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Acta, Lectures on the Scientific Basi8 of Medicine, vol. 6, p. 198. 4, 301. Ed. by Fraser, F. R. London: Athlone Press. Mahler, H. R., Wakil, S. J. & Bock, R. M. (1953). J. biol. Whittaker, V. P. & Wijesundera, S. (1952). Biochem. J. 51, Chem. 204, 453. 348. Controlled Biosynthesis of Actinomycin with Sarcosine BY E. KATZ AND W. A. GOSS In8titute of Microbiology, Rutger8, The State Univer8ity, New Bru?nwick, New Jer8ey, U.S.A. (Received 3 April 1959) The actinomycins represent a family of chromo- New actinomycins were formed by S. chryaomallus peptide antibiotics which differ solely in the nature when DL-isoleucine or sarcosine was added to the of the amino acids present in the peptides of the medium (Schmidt-Kastner, 1956a). In our Lab- molecule (Fig. 1). It has been established that an oratory it has been determined that S. antiobiticus actinomycin-producing organism generally syn- forms at least one new actinomycin with sarcosine thesizes a mixture of these substances. For and several new compounds when DL-pipecolic acid example, Streptomyces antibioticu8 forms a mixture is used. consisting of actinomycins I-V; occasionally trace During an investigation of the role of amino amounts of a sixth component are produced acids on actinomycin synthesis by S. antibioticus, it (Katz & Goss, 1958). Streptomyces chry8omallu8 was observed that sarcosine selectively stimulated produces actinomycins IV, VI and VII (Schmidt- the production of actinomycins II and III (Fig. 2). Kastner, 1956b). These components, normally synthesized in trace The quantitative and qualitative nature of the amounts, represented approximately 60 % of the actinomycin mixture synthesized can be modified actinomycin mixture formed under the nutritional to a considerable extent; in particular, the nitrogen conditions employed. By paper-chromatography source supplied has been shown to have a profound techniques, it was established that actinomycin II influence on its composition. Actinomycin IV in- contains threonine, valine, sarcosine, and N- creased from 10 to 83 % ofthe mixture produced by methylvaline, whereas actinomycin III possesses, S. chry8omallu8 when DL-valine was added to the in addition, one-half the amount ofproline found in medium (Schmidt-Kastner, 1956b); hydroxy-L- actinomycin IV (Katz, Goss & Pugh, 1958). proline brought about an increase in synthesis of Recently Johnson & Mauger (1959) obtained actinomycin I from 6-7 to 31 % of the actinomycins quantitative data showing that 4 moles of sarcosine produced by S. antibioticu, (Katz & Goss, 1958). and no proline are present in actinomycin II, and VoI. 73 CONTROLLED BIOSYNTHESIS OF ACTINOMYCIN 459 that 3 moles of sarcosine and only 1 mole of proline Chemical Co., Norwich, N.Y., U.S.A.), 25 g., Bacto beef are in actinomycin III. They proposed for actino- extract 10 g., tap water 11., pH 7 0, was employed for mycins II and III the structures indicated in the growth of the organism. legend of Fig. 1. Production medium. A chemically defined medium con- taining L-glutamic acid 2-0 g., D-galactose 10.0 g., K2HPO4 Schmidt-Kastner (1956b) has suggested that 1.0 g., MgSO4,7H20 0-025 g., CaCl2,2H20 0-025 g., ZnSO4, exogenous sarcosine interfered with the incorpora- 7H2O 0-025 g., FeSO4,7H20 0-025 g., distilled water 1 1., tion of proline into certain actinomycin peptides. pH 7-2-7-3, was used for actinomycin production. Amounts The results obtained in our studies provide evidence (100 ml.) of medium were distributed into 250 ml. Erlen- for the view that sarcosine competes with proline meyer flasks and sterilized at 15 lb./in.2 pressure and 1210 and replaces it in certain actinomycin peptides. for 15 min. D-Galactose was autoclaved separately in a similar manner and added to the medium just before in- oculation. Production of actinomycin was carried out in EXPERIMENTAL shaken cultures (240 rev./min.) at 280. Aqueous solutions Culture. Streptomyces antibioticu8, strain 3720, was used of sarcosine and other amino acids generally were added at throughout the investigation. The procedure for prepara- the onset of actinomycin synthesis (1-3 days after inocula- tion of an inoculum has been described previously (Goss & tion), unless specified otherwise. Katz, 1957). An N-Z amine medium consisting of N-Z Determination of actinomycin potency. The antibiotic amine A (a pancreatic digest of casein prepared by Sheffield titre of culture filtrates was determined by a paper-disk method of bioassay (Goss & Katz, 1957). H3C CH3 H3C H3 fH fH CO CH (L) (L) CH CO N-CR3 N-CH3 Sar Sar L-Pro L-Pro D-Val D-Val CO CO ) H3C-tH RH (L) (L) Rt-CH3 NH NH N H2 0 CH3 CR3 Fig. 1. Actinomycin IV. Sequence of amino acids: L- Fig. 2. Circular paper chromatogram showing the actino- threonine, D-vahne (D-Val), L-prohne (L-Pro), sarcosine mycin mixtures produced by Streptomyces antibioticus. (Sar), N-methyl-L-valine (Brockmann et al. 1956; Solvent system: 10% aqueous solution of sodium o- Bullock & Johnson, 1957). Actinomycin I. 1 mole of cresotinate-di-n-butyl ether-8-tetrachloroethane (4:3: 1, proline is replaced by 1 mole of hydroxyproline (Brook- by vol.). Sequence of actinomycins is from centre to mann & Pampus, 1955). Actinomycin II. 2 moles of periphery; in all cases, the first zone just beyond the proline are replaced by 2 moles of sarcosine (Johnson & origin constitutes biologically-inactive coloured material. Mauger, 1959). Actinomycin III. 1 mole of proline is A, Actinomycin mixture produced in the glutamic acid- replaced by 1 mole of sarcosine (Johnson & Mauger, galactose medium; actinomycins I II (trace), III 1959). Actinomycin V. 1 mole of proline is replaced by (trace), IV and V; B, actinomycin mixture produced in 1 mole of 4-oxoproline (Brockmann & Manegold, 1958). the glutamic acid-galactose medium plus sarcosine Actinomycin VI. 1 mole ofD-valine is replaced by 1 mole (250,ug./ml.); actinomycins I, II, III, IV, an unidentified of D-alloisoleucine (Brockmann et al. 1956). Actinomycin component and V; C, actinomycin mixture produced in VII. 2 moles of D-valine are replaced by 2 moles of D- the glutamic acid-galactose medium plus L-valine alloisoleucine (Brockmann et al. 1956). (1000 pg./ml.); same as in A. 460 E. KATZ AND W. A. GOSS I959 Extraction of actinomycin. After a given period of incu- of the washed mycelium was then inoculated into flasks of bation, the actinomycin in 500 ml. of a fermentation glutamic acid-galactose medium and allowed to grow for a broth from a replicate series of flasks was extracted three 48 hr. period. The mycelium was harvested by centrifuging, times with butan-l-ol [20, 10, and 5% (v/v) respectively]. washed twice in 0-09 % NaCl soln. and finally suspended in The extracts were combined and the butan-l-ol was water; the final amount of mycelium was 8 mg./ml. A removed by distillation in vacuo. The crude actinomycin 20 ml. amount of this suspension was inoculated into each residue was recovered in a small volume of acetone and of a duplicate series of 250 ml. Erlenmeyer flasks contain- used directly for circular paper chromatography. If ing 20 ml. of m/15-phosphate buffer, 30 ml. of water and necessary, the material could be recovered by evaporation 10 ml. of an aqueous solution of sarcosine or L-glutamic of the acetone and then stored in the dry state in the dark acid (final concentration, 1 mM). In place of substrate, 10 ml. until used. of water was added to the control flasks. The flasks were Extraction of actinomycin from the mycelium of S. shaken at 240 rev./min. at 280. The antibiotic titre of antibioticus was accomplished in the following manner: the culture filtrates was determined daily by the disk-assay mycelium in a sample of broth was collected by suction method.