Accumulation of Xanthosine by Auxotrophic Mutants of Bacillus Subtilis ??

[Agr. Biol. Chem., Vol. 30, No. 6, p. 605•`610, 1966]

Accumulation of Xanthosine by Auxotrophic Mutants of Bacillus subtilis_??_

By Masao FUJIMOTO, Kazuo UCHIDA, Morio SUZUKI and Hiroshi YOSHINO

Microbial Laboratory of Yamasa Shoyu Co., Ltd., Choshi Received January 8, 1966

Several guanineless mutants derived from Bacillus subtilis JAM 1145 were found to accumulate xanthosine in the culture broth. Further mutation of the guanineless mutants to adenine dependence led to remarkable increase in the accumulation of xanthosine. One of the guanine-adenine doubleless mutants, strain Gu-Ad-3-35, accumulated 8.9g of xanthosine per liter. Xanthosine was isolated in a crystalline form from the culture broth by a procedure involving charcoal treatment and ion-exchange chromatography.

INTRODUCTION and with the isolation of pure xanthosine Magasanik and Brook1) first identified a from the culture broth. product of metabolism of a guanineless mutant MATERIALS AND METHODS of Aerobacter aerogenes as xanthosine, and Naka- Microorganism. Bacillus subtilis IAM 1145** was yama et al.2) developed a complex medium used as the starting strain for the artificial mutation. in which a large amount of xanthosine is Induction of mutation and isolation of mutants. produced by this mutant. Misawa et al.3) Spores from 12-days' culture of Bacillus subtilis IAM and Demain et al.4) reported the accumula 1145 on a nutrient agar slant were washed twice tion of xanthosine-5'-monophosphate by auxo with distilled water and then suspended in 0.85% trophic mutants of Micrococcus glutamicus and sodium chloride solution. The suspension containing coryneform bacterium, respectively. 106 to 107 spores per ml was exposed to ultraviolet In the course of our investigations on pro light (Toshiba sterilizing lamp, 15W, 300mA) at the duction of nucleic acid-related compounds by distance of 30cm, for 7 minutes. Guanineless auxo microorganisms, several guanineless mutants trophs were detected by replica plating. Two of these mutants accumulating xanthosine (strain Gu-3, derived from Bacillus substilis IAM 1145 were strain Gu-l0) were chosen for further mutation to found to accumulate xanthosine in the culture adenine dependence by the same technique. broth,* and further mutation of the guanine Media. The complete medium for master plates of less mutants to adenine dependence was found replica plating was composed of the following com to result in increased production of xanthosine. ponents: beef extract 5g, polypepton 5g, yeast ex The present paper mainly deals with the tract 5g, casein hydrolyzate 5g, NaCl 3g, agar 18g, accumulation of xanthosine by the guanine- and distilled water 1000ml. The pH was adjusted adenine doubleless mutants of Bacillus subtilis, to 7.0. This medium was also used for maintenance of cultures. The composition of the minimal medium _??_ Presented at the Meeting of Kanto Division of the Agricultural Chemical Society of Japan, Tokyo Feb. 5, 1966. was the same as that of Gray and Tatum's minimal 1) B. Magasanik and M.S. Brook, J. Biol. Chem., 206, 83 medium.5) The production medium was composed (1954). 2) K. Nakayama, T. Suzuki. Z. Sato and S. Kinoshita, ** This strain was kindly supplied by Dr. H. Iizuka, In Amino Acid and Nucleic Acid (in Japanese), 8, 88 (1963). 3) M. Misawa, T. Nara, K. Udagawa, S. Abe and S. stitute of Applied Microbiology, the University of Tokyo. Kinoshita, This Journal, 28,690 (1964). 4) A. L. Demain, M. Jackson, R. A. Vitali, D. Hendlin and * Xanthosine fermentation by auxotrophic mutants of T. A. Jacob, Appl. Microbiol., 13, 757 (1965). 5) C. H. Gray and E. L. Tatum, Proc. Natl. Acad. Sci. U.S., Bacillus subtilis or Escherichia coli was published: S. Okumura 30, 404 (1944). et al., Japanese Patent Publication No. 24517 (1965). 606 Masao FUJIMOTO, Kazuo UCHIDA, Morio SUZUKI and Hiroshi YOSHINO

of the following components: sodium citrate 12 g, hypoxanthine, xanthine, their ribosides and NH4Cl 10g, KH2PO4 5g, KCl 1.5g, MgSO4•E7H2O ribotides failed to support the growth of these 0.5g, CaCl2•E2H2O 0.15g, glucose 100g (autoclaved mutants. separately) and defatted soybean extract 1000ml (5% Their productivity of nucleic acid-related weight of defatted soybean was extracted with 0.1% compounds in the culture broth was examined. NaOH solution at 95°C for 15 minutes). The pH The results (Table I) show that the guanine was adjusted to 6.5. Twenty-five ml of the medium less mutants were capable of accumulating was prepared in a 500-m1 Erlenmeyer flask. This xanthosine and xanthine. The amount of the medium was also used as the inoculum medium.

Cultivation. Culture on an agar slant was trans former was 6 to 9 times more than that of ferred to flasks containing the inoculum medium. the latter. These seed flasks were incubated for 20 hrs. on a

rotary shaker at 28•Ž. Production medium was TABLE I. PRODUCTION OF XANTHINE DERIVATIVES inoculated with 0.25 ml of the seed culture and was IN VARIOUS GUANINELESS MUTANTS shaken at 28•Ž. Chemicals. Defatted soybean was purchased from

Nisshin Seifun Company. Xanthosine was prepared by the deamination of guanosine with nitrous acid by the method of Leven.6) Xanthine, adenine and guanosine (Daiichi Seiyaku Company); inosine (Nutritional

Biochemicals Corporation); and D-ribose (Tokyo Kasei Kogyo Company) were commercial preparations. Analytical methods. Determination of purine

bases and nucleosides accumulated in the culture broth was carried out as follows. The supernatant of the culture broth was subjected to ascending paper

chromatography by use of Toyo Roshi's No.53A filter

paper and the solvent system: n-butanol, acetic acid, water (4:1:1). After developing and drying of the

chromatograms, the separated spots were located as The amounts of products were determined after 6 days of dark shadows on the paper illuminated with ultra- cultivation in the fermentation medium at 28•Ž.

violet light. The RF values of xanthosine, inosine, xanthine and hypoxanthine were 0.20, 0.25, 0.34 and TABLE II. COMPARISON BETWEEN GUANINELESS 0.40, respectively. These materials were eluted from MUTANTS AND GUANINE-ADENINE DOUBLELESS

the paper with distilled water and were determined MUTANTS IN XANTHOSINE PRODUCTION spectrophotometrically comparing with the authentic

preparations. Residual sugar was analyzed by the method of Lane.7) Growth was estimated by measur

ing the turbidity at the wavelength of 660mƒÊ. Identification of xanthosine will be described in detail in "Results''

RESULTS Isolation of xanthosine-producing mutants. Fifteen auxotrophic mutants, which required guanine, guanosine or 5'-guanylic acid for growth, were derived from Bacillus subtilis I AM 1145 by ultraviolet irradiation. Adenine, Each mutant was cultivated in the fermentation medium 6) P. A. Leven, J. Biol. Chem., 55, 439 (1923). containing the supplement indicated to accumulate xantho 7) J. H. Lane and L. Eynon, J. Soc. Chem. Ind., 17, 32T sine effectively. The amounts of product were determined (1923): 19.150T (1925). after 7 days of cultivation at 28•Ž. Accumulation of Xanthosine by Auxotrophic Mutants of Bacillus subtilis 607

For the purpose of effective accumulation mg of guanosine to one liter of the medium, of xanthosine, further mutation of two gua strain Gu-Ad-3-35 accumulated a large amount nineless mutants was attempted. Several of xanthosine almost exclusively, while, in the mutants, which required adenine, adenosine addition of 250mg of adenine alone to one or 5'-adenylic acid besides guanine derivatives, liter of the medium, it accumulated inosine were obtained from the guanineless mutants, much more than xanthosine. strain Gu-3 and Gu-10. The accumulation Time course of fermentation. Xanthosine fer of xanthosine in those guanine-adenine double- mentation was studied as a function of time. less mutants was compared with that in the A typical chemical change in the medium original guanineless mutants (Table II). As during fermentation by strain Gu-Ad-3-35 is a result, it was found that further mutation given in Fig. 1. The bacteria grew linearly of a guanineless mutant to adenine dependence until the fourth day and then decreased led to marked increase in the accumulation gradually. Glucose, whose initial concentra of xanthosine. On the other hand, it was tion was 10%, was consumed gradually to 4% also observed that the guanine-adenine double- for seven days. The pH of the medium was less mutants acquired the ability to accumu maintained in the range of 6.2-7.0 during late xanthine derivatives and hypoxanthine the course of fermentation. derivatives simultaneously. The ratio of the At the early stage the accumulation level accumulation level of xanthine derivatives to of hypoxanthine derivatives was much higher that of hypoxanthine derivatives in a guanine- than that of xanthine derivatives, while at adenine doubleless mutant depended on the the late stage the rapid increase in the ac initial guanine and adenine content of the cumulation of xanthosine occurred with the fermentation medium. As shown in Table III, rapid decrease in the accumulation of hypo in the addition of 50 mg of adenine and 150 xanthine derivatives. After seven days of cultivation 8.9g per liter of xanthosine was TABLE III. EFFECT OF ADENINE AND GUANOSINE ON THE ACCUMULATION OF HYPOXANTHINE DERIVATIVES AND XANTHINE DERIVATIVES IN GUANINE-ADENINE DOUBLELESS MUTANT, STRAIN Gu-Ad-3-35

FIG. 1. Time Course of Xanthosine Fermentation in Strain Gu-Ad-3-35.

The amounts of products were determined after 6 days of cutivation at 28°C. 608 Masao FUJIMOTO, Kazuo UCHIDA, Morio SUZUKI and Hiroshi YOSHINO

accumulated, whereas hypoxanthine deriva crystal were compared with those of the tives were not detected at all. authentic xanthosine as follows:

Separation and identification of the fermenta 1) Ultraviolet absorption spectra and ultra- tion product. One hundred ml of the culture violet extinction. Ultraviolet absorption spectra broth (containing 800 mg of xanthosine), which at various pH values are shown in Fig. 2. was obtained by cultivating a guanine-adenine The spectra of authentic xanthosine were doubleless mutant, strain Gu-Ad-3-35, in two confirmed to be quite the same as these 500-ml Erlenmeyer flasks each of which con spectra. Aqueous solution of this crystal tained 50ml of the fermentation medium (1 mg/ml) and its acid hydrolyzate prepared supplemented with 50mg/l of adenine and by adding 0.11111of 10 N H2SO4 to the aqueous 150mg/1 of guanosine for seven days, was solution, followed by heating the mixture in centrifuged for removal of bacterial cells. a boiling water bath for one hour, were diluted The supernatant fluid was adjusted to pH 2.0 1 : 100 and the pH was adjusted to 6.2 with with 5% perchloric acid and the resulting 0.1 M phosphate buffer. As shown in Table

precipitate was centrifuged off. After neu IV, their ultraviolet extinctions were in accord tralization with KOH, the supernatant was added with 10% Ba(OH)2 for removal of inorganic phosphate and the resulting pre cipitate was centrifuged off. The clear supernatant was adjusted to pH 4.0 and then applied to a charcoal column, with a diameter of 3 cm and 20 cm in height. The column was washed with water until the eluate was free of impurities. The ultraviolet-absorbing materials were eluted with an aqueous solu tion containing 50% ethanol and 1.5% NH4OH at a flow rate of 1.0 ml per minute. The eluate was concentrated to 20 ml in vacuo at 50•Ž. The concentrated fluid was adjusted to pH 8.5 with NaOH, followed by dilution FIG. 2. Ultraviolet Absorption Spectra of the Fermentation Product. to 120ml with 0.01 M Na2B4O7 solution, and was applied to a column of Dowex-l-Cl (10, 50•`100 mesh, 1•~20cm). The column was first washed with water until the eluate was TABLE IV. ULTRAVIOLET EXTINCTION OF FER MENTATION PRODUCT AND ITS ACID HYDRO free of xanthine. The adsorbed xanthosine LYZATE AT pH 6.2 was then eluted with 0.001N HCl at a flow rate of 1.0 ml per minute. After neutraliza tion with NaOH, the xanthosine fraction

(160ml) was concentrated in vacuo to a volume of approximately 20ml. After allowing to stand at 5°C for one day, long needle-shaped crystals formed were collected, washed with ethanol and dried in vacuo at 60•Ž for 4 hours. Crystals were recrystallized two times in the same way. The yield was 320mg. Chemical and physical properties of this * Authentic preparation Accumulation of Xanthosine by Auxotrophic Mutants of Bacillus subtilis 609

TABLE V. RF VALUES OF FERMENTATION PRODUCT AND ITS ACID HYDROLYZATES

* Authentic preparation Compositions of solvent systems (a), (b) and (c) are described in the text .

FIG. 3. Infrared Absorption Spectra of the Fermentation Product. (Nujol)

A: Product B: Authentic xanthosine with those of the authentic xanthosine and its xanthine and D-ribose. acid hydrolyzate. 3) Molar ratio of base to pentose. Pentose 2) R,, value. The RF values of this crystal content of this crystal was determined by the and the acid hydrolyzates were determined orcinol method9) with D-ribose as the standard. by ascending paperchromatography in three The xanthine content was determined by the solvent systems; (a) n-butanol, acetic acid extinction of the acid hydrolyzate at pH 6.2 and water (4:1:1); (b) isobutyric acid, 15N at 267.5 mi with xanthine as the standard. NH4OH and water (66:1:33); (c) the upper The molar ratio of base to pentose was found layer of a mixture of n-butanol, ethanol and to be approximately 1.0. water (4:1:5). The acid hydrolyzate was 4) Infrared absorption spectra. The infrared neutralized on the paper by exposure to am absorption spectrum of the sample was quite monia vapor. Xanthine derivatives on the identical with that of the authentic xanthosine. chromatograms were located by inspection of The results are recorded in Fig. 3. From the the paper with an ultraviolet lamp, while above results this crystal was identified as reducing sugar on the chromatograms was xanthosine. located by spraying with aniline hydrogen DISCUSSION phthalate.8) As shown in Table V, the result indicates that the fermentation product is The present results demonstrated that several xanthosine and its acid hydrolyzates are guanineless mutants of Bacillus subtilis were

8) S. M. Partridge, Nature, 164, 443 (1949). 9) A. H. Brown, Arch. Biochem., 11, 269 (1946). 610 Masao FUJIMOTO, Kazuo UCHIDA, Morio SUZUKI and Hiroshi YOSHINO capable of producing xanthosine in the culture fore, it may be natural that the level of xan broth and further mutation to adenine de thosine accumulation of a guanine-adenine pendence led to a marked increase in its pro doubleless mutant is much higher than that duction. The accumulation of xanthosine in of a guanineless mutant. a guanineless mutant might be ascribed to a Microbial production of xanthosine in high genetic block of XMP aminase, responsible yields is of special interest from viewpoint of for the conversion of XMP to GMP, as re- applied microbiology, since xanthosine can be ported by Magasanik. converted by phosphorylation to XMP whose 1)Induction of specific adenine requirement sodium salt has been known as a flavoring in a guanineless mutant resulted in cultures agent. capable of producing hypoxanthine derivatives 11) Acknowledgements. The authors wish to in addition to xanthine derivatives. On the thank Mr. M. Kibi and Dr. A. Kuninaka, other hand, the results presented in Fig. 1 clearly indicate that hypoxanthine derivatives research members of Microbial Laboratory of accumulated at the early stage are converted Yamasa Shoyu Co., Ltd., for their encourage to xanthosine during cultivation. This finding ments and advices throughout this work. is in accord with the fact that exogenous Thanks are also due to Mr. M. Morozumi, Mr. Y. Midorikawa and Mr. S. Miyakawa of hypoxanthine derivatives are converted to xanthosine by growing culture of a guanine this laboratory for their assistance in carrying less mutant as reported previously.10) There out the experiments.

10) M. Fujimoto and K. Uchida, This Journal, 29, 1150 11) A. Kuninaka, J. Agr. Chem. Soc. Japan, 34, 489 (1960). (1965).