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Isolation of l -methionine-enriched mutant of a methylotrophic yeast, Candida biodinii No. 2201
[J. Ferment. Technol., Vol. 66, No. 2, 153-158. 1988]
Isolation of L-Methionine-Enriched Mutant of a Methylotrophic Yeast, Candida boidinii No. 2201 YOSHIKI TAN1.1, WANG-JIN LIM 1'2, and H A N - C H u L Y A N G ~
Research Centerfor Cell and Tissue Culture, Faculty of Agriculture, Kyoto University, Kyoto 606, Japant ; Department of Food Technology, Korea University, Seoul 132, Korea~
Six strains of methylotrophic yeast were examined for production of L-methionineenriched cells. Candidaboidinii (Kloeckera sp.) No. 2201, which accumulated 0.54 rag/gdry cell weight (DCW) of free L-methionine (pool methionine), was selected as the parental strain for breeding L-methionine-rich mutants. Ethionine-resistant mutants were derived from the strain by UV irradiation. A mutant strain, E500-78, which was resistant to 500 #g/ml of DL-ethionine, accumulated 6.02 mg/g-DCW of pool methionine. The culture conditions for mutant strain E500-78 to increase pool methionine accumulation were optimized. As a result, the mutant strain accumulated 8.80 mg/g-DCW of pool methionine and contained 16.02 mg/g-DCW total methionine.
M e t h a n o l has received m u c h a t t e n t i o n as a n e w raw m a t e r i a l for single cell p r o t e i n p r o d u c t i o n because of its relatively low cost a n d attractive biotechnological features (e.g., m i s c i b i l i t y w i t h w a t e r a n d reduced risks of c o n t a m i n a t i o n ) . 1 , 2) A n u m b e r of yeast bel o n g i n g to the Hansenula, Pichia, Torulo~Osis a n d Candida have b e e n reported to be c a p a b l e of u t i l i z i n g m e t h a n o l as their sole c a r b o n energy source. 8-5) These yeast have 4 5 - 6 0 % p r o t e i n a n d 2 . 8 - 7 % n u c l e i c acid contents. 5) T h e low n u c l e i c acid c o n t e n t is a n a d v a n t a g e for use as a h u m a n food. However, a p r o b l e m w i t h m e t h y l o t r o p h i c yeast as a source of d i e t a r y p r o t e i n lies i n their relatively low c o n t e n t of a n essential a m i n o acid, L-methionine. T h e m e t h i o n i n e c o n t e n t of yeast c a n be increased b y e x p a n d i n g the i n t r a c e l l u l a r pool of free L - m e t h i o n i n e (pool m e t h i o n i n e ) b y regulatory m u t a t i o n s . L - M e t h i o n i n e overp r o d u c t i o n b y ethionine-resistant m u t a n t s has b e e n reported for Saccharomyces cerevisiae, s) Saccharomycopsis lipolytica 7) a n d a n n-paraffinu t i l i z i n g yeast, Candida petrophilum. 8) How* Corresponding author
ever, there has b e e n no report on the prod u c t i o n of L - m e t h i o n i n e b y m e t h y l o t r o p h i c yeast. T h i s p a p e r describes the isolation of a n ethionine-resistant m u t a n t from a methylotrophic yeast, Candida boidinii (Kloeckera sp.) No. 2201, a n d the p r o d u c t i o n of m e t h i o n i n e enriched m u t a n t cells. M a t e r i a l s and M e t h o d s Microorganisms and chemicals Methylotrophic yeasts used were strains preserved in our laboratory. All chemicals were obtained from usual commercial sources and used without any purification procedure. Media M 1 3 1 medium contained 2% (v/v) methanol, 0.4g NHiC1, 0.I g KI-I~PO4, 0.05 g MgSO4"7H~O and 0.2 g yeast extract in 100 ml of tap water, pH 6.0. The minimal medium (M medium) was the same as the M131 medium except that 20 #g thiamin.HC1 and 0.2/*g biotin were added instead of yeast extract. The optimal synthetic medium (OS medium) contained 1.5% (v/v) methanol, 0.8 g (NI-I4)~SO4, 0.1g KH~PO4, 0.2g 1VIgSO4.7H20, 0.2 g ZnSO,.7I-I~O, 20#g thiamin.HC1 and 0.2/~g biotin in 100 ml of deionized water, pI-t 5.5. Cultivation Y e a s t cells were transferred from an M131 medium agar slant to 5 ml of the medium for preculture which contained 1% glucose instead of
154
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[J. Ferment. Technol.,
methanol as the carbon source. The preculture was photometer 220A while referring to a standard DCW carried out at 28°C for 24 h with reciprocal shaking curve. (300 rpm). The culture broth of 2 ml was then inoculated into a 500-ml shaking flask containing Results and Discussion 100 ml of M131 medium. The cultivation was carried out at 28°C for 96 h on a reciprocal shaker at Selection of a methylotrophic yeast 100 rpm. having a high intracellular pool of Isolation of mutants Cells grown on M L - m e t h i o n i n e The ability of methylomedium for 24 h were harvested and washed twice t r o p h i c y e a s t to a c c u m u l a t e p o o l m e t h i o n i n e with physiological saline. Washed cell suspension w a s i n v e s t i g a t e d i n six s t r a i n s ( T a b l e 1). in physiological saline was adjusted to 2 × 107 colony A m o n g t h e s t r a i n s t e s t e d , C. boidinii N o . 2201 forming units per ml (CFU/ml), irradiated with U V showed the highest contents of pool and total light to produce a 99% decrease in viability, and then Since the yeast does not usually spread on plates containing M medium supplemented m e t h i o n i n e . with nL-ethionine. Resistant colonies were selected e x c r e t e a m i n o a c i d s i n t o t h e c u l t u r e m e d i u m , would and re-streaked on the same medium followed by t h e i n c r e a s e i n t o t a l m e t h i o n i n e incubation for 120 h at 28°C. The mutants were then r e f l e c t a n i n c r e a s e i n t h e m e t h i o n i n e p o o l cultured in 1V[131 medium for 96 h in order to de- c o n t e n t . T h e r e f o r e , C. boidinii N o . 2 2 0 1 , termine the L-methionine content. w h i c h h a d t h e l a r g e s t p o o l size o f L - m e t h i o L-Methionlne assay The cells were harvested n i n e (0.54mg/g-DCW), w a s s e l e c t e d as and washed twice with deionized water by centrifu- t h e p a r e n t a l s t r a i n for t h e b r e e d i n g o f Lgation. Three-milliliter aliquots of the cell suspension, methionine-rich mutants. containing 10 mg cells as dry cell weight (DCW), were Isolation of ethionine-resistant muheated for 20 min at 100°C (boiling water bath) in There have been several reports a capped tube. The amount of L-methionine in the t a n t s w h i c h d e s c ribed that the ethionine-resistant supernatant obtained by centrifugation was determined m u t a t i o n c a used an alteration in the reguand designated as pool methionine. The total methioof L-methionine b i o s y n t h e s i s . 10-1~) nine content was determined as follows: Cells (15 mg l a t i o n DCW) were suspended in 3 m l of 6 N HC1 in an T h e r e f o r e , a t t e m p t s w e r e m a d e to d e r i v e ampoule. The ampoule was sealed in vacuo and e t h i o n i n e - r e s i s t a n t m u t a n t s f r o m C. boidinii placed in an oven at 105°C for 24 h. After hydrolysis N o . 2201 i n o r d e r to o b t a i n L - m e t h i o n i n e - r i c h the acid was evaporated to dryness in vacuo. The m u t a n t s . Ethionine-resistant mutants were residue was dissolved in 3 ml of 0.02 M Na2t-IPO4 first d e r i v e d f r o m t h e w i l d - t y p e s t r a i n b y U V and assayed for L-methionine. i r r a d i a t i o n a n d t h e i r a b i l i t i e s to a c c u m u l a t e The amount of L-methionine was determined by A mutant a turbidimetric microbiological assay using Pediococcus p o o l m e t h i o n i n e w e r e e x a m i n e d . s t r a i n , E 4 0 0 4 9 , r e s i s t a n t to 4 0 0 p g/ml of acidilacti (Leuconostoc mesenteroides P60) ATCC 80429) DL-ethionine, accumulated 4.25 mg/g-DCW as the assay organism. Growth measurement Growth was determined o f p o o l m e t h i o n i n e ( T a b l e 2). T h e m u t a n t turbidimetrically at 610 nm using a Hitachi spectro- s t r a i n w a s t h e n i r r a d i a t e d w i t h U V l i g h t a n d Table 1.
L-Methionine contents of the methylotrophic yeast.
Strain
L-Methionine content Growth (mg/g-DCW) (g-DCW//) ........................... Pool Total
Torulopsis glabrata IFO 0005 Pichia pastoris IFO 0948 Candida boidinii A K U 4618 Gandida boidinii No. 2201
0. 67 3. 36 3. 80 3. 86
0. 18 0. 25 0. 44 0. 54
5.04 5.60 4. 54 6.20
Hansenula polymorpha DL-1 Candida sp. 25A
4. 95 3. 46
0. 12 0.36
5.00 6. 12
Cultivation was carried out in MI31 medium.
Vol. 66, 1988]
L-Methionine-Enriched Cells of a Methylotrophic Yeast
Table 2. Accumulation of pool methionine by ethionine-resistant mutants of C. boidinii No. 2201. Strain E300-22 E400-18 E400-37 E400-49 E500-22 Wild type
Growth (g-DCW//)
Pool methionine (mg/g-DCW)
3.54 3.49 3. 13 3.50 3. 38 3.45
I. 84 1.54 1.88 4. 25 2.02 0. 60
Cultivation was carried out in M131 medium. Table 3. Accumulation of pool methionine by ethionine-highly resistant mutants derived from strain E400-49. Strain
(A)
,
q
(B) '
155
'
(C) '
'|
(D)
'
'
/ /
E
/
1'/ 418
96
0
e~
418 9'6 0 4'8 9'6 Cultivation time (h)
4'8
Fig. 1. Effect of the carbon source on pool methionine accumulation by mutant strain E500-78. Cultivation was carried out in M131 medium and M131 media containing 20//o (w/v) glucose (B), glycerol (C) or ethanol (D) instead of methanol
(A).
Growth (g-DCWfl)
Pool methionine (mg/g-DCW)
content of mutant strain E500-78 with that of the wild strain. The m u t a n t strain had
3. 46 3. 28 3. 33 3.31 3.55
5. 02 6. 12 5. 16 5. 54 3.96
about a 90% higher content of total methionine than did the wild type strain, and an increased portion of the total methionine in the mutant strain corresponded approxim a t e l y to t h e i n c r e a s e i n t h e p o o l m e t h i o n i n e .
Cultivation was carried out in M131 medium.
M u t a n t s t r a i n E500-78 c u l t u r e c o n d i tions for pool rnethlonlne accumulation
E500-65 E500-78 E600-111 E800-179 E400-49
plated out on M media containing 500, 600 or 8 0 0 p g / m l of DL-ethionine, respectively. As shown in T a b l e 3, several mutants isolated from these plates were found to accumulate a larger amount of pool methionine than m u t a n t strain E400-49. M u t a n t strain E500-78, one of these mutants, was used throughout the following experiments. T a ble 4 shows a comparison of the L-methionine Table 4. Comparison of L-methionine content between the wild type strain and mutant strain E500-78. Strain
L-Methionine content (mg/g-DCW) . . . . . . . Pool Total
Wild type
0. 58
6. 60
E500-78
6.02
12.45
Cultivation was carried out in M:131 medium.
C u l t u r e c o n d i t i o n s w e r e i n v e s t i g a t e d for m u t a n t s t r a i n E 5 0 0 - 7 8 i n o r d e r to e n h a n c e pool methionine accumulation. As shown i n F i g . 1, m e t h a n o l w a s s u p e r i o r to o t h e r c a r b o n s o u r c e s w i t h r e s p e c t to p o o l m e t h i o -
4 g
E o. 0'.40.'SO
01.4 0180 014 (NHa)2SO 4 cone. (%)
0.~8 0
0.4
0.8
Fig. 2. Effects of the concentrations of methanol and (NH4)zSO4 on pool methionine accumulation by mutant strain E500-78. Cultivation was carried out in M medium supplemented with 0.2% yeast extract containing 1% (A), 1.5% (B), 2.0% (C) or 2.5% (D) methanol and the indicated m o u n t s of (NH4),SO4.
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[J. Ferment. Technol.,
Table 5. Effect of metal salts on pool methionine accumulation by mutant strain E500-78. Metal salt
Growth Pool methionine (g-DCW/I) (mg/g-DCW)
(%)
None MnSO4.4-6H,O ZnSO4"7Ff20 FeCIn-6H20 CaC12 ZnC12 NaC1
0. 005 0. 005 0. 005 0. 005 0. 005 0. 040
1.28 1.66 2.66 1.58 1.44 2.62 2.48
0. 68 0. 62 3. 02 0. 70 0.68 2.78 0. 38
(NH4)eMo7024"4H~O CuSO4"5H~O
0. 001 O. 001
2.30 O. 64
0. 72 --
Cultivation was carried out in M medium supplemented with the repective metal salt.
nine accumulation. In methylotrophic bacteria, 14) the m e t h y l g r o u p of m e t h i o n i n e is synthesized from methanol via formaldehyde and 5,10-methylenetetrahydrofolate. It is possible that this yeast has a high formation activity of the methyl donor when grown on methanol. Among inorganic nitrogen compounds tested here, (NH4),SO4 was the most suitable for pool methionine accumulation. Figure 2 demonstrates the effects of concentrations of methanol and (NH4)2SO4 on pool methionine accumulation. When 1.5% methanol and 0.8% (NH4),SO4 were added, the content of pool methionine reached the maximum value of 8.40mg/g-DCW. Ta-
ble 5 shows the effects o f m e t a l ions on g r o w t h an d pool m e t h i o n i n e a c c u m u l a t i o n . All m e t a l ions tested except C u 2+ s t i m u l a t e d cell g r o w t h , however, only Z n 2+ n o t i c e a b l y stimulated pool m e t h i o n i n e a c c u m u l a t i o n . T h e effect of the M g S O 4 . 7 H , O c o n c e n t r a t i o n on pool m e t h i o n i n e a c c u m u l a t i o n was also e x a m i n e d . T h e o p t i m u m c o n c e n t r a t i o n of M g S O 4 . 7 H 2 0 was 0 . 2 % (data not shown). F i g u r e 3 shows the effect of the Z n S O 4 " 7 H 2 0 c o n c e n t r a t i o n on pool m e t h i o n i n e a c c u m u l a t i o n in the presence or absence o f 0 . 2 % MgSO4.7H20. T h e m a x i m u m co n t en t of pool m e t h i o n i n e was observed in the cu l t u r e to w h i c h 0 . 2 % M g S O 4 . 7 H , O an d 0 . 2 % Z n S O 4 . 7 H , O h a d been added. However, in the absence o f M g S O 4 . 7 H 2 0 , ZnSO4. 7 H 2 0 i n h i b i t e d g r o w t h at concentrations Table 6. Effect of yeast extract on pool methionine accumulation by mutant strain E500-78.
,
a.
2
,c \.\ o
0.10
0.15 0.20 ZnSO 4 conc. (%)
0125
0'.30
Fig. 3. Effect of the ZnSO4'7I-I~O concentration on pool methionine accumulation by mutant strain E500-78. Cultivation was carried out in OS medium with (-O-A-) or without (-O-A-) MgSO4-7H,O and with different concentrations of ZnSO4"7H20.
Yeast extract (%)
Growth (g-DCW//)
Pool methionine (mg/g-DCW)
0 O. 02 0. 05 0. 10 0. 15 0. 20
2.26 2.82 3. 06 3. 18 3.28 3.44
6. 06 7.22 8. 90 7.88 6. 65 6.22
Cultivation was carried out in OS medium supplemented with yeast extract as indicated.
Vol. 66, 1 9 8 8 ]
L-Methionine-Enriched Cells of a Methylotrophic Yeast
h i g h e r t h a n 0 . 1 % . Based on these results, we p r e p a r e d a n O S m e d i u m (see Materials and Methods) w i t h w h i c h to f u r t h e r i n v e s t ig a t e the effect o f a d d i n g v a r i o u s c o n c e n t r a t i o n s o f yeast e x t r a c t in o r d e r to c o m e u p w i t h a b e t t e r m e d i u m composition. I t was found t h a t yeast e x t r a c t at a c o n c e n t r a t i o n o f 0 . 0 5 % resulted in the m a x i m u m c o n t e n t of pool m e t h i o n i n e , w h i l e a h i g h e r level o f yeast extract supported m a x i m u m growth (Table 6). T h e r e f o r e , O S m e d i u m s u p p l e m e n t e d
157
w i t h 0 . 0 5 % yeast e x t r a c t was used for s t u d y i n g the t i m e course o f L - m e t h i o n i n e c o n t e n t d u r i n g c u l t i v a t i o n as described below.
Effects of various additives on pool methlonine accumulation by mutant strain
E500-78
I n c o n n e c t i o n w i t h the
s t i m u l a t o r y effect of yeast extract, the effects o f a d d i n g a m i n o acids were investigated ( T a b l e 7). DL-Homocysteine, a d i r e c t precursor o f L-methionine, i n h i b i t e d growth, w h i l e L-aspartic acid an d L-homoserine
Table 7. Effect of amino acids on pool methionine accumulation by mutant strain E500-78. Amino acid None L-Aspartic acid L-Lysine.HCl n-Homoserine n-Threonine DL-Homocysteine n-Serine
Growth (g-DCW//)
Pool methionine (mg/g-DCW) 5. 96
1.0
2. 15 3.22 2. 90 2. 62 2.66 1.72 2.56 2.64 2.86
2.0
2.82
(mg/ml)
1.0 1.0 1.0 1.0 0. 1 1.0
2.0 Glycine
5. 32 4. 68 5. 40 5. 92 2.04 6.22 6.08 6.54 6.99
Cultivation was carried out in OS medium supplemented with tile amino acids as indicated. Table 8. Effect of vitamins on pool methionine accumulation by mutant strain E500-78. Vitamin None Riboflavin Ca-pantothenate Nicotinic acid p-Aminobenzoi c acid Inositol Pyridoxine.HCl Folic acid Cyanocobalamin
(pg/ml)
0. 2 0. 2 0. 2 0. 2 2.0 0. 2 2.0 0. 02 0. 2 0.01 0. 1
Growth Pool methionine (g-DCW/I) (mg/g-DCW) 2. 25 2. 55 2.60 2.56 2. 64 2.76 2.28 2.22 2.30 2. 28 2.28
2.22
5. 82 5.90 5.75 5.72 5.95 5.88 6.05 6.60 6.38 6. 86 5. 80 5. 64
Cultivation was carried out in OS medium supplemented with the vitamins as indicated.
158
T~x, LIM,and Y~G
did not affect pool methionine accumulation. However, L-serine and glycine stimulated pool methionine accumulation. These findings suggest that the reaction of L-serineglycine interconversion 18) was involved in the biogenesis of the methyl group of Lmethionine in this yeast. Various vitamins were also added to the culture m e d i u m (Table 8). A m o n g the vitamins tested, pyridoxine and folic acid stimulated pool methionine accumulation. Pyridoxine and folic acid are k n o w n to function as cofactors d u r i n g synthesis of the methionine methyl group. T h e addition of c y a n o c o b a l a m i n did not stimulate pool methionine a c c u m u lation. I n Candida utilis TM and S. cerevisiae, TM the involvement of v i t a m i n Bl~-independent enzymes in homocysteine methylation, the final step of methionine biosynthesis, has been reported.
tained by cells at the late-stationary phase (96 h) when pool methionine reached the maximum value of 8.80 m g / g - D C W . Thereafter, total methionine gradually decreased in parallel to the decrease in pool methionine. T h e p H value of the culture broth fell rapidly as the yeast grew, and then rose gradually during the later period. References
1) Oki, T., Kitai, A., Kouno, K., Ozaki, A.: 3.. Gen. Appl. Microbiol., 19, 79 (1973). 2) Cooney, C.L.: Proceedings of the International Symposium on Microbial Growth on Cl-Compounds,
3) 4) 5)
Time course of h-methionine content in mutant strain E500-78 T h e time
courses of pool a n d total methionine content in m u t a n t strain E500-78 were studied using the O S m e d i u m supplemented with 0.05% yeast extract. As shown in Fig. 4, the m a x i m u m total methionine content was 16.02 m g / g - D C W , a value w h i c h was ati"
6) 7) 8) 9)
(
t0)
°~ ~ 1.. •
CllC
•
o
~o
~
11) 12)
. ?
o--~
13) 14) 24
4'6
~2
9'e
t~o
Cultivation time (h)
Fig. 4. Changes in the pool and total methionine contents of mutant strain E500-78 during cultivation. Cultivation was carried out in OS medium supplemented with 0.05% yeast extract.
15) 16)
The Society of Fermentation Technology, Tokyo, p. 183 (1975). Cooney, C. L., Levine, D.W.: Single-CeU Protein II, p. 402, MIT Press, MA (1975). Cooney, C. L., Makiguchi, N. : Biotechnol. Bioeng. Symposium, 7, 65 (1977). Tani, Y. : Methylotrophs: Microbiology, Biochemistry and Genetics, (ifou, C.T.), p. 55, CRC Press, Florida (1984). Cherest, H., Surdin-Kerjan, Y., Antoniewski, J., de Robichon-Szulmajster, H.: J. Bacteriol., 115, 1084 (1973). Morzycka, E., Sawnor-Korszynska, D., Paszewski, A., Grabski, J., Raczynska-Bojanowska, K. : Appl. Environ. Microbiol., 32, 125 (1976). Komatsu, K., Yamada, T., Kodaira, R.: J. Ferment. Technol., 52, 93 (1974). Steele, B. F., Sawberlich, If. E., Reynolds, M.S., Bauman, C.A.: J. Biol. Chem., 177, 533 (1949). Cherest, H., Eichler, F., de Robichon-Szulmajaster, H.: J. Bacteriol., 97, 328 (1969). Cherest, Yr., Talbot, G., de Robichon-Szulmajaster, I-[. : Biochem. Biophys. Res. Commun., 32, 723 (1968). Cberest, If., Talbot, G., de Robichon-Szulmajaster, H.: J. Bacteriol., 102, 448 (1970). Blakley, R.L.: Biochemistry of Folic Acid and Related Pteridines, p. 267, Elsevier/North Holland, Amsterdam (1969). Morinaga, Y., Tani, Y., Yamada, H.: Agric. Biol. Chem., 48, 143 (1984). Salem, A.R., Foster, M.A.: Bioehem. J., 127, 845 (1972). Burton, E., Selhub, J., Sakami, W." Biochem. J., 111,793 (1969). (Received August 28, 1987)
Isolation of L-Methionine-Enriched Mutant of a Methylotrophic Yeast, Candida boidinii No. 2201 YOSHIKI TAN1.1, WANG-JIN LIM 1'2, and H A N - C H u L Y A N G ~
Research Centerfor Cell and Tissue Culture, Faculty of Agriculture, Kyoto University, Kyoto 606, Japant ; Department of Food Technology, Korea University, Seoul 132, Korea~
Six strains of methylotrophic yeast were examined for production of L-methionineenriched cells. Candidaboidinii (Kloeckera sp.) No. 2201, which accumulated 0.54 rag/gdry cell weight (DCW) of free L-methionine (pool methionine), was selected as the parental strain for breeding L-methionine-rich mutants. Ethionine-resistant mutants were derived from the strain by UV irradiation. A mutant strain, E500-78, which was resistant to 500 #g/ml of DL-ethionine, accumulated 6.02 mg/g-DCW of pool methionine. The culture conditions for mutant strain E500-78 to increase pool methionine accumulation were optimized. As a result, the mutant strain accumulated 8.80 mg/g-DCW of pool methionine and contained 16.02 mg/g-DCW total methionine.
M e t h a n o l has received m u c h a t t e n t i o n as a n e w raw m a t e r i a l for single cell p r o t e i n p r o d u c t i o n because of its relatively low cost a n d attractive biotechnological features (e.g., m i s c i b i l i t y w i t h w a t e r a n d reduced risks of c o n t a m i n a t i o n ) . 1 , 2) A n u m b e r of yeast bel o n g i n g to the Hansenula, Pichia, Torulo~Osis a n d Candida have b e e n reported to be c a p a b l e of u t i l i z i n g m e t h a n o l as their sole c a r b o n energy source. 8-5) These yeast have 4 5 - 6 0 % p r o t e i n a n d 2 . 8 - 7 % n u c l e i c acid contents. 5) T h e low n u c l e i c acid c o n t e n t is a n a d v a n t a g e for use as a h u m a n food. However, a p r o b l e m w i t h m e t h y l o t r o p h i c yeast as a source of d i e t a r y p r o t e i n lies i n their relatively low c o n t e n t of a n essential a m i n o acid, L-methionine. T h e m e t h i o n i n e c o n t e n t of yeast c a n be increased b y e x p a n d i n g the i n t r a c e l l u l a r pool of free L - m e t h i o n i n e (pool m e t h i o n i n e ) b y regulatory m u t a t i o n s . L - M e t h i o n i n e overp r o d u c t i o n b y ethionine-resistant m u t a n t s has b e e n reported for Saccharomyces cerevisiae, s) Saccharomycopsis lipolytica 7) a n d a n n-paraffinu t i l i z i n g yeast, Candida petrophilum. 8) How* Corresponding author
ever, there has b e e n no report on the prod u c t i o n of L - m e t h i o n i n e b y m e t h y l o t r o p h i c yeast. T h i s p a p e r describes the isolation of a n ethionine-resistant m u t a n t from a methylotrophic yeast, Candida boidinii (Kloeckera sp.) No. 2201, a n d the p r o d u c t i o n of m e t h i o n i n e enriched m u t a n t cells. M a t e r i a l s and M e t h o d s Microorganisms and chemicals Methylotrophic yeasts used were strains preserved in our laboratory. All chemicals were obtained from usual commercial sources and used without any purification procedure. Media M 1 3 1 medium contained 2% (v/v) methanol, 0.4g NHiC1, 0.I g KI-I~PO4, 0.05 g MgSO4"7H~O and 0.2 g yeast extract in 100 ml of tap water, pH 6.0. The minimal medium (M medium) was the same as the M131 medium except that 20 #g thiamin.HC1 and 0.2/*g biotin were added instead of yeast extract. The optimal synthetic medium (OS medium) contained 1.5% (v/v) methanol, 0.8 g (NI-I4)~SO4, 0.1g KH~PO4, 0.2g 1VIgSO4.7H20, 0.2 g ZnSO,.7I-I~O, 20#g thiamin.HC1 and 0.2/~g biotin in 100 ml of deionized water, pI-t 5.5. Cultivation Y e a s t cells were transferred from an M131 medium agar slant to 5 ml of the medium for preculture which contained 1% glucose instead of
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[J. Ferment. Technol.,
methanol as the carbon source. The preculture was photometer 220A while referring to a standard DCW carried out at 28°C for 24 h with reciprocal shaking curve. (300 rpm). The culture broth of 2 ml was then inoculated into a 500-ml shaking flask containing Results and Discussion 100 ml of M131 medium. The cultivation was carried out at 28°C for 96 h on a reciprocal shaker at Selection of a methylotrophic yeast 100 rpm. having a high intracellular pool of Isolation of mutants Cells grown on M L - m e t h i o n i n e The ability of methylomedium for 24 h were harvested and washed twice t r o p h i c y e a s t to a c c u m u l a t e p o o l m e t h i o n i n e with physiological saline. Washed cell suspension w a s i n v e s t i g a t e d i n six s t r a i n s ( T a b l e 1). in physiological saline was adjusted to 2 × 107 colony A m o n g t h e s t r a i n s t e s t e d , C. boidinii N o . 2201 forming units per ml (CFU/ml), irradiated with U V showed the highest contents of pool and total light to produce a 99% decrease in viability, and then Since the yeast does not usually spread on plates containing M medium supplemented m e t h i o n i n e . with nL-ethionine. Resistant colonies were selected e x c r e t e a m i n o a c i d s i n t o t h e c u l t u r e m e d i u m , would and re-streaked on the same medium followed by t h e i n c r e a s e i n t o t a l m e t h i o n i n e incubation for 120 h at 28°C. The mutants were then r e f l e c t a n i n c r e a s e i n t h e m e t h i o n i n e p o o l cultured in 1V[131 medium for 96 h in order to de- c o n t e n t . T h e r e f o r e , C. boidinii N o . 2 2 0 1 , termine the L-methionine content. w h i c h h a d t h e l a r g e s t p o o l size o f L - m e t h i o L-Methionlne assay The cells were harvested n i n e (0.54mg/g-DCW), w a s s e l e c t e d as and washed twice with deionized water by centrifu- t h e p a r e n t a l s t r a i n for t h e b r e e d i n g o f Lgation. Three-milliliter aliquots of the cell suspension, methionine-rich mutants. containing 10 mg cells as dry cell weight (DCW), were Isolation of ethionine-resistant muheated for 20 min at 100°C (boiling water bath) in There have been several reports a capped tube. The amount of L-methionine in the t a n t s w h i c h d e s c ribed that the ethionine-resistant supernatant obtained by centrifugation was determined m u t a t i o n c a used an alteration in the reguand designated as pool methionine. The total methioof L-methionine b i o s y n t h e s i s . 10-1~) nine content was determined as follows: Cells (15 mg l a t i o n DCW) were suspended in 3 m l of 6 N HC1 in an T h e r e f o r e , a t t e m p t s w e r e m a d e to d e r i v e ampoule. The ampoule was sealed in vacuo and e t h i o n i n e - r e s i s t a n t m u t a n t s f r o m C. boidinii placed in an oven at 105°C for 24 h. After hydrolysis N o . 2201 i n o r d e r to o b t a i n L - m e t h i o n i n e - r i c h the acid was evaporated to dryness in vacuo. The m u t a n t s . Ethionine-resistant mutants were residue was dissolved in 3 ml of 0.02 M Na2t-IPO4 first d e r i v e d f r o m t h e w i l d - t y p e s t r a i n b y U V and assayed for L-methionine. i r r a d i a t i o n a n d t h e i r a b i l i t i e s to a c c u m u l a t e The amount of L-methionine was determined by A mutant a turbidimetric microbiological assay using Pediococcus p o o l m e t h i o n i n e w e r e e x a m i n e d . s t r a i n , E 4 0 0 4 9 , r e s i s t a n t to 4 0 0 p g/ml of acidilacti (Leuconostoc mesenteroides P60) ATCC 80429) DL-ethionine, accumulated 4.25 mg/g-DCW as the assay organism. Growth measurement Growth was determined o f p o o l m e t h i o n i n e ( T a b l e 2). T h e m u t a n t turbidimetrically at 610 nm using a Hitachi spectro- s t r a i n w a s t h e n i r r a d i a t e d w i t h U V l i g h t a n d Table 1.
L-Methionine contents of the methylotrophic yeast.
Strain
L-Methionine content Growth (mg/g-DCW) (g-DCW//) ........................... Pool Total
Torulopsis glabrata IFO 0005 Pichia pastoris IFO 0948 Candida boidinii A K U 4618 Gandida boidinii No. 2201
0. 67 3. 36 3. 80 3. 86
0. 18 0. 25 0. 44 0. 54
5.04 5.60 4. 54 6.20
Hansenula polymorpha DL-1 Candida sp. 25A
4. 95 3. 46
0. 12 0.36
5.00 6. 12
Cultivation was carried out in MI31 medium.
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L-Methionine-Enriched Cells of a Methylotrophic Yeast
Table 2. Accumulation of pool methionine by ethionine-resistant mutants of C. boidinii No. 2201. Strain E300-22 E400-18 E400-37 E400-49 E500-22 Wild type
Growth (g-DCW//)
Pool methionine (mg/g-DCW)
3.54 3.49 3. 13 3.50 3. 38 3.45
I. 84 1.54 1.88 4. 25 2.02 0. 60
Cultivation was carried out in M131 medium. Table 3. Accumulation of pool methionine by ethionine-highly resistant mutants derived from strain E400-49. Strain
(A)
,
q
(B) '
155
'
(C) '
'|
(D)
'
'
/ /
E
/
1'/ 418
96
0
e~
418 9'6 0 4'8 9'6 Cultivation time (h)
4'8
Fig. 1. Effect of the carbon source on pool methionine accumulation by mutant strain E500-78. Cultivation was carried out in M131 medium and M131 media containing 20//o (w/v) glucose (B), glycerol (C) or ethanol (D) instead of methanol
(A).
Growth (g-DCWfl)
Pool methionine (mg/g-DCW)
content of mutant strain E500-78 with that of the wild strain. The m u t a n t strain had
3. 46 3. 28 3. 33 3.31 3.55
5. 02 6. 12 5. 16 5. 54 3.96
about a 90% higher content of total methionine than did the wild type strain, and an increased portion of the total methionine in the mutant strain corresponded approxim a t e l y to t h e i n c r e a s e i n t h e p o o l m e t h i o n i n e .
Cultivation was carried out in M131 medium.
M u t a n t s t r a i n E500-78 c u l t u r e c o n d i tions for pool rnethlonlne accumulation
E500-65 E500-78 E600-111 E800-179 E400-49
plated out on M media containing 500, 600 or 8 0 0 p g / m l of DL-ethionine, respectively. As shown in T a b l e 3, several mutants isolated from these plates were found to accumulate a larger amount of pool methionine than m u t a n t strain E400-49. M u t a n t strain E500-78, one of these mutants, was used throughout the following experiments. T a ble 4 shows a comparison of the L-methionine Table 4. Comparison of L-methionine content between the wild type strain and mutant strain E500-78. Strain
L-Methionine content (mg/g-DCW) . . . . . . . Pool Total
Wild type
0. 58
6. 60
E500-78
6.02
12.45
Cultivation was carried out in M:131 medium.
C u l t u r e c o n d i t i o n s w e r e i n v e s t i g a t e d for m u t a n t s t r a i n E 5 0 0 - 7 8 i n o r d e r to e n h a n c e pool methionine accumulation. As shown i n F i g . 1, m e t h a n o l w a s s u p e r i o r to o t h e r c a r b o n s o u r c e s w i t h r e s p e c t to p o o l m e t h i o -
4 g
E o. 0'.40.'SO
01.4 0180 014 (NHa)2SO 4 cone. (%)
0.~8 0
0.4
0.8
Fig. 2. Effects of the concentrations of methanol and (NH4)zSO4 on pool methionine accumulation by mutant strain E500-78. Cultivation was carried out in M medium supplemented with 0.2% yeast extract containing 1% (A), 1.5% (B), 2.0% (C) or 2.5% (D) methanol and the indicated m o u n t s of (NH4),SO4.
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[J. Ferment. Technol.,
Table 5. Effect of metal salts on pool methionine accumulation by mutant strain E500-78. Metal salt
Growth Pool methionine (g-DCW/I) (mg/g-DCW)
(%)
None MnSO4.4-6H,O ZnSO4"7Ff20 FeCIn-6H20 CaC12 ZnC12 NaC1
0. 005 0. 005 0. 005 0. 005 0. 005 0. 040
1.28 1.66 2.66 1.58 1.44 2.62 2.48
0. 68 0. 62 3. 02 0. 70 0.68 2.78 0. 38
(NH4)eMo7024"4H~O CuSO4"5H~O
0. 001 O. 001
2.30 O. 64
0. 72 --
Cultivation was carried out in M medium supplemented with the repective metal salt.
nine accumulation. In methylotrophic bacteria, 14) the m e t h y l g r o u p of m e t h i o n i n e is synthesized from methanol via formaldehyde and 5,10-methylenetetrahydrofolate. It is possible that this yeast has a high formation activity of the methyl donor when grown on methanol. Among inorganic nitrogen compounds tested here, (NH4),SO4 was the most suitable for pool methionine accumulation. Figure 2 demonstrates the effects of concentrations of methanol and (NH4)2SO4 on pool methionine accumulation. When 1.5% methanol and 0.8% (NH4),SO4 were added, the content of pool methionine reached the maximum value of 8.40mg/g-DCW. Ta-
ble 5 shows the effects o f m e t a l ions on g r o w t h an d pool m e t h i o n i n e a c c u m u l a t i o n . All m e t a l ions tested except C u 2+ s t i m u l a t e d cell g r o w t h , however, only Z n 2+ n o t i c e a b l y stimulated pool m e t h i o n i n e a c c u m u l a t i o n . T h e effect of the M g S O 4 . 7 H , O c o n c e n t r a t i o n on pool m e t h i o n i n e a c c u m u l a t i o n was also e x a m i n e d . T h e o p t i m u m c o n c e n t r a t i o n of M g S O 4 . 7 H 2 0 was 0 . 2 % (data not shown). F i g u r e 3 shows the effect of the Z n S O 4 " 7 H 2 0 c o n c e n t r a t i o n on pool m e t h i o n i n e a c c u m u l a t i o n in the presence or absence o f 0 . 2 % MgSO4.7H20. T h e m a x i m u m co n t en t of pool m e t h i o n i n e was observed in the cu l t u r e to w h i c h 0 . 2 % M g S O 4 . 7 H , O an d 0 . 2 % Z n S O 4 . 7 H , O h a d been added. However, in the absence o f M g S O 4 . 7 H 2 0 , ZnSO4. 7 H 2 0 i n h i b i t e d g r o w t h at concentrations Table 6. Effect of yeast extract on pool methionine accumulation by mutant strain E500-78.
,
a.
2
,c \.\ o
0.10
0.15 0.20 ZnSO 4 conc. (%)
0125
0'.30
Fig. 3. Effect of the ZnSO4'7I-I~O concentration on pool methionine accumulation by mutant strain E500-78. Cultivation was carried out in OS medium with (-O-A-) or without (-O-A-) MgSO4-7H,O and with different concentrations of ZnSO4"7H20.
Yeast extract (%)
Growth (g-DCW//)
Pool methionine (mg/g-DCW)
0 O. 02 0. 05 0. 10 0. 15 0. 20
2.26 2.82 3. 06 3. 18 3.28 3.44
6. 06 7.22 8. 90 7.88 6. 65 6.22
Cultivation was carried out in OS medium supplemented with yeast extract as indicated.
Vol. 66, 1 9 8 8 ]
L-Methionine-Enriched Cells of a Methylotrophic Yeast
h i g h e r t h a n 0 . 1 % . Based on these results, we p r e p a r e d a n O S m e d i u m (see Materials and Methods) w i t h w h i c h to f u r t h e r i n v e s t ig a t e the effect o f a d d i n g v a r i o u s c o n c e n t r a t i o n s o f yeast e x t r a c t in o r d e r to c o m e u p w i t h a b e t t e r m e d i u m composition. I t was found t h a t yeast e x t r a c t at a c o n c e n t r a t i o n o f 0 . 0 5 % resulted in the m a x i m u m c o n t e n t of pool m e t h i o n i n e , w h i l e a h i g h e r level o f yeast extract supported m a x i m u m growth (Table 6). T h e r e f o r e , O S m e d i u m s u p p l e m e n t e d
157
w i t h 0 . 0 5 % yeast e x t r a c t was used for s t u d y i n g the t i m e course o f L - m e t h i o n i n e c o n t e n t d u r i n g c u l t i v a t i o n as described below.
Effects of various additives on pool methlonine accumulation by mutant strain
E500-78
I n c o n n e c t i o n w i t h the
s t i m u l a t o r y effect of yeast extract, the effects o f a d d i n g a m i n o acids were investigated ( T a b l e 7). DL-Homocysteine, a d i r e c t precursor o f L-methionine, i n h i b i t e d growth, w h i l e L-aspartic acid an d L-homoserine
Table 7. Effect of amino acids on pool methionine accumulation by mutant strain E500-78. Amino acid None L-Aspartic acid L-Lysine.HCl n-Homoserine n-Threonine DL-Homocysteine n-Serine
Growth (g-DCW//)
Pool methionine (mg/g-DCW) 5. 96
1.0
2. 15 3.22 2. 90 2. 62 2.66 1.72 2.56 2.64 2.86
2.0
2.82
(mg/ml)
1.0 1.0 1.0 1.0 0. 1 1.0
2.0 Glycine
5. 32 4. 68 5. 40 5. 92 2.04 6.22 6.08 6.54 6.99
Cultivation was carried out in OS medium supplemented with tile amino acids as indicated. Table 8. Effect of vitamins on pool methionine accumulation by mutant strain E500-78. Vitamin None Riboflavin Ca-pantothenate Nicotinic acid p-Aminobenzoi c acid Inositol Pyridoxine.HCl Folic acid Cyanocobalamin
(pg/ml)
0. 2 0. 2 0. 2 0. 2 2.0 0. 2 2.0 0. 02 0. 2 0.01 0. 1
Growth Pool methionine (g-DCW/I) (mg/g-DCW) 2. 25 2. 55 2.60 2.56 2. 64 2.76 2.28 2.22 2.30 2. 28 2.28
2.22
5. 82 5.90 5.75 5.72 5.95 5.88 6.05 6.60 6.38 6. 86 5. 80 5. 64
Cultivation was carried out in OS medium supplemented with the vitamins as indicated.
158
T~x, LIM,and Y~G
did not affect pool methionine accumulation. However, L-serine and glycine stimulated pool methionine accumulation. These findings suggest that the reaction of L-serineglycine interconversion 18) was involved in the biogenesis of the methyl group of Lmethionine in this yeast. Various vitamins were also added to the culture m e d i u m (Table 8). A m o n g the vitamins tested, pyridoxine and folic acid stimulated pool methionine accumulation. Pyridoxine and folic acid are k n o w n to function as cofactors d u r i n g synthesis of the methionine methyl group. T h e addition of c y a n o c o b a l a m i n did not stimulate pool methionine a c c u m u lation. I n Candida utilis TM and S. cerevisiae, TM the involvement of v i t a m i n Bl~-independent enzymes in homocysteine methylation, the final step of methionine biosynthesis, has been reported.
tained by cells at the late-stationary phase (96 h) when pool methionine reached the maximum value of 8.80 m g / g - D C W . Thereafter, total methionine gradually decreased in parallel to the decrease in pool methionine. T h e p H value of the culture broth fell rapidly as the yeast grew, and then rose gradually during the later period. References
1) Oki, T., Kitai, A., Kouno, K., Ozaki, A.: 3.. Gen. Appl. Microbiol., 19, 79 (1973). 2) Cooney, C.L.: Proceedings of the International Symposium on Microbial Growth on Cl-Compounds,
3) 4) 5)
Time course of h-methionine content in mutant strain E500-78 T h e time
courses of pool a n d total methionine content in m u t a n t strain E500-78 were studied using the O S m e d i u m supplemented with 0.05% yeast extract. As shown in Fig. 4, the m a x i m u m total methionine content was 16.02 m g / g - D C W , a value w h i c h was ati"
6) 7) 8) 9)
(
t0)
°~ ~ 1.. •
CllC
•
o
~o
~
11) 12)
. ?
o--~
13) 14) 24
4'6
~2
9'e
t~o
Cultivation time (h)
Fig. 4. Changes in the pool and total methionine contents of mutant strain E500-78 during cultivation. Cultivation was carried out in OS medium supplemented with 0.05% yeast extract.
15) 16)
The Society of Fermentation Technology, Tokyo, p. 183 (1975). Cooney, C. L., Levine, D.W.: Single-CeU Protein II, p. 402, MIT Press, MA (1975). Cooney, C. L., Makiguchi, N. : Biotechnol. Bioeng. Symposium, 7, 65 (1977). Tani, Y. : Methylotrophs: Microbiology, Biochemistry and Genetics, (ifou, C.T.), p. 55, CRC Press, Florida (1984). Cherest, H., Surdin-Kerjan, Y., Antoniewski, J., de Robichon-Szulmajster, H.: J. Bacteriol., 115, 1084 (1973). Morzycka, E., Sawnor-Korszynska, D., Paszewski, A., Grabski, J., Raczynska-Bojanowska, K. : Appl. Environ. Microbiol., 32, 125 (1976). Komatsu, K., Yamada, T., Kodaira, R.: J. Ferment. Technol., 52, 93 (1974). Steele, B. F., Sawberlich, If. E., Reynolds, M.S., Bauman, C.A.: J. Biol. Chem., 177, 533 (1949). Cherest, H., Eichler, F., de Robichon-Szulmajaster, H.: J. Bacteriol., 97, 328 (1969). Cherest, Yr., Talbot, G., de Robichon-Szulmajaster, I-[. : Biochem. Biophys. Res. Commun., 32, 723 (1968). Cberest, If., Talbot, G., de Robichon-Szulmajaster, H.: J. Bacteriol., 102, 448 (1970). Blakley, R.L.: Biochemistry of Folic Acid and Related Pteridines, p. 267, Elsevier/North Holland, Amsterdam (1969). Morinaga, Y., Tani, Y., Yamada, H.: Agric. Biol. Chem., 48, 143 (1984). Salem, A.R., Foster, M.A.: Bioehem. J., 127, 845 (1972). Burton, E., Selhub, J., Sakami, W." Biochem. J., 111,793 (1969). (Received August 28, 1987)