MICROBIAL PRODUCTION OF L-LYSINE

Transcription

1 J. Gen. Appl. Microbiol., 17, (1971) MICROBIAL PRODUCTION OF L-LYSINE IV. SELECTION OF LYSINE-PRODUCING MUTANTS FROM BRE VIBACTERI UM FLA V UM BY DETECTING THREONINE SENSITIVITY OR HALO-FORMING METHOD KONOSUKE SANG AND ISAMU SHIIO Central Research Laboratories, Ajinomoto Co., Inc., Kawasaki, Japan (Received June 23, 1970) Mutants sensitive to fairly low concentration of threonine or methionine were derived from Brevibacterium flavum No (ATCC 14067). These mutants were classified into three by the growth inhibition by threonine or methionine ; type A was inhibited only by threonine, type B only by methionine, and type C by both threonine and methionine. Further, every type of mutants was divided into two subtypes by their growth response ; one whose growth was inhibited by threonine (or methionine) was to be recovered in the coexistence of methionine (or threonine), and the other could not be recovered. Among these six types of mutants, one whose growth was inhibited by either methionine or threonine but could be recovered by threonine or methionine, respectively, was found to produce much lysine. This character is similar to that of the threonine-sensitive and methionine-sensitive revertant derived from a homoserine auxotroph lacking homoserine dehydrogenase activity. Homoserine dehydrogenase level of these revertants and the above-mentioned lysine-producing mutants were similar, being about 3% of the original strain, No. 2247, but a little difference was observed in the minimal inhibiting concentration of threonine or methionine and the kind of agents diminishing the inhibition by threonine or methionine. Furthermore, about one-half of threonine auxotrophs which were derived from the above-mentioned lysine-producing mutants sensitive to either threonine or methionine were found to produce larger amounts of lysine than the above parental mutants. A modified microbiological assay procedure using lysine auxotroph was introduced to isolate non-auxotrophic lysine-producing mutants directly from No However, no mutants other than the two known types of lysineproducer (threonine-sensitive, methionine-sensitive mutants and S-(2-aminoethyl)-L-cysteine-resistant mutants) were found through this procedure. 97

2 98 SANG AND SHIIO VOL. 17 The preceding paper (1) reported that some revertants having a low level of homoserine dehydrogenase [EC ] (HDH), derived from a homoserine auxotroph of Brcvibaeterium flavum, produced much lysine. Although these revertants grew in a minimal medium as well as the original strain they unexpectedly accumulated a large amount of lysine. It was also found that these revertants had a unique character of being sensitive in growth to threonine or methionine. In other words, their growth on a minimal medium was more strongly inhibited than that of the original wild strain No by the addition of threonine or methionine. Therefore, it will be possible to isolate such mutants directly from the wild strain. This paper describes the isolation of such sensitive mutants and their characters in comparison with those of the revertants. It is also considered to be quite possible to isolate all the lysine-producing mutants in this condition by the halo-forming method. Some new mutants, which have never been isolated because of their unknown characters, could be expected to be obtained by this method. It is concluded, however, that lysine-producing non-auxotrophic mutant other than the threonine-sensitive, methionine-sensitive mutants or S-(2-aminoethyl)-L-cysteine-resistant (AECresistant) mutants might be difficult to isolate. MATERIALS AND METHODS Strains. Brevibacterium flavum No (ATCC 14067) was the parental strain of threonine-sensitive, methionine-sensitive mutants. D 5-6, one of the typical threonine-sensitive, methionine-sensitive and lysine-producing mutants was the parental strain of threonine auxotrophs. H-1013 (h~ moserine auxotroph), T-36 (threonine auxotroph), M-78 (methionine auxotroph), S-20 (threonine-sensitive, methionine-sensitive, and lysine-producing revertant of H-1013), and FA 1-30 and FA (AEC-resistant lysine producer) were all mutants of No No was also used as the parental strain in the experiment to isolate lysine-producing mutants by the modified microbiological assay procedure, using Lys-1, a lysine auxotroph of No. 2247, as the indicator strain. Media. The following media were employed. Medium A : 10 g polypeptone, 10 g yeast extract, 5 g NaCI, and 1,000 ml tap water, adjusted to ph 7.2. Medium B: 5 g glucose, 1.5 g (NH4)2SO4, 1.5 g urea, 1 g KH2PO4, 3 g K2HPO4, 0.1 g Mg5O4.7 H2O, g CaCl2. 2 H2O, 30 ig d-biotin, 100 jig thiamine HCI, 1 ml trace element solution, and 1,000 ml distilled water. The ph was 7.0 after sterilization. The trace element solution consisted of 8,800 mg ZnSO4 H2O, 970 mg FeCl3.6H2O, 393 mg CuSO4.5H2O, 88 mg Na2B4O 7H2O, 72 mg MnCl2.4H2O, 37 mg (NH4)6Mo7O2 H2O, and 1,000 ml distilled water. Medium B-1 was made up of medium B and 1% growth factor solution to promote the growth of BreJibacterium. The growth factor solution was a supernatant from a 48-hr culture broth in which Bacillus megaterium sp. (AJ

3 1971 L-Lysine Production by Mutants , IAM 1030) had been grown. The medium used for this bacterium was medium B. The supernatant contained no detectable amino acids or nucleic acids, but promoted the growth of No very much. Medium C : 100 g glucose, 40 g (NH4)2S04i 1 g KH2P04, 0.4 g MgSO4.7H20,. 10 mg FeSO4.7H20, 7 mg MnCl2.4H20, 300,ug d-biotin, 200 ~cg thiamine HCI, 2 ml Mieki (amino acid mixture), 600 mg L-threonine, 200 mg L-methionine, 50 g CaCO3, and 1,000 ml distilled water. The ph was 7.5 after sterilization. Medium C-1: Both L-threonine and L-methionine omitted from medium C. Medium C-2: L-Threonine and L-methionine, and Mieki omitted from medium C. Medium D : 36 g glucose, 10 g urea, 1 g KH2P04, 0.4 g MgS04.7H20, 10 mg FeSO4. 7H20, 7 mg MnCl2.4H20, 30 jig d-biotin, 100 pg thiamine HCI, 1 ml Mieki, and 1,000 ml distilled water. The ph was 6.8 after sterilization. Medium D-1 consisted of medium D and 0.3 g L-homoserine. This concentration of L-homoserine was one-half of that necessary for the maximal growth of H Isolation of mutants. Intermediate culture after mutagenesis to stabilize the mutation was omitted to obtain mutually independent mutants. Exponentially growing B. flavum No cells in medium A were washed by centrifugation, resuspended in 0.1 M phosphate buffer (ph 7.0), and treated with 2 mg/ml of N-methyl-N-nitro-N'-nitrosoguanidine (NG) at 0 for 30 mils, with 0.5 M ethylmethanesulfonate (EMS) at 30 for 40 min with shaking, or with mg/ml diethyl sulfate (DES) at 30 for 45 min with shaking. The cells were then washed and inoculated on a solid medium A supplemented with 0.2% glucose after appropriate dilution. After incubation for 3-4 days, colonies that appeared were replicated onto media B-1 and B-1 supplemented with L-threonine (or L-methionine), then various sensitive mutants were isolated by the screening method shown in Table 1. In the microbiological assay method (2), colonies were stamped onto medium B-1 on which lysine auxotroph Lys-1 had been previously spread over, and incubated for 1-2 days at 30, and halo-formers were picked up. When the S-(2-aminoethyl)-L-cysteine-resistant mutants (3) were employed as models, the coincidence of halo-forming ability and lysine-producing ability was not so strict (Fig. 1). Cylinder-plate method was used to test the sensitivity to threonine or methionine (1). Other conditions. The assay procedure of growth inhibition and restoration, lysine-producing ability, and enzyme activity were the same as described in the previous paper (1, 3). Alanine, valine, and proline were estimated by microbioassay. RESULTS Isolation of threonine- or methionine-sensitive mutants Replica method was applied to NG-treated 19,000 colonies and grouping

4 100 SANO AND SHIIO VOL. 17 Table 1. Selection method of threonine- or methionine-sensitive mutants. Expected growth response of various mutants on replicated plates is shown. The symbol + or - indicates that growth can or cannot be seen. Abbreviations : Ts ; threonine-sensitive, M ; methionine-sensitive, thr- ; threonine auxotroph, met- ; methionine auxotroph, hse- ; homoserine auxotroph. of the mutants was done according to the type shown in Table 1. Ninetythree mutants were obtained, named D2-group, and divided into six types as shown in Table 1; Type 1 (14 strains), Type 2 (21 strains), Type 3 (2 strains), Type 4 (11 strains), Type 5 (19 strains), and Type 6 (26 strains). A modified microbiological assay using lysine auxotroph was applied and 35 large halo-formers were obtained from 21,000 colonies as shown in Table 2. Twenty of the large halo-formers were Type 1. The remainder was named X-type mutants and used in further investigations. Lysine production by sensitive mutants Lysine-producing abilities of D2-group mutants were estimated by cultivation in 3 ml of medium C-1 at 30 for 72 hr. The result is shown in Fig. 2. All the five strains which produced more than 5 g/liter of lysine monohydrochloride belonged to Type 1, sensitive to threonine and sensitive to methionine, and their growth was restored by the coexistence of threonine and methionine. Maximum amount of lysine monohydrochloride produced was 17.2 g/liter which is 10 times more than that of No (1.5 g,/liter).

5 971 L-Lysine Production by Mutants 101 Fig. 1. Relationship between the amount of lysine produced and the diameter of halo. AEC-resistant mutants, which were selected as a lysine analog (AEC) resistant mutants and some of which were reported as good producers of lysine (3), were cultivated for 72 hr in 20 ml of medium C-1 or 48 hr on medium B-1 plate with lysine auxotroph. The most powerful lysine producer of threonine-sensitive, methioninesensitive mutant was D5-6 which accumulated 25 g/liter of lysine monohydrochloride in 20 ml of medium C-1 in 72-hr cultivation. In Fig. 3, time course of lysine production by D5-6 in minimal medium C-2 is shown. Properties of the sensitive mutants Mutant strain D5-6 was able to grow very well on minimal medium, as shown in Fig. 4. The growth was inhibited by the addition of L-methionine and, somewhat to a lesser degree, by L-threonine (Fig. 5, A, B). L-Serine or L-isoleucine inhibited the growth to some extent but L-valine and L-leucine did not (Fig. 5, C, D, E, F). Low concentration of L-methionine (50 pg/ml) or L-threonine (50 pg/ml) completely restored the growth of D5-6 from the inhibition by 100 beg/ml of L-threonine or L-methionine. These behaviors were quite similar to those of strain S-20, a revertant from homoserine-auxotrophic strain H-1013 (1).

6 102 SANO AND SHIIO VOL. 17' ability After by Table 2. Isolation of lysine-producing mutants directly No. 2247, the wild strain. mutagenesis, colonies that appeared were tested for the modified microbiological assay. from lysine-producing Fig. 2. Production of lysine by threonine- and/or methionine-sensitive mutants. Cultivation was carried out at 30 for 72 hr in 3 ml of medium C-1. Lysine was estimated by colorimetry (6). Typing was done as described in Table 1. Then examination was made on other amino acids, nucleic acid constituents,, and vitamins by the cylinder-plate method to see whether they can restore the growth inhibited by threonine or by methionine. Experimental conditions were the same as previously described (1). In liquid medium B4, growth inhibition of D5-6 by 100 pg/ml of L-threonine was restored by 25% in the

7 1971 L-Lysine Production by Mutants 103 Fig. 3. Time course of lysine production in minimal medium by sensitive, methionine-sensitive mutant, D5-6. Twenty ml of medium C-2 was seeded, dispensed in a shake cultivated aerobically at 30. At appropriate time intervals, samples and assayed. L-Lysine HCl was estimated by the enzymic method. Lysine. HCI, -- x-- O.D., ---A-- ph. threonineflask, and were taken -0- L- presence of 25 pg/ml of niacin. Niacin also partially restored the growth inhibited by 100 pg/ml of L-threonine. HDH activities of three threonine-sensitive, methionine-sensitive mutants were assayed by the previously described method (1). As shown in Table 3, the activities of the mutants were all about 3 % of No The activities were very low, but their presence was evident from the fact that the activities decreased further by heat treatment or addition of threonine (Table 4).

8 104 SANG AND SHIIO VOL. 17 Fig. 4. Effect of homoserine on the growth of threonine-sensitive, methioninesensitive mutant. Cells for inoculation were at late log phase growing in complete medium A supplemented with 0.2% glucose. Then 4 ml of medium B-1 was seeded with these washed cells at the concentration of about 4x107 cells/ml. They were cultivated aerobically at 30 and its turbidity estimated. -- x -- no addition of homoserine, pg/ml L-homoserine added. (A) No. 2247, (B) H-1013 (homoserine auxotroph), (C) D5-6 (threonine-sensitive, methionine-sensitive mutant). Derivation of threonine auxotrophs from the threonine-sensitive strain, and characters of auxotrophs Threonine auxotrophs were derived from D5-6. From 7700 diethyl sulfatetreated colonies, 29 threonine auxotrophs were selected by the replica method. During a 72-hr cultivation in medium C, the threonine auxotrophs accumulated L-lysine monohydrochloride as shown in Fig. 6. The distribution pattern of the lysine production is similar to that of the threonine auxotrophs of S-20 or S-13 (1). Maximum and average amounts of L-lysine monohydrochloride were 35.0 and 21.3 g/liter, respectively. Parental strain D5-6 produced 25.4 g/liter. Effect of L-threonine concentration on the growth of DT-16-1, a typical strain, was studied and is shown in Fig. 7. The unique pattern of threoninesensitive and threonine-requiring mutant can be seen.

9 1971 L-Lysine Production by Mutants 105 Fig. 5. Growth inhibition by various amino acids. Conditions were the same as indicated in Fig. 4. Initial cell concentration was 2 x 10, ml D5--6 at 28 hr (estimated time), -- x -- No at 16 hr. (A) L-methionine, (B) L-threonine, (C) L-isoleucine, (D) L-serine, (E) L-valine, (F) L-leucine.

10 106 SANO AND Smio VOL. 17 Table 3. HDH activity of threonine or methionine sensitive mutant. Experimental procedure was the same as described in the previous paper (1). Table 4. Effect of heat treatment and L-threonine on HDH of D5-6, a threonine- or methionine-sensitive mutant. Experimental procedure was the same as described in the previous paper (1). Prototrophic lysine producers other than the sensitive mutants As shown in Table 2, about one-half of prototrophic lysine producers selected by the modified microbiological assay were not sensitive to threonine or methionine and were named X-type mutants. Some characters of these 15 X-type mutants were studied in comparison with AEC-resistant prototrophic lysine poducers which had been reported (3). The amount of lysine accumulated in medium C-1 during 72-hr cultivation is shown in the second column of Table 5. Relationship between the diameter of halo in the modified microbiological assay method and the amount of lysine accumulated in medium C-1 are shown in Fig, 8. Unexpectedly close correlation was observed. The effect of L-threonine on lysine accumulation was investigated andd the result is shown in the third column of Table 5. From these data, X- type mutants were divided into two groups. Lysine production of the first

11 1971 L-Lysine Production by Mutants 107 Fig. 6. Lysine production by the threonine auxotrophs derived from the threonine-sensitive, methionine-sensitive mutant, D5-6. Experimental conditions were the same as in Fig. 3. Medium C was used. Fig. 7. Growth response of the threonine-sensitive, threoninerequiring mutant, DT-16-1, to L-threonine. Cells grown on medium A supplemented with 0.2% glucose for 24 hr were seeded at the concentration of about 2 x 108 cells/ml. Other conditions were the same as in Fig. 4. T-36 (threonine auxotroph) was used as control.

12 108 SANG AND SHIIO VOL. 17 Table 5. Lysine production by X-type mutants in medium plus L-threonine. C-1 or C-1 Experimental conditions were the same as described in Fig. 3. group was scarcely affected by the addition of threonine but that of the second was strongly affected. The mutant strains X-15 and X-3 were selected as the representatives of each group and used in further investigations. Four weak producers of lysine were omitted from this grouping. The effect of threonine on lysine production by X-15 whose lysineproducing ability was unaffected by 1.5 mg/ml of L-threonine (Table 5) was studied in detail. As shown in Fig. 9, the lysine production of T-36, a threonine auxotroph of No. 2247, was strongly inhibited by the addition of a rather high concentration of L-threonine, while X-15 was not affected even at the concentration of 3 mg/ml. This character of X-15 is similar to that of the AEC-resistant mutant (3) and suggested that its mutation site might be aspartokinase [EC ] (AK), as observed in that of FA1-30 (4) or FA Then, effect of AEC plus threonine on the growth of X-15 and X-3 was tested in medium C-2, as shown in Fig. 10. As expected from the results mentioned above the growth of X-3 and No were drastically reduced by the addition of AEC and threonine, while X-15 and FAl-30 were rather resistant.

13 1971 L-Lysine Production by Mutants lob of Fig. halo 8. Relationship between the amount formed by X-type mutants. of lysine produced and the diameter Experimental conditions were the same as in Fig. 1. lysine Fig. 9. Effect producer. of?l-threonine on lysine accumulation by X-15, a prototrophic Experimental conditions were the C O.D., - - L-Lysine HCI. same as in Fig. 3. Basal medium used was

14 110 SANO AND SHuo VOL. 17 Fig. 10. Growth response of X-type mutants on AEC and threonine. Experimental conditions were essentially the same as described in Fig. 4, except that the medium used was C-2 and growth estimation was by optical density at 562 rn. The values of X-3, X-15, No. 2247, and FA 1-30 in basal medium C-2 were 34.5 (71 hr), 31.8 (47 hr), 35.5 (47 hr), and 28.8 (47 hr), respectively. AK activities were assayed using crude extracts of cells grown in medium C-1 for 23 hr. As shown in Fig, ii, AK of X-15 is remarkably desensitized against the concerted feedback inhibition by lysine plus threonine, as observed in those of AEC-resistant mutants, FA1-30 and FA The mutation site of X-15, therefore, might be AK. In contrast, AK activity of X-3 was inhibited, as well as that of No In addition, the specific activity of AK of X-3 was similar to that of No It might be concluded from these findings that AK activity of X-3 is not changed. Further investigation on X-3, "a new type lysine producer" X-3 could be called a new type lysine producer because of its unique characters, that is, prototrophy, the same level of sensitivity to AEC plus threonine, threonine, and methionine, and activity of AK as the parental strain. The sensitivity of X-3 to threonine or methionine was further examined, since it had been examined only by the cylinder-plate method. The effect of threonine or methionine on the growth of X-3 and No was studied with liquid medium B-l. As shown in Fig. 12, X-3 was obviously more sensitive than No. 2247, though the sensitiveness of X-3 was much less than that of 5-20, a typical sensitive mutant. Since the growth of X-3 was not affected

15 1971 L-Lysine Production by Mutants 111 Fig. 11. Effect of lysine =._id threonine on aspartokinase of X-type mutants. Cells grown for 23 hr i~ iedium C-1 were disrupted with glass beads for 1 min and the cell extract was ti aced with 5 volumes of saturated solution of (NH4)25O4 for 20 min. After centrifugation, the precipitate was resuspended in 0.1 N1 Tris-H2504 buffer (ph 7.5) and used as enzyme solution. Other conditions were the same as described in the previous paper (1). Specific activities of X-3, X-15, No. 2247, FA 1-30, and FA were 129, 102, 135, 103, and 100, respectively. by a concentration lower than 10 mg/ml of L-threonine or L-methionine, it might be reasonable to define it as an insensitive strain by the cylinder-plate method. The sensitiveness of X-3 to threonine or methionine suggested that HDH activity of X-3 might be fairly low. Then, the enzyme activity was assayed as given in Table 6. As expected, HDH activity was found to be reduced by 41%. DISCUSSION Threonine- or methionine-sensitive mutants whose growth in a minimal medium was inhibited by the addition of a rather low concentration of threonine or methionine were derived directly from Brevibacterium flavum wild strain No and their lysine-producing abilities were tested. All of the excellent lysine producers were characterized to be inhibited by threonine or methionine and, at the same time, the inhibited growth was restored by the coexistence of methionine or threonine. The HDH activities of these

16 112 SANO AND SHIIO VOL. 17 Fig. 12. Threonine or methionine sensitivity of X-3, a prototrophic lysine producer. Experimental conditions were the same as in Fig. 4. In basal medium, the growth of No. 2247, X-3, and S-20 were 355 (12 hr), 322 (17 hr), and 350 (15 hr), respectively. Growth was estimated by Shimadzu-Kotaki nepherometer. Table 6. HDH activity of X-3, a prototrophic lysine producer. Experimental conditions were Enzyme sources were identical to the same as described those described in Fig. in the 11. previous paper (1). sensitive mutants were all about 3% of No. 2247, as observed in S-20, a lysine-producing revertant of a homoserine auxotroph. The fact that these lysine-producing mutants selected by two different methods are very similar in all the qualities described above suggests that these qualities are closely related and probably originate in one gene mutation. This idea supports the explanation about the relationship between lysine-producing ability and threonine or methionine sensitivity described in the previous paper (1). From above view, the method of selecting the threonine- or methioninesensitive lysine producer directly from the wild strain may be better than the method using reversion of an auxotroph which requires two-step mutation treatments, making the auxotroph and making the revertant. In fact, frequency of the appearance of the homoserine auxotroph and lysine producers among the prototrophic revertants was very low in B, flavum No

17 1971 L-Lysine Production by Mutants 113 Furthermore, as a parental strain of revertants, the homoserine auxotroph having long deletion cannot be used because of the difficulty of its reversion. However, there may be some difference between the genetical alterations of HDH in the sensitive mutants obtained by these two processes. Namely, the directly derived mutants may have only one mutation site which is related to HDH activity, while the revertants may have one or two different sites. In the case of revertants, the second mutation might occur at the same site of initial mutation and first caused HDH inactivity, or at a different, new site, a suppressor (5). As reported previously, there are three types of lysine producers, homoserine and threonine auxotrophs, threonine- or methionine-sensitive mutants, and AEC-resistant mutants (1, 3, 6). It will be very interesting to see whether any other type of lysine producer exists or not. The modified microbiological assay method, which can easily detect the lysine-producing ability on agar plate, must theoretically be the broadest screening method, and any type of mutant which can produce lysine under these experimental conditions can be expected to be obtained. In fact, many halo-fors mers were derived from No Though most of these lysine-producers belonged to either threonine- or m.ethionine-sensitive mutants or AEC-resistant mutants, there were a few mutants which could be assumed as a new type. A typical one of them, X-3, has some unique characters different from any other lysine producers ; it can grow on minimal medium and its growth is inhibited by AEC plus threonine, but not by threonine or methionine by the cylinder-plate method, and it can produce much lysine in a minimal medium and the production is inhibited by excess of threonine. The character of AK was not virtually different from that of No However, 41% reduction in HDH activity was observed, and weak but distinct inhibition of growth by threonine or methionine was discovered by a detailed test in a liquid medium. Consequently, the usual lysine producers obtained rather easily by onestep mutation treatment may belong to one of the three types ; auxotrophs, threonine- or methionine-sensitive mutants, and AEC-resistant mutants. The authors are indebted to the Director, Mr. K. Akino and Dr. T. Tsunoda of this company for the helpful encouragement during the course of this work, and they also thank Mr. M. Kasai :for his technical assistance. REFERENCES 1) I. SHilo and K..SANO, J. Gen. App!. Microbiol., 15, 267 (1969). 2) AL. BACHARACH, Nature, 160, 640 (1947). 3) K. SANG and L SHIM, J. Gen. Appl. Mierobiol., 16, 373 (1970). 4) I. SHIID, R. MIYAJIMAA and K. SANO, J. Biochem. (Tokyo), 68, 701 (1970). 5) A. GAREN and 0. SIDDI i, Genetics, 48, 1121 (1962). t) K. SAND and I. SHIIO, J. Gen. App!. Microbiol., 13, 349 (1967).