PREPARATION OF 6-AMINOCAPROIC ACID FROM 5-FORMYL VALERIC ACID

Abstract

The invention relates to a method for preparing 6-aminocaproic acid (hereinafter also referred to as ‘6-ACA’) using a biocatalyst. The invention further relates to a method for preparing ε-caprolactam (hereafter referred to as ‘caprolactam’) by cyclising such 6-ACA. The invention further relates to a host cell, a micro-organism, or a polynucleotide which may be used in the preparation of 6-ACA or caprolactam.

Claims

1. (canceled)

2. Method for preparing 6-aminocaproic acid, wherein the 6-aminocaproic acid is prepared from 5-formylpentanoate, using a recombinant host cell comprising at least one biocatalyst, wherein the biocatalyst is an enzyme capable of catalysing a transamination and/or a reductive amination selected from the group of aminotransferases (E.C. 2.6.1) and amino acid dehydrogenases (E.C.1.4.1).

3-4. (canceled)

5. Method according to claim 2, wherein the aminotransferase or amino acid dehydrogenase is selected from the group of β-aminoisobutyrate:α-ketoglutarate aminotransferases, β-alanine aminotransferases, aspartate aminotransferases, 4-amino-butyrate aminotransferases (EC 2.6.1.19), L-lysine 6-aminotransferase (EC 2.6.1.36), 2-aminoadipate aminotransferases (EC 2.6.1.39), 5-aminovalerate aminotransferases (EC 2.6.1.48), 2-aminohexanoate aminotransferases (EC 2.6.1.67), lysine:pyruvate 6-aminotransferases (EC 2.6.1.71), and lysine-6-dehydrogenases (EC 1.4.1.18).

6. Method according to claim 5, wherein the enzyme is selected from the group of enzymes capable of catalysing a transamination and/or a reductive amination from an organism selected from the group of Vibrio; Pseudomonas; Bacillus; Mercurialis; Asplenium; Ceratonia; mammals; Neurospora; Escherichia; Thermus; Saccharomyces; Brevibacterium; Corynebacterium; Proteus; Agrobacterium; Geobacillus; Acinetobacter; Ralstonia; Salmonella; Rhodobacter and Staphylococcus, in particular from an organism selected from the group of Bacillus subtilis, Bacillus weihenstephanensis, Rhodobacter sphaeroides, Staphylococcus aureus, Legionella pneumophila, Nitrosomonas europaea, Neisseria gonorrhoeae, Pseudomonas syringae, Rhodopseudomonas palustris, Vibrio fluvialis and Pseudomonas aeruginosa.

7. Method according to claim 6, wherein an aminotransferase is used comprising an amino acid sequence according to Sequence ID 2, Sequence ID 5, Sequence ID 8, Sequence ID 12, Sequence ID 15, Sequence ID 17, Sequence ID 19, Sequence ID 21, Sequence ID 23, Sequence ID 25, Sequence ID 27, Sequence ID 29, Sequence ID 65, Sequence ID 67, Sequence ID 69 or a homologue of any of these sequences.

8-15. (canceled)

16. Method for preparing caprolactam, comprising cyclising the 6-aminocaproic acid prepared by the method of claim 2 in the presence of superheated steam, thereby forming caprolactam.

17. A recombinant host cell comprising a nucleic acid sequence encoding an enzyme with 5-formylpentanoate aminotransferase activity.

18. A recombinant host cell according to claim 17, comprising a nucleic acid sequence encoding an enzyme with 5-formylpentanoate aminotransferase comprising an amino acid sequence according to Sequence ID 2, Sequence ID 5, Sequence ID 8, Sequence ID 65 Sequence ID 67, Sequence ID 69 or a homologue thereof.

19-22. (canceled)

23. A recombinant host cell according to claim 18, wherein the host cell is selected from the group of Aspergillus, Penicillium, Saccharomyces, Kluyveromyces, Pichia, Candida, Hansenula, Bacillus, Corynebacterium, and Escherichia.

24. A micro-organism according to claim 23 comprising DNA containing a nucleic acid sequence selected from the group of sequences represented by any sequence selected from the group of Sequence ID 1, Sequence ID 3, Sequence ID 4, Sequence ID 6, Sequence ID 7, Sequence ID 64, Sequence ID 66, Sequence ID 68 and functional analogues thereof.

25. (canceled)

Description

EXAMPLES

[0161] General Methods

[0162] Molecular and Genetic Techniques

[0163] Standard genetic and molecular biology techniques are generally known in the art and have been previously described (Maniatis et al. 1982 “Molecular cloning: a laboratory manual”. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Miller 1972 “Experiments in molecular genetics”, Cold Spring Harbor Laboratory, Cold Spring Harbor; Sambrook and Russell 2001 “Molecular cloning: a laboratory manual” (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press; F. Ausubel et al, eds., “Current protocols in molecular biology”, Green Publishing and Wiley Interscience, New York 1987).

[0164] Plasmids and Strains

[0165] pBAD/Myc-His C was obtained from Invitrogen (Carlsbad, Calif., USA). Plasmid pBAD/Myc-His-DEST constructed as described in WO2005/068643, was used for protein expression. E. coli TOP10 (Invitrogen, Carlsbad, Calif., USA) was used for all cloning procedures and for expression of target genes.

[0166] Media

[0167] LB medium (10 g/l tryptone, 5 g/l yeast extract, 5 g/l NaCl) was used for growth of E. coli. Antibiotics (50 μg/ml carbenicillin) were supplemented to maintain plasmids. For induction of gene expression under control of the P.sub.BAD promoter in pBAD/Myc-His-DEST derived plasmids, L-arabinose was added to a final concentration of 0.2% (w/v).

[0168] Identification of Plasmids

[0169] Plasmids carrying the different genes were identified by genetic, biochemical, and/or phenotypic means generally known in the art, such as resistance of transformants to antibiotics, PCR diagnostic analysis of transformant or purification of plasmid DNA, restriction analysis of the purified plasmid DNA or DNA sequence analysis.

[0170] HPLC-MS Analysis Method for the Determination of 5-FVA

[0171] 5-FVA was detected by selective reaction monitoring (SRM)-MS, measuring the transition m/z 1294.fwdarw.83. Concentrations for 5-FVA were calculated by measuring the peak area of the 5-FVA peak eluting at approximately 6 min. Calibration was performed by using an external standard procedure. All the LC-MS experiments were performed on an Agilent 1200 LC system, consisting of a quaternary pump, autosampler and column oven, coupled with an Agilent 6410 QQQ triple quadrupole MS.

[0172] Lc Conditions: [0173] Column: 50×4.6 mm Nucleosil C18, 5 μm (Machery & Nagel) pre column coupled to a 250×4.6 mm id. Prevail C18, 5 μm (Alltech) [0174] Column temperature: room temperature [0175] Eluent: A: water containing 0.1% formic acid

[0176] B: acetonitrile containing 0.1% formic acid [0177] Gradient:

TABLE-US-00003 time (min) % eluent B  0 10  6 50  6.1 10 11 10 [0178] Flow: 1.2 ml/min, before entering the MS the flow is split 1:3 [0179] Injection volume: 2 μl [0180] MS conditions: [0181] Ionisation: negative ion electrospray

[0182] source conditions: ionspray voltage: 5 kV [0183] temperature: 350° C. [0184] fragmentor voltage and collision energy optimized [0185] Scan mode: selective reaction mode: transition m/z 129.fwdarw.83

[0186] HPLC-MS Analysis for the Determination of AAP

[0187] AAP was detected by selected ion monitoring (SIM)-MS, measuring the protonated molecule for AAP with m/z 176. Concentrations for AAP were calculated by measuring the peak area of the AAP peak eluting at a retention time of 2.7 minutes in the samples. Calibration was performed by using an external standard procedure. All the LC-MS experiments were performed on an Agilent 1100 LC system consisting of a quaternary pump, degasser, autosampler and column oven, coupled with an API 2000 triple quadrupole MS (Applied Biosystems).

[0188] LC conditions were as follows:

Column: 50*4 Nucleosil C18, 5 μm (Macherey-Nagel)+250×4.6 Prevail C18, 5 μm (Alltech), both at room temperature (RT)
Eluent: A=0.1% (v/v) formic acid in ultrapure water

[0189] B=0.1% (v/v) formic acid in acetonitrile (pa, Merck)

Flow: 1.2 ml/min, before entering the MS the flow was split 1:3
Gradient: The gradient was started at t=0 minutes with 90% (v/v) A and changed within 6 minutes to 50% (v/v) A. At 6.1 minutes the gradient was changed to the original condition.
Injection volume: 2 μl
MS conditions: Positive ion electrospray was used for ionization
Detection: in SIM mode on m/z 176, with a dwell time of 100 msec.

[0190] HPLC-MS Analysis for the Determination of 6-ACA

[0191] Calibration:

[0192] The calibration was performed by an external calibration line of 6-ACA (m/z 132.fwdarw.m/z 114, Rt 7.5 min). All the LC-MS experiments were performed on an Agilent 1100, equipped with a quaternary pump, degasser, autosampler, column oven, and a single-quadrupole MS (Agilent, Waldbronn, Germany). The LC-MS conditions were: [0193] Column: 50*4 Nucleosil (Mancherey-Nagel)+250×4.6 Prevail C18 (Alltech), both at room temperature (RT) [0194] Eluent: A=0.1 (v/v) formic acid in ultrapure water

[0195] B=Acetonitrile (pa, Merck) [0196] Flow: 1.0 ml/min, before entering the MS the flow was split 1:3 [0197] Gradient: The gradient was started at t=0 minutes with 100% (v/v) A, remaining for 15 minutes and changed within 15 minutes to 80% (v/v) B (t=30 minutes). From 30 to 31 minutes the gradient was kept at constant at 80% (v/v) B. [0198] Injection volume: 5 μl [0199] MS detection: ESI(+)-MS [0200] The electrospray ionization (ESI) was run in the positive scan mode with the following conditions; m/z 50-500, 50 V fragmentor, 0.1 m/z step size, 350° C. drying gas temperature, 10 L N.sub.2/min drying gas, 50 psig nebuliser pressure and 2.5 kV capillary voltage.

[0201] Cloning of Target Genes

[0202] Design of Expression Constructs

[0203] attB sites were added to all genes upstream of the ribosomal binding site and start codon and downstream of the stop codon to facilitate cloning using the Gateway technology (Invitrogen, Carlsbad, Calif., USA).

[0204] Gene Synthesis and Construction of Plasmids

[0205] Synthetic genes were obtained from DNA2.0 and codon optimised for expression in E. coli according to standard procedures of DNA2.0. The aminotransferase genes from Vibrio fluvialis JS17 [SEQ ID No. 1] and Bacillus weihenstephanensis KBAB4 [SEQ ID No. 4] encoding the amino acid sequences of the V. fluvialis JS17 ω-aminotransferase [SEQ ID No. 2] and the B. weihenstephanensis KBAB4 aminotransferase (ZP_01186960) [SEQ ID No. 5], respectively, were codon optimised and the resulting sequences [SEQ ID No. 3] and [SEQ ID No. 6] were obtained by DNA synthesis.

[0206] The decarboxylase genes from Escherichia coli [SEQ ID No. 30], Saccharomyces cerevisiae [SEQ ID No. 33], Zymomonas mobilis [SEQ ID No. 36], Lactococcus lactis [SEQ ID No. 39], [SEQ ID No. 42], and Mycobacterium tuberculosis [SEQ ID No. 45] encoding the amino acid sequences of the V. fluvialis JS17 w-aminotransferase [SEQ ID No. 3], the B. weihenstephanensis KBAB4 aminotransferase (ZP_01186960) [SEQ ID No. 6], the Escherichia coli diaminopimelate decarboxylase LysA [SEQ ID No. 31], the Saccharomyces cerevisiae pyruvate decarboxylase Pdc [SEQ ID No. 34], the Zymomonas mobilis pyruvate decarboxylase Pdcl472A [SEQ ID No. 37], the Lactococcus lactis branched chain alpha-keto acid decarboxylase KdcA [SEQ ID No. 40] and alpha-ketoisovalerate decarboxylase KivD [SEQ ID No. 43], and the Mycobacterium tuberculosis alpha-ketoglutarate decarboxylase Kgd [SEQ ID No. 46], respectively, were also codon optimised and the resulting sequences [SEQ ID No. 32], [SEQ ID No. 35], [SEQ ID No. 38], [SEQ ID No. 41], [SEQ ID No. 44], and [SEQ ID No. 47] were obtained by DNA synthesis, respectively.

[0207] The gene constructs were cloned into pBAD/Myc-His-DEST expression vectors using the Gateway technology (Invitrogen) via the introduced attB sites and pDONR201 (Invitrogen) as entry vector as described in the manufacturer's protocols (www.invitrogen.com). This way the expression vectors pBAD-Vfl_AT and pBAD-Bwe_AT were obtained, respectively. The corresponding expression strains were obtained by transformation of chemically competent E. coli TOP10 (Invitrogen) with the respective pBAD-expression vectors.

[0208] Cloning by PCR

[0209] Various genes encoding a biocatalyst were amplified from genomic DNA by PCR using PCR Supermix High Fidelity (Invitrogen) according to the manufacturer's specifications, using primers as listed in the following table.

TABLE-US-00004 TABLE 2 gene enzyme primer Sequence Sequence Sequence origin of gene ID ID ID's Pseudomonas  7  8  9&10 aeruginosa Pseudomonas 26 27 60&61 aeruginosa Pseudomonas 66 67 72&73 aeruginosa Pseudomonas 68 69 74&75 aeruginosa Bacillus subtilis 14 15 48&49 Bacillus subtilis 16 17 50&51 Bacillus subtilis 64 65 70&71 Rhodobacter 18 19 52&53 sphaeroides Legionella 20 21 54&55 pneumophilia Nitrosomas europaea 22 23 56&57 Neisseria 24 25 58&59 gonorrhoeae Rhodopseudomonas 28 29 62&63 palustris

[0210] PCR reactions were analysed by agarose gel electrophoresis and PCR products of the correct size were eluted from the gel using the QIAquick PCR purification kit (Qiagen, Hilden, Germany). Purified PCR products were cloned into pBAD/Myc-His-DEST expression vectors using the Gateway technology (Invitrogen) via the introduced attB sites and pDONR-zeo (Invitrogen) as entry vector as described in the manufacturer's protocols. The sequence of genes cloned by PCR was verified by DNA sequencing. This way the expression vectors pBAD-Pae-_gi9946143_AT, pBAD-Bsu_gi16078032_AT, pBAD-Bsu_gi16080075 AT, pBAD-Bsu_gi16077991_AT, pBAD-Rsp_AT, pBAD-Lpn_AT, pBAD-Neu_AT, pBAD-Ngo_AT, pBAD-Pae_gi9951299_AT, pBAD-Pae_gi9951072_AT, pBAD-Pae_gi9951630_AT and pBAD-Rpa_AT were obtained. The corresponding expression strains were obtained by transformation of chemically competent E. coli TOP10 (Invitrogen) with the pBAD constructs.

[0211] Growth of E. coli for Protein Expression

[0212] Small scale growth was carried out in 96-deep-well plates with 940 μl media containing 0.02% (w/v) L-arabinose. Inoculation was performed by transferring cells from frozen stock cultures with a 96-well stamp (Kühner, Birsfelden, Switzerland). Plates were incubated on an orbital shaker (300 rpm, 5 cm amplitude) at 25° C. for 48 h. Typically an OD.sub.620 nm of 2-4 was reached.

[0213] Preparation of Cell Lysates

[0214] Preparation of Lysis Buffer

[0215] The lysis buffer contained the following ingredients:

TABLE-US-00005 TABLE 3 1M MOPS pH 7.5 5 ml DNAse I grade II (Roche) 10 mg Lysozyme 200 mg MgSO.sub.4.7H.sub.2O 123.2 mg dithiothreitol (DTT) 154.2 mg H.sub.2O (MilliQ) Balance to 100 ml

[0216] The solution was freshly prepared directly before use.

[0217] Preparation of Cell Free Extract by Lysis

[0218] Cells from small scales growth (see previous paragraph) were harvested by centrifugation and the supernatant was discarded. The cell pellets formed during centrifugation were frozen at −20° C. for at least 16 h and then thawed on ice. 500 μl of freshly prepared lysis buffer were added to each well and cells were resuspended by vigorously vortexing the plate for 2-5 min. To achieve lysis, the plate was incubated at room temperature for 30 min. To remove cell debris, the plate was centrifuged at 4° C. and 6000 g for 20 min. The supernatant was transferred to a fresh plate and kept on ice until further use.

[0219] Preparation of Cell Free Extract by Sonification

[0220] Cells from medium scales growth (see previous paragraph) were harvested by centrifugation and the supernatant was discarded. 1 ml of potassium phosphate buffer pH7 was added to 0.5 g of wet cell pellet and cells were resuspended by vigorously vortexing. To achieve lysis, the cells were sonicated for 20 min. To remove cell debris, the lysates were centrifuged at 4° C. and 6000 g for 20 min. The supernatant was transferred to a fresh tube and frozen at −20° C. until further use.

[0221] Preparation of 5-Formylpentanoic Acid by Chemical Hydrolysis of Methyl 5-Formylpentanoate

[0222] The substrate for the aminotransferase reaction i.e. 5-formylpentanoic acid was prepared by chemical hydrolysis of methyl 5-formylpentanoate as follows: a 10% (w/v) solution of methyl 5-formylpentanoate in water was set at pH 14.1 with NaOH. After 24 h of incubation at 20° C. the pH was set to 7.1 with HCl.

[0223] Enzymatic Reactions for Conversion of 5-Formylpentanoic Acid to 6-ACA

[0224] Unless specified otherwise, a reaction mixture was prepared comprising 10 mM 5-formylpentanoic acid, 20 mM racemic α-methylbenzylamine, and 200 μM□ pyridoxal 5′-phosphate in 50 mM potassium phosphate buffer, pH 7.0. 100 μl of the reaction mixture were dispensed into each well of the well plates. To start the reaction, 20 μl of the cell free extracts were added, to each of the wells. Reaction mixtures were incubated on a shaker at 37° C. for 24 h. Furthermore, a chemical blank mixture (without cell free extract) and a biological blank (E. coli TOP10 with pBAD/Myc-His C) were incubated under the same conditions. Samples were analysed by HPLC-MS. The results are summarised in the following table.

TABLE-US-00006 TABLE 4 6-ACA formation from 5-FVA in the presence of aminotransferases 6-ACA concentration Biocatalyst [mg/kg] E. coli TOP10/pBAD-VfI_AT 43* E. coli TOP10/pBAD-Pae_AT 930 E. coli TOP10/pBAD-Pae_AT 25* E. coli TOP10/pBAD-Bwe_AT 24* E. coli TOP10/pBAD-Bsu_gi16077991_AT 288 E. coli TOP10/pBAD-Pae_gi9951072_AT 1087 E. coli TOP10/pBAD-Pae_gi9951630_AT 92 E. coli TOP10 with pBAD/Myc-His C 0.6 (biological blank) None (chemical blank) n.d. n.d.: not detectable *method differed in that 10 μl cell free extract was used instead of 20 μl, the pyridoxal-5′-phosphate concentration was 50 μM instead of 200 μM and the reaction mixture volume in the wells was 190 μl instead of 100 μl.

[0225] It is shown that 6-ACA is formed from 5-FVA in the presence of an aminotransferase.

[0226] Enzymatic Reactions for Conversion of AKP to 5-Formylpentanoic Acid

[0227] A reaction mixture was prepared comprising 50 mM AKP, 5 mM magnesium chloride, 100 μM□ pyridoxal 5′-phosphate (for LysA) or 1 mM thiamine diphosphate (for all other enzymes) in 100 mM potassium phosphate buffer, pH 6.5. 4 ml of the reaction mixture were dispensed into a reaction vessel. To start the reaction, 1 ml of the cell free extracts obtained by sonification were added, to each of the wells. In case of the commercial oxaloacetate decarboxylase (Sigma-Aldrich product number 04878), 50 U were used. Reaction mixtures were incubated with a magnetic stirrer at 37° C. for 48 h. Furthermore, a chemical blank mixture (without cell free extract) and a biological blank (E. coli TOP10 with pBAD/Myc-His C) were incubated under the same conditions. Samples from different time points during the reaction were analysed by HPLC-MS. The results are summarised in the following table.

TABLE-US-00007 TABLE 5 5-FVA formation from AKP in the presence of decarboxylases 5-FVA concentration [mg/kg] Biocatalyst 3 h 18 h 48 h E. coli TOP10/pBAD-LysA  150  590  720 E. coli TOP10/pBAD-Pdc 1600 1700 1300 E. coli TOP10/pBAD-Pdcl472A 2000 2000 1600 E. coli TOP10/pBAD-KdcA 3300 2300 2200 E. coli TOP10/pBAD-KivD  820 1400 1500 Oxaloacetate decarboxylase n.d.   6  10 E. coli TOP10 with pBAD/Myc- n.d. n.d. n.d. His C (biological blank) None (chemical blank) n.d. n.d. n.d. n.d.: not detectable

[0228] It is shown that 5-FVA is formed from AKP in the presence of a decarboxylase.

[0229] Enzymatic Reactions for Conversion of AKP to 6-ACA in Presence of Recombinant Decarboxylase

[0230] A reaction mixture was prepared comprising 50 mM AKP, 5 mM magnesium chloride, 100 μM□ pyridoxal 5′-phosphate (for LysA) or 1 mM thiamine diphosphate (for all other tested biocatalysts) in 100 mM potassium phosphate buffer, pH 6.5. 4 ml of the reaction mixture were dispensed into a reaction vessel. To start the reaction, 1 ml of the cell free extracts were added, to each of the wells. Reaction mixtures were incubated with a magnetic stirrer at 37° C. for 48 h. Furthermore, a chemical blank mixture (without cell free extract) and a biological blank (E. coli TOP10 with pBAD/Myc-His C) were incubated under the same conditions. Samples from different time points during the reaction were analysed by HPLC-MS. The results are summarised in the following table.

TABLE-US-00008 TABLE 6 6-ACA formation from AKP in the presence of decarboxylases 6-ACA concentration [mg/kg] Biocatalyst 3 h 18 h 48 h E. coli TOP10/pBAD-LysA n.a. 0.01 0 E. coli TOP10/pBAD-Pdc 0.1 0.3 n.a. E. coli TOP10/pBAD-Pdcl472A 0.03 0.1 0.2 E. coli TOP10/pBAD-KdcA 0.04 0.1 0.3 E. coli TOP10/pBAD-KivD n.a. 0.3 0.6 E. coli TOP10 with pBAD/Myc- n.d. n.d. n.d. His C (biological blank) None (chemical blank) n.d. n.d. n.d. n.a. = not analysed n.d. = not detectable

[0231] It is shown that 6-ACA is formed from AKP in the presence of a decarboxylase. It is contemplated that the E. coli contained natural 5-FVA aminotransferase activity.

[0232] Enzymatic Reactions for Conversion of AKP to 6-ACA in Presence of Recombinant Decarboxylase and Recombinant Aminotransferase

[0233] A reaction mixture was prepared comprising 50 mM AKP, 5 mM magnesium chloride, 100 μM□ pyridoxal 5′-phosphate, 1 mM thiamine diphosphate and 50 mM racemic α-methylbenzylamine in 100 mM potassium phosphate buffer, pH 6.5. 1.6 ml of the reaction mixture were dispensed into a reaction vessel. To start the reaction, 0.2 ml of the decarboxylase containing cell free extract and 0.2 ml of the aminotransferase containing cell free extract were added, to each of the reaction vessels. Reaction mixtures were incubated with a magnetic stirrer at 37° C. for 48 h. Furthermore, a chemical blank mixture (without cell free extract) and a biological blank (E. coli TOP10 with pBAD/Myc-His C) were incubated under the same conditions. Samples from different time points during the reaction were analysed by HPLC-MS. The results are summarised in the following table.

TABLE-US-00009 TABLE 7 6-ACA formation from AKP in the presence of a recombinant decarboxylase and a recombinant aminotransferase 6-ACA concentration [mg/kg] after 48 hours E. coli TOP10/ AT E. coli TOP10/ E. coli TOP10/ pBAD-PAE_ DC pBAD-Vfl-AT pBAD-Bwe-AT gi9946143_AT E. coli TOP10/ 183.4 248.9 117.9 pBAD-Pdc E. coli TOP10/ 458.5 471.6 170.3 pBAD-Pdc1472A E. coli TOP10/ 497.8 497.8 275.1 pBAD-KdcA E. coli TOP10/ 510.9 510.9 314.4 pBAD-KivD AT = aminotransferase DC = decarboxylase

[0234] In the chemical blank and in the biological blank no 6-ACA was detectable.

[0235] Further, the results show that compared to the example wherein a host-cell with only recombinant decarboxylase (and no recombinant aminotransferase) the conversion to 6-ACA was improved.

[0236] Construction of Plasmids for Expression of Aminotransferases and Decarboxylases in S. cerevisiae

[0237] The aminotransferase gene from Vibrio fluvialis JS17 encoding the amino acid sequence of the V. fluvialis JS17 ω-aminotransferase [SEQ ID No. 2] was amplified by PCR from pBAD-Vfl_AT [SEQ ID No. 3] using Phusion DNA polymerase (Finnzymes) according to the manufacturers specifications and using specific primers [SEQ ID No. 76 & 77].

[0238] The aminotransferase gene from Pseudomonas aeruginosa [SEQ ID No. 7] coding for P. aeruginosa aminotransferase [SEQ ID No. 8] was amplified from pBAD-Pae_AT by PCR using Phusion DNA polymerase (Finnzymes) according to the manufacturers specifications and using specific primers [SEQ ID No. 78 & 79].

[0239] The resulting PCR products were cloned into vector pAKP-41 using SpeI and BamHI restriction enzymes resulting in vectors pAKP-79 and pAKP-80 respectively, which now contain the aminotransferase gene under the S. cerevisiae gal10 promoter and the S. cerevisiae adh2 terminator.

[0240] The decarboxylase gene from Saccharamyces cerevisiae [SEQ ID No. 33] coding for Saccharamyces cerevisiae pyruvate decarboxylase Pdc [SEQ ID No. 34] was amplified from pBAD-Pdc by PCR using Phusion DNA polymerase (Finnzymes) according to the manufacturers specifications and using specific primers [SEQ ID No 80 & 81].

[0241] The decarboxylase gene from Lactococcus lactis [SEQ ID No. 39] coding for Lactococcus lactis branched chain alpha-keto acid decarboxylase KdcA [SEQ ID No. 40] was amplified from pBAD-KdcA by PCR using Phusion DNA polymerase (Finnzymes) according to the manufacturers specifications and using specific primers [SEQ ID No 82 & 83].

[0242] The resulting PCR products were cloned into vector pAKP-44 using AscI and BamHI restriction enzymes resulting in vectors pAKP-81 and pAKP-82 respectively, which now contain the decarboxylase gene under the S. cerevisiae gal2 promoter and the S. cerevisiae pma1 terminator.

[0243] Plasmids pAKP-79 and pAKP-80 were restriction enzyme digested with SacI and XbaI and plasmids pAKP-81 and pAKP-82 were restriction enzyme digested with SalI and XbaI. A SacI/XbaI aminotransferase fragment was combined with a SalI/XbaI decarboxylase fragment into the S. cerevisiae low copy episomal vector pRS414, which was restriction enzyme digested with SalI and SacI.

[0244] The resulting plasmids were obtained:

pAKP-85: Pgal10-Pae_AT-Tadh2 Pgal2-Pdc_DC-Tpma1
pAKP-86: Pgal10-Pae_AT-Tadh2 Pgal2-KdcA_DC-Tpma1
pAKP-87: Pgal10-Vfl_AT-Tadh2 Pgal2-Pdc_DC-Tpma1
pAKP-88: Pgal10-Vfl_AT-Tadh2 Pgal2-KdcA_DC-Tpma1

[0245] Transformation and Growth of S. cerevisiae

[0246] S. cerevisiae strain CEN. PK113-3C was transformed with 1 μg of plasmid DNA according to the method as described by Gietz and Woods (Gietz, R. D. and Woods, R. A. (2002). Transformation of yeast by the Liac/SS carrier DNA/PEG method. Methods in Enzymology 350: 87-96). Cells were plated on agar plates with 1× Yeast Nitrogen Base without amino acids and 2% glucose.

[0247] The resulting strains were grown aerobically at 30° C. for 48 hour in Verduyn minimal medium containing 0.05% glucose and 4% galactose.

[0248] Preparation of Cell Free Extract

[0249] 1 ml of potassium phosphate buffer (pH 7) was added to 0.5 g of the cell pellet. This mixture was added to a 2 ml eppendorf tube which contained 0.5 g of glassbeads with a diameter of 0.4-0.5 mM. Samples were vigorously shaken with an eppendorf shaker (IKA VIBRAX-VXR) for 20 s. The resulting cell free extract was centrifuged for 5 minutes at 14000 rpm and 4° C. The supernatant was used for enzyme activity assays.

[0250] Enzymatic Reactions for Conversion of AKP to 6-ACA in Presence of Decarboxylase and Aminotransferase Co-Expressed in S. cerevisiae

[0251] A reaction mixture was prepared comprising 50 mM AKP, 5 mM magnesium chloride, 100 μM□ pyridoxal 5′-phosphate, 1 mM thiamine diphosphate and 50 mM racemic α-methylbenzylamine in 100 mM potassium phosphate buffer, pH 6.5. 1.6 ml of the reaction mixture were dispensed into a reaction vessel. To start the reaction, 0.4 ml of the cell free extract from S. cerevisiae containing decarboxylase and aminotransferase were added, to each of the reaction vessels. Reaction mixtures were incubated with a magnetic stirrer at 37° C. Furthermore, a chemical blank mixture (without cell free extract) and a biological blank (S. cerevisiae) were incubated under the same conditions. Samples, taken after 19 hours of incubation, were analysed by HPLC-MS. The results are summarised in the following table.

TABLE-US-00010 TABLE 8 6-ACA formation from AKP using a micro-organism as a biocatalyst 6-ACA concentration Biocatalyst [mg/kg] S. cerevisiae pAKP-85 63 S. cerevisiae pAKP-86 226 S. cerevisiae pAKP-87 1072 S. cerevisiae pAKP-88 4783 S. cerevisiae 3.9 (biological blank) None (chemical blank) 1.3

[0252] Enzymatic Reactions for Conversion of Alpha-Ketopimelic Acid to Alpha-Aminopimelic Acid

[0253] A reaction mixture was prepared comprising 10 mM alpha-ketopimelic acid, 20 mM L-alanine, and 50 μM□ pyridoxal 5′-phosphate in 50 mM potassium phosphate buffer, pH 7.0. 800 μl of the reaction mixture were dispensed into each well of the well plates. To start the reaction, 200 μl of the cell lysates were added, to each of the wells. Reaction mixtures were incubated on a shaker at 37° C. for 24 h. Furthermore, a chemical blank mixture (without cell free extract) and a biological blank (E. coli TOP10 with pBAD/Myc-His C) were incubated under the same conditions. Samples were analysed by HPLC-MS. The results are summarised in the following table.

TABLE-US-00011 TABLE 9 AAP formation from AKP in the presence of aminotransferases AAP concentration [mg/kg] Biocatalyst (after 24 hrs) E. coli TOP10/pBAD-Vfl_AT 3.7 E. coli TOP10/pBAD-Psy_AT 15.8 E. coli TOP10/pBAD-Bsu_ 11.2 gi16078032_AT E. coli TOP10/pBAD-Rsp_AT 9.8 E. coli TOP10/pBAD-Bsu_ 4.6 gi16080075_AT E. coli TOP10/pBAD-Lpn_AT 5.4 E. coli TOP10/pBAD-Neu_AT 7.7 E. coli TOP10/pBAD-Ngo_AT 5.1 E. coli TOP10/pBAD-Pae_gi9951299_AT 5.6 E. coli TOP10/pBAD-Rpa_AT 5.4 E. coli TOP10 with pBAD/Myc-His 1.4 C (biological blank) None (chemical blank) 0

[0254] It is shown that the formation of AAP from AKP is catalysed by the biocatalyst.

[0255] Chemical Conversion of AAP to Caprolactam

[0256] To a suspension of 1.5 grams of D,L-2-aminopimelic acid in 21 ml cyclohexanone, 0.5 ml of cyclohexenone was added. The mixture was heated on an oil bath for 20 h at reflux (approximately 160° C.). After cooling to room temperature the reaction mixture was decanted and the clear solution was evaporated under reduced pressure. The remaining 2 grams of brownish oil were analyzed by .sup.1H-NMR and HPLC and contained 0.8 wt % caprolactam and 6 wt % of cyclic oligomers of caprolactam.