ANTIMICROBIL AGENTS AND CHzMOTHRAPY, Nov. 1977, P. 571-572 Copyright C 1977 American Society for Microbiology
Vol. 12, No. 5 Printed in U.S.A.
Chartreusin: Production and Microbiological Assay LADISLAV J. HANKA* AND S. A. GERPHEIDE
Cancer Research Laboratories, The Upjohn Company, Kalamazoo, Michigan 49001 Received for publication 18 July 1977
Chartreusin was produced in the fermentation liquors of Streptomyces chartreusis at peak concentrations of 200 to 300 ,ug/ml. The titers could be increased by 200 to 300% or more by incorporating r.-fucose, a part of the chartreusin molecule, into the fermentation media. A microbiological assay with Sarcina lutea could detect concentrations of the drug of 0.5 to 1.0 ,ug/ml. Microbiological assay. Since no practical microChartreusin (Fig. 1) is an antimicrobial antibiotic that was first described by Leach et al. biological assay was available, a large number of in 1953 (3). Recently, chartreusin was shown microorganisms were tested for susceptibility to chartreusin. The most susceptible organism apto inhibit experimental tumors in animals (4). peared a strain of Sarcina lutea, UC-3383. A Such results generated renewed interest in this numbertoofbetechniques were investigated before a drug. satisfactory assay was developed. (The drug is Much of the fermentation technology has poorly soluble in water; it is unstable in solution changed during the 23 years since the drug under some conditions; and it is apparently bound was first discovered. Accordingly, a considera- to the paper disks commonly used in microbiological ble amount of work was done recently both on assay.) The drug is first dissolved in 0.01 N NaOH the fernentation and in the development of a to a concentration of 1 mg/ml. It is then further buffer to obtain suitable microbiological assay. The pertinent diluted in 0.1 M, pH 7.0 phosphate and 0.62 ,ug/ml, concentrations of 10.0, 5, 2.5, 1.25, results are reported herein. which are used for the construction of the standard curve. These solutions are kept frozen in the dark and are prepared fresh every week. MATERIALS AND METHODS microorganism used for assay was cultivated Fermentation. The inocula of Streptomyces char- in The a synthetic medium of the following composition: treusis NRRL-2287 were frozen plugs made from a Na1HPO4 * 7H10, 2 g; KH2PO4, 0.5 g; (NH4)2SO4, 1 g; petri plate heavy surface growth on agar and kept MgSO4, 0.1 g; glucose, 2 g; distilled water, 1 liter. in a liquid nitrogen storage tank. The frozen plugs After autoclaving, medium was further supplewere used to inoculate the medium, which consisted mented aseptically this with sterile solutions of: glycine, of (grams per liter): cerelose, 10; corn steep liquor 20 mg; valine, 20 mg; 20 mg; glutamine, (Corn Products Co., Englewood Cliffs, N.J.), 10; 20 mg; cysteine, 20 mg;isoleucine, 20 mg; metallic adenosine, Pharmamedia (Traders Oil Mill Co., Fort Worth, stock solution (1), 1 ml; vitamin mixture (EaTex.), 2; and liquid peptone (Wilson Protein Tech- ion 100 x; Microbiological Associates, Bethesda, nology, Calumet City, I1.), 10. The cultivation was gle, Md.), 1 ml. This medium was inoculated with S. done for 48 h in 500-ml nonstippled flasks (each lutea and cultivated in Erlenmeyer flasks for 16 h containing 100 ml of medium) on a rotary shaker on a reciprocating shaker at 32°C. This culture was (250 rpm) at 28°C. This inoculum was used to seed to inoculate (at a rate of 0.2 ml/100 ml) the the production medium at the rate of 5% (vol/vol). used of the following composition: Na2HPO4The production medium was a slightly modified assay agar 0.2 g; nutrient broth (Difco), 4 g; 3 g; KH2PO4, 7H20, medium described previously by Calhoun and John1 g; agar, 15 g; distilled water, 1 liter. The son (1); it contained (grams per liter): lactose, 20; glucose, starch, 15; soybean meal (Lauhoff Grain Co., Dan- inoculated agar was dispensed into plastic petri ville, Ill.), 15; distillers solubles (Brown and Forman, Louisville, Ky.), 20; corn steep liquor, 5; NaCl, H3C / 0 5; CaCO3, 8; and lard oil, 10 ml/liter. The fermenta0 HO 0= tion was carried out in nonstippled flasks at 28°C. 0 CH3 S OH Since the chartreusin molecule contains two mon/ o D-FUCOSE it HO D-fucose and appeared -digitalose, osaccharides, reasonable to examine the effect of incorporating 0 these compounds into the fermentation media on Ha o the titer of the drug. Since D-digitalose was not CH3 HO0 D-DIGITALOSE readily available, only D-fucose was evaluated in H3COI these studies. It was used at three levels (20, 30, OH and 40 g/liter) to replace lactose in the production FIG. 1. Structure of chartreusin (2, 5). medium. 571
572
ANTiMICROB. AGENTS CHEMOTHZR.
HANKA AND GERPHEIDE
TABLE 1. Fermentation of chartreusin in a 500-ml flask incuof Length Con(n of drug pH (g/ml)
bation (h)
Vol. 12, No. 5 Printed in U.S.A.
Chartreusin: Production and Microbiological Assay LADISLAV J. HANKA* AND S. A. GERPHEIDE
Cancer Research Laboratories, The Upjohn Company, Kalamazoo, Michigan 49001 Received for publication 18 July 1977
Chartreusin was produced in the fermentation liquors of Streptomyces chartreusis at peak concentrations of 200 to 300 ,ug/ml. The titers could be increased by 200 to 300% or more by incorporating r.-fucose, a part of the chartreusin molecule, into the fermentation media. A microbiological assay with Sarcina lutea could detect concentrations of the drug of 0.5 to 1.0 ,ug/ml. Microbiological assay. Since no practical microChartreusin (Fig. 1) is an antimicrobial antibiotic that was first described by Leach et al. biological assay was available, a large number of in 1953 (3). Recently, chartreusin was shown microorganisms were tested for susceptibility to chartreusin. The most susceptible organism apto inhibit experimental tumors in animals (4). peared a strain of Sarcina lutea, UC-3383. A Such results generated renewed interest in this numbertoofbetechniques were investigated before a drug. satisfactory assay was developed. (The drug is Much of the fermentation technology has poorly soluble in water; it is unstable in solution changed during the 23 years since the drug under some conditions; and it is apparently bound was first discovered. Accordingly, a considera- to the paper disks commonly used in microbiological ble amount of work was done recently both on assay.) The drug is first dissolved in 0.01 N NaOH the fernentation and in the development of a to a concentration of 1 mg/ml. It is then further buffer to obtain suitable microbiological assay. The pertinent diluted in 0.1 M, pH 7.0 phosphate and 0.62 ,ug/ml, concentrations of 10.0, 5, 2.5, 1.25, results are reported herein. which are used for the construction of the standard curve. These solutions are kept frozen in the dark and are prepared fresh every week. MATERIALS AND METHODS microorganism used for assay was cultivated Fermentation. The inocula of Streptomyces char- in The a synthetic medium of the following composition: treusis NRRL-2287 were frozen plugs made from a Na1HPO4 * 7H10, 2 g; KH2PO4, 0.5 g; (NH4)2SO4, 1 g; petri plate heavy surface growth on agar and kept MgSO4, 0.1 g; glucose, 2 g; distilled water, 1 liter. in a liquid nitrogen storage tank. The frozen plugs After autoclaving, medium was further supplewere used to inoculate the medium, which consisted mented aseptically this with sterile solutions of: glycine, of (grams per liter): cerelose, 10; corn steep liquor 20 mg; valine, 20 mg; 20 mg; glutamine, (Corn Products Co., Englewood Cliffs, N.J.), 10; 20 mg; cysteine, 20 mg;isoleucine, 20 mg; metallic adenosine, Pharmamedia (Traders Oil Mill Co., Fort Worth, stock solution (1), 1 ml; vitamin mixture (EaTex.), 2; and liquid peptone (Wilson Protein Tech- ion 100 x; Microbiological Associates, Bethesda, nology, Calumet City, I1.), 10. The cultivation was gle, Md.), 1 ml. This medium was inoculated with S. done for 48 h in 500-ml nonstippled flasks (each lutea and cultivated in Erlenmeyer flasks for 16 h containing 100 ml of medium) on a rotary shaker on a reciprocating shaker at 32°C. This culture was (250 rpm) at 28°C. This inoculum was used to seed to inoculate (at a rate of 0.2 ml/100 ml) the the production medium at the rate of 5% (vol/vol). used of the following composition: Na2HPO4The production medium was a slightly modified assay agar 0.2 g; nutrient broth (Difco), 4 g; 3 g; KH2PO4, 7H20, medium described previously by Calhoun and John1 g; agar, 15 g; distilled water, 1 liter. The son (1); it contained (grams per liter): lactose, 20; glucose, starch, 15; soybean meal (Lauhoff Grain Co., Dan- inoculated agar was dispensed into plastic petri ville, Ill.), 15; distillers solubles (Brown and Forman, Louisville, Ky.), 20; corn steep liquor, 5; NaCl, H3C / 0 5; CaCO3, 8; and lard oil, 10 ml/liter. The fermenta0 HO 0= tion was carried out in nonstippled flasks at 28°C. 0 CH3 S OH Since the chartreusin molecule contains two mon/ o D-FUCOSE it HO D-fucose and appeared -digitalose, osaccharides, reasonable to examine the effect of incorporating 0 these compounds into the fermentation media on Ha o the titer of the drug. Since D-digitalose was not CH3 HO0 D-DIGITALOSE readily available, only D-fucose was evaluated in H3COI these studies. It was used at three levels (20, 30, OH and 40 g/liter) to replace lactose in the production FIG. 1. Structure of chartreusin (2, 5). medium. 571
572
ANTiMICROB. AGENTS CHEMOTHZR.
HANKA AND GERPHEIDE
TABLE 1. Fermentation of chartreusin in a 500-ml flask incuof Length Con(n of drug pH (g/ml)
bation (h)
