ARTICLE IN PRESS Microbiological Research 160 (2005) 243—248
Effects of nutrients on the production of AK-111-81 macrolide antibiotic by Streptomyces hygroscopicus V. Gesheva, V. Ivanova, R. Gesheva Institute of Microbiology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria Accepted 16 January 2004
KEYWORDS Nonpolyenic macrolide antibiotic; Streptomyces hygroscopicus; Nutrient effects
Summary The influence of different carbon and nitrogen sources on the production of AK-111-81 nonpolyenic macrolide antibiotic by Streptomyces hygroscopicus 111-81 was studied. Substitution of glucose with lactose or glycerol significantly affected maximal antibiotic AK-111-81 productivity as the growth rate was close to that of the basal fermentation medium. Addition of ammonium succinate to the fermentation medium markedly increased the antibiotic productivity as the growth rate was low. Divalent ions as Mn2+, Cu2+, Fe2+ stimulated AK-111-81 antibiotic biosynthesis. These results allow us to develop a new fermentation medium showing 6-fold increase of AK-111-81 antibiotic formation compared with the basal fermentation medium. & 2005 Elsevier GmbH. All rights reserved.
Introduction Nature and concentration of some components in fermentation medium have a marked effect on antibiotic production (Aharonowitz and Demain, 1978; Martin and Demain, 1980; Chatterjee and Vining 1981; Omura and Tanaka, 1986; Doull and
Vining, 1990; Lebrihi et al., 1992; Cheng et al., 1995; Lee et al., 1997; Coisne et al., 1999; Parekh et al., 2000; Sanchez and Demain, 2002). Influence of particular nutrients on the antibiotic biosynthesis is determined by the chemical structures of antibiotic substances (Pereda et al., 1998). Thus some nitrogen sources may incorporate in
Abbreviations: AK-111-81, nonpolyenic macrolide antibiotic; 1-2, 4-5, niphimycin derivatives; 3, demalonylniphimycin; Az, azalomycin B; IM-111-81, polyether antibiotic IM-111-81; I, stereoisomer of nigericin; II, nigericin; III, acetate nigericin; IV, dehydrooxynigericin Corresponding author. E-mail address: [email protected]
(V. Gesheva). 0944-5013/$ - see front matter & 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2004.06.005
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antibiotic molecules as precursors or their amino groups transfer to specific intermediate products (Aharonowitz, 1980; Martin and Demain, 1980; Omura and Tanaka, 1986; Doull and Vining, 1990; Cheng et al., 1995). Nutrient deficiency is responsible for onset of antibiotic biosynthesis (Demain et al., 1983; Doull and Vining, 1990; Sanchez and Demain, 2002). When carbon or nitrogen source is a limiting factor, growth is rapidly reduced and antibiotic biosynthesis takes place in the stationary phase. In other cases, antibiotic production is associated with the growth phase. Streptomyces hygroscopicus strain 111-81 produces the nonpolyenic macrolide antibiotic AK-11181 (Gesheva et al., 1994). It possesses antibacterial and antifungal activity (Gesheva et al., 1977). AK111-81 comprises 5 major and 2 minor components. The component 3 is determined as demalonylniphimycin (Ivanova and Schlegel, 1998). Its structure shows a macrocyclic polyhydroxyl lactone, a 6membered inner-molecular hemiketal ring and a side chain with a terminal quanidine group (Fig. 1). The strain 111-81 forms also the polyether antibiotic IM-111-81 consisting of 4 components, nigericin and its derivatives (Gesheva et al., 1981; Ivanova et al., 1984) and azalomycin B (Gesheva et al.,1994). Little is known about effects of fermentation medium components on the biosynthesis of nonpolyenic macrolide antibiotics (Georgieva-Borisova, 1974; Moncheva et al., 1997). The aim of this study is to observe the influence of different nutrients on AK-111-81 production.
Materials and methods Media and growth conditions Inoculation medium contained (g/l): soy meal, 20; glucose, 30; K2HPO4, 0.5; NaCl, 0.5; CaCO3, 6; pH was adjusted to 6.8 before sterilization. Basal production medium contained the following components (g/l): glucose, 25; soy meal, 5; NaCl, 5; K2HPO4, 0.6; MgSO4, 0.5; CaCO3, 1.0. Carbohydrates (fructose, sucrose, lactose, glycerol, starch and their mixtures at a concentration of 2.5% (w/w) were added to basal fermentation medium in which glucose was omitted. Ammonia salts (NH4NO3, (NH4)2SO4, NH4Cl, KNO3, ammonium acetate, ammonium succinate), L-amino acids (lysine, arginine) and casein hydrolysate were added at a concentration of 0.15% to basal fermentation medium. The mineral salts CuSO4 5H2O, (NH4)6Mo7O24 4H2O, MnCL2 4H2O, FeSO4 7H2O, ZnSO4 7H2O were supplemented in different concentrations to basal production medium. Cultures were grown in 500 ml Erlenmeyer flasks on a rotary shaker (220 rpm) at 28 1C. The fermentation medium was inoculated with 5% of a preculture after 40 h growth and incubated for 144 h. Multifactorial experiments were performed according to Maximov (1980).
Antibiotic assay and chromatography Antifungal activity of methanol extracts from culture broths were assayed against Candida utilis, using as standard nonpolyenic macrolide antibiotic
Fig. 1. Demalonylniphimycin.
ARTICLE IN PRESS Effects of nutrients on the production of AK-111-81 macrolide antibiotic by S. hygroscopicus AK-111-81 isolated by us. Component composition of AK-111-81, polyether IM-111-81 and azalomycin B were monitored by thin-layer chromatography (TLC) on silica gel 60 F254 (Merck) with chloroform–methanol–water, 2:2:1(v/v/v), as mobile phase. Antibiotics were visualised by spraying with a solution of 3% vanillin in 1.5% sulfuric acid in ethanol followed by heating at 110 1C for 3–5 min. Growth and biosynthetic potential of S. hygroscopicus with respect to antifungal antibiotic AK-111-81 were determined by maximal specific growth rate (Vmax) and maximal antibiotic productivity (Pmax) according to Vinogradova et al. (1985). Vmax and Pmax were calculated as follows: V max ¼
X max 100 , tmax
where Xmax is the maximal biomass in 100 ml culture broth and tmax is the time to reach Xmax: P max ¼
AZ2 AZ1 , ðX Z1 þ Z Z2 Þ=2 Dz
where AZ2 and AZ1 are antibiotic amounts at the time points z2 and z1, X Z2 and X Z1 are biomasses at the time points z2 and z1. Biomass was determined by weighing to constant dry weight after drying at 105 1C.
Table 1. Influence of carbon and nitrogen sources on growth and antibiotic productivity of S. hygroscopicus 111-81 Source
Vmax (mg/ml h)
Pmax (mg/mg h)
2.5% Fructose Sucrose Lactose Glycerol Starch Glucose and lactose Glucose and sucrose Glucose and glycerol
5.60 2.55 4.84 4.78 4.16 17.91 4.30 6.22
0.36 0.08 11.85 7.57 3.58 0.36 2.14 0.07
0.15% Ammonium nitrate Ammonium sulphate Ammonium chloride Ammonium succinate Ammonium acetate Potassium nitrate Lysine Arginine Casein hydrolysate
6.30 8.80 6.60 3.20 5.90 10.55 17.27 13.18 20.83
1.85 1.86 0.30 17.68 4.55 0.80 0.01 2.72 1.76
Basal fermentation medium
Vmax, maximum specific growth rate; Pmax, maximum productivity of AK-111-81 antibiotic. Basal fermentation medium contained 2.5% glucose and 0.5% soy meal.
Results and discussion Growth and antibiotic production on synthetic media were unsatisfactory. Consequently we used a medium with a minimal amount of soy meal (0.5%), which supports both, growth and antibiotic productivity.
Influence of carbon sources on growth and AK-111-81 antibiotic production When fructose, sucrose, glucose plus glycerol or lactose were added, low values of Pmax were obtained (Table 1). Vmax for these substrates varies from 2.55 to 17.91 which indicates that there is no correlation between Vmax and Pmax. The carbohydrates listed above decreased AK-111-81 biosynthesis. From TLC analysis it was established that some components of AK-111-81 have been formed. The carbohydrate mixtures favoured the biosynthesis of azalomycin B, while glucose, glycerol, sucrose, fructose, starch favoured the production of some components of polyether IM-111-81 and azalomycin B (Fig. 2). Glycerol had a weak stimulation effect on AK111-81 biosynthesis, as Pmax was 7.57. In contrast,
glycerol was a good source for niphimycin production (Georgieva-Borisova, 1974). Smith and Chater (1988) have shown that glycerol induction and glucose repression act at the level of transcription in S. coelicolor A3 (2). The best carbon source for AK-111-81 synthesis was lactose with a Pmax value of 11.85. Similarly Vinogradova et al. (1985) detected a high level of heliomycin on lactose. Sanchez and Demain (2002) have reported positive effects of lactose on biosynthesis of penicillin, enniatin and erythromycin. Chatterjee and Vining (1981) showed that lactose usage was mediated by a lactose-inducible b-galactosidase subject to glucose catabolite repression.
Effects of nitrogen sources on the growth and biosynthesis of AK-111-81 Depending on the biosynthetic pathways involved, nitrogen sources may affect antibiotic formation. Inorganic salts (Table 1) in the medium inhibited the AK-111-81 production as Pmax values were lower than Pmax for the basal fermentation
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Fig. 2. Influence of carbon source to antibiotic production of Streptomyces hygroscopicus 111-81 at 96 h. (a) glucose; (b) glycerol; (c) sucrose; (d) fructose; (e) lactose; (f) starch; (g) glucose and glycerol; (h) glucose and lactose; (i) glucose and sucrose.
Fig. 3. Influence of nitrogen source to antibiotic production of Streptomyces hygroscopicus 111-81: (a and b) ammonium nitrate 96 and 120 h, respectively; (c and d) ammonium acetate 96 and 120 h, respectively; (e) ammonium succinate 96 h; (f) lysine 96 h; (g) arginine 96 h; (h) sodium nitrate 96 h; (i) ammonium sulphate 96 h; (j) ammonium chloride 96 h; (k) casein hydrolyzate 96 h; (l) basal fermentation medium with 0.5% soy meal 96 h.
medium. It was noted by Aharonowitz (1980), Martin and Demain (1980) and Sanchez and Demain (2002) that ammonium salts did not favour biosynthesis of novobiocin, actinomycin, neomycin, kanamycin and others, but for rapamycin ammonium sulfate was the best nitrogen source (Lee et al. 1997). Ammonium nitrate, sulfate, chloride, acetate and arginine stimulate the formation of some components of IM-111-81 and azalomycin B (Fig. 3). The highest Pmax for AK-111-81 antibiotic is reached after addition of ammonium succinate. It has a strong effect on AK-111-81 synthesis at low growth rate. TLC showed that on ammonium
succinate supplemented medium S. hygroscopicus 111-81 synthesised components 1–5 of AK-111-81, while azalomycin B and IM-111-81 were present only in trace amounts (Fig. 3). Malonate, methylmalonate and ethylmalonate are used for polyketide biosynthesis (Pereda et al., 1998). Succinate might be incorporated in biosynthetic paths for the formation of elongation units of the oligoketide chain (methyl-CoA and methylmalonyl-CoA) and might also serve as precursor of the nonpolyenic macrolide AK-111-81, a polyketides antibiotic. Lysine, arginine or casein hydrolysate allow significant growth of strain 111-81 although Pmax values are low. TLC data indicate that on medium
ARTICLE IN PRESS Effects of nutrients on the production of AK-111-81 macrolide antibiotic by S. hygroscopicus Table 2.
CuSO4 5H2O a (mg/ml)
FeSO4 7H2O b (mg/ml)
MnCl2 4H2O c (mg/ml)
Antifungal activity(mg/ml) 96 h 120 h
1 2 3 4 5 6 7 8 9
0.1 0.05 0.1 0.05 0.1 0.05 0.1 0.05 0.07
400 400 300 300 400 400 300 300 350
10 10 10 10 5 5 5 5 7
1800 370 1700 780 1000 1000 1100 2420 1060
with lysine or casein hydrolysate, components 1 and 2 of AK-111-81 and also azalomycin B were synthesised. Arginine favours formation of components 1–4 of AK-111-81.
Influence of mineral ions on antibiotic production of AK-111-81 Preliminary investigations on the effects of several ions indicate that Cu2+, Mn2+, Mo2+, Zn2+, and Fe2+ affect AK-111-81 biosynthesis depending on their concentration. Divalent ions stimulate the production of polyenes (Liu et al.,1975; Solivery et al., 1988; Lee et al., 1997). Georgieva-Borisova (1974) had shown that Fe2+ and Mn2+ favour niphimycin production. In our case, the highest yields of AK-111-81 have been achieved in the presence of FeSO4 7H2O (300–400 mg/ml), CuSO4 5H2O (0.05–0.07 mg/ml) and MnCl2 4H2O (5-10 mg/ml). TLC-analysis detected that Cu2+, Fe2+ and Mn2+ stimulated the biosynthesis of components 1–5 of AK-111-81, whereas azalomycin B is present only in traces. To determine the optimal concentrations of microelements, multifactorial experiments were carried out. The results showed that the best variant is No. 8 which contained the mineral salts at low levels (Table 2). The antifungal activity obtained at 96 h was 2420 mg/ml.
Kinetic of fermentation parameters of S. hygroscopicus 111-81 A culture was grown in a medium containing 25 g/ l lactose, 5 g/l soy meal, 1.5 g/l ammonium succinate, 1 g/l CaCO3, and 1 ml trace element solution with 5 mg/ml MnCl2 4H2O, 0.05 mg/ml CuSO4 5H2O, and 300 mg/ml FeSO4 7H2O. The biomass increased rapidly during the first 24 h, and declined again after 72 h (Table 3). The maximum
800 830 1080 560 500 610 1040 1700 660
Table 3. Kinetics of fermentation parameters of S. hygroscopicus 111-81 Hour
Residual glucose (g/ 100 m)
Cell dry AK-111-81 biomass (g/l) antibiotic activity (mg/ ml)
24 48 72 96 120 144
1.17 0.99 0.95 0.81 0.64 0.36
15.6 16.0 16.4 12.2 8.4 8.0
830 1200 2570 3650 3240 2870
of antifungal activity was reached with 3650 mg/ml, when the cell biomass had decreased. TLC showed that the strain 111-81 synthesized AK-111-81, while the antibiotics IM-111-81 and azalomycin B were present only in traces. The improved medium gave rise to a 6-fold increase of AK-111-81 activity when compared with that of the basal fermentation medium ranging around 600 mg/ml.
References Aharonowitz, Y., 1980. Nitrogen metabolite regulation of antibiotic biosynthesis. Annu. Rev. Microbiol. 34, 209–234. Aharonowitz, Y., Demain, A.L., 1978. Carbon catabolite regulation of cephalosporin production in Streptomyces clavuligerus. Antimicrob. Agents Chemother. 14, 159–164. Chatterjee, S., Vining, L.C., 1981. Nutrient utilization in actinomycetes. Induction of a-glucosidases in Streptomyces venezulae. Can. J. Microbiol. 27, 639–645. Cheng, J.R., Fang, A., Demain, A.L., 1995. Effect of amino acids on rapamycin biosynthesis in Streptomyces hygroscopicus. Appl. Microbiol. Biotechnol. 43, 1096–1098.
ARTICLE IN PRESS 248 Coisne, S., Bechet, M., Blondeau, R., 1999. Actinorhodin production by Streptomyces coelicolor A3(2) in ironrestricted media. Lett. Appl. Microbiol. 28, 199–202. Demain, A.L., Aharonowitz, Y., Martin, J.F., 1983. Metabolite control of secondary biosynthetic pathways. In: Vining, L.C. (Ed.), Biochemistry and Genetic Regulation of Commercially Important Antibiotics. Addison-Wesley, London, pp. 49–67. Doull, J.L., Vining, L.C., 1990. Nutritional control of actinorhodin production of Streptomyces coelicolor A3(2); suppressive effect of nitrogen and phosphate. Appl. Microbiol. Biotechnol. 32, 449–454. Georgieva-Borisova, J.Ch., 1974. Taxonomic characteristics of strain Actinomyces hygroscopicus B-255 and conditions for its antibiotic production. Ph. D. Thesis, Sofia. Gesheva, V., Ivanova, V., Gesheva, R., 1994. Biological characteristics and antibiotic production of Streptomyces hygroscopicus strains. Actinomycetes 5, 57–63. Gesheva, R.L., Ivanova, V.B., Saharieva, M., Rachev, R., Rusev, P.P., Popov, K.B., Tzvetkova, R.Ch., Belomusova, D.K., Sevrieva, V.S., Gushterova, A.G., Thrum, H., Schlegel, R., Kleinwachter, W., Schade, W., 1981. Bulgarian Patent No. 32,645. Gesheva, R., Panajotov, P., Darakchieva, M., Naumova, M., Tsankov, H., Mladenov, M., Karajova, J., 1977. Actinomycetes antagonists of Fusarium graminearum schw., responsible for fusariosis of wheat. Prilojna Mikrobiol. 7, 76–81. Ivanova, V., Gesheva, R., Panajotov, P., Belomusova, D., 1984. Isolation, purification and separation of polyether antibiotic complex from Streptomyces hygroscopicus 111-81. In: Toshkov, A. (Ed.), Successes in Industrial and Infection Microbiology. BAN, Sofia, pp. 27–34. Ivanova, V., Schlegel, R., 1998. Structure elucidation of the antibiotic demalonylniphimycin by two-dimensial NMR techniques. Actinomycetes 8, 1–9. Lebrihi, A., Lamsaif, D., Lefebvre, G., Germain, P., 1992. Effect of ammonium salts ions on spiramycin biosynthesis in Streptomyces ambofaciens. Appl. Microbiol. Biotechnol. 37, 382–387. Lee, M.C., Kojima, J., Demain, A.L., 1997. Effect of nitrogen source on biosynthesis of rapamycin by
V. Gesheva et al. Streptomyces hygroscopicus. J. Ind. Microbiol. Biotechnol. 19, 83–86. Liu, C.M., McDaniel, L.E., Schafferer, C.P., 1975. Factors affecting the production of candicidin. Antimicrob. Agents Chemother. 7, 196–202. Martin, J.F., Demain, A.L., 1980. Control of antibiotic biosynthesis. Microbiol. Rev. 44, 230–251. Maximov, V.N., 1980. Multifactorial Experiments in Biology. Moscow State University Publ., Moscow. Moncheva, P., Danova, S., Antonova, S., Ivanova, I., 1997. Physiological role of extracellular proteases and Ca2+ in the differentiation processes and antibiotic formation by Streptomyces albogriseolus. Antibiot. Chimiotherapia 42, 14–19. Omura, S., Tanaka, J., 1986. Biosynthesis of tylosin and its regulation by ammonium and phosphate. In: Kleinkauf, H., von Do ¨hren, H., Dormaner, H., Nesmann, G. (Eds.), Regulation of Secondary Metabolites. VCH Publishers Inc., Berlin, pp. 306–332. Parekh, S., Vinci, V.A., Strobel, K.J., 2000. Improvement of microbial strains and fermentation processes. Appl. Microbiol. Biotechnol. 54, 287–301. Pereda, A., Summers, K.G., Stassi, D.L., Ruan, X., Katz, L., 1998. The loading domain of the erythromycin polyketide synthase is not essential for erythromycin biosynthesis in Saccharopolyspora erytraea. Microbiology 144, 543–553. Sanchez, S., Demain, A.L., 2002. Metabolic regulation of fermentation processes. Enz. Microb. Technol. 31, 895–906. Smith, C.P., Chater, K.F., 1988. Cloning and transcriptional analysis of the entire glycerol utilization (gylABX) operon of Streptomyces coelicolor A3(2) and identification of a closely associated transcription unit. Mol. Gen. Genet. 211, 129–137. Solivery, S., Mendosa, A., Arias, M.E., 1988. Effect of different nutrients on the production of polyene antibiotics PA-5 and PA-7 by Streptoverticillium sp. 43/16 in chemically defined medium. Appl. Microbiol. Biotechnol. 28, 254–257. Vinogradova, K.A., Kirilova, N.P., Sokolova, Z.G., Nikolau, P.A., Shalgina, M.V., Skvortsova, G.N., Polin, A.N., 1985. Regulation of heliomycin biosynthesis by carbon sources. Antibiot. Med. Technol. 30, 264–270.