GB2130216A - Enzymatic synthesis of L-serine - Google Patents

Abstract

A method of synthesizing L-serine from glycine and formaldehyde in the presence of biocatalytic amounts of serine hydroxymethyltransferase and tetrahydrofolate is disclosed. The serine hydroxymethyl- transferase may be amplified by cloning the gene therefor into a plasmid, and transforming a host cell, preferably Escherichia coli, Salmonella tymphimurium or Klebsiella aerogenes therewith.

Description

SPECIFICATION Enzymatic synthesis of L-serine Background of the invention This invention relates to a method of synthesizing the amino acid L-serine. The amino acid Lserine is a valuable commercial product that is employed in hyperalimentation and nutritional compositions and is also used as an intermediate or starting material in certain synthetic processes.

There thus is a demand for L-serine, and various processes have been developed for producing it. A number of these processes involve the production of L-serine by fermentation. See, for example, Morinaga, Y., petal., Agric. Biol. Chem. 45(6) 1419--24 (1981); Morinaga, Y., et al., Agric. Biol. Chem.

45(6)1425-1430 (1981). Also, U.S.P. 3,61 6,224, issued to Shiio et al., (1971), discloses a process for producing various amino acids, including serine, by fermentation wherein strains of certain bacteria are cultured on media containing methanol as the source of assimilable carbon. See also U.S.P.

3,943,038, issued to Morinaga et al. (1976), which discloses a method for making serine and other amino acids by culturing specific strains of various bacteria in an aqueous culture medium in the presence of oxygen, hydrogen, and carbon dioxide.

A common shortcoming of the fermentation methods taught by the prior art is that the concentration of L-serine produced in the broth and recovered is relatively low, even after relatively long fermentations.

Serine also can be produced by chemical synthesis processes. See, for example, Kanedo (ed.), Synthetic Production and Utilization of Amino Acids, Halsted Press Books (1974). Often, these chemical procedures produce serine as a racemic mixture of the D and L optical isomers or as the less preferred D-isomer. DL-mixtures must be resolved with methods that utilize D-serine catabolizing bacteria, serine racemase, by L-amino acid acylase, fractional crystallization of serine derivatives, or similar methods, thus adding to the cost of the product.

It is known that L-serine can be produced from glycine. Biological production of L-serine from glycine has been accomplished with several microorganisms. See, For example, Tanaka, Y., et al., J.

Ferment. Technol. 59:447 (1981). As with the fermentation procedures, a drawback of this method has been that the yield of L-serine typically is not very high.

There thus is a need for a method of synthesizing L-serine in high yields. It therefore is an object of this invention to develop a method of synthesizing L-serine wherein the serine is produced in high concentrations in a solution from which it can be efficiently recovered.

It also is an object of this invention to provide an enzymatic means of producing L-serine wherein the reaction can be conducted in a batch or immobilized system.

Summary of the invention It has been discovered that L-serine can be synthesized efficiently from glycine and formaldehyde in the presence of biocatalytic amounts of the enzyme serine hydroxymethyltransferase and the cofactor tetrahydrofolate under serine-producing conditions. The enzyme can be present in whole cells, as a crude extract, or as a purified enzyme and can be in immobilized or non-immobilized form.

Detailed description of the invention It is known that the enzyme serine hydroxymethyltransferase (hereinafter referred to as SHMT) catalyses the cleavage of serine to glycine in a reaction that is dependent upon the co-factors pyridoxal-5'-phosphate and tetrahydrofolate. The reaction yields glycine and methylene tetrahydrofolate. See Schirck, L, Advances in Enzymology 53:83 (1982). It now has been discovered that the SHMT enzyme may be used efficiently as a biocatalyst to produce L-serine from glycine and formaldehyde. The reaction takes place in the presence of the co-factor tetrahydrofolate (THF). The source of the THF may be the source of the SHMT, for THF is found in microorganism cells which contain SHMT, or the THF may be added from an exogenous source. One advantage of this method is that it produces only the L-serine optical isomer.Another advantage is that after the reaction has been run the output from the reactor contains only glycine and L-serine rather than a complex mixture as one would expect to find in a fermentation broth or as the result of chemical synthesis.

It is surprising that L-serine can be synthesized from glycine and formaldehyde, for formaldehyde is known to chemically react with proteins and cause inactivation of enzymes. French, D., et al., Advances in Protein Chemistry 2:277-335 (1945). In fact, SHMT is inactivated rapidly by formaldehyde. It has been discovered, however, that the enzyme can be protected by adding excess tetrahydrofolate to the reaction mixture. The THF reacts with the formaldehyde, thus protecting the enzyme.

Chemical modification of SHMT also provides stability and protection from formaldehyde inactivation. For example, it has been found that reaction of imidoesters with amino groups (see Means, G., et al., Chemical Modification of Proteins, Holden-Day, Inc., 1971) on the enzyme surface allows the enzyme to function in the presence of higher concentrations of formaldehyde than the nonmodified enzyme.

The enzymatic pathway believed to account for the synthesis of L-serine from glycine is shown below: formaldehyde+THF

methylene-THF SHMT methylene THF+glycine

L-serin+THF It is preferred that the reaction be carried out under anaerobic conditions, such as in a nitrogen atmosphere, to prevent oxidation of the THF.

The substrates, the SHMT, and the cofactor, THF, can be reacted together in a variety of ways.

Although the order in which the reactants and catalysts are introduced are not critical, it is preferred that the glycine and formaldehyde are added slowly to SHMT in the presence of THF. The reaction is run under L-serine producing conditions. When the E. coli SHMT gene (glyA) is used as the source of the SHMT, these conditions generally include a reaction temperature of from about 4 to about 600C and a pH in the range of about 4 to about 11. The preferred reaction conditions include carrying out the reaction at a temperature of from about 20 to about 450C and a pH of about 6 to 8.5. If the temperature is below about 40C, the reaction time is slowed considerably, and if the temperature rises above about 600C the enzyme can be denatured. Similarly, at a pH below about 4 or higher than about 11 , the enzyme can be inactivated.SHMT is an enzyme central in the metabolizm of microorganisms and higher organisms; thus, there are many potential sources of the enzyme. The ranges of conditions under which the reaction to produce L-serine is run are related to the source of the enzyme used. For example, enzymes obtained from the thermophilic microorganisms could be used at a higher temperature than is possible when the enzyme source is E. coli.

The serine hydroxymethyltransferase may be in the form of whole cells, a crude extract, or as a purified enzyme. The enzyme may be used in immobilized or nonimmobilized form. The enzyme is used in amounts sufficient to catalyze the reaction.

The enzyme may be obtained from microorganisms that have been modified using conventional genetic engineering techniques to produce it in high yields. See Stauffer, G., et al., Gene 15:63-72 (1981). The SHMT gene (glyA) may be isolated and cloned into a plasmid which then can be used to transform suitable host cells resulting in high level SHMT expression. Mutant microorganisms which have been modified in their methionine metabolism also will overproduce SHMT. See Stauffer, G. V., and Brenchiey, J. E., Genetics 88, 221 (1978) and Stauffer, G. V., and Brenchley, J. E.,J. Bacteriol.

129, 740 (1977). Using gene cloning techniques the enzyme activity has been increased as much as twenty fold and can represent more than ten percent of the soluble protein of the cell. An E. coli strain (Gx 1 703) transformed with such a plasmid, specifically, a derivative of pBR322 into which the glyA gene has been cloned, pGx1 22, has been deposited with the Northern Regional Research Laboratory in Peoria, Illinois, as NRRL No. B-1 521 5 (October 22, 1 982). A Klebsiella aerogenes strain (Gx1 704) transformed with a similar but smaller plasmid with an alteration causing high copy number, pGx1 39, has been deposited with the American Type Culture Collection in Rockville, Maryland as ATCC No.

39214, and a Salmonella typhimurium strain (Gx1 682) transformed with pGx1 39 has been deposited as ATCC No. 3921 5 (both deposited October 22, 1 982).

Further, when the gene is taken from a source such as E. coli it can be modified by random mutagenesis or site directed mutagenesis to produce an enzyme with improved stability. Alternatively, the gene can be completely chemically synthesized with multiple changes to improve the enzyme's stability during the disclosed process.

Using whole cells can provide a source of THF. If desired, additional THF may be added to reach saturation levels, which are dependent upon pH and temperature. For example, at pH of about 7.5 and a reaction temperature of about 370C, in an aqueous solution, THF may be added to reach a concentration much greater than 50 mM. The pH must be adjusted while THF is dissolving. If the SHMT is added as either a crude extract or a purified enzyme, an independent source of THF is needed.

The amount of THF which can be added varies with the temperature, solvent conditions and pH at which the reaction is run.

It has been discovered that THF retains its activity towards SHMT when immobilized.

Immobilizing THF by attaching it to a support which can be retained inside a bioreactor used for carrying out the reaction is advantageous, for it enhances repeated use of the co-factor after the synthesis of L-serine is complete. For example, THF can be immobilized with soluble polymers, such as dextran, polyethylene glycol, or polyethyleneimine. Immobilization takes place by means of covalent attachment in the first two instances and by ionic interaction in the third. Covalent attachment generally occurs through the carboxy groups of the THF with an amino group of the support.

Alternatively, using similar attachment methods, the THF can also be attached to an insoluble support.

Alternatively, if serine is synthesized in a batch process, it is possible to recycle the THF. For example, after the L-serine has been synthesized the reaction solution can be passed through activated charcoal or an ion exchange column, which will retain and separate the THF from the L-serine product solution. The THF then can be released and neutralized. Alternatively, the THF may be modified by covalent attachment to a small molecule, such as a glucosamine to facilitate recovery of the cofactor.

After the L-serine has been synthesized, the reactor solution is passed over a borate column. The borate binds only to the THF-glucosamine, and the modified THF can be released and recycled.

Glycine and formaldehyde preferably are added to the tetrahydrofolate-SHMT mixture. The amount of glycine which can be added varies with the pH, temperature, and solvent conditions of the reaction, but it generally can be added until the saturation level is reached.

As stated previously, formaldehyde can be highly toxic to SHMT. The formaldehyde therefore, generally is added slowly to the other components, and its addition is regulated. The formaldehyde is added in an amount sufficient to retain enzymatic activity, generally to maintain a concentration of less than about 10 mM higher than the THF concentration used, although the formaldehyde may be added to maintain a concentration as high as about 50 mM greater than the THF concentration used. The greater the concentration of THF in the system, the higher the formaldehyde concentration can be maintained, for the THF reacts favorably with the formaldehyde, thus protecting the enzyme. The mechanism of the reaction of THF and formaldehyde is described by Kallen, R. G., et al., J. Biol. Chem.

241 (24) 5851-5863 (1966). The greater the enzyme activity in the reactor the faster the formaldehyde is added to maintain the desired concentration.

It also has been discovered that it may be advantageous to add a second SHMT co-factor, pyridoxal 5'-phosphate, to the reactants. Pyridoxal 5'-phosphate is tightly bound to the enzyme. If Lserine is synthesized in a reactor over an extended period of time, the pyridoxal phosphate may be lost or inactivated, in which case additional pyridoxal phosphate may be added. Loss of the co-factor during the synthesis is indicated by loss of yellow color of the reaction solution and loss of activity by the serine hydroxymethyltransferase. The concentration of pyridoxal phosphate added to the reaction can vary from 0 to about 20 mM, as needed, and preferably is from about 0.1 mM to about 1 mM.

The addition of excess pyridoxal phosphate can serve an additional function. It has been discovered that in some microorganisms which have been transformed by plasmids containing the glyA gene and which express high levels of SHMT the addition of pyridoxal 5'-phosphate is necessary to saturate the SHMT present and enhance the enzyme activity observed. One such microorganism is Klebsiella aerogenes containing the plasmid pGxl 39 identified above.

The synthesis reaction can be conducted in the presence of any non-deleterious solvent.

Examples of such solvents include ethanol, methanol, isopropanol and dioxane.

The following examples are intended to further illustrate this invention.

Example 1 Bacterial strains with and without plasmid pGx1 39 were grown in LB medium (1 0 g/liter Bacto tryptone, 5 g/liter yeast extract 5 g/liter NaCI) or minimal medium (10.5 g/liter K2HPO4, 4.5 g/liter KH2PO4, 1.0 g/liter (NH4)2SO4 and 0.5 liter sodium citrate. 2H2O) supplemented with 0.4% glucose or lactose. Amino acids were added to 20 jug/ml and vitamins to 1 ssg/ml where indicated. The specific activity of serine hydroxymethyl transferase is expressed as nmoles p-phenyl serine converted to benzaldehyde and glycine per minute per mg of extractable protein after sonication of cells.

Assays were in 50 mM phosphate buffer at pH 7.3 with 0.1 mM pyridoxal phosphate and at 35 mM p-phenylserine at 200 C. The appearance of benzaldehyde was monitored at 279 nM in a recording spectrophotometer.

SHMT specific activity (nmoles/min/mg) Strain Minimal E. coli Plasmid LB medium medium Supplement GX1698 39 Nod.* GX1671 pGxl39 221 N.D.

GX1703 pGxl39 141 533 glucose, phenylalanine, thiamine GX1703 pGxl22 218 682 glucose, phenylalanine, thiamine Salmonella typhimurium LT2 GX1682 28 10 glucose, tryptophan 17 lactose, tryptophan GX1682 pGx139 152 133 glucose, tryptophan 306 lactose, tryptophan Klebsiella aerogenes GX1704 - 36 N.D.

GX1704 pGxl39 590 114 glucose 223 108 lactose Strain Genotype E. coli GX1698 trpEA, tna2, serB, laclq ts 402 GX1671 thi, ara, strR, glyA, [serB trpR], laclql, lacZ: :tn5 GX1 703 glyA, pheA, thi, lac, ara, strR Salmonella typhimurium LT2 GX1682 trpBEDC43 F' laclq ts420 pro Klebsiella aerogenes GX1704 lsd (L-serine deaminase mutant) *not determined E. collstrain GX1671 which contains plasmid pGx1 39, is a representative additional strain that has been used as the source of SHMT in the method of this invention Example 2 One colony of Escherichia coli strain GXl 703 (containing pGx1 22), which has been deposited as NRRL No. B-1 521 5 was inoculated into 100 ml of culture medium I described below, and was shaken at 370C overnight.

Culture medium I: K2HPO4 10.5 g/l KH2PO4 4.5 g/l (NH4)2So4 1 9/1 Sodium citrate-2H20 0.5 g/l phenylalanine 20,uglml Vitamin B1 1 yg/ml Ampicillin 100 yg/ml MgSO4 2 mM FeSO4 5 mg/ml Cells were collected by centrifugation of cell culture medium and used as enzyme source. Glycine (12 mM, final concentration) was mixed with tetrahydrofolate (5 mM, final concentration) pyridoxalphosphate (1 mM), and formaldehyde (10 mM, final concentration) under a nitrogen blanket. The pH was maintained at 7.6 with 0.1 M potassium phosphate buffer and the final volume of this reaction mixture was 100 ml.Reaction was started by mixing the above mentioned reaction solution and cells at 370C with shaking. After 8 hours, 5 mM serine was produced (the yield was 45% based on glycine).

All of the enzyme activity was retained. Glycine and serine concentrations were determined using high performance liquid chromatography.

Example 3 One colony of Klebsiella aerogenes strain GX1 704 (containing pGxl 39), which has been deposited as ATCC No. 39214, was inoculated into 100 ml of culture medium II described below and was shaken at 300C overnight.

Culture medium II.

Tryptone 10 g/l NaCI lOg/I Yeast extract 5 g/l Glucose lOg/I Cells were collected by centrifugation of cell culture medium and used as the enzyme source.

The procedure of Example 2 for serine production was followed with the exception that Klebsiella aerogenes was used in place of E. coli. After 8 hours, 7 mM serine was produced (the yield was 64% based on gylcine). All the enzyme activity was retained.

Example 4 The procedure of Example 2 was followed with the exception that partially purified serine hydroxylmethyltransferase from E. coli (strain GX1703 was used. Cells were broken by sonification at 40C. The supernatant collected after centrifugation was mixed with ammonium sulfate (50% saturation) at 40C and pH 7.5. After removing the solid by the centrifugation, the enzyme was forced out of the solution by increasing the ammonium sulfate content to 100% saturation. The enzyme was collected by centrifugation and dialyzed.10 mM serine was produced after 4 hours of reaction. The yield was 90% based on glycine. All enzymatic activity was retained.

Example 5 The procedure of Example 4 was followed with the exception that initial glycine concentration was 340 mM and formaldehyde (original concentration of 2 M) was introduced at a rate of 1 ml per hour. After 12 hours, 119 mM serine was produced.

Example 6 The procedure of Example 4 was followed with the exception that glycine (original concentration of 2 M) and formaldehyde (original concentration of 2 M) were introduced at the same rate of 1 ml per hour. After 5 hours, 57 mM serine was produced.

Example 7 The procedure of Example 4 was followed with the exception that THF was modified. Dextran (5 g, Pharmacia T40) was dissolved in water to a final concentration of 5%. Dextran solution was oxidized by 0.1 M Na 104 for 1 hour at room temperature. Oxidized dextran was precipitated out by adding ethanol to 60% (v/v). This step was repeated twice. Oxidized dextran was dissolved in 100 ml of 0.2 M 1.6-hexanediamine (HMD) at pH 9.0. Sodium borohydride (0.2 g) was added after 30 and 60 minutes respectively. HMD-dextran was dialyzed against water overnight and lypholized. THF (5 mM) was mixed with 10 mM 1 -ethyl-3-(3-dimethylaminopropyl) carbodiimide and 0.5% HMD-dextran at pH 7.0 under nitrogen atmosphere. The precipitate formed was collected by centrifugation and was washed twice with 0.5 M NaCI solution. The solid THF was mixed with reaction mixture as described in Example 2. After 3 hours of reaction , 7 mM serine was produced.

Claims (35)

Claims

1. A method of synthesizing L-serine comprising, reacting glycine and formaldehyde in the presence of biocatalytic amounts of serine hydroxymethyltransferase and tetrahydrofolate under Lserine producing conditions.

2. A method according to claim 1 wherein the temperature is maintained at about 40 to about 600C and the pH is within the range of about 4 to about 1 1.

3. A method according to claim 2 wherein the temperature is within the range of about 200 to about 450C and the pH is within the range of about 6 to about 8.5.

4. A method according to claim 1 wherein the serine hydroxymethyltransferase is contained in whole cells.

5. A method according to claim 1 wherein the serine hydroxymethyltransferase is in the form of a crude extract.

6. A method according to claim 1 wherein the serine hydroxymethyltransferase is in the form of a purified enzyme.

7. A method according to claim 1 wherein the serine hydroxymethyltransferase is immobilized.

8. A method according to claim 1 wherein the reaction is carried out as a batch system.

9. A method according to claim 1 wherein the reaction is carried out as a continuous system.

1 0. A method according to claim 1 wherein the source of tetrahydrofolate is whole microbial cells containing serine hydroxymethyltransferase.

11. A method according to claim 10 wherein additional tetrahydrofolate is added to the reaction system to increase the tetrahydrofolate concentration to a maximum of the saturation level.

12. A method according to claim 1 wherein the concentration of tetrahydrofolate is within the range of about .15 to about 50 mM.

1 3. A method according to claim 1 wherein the tetrahydrofolate is immobilized.

1 4. A method according to claim 13 wherein the tetrahydrofolate is immobilized with a soluble polymer.

1 5. A method according to claim 1 3 wherein the tetrahydrofolate is immobilized by attachment to a solid support.

1 6. A method according to claim 1 wherein the tetrahydrofolate is modified in such a way that it can be recycled.

1 7. A method according to claim 1 wherein glycine can be added until the reaction solution is saturated with glycine.

18. A method according to claim 1 wherein formaldehyde is added to a maximum concentration of about 30 mM to about 50 mM greater than the THF concentration.

1 9. A method according to claim 1 8 wherein the concentration of formaldehyde is brought to a steady state of about 10 mM greater than the THF concentration.

20. A method according to claim 1 wherein pyridoxal phosphate is added to the reactants at a concentration of O to about 20 mM.

21. A method according to claim 20 wherein the pyridoxal phosphate concentration ranges from about 0.1 to 1.0 mM.

22. A method according to claim 1 wherein the source of SHMT is Escherichia coli strain GX1703, containing plasmid pGx1 22, deposited with the Northern Regional Research Laboratory as NRRL No. B-15215.

23. A method according to claim 1 wherein the source of SHMT is Salmonella typhimurium strain Gxl 682, containing plasmid pGx1 39, deposited with the American Type Culture Collection as ATCC No.39215.

24. A method according to claim 1 wherein the source of SHMT is Klebsiella aerogenes strain GX1704, containing plasmid pGx1 39, deposited with the American Type Culture Collection as ATCC No.39214.

25. A method according to claim 1 wherein the serine hydroxymethyl transferase activity in cells has been amplified by genetic manipulation.

26. A method according to claim 1 wherein the serine hydroxymethyl transferase activity in cells has been amplified by cloning the serine hydroxymethyltransferase gene into a plasmid and transforming a host cell with that plasmid to overproduce serine hydroxymethyl transferase.

27. A method according to claim 1 wherein the serine hydroxymethyl transferase is obtained from any biological source.

28. A method according to claim 1 wherein the serine hydroxymethyltransferase gene has been altered by random mutagenesis or site directed mutagenesis to increase the enzyme's stability.

29. A method according to claim 1 wherein the serine hydroxymethyltransferase enzyme is chemically modified to increase enzyme stability.

30. A method according to claim 29 wherein the enzyme is modified by reaction with imidoesters.

31. A substantially biologicaily pure culture of Escherichia collstrain Gel 703 containing plasmid pGx122.

32. A substantially biologicaliy pure culture of Salmonella typhimurium strain GX1682 containing plasmid pGx139.

33. A substantially biologically pure culture of Klebsiella aerogenes strain GX1704 containing pGx1 39.

34. A method of production of L-serine substantially as hereinbefore described with reference to any of the Examples.

35. L-serine whenever produced by a process as claimed in any of the preceding claims.