Working with Streptomyces sp. MA406-A-1, effects of addition of various amino acids on the production of formycin were examined. In replacement culture, it was confirmed that lysine, aspartate or glutamate stimulates the production of formycin and carbon molecules in these amino acids incorporated efficiently into formycin. It is estimated that biosynthesis of formycin in this organism is closely related to lysine metabolism but not to biosynthesis of purine nucleosides. Formycin was originally isolated as an antitumor antibiotic from the culture filtrates of Nocardia interforma1) and identified as 7-amino-3-/3-D-ribofuranosyl-lH-pyrazolo-[4, 3d] pyrimi-dine2-7). This antibiotic, having been also found in cultured broth of Streptomyces lavendulae8) or S. gummaensis9), is one of microbial products which contain an unusual C-riboside linkage in their molecules. The other natural C-riboside products of microorganisms are formycin B2'8.10) oxoformycin B11,12), pseudouridine13-15), showdomycin16-18) pyrazomycin19-22) and minimy-cin23,24). Formycin, formycin B and oxoformycin B are pyrazolopyrimidine nucleosides and structural analogs of adenosine, inosine and xanthosine, respectively. Formycin has been found

1. Two carbons, carbon-2 and one of carbons-3 to -5, of lysine seemed likely to be incorporated into one of carbon of the chromophore moiety of formycin.2. From the results of radioisotopic studies with glutamate, r-amino-n-butyrate or organic acids related to tricarboxylic acid cycle, the important role of glutamate in the biosynthesis of formycin was strongly suggested.3. The incorporation of nitrogen(s) of lysine into three nitrogens, including two nitrogens of pyrazole ring, of formycin was suggested by mass spectroscopy. 4. Ribose was estimated as a direct precursor for the ribosyl moiety of formycin, whereas the biosynthesis of ribose was shown to occur via the pathway other than hexose monophosphate shunt or glucuronate pathway.5. In replacement culture, the salvage synthesis of formycin from its chromophore moiety was not observed and it was also evident that the chromophore moiety or pyrazofurin (pyrazomycin) inhibited the biosynthesis of formycin.There have been several investigations on the biosynthesis of C-riboside nucleosides, i.e., pseudouridine, showdomycin, minimycin, pyrazofurin (pyrazomycin) and the formycin family. In the biosynthesis of pseudouridine, the intramolecular rearrangement in tRNA of uridylate to pseudouridylate has been strongly suggested.1-6) The biosynthesis of showdomycin has been well established and confirmed to occur by the condensation of ribose and an asymmetric four-carbon dicaboxylic acid.7-10)Recently, studies on the biosynthesis of minimycin revealed a novel mechanism whereby C-riboside linkage is formed from sedoheptulose 7-phosphate (or eight-carbon branched chain sugar phosphate).11) C-Riboside linkage of pyrazofurin was also believed to be formed by this mechanism.11)The biosynthesis of formycin by Nocardia interforma has been proposed to occur via a pathway other than that of biosynthesis of purine nucleotide while exogeneously added ribose was estimated to be incorporated into the ribosyl moiety of formycin.12) It was also reported that formycin may be formed from formycin 5'-monophosphate then deaminated to formycin B.13) Using Streptomyces sp.MA406-A-1, a formycin-producing strain, we reported that carbon atom from lysine, glutamate and/or aspartate was incorporated into formycin with high efficiency, and that novel enzyme(s) catalyzing the amination of formycin B to formycin was present in this organism. 14,15) To elucidate the more detailed mechanism of formycin biosynthesis, we examined the incorporation and the distribution of various labeled compounds into formycin and this paper presents the results of these studies.

It has been reported by several investigators that most strains of heterolactic bacteria are capable of utilizing oxygen and produce lactate, acetate, and carbon dioxide, as major products from glucose under aerobic conditions (1-5). This paper describes the remarkable effect of oxygen on glucose dissimilation in resting cells of heterolactic bacteria. All the organisms used in this study were grown under stationary conditions at 30° in a medium containing (in per cent), glucose, 2.0; anhydrous sodium acetate, 2.0; peptone, 1.0; meat extract, 1.0; NaCI, 0.5; yeast extract, 0.2; and MnSO4.7H2O, 0.05 (pH 7.0). The cells were harvested late in logarithmic growth phase and washed three times with cold distilled water. Aerobic dissimilation of glucose was carried out in 100-m1 Erlenmeyer flasks on a reciprocating shaker operating at 145 strokes (2 cm) per min, and stationary (semi-anaerobic) dissimilation of glucose was carried out in 15-m1 test tubes. Each flask or tube contained the following substances : 500 imoles of glucose, 5 cimoles of MgSO4, 5 ml of 0.1 M phosphate buffer (pH 7.0), and 40-50 mg (dry weight) of washed cells. All incubations were carried out at 30°. Samples were taken during incubation, cooled rapidly by immersing the test tube or flask in ice, and centrifuged. The supernatant fluids thus obtained were analyzed by spectrophotometry for glucose (6), lactate (7), acetate (8), and ethanol (9) according to the indicated procedures. The results obtained after 90 min of incubation are summarized in Fig. 1. From these results, it is worth noting that the resting cells of Lactobacillus brevis and L. buchneri lose their fermentative activities under stationary conditions, while they can dissimilate glucose under aerobic conditions. These phenomena were also confirmed after 120 or 150 min of incubation.

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