Patent Publication Number: US-4321329-A

Title: Saccharopolyspora culture

Description:
This is a division of application Ser. No. 081,211, filed Oct. 2, 1979, now U.S. Pat. No. 4,293,651. 
    
    
     BRIEF SUMMARY OF THE INVENTION 
     The process of the invention utilizes the biologically pure culture of the novel microbe Saccharopolyspora hirsuta strain 367, NRRL 12045, to produce the useful antibiotic U-59,760. The physical and chemical characteristics of this antibiotic cannot be distinguished from the antibiotic known as compound 47444 which is disclosed in U.S. Pat. No. 4,148,883. The production of the antibiotic in U.S. Pat. No. 4,148,883 is accomplished by an entirely different microbe which is classified as a Nocardia. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The microorganism used for the production of antibiotic U-59,760, as described herein, is a biologically pure culture of Saccharopolyspora hirsuta strain 367, NRRL 12045. 
     A subculture of this microorganism can be obtained from the permanent collection of the Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Ill., U.S.A. Its accession number in this depository is NRRL 12045. It should be understood that the availability of the culture does not constitute a license to practice the subject invention in derogation of patent rights granted with the subject instrument by governmental action. 
     The microorganism of this invention was studied and characterized by Alma Dietz and Grace P. Li of The Upjohn Company Research Laboratories. 
     An actinomycete isolated in the Upjohn soils screening laboratory has been characterized and found to have the macroscopic, microscopic and whole cell hydrolysate properties of the genus Saccharopolyspora [Iwasaki, A., N. Itoh, and T. Mori, 1979. A new broad-spectrum aminoglycoside antibiotic complex, sporaricin. II. Taxonomic studies on the sporaricin producing strain Saccharopolyspora hirsuta subsp. kobensis nov. subsp. J. Antibiotics. 32: 180-186.] [Lacey, J. and M. Goodfellow. 1975. A novel actinomycete from sugar-cane bagasse: Saccharopolyspora hirsuta gen. et sp. novl. J. Gen. Microbiol. 88: 75-87]. A comparison with the type culture Saccharopolyspora hirsuta ATCC 27875 is given in Tables 1 and 2. A comparison with the type culture and with the subspecies, Saccharopolyspora hirsuta ss. kobensis ATCC 20501 [Iwasaki, A. supra] based on data published for these strains is given in Table 3. 
     Unique properties of the genus Saccharopolyspora are its butyrous or gelatinous-type vegetative growth, sparse aerial growth which is best studied after 21 days incubation, yellow to orange to orange-tan vegetative growth and pigment production, spore chains developing in a sheath with unique hair tufts with truncated bases and smooth areas between the tufts, the presence of meso-diaminopimelic acid, arabinose and galactose in whole cell hydrolysates and in cell wall preparations. L-diaminopimelic acid was detected in addition to meso-DAP in our cell wall preparations. 
     No antibiotic production has been reported for the type culture; the subspecies kobensis produces the antibiotic complex sporaricin (KA-6606) [Iwasaki, A. supra]. The new isolate is distinguished by the production of antibiotic U-59,760 and U-59,761. The only significant difference among the strains is in their antibiotic production capability. Therefore, the new strain is designated Saccharopolyspora hirsuta strain 367. 
     The taxonomic methods used herein were those cited in Dietz [Dietz, A. 1954. Ektachrome transparencies as aids in actinomycete classification. Ann N.Y. Acad. Sci. 60: 152-154.] [Dietz, A. 1967. Streptomyces steffisburgensis sp. n. J. Bacteriol. 94: 2022-2026.], Dietz and Mathews [Dietz, A., and J. Mathews. 1971. Classification of Streptomyces spore surfaces into five groups. Appl. Microbiol. 21: 527-533.], Becker et al. [Becker, B., M. P. Lechevalier, and H. A. Lechevalier. 1966. Chemical composition of cell wall preparations from strains of various form-genera of aerobic actinomycetes. Appl. Microbiol. 13 236-243.], Lechevalier and Lechevalier [Lechevalier, H. A., and M. P. Lechevalier. 1970. A critical evaluation of the genera of aerobic actinomycetes, p. 393-405. In H. Prauser (ed.), The Actinomycetales. Gustav Fisher, Jena.] [Lechevalier, M. P., and H. A. Lechevalier. 1970. Chemical composition as a criterion in the classification of aerobic actinomycetes. Int. J. Syst. Bacteriol. 20: 435-443.], and Shirling and Gottlieb [Shirling, E. B., and D. Gottlieb. 1966. Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol. 16: 313-340.]. 
     Color characteristics: Aerial mycelium gray to gray-pink (very sparse at 14 days). Melanin negative. Appearance on Ektachrome is given in Table 1. Surface colors of the vegetative growth were determined to be red (R) or yellow (Y) by matching the color with the chips in the Tresner and Backus [Tresner, H. D., and E. J. Backus. 1963. System of color wheels for streptomycete taxonomy. Appl. Microbiol. 11: 335-338.] color wheels. 
     Microscopic characteristics: Spore masses and spore chains, intact and fragmented, and flexuous or loosely spiraled chains, as observed by Scanning Electron Microscopy, appear to develop in a sheath which is at first almost smooth; then unique tufts of hairs (triangular at the base) are formed. The surface between the tufts is smooth. Finally, the sheath becomes covered with fine long hairs. The long sheaths are tangled and appear to arise from, or fuse to form pseudosporangia. As the chains come in contact with the substrate the spores become smooth in outline with a depressed or ridged appearance. &#34;Empty&#34; hyphae are noted in the developed chains. Microscopic characteristics are best noted after 21 days incubation at 28°-37° C. Nocardioform fragmentation may be seen in 21 day cultures. 
     Growth on carbon compounds: Under the test conditions of Shirling and Gottlieb [supra] growth was good on the postive control (basal medium plus D-glucose), sucrose, D-mannitol, D-fructose and raffinose. Growth was doubtful on L-arabinose and D-xylose. There was no growth on the negative control (basal medium only), inositol, rhamnose, and cellulose. 
     Whole cell analysis: meso-Diaminopimelic acid, arabinose and galactose were detected. 
     Cell wall analysis: meso-Diaminopimelic acid, L-diaminopimelic acid, arabinose and galactose were found. 
     Cultural and biochemical characteristics: See Table 2. 
     Temperature: There was no to very poor growth at 18° C. on Bennett&#39;s and maltose-tryptone agars and poor growth on Czapek&#39;s sucrose agar. Growth was good at 24°-45° C. There was no growth at 55° C. 
     
                                           TABLE 1                                 
__________________________________________________________________________
Color characteristics* of Saccharopolyspora hirsuta ATCC 27875 and        
S. hirsuta strain 367 on Ektachrome                                       
                 S. hirsuta ATCC 27875                                    
                                S. hirsuta strain 367                     
Agar Medium                                                               
         Determination                                                    
                 Chip                                                     
                    Color       Chip                                      
                                   Color                                  
__________________________________________________________________________
Bennett&#39;s                                                                 
         S       71 moderate orange yellow                                
                                71 moderate orange yellow                 
         R       71 moderate orange yellow                                
                                71 moderate orange yellow                 
Czapek&#39;s sucrose                                                          
         S       74 strong yellowish brown                                
                                67 brilliant orange yellow                
         R       72 dark orange yellow                                    
                                67 brilliant orange yellow                
Maltose-tryptone                                                          
         S       71 moderate orange yellow                                
                                71 moderate orange yellow                 
         R       71 moderate orange yellow                                
                                71 moderate orange yellow                 
Peptone-iron                                                              
         S       68 strong orange yellow                                  
                                68 strong orange yellow                   
         R       68 strong orange yellow                                  
                                68 strong orange yellow                   
0.1% Tyrosine                                                             
         S       73 pale orange yellow                                    
                                71 moderate orange yellow                 
         R       73 pale orange yellow                                    
                                71 moderate orange yellow                 
Casein   S       73 pale orange yellow                                    
                                70 light orange yellow                    
         R       73 pale orange yellow                                    
                                73 pale orange yellow                     
__________________________________________________________________________
 *Color was determined by comparison with NBS color chips (SP 440. Color: 
 Universal Language and Dictionary of Names. U.S. Government Printing     
 Office, Washington, DC 20402. SRM 2106. ISCCNBS Centroid Color Charts.   
 Office of Standard Reference Material, Room B311, Chem. Building, Nationa
 Bureau of Standards, Washington, DC 20234. S = Surface R = Reverse       
 
    
     
                                           TABLE 2                                 
__________________________________________________________________________
Cultural and biochemical characteristics of Saccharopolyspora hirsuta     
strains.                                                                  
Agar medium                                                               
          Determination                                                   
                  S. hirsuta ATCC 27875                                   
                               S. hirsuta strain 367                      
__________________________________________________________________________
Peptone-iron                                                              
          S       Colorless (V)                                           
                               Colorless or light tan                     
                               wrinkled (V)                               
          R       Pale yellow  Orange-tan                                 
          P       --           Pale tan to orange-tan                     
          O       Melanin negative                                        
                               Melanin negative                           
Calcium malate                                                            
          S       Trace white (A)                                         
                               Trace gray (A)                             
          R       Cream        Pale gray cream                            
          P       --           --                                         
          O       Malate solubilized                                      
                               Malate slightly solubilized                
Glucose asparagine                                                        
          S       Colorless to light tan (V)                              
                               Light tan (V)                              
          R       Orange       Orange-tan                                 
          P       Orange       Orange-tan                                 
Skim milk S       Tan (V)      Orange-brown (V)                           
          R       Orange-tan   Dark orange-tan                            
          P       Orange-tan   Deep orange-tan                            
          O       Casein solubilized                                      
                               Casein solubilized                         
Tyrosine  S       Tan (V)      Orange-tan (V)                             
          R       Orange       Orange                                     
          P       Orange       Orange                                     
          O       Tyrosine solubilized                                    
                               Tyrosine solubilized                       
Xanthine  S       Colorless (V)                                           
                               Pale tan (V)                               
          R       Pale yellow  Pale tan                                   
          P       Pale yellow  Pale tan-peach                             
          O       Xanthine solubilized                                    
                               Xanthine solubilized                       
                  around growth                                           
Nutrient starch                                                           
          S       Colorless (V)                                           
                               Pale tan (V)                               
          R       Pale yellow  Pale tan                                   
          P       Very pale yellow                                        
                               Very pale tan                              
          O       Starch hydrolyzed                                       
                               Starch hydrolyzed                          
Yeast extract-malt                                                        
          S       Colorless (V)                                           
                               Pale tan (V)                               
extract   R       Orange       Pale-orange-tan                            
          P       Orange       Orange                                     
Peptone-yeast                                                             
          S       Colorless (V) with very                                 
                               Colorless (V)                              
extract-iron      slight trace white (A)                                  
(ISP-6)   R       Yellow-tan   Light orange-tan to tan                    
          O       Melanin negative                                        
                               Melanin negative                           
Tyrosine (ISP-7)                                                          
          S       Trace white (A)                                         
                               Very pale orange (V)                       
          R       Yellow tan   Very pale orange                           
          O       Melanin negative                                        
                               Melanin negative                           
Gelatin                                                                   
Plain     S       Trace white (A) on                                      
                               Colorless to pale yellow (V)               
                  colorless surface                                       
                  pellicle                                                
          P       None         None to trace pale yellow                  
          O       Gelatin liquefied                                       
                               Gelatin liquefied                          
Nutrient  S       Trace white (A) on                                      
                               Trace colorless (V) to trace               
                  yellow (V) pellicle                                     
                               white (A) on (V)                           
          P       None         None                                       
          O       Gelatin liquefied                                       
                               Gelatin liquefied                          
Broth                                                                     
Synthetic nitrate                                                         
          S       No surface growth                                       
                               Trace white (A) on colorless (V)           
          P       None         None                                       
          O       Compact bottom growth                                   
                               Compact to flocculent bottom               
                  No reduction growth                                     
                               No reduction                               
Nutrient nitrate                                                          
          S       Trace white (A) on color-                               
                               Trace white (A) on colorless               
                  less surface pellicle                                   
                               surface ring                               
          P       None         None                                       
          O       Flocculent bottom growth;                               
                               Poor colorless compact bottom              
                  No reduction growth; No reduction                       
Litmus milk                                                               
          S       Pale gray (A) on yellow-                                
                               Blue-gray surface ring                     
                  tan surface ring                                        
          P       None         Deep red-tan                               
          O       Litmus not reduced                                      
                               Peptonization                              
                  pH 6.7-7.3   pH 7.6-7.8                                 
__________________________________________________________________________
 S = Surface                                                              
 O = Other characteristics                                                
 R = Reverse                                                              
 (V) = Vegetative Growth                                                  
 P = Pigment                                                              
 (A) = Aerial growth                                                      
 
    
     
                                           TABLE 3                                 
__________________________________________________________________________
Comparision of Saccharopolyspora hirsuta strains                          
Tests        S. hirsuta strain 367                                        
                        S. hirsuta ATCC 27875                             
                                    S. hirsuta kobense ATCC               
__________________________________________________________________________
                                    20501                                 
Physiological Properties                                                  
Temperature  Growth from 18-45C                                           
                        Growth from 25-45C                                
                                    Growth from 18-45C                    
Optimum Temperature                                                       
             28-45C     37-40C      37-42C                                
Gelatin liquefaction                                                      
             Complete   Positive at 27C                                   
                                    Positive at 27C                       
Starch hydrolysis                                                         
             Positive   Positive    Positive                              
Action on milk                                                            
             No coagulation                                               
                        No coagulation                                    
                                    No coagulation                        
             Peptonization                                                
                        Peptonization                                     
                                    Peptonization                         
Melanoid pigment                                                          
             Negative   Negative    Negative                              
production                                                                
Nitrate reduction                                                         
             Negative   Negative    Positive                              
Carbon Source Utilization                                                 
L-Arabinose  ±       -           -                                     
D-Xylose     ±       +           -                                     
L-Rhamnose   -          +           -                                     
D-Glucose    +          +           +                                     
D-Fructose   ++         +           +                                     
Sucrose      +          +           +                                     
Raffinose    ++         +           +                                     
Inositol     -          +           -                                     
D-Mannitol   ++         +           +                                     
__________________________________________________________________________
 
    
     The compound of the invention process is produced when the elaborating organism is grown in an aqueous nutrient medium under submerged aerobic conditions. It is to be understood, also, that for the preparation of limited amounts surface cultures and bottles can be employed. The organism is grown in a nutrient medium containing a carbon source, for example, an assimilable carbohydrate, and a nitrogen source, for example, an assimilable nitrogen compound or proteinaceous material. Preferred carbon sources include glucose, brown sugar, sucrose, glycerol, starch, cornstarch, lactose, dextrin, molasses, and the like. Preferred nitrogen sources include cornsteep liquor, yeast, autolyzed brewer&#39;s yeast with milk solids, soybean meal, cottonseed meal, cornmeal, milk solids, pancreatic digest of casein, fish meal, distillers&#39; solids, animal peptone liquors, meat and bone scraps, and the like. Combinations of these carbon and nitrogen sources can be used advantageously. Trace metals, for example, zinc, magnesium, manganese, cobalt, iron, and the like, need not be added to the fermentation media since tap water and unpurified ingredients are used as components of the medium prior to sterilization of the medium. 
     Production of the compound by the invention process can be effected at any temperature conducive to satisfactory growth of the microorganism, for example, between about 18° and 40° C., and preferably between about 20° and 28° C. Ordinarily, optimum production of the compound is obtained in about 3 to 15 days. The final pH of the fermentation is dependent, in part, on the buffers present, if any, and in part on the initial pH of the culture medium. 
     When growth is carried out in large vessels and tanks, it is preferable to use the vegetative form, rather than the spore form, of the microorganism for inoculation to avoid a pronounced lag in the production of the compound and the attendant inefficient utilization of the equipment. Accordingly, it is desirable to produce a vegetative inoculum in a nutrient broth culture by inoculating this broth culture with an aliquot from a soil, liquid N 2  agar plug, or a slant culture. When a young, active vegetative inoculum has thus been secured, it is transferred aseptically to large vessels or tanks. The medium in which the vegetative inoculum is produced can be the same as, or different from, that utilized for the production of the compound, so long as a good growth of the microorganism is obtained. 
     A variety of procedures can be employed in the isolation and purification of the compound produced by the subject invention from fermentation beers. Isolation can be accomplished by extraction with solvents such as methylene chloride, acetone, butanol, ethyl acetate and the like; and silica gel chromatography can be used to purify crude preparations of the antibiotic. 
     In a preferred recovery process, the compound produced by the subject process is recovered from the culture medium by separation of mycelia and undissolved solids by conventional means, such as by filtration or centrifugation, and solvent extraction of both mycelial cake or clarified broth. The clarified broth can be extracted with a suitable solvent, for example, methylene chloride (preferred), ethylacetate, butanol, and MIBK. The extract can be evaporated under reduced pressure to a concentrate and the antibiotic recovered from this concentrate by subjecting the concentrate to chromatography over silica gel and eluting with methanol and methylene chloride. 
    
    
     The following examples are illustrative of the process of the invention, but are not to be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted. 
     EXAMPLE 1 
     Part A. Fermentation 
     A biologically pure culture Saccharopolyspora hirsuta strain 367, NRRL 12045, is used to inoculate 500-ml Erlenmeyer seed flasks containing 100 ml of sterile medium consisting of the following ingredients: 
     
         ______________________________________                                    
Glucose monohydrate  25 g/l                                               
Pharmamedia*         25 g/l                                               
Tap water q.s.        1 liter                                             
______________________________________                                    
 *Pharmamedia is an industrial grade of cottonseed flour produced by      
 Traders Oil Mill Company, Fort Worth, Texas.                             
 
    
     The seed medium presterilization pH is 7.2. The seed inoculum is grown for three days at 28° C. on a Gump rotary shaker operating at 250 rpm and having a 21/2 inch stroke. 
     After three days incubation, the seed medium is used to inoculate (the inoculation rate is 5 ml of seed inoculum per 100 ml of fermentation medium) a series of 500-ml Erlenmeyer flasks containing sterile fermentation medium consisting of the following ingredients: 
     
         ______________________________________                                    
Glucose monohydrate 10       g/l                                          
Dextrin             20       g/1                                          
Corn steep liquor   2.5      g/l                                          
NH.sub.4 NO.sub.3   3.0      g/l                                          
NaCl                2.0      g/l                                          
CaCO.sub.3          5.0      g/l                                          
ph-7.2 (presterilization)                                                 
______________________________________                                    
 
    
     The fermentation flasks are incubated at a temperature of 28° C. on a Gump torary shaker operating at 250 rpm nd having a 21/2 inch stroke. Harvest is usually after about 5 days of fermentation. A typical 5 day fermentation has the following titers of antibiotic in the fermentation broth: 
     
         ______________________________________                                    
Day         S. lutea Assay, Bu/ml                                         
______________________________________                                    
2            8.0                                                          
3           10.4                                                          
4           10.4                                                          
5           11.2                                                          
______________________________________                                    
 
    
     In the assay results, a biounit (BU) is defined as the concentration of the antibiotic which gives a 20 mm zone of inhibition under the standard assay condition. Thus, if, for example, a fermentation beer has to be diluted 1/100 to give a 20 mm zone of inhibition, the potency of such beer is 100 Bu/ml. 
     B. Recovery and Purification 
     The whole beer (ca. 5,000 l) from a fermentation, as described above, is adjusted to pH 7.3 with NaOH and filtered on a 30 inch filter press using diatomaceous earth as a filter aid. During the filtration operation wash water is applied to the filter cake. From the filtration operation is recovered 5,500 l of clear fermentation broth which is then extracted twice with methylene chloride (1,400 l each time) to give a total of 2.800 l of solvent extract. This solvent extract is concentrated in vacuo to 10 l. Assay on a standard S. lutea disc plate assay gives a value of 2424 Bu/ml. 
     The extract concentrate described above (9 l), is chromatographed over a column containing 9 kg of silica gel (E. Merck-silica gel 7734). The column is eluted as follows: 
     20 liters methylene chloride; then 40 liters 2% methanol in methylene chloride; then 150 liters 5% methanol in methylene chloride; then 100 liters 10% methanol in methylene chloride. Four liter fractions are collected after an 80 liter forerun. Fractions 5-8 contain antibiotic U-59,760 having the following characteristics: 
     Elemental Analysis: C, 64.95, H, 7.42, N, 2.49 
     Molecular Weight: 515.2482 (Mass spectroscopy) (C 28  H 37  NO 8 ). 
     Optical Rotation: [a] D   25  +53° (C, 0.0848 MeOH). 
     Solubilities: Soluble in methanol, ethanol and methylene chloride, and relatively insoluble in petroleum ether and cyclohexane. 
     Infrared Absorption Spectrum: Antibiotic U-59,670 has a characteristic infrared absorption spectrum in a mineral oil mull. Peaks are observed at the following wave lengths expressed in reciprocal centimeters. 
     
         ______________________________________                                    
Band Frequency                                                            
(Wave Numbers)     Intensity                                              
______________________________________                                    
3459               S                                                      
3330               S, sh.                                                 
3139               M, sh.                                                 
3025               M                                                      
2957               S                                                      
2922               S                                                      
2855               S                                                      
2725               W                                                      
1725               S, sh.                                                 
1704               S                                                      
1688               S                                                      
1554               M                                                      
1455               S                                                      
1411               S                                                      
1377               S                                                      
1354               S                                                      
1339               S                                                      
1315               S, sh.                                                 
1307               S                                                      
1276               S                                                      
1265               S, sh.                                                 
1240               S                                                      
1205               M                                                      
1169               S                                                      
1109               S                                                      
1082               S                                                      
1060               S                                                      
1045               S                                                      
1031               S                                                      
1010               S                                                      
968                S                                                      
956                S                                                      
949                S                                                      
937                M                                                      
909                M                                                      
882                M                                                      
870                M, sh.                                                 
857                M                                                      
835                M                                                      
794                M                                                      
764                S                                                      
747                S                                                      
719                M                                                      
699                M                                                      
677                M                                                      
638                M                                                      
______________________________________                                    
 Key: S = Strong, M = Medium, W =0 Weak, sh. = shoulder                   
 
    
     
         ______________________________________                                    
Antimicrobial spectrum:                                                   
                    Inhibition                                            
Organism            Zone Size (mm).sup.1                                  
______________________________________                                    
B. subtilis UC 564  0                                                     
B. subtilis UC 6033 16                                                    
S. aureus UC 70     36                                                    
S aureus UC 3665    42                                                    
S. aureus UC 6029   38                                                    
S. lutea UC 130     42                                                    
S. lutea UC 3383    41                                                    
S. lutea sens       45                                                    
K. pneumoniae UC 58 32                                                    
E. coli UC 51       0                                                     
S. schottmuelleri UC 126                                                  
                    0                                                     
P. vulgaris UC 93   0                                                     
P. aeruginosa UC 95 0                                                     
M. avium UC 159     30                                                    
P. oxalicum UC 1268 0                                                     
S. pastorianus UC 1342                                                    
                    0                                                     
R. sphaeroides UC 3238                                                    
                    21                                                    
S. pyogenes UC 6055 16                                                    
B. fragilis UC 6513 37                                                    
C. perfringens UC 6509                                                    
                    48                                                    
______________________________________                                    
 
    
     &#34;UC&#34; is a registered trademark of The Upjohn Company culture collection. These cultures can be obtained from The Upjohn Company in Kalamazoo, Mich., upon request. 
     Since antibiotic U-59,760 is active against S. aureus, it can be used to disinfect washed and stacked food utensils contaminated with this bacteria. Also, it can be used as a disinfectant on various dental and medical equipment contaminated with S. aureus. Further, antibiotic U-59,760 can be used as a bacteriostatic rinse for laundered clothes, and for impregnating papers and fabrics; and, it is also useful for suppressing the growth of sensitive organisms in plate assays, and other microbiological media.