Mevalonate diphosphate decarboxylase variant

Abstract

Described are mevalonate diphosphate decarboxylase variants having improved activity in the conversion of 3-phosphonoxyisovaleric acid to isobutene. Such variants can be used in methods for the biological production of isobutene from 3-hydroxyisovaleric acid or from 3-hydroxy-3-methylbutyric acid, for the biological production of 3-methylbut-3-en-1-ol from mevalonic acid or from mevalonic acid-3-phosphate, or for the biological production of 1,3-butadiene from 3-hydroxypent-4-enoic acid or from 3-phosphonoxypent-4-enoic acid. Also described is an enzyme characterized in that it is capable of converting 3-phosphonoxyisovaleric acid to isobutene at a k ‑1 greater than 0.1 s cat -1.

Description

[0001] The present invention relates to mevalonate diphosphate decarboxylase variants having improved activity in the conversion of 3-phosphonoxyisovalerate to isobutene. Additionally, the present invention relates to enzymes characterized in that they are capable of converting 3-phosphonoxyisovalerate at a k -1 greater than 0.1 s cat to isobutene.

[0002] Many chemical compounds currently originate from petrochemicals. Olefins such as ethylene, propylene, different butenes or other pentenes, for example, are used in the plastics industry for producing polypropylene or polyethylene, for example, and in other fields of the chemical industry and in the fuel field. In the past few years, the biological production of plastics (“bioplastics”) and biofuels has become a booming area, due to economic concerns related to oil prices and global (carbon-neutral products) and local (waste management) environmental considerations. Therefore, there is a need for efficient enzymes for producing olefins such as isobutene.

[0003] WO 2010 / 001078 describes a method for producing olefins such as isobutene by the enzymatic conversion of 3-hydroxyalkanoic acids with an enzyme having decarboxylase activity, such as mevalonate diphosphate (MDP) decarboxylase. This method is advantageous because it helps avoid the use of petroleum products, helps reduce the cost of producing plastics and fuels, and can have a significant global environmental impact by allowing carbon to be stored in solid form. It can be shown that mevalonate diphosphate decarboxylase is capable of using substrates other than its natural substrate mevalonate diphosphate, in particular 3-hydroxyalkanoic acids, and converting them to the final olefin. Mevalonate diphosphate (MDP) decarboxylase (enzyme nomenclature EC 4.1.1.33) is an enzyme involved in cholesterol biosynthesis. This enzyme has been isolated from a variety of organisms including animals, fungi, yeast and some bacteria. It can also be expressed by some plants (Lalitha et al., Phytochemistry 24(11), (1985), 2569-2571). Many genes encoding this enzyme have been cloned and sequenced. These enzymes are generally composed of 300 to 400 amino acids and use ATP as a cosubstrate, which is converted to ADP and inorganic phosphate during the reaction. In the first step, a phosphate group is transferred from the ATP molecule to the tertiary alcohol of mevalonate diphosphate, releasing ADP. The reaction intermediate phosphorylated at the 3-hydroxy group undergoes elimination of the phosphate group and decarboxylation in the second step, releasing isopentenyl diphosphate under physiological conditions.

[0004] MDP decarboxylase has been isolated from a variety of different eukaryotic and prokaryotic organisms and has been analyzed and characterized in detail. In addition, a variety of mutants have been generated to identify amino acid residues that may play an important role in the enzymatic activity of the enzyme. For example, Alvear et al. (Biochemistry 21(1982), 4646-4650) described the purification and characterization of avian liver MDP decarboxylase, and Dhe-Paganon et al. (Biochemistry 33(1994), 13355-13362) described the mechanism of the reaction catalyzed by MDP decarboxylase. Berges et al. (J. Bacteriol. 179(1997), 4664-4670) reported mutations in the MDP decarboxylase of Saccharomyces cerevisiae (S. cerevisiae) that result in thermosensitivity. Krepkiy and Miziorko (Protein Sci. 13(2004), 1875-1881) identified the active site residues in the MDP decarboxylase of yeast and analyzed mutations that result in decreased activity. Similarly, Krepkiy and Miziorko (Biochemistry 44(2005), 2671-2677) studied the conserved serine residues located in the proposed interdomain active site cleft of MDP decarboxylase for their relevance and could confirm that mutation of any of these serine residues results in decreased or loss of activity. In addition, Qiu et al. (Bioorganic & medicinal Chemistry Letters 17(2007), 6164-6168) analyzed (rat) MDP decarboxylase and reported a variety of mutants that result in decreased or loss of activity. Voynova et al. (Arch. Biochem. Biophys. 480(2008), 58-67) characterized human MDP decarboxylase and identified several amino acid residues that result in decreased or loss of enzyme activity.

[0005] In addition, crystal structures of several MDP decarboxylases and MDP decarboxylase mutants from different sources have been established, such as the crystal structures of the following enzymes: Staphylococcus epidermidis enzyme (Barta et al., J. Biol. Chem. 286 (2011), 23900-23910; Barta et al., Biochemistry 51 (2012), 5611-5621; PDB accession numbers 3QT5-6-7-8 and 4DPX, 4DPY, 4DPU, 4DPT, 4DU8, 4DU7 and 4DPW), Trypanosoma brucei and Staphylococcus aureus enzymes (Byres et al., J. Mol. Biol. 371 (2007), 540-553; PDB accession numbers 2HKE, 2HK2, 2HK3), human enzyme (Voynova et al., (Arch. Biochem. Biophys. 480 (2008), 58-67; PDB accession number 3D4J), Streptococcus pyogenes enzyme (PDB accession number 2GS8), mouse enzyme (PDB accession number 3F0N), Legionella pneumophila enzyme (PDB accession number 3LTO) and Saccharomyces cerevisiae enzyme (Bonanno et al., Proc. Natl. Acad. Sci. USA. 98 (2001), 12896-12901; PDB accession number 1FI4).

[0006] Lefurgy et al. (J. Biol. Chem. 285 (2010), 20654-20663) analyzed the ligand-binding pocket of the MDP decarboxylase of Streptococcus pneumoniae by using a series of MDP analogs, and Weerasinghe and Dassanayake (J. Mol. Model. 16 (2010) 489-498) reported simulating the structural and functional properties of the Saccharomyces cerevisiae MDP decarboxylase using the wild-type enzyme and heat-sensitive mutants described in Berges et al. (J. Bacteriol. 179 (1997), 4664-4670).

[0007] Byres et al. (J. Mol. Biol. 371 (2007), 540-553) compared the crystal structures of different MDP decarboxylases, particularly those from Trypanosoma brucei (T. brucei), Staphylococcus aureus and Saccharomyces cerevisiae and observed that the overall architecture of the enzyme is conserved.

[0008] WO 2010 / 001078 discloses in particular that 3-hydroxy-3-methylbutyric acid (or 3-hydroxyisovaleric acid) can be converted into isobutene by a decarboxylase, in particular an MDP decarboxylase. In this case, the reaction intermediate is 3-phosphonyloxyisovaleric acid, which is further converted into isobutene in the second part of the reaction. Gogerty et al. (Appl. Environ. Microbiol. 76 (2010), 8004-8010) also reported the formation of isobutene from 3-hydroxy-3-methylbutyric acid using an MDP decarboxylase from Saccharomyces cerevisiae and showed that mutations at residues 145 and 74 of this enzyme (inside or close to the proposed active site of this enzyme) led to an increase in the conversion of 3-hydroxy-3-methylbutyric acid to isobutene. However, for commercial applications, the level of isobutene produced is still too low.

[0009] Subsequent work has shown that different MDP decarboxylases can display different efficiencies with respect to catalyzing the first and second steps of the reaction as described above, with some MDP decarboxylases showing high activity in the first step and others showing high activity in the second step. Thus, it has been proposed to combine two MDP decarboxylases that respectively show high activity in the first and in the second steps of the reaction, thereby optimizing the overall enzymatic reaction (WO 2012 / 052427).

[0010] However, although this method allows the production of alkenes by the enzymatic conversion of 3-hydroxyalkanoic acids (e.g., the production of isobutene from 3-hydroxyisovaleric acid), improvements are still needed, particularly in terms of further increasing the efficiency of the method so as to make it more suitable for industrial purposes.

[0011] This application addresses this need by providing embodiments as defined in the claims.

[0012] In particular, the present invention provides an enzyme, characterized in that it is capable of converting 3-phosphonyloxyisovaleric acid at a k -1 of more than 0.01 s -1 or 0.1 s -1 Preferably more than 1 s -1 More preferably more than 10 s 2 s -1 And even more preferably more than 10 3 s -1 of catConverted to isobutene. Preferably, such an enzyme is a mevalonate diphosphate decarboxylase variant, and even more preferably, such an enzyme has an amino acid sequence showing more than 60% sequence homology with the amino acid sequence shown in SEQ ID NO: 1. Preferably, the enzyme of the present invention is a non-naturally occurring enzyme. This means that they are substantially different from naturally occurring enzymes, especially in terms of their primary structure, i.e., the amino acid sequence. Therefore, they show amino acid sequences that do not exist in nature. Preferably, such non-naturally occurring enzymes are also different from naturally occurring enzymes in that they have higher enzymatic activity towards the reactions described herein.

[0013] Accordingly, the present invention particularly provides variants of mevalonate diphosphate decarboxylase that show improved activity in converting 3-phosphonoxyisovalerate to isobutene, thus allowing a substantial increase in the efficiency of producing isobutene from 3-hydroxyisovalerate.

[0014] In the context of the present invention, the term "mevalonate diphosphate decarboxylase" refers to an enzyme that naturally has the ability to convert mevalonate diphosphate to isopentenyl diphosphate and is classified as EC 4.1.1.33. The term "mevalonate diphosphate decarboxylase" also encompasses enzymes that are classified as mevalonate diphosphate decarboxylase and act on mevalonate monophosphate, i.e., the enzyme is a mevalonate monophosphate decarboxylase. An example of such an enzyme is the enzyme from Roseiflexus sp. (strain RS-1) (Uniprot accession number: A5V173) that is classified as mevalonate diphosphate decarboxylase. Since it has been reported that some bacteria from the genus Roseiflexus, such as Roseiflexus castenholzii, have an alternative mevalonate pathway involving the action of mevalonate monophosphate decarboxylase, the enzyme from Roseiflexus sp. can act as a mevalonate monophosphate decarboxylase. Additionally, a third mevalonate pathway has recently been described in T. acidophilum. In this pathway, the formation of isopentenyl monophosphate may occur via mevalonate-3,5-pyrophosphate (MVA-3,5-PP). Two genes capable of involving the decarboxylation of this intermediate are Ta0461 and Ta0893. These genes can be related to as IPR005935 (http: / / www.ebi.ac.uk / interpro / entry / IPR005935)is related to the diphosphomevalonate decarboxylase family referenced in the InterPro database, but these two enzymes can act as mevalonate-3,5-bisphosphate decarboxylase. Thus, the term "mevalonate diphosphate decarboxylase" also encompasses such enzymes. The present invention relates to variants derived from mevalonate diphosphate decarboxylase. Variants of such an enzyme according to the present invention are characterized in that they are derived from an MDP decarboxylase having the amino acid sequence shown in SEQ ID NO: 1 or having a related sequence (at least 40% identical, preferably at least 50% identical, even more preferably at least 60% or at least 90% identical) and in which the mutations are made at one or more positions as shown below, and are characterized in that they show the ability to convert 3-phosphonoxyisovalerate into isobutene and that they can effect this conversion with improved activity. In a preferred embodiment, the variants according to the present invention are derived from a sequence showing at least 80% sequence identity with SEQ ID NO: 1 and in which one or more substitutions and / or deletions and / or insertions have been made at the positions shown below.

[0015] Given the high conservation among MDP decarboxylases, the teachings of the present invention are not limited to the enzyme of Streptococcus mitis (represented by SEQ ID NO: 1) but can be extended to MDP decarboxylases from other organisms. Thus, the present invention also relates to variants of MDP decarboxylase, said variants being derived from an enzyme that is structurally related to the Streptococcus mitis sequence and that shows one or more substitutions and / or deletions and / or insertions at positions corresponding to any of the positions shown below. The term "structurally related" refers to an MDP decarboxylase that shows at least n% sequence identity with the sequence shown in SEQ ID NO: 1, where n is an integer between 40 and 100, preferably 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99. In a preferred embodiment, the structurally related MDP decarboxylase is of prokaryotic origin. These variants are characterized in that they show the ability to convert 3-phosphonoxyisovalerate into isobutene and that they can effect this conversion with improved activity compared to the enzyme from which these variants are derived. Even more preferably, such variants also show improved activity when compared to the enzyme having the amino acid sequence shown in SEQ ID NO: 1.

[0016] Thus, in one embodiment, a variant of the MDP decarboxylase according to the invention has or preferably is derived from a sequence that is at least n% identical to SEQ ID NO: 1 and has one or more substitutions and / or deletions and / or insertions at the positions shown below, where n is an integer between 40 and 100, preferably 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99. When the sequences being compared do not have the same length, the degree of identity refers to the percentage of amino acid residues in the shorter sequence that are identical to amino acid residues within the longer sequence or to the percentage of amino acid residues in the longer sequence that are identical to amino acid residues within the shorter sequence. Preferably, it refers to the percentage of amino acid residues in the shorter sequence that are identical to amino acid residues within the longer sequence. The degree of sequence identity can be determined according to methods well known in the art, preferably using a suitable computer algorithm such as CLUSTAL.

[0017] When using the Clustal analysis method to determine whether a particular sequence is identical to a reference sequence, for example, at least 40%, 50%, 60% or 90%, for the comparison of amino acid sequences, the default settings can be used or the settings are preferably as follows: Matrix: blosum 30; Open gap penalty: 10.0; Extension gap penalty: 0.05; Delay divergence: 40; Gap separation distance: 8. For nucleotide sequence comparison, the extension gap penalty is preferably set to 5.0.

[0018] In a preferred embodiment, ClustalW2 is used for the comparison of amino acid sequences. In the case of pairwise comparison / alignment, the following settings are preferably selected: Protein weight matrix: BLOSUM 62; Gap open: 10; Gap extension: 2. In the case of multiple comparison / alignment, the following settings are preferably selected: Protein weight matrix: BLOSUM 62; Gap open: 10; Gap extension: 2; Gap distance: 5; No end gaps.

[0019] Preferably, the degree of identity is calculated over the entire length of the sequence.

[0020] Examples of MDP decarboxylases showing at least 60%, particularly between 60% and 80%, sequence identity with SEQ ID NO: 1 are shown in the table below.

[0021] Table 1

[0022]

[0023]

[0024] Examples of MDP decarboxylases showing at least 80%, in particular between 80% and 90% sequence identity with SEQ ID NO: 1 are shown in the table below.

[0025] Table 2

[0026]

[0027]

[0028] Examples of MDP decarboxylases showing at least 90%, in particular between 90% and 100% sequence identity with SEQ ID NO: 1 are shown in the table below.

[0029] Table 3

[0030]

[0031] Amino acid residues located at positions corresponding to the positions shown below in the amino acid sequence shown in SEQ ID NO: 1 can be identified by the person skilled in the art by methods known in the art. For example, such amino acid residues can be identified by aligning the sequence in question with the sequence shown in SEQ ID NO: 1 and by determining the positions corresponding to the above positions of SEQ ID NO: 1. The alignment can be carried out by means and methods known to the person skilled in the art, for example by using known computer algorithms such as the Lipman - Pearson method (Science 227 (1985), 1435) or the CLUSTAL algorithm. Preferably, in such an alignment, the maximum homology is assigned to the conserved amino acid residues present in the amino acid sequence.

[0032] In a preferred embodiment, ClustalW2 is used for the comparison of amino acid sequences. In the case of pairwise comparison / alignment, the following settings are preferably selected: Protein weight matrix: BLOSUM 62; Gap opening: 10; Gap extension: 2. In the case of multiple comparison / alignment, the following settings are preferably selected: Protein weight matrix: BLOSUM 62; Gap opening: 10; Gap extension: 2; Gap distance: 5; No end gaps.

[0033] When aligning the amino acid sequences of MDP decarboxylases by means of such a method, the positions of the corresponding amino acid residues can be determined in each MDP decarboxylase regardless of the insertions or deletions present in the amino acid sequence. Examples of the alignment are provided in Figures 10 to 12 below.

[0034] In the context of the present invention, "substituted with another amino acid residue" means that the corresponding amino acid residue at the indicated position can be substituted with any other possible amino acid residue, for example, a naturally occurring amino acid or a non-naturally occurring amino acid (Brustad and Arnold, Curr. Opin. Chem. Biol. 15 (2011), 201-210), preferably with an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Preferred substitutions at certain positions are further indicated below. Additionally, the term "substituted" or "substitution" also means that the corresponding amino acid residue at the indicated position is modified.

[0035] Such modifications include naturally occurring modifications and non-naturally occurring modifications. Naturally occurring modifications include, but are not limited to, eukaryotic post-translational modifications such as attachment of functional groups (e.g., acetate, phosphate, hydroxyl, lipid (myristoylation of glycine residue) and sugar (e.g., glycosylation of arginine, asparagine, etc.). Naturally occurring modifications also encompass chemical structure changes resulting from citrullination, carbamylation, and disulfide bond formation between cysteine residues; attachment of cofactors (FMN or FAD that can be covalently attached) or attachment of peptides (e.g., ubiquitination or SUMOylation).

[0036] Non-naturally occurring modifications include, for example, in vitro modifications such as biotinylation of lysine residues or inclusion of non-canonical amino acids (see Liu and Schultz, Annu. Rev. Biochem. 79 (2010), 413-44 and Wang et al., Chem. Biol. 2009 March 27; 16(3), 323-336; doi:101016 / jchembiol.2009.03.001).

[0037] In the context of the present invention, "deleted" or "deletion" means the absence of an amino acid at the corresponding position.

[0038] In the context of the present invention, "inserted" or "insertion" means the insertion of one or two, preferably one, amino acid residue at the corresponding position, preferably in front of the indicated position.

[0039] Accordingly, the present invention relates to mevalonate diphosphate decarboxylase variants that show improved activity in converting 3-phosphonatooxyisovalerate to isobutene relative to the corresponding mevalonate diphosphate decarboxylase from which they are derived, wherein the mevalonate diphosphate decarboxylase variants are characterized by showing one or more substitutions, deletions and / or insertions compared to the corresponding sequence from which the variant is derived and wherein these substitutions, deletions and / or insertions occur at one or more positions corresponding to positions 282, 9, 11, 16, 24, 28, 42, 43, 45, 53, 66, 77, 80, 91, 105, 116, 118, 120, 121, 122, 123, 129, 134, 141, 159, 160, 161, 173, 177, 180, 186, 215, 238, 241, 242, 248, 251, 253, 258, 264, 279, 291, 293, 297, 299, 303, 307, 308, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267 and 315 in the amino acid sequence shown in SEQ ID NO: 1. "Corresponding" to these positions means corresponding to any of these positions in the relevant sequence.

[0040] In a preferred embodiment, the mevalonate diphosphate decarboxylase from which the variant is derived is a mevalonate diphosphate decarboxylase that shows the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 40%, preferably at least 50% and even more preferably at least 60%, or most preferably at least 90% sequence identity to SEQ ID NO: 1.

[0041] Accordingly, in one embodiment, the invention relates to variants of mevalonate diphosphate decarboxylase having an amino acid sequence as set forth in SEQ ID NO: 1 or an amino acid sequence having at least 40%, 50%, 60% or 90% sequence identity with SEQ ID NO: 1, wherein one or more amino acid residues at positions 9, 11, 16, 24, 28, 42, 43, 45, 53, 66, 77, 80, 91, 105, 116, 118, 120, 121, 122, 123, 129, 134, 141, 159, 160, 161, 173, 177, 180, 186, 215, 238, 241, 242, 248, 251, 253, 258, 264, 279, 282, 291, 293, 297, 299, 303, 307, 308, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267 and 315 as shown in the amino acid sequence set forth in SEQ ID NO: 1 or at positions corresponding to any of these positions in a related sequence are replaced or deleted with another amino acid residue or show an insertion at at least one of these positions and wherein the mevalonate diphosphate decarboxylase has improved activity in converting 3-phosphonoxyisovalerate to isobutene. In a preferred embodiment, the deletion, insertion or substitution is at a position selected from positions 9, 11, 42, 43, 45, 66, 77, 116, 118, 120, 121, 123, 129, 134, 159, 160, 173, 177, 186, 251, 253, 282, 293, 297, 299, 303, 307 and 308.

[0042] The inventors have found that the activity of mevalonate diphosphate decarboxylase in catalyzing the second step of the above conversion of 3-hydroxyisovalerate to isobutene, i.e., the conversion of 3-phosphonoxyisovalerate to isobutene, can be significantly improved by mutating the MDP decarboxylase at certain positions. They used the enzyme from Streptococcus mitis as a model enzyme, the sequence of which is shown in SEQ ID NO: 1. The single mutations identified led to an increase in activity of up to more than 300% compared to the unmutated Streptococcus mitis enzyme sequence (represented by SEQ ID NO: 1). Combinatorial mutations led to a further increase in activity of up to 750%, and additional mutations identified in addition to these combinations allowed for a further increase of 16-fold.

[0043] In particular, the inventors have found that substitution at positions 9, 11, 16, 24, 28, 42, 43, 45, 53, 66, 77, 80, 91, 105, 116, 118, 120, 121, 122, 123, 129, 134, 141, 159, 160, 161, 173, 177, 180, 186, 215, 238, 241, 242, 248, 251, 253, 258, 264, 279, 282, 291, 293, 297, 299, 303, 307, 308, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267 or 315, or substitution at combinations of these positions, results in a substantial increase in the ability of the enzyme to convert 3-phosphonoxyisovalerate into isobutene.

[0044] As shown above, variants of the MDP decarboxylase according to the invention are characterized in that they show increased activity in converting 3-phosphonoxyisovalerate into isobutene when compared to the MDP decarboxylase from which the variant is derived. Thus, in the case of a variant derived from the MDP decarboxylase of Streptococcus mitis having the amino acid sequence shown in SEQ ID NO: 1, the variant shows increased activity in converting 3-phosphonoxyisovalerate into isobutene when compared to the MDP decarboxylase shown in SEQ ID NO: 1. When the variant is derived from an MDP decarboxylase that is structurally related to the MDP decarboxylase of Streptococcus mitis as defined above, the variant shows increased activity in converting 3-phosphonoxyisovalerate into isobutene when compared to the corresponding starting sequence into which the corresponding mutation has been introduced. In a particularly preferred embodiment, such variants also show increased activity in converting 3-phosphonoxyisovalerate into isobutene when compared to the MDP decarboxylase shown in SEQ ID NO: 1. The activity of converting 3-phosphonoxyisovalerate into isobutene can be determined by methods known to those skilled in the art. In one embodiment, this activity is determined as described in the examples attached herein. In a specific embodiment, this activity can be measured in particular by using the following assay:

[0045] Transform nucleic acid molecules encoding various enzymes into Escherichia coli, such as Escherichia coli BL21DE3 and express the enzymes in Escherichia coli. Subsequently, the activity of the enzymes can be measured using the following

[0046] - crude cell lysates (i.e., centrifuging the cell culture, resuspending the cell pellet in a hypotonic buffer, sonicating, or not sonicating but not centrifuging); or

[0047] - Soluble fraction (i.e., the supernatant obtained after centrifuging the crude cell lysate); or

[0048] - Purified protein (i.e., the recombinant protein present in the soluble fraction and captured on a resin, for example, using chromatography).

[0049] The activity of the expressed enzyme was tested as follows:

[0050] The reaction mixture was prepared in a glass GC vial by mixing the crude cell lysate, soluble fraction, or purified protein as described above with 3 - phosphonyloxyisovaleric acid to a final concentration of 50 mM Tris pH 7. ATP, MgCl 2 and KCl were provided as cofactors.

[0051] The vial was sealed completely and the reaction mixture was incubated at 37 °C for an appropriate time (e.g., 24 hours). Isobutene produced by the reaction was determined by gas chromatography by injecting the gas phase obtained from the sample vial into a gas chromatograph. Controls in the assay were cultures of bacteria with untransformed bacteria, bacteria transformed with an empty expression vector, and bacteria expressing the corresponding starting enzyme (e.g., the Streptococcus mitis enzyme showing the amino acid sequence as shown in SEQ ID NO: 1):.

[0052] The 3 - phosphonyloxyisovaleric acid used in the assay can be prepared in different ways. One possibility is to provide this compound by the following reaction:

[0053] 0.063 mg / ml of purified Thermophilus acidophilum MDP decarboxylase was mixed with 50 mM hydroxyisovaleric acid, 40 mM ATP in 50 mM Tris - Cl pH 7, 20 mM KCl, 20 mM MgCl 2 and incubated at 45 °C for 24 hours. This enzymatically prepared 3 - phosphonyloxyisovaleric acid substrate was used in the reaction mixture.

[0054] Another preferred possibility is to chemically synthesize the 3 - phosphonyloxyisovaleric acid used in the assay. Figure 9 The synthetic scheme is shown in.

[0055] If chemically synthesized 3 - phosphonyloxyisovaleric acid is used in the assay, the concentration is preferably between 3 mM and 8 mM, and preferably, it is 5 mM.

[0056] In a preferred assay for examining enzyme activity, a purified enzyme is used. In this case, the reaction mixture contains 500 μg of the purified enzyme in 50 mM Tris HCl pH 7.5 and 3 - phosphonyloxyisovaleric acid (provided as described above).

[0057] Ensure the presence of cofactors such as ATP, MgCl2 and KCl. The volume was adjusted to 500 μl with 50 mM Tris HCl pH 7.5 and the reaction was incubated at 37 °C for 15 hours. Subsequently, the isobutene produced was determined by gas chromatography.

[0058] In one embodiment, 3-phosphonyloxyisovaleric acid is prepared enzymatically as described above.

[0059] In a particularly preferred embodiment, 3-phosphonyloxyisovaleric acid is chemically synthesized. In this case, the reaction mixture contains:

[0060] 200 μg of purified enzyme

[0061] 3-phosphonyloxyisovaleric acid

[0062] 5 mM ATP, 20 mM KCl, 10 mM MgCl 2 and 50 mM Tris-Cl pH 7.5.

[0063] To test the enzyme activity, varying concentrations of 3-phosphonyloxyisovaleric acid were used, preferably the following concentrations: 0.625 mM, 1.25 mM, 2.5 mM, 5 mM, 10 mM, 20 mM, 40 mM, 80 mM and 160 mM. A concentration of 5 mM of 3-phosphonyloxyisovaleric acid is particularly preferred.

[0064] The volume was adjusted to 500 μl with 50 mM Tris HCl pH 7.5 and the reaction was incubated at 37 °C for 15 hours. Subsequently, the isobutene produced was determined by gas chromatography.

[0065] An example of this assay for determining the activity of the enzyme to convert 3-phosphonyloxyisovaleric acid to isobutene is given in Example 1 of the Examples section below.

[0066] In the context of the present invention, "improved activity" means that the activity of the MDP decarboxylase in question is at least 10%, preferably at least 20%, more preferably at least 30% or 50%, even more preferably at least 70% or 80% and particularly preferably at least 90% or 100% higher than the activity of the MDP decarboxylase from which the variant is derived, preferably higher than the activity of the MDP decarboxylase of Streptococcus mitis represented by SEQ ID NO: 1. In an even more preferred embodiment, the improved activity can be at least 150%, at least 200%, at least 300%, at least 750% or at least 1000% higher than the activity of the MDP decarboxylase from which the variant is derived, preferably higher than the activity of the Streptococcus mitis MDP decarboxylase represented by SEQ ID NO: 1. In a particularly preferred embodiment, the activity is measured using an assay with purified enzyme and chemically synthesized 3-phosphonoxyisovaleric acid as described above. The improved activity of the variant can be measured as higher isobutene production over a given time under defined conditions compared to the parental enzyme. This improved activity can be due to a higher kcat value. It can also be due to a lower Km value. It can also be due to a higher kcat / Km value. The degree of improvement can be measured as the improvement in isobutene production. The degree of improvement can also be measured in terms of kcat improvement, kcat / Km improvement or Km reduction.

[0067] According to one embodiment, the MDP decarboxylase of the present invention has an amino acid sequence, wherein

[0068] (1) the amino acid residue at position 1 in the amino acid sequence shown in SEQ ID NO: 1 or at the position corresponding to this position in the relevant sequence is deleted or replaced with leucine; and / or

[0069] (2) the amino acid residue at position 2 in the amino acid sequence shown in SEQ ID NO: 1 or at the position corresponding to this position in the relevant sequence is deleted or replaced with histidine; and / or

[0070] (3) the amino acid residue at position 9 in the amino acid sequence shown in SEQ ID NO: 1 or at the position corresponding to this position in the relevant sequence is deleted or replaced with leucine; and / or

[0071] (4) the amino acid residue at position 11 in the amino acid sequence shown in SEQ ID NO: 1 or at the position corresponding to this position in the relevant sequence is deleted or replaced with cysteine, glutamate or phenylalanine, preferably cysteine; and / or

[0072] (5) the amino acid residue at position 16 in the amino acid sequence shown in SEQ ID NO: 1 or at the position corresponding to this position in the relevant sequence is deleted or replaced with leucine; and / or

[0073] (6) Deleting or replacing the amino acid residue at position 23 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with leucine; and / or

[0074] (7) Deleting or replacing the amino acid residue at position 24 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with arginine, serine or leucine; and / or

[0075] (8) Deleting or replacing the amino acid residue at position 28 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with lysine or alanine; and / or

[0076] (9) Deleting or replacing the amino acid residue at position 31 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with serine; and / or

[0077] (10) Deleting or replacing the amino acid residue at position 42 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with alanine or leucine; and / or

[0078] (11) Deleting or replacing the amino acid residue at position 43 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with leucine; and / or

[0079] (12) Deleting or replacing the amino acid residue at position 45 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with leucine, phenylalanine, methionine or valine, preferably leucine; and / or

[0080] (13) Deleting or replacing the amino acid residue at position 53 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with valine; and / or

[0081] (14) Deleting or replacing the amino acid residue at position 57 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with serine; and / or

[0082] (15) Deleting or replacing the amino acid residue at position 58 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with threonine; and / or

[0083] (16) Deleting or replacing the amino acid residue at position 66 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with histidine; and / or

[0084] (17) Deleting or replacing the amino acid residue at position 75 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with isoleucine; and / or

[0085] (18) Deleting or replacing the amino acid residue at position 77 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with asparagine or arginine; and / or

[0086] (19) Deleting or replacing the amino acid residue at position 80 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with glycine; and / or

[0087] (20) Deleting or replacing the amino acid residue at position 86 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with glutamine; and / or

[0088] (21) Deleting or replacing the amino acid residue at position 87 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with glutamate; and / or

[0089] (22) Deleting or replacing the amino acid residue at position 91 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with histidine; and / or

[0090] (23) Deleting or replacing the amino acid residue at position 105 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with alanine; and / or

[0091] (24) Deleting or replacing the amino acid residue at position 111 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with methionine; and / or

[0092] (25) Deleting or replacing the amino acid residue at position 116 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with arginine, isoleucine, leucine, serine or methionine, preferably arginine or isoleucine; and / or

[0093] (26) Deleting or substituting the amino acid residue at position 118 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with leucine or tryptophan; and / or

[0094] (27) Deleting or substituting the amino acid residue at position 120 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with asparagine, leucine, arginine, isoleucine or valine, preferably asparagine, leucine, arginine or isoleucine; and / or

[0095] (28) Deleting or substituting the amino acid residue at position 121 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with arginine, leucine, tryptophan, phenylalanine, tyrosine, asparagine or lysine, preferably arginine or phenylalanine; and / or

[0096] (29) Substituting the amino acid residue at position 122 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with methionine or tyrosine; and / or

[0097] (30) Deleting or substituting the amino acid residue at position 123 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with methionine or arginine; and / or

[0098] (30) Deleting or substituting the amino acid residue at position 129 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with proline or valine; and / or

[0099] (32) Deleting or substituting the amino acid residue at position 134 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with glycine; and / or

[0100] (33) Deleting or substituting the amino acid residue at position 139 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with cysteine or alanine; and / or

[0101] (34) Deleting or substituting the amino acid residue at position 141 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with proline, cysteine, glycine or threonine; and / or

[0102] (35) Deleting or replacing the amino acid residue at position 142 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with alanine; and / or

[0103] (36) Deleting or replacing the amino acid residue at position 159 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with leucine; and / or

[0104] (37) Deleting or replacing the amino acid residue at position 160 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with valine; and / or

[0105] (38) Deleting or replacing the amino acid residue at position 161 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with arginine; and / or

[0106] (39) Deleting or replacing the amino acid residue at position 164 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with glutamine; and / or

[0107] (40) Deleting or replacing the amino acid residue at position 166 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with serine; and / or

[0108] (41) Deleting or replacing the amino acid residue at position 173 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with cysteine; and / or

[0109] (42) Deleting or replacing the amino acid residue at position 177 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with valine or cysteine, preferably valine; and / or

[0110] (43) Deleting or replacing the amino acid residue at position 179 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with lysine or leucine; and / or

[0111] (44) Deleting or replacing the amino acid residue at position 180 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with proline; and / or

[0112] (45) Deleting or replacing the amino acid residue at position 182 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with glutamic acid; and / or

[0113] (46) Deleting or replacing the amino acid residue at position 186 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with histidine, leucine, valine, isoleucine or asparagine; and / or

[0114] (47) Deleting or replacing the amino acid residue at position 188 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with cysteine; and / or

[0115] (48) Deleting or replacing the amino acid residue at position 198 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with aspartic acid; and / or

[0116] (49) Deleting or replacing the amino acid residue at position 204 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with leucine; and / or

[0117] (50) Deleting or replacing the amino acid residue at position 205 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with histidine; and / or

[0118] (51) Deleting or replacing the amino acid residue at position 208 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with leucine; and / or

[0119] (52) Deleting or replacing the amino acid residue at position 215 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with alanine; and / or

[0120] (53) Deleting or replacing the amino acid residue at position 221 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with glutamic acid; and / or

[0121] (54) Deleting or replacing the amino acid residue at position 227 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with lysine; and / or

[0122] (55) Deleting or substituting the amino acid residue at position 231 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with glutamine or leucine; and / or

[0123] (56) Deleting or substituting the amino acid residue at position 238 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with arginine, glutamic acid or lysine; and / or

[0124] (57) Deleting or substituting the amino acid residue at position 241 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with methionine or isoleucine; and / or

[0125] (58) Deleting or substituting the amino acid residue at position 242 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with alanine or glutamic acid; and / or

[0126] (59) Deleting or substituting the amino acid residue at position 246 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with glutamic acid; and / or

[0127] (60) Deleting or substituting the amino acid residue at position 248 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with threonine; and / or

[0128] (61) Deleting or substituting the amino acid residue at position 251 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with methionine, phenylalanine or valine, preferably methionine; and / or

[0129] (62) Deleting or substituting the amino acid residue at position 252 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with glutamic acid; and / or

[0130] (63) Deleting or substituting the amino acid residue at position 253 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with valine or isoleucine; and / or

[0131] (64) Deleting or substituting the amino acid residue at position 255 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with glutamic acid; and / or

[0132] (65) Deleting or replacing the amino acid residue at position 258 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with leucine; and / or

[0133] (66) Deleting or replacing the amino acid residue at position 264 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with glutamine; and / or

[0134] (67) Deleting or replacing the amino acid residue at position 267 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with arginine; and / or

[0135] (68) Deleting or replacing the amino acid residue at position 279 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with alanine; and / or

[0136] (69) Deleting or replacing the amino acid residue at position 282 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with cysteine, serine, glutamate, glycine, glutamine, threonine, valine, alanine or aspartic acid; and / or

[0137] (70) Deleting or replacing the amino acid residue at position 291 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with aspartic acid; and / or

[0138] (71) Deleting or replacing the amino acid residue at position 293 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with phenylalanine or tryptophan; and / or

[0139] (72) Deleting or replacing the amino acid residue at position 297 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with cysteine or leucine, preferably cysteine; and / or

[0140] (73) Deleting or replacing the amino acid residue at position 299 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with proline or lysine; and / or

[0141] (74) Deleting or replacing the amino acid residue at position 303 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with methionine; and / or

[0142] (75) Deleting or replacing the amino acid residue at position 307 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with histidine; and / or

[0143] (76) Deleting or replacing the amino acid residue at position 308 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with serine; and / or

[0144] (77) Deleting or replacing the amino acid residue at position 315 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence with serine.

[0145] The present invention also relates to variants as defined in (1) to (77) above, wherein the amino acid residue referred to as replacing the amino acid residue at the position in SEQ ID NO: 1 is not this specific amino acid residue, but an amino acid residue conservative relative to the indicated replacement amino acid.

[0146] Whether an amino acid is conservative relative to another amino acid can be determined by means and methods known in the art. One possibility is the PAM250 matrix; alternatively, the Blosum Family matrix can be used.

[0147] In one embodiment, the present invention relates to variants of MDP decarboxylase having an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having at least 40%, 50%, 60% or 90% sequence identity with SEQ ID NO: 1, wherein the amino acid residue at position 282 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence is replaced or deleted with another amino acid residue. In a preferred embodiment, the present invention relates to such variants wherein at least one other amino acid residue is replaced or deleted at a position selected from positions 9, 11, 16, 24, 28, 42, 43, 45, 53, 66, 77, 80, 91, 105, 116, 118, 120, 121, 122, 123, 129, 134, 141, 159, 160, 161, 173, 177, 180, 186, 215, 238, 241, 242, 248, 251, 253, 258, 264, 279, 291, 293, 297, 299, 303, 307, 308, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267 and 315, preferably selected from positions 9, 11, 16, 24, 28, 42, 45, 53, 80, 91, 105, 116, 118, 120, 121, 122, 123, 129, 141, 159, 161, 173, 177, 180, 215, 238, 241, 242, 248, 251, 253, 264, 279, 291, 293, 297, 299, 303, 307, 308 and 315, even more preferably selected from positions 9, 11, 42, 45, 116, 118, 120, 121, 122, 123, 129, 177, 251, 253, 264, 293, 297 and 303.

[0148] In a specific embodiment, the amino acid residue at position 282 in the amino acid sequence shown in SEQ ID NO: 1 or at the position corresponding to this position in a related sequence is replaced with cysteine, serine, glutamic acid, glycine, glutamine, threonine, valine, alanine or aspartic acid, preferably with cysteine. In another specific embodiment, the substitution at any of the positions 9, 11, 16, 24, 28, 42, 43, 45, 53, 66, 77, 80, 91, 105, 116, 118, 120, 121, 122, 123, 129, 134, 141, 159, 160, 161, 173, 177, 180, 186, 215, 238, 241, 242, 248, 251, 253, 258, 264, 279, 291, 293, 297, 299, 303, 307, 308, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267 and 315 is as shown above.

[0149] In one embodiment, the present invention relates to variants of MDP decarboxylase having an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having at least 40%, 50%, 60% or 90% sequence identity with SEQ ID NO: 1, wherein the amino acid residue at position 121 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence is deleted or replaced with another amino acid residue, preferably arginine, leucine, lysine or phenylalanine. In a preferred embodiment, the present invention relates to such variants wherein at least one other amino acid residue is deleted or replaced at a position selected from positions 9, 11, 16, 24, 28, 42, 43, 45, 53, 66, 77, 80, 91, 105, 116, 118, 120, 122, 123, 129, 134, 141, 159, 160, 161, 173, 177, 180, 186, 215, 238, 241, 242, 248, 251, 253, 258, 264, 279, 282, 291, 293, 297, 299, 303, 307, 308, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267 and 315, preferably selected from positions 11, 16, 24, 28, 45, 53, 80, 91, 105, 116, 118, 120, 123, 141, 159, 161, 173, 177, 180, 215, 238, 241, 242, 248, 258, 279, 282, 291, 297, 299, 303, 307, 308 and 315. The substitution at any of these positions is preferably one of those listed above.

[0150] The invention also relates to variants of MDP decarboxylase, said variants having an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having at least 40%, 50%, 60% or 90% sequence identity with SEQ ID NO: 1, wherein the amino acid residue at position 282 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence is deleted or replaced with another amino acid residue, wherein the amino acid residue at position 121 in the amino acid sequence shown in SEQ ID NO: 1 or at the corresponding position in a related sequence is deleted or replaced with another amino acid residue and wherein at least one other amino acid residue is replaced at a position selected from positions 9, 11, 16, 24, 28, 42, 43, 45, 53, 66, 77, 80, 91, 105, 116, 118, 120, 122, 123, 129, 134, 141, 159, 160, 161, 173, 177, 180, 186, 215, 238, 241, 242, 248, 251, 253, 258, 264, 279, 291, 293, 297, 299, 303, 307, 308, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267 and 315, preferably selected from positions 11, 16, 24, 28, 45, 53, 80, 91, 105, 116, 118, 120, 123, 141, 159, 161, 173, 177, 180, 215, 238, 241, 242, 248, 258, 279, 291, 297, 299, 303, 307, 308 and 315.

[0151] In another specific embodiment, the amino acid residue at position 121 in the amino acid sequence shown in SEQ ID NO: 1 or at the position corresponding to this position in a related sequence is replaced with arginine, leucine, lysine, and phenylalanine. In yet another preferred embodiment, the substitution at any of the positions 9, 11, 16, 24, 28, 42, 43, 45, 53, 66, 77, 80, 91, 105, 116, 118, 120, 122, 123, 129, 134, 141, 159, 160, 161, 173, 177, 180, 186, 215, 238, 241, 242, 248, 251, 253, 258, 264, 279, 291, 293, 297, 299, 303, 307, 308, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267, and 315 is as those shown above.

[0152] In a preferred embodiment, the variant of the present invention is characterized in that it contains at least three deletions, substitutions, and / or insertions, wherein one deletion / substitution is at position 282 in the amino acid sequence shown in SEQ ID NO: 1 or at the position corresponding to this position in a related sequence, another deletion / substitution is at position 121 in the amino acid sequence shown in SEQ ID NO: 1 or at the position corresponding to this position in a related sequence and at least one other deletion / substitution is at a position selected from positions 11, 45, 116, 120, or 177 of SEQ ID NO: 1 or at the position corresponding to any of these positions.

[0153] Preferably, SEQ ID NO: 1 is modified at least by deletion or substitution at the following positions or at the positions corresponding to any of these positions in a related sequence as defined above:

[0154] 45, 121, and 282; or

[0155] 11, 121, and 282; or

[0156] 116, 121, and 282; or

[0157] 121, 177, and 282; or

[0158] 120, 121, and 282; or

[0159] 173, 282, and 297.

[0160] Substitution at the indicated positions is preferably those as shown above.

[0161] Particularly preferred variants having three mutations show the following substitutions in SEQ ID NO: 1:

[0162] E45L-Y121R-K282C

[0163] K282C-Y121R-Y11E

[0164] K116I-Y121R-K282C

[0165] Y121R-E177V-K282C

[0166] A120R-Y121L-K282C

[0167] M173C-K282C-F297L

[0168] or corresponding substitutions at the corresponding positions in structurally related sequences as defined above.

[0169] The present invention also relates to variants of MDP decarboxylase having an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having at least 40%, 50%, 60% or 90% sequence identity with SEQ ID NO: 1, wherein the amino acid residue at position 297 in the amino acid sequence shown in SEQ ID NO: 1 or at the position corresponding to this position in a related sequence is replaced with another amino acid residue, wherein the amino acid residue at position 297 in the amino acid sequence shown in SEQ ID NO: 1 or at the position corresponding to this position in a related sequence is replaced with another amino acid residue and wherein the amino acid residue at position 173 in the amino acid sequence shown in SEQ ID NO: 1 or at the position corresponding to this position in a related sequence is replaced with another amino acid residue. In a specific embodiment, the amino acid residue at position 282 in the amino acid sequence shown in SEQ ID NO: 1 or at the position corresponding to this position in a related sequence is replaced with cysteine, serine, glutamic acid, glycine, glutamine, threonine, valine, alanine or aspartic acid, preferably with cysteine. In another specific embodiment, the amino acid residue at position 297 in the amino acid sequence shown in SEQ ID NO: 1 or at the position corresponding to this position in a related sequence is replaced with leucine. In yet another embodiment, the amino acid residue at position 173 in the amino acid sequence shown in SEQ ID NO: 1 or at the position corresponding to this position in a related sequence is replaced with cysteine.

[0170] In another embodiment, a variant of the invention is characterized in that it contains at least four deletions and / or substitutions at positions selected from positions 9, 11, 16, 24, 28, 42, 43, 45, 53, 66, 77, 80, 91, 105, 116, 118, 120, 122, 123, 129, 134, 141, 159, 160, 161, 173, 177, 180, 186, 215, 238, 241, 242, 248, 251, 253, 258, 264, 279, 291, 293, 297, 299, 303, 307, 308, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267 and 315 in the amino acid sequence shown in SEQ ID NO: 1, preferably at positions selected from positions 9, 11, 16, 24, 28, 45, 53, 80, 91, 105, 118, 121, 123, 141, 159, 161, 173, 177, 180, 215, 238, 241, 242, 248, 258, 279, 282, 291, 297, 299, 303, 307, 308 and 315 or at positions corresponding to any of these positions in the related sequences as defined above. Preferably, one of said deletions / substitutions is at position 303 of SEQ ID NO: 1 or at the position corresponding to this position in the related sequences as defined above. More preferably, this position is replaced by methionine. In another embodiment, the substitutions at the remaining three positions are effected at positions selected from positions 45, 121, 173, 282, 307 and 308 or at positions corresponding to any of these positions in the related sequences as defined above.

[0171] Preferably modify at least the following positions in SEQ ID NO: 1 or the positions corresponding to any of these positions in the related sequences as defined above by deletion or substitution:

[0172] 121, 282, 303 and 308; or

[0173] 173, 303, 307 and 308; or

[0174] 45, 173, 282 and 303.

[0175] Preferably, the substitution at position 45 is valine, the substitution at position 121 is arginine, the substitution at position 173 or 282 is cysteine, the substitution at position 307 is histidine and the substitution at position 308 is serine. Most preferably, the following combinations of substitutions are: Y121R-K282C-L303M-T308S; M173C-L303M-K307H-T308S; E45V-M173C-K282C-L303M or the corresponding substitutions at the corresponding positions in a structurally related sequence as defined above.

[0176] In another embodiment, a variant according to the invention is characterized in that it contains at least five deletions and / or substitutions in the amino acid sequence shown in SEQ ID NO: 1 at positions selected from positions 9, 11, 16, 24, 28, 42, 43, 45, 53, 66, 77, 80, 91, 105, 116, 118, 120, 122, 123, 129, 134, 141, 159, 160, 161, 173, 177, 180, 186, 215, 238, 241, 242, 248, 251, 253, 258, 264, 279, 291, 293, 297, 299, 303, 307, 308, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267 and 315, preferably selected from positions 9, 11, 16, 24, 28, 45, 53, 80, 91, 105, 118, 121, 123, 141, 159, 161, 173, 177, 180, 215, 238, 241, 242, 248, 258, 279, 282, 291, 297, 299, 303, 307, 308 and 315 or at positions corresponding to any of these positions in a related sequence as defined above. Preferably, one of these substitutions is at position 303 of SEQ ID NO: 1 or at a position corresponding to this position in a related sequence as defined above. More preferably, this position is replaced by methionine. In another embodiment, the deletions / substitutions at the remaining three positions are effected at positions selected from positions 9, 11, 118, 121, 159, 173, 282, 307 and 308 or at positions corresponding to any of these positions in a related sequence as defined above.

[0177] Preferably modify SEQ ID NO: 1 at the following positions or at positions corresponding to any of these positions in a related sequence as defined above, at least by deletion or substitution:

[0178] 121, 173, 282, 303, and 308; or

[0179] 159, 173, 303, 307, and 308; or

[0180] 9, 11, 303, 307, and 308; or

[0181] 118, 121, 173, 282, and 303.

[0182] Preferably, the substitution at position 9 is leucine, the substitution at position 11 is phenylalanine, the substitution at position 118 is leucine, the substitution at position 121 is arginine, the substitution at position 159 is leucine, the substitution at position 173 or 282 is cysteine, the substitution at position 307 is histidine and the substitution at position 308 is serine.

[0183] Particularly preferred variants with five mutations show the following substitutions in SEQ ID NO: 1:

[0184] Y121R - M173C - K282C - L303M - T308S

[0185] E159L - M173C - L303M - K307H - T308S

[0186] R9L - Y11F - L303M - K307H - T308S

[0187] C118L - Y121R - M173C - K282C - L303M

[0188] or the corresponding substitutions at the corresponding positions in structurally related sequences as defined above.

[0189] In another embodiment, a variant according to the invention is characterized in that it contains at least six deletions and / or substitutions at positions selected from positions 9, 11, 16, 24, 28, 42, 43, 45, 53, 66, 77, 80, 91, 105, 116, 118, 120, 122, 123, 129, 134, 141, 159, 160, 161, 173, 177, 180, 186, 215, 238, 241, 242, 248, 251, 253, 258, 264, 279, 291, 293, 297, 299, 303, 307, 308, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267 and 315 in the amino acid sequence shown in SEQ ID NO: 1, preferably at positions selected from positions 16, 24, 28, 45, 53, 80, 91, 105, 118, 121, 123, 141, 159, 161, 173, 177, 180, 215, 238, 241, 242, 248, 258, 279, 282, 291, 297, 299, 303, 307, 308 and 315 or at positions corresponding to any of these positions in the related sequences as defined above. Preferably, one of these deletions / substitutions is at position 282 of SEQ ID NO: 1 or at the position corresponding to this position in the related sequences as defined above. More preferably, this position is replaced by cysteine. In another embodiment, one of these deletions / substitutions is at position 173 of SEQ ID NO: 1 or at the position corresponding to this position in the related sequences as defined above. More preferably, this position is replaced by cysteine. In another embodiment, the deletions / substitutions at the remaining four positions are effected at positions selected from positions 45, 121, 159, 215, 297, 303 and 308 or at positions corresponding to any of these positions in the related sequences as defined above.

[0190] Preferably, SEQ ID NO: 1 is modified at least by deletion or substitution at the following positions or at positions corresponding to any of these positions in the related sequences as defined above:

[0191] 121, 159, 173, 282, 303 and 308; or

[0192] 121, 159, 173, 215, 282 and 303; or

[0193] 45, 159, 173, 282, 297 and 308.

[0194] Preferably, the substitution at position 45 is leucine, the substitution at position 121 is arginine, the substitution at position 159 is leucine, the substitution at position 215 is alanine, the substitution at position 297 is leucine, the substitution at position 303 is methionine and the substitution at position 308 is serine. Most preferably the following substitution combinations:

[0195] Particularly preferred variants with six mutations show the following substitutions in SEQ ID NO: 1:

[0196] Y121R-E159L-M173C-K282C-L303M-T308S

[0197] Y121R-E159L-M173C-V215A-K282C-L303M

[0198] E45L-E159L-M173C-K282C-F297L-T308S

[0199] or the corresponding substitutions at the corresponding positions in structurally related sequences as defined above.

[0200] In another embodiment, the variant according to the invention is characterized in that it contains at least seven substitutions at positions selected from positions 9, 11, 16, 24, 28, 42, 43, 45, 53, 66, 77, 80, 91, 105, 116, 118, 120, 122, 123, 129, 134, 141, 159, 160, 161, 173, 177, 180, 186, 215, 238, 241, 242, 248, 251, 253, 258, 264, 279, 291, 293, 297, 299, 303, 307, 308, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267 and 315 in the amino acid sequence shown in SEQ ID NO: 1, preferably selected from positions 16, 24, 28, 45, 53, 80, 91, 105, 118, 121, 123, 141, 159, 161, 173, 177, 180, 215, 238, 241, 242, 248, 258, 279, 282, 291, 297, 299, 303, 307, 308 and 315, or at positions corresponding to any of these positions in the relevant sequences as defined above. Preferably, one of these deletions / substitutions is at position 282 of SEQ ID NO: 1 or at a position corresponding to this position in the relevant sequences as defined above. More preferably, this position is replaced by cysteine. In another embodiment, one of these deletions / substitutions is at position 173 of SEQ ID NO: 1 or at a position corresponding to this position in the relevant sequences as defined above. More preferably, this position is replaced by cysteine. In another embodiment, one of these deletions / substitutions is at position 121 of SEQ ID NO: 1 or at a position corresponding to this position in the relevant sequences as defined above. More preferably, this position is replaced by arginine. In another embodiment, one of these deletions / substitutions is at position 303 of SEQ ID NO: 1 or at a position corresponding to this position in the relevant sequences as defined above. More preferably, this position is replaced by methionine. In another embodiment, the deletions / substitutions at the remaining three positions are effected at positions selected from positions 45, 118, 159, 177, 242, 297, 307, 308 and 315, or at positions corresponding to any of these positions in the relevant sequences as defined above.

[0201] Preferably, SEQ ID NO: 1 is modified at least by deletion or substitution at the following positions or at positions corresponding to any of these positions in the relevant sequences as defined above:

[0202] 121, 159, 173, 282, 303, 307, and 308; or

[0203] 45, 121, 159, 173, 282, 303, and 308; or

[0204] 121, 159, 173, 282, 297, 303, and 308; or

[0205] 118, 121, 159, 173, 282, 303, and 308; or

[0206] 121, 159, 173, 177, 282, 303, and 308; or

[0207] 121, 159, 173, 242, 282, 303, and 308; or

[0208] 118, 121, 159, 173, 282, 303, and 315; or

[0209] 118, 121, 159, 173, 177, 282, and 303;

[0210] 45, 121, 173, 282, 297, 303, and 308; or

[0211] 45, 118, 121, 173, 282, 303, and 308; or

[0212] 45, 121, 159, 173, 282, 297, and 303.

[0213] Preferably, the substitution at position 45 is valine, the substitution at position 118 is leucine or tryptophan, the substitution at position 159 is leucine, the substitution at position 177 is cysteine, the substitution at position 242 is alanine, the substitution at position 297 is leucine, the substitution at position 307 is histidine and the substitution at position 308 or 315 is serine. Most preferably the following substitution combinations:

[0214] Particularly preferred variants with seven mutations are shown in SEQ ID NO: 1 with the following substitutions:

[0215] Y121R-E159L-M173C-K282C-L303M-K307H-T308S

[0216] E45V-Y121R-E159L-M173C-K282C-L303M-T308S

[0217] Y121R-E159L-M173C-K282C-F297L-L303M-T308S

[0218] C118L-Y121R-E159L-M173C-K282C-L303M-T308S

[0219] Y121R-E159L-M173C-E177C-K282C-L303M-T308S

[0220] Y121R-E159L-M173C-T242A-K282C-L303M-T308S

[0221] C118L-Y121R-E159L-M173C-K282C-L303M-G315S

[0222] C118W-Y121R-E159L-M173C-E177C-K282C-L303M

[0223] C118L-Y121R-E159L-M173C-E177C-K282C-L303M

[0224] E45V-Y121R-M173C-K282C-F297L-L303M-T308S

[0225] E45V-C118L-Y121R-M173C-K282C-L303M-T308S

[0226] E45V-Y121R-E159L-M173C-K282C-F297L-L303M

[0227] or a corresponding substitution at the corresponding position in a structurally related sequence as defined above.

[0228] In another embodiment, the variant according to the invention is characterized in that it contains at least 8 or at least 9 deletions and / or substitutions at positions selected from positions 9, 11, 16, 24, 28, 42, 43, 45, 53, 66, 77, 80, 91, 105, 116, 118, 120, 122, 123, 129, 134, 141, 159, 160, 161, 173, 177, 180, 186, 215, 238, 241, 242, 248, 251, 253, 258, 264, 279, 291, 293, 297, 299, 303, 307, 308, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267 and 315 in the amino acid sequence shown in SEQ ID NO: 1, preferably at positions selected from positions 16, 24, 28, 45, 53, 80, 91, 105, 118, 121, 123, 141, 159, 161, 173, 177, 180, 215, 238, 241, 248, 258, 279, 282, 291, 297, 299, 303, 308 and 315 or at positions corresponding to any of these positions in the related sequences as defined above. Preferably, one of these deletions / substitutions is at position 282 of SEQ ID NO: 1 or at a position corresponding to this position in the related sequences as defined above. More preferably, this position is replaced by cysteine. In another embodiment, one of these deletions / substitutions is at position 173 of SEQ ID NO: 1 or at a position corresponding to this position in the related sequences as defined above. More preferably, this position is replaced by cysteine. In another embodiment, one of these deletions / substitutions is at position 121 of SEQ ID NO: 1 or at a position corresponding to this position in the related sequences as defined above. More preferably, this position is replaced by arginine. In another embodiment, one of these deletions / substitutions is at position 303 of SEQ ID NO: 1 or at a position corresponding to this position in the related sequences as defined above. More preferably, this position is replaced by methionine. In another embodiment, the deletions / substitutions at the remaining four and five positions are achieved at positions selected from positions 24, 45, 80, 118, 123, 159, 177, 215, 258, 297, 308 and 315 or at positions corresponding to any of these positions in the related sequences as defined above.

[0229] Preferably, modify SEQ ID NO: 1 at least by deletion or substitution at the following positions or at positions corresponding to any of these positions in a related sequence as defined above:

[0230] 80, 121, 159, 173, 282, 303, 308 and 315; or

[0231] 24, 121, 159, 123, 173, 258, 282 and 303; or

[0232] 118, 121, 159, 173, 177, 215, 282 and 303; or

[0233] 24, 118, 121, 159, 173, 177, 282 and 303; or

[0234] 45, 80, 121, 173, 282, 297, 303 and 308; or

[0235] 45, 121, 159, 173, 177, 282, 303 and 308; or

[0236] 118, 121, 159, 173, 177, 282, 297, 303 and 308; or

[0237] 118, 121, 159, 173, 177, 215, 282, 297 and 303; or

[0238] 80, 118, 121, 159, 173, 177, 282, 303 and 315; or

[0239] 24, 45, 121, 123, 173, 282, 297, 303 and 308; or

[0240] 45, 121, 159, 173, 177, 215, 258, 282 and 303.

[0241] Preferably, the substitution at position 24 is arginine, the substitution at position 45 is valine or leucine, the substitution at position 80 is glycine, the substitution at position 118 is leucine or tryptophan, the substitution at position 123 is arginine, the substitution at position 159 is leucine, the substitution at position 177 is cysteine, the substitution at position 215 is alanine, the substitution at position 258 is leucine, the substitution at position 297 is leucine and the substitution at position 308 or 315 is serine.

[0242] Particularly preferred variants having 8 or 9 mutations show the following substitutions in SEQ ID NO: 1:

[0243] D80G-Y121R-E159L-M173C-K282C-L303M-T308S-G315S

[0244] K24R-121R-E159L-K123R-M173C-M258L-K282C-L303M

[0245] C118L-Y121R-E159L-M173C-E177C-V215A-K282C-L303M

[0246] K24R-C118L-Y121R-E159L-M173C-E177C-K282C-L303M

[0247] E45V-D80G-Y121R-M173C-K282C-F297L-L303M-T308S

[0248] E45L-Y121R-E159L-M173C-E177C-K282C-L303M-T308S

[0249] C118L-Y121R-E159L-M173C-E177C-K282C-F297L-L303M-T308S

[0250] C118L-Y121R-E159L-M173C-E177C-V215A-K282C-F297L-L303M

[0251] D80G-C118L-Y121R-E159L-M173C-E177C-K282C-L303M-G315S

[0252] K24R-E45V-Y121R-K123R-M173C-K282C-F297L-L303M-T308S

[0253] E45V-Y121R-E159L-M173C-E177C-V215A-M258L-K282C-L303M

[0254] or a corresponding substitution at the corresponding position in a structurally related sequence as defined above.

[0255] In another embodiment, a variant according to the invention is characterized in that it has at least 10 substitutions at positions selected from positions 9, 11, 16, 24, 28, 42, 43, 45, 53, 66, 77, 80, 91, 105, 116, 118, 120, 122, 123, 129, 134, 141, 159, 160, 161, 173, 177, 180, 186, 215, 238, 241, 242, 248, 251, 253, 258, 264, 279, 291, 293, 297, 299, 303, 307, 308, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267 and 315 in the amino acid sequence shown in SEQ ID NO: 1, preferably at positions selected from positions 16, 24, 28, 45, 53, 91, 105, 118, 121, 141, 159, 161, 173, 177, 180, 215, 238, 241, 248, 279, 282, 291, 297, 299, 303 and 308 or at positions corresponding to any of these positions in a related sequence as defined above. In one embodiment, the positions of the deletions / substitutions are positions 45, 118, 121, 159, 173, 215, 282, 297, 303 and 308. Preferably, the substitution at position 45 is valine, the substitutions at positions 118, 159 and 297 are leucine, the substitution at position 121 is arginine, the substitution at position 173 or 282 is cysteine, the substitution at position 215 is alanine, the substitution at position 303 is methionine and the substitution at position 308 is serine. Thus, a particularly preferred variant having 10 mutations shows the following substitutions in SEQ ID NO: 1:

[0256] E45V-C118L-Y121R-E159L-M173C-V215A-K282C-F297L-L303M-T308S

[0257] or corresponding substitutions at the corresponding positions in a structurally related sequence as defined above.

[0258] In another embodiment, the variant according to the invention is characterized in that it contains at least 11 deletions and / or substitutions at positions selected from positions 9, 11, 16, 24, 28, 42, 43, 45, 53, 66, 77, 80, 91, 105, 116, 118, 120, 122, 123, 129, 134, 141, 159, 160, 161, 173, 177, 180, 186, 215, 238, 241, 242, 248, 251, 253, 258, 264, 279, 291, 293, 297, 299, 303, 307, 308, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267 and 315 in the amino acid sequence shown in SEQ ID NO: 1, preferably at positions selected from positions 16, 24, 28, 53, 91, 105, 118, 121, 141, 159, 161, 173, 177, 180, 238, 241, 248, 279, 282, 291, 297, 299, 303 and 308 or at positions corresponding to any of these positions in the related sequences as defined above. In one embodiment, the positions of the deletions / substitutions are positions 24, 118, 121, 159, 173, 177, 282, 291, 297, 303 and 308. Preferably, the substitution at position 24 or 121 is arginine, the substitution at positions 118, 159 and 297 is leucine, the substitution at positions 173, 177 or 282 is cysteine or valine, the substitution at position 291 is aspartic acid, the substitution at position 303 is methionine and the substitution at position 308 is serine. Thus, a particularly preferred variant with 11 mutations shows the following substitutions in SEQ ID NO: 1:

[0259] K24R-C118L-Y121R-E159L-M173C-E177C-K282C-E291D-F297L-L303M-T308S

[0260] or the corresponding substitutions at the corresponding positions in the structurally related sequences as defined above.

[0261] The mutant containing these 11 substitutions is also referred to as "F9" in the context of the appended Examples section.

[0262] In a preferred embodiment, a variant of the invention is characterized in that it contains at least 10, preferably at least 11, deletions and / or substitutions at positions selected from positions 24, 118, 121, 159, 173, 177, 282, 291, 297, 303 and 308 in SEQ ID NO: 1 or at positions corresponding to any of these positions in a related sequence as defined above, preferably substitutions as in variant F9, and it additionally contains at least one other deletion / substitution at positions selected from positions 16, 28, 53, 91, 105, 141, 161, 180, 238, 241, 248, 279, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267 and 299 in SEQ ID NO: 1 or at positions corresponding to any of these positions in a related sequence as defined above. Preferably, the substitutions at any of these positions are as defined above.

[0263] In one embodiment, at least one other deletion / substitution is at position 16 and 105 in SEQ ID NO: 1 or at positions corresponding to any of these positions in a related sequence as defined above.

[0264] In another embodiment, at least one other deletion / substitution is at positions 16, 141 and 241 in SEQ ID NO: 1 or at positions corresponding to any of these positions in a related sequence as defined above.

[0265] In another embodiment, at least one other deletion / substitution is at positions 16, 141, 241 and 248 in SEQ ID NO: 1 or at positions corresponding to any of these positions in a related sequence as defined above.

[0266] In another embodiment, at least one other deletion / substitution is at positions 141, 241 and 248 in SEQ ID NO: 1 or at positions corresponding to any of these positions in a related sequence as defined above.

[0267] In another embodiment, at least one other deletion / substitution is at positions 16, 91, 141, 241 and 248 in SEQ ID NO: 1 or at positions corresponding to any of these positions in a related sequence as defined above.

[0268] In another embodiment, at least one other deletion / substitution is at position 16, 91, 141, 241, 248, and 299 in SEQ ID NO: 1 or at a position corresponding to any of these positions in the related sequences as defined above.

[0269] In another embodiment, at least one other deletion / substitution is at position 16, 91, 141, 241, 248, 299, and 28 in SEQ ID NO: 1 or at a position corresponding to any of these positions in the related sequences as defined above.

[0270] In another embodiment, at least one other deletion / substitution is at position 16, 91, 141, 241, 248, 299, and 28 in SEQ ID NO: 1 or at a position corresponding to any of these positions in the related sequences as defined above.

[0271] In another embodiment, at least one other deletion / substitution is at position 16, 141, 241, 248, and 28 in SEQ ID NO: 1 or at a position corresponding to any of these positions in the related sequences as defined above.

[0272] In another embodiment, at least one other deletion / substitution is at position 16, 141, 241, 248, 28, and 180 in SEQ ID NO: 1 or at a position corresponding to any of these positions in the related sequences as defined above.

[0273] In another embodiment, at least one other deletion / substitution is at position 16, 141, 241, 248, 28, 53, and 180 in SEQ ID NO: 1 or at a position corresponding to any of these positions in the related sequences as defined above.

[0274] In another embodiment, at least one other deletion / substitution is at position 16, 141, 241, 248, 28, 180, and 238 in SEQ ID NO: 1 or at a position corresponding to any of these positions in the related sequences as defined above.

[0275] In another embodiment, at least one other deletion / substitution is at position 16, 141, 241, 248, 28, 180, and 279 in SEQ ID NO: 1 or at a position corresponding to any of these positions in the related sequences as defined above.

[0276] In another embodiment, at least one other deletion / substitution is at position 16, 141, 241, 248, 28, 180, and 161 in SEQ ID NO: 1 or at a position corresponding to any of these positions in a related sequence as defined above.

[0277] Particularly preferred variants show at least 10, preferably at least 11 deletions and / or substitutions at positions selected from positions 24, 118, 121, 159, 173, 177, 282, 291, 297, 303, and 308 in SEQ ID NO: 1, preferably substitutions as in variant F9, or corresponding substitutions at the corresponding positions in a structurally related sequence as defined above, and additionally show the following substitutions in SEQ ID NO: 1:

[0278] S141P

[0279] S141T

[0280] S105A

[0281] Q299K

[0282] I16L

[0283] S248T

[0284] K241M

[0285] I16L - S105A

[0286] S141P - K241M - S248T

[0287] I16L - R91H - S141P - K241M - S248T

[0288] I16L - S141P

[0289] I16L - R91H - S141P - K241M - S248T Q299K

[0290] I16L - S141P - K241M

[0291] I16L - S141P - K241M - S248T

[0292] I16L - R91H - S141P - K241M - S248T - Q299K - M28K

[0293] I16L - R91H - S141P - K241M - S248T - Q299K - M28A

[0294] I16L-R91H-S141P-K241M-S248T-Q299K-K180P

[0295] I16L-S141P-K241I-S248T

[0296] I16L-S141P-K241I-S248T-M28K

[0297] I16L-S141P-K241I-S248T-M28K-K180P

[0298] I16L-S141P-K241I-S248T-M28K-T53V-K180P

[0299] I16L-S141P-K241I-S248T-M28K-K180P-A238K

[0300] I16L-S141P-K241I-S248T-M28K-K180P-A238R

[0301] I16L-S141P-K241I-S248T-M28K-K180P-C282V

[0302] I16L-S141P-K241I-S248T-M28K-K180P-P279A

[0303] I16L-S141P-K241I-S248T-M28K-K180P-Y161R

[0304] D2H

[0305] M42L-D87E-S139C-R186L-K231Q

[0306] E164Q-R186V-D252E

[0307] D87E-S139C-R186L-K231Q

[0308] R186V-Q267R

[0309] S139C-R186I

[0310] L111M-F122Y-R186L

[0311] M75I-R186V

[0312] S139A-S141C

[0313] K179K-R186V

[0314] R186V

[0315] A57S - A58T - K77R - R186V

[0316] L111M - R186L

[0317] R186L

[0318] A31S - R186V

[0319] S139A - S141G

[0320] M75I - R186L - S308T

[0321] R186L - S308T

[0322] R186I

[0323] L111M - R186V - S308T

[0324] R186V - D221E

[0325] L111M - R186V

[0326] M1L - L111M - R186V - S308T

[0327] R186N

[0328] R24S - G86Q - R186I

[0329] I16L - S141P - K241I - K180P - E227K

[0330] I16L - S141P - K241I - K180P - D291E - M303L

[0331] S141P - K241I - S248T - K180P - R24K

[0332] I16L - S141P - K241I - S248T - L297F

[0333] I16L - S141P - K241I - S248T - M28K - K180P - L188C - L297F - A246E

[0334] I16L - S141P - K241I - S248T - M28K - K180P - L188C - L297F - T242E

[0335] I16L-S141P-K241I-S248T-M28K-K180P-L188C-L297F-Y255E

[0336] I16L-S141P-K241I-S248T-M28K-K180P-L188C-L297F-T198D

[0337] I16L-S141P-K241I-S248T-M28K-K180P-L188C-L297F-K23L

[0338] I16L-S141P-K241I-S248T-M28K-K180P-L188C-L297F-K179L

[0339] I16L-S141P-K241I-S248T-M28K-K180P-L188C-L297F-K231L

[0340] I16L-S141P-K241I-S248T-M28K-K180P-L188C-L297F-P182E

[0341] I16L-S141P-K241I-S248T-M28K-K180P-L188C-L297F-A238E

[0342] I16L-S141P-K241I-S248T-M28K-K180P-L188C-L297F-K208L

[0343] I16L-S141P-K241I-S248T-M28K-K180P-L188C-L297F-R204L

[0344] I16L-S141P-K241I-S248T-M28K-K180P-L188C-L297F-R24L

[0345] I16L-S141P-K241I-S248T-M28K-K180P-L188C-L297F

[0346] I16L-S141P-K241I-K180P-D291E

[0347] I16L-S141P-K241I-S248T-K180P-Q267R-R24K-L118C-L159E

[0348] I16L-S141P-K241I-S248T-M28K-K180P-L188C-L297F-S142A

[0349] I16L-S141P-K241I-S248T-M28K-K180P-L159E-D291E

[0350] I16L-S141P-K241I-S248T-K180P-G166S-R24K

[0351] I16L-S141P-K241I-S248T-K180P-R24K

[0352] I16L-S141P-K241I-S248T-M28K-K180P-L188C-L297F-Q205H

[0353] or a corresponding substitution at the corresponding position in a structurally related sequence as defined above.

[0354] The inventors also constructed a computer 3D model of the Streptococcus mitis enzyme represented by the amino acid sequence shown in SEQ ID NO: 1 (see Figure 6 ) to locate the mutated residues in the three-dimensional structure. The structure of the enzyme is characterized by a conical fold, where the N-terminal region is oriented normal to the relatively flat C-terminal region that contains five α-helices. The active site is a positively charged cleft formed between the N-domain and the C-terminal domain, with an ATP-binding P-loop nearby. The 3D structure of the Streptococcus mitis enzyme is extremely similar to the 3D structure of the MDP decarboxylase of Streptococcus pyogenes (see Figure 6 ), whose amino acid sequence shows 69% sequence identity with the Streptococcus mitis enzyme (SEQ ID NO: 1). The 3D structure is also quite similar to the 3D structures reported by Byres et al. (J. Mol. Biol. 371 (2007), 540-553) for the MDP decarboxylases of S. brucei and Staphylococcus aureus. Figure 7 Sequences showing the MDP decarboxylases, with secondary structures (β-sheets and α-helices) and some mutated residues highlighted.

[0355] It has been demonstrated that the mutations identified in the present invention directly affect the rate at which the enzyme produces isobutene from 3-phosphonoxyisovalerate; however, these mutations may confer other properties on the enzyme that have not been fully characterized. For example, several substitutions replace wild-type residues (Y11, M173, and K282) with cysteine residues. In the improved mutants, residues 2B4, M173C, and K282C are close enough in the 3D modeled structure to suggest that creation of a disulfide bridge is possible. Such a bond could stabilize the overall structure of the enzyme. To support this possibility, some preliminary data show that some mutants are more resistant to heat denaturation than the wild-type enzyme.

[0356] Similarly, these mutations may affect the stability of the protein to pH, the optimal temperature, and the oligomeric state. Preliminary data collected on the effect of pH on isobutene production suggest that some mutant variants are more active at acidic pH compared to the wild-type enzyme.

[0357] A set of identified positions is located in the N-terminal domain of the protein, specifically positions 1, 2, 9, 11, 42, 43, 45, 57, 58, 75, 77, 80, 86, 87, 91, 116, 118, 120, 121, 122, 123, 129, and 134.

[0358] Analysis reveals that residues 77, 80, 116, 118, 120, 121, 122, 123, 129, and 134 are located on α-helices N°1, 2, and 3 in the N-terminal domain and are the furthest from the substrate-binding pocket. Residues 9, 11, 42, 43, 45, and 91 are adjacent to these α-helices and, in particular, they are located in the β-sheet on β-strands 1 and β-strand 4 that are directly adjacent to each other (see Figure 8 ). Thus, all of these residues are located at the N-terminus, are close to each other in the 3D structure, and do not show any structural proximity to the active site of the enzyme. Thus, in a preferred embodiment, the present invention relates to variants of mevalonate diphosphate decarboxylase having an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having at least 40%, 50%, 60%, or 90% sequence identity to SEQ ID NO: 1, wherein one or more amino acid residues at positions selected from positions 9, 11, 42, 43, 45, 77, 80, 91, 116, 118, 120, 121, 122, 123, 129, and 134 or positions corresponding to any of these positions in the amino acid sequence shown in SEQ ID NO: 1 are replaced with another amino acid residue and wherein the mevalonate diphosphate decarboxylase has improved activity in converting 3-phosphonoxyisovalerate into isobutene.

[0359] Another set of identified positions, particularly positions 159, 160, 164, 166, 173, 177, 179, 198, 204, 205, 208, 221, 227, 231, 238, 241, 242, 246, 248, 251, 252, 253, 255, 258, 264, 267, 282, 291, 293, 297, 299, 303, 307 and 308, are located in the C-terminal domain, and most of these positions are located in α-helices and β-strands, particularly in α-helices 8 and 9 and in β-strands 9 to 12. Mutations at positions 238, 241, 242, 248, 253, 258, 264, 291, 293, 297, 299, 303, 307 and 308 (i.e., in the 9th α-helix and the 12th β-strand) are hypothesized to result in a more stable conformation that stabilizes the entire structure. Accordingly, in a preferred embodiment, the invention relates to variants of mevalonate diphosphate decarboxylase having the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 40%, 50%, 60% or 90% sequence identity to SEQ ID NO: 1, wherein one or more amino acid residues at positions selected from positions 159, 160, 173, 177, 238, 241, 242, 248, 251, 253, 258, 264, 282, 291, 293, 297, 299, 303, 307 and 308, more preferably positions 238, 241, 242, 248, 253, 258, 264, 291, 293, 297, 299, 303, 307 and 308, of the amino acid sequence shown in SEQ ID NO: 1 or positions corresponding to any of these positions in a related sequence are replaced with another amino acid residue and wherein the mevalonate diphosphate decarboxylase has improved activity in converting 3-phosphonoxyisovalerate to isobutene.

[0360] In a specific embodiment, it is preferred that one or more amino acid residues at positions selected from positions 293, 297, 299, 303, 307 and 308 of the amino acid sequence shown in SEQ ID NO: 1 or positions corresponding to any of these positions in a related sequence are replaced with another amino acid residue. These residues are located in the most C-terminal part of the enzyme (within α-helix 9 and β-strand 12) and may result in a more stable conformation that stabilizes the entire structure.

[0361] The third set of identified positions is located at or near the binding pocket of the enzyme. This set encompasses positions 16, 23, 24, 28, 31, 111, 139, 141, 142, 182, 186, 188, 279, and 282. The MDP binding pocket is defined by regions 13 - 22 and 97 - 107 that form the ATP binding site and particularly includes the P-loop (P99 - S107). The other side consists of region 274 - 280 where the catalytic base D276 is present and region 136 - 146 that contains R144 which drives the final decarboxylation step of the substrate. Position S105 is within the phosphate-binding loop (consensus GHMP kinase in Streptococcus mitis P-Xaa-GLSASAA->PTAAGLSSSSS). Interestingly, compared to the wild-type Streptococcus mitis MDP P-loop sequence, the mutations that improve IBN production are substitutions to Ala that are closer to the consensus P-binding loop sequence. S141 is closely located to the R144 residue and its hydroxyl group interacts with the enzyme's natural substrate and is thus thought to be important in determining enzyme specificity. Substitution of S141 with threonine having a bulkier side chain may potentially facilitate the interaction of the enzyme with the monophosphorylated substrate PIV of smaller size. Substitution to proline would significantly alter the structure of this region and may better accommodate this unnatural substrate PIV. I16, K24R, and M28 are adjacent to K22 which interacts with the phosphate group of the natural substrate; altering this residue may affect the structure of this loop and cause K22 to be closer to the substrate. The best-performing mutants carry K24R that changes the side-chain length while increasing the positive charge environment of the binding pocket and the mutation M28K that increases the positive charge. P279 and K282 are close to the catalytic base D276 and R186 is adjacent to S185 which interacts with the MVAPP substrate.

[0362] Accordingly, in a preferred embodiment, the present invention relates to a variant of mevalonate diphosphate decarboxylase having an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having at least 40%, 50%, 60%, or 90% sequence identity with SEQ ID NO: 1, wherein one or more amino acid residues at positions selected from positions 16, 24, 28, 141, 186, 279, and 282 in the amino acid sequence shown in SEQ ID NO: 1 or corresponding positions in related sequences are replaced with another amino acid residue and wherein said mevalonate diphosphate decarboxylase has improved activity in converting 3-phosphonoxyisovalerate to isobutene.

[0363] The present invention also relates to a method for providing a mevalonate diphosphate decarboxylase variant, wherein the variant shows improved activity in converting 3-phosphonoxyisovalerate into isobutene, the method comprising the step of introducing one or more changes in the sequence of mevalonate diphosphate decarboxylase, wherein the (one or more) change(s) is / are introduced at one or more amino acid positions corresponding to the amino acid positions selected from positions 282, 9, 11, 16, 24, 28, 42, 43, 45, 53, 66, 77, 80, 91, 105, 116, 118, 120, 121, 122, 123, 129, 134, 141, 159, 160, 161, 173, 177, 180, 186, 215, 238, 241, 242, 248, 251, 253, 258, 264, 279, 291, 293, 297, 299, 303, 307, 308, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267 and 315 in the amino acid sequence shown in SEQ ID NO: 1. "Corresponding to" means corresponding to any of these positions in the relevant sequence.

[0364] For a preferred embodiment of the mevalonate diphosphate decarboxylase to be mutated according to this method, the same applies as has been set out above.

[0365] In a preferred embodiment, the mevalonate diphosphate decarboxylase from which the variant is derived is a mevalonate diphosphate decarboxylase showing the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 40%, 50%, 60% or 90% sequence identity with SEQ ID NO: 1 or any of the preferred degrees of sequence identity as described above.

[0366] Furthermore, with regard to the degree of improvement in activity and the preferred embodiments of the changes to be introduced, the same applies as has been set out above.

[0367] The MDP decarboxylase of the present invention can be fused with a homologous or heterologous polypeptide or protein, enzyme, substrate or tag to form a fusion protein. The fusion protein of the present invention will have the same improved activity as the MDP decarboxylase of the present invention. Polypeptides, enzymes, substrates or tags that can be added to another protein are known in the art. They can be used for purifying or detecting the protein of the present invention. For example, tags that can be used for detection and / or purification are, for example, FLAG tag, His6 tag or streptavidin tag. Tags that improve enzyme solubility or stability are MBP or ATS. Alternatively, the protein of the present invention can be fused with an enzyme such as luciferase to detect or localize the protein. Other fusion partners include, but are not limited to, bacterial β-galactosidase, trpE, protein A, β-lactamase, α-amylase, alcohol dehydrogenase or yeast α mating factor.

[0368] It is also conceivable to remove polypeptides, enzymes, substrates or tags from the protein of the present invention, for example, after purification.

[0369] Fusion proteins can generally be produced by recombinant nucleic acid methods or synthetic polypeptide methods known in the prior art.

[0370] The present invention also relates to nucleic acid molecules encoding the enzymes of the present invention, more preferably variants of the MDP decarboxylase of the present invention, and to vectors containing said nucleic acid molecules. Vectors that can be used according to the present invention are known in the art. The vector can also contain expression control sequences operably linked to the nucleic acid molecule of the present invention contained in the vector. These expression control sequences can be suitable for ensuring transcription and synthesis of translatable RNA in bacteria or fungi. Expression control sequences can be, for example, promoters. The promoter used for ligation with the nucleic acid molecule of the present invention can be homologous or heterologous with respect to its origin and / or with respect to the gene to be expressed. Suitable promoters are, for example, promoters that cause constitutive expression by themselves. However, promoters that are activated only at time points determined by external influences can also be used. In this case, artificial and / or chemically inducible promoters can be used.

[0371] Preferably, the vector of the present invention is an expression vector. Expression vectors have been widely described in the literature. Generally, they contain not only a selectable marker gene and an origin of replication ensuring replication in the selected host, but also a bacterial or viral promoter, and in most cases a transcription termination signal. There is usually at least one restriction site or multiple cloning site between the promoter and the termination signal that enables the insertion of a coding DNA sequence. If active in the selected host organism, the DNA sequence that naturally controls the transcription of the corresponding gene can be used as the promoter sequence. However, this sequence can also be exchanged for other promoter sequences. Promoters that ensure constitutive gene expression and inducible promoters that allow intentional control of gene expression can be used. Bacterial and viral promoter sequences with these properties are detailed in the literature. Regulatory sequences for expression in microorganisms (e.g., Escherichia coli, Saccharomyces cerevisiae) are well described in the literature. Promoters that allow particularly high expression of downstream sequences are, for example, the T7 promoter (Studier et al., Methods in Enzymology 185 (1990), 60-89), lacUV5, trp, trp-lacUV5 (DeBoer et al., cited in Rodriguez and Chamberlin (eds.), Promoters, Structure and Function; Praeger, New York, (1982), 462-481; DeBoer et al., Proc. Natl. Acad. Sci. USA (1983), 21-25), lp1, rac (Boros et al., Gene 42 (1986), 97-100). Inducible promoters are preferably used for the synthesis of polypeptides. These promoters often result in higher polypeptide yields than constitutive promoters. To obtain the optimal amount of polypeptide, a two-stage method is often used. First, the host cells are cultured under optimal conditions until a relatively high cell density is reached. In the second step, transcription is induced according to the type of promoter used. In this regard, the tac promoter is particularly suitable, which can be induced by lactose or IPTG (= isopropyl-β-D-thiogalactopyranoside) (deBoer et al., Proc. Natl. Acad. Sci. USA 80 (1983), 21-25). Transcription termination signals are also described in the literature.

[0372] Preferably, the nucleic acid molecules according to the invention are non-naturally occurring nucleic acid molecules, i.e., molecules that do not exist in nature. Such non-naturally occurring molecules differ significantly from naturally occurring nucleic acid molecules, for example, due to differences in their structure. For example, such non-naturally occurring nucleic acid molecules can encode non-naturally occurring enzymes as described above. The vectors according to the invention are also preferably non-naturally occurring vectors, for example, due to the presence of non-naturally occurring nucleic acid molecules or due to combinations of elements that do not occur in nature in such combinations.

[0373] Furthermore, the invention relates to a host cell comprising the vector according to the invention.

[0374] In a preferred embodiment, the host cell according to the invention is a microorganism, in particular a bacterium or a fungus. In a more preferred embodiment, the host cell according to the invention is Escherichia coli, a Clostridium bacterium or a yeast cell, such as Saccharomyces cerevisiae. In another preferred embodiment, the host cell is a plant cell or a non-human animal cell.

[0375] Transformation of the host cell with the vector according to the invention can be carried out by standard methods described, for example, in Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA; Methods in Yeast Genetics, A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, 1990. The host cell is cultured in a nutrient medium that meets the requirements of the specific host cell used (in particular with respect to pH value, temperature, salt concentration, aeration, antibiotics, vitamins, trace elements, etc.). Such a host cell according to the invention is preferably a non-naturally occurring host cell, i.e., a host cell that does not exist in nature. Such a non-naturally occurring host cell differs from a naturally occurring cell due to the modified nucleic acid molecule or vector as described above.

[0376] The invention also relates to the use of the MDP decarboxylase according to the invention or a host cell comprising said MDP decarboxylase for the conversion of 3-hydroxyisovaleric acid or for the conversion of 3-phosphonyloxyisovaleric acid into isobutene.

[0377] Furthermore, the invention relates to a method for producing isobutene from 3-hydroxyisovaleric acid or from 3-phosphonyloxyisovaleric acid, the method comprising the steps of culturing the host cell according to the invention in a suitable medium and recovering the isobutene.

[0378] As previously described, MDP decarboxylase is capable of catalyzing the conversion of mevalonic acid into 3-methylbut-3-en-1-ol via the intermediate mevalonate-3-phosphate (see WO 2011 / 076261). The inventors could show that the MDP decarboxylase variants according to the invention are also capable of catalyzing this conversion and in particular the conversion of mevalonate-3-phosphate into 3-methylbut-3-en-1-ol. Accordingly, the invention also relates to the use of an MDP decarboxylase variant according to the invention and as described hereinabove or a microorganism expressing such a variant for the conversion of mevalonic acid or mevalonate-3-phosphate into 3-methylbut-3-en-1-ol.

[0379] Furthermore, the invention relates to a method for producing 3-methylbut-3-en-1-ol from mevalonic acid or from mevalonate-3-phosphate, said method comprising the steps of cultivating a host cell according to the invention in a suitable culture medium and recovering said 3-methylbut-3-en-1-ol.

[0380] It has also been described that MDP decarboxylase is capable of catalyzing the conversion of 3-hydroxypent-4-enoic acid into 1,3-butadiene via the intermediate 3-phosphonoxypent-4-enoic acid (see PCT / EP 2012 / 075921). The inventors could show that the MDP decarboxylase variants of the invention are also capable of catalyzing this conversion, in particular the conversion of 3-phosphonoxypent-4-enoic acid into 1,3-butadiene. Accordingly, the invention also relates to the use of an MDP decarboxylase variant according to the invention and as described hereinabove or a microorganism expressing such a variant for the conversion of 3-hydroxypent-4-enoic acid or 3-phosphonoxypent-4-enoic acid into 1,3-butadiene.

[0381] Furthermore, the invention relates to a method for producing 1,3-butadiene from 3-hydroxypent-4-enoic acid or from 3-phosphonoxypent-4-enoic acid, said method comprising the steps of cultivating a host cell according to the invention in a suitable culture medium and recovering said 1,3-butadiene.

[0382] In the above methods, the microorganism is cultivated under suitable culture conditions allowing the enzymatic reaction of the MDP decarboxylase of the invention to take place. The specific culture conditions depend on the particular microorganism used, but are well known to the person skilled in the art. The culture conditions are generally selected in such a way that they allow the expression of the gene encoding the MDP decarboxylase of the invention. A variety of methods are known to the person skilled in the art for improving and finely regulating the expression of certain genes at certain stages of the culture, such as inducing gene expression by chemical inducers or by temperature variations.

[0383] In another embodiment, the above method of the present invention comprises the steps of: providing an organism, preferably a microorganism carrying each enzymatic activity in the form of a (cell) culture, preferably a liquid cell culture, and the subsequent step of cultivating said organism, preferably the microorganism, in a fermenter (often also referred to as a bioreactor) under suitable conditions allowing the expression of each enzyme, and further comprises the step of effecting the enzymatic conversion of the method of the present invention as described above herein. Suitable fermenter or bioreactor devices and fermentation conditions are known to those skilled in the art. A bioreactor or fermenter refers to any fabricated or engineered device or system known in the art that supports a biologically active environment. Thus, a bioreactor or fermenter can be a vessel in which a chemical / biochemical process such as the method of the present invention is carried out, said chemical / biochemical process involving an organism, preferably a microorganism and / or a biochemical active substance, i.e., the above (one or more) enzymes derived from such an organism or organisms carrying the above (one or more) enzymes. In a bioreactor or fermenter, such a process can be aerobic or anaerobic. These bioreactors are commonly cylindrical and can have a range of sizes from several liters to several hundred cubic meters and are often made of stainless steel. In this regard, without being bound by theory, a fermenter or bioreactor can be designed in such a way that it is suitable for cultivating an organism, preferably a microorganism, in, for example, batch culture, fed-batch culture, perfusion culture or chemostat culture, all of which culture methods are generally known in the art.

[0384] The culture medium can be any medium suitable for culturing various organisms or microorganisms.

[0385] The method according to the present invention further comprises the step of collecting the gaseous product, i.e., isobutene, degassing the reaction, i.e., recovering the product, for example, degassed from the culture. Thus, in a preferred embodiment, the method is carried out in the presence of a system for collecting isobutene in gaseous form during the reaction.

[0386] As a matter of fact, short-chain alkenes such as isobutene are gaseous at room temperature and atmospheric pressure. The method of the present invention thus does not require the extraction of the product from the liquid culture medium, which step is always a very expensive step when carried out on an industrial scale. The evacuation and storage of gaseous hydrocarbons and their possible subsequent physical separation and chemical conversion can be carried out according to methods known to those skilled in the art.

[0387] The present invention is further described with reference to the following non-limiting figures and examples.

[0388] Figure 1 : General map of the directed evolution scheme

[0389] Figure 2 : General schematic overview of the screening process

[0390] Figure 3Results obtained by analyzing the amount of isobutene produced from a subset of mutants from Table 1 using an activity assay employing purified protein

[0391] Figure 4: A set of substitutions at positions K282 and Y121 that improve the activity of converting 3-phosphonoxyisovalerate to isobutene

[0392] a) Mutation analysis at position K282 showed that conservative substitutions can have a similar effect on isobutene production. That is, substitution of K282 with the nucleophilic S or C or the hydrophobic V and A increases the activity of the enzyme (assay established using clarified cell lysates - n = 3);

[0393] b) Mutation analysis at position Y121 showed that conservative substitutions can have a similar effect on isobutene production (assay established using clarified cell lysates - n = 3):

[0394] Figure 5 : Plotting the isobutene production rate (moles of isobutene / mole of enzyme / second) as a function of 3-phosphonoxyisovalerate concentration and fitting the curve using the Michealis Menten equation (V = (Vmax*(substrate)) / (Km+(substrate))

[0395] Figure 6 : The 3D structure of the MDP decarboxylase of Streptococcus mitis (SEQ ID NO: 1) and the 3D structure of the MDP decarboxylase from Streptococcus pyogenes MDD that shows 69% sequence identity with SEQ ID NO: 1. The 3D structure was built using the MODELER algorithm (version 3.0, Accelrys, San Diego, CA) implemented in the Discovery Studio platform. 100 structures were generated and selected based on total energy minimization and analysis of the structural quality of the models according to the MODELER probability density function. The selected structures were used for further study. Computer mutagenesis was performed in Discovery studio 3.0 to visualize the mutations in the 2B4 variant.

[0396] Figure 7 : The sequence of the MDP decarboxylase, with secondary structures (β-sheets and α-helices) and mutated residues highlighted.

[0397] Figure 8 : (A) The structure of mevalonate diphosphate decarboxylase is characterized by a conical fold, where the N-terminal region is oriented normal to the relatively flat C-terminal region containing 5 α-helices. The active site is a positively charged cleft formed between the N domain and the C-terminal domain, with an ATP-binding P-loop nearby.

[0398] The mutations were classified according to their position in the structure: residues are visualized in yellow.

[0399] (B) A 3-D structure showing the positions of residues 9, 11, 42, 43, and 45 on β-strand 1 and β-strand 4 that are directly adjacent to each other.

[0400] (C) Residues in the C-terminal domain: Most of these residues are located within the region defined by α-helices 8-9 and β-strands 9-12. Mutations targeting the most C-terminal residues L293, F297, Q299, L303, K307, and T308 (the 9th α-helix and the 12th β-strand) may produce a more stable conformation that stabilizes the entire structure.

[0401] Figure 9 : Scheme for the chemical synthesis of 3-phosphonyloxyisovaleric acid

[0402] Figure 10 : Sequence alignment of SEQ ID NO: 1 with an MDP decarboxylase showing 60% to 80% sequence identity to SEQ ID NO: 1.

[0403] Figure 11 : Sequence alignment of SEQ ID NO: 1 with an MDP decarboxylase showing 80% to 90% sequence identity to SEQ ID NO: 1.

[0404] Figure 12 : Sequence alignment of SEQ ID NO: 1 with an MDP decarboxylase showing 90% to 100% sequence identity to SEQ ID NO: 1.

[0405] Figure 13 : Apparent kcat of MDP decarboxylase variants F9, F9-S141P, F9-116L-R91H-S141P-K241M-S248T-Q299K.

[0406] Figure 14 : Conversion of mevalonate-3-phosphate to 3-methylbut-3-en-1-ol by several mutants.

[0407] Figure 15 : Showing the production of 1,3-butadiene from (R)-3-hydroxypent-4-enoic acid in the following enzymatic assay:

[0408] Assay A: Without enzyme

[0409] Assay B: In the presence of 0.5 mg of Th. acidophilum MDP decarboxylase mutant L200E

[0410] Assay C: A combined assay containing 0.5 mg of Th. acidophilum MDP decarboxylase mutant L200E and 5 mg of Streptococcus mitis MDP decarboxylase mutant 2B4.

[0411] Figure 16 : Showed production of 1,3-butadiene from (R)-3-hydroxy-4-pentenoic acid in the following enzymatic assays:

[0412] Assay A: Without enzyme

[0413] Assay B: In the presence of 0.5 mg of Th. acidophilum MDP decarboxylase mutant L200E

[0414] Assay C: A combined assay containing 0.5 mg of Th. acidophilum MDP decarboxylase mutant L200E and 5 mg of S. mitis MDP decarboxylase mutant F9. Example

[0415] Materials and methods

[0416] Methods used to construct and select mutants

[0417] a. Directed evolution strategy

[0418] The enzyme S. mitis MDP decarboxylase is capable of catalyzing, among other reactions, the phosphorylation of 3-hydroxyisovaleric acid to 3-phosphonoxyisovaleric acid and the decarboxylation of 3-phosphonoxyisovaleric acid to isobutene. A directed evolution protocol was used to specifically improve the rate of conversion of 3-phosphonoxyisovaleric acid to isobutene by S. mitis MDP decarboxylase. This method consists of: (1) generating an initial collection of single-site mutants of S. mitis MDP decarboxylase, (2) designing an assay system to test the activity of these enzyme variants, (3) using this activity assay to screen the collection of mutants to identify mutants with improved activity compared to the activity of the wild-type S. mitis MDP decarboxylase, (4) using the best mutants identified during previous rounds of evolution as starting material for additional rounds of evolution (library construction and screening) (see Figure 1 ). This method led to the identification and characterization of a set of mutants with increased activity compared to the wild-type enzyme. During successive rounds of evolution, a series of molecular biology techniques were used to generate the libraries and collections of mutants to be screened. The activity assay typically consists of several steps to eliminate false negatives or assay artifacts among the initial positive hits and thus retain only true leads. The activity assay was accordingly modified to increase the activity rate of the isolated mutants to adjust its sensitivity and throughput.

[0419] b. Construction of S. mitis MDP decarboxylase mutants

[0420] Using a series of standard molecular biology techniques, polynucleotide sequences encoding different mutants identified during the evolution of Streptococcus mitis MDP decarboxylase were generated. All of these techniques used codon-optimized polynucleotide sequences for expression in Escherichia coli as templates (see SEQ ID NO: 4). Sequence optimization has been done by Geneart using its GeneOptimizer software.

[0421] Various PCR-based techniques known in the art were used to construct single-point mutants. To generate enzyme variants carrying multiple mutations (at least two mutations), PCR-based techniques or other methods known in the art were used to introduce these mutations.

[0422] After mutagenesis, the mutated polynucleotide sequences were inserted into an expression vector (for recombinant protein production and screening in Escherichia coli) using standard ligation-based subcloning techniques, whole plasmid PCR extension methods, or ligation-independent cloning techniques (LIC; Life Technology recombinant technology).

[0423] c. Selection of enzyme mutants with increased activity

[0424] Screening method description (see Figure 2 ):

[0425] The DNA library or collection of mutants inserted into the expression vector was transformed into the commercially available recombinant protein-expressing Escherichia coli strain BL21DE3. The DNA library transformation reaction was spread onto LB ampicillin plates and the isolated clones were used to inoculate starter cultures. When transforming a collection of mutants, each clone was transformed individually, spread out, and the individual clones were used to inoculate starter cultures; or the transformation mixture of each independent clone was directly used to inoculate starter cultures. Protein expression was carried out using the autoinduction medium (ZYM-5250) as described by Studier F.W (Protein Expr. Purif. 41 (2005), 207 - 234), where the initial stage of cell growth was at 37 °C for 6 hours and the induction stage was overnight at 28 °C. After transformation, all subsequent steps were carried out in microtiter plates or deep well plates. The cell cultures were centrifuged and the pellets were stored at -80 °C for at least 1 hour. Subsequently, the cell pellets were resuspended in a small volume of buffer and lysed by sonication, followed by centrifugation to remove cell debris.

[0426] Screening for variants with increased activity that convert 3-phosphonyloxyisovaleric acid to isobutene

[0427] To test the activity of the mutant enzyme in catalyzing the conversion of 3-phosphonyloxyisovalerate to isobutene, reaction mixtures were prepared in glass GC sample vials by mixing cell lysates (supernatants), 3-phosphonyloxyisovalerate substrate in 50 mM Tris pH 7 final concentration. 3-Phosphonyloxyisovalerate has been prepared enzymatically by incubating 3-hydroxyisovalerate, purified Thermophilus acidophilum MDP decarboxylase and cofactors (ATP, MgCl 2 , KCl) at 37 °C for 24 h. The reaction mixtures were incubated at 37 °C for varying lengths of time and the gases were injected into a gas chromatograph along with appropriate references (calibration isobutene, wild-type enzyme, negative control, etc.). After analyzing the GC chromatograms, mutants showing at least a 20% increase in activity were selected and subjected to a second round of screening, which followed the same conditions as the primary screening.

[0428] The final step in the selection process involves the production and purification of the top hits, i.e., enzyme variants with the highest improved activity verified by primary and secondary screening, and testing their activity at different substrate concentrations. The verified hits are further characterized and the kcat and Km of the reactions are calculated.

[0429] Modifications to the screening process:

[0430] As previously mentioned, the screening protocol was modified regularly as new improved mutants were identified. The major modifications to the protocol covered the following points:

[0431] - Cell culture conditions : Inoculation mode (using isolated clones or transformation reactions), media volume (200 μl to 5 ml), 24-deep well plates, 96-deep well and standard 96-well microplates, agitation varied according to the type of shaking incubator.

[0432] - Cell lysis : Type and volume of buffer used, sonication or no sonication, centrifugation or no centrifugation (crude lysates can be used effectively)

[0433] - Set-up of the enzymatic reaction:

[0434] ο Enzymatic preparation of 3-phosphonyloxyisovalerate before the assay (concentration of T. acidophilum enzyme, cofactors, HIV... variables, and incubation time and temperature)

[0435] ο Combine the phosphorylation of 3-hydroxyisovalerate and the decarboxylation of 3-phosphonyloxyisovalerate to isobutene in one tube: Streptococcus mitis MDD mutant combined with purified T. acidophilum and 3-hydroxyisovalerate. The reaction was incubated and the presence and amount of isobutene were determined.

[0436] ο The chemically synthesized pure 3-phosphonyloxyisovaleric acid compound is used in the assay. According to Figure 9 the protocol described in

[0437] ο Stirring the enzymatic reaction during incubation results in a 5-fold increase in signal

[0438] - The number of steps in the screening can vary

[0439] - The number of replicates analyzed for each clone varies (1 reaction per clone in the primary screening, and the clones can be tested in duplicate, triplicate, or more in subsequent screenings)

[0440] - The GC analysis method can vary: column type, type of vial and septum, and method (oven, injector, detector, temperature, analysis time... )

[0441] Example 1: Identification of single-point mutants of Streptococcus mitis MDP decarboxylase with increased activity in the reaction converting 3-phosphonyloxyisovaleric acid to isobutene

[0442] Using standard molecular biology techniques, a collection of 2,632 single-point mutants of Streptococcus mitis MDP decarboxylase was prepared. Saturation mutagenesis aimed to systematically replace all 317 amino acids of Streptococcus mitis MDP decarboxylase with 19 non-wild-type amino acids. The average number of substitutions at each position was 8 out of 19 possibilities. The coding sequence of each single mutant was subcloned into an expression vector to allow the production of a recombinant mutant enzyme with a 6His tag at the N-terminus in Escherichia coli.

[0443] The Escherichia coli strain BL21DE3 expressing recombinant proteins was transformed with an expression vector encoding a mutant enzyme, an empty expression vector (negative control), and an expression vector encoding a wild-type enzyme (positive control). To accelerate the process, the transformation was carried out in 96-well plates. Briefly, 2 μl of each mutant and each control DNA plasmid minipreparation per well was transferred to a 96-well 0.2 ml pCR reaction plate, followed by the addition of 40 μl / well of chemically competent Escherichia coli BL21DE3 cells. The plate was incubated on ice for 15 minutes, followed by heat shock at 42 °C for 1 minute in an AB2720 thermal cycler. Subsequently, the plate was immediately placed on ice and cooled for 1 minute, followed by the addition of 1 ml of sterile Luria-Bertani medium (10 g / l tryptone, 5 g / l yeast extract, 10 g / l NaCl, pH 7). The plate was sealed using a breathable adhesive film and incubated at 37 °C at 200 rpm in an Infors Minitron rotary shaker for 45 minutes. Subsequently, 50 μl of the transformation mixture was used to inoculate 0.5 ml of Luria-Bertani medium supplemented with 100 μg / ml ampicillin placed in a 96-deep well plate. The plates were sealed and incubated at 37 °C at 200 rpm overnight. Bacterial stocks were prepared by mixing 100 μl of the overnight starter culture with 35 μl of sterile 50% glycerol in a 96-well plate and storing at -80 °C until further use.

[0444] To produce the recombinant mutant enzymes, 1 ml of sterile autoinduction medium (Studier F.W, Protein Expr. Purif. 41 (2005), 207-234) supplemented with the appropriate antibiotic was dispensed into the wells of a 96-deep well plate and the wells were inoculated with 10 μl of the thawed glycerol stock. Each plate contained approximately 70 to 80 different mutant enzymes, 8 negative controls (empty expression vector), and 8 wild-type enzyme clones used as references. The plate was incubated at 37 °C at 1000 rpm in a Heidolph Titramax rotary shaker for 6 hours, followed by further incubation at 28 °C at 1000 rpm overnight. The bacterial cells were pelleted by centrifugation at 3200 x g at 4 °C for 20 minutes. The cell pellet was stored at -80 °C.

[0445] The pellet was thawed on ice for 5 to 10 minutes and resuspended in 250 μl of resuspension buffer (50 mM Tris-Cl pH 7, 20 mM KCl, 10 mM MgCl 2, in 10% glucose, 1 μl / ml Merck-Novagen Lysonase). The cell suspension was incubated at room temperature for 15 minutes and then on ice for 30 minutes. Bacterial cell lysis was performed by sonication of these cell suspensions with 4 five-minute pulses in an Advatange Lab ultrasonic water bath filled with ice and water (resting on ice for 5 minutes between pulses). The cell lysate was then centrifuged at 3200 x g for 20 minutes at 10 °C to pellet cell debris and 240 μl of the supernatant was transferred into a new plate. An enzymatic reaction was set up in an Agilent 2 ml glass vial by mixing 200 μl of the supernatant with 300 μl of 3-phosphonoxyisovaleric acid substrate. The vial was sealed completely using a crimp cap (PTFE-silicon-PTFE coated), incubated in a water bath at 37 °C for 24 hours and stored at -20 °C, after which it was analyzed by gas chromatography. To prepare the 3-phosphonoxyisovaleric acid substrate, 0.063 mg / ml of purified Thermophilus acidophilum MDP decarboxylase was mixed with 50 mM hydroxyisovaleric acid, 40 mM adenosine triphosphate in 50 mM Tris-Cl pH 7, 20 mM KCl, 20 mM MgCl 2 and incubated at 45 °C for 24 hours. This enzymatically prepared 3-phosphonoxyisovaleric acid substrate was aliquoted and stored at -20 °C until further use.

[0446] Quantify isobutene produced by the enzymatic reaction in the presence of Streptococcus mitis MDP decarboxylase by gas chromatography. Thaw the vial rapidly at 30 °C for 30 minutes and place it on an automated sampler mounted on a Varian GC-430 system equipped with a Varian CP SilicaPlot column (30 m x 0.32 mm), an injection port, and a flame ionization detector (FID). Set the sampler to inject 100 μl of headspace gas. For the GC analytical method used to detect isobutene, set the oven temperature to 185 °C, the injection port temperature to 150 °C, the split ratio 4:1, and the FID detector to 250 °C. The GC uses nitrogen as the carrier gas (constant flow rate 1.5 ml / min) and a mixture of air (flow rate 28 ml / min) and hydrogen (300 ml / min) for the FID detection system. The duration of the analysis is approximately 3 minutes per sample and under these conditions, isobutene is observed to elute at 2.5 minutes. Prior to starting the analysis, inject a sample of commercially purchased pure isobutene to calibrate the GC system and determine the retention time of isobutene. After the analysis, process the chromatogram using Galaxy software; integrate the area under the peak for each mutant and compare it to the wild-type enzyme. According to the protocol described above, perform a second test on mutants that show at least a 10 - 15% increase in isobutene production compared to the amount produced by the wild-type enzyme to eliminate false positives. Finally, test all mutant enzymes that have been selected through these two rounds of screening again using a normalized amount of purified protein. Briefly, the bacterial glycerol stock of the selected mutant enzyme is used to inoculate 7 ml of LB-Amp. 2 ml of this starter culture is used to inoculate 200 ml of autoinduction medium and plasmid DNA is extracted from 5 ml of the culture remainder. Sequence the plasmid DNA to confirm the presence and type of the mutation. Bacterial expression for the production of the mutant enzyme is carried out as previously described and the N-terminal 6His-tagged mutant enzyme is purified from the pellet using the Macherey-Nagel Protino purification kit according to the user manual. Determine the activity by mixing in a 2 ml GC vial: 500 μg of purified enzyme, 300 μl of 3-phosphonoxyisovaleric acid substrate, and adjust the volume to 500 μl with 50 mM Tris-Cl pH 7. Incubate the reaction at 37 °C for 24 hours and terminate it by freezing the sample at -20 °C. Determine the amount of isobutene produced by GC analysis.

[0447] This screening procedure led to the identification of 71 mutations that confer increased isobutene production activity on Streptococcus mitis MDP decarboxylase. Table 4 lists the positions and types of substitutions that have been identified. Figure 3 Show the results obtained by analyzing the amount of isobutene produced from a subset of mutants from Table 4 using an activity assay with purified protein. Figure 4a ) and Figure 4b)Display additional data.

[0448]

[0449] Example 2: Characterization of Variants of Streptococcus mitis MDP Decarboxylase with High Conversion Rates

[0450] A collection of Streptococcus mitis MDP decarboxylase variants has been generated using PCR-based techniques by combining the selection of single point mutations that confer a highly increased activity (at least 50% increase compared to the activity of the wild-type enzyme). Additional diversity has also been generated by random mutagenesis. The amount of isobutene produced by these variants was determined in an enzymatic assay in which 500 μg of purified enzyme was mixed with 300 μl of 3-phosphonovaleric acid substrate in 50 mM Tris-Cl pH 7 buffer. After a 24-hour incubation time at 37 °C, the reaction was terminated by freezing the samples at -20 °C and the amount of isobutene produced was determined by GC analysis. For GC headspace measurements, 100 μl of headspace gas was injected into the injection port of a Varian GC-430 system equipped with a Varian CP SilicaPlot column (30 m x 0.32 mm) and an FID. The GC analysis method used to detect isobutene was characterized by an oven temperature of 185 °C, an injection port temperature of 150 °C, a split ratio of 1:10, and an FID detector temperature of 250 °C. Nitrogen was used as the carrier gas (constant flow rate of 1.5 ml / min) and a mixture of air (air flow of 28 ml / min) and hydrogen (300 ml / min) was used to supply the FID detection system.

[0451] Multiple variants showing up to 11 position mutations have been identified, and these variants showed increased activity in the activity assay. Different variants are shown in the following table.

[0452] Table 5: Double mutants

[0453] Mutation % increase in activity compared to the wild-type enzyme K282CM42A 483 K282CL264Q 453 K282CL303M 510 K282CC118L 465 K282CA253i 493 K282CA120N 507 K282CE45M 516 K282CF297L 471 K282CY11C 508 K282CY121R 400 K282CS129V 514 K282CA120L 435 K282CK116L 440 K282R9L 428 K282CY11E 270 K282CE177V 493 K282CK116R 479 K282CA120I 205 K282CK116M 576 K282CT251M 452 K282CL293F 375 K282CK123M 178 K282CF122M 233 K282CY121L 290

[0454] Mutation % increase in activity compared to the wild-type enzyme E45LY121RK282C 420 K282CY121RY11E 230 K116IY121RK282C 147 Y121RE177VK282C 524 A120RY121LK282C 184 M173CK282CF297L 500

[0455] Mutation % increase in activity compared to the wild-type enzyme Y121RK282CL303MT308S 377 M173CL303MK307HT308S 480 E45VM173CK282CL303M 500

[0456] Mutation % increase in activity compared to the wild-type enzyme Y121RM173CK282CL303MT308S 507 E159LM173CL303MK307HT308S 480 R9LY11FL303MK307HT308S 440 C118LY121RM173CK282CL303M 500

[0457] Mutation % increase in activity compared to the wild-type enzyme Y121RE159LM173CK282CL303MT308S 558 Y121RE159LM173CV215AK282CL303M 500 E45LE1 59LM1 73CK282CF297LT308S 550

[0458] Table 10: Combinations of seven mutations

[0459]

[0460]

[0461] Table 11: Combinations of eight mutations

[0462]

[0463] Table 12: Combinations of nine mutations

[0464]

[0465] Table 13: Combinations of ten mutations

[0466]

[0467] Table 14: Combinations of eleven mutations

[0468]

[0469] Two variants named 2B4 (SEQ ID NO: 2) and F9 (SEQ ID NO: 3) with highly increased activity in the assay were selected for further characterization. Compared with the wild-type enzyme, the 2B4 and F9 protein sequences contain 6 and 11 mutations, respectively (see Table 15). F9 carries two new mutations, K24R and E291D, not identified in the original single-point mutation screen.

[0470]

[0471] Table 15: Mutations in variants 2B4 and F9

[0472] The Michaelis Menten kcat and Km steady-state kinetic constants of these two variants were determined as follows: In a GC vial, 200 μg of purified 2B4, F9, or wild-type enzyme, a series of 0 to 320 mM chemically synthesized 3-phosphonyloxyisovaleric acid, 5 mM ATP, 20 mM KCl, 10 mM MgCl 2A series of enzymatic reactions were established with 50 mM Tris-Cl pH 7.5. The vials were sealed and incubated at 37 °C for 15 h, after which the isobutene produced by GC was analyzed as previously described. Previous experiments had determined that the rate of isobutene production was constant during the first 20 h of the enzymatic reaction and thus the rate of isobutene production per hour measured after 15 h of incubation was equal to the initial rate of isobutene production at the start of the reaction. To quantify the absolute amount of isobutene produced by this reaction, a GC was calibrated using a series of concentrations of pure isobutene (0 to 10,000 ppm). The calibration table was found to be linear within this range of isobutene concentrations. The isobutene production rate (moles of isobutene / mole of enzyme / second) was plotted as a function of the 3-phosphonoxyisovalerate concentration and the curve was fitted using the Michealis Menten equation (V = (Vmax*(substrate)) / (Km+(substrate))) to extract the kcat (s-1) and Km values (mM) summarized in Table 16. As Figure 5 and shown in Table 16, variants 2B4 and F9 have a higher kcat than the wild-type enzyme.

[0473] Table 16: Summary of kcat (s-1) and Km (mM) values for variants 2B4 and F9

[0474]

[0475] Example 3: Identification of variants of Streptococcus mitis MDP decarboxylase with further increased activity in the reaction converting 3-phosphonoxyisovalerate to isobutene

[0476] Additional MVD variants with further enhanced activity in converting 3-phosphonoxyisovalerate to isobutene were identified by successive rounds of mutagenesis, site-directed mutagenesis recombination, and in vitro and / or in vivo screening assays. A list of these MVD variants is provided in Table 17 below.

[0477] Table 17

[0478]

[0479]

[0480] The fold increase is the ratio of the activity of the MDP decarboxylase variant to the activity of the wild-type MDP decarboxylase. The fold increase was determined for one substrate concentration (1 or 2 mM PIV for in vitro assays and 500 mM acetone for in vivo assays). The enzyme amounts were not normalized, but these MVD variants were expressed in similar amounts as observed by SDS-PAGE analysis of cell lysates.

[0481] The template for mutagenesis was a variant named "F9", which corresponded to the Streptococcus mitis MDP decarboxylase of SEQ ID NO: 1 with the following mutations: K24R C118L Y121R E159L M173C E177C K282C E291D F297L L303M T308S (see SEQ ID NO: 3). The in vitro screening assay used was the one described in section c of the above materials and methods, preferably an in vitro assay involving the use of cell lysates.

[0482] For in vivo testing, yet another in vivo assay was developed. This assay was based on the use of a bacterial strain transformed with an expression vector containing the coding sequences and resulting in the production of three enzymes involved in the metabolic pathway for the conversion of acetone to isobutene; namely, the house mouse (M. musculus) HMG-CoA synthase (hereinafter referred to as HIV synthase) was used for the production of 3-hydroxyisovaleric acid (HIV); the T. acidophilum MDP decarboxylase (hereinafter referred to as HIV phosphorylase) was used to phosphorylate 3-hydroxyisovaleric acid to 3-phosphonoxyisovaleric acid (PIV), and the Streptococcus mitis MDP decarboxylase variant to be tested (hereinafter referred to as PIV decarboxylase) was used to convert PIV to isobutene (IBN) by decarboxylation. This strain was first cultured overnight in a shaker at 30 °C / 1000 rpm in 1 ml of autoinduction medium to produce the three recombinant enzymes. Subsequently, the cell pellet containing the three overexpressed recombinant enzymes was resuspended in 500 μl of minimal medium supplemented with 500 mM acetone and incubated for an additional 16 hours in a shaker set at 37 °C / 1000 rpm. During this second step, the HIV synthase catalyzed the condensation of acetone (from the medium) with the cell's acetyl-CoA to form HIV, which was then sequentially converted to PIV and IBN by the HIV phosphorylase and PIV decarboxylase using the cell's ATP. Subsequently, the IBN produced was quantified by gas chromatography using the same method as described for the in vitro screening assay in section c of the materials and methods chapter.

[0483] The main advantages of this assay were as follows: (1) the production of IBN occurred inside the cell, (2) cell metabolites (ATP and acetyl-CoA) and cell cofactors were utilized, and only acetone was added to the reaction (acetone was a non-limiting factor in this pathway due to the efficiency of the Clostridium acetone pathway), (3) the increase in IBN production was measured in the context of the complete IBN production pathway and not only considered the cooperation of the three target enzymes but also the presence of potential endogenous inhibitors or competitors and low substrate concentrations, (4) the enzymes were mostly in their native form and had not undergone any form of purification process extraction, which might denature these enzymes and adversely affect their activity.

[0484] For certain of these variants, the fold increase in activity has also been determined over a range of amounts of the MDP decarboxylase variant to calculate kinetic parameters (apparent kcat and Km). The fold increase in apparent kcat generally parallels the apparent kcat determined in the screening assay (see Figure 13 ). In Figure 13 , the fold increase in F9-S141P kcat is approximately x3.5 of F9 kcat compared to x3 determined in the screening assay; the fold increase in F9-I16L-R91H-S141P-K241M-S248T-Q299K kcat is approximately x5.5 of F9 kcat compared to x3.7 determined in the screening assay.

[0485] Example 4: Variants of the Streptococcus mitis MDP decarboxylase also show increased activity in catalyzing the conversion of mevalonate-3-phosphate to 3-methylbut-3-en-1-ol

[0486] The ability of the MDP decarboxylase variants to convert mevalonate-3-phosphate to 3-methylbut-3-en-1-ol was evaluated using a coupled enzyme assay. This assay combines the sequential activities of two MDP decarboxylases: (1) T. acidophilum MDP decarboxylase (L200E mutant) catalyzes the phosphorylation of mevalonate to mevalonate-3-phosphate; (2) Streptococcus mitis MDP decarboxylase catalyzes the conversion of mevalonate-3-phosphate to 3-methylbut-3-en-1-ol. This assay was established using 200 mM R,S-sodium mevalonate substrate, 2 mg / ml of the Streptococcus mitis MDP decarboxylase variant, and 0.1 mg / ml of T. acidophilum MDP decarboxylase (L200E mutant) in 50 mM Tris-HCl pH 7.5, 10 mM MgCl 2 , 20 mM KCl, 40 mM ATP. Negative controls without enzyme or with either enzyme were also prepared. The reaction mixture was incubated at 37 °C in a sealed glass bottle for 24 hours. 3-Methylbut-3-en-1-ol was extracted by mixing 50 μl of the reaction mixture with 100 μl of ethyl acetate. The 100 μl ethyl acetate upper phase was transferred to a clean bottle and analyzed by gas chromatography. Commercial 3-methylbut-3-en-1-ol was used as a reference. Samples were analyzed on a Varian GC-430 gas chromatograph equipped with a flame ionization detector (FID). A 1 μl sample was analyzed using the following temperature gradient on a DB-WAX column (30 m, 0.32 x 0.50 μm, Agilent): 2 minutes at 60 °C, temperature ramp to 220 °C (20 °C / minute), and a final 10 minutes at 220 °C. Under these conditions, the retention time of 3-methylbut-3-en-1-ol is 7.38 minutes.

[0487] The mutations I160N, R186H, and R91H were observed to also confer increased activity of the Streptococcus mitis MDP decarboxylase to convert mevalonate-3-phosphate to 3-methylbut-3-en-1-ol (see Figure 14 ). The increased activity is specific to these particular mutations, as increased production was detected on the "2B4" or "F9" variant templates.

[0488] Example 5: Production of butadiene from 3-hydroxypent-4-enoic acid catalyzed by mutant 2B4 of MDP decarboxylase from Streptococcus mitis

[0489] Mutant 2B4 was described in Example 2.

[0490] (R)-3-Hydroxypent-4-enoic acid was synthesized as required by a company specializing in custom synthesis (Syntheval, France).

[0491] The enzymatic reaction was carried out under the following conditions:

[0492] 50 mM Tris-HCl pH 7.5

[0493] 0 - 200 mM (R)-3-hydroxypent-4-enoic acid ("R" HPA)

[0494] 50 mM ATP

[0495] 20 mM MgCl 2

[0496] 20 mM KCl

[0497] The pH was adjusted to 7.5

[0498] Each assay was initiated by adding the specific purified enzyme to 0.5 ml of the reaction mixture. The assays were then incubated in 2 ml sealed vials (Interchim) at 37 °C with shaking. Control reactions were carried out in parallel. After 20 hours of incubation, butadiene production was analyzed as follows. 1 ml of the gas phase of each assay was collected and directly injected into a gas chromatograph GC-450 equipped with a flame ionization detector (FID). Nitrogen was used as the carrier gas at a flow rate of 1.5 ml / min. The volatile compounds were chromatographically separated using the isothermal mode at 130 °C on a RT-alumina bonded / Na 2 SO 4 column (30 m, 0.32 mm ID, 5 μm) (Restek). The enzymatic reaction products were identified by comparison with a 1,3-butadiene standard (Sigma). Under these GC conditions, the retention time of butadiene was 7.4 minutes.

[0499] Figure 15Results shown: No 1,3-butadiene formation was observed in the absence of substrate. GC analysis of the enzyme-free reaction showed only trace amounts of butadiene from the thermal decomposition of 3-hydroxy-4-pentenoic acid. Catalytic tests showed a significant increase in butadiene production in the presence of mutant 2B4 of MDP decarboxylase from Streptococcus mitis.

[0500] Example 6: Production of butadiene from 3-hydroxy-4-pentenoic acid catalyzed by mutant F9 of MDP decarboxylase from Streptococcus mitis

[0501] Mutant F9 is described in Example 2.

[0502] The enzymatic reaction was carried out under the following conditions:

[0503] 50 mM Tris-HCl pH 7.5

[0504] 0 - 200 mM “R” 3-hydroxy-4-pentenoic acid (“R” HPA)

[0505] 50 mM ATP

[0506] 20 mM MgCl 2

[0507] 20 mM KCl

[0508] The pH was adjusted to 7.5.

[0509] Each assay was initiated by adding a specific enzyme to 0.5 ml of the reaction mixture. The assays were then incubated in 2 ml sealed vials (Interchim) at 37 °C with shaking. Control reactions were carried out in parallel. After 20 h of incubation, butadiene production was evaluated according to the method described in Example 5.

[0510] Figure 16 Results shown: No 1,3-butadiene formation was observed in the absence of substrate. GC analysis of the enzyme-free reaction showed only trace amounts of butadiene from the thermal decomposition of 3-hydroxy-4-pentenoic acid. Catalytic tests showed a significant increase in butadiene production in the presence of mutant F9 of MDP decarboxylase from Streptococcus mitis.

[0511] Example 7: Identification of variants of MDP decarboxylase from Streptococcus mitis with further increased activity in the reaction converting 3-phosphooxyisovalerate to isobutene

[0512] Additional MVD variants with further enhanced activity in the conversion of 3-phosphooxyisovalerate to isobutene were identified by successive rounds of directed or random mutagenesis, site-directed recombination, and in vitro and / or in vivo screening assays. A list of these MVD variants is provided in Table 18 below.

[0513] Table 18

[0514]

[0515]

[0516]

[0517] The fold increase is the ratio of the activity of the MDP decarboxylase variant to the activity of the "F9" variant of MDP decarboxylase. The "F9" variant corresponds to the Streptococcus mitis MDP decarboxylase of SEQ ID NO: 1 having the following mutations: K24R-C118L-Y121R-E159L-M173C-E177C-K282C-E291D-F297L-L303M-T308S (see SEQ ID NO: 3). The fold increase is determined for one substrate concentration (2 or 6 mM PIV in in vitro assays and 10 mM HIV in in vivo assays). The enzyme amounts are not normalized, but these MVD variants are expressed in similar amounts as observed by SDS-PAGE analysis of cell lysates.

[0518] The in vitro screening assay used in Example 7 herein is described in Section c of the above Materials and Methods, where preferably, in vitro assays involving cell lysates have been used and the assays have preferably been further miniaturized in 384 deep well microtiter plates.

[0519] For in vivo testing, yet another in vivo screening assay has been developed as outlined below. This assay is based on a bacterial strain transformed with an expression vector that contains a coding sequence and results in the production of the last two enzymes involved in the metabolic pathway for converting acetone to isobutene. More specifically, to produce 3-phosphonoxyisovalerate (PIV) from 3-hydroxyisovalerate (HIV), T. acidophilum MDP decarboxylase (hereinafter referred to as HIV phosphorylase) is used, and to convert PIV to isobutene (IBN), a Streptococcus mitis MDP decarboxylase variant to be tested (hereinafter referred to as PIV decarboxylase) is used. This strain is first cultured in a shaker at 30 °C, 700 rpm, in 300 μL of autoinduction medium in a 384 deep well microtiter plate for 24 hours to produce two types of recombinant enzymes. Subsequently, the cell pellet containing these two overexpressed recombinant enzymes is resuspended in 50 μL of minimal medium supplemented with 10 mM HIV and further incubated in a shaker at 30 °C, 700 rpm for an additional 4 hours. During this step, HIV phosphorylase catalyzes the phosphorylation of HIV to PIV using cellular ATP, and the PIV is then converted to IBN by the PIV decarboxylase variant. Subsequently, the IBN produced is quantified by gas chromatography using the same method as described for the in vitro screening assay in Section c of the above Materials and Methods chapter.

Claims

1. A variant of mevalonate diphosphate decarboxylase that, relative to the corresponding mevalonate diphosphate decarboxylase from which it is derived, shows improved activity in converting 3-phosphonoxyisovalerate to isobutene, wherein the mevalonate diphosphate decarboxylase variant is characterized by a cysteine, serine, glutamate, glycine, glutamine, threonine, valine, alanine, or aspartate substitution at position 282 in the amino acid sequence shown in SEQ ID NO:

1.

2. The mevalonate diphosphate decarboxylase variant of claim 1, wherein at least one additional amino acid residue is substituted at a position selected from positions 9, 11, 16, 24, 28, 42, 45, 53, 80, 105, 116, 118, 120, 121, 122, 123, 129, 134, 141, 159, 161, 173, 177, 180, 186, 215, 238, 241, 242, 248, 251, 253, 264, 279, 291, 293, 297, 299, 303, 307, 308, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267, and 315 in SEQ ID NO:

1.

3. The mevalonate diphosphate decarboxylase variant of claim 1 or 2, wherein: (1) the amino acid residue at position 16 in the amino acid sequence shown in SEQ ID NO:1 is replaced with leucine; and / or (2) the amino acid residue at position 23 in the amino acid sequence shown in SEQ ID NO:1 is replaced with leucine; and / or (3) the amino acid residue at position 24 in the amino acid sequence shown in SEQ ID NO:1 is replaced with arginine, serine, or leucine; and / or (4) the amino acid residue at position 28 in the amino acid sequence shown in SEQ ID NO:1 is replaced with lysine or alanine; and / or (5) the amino acid residue at position 31 in the amino acid sequence shown in SEQ ID NO:1 is replaced with serine; and / or (6) the amino acid residue at position 105 in the amino acid sequence shown in SEQ ID NO:1 is replaced with alanine; and / or (7) the amino acid residue at position 111 in the amino acid sequence shown in SEQ ID NO:1 is replaced with methionine; and / or (8) the amino acid residue at position 134 in the amino acid sequence shown in SEQ ID NO:1 is replaced with glycine; and / or (9) the amino acid residue at position 139 in the amino acid sequence shown in SEQ ID NO:1 is replaced with cysteine or alanine; and / or (10) the amino acid residue at position 141 in the amino acid sequence shown in SEQ ID NO:1 is replaced with proline, cysteine, glycine, or threonine; and / or (11) Substitute the amino acid residue at position 142 in the amino acid sequence shown in SEQ ID NO:1 with alanine; and / or (12) Substitute the amino acid residue at position 182 in the amino acid sequence shown in SEQ ID NO:1 with glutamic acid; and / or (13) Substitute the amino acid residue at position 186 in the amino acid sequence shown in SEQ ID NO:1 with histidine, leucine, valine, isoleucine or asparagine; and / or (14) Substitute the amino acid residue at position 188 in the amino acid sequence shown in SEQ ID NO:1 with cysteine; and / or (15) Substitute the amino acid residue at position 279 in the amino acid sequence shown in SEQ ID NO:1 with alanine.

4. A mevalonate diphosphate decarboxylase variant according to claim 1 or 2, wherein the amino acid residue at position 282 in the amino acid sequence shown in SEQ ID NO:1 is substituted with cysteine, serine, glutamic acid, glycine, glutamine, threonine, valine, alanine or aspartic acid, and wherein the amino acid residue at position 121 in the amino acid sequence shown in SEQ ID NO:1 is substituted with arginine.

5. A mevalonate diphosphate decarboxylase variant according to claim 1 or 2, the amino acid sequence of which is as shown in SEQ ID NO:1, wherein the following substitutions have been effected: K24R-C118L-Y121R-E159L-M173C-E177C-K282C-E291D-F297L-L303M-T308S.

6. A mevalonate diphosphate decarboxylase variant according to claim 5, which variant shows the following additional substitutions: S141P-I16L-K241I-S248T-M28K-K180P.

7. A mevalonate diphosphate decarboxylase variant according to claim 5, which variant shows the following additional substitutions: S141P-I16L-R91H-K241M-S248T-Q299K.

8. A mevalonate diphosphate decarboxylase variant according to claim 1 or 2, the amino acid sequence of which is as shown in SEQ ID NO:1, wherein the following substitutions have been effected: Y121R-E159L-M173C-K282C-L303M-T308S.

9. A nucleic acid molecule encoding a mevalonate diphosphate decarboxylase variant according to any one of claims 1 to 8.

10. A vector comprising the nucleic acid molecule of claim 9.

11. A host cell comprising the vector of claim 10.

12. A method for providing a variant of mevalonate diphosphate decarboxylase, wherein the variant shows improved activity in the conversion of 3-phosphonooxyisovalerate to isobutene, the method comprising the step of substituting the amino acid at position 282 in the amino acid sequence shown in SEQ ID NO:1 with cysteine, serine, glutamic acid, glycine, glutamine, threonine, valine, alanine or aspartic acid.

13. The method of claim 12, wherein the implemented changes further include the changes described in claim 2.

14. Use of a mevalonate diphosphate decarboxylase according to any one of claims 1 to 8 or a host cell according to claim 11 for converting 3-hydroxyisovaleric acid or 3-phosphonyloxyisovaleric acid into isobutene.

15. A method for producing isobutene from 3-hydroxyisovaleric acid or from 3-phosphonyloxyisovaleric acid, the method comprising the steps of: (i) culturing the host cell of claim 11 in a suitable medium; and (ii) recovering the produced isobutene.

16. Use of a mevalonate diphosphate decarboxylase according to any one of claims 1 to 8 or a host cell according to claim 11 for converting mevalonic acid or mevalonate 3-phosphate into 3-methylbut-3-en-1-ol.

17. A method for producing 3-methylbut-3-en-1-ol from mevalonic acid or from mevalonate 3-phosphate, the method comprising the steps of: (i) culturing the host cell of claim 11 in a suitable medium; and (ii) recovering the produced 3-methylbut-3-en-1-ol.

18. Use of a mevalonate diphosphate decarboxylase according to any one of claims 1 to 8 or a host cell according to claim 11 for converting 3-hydroxypent-4-enoic acid or 3-phosphonyloxypent-4-enoic acid into 1,3-butadiene.

19. A method for producing 1,3-butadiene from 3-hydroxypent-4-enoic acid or from 3-phosphonyloxypent-4-enoic acid, the method comprising the steps of: (i) culturing the host cell of claim 11 in a suitable medium; and (ii) recovering the produced 1,3-butadiene.

Images