PRODUCTION OF 3-HYDROXYBUTYRATE

Abstract

There is provided amicrobial cell which is capable of producing acetoacetate, 3-hydroxybutyrate and/or 3-hydroxybutyrate variants, wherein the cell is genetically modified to comprise an increased expression relative to its wild type cell of: an enzyme E.sub.1 capable of catalysing the conversion of acetyl-CoA to acetoacetyl-CoA; an enzyme E.sub.2 capable of catalysing the conversion of acetoacetyl-CoA to acetoacetate; and an enzyme E.sub.3 capable of catalysing the conversion of acetoacetate to 3-hydroxybutyrate and/or variants thereof.

Claims

1. A microbial cell which is capable of producing acetoacetate, 3-hydroxybutyrate and/or 3-hydroxybutyrate variants, wherein the cell is genetically modified to comprise an increased expression relative to its wild type cell of: an enzyme E.sub.1 capable of catalysing the conversion of acetyl-CoA to acetoacetyl-CoA; an enzyme E.sub.2 capable of catalysing the conversion of acetoacetyl-CoA to acetoacetate; and an enzyme E.sub.3 capable of catalysing the conversion of acetoacetate to 3-hydroxybutyrate and/or variants thereof and wherein the genetically modified cell has reduced or no expression of acetoacetate decarboxylase (Adc; EC 4.1.1.4).

2. The cell according to claim 1, wherein E.sub.1 is a thiolase, E.sub.2 is an acetoacetate CoA transferase and/or E.sub.3 is a secondary alcohol dehydrogenase.

3. The cell according to claim 1, wherein the thiolase (E.sub.1) is from C. acetobutylicum.

4. The cell according to claim 1, wherein the acetoacetate CoA transferase (E.sub.2) is from C. acetobutylicum,

5. The cell according to claim 1, wherein the secondary alcohol dehydrogenase (E.sub.3) is from C. beijerinckii.

6. The cell according to claim 1, wherein E.sub.1 comprises 60% sequence identity with SEQ ID NO: 2, E.sub.2 comprises 60% sequence identity with SEQ ID NO: 4 or 6 and/or E.sub.3 comprises 60% sequence identity with SEQ ID NO: 8.

7. The cell according to claim 1, wherein E.sub.1 comprises SEQ ID NO: 2, E.sub.2 comprises SEQ ID NO: 4 and 6 and E.sub.3 comprises SEQ ID NO: 8.

8. The cell according to claim 1, wherein the cell is an acetogenic cell.

9. The cell according to claim 8, wherein the acetogenic cell is selected from the group consisting of Clostridium autothenogenum DSMZ 19630, Clostridium ragsdahlei ATCC no. BAA-622, Clostridium autoethanogenum, Moorella sp HUC22-1, Moorella thermoaceticum, Moorella thermoautotrophica, Rumicoccus productus, Acetoanaeroburn, Oxobacter pfennigii, Methanosarcina barkeri, Methanosarcina acetivorans, Carboxydothermus, Desulfotomaculum kutznetsovii, Pyrococcus, Peptostreptococcus, Butyribacterium methylotrophicum ATCC 33266, Clostridium formicoaceticum, Clostridium butyricum, Lactobacillus deibrukiis, Propionibacterium acidoproprionici, Proprionispera arboris, Anaerobierspirillum succiniproducens, Bacterioides amylophilus, Becterioides ruminicola, Thermoanaerobacter kivui, Acetobacterium woodii, Acetoanaerobium notera, Clostridium aceticum, Butyribacterium methylotrophicum, Moorella thermoacetica, Eubacterium limosum, Peptostreptococcus productus, Clostridium ljungdahlii, Clostridium ATCC 29797 and Clostridium carboxidivorans.

10. The cell according to claim 1, wherein the cell is Clostridium ljungdahlii or Clostridium autothenogenum DSMZ 10061.

11. A method of producing acetoacetate, 3-hydroxybutyrate and/or 3-hydroxybutyrate variants, the method comprising contacting a recombinant microbial cell according to claim 1 with a medium comprising a carbon source.

12. The method according to claim 11, wherein the carbon source comprises CO.sub.2 and/or CO.

13. The method according to claim 11, wherein the carbon source comprises 50% or more H.sub.2.

14. Use of the cell according to claim 1 for the production of acetoacetate, 3-hydroxybutyrate and/or 3-hydroxybutyrate variants.

Description

BRIEF DESCRIPTION OF FIGURES

[0068] FIG. 1 is an illustration of plasmid backbone pSOS95

EXAMPLES

[0069] 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

Generation of Genetically Modified Acetogens for the Formation of 3HB via Acetoacetate

[0070] The genes Thiolase (thl) from C. acetobutylicum ATTC 824, Acetoacetat-transferase (ctfAB) from C. acetobutylicum ATTC 824 and the secondary alcohol dehydrogenase (sadh) from C. beijerinckii DSM 6423 were inserted into the vector pEmpty. This plasmid is based on the plasmid backbone pSOS95 (FIG. 1). To use pSOS95, it was digested with BamHI and KasI. This removes the operon ctfA-ctfB-adc, but leaves the thl promoter and the rho-independent terminator of adc. The transformation of C. ljungdahlii and C. autoethanogenum was done as disclosed in Leang et al. 2013. The nucleotide sequences of the enzymes used are SEQ ID NOs:1, 3 (ctfA), 5 (ctfB) and 7 respectively. These sequences were transformed to be controlled by a thiolase promotor and integrated as a single operon into the vector backbone. The created vector was named pTCtS. The vector pTCts was then used to modify C. ljungdahlii and C. autoethanogenum using a method disclosed in Leang et al. 2013. The modified C. ljungdahlii strain was named C. ljungdahlii pTCtS. The modified C. autoethanogenum strain was named C. autoethanogenum pTCtS.

Example 2

[0071] Fermentation of 3HB Strain on H.sub.2 and CO.sub.2 Showing Acetoacetate and 3HB Production.

[0072] For cell culture of C. ljungdahlii pTCts 5 mL of the culture were anaerobically grown in 500 ml of medium (ATCC1754 medium: pH 6.0; 20 g/L MES; 1 g/L yeast extract, 0.8 g/L NaCl, 1 g/L NH4Cl, 0.1 g/L KCl, 0.1 g/L KH2PO4, 0.2 g/L MgSO4x 7 H2O; 0.02 g/L CaCl.sub.22H.sub.2O; 20 mg/L nitrilotriacetic acid 10 mg/L MnSO.sub.4H.sub.2O; 8 mg/L (NH.sub.4).sub.2Fe(SO.sub.4).sub.26H.sub.2O; 2 mg/L CoCl.sub.26H.sub.2O; 2 mg/L ZnSO.sub.47H.sub.2O; 0.2 mg/L CuCl.sub.22H.sub.2O; 0.2 mg/L Na.sub.2MoO.sub.42H.sub.2O; 0.2 mg/L NiCl.sub.26H.sub.2O; 0.2 mg/L Na.sub.2SeO.sub.4; 0.2 mg/L Na.sub.2WO.sub.42H.sub.2O; 20 g/L d-Biotin, 20 g/L folic acid, 100 g/L pyridoxine-HCl; 50 g/L thiamine-HClH.sub.2O; 50 g/L riboflavin; 50 g/L nicotinic acid, 50 g/L Ca-pantothenate; 1 g/L vitamin B12 ; 50 g/L p-aminobenzoate; 50 g/L lipoic acid, approximately 67.5 mg/L NaOH) with about 400 mg/L L-cysteine hydrochloride and 400 mg/L Na.sub.2S9H.sub.2O, given 100 mg/L erythromycin). Cultivation was carried out in duplicate into 1 L glass bottles with a premixed gas mixture composed of 67% H.sub.2, 33% CO.sub.2 in an open water bath shaker at 37 C., 150 rpm and aeration of 3 L/h for 70.3 h.

[0073] The gas entered the medium through a filter with a pore size of 10 microns, which was mounted in the middle of the reactor, at a gassing tube. When sampling each 5 ml sample was removed for determination of OD.sub.600 nm, pH and the product range. The determination of the product concentration was performed by semi-quantitative 1 H-NMR spectroscopy. As an internal quantification standard sodium trimethylsilylpropionate was used (T(M) SP). A culture produced 7 ppm acetoacetate and 26 ppm 3-HB in 70.3 hours. The other culture produced 24 ppm acetoacetate and 104 ppm 3-HB in 70.3 hours.

Example 3

[0074] Fermentation of Vector Control Strain with No Production of Acetoacetate or 3HB

[0075] For cell culture of C. ljungdahlii pEmpty 5 mL of the culture were anaerobically grown in 500 ml of medium (ATCC1754 medium: pH 6.0; 20 g/L MES; 1 g/L yeast extract, 0.8 g/L NaCl, 1 g/L NH.sub.4Cl, 0.1 g/L KCl, 0.1 g/L KH.sub.2PO.sub.4, 0.2 g/L MgSO.sub.47H.sub.2O; 0.02 g/L CaCl.sub.22H.sub.2O; 20 mg/L nitrilotriacetic acid 10 mg/L MnSO.sub.4H.sub.2O; 8 mg/L (NH.sub.4).sub.2Fe(SO.sub.4).sub.26H.sub.2O; 2 mg/L CoCl.sub.26H.sub.2O; 2 mg/L ZnSO.sub.47H.sub.2O; 0.2 mg/L CuCl.sub.22H.sub.2O; 0.2 mg/L Na.sub.2MoO.sub.42H.sub.2O; 0.2 mg/L NiCl.sub.26H.sub.2O; 0.2 mg/L Na.sub.2SeO.sub.4; 0.2 mg/L Na.sub.2WO.sub.42H.sub.2O; 20 g/L d-Biotin, 20 g/L folic acid, 100 g/L pyridoxine-HCl; 50 g/L thiamine-HClH.sub.2O; 50 g/L riboflavin; 50 g/L nicotinic acid, 50 g/L Ca-pantothenate; 1 g/L vitamin B12 ; 50 g/L p-aminobenzoate; 50 g/L lipoic acid, approximately 67.5 mg/L NaOH) with about 400 mg/L L-cysteine hydrochloride and 400 mg/L Na.sub.2S9H.sub.2O, given 100 mg/L erythromycin). Cultivation was carried out in duplicate into 1 L glass bottles with a premixed gas mixture composed of 67% H.sub.2, 33% CO.sub.2 in an open water bath shaker at 37 C., 150 rpm and aeration of 3 L/h for 70.3 h.

[0076] The gas entered the medium through a filter with a pore size of 10 microns, which was mounted in the middle of the reactor, at a gassing tube. When sampling each 5 ml sample was removed for determination of OD.sub.600 nm, pH and the product range. The determination of the product concentration was performed by semi-quantitative 1 H-NMR spectroscopy. As an internal quantification standard sodium trimethylsilylpropionate was used (T(M) SP). Neither acetoacetate nor 3HB was produced.

Example 4

[0077] Fermentation of C. autoethanogenum pTCtS on CO, H.sub.2 and CO.sub.2 Showing 3HB Production.

[0078] For cell culture of C. autoethanogenum pTCts 5 mL of the culture was anaerobically grown in 500 ml of medium (ATCC1754 medium: pH 6.0; 20 g/L MES; 1 g/L yeast extract, 0.8 g/L NaCl, 1 g/L NH.sub.4Cl, 0.1 g/L KCl, 0.1 g/L KH.sub.2PO.sub.4, 0.2 g/L MgSO.sub.47H.sub.2O; 0.02 g/L CaCl.sub.22H.sub.2O; 20 mg/L nitrilotriacetic acid 10 mg/L MnSO.sub.4H.sub.2O; 8 mg/L (NH.sub.4).sub.2Fe(SO.sub.4).sub.26H.sub.2O; 2 mg/L CoCl.sub.26H.sub.2O; 2 mg/L ZnSO.sub.47H.sub.2O; 0.2 mg/L CuCl.sub.22H.sub.2O; 0.2 mg/L Na.sub.2MoO.sub.42H.sub.2O; 0.2 mg/L NiCl.sub.26H.sub.2O; 0.2 mg/L Na.sub.2SeO.sub.4; 0.2 mg/L Na.sub.2WO.sub.42H.sub.2O; 20 g/L d-Biotin, 20 g/L folic acid, 100 g/L pyridoxine-HCl; 50 g/L thiamine-HClH.sub.2O; 50 g/L riboflavin; 50 g/L nicotinic acid, 50 g/L Ca-pantothenate; 1 g/L vitamin B12; 50 g/L p-aminobenzoate; 50 g/L lipoic acid, approximately 67.5 mg/L NaOH) with about 400 mg/L L-cysteine hydrochloride and 400 mg/L Na.sub.2S9H.sub.2O, given 100 mg/L erythromycin). Cultivation was carried out in duplicate into 1 L glass bottles with a premixed gas mixture composed of 55% CO, 20% Hz, 10% CO.sub.2 and 15% N.sub.2 in an open water bath shaker at 37 C., 150 rpm and aeration of 3 L/h for 7 days.

[0079] The gas entered the medium through a filter with a pore size of 10 microns, which was mounted in the middle of the reactor, in a gassing tube. When sampling, 5 ml samples were removed for determination of OD.sub.600, pH and the product range. The determination of the product concentration was performed by semi-quantitative 1 H-NMR spectroscopy. As an internal quantification standard sodium trimethylsilylpropionate was used (T(M) SP). The modified strain produced 40 ppm 3-HB in 7 days.

Example 5

[0080] Fermentation of C. autoethanogenum Vector Control Strain with No Production of Acetoacetate or 3HB

[0081] For cell culture of C. autoethanogenum pEmpty 5 mL of the culture was anaerobically grown in 500 ml of medium (ATCC1754 medium: pH 6.0; 20 g/L MES; 1 g/L yeast extract, 0.8 g/L NaCl, 1 g/L NH.sub.4Cl, 0.1 g/L KCl, 0.1 g/L KH.sub.2PO.sub.4, 0.2 g/L MgSO.sub.47H.sub.2O; 0.02 g/L CaCl.sub.22H.sub.2O; 20 mg/L nitrilotriacetic acid 10 mg/L MnSO.sub.4H.sub.2O; 8 mg/L (NH.sub.4).sub.2Fe(SO.sub.4).sub.26H.sub.2O; 2 mg/L CoCl.sub.26H.sub.2O; 2 mg/L ZnSO.sub.47H.sub.2O; 0.2 mg/L CuCl.sub.22H.sub.2O; 0.2 mg/L Na.sub.2MoO.sub.42H.sub.2O; 0.2 mg/L NiCl.sub.26H.sub.2O; 0.2 mg/L Na.sub.2SeO.sub.4; 0.2 mg/L Na.sub.2WO.sub.42H.sub.2O; 20 g/L d-Biotin, 20 g/L folic acid, 100 g/L pyridoxine-HCl; 50 g/L thiamine-HClH.sub.2O; 50 g/L riboflavin; 50 g/L nicotinic acid, 50 g/L Ca-pantothenate; 1 g/L vitamin B12 ; 50 g/L p-aminobenzoate; 50 g/L lipoic acid, approximately 67.5 mg/L NaOH) with about 400 mg/L L-cysteine hydrochloride and 400 mg/L Na.sub.2S9H.sub.2O, given 100 mg/L erythromycin). Cultivation was carried out in duplicate in 1 L glass bottles with a premixed gas mixture composed of 55% CO, 20% Hz, 10% CO.sub.2 an open water bath shaker at 37 C., 150 rpm and aeration of 3 L/h for 7 days.

[0082] The gas entered the medium through a filter with a pore size of 10 microns, which was mounted in the middle of the reactor, in a gassing tube. When sampling, 5 ml samples were removed for determination of OD.sub.600 nm, pH and the product range. The determination of the product concentration was performed by semi-quantitative 1 H-NMR spectroscopy. As an internal quantification standard sodium trimethylsilylpropionate was used (T(M) SP). Neither acetoacetate nor 3HB was produced.

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