Byosynthetic Production of Acyl Amino Acids

Abstract

The present invention relates to a cell for producing acyl glycinates wherein the cell is genetically modified to comprise at least a first genetic mutation that increases the expression relative to the wild type cell of an amino acid-N-acyl-transferase, at least a second genetic mutation that increases the expression relative to the wild type cell of an acyl-CoA synthetase, and at least a third genetic mutation that decreases the expression relative to the wild type cell of at least one enzyme selected from the group consisting of an enzyme of the glycine cleavage system, glycine hydroxymethyltransferase (GlyA) and threonine aldolase (LtaE).

Claims

1-15. (canceled)

16. A cell for producing acyl glycinates, wherein said cell is genetically modified to comprise: a) at least a first genetic mutation that, relative to the wild type cell, increases the expression of an amino acid-N-acyl-transferase; b) at least a second genetic mutation that, relative to the wild type cell, increases the expression of an acyl-CoA synthetase; and c) at least a third genetic mutation that, relative to the wild type cell, decreases the expression of at least one enzyme selected from the group consisting of: an enzyme of the glycine cleavage system; glycine hydroxymethyltransferase (GlyA); threonine aldolase (LtaE); threonine dehydrogenase (Tdh); 2-Amino-3-Ketobutyrate CoA-Ligase (Kbl); and allothreonine dehydrogenase (YdfG).

17. The cell of claim 16, wherein said cell comprises a mutation in an enzyme from the glycine cleavage system selected from the group consisting of: glycine cleavage system T protein; glycine cleavage system H protein; and glycine cleavage system P protein.

18. The cell of claim 16, wherein the third genetic mutation decreases the expression relative to the wild type cell of the glycine hydroxymethyltransferase (GlyA), the threonine aldolase (LtaE), the glycine cleavage system T protein, the glycine cleavage system H protein and the glycine cleavage system P protein.

19. The cell of claim 18, wherein the glycine cleavage system T protein has 85% sequence identity to SEQ ID NO:58, the glycine cleavage system H protein has 85% sequence identity to SEQ ID NO:59, and the glycine cleavage system P protein has 85% sequence identity to SEQ ID NO:60.

20. The cell of claim 16, wherein the glycine hydroxymethyltransferase (GlyA) has 85% sequence identity to SEQ ID NO:61 and the threonine aldolase (LtaE) has 85% sequence identity to SEQ ID NO:62.

21. The cell of claim 16, wherein the cell has a reduced fatty acid degradation capacity relative to the wild type cell.

22. The cell of claim 21, wherein the reduced fatty acid degradation capacity is a result of a decrease in expression, relative to the wild type cell, of at least one enzyme selected from the group consisting of: acyl-CoA dehydrogenase; 2,4-dienoyl-CoA reductase; enoyl-CoA hydratase; and 3-ketoacyl-CoA thiolase.

23. The cell of claim 16, wherein the amino acid-N-acyl-transferase has 85% sequence identity to SEQ ID NO:63 or SEQ ID NO:64 and the acyl-CoA synthetase has 85% sequence identity to SEQ ID NO:65.

24. The cell according to claim 16, wherein said cell is a bacterial cell.

25. The cell of claim 16, wherein the cell is E. coli.

26. The cell of claim 16, wherein the cell is further genetically modified to comprise a fourth genetic mutation, wherein, compared to the wild type cell, said fourth mutation increases the expression of at least one transporter protein.

27. The cell of claim 26, wherein the transporter protein is selected from the group consisting of FadL and AlkL.

28. The cell of claim 16, wherein the acyl glycinate is lauroylglycinate.

29. The cell of claim 16, wherein the cell is capable of making proteinogenic amino acids or fatty acids.

30. The cell of claim 16, wherein said cell is a bacterium and: a) the amino acid-N-acyl-transferase comprises the sequence of SEQ ID NO:63 or SEQ ID NO:64; b) the acyl-CoA synthetase comprises the sequence of SEQ ID NO:65; and c) the third genetic mutation decreases the expression of at least one enzyme selected from the group consisting of: a glycine cleavage system T protein comprising the sequence of SEQ ID NO:58; a glycine cleavage system H protein comprising the sequence of SEQ ID NO:59; a glycine cleavage system P protein comprising the of SEQ ID NO:60; a glycine hydroxymethyltransferase (GlyA) comprising the sequence of SEQ ID NO:61; and a threonine aldolase (LtaE) comprising the sequence of SEQ ID NO:62.

31. The cell of claim 30, wherein the cell is E. coli.

32. The cell of claim 30, wherein the cell has a reduced fatty acid degradation capacity relative to the wild type cell.

33. The cell of claim 30, wherein the cell is further genetically modified to comprise a fourth genetic mutation, wherein, compared to the wild type cell, said fourth mutation increases the expression of at least one transporter protein selected from the group consisting of FadL and AlkL.

34. The cell of claim 30, wherein the acyl glycinate is lauroylglycinate.

35. A method for producing acyl amino acids, comprising contacting an amino acid and a fatty acid or an acyl CoA thereof with the cell of claim 16.

Description

BRIEF DESCRIPTION OF FIGURES

[0130] The inventions are further illustrated by the following figures and non-limiting examples from which further embodiments, aspects and advantages of the present invention may be taken.

[0131] FIG. 1 is a graph depicting a total ion chromatogram of the 48 h sample from the E. coli W3110 fadE pJ294{Ptac}[synUcTE]/pCDF{Ptac}[hGLYAT2(co_Ec)_fadD_Ec] fermentation.

[0132] FIG. 2 is a graph depicting a total ion chromatogram of the 48 h sample from the E. coli W3110 fadE pJ294{Ptac}[synUcTE]/pCDF{Ptac}[hGLYAT3(co_Ec)_fadD_Ec] fermentation.

[0133] FIG. 3 is a graph depicting a total ion chromatogram of the 48 h sample from the E. coli W3110 fadE pJ294{Ptac}[synUcTE]/pCDFDuet-1 fermentation (negative control).

[0134] FIG. 4 is a graph showing the production of lauroylglycinate by E. coli strains W3110 fadE pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1].

[0135] FIG. 5 is a graph showing the production of lauroylglycinate by E. coli strain W3110 fadE gcvTHP pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1].

[0136] FIG. 6 is a tree showing the percentage sequence identity of GLYAT2 in the various organisms tested in Example 18.

[0137] FIG. 7 is an illustration showing the metabolic pathways of glycine.

EXAMPLES

[0138] The foregoing describes preferred embodiments, which, as will be understood by those skilled in the art, may be subject to variations or modifications in design, construction or operation without departing from the scope of the claims. These variations, for instance, are intended to be covered by the scope of the claims.

Sequence ID NOs:

[0139] Throughout this application a range of SEQ ID NOs are used. These are shown in Table 3 below.

TABLE-US-00003 TABLE 3 Sequences used in the Examples. SEQ ID NO: Comment 1 Umbellularia californica synUcTE (an acyl-CoA thioesterase) gene (codon-optimized) 2 tac promoter 3 Vector pJ294[Ptac-synUcTE], see example 1 4 Homo sapiens genes hGLYAT2 (an amino acid N-acyl transferase) 5 Homo sapiens genes hGLYAT3 (another amino acid N-acyl transferase) 6 Escherichia coli fadD (an acyl-CoA synthetase) 7 Vector pCDF[atfA1_Ab(co_Ec)-fadD_Ec], see example 2 8 Vector pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec], see example 2 9 Vector pCDF{Ptac}[hGLYAT3(co_Ec)-fadD_Ec], see example 2 10 alkL (an importer facilitating transport of hydrophobic acyl across cell membranes) gene, see example 3 11 lacuv5 promoter, see example 3 12 Vector pCDF[alkLmod1], see example 3 13 Vector pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1], see example 3 14 Vector pET-28b, see example 10 15 Vector pET-28b{Ptac}[hGLYAT2(co_Ec)], see example 10 16 pET-28b{Ptac}[hGLYAT3(co_Ec)], see example 10

Example 1

[0140] Generation of an Expression Vector for the Umbellularia californica Gene synUcTE

[0141] To generate an expression vector for the Umbellularia californica synUcTE gene (SEQ ID NO:1), which encodes the Umbellularia californica acyl CoA-thioesterase, this gene was codon-optimized for expression in Escherichia coli. The gene was synthesized together with a tac promoter (SEQ ID N0:2), and, simultaneously, one cleavage site was introduced upstream of the promoter and one cleavage site downstream of the terminator. The synthesized DNA fragment Ptac-synUcTE was digested with the restriction endonucleases BamHI and NotI and ligated into the correspondingly cut vector pJ294 (DNA2.0 Inc., Menlo Park, Calif., USA). The finished E. coli expression vector was referred to as pJ294[Ptac-synUcTE] (SEQ ID NO:3).

Example 2

[0142] Generation of Vectors for Coexpression of Escherichia coli fadD with Either the Homo sapiens Genes hGLYAT3 and hGLYAT2

[0143] To generate vectors for the coexpression of the Homo sapiens genes hGLYAT2 (SEQ ID NO:4) or hGYLAT3 (SEQ ID NO:5), which encodes human glycine-N-acyltransferase, with Escherichia coli fadD (SEQ ID NO:6), which encodes the E. coli acyl-CoA synthetase, the genes hGLYAT2 and hGLYAT3 were codon-optimized for expression in Escherichia coli and synthesized. The synthesized DNA fragments were digested with the restriction endonucleases SacII and Eco47III and ligated into the correspondingly cut pCDF[atfA1_Ab(co_Ec)-fadD_Ec] (SEQ ID NO:7) with removal of the aftAl gene. The sequence segments which were additionally removed in this process were cosynthesized during gene synthesis. The vector is a pCDF derivative which already comprises a synthetic tac promoter (SEQ ID NO:2) and the Escherichia coli fadD gene. The resulting expression vectors were named pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec] (SEQ ID NO:8) and pCDF{Ptac}[hGLYAT3(co_Ec)-fadD_Ec] (SEQ ID NO:9).

Example 3

[0144] Generation of Vectors for the Coexpression of the Homo sapiens hGLYAT2, Escherichia coli fadD and Pseudomonas putida alkL Genes

[0145] To generate vectors for the coexpression of the hGLYAT2 genes with a modified Pseudomonas putida alkL gene, which encodes AlkL, an outer membrane protein that facilitates the import of hydrophobic substrates into a cell, the alkL gene (SEQ ID NO:10) was amplified together with the lacuv5 promoter (SEQ ID NO:11) from the plasmid pCDF[alkLmod1] (SEQ ID NO:12) by means of sequence-specific oligonucleotides. The PCR products were cleaved with the restriction endonucleases BamHI and NsiI and ligated into the correspondingly cleaved vector pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec] (SEQ ID NO:8). The correct insertion of the target genes was checked by restriction analysis and the authenticity of the introduced genes was verified by DNA sequencing. The resulting expression vector was named pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1] (SEQ ID NO:13).

[0146] The following parameters were used for PCR: 1: initial denaturation, 98 C., 3:00 min; 35 denaturation, 98 C., 0:10 min; annealing, 65 C., 0:20 min; elongation, 72 C., 0:17 min; 1: final elongation, 72 C., 10 min. For amplification the Phusion High-Fidelity Master Mix from New England Biolabs (Frankfurt) was used according to manufacturer's manual. 50 l of the PCR reaction were analyzed on a 1% TAE agarose gel. Procedure of PCR, agarose gel electrophoresis, ethidium bromide staining of DNA and determination of PCR fragment size were carried out known to those skilled in the art.

Example 4

[0147] Generation of an E. coli Strain with Deletion in the fadE Gene, which Strain Overexpresses the Umbellularia californica synUcTE, Escherichia coli fadD and Homo sapiens hGLYAT2 and hGLYAT3 Genes

[0148] To generate E. coli strains which coexpress the Umbellularia californica synUcTE in combination with the Escherichia coli fadD and Homo sapiens hGLYAT2 or Homo sapiens hGLYAT3 genes, the strain E. coli W3110 fadE was transformed with the plasmids pJ294{Ptac}[synUcTE] and pCDF{Ptac}[hGLYAT2(co_Ec)_fadD_Ec] or pCDF{Ptac}[hGLYAT3(co_Ec)_fadD_Ec] by means of electroporation and plated onto LB-agar plates supplemented with spectinomycin (100 g/ml) and ampicillin (100 g/ml). Transformants were checked for the presence of the correct plasmids by plasmid preparation and analytic restriction analysis. The strains E. coli W3110 fadE pJ294{Ptac}[synUcTE]/pCDF{Ptac}[hGLYAT2(co_Ec)_fadD_Ec] and E. coli W3110 fadE pJ294{Ptac}[synUcTE]/pCDF{Ptac}[hGLYAT3(co_Ec)_fadD_Ec] were generated thus.

Example 5

[0149] Generation of an E. coli Strain with Deletion in the fadE Gene, which Strain Overexpresses the Escherichia coli fadD and Either Homo sapiens hGLYAT2 or hGLYAT3 Genes

[0150] To generate E. coli strains which overexpress the Escherichia coli fadD gene in combination with the Homo sapiens hGLYAT2 or hGLYAT3 genes, electrocompetent cells of E. coli strain W3110 fadE were generated. E. coli W3110 fadE was transformed with the plasmid pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1] and plated onto LB-agar plates supplemented with spectinomycin (100 g/ml). Transformants were checked for the presence of the correct plasmids by plasmid preparation and analytic restriction analysis. The strain generated thus was named E. coli W3110 fadE pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1].

Example 6

[0151] Production of Fatty Acid/Amino Acid Adducts by E. coli Strains with Deletion in the fadE Gene, which Strains Overexpress the synUcTE and fadD Genes in Combination with Either hGLYAT2 or hGLYAT3

[0152] The strains generated in Example 4 were used to study their ability to produce fatty acid/amino acid adducts. Starting from a 80 C. glycerol culture, the strains to be studied were first plated onto an LB-agar plate supplemented with 100 g/ml ampicillin and 100 g/ml spectinomycin and incubated overnight at 37 C. Starting from a single colony in each case, the strains were then grown as a 5-ml preculture in Luria-Bertani broth, Miller (Merck, Darmstadt) supplemented with 100 g/mlampicillin and 100 g/ml spectinomycin. The further culture steps were performed in M9 medium. The medium, composed of 38 mM disodium hydrogenphosphate dihydrate, 22 mM potassium dihydrogenphosphate, 8.6 mM sodium chloride, 37 mM ammonium chloride, 2% (w/v) glucose, 2 mM magnesium sulphate heptahydrate (all chemicals from Merck, Darmstadt) and 0.1% (v/v) trace element solution, was brought to pH 7.4 with 25% strength ammonium hydroxide solution. The trace element solution added, composed of 9.7 mM manganese(II) chloride tetrahydrate, 6.5 mM zinc sulphate heptahydrate, 2.5 mM sodium-EDTA (Titriplex III), 4.9 mM boric acid, 1 mM sodium molybdate dihydrate, 32 mM calcium chloride dihydrate, 64 mM iron(II) sulphate heptahydrate and 0.9 mM copper(II) chloride dihydrate, dissolved in 1 M hydrochloric acid (all chemicals from Merck, Darmstadt) was filter-sterilized before being added to the M9 medium. 20 ml of M9 medium supplemented with 100 g/ml spectinomycin and 100 g/mlampicillin were introduced into baffled 100-ml Erlenmeyer flasks and inoculated with 0.5 ml preculture. The flasks were cultured at 37 C. and 200 rpm in a shaker-incubator. After a culture time of 8 hours, 50 ml of M9 medium supplemented with 100 g/ml spectinomycin and 100 g/mlampicillin were introduced into a baffled 250-ml Erlenmeyer flask and inoculated with the 10-ml culture to achieve an optical density (600 nm) of 0.2. The flasks were cultured at 37 C. and 200 rpm in a shaker-incubator. When an optical density (600 nm) of 0.7 to 0.8 was reached, gene expression was induced by addition of 1 mM IPTG. The strains were cultured for a further 48 hours at 30 C. and 200 rpm. Simultaneously with the induction, 1 g/l glycine was added to some of the cultures. During culturing, samples were taken, and fatty acid/amino acid adducts present were analysed. The results are shown in FIGS. 1 and 2. It has been possible to demonstrate that both E. coli strain W3110 fadE pJ294{Ptac}[synUcTE]/pCDF{Ptac}[hGLYAT2(co_Ec)_fadD_Ec] and E. coli strain W3110 fadE pJ294{Ptac}[synUcTE]/pCDF{Ptac}[hGLYAT3(co_Ec)_fadD_Ec] are capable of forming various fatty acid/amino acid adducts, for example lauroyl-glutamic acid, from glucose. By contrast, no such adducts can be found in a cell that, as a negative control, lacks the plasmids (FIG. 3). It appears that slight sequence variations, for example amino acid substitutions that distinguish hGLYAT2 from hGLYAT3, do not compromise the ability of the cell to make the fatty acid/acyl amino acid adducts as shown in Table 4.

TABLE-US-00004 TABLE 4 Quantitative determination of fatty acid glycinates after 48 h culture time C.sub.lauroylglycinate C.sub.myristoylglycinate Strain [mg/L] [mg/L] E. coli W3110 fadE pJ294{Ptac}[synUcTE]/ 111 <2 pCDF{Ptac}[hGLYAT2(co_Ec)_fadD_Ec] E. coli W3110 fadE pJ294{Ptac}[synUcTE]/ 121 2.8 pCDF{Ptac}[hGLYAT2(co_Ec)_fadD_Ec] + 1 g/L glycine E. coli W3110 fadE pJ294{Ptac}[synUcTE]/ n.d. n.d. pCDFDuet-1

Example 7

Chromatographic Quantification of Products by HPLC/MS

[0153] The quantification of N-lauroylglycine, N-myristoylglycine and N-palmitoylglycine and the detection of other acylamino acids in fermentation samples was performed by HPLC-ESI/MS. The quantification was performed with the aid of an external calibration (approx. 0.1-50 mg/l) for the three target compounds in the single ion monitoring mode (SIM). In parallel, a scan was carried out over a mass range m/z=100-1000 so as to identify further acylamino acids.

[0154] The samples for the determination of the fatty acid glycinates were prepared as follows: 800 l of solvent (acetone) and 200 l of sample were pipetted into a 2-ml reaction vessel. The mixture was shaken in a Retsch mill for 1 minute at 30 Hz and then centrifuged for 5 min at approximately 13 000 rpm. The clear supernatant was removed using a pipette and, after suitable dilution with diluent (80% acetonitrile/20% water+0.1% formic acid), analyzed. The calibration standards used were likewise dissolved and diluted in this diluent.

[0155] The following equipment was employed: [0156] Surveyor HPLC system (Thermo Scientific, Waltham, Mass., USA) composed of MS pump, Autosampler Plus and PDA Detector Plus [0157] Mass spectrometer TSQ Vantage with HESI II source (Thermo Scientific, Waltham, Mass., USA) [0158] HPLC column: 1002 mm Pursuit XRS Ultra C8; 2.8 m (Agilent, Santa Clara, Calif., USA)

Chemicals:

[0159] Water from a Millipore system [0160] Acetonitrile for HPLC (Merck AG, Darmstadt, Germany) [0161] Formic acid, p.a. grade (Merck, Darmstadt, Germany) [0162] N-propanol Lichrosolv (Merck, Darmstadt, Germany) [0163] N-lauroylglycine 99% (Chem-Impex International, Wood Dale, Ill., USA) [0164] N-myristoylglycine >98% (Santa Cruz Biotechnology, Texas, USA) [0165] N-palmitoylglycine >99% (provenance unknown)

[0166] The HPLC separation was carried out using the abovementioned HPLC column. The injection volume amounted to 2 l, the column temperature to 40 C., the flow rate to 0.3 ml/min. The mobile phase consisted of Eluent A (0.1% strength (v/v) aqueous formic acid) and Eluent B (75% acetonitrile/25% n-propanol (v/v) with 0.1% (v/v) formic acid). The following gradient profile was used:

TABLE-US-00005 TABLE 5 Gradient profile used in Example 7. Time Eluent A Eluent B [min] [%] [%] 0 90 10 1 90 10 20 5 95 25 5 95

[0167] The HPLC/MS analysis was carried out under positive ionization mode with the following parameters of the ESI source:

TABLE-US-00006 Spray Voltage: 3500 V Vaporizer Temperature: 50 C. Sheath Gas Pressure: 40 Aux. Gas Pressure: 10 Capillary Temperature: 250 C. Sprayer Distance: Ring C

[0168] Detection and quantification of the three analytes were performed by single ion monitoring (SIM) with the following parameters shown in Table 6.

TABLE-US-00007 TABLE 6 Parameters used in SIM of Example 7. Ion [M + H] Scan range Scan time Resolution Analyte [m/z] [m/z] [ms] Q3 N-lauroylglycine 258.2 0.002 50 0.7 N-myristoylglycine 286.2 0.002 50 0.7 N-palmitoylglycine 314.2 0.002 50 0.7

Example 8

[0169] Production of Fatty Acid Amino Acid Adducts by E. coli Strains with Deletion in the fadE Gene, which Strains Overexpress the synUcTE and fadD Genes in Combination with hGLYAT2 or hGLYAT3 in a Parallel Fermentation System

[0170] The strains generated in Example 4 were used for studying their ability to produce fatty acid amino acid adducts from glucose. For this purpose, the strain was cultured both in a shake flask and in a fed-batch fermentation. The fermentation was carried out in a parallel fermentation system from DASGIP with 8 bioreactors.

[0171] The production cells were prepared as described in Example 6.

[0172] The fermentation was performed using 1 l reactors equipped with overhead stirrers and impeller blades. pH and pO.sub.2 were measured online for process monitoring. OTR/CTR measurements served for estimating the metabolic activity and cell fitness, inter alia.

[0173] The pH electrodes were calibrated by means of a two-point calibration using standard solutions of pH 4.0 and pH 7.0, as specified in DASGIP's technical instructions. The reactors were provided with the necessary sensors and connections as specified in the technical instructions, and the agitator shaft was fitted. The reactors were then charged with 300 ml of water and autoclaved for 20 min at 121 C. to ensure sterility. The pO.sub.2 electrodes were connected to the measuring amplifiers and polarized overnight (for at least 6 h). Thereafter, the water was removed under a clean bench and replaced by M9 medium (pH 7.4) composed of KH.sub.2PO.sub.4 3.0 g/l, Na.sub.2HPO.sub.4 6.79 g/l, NaCl 0.5 g/l, NH.sub.4Cl 2.0 g/l, 2 ml of a sterile 1 M MgSO.sub.4*7H.sub.2O solution and 1 ml/l of a filter-sterilized trace element stock solution (composed 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 the carbon source (added by metering in 30 ml/l of a sterile feed solution composed of 500 g/l glucose, 1.3% (w/v) MgSO.sub.4*7H.sub.2O) supplemented with 100 mg/l spectinomycin and 3 ml/l DOW1500.

[0174] Thereafter, the pO.sub.2 electrodes were calibrated to 100% with a one-point calibration (stirrer: 400 rpm/aeration: 10 sl/h air), and the feed, correction agent and induction agent lines were cleaned by cleaning in place as specified in the technical instructions. To this end, the tubes were rinsed first with 70% ethanol, then with 1 M NaOH, then with sterile fully-demineralized water and, finally, filled with the respective media.

[0175] Using the E. coli strain of Example 4, a dilution streak was first performed with a cryoculture on an LB agar plate supplemented with 100 mg/l spectinomycin, and the plate was incubated for approximately 16 h at 37 C. LB medium (10 ml in a 100-ml baffle flask) supplemented with 100 mg/l spectinomycin was then inoculated with a single colony and the culture was grown overnight at 37 C. and 200 rpm for approximately 16 h. Thereafter, this culture was used for a second preculture stage with an initial OD of 0.2 in 50 ml of M9 medium, composed of KH.sub.2PO.sub.4 3.0 g/l, Na.sub.2HPO.sub.4 6.79 g/l, NaCl 0.5 g/l, NH.sub.4Cl 2.0 g/l, 2 ml of a sterile 1 M MgSO.sub.4*7H.sub.2O solution and 1 ml/l of a filter-sterilized trace element stock solution (composed 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) supplemented with 20 g/l glucose as carbon source (added by metering in 40 ml/l of a sterile feed solution composed of 500 g/l glucose) together with the above-described antibiotics was transferred into a 500-ml baffle flask and incubated for 8-12 h at 37 C./200 rpm.

[0176] To inoculate the reactors with an optical density of 0.1, the 00600 of the second preculture stage was measured and the amount of culture required for the inoculation was calculated. The required amount of culture was placed into the heated and aerated reactor with the aid of a 5-ml syringe through a septum.

[0177] The standard program shown in Table 7a-c was used:

TABLE-US-00008 TABLE 7 Standard program for use of heated and aerated reactor in Example 8 a) DO controller pH controller 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 b) XO2 F (gas (gas N (Rotation) from to mixture) from to flow) from to Growth and 0% 30% Growth and 0% 100% Growth and 15% 80% biotransformation 400 rpm 1500 rpm biotransformation 21% 21% biotransformation 6 sl/h 72 sl/h c) Script Trigger fires 31% DO (1/60 h) Induction 2 h after the feed IPTG start Feed trigger 50% DO Feed rate 3 [ml/h]

[0178] The pH was adjusted unilaterally to pH 7.0 with 12.5% strength ammonia solution. During the growth phase and the biotransformation, the dissolved oxygen (pO.sub.2 or DO) in the culture was adjusted to at least 30% via the stirrer speed and the aeration rate. After the inoculation, the DO dropped from 100% to these 30%, where it was maintained stably for the remainder of the fermentation.

[0179] The fermentation was carried out as a fed batch, the feed start as the beginning of the feed phase with 5 g/l*h glucose feed, composed of 500 g/l glucose, 1.3% (w/v) MgSO.sub.4*7H.sub.2O, being triggered via the DO peak which indicates the end of the batch phase. From the feed start onwards, the temperature was reduced from 37 C. to 30 C. 2 h after the feed start, the expression was induced with 1 mM IPTG.

[0180] To quantify lauroyl, myristoyl and palmitoyl glycinate, samples were taken 47 h and 64 h after the start of the fermentation. These samples were prepared for analysis, and analyzed as described in Example 7.

[0181] It has been possible to demonstrate that strain E. coli W3110 fadE pJ294{Ptac}[synUcTE]/pCDF{Ptac}[hGLYAT2(co_Ec)_fadD_Ec] is capable of forming lauroyl glycinate from glucose.

TABLE-US-00009 TABLE 8 Quantification of fatty acid glycinates after 47 and 64 h fermentation time. Ion [M + H] Scan range Scan time Resolution Analyte [m/z] [m/z] [ms] Q3 N-Lauroylglycine 258.2 0.002 50 0.7 N-Myristoylglycine 286.2 0.002 50 0.7 N-Palmitoylglycine 314.2 0.002 50 0.7

TABLE-US-00010 TABLE 9 Production of fatty acids after 47 and 64 hours' fermentation time. (n.d.: not determined) Fermentation time [h] C.sub.Lauroyl glycinate [g/l] C.sub.Myristoyl glycinate [g/l] C.sub.Palmitoyl glycinate [g/l] C.sub.Glycine [g/l] C.sub.Octanoic acid [g/l] C.sub.Decanoic acid [g/l] 47 0.63 0.07 n.d. n.d. n.d. 0.001 64 1.06 0.11 n.d. n.d. n.d. n.d. Fermentation time [h] C.sub.Lauric acid [g/l] C.sub.Myristic acid [g/l] C.sub.Palmitoleic acid [g/l] C.sub.Palmitic acid [g/l] C.sub.Oleic acid [g/l] C.sub.Stearic acid [g/l] 47 n.d. n.d. n.d. 0.002 0.006 0.001 64 0.001 n.d. n.d. n.d. 0.002 n.d.

Example 9

[0182] The strain of Example 5 was fermented in a fed-batch fermentation to study the ability of linking lauric acid and glycine to give lauroyl glycinate. This fermentation was carried out in a parallel fermentation system from DASGIP with 8 bioreactors.

[0183] The experimental setting was as described in Example 8 except that 100 g/l glycine in demineralized water and 100 g/l laurate in lauric acid methyl ester were fed rather than glucose. To quantify lauroyl, myristoyl and palmitoyl glycinate in fermentation samples, samples were taken 23 h and 42 h after the start of the fermentation. These samples were prepared for analysis, and analyzed as described in Example 7. The results are shown in Tables 10 and 11.

[0184] It has been possible to demonstrate that the strain E. coli W3110 fadE pCDF{Ptac}[hGLYAT2(co_Ec)_fadD_Ec] {Plavuv5} [alkLmod1] is capable of linking lauric acid and glycine and of producing lauroyl glycinate.

TABLE-US-00011 TABLE 10 Production of lauroyl glycinate after fermentation for 23 and 42 hours with feeding of lauric acid and glycine. Fermentation time [h] C.sub.Lauroyl glycinate [g/l] C.sub.Myristoyl glycinate [g/l] C.sub.Palmitoyl glycinate [g/l] C.sub.Glycine [g/l] C.sub.Octanoic acid [g/l] C.sub.Decanoic acid [g/l] 21 1.02 n.d. n.d. n.d. n.d. n.d. 40 1.78 n.d. n.d. n.d. n.d. n.d. Fermentation time [h] C.sub.Lauric acid [g/l] C.sub.Myristic acid [g/l] C.sub.Palmitoleic acid [g/l] C.sub.Palmitic acid [g/l] C.sub.Oleic acid [g/l] C.sub.Stearic acid [g/l] 21 6.38 n.d. n.d. n.d. n.d. n.d. 40 6.79 n.d. n.d. n.d. n.d. n.d.

TABLE-US-00012 TABLE 11 Production of lauroyl glycinate after fermentation for 23 and 42 hours without feeding of lauric acid and glycine (negative control). Fermentation time [h] C.sub.Lauroyl glycinate [g/l] C.sub.Myristoyl glycinate [g/l] C.sub.Palmitoyl glycinate [g/l] C.sub.Glycine [g/l] C.sub.Octanoic acid [g/l] C.sub.Decanoic acid [g/l] 21 n.d. n.d. n.d. n.d. n.d. n.d. 40 n.d. n.d. n.d. n.d. n.d. n.d. Fermentation time [h] C.sub.Lauric acid [g/l] C.sub.Myristic acid [g/l] C.sub.Palmitoleic acid [g/l] C.sub.Palmitic acid [g/l] C.sub.Oleic acid [g/l] C.sub.Stearic acid [g/l] 21 n.d. n.d. n.d. n.d. n.d. n.d. 40 n.d. n.d. n.d. n.d. n.d. n.d.

Example 10

[0185] Generation of Vectors for Expression of the Homo sapiens Genes hGLYAT3 and hGLYAT2 in E. coli Strains Producing Fatty Acids Via Malonyl-CoA and Acetyl-CoA

[0186] To generate vectors for the expression of the Homo sapiens genes hGLYAT2 (SEQ ID NO: 4) or hGYLAT3 (SEQ ID NO: 5) in the fatty acid producing strains listed in Table 3 below (from Table 3.2 of WO2014026162A1), the genes hGLYAT2 and hGYLAT3 were first amplified. The gene hGLYAT2 was amplified from the plasmid pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1] (SEQ ID NO:13) and the hGYLAT3 gene was amplified from the plasmid pCDF{Ptac}[hGLYAT3(co_Ec)-fadD_Ec] (SEQ ID NO:9) by means of sequence-specific oligonucleotides. The PCR products were cleaved with the restriction endonucleases NotI and Sacl and ligated in the correspondingly cleaved vector pET-28b (SEQ ID NO:14). The correct insertion of the target genes was checked by restriction analysis and the authenticity of the introduced genes was verified by DNA sequencing. The resulting expression vectors were named pET-28b{Ptac}[hGLYAT2(co_Ec)] (SEQ ID NO:15) and pET-28b{Ptac}[hGLYAT3(co_Ec)] (SEQ ID NO:16).

Example 11

[0187] Production of Fatty Acid Amino Acid Adducts Via Malonyl-CoA and Acetyl-CoA by Strains Overexpressing hGLYAT2 or hGLYAT3 in a Shake Flask Experiment

[0188] The vectors produced according to Example 6 were then used to generate a microorganism strain from Table 12 below (OPX Biotechnologies Inc., USA) using any transformation method known in the art. In particular, the methods provided in section IV of WO2014026162A1 were used.

[0189] The strains generated were used for studying their ability to produce fatty acids, in particular amino acid adducts from glucose. For this purpose, the strains were transformed with the vectors pET-28b{Ptac}[hGLYAT2(co_Ec)] (SEQ ID NO:15) and pET-28b{Ptac}[hGLYAT3(co_Ec)] (SEQ ID NO:16) and cultured in shake flasks (Subsection C of section IV of WO2014026162A1). Strain BXF_031 (OPX Biotechnologies Inc., USA) harbouring the empty vector pET-28b was used as a control.

[0190] Triplicate evaluations were performed. Briefly, overnight starter cultures were made in 50 ml of Terrific Broth including the appropriate antibiotics and incubated 16-24 hours at 30 C., while shaking at 225 rpm. These cultures were used to inoculate 150 ml cultures of each strain in SM11 minimal medium to an OD.sub.600 of 0.8 and 5% TB culture carryover as starting inoculum, and antibiotics. 1 L SM11 medium consists of: 2 ml FM10 Trace Mineral Stock, 2.26 ml 1M MgSO.sub.4, 30 g glucose, 200 mM MOPS (pH 7.4), 1 g/L yeast extract, 1.25 ml VM1 Vitamin Mix, 0.329 g K.sub.2HPO.sub.4, 0.173 g KH.sub.2PO.sub.4, 3 g (NH.sub.4).sub.2SO.sub.4, 0.15 g citric acid (anhydrous); FM10 Trace Mineral Stock consists of: 1 ml of concentrated HCl, 4.9 g CaCl.sub.2*2H.sub.2O, 0.97 g FeCl.sub.3*6H.sub.2O, 0.04 g CoCl.sub.2*6H.sub.2O, 0.27 g CuCl.sub.2*2H.sub.2O, 0.02 g ZnCl.sub.2, 0.024 g Na.sub.2MoO.sub.4*2H.sub.2O, 0.007 g H.sub.3BO.sub.3, 0.036 g MnCl.sub.2*4H.sub.2O, Q.S. with DI water to 100 ml; VM1 Vitamin Mix Solution consists of: 5 g Thiamine, 5.4 g Pantothenic acid, 6.0 g Niacin, 0.06 g, Q.S. with DI water to 1000 ml. All ingredients for the culture mediums used in this example are provided in (Subsection A of section IV of WO2014026162A1).

[0191] Cultures were incubated for 2 hours at 30 C., while shaking at 225 rpm. After 2 hours, the cells were washed with SM11 (SM11 medium without phosphate). Cells were twice spun down (4,000 rpm, 15 min), the supernatant decanted, the pellet re-suspended in 150 ml of SM11 (SM11 medium without phosphate). The cultures were used to inoculate 350 ml of each strain in SM11 (no phosphate). The cultures were grown at 30 C. for approximately 2 h to an 00600 of 1.0-1.5 after 2 h cells and shifted to 37 C. and samples removed periodically for product measurement over the course of 72 hrs.

[0192] The quantification of N-lauroylglycine, N-myristoylglycine and N-palmitoylglycine and the detection of other acylamino acids in fermentation samples was performed by HPLC-ESI/MS.

TABLE-US-00013 TABLE 12 List of microorganism strains that were used to introduce the genes hGLYAT2 or hGLYAT3 in Examples 10 and 11. The method of production and the sequences of the strains are provided in Table 3.2 of WO2014026162A1 (OPX Biotechnologies Inc., USA). Strain SEQ ID designation Host Plasmid NOs. BXF_0012 BX_864 1)pBMT-3_ccdAB 17 BXF_0013 BX_864 1)pBMT-3_ccdAB_P.sub.T7-tesA 18 BXF_0014 BX_864 1)pBMT-3_ccdAB_P.sub.T7-nphT7-hbd-crt-ter 19 BXF_0015 BX_864 1)pBMT-3_ccdAB_P.sub.T7-tesA_PT7-nph.sub.T7-hbd-crt-ter 20 BXF_0020 BX_860 1)pBMT-3_ccdAB_PT7-tesA_PT7-nphT7-hbd-crt-ter 20 BXF_0021 BX_876 1)pBMT-3_ccdAB_PT7-tesA_PT7-nphT7-hbd-crt-ter 20 BXF_0022 BX_874 1)pBMT-3_ccdAB 17 BXF_0023 BX_874 1)pBMT-3_ccdAB_PT7-tesA 18 BXF_0024 BX_874 1)pBMT-3_ccdAB_PT7-tesA_PT7-nphT7-hbd-crt-ter 20 BXF_0025 BX_875 1)pBMT-3_ccdAB 17 BXF_0026 BX_875 1)pBMT-3_ccdAB_PT7-tesA 18 BXF_0027 BX_875 1)pBMT-3_ccdAB_PT7-tesA_PT7-nphT7-hbd-crt-ter 20 BXF_0028 BX_878 1)pBMT-3ccdAB-T7-tesA-PT7_nphT7_hbd_crt_ter 20 BXF_0028 BX_878 1)pBMT-3_ccdAB_PT7-tesA_PT7-nphT7-hbd-crt-ter 20 BXF_0029 BX_879 1)pBMT-3_ccdAB_PT7-tesA_PT7-nphT7-hbd-crt-ter 20 BXF_0030 BX_881 1)pBMT-3_ccdAB_PT7-tesA_PT7-nphT7-hbd-crt-ter 20 BXF_0031 BX_864 1)pBMT-3_ccdAB_PT7-tesA_PT7-nphT7-hbd-crt-ter 20 2)pET-28b(empty vector) 21 BXF_0033 BX_878 1)pBMT-3_ccdAB_PT7-nphT7-hbd-crt-ter 19 BXF_0034 BX_879 2)pBMT-3_ccdAB_PT7-nphT7-hbd-crt-ter 19

Example 12

[0193] Generation of a Vector for Deletion of the gcvTHP Operon in Escherichia coli W3110 fadE

[0194] To generate a vector for the deletion of the gcvTHP operon of E. coli W3110, which encodes a glycine cleavage system (GcvT: aminomethyltransferase,tetrahydrofolate-dependent, subunit (T protein) of glycine cleavage complex; GcvH: glycine cleavage complex lipoylprotein; GcvP: glycine decarboxylase, PLP-dependent, subunit (protein P) of glycine cleavage complex), approx. 500 bp upstream and downstream of the GcvTHP operon were amplified via PCR. The upstream region of GcvTHP was amplified using the oligonucleotides o-MO-40 (SEQ ID NO:22) and o-MO-41 (SEQ ID NO:23) The downstream region of gcvTHP was amplified using the oligonucleotides o-MO-42 (SEQ ID NO:24) and o-MO-43 (SEQ ID NO:25). The PCR procedure is described above in Example 3.

[0195] In each case PCR fragments of the expected size could be amplified (PCR 1,553 bp, (SEQ ID NO:26); PCR 2,547 bp, SEQ ID NO:27). The PCR samples were separated via agarose gel electrophoresis and DNA fragments were isolated with QiaQuick Gel extraction Kit (Qiagen, Hilden). The purified PCR fragments were cloned into the vector pKO3 (SEQ ID NO:28), and cut with BamHI using the Geneart Seamless Cloning and Assembly Kit (Life Technologies, Carlsbad, Calif., USA). The assembled product was transformed into chemically competent E. coli DH5 cells (New England Biolabs, Frankfurt). Procedure of PCR purification, in-vitro cloning and transformation were carried out according to manufacturer's manual. The correct insertion of the target genes was checked by restriction analysis and the authenticity of the introduced DNA fragments was verified by DNA sequencing. The resulting knock-out vector was named pKO3 delta gcvTHP (SEQ ID NO:29).

[0196] The construction of strain E. coli W3110 fadE gcvTHP was carried out with the help of pKO3 delta gcvTHP using the method described in Link et al., 1997. The DNA sequence after deletion of gcvTHP is SEQ ID NO:30. The E. coli strain W3110 fadEgcvTHP was transformed with the plasmid pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1] (SEQ ID NO:13, Example 3) by means of electroporation and plated onto LB-agar plates supplemented with spectinomycin (100 g/mL). Transformants were checked for the presence of the correct plasmids by plasmid preparation and analytic restriction analysis. The resulting strain was named E. coli W3110 fadEgcvTHP pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1].

Example 13

[0197] Production of Lauroylglycinate by E. coli Strains with Deletion in the fadE or fadE/gcvTHP Gene, Overexpressing the hGLYAT2, fadD and alkL Genes

[0198] The strain generated in Example 1 was used to study its ability to produce more lauroylglycinate, in comparison to the reference strain without gcvTHP deletion.

[0199] Starting from a 80 C. glycerol culture, the strains to be studied were first plated onto an LB-agar plate supplemented with 100 g/mL spectinomycin and incubated overnight at 37 C. Starting from a single colony in each case, the strains were then grown as a 5-mL preculture in LB-broth, Miller (Merck, Darmstadt) supplemented with 100 g/mL spectinomycin. The further culture steps were performed in M9-FIT medium. The medium, composed of 38 mM disodium hydrogenphosphate dihydrate, 22 mM potassium dihydrogenphosphate, 8.6 mM sodium chloride, 37 mM ammonium chloride, 2 mM magnesium sulphate heptahydrate (all chemicals from Merck, Darmstadt), 5% (w/v) maltodextrin solution (dextrose equivalent 13.0-17.0, Sigma Aldrich, Taufkirchen), 1% (w/v) amyloglycosidase from Aspergillus niger (Sigma-Aldrich, Taufkirchen), 1 drop Delamex 180 (Bussetti & Co, Wien) and 0.1% (v/v) trace element solution, was brought to pH 7.4 with 25% strength ammonium hydroxide solution. The trace element solution added, composed of 9.7 mM manganese(II) chloride tetrahydrate, 6.5 mM zinc sulphate heptahydrate, 2.5 mM sodium-EDTA (Titriplex III), 4.9 mM boric acid, 1 mM sodium molybdate dihydrate, 32 mM calcium chloride dihydrate, 64 mM iron(II) sulphate heptahydrate and 0.9 mM copper(II) chloride dihydrate, dissolved in 1 M hydrochloric acid (all chemicals from Merck, Darmstadt) was filter-sterilized before being added to the M9 medium. 20 mL of M9 medium supplemented with 100 g/mL spectinomycin were introduced into baffled 100-mL Erlenmeyer flasks and inoculated with 0.5 mL preculture. The flasks were cultured at 37 C. and 200 rpm in a shaker-incubator. After a culture time of 8 hours, 50 mL of M9 medium supplemented with 100 g/mL spectinomycin and were introduced into a baffled 250-mL Erlenmeyer flask and inoculated with the 10-mL culture to achieve an optical density (600 nm) of 0.1. The flasks were cultured at 37 C. and 200 rpm in a shaker-incubator. When an optical density (600 nm) of 0.6 to 0.8 was reached, gene expression was induced by addition of 1 mM IPTG. The strains were cultured for a further 48 hours at 37 C. and 200 rpm. 1-3 h after the induction, 6 g/L glycine and 6 g/L lauric acid (dissolved in lauric acid methyl ester) were added to the cultures. After 0 h and 24 h cultivation samples were taken, and lauroylglycinate, lauric acid and glycine present were analysed. The results are shown in FIGS. 4 and 5. It has been possible to demonstrate that both E. coli strains W3110 fadE pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1] and E. coli strain W3110 fadE fadE pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1] are capable of forming lauroylglycinate. But in the new strain background fadE gcvTHP higher amounts of lauroylglycinate were synthesized and higher amounts of glycine are detected. It appears that less glycine is metabolized in the gcvTHP background and can be used for lauroylglycinate synthesis.

Example 14

[0200] Generation of a Vector for Deletion of the glyA Gene in Escherichia coli W3110 fadE

[0201] To generate a vector for the deletion of the glyA-gene encoding a component of the Glycine hydroxymethyltransferase of E. coli W3110 approx. 500 bp upstream and downstream of the glyA-gene were amplified via PCR. The upstream region of glyA was amplified using the oligonucleotides o-MO-44 (SEQ ID NO:31) and o-MO-45 (SEQ ID NO:32). The downstream region of glyA was amplified using the oligonucleotides o-MO-46 (SEQ ID NO:33) and o-MO-47 (SEQ ID NO:34).

[0202] In each case PCR fragments of the expected size could be amplified (PCR 1,546 bp, (SEQ ID NO:35); PCR 2,520 bp, SEQ ID NO:36). The PCR samples were separated via agarose gel electrophoresis and DNA fragments were isolated with QiaQuick Gel extraction Kit (Qiagen, Hilden). The purified PCR fragments were assembled via a crossover PCR. The generated fragment was purified and subcloned into the cloning vector pCR-Blunt IITOPO (Life technologies) according to manufacturer's manual. To clone the fragment into the target plasmid pKO3 (SEQ ID NO:28) it was amplified with flanking BamHI restriction sites, using the oligonucleotides o-MO-52 (SEQ ID NO:37) and o-MO-53 (SEQ ID NO:38). The purified, BamHI cleaved PCR 3 fragment (SEQ ID NO:39) was ligated into the correspondingly cleaved vector pKO3 (SEQ ID N0:28). The assembled product was transformed into chemically competent E. coli DH5 cells (New England Biolabs, Frankfurt). Procedure of PCR purification, in-vitro cloning and transformation were carried out according to manufacturer's manual. The correct insertion of the target genes was checked by restriction analysis and the authenticity of the introduced genes was verified by DNA sequencing. The resulting knock-out vector was named pKO3 delta glyA (SEQ ID NO:40).

[0203] The construction of strain E. coli W3110 fadE glyA was carried out with the help of pKO3 delta GlyA using the method described in Link et al., 1997. SEQ ID NO:41 is the DNA sequence after deletion of glyA. The E. coli strain W3110 fadE gcvTHP was transformed with the plasmid pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1] (SEQ ID NO:13 from Example 3), by means of electroporation and plated onto LB-agar plates supplemented with spectinomycin (100 g/mL). Transformants were checked for the presence of the correct plasmids by plasmid preparation and analytic restriction analysis. The resulting strain was named E. coli W3110 fadE glyA pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1].

Example 15

[0204] Generation of a Vector for Deletion of the taE Gene in Escherichia coli W3110 fadE

[0205] To generate a vector for the deletion of the ItaE-gene encoding the L-allo-threonine aldolase of E. coli W3110 approximately 500 bp upstream and downstream of the ItaE were amplified via PCR as described above. The upstream region of ItaE was amplified using the oligonucleotides ItaE-UP_fw (SEQ ID NO:42) and ItaE-UP-XhoI_rev (SEQ ID NO:43). The downstream region of ItaE was amplified using the oligonucleotides ItaE-DOWN_fw (SEQ ID NO:44) and ItaE-DOWN_rev (SEQ ID NO:45).

[0206] In each case PCR fragments of the expected size could be amplified (PCR 4,550 bp, (SEQ ID NO:46); PCR 5, 536 bp, SEQ ID NO:47). The PCR samples were separated via agarose gel electrophoresis and DNA fragments were isolated with QiaQuick Gel extraction Kit (Qiagen, Hilden). The purified PCR fragments were assembled via a crossover PCR. The generated fragment was purified and cloned into the cloning vector pCR-Blunt IITOPO (Life technologies) according to manufacturer's manual. To clone the fragment into the target plasmid pKO3 (SEQ ID NO:28) it was amplified with flanking BamHI restriction sites, using the oligonucleotides o-MO-54 (SEQ ID NO:48) and o-MO-55 (SEQ ID NO:49). The purified, BamHI cleaved PCR 6 fragment (SEQ ID NO:50) was ligated into the correspondingly cleaved vector pKO3 (SEQ ID N0:28). The assembled product was transformed into chemically competent E. coli DH5 cells (New England Biolabs, Frankfurt). Procedure of PCR purification, in-vitro cloning and transformation were carried out according to manufacturer's manual. The correct insertion of the target genes was checked by restriction analysis and the authenticity of the introduced genes was verified by DNA sequencing. The resulting knock-out vector was named pKO3 delta ItaE (SEQ ID NO:51).

[0207] The construction of strain E. coli W3110 fadE ItaE was carried out with the help of pKO3 delta ItaE using the method described in Link et al., 1997. The DNA sequence after deletion of ItaE is described in SEQ ID NO:52). The E. coli strain W3110 fadE ItaE was transformed with the plasmid pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1] (SEQ ID NO:13 from Example 3), by means of electroporation and plated onto LB-agar plates supplemented with spectinomycin (100 g/mL). Transformants were checked for the presence of the correct plasmids by plasmid preparation and analytic restriction analysis. The resulting strain was named E. coli W3110 fadEItaE pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1].

Example 16

LC-ESI/MS.SUP.2.-Based Quantification of Lauric Acid

[0208] Quantification of lauric acid in fermentation samples was carried out by means of LC-ESI/MS.sup.2 on the basis of an external calibration for lauric acid (0.1-50 mg/L) and by using the internal standard d3-LS.

[0209] The following instruments were used: [0210] HPLC system 1260 (Agilent; Bblingen) with Autosampler (G1367E), binary pump (G1312B) and thermo-statted column (G1316A) [0211] Mass spectrometer TripelQuad 6410 (Agilent; Bblingen) with ESI source [0212] HPLC column: Kinetex C18, 1002.1 mm, particle size: 2.6 m, pore size 100 (Phenomenex; Aschaffenburg) [0213] Pre-column: KrudKatcher Ultra HPLC In-Line Filter; 0.5 m filter depth and 0.004 mm inner diameter (Phenomenex; Aschaffenburg)

[0214] The samples were prepared by pipetting 1900 L of solvent (80% (v/v) ACN, 20% double-distilled H.sub.2O (v/v), +0.1% formic acid) and 100 L of sample into a 2 mL reaction vessel. The mixture was vortexed for approx. 10 seconds and then centrifuged at approx. 13 000 rpm for 5 min. The clear supernatant was removed using a pipette and analysed after appropriate dilution with a diluent (80% (v/v) ACN, 20% double-distilled H.sub.2O (v/v), +0.1% formic acid). In each case, 100 L of ISTD were added to 900 l of sample (10 l with a sample volume of 90 l).

[0215] HPLC separation was carried out using the above-mentioned column and pre-column. The injection volume is 1.0 l, the column temperature 50 C., 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 gradient shown it Table 4 was utilized:

TABLE-US-00014 TABLE 13 Concentrations of Eluent A and B used in Example 12 Time Eluent A Eluent B [min] [%] [%] 0 85 15 1 85 15 5 2 98 8 2 98 8.1 85 15 12 85 15

[0216] ESI-MS.sup.2 analysis was carried out in positive mode with the following parameters of the ESI source: [0217] Gas temperature 320 C. [0218] Gas flow 11 L/min [0219] Nebulizer pressure 50 psi [0220] Capillary voltage 4000 V

[0221] Detection and quantification of lauric acid was carried out with the following MRM parameters.

TABLE-US-00015 TABLE 14 MRM parameters used in detection and quantification of lauric acid Precursor ion Product ion Collision energy Analyte [m/z] [m/z] [eV] LS 201.1 201.1 110 d3-LS 204.1 204.1 110

Example 17

Detection of Glycine

[0222] Detection of glycine was performed via derivatization with ortho-phthaldialdehyde (OPA) and UV/VIS detection using an Agilent 1200 HPLC system.

[0223] 200 L of a homogeneous fermentation broth simple was mixed with 1800 L of 30% (v/v) 1-propanol, vortexed for 10 s and subsequently centrifuged at 13,000g for 5 min. The supernatant was removed and used for HPLC analysis using the following parameters:

TABLE-US-00016 Mobile phase: Eluent A 2.5 mL acetic acid per 1 L distilled water, pH adjustment with NaOH @ 6.0 Eluent B Methanol Column: Luna 5 C8 100 A (100 4.6 mm); Phenomenex Column oven 40 C. temperature: Flow: 1.0 mL/min Gradient: Time % B Flow Max. Press. 0.0 30.0 1.0 400 1.0 30.0 1.0 400 17.0 90.0 1.0 400 19.5 90.0 1.0 400 19.6 30.0 1.0 400 20.5 30.0 1.0 400 Run time: 22 min Detector: DAD 334 nm Spectrum Store: all Range: 200-400 nm step 2 nm FLD (excitation @ 330 nm; emission @ 450 nm, PMT gain 13) Derivatization: automatically with injection program: Inject Programm # Command 1 DRAW 4.5 L from Vial 1*, def. speed, def. offset 2 DRAW 1.5 L from sample, def. speed, def. offset 3 DRAW 0.5 L from air, def. speed 4 NEEDLE wash in flush Port. 15.0 sec 5 DRAW 4.5 L from Vial 1, def. speed, def. offset 6 MIX 11.0 L in seat, def. speed, 1 times 7 WAIT 1.00 min 8 INJECT 9 WAIT 0.50 min 10 VALVE bypass 11 Draw 100.0 L from Vial 2*, def. speed, def. offset 12 Eject 100.0 L from Vial 2, def. speed, def. offset 13 Valve mainpass *vial 1 contains OPA reagent (see below); vial 2 contains water

Preparation of OPA Reagent

[0224] 100 mg o-phthaldialdehyde was dissolved in 1 ml methanol and subsequently 0.4 mM borate buffer (pH 10.4) was added to give 10 mL. Subsequently, 50 L mercaptoethanol was added and the reagent stored at 4 C. Additional 10 L mercaptoethanol was added before use.

Preparation of Borate Buffer (0.4 mM H.sub.3BO.sub.4):

[0225] 38.1 g Na.sub.2B.sub.4O.sub.7*10 H.sub.2O (0.1 mol) were dissolved in 1 L distilled water and the pH adjusted to 10.4 M NaOH auf 10.4 eingestellt. Subsequently, 1 mL 25% Brij35 (v/v) was added.

TABLE-US-00017 Retention time: Glycine: 7.153 min

Example 18

[0226] Generation of Vectors for the Expression of hGLYAT2-Homologs

[0227] To generate vectors for the expression of N-acyltransferases of different organisms, variants found in the NCBI databases with homology to HGLYAT2 were synthesized and codon-optimized for E. coli. These were glycine N-acyltransferase-like protein 2 isoform 1 of Nomascus leucogenys (NI, XP_003275392.1, SEQ ID NO:53), glycine N-acyltransferase-like protein 2 of Saimiri boliviensis (Sb, XP_003920208.1, SEQ ID NO:54), glycine-N-acyltransferase-like 2 of Felis catus (Fc, XP_003993512.1, SEQ ID NO:55), glycine N-acyltransferase-like protein 2 of Bos taurus (Bt, NP_001178259.1, SEQ ID NO:56), and glycine N-acyltransferase of Mus musculus (Mm, NP_666047.1, SEQ ID NO:57).

[0228] The hGLYAT2-gene of pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1] (SEQ ID NO:13 of Example 3) was replaced by this variants as follows: The synthesized DNA fragments were digested with the restriction endonucleases BamHI and AsiSI and ligated into the correspondingly cut pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1].

[0229] The correct insertion of the target genes was checked by restriction analysis and the authenticity of the introduced genes was verified by DNA sequencing. The resulting expression vectors were named: [0230] pCDF{Ptac}[GLYAT_NI(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1] [0231] pCDF{Ptac}[GLYAT_Sb(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1] [0232] pCDF{Ptac}[GLYAT_Fc(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1] [0233] pCDF{Ptac}[GLYAT_Bt(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1] [0234] pCDF{Ptac}[GLYAT_Mm(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1]

Example 19

[0235] Production of Lauroylglycinate by E. coli Strains with Deletion in the fadE Gene, Overexpressing the hGLYAT-Variants, fadD and alkL Genes

[0236] The strains generated in Example 18 were used to study their ability to produce lauroylglycinate, in comparison to the reference strain expressing hGLYAT2 harbouring the plasmid pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1], applying the protocol described in Example 13.

[0237] 1-3 h after the induction, 6 g/L glycine and 6 g/L lauric acid (dissolved in lauric acid methyl ester) were added to the cultures. After cultivation time of 48 h the entire broth of a shake flask was extracted with Acetone (ratio 1:2). Further sample treatment is described in Example 7. Samples were taken, and lauroylglycinate, lauric acid and glycine present were analysed. The results are shown in Table 15.

[0238] All strains except the none-plasmid control produced lauroylglycinate in amounts between 0.44 and 2109.8 mg/L.

TABLE-US-00018 TABLE 15 Quantitative determination of lauroylglycinate after a cultivation time of 48 h in strains of E. coli W3110 fadE harboring different plasmids. Each strain was fed with 6 g/L glycine and 6 g/L lauric acid C.sub.lauroylglycinate strain plasmid [mg/L] E. coli 0.0 W3110 pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1] 2109.8 fadE pCDF{Ptac}[GLYAT_Fc(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1] 1.1 pCDF{Ptac}[GLYAT_Sb(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1] 1.5 pCDF{Ptac}[GLYAT_Mm(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1] 0.5 pCDF{Ptac}[GLYAT_Nl(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1] 209.7 pCDF{Ptac}[GLYAT_Bt(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1] 0.4

Example 20

Construction of Mutants Defect in Different Glycine-Metabolizing Pathways

[0239] With the Knock out-plasmid described in Examples 12, 14 and 15, different mutants were constructed. The construction of each strain was performed with the help of the plasmids pKO3 delta ItaE (SEQ ID NO:51), pKO3 delta GlyA (SEQ ID NO: 40) and pKO3 delta gcvTHP (SEQ ID NO:29) using strain E. coli W3110 fadE with the method described in Link et al., 1997. The E. coli strains W3110 fadE gcvTHP ItaE, W3110 fadE gcvTHP glyA, W3110 fadE glyA ItaE and W3110 fadE gcvTHP ItaE glyA were each transformed with the plasmid pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1] (SEQ ID NO:13 from Example 3), by means of electroporation and plated onto LB-agar plates supplemented with spectinomycin (100 g/mL). Transformants were checked for the presence of the correct plasmids by plasmid preparation and analytic restriction analysis. The resulting strains were named E. coli W3110 fadE gcvTHP ItaE pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1], E. coli W3110 fadE gcvTHP glyA pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1], E. coli W3110 fadE glyA ItaE pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1] and W3110 fadE gcvTHP ItaE glyA pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1].

Example 21

[0240] Generation of a Vector for Expression of the Porin-Gene fadL Instead of alkL in Escherichia coli W3110 fadE Overexpressing the hGLYAT2 and fadD

[0241] To generate a vector for the expression of fadL instead of alkL, the fadL gene was amplified from chromosomal DNA of E. coli W3110 by means of sequence-specific oligonucleotides fadL_ec-fp and fadL_EC_rp (SEQ IDs No. 66 and 67). The promoter region (Placuv5) was amplified from the target vector pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1] (SEQ ID NO:13) using oligonucleotides upstream_fp and upstream_rp (SEQ IDs No. 68 and 69). The fragments were fused using PCR and cloned to target vector opened NsiI/BamHI using the Geneart Seamless Cloning and Assembly Kit (Life Technologies, Carlsbad, Calif., USA). The assembled product was transformed into chemically competent E. coli DH5 cells (New England Biolabs, Frankfurt).

[0242] The correct insertion of the target genes was checked by restriction analysis and the authenticity of the introduced genes was verified by DNA sequencing. The resulting expression vector was named pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[fadL] (SEQ ID NO:70).

[0243] The E. coli strain W3110 fadE gcvTHP generated in Example 12 was transformed with the plasmid pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[fadL] by means of electroporation and plated onto LB agar plates supplemented with spectinomycin (100 g/mL). Transformants were checked for the presence of the correct plasmid by plasmid preparation and analytic restriction analysis. The resulting strain was named E. coli W3110 fadE gcvTHP pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[fadL].

Example 22

[0244] Generation of a Vector for Deletion of the Kbl Gene in Escherichia coli W3110 fadE

[0245] To generate a vector for the deletion of the kbl-gene encoding the 2-Amino-3-Ketobutyrate CoA-Ligase of E. coli W3110 approx. 500 bp upstream and downstream of the kbl-gene were amplified via PCR. The upstream region of kbl was amplified using the oligonucleotides 1960_up_fp (SEQ ID NO:71) and 1960_up_rp (SEQ ID NO:72). The downstream region of kbl was amplified using the oligonucleotides 1960_down_fp (SEQ ID NO:73) and 1960_down_rp (SEQ ID NO:74).

[0246] In each case PCR fragments of the expected size could be amplified. The PCR samples were separated via agarose gel electrophoresis and DNA fragments were isolated with QiaQuick Gel extraction Kit (Qiagen, Hilden). The purified PCR fragments were assembled via a crossover PCR. The generated fragment was purified, BsaI and SalI cleaved and ligated into the correspondingly cleaved vector pKO3 (SEQ ID NO:28). The assembled product was transformed into chemically competent E. coli DH5 cells (New England Biolabs, Frankfurt). Procedure of PCR purification, in-vitro cloning and transformation were carried out according to manufacturer's manual. The correct insertion of the target genes was checked by restriction analysis and the authenticity of the introduced genes was verified by DNA sequencing. The resulting knock-out vector was named pKO3 delta kbl (SEQ ID NO:75).

[0247] The construction of strain E. coli W3110 fadE kbl was carried out with the help of pKO3 delta kbl using the method described in Link et al., 1997. SEQ ID NO:76 is the DNA sequence after deletion of kbl. The E. coli strain W3110 fadE gcvTHP was transformed with the plasmid pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1] (SEQ ID NO:13 from Example 3), by means of electroporation and plated onto LB-agar plates supplemented with spectinomycin (100 g/mL). Transformants were checked for the presence of the correct plasmids by plasmid preparation and analytic restriction analysis. The resulting strain was named E. coli W3110 fadE kbl pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1].

Example 23

[0248] Generation of Strains which Overexpress the Escherichia coli fadD and Homo sapiens hGLYAT2 Under Control of the Ptrc Promoter Instead of Ptac

[0249] To generate E. coli strains which overexpress the Escherichia coli fadD gene in combination with the Homo sapiens hGLYAT2 under control of the Ptrc promoter (Brosius et al. 1985), the promoter region of plasmid pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1] was changed to the sequence of the trc-promoter by PCR and usage of sequence-specific oligonucleotides. The new plasmid was named pCDF{Ptrc}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1] SEQ ID NO:77

[0250] Analogous to the description in Examples 12 and 20, the following E. coli W3110 strains were constructed: [0251] fadE gcvTHP pCDF{Ptrc}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1], [0252] fadEgcvTHP ItaE pCDF{Ptrc}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1], [0253] fadE glyA ItaE pCDF{Ptrc}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1], [0254] fadE gcvTHP kbl pCDF{Ptrc}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1], [0255] fadE gcvTHP ItaE kbl pCDF{Ptrc}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1] and [0256] fadE gcvTHP ItaE glyA pCDF{Ptrc}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1].

Example 24

[0257] Production of Acyl Glycinate by E. coli Strains from Hydrolysed Coconut Oil and Glycine

[0258] The E. coli strain W3110 fadE pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1] generated as described in Example 5 was fermented in a fed-batch fermentation to study the ability of linking fatty acids from hydrolysed coconut oil and glycine to give acyl amino acids, e.g. lauroylglycinate. This fermentation was carried out in a parallel fermentation system from DASGIP with 8 bioreactors.

[0259] The experimental setting was as described in Example 8 except for the following modifications: Fatty acids from hydrolysed coconut oil are solid at 30 C. and cannot be used as a fluidic feed in a microbial fermentation process. To overcome this problem the temperature shift from 37 C. to 30 C. before induction of the heterologous genes was omitted. The entire process was run at 37 C. from start to finish. Induction was triggered 2 h after feedstart.

[0260] Biotransformation was started 7 h after induction by adding 10 g fatty acids from hydrolysed coconut oil and 100 mL of a 100 g/L aqueous solution of glycine, yielding 24 g/L of each substrate referring to the whole fermentation broth.

[0261] To monitor the conversion samples were taken after 47 h and 65 h of biotransformation and analysed as described in Example 7. The results are shown in Table 16.

TABLE-US-00019 TABLE 16 Concentrations of different Acyl glycinates, free fatty acids and glycine after 47 h and 65 h of biotransformation. Fermentation time [h] C.sub.Lauroyl glycinate [g/l] C.sub.Myristoyl glycinate [g/l] C.sub.Palmitoyl glycinate [g/l] C.sub.Glycine [g/l] C.sub.Octanoic acid [g/l] C.sub.Decanoic acid [g/l] 47 7.4 1.1 0.4 0.0 0.1 0.01 65 5.7 0.8 0.4 0.0 0.0 0.0 Fermentation time [h] C.sub.Lauric acid [g/l] C.sub.Myristic acid [g/l] C.sub.Palmitoleic acid [g/l] C.sub.Palmitic acid [g/l] C.sub.Oleic acid [g/l] C.sub.Stearic acid [g/l] 47 1.0 1.0 0.0 0.5 0.8 n.d. 65 0.8 0.7 0.0 0.3 0.5 n.d.

[0262] It was shown, that E. coli W3110 fadE pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1] is able to link not only lauric acid but also fatty acids of other chain lengths from hydrolysed coconut oil to glycine forming Acyl glycinates.

Example 25

[0263] Production of Acyl Glycinate by E. coli Strains with Deletions in Glycine Metabolizing Pathways from Hydrolysed Coconut Oil and Glycine

[0264] The E. coli strains as generated in Examples 12 and 23, E. coli W3110 [0265] fadE gcvTHP pCDF{Ptac}[hGLYAT2(co_Ec)/fadD_Ec] {Plavuv5}[alkLmod1] [0266] fadE gcvTHP pCDF{Ptrc}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1] [0267] fadE gcvTHP ItaE pCDF{Ptrc}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1] [0268] fadE glyA pCDF{Ptrc}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[alkLmod1]
were cultivated and biotransformation carried out as described in Example 24 and compared to the results of Example 24. To monitor the conversion samples were taken after 47 h and 65 h of biotransformation and analysed as described in Example 7. The results are shown in Table 17 to 20.

TABLE-US-00020 TABLE 17 Concentrations of different Acyl glycinates, free fatty acids and glycine after 47 h and 65 h of biotransformation using E. coli W3110 fadE gcvTHP pCDF{Ptac} [hGLYAT2(co_Ec)- fadD_Ec]{Placuv5}[alkLmod1] as biocatalyst. Fermentation time [h] C.sub.Lauroyl glycinate [g/l] C.sub.Myristoyl glycinate [g/l] C.sub.Palmitoyl glycinate [g/l] C.sub.Glycine [g/l] C.sub.Octanoic acid [g/l] C.sub.Decanoic acid [g/l] 47 9.3 2.4 0.9 9.7 0.0 0.0 65 6.6 1.7 0.6 7.0 0.0 0.0 Fermentation time [h] C.sub.Lauric acid [g/l] C.sub.Myristic acid [g/l] C.sub.Palmitoleic acid [g/l] C.sub.Palmitic acid [g/l] C.sub.Oleic acid [g/l] C.sub.Stearic acid [g/l] 47 0.0 0.1 0.0 0.2 0.9 n.d. 65 0.0 0.0 0.0 0.0 0.3 n.d.

TABLE-US-00021 TABLE 18 Concentrations of different Acyl glycinates, free fatty acids and glycine after 47 h and 65 h of biotransformation using E. coli W3110 fadE gcvTHP pCDF{Ptrc} [hGLYAT2(co_Ec)- fadD_Ec]{Placuv5}[alkLmod1] as biocatalyst. Fermentation time [h] C.sub.Lauroyl glycinate [g/l] C.sub.Myristoyl glycinate [g/l] C.sub.Palmitoyl glycinate [g/l] C.sub.Glycine [g/l] C.sub.Octanoic acid [g/l] C.sub.Decanoic acid [g/l] 47 10 2.8 0.9 10.5 0.0 0.0 65 7.5 2.1 0.7 8.4 0.0 0.0 Fermentation time [h] C.sub.Lauric acid [g/l] C.sub.Myristic acid [g/l] C.sub.Palmitoleic acid [g/l] C.sub.Palmitic acid [g/l] C.sub.Oleic acid [g/l] C.sub.Stearic acid [g/l] 47 0.0 0.0 0.0 0.1 0.5 n.d. 65 0.0 0.0 0.0 0.0 0.2 n.d.

TABLE-US-00022 TABLE 19 Concentrations of different Acyl glycinates, free fatty acids and glycine after 47 h and 65 h of biotransformation using E. coli W3110 fadE gcvTHP ltaE pCDF{Ptrc}[hGLYAT2(co_Ec)- fadD_Ec]{Placuv5}[alkLmod1] as biocatalyst. Fermentation time [h] C.sub.Lauroyl glycinate [g/l] C.sub.Myristoyl glycinate [g/l] C.sub.Palmitoyl glycinate [g/l] C.sub.Glycine [g/l] C.sub.Octanoic acid [g/l] C.sub.Decanoic acid [g/l] 47 9.7 2.9 1.3 0.0 0.0 0.0 65 8.5 2.6 1.2 0.0 0.0 0.0 Fermentation time [h] C.sub.Lauric acid [g/l] C.sub.Myristic acid [g/l] C.sub.Palmitoleic acid [g/l] C.sub.Palmitic acid [g/l] C.sub.Oleic acid [g/l] C.sub.Stearic acid [g/l] 47 0.0 0.0 0.0 0.0 0.0 n.d. 65 0.0 0.0 0.0 0.0 0.0 n.d.

TABLE-US-00023 TABLE 20 Concentrations of different Acyl glycinates, free fatty acids and glycine after 47 h and 65 h of biotransformation using E. coli W3110 fadE glyA pCDF{Ptrc}[hGLYAT2(co_Ec)- fadD_Ec]{Placuv5}[alkLmod1 as biocatalyst. Fermentation time [h] C.sub.Lauroyl glycinate [g/l] C.sub.Myristoyl glycinate [g/l] C.sub.Palmitoyl glycinate [g/l] C.sub.Glycine [g/l] C.sub.Octanoic acid [g/l] C.sub.Decanoic acid [g/l] 47 10 3 1.3 9.5 0.0 0.0 65 8.5 2.5 1.1 8.6 0.0 0.0 Fermentation time [h] C.sub.Lauric acid [g/l] C.sub.Myristic acid [g/l] C.sub.Palmitoleic acid [g/l] C.sub.Palmitic acid [g/l] C.sub.Oleic acid [g/l] C.sub.Stearic acid [g/l] 47 0.0 0.0 0.0 0.0 0.0 n.d. 65 0.0 0.0 0.0 0.0 0.0 n.d.

[0269] It was shown, that the gcvTHP mutation reduces the glycine loss caused by the natural metabolism of E. coli significantly as already shown in Example 13. The glycine concentration at the end of the biotransformation is >7 g/L (respectively >8 g/L) for the mutants as shown in Table 17 (respectively Table 18). For E. coli lacking this mutation the glycine concentration at the end of the biotransformation is 0 g/L as shown in Table 16.

[0270] An additional mutation blocking a second glycine metabolising pathway (ItaE) does not enhance the effect of the gcvTHP mutation. Surprisingly suppresses ItaE the desired effect of gcvTHP as shown in Table 19.

[0271] It was shown, that the glyA mutation also reduces the glycine loss caused by the natural metabolism of E. coli significantly. The glycine concentration at the end of the biotransformation is >8 g/L for the mutant as shown in Table 20.

Example 26

[0272] Production of Acyl Glycinate by E. coli Strains from Hydrolysed Coconut Oil and Glycine Using the Alternative Porine FadL

[0273] The E. coli W3110 strains as generated in Example 12 and 23, [0274] fadE gcvTHP pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec] [0275] fadE gcvTHP pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec]{Placuv5}[fadL]
were grown and biotransformed as described in Example 24. The conversion samples were taken after 47 h and 65 h of biotransformation and analysed as described in Example 7. Then results are shown in Tables 21 and 22.

TABLE-US-00024 TABLE 21 Concentrations of different Acyl glycinates, free fatty acids and glycine after 47 h and 65 h of biotransformation using E. coli W3110 fadE gcvTHP pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec] as biocatalyst. Fermentation time [h] C.sub.Lauroyl glycinate [g/l] C.sub.Myristoyl glycinate [g/l] C.sub.Palmitoyl glycinate [g/l] C.sub.Glycine [g/l] C.sub.Octanoic acid [g/l] C.sub.Decanoic acid [g/l] 47 4.4 0.5 0.0 12.7 0.1 0.0 65 3.9 0.4 0.0 10.8 0.0 0.0 Fermentation time [h] C.sub.Lauric acid [g/l] C.sub.Myristic acid [g/l] C.sub.Palmitoleic acid [g/l] C.sub.Palmitic acid [g/l] C.sub.Oleic acid [g/l] C.sub.Stearic acid [g/l] 47 3.4 2.0 0.0 1.0 1.0 n.d. 65 3.3 1.9 0.0 0.8 0.9 n.d.

TABLE-US-00025 TABLE 22 Concentrations of different Acyl glycinates, free fatty acids and glycine after 47 h and 65 h of biotransformation using E. coli W3110 fadE gcvTHP pCDF{Ptac}[hGLYAT2(co_Ec)- fadD_Ec]{Placuv5}[fadL] as biocatalyst. Fermentation time [h] C.sub.Lauroyl glycinate [g/l] C.sub.Myristoyl glycinate [g/l] C.sub.Palmitoyl glycinate [g/l] C.sub.Glycine [g/l] C.sub.Octanoic acid [g/l] C.sub.Decanoic acid [g/l] 47 7.8 2.4 1.1 9.4 0.0 0.0 65 7.2 2.2 1.0 7.8 0.0 0.0 Fermentation time [h] C.sub.Lauric acid [g/l] C.sub.Myristic acid [g/l] C.sub.Palmitoleic acid [g/l] C.sub.Palmitic acid [g/l] C.sub.Oleic acid [g/l] C.sub.Stearic acid [g/l] 47 0.8 0.2 0.0 0.0 0.2 n.d. 65 0.5 0.1 0.0 0.0 0.0 n.d.

[0276] It was shown that a porine is important for good biocatalyst performances. No porine like in the strain E. coli W3110 fadE gcvTHP pCDF{Ptac}[hGLYAT2(co_Ec)-fadD_Ec] leads to significant amounts of residual fatty acids in the fermentation broth at the end of biotransformation carried out under chosen conditions (Table 21). AlkL used as a porine led to almost full conversion of all the fatty acids from hydrolized coconut oil (Table 17). It was also shown that FadL can be used as a porine like AlkL leading to almost full conversion of fatty acids subjected to biotransformation.

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