[Agr. Biol. Chem., Vol. 33, No. 3, p. 370•`376, 1969]

Production of Nucleic Acid-Related Substances by Fermentative Processes

Part XXII. Fermentative Production of 5'-Xanthylic Acid by a Guanine Auxotroph of Brevibacterium ammoniagenes

By Masanaru MISAWA,Takashi NARA, Kiyoshi UDAGAWA Shigeo ABE and Shukuo KINOSHITA TokyoResearch Laboratory, Kyowa Hakko Kogyo Co., Ltd., Machida-shi, Tokyo ReceivedAugust 5, 1968

A guanine-requiringmutant of Brevibacteriumammoniagenes ATCC 6872 accumulated a large amonnt of 5'-xanthosine-monophosphate(abbreviated as XMP). The quantity of XMP accumulatedby the strain was affectedsignificantly by guanine levelsin the medium. The suppressionof XMP accumulationby an excessiveaddition of guanine compoundswas recoveredby the supply of casaminoacids in the medium. An enzyme in the pathway of de novaXMP synthesis,IMP dehydrogenase(IMP: NAD oxidoreductase,EC 1.2.1.14),was repressedand inhibited by guanine compounds. The facts that an exogenousxanthine was not convertedto XMP by the growingcells and that the activity of XMP-pyrophosphorylasewas very low or deficient suggest that XMP accumulation by the strain would be probably due to the direct excretionof the nucleotidefrom the cells.

As reported previously,1) a guanineless or production of nucleotides, it was observed that a guanine-adenine doubleless mutant of Coryne guanine-requiring mutants of Brevibacterium bacterium glutamicum (a synonym of Micrococcus ammoniagenes accumulated larger amounts of glutamicus) accumulated a large amount of 5'- 5'-XMP than those of C. glutamicum. xanthylic acid (abbreviated as XMP) in the The present paper describes 5'-XMP pro culture medium. duction by a mutant of Brev. ammoniagenes and Demain et al.2) have recently reported similar enzymatic studies involved in the nucleotide results with a mutant of C. glutamicum. synthesis. During further investigation on the microbial MATERIALS AND METHODS Abbreviation XMP: 5'-xanthosine monophosphate; NAD: nicotin amide adenine dinucleotide; IMP: Microorganism used. A guanine-requiring mutant 5'-inosine monophosphate; GMP: 5'-guanosine mono- (KY 7450) of Brevibacterium ammoniagenes was used phosphate; GDP: 5'-guanosine diphosphate; GTP: 5'-guanosine triphosphate; ATP: 5'-adenosine tripho- exclusively in these experiments. sphate; 2, 6-DAP: 2, 6-diaminopurine; Hx: hypoxan- This mutant was derived from Brev. ammoniagenes thine; DCW: dry cell weight; R-5-P: ribose-5- ATCC 6872 treated with diethyl sulfate. phosphate.1)M . Misawa, T. Nara, K. Udagawa, S. Abe and Fermentation techniques. The most suitable medium S. Kinoshita, Agr. Biol. Chem., 28, 688 (1964). 2) A. L. Demain, M. Jackson, R. A. Vitali, D. for the selection of u mutant for XMP production Hendlin and T. A. Jacob, Appl. Microbiol., 13, 757 was the same as that described previously.1) (1965). Chemically defined media based on those for 5'- Production of Nucleic Acid-Related Substances by Fermentative Processes. Part XXII 371

inosinic acid production by B. ammoniagenessl were as follows: Seed medium: 2.0% of glucose, 0.4%% of (HN4)2SO4,

0.25% of NaCl, 0.03% of MgSO4•E7aq, 0.1% of K2HPO4, 0.01% of FeSO4•E7 aq, 2.0% of casamino

acids, 30ƒÊg/liter of biotin, 2.5ƒÊg/ml of Ca-D-panto- thenate, 2.5ƒÊg/ml of thiamine. HCl and 20ƒÊg/ml of

guamine. Urea (0.1%) was autoclaved separately and added to the medium before inoculation. Fermentation media (A) and (B):

(A) glucose 10.0%, KH2PO4 1.0%, K2HPO4 1.0%, MgSO4•E7aq 1.0%, CaC12•E2aq 0.01%, biotin 30ƒÊg/ liter, Ca-D-pantothenate 10ƒÊg/ml, thiamine. HC1 2ƒÊg / ml and urea 0.6%.

pH was adjusted to 8.0•`8.2 with 5 N-NaOH before FIG. 1. Growth Responses of KY 7450 Strain to sterilization. Urea was autoclaved separately. Guanine Compounds. (B) glucose 10.0%, KH2PO4 0.2%, K2HPO4 0.2%, MgSO4•E7aq 0.4%, (NH4)2SO4 2.0%, CaCO3 2.0%,

biotin 30ƒÊg/liter, Ca-D-pantothenate 10ƒÊg/ml and Medium: Glucose 2.0%, (NH4)2SO4 0.4%, thiamine.HCl 2ƒÊg/ml, pH was adjusted to 8.0•`8.2 casamino acids 1.00%, K2HPO4 0.1%, urea 0.1%, with 5 N-NaOH before sterilization. vitamin mixture 1ml/100 ml. MgSO4 7aq 0.03%, Other cultivation methods were the same as in the FeSO4. 7aq 0.01% and CaC12. 2aq 0.01%. previous report. Incubation: 30•Ž for 2 days. DCW: Dried cell weight. Enzyme assay. The cells grown in the fermenta tion medium for 3 days were harvested by centrifuga tion, washed twice with saline and suspended in M/10 RESULTS phosphate buffer. Cell free extracts were prepared by treating the cell suspensions with a 10kc Sonic oscill Growth response of KY 7450 strain ator for 20min, and the supernatants of the disrupted Among several guanine-requiring mutants sonicate were employed as enzyme preparation. of Brevibacterium ammoniagenes ATCC 6872, Protein content in enzyme preparations was esti strain KY 7450 was selected for the investiga mated by measuring optical density at 280 mƒÊ.41 tion of XMP fermentation, because of its high IMP dehydrogenase activity: A modified method XMP productivity. of Magasanik's5) was used. One unit of the enzyme The growth responses of KY 7450 strain to was represented by 0.001 increase of the optical guanine compounds and 2, 6-diaminopurine density at 290mƒÊ with 1mg of protein per minute. are shown in Fig. 1. The results were similar

Nucleotide pyrophosphorylase activity: The assay to those of C. glutamicum KY 9978 strain.7) method was the same as previously described.6) Guanine supported the growth most efficiently,

Quantitative estimations of XMP, xanthine and bac and 2, 6-diaminopurine was not utilized as terial growth were carried out by the same methods guanine source by this strain, while Aerobacter as reported previously.1) aerogenes8) utilized 2,6-DAP instead of guanine. Adenine, hypoxanthine, their ribosides and 3) T. Nara, M. Misawa and S. Kinoshita, Agr. Biol. Chem., 32, 1153 (1968). ribotides failed to support the growth of KY 4) J. G. Flakes "Methods in Enzymology" vol. VI. 7450 strain. Academic Press, New York and London, 1963, p. 136. 5) B. Magasanik, "Methods in Enzymology" vol. VI. Academic Press, New York and London, 1963, 7) M. Misawa, T. Nara and S. Kinoshita, Agr. p. 107. Biol. Chem., 28, 694 (1964). 6) T. Nara, M. Misawa, T. Komuro and S. 8) B. Magasanik and M. S. Brooke, J. Biol. Chem., Kinoshita, Agr. Biol. Chem., 31, 1224 (1967). 206, 83 (1954). 372 M. MISAWA, T. NARA, K. UDAGAWA, S. ABE and S. KINOSHITA

FIG. 2. Effect of Casamino Acids in Fermentation Medium on Growth and XMP Production.

Fermentation medium: (A) + Guanine 50ƒÊg/ ml.•œ•\•œ XMP (mg/ml) •›•\•› DCW (mg/ml) Incubation: 30•Ž, 5 days

XMP production As described in Materials and Methods, chemically defined media were decided for XMP production by KY 7450 strain. Supple ment of casamino acids to the fermentation FIG. 3. Effect of Guanine and Casamino Acids in medium stimulated both XMP accumulation Fermentation Medium on Growth and XMP and cell growth as shown in Fig. 2. The Production. quantities of guanine in the media exhibited Fermentation medium: (A)_??_ remarkable effects on XMP prodution and on casamino acids 0%•›•\•› cell growth. Addition of more than 50ƒÊg/ml casamino acids 0.50% of guanine increased cell mass, but decreased Incubation: 5 days at 30•Ž•¢•\•¢ the amounts of the products. However, the casamino acids 0.25%•œ•\•œ casamino acids 1.00% amounts of casamino acids in the media excerted a marked effect on the guanine level for obtaining a maximal yield of XMP. Figure 4-(B) shows the result of fermenta The results are summarized in Fig. 3. tion in a medium most suitable for XMP It is of interest that the supression of XMP accumulation. After 6 day's culture, about accumulation by excessive guanine was re 4.0mg/ml of XMP were accumulated in the covered by the supplement of casamino acids medium and the dried cell weight was ap- in the media. proximately 10mg/ml. The time courses in Time courses of XMP fermentation in the the excessive addition of guanine are presented media which contained various amounts of in Fig. 4-(C) and (D). In both cases, the cell guanine are shown in Fig. 4. growth and sugar consumption proceeded at Although cell growth and XMP production a rapid rate and the nucleotide production were observed even in the absence of guanine, was suppressed. they were of low levels as shown in Fig. 4- Thus, guanine content in the fermentation (A). The consumption of glucose proceeded medium affected remarkably the chemical at a slow rate. changes of the fermentation. Production of Nucleic Acid-Related Substances by Fermentative Processes . Part XXII 373

FIG. 4. Time Courses of XMP Fermentation with Br. ammoniagenes KY 7450 Strain under Various Concentrations of Guanine.

The product was isolated and identified TABLE I. EFFECT OF Mn2+ ON chemically and enzymatically as 5'-XMP by XMP FERMENTATION the methods described in the previous report."

Effect of manganese In previous reports, 3,61 the authors showed that manganese levels in the medium exhibited a remarkable effect on IMP, guanine nucleo Fermentation medium: (B) 1.0% of yeast extract was added in stead of both thiamine and pantothenate. tides production by Brev. ammoniagenes and "Supernatant of broth" was obtained by centrifu other bacteria. In these fermentations, exces gation of the culture broth at 3000rpm for 20min. sive levels of Mn2+ stimulated bacterial growth and inhibited accumulation of these nucleo into the medium. tides markedly. Under a suboptimal level of In XMP fermentation by Brev. ammoniagenes Mn2+ large amounts of these nucleotides were excessive levels of Mn2+ in the fermentation accumulated and bacterial cells were elongated medium did not result in alterations of cellular or swollen and the supernatant of the broth form, but stimulated markedly XMP formation became turbid since cell protein was solubilized (Fig. 5 and Table I). This is in striking 374 M. MISAWA, T. NARA, K. UDAGAWA, S. ABE and S. KINOSHITA

contrast with other nucleotide fermentations with Brev. ammoniagenes.

Regulation of IMP dehydrogenaseby guanine compounds As mentioned above the excessive addition of guanine to fermentation medium reduced the yields of XMP greatly. It is very likely that IMP dehydrogenase, an enzyme in de novo guanine biosynthesis pathway, might be regulated by guanine compounds in our strain. The cell-free extracts from the cells culti vated for 3 days in medium B containing various amounts of guainine were prepared as described in Materials and Methods, and the activity of IMP dehydrogenase was assayed. As shown in Fig. 6, the maximum enzyme activity was obtained by the cells which were

FIG. 6. Effect of Guanine Concentration in the Medium on IMP-dehydrogenase Activity, XMP Accumulation and Growth of KY 7450 Strain.

•œ•\•œ IMP dehydrogenase activity, unit (3-Day Culture)•›•\•› XMP accumulated, mg/ml (5-Day Cul- ture)•› ---•› DCW, mg/ml (3-Day Culture) Medium: (B), described in Materials and Methods Reaction mixture for IMP dehydrogenase assay: 100ƒÊmoles of tris-HCl, 5ƒÊmoles of reduced gluta thione, 100ƒÊmoles of KC1, 3ƒÊmoles of 5•L-IMP, FIG. 5. Cell From of Brev. ammoniagenes. 2.5ƒÊmoles of DPN and cell-free extract of KY Cell: Cultured in the fermentation medium 7450 strain (3mg as Protein) described under Table I for 5 days. Phase contrast Total volume 3.0ml, pH 7.55; Incubated at microscope was used. 25•Ž for 15min. Production of Nucleic Acid-Related Substances by Fermentative Processes. Part XXII 375

TABLE II. INHIBITION OF IMP DEHYDROGENASE TABLE III. ACTIVITY OF XMP- AND GMP- ACTIVITY BY 5'-GMP PYROPHOSPHORYLASES OF Brevibacterium.

Reaction mixture: 100ƒÊmoles of tris-HCl, 5ƒÊmoles of reduced glutathione, 100ƒÊmoles of KCl, 3 or 1.5ƒÊ moles of 5•L-IMP, 2.5ƒÊmoles of DPN, and cell-free Both enzyme sources were prepared by the same extract of KY 7450 strain (3mg as protein). Total methods as described in the previous report.6) KY volume 3.0ml, pH 7.55; Incubated at 25•Ž for 15min. 3454 was incubated in the medium described in the

same report6) and KY 7450 was in (A) medium for 3 days. cultivated in a medium containing 12.5ƒÊg/ml Reaction mixture: 8-14C-bases, ca. 2 x l04-4 x 104 of guanine. However an optimal level of cpm bases 0.5ƒÊmoles, Nat-ATP 0.5ƒÊmoles, Na-R-5-P guanine for XMP accumulation was 50ƒÊg/ml 10ƒÊmoles, MgCl2. 2aq 5ƒÊmoles, phosphate 5ƒÊmoles and the enzyme activity was rather repressed and enzymes (4mg as protein). Total volume 1.0ml, pH 7.4; Incubated at 37•Ž at that level. This enzyme was also inhibited for 30min. by 5'-guanosine monophosphate as demon strated in Table II. exception of the activity for hypoxanthine by an extracellular enzyme source. Salvage synthesis of XMP The enzyme activities per ml of broth of It was previously shown that wild type strains KY 7450 strain were lower than those of the of Micrococcus sodonensis,6) Arthrobacter citreuss6) parent strain, due to lower growth level of and Brev. ammoniagenes91 were able to accumu the mutant. late an appreciable amount of IMP from The order of the strength of nucleotide hypoxanthine added to the medium. Such pyrophosphorylase activities of the both strainss nucleotide accumulation was suggested to be are as follows: catalyzed through the action of two salvages guanine>hypoxanthine>adenine>xanthine enzymes, 5-phosphoribose pyrophosphokinase (as substrate). and IMP pyrophosphorylase.6,l0) When xanthine was used as a substrate, the However, any significant increase in the enzyme activity was the lowest in both strains. nucleotide production by KY 7450 was not As the details will be described in the suc observed when large amounts of purine base, ceeding report, non-exacting purine auxotrophs such as adenine, hypoxanthine, or xanthine, of Brev. ammoniagenes which grow with both were added to the medium. adenine and guanine, were not able to grow The activities of nucleotide pyrophosphory in the presence of both adenine and xanthine. lases were determined as shown in Table III. Furthermore, a number of experiments showed The comparison of the enzyme activities per that Brev. ammoniagenes ATCC 6872, a parent mg protein in KY 7450 and in KY 3454 strain of KY 7450, was unable to accumulate (ATCC 6872), a parent strain of KY 7450, did XMP from xanthine. Such facts suggest this not show any remarkable differences with the strain does not have XMP pyrophosphorylase. 9) T. Nara, M. Misawa and S. Kinoshita, Agr. Biol. Chem., 32, 561 (1968). 10) T. Nara, M. Misawa, T. Komuro and S. Kinoshita, ibid., 33, 358 (1969). 376 M. MISAWA, T. NARA, K. UDAGAWA, S. ABE and S. KINOSHITA

DISCUSSION lation by KY 7450, the following two pathways would be considered: The quantities of guanine in the fermenta (1) direct excretion of XMP produced de tion medium markedly affected the accumu novo inside of the cells or (2) extracellular lation of XMP by a guanine-requiring mutant synthesis of XMP from xanthine excreted of Brevibacterium ammoniagenes as had been from the cells by XMP-pyrophosphorylase, as noted for Corynebacterium glutamicum.11 Casa was reported in IMP accumulations by wild mino acids in the medium recovered the sup strains of M. sodonensis6) and Brev. ammonia pression of nucleotide accumulation which was genes.l0) caused by an excessive addition of guanine. However, xanthine supplemented to the Under a constant concentration of guanine, fermentation medium was not converted to the increase of casamino acids level in the XMP, in contrast with the previous data medium led to the accumulation of more XMP. showing the salvage synthesis of IMP from Hx This finding suggests that amino acid in the by both a wild strain and an adenineless medium might release enzyme inhibition or mutant of Brev. ammoniagenes.3) In addition, repression by guanine compounds. XMP pyrophosphorylase was found of a very It is of interest that a maximal enzyme low level or deficient in the mutants employed. activity of IMP dehydrogenase was obtained These results suggest that XMP accumula by cells incubated in a medium containing tion would be probably due to the direct 12.5ƒÊg/ml of guanine, although maximal yield excretion of the nucleotide from the cells. of XMP was obtained in the presence of 50ƒÊ This is in a marked contrast with IMP fer g/ml of guanine. The guanine concentration mentation with an adenineless mutant of in the medium may affect not only IMP Brev. ammoniagenes where IMP appears to be dehydrogenase activity, but also other enzymes resynthesized by IMP-pyrophosphorylase from in XMP biosynthesis. Hx excreted in an earlier stage of growth.10) As probable mechanisms of XMP accumu