3-Methylcrotonate decarboxylase (MDC) variants

Abstract

Kind Code: A1 Abstract: 3-methylcrotonic acid decarboxylase (MDC) variants that exhibit improved activity in converting 3-methylcrotonic acid to isobutene are described, as well as methods for the production of isobutene using such enzyme variants.

Description

Description of the invention

[0001] Kind Code: A1 Abstract: 3-methylcrotonic acid decarboxylase (MDC) variants that exhibit improved activity in converting 3-methylcrotonic acid to isobutene are described, as well as methods for the production of isobutene using such enzyme variants.

[0002] Many chemical compounds are currently obtained from petrochemicals: alkenes (such as ethylene, propylene, the various butenes or pentenes, etc.) are used in the plastics industry, for example to produce polypropylene or polyethylene, and in other areas of the chemical and fuel industries.

[0003] Butylene exists in four forms, one of which, isobutene (also called isobutylene), is included in the composition of methyl tert-butyl ether (MTBE), an anti-knock additive for automotive fuel. Isobutene can also be used to produce isooctene, which can then be reduced to isooctane (2,2,4-trimethylpentane). Isooctane has a very high octane number, making it the fuel of choice for so-called "gasoline" engines. Alkenes such as isobutene are currently produced by catalytic cracking of petroleum products (or, in the case of hexene, by Fischer-Tropsch derivatives from coal or gas). For this reason, production costs are closely linked to oil prices. Furthermore, catalytic cracking can involve considerable technical difficulties that increase the complexity of the process and the production costs.

[0004] The biological production of alkenes such as isobutene is needed in the context of sustainable industrial operations in harmony with geochemical cycles. First generation biofuels existed in the fermentative production of ethanol, since fermentation and distillation processes already existed in the food processing industry. The production of second generation biofuels is in the exploratory stage, which includes in particular the production of long-chain alcohols (butanols and pentanols), terpenes, linear alkanes and fatty acids. Two recent reviews provide an overview of research in this field: Ladygina et al. (Process Biochemistry 41 (2006), 1001) and Wackett (Current Opinions in Chemical Biology 21 (2008), 187).

[0005] Various pathways for the enzymatic production of isobutene have been described, see for example Fujii et al. (Appl. Environ. Microbiol. 54 (1988), 583); Gogerty et al. (Appl. Environm. Microbiol. 76 (2010), 8004-8010) and van Leeuwen et al. (Appl. Microbiol. Biotechnol. 93 (2012), 1377-1387) and WO 2010 / 001078.

[0006] In addition to these pathways, there is also an alternative route for providing isobutene, utilizing the enzymatic conversion of 3-methylcrotonic acid to isobutene by decarboxylation, a chemical reaction that removes a carboxy group to release carbon dioxide (CO2).

[0007] The decarboxylation of 3-methylcrotonic acid has already been suggested in US Patent Application Publication No. 2009 / 0092975, but there is no experimental evidence for this transformation. US Patent Application Publication No. 2009 / 0092975 describes a nucleic acid sequence called PAD1 from Saccharomyces cerevisiae, which is disclosed to encode a decarboxylase enzyme. It is suggested that this enzyme is useful as a selection marker in recombinant organisms, while it is described that a "weak acid" may be used as a selection agent. 3-methylcrotonic acid is mentioned as a potential "weak acid" among many others.

[0008] However, it was later discovered that the PAD1 does not in fact confer decarboxylase activity.

[0009] Indeed, the bacterial ubiD and ubiX genes, or the homologous eukaryotic fdc1 and pad1 genes, are thought to be involved in nonoxidative reversible decarboxylation. The combined action of phenylacrylic acid decarboxylase (PAD) and ferulic acid decarboxylase (FDC) is thought to be essential for the decarboxylation of phenylacrylic acid in Saccharomyces cerevisiae (J. Biosci. Bioeng. 109, (2010), 564-569; AMB Express, 5:12 (2015) 1-5; ACS Chem. Biol. 10 (2015), 1137-1144).

[0010] The above mentioned family of enzymes, previously described as phenylacrylic acid decarboxylases (PADs), have recently been characterized no longer as decarboxylases but as flavin prenyltransferases. Fdc1 (not a PAD) has been shown to be the sole source of the reversible decarboxylase activity and to require a new type of cofactor, namely a prenylated flavin, synthesized by the related UbiX (or Pad1) protein. Thus, the true enzymatic activity of this PAD enzyme was identified as the conversion of the flavin mononucleotide (FMN) cofactor by its prenyl moiety (derived from dimethyl-allyl-phosphate or dimethyl-allyl-pyrophosphate, termed DMAP or DMAPP). This reaction is illustrated in Figure 1A.

[0011] Thus, in contrast to the prior art thinking, the true decarboxylase is ferulic acid decarboxylase (FDC) accompanied by modified FMN (prenylated-FMN). The reaction is shown in FIG. 1B. This mechanism of ferulic acid decarboxylase (FDC) accompanied by modified FMN (prenylated-FMN), the latter provided by PAD enzymes, has been recently described and involves a surprising enzymatic mechanism, namely, α,β-unsaturated acid decarboxylation via 1,3-dipolar cycloaddition. Moreover, the structure of this FDC decarboxylase has recently been elucidated (Nature 522 (2015), 497-501; Nature, 522 (2015), 502-505; Appl. Environ. Microbiol. 81 (2015), 4216- 4223).

[0012] WO 2017 / 191239 and WO 2020 / 007886 describe enzyme variants based on the FDC enzymes of Hypocrea atroviridis and Streptomyces sp. 769 that show improved capacity in converting 3-methylcrotonic acid to isobutene.

[0013] Although the above means and processes make it possible to produce isobutene from 3-methylcrotonic acid, there is still a need for improvements, in particular with regard to further increasing the efficiency of the process to make it more suitable for industrial purposes.

[0014] The present application addresses this need by providing embodiments defined in the claims.

[0015] Thus, the present invention provides a variant of 3-methylcrotonic acid decarboxylase (MDC), as defined in the claims, which exhibits improved activity in converting 3-methylcrotonic acid to isobutene over the corresponding MDC from which it is derived.

[0016] An improved enzyme variant, or an enzyme variant capable of catalyzing a reaction with enhanced activity, is defined as an enzyme variant that differs from the wild-type enzyme such that, as a result of catalyzing the conversion of 3-methylcrotonic acid to isobutene, the specific activity of the enzyme variant is higher than the specific activity of the wild-type enzyme for at least one given concentration of 3-methylcrotonic acid (preferably greater than 0M and up to 1M of any 3-methylcrotonic acid). Specific activity is defined as the number of moles of substrate converted to moles of product per unit time and mole of enzyme. K cat (Turnover number) is the specific activity at saturating concentrations of substrate.

[0017] In the context of the present invention, "improved activity" means that the activity of the enzyme 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%, particularly preferably at least 90% or 100% higher than the activity of the enzyme from which the variant is derived, preferably higher than the activity of the enzyme represented by SEQ ID NO: 1. In an even more preferred embodiment, improved activity means at least 150%, at least 200%, at least 300%, at least 750% or at least 1000% higher than the activity of the corresponding enzyme from which the variant is derived, preferably higher than the activity of the enzyme represented by SEQ ID NO: 1. In a particularly preferred embodiment, the activity is measured by using an assay with purified enzyme and chemically synthesized substrate, as described below. The improved activity of the variant can be measured as a higher production of isobutene in a given time period compared to the parent enzyme under defined conditions. This improved activity can be due to a higher turnover number, e.g. a higher kcat value. It can also be due to a lower Km value. This may also be due to a higher kcat / Km value. Additionally, this may also be due to a higher solubility or stability of the enzyme. The degree of improvement may be measured as an improvement in isobutene production. The degree of improvement may also be measured by an improvement in kcat, an improvement in kcat / Km, or a decrease in Km, the production of soluble protein, or protein stability.

[0018] In another embodiment, the enzyme variants provided herein are capable of converting 3-methylcrotonic acid to isobutene with an activity at least 1.10 times higher than the turnover rate of the corresponding wild-type enzyme having the amino acid sequence set forth in SEQ ID NO: 1. In a more preferred embodiment, the enzyme variants capable of converting 3-methylcrotonic acid to isobutene have a turnover rate (i.e., k) that is at least 2 times, at least 3 times, at least 5 times, or even at least 10 times higher than the turnover rate of the corresponding wild-type enzyme having the amino acid sequence set forth in SEQ ID NO: 1.cat In even more preferred embodiments, the turnover rate is at least 100 times, or even at least 500 times, higher than the turnover rate of the corresponding wild-type enzyme having the amino acid sequence shown in SEQ ID NO:1.

[0019] Such enzyme variants are obtained by mutating specific positions in the amino acid sequence of MDC, and the variants obtained by such mutations show improved activity in catalyzing the conversion of 3-methylcrotonic acid to isobutene. The activity of the enzyme capable of converting 3-methylcrotonic acid to isobutene can be measured by methods known to those skilled in the art. In one embodiment, this activity is measured as described in the examples attached hereto. In a particular embodiment, this activity can be measured by incubating in vitro a cell lysate containing the enzyme, preferably an overexpressed recombinant protein. Alternatively, purified enzymes can be used or in vivo assays can be used.

[0020] More specifically, the activity of MDC variants for the conversion of 3-methylcrotonic acid to isobutene can be assessed by purified protein-based enzymatic in vitro assays and detection of isobutene by gas chromatography.

[0021] The MDC variants to be tested can be provided according to the following protocol: the MDC to be tested is subcloned into the pET25 (Novagen) expression vector or is fused with a 5' or 3' polynucleotide tag encoding a 6His purification tag and then cloned into the pET25 expression vector. The assay is based on the use of a bacterial strain (BL21(DE3), Novagen) transformed with two expression vectors directing the production of the last two enzymes involved in the metabolic pathway that converts 3-methylcrotonic acid (3MC) to isobutene, namely the flavin prenyltransferase UbiX protein from E. coli cloned into the pRSFDuet™ (Novagen) expression vector, and the considered MDC variant cloned into one of the expression vectors mentioned above. The strain is first plated on an LB agar plate supplemented with the appropriate antibiotic. The cells are grown overnight at 32° C. until individual colonies reach the desired size. Single colonies are then picked and individually transferred to 50 μL of liquid LB medium supplemented with the appropriate antibiotic. Cells are grown at 34° C. with shaking for 21 hours. The LB cultures are used to produce the two recombinant enzymes by inoculating 300 μL into autoinduction medium (Studier FW, Prat. Exp. Pur. 41, (2005), 207-234) in 384-well deep-well microplates supplemented with the appropriate antibiotic and grown for 24 hours at 34° C. in a shaking incubator set at 700 rpm and 85% humidity. The cell pellet containing these two overexpressed recombinant enzymes is then resuspended in 30 μL of lysis mixture (pH 7.5, phosphate 50 mM, NaCl 20 mM, MgCl2 2 mM, lysozyme 1 mg / mL, DNase 0.03 mg / mL) and incubated for 1 hour at 34° C. in a shaking incubator at 700 rpm.This mixture is then supplemented with 10 μL of reaction mixture (final composition: pH 7.5, phosphate 50 mM, NaCl 20 mM, MgCl2 2 mM, lysozyme 0.75 mg / mL, DNase 0.0225 mg / mL, KCl 100 mM) supplemented with 1–200 mM (final) 3MC and incubated for another 1–4 h at 34 °C and 700 rpm in a shaking incubator. During this step, the MDC enzyme catalyzes the decarboxylation of 3MC to isobutene (IBN). After inactivation at 80 °C or 90 °C for 5–10 min, the IBN produced is quantified by gas chromatography as follows: 100 μL of headspace gas from each enzymatic reaction is injected into a Brucker GC-450 system equipped with a flame ionization detector (FID). The compounds present in the samples are analyzed using an RTX-1 column at 1 mL.min. -1 The mixture is separated by chromatography at 100° C. with a constant flow of nitrogen as the carrier gas. Upon injection, the peak area of ​​isobutene is calculated.

[0022] By providing the above enzyme variant, the present invention makes it possible to dramatically improve the efficiency of producing isobutene from 3-methylcrotonic acid.

[0023] The term "3-methylcrotonic acid decarboxylase (MDC)" refers to an enzyme capable of catalyzing the decarboxylation of 3-methylcrotonic acid to isobutene. Decarboxylation is a chemical reaction that removes a carboxyl group to release carbon dioxide. This activity can be measured by methods known in the art and as described above. In a preferred embodiment, MDC is ferulic acid decarboxylase (FDC) or is derived from such an enzyme. FDC belongs to the enzyme class EC4.1.1.-. As mentioned above, it has been originally described that FDC coupled with modified FMN (prenylated-FMN) can catalyze α,β-unsaturated decarboxylation via 1,3-dipolar cycloaddition, more specifically, the decarboxylation of 3-methylcrotonic acid to isobutene. Thus, in the context of the present invention, the term FDC relates to an enzyme capable of catalyzing the decarboxylation of 3-methylcrotonic acid to isobutene, preferably when provided with prenylated FMN. In a preferred embodiment, the enzyme can be classified as a UbiD-like enzyme, meaning that it has the same enzymatic activity as UbiD (decarboxylation of α,β-unsaturated carboxylic acids using the same cofactors), but different substrate preference. Unlike UbiD, FDC is not involved in the biosynthesis of ubiquinone.

[0024] FDC enzymes have been described, for example, in Saccharomyces cerevisiae, Enterobacter sp., Bacillus pumilus, Aspergillus niger or Candida dubliniensis. Thus, in a preferred embodiment, the FDC is derived from Saccharomyces cerevisiae (UniProt Accession No. Q03034), Enterobacter sp. (UniProt Accession No. V3P7U0), Bacillus pumilus (UniProt Accession No. Q45361), Aspergillus niger (UniProt Accession No. A2R0P7) or Candida dubliniensis (UniProt Accession No. B9WJ66). In a more preferred embodiment, the FDC is 3-polyprenyl-4-hydroxybenzoate decarboxylase (UbiD).3-Polyprenyl-4-hydroxybenzoate decarboxylases are found in, for example, Hypocrea atroviridis, Sphaerulina musiva, Penicillinum requeforti, Fusarium oxysporum f. sp. lycopersici, Saccharomyces kudriavzevii, Saccharomyces cerevisiae, Aspergillus parasiticus, Candida albicans, Grosmannia clavigera, Escherichia coli, Bacillus megaterium, and the like. megaterium, Methanothermobacter sp. CaT2 or Mycobacterium chelonae 1518.Thus, in a more preferred embodiment, the FDC enzyme variant capable of catalyzing the decarboxylation of 3-methylcrotonic acid to isobutene is selected from the group consisting of Hypocrea atroviridis (UniProt accession number G9NLP8), Sphaerulina musiva (UniProt accession number M3DF95), Penicillinum roqueforti (UniProt accession number W6QKP7), Fusarium oxysporum f. sp. lycopersici (UniProt accession number W9LTH3), Saccharomyces kudriavzevii (UniProt accession number J8TRN5), Saccharomyces cerevisiae (UniProt accession number H8TRN6), Saccharomyces cerevisiae (UniProt accession number H8TRN5), Saccharomyces cerevisiae (UniProt accession number H8TRN7), Saccharomyces cerevisiae (UniProt accession number H8TRN9), Saccharomyces cerevisiae (UniProt accession number H8TRN1 ... cerevisiae, Aspergillus parasiticus, Candida albicans, Grosmannia clavigera, Escherichia coli (UniProt accession number P0AAB4), Bacillus megaterium (UniProt accession number D5DTL4), Methanothermobacter sp. CaT2 (UniProt accession number T2GKK5) or 3-polyprenyl-4-hydroxybenzoate decarboxylase (UbiD) from Mycobacterium chelonae 1518 (UniProt accession number X8EX86). Preferably, MDC is an enzyme that is associated with and / or dependent on flavin prenyltransferase.As mentioned above, the enzymatic conversion of 3-methylcrotonic acid to isobutene using a prenylated FMN-dependent decarboxylase is preferably associated with a flavin prenyltransferase and relies on a two-step reaction catalyzed by two enzymes, namely a prenylated FMN-dependent decarboxylase (which catalyzes the actual decarboxylation of 3-methylcrotonic acid to isobutene) and an associated flavin prenyltransferase which provides a modified flavin cofactor. The flavin cofactor can preferably be FMN or FAD. FMN (flavin mononucleotide, also called riboflavin-5'-phosphate) is a biomolecule generated from riboflavin (vitamin B2) by the enzyme riboflavin kinase and serves as a prosthetic group for various reactions. FAD (flavin adenine dinucleotide) is a redox cofactor, more specifically a prosthetic group, and is involved in several important reactions in metabolism. Flavin prenyltransferases that can be associated with the MDC variants of the present invention are described in more detail below.

[0025] The present invention now provides improved variants of the enzyme capable of converting 3-methylcrotonic acid to isobutene. Using the FDC of Yersinia frederiksenii, shown in SEQ ID NO: 1, as a model enzyme, the inventors were able to show that it is possible to provide variants of this enzyme that show enhanced activity for the conversion of 3-methylcrotonic acid to isobutene.

[0026] The model enzyme used by the present inventors, namely FDC of Yersinia frederiksenii, has the amino acid sequence shown in SEQ ID NO:1.

[0027] In a preferred embodiment, the variants of the invention are characterized by the fact that they are derived from MDC, more preferably from an MDC having the amino acid sequence shown in SEQ ID NO: 1 or a highly related sequence (at least 55% identical), with mutations occurring at one or more of the positions indicated above, and that they exhibit the ability to convert 3-methylcrotonic acid to isobutene, and that they can do so with improved activity. In a preferred embodiment, the variants according to the invention are derived from a sequence that exhibits at least 60%, more preferably at least 70%, even more preferably at least 80% sequence identity to SEQ ID NO: 1, with one or more substitutions and / or deletions and / or insertions occurring at the positions indicated herein.

[0028] However, the teachings of the present invention are not limited to the MDC enzyme of Yersinia frederiksenii shown in SEQ ID NO: 1, which has been used as a model enzyme, but can be extended to MDC enzymes from other organisms, or to enzymes that are structurally related to SEQ ID NO: 1, such as truncated variants of the enzyme. Thus, the present invention also relates to variants of MDC that are structurally related to the Yersinia frederiksenii sequence (SEQ ID NO: 1) and that exhibit one or more substitutions and / or deletions and / or insertions at positions corresponding to any of the positions shown herein below. The term "structurally related" refers to an MDC that exhibits at least n% sequence identity to the sequence shown in SEQ ID NO:1, where n is an integer between 55 and 100, and preferably 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99. In a preferred embodiment, the structurally related MDC is derived from a bacterium, more preferably a Gram-negative bacterium, and even more preferably from the genus Yersinia.

[0029] Thus, in one embodiment a variant of MDC according to the invention has a sequence (or preferably is derived from) that is at least n% identical to SEQ ID NO:1, where n is an integer between 55 and 100, preferably 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99, and has substitutions and / or deletions and / or insertions at the positions indicated herein. When the sequences to be 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 the amino acid residues in the longer sequence, or refers to the percentage of amino acid residues in the shorter sequence that are identical to the amino acid residues in the longer sequence.Preferably, it refers to the percentage of amino acid residues in the shorter sequence that are identical to the amino acid residues in 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.

[0030] When using the Clustal analysis method to determine whether a particular sequence is, for example, at least 55% identical to a reference sequence, the default settings can be used, or the settings are preferably as follows: for amino acid sequence comparisons, matrix: blosum 30; open gap penalty: 10.0; extension gap penalty: 0.05; delayed branching: 40; gap separation distance: 8. For nucleotide sequence comparisons, the extension gap penalty is preferably set to 5.0.

[0031] In a preferred embodiment, ClustalW2 is used for comparing amino acid sequences. For pairwise comparison / alignment, the following settings are preferably selected: protein weight matrix: BLOSUM 62; gap open: 10; gap extension: 0.1. For multiple comparison / alignment, the following settings are preferably selected: protein weight matrix: BLOSUM 62; gap open: 10; gap extension: 0.2; gap distance: 5; no end gap. Preferably, the degree of identity is calculated over the full length of the sequence.

[0032] The amino acid residues located at positions in the amino acid sequence shown in SEQ ID NO:1 that correspond to the positions shown herein below can be identified by those 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 identifying positions that correspond to the positions shown above or below SEQ ID NO:1. Alignment can be performed by means and methods known to those skilled in the art, for example by using known computer algorithms such as the Lippmann-Pearson method (Science 227 (1985), 1435) or the CLUSTAL algorithm. In such an alignment, it is preferred to assign maximum homology to conserved amino acid residues present in the amino acid sequence.

[0033] In a preferred embodiment, ClustalW2 is used for comparing amino acid sequences. For pairwise comparison / alignment, the following settings are preferably selected: protein weight matrix: BLOSUM 62; gap open: 10; gap extension: 0.1. For multiple comparison / alignment, the following settings are preferably selected: protein weight matrix: BLOSUM 62; gap open: 10; gap extension: 0.2; gap distance: 5; no end gap.

[0034] When the amino acid sequences of the MDCs are aligned by such methods, the positions of corresponding amino acid residues can be determined in each of the MDCs, regardless of any insertions or deletions that may be present in the amino acid sequences.

[0035] In the context of the present invention, "substituted with another amino acid residue" means that each amino acid residue at the indicated position can be substituted with any other possible amino acid residue, for example a naturally occurring or non-naturally occurring amino acid (Brustad and Arnold, Curr. Opin. Chem. Biol. 15 (2011), 201-210), preferably with an amino acid residue selected from the group consisting of 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 for specific positions are further indicated below. Furthermore, the term "substituted" or "substitution" also means that each amino acid residue at the indicated position is modified.

[0036] Such modifications include naturally occurring modifications and non-naturally occurring modifications.Naturally occurring modifications include, but are not limited to, post-translational modifications in eukaryotes, such as the attachment of functional groups (e.g., acetate groups, phosphate groups, hydroxyl groups, lipids (myristoylation of glycine residues)) and carbohydrates (e.g., glycosylation of arginine, asparagine, etc.).Naturally occurring modifications also include changes in chemical structure due to citrullination, carbamylation, and disulfide bond formation between cysteine ​​residues, attachment of cofactors (FMN or FAD that can be covalently bound), or attachment of peptides (e.g., ubiquitination or SUMOylation).

[0037] Non-naturally occurring modifications include, for example, in vitro modifications, such as biotinylation of lysine residues or the inclusion of non-standard amino acids (see Liu and Schultz, Annu. Rev. Biochem. 79 (2010), 413-44 and Wang et al., Chem. Bio. 2009 March 27; 16 (3), 323-336; doi:101016 / jchembiol.2009.03.001).

[0038] In the context of the present invention, "deleted" or "deletion" means that an amino acid at the corresponding position is deleted.

[0039] In the context of the present invention, "inserted" or "insertion" means that at each position, one or two, preferably one, amino acid residues are inserted after the indicated position.

[0040] The present invention relates to variants of 3-methylcrotonic acid decarboxylase (MDC), which exhibit improved activity in converting 3-methylcrotonic acid to isobutene over the corresponding MDC from which it is derived, and which exhibit one or more substitutions, deletions and / or insertions compared to the corresponding sequence from which it is derived, and these substitutions, deletions and / or insertions are at positions 7, 17, 27, 33, 35, 45, 46, 48, 51, 140, 144, 183, 184, 185, 222, 227, 284, 285, 286, 287, 288, 289, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362 9, 290, 292, 321, 322, 327, 329, 330, 331, 337, 338, 355, 365, 378, 380, 384, 387, 388, 389, 391, 392, 393, 394, 395, 419, 424, 425, 428, 429, 431, 432, 434, 436, 437, 438, 439, 443, 444, 446, 447, 448, 449, 451, 453, 457, 458, 459, 460, 462, 464, 465, 467, 468, 470 and 471.

[0041] In a preferred embodiment, the present invention relates to an MDC variant having an amino acid sequence as set forth in SEQ ID NO:1 or an amino acid sequence having at least 55% sequence identity to SEQ ID NO:1, wherein the variant is selected from the group consisting of 7, 17, 27, 33, 35, 45, 46, 48, 51, 140, 144, 183, 184, 185, 222, 227, 284, 285, 286, 287, 288, 289, 290, 292, 321, 322, 327, 329, 330, 331, 337, 338, 355, 365, 378, 380, 384, 387, 388, 389, 391, 392, 393, 394, 395, 419, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, , 428, 429, 431, 432, 434, 436, 437, 438, 439, 443, 444, 446, 447, 448, 449, 451, 453, 457, 458, 459, 460, 462, 464, 465, 467, 468, 470 and 471, or a position corresponding to any of these positions, has been substituted with another amino acid residue or deleted, or an insertion has occurred at one or more of these positions, wherein said MDC variant shows improved activity in converting 3-methylcrotonic acid to isobutene.

[0042] According to one embodiment, the invention relates to any of the above MDC variants having an amino acid sequence as shown in SEQ ID NO:1 or an amino acid sequence having at least 55% sequence identity to SEQ ID NO:1, (1) the amino acid residue at position 7 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with cysteine, and / or (2) the amino acid residue at position 17 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with serine, and / or (3) the amino acid residue at position 27 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with asparagine, and / or (4) the amino acid residue at position 33 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with proline, and / or (5) the amino acid residue at position 35 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with valine, and / or (6) The amino acid residue at position 45 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine or glutamine, and / or (7) The amino acid residue at position 46 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine or isoleucine, and / or (8) The amino acid residue at position 48 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with methionine, and / or (9) The amino acid residue at position 51 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with aspartic acid, and / or (10) The amino acid residue at position 140 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with leucine, and / or (11) The amino acid residue at position 144 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with serine, and / or (12) The amino acid residue at position 183 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with tyrosine, and / or (13) The amino acid residue at position 184 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine or serine, and / or (14) The amino acid residue at position 185 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with threonine, and / or (15) The amino acid residue at position 122 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with valine, and / or (16) The amino acid residue at position 227 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with leucine or tyrosine, and / or (17) The amino acid residue at position 284 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with aspartic acid or glutamic acid, or a proline is inserted therein; and / or (18) The amino acid residue at position 285 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine, histidine, isoleucine, leucine, methionine, proline, serine, threonine, or valine, and / or (19) The amino acid residue at position 286 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine, cysteine, aspartic acid, glycine, histidine, asparagine, serine, or tryptophan, and / or (20) The amino acid residue at position 287 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with isoleucine, lysine, proline, glutamine, arginine, serine, threonine, or valine, or a serine is inserted therein; and / or (21) The amino acid residue at position 288 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with asparagine or threonine, and / or (22) The amino acid residue at position 289 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine, cysteine, histidine, methionine, glutamine, arginine, tryptophan, or tyrosine, and / or (23) The amino acid residue at position 290 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with glutamic acid, and / or (24) The amino acid residue at position 292 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with leucine or valine, and / or (25) The amino acid residue at position 321 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with threonine or valine, and / or (26) The amino acid residue at position 322 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with threonine, and / or (27) The amino acid residue at position 327 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with serine, and / or (28) The amino acid residue at position 329 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with serine, and / or (29) The amino acid residue at position 330 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with valine, and / or (30) The amino acid residue at position 331 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with glycine, and / or (30) The amino acid residue at position 331 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with glycine, and / or (31) The amino acid residue at position 337 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with threonine, and / or (32) The amino acid residue at position 338 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with glycine, and / or (33) The amino acid residue at position 355 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with cysteine, threonine, or valine, and / or (34) The amino acid residue at position 365 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with isoleucine, methionine, serine, or threonine, and / or (35) The amino acid residue at position 378 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with methionine, and / or (36) The amino acid residue at position 380 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with valine, and / or (37) The amino acid residue at position 384 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine, glycine, leucine, serine, or threonine, and / or (38) The amino acid residue at position 387 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine, and / or (39) The amino acid residue at position 388 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with methionine, glutamine or serine, and / or (40) The amino acid residue at position 389 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine, histidine, or glutamine, and / or (41) The amino acid residue at position 391 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with isoleucine or leucine, and / or (42) The amino acid residue at position 392 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with tyrosine, and / or (43) The amino acid residue at position 393 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine, and / or (44) The amino acid residue at position 394 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine, cysteine, glycine, histidine, leucine, glutamine, or arginine, and / or (45) The amino acid residue at position 395 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with valine, and / or (46) The amino acid residue at position 419 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine or proline, and / or (47) An alanine, aspartic acid, leucine, serine or tryptophan is inserted at position 424 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, and / or (48) A tryptophan is inserted at position 425 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position. (49) The amino acid residue at position 428 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with methionine or threonine, and / or (50) The amino acid residue at position 429 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with alanine, aspartic acid or valine, or glycine or asparagine is inserted therein; and / or (51) The amino acid residue at position 431 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with alanine, cysteine, lysine, methionine, asparagine, proline, threonine or valine, or glycine, leucine or asparagine is inserted therein; and / or (52) The amino acid residue at position 432 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine, phenylalanine or tryptophan, and / or (53) The amino acid residue at position 434 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with phenylalanine, methionine, valine, or tryptophan; and / or (54) The amino acid residue at position 436 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine, methionine, asparagine, glutamine, threonine, or tryptophan; and / or (55) The amino acid residue at position 437 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with phenylalanine, glycine, isoleucine, leucine, methionine, threonine, or valine, and / or (56) The amino acid residue at position 438 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with leucine, methionine or tryptophan, or an aspartic acid is inserted therein; and / or (57) The amino acid residue at position 439 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with leucine or proline, and / or (58) The amino acid residue at position 443 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with histidine, or an alanine, glycine, leucine, arginine, threonine or valine is inserted therein; and / or (59) The amino acid residue at position 444 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with phenylalanine, isoleucine, leucine, methionine, asparagine, arginine, threonine or valine, or glutamic acid, glycine, histidine, lysine, leucine, asparagine, proline, arginine, serine, threonine or valine is inserted therein; and / or (60) The amino acid residue at position 446 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with aspartic acid, glycine or proline, and / or (61) The amino acid residue at position 447 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with phenylalanine, histidine, isoleucine, threonine or tryptophan, or lysine, leucine, proline or tyrosine is inserted therein; and / or (62) The amino acid residue at position 448 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with cysteine, glutamic acid, threonine or valine, or a serine is inserted therein; and / or (63) The amino acid residue at position 449 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with asparagine or tryptophan, or valine or serine is inserted therein; and / or (64) The amino acid residue at position 451 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with serine, and / or (65) The amino acid residue at position 453 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with leucine, and / or (66) The amino acid residue at position 457 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine, and / or (67) The amino acid residue at position 458 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with cysteine, glutamic acid, isoleucine, leucine, methionine, glutamine or arginine, or glycine is inserted therein; and / or (68) The amino acid residue at position 459 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with glycine, leucine, proline, arginine, or threonine, and / or (69) The amino acid residue at position 460 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with glycine, histidine, isoleucine, leucine, methionine, valine, or tyrosine, and / or (70) The amino acid residue at position 462 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with alanine, cysteine, isoleucine, proline, arginine, threonine or valine, or an alanine is inserted therein; and / or (71) The amino acid residue at position 464 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with proline, and / or (72) The amino acid residue at position 465 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine, isoleucine, or valine, and / or (73) The amino acid residue at position 467 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with tryptophan, and / or (74) The amino acid residue at position 468 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with leucine, and / or (75) The amino acid residue at position 470 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with glycine, serine, or threonine, and / or (76) The amino acid residue at position 471 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with phenylalanine. Regarding any of the above MDC variants.

[0043] The present invention also relates to variants as defined in (1) to (76) above, wherein the amino acid residue shown as substituting for the amino acid residue at that position in SEQ ID NO:1 or inserted at a particular position is not the particular amino acid residue, but an amino acid residue that is conservative to the indicated substituted amino acid.

[0044] Whether an amino acid is conservative with respect to another amino acid can be determined according to means and methods known in the art, as described herein above. One possibility is the PAM 250 matrix, or alternatively, matrices of the Blosum family can be used.

[0045] The present invention also relates to a nucleotide sequence comprising an amino acid sequence as set forth in SEQ ID NO:1 or an amino acid sequence having at least 55% sequence identity to SEQ ID NO:1, the nucleotide sequence being selected from the group consisting of nucleotides at positions 7, 17, 27, 33, 35, 45, 46, 48, 51, 140, 144, 183, 184, 185, 222, 227, 284, 285, 286, 287, 288, 289, 290, 292, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 38 22, 327, 329, 330, 331, 337, 338, 355, 365, 378, 380, 384, 387, 388, 389, 391, 392, 393, 394, 395, 419, 424, 425, 428, 429, 431, 432, 434, 436, 437, 438, 439, 443, 444, 446, 447, 448, 449, 451, 453, 457, 458, 459, 46 The present invention also relates to MDC variants as described herein above, wherein one or more amino acid residues at positions selected from the group consisting of 0, 462, 464, 465, 467, 468, 470 and 471, or positions corresponding to any of these positions, have been substituted with another amino acid residue or deleted, or an insertion has occurred at one or more of these positions, and further wherein the MDC variants further exhibit at least one modification at a position selected from the group consisting of positions 6, 15, 25, 39, 43, 72, 89, 126, 141, 189, 223, 224, 272, 283, 332, 333, 336, 361, 366, 368, 379, 382, ​​385, 418, 421, 422, 423, 426, 430, 440, 441, 445, 461, 463 and 469 of the amino acid sequence set forth in SEQ ID NO:1.

[0046] According to one embodiment, an MDC variant as described herein above further exhibits at least one modification at a position selected from the group consisting of positions 6, 15, 25, 39, 43, 72, 89, 126, 141, 189, 223, 224, 272, 283, 332, 333, 336, 361, 366, 368, 379, 382, ​​385, 418, 421, 422, 423, 426, 430, 440, 441, 445, 461, 463 and 469 of the amino acid sequence set forth in SEQ ID NO:1, or at a position corresponding to any of these positions, is an MDC variant, wherein (1) the amino acid residue at position 6 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with methionine, and / or (2) the amino acid residue at position 15 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with histidine or leucine, and / or (3) the amino acid residue at position 25 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with leucine, and / or (4) the amino acid residue at position 39 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with glycine, and / or (5) the amino acid residue at position 43 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with glutamic acid, and / or (6) the amino acid residue at position 72 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with lysine, and / or (7) The amino acid residue at position 89 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with asparagine, and / or (8) The amino acid residue at position 126 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with lysine, and / or (9) The amino acid residue at position 141 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with cysteine, leucine, glutamine, serine, or tyrosine, and / or (10) The amino acid residue at position 189 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with cysteine, and / or (11) The amino acid residue at position 223 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with tyrosine, and / or (12) The amino acid residue at position 224 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine, proline, or glutamine, and / or (13) The amino acid residue at position 272 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with isoleucine, lysine, leucine, or arginine, and / or (14) The amino acid residue at position 283 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with cysteine, phenylalanine, histidine, leucine, glutamine, serine, threonine, or valine, and / or (15) The amino acid residue at position 332 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with histidine, arginine, or serine, and / or (16) The amino acid residue at position 333 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with methionine, and / or (17) The amino acid residue at position 336 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with histidine, asparagine, or glutamine, and / or (18) The amino acid residue at position 361 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with lysine, and / or (19) The amino acid residue at position 3665 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine, and / or (20) The amino acid residue at position 368 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with serine, and / or (21) The amino acid residue at position 379 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with isoleucine, and / or (22) The amino acid residue at position 382 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine or leucine, and / or (23) The amino acid residue at position 385 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with aspartic acid, phenylalanine, glycine, leucine, proline, arginine, serine, threonine, or tryptophan; and / or (24) The amino acid residue at position 418 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with valine, and / or (25) The amino acid residue at position 421 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with phenylalanine, lysine, leucine, threonine, valine or tryptophan, or aspartic acid, glutamic acid, glycine, isoleucine, proline, arginine, serine or threonine is inserted therein; and / or (26) The amino acid residue at position 422 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with aspartic acid, phenylalanine, glycine, histidine, leucine, glutamine, serine, or valine, or a lysine is inserted therein; and / or (27) The amino acid residue at position 423 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with cysteine ​​or glycine, and / or (28) The amino acid residue at position 426 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with histidine, isoleucine, leucine, threonine, valine, tryptophan or tyrosine, or leucine is inserted therein; and / or (29) The amino acid residue at position 430 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with alanine, lysine, asparagine, proline, arginine, serine, threonine or valine, or cysteine, glycine, histidine, isoleucine, leucine, asparagine, glutamine, arginine or serine is inserted therein; and / or (30) The amino acid residue at position 440 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with aspartic acid or isoleucine, and / or (31) The amino acid residue at position 441 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with aspartic acid, glycine, histidine, isoleucine, leucine, proline, threonine, or valine, and / or (32) The amino acid residue at position 445 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with alanine, glutamine or threonine, or glutamine is inserted therein; and / or (33) The amino acid residue at position 461 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine, aspartic acid, glutamic acid, glycine, asparagine, serine, threonine, valine, or tyrosine, and / or (34) The amino acid residue at position 463 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with glutamic acid, phenylalanine, glycine, isoleucine, leucine, methionine, proline, serine, threonine, or valine, and / or (35) The amino acid residue at position 469 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine, aspartic acid, glutamic acid, histidine, asparagine, serine, or threonine. It is an MDC variant.

[0047] The present invention also relates to variants as defined in (1) to (35) above, wherein the amino acid residue shown as substituting for the amino acid residue at that position in SEQ ID NO:1 is not that specific amino acid residue, but an amino acid residue that is conservative to the indicated substitute amino acid.

[0048] Whether an amino acid is conservative with respect to another amino acid can be determined according to means and methods known in the art, as described herein above. One possibility is the PAM 250 matrix, or alternatively, matrices of the Blosum family can be used.

[0049] In a preferred embodiment, the present invention relates to a variant of 3-methylcrotonate decarboxylase (MDC) that exhibits improved activity over the 3-methylcrotonate decarboxylase (MDC) of SEQ ID NO: 1, in particular in converting 3-methylcrotonate to isobutene, (a) it has at least 55% sequence identity to the sequence shown in SEQ ID NO:1; and (b) it is located at positions 7, 17, 27, 33, 35, 45, 46, 48, 51, 140, 144, 183, 184, 185, 222, 227, 284, 285, 286, 287, 288, 289, 290, 292, 321, 322, 327, 329, 330, 331, 337, 338, 355, 365, 378, 380, 384, 387, 388, 389, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 420, 421, 422, 423, 424, 425, 426, 427, 428, 430, 431, 437, 438, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 46 93, 394, 395, 419, 428, 429, 431, 432, 434, 436, 437, 438, 439, 443, 444, 446, 447, 448, 449, 451, 453, 457, 458, 459, 460, 462, 464, 465, 467, 468, 470 and 471, or at a position corresponding to any of these positions; and / or It exhibits one or more deletions at positions selected from the group consisting of positions 422, 458, 459, 460, 461, 462 and 463 of the amino acid sequence shown in SEQ ID NO:1, or at positions corresponding to any of these positions, and / or that it exhibits one or more insertions at positions selected from the group consisting of positions 284, 287, 421, 422, 424, 425, 426, 429, 430, 431, 438, 443, 444, 445, 447, 448, 449, 458 and 462 of the amino acid sequence set forth in SEQ ID NO:1, or at positions corresponding to any of these positions; The present invention relates to a 3-methylcrotonate decarboxylase (MDC) characterized by the following:

[0050] With regard to preferred modifications at the various positions the same applies as above, particularly with regard to substitutions and insertions.

[0051] In a preferred embodiment, the MDC variant according to the invention, which exhibits improved activity in converting 3-methylcrotonic acid to isobutene, is characterized in that it comprises at least one deletion, substitution and / or insertion, which deletion / insertion / substitution is at position 432 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position. Preferably, such variants further have one or more substitutions, deletions and / or insertions compared to the corresponding sequence from which they are derived, and these substitutions, deletions and / or insertions are at positions 7, 17, 27, 33, 35, 45, 46, 48, 51, 140, 144, 183, 184, 185, 222, 227, 284, 285, 286, 287, 288, 289, 290, 292, 321, 322, 327, 329, 330, 331, 337, 338, 355, 365, 378, 380, 384, 387, 388, 389, 391, 392, 393, 394, 395, 4 19, 424, 425, 428, 429, 431, 434, 436, 437, 438, 439, 443, 444, 446, 447, 448, 449, 451, 453, 457, 458, 459, 460, 462, 464, 465, 467, 468, 470, 471, 6, 15, 25, 39, 43, 72, and 469.

[0052] Regarding preferred embodiments of substitutions, deletions and / or insertions at specific positions the same applies as above.

[0053] In another preferred embodiment, the MDC variant according to the invention, exhibiting improved activity in converting 3-methylcrotonic acid to isobutene, is characterized in that it comprises at least one deletion, substitution and / or insertion, which deletion / insertion / substitution is at position 434 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position. Preferably, such variants further have one or more substitutions, deletions and / or insertions compared to the corresponding sequence from which they are derived, and these substitutions, deletions and / or insertions are at positions 7, 17, 27, 33, 35, 45, 46, 48, 51, 140, 144, 183, 184, 185, 222, 227, 284, 285, 286, 287, 288, 289, 290, 292, 321, 322, 327, 329, 330, 331, 337, 338, 355, 365, 378, 380, 384, 387, 388, 389, 391, 392, 393, 394, 395, 4 19, 424, 425, 428, 429, 431, 432, 436, 437, 438, 439, 443, 444, 446, 447, 448, 449, 451, 453, 457, 458, 459, 460, 462, 464, 465, 467, 468, 470, 471, 6, 15, 25, 39, 43, 72, and 469.

[0054] With regard to preferred embodiments of substitutions, deletions and / or insertions at specific positions the same applies as described herein above.

[0055] In a further preferred embodiment, the MDC variant according to the invention, exhibiting improved activity in converting 3-methylcrotonic acid to isobutene, is characterized in that it comprises at least one deletion, substitution and / or insertion, which deletion / insertion / substitution is at position 436 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position. Preferably, such variants further have one or more substitutions, deletions and / or insertions compared to the corresponding sequence from which they are derived, and these substitutions, deletions and / or insertions are at positions 7, 17, 27, 33, 35, 45, 46, 48, 51, 140, 144, 183, 184, 185, 222, 227, 284, 285, 286, 287, 288, 289, 290, 292, 321, 322, 327, 329, 330, 331, 337, 338, 355, 365, 378, 380, 384, 387, 388, 389, 391, 392, 393, 394, 395, 4 19, 424, 425, 428, 429, 431, 432, 434, 437, 438, 439, 443, 444, 446, 447, 448, 449, 451, 453, 457, 458, 459, 460, 462, 464, 465, 467, 468, 470, 471, 6, 15, 25, 39, 43, 72, and 469.

[0056] With regard to preferred embodiments of substitutions, deletions and / or insertions at specific positions the same applies as described herein above.

[0057] In a further preferred embodiment, the MDC variant according to the invention, exhibiting improved activity in converting 3-methylcrotonic acid to isobutene, is characterized in that it comprises at least one deletion, substitution and / or insertion, which deletion / insertion / substitution is at position 387 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position. Preferably, such variants further have one or more substitutions, deletions and / or insertions compared to the corresponding sequence from which they are derived, and these substitutions, deletions and / or insertions are at positions 7, 17, 27, 33, 35, 45, 46, 48, 51, 140, 144, 183, 184, 185, 222, 227, 284, 285, 286, 287, 288, 289, 290, 292, 321, 322, 327, 329, 330, 331, 337, 338, 355, 365, 378, 380, 384, 388, 389, 391, 392, 393, 394, 395, 419, 420, 430, 431, 432, 433, 434, 435, 436, 437, 438, 440, 442, 444, 446, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 24, 425, 428, 429, 431, 432, 434, 436, 437, 438, 439, 443, 444, 446, 447, 448, 449, 451, 453, 457, 458, 459, 460, 462, 464, 465, 467, 468, 470, 471, 6, 15, 25, 39, 43, 72, and 469.

[0058] In a more preferred embodiment, the MDC variant according to the invention exhibiting improved activity in converting 3-methylcrotonic acid to isobutene is characterized in that it contains at least two deletions, substitutions and / or insertions, one deletion / insertion / substitution at or corresponding to position 432 of the amino acid sequence shown in SEQ ID NO:1 and another deletion / insertion / substitution at or corresponding to position 434 of the amino acid sequence shown in SEQ ID NO:1. Preferably, such variants further have one or more substitutions, deletions and / or insertions compared to the corresponding sequence from which they are derived, and these substitutions, deletions and / or insertions are at positions 7, 17, 27, 33, 35, 45, 46, 48, 51, 140, 144, 183, 184, 185, 222, 227, 284, 285, 286, 287, 288, 289, 290, 292, 321, 322, 327, 329, 330, 331, 337, 338, 355, 365, 378, 380, 384, 387, 388, 389, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 430, 431, 432, 433, 434, 435, 436, 437, 438, 440, 441, 442, 443, 444, 445, 446, 448, 5, 419, 424, 425, 428, 429, 431, 436, 437, 438, 439, 443, 444, 446, 447, 448, 449, 451, 453, 457, 458, 459, 460, 462, 464, 465, 467, 468, 470, 471, 6, 15, 25, 39, 43, 72, 8 9, 126, 141, 189, 223, 224, 272, 283, 332, 333, 336, 361, 366, 368, 379, 382, ​​385, 418, 421, 422, 423, 426, 430, 440, 441, 445, 461, 463 and 469. It is particularly preferred that such variants containing alterations at positions 432 and 434 of the amino acid sequence set forth in SEQ ID NO:1 or at a position corresponding to these positions further exhibit a substitution, deletion or substitution at position 430 of the amino acid sequence set forth in SEQ ID NO:1 or at a position corresponding to this position.

[0059] It is also particularly preferred that such variants containing an alteration at positions 432 and 434 of the amino acid sequence shown in SEQ ID NO:1 or at positions corresponding to these positions further exhibit a substitution, deletion or substitution at position 436 of the amino acid sequence shown in SEQ ID NO:1 and or at a position corresponding to this position.

[0060] It is even more preferred that such variants containing modifications at positions 432 and 434 of the amino acid sequence set forth in SEQ ID NO:1 or at positions corresponding to these positions further exhibit substitutions, deletions or substitutions at positions 430 and 436 of the amino acid sequence set forth in SEQ ID NO:1 or at positions corresponding to these positions.

[0061] With regard to preferred embodiments of substitutions, deletions and / or insertions at specific positions the same applies as described herein above.

[0062] In a particularly preferred embodiment, the MDC variant according to the invention which shows an improved activity in converting 3-methylcrotonic acid to isobutene contains at least one deletion, substitution and / or insertion, which deletion / insertion / substitution is at position 430 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, and which comprises a deletion, substitution and / or insertion at positions 7, 17, 27, 33, 35, 45, 46, 48, 51, 140, 144, 183, 184, 185, 222, 227, 284, 285, 286, 287, 288, 289, 290, 292, 321, 322, 327, 329, 330, 331, 337, 338, 355, 365, 378, 380, 384, 387, 388, 390, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 430, 431, 432, 433, 434, 435, 436, 437, 438, 440, 441, 442, 443, 444, 445, 44 , 389, 391, 392, 393, 394, 395, 419, 424, 425, 428, 429, 431, 432, 434, 436, 437, 438, 439, 443, 444, 446, 447, 448, 449, 451, 453, 457, 458, 459, 460, 462, 464, 465, 467, 468, 470, 471, 6, 15, 25, 39, 43, 72, 8 9, 126, 141, 189, 223, 224, 272, 283, 332, 333, 336, 361, 366, 368, 379, 382, ​​385, 418, 421, 422, 423, 426, 440, 441, 445, 461, 463 and 469, or at positions corresponding to these positions. In a preferred embodiment, the at least two additional positions are 432 and 434. In another preferred embodiment, the at least two additional positions are 432, 434 and 436.

[0063] With regard to preferred embodiments of substitutions, deletions and / or insertions at specific positions the same applies as described herein above.

[0064] In a preferred embodiment, an MDC variant according to the invention that exhibits improved activity in converting 3-methylcrotonic acid to isobutene is characterized in that it comprises two or more modifications. Preferred combinations of positions at which such modifications are present are set out in Table 1 below. Thus, in a preferred embodiment, the MDC variant exhibits modifications at two or more positions (in the amino acid sequence set out in SEQ ID NO: 1 or at positions corresponding to these positions) as reflected in the positions set out for any one of the variants with multiple modifications set out in Table 1.

[0065] For example, Table 1 shows the variant "K391I-L432W-L434M." Thus, an MDC variant according to the invention in one preferred embodiment contains three alterations present at positions 391, 432 and 434 (in the amino acid sequence shown in SEQ ID NO: 1 or at positions corresponding to these positions). Alternative combinations of positions are reflected in the other variants listed in Table 1.

[0066] In a more preferred embodiment, the MDC variants containing two or more alterations at the positions indicated for any of the variants listed in Table 1 contain the alterations shown in Table 1.

[0067] Thus, for example, an MDC variant exhibiting alterations at positions 391, 432 and 434, in one embodiment, preferably exhibits a K to I substitution at position 391, an L to W substitution at position 432, and an L to M substitution at position 434.

[0068] [Table 1-1]

[0069] [Table 1-2]

[0070]

Table 1-3

[0071]

Table 1-4

[0072]

Table 1-5

[0073]

Table 1-6

[0074]

Table 1-7

[0075]

Table 1-8

[0076]

Table 1-9

[0077]

Table 1-10

[0078]

Table 1-11

[0079]

Table 1-12

[0080]

Table 1-13

[0081]

Table 1-14

[0082]

Table 1-15

[0083]

Table 1-16

[0084]

Table 1-17

[0085]

Table 1-18

[0086]

Table 1-19

[0087]

Table 1-20

[0088] Preferably, any of the above variants having multiple mutations further have one or more substitutions, deletions and / or insertions compared to the corresponding sequence from which they are derived, and these substitutions, deletions and / or insertions are at positions 7, 17, 27, 33, 35, 45, 46, 48, 51, 140, 144, 183, 184, 185, 222, 227, 284, 285, 286, 287, 288, 289, 290, 292, 321, 322, 327, 329, 330, 331, 337, 338, 355, 365, 378, 380, 384, 387, 388, 389, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 420, 421, 422, 423, 424, 425, 426, 427, 428, 430, 431, 432, 433, 434, 435, 436, 437, 438, 440, 441, 442, 443, 444, 445, 446, 44 94, 395, 419, 424, 425, 428, 429, 431, 432, 434, 436, 437, 438, 439, 443, 444, 446, 447, 448, 449, 451, 453, 457, 458, 459, 460, 462, 464, 465, 467, 468, 470, 471, 6, 15, 25, 39, and 469.

[0089] The present invention also provides a method for providing a variant of MDC that exhibits improved activity in converting 3-methylcrotonic acid to isobutene, comprising the step of making one or more changes in the sequence of MDC, the changes being at positions 7, 17, 27, 33, 35, 45, 46, 48, 51, 140, 144, 183, 184, 185, 222, 227, 284, 285, 286, 287, 288, 289, 290, 292, 321, 322, 327, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 392, 393, 394, 395 The present invention also relates to methods in which the amino acid sequence "corresponding to" occurs at one or more amino acid positions selected from the group consisting of amino acid positions corresponding to 337, 338, 355, 365, 378, 380, 384, 387, 388, 389, 391, 392, 393, 394, 395, 419, 424, 425, 428, 429, 431, 432, 434, 436, 437, 438, 439, 443, 444, 446, 447, 448, 449, 451, 453, 457, 458, 459, 460, 462, 464, 465, 467, 468, 470 and 471. "Corresponding to" means corresponding to any of these positions in the relevant sequence.

[0090] With regard to preferred embodiments of MDC to be mutated according to such methods, the same applies as described herein above.

[0091] In a preferred embodiment, the MDC from which the MDC variant is derived is an MDC exhibiting an amino acid sequence as set forth in SEQ ID NO:1, or an amino acid sequence having at least 55%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% sequence identity to SEQ ID NO:1, or any of the preferred degrees of sequence identity specified herein above.

[0092] Moreover, with regard to the degree of improvement in activity and preferred embodiments of the changes that occur, the same applies as described herein above.

[0093] The alterations which occur at any of the above positions are substitutions, deletions and / or insertions as defined herein above.

[0094] The MDC variants of the invention can be fused with homologous or heterologous polypeptides or proteins, enzymes, substrates or tags to form fusion proteins. The fusion proteins according to the invention are believed to have the same improved activity as the MDC variants of the invention. Polypeptides, enzymes, substrates or tags that can be added to another protein are known in the art. They can be useful for purifying or detecting the proteins of the invention. For example, tags that can be used for detection and / or purification are, for example, FLAG-tags, His6-tags or Strep-tags. Alternatively, the proteins of the invention can be fused with enzymes such as luciferase for detection or localization of said proteins. Other fusion partners include, but are not limited to, bacterial β-galactosidase, trpE, protein A, β-lactamase, alpha amylase, alcohol dehydrogenase or yeast alpha mating factor. It can also be envisaged that the polypeptides, enzymes, substrates or tags are removed from the proteins of the invention, for example after purification. Fusion proteins can typically be made by either recombinant nucleic acid methods or synthetic polypeptide methods known in the art.

[0095] The present invention further relates to nucleic acid molecules encoding the MDC variants of the present invention and vectors comprising said nucleic acid molecules. Vectors that can be used according to the present invention are known in the art. The vectors can further comprise expression control sequences operably linked to the nucleic acid molecules 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. The expression control sequence can be, for example, a promoter. The promoters for use in connection with the nucleic acid molecules of the present invention can be homologous or heterologous with respect to their origin and / or the gene to be expressed. Suitable promoters are, for example, promoters that serve for constitutive expression. However, promoters that are only activated at times determined by external influences can also be used. Artificial and / or chemically inducible promoters can be used in this context.

[0096] Preferably, the vector of the present invention is an expression vector. Expression vectors are widely described in the literature. Usually, they contain a bacterial or viral promoter and, in most cases, a termination signal for transcription, as well as a selection marker gene and an origin of replication that ensures replication in the selected host. Between the promoter and the termination signal, there is generally at least one restriction site, or a polylinker that allows the insertion of a coding DNA sequence. The DNA sequence that naturally controls the transcription of the corresponding gene can be used as the promoter sequence, provided that it is active in the selected host organism. However, this sequence can also be exchanged for other promoter sequences. It is possible to use promoters that ensure constitutive expression of the gene and inducible promoters that allow deliberate control of the expression of the gene. Bacterial and viral promoter sequences with these characteristics are described in detail in the literature. Regulatory sequences for expression in microorganisms (e.g., E. coli, S. 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., 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 optimal amounts of polypeptide, a two-step process is often used. First, the host cells are cultured under optimal conditions to a relatively high cell density.In the second step, transcription is induced depending on the type of promoter used. In this respect, the tac promoter, which can be induced by lactose or IPTG (=isopropyl-β-D-thiogalactopyranoside), is particularly suitable (deBoer et al., Proc. Natl. Acad. Sci. USA 80 (1983), 21-25). Transcription termination signals are also described in the literature.

[0097] In addition, the present invention relates to a host cell comprising the nucleic acid molecule or vector of the present invention.

[0098] In a preferred embodiment, the host cell according to the present invention is a microorganism, in particular a bacterium or a fungus. In a more preferred embodiment, the host cell of the present invention is an E. coli, a bacterium of the genus Clostridium, or a yeast cell such as S. cerevisiae. In another preferred embodiment, the host cell is a plant cell or a non-human animal cell.

[0099] Transformation with the vectors according to the invention can be carried out by standard methods, as 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 cells are cultured in a nutrient medium which meets the requirements of the particular host cells used, in particular with regard to pH value, temperature, salt concentration, aeration, antibiotics, vitamins, trace elements, etc.

[0100] As mentioned above, the enzymatic conversion of 3-methylcrotonic acid to isobutene using MDC is preferably carried out in the presence of flavin prenyltransferase and relies on two consecutive step reactions catalyzed by two enzymes, namely MDC (which catalyzes the actual decarboxylation of 3-methylcrotonic acid to isobutene) and flavin prenyltransferase, which provides a modified flavin cofactor. The flavin cofactor can preferably be FMN or FAD. FMN (flavin mononucleotide; also called riboflavin-5'-phosphate) is a biomolecule generated from riboflavin (vitamin B2) by the enzyme riboflavin kinase and serves as a prosthetic group for various reactions. FAD (flavin adenine dinucleotide) is a redox cofactor, more specifically a prosthetic group, and is involved in several important reactions in metabolism. Thus, in a preferred embodiment, in the production of isobutene from 3-methylcrotonic acid, which comprises incubating an MDC variant of the invention with 3-methylcrotonic acid, a flavin prenyltransferase is present which in a first step modifies a flavin cofactor (FMN or FAD) into a (modified) flavin-derived cofactor. The flavin prenyltransferase prenylates the flavin ring of the flavin cofactor (FMN or FAD) to give a (modified) prenylated flavin cofactor. This reaction is shown diagrammatically in Figure 1A.

[0101] In the second step, the actual conversion of 3-methylcrotonic acid to isobutene is catalyzed by the MDC variant via a 1,3-dipolar cycloaddition-based mechanism, in which the FMN-dependent decarboxylase uses a prenylated flavin cofactor (FMN or FAD) provided by an associated flavin prenyltransferase, as shown diagrammatically in Figure 1B.

[0102] Therefore, preferably, the host cell of the present invention is a cell that expresses a flavin prenyltransferase that can modify flavin cofactors (FMN or FAD) into (modified) flavin-derived cofactors.In a preferred embodiment, the host cell is a cell that naturally (endogenously) expresses flavin prenyltransferase.In another preferred embodiment, the host cell is a cell that recombinantly expresses flavin prenyltransferase, for example, by introducing a nucleic acid molecule that codes for flavin prenyltransferase or a vector that contains such a nucleic acid molecule.

[0103] In a preferred embodiment, said flavin prenyltransferase, which modifies flavin cofactors (FMN or FAD) into (modified) flavin-derived cofactors, is a phenylacrylic acid decarboxylase (PAD)-type protein or the closely related prokaryotic enzyme UbiX, an enzyme involved in ubiquinone biosynthesis in prokaryotes.

[0104] In Escherichia coli, the protein UbiX (also called flavin prenyltransferase) has been shown to be involved in the third step of ubiquinone biosynthesis.

[0105] [ka]

[0106] Thus, in a preferred embodiment, the modification of flavin cofactors (FMN or FAD) to the corresponding (modified) flavin-derived cofactors is catalyzed by FMN-containing proteins, phenylacrylic acid decarboxylases (PADs). Enzymes involved in the modification of flavin cofactors (FMN or FAD) to the corresponding modified flavin-derived cofactors were originally annotated as decarboxylases (EC 4.1.1.-). Currently, some phenylacrylic acid decarboxylases (PADs) are annotated as flavin prenyltransferases, EC 2.5.1.-.

[0107] In a more preferred embodiment, the conversion of 3-methylcrotonic acid to isobutene uses a phenylacrylic acid decarboxylase (PAD) type protein as a flavin prenyltransferase that modifies a flavin cofactor (FMN or FAD) to the corresponding (modified) flavin-derived cofactor, said phenylacrylic acid decarboxylase (PAD) type protein being derived from Candida albicans (Uniprot accession number Q5A8L8), Aspergillus niger (Uniprot accession number A3F715), Saccharomyces cerevisiae (Uniprot accession number P33751) or Cryptococcus gattii (Uniprot accession number E6R9Z0).

[0108] In a preferred embodiment, the phenylacrylic acid decarboxylase (PAD) type protein used in the method of the invention is a phenylacrylic acid decarboxylase (PAD) type protein from Candida albicans (Uniprot accession number Q5A8L8; SEQ ID NO: 3), Aspergillus niger (Uniprot accession number A3F715; SEQ ID NO: 4), Saccharomyces cerevisiae (Uniprot accession number P33751; SEQ ID NO: 5) or Cryptococcus gattii (Uniprot accession number E6R9Z0; SEQ ID NO: 6), having the amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0109] In a preferred embodiment of the present invention, the phenylacrylic acid decarboxylase (PAD) type protein is an enzyme comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 6, or a sequence that is at least n% identical to any of SEQ ID NOs: 3 to 6, where n is an integer of 10 to 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99, and has an enzymatic activity of modifying a flavin cofactor (FMN or FAD) into a corresponding (modified) flavin-derived cofactor.

[0110] With regard to the determination of sequence identity the same applies as above.

[0111] In another preferred embodiment, the modification of flavin cofactors (FMN or FAD) to the corresponding (modified) flavin-derived cofactors is catalyzed by flavin prenyltransferases, also called UbiX (originally annotated as EC 4.1.1.-). As mentioned above, the enzymes involved in the modification of flavin cofactors (FMN or FAD) to the corresponding modified flavin-derived cofactors were originally annotated as decarboxylases. Several phenylacrylic acid decarboxylases (PADs) are now annotated as flavin prenyltransferases, EC 2.5.1.-.

[0112] In a more preferred embodiment, the conversion of 3-methylcrotonic acid to isobutene uses as flavin prenyltransferase a flavin prenyltransferase (also called UbiX) which modifies a flavin cofactor (FMN or FAD) to the corresponding (modified) flavin-derived cofactor, said flavin prenyltransferase (also called UbiX) originating from Escherichia coli (Uniprot accession number P0AG03), Bacillus subtilis (Uniprot accession number A0A086WXG4), Pseudomonas aeruginosa (Uniprot accession number A0A072ZCW8) or Enterobacter sp. DC4 (Uniprot accession number W7P6B1).

[0113] In an even more preferred embodiment, the flavin prenyltransferase (also called UbiX) used in the method of the invention is a flavin prenyltransferase (also called UbiX) from Escherichia coli (Uniprot accession number P0AG03; SEQ ID NO:2), Bacillus subtilis (Uniprot accession number A0A086WXG4; SEQ ID NO:7), Pseudomonas aeruginosa (Uniprot accession number A0A072ZCW8; SEQ ID NO:8) or Enterobacter sp. DC4 (Uniprot accession number W7P6B1; SEQ ID NO:9), having the amino acid sequences set out in SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively.

[0114] In a preferred embodiment of the present invention, the flavin prenyltransferase is an enzyme comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 7 to 9, or a sequence that is at least n% identical to any of SEQ ID NOs: 2 and 7 to 9, where n is an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99, and has the enzymatic activity of modifying a flavin cofactor (FMN or FAD) into the corresponding (modified) flavin-derived cofactor. The same applies as above with regard to the determination of sequence identity.

[0115] The present invention also relates to a method for producing isobutene from 3-methylcrotonic acid, comprising the step of incubating an MDC variant of the invention with 3-methylcrotonic acid under conditions allowing said conversion (preferably further in the presence of a flavin prenyltransferase as described above) or cultivating a host cell of the invention expressing an MDC variant (preferably further expressing a flavin prenyltransferase as described above) in a suitable medium and recovering the isobutene produced.

[0116] It is also conceivable to use in such a method not only one enzyme according to the invention but also a combination of two or more enzymes.

[0117] The present invention also relates to the use of an MDC variant as described above or a host cell of the present invention for converting 3-methylcrotonic acid to isobutene, preferably in the presence of a flavin prenyltransferase or in the presence of a host co-expressing a flavin prenyltransferase as described herein above.Furthermore, in a further embodiment, the present invention relates to a method for producing isobutene from 3-methylcrotonic acid by contacting the latter with an MDC variant of the present invention, preferably in the presence of a flavin prenyltransferase, or with a host cell comprising a nucleic acid molecule encoding an MDC variant of the present invention, said host cell preferably expressing a flavin prenyltransferase.Thus, in a preferred embodiment, the present invention relates to a method for converting 3-methylcrotonic acid to isobutene, comprising the steps of: (i) culturing the above-mentioned host cell of the present invention in a suitable medium; and (ii) achieving the production of isobutene from 3-methylcrotonic acid.

[0118] Thus, in a preferred embodiment, the invention relates to methods and uses utilising a host cell of the invention which expresses an MDC variant of the invention and preferably further expresses a flavin prenyltransferase as described herein above.

[0119] In another preferred embodiment, such a host cell is an organism capable of producing 3-methylcrotonic acid.

[0120] In another preferred embodiment, the method according to the invention is carried out in culture in the presence of an organism, preferably a microorganism, which produces the enzyme variant of the invention, preferably also produces flavin prenyltransferase. In one such embodiment of the invention, an organism, preferably a microorganism, which produces the enzyme of the invention, preferably also produces flavin prenyltransferase, is used. In a preferred embodiment, the (micro)organism is recombinant in that the enzyme produced by the host is heterologous to the production host. Thus, the method can be carried out directly in the culture medium without the need to separate or purify the enzyme. In a particularly advantageous manner, a (micro)organism with the natural or artificial property of endogenously producing 3-methylcrotonic acid is used to produce isobutene directly from the substrate already present in the culture, which is a solution.

[0121] In the context of the above methods and uses, the microorganism is cultured under suitable culture conditions that allow the occurrence of the enzymatic reaction of the MDC variant of the present invention (and preferably also flavin prenyltransferase as described above). The specific culture conditions depend on the specific microorganism used, but are well known to those skilled in the art. The culture conditions are generally selected in such a way that they allow the expression of the gene encoding the MDC variant of the present invention (and preferably also flavin prenyltransferase as described above). Various methods are known to those skilled in the art for improving and fine-tuning the expression of a particular gene at a particular stage of culture, such as induction of gene expression by chemical inducers or temperature changes.

[0122] In another embodiment, the method of the invention as described above comprises the steps of providing an organism, preferably a microorganism, having the respective enzymatic activity, in the form of a (cell) culture, preferably in the form of a liquid cell culture, followed by culturing the organism, preferably the microorganism, in a fermenter (often also called bioreactor) under suitable conditions allowing the expression of the respective enzyme, and further performing the enzymatic conversion of the method of the invention as described herein above. Suitable fermenter or bioreactor equipment and fermentation conditions are known to the skilled person. Bioreactor or fermenter refers to any manufactured or designed equipment 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 invention, is carried out, involving organisms, preferably microorganisms and / or biochemically active substances, i.e. the enzymes mentioned above originating from such organisms or from organisms carrying the enzymes mentioned above. In a bioreactor or fermenter, this process can be either aerobic or anaerobic. These bioreactors are generally cylindrical, range in size from liters to several hundred cubic meters, and are often made of stainless steel. Without being bound by theory in this regard, the fermenter or bioreactor can be designed in a manner suitable for culturing organisms, preferably microorganisms, e.g. in batch culture, fed-batch culture, perfusion culture or chemostate-culture, all of which are commonly known in the art.

[0123] The culture medium may be any culture medium suitable for culturing the respective organism or microorganism.

[0124] In yet another embodiment, the method according to the invention can be carried out in vitro, for example in the presence of an isolated enzyme comprising an MDC variant of the invention (and preferably a flavin prenyltransferase as described above) or a cell lysate comprising an enzyme or a partially purified enzyme preparation, in vitro preferably meaning a cell-free system.

[0125] In one embodiment, the enzymes used in the method are used in purified form, however, such methods can be costly due to the high costs of enzyme and substrate production and purification.

[0126] Thus, in another preferred embodiment, the enzymes used in the method are present in the reaction as unpurified extracts or in the form of unlysed bacteria to save on protein purification costs, however the costs associated with such methods can still be very high due to the costs of producing and purifying the substrates.

[0127] In an in vitro reaction, the native or recombinant, purified or unpurified enzyme is incubated in the presence of a substrate under physicochemical conditions that allow the enzyme to be active, and the incubation is allowed to proceed for a time sufficient to allow the production of the desired product as described above. At the end of the incubation, the presence of isobutene is optionally measured by using any detection system known to those skilled in the art, such as gas chromatography or a colorimetric test to measure the formation of isobutene.

[0128] In a particularly preferred embodiment of the invention, the method is carried out in vitro and the enzyme is immobilized. Means and methods for immobilizing enzymes on various supports are well known to those skilled in the art.

[0129] The process according to the invention further comprises a step of recovering the gaseous product, i.e. isobutene, by degassing it from the reaction, i.e. recovering the product by degassing it from the culture, for example. Thus, in a preferred embodiment, the process is carried out in the presence of a system for recovering the isobutene in gaseous form during the reaction.

[0130] In fact, isobutene is in gaseous state at room temperature and atmospheric pressure. Moreover, isobutene is also in gaseous state under the culture conditions at 37°C. Therefore, the method according to the invention does not require extraction of isobutene from the liquid culture medium, which is always a very costly step on an industrial scale. The evacuation and storage of gaseous hydrocarbons, especially isobutene, and their possible subsequent physical separation and chemical conversion can be carried out according to any method known to those skilled in the art.

[0131] Finally, the present invention relates to a composition comprising an MDC variant of the invention, a nucleic acid molecule of the invention, a vector of the invention or a host cell of the invention. With regard to the MDC variant, nucleic acid molecule, vector or host cell, the same applies as described above in connection with the method according to the invention.

[0132] Numerous documents, including patent applications, are cited herein. The disclosures of these documents, although not believed to be relevant to the patentability of the present invention, are incorporated herein by reference in their entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. [Brief description of the drawings]

[0133] [Figure 1A] 1 shows a schematic reaction diagram of the enzymatic prenylation of flavin mononucleotide (FMN) to the corresponding modified (prenylated) flavin cofactor. [Figure 1B] Schematic reaction diagram for the enzymatic conversion of 3-methylcrotonic acid to isobutene.

[0134] The invention will now be described by reference to the following examples, which are merely illustrative and should not be construed as limiting the scope of the invention. EXAMPLES

[0135] [Example 1] Identification of a variant of ferulic acid decarboxylase (UbiD-like) enzyme from Yersinia frederiksenii with enhanced activity for the conversion of 3-methylcrotonic acid (3MC) to isobutene (IBN) The Yersinia frederiksenii ferulic acid decarboxylase enzyme (UniProt ID A0A0T9UUQ9, RefSeq WP_050108772.1; SEQ ID NO:1) can catalyze, among other reactions, the decarboxylation of 3-methylcrotonic acid (3MC) to isobutene (IBN). To specifically improve the catalytic efficiency of this reaction, a directed evolution approach was used. This approach consisted of (1) designing an assay system to test the activity of enzyme variants, (2) generating a collection of single- or multi-point mutants of Yersinia frederiksenii ferulic acid decarboxylase, and (3) using an activity assay to screen the collection of mutants with the aim of identifying variants with improved activity compared to that of wild-type Yersinia frederiksenii ferulic acid decarboxylase.

[0136] This approach led to the identification and characterization of a collection of mutants with enhanced activity compared to the wild-type enzyme.

[0137] material and method a) Cloning of the ferulic acid decarboxylase enzyme Polynucleotide sequences encoding the various variants identified during the evolution of the Yersinia frederiksenii ferulic acid decarboxylase enzyme were generated using a series of standard molecular biology techniques, all of which used as templates polynucleotide sequences that were codon-optimized for expression in Escherichia coli.

[0138] In particular, all enzyme encoding polynucleotide sequences were codon-optimized for expression in Escherichia coli and then chemically synthesized, and then cloned into the pET25 (Novagen) expression vector or fused with a 5' or 3' polynucleotide tag encoding a 6His purification tag before cloning into the pET25 expression vector.

[0139] b) Construction of Ferulic Acid Decarboxylase Mutants Polynucleotide sequences encoding the various variants identified during the evolution of the selected ferulic acid decarboxylase enzyme were generated using a series of standard molecular biology techniques.

[0140] Various PCR-based techniques known in the art were used to construct single point mutants. For the generation of enzyme variants with multiple mutations (at least two mutations), these mutations were introduced using either PCR-based techniques or other methods known in the art.

[0141] After mutagenesis, the mutated polynucleotide sequences were inserted into the pET25 expression vector with or without fusion to a tag as described above using either standard ligase-based subcloning procedures, whole plasmid extension by PCR, or ligase-independent cloning procedures.

[0142] c) Selection of enzyme mutants with enhanced activity In vitro assay in 384-well deep-well microplates (VITRO384) based on exogenous 3-methylcrotonic acid (3MC)

[0143] The assay is based on the use of a bacterial strain (BL21(DE3), Novagen) transformed with two expression vectors directing the production of the last two enzymes involved in the metabolic pathway that converts 3MC to isobutene: the flavin prenyltransferase UbiX protein from E. coli cloned into the pRSFDuet™ (Novagen) expression vector, and the variant ferulic acid decarboxylase from Yersinia frederiksenii cloned into one of the above expression vectors. The strain is first plated on an LB agar plate supplemented with the appropriate antibiotic. The cells are grown overnight at 32°C until individual colonies reach the desired size. Single colonies are then picked and transferred individually into 50 μL of liquid LB medium supplemented with the appropriate antibiotic. The cells are grown at 34°C for 21 hours with shaking. The LB culture is used to produce the two recombinant enzymes by inoculating 300 μL into autoinduction medium (Studier FW, Prat. Exp. Pur. 41, (2005), 207-234) in 384-well deep-well microplates supplemented with the appropriate antibiotics and growing for 24 hours at 34° C. in a shaking incubator set at 700 rpm and 85% humidity. The cell pellet containing the two overexpressed recombinant enzymes is then resuspended in 30 μL of lysis mixture (pH 7.5, phosphate 50 mM, NaCl 20 mM, MgCl2 2 mM, lysozyme 1 mg / mL, DNase 0.03 mg / mL) and incubated for 1 hour at 34° C. in a shaking incubator at 700 rpm. This mixture is then supplemented with 10 μL of reaction mixture (final composition: pH 7.5, phosphate 50 mM, NaCl 20 mM, MgCl2 2 mM, lysozyme 0.75 mg / mL, DNase 0.0225 mg / mL, KCl 100 mM) supplemented with 1-200 mM (final) 3MC and incubated for an additional 1-4 hours at 34 °C and 700 rpm in a shaking incubator. During this step, the MDC enzyme catalyzes the decarboxylation of 3MC to IBN.After inactivation at 80 or 90 °C for 5-10 min, the IBN produced is quantified by gas chromatography as follows: 100 μL of headspace gas from each enzymatic reaction is injected into a Brucker GC-450 system equipped with a Flame Ionization Detector (FID). Compounds present in the sample are analyzed using an RTX-1 column at 1 mL.min. -1 The mixture was separated by chromatography at 100° C. with a constant flow of nitrogen as the carrier gas. Upon injection, the peak area of ​​isobutene was calculated.

[0144] d) Identification of a variant of Yersinia frederiksenii ferulic acid decarboxylase with enhanced activity in the conversion of 3-methylcrotonic acid to isobutene The ferulic acid decarboxylase gene (UniProt ID A0A0T9UUQ9, RefSeq WP_050108772.1, SEQ ID NO:1) from Yersinia frederiksenii, which exhibits the ability to catalyze the conversion reaction of 3MC to IBN, was subjected to directed mutagenesis with the aim of generating single point mutations, multiple mutations, deletions or insertion variants. Each of these variants was then tested for their enhanced activity to convert 3MC to IBN using the assay described above. 841 variants exhibited enhanced ability to convert 3MC to IBN.

[0145] A list of improved variants is provided in Table 2 below. The activity enhancement is noted relative to the wild type enzyme ("+" indicates mildly enhanced activity and "++++" indicates highly enhanced activity). The mutations are noted as follows: R387A means that the wild type amino acid arginine (R) at position 387 is replaced with alanine (A), dG462 means that the wild type amino acid glycine (G) at position 462 is deleted, and i443aR means that an arginine (R) is inserted after the amino acid at position 443 (if a second amino acid is inserted it is annotated with i443b).

[0146] [Table 2-1]

[0147] [Table 2-2]

[0148] [Table 2-3]

[0149] [Table 2-4]

[0150] [Table 2-5]

[0151] [Table 2-6]

[0152] [Table 2-7]

[0153]

Table 2-8

[0154]

Table 2-9

[0155]

Table 2-10

[0156]

Table 2-11

[0157]

Table 2-12

[0158]

Table 2-13

[0159]

Table 2-14

[0160]

Table 2-15

[0161]

Table 2-16

[0162]

Table 2-17

[0163] [Table 2-18]

[0164] [Table 2-19]

[0165] [Table 2-20]

[0166] A list of mutations (substitutions) involved in Yersinia frederiksenii ferulic acid decarboxylase variants with increased activity is provided in Table 3.

[0167] A list of deletions resulting in variants of Yersinia frederiksenii ferulic acid decarboxylase with increased activity is provided in Table 4.

[0168] A list of insertions that result in variants of Yersinia frederiksenii ferulic acid decarboxylase with increased activity is provided in Table 5.

[0169] [Table 3-1]

[0170] [Table 3-2]

[0171] [Table 3-3]

[0172] [Table 3-4]

[0173]

Table 3-5

[0174]

Table 4

[0175]

Table 5

Claims

1. A variant of 3-methylcrotonate decarboxylase (MDC), which exhibits improved activity in converting 3-methylcrotonate to isobutene over the corresponding MDC from which it is derived, having an amino acid sequence as set forth in SEQ ID NO:1 or an amino acid sequence having at least 55% sequence identity to SEQ ID NO:1, comprising the amino acid sequence at positions 7, 17, 27, 33, 35, 45, 46, 48, 51, 140, 144, 183, 184, 185, 222, 227, 284, 285, 286, 287, 288, 289, 290, 292, 321, 322, 327, 329, 330, 331, 337, 338, 355 of the amino acid sequence as set forth in SEQ ID NO:1; A variant of MDC in which one or more amino acid residues at a position selected from the group consisting of: 365, 378, 380, 384, 387, 388, 389, 391, 392, 393, 394, 395, 419, 424, 425, 428, 429, 431, 432, 434, 436, 437, 438, 439, 443, 444, 446, 447, 448, 449, 451, 453, 457, 458, 459, 460, 462, 464, 465, 467, 468, 470 and 471, or a position corresponding to any of these positions, has been substituted with another amino acid residue or deleted, or an insertion has occurred at one or more of these positions.

2. The MDC variant described in claim 1, having an amino acid sequence as shown in SEQ ID NO:1 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

1.

3. (1) the amino acid residue at position 7 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with cysteine, and / or (2) the amino acid residue at position 17 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with serine, and / or (3) the amino acid residue at position 27 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with asparagine, and / or (4) The amino acid residue at position 33 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with proline, and / or (5) the amino acid residue at position 35 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with valine, and / or (6) The amino acid residue at position 45 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine or glutamine, and / or (7) The amino acid residue at position 46 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine or isoleucine, and / or (8) The amino acid residue at position 48 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with methionine, and / or (9) The amino acid residue at position 51 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with aspartic acid, and / or (10) The amino acid residue at position 140 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with leucine, and / or (11) The amino acid residue at position 144 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with serine, and / or (12) The amino acid residue at position 183 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with tyrosine, and / or (13) The amino acid residue at position 184 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine or serine, and / or (14) The amino acid residue at position 185 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with threonine, and / or (15) The amino acid residue at position 122 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with valine, and / or (16) The amino acid residue at position 227 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with leucine or tyrosine, and / or (17) The amino acid residue at position 284 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with aspartic acid or glutamic acid, or a proline is inserted therein; and / or (18) The amino acid residue at position 285 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine, histidine, isoleucine, leucine, methionine, proline, serine, threonine, or valine, and / or (19) The amino acid residue at position 286 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine, cysteine, aspartic acid, glycine, histidine, asparagine, serine, or tryptophan, and / or (20) The amino acid residue at position 287 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with isoleucine, lysine, proline, glutamine, arginine, serine, threonine or valine, or a serine is inserted therein; and / or (21) The amino acid residue at position 288 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with asparagine or threonine, and / or (22) The amino acid residue at position 289 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine, cysteine, histidine, methionine, glutamine, arginine, tryptophan, or tyrosine, and / or (23) The amino acid residue at position 290 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with glutamic acid, and / or (24) The amino acid residue at position 292 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with leucine or valine, and / or (25) The amino acid residue at position 321 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with threonine or valine, and / or (26) The amino acid residue at position 322 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with threonine, and / or (27) The amino acid residue at position 327 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with serine, and / or (28) The amino acid residue at position 329 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with serine, and / or (29) The amino acid residue at position 330 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with valine, and / or (30) The amino acid residue at position 331 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with glycine, and / or (30) The amino acid residue at position 331 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with glycine, and / or (31) The amino acid residue at position 337 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with threonine, and / or (32) The amino acid residue at position 338 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with glycine, and / or (33) The amino acid residue at position 355 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with cysteine, threonine or valine, and / or (34) The amino acid residue at position 365 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with isoleucine, methionine, serine, or threonine, and / or (35) The amino acid residue at position 378 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with methionine, and / or (36) The amino acid residue at position 380 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with valine, and / or (37) The amino acid residue at position 384 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine, glycine, leucine, serine, or threonine, and / or (38) The amino acid residue at position 387 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine, and / or (39) The amino acid residue at position 388 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with methionine, glutamine or serine, and / or (40) The amino acid residue at position 389 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine, histidine, or glutamine, and / or (41) The amino acid residue at position 391 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with isoleucine or leucine, and / or (42) The amino acid residue at position 392 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with tyrosine, and / or (43) The amino acid residue at position 393 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine, and / or (44) The amino acid residue at position 394 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine, cysteine, glycine, histidine, leucine, glutamine or arginine, and / or (45) The amino acid residue at position 395 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with valine, and / or (46) The amino acid residue at position 419 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine or proline, and / or (47) An alanine, aspartic acid, leucine, serine or tryptophan is inserted at position 424 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, and / or (48) A tryptophan is inserted at position 425 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position. (49) The amino acid residue at position 428 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with methionine or threonine, and / or (50) The amino acid residue at position 429 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with alanine, aspartic acid or valine, or glycine or asparagine is inserted therein; and / or (51) The amino acid residue at position 431 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with alanine, cysteine, lysine, methionine, asparagine, proline, threonine or valine, or glycine, leucine or asparagine is inserted therein; and / or (52) The amino acid residue at position 432 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine, phenylalanine or tryptophan, and / or (53) The amino acid residue at position 434 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with phenylalanine, methionine, valine, or tryptophan, and / or (54) The amino acid residue at position 436 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine, methionine, asparagine, glutamine, threonine or tryptophan, and / or (55) The amino acid residue at position 437 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with phenylalanine, glycine, isoleucine, leucine, methionine, threonine, or valine, and / or (56) The amino acid residue at position 438 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with leucine, methionine or tryptophan, or an aspartic acid is inserted therein; and / or (57) The amino acid residue at position 439 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with leucine or proline, and / or (58) The amino acid residue at position 443 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with histidine, or alanine, glycine, leucine, arginine, threonine or valine is inserted therein; and / or (59) The amino acid residue at position 444 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with phenylalanine, isoleucine, leucine, methionine, asparagine, arginine, threonine or valine, or glutamic acid, glycine, histidine, lysine, leucine, asparagine, proline, arginine, serine, threonine or valine is inserted therein; and / or (60) The amino acid residue at position 446 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with aspartic acid, glycine or proline, and / or (61) The amino acid residue at position 447 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with phenylalanine, histidine, isoleucine, threonine or tryptophan, or lysine, leucine, proline or tyrosine is inserted therein; and / or (62) The amino acid residue at position 448 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with cysteine, glutamic acid, threonine or valine, or a serine is inserted therein; and / or (63) The amino acid residue at position 449 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with asparagine or tryptophan, or valine or serine is inserted therein; and / or (64) The amino acid residue at position 451 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with serine, and / or (65) The amino acid residue at position 453 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with leucine, and / or (66) The amino acid residue at position 457 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine, and / or (67) The amino acid residue at position 458 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with cysteine, glutamic acid, isoleucine, leucine, methionine, glutamine or arginine, or arginine or glycine is inserted therein; and / or (68) The amino acid residue at position 459 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with glycine, leucine, proline, arginine or threonine, and / or (69) The amino acid residue at position 460 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with glycine, histidine, isoleucine, leucine, methionine, valine, or tyrosine, and / or (70) The amino acid residue at position 462 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with alanine, cysteine, isoleucine, proline, arginine, threonine or valine, or an alanine is inserted therein; and / or (71) The amino acid residue at position 464 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with proline, and / or (72) The amino acid residue at position 465 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine, isoleucine, or valine, and / or (73) The amino acid residue at position 467 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with tryptophan, and / or (74) The amino acid residue at position 468 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with leucine, and / or (75) The amino acid residue at position 470 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with glycine, serine, or threonine, and / or (76) The amino acid residue at position 471 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with phenylalanine. The MDC variant of claim 1.

4. 4. The MDC variant of any one of claims 1 to 3, further exhibiting at least one modification at position 6, 15, 25, 39, 43, 72, 89, 126, 141, 189, 223, 224, 272, 283, 332, 333, 336, 361, 366, 368, 379, 382, ​​385, 418, 421, 422, 423, 426, 430, 440, 441, 445, 461, 463, 469 of the amino acid sequence set forth in SEQ ID NO:1, or a position corresponding to any of these positions.

5. The variant further exhibits at least one modification at position 6, 15, 25, 39, 43, 72, 89, 126, 141, 189, 223, 224, 272, 283, 332, 333, 336, 361, 366, 368, 379, 382, ​​385, 418, 421, 422, 423, 426, 430, 440, 441, 445, 461, 463, 469 of the amino acid sequence set forth in SEQ ID NO:1, or a position corresponding to any of these positions; (1) the amino acid residue at position 6 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with methionine, and / or (2) the amino acid residue at position 15 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with histidine or leucine, and / or (3) the amino acid residue at position 25 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with leucine, and / or (4) the amino acid residue at position 39 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with glycine, and / or (5) the amino acid residue at position 43 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with glutamic acid, and / or (6) the amino acid residue at position 72 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with lysine, and / or (7) The amino acid residue at position 89 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with asparagine, and / or (8) The amino acid residue at position 126 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with lysine, and / or (9) The amino acid residue at position 141 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with cysteine, leucine, glutamine, serine, or tyrosine, and / or (10) The amino acid residue at position 189 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with cysteine, and / or (11) The amino acid residue at position 223 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with tyrosine, and / or (12) The amino acid residue at position 224 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine, proline, or glutamine, and / or (13) The amino acid residue at position 272 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with isoleucine, lysine, leucine, or arginine, and / or (14) The amino acid residue at position 283 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with cysteine, phenylalanine, histidine, leucine, glutamine, serine, threonine, or valine, and / or (15) The amino acid residue at position 332 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with histidine, arginine or serine, and / or (16) The amino acid residue at position 333 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with methionine, and / or (17) The amino acid residue at position 336 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with histidine, asparagine, or glutamine, and / or (18) The amino acid residue at position 361 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with lysine, and / or (19) The amino acid residue at position 3665 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine, and / or (20) The amino acid residue at position 368 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with serine, and / or (21) The amino acid residue at position 379 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with isoleucine, and / or (22) The amino acid residue at position 382 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine or leucine, and / or (23) The amino acid residue at position 385 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with aspartic acid, phenylalanine, glycine, leucine, proline, arginine, serine, threonine or tryptophan, and / or (24) The amino acid residue at position 418 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with valine, and / or (25) The amino acid residue at position 421 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with phenylalanine, lysine, leucine, threonine, valine or tryptophan, or aspartic acid, glutamic acid, glycine, isoleucine, proline, arginine, serine or threonine is inserted therein; and / or (26) The amino acid residue at position 422 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with aspartic acid, phenylalanine, glycine, histidine, leucine, glutamine, serine or valine, or a lysine is inserted therein; and / or (27) The amino acid residue at position 423 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with cysteine ​​or glycine, and / or (28) The amino acid residue at position 426 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with histidine, isoleucine, leucine, threonine, valine, tryptophan or tyrosine, or leucine is inserted therein; and / or (29) The amino acid residue at position 430 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with alanine, lysine, asparagine, proline, arginine, serine, threonine or valine, or cysteine, glycine, histidine, isoleucine, leucine, asparagine, glutamine, arginine or serine is inserted therein; and / or (30) The amino acid residue at position 440 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with aspartic acid or isoleucine, and / or (31) The amino acid residue at position 441 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with aspartic acid, glycine, histidine, isoleucine, leucine, proline, threonine, or valine, and / or (32) The amino acid residue at position 445 of the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position is deleted or substituted with alanine, glutamine or threonine, or glutamine is inserted therein; and / or (33) The amino acid residue at position 461 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine, aspartic acid, glutamic acid, glycine, asparagine, serine, threonine, valine, or tyrosine, and / or (34) The amino acid residue at position 463 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with glutamic acid, phenylalanine, glycine, isoleucine, leucine, methionine, proline, serine, threonine, or valine, and / or (35) The amino acid residue at position 469 of the amino acid sequence shown in SEQ ID NO:1 or a position corresponding to this position is deleted or substituted with alanine, aspartic acid, glutamic acid, histidine, asparagine, serine or threonine. The MDC variant according to any one of claims 1 to 4.

6. A nucleic acid molecule encoding the MDC variant of any one of claims 1 to 5.

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

8. A host cell comprising the nucleic acid molecule of claim 6 or the vector of claim 7.

9. Use of an MDC variant according to any one of claims 1 to 5 or a host cell according to claim 8 for the conversion of 3-methylcrotonic acid to isobutene.

10. A method for producing isobutene from 3-methylcrotonic acid by incubating the acid with an MDC variant according to any one of claims 1 to 5.

11. The method of claim 10, wherein the enzymatic conversion is carried out in vitro.

12. A composition comprising a variant of MDC according to any one of claims 1 to 5, a nucleic acid molecule according to claim 6, a vector according to claim 7 or a host cell according to claim 8.

13. A composition comprising a variant of MDC according to any one of claims 1 to 5, a nucleic acid molecule according to claim 6, a vector according to claim 7 or a host cell according to claim 8, and 3-methylcrotonic acid.

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