HIV kinase variants

Abstract

Described are HIV kinase variants showing an improved activity in converting 3-hydroxyisovalerate (HIV) into 3-phosphonoxyisovalerate (PIV), methods for the production of PIV using such enzyme variants as well as methods for the production isobutene in a subsequent reaction.

Claims

1. A variant of an 3-hydroxyisovalerate (HIV) kinase showing an improved activity in converting 3-hydroxyisovalerate (HIV) into 3-phosphonoxyisovalerate (PIV) over the corresponding HIV kinase from which it is derived, wherein the HIV kinase variant has at least 80% sequence identity to SEQ ID NO:1, and in which the HIV kinase variant comprises one or more substitutions, deletions or insertions at one or more of the positions corresponding to positions 3, 5, 6, 7, 8, 9, 14, 16, 17, 23, 24, 25, 26, 32, 43, 44, 45, 46, 49, 51, 52, 54, 56, 59, 60, 64, 65, 67, 70, 71, 73, 74, 75, 76, 77, 80, 81, 82, 83, 88, 89, 90, 91, 92, 93, 94, 98, 99, 115, 118, 119, 121, 123, 127, 133, 135, 136, 137, 141, 147, 158, 160, 172, 174, 175, 178, 180, 183, 184, 186, 187, 189, 197, 202, 203, 205, 206, 208, 211, 217, 240, 248, 252, 256, 259, 260, 285, 289, 295, 296, 302, 303, 307, 311, 313, 315, 316 and 318 in the amino acid sequence of SEQ ID NO:1.

2. The HIV kinase variant of claim 1, wherein said HIV kinase variant has at least 90% sequence identity to SEQ ID NO:1.

3. The HIV kinase variant of claim 1, wherein (1) the amino acid residue at position 3 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with histidine; and/or (2) the amino acid residue at position 5 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with alanine or phenylalanine; and/or (3) the amino acid residue at position 6 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with leucine; and/or (4) the amino acid residue at position 7 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with leucine, glutamine or valine; and/or (5) the amino acid residue at position 8 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with cysteine or threonine; and/or (6) the amino acid residue at position 9 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with histidine; and/or (7) the amino acid residue at position 14 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with valine; and/or (8) the amino acid residue at position 16 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with isoleucine; and/or (9) the amino acid residue at position 17 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with isoleucine or leucine; and/or (10) the amino acid residue at position 23 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with threonine; and/or (11) the amino acid residue at position 24 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with aspartic acid; and/or (12) the amino acid residue at position 25 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with leucine, arginine or serine; and/or (13) the amino acid residue at position 26 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with phenylalanine; and/or (14) the amino acid residue at position 32in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with aspartic acid; and/or (15) the amino acid residue at position 43 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with glycine or serine; and/or (16) the amino acid residue at position 44 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with aspartic acid; and/or (16) the amino acid residue at position 45 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with isoleucine; and/or (18) the amino acid residue at position 46 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with valine; and/or (19) the amino acid residue at position 49 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with aspartic acid, glycine or serine; and/or (20) the amino acid residue at position 51in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with histidine; and/or (21) the amino acid residue at position 52 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with phenylalanine, leucine or methionine; and/or (22) the amino acid residue at position 54 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with arginine; and/or (23) the amino acid residue at position 56 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with serine; and/or (24) the amino acid residue at position 59 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with arginine; and/or (25) the amino acid residue at position 60 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with histidine or tryptophan; and/or (26) the amino acid residue at position 64 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with glutamic acid; and/or (27) the amino acid residue at position 65 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with aspartic acid or glutamic acid; and/or (28) the amino acid residue at position 67 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with cysteine or asparagine; and/or (29) the amino acid residue at position 70 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with leucine or valine; and/or (30) the amino acid residue at position 71 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with glycine or isoleucine; and/or (31) the amino acid residue at position 73 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with proline; and/or (32) the amino acid residue at position 74 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with proline; and/or (33) the amino acid residue at position 75 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with glycine; and/or (34) the amino acid residue at position 76 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with proline; and/or (35) the amino acid residue at position 77 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with proline; and/or (36) the amino acid residue at position 80 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with alanine or threonine; and/or (37) the amino acid residue at position 81 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with glutamic acid, glycine, glutamine, arginine or threonine; and/or (38) the amino acid residue at position 82 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with aspartic acid; and/or (39) the amino acid residue at position 83 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with asparagine; and/or (40) the amino acid residue at position 88 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with threonine; and/or (41) the amino acid residue at position 89 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with glycine or serine; and/or (42) the amino acid residue at position 90 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with proline or serine; and/or (43) the amino acid residue at position 91 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with glycine, proline or serine; and/or (44) the amino acid residue at position 92 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with threonine; and/or (45) the amino acid residue at position 93 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with isoleucine; and/or (46) the amino acid residue at position 94 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with alanine or serine; and/or (47) the amino acid residue at position 98 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with threonine; and/or (48) the amino acid residue at position 99 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with lysine; and/or (49) the amino acid residue at position 115 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with valine; and/or (50) the amino acid residue at position 118 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with threonine; and/or (51) the amino acid residue at position 119 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with valine; and/or (52) the amino acid residue at position 121 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with alanine; and/or (53) the amino acid residue at position 123 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with arginine or asparagine; and/or (54) the amino acid residue at position 127 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with asparagine; and/or (55) the amino acid residue at position 133 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with serine; and/or (56) the amino acid residue at position 135 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with cysteine; and/or (57) the amino acid residue at position 136 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with glycine; and/or (58) the amino acid residue at position 137 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with glutamic acid, proline or serine; and/or (59) the amino acid residue at position 141 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with alanine or glycine; and/or (60) the amino acid residue at position 147 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with leucine; and/or (61) the amino acid residue at position 158 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with serine; and/or (62) the amino acid residue at position 160 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with valine; and/or (63) the amino acid residue at position 172 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with methionine; and/or (64) the amino acid residue at position 174 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with phenylalanine; and/or (65) the amino acid residue at position 175 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with threonine; and/or (66) the amino acid residue at position 178 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with asparagine; and/or (67) the amino acid residue at position 180 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with cysteine, glutamic acid, glycine, leucine, arginine or threonine; and/or (68) the amino acid residue at position 183 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with proline; and/or (70) the amino acid residue at position 186 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with methionine; and/or (71) the amino acid residue at position 187 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with glutamic acid, glycine, methionine or valine; and/or (72) the amino acid residue at position 189 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with glutamic acid or serine; and/or (73) the amino acid residue at position 197 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with cysteine, lysine or leucine; and/or (74) the amino acid residue at position 202 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with serine; and/or (75) the amino acid residue at position 203 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with serine; and/or (76) the amino acid residue at position 205 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with isoleucine or arginine; and/or (77) the amino acid residue at position 206 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with arginine; and/or (78) the amino acid residue at position 208 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with threonine; and/or (79) the amino acid residue at position 211 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with lysine or arginine; and/or (80) the amino acid residue at position 217 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with methionine; and/or (81) the amino acid residue at position 240 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with glycine; and/or (82) the amino acid residue at position 248 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with threonine; and/or (83) the amino acid residue at position 252 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with lysine or serine; and/or (84) the amino acid residue at position 256 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with arginine; and/or (85) the amino acid residue at position 259 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with asparagine; and/or (86) the amino acid residue at position 260 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with phenylalanine or histidine; and/or (87) the amino acid residue at position 285 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with cysteine; and/or (88) the amino acid residue at position 289 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with valine; and/or (89) the amino acid residue at position 295 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with asparagine; and/or (90) the amino acid residue at position 296 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with threonine; and/or (91) the amino acid residue at position 302 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with arginine; and/or (92) the amino acid residue at position 303 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with threonine; and/or (93) the amino acid residue at position 307 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with histidine; and/or (94) the amino acid residue at position 311 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with cysteine, proline or glutamine; and/or (95) the amino acid residue at position 313 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with leucine, arginine, serine, threonine, valine or tyrosine; and/or (96) the amino acid residue at position 315 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with histidine, lysine or threonine; and/or (97) the amino acid residue at position 316 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with histidine; and/or (98) the amino acid residue at position 318 in the amino acid sequence of SEQ ID NO:1 is deleted or substituted with aspartic acid.

4. The HIV kinase variant of claim 1, wherein the HIV kinase variant further comprises at least one deletion, substitution and/or insertion at position 200 in the amino acid sequence of SEQ ID NO:1 or at a position corresponding to this position from which the HIV kinase variant is derived.

5. The HIV kinase variant of claim 4, wherein said HIV kinase variant has at least 90% sequence identity to SEQ ID NO:1.

6. A nucleic acid molecule encoding the HIV kinase variant of claim 1.

7. A vector comprising the nucleic acid molecule of claim 6.

8. An isolated host cell comprising the vector of claim 7.

9. A method for producing PIV from HIV by employing HIV with the HIV kinase variant of claim 1.

10. A method for producing isobutene (IBN) from HIV comprising the method of claim 9 and further the step of converting the thus produced PIV into IBN by a dephosphorylation/decarboxylation reaction.

11. The method according to claim 9, further comprising providing the HIV by the enzymatic conversion of acetone into said HIV.

12. The method of claim 9, wherein the enzymatic conversion is carried out in vitro.

13. A composition comprising a variant of an HIV kinase of claim 1.

14. The method of claim 9, wherein the enzymatic conversion is carried out in an isolated host cell.

15. A composition comprising the nucleic acid molecule of claim 6.

16. A composition comprising the vector of claim 7.

17. A composition comprising the isolated host cell of claim 8.

18. The HIV kinase variant of claim 2, wherein the HIV kinase variant further comprises at least one deletion, substitution and/or insertion at position 200 in the amino acid sequence of SEQ ID NO:1.

19. The HIV kinase variant of claim 3, wherein the HIV kinase variant further comprises at least one deletion, substitution and/or insertion at position 200 in the amino acid sequence of SEQ ID NO:1.

Description

(1) 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.

(2) FIG. 1: shows a two-step pathway for the decarboxylation of 3-hydroxyisovalerate (HIV) into isobutene. HIV is first phosphorylated into 3-phosphonoxyisovalerate (PIV). PIV is then dephosphorylated/decarboxylated into isobutene.

(3) FIG. 2: 3-phosphonoxyisovalerate (PIV) production rate of two Thermoplasma acidophilum mevalonate kinase variants as a function of 3-hydroxyisovalerate (HIV) concentration. Knowing the concentration Et of each enzyme, the Michaelis-Menten approximation is used to fit the experimental data in order to compute the Michaelis constant Km, the maximum production rate Vm, the catalytic rate constant kcat and the catalytic efficiency (kcat/Km).

(4) 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

Directed Evolution Strategy

(5) The enzyme Thermoplasma acidophilum mevalonate kinase (SEQ ID NO:1) is capable of catalysing, amongst other reactions, the phosphorylation of 3-hydroxyisovalerate (HIV) into 3-phosphonoxyisovalerate (PIV) as described in WO2012052427. A directed evolution approach was used in order to specifically improve the catalytic efficiency of this reaction. This approach consisted in (1) the design of assay systems to test the activity of enzyme variants, (2) the generation of collections of single point or multiple mutants for T. acidophilum mevalonate kinase, (3) the use of the activity assays to screen the collection of mutants in order to identify with improved activity compared to the activity of the wild type T. acidophilum mevalonate kinase. Different cycles of evolution are repeated several times (steps (2) and (3)) using improved variants as the new seed for the generation of new variants.

(6) This approach led to the identification and characterization of a collection of mutants with increased activity compared to the wild type enzyme.

Example 2

Construction of Thermoplasma acidophilum Mevalonate Kinase Enzyme Mutants

(7) The polynucleotide sequences coding for the different mutants identified during the evolution of the Thermoplasma acidophilum mevalonate kinase enzyme were generated using a range of standard molecular biology techniques. All these techniques used a codon-optimised polynucleotide sequence for expression in Escherichia coli as template. The sequence optimisation has been done by Geneart using their GeneOptimizer software.

(8) Different PCR-based techniques known in the art were used for the construction of single-point mutants. For the generation of enzyme variants bearing multiple mutations (at least two mutations), either PCR-based techniques or other methods known in the art were used to introduce these mutations.

(9) Following mutagenesis, the mutated polynucleotide sequence was inserted into an expression vector (used for recombinant protein production in E. coli and screening) either using standard ligase-based subcloning techniques, whole plasmid extension by PCR or ligase-independent cloning techniques.

Example 3

Methods for the Identification of the Enzyme Mutants with Increased Activity

(10) Three different screening methods were developed and used during the evolution of the Thermoplasma acidophilum mevalonate kinase enzyme which are summarized in the following.

(11) 1) In Vitro Coupled Assay on a HIV Substrate Substrate (IN VITRO)

(12) The Thermoplasma acidophilum mevalonate kinase enzyme variants were cloned in the pET 25b vector (Novagen). A stretch of 6 histidine codons was inserted after the methionine initiation codon to provide an affinity tag for purification. Competent E. coli BL21 (DE3) cells (Novagen) were transformed in 96-well plates with these vectors according to the heat shock procedure and plated out onto LB-agar plates supplemented with the appropriate antibiotic. Cells were grown overnight at 30 C. until individual colonies reach the desired size. Single colonies were then picked and individually transferred into 500 L of liquid LB medium supplemented with the appropriate antibiotic. Cell growth is carried out with shaking for 20 hours at 30 C. The LB cultures were used to inoculate 1 mL of of auto-induction medium (Studier F W, Prat. Exp. Pur. 41, (2005), 207-234) supplemented with the appropriate antibiotic and grown in a shaking incubator set at 1000 rpm and 85% humidity 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. The pellets were thawed on ice and resuspended in 200 L ml of BugBuster (Millipore) and one microliter of lysonase (Novagen) was added. Cells were incubated 10 minutes at room temperature and then returned to ice for 20 minutes. 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 multi-96 Ni-IDA microplates (Macherey-Nagel) allowing adsorption of 6-His tagged proteins. Columns were washed and the enzymes of interest were eluted with 400 L of the supplied elution buffer. Eluates were then concentrated by centrifugation, washing and resuspension in 120 L of 50 mM Tris/HCl pH 7. Protein concentrations were quantified using a Nanodrop 1000 (ThermoScientific).

(13) Thermoplasma acidophilum mevalonate kinase phosphorylates HIV into PIV by an ATP to ADP conversion. It is thus possible to follow the rate of conversion of HIV into PIV by measuring the production of ADP, by assays known to the person skilled in the art. The release of ADP was quantified using a pyruvate kinase/lactate dehydrogenase coupled assay. Briefly, in the presence of ADP and the pyruvate kinase (PK), phosphoenolpyruvate (PEP) is converted into pyruvate, which is then converted into lactate by the lactate dehydrogenase (LDH). This last reaction requires a molecule of NADH which is oxidised in NAD.sub.+. The rate of NADH oxidation, proportional to the ADP production, is followed by the rate of absorbance decrease at 340 nm. The enzymatic reaction is carried out at 40 C. in a 50 mM Tris-HCl pH7 buffer, with 10 mM MgCl2, 100 mM KCl, 0.4 mM NADH, 1 mM PEP, 1.5 U/mL PK and 3 U/mL LDH, 5 mM ATP, the mevalonate kinase variant at 0.05 mg/mL and different concentrations of HIV ranging from 0 to 20 mM. By following the absorbance at 340 nm, an initial velocity of conversion can be calculated and using the Michaelis-Menten approximation, the enzyme catalytic efficiency can be computed.

(14) 2) In Vivo Coupled Assay on an Acetone Substrate (IN VIVO 1)

(15) For the in vivo testing a screening assay was developed. This coupled assay is based on the use of a bacterial strain transformed with an expression vector that contains the coding sequences and lead to the production of three enzymes involved in a three-step metabolic pathway converting acetone to isobutene.

(16) In this pathway, the first step is the production of 3-hydroxyisovalerate (HIV) from acetone. Condensation of acetone and acetyl-CoA into 3-hydroxyisovalerate by HMG-CoA Synthases has been described previously (WO2011032934). In this study, variants of the Mus musculus HMG-CoA Synthase (referred to in the following as HIV synthase) were used. Variants of HIV synthase have previously been described in WO2015/101493. In this assay, acetone is exogenously provided while acetyl-CoA is provided by the E. coli strain. The second step is the phosphorylation of HIV into PIV. The Thermoplasma acidophilum mevalonate kinase variants were used to catalyze this step. The third step is the decarboxylation of 3-phosphonoxyisovalerate into isobutene (IBN), catalyzed by a mevaonate diphosphate decarboxylase, as described in WO2012052427. Variants of the Streptococcus mitis MDP decarboxylase (referred to in the following as PIV decarboxylase), described in WO2015004211, were used.

(17) This strain is first plated out onto LB-agar plates supplemented with the appropriate antibiotic. Cells were grown overnight at 30 C. until individual colonies reach the desired size. Single colonies were then picked and individually transferred into either 50 or 500 L of liquid LB medium supplemented with the appropriate antibiotic. Cell growth is carried out with shaking for 20 hours at 30 C. The LB cultures were used to inoculate 300 L in 384 deepwell microplates or 1 mL in 96 deepwell microplates of auto-induction medium (Studier F W, Prat. Exp. Pur. 41, (2005), 207-234) supplemented with the appropriate antibiotic and grown in a shaking incubator set at 700 rpm and 85% humidity for 24 h at 30 C. in order to produce the three types of recombinant enzymes. The cell pellet containing these three overexpressed recombinant enzymes is then resuspended in 50 L (or 500 L) of minimum medium supplemented with 250 mM or 500 mM acetone and incubated for a further 16 hours in a shaking incubator at 37 C., 700 rpm. During this step, HIV synthase catalyses the condensation of acetone with the cellular acetyl-CoA into HIV, which is then converted into PIV by the Thermoplasma acidophilum mevalonate kinase variants tested. The PIV decarboxylase finally catalyses the conversion of PIV into IBN. After 5 min inactivation at 80 C., the IBN produced is quantified by gas chromatography as followed. 100 L of headspace gases from each enzymatic reaction are injected in a Brucker GC-450 system equipped with a Flame Ionization Detector (FID). Compounds present in samples were separated by chromatography using a RTX-1 columns at 100 C. with a 1 mL/min constant flow of nitrogen as carrier gas. Upon injection, peak areas of isobutene were calculated.

(18) 3) In Vivo Assay Based on Exogenous HIV (IN VIVO 2)

(19) A second in vivo screening assay was developed. This assay is based on the use of a bacterial strain transformed with an expression vector that contain the coding sequences and lead to the production of the last two enzymes involved in a metabolic pathway converting HIV to isobutene. The reactions involved in this pathway are the two last reactions of the pathway used in the IN VIVO 1 assay: phosphorylation of HIV into PIV and decarboxylation of PIV into isobutene. The same enzymes were used as for the IN VIVO 1 assay.

(20) This strain is first plated out onto LB-agar plates supplemented with the appropriate antibiotic. Cells were grown overnight at 30 C. until individual colonies reach the desired size. Single colonies were then picked and individually transferred into 50 l of liquid LB medium supplemented with the appropriate antibiotic. Cell growth is carried out with shaking for 20 hours at 30 C. The LB cultures were used to inoculate 300 L in 384 deepwell microplates of auto-induction medium (Studier F W, Prat. Exp. Pur. 41, (2005), 207-234) supplemented with the appropriate antibiotic and grown in a shaking incubator set at 700 rpm and 85% humidity for 24 h at 30 C. in order to produce the two types of recombinant enzymes. The cell pellet containing these two overexpressed recombinant enzymes is then resuspended in 30 L of minimum medium supplemented with 10 mM HIV and incubated for a further 4 or 16 hours in a shaking incubator at 37 C., 700 rpm. During this step, the Thermoplasma acidophilum mevalonate kinase variants catalyse the phosphorylation of HIV into PIV. The PIV decarboxylase finally catalyses the conversion of PIV into IBN. After 5 min inactivation at 80 C., the IBN produced is quantified by gas chromatography as followed. 100 L of headspace gases from each enzymatic reaction are injected in a Brucker GC-450 system equipped with a Flame Ionization Detector (FID). Compounds present in samples were separated by chromatography using a RTX-1 columns at 100 C. with a 1 mL/min constant flow of nitrogen as carrier gas. Upon injection, peak areas of isobutene were calculated.

(21) 4) Selection Based Assays (SELECTION 1 and SELECTION 2)

(22) In order to efficiently select improved variants from the high diversity libraries and filter out non-working variants, a negative selection assay was designed. It has been shown that, in the presence of HIV in the culture medium, the growth rate of a strain was inversely proportional to the Thermoplasma acidophilum mevalonate kinase variant catalytic efficiency. This property was used for selecting the best active/best performing variants using the following protocol.

(23) The Thermoplasma acidophilum mevalonate kinase enzyme variants were cloned in the pET 25b vector (Novagen). Competent E. coli BL21 (DE3) cells (Novagen) were transformed in 96-well plates with these vectors according to the heat shock procedure and plated out onto LB-agar plates supplemented with the appropriate antibiotic. Cells were grown overnight at 30 C. until individual colonies reach the desired size. Single colonies were then picked and individually transferred into 1200 L of liquid LB medium supplemented with the appropriate antibiotic. Cell growth is carried out with shaking for 22 hours at 30 C. The LB cultures were used to inoculate 150 L of selection medium (LB, appropriate antibiotic, 0.1 mM IPTG, 10 mM HIV) and grown in a shaking incubator set at 700 rpm and 85% humidity for 5 h at 30 C. The optical density (OD) of each well is measured and compared against a reference. Only the wells presenting an OD inferior to the reference are chosen. The variants then selected are either tested directly according to the in vitro assay described in 1) or subcloned into the appropriate vector and screened according to the in vivo assay described in 3). The selected variants are summarized in the below Tables as results of these selection-based assays SELECTION 1 and SELECTION 2, respectively.

(24) 5) In Vivo Assay Based on Exogenous HIV (IN VIVO 3)

(25) A third in vivo screening assay was developed. This assay is based on the use of a bacterial strain transformed with an expression vector that contains the coding sequences and lead to the production of the last two enzymes involved in the metabolic pathway converting HIV to isobutene; namely the Thermoplasma acidophilum mevalonate kinase variants were used for the phosphorylation of HIV into PIV and the above-mentioned Streptococcus mitis MDP decarboxylase was used for the conversion of 3-phosphonoxyisovalerate into isobutene (IBN) (referred to in the following as PIV decarboxylase).

(26) This strain is first plated out onto LB-agar plates supplemented with the appropriate antibiotic. Cells were grown overnight at 30 C. until individual colonies reached the desired size. Single colonies were then picked and individually transferred into 200 L of liquid LB medium supplemented with the appropriate antibiotic. Cell growth is carried out with shaking for 20 hours at 30 C. The LB cultures were used to inoculate 1400 L in 96 deepwell microplates of auto-induction medium (Studier F W, Prat. Exp. Pur. 41, (2005), 207-234) supplemented with the appropriate antibiotic and grown in a shaking incubator set at 700 rpm and 85% humidity for 24 h at 30 C. in order to produce the two types of recombinant enzymes. The cell pellet containing these two overexpressed recombinant enzymes was then resuspended in 400 L of minimum medium supplemented with 10 mM HIV and incubated for a further 1 or 2 hours in a shaking incubator at 30 C., 700 rpm. During this step, the Thermoplasma acidophilum mevalonate kinase variants catalyse the phosphorylation of HIV into PIV. The PIV decarboxylase finally catalyses the conversion of PIV into IBN. After 5 min inactivation at 80 C., the IBN produced is quantified by gas chromatography as follows. 100 L of headspace gases from each enzymatic reaction were injected in a Brucker GC-450 system equipped with a Flame Ionization Detector (FID). Compounds present in samples were separated by chromatography using a RTX-1 columns at 100 C. with a 1 mL.min-1 constant flow of nitrogen as carrier gas. Upon injection, peak areas of isobutene were calculated.

Example 4

Identification of Variants of Thermoplasma acidophilum Mevalonate Kinase with further Increased Activity for the Reaction of Conversion of 3-hydroxyisovalerate into 3-phosphonoxyisovalerate

(27) A collection of single-point mutants and multiple mutants was created by successive rounds of mutagenesis, recombination of single and multiple mutations, using the Thermoplasma acidophilum mevalonate kinase (SEQ ID NO:1) as the template. Several variants with an enhanced activity in converting 3-hydroxyisovalerate into 3-phosphonoxyisovalerate have been identified through in vitro and/or in vivo screening assays as described above. The increase in activity is described relative to the wild-type enzyme (with + representing a low increase in activity and ++++++ and up to +++++++ representing a high increase in activity). The activity considered is the increased production of 3-phosphonoxyisovalerate from 3-hydroxyisovalerate as measured directly (see the above-described IN VITRO and SELECTION assays), through the successive conversion of acetone to 3-hydroxyisovalerate, subsequently to 3-phosphonoxyisovalerate and finally to isobutene (see the above-described IN VIVO 1 assay) or by the combined conversion of 3-hydroxyisovalerate into isobutene (through 3-phosphonoxyisovalerate) (see the above-described IN VIVO 2 and IN VIVO 3 assay). The list of these variants is presented in the following Table 1, the list of individual mutations in Table 2 and the list of the positions presenting an increase in activity, either as single-point mutations or in combination with others, is presented in Table 3.

(28) TABLE-US-00002 TABLE 1 List of Thermoplasma acidophilum mevalonate kinase variants presenting an increase in 3-phosphonoxyisovalerate production from 3-hydroxyisovalerate Activity relative to the wild-type Screening Mutations enzyme Assay used E252S + IN VITRO H59R + IN VITRO I6L + IN VITRO L200E + IN VITRO L200E-I6L + IN VITRO L200E-S240G + IN VITRO L200T + IN VITRO S240G + IN VITRO V26F + IN VITRO L200E-A137S ++ IN VIVO 2 L200E-E285C ++ IN VIVO 1 L200E-E49D-V77P-R315K ++ IN VIVO 1 L200E-E49S-V74P ++ IN VIVO 1 L200E-F89G ++ IN VIVO 2 L200E-G90P ++ IN VIVO 2 L200E-H211K ++ IN VIVO 1 L200E-H211R ++ IN VIVO 1 L200E-L135C ++ IN VIVO 1 L200E-P25R ++ IN VIVO 1 L200E-R197K ++ IN VIVO 1 L200E-R197L ++ IN VIVO 1 L200E-R307H ++ IN VIVO 2 L200E-R315K ++ IN VIVO 1 L200E-R76P ++ IN VIVO 1 L200E-S67N-R197K ++ IN VIVO 1 L200E-S73P-V74P ++ IN VIVO 2 L200E-S82D ++ IN VIVO 1 L200E-V74P ++ IN VIVO 1 L200E-V74P-A137P ++ IN VIVO 2 L200E-V74P-E285C ++ IN VIVO 1 L200E-V74P-F89S ++ IN VIVO 2 L200E-V74P-G90P-T91S ++ IN VIVO 2 L200E-V74P-G90S-T91G ++ IN VIVO 2 L200E-V74P-G90S-T91P ++ IN VIVO 2 L200E-V74P-G90S-T91S ++ IN VIVO 2 L200E-V74P-H211K ++ IN VIVO 1 L200E-V74P-H211R ++ IN VIVO 1 L200E-V74P-L135C ++ IN VIVO 1 L200E-V74P-R136G ++ IN VIVO 2 L200E-V74P-R197L ++ IN VIVO 1 L200E-V74P-R315H ++ IN VIVO 1 L200E-V74P-R315K ++ IN VIVO 1 L200E-V74P-T91G ++ IN VIVO 2 L200E-V74P-T91P ++ IN VIVO 2 L200E-V74P-T91S ++ IN VIVO 2 L200E-V74P-W313T ++ IN VIVO 1 L200E-V77P ++ IN VIVO 1 L200E-V77P-H211R ++ IN VIVO 1 L200E-V77P-R197L ++ IN VIVO 1 L200E-V77P-R315H ++ IN VIVO 1 L200E-V77P-R315K ++ IN VIVO 1 L200E-V77P-W313R ++ IN VIVO 1 L200E-V77P-W313T ++ IN VIVO 1 L200E-V77P-W313Y ++ IN VIVO 1 L200E-W313R ++ IN VIVO 1 L200E-W313T ++ IN VIVO 1 L200E-W313Y ++ IN VIVO 1 L200E-Y81E ++ IN VIVO 1 L200E-Y81T ++ IN VIVO 1 L200E-R76P-L135C-R197L-H211R +++ IN VIVO 1 L200E-R76P-Y81T-L135C-R197L-H211R +++ IN VIVO 1 L200E-R76P-Y81T-R197L-H211R +++ IN VIVO 1 L200E-V74P-V77P-H211K +++ IN VIVO 1 L200E-V74P-V77P-H211R +++ IN VIVO 1 L200E-V74P-V77P-L135C-R197L-H211R +++ IN VIVO 1 L200E-V74P-V77P-R197L-H211R +++ IN VIVO 1 L200E-V77P-H211K-W313T +++ IN VIVO 1 L200E-V77P-L135C-H211K +++ IN VIVO 1 L200E-V77P-L135C-H211R +++ IN VIVO 1 L200E-V77P-R197C-H211R +++ IN VIVO 1 L200E-V77P-V94S-L135C-R197L-H211R +++ IN VIVO 1 L200E-L135C-H211R-R76P-Y81T-R197L ++++ IN VIVO 1 L200E-V77P-L135C-H211R-R197L-R80A-Y81R ++++ IN VIVO 1 L200E-V77P-L135C-H211R-R80A-Y81Q ++++ IN VIVO 1 L200E-V77P-L135C-H211R-R80A-Y81R ++++ IN VIVO 1 L200E-V77P-L135C-H211R-R80T-Y81R ++++ IN VIVO 1 L200E-V77P-L135C-H211R-S141A ++++ IN VIVO 1 L200E-V77P-L135C-H211R-S141G ++++ IN VIVO 1 L200E-V77P-L135C-H211R-T65D ++++ IN VIVO 1 L200E-V77P-L135C-H211R-T65E ++++ IN VIVO 1 L200E-V77P-L135C-H211R-V74P-R197L ++++ IN VIVO 1 L200E-V77P-L135C-H211R-V74P-R197L-R80A-Y81R ++++ IN VIVO 1 L200E-V77P-L135C-H211R-V74P-R197L-R80T-Y81R ++++ IN VIVO 1 L200E-V77P-L135C-H211R-V74P-R80T-Y81R ++++ IN VIVO 1 L200E-V77P-L135C-H211R-Y183D ++++ IN VIVO 1 L200E-V77P-L135C-H211R-Y3H ++++ IN VIVO 1 L200E-V74P-R197L-H211R-E49G-T91S +++++ IN VIVO 2 L200E-V74P-R197L-H211R-G90P-T91S-L135C-A137P +++++ IN VIVO 2 L200E-V74P-R197L-H211R-H32D-S73P-Y81R-G90S-T91S- +++++ IN VIVO 2 L135C-R136G L200E-V74P-R197L-H211R-L135C +++++ IN VIVO 2 L200E-V74P-R197L-H211R-R80A-G90S-T91G-R307H +++++ IN VIVO 2 L200E-V74P-R197L-H211R-R80A-L135C-R307H +++++ IN VIVO 2 L200E-V74P-R197L-H211R-R80A-T91G-R307H +++++ IN VIVO 2 L200E-V74P-R197L-H211R-R80A-Y81R-T91G +++++ IN VIVO 2 L200E-V74P-R197L-H211R-S73P-Y81R-L135C +++++ IN VIVO 2 L200E-V74P-R197L-H211R-Y81R-A137S +++++ IN VIVO 2 L200E-V74P-R197L-H211R-Y81R-G90S-T91S-R307H +++++ IN VIVO 2 L200E-V74P-R197L-H211R-Y81R-G90S-T91S-T93I-L135C- +++++ IN VIVO 2 R307H L200E-V74P-R197L-H211R-Y81R-L135C-R307H +++++ IN VIVO 2 L200E-V74P-R197L-H211R-Y81R-T91G-A137S-R307H +++++ IN VIVO 2 L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-A137E +++++ SELECTION 2 L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-A208T +++++ SELECTION 1 L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-A23T- +++++ SELECTION 2 V94A-K123N L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-C43S +++++ SELECTION 2 L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-D70V- +++++ SELECTION 2 A88T-K92T L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-F158S +++++ SELECTION 1 L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-G121A- +++++ SELECTION 1 S259N L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-G44D +++++ SELECTION 2 L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-G44D- +++++ SELECTION 2 K205I L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-G7V- +++++ SELECTION 2 S45I-E49D-S67C-A160V-K303T L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-I46V +++++ SELECTION 2 L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-K256R- +++++ SELECTION 2 K302R L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-K303T +++++ SELECTION 2 L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-N133S +++++ SELECTION 2 L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-N24D +++++ SELECTION 1 L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-N24D- +++++ IN VIVO 1 S141A-Q184P-A208T L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-P25L- +++++ SELECTION 2 N71I-F147L-K302R L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-Q184P +++++ SELECTION 1 L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-Q99K +++++ SELECTION 2 L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141A +++++ IN VIVO 1 L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G +++++ IN VIVO 1 L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- +++++ IN VIVO 1 Q184P-A208T L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S83N- +++++ SELECTION 2 I295N L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S8T- +++++ SELECTION 2 I115V L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-T65E +++++ IN VIVO 1 L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-T65E- +++++ IN VIVO 1 S141A-Q184P-A208T L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-Y260H +++++ SELECTION 2 L200E-V74P-R197L-H211R-C43S-G44D-R80A-Y81R-N133S- ++++++ IN VIVO 2 S141G-Y260H-R307H L200E-V74P-R197L-H211R-C43S-L135C-S141G ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-C43S-Q99K-N133S-L135C-R136G- ++++++ IN VIVO 2 S141A-K303T L200E-V74P-R197L-H211R-C43S-Q99K-N133S-L135C-R136G- ++++++ IN VIVO 2 S141G-K303T-R307H L200E-V74P-R197L-H211R-C43S-Q99K-N133S-L135C-R136G- ++++++ IN VIVO 2 S141G-Y260H-K303T-R307H L200E-V74P-R197L-H211R-C43S-Q99K-N133S-S141G-Y260H- ++++++ IN VIVO 2 K303T L200E-V74P-R197L-H211R-C43S-R80A-Q99K-L135C-S141G- ++++++ IN VIVO 2 R307H L200E-V74P-R197L-H211R-C43S-R80A-Q99K-N133S-L135C- ++++++ IN VIVO 2 S141G-K303T L200E-V74P-R197L-H211R-C43S-R80A-Q99K-N133S-S141G- ++++++ IN VIVO 2 Y260H-K303T L200E-V74P-R197L-H211R-C43S-R80A-T91S-Q99K-L135C- ++++++ IN VIVO 2 R136G-S141G-K303T L200E-V74P-R197L-H211R-C43S-R80A-Y81R-N133S-S141G- ++++++ IN VIVO 2 K303T-R307H L200E-V74P-R197L-H211R-C43S-R80A-Y81R-Q99K-N133S- ++++++ IN VIVO 2 S141G-K303T L200E-V74P-R197L-H211R-C43S-R80A-Y81R-T91S-Q99K- ++++++ IN VIVO 2 L135C-R136G-S141G-Y260H-K303T L200E-V74P-R197L-H211R-C43S-R80A-Y81R-T91S-Q99K- ++++++ IN VIVO 2 L135C-S141A-Y260H-K303T L200E-V74P-R197L-H211R-C43S-R80A-Y81R-T91S-Q99K- ++++++ IN VIVO 2 N133S-L135C-R136G-S141G-Y260H-K303T L200E-V74P-R197L-H211R-C43S-S141G-R307H ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-C43S-T91S-N133S-S141G-Y260H- ++++++ IN VIVO 2 K303T L200E-V74P-R197L-H211R-C43S-Y81R-L135C-R136G-S141G- ++++++ IN VIVO 2 Y260H-K303T L200E-V74P-R197L-H211R-C43S-Y81R-Q99K-N133S-S141G- ++++++ IN VIVO 2 Y260H-K303T L200E-V74P-R197L-H211R-C43S-Y81R-S141G-E252K-Y260H- ++++++ IN VIVO 2 R307H L200E-V74P-R197L-H211R-C43S-Y81R-S141G-Y260H-K303T ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-C43S-Y81R-T91S-L135C-S141A- ++++++ IN VIVO 2 Y260H-K303T L200E-V74P-R197L-H211R-C43S-Y81R-T91S-N133S-R136G- ++++++ IN VIVO 2 S141G-K303T-R307H L200E-V74P-R197L-H211R-C43S-Y81R-T91S-N133S-S141G- ++++++ IN VIVO 2 Y260H-K303T-G311C L200E-V74P-R197L-H211R-C43S-Y81R-T91S-Q99K-L135C- ++++++ IN VIVO 2 R136G-S141G-K303T L200E-V74P-R197L-H211R-G44D-Q99K-S141G-Y260H-R307H ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-G44D-R80A-N133S-L135C-S141G- ++++++ IN VIVO 2 K303T L200E-V74P-R197L-H211R-G44D-R80A-Q99K-L135C-S141G- ++++++ IN VIVO 2 K303T L200E-V74P-R197L-H211R-G44D-R80A-S141G-Y260H-R307H ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-G44D-R80A-T91S-Q99K-N133S- ++++++ IN VIVO 2 L135C-S141G-Y260H-R307H L200E-V74P-R197L-H211R-G44D-R80A-Y81R-N133S-S141G- ++++++ IN VIVO 2 R307H L200E-V74P-R197L-H211R-G44D-R80A-Y81R-T91S-N133S- ++++++ IN VIVO 2 L135C-S141A-A180T-Y260H-K303T L200E-V74P-R197L-H211R-G44D-S73P-Y81R-T91S-Q99K- ++++++ IN VIVO 2 L135C-S141G-Y260H-K303T L200E-V74P-R197L-H211R-G44D-T91S-K123R-L135C-S141G- ++++++ IN VIVO 2 Y260H-K303T L200E-V74P-R197L-H211R-G44D-T91S-N133S-S141G-Y260H- ++++++ IN VIVO 2 K303T L200E-V74P-R197L-H211R-G44D-T91S-Q99K-A118T-N133S- ++++++ IN VIVO 2 L135C-S141G-S175T-Y260H-K303T L200E-V74P-R197L-H211R-G44D-T91S-Q99K-L135C-S141G- ++++++ IN VIVO 2 K303T L200E-V74P-R197L-H211R-G44D-T91S-Q99K-L135C-S141G- ++++++ IN VIVO 2 Y260H-K303T-R307H L200E-V74P-R197L-H211R-G44D-T91S-Q99K-L135C-S141G- ++++++ IN VIVO 2 Y260H-R307H L200E-V74P-R197L-H211R-G44D-T91S-S141G-R307H ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-G44D-Y81R-Q99K-L135C-S141G- ++++++ IN VIVO 2 Y260H-K303T-R307H L200E-V74P-R197L-H211R-G44D-Y81R-Q99K-N133S-R136G- ++++++ IN VIVO 2 S141G-Y260H-K303T L200E-V74P-R197L-H211R-G44D-Y81R-S141G-R307H ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-G44D-Y81R-S141G-Y260H ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-G44D-Y81R-S141G-Y260H-R307H ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-G44D-Y81R-T91S-L135C-S141G- ++++++ IN VIVO 2 K303T-R307H L200E-V74P-R197L-H211R-G44D-Y81R-T91S-L135C-S141G- ++++++ IN VIVO 2 Y260H-K303T L200E-V74P-R197L-H211R-G44D-Y81R-T91S-Q99K-N133S- ++++++ IN VIVO 2 S141G-Y260H L200E-V74P-R197L-H211R-G44D-Y81R-T91S-Q99K-S141G- ++++++ IN VIVO 2 Y260H-K303T L200E-V74P-R197L-H211R-G90P-T91S-L135C-S141A ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-G90S-T91S-S141A-R307H ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-L135C-A137S-R307H ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-L135C-A137S-S141A-R307H ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-L135C-R136G-S141A ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-L135C-S141A ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-L135C-S141A-A289V ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-L135C-S141A-R307H ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-R80A-D127N-L135C-S141A ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-R80A-G90S-T91G-L135C-S141A ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-R80A-G90S-T91S-S141A-R307H ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-R80A-L135C-R136G-S141A ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-R80A-T91G-A137S-S141A-R307H ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-R80A-Y81R-A137P-S141A-R307H ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-R80A-Y81R-L135C-S141A ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-R80A-Y81R-L135C-S141A-R307H ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-S141A ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-S73P-R80A-L135C-R307H ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-S73P-R80A-T91S-L135C-A137S- ++++++ IN VIVO 2 S141A-R307H L200E-V74P-R197L-H211R-S73P-R80A-Y81R-L135C-S141A- ++++++ IN VIVO 2 R307H L200E-V74P-R197L-H211R-S73P-T91G-L135C-S141A-R307H ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-S73P-T91S-L135C-A137P-S141A- ++++++ IN VIVO 2 R307H L200E-V74P-R197L-H211R-T91G-L135C-R136G-S141A ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-T91P-L135C-A137S-S141A ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-T91P-L135C-S141A-R307H ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-T91S-A137P-S141A ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-Y81R-A137P-S141A ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-Y81R-S141A ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-Y81R-S141A-R307H ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-Y81R-T91G-L135C-A137S ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-Y81R-T91G-S141A ++++++ IN VIVO 2 L200E-V74P-R197L-H211R-Y81R-T91S-S141A ++++++ IN VIVO 2 L200E-V77P-L135C-H211R-V74P-R80A-Y81G-R197L-S141G ++++++ IN VIVO 2 L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141A- ++++++ IN VIVO 2 C43S-Q99K-N133S-K303T L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141A- ++++++ IN VIVO 2 C43S-Q99K-N133S-Y260H-K303T L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141A- ++++++ IN VIVO 2 G44D-N133S-K303T L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141A- ++++++ IN VIVO 2 K303T L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 A178N L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 A180C L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 A180E L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 A180G L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 A180L L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 A180R L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 A54R L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 C43G L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 C43S-G44D-N133S-K303T L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 C43S-N133S-K303T L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 C43S-N133S-Y260H-K303T L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 C43S-Q99K-N133S-K303T L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 C43S-Q99K-N133S-Y260H-K303T-S141G L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 D189E L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 D189S L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 D70L L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 E217M L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 E296T L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 E56S L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 G311P L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 G311Q L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 G44D-N133S-K303T L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 G64E L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 G7L L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 G7Q L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 I119V L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 I52F-P202S L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 I52L L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 I52M L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 I60H L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 I60W L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 N133S L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 N133S-K303T L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 N133S-Y260H-K303T L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 N248T L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 N71G L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 P187E L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 P187G L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 P187M L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 P187V L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 R315T L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 R75G L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 S172M L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 S5A L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 S5F L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 S8C L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 S98T L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 T9H L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 V17I L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 V17L-P25S L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 W313L L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 W313S L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 W313V L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 Y174F L200E-V77P-L135C-H211R-V74P-R80A-Y81R-R197L-S141G- ++++++ IN VIVO 2 Y260F L200E-V74P-V77P-R80A-Y81R-L135C-S141G-R197L-H211R- +++++++ IN VIVO 3 G7L-I52L-S172M-W313V L200E-V74P-V77P-R80A-Y81R-L135C-S141G-R197L-H211R- +++++++ IN VIVO 3 V17L-P25S-D70L-S98T-G311P L200E-V74P-V77P-R80A-Y81R-L135C-S141G-R197L-H211R- +++++++ IN VIVO 3 G7Q-V17L-I60H L200E-V74P-V77P-R80A-Y81R-L135C-S141G-R197L-H211R- +++++++ IN VIVO 3 G7Q-I52L L200E-V74P-V77P-R80A-Y81R-L135C-S141G-R197L-H211R- +++++++ IN VIVO 3 G7Q-I52L-W313L L200E-V74P-V77P-R80A-Y81R-L135C-S141G-R197L-H211R- +++++++ IN VIVO 3 D70L-S98T-R315H L200E-V74P-V77P-R80A-Y81R-L135C-S141G-R197L-H211R- +++++++ IN VIVO 3 D70L-S98T-G311P L200E-V74P-V77P-R80A-Y81R-L135C-S141G-R197L-H211R- +++++++ IN VIVO 3 G7Q-I52L-S172M-W313L L200E-V74P-V77P-R80A-Y81R-L135C-S141G-R197L-H211R- +++++++ IN VIVO 3 I52L-S98T-Y174F-G311P L200E-V74P-V77P-R80A-Y81R-L135C-S141G-R197L-H211R- +++++++ IN VIVO 3 P25S-I60H-G311P L200E-V74P-V77P-R80A-Y81R-L135C-S141G-R197L-H211R- +++++++ IN VIVO 3 S5A-G7Q-V17L-P25S-D70L-Y174F L200E-V74P-V77P-R80A-Y81R-L135C-S141G-R197L-H211R- +++++++ IN VIVO 3 V17L-P25S-D70L-S98T L200E-V74P-V77P-R80A-Y81R-L135C-S141G-R197L-H211R- +++++++ IN VIVO 3 P25S-S98T-G311P L200E-V74P-V77P-R80A-Y81R-L135C-S141G-R197L-H211R- +++++++ IN VIVO 3 I60H L200E-V74P-V77P-R80A-Y81R-L135C-S141G-R197L-H211R- +++++++ IN VIVO 3 P25S-I52L-S98T-Y174F L200E-V74P-V77P-R80A-Y81R-L135C-S141G-R197L-H211R- +++++++ IN VIVO 3 G7Q-P25S-I52L L200E-V74P-V77P-R80A-Y81R-L135C-S141G-R197L-H211R- +++++++ IN VIVO 3 P25S-I52L-S98T-G311P L200E-V74P-V77P-R80A-Y81R-L135C-S141G-R197L-H211R- +++++++ IN VIVO 3 P25S-I119V-W313S L200E-V74P-V77P-R80A-Y81R-L135C-S141G-R197L-H211R- +++++++ IN VIVO 3 P25S-S98T L200E-V74P-V77P-R80A-Y81R-L135C-S141G-R197L-H211R- +++++++ IN VIVO 3 G7Q-P25S-I60H L200E-V74P-V77P-R80A-Y81R-L135C-S141G-R197L-H211R- +++++++ IN VIVO 3 P25S-I60H L200E-V74P-V77P-R80A-Y81R-L135C-S141G-R197L-H211R- +++++++ IN VIVO 3 I52L-S98T L200E-V74P-V77P-R80A-Y81R-L135C-S141G-R197L-H211R- +++++++ IN VIVO 3 S98T-I119V-Y174F L200E-V74P-V77P-R80A-Y81R-L135C-S141G-R197L-H211R- +++++++ IN VIVO 3 V17L-R51H L200E-V74P-V77P-R80A-Y81R-L135C-S141G-R197L-H211R- +++++++ IN VIVO 3 S98T-G311P-W313S L200E-V74P-V77P-R80A-Y81R-L135C-S141G-R197L-H211R- +++++++ IN VIVO 3 I52L L200E-C43S-V74P-R80A-Y81R-N133S-S141G-R197L-H211R- +++++++ IN VIVO 3 K303T-R307H-G7Q-I52L L200E-C43S-V74P-R80A-Y81R-N133S-S141G-R197L-H211R- +++++++ IN VIVO 3 K303T-R307H-G7L-I52L-S172M-W313V-N186M L200E-C43S-V74P-R80A-Y81R-N133S-S141G-R197L-H211R- +++++++ IN VIVO 3 K303T-R307H-A203S-K205R-K206R L200E-C43S-V74P-R80A-Y81R-N133S-S141G-R197L-H211R- +++++++ IN VIVO 3 K303T-R307H-I14V-V16I-V17I L200E-C43S-V74P-R80A-Y81R-N133S-S141G-R197L-H211R- +++++++ IN VIVO 3 K303T-R307H-R315H-R316H-E318D

(29) TABLE-US-00003 TABLE 2 List of mutations involved in the variants of Thermoplasma acidophilum mevalonate kinase with increased activity Mutant Wild-Type Amino Acid Sequence Number Mutation Y3H Y 3 H S5A S 5 A S5F S 5 F I6L I 6 L G7L G 7 L G7Q G 7 Q G7V G 7 V S8C S 8 C S8T S 8 T T9H T 9 H I14V I 14 V V16I V 16 I V17I V 17 I V17L V 17 L A23T A 23 T N24D N 24 D P25L P 25 L P25R P 25 R P25S P 25 S V26F V 26 F H32D H 32 D C43G C 43 G C43S C 43 S G44D G 44 D S45I S 45 I I46V I 46 V E49D E 49 D E49G E 49 G E49S E 49 S R51H R 51 H I52F I 52 F I52L I 52 L I52M I 52 M A54R A 54 R E56S E 56 S H59R H 59 R I60H I 60 H I60W I 60 W G64E G 64 E T65D T 65 D T65E T 65 E S67C S 67 C S67N S 67 N D70L D 70 L D70V D 70 V N71G N 71 G N71I N 71 I S73P S 73 P V74P V 74 P R75G R 75 G R76P R 76 P V77P V 77 P R80A R 80 A R80T R 80 T Y81E Y 81 E Y81G Y 81 G Y81Q Y 81 Q Y81R Y 81 R Y81T Y 81 T S82D S 82 D S83N S 83 N A88T A 88 T F89G F 89 G F89S F 89 S G90P G 90 P G90S G 90 S T91G T 91 G T91P T 91 P T91S T 91 S K92T K 92 T T93I T 93 I V94A V 94 A V94S V 94 S S98T S 98 T Q99K Q 99 K I115V I 115 V A118T A 118 T I119V I 119 V G121A G 121 A K123N K 123 N K123R K 123 R D127N D 127 N N133S N 133 S L135C L 135 C R136G R 136 G A137E A 137 E A137P A 137 P A137S A 137 S S141A S 141 A S141G S 141 G F147L F 147 L F158S F 158 S A160V A 160 V S172M S 172 M Y174F Y 174 F S175T S 175 T A178N A 178 N A180C A 180 C A180E A 180 E A180G A 180 G A180L A 180 L A180R A 180 R A180T A 180 T Y183D Y 183 D Q184P Q 184 P N186M N 186 M P187E P 187 E P187G P 187 G P187M P 187 M P187V P 187 V D189E D 189 E D189S D 189 S R197C R 197 C R197K R 197 K R197L R 197 L L200E L 200 E L200T L 200 T P202S P 202 S A203S A 203 S K205I K 205 I K205R K 205 R K206R K 206 R A208T A 208 T H211K H 211 K H211R H 211 R E217M E 217 M S240G S 240 G N248T N 248 T E252K E 252 K E252S E 252 S K256R K 256 R S259N S 259 N Y260F Y 260 F Y260H Y 260 H E285C E 285 C A289V A 289 V I295N I 295 N E296T E 296 T K302R K 302 R K303T K 303 T R307H R 307 H G311C G 311 C G311P G 311 P G311Q G 311 Q W313L W 313 L W313R W 313 R W313S W 313 S W313T W 313 T W313V W 313 V W313Y W 313 Y R315H R 315 H R315K R 315 K R315T R 315 T R316H R 316 H E318D E 318 D

(30) TABLE-US-00004 TABLE 3 List of the positions modified in the variants of Thermoplasma acidophilum mevalonate kinase with increased activity Position Wild-Type Amino Acid Mutations 3 Y H 5 S A, F 6 I L 7 G L, Q, V 8 S C, T 9 T H 14 I V 16 V I 17 V I, L 23 A T 24 N D 25 P L, R, S 26 V F 32 H D 43 C G, S 44 G D 45 S I 46 I V 49 E D, G, S 51 R H 52 I F, L, M 54 A R 56 E S 59 H R 60 I H, W 64 G E 65 T D, E 67 S C, N 70 D L, V 71 N G, I 73 S P 74 V P 75 R G 76 R P 77 V P 80 R A, T 81 Y E, G, Q, R, T 82 S D 83 S N 88 A T 89 F G, S 90 G P, S 91 T G, P, S 92 K T 93 T I 94 V A, S 98 S T 99 Q K 115 I V 118 A T 119 I V 121 G A 123 K N, R 127 D N 133 N S 135 L C 136 R G 137 A E, P, S 141 S A, G 147 F L 158 F S 160 A V 172 S M 174 Y F 175 S T 178 A N 180 A C, E, G, L, R, T 183 Y D 184 Q P 186 N M 187 P E, G, M, V 189 D E, S 197 R C, K, L 200 L E, T 202 P S 203 A S 205 K I, R 206 K R 208 A T 211 H K, R 217 E M 240 S G 248 N T 252 E K, S 256 K R 259 S N 260 Y F, H 285 E C 289 A V 295 I N 296 E T 302 K R 303 K T 307 R H 311 G C, P, Q 313 W L, R, S, T, V, Y 315 R H, K, T 316 R H 318 E D

Example 5

In Vitro Characterization of Thermoplasma acidophilum Mevalonate Kinase Variants with Increased Activity for the Reaction of Conversion of 3-hydroxyisovalerate into 3-phosphonoxyisovalerate

(31) The activity of Thermoplasma acidophilum mevalonate kinase variants for the conversion of 3-hydroxyisovalerate (HIV) into 3-phosphonoxyisovalerate (PIV) can be assessed by an enzymatic in vitro assay based on purified proteins and on the detection of PIV by High-Performance Liquid Chromatography (HPLC).

(32) The Thermoplasma acidophilum mevalonate kinase enzyme variants were cloned in the pET 25b vector (Novagen). A stretch of 6 histidine codons was inserted after the methionine initiation codon to provide an affinity tag for purification and a unique cleavage site for the TEV protease (ENLYFQG) in order to remove the affinity tag from the purified protein. Competent E. coli BL21 (DE3) cells (Novagen) were transformed with these vectors according to the heat shock procedure and plated out onto LB-agar plates supplemented with the appropriate antibiotic. Cells were grown overnight at 30 C. until individual colonies reach the desired size. Single colonies were then picked and individually transferred into 5 mL of liquid LB medium supplemented with the appropriate antibiotic. Cell growth is carried out with shaking for 16 hours at 30 C. The LB cultures were used to inoculate 1 L of of auto-induction medium (Studier F W, Prat. Exp. Pur. 41, (2005), 207-234) supplemented with the appropriate antibiotic and grown in a shaking incubator set at 170 rpm and 85% humidity 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., 4000 rpm for 20 min and the pellets were frozen at 80 C. The pellets were thawed on ice and resuspended in 40 ml of BugBuster (Millipore) and four microliter of lysonase (Novagen) was added. Cells were incubated 10 minutes at room temperature and then returned to ice for 20 minutes. The bacterial extracts were then clarified by centrifugation at 4 C., 10,000 rpm for 30 min. The clarified bacterial lysates were loaded on Protino Ni-IDA columns (Macherey-Nagel) allowing adsorption of 6-His tagged proteins. Columns were washed and the enzymes of interest were eluted with 6 mL of the supplied elution buffer. Eluates were then concentrated and desalted by centrifugation, washing and resuspension in 1 mL of 100 mM Tris/HCl pH 7.5, 50 mM NaCl, 5% glycerol. Protein concentrations were quantified using a Nanodrop 1000 (ThermoScientific). Cleavage of the affinity tag was then performed by adding 100 U TEV protease (Invitrogen) per 1 g of purified protein and incubated overnight at 4 C. The uncleaved proteins were separated by affinity chromatography using an Akta Purifier and a HisTrap HP 5 mL column (GE Healthcare Life Sciences) using standard protocol. The cleaved proteins were collected in the flow-through and concentrated by centrifugation, washing and resuspension in 100 L of 50 mM Tris/HCl pH 7.5 on Amicon Ultra 4 mL with a 10 kDa cut-off (Merck Millipore). Protein concentrations were quantified using a Nanodrop 1000 (ThermoScientific).

(33) The enzymatic assay for quantifying the conversion of HIV into PIV was carried out at 37 C. in a 50 mM Tris/HCl pH 7.5 buffer with 10 mM MgCl2, 10 mM NaCl, 20 mM ATP, 0.1 mg/mL enzyme and different concentration of HIV ranging from 0 to 128 mM. After 30 min, the reaction was stopped by incubating at 80 C. for 5 min. The rate of PIV production was quantified by HPLC analyses performed using a 1260 Infinity LC System (Agilent), equipped with a refractometer detector and a column heating module. 2 L sample was separated on a Polaris 150 column (1502 mm, 5 m particle size, column temperature 30 C.). The mobile phase consisted of 16 mM sulphuric acid in water with 1% methanol and was run with a flow rate of 1.5 mL/min. Retention time of HIV and PIV was 4.8 and 3.5 min, respectively. Commercial HIV and PIV were used as reference. From the rate of PIV production, and using the Michaelis-Menten approximation, the enzyme catalytic efficiency can then be computed.