Polsinelli, M., and M. Beretta. 1966. Genetic recombina- tion in crosses between Streptomyces aureofaciens and. Streptomyces rimosus. J. Bacteriol. 91:63-68.
Vol. 45, No. 1
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1983, p. 350-353
0099-2240/83/010350-04$02.00/0
Production of Rosamicin: Improvement of Synthetic Medium J. W. KWAK, K. S. KIM, AND DEWEY D. Y. RYU* The Korea Advanced Institute of Science, Chung-Ryang-Ri, Seoul, Korea 0
Received 6 July 1982/Accepted 28 September 1982
Rosamicin is one of the important macrolide antibiotics that has clinical efficacy and broad-spectrum antibacterial activity. Using a mutant strain of Micromonospora rosaria (NRRL 3718), a chemically defined medium was developed, and some fermentation conditions that are important to rosamicin biosynthesis were optimized to achieve rosamicin productivity of 230 jig/ml. Soluble starch and Lasparagine were found to be the best carbon and nitrogen sources, and a stimulative effect of magnesium and zinc ions was also found. The medium developed contains: soluble starch, 4%; L-asparagine, 0.15%; K2HPO4, 0.075%; CaCO3, 0.6%; MgSO4 * 7H20, 0.05%; FeSO4 * 7H20, 10-4 M; CuSO4 * 5H20, 10-5 M; ZnSO4 * 7H20, 10-5 M; and MnSO4 * (4-6)H20, 10-6 M. The required air supply was about 40 mmol of 02 liter-1 * h-1 atm-1, and the favorable culture temperature was 28 to 29°C. -
with a 2-in. (ca. 5-cm) stroke. Fermentation samples were taken after 5 days of cultivation for the growth analysis and after 6 days of cultivation for the antibiotic analysis and analyzed for pH, mycelial dry weight, and antibiotic potency. Since the culture media contained calcium carbonate, a procedure reported by Wagman and Weinstein (11) which insured complete decomposition and solubilization of residual calcium carbonate was employed. The paper disk susceptibility assay was used as the activity test for rosamicin with Staphylococcus aureus ATCC 6538P as a test organism (3, 4). This test organism was grown on 0.8% nutrient broth medium at 33°C for 14 h, and 0.1 ml of test organism was spread on a nutrient agar plate. A filter paper disk, which was impregnated with a measured amount of sample and dried at room temperature, was placed on the agar plate and incubated for 24 h. The rosamicin standard used was in the range of 0 to 30 ,ug/ml, and the sample was assayed after appropriate dilution. Almost all rosamicin titers reported represent mean values of duplicate flask culture experiments, and the deviation was found to be less than 6%. When deviation was greater than 6%, the duplicate flask culture experiment was repeated, and mean values were used. The rosamicin standard was obtained from Schering Corp. (Bloomfield, N.J.). The standard calibration curve of rosamicin activity was prepared each time when samples had to be assayed, although only a slight variation of the standard curve was observed each time. The deviation of each standard assay point from the calibration curve was within 4%. For the selection of carbon and nitrogen
To investigate the effect of medium components on antibiotic production and further improve the level of antibiotic biosynthesis it was necessary to develop a chemically defined medium. The fermentation medium reported by Wagman et al. (10) was a complex medium, and there has not been any report on a chemically defined fermentation medium suitable for rosamicin fermentation research. In this paper, the results of our studies related to the development of a chemically defined medium, and optimal fermentation conditions found for the production of rosamicin are presented. A mutant strain of Micromonospora rosaria (NRRL 3718) was used throughout this experiment. This mutant strain was isolated as a superior strain from UV treatment. It is now available as M. rosaria (KAIS 3718) from our laboratory. Inoculum was prepared in submerged culture with a medium containing: beef extract, 0.3%; tryptone, 0.5%; glucose, 0.1%; soluble starch, 2.4%; yeast extract, 0.5%; and calcium carbonate, 0.2%. The culture was incubated at 30°C for 96 h and transferred to a second-stage culture containing the same medium, and it was cultivated for another 72 h. The mycelial inoculum culture was centrifuged at 5,000 rpm, and the cake was washed twice with sterile distilled water adjusted to pH 7.2. It was brought up to its original volume with sterile distilled water (pH 7.2), and 3 ml of inoculum was transferred into a 70-ml portion of the synthetic medium in a 500-ml flask. The pH of all synthetic media was adjusted to 7.2, and the media were routinely sterilized at 121°C for 15 min. The fermentation cultures were incubated for 6 days at 280 rpm and 28°C on a rotary shaker 350
VOL. 45, 1983
NOTES
351
TABLE 1. Effect of carbon source on rosamicin productiona Carbon sourceb
Final pH
Mycelial dry wt (mg per ml of broth) 6.6
Specific rate of
potency Rosamicin per ml of broth) (pLg
production rosamicin (mg per g of cell per batch cycle)
7.6 50 8.1 Dextran 9.0 47 5.2 7.1 Glucose 0 0 0.6 8.2 Glycerol 0 0 0.8 7.9 Lactose 8.8 51 5.8 7.0 Maltose 7.9 56 7.1 8.1 Soluble starch 22.6 43 1.9 5.8 Sucrose 6.3 36 5.7 8.1 Glucose (1%) and soluble starch (3%) a 0.05%; 7H20, MgSO4 0.2%; K2HPO4, 0.1%; CaCO3, 0.3%; KNO3, Basal medium: L-asparagine, 0.15%; FeSO4 7H20, 0.0028%; MnSO4 (4-6)H20, 0.00026%; CuSO4 * 5H20, 0.00025%; ZnSO4, 0.0003%; NaCl, 0.01%. Culture conditions: 280 rpm, 28°C. Culture samples for determination of mycelial dry weight and rosamicin potency were taken at days 5 and 6, respectively. b Concentration of the carbon source used was 4% (wtlvol). -
sources, the mineral salts reported by Wagman and Weinstein (11) were slightly modified and used in the synthetic media. The modified mineral composition used in the media are shown in the tables where appropriate. Soluble starch was found to be the best carbon source in terms of the cell growth and antibiotic production (56 + 4 pLg/ml), and dextrin, glucose, maltose, and sucrose gave relatively good antibiotic yield (Table 1). Glycerol and lactose did not
yield any rosamicin. Although the specific rate of rosamicin production was 22.6 mg per g of cell per batch cycle and highest when sucrose was used as a carbon source, soluble starch was selected as the best carbon source based on the highest yield of rosamicin, 56 mg of rosamicin per liter. Among the various nitrogen sources tested, Lasparagine was by far the best (Table 2). The rosamicin broth potency reached 80 ,ug/ml. Al-
TABLE 2. Effect of nitrogen sources on rosamicin productiona Nirgnsourceb Nitrogen source
Final b Mycelial dry wt Final (mycperialdrfw pH broth)
Inorganic KNO3 NaNO3 NaNO2 NH4Cl
Organic Urea
Amino acids L-Arginine L-Asparagine L-Aspartic acid L-Glutamine L-Glutamic acid L-Glycine L-Leucine L-Isoleucine DL-Methionine L-Tyrosine L-Asparagine (0.15%) and KNO3 (0.3%) L-Asparagine (0.15%) and L-arginine
Rosamicin potency Rosamicin potency broth) per ml of
Specific rate of rosamicin of cell per (mg per g production batch cycle)
(ptg
7.5
0.7
2.8
4
7.5 6.9 6.0
0.8 0.3 0.7
5.0 0 4.5
6.3 0 6.4
6.8
2.2
2.8
1.3
7.3 7.1 8.1 6.3 7.2 6.8 6.5 6.6 7.0 6.7 7.5 6.9
3.7 7.7 2.6 2.3
30 81 1.5 22 5.4 0 19 26 1.1 2.8 56 64
8.1 10.5 0.6 9.6 1.0 0.0 17.3 28.9 2.8 0.7 9.2 10.2
5.3 0.4 1.1 0.9 0.4 3.8 6.1 6.3
(0.3%)
a Basal medium: soluble starch, 4%; the others are the same Culture conditions: the same as in Table 1. b Nitrogen source used was 0.45% (wtlvol).
as
in Table 1 except for the
nitrogen source.
352
APPL. ENVIRON. MICROBIOL.
NOTES
TABLE 3. Effect of metal ions on rosamicin productiona Metal ions
Final pH
All
7.0
Mycelial (mg per otdry wt of broth) 7.1
potency Rosamicin broth) (pug per ml of
production Specific rosamicin cycle) g ofofcell per batch (mg perrate
119
16.8
Without 0.53 1 1.9 6.6 Mg2+ 11.1 6.1 88 6.8 Fe2+ 14.9 101 7.0 6.8 Cu2+ 2.1 7 6.5 3.4 Zn2+ 13.5 96 7.1 7.1 Mn2+ 26 169 6.9 6.5 Na+ a Basal medium: KH2PO4, 0.075%; CaCO3, 0.6%; soluble starch, 4%; others are the same as in Table 1 except for the metal ions. For culture conditions, see Table 1.
though isoleucine gave the highest specific rate of rosamicin production, the yield was only 26 mg of rosamicin per liter. When soluble starch was used as a carbon source, mycelial growth and rosamicin production increased with concentration, but no more increments were observed beyond a concentration of 4% starch. At this starch concentration, the highest specific rate of rosamicin production observed was 14.3 mg per g of cell per batch cycle, and the final potency was 103 pVg/ml. The optimal concentration of L-asparagine was determined as 0.15% at which the highest rosamicin yield, 230 ,ug/ml, was achieved. The specific rate of rosamicin production was highest at 0.1% L-asparagine. Rosamicin production was found to be very sensitive to L-asparagine. The maximum specific rate of rosamicin production observed was 59.5 mg of rosamicin per g of cell per batch cycle. The enhancement of productivity by amino acid supplementation appears to be very important. The effect of phosphate ion on rosamicin production was studied. Mycelial growth and antibiotic production increase with the concentration of phosphate ion up to 0.075%, but decrease beyond 0.075% phosphate concentration. This phenomenon shows that phosphate ion not only stimulates rosamicin production at low concentration but also suppresses it at high concentration. Metal ions of Mg, Fe, Cu, Zn, Mn, and Na have been known to be important in some macrolide fermentations (8, 9, 11). The effects of these metal ions on rosamicin yield were evaluated (Table 3). Magnesium and zinc ions seemed to have the most significant effect on both mycelial growth and rosamicin production. In the absence of sodium ion, rosamicin potency increased by 42% up to 169 ,ug per ml of broth. The optimal initial pH of the medium was found to be 7.2, and this pH was used throughout this work.
Rosamicin production was also found to be sensitive to the amount of oxygen supplied. At an agitation speed of 280 rpm, the oxygen absorption rate measured was 40 mmol of oxygen per liter-1 * h-1 * atm-, and a rosamicin potency of 230 ,ug/ml was achieved. The sulfite oxidation method was used to determine the oxygen absorption rates in the flasks under the same operating conditions as were used in the fermentation experiment. Two ranges of fermentation temperature were compared for mycelial growth and rosamicin production. Rosamicin production was better at the lower range, 28 to 29°C, as compared with the higher range, 31 to 32°C, while the mycelial growth was slightly better at the high temperature range (7.8 mg/ml at 31 to 32°C versus 7.1 mg/ml at 28 to 29°C). For the development of a chemically defined medium for rosamicin production, several medium components were tested. The best carbon and nitrogen sources selected were soluble starch and L-asparagine. This observation is in agreement with others (7) that carbon sources which can be assimilated relatively slowly are most suitable for the fermentation of secondary metabolites (i.e., polysaccharides are better sources than monosaccharides). Our results on the stimulatory effect of L-asparagine on macrolide antibiotic production have not been reported previously. It is interesting to note that this microorganism did not utilize efficiently glycerol and lactose as a carbon source or L-glycine, DL-methionine, and L-aspartic acid as a sole nitrogen source for rosamicin production. DL-methionine was reported to be a precursor of rosamicin (2), but it inhibited both growth and antibiotic production. Sucrose, L-leucine, and L-isoleucine seemed to suppress the cell growth but gave relatively high specific productivities (amount of rosamicin produced per gram of cell per batch cycle).
VOL. 45, 1983
NOTES
TABLE 4. Chemically defined fermentation medium developed for production of rosamicin and defined medium for erythromycina Amt (g/liter) in:
Component
Rosamicin
Erythromycin
medium
medium
Soluble starch b 40 L-Asparagine 1.5 CaCO3 6.0 3 K2HPO4 0.75 2.5 MgSO4 * 7H20 0.5 0.5 FeSO4 * 7H20 0.028 0.02 CuSO4 5H20 0.0025 ZnSO4- 7H20 0.003 0.05 MnSO4' (4-6)H20 0.0026 NaCI 5.0 MnCl2 * 4H20 0.001 CoC12 6H20 0.001 Sucrose 68.4 Glycine 7.5 L-Tyrosine 1.8 a The rosamicin medium was determined in this study, while the erythromycin medium was determined by Porter (9). b , Not used. -
The inability to utilize glycerol, lactose, and Lglycine suggests the absence or repression of such enzymes as glycerol kinase, ,B-galactosidase, and glycine synthase in this organism or a permeability problem. The reason why this strain cannot utilize DL-methionine and L-aspartic acid has not been determined. This high production of antibiotic by sucrose, L-leucine, and L-isoleucine in spite of very low cell yield suggests a possibility that these compounds may induce the biosynthesis of rosamicin synthetase or may activate these enzymes. L-Tyrosine may act as a repressor for the biosynthesis of these enzymes or as an inhibitor of the activities of these enzymes in antibiotic production, based on the observation that very low production of rosamicin was achieved in spite of relatively
good growth. The fermentative production of some polypeptide macrolide antibiotics is subject to regulation by nitrogen metabolism and phosphate, which can decrease antibiotic yield at a high concentration of these compounds (5). Nitrogen metabolite repression affects the biosynthesis of enzymes that act on nitrogen-containing substrates, and high energy charge produced from
353
phosphate may inhibit some enzymes involved in secondary metabolism (1). M. rosaria required the divalent metal ions, magnesium, iron, copper, and zinc, for the production of rosamicin. Of these, magnesium and zinc ions were essential to both growth and antibiotic production. These metals can probably activate antibiotic synthetase and stabilize some enzyme-substrate complexes participating in antibiotic biosynthesis; for example, magnesium ion may activate acetyl coenzyme A carboxylase. The requirement of oxygen for production of rosamicin was also important. The maximal production was achieved at about 40 mmol of 02 per liter * per h * per atm. The chemically defined medium developed and optimized based on our results is shown in Table 4. The defined medium for rosamicin appears to be similar to that for erythromycin, although they differ significantly in terms of medium components and their amounts (Table 4). By using the medium, the rosamicin productivity achieved was as high as 230 ,ug per ml of broth. Our results are comparable to those reported by Wagman et al. (10). LITERATURE CITED 1. Drew, S. W., and A. L. Demain. 1977. Effect of primary metabolites on secondary metabolism. Annu. Rev. Microbiol. 31:343-356. 2. Ganguly, A. K., B. K. Lee, R. Brambilla, R. Condon, and 0. Sarre. 1976. Biosynthesis of rosamicin. J. Antibiot. 29:976-977. 3. Grove, D. C., and W. A. Randall. 1955. Assay methods of antibiotics, a laboratory manual. Medical Encyclopedia, New York. 4. Lorian, V. 1980. Antibiotics in laboratory medicine, p. 2454. The Williams & Wilkins Co., Baltimore. 5. Martin, J. F. 1979. Nonpolyene macrolide antibiotics, p. 239-291. In A. H. Rose (ed.), Secondary products of metabolism. Academic Press, Inc., London. 6. Martin, J. F., and A. L. Demain. 1980. Control of antibiotic biosynthesis. Microbiol. Rev. 44:230-251. 7. Martin, J. F., and L. E. McDaniel. 1977. Production of polyene macrolide antibiotics. Adv. Appl. Microbiol. 21:1-52. 8. Polsinelli, M., and M. Beretta. 1966. Genetic recombination in crosses between Streptomyces aureofaciens and Streptomyces rimosus. J. Bacteriol. 91:63-68. 9. Porter, J. N. 1975. Cultural conditions for antibioticproducing microorganisms. Methods Enzymol. 43:3-23. 10. Wagman, G. H., J. A. Waitz, J. Marquez, A. Murawski, E. M. Oden, R. T. Testa, and M. J. Weinstein. 1972. A new Micromonospora-produced macrolide antibiotic, rosamicin. J. Antibiot. 25:641-646. 11. Wagman, G. H., and M. J. Weinstein. 1966. A chemically defined fermentation medium for the growth of Micromonospora purpurea. Biotechnol. Bioeng. 8:259-273.
