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Efficient conversion of glycine to l -serine by a glycine-resistant mutant of a methylotroph using Co2+ as an inhibitor of l -serine degradation
[J. Ferment. Technol., Vol. 65, No. 5, 563-567. 1987]
Efficient Conversion of Glycine to L-Serine by a GlycineResistant Mutant of a Methylotroph Using Co s÷ as an Inhibitor of L-Serine Degradation MAYUMI WATANABE, YASUSHI MORINAGA, TOMOHARU TAKENOUCHI, a n d HITOSHI ENEI
Central Research Laboratories of Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210, Japan
A glyJcine-resistant mutant, no. 18, which was not lysed by glycine, was obtained from a n L-serine-producing mutant, $395 (temperature-sensitive, 0-methylserineresistant), of a facultative methylotroph, Pseudomonas MS31. The mutant stably produced L-serine from glycine. The properties of the enzymes involved in the synthesis and degradation of L-serine were investigated in the wild-type strain MS31 and the L-serine-producing mutants. M u t a n t derivation had no effect on the activities of methanol dehydrogenase or serine hydroxymethyltramferase, which are involved in L-serine synthesis. O n the other h a n d , the activity of L-serine dehydratase (SDH), which degrades L-serine, was reduced in the mutants. Cobalt (Co s+) inhibited SDH activity and its addition (6.5 raM) to the L-serine production culture significantly stimulated L-serine accumulation up to 14.9 mg/ml. The results suggest that blocking of SDH is important for the efficient production of L-serine from glycine by methylotrophs.
Recently, L-serine production by methylotrophs has attracted much attention because of the high yield of L-serine from glycine, a precursor. 1) We previously reported Lserine production by a facultative methylotroph, Ps~udornonas MS31,s~ and its derivative mutants.3, 4) However, they were highly sensitive to glycine and this sometimes affected L-serine productivity. Although the serine degradation activity of these mutants was low compared with the wild-type strain, the remaining activity was still unfavorable for L-serine production. To overcome these two problems, we bred a glycine-resistant mutant and improved the cultivation conditions. Furthermore, we investigated the enzymatic properties of these L-serineproducers, especially of the serine degrading enzyme, L-serine dehydratase. Corresponding author: Mayumi Watanabe
Materials and Methods Microorganisms PseudomonasMS312) and its mutants3, 4) wereused: ts162,*) a temperature-sensitive mutant derived from MS31 ; $395, 4) an O-methylserineresistant mutant derived from ts162; and no. 18, a glycine-resistant mutant. Cultivation The media were those described previously, s)
Cultivation in a 500-ml shaking flasks)
was started at 30°C with 1.0~/o (v/v) methanol. After 16, 24, and 40 h of cultivation, 1.0, 2.0, and 1.0% (v/v) methanol was added respectively. After 4 8 h of cultivation, 15 mg/ml glycine and 4% (v/v) methanol were added, and the cultivation temperature was increased to 340C. The L-serine produced was measured after 72 to 96 h of cultivation. Mutant derivation $395 was treated with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) as described previously. 8) After suitable dilution, the cell suspension was spread on agar plates (BM medium s) without yeast extract) containing 0.5 to 2 mg/ml glycine, followed by culturing for 5-10 days at 30°C. Colonies that appeared were regarded as glycineresistant mutants.
564
~¥ATANAB~-et al.
Enzyme preparation and assay Ceils were grown and harvested as described previously2) except that the buffer concentration was 0.05 M. The cells were resuspended in the same buffer containing 1 mM dithiothreitol (DTT), disrupted by sonication, and centrifuged at 10,000×g for 10 min. The supernatant was used as the cell-free extract. Dye-linked methanol dehydrogenase (EC 1.1.99.8), serine-glyoxylate alninotransferase (EC 2.6.1.45) and serine-pyruvate aminotransferase (EC 2.6.1.51) were assayed.o-) Serine hydroxymethyltransferase (EC 2.1.2.1) was assayed by measuring the rate of glycine-dependent disappearance of formaldehyde, s) Protein was measured by the method of Lowry et al. 8) L-Serine dehydratase (EC 4.2.1.13) was assayed using toluene-treated whole cells by measuring the rate of serine-dependent appearance ofpyruvate. 7) Analyses Amino acids were measured by an amino acid analyzer (model JLC-200A, JEOL). Inorganic ammonia in cultured broth was measured by the method of Russell. s) The methanol concentration was measured by gas chromatography. Cell growth was measured as described previously.°-) Chemicals Reagents used for enzyme assays were purchased from the Sigma Co. Results Derivation of a glycine-resistant mut a n t , n o . 18 T h e a d d i t i o n of glycine to the m e d i u m reduced the cell viability of temperature-sensitive m u t a n t ts162 a n d the 0-methylserine-resistant m u t a n t $395, a n d increased the p r o t e i n c o n t e n t i n the supern a t a n t of the culture b r o t h ( T a b l e 1). This was considered to be due to cell lysis by a d d e d glycine. Therefore, we tried to o b t a i n glycine-resistant m u t a n t s from strain $395.
[J. Ferment. Technol., 10
No.18
0
5
10
L-Ser (gB) Fig. 1. L-Serine productivity among glycine-resistant mutants. The shadow represents the L-serine productivity of the parent strain $395. T h e m i n i m u m c o n c e n t r a t i o n for i n h i b i t i o n of growth of $395 b y glycine was 0.5 m g / m l . $395 was treated with N T G a n d glycineresistant colonies were isolated o n plates c o n t a i n i n g 0.5 to 2 m g / m l glycine. A m o n g the 28 m u t a n t s obtained, strain no. 18 was resistant to 1 m g / m l glycine a n d a c c u m u l a t e d the most L-serine (Fig. 1). T h e cultivation of these cells with glycine did not affect cell viability a n d did n o t increase the extracellular p r o t e i n ( T a b l e 1). This m u t a n t showed stable L-serine p r o d u c t i o n c o m p a r e d with the p a r e n t strain. Alteration of enzyme activities during mutant derivation T h e activities of the two key enzymes involved i n L-serine prod u c t i o n from m e t h a n o l a n d glycine, m e t h a n o l dehydrogenase ( M D H ) a n d serine hydroxy-
Table 1. Effectsof glycine of cell viability and extracellular protein content. Strain
Extracellular protein (mg/ml)*
Viable cells (/ml)
48 h
72 h
48 h
72 h
$395
0. 5
0. 9
6.5× 109
6.0× l0 s
No. 18
0.5
0.6
6.5)<109
8.2×109
The primary cultur£-w~ i~n the production medium for48 h as-described in M a r c e l s and Methods. Then glycine (15 mg/ml) and methanol (4%) were added to the medium and cultivation was continued for 24 h. * The protein in the supernatant of cultured broth was measured by the method of Lowry et aL e)
Vol. 65, 1987]
Conversion of Glycine to L-Serine
Table 2. Alterationof MDH and SHMT activities. Relative activities
Strain
MDH
SHMT
100
100
ts162
69
80
$395
77
76
101
90
MS31 (wild-type)
No. 18
Specific activities of MDH (68.3 nmol/min/mg protein) and SHMT (40.7 nmol/min/mg protein) of MS31 were taken as 100. methyltransferase (SHMT), were examined in the wild-type strain MS31 and its mutants (Table 2). The activities of the two enzymes were not increased in the mutants that acquired -the higher L-serine producing ability. Activities of the enzymes involved in Lserine degradation were assayed. We previously reported s) no L-serine dehydrata.se (SDH) activity, which converts L-serine to pyruvate and ammonia, in the crude extracts of Psntdomonas MS31. However, in this study S D H activity could be detected using toluene-treated cells. As shown in Table 3, the activities of SDH and serine-glyoxylate aminotransferase (SGAT) in the temperaturesensitive mutant ts162 were lower than the wild-type strain. Especially, the level of SDH in ts162 was only 16% of that of the wild-type strain. O n the other hand, the Table 3. Alterationof activities of enzymes involved in L-serine degradation. Strain
Relative activities
activity of serine-pyruvate aminotransferase (SPAT) did not alter during mutant derivation. Decreased SGAT activity in ts162 recovered to the level observed in the wildtype after the ceils acquired 0-methylserineresistance and glycine-resistance, therefore the low SGAT level was not essential for the potent L-serine production. On the other hand, the S D H level remained low during the further mutation, suggesting the necessity of a low SDH level for higher L-serine production. Effects
SPAT
SDH
100
100
100
ts162
42
98
16
L-serine
dehydratase
on
P~
~
4 2,
_ . - - - - - - -
....
.............
-
Z
"10 <
$395
77
102
30
113
105
28
h 0
No. 18
of
L-serine degradation As shown in Fig. 2, the L-serine accumulation became maxim u m after the addition of the glycine (15 mg/ ml) to the medium, and the further incubation resulted in a decrease of L-serine concentration with the increased accumulation of ammonium ions and alanine. However, when chloramphenicol (1 mg/ml) was added to the medium together with glycine, no decrease of L-serine was observed (Fig. 2). This indicated that L-serine was degraded by enzyme(s) induced by the addition of glycine. We investigated the effect of Lserine- or glycine-addition on the levels of enzymes that were involved in L-serine
g5
SGAT
MS31 (wild-type)
565
Specific activities of SGAT (398 nmol/min/mg protein), SPAT (23.5 nmol/min/mg protein) and SDH (17.8nmol/35min[0.1 ml of I-O.D. suspension of cells) of MS31 were taken as 100.
14
4'8
Cultivation time (h)
Fig. 2. Effects of chloramphenicol on accumulation of L-serine, L-alanine, and ammonia. Kinetics of accumulation of the products after addition of glycine (15mg/ml) to the 48 h-grown culture are shown. Symbols: O0, L-serine; A&, Lalanine; Vll, ammonia; open symbols, control; closed symbols, 1 mg/ml chloramphenicol added.
566
X~TATANABEet al.
[,]. Ferment. Technol.,
metabolism. The level of SDH was increased 1.8- and 1.7-fold, respectively, by the addition of L-serine and glycine, but the activities of other enzymes involved in the L-serme ~c ' metabolism in Pseudomonas MS31, such as SGAT and SPAT, were negligibly affected. >, \\ The generation of ammonium ions in accordance with L-serine disappearance suggested that deaminating cleavage of L-serine by SDH was the main pathway of L-serine degradation. To confirm the role of SDH in the L-serine / fl. V_.-------'"--'-=--" .... 0 24 0 2'4 ..... degradation, we tested the effect of L-serine Cultivation time (h) degradation inhibitors on SDH activity. We previously found that Co ~+ and Ni °+ Fig. 4. Course o£ L-serine p r o d u c t i o n by strain no. 18 in the absence and presence of Co ~+. Kinetics strongly inhibited the L-serine degradation after addition of glycine (12mg/ml: control, by the intact cells of Pseudomonas MS31.0) 13 mg/ml: Co ~+ added) to the 48-h grown culture As expected, the addition of 1 m M Co o+ and are shown. Symbols: Q, L-serine; A, L-alanine; I m M NO+ to the SDH assay mixture inhibitl , glycine. ed SDH activity by 82% and 71%, respectively. Thus, the L-serine degradation L-serine accumulation became maximum in Pseudomonas MS31 involves SDH. (12.7mg/ml) 3 0 h after the addition of L-Serine p r o d u c t i o n in t h e p r e s e n c e o£ Co 2+ To avoid L-serine degradation, glycine, while in the presence of 6.5 m M we optimized the conditions for L-serine Co 2+, the maximum accumulation was production by mutant no. 18 (Fig. 3). 14.9 mg/ml at 2 4 h of cultivation with no After cultivation in methanol medium tor further decrease in the L-serine concentration 48 h, various concentrations of Co o+ were at longer incubation periods. The yield added together with glycine (15 mg/ml). coefficient of L-serine from consumed glycine The optimum concentration of Co o+ was increased from 78 to more than 90% 6.5 mM, at which L-serine accumulation was (tool/tool) by the addition of Co 2+. increased by 250/0. Figure 4 shows the LDiscussion serine production kinetics in the presence and absence of Co 2+. Without Co o+, the The acquisition of glycine resistance by mutant no. 18 appears to involve changes in its cell wall or cell wall synthesizing mechaf"~. 7.5 nism, because the cells were resistant to lysis by glycine. Glycine is known to cause cell lysis by being misincorporated into peptide ,- 5.C ¢D glycan. Glycine mimics D-alanine, which is an essential constituent of peptide glycan. 2.6 Yamada et al. reported that in an obligate methylotroph, Hyphomicrobium methylovolum 0 # 1~0-3 I'0-~ KM146, the specific activities of the key Co ~* (M) enzymes of methanol assimilation, methanol Fig. 3. Effects of Co 2+ concentration on L-serine production by strain no. 18. After 48 h of culti- dehydrogenase and serine hydroxymethylvation, 15 mg/ml glycine and various concentration transferase, were elevated by the acquisition of Co ~+ were added and cultivation was continued of glycine resistance, and these elevated for 20 h. enzyme activities enhanced L-serine pro-
Vol. 65, 1987]
Conversion of Glycine to L-Serine
duction.9) However in our glycine-resistant m u t a n t no. 18, the activities of the two enzymes were not elevated. T h e enhanced L-serine production by this mutant, therefore, was likely to be due to resistance against cell lysis caused by glycine. O u r previous work showed the importance of blocking L-serine degradation for the improvement of L-serine production by Pseudomonas MS31. 2) However, the precise mechanism of L-serine degradation in this methylotroph has not been elucidated. In this study, we found that S D H induced by L-serine and/or glycine was mainly responsible for the L-serine degradation in Pseudomonas MS31. This enzyme was quite unstable so that hardly any activity was detected in the conventionally prepared cell-free extract of the cells2); however it could be detected in toluene-treated ceils. We think that blocking S D H is essential for the high accumulation of L-serine for the following two reasons. First, the significant decrease in S D H activity in the m u t a n t ts162 caused a 10-fold increase in L-serine. Furthermore, it was reported that the decrease in S D H activity contributed to the increased L-serine accumulation by mutants of a heterotrophic L-serine producer, Corynebacterium glycinophilum.lO) Second, COS+, an S D H inhibitor, enhanced the L-serine accumulation significantly. T h e mechanism of inhibition by Co~+ is not known, but the
567
findings that the S D H of Pseudomonas MS31 was activated by Fe*+ (data not shown) suggest that Fe*+ plays an essential role in the enzyme action and Co 2+, having similar ionic properties as Fe*+, disturbs the enzyme's function. Acknowledgments
The authors wish to thank Dr. H. Okada and Dr. R. Tsugawa for their encouragement, and Dr. A. Yokota for his helpful discussions. References
1) Morinaga, Y., Yamada, H.: Biotechnology of Amino Acid Production, (Aida, K., Chibata, I., Nakayama, K., Takinami, K., Yamada, H.), p. 207, Elsevier, Tokyo (1986). 2) Morinaga, Y., Yamanaka, S., Takinami, K.: Agric. Biol. Chem., 45, 1419 (1981). 3) Morinaga, Y., Yamanaka, S., Takinami, K.: Agric. Biol. Chem., 45, 1425 (1981). 4) Morinaga, Y., Yamanaka, S., Takinami, K.: Agric. Biol. Chem., 47, 2113 (1983). 5) Akhtar, M., E1-Obeid, H.A.: Biochem. Biophys. Acta, 258, 791 (1972). 6) Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J.: J. Biol. Chem., 193, 265 (1951). 7) Isenberg, S., Newman, E.B.: J. Bucteriol., 118, 53 (1974). 8) Russell,J. A.: J. Biol. Chem., 156, 457 (1944). 9) Yaxaada, H., Miyazaki, S.S., Izumi, Y.: Agric. Biol. Chem., 50, 17 (1986). 10) Kubota, K.: Agric. Biol. Chem., 49, 7 (1985). (Received May 20, 1987)
Efficient Conversion of Glycine to L-Serine by a GlycineResistant Mutant of a Methylotroph Using Co s÷ as an Inhibitor of L-Serine Degradation MAYUMI WATANABE, YASUSHI MORINAGA, TOMOHARU TAKENOUCHI, a n d HITOSHI ENEI
Central Research Laboratories of Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210, Japan
A glyJcine-resistant mutant, no. 18, which was not lysed by glycine, was obtained from a n L-serine-producing mutant, $395 (temperature-sensitive, 0-methylserineresistant), of a facultative methylotroph, Pseudomonas MS31. The mutant stably produced L-serine from glycine. The properties of the enzymes involved in the synthesis and degradation of L-serine were investigated in the wild-type strain MS31 and the L-serine-producing mutants. M u t a n t derivation had no effect on the activities of methanol dehydrogenase or serine hydroxymethyltramferase, which are involved in L-serine synthesis. O n the other h a n d , the activity of L-serine dehydratase (SDH), which degrades L-serine, was reduced in the mutants. Cobalt (Co s+) inhibited SDH activity and its addition (6.5 raM) to the L-serine production culture significantly stimulated L-serine accumulation up to 14.9 mg/ml. The results suggest that blocking of SDH is important for the efficient production of L-serine from glycine by methylotrophs.
Recently, L-serine production by methylotrophs has attracted much attention because of the high yield of L-serine from glycine, a precursor. 1) We previously reported Lserine production by a facultative methylotroph, Ps~udornonas MS31,s~ and its derivative mutants.3, 4) However, they were highly sensitive to glycine and this sometimes affected L-serine productivity. Although the serine degradation activity of these mutants was low compared with the wild-type strain, the remaining activity was still unfavorable for L-serine production. To overcome these two problems, we bred a glycine-resistant mutant and improved the cultivation conditions. Furthermore, we investigated the enzymatic properties of these L-serineproducers, especially of the serine degrading enzyme, L-serine dehydratase. Corresponding author: Mayumi Watanabe
Materials and Methods Microorganisms PseudomonasMS312) and its mutants3, 4) wereused: ts162,*) a temperature-sensitive mutant derived from MS31 ; $395, 4) an O-methylserineresistant mutant derived from ts162; and no. 18, a glycine-resistant mutant. Cultivation The media were those described previously, s)
Cultivation in a 500-ml shaking flasks)
was started at 30°C with 1.0~/o (v/v) methanol. After 16, 24, and 40 h of cultivation, 1.0, 2.0, and 1.0% (v/v) methanol was added respectively. After 4 8 h of cultivation, 15 mg/ml glycine and 4% (v/v) methanol were added, and the cultivation temperature was increased to 340C. The L-serine produced was measured after 72 to 96 h of cultivation. Mutant derivation $395 was treated with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) as described previously. 8) After suitable dilution, the cell suspension was spread on agar plates (BM medium s) without yeast extract) containing 0.5 to 2 mg/ml glycine, followed by culturing for 5-10 days at 30°C. Colonies that appeared were regarded as glycineresistant mutants.
564
~¥ATANAB~-et al.
Enzyme preparation and assay Ceils were grown and harvested as described previously2) except that the buffer concentration was 0.05 M. The cells were resuspended in the same buffer containing 1 mM dithiothreitol (DTT), disrupted by sonication, and centrifuged at 10,000×g for 10 min. The supernatant was used as the cell-free extract. Dye-linked methanol dehydrogenase (EC 1.1.99.8), serine-glyoxylate alninotransferase (EC 2.6.1.45) and serine-pyruvate aminotransferase (EC 2.6.1.51) were assayed.o-) Serine hydroxymethyltransferase (EC 2.1.2.1) was assayed by measuring the rate of glycine-dependent disappearance of formaldehyde, s) Protein was measured by the method of Lowry et al. 8) L-Serine dehydratase (EC 4.2.1.13) was assayed using toluene-treated whole cells by measuring the rate of serine-dependent appearance ofpyruvate. 7) Analyses Amino acids were measured by an amino acid analyzer (model JLC-200A, JEOL). Inorganic ammonia in cultured broth was measured by the method of Russell. s) The methanol concentration was measured by gas chromatography. Cell growth was measured as described previously.°-) Chemicals Reagents used for enzyme assays were purchased from the Sigma Co. Results Derivation of a glycine-resistant mut a n t , n o . 18 T h e a d d i t i o n of glycine to the m e d i u m reduced the cell viability of temperature-sensitive m u t a n t ts162 a n d the 0-methylserine-resistant m u t a n t $395, a n d increased the p r o t e i n c o n t e n t i n the supern a t a n t of the culture b r o t h ( T a b l e 1). This was considered to be due to cell lysis by a d d e d glycine. Therefore, we tried to o b t a i n glycine-resistant m u t a n t s from strain $395.
[J. Ferment. Technol., 10
No.18
0
5
10
L-Ser (gB) Fig. 1. L-Serine productivity among glycine-resistant mutants. The shadow represents the L-serine productivity of the parent strain $395. T h e m i n i m u m c o n c e n t r a t i o n for i n h i b i t i o n of growth of $395 b y glycine was 0.5 m g / m l . $395 was treated with N T G a n d glycineresistant colonies were isolated o n plates c o n t a i n i n g 0.5 to 2 m g / m l glycine. A m o n g the 28 m u t a n t s obtained, strain no. 18 was resistant to 1 m g / m l glycine a n d a c c u m u l a t e d the most L-serine (Fig. 1). T h e cultivation of these cells with glycine did not affect cell viability a n d did n o t increase the extracellular p r o t e i n ( T a b l e 1). This m u t a n t showed stable L-serine p r o d u c t i o n c o m p a r e d with the p a r e n t strain. Alteration of enzyme activities during mutant derivation T h e activities of the two key enzymes involved i n L-serine prod u c t i o n from m e t h a n o l a n d glycine, m e t h a n o l dehydrogenase ( M D H ) a n d serine hydroxy-
Table 1. Effectsof glycine of cell viability and extracellular protein content. Strain
Extracellular protein (mg/ml)*
Viable cells (/ml)
48 h
72 h
48 h
72 h
$395
0. 5
0. 9
6.5× 109
6.0× l0 s
No. 18
0.5
0.6
6.5)<109
8.2×109
The primary cultur£-w~ i~n the production medium for48 h as-described in M a r c e l s and Methods. Then glycine (15 mg/ml) and methanol (4%) were added to the medium and cultivation was continued for 24 h. * The protein in the supernatant of cultured broth was measured by the method of Lowry et aL e)
Vol. 65, 1987]
Conversion of Glycine to L-Serine
Table 2. Alterationof MDH and SHMT activities. Relative activities
Strain
MDH
SHMT
100
100
ts162
69
80
$395
77
76
101
90
MS31 (wild-type)
No. 18
Specific activities of MDH (68.3 nmol/min/mg protein) and SHMT (40.7 nmol/min/mg protein) of MS31 were taken as 100. methyltransferase (SHMT), were examined in the wild-type strain MS31 and its mutants (Table 2). The activities of the two enzymes were not increased in the mutants that acquired -the higher L-serine producing ability. Activities of the enzymes involved in Lserine degradation were assayed. We previously reported s) no L-serine dehydrata.se (SDH) activity, which converts L-serine to pyruvate and ammonia, in the crude extracts of Psntdomonas MS31. However, in this study S D H activity could be detected using toluene-treated cells. As shown in Table 3, the activities of SDH and serine-glyoxylate aminotransferase (SGAT) in the temperaturesensitive mutant ts162 were lower than the wild-type strain. Especially, the level of SDH in ts162 was only 16% of that of the wild-type strain. O n the other hand, the Table 3. Alterationof activities of enzymes involved in L-serine degradation. Strain
Relative activities
activity of serine-pyruvate aminotransferase (SPAT) did not alter during mutant derivation. Decreased SGAT activity in ts162 recovered to the level observed in the wildtype after the ceils acquired 0-methylserineresistance and glycine-resistance, therefore the low SGAT level was not essential for the potent L-serine production. On the other hand, the S D H level remained low during the further mutation, suggesting the necessity of a low SDH level for higher L-serine production. Effects
SPAT
SDH
100
100
100
ts162
42
98
16
L-serine
dehydratase
on
P~
~
4 2,
_ . - - - - - - -
....
.............
-
Z
"10 <
$395
77
102
30
113
105
28
h 0
No. 18
of
L-serine degradation As shown in Fig. 2, the L-serine accumulation became maxim u m after the addition of the glycine (15 mg/ ml) to the medium, and the further incubation resulted in a decrease of L-serine concentration with the increased accumulation of ammonium ions and alanine. However, when chloramphenicol (1 mg/ml) was added to the medium together with glycine, no decrease of L-serine was observed (Fig. 2). This indicated that L-serine was degraded by enzyme(s) induced by the addition of glycine. We investigated the effect of Lserine- or glycine-addition on the levels of enzymes that were involved in L-serine
g5
SGAT
MS31 (wild-type)
565
Specific activities of SGAT (398 nmol/min/mg protein), SPAT (23.5 nmol/min/mg protein) and SDH (17.8nmol/35min[0.1 ml of I-O.D. suspension of cells) of MS31 were taken as 100.
14
4'8
Cultivation time (h)
Fig. 2. Effects of chloramphenicol on accumulation of L-serine, L-alanine, and ammonia. Kinetics of accumulation of the products after addition of glycine (15mg/ml) to the 48 h-grown culture are shown. Symbols: O0, L-serine; A&, Lalanine; Vll, ammonia; open symbols, control; closed symbols, 1 mg/ml chloramphenicol added.
566
X~TATANABEet al.
[,]. Ferment. Technol.,
metabolism. The level of SDH was increased 1.8- and 1.7-fold, respectively, by the addition of L-serine and glycine, but the activities of other enzymes involved in the L-serme ~c ' metabolism in Pseudomonas MS31, such as SGAT and SPAT, were negligibly affected. >, \\ The generation of ammonium ions in accordance with L-serine disappearance suggested that deaminating cleavage of L-serine by SDH was the main pathway of L-serine degradation. To confirm the role of SDH in the L-serine / fl. V_.-------'"--'-=--" .... 0 24 0 2'4 ..... degradation, we tested the effect of L-serine Cultivation time (h) degradation inhibitors on SDH activity. We previously found that Co ~+ and Ni °+ Fig. 4. Course o£ L-serine p r o d u c t i o n by strain no. 18 in the absence and presence of Co ~+. Kinetics strongly inhibited the L-serine degradation after addition of glycine (12mg/ml: control, by the intact cells of Pseudomonas MS31.0) 13 mg/ml: Co ~+ added) to the 48-h grown culture As expected, the addition of 1 m M Co o+ and are shown. Symbols: Q, L-serine; A, L-alanine; I m M NO+ to the SDH assay mixture inhibitl , glycine. ed SDH activity by 82% and 71%, respectively. Thus, the L-serine degradation L-serine accumulation became maximum in Pseudomonas MS31 involves SDH. (12.7mg/ml) 3 0 h after the addition of L-Serine p r o d u c t i o n in t h e p r e s e n c e o£ Co 2+ To avoid L-serine degradation, glycine, while in the presence of 6.5 m M we optimized the conditions for L-serine Co 2+, the maximum accumulation was production by mutant no. 18 (Fig. 3). 14.9 mg/ml at 2 4 h of cultivation with no After cultivation in methanol medium tor further decrease in the L-serine concentration 48 h, various concentrations of Co o+ were at longer incubation periods. The yield added together with glycine (15 mg/ml). coefficient of L-serine from consumed glycine The optimum concentration of Co o+ was increased from 78 to more than 90% 6.5 mM, at which L-serine accumulation was (tool/tool) by the addition of Co 2+. increased by 250/0. Figure 4 shows the LDiscussion serine production kinetics in the presence and absence of Co 2+. Without Co o+, the The acquisition of glycine resistance by mutant no. 18 appears to involve changes in its cell wall or cell wall synthesizing mechaf"~. 7.5 nism, because the cells were resistant to lysis by glycine. Glycine is known to cause cell lysis by being misincorporated into peptide ,- 5.C ¢D glycan. Glycine mimics D-alanine, which is an essential constituent of peptide glycan. 2.6 Yamada et al. reported that in an obligate methylotroph, Hyphomicrobium methylovolum 0 # 1~0-3 I'0-~ KM146, the specific activities of the key Co ~* (M) enzymes of methanol assimilation, methanol Fig. 3. Effects of Co 2+ concentration on L-serine production by strain no. 18. After 48 h of culti- dehydrogenase and serine hydroxymethylvation, 15 mg/ml glycine and various concentration transferase, were elevated by the acquisition of Co ~+ were added and cultivation was continued of glycine resistance, and these elevated for 20 h. enzyme activities enhanced L-serine pro-
Vol. 65, 1987]
Conversion of Glycine to L-Serine
duction.9) However in our glycine-resistant m u t a n t no. 18, the activities of the two enzymes were not elevated. T h e enhanced L-serine production by this mutant, therefore, was likely to be due to resistance against cell lysis caused by glycine. O u r previous work showed the importance of blocking L-serine degradation for the improvement of L-serine production by Pseudomonas MS31. 2) However, the precise mechanism of L-serine degradation in this methylotroph has not been elucidated. In this study, we found that S D H induced by L-serine and/or glycine was mainly responsible for the L-serine degradation in Pseudomonas MS31. This enzyme was quite unstable so that hardly any activity was detected in the conventionally prepared cell-free extract of the cells2); however it could be detected in toluene-treated ceils. We think that blocking S D H is essential for the high accumulation of L-serine for the following two reasons. First, the significant decrease in S D H activity in the m u t a n t ts162 caused a 10-fold increase in L-serine. Furthermore, it was reported that the decrease in S D H activity contributed to the increased L-serine accumulation by mutants of a heterotrophic L-serine producer, Corynebacterium glycinophilum.lO) Second, COS+, an S D H inhibitor, enhanced the L-serine accumulation significantly. T h e mechanism of inhibition by Co~+ is not known, but the
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findings that the S D H of Pseudomonas MS31 was activated by Fe*+ (data not shown) suggest that Fe*+ plays an essential role in the enzyme action and Co 2+, having similar ionic properties as Fe*+, disturbs the enzyme's function. Acknowledgments
The authors wish to thank Dr. H. Okada and Dr. R. Tsugawa for their encouragement, and Dr. A. Yokota for his helpful discussions. References
1) Morinaga, Y., Yamada, H.: Biotechnology of Amino Acid Production, (Aida, K., Chibata, I., Nakayama, K., Takinami, K., Yamada, H.), p. 207, Elsevier, Tokyo (1986). 2) Morinaga, Y., Yamanaka, S., Takinami, K.: Agric. Biol. Chem., 45, 1419 (1981). 3) Morinaga, Y., Yamanaka, S., Takinami, K.: Agric. Biol. Chem., 45, 1425 (1981). 4) Morinaga, Y., Yamanaka, S., Takinami, K.: Agric. Biol. Chem., 47, 2113 (1983). 5) Akhtar, M., E1-Obeid, H.A.: Biochem. Biophys. Acta, 258, 791 (1972). 6) Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J.: J. Biol. Chem., 193, 265 (1951). 7) Isenberg, S., Newman, E.B.: J. Bucteriol., 118, 53 (1974). 8) Russell,J. A.: J. Biol. Chem., 156, 457 (1944). 9) Yaxaada, H., Miyazaki, S.S., Izumi, Y.: Agric. Biol. Chem., 50, 17 (1986). 10) Kubota, K.: Agric. Biol. Chem., 49, 7 (1985). (Received May 20, 1987)