INCREASED RUBRADIRIN YIELDS

Abstract: Rubradirin bioactivity production yields are increased by incorporating into the rubradirin producing fermentation medium a nonionic adsorbent resin in a size and form suitable for agitated distribution in said fermenting medium for a time sufficient to increase the rubradirin bioactivity titer in that medium. The rubradirin bioactivity titer is also increased by incorporating into said fermentation medium a vegetable oil such as corn oil, peanut oil, cottonseed oil and soybean oil.

Description: INCREASED RUBRADIRIN YIELDS FIELD OF THE INVENTION This invention relates to fermentation processes or methods for producing antibiotics, specifically rubradirin and compounds associ- ated therewith, in fermentation production methods. More particu¬ larly, this invention provides a method or process for increasing the amounts (yields) of rubradirin generated in fermentation media during production methods, involving the use of (1) commercially available nonionic, pH neutral resins and/or (2) vegetable oils, both added to the fermentation medium. BACKGROUND OF THE INVENTION AND INFORMATION DISCLOSURE STATEMENT The antibiotic rubradirin, previously known as rubradirin A, was patented in Johnson, et al. , U.S. Patent 3,335,057 where it was claimed by its chemical and biological properties. The chemical structure was not specifically known at the time the application for that patent was filed. In two publications: (a) B.K. Bhuyan, et al., Antimicrobial Agents and Chemotherapy - 1964. (1965), "Rubradirin - A New Antibi¬ otic, I. Fermentation and Biological Properties," pp. 91-96; and (b) C.E. Meyer, ibid., "Rubradirin - A New Antibiotic, II. Isolation and Characterization," pp. 97-99, the details for the early fermentation of rubradirin and its antibacterial properties and physical chemical properties were disclosed. Hoeksema U.S. Patent 4,107,295 disclosed and claimed the antibacterial compound and a process for making rubradirin B by its physical chemical properties and disclosed a tentative chemical structural formula therefor. Hoeksema U.S. Patent 4,107,296 disclosed and claimed rubradirin C by its chemical and biological properties. Hoeksema U.S. Patents 4,137,410 and 4,171,437 disclosed and claimed some degradation compound per se products of rubradirin and rubradirin B by chemical structure, and acylates thereof, which com¬ pounds are disclosed as being useful as antibacterial agents, as ultraviolet (UV) light absorbers and as anti-ultraviolet light addi- tives to plastic compositions. The chemical structure of rubradirin and rubradirin B, rubra¬ dirin C and some of their degradation products, are now more recently set forth in J . Am. Che . Soc. 104:5173 (1982) by H. Hoeksema, et al. In a book series entitled Antibiotics. VI. Modes and Mechan¬ isms of Microbial Growth Inhibitors. (1983) in a chapter on "Rubra¬ dirin," by F. Reusser, the chemistry and biological functions of rubradirin are reviewed. Reusser, et al. , U.S. Patent 4,749,568, disclosed and claimed that rubradirin can be used to treat rubradirin-sensitive, methacil- lin-resistant Staphylococcus infections. Marshall, Little, and Johnson in "A New Process and Organism for the Fermentation Production of Volonomycin," The Journal of Antibi- otics XXXIV:902-904 (July 1981) disclose the production of the antibiotic, PAULOMYCIN, from Streptomyces paulus. The production of antibiotic was increased when the fermentation incorporated from one to 12 grams per liter of a resin, XAD-2 (Amberlite) , in the growth medium. Marshall, McWethy, Visser, Cialdella and Laborde in "Current Fermentation Technology for the Production of Antibiotics from Actinomycetes: The Example of Paulomycin," Developments in Indus¬ trial Microbiology 28:105-114 (1987) also disclose increased produc¬ tion of antibiotic when the fermentation incorporates XAD-2 resin in the growth medium. However, the use of resins in one antibiotic fermentation medium does not predict what the effect on antibiotic compound yield will be when using such a resin in another fermentation procedure. To date, the commercial development of rubradirin has bee discouraged or impaired by the lack of know-how to produce it i acceptable amounts or recoverable yields. OBJECTS OF THE INVENTION It is an object of this invention to provide a process or metho for improving the fermentation production yields of rubradirin an related fermentation derived compounds using commercially available nonionic resins in the mixture during the fermentation or incubatio period. It is another object of this invention to further improve th production yields of rubradirin and related fermentation derive compounds by adding vegetable oils to the fermentation mixture i addition to the nonionic resin in the mixture. SUMMARY OF THE INVENTION Briefly, this invention provides a method or process fo improving the weight yield of rubradirin or rubradirin-like compounds which respond like rubradirin by the assay method used to determine the maximum or optimum economical time to recover the yield of rubra- dirin-like compounds produced in that fermentation mixture comprising fermenting the rubradirin producing microorganisms in an aqueous aerobic fermentation medium in the presence of a nonionic adsorbent resin. Further, higher assay numbers indicating higher yields of rubra¬ dirin and rubradirin acting compounds are also observed when the aqueous fermentation recipe mixture also contains a plant-derived vegetable oil such as corn oil, peanut oil, cottonseed oil, or pre¬ ferably soybean oil. More particularly, this invention provides a process or method for improving the fermentation yields of rubradirin bioactivity in an aerated fermenting mixture in the presence of a rubradirin producing microorganism which comprises fermenting the rubradirin producing microorganism in an aerated growth medium containing a nonionic adsorbent resin in a size and form suitable for agitated distribution in said fermenting medium, for a time sufficient to increase the rubradirin yield in that medium over such a fermentation yield measured in the absence of said resin in the fermentation medium. We prefer to add the resin to the fermenting mixture about 20 to 25 hours after the fermentation process is allowed to begin. Another aspect of this process invention also provides a further improvement wherein the fermentation growth medium recipe also includes an amount of an economical vegetable oil such as corn oil, peanut oil, cottonseed oil, buc preferably soybean oil, in an amount sufficient to increase the rubradirin bioactivity yield of such medium over the yield obtained in the absence of said vegetable oil. DETAILED DESCRIPTION OF THE INVENTION Aqueous fermentation media and microorganisms that can be fer¬ mented therein to produce rubradirin and related compounds are described, e.g., in Johnson, et al., U.S. Patent Nos. 3,335,057 and 4,107,296. To these fermentation recipe mixtures this invention adds an amount of the nonionic resin effective to enhance the yield of rubradirin and related compounds over the term of the fermentation run, usually 2 to 4 days. The resin can be added to the initial fermentation recipe as it is being prepared and then sterilized. Preferably, however, the resin is added as a sterile aqueous slurry to the fermentation mixture after fermentation has proceeded for a period of time. As a further option, we prefer to add vegetable oil, as indi¬ cated above, preferably soybean oil, in an amount optimally effective to further increase the yield of the rubradirin and compounds that react like rubradirin in the fermentation mixture assay method being used. Generally from about 5 to about 20 ml/liter of fermentation mixture of the plant oil, e.g., soybean oil, is sufficient. Prefer¬ ably about 8 to 12 ml of soybean oil/liter of fermentation oil is used. The term "rubradirins" used herein to define the yield of the fermentation generally includes rubradirin (U.S. Patent 3,335,057), rubradirin B (U.S. Patent 4,107,295), rubradirin C (U.S. Patent 4,107,296), rubradirin aglycone (J. Antibiotics 32(7) :773-776. July 1979), depending upon whether the mixture has been exposed to light, as well as more recently discovered compounds protorubradirin, protorubradirin D and rubradirin D, which are claimed in a separate application and whose chemical structures have not been completely elucidated but which react as a "rubradirin" in one or more of the biological or chemical assays used to measure the yield or content of the desired rubradirins as product of the process. Examples of the nonionic resins that are used in the fermenta¬ tion mixtures according to this invention can be obtained from commercial sources. The resins are generally obtained in ball or bead shapes and can generally be washed with acetone, then with methanol and then with water to ensure cleanliness, and added directly into the initial fermentation medium which is to be used, and then sterilized in that medium, and stirred in that medium to obtain at least some degree of distribution in the mixture. Prefera¬ bly, however, as exemplified by the examples, we prefer to mix or distribute the nonionic resin beads or particles into an appropriate aqueous mixture or into a portion of aqueous fermentation medium, and sterilized in such mixture or medium, and then to add that sterilized nonionic resin mixture or bead containing medium to the ongoing fermentation mixture in one or more increments or "shots" preferably starting about 15 to 25 hours after the fermentation has been allowed to progress. Examples of commercially available nonionic adsorbent resins, which are generally available in bead, particle or chip form, include the translucent or white colored resins in wet or dry bead form available under trade names such as Amberlite® XAD-4, XAD-16, XAD- 1180 and XAD-2000 brands of bead resin, available from Rohm and Haas Company, Philadelphia, PA, USA, as well as similar active resins available under such names as the Diaion HP-20 and HP-21 resins of the Mitsubishi Chemicals, Ltd of Tokyo, Japan. For reasons of cost and optimum effectiveness in our fermentation media, we prefer to use the Amberlite® XAD-16 resin in our process. Such resin is described in product literature as having a white, wet bead appearance, a porosity (by nitrogen desorption method) of 1.82 cc/g, a percent porosity of 66, a total surface area of 860 square meters/g (by the N2, BET method), an average pore diameter of 144 Angstroms, and particle size (U.S. Sieve Series) as follows: Size Percent +20 Mesh 0-2 -20 to +35 Mesh 40-60 -35 to +50 Mesh 33-53 -50 Mesh 2-10 However, the other above types of resin can be used. Other useful nonionic adsorbent resins which can be used include Amberlite ® XAD-2 (a polymer of styrene) , XAD-7 (a polymer of an acrylic ester) , products of the Rohm and Haas Company of Philadel¬ phia, PA, USA. The optional vegetable oil, preferably soybean oil, can be added to the initial fermentation mixture in amounts sufficient to increase the assay yield of rubradirin and rubradirin-like reacting compounds in that fermentation medium. We have done fermentation runs using about 10 ml of soybean oil per liter of fermentation medium but the optimum amounts thereof over time have not yet been determined. We expect that amounts of vegetable oil, such as soybean oil, will be effective to increase the rubradirin yields relative to control (no vegetable oil) fermentations when such vegetable oil is used at a range of from about 5 to about 20 ml of such vegetable oil per liter of fermentation medium. The optimum effect on yield by such vegetable oil will probably vary depending on the choice of the vegetable oil, its concentration in the medium, and time of its presence and the length of time of the fermentation, in addition to the choice and amount of the nonionic adsorbent resin that is used in the fermenta- tion medium. The process of this invention produces rubradirin and rubra¬ dirin-like compounds in fermenting media when inoculated with the organism Streptomvces achromogenes v. rubradiris (UC ® 8051) . A culture of this living organism was deposited on 4 May 1988 in compliance with the terms of the Budapest Treaty at the Agricultural Research Patent Culture Collection, Microbial Properties Research, Northern Regional Research Center, Peoria, IL, USA, deposit number NRRL 18366. The progressive increase in the fermentation medium for the growth of this organism is done as follows: The contents of a frozen ampoule of the above organism are inoculated into a commercially available bacterium growth seed mediu (GS-7), and the inoculated medium is incubated for 3 days at 28 β C. Primary seed culture, as above, is placed in one liter shake flasks containing 300 ml of GS-7 medium (Pharmamedia ® trader's pro¬ tein), 25 g, glucose, 25 g, and polypropylene glycol, 0.2 ml per liter at pH 7.2) are inoculated with three loops of the Streptomvces achromogenes v. rubradiris (UC ® 8051) in the form of a soil stoc culture or from a 0.5 ml vegetative ampoule, sample thereof, and the the shake flasks are incubated for 72 hours on a rotary shaker at 28°C. Secondary seed vessels containing 250 liters of the commercial seed medium (GS-7) are inoculated with a single shake flask culture containing the 300 ml of primary seed medium from above. The secondary seed vessel fermentation is conducted at 28°C with a agitation rate of 280 rpm, an aeration rate of 200 standard liters per minute (slm) while maintaining a back or head pressure of 7 psig in the vessel over an incubation time of 48 hours. For antibiotic compound preparation 250 liter fermentor tanks containing aqueous media of the following ingredients are used to exemplify the merit of this process invention. Component Distiller's Solubles Corn Starch Corn Steep Liquor Sodium Nitrate pH Adjustment to 7.2 with NaOH solution and then add Calcium Carbonate^ 5 3 5 Soybean Oil Polypropylene Glycol 3 0.2 ml 0.5 ml 0.2 ml Resin -- 60 ml 4 60 ml 5 1. Solulac® , Grain Processing Corporation 2. Q-100, Huber 3. Ucon ® LB-625, Union Carbide 4. DIAION ® HP-21 Resin, Mitsubishi, sterilized in the initial fermentation mix 5. DIAION ® HP-21 Resin, supra. added at 20 hours, in sterilized aqueous mixture 6. DIAION ® HP-21 Resin, supra, or Amberlite ® XAD-16 (Rohm and Haas) added at 20-23 hours of fermentation The antibiotic production fermentors are inoculated with 12.5 liters of secondary seed culture prepared as above and incubated for approximately 90 hours at 28"C. Other conditions are agitation rate, 280 rpm, aeration rate, 200 slm, back pressure, 7 psig, and pH con- trol at 7.5 with sulfuric acid. The bioactivity of the rubradirin and rubradirin compounds which act like rubradirin in the bioassay of samples of the whole fermenta¬ tion mixtures can be used to determine the relative yields of such antibiotic compounds in these fermentation media. A biounit assay using the organism Micrococcus luteus (UC ® 130) as the test organism, was compared against rubradirin (formerly the rubradirin A of Johnson, et al. , U.S. Patent 3,335,057) as a standard. In this test, one biounit of anti-M. luteus activity is defined as the amount of antibiotic that when applied to a 12.7 mm paper disc (Schleicher and Schuell No. 740-E) produces a zone of growth inhibition of 20 mm when applied onto a seeded agar culture. To relate biounit potency con¬ tained in the fermentation sample to the amounts of rubradirin pro¬ duced, such bioassays are performed in the presence of side-by-side standards of known rubradirin samples. Because of variation observed in the biounit assay, discs containing known amounts of rubradirin are used with every assay tray. Aliquots (2 ml) of whole fermentation media suspensions are taken at 2, 3 and 4 days and are mixed with an equal volume of ace¬ tone. The mixtures are allowed to stand for 15 minutes and then are agitated for one minute before centrifugation at 5,000 x g for 5 minutes. The supernatant fluids are then assayed for anti-M. luteus activity following serial dilution (2-fold increments) in 50% aqueous acetone buffered with 50 mM (millimolar) potassium phosphate, pH 7. Such dilutions are routinely assayed between 4- and 2096-fold. The buffered acetone solvent have no antibacterial activity on this assay. The assay trays are developed by incubation at 32°C. Following this primary dilution in acetone, further dilutions (2-fold increments) are made in deionized H2O. Each resulting solu¬ tion assay is performed in the presence of the first 6 aqueous dilu- tions of the rubradirin standard. Assay discs containing diluted rubradirin are placed on the seeded trays before drying. Dilutions of samples are made until the inhibition zone on the plate is small enough to measure for antibiotic concentration and then back calcula- tions are made to determine the quantity of rubradirin in the orig¬ inal sample. Table 2 herebelow sets forth the comparative yield results in fermentation production of rubradirin components by Streptomvces achromogenes v. rubradiris (UC ® 8051) in shake flask size fermentation runs, where the indicated commercially available resins are added as part of the initial media. TABLE 2 The Effects of Resins on the Production of Rubradirin Components by S. achromogenes v. rubradiris UC ® 8051 Rubradirin Produced (μg/ml) Resin Level of Resin None XAD-2 XAD-2 XAD-7 XAD-7 HP-20 HP-20 HP-21 HP-21 Initial attempts to scale up the rubradirin value yields from shaker flask size runs to pilot plant tank size runs, using these nonionic adsorbent resins in the fermentation media, where the resin is added as part of the initial media being prepared and sterilized are better than (non-resin containing) control but somewhat lower than desired; ranging around 30 micrograms/ml at 69 hour by bioassay analysis. The use of these resins should include pH control at about pH 7.5. The process can be run with or without the vegetable oil, e.g., soybean oil, component in the mixture. We discovered in later runs that if the resin is added as a separate, sterile aqueous mixture at about 20 to 23 hours, the yiel of rubradirins in the fermentation suspension samples increase to about 52 micrograms/ml by 92 hours with a pH control at 7.5. This yield is approximately five-fold the rubradirin yields in the control vessel without the resin. Then, additional fermentation runs are done with different non¬ ionic adsorbent resins, and, in some cases, with the addition of soy- bean oil into the fermentation media. The pH of these mixtures are controlled at about 7.5 and the resins are added separately as sterile aqueous/resin mixtures at about 20-23 hours of incubation of the fermenting media. Yields of the rubradirins have been increased up to about 300 to 346 μg/liter by the use of these resins in the fermentation media, particularly when combined with the use of the vegetable oil, e.g., the soybean oil, in the fermentation media. Both of the Mitsubishi HP-21 and the Rohm and Haas Amberlite ® XAD-16 resins, added at about 23 hours of incubation time, used with soybean oil, produce fermenta- tion mixtures containing more acceptable yield amounts of rubradirin- type compounds, and these are presently our preferred resins to use in this process. The following table summarizes the rubradirin bioactivity obtained in pilot fermentation operations under variable conditions of operations of the process. TABLE 3 Rubradirin Bioactivity in Pilot Fermentors (in μg/ml units) Fermentation Runs Hours Control 1 2 3 4 5 44 11 8 51 58 41 46 70 6 30 41 107 154 246 90 4 22 52 113 326 346 Resin None HP-21 HP-21 HP-21 HP-21 XAD-16 mix shot shot shot shot Soybean oil None None None None lOml/1 lOml/1 pH Control 7.5 8.5 7.5 7.5 7.5 7.5 Also, due to organism seed variability in pilot tank fermenta¬ tions employing soil stock samples of the organism, and the effect such source of the organism seems to have on the metabolic profile and rubradirin antibiotic final liters, we prefer to use a frozen vegetative ampoule source of the organism, which seems to provide substantially enhanced yields of rubradirin in these fermentative media. 5 We conclude from these experiments that incorporation of pH neutral resins, such as the HP-21 or XAD-16 resins into the rubra¬ dirin production fermentation media results in significant improve¬ ments of rubradirin bioactivity. Other factors contributing to yield improvements of the rubradirin production media are a) addition of 10 the resin as a separate sterilized, e.g., a steam sterilized, addi¬ tion or shot at approximately 20-23 hours, b) inclusions of a vege¬ table oil, such as soybean oil, into the fermentation mixture, and c) control of the pH close to pH 7.5 with acid. To harvest the rubradirins from the fermentation mixture, the 15 fermentation mixture is filtered through an appropriately sized screen such as 100 to 200 mesh size openings. The filtrate contains the bulk of the organism cells and the growth medium components, the solids on the filter are mostly the resin, probably in the form of beads, having adsorbed thereon the bulk of the rubradirins. 20 The resin beads or other resin particulate form, filtered from the fermentation liquor, and protected from light, can be transported to where the resin is to be treated to recover the rubradirins. For example, about a 1.5 liter portion or batch of HP-21 (Mitsubishi) resin from such a fermentation, a dark brown resin, can 25 be placed in a 4 liter sintered glass or equivalent funnel and excess water can be removed under suction. Fatty material adhering to the resin can be removed by washing with a cyclohexane/methylene chloride mixture. For example, for the above 1.5 liter volume of resin beads can be batch washed with a cyclohexane/methylene chloride (4:1, v/v, 30 3x2 liters) first under gravity and then under suction to remove adhering fatty material. These washes can be discarded. The washed resin can then be eluted with ethyl acetate to remove the rubradirin * and related compounds into the ethyl acetate. For the 1.5 liters of resin beads we used about 10 liters of ethyl acetate. After separat- 35 ing the resulting solution from the beads, the ethyl acetate solvent can be removed, e.g., under a vacuum extractor to leave the crude rubradirins as a residue. The residue can be taken up in a small volume of ethyl acetate and the resulting concentrated solution can be diluted with a hydrocarbon liquid such as hexane, heptane or a commercial hexane mixture such as Skellysolve ® B which precipitates the rubradirins as a reddish powder, which powder can be separated by filtration and then dried in vacuo. This dried material contains all the rubradirin components. This dried solid can then be further purified into its different compound components by preparative chromatography procedures to iso¬ late the respective rubradirin compounds. The rubradirins are amor¬ phous materials with melting points above 300°C, and purity is best established by liquid chromatography or spectrometrically. In our above batch example, the dried reddish powder solid is dissolved in 20 ml of tetrahydrofuran (THF) and purified by preparative reversed phase chromatography procedures, using commercially available PrePak 500/C-18 chromatography columns with a Waters Prep 500 Instrument. The THF solution is transferred via pumps to two C-18 chromatography columns in series, pre-equilibrated with mobile liquid phase (0.05 M) aqueous ammonium acetate adjusted to pH 4.0 by the addition of a glacial acetic acid/THF, 1:1 v/v mixture and rinsed in, using an additional 30 ml. Elution of the column contents commences using the same 1:1 solvent mixture system isocratically (same proportions throughout the elution), at a flow rate of 150 ml/minute, monitoring at 365 nm (UV wave length) . Appropriate fractions of the liquid from the column are com¬ bined, and volatile material (solvents, etc.) are removed on a rotating evaporator at 30°C using a water pump pressure reducer, to give an aqueous suspension of a red solid. The solid is removed by filtration under suction on a sintered glass funnel and washed with water, and dissolved in chloroform. After drying the resulting solution using sodium sulfate dehydrator, a four-fold volume of Skellysolve B hexanes is added with stirring using a magnetic stir bar, to precipitate the rubradirin solids. The precipitate is •collected under suction on a sintered glass filter funnel and dried in a vacuum desiccator. Fractions 1 to 3 inclusive give 657.4 mg rich in a new "10.7 min." component, believed to be a mixture of rubradirin D and protorubradirin D, which are claimed in PCT/US 88/01493, filed 12 May 1988. Fraction 4 gives 417.4 mg of slightly impure rubradirin. Essen- tially pure rubradirin can be isolated from the 1.2846 g of material contained in fractions 5 to 7. The rubradirins produced by the fermentation process of this invention, and separated from the fermentation media, and thereafter from the resin particles or beads and purified can be used as an antibiotic in human and warm-blooded animal patients to treat rubra¬ dirin-sensitive, methicillin-resistant Staphylococcus infections, as described, for example, in the above referenced Reusser, et al. , U.S. Patent No. 4,749,568.

Claims: CLAIMS 1. A process for improving the yield of rubradirins in an aerated fermenting mixture in the presence of a rubradirin producing microor¬ ganism which comprises fermenting the rubradirin producing microor- ganism in an aerated growth medium containing a nonionic, pH neutral, adsorbent resin in a size and form suitable for agitated distribution in said fermenting medium for a time sufficient to increase the rubradirin bioactivity titer in that medium over such a fermentation titer measured in the absence of said resin. 2. A process as defined in claim 1 wherein said resin is added to the fermenting mixture about 20 to 25 hours after the fermentation process is allowed to begin. 3. A process as defined in claim 1 which also includes in the fermentation medium an amount of a vegetable oil selected from the group consisting of corn oil, peanut oil, cottonseed oil and soybean oil in an amount sufficient to increase the rubradirin bioactivity titer of such medium over such a titer obtained in the absence of said vegetable oil. 4. A process according to claim 3 wherein the vegetable oil is soybean oil.

Inventors: BANNISTER BRIAN, CIALDELLA JOYCE I, DOLAK LESTER A, MARSHALL VINCENT P, MCWETHY STEVEN J, BANNISTER, BRIAN, CIALDELLA, JOYCE, I., DOLAK, LESTER, A., MARSHALL, VINCENT, P., MCWETHY, STEVEN, J.
Applicants: UPJOHN CO, THE UPJOHN COMPANY
Application year: 1989
Main classification: C12P17
IPC codes: C12N1, C12P17, C12R1