J. Gen. Appl. Microbiol., 45, 169–176 (1999)

Accumulation of and N-glucosylanthranilic acid by a Corynebacterium glutamicum mutant resistant to DL- hydroxamate

Kazumi Araki,* Hiroaki Takakura, Yoshiro Miyajima, Yoshiteru Akashi, Kensuke Kawanishi, Shingo Kakita,1 and Yasuo Kondo2,†

Department of Food Technology, 2Department of Life Science and Technology, Faculty of Engineering, University of East Asia, Shimonoseki 751–8503, Japan 1Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Machida, Tokyo 194–8533, Japan

(Received March 5, 1999; Accepted August 30, 1999)

During a study on the effect of DL-serine hydroxamate on Corynebacterium glutamicum (JCM1318, a wild strain), a mutant resistant to the drug, strain TO3002, was isolated. This mutant accumulated five Ehrlich’s reagent positive fluorescent substances in the culture medium. Two major and one minor fluorescent products were isolated by preparative high-performance liquid chromatography following charcoal column chromatography from the culture supernatant. One major product was identified as anthranilic acid whose molecular ion was confirmed to be 137 by a measurement of liquid chromatography- (LC-MS), and NMR spectrum coincided with that of an- thranilic acid. LC-MS spectra of another major and the minor product showed that they had the same molecular weight of 299. This major product was supported to be N-glucosylanthranilic acid (N-o-carboxyphenyl-1-b-glucosylamine) by two-dimensional 1H and 13C NMR analyses. The minor product was speculated to be an Amadori compound derived from N-glucosylanthranilic acid. N- Glucosylanthranilic acid accumulated in the early phase, then decreased in the late phase of the culture. In contrast, the accumulation of anthranilic acid increased remarkably in the late phase of the . Based on this phenomenon, it was assumed that N-glucosylanthranilic acid once accumulated was decomposed to form anthranilic acid, at least in large part, with the progress of fermentation. The strain TO3002 showed a leaky requirement for L- or indole (but did not for anthranilic acid) and resistance to DL-serine hydroxamate.

Key Words——anthranilic acid; carboxyphenylglucosylamine; Corynebacterium glutamicum; glucosylan- thranilic acid; hydroxamate; serine; tryptophan

Some hydroxamates have been known serine, although the amount of the excretion was to antagonize the amino acid and are used as a tool small (Tosa and Pizer, 1971). for the induction of the mutant overproducing the cor- Having an interest in inducing an L-serine producer, responding amino acid. For example, L- pro- we had investigated the effect of DL-serine hydroxa- ducer and L- producer have been isolated mate on Corynebacterium glutamicum, the mutants of as the mutant resistant to the hydroxamate in Bacillus which have been widely used for the production of subtilis (Kisumi et al., 1971a) and Serratia marcescens many amino acids (Kinoshita, 1985). During this re- (Kisumi et al., 1971b), respectively. A serine hydroxa- search we found a mutant that accumulated an- mate-resistant mutant of Escherichia coli excreted L- thranilic acid and N-glucosylanthranilic acid (N-o-car- boxyphenyl-1-b-glucosylamine) in high amounts and * Address reprint requests to: Dr. Kazumi Araki, Department of three minor unidentified fluorescent substances. Food Technology, Faculty of Engineering, University of East Asia, N-Phosphoribosylanthranilic acid has been known 2–1 Ichinomiya-gakuencho, Shimonoseki 751–8503, Japan. as an intermediate in the tryptophan biosynthetic path- E-mail: [email protected] way (Crawford, 1987; Somerville, 1983). However, the † Present address: Department of Bioresource Science, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obi- N-glucosylated one had not been known as the inter- hiro, Hokkaido 080–8555, Japan. mediate. The present paper describes the nature of 170 ARAKI et al. Vol. 45 the mutant and the identification of the two major fluo- Japan). The nutritional requirement for the mutant rescent products accumulated in the culture medium strain was tested on agar plates similar to the way de- by the high-resolution instrumental analyses. scribed above, except that a large agar plate (25 ml in a 90 mm diameter petri dish) was used, and each Materials and Methods (0.05 ml) of the solutions to be tested was added to a round well (7 mm diameter) made on a cor- Microorganisms. C. glutamicum JCM1318 (ATCC ner of the agar plate. The requirement was preliminar- 13032, a wild strain) and its DL-serine hydroxamate-re- ily tested on mixtures of amino acids (2 mg/ml for sistant mutant, TO3002, were used. each), base components of nucleic acids (1 mg/ml for Isolation of the mutant. The cells of the parent each), and vitamins. The composition of the vitamin strain grown overnight on an agar slant (10 g of mixture was as follows: 1 mg of calcium pantothenate, polypeptone, 10 g of meat extract, 5 g of yeast extract, pyridoxine · HCl, riboflavin, p-aminobenzoic acid and 5g of NaCl, and 20 g of agar/L, pH 7) were sus- nicotinic acid, and 0.3 mg of cobalamin and folic pended in 1 ml of 0.2 M Tris-maleic acid-0.2 N NaOH acid/100 ml. The bacterial cells, grown overnight on buffer (pH 6) containing 300 mg/ml of N-methyl-N- the agar slant, were inoculated in 102 to 103 cell nitro-N-nitrosoguanidine to make a cell concentration number/plate and incubated at 30°C for 3 days (un- of 109 cells/ml and stood for 30 min at room tempera- less otherwise stated), and the colony formation was ture. After washing through centrifugation, they were observed. resuspended in the same buffer. An aliquot (0.05 ml) Fractionation of the culture filtrate by a charcoal col- was plated on 8 ml of a minimal agar medium contain- umn. To obtain the enriched fraction with fluorescent ing 500 mg/ml of DL-serine hydroxamate in a small petri substance, 170 ml of the culture supernatant of dish (40 mm diameter) and incubated at 30°C for 6 TO3002 was fractionated by a charcoal column days. The composition of the minimal medium was as (25 mm diameter and 120 mm high). After it was follows: 10 g of glucose, 2 g of (NH4)2SO4, 1.5 g of washed with the void volume of water and subse- KH2PO4, 0.5 g of K2HPO4, 0.5 g of MgSO4 ·7H2O, quently 10% (v/v) acetone, the column was eluted 0.01 g of FeSO4 ·7H2O, 0.007 g of MnSO4 ·5H2O, 0.1 g with 120 ml each of 50% (v/v) acetone and 80% (v/v) of NaCl, 30 mg of biotin, 1,000 mg of thiamine · HCl, acetone at a rate of 3 ml/min. Every 50 ml fraction was

1ml of a mineral salt solution (5.3 g of CaCl2 ·2H2O, collected, then concentrated in vacuo to 1/10 volume 40 mg of CoCl2 and CuSO4 ·5H2O, 30 mg of H3BO3, and stored at 20°C 47 mg of Na2MoO4, and 200 mg of ZnSO4 ·7H2O/L), Paper chromatography and thin-layer chromatogra- and 20 g of agar/L (pH 7). Thirty of the larger colonies phy of the fluorescent substances. The enriched that had appeared were isolated and applied to the fraction with fluorescent substances obtained was production test. separated by paper chromatography on Toyo Roshi Culture method for the production of anthranilic acid No. 50 filter paper and thin-layer chromatography on and other fluorescent substances. Six milliliters of a cellulose plate (SF Cellulose plate, Funakoshi Co., fermentation medium in a 300 ml Erlenmeyer flask Ltd., Tokyo, Japan). A solvent system of n-butanol/ were inoculated with an overnight culture (0.6 ml), acetone/diethylamine/water (10 : 10 : 2 : 5, by volume) which was grown on an incubator at room tempera- was used. The fluorescent spots were located under ture and incubated on a reciprocal shaker with a rota- the light at 330 nm. tion speed of 120 rpm, oscillation of 9 cm, at 30°C for High-performance liquid chromatography (HPLC)- 40 h, unless otherwise stated. The seed medium con- mass spectrometry (LC-MS) analysis. The fraction tained 10 g of glucose, 5 g of yeast extract, 10 g of obtained from the above charcoal column chromatog- peptone, and 5 g of NaCl/L (pH 7). The fermentation raphy was analyzed by LC-MS. The HPLC system medium contained 120 g of glucose, 20 g of used for LC-MS was a Shimadzu 10ADvp gradient liq-

(NH4)2SO4, 1 g of yeast extract, 1.5 g of KH2PO4, 0.5 g uid chromatograph equipped with a UV detector of K2HPO4, 0.5 g of MgSO4 ·7H2O, 0.01 g of (Shimadzu Seisakusho Co., Kyoto, Japan). The col- FeSO4 ·7H2O, 0.007 g of MnSO4 ·5H2O, 30 mg of biotin, umn used was an Inertsil SIL (GL Science Inc., Tokyo, 1,000 mg of thiamine · HCl, and 20 g of CaCO3/L (pH Japan), 4.6 mm i.d. 150 mm. Samples were eluted by 7). the linear gradient time program from solvent A = Growth test. The effect of DL-serine hydroxamate dichloromethane/methanol/water (90 : 10 : 0, by vol- on the bacterial growth was tested by culturing them ume) to solvent Bdichloromethane/methanol/water on the minimal agar medium (8 ml in the 40 mm diam- (68 : 28 : 4, by volume) for 20 min. The other HPLC eter petri dish) with the supplements of the drug. The conditions were as follows: flow rate, 1.0 ml/min; col- hydroxamate was used after sterilization by filtration umn temperature, 27°C; detection, UV at 330 nm. with a filter (0.45 mm pore size, NALGENE Co., Tokyo, Mass spectra were taken with a JEOL JMS 700 re- 1999 Accumulation of anthranilic acid and N-glucosylanthranilic acid 171 verse-geometry double-focusing mass spectrometer (JEOL Ltd., Tokyo, Japan) equipped with an atmos- pheric pressure chemical ionization (APCI) source. The accelerating voltage and the ring lens voltage were set at 5 kV and 50 V, respectively. The vaporizer was operated at 500°C, and the corona voltage for APCI was set at 5 kV. High-purity nitrogen was used as the nebulizer gas. Mass spectra were obtained from 50 to 1,000 Da with a scan time of 4 s. An APCI source allows direct introduction of HPLC column ef- fluent at the rate of 1.0 ml/min. Isolation of the fluorescent substances. The isola- tion of fluorescent substances was carried out in two different ways. First, fluorescent substance enriched fraction was subjected to a preparative-paper chro- matography in the same manner as above. After de- velopment, the product was extracted and isolated with 100 mM potassium phosphate buffer (pH 7.7) from each strip of spot under a light at 365 nm on the Fig. 1. A cellulose thin-layer chromatogram of the fluorescent paper chromatogram. Second, fluorescent substances substances from the culture filtrate of C. glutamicum mutant were separated by preparative-HPLC using a JASCO TO3002. Solvent system, n-butanol/acetone/diethylamine/water (10 : 10 : apparatus (Japan Spectroscopic Co., Tokyo, Japan), 2:5, by volume); detection, UV irradiation at 330 nm; lane 1, fluo- which was composed of the same system as the rescent substances from the culture filtrate; lane 2, authentic stan- Shimadzu instrument. Other chromatographic condi- dard of anthranilic acid. For details of the spots, see text. tions were also the same as the LC-MS measure- ment. The effluent of peak monitoring by a UV detec- sistant colonies clearly appeared on the agar medium tor was recovered directly from the outlet of the detec- supplemented with 500 mg/ml of the drug at a fre- tor. quency of 106 in 5 days incubation. Nuclear magnetic resonance (NMR) measure- ments. NMR spectra [1H NMR, 13C NMR, DEPT, 1H- Accumulation of fluorescent substances by a DL-serine 1H COSY, NOESY, HSQC, pulsed field gradient (PFG) hydroxamate-resistant mutant HMBC] were measured on Bruker DMX500 or JEOL It has often been realized that a mutant resistant to JNM-A400 at 500 or 400 MHz for 1H and 125 or a drug antagonizing a metabolite overproduces the 100 MHz for 13C. metabolite in the culture medium (Araki and Ozeki, Reagents. Anthranilic acid, charcoal, L-, 1992; Nakayama, 1982). Actually, a serine hydroxa- indole, L-serine, and L-tryptophan were purchased mate-resistant E. coli mutant has a 3-phosphoglycer- from Wako Pure Chemical Co. (Osaka, Japan). DL- ate dehydrogenase (EC that is insensitive to Serine hydroxamate was purchased from Sigma (St. end-product inhibition and that excretes L-serine (Tosa Louis, MO, USA). Other reagents and chemicals were and Pizer, 1971). In this respect, the isolated mutants commercially available extrapure products. of C. glutamicum resistant to DL-serine hydroxamate were expected to produce L-serine. So we cultured Results these mutants obtained in the preceding experiment. Contrary to expectation, no such mutant was found. Inhibition of DL-serine hydroxamate on the growth of Instead, we found a novel mutant, TO3002, which ac- C. glutamicum and isolation of the DL-serine hydroxa- cumulated five fluorescent substances in the culture mate-resistant mutants medium. Figure 1 shows a cellulose thin-layer chro- Having an interest in inducing an L-serine producer matogram of the products detected as blue fluores- from C. glutamicum, we first investigated the effect of cent spots by UV irradiation. Two major spots of X1 DL-serine hydroxamate on the growth of the bac- and X3 and a minor spot of X2 are exemplified in Fig. terium. An agar culture test revealed that the drug in- 1. Two other fluorescent spots below those spots were hibited the growth of the wild strain JCM1318 at con- occasionally observed as can be faintly seen in the centrations of 250 mg/ml or more. chromatogram, indicated by arrows. These spots were Subsequently, we tried to isolate DL-serine hydroxa- all positive to Ehrlich’s reaction (data not shown). The mate-resistant mutants from C. glutamicum as de- former three substances were studied in more detail. scribed in the MATERIALS AND METHODS section. The re- 172 ARAKI et al. Vol. 45

Fig. 2. An HPLC separation of the fluorescent substances in a concentrated fraction of a charcoal column chromatography. The chromatogram was recorded on a UV detector at 330 nm. Peaks I, II, and III correspond to spots X1, X2, and X3, re- spectively, in Fig. 1.

Identification of the fluorescent substances employed as a sample for NMR measurement. LC-MS analysis NMR spectra Figure 2 shows an HPLC chart of the fluorescent 1H and 13C NMR data of products X1 and X3 are substances in a concentrated fraction of a charcoal shown in Table 1. X1 was identified with anthranilic column chromatography. Peak I (retention time 2.47 acid, by comparison of 1H and 13C NMR spectra, with min) and peak III (retention time 9.49 min) as major those of an authentic sample. Analyses of the 1H-1H peaks and a peak II (retention time 7.34 min) are COSY, HSQC, and PFG HMBC spectra of X3 re- recorded on the chromatogram. The APCI-MS spec- vealed the presence of an anthranilic acid moiety and trum of peak I showed a molecular ion at m/z 138 a pyranosylamine moiety. The coupling constants and (MH); relative intensity 100% and its dehydration the NOEs data were studied to determine the struc- ion at m/z 120; relative intensity 33%. Peak III showed ture of the pyranosylamine moiety. The vicinal cou- a molecular ion at m/z 300 (MH); relative intensity pling constants between 1-H and 2-H, 2-H and 3-H, 100%. Peak II showed the same molecular ion as and 3-H and 4-H were 8.6 Hz, 8.8 Hz, and 8.8 Hz, re- peak III. spectively, so 1-H, 2-H, 3-H, and 4-H were all iden- Light absorption spectra tified to axial. In the NOESY spectrum, the NOEs ob- The light absorption spectra of each substance, X1 served between 1-H and 3-H and 1-H and 5-H re- to X3, isolated by the preparative-paper chromatogra- vealed that 1-H, 3-H, and 5-H were in the same phy, were measured by a spectrophotometer (Model plane. Therefore the pyranosylamine moiety of X3 UV-240, Shimadzu) after dilution with the appropriate was confirmed to the b-glucosylamine. Furthermore, volume of 100 mM phosphate buffer (pH 7.7). The the correlation from 1-H to C-2 in the PFG HMBC spectrum of X1 had two peaks (238 and 306 nm), spectrum showed that the glucosylamine moiety was which are close to those reported for anthranilic acid linked to the anthranilic acid moiety through the amino (240 and 310 nm) (Doy and Gibson, 1959) and coin- group of the anthranilic acid moiety. The results of cided with that of authentic anthranilic acid, as shown NMR analysis of the product X3 are condensed into in Fig. 3A. The spectra of X2 and X3 also had two the assignment figure as shown in Fig. 4. peaks, at 245 and 313 nm (Fig. 3B), and at 245 and From the data of the NMR experiment, LC-MS 315 nm (Fig. 3C), respectively. These spectra resem- analysis, and UV measurement, we identified that the bled those reported for N-o-carboxyphenylgly- products X1 and X3 are anthranilic acid and N-o-car- cosamines, anthranilic acid derivatives (Doy and Gib- boxyphenyl-1-b-glucosylamine, respectively. son, 1959). According to the thin-layer chromatographic analy- Time-course for accumulation of anthranilic acid and sis of each isolated peak by the preparative-HPLC, N-glucosylanthranilic acid using JASCO system, peaks I, II, and III in Fig. 2 were Thus identified substances of X1 and X3 as an- confirmed to correspond with the spots X1, X2, and thranilic acid and N-glucosylanthranilic acid, respec- X3 in Fig. 1, respectively. Each isolated product was tively, were determined by measurement of an ab- 1999 Accumulation of anthranilic acid and N-glucosylanthranilic acid 173

Fig. 3. Light absorption spectra of fluorescent substances accumulated by C. glutamicum mutant TO3002. The spectra were measured with the samples prepared as described in the MATERIALS AND METHODS section after dilution with 100 mM potassium phosphate buffer (pH 7.7). A, X1 (– – –) and authentic anthranilic acid (——); B, X2; C, X3.

Table 1. 1H and 13C NMR data of substances X1 and X3.

X1 X3

Position Proton Carbon Proton Carbon

Chemical shiftsa J (Hz) Chemical shiftsb Chemical shiftsa J (Hz) Chemical shiftsb

1110.3 116.7 2 152.8 150.5 3 6.71 dd, 1.1, 8.3 116.6 6.93 d, 8.0 114.1 4 7.21 ddd, 1.6, 8.3, 7.0 135 7.3 ddd, 1.6, 8.0, 7.1 134.1 5 6.55 ddd, 1.1, 7.0, 8.0 117.7 6.68 dd, 7.1, 7.9 117.7 6 7.79 dd, 1.6, 8.0 132.7 7.89 dd, 1.6, 7.9 132.8 1-COOH 171.6 173.9 1 4.61 d, 8.6 86 2 3.36 m 75 3 3.46 dd, 8.8, 8.8 79.2 4 3.33 m 71.9 5 3.4 m 78.5 6 3.68 m 62.8 3.85 m

X1 and X3 were dissolved in CD3OD and analyzed at 30°C by a nuclear magnetic resonance (NMR) spectrometer, Bruker DMX500 at 500/125 MHz, or JEOL JNM-A400 at 400/100 MHz. The assignments of 1H and 13C NMR spectra were done according to the methods of DEPT, 1H-1H COSY, HSQC, PFG HMBC, and NOESY. a Chemical shifts are shown with reference to residual CHD2OD as 3.30 ppm. b Chemical shifts are shown with reference to CD3OD as 49.0 ppm. sorption at 310 and 317 nm, respectively. Molecular mined in the same manner as N-glucosylanthranilic absorption coefficients 2,980 for anthranilic acid and acid was, and its amount was expressed as N-gluco- 3,740 for N-glucosylanthranilic acid (Doy and Gibson, sylanthranilic acid equivalent in Figs. 5 and 6. 1959) were used for the calculation. X2 was deter- We investigated the time course for accumulation of 174 ARAKI et al. Vol. 45

Fig. 4. NMR experiments of a fluorescent substance X3 accumulated by C. glutamicum mutant TO3002. ←→, NOESY;→ , PGF HMBC.

Fig.⦆5.⦆⦆Time course of the accumulation of fluorescent sub- stances by C. glutamicum mutant TO3002. Fig. 6. The effect of L-tryptophan on the accumulation of fluo- Fermentation was carried out in five flasks, and one culture at the rescent substances by C. glutamicum mutant TO3002. indicated cultivation time was assayed for the accumulation of the Fermentation was carried out with the fermentation medium sup- fluorescent substances. The concentration of X2 is indicated as that plemented with L-tryptophan at concentrations indicated. For meth- of N-glucosylanthranilic acid. For the methods of cultivation, deter- ods for the cultivation and determination of the fluorescent sub- mination of the products, and other details, see the MATERIALS AND stances, see the MATERIALS AND METHODS section. , Anthranilic METHODS section. , Anthranilic acid; , X2; , N-glucosylan- acid; , X2; , N-glucosylanthranilic acid. thranilic acid.

Inhibition of the accumulation of the fluorescent sub- the three fluorescent substances X1 to X3. As exem- stances by L-tryptophan plified in Fig. 5, the accumulation of N-glucosylan- As shown in Fig. 6, the accumulation of the fluores- thranilic acid increased in the early phase and de- cent substances X1 to X3 by TO3002 was inhibited by creased rapidly in the prolonged incubation. In con- the addition of L-tryptophan to the medium at 200 mg/ml trast, the accumulation of anthranilic acid increased in or more. Other natural amino acids including L-serine, the late phase of the culture. These features of the L-, and L- did not inhibit the ac- fermentation supported a thought that N-glucosylan- cumulation when these were added singly at 500 thranilic acid accumulated in the early phase was mg/ml. transformed to anthranilic acid, at least in large part, with the progress of fermentation. The maximal accu- Nutritional requirement and DL-serine hydroxamate re- mulated concentration of anthranilic acid and N-gluco- sistance of the strain TO3002 sylanthranilic acid was approximately equimolar, 10 Anthranilic acid must be a precursor for L-trypto- and 9 mM, respectively. phan synthesis as will be discussed below in C. glu- tamicum. Depending on this premise, the accumula- 1999 Accumulation of anthranilic acid and N-glucosylanthranilic acid 175

ence of colony size could not be distinguished with or without the nutritional supplement. L-Serine and an- thranilic acid did not affect the colony formation (data not shown). Under the same condition, the parent strain JCM1318 was not affected on its growth by the substances that were effective on TO3002. TO3002 showed a distinct resistance to DL-serine hydroxamate at 250 µg/ml in comparison with the par- ent strain JCM1318, as shown in Fig. 8.


Anthranilic acid synthetase (EC of C. glu- Fig. 7. Requirement for L-tryptophan or indole of C. glutamicum tamicum has been known to be subject to the end- mutant TO3002. product inhibition and repression by L-tryptophan The bacterial cells were cultured on a minimal agar medium sup- (Hagino and Nakayama, 1975). In fact, the accumula- plemented with L-tryptophan (A), indole (B), or water as control (C) in a well made on a corner of the plate. The colonies formed after tion of anthranilic acid [and its glucosylated deriva- the culture for 36 h at 30°C are given. For details of the experiment, tive(s)] was inhibited by exogenously added L-trypto- see the MATERIALS AND METHODS section. phan. Moreover, all six tryptophan biosynthetic en- zymes, including N-phosphoribosylanthranilic acid iso- merase and indoleglycerol phosphate synthase (EC, have been determined in a strain of Bre- vibacterium flavum (Shiio and Sugimoto, 1978; Sugi- moto and Shiio, 1977). This bacterium is a coryneform bacterium that is closely related to the strain studied here (Kinoshita, 1985). Therefore the terminal reactions leading from anthranilic acid and N- phosphopentosylanthranilic acids as intermediates to indole (or its derivative), then L-tryptophan, must be operating as the tryptophan biosynthetic pathway in C. glutamicum as found in other microorganisms (Craw- ford, 1987; Somerville, 1983; Yanofsky, 1956). Fig. 8. Resistance to DL-serine hydroxamate of C. glutamicum N-Glucosylated anthranilic acid has not been found mutant TO3002. as the intermediate member in the pathway. Based on The cells of strain TO3002 and the parent strain JCM1318 were the tryptophan biosynthetic pathway premised above cultured for 70 h at 30°C on minimal agar plates containing 0, 250, 500, 750, or 1,000 µg/ml of DL-serine hydroxamate besides 30 µg/ml and the auxotrophic nature of the mutant TO3002, it of L-tryptophan. The colonies formed were visible only on the plate should be considered that a genetic block at an an- without the drug for the parent strain (A1) and on the plates without thranilic acid metabolizing [e.g., anthranilate (A2) and with 250 µg/ml of the drug (B2) for the mutant. These fea- phosphoribosyl (EC] caused the tures of the growth are given in comparison with that of JCM1318, overproduction of anthranilic acid, and anthranilic acid which was inhibited by the drug at 250 µg/ml (B1). was then glucosylated by some mechanism inside or outside the cells to accumulate in the culture. How- tion of anthranilic acid and its derivative might be due ever, in this case why the accumulation of N-glucosyl- to some defect in the enzyme activity between an- anthranilic acid preceded that of anthranilic acid re- thranilic acid and the end product in the L-tryptophan mains as a pending program. Whether the trait of DL- biosynthetic pathway, because the block would cause serine hydroxamate resistance essentially concerns a release of the feedback regulation exerted on it. Ac- these phenomena is also obscure. This is, however, cording to the investigation of the nutritional require- possible because 2 of 30 mutants accumulated the ment of TO3002, it was revealed that the mutant had fluorescent substances in a separate experiment a leaky requirement for L-tryptophan or indole. That is, traced in the same manner as described above. the mutant made distinct colonies around L-tryptophan The N-glucosylanthranilic acid once accumulated in or indole placed on the corner of the minimal agar the culture medium may be nonenzymatically trans- plate in the 36-h incubation, as shown in Fig. 7. In the formed to anthranilic acid or rearranged into an prolonged incubation for 45 h, the colonies grew so Amadori compound, depending on the acidity of the largely on the whole area of the plate that the differ- culture as has been known with N-ribosylanthranilic 176 ARAKI et al. Vol. 45 acid (Doy and Gibson, 1959). Although a minor prod- oxyribulose, a compound formed by mutant strains of Aerobac- uct, X2, was like an anthranilic acid analog because ter aerogenes and Escherichia coli blocked in the its chemical shifts were similar to those of anthranilic of tryptophan. Biochem. J., 72, 586–597. 1 Hagino, H. and Nakayama, K. (1975) The biosynthetic control in acid by the analysis of H NMR, determinative signals producing mutants of Corynebacterium 13 from C NMR have not been obtained because of a glutamicum. Agric. Biol. Chem., 39, 351–361. small amount of the sample (unstable when isolated). Kinoshita, S. (1985) Glutamic acid bacteria. In Biology of Industrial However, the substance is likely to be the Amadori Microorganisms, ed. by Demain, A. L. and Solomon, N. A., The compound corresponding to N-glucosylanthranilic Benjamin/Cummings Publishing Co., Inc., London, Don Mills, Sydney, Tokyo, pp.115–142. acid, based on the data of LC-MS measurement (mo- Kisumi, M., Kato, J., Sugiyama, M., and Chibata, I. (1971a) Produc- lecular weight, 299) and Ehrlich’s reaction positive- tion of L-arginine hydroxamate-resistant mutants of Bacillus ness and the light absorption spectrum. subtilis. Appl. Microbiol., 22, 987–991. It is of our interest to know whether N-glucosylan- Kisumi, M., Komatubara, S., Sugiura, M., and Chibata, I. (1971b) thranilic acid has some physiological role such as par- Isoleucine hydroxamate, an isoleucine antagonist. J. Bacteriol., 107, 741–745. ticipation in the tryptophan biosynthetic pathway, be- Nakayama, K. (1982) Amino acids. In Prescott & Dunn’s Industrial sides the pending problems discussed above. Microbiology, 4th ed., ed. by Read, G., Avi Publishing Co. Inc., Westport, pp. 748–801. We thank Mr. Y. Takahashi, MS Application Laboratory, Applica- Shiio, I. and Sugimoto, S. (1978) Altered regulatory mechanisms for tion and Research Center, JEOL Ltd., for measurements of LC-MS tryptophan synthesis in fluorotryptophan-resistant mutants of and for helpful advice about MS analysis. Brevibacterium flavum. J. Biochem., 83, 879–886. Somerville, R. L. (1983) Tryptophan: Biosynthesis, regulation, and References large scale production. In Amino Acids: Biosynthesis and Ge- netic Regulation, ed. by Herrman, K. M. and Somerville, R. Araki, K. and Ozeki, T. (1992) Amino acid: Survey. In Kirk-Osmer L., Addison-Wesley Publishing Co., London, Amsterdam, Don Encyclopedia of Chemical Technology, 4th ed., Vol. 2, John Mills, Ontario, Sydney, Tokyo, pp. 351–378. Wiley & Sons, Inc., New York, pp. 504–571. Sugimoto, S. and Shiio, I. (1977) of tryptophan synthetic Crawford, I. P. (1987) Synthesis of tryptophan from chorismate: pathway in Brevibacterium flavum. J. Biochem., 81, 823–833. Comparative aspect. In Methods in Enzymology, Vol. 142, ed. Tosa, T. and Pizer, L. I. (1971) Biochemical bases for the an- by Kaufman, S., Academic Press Inc., Orlando, Sandiego, New timetabolite action of L-serine hydroxamate. J. Bacteriol., 106, York, Austin, Boston, London, Sydney, Tokyo, Toronto, pp. 972–982. 293–300. Yanofsky, C. (1956) The enzymatic conversion of anthranilic acid to Doy, C. H. and Gibson, F. (1959) 1-(o-Carboxyphenylamino)-1-de- indole. J. Biol. Chem., 223, 171–184.