Method for the enzymatic production of isoprenol using mevalonate as a substrate

Abstract

Described is a method for generating isoprenol through a biological process. More specifically, described is a method for producing isoprenol from mevalonate.

Claims

1. A multicellular organism or a microorganism comprising: (i) a first heterologous enzyme comprising an amino acid sequence at least 80% identical to the amino acid sequence shown in SEQ ID NO: 16 wherein said first heterologous enzyme converts mevalonate into mevalonate 3-phosphate; and (ii) a second heterologous enzyme being different from the first enzyme and comprising an amino acid sequence at least 80% identical to the amino acid sequence shown in SEQ ID NO: 10 wherein said second heterologous enzyme converts said mevalonate 3-phosphate into isoprenol, wherein the multicellular organism is a fungus, yeast, microalgae or plant, wherein the multicellular organism or microorganism is genetically modified to overproduce mevalonate and wherein the production of isoprenol by use of the combination of the first and second enzyme is higher than the production of isoprenol achieved by either enzyme alone or the addition of the production which either enzyme achieves in isolation.

2. The multicellular organism or a microorganism of claim 1, wherein (i) the first heterologous enzyme is selected from: (A) a protein comprising the amino acid sequence as shown in SEQ ID NO: 2; and (B) a protein comprising the amino acid sequence as shown in SEQ ID NO: 16.

3. The multicellular organism or a microorganism of claim 1, wherein (ii) the second heterologous enzyme is selected from: (A) a protein comprising the amino acid sequence as shown in SEQ ID NO: 10; (B) a protein comprising the amino acid sequence as shown in SEQ ID NO: 13; and (C) a protein comprising the amino acid sequence as shown in SEQ ID NO: 19.

4. The multicellular organism or a microorganism of claim 1 wherein (i) the first heterologous enzyme is selected from: (A) a protein comprising the amino acid sequence as shown in SEQ ID NO: 2; and (B) a protein comprising the amino acid sequence as shown in SEQ ID NO: 16; and (ii) the second heterologous enzyme is selected from: (A) a protein comprising the amino acid sequence as shown in SEQ ID NO: 10; (B) a protein comprising the amino acid sequence as shown in SEQ ID NO: 13; and (C) a protein comprising the amino acid sequence as shown in SEQ ID NO: 19.

5. A composition comprising the multicellular organism or a microorganism of claim 1.

6. A method of producing isoprenol comprising: (i) culturing the multicellular organism or microorganism of claim 1 for a sufficient period of time to allow for the conversion of the mevalonate to isoprenol and (ii) recovering said isoprenol.

7. A composition comprising the multicellular organism or a microorganism of claim 2.

8. The method of claim 6 wherein the first heterologous enzyme is selected from: (A) a protein comprising the amino acid sequence as shown in SEQ ID NO: 2; and (B) a protein comprising the amino acid sequence as shown in SEQ ID NO: 16.

9. A composition comprising the multicellular organism or a microorganism of claim 3.

10. The method of claim 6 wherein the second heterologous enzyme is selected from: (A) a protein comprising the amino acid sequence as shown in SEQ ID NO: 10; (B) a protein comprising the amino acid sequence as shown in SEQ ID NO: 13; and (C) a protein comprising the amino acid sequence as shown in SEQ ID NO: 19.

11. A composition comprising the multicellular organism or a microorganism of claim 4.

12. The method of claim 6 wherein the first heterologous enzyme is selected from: (A) a protein comprising the amino acid sequence as shown in SEQ ID NO: 2; and (B) a protein comprising the amino acid sequence as shown in SEQ ID NO: 16; and wherein said second heterologous enzyme is selected from: (A) a protein comprising the amino acid sequence as shown in SEQ ID NO: 10; (B) a protein comprising the amino acid sequence as shown in SEQ ID NO: 13; and (C) a protein comprising the amino acid sequence as shown in SEQ ID NO: 19.

13. The method of claim 6, wherein the method further comprises: (i) providing a composition comprising the first enzyme, the second enzyme and mevalonate; and (ii) recovering said isoprenol.

14. The method of claim 6 wherein the method is carried out with ATP, dATP, ADP, AMP, an NTP other than ATP, a dNTP or pyrophosphate as co-substrate.

15. The method of claim 6, further comprising the step of converting isoprenol into isoprene.

16. The method of claim 6, further comprising the step of converting isoprenol into isoamyl alcohol.

17. The multicellular organism or the microorganism of claim 1, wherein the multicellular organism or the microorganism is genetically modified to comprise the genes encoding the mevalonate pathway.

18. The multicellular organism or the microorganism of claim 1, wherein the multicellular organism or the microorganism is genetically modified to comprise the genes encoding thiolase, HMG-CoA synthase, and HMG-CoA reductase.

19. The method of claim 6, wherein the multicellular organism or the microorganism is genetically modified to comprise the genes encoding the mevalonate pathway.

20. The method of claim 6, wherein the multicellular organism or the microorganism is genetically modified to comprise the genes encoding thiolase, HMG-CoA synthase, and HMG-CoA reductase.

Description

(1) Moreover, the present invention also relates to a method for producing isoamyl alcohol from mevalonate comprising the method for producing isoprenol according to the invention as described above and further comprising the step of converting the produced isoprenol into isoamyl alcohol. The conversion of isoprenol into isoamyl alcohol can be achieved by means and methods known to the person skilled in the art. In particular, the respective reaction is a hydrogenation reaction.

(2) FIG. 1 Chemical structure of mevalonic acid.

(3) FIG. 2 Reaction of diphosphomevalonate decarboxylase on the physiological substrate 5-diphosphomevalonate and on the precursor mevalonate.

(4) FIG. 3 Structure of phosphono-phosphate and phosphonoamido-phosphate

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

(6) FIG. 5 Plot of the rate as a function of substrate concentration for the phosphotransferase reaction catalyzed by Th. acidophilum MDP decarboxylase (mutant L200E). Initial rates were computed from the kinetics over the 20 first minutes of the reaction.

(7) FIG. 6 Electrospray MS spectrums of mevalonate phosphorylation reaction catalyzed by MDP decarboxylase from Th. acidophilum (a), control assay without enzyme (b).

(8) FIG. 7 Screening of MDP decarboxylases in a complementation assay. Peak area ratios were obtained by dividing the isoprenol peak area of each enzymatic assay by the peak area of the sample without enzyme (background noise).

(9) FIG. 8 Combined effect of MDP decarboxylase enzymes from Th. acidophilum and S. mitis for converting mevalonate into isoprenol.

(10) FIG. 9 Mass spectrums of commercial isoprenol (a) and isoprenol produced from mevalonate by combining action of two enzymes (b). The characteristic ions 68 and 56 representing, respectively, the loss of H20 and CH2O were observed in both spectrums.

(11) FIG. 10 Plot of the rate of isoprenol production as a function of the S. gordonii MDP decarboxylase concentration.

(12) FIG. 11 Combined effect of MDP decarboxylase enzymes from Th. acidophilum and S. tokodaii for converting mevalonate into isoprenol.

(13) FIG. 12 Combined effect of MDP decarboxylase enzymes from Th. acidophilum and D. discoideum for converting mevalonate into isoprenol.

(14) 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

(15) A set of genes encoding representatives of the diphosphomevalonate decarboxylase (MDP decarboxylase) family across eukaryotic, prokaryotic and archaeal organisms was constructed and tested to identify the most active candidates for improving isoprenol production.

(16) Cloning, Bacterial Cultures and Expression of Proteins

(17) 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 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 h at 37 C. and protein expression was continued at 28 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.

(18) Protein Purification and Concentration

(19) 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 thus purified varied from 50% to 90%.

Example 2

Characterization of the Phosphotransferase Activity

(20) The release of ADP that is associated with isoprenol production from mevalonate was quantified using the pyruvate kinase/lactate dehydrogenase coupled assay (FIG. 4). The MDP decarboxylases from P. torridus phylum and S. mitis enzyme were evaluated for their ability to phosphorylate mevalonate, releasing ADP.

(21) The studied enzymatic reaction was carried out under the following conditions at 40 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 1.5 U/ml Lactate dehydrogenase 3 U/ml Pyruvate kinase 0 to 5 mM R,S-sodium Mevalonate The pH was adjusted to 7.5.

(22) Each assay was started by addition of particular enzyme (at a concentration from 0.025 to 1 mg/ml) and the disappearance of NADH was monitored by following the absorbance at 340 nm.

(23) FIG. 5 shows an example of a Michaelis-Menten plot corresponding to the data collected for the Th. acidophilum (L200E) enzyme. The kinetic parameters are shown in the following Table 1.

(24) TABLE-US-00002 TABLE 1 k.sub.cat/K.sub.M, Enzyme K.sub.M, mM k.sub.cat, s.sup.1 mM.sup.1 s.sup.1 Ferroplasma acidarmanus 0.62 1.5 2.5 Picrophilus torridus 0.32 1.2 3.8 Thermoplasma volcanium 0.25 1.1 4.4 Thermoplasma acidophilum 0.32 1.5 4.7 Mutant L200E of Thermoplasma 0.50 2.5 5.0 acidophilum enzyme Streptococcus mitis 0.20 2 10.sup.3 0.01

(25) Assays with MDP decarboxylases from the P. torridus phylum as well as Streptococcus mitis enzyme gave rise to a reproducible increase in ADP production in the presence of mevalonate. The enzymes from the P. torridus phylum displayed higher phosphotransferase activities than the Streptococcus mitis enzyme.

Example 3

Analysis of Mevalonate Phosphorylation by Mass Spectrometry

(26) The mevalonate phosphorylation reactions were run under the following conditions: 50 mM Tris HCl pH 7.5 10 mM MgCl.sub.2 20 mM KCl 40 mM ATP 200 mM R,S-sodium Mevalonate

(27) The assays were initiated by adding purified MDP decarboxylase (0.2 mg/ml) and incubated at 37 C. Control reactions were performed in which either no enzyme was added, or no substrate was added. Following incubation assays were processed by mass spectrometry analysis in negative ion mode. Typically, an aliquot of 80 l reaction was removed, centrifuged and the supernatant was transferred to a clean vial. The product was then extracted with equal volume of ethyl acetate and diluted 1:5 (20%, vol/vol) with methanol. An aliquot of 10 l was directly injected into mass spectrometer. Detection was performed by a PE SCIEX API 2000 quadrupole spectrometer interfaced to an electrospray ionisation (ESI) source. MS analysis showed an [MH].sup. ion at m/z=227.20, corresponding to mevalonate 3-phosphate (3-phosphonoxy-3-methyl-5-hydroxypentanoate), from the complete enzymatic assay (FIG. 6a), but not from the control (FIG. 6b).

Example 4

Identification of Enzyme Combinations Leading to an Increased Isoprenol Production from Mevalonate

(28) MDP decarboxylases were evaluated using a complementation assay. Th. acidophilum MDP decarboxylase (mutant L200E) was incubated together with each tested enzyme from the library.

(29) The combined enzymatic assay was carried out under the following conditions: 50 mM Tris HCl pH 7.5 10 mM MgCl.sub.2 20 mM KCl 40 mM ATP 200 mM R,S-sodium Mevalonate The pH was adjusted to 7.5

(30) 0.01 mg of the Th. acidophilum enzyme and 0.5 mg of the MDP decarboxylase to be tested were added to 0.1 ml of reaction mixture. Reaction mixture containing only 0.51 mg of the Th. acidophilum MDP decarboxylase (L200E) was used as reference. The assays were incubated without shaking at 37 C. for 24 h in a sealed vial (Interchim). The isoprenol production was analyzed as follows. An aliquot of 50 l of liquid phase was removed and mixed with 100 l of ethyl acetate. 100 l of the upper ethyl acetate phase was transferred to a clean vial for analysis by gas chromatography. Commercial isoprenol was used as reference. The samples were analyzed on a Varian GC-430 gas chromatograph equipped with a flame ionization detector (FID). A 1 l sample was separated on the DB-WAX column (30 m, 0.320.50 m, Agilent) using the following gradient: 60 C. for 2 minutes, increasing the temperature at 20 C./minute to a temperature of 220 C. and hold at final temperature for 10 minutes. The retention time of isoprenol in these conditions was 7.38 min.

(31) This screening procedure led to the identification of several archaeal, prokaryotic and eukaryotic MDP decarboxylases increasing the isoprenol production yield in combined assay (FIG. 7). The highest production of isoprenol was observed with enzymes from the Streptococcus genus, in particular with S. mitis MDP decarboxylase, and with the S. cerevisiae MDP decarboxylase.

Example 5

Detailed Study of Isoprenol Production from Mevalonate by Combining MDP Decarboxylase from Th. Acidophilum and MDP Decarboxylase from S. Mitis

(32) 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 40 mM ATP 200 mM R,S-sodium Mevalonate

(33) 0.01 mg of purified MDP decarboxylase from Th. acidophilum (mutant L200E) and 0.2 mg of purified MDP decarboxylase from S. mitis were added to 0.1 ml of reaction mixture. Control reactions were performed in which either no enzyme was added, or no ATP was added.

(34) To validate the combined action of two enzymes, a series of additional controls were carried out. In one assay, MDP decarboxylase from S. mitis (0.21 mg) was the only enzyme using as the catalyst. In the other experiment, 0.21 mg of the Th. acidophilum (L200E) enzyme was added, lacking S. mitis decarboxylase. The assays were incubated in a sealed vial (Interchim) without shaking for 24 hours at 37 C. Isoprenol extraction and analysis were performed according to the procedure described in Example 4.

(35) The highest production of isoprenol was observed in the reaction mixture contained decarboxylase from S. mitis and decarboxylase from Th. acidophilum (FIG. 8). This indicated that the two enzymes present in the assay were performing complementarily the two steps of reaction producing isoprenol from mevalonate: transfer of the terminal phosphoryl group from ATP to the C3-oxygen of mevalonate followed by combined dephosphorylation-decarboxylation of the intermediate mevalonate 3-phosphate.

(36) Gas chromatography-mass spectrometry (GC/MS) was then used to confirm the identity of the product detected by gas chromatography with flame ionization. The samples were analyzed on a Varian 3400Cx gas chromatograph equipped with Varian Saturn 3 mass selective detector. A mass spectrum of isoprenol obtained by enzymatic conversion of mevalonate was similar to this of commercial isoprenol (FIGS. 9a and 9b).

Example 6

Effect of Enzyme Concentration on Isoprenol Production Yield

(37) The effect of S. mitis MDP decarboxylase concentration was assessed under the following conditions: 50 mM Tris HCl pH 7.5 10 mM MgCl.sub.2 20 mM KCl 40 mM ATP 200 mM R,S-Mevalonate

(38) 0.01 mg of purified MDP decarboxylase from Th. acidophilum (mutant L200E) and a varying amount (from 0 to 0.4 mg) of purified MDP decarboxylase from S. mitis were added to 0.1 ml of reaction mixture. The mixtures were then incubated without shaking at 37 C. for 24 h in a sealed vial. Isoprenol extraction and analysis were performed according to the procedure described in Example 4. Increasing the S. mitis enzyme concentration resulted in an increase of the amount of produced isoprenol (FIG. 10).

Example 7

Detailed Study of Isoprenol Production from Mevalonate by Combining MDP Decarboxylase from Th. Acidophilum and MDP Decarboxylase from S. Tokodaii

(39) The studied reaction was carried out under the following conditions: 50 mM Tris-HCl pH 7.5 10 mM MgCl.sub.2 20 mM KCl 40 mM ATP 200 mM R,S-sodium mevalonate

(40) 0.01 mg of purified MDP decarboxylase from Th. acidophilum (L200E) and 0.4 mg MDP of decarboxylase from S. tokodaii were added to 0.1 ml of reaction mixture. A series of controls were performed in parallel under the same conditions. In one assay with MDP decarboxylase from S. tokodaii (0.41 mg) alone, containing no enzyme from Th. acidophilum was performed. In the other experiment, 0.41 mg of the Th. acidophilum (L200E) enzyme was added to the reaction mixture, lacking S. tokodaii decarboxylase.

(41) The assays were incubated in sealed vials (Interchim) for 24 hours at 37 C. Isoprenol extraction was performed according to the procedure described in Example 4. Commercial isoprenol was used as reference.

(42) Isoprenol production was then analyzed by gas-chromatography using Bruker 430-GC gas chromatograph equipped with flame ionization detector (FID) according to the following procedure:

(43) 5 l of sample was separated on DB-WAX column (30 m, 0.32 mm0.50 m, Agilent Technologies) using the gradient described in Example 4.

(44) The highest production of isoprenol was observed in the reaction mixture contained MDP decarboxylase S. tokodaii and MDP decarboxylase Th. acidophilum (FIG. 11). This indicates that the combination of two enzymes significantly increases isoprenol yield.

Example 8

Detailed Study of Isoprenol Production from Mevalonate by Combining MDP Decarboxylase from Th. Acidophilum and MDP Decarboxylase from D. Discoideum

(45) The studied assay was carried out according the protocol described in Example 7.

(46) 0.01 mg of purified MDP decarboxylase from Th. acidophilum (L200E) and 0.4 mg MDP of decarboxylase from D. discoideum were added to 0.1 ml of reaction mixture. A series of control were performed in parallel under the same conditions. Assay with MDP decarboxylase from D. discoideum (0.41 mg) alone, containing no enzyme from Th. acidophilum was performed. In the other experiment, 0.41 mg of the Th. acidophilum (L200E) enzyme was added to the reaction mixture, lacking D. discoideum decarboxylase.

(47) Isoprenol production was analyzed as described in Example 7.

(48) The highest production of isoprenol was observed in the reaction mixture contained MDP decarboxylase D. discoideum and MDP decarboxylase Th. acidophilum (FIG. 12). Thus, higher isoprenol yield can be achieved by combining action of two enzymes on mevalonate.