Patent Application
20160160204
The present invention relates to mevalonate diphosphate decarboxylase variants having improved activity in converting 3-phosphonoxyisovalerate into isobutene. Moreover, the present invention relates to enzymes which are characterized in that they are capable of converting 3-phosphonoxyisovalerate into isobutene with a kcat of more than 0.1 s−1.
A large number of chemical compounds are currently derived from petrochemicals. Alkenes (such as ethylene, propylene, the different butenes, or else the pentenes, for example) are used in the plastics industry, for example for producing polypropylene or polyethylene, and in other areas of the chemical industry and that of fuels. Over the past years, the bioproduction of plastics (“bioplastics”) and biofuels has become a thriving field due to economic concerns linked to the price of oil, and to environmental considerations that are both global (carbon-neutral products) and local (waste management). Thus, there is a need for efficient enzymes for producing alkenes such as isobutene.
WO 2010/001078 describes a process for producing alkenes, such as isobutene, by enzymatic conversion of 3-hydroxyalkanoic acids with an enzyme having the activity of a decarboxylase, for example a mevalonate diphosphate (MDP) decarboxylase. Such a method is advantageous because it helps to avoid the use of petroleum products, to lower the costs of producing plastics and fuels and can have a considerable global environmental impact by allowing carbon to be stored in solid form. It could be shown that mevalonate diphosphate decarboxylase is capable of using substrates other than its natural substrate mevalonate diphosphate, in particular 3-hydroxyalkanoic acids, and convert them into terminal alkenes. Mevalonate diphosphate (MDP) decarboxylase (enzyme nomenclature EC 4.1.1.33) is an enzyme involved in cholesterol biosynthesis. The enzyme has been isolated from a variety of organisms including animals, fungi, yeasts and some bacteria. It can also be expressed by some plants (Lalitha et al., Phytochemistry 24 (11), (1985), 2569-2571). Many genes encoding this enzyme have been cloned and sequenced. These enzymes are generally composed of 300 to 400 amino acids and use ATP as co-substrate, which is converted during the reaction to ADP and inorganic phosphate. The phosphate group is transferred in a first step from the ATP molecule to the tertiary alcohol of mevalonate diphosphate, releasing ADP. The reaction intermediate, which is phosphorylated on the 3-hydroxyl group, undergoes in a second step elimination of the phosphate group and decarboxylation, in the physiological case releasing isopentenyl diphosphate.
MDP decarboxylases have been isolated from a multitude of different organisms, eukaryotic and prokaryotic, and have been analyzed and characterized in detail. Also, various mutants have been produced in order to identify the amino acid residues which may play a crucial role in the enzymatic activity of the enzyme. For example, Alvear et al. (Biochemistry 21 (1982), 4646-4650) describe the purification and characterization of avian liver MDP decarboxylase and Dhe-Paganon et al. (Biochemistry 33 (1994), 13355-13362) describe the mechanism of the reaction catalyzed by MDP decarboxylase. Berges et al. (J. Bacteriol. 179 (1997), 4664-4670) reported on a mutation of the S. cerevisiae MDP decarboxylase which leads to thermosensitivity. Krepkiy and Miziorko (Protein Sci. 13 (2004), 1875-1881) identified active site residues in MDP decarboxylase of yeast and analysed mutations which led to a reduction in activity. Similarly, Krepkiy and Miziorko (Biochemistry 44 (2005), 2671-2677) investigated conserved serine residues which are located in a proposed interdomain active site cleft of MDP decarboxylase as to their relevance and could show that mutation of any of these serine residues leads to a reduction or loss of activity. Also Qiu et al. (Bioorganic & medicinal Chemistry Letters 17 (2007), 6164-6168) analyze MDP decarboxylase (of rat) and report on various mutants which lead to a reduction or loss of activity. Voynova et al. (Arch. Biochem. Biophys. 480 (2008), 58-67) characterized the human MDP decarboxylase and identified several amino acid residues which lead to a decrease or loss of enzyme activity.
Moreover, the crystal structure of several MDP decarboxylases and MDP decarboxylase mutants from different origins has been established, e.g. for the Staphylococcus epidermidis enzyme (Barta et al., J. Biol. Chem. 286 (2011), 23900-23910; Barta et al., Biochemistry 51 (2012), 5611-5621; PDB accession numbers 3QT5-6-7-8 and 4DPX, 4DPY, 4DPU, 4DPT, 4DU8, 4DU7 and 4DPW), for the Trypanosoma brucei and Staphylococcus aureus enzymes (Byres et al. J. Mol. Biol. 371 (2007), 540-553; PDB accession number 2HKE, 2HK2, 2HK3), the human enzyme (Voynova et al. (Arch. Biochem. Biophys. 480 (2008), 58-67; PDB accession number 3D4J), the Streptococcus pyogenes enzyme (PDB accession number 2GS8), the mouse enzyme (PDB accession number 3FON), the Legionella pneumophila enzyme (PDB accession number 3LTO) and the S. cerevisiae enzyme (Bonanno et al., Proc. Natl. Acad. Sci. USA. 98 (2001), 12896-12901; PDB accession number 1Fl4).
Lefurgy et al. (J. Biol. Chem. 285 (2010), 20654-20663 analyzed the ligand binding pocket of MDP decarboxylase of Streptococcus pneumoniae by using a series of MDP analogues and Weerasinghe and Dassanayake (J. Mol. Model. 16 (2010) 489-498) reported on the simulation of structural and functional properties of MDP decarboxylase of S. cerevisiae using the wild-type enzyme and the thermosensitive mutant described in Berges et al. (J. Bacteriol. 179 (1997), 4664-4670).
Byres et al. (J. Mol. Biol. 371 (2007), 540-553) compared the crystal structures of different MDP decarboxylases, in particular of those from T. brucei, S. aureus and S. cerevisiae and observe that the overall architecture of the enzyme remains conserved.
WO 2010/001078 discloses inter alia that it is possible to convert 3-hydroxy-3-methylbutyrate (or 3-hydroxyisovalerate) into isobutene by a decarboxylase, in particular an MDP decarboxylase. In this case, the reaction intermediate is 3-phosphonoxyisovalerate which is further converted in the second part of the reaction into isobutene. Gogerty et al. (Appl. Environ. Microbiol. 76 (2010), 8004-8010) also report on the formation of isobutene from 3-hydroxy-3-methylbutyrate using an MDP decarboxylase from S. cerevisiae and show that mutations at residues 145 and 74 of this enzyme, which are located within or close to the proposed active site of this enzyme, lead to an increase of the conversion of 3-hydroxy-3-methylbutyrate into isobutene. However, the level of production of isobutene achieved is still too low for commercial application.
Later works have shown that different MDP decarboxylases may show different efficiencies as regards the catalysis of the first and the second step of the reaction as described above, with some MDP decarboxylases showing a high activity in the first step and others showing a high activity in the second step. Therefore, it had been proposed to combine two MDP decarboxylases which show a high activity in the first and in the second step of the reaction, respectively, so as to optimize the overall enzymatic reaction (WO2012/052427).
However, although such a method allows to produce alkenes by enzymatically converting 3-hydroxyalkanoates (e.g. isobutene from 3-hydroxyisovalerate), there is still a need for improvements, in particular as regards a further increase in efficiency of the process so as to make it more suitable for industrial purposes.
The present application addresses this need by providing the embodiments as defined in the claims.
In particular, the present invention provides enzymes which are characterized in that they are capable of converting 3-phosphonoxyisovalerate into isobutene with a kcat of more than 0.01 s−1 or 0.1 s−1, preferably of more than 1 s−1, more preferably of more than 10 s−1, and even more preferably of more than 102 s−1 or most preferably of more than 103 s−1. Preferably such enzymes are variants of a mevalonate diphosphate decarboxylase, and even more preferably such enzymes have an amino acid sequence which shows more than 60% sequence homology to the amino acid sequence shown in SEQ ID NO:1. It is preferred that the enzymes according to the invention are non-naturally occurring enzymes. This means that they substantially differ from naturally occurring enzymes, in particular as regards their primary structure, i.e., the amino acid sequence. Thus, they show an amino acid sequence which does not occur in nature. It is preferable that such non-naturally occurring enzymes also differ from naturally occurring enzymes insofar that they have a higher enzymatic activity for the reactions described herein.
Thus, the present invention provides in particular variants of mevalonate diphosphate decarboxylase which show an improved activity as regards the conversion of 3-phosphonoxyisovalerate into isobutene thereby allowing to dramatically increase the production efficiency of isobutene from 3-hydroxyisovalerate.
The term “mevalonate diphosphate decarboxylase” in the context of the present invention refers to an enzyme which naturally has the ability to convert mevalonate diphosphate into isopentenyl diphosphate and which is classified as EC 4.1.1.33. The term “mevalonate diphosphate decarboxylase” also covers enzymes which are classified as a mevalonate diphosphate decarboxylase and which act on mevalonate monophosphate, i.e. which are mevalonate monophosphate decarboxylases. An example for such an enzyme is the enzyme from Roseiflexus sp. (strain RS-1) (Uniprot Accession number: A5V173) which is classified as a mevalonate diphosphate decarboxylase. Since some bacteria from Roseiflexus genus, e.g. Roseiflexus castenholzii, have been reported to have an alternative mevalonate pathway which involves the action of a mevalonate monophosphate decarboxylase an enzyme from Roseiflexus sp. may act as a mevalonate monophosphate decarboxylase. Additionally, a third mevalonate pathway has recently been described in T. acidophilum. In this pathway, the formation of isopentenyl monophosphate probably occurs through mevalonate-3,5-pyrophosphate (MVA-3,5-PP). The two genes which can be implicated in the decarboxylation of this intermediate are Ta0461 and Ta0893. These genes can be associated to the family of diphosphomevalonate decarboxylase referenced in InterPro database as IPR005935 (http://www.ebi.ac.uk/interpro/entrv/IPR005935), but these two enzymes may act as mevalonate-3,5-pyrophosphate decarboxylase. Thus, the term “mevalonate diphosphate decarboxylase” also covers such enzymes. The present invention relates to variants which are derived from a mevalonate diphosphate decarboxylase. The variants of such an enzyme according to the present invention are characterized by the feature that they are derived from an MDP decarboxylase having the amino acid sequence shown in SEQ ID NO:1 or having a related sequence (at least 40% identical, preferably at least 50% identical, even more preferably at least 60% or at least 90% identical) and in which mutations are effected at one or more of the positions as indicated herein below and by the feature that they show the ability to convert 3-phosphonoxyisovalerate into isobutene and that they can do this with an improved activity. In a preferred embodiment the variant according to the present invention is derived from a sequence which shows at least 80% sequence identity to SEQ ID NO:1 and in which one or more substitutions and/or deletions and/or insertions at the positions indicated herein below have been effected.
Given the high conservation between MDP decarboxylases, the teaching of the present invention is not restricted to the enzyme of S. mitis (represented by SEQ ID NO: 1) but can be extended to MDP decarboxylases from other organisms. Thus, the present invention also relates to variants of MDP decarboxylase which are derived from enzymes which are structurally related to the S. mitis sequence and which show one or more substitutions and/or deletions and/or insertions at positions corresponding to any of the positions as indicated herein-below. The term “structurally related” refers to MDP decarboxylases which show a sequence identity of at least n % to the sequence shown in SEQ ID NO: 1 with n being an integer between 40 and 100, preferably 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99. In a preferred embodiment the structurally related MDP decarboxylase is of prokaryotic origin. These variants are characterized by the feature that they show the ability to convert 3-phosphonoxyisovalerate into isobutene and that they can do this with an improved activity when compared to the enzyme from which these variants are derived. Even more preferably such variants also show an improved activity when compared to the enzyme having the amino acid sequence shown in SEQ ID NO:1.
Thus, in one embodiment the variant of MDP decarboxylase according to the present invention has or preferably is derived from a sequence which is at least n % identical to SEQ ID NO:1 with n being an integer between 40 and 100, preferably 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99, and it has (a) substitution(s) and/or (a) deletion(s) and/or (an) insertion(s) at a position as indicated below. When the sequences which are compared do not have the same length, the degree of identity either refers to the percentage of amino acid residues in the shorter sequence which are identical to amino acid residues in the longer sequence or to the percentage of amino acid residues in the longer sequence which are identical to amino acid residues in the shorter sequence. Preferably, it refers to the percentage of amino acid residues in the shorter sequence which are identical to amino acid residues in the longer sequence. The degree of sequence identity can be determined according to methods well known in the art using preferably suitable computer algorithms such as CLUSTAL.
When using the Clustal analysis method to determine whether a particular sequence is, for instance, at least 40%, 50% or 60% or 90% identical to a reference sequence default settings may be used or the settings are preferably as follows: Matrix: blosum 30; Open gap penalty: 10.0; Extend gap penalty: 0.05; Delay divergent: 40; Gap separation distance: 8 for comparisons of amino acid sequences. For nucleotide sequence comparisons, the Extend gap penalty is preferably set to 5.0.
In a preferred embodiment ClustalW2 is used for the comparison of amino acid sequences. In the case of pairwise comparisons/alignments, the following settings are preferably chosen: Protein weight matrix: BLOSUM 62; gap open: 10; gap extension: 0.1. In the case of multiple comparisons/alignments, the following settings are preferably chosen: 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 complete length of the sequence.
Examples for MDP decarboxylases which show a sequence identity of at least 60%, in particular between 60% and 80%, to SEQ ID NO:1 are shown in the following Table.
Streptococcus
anginosus
Streptococcus
gordonii
Streptococcus
sanguinis
Streptococcus
australis
Streptococcus
agalactiae
Streptococcus
gallolyticus
Streptococcus
uberis (strain
Streptococcus
equinus ATCC
Streptococcus
pyogenes
Streptococcus
pyogenes
Leptotrichia
goodfellowii
Examples for MDP decarboxylases which show a sequence identity of at least 80%, in particular between 80% and 90%, to SEQ ID NO:1 are shown in the following Table.
Streptococcus
peroris
Streptococcus
infantis
Streptococcus
infantis
Examples for MDP decarboxylases which show a sequence identity of at least 90%, in particular between 90% and 100%, to SEQ ID NO:1 are shown in the following Table.
Streptococcus
mitis SK321
Streptococcus
Streptococcus
sanguinis
Streptococcus
oralis ATCC
Streptococcus
pneumonia
Amino acid residues located at a position corresponding to a position as indicated herein-below in the amino acid sequence shown in SEQ ID NO:1 can be identified by the skilled person 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 the positions which correspond to the above indicated positions of SEQ ID NO:1. The alignment can be done with means and methods known to the skilled person, e.g. by using a known computer algorithm such as the Lipman-Pearson method (Science 227 (1985), 1435) or the CLUSTAL algorithm. It is preferred that in such an alignment maximum homology is assigned to conserved amino acid residues present in the amino acid sequences.
In a preferred embodiment ClustalW2 is used for the comparison of amino acid sequences. In the case of pairwise comparisons/alignments, the following settings are preferably chosen: Protein weight matrix: BLOSUM 62; gap open: 10; gap extension: 0.1. In the case of multiple comparisons/alignments, the following settings are preferably chosen: Protein weight matrix: BLOSUM 62; gap open: 10; gap extension: 0.2; gap distance: 5; no end gap.
When the amino acid sequences of MDP decarboxylases are aligned by means of such a method, regardless of insertions or deletions that occur in the amino acid sequences, the positions of the corresponding amino acid residues can be determined in each of the MDP decarboxylases. Examples of alignments are provided in
In the context of the present invention, “substituted with another amino acid residue” means that the respective amino acid residues at the indicated position can be substituted with any other possible amino acid residues, e.g. naturally occurring amino acids or non-naturally occurring amino acids (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 certain positions are indicated further below. Moreover, the term “substituted” or “substitution” also means that the respective amino acid residue at the indicated position is modified.
Such modifications include naturally occurring modifications and non-naturally occurring modifications. Naturally occurring modifications include but are not limited to eukaryotic post-translational modification, such as attachment of functional groups (e.g. acetate, phosphate, hydroxyl, lipids (myristoylation of glycine residues) and carbohydrates (e.g. glycosylation of arginine, asparagines etc.). Naturally occurring modifications also encompass the change in the chemical structure by citrullination, carbamylation and disulphide bond formation between cysteine residues; attachment of co-factors (FMN or FAD that can be covalently attached) or the attachement of peptides (e.g. ubiquitination or sumoylation).
Non-naturally occurring modifications include, e.g., in vitro modifications such as biotinylation of lysine residue or the inclusion of non-canonical amino acids (see Liu and Schultz, Annu. Rev. Biochem. 79 (2010), 413-44 and Wang et al., Chem. Bio. 2009 Mar. 27; 16 (3), 323-336; doi:101016/jchembiol.2009.03.001).
In the context of the present invention, “deleted” or “deletion” means that the amino acid at the corresponding position is deleted.
In the context of the present invention, “inserted” or “insertion” means that at the respective position one or two, preferably one amino acid residue is inserted, preferable in front of the indicated position.
Thus, the present invention relates to a variant of a mevalonate diphosphate decarboxylase showing an improved activity in converting 3-phosphonoxyisovalerate into isobutene over the corresponding mevalonate diphosphate decarboxylase from which it is derived, wherein the mevalonate diphosphate decarboxylase variant is characterized in that it shows one or more substitutions, deletions and/or insertions in comparison to the corresponding sequence from which it is derived and wherein these substitutions, deletions and/or insertions occur at one or more of the positions corresponding to positions 282, 9, 11, 16, 24, 28, 42, 43, 45, 53, 66, 77, 80, 91, 105, 116, 118, 120, 121, 122, 123, 129, 134, 141, 159, 160, 161, 173, 177, 180, 186, 215, 238, 241, 242, 248, 251, 253, 258, 264, 279, 291, 293, 297, 299, 303, 307, 308, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267, and 315 in the amino acid sequence shown in SEQ ID NO:1. “Corresponding to” these positions means corresponding to any of these positions in a related sequence.
In a preferred embodiment the mevalonate diphosphate decarboxylase from which the variant is derived is a mevalonate diphosphate decarboxylase which shows the amino acid sequence as shown in SEQ ID NO:1 or an amino acid sequence having at least 40%, preferably at least 50% and even more preferably at least 60%, or most preferred at least 90% sequence identity to SEQ ID NO:1.
Accordingly, in one embodiment, the present invention relates to a variant of a mevalonate diphosphate decarboxylase having an amino acid sequence as shown in SEQ ID NO:1 or an amino acid sequence having at least 40%, 50%, 60% or 90% sequence identity to SEQ ID NO:1, wherein one or more amino acid residues at a position selected from the group consisting of positions 9, 11, 16, 24, 28, 42, 43, 45, 53, 66, 77, 80, 91, 105, 116, 118, 120, 121, 122, 123, 129, 134, 141, 159, 160, 161, 173, 177, 180, 186, 215, 238, 241, 242, 248, 251, 253, 258, 264, 279, 282, 291, 293, 297, 299, 303, 307, 308, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267, and 315 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to any of these positions in a related sequence, are substituted with another amino acid residue or deleted or show an insertion at at least one of these positions and wherein said mevalonate diphosphate decarboxylase has improved activity in converting 3-phosphonoxyisovalerate into isobutene. In a preferred embodiment the deletion, insertion or substitution is at a position selected from the group consisting of positions 9, 11, 42, 43, 45, 66, 77, 116, 118, 120, 121, 123, 129, 134, 159, 160, 173, 177, 186, 251, 253, 282, 293, 297, 299, 303, 307 and 308.
The present inventors have found that the activity of a mevalonate diphosphate decarboxylase to catalyze the second step of the above described conversion of 3-hydroxyisovalerate into isobutene, i.e. the conversion of 3-phosphonoxyisovalerate into isobutene, can dramatically be improved by mutating the MDP decarboxylase enzyme at certain positions. They used as a model enzyme the enzyme from S. mitis, the sequence of which is shown in SEQ ID NO: 1. The identified single mutations lead to an increase of activity of up to more than 300% when compared to the unmutated sequence of the S. mitis enzyme (represented by SEQ ID NO: 1). Combining mutations led to a further increase of up to 750% and the identification of additional mutations in addition to these combinations allowed to achieve a further 16-fold increase.
In particular, the present inventors found that substitutions at positions 9, 11, 16, 24, 28, 42, 43, 45, 53, 66, 77, 80, 91, 105, 116, 118, 120, 121, 122, 123, 129, 134, 141, 159, 160, 161, 173, 177, 180, 186, 215, 238, 241, 242, 248, 251, 253, 258, 264, 279, 282, 291, 293, 297, 299, 303, 307, 308, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267, or 315 or substitutions at combinations of these positions lead to a drastic increase in the enzyme's ability to convert 3-phosphonoxyisovalerate into isobutene.
As indicated above, the variants of an MDP decarboxylase according to the present invention are characterized in that they show an increased activity in converting 3-phosphonoxyisovalerate into isobutene when compared to the MDP decarboxylase from which they are derived. Thus, in the case where the variant is derived from the MDP decarboxylase of S. mitis having the amino acid sequence shown in SEQ ID NO: 1, the variant shows an increased activity in converting 3-phosphonoxyisovalerate into isobutene when compared to the MDP decarboxylase shown in SEQ ID NO: 1. When the variant is derived from an MDP decarboxylase which is structurally related to the MDP decarboxylase of S. mitis as defined herein above, the variant shows an increased activity in converting 3-phosphonoxyisovalerate into isobutene when compared to the corresponding starting sequence into which the corresponding mutations have been introduced. In a particularly preferred embodiment such variants show also an increased activity in converting 3-phosphonoxyisovalerate into isobutene when compared to the MDP decarboxylase shown in SEQ ID NO: 1. The activity of converting 3-phosphonoxyisovalerate into isobutene may be determined by methods known to the person skilled in the art. In one embodiment, this activity is determined as described in the Examples appended hereto. In a particular embodiment this activity can be measured by using in particular the following assay:
A nucleic acid molecule encoding the respective enzyme is transformed into E. coli, such as E. coli BL21 DE3 and the enzyme is expressed in E. coli. The activity of the enzyme can then be measured using either
The activity of the expressed enzyme is tested as follows:
A reaction mixture is prepared in glass GC vials by mixing the crude cell lysate, the soluble fraction or the purified protein as described above with 3-phosphonoxyisovalerate in 50 mM Tris pH 7 final concentration. ATP, MgCl2 and KCl are provided as cofactors.
The vials are hermetically sealed and the reaction mixture is incubated for an appropriate time (e.g. 24 h) at 37° C. The isobutene produced by the reaction is determined by gas chromatography by injecting the gas phase obtained from the vial into a gas chromatograph. Controls in the assay are culture with untransformed bacteria, bacteria transformed with an empty expression vector and bacteria expressing the corresponding starting enzyme, e.g. the S. mitis enzyme showing the amino acid sequence as shown in SEQ ID NO:1.
The 3-phosphonoxyisovalerate used in the assay may be prepared in different ways. One possibility is to provide this compound by the following reaction:
0.063 mg/ml of purified Thermophilus acidophilum MDP decarboxylase is mixed with 50 mM hydroxyisovalerate, 40 mM ATP in a 50 mM Tris-Cl pH 7, 20 mM KCl, 20 mM MgCl2 and incubated for 24 hours at 45° C. This enzymatically prepared 3-phosphonoxyisovalerate substrate is used in the reaction mixture.
Another, preferred possibility is that the 3-phosphonoxyisovalerate used in the assay is chemically synthesized. The synthesis scheme is shown in
If chemically synthesized 3-phosphonoxyisovalerate is used in the assay, the concentration is preferably between 3 and 8 mM, preferably it is 5 mM.
In a preferred assay for testing the enzyme activity purified enzyme is used. In this case, the reaction mixture contains
500 μg purified enzyme in 50 mM Tris HCl pH 7.5 and
3-phosphonoxyisovalerate (provided as described above)
It is made sure that cofactors such as ATP, MgCl2 and KCl are present. The volume is adjusted to 500 μl with 50 mM Tris HCl pH 7.5 and the reaction is incubated for 15 hours at 37° C. Subsequently the isobutene produced is determined by gas chromatography.
In one embodiment, the 3-phosphonoxyisovalerate is prepared enzymatically as described above.
In a particularly preferred embodiment the 3-phosphonoxyisovalerate is chemically synthesized. In this case, the reaction mixture contains:
200 μg purified enzyme
3-phosphonoxyisovalerate
5 mM ATP, 20 mM KCl, 10mM MgCl2 and 50 mM Tris-Cl pH 7.5
In order to test the activity of the enzyme varying concentrations of 3-phosphonoxyisovalerate are used, preferably the following concentrations: 0.625 mM, 1.25 mM, 2.5 mM, 5 mM, 10, mM, 20 mM, 40 mM, 80 mM and 160 mM. It is particularly preferred that the concentration of 3-phosphonoxyisovalerate is 5 mM.
The volume is adjusted to 500 μl with 50 mM Tris HCl pH 7.5 and the reaction is incubated for 15 hours at 37° C. Subsequently the isobutene produced is determined by gas chromatography.
Examples for such an assay to determine the activity of an enzyme to convert 3-phosphonoxyisovalerate into isobutene are given in the Example section below in Example 1.
In the context of the present invention, an “improved activity” means that the activity of the MDP decarboxylase in question is at least 10%, preferably at least 20%, more preferably at least 30% or 50%, even more preferably at least 70% or 80% and particularly preferred at least 90% or 100% higher than that of the MDP decarboxylase from which the variant is derived, preferably higher than that of the MDP decarboxylase of S. mitis represented by SEQ ID NO:1. In even more preferred embodiments the improved activity may be at least 150%, at least 200%, at least 300%, at least 750% or at least 1000% higher than that of the corresponding MDP decarboxylase from which the variant is derived, preferably higher than that of the MDP decarboxylase of S. mitis 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 3-phosphonoxyisovalerate as described herein-above. The improved activity of a variant can be measured as a higher isobutene production in a given time under defined conditions, compared with the parent enzyme. This improved activity can result from a higher kcat value. It can also result from a lower Km value. It can also result from a higher kcat/Km value. The degree of improvement can be measured as the improvement in isobutene production. The degree of improvement can also be measured in terms of kcat improvement, of kcat/Km improvement, or in terms of Km decrease.
According to one embodiment, the MDP decarboxylase of the present invention has an amino acid sequence in which
The invention also relates to variants as defined in (1) to (77) hereinabove, wherein the amino acid residue indicated as substituting the amino acid residue at the position in SEQ ID NO: 1 is not that particular amino acid residue but an amino acid residue which is conservative in relation to the indicated substituting amino acid. Whether an amino acid is conservative with respect to another amino acid can be judged according to means and methods known in the art. One possibility is the PAM 250 matrix; alternatively, the Blosum Family Matrices can be used.
In one embodiment the present invention relates to a variant of a MDP decarboxylase having an amino acid sequence as shown in SEQ ID NO:1 or an amino acid sequence having at least 40%, 50%, 60% or 90% sequence identity to SEQ ID NO:1, wherein the amino acid residue at position 282 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in a related sequence is substituted with another amino acid residue or is deleted. In a preferred embodiment the present invention relates to such a variant in which at least one further amino acid residue is substituted or deleted at a position selected from the group consisting of positions 9, 11, 16, 24, 28, 42, 43, 45, 53, 66, 77, 80, 91, 105, 116, 118, 120, 121, 122, 123, 129, 134, 141, 159, 160, 161, 173, 177, 180, 186, 215, 238, 241, 242, 248, 251, 253, 258, 264, 279, 291, 293, 297, 299, 303, 307, 308, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267, and 315, preferably selected from the group consisting of positions 9, 11, 16, 24, 28, 42, 45, 53, 80, 91, 105, 116, 118, 120, 121, 122, 123, 129, 141, 159, 161, 173, 177, 180, 215, 238, 241, 242, 248, 251, 253, 264, 279, 291, 293, 297, 299, 303, 307, 308 and 315, even more preferably selected from the group consisting of positions 9, 11, 42, 45, 116, 118, 120, 121, 122, 123, 129, 177, 251, 253, 264, 293, 297 and 303.
In one particular embodiment, the amino acid residues at position 282 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in a related sequence is substituted with cystein, serine, glutamic acid, glycine, glutamine, threonine, valine, alanine or aspartic acid, preferably with cystein. In another particular embodiment, the substitutions at any one of positions 9, 11, 16, 24, 28, 42, 43, 45, 53, 66, 77, 80, 91, 105, 116, 118, 120, 121, 122, 123, 129, 134, 141, 159, 160, 161, 173, 177, 180, 186, 215, 238, 241, 242, 248, 251, 253, 258, 264, 279, 291, 293, 297, 299, 303, 307, 308, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267, and 315 are as indicated herein-above.
In one embodiment the present invention relates to a variant of a MDP decarboxylase having an amino acid sequence as shown in SEQ ID NO:1 or an amino acid sequence having at least 40%, 50%, 60% or 90% sequence identity to SEQ ID NO:1, wherein the amino acid residues at position 121 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in a related sequence is deleted or substituted with another amino acid residue, preferably arginine, leucine, lysine or phenylalanine. In a preferred embodiment the present invention relates to such a variant in which at least one further amino acid residue is deleted or substituted at a position selected from the group consisting of positions 9, 11, 16, 24, 28, 42, 43, 45, 53, 66, 77, 80, 91, 105, 116, 118, 120, 122, 123, 129, 134, 141, 159, 160, 161, 173, 177, 180, 186, 215, 238, 241, 242, 248, 251, 253, 258, 264, 279, 282, 291, 293, 297, 299, 303, 307, 308, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267, and 315, preferably selected from the group consisting of positions 11, 16, 24, 28, 45, 53, 80, 91, 105, 116, 118, 120, 123, 141, 159, 161, 173, 177, 180, 215, 238, 241, 242, 248, 258, 279, 282, 291, 297, 299, 303, 307, 308 and 315. The substitutions at any of these positions are preferably those as listed herein-above.
The present invention also relates to a variant of a MDP decarboxylase having an amino acid sequence as shown in SEQ ID NO:1 or an amino acid sequence having at least 40%, 50%, 60% or 90% sequence identity to SEQ ID NO:1, wherein the amino acid residues at position 282 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in a related sequence is deleted or substituted with another amino acid residue, wherein the amino acid residue at position 121 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in a related sequence is deleted or substituted with another amino acid residue and wherein at least one further amino acid residue is substituted at a position selected from the group consisting of positions 9, 11, 16, 24, 28, 42, 43, 45, 53, 66, 77, 80, 91, 105, 116, 118, 120, 122, 123, 129, 134, 141, 159, 160, 161, 173, 177, 180, 186, 215, 238, 241, 242, 248, 251, 253, 258, 264, 279, 291, 293, 297, 299, 303, 307, 308, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267, and 315, preferably selected from the group consisting of positions 11, 16, 24, 28, 45, 53, 80, 91, 105, 116, 118, 120, 123, 141, 159, 161, 173, 177, 180, 215, 238, 241, 242, 248, 258, 279, 291, 297, 299, 303, 307, 308 and 315.
In another particular embodiment, the amino acid residue at position 121 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in a related sequence is substituted with arginine, leucine, lysine and phenylalanine. In a further preferred embodiment, the substitutions at any one of the positions 9, 11, 16, 24, 28, 42, 43, 45, 53, 66, 77, 80, 91, 105, 116, 118, 120, 122, 123, 129, 134, 141, 159, 160, 161, 173, 177, 180, 186, 215, 238, 241, 242, 248, 251, 253, 258, 264, 279, 291, 293, 297, 299, 303, 307, 308, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267, and 315 are those as indicated herein-above.
In a preferred embodiment the variant according to the invention is characterized in that it contains at least three deletions and/or substitutions, wherein one deletion/substitution is at position 282 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in a related sequence, another deletion/substitution is at position 121 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in a related sequence and at least one further deletion/substitution is at a position selected from the group consisting of positions 11, 45, 116, 120 or 177 of SEQ ID NO: 1 or a position corresponding to any of these positions.
It is preferred that the following positions in SEQ ID NO:1 or the positions corresponding to any of these positions in a related sequence in a related sequence as defined above are at least modified by deletion or substitution:
45, 121 and 282; or
11, 121 and 282; or
116, 121 and 282; or
121, 177 and 282; or
120, 121 and 282; or
173, 282 and 297.
The substitutions at the indicated positions are preferably those as indicated herein-above.
Particularly preferred variants with three mutations show the following substitutions in SEQ ID NO:1:
E45L-Y121 R-K282C
K282C-Y121R-Y11E
K116I-Y121R-K282C
Y121R-E177V-K282C
A120R-Y121L-K282C
M173C-K282C-F297L
or the corresponding substitutions at the corresponding positions in a structurally related sequence as defined above.
The present invention also relates to a variant of a MDP decarboxylase having an amino acid sequence as shown in SEQ ID NO:1 or an amino acid sequence having at least 40%, 50%, 60% or 90% sequence identity to SEQ ID NO:1, wherein the amino acid residues at position 282 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in a related sequence is substituted with another amino acid residue, wherein the amino acid residues at position 297 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in a related sequence is substituted with another amino acid residue and wherein the amino acid residues at position 173 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in a related sequence is substituted with another amino acid residue. In a particular embodiment, the amino acid residues at position 282 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in a related sequence is substituted with cystein, serine, glutamic acid, glycine, glutamine, threonine, valine, alanine or aspartic acid, preferably with cystein. In another particular embodiment, the amino acid residue at position 297 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position in a related sequence is substituted with leucine. In a further embodiment the amino acid residues at position 173 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding in a related sequence to this position is substituted with cysteine.
In another embodiment the variant according to the invention is characterized in that it contains at least four deletions and/or substitutions at positions selected from the group consisting of positions 9, 11, 16, 24, 28, 42, 43, 45, 53, 66, 77, 80, 91, 105, 116, 118, 120, 122, 123, 129, 134, 141, 159, 160, 161, 173, 177, 180, 186, 215, 238, 241, 242, 248, 251, 253, 258, 264, 279, 291, 293, 297, 299, 303, 307, 308, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267, and 315, preferably selected from the group consisting of positions 9, 11, 16, 24, 28, 45, 53, 80, 91, 105, 118, 121, 123, 141, 159, 161, 173, 177, 180, 215, 238, 241, 242, 248, 258, 279, 282, 291, 297, 299, 303, 307, 308 and 315 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to any of these positions in a related sequence as defined above. Preferably, one of the deletions/substitutions is at position 303 of SEQ ID NO:1 or at a position corresponding to this position in a related sequence as defined above. More preferably, this position is replaced by a methionine. In another embodiment the substitutions at the remaining three positions are effected at positions selected from the group consisting of positions 45, 121, 173, 282, 307 and 308 or at a position corresponding to any of these positions in a related sequence as defined above.
It is preferred that the following positions in SEQ ID NO:1 or the positions corresponding to any of these positions in a related sequence as defined above are at least modified by deletion or substitution:
121, 282, 303 and 308; or
173, 303, 307 and 308; or
45, 173, 282 and 303.
Preferably, the substitution at position 45 is valine, the substitution at position 121 is an arginine, the substitution at position 173 or 282 is a cysteine, the substitution at position 307 is a histidine and the substitution at position 308 is a serine. Most preferred are the following combinations of substitutions: Y121R-K282C-L303M-T308S; M173C-L303M-K307H-T308S; E45V-M173C-K282C-L303M or the corresponding substitutions at the corresponding positions in a structurally related sequence as defined above.
In another embodiment the variant according to the invention is characterized in that it contains at least five deletions and/or substitutions at positions selected from the group consisting of positions 9, 11, 16, 24, 28, 42, 43, 45, 53, 66, 77, 80, 91, 105, 116, 118, 120, 122, 123, 129, 134, 141, 159, 160, 161, 173, 177, 180, 186, 215, 238, 241, 242, 248, 251, 253, 258, 264, 279, 291, 293, 297, 299, 303, 307, 308, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267, and 315, preferably selected from the group consisting of positions 9, 11, 16, 24, 28, 45, 53, 80, 91, 105, 118, 121, 123, 141, 159, 161, 173, 177, 180, 215, 238, 241, 242, 248, 258, 279, 282, 291, 297, 299, 303, 307, 308 and 315 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to any of these positions in a related sequence as defined above. Preferably, one of these substitutions is at position 303 of SEQ ID NO:1 or at a position corresponding to this position in a related sequence as defined above. More preferably, this position is replaced by a methionine. In another embodiment the deletions/substitutions at the remaining three positions are effected at positions selected from the group consisting of positions 9, 11, 118, 121, 159, 173, 282, 307 and 308 or at a position corresponding to any of these positions in a related sequence as defined above.
It is preferred that the following positions in SEQ ID NO:1 or the positions corresponding to any of these positions in a related sequence as defined above are at least modified by deletion or substitution:
121, 173, 282, 303 and 308; or
159, 173, 303, 307 and 308; or
9, 11, 303, 307 and 308; or
118, 121, 173, 282 and 303.
Preferably the substitution at position 9 is leucine, the substitution at position 11 is phenylalanine, the substitution at position 118 is leucine, the substitution at position 121 is an arginine, the substitution at position 159 is leucine, the substitution at position 173 or 282 is a cysteine, the substitution at position 307 is a histidine and the substitution at position 308 is a serine.
Particularly preferred variants with five mutations show the following substitutions in SEQ ID NO:1:
Y121R-M173C-K282C-L303M-T308S
E159L-M173C-L303M-K307H-T308S
R9L-Y11F-L303M-K307H-T308S
C118L-Y121R-M173C-K282C-L303M
or the corresponding substitutions at the corresponding positions in a structurally related sequence as defined above.
In another embodiment the variant according to the invention is characterized in that it contains at least six deletions and/or substitutions at positions selected from the group consisting of positions 9, 11, 16, 24, 28, 42, 43, 45, 53, 66, 77, 80, 91, 105, 116, 118, 120, 122, 123, 129, 134, 141, 159, 160, 161, 173, 177, 180, 186, 215, 238, 241, 242, 248, 251, 253, 258, 264, 279, 291, 293, 297, 299, 303, 307, 308, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267, and 315, preferably selected from the group consisting of positions 16, 24, 28, 45, 53, 80, 91, 105, 118, 121, 123, 141, 159, 161, 173, 177, 180, 215, 238, 241, 242, 248, 258, 279, 282, 291, 297, 299, 303, 307, 308 and 315 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to any of these positions in a related sequence as defined above. Preferably, one of these deletions/substitutions is at position 282 of SEQ ID NO:1 or at a position corresponding to this position in a related sequence as defined above. More preferably, this position is replaced by a cysteine. In another embodiment, one of these deletions/substitutions is at position 173 of SEQ ID NO:1 or at a position corresponding to this position in a related sequence as defined above. More preferably, this position is replaced by a cysteine. In another embodiment the deletions/substitutions at the remaining four positions are effected at positions selected from the group consisting of positions 45, 121, 159, 215, 297, 303 and 308 or at a position corresponding to any of these positions in a related sequence as defined above.
It is preferred that the following positions in SEQ ID NO:1 or the positions corresponding to any of these positions in a related sequence as defined above are at least modified by deletion or substitution:
121, 159, 173, 282, 303 and 308; or
121, 159, 173, 215, 282 and 303; or
45, 159, 173, 282, 297 and 308.
Preferably, the substitution at position 45 is leucine, the substitution at position 121 is an arginine, the substitution at position 159 is leucine, the substitution at position 215 is alanine, the substitution at position 297 is leucine, the substitution at position 303 is a methionine and the substitution at position 308 is a serine. Most preferred are the following combinations of substitutions:
Particularly preferred variants with six mutations show the following substitutions in SEQ ID NO:1:
Y121R-E159L-M173C-K282C-L303M-T308S
Y121R-E159L-M173C-V215A-K282C-L303M
E45L-E159L-M173C-K282C-F297L-T308S
or the corresponding substitutions at the corresponding positions in a structurally related sequence as defined above.
In another embodiment the variant according to the invention is characterized in that it contains at least seven substitutions at positions selected from the group consisting of positions 9, 11, 16, 24, 28, 42, 43, 45, 53, 66, 77, 80, 91, 105, 116, 118, 120, 122, 123, 129, 134, 141, 159, 160, 161, 173, 177, 180, 186, 215, 238, 241, 242, 248, 251, 253, 258, 264, 279, 291, 293, 297, 299, 303, 307, 308, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267, and 315, preferably selected from the group consisting of positions 16, 24, 28, 45, 53, 80, 91, 105, 118, 121, 123, 141, 159, 161, 173, 177, 180, 215, 238, 241, 242, 248, 258, 279, 282, 291, 297, 299, 303, 307, 308 and 315 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to any of these positions in a related sequence as defined above. Preferably, one of these deletions/substitutions is at position 282 of SEQ ID NO:1 or at a position corresponding to this position in a related sequence as defined above. More preferably, this position is replaced by a cysteine. In another embodiment, one of these deletions/substitutions is at position 173 of SEQ ID NO:1 or at a position corresponding to this position in a related sequence as defined above. More preferably, this position is replaced by a cysteine. In another embodiment, one of these deletions/substitutions is at position 121 of SEQ ID NO:1 or at a position corresponding to this position in a related sequence as defined above. More preferably, this position is replaced by an arginine. In another embodiment, one of these deletions/substitutions is at position 303 of SEQ ID NO:1 or at a position corresponding to this position in a related sequence as defined above. More preferably, this position is replaced by a methionine. In another embodiment the deletions/substitutions at the remaining three positions are effected at positions selected from the group consisting of positions 45, 118, 159, 177, 242, 297, 307, 308 and 315 or at a position corresponding to any of these positions in a related sequence as defined above.
It is preferred that the following positions in SEQ ID NO:1 or the positions corresponding to any of these positions in a related sequence as defined above are at least modified by deletion or substitution:
121, 159, 173, 282, 303, 307 and 308; or
45, 121, 159, 173, 282, 303 and 308; or
121, 159, 173, 282, 297, 303 and 308; or
118, 121, 159, 173, 282, 303 and 308; or
121, 159, 173, 177, 282, 303 and 308; or
121, 159, 173, 242, 282, 303 and 308; or
118, 121, 159, 173, 282, 303 and 315; or
118, 121, 159, 173, 177, 282 and 303;
45, 121, 173, 282, 297, 303 and 308; or
45, 118, 121, 173, 282, 303 and 308; or
45, 121, 159, 173, 282, 297 and 303.
Preferably, the substitution at position 45 is valine, the substitution at position 118 is leucine or tryptophan, the substitution at position 159 is leucine, the substitution at position 177 is cysteine, the substitution at position 242 is alanine, the substitution at position 297 is leucine, the substitution at position 307 is a histidine and the substitution at position 308 or 315 is a serine. Most preferred are the following combinations of substitutions:
Particularly preferred variants with seven mutations show the following substitutions in SEQ ID NO:1:
Y121R-E159L-M173C-K282C-L303M-K307H-T308S
E45V-Y121R-E159L-M173C-K282C-L303M-T308S
Y121R-E159L-M173C-K282C-F297L-L303M-T308S
C118L-Y121R-E159L-M173C-K282C-L303M-T308S
Y121R-E159L-M173C-E177C-K282C-L303M-T308S
Y121R-E159L-M173C-T242A-K282C-L303M-T308S
C118L-Y121R-E159L-M173C-K282C-L303M-G315S
C118W-Y121R-E159L-M173C-E177C-K282C-L303M
C118L-Y121R-E159L-M173C-E177C-K282C-L303M
E45V-Y121R-M173C-K282C-F297L-L303M-T308S
E45V-C118L-Y121R-M173C-K282C-L303M-T308S
E45V-Y121R-E159L-M173C-K282C-F297L-L303M
or the corresponding substitutions at the corresponding positions in a structurally related sequence as defined above.
In another embodiment the variant according to the invention is characterized in that it contains at least eight or at least nine deletions and/or substitutions at positions selected from the group consisting of positions 9, 11, 16, 24, 28, 42, 43, 45, 53, 66, 77, 80, 91, 105, 116, 118, 120, 122, 123, 129, 134, 141, 159, 160, 161, 173, 177, 180, 186, 215, 238, 241, 242, 248, 251, 253, 258, 264, 279, 291, 293, 297, 299, 303, 307, 308, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267, and 315, preferably selected from the group consisting of positions 16, 24, 28, 45, 53, 80, 91, 105, 118, 121, 123, 141, 159, 161, 173, 177, 180, 215, 238, 241, 248, 258, 279, 282, 291, 297, 299, 303, 308 and 315 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to any of these positions in a related sequence as defined above. Preferably, one of these deletions/substitutions is at position 282 of SEQ ID NO:1 or at a position corresponding to this position in a related sequence as defined above. More preferably, this position is replaced by a cysteine. In another embodiment, one of these deletions/substitutions is at position 173 of SEQ ID NO:1 or at a position corresponding to this position in a related sequence as defined above. More preferably, this position is replaced by a cysteine. In another embodiment, one of these deletions/substitutions is at position 121 of SEQ ID NO:1 or at a position corresponding to this position in a related sequence as defined above. More preferably, this position is replaced by an arginine. In another embodiment, one of these deletions/substitutions is at position 303 of SEQ ID NO:1 or at a position corresponding to this position in a related sequence as defined above. More preferably, this position is replaced by a methionine. In another embodiment the deletions/substitutions at the remaining four and five positions, respectively, are effected at positions selected from the group consisting of positions 24, 45, 80, 118, 123, 159, 177, 215, 258, 297, 308 and 315 or at a position corresponding to any of these positions in a related sequence as defined above.
It is preferred that the following positions in SEQ ID NO:1 or the positions corresponding to any of these positions in a related sequence as defined above are at least modified by deletion or substitution:
80, 121, 159, 173, 282, 303, 308 and 315; or
24, 121, 159, 123, 173, 258, 282 and 303; or
118, 121, 159, 173, 177, 215, 282 and 303; or
24, 118, 121, 159, 173, 177, 282 and 303; or
45, 80, 121, 173, 282, 297, 303 and 308; or
45, 121, 159, 173, 177, 282, 303 and 308; or
118, 121, 159, 173, 177, 282, 297, 303 and 308; or
118, 121, 159, 173, 177, 215, 282, 297 and 303; or
80, 118, 121, 159, 173, 177, 282, 303 and 315; or
24, 45, 121, 123, 173, 282, 297, 303 and 308; or
45, 121, 159, 173, 177, 215, 258, 282 and 303.
Preferably, the substitution at position 24 is arginine, the substitution at position 45 is valine or leucine, the substitution at position 80 is glycine, the substitution at position 118 is leucine or tryptophan, the substitution at position 123 is arginine, the substitution at position 159 is leucine, the substitution at position 177 is cysteine, the substitution at position 215 is alanine, the substitution at position 258 is leucine, the substitution at position 297 is leucine and the substitution at position 308 or 315 is a serine.
Particularly preferred variants with eight or nine mutations show the following substitutions in SEQ ID NO:1:
D80G-Y121R-E159L-M173C-K282C-L303M-T308S-G315S
K24R-121R-E159L-K123R-M173C-M258L-K282C-L303M
C118L-Y121R-E159L-M173C-E177C-V215A-K282C-L303M
K24R-C118L-Y121R-E159L-M173C-E177C-K282C-L303M
E45V-D80G-Y121R-M173C-K282C-F297L-L303M-T308S
E45L-Y121R-E159L-M173C-E177C-K282C-L303M-T308S
C118L-Y121R-E159L-M173C-E177C-K2820-F297L-L303M-T308S
C118L-Y121R-E159L-M173C-E177C-V215A-K282C-F297L-L303M
D80G-C118L-Y121R-E159L-M173C-E177C-K282C-L303M-G315S
K24R-E45V-Y121R-K123R-M173C-K282C-F297L-L303M-T308S
E45V-Y121R-E159L-M173C-E177C-V215A-M258L-K282C-L303M
or the corresponding substitutions at the corresponding positions in a structurally related sequence as defined above.
In another embodiment the variant according to the invention is characterized in that it contains at least ten substitutions at positions selected from the group consisting of positions 9, 11, 16, 24, 28, 42, 43, 45, 53, 66, 77, 80, 91, 105, 116, 118, 120, 122, 123, 129, 134, 141, 159, 160, 161, 173, 177, 180, 186, 215, 238, 241, 242, 248, 251, 253, 258, 264, 279, 291, 293, 297, 299, 303, 307, 308, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267, and 315, preferably selected from the group consisting of positions 16, 24, 28, 45, 53, 91, 105, 118, 121, 141, 159, 161, 173, 177, 180, 215, 238, 241, 248, 279, 282, 291, 297, 299, 303 and 308 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to any of these positions in a related sequence as defined above. In one embodiment the deleted/substituted positions are positions 45, 118, 121, 159, 173, 215, 282, 297, 303 and 308. Preferably, the substitution at position 45 is valine, the substitution at position 118, 159 and 297 is leucine, the substitution at position 121 is arginine, the substitution at position 173 or 282 is cysteine, the substitution at position 215 is alanine, the substitution at position 303 is methionine and the substitution at position 308 is a serine. Thus, a particularly preferred variant with ten mutations shows the following substitutions in SEQ ID NO:1:
E45V-C118L-Y121R-E159L-M173C-V215A-K282C-F297L-L303M-T308S
or the corresponding substitutions at the corresponding positions in a structurally related sequence as defined above.
In another embodiment the variant according to the invention is characterized in that it contains at least eleven deletions and/or substitutions at positions selected from the group consisting of positions 9, 11, 16, 24, 28, 42, 43, 45, 53, 66, 77, 80, 91, 105, 116, 118, 120, 122, 123, 129, 134, 141, 159, 160, 161, 173, 177, 180, 186, 215, 238, 241, 242, 248, 251, 253, 258, 264, 279, 291, 293, 297, 299, 303, 307, 308, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267, and 315, preferably selected from the group consisting of positions 16, 24, 28, 53, 91, 105, 118, 121, 141, 159, 161, 173, 177, 180, 238, 241, 248, 279, 282, 291, 297, 299, 303 and 308 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to any of these positions in a related sequence as defined above. In one embodiment the deleted/substituted positions are positions 24, 118, 121, 159, 173, 177, 282, 291, 297, 303 and 308. Preferably, the substitution at position 24 or 121 is arginine, the substitution at position 118, 159 and 297 is leucine, the substitution at position 173, 177 or 282 is cysteine or valine, the substitution at position 291 is aspartate, the substitution at position 303 is methionine and the substitution at position 308 is a serine. Thus, a particularly preferred variant with eleven mutations shows the following substitutions in SEQ ID NO:1:
K24R-C118L-Y121R-E159L-M173C-E177C-K282C-E291D-F297L-L303M-T308S
or the corresponding substitutions at the corresponding positions in a structurally related sequence as defined above.
A mutant containing these 11 substitutions is also referred to as “F9” in the context of the appended Example section.
In a preferred embodiment, the variant to the invention is characterized in that it contains at least ten, preferably at least eleven deletions and/or substitutions at positions selected from the group consisting of position 24, 118, 121, 159, 173, 177, 282, 291, 297, 303 and 308 in SEQ ID NO:1 or the positions corresponding to any of these positions in a related sequence as defined above, preferably substitutions as in the variant F9, and that it contains in addition at least one further deletion/substitution at a position selected from the group consisting of positions 16, 28, 53, 91, 105, 141, 161, 180, 238, 241, 248, 279, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267, and 299 in SEQ ID NO:1 or the positions corresponding to any of these positions in a related sequence as defined above. Preferably substitutions at any of these positions are as defined herein-above.
In one embodiment the at least one further deletion/substitution is at positions 16 and 105 or at positions 16 and 141 in SEQ ID NO:1 or the positions corresponding to any of these positions in a related sequence as defined above.
In another embodiment the at least one further deletion/substitution is at positions 16, 141 and 241 in SEQ ID NO:1 or the positions corresponding to any of these positions in a related sequence as defined above.
In another embodiment the at least one further deletion/substitution is at positions 16, 141, 241 and 248 in SEQ ID NO:1 or the positions corresponding to any of these positions in a related sequence as defined above.
In another embodiment the at least one further deletion/substitution is at positions 141, 241 and 248 in SEQ ID NO:1 or the positions corresponding to any of these positions in a related sequence as defined above.
In another embodiment the at least one further deletion/substitution is at positions 16, 91, 141, 241 and 248 in SEQ ID NO:1 or the positions corresponding to any of these positions in a related sequence as defined above.
In another embodiment the at least one further deletion/substitution is at positions 16, 91, 141, 241, 248 and 299 in SEQ ID NO:1 or the positions corresponding to any of these positions in a related sequence as defined above.
In another embodiment the at least one further deletion/substitution is at positions 16, 91, 141, 241, 248, 299 and 28 in SEQ ID NO:1 or the positions corresponding to any of these positions in a related sequence as defined above.
In another embodiment the at least one further deletion/substitution is at positions 16, 91, 141, 241, 248, 299 and 28 in SEQ ID NO:1 or the positions corresponding to any of these positions in a related sequence as defined above.
In another embodiment the at least one further deletion/substitution is at positions 16, 141, 241, 248 and 28 in SEQ ID NO:1 or the positions corresponding to any of these positions in a related sequence as defined above.
In another embodiment the at least one further deletion/substitution is at positions 16, 141, 241, 248, 28 and 180 in SEQ ID NO:1 or the positions corresponding to any of these positions in a related sequence as defined above.
In another embodiment the at least one further deletion/substitution is at positions 16, 141, 241, 248, 28, 53 and 180. in SEQ ID NO:1 or the positions corresponding to any of these positions in a related sequence as defined above
In another embodiment the at least one further deletion/substitution is at positions 16, 141, 241, 248, 28, 180 and 238 in SEQ ID NO:1 or the positions corresponding to any of these positions in a related sequence as defined above.
In another embodiment the at least one further deletion/substitution is at positions 16, 141, 241, 248, 28, 180 and 279 in SEQ ID NO:1 or the positions corresponding to any of these positions in a related sequence as defined above.
In another embodiment the at least one further deletion/substitution is at positions 16, 141, 241, 248, 28, 180 and 161 in SEQ ID NO:1 or the positions corresponding to any of these positions in a related sequence as defined above.
Particularly preferred variants show at least ten, preferably at least eleven deletions and/or substitutions at positions selected from the group consisting of position 24, 118, 121, 159, 173, 177, 282, 291, 297, 303 and 308, preferably substitutions as in the variant F9, in SEQ ID NO:1 or the corresponding substitutions at the corresponding positions in a structurally related sequence as defined above, and show in addition the following substitution(s) in SEQ ID NO:1:
S141P
S141T
S105A
Q299K
I16L
S248T
K241M
116L-S105A
S141P-K241M-S248T
I16L-R91H-S141P-K241M-S248T
I16L-S141P
I16L-R91H-S141P-K241M-S248T Q299K
I16L-S141P-K241M
I16L-S141 P-K241M-S248T
I16L-R91H-S141P-K241M-S248T-Q299K-M28K
I16L-R91H-S141P-K241M-S248T-Q299K-M28A
I16L-R91H-S141P-K241M-S248T-Q299K-K180P
I16L-S141P-K241I-S248T
I16L-S141P-K241I-S248I-M28K
I16L-S141P-K241I-S248T-M28K-K180P
I16L-S141P-K241I-S248T-M28K-T53V-K180P
I16L-S141P-K241I-S248T-M28K-K180P-A238K
I16L-S141P-K241I-S248T-M28K-K180P-A238R
I16L-S141P-K241I-S248T-M28K-K180P-C282V
I16L-S141P-K241I-S248T-M28K-K180P-P279A
I16L-S141P-K241I-S248T-M28K-K180P-Y161R
D2H
M42L-D87E-S139C-R186L-K231Q
E164Q-R186V-D252E
D87E-S139C-R186L-K231Q
R186V-Q267R
S139C-R186I
L111M-F122Y-R186L
M75I-R186V
S139A-S141C
K179K-R186V
R186V
A57S-A58T-K77R-R186V
L111M-R186L
R186L
A31S-R186V
S139A-S141G
M75I-R186L-S308T
R186L-S308T
R186I
L111M-R186V-S308T
R186V-D221E
L111M-R186V
M1L-L111M-R186V-S308T
R186N
R24S-G86Q-R186I
I16L-S141P-K241I-K180P-E227K
I16L-S141P-K241I-K180P-D291E-M303L
S141P-K241I-S248T-K180P-R24K
I16L-S141P-K241I-S248T-L297F
I16L-S141P-K241I-S248T-M28K-K180P-L188C-L297F-A246E
I16L-S141P-K241I-S248T-M28K-K180P-L188C-L297F-T242E
I16L-S141P-K241I-S248T-M28K-K180P-L188C-L297F-Y255E
I16L-S141P-K241I-S248T-M28K-K180P-L188C-L297F-T198D
I16L-S141P-K241I-S248T-M28K-K180P-L188C-L297F-K23L
I16L-S141P-K241I-S248T-M28K-K180P-L188C-L297F-K179L
I16L-S141P-K241I-S248T-M28K-K180P-L188C-L297F-K231L
I16L-S141P-K241I-S248T-M28K-K180P-L188C-L297F-P182E
I16L-S141P-K241I-S248T-M28K-K180P-L188C-L297F-A238E
I16L-S141P-K241I-S248T-M28K-K180P-L188C-L297F-K208L
I16L-S141P-K241I-S248T-M28K-K180P-L188C-L297F-R204L
I16L-S141P-K241I-S248T-M28K-K180P-L188C-L297F-R24L
I16L-S141P-K241I-S248T-M28K-K180P-L188C-L297F
I16L-S141P-K241I-K180P-D291E
I16L-S141P-K241I-S248T-K180P-Q267R-R24K-L118C-L159E
I16L-S141P-K241I-S248T-M28K-K180P-L188C-L297F-S142A
I16L-S141P-K241I-S248T-M28K-K180P-L159E-D291E
I16L-S141P-K241I-S248T-K180P-G166S-R24K
I16L-S141P-K241I-S248T-K180P-R24K
I16L-S141P-K241I-S248T-M28K-K180P-L188C-L297F-Q205H
or the corresponding substitution(s) at the corresponding positions in a structurally related sequence as defined above.
The present inventors also constructed a computer 3D model of the S. mitis enzyme represented by the amino acid sequence shown in SEQ ID NO: 1 (see
It has been demonstrated that the mutations identified in the present invention have a direct effect on the rate of production of isobutene from 3-phosphonoxyisovalerate by the enzyme; however it is possible that these mutations confer to the enzyme other properties that have not been fully identified yet. For example, several substitutions replace wild type residues with cysteine residues (Y11, M173 and K282). In the improved mutants residues 2B4, M173C and K282C are close enough in the 3D modeled structure to suggest that the creation of a disulphide bridge is possible. Such a bond could stabilize the overall structure of the enzyme. To support this, some preliminary data indicates that some mutants are more resistant to thermal denaturation than the wild type enzyme.
Similarly these mutations may have an effect on the stability to pH, optimal temperature and the oligomeric state of the proteins. Preliminary data collected on the effect of pH on isobutene production indicate that some mutant variants are more active at acidic pH compared to the wild type enzyme.
One group of the identified positions are located in the N-terminal domain of the protein, in particular positions 1, 2, 9, 11, 42, 43, 45, 57, 58, 75, 77, 80, 86, 87, 91, 116, 118, 120, 121, 122, 123, 129 and 134.
An analysis revealed that residues 77, 80, 116, 118, 120, 121, 122, 123, 129 and 134 are located on the alpha helices No 1, 2 and 3 in the N-terminal domain and are situated most distally from the substrate binding pocket. Residues 9, 11, 42, 43, 45 and 91 are located in close proximity to these alpha helices and in particular, they are located in the beta sheet on beta strands 1 and 4 that are directly adjacent to each other (see
Another group of the identified positions, in particular positions 159, 160, 164, 166, 173, 177, 179, 198, 204, 205, 208, 221, 227, 231, 238, 241, 242, 246, 248, 251, 252, 253, 255, 258, 264, 267, 282, 291, 293, 297, 299, 303, 307 and 308, are located in the C-terminal domain and most of these positions are located in alpha helices and in beta strands, in particular in alpha helices 8 and 9 and in beta strands 9 to 12. It is assumed that mutations at positions 238, 241, 242, 248, 253, 258, 264, 291, 293, 297, 299, 303, 307 and 308 (i.e. in the alpha helix no 9 and in beta-strand no 12) might create a more stable conformation that stabilizes the whole structure. Thus, in one preferred embodiment, the present invention relates to a variant of a mevalonate diphosphate decarboxylase having an amino acid sequence as shown in SEQ ID NO:1 or an amino acid sequence having at least 40%, 50%, 60% or 90% sequence identity to SEQ ID NO:1, wherein one or more amino acid residues at a position selected from the group consisting of positions 159, 160, 173, 177, 238, 241, 242, 248, 251, 253, 258, 264, 282, 291, 293, 297, 299, 303, 307 and 308, more preferably selected from the group consisting of positions 238, 241, 242, 248, 253, 258, 264, 291, 293, 297, 299, 303, 307 and 308 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to any of these positions in a related sequence, are substituted with another amino acid residue and wherein said mevalonate diphosphate decarboxylase has improved activity in converting 3-phosphonoxyisovalerate into isobutene.
In one particular embodiment it is preferred that one or more amino acid residues at a position selected from the group consisting of positions 293, 297, 299, 303, 307 and 308 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to any of these positions in a related sequence, are substituted with another amino acid residue. These residues are located in the most C-terminal part of the enzyme (in alpha helix 9 and beta strand 12) and might create a more stable confirmation that stabilizes the whole structure.
A third group of identified positions is located in or close to the binding pocket of the enzyme. This group encompasses positions 16, 23, 24, 28, 31, 111, 139, 141, 142, 182, 186, 188, 279 and 282. The MDP binding pocket is delimited by regions 13-22 and 97-107 that form the ATP binding site and includes in particular the P-loop (P99-S107). Another side consists of the region 274-280 where the catalytic base D276 is located and the region 136-146 which includes R144 that drives the final decarboxylation step of the substrate. Position S105 lies within the phosphate binding loop (consensus GHMP kinase P-Xaa-GLSASAA→PTAAGLSSSSS in S. mitis). Interestingly the mutation that improves the production of IBN is a substitution to Ala that is closer to the consensus P-binding loop sequence compared to the wild type S. mitis MDP P-loop sequence. S141 is closely located to the R144 residue and its hydroxyl group interacts with the natural enzyme substrate and is thus believed to be important to determine the enzyme specificity. Substitution of S141 with threonine which has a bulkier side chain could potentially facilitate the interaction of the enzyme with the smaller sized mono-phosphorylated substrate PIV. Substitution to proline would drastically alter the structure of the region and might better accommodate this non-natural substrate PIV. I16, K24R and M28 are neighboring K22 which interacts with the phosphate groups of the natural substrate; altering this residues could affect the structure of this loop and bring K22 closer to the substrate. The best performing mutants harbor the K24R which alters the length of the side chains while increasing the positively charged environment of the binding pocket and the mutation M28K which adds a positive charge. P279 and K282 are close to the catalytic base D276 and R186 is neighboring S185 which interacts with the MVAPP substrate.
Thus, in one preferred embodiment, the present invention relates to a variant of a mevalonate diphosphate decarboxylase having an amino acid sequence as shown in SEQ ID NO:1 or an amino acid sequence having at least 40%, 50%, 60% or 90% sequence identity to SEQ ID NO:1, wherein one or more amino acid residues at a position selected from the group consisting of positions 16, 24, 28, 141, 186, 279 and 282 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to any of these positions in a related sequence, are substituted with another amino acid residue and wherein said mevalonate diphosphate decarboxylase has improved activity in converting 3-phosphonoxyisovalerate into isobutene.
The present invention also relates to a method for providing a variant of a mevalonate diphosphate decarboxylase wherein said variant shows an improved activity of converting 3-phosphonoxyisovalerate into isobutene said method comprising the step of effecting one or more changes in the sequence of the mevalonate diphosphate decarboxylase wherein said change(s) is/are effected at one or more amino acid positions selected from the group consisting of the amino acid positions corresponding to positions 282, 9, 11, 16, 24, 28, 42, 43, 45, 53, 66, 77, 80, 91, 105, 116, 118, 120, 121, 122, 123, 129, 134, 141, 159, 160, 161, 173, 177, 180, 186, 215, 238, 241, 242, 248, 251, 253, 258, 264, 279, 291, 293, 297, 299, 303, 307, 308, 1, 2, 23, 31, 57, 58, 75, 86, 87, 111, 139, 142, 164, 166, 179, 182, 188, 198, 204, 205, 208, 221, 227, 231, 246, 252, 255, 267, and 315 in the amino acid sequence shown in SEQ ID NO:1. “Corresponding to” means corresponding to any of these positions in a related sequence.
As regards the preferred embodiments of a mevalonate diphosphate decarboxylase to be mutated according to such a method, the same applies as has been set forth herein-above.
In one preferred embodiment the mevalonate diphosphate decarboxylase from which the variant is derived is a mevalonate diphosphate decarboxylase which shows the amino acid sequence as shown in SEQ ID NO:1 or an amino acid sequence having at least 40%, 50%, 60% or 90% sequence identity to SEQ ID NO:1 or any of the preferred degrees of sequence identity as specified herein above.
Moreover, as regards preferred embodiments of the degree of improvement in activity and the changes to be effected, the same applies as described herein above.
The MDP decarboxylase of the present invention can be fused to a homologous or heterologous polypeptide or protein, an enzyme, a substrate or a tag to form a fusion protein. Fusion proteins in accordance with the present invention will have the same improved activity as the MDP decarboxylases of the present invention. Polypeptides, enzymes, substrates or tags that can be added to another protein are known in the art. They may be useful for purifying or detecting the proteins of the invention. For instance, tags that can be used for detection and/or purification are e.g. FLAG-tag, His6-tag or a Strep-tag. Tags for improving solubility or stability of the enzyme are MBP or ATS. Alternatively, the protein of the invention can be fused to an enzyme e.g. luciferase, for the detection or localisation of said protein. 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 is also conceivable that the polypeptide, enzyme, substrate or tag is removed from the protein of the invention after e.g. purification.
Fusion proteins can typically be made by either recombinant nucleic acid methods or by synthetic polypeptide methods known in art.
The present invention further relates to a nucleic acid molecule encoding the enzymes, more preferably the MDP decarboxylase variants of the present invention and to a vector comprising said nucleic acid molecules. Vectors that can be used in accordance with 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 vectors. These expression control sequences may be suited to ensure transcription and synthesis of a translatable RNA in bacteria or fungi. Expression control sequences can for instance be promoters. Promoters for use in connection with the nucleic acid molecules of the present invention may be homologous or heterologous with regard to its origin and/or with regard to the gene to be expressed. Suitable promoters are for instance promoters which lend themselves to constitutive expression. However, promoters which are only activated at a point in time determined by external influences can also be used. Artificial and/or chemically inducible promoters may be used in this context.
Preferably, the vector of the present invention is an expression vector. Expression vectors have been widely described in the literature. As a rule, they contain not only a selection marker gene and a replication-origin ensuring replication in the host selected, but also a bacterial or viral promoter, and in most cases a termination signal for transcription. Between the promoter and the termination signal there is in general at least one restriction site or a polylinker which enables the insertion of a coding DNA sequence. The DNA sequence naturally controlling the transcription of the corresponding gene can be used as the promoter sequence, if 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 ensuring constitutive expression of the gene and inducible promoters which permit a deliberate control of the expression of the gene. Bacterial and viral promoter sequences possessing these properties are described in detail in the literature. Regulatory sequences for the expression in microorganisms (for instance E. coli, S. cerevisiae) are sufficiently described in the literature. Promoters permitting a particularly high expression of a downstream sequence are for instance 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), Ip1, rac (Boros et al., Gene 42 (1986), 97-100). Inducible promoters are preferably used for the synthesis of polypeptides. These promoters often lead to higher polypeptide yields than do constitutive promoters. In order to obtain an optimum amount of polypeptide, a two-stage process is often used. First, the host cells are cultured under optimum conditions up to a relatively high cell density. In the second step, transcription is induced depending on the type of promoter used. In this regard, a tac promoter is particularly suitable which can be induced by lactose or IPTG (=isopropyl-1-D-thiogalactopyranoside) (deBoer et al., Proc. Natl. Acad. Sci. USA 80 (1983), 21-25). Termination signals for transcription are also described in the literature.
Preferably, the nucleic acid molecules according to the invention are non-naturally occurring nucleic acid molecules, i.e., they are molecules which do not occur in nature. Such non-naturally occurring molecules differ significantly from naturally occurring nucleic acid molecules due to a difference, e.g., in structure. For example, such a non-naturally occurring nucleic acid molecule may encode a non-naturally occurring enzyme as described herein-above. Also a vector according to the present invention is preferably a non-naturally occurring vector, e.g., due to the presence of a non-naturally occurring nucleic acid molecule or due to the combination of elements which do not occur in such a combination in nature.
In addition, the present invention relates to a host cell comprising the vector of the present invention.
In a preferred embodiment, the host cell according to the presenting invention is a microorganism, in particular a bacterium or a fungus. In a more preferred embodiment, the host cell of the present invention is 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.
The transformation of the host cell with a vector according to the invention can be carried out by standard methods, as for instance described in Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, N.Y., USA; Methods in Yeast Genetics, A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, 1990. The host cell is cultured in nutrient media meeting the requirements of the particular host cell used, in particular in respect of the pH value, temperature, salt concentration, aeration, antibiotics, vitamins, trace elements etc. Such a host cell according to the present invention is preferably a non-naturally occurring host cell, i.e., a host cell which does not occur in nature. Such a non-naturally occurring host cell differs from a naturally occurring cell due to a modification with a nucleic acid molecule or vector as described herein-above.
The present invention also relates to the use of the MDP decarboxylase of the present invention or a host cell comprising said MDP decarboxylase for the conversion of 3-hydroxyisovalerate or of 3-phosphonoxyisovalerate into isobutene.
In addition, the present invention relates to a method for producing isobutene from 3-hydroxyisovalerate or from 3-phosphonoxyisovalerate comprising the steps of: Culturing the host cell of the present invention in a suitable medium and recovering said isobutene.
It has been described previously that the MDP decarboxylase is able to catalyze the conversion of mevalonate into isoprenol via the intermediate mevalonate-3-phosphate (see WO 2011/076261). The inventors could show that also the MDP decarboxylase variants according to the present invention are able to catalyze this conversion and in particular the conversion of mevalonate-3-phosphate into isoprenol. Therefore, the present invention also relates to the use of an MDP decarboxylase variant according to the present invention and as described herein above or of a microorganism expressing such a variant for the conversion of mevalonate or mevalonate-3-phosphate into isoprenol.
In addition, the present invention relates to a method for producing isoprenol from mevalonate or from mevalonate-3-phosphate comprising the steps of: Culturing the host cell of the present invention in a suitable medium and recovering said isoprenol.
It has also been described that the MDP decarboxylase is able to catalyze the conversion of 3-hydroxypent-4-enoate into 1,3-butadiene (see PCT/EP2012/075921) via the intermediate 3-phosphonoxypent-4-enoate. The inventors could show that also the MDP decarboxylase variants according to the present invention are able to catalyze this conversion, in particular the conversion of 3-phosphonoxypent-4-enoate into 1,3-butadiene. Therefore, the present invention also relates to the use of an MDP decarboxylase variant according to the present invention and as described herein above or of a microorganism expressing such a variant for the conversion of 3-hydroxypent-4-enoate or of 3-phosphonoxypent-4-enoate into 1,3-butadiene.
In addition, the present invention relates to a method for producing 1,3-butadiene from 3-hydroxypent-4-enoate or from 3-phosphonoxypent-4-enoate comprising the steps of: Culturing the host cell of the present invention in a suitable medium and recovering said 1,3-butadiene.
In the above described methods, the microorganisms are cultivated under suitable culture conditions allowing the occurrence of the enzymatic reaction of the MDP decarboxylases of the present invention. The specific culture conditions depend on the specific microorganism employed but are well known to the person skilled in the art. The culture conditions are generally chosen in such a manner that they allow the expression of the genes encoding the MDP decarboxylases of the present invention. Various methods are known to the person skilled in the art in order to improve and fine-tune the expression of certain genes at certain stages of the culture such as induction of gene expression by chemical inducers or by a temperature shift.
In another embodiment, the above described methods of the invention comprise the step of providing the organism, preferably the microorganism carrying the respective enzyme activity or activities in the form of a (cell) culture, preferably in the form of a liquid cell culture, a subsequent step of cultivating the organism, preferably the microorganism in a fermenter (often also referred to a bioreactor) under suitable conditions allowing the expression of the respective enzyme and further comprising the step of effecting an enzymatic conversion of a method of the invention as described herein above. Suitable fermenter or bioreactor devices and fermentation conditions are known to the person skilled in the art. A bioreactor or a fermenter refers to any manufactured or engineered device or system known in the art that supports a biologically active environment. Thus, a bioreactor or a fermenter may be a vessel in which a chemical/biochemical process like the method of the present invention is carried out which involves organisms, preferably microorganisms and/or biochemically active substances, i.e., the enzyme(s) described above derived from such organisms or organisms harboring the above described enzyme(s). In a bioreactor or a fermenter, this process can either be aerobic or anaerobic. These bioreactors are commonly cylindrical, and may range in size from litres to hundreds of cubic meters, and are often made of stainless steel. In this respect, without being bound by theory, the fermenter or bioreactor may be designed in a way that it is suitable to cultivate the organisms, preferably microorganisms, in, e.g., a batch-culture, feed-batch-culture, perfusion culture or chemostate-culture, all of which are generally known in the art.
The culture medium can be any culture medium suitable for cultivating the respective organism or microorganism.
The method according to the invention furthermore comprises the step of collecting gaseous products, i.e. isobutene, degassing out of the reaction, i.e. recovering the products which degas, e.g., out of the culture. Thus in a preferred embodiment, the method is carried out in the presence of a system for collecting isobutene under gaseous form during the reaction.
As a matter of fact, short alkenes such as isobutene adopt the gaseous state at room temperature and atmospheric pressure. The method according to the invention therefore does not require extraction of the product from the liquid culture medium, a step which is always very costly when performed at industrial scale. The evacuation and storage of the gaseous hydrocarbons and their possible subsequent physical separation and chemical conversion can be performed according to any method known to one of skill in the art.
The present invention is further described by reference to the following non-limiting figures and examples.
Materials and Methods
Methods Used to Construct and Select Mutations
a. Directed Evolution Strategy
The enzyme Streptococcus mitis MDP decarboxylase is capable of catalyzing, amongst other reactions, the reaction of phosphorylation of 3-hydroxyisovalerate into 3-phosphonoxyisovalerate and the reaction of decarboxylation of 3-phosphonoxyisovalerate into isobutene. A directed evolution approach was used in order to specifically improve the rate of conversion of 3-phosphonoxyisovalerate into isobutene by S. mitis MDP decarboxylase. This approach consisted in (1) the generation of an initial collection of single point mutants for S. mitis MDP decarboxylase, (2) the design of assay systems to test the activity of these enzyme variants, (3) the use of the activity assays to screen the collection of mutants in order to identify mutants with improved activity compared to the activity of the wild type S. mitis MDP decarboxylase, (4) additional rounds of evolution (library construction and screening) using as starting material the best mutants identified during the previous round of evolution (see
b. Construction of Streptococcus mitis MDP Decarboxylase Enzyme Mutants
The polynucleotide sequences coding for the different mutants identified during the evolution of the Streptococcus mitis MDP decarboxylase enzyme were generated using a range of standard molecular biology techniques. All these techniques used a codon-optimised polynucleotide sequence for expression in Escherichia coli as template (see SEQ ID NO: 4;). The sequence optimization has been done by Geneart using their GeneOptimizer software.
Different PCR-based techniques known in the art were used for the construction of single point mutants. For the generation of enzyme variants bearing multiple mutations (at least two mutations), either PCR-based techniques or other methods known in the art were used to introduce these mutations.
Following mutagenesis, the mutated polynucleotide sequence was inserted into an expression vector (used for recombinant protein production in E. coli and screening) either using standard ligase-based subcloning techniques, whole plasmid extension by PCR or ligase-independent cloning techniques (LIC; Life Technology Gateway® recombinant technology),
c. Selection of the Enzyme Mutants with Increased Activity
Description of the Screening Method (see
The DNA library or the collection of mutants inserted into the expression vector was transformed into the commercially available recombinant protein expression E. coli strain BL21 DE3. The DNA library transformation reaction was plated out onto LB Ampicillin plates and isolated clones were used to inoculate a starter culture. When transforming a collection of mutants, each clone was individually transformed, plated out and an isolated clone used to inoculate a starter culture; or the transformation mixture for each individual clone was directly used to inoculate a starter culture. The protein expression was carried out using an auto-induction medium (ZYM-5250) as described in Studier F. W (Protein Expr. Purif. 41 (2005), 207-234) with an initial cell growth phase for 6 hours at 37° C. and an induction stage overnight at 28° C. After the transformation, all subsequent steps were carried out in microplates or deepwell plates. The cell cultures were centrifuged and the pellets were stored for at least 1 hour at −80° C. The cell pellets were then resuspended in a small volume of buffer and lysed by sonication before being subjected to centrifugation for the removal of cell debris.
Screening for Variants with an Increased Activity of Converting 3-phosphonoxyisovalerate into Isobutene
To test the activity of the mutant enzyme to catalyze the conversion of 3-phosphonoxyisovalerate into isobutene, a reaction mix was prepared in glass GC vials by mixing cell lysates (supernatant), 3-phosphonoxyisovalerate substrate in 50 mM Tris pH 7 final concentration. 3-phosphonoxyisovalerate has been prepared enzymatically by incubating 3-hydroxyisovalerate, purified Thermoplasma acidophilum MDP decarboxylase and cofactors (ATP, MgCl2, KCl) at 37° C. for 24 hours. The reaction mixture was incubated for varying length of time at 37° C. and the gas phase was injected in a gas chromatograph alongside the appropriate references (isobutene for calibration, wild type enzymes, negative controls . . . ). Following analysis of the GC chromatograms, mutants showing an activity increased by at least 20% were selected and subjected to a second round of screening which followed the same conditions as the primary screen.
The final step in the selection process included the production and purification of the top hits, i.e. enzyme variants with the highest improved activity validated by the primary and secondary screens and their activity was tested with different substrate concentrations. Confirmed hits were further characterized and the kcat and Km for the reaction was calculated.
Modification of the Screening Process:
As mentioned before, the screening protocol was regularly modified as new improved mutants were identified. The main modification of the protocols covers the following points:
A collection of 2632 single point mutants of S. mitis MDP decarboxylase was prepared using standardmolecular biology techniques. Saturation mutagenesis aimed at substituting systematically all the 317 amino acids of S. mitis MDP decarboxylase with the 19 non wild type amino acids. The average number of substitutions per position was 8 out of the 19 possible. The coding sequence for each individual mutant was sub-cloned in an expression vector to allow the production of an N-terminal 6-His-tagged recombinant mutant enzyme in E. coli.
The recombinant protein expression E. coli strain BL21 DE3 was transformed with the expression vectors encoding the mutant enzymes, the empty expression vector (negative control) and the expression vector encoding the wild type enzyme (positive control). To speed up the process, the transformation was carried out in a 96-well plate. Briefly, 2 μl of DNA plasmid miniprep of each mutant and each control was transferred per well into a 96-well 0.2 ml PCR reaction plate before addition of 40 μl/well of chemically competent BL21 DE3 E. coli cells. The plate was incubated on ice for 15 minutes before carrying out a heatshock for 1 minute at 42° C. in the AB 2720 Thermal cycler. The plate was then immediately placed on ice and cooled for 1 minute before 1 ml of sterile Luria-Bertani medium was added (10 g/l Tryptone, 5 g/l yeast extract, 10 g/l NaCl, pH 7). The plate was sealed using gas-permeable adhesive film and incubated for 45 minutes at 37° C. at 200 rpm in an Infors Minitron orbital shaker. 50 μl of the transformation mixture was then used to inoculate 0.5 ml of Luria-Bertani medium supplemented with 100 μg/ml ampicillin placed in a 96 deep-well plate. These plates were sealed and incubated overnight at 37° C. at 200 rpm. Bacterial stocks were prepared in a 96-well plate by mixing 100 μl of the overnight starter cultures with 35 μl sterile 50% glycerol and stored at −80° C. until further use.
For the production of the recombinant mutant enzyme, 1 ml of sterile autoinduction medium (Studier F. W, Protein Expr. Purif. 41 (2005), 207-234) supplemented with the appropriate antibiotic, was distributed in the wells of a 96-deep well plate and said wells were inoculated with 10 μl of thawed glycerol stocks. Each plate contained approximately 70 to 80 distinct mutant enzymes, 8 negative controls (empty expression vector) and 8 wild type enzyme clones for use as a reference. Plates were incubated for 6 hours at 37° C. at 1000 rpm in a Heidolph Titramax orbital shaker followed by a further overnight incubation at 28° C. at 1000 rpm. Bacterial cells were pelleted by centrifugation for 20 minutes at 3200×g at 4° C. Cell pellets were stored at −80° C.
Pellets were thawed on ice for 5 to 10 minutes and were resuspended in 250 μl of resuspension buffer (50 mM Tris-Cl pH7, 20 mM KCl, 10 mM MgCl2, 10% glucose, 1 μl/ml Merck-Novagen Lysonase). Cell suspensions were incubated at room temperature for 15 minutes and on ice for 30 minutes. Bacterial cells lysis was carried out by sonicating these cell suspensions for 4 pulses of 5 minutes in an Advatange Lab ultrasonic water bath filled with ice and water (5 minutes of rest on ice between pulses). Cell lysates were then centrifuged for 20 minutes at 10° C. at 3200×g to pellet cell debris and 240 μl of the supernatants were transferred into a fresh plate. The enzymatic reaction was set up in Agilent 2 ml glass vials by mixing 200 μl of the supernatant with 300 μl of 3-phosphonoxyisovalerate substrate. The vials were hermetically sealed using crimp caps (PTFE-silicon-PTFE coated), incubated in a waterbath for 24 hours at 37° C. and stored at −20° C. prior to analysis by gas chromatography. To prepare 3-phosphonoxyisovalerate substrate, 0.063 mg/ml of purified Thermophilus acidophilum MDP decarboxylase was mixed with 50 mM hydroxyisovalerate, 40 mM Adenosine tri-phosphate in a 50 mM Tris-Cl pH7, 20 mM KCl, 20 mM MgCl2 and incubated for 24 hours at 45° C. This enzymatically prepared 3-phosphonoxyisovalerate substrate was aliquoted and stored at −20° C. until further use.
The isobutene produced by the enzymatic reaction in the presence of S. mitis MDP decarboxylase was quantified by gas chromatography. The vials were thawed quickly at 30° C. for 30 minutes and placed on an automated sampler mounted onto a Varian GC-430 system equipped with a Varian CP SilicaPlot column (30 m×0.32 mm), one injection port and one Flame Ionization detector (FID). The sampler was set up to inject 100 μl of headspace gas. For the GC analysis method used to detect isobutene, oven temperature was set to 185° C., injector port temperature was set to 150° C. with a split ratio of 4:1 and the FID detector to 250° C. The GC uses nitrogen as carrier gas (constant flow of 1.5 ml/min) and a mixture of air (air flow 28 ml/min) and hydrogen (300 ml/min) for the FID detection system. The duration of the analysis was approx. 3 minutes per sample and under these conditions isobutene was observed to elute at 2.5 min. A sample of commercially bought pure isobutene was injected prior to the start of the analysis to calibrate the GC system and to determine the retention time for isobutene. Following the analysis, chromatograms were processed using Galaxy software; the area under the peak was integrated for each mutant and compared to that of the wild type enzyme. Mutants showing an increase of at least 10-15% of isobutene production compared to the amount produced by the wild type enzyme were tested for a second time according to the protocol described above in order to eliminate false positives. Finally all mutant enzymes that have been selected through these two rounds of screening were tested once again using normalized quantities of purified protein in the assay. Briefly, the bacterial glycerol stocks of the selected mutant enzymes were used to inoculate 7 ml of LB-Amp. 2 ml of this starter culture was used to inoculate 200 ml of autoinduction medium and the plasmid DNA was extracted from the 5 ml of culture leftover. The plasmid DNA was sequenced to confirm the presence and the type of a mutation. The bacterial expression for the production of the mutant enzymes was carried out as previously described and the N-terminal 6 His-tagged mutant enzymes were purified from the pellet using Macherey-Nagel Protino purification kit according the users' manual. The activity was determined by mixing in a 2 ml GC vial: 500 μg of purified enzyme, 300 μl of 3-phosphonoxyisovalerate substrate and the volume was adjusted to 500 μl with 50 mM Tris-Cl pH7. The reaction was incubated for 24 hours at 37° C. and stopped by freezing the samples at −20° C. The quantity of isobutene produced was determined by GC analysis.
This screening procedure led to the identification of 71 mutations which confer S. mitis MDP decarboxylase an increased activity of isobutene production. Table 4 lists the position and type of substitutions that have been identified.
A collection of S. mitts MDP decarboxylase variants has been created by combining a selection of single point mutations which confer high increase in activity (increase of at least 50% compared with the activity of the wild type enzyme) using PCR-based techniques. Additional diversity was also created by random mutagenesis. The quantity of isobutene produced by these variants was determined in an enzymatic assay whereby 500 μg of purified enzyme was mixed to 300 μl of 3-phosphonoisovalerate substrate in 50 mM Tris-Cl pH7 buffer. Following a 24-hour incubation period at 37° C., the reaction was stopped by freezing the samples at −20° C. and the quantity of isobutene produced was determined by GC analysis. For the GC headspace assay, 100 μl of the headspace gas was injected in the injector port of a Varian GC-430 system equipped with a Varian CP SilicaPlot column (30 m×0.32 mm) and a FID. The GC analysis method used to detect isobutene is characterized by an oven temperature at 185° C., injector port temperature at 150° C. with a split ratio of 1:10 and the FID detector temperature at 250° C. Nitrogen was used as carrier gas (constant flow of 1.5 ml/min) and a mixture of air (air flow 28 ml/min) and hydrogen (300 ml/min) were used to supply the FID detection system.
A number of variants showing mutations of up to 11 positions have been identified which show an increased activity in the activity assay. The different variants are shown in the following Tables.
Two variants named 2B4 (SEQ ID NO:2) and F9 (SEQ ID NO:3) as having high increase in activity in the assay were selected for further characterization. The 2B4 and F9 protein sequence contain 6 and 11 mutations respectively compared to the wild type enzyme (see Table 15). F9 carries two novel mutations, K24R and E291D that were not identified in the original single point mutations screen.
Michaelis Menten kcat and Km steady-state kinetics constants were determined for these two variants as follows: a series of enzymatic reactions were set up in GC vials with 200 μg of purified 2B4, F9 or the wild type enzyme, a range of 0 to 320 mM of chemically synthesized 3-phosphonooxyisovalerate, 5 mM ATP, 20 mM KCl, 10 mM MgCl2 and 50 mM Tris-Cl pH 7.5. The vials were sealed and incubated for 15 hours at 37° C. before analysing the isobutene produced by GC as previously described. Previous experiments had determined that the rate of isobutene production in this experimental set up was constant in the 20 first hours of the enzymatic reaction and thus the rate of isobutene produced per hour as determined after 15-hour incubation is equal to the initial rate of isobutene production at the beginning of the reaction. In order to quantify the absolute amount of isobutene produced by the reaction, the GC was calibrated using a range of concentration of pure isobutene (0 to 10,000 ppm). The calibration table was found to be linear in this range of isobutene concentration. The production rate of isobutene (mole of isobutene /mole enzyme/sec) were plotted as a function of the concentration of 3-phosphonooxyisovalerate and the curve was fitted using Michealis Menten equation (V=(Vmax*(substrate))/(Km+(substrate)) (
Additional MVD variants with a further enhanced activity in converting 3-phosphonoxyisovalerate into isobutene were identified through successive rounds of mutagenesis, recombination of point mutations and in vitro and/or in vivo screening assay. The list of these MVD variants is presented in the following Table 17.
The fold increase is the ratio of MDP decarboxylase variant activity over wild type MDP decarboxylase activity. The fold increase is determined for one substrate concentration (1 or 2 mM PIV for in vitro assay and 500 mM acetone for in vivo assay). The quantity of the enzyme is not normalised but these MVD variants are expressed in similar amounts as observed on SDS-PAGE analysis of cell lysates.
The template for mutagenesis was the variant named “F9” which corresponds to the S. mitis MDP decarboxylase of SEQ ID NO:1 with the following mutations: K24RC118LY121RE159LM173CE177CK282CE291DF297LL303MT308S (see SEQ ID NO: 3). The in vitro screening assay used was the one described herein-above section c of Materials and Methods, preferably an in vitro assay involving the use of cell lysate.
For the in vivo testing a further in vivo assay was developed. This assay is based on the use of a bacterial strain transformed with an expression vector that contains the coding sequences and lead to the production of the 3 enzymes involved in the metabolic pathway converting acetone to isobutene; namely for the production of 3-hydroxyisovalerate (HIV) the M. musculus HMG-CoA synthase (referred to in the following as HIV synthase) was used; for the phosphorylation of 3-hydroxyisovalerate into 3-phosphonoxyisovalerate (PIV) the T. acidophilum MDP decarboxylase was used (referred to in the following as HIV phosphorylase) and for the conversion of PIV into isobutene (IBN) by decarboxylation the S. mitis MDP decarboxylase variant to be tested was used (referred to in the following as PIV decarboxylase). This strain is first cultured, overnight in a shaking incubator at 30° C./1000 rpm, in 1 ml of auto-induction culture medium to produce the three types of recombinant enzymes. The cell pellet containing these three overexpressed recombinant enzymes is then resuspended in 500 μl of minimum medium supplemented with 500 mM acetone and incubated for a further 16 hours in a shaking incubator set at 37° C./1000 rpm. During this second step, HIV synthase catalyses the condensation of acetone (from the medium) with the cellular acetyl CoA into HIV which is then sequentially converted into PIV and IBN by the HIV phosphorylase and the PIV decarboxylase using the cellular ATP. The IBN produced is then quantified by gas chromatography using the same method as the in vitro screening assay described in section c of Materials and Methods.
The main advantages of this assay are as follows: (1) the production of IBN occurs within the cell, (2) cellular metabolites (ATP and acetylCoA) and cellular co-factors are used, only acetone is added to the reaction (acetone being a none limiting factor in this pathway due to the efficiency of the clostridial acetone pathway) (3) the increase in IBN production is measured in the context of the complete IBN production pathway and takes into account not only the cooperation of the three enzymes of interests, but also the presence of potential endogenous inhibitors or competitors and low substrate concentrations, (4) enzymes are in their most native form and are not subjected to any form of extraction of a purification process that could denature them and negatively affect their activity.
For some above described variants, the fold increase in activity has also been determined over a range of substrate concentrations in order to calculate kinetic parametres (apparent kcat and Km) using equal quantities of MDP decarboxylase variants. The fold increase for the apparent kcat is generally similar to that determined in the screening assays (see
The ability of MDP decarboxylases variants to convert mevalonate-3-phosphate into isoprenol was evaluated using a coupled-enzymatic assay. This assay combines the sequential activities of two MDP decarboxylases, (1) T. acidophilum MDP decarboxylase (L200E mutant) catalyses the phosphorylation of mevalonate into mevalonate-3-phosphate; (2) S. mitis MDP decarboxylase catalyses the conversion of mevalonate-3-phosphate into isoprenol. This assay was set up in 50 mM Tris-HCl pH7.5, 10 mM MgCl2, 20 mM KCl, 40 mM ATP using 200 mM R,S-sodium mevalonate substrate, 2 mg/ml of S. mitis MDP decarboxylase variants and 0.1 mg/ml of T. acidophilum MDP decarboxylase (L200E mutant). Negative controls were also prepared without enzymes or with either enzyme. The reaction mixture was incubated in a sealed glass vial for 24 hours at 37° C. Isoprenol was extracted by mixing 50 μl of the reaction mix to 100 μl of ethyl acetate. 100 μl of the upper ethyl acetate phase was transferred to a clean vial and analysed by Gas chromatography. Commercial isoprenol was used as a reference. The samples were analysed on a Varian GC-430 gas chromatograph equipped with a flame ionization detector (FID). A 1 μl sample was analysed on a DB-WAX column (30 m, 032×0.50 μm, Agilent) using the following temperature gradient: 2 minutes at 60° C., temperature ramp to 220° C. (20° C./min) and finally 10 minutes at 220° C. In these conditions the retention time of isoprenol was 7.38 min.
It was observed that mutations I160N, R186H, R91H also confer an increase in the activity of S. mitis MDP decarboxylase to convert mevalonate-3-phosphate into isoprenol (see
The mutant 2B4 is described in Example 2.
(R)-3-hydroxypent-4-enoic acid was synthesized upon request by a company specialized in custom synthesis (Syntheval, France)
The enzymatic reactions were performed under the following conditions:
50 mM Tris-HCl pH 7.5
0-200 mM (R)-3-hydroxypent-4-enoic acid (“R” HPA)
50 mM ATP
20 mM MgCl2
20 mM KCl
The pH was adjusted to 7.5
Each assay was started by the addition of a particular purified enzyme to 0.5 ml of reaction mixture. The assays were then incubated with shaking at 37° C. in a 2 ml sealed vial (Interchim). Control reactions were run in parallel. After 20 hours of incubation the butadiene production was analyzed as follows. One ml of the gaseous phase of each assay was collected and directly injected into a Gas Chromatograph GC-450 (Brucker) equipped with a Flame Ionization Detector (FID). Nitrogen was used as carrier gas with a flow rate of 1.5 ml/min. Volatile compounds were chromatographically separated on Rt-Alumina Bond/Na2SO4 column (30 m, 0.32 mm ID, 5 μm) (Restek) using an isothermal mode at 130° C. The enzymatic reaction product was identified by comparison with 1,3-butadiene standard (Sigma). Under these GC conditions, the retention time for butadiene was 7.4 min.
The results are shown in
The mutant F9 is described in Example 2.
The enzymatic reactions were performed under the following conditions:
50 mM Tris-HCl pH 7.5
0-200 mM “R” 3-hydroxypent-4-enoic acid (“R” HPA)
50 mM ATP
20 mM MgCl2
20 mM KCl
The pH was adjusted to 7.5
Each assay was started by the addition of a particular enzyme to 0.5 ml of reaction mixture. The assays were then incubated with shaking at 37° C. in a 2 ml sealed vial (Interchim). Control reactions were run in parallel. After 20 hours of incubation the butadiene production was analyzed according to the procedure described in Example 5.
The results are shown in
Additional MVD variants with a further enhanced activity in converting 3-phosphonoxyisovalerate into isobutene were identified through successive rounds of directed or random mutagenesis, recombination of points mutations and in vitro and/or in vivo screening assay. The list of these MVD variants is presented in the following Table 18.
The fold increase is the ratio of MDP decarboxylase variant activity over the “F9” variant MDP decarboxylase activity. The “F9” variant corresponds to the S. mitis MDP decarboxylase of SEQ ID NO:1 with the following mutations: K24R-C118L-Y121R-E159L-M173C-E177C-K282C-E291D-F297L-L303M-T308S (see SEQ ID NO:3). The fold increase is determined for one substrate concentration (2 or 6 mM Ply in the in vitro assay and 10 mM HIV in the in vivo assay). The quantity of the enzyme is not normalized but these MVD variants are expressed in similar amounts as observed on SDS-PAGE analysis of cell lysates.
The in vitro screening assay used in present Example 7 is described herein-above in section c of Materials and Methods, wherein, preferably, an in vitro assay involving cell lysates has been used, while the assay has preferably been further miniaturized in 384 deepwell microplates.
For the in vivo testing a further in vivo screening assay has been developed as outlined in the following. This assay is based on the use of a bacterial strain transformed with an expression vector containing the coding sequences and leading to the production of the last two enzymes involved in the metabolic pathway converting acetone to isobutene. More specifically, for the production of 3-phosphonoxyisovalerate (PIV) from the 3-hydroxyisovalerate (HIV), the T. acidophilum MDP decarboxylase was used (referred to in the following as HIV phosphorylase) and, for the conversion of PIV into isobutene (IBN), the S. mitits MDP decarboxylase variant to be tested was used (referred to in the following as PIV decarboxylase). This strain is first cultured for 24 hours in a shaking incubator at 30° C., 700 rpm, in 300 μL of auto-induction culture medium in 384 deepwell microplates, in order to produce the two types of recombinant enzymes. The cell pellet containing these two overexpressed recombinant enzymes was then resuspended in 50 μL of minimum medium supplemented with 10 mM HIV and incubated further for another four hours in a shaking incubator at 30° C., 700 rpm. During this step, HIV phosphorylase catalyses the phosphorylation of HIV with cellular ATP into Ply, which is then converted into IBN by the PIV decarboxylase variants. The IBN produced is then quantified by gas chromatography using the same method as the in vitro screening assay described above in section c of Materials and Methods.
| Number | Date | Country | Kind |
|---|---|---|---|
| 13175790.8 | Jul 2013 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2014/064767 | 7/9/2014 | WO | 00 |
