Biosynthesis of Streptomycin

A natural precursor (L) of streptomycin which had no antibiotic potency was obtained from mycelium suspension of Streptomyces griseus in glucose solution and was transformed to streptomycin by H enzyme obtained from mycelium of the organism. This transforming reaction was carried out most effectively at slightly alkaline pH and inhibited by inorganic phosphate and ethylenediaminetetraacetate. L component was considered to be a phosphorylated compound and liberation of the phosphoric acid was essential for L component to be transformed to streptomycin. This transformation was performed not only by H enzyme but also by intestinal alkaline phosphatase, although some difference in the reaction mechanism was supposed to be between those two enzymes.     

Biosynthesis of Streptomycin

A method for the accumulation of the streptomycin precursor (L) in the culture broth of Streptomyces griseus was developed and the precursor was successfully isolated from the broth.

When the microorganism was cultured under shaking in the glucose-meat extract-peptone medium (0.5% glucose, 0.2% yeast extract, 0.2% meat extract, 0.4% peptone, 0.5% sodium chloride, 0.025% magnesium sulfate, pH 7.0), the accumulation of the precursor in the broth was induced by the addition of supplementary glucose (e.g., 2 g glucose per 100 ml broth) 24 hr after inoculation followed by further cultivation for 48 hr. Increased accumulation of L component was obtained merely by increasing glucose content in the culture medium (e.g., 5% glucose-containing medium in the above-indicated one) instead of glucose supplement on the way of fermentation. For the accumulation of a large amount of L component in a culture broth, it looked to be necessary for pH value of the broth to be maintained between 6 and 7 during fermentation.

L component was isolated from the culture broth by adsorption on Amberlite IRC-50 and elution with 2% NaCl solution. The L component was separated on this column from contaminated streptomycin which requires 5% NaCl solution to be eluted. The L component in the 2% NaCl eluate was adsorbed on active carbon at neutral or slightly alkaline pH and eluted with 95% methanol at acidic pH, Partially purified L component precipitated as hydrochloride by addition of acetone to the methanol extract which had been concentrated in vacuo.     

Biosynthesis of Streptomycin

A precursor system for formation of streptomycin was investigated with a cell-free supernatant obtained from suspension of young mycelium of Streptomyces griseus in a nongrowth medium containing only glucose and sodium chloride. When the supernatant was kept at a slightly alkaline condition for a day, a remarkable development of antibiotic potency was observed, while the supernatant itself had a very weak potency. It was made clear by column chromatography with Sephadex G-25, CM-cellulose and DEAE-cellulose that materials required for incearse of antibiotic potency in the supernatant consisted of a cationic component with low molecular weight and an anionic one with high molecular weight. Although each of the components showed little change in antibiotic potency, the mixture of them gave rise to a remarkable increase in antibiotic potency at a slightly alkaline condition. Thus, these two components were considered to construct the precursor system appearing in the supernatant and to be able to react in a cell-free state creating the antibiotic potency.

The optimum pH for the reaction occuring in the supernatant was about 9. This reaction was inhibited by phosphate or ethylenediaminetetraacetate, but not by arsenate. The precursor system was stable at and below 50°C.     

Studies on the Biosynthesis of Streptomycin

Myo-inositol, especially in combination with arginine, enhances streptomycin production. Compounds which show structural relationship with myo-inositol are ineffective.

Myo-inositol decreases the incorporation of C¹⁴-glucose into streptomycin, particularly into streptidine. This effect suggests that myo-inositol is a precursor of the streptidine ring.

Methionine stimulates antibiotic production in a synthetic medium but proves to be unfavorable in a complex medium.

The γ- and δ-isomers of hexachlorocyclohexane inhibit streptomycin formation.

The formation of streptomycin by washed mycelium was studied. Essentially the same results were here obtained as with growing cultures.

     

Transfer of Streptomycin Biosynthesis Gene Clusters within Streptomycetes Isolated from Soil

Streptomyces strains isolated from soil were found to possess various numbers of genes from the streptomycin biosynthesis cluster. The strains missing genes from the cluster also lacked the ability to produce streptomycin. Two of the isolates which contain only part of the cluster are apparently recipients of a gene transfer event. The implications for the role of gene transfer in antibiotic evolution are discussed.     

Streptomycin

Summary Streptomyces griseus DTH-2 was grown in 5 1 fermentors on complex media containing calcium carbonate as a buffering agent. It was shown that automatic pH control (4N KOH) could substitute the calcium carbonate giving higher yields of streptomycin. The yield was further increased by omitting inorganic phosphate from the medium and by differential addition of glucose/ammonium sulphate during the fermentation. The maximal yield obtained was 8.5 g of streptomycin per liter.     

Streptomycin and lysozyme

Résumé Les microorganismes sensibles au lysozyme ne subissent pas l'agglutination par la streptomycine. Inversement, la streptomycine empêche la clarification des suspensions microbiennes par le lysozyme. Cet effet est attribuable à la prévention de la dissolution des noyaux. Les mutants résistants à la streptomycine restent sensibles au lysozyme et se comportent, en présence de streptomycine plus lysozyme, comme les souches-mères.

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Lysozyme-sensitive microorganisms are not subject to streptomycin-agglutination. Conversely, streptomycin inhibits the clarification of bacterial suspensions by lysozyme. This effect is mainly due to a prevention of nuclear dissolution. Streptomycin-resistant mutants remain lysozyme-sensitive and behave towards streptomycin plus lysozyme as do parent-cells.