Hungary. According to a preferred method of the invention the Pseudomonas sp , bacterium strain No. 19/26 deposited under accession No. NCAIM(P)B 001235 is used for the manufacture of substantially pure pseudomonic acid A. The selected strain can use peptone, beef extract, com steep liquor, yeast extract, casein and soy-bean meal as nitrogen sources. Beside; the above nitrogen sources, carbon sources such as glucose, glycerol and sunflower oil can be applied in different combinations. Althouuli the medium components of natural origin contain mineral salts, it is advantageous to add some mineral salts (e.g. ammonium, calcium, iron, zinc, copper, magnesium, manganese, sodium or potassium salts) to the seed culture medium. Magnesium sulfate, manganese dichloride, ferrous sulfate, zinc chloride, copper II sulfate, ammonium sulfate, potassium dihydrogen phosphate, sodium chloride and calcium carbonate are preferred. Maintenance of the strain is carried out on siant agar medium containing peptone-casein at 4°C, freshly transferring it every two weeks. To maintain the pseudomonic acid A productivity, the strain can be stored properly both by deep-freezing the culture or in lyophilized form. In the course of the fermentation, the Pseudomonas sp. No. 19/26 strain is seeded into a suitable medium and cultivated in submerged and aerated fermentation conditions. The pH of the culture medium is preferably set to a neutral value (pH = about 7.0), the temperature of the cultivation between is about 20°C and about 30°C, preferably between about 24nC and about 26°C. Depending on the fermentation conditions the maximum value of the antibiotic productivity can be reached after 50-60 hours. The pseudomonic acid complex being formed in the course of the fermentation comprises substantially pure pseudomonic acid A, although as a result of the biosynthesis, small quantities of the pseudomonic acid B of formula (II) OH and pseudomonic acid C of formula (HI) are also formed. (III) The antibacterial activity of the pseudomonic acid antibiotic complex is determined by agar diffusion microbial mithod. The medium is a beef-extract-peptone-glucobe-containing agar, its pH is 6.5 and the test organism is Bacillus subtilis ATCC 6633. The activity value obtained by the microbiological method represents primarily the quantity of the pseudomonic acid A, since the quantity of the other pseudomonic acid components is very small, and relating to the component A their specific activity against the test-mi ~roorganism—especially in the ^ase of the component B—is considerably weaker. In the course of the fermentation the exact quantity of the pseudomonic acid A and the accompanying minor components in the fermentation broth are determined by high pressure liquid chromatography (HPLC) in which the supernatant of the ultrasonic treated and centrifuged sample of the broth diluted to twice by ethanol is investigated (equipment: LKB 2248 pump, LKB 2141 UV detector (analysis at 222 nm), column: Nucleosil C„ 10 pirn (BST), eluent: mixture (35:65) of acetonitrile and 0.1 M NH4HYP04 solution (pH:5.0), flow rate: 1.2 ml/min), Retention times: pseudomonic acid A (PSA) is 8.5 min, pseudomonic acid B (PSB) is 6.0 min, and pseudomonic acid C (PSC) is 22.5 min. The HPLC chromatogram of the pseudomonic acid antibiotic complex biosynthesized by Pseudomonas sp. No 19/26 strain can be seen in Figure 1. This chromatogram indicates that the pseudomonic acid complex obtained by the fermentation acid No. 19/26 strain of substantially pure pseudomonic acid A. The total quantity of the pseudomonic acid B and pseudomonic acid C components is below 5%. In this way it became evident that the Pseudomonas sp. No. 19/26 strain is able to biosynthesize the antibiotic in a more favorable composition, than the pseudomonic acid complex producing- Pseudomonas fluorescens strains published earlier. This fact is advantageous for industrial production The process according to the present invention is illustrated by the example below. However, the present invention should not be construed as limited thereby. example Pseudomonas sp. No. 19/26 strain was maintained on PCA marked slant agar medium. Composition of the PCA medium was the following: beef extract 3g peptone 5g agar 15g diluted up to 1000 ml with distilled water. The pH of the medium was 7.0-7.2. Seeded slant agar was incubated at 25°C for 24 hours, and the cells were suspended in 5 ml normal saline solution (cell number in the suspension: 10"- 10"°/ml). 1 mi of the suspension obtained was seeded into 100 ml 1-21 marked seed culture medium sterilized in a 500 ml Erlenmeyer flask. Composition of 1-21 medium was the following. glucose 10g glycerol 5g corn steep liquor 3g ammonium sulfate 2g potassium dihydrogen phosphate 0.4g magnesium sulfate-water (1:7) 04g manganese dichloride-water (1:2) 003g calcium carbonate 4g sunflower oil 2g diluted up to 1000 ml with tap water. The pH of the medium is set to 7.0 before sterilization. The flask containing the seeded medium was shaken on a shaking table (260 RPM, amplitude 10 cm) at 25°C for 18-20 hours. After this 50 ml (1%) of the shaken culture was seeded into 5 liter medium with mark E-5 sterilized at 121°C for 45 min in a 10 liter jar fermentor. Composition of E-5 medium was the following. diluted up to 5 liters with tap water. (*Glucose was sterilized in 50% solution for 30 min, and added into the medium .~6ether u ith the seeding material.) The pH of the medium was set to 7.0 pefore sterilization. The cultivation was stirred and aerated for 50-60 hours. The temperature of the cultivation was 25Co strring tate was 500 RPM airflow rate was 200 litters/how. As an antifoaming agent, sunflower oil was used in minimal quantity (about 20-30 ml per fermentor). The biosynthesis of the pseudomonic acid complex was started at hours 8-10 of the fermentation and the maximal antibiotic concentration was experienced at hours 55-60 hours of the culture. Isolation of the pseudomonic acid A from the fermentation broth was carried out as follows: After finishing the fermentation, the 4.5 liter culture broth obtained was centrifuge^, then the pH of the supernatant (4.06 liter) was adjusted to 4.5 with diluted (20%) sulfuric acid. The acidified liquor was then twice extracted by 2.03 liters ethyl acetate. The phases were separated and a sharp phase was prepared from the emulsive organic phase by centrifugation. The combined extract was evaporated in vacuum. The crude product obtained (2.45 g) was dissolved in a 25 ml mixture of chloroform-methanol-99 5% acetic acid (93:5:2), and the solution obtained was loaded on the column (hei«ht:diameter = 28 5) prepared from 245 g Kieselgel 60 (particle size: 0.063-0.2 mm, Reanal) and" with the above solvent mixture. Elution was carried out with the above solvent mixture. In the course of the elution 50 ml fractions were collected and the pseudomonic acid A content of the fractions was analyzed by thin layer chromatography using Kieselgel 60 (DC-Alufolien: 105554, Merck) adsorbent a.td chloroform-methanol-99 5% acetic acid (90:8:2) developing solvent mixture. Fractions 34-50 eluted from the Kieselgel 6C column contained pseudomonic acid A. These fractions were combined (850 ml) and meanwhile cooling 280 ml water was added to the solution obtained. After this the pH of the solution mixture was adjusted to 4.5 v. ith IN aqueous sodium hydroxide solution. The organic solution was separated from the water phase, then the water phase was extracted again by 280 ml chloroform. The combined extract was evaporated in vacuum, thus pure pseudomonic acid A could be obtained. The column chromatographic fractions 14-15 contained pseudomonic acid C, while fraction 54 contained pseudomonic acid B, from the minor components could be recovered in pure form by the above written procedure. The spectroscopic characterization of the isolated pseudomonic acid components was described using the numbering system of structural formula (I), shown above. Spectroscopic characterization of the pseudomonic acid A: Ultraviolet spectrum (10µg/ml, in 95% ethanol solution): Infrared spectrum "H-NMRspectrun 5 [ppm j, Coupling constant (Hz) Assignment (integral), multiplicity 1.33-1.43 (9H)m 1.22 (3H)d 0.94 (3H)d 12-H; 4"-H2; 5"-H2"; 6"-H2; 7-H2 14-H3 I7-H3 ,3C-NMR spectrum (CDCl., solution, 6^^ = 0): * interchangeable assignments Chemical ionization (CI) mass spectrum: Characteristic spectral data: Spectroscopic characterization of the pseudomonic acid B: Ultraviolet spectrum (10µg/ml, in 95% ethanol solution)λmax = 222nm Spectroscopic characterization of the pseudomonic acid C; Ultraviolet spectrum (lOng/ml, in 95% ethanol solution); λmax= 222 nm 14 5 [ppm] (integral), multiplicity Coupling constant (Hz) Assignment Although certain presently preferred embodiments of the invention have been described herein, it will be apparent to those skilled in the art to which the invention pertains that variations and modifications of the describe embodiment may be made without departing from the spirit and scope of the invention. Accordingly, it is intended that the invention be limited only to the extent required by the appended claims ind the applicable rules of the law. WE CLAIM: 1. A process for the preparation of pseudomonic acid A having the steps of providing a biologically pure culture of Pseudomonas sp. bacterium strain No. 19/26 deposited under accession No. NCAIM(P)B 001235 in the National Collection of Agricultural and Industrial Microorganisms, Budapest, Hungary, and fermentation of the Pseudomonas culture such that pseudomonic acid A is formed, isolating the formed pseudomonic acid A. 2. A process as claimed in claim 1 wherein said Pseudomonas bacterium strain is a mutant or a variant which retains the ability to produce pseudomonic acid A. 3. A process for the preparation of pseudomonic acid A comprising the steps of cultivating on a medium comprising at least one organic nitrogen or carbon source, in submerged aerated conditions, a Pseudomonas bacterium strain capable of the biosynthesis of pseudomonic acid A. 4. The process as claimed in claim 1 or 2, wherein said at least one organic nitrogen or carbon source is selected from the group consisting of glucose, glycerol, sunflower oil, casein, soybean meal, peptone, beef extract, corn steep liquor, and yeast extract. 5. The process as claimed in claim 1 or 2, wherein said medium contains at least one mineral salt. 6. The process as claimed in claim 5, wherein said at least one mineral salt is selected from the group consisting of ammonium, calcium, iron, zinc, copper, magnesium, manganese, sodium and potassium salts, and mixtures thereof. 7. The process as claimed in claim 6, wherein said at least one mineral salt is selected from the group consisting of ammonium sulfate, calcium carbonate, ferrous sulfate, zinc chloride, copper II sulfate, magnesium sulfate, manganese dichloride, sodium chloride, and potassium dihydrogen phosphate, and mixtures thereof. 8. The process as claimed in any of preceding claims 1 to 5, wherein said fermenting is carried out at a temperature between about 20°C and about 30°C. 9. The process of claim 1, wherein said fermenting is carried out at a temperature of between 24°C and 26°C. -18-

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