Biotechnological production of diols

Abstract

The present invention relates to a method for the stereo selective production of a trans-diol or a cis-diol or a hydroxyketone comprising the step(s) (i) conversion of a trans-diol or a cis-diol to a hydroxyketone and/or (ii) conversion of a hydroxyketoneto a cis-diol or a trans-diol, catalyzed by an enzyme, which is encoded by a nucleic acid sequence of SEQ ID NO: or wherein the enzyme comprises an amino acid sequence of SEQ ID NO: 2. The present invention also relates to the use of an enzyme encoded by a nucleic acid sequence of SEQ ID NO: 1 or wherein the enzyme comprises an amino acid sequence of SEQ ID NO: 2 for the conversion of a trans-diol to a cis-diol or for the conversion of a trans-diol or a cis-diol to a hydroxy ketone and/or the conversion of a hydroxyketone to a trans-diol or a cis-diol.

Claims

1. A method for the production of a trans-diol or a cis-diol or a hydroxyketone comprising the step(s) (i) conversion of a trans-diol or a cis-diol to a hydroxyketone catalyzed by an enzyme, which is encoded by a nucleic acid sequence of SEQ ID NO: 1 or a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 1, or wherein the enzyme comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2, and/or (ii) conversion of a hydroxyketone to a trans diol or a cis-diol catalyzed by an enzyme as defined in step (i); and wherein the two hydroxyl groups of the trans-diol and/or the cis-diol is/are attached to an aliphatic ring system comprising 2 to 4 bridged or non-bridged rings; wherein the trans-diol is (8R,9R)-trans-cedrene diol, the cis-diol is (8R,9S)-cis-cedrene diol and the hydroxyketone is cedrene hydroxyketone.

2. The method of claim 1 for the production of a cis-diol comprising the step(s) (i) conversion of a trans-diol to a hydroxyketone catalyzed by an enzyme, which is encoded by a nucleic acid sequence of SEQ ID NO: 1 or a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 1, or wherein the enzyme comprises an amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2, and (ii) conversion of a hydroxyketone, optionally the hydroxyketone obtained in step (i), to a cis-diol catalyzed by an enzyme as defined in step (i).

3. The method of claim 1 for the production of (8R,9S)-cis-cedrene diol, wherein in step (i) (8R,9R)-trans-cedrene diol is converted to cedrene hydroxyketone and in step (ii) cedrene hydroxyketone is converted to (8R,9S)-cis-cedrene diol.

4. The method of claim 1, wherein the same enzyme catalyzes the conversion in step (i) and step (ii).

5. The method of claim 1, wherein a cofactor is used for the reaction of the enzyme and wherein the cofactor is selected from the group consisting of NAD, NADP, FAD and PQQ.

6. The method of claim 5, wherein the cofactor is regenerated by an enzymatic regeneration system or wherein the enzyme is the same in step (i) and step (ii) and the cofactor is regenerated by the enzyme.

Description

Example 1: Production of a gene construct for limonene-1,2-epoxide hydrolase

(1) Cloning of limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis can be performed by standard methods. The limA gene with the entry number Q9ZAG3 (Uniport) is used (GenBank accession code: CAA77012.1). The coding sequence is represented by SEQ ID NO: 3 and the amino acid sequence of the translated protein is represented by SEQ ID NO: 4. Commercially available expression vectors can be used. For example, the pET3a plasmid by Novagen is suitable. It comprises an N-terminal T7 tag and a BamH I restriction site. The Lac operon is used as promotor. The plasmid includes an ampicillin resistance gene as selection marker. Host organisms for the expression can be selected from any state of the art systems. For example, BL21 Escherichia coli strains can be used.

(2) As a construct for cloning, a sequence according to SEQ ID NO: 5 can be used, which comprises the added BamHI site GGATCC for cloning into the pet3a vector. Furthermore, the construct comprises the Ndel site CATATG for cloning into the pet3a vector. As stop codon, TAA is used.

Example 2: Transformation of Escherichia coli BL21

(3) The transformation is performed according to standard methods, e.g. heat transformation as described by Sambrook and Russel (Sambrook, J.; Russell, R. W. (2001): Molecular cloning: a laboratory manual, 3rd ed. Cold spring harbor laboratory press, cold spring harbor, N.Y.).

Example 3: Production of biomass comprising an inducible limonene-1,2-epoxide hydrolase

(4) An auto-induction medium is used (e.g. Studier, Protein Expression and Purification, 41 (2005), 207-234). Composition of the base medium for 100 mL: dissolve 1.0 g tryptone, 0.67 g Na.sub.2HPO.sub.47H.sub.2O (25 mM), 0.34 g KH.sub.2PO.sub.4 (25 mM), 0.27 g NH.sub.4Cl (50 mM), 0.07 g Na.sub.2SO.sub.4 (5 mM), 0.5 g yeast extract in deionized water. The pH is adjusted to 8.8. Subsequently, the medium is sterilized at 121 C. for 20 minutes at 1.013 bar overpressure.

(5) Composition carbohydrate supplement: 100 mL carbohydrate supplement (20fold) comprise: dissolve 10.0 g lactose (0.2%), 2.75 g glucose x H.sub.2O (0.05%), 25.0 g glycerol (0.5%) in deionized water. Autoclave at 121 C. for 20 minutes at 1,013 bar overpressure. 2 mL of the carbohydrate supplement are used for 100 mL auto-induction medium.

(6) Magnesium sulfate solution 1M: dissolve 24.65 g magnesium sulfate in 100 mL deionized water. Autoclave at 121 C. for 20 minutes at 1,013 bar overpressure. 0.2 mL (2 mM) of the magnesium sulfate solution are used for 100 mL auto-induction medium.

(7) Trace mineral solution: 100 mL trace mineral solution (1.000fold) comprise: 50 mM FeCl.sub.3, 20 mM CaCl.sub.2), 10 mM MnCl.sub.2, 10 mM ZnSO.sub.4, 2 mM CoCl.sub.2, 2 mM CuCl.sub.2, 2 mM NiCl.sub.2, 2 mM Na.sub.2MoO.sub.4, 2 mM Na.sub.2SeO.sub.3, 2 mM H.sub.3BO.sub.3. Dissolve the metals, except iron chloride, according to the following list separately in ca. 60 mM HCl. Dissolve iron chloride 0.1 M in 50 mL 100 fold diluted concentrated hydrochloric acid.

(8) TABLE-US-00001 50 ml 0.1M FeCl.sub.36H2O 2.70 g/100 ml 2 ml 1.0M CaCl.sub.22H2O 15.8 g/100 ml 1 ml 1.0M MnCl.sub.24H2O 19.8 g/100 ml 1 ml 1.0M ZnSO.sub.47H2O 28.8 g/100 ml 1 ml 0.2M CoCl.sub.26H2O 4.76 g/100 ml 2 ml 0.1M CuCl.sub.22H2O 1.70 g/100 ml 1 ml 0.2M NiCl.sub.26H2O 4.76 g/100 ml 2 ml 0.1M Na.sub.2MoO.sub.42H2O 2.42 g/100 ml 2 ml 0.1M Na.sub.2SeO.sub.3 1.73 g/100 ml 2 ml 0.1M H.sub.3BO.sub.3 0.62 g/100 ml

(9) Sterile filtrate with a 0.45 m membrane filter. 0.02 mL of the trace mineral solution are used for 100 mL auto-induction medium.

(10) Ampicillin solution: dissolve 500 mg sodium ampicillin in 10 mL deionized water. Sterile filtrate with a 0.45 m membrane filter. 0.2 mL of the ampicillin solution are used for 100 mL auto-induction medium (100 g/mL).

(11) An Escherichia coli BL21 strain is used, which carries a IPTG or lactose inducible gene of a epoxide hydrolase on a pet3a-Plasmid. An inoculation loop of Escherichia coli BL21 of a well grown dYT agar plate (24 hours at 30 C.) is used for inoculation of 100 mL auto-induction medium with 100 g ampicillin in a 500 mL Erlenmeyer flask with baffle. The cultivation is performed at 30 C. and 120 U/min for 18 hours. Typically, the optical density is at 6-9 (600 nm) at this point. Composition of the dYT medium: 5 g/L NaCl; 16.0 g/L tryptone; 10.0 g yeast extract. The pH is adjusted to 7. Autoclave at 121 C. for 20 minutes at 1,013 bar overpressure.

Example 4: Biotransformation with a limonene-1,2-epoxide hydrolase containing biomass

(12) The culture broth of example 3 is used. 100 mg (-)--cedrene epoxid are added to 100 mL culture broth. Biotransformation is performed at 30 C. and 120 U/min. Optionally, cell membrane damaging agents such as Triton X-100 (t-Octylphenoxypolyethoxyethanol), EDTA or organic solvents can be added, which accelerate uptake of the epoxide. The conversion is monitored by gas chromatography. After 96 hours, the conversion is 33.9% trans-cedrene diol.

Example 5: Production of a limonene-1,2-epoxide hydrolase containing raw extract

(13) The culture broth of example 3 is used. The culture broth is centrifuged at 4.000 g for 5 minutes in falcon tubes. The supernatant is discarded and the biomass is resuspended in 10 mL 50 mM phosphate buffer (pH 7). Subsequently, the cell disruption is performed by sonification in a falcon tube with a Bandelin Sonoplus UW2200 Sonifier 15 minutes, 40% performance, cycle 1 in an ice bath. The raw extract is centrifuged at 12.500 g for 10 minutes at 5 C. The epoxide hydrolase containing supernatant is removed and stored in an ice bath. The residue is discarded.

Example 6: Biotransformation with limonene-1,2-epoxide hydrolase containing raw extract

(14) The culture broth of example 3 is used. 100 L (-)--cedrene epoxide are added to the culture broth. The biotransformation is performed at 30 C. and 120 U/min in a 500 mL Erlenmeyer flask with baffle. The conversion is monitored by gas chromatography. After 96 hours, the conversion is 62.9% trans-(8R,9R)-cedrene diol. Other products are not present.

Example 7: Biotransformation with resting cells, which have expressed a limonene-1,2-epoxide hydrolase

(15) The culture broth of example 3 is centrifuged at 4.000 g for 5 minutes in falcon tubes. The supernatant is discarded and the biomass is resuspended is 100 mL 50 mM phosphate buffer (pH 7). The cultivation is performed at 30 C. and 120 U/min. After 8 hours, 100 L (-)--cedrene epoxide and 10 mg Triton X-100 (t-Octylphenoxypolyethoxyethanol) are added to the resting cells. Optionally, membrane damaging agents such as EDTA or organic solvents can be added. The biotransformation is performed at 30 C. and 120 U/min. The conversion is monitored by gas chromatography. After 72 hours, the conversion is 27.5% trans-(8R,9R)-cedrene diol. Other products are not present.

Example 8: Production of a hydroxysteroid dehydrogenase gene construct

(16) Cloning of hydroxysteroid dehydrogenase from C. testosteroni can be performed by standard methods. The hsdA gene with the entry number P80702 (Uniport) is used. The coding sequence is represented by SEQ ID NO: 1 and the amino acid sequence of the translated protein is represented by SEQ ID NO: 2. Commercially available expression vectors can be used. For example, the pet3a plasmid by Novagen is suitable. It comprises an N-terminal T7 tag and a BamH I restriction site. The Lac operon is used as promotor. The plasmid includes an ampicillin resistance gene as selection marker. Host organisms for the expression can be selected from any state of the art systems. For example, BL21 Escherichia coli strains can be used.

(17) As a construct for cloning, a sequence according to SEQ ID NO: 6 can be used, which additionally comprises the BamHI site GGATCC for cloning into the pet3a vector. In addition, the construct comprises the Ndel site CATATG for cloning into the pet3a vector. As stop codon, TGA is used.

Example 9: Transformation of Escherichia coli BL21

(18) The transformation is preformed according to standard methods, e.g. heat transformation as described by Sambrook and Russel (Sambrook, J.; Russell, R. W. (2001): Molecular cloning: a laboratory manual, 3rd ed. Cold spring harbor laboratory press, cold spring harbor, N.Y.).

Example 10: Production of a biomass with an inducible hydroxyl steroid dehydrogenase

(19) An auto-induction medium is used (e.g. Studier, Protein Expression and Purification, 41 (2005), 207-234). Composition of the base medium for 100 mL: dissolve 1.0 g tryptone, 0.67 g Na.sub.2HPO.sub.47H.sub.2O (25 mM), 0.34 g KH.sub.2PO.sub.4 (25 mM), 0.27 g NH.sub.4Cl (50 mM), 0.07 g Na.sub.2SO.sub.4 (5 mM), 0.5 g yeast extract in deionized water. The pH is adjusted to 8.8. Subsequently, the medium is sterilized at 121 C. for 20 minutes at 1.013 bar overpressure.

(20) Composition carbohydrate supplement: 100 mL carbohydrate supplement (20fold) comprise: dissolve 10.0 g lactose (0.2%), 2.75 g glucose x H.sub.2O (0.05%), 25.0 g glycerol (0.5%) in deionized water. Autoclave at 121 C. for 20 minutes at 1,013 bar overpressure. 2 mL of the carbohydrate supplement are used for 100 mL auto-induction medium.

(21) Magnesium sulfate solution 1M: dissolve 24.65 g magnesium sulfate in 100 ml deionized water. Autoclave at 121 C. for 20 minutes at 1,013 bar overpressure. 0.2 mL (2 mM) of the magnesium sulfate solution are used for 100 mL auto-induction medium.

(22) Trace mineral solution: 100 mL trace mineral solution (1.000fold) comprise: 50 mM FeCl.sub.3, mM CaCl.sub.2), 10 mM MnCl.sub.2, 10 mM ZnSO.sub.4, 2 mM CoCl.sub.2, 2 mM CuCl.sub.2, 2 mM NiCl.sub.2, 2 mM Na.sub.2MoO.sub.4, 2 mM Na.sub.2SeO.sub.3, 2 mM H.sub.3BO.sub.3. Dissolve the metals, except iron chloride, according to the following list separately in ca. 60 mM HCl. Dissolve iron chloride 0.1 M in 50 mL 100fold diluted concentrated hydrochloric acid.

(23) TABLE-US-00002 50 ml 0.1M FeCl.sub.36H2O 2.70 g/100 ml 2 ml 1.0M CaCl.sub.22H2O 15.8 g/100 ml 1 ml 1.0M MnCl.sub.24H2O 19.8 g/100 ml 1 ml 1.0M ZnSO.sub.47H2O 28.8 g/100 ml 1 ml 0.2M CoCl.sub.26H2O 4.76 g/100 ml 2 ml 0.1M CuCl.sub.22H2O 1.70 g/100 ml 1 ml 0.2M NiCl.sub.26H2O 4.76 g/100 ml 2 ml 0.1M Na.sub.2MoO.sub.42H2O 2.42 g/100 ml 2 ml 0.1M Na.sub.2SeO.sub.3 1.73 g/100 ml 2 ml 0.1M H.sub.3BO.sub.3 0.62 g/100 ml

(24) Sterile filtrate with a 0.45 m membrane filter. 0.02 mL of the trace mineral solution are used for 100 mL auto-induction medium.

(25) Ampicillin solution: dissolve 500 mg sodium ampicillin in 10 mL deionized water. Sterile filtrate with a 0.45 m membrane filter. 0.2 mL of the ampicillin solution are used for 100 mL auto-induction medium (100 g/mL).

(26) An Escherichia coli BL21 strain is used, which carries a IPTG or lactose inducible gene of a 3-a-hydroxysteroid dehydrogenase on a pet3a-Plasmid. An inoculation loop of Escherichia coli BL21 of a well grown dYT agar plate (24 hours at 30 C.) is used for inoculation of 100 mL auto-induction medium with 100 g ampicillin in a 500 mL Erlenmeyer flask with baffle. The cultivation is performed at 30 C. and 120 U/min for 18 hours. Typically, the optical density is at 6-9 (600 nm) at this point. Composition of the dYT medium: 5 g/L NaCl; 16.0 g/L tryptone; 10.0 g yeast extract. The pH is adjusted to 7. Autoclave at 121 C. for 20 minutes at 1,013 bar overpressure.

Example 11: Biotransformation with a hydroxysteroid dehydrogenase containing biomass

(27) The culture broth of example 10 is used. 50 mg trans-cedrene diol are added to 100 ml culture broth. Biotransformation is performed at 30 C. and 120 U/min. Optionally, cell wall or membrane damaging agents such as Triton X-100 (t-Octylphenoxypolyethoxy-ethanol), EDTA, lysozym or organic solvents can be added, which accelerate substrate uptake. The conversion is monitored by gas chromatography. After 24 hours, the conversion is 45% cedrene hydroxyketone.

Example 12: Production of a hydroxysteroid dehydrogenase containing raw extract

(28) The culture broth of example 10 is used. The culture broth is centrifuged at 4.000 g for 5 minutes in falcon tubes. The supernatant is discarded and the biomass is resuspended in 10 mL 200 mM phosphate buffer (pH 8). Subsequently, the cell disruption is performed by sonification in a falcon tube with a Bandelin Sonoplus UW2200 Sonifier 15 minutes, 40% performance, cycle 1 in an ice bath. The raw extract is centrifuged at 12.500 g for 10 minutes at 5 C. The dehydrogenase containing supernatant is removed and stored in and ice bath. The residue is discarded.

(29) The culture broth can be concentrated by methods known to the skilled person, such as e.g. membrane filtration, precipitation with salts (e.g. ammonium sulfate) or crystallization. It is also possible to use chromatographic methods for concentration of the 3--hydroxysteroid dehydrogenase. A good purification method is affinity chromatography. In this method, a modified 3--hydroxysteroid dehydrogenase is used, which carries a 6-histidine residue at the N-terminus. This is achieved on genetic level by a corresponding extension of the 3--hydroxysteroid dehydrogenase gene.

Example 13: Synthesis of cedrene hydroxyketone from (8R,9R)-trans-cedrene diol with a hydroxysteroid dehydrogenase containing raw extract

(30) A culture broth from example 10 is used. 20 mg trans-cedrene diol, 2 g/L Tween 80 (Polyethylene glycol sorbitan monooleate) and 25 L ampicilin solution (50 mg/mL) are added to 10 mL culture broth (protein content 18 g/L) in a 50 mL falcon tube with a magnetic stir bar. The biotransformation is performed at 30 C., but it can be higher or lower. The conversion is monitored by gas chromatography. After 21 hours, the conversion is 76.1% cedrene hydroxyketone. No other products are present.

Example 14: Stereo selective reduction of cedrene hydroxyketone to a (8R,9S)-cis-cedrene diol

(31) A culture broth from example 10 is used. The biotransformation is performed at 30 C., but it can be higher or lower. 200 mg cedrene hydroxyketone, 2 g/L Tween 80 (Polyethylene glycol sorbitan monooleate), 10 mg protease inhibitor 8830 (Sigma Aldrich) and 100 L ampicillin solution (50 mg/mL) are added to 120 mL culture broth (protein content 15.9 g/L) in a 250 mL Schott flask with a magnetic stir bar. 20 mg NAD and 3.3 g glucose monohydrate are added to the solution. Subsequently, 4.4 mg commercial glucose dehydrogenase (activity >25U/mg) in 1 mL buffer is added. The conversion proceeds at a constant pH 7 with automatic addition of 1 M lye. After 27.5 and 47.5 hours, another 20 mg NAD are added. After 30.5 hours, an additional 1.5 g glucose monohydrate are added. The conversion is monitored by gas chromatography. After 71.5 hours, the conversion is 84.9% cis-cedrene diol. No other products are present.

Example 15: Direct stereo selective conversion of (8R,9R)-trans-cedrene diol to (8R,9S)-cis-cedrene diol with a hydroxysteroid dehydrogenase containing raw extract

(32) A culture broth from example 10 is used. The biotransformation is performed at 40 C., but it can be higher or lower. 500 mg trans-cedrene diol and 500 mg cedrene hydroxyketone, 2 g/L Tween 80 (Polyethylene glycol sorbitan monooleate), 10 mg protease inhibitor 8830 (Sigma Aldrich) and 100 L ampicillin solution (50 mg/mL) are added to 50 mL culture broth (protein content 25 g/L) in a 250 mL Schott flask with a magnetic stir bar. 120 mg NAD are added to the solution. After 16 hours, another 500 mg trans-cedrene diol are added. After 66 hours, an additional 60 mg NAD are added. The conversion is monitored by gas chromatography. After 95.5 hours, the trans-cedrene diol is entirely converted. The product mixture comprises 29.9% cis-cedrene diol and 70.1% cedrene hydroxyketone.

(33) Sequences:

(34) SEQ ID NO: 1: coding region of the hedA gene from Comamonas testosteroni SEQ ID NO: 2: translated sequence of hedA gone from Comamonas testosteroni SEQ ID NO: 3: coding region of the limA gone from Rhodococcus erythropolis SEQ ID NO: 4: translated sequence of limA gene from Rhodococcus erythropolis SEQ ID NO: 5: construct of limA for cloning into pet3a vector used in example 1 SEQ ID NO: 6 construct of hedA for cloning into pet3a vector used in example 8