Process for the production of isoprenol from mevalonate employing a diphosphomevalonate decarboxylase

Abstract

Described is a method for the enzymatic production of isoprenol using mevalonate as a substrate and enzymatically converting it by a decarboxylation step into isoprenol as well as the use of an enzyme which is capable of catalyzing the decarboxylation of mevalonate for the production of isoprenol from mevalonate. Furthermore described is the use of mevalonate as a starting material for the production of isoprenol in an enzymatically catalyzed reaction. Also disclosed is a method for the production of isoprene comprising the method for the production of isoprenol using mevalonate as a substrate and enzymatically converting it by a decarboxylation step into isoprenol and further comprising the step of converting the produced isoprenol into isoprene as well as a method for the production of isoamyl alcohol comprising the method for the production of isoprenol using mevalonate as a substrate and enzymatically converting it by a decarboxylation step into isoprenol and further comprising the step of converting the produced isoprenol into isoamyl alcohol.

Claims

1. A method of producing isoprenol comprising: converting mevalonate into isoprenol by the enzyme of SEQ ID NO:2.

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

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

4. The method of claim 1 wherein ATP, a rNTP, a dNTP, a polyphosphate or pyrophosphate, or a mixture of any of these compounds is added.

5. The method of claim 1 further comprising culturing a recombinant microorganism or a plant cell overexpressing the enzyme to convert mevalonate into isoprenol.

6. The method of claim 5, wherein the recombinant microorganism is a bacterium, a fungus, a yeast or a microalgae.

7. The method of claim 5, wherein the method is carried out in a plant cell.

8. The method of claim 5, wherein the recombinant microorganism or plant cell has been genetically modified with a heterologous polynucleotide encoding the enzyme.

9. The method of claim 5, wherein the recombinant microorganism or plant cell has been genetically modified with a promoter that overexpresses the enzyme.

10. The method of claim 1 further comprising recovering isoprenol.

11. The method of claim 1 further comprising converting the isoprenol into isoprene.

12. The method of claim 11 further comprising recovering isoprene.

13. The method of claim 1 further comprising converting the isoprenol into isoamyl alcohol.

14. The method of claim 13 further comprising recovering isoamyl alcohol.

15. A method of producing isoprenol comprising converting mevalonate into isoprenol by an enzyme at least 95% identical to the amino acid sequence of SEQ ID NO: 2, wherein the enzyme accepts mevalonate as a substrate and catalyzes a decarboxylation reaction to produce isoprenol.

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

17. The method of claim 15 wherein ATP, a rNTP, a dNTP, a polyphosphate or pyrophosphate, or a mixture of any of these compounds is added.

18. The method of claim 15 further comprising culturing a recombinant microorganism or a plant cell overexpressing the enzyme to convert mevalonate into isoprenol.

19. The method of claim 18, wherein the recombinant microorganism is a bacterium, a fungus, a yeast or a microalgae.

20. The method of claim 18, wherein the method is carried out in a plant cell.

21. The method of claim 18, wherein the recombinant microorganism or plant cell has been genetically modified with a heterologous polynucleotide encoding the enzyme.

22. The method of claim 18, wherein the recombinant microorganism or plant cell has been genetically modified with a promoter that overexpresses the enzyme.

23. The method of claim 15 further comprising recovering isoprenol.

24. The method of claim 15 further comprising converting the isoprenol into isoprene.

25. The method of claim 24 further comprising recovering isoprene.

26. The method of claim 15 further comprising converting the isoprenol into isoamyl alcohol.

27. The method of claim 26 further comprising recovering isoamyl alcohol.

Description

(1) FIG. 1: shows the chemical structure of mevalonate

(2) FIG. 2: shows the reaction of diphosphomevalonate decarboxylase on the physiological substrate diphosphomevalonate and on the precursor mevalonate

(3) FIG. 3: shows an example of screening of enzyme library for mevalonate decarboxylase activity by following inorganic phosphate production. The control reaction was carried out with extract of E. coli BL21(DE3) transformed with pET 22b lacking MDP decarboxylase gene.

(4) FIG. 4: FIG. 4(a) shows the results of optimisation of P. torridus MDP decarboxylase expression in E. coli. SDS-PAGE analysis of samples of proteins obtained from the expression of native P. torridus MDP decarboxylase DNA sequence (lanes 1 to 3) and of optimized gene (lanes 4 to 6). FIG. 4(b) shows Mevalonate decarboxylation activity of crude lysate of E. coli obtained from the expression of native P. torridus MDP decarboxylase DNA sequence and of optimized gene. Control reaction was carried out with extract of E. coli BL21(DE3) transformed with pET 22b lacking MDP decarboxylase gene. The enzyme activity was detected via inorganic phosphate production measurement.

(5) FIG. 5: shows the comparison of mevalonate decarboxylation activity among MDP decarboxylases from the Picrophilus/Thermoplasma phylum. The enzyme activity was detected by means of inorganic phosphate production measurement.

(6) FIG. 6: shows product formation as function of mevalonate concentration. The product formation was followed by permanganate assay.

(7) FIG. 7: shows the structure of phosphono-phosphate and phosphonamido-phosphate.

(8) The following Examples serve to illustrate the invention.

Example 1: Screening of a Library of MDP Decarboxylase for Mevalonate Decarboxylation Activity

(9) A library of 63 genes encoding enzymes of the MDP decarboxylase family was constructed and tested for activity on mevalonate as substrate.

(10) Cloning, Bacterial Cultures and Expression of Proteins

(11) The genes encoding mevalonate diphosphate (MDP) decarboxylase EC 4.1.1.33 were cloned in the pET 25b vector (Novagen) in the case of eukaryotic genes and in pET 22b (Novagen) in the case of prokaryotic genes. A stretch of 6 histidine codons (6 histidine disclosed as SEQ ID NO: 22) 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) at 30 C. in terrific broth (TB) medium containing 0.5 M sorbitol, 5 mM betain, 100 g/ml ampicillin until reaching an OD at 600 nm comprised between 0.8 and 1. Isopropyl-B-D-thiogalactopyranoside (IPTG) was then added to a final concentration of 1 mM and protein expression was continued at 20 C. overnight (approximately 16 h). The cells were collected by centrifugation at 4 C., 10,000 rpm for 20 min and the pellets were frozen at 80 C.

(12) Cell Lysis

(13) The pellets from 12 ml of culture cells were thawed on ice and resuspended in 1 ml of 50 mM Tris/HCl pH 7.4, containing 20 mM KCl, 0.5 mM DTT, 5 mM MgCl.sub.2. One microliter of lysonase (Novagen) was added. Cells were incubated for 10 minutes at room temperature and then returned to ice for 20 minutes. Cell lysis was completed by sonication for 15 seconds. The bacterial extracts were then clarified by centrifugation at 4 C., 10,000 rpm for 20 min.

(14) Enzymatic Reactions

(15) The desired enzymatic reaction (conversion of mevalonate into isoprenol) was tested as follows.

(16) The reaction medium contained 100 mM mevalonate, 40 mM ATP, 10 mM MgCl.sub.2, 20 mM KCl, 0.5 mM DTT and enzyme preparation varying from 0.01 to 0.05 mg/ml of protein. 50 mM sodium citrate was used in the range of pH from 4 to 6, and 50 mM Tris-HCl for pH 7 and 7.5. Enzyme-free control assays were carried out in parallel. After 72 h incubation, inorganic phosphate was quantified colorimetrically according to the ammonium molybdate method (Gawronski J D, Benson D R, Anal. Biochem. 327 (2004) 114-118). A 50 l sample (containing not more than 0.5 mole of phosphate) was mixed with 150 l of ammonium molybdate reagent containing 50% v/v acetone, 1.25 N H.sub.2SO.sub.4, 2.5 mM (NH.sub.4).sub.6Mo.sub.7O.sub.24 and then with 10 l 1 M citric acid. The mixture was incubated for 2 minutes at room temperature. The absorbance of ammonium phosphomolybdate formed was measured at 355 nm and the quantity of inorganic phosphate estimated using a calibration curve obtained with potassium phosphate.

(17) The results are shown in FIG. 3.

(18) During the initial screening, only assays using the recombinant strain expressing the genetic construct inferred from Picrophilus torridus MDP decarboxylase sequence gave rise to a reproducible increase in phosphate production over the background level.

Example 2: Optimisation of P. torridus MDP Decarboxylase Expression in E. Coli

(19) The initial level of enzyme expression in E. coli BL21 was low, as judged from the faint band visible on SDS-PAGE gels. The Codon Optimization Index (CAI) of the native sequence for expression in E. coli measured with the Optimizer program available at http://genomes.urv.es/OPTIMIZER/, as based on the method of Sharp and Li (Nucl. Acids Res. 15 (1987), 1281-1295) gave a value as low as 0.23.

(20) A gene sequence coding for an identical protein but containing codons better adapted for expression in E. coli was generated. It featured a CAI of 0.77.

(21) The native sequence and the optimized sequence are shown in SEQ ID NO: 20 (native sequence of P. torridus (AAT43941) MDP decarboxylase including the His-tag) and SEQ ID NO: 21 (optimized sequence of P. torridus (AAT43941) MDP decarboxylase including the His-tag). The optimized sequence was synthesized by oligonucleotide concatenation and cloned in a pET25b expression vector. After transformation of E. coli strain BL21(DE3) and induction, the proteins were produced and analyzed on a gel as described according to the protocol described in Example 1. The same protocol was carried out with the native sequence for comparison.

(22) Expression levels using either the native nucleotide sequence or the sequence optimized for expression in E. coli were compared. The results in FIG. 4a show that the protein (arrow) corresponding to the optimized gene was clearly visible on the gel in the non-purified cell lysate (lane 4), which indicates a very notable increase in expression.

(23) The expression of the protein was improved such that the crude lysate obtained with the optimized sequence contained a higher enzyme activity with mevalonate as substrate, as shown in FIG. 4b.

Example 3: Characterization of the Reaction Using the Optimized P. torridus MDP Decarboxylase

(24) The recombinant enzyme was purified as follows:

(25) Protein Purification and Concentration

(26) The pellets from 150 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) was 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-IDA column (Macherey-Nagel) allowing adsorption of 6-His tagged proteins (6-His disclosed as SEQ ID NO: 22) 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 250 l 50 mM Tris-HCl pH 7.4 containing 0.5 mM DTT and 5 mM MgCl.sub.2. Protein concentrations were quantified according to the Bradford method.

(27) The purity of proteins thus purified was estimated as approximately 90%.

(28) The activity of the enzyme was confirmed and further analyzed using a range substrate: The conversion rate was shown to increase with the concentration of mevalonate (FIG. 6).

Example 4: Optimization of Reaction Conditions by Using a Cofactor

(29) The same reaction as that described in Example 1 is carried out using purified preparations of optimized P. torridus MDP decarboxylase. In one of the samples, the phosphono-phosphate or phosphonamido-phosphate (FIG. 7) is added as cofactor at the concentration of 100 mM. The conversion of mevalonate is observed using the colorimetric assay described in Example 1. It is found that when a cofactor is present, the amount of ATP consumed over time is markedly higher.

Example 5: Screening of a Library of MDP Decarboxylase Homologs from the P. torridus Phylum

(30) Sequence of MDP decarboxylase enzymes inferred from the genomes of Thermoplasma volcanium (accession number Q97BY2) and Thermoplasma acidophilum (accession number Q9H1N1) were generated as in Example 1. Proteins were purified as described in Example 3 and assayed using the assay described in Example 1. A significant increase in phosphate production was observed from these vials, indicating that these enzymes were also active toward mevalonate. Results are shown in FIG. 5.

Example 6: Method for Synthesizing Isoprenol from Glucose

(31) E. coli K12 is transformed with an expression plasmid, carrying the genes of thiolase, HMG-CoA synthase and HMG-CoA reductase from Saccharomyces cerevisiae in order to overproduce mevalonate.

(32) The strain is further transformed with a second, compatible expression plasmid carrying the optimized gene encoding the His-tagged version of MDP decarboxylase from Picrophilus torridus.

(33) The resulting recombinant bacteria are then incubated in a fermenter in a mineral nutrient medium containing glucose, in the presence of oxygen and under moderate stirring. A significant production of isoprenol is measured using TLC or GC/MS analysis as follows:

(34) TLC Analysis

(35) For TLC analysis an aliquot of reaction medium is spotted on a silica-coated plate and chromatographed using as eluant ethyl acetate/heptane 1/1 v/v. Mevalonate, isoprenol, ATP, ADP are used as internal standards. After drying, plates are sprayed with alkaline KMnO4 reagent. R.sub.f for isoprenol is found to be 0.57.

(36) GC/MS Analysis

(37) An aliquot of 10 l of reaction medium is centrifuged and the supernatant is transferred to a clean vial for isoprenol detection by GC/MS. 1 L sample is separated by GC using a DB-5 column and the presence of isoprenol is monitored by mass spectrometry.

Example 7: Measurement of Mevalonate Decarboxylase Activity and 3-methyl-3-buten-1-ol(isoprenol) production

(38) Mevalonate is prepared from mevanolactone (Sigma) by hydrolysis with NaOH according to Campos et al. (Biochem. J. 2001, 353, 59-67).

(39) The complete assay for mevalonate decarboxylation contains reaction buffer, 100 mM mevalonate, 40 mM ATP, 10 mM MgCl.sub.2, 20 mM KCl, 0.5 mM DTT and enzyme preparation at a concentration ranging from 0.01 to 0.05 mg/ml of protein. 50 mM sodium citrate is used in the range of pH from 4 to 6, and 50 mM Tris-HCl for pH 7 and 7.5. Control reactions are carried out in the absence of enzyme, substrate or co-factor.

(40) The progress of isoprenol production is followed by analyzing aliquots taken at successive time intervals from a reaction mixture incubated at 37 C. by thin-layer chromatography (TLC), gas chromatography/mass spectrometry (GC/MS) and product determination by permanganate assay. In parallel, the release of inorganic phosphate is quantified by ammonium molybdate method.

(41) Permanganate Assay

(42) The formation of products containing double-bonds is followed by oxidization with alkaline potassium permanganate solution, resulting in increase of absorbance at 420 nm.

(43) To an aliquot of reaction mixture diluted with H.sub.2O to 120 l, 80 l of permanganate reagent, containing 5 mM KMnO.sub.4 and 50 mM NaOH, is added. The mixture is kept at room temperature for 20 min and the absorbance at 420 nm is measured. The calibration curve is prepared using commercial isoprenol.

(44) Inorganic Phosphate Quantification

(45) Inorganic phosphate concentration is measured by spectroscopic colorimetry according to the ammonium molybdate method (Gawronski J D, Benson D R, Anal. Biochem. 327 (2004) 114-118). A 50 l aliquot from the reaction assay (containing not more than 0.5 mole of phosphate) is mixed with 150 l ammonium molybdate reagent, containing 50% volume acetone, 1.25 N H.sub.2SO.sub.4, 2.5 mM (NH.sub.4).sub.6Mo.sub.7O.sub.24 and then with 10 l 1 M citric acid. The mixture is then incubated for 2 minutes at room temperature. The absorbance of ammonium phosphomolybdate formed was measured at 355 nm and the quantity of inorganic phosphate estimated using a calibration curve obtained with potassium phosphate.