Process for Producing L-Methionine from Methional

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(11) EP 3 330 380 A1

(12) EUROPEAN PATENT APPLICATION

(43) Date of publication: (51) Int Cl.: 06.06.2018 Bulletin 2018/23 C12P 13/06 (2006.01) C12P 13/08 (2006.01) C12P 13/12 (2006.01) C12P 13/22 (2006.01) (2006.01) (21) Application number: 16202157.0 C12P 7/40

(22) Date of filing: 05.12.2016

(84) Designated Contracting States: • Technische Universität München AL AT BE BG CH CY CZ DE DK EE ES FI FR GB 80290 München (DE) GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR (72) Inventors: Designated Extension States: • SKERRA, Arne BA ME 85354 Freising (DE) Designated Validation States: • EISOLDT, Lukas MA MD 85356 Freising (DE)

(71) Applicants: • Evonik Degussa GmbH 45128 Essen (DE)

(54) PROCESS FOR PRODUCING L-METHIONINE FROM METHIONAL

(57) The present invention relates to a method for hyde and carbon dioxide and, in particular, L-methionine the biocatalytic synthesis of proteinogenic L-amino acids, from 3-methylthio-propanal ("methional") and carbon di- such as L-alanine, L-valine, L-methionine, L-leucine, oxide. L-isoleucine or L-phenylalanine from a respective alde- EP 3 330 380 A1

Printed by Jouve, 75001 PARIS (FR) EP 3 330 380 A1

Description

[0001] The present invention relates to a method for the biocatalytic synthesis of proteinogenic L-amino acids, such as L-alanine, L-valine, L-methionine, L-leucine, L-isoleucine or L-phenylalanine from a respective aldehyde and carbon 5 dioxide as well as an amino donor and, in particular, L-methionine from 3-methylthio-propanal ("methional") and carbon dioxide as well as an amino donor such as ammonia. [0002] α-Amino acids are usually synthesized by the Strecker amino acid synthesis which involves the treatment of an aldehyde with potassium cyanide and ammonia leading to an α-amino nitrile as an intermediate. Hydrolysis of the nitrile into acid then yields the α-amino acid. This chemical synthesis gives a racemic mixture of D- and L-amino acids. 10 Proteinogenic α-amino acids may also be produced by fermentation of microorganisms leading preferentially to L-amino acids. [0003] The amino acid methionine is currently industrially produced worldwide in large amounts and is of considerable commercial importance. Methionine is employed in many fields, such as pharmaceutical, health and fitness products, but particularly as additive in many feedstuffs for various livestock, where both the racemic and the enantiomerically 15 pure L-stereoisomer of methionine may be used. [0004] At the industrial scale, methionine is produced chemically via the Bucherer-Bergs reaction, which is a variant of the Strecker synthesis. The starting substance, 3-methylthio-propanal ("methional"), is usually prepared from propenal (acrolein) and methanethiol (methyl mercaptan). Methional, hydrogen cyanide, ammonia and carbon dioxide are reacted to give 5-(2-methylmercaptoethyl) hydantoin, which is subsequently hydrolyzed by alkali to give the alkali metal D,L- 20 methionate. The D,L-methionine is then liberated by neutralization with acid (US 5,770,769). However, the need to use hydrogen cyanide is a disadvantage of this process. Owing to the high toxicity of hydrogen cyanide, outlay on safety must be high for the reaction. Another great disadvantage are the high amounts of ammonium sulfate that is formed by purification of the hydrogen cyanide with sulfuric acid prior to its use for methionine hydantoin synthesis. There is therefore a need for an HCN-free process for the production of methionine. 25 [0005] Various other methods can also be used to prepare methionine, for example, the amidocarbonylation of methional in the presence of an amide, carbon monoxide and of a transition metal carbonyl catalyst (WO 2005/121068 A1, WO 2005/121079 A1), the hydrolysis of proteins or the fermentation of microorganisms producing methionine. [0006] As mentioned above, in chemical synthesis, like all other α-amino acids, methionine usually is produced as a racemic mixture of D- and L-methionine. However, pharmaceutical or medical applications often require the chiral pure 30 L-amino acid, in particular L-methionine. L-Methionine may be produced either by conversion of the D,L-racemate into pure enantiomers or by fermentation of suitable microorganisms. However, a major drawback of the complete fermentation of L-methionine in microorganisms is the huge amount of nutrients and/or energy required, in particular for the reduction of sulfur to be introduced into the methionine molecule, which is usually added in the form of sulfate to the fermentation media due to the toxic effects of reduced sulfur compounds (such as methanethiol) on the methionine- 35 producing microorganisms. Whereas the direct synthesis of L-methionine starting from aspartate requires 1 ATP and 2 NADPH molecules, the incorporation of sulfur from inorganic sulfate additionally consumes 2 ATP, 1 GTP and 4 NADPH molecules. Therefore, the energy balance would be tremendously improved if a reduced sulfur compound (e.g. methanethiol) could be used for the L-methionine synthesis (Wilke (2014) Appl. Microbiol. Biotechnol. 98, 9893-9914). [0007] To solve this problem, a two-stage biotechnological method for preparing L-methionine was proposed by Kim 40 et al. (WO 2008/013432 A1). In a first step, an L-methionine precursor, O-succinyl-L-homoserine or O-acetyl-L-homoserine, is obtained by means of recombinant microorganisms and accumulated in the culture broth. In the second step, the L-methionine precursor is reacted with methanethiol in the presence of a protein having O-succinyl-L-homoserine sulfhydrylase activity or O-acetyl-L-homoserine sulfhydrylase activity to give L-methionine and the corresponding car- boxylic acid, i.e. acetate or succinate. However, in this enzymatic reaction, equimolar amounts of acetate or succinate 45 are formed in addition to L-methionine. When choosing O-acetyl-L-homoserine as L-methionine precursor, for example, this leads to high acetate concentration in the course of the reaction, particularly at industrial scale. However, acetate cannot be completely removed from the L-methionine product with acceptable effort. Accordingly, Hong et al. (WO 2012/091479 A2) proposed numerous methods to remove and to reuse the relatively large amounts of acetate generated in the second stage of the L-methionine production process from the L-methionine product. 50 [0008] Several attempts have been undertaken to use carbon dioxide as C1 building block for carbon chain extension. Miyazaki et al. (Chem. Commun., 2001, 1800-1801) reported the successful synthesis of pyruvic acid from acetaldehyde in the presence of pyruvate decarboxylase and carbon dioxide as C1 building block making use of the reverse enzymatic reaction. The reaction requires a large excess of carbon dioxide in order to drive the equ ilibrium into the opposite direction of decarboxylation. A multienzyme catalytic system including a cofactor regeneration cycle that uses carbon dioxide and 55 ethanol to produce L-lactate via acetaldehyde and pyruvic acid was proposed by Tong et al. (Biotechnol. Bioeng. (2011) 108, 465-469). [0009] Wichmann et al. (Biotechnol. Bioeng. (1981) 23, 2789-2802) proposed for the reductive amination of 2-oxo-4- methylpentanoic acid (α-ketoisocaproate) to L-leucine catalyzed by L-leucine dehydrogenase (LeuDH), an NADH-de-

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pendent enzyme, a biocatalytic NADH regeneration system using formate and formate dehydrogenase for the regeneration of NADH. [0010] The object of the present invention is to provide a method for producing L-amino acids, such as L-alanine, L- valine, L-methionine, L-leucine, L-isoleucine and L-phenylalanine that uses carbon dioxide instead of cyanide as C1 5 building block and thus avoids the formation of high amounts of ammonium sulfate and that directly leads to the enantiomerically pure L-amino acid. [0011] This object is achieved by a method for producing an L-amino acid, comprising a step of reacting a mixture comprising an aldehyde, carbon dioxide, a decarboxylase, its corresponding cofactor and (a) at least one donor amino acid and an aminotransferase and/or (b) NADH, ammonia and/or an ammonium salt and an amino acid dehydrogenase 10 to form the L-amino acid or a salt thereof. [0012] Preferred L-amino acids that can be synthesized from their starting aldehydes by the method according to the presentinvention areL-alanine fromethanal (acetaldehyde),L-valine from2-methyl propanal, L-methionine from 3-(methylthio) propanal (methional), L-leucine from 3-methyl butanal, L-isoleucine from 2-methyl butanal and L-phenylalanine from 2-phenyl ethanal (phenyl acetaldehyde). 15 [0013] In a preferred embodiment of the present invention, the object is achieved by a method for producing L- methionine (L-Met), comprising a step of reacting a mixture of 3-(methylthio)-propanal (methional), carbon dioxide, a decarboxylase, its corresponding cofactor and (a) at least one donor amino acid and an aminotransferase and/or (b) NADH, ammonia and/or an ammonium salt and an amino acid dehydrogenase to form L-methionine or a salt thereof. [0014] Without willing to be bound by theory, it is thought that, initially, the decarboxylase in the mixture catalyzes its 20 reverse reaction, i.e. the carboxylation of the aldehyde, e.g. methional, with carbon dioxide (CO2), which leads to the intermediate α-keto acid (2-oxo acid), e.g. 4-methylthio-2-oxobutanoic acid (MTOB). Subsequently, the α-carbonyl group of the α-keto acid, e.g. 2-MTOB, is exchanged by an amino group in a stereospecific reaction catalyzed by an aminotransferase to yield the L-amino acid, such as L-methionine (Figure 1A). This reaction requires the presence of a donor amino acid (e.g. L-glutamine) which itself is converted to the respective α-keto acid. Alternatively, the conversion 25 of the α-keto acid of said L-amino acid, e.g. MTOB, to L-methionine, can be achieved by an amino acid dehydrogenase (e.g. LeuDH or phenylalanine dehydrogenase, PheDH) that catalyzes the reductive amination of theα -keto acid, e.g. of MTOB, under the consumption of NADH with NH3 as amino donor (Figure 1 B). [0015] The cofactor of the decarboxylase comprises thiamine pyrophosphate. Suitable decarboxylases that catalyze

the carboxylation of methional with CO2 are, for example, the pyruvate decarboxylase PDC1, which originates from 30 Saccharomyces cerevisiae, the phenylpyruvate decarboxylase ARO10, which originates from Saccharomyces cerevisiae, and the branched chain decarboxylase KdcA, which originates from Lactococcus lactis, as well as mutants and variants of these decarboxylase having decarboxylase activity (Table 1). The carbon dioxide is preferably applied to the reaction mixture at a pressure from 10 to 7400 kPa (from 0,1 to 74 bar), preferably from 100 to 1000 kPa (1 to 10 bar), more preferable from 200 to 800 kPa (2 to 8 bar). 35 [0016] Aminotransferases that are particularly suitable for variant (a) of the method according to the present invention, e.g. the transfer of the amino group from the donor amino acid to the α-carbonyl group of MTOB, are, for example, the methionine aminotransferase YbdL, which originates from E. coli, and the aromatic aminotransferase Aro8, which originates from Saccharomyces cerevisiae, as well as mutants and variants of these aminotransferases having aminotransferase activity (Table 1). Preferably, the donor amino acid is different from the L-amino acid to be formed. Preferred 40 donor amino acids for this variant of the method according to the present invention are at least one L-amino acid, selected from the group consisting of L-glutamine, L-glutamate, L-alanine, L-phenylalanine, L-tyrosine, L-leucine, L-isoleucine, L-histidine and L-tryptophan.. [0017] Amino acid dehydrogenases that are particularly suitable for variant (b) of the method according to the present invention, e.g. the reductive amination of MTOB formed from methional under the consumption of NADH and NH 3, are, 45 for example, the leucine dehydrogenase LeuDH, which originates fromBacillus sphaericus, and the phenylalanine dehydrogenase PheDH, which originates from Thermoactinomyces intermedius, as well as well as mutants and variants of these amino acid dehydrogenases having amino acid dehydrogenase activity (Table 1).

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Table 1: Enzymes suitable for the synthesis of L-Methionine from Methional. SEQ Enzyme class and example Organism Modifications ID Reaction 5 No. Decarboxylase EC 4.1.1 Pyruvate decarboxylase: Pdc1 (P06169; Saccharomyces C-terminal Killenberg-Jabs et al. (1997) Biochemistry 1 cerevisiae His -tag 10 36, 1900-1905) 6 Phenylpyruvate decarboxylase: Aro10 ΔK635; C- Saccharomyces Carboxylation of (Q06408;Kneen et al. (2011) FEBS J. 278, terminal 3 cerevisiae methional to MTOB 1842-1853) His6-tag 15 Branched chain decarboxylase: KdcA C-terminal (Q6QBS4;Yep et al. (2006) Bioorg. Chem. Lactococcus lactis 5 His -tag 34, 325-336) 6 Aminotransferase 2.6.1.87 & .6.1.57

20 Methionine aminotransferase: YbdL Escherichia coli K12 N-terminal Transamination of (P77806; Dolzan et al. (2004) FEBS Lett. 7 MG1655 His -tag MTOB to L-Met 571, 141-146) 6 Aromatic aminotransferase: Aro8 from Saccharomyces N-terminal Transamination of (P53090; Bulfer et al. (2013) Protein Sci. 9 25 cervisiae His -tag MTOB to L-Met 22,1417-1424) 6 Amino Acid dehydrogenase EC 1.4.1.9 Leucine dehydrogenase: LeuDH (Li et al. N-terminal (2009) Appl. Biochem. Biotechnol. 158, Bacillus sphaericus 11 His -tag 30 343-351) 6 Reductive amination of MTOB Phenylalanine dehydrogenase: PheDH Thermoactinomyces N-terminal to L-Met (P22823; Takada et al. (1991) J. Biochem. 13 intermedius His -tag 109, 371-376) 6 35 [0018] Without willing to be bound by theory, during the two-step enzymatic synthesis of the L-amino acid from an aldehyde, such as L-methionine from methional, catalyzed by a decarboxylase (e.g. ARO10 or KdcA) and an amino acid dehydrogenase (e.g. LeuDH or PheDH) the cosubstrate NADH is consumed by the dehydrogenase for reduction of the α-carbonyl group of the α-keto acid, e.g. MTOB. 40 [0019] In order to recycle NADH from its oxidized form+ in NAD situ, a formate dehydrogenase (e.g. + CboFDH(C23A/F285S)) can be employed (Figure 1C). This enzyme oxidizes formate with NAD to yield CO2, which may also serve as substrate for the carboxylation reaction of the aldehyde, e.g methional, as well as the cosubstrate NADH (Schütte et al. (1976) Eur. J. Biochem. 62, 151-160; Wichmann et al. (1981) Biotechnol. Bioeng. 23, 2789-2802). [0020] Therefore, in a particular embodiment of the method according to the present invention the reaction mixture 45 comprising an aldehyde, carbon dioxide, a decarboxylase, its corresponding cofactor and NADH, ammonia and/or an ammonium salt and an amino acid dehydrogenase and, optionally, at least one donor amino acid and an aminotransferase, further comprises formic acid or a salt thereof and a formate dehydrogenase. [0021] NADH regeneration can be achieved, for example, by a formate dehydrogenase, such as PseFDH from Pseu- domonas sp. 101 (Egorov et al. (1979) Eur. J. Biochem. 99, 569-576) or formate dehydrogenase from Candida boidinii 50 (CboFDH; 013437; Schütte et al. (1976) Eur. J. Biochem. 62, 151-160) or its mutant CboFDH(C23A/F285S), under consumption of formate and release of CO2. [0022] Therefore, in a preferred embodiment of the method according to the present invention the formate dehydrogenase originates from Pseudomonas sp. or from Candida sp., as well as mutants and variants of these formate dehydrogenases having formate dehydrogenase activity. 55

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Description of the figures

[0023]

5 Figure 1A: Scheme for the two-step biocatalytic synthesis of L-methionine from 3-(methylthio)propanal (methional) involving a decarboxylase and an aminotransferase.

Figure 1B: Scheme for the two-step biocatalytic synthesis of L-methionine from 3-(methylthio)propanal (methional) involving a decarboxylase and an amino acid dehydrogenase. 10 Figure 1C: Scheme for the two-step biocatalytic synthesis of L-methionine from 3-(methylthio)propanal (methional) in the presence of a biocatalytic NADH regeneration system.

Figure 2A: Detection of the reaction product L-methionine using HPLC analytics. The production of L-methionine 15 was verified in a 5 mL sample from Example 4 via HPLC analytics using a C18 column (Gemini C18, 4.6x15 mm, 3 mm, 110 A) and isocratic elution in 4 % (v/v) aqueous acetonitrile supplemented with 1 % v/v phosphoric acid. Methional and L-methionine were detected according to their absorption at 210 nm. L-Methionine synthesis for 105

min under 2 bar (200 kPa) CO2 in the presence of 5 mM KdcA, 5 mM YbdL, 4 mM methional, 50 mM L-glutamine. The dottedtrace correspondsto an L-methioninestandard with defined concentration. Slight variations in the retention 20 time correspond to the typical experimental error between repeated chromatography runs.

Figure 2B: Control reactions under the same conditions as in (a) but omitting KdcA, YbdL or methional, respectively.

Figure 2C: Increase of the L-methionine yield from 3 %, as shown in (a), to 12.5 % after optimization of reaction 25 conditions using rising KdcA concentration: 5 or 10 or 20 mM KdcA, 5 mM YbdL, 4 mM methional, 50 mM L-glutamine; reaction for 105 min under 2 bar (200 kPa) CO2.

Figure 3A: Detection of the reaction product L-methionine using HPLC analytics. The production of L-methionine was verified in a 5 mL sample from Example 5 via HPLC analytics using a C18 column (Gemini C18, 4.6x15 mm, 30 3 mm, 110 A) and isocratic elution in 4 % (v/v) aqueous acetonitrile supplemented with 1 % v/v phosphoric acid. Methional and L-methionine were detected according to their absorption at 210 nm. L-Methionine synthesis for 45 min under 2 bar (200 kPa) CO2 in the presence of 10 mM KdcA, 5 mM LeuDH, 4 mM methional, 4 mM NADH.

Figure 3B: Control reactions under the same conditions as in (a) but omitting KdcA, LeuDH, methional or NADH, 35 respectively.

Figure 3C: Increase of the L-methionine yield from 1.5 %, as shown in (a), to 3 % using doubled LeuDH concentration; reaction for 45 min under 2 bar (200 kPa) CO2.

40 Examples

Example 1: Production of decarboxylases in E. coli

[0024] The gene for a pyruvate decarboxylase (Pdc1; SEQ ID NO: 1; P06169; Killenberg-Jabs et al. (1997) Biochemistry 45 36, 1900-1905) and a phenylpyruvate decarboxylase (Aro10; SEQ ID NO: 3; Q06408; Kneen et al. (2011) FEBS J. 278, 1842-1853), both from Saccharomyces cerevisiae, as well as the gene for a branched chain decarboxylase (KdcA) from Lactococcus lactis (SEQ ID NO: 5; Q6QBS4; Yep et al. (2006) Bioorg. Chem. 34, 325-336) were synthesized with optimal codon usage for expression in E. coli (Geneart, Regensburg, Germany) and subsequently cloned on the expression vector pET21 (Novagen, Madison, WI) using the restriction enzymesNde I and XhoI. The three resulting expression 50 plasmids pET21-Pdc1, pET21-Aro10 and pET21-KdcA, respectively, which also encoded a carboxy-terminal His6-tag for each of the enzymes, were verified by DNA sequencing of the cloned structural genes (Eurofins Genomics, Ebersberg, Germany). [0025] After chemical transformation of E. coli BL21 cells (Studier and Moffatt (1986) J. Mol. Biol. 189, 113-130)

according to the CaCl2-method (Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor 55 Laboratory Press) with these expression plasmids, the enzymes Pdc1, Aro10 and KdcA were individually produced under control of the T7 promoter (Studier and Moffatt, ibid.). To this end, transformed bacteria were grown in 2 liter cultures in LB medium supplemented with 100m g/ml ampicillin at 30° C upon shaking until an OD550 of 0.3-0.4 was reached. After lowering of the temperature during 45-60 min to 22°C, recombinant gene expression was induced at

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OD550 = 0.6-0.8 for 5 h at 22 °C by addition of 0.01 mM isopropylβ-D-1-thiogalactopyranoside (IPTG). Finally, the bacteria were harvested by centrifugation (10 min, 6000 rpm, 4 °C) and the cell paste was frozen at -20 °C. [0026] All decarboxylases were purified using a two-step strategy comprising an immobilized metal ion affinity chromatography (IMAC) followed by a size exclusion chromatography (SEC). Therefore, the cells were resuspended in 3 ml 5 300 mM NaCl, 1 mM MgSO4, 0.1 mM thiamine pyrophosphate (ThDP), 20 mM PIPES/NaOH pH 7.0 per 1 g wet weight and then disrupted mechanically using a French pressure cell (SLM Aminco, Rochester, NY). The homogenate was centrifuged (30 min, 18000 rpm, 4 °C), and the complete supernatant was applied to a 5 ml bed volume HisTrap HP

column (GE Healthcare, Munich, Germany) charged with Ni(II) ions using 300 mM NaCl, 1 mM MgSO 4, 0.1 mM ThDP, 20 mM PIPES/NaOH pH 7.0 as running buffer. The bound decarboxylase was eluted by a linear concentration gradient 10 of 0 to 500 mM imidazole/HCl in running buffer. Main fractions containing the decarboxylase were identified by Com- massie-stained SDS-PAGE and concentrated to a final volume of 2-2.5 ml using a centrifugal filter unit with a nominal molecular weight limit (NMWL) of 30 kDa (Merck, Darmstadt, Germany). The concentrated sample was further purified via SEC using a 120 ml bed volume HiLoad Superdex 200 16/60 column (GE Healthcare) in the presence of 500 mM

NaCl, 1 mM MgSO4, 0.5 mM ThDP, 20 mM PIPES/NaOH pH 7.0. 15 [0027] As result, all three decarboxylases were obtained with >90 % purity as confirmed by Commassie-stained SDS- PAGE analysis. The yield was approximately 50 mg, 10 mg and 30 mg per 1 liter culture volume for Pdc1, Aro10 and KdcA, respectively.

Example 2: Production of aminotransferases in E. coli 20 [0028] The gene for a methionine aminotransferase (YbdL) from E. coli (SEQ ID NO: 7; P77806; Dolzan et al. (2004) FEBS Lett. 571, 141-146) was amplified from E. coli K12 MG1655 using suitable primers and cloned on the expression vector pASK-IBA35(+) (IBA, Göttingen, Germany) using the restriction enzymes Kasl and HindIII. The resulting expres-

sion plasmid pASK-IBA35(+)-YbdL, also encoding an amino-terminal His 6-tag for YbdL, was verified by DNA sequencing 25 of the cloned structural gene (Eurofins Genomics). [0029] The gene for an aromatic aminotransferase (Aro8) fromSaccharomyces cervisiae (SEQ ID NO: 9; P53090; Bulfer et al. (2013) Protein Sci. 22, 1417-1424) was synthesized with optimal codon usage for expression E.in coli (Geneart) and cloned on the expression vector pASK-IBA35(+) using the restriction enzymes Kasl andHind III. The

resulting expression plasmid pASK-IBA35(+)-Aro8, also encoding an amino-terminal His 6-tag for Aro8, was verified by 30 DNA sequencing of the cloned structural gene (Eurofins Genomics). [0030] Both enzymes, YbdL and Aro8, were produced in E. coli BL21 under control of the tet promoter (Skerra (1994) Gene 151, 131-135). Therefore, E. coli BL21 cells were transformed according to the CaCl 2-method (Sambrook et al., ibid.) with the corresponding expression plasmid and subsequently grown in 2 liter LB medium supplemented with 100 mg/ml ampicillin at 30 °C upon shaking until an OD550 = 0.3-0.4 was reached. Then, the temperature was reduced to 35 22 °C during 45-60 min and recombinant gene expression was induced with 0.2 mg/l anhydrotetracycline (aTc; Acros, Geel, Belgium). After 5 h at 22°C the bacteria were harvested by centrifugation (10 min, 6000 rpm, 4 °C) and frozen at -20 °C. [0031] To purify the aminotransferases, the cells containing each recombinant protein were resuspended in 3 ml 500 mM NaCl, 40 mM Tris/HCl pH 7.4 per 1 g wet weight. Then, the bacteria were disrupted mechanically in a French 40 pressure cell. The homogenate was centrifuged (30 min, 18000 rpm, 4 °C) and the entire supernatant was applied to a 5 ml bed volume HisTrap HP column (GE Healthcare) charged with Ni(II) ions using 500 mM NaCl, 40 mM Tris/HCl pH 7.4 as running buffer. The bound aminotransferase was eluted by a linear concentration gradient of 0 to 500 mM imidazole/HCl in running buffer. Main fractions containing the aminotransferase were identified by Coomassie-stained SDS-PAGE and concentrated to a final volume of 4-5 ml using a centrifugal filter unit with a NMWL of 30 kDa. In a 45 second step, the concentrated sample was purified by SEC using a 320 ml bed volume HiLoad Superdex 200 26/60 column in the presence of 500 mM NaCl, 20 mM Tris/HCl pH 7.4. [0032] Both aminotransferases were obtained with >90 % purity as confirmed by SDS-PAGE analysis with a yield of 18 mg/l for YbdL and 47 mg/l for Aro8.

50 Example 3: Production of amino acid dehydrogenases in E. coli

[0033] The gene for the leucine dehydrogenase fromBacillus sphaericus (LeuDH; SEQ ID NO: 11; Li et al. (2009) Appl. Biochem. Biotechnol. 158, 343-351) and the gene for the Phenylalanine dehydrogenase from Thermoactinomyces intermedius (PheDH; SEQ ID NO: 13; P22823; Takada et al. (1991) J. Biochem. 109, 371-376) were synthesized with 55 optimal codon usage for expression in E. coli (Geneart) and cloned on the expression vector pASK-IBA35(+) using the restriction enzymes Kasl andHind III. The resulting expression plasmids pASK-IBA35(+)-LeuDH and pASK- IBA35(+)-PheDH, respectively, both also encoding an amino-terminal His 6-tag, were verified by DNA sequencing of the cloned structural gene (Eurofins Genomics).

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[0034] LeuDH as well as PheDH were produced in E. coli BL21 under the same conditions as the aminotransferases described herein above in Example 2 with the exception that the culture was incubated for 5 h at 30°C (instead of 22°C) after induction with aTc. [0035] For purification of both amino acid dehydrogenases the bacterial paste was resuspended in 3 ml 100 mM NaCl, 5 50 mM Tris/HCl pH 8.0 per 1 g wet cell mass and disrupted mechanically using a French pressure cell. After centrifugation (30 min, 18000 rpm, 4 °C), the supernatant was applied to a 5 ml bed volume HisTrap HP column (GE Healthcare) charged with Ni(II) ions using 100 mM NaCl, 50 mM Tris/HCl pH 8.0 as running buffer. The bound amino acid dehydrogenase was eluted by a linear concentration gradient of 0 to 500 mM imidazole/HCl in running buffer. Main fractions containing the amino acid dehydrogenase were identified by Coomassie-stained SDS-PAGE and concentrated to a final 10 volume of 4-5 ml using a centrifugal filter unit with a NMWL of 30 kDa (Merck). In a second step, the concentrated sample was purified by SEC using a 320 ml bed volume HiLoad Superdex 200 26/60 column in the presence of 300 mM NaCl, 20 mM Tris/HCl pH 8.0. [0036] The LeuDH and PheDH were obtained in yields of 7.5 mg/l and 19 mg/l, respectively. High purity of >95 % was confirmed by Commassie-stained SDS-PAGE analysis. 15 Example 4: Synthesis of L-methionine from 3-(methylthio)propanal (methional) by a two-step biocatalytic reaction involving a decarboxylase and an aminotransferase.

[0037] To synthesize L-methionine in a two-step biocatalytic reaction (Fig. 1A), the purified decarboxylase KdcA and 20 the aminotransferase YbdL were mixed with the following reagents in a 10 ml pressure reactor (Tinyclave steel; Büchi, Uster, Switzerland) to a final volume of 1 ml:

Reagent/enzyme Final concentration

25 NaHCO3 200 mM ThDP 0.5 mM

MgSO4 1 mM KdcA 5 mM 30 YbdL 5 mM L-Glutamine 50 mM Methional 4 mM

[0038] The reaction was started by the addition of the substrate methional and application of 2 bar (200 kPa) CO2. The initial pH of the mixture was 8, which shifted to ca. 6.5 upon application of CO 2 (as measured with a fixed-color pH indicator stick; Carl Roth, Karlsruhe, Germany). After 1 h incubation the mixture was collected from the reactor and centrifuged for 5 min at 13400 rpm in a bench top centrifuge to remove precipitated protein. Using the clear supernatant, 40 product formation was analyzed by HPLC using a C18 column (Gemini C18, 4.6x15 mm, 3 mm, 110 A; Phenomenex, Aschaffenburg, Germany) with isocratic elution in 4 % (v/v) aqueous acetonitrile supplemented with 1 % (v/v) phosphoric acid. [0039] Compared to control reactions with omission of KdcA, YbdL or methional, respectively (Fig. 2B), the chromatograms of the two-step biocatalytic synthesis in the presence of the decarboxylase (e.g. KdcA) and the aminotransferase 45 (e.g. YbdL) as well as a donor amino acid (e.g. L-glutamine) clearly demonstrated that L-methionine was produced from methional (Fig. 2A). By increasing the concentration of the decarboxylase to 20 mM, the L-methionine yield was improved from 3 % to 12.5 % (Fig. 2C).

Example 5: Synthesis of L-methionine from methional by a two-step biocatalytic reaction involving a decarbox- 50 ylase and an amino acid dehydrogenase.

[0040] To synthesize L-methionine in a two-step biocatalytic reaction without the need for an amino donor cosubstrate (Fig. 1 B), the purified decarboxylase KdcA and the amino acid dehydrogenase LeuDH were mixed with the following reagents in a 10 ml pressure reactor (Tinyclave steel) to a final volume of 1 ml: 55 Reagent/enzyme Final concentration

NH4HCO3 500 mM

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(continued)

Reagent/enzyme Final concentration ThDP 0.5 mM 5 MgSO4 1 mM KdcA 10 mM LeuDH 5 mM 10 NADH 4 mM Methional 4 mM

[0041] Like in Example 4, the reaction was started by the addition of methional and application of 2 bar (200 kPa) 15 CO2. The initial pH of the mixture was 8 and shifted to ca. 7 upon application of CO2 (as measured with a fixed-color pH indicator stick). After 45 min incubation the mixture was recovered from the reactor and analyzed using HPLC as described in Example 4. [0042] Compared to a control reaction with omission of the decarboxylase (e.g. KdcA), the amino acid dehydrogenase (e.g. LeuDH) or of NADH, respectively (Fig. 3B), the chromatograms of the two-step biocatalytic synthesis in the presence 20 of a decarboxylase (e.g. KdcA) and an amino acid dehydrogenase (e.g. LeuDH) as well as NADH and an ammonium salt clearly demonstrated that L-methionine was produced from methional (Fig. 3A). By doubling the concentration of the amino acid dehydrogenase to 10 mM, the L-methionine yield was improved from 1.5 % to 3 % (Fig. 3C).

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Claims

1. A method for producing an L-amino acid, comprising a step of reacting a mixture comprising an aldehyde, carbon 30 dioxide, a decarboxylase, its corresponding cofactor and

(a) at least one donor amino acid and an aminotransferase and/or (b) NADH, ammonia and/or an ammonium salt and an amino acid dehydrogenase to form said L-amino acid or a salt thereof. 35 2. The method as claimed in claim 1, wherein the L-amino acid to be formed is selected from the group consisting of L-alanine, L-valine, L-methionine, L-leucine, L-isoleucine and L-phenylalanine.

3. The method as claimed in any of claims 1 or 2, wherein the L-amino acid to be formed is L-methionine and the 40 aldehyde is 3-(methylthio)-propanal (methional).

4. The method as claimed in any of claims 1 to 3, wherein the cofactor comprises thiamine pyrophosphate.

5. The method as claimed in any of claims 1 to 4, wherein the decarboxylase is selected from the group consisting of 45 pyruvate decarboxylase PDC1, which originates from Saccharomyces cerevisiae, phenylpyruvate decarboxylase ARO10, which originates from Saccharomyces cerevisiae, and branched chain decarboxylase KdcA, which originates from Lactococcus lactis, as well as mutants and variants of these decarboxylase having decarboxylase activity.

6. The method as claimed in any of claims 1 to 5, wherein the aminotransferase is selected from the group consisting 50 of methionine aminotransferase YbdL, which originates from E. coli, and aromatic aminotransferase Aro8, which originates from Saccharomyces cerevisiae, as well as mutants and variants of these aminotransferases having aminotransferase activity.

7. The method as claimed in any of claims 1 to 6, wherein the amino acid dehydrogenase is selected from the group 55 consisting of leucine dehydrogenase LeuDH, which originates from Bacillus sphaericus, and phenylalanine dehydrogenase PheDH, which originates from Thermoactinomyces intermedius, as well as well as mutants and variants of these amino acid dehydrogenases having amino acid dehydrogenase activity.

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8. The method as claimed in any of claims 1 to 7, wherein the donor amino acid is different from the L-amino acid to be formed and at least one L-amino acid is selected from the group consisting of L-glutamine, L-glutamate, L-alanine, L-phenylalanine, L-tyrosine, L-leucine, L-isoleucine, L-histidine and L-tryptophan.

5 9. The method as claimed in any of claims 1 to 8, wherein the carbon dioxide is applied to the mixture at a pressure from 10 to 7400 kPa.

10. The method as claimed in any of claims 1 to 9, wherein the mixture further comprises formic acid or a salt thereof and a formate dehydrogenase. 10 11. The method as claimed in claim 9, wherein the formate dehydrogenase is selected from the group consisting of formate dehydrogenase from Pseudomonas sp. and formate dehydrogenase from Candida sp. as well as mutants and variants of these formate dehydrogenases having formate dehydrogenase activity.

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description

• US 5770769 A [0004] • WO 2008013432 A1, Kim [0007] • WO 2005121068 A1 [0005] • WO 2012091479 A2, Hong [0007] • WO 2005121079 A1 [0005]

Non-patent literature cited in the description

•WILKE. Appl. Microbiol. Biotechnol., 2014, vol. 98, • BULFER et al. Protein Sci., 2013, vol. 22, 1417-1424 9893-9914 [0006] [0017] [0029] • MIYAZAKI et al. Chem. Commun., 2001, 1800-1801 • LI et al. Appl. Biochem. Biotechnol., 2009, vol. 158, [0008] 343-351 [0017] [0033] • TONG et al. Biotechnol. Bioeng., 2011, vol. 108, • TAKADA et al. J. Biochem., 1991, vol. 109, 371-376 465-469 [0008] [0017] [0033] • WICHMANN et al. Biotechnol. Bioeng., 1981, vol. 23, • SCHÜTTE et al. Eur. J. Biochem., 1976, vol. 62, 2789-2802 [0009] [0019] 151-160 [0019] [0021] • KILLENBERG-JABS et al. Biochemistry, 1997, vol. • EGOROV et al. Eur. J. Biochem., 1979, vol. 99, 36, 1900-1905 [0017] [0024] 569-576 [0021] • KNEEN et al. FEBS J., 2011, vol. 278, 1842-1853 • Eurofins Genomics [0024] [0017] [0024] •STUDIER; MOFFATT. J. Mol. Biol., 1986, vol. 189, • YEP et al. Bioorg. Chem., 2006, vol. 34, 325-336 113-130 [0025] [0017] [0024] • SAMBROOK et al. Molecular Cloning: A Laboratory • DOLZAN et al. FEBS Lett., 2004, vol. 571, 141-146 Manual. Cold Spring Harbor Laboratory Press, 1989 [0017] [0028] [0025] • SKERRA. Gene, 1994, vol. 151, 131-135 [0030]