Vargha László et al. (szerk.): Beszámoló a Gyógyszeripari Kutató Intézet 10 éves működéséről 1950-1959 (Budapest, 1969)
dr. Horváth István: Anyagcsere vizsgálatok és oxitetraciklin termelési kísérletek Streptomyces rimosusszal
EXPERIMENTS ON STREPTOMYCES RIMOSUS, CONCERNING ITS METABOLISM AND OXYTETRACYCLINE PRODUCTION After a large scale production of penicillin, streptomycin and synthetical chloramphenicol has been settled in Hungary, the actual task standing in front of the research of antibiotics was the elaboration of production methods for a member of the tetracycline group. Laboratory experiments of yielding Chlortetracycline from Streptomyces aureofaciens were going on successfully, but pilot plant production staggered on the iron sensibility of fermentation. Thus, to our pharmaceutical industry, equipped then with iron fermenters only, it seemed more prospective to realize the production of Oxytetracycline derived from Streptomyces rimosus. This concept found support shortly in the experiments carried out. The principal laboratory experiments, commenced in August of 1954, were successfully reiterated, next month already, in the pilot plant, and after release of a probatory charge by the C'hinoin Works in December, it was begun with the final large scale production by May 1955. The experiments rim along the lines and methodology known as extensively used in antibiotic research. The initial yield of 600 pig ml of Oxytetracycline rose, as a result of strain and media experimentation, to 2200 jug ml. The antibiotic producing strain proved to be very stable in that respect, but further attempts to heighten its productivity failed. Several thousands of variants, isolated after treatment with 1 Y irradiation or X-mustard. were but without success tried. An observation made by chance in the pilot plant, however, helped the production rate to rise to 4000 pg ml. Namely, it has turned out that media of dense consistence which do not display elevated production in the laboratory, do so among circumstances prevailing in the pilot plant. This comprehensive study confines itself to some experimental results to be regarded as new. The optimal medium to produce the antibiotic by Streptomyces rimosus has a high content of oil. We made the observation that the iron toxicity was related to the saturation grade of that oil, by the reason, that iron ions had catalytic effect on arising of oil peroxides which, on their side, were poisonous to the fermentation process. Beside the oil content of the medium, also the unsaturation grade of the former seemed to play a part, hence palm oil is the most suitable one for antibiotic production. Further, methylene blue was observed to cope with the oil peroxides’toxicity. All these findings of ours offered some assurance against unfavourable changes in productivity. In order to become familiar with the peculiarities of the producing strain, we carried out experiments with synthetic media, too. These resulted in elucidation of some factors of Oxytetracycline synthesis, and, what is more, also the process of synthesis could find explanation by them to a certain degree. Some of our observations concerning the intermediary metabolism of amino acids likely have biochemical interest in general. Thus, whilst the production of Oxytetracycline in a natural medium (which contains oil, as mentioned), is independent of phosphate ion contentration, in a synthetic one with glucose as energy source) it is not. Although several enzymes involved in intermediary glucose metabolism are known to be phosphate-dependent, like Streptomyces rimosus itself, our metabolic studies did not end with the conclusion that the process of synthesis can primarily be explained in that way. The synthesis of Oxytetracycline molecule goes rather through active acetate. Along with the decline of antibiotic production by Streptomyces rimosus, morphological changes occur and the metabolic activity of the organism rises. Regression of productivity is first seen on a medium including serine and glucose. This might be in connection with the content of the organism on highly active and constitutive serine desaminase. On a medium incorporating glycine and glucose there is alanine synthesis to a considerable degree observable, this on phosphate concentrations coincident with the optimum production rate of Oxytetracycline. In cell-free extracts this synthesis is partly due to the presence of coenzyme A. Further, on a medium of threonine and glucose content isoleucine is produced, and it comes to this transformation in cell-free extracts, too. Ihese observations furnish guide to the enzyme-chemical study of amino acids with branched carbon-chain. DR. ISTVÁN HORVÁTH 12* 179
