Production of Nucleic Acid-related Substances

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1 APPLID MICROBIOLOGY, JUly 1968, p Vol. 16, No. 7 Copyright 1968 American Society for Microbiology Printed in U.S.A. Production of Nucleic Acid-related Substances by Fermentative Processes XIX. Accumulation of 5'-Inosinic Acid by a Mutant of Brevibacterium ammoniagenes AKIRA FURUYA, SHIGO AB, AND SHUKUO KINOSHITA Tokyo Research Laboratories, Kyowa Fermentation Industry Co., Ltd., Machida-shi, Tokyo, Japan Received for publication 19 March 1968 The accumulation of 5'-inosinic acid (IMP) by a mutant, KY 13102, induced from Brevibacterium ammoniagenes ATCC 6872 by ultraviolet light irradiation, was examined. Although growth was stimulated by adenine or adenosine, the microorganism showed fair growth in the medium containing amino acids but no adenine. Among six kinds of natural nutrients tested, meat extract and Casamino Acids were suitable for the accumulation of IMP. Manganese ion strongly affected growth, the accumulation of IMP and hypoxanthine, and cell morphology. Among amino acids tested, L-methionine, L-proline, and L-valine stimulated IMP accumulation. In the medium containing g of L-proline per liter, 12.8 mg of IMP per ml was accumulated. The mechanism of IMP accumulation by the mutant is discussed. Industrial fermentative processes for production of 5'-inosinic acid (IMP), an important flavoring agent, have been extensively studied in recent years (3, 9). The procedures reported hitherto may be grouped as follows. (i) IMP was isolated from yeast ribonucleic acid after digestion by microbial phosphodiesterase (2, 4, 10) and deamination. (ii) Inosine was first produced by a fermentative process using adenine auxotrophic mutants of Bacillus subtilis (1, 6, 11) and was phosphorylated to yield IMP by chemical procedures (T. Hashizume and I. Yama, Proc. Ann. Meeting Agr. Chem. Soc. Japan, p. 138, 1962; T. Kato et al., Proc. Ann. Meeting Agr. Chem. Soc. Japan, p. 124, 1963). (iii) IMP was produced fermentatively from hypoxanthine by "salvage biosynthesis" using Micrococcus sodonensis and Arthrobacter citreus (7) and Brevibacterium ammoniagenes (T. Nara, M. Misawa, and S. Kinoshita, Symp. Amino Acid Nucleic Acid, p. 18, 1967). (iv) IMP was directly accumulated in culture broths of adenine auxotrophic mutants of M. glutamicus (5) and B. ammoniagenes (8) and was formed from carbon and nitrogen sources via de novo purine nucleotide synthetic pathway. The procedures for direct production of IMP from carbon and nitrogen sources have been reported in a few papers (5, 8) but high yields of the acid have not been attained. We have been conducting induction of biochemical mutants from B. ammoniagenes using various mutagens and have obtained a mutant, induced by ultraviolet light irradiation, which accumulates large amounts of IMP. The growth response of the mutant strain and the factors affecting IMP accumulation and growth have been examined. The largest amount of IMP, 12.8 mg per ml, was obtained after cultivation at 30 C for 7 days. MATRIALS AND MTHODS Strains. B. ammoniagenes ATCC 6872 was used as the parent strain. The IMP-accumulating mutant KY was prepared from ATCC 6872 by ultraviolet light irradiation. Media. The slant medium for maintenance of the cultures contained (w/v) 1% meat extract, 1% peptone, 0.5% yeast extract, 0.3% NaCl, and 2% agar (ph 7.3). Inoculum medium was made of (w/v) 2% glucose, 1% peptone, 1% yeast extract, and 0.3% NaCl (ph 7.3). The basal accumulation medium was composed of the following components per liter: glucose, g; KH2PO4, 10.0 g; K2HPO4, 10.0 g; MgSO4.7H20, 10.0 g; CaCl2-2H20, g; thiamine, mg; calcium D-pantothenate, 10 mg; biotin, 30 ug; and urea, 6.0 g (sterilized separately). The ph was adjusted to 8.3 with 5 N NaOH solution. The inoculum and accumulation media were sterilized at 120 C for 15 min. The minimal medium for determination of nutritional requirements of the mutant contained the following components per liter: glucose, 20.0 g; urea, 2.0 g; (NH4)2SO4, 3.0 g; KH2PO4 (sterilized separately), g; K2HPO4 (sterilized separately), 3.0 g; MgSO4*7H20, 0.3 g; CaCl2*2H20, 10 mg; FeSO4 D

2 982 FURUYA, AB, AND KINOSHITA APPL. MICROBIOL. TABL 1. Growth response of KY strain in the minimal medium Compound Amt Growtha Compound Amt Growtha mg/liter mg/ml mg/mi mg/ml None.0.82 Adenosine Amino acid mixtureb '-Adenylic acid Base mixturec Guanine Vitamin mixtured Hypoxanthine Adenine Xanthine Uracil a The organism was cultivated at 30 C for 48 hr on a shaking machine. Growth is expressed as dry cell weight. b ighteen kinds of L-amino acids were contained at the concentration indicated. c Adenine, guanine, hypoxanthine, xanthine, uracil, cytosine, and thymine were contained at the concentration indicated. d The amount is given in milliliters per liter. See Materials and Methods. 1- co c: 0 cl I.( 0. 1( ))21 Time in hours FIG. 1. Growth response of KY strain in the minimal medium supplemented with Casamino Acids. Symbols: *, addition of adenine, 20 mg/liter; *, guanine, 20 mg/liter; *, hypoxanihine, 20 mg/liter; and V, none. 7H20, 10 mg; ZnSO4-7H20, 1 mg; MnCl2-4H20, 3.6 mg; cysteine, 20 mg; thiamine, 5 mg; calcium D-pantothenate, 10 mg; and biotin, 30 Ag (ph 7.3). The amino acid mixture contained 1 g per liter each of L-alamine, L-argmiine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L- tyrosine, and L-valine. The vitamin mixture contained the following compounds in 100 ml: thiamine, 12.5 mg; riboflavine, 25 mg; p-aminobenzoic acid, 5.0 mg; nicotinic acid, 5.0 mg; pyridoxine, 5.0 mg; folic acid, 5.0 mg; choline, 100 mg; inositol, 50 mg; vitamin K, 50 mg; and vitamin B12, 0.5 yg. Cultivation. For accumulation of IMP, the organism was cultivated as follows. Growth from slants, incubated at 30 C overnight, was transferred to 250-ml rlenmeyer flasks containing 20 ml of the inoculum medium. These seed flasks were incubated for 24 hr on a rotary shaking machine (220 rev/min) at 30 C. Accumulation flasks containing 20 ml of the medium were inoculated with 2.0 ml of the seed growth and were incubated for 5 to 8 days on the shaking machine at 30 C. For the determination of nutritional requirements of the mutant, cells from slants incubated at 30 C over night were washed twice with physiological saline solution, inoculated to the minimal media supplemented with various compounds, and incubated on a reciprocal shaking machine (300 oscillations/min) for 48 hr at 30 C or on a Monod shaker (40 strokes/min) at 30 C. Assay. IMP and hypoxanthine were separated by paper chromatography and measured spectrophotometrically. Portions of 10 to 20,uliters of centrifuged broth were spotted on paper (Toyo Roshi no. 51A) and were developed in the following system in the ascending direction: isobutyric acid-glacial acetic acid-i N NH4OH (10: 1: 5). After drying the chromatograms, IMP and hypoxanthine were detected with ultraviolet light and were extracted with 5 ml of boiling water for 30 min. The amounts of IMP and hypoxanthine in extracts were determined by measuring optical densities at 250 m,. All IMP values in this paper were as IMP-Na2 7-5H20. Cell growth was estimated by measuring the optical density at 660 m,u of samples diluted with 0.4 N HCI solution and is expressed in dry cell weight calculated from a standard curve prepared previously. RSULTS Growth response of KY The nutritional requirements of the mutant were examined. As shown in Table 1, growth was stimulated by adenine and adenosine. Among adenine derivatives, 5'-adenylic acid showed little effect on growth. It is noteworthy that the microorganism

3 VOL. 16, 1968 ACCUMULATION OF 5'-INOSINIC ACID 983 Peptone e i20_ S toi0- Casamino Acids _h 20_ -10 to.14 CZ 5-0- I U r~~~~~~~~~~~~~~~~~~~~ a) :v.,1 lo - 1- w )I I X Corrn Steep Liq uor %I ;--- 1 I I I % FIG. 2. Suitability oj natural nutrients for IMP accumulation. Symbols: *, dry cell weighlt; 0, IMP; and 0, Hx (hypoxanthine). showed a little growth in the minimal medium and mg per ml of 1 % solution of each nutrient). In fair growth in the medium supplemented with the meat extract and yeast extract, 0.3 mg and 0.2 mg amino acid mixture. From these results, the of IMP per ml of 1% solution were detected, microorganism appeared to be a leaky mutant respectively, by paper chromatographic analysis. whose growth was stimulated by adenine or The results of the cultivation of the mutant in the adenosine. media containing 10 mg of adenine per liter and For further confirmation of this point, minimal these natural nutrients at 30 C for 6 days are medium supplemented with 0.5% Casamino shown in Fig. 2. Casamino Acids and meat ex- Acids (Difco) was used. The organism was culti- tract supported IMP accumulation. On addition vated on a Monod shaker at 30 C and growth was of 1% of either nutrient, 8 to 9 mg of IMP per measured at intervals of 2 hr (Fig. 1). Although ml was accumulated, and the growth corregrowth was stimulated markedly by adenine, the sponded roughly to half that obtained in media Casamino containing excess amounts of these nutrients. oanidsmgrewiumi tha airlycfast atenint. The results with peptone were very similar to Acidmeiumtyof inatheaabsrienefofdine. Acc those with N-Z-Amine. When either of these Suitability of natural nutrients for IMP Accumu- nutrients was added at a concentration of 0.25 %, lation. The effects of natural nutrients on growth IMP accumulation was slightly increased; howof the mutant and on IMP accumulation were ever, further addition resulted in reduced amounts examined. Six kinds of natural nutrients [peptone, of IMP and increased growth. On the other hand, Casamino Acids, N-Z-Amine (Sheffield Chemi- yeast extract and corn steep liquor inhibited cal, Norwich, N.Y.), yeast extract, meat extract IMP accumulation but stimulated growth. Parand corn steep liquor] were used. The content of ticularly, growth was favored with increased IMP in peptone, Casamino Acids, N-Z-Amine, yeast extract and reached 24 mg of dry cell and corn steep liquor was negligible (less than weight per ml at the maximum. S- S 0,- 0, U

4 984 FURUYA, AB, AND KINOSHITA APPL. MICROBIOL. The small amount of corn steep liquor, 0.25 %, caused a drastic decrease in IMP accumulation. ffects of adenine. As described above, growth of the mutant was stimulated by adenine. Therefore, the role of adenine in accumulation of IMP was examined. Adenine was added at various concentrations to medium containing 1% meat extract. As shown in Fig. 3, on addition of 10 mg of adenine per liter, IMP accumulation increased slightly. Further addition of adenine reduced accumulation of the acid. ffects of manganese ion. It was reported by Nara et al. (7) that manganese ion (Mn++) strongly affected IMP formation from hypoxanthine by the purine salvage pathway in M. sodonensis. Manganese ion also showed pronounced effects on the formation of IMP by Brevibacterium ammoniagenes (12). The effects of Mn++, therefore, on the accumulation of IMP by KY were examined. Manganese ion at concentrations of 0 to 15,g per liter was added to the accumulation medium containing 10 mg of adenine per liter and 0.75 or 1.25% meat extract. As shown in Figure 4, the addition of 5,ug of Mn++ per liter to the 0.75% meat extract medium caused a small decrease in accumulation of IMP, whereas the IMP level was reduced from 10 mg per ml to 8 mg per ml on addition of the same amount of Mn++ to the 1.25% meat extract medium. On addition of 10,ug of Mn++ per liter, accumulation of the acid was decreased drastically in both media. From these results, it is evident that Mn++ had strong effects on IMP accumulation by the mutant. An interesting point in this experiment is that the 4 1Q Adenine mg/liter FIG. 3. ffect ofadenine on IMP accumulation. The organism was cultivated at 30 C for 6 days. Symbols: U, dry cell weight; 0, IMP; and 0, Hx (hypoxanthine). A, -.- \\k X~~~~~~0 l l l~~~~l Mn++ pg/liter FIG. 4..#kt ofmanganese ion in the meat extract medium. The organism was cultivated at 30 Cfor 7 days. Symbols: solid lines are used with meat extract as 1.25% of the mediwn and dashed lines with meat extract as 0.75% of the mediwn; 0, dry cell weight; *, IMP; and 0, Hx (hypoxanthine). addition of excess amounts of Mn++ caused a drastic decrease in IMP accumulation, followed by an increase of hypoxanthine concentration corresponding to the decrease in IMP. That is, excess amounts of Mn++ resulted in accumulation of hypoxanthine in preference to IMP. To make clear the effects of Mn++, this ion at concentrations of 0 to 50,ug per liter was added to the basal accumulation medium containing 10 mg of adenine per liter but no meat extract. As shown in Fig. 5, at low concentrations of Mn++, growth of the organism was very poor, indicating that Mn++ is essential for cell production. As the Mn++ concentration was increased, growth was stimulated, reaching a maximal level on addition of 20,ug of Mn++ per liter. The accumulation of IMP, however, was maximal on addition of 10,ug of Mn++ per liter, and further addition reduced the accumulation of IMP. The optimal concentration range of Mn++ for IMP accumulation was very narrow, about 10,ug per liter. At the concentration of Mn++ supporting maximal IMP accumulation, growth reached about half its maximal level. From microscopic observations, it was found that Mn also affected cell morphology. At low concentrations of Mn++, the cells showed fairly

5 VOL. 16, 1968 ACCUMULATION OF 5'-INOSINIC ACID U- w Ui IC 5- t a CJ a Mn pg/liter ( added ) FIG. 5. ffect ofmanganese ion in the basal accumulation medium. The organism was cultivated at 30 C for 7 days. Symbols: *, dry cell weight; 0, IMP; and 0, Hx (hypoxanthine). slender shapes, and, at concentrations of the ion suitable for IMP accumulation, a number of elongated and swollen cells (Fig. 6A) were observed. As the Mn+ concentration increased, elongated and swollen cells disappeared, and small rod to ellipsoidal cells increased in number (Fig. 6B). ffects of amino acid. The results shown in Fig. 2 suggest that amino acids stimulated accumulation of IMP. ighteen kinds of L-amino acids (except for DL-serine) were added at concentrations of or g per liter to the accumulation medium containing % meat extract and 10 mg of adenine per liter. Table 2 shows the effect of the amino acids on IMP accumulation and growth. On addition of g of methionine per liter, g of proline per liter, or g of valine per liter, accumulation of IMP was stimulated. The highest yield of IMP, mg per ml, was obtained in the medium containing g of proline per liter. On the other hand, g of leucine per liter or 0.2 to 2.0 g of DL-serine per liter acted repressively on IMP accumulation. Valine, which was stimulatory at a concentration of g/liter, inhibited IMP accumulation at a concentration of 1 g/liter. Time course of the IMP accumulation. The time course of IMP accumulation by the mutant in the accumulation medium containing % meat extract and 10 mg of adenine per liter is shown in Fig. 7. With the initiation of the growth, hypoxanthine accumulated and reached the maximal value, 1.75 mg per ml, on the third day. Rapid IMP accumulation occurred after 3 days, accompanying the decrease of hypoxanthine. The elongated and swollen cells, as shown in Fig. 6A, 0 FIG. 6. Cells cultivated at 30 Cfor 7 days in the basal accumulation medium supplemented with (A) IOg/ 10 liter ofmn++ or (B) 50 ug/liter ofmn++. appeared after 3 days. Finally, 11.2 mg of IMP per ml was accumulated after 8 days of cultivation. DISCUSSION KY showed fair growth in the medium supplemented with amino acids but no adenine, as shown in Table 1 and Fig. 1. Growth was not caused by reversion to prototrophy. The mutant did not require specific amino acids for growth. It

6 986 FURUYA, AB, AND KINOSHITA APPL. MICROBIOL. TABL 2. ffect ofamino acids on IMP accumulation Amino Acid Amino acid mixtureb Glutamic acid Histidine Methionine Cystine Isoleucine Leucine Proline DL-Serine Valine None Amt glliier ph Growtha mg/ml IMP mg/ml Hypoxanthine mg/ml a The organism was cultivated at 30 C for 7 days. Growth is expressed as dry cell weight. bighteen kinds of L-amino acids were added at the concentration indicated. has been found that growth of the parent strain ATCC 6872 is also stimulated by amino acids (unpublished data). Therefore, the stimulated growth of the mutant by amino acids may be due to a common character in both strains. The natural nutrients tested showed characteristic effects on IMP accumulation as well as on growth. The effects of each natural nutrient would be determined chiefly by its content of adenine, amino acids, and Mn++. It is entirely conceivable that the concentration of Mn++ in corn steep liquor is relatively high compared with that of the other nutrients. Therefore, the addition of a small amount of corn steep liquor resulted in elevation of the Mn++ concentration of the medium and caused the drastic decrease in IMP accumulation. The effects of amino acids were examined in the medium containing 1 % meat extract; therefore, the influence of each amino acid was not clear. t..) -L tj *_ c.: ') Gw i. _--s - -4 C,i, r;-i -i._ 1 -l I.. N P A A 46. I v. a- V- W'I I.. F A 4 -. L. I i; /~~~~~~~~~~~ t-) V '-I- U-~~~~~~- *0- IF,' FIG. 7. Time course of tlte IMP accumulation. Symbols: *, dry cell weight; *, IMP; 0, Hx (hzypoxanithine); A, ph; and V, residual sugar. Further studies should be done in the basal accumulation medium without addition of meat extract. Nara et al. (8) observed accumulation of hypoxanthine and its conversion to IMP in the cultivation of an adenine auxotroph of B. ammoniagenes. In strain KY 13102, this conversion seems to occur later in the growth cycle. By calculation, the amount of hypoxanthine which disappears (1.35 mg/ml) would account for about 5.2 mg of IMP per ml formed by salvage biosynthesis. Therefore, assuming no more hypoxanthine was produced after 3 days, de novo biosynthesis of IMP would occur at the same time as the formation of the acid by salvage synthesis. LITRATUR CITD 1. Aoki, R., H. Momose, Y. Kondo, N. Muramatsu, and Y. Tsuchiya Studies on inosine fermentation-production of inosine by mutants of Bacillus subtilis. I. Isolation and characterization of inosine producing mutants. J. Gen. Appl. Microbiol. 9: Hasegawa, Y., T. Nakai, Y. Fujimura, Y. Kaneko, and S. Doi Studies on nucleases produced by microorganisms. I. Production of phosphodiesterase (5'-nucleotide forming) by Pellicularia sp. Nippon Nogeikagaku Kaishi 38: Kinoshita, S Reviews on the fermentative productions of amino acids and nucleic acidrelated substances. Hakko Kyokaishi 22:

7 VOL. 16, 1968 ACCUMULATION OF 5'-INOSINIC ACID Kuninaka, A., S. Otsuka, Y. Kobayashi, and K. Sakaguchi Studies on 5'-phosphodiesterases in microorganisms. I. Formation of nucleoside-5'-monophosphates from yeast ribonucleic acid by Penicillium citrinum. Bull. Agr. Chem. Soc. Japan 23: Nakayama, K., T. Suzuki, Z. Sato, and S. Kinoshita Production of nucleic acid-related substances by fermentative processes. V. Accumulation of inosinic acid by an adenine auxotroph of Micrococcus glutamicus. J. Gen. Appl. Microbiol. 10: Nara, T., M. Misawa, K. Nakayama, and S. Kinoshita Production of nucleic acid related substances by fermentative processes. III. Accumulation of inosine by adenine requiring mutants of microorganisms. Amino Acid and Nucleic Acid, No 8: Nara, T., M. Misawa, T. Komuro, and S. Kinoshita Production of nucleic acid-related substances by fermentative processes. XII Accumulation of inosinic acid by Micrococcus sodonensis and Arthrobacter citreus. Amino Acid and Nucleic Acid, No 15: Nara, T., M. Misawa, and S. Kinoshita Production of nucleic acid-related substances by fermentative processes. XIII. Fermentative production of 5'-inosinic acid by an adenine auxotroph of Brevibacterium ammoniagenes. Agr. Biol. Chem. (Tokyo) 31: Ogata, K Reviews on the productions of nucleic acids and related substances. Amino Acid and Nucleic Acid, No 8: Sugino, Y., M. Yoneda, and I. Suhara Degradation of nucleic acid and their related compounds by microbial enzymes. I. On the distribution of extracellular enzymes capable of degrading ribonucleic acid into 5'-mononucleotides in microorganisms. Agr. Biol. Chem. (Tokyo) 27: Uchida, K., A. Kuninaka, H. Yoshino, and M. Kubi Fermentative production of hypoxanthine derivatives. Agr. Biol. Chem. (Tokyo) 25: Downloaded from on April 30, 2018 by guest