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

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1 I [Agr. Biol. Chem., Vol. 36, No. 7, p , 1972] Microbial Production of L-Threonine Part III. Production by Methionine and Lysine Auxotrophs Derived from ƒ -Amino-ƒÀ-hydroxyvaleric Acid Resistant Mutants of Brevibaclerium flavum By Shigeru NAKAMORI and Isamu SHIIO Central Research Laboratories of Ajinomoto Co., Inc., Kawasaki, Japan Received December 22, 1971 L-Threonine production by strain BB-69, which was derived from Brevibacterium flavum No as a ƒ -amino-ƒà-hydroxyvaleric acid resistant mutant and produced about 12 g/liter of L-threonine, was reduced by the addition of L-lysine or L-methionine in the culture medium. Many of lysine auxotrophs but not methionine auxotrophs derived from strain B-2, which produced about 7g/liter of L-threonine, produced more L-threonine than the parental strain. Except only one methionine auxotroph (BBM-2l), none of lysine and methionine auxotrophs derived from BB-69 produced more L-threonine than the parental strain. Homoserine dehydrogenase of crude extract from strain B-2 was inhibited by L- threonine more strongly than that from BB-69. Strain BBM-21, a methionine auxotroph derived from BB-69, produced about 18g/liter of L-threonine, 500 more than BB-69, while accumulation of homoserine decreased remarkably as compared with BB-69. L-Threonine production by BBM-21 was increased by the addition of L-homoserine, a precursor of L- threonine, while that by BB-69 was not. No difference was found among BBM-21, BB-69 and No in the degree of inhibition of homoserine kinase by L-threonine. L-Threonine production by revertants of BBM-21, that is, mutants which could grow without methionine, were all lower than that of BBM-21. Correlation between L-threonine production and methionine or lysine auxotrophy was discussed. In the previous papers, microbial production of L-threonine has been reported with ƒ -aminoƒà-hydroxyvaleric acid resistant mutants of Escherichia coli, Brevibacterium flavum and Coryne bacterium acetoacidophilum. In E. coli, about 2 g/liter of L-threonine were accumulated with AHV resistant mutants, and the amount of L-threonine increased to about 4.7, 3.8 and 6 g/liter with isoleucine, methionine, and iso leucine plus methionine auxotrophs derived from the resistant strains, respectively. On the other hand, g/liter of L-threonine were The abbreviations used through this paper include ƒ -amino-ƒà-hydroxyvaleric acid; AHV, N-methyl-N Lnitrosoguanidine; NG. accumulated by methionine and methionine plus isoleucine auxotrophs derived from AHV sensitive strains, and these accumulations of L-threonine were discussed from the view of regulatory mechanisms of L-threonine biosyn thesis in E. coli.1) AHV resistant mutants from glutamate producing bacteria produced larger amounts of L-threonine than those from E. coli, and the best producer BB-69 derived from Br. flavum accumulated about 13 g/liter, and C-553 derived from C. acetoacidophilum did about 6 g/liter, respectively, in media containing 100/ glucose, inorganic salts, and some other com ponents." Moreover, homoserine dehydro genase of strain BB-69 has been found to be

2 1210 S. NAKAMORI and I. SHIIO different from that of the parental strain in the sensitivity to feedback inhibition by L- threonine; the enzyme from BB-69 was about 1,000 fold insensitive to feedback inhibition by L-threonine as compared with that from the parental strain." Therefore, L-threonine biosynthesis in Br. flavum seems to be controlled mainly through feedback inhibition of homoserine dehydro genase by L-threonine. However, since feedback inhibition of aspartokinase by L-threonine plus L-lysine, and repression of homoserine dehydrogenase and homoserine kinase by L- methionine,4,5) have also been found, it is likely that lysine and methionine may affect the production of L-threonine. Thus, for the better production of L-threonine it seems useful to remove these effects, if present. The present paper deals with effects of these amino acids, the derivation of lysine and methionine auxotrophs from the AHV resistant strains, various conditions for L-threonine pro duction by these mutants, and with some properties of L-threonine biosynthetic enzymes of the mutants. microbioassay using Streptococcus faecalis (L-threonine), Leuconostoc mesenteroides (L-isoleucine, L-valine and L- leucine), and Leuconostoc citrovorum (L-proline and DLalanine). Homoserine was estimated by colorimetric determination at 570 mis of ethanol solution of homo serine spot on paper chromatograms obtained as described in the previous paper.2) Enzyme assay. Crude enzyme solutions were pre pared by the same methods as described previously6) from cells cultured in media T-2 or T-2 containing L-methionine at 30 Ž for 40 hr. Assay of homoserine dehydrogenase and homoserine kinase were the same as described previously.6,7) Selection of lysine and methionine auxotrophs. Cells of strain B-2 or BB-69 of late logarithmic phase cul tured in media 1 were contacted with 2 mg/ml of NG dissolved in 0.1M K-phosphate buffer (ph 7.0) for 10 to 20 min in ice baths. After washing twice with the buffer, cells were spread onto medium 1 plate and cultured at 30 C for 3 to 4 days. Appeared colonies were replicated onto medium 2 and medium 2 con taining 200ƒÊ g/ml of L-lysine and L-methionine, respec tively. Lysine and methionine auxotrophs were selected from the colonies which could grow on the latter medium but not on the former, and used for further studies. MATERIALS AND METHODS Bacterial strains. Brevibacterium flavum BB-69 and B-2, which were L-threonine producers derived from Br. favum No as AHV resistant mutants by treat ment with N-methyl-N L-nitro-N-nitrosoguanidine.2) Chemicals. NG was the product of Aldrich Chem. Corp., and all amino acids except DL-diaminopimelic acid were the products of Ajinomoto Co. Inc. DL- Diaminopimelic acid was purchased from Cyclo Chemical Corp. Culture media. Medium 1, medium 2 and medium T-2 were described previously.2) Amino acids were supplemented to medium 2 or medium T-2 at final concentrations 100 to 1,000ƒÊg/ml, when required. RESULTS Effect of amino acids on the production of L- threonine Strain BB-69 was cultured in medium T-2 containing 500 or 1,000ƒÊg/ml of amino acids to examine effects of amino acids on the L- threonine production. No effect was seen by the addition of amino acids except L-methionine and L-lysine (Table I). At high concentrations of L-methionine and L-lysine, the levels of L-threonine accumula tions were reduced to two-third or a half, as compared with no additions. Culture conditions. Culture conditions were the same as described in the previous paper.2) Determination of amino acids. Determination of amino acids, except homoserine, was conducted by Derivation of lysine and methionine auxotrophs from AHV resistant strains Since additions of L-lysine and L-methionine reduced L-threonine accumulation by strain

3 Microbial Production of L-Threonine. Part TABLE I. EFFECT OF AMINO ACIDS ON L-THREONINE PRODUCTION BY STRAIN BB-69 A loopful of BB-69 cells grown on medium 1 agar slant for 2 days at 30 C was inoculated into 20 ml of media T-2 containing various concentrations of amino acids, and amounts of L-threonine in broth were determined after 72 hr cultivation on a shaker. FIG. 1. Distribution of Lysine and Methionine Auxotrophs Derived from AHV Resistant Mutants with Respect to L-Threonine Production. Lysine auxotrophs derived from B-2 (a) and BB-69 (b), and methionine auxotrophs from B-2 (c) and BB-69 (d) were cultured in media T-2 containing g,ml of L-lysine.HCl and 200 Đg/ml of L-methionine, respectively, as the same methods in Table I. The arrows indicate the amounts of L-threonine produced by parental strains B-2 (a, c) and BB-69 (b, d). methionine auxotrophs derived from B-2 a) DL-Diaminopimelic acid. BB-69, methionine and lysine auxotrophs were derived from AHV resistant strains in order to culture L-threonine producing strains under limiting conditions of L-lysine and L-me thionine. Sixteen lysine auxotrophs and 30 methionine auxotrophs from strain B-2, and 5 lysine auxotrophs and 76 methionine auxo trophs from BB-69 were isolated. All these mutants were examined for their L-threonine productivities using medium T-2 containing 500 Đg/ml of L-lysine. HCl, or 200 Đg/ml of L- methionine, and distribution of these auxo trophs with respect to their L-threonine pro duction was shown in Fig. 1. Many of lysine auxotrophs but none of showed higher yields of L-threonine than the parental strain, and the best producer, BL-14, accumulated 10.4 g/liter of L-threonine. On the other hand, all the mutants from strain BB-69, except only one methionine auxotroph, showed lower levels of L-threonine production than the parental strain. Comparison of homoserine dehydrogenase from B-2 with that from BB-69 Difference was observed between lysine auxotrophs derived from B-2 and BB-69 in the production of L-threonine, that is, many of lysine auxotrophs from B-2 but none from BB-69 produced more L-threonine than the parental strains. In order to elucidate the dif ference, some properties of homoserine dehy-

4 1212 S. NAKAMORI and I. SHIIO TABLE II. COMPARISON OF PROPERTIES OF HOMO SERINE DEHYDROGENASE FROM STRAIN No. 2247, BB-69 AND B-2 Preparations and assays of enzymes were conducted as described in Methods. Enzyme levels were expressed as ƒêmoles of NADPH oxidized/min/mg of protein. L-Threonine production by a methionine auxotroph derived from BB-69 One of methionine auxotrophs from BB-69, strain BBM-21, accumulated more L-threonine than the parental strain when cultured in medium T-2 containing 200ƒÊg/ml of L-me thionine. Since the growth of this strain responded also to cystathionine, an early step of methionine biosynthesis might be blocked in the strain. This strain was cultured further at various concentrations of L-methionine to determine the optimum amount of L-me thionine for the better production of L-threo nine. As shown in Table III, the maximum production of L-threonine (17.5 g/liter), which was 50 o more than that with the parental strain, was obtained when the initial concen tration of L-methionine was 750-1,000 ƒêg/ml, and further addition of L-methionine decreased the level of L-threonine. The maximum drogenase from both strains were investigated. Crude enzyme solutions were prepared from the cells cultured in medium T-2 for 40 hr at 30 C, and assayed as described in Methods. As shown in Table II, although no dif ference was found in the enzyme levels of crude extracts between B-2 and BB-69, degree of inhibition of the enzyme by L-threonine was lower in enzyme from BB-69 than that from B-2. Thus, the reactivity of homoserine dehydrogenase of BB-69 may be better than that of B-2 under L-threonine producing conditions. growth was obtained at more than 250ƒÊg/ml of L-methionine. Other methionine auxo trophs derived from BB-69 were cultured in medium T-2 containing 1,000 tig/ml of L- methionine, but none produced more L-threo nine than the parental strain. Accumulation of other amino acids by strain BBM- 21 Various amino acids were accumulated by strain BBM-21 when cultured in media T-2 containing various concentrations of L-me thionine. These amounts were compared with TABLE III. ACCUMULATION OF AMINO ACIDS BY STRAIN BBM-21 AND BB-69 BBM-21 and BB-69 were cultured in media T-2 containing various concentrations of L-methionine, and amino acids in culture broth were determined by microbioassay as described in Methods.

5 Microbial Production of L-Threonine. Part III 1213 those from BB-69 cultured in medium T-2 (Table III). When BBM-21 was cultured with addition of 1,000ƒÊg/ml of L-methionine, L- isoleucine increased, while homoserine decreased remarkably in comparison with those with the parental strain. L-Proline was newly accumulated by the methionine auxotroph. Effect of aspartate related compounds on L-threonine production by BB-69 and BB-21 Strain BB-69 and BBM-21 were cultured by adding various concentrations of aspartate related compounds to medium T-2 or T-2 containing 1,000 tag/ml of L-methionine to in vestigate the effect of these compounds on the accumulation of L-threonine. Results shown in Fig. 2 indicated that addition of fumarate, L-aspartate and L-threonine had no effect, and of L-lysine had reductive effects in both cases. Addition of L-homoserine had also no effect in BB-69, while it resulted in the increase of L-threonine accumulation in BBM-21. Properties of homoserine kinase of strain BBM-21 Since homoserine accumulation was smaller in BBM-21 than BB-69, and larger part of L- homoserine added was converted to L-threonine by BBM-21 than by BB-69, the increase of L-threonine may be accounted for by higher activities of L-threonine synthetic enzymes in BBM-21 as compared with BB-69. Thus, the activities of homoserine kinase of both strains at various conditions were examined. Sonic extracts were prepared from the cells of Br. flavum No. 2247, BB-69 and BBM-21 cultured at 30 C for 40hr in media T-2 containing various levels of L-methionine, and assayed for homoserine kinase. The results shown in Tables IV and V in dicated that, unexpectedly, no difference was observed in the levels of inhibition of homo serine kinase by L-threonine among three strains, and further, no remarkable repression of homoserine kinase by L-methionine was observed with all the strains under these culture conditions. FIG. 2. Effect of Aspartate-related Compounds on the Productions of L-Threonine by Strain BB-69 and BBM-21. Aspartate-related compounds (a-e) were added to medium T-2 in the case of BB-69, and to medium T-2 containing 1.0 mg/ml of L-methionine in the case of BBM-21. Other conditions were same as described in Table I. - BB-69, O-O BBM-21.

6 1214 S. NAKAMORI and I. SHIM TABLE IV. INHIBITION OF HOMOSERINE DEHY DROGENASE BY L-THREONINE Preparation and assay of the enzyme were con ducted as described in the text and Methods. Specific activity was expressed as Đmoles of NADH oxidized/ min/mg of protein. FIG. 3. Distribution of Revertants of BBM-21 with Respect to L-Threonine Production. Thirty-two revertants isolated were cultured in medium T-2 at 30 C for 66 hr and the amounts of L-threonine were determined. The arrows show the amounts of L-threonine produced by the parental strains, BBM-21, - BB-69. TABLE V. REPRESSION OF HOMOSERINE KINASE BY L-METHIONINE Preparation and assay of the enzyme were con ducted as described in the text and Methods. Specific activity was expressed as Đmoles of NADH oxidized/ min/mg of protein. derived, and were cultured in medium T-2 to know whether methionine auxotrophy had an important role in the higher L-threonine production by BBM-21 than by BB-69. Spontaneous revertants were selected on medium 2 plate by spreading the cells of BBM-21 which had been cultured overnight in medium 1 at 30 C and washed twice with 0.1M k-phosphate buffer (ph 7.0) by centrifuga tion. After culturing the plates for 5 days at 30 C, appeared colonies were picked up and cultured in medium T-2. As shown in Fig. 3, the levels of L-threonine production by these revertants were all lower than that of BBM-21, and that of BB-69. These results indicated that methionine auxo trophy may be correlated with the higher production of L-threonine in strain BBM-21. DISCUSSION L-Threonine production by revertants of strain BBM-21 Revertants of strain BBM-21, i.e. mutants which could grow without L-methionine were L-Threonine producing strains were obtained from glutamate producing bacteria as AHV resistant mutants," among which strain BB- 69 from Br. flavum No was selected as

7 Microbial Production of L-Threonine. Part III 1215 the best producer. These accumulations were explained by the genetical desensitization of homoserine dehydrogenase to the feedback inhibition by L-threonine, which might control L-threonine biosynthesis in the wild type strain.3) Trials for the better production of L-threonine than that by the parental strains have been made. In Br. flavum, feedback inhibition of aspartokinase by L-threonine plus L-lysine, and repression of homoserine dehydrogenase and homoserine kinase by L-methionine had been found."" In the present study, it was shown that L-threonine production by strain BB-69 was reduced by the addition of L-lysine or L- methionine. Thus, for the better production of L-threonine, it was expected to be useful to derive lysine or methionine auxotrophs from AHV resistant-threonine producing strains, and to culture them under limiting conditions of these amino acids, to remove these negative effects of L-lysine or L-methionine. Actually, many of lysine auxotrophs but none of methionine auxotrophs derived from B-2 produced larger amount of L-threonine (maximally 10 g/liter) than the parental strain, which produced about 7 g/liter of L-threonine. On the other hand, no better producer of L- threonine than the parental strain was found in auxotrophs from BB-69 except only one methionine auxotroph (BBM-21). Strain BBM-21 produced about 18 g/liter of L-threonine, 5000 more than BB-69, while ac cumulation of homoserine, a precursor of L- threonine, decreased remarkably as compared with those of BB-69. Moreover, L-threonine production of BBM-21 increased by the addi tion of L-homoserine, while that of BB-69 was not. These results suggested that the enzymes which converted L-homoserine to L-threonine might be activated in BBM-21 more than in BB-69. Thus, homoserine kinase of crude extracts from these strains at various condi tions was investigated. As to inhibition of the enzyme by L-threonine, no difference was found among BBM-21, BB-69 and No It had already been shown that threonine synthetase, which catalyzed the formation of L-threonine from phosphohomoserine, was not inhibited by L-threonine in Br. flavum. Thus, the enzymatic alteration, which correlated with the increase of L-threonine production by strain BBM-21, could not be found. Difference of L-threonine production be tween BB-69 and BBM-21 may be interpreted by the difference in the transport systems of L-homoserine. In strain BBM-21, ability to maintain or incorporate L-homoserine may be larger than that of BB-69. Accumulation of homoserine by BB-69 may be explained by lesser ability to maintain homoserine inside the cells than that of BBM-21. No increase of L-threonine by the addition of L-homoserine in BB-69 may be explained by lesser ability to incorporate exogenous L-homoserine into the cells than in BBM-21. These characteristics may be related with methionine auxotrophy in BBM-21. Many of lysine auxotrophs derived from B-2 produced more L-threonine than the parental strain, while none from BB-69 did. This difference might be interpreted by the following presumptions. From the results shown in Table II, homoserine dehydrogenase from BB-69 seems to have better reactivity than that from B-2 under L-threonine produc ing conditions. As shown previously," in Br. flavum, the level of homoserine dehydrogenase in the cell extracts was 15 fold larger than di hydrodipicolinate synthetase, which catalyzed conversion of the same substrate, aspartate-Ĉsemialdehyde, to L-lysine. Although this dif ference would be controlled by feedback in hibition of homoserine dehydrogenase by L- threonine, and adequate L-lysine would be supplied in the parental strain, this might result in a limiting supply of L-lysine in BB- 69, in which homoserine dehydrogenase was desensitized to feedback inhibition by L- threonine.s" Thus, limitation of L-lysine by using lysine auxotrophs may have no further

8 1216 S. NAKAMORI and I. SHIIO effect on L-threonine production, though L- threonine production was reduced by the ad dition of excess L-lysine, mainly through feedback inhibition of aspartokinase by L-lysine plus L-threonine. On the other hand, in strain B-2, as homoserine dehydrogenase was "par tially" desensitized to feedback inhibition, L- lysine would be supplied adequately enough to partially inhibit aspartokinase, and therefore, lysine auxotrophs would produce more L-threonine than the parental strain by limiting the amount of L-lysine. No better producer of L-threonine than the parental strains was found in all of methionine auxotrophs derived from B-2, and in many of those from BB-69. Presumably, pool of L- methionine in the cells would not be so large as to repress homoserine dehydrogenase, a common enzyme for the synthesis of L-me thionine, L-threonine and L-isoleucine. Therefore, limitation of L-methionine by using me thionine auxotrophs would have no effect on L-threonine production, though L-threonine production was reduced by the addition of excess amount of L-methionine, mainly through repression of homoserine dehydrogenase by L - methionine. Since all of methionine revertants of BBM-21 produced lower levels of L-threonine than the parental strain, methionine auxo trophy had an important role in the produc tion of L-threonine in strain BBM-21, presuma bly because of the changes in the transport system of L-homoserine. Acknowledgement. The authors indebted to Director Dr. T. Tsunoda and Assistant Director Dr. M. Taka hashi of the laboratories for their encouragements. They also wish to thank Mr. Z. Terahara and Miss K. Furuya for the skillful technical assistance. REFERENCES 1) I. Shiio and S. Nakamori, Agr. Biol. Chem., 33, 1152 (1969). 2) I. Shiio and S. Nakamori, ibid., 34, 448 (1970). 3) I. Shiio, R. Miyajima and. S. Nakamori, J. Biochem., 68, 859 (1970). 4) I. Shiio and R. Miyajima, ibid., 65, 849 (1969). 5) R. Miyajima and I. Shiio, Agr. Biol. Chem., 35, 424 (1971). 6) R. Miyajima, S. Otsuka and I. Shiio, J. Biocheṃ, 63, 139 (1968). 7) R. Miyajima and 1. Shiio, ibid., 71, 219 (1972). 8) R. Miyajima and I. Shiio, ibid., 68, 311 (1970).