Enzymatic method for producing 2-hydroxy-4-methylmercaptobutanoic acid (MHA)

Abstract

The invention relates to an enzymatic method for producing 2-hydroxy-4-methylmercaptobutanoic acid from 3-methylthio-propanal (3-methylmercaptopropanal (MMP) or methional) and carbon dioxide.

Claims

1. A method for producing D- or L-2-hydroxy-4-methylmercaptobutanoic acid (MHA), comprising reacting a mixture comprising: 3-(methylthio)-propanal (methional); carbon dioxide; a decarboxylase (EC 4.1.1); a corresponding cofactor of the decarboxylase; an alcohol dehydrogenase (EC 1.1.1); and NADH or NADPH, to form D- or L-2-hydroxy-4-methylmercaptobutanoic acid (MHA) or a salt thereof.

2. The method of claim 1, wherein the cofactor comprises thiamine pyrophosphate.

3. The method of claim 1, wherein the decarboxylase is selected from the group consisting of 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.

4. The method of claim 1, wherein said method is for producing D-2-hydroxy-4-methylmercapto-butanoic acid (D-MHA), and wherein the alcohol dehydrogenase is a D-hydroxyisocaproate dehydrogenase.

5. The method of claim 4, wherein the D-hydroxyisocaproate dehydrogenase is D-HicDH from Lactobacillus casei.

6. The method of claim 1, where said method is for producing L-2-hydroxy-4-methylmercapto-butanoic acid (L-MHA), and wherein the alcohol dehydrogenase is a L-hydroxyisocaproate dehydrogenase.

7. The method of claim 6, wherein the L-hydroxyisocaproate dehydrogenase is L-HicDH from Lactobacillus confusus.

8. The method of claim 1, wherein the carbon dioxide is applied to the mixture at a pressure from 10 to 7400 kPa.

9. The method of claim 1, wherein the mixture further comprises formic acid or a salt thereof and a formate dehydrogenase (EC 1.17.1.9).

10. The method of claim 9, wherein the formate dehydrogenase is selected from the group consisting of a formate dehydrogenase from Pseudomonas sp. and a formate dehydrogenase from Candida sp.

11. The method of claim 2, wherein the decarboxylase is selected from the group consisting of 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.

12. The method of claim 11, wherein said method is for producing D-2-hydroxy-4-methylmercapto-butanoic acid (D-MHA), and wherein the alcohol dehydrogenase is a D-hydroxyisocaproate dehydrogenase.

13. The method of claim 12, wherein the D-hydroxyisocaproate dehydrogenase is D-HicDH from Lactobacillus casei.

14. The method of claim 13, wherein the carbon dioxide is applied to the mixture at a pressure from 10 to 7400 kPa.

15. The method of claim 14, wherein the mixture further comprises formic acid or a salt thereof and a formate dehydrogenase (EC 1.17.1.9).

16. The method of claim 15, wherein the formate dehydrogenase is selected from the group consisting of a formate dehydrogenase from Pseudomonas sp. and a formate dehydrogenase from Candida sp.

17. The method of claim 2, where said method is for producing L-2-hydroxy-4-methylmercapto-butanoic acid (L-MHA), and wherein the alcohol dehydrogenase is a L-hydroxyisocaproate dehydrogenase.

18. The method of claim 17, wherein the L-hydroxyisocaproate dehydrogenase is L-HicDH from Lactobacillus confusus.

19. The method of claim 18, wherein the decarboxylase is selected from the group consisting of 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.

20. The method of claim 19, wherein the carbon dioxide is applied to the mixture at a pressure from 10 to 7400 kPa and wherein the mixture further comprises formic acid or a salt thereof and a formate dehydrogenase (EC 1.17.1.9).

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: Scheme for the two-step biocatalytic synthesis of a methionine-hydroxy-analog (MHA) from 3-(methylthiopropanal (methional) in the presence of a decarboxylase, an alcohol dehydrogenase and optionally a formate dehydrogenase as a biocatalytic NADH regeneration system.

(2) First, under a CO.sub.2 atmosphere a decarboxylase (e.g., KdcA, Pdc1, Aro10) is employed to catalyze the reverse reaction, i.e. the carboxylation of methional, which leads to the intermediate product 4-methylthio-2-oxobutanoic acid (MTOB). Second, the -carbonyl group of MTOB is reduced to the hydroxyl group in a stereospecific and NADH- (or, alternatively, NADPH-) dependent reaction catalyzed by an alcohol dehydrogenase (e.g., D/L-HicDH) to yield D- or L-MHA. Optionally, NADH regeneration can be achieved, for example, by a formate dehydrogenase (such as CboFDH(C23A/F285S)/PseFDH) under consumption of formate and generation of CO.sub.2.

(3) FIG. 2a: Detection of the reaction product D-methionine-hydroxy-analog (D-MHA) using HPLC analytics. The production of D-MHA was verified in a 5 L sample from Examples 4 and 5 via HPLC analytics using a C18 column (Gemini C18, 4.615 mm, 3 m, 110 ) and isocratic elution in 4% (v/v) aqueous acetonitrile supplemented with 1% v/v phosphoric acid. Methional and MHA were detected according to their absorption at 210 nm. D-MHA synthesis for 1 h under 2 bar CO.sub.2 in the presence of 10 M KdcA, 1 M D-HicDH, 4 mM methional, 2 mM NADH. The dotted trace corresponds to an MHA standard with defined concentration.

(4) FIG. 2b: Control reactions under the same conditions as in (a) but omitting KdcA, D-HicDH, methional or NADH, respectively.

(5) FIG. 2c: Increase of the D-MHA yield, from 3% as shown in (a), to 23%, after optimization of reaction conditions using twice the KdcA concentration, an eightfold reaction time and a fourfold CO.sub.2 pressure: 20 M KdcA, 1 M D-HicDH, 4 mM methional, 4 mM NADH; catalysis for 8 h under 8 bar CO.sub.2.

(6) FIG. 2d: Under limiting NADH concentration, the presence of a biocatalytic NADH regeneration system increases the final D-MHA concentration: 10 M KdcA, 0.5 M D-HicDH, 10 M CboFDH(C23A/F285S), 4 mM methional, 80 M NADH, 25 mM formate; catalysis for 1.5 h under 8 bar CO.sub.2. An enlargement of the MHA peak is shown in the inset.

(7) FIG. 3a: Detection of the reaction product L-methionine-hydroxy-analog (L-MHA) using HPLC analytics. The production of L-MHA was verified in a 5 L sample from Example 6 via HPLC analytics using a C18 column (Gemini C18, 4.615 mm, 3 m, 110 ) and isocratic elution in 4% (v/v) aqueous acetonitrile supplemented with 1% v/v phosphoric acid. Methional and MHA were detected according to their absorption at 210 nm. L-MHA synthesis for 45 min under 8 bar CO.sub.2 in the presence of 20 M KdcA, 0.5 M L-HicDH, 4 mM Methional, 4 mM NADH. The dotted trace corresponds to an MHA standard with defined concentration.

(8) FIG. 3b: Control reactions under the same conditions as in (a) but omitting KdcA, L-HicDH, methional or NADH, respectively.

EXAMPLES

Example 1: Production of Decarboxylase in E. coli

(9) The gene for a pyruvate decarboxylase (Pdc1; SEQ ID NO: 1; P06169; Killenberg-Jabs et al. (1997) Biochemistry 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, Wis.) using the restriction enzymes NdeI and XhoI. The three resulting expression plasmids pET21-Pdc1, pET21-Aro10 and pET21-KdcA, respectively, which also encoded a carboxy-terminal His.sub.6-tag for each of the enzymes, were verified by DNA-sequencing of the cloned structural gene (Eurofins Genomics, Ebersberg, Germany).

(10) After chemical transformation of E. coli BL21 cells (Studier and Moffatt (1986) J. Mol. Biol. 189, 113-130) according to the CaCl.sub.2-method (Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press) with these expression plasmids, Pdc1 (SEQ ID NO: 2), Aro10 (SEQ ID NO: 4) and KdcA (SEQ ID NO: 6) were individually produced under control of the T7 promoter (Studier and Moffatt (1986) J Mol Biol 189, 113-130). To this end, bacteria were grown in 2 liter cultures in LB medium supplemented with 100 g/ml ampicillin at 30 C. upon shaking until an OD.sub.550 of 0.3-0.4 was reached. After reduction of the temperature during 45-60 min to 22 C., recombinant gene expression was induced at OD.sub.550=0.6-0.8 for 5 h at 22 C. by addition of 0.01 mM isopropyl.sup.-D-1-thiogalactopyranoside (IPTG). Finally, the bacteria were harvested by centrifugation (10 min, 6000 rpm, fixed angle rotor, 4 C.) and the cell paste was frozen at 20 C.

(11) 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 300 mM NaCl, 1 mM MgSO.sub.4, 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, N.Y.). The homogenate was centrifuged (30 min, 18000 rpm, fixed angle rotor, 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.sub.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 of 0 to 500 mM imidazole/HCl in running buffer. Main fractions containing the decarboxylase were identified by Commassie-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 MgSO.sub.4, 0.5 mM ThDP, 20 mM PIPES/NaOH pH 7.0.

(12) 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 Alcohol Dehydrogenase in E. coli

(13) The gene for a D-hydroxyisocaproate dehydrogenase (D-HicDH) from Lactobacillus casei (SEQ ID NO: 7; P17584; Hummel et al. (1985) Appl. Microbiol. Biotechnol. 21, 7-15) was synthesized with optimal codon usage for expression in E. coli (Geneart) and cloned on the expression vector pASK-IBA35(+) (IBA, Gottingen, Germany) using the restriction enzymes KasI and HindIII. The resulting expression plasmid pASK-IBA35(+)-D-HicDH, also encoding an amino-terminal His.sub.6-tag for the D-HicDH, was verified by DNA-sequencing of the cloned structural gene (Eurofins Genomics). The gene for a L-hydroxyisocaproate dehydrogenase (L-HicDH) from Lactobacillus confusus (SEQ ID NO: 9; P14295; Schtte et al. (1984) Appl. Microbiol. Biotechnol. 19, 167-176) was synthesized with optimal codon usage for expression in E. coli (Geneart). As an amino-terminal His.sub.6-tag would disrupt the tetramer formation of L-HicDH, the synthesized gene (SEQ ID NO: 9) was cloned on the expression vector pASK75(T7RBS)his using the restriction enzymes NdeI and Eco47III. The resulting expression plasmid pASK75(T7RBS)L-HicDH-his, encoding the L-HicDH with a carboxy-terminal His.sub.6-tag, was verified by DNA-sequencing of the cloned structural gene (Eurofins Genomics).

(14) Both enzymes, the D-HicDH (SEQ ID NO: 8) and the L-HicDH (SEQ ID NO: 10), were produced in E. coli BL21 under the control of the tet promoter (Skerra (1994) Gene 151, 131-135). Therefore, E. coli BL21 cells were transformed according to the CaCl.sub.2-method (Sambrook et al., ibid.) with the corresponding expression plasmid and subsequently grown in 21 LB medium supplemented with 100 g/ml ampicillin at 30 C. upon shaking until an OD.sub.550=0.3-0.4 was reached. Then, for production of the D-HicDH the temperature was reduced to 22 C. during 45-60 min, while for the production of the L-HicDH the temperature was kept at 30 C. In both cases, the recombinant gene expression was induced with 0.2 mg/l anhydrotetracycline (aTc; Acros, Geel, Belgium) at OD.sub.550=0.6-0.8. After 5 h at 22 C./30 C. the bacteria were harvested by centrifugation (10 min, 6000 rpm, fixed angle rotor, 4 C.) and frozen at 20 C.

(15) To purify both dehydrogenases, the cells containing the D-HicDH were resuspended in 3 ml 150 mM NaCl, 50 mM PIPES pH 7.0 per 1 g wet weight while the cells containing L-HicDH were resuspended in 3 ml 300 mM NaCl, 50 mM KP.sub.i pH 7 per 1 g wet weight. Then the bacteria were disrupted mechanically in a French pressure cell. The homogenate was centrifuged (30 min, 18000 rpm, fixed angle rotor, 4 C.) and the entire supernatant was applied to a 5 ml bed volume HisTrap HP column charged with Ni(II) ions using 150 mM NaCl, 50 mM PIPES pH 7.0 for the D-HicDH and 300 mM NaCl, 50 mM KP.sub.i pH 7 for the L-HicDH, respectively, as running buffer. The bound dehydrogenase was eluted by a linear concentration gradient of 0 to 500 mM imidazole/HCl in running buffer. Main fractions containing the dehydrogenase were identified by Commassie-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 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 150 mM NaCl, 50 mM PIPES pH 7.0 and 300 mM NaCl, 20 mM KP.sub.i pH 6.5 for D-HicDH and L-HicDH, respectively.

(16) Both alcohol dehydrogenases were obtained with >90% purity as confirmed by SDS-PAGE analysis with a yield of 7 mg/l for D-HicDH and >47 mg/l for L-HicDH.

Example 3: Production of Formate Dehydrogenase in E. coli

(17) The gene for the formate dehydrogenase from Pseudomonas sp. 101 (PseFDH; SEQ ID NO: 11; P33160; Egorov et al. (1979) Eur. J. Biochem. 99, 569-576) was synthesized with optimal codon usage for expression in E. coli (Geneart) and cloned on the expression vector pASK-IBA35(+) using the restriction enzymes KasI and HindIII. Also, the gene for the formate dehydrogenase from Candida boidinii (CboFDH; 013437; Schtte et al. (1976) Eur. J. Biochem. 62, 151-160) was synthesized with optimal codon usage for expression in E. coli (Geneart) carrying two amino acid exchanges, C23A and F285S, to potentially enhance stability and activity. Substitution of Cys23 with its reactive thiol side chain by Ala should stabilize the enzyme against oxidation in a similar manner as the previously described mutation C23S (Slusarczyk et al. (2000) Eur. J. Biochem, 267, 1280-1287). The substitution of Phe285 by Ser was previously shown to enhance the enzyme activity (Felber (2001) Doctoral Thesis, Heinrich-Heine University Dusseldorf; US 20030157664 A1). The resulting gene coding for CboFDH(C23A/F285S) (SEQ ID NO: 13) was cloned on pASK-IBA35(+) as described above for the PseFDH. The resulting expression plasmids pASK-IBA35(+)-CboFDH and pASK-IBA35(+)-PseFDH, respectively, both also encoding an amino-terminal His.sub.6-tag, were verified by DNA-sequencing of the cloned structural gene (Eurofins Genomics).

(18) PseFDH (SEQ ID NO: 12) as well as CboFDH(C23A/F285S) (SEQ ID NO: 14) were produced in E. coli BL21 under the same conditions as the alcohol dehydrogenase D-HicDH described herein above in Example 2.

(19) For the purification of both FDHs the bacterial paste was resuspended in 3 ml per 1 g wet cell mass in 300 mM NaCl, 50 mM KP.sub.i pH 7.5 and disrupted mechanically using a French pressure cell. After centrifugation (30 min, 18000 rpm, fixed angle rotor, 4 C.), the entire supernatant was applied to a 5 ml bed volume HisTrap HP column charged with Ni(II) ions using 300 mM NaCl, 50 mM KP.sub.i pH 7.5 as running buffer. The bound FDH was eluted by a linear concentration gradient of 0 to 500 mM imidazole/HCl in running buffer. The eluted protein was concentrated using a centrifugal filter unit with a NMWL of 30 kDa. 4 ml protein solution containing approximately 50 mg PseFDH, or 6 ml containing about 130 mg CboFDH(C23A/F285S), were loaded on a 320 ml bed volume HiLoad Superdex 200 26/60 column for SEC in the presence of 300 mM NaCl, 20 mM KP.sub.i pH 7.5.

(20) The formate dehydrogenases PseFDH and CboFDH(C23A/F285S) were obtained in yields of 19 mg/l and 45 mg/1, respectively. High purity of >95% was confirmed by Commassie-stained SDS-PAGE analysis.

Example 4: Synthesis of the D-Methionine-Hydroxy-Analog (D-MHA) from 3-(Methylthio)Propanal (Methional) by a Two-Step Biocatalytic Reaction Involving a Decarboxylase and an Alcohol Dehydrogenase

(21) To synthesize D-MHA in the proposed two-step biocatalytic reaction (FIG. 1), the purified decarboxylase KdcA and the alcohol dehydrogenase D-HicDH were mixed with the following reagents in a 10 ml pressure reactor (Tinyclave steel; Biichi, Uster, Switzerland) to a final volume of 1 ml:

(22) TABLE-US-00002 Reagent/enzyme Final concentration NaHCO.sub.3 200 mM ThDP 0.5 mM MgCl.sub.2 1 mM KdcA 10 M D-HicDH 1 M NADH 2 mM Methional 4 mM

(23) The carboxylation reaction under catalysis of the decarboxylase KdcA was started by the addition of the substrate methional and application of 200 kPa (2 bar) CO.sub.2. The initial pH of the mixture was 8, which shifted to ca. 6.5 upon application of CO.sub.2 (as measured with a fixed-color-pH indicator stick (Carl Roth, Karlsruhe, Germany) in a sample). After 1 h incubation the mixture was recovered from the reactor and centrifuged for 5 min at 13400 rpm in a bench top centrifuge to remove precipitated proteins. In the clear supernatant, product formation was analyzed by HPLC using a C18 column (Gemini C18, 4.615 mm, 3 m, 110 A; Phenomenex, Aschaffenburg, Germany) with isocratic elution in 4% (v/v) aqueous acetonitrile supplemented with 1% (v/v) phosphoric acid.

(24) Compared to control reactions with omission of KdcA, D-HicDH, methional or NADH, respectively (FIG. 2b), the chromatograms of the two-step biocatalytic synthesis in the presence of the decarboxylase (e.g. KdcA) and the dehydrogenase (e.g. D-HicDH) clearly demonstrated that D-MHA was produced from methional under the chosen reaction conditions (FIG. 2a). By elongating the reaction time to 8 h, increasing the CO.sub.2 pressure to 800 kPa (8 bar) and doubling the KdcA concentration to 20 M the D-MHA yield was improved from 3% to 23% (FIG. 2c).

Example 5: Synthesis of D-MHA from Methional by a Two-Step Biocatalytic Reaction Involving a Decarboxylase and an Alcohol Dehydrogenase in the Presence of a Biocatalytic NADH Regeneration System

(25) During the two-step enzymatic synthesis of D-MHA from methional catalyzed by a decarboxylase (e.g. KdcA) and an alcohol dehydrogenase (e.g. D-HicDH) the cosubstrate NADH is consumed by the dehydrogenase for reduction of the -carbonyl group of MTOB. In order to recycle NADH from its oxidized form NAD.sup.+ in situ a formate dehydrogenase (e.g. CboFDH(C23A/F285S)) can be employed. This enzyme oxidizes formate with NAD.sup.+ as cosubstrate to yield CO.sub.2, which may also serve as substrate for the carboxylation reaction of methional, as well as NADH (Schtte et al. (1976) Eur. J. Biochem. 62, 151-160; Wichmann et al. (1981) Biotechnol. Bioeng. 23, 2789-2802).

(26) This three-enzyme coupled reaction was performed under a limiting concentration of NADH (80 M), which was added to a reaction mixture in a 10 ml pressure reactor (Tinyclave steel) containing the following reagents in a final volume of 1 ml:

(27) TABLE-US-00003 Reagent/enzyme Final concentration NaHCO.sub.3 200 mM ThDP 0.5 mM MgCl.sub.2 1 mM KdcA 10 M D-HicDH 0.5 M CboFDH(C23A/F285S) 10 M NADH 80 M NaHCO.sub.3 25 mM Methional 4 mM

(28) As in Example 4 the reaction was started by the addition of methional and application of 800 kPa (8 bar) CO.sub.2. The initial pH of the mixture was 8 and shifted to ca. 6.5 upon application of CO.sub.2 (as measured with a fixed-color-pH indicator stick in a sample). After 1.5 h incubation, the mixture was recovered from the reactor and analyzed using HPLC as described in Example 4. The resulting chromatogram showed a significantly increased D-MHA peak (114 M) compared to the control reaction (38 M) in which the substrate formate of the formate dehydrogenase was omitted (FIG. 2 D).

(29) Thus, the addition of a formate dehydrogenase together with its substrate to the D-MHA-forming reaction, involving a decarboxylase (e.g., KdcA) and a NADH dependent alcohol dehydrogenase (e.g. D-HicDH), can compensate for limiting NADH concentrations and regenerate this cosubstrate.

Example 6: Synthesis of the L-Methionine-Hydroxy-Analog (L-MHA) from Methional by a Two-Step Biocatalytic Reaction Involving a Decarboxylase and an Alcohol Dehydrogenase

(30) To synthesize L-MHA in the proposed two-step biocatalytic reaction (FIG. 1), the purified decarboxylase (e.g. KdcA) and the alcohol dehydrogenase (e.g. L-HicDH) were mixed with the following reagents in a 10 ml pressure reactor (Tinyclave steel) to a final volume of 1 ml:

(31) TABLE-US-00004 Reagent/enzyme Final concentration NaHCO.sub.3 200 mM ThDP 0.5 mM MgCl.sub.2 1 mM KdcA 20 M L-HicDH 500 nM NADH 4 mM Methional 4 mM

(32) The reaction was started by the addition of methional and application of 800 kPa (8 bar) CO.sub.2 as described herein above in Examples 4 and 5. Upon the application of CO.sub.2 the initial pH of 8 was shifted to 6.5 as measured with a fixed-color-pH indicator stick in a sample.

(33) After 45 min at 800 kPa (8 bar), the mixture was recovered from the autoclave and centrifuged for 5 min at 13400 rpm in a bench top centrifuge to remove precipitated proteins. In the cleared supernatant, product formation was analyzed by HPLC using a C18 column as described in Example 4.

(34) The chromatograms for the two-step biocatalytic synthesis of L-MHA from methional via combined action of a decarboxylase (e.g. KdcA) and a dehydrogenase (e.g. L-HicDH; FIG. 3 A) and for control reactions with KdcA, L-HicDH, methional or NADH, respectively, omitted (FIG. 3 B) clearly demonstrated that L-MHA was specifically synthesized from methional under the chosen reaction conditions only if both enzymes, a decarboxylase, e.g. KdcA, and an alcohol dehydrogenase, e.g. L-HicDH, as well as the cofactor NADH were present.

(35) All references cited herein are fully incorporated by reference. Having now fully described the invention, it will be understood by one of skill in the art that the invention may be performed within a wide and equivalent range of conditions, parameters and the like, without affecting the spirit or scope of the invention or any embodiment thereof.