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1 Agric. Biol Chem., 51 (8), , Production of L-Phenylalanine by a Mutant of Brevibacterium lactofermentum 2256 Takayasu Tsuchida, Koji Kubota, Yasushi Morinaga, Hiroshi Matsui, Hitoshi Enei and Fumihiro Yoshinaga Central Research Laboratories of Ajinomoto Co., Inc., Kawasaki 210, Japan Received January 29, 1987 Excellent L-phenylalanine producers were screened from among tyrosine, methionine doubly auxotrophic and antimetabolite resistant mutants derived from a L-glutamic -acid-producing bacterium; Brevibacterium lactofermentum A selected mutant, Brevibacterium lactofermentum No. 123, produced 21.7mg/ml of l- phenylalanine in 72 hr when13%glucose was supplied as a carbon source. The accumulation of l- phenylalanine by strain No. 123 became 5.17-fold higher than that by parental strain No This yield was the highest among those so far reported. The addition of fumaric acid to the culture mediumpromoted the L-phenylalanine production. Accumulation of other amino acids in the culture broth was negligible compared with that of the main product, L-phenylalanine. Excretion of L-phenylalanine by tyrosine auxotrophs has been shown in Escherichia colip Accumulation of L-phenylalanine in the culture mediumwas also reported in Corynebacterium tyrosine auxotrophs.2' 3) Phenylalanine analogue resistant mutants also excrete L-phenylalanine into the culture medium. /?-2-Thienylalanihe,4) ^-fluorophenylalanine5) or o-fluorophenylalanine5) resistant mutants derived from Escherichia coli, /?-fluorophenylalanine6) or /?-2-thienylalanine7) resistant mutants derived from Bacillus subtilis, /?-2-thienylalanine8) resistant mutants derived from Pseudomonas aeruginosa, p- fluorophenylalanine9) resistant mutants derived from Brevibacterium lactofermentum, p- fluorophenylalanine9) resistant mutants derived from Corynebacterium acetoacidophilum, /?-fluorophenylalanine9) resistant mutants derived from Arthrobacter citreus, m-fluorophenylalanine resistant mutants derived from Candida lipolitica and /?-fluorophenylalanine10) resistant mutants derived from Corynebacterium glutamicum were reported to excrete L-phenylalanine. However, the amounts of L-phenylalanine accumulatedby these mutants are comparatively small from an industrial point of view. The present paper deals with the production of a large amount of L-phenylalanine, which is adequate from a commercial viewpoint, by a tyrosine, methionine doubly auxotrophic, dl- /?-ftuorophenylalanine, DL-/?-3-thienylalanine, adenine and DL-5-methyltryptophan resistant, nitrosoguanidine-treated mutant derived from Brevibacterium lactofermentum 2256, a glutamic acid producing bacterium.11} In this study, we succeeded in obtaining a L-phenylalanine producer that gave a higher yield than any other so far reported. MATERIALS AND METHODS Microorganisms and culture medium. A tyrosine, methionine doubly auxotrophic mutant, No. 808, was derived from Brevibacterium lactofermentum 2256 (ATCCNo ). Strain 2256 is a typical bacterium that produces l- glutamic acid in the presence of a limited concentration of biotin, which is required for its growth. Analogue resistant

2 2096 T. Tsuchida et al. Table I. Compositions of the Media Anamino acid mixture prepared from soybean hydrolysate. mutants were derived from No. 808 by nitrosoguanidine treatment. The compositions of the seed medium, the minimal mediumand the two types of standard mediumfor l- phenylalanine production are given in Table I. Culture for h-phenylalanine production. A loopful of refreshed bacterial cells was inoculated into 3 ml of standard medium I or 20ml of standard medium I or II in a "Sakaguchi" flask, that had been autoclaved at 1 10 C for 5min. Cultivation was carried out at 31.5 C for 72hr on a reciprocal shaker for L-phenylalanine production. The culture broth was then subjected to various analyses. Analytical methods. Bacterial growth was measured, using a Hirama Colorimeter Model ll-b, as the optical density at 562nm (O.D. 562) of the culture broth diluted 26 times after CaCO3 had been dissolved with HCL Glucose was determined by the method of Fehling Lehmann Schoorl.12) Quantitative analysis of l- phenylalanine was carried out by a microbiological assay involving Leuconostoc mesenteroides ATCC8042. Amino acid analysis was carried out with a Hitachi Amino Acid Analyzer Model KLA-2. Mutation and selection of mutants. Overnight grown cells in 5ml of the complete mediumwere harvested, washed with 1/15m phosphate buffer (ph 7.0) and then resuspended in an equal volume of the same buffer containing 200/ig/ml of N-methyl-TV'-nitro-./V-nitrosoguanidine.13) After 30min incubation at 31.5 C with shaking, the treated cells were washed twice with and then resuspended in the same buffer. The number of viable cells decreased from 1.1 x 109 to 1.0x 107 perml with the nitrosoguanidine treatment. The diluted cell suspension was spread on minimal plates containing various amounts of analogues. Cultivation was carried out for 5 days at 31.5 C. The colonies that appeared were picked up and checked again for the analogue resistant character. Chemicals. All natural amino acids were products of Ajinomoto Co., Inc. TV-Methyl-N'-nitro-TV-nitrosoguanidine was purchased from Aldrich Chemical Co. DL-/?-3-Thienylalanine, adenine and 5-methyltryptophan were purchased from Sigma Chemical Co. RESULTS Production of L-phenylalanine by an auxotrophic and analogue resistant mutant of Brevibacterium lactofermentum 2256 L-Phenylalanine producing mutants were

3 Fermentative Production of L-Phenylalanine 2097 % 2 ^ 3 ^ 4 ^ 5 L-Phenylalanine produced (mg/ml) Fig. 1. Pattern of L-Phenylalanine Accumulation by /7-Fluoro-DL-phenylalanine Resistant Mutants. Each fermentation was carried out in a large test tube containing 4ml of the standard mediumat 31.5 C for 72 hr. The arrow indicates the L-phenylalanine production by parent strain No screened on a test tube scale from auxotrophic mutants in the authors' culture collection. A strain which produced 4.2mg/ml of L-phenylalanine in standard mediumi was selected. This strain, No. 808, is a tyrosine, methionine doubly auxotrophic mutant that was derived from Brevibacterium lactofermentum 2256 (ATCC No ) by mutational treatment involving X-ray irradiation (met") arid nitrosoguanidine treatment (tyr"). Nitrosoguanidine-treated cells of Brevibacterium lactofermentum No. 808 were spread on a minimal plate containing SOO^g/ml of /?-fluoro-dl-phenylalanine. Resistant mutants appeared at a frequency of 2.9x 10~5. Figure 1 shows the pattern of L-phenylalanine accumulation by the /?-fluoro-dl-phenylalanine resistant mutants. The best producer, strain No. 84, accumulated 5.7mg/ml of L-phenylalanine in the standard medium. It was reported that DL-^-3-thienylalanine14) was a metabolic analogue of L-phenylalanine in Saccharomyces cerevisiae and Escherichia coli. We found that the growth of Brevibacterium lactofermentum No. 84 in the minimal mediumwas inhibited by the addition of DL-jS-3-thienylalanine. From the above results, we expected that the yield of l- phenylalanine would be improved through the derivation of DL-/?-3-thienylalanine resistant mutants from strain No. 84. Thus, we tried to induce resistant mutants on the minimal medium supplemented with 10,000jug/ml of dl- /?-3-thienylalanine. The 300 mutants obtained were examined for L-phenylalanine productivity in standard medium I. The best strain, 70-6, produced 8.8 mg/ml of L-phenylalanine, which was a 29.4% higher yield than that (6.8mg/ml) in the case of the parent strain, No. 84. Improvement of L-phenylalanine producer No through the derivation of an adenine and 5-methyl-DL-tryptophan resistant mutant Improvement of L-phenylalanine producer No through the derivation of adenine or metabolic analogue, other than phenylalanine analogues, resistant mutants was conducted. Adenine, sulfaguanidine and 2-thiazolealanine inhibited the growth of strain No at concentrations of 5000, 200 and 1500 jitg/ml in the minimal medium, respectively. As shown in Table II, four strains of adenine or 2-thiazolealanine resistant mutants were found to produce increased levels of l- phenylalanine compared with the parent strain, No The amount of L-phenylalanine produced by the best strain, No. 7~12,

4 2098 T. Tsuchida et al. Jjj 10^^"^ T _* -i.o_ C <u jc ^ ~ Q o I lo « Fumaric acid added (%) Fig. 2. Effect of Fumaric Acid on the L-Phenylalanine Production by Strain T-222. The cultivation was carried out at 31 C for 72hr in standard medium II. O-O, L-phenylalanine productivity; O-à"- growth of T-222. g Table II. Improvementof an l-phenylalanine Producing Strain through the Derivation of Metabolic Analogue, Other than Phenylalanine Analogues, Resistant Mutants 248 w ^ 50- ^L_^ >-T L -p ~ o 30- X! e = j Adenine resistant mutant 7~ 12 was derived from 70-6, which accumulated 8.0mg/ml of l- phenylalanine in this experiment. was 9.3mg/ml in standard medium I. To strengthen the biosynthetic pathway for aromatic amino acids, 5-methyl-DL-tryptophan resistant mutants were derived from strain No. 7~ 12. Wederived mutants on minimal medium plates supplemented with 200 /ig/ml of 5-methyl-DL-tryptophan. Among methyl-DL-tryptophan resistant mutants, the best strain, T-222, accumulated 12.5 mg/ml of L-phenylalanine in the culture medium L-Phenylalanine accumulated (mg/ml) Fig. 3. Distribution of L-Phenylalanine Productivity in NTG Treated Mutants Derived from T-222. Each test strain was cultivated at 31 C in 3 ml of standard medium II in a test tube. After 72hr, the amount of l- phenylalanine accumulated in the culture broth was measured. The arrow indicates the amount of L-phenylalanine accumulated by parent strain T-222 under the same experimental conditions. Effects offumaric acid and other mutations on L-phenylalanine production As to the additive effects of organic acids on L-phenylalanine production, it was found that the addition of fumaric acid to the standard medium enhanced the productivity of l- phenylalanine. We investigated in detail the optimum concentration of fumaric acid added to the standard medium. The maximumproduction of L-phenylalanine was observed with 1.2~ 1.6% offumaric acid, as shown in Fig. 2.

5 i Fermentative Production of L-Phenylalanine 2099 ~z^*^z- ~20- ~ 80- >y S^0*^ Hi. W \. js en u \. y^ 15- di60- \ / _i c c P Xa ^" ^10- to40- y^-*^*"x à" à" à" -] q S5" 20- X/^ Nv " '5^ 01 L^^^ I I I I I^^-^^A Io Time in hours Fig. 4. Time Course of L-Phenylalanine Production. The cultivation was carried out at 31.5 C for 72hr in standard mediumii. O-O, L-phenylalanine productivity; à"-#, growth of strain 123; A-A, residual glucose; A-A, ph. L-Phenylalanine was produced at a concentration of 17.5mg/ml when the culture was performed in standard mediumii containing 13% of glucose and 1.2% of fumaric acid. In the course of improvement of L-phenylalanine producers, we observed that the morphology of colonies after nitrosoguanidine treatment grown on bouillon agar plates varied as to size, colour and shape. Based on the assumption that these colonies are different as to the ability of L-phenylalanine production, we further attempted to screen L-phenylalanine producers from among nitrosoguanidine-treated cells. The best producer, No. 123, accumulated 21.7mg/ml of l- phenylalanine in standard medium II. Figure 3 shows the L-phenylalanine productivity distribution of nitrosoguanidine-treated cells. Time course of L-phenylalanine production An example of the time course of fermentation by strain No. 123 is presented in Fig. 4. During the course of fermentation in standard medium II, the amount of L-phenylalanine increased in parallel with glucose consumption. From 13% of glucose, 21.7mg/ml of l- phenylalanine was finally obtained at 72hr cultivation. The amounts of other amino acids accu- Table III. The Amounts of Amino Acids Accumulated in the Culture Broth The fermentation was carried out at 31.5 C for 72 hr in standard medium II with strain 123. Amino acid analysis was carried out with an amino acid analyzer. mulated in the culture were negligible compared with that of the main product, L- phenylalanine: 2.5 mg of lysine, 0.7mg of alanine and 0.7 mg ofvaline per ml, and much less of other amino acids, as shownin Table III. The genealogy of strain No. 123, the finally selected mutant strain, and of the mutants which were employed as parent strains, is shown in Fig. 5, together with their L- phenylalanine productivities.

2100 T. Tsuchida et al. Fig. 5. The Process of Improvement ofl-phenylalanine Production. Each fermentation was carried out at 31 C for 72hr in fermentation medium II or III.

6 2100 T. Tsuchida et al. Fig. 5. The Process of Improvement ofl-phenylalanine Production. Each fermentation was carried out at 31 C for 72hr in fermentation medium II or III. ** Addition of acetic acid and fumaric acid. *2 Addition of an increased amount of L-tyrosine. *3 Addition of an increased amount of glucose and fumaric acid. *4 Nitrosoguanidine treatment. DISCUSSION The fermentative production of L-phenylalanine by auxotrophic mutants or analogue resistant mutants was reported in several papers.1~6) However, the L-phenylalanine productivities of these mutants were comparatively low, because of the long biosynthetic pathway and the complicated regulation mechanisms for aromatic amino acids. From an industrial point of view, it is important to improve the L-phenylalanine productivity from carbon sources. Wefound that the phenylalanine productivity of Brevibacterium lactofermentum No. 808, a tyrosine, methionine double auxotrophic mutant, could be improved in a stepwise manner by the successive addition of such resistant markers as DL-/>-fluorophenylalanine, DL-/?-3-thienylalanine, adenine and dl- 5-methyltryptophan. Nitrosoguanidine treatment and the addition of a large amount of glucose and fumaric acid were also effective for improvement of the L-phenylalanine production. The amount of L-phenylalanine produced from 13% glucose by the finally selected mutant strain, No. 123, reached 21.7mg/ml after 72 hr cultivation in a standard medium (Fig. 4), the yield thus being approximately 16.6% from glucose in weight. DL-/?-3-Thienylalanine resistant mutant strain 70-6, derived from Brevibacterium lactofermentum No. 808, accumulated 8.8 mg/ml of L-phenylalanine in the culture medium. When investigating the microbiological properties of DL-jS-3-thienylalanine, Dittmer found that it was a phenylalanine antagonist in Saccharomyces cerevisiae and EscheYichia coli.14) The authors found that DL-/?-3-thienylalanine was also a phenylalanine antagonist in Brevibacterium lactofermentaum 2256, and resistant mutants derived from No excreted 1.0~2.0mg/ml of L-phenylalanine and /or L-tyrosine. An adenine resistant mutant accumulated more L-phenylalanine than an adenine sensitive strain. The production of L-amino acids such as L-proline15) and L-leucine16) by Brevibacterium mutants has been reported to decrease on the addition of adenine. The effect of adenine on the amino acid biosynthesis will be described in a separate paper. DL-5-Methyltryptophan resistant mutant strain T-222 showed increased L-phenylalanine productivity compared with parent strain No This suggests that the L-phenylalanine production by strain No is controlled by L-tryptophan, which is known as an effector concerned in phenylalanine, tyrosine and tryptophan biosynthesis in many bacteria. L-Phenylalanine production was enhanced by the addition of fumaric acid to the standard medium. This stimulative effect of fumaric acid is under study using L-phenylalanine producers obtained. Acknowledgments. The authors are indebted to Drs. T. Tsunoda, T. Akashi, H. Okada and S. Okumura of this Company for their encouragement. REFERENCES 1) B. D. Davis, Experientia, 6, 41 (1950). 2) K. Nakayama, Z. Sato and S. Kinoshita, Nippon Nogeikagaku Kaishi, 35, 142 (1961). 3) Y. Tokoro, K. Oshima, M. Okii, K. Yamaguchi, K.

7 Fermentative Production of L-Phenylalanine 2101 Tanaka and S. Kinoshita, Acid, No. 21, 49 (1970). Amino Acid and Nucleic 4) E. A. Adelberg, J. Bacterial, 76, 326 (1958). 5) S. W. Kim and J. Pittard, /. Bacterial, 106, 784 (1971). 6) M. Polsinelli, Giorm. Microbiol, 13, 99 (1965). 7) J. H. Coats 4948 (1967). and E. W. Nester, J. Biol. Chern., 242, 8) D. H. Calhown and R. A. Jensen, J. Bacteriol, 109, 365 (1972). 9) S. Okumura, S. Otsuka, A. Yamanoi, F. Yoshinaga, T. Honda, K. Kubota 3,660,235 (1972). and T. Tsuchida, U. S. Patent H. Hagino and K. Nakayama, Agric. Biol. Chem., 38, 157 (1974). S. Okumura, R. Tsugawa, K. Kono, T. Matsui and N. Miyachi, Agric. Biol. Chem., 36, 141 (1962). N. Schoorl, Arch. Suikerind., 24, 1967 (1916). A. Eisenstak, R. Eisenstark and R. Van Sickle, Mutation Res., 2, 1 (1965). J. Dittmer, /. Amer. Chem. Soc, 71, 1205 (1949). F. Yoshinaga, Y. Yoshihara, S. Okumura and N. Katsuya, J. Gen. Appl. Microbiol, 13, 25 (1967). T. Tsuchida, F. Yoshinaga, K. Kubota, H. Momose and S. Okumura, Agric. Biol. Chem., 38, 1907 (1974).

Microbial Production of L-Threonine. Part III. Production by Methionine and Lysine Auxotrophs. Derived from ƒ -Amino-ƒÀ-hydroxyvaleric Acid Resistant

I [Agr. Biol. Chem., Vol. 36, No. 7, p. 12091216, 1972] Microbial Production of L-Threonine Part III. Production by Methionine and Lysine Auxotrophs Derived from ƒ -Amino-ƒÀ-hydroxyvaleric Acid Resistant