ACYL AMINO ACID PRODUCTION

Abstract

The present invention relates to a microbial cell for producing at least one acyl amino acid, wherein the cell is genetically modified to comprise; a first genetic mutation that enables the cell to produce at least one acyl amino acid and; a second genetic mutation that enables the cell to decrease glutamate breakdown relative to the wild type cell.

Claims

1-15. (canceled)

16. A microbial cell for producing at least one acyl amino acid, wherein the cell is genetically modified to comprise: a) a first genetic mutation that enables the cell to produce the acyl amino acid; and b) a second genetic mutation that results in a decrease in activity relative to a wild type cell of at least one enzyme involved in glutamate breakdown.

17. The microbial cell of claim 16, wherein the acyl amino acid is N-acyl glutamate or lauroyl glutamate.

18. The microbial cell of claim 16, wherein the first genetic mutation results in the cell having increased expression of (i) an amino acid-N-acyl-transferase (E.sub.1) and (ii) acyl-CoA synthetase (E.sub.2).

19. The microbial cell of claim 18, wherein the amino acid-N-acyl-transferase (E.sub.1) is a glycine-N-acyl transferase (E.sub.1a) that is capable of producing N-acyl glutamate.

20. The microbial cell of claim 16, wherein the enzyme involved in glutamate breakdown is selected from the group consisting of E.sub.11-E.sub.28.

21. The microbial cell of claim 20, wherein the enzyme involved in glutamate breakdown is selected from the group of enzymes consisting of: (i) E.sub.11; (ii) E.sub.12, E.sub.13, and E.sub.14; (iii) E.sub.12, E.sub.13, and E.sub.15; (iv) E.sub.16; (v) E.sub.12, E.sub.17, E.sub.18, E.sub.19, E.sub.20, E.sub.21, E.sub.22, and E.sub.23; (vi) E.sub.24, E.sub.25, E.sub.26, and E.sub.27; and (vii) E.sub.28.

22. The microbial cell of claim 16, wherein the cell further comprises a genetic mutation in at least one enzyme selected from the group consisting of: (i) an enzyme (E.sub.3) capable of uptake of glutamate; (ii) an enzyme (E.sub.4) capable of interconverting acyl-CoAs and acyl-ACPs; and (iii) an enzyme (E.sub.5) capable of uptake of at least one fatty acid.

23. The microbial cell of claim 22, wherein: a) E.sub.3 is a glutamate-translocating ABC transporter or permease; b) E.sub.4 is acyl-CoA:ACP transacylase; and c) E.sub.5 is AlkL and/or FadL.

24. The microbial cell of claim 18, wherein E.sub.1 comprises SEQ ID NO:4 or a variant thereof; and/or E.sub.2 comprises SEQ ID NO:1 or a variant thereof.

25. The microbial cell of claim 16, wherein the cell is capable of making proteinogenic amino acids and/or fatty acids.

26. The microbial cell of claim 16, wherein the cell has a further genetic mutation that enables the cell to have increased expression of acyl-CoA thioesterase (E.sub.10).

27. The microbial cell of claim 18, wherein the first genetic mutation results in the cell having increased expression of (i) an amino acid-N-acyl-transferase (E.sub.1) and (ii) acyl-CoA synthetase (E.sub.2).

28. The microbial cell of claim 27, wherein the amino acid-N-acyl-transferase (E.sub.1) is a glycine-N-acyl transferase (E.sub.1a) that is capable of producing N-acyl glutamate.

29. The microbial cell of claim 28, wherein the enzyme involved in glutamate breakdown is selected from the group of enzymes consisting of: (i) E.sub.11; (ii) E.sub.12, E.sub.13, and E.sub.14; (iii) E.sub.12, E.sub.13, and E.sub.15; (iv) E.sub.16; (v) E.sub.12, E.sub.17, E.sub.18, E.sub.19, E.sub.20, E.sub.21, E.sub.22, and E.sub.23; (vi) E.sub.24, E.sub.25, E.sub.26, and E.sub.27; and (vii) E.sub.28.

30. The microbial cell of claim 29, wherein the cell further comprises a genetic mutation in at least one enzyme selected from the group consisting of: (i) an enzyme (E.sub.3) capable of uptake of glutamate; (ii) an enzyme (E.sub.4) capable of interconverting acyl-CoAs and acyl-ACPs; and (iii) an enzyme (E.sub.5) capable of uptake of at least one fatty acid.

31. The microbial cell of claim 30, wherein: a) E.sub.3 is a glutamate-translocating ABC transporter or permease; b) E.sub.4 is acyl-CoA:ACP transacylase; and c) E.sub.5 is AlkL and/or FadL.

32. A method of producing at least one acyl amino acid, comprising contacting the microbial cell of claim 16, with at least one fatty acid and/or amino acid.

33. The method of claim 32, wherein the amino acid is glutamic acid and the acyl amino acid is N-acyl glutamate and/or lauroyl glutamate.

34. The method of claim 33, wherein the first genetic mutation in said microbial cell results in the cell having increased expression of (i) an amino acid-N-acyl-transferase (E.sub.1) and (ii) acyl-CoA synthetase (E.sub.2).

35. The method of claim 34, wherein the amino acid-N-acyl-transferase (E.sub.1) is a glycine-N-acyl transferase (E.sub.1a) that is capable of producing N-acyl glutamate and the enzyme involved in glutamate breakdown is selected from the group of enzymes consisting of: (i) E.sub.11; (ii) E.sub.12, E.sub.13, and E.sub.14; (iii) E.sub.12, E.sub.13, and E.sub.15; (iv) E.sub.16; (v) E.sub.12, E.sub.17, E.sub.18, E.sub.19, E.sub.20, E.sub.21, E.sub.22, and E.sub.23; (vi) E.sub.24, E.sub.25, E.sub.26, and E.sub.27; and (vii) E.sub.28.

Description

EXAMPLES

[0114] 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.

Example 1

[0115] Generation of Vectors for Deletion of Glutamate Degrading Pathway-Enzymes in Escherichia Coli W3110 fadE

[0116] To reduce glutamate degradation, enzymes of different degradation pathways (I, II, III and IV as provided above) is deleted (Table 1).

TABLE-US-00001 TABLE 1 list of deleted glutamate degrading enzymes Degradation SEQ ID Pathway Enzyme EC-No. Name NO: I and V E.sub.11 1.4.1.4 Glutamate dehydrogenase 18 (gdhA) II and III E.sub.12 4.1.1.15 Glutamate Decarboxylase 19, 20 (gadA/gadB) IV E.sub.16 2.6.1.1 glutamate:aspartate 21 transaminase (aspC)
Generation of a Vector for Deletion of the gdhA in Escherichia Coli W3110 fadE

[0117] To generate a vector for the deletion of gdhA of E. coli W3110, approximately 500 bp upstream and downstream of gdhA is amplified via PCR. The upstream region of gdhA is amplified using the oligonucleotides gdhA_Up_fw (SEQ ID NO:9) and gdhA_Up_rev (SEQ ID NO: 10). The downstream region of gdhA is amplified using the oligonucleotides gdhA-DOWN_fw (SEQ ID NO: 11) and gdhA-DOWN_rev (SEQ ID NO: 12).

[0118] The following parameters are used for PCR: 1: initial denaturation, 98 C., 3:00 min; 35denaturation, 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) is used according to manufacturer's manual. 50 L of the PCR reaction is analyzed on a 1% TAE agarose gel. Procedure of PCR, agarose gel electrophoresis, ethidium bromide staining of DNA and determination of PCR fragment size is carried out as known to those skilled in the art.

[0119] In each case PCR fragments of the expected size is amplified (PCR 1, 523 bp, (SEQ ID NO: 13); PCR 2, 544 bp, SEQ ID NO: 14). The PCR samples are separated via agarose gel electrophoresis and DNA fragments are isolated with QiaQuick Gel extraction Kit (Qiagen, Hilden). The purified PCR fragments are cloned into the Notl cut vector pKO3 (SEQ ID NO:15), using the NEBuilder HiFi DNA Assembly Cloning Kit (New England Biolabs, Frankfurt). The assembled product is transformed into NEB 10-beta electrocompetent E. coli cells (New England Biolabs, Frankfurt). Procedure of PCR purification, in-vitro cloning and transformation are carried out according to manufacturer's manual. The correct insertion of the target genes is checked by restriction analysis and the authenticity of the introduced DNA fragments is verified by DNA sequencing. The resulting knock-out vector is named pKO3_KO-gdhA (SEQ ID NO:16).

[0120] The construction of strain E. coli W3110 fadEAgdhA is carried out with the help of pKO3_KO-gdhA using the method described in Link et al., 1997. The DNA sequence after the deletion step of gdhA is SEQ ID NO: 17.

[0121] Generation of a vector for deletion of the gadA/gadB and aspC in Escherichia coli W3110 fadE To generate vectors for the deletion of enzymes of the degradation pathways II to IV approximately 500 bp upstream and downstream of the corresponding genes listed in Table 1 (gadA/gadB and aspC) are amplified via PCR and the pKO3-vectors are constructed analogous as described for pKO3_KO-gdhA and named pKO3_KO-gadA/gadB and pKO3_KO-aspC respectively.

Transformation of Vectors into E. Coli Cells

[0122] The E. coli strains W3110 fadEAgdhA, fadEgadA/gadB and fadEgdhA are each transformed with the plasmid of Example 2 of WO2015/028423 pJ294{Ptac}[synUcTE]/pCDF{Ptac}[hGLYAT3(co_Ec).sub. fadD_Ec] by means of electroporation and plated onto LB-agar plates and is supplemented with spectinomycin and ampicillin (both 100 g/mL). Transformants are checked for the presence of the correct plasmids by plasmid preparation and analytic restriction analysis as known in the art. The resulting strains were named: [0123] 1. E. coli W3110 fadEgdhA pJ294{Ptac}[synUcTE]/pCDF{Ptac}[hGLYAT3(co_Ec)_fadD_Ec], [0124] 2. E. coli W3110 fadEgadA/gadB pJ294{Ptac}[synUcTE]/pCDF{Ptac}[hGLYAT3(co_Ec)_fadD_Ec] and [0125] 3. E. coli W3110 fadEaspC pJ294{Ptac}[synUcTE]/pCDF{Ptac}[hGLYAT3(co_Ec)_fadD_Ec].

Example 2

[0126] Production of Lauroyl Glutamate by E. Coli Strains Expressing hGLYAT3

[0127] The strains generated in Example 1 may be used to study their ability to produce fatty acid/amino acid adducts, as described in Example 9 of WO2015/028423. The results may be compared to the results of culturing the strains of Example 4 of WO2015/028423. A brief description of determining the results (i.e. measuring fatty acid/amino acid adduct production) is provided below. Starting from a 80 C. glycerol culture, the strains to be studied are first plated on 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 are then grown as a 5 mL preculture in Luria-Bertani broth, Miller (Merck, Darmstadt) supplemented with 100 g/mL ampicillin and 100 g/mL spectinomycin. The further culture steps are performed in M9 medium (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, is brought to pH 7.4 with 25% strength ammonium hydroxide solution). The trace element solution to be 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, to be dissolved in 1 M hydrochloric acid (all chemicals from Merck, Darmstadt), is filter-sterilized before being added to the M9 medium. 20 mL of M9 medium is supplemented with 100 g/mL spectinomycin and 100 g/mL ampicillin is introduced into baffled 100-mL Erlenmeyer flasks and is inoculated with 0.5 mL preculture. The flasks are cultured at 37 C. and 200 rpm in a shaker-incubator. After a culture time of 8 hours, 50 mL of M9 medium is supplemented with 100 g/mL spectinomycin and 100 g/mL ampicillin is introduced into a baffled 250-mL Erlenmeyer flask and is inoculated with the 10 mL culture to achieve an optical density (00600) of 0.2. The flasks are incubated at 37 C. and 200 rpm in a shaker-incubator. When an OD.sub.600 of 0.7 to 0.8 is reached, gene expression is induced by addition of 1 mM IPTG. The induced strains are cultured for a further 48 hours at 30 C. at 200 rpm. During culturing, samples are taken, and the fatty acid/amino acid adducts present are analysed. It was demonstrated that E. coli strains W3110 fadE pJ294{Ptac}[synUcTE]/pCDF{Ptac}[hGLYAT3(co_Ec)_fadD_Ec] was 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 lacks the plasmids (negative control). All strains of Example 1 produce more lauroyl glutamic acid compared to strain W3110 fadE pJ294{Ptac}[synUcTE]/pCDF{Ptac}[hGLYAT3(co_Ec)_fadD_Ec], due to the reduced capability of glutamate degradation.

Example 3

[0128] Production of Lauroyl Glutamate by E. Coli Strains Expressing hGLYAT3 in a Parallel Fermentation System

[0129] The strains generated in Example 1, E. coli W3110 fadEgdhA pJ294{Ptac}[synUcTE]/pCDF{Ptac}[hGLYAT3(co_Ec)_fadD_Ec], E. coli W3110 fadEgadA/gadB pJ294{Ptac}[synUcTE]/pCDF{Ptac}[hGLYAT3(co_Ec)_fadD_Ec] and E. coli W3110 fadEaspC pJ294{Ptac}[synUcTE]/pCDF{Ptac}[hGLYAT3(co_Ec)_fadD_Ec] are fermented in a fed-batch fermentation to study the ability of linking lauric acid and glutamic acid to produce more lauroyl glutamate compared to the strain of Example 4 of WO2015/028423 (where no second genetic mutation that results in a decrease in activity relative to a wild type cell of at least one enzyme involved in glutamate breakdown is present). This fermentation is carried out in a parallel fermentation system from DASGIP (https://online-shop.eppendorf.de/DE-de/Bioprozesstechnik-44559/Bioprozess-Systeme-60767/DASGIP-Parallele-Bioreaktorsysteme-PF-133597.html) with 8 bioreactors as described in Example 9 of WO2015/028423 except that 100 g/L glutamic acid instead of glycine is used. To quantify lauroyl, myristoyl and palmitoyl glutamate in the fermentation, samples are taken 23 h and 42 h after the start of the fermentation. These samples are prepared for analysis, and analysed using chromatography as described in Example 7 of WO2015/028423.

[0130] During the fermentation the strains of Example 1, produce more lauroyl-glutamic acid compared to the strain W3110 fadE pJ294{Ptac}[synUcTE]/pCDF{Ptac}[hGLYAT3(co_Ec)_fadD_Ec.

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