Producing amines and diamines from a carboxylic acid or dicarboxylic acid or a monoester thereof

Abstract

The invention relates to a whole-cell catalyst which expresses a recombinant α-dioxygenase or the combination of a recombinant fatty acid reductase and a phosphopantetheinyl transferase which phosphopantetheinylates the fatty acid reductase, and which expresses, in addition to the α-dioxygenase and/or the combination of fatty acid reductase and phosphopantetheinyl transferase, a transaminase, wherein the phosphopantetheinyl transferase and/or transaminase is preferably recombinant; and also to a process for converting a carboxylic acid or dicarboxylic acid or a monoester thereof to an amine or diamine, comprising the steps of contacting the carboxylic acid or dicarboxylic acid or the monoester thereof with a phosphopantetheinylated fatty acid reductase or an α-dioxygenase and contacting the product with a transaminase.

Claims

1. A whole-cell catalyst, comprising heterologous expression of: a combination of a recombinant fatty acid reductase and a phosphopantetheinyl transferase capable of phosphopantetheinylating the fatty acid reductase; and a transaminase, wherein the recombinant fatty acid reductase and the phosphopantetheinyl transferase, and the transaminase, are expressed in the whole-cell catalyst, and wherein the whole-cell catalyst is a microorganism.

2. The whole-cell catalyst according to claim 1, further comprising: an amino acid dehydrogenase expressed in the whole-cell catalyst.

3. The whole-cell catalyst according to claim 1, further comprising: an alkane hydroxylase expressed in the whole-cell catalyst.

4. The whole-cell catalyst according to claim 1, further comprising: a polypeptide of the AlkL family expressed in the whole-cell catalyst.

5. The whole-cell catalyst according to claim 1, further comprising: an alcohol dehydrogenase expressed in the whole-cell catalyst.

6. The whole-cell catalyst according to claim 1, wherein an activity of at least one enzyme involved in β-oxidation is reduced as compared to an activity of the at least one enzyme in the wild type of the whole-cell catalyst.

7. The whole-cell catalyst according to claim 1, wherein an activity of BioH is reduced or elevated as compared to an activity of BioH in the wild type of the whole-cell catalyst.

8. The whole-cell catalyst according to claim 1, wherein an activity of FadL is elevated as compared to an activity of FadL in the wild type of the whole-cell catalyst.

9. A reaction mixture, comprising: the whole-cell catalyst according to claim 1 in an aqueous solution; and a carboxylic acid, a dicarboxylic acid, or a monoester thereof having the formula (I):
R.sup.1-A-COOR.sup.2  (I), wherein R.sup.1 is —H or COOR.sup.3, R.sup.2 and R.sup.3 are each independently selected from the group consisting of H, methyl, ethyl and propyl, with the proviso that at least one of the radicals R.sup.2 and R.sup.3 is H, and A is an unbranched, branched, linear, cyclic, substituted or unsubstituted hydrocarbon group having at least four carbons.

10. The whole-cell catalyst according to claim 1, wherein at least one of the phosphopantetheinyl transferase and the transaminase is recombinant.

11. The whole-cell catalyst according to claim 1, further comprising: at least one of a recombinant amino acid dehydrogenase, a recombinant alkane hydroxylase, a recombinant polypeptide of the AlkL family, and a recombinant alcohol dehydrogenase, expressed in the whole-cell catalyst.

12. The whole-cell catalyst according to claim 1, wherein the recombinant fatty acid reductase is encoded by a gene having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 1, the phosphopantetheinyl transferase is encoded by a gene having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 2, and the transaminase is encoded by a gene having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 12.

13. The whole-cell catalyst according to claim 1, further comprising: an alanine dehydrogenase expressed in the whole-cell catalyst and having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 11, wherein the recombinant fatty acid reductase is encoded by a gene having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 1, the phosphopantetheinyl transferase is encoded by a gene having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 2, and the transaminase is encoded by a gene having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 12.

Description

(1) The present invention is more particularly described by the following FIGURES and non-limiting examples from which further features, embodiments, aspects and advantages of the present invention may be discerned.

(2) FIG. 1: Detection of monoamines and diamines in the fermentation broth of the strain E. coli W3110 pACYC{Placuv5}[carA_Ms-npt_Noc]/pJ281_alaDH_B.s._TA_C.v.(ct) after a 21.75 h process time.

EXAMPLE 1

(3) Producing an Expression Vector for the Expression of the Genes MSMEG_2956 from Mycobacterium smegmatis and npt from Nocardia sp.

(4) To produce vectors for the coexpression of MSMEG_2956 (carA, SEQ ID No. 1) from Mycobacterium smegmatis encoding a fatty acid reductase (YP_887275.1) and with npt (SEQ ID No. 2) from Nocardia sp. encoding a phosphopantetheinyl transferase (AB183656.1) which phosphopantetheinylates the fatty acid reductase, both genes were amplified by means of PCR with insertion of homologous regions for recombinant cloning. In this connection, genomic DNA from the donor organism for the amplification of the gene MSMEG_2956 and a synthesized DNA fragment for the amplification of the gene npt served as template. The genes are under the control of a lacuv5 promoter (SEQ ID No. 3), which was likewise amplified by means of PCR proceeding from an available vector with insertion of homologous regions for the recombinant cloning.

(5) In this connection, the following oligonucleotides were used:

(6) TABLE-US-00001 Plac_H1_fw: (SEQ ID Nr. 4) 5′-TTATGCGACTCCTGCTGGCTATGGTGGGATTTCC-3′ Plac_H2_rv: (SEQ ID Nr. 5) 5′-GATCGTCATATGCCACTCTCCTTGGTTCC-3′ carA_H2_fw: (SEQ ID Nr. 6) 5′-TGGCATATGACGATCGAAACGCGCG-3′ carA_H3_rv: (SEQ ID Nr. 7) 5′-TCCTTCTCTTACAGCAATCCGAGCATCT-3′ npt_H3_fw: (SEQ ID Nr. 8) 5′-GCTGTAAGAGAAGGAGTTCTATCATGATCGAG-3′ npt_H4_rv: (SEQ ID Nr. 9) 5′-GCAGCCTAGGTTAATTTATCAGGCGTACGCGATCG-3′

(7) The following parameters were used for the PCR for the amplification of the P.sub.lacuv5 and the gene npt: 1×: initial denaturation, 98° C., 0:30 min; 35×: denaturation, 98° C., 0:10 min, annealing, 55° C., 0:20 min; elongation, 72° C., 0:15 min; 1×: terminal elongation, 72° C., 10 min. For the amplification of the gene MSMEG_2956, the following parameters were used: 1×: initial denaturation, 98° C., 0:30 min; 35×: denaturation, 98° C., 0:10 min, annealing, 65° C., 0:20 min; elongation, 72° C., 1 min; 1×: terminal elongation, 72° C., 10 min. For the amplification, the Phusion™ High-Fidelity Master Mix from New England Biolabs (Frankfurt) was used according to the recommendations from the manufacturer. In each case, 50 μl of the PCR reactions were then resolved on a 1% strength TAE agarose gel. The PCR, the agarose gel electrophoresis, ethidium bromide staining of the DNA and determination of the PCR fragment sizes were performed in the manner known to the person skilled in the art. In all cases, PCR fragments of the expected size could be amplified. These were 325 base pairs for P.sub.lacuv, 5, 3.5 kilobase pairs for MSMEG_2956 and 718 base pairs for npt. To isolate the DNA from the agarose gel, the target DNA was cut out of the gel using a scalpel and purified using the QiaQuick Gel extraction Kit in accordance with the manufacturer's instructions (Qiagen, Hilden). The purified PCR products were cloned into an EcoNI- and PacI-cut pACYCDuet-1 vector (Merck, Darmstadt) by means of recombination using the Geneart® Seamless Cloning and Assembly Kit in accordance with the manufacturer's instructions (Life Technologies, Carlsbad, Calif., USA). Chemically competent E. coli DH10β (New England Biolabs, Frankfurt) were transformed in the manner known to the person skilled in the art. The correct insertion of the target genes was checked by restriction analysis and the authenticity of the inserted genes confirmed by DNA sequencing. The finished expression vector was referred to as pACYC{Placuv5}[carA_Ms-npt_Noc] (SEQ ID No. 10).

EXAMPLE 2

(8) Producing an Expression Vector for the Coexpression of the Genes ald from Bacillus subtilis and Cv2025 from Chromobacterium violaceum

(9) To produce an E. coli expression vector for the genes ald (SEQ ID No. 11) from Bacillus subtilis encoding an alanine dehydrogenase (NP_391071.1) and Cv_2025 (SEQ ID No. 12) from Chromobacterium violaceum encoding a transaminase (NP_901695.1), the gene ald from Bacillus subtili s was, in exchange for the gene ald from Bacillus sphaericus, cloned into the E. coli expression vector pJ281_alaD_Bsp_TA_C.v.(ct) (sequence and production, cf. example 1 in WO/2013/024114 and SEQ ID No. 17 listed therein). The gene ald from Bacillus subtilis was amplified by PCR from chromosomal DNA from the strain Bacillus subtilis str. 168. In this connection, the following oligonucleotides were used:

(10) TABLE-US-00002 alaDH_pCR22_fw: (SEQ ID No. 13) 5′-ATGATCATAGGGGTTCCTAAAGAG-3′ alaDH_pCR22_rev: (SEQ ID No. 14) 5′-TTAAGCACCCGCCACAGATG-3′

(11) The following parameters were used for the PCR: 1×: initial denaturation, 98° C., 0:30 min; 35×: denaturation, 98° C., 0:10 min, annealing, 65° C., 0:30 min; elongation, 72° C., 0:20 min; 1×: terminal elongation, 72° C., 10 min. For the amplification, the Phusion™ High-Fidelity Master Mix from New England Biolabs (Frankfurt) was used according to the recommendations from the manufacturer. In each case, 50 μl of the PCR reactions were then resolved on a 1% strength TAE agarose gel. The PCR, the agarose gel electrophoresis, ethidium bromide staining of the DNA and determination of the PCR fragment sizes were performed in the manner known to the person skilled in the art. The PCR fragment showed the expected size of 1137 base pairs and was purified from the PCR volume using the Quick PCR Purification Kit from Qiagen (Hilden) in accordance with the information from the manufacturer. For the ligation of the PCR product to the vector, 5′-phosphates were attached to the PCR product using the polynucleotide kinase (New England Biolabs, Frankfurt). In this connection, the recommendation from the manufacturer was followed.

(12) The vector was digested with the restriction endonucleases HindIII and NdeI, and as a result, the gene present, Bacillus sphaericus ald, was removed. The restriction digest volume was resolved on a 1% strength TAE agarose gel. It was possible to identify two bands of sizes 5696 bp and 1124 bp. To isolate the vector DNA from the agarose gel, the DNA band of 5696 bp was isolated from the gel using a scalpel and purified using the Quick Gel Extraction Kit from Qiagen (Hilden) in accordance with the information from the manufacturer. To generate blunt ends, the 5′-overhangs of the purified vector DNA were filled using the Klenow fragment of DNA polymerase I (New England Biolabs, Frankfurt). In this connection, the information from the manufacturer was followed. The DNA fragment Bacillus subtilis ald with 5′-phosphate residues was ligated into the vector having blunt ends. The finished E. coli expression vector was referred to as pJ281_alaDH_B.s._TA_C.v.(Ct) (SEQ ID No. 15).

EXAMPLE 3

(13) Producing an E. coli strain Overexpressing the Genes MSMEG_2956 from Mycobacterium smegmatis and npt from Nocardia sp., ald from Bacillus subtilis and Cv2025 from Chromobacterium violaceum

(14) To generate an E. coli strain coexpressing the genes MSMEG_2956 from Mycobacterium smegmatis encoding a fatty acid reductase (YP_887275.1) and npt from Nocardia sp. encoding a phosphopantetheinyl transferase (ABI83656.1) which phosphopantetheinylates the fatty acid reductase, in combination with the genes ald from Bacillus subtilis encoding an alanine dehydrogenase (NP_391071.1) and Cv2025 from Chromobacterium violaceum encoding a transaminase (NP_901695.1), the strain E. coli W3110 was transformed with the plasmids pACYC{Placuv5}[carA_Ms-npt_Noc] (SEQ ID No. 10) and pJ281_alaDH_B.s._TA_C.v.(ct) (SEQ ID No. 15) by means of electroporation and plated out on LB agar plates containing chloramphenicol (50 μg/ml) and kanamycin (50 μg/ml). Transformants were checked as regards the presence of the correct plasmids by plasmid preparation and analytical restriction analysis. The strain generated was referred to as E. coli W3110 pACYC{Placuv5}[carA_Ms-npt_Noc]/pJ281_alaDH_B.s._TA_C.v.(ct). The strain was used to investigate its capability for the production of dodecanediamine proceeding from dodecanedioic acid and dodecylamine proceeding from dodecanoic acid. The gene product CarA, a fatty acid reductase which is activated by the overexpressed phosphopantetheinyl transferase npt, converts the substrate dodecanoic acid or dodecanedioic acid to the respective aldehyde or dialdehyde. The function of the gene product Cv_2505 is that of converting the (di)aldehyde terminally to the dodecylamine or dodecanediamine. The alanine amino donor required for the amination reaction is provided from pyruvate by the gene product ald.

EXAMPLE 4

(15) Production of Dodecanediamine and Dodecylamine by E. coli Strains Containing an Expression Vector for the Genes MSMEG_2956 from Mycobacterium smegmatis and npt from Nocardia sp. in Combination with an Expression Vector for the Genes ald from Bacillus subtilis and Cv_2025 from Chromobacterium violaceum

(16) The strain generated in example 3 was used to investigate its capability in relation to the production of dodecylamine and dodecanediamine. The biotransformation of dodecanoic acid and dodecanedioic acid to dodecylamine and dodecanediamine, respectively, was carried out in the 8-fold parallel fermentation system from DASGIP. The procedure for this was as follows: For the fermentation, 1 L reactors were used. The pH probes were calibrated by means of a two-point calibration with measurement solutions of pH 4.0 and pH 7.0. The reactors were filled with 300 mL of drinking water and autoclaved for 20 min at 121° C. in order to ensure sterility. Then, the pO2 probes were polarized on the DASGIP system overnight (for at least 6 h). The next morning, the water was removed under the clean bench and replaced with 300 mL of high-cell-density medium containing 50 mg/L chloramphenicol and 50 mg/L kanamycin. Subsequently, the pO2 probes were calibrated using a single-point calibration (stirrer: 400 rpm/aeration: 10 sL/h air) and the feed, correcting agent and induction agent paths were cleaned by means of clean-in-place. To this end, the hoses were flushed with 70% ethanol, then with 1 M NaOH, then with sterile demineralized water and finally filled with the particular media.

(17) The E. coli strain producing dodecanediamine and dodecylamine was firstly cultured from the cryogenic culture in LB medium (25 mL in a 100 mL baffled flask) containing the aforementioned antibiotics overnight at 37° C. and 200 rpm, for about 18 h. Then, 2 mL of the culture were inoculated into high-cell-density medium (15 g/L glucose (30 mL/L of a separately autoclaved 500 g/L stock solution containing 1% MgSO.sub.4*7H.sub.2O and 2.2% NH.sub.4Cl), 1.76 g/L (NH.sub.4).sub.2SO4, 19.08 g/L K.sub.2HPO.sub.4, 12.5 g/L KH.sub.2PO.sub.4, 6.66 g/L yeast extract, 2.24 g/L trisodium citrate dihydrate, ammonium ferric citrate solution, 17 mUL of a separately autoclaved 1% strength stock solution, trace element solution, 5 mUL separately autoclaved stock solution (36.50 g/L HCl (37%), 1.91 g/L MnCl.sub.2*4H.sub.2O, 1.87 g/L ZnSO.sub.4*7H.sub.2O, 0.84 g/L ethylenediaminetetraacetic acid dihydrate, 0.30 g/L H.sub.3BO.sub.3, 0.25 g/L Na.sub.2MoO.sub.4*2H.sub.2O, 4.70 g/L CaCl.sub.2*2H.sub.2O, 17.80 g/L FeSO.sub.4*7H.sub.2O, 0.15 g/L CuCl.sub.2*2H.sub.2O)) (25 mL in a 100 mL baffled flask) containing the aforementioned antibiotics and incubated at 37° C./200 rpm for a further 5.5 h. The reactors were inoculated at an optical density of 0.1 by an appropriate volume of the pre-culture being filled into a 5 mL syringe (under sterile conditions) and the reactors being inoculated by means of a needle across a septum covered with 70% ethanol.

(18) The following standard program was used:

(19) TABLE-US-00003 DO regulator pH regulator Preset 0% Preset 0 ml/h P 0.1 P 5 Ti 300 s Ti 200 s Min  0% Min 0 mlL/h Max 100% Max 40 mL/h

(20) TABLE-US-00004 XO2 F (gas N (gas flow (rotation) from to mixture) from to rate) from to Growth 0% 30% Growth  0% 100% Growth 15% 80% and 400 1500 and 21%  21% and  6 72 biotrans- rpm rpm biotrans- biotrans- sL/h sL/h form- form- form- ation ation ation

(21) TABLE-US-00005 Script Trigger 31% DO (1/60 h) activated IPTG 2 h after feed induction start Feed trigger 50% DO Feed rate 3 [mL/h]

(22) The experiment carried out can be divided into two phases: growth, during which the cells are to reach a certain optical density, and subsequent biotransformation, in which, after addition of the substrates dodecanoic acid, oleic acid and dodecanedioic acid, a conversion to dodecylamine, oleylamine and dodecanediamine, respectively, is to take place by enzymes formed during expression. The pH levels were unilaterally adjusted to pH 6.8 using ammonia (12.5%). During growth and biotransformation, the dissolved oxygen (DO) in the culture was adjusted at 30% by means of stirrer speed and aeration rate. The fermentation was carried out as a fed batch, with the feed start, 5 g/Lh glucose feed (500 g/L glucose containing 1% MgSO.sub.4*7H.sub.2O and 2.2% NH.sub.4Cl), being triggered via a DO peak. With the feed start, the temperature was also lowered from previously 37° C. to 30° C. The expression of the transaminase, alanine dehydrogenase, carboxylic acid reductase and phosphopantetheinyl transferase was induced 2 h after the feed start by the automated addition of 1 mM IPTG. Before the start of biotransformation, the optical density of the culture broths was determined.

(23) The start of the biotransformation phase took place 1 h or 12 h after the feed start. To this end, 150 mL or 75 mL of a mixture of dodecanoic acid or dodecanedioic acid and oleic acid (technical-grade 90%) were added as a batch to the fermentation broth. To provide an amino group donor for the transaminase, 5 mL of a 3 M ammonium sulphate solution were added to the fermentation broth 30 minutes before the biotransformation start. For sampling, 2 mL of fermentation broth were removed from the tank and a portion thereof was diluted 1/20 in a and in a mixture of 80% acetonitrile, 20% water and 0.1% formic acid and extracted. Samples were taken from all reactors at 1.25 h, 2.75 h, 4.25 h, 18.25 h, and 21.75 h after the start of biotransformation. The conversion rates for oxygen (OTR=oxygen transfer rate) and carbon (CTR=carbon transfer rate) were determined during the fermentation via the waste-gas analyses on the DASGIP systems. The fermentation was ended 21.75 h after the start of biotransformation. The stirrer, the aeration system, the temperature control and pH control were turned off and the tanks were left to stand undisturbed for 5-10 minutes.

(24) HPLC-ESI/MS Scan Method

(25) The samples were qualitatively assessed by means of HPLC/MS coupling with high-resolution MS detection in the scan mode.

(26) The following instruments were used here: Accela HPLC system (Thermo Scientific, Waltham, Mass., USA) with autosampler, quaternary pump, PDA detector and column oven LTQ-FT mass spectrometer (Thermo Scientific, Waltham, Mass., USA) with ESI source HPLC column: Kinetex C18, 100×2.1 mm, particle size: 2.6 μm, pore size 100 Å (Phenomenex; Aschaffenburg)

(27) The samples were prepared by pipetting 1950 μl of solvent (80% (v/v) acetonitrile, 20% double-distilled H.sub.2O (v/v), +0.1% formic acid) and 50 μl of sample into a 2 ml reaction vessel. The mixture was vortexed for about 10 seconds and then centrifuged at about 13 000 rpm for 5 min. The clear supernatant was removed using a pipette.

(28) The HPLC separation was carried out with the aforementioned HPLC column. The injection volume was 0.5 μL, the column temperature 40° C., and the flow rate 0.3 mL/min. The mobile phase was composed of eluent A (0.02% strength (v/v) aqueous trifluoroacetic acid) and eluent B (acetonitrile with 0.015% (v/v) trifluoroacetic acid). The following gradient profile was used:

(29) TABLE-US-00006 Time [min] Eluent A [%] Eluent B [%] 0 98 2 2 98 2 17 2 98 32 2 98

(30) The ESI-MS analysis was carried out in the positive mode with the following parameters of the ESI source:

(31) TABLE-US-00007 ESI voltage: 4 kV Capillary temperature 300° C. Sheath gas flow 40 Aux gas flow 5 Sweep gas flow 3

(32) The detection was carried out within a mass range of m/z=100 to 1000. The mass spectrometry resolution was R=100 000.

(33) The results are shown in the table below.

(34) TABLE-US-00008 MS intensity [−] Substrate Induction Dodecanediamine Dodecylamine Oleylamine LA/oleic acid (150 ml) 1 mM IPTG (12 h) n.d. 1,160,000 23,700 DDA/oleic acid (75 ml) 1 mM IPTG in 742,000 n.d. 18,000 H.sub.2O/ethanol (12 h) DDA/oleic acid (150 ml) 1 mM IPTG (1 h) 30,700 n.d. n.d. DDA/oleic acid (150 ml) 1 mM IPTG (12 h) 23,500 n.d. n.d. Qualitative detection of monoamines and diamines in the fermentation broth of the strain E. coli W3110 pACYC{Placuv5}[carA_Ms-npt_Noc]/pJ281_alaDH_B.s._TA_C.v.(ct) after a 21.75 h process time (n.d. = not detectable, LA = lauric acid, DDA = dodecanedioic acid).

(35) Further data are illustrated in FIG. 1.

(36) 1,12-dodecanediamine and dodecylamine are quantitatively determined by means of HPLC/UV measurement, after derivatization by means of ortho-phthaldialdehyde. The methanolic supernatant was measured. The most important chromatographic parameters are summarized in the following table.

(37) TABLE-US-00009 Column Luna 5u C8, 100 Å, 150 × 4.60 mm (Phenomenex; Aschaffenburg) HPLC system Agilent 1200 Eluent A 2.5 mL of acetic acid (100%) to 1 L of double-distilled water, pH adjustment with sodium hydroxide solution to pH 6.0 Eluent B methanol Column temp. 40° C. Flow rate 1 mL/min Gradient 0.0-1 min: 30.0% B, 1.0-17.0 min: 90.0% B, 17-19.5 min: 90.0% B, 19.6- 20.5 min: 30.0% B Detector DAD, 334 nm Derivatization/ Automatic derivatization by means of injector program, 1 μL of sample is injection reacted with 9 μL of derivatization reagent; composition of derivatization volume reagent: 10 g/L o-phthaldialdehyde dissolved in borate buffer (0.4 mol/L), with addition of mercaptoethanol (5 mL/L) and methanol (100 mL/L) Calibration External calibration, measurement range 50-1000 mg/L, 5-point calibration, calibration before and after the sample series, averaging via both calibration series, quadratic regression

(38) The results are shown in the tables which follow.

(39) TABLE-US-00010 Dodecylamine Dodecanediamine Substrate Induction [mg/L] [mg/L] DDA/oleic acid 1 mM IPTG in n.d. 40.5 (75 ml) H.sub.2O/ethanol (12 h) DDA/oleic acid 1 mM IPTG (1 h) n.d. 3.1 (150 mL) DDA/oleic acid 1 mM IPTG (12 h) n.d. <1*.sup.) (150 ml) LA/oleic acid 1 mM IPTG (12 h) 11.6 n.d. (150 ml) Quantification of monoamines and diamines in the fermentation broth of the strain E. coli W3110 pACYC{Placuv5}[carA_Ms-npt_Noc]/pJ281_alaDH_B.s._TA_C.v.(ct) after a 21.75 h process time (n.d. = not detectable, *.sup.)lower than the detection limit, DDA = dodecanedioic acid, LA = lauric acid).

(40) It was shown that the strains are capable of producing, from dodecanoic acid, dodecanedioic acid and oleic acid, the respective amines dodecylamine, dodecanediamine and oleylamine.

EXAMPLE 5

(41) Producing an Expression Vector for the Expression of the Gene αDOX Encoding an α-dioxygenase from Oryza sativa

(42) To produce a vector for the expression of αDOX (Os12g0448900, SEQ ID No. 16) from Oryza sativa encoding an α-dioxygenase (NP_001066718.1), the gene was codon-optimized for expression in Escherichia coli, synthesized and, at the same time, an upstream NdeI restriction site and a downstream AvrII restriction site were introduced. The synthesized DNA fragment was digested with the restriction endonucleases NdeI and AvrII and ligated into the correspondingly cut vector pACYC{Placuv5}[carA_Ms-npt_Noc] (SEQ ID No. 10) with removal of the genes carA_Ms and npt_Noc. The lacuv5 promoter (SEQ ID No. 3) present in the vector was retained. The finished vector was referred to as pACYC{Placuv5}[DOX_Os(co_Ec)] (SEQ ID No. 17). The vector pACYC is an E. coli vector which mediates chloramphenicol resistance and also bears a p15A origin of replication and thus has a low copy number (10-15 copies per cell).

EXAMPLE 6

(43) Producing an E. coli Strain having a Deletion in the Gene bioH, Overexpressing the Genes αDOX from Oryza sativa, ald from Bacillus subtilis and Cv2025 from Chromobacterium violaceum

(44) To generate an E. coli strain which coexpresses the gene αDOX from Oryza sativa encoding an α-dioxygenase (NP_001066718.1) in combination with the genes ald (SEQ ID No. 11) from Bacillus subtilis encoding an alanine dehydrogenase (NP_391071.1) and Cv2025 (SEQ ID No. 12) from Chromobacterium violaceum encoding a transaminase (NP_901695.1), the strain E. coli W3110 ΔbioH (production: see EP12007663, example 1) was transformed with the plasmids pACYC{Placuv5}[DOX_Os(co_Ec)] (SEQ ID No. 17) and pJ281_alaDH_B.s._TA_C.v.(ct) (SEQ ID No. 15) by means of electroporation and plated out on LB agar plates containing chloramphenicol (50 μg/ml) and kanamycin (50 μg/ml). Transformants were checked as regards the presence of the correct plasmids by plasmid preparation and analytical restriction analysis. The strain generated was referred to as E. coli ΔbioH pACYC{Placuv5}[DOX_Os(co_Ec)]/pJ281_alaDH_B.s._TA_C.v.(ct).

(45) The strain was used to investigate its capability for the production of methyl aminoundecanoate proceeding from methyl dodecanedioate.

EXAMPLE 7

(46) Production of Methyl Aminoundecanoate Proceeding from Methyl Dodecanedioate by an E. coli Strain Containing an Expression Vector for the Gene αDOX from Oryza sativa in Combination with an Expression Vector for the Genes ald from Bacillus subtilis and Cv_2025 from Chromobacterium violaceum

(47) The strain described in example 8 was used to investigate its capability in relation to the production of methyl aminoundecanoate. The procedure for this was as follows:

(48) The strain under investigation was firstly spread out on an LB agar plate containing 50 μg/ml chloramphenicol and 50 μg/ml kanamycin and incubated overnight at 37° C. As control, the strain E. coli W3110 αbioH was additionally spread out on an LB agar plate not containing antibiotics. The strains were then cultured in Luria-Bertani broth, Miller (Merck, Darmstadt) containing 50 μg/ml chloramphenicol and 50 μg/ml kanamycin (for the plasmid-bearing strain) as a 20 ml pre-culture from a single colony in each case. As main culture, 100 ml of LB broth containing 50 μg/ml chloramphenicol and 50 μg/ml kanamycin were initially charged into a 500 ml Erlenmeyer flask containing baffles and inoculated with 2 ml from the pre-culture. Culturing was firstly carried out at 37° C. and 200 rpm in an incubator shaker. Upon attainment of an optical density (600 nm) of 0.5-0.7, the gene expression was induced by addition of 1 mM IPTG. Further culturing was carried out overnight at 22° C. and 200 rpm. The following day, the cultures were harvested by a 10-minute centrifugation at 4° C. and 5525×g. The supernatant was discarded and the cell pellet washed in 200 mM potassium phosphate buffer (pH 7.5). The cell pellet was lastly taken up in 200 mM potassium phosphate buffer containing 50 mM ammonium chloride and 0.5% (w/v) glucose, and so an OD (600 nm) of 20 was attained. 12.5 mM methyl dodecanedioate (abcr, Karlsruhe) in ethanol were added to the cell suspension and gently shaken for 4 hours at 30° C. and 300 rpm. During the incubation, samples were taken at the times 0 min, 60 min, 120 min, 180 min and 240 min and extracted in a mixture of 80% acetonitrile, 20% water and 0.1% formic acid. The supernatant was analysed by means of HPLC/MS analysis. The results are shown in the table below.

(49) TABLE-US-00011 Peak area Time Methyl Strain [min] aminoundecanoate E. coli W3110 ΔbioH 0 n.d. 120 n.d. 180 n.d. 240 n.d. E. coli W3110 ΔbioH pACYC{Placuv5}[DOX]/ 0 n.d. pJ281_alaDH_B.s._TA_C.v.(ct) 120 57616 180 371989 240 1764605 Production of methyl aminoundecanoate with E. coli W3110 ΔbioH overexpressing αDOX from Oryza sativa, ald from Bacillus subtilis and Cv_2025 from Chromobacterium violaceum. Peak areas are specified (n.d. = not detectable).

(50) It was possible to show that the strain E. coli W3110 ΔbioH pACYC{Placuv5}[DOX]/pJ281_alaDH_B.s._TA_C.v.(ct) is capable of forming methyl aminoundecanoate proceeding from methyl dodecanedioate.