Production of 1,3-dienes by enzymatic conversion of 3-hydroxyalk 4-enoates and/or 3-phosphonoxyalk-4-enoates

Abstract

The present invention relates to a method for generating 1,3-diene compounds through a biological process. More specifically, the invention relates to a method for producing 1,3-diene compounds (for example butadiene or isoprene) from molecules of the 3-hydroxyalk-4-enoate type or from 3-phosphonoxyalk-4-enoates.

Claims

1. A method for the production of a 1,3-diene compound that comprises converting a 3-hydroxyalk-4-enoate with a diphosphomevalonate decarboxylase (EC 4.1.1.33) into a 1,3-diene compound, wherein the 3-hydroxyalk-4-enoate has the general formula of C.sub.n+1H.sub.2nO.sub.3 with 3<n<7 and comprises a 3-hydroxypent-4-enoate as a common motif and optionally a methyl substitution on carbon 3 and carbon 4.

2. The method of claim 1 wherein the 3-hydroxyalk-4-enoate is 3-hydroxypent-4-enoate and the produced 1,3-diene compound is 1,3-butadiene.

3. The method of claim 1 wherein the 3-hydroxyalk-4-enoate is 3-hydroxy-4-methylpent-4-enoate or 3-hydroxy-3-methylpent-4-enoate and the produced 1,3-diene compound is isoprene.

4. The method of claim 1 wherein the diphosphomevalonate decarboxylase comprises the amino acid sequence of SEQ ID NOs: 1 to 19 or 22 to 29.

5. The method of claim 4 wherein the diphosphomevalonate decarboxylase comprises the amino acid sequence of SEQ ID NO: 6, 16, 17, 18 or 19.

6. The method of claim 1, wherein (i) a first diphosphomevalonate decarboxylase converts the 3-hydroxyalk-4-enoate into the corresponding 3-phosphonoxyalk-4-enoate; and (ii) a second diphosphomevalonate decarboxylase being different from the first diphosphomevalonate decarboxylase which converts said 3-phosphonoxyalk-4-enoate into said 1,3-diene compound by a decarboxylation reaction.

7. The method of claim 6 wherein the first diphosphomevalonate decarboxylase is a protein comprising the amino acid sequence as shown in SEQ ID NO: 18.

8. The method of claim 6 wherein the second diphosphomevalonate decarboxylase is a protein comprising the amino acid sequence as shown in SEQ ID NO: 24.

9. A method for the production of a 1,3-diene compound comprising: (i) converting a 3-hydroxyalk-4-enoate into a 3-phosphonoxyalk-4-enoate by a disphosphomevalonate decarboxylase (EC 4.1.1.33); and (ii) converting 3-phosphonoxyalk-4-enoate into a 1,3-diene compound by a terpene synthase; wherein 3-hydroxyalk-4-enoate has the general formula of C.sub.n+1 H.sub.2nO.sub.3 with 3<n<7 and comprises a 3-hydroxypent-4-enoate as common motif and optionally a methyl substitution on carbon 3 and carbon 4.

10. The method of claim 9, wherein said terpene synthase is an isoprene synthase (EC 4.2.3.27).

11. A method for producing a 1,3-diene compound comprising enzymatically converting a 3-phosphonoxyalk-4-enoate into the corresponding 1,3-diene compound by a terpene synthase; wherein 3-hydroxyalk-4-enoate has the general formula of C.sub.n+1 H.sub.2nO.sub.3 with 3<n<7 and comprises a 3-hydroxypent-4-enoate as common motif and optionally a methyl substitution on carbon 3 and carbon 4.

12. The method of claim 11, wherein said terpene synthase is an isoprene synthase (EC 4.2.3.27).

13. The method of claim 11 wherein the 3-phosphonoxyalk-4-enoate is 3-phosphonoxypent-4-enoate and the produced 1,3-diene is 1,3-butadiene.

14. The method of claim 11 wherein the 3-phosphonoxyalk-4-enoate is 3-phosphonoxy-4-methylpent-4-enoate or 3-phosphonoxy-3-methylpent-4-enoate and the produced 1,3-diene is isoprene.

15. The method of claim 1 which is carried out in vitro.

16. The method of claim 1 wherein a co-substrate is added.

17. The method of claim 16 wherein the co-substrate is ATP, a rNTP, a dNTP, a polyphosphate or pyrophosphate, or a mixture of any of these compounds.

18. The method of claim 1 wherein the method is carried out by making use of a microorganism producing said enzyme or said enzymes.

19. The method of claim 18, wherein the microorganism is capable of producing a 3-hydroxyalk-4-enoate and/or a 3-phosphonoxyalk-4-enoate.

20. The method of claim 6 wherein the first diphosphomevalonate decarboxylase is: (A) a protein comprising the amino acid sequence as shown in SEQ ID NO: 16; (B) a protein comprising the amino acid sequence as shown in SEQ ID NO: 17; or (C) a protein comprising the amino acid sequence as shown in SEQ ID NO: 19.

21. The method of claim 6 wherein the second diphosphomevalonate decarboxylase is: (A) a protein comprising the amino acid sequence as shown in SEQ ID NO: 12; (B) a protein comprising the amino acid sequence as shown in SEQ ID NO: 22; (C) a protein comprising the amino acid sequence as shown in SEQ ID NO: 23; (D) a protein comprising the amino acid sequence as shown in SEQ ID NO: 1; (E) a protein comprising the amino acid sequence as shown in SEQ ID NO: 7; (F) a protein comprising the amino acid sequence as shown in SEQ ID NO: 25; (G) a protein comprising the amino acid sequence as shown in SEQ ID NO: 26; (H) a protein comprising the amino acid sequence as shown in SEQ ID NO: 27; (I) a protein comprising the amino acid sequence as shown in SEQ ID NO: 28; or (J) a protein comprising the amino acid sequence as shown in SEQ ID NO: 29.

22. The method of claim 9, wherein said terpene synthase is a monoterpene synthase, an alpha-farnesene synthases (EC 4.2.3.46), a beta-farnesene synthase (EC 4.2.3.47), a myrcene/(E)-beta-ocimene synthase (EC 4.2.3.15) or a pinene synthase (EC 4.2.3.14).

23. The method of claim 11, wherein said terpene synthase is a monoterpene synthase, an alpha-farnesene synthases (EC 4.2.3.46), a beta-farnesene synthase (EC 4.2.3.47), a myrcene/(E)-beta-ocimene synthase (EC 4.2.3.15) or a pinene synthase (EC 4.2.3.14).

24. The method of claim 9 wherein the diphosphomevalonate decarboxylase comprises the amino acid sequence of SEQ ID NOs: 1 to 19 or 22 to 29.

Description

(1) FIG. 1 shows the reaction catalyzed by a diphosphomevalonate decarboxylase using diphosphomevalonate (A) or using a 3-hydroxyalk-4-enoate (B) as a substrate.

(2) FIG. 2 shows the reaction catalyzed by a diphosphomevalonate decarboxylase using 3-hydroxypent-4-enoate as a substrate leading to the production of 1,3-butadiene.

(3) FIG. 3 shows the reaction catalyzed by a diphosphomevalonate decarboxylase using 3-hydroxy-4-methylpent-4-enoate or 3-hydroxy-3 methylpent-4-enoate as a substrate leading to the production of isoprene.

(4) FIG. 4 shows a scheme of the ADP quantification assay, monitoring NADH consumption by the decrease of absorbance at 340 nm.

(5) FIG. 5 shows a mass spectrum of the enzymatic assay for 3-hydroxypent-4-enoate phosphorylation.

(6) FIG. 6 shows a mass spectrum of the enzyme-free control assay for 3-hydroxypent-4-enoate phosphorylation.

(7) FIG. 7 shows the plot of the velocity as a function of substrate concentration for the phosphotransferase reaction catalyzed by the mutant L200E of MDP decarboxylase from Th. acidophilum. Initial rates were computed from the kinetics over the 30 first minutes of the reaction.

(8) FIG. 8 shows the production of 1,3-butadiene from 3-phosphonoxypent-4-enoate in the absence and presence of isoprene synthase from Pueraria montana var. lobata.

(9) 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.

EXAMPLES

Example 1

Cloning, Expression and Purification of Enzymes

(10) Cloning, Bacterial Cultures and Expression of Proteins.

(11) The genes encoding studied enzymes were cloned in the pET 25b or pET 22b vectors (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 these vectors 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 28 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 315 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

Characterization of the 3-hydroxypent-4-enoate Phosphorylation Activity

(14) The release of ADP that is associated with 1,3-butadiene production from 3-hydroxypent-4-enoate, was quantified using the pyruvate kinase/lactate dehydrogenase coupled assay (FIG. 4). The purified mevalonate diphosphate decarboxylases from Th. acidophilum, Th. volcanium and S. mitis were evaluated for their ability to phosphorylate 3-hydroxypent-4-enoate releasing ADP.

(15) The studied enzymatic reaction was carried out under the following conditions at 37 C.: 50 mM Tris-HCl pH 7.5 10 mM MgCl.sub.2 100 mM KCl 5 mM ATP 0.4 mM NADH 1 mM Phosphoenolpyruvate 3 U/ml Lactate dehydrogenase 1.5 U/ml Pyruvate kinase 50 mM 3-Hydroxypent-4-enoate
The pH was adjusted to 7.5

(16) Each assay was started by the addition of a particular enzyme (at a concentration from 0.05 to 1 mg/ml) and the disappearance of NADH was monitored by following the absorbance at 340 nm. Assays with mevalonate diphosphate (MDP) decarboxylases gave rise to a reproducible increase in ADP production in the presence of 3-hydroxypent-4-enoate. The enzymes from the Thermoplasma phylum displayed higher phosphotransferase activity than decarboxylase from S. mitis (Table 3).

(17) TABLE-US-00003 TABLE 3 Mevalonate diphosphate decarboxylase Activity, nmol/min/mg protein Th. acidophilum (mutant L200E) 138 Th. volcanium 114 S. mitis 0.52

(18) Mass spectrometry was then applied to confirm the formation of 3-phosphonoxypent-4-enoate in the assay with mutant L200E of MDP decarboxylase from Th. acidophilum.

Example 3

Mass Spectrometry Analysis of the Phosphorylation Reaction

(19) The desired enzymatic reactions were carried out under the following conditions: 50 mM Tris-HCl pH 7.5 10 mM MgCl.sub.2 10mM KCl 20 mM 3-hydroxypent-4-enoate 20 mM ATP 0.2 mg/ml purified MDP decarboxylase from Th. acidophilum (mutant L200E).

(20) The control reactions without enzyme, without substrate and without ATP were run in parallel. The assays were incubated overnight without shaking at 37 C. Typically, an aliquot of 200 l reaction was removed, centrifuged and the supernatant was transferred into a clean vial. The MS spectra were obtained on Esquire 3000 (Bruker) Ion Trap Mass Spectrometer with Electrospray Ionization Interface in negative ion mode. The presence of 3-phosphonoxypent-4-enoate was evaluated. MS analysis showed an [M-H].sup. ion at m/z 194.9 corresponding to 3-phosphonoxypent-4-enoate from the sample containing the enzyme but not from the negative controls (FIGS. 5, 6).

Example 4

Kinetic Parameters of Reaction of 3-hydroxypent-4-enoate Phosphorylation

(21) Kinetic parameters were determined by varying substrate concentration between 0 and 30 mM under assay conditions, described in example 2.

(22) FIG. 7 shows an example of a Michaelis-Menten plot corresponding to the data collected for the mutant L200E of MDP decarboxylase from Th. acidophilum. This enzyme was found to have a K.sub.M of 3.7 mM and a k.sub.cat of 0.09 sec.sup.1.

Example 5

Butadiene production from 3-hydroxypent-4-enoate

(23) The desired enzymatic reaction was carried out under the following conditions: 50 mM Tris HCl pH 7.5 10 mM MgCl.sub.2 20 mM KCl 50 mM ATP 200 mM 3-hydroxypent-4-enoate
The pH was adjusted to 7.5

(24) Each assay was started by the addition of a particular enzyme to 0.5 ml of reaction mixture. The assays were then incubated with shaking at 37 C. in a 2 ml sealed vial (Interchim). Control reactions were run in parallel. After 48 hours of incubation the reaction mixtures were analyzed as follows. To 0.5 mL of each sample, 0.125 ml of heptane were added and the sample was incubated at 25 C. for 1 hour with shaking. The upper heptane phase was analyzed by gas chromatography (GC) on a Varian 430-GC chromatograph equipped with a FID detector. A 1 L sample was separated on the GC using an Rt-Alumina BOND/Na.sub.2SO.sub.4 column (Restek) and nitrogen carrier gas. The oven cycle for each sample was 130 C. for 10 minutes, increasing temperature at 20 C./minute to a temperature of 200 C., and a hold at 200 C. for 10 minutes. The total run time was 23.5 minutes The enzymatic reaction product was identified by comparison with commercial 1,3-butadiene (Sigma). The results of butadiene production are presented in Table 4.

(25) TABLE-US-00004 TABLE 4 1,3-Butadiene peak area, Assay arbitrary units Without substrate 0 Without enzyme 0.8 With 11 mg/ml of purified 1.0 S. mitis MDP decarboxylase Combining assay with 1 mg/ml 1.6 Th. acidophilum enzyme (mutant L200E) and 10 mg/ml S. mitis enzyme

(26) The formation of 1,3-butadiene observed in the assay without enzyme is probably due to the spontaneous decomposition of 3-hydroxypent-4-enoate. The addition of MDP decarboxylase from S. mitis led to a 1.25-fold increase of butadiene production after 48 h of incubation. The highest production of isobutene was observed in the assay combining the MDP decarboxylase from Th. acidophilum and the MDP decarboxylase from S. mitis. This indicated that the two enzymes present in the assay were performing complementarily the two steps of reaction producing butadiene from 3-hydroxypent-4-enoate: transfer of the terminal phosphoryl group from ATP to the C3-oxygen of 3-hydroxypent-4-enoate followed by combined dephosphorylation-decarboxylation of the intermediate 3-phosphonoxypent-4-enoate.

Example 6

Butadiene Production from 3-phosphonoxypent-4-enoate

(27) 3-phosphonoxypent-4-enoate is synthesized by company specialized in custom synthesis, Syntheval (France).

(28) The studied enzymatic reactions are carried out under the following conditions at 37 C.: 50 mM Tris-HCl pH 7.5 10 mM MgCl.sub.2 0-20 mM KCl 5 mM ATP 0-250 mM 3-phosphonoxypent-4-enoate
The pH is adjusted to 7.5

(29) The reaction is initiated by the addition of a particular enzyme to 0.5 ml of reaction mixture. The free-enzyme control reactions are carried out in parallel. The assays are incubated with shaking at 37 C. in a 2 ml sealed vial (Interchim). The production of butadiene is measured by analyzing aliquots sampled over a 72 hour incubation period. Volatile compounds in the headspace of reaction mixture are collected and directly injected into a Varian 430-GC chromatograph equipped with a flame ionization detector and an Rt-Alumina BOND/Na.sub.2SO.sub.4 column (Restek). Additionally, 1,3-butadiene production is monitored by analysis of reaction mixture using gas chromatography as described in example 5. Commercial 1,3-butadiene is used as reference.

Example 7

Characterization of the 3-hydroxy-3-methylpent-4-enoate Activity

(30) The release of ADP associated with isoprene production from 3-hydroxy-3-methylpent-4-enoate is quantified using the pyruvate kinase/lactate dehydrogenase coupled assay (FIG. 4). The purified mevalonate diphosphate decarboxylases are evaluated for their ability to phosphorylate this substrate releasing ADP.

(31) The studied enzymatic reactions are carried out under the following conditions at 37 C.: 50 mM Tris-HCl pH 7.5 10 mM MgCl.sub.2 100 mM KCl 5 mM ATP 0.4 mM NADH 1 mM Phosphoenolpyruvate 3 U/ml Lactate dehydrogenase 1.5 U/ml Pyruvate kinase 50 mM 3-Hydroxy-3-methylpent-4-enoate

(32) Each assay is started by the addition of a particular enzyme (at a concentration from 0.05 to 1 mg/ml) and the disappearance of NADH is monitored by following the absorbance at 340 nm.

Example 8

Characterization of the 3-hydroxy-4-methylpent-4-enoate Phosphorylation Activity

(33) The release of ADP associated with isoprene production from 3-hydroxy-4-methylpent-4-enoate is quantified using the pyruvate kinase/lactate dehydrogenase coupled assay (FIG. 4). The purified mevalonate diphosphate decarboxylases are evaluated for their ability to phosphorylate this substrate releasing ADP.

(34) The studied enzymatic reactions are carried out under the following conditions at 37 C.: 50 mM Tris-HCl pH 7,5 10 mM MgCl.sub.2 100 mM KCl 5 mM ATP 0.4 mM NADH 1 mM Phosphoenolpyruvate 3 U/ml Lactate dehydrogenase 1.5 U/ml Pyruvate kinase 50 mM 3-Hydroxy-4-methylpent-4-enoate

(35) Each assay is started by the addition of a particular enzyme (at a concentration from 0.05 to 1 mg/ml) and the disappearance of NADH is monitored by following the absorbance at 340 nm.

Example 9

Isoprene Production from 3-hydroxy-3-methylpent-4-enoate

(36) The studied enzymatic reactions are carried out under the following conditions at 37 C.: 50 mM Tris-HCl pH 7.5 10 mM MgCl.sub.2 20 mM KCl 5 mM ATP 0-200 mM 3-hydroxy-3-methylpent-4-enoate
The pH is adjusted to 7.5

(37) The reaction is initiated by the addition of one or two particular enzyme(s) to 0.5 ml of reaction mixture. The enzyme-free control reactions are carried out in parallel. The assays are incubated with shaking at 37 C. in a 2 ml sealed vial (Interchim). The gas present in the headspace is collected and analyzed by gas chromatography coupled with a flame ionization detector. The enzymatic reaction product is identified by comparison with commercial isoprene (Sigma).

Example 10

Isoprene Production from 3-hydroxy-4-methylpent-4-enoate

(38) The studied enzymatic reactions are carried out under the following conditions at 37 C.: 50 mM Tris-HCl pH 7.5 10 mM MgCl.sub.2 20 mM KCl 5 mM ATP 0-200 mM 3-hydroxy-4-methylpent-4-enoate
The pH is adjusted to 7.5

(39) The reaction is initiated by the addition of one or two particular enzymes to 0.5 ml of reaction mixture. The enzyme-free control reactions are carried out in parallel. The assays are incubated with shaking at 37 C. in a 2 ml sealed vial (Interchim). The gas present in the headspace is collected and analyzed by gas chromatography coupled with a flame ionization detector. The enzymatic reaction product is identified by comparison with commercial isoprene (Sigma).

Example 11

1,3-butadiene Production from 3-phosphonoxypent-4-enoate by Using a Terpene Synthase

(40) The sequence of the isoprene synthase inferred from the genome from Pueraria montana var. lobata (Uniprot Q6EJ97) was generated by oligonucleotide concatenation to fit the codon usage of E. coli. The amino acid sequence of the enzyme is shown in SEQ ID NO: 30. 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 pET 25b(+) expression vector (the vector was constructed by GeneArt AG). The corresponding enzyme was expressed in E. coli and purified as described in Example 1.

(41) The reactions were performed in sealed vials. The total volume was 0.5 ml. Final concentrations were 5 mg/ml enzyme, 50 mM 3-phosphonoxypent-4-enoate, 4 mM DTT, 50 mM MgCl.sub.2, 50 mM KCl, 50 mM Tris-HCl buffer pH 7.5. The incubation was carried out at 37 C. for 24 h. The control reactions without enzyme or without substrate were performed in parallel under the same conditions.

(42) One ml of the gaseous phase of the reaction was collected and analyzed by Gas-Chromatography with Flame Ionization Detector (GC-FID) ((Brucker GC 450) using a RTX-alumina column (Varian), with an isocratic elution at 130 C. and nitrogen as carrier gas at flow rate of 1.5 ml/min. The retention of commercial 1,3-butadiene (Sigma) in these conditions was 7.4 min.

(43) Results: No formation of 1,3-butadiene was observed without substrate. The GC analysis of reactions without enzyme and with non-relevant enzyme showed only traces of butadiene resulted from the thermal decomposition of the 3-phosphonoxypent-4-enoate. The catalytic tests showed a significant increase of butadiene production in the presence of purified isoprene synthase from Pueraria montana var. lobata. The ratio of butadiene produced after 24 hours incubation in the presence of isoprene synthase versus butadiene produced in the absence of enzyme is about 5 fold judging from butadiene peak areas (FIG. 8). These results clearly indicate that a terpene synthase such as isoprene synthase catalyzes the conversion of a 3-phosphonoxyalk-4-enoate to a 1,3-diene, in particular 3-phosphonoxypent-4-enoate to 1,3-butadiene.