Enzymatic omega-oxidation and omega-amination of fatty acids

Abstract

The invention relates to a method for oxidizing a fatty acid or an ester thereof of formula (I) H3C (CH2)n-COOR, wherein R is selected from the group that comprises H, methyl, ethyl, propyl, and butyl, wherein n is 0 to 30, preferably 6 to 24, comprising the step of oxidizing the fatty acid or the ester thereof by contacting the fatty acid or the ester thereof with a cytochrome P450 monooxygenase of the CYP153 family in the presence of molecular oxygen and NAD(P)H and a whole-cell catalyst that expresses a recombinant cytochrome P450 monooxygenase of the CYP153 family, a recombinant alcohol dehydrogenase, a recombinant transaminase, and optionally one or more than one recombinant enzyme from the group comprising alanine dehydrogenase, ferredoxin, and ferredoxin reductase, and the use of said whole-cell catalyst to oxidize a fatty acid or an ester thereof.

Claims

1. A method for oxidizing a fatty acid or an ester of the formula (I):
H.sub.3C(CH.sub.2).sub.nCOOR(I), where R is H, methyl, ethyl, propyl or butyl, and n is an integer from 6 to 30, the method comprising: a) contacting the fatty acid or the ester with a cytochrome P450 monooxygenase of the CYP153 family in the presence of molecular oxygen, NAD(P)H, and an electron donor, such that the fatty acid or the ester is oxidized and at least one fatty acid alcohol is produced, wherein the cytochrome P450 monooxygenase of the CYP153 family comprises a peptide having the amino acid sequence of SEQ ID NO: 21; b) contacting the fatty acid alcohol produced in a) with an alcohol dehydrogenase, such that the fatty acid alcohol reacts with the alcohol dehydrogenase, and aldehyde or ketone is produced; and c) contacting the aldehyde or ketone produced in b) with a transaminase in the presence of an amine donor, such that the aldehyde or ketone is aminated, wherein the contacting in a), b), and c) occurs in the presence of a cell which is genetically transformed to have a vector having a gene encoding the cytochrome P450 monooxygenase of the CYP153 family and at least one additional gene selected from the group consisting of a gene encoding the alcohol dehydrogenase and a gene encoding the transaminase, such that the cell expresses the cytochrome P450 monooxygenase of the CYP153 family, and at least one of the alcohol dehydrogenase and the transaminase.

2. The method according to claim 1, wherein the alcohol dehydrogenase is selected from the group consisting of a NAD(P).sup.+-dependent alcohol dehydrogenase, an alcohol dehydrogenase from Pseudomonas putida comprising the amino acid sequence of SEQ ID NO: 46 or a variant thereof, wherein the variant of the alcohol dehydrogenase from Pseudomonas putida has at least 70% homology to the amino acid sequence of SEQ ID NO: 46, a flavin-containing alcohol dehydrogenase from Candida tropicalis comprising the amino acid sequence of SEQ ID NO: 40 or a variant thereof, wherein the variant of the flavin-containing alcohol dehydrogenase from Candida tropicalis has at least 70% homology to the amino acid sequence of SEQ ID NO: 40, and a flavin-containing alcohol dehydrogenase from Candida cloacae comprising the amino acid sequence of SEQ ID NO: 68 or a variant thereof, wherein the variant of the flavin-containing alcohol dehydrogenase from Candida cloacae has at least 70% homology to the amino acid sequence of SEQ ID NO: 68.

3. The method according claim 1, wherein the electron donor is at least one of a ferredoxin reductase and a ferredoxin.

4. The method according to claim 1, wherein the contacting in c) is carried out in the presence of an alanine dehydrogenase, ammonia, and NADH, and the alanine dehydrogenase is an alanine dehydrogenase from Bacillus subtilis subsp. subtilis str. 168 having the amino acid sequence of SEQ ID NO: 22 or an alanine dehydrogenase having an amino acid sequence having at least 70% homology to the amino acid sequence of SEQ ID NO: 22.

5. The method according to claim 1, wherein the electron donor is at least one of a ferredoxin reductase and a ferredoxin, and the contacting in c) is carried out in the presence of an alanine dehydrogenase, ammonia, and NAD(P)H.

6. The method according to claim 5, wherein the vector further has a gene encoding the ferredoxin reductase, a gene encoding the ferredoxin, or both, such that the cell further expresses the ferredoxin reductase, the ferredoxin, or both.

7. The method according to claim 1, wherein the cell further expresses an AlkL polypeptide having the amino acid sequence of SEQ ID NO: 1, SEQ NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ NO: 9, or SEQ ID NO: 11.

8. The method according to claim 1, wherein the cell has a reduced activity of an enzyme which catalyzes at least one reaction of -oxidation of a fatty acid, compared to an activity in a corresponding wildtype cell, and the enzyme is at least one selected from the group consisting of FadA, FadB, FadD, FadL, and FadE from Escherichia coli.

9. The method according to claim 1, wherein the contacting in c) is carried out in the presence of an alanine dehydrogenase, ammonia, and NAD(P)H.

10. The method according to claim 1, wherein the cytochrome P450 monooxygenase of the CYP153 family has a peptide having the amino acid sequence of SEQ ID NO: 19 or a peptide having an amino sequence having 90% or more of homology to the amino acid sequence of SEQ ID NO: 19.

11. The method according to claim 10, wherein the fatty acid or the ester comprises methyl laurate.

12. The method according to claim 10, wherein the fatty acid or the ester comprises methyl laurate, and the electron donor is at least one of a ferredoxin reductase and a ferredoxin.

Description

EXAMPLE 1

Preparation of Expression Vectors for the Genes CYP153, Fd and FdOR from Alcanivorax borkumensis SK2 and alkL from Pseudomonas oleovorans

(1) To prepare an E. coli expression vector for the genes CYP153 (SEQ ID No. 20), Fd (SEQ ID No. 16) and FdOR (SEQ ID No. 14) from Alcanivorax borkumensis, as well as the gene alkL (SEQ ID No. 2) from Pseudomonas oleovorans, the genes were cloned under the control of the alkB promotor (SEQ ID No. 50) in the plasmid pCOM10. The different DNA fragments were amplified by inserting homologous regions for recombination cloning. The template used was the respective chromosomal DNA.

(2) The following oligonucleotides were used for the amplification of the respective fragments:

(3) TABLE-US-00002 Fd_CYP153 pHg-LL-07: (SEQIDNo.51) 5-TTAATAAAAATTGGAGTACAGACTTTTGGTAGGAGAATGC-3 pHg-LL-08: (SEQIDNo.52) 5-CCTTGGGCTTATTTTTTAGCCGTCAACTTAAC-3 FdOR pHg-LL-09: (SEQIDNo.53) 5-AAAAATAAGCCCAAGGCACAGATAAAGAGAGA-3 pHg-LL-10: (SEQIDNo.54) 5-TAGATCCTTCAGATCAAAGACTTTAATTCAAC-3 alkL pHg-LL-11: (SEQIDNo.55) 5-TGATCTGAAGGATCTAGGAACCAAGGAGAGTG-3 pHg-LL-06: (SEQIDNo.56) 5-CTTGGCTGCAGGTCGATTAGAAAACATATGACGCACCAAG-3

(4) The following parameters were used for the PCR:

(5) TABLE-US-00003 Fd-CYP153: Denaturation: 98 C. 30 s Denaturation: 98 C. 10 s 35x Annealing: 62 C. 20 s 35x Elongation: 72 C. 1:10 min 35x Final elongation: 72 C. 10 min FdOR Denaturation: 98 C. 30 s Denaturation: 98 C. 10 s 35x Annealing: 53 C. 20 s 35x Elongation: 72 C. 55 s 35x Final elongation: 72 C. 10 min alkL Denaturation: 98 C. 30 s Denaturation: 98 C. 10 s 25x Annealing: 65 C. 20 s 25x Elongation: 72 C. 18 s 25x Final elongation: 72 C. 10 min

(6) For the amplification, the Phusion High-Fidelity Master Mix from New England Biolabs (Frankfurt) was used according to the manufacturer's recommendations. In each case, 50 l of the PCR reactions were then separated on a 1% strength TAE agarose gel. The implementation of the PCR, of the agarose gel electrophoresis, of the ethidium bromide staining of the DNA and determination of the PCR fragment sizes was performed in the manner known to the person skilled in the art. In all cases, PCR fragments of the expected size could be amplified (Fd-CYP153: 1800 bp; FdOR: 1276 bp; alkL: 745 bp). For isolating and purifying the DNA, the PCR products were cut out of a preparative 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 using the Geneart Seamless Cloning and Assembly Kit in accordance with the manufacturer's instructions (Life Technologies, Carlsbad, Calif., USA) into a pCOM10 vector cleaved with EcoRI-HF and SalI (SEQ ID No. 57) behind the alkB promotor (SEQ ID No. 50). The transformation of chemically competent E. coli 10 beta cells (New England Biolabs, Frankfurt) was carried out 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 resulting expression vector was referred to as pCOM[Ab_Fd/CYP153-2/FdOR/alkL] (SEQ ID No. 58).

EXAMPLE 2

Preparation of Expression Vectors for the Genes CYP52A12 and OR from Candida tropicalis, and Also alkL from Pseudomonas oleovorans

(7) To prepare an E. coli expression vector for the genes CYP52A12 (SEQ ID No. 60) and OR (SEQ ID No. 64) from Candida tropicalis, and also the gene alkL (SEQ ID No. 2) from Pseudomonas oleovorans, the genes CYP52A12 and OR were codon-optimized for the expression in Escherichia coli in silico and synthesized together with the gene alkL as operon. During the synthesis, cleavage sites for AscI and SalI were inserted upstream of the CYP52A12 gene and downstream of the alkL gene. The synthesized DNA fragment CYP52A12 OR alkL was digested with the restriction endonucleases AscI and SalI, ligated into the correspondingly cleaved vector pCOM10 and the product was transformed into chemically competent E. coli 10 beta cells (New England Biolabs, Frankfurt). The finished vector was referred to as pCOM10-Ct CYP52A12_co plus OR_co (SEQ ID No. 65).

EXAMPLE 3

Preparation of Expression Vectors for the Genes CYP52A17 and OR from Candida tropicalis, and alkL from Pseudomonas oleovorans

(8) To prepare an E. coli expression vector for the genes CYP52A17 (SEQ ID No. 62), and OR (SEQ ID No. 64) from Candida tropicalis, and also the gene alkL (SEQ ID No. 2) from Pseudomonas oleovorans, the genes CYP52A17 and OR were codon-optimized for the expression in Escherichia coli in silico and synthesized together with the gene alkL as operon. During the synthesis, cleavage sites for AscI and SalI were inserted upstream of the CYP52A17 gene and downstream of the alkL-gene. The synthesized DNA fragment CYP52A12 OR alkL was digested with the restriction endonucleases AscI and SalI, ligated into the correspondingly cleaved vector pCOM10 and the product was transformed into chemically competent E. coli 10 beta cells (New England Biolabs, Frankfurt). The finished vector was referred to as pCOM10-Ct CYP52A17_co plus OR_co (SEQ ID No. 66).

EXAMPLE 4

Production of Methyl Hydroxylaurate by an E. coli Strain with Expression Vectors for the Genes CYP153, Fd and FdOR from Alcanivorax borkumensis SK2 and alkL from Pseudomonas oleovorans, or for the Genes CYP52A17 and OR from Candida tropicalis and alkL from Pseudomonas oleovorans

(9) To produce an E. coli strain with the expression vector pCOM[Ab_Fd/CYP153-2/FdOR/alkL] or pCOM10-Ct CYP52A17_co plus OR_co, electrocompetent cells of E. coli W3110 were prepared. This was carried out in a manner known to the person skilled in the art. E. coli W3110 was transformed in each case with one of the two listed plasmids and plated out onto LB-agar plates with kanamycin (50 g/ml). Transformants were checked as regards the presence of the correct plasmids by plasmid preparation and analytical restriction analysis. The following strains were constructed in this way: E. coli W3110 pCOM[Ab_Fd/CYP153-2/FdOR/alkL] E. coli W3110 pCOM10-Ct CYP52A17_co plus OR_co

(10) The strains were subjected to a fed-batch fermentation in order to investigate their ability to produce methyl hydroxylaurate, methyl oxolaurate and methyl carboxylaurate from methyl laurate. This was carried out in an 8-fold parallel fermentation system from DASGIP.

(11) For the fermentation, 1 l reactors were used which were equipped with overhead stirrers and impeller turbines. To monitor the process, pH and pO.sub.2 were measured online. OTR/CTR measurements served inter alia for estimating the metabolic activity and fitness of the cells.

(12) The pH probes were calibrated by means of a two-point calibration with measurement solutions of pH 4.0 and pH 7.0 according to technical reference of DASGIP. The reactors were provided according to technical reference with the required sensors and connections and the stirrer shaft was installed. The reactors were then filled with 300 ml of water and autoclaved for 20 min at 121 C. in order to ensure sterility. The pO.sub.2 probes were polarized overnight (at least 6 h) following connection to the measurement amplifier. The water was then removed under the clean bench and replaced by high-cell-density medium consisting of (NH.sub.4).sub.2SO4 1.76 g/l, K.sub.2HPO.sub.4 19.08 g/l, KH.sub.2PO.sub.4 12.5 g/l, yeast extracts 6.66 g/l, trisodium citrate dihydrate 11.2 g/l, 17 ml/l of a filter-sterilized 1% strength ammonium iron citrate solution, and 5 ml/l of a filter-sterilized trace element stock solution (consisting of HCl (37%) 36.50 g/l, MnCl.sub.2*4H.sub.2O 1.91 g/l, ZnSO.sub.4*7H.sub.2O 1.87 g/l, ethylenediaminetetraacetic acid dihydrate 0.84 g/l, H.sub.3BO.sub.3 0.30 g/l, Na.sub.2MoO.sub.4*2H.sub.2O 0.25 g/l, CaCl.sub.2*2H.sub.2O 4.70 g/l, FeSO.sub.4*7H.sub.2O 17.80 g/l, CuCl.sub.2*2H.sub.2O 0.15 g/l) with 15 g/l glucose as carbon source (added by metered addition of 30 ml/l of a sterile feed solution consisting of 500 g/l glucose, 1% (w/v) MgSO.sub.4*7H.sub.2O and 2.2% (w/v) NH.sub.4Cl) with 50 mg/l kanamycin.

(13) Subsequently, the pO.sub.2 probes were calibrated using a single-point calibration (stirrer: 600 rpm/gassing: 10 sL/h air) to 100% and the feed, correction agent and induction agent stretches were cleaned by means of cleaning-in-place according to technical reference. For this, the tubes were firstly flushed with 70% ethanol, then with 1 M NaOH, then with sterile demineralized water and finally filled with the respective media.

(14) All of the aforementioned E. coli strains were cultured firstly from a cryoculture in LB medium (25 ml in a 100 ml chicane flask) with 50 mg/l kanamycin overnight at 37 C. and 200 rpm for about 18 h. Then, 2 ml of this culture were transferred for a second preculture stage into 25 ml of high-cell-density medium consisting of (NH.sub.4).sub.2SO.sub.4 1.76 g/L, K.sub.2HPO.sub.4 19.08 g/l, KH.sub.2PO.sub.4 12.5 g/l, yeast extract 6.66 g/l, trisodium citrate dihydrate 11.2 g/l, 17 ml/l of a filter-sterilized 1% strength ammonium iron citrate solution, and 5 ml/l of a filter-sterilized trace element stock solution (consisting of HCl (37%) 36.50 g/l, MnCl.sub.2*4H.sub.2O 1.91 g/l, ZnSO.sub.4*7H.sub.2O 1.87 g/l, ethylenediaminetetraacetic acid dihydrate 0.84 g/l, H.sub.3BO.sub.3 0.30 g/l. Na.sub.2MoO.sub.4*2H.sub.2O 0.25 g/l, CaCl.sub.2*2H.sub.2O 4.70 g/l, FeSO.sub.4*7H.sub.2O 17.80 g/l, CuCl.sub.2*2H.sub.2O 0.15 g/l) with 15 g/l glucose as carbon source (added by metered addition of 30 ml/l of a sterile feed solution consisting of 500 g/l glucose, 1% (w/v) MgSO.sub.4*7H.sub.2O and 2.2% (w/v) NH.sub.4Cl) with the already described antibiotics in a 100 ml shake flask and incubated at 37 C./200 rpm for a further 6 h.

(15) In order to inoculate the reactors with an optical density of 0.1, the OD.sub.600 of the second preculture stage was measured and the amount of culture required for the inoculation was calculated. The required amount of culture was added with the help of a 5 ml syringe through a septum into the heat-treated and aerated reactor.

(16) The following standard program was used:

(17) TABLE-US-00004 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 ml/h max 100% max 40 ml/h

(18) TABLE-US-00005 XO2 (gas F (gas N (Rotation) from to mixture) from to flow rate) from to growth and 0% 30% growth and 0% 100% growth and 15% 80% biotransfor- 400 rpm 1500 rpm biotransfor- 21% 21% biotransfor- 6 sL/h 72 sL/h mation mation mation

(19) TABLE-US-00006 Script Trigger 31% DO (1/60h) sharp Induction 10 h after feed DCPK start Feed trigger 50% DO Feed rate 3 [ml/h]

(20) The pH was regulated to pH 6.8 on one side with 12.5% strength ammonia solution. During cultivation and biotransformation, the dissolved oxygen (pO.sub.2 or DO) in the culture was regulated to at least 30% by means of stirrer feed and gassing rate. Following inoculation, the DO dropped from 100% to this 30%, where it was kept stable for the remainder of the fermentation.

(21) The fermentation was carried out as fed-batch, where the feed start was triggered as delivery to the feed phase with 5 g/l*h glucose feed, consisting of 500 g/l glucose, 1% (w/v) MgSO.sub.4*7H.sub.2O and 2.2% (w/v) NH.sub.4Cl, via the DO peak inducing the end of the batch phase. With feed start, the temperature of 37 C. was lowered to 30 C. 10 h after feed start, the expression of the oxidation genes was induced with 0.025% (v/v) DCPK. The start of the methyl hydroxylaurate production (=start of the biotransformation) was carried out 14 h after feed start. For this purpose, 150 ml of a mixture of methyl laurate and oleic acid (technical-grade 90%) were added as batch to the fermentation broth.

(22) To quantify LSME and HLS in fermentation samples, samples were taken 1/2/4/20/22 h after the start of biotransformation. These samples were prepared for analysis. (see LC-ESI/MS.sup.2-based quantification of products).

(23) LC-ESI/MS.sup.2-Based Quantification of Products

(24) The quantification of LSME and HCL in fermentation samples was carried out by means of LC-ESI/MS.sup.2 by reference to an external calibration for all analytes (0.1-50 mg/l) and using the internal standard aminoundecanoic acid (AUD for HLSME), and d3-LSME (for LSME).

(25) The following instruments were used here: HPLC system 1260 (Agilent; Bblingen) with autosampler (G1367E), binary pump (G1312B) and column oven (G1316A) Mass spectrometer TripelQuad 6410 (Agilent; Bblingen) with ESI source HPLC column: Kinetex C18, 1002.1 mm, particle size: 2.6 m, pore size 100 (Phenomenex; Aschaffenburg) Precolumn: KrudKatcher Ultra HPLC In-Line Filter; 0.5 m filter depth and 0.004 mm internal diameter (Phenomenex; Aschaffenburg)

(26) The samples were prepared by pipetting 1900 l of solvent (80% (v/v) acetonitrile, 20% double-distilled H.sub.2O (v/v), +0.1% formic acid) and 100 l sample in 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 and, after appropriate dilution, analysed with diluents (80% (v/v) ACN, 20% double-distilled. H.sub.2O (v/v), +0.1% formic acid). 100 L of ISTD were pipetted into each 900 L sample (10 L for a sample volume of 90 L).

(27) The HPLC separation was carried out with the aforementioned column and precolumn. The injection volume was 0.7 L, the column temperature 50 C., the flow rate 0.6 mL/min. The mobile phase consisted of eluent A (0.1% strength (v/v) aqueous formic acid) and eluent B (acetonitrile with 0.1% (v/v) formic acid). The following gradient profile was used:

(28) TABLE-US-00007 Time [min] Eluent A [%] Eluent B [%] 0 77 23 0.3 77 23 0.4 40 60 2.5 40 60 2.6 2 98 5.5 2 98 5.6 77 23 9 77 23

(29) The ESI-MS.sup.2 analysis was carried out in the positive mode with the following parameters of the ESI source: Gas temperature 280 C. Gas flow rate 11 L/min Nebulizing pressure 50 psi Capillary voltage 4000 V

(30) The detection and quantification of the compounds DDS, DDSME, HLS, HLSME, OLS, OLSME was carried out with the following MRM parameters, with in each case a product ion being used as qualifier and one as quantifier

(31) TABLE-US-00008 Precursor ion Product ion Residence Analyte [m/z] [m/z] time [ms] Collision energy [eV] HLSME 231.3 181.2 15 2 HLSME 231.3 163.2 25 5

(32) The analyte LSME was detected in the SIM mode (m/z 201 and 215).

(33) It was able to be shown that the strain E. coli W3110 pCOM[Ab_Fd/CYP153-2/FdOR/alkL] is able to form methyl w-hydroxylaurate from methyl laurate. The strain E. coli W3110 pCOM10-Ct CYP52A17_co plus OR_co was able to convert methyllaurate to methyl -hydroxylaurate or further oxidation products only to a considerably lesser extent.

(34) The concentrations of methyl laurate and methyl -hydroxylaurate are given after a fermentation time of 22 hours.

(35) TABLE-US-00009 C (Lauric acid) C (-Hydroxyacid methylester methylester) Strain [g/L] [g/L] E. coli W3110 pCOM[Ab_Fd/ 88.1 4.35 CYP153-2/FdOR/alkL] E. coli W3110 pCOM10-Ct 106.9 <0.1 CYP52A17_co plus OR_co

EXAMPLE 5

Prophetic

Production of Methyl Hydroxylaurate by an E. coli Strain with Expression Vectors for the Genes CYP153, Fd and FdOR from Alcanivorax Borkumensis SK2 and alkL from Pseudomonas oleovorans or for the Genes CYP52A12 and OR from Candida tropicalis and alkL from Pseudomonas oleovorans

(36) To produce an E. coli strain with the expression vector pCOM[Ab_Fd/CYP153-2/FdOR/alkL] or pCOM10-Ct CYP52A12_co plus OR_co, electrocompetent cells of E. coli W3110 are prepared. This is carried out in a manner known to the person skilled in the art. E. coli W3110 is transformed in each case with one of the two listed plasmids and plated out onto LB-agar plates with kanamycin (50 g/ml). Transformants are tested as regards the presence of the correct plasmids by plasmid preparation and analytical restriction analysis. The following strains are constructed in this way: E. coli W3110 pCOM[Ab_Fd/CYP153-2/FdOR/alkL] E. coli W3110 pCOM10-Ct CYP52A12_co plus OR_co

(37) The strains are subjected to a fed-batch fermentation in order to investigate their ability to produce HLSME. This is carried out in an 8-fold parallel fermentation system from DASGIP. 1 L reactors equipped with overhead stirrers and impeller turbines are used for the fermentation. pH and pO.sub.2 are measured online for monitoring the process. OTR/CTR measurements serve inter alia to estimate the metabolic activity and fitness of the cells.

(38) The pH probes are calibrated by means of a two-point calibration with measurement solutions of pH 4.0 and pH 7.0 according to technical reference from DASGIP. The reactors are provided according to technical reference with the required sensors and connections and the stirrer shaft is installed. Then, the reactors are filled with 300 mL of water and autoclaved for 20 min at 121 C. in order to ensure sterility. The pO.sub.2 probes are polarized overnight (at least 6 h) following connection to the measurement amplifier. The water is then removed under the clean bench and replaced by high-cell-density medium consisting of (NH.sub.4).sub.2SO4 1.76 g/L, K.sub.2HPO.sub.4 19.08 g/L, KH.sub.2PO.sub.4 12.5 g/L, yeast extract 6.66 g/L, trisodium citrate dihydrate 11.2 g/L, 17 mL/L of a filter-sterilized 1% strength ammonium iron citrate solution, and 5 mL/L of a filter-sterilized trace element strain solution (consisting of HCl (37%) 36.50 g/L, MnCl.sub.2*4H.sub.2O 1.91 g/L, ZnSO.sub.4*7H.sub.2O 1.87 g/L, ethylenediaminetetraacetic acid dihydrate 0.84 g/L, H.sub.3BO.sub.3 0.30 g/L. Na.sub.2MoO.sub.4*2H.sub.2O 0.25 g/L, CaCl.sub.2*2H.sub.2O 4.70 g/L, FeSO.sub.4*7H.sub.2O 17.80 g/L, CuCl.sub.2*2H.sub.2O 0.15 g/L) with 15 g/L glucose as carbon source (added by metered addition of 30 mL/L of a sterile feed solution consisting of 500 g/L glucose, 1% (w/v) MgSO.sub.4*7H.sub.2O and 2.2% (w/v) NH.sub.4Cl) with 50 mg/L kanamycin.

(39) Subsequently, the pO.sub.2 probes are calibrated with a single-point calibration (stirrer: 600 rpm/gassing: 10 sL/h air) to 100%, and the feed, correcting agent and induction agent stretches are cleaned by means of cleaning-in-place according to technical reference. For this, the tubes are first flushed with 70% ethanol, then with 1 M NaOH, then with sterile demineralized water, and finally filled with the respective media.

(40) All of the aforementioned E. coli strains are first cultivated from a cryoculture in LB medium (25 mL in a 100 mL shake flask) with 50 mg/L kanamycin overnight at 37 C. and 200 rpm for about 18 h. Then, 2 mL of this culture are transferred for a second preculture stage in 25 mL of high-cell-density medium consisting of (NH.sub.4).sub.2SO.sub.4 1.76 g/L, K.sub.2HPO.sub.4 19.08 g/L, KH.sub.2PO.sub.4 12.5 g/L, yeast extract 6.66 g/L, trisodium citrate dihydrate 11.2 g/L, 17 mL/L of a filter-sterilized 1% strength ammonium iron citrate solution, and 5 mL/L of a filter-sterilized trace element strain solution (consisting of HCl (37%) 36.50 g/L, MnCl.sub.2*4H.sub.2O 1.91 g/L, ZnSO.sub.4*7H.sub.2O 1.87 g/L, ethylenediaminetetraacetic acid dihydrate 0.84 g/L, H.sub.3BO.sub.3 0.30 g/L. Na.sub.2MoO.sub.4*2H.sub.2O 0.25 g/L, CaCl.sub.2*2H.sub.2O 4.70 g/L, FeSO.sub.4*7H.sub.2O 17.80 g/L, CuCl.sub.2*2H.sub.2O 0.15 g/L) with 15 g/L glucose as carbon source (added by metered addition of 30 mL/L of a sterile feed solution consisting of 500 g/L glucose, 1% (w/v) MgSO.sub.4*7H.sub.2O and 2.2% (w/v) NH.sub.4CI) with the already described antibiotics in a 100 mL shake flask and incubated at 37 C./200 rpm for a further 6 h.

(41) In order to inoculate the reactors with an optical density of 0.1, the OD.sub.600 of the second preculture stage is measured and the amount of culture required for the inoculation is calculated. The amount of culture required is added with the help of a 5 mL syringe through a septum into the heat-treated and aerated reactor.

(42) The following standard program is used:

(43) TABLE-US-00010 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 mL/h max 100% max 40 mL/h

(44) TABLE-US-00011 XO2 (gas F (gas N (rotation) from to mixture) from to flow rate) from to Growth and 0% 30% Growth and 0% 100% Growth and 15% 80% biotransfor- 400 rpm 1500 rpm biotransfor- 21% 21% biotransfor- 6 sL/h 72 sL/h mation mation mation

(45) TABLE-US-00012 Script Trigger 31% DO (1/60 h) sharp Induction 10 h after feed DCPK start Feed trigger 50% DO Feed rate 3 [mL/h]

(46) The pH is regulated to pH 6.8 on one side with 12.5% strength ammonia solution. During cultivation and biotransformation, the dissolved oxygen (pO.sub.2 or DO) in the culture is regulated to at least 30% via stirrer speed and gassing rate. Following inoculation, the DO drops from 100% to this 30%, where it is kept stable for the remainder of the fermentation.

(47) The fermentation is carried out as fed batch, where the feed start is triggered as entry to the feed phase with 5 g/L*h glucose feed, consisting of 500 g/L glucose, 1% (w/v) MgSO.sub.4*7H.sub.2O and 2.2% (w/v) NH.sub.4Cl, via the DO peak indicating the end of the batch phase. With feed start, the temperature is lowered from 37 C. to 30 C. 10 h after feed start, the expression of the oxidation genes is induced with 0.025% (v/v) DCPK. The start of the methyl hydroxylaurate production (=start of the biotransformation) takes place 14 h after feed start. For this, 150 mL of a mixture of methyl laurate and oleic acid (technical-grade 90%) were added as batch to the fermentation broth.

(48) For quantification of LSME and HLSME, fermentation samples are taken 1/2/4/20/22 h after the start of biotransformation. These samples are prepared for analysis. (See LC-ESI/MS.sup.2-based quantification of products).

(49) LC-ESI/MS.sup.2-Based Quantification of Products.

(50) The quantification of LSME and HLSME in fermentation samples takes place by means of LC-ESI/MS.sup.2 by reference to an external calibration for all analytes (0.1-50 mg/L) and using the internal standard aminoundecanoic acid (AUD for HLSME) and d3-LSME (for LSME).

(51) The following equipment is used here: HPLC system 1260 (Agilent; Bblingen) with autosampler (G1367E), binary pump (G1312B) and column oven (G1316A) Mass spectrometer TripelQuad 6410 (Agilent; Bblingen) with ESI source HPLC column: Kinetex C18, 1002.1 mm, particle size: 2.6 m, pore size 100 (Phenomenex; Aschaffenburg) Precolumn: KrudKatcher Ultra HPLC In-Line Filter; 0.5 m filter depth and 0.004 mm internal diameter (Phenomenex; Aschaffenburg)

(52) The samples are prepared by pipetting 1900 L of solvent (80% (v/v) of acetonitrile, 20% double-distilled H.sub.2O (v/v), +0.1% formic acid) and 100 L of sample in a 2-mL reaction vessel. The mixture is vortexed for about 10 seconds and then centrifuged at about 13 000 rpm for 5 min. The clear supernatant is removed using a pipette and analysed following appropriate dilution with diluent (80% (v/v) ACN, 20% double-distilled H.sub.2O (v/v), +0.1% formic acid). 100 L of ISTD are pipetted in for each 900 L of sample (10 L for a sample volume of 90 L).

(53) The HPLC separation takes place with the aforementioned column or precolumn. The injection volume is 0.7 L, the column temperature is 50 C., and the flow rate is 0.6 mL/min. The mobile phase consists of eluent A (0.1% strength (v/v) aqueous formic acid) and eluent B (acetonitrile with 0.1% (v/v) formic acid). The following gradient profile is used:

(54) TABLE-US-00013 Time [min] Eluent A [%] Eluent B [%] 0 77 23 0.3 77 23 0.4 40 60 2.5 40 60 2.6 2 98 5.5 2 98 5.6 77 23 9 77 23

(55) The ESI-MS.sup.2 analysis takes place in the positive mode with the following parameters of the ESI source: Gas temperature 280 C. Gas flow rate 11 L/min Nebulizer pressure 50 psi Capillary voltage 4000 V

(56) The detection and quantification of the compound HLSME takes place with the following MRM parameters, with in each case one product ion being used as qualifier and one as quantifier

(57) TABLE-US-00014 Precursor ion Product ion Residence Analyte [m/z] [m/z] time [ms] Collision energy [eV] HLSME 231.3 181.2 15 2 HLSME 231.3 163.2 25 5

(58) The analyte is detected in the SIM mode (m/z 201 and 215).

(59) It is found that the strain E. coli W3110 pCOM[Ab_Fd/CYP153-2/FdOR/alkL] is able to form methyl w-hydroxylaurate from methyl laurate. The strain E. coli W3110 pCOM10-Ct CYP52A12_co plus OR_co can convert methyl laurate to methyl -hydroxylaurate or other oxidation products only to a lesser extent.

(60) The features of the invention disclosed in the preceding description, the claims and the examples may be essential both individually and also in any desired combination for realizing the invention in its various embodiments.