Abstract:
This disclosure describes a novel process for the production of the known antibiotic nosiheptide using a new strain of Streptomyces glaucogriseus and mutants thereof.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of Invention 
     The present invention relates to a novel process for the production of the known antibiotic nosiheptide using a new strain of Streptomyces glaucogriseus and mutants thereof. 
     2. Description of the Prior Art 
     The applicants are not aware of any prior art patents or publications which, in their respective judgment, should be deemed to anticipate or render obvious the process or the microorganism described and claimed herein. By way of background, U.S. Pat. No. 3,155,581 to Rhone-Poulenc S. A. is cited, wherein the product nosiheptide (designated therein as antibiotic 9671-RP) and a method for its manufacture by the aerobic cultivation of the microorganism Streptomyces actuosus NRRL 2954 are claimed. The utility of nosiheptide as an antibacterial agent is disclosed in that patent. Also, Pascard, C., et al., J. A. C. S. 99:19 (Sept. 14, 1977) discloses the structure of nosiheptide. 
     BRIEF SUMMARY OF THE INVENTION 
     The instant invention relates to a new strain of Streptomyces glaucogriseus, sp. nov., and mutants thereof derived spontaneously or by conventional mutagenic or recombinant techniques. This invention further concerns a new process for the production of the antibiotic nosiheptide by fermentation, under controlled conditions, as well as the methods for recovery and concentration thereof from crude solutions. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     In accordance with the present invention, there is provided a new process for the production of the known antibiotic nosiheptide by the cultivation, under controlled conditions, of a new strain of Streptomyces glaucogriseus, sp. nov. 
     This new antibiotic-producing strain was isolated from a soil sample collected in Bernalillo County, N. Mex. and is maintained in the culture collection of the Lederle Laboratories Medical Research Division, American Cyanamid Company, Pearl River, N.Y. 10965, as culture number BP-189. A viable culture of this new microorganism has been deposited with the Culture Collection Laboratory, Northern Utilization Research and Development Division, U.S. Department of Agriculture, Peoria, Ill. 61604, and has been added to its permanent collection. It is freely available to the public from this depository under its accession number NRRL 12514. 
     The following is a general description of the microorganism Streptomyces glaucogriseus NRRL 12514, based on diagnostic characteristics observed. Observations were made of the cultural, physiological and morphological features of the organism in accordance with the methods detailed by E. B. Shirling and D. Gottlieb, Methods for the Characterization of Streptomyces species, Internat. J. Syst. Bacteriol. 16:313-340 (1966). Media used in the study were selected from those recommended for the taxonomic study of actinomycetes and soil bacteria by T. G. Pridham, et al., A Selection of Media for Maintenance and Taxonomic Study of Streptomycetes, Antibiotics Ann. 947-953 (1956-1957) and R. E. Gordon, The Taxonomy of Soil Bacteria, THE ECOLOGY OF SOIL BACTERIA, pp. 293-321 (T. G. R. Gray and D. Parkinson eds.), Liverpool University Press, Liverpool, England (1967), respectively, Chemical composition of the cell walls of the culture was determined using the method of H. A. Lechevalier, et al., Chemical Composition as a Criterion in the Classification of Actinomycetes, Adv. Appl. Microbiol. 14:47-72 (1971), as modified by J. L. Staneck and G. D. Roberts, Simplified Approach to Identification of Aerobic Actinomycetes by Thin-layer Chromatography, Appl. Microbiol. 28:226-234 (1974). Underscored descriptive colors are taken from K. L. Kelly and D. B. Judd, Color, Universal Language and Dictionary of Names, Nat. Bur. Stand. (U.S.) Spec. Publ. 440, Washington, D.C. (1976) and the accompanying Inter-Society Color Council, National Bureau of Standards Centroid Color Charts. Details on the following general description of the culture are recorded in Tables I-V below. 
     MICROMORPHOLOGY 
     Spores are formed in long spiral chains (Spira) on aerial sporophores. The spores are ovoid (0.6-0.8 micro×1.0-1.2 micron) and the surface of the mature spores is ornamented with spines approximately 200 nanometers in length. 
     CELL WALL COMPOSITION 
     Whole cell hydrolysates of this culture contain the L,L-isomer of diaminopimelic acid, placing it in the Type I cell wall group of Lechevalier, et al. (1971). This is typical of all Streptomyces species. 
     AMOUNT OF GROWTH 
     Good growth is observed on most media; moderate growth is observed on asparagine-dextrose agar, Benedict&#39;s agar, Czapek&#39;s-casamino acid agar, Emerson agar and glycerol-casein agar; poor growth is observed on nutrient agar. 
     AERIAL MYCELIUM AND/OR EN MASSE SPORE COLOR 
     Aerial mycelium is white; spore masses are blue shades ranging from 189. bluish white to 190. light bluish gray on most media but are green shades ranging from 153. greenish white to 154. light greenish gray on several other media. Sporulation is absent on Benedict&#39;s and Emerson agar, but ranges from sparse to heavy on other media. 
     SOLUBLE PIGMENTS 
     Produced on all media evaluated except nutrient agar; colors range from yellow through orange and red to brown, depending upon the medium. Dark wine-red soluble pigments are produced on oatmeal and malt extract media. 
     REVERSE COLOR 
     In brown shades, ranging from grayish brown to dark reddish-brown. 
     PHYSIOLOGICAL REACTIONS 
     Nitrates not reduced to nitrites; moderate liquefaction of gelatin in 14 days; litmus milk weakly peptonized in 14 days; melanoid pigments produced on both peptone-yeast extract-iron agar and tyrosine medium. Hydrolysis of adenine, hypoxanthine, and tyrosine, but not guanine or xanthine in 7 days. Carbohydrate utilization as per the method of T. G. Pridham and D. Gottlieb, The Utilization of Carbon Compounds by Some Actinomycetales as an Aid for Species Determination, J. Bacteriol., 56:107-114 (1948): good utilization of adonitol, galactose, glucose, inositol, maltose, melibiose, rhamnose and salicin; moderate utilization of arabinose, fructose, lactose, mannitol, mannose, raffinose, ribose, sucrose, trehalose, and xylose; poor utilization of dulcitol; no utilization of glycerol or melezitose. Growth observed at 10% but not 13% NaCl. 
     Streptomyces glaucogriseus BP-189 was compared with Streptomyces reference cultures from the Lederle Culture Collection which have similar characteristics, i.e., blue spores (B), chromogenic (C+), and having spiral spore chains (S) with spiny spores (SPY). The Rhone-Poulenc culture Streptomyces actuosus, a gray, smooth-spored (GY; SM) strain which produces nosiheptide, was also examined. 
     Isolate BP189, which is a blue-series streptomycete, bears no resemblance to Streptomyces actuosus NRRL 2757, a gray-series streptomycete, other than in the production of the antibiotic nosiheptide. Moreover, this isolate does not closely resemble any of the described spiny, blue-spored Streptomyces species whose spores are borne in spiral chains, including Streptomyces afghaniensis ATCC 23871, the taitomycin producer. Thus it was designated as a new species of the blue-series Streptomyces to be named Streptomyces glaucogriseus, sp. nov., because of the blue-gray to green-gray color of the spore masses on many media. 
     The observations of these cultures, made after 14 days of growth on yeast-extract-malt-extract agar, are recorded in Table I. 
     
                                           TABLE 1__________________________________________________________________________Morphological Comparisons of Streptomyces glaucogriseus NRRL 12514with Other Reference Cultures                  SolubleCulture      Spore Color                  Pigment                       Reverse Color                                Growth__________________________________________________________________________GY;S;C+;SMRC50 S. actuosus        Medium gray                  Orange-                       Grayish brown                                GoodNRRL 2757              brownB;S;C+;SPYRC51 S. afghaniensis        Bluish white                  Orange-                       Dark reddish-                                GoodATCC 23871             red  brownBB643 S. azureus        Light bluish-gray                  None Light yellowish-                                GoodATCC 14921                  brownAE4 S. chartreusis        Pale blue Yellow                       Brownish-orange                                GoodATCC 14922BE821 S. chartreusis        Grayish-green                  Orange-                       Moderate brown                                GoodNRRL B-2199            yellowBB102 S. coeruleofuscus        Light bluish-gray                  Reddish-                       Brownish-orange                                GoodPSA 187                brownBB103 S. coeruleorubidus        Very pale blue                  Orange                       Light brown                                GoodATCC 13740BB104 S. coerulescens        Pale blue Yellow                       Light yellowish-                                GoodPSA 168                     brownBP189 S. glaucogriseus        Light bluish-gray to                  Dark red                       Moderate reddish-                                Good        light greenish-gray                       brown__________________________________________________________________________ 
    
     
                                           TABLE II__________________________________________________________________________CULTURAL CHARACTERISTICS OFStreptomyces glaucogriseus NRRL 12514INOCULATION       INCUBATION 14 days         TEMPERATURE 28° C.     AMOUNT OF             AERIAL MYCELIUMMEDIUM    GROWTH  AND/OR SPORES  SOLUBLE PIGMENT                                        REVERSE COLOR                                                   REMARKS__________________________________________________________________________Asparagine     Moderate             White aerial mycelia becoming                            Orange-yellow                                        53.                                           moderateDextrose Agar     153. greenish-white. Sporu-   orange             lation light.Bennett&#39;s Agar     Good    White aerial mycelia becoming                            Red-brown   47.                                           dark             190. light bluish gray.       grayish             Sporulation heavy             reddish                                           brownBenedict&#39;s Agar     Moderate             Sparse white aerial mycelia.                            Yellow      89.                                           pale             No sporulation.               yellowCzapek&#39;s Solution     Good    White to 92. yellowish white                            Red-brown   39.                                           grayishAgar              aerial mycelia becoming       reddish             189. bluish white. Moderate   orange             sporulation.Czapek&#39;s-Casamino     Moderate             White aerial mycelia becoming                            Red-brown   39.                                           grayishAcid Agar         190. light bluish gray.       reddish             Moderate sporulation.         orangeEmerson&#39;s Agar     Poor to No aerial mycelia; substrate                            Brown       56.                                           deep     moderate             mycelia 92. yellowish white   brownGlycerol-Casein     Moderate             White aerial mycelia becoming                            Brownish    76.                                           lightAgar              153. greenish white to 189.   yellowish             bluish white. Moderate        brown             sporulation.Hickey-Tresner     Good    White aerial mycelia becoming                            Reddish-brown                                        47.                                           darkAgar              153. greenish white to 154.   grayish             light grayish green.          reddish             Sporulation heavy.            brownInorganic-Salts     Good    White aerial mycelia becoming                            Red         40.                                           strongStarch Agar       153. greenish white to        reddish             154. light greenish gray.     brown             Moderate sporulation.Nutrient Agar     Poor    Sparse white aerial mycelia                            None           Colorless             with scattered patches of             184. very pale blue             sporulation.Oatmeal Agar     Good    White aerial mycelia be-                            Dark red    47.                                           dark             coming 153. greenish white    grayish             to 154. light greenish gray.  reddish             Sporulation heavy.            brownTomato Paste-     Good    White aerial mycelia be-                            Dark red    47.                                           darkOatmeal Agar      coming 154. light greenish    grayish             gray to 190. light bluish     reddish             gray. Heavy sporulation.      brownYeast Extract-     Good    White aerial mycelia be-                            Dark red    43.                                           moderateMalt Extract Agar coming 190. light bluish      reddish             gray to 154. light greenish   brown             gray. Sporulation heavy.__________________________________________________________________________ 
    
     
                                           TABLE III__________________________________________________________________________MICROMORPHOLOGY OFStreptomyces glaucogriseus NRRL 12514                                                    SPOREMEDIUM AERIAL MYCELIUM AND/OR SPORIFEROUS STRUCTURES                                   SPORE SHAPE                                            SPORE SIZE                                                    SURFACE__________________________________________________________________________Yeast Extract  Spore chains arise as spiral chains from                                   Ovoid to 0.6-0.8 μm  aerial sporophores (Spira)       spherical                                            × Spiny                                            1.0-1.2__________________________________________________________________________                                            μm 
    
     
                                           TABLE IV__________________________________________________________________________MISCELLANEOUS PHYSIOLOGICAL REACTION OFStreptomyces glaucogriseus NRRL 12514INOCULATION                         TEMPERATURE 28° C.MEDIUM    INCUBATION PERIOD                  AMOUNT OF GROWTH                               PHYSIOLOGICAL REACTION                                                   REMARKS__________________________________________________________________________Peptone-Yeast  48            hours Good         Strong production of melanoidExtract-Iron Agar                   pigments(ISP-6)Tyrosine Medium          48            hours Good         Moderate production of melanoid(ISP-7)                             pigmentsLitmus Milk    7 days  Good         Slight peptonization          14            days  Good         Slight peptonizationNutrient Gelatin          7 days  Good         Slight hydrolysis          14            days  Good         Moderate hydrolysisOrganic Nitrate          7 days  Good         NegativeBroth          14            days  Good         NegativeNaCl Tolerance 14            days               Tolerates 10% but not 13% NaClAgarAdenine Agar   7 days  Good         HydrolysisGuanine Agar   7 days  Good         No HydrolysisHypoxanthine Agar          7 days  Good         HydrolysisTyrosine Agar  7 days  Good         Hydrolysis; melanoid pigment                               productionXanthine Agar  7 days  Good         No hydrolysis__________________________________________________________________________ 
    
     
                       TABLE V______________________________________Carbon source utilization pattern ofStreptomyces glaucogriseus NRRL 12514Incubation: 14 days           Temperature: 28° C.Carbon Source   Utilization*______________________________________Adonitol        3l-Arabinose     2Dulcitol        1Fructose        2d-Galactose     3d-Glucose       3Glycerol        0i-Inositol      3Lactose         2Maltose         3d-Mannitol      2d-Mannose       2d-Melezitose    0d-Melibiose     3d-Raffinose     2l-Rhamnose      3d-Ribose        2Salicin         3Sucrose         2d-Trehalose     2d-Xylose        2Negative Control           0______________________________________ *3 = Good utilization 2 = Fair utilization 1 = Poor utilization 0 = No utilization 
    
     It is to be understood that for the production of the antibacterial agent nosiheptide, the present invention is not limited to this particular organism or to organisms fully answering the above growth and microscopic characteristics, which are given for illustrative purposes only. In fact, it is desired and intended to include in the term &#34;Steptomyces glaucogriseus, sp. nov., NRRL 12514&#34; the natural (spontaneous) mutants of this organism as well as induced mutants produced from this organism by various mutagenic means known to those skilled in the art, such as exposure to X-ray radiation, ultraviolet irradiation, nitrogen mustard, actinophages, nitrosamines and the like. It is also desired and intended to include inter- and intraspecific genetic recombinants produced by genetic techniques known to those skilled in the art, such as, for example, conjugation, transduction, and genetic engineering techniques. 
     FERMENTATION PROCESS 
     Cultivation of Streptomyces glaucogriseus NRRL 12514 may be carried out in a wide variety of liquid media. Media which are useful for the production of this antibacterial agent include an assimilable source of carbon such as starch, sugar, molasses, glycerol, etc.; an assimilable source of nitrogen such as protein, protein hydrolysate, polypeptides, amino acids, corn steep liquor, etc.; and inorganic anion and cation salts, such as potassium, sodium, ammonium, calcium, sulfate, carbonate, phosphate, chloride, etc. Trace elements such boron, molybdenum, copper, etc. are supplied as impurities of other constituents of the media. Aeration in tanks, bottles and flasks is provided by forcing sterile air through or onto the surface of the fermenting medium. Further agitation in tanks is provided by a mechanical impeller. An anti-foaming agent such as lard oil or silicone defoamer may be added as needed. 
     INOCULUM PREPARATION 
     Shaker flask inoculum of Streptomyces glaucogriseus NRRL 12514 is prepared by inoculating 100 ml portions of sterile liquid medium in 500 ml flasks with scrapings or washings of spores from an agar slant of the culture. The following is an example of a suitable seed medium: 
     
         ______________________________________Corn starch       1.2%Dextrose          0.6%Beef extract      0.3%Yeast extract     0.5%Bacto®-tryptone.sup.1             0.5%Calcium carbonate 0.2%______________________________________ [.sup.1 A peptone, registered trademark of Difco Laboratories, Detroit, Michigan]- 
    
     The above ingredients are added to an appropriate amount of water, the pH is adjusted to 7.5 with an alkali metal hydroxide and the mixture is sterilized prior to inoculation. These flasks are incubated at 24°-35° C., preferably 28° C., with agitation at 210 r.p.m. for 40-56 hours and are then used to inoculate 12 liters of the same sterile medium in a bottle, which after incubation with aeration by a sterile air flow of 2.0 liters per minute at 24°-35° C., preferably 28° C., for 40-56 hours is used to inoculate 300 liters of the same sterile medium in a seed tank. This inoculum is incubated at 24°-35° C., preferably 28° C., for 24 hours with aeration by a sterile air flow of 150 liters per minute and impeller agitation at 200 r.p.m. and then used to inoculate tank fermentors. 
     TANK FERMENTATION 
     The following is a suitable medium for the production of nosiheptide in fermentation tanks: 
     
         ______________________________________  Corn starch           3.0%  Molasses 2.0%  Soy peptone           0.75%  Yeast extract           0.25%______________________________________ 
    
     The appropriate portions are mixed with sufficient water to make 3000 liters, sterilized, adjusted to pH 7.0-7.6, preferably pH 7.2, and inoculated with 3 to 10% of inoculum prepared as described above. Sterile aeration is supplied at 1650 liters of air per minute and the mixture is agitated by an impeller driven at 100 r.p.m. Defoamer is added when necessary. The temperature is maintained at 24°-35° C., preferably 28° C. The fermentation is continued for 40-140 hours, at which time the mash is harvested. 
     The invention will be described in greater detail in conjunction with the following non-limiting examples. 
     EXAMPLE 1 
     Inoculum Preparation 
     An inoculum medium is prepared according to the following formulation: 
     
         ______________________________________Corn starch           3,600  gDextrose              1,800  gBeef extract          900    gYeast extract         1,500  gBacto®-tryptone   1,500  gCalcium carbonate     600    gWater qs ad           300    liters______________________________________ 
    
     This medium is adjusted to pH 7.5 with the addition of 75 ml of 6 N sodium hydroxide and then sterilized at 120° C. for 60 minutes. 
     Washed or scraped spores from an agar slant of Streptomyces glaucogriseus NRRL 12514 are used to inoculate 500 ml flasks containing 100 ml of the above sterile medium. The flasks are placed on a rotary shaker and agitated at 180 r.p.m. at 28° C. for 48 hours. The resulting flask inoculum is transferred to a 12-liter bottle containing the same sterile medium and incubated at 28° C. for 48 hours with sterile aeration. The resulting bottle inoculum is used to inoculate a tank containing 300 liters of the same sterile medium. The tank is then incubated at 28° C. for 24 hours with impeller agitation at 200 r.p.m. and aeration supplied by a sterile air flow of 150 liters per minute, producing a tank inoculum. 
     EXAMPLE 2 
     Fermentation 
     A fermentation medium is prepared according to the following formula: 
     
         ______________________________________Corn starch          90     KgMolasses             60     KgSoy peptone          22.5   KgYeast extract        7.5    KgWater to             3,000  liters______________________________________ 
    
     This fermentation medium is adjusted to pH 7.2 with 6 N sodium hydroxide, sterilized at 120° C. for 60 minutes and then inoculated with 300 liters of the tank inoculum prepared in Example 1. The fermentation is carried out at 28° C., using Hodag FD-82® [a silicone antifoam, registered trademark of Hodag Chemical Corp., Skokie, Ill.] as a defoaming agent. Sterile aeration is supplied at 1650 liters of sterile air per minute. The mash is agitated by impellers driven at 100 r.p.m. At the end of approximately 120 hours of fermentation time, the mash is harvested. 
     EXAMPLE 3 
     Preliminary Isolation of Nosiheptide 
     A fermentation is carried out as described in Example 2. The 2700 liters of harvest mash is divided into two equal volumes. To each is added an equal volume of methylene chloride. The methylene chloride is cycled from the bottom of each container down through the mash for a period of 2 hours. The phases are allowed to separate for 11/2-2 hours and then the clear methylene chloride extract is withdrawn. This extract is concentrated in several stages to a syrup, giving 876 g of the crude product. 
     EXAMPLE 4 
     Isolation of Nosiheptide 
     The 876 g of crude product from Example 3 is triturated with three pints of ether for 1/2 hour. The ether is decanted through a filter and the filter is washed with ether. The residue on the funnel and the residue in the trituration flask are dissolved in methanol. The methanol solutions are combined and concentrated in vacuo, giving 152 g of a semi-solid. 
     A column with a diameter of 8.5 cm is packed to a height of 75 cm with silica gel. The 152 g of semi-solid is dissolved in 300 ml of methylene chloride: ethyl acetate (1:1) and allowed to seep into the column. The column is developed with 4 liters of ethyl acetate: methylene chloride (1:1) and then methylene chloride: ethyl acetate:methanol (6:6:1). Fractions of 90 ml each are collected, and checked for activity by bioautography against Straphylococcus aureus 209P. Fractions 142-207 are combined and lyophilized, giving 22 g of solid. 
     A column with a diameter of 8.5 cm is packed to a height of 82 cm with silica gel. The 22 g of solid is dissolved in 200 ml of chloroform:ethyl acetate (1:1) and allowed to seep into the column. The column is first eluted with 3 liters of chloroform:ethyl acetate (1:1), collecting fractions of 65 ml each. The eluting solvent is changed to ethyl acetate:ethanol (95:5) for the remaining 65 ml fractions. A total of 160 fractions are collected, checking for activity by bioautography. Fractions 78-102 are combined and concentrated in vacuo to a 50 ml suspension. This suspension is filtered and the solid is washed with ether and are dried, giving 864 mg of nosiheptide. 
     A 133 mg portion of the above amorphous solid is dissolved in 5 ml of glacial acetic acid and then filtered. A 0.5 ml portion of acetone is added to the filtrate and this solution is chilled at 4° C. overnight. The resulting suspension is filtered cold and the solid washed with ether and dried giving 93 mg of crystalline nosiheptide.