PROCESS FOR PRODUCING THIENAMYCIN EMPLOYING STREPTROMYCES CATTLEYA CELLS IMMOBILIZED TO KIESELGUHR

Abstract: The antibiotic, thienamycin, is produced by fermentation using Streptomyces cattleye cells immobilized to Kieselguhr.

Description: TITLE OF THE INVENTION PROCESS FOR PRODUCING THIENAMYCIN EMPLOYING STREPTOMYCES CATTLEYA CELLS IMMOBILIZED TO KIESELGUHR The instant invention relates to a new and useful process for preparing the antibiotic, thienamycin. More particularly, the instant invention relates to a process for thienamycin wherein cells of a thienamycin producing microorganism are immobilized on Kieselguhr and the cells so immobilized are employed in the production of thienamycin by fermentation techniques. The antibiotic, thienamycin, is a uniquely structured -lactam compound of the formula: EMI1.1 As will be noted, thienamycin is structurally characterized by having the unique trans ss-configuration at the hydroxyethyl group at the 6-position. Thienamyrin may be obtained as a natural product by fermentation of a thienamycin producing strain of Streptomyces cattleya by techniques already fully described, such as, for example, United States Patent No. 3,950,357, issued April 13, 1976, wherein its antibiotic activity in animal and human therapy and in inanimate systems also is disclosed. Preparation of thienamycin by classical fermentation techniques employing a thienamycin producing strain of Streptomyces cattleya, such as, for example, Streptomyces cattleya NRRL 8057 (Northern Regional Research Laboratories, Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Ill., as disclosed in the above-identified U.S. patent, although successfully producing the antibiotic, never-the-less presents certain difficulties inherent in such classical fermentation processes. Included among these are relatively low productivity due in part to feedback inhibition phenomenum and degradation of product due to complex media composition used in cell growth. Further, the high level of impurities in the fermentation broth resulting from the complex media employed for cell growth makes isolation and purification of the thienamycin produced difficult and expensive. Also, since optimum conditions for growth of cells may be quite different from those required for product formation, selection of appropriate media and conditions for production of a product becomes complex and difficult. The process of the instant invention eliminates or materially decreases those difficulties. The instant invention is based upon applicants' discovery that cells of a thienamycin producing strain of Streptomyces cattleya can be immobilized to Kieselguhr and employed in the production of thienamycin by fermentation techniques with significant advantages over classical fermentation techniques. The cells of Streptomyces cattleya are immobilized by growing in submerged culture in the presence of Kieselguhr. Kieselguhr is composed essentially of fossilized shells of diatoms produced amorphous silica and is characterized by a high internal pore volume. During growth of Streptomyces cattleya in the presence of Kieselguhr the mycelium of the microorganism grows into the pores of the Kieselguhr and becomes tightly attached to the Kieselguhr. The cells so immobilized may be maintained in a productive state by intermittent decanting and feeding of a production medium throughout a prolonged period extending for as long as 190 days. Applicants have found that thienamycin production as described above results in significant advantages as compared to classical fermentation techniques. Notably, the process of the instant invention separates the growth stage of the cells from the production stage thus allowing conditions to be optimized for product formation with consequent increase in overall productivity. The process allows for extended productive operation, as long as 190 days, as opposed to 5 days for conventional batch fermentation techniques. Intermittent or continuous removal of product and replinishment of production medium decreases feedback inhibition and product degradation. Also, the product stream, being largely freed from suspended cellular material and unused nutrients, is much purer and final product separation and purification is less difficult and cheaper. Production of thienamycin according to the process of this invention may be carried out as follows: KIESELGUHR PREPARATION Commercially available Kieselguhr qualities such as Johns-Manville celite, for example, Celite 560 (Johns Manville, New York, New York), are treated to eliminate fines by floatation. The Kieselguhr is suspended in water, aqitated vigorously and allowed to settle for ten seconds, then decanted. This process is repeated several times until the Kieselguhr settles to a clear supernatant within ten seconds. The Kieselguhr is then dried to a constant weight and is used without further treatment in preparing immobilized cells. Desirably the Kieselguhr will have a particle diameter of from 100 to 900 microns. INOCULUM DEVELOPMENT The stock culture of the producing microorganism may be maintained either in the lyophilized state or as frozen vegetative mycelium. The inoculum for production is developed by suspending the appropriate stock culture in sterile seed medium and incubating at 28-34 C for 1 to 18 hours (typical seed media are illustrated in the appended Examples). Conveniently, the incubation is carried out on a rotary shaker with a two inch throw rotating at 220 r.p.m. GROWTH AND IMMOBILIZATION Growth as well as production may be carried out at any volume depending upon production requirements. Typically, for small scale runs, fermentation is carried out in submerged culture in 250 ml non-baffled Erlenmeyer flasks containing 20.0 ml. of a suitable growth medium (illustrated in the appended Examples) and 3.0 g of Kieselguhr. The growth medium is sterilized by conventional procedures at 1200C for 15 minutes. Growth is initiated by introducing a measured quantity of the inoculum described above. After inoculation, the culture is incubated for 24 to 96 hours at 28 to 370C on a rotary shaker with a two inch throw rotating at 220 rOp.m. During the growth stage, the mycelia grow into the pores of the Kieselguhr and is entrapped thus becoming immobilized in the Kieselguhr matrix. PRODUCTION STAGE The immobilized cells are harvested by decanting the spent growth medium. An appropriate production medium (illustrated in the appended Examples) then is added to the immobilized cells and the culture is incubated at 28-37 C for 24 hours. The product, which is water soluble, is contained in the medium fraction and is harvested by decanting and fresh production medium is added to the immobilized cells to replace the volume decanted. Harvest of product is continued on a periodic basis usually from 4 to 24 hours. Where harvest is conducted on a continuous basis, addition of fresh media and removal of spent media conveniently is adjusted so as to effect a complete turnover of reactor volume in a 4-24 hour period. Duration and productivity of the immobilized cells will vary with the producion medium. Immobilized cultures prepared as described above have been shown to produce for periods exceeding 190 days. The course of the fermentation is monitored by pH measurement and by product accumulation. Isolation of product from the decanted medium is accomplished by techniques already fully described in the art. Such techniques form no part of the instant invention. The media employed in the growth and production stages described above may vary widely in composition. In general, however, the antibiotic is produced during the aerobic fermentation of the producing microorganism in aqueous nutrient media. Aqueous media, such as those employed for the production of other antibiotics, are suitable for producing thienamycin. Such media contain sources of carbon, nitrogen and inorganic salts assimilable by the microorganism. Typically, carbohydrates such as sugars, for example, glucose, fructose, maltose, sucrose, xylose, mannitol and the like and starches such as grain, for example, oats, rye, cornstarch, corn meal and the like can be used, either alone or in combination, as sources of assimilable carbon in the nutrient medium. The exact quality of carbohydrate utilized in the medium depends in part on the nature of the other components of the medium, but, in general, the amount of carbohydrate usually varies between about 0.18 to about 7% by weight of the medium. Many proteinaceous materials may be used as nitrogen sources in the fermentation. Suitable nitrogen sources will include, for example, yeast hydrolysates, primary yeast, soy bean meal, cottonseed flour, hydrolysates of casein, cornsteep liquor, distilleres solubles, tomato paste and the like The nitrogen sources, either alone or in combination, are used in amounts ranginq from about 0.005% to about 6% by weight of the medium. Among the nutrient inorganic salts which can be employed in the media are the customary salts capable of yielding sodium, potassium, ammonium, calcium, phosphate, sulfate, chloride, carbonate and the like ions. Also trace metals may be included such as, for example, cobalt, manganese, iron, magnesium and the like. Such media, qenerally, will display a pH ranging from about 6 to about 8. The best mode contemplated by applicants for carrying out their invention is further illustrated in the following working Examples. No limitation, however, is intended except as defined in the appended claims. EXAMPLE 1 A Stage - Stock Culture: Streptomyces cattleya NRRL 8057 1. Vessel: 2 ml. glass vial containing 2.0 ml frozen vegetative mycelium 2. Medium: same as Example 2 3. Inoculum: same as Example 2 4. Incubation: same as Example 2 5. Storage: same as Example 2 B-Stage - Same as Example 2 C Stage - Same as Example 2 D Stage - (Growth): same as Example 2 Stage - (Production) 1. Vessel: same as Example 2 2. Medium: 100 ml production medium liter Acid Hydolized Casein - 0.460 (Vitamin free - Salt free) Sodium Lactate (60%) 2.4 Primary Yeast (Yeast Products, 1.0 Inc. - Clifton, N.J.) KH2PO4 0.250 MgSO4.7H2O 0.0625 CaCl2 (anh.) + 0.0425 FeSO4.7H2O 0.00125 CuSO4.5H2O 0.000125 MnCl2.4H2O 0.000125 CoCl2.6H2O 0.001 MES Buffer 14.6 pH 6.5 3. Inoculum: same as Example 2 4. Incubation: same as Example 2 50 Harvest: same as Example 2 6. Productivity/ml. Kieselguhr (Celite)/Day: 34.5 mcg 7. Duration of Productivity: 6 days 8. Production medium changed (day 6) to 100 ml of: same as Example 2 9. Incubation same as Example 2 10. Harvest: same as Example 2 11. Productivity/ml. Kieselguhr (Celite)/Day: 55.8mcg 12. Duration of Productivity: 8 days EXAMPLE 2 THIENAMYCIN PRODUCTION A Stage - Stock Culture: Streptomyces Cattleya 1. Vessel: 2 ml. glass vial containing 2.0 ml frozen vegetative mycelium 2. Medium: g./liter Sucrose 30.0 Distillers Solubles (B & F) 15.0 Ardamine YEP (Yeast Products, 5.0 Inc., Clifton, N.J.) Corn Gluten Meal 5.0 Distilled Water pH 7.5 (with NaOH) 3. Inoculum: 2 ml. of 24 hour culture 4. Incubation: none 5. Storage: -800C indefinitely B Stage 1. Vessel: 250 ml. 3-baffled Erlenmeyer flask 2. Medium: 50 ml. seed medium g./liter Primary Yeast 10.0 Ardamine YEP (Yeast Products, 10.0 Inc., Clifton, N.J.0 Cerelose 10.0 Tap Water pH 7.0 3. Inoculum: 1.0 ml. A Stage 4. Incubation: 280C for 24 hours on rotary shaker with two-inch throw at 220 r.p.m. C Stage 1. Vessel: 250 ml. 3 baffled Erlenmeyer flask 2. Medium: 50 ml. seed medium g./liter Mono Sodium Glutamate H2O 5.0 NH4C1 1.5 K2HPo4 2.0 Inositol 0.4 NaCl 0.5 MgSO4.7H2O 0.5 FeSO4.7H2O 0.025 ZnSO4.7H2O 0.010 Paraamino Benzoic Acid 0.0001 CoCl2.6H2O 0.010 Glycerin 10.0 Ardamine YEP 15.0 Distilled Water pH 7.0 Then add CaCO3 0.250 3. Inoculum: 2.0 ml. B Stage 4. Incubation: same as B Stage D Stage - (Growth) 1. Vessel: 250 ml. no-baffled Erlenmeyer flask 2. Medium 20 ml. growth medium + 3.0 g.Kieselguhr (Celite) liter Mono Sodium Glutamate.R2O 3.75 L-Isoleucine 2.4 NH4C1 0.75 K2HP04 1.0 CoCl2.6H2o 0.010 MgSO4. 7R2O 0.5 FeSO4.7H2O 0.025 Glycerin 10.0 MES Buffer (2-[N-morpholino] 14.6 etbanesulfonic Acid) - (Sigma Chemical co., St. Louis, Mo.) Distilled Water pH 7.0 3. Inoculum 2 ml. C Stage 4° Incubation: 280C for 72 hours on rotary shaker with two inch throw at 220 r.pm. E Stage - (Production) 1. Vessel: 250 ml. non-baffled Erlenmeyer flask 20 Medium: 100 ml. production medium g./liter Mono Sodium Glutamate.H2O 0.920 Sodium Lactate (60%) 2.4 L-Isoleucine 0.590 KH2PO4 0.500 MgSO4.7H2O 0.125 CaCl2 (anhyd.) 0.085 FeSO4.7H2O 0.0025 CuSO4.5H2O 0.00025 MnCl2.4H2O 0.00025 CoCl2.6H2O 0.001 MES Buffer 14.6 Distilled Water pH 6.5 3. Inoculum: immobilized cells from D Stage 4. Incubation: 340C for 24 hours on rotary shaker with two inch throw at 220 r.p.m. 5. Harvest: harvesting daily by decanting spent medium containing thienamycin 6. Productivity/ml. Kieselguhr (Celite)/Day: 130 mcg 7. Duration of Productivity: 14 days EXAMPLE 3 THIENAMYCIN PRODUCTION A Stage - Stock Culture: Streptomyces Cattleva 1. Vessel: 2 ml. glass vial containing 2.0 ml. frozen vegetative mycelium 2. Medium: same as Example 2 3. Inoculation: 2.0 ml. of 24 hour culture 4. Incubation: none 5. Storage: -800C indefinitely B-Stage 1. Vessel: 250 ml. 3 baffled Erlenmeyer flask 2. Medium: same as Example 2 (C Stage) 3. Inoculum: 1.0 ml. A Stage 4. Incubation: 280C for 24 hours on rotary shaker with two inch throw at 220 r.p.m. C Stage - (Growth) lo Vessel: 250 m10 non-baffled Erlenmeyer flask 2. Medium: 20 ml. growth medium + 3.0 g. Kieselguhr (Celite) g./liter Corn Steep Liquor 5.0 Tomato Paste 40.0 Oat Flour 10.0 Glucose 10.0 Trace Element &num;2* 10.0 pH 608 3. Inoculum: 2.0 ml. B Stage 4O Incubation: 72 hours at 280C on rotary shaker with two inch throw at 220 r.p.m. D Stage - (Production) 1. Vessel: 250 ml. non-baffled Erlenmeyer flask 2O Medium same as Example 2 3. Inoculum: immobilized cells from C Stage 4. Incubation: 28 C for 24 hours or rotary shaker with two inch throw at 220 r.p.m. 5. Harvest: same as Example 2 (E Stage) 6. Productivity/ml. Kieselguhr (Celite)/Day: 130 mcg. 7. Duration of Productivity: 14 days *Trace Element 12 mg./liter FeSO4.7H2O 10.0 MnSO4.4H2O 10.0 CuCl2.2H2O 0.25 CaCl2 1.0 H3BO3 0.56 (NH4)6MoO2.4H2O 0.19 ZnSO4.7H2O 2.0 EXAMPLE 4 THIENAMYCIN PRODUCTION A Stage - same as Example 3 B Stage - same as Example 3 C Stage - (Growth) 1. Vessel: same as Example 3. 2. Medium: 20 ml. growth medium + 3.0 g. Kiesel guhr (Celite). g./liter Yeast Extract 10.0 Dextrose 10.0 Agar 15.0 pH 7.0 3. Inoculum: 2.0 ml B Stage 4. Incubation: same as Example 3 D Stage = (Production) 1. Vessel: same as Example 3 2. Medium: same as Example 3 3O Inoculum: same as Example 3 4. Incubation: same as Example 3 5. Harvest same as Example 3 6. Productivity/ml.Kieselguhr (Celite)/Day 120 mcg. 7. Duration of Productivity: 14 days EXAMPLE 5 THIENAMYCIN PRODUCTION A Staqe - same as Example 3 B Stage - same as Example 3 C Stage - (Growth) 1. Vessel: same as Example 3 2. Medium: 20.0 ml BACTO Nutrient Broth (Difco Laboratories, Detroit, Mich.) + 3.0 g. Celite 3. Inoculum: same as Example 3 4. Incubation: same as Example 3 D Stage (Production) 1. Vessel: same as Example 3 2. Medium: same as Example 3 3. Inoculum: same as Example 3 4. Incubation: same as Example 3 5. Harvest: same as Example 2 6. Productivity/ml Kieselguhr CCe1ite)Day: 120 mcg. 7. Duration of Productivity: 14 days EXAMPLE 6 THIENAMYCIN PRODUCTION A Stage - same as Example 2 B Stage - same as Example 2 (C Stage) C Stage - (Growth) same as Example 2 D Stage - (Production) 1. Vessel: same as Example 2 2. Medium: 100.0 ml production medium g./liter Sodium Lactate (60%) 2.4 Ardamine pH (Yeast Products, 2.0 Inc., Clifton, N. J.) MES Buffer 14.6 Distilled Water pH 6.5 3. Inoculum: same as Example 2 4. Incubation: same as Example 2 5. Harvest: same as Example 2 6. Productivity/ml. Kieselguhr (Celite)/Day: 60 mcg. 7. Duration of Productivity: 33 days EXAMPLE 7 A Stage - same as Example 2 B Stage - same as Example 2 D Stage - (Growth) - same as Example 2 Stage - (Production) 1. Vessel: Same as Example 2 2. Medium: 100 ml production medium g./liter Primary Yeast 2.0 Sodium Lactate (60%) 2.4 MES Buffer 14.6 Distilled Water pH 6.5 3. Inoculum: same as Example 2 4. Incubation: same as Example 2 5. Harvest: same as Example 2 6. Productivity/ml. Kieselguhr (Celite)/Day: 88.7 mcg 7. Duration of Productivity: 32 days 80 Production medium changed (day 32) to 100 ml of: g./liter Ardamine pH 2.0 Sodium Lactate (60%) 2.4 MES Buffer 14.6 Distilled Water pH 6.5 9. Incubation same as Example 2 10. Harvest: same as Example 2 11. Productivity/ml. Kieselguhr (Celite)/Day: 53.6 mcg 12. Duration of Productivity: 14 days 13. Production medium changed (day 46) to 100 ml of: g./liter Acid Hydrolyzed Casein - 0.92 (Vitamin free - Salt free) Sodium Lactate (60%) 2.4 KE2PO4 2.0 MgSO4.7H2O 0.5 CaCl2 (anh.) 0.34 FeSO4.7H2O 0.01 CuSO4. 5H2O 0.001 MnCl2. 4H2O 0.001 CoCl2.6H2o 0.01 MES Buffer " 14.6 Distilled Water pH 6.5 14. Incubation: same as Example 2 15. Harvest: same as Example 2 16. Productivity/ml. Kieselguhr (Celite)/Day: 68.3 mcg 17. Duration of Productivity: 8 days 18. Production medium changed (day 54) to 100 ml of: g./liter NZ Amine Type E (Humko - 0.920 Sheffield - Norwich, N.Y.) Sodium Lactate (60%) 2.4 KH2PO4 0.5 MgSO4.7H2O 0.125 CaCl2 (anh.) 0.085 FeSO4.7H2O 0.0025 CuSO4. 5H2O 0.00025 MnCl2.4H2O 0.00025 CoCl2.6H2O 0.010 MES Buffer 14.6 Distilled Water pH 6.5 19. Incubation: same as Example 2 20. Harvest: same as Example 2 21. Productivity/ml. Kieselguhr (Celite)/Day: 27.0 mcg 22. Duration of Productivity: 8 days 23. Production medium changed (day 62) to 100 ml of: same as Step 2 24. Incubation: same as Example 2 25. Harvest: same as Example 2 26. Productivity/ml.Kieselguhr. (Celite)/Day: 69.0 mcg 27, Duration of Productivity: 25 days 28. Production medium changed (day 86) to 100 ml of: g./liter Acid Hydrolyzed Casein - 0.460 (Vitamin free - Salt free) Sodium Lactate (608) 2.4 Primary Yeast 1.0 KH2PO4 0.250 MgSO4.7H2O 0.0625 CaCl2 (anh.) 0.425 FeSO4. 7H2O 0.00125 CuSO4.5H2O 0.000125 CoC12.6H2O 0.005 MnC12.4H2O 0.000125 MES Buffer - 14.6 Distilled Water pH 6.5 29. Incubation: same as Example 2 30. Harvest: same as Example 2 31. Productivity/ml.Kieselguhr (Celite)/Day: 37.2 mcg 32. Duration of Productivity: 11 days 33. Production medium changed (day 98) to MES buffer without nutrient supplement 34. Incubation: same as Example 2 35. Harvest: same as Example 2 36. Productivity/ml. Kieselguhr (Celite)/Day: 0 after 4 days 37. Duration of Productivity: 13 days 38. Production medium changed (day 111) to 100 ml of: same as Step 28 39. Incubation: same as Example 2 40. Harvest: same as Example 2 41. Productivity/ml. Kieselguhr (Celite)/Day: increased over a five day period to 24o7 mcg 42. Duration of Productivity: 3 days 43. Production medium changed (day 119) to 100 ml of: g./liter Monosodium Glutamate 0.920 L-Isoleucine 0.590 Sodium Lactate (60%) 2.4 KH2PO4 2.0 MgSO4.7H2O 0.5 CaCl2 (anh.) 0.34 FeSO4.7H2O 0.01 CuSO4. 5H2O 0.001 MnCL2.4H2O 0.001 CoCl2.6H2O 0.01 MES Buffer 14.6 Distilled Water pH 6.5 44. Incubation: same as Example 2 45. Harvest: same as Example 2 46. Productivity/ml.Kieselguhr (Celite)/Day: 77 mcg 47. Duration of Productivity: 25 days 48. Production medium changed (day 144) to 100 ml of: g./liter Acid Hydrolyzed Casein - 0.460 (Vitamin free - Salt free) Sodium Lactate (60%) 2.4 Primary Yeast 1.0 KH2PO4 0.250 MgSO4.7H2O 0.0625 CaCl2 (anh.) 0.0425 FeSO4.7H2O 0.00125 CuSO4.5H2O 0.000125 CoC12.6H20 0.001 MnCl2.4H2O 0.000125 MES Buffer 14.6 pH 6.5 49. Incubation: same as Example 2 50. Harvest: same as Example 2 51. Productivity/ml. Kieselguhr (Celite)/Day: 50.0 mcg 52. Duration of Productivity: 21 days 53. Production medium changed (day 165) to 100 ml of: g./liter Monosodium Glutamate 0.920 L-Isoleucine 0.590 Sodium Lactate (60%) 2.4 KH2PO4 2.0 MgSO4.7H2O 0.5 CaCl2 (anh.) 0.34 FeSO4.7H2O 0.01 CuSO4.5H2O 0.001 MnCl2.4H2O 0.001 CoCl2.6H2O 0.001 MES Buffer Distilled Water pH 6.5 54. Incubation: same as Example 2 55. Harvest: same as Example 2 56. Productivity/ml. Kieselguhr (Celite)/Day: 72.9 mcg 57. Duration of Productivity: 15 day 1 58. Production medium changed (day 180) to 100 ml of MES buffer without nutrient supplement 59. Incubation: same as Example 2 60. Harvest: same as Example 2 61. Productivity/ml. Kieselguhr (Celite)/Day: decreased to 0 over a three day period 62. Duration of Productivity: 4 days 63. Production medium changed (day 187) to 100 ml of: same as Step 53 64. Incubation: same as Example 2 65. Harvest: same as Example 2 66. Productivity/ml. Kieselguhr (Celite)/Day: 60.1 mcg 67. Duration of Productivity: 5 days 68. Discontinued on day 193 The subject matter which applicants regard as their invention is particularly pointed out and distinctly claimed as follows:

Claims: WHAT IS CLAIMED IS: 1. A process for producing thienamycin by immobilized cell fermentation which comprises: (a) growing Streptomyces cattleya in submerged culture in a nutrient medium in the presence of Kieselguhr until the mycelia of the growing cells grows into the Kieselguhr pores; (b) separating the immobilized cells so produced from the growth medium; (c) maintaining the immobilized cells in a productive state in the presence of a production nutrient medium; and (d) withdrawing a portion of the production medium containing produced thienamycin and replacing the withdrawn medium with an equivalent quantity of fresh production medium. 2. The process of Claim 1 wherein the Kieselguhr has a particle diameter between 100 and 900 microns. 3. The process of Claim 2 wherein the withdrawal and replacement of production media is made on a 4-24 hour cycle. 49 The process of Claim 3 wherein the withdrawal is intermittent. 5. The process of Claim 3 wherein the withdrawal is continuous.

Inventors: BAKER, EDWARD E., BUCKLAND, BARRY C., PREVOZNAK, RICHARD
Applicants: MERCK & CO. INC.
Application year: 1983
Main classification: C12P17
IPC codes: C12P17, C12N11, C12R1