Production of alkenes from 3-hydroxy-1-carboxylic acids via 3-sulfonyloxy-1-carboxylic acids
09803220 · 2017-10-31
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Inventors
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Abstract
The application describes a method for producing alkenes (for example propylene, ethylene, 1-butylene, isobutylene, isoamylene, butadiene or isoprene) from 3-hydroxy-1-carboxylic acids via 3-sulfonyloxy-1-carboxylic acids.
Claims
1. A method for producing an alkene from a 3-hydroxy-1-carboxylic acid comprising enzymatically converting, using a sulfotransferase, a 3-hydroxy-1-carboxylic acid of the following general formula I: ##STR00053## into a 3-sulfonyloxy-1-carboxylic acid of the following general formula II: ##STR00054## and then converting the 3-sulfonyloxy-1-carboxylic acid by thermal conversion into an alkene of the following general formula III: ##STR00055## wherein R.sup.1 and R.sup.3 are independently selected from hydrogen (—H), methyl (—CH3), ethyl (—CH2-CH3), isopropyl (—CH2(CH3)2), vinyl (—CH═CH2) and isopropenyl (—C(CH3)=CH2) and in which R.sup.2 and R.sup.4 are independently selected from hydrogen (—H) and methyl (—CH3).
2. The method of claim 1, wherein the sulfotransferase is selected from alcohol sulfotransferase (EC 2.8.2.2), steroid sulfotransferase (EC 2.8.2.15), scymnol sulfotransferase (EC 2.8.2.32) flavonol 3-sulfotransferase (EC 2.8.2.25), retinol sulfotransferase/dehydratase, polyketide synthase (PKS) sulfotransferase or an olefin synthase (OLS) sulfotransferase.
3. The method of claim 1, wherein the method is carried out in vitro.
4. The method of claim 1, wherein the method is carried out in the presence of a microorganism or a plant producing the sulfotransferase.
5. The method of claim 1, further comprising a step of collecting the alkene produced in a gaseous state.
6. The method of claim 1 wherein the method is carried out in the presence of a composition comprising the sulfotransferase and a 3-hydroxy-1-carboxylic acid of formula I.
7. The method of claim 1, wherein the thermal conversion is carried out at a temperature of 30° C. or higher.
8. The method of claim 1, wherein the thermal conversion is carried out at a temperature of 37° C. or higher.
9. The method of claim 1, wherein the thermal conversion is carried out at a temperature of 40° C. or higher.
10. The method of claim 1, wherein the thermal conversion is carried out at a temperature of 45° C. or higher.
11. The method of claim 1, wherein the thermal conversion is carried out at a temperature of 50° C. or higher.
12. The method of claim 1, wherein the thermal conversion is carried out at a temperature of 55° C. or higher.
13. The method of claim 1, wherein the thermal conversion is carried out at a temperature of 60° C. or higher.
14. The method of claim 1, wherein the thermal conversion is carried out at a temperature of 65° C. or higher.
15. The method of claim 1, wherein the method is carried out in the presence of a mesophilic microorganism producing the sulfotransferase.
16. The method of claim 15, wherein the mesophilic organism is E. coli.
Description
(1) Other aspects and advantages of the invention will be described in the following examples, which are given for purposes of illustration and not by way of limitation.
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(9) The following Examples serve to illustrate the invention.
EXAMPLES
Example 1: Cloning, Expression and Purification of Enzymes
(10) Cloning and Bacterial Culture
(11) The genes encoding the enzymes of interest were cloned in the pET 25b (+) vector. (Novagen). A stretch of 6 histidine codons was inserted after the methionine initiation codon to provide an affinity tag for purification. Competent E. coli BL21(DE3) cells (Novagen) were transformed with this vector according to the heat shock procedure. The transformed cells were grown with shaking (160 rpm) on ZYM-5052 auto-induction medium (Studier F W, Prot. Exp. Pur. 41, (2005), 207-234) for 6 hours at 37° C. and protein expression was continued at 18° C. overnight (approximately 16 hours). The cells were collected by centrifugation at 4° C., 10,000 rpm for 20 min and the pellets were frozen at −80° C.
(12) Protein Purification and Concentration
(13) The pellets from 200 ml of culture cells were thawed on ice and resuspended in 5 ml of Na.sub.2HPO.sub.4 pH 8 containing 300 mM NaCl, 5 mM MgCl.sub.2 and 1 mM DTT. Twenty microliters of lysonase (Novagen) were added. Cells were incubated 10 minutes at room temperature and then returned to ice for 20 minutes. Cell lysis was completed by sonication for 3×15 seconds. The bacterial extracts were then clarified by centrifugation at 4° C., 10,000 rpm for 20 min. The clarified bacterial lysates were loaded on PROTINO-1000 Ni-TED column (Macherey-Nagel) allowing adsorption of 6-His tagged proteins. Columns were washed and the enzymes of interest were eluted with 4 ml of 50 mM Na.sub.2HPO.sub.4 pH 8 containing 300 mM NaCl, 5 mM MgCl.sub.2, 1 mM DTT, 250 mM imidazole. Eluates were then concentrated and desalted on Amicon Ultra-4 10 kDa filter unit (Millipore) and resuspended in 0.25 ml 50 mM Tris-HCl pH 7.5 containing 0.5 mM DTT and 5 mM MgCl.sub.2. Protein concentrations were quantified by direct UV 280 nm measurement on the NanoDrop 1000 spectrophotometer (Thermo Scientific). The purity of proteins varied from 70% to 90%.
Example 2: Mass Spectrometry Analysis of the 3-hydroxy-1-carboxylic acid Sulfurylation Reaction Using PAPS as Co-Substrate
(14) The enzymatic reactions were carried out under the following conditions:
(15) 50 mM Tris-HCl pH 7.5
(16) 5 mM DTT
(17) 100 mM NaCl
(18) 5-50 mM PAPS
(19) 5-100 mM 3-hydroxy-1-carboxylic acid
(20) 1-5 mg/ml purified sulfotransferase
(21) The reactions are initiated with the addition of purified sulfotransferase and incubated at 37° C. Control assays are performed in which either no enzyme is added, or no co-factor is added. Following incubation the samples are processed by mass spectrometry analysis. An aliquot of 50-200 μl reaction is removed, centrifuged and the supernatant is transferred to a clean vial. MS analyses are performed on a PE SCIEX API 2000 quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source.
Example 3: Mass Spectrometry Analysis of the 3-hydroxy-1-carboxylic acid Sulfurylation Reaction Using APS as Co-Substrate
(22) The enzymatic reactions were carried out under the following conditions:
(23) 50 mM Tris-HCl pH 7.5
(24) 5 mM DTT
(25) 100 mM NaCl
(26) 5-50 mM APS
(27) 5-100 mM 3-hydroxy-1-carboxylic acid
(28) 1-5 mg/ml purified sulfotransferase
(29) The reactions are initiated with the addition of purified sulfotransferase and incubated at 37° C. Control assays are performed in which either no enzyme is added, or no co-factor is added. Following incubation the samples are processed by mass spectrometry analysis. An aliquot of 50-200 μl reaction is removed, centrifuged and the supernatant is transferred to a clean vial. MS analyses are performed on a PE SCIEX API 2000 quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source.
Example 4: Mass Spectrometry Analysis of the 3-hydroxybutyrate sulfurylation Reaction
(30) Sequence of sulfotransferase domain of polyketide synthase CurM (PKS ST) inferred from the genome of Moorea producens (formerly Lyngbya majuscala) 19L was generated by oligonucleotide concatenation to fit the codon usage of E. coli. A stretch of 6 histidine codons was inserted after the methionine initiation codon to provide an affinity tag for purification. The gene thus synthesized was cloned in a pET25b(+) expression vector (the vectors were constructed by GENEART AG). After transformation of the E. coli strain BL21 (DE3), the protein was produced according to the procedure described in Example 1 and sulfotransferase activity was assayed under the following conditions
(31) 50 mM Tris-HCl pH 7.5
(32) 5 mM DTT
(33) 100 mM NaCl
(34) 5 mM PAPS
(35) 5 mM (R,S)-3-hydroxybutyrate
(36) 5 mg/ml purified sulfotransferase (ST) CurM.
(37) Control reaction was performed using the same reaction mixture without addition of the enzyme. The assays were incubated for 1.5 hours at 37° C. An aliquot of 200 μl of each assay was then removed, filtered through a 0.45 μm filter and the filtrated solution was transferred into a clean vial. MS analyses were performed in negative electrospray mode by direct injection of sample using PE SCIEX API 2000 quadrupole instrument. The presence of 3-sulfonyloxybutyrate was evaluated. Mass spectra of enzymatic reaction showed characteristic peaks at m/z values of 182.8 and 90.9 (
(38) Thus, the sulfotransferase CurM (PKS sulfotransferase ST) catalyzes transfer of the sulfate group from PAPS onto hydroxyl group of 3-hydroxybutyrate.
Example 5: Mass Spectrometry Analysis of the 3-hydroxy-3-methylbutyrate Sulfurylation Reaction Using PAPS as Co-Substrate
(39) The enzymatic reactions were carried out under the following conditions:
(40) 50 mM Tris-HCl pH 7.5
(41) 5 mM DTT
(42) 100 mM NaCl
(43) 5-50 mM PAPS
(44) 5-50 mM 3-hydroxy-3-methylbutyrate
(45) 1-5 mg/ml purified sulfotransferase
(46) The reactions are initiated with the addition of purified sulfotransferase and incubated at 37° C. Control assays are performed in which either no enzyme is added, or no co-factor is added. Following incubation the samples are processed by mass spectrometry analysis. An aliquot of 50-200 μl reaction is removed, centrifuged and the supernatant is transferred to a clean vial. MS analyses are performed on a PE SCIEX API 2000 quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source.
Example 6: Thermal Conversion of 3-sulfonyloxy-1-carboxylic acids to Corresponding Alkenes
(47) Thermal conversion of 3-sulfonyloxy-1-carboxylic acids to alkenes is carried out under the following conditions:
(48) 50 mM Tris-HCl pH 7.5
(49) 50 mM 3-sulfonyloxy-1-carboxylic acid
(50) Assays are incubated with shaking at 37-65° C. for 2-72 hours in 2 ml sealed glass vials (Interchim). At the end of incubation one ml of the gaseous phase is collected and injected into a gas chromatograph Varian 430-GC equipped with a flame ionization detector (FID). Alkenes are identified by comparison with standard.
Example 7: Thermal Conversion of 3-sulfonyloxybutyrate to Propylene
(51) Disodium (R,S)-3-sulfonyloxybutyrate was synthesized upon request by company specialized in custom synthesis, Syntheval (France).
(52) The thermal conversion assays containing, in a total volume of 0.5 mL, 50 mM 3-sulfonyloxybutyrate and 50 mM Tris-HCl pH 7.5, were incubated in 2 ml sealed glass vials (Interchim) at 37° C. and 65° C. with shaking. Propylene production at different incubation times (from 0 to 90 h) was analyzed as follows. One ml of the gaseous phase was collected and injected into a gas chromatograph Varian 430-GC equipped with FID detector. Nitrogene was used as carrier gas with a flow rate of 1.5 ml/min. Volatile compounds were separated on RT-Alumina Bond/Na2SO4 column (Restek) using an isothermal mode at 130° C. Gaseous product of thermal decomposition of disodium (R,S)-3-sulfonyloxybutyrate was identified by comparison with propylene standard (Sigma, Aldrich). Under these GC conditions, the retention time for propylene was 3.2 min. Significant production of propylene was observed at 65° C. as well at 37° C. (
Example 8: Thermal Conversion of 3-sulfonyloxy-3-methylbutyrate to Isobutene
(53) 3-sulfonyloxy-3-methylbutyrate is synthesized upon request by company specialized in custom synthesis, Syntheval (France).
(54) The thermal conversion assays containing, in a total volume of 0.5 ml, 50 mM 3-sulfonyloxybutyrate and 50 mM Tris-HCl pH 7.5, are incubated in 2 ml sealed glass vials (Interchim) at 37° C. and 65° C. with shaking. Isobutene production at different incubation times is analyzed as follows. One ml of the gaseous phase is collected and injected into a gas chromatograph Varian 430-GC equipped with FID detector. Nitrogene is used as carrier gas with a flow rate of 1.5 ml/min. Volatile compounds are separated on RT-Alumina Bond/Na2SO4 column (Restek) using an isothermal mode at 130° C. Gaseous product of thermal decomposition of 3-sulfonyloxy-3-methylbutyrate is identified by comparison with isobutene standard (Sigma, Aldrich).
Example 9: Enzyme Catalyzed Conversion of 3-sulfonyloxy-1-carboxylic acids to Corresponding Alkenes
(55) Enzyme catalyzed conversion of 3-sulfonyloxy-1-carboxylic acids to alkenes is carried out under the following conditions:
(56) 50 mM Tris-HCl pH 7.5
(57) 50 mM 3-sulfonyloxy-1-carboxylic acid
(58) 10 mM MgCl.sub.2
(59) 5-10 mg/ml enzyme
(60) Assays are incubated with shaking at 37-42° C. for 2-72 h in 2 ml sealed glass vials (Interchim). At the end of incubation one ml of the gaseous phase is collected and injected into a gas chromatograph Varian 430-GC equipped with a flame ionization detector (FID). Alkenes are identified by comparison with standards.
Example 10: Enzyme Catalyzed Conversion of 3-sufonyloxybutyrate into Propylene
(61) Disodium (R,S)-3-sulfonyloxybutyrate was synthesized upon request by the custom chemist Syntheval.
(62) Enzyme catalyzed conversion of 3-sufonyloxybutyrate into propylene is carried out under the following conditions:
(63) 50 mM Tris-HCl pH 7.5
(64) 0-100 mM Disodium (R,S)-3-sulfonyloxybutyrate
(65) 10 mM MgCl.sub.2
(66) 5-10 mg/ml enzyme
(67) Assays are incubated with shaking at 37-42° C. for 2-72 h in 2 ml sealed glass vials (Interchim). At the end of incubation one ml of the gaseous phase is collected and injected into a gas chromatograph Varian 430-GC equipped with a flame ionization detector (FID). Propylene is identified by comparison with propylene standard (Sigma-Aldrich).
Example 11: Enzyme Catalyzed Conversion of 3-sulfonyloxy-3-methylbutyrate into Isobutene
(68) 3-sulfonyloxy-3-methylbutyrate is synthesized upon request by the custom chemist Syntheval.
(69) Enzyme catalyzed conversion of 3-sulfonyloxy-3-methylbutyrate into isobutene is carried out under the following conditions:
(70) 50 mM Tris-HCl pH 7.5
(71) 0-100 mM 3-sulfonyloxy-3-methylbutyrate
(72) 10 mM MgCl.sub.2
(73) 5-10 mg/ml enzyme
(74) Assays are incubated with shaking at 37-42° C. for 2-72 h in 2 ml sealed glass vials (Interchim). At the end of incubation one ml of the gaseous phase is collected and injected into a gas chromatograph Varian 430-GC equipped with a flame ionization detector (FID). Isobutene is identified by comparison with isobutene standard (Sigma-Aldrich).