Production of alkenes from 3-hydroxycarboxylic acids via 3-hydroxycarboxyl-nucleotidylic acids
10017787 ยท 2018-07-10
Assignee
Inventors
Cpc classification
C12P5/007
CHEMISTRY; METALLURGY
C12P5/026
CHEMISTRY; METALLURGY
International classification
C12P5/00
CHEMISTRY; METALLURGY
Abstract
The application describes a method for producing alkenes (for example propylene, ethylene, 1-butylene, isobutylene, isoamylene, butadiene or isoprene) from 3-hydroxycarboxylic acids via 3-hydroxycarboxyl-nucleotidylic acids.
Claims
1. A method for producing an alkene from a 3-hydroxycarboxylate wherein a 3-hydroxycarboxylate of the following general formula I: ##STR00054## wherein R.sup.1 and R.sup.3 are independently selected from hydrogen (H), methyl (CH.sub.3), ethyl (CH.sub.2CH.sub.3), isopropyl (CH.sub.2(CH.sub.3).sub.2), vinyl (CHCH.sub.2) and isopropenyl (C(CH.sub.3)CH.sub.2) and in which R.sup.2 and R.sup.4 are independently selected from hydrogen (H) and methyl (CH.sub.3), is, in a first step, enzymatically converted using an adenylate forming enzyme (EC 6.2.1) together with a co-substrate of the following formula II: ##STR00055## wherein X is selected from the group consisting of OPO.sub.3H.sub.2 monophosphate, OPO.sub.2HOPO.sub.3H.sub.2 diphosphate, and OSO.sub.3H sulfate, and wherein Y is selected from the group consisting of OH hydroxyl and OPO.sub.3H.sub.2 monophosphate, and wherein Z is a nucleobase selected from the group consisting of adenine, guanine, thymine, cytosine, uracil and hypoxanthine, and wherein W is selected from the group consisting of hydrogen (H) hydroxyl (OH), into a 3-hydroxycarboxyl-nucleotidylate of the following general formula III: ##STR00056## wherein R.sup.1, R.sup.2, R.sup.3 and R.sup.4 have the same meaning as specified above in connection with formula I and wherein W, Y and Z have the same meaning as specified above in connection with formula II, and in that the thus produced 3-hydroxycarboxyl-nucleotidylate is subsequently enzymatically converted, using a recombinant OleC protein, into an alkene of the following general formula IV: ##STR00057## wherein R.sup.1, R.sup.2, R.sup.3 and R.sup.4 have the same meaning as specified above in connection with formula I.
2. The method of claim 1, wherein the adenylate forming enzyme is selected from the group consisting of: an AMP-dependent synthetase and ligase an adenylation domain of a non-ribosomal peptide synthetase (NRPS); an acyl- or aryl-CoA synthetases; and an adenylation domain of a polyketide synthase (PKS).
3. The method of claim 1, wherein the adenylate forming enzyme is selected from the group consisting of: acetate:CoA ligase (AMP forming) (EC 6.2.1.1); butanoate:CoA ligase (AMP forming) (EC 6.2.1.2); long-chain fatty acid:CoA ligase (AMP-forming) (EC 6.2.1.3); 4-Coumarate-CoA ligase (EC 6.2.1.12); long-chain-fatty-acid:[acyl-carrier protein] ligase (AMP-forming) (EC 6.2.1.20); 4-chlorobenzoate:CoA ligase (EC 6.2.1.33); and 3-hydroxypropionate:CoA ligase (AMP-forming) (EC 6.2.1.36).
4. The method of claim 1, wherein the method is carried out in vitro.
5. The method of claim 1, wherein the method is carried out in the presence of a microorganism producing the adenylated forming enzyme (EC 6.2.1).
6. The method according to claim 1, comprising a step of collecting the gaseous alkene degassing out of the reaction.
7. The method of claim 1, wherein the co-substrate is selected from ATP, UTP, CTP, GTP, ITP, ADP, CDP, GCP, UDP, IDP, dATP, dCTP, dGTP, dTTP, dITP, 3-phosphoadenosin-5-phosphosulfate (PAPS), or adenosin-5-phosphosulfate (APS).
8. The method of claim 1, wherein the adenylate forming enzyme is a polypeptide comprising the amino acid sequence of SEQ ID NO:1.
9. The method of claim 1, wherein the adenylate forming enzyme is a polypeptide comprising the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
10. The method of claim 1, wherein the OleC protein comprises the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.
11. The method of claim 5, wherein the microorganism is selected from bacteria, yeasts, fungi or mold.
12. The method of claim 11, wherein the microorganism is a thermophilic bacterium or an anaerobic bacterium.
13. The method of claim 12, wherein the bacterium is of the genus Thermus, Thermoplasma, or Clostridiae.
14. The method of claim 11, wherein the bacterium is Escherichia coli, Alcaligenes eutrophus, or Bacillus megaterium.
15. The method of claim 11, wherein the fungus is of the genus Saccharomyces, Schizosaccharomyces, Aspergillus, Trichoderma, Pichia, or Kluyveromyces.
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. Each publication, patent, patent application or other document cited in this application is hereby incorporated by reference in its entirety.
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(38) It is to be understood that the present invention specifically relates to each and every combination of features and process parameters described herein, including any combination of general and/or preferred features/parameters. In particular, the invention specifically relates to all combinations of preferred features (including all degrees of preference) of the process provided herein.
(39) In this specification, a number of documents including patent applications are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.
(40) The invention will now be described by reference to the following examples which are merely illustrative and are not to be construed as a limitation of the scope of the present invention.
EXAMPLES
Example 1: Cloning, Expression and Purification of Enzymes
(41) Gene Synthesis, Cloning and Expression of Recombinant Proteins
(42) The sequences of the studied enzymes inferred from the genomes of prokaryotic and eukaryotic organisms were generated by oligonucleotide concatenation to fit the codon usage of E. coli (genes were commercially synthesized by GeneArt). A stretch of 6 histidine codons was inserted after the methionine initiation codon to provide an affinity tag for purification. The genes thus synthesized were cloned in a pET-25b(+) expression vector (vectors were constructed by GeneArt), except for Nocardia iowensis carboxylic acid reductase gene.
(43) Competent E. coli BL21(DE3) cells (Novagen) were transformed with these vectors according to standard heat shock procedure. The transformed cells were grown with shaking (160 rpm) using ZYM-5052 auto-induction medium (Studier F W, Prot. Exp. Pur. 41, (2005), 207-234) for 6 h at 37 C. and protein expression was continued at 28 C. or 18 C. overnight (approximately 16 h). The cells were collected by centrifugation at 4 C., 10,000 rpm for 20 min and the pellets were stored at 80 C.
(44) The gene coding for the carboxylic acid reductase from Nocardia iowensis (Uniprot Q6RKB1) was codon-optimized by GeneArt (Life Technologies). The gene construction provided by GeneArt was flanked by PacI and NotI restriction sites and provided within master vector pMK. The gene thus synthesized was then subcloned into a modified version of pUC18 (New England Biolabs), containing a modified Multiple Cloning Site (MCS) (WO 2013/007786).
(45) Competent MG1655 E. coli cells were transformed with this vector using standard heat shock procedure. The transformed cells were grown in LB-ampicillin medium for 24 h at 30 C., 160 rpm shaking.
(46) The cells were collected by centrifugation at 4 C., 10,000 rpm for 20 min and the pellets were stored at 80 C.
(47) Protein Purification and Concentration
(48) The pellets from 200 ml of culture cells were thawed on ice and resuspended in 5 ml of 50 mM Tris-HCl buffer pH 7.5 containing 500 mM NaCl, 10 mM MgCl.sub.2, 10% glycerol, 10 mM imidazole 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 230 seconds. The bacterial extracts were then clarified by centrifugation at 4 C., 10,000 rpm for 20 min. The clarified bacterial lysates were loaded onto a PROTINO-2000 Ni-TED column (Macherey-Nagel) allowing adsorption of 6-His tagged proteins. Columns were washed and the enzymes of interest were eluted with 6 ml of 50 mM Tris-HCl buffer pH 7.5 containing 300 mM NaCl, 10% glycerol, 1 mM DTT, 250 mM imidazole. Eluates were then concentrated, desalted on Amicon Ultra-4 10 kDa filter unit (Millipore) and enzymes were resuspended in solution containing 50 mM Tris-HCl pH 7.5, containing 100 mM NaCl, 10% glycerol, 1 mM DTT. In the case of the OleC enzymes this resuspension buffer was supplemented with 1 mM AMP.
(49) Protein concentrations were quantified by direct UV 280 nm measurement on the NanoDrop 1000 spectrophotometer (Thermo Scientific). The purity of proteins thus purified varied from 70% to 90% as estimated by SDS-PAGE analysis.
Example 2: Continuous Spectrophotometric Assay for Adenylation Enzyme Activity with 3-Hydroxypropionate as a Substrate and ATP as a Co-Substrate
(50) The genes coding for the adenylate-forming enzymes were synthesized and the corresponding enzymes were further produced according to the procedure described in Example 1. 3-hydroxypropionic acid (TCI) stock solution was prepared in water with the pH adjusted to 7.5 with 1 M NaOH. The release of diphosphate which is associated with 3-hydroxypropionyl-adenylate formation from 3-hydroxypropionate was quantified using the EnzCheck Pyrophosphatase Assay Kit (E6645, Life Technologies).
(51) In this assay, diphosphate was hydrolyzed to inorganic phosphate by inorganic pyrophosphatase and phosphate production coupled to phosphorolysis of the 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG), catalyzed by the enzyme purine nucleoside phosphorylase (PNP). The chromophoric product, 2-amino-6-mercapto-7-methylpurine, was monitored by absorbance at 360 nm (
(52) Standard reaction mixture contained:
(53) 100 mM Tris-HCl pH 7.5
(54) 5 mM 3-hydroxypropionate
(55) 2 mM MgCl.sub.2
(56) 0.1 mM DTT
(57) 2 mM ATP
(58) 0.1 mg/ml of studied enzyme
(59) 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG), purine nucleoside phosphorylase (PNP) and inorganic pyrophosphatase were added to the reaction mix according to the procedure described in the EnzCheck Pyrophosphatase Assay Kit. Control reactions were performed in which either no adenylate-forming enzyme was added, or no 3-hydroxypropionate was added. Each reaction was started by the addition of ATP. Reactions were performed in 96-well plates at 37 C.
(60) Each sample was continuously monitored for the increase of 2-amino-6-mercapto-7-methylpurine at 360 nm on a SpectraMax Plus 384 UV/Vis Microplate Reader (Molecular Devices).
(61) Several enzymes showed adenylation activity with 3-hydroxypropionate (
Example 3: Continuous Spectrophotometric Assay for Adenylation Enzyme Activity with 3-Hydroxyvalerate as a Substrate and ATP as a Co-Substrate
(62) Spectrophotometric assay was performed according to the procedure described in Example 2. R-3-hydroxyvaleric acid was purchased from EMPA (Switzerland). R-3-hydroxyvaleric acid stock solution was prepared in water with the pH adjusted to 7.5 with 1 M NaOH. The composition of the reaction mixture was the same as that described in Example 2 using 5 mM R-3-hydroxyvalerate as a substrate instead of 3-hydroxypropionate. Control reactions were performed in which either no adenylate-forming enzyme was added, or no 3-hydroxyvalerate was added. Each reaction was started by the addition of ATP. Reactions were performed in 96-well plates at 37 C. Each sample was continuously monitored for the increase of 2-amino-6-mercapto-7-methylpurine at 360 nm on a SpectraMax Plus 384 UV/Vis Microplate Reader (Molecular Devices). Several enzymes demonstrated adenylation activity with 3-hydroxyvalerate (
Example 4: Continuous Spectrophotometric Assay for Adenylation Enzyme Activity with 3-Hydroxypent-4-Enoate as a Substrate and ATP as a Co-Substrate
(63) Spectrophotometric assay was performed according to the procedure described in Example 2. (R,S)-3-hydroxypent-4-enoic acid (Epsilon Chimie) stock solution was prepared in water with the pH adjusted to 7.5 with 1 M NaOH. The composition of the reaction mixture was the same as that described in Example 2 using 5 mM (R,S)-3-hydroxypent-4-enoate as a substrate instead of 3-hydroxypropionate. Control reactions were performed in which either no adenylate-forming enzyme was added, or no 3-hydroxypent-4-enoate was added. Reactions were performed in 96-well plates at 37 C. Each sample was continuously monitored for the increase of 2-amino-6-mercapto-7-methylpurine at 360 nm on a SpectraMax Plus 384 UV/Vis Microplate Reader (Molecular Devices). Several enzymes demonstrated adenylation activity with 3-hydroxypent-4-enoate (
Example 5: Continuous Spectrophotometric Assay for Adenylation Enzyme Activity with 3-Hydroxyisovalerate as a Substrate and ATP as a Co-Substrate
(64) Spectrophotometric assay was performed according to the procedure described in Example 2. 3-hydroxyisovaleric acid (3-hydroxy-3-methylbutyric acid) (TCI) stock solution was prepared in water with the pH adjusted to 7.5 with 1 M NaOH. The composition of the reaction mixture was the same as that described in Example 2 using 5 mM 3-hydroxyisovalerate as a substrate instead of 3-hydroxypropionate. Control reactions were performed in which either no adenylate-forming enzyme was added, or no 3-hydroxyisovalerate was added. Reactions were performed in 96-well plates at 37 C. Each sample was continuously monitored for the increase of 2-amino-6-mercapto-7-methylpurine at 360 nm on a SpectraMax Plus 384 UV/Vis Microplate Reader (Molecular Devices). Several enzymes demonstrated adenylation activity with 3-hydroxyisovalerate (
Example 6: Continuous Spectrophotometric Assay for Adenylation Enzyme Activity with 3-Hydroxybutyrate as a Substrate and ATP as a Co-Substrate
(65) Spectrophotometric assay was performed according to the procedure described in Example 2. (R,S)-3-hydroxybutyric acid (Sigma-Aldrich) stock solution was prepared in water with the pH adjusted to 7.5 with 1 M NaOH. The composition of the reaction mixture was the same as that described in Example 2 using 5 mM (R,S)-3-hydroxybutyrate as a substrate instead of 3-hydroxypropionate. Control reactions were performed in which either no adenylate-forming enzyme was added, or no 3-hydroxybutyrate was added. Reactions were performed in 96-well plates at 37 C. Each sample was continuously monitored for the increase of 2-amino-6-mercapto-7-methylpurine at 360 nm on a SpectraMax Plus 384 UV/Vis Microplate Reader (Molecular Devices). Several enzymes demonstrated adenylation activity with 3-hydroxybutyrate (
Example 7: Study of Adenylation of 3-Hydroxybutyrate with ATP or ADP as Co-Substrates
(66) Spectrophotometric assay was performed according to the procedure described in Example 2. The specificity of acyl-CoA synthase from M. algicola and AMP-dependent synthetase/ligase from Burkholderia sp. with respect to co-substrates was analyzed. Standard reaction contained:
(67) 100 mM Tris-HCl pH 7.5
(68) 5 mM (R,S)-3-hydroxybutyrate
(69) 2 mM MgCl.sub.2
(70) 0.1 mM DTT
(71) 0-3.2 mM ATP or ADP
(72) 0.1 mg/ml of purified enzyme
(73) 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG), purine nucleoside phosphorylase (PNP) and inorganic pyrophosphatase were added to the reaction mix according to the procedure described in the EnzCheck Pyrophosphatase Assay Kit. A reaction mixture without inorganic pyrophosphatase was used for assays with ADP as co-substrate. Control assays were performed in which either no enzyme was added, or no 3-hydroxybutyrate was added. Each assay was started with the addition of co-substrate (ATP or ADP). Each sample was continuously monitored for the increase of 2-amino-6-mercapto-7-methylpurine at 360 nm on a SpectraMax Plus 384 UV/Vis Microplate Reader (Molecular Devices).
(74) Plots of velocity of 2-amino-6-mercapto-7-methylpurine formation as a function of co-substrates concentration are shown on
Example 8: Mass Spectrometry Analysis of the Enzyme-Catalyzed Adenylation Reaction of Different 3-Hydroxycarboxylates
(75) The studied enzymatic reactions were carried out under the following conditions:
(76) 50 mM Tris-HCl pH 7.5
(77) 2 mM 3-hydroxycarboxylate
(78) 2 mM ATP
(79) 20 mM MgCl.sub.2
(80) 100 mM NaCl
(81) 1 mM DTT
(82) 2 mg/ml purified AMP-dependant synthase/ligase from Burkholderia sp.
(83) Each reaction was started by addition of ATP and incubated for 40 minutes at 37 C. Following incubation reactions mix were analyzed by mass spectrometry (MS) using negative ion mode. Typically, an aliquot of each assay was removed every 15 minutes, centrifuged and transferred into a clean vial. An aliquot of 5 l was then directly injected into mass spectrometer. Detection was performed by a PE SCIEX API 2000 quadrupole spectrometer interfaced to an electrospray ionisation (ESI) source. Mass spectra of the enzymatic reactions using 3-hydroxybutyrate, 3-hydroxyvalerate, 3-hydroxyisovalerate and 3-hydroxypent-4-enoate as substrates are presented in
Example 9: Kinetic Parameters of the Reactions of Adenylation of 3-Hydroxycarboxylates Catalyzed by AMP-Dependent Synthetase/Ligase from Marinobacter aquaeolei
(84) Kinetic parameters were determined by using the spectrophotometric assay described in Example 2. Reaction mixture for the assay of adenylation activity contained:
(85) 100 mM Tris-HCl pH 7.5
(86) 0-10 mM 3-hydroxycarboxylate
(87) 2 mM ATP
(88) 2 mM MgCl.sub.2
(89) 0.1 mM DTT
(90) 0.1 mg/ml of purified AMP-dependent synthetase/ligase from M. aquaeolei
(91) 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG), purine nucleoside phosphorylase (PNP) and inorganic pyrophosphatase were added to the reaction mix according to the procedure described in the EnzCheck Pyrophosphatase Assay Kit. Kinetic parameters for adenylation reaction with different 3-hydroxycarboxylates are shown in Table 2.
(92) TABLE-US-00002 TABLE 2 Substrate K.sub.M, mM k.sub.cat 10.sup.3, s.sup.1 3-hydroxypropionate 5 40 (R,S)-3-hydroxybutyrate 2 30 3-hydroxyisovalerate 10 7 R-3-hydroxyvalerate 4 12 (R,S)-3-hydroxypent-4- 3 23 enoate
Example 10: Analysis of Propylene Production from 3-Hydroxybutyrate by the Combined Action of AMP-Dependent Synthetase/Ligase from Marinobacter aquaeolei and OleC Proteins
(93) The studied enzymes were produced and purified according to the procedure described in Example 1. The studied reaction was carried out under the following conditions
(94) 50 mM Tris-HCl pH 7.5
(95) 50 mM (R,S)-3-hydroxybutyrate
(96) 10 mM ATP
(97) 20 mM MgCl.sub.2
(98) 100 mM NaCl
(99) 1 mM DTT
(100) 2 mg/ml purified AMP-dependent synthetase/ligase from M. aquaeolei
(101) 2 mg/ml purified OleC protein from Shewanella amazonensis or from Chloroflexus aurantiacus
(102) Reaction volume was 0.3 ml.
(103) For the no enzymes control, buffer was used in place of enzymes.
(104) Controls reactions without ATP were realized in parallel.
(105) The reaction mixtures were incubated in 2 ml sealed vials (Interchim) for 18 hours at 37 C. with shaking. Propylene production was analyzed by Gas Chromatography (GC) using Bruker 450-GC gas chromatograph equipped with Flame Ionization Detector (FID). Nitrogen was used as carrier gas with a flow rate of 6 ml/min. Volatile compounds were chromatographically separated on GS-Alumina column (30 m0.53 mm ID) (Agilent) using an isothermal mode at 130 C. The enzymatic reaction product were identified by comparison with standard of propylene (Sigma-Aldrich), the retention time of propylene in these conditions was 1.57 min.
(106) A significant production of propylene from 3-hydroxybutyrate was observed in enzymatic reactions contained AMP-dependent synthetase/ligase from M. aquaeolei and OleC protein (Table 3). No propylene signal was observed in controls reactions described above (
(107) TABLE-US-00003 TABLE 3 Propylene peak area, arbitrary Reaction units Control reaction without enzymes 0 AMP-dependent synthetase/ligase from M. aquaeolei alone 0 AMP-dependent synthetase/ligase from M. aquaeolei + OleC 50.8 protein from S. amazonensis AMP-dependent synthetase/ligase from M. aquaeolei + OleC 3.4 from C. aurantiacus
(108) These data indicated that the two enzymes present in the assay were performing complementarily the two steps of reaction to produce propylene from 3-hydroxybutyrate: transfer of the adenylyl group of ATP to the carboxyl group of 3-hydroxybutyrate followed by combined deadenylation/decarboxylation of the reaction intermediate into propylene.
Example 11: Analysis of Propylene Production from 3-Hydroxybutyrate by the Combined Action of Carboxylic Acid Reductase from Nocardia iowensis and OleC Protein from Shewanella amazonensis
(109) The studied reaction was carried out under the following conditions
(110) 50 mM Tris-HCl pH 7.5
(111) 10 mM (R,S)-3-hydroxybutyrate
(112) 2 mM ATP
(113) 25 mM MgCl.sub.2
(114) 100 mM NaCl
(115) 1 mM DTT
(116) 2 mg/ml purified carboxylic acid reductase from N. iowensis
(117) 2 mg/ml purified OleC protein from S. amazonensis
(118) Reaction volume was 0.3 ml.
(119) For the no enzymes control, buffer was used in place of enzymes.
(120) The reactions were incubated in 2 ml sealed vials (Interchim) for 3 hours at 37 C. and then stopped by 1-minute incubation at 80 C. Propylene production was analyzed according to GC-FID procedure described in Example 10. A significant production of propylene from 3-hydroxybutyrate was observed in enzymatic reactions containing carboxylic acid reductase N. iowensis and OleC protein from S. amazonensis. Propylene peak area was measured to be 16.7 arbitrary units. No propylene signal was observed in the control reaction.
Example 12: Analysis of Propylene Production from 3-Hydroxybutyrate by the Combined Action of AMP-Dependent Synthetase/Ligase from Burkholderia sp and OleC Proteins from Shewanella Genus
(121) The studied reaction was carried out under the following conditions
(122) 50 mM Tris-HCl pH 7.5
(123) 10 mM (R,S)-3-hydroxybutyrate
(124) 2 mM ATP
(125) 25 mM MgCl.sub.2
(126) 100 mM NaCl
(127) 1 mM DTT
(128) 2 mg/ml purified AMP-dependent synthetase/ligase from Burkholderia sp.
(129) 2 mg/ml purified OleC protein from S. amazonensis or S. loihica
(130) Reaction volume was 0.3 ml.
(131) The reactions were incubated in 2 ml sealed vials (Interchim) for 3 hours at 37 C. and then stopped by 1-minute incubation at 80 C.
(132) Propylene production was analyzed according to GC-FID procedure described in Example 10. A significant propylene production was observed in coupled enzyme reactions (
Example 13: Analysis of 1-Butene Production from 3-Hydroxyvalerate by the Combined Action of AMP-Dependent Synthetase/Ligase from Marinobacter aquaeolei and the OleC Protein from Shewanella amazonensis
(133) The studied reaction was carried out under the following conditions:
(134) 50 mM Tris-HCl pH 7.5
(135) 10 mM R-3-hydroxyvalerate
(136) 4 mM ATP
(137) 20 mM MgCl.sub.2
(138) 100 mM NaCl
(139) 1 mM DTT
(140) 2 mg/ml purified AMP-dependent synthetase and ligase from M. aquaeolei
(141) 2 mg/ml purified OleC protein from S. amazonensis
(142) Reaction volume was 0.3 ml.
(143) For the no enzymes control, buffer was used in place of enzymes.
(144) The assays were incubated in 2 ml sealed vials (Interchim) for 16 hours at 37 C. with shaking.
(145) 1-Butene production was then analyzed by Gas Chromatography (GC) using Bruker 450-GC gas chromatograph equipped with Flame Ionization Detector (FID). Nitrogen was used as carrier gas with a flow rate of 6 ml/min. Volatile compounds were chromatographically separated on GS-Alumina column (30 m0.53 mm ID) (Agilent) using an isothermal mode at 130 C. The enzymatic reaction product were identified by comparison with standard of 1-butene (Sigma-Aldrich), the retention time of 1-butene in these conditions was 2.65 min.
(146) A significant production of 1-butene from 3-hydroxyvalerate was observed in the enzymatic reaction, containing the AMP-dependent synthetase/ligase from M. aquaeolei and the OleC protein from S. amazonensis. 1-Butene peak area was measured to be 32 arbitrary units. No 1-butene signal was observed in control reaction without enzyme.
(147) These data indicated that the two enzymes present in the assay were performing complementarily the two steps of reaction to produce 1-butene from 3-hydroxyvalerate: transfer of the adenylyl group of ATP to the carboxyl group of 3-hydroxyvalerate followed by combined deadenylation/decarboxylation of the reaction intermediate into 1-butene.
Example 14: Analysis of 1-Butene Production from 3-Hydroxyvalerate by the Combined Action of Carboxylic Acid Reductase from Nocardia iowensis and OleC Protein from Shewanella amazonensis
(148) The studied reaction was carried out under the following conditions:
(149) 50 mM Tris-HCl pH 7.5
(150) 10 mM R-3-hydroxyvalerate
(151) 2 mM ATP
(152) 25 mM MgCl.sub.2
(153) 100 mM NaCl
(154) 1 mM DTT
(155) 2 mg/ml purified carboxylic acid reductase from N. iowensis
(156) 2 mg/ml purified OleC protein from S. amazonensis
(157) Reaction volume was 0.3 ml.
(158) For the no enzymes control, buffer was used in place of enzymes.
(159) The reactions were incubated in 2 ml sealed vials (Interchim) for 3 hour at 37 C. and then stopped by 1-minute incubation at 80 C.
(160) 1-Butene production was then analyzed according to GC-FID procedure described in Example 13. Chromatograms of enzyme-catalyzed reaction and control reaction are shown on
Example 15: Analysis of 1-Butene Production from 3-Hydroxyvalerate by the Combined Action of AMP-Dependent Synthetase/Ligase from Burkholderia sp and OleC Proteins from Shewanella Phylum
(161) The studied reaction was carried out under the following conditions
(162) 50 mM Tris-HCl pH 7.5
(163) 10 mM R-3-hydroxyvalerate
(164) 2 mM ATP
(165) 25 mM MgCl.sub.2
(166) 100 mM NaCl
(167) 1 mM DTT
(168) 2 mg/ml purified AMP-dependent synthetase/ligase from Burkholderia sp.
(169) 2 mg/ml purified OleC protein from S. amazonensis or S. loihica
(170) Reaction volume was 0.3 ml. The assays were incubated as described in Example 14 and analyzed according to GC-FID procedure described in Example 13.
(171) TABLE-US-00004 TABLE 4 1-butene peak area, Reaction arbitrary units Control reaction without enzymes 1 Reaction containing AMP-dependent 50 synthetase/ligase from Burkholderia sp.and OleC from S. amazonensis Reaction containing AMP-dependent 51 synthetase/ligase from Burkholderia sp. and OleC from S. loihica
Example 16: Analysis of 1,3-Butadiene Production from 3-Hydroxypent-4-Enoate by the Combined Action of AMP-Dependent Synthetase/Ligase from Marinobacter aquaeolei and OleC Proteins
(172) The studied reaction was carried out under the following conditions
(173) 50 mM Tris-HCl pH 7.5
(174) 50 mM (R,S)-3-hydroxypent-4-enoate
(175) 10 mM ATP
(176) 20 mM MgCl.sub.2
(177) 100 mM NaCl
(178) 1 mM DTT
(179) Reaction volume was 0.3 ml
(180) 0.6 mg of AMP-dependent synthetase/ligase from M. aquaeolei and 0.6 mg of OleC protein were added to 0.3 ml of reaction mixture. A reaction mix containing only 0.6 mg of AMP-dependent synthetase/ligase from M. aquaeolei was used as reference.
(181) The reactions were incubated in 2 ml sealed vials (Interchim) for 18 hours at 37 C. with shaking. 1,3-butadiene production was analyzed by Gas Chromatography (GC) using Bruker 450-GC gas chromatograph equipped with Flame Ionization Detector (FID). Nitrogen was used as carrier gas with a flow rate of 6 ml/min. Volatile compounds were chromatographically separated on GS-Alumina column (30 m0.53 mm ID) (Agilent) using an isothermal mode at 130 C. The enzymatic reaction product were identified by comparison with standard of 1,3-butadiene (Sigma-Aldrich), the retention time of 1,3-butadiene in these conditions was 3.22 min.
(182) A significant production of 1,3-butadiene was observed in the enzymatic reactions, containing AMP-dependent synthetase/ligase from M. aquaeolei and OleC protein. A negligible signal of 1,3-butadiene corresponding to the spontaneous decomposition of 3-hydroxypent-4-enoate was observed in control reaction (
(183) These data indicated that the two enzymes present in the assay were performing complementarily the two steps of reaction to produce 1,3-butadiene from 3-hydroxypent-4-enoate: transfer of the adenylyl group of ATP to the carboxyl group of 3-hydroxypent-4-enoate followed by combined deadenylation/decarboxylation of the reaction intermediate into butadiene.
Example 17: Analysis of 1,3-Butadiene Production from 3-Hydroxypent-4-Enoate by Combining Carboxylic Acid Reductase from Nocardia iowensis and OleC Protein from Shewanella amazonensis
(184) The studied reaction was carried out under the following conditions:
(185) 50 mM Tris-HCl pH 7.5
(186) 10 mM (R,S)-3-hydroxypent-4-enoate
(187) 2 mM ATP
(188) 25 mM MgCl.sub.2
(189) 100 mM NaCl
(190) 1 mM DTT
(191) 2 mg/ml purified carboxylic acid reductase from N. iowensis
(192) 2 mg/ml purified OleC protein from S. amazonensis.
(193) Reaction volume was 0.3 ml.
(194) For the no enzymes control, buffer was used in place of enzymes. The reactions were incubated in 2 ml sealed vials (Interchim) for 3 hours at 37 C. and the reactions were stopped by 1-minute incubation at 80 C. 1,3-Butadiene production was analyzed according to GC-FID procedure described in Example 16. A significant quantity of butadiene was produced in the enzymatic reaction. A background level of butadiene was observed in the enzyme-free control reaction due to the spontaneous decomposition of 3-hydroxypent-4-enoate (
Example 18: Analysis of 1,3-Butadiene Production from 3-Hydroxypent-4-Enoate by Combining Action of AMP-Dependent Synthetase/Ligase from Burkholderia Sp and OleC Proteins from Shewanella Phylum
(195) The studied reaction was carried out under the following conditions
(196) 50 mM Tris-HCl pH 7.5
(197) 10 mM (R,S)-3-hydroxypent-4-enoate
(198) 2 mM ATP
(199) 25 mM MgCl.sub.2
(200) 100 mM NaCl
(201) 1 mM DTT
(202) 2 mg/ml purified AMP-dependent synthetase/ligase from Burkholderia sp
(203) 2 mg/ml purified OleC protein from S. amazonensis or S. loihica
(204) Reaction volume was 0.3 ml. The assays were incubated and analyzed according to the procedure described in Example 16. A significant production of 1,3-butadiene was observed in coupled enzymatic reactions, a negligible signal of butadiene was observed in control reaction without enzymes due to the spontaneous decomposition of 3-hydroxypent-4-enoate (Table 5).
(205) TABLE-US-00005 TABLE 5 1,3-butadiene peak Reaction area, arbitrary units Control reaction without enzymes 10 Reaction containing AMP-dependent 559 synthetase/ligase from Burkholderia sp. and OleC from S. amazonensis Reaction containing AMP-dependent 1550 synthetase/ligase from Burkholderia sp.and OleC from S. loihica
Example 19: Analysis of Isobutene Production from 3-Hydroxyisovalerate by the Combined Action of AMP-Dependent Synthetase/Ligase from Marinobacter aquaeolei and OleC Proteins
(206) The studied reaction was carried out under the following conditions
(207) 50 mM Tris-HCl pH 7.5
(208) 50 mM 3-hydroxyisovalerate
(209) 10 mM ATP
(210) 20 mM MgCl.sub.2
(211) 100 mM NaCl
(212) 1 mM DTT
(213) Reaction volume was 0.3 ml
(214) 0.6 mg of AMP-dependent synthetase/ligase from M. aquaeolei and 0.6 mg of OleC protein were added to 0.3 ml of reaction mixture. A reaction mixture containing only 0.6 mg of AMP-dependent synthetase/ligase from M. aquaeolei was used as reference. The assays were incubated in 2 ml sealed vials (Interchim) for 18 hours at 37 C. with shaking. Isobutene production was analyzed by Gas Chromatography (GC) using Bruker 450-GC gas chromatograph equipped with Flame Ionization Detector (FID). Nitrogen was used as carrier gas with a flow rate of 6 ml/min. Volatile compounds were chromatographically separated on GS-Alumina column (30 m0.53 mm ID) (Agilent) using an isothermal mode at 130 C. The enzymatic reaction product were identified by comparison with standard of isobutene (Sigma-Aldrich), the retention time of isobutene in these conditions was 2.40 min.
(215) A significant production of Isobutene was observed in combined enzymatic reactions, contained the AMP-dependent synthetase/ligase from M. aquaeolei and OleC protein (Table 6). A negligible signal of isobutene corresponding to spontaneous decomposition of 3-hydroxyisovalerate was observed in control assay without enzyme.
(216) TABLE-US-00006 TABLE 6 Isobutene peak area, Reaction arbitrary units Control reaction without enzymes 341 Reaction containing AMP-dependent 414 synthetase/ligase from. M. aquaeolei alone Reaction containing AMP-dependent 113259 synthetase/ligase from M. aquaeolei and OleC from S. amazonensis Reaction containing AMP-dependent 4824 synthetase/ligase from M. aquaeolei and OleC from C. aurantiacus Reaction containing AMP-dependent 1419 synthetase/ligase from M. aquaeolei and OleC from S. maltophilia
(217) An example of chromatogram obtained for the coupled reaction with enzyme from M. aquaeolei and OleC protein from S. amazonensis is shown in
(218) These data indicated that the two enzymes present in the assay were performing complementarily the two steps of reaction to produce isobutene from 3-hydroxyisovalerate: transfer of the adenylyl group of ATP to the carboxyl group of 3-hydroxyisovalerate followed by combined deadenylation/decarboxylation of the reaction intermediate into isobutene.
Example 20: Study of Isobutene Production as a Function of ATP Concentration
(219) The studied reaction was carried out under the following conditions
(220) 50 mM Tris-HCl pH 7.5
(221) 40 mM 3-hydroxyisovalerate
(222) 0-32 mM ATP
(223) 25 mM MgCl.sub.2
(224) 100 mM NaCl
(225) 1 mM DTT
(226) 2 mg/ml purified adenylate-forming enzyme
(227) 2 mg/ml purified OleC protein from S. amazonensis
(228) The reactions mix were incubated in 2 ml sealed vials (Interchim) for 3 hours at 37 C. and the reactions were stopped by 1-minute incubation at 80 C. The isobutene production as function of ATP is shown in
Example 21: Study of Isobutene Production as a Function of ADP Concentration
(229) The studied reactions were performed according to the protocol described in Example 20 using ADP as co-substrate instead of ATP. The isobutene production as function of ADP is shown in