Method for producing L-methionine

Abstract

The present invention relates to a method for producing L-methionine in which a microorganism is cultured in the presence of L-homoserine and methyl mercaptan, a salt of the same or dimethyl disulfide whereby the L-methionine is accumulated in the culture medium.

Claims

1. A method of producing L-methionine, comprising: culturing a microorganism selected from the group consisting of: Escherichia coli, Corynebacterium glutamicum, and Corynebacterium humireducens in a culture medium which, at the start of culturing, comprises L-homoserine and a sulphur source selected from the group consisting of: methyl mercaptan, a methyl mercaptan salt and dimethyl disulfide, wherein said culturing results in the accumulation of said L-methionine in said culture medium, and wherein: the microorganism has been genetically engineered to increase, compared to the microorganism prior to genetic engineering, the activity of: i) an enzyme having L-homoserine O-acetyltransferase activity and comprising the amino acid sequence of SEQ ID NO:2; and ii) an enzyme having O-acetyl-L-homoserine sulfhydrylase activity and comprising the amino acid sequence of SEQ ID NO:4; by increasing the copy number of nucleotide sequences encoding the enzymes and/or due to nucleotide sequences encoding the enzymes being in functional linkage to a promoter that increases expression of the enzymes; and during culturing, acetate formed as a result of conversion of O-acetyl-L-homoserine to L-methionine is reused by the microorganism that has been genetically engineered.

2. The method of claim 1, wherein said L-homoserine O-acetyltransferase activity is encoded by a gene comprising the coding sequence of SEQ ID NO:1.

3. The method of claim 1, wherein said O-acetyl-L-homoserine sulfhydrylase activity is encoded by a gene comprising the coding sequence of SEQ ID NO:3.

4. The method of claim 1, wherein the increase in the activity of both the L-homoserine O-acetyltransferase and the O-acetyl-L-homoserine sulfhydrylase is due to an increase in the copy number of nucleotide sequences encoding these enzymes.

5. The method of claim 1, wherein the increase in the activity of both the L-homoserine O-acetyltransferase and the O-acetyl-L-homoserine sulfhydrylase is due to nucleotide sequences encoding these enzymes being in functional linkage to a promoter that increases expression.

6. The method of claim 5, wherein said promoter is selected from the group consisting of: a tact promoter (PtacI) comprising the sequence of SEQ ID NO:5; a glucose dependent deo promoter; a tac promoter; a lac promoter; a trp promoter; an Escherichia coli lac operon inducible by lactose or isopropyl ß-D-thiogalactopyranoside; systems using arabinose or rhamnose as inducers; an Escherichia coli cspA promoter; a Lambda PL promoter; and an osmB promoter.

7. The method of claim 1, wherein at the start of culturing, homoserine is present in the culture medium at about 3.5 g/l to about 6.6 g/l.

8. The method of claim 1, wherein the amino acid sequence of the enzyme having L-homoserine O-acetyltransferase activity, consists essentially of the amino acid sequence of SEQ ID NO:2.

9. The method of claim 1, wherein the amino acid sequence of the enzyme having L-homoserine O-acetyltransferase activity, consists of the amino acid sequence of SEQ ID NO:2.

10. The method of claim 1, wherein the amino acid sequence of the enzyme having O-acetyl-L-homoserine sulfhydrylase activity consists essentially of the amino acid sequence of SEQ ID NO:4.

11. The method of claim 1, wherein the amino acid sequence of the enzyme having O-acetyl-L-homoserine sulfhydrylase activity consists of the amino acid sequence of SEQ ID NO:4.

12. The method of claim 8, wherein the amino acid sequence of the enzyme having O-acetyl-L-homoserine sulfhydrylase activity consists essentially of the amino acid sequence of SEQ ID NO:4.

13. The method of claim 9, wherein the amino acid sequence of the enzyme having O-acetyl-L-homoserine sulfhydrylase activity consists of the amino acid sequence of SEQ ID NO:4.

14. The method of claim 13, wherein the increase in activity of both the enzyme of i) and the enzyme of ii) is due to an increase in the copy number of nucleotide sequences encoding said enzyme.

15. The method of claim 13, wherein the increase in activity of both the enzyme of i) and the enzyme of ii) is due to nucleotide sequences encoding said enzyme being in functional linkage to a promoter that increases expression.

16. The method of claim 15, wherein said promoter is selected from the group consisting of: a tacI promoter (PtacI) comprising the sequence of SEQ ID NO:5; a glucose dependent deo promoter; a tac promoter; a lac promoter; a trp promoter; an Escherichia coli lac operon inducible by lactose or isopropyl ß-D-thiogalactopyranoside; systems using arabinose or rhamnose as inducers; an Escherichia coli cspA promoter; a Lambda PL promoter; and an osmB promoter.

Description

(1) FIG. 1 Shows the pMW218 plasmid map.

(2) FIG. 2: Shows the pMW218_Ptac-metX_Ptac-metY plasmid map.

(3) FIG. 3: Shows the catalytic conversion of L-homoserine and acetyl-CoA by the cell homogenates of MG1655/pMW218 or MG1655/pMW218_Ptac-metX_Ptac-metY.

(4) FIG. 4: Shows the catalytic conversion of sodium methyl mercaptide in the presence of O-acetylhomoserine and pyridoxal 5′-phosphate (PLP) by the O-acetylhomoserine sulfhydrylase enzyme (MetY). Comparison of the cell homogenates of MG1655/pMW218 and MG1655/pMW218_Ptac-metX_Ptac-metY is shown with the respective total protein concentrations used.

EXAMPLES

(5) 1) Preparation of an Enterobacterium Heterologously Expressing the Genes for an L-Homoserine O-Acetyltransferase and a Sulfhydrylase of a Corynebacteria Species

(6) On the basis of the genome sequence of Corynebacterium glutamicum (ATCC13032) NC_003450, the gene sequences metX (SEQ ID NO:1) and metY (SEQ ID NO:3), which encode the L-homoserine O-acetyltransferase having the amino acid sequence according to SEQ ID NO:2 and the O-acetyl-L-homoserine sulfhydrylase having the amino acid sequence according to SEQ ID NO:4 respectively, both with upstream promoter PtacI (SEQ ID NO:5) (H. A. deBoer et al., Proc. Natl. Acad. Sci. USA, Vol. 80, 21-25, January 1983, Biochemistry) from Life Technologies Invitrogen GeneArt (Germany), were synthesized (SEQ ID NO:6).

(7) In this SEQ ID NO:6, the PtacI promoter is from base pair 407-447, the gene sequence of metX from 502-1638, the PtacI promoter again from 1645-1685 and the gene sequence of metY from 1742-3055.

(8) Subsequently, the cloning of this synthetic sequence was carried out via the restriction sites BssHII and BglI in the vector sequence pMW218 (Accession Number: AB005477) (Nippon Gene, Toyama, Japan) (FIG. 1). The plasmid pMW218_Ptac-metX_Ptac-metY is formed therefrom (FIG. 2). To analyze the pMW218_Ptac-metX_Ptac-metY plasmid, DNA sequencing was additionally carried out by Eurofins MWG Operon. The DNA sequences obtained were checked with respect to correctness using the Clone Manager software so that the nucleotide sequence SEQ ID NO:6 was confirmed.

(9) The plasmids pMW218 and pMW218_Ptac-metX_Ptac-metY have been transformed in each case in the Escherichia coli K-12 strain MG1655 (DSM No. 18039). The transformants were subsequently cultured on LB medium agar plates with 50 μg/ml kanamycin such that the MG1655/pMW218 and MG1655/pMW218_Ptac-metX_Ptac-metY strains could be generated. In each case a colony has been selected which was inoculated in each case into 10 ml of LB medium with 50 μg/ml kanamycin, and was cultured at 37° C., 200 rpm for 6 hours. Subsequently, 10 ml of medium A [25 g/l ammonium sulphate; 1 g/l magnesium sulphate heptahydrate; 2 g/l potassium dihydrogen phosphate; 0.03 g/l iron heptahydrate; 0.02 g/l manganese sulphate monohydrate; 20 g/l glucose monohydrate; 30 g/l calcium carbonate; 0.05 g/l kanamycin; 0.025 g/l pyridoxal phosphate (PLP); 0.0024 g/l isopropyl-β-D-thiogalactopyranoside (IPTG)] were inoculated with 200 μl of the growth cell culture and incubated at 37° C., 200 rpm for 16 h. These cell cultures were diluted with 10 ml of fresh medium A in a 100 ml flask to an OD of 2 and were further cultured under identical conditions until an OD of about 5 had been attained (circa 3-4 h). Subsequently, these cells, which are in the exponential growth phase and have homoserine O-acetyltransferase (MetX) and sulfhydrylase (MetY) activity, can be used for the biotransformation. Biotransformation is understood to mean a substance conversion, in which whole living cells, fixed cells or isolated free or carrier-linked enzymes or the combination of the above are used.

(10) 2) Detection of the Enzymatic Activities of L-Homoserine O-Acetyltransferase and Acetyl-L-Homoserine Sulfhydrylase

(11) 10 ml of LB medium with 50 μg/ml kanamycin have been inoculated in each case with a single colony of the MG1655/pMW218 and MG1655/pMW218_Ptac-metX_Ptac-metY strains and have been cultured at 37° C., 200 rpm for 6 hours. Subsequently, 10 ml of medium A (see Example 1) were inoculated with 200 μl of the growth cell culture and incubated at 37° C., 200 rpm for 16 h. The cell cultures were then each harvested (8 ml normalized to an OD=1), the supernatants removed by centrifugation (20 min, 4000 rpm, 4° C.) and the pelleted cells were washed twice with 800 μl of 0.1 M potassium phosphate buffer (pH 7.5) and taken up in 1 ml of buffer. The mechanical cell disruption was carried out in a FastPrep FP120 instrument (QBiogene, Heidelberg), wherein the cells were shaken three times for 20 s at 6.5 m/s in digestion vessels with 300 mg of glass beads (Ø 0.2-0.3 mm). The crude extract was then centrifuged at 12 000 rpm, 4° C., 20 min, in order to remove undigested cells and cell debris. The total amount of protein was determined using the Bio-Rad protein quantification assay (Bio-Rad, USA). The cell homogenate was then used for the enzymatic detection of the cytoplasmatic L-homoserine O-acetyltransferase and acetyl-L-homoserine sulfhydrylase activity.

(12) 2a) Detection of the Cytoplasmatic Activity of MetX (L-Homoserine O-Acetyltransferase)

(13) The reaction, which the enzyme L-homoserine O-acetyltransferase (MetX) [EC2.3.1.31] catalyzes, is the conversion of L-homoserine and acetyl-CoA to O-acetyl-L-homoserine and CoA. With the aid of a DTNB solution (5,5′-dithiobis-2-nitrobenzoic acid, “Ellmans reagent”, Sigma Aldrich, Germany) the progress of this reaction can be recorded by measurements of absorption at 412 nm, since DTNB forms a yellow substance with the SH group of CoA (S. Yamagata Journal of Bacteriology 169, No. 8 (1987) 3458-3463). The photometric MetX enzyme assay was conducted at 37° C., in which calibration was previously carried out using CoA concentrations between 0-200 μM. Each preparation was conducted in a 0.2 ml reaction mixture with 100 mM potassium phosphate buffer (pH 7.5), 0.65 mM DNTB [100 μl of a 1.3 mM DTNB stock], 0.13 mM acetyl-CoA [30 μl of a 0.886 mM acetyl-CoA stock, Sigma Aldrich, Germany], 10 mM L-homoserine [20 μl of a 100 mM L-homoserine stock, Sigma Aldrich, Germany] and the specified protein concentration of 0.012 mg/ml, or 0.024 mg/ml of the respective cell homogenate.

(14) Since acetyl-CoA is used within the cell for various biosyntheses, diverse enzymes are present in the cytoplasm which catalyze the cleavage of acetyl-CoA to CoA, such that the difference between the cell homogenates with and without MetX needs to be considered.

(15) It was observed as a result of the enzyme assay that the DNTB absorption increase of the cell homogenate of MG1655/pMW218_Ptac-metX_Ptac-metY was constantly above that of MG1655/pMW218 over the time course (FIG. 3). This confirms that MetX here is catalytically active as an additional enzyme. Comparison of the slopes of the initial linear regions of the recorded curves shows activities in the cell homogenate of MG1655/pMW218 of around 580 μmol/min per g of total protein and in MG1655/pMW218_Ptac-metX_Ptac-metY around 730 μmol/min per g of total protein. The difference therefore is the specific activity of the L-homoserine O-acetyltransferase (MetX) at around 150 units per g of total protein (1 unit=1 μmol substrate conversion/min).

(16) 2b) Detection of the Cytoplasmic Activity of MetY (O-Acetyl-L-Homoserine Sulfhydrylase)

(17) The reaction, which the enzyme O-acetyl-L-homoserine sulfhydrylase (MetY) [EC 2.5.1.49] catalyzes, is the conversion of O-acetyl-L-homoserine with methanethiol (MC) in the presence of pyridoxal 5′-phosphate (PLP) to give L-methionine and acetate. As described in Example 2a, the progress of this reaction can be determined by means of DTNB absorption measurements at 412 nm, since DTNB reacts with the SH group of unreacted methyl mercaptan to give a yellow substance. For this purpose, the two strains MG1655/pMW218 and MG1655/pMW218_Ptac-metX_Ptac-metY were prepared as cell homogenates as described above and the decrease or the conversion of the substrate sodium methyl mercaptide was measured in the subsequent enzyme assay.

(18) Each preparation was conducted at 37° C. in a 1 ml reaction mixture with 100 mM potassium phosphate buffer (pH 7.5), 2 mM sodium methyl mercaptide (NaMC) [10 μl of a 200 mM NaMC stock], 3 mM OAH HCl [30 μl of a 100 mM OAH HCl stock] and 0.01 mM PLP [10 μl of a 1 mM PLP stock] with the respective cell homogenate at a total protein concentration of 0.012 g/l; 0.024 g/l, or 0.048 g/l. Following the time-limited enzymatic reaction, the photometric measurement of the NaMC content by means of DTNB was conducted, wherein a calibration was previously carried out using MC concentrations between 0-200 μM. For this purpose, 180 μl of a DTNB solution (4 mg/ml) were added to each 20 μl of the enzymatic reaction mixture and subsequently measured at 412 nm.

(19) The presence of the cell homogenate of the MG1655/pMW218_Ptac-metX_Ptac-metY strain leads to the decrease of the NaMC being catalyzed significantly faster than in the presence of the cell homogenate of MG1655/pMW218, due to the enzyme activity of MetY, depending on the total protein concentration (FIG. 4). The decrease of NaMC likewise occurring, but more weakly, in the preparations with the cell homogenate of MG1655/pMW218 is independent of the amount of protein used. An identical decrease could also be observed in a preparation without cell homogenate and is due to the chemically dependent outgassing of the methyl mercaptan occurring from the solutions. From the difference in the slopes in the linear range, a specific MetY sulfhydrylase activity around 1500 units per g of total protein (1 unit=1 μmol substrate conversion/min) can be calculated, which is on the same level as that from the MetX enzyme.

(20) 3) Detection of the Cellular Biotransformation of L-Homoserine and Sodium Methyl Mercaptide to Give L-Methionine

(21) The MG1655/pMW218 and MG1655/pMW218_Ptac-metX_Ptac-metY strains have been cultured as described in Example 1 and then, in the exponential phase of each preparation, have been adjusted to an OD600 of around 7.

(22) The biotransformation was then carried out in 100 ml shaking flasks at 37° C., 200 rpm over a time period of 0, 2, 4 and 24 h. Each preparation was conducted in 10 ml of medium A with 6.5 g/l L-homoserine [500 μl of a 100 g/l homoserine stock] (Sigma Aldrich, Germany), 3 g/l NaMC [500 μl of a 6% NaMC stock] and 12 g/l KH.sub.2PO.sub.4 [600 μl of a 200 g/l KH.sub.2PO.sub.4 stock].

(23) The conversion of L-homoserine with methyl mercaptan to give L-methionine was conducted using the MG1655/pMW218_Ptac-metX_Ptac-metY strain, whereas no L-methionine was synthesized using the MG1655/pMW218 strain (Table 1). The various yields based on the amounts of NaMC initially charged and amounts of L-homoserine consumed are based on an equal stoichiometry of both substrates present at the start but subsequent naturally occurring evaporation of the methyl mercaptan.

(24) TABLE-US-00001 TABLE 1 Comparison of the biotransformations of 6.5 g/l synthetic L-homoserine and 3 g/l sodium methyl mercaptide using the MG1655/pMW218 and MG1655/pMW218_Ptac-metX_Ptac-metY strains. The L-methionine titre obtained over the time course and the related yields are shown, based on the amount of NaMC pulsed at the start and the amount of L-homoserine (L-HS) consumed. 0 h 2 h 4 h 24 h MG1655/pMW218 L-Met (g/l) <0.005 <0.005 <0.005 <0.005 MG1655/pMW218_Ptac- metX_Ptac-metY L-Met (g/l) <0.005 0.810 0.980 1.500 L-Homoserine (g/l) 6.580 4.220 4.020 3.560 L-Met/initially charged NaMC 0% 12.7% 15.3% 23.5% (mol/mol) L-Met/consumed L-HS (mol/mol) 0% 27.4% 30.6% 39.7%

(25) Furthermore, a biotransformation using the MG1655/pMW218_Ptac-metX_Ptac-metY strain has been carried out, in which deuterated NaMC (D.sub.3CSNa) was used in place of the NaMC stock from Sigma Aldrich. This was prepared by introducing CD.sub.3SD (Sigma-Aldrich, 98 atom % D) into an equimolar amount of aqueous sodium hydroxide solution. (Alternatively, it can be prepared according to J. Voss et al., Phosphorous, Sulfur and Silicon and the Related Elements, 2012, 187, 382 from thiourea and CD.sub.3I.) Analysis of the solution after 24 h reaction by LC-MS showed a ratio of methionine to methionine-d-3 of 1:200. It could be detected, therefore, that the methionine formed in the biotransformation is formed exclusively by the incorporation of externally supplied methyl mercaptan.

(26) 4) Conversion of L-Homoserine, Produced by Fermentation, to L-Methionine Via a Biotransformation

(27) On the basis of the biotransformation of synthetic L-homoserine to L-methionine conducted in Example 3a, the biotransformation of L-homoserine produced by fermentation has also been investigated. The concentration of the L-homoserine broth produced by fermentation was 10 g/l. The MG1655/pMW218_Ptac-metX_Ptac-metY strain has been cultured as in Example 1 and the biotransformation conducted in the exponential phase at an OD of 5 in the presence of 5 g/l L-homoserine produced by fermentation and as described in Example 3a for 2, 4 and 24 h. As shown in Table 2, after two hours' biotransformation around 7%, after four hours around 12% and after 24 hours around 45% of the substrates L-homoserine or NaMC were converted to L-methionine, which was reflected in a maximum titre of around 2.9 g/l L-methionine.

(28) TABLE-US-00002 TABLE 2 L-methionine and the related yields formed by the biotransformation of 5 g/l L-homoserine produced by fermentation and 3 g/l sodium methyl mercaptide by the MG1655/pMW218_Ptac-metX_Ptac-metY strain. Time (h) 0 2 4 24 L-Met (g/l) <0.005 0.45 0.79 2.87 L-Homoserine from fermentation (g/l) 5.26 4.74 4.24 2.15 L-Met/initially charged NaMC (mol/mol) 0%  7% 12% 45% L-Met/consumed L-HS (mol/mol) 0% 69% 62% 74%
5) Cellular Recycling of Acetate During the Biotransformation

(29) To investigate the amounts of acetate formed in the biotransformation of L-homoserine and methyl mercaptan, preparations using the MG1655/pMW218 and MG1655/pMW218_Ptac-metX_Ptac-metY strains in the presence of 5 g/l L-homoserine and 3 g/l sodium methyl mercaptide and 12 g/l KH.sub.2PO.sub.4 [600 μl of a 200 g/l KH.sub.2PO.sub.4 stock] were documented over four hours with respect to their acetate content.

(30) The MG1655/pMW218 and MG1655/pMW218_Ptac-metX_Ptac-metY strains have been prepared as described in Example 1, so that an exponential culture having a starting OD of around 3 was used for the respective 10 ml preparations in 100 ml flasks as described in Example 3b.

(31) The acetate concentrations which are formed during the biotransformation of 5 g/l L-homoserine and 3 g/l sodium methyl mercaptide by the MG1655/pMW218 and MG1655/pMW218_Ptac-metX_Ptac-metY strains are documented in Table 3. L-methionine is formed only in the preparation with the strain heterologously expressing the metX and metY genes, whereas in the preparation with the control strain MG1655/pMW218 no L-methionine was detectable.

(32) Within the first four hours around 11 mM acetate are formed due to the experimental parameters in the control preparation, whereas in the biotransformation around 17 mM acetate and 7 Mm L-methionine are formed. The excess of acetate measured in the biotransformation preparation which gives rise to the difference is thus 6 mM. Due to the additional methionine synthesis with an equimolar production of acetate and methionine, which is not described for non-cellular systems (WO 2008/013432 A1), this value would be 7 mM. Therefore, a cellular recycling of the acetate formed in the L-methionine synthesis in the biotransformation could be detected. The additional acetate resulting from the biotransformation has therefore obviously been partly recycled by the acetyl-CoA synthetase (Acs) to acetyl-CoA.

(33) TABLE-US-00003 TABLE 3 Formation and recycling of acetate in biotransformation preparations with the MG1655/pMW218 and MG1655/pMW218_Ptac-metX_Ptac-metY strains in the presence of 5 g/l L-homoserine and 3 g/l sodium methyl mercaptide. Amount of MG1655/ MG1655/ acetate pMW_Ptac- pMW_Ptac- MG1655/ expected metX_Ptac- metX_Ptac- pMW218 Excess (equimolar Recycled Time metY L-Met metY Acetate Acetate acetate to L-Met) acetate [h] [mM] [mM] [mM] [mM] [mM] [mM] 0 <0.03 <0.08 <0.08 0.00 0.00 0.00 2 4.76 10.29 7.54 2.75 4.76 2.01 4 7.37 17.45 11.22 6.22 7.37 1.15
6) External Addition of L-Homoserine

(34) The MG1655/pMW218 and MG1655/pMW218_Ptac-metX_Ptac-metY strains have been prepared as described in Example 1, so that an exponential culture having a starting OD of around 3 was used for the respective 10 ml preparations in 100 ml flasks as described in Example 3b. To the respective preparations followed at the 0 h time point firstly no addition, the addition of 5 g/l L-homoserine, the addition of 3 g/l sodium methyl mercaptide with 12 g/l KH.sub.2PO.sub.4 [600 μl of a 200 g/l KH.sub.2PO.sub.4 stock] and the addition of 5 g/l L-homoserine with 3 g/l sodium methyl mercaptide and 12 g/l KH.sub.2PO.sub.4 [600 μl of a 200 g/l KH.sub.2PO.sub.4 stock].

(35) The L-methionine and L-homoserine titres of the preparations were determined at the time points 0, 2, 4 and 6 h.

(36) TABLE-US-00004 TABLE 4 L-methionine and L-homoserine titres after the external addition of 5 g/l L-homoserine (L-HS) to the biotransformation preparations using the MG1655/pMW218 and MG1655/pMW218_Ptac-metX_Ptac-metY strains in the presence and absence of 3 g/l sodium methyl mercaptide (Na-MC). MG1655/pMW_Ptac- MG1655/ metX_Ptac-metY pMW218 Time Addition Na-MC L-Methionine L-Homoserine L-Methionine L-Homoserine [h] L-HS addition [g/l] [g/l] [g/l] [g/l] 0 — — <0.005 <0.005 <0.005 <0.005 2 — — <0.005 <0.005 <0.005 <0.005 4 — — <0.005 <0.005 <0.005 <0.005 6 — — <0.005 <0.005 <0.005 <0.005 24 — — <0.005 <0.005 <0.005 <0.005 0 5 g/l — <0.005 5.38 <0.005 5.34 2 — — <0.005 4.09 <0.005 5.08 4 — — <0.005 2.35 <0.005 4.77 6 — — <0.005 1.51 <0.005 4.46 24 — — <0.005 <0.005 <0.005 3.52 0 — 3 g/l <0.005 <0.005 <0.005 <0.005 2 — — <0.005 <0.005 <0.005 <0.005 4 — — 0.22 <0.005 <0.005 <0.005 6 — — 0.27 <0.005 <0.005 <0.005 24 — — 0.28 <0.005 <0.005 <0.005 0 5 g/l 3 g/l <0.005 5.01 <0.005 5.31 2 0.71 4.44 <0.005 5.09 4 1.10 4.11 <0.005 4.89 6 1.24 4.06 <0.005 4.65 24 1.46 3.56 <0.005 3.98
7) Biotransformation of L-Homoserine and Dimethyl Disulfide (DMDS) or of L-Homoserine and Sodium Methylmercaptide (NaMC) to Give L-Methionine

(37) The MG1655/pMW218_Ptac-metX_Ptac-metY strain has been cultured as described in Example 1 and then, in the exponential phase of each preparation, has been adjusted to an OD600 of around 10.

(38) The biotransformation was then carried out in 100 ml shaking flasks at 37° C., 200 rpm over a time period of 0, 24 and 48 h. Each preparation was conducted in 10 ml of medium A (see Example 1) with 5.0 g/l L-homoserine and 12 g/l KH.sub.2PO.sub.4 [600 μl of a 200 g/l KH.sub.2PO.sub.4 stock] and the quantities of the sulphur source (i.e. NaMC or DMDS) as provided in Table 5. The control did not contain any sulphur source (i.e. no NaMC and no DMDS).

(39) TABLE-US-00005 TABLE 5 Comparison of the results of the biotransformation with NaMC, control (no sulphur source) and DMDS at different concentrations and reaction time periods Sulphur source L- concentration, L-Homoserine Methionine O-Acetyl-L- reaction time [g/l] [g/l] homoserine [g/l] 1.0 g/L NaMC, 24 h 3.52 1.64 <0.005 Control, 0 h 4.9 <0.005 <0.005 Control, 24 h 1.48 <0.005 3.74 0.5 g/L DMDS, 24 h 2.3 0.2 2.82   1 g/L DMDS, 24 h 2.48 0.32 2.34   1 g/L DMDS, 48 h 2.52 0.46 2.06   2 g/L DMDS, 24 h 3.3 0.42 1.38