WO2017022856A1 - Method for producing isoprene monomer - Google Patents

Abstract

The invention provides an alternative method for producing an isoprene monomer. Specifically, the present invention is a method for producing an isoprene monomer including the production of an isoprene monomer from dimethylallyl diphosphate in the presence of proteins such as (A) and (B) below: (A) a protein including a full-length amino acid sequence of SEQ ID NO: 2, and (B) a protein including a partial amino acid sequence comprising amino acid residues 26-581 in the amino acid sequence of SEQ ID NO: 2 and also including a starting amino acid residue at the N-terminus.

Description

Method for producing isoprene monomer

The present invention relates to a method for producing an isoprene monomer.

Natural rubber is a very important raw material in the tire industry and rubber industry. As demand grows in the future due to motorization centering on emerging countries, it is difficult to expand farms due to competition with deforestation and palm cultivation. Therefore, the supply-demand balance is expected to become tight. Synthetic polyisoprene is an alternative material to natural rubber, but the raw material monomer (isoprene (2-methyl-1,3-butadiene)) is obtained mainly by extraction from the C5 fraction obtained by naphtha cracking. . However, in recent years, the production of isoprene has been decreasing with the lightening of the cracker feed, and there is concern about the supply. In recent years, it is strongly affected by fluctuations in oil prices, and in order to stably secure isoprene monomers, establishment of a system for producing isoprene at low cost from non-petroleum resources is desired.

In response to such a demand, dimethylallyl diphosphate (DMAPP) is obtained using a transformant obtained by incorporating an isolated isoprene synthase gene derived from kuzu or poplar and a mutant thereof into a fungus for fermentation production. ) Has been disclosed (see Patent Documents 1 and 2).

Moreover, there exists the following as a prior art which described the relationship between isoprene synthase and Mg ion.
Non-Patent Document 1 analyzes the dependence of isoprene synthase on the Mg ion concentration using a fraction extracted from a Velvet bean plant and roughly purified.
Non-Patent Document 2 describes that the optimal bivalent cation requirement is 20 mM for the relationship between the isoprene synthase derived from Populus x canescens and the divalent cation.
Patent Document 4 reports a change in the amount of isoprene produced at two Mg ion concentrations (10 mM and 20 mM) for isoprene synthase derived from peanut.
Patent Documents 1 to 5 can be cited as reports showing a method for producing isoprene by fermentation using isoprene synthase.

International Publication No. 2013/179722 Special table 2011-505841 gazette Special table 2011-518564 International Publication No. 2013/166320 International Publication No. 2013/016591

The present invention aims to provide an alternative method for producing isoprene monomers.

As a result of intensive studies, the present inventors have found that isoprene synthase derived from Ginkgo biloba exhibits higher activity at a lower Mg ion (Mg ²⁺ ) concentration than the previously reported isoprene synthase. Physiological Mg ion concentration in microorganisms may vary depending on the type of microorganism, and as far as the present inventors know, it seems that an accurate value is not reported. However, conventional isoprene synthase exhibits high activity. Expected to be lower than ion concentration. In fact, when the present inventors expressed isoprene synthase derived from Ginkgo biloba in a microorganism, it showed higher activity than conventional isoprene synthase. As described above, the present inventors have isolated and identified an isoprene synthase derived from Ginkgo biloba that can exhibit higher activity than conventional isoprene synthase under the condition of physiological Mg ion concentration in the microorganism, and the isoprene The inventors found that isoprene monomers can be efficiently produced by using synthase, and have completed the present invention.

That is, the present invention is as follows.
[1] A method for producing an isoprene monomer, comprising producing an isoprene monomer from dimethylallyl diphosphate in the presence of a protein selected from the group consisting of the following (A) to (F):
(A) a protein comprising the full-length amino acid sequence of SEQ ID NO: 2;
(B) a protein comprising a partial amino acid sequence consisting of amino acid residues 26 to 581 in the amino acid sequence of SEQ ID NO: 2 and further comprising a starting amino acid residue at the N-terminus;
(C) a protein comprising an amino acid sequence having 90% or more identity with the full-length amino acid sequence of SEQ ID NO: 2 and having isoprene synthesis activity;
(D) an amino acid sequence having 90% or more identity with a partial amino acid sequence consisting of amino acid residues 26 to 581 in the amino acid sequence of SEQ ID NO: 2, further comprising a starting amino acid residue at the N-terminus; A protein having isoprene synthesis activity;
(E) a protein comprising an amino acid sequence in which one or several amino acid residues are deleted, substituted, added or inserted in the full-length amino acid sequence of SEQ ID NO: 2 and having isoprene synthesis activity; and (F) a sequence Including an amino acid sequence in which one or several amino acid residues are deleted, substituted, added or inserted in the partial amino acid sequence consisting of amino acid residues 26 to 581 in the amino acid sequence of No. 2, and at the N-terminus A protein further comprising a starting amino acid residue and having isoprene synthesis activity.
[2] The method according to [1], wherein the protein is derived from Ginkgo biloba.
[3] The method according to [1] or [2], wherein isoprene monomer is produced by culturing the transformant producing the protein.
[4] The method according to any one of [1] to [3], wherein dimethylallyl diphosphate is supplied from a carbon source in the medium by culturing the transformant.
[5] The transformant is prepared by introducing an expression vector containing a polynucleotide selected from the group consisting of (a) to (g) below into a host cell, [3] or [3] Method 4):
(A) a polynucleotide comprising the full-length base sequence of SEQ ID NO: 1;
(B) a polynucleotide comprising a partial base sequence consisting of nucleotide residues 76 to 1746 in the base sequence of SEQ ID NO: 1 and further comprising a start codon at the 5 ′ end;
(C) a polynucleotide encoding a protein comprising a base sequence having 90% or more identity with the full-length base sequence of SEQ ID NO: 1 and having isoprene synthesis activity;
(D) a base sequence having 90% or more identity to a partial base sequence consisting of nucleotide residues 76 to 1746 in the base sequence of SEQ ID NO: 1, further including a start codon at the 5 ′ end, and isoprene A polynucleotide encoding a protein having synthetic activity;
(E) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the full-length base sequence of SEQ ID NO: 1 and encodes a protein having isoprene synthesis activity;
(F) hybridizing under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to a partial nucleotide sequence comprising nucleotide residues 76 to 1746 in the nucleotide sequence of SEQ ID NO: 1, and starting codon at the 5 ′ end And (g) a degenerate variant of a polynucleotide selected from the group consisting of (a) to (f).
[6] The method according to [5], wherein the host has an ability to synthesize dimethylallyl diphosphate by the methylerythritol phosphate pathway.
[7] The method according to [6], wherein the host is an Escherichia bacterium.
[8] The method according to [7], wherein the host is Escherichia coli.
[9] The method according to [5], wherein the host has an ability to synthesize dimethylallyl diphosphate by the mevalonate pathway.
[10] The method according to [9], wherein the host is a Pantoea bacterium.
[11] The method of [10], wherein the host is Pantoea ananatis.
[12] A method for producing an isoprene polymer comprising the following (I) and (II):
(I) producing an isoprene monomer by any one of the methods [1] to [11];
(II) polymerizing isoprene monomers to produce isoprene polymers.
[13] A protein selected from the group consisting of (A) to (F) below:
(A) a protein comprising the full-length amino acid sequence of SEQ ID NO: 2;
(B) a protein comprising a partial amino acid sequence consisting of amino acid residues 26 to 581 in the amino acid sequence of SEQ ID NO: 2 and further comprising a starting amino acid residue at the N-terminus;
(C) a protein comprising an amino acid sequence having 90% or more identity with the full-length amino acid sequence of SEQ ID NO: 2 and having isoprene synthesis activity;
(D) an amino acid sequence having 90% or more identity with a partial amino acid sequence consisting of amino acid residues 26 to 581 in the amino acid sequence of SEQ ID NO: 2, further comprising a starting amino acid residue at the N-terminus; A protein having isoprene synthesis activity;
(E) a protein comprising an amino acid sequence in which one or several amino acid residues are deleted, substituted, added or inserted in the full-length amino acid sequence of SEQ ID NO: 2 and having isoprene synthesis activity; and (F) a sequence Including an amino acid sequence in which one or several amino acid residues are deleted, substituted, added or inserted in the partial amino acid sequence consisting of amino acid residues 26 to 581 in the amino acid sequence of No. 2, and at the N-terminus A protein further comprising a starting amino acid residue and having isoprene synthesis activity.
[14] A polynucleotide selected from the group consisting of (a) to (g) below:
(A) a polynucleotide comprising the full-length base sequence of SEQ ID NO: 1;
(B) a polynucleotide comprising a partial base sequence consisting of nucleotide residues 76 to 1746 in the base sequence of SEQ ID NO: 1 and further comprising a start codon at the 5 ′ end;
(C) a polynucleotide encoding a protein comprising a base sequence having 90% or more identity with the full-length base sequence of SEQ ID NO: 1 and having isoprene synthesis activity;
(D) a base sequence having 90% or more identity to a partial base sequence consisting of nucleotide residues 76 to 1746 in the base sequence of SEQ ID NO: 1, further including a start codon at the 5 ′ end, and isoprene A polynucleotide encoding a protein having synthetic activity;
(E) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the full-length base sequence of SEQ ID NO: 1 and encodes a protein having isoprene synthesis activity;
(F) hybridizing under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to a partial nucleotide sequence comprising nucleotide residues 76 to 1746 in the nucleotide sequence of SEQ ID NO: 1, and starting codon at the 5 ′ end And (g) a degenerate variant of a polynucleotide selected from the group consisting of (a) to (f).
[15] An expression vector comprising a polynucleotide encoding the protein of [13], or a polynucleotide of [14], and a heterologous promoter operably linked thereto.
[16] A host cell comprising the expression vector according to [15].
[17] A method for producing a protein, comprising producing the protein using the host cell of [16].

According to the present invention, an excellent isoprene monomer production system can be established.

FIG. 1 shows a map of the pAH162-Para-mvaES plasmid. FIG. 2 is a diagram showing a map of pAH162-KKDyI-ispS (K). FIG. 3 is a view showing a map of pAH162-Ptac-ispS (M) -mvk (Mma). FIG. 4 is a diagram showing the construction of ΔampC :: KKDyI-ispS (K) chromosome variant. A) λRed dependent substitution of the ampC gene with the attLphi80-kan-attRphi80 PCR-generated DNA fragment. B) phi80Int dependent integration of pAH162-KKDyI-ispS (K) plasmid. C) phi80Int / Xis-dependent removal of the vector portion of pAH162-KKDyI-ispS (K). FIG. 5 is a diagram showing the construction of ΔampH :: Para-mvaES chromosome variant. A) λRed dependent replacement of the amph gene with attLphi80-kan-attRphi80 PCR-generated DNA fragment. B) phi80Int dependent integration of pAH162-Para-mvaES plasmid. C) phi80Int / Xis-dependent removal of the vector portion of pAH162-Para-mvaES. FIG. 6 is a diagram showing the construction of a Δcrt :: KKDyI-ispS (K) variant of pEA320 megapramide. A) P. coli placed in pEA320 megaplasmid. Ananatis crt locus structure. B) λRed dependent substitution of the crt operon with the attLphi80-kan-attRphi80 PCR-generated DNA fragment. C) phi80Int dependent integration of pAH162-Ptac-ispS (M) -mvk (Mma) plasmid. D) phi80Int / Xis-dependent removal of the vector portion of pAH162-Ptac-ispS (M) -mvk (Mma). FIG. 7 is a diagram showing the analysis results of the molecular masses of IspSU and IspSM by SDS-PAGE. FIG. 8 is a diagram showing a pAH162-Ptac-integrated expression vector. FIG. 9 is a diagram showing a map of pAH162-Ptac-mvk (M. palidicola).

<Isoprene synthase>
The present invention provides a protein (isoprene synthase) having isoprene synthesis activity.

Isoprene synthase (EC: 4.2.2.37) is an enzyme that converts dimethylallyl diphosphate (DMAPP) to isoprene. The present inventors have found that isoprene synthase derived from Ginkgo biloba exhibits higher activity under low Mg ion (Mg ²⁺ ) concentration conditions than reported isoprene synthase. When compared with sequences excluding the chloroplast translocation signal, the identity (%) between the ginseng-derived isoprene synthase and the known isoprene synthase is as follows.

In one embodiment, the protein of the invention is:
(A) a protein comprising the full-length amino acid sequence of SEQ ID NO: 2; and (B) a partial amino acid sequence consisting of amino acid residues 26 to 581 in the amino acid sequence of SEQ ID NO: 2, and the starting amino acid residue at the N-terminus Further comprising a protein.
In the amino acid sequence of SEQ ID NO: 2, the amino acid sequence consisting of amino acid residues 1 to 25 encodes a putative chloroplast translocation signal, and the amino acid sequence consisting of amino acid residues 26 to 581 is mature isoprene. It can encode a synthase.
In nature, the chloroplast translocation signal containing the N-terminal methionine residue (starting amino acid residue) is removed after chloroplast translocation. Therefore, despite the removal of the chloroplast translocation signal, the protein (B) containing the starting amino acid residue at the N-terminus, and the proteins (D) and (F) described later are naturally occurring proteins. is not. The amino acid sequence encoding the protein (B) is the same as the amino acid sequence of SEQ ID NO: 4.

As used herein, the term “starting amino acid residue” refers to an amino acid residue encoded by a start codon. Examples of the amino acid residue encoded by the start codon include a methionine residue, a valine residue, an isoleucine residue, and a leucine residue, and a methionine residue is preferable.

In another embodiment, the protein of the present invention is a homologous protein of the protein of (A) or (B). Examples of such homologous proteins include the following:
(C) a protein comprising an amino acid sequence having 90% or more identity with the full-length amino acid sequence of SEQ ID NO: 2 and having isoprene synthesis activity; and (D) amino acids 26 to 581 in the amino acid sequence of SEQ ID NO: 2 A protein comprising an amino acid sequence having 90% or more identity with a partial amino acid sequence consisting of residues, further comprising a starting amino acid residue at the N-terminus, and having isoprene synthesis activity.
The percent identity with the amino acid sequence may be 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more.

The homology (that is, identity or similarity) between the amino acid sequence described above and the base sequence described below is described in, for example, the algorithm BLAST (Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)) by Karlin and Altschul, Pearson. FASTA (Methods Enzymol., 183, 63 (1990)). Based on this algorithm BLAST, programs called BLASTP and BLASTN have been developed (see http://www.ncbi.nlm.nih.gov), and these programs are used with default settings for homology. You may calculate. In addition, as homology, for example, using the GENETYX software version 7.0.9 of GENETICS, which employs the Lipman-Pearson method, using the full length of the polypeptide part encoded by the ORF, the unit size to compare A numerical value obtained when the percentity is calculated by the percentage with the setting of = 2 may be used. Alternatively, homology was obtained using the default parameters (Gap penalty = 10, Extend penalty = 0.5, Matrix = EBLOSUM62) in the NEEDLE program (J Mol Biol 1970; 48: 443-453) search. It may be a value (Identity). As the homology, the lowest value among the values derived by these calculations may be adopted.

In yet another embodiment, the protein of the present invention is a modified protein of the protein of (A) or (B). Examples of such modified proteins include the following:
(E) a protein comprising an amino acid sequence in which one or several amino acid residues are deleted, substituted, added or inserted in the full-length amino acid sequence of SEQ ID NO: 2 and having isoprene synthesis activity; and (F) a sequence Including an amino acid sequence in which one or several amino acid residues are deleted, substituted, added or inserted in the partial amino acid sequence consisting of amino acid residues 26 to 581 in the amino acid sequence of No. 2, and at the N-terminus A protein further comprising a starting amino acid residue and having isoprene synthesis activity.

In the above protein, one or several amino acid residues can be modified by 1, 2, 3 or 4 mutations selected from the group consisting of deletion, substitution, addition and insertion of amino acid residues. The amino acid residue mutation may be introduced into one region in the amino acid sequence, or may be introduced into a plurality of different regions. The term “one or several” refers to a number that does not significantly impair the activity of the protein. The number represented by the term “one or several” is, for example, 1 to 100, preferably 1 to 80, more preferably 1 to 50, 1 to 30, 1 to 20, 1 to 10 or 1 to 5 (eg, 1, 2, 3, 4, or 5).

As used herein, the term “isoprene synthesis activity” refers to an enzyme activity that produces isoprene from dimethylallyl diphosphate (DMAPP). When the isoprene synthesis activity of the protein of the present invention is measured in the same reaction solution (eg, reaction solution containing 50 mM Tris-HCl (pH 8.0) and 4 mM DMAPP), the protein of (A) or (B) above. It preferably has an activity of 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more of the isoprene synthesis activity. Moreover, the protein of the present invention can preferably exhibit high isoprene synthesis activity under low-concentration Mg ion conditions. For example, the protein of the present invention can exhibit higher isoprene synthesis activity under 10 mM Mg ion concentration conditions than under 20 mM Mg ion concentration conditions. For example, the ratio of isoprene synthesis activity “(isoprene synthesis activity measured in a reaction solution containing 10 mM Mg ions) / (isoprene synthesis activity measured in a reaction solution containing 20 mM Mg ions)” is about 1.1. About 1.2 or more, about 1.3 or more, about 1.4 or more, about 1.5 or more, about 1.6 or more, about 1.7 or more, about 1.8 or more, or about 1.9 or more It may be. The ratio is not particularly limited, but may be about 3.0 or less, about 2.5 or less, or about 2.0 or less.

The protein of the present invention may have mutations introduced at sites in the catalytic domain and at sites other than the catalytic domain as long as the target activity can be maintained. The positions of amino acid residues to which mutations can be introduced that can retain the desired activity will be apparent to those skilled in the art. Specifically, a person skilled in the art compares 1) the amino acid sequences of a plurality of proteins having the same type of activity (eg, the amino acid sequence of SEQ ID NO: 2 or 4 and the amino acid sequence of other isoprene synthase), and 2) relative The regions that are conserved and the relatively unconserved regions, then 3) from the relatively conserved regions and the relatively unconserved regions, respectively, Areas that can play a role and areas that cannot play an important role in function can be predicted, so that the correlation between structure and function can be recognized. Therefore, those skilled in the art can specify the position of an amino acid residue to which a mutation may be introduced in the amino acid sequence of isoprene synthase.

When an amino acid residue is mutated by substitution, the amino acid residue substitution may be a conservative substitution. As used herein, the term “conservative substitution” refers to the replacement of a given amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are well known in the art. For example, such families include amino acids having basic side chains (eg, lysine, arginine, histidine), amino acids having acidic side chains (eg, aspartic acid, glutamic acid), amino acids having uncharged polar side chains (Eg, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (eg, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chain Amino acids (eg, threonine, valine, isoleucine), amino acids having aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine), amino acids having side groups containing hydroxyl groups (eg, alcoholic, phenolic) ( Example, serine, thread Nin, tyrosine), and amino acids (e.g. having sulfur-containing side chains, cysteine, methionine) and the like. Preferably, the conservative substitution of amino acids is a substitution between aspartic acid and glutamic acid, a substitution between arginine and lysine and histidine, a substitution between tryptophan and phenylalanine, and between phenylalanine and valine. Or a substitution between leucine, isoleucine and alanine, and a substitution between glycine and alanine.

The protein of the present invention may also be a fusion protein linked to a heterologous moiety via a peptide bond. Examples of such heterogeneous moieties include peptide components that facilitate the purification of the target protein (eg, tag sections such as histidine tag, Strep-tag II, etc .; purification of target proteins such as glutathione-S-transferase, maltose binding protein, etc. ), Peptide components that improve the solubility of the target protein (eg, Nus-tag), peptide components that act as chaperones (eg, trigger factor), peptide components that have other functions (eg, full-length protein or its protein) Some), as well as linkers.

The proteins of the invention can also be post-translationally modified (eg, glycosylated) proteins, or post-translationally unmodified (eg, non-glycosylated) proteins, depending on the type of host cell in which production of the protein of the invention is desired. There may be. Since the protein of the present invention is derived from the eukaryotic ginkgo, it is considered that it may be post-translationally modified (eg, glycosylated) in nature. In prokaryotes, proteins are not post-translationally modified, and in animal cells, plant cells, insect cells (eg, Sf9 cells), and lower eukaryotes (eg, yeast), the post-translational modification of proteins is very different. Are generally known [eg, Bretthauser et al. Biotechnol. Appl. Biochem. (1999), 30 (19), 3-200]. Therefore, when a protein derived from a plant cell in nature is produced in such a heterologous host cell, it is considered that a post-translational unmodified protein or a protein having a different post-translational modification that cannot occur in nature can be obtained. It is done.

Examples of the protein of the present invention as described above include proteins derived from Ginkgo biloba, naturally occurring homologues, or artificially produced mutant proteins. The mutant protein can be obtained, for example, by introducing a mutation into DNA encoding the target protein and producing the mutant protein using the obtained mutant DNA. Examples of the mutagenesis method include site-specific mutagenesis and random mutagenesis treatment (eg, treatment with a mutagen and ultraviolet irradiation).

<Polynucleotide encoding isoprene synthase>
The present invention provides a polynucleotide encoding isoprene synthase. The polynucleotide of the present invention may be DNA or RNA, but is preferably DNA. The polynucleotide of the present invention may be derived from Ginkgo biloba. The polynucleotide of the present invention is not particularly limited as long as it encodes the protein of the present invention. For example, the polynucleotide described below may be used.

In one embodiment, the polynucleotide of the present invention is:
(A) a polynucleotide comprising the full-length base sequence of SEQ ID NO: 1; and (b) a partial base sequence consisting of nucleotide residues 76 to 1746 in the base sequence of SEQ ID NO: 1, and a start codon at the 5 ′ end A polynucleotide further comprising:
The base sequence of SEQ ID NO: 1 encodes the amino acid sequence of SEQ ID NO: 2. In the nucleotide sequence of SEQ ID NO: 1, the nucleotide sequence consisting of the 1st to 75th nucleotide residues encodes the amino acid sequence of the putative chloroplast transition signal, and the nucleotide sequence consisting of the 76th to 1746th nucleotide residues is Can encode the amino acid sequence of mature isoprene synthase.
The base sequence of SEQ ID NO: 1 corresponds to cDNA. This is because the polynucleotide (a) is derived from Eukaryote (plant), Ginkgo biloba. Therefore, the polynucleotide (a) and the polynucleotides (c) and (e) described below are not naturally occurring polynucleotides.
Further, in nature, the base sequence consisting of nucleotide residues 76 to 1746 in the base sequence of SEQ ID NO: 1 is not linked to the start codon. Therefore, the polynucleotide of (b) containing the initiation codon at the 5 ′ end, and the polynucleotides of (d) and (f) described below, even though the polynucleotide encoding the chloroplast translocation signal has been removed Is not a naturally occurring polynucleotide. The base sequence encoding the polynucleotide of (b) is the same as the base sequence of SEQ ID NO: 3.

As used herein, the term “start codon” refers to a codon that specifies the start of protein synthesis. Examples of the start codon include, for example, a codon encoding a methionine residue (eg, AUG), a codon encoding a valine residue (eg, GUG), a codon encoding an isoleucine residue (eg, AUA), and a leucine residue Are codons (eg, UUG), and a codon (eg, AUG) encoding a methionine residue is preferred. When the polynucleotide of the present invention is RNA, nucleotide residue “U” should be used as described above. However, when the polynucleotide of the present invention is DNA, “T” is substituted for nucleotide residue “U”. Should be used.

In another embodiment, the polynucleotide of the present invention is a homologous polynucleotide of the polynucleotide of (a) or (b). Examples of such homologous polynucleotides include the following:
(C) a polynucleotide comprising a base sequence having 90% or more identity with the full-length base sequence of SEQ ID NO: 1 and encoding a protein having isoprene synthesis activity; and (d) 76 in the base sequence of SEQ ID NO: 1 A polynucleotide encoding a protein comprising a partial base sequence consisting of a partial base sequence consisting of the 1746th nucleotide residue and having 90% or more identity, further comprising a start codon at the 5 ′ end, and having isoprene synthesis activity.
The percent identity with the base sequence may be 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more.

In yet another embodiment, the polynucleotide of the present invention is an analog polynucleotide of the polynucleotide of (a) or (b). Examples of such analog polynucleotides include the following:
(E) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the full-length base sequence of SEQ ID NO: 1 and encodes a protein having isoprene synthesis activity; and (f) SEQ ID NO: 1 And a polynucleotide comprising a nucleotide sequence complementary to a partial nucleotide sequence consisting of nucleotide residues 76 to 1746 in the nucleotide sequence under the stringent conditions, further comprising a start codon at the 5 ′ end, and isoprene A polynucleotide encoding a protein having synthetic activity.

As used herein, the term “stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, stringent conditions include hybridization at about 45 ° C. in 6 × SSC (sodium chloride / sodium citrate), followed by 50 × 65 ° C. in 0.2 × SSC, 0.1% SDS. 1 or 2 or more washing | cleaning is mentioned.

In yet another embodiment, the polynucleotide of the invention is a degenerate polynucleotide. Examples of such degenerate polynucleotides include the following:
(G) A degenerate variant of a polynucleotide selected from the group consisting of (a) to (f).

As used herein, the term “degenerate variant” refers to another codon in which at least one codon encoding a given amino acid residue in a polynucleotide before mutation encodes the same amino acid residue. Refers to a polynucleotide variant that has been altered to. Since such a degenerate mutant is a mutant based on a silent mutation, the protein encoded by the degenerate mutant is the same as the protein encoded by the polynucleotide before the mutation.

Preferably, a degenerate variant is a polynucleotide variant in which the codons have been altered to match the codon usage of the host cell into which it is to be introduced. When a gene is expressed in a heterologous host cell (eg, a microorganism), the corresponding tRNA molecular species are not sufficiently supplied due to the difference in codon usage, resulting in decreased translation efficiency and / or incorrect translation (eg, translation). Stop) may occur. For example, in Escherichia coli, the low frequency codons shown in Table 2 are known.

Therefore, in the present invention, a degenerate mutant that matches the codon usage of the host cell (eg, microorganism) as described below can be used. For example, in the degenerate mutant of the present invention, the codon encoding one or more amino acid residues selected from the group consisting of arginine residues, glycine residues, isoleucine residues, leucine residues, and proline residues is changed. It may be what was done. More specifically, the degenerate mutant of the present invention has one or more codons selected from the group consisting of low frequency codons (eg, AGG, AGA, CGG, CGA, GGA, AUA, CUA, and CCC). It may have been changed. Preferably, the degenerate variant may comprise one or more (eg, 1, 2, 3, 4 or 5) codon changes selected from the group consisting of:
i) Change of at least one codon selected from the group consisting of four codons encoding Arg (AGG, AGA, CGG, and CGA) to another codon encoding Arg (CGU or CGC);
ii) changing one codon (GGA) encoding Gly to another codon (GGG, GGU, or GGC);
iii) changing one codon (AUA) encoding Ile to another codon (AUU, or AUC);
iv) changing one codon (CUA) encoding Leu to another codon (UUG, UUA, CUG, CUU, or CUC); and v) one codon (CCC) encoding Pro. Change to another codon (CCG, CCA, or CCU).
When the degenerate variant of the present invention is RNA, the nucleotide residue “U” should be used as described above. However, when the degenerate variant of the present invention is DNA, the nucleotide residue “U” is substituted. “T” should be used. The number of nucleotide residue mutations for adapting to the codon usage of the host cell is not particularly limited as long as it encodes the same protein before and after the mutation. For example, 1 to 500, 1 to 400, 1 to 300 1 to 200, or 1 to 100.

Identification of low-frequency codons can be easily performed based on any host cell type and genome sequence information by using a technique known in the art. Thus, a degenerate variant may include a change from a low frequency codon to a non-low frequency codon (eg, a high frequency codon). In addition, there are known methods for designing mutants in consideration of not only low-frequency codons but also factors such as suitability of the production strain to the genomic GC content (Alan Villabos et al., Gene Designer: a synthetic). biology tool for constructing artificial DNA segments, BMC Bioinformatics. 2006 Jun 6; 7: 285.), such a method may be used. As described above, the above-described mutant can be appropriately prepared according to the type of any host cell into which it can be introduced (for example, a microorganism as described below). For example, a host commonly used for fermentation production of heterologous proteins. It may be made to fit the cell type. Examples of host cells widely used for fermentative production of heterologous proteins include any microorganism described below, and bacteria and yeasts are preferred. Examples of such bacteria and yeasts include bacteria belonging to the genus Bacillus such as Bacillus subtilis, bacteria belonging to the genus Corynebacterium such as Corynebacterium glutamicum, and Escherichia coli. Escherichia bacteria such as Escherichia coli, Pantoea bacteria such as Pantoea ananatis, Enterobacter cerevisiae such as Enterobacter aerogenes (Saccha Omyces cerevisiae) Saccharomyces (Saccharomyces) genus, and the like, and Schizosaccharomyces pombe (Schizosaccharomyces pombe) Schizosaccharomyces (Schizosaccharomyces such) include yeasts of the genus. Therefore, consideration of factors such as changing the low frequency codons to non-low frequency codons in such microorganisms (eg, bacteria, yeast) and suitability for genomic GC content is preferable.

<Expression vector>
The present invention provides an expression vector. The expression vector of the present invention contains a polynucleotide encoding the protein of the present invention or the polynucleotide of the present invention.

The expression vector of the present invention may further contain a heterologous promoter operably linked to the polynucleotide. The term “heterologous promoter” refers to a promoter other than the natural promoter of the ginkgo beetle-derived isoprene synthase gene. Accordingly, examples of heterologous promoters include promoters of ginkgo bean-derived genes other than ginkgo beetle-derived isoprene synthase gene, promoters derived from other organisms (eg, microorganisms, animals, insects, and plants) other than ginkgo beetle, promoters derived from viruses, and artificial synthesis A promoter is mentioned. As the heterologous promoter, a promoter widely used for heterologous protein production may be used. Examples of such promoters include PhoA promoter, PhoC promoter, T7 promoter, T5 promoter, T3 promoter, lac promoter, trp promoter, trc promoter, tac promoter, PR promoter, PL promoter, SP6 promoter, arabinose inducible promoter, cold A shock promoter and a tetracycline inducible promoter are mentioned.

The expression vector of the present invention may further contain a terminator downstream of the polynucleotide. Examples of such terminators include T7 terminator, fd phage terminator, T4 terminator, tetracycline resistance gene terminator, and Escherichia coli trpA gene terminator.

The expression vector of the present invention may further contain a ribosome binding site (eg, Shine-Dalgarno sequence) upstream of the start codon.

The expression vector of the present invention may further contain a polynucleotide encoding a drug resistance gene. Examples of drug resistance genes include resistance genes for drugs such as tetracycline, ampicillin, kanamycin, hygromycin, and phosphinothricin.

Examples of the expression vector of the present invention include a cell-based vector for expressing a protein in a host and a cell-free vector utilizing a protein translation system. The expression vector can also be a plasmid, viral vector, phage, integrative vector, or artificial chromosome. An integrative vector may be a type of vector that is integrated entirely into the genome of the host cell. Alternatively, the integrative vector may be a vector in which only a part (eg, an expression unit described later) is integrated into the host cell genome. The expression vector may further be a DNA vector or an RNA vector.
As the cell-based vector, a known expression vector suitable for the host is used. Examples of such vectors include pUC (eg, pUC19, pUC18), pSTV, pBR (eg, pBR322), pHSG (eg, pHSG299, pHSG298, pHSG399, pHSG398), RSF (eg, RSF1010), pACYC (eg, PACYC177, pACYC184), pMW (eg, pMW119, pMW118, pMW219, pMW218), pQE (eg, pQE30), and derivatives thereof.
Examples of the cell-free vector include wheat-cell-free protein synthesis vectors such as an expression vector having a T7 or T3 promoter and a pEU-based plasmid having an SP6 promoter.

<Transformant>
The transformant of the present invention is a host cell capable of producing the protein of the present invention or expressing the polynucleotide of the present invention to produce the protein. Specifically, the transformant of the present invention is a host cell containing an expression unit containing the polynucleotide of the present invention. The expression unit containing the polynucleotide of the present invention refers to the expression of the protein encoded by the polynucleotide of the present invention, including the polynucleotide of the present invention and a promoter operably linked thereto (eg, homologous promoter, heterologous promoter). A unit that makes possible. Examples of the host cell containing the expression unit containing the polynucleotide of the present invention include a host cell containing the expression vector of the present invention. The host cell is not particularly limited as long as it can express the protein of the present invention. The host cell may be homologous or heterologous to the protein of the present invention and the polynucleotide of the present invention, but is preferably heterologous. The host cell may also be homologous or heterologous to the promoter, but is preferably heterologous. Examples of host cells include animal cells, plant cells, insect cells, and microorganisms, with microorganisms being preferred. Examples of the microorganism include bacteria such as bacteria belonging to the family Enterobacteriaceae and fungi. The bacterium may be a gram positive bacterium or a gram negative bacterium.

Examples of Gram-positive bacteria include Bacillus bacteria, Listeria bacteria, Staphylococcus bacteria, Streptococcus bacteria, Enterococcus bacteria, Clostridium bacteria, Clostridium bacteria, and Clostridium bacteria. Examples include bacteria belonging to the genus Corynebacterium and bacteria belonging to the genus Streptomyces, and bacteria belonging to the genus Bacillus and bacteria belonging to the genus Corynebacterium are preferred.
Examples of Bacillus bacteria include Bacillus subtilis, Bacillus anthracis, Bacillus cereus, and the like, and Bacillus subtilis is more preferable.
Examples of bacteria belonging to the genus Corynebacterium include Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium, and Corynebacterium. Is more preferable.

Examples of the gram-negative bacteria include Escherichia bacteria, Pantoea bacteria, Salmonella bacteria, Vibrio bacteria, Serratia bacteria, Enterobacter bacteria, and the like. Among them, Escherichia bacteria, Pantoea bacteria, Enterobacter bacteria are preferable.
As the bacterium belonging to the genus Escherichia, Escherichia coli is preferable.
Examples of the genus Pantoea include Pantoea ananatis, Pantoea stewartii, Pantoea agglomerans, Pantoea citrea, and Pantoea citrea Pantoea ananatis) and Pantoea citrea are preferred. Further, as the Pantoea bacterium, strains exemplified in European Patent Application Publication No. 09522121 may be used. As typical strains of the genus Pantoea, for example, Pantoea ananatis AJ13355 strain (FERM BP-6614), Pantoea ananatis AJ13356 strain (FERM BP-6615), and Pantoea disclosed in European Patent Application No. 0952212 are disclosed. Ananatis SC17 (0) strain. The Pantoea Ananatis SC17 (0) strain was established on September 21, 2005, in the Russian National Collection of Industrial Microorganisms (VKPM), GNII Genetika (adress: 11). 1) Registration number VKPM B-9246 in Moscow, 1 Dorozhny proezd.
Examples of Enterobacter bacteria include Enterobacter agglomerans, Enterobacter aerogenes, and the like, with Enterobacter aerogenes being preferred. Further, as the Enterobacter bacterium, a strain exemplified in European Patent Application Publication No. 09522121 may be used. As representative strains of Enterobacter bacteria, for example, Enterobacter agglomerans ATCC 12287 strain, Enterobacter aerogenes ATCC 13048 strain, Enterobacter aerogenes NBRC 12010 strain (Sakai Set et al., Biotechnol. Bioeng., Vol. 98, pp. 340-348, 2007) and Enterobacter aerogenes AJ11037 (FERM BP-10955). Enterobacter aerogenes AJ110737 is an independent administrative agency, National Institute of Advanced Industrial Science and Technology (currently National Institute of Advanced Industrial Science and Technology), Patent Biological Deposit Center (Tsukuba, Ibaraki, 305-8856, Japan) Deposited as FERM P-21348 in 1st, 1-Chome, 1-City, City East, and transferred to an international deposit based on the Budapest Treaty on March 13, 2008, and received the FERM BP-10955 receipt number. .

Examples of the fungi include the genus Saccharomyces, the genus Schizosaccharomyces, the genus Yarrowia, the genus Trichoderma, the genus Aspergillus, the genus Aspergillus, Preferred are microorganisms of the genus Saccharomyces, the genus Schizosaccharomyces, the genus Yarrowia, or the genus Trichoderma.
The Saccharomyces (Saccharomyces) microorganisms of the genus, Saccharomyces carlsbergensis (Saccharomyces carlsbergensis), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Saccharomyces Deer statics (Saccharomyces diastaticus), Saccharomyces Dougurashi (Saccharomyces douglasii), Saccharomyces Kuruibera (Saccharomyces kluyveri), Saccharomyces norbensis, Saccharomyces obiformis, and Saccharomyces s. -Saccharomyces cerevisiae is preferred.
As a microorganism belonging to the genus Schizosaccharomyces, Schizosaccharomyces pombe is preferable.
As a microorganism belonging to the genus Yarrowia, Yarrowia lipolytica is preferable.
The Trichoderma (Trichoderma) microorganisms of the genus, Trichoderma Harujianumu (Ttichoderma harzianum), Trichoderma Koningi (Trichoderma koningii), Trichoderma Rongiburakiamu (Trichoderma longibrachiatum), Trichoderma reesei (Trichoderma reesei), Trichoderma viride (Trichoderma viride) are And Trichoderma reesei is preferred.
In addition, the host used in the present invention includes dimethylallyl diphosphate by the mevalonate (MVA) pathway and / or methylerythritol phosphate (MEP) pathway involved in the synthesis of dimethylallyl diphosphate which is a substrate for isoprene synthase. It is not particularly limited as long as it has the ability to synthesize (DMAPP), and may be an insect cell, an animal cell, a plant cell, or the like. For example, Escherichia bacteria such as Escherichia coli may inherently have the ability to synthesize dimethylallyl diphosphate through the methylerythritol phosphate pathway. In addition, Pantoea bacteria such as Pantoea ananatis may inherently have the ability to synthesize dimethylallyl diphosphate by the mevalonate pathway.
For the expression “ability to synthesize dimethylallyl diphosphate (DMAPP)”, see Michele CY Chang and Jay D Keasling, Nature Chemical Biology 2,674-681 (2006).
For the expression “mevalonic acid (MVA) pathway”, see Kuzuyama T and Seto H, Proc Jpn Acad Ser B Phys Biol Sci. 88, 41-52 (2012); Miziorko HM, Arch Biochem Biophys. 505, 131-143 (2011).
For the expression “methylerythritol phosphate (MEP) pathway”, see Kuzuyama T and Seto H, Proc Jpn Acad Ser B Phys Biol Sci. 88, 41-52 (2012); Grawert T et al. , Cell Mol Life Sci. 68, 3797-3814 (2011).

In the transformant of the present invention, the pathway for synthesizing dimethylallyl diphosphate (DMAPP), which is a substrate for isoprene synthase, may be further enhanced. For such enhancement, an isopentenyl diphosphate delta isomerase expression vector having the ability to convert isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP) was introduced into the transformant of the present invention. May be. In addition, an expression vector for one or more enzymes involved in the mevalonate pathway and / or methylerythritol phosphate pathway associated with the production of IPP and / or DMAPP may be introduced into the transformant of the present invention. Such enzyme expression vectors may be plasmids or integrative vectors. Such enzyme expression vectors may also be DNA vectors or RNA vectors. Such an enzyme expression vector further expresses a plurality of (eg, 1, 2, 3 or 4 or more) enzymes involved in the mevalonate pathway and / or the methylerythritol phosphate pathway. For example, it may be an expression vector for polycistronic mRNA. The origin of one or more enzymes involved in the mevalonate pathway and / or the methylerythritol phosphate pathway may be homologous or heterologous to the host. When the origin of the enzyme involved in the mevalonate pathway and / or the methylerythritol phosphate pathway is heterologous to the host, for example, the host is a bacterium as described above (eg, Escherichia coli), and mevalonic acid At least one enzyme involved in the pathway may be derived from a fungus (eg, Saccharomyces cerevisiae). In addition, when the host inherently produces an enzyme involved in the methylerythritol phosphate pathway, one or more expression vectors introduced into the host are involved in the mevalonate pathway (eg, 1 type, 2 types). (3 types or 4 types or more) of enzymes may be expressed.

As isopentenyl diphosphate delta isomerase (EC: 5.3.3.2), for example, Idi1p (ACCESSION ID NP_015208), AT3G02780 (ACCESSION ID NP_186927), AT5G16440 (ACCESSION ID NP_197165N, ID_197165N, P_197165) ).

Examples of enzymes involved in the mevalonate (MVA) pathway include, for example, mevalonate kinase (EC: 2.7.1.36; Example 1, Erg12p, ACCESSION ID NP — 013935; Example 2, AT5G27450, ACCESSION ID NP — 001190411), phosphomevalonic acid Kinase (EC: 2.7.4.2; Example 1, Erg8p, ACCESSION ID NP_013947; Example 2, AT1G31910, ACCESSION ID NP_001185124), diphosphomevalonate decarboxylase (EC: 4.1.1.33; Example 1, Mvd1p , ACCESSION ID NP_014441; Example 2, AT2G38700, ACCESSION ID NP_181404; Example 3, AT3G54250, A CESSION ID NP_566995), acetyl-CoA-C-acetyltransferase (EC: 2.3.1.9; Example 1, Erg10p, ACCESSION ID NP_015297; Example 2, AT5G47720, ACCESSION ID NP_001032028; Example 3, AT5G4856ID, 941 ), Hydroxymethylglutaryl-CoA synthase (EC: 2.3.3.10; Example 1, Erg13p, ACCESSION ID NP_013580; Example 2, AT4G11820, ACCESSION ID NP_192919; Example 3, MvaS, ACCESSION ID AAG02438), hydroxymethyl Glutaryl-CoA reductase (EC: 1.1 1.34; Example 1, Hmg2p, ACCESSION ID NP_013555; Example 2, Hmg1p, ACCESSION ID NP_013636; Example 3, AT1G76490, ACCESSION ID NP_177775; Example 4, AT2G17370, ACCESSION ID.NP. Example, MvaA, ACCESSION ID P13702), acetyl-CoA-C-acetyltransferase / hydroxymethylglutaryl-CoA reductase (EC: 2.3.1.9/1.1.1.34, example, MvaE, ACCESSION ID) AAG02439).

Examples of enzymes involved in the methylerythritol phosphate (MEP) pathway include 1-deoxy-D-xylulose-5-phosphate synthase (EC: 2.2.1.7, Example 1, Dxs, ACCESSION ID NP_414954; Example 2, AT3G21500, ACCESSION ID NP_566686; Example 3, AT4G15560, ACCESSION ID NP_193291; Example 4, AT5G11380, ACCESSION ID NP_001078570), 1-deoxy-D-xylulose-5-phosphate reductoisomerase (EC: 1.1. 1.267; Example 1, Dxr, ACCESSION ID NP_414715; Example 2, AT5G62790, ACCESSION ID NP_001190600), 4 Diphosphocytidyl-2-C-methyl-D-erythritol synthase (EC: 2.7.7.60; Example 1, IspD, ACCESSION ID NP_417227; Example 2, AT2G02500, ACCESSION ID NP_565286), 4-diphosphocytidyl-2-C- Methyl-D-erythritol kinase (EC: 2.7.1.148; Example 1, IspE, ACCESSION ID NP_415726; Example 2, AT2G26930, ACCESSION ID NP_180261), 2-C-methyl-D-erythritol-2,4- Cycloniphosphate synthase (EC: 4.6.1.12; Example 1, IspF, ACCESSION ID NP_417226; Example 2, AT1G63970, ACCESSION ID NP 564819), 1-hydroxy-2-methyl-2- (E) -butenyl-4-nitrophosphate synthase (EC: 1.1.77.1; Example 1, IspG, ACCESSION ID NP — 417010; Example 2, AT5G60600, ACCESSION ID NP_001119467), 4-hydroxy-3-methyl-2-butenyl diphosphate reductase (EC: 1.17.1.2; Example 1, IspH, ACCESSION ID NP_414570; Example 2, AT4G34350, ACCESSION ID NP_567965) It is done.

The introduction (transformation) of an expression vector into which a gene has been incorporated into a host can be performed using a conventionally known method. For example, a competent cell method using microbial cells treated with calcium, an electroporation method, and the like can be mentioned. In addition to a plasmid vector, a phage vector may be used to infect and introduce into a microbial cell.

Furthermore, in the transformant of the present invention, a gene encoding a mevalonate pathway or methylerythritol phosphate pathway enzyme that synthesizes dimethylallyl diphosphate, which is a substrate for isoprene synthase, may be introduced.
Such enzymes include 1-deoxy-D-xylose-5-phosphate synthase that converts pyruvate and D-glyceraldehyde-3-phosphate to 1-deoxy-D-xylose-5-phosphate; isopentenyl Examples include isopentyl diphosphate isomerase that converts diphosphate to dimethylallyl diphosphate.

In the transformant of the present invention, one or a plurality of (eg, 2, 3, 4 or 5) specific genomic regions (eg, coding or non-coding regions) may be disrupted. Such genomic regions include, for example, the crt operon (encoding isoprenoid biosynthetic pathways such as polyprenyl synthetase, beta-carotene hydroxylase, phytoene synthase, phytoene dehydrogenase, lycopene cyclase), amp gene (eg, ampC gene, Or ampH gene). For example, the destruction of the crt operon can be advantageous because it can suppress the production of isoprenoid compounds. The disruption of the amp gene can be advantageous as an ampicillin resistance gene (drug resistance selection marker) becomes available.
The term “disruption” for a gene means that the gene coding region has been modified to reduce or completely eliminate the function or expression of the protein encoded by the gene. The term “disruption” for an operon means that the genomic region corresponding to the operon has been modified to reduce or completely eliminate the function of the operon. When the function of an operon that can act as an enhancer (eg, the crt operon described above) is reduced or completely lost, the expression of a protein encoded by a gene operably linked to the operon can be reduced or completely lost. On the other hand, when the function of an operon that can act as a repressor is reduced or completely eliminated, the expression of a protein encoded by a gene operably linked to the operon can be improved. Such modifications include, but are not limited to, insertions, deletions and exchanges, for example.
The genomic region may be destroyed, for example, by introducing the above-described gene (eg, isoprene synthase gene) into the genomic region according to a known gene targeting method.

Isoprene synthase may be purified from the transformant of the present invention. Examples of the purification method include a salting-out method and a method using various chromatographies. When the isoprene synthase is designed to include a heterologous moiety such as a histidine tag, the isoprene synthase can be purified by an affinity column using a metal such as nickel or a substance having affinity for the heterogeneous moiety. In addition, the purity of the protein of the present invention can be increased by appropriately combining methods such as ion exchange chromatography and gel filtration chromatography.

<Method for producing isoprene monomer and isoprene polymer>
The present invention provides a method for producing an isoprene monomer comprising producing an isoprene monomer from dimethylallyl diphosphate (DMAPP) in the presence of the protein of the present invention.

In one embodiment, the method for producing the isoprene monomer of the present invention can be performed using the protein itself of the present invention. A natural protein or a recombinant protein can be used as the protein of the present invention. The recombinant protein can be obtained, for example, using a cell-free vector or from the transformant of the present invention. The protein of the present invention can be used as an unpurified, crudely purified or purified protein. As the reaction system, for example, a buffer solution (eg, pH 6.0 to 9.0) containing the protein of the present invention, a substrate (DMAPP) and Mg ions can be used. The Mg ion (Mg ²⁺ ) concentration is not particularly limited, but the protein of the present invention can exhibit high activity at a low Mg ion concentration, and thus, for example, the reaction is performed at a Mg ion concentration of 15 mM or less, 12 mM or less, or 10 mM or less. It may be broken.

In another embodiment, the method for producing an isoprene monomer of the present invention can be performed using the transformant of the present invention. Since the transformant of the present invention can produce isoprene monomer mainly as an outgas from a carbon source in the medium, the isoprene monomer can be recovered by collecting the gas generated by the transformant. . Dimethylallyl diphosphate, which is a raw material for the isoprene monomer, is synthesized by the mevalonate pathway or the methyl erythritol phosphate pathway in the host, using the carbon source in the medium as the basis. Alternatively, dimethylallyl diphosphate may be added to the medium.

The medium for culturing the transformant of the present invention preferably contains a carbon source for conversion to mevalonic acid or isoprene. Examples of the carbon source include carbohydrates such as monosaccharides, disaccharides, oligosaccharides and polysaccharides; invert sugar obtained by hydrolyzing sucrose; glycerol; 1 carbon number such as methanol, formaldehyde, formate, carbon monoxide and carbon dioxide Compounds (hereinafter referred to as C1 compounds); oils such as corn oil, palm oil and soybean oil; acetates; animal fats; animal oils; fatty acids such as saturated fatty acids and unsaturated fatty acids; lipids; phospholipids; glycerolipids; Glycerin fatty acid esters such as glyceride, diglyceride, and the like; polypeptides such as microbial proteins and plant proteins; renewable carbon sources such as hydrolyzed biomass carbon sources; yeast extracts; or combinations thereof. As the nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, organic nitrogen such as soybean hydrolysate, ammonia gas, aqueous ammonia and the like can be used. As an organic trace nutrient source, it is desirable to contain an appropriate amount of a required substance such as vitamin B1, L-homoserine or a yeast extract. In addition to these, a small amount of potassium phosphate, magnesium sulfate, iron ion, manganese ion or the like is added as necessary. The medium used in the present invention may be a natural medium or a synthetic medium as long as it contains a carbon source, a nitrogen source, inorganic ions, and other organic trace components as required.

Examples of monosaccharides include trioses such as ketotriose (dihydroxyacetone) and aldtriose (glyceraldehyde); tetroses such as ketotetrose (erythrulose) and aldetetrose (erythrose and threose); ketopentose (ribulose and xylulose) Pentose such as ribose, arabinose, xylose, lyxose), deoxy sugar (deoxyribose); ketohexose (psicose, fructose, sorbose, tagatose), aldohexose (allose, altrose, glucose, mannose, gulose, idose, galactose, talose ), Deoxy sugars (fucose, fucrose, rhamnose) and other hexoses; heptose such as sedoheptulose; Scan, galactose, C6 sugars such as glucose; xylose, carbohydrates C5 sugars arabinose and the like are preferable.
Examples of the disaccharide include sucrose, lactose, maltose, trehalose, tunulose, and cellobiose, and sucrose and lactose are preferable.
Examples of oligosaccharides include trisaccharides such as raffinose, melezitose, and maltotriose; tetrasaccharides such as acarbose and stachyose; and other oligosaccharides such as fructooligosaccharide (FOS), galactooligosaccharide (GOS), and mannan oligosaccharide (MOS). Can be mentioned.
Examples of the polysaccharide include glycogen, starch (amylose, amylopectin), cellulose, dextrin, and glucan (β1,3-glucan), and starch and cellulose are preferable.

Microbial proteins include polypeptides derived from yeast or bacteria.
Examples of plant proteins include polypeptides derived from soybean, corn, canola, jatropha, palm, peanut, sunflower, coconut, mustard, cottonseed, palm kernel oil, olive, safflower, sesame and flaxseed.

Examples of lipids include substances containing one or more saturated or unsaturated fatty acids of C4 or higher.

The oil contains one or more saturated or unsaturated fatty acids of C4 or higher, and is preferably a lipid that is liquid at room temperature. Soybean, corn, canola, jatropha, palm, peanut, sunflower, coconut, mustard, cottonseed, palm kernel oil, Examples thereof include olives, safflowers, sesame, flaxseed, oily microbial cells, peanuts, or combinations of two or more thereof.

Examples of the fatty acid include compounds represented by the formula RCOOH (“R” represents a hydrocarbon group).
The unsaturated fatty acid is a compound having at least one carbon-carbon double bond in “R”, and examples thereof include oleic acid, vaccenic acid, linoleic acid, palmitate acid, and arachidonic acid.
The saturated fatty acid is a compound in which “R” is a saturated aliphatic group, and examples thereof include docosanoic acid, icosanoic acid, octadecanoic acid, hexadecanoic acid, tetradecanoic acid, and dodecanoic acid.
Among these, fatty acids containing one or more C2 to C22 fatty acids are preferred, and those containing C12 fatty acids, C14 fatty acids, C16 fatty acids, C18 fatty acids, C20 fatty acids and C22 fatty acids are more preferred.
Examples of the carbon source include salts of these fatty acids, derivatives, and salts of derivatives. Examples of the salt include lithium salt, potassium salt, sodium salt and the like.

Also, examples of the carbon source include lipids, oils, fats and oils, fatty acids, combinations of glycerin fatty acid esters and carbohydrates such as glucose.

Renewable carbon sources include hydrolyzed biomass carbon sources.
Biomass carbon sources include cellulosic substrates such as wood, paper, and pulp waste, foliate plants, and pulp; parts of plants such as stalks, grains, roots, and tubers.
Examples of plants used as biomass carbon sources include corn, wheat, rye, sorghum, triticate, rice, millet, barley, cassava, peas and other legumes, potatoes, sweet potatoes, bananas, sugar cane, tapioca and the like.

When a renewable carbon source such as biomass is added to the cell culture medium, pretreatment is preferably performed. Examples of the pretreatment include enzymatic pretreatment, chemical pretreatment, and a combination of enzymatic pretreatment and chemical pretreatment.
It is preferred to hydrolyze all or part of the renewable carbon source before adding it to the cell culture medium.

Further, examples of the carbon source include yeast extract or a combination of yeast extract and another carbon source such as glucose, and a combination of yeast extract and C1 compound such as carbon dioxide or methanol is preferable.

In the present invention, as a method for culturing a transformant, cells are preferably cultured in a standard medium containing physiological saline and a nutrient source.
The culture medium is not particularly limited, and examples thereof include commercially available conventional culture media such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth, and Yeast medium (YM) broth. A medium suitable for cultivation of a specific host can be appropriately selected and used.

In addition to a suitable carbon source, the cell culture medium includes suitable minerals, salts, cofactors, buffers, and ingredients known to those skilled in the art to be suitable for cultivation or to promote isoprene production. It is desirable.
In the transformant of the present invention, it is preferable to add sugar, metal salt, antibacterial substance and the like to the medium in order to maintain the expression of the target protein.

The culture conditions for the transformant of the present invention are not particularly limited as long as the target protein can be expressed, and standard cell culture conditions can be used.
The culture temperature is preferably 20 ° C. to 37 ° C., the gas composition is preferably a CO ₂ concentration of about 6% to about 84%, and a pH of about 5 to about 9.
Further, it is preferable to perform the culture under aerobic, anoxic, or anaerobic conditions depending on the properties of the host.

Examples of the culture method include a method of culturing the transformant using a known fermentation method such as a batch culture method, a fed-batch culture method, or a continuous culture method.

The batch culture method is a method in which a medium composition is charged at the start of fermentation, a host is inoculated into the medium, and the transformant is cultured while controlling pH, oxygen concentration, and the like. In culturing a transformant by a batch culture method, the transformant goes from a gentle induction phase to a logarithmic growth phase and finally to a stationary phase where the growth rate decreases or stops. Isoprene is produced by transformants in the logarithmic growth phase or stationary phase.

The fed-batch culture method is a method in which a carbon source is gradually added as the fermentation process proceeds in addition to the batch method described above. The fed-batch culture method is effective when the metabolism of the transformant tends to be suppressed by catabolite suppression and it is preferable to limit the amount of the carbon source in the medium. The fed-batch culture method can be performed using a limited or excessive amount of a carbon source such as glucose.

The continuous culture method is a culture method in which a predetermined amount of culture medium is continuously supplied to a bioreactor at a constant rate, and at the same time, the same amount of culture medium is withdrawn. In the continuous culture method, the culture can be kept at a constant high density, and the transformant in the culture solution is mainly in the logarithmic growth phase.
By appropriately replacing a part or all of the medium, it is possible to supplement nutrients and prevent accumulation of metabolic byproducts and dead cells that may adversely affect the growth of the transformant.

When the expression vector has an inducible promoter such as lac promoter, an inducing agent such as IPTG (isopropyl-β-thiogalactopyranoside) is added to the culture solution to express the target protein. May be induced.

Examples of a method for evaluating the production amount of isoprene monomer obtained by culturing the transformant of the present invention include a method in which a gas phase is collected by a headspace method and the gas phase is analyzed by a gas chromatography method.
Specifically, the isoprene monomer in the headspace when the culture medium containing the transformant is cultured while shaking in a sealed vial is analyzed using standard gas chromatography. Next, using the calibration curve, the area obtained from the gas chromatography measurement curve is converted into the isoprene monomer production amount of the transformant.

As a method for recovering the isoprene monomer obtained by culturing the transformant of the present invention, gas stripping, fractional distillation, or desorption of the isoprene monomer adsorbed on the solid phase from the solid phase by heat or vacuum, or Examples include extraction with a solvent.
In gas stripping, isoprene gas is continuously removed from the outgas. Such isoprene gas can be removed by various methods, such as adsorption to a solid phase, separation into a liquid phase, or a method of directly condensing isoprene gas.

The recovery of isoprene monomer can be performed in one stage or in multiple stages. When the isoprene monomer is recovered in one stage, the separation of the isoprene monomer from the outgas and the conversion of the isoprene monomer into a liquid phase are simultaneously performed. Isoprene monomer can also be condensed directly from the outgas into a liquid phase. When the isoprene monomer is recovered in multiple stages, the separation of the isoprene monomer from the off-gas and the conversion of the isoprene monomer into a liquid phase are sequentially performed. For example, a method in which isoprene monomer is adsorbed on a solid phase and extracted from the solid phase with a solvent can be mentioned.

Furthermore, the method for recovering the isoprene monomer may include a step of purifying the isoprene monomer. Examples of the purification step include separation from a liquid phase extract by distillation and various chromatographies.

The present invention also provides a method for producing an isoprene polymer. The method for producing the isoprene polymer of the present invention includes the following (I) and (II):
(I) producing an isoprene monomer by the method of the present invention;
(II) polymerizing isoprene monomers to produce isoprene polymers.

Step (I) can be performed in the same manner as the above-described method for producing the isoprene monomer of the present invention. The polymerization of the isoprene monomer in the step (II) can be performed by any method known in the art (eg, organic chemical synthesis method).

Next, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the following examples.

Example 1: P.I. Construction of ananatis AG10265 1-1) Construction of pMW-Para-mvaES-Trp 1-1-1) Chemical synthesis of enterococcus faecalis mvaE gene And the amino acid sequence are already known (base sequence ACCESSION numbers: AF29009.1, (1479..3890), amino acid sequence ACCESSION number: AAG02439) (J. Bacteriol. 182 (15), 4319-4327 (2000). )). The amino acid sequence of Enterococcus faecalis-derived mvaE protein and the nucleotide sequence of the gene are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. The mvaE gene was transformed into E. coli. E. coli for efficient expression in E. coli. An mvaE gene optimized for E. coli codon usage was designed and named EFmvaE. This base sequence is shown in SEQ ID NO: 7. The mvaE gene was chemically synthesized and then cloned into pUC57 (GenScript), and the resulting plasmid was named pUC57-EFmvaE.

1-1-2) Chemical synthesis of Enterococcus faecalis-derived mvaS gene The base sequence of Enterococcus faecalis-derived mvaS encoding hydroxymethyl-CoA synthase, and the amino acid sequence thereof are already known (base number: ACFSON. (142 .. 1293), ACCESSION number of amino acid sequence: AAG02438) (J. Bacteriol. 182 (15), 4319-4327 (2000)). The amino acid sequence of Enterococcus faecalis-derived mvaS protein and the base sequence of the gene are shown in SEQ ID NO: 8 and SEQ ID NO: 9, respectively. The mvaS gene was transformed into E. coli. E. coli for efficient expression in E. coli. An mvaS gene optimized for E. coli codon usage was designed and named EFmvaS. This base sequence is shown in SEQ ID NO: 10. The mvaS gene was chemically synthesized and then cloned into pUC57 (GenScript), and the resulting plasmid was named pUC57-EFmvaS.

1-1-3) Construction of arabinose-inducible mvaES expression vector An arabinose-inducible mevalonate pathway upstream gene expression vector was constructed by the following procedure. E. coli by PCR using the plasmid pKD46 as a template and the synthetic oligonucleotides shown in SEQ ID NO: 11 and SEQ ID NO: 12 as primers. A PCR fragment containing Para comprising the araC and araBAD promoter sequences from E. coli was obtained. A PCR fragment containing the EFmvaE gene was obtained by PCR using the plasmid pUC57-EFmvaE as a template and the synthetic oligonucleotides shown in SEQ ID NO: 13 and SEQ ID NO: 14 as primers. A PCR fragment containing the EFmvaS gene was obtained by PCR using the plasmid pUC57-EFmvaS as a template and the synthetic oligonucleotides shown in SEQ ID NO: 15 and SEQ ID NO: 16 as primers. A PCR fragment containing the Ttrp sequence was obtained by PCR using the plasmid pSTV-Ptac-Ttrp as a template and the synthetic oligonucleotides shown in SEQ ID NO: 17 and SEQ ID NO: 18 as primers. Prime Star polymerase (manufactured by Takara Bio Inc.) was used for PCR to obtain these four PCR fragments. The reaction solution was prepared according to the composition attached to the kit, and 30 cycles of reaction at 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, and 72 ° C. for 1 minute / kb were performed. Synthetic oligonucleotides shown in SEQ ID NO: 11 and SEQ ID NO: 14 using a PCR product containing purified Para and a PCR product containing EFmvaE gene as a template, and a PCR product containing purified EFmvaS gene and a PCR product containing Ttrp as a template. PCR was performed using 15 and the synthetic oligonucleotide shown in SEQ ID NO: 18 as primers. As a result, PCR products containing Para and EFmvaE genes, EFmvaS and Ttrp were obtained. Plasmid pMW219 (Nippon Gene) was digested with SmaI according to a conventional method. A PCR product containing pMW219 and purified Para and EFmvaE genes, and a PCR product containing EFmvaS gene and Ttrp after SmaI digestion were ligated using In-Fusion HD Cloning Kit (Clontech). The resulting plasmid was named pMW-Para-mvaES-TTrp.

1-2) Construction of pTrc-KKDyI-ispS (K) First, construction of an expression vector including a sequence in which mevalonate kinase, phosphomevalonate kinase, diphosphomevalonate decarboxylase and isopentenyl diphosphate delta isomerase are linearly arranged is performed. The in-fusion cloning method was used. Mevalonate kinase and phosphomevalonate kinase sequences were amplified by the PCR method using pUC-mvk-pmk (see Example 7-2 of WO2013 / 179722A1) as a template and using primers consisting of the nucleotide sequences of SEQ ID NOS: 19-22. PCR using pTWV-dmd-yidi (see Example 7-2 of WO2013 / 179722A1) as a template and a primer comprising the nucleotide sequences of SEQ ID NOS: 19 to 22 and diphosphomevalonate decarboxylase and isopentenyl diphosphate delta isomerase After amplification by the method, an expression plasmid was constructed in which four enzyme genes were arranged in a linear form by cloning into the pTrcHis2B vector by the In-fusion cloning method. PrimeSTAR HS DNA polymerase sold by Takara Bio Inc. is used as a PCR enzyme, 98 ° C., 2 minutes, (98 ° C., 10 seconds, 52 ° C., 5 seconds, 72 ° C., 1 minute / kb) × 30 cycles The reaction was conducted at 72 ° C. for 10 minutes. The PCR fragment was inserted into the pTrcHis2B vector treated with restriction enzymes NcoI and PstI by the in-fusion cloning method to construct an expression vector. E. E. coli JM109 was transformed and a clone having the target sequence length was selected, followed by plasmid extraction according to a conventional method to confirm the sequence. The constructed expression vector was named pTrc-KKDyI (α). The base sequence of pTrc-KKDyI (α) is shown in SEQ ID NO: 23.

Subsequently, the construction of the plasmid pTrc-KKDyI-ispS (K) in which IspS (K) was added to the obtained pTrc-KKDyI (α) (SEQ ID NO: 23) was performed by the following procedure.
pTrc-KKDyI (α) was digested with the restriction enzyme PstI (Takara Bio Inc.) to obtain pTrc-KKDyI (α) / PstI. PCR was carried out using Prime Star Polymerase (manufactured by Takara Bio Inc.) using pUC57-ispSK as a template, pTrcKKDyIkSS_6083-10-1 (SEQ ID NO: 24) and pTrcKKDyIkSA_6083-10-2 (SEQ ID NO: 25) as primers. The reaction solution was prepared according to the composition attached to the kit, and the reaction was carried out for 30 cycles of 98 ° C. for 10 seconds, 54 ° C. for 20 seconds, and 68 ° C. for 120 seconds. As a result, a PCR product containing the IspSK gene was obtained. Thereafter, the purified IspSK gene fragment and pTrc-KKDyI (α) / PstI were ligated using In-Fusion HD Cloning Kit (Clontech). The obtained plasmid was designated as pTrc-KKDyI-ispS (K) (SEQ ID NO: 26).

1-3) Construction of pSTV-Ptac-ispS-Mmamvk 1-3-1) Chemical Synthesis of Methanosarcina mazei Mevalonate Kinase From Methanosarcina mazei Go1, the base sequence of mevalonate kinase and the amino acid sequence are already known ( ACCESSION number of the nucleotide sequence: NC — 000039.1.1 (2101873 ... 2102778, LOCUS TAG MM — 1762, amino acid sequence number: NP — 633786.1)). The amino acid sequence of the MVK protein derived from Methanosarcina mazei and the base sequence of the gene are shown in SEQ ID NO: 27 and SEQ ID NO: 28, respectively. The MVK gene was transformed into E. coli. E. coli for efficient expression in E. coli. An MVK gene optimized for E. coli codon usage was designed and named Mmamvk. The base sequence of Mmamvk is shown in SEQ ID NO: 29. The Mmamvk gene was chemically synthesized and then cloned into pUC57 (GenScript), and the resulting plasmid was named pUC57-Mmamvk.

1-3-2) Pueraria montana var. Construction of isobarene synthase from lobata and MVK gene expression E. A plasmid for expressing the IspSK gene and the Mmamvk gene in E. coli was constructed by the following procedure. PCR was performed using Prime Star polymerase (manufactured by Takara Bio Inc.) using pUC57-IspSK as a template and a synthetic oligonucleotide consisting of the nucleotide sequences of SEQ ID NO: 30 and SEQ ID NO: 31 as primers. The reaction solution was prepared according to the composition attached to the kit, and 40 cycles of reaction at 98 ° C. for 10 seconds, 54 ° C. for 20 seconds, and 68 ° C. for 120 seconds were performed. As a result, a PCR product containing the IspSK gene was obtained. Similarly, pSTV28-Ptac-Ttrp was subjected to PCR using Prime Star polymerase (manufactured by Takara Bio Inc.) using a synthetic oligonucleotide consisting of the nucleotide sequences of SEQ ID NO: 32 and SEQ ID NO: 33 as a primer. The reaction solution was prepared according to the composition attached to the kit, and 40 cycles of reaction at 98 ° C. for 10 seconds, 54 ° C. for 20 seconds, and 68 ° C. for 210 seconds were performed. As a result, a PCR product containing pSTV28-Ptac-Ttrp was obtained. Thereafter, the purified IspSK gene fragment and the pSTV28-Ptac-Ttrp PCR product were ligated using In-Fusion HD Cloning Kit (Clontech). The obtained plasmid for expressing the IspSK gene was named pSTV28-Ptac-IspSK. Next, PCR was performed using Prime Star polymerase (manufactured by Takara Bio Inc.) using pUC57-Mmamvk as a template and a synthetic oligonucleotide consisting of the nucleotide sequences of SEQ ID NOs: 34 and 35 as primers. The reaction solution was prepared according to the composition attached to the kit, and 30 cycles of reaction at 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, and 72 ° C. for 1 minute / kb were performed. As a result, a PCR product containing the Mmamvk gene was obtained. Similarly, pSTV28-Ptac-IspSK was subjected to PCR using Prime Star polymerase (manufactured by Takara Bio Inc.) with a synthetic oligonucleotide consisting of the nucleotide sequences of SEQ ID NO: 36 and SEQ ID NO: 37 as primers. The reaction solution was prepared according to the composition attached to the kit, and 30 cycles of reaction at 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, and 72 ° C. for 1 minute / kb were performed. As a result, a PCR product containing pSTV28-Ptac-IspSK was obtained. Thereafter, the purified Mmamvk gene and the pSTV28-Ptac-IspSK PCR product were ligated using In-Fusion HD Cloning Kit (Clontech). The obtained plasmid for expression of IspSK gene and Mmamvk gene was named pSTV28-Ptac-ispSK-Mmamvk.

1-4) Construction of pMW-Ptac-Mclmvk-Ttrp Next, an MVK expression plasmid (pMW-Ptac-mvk-Ttrp) was constructed.
First, the base sequence and amino acid sequence of Methanosaeta concilii-derived mevalonate kinase are already known (base sequence ACCESSION: NC — 01546.16.1 (2189055.10.22190004, LOCUS TAG MCON — 2559), amino acid sequence ACCESSION number: YP_84. 1 (GenPept)). The amino acid sequence of MVK protein derived from Methanosaeta concilii and the base sequence of the gene are shown in SEQ ID NO: 38 and SEQ ID NO: 39, respectively. The MVK gene was transformed into E. coli. E. coli for efficient expression in E. coli. An MVK gene optimized for the codon usage of E. coli was designed and named Mclmvk. The base sequence of Mclmvk is shown in SEQ ID NO: 40. The Mclmvk gene was chemically synthesized and then cloned into pUC57 (GenScript), and the resulting plasmid was named pUC57-Mclmvk.

PCR was performed using Prime Star polymerase (manufactured by Takara Bio Inc.) using pUC57-Mclmvk as a template and a synthetic oligonucleotide consisting of base sequences of Mcl_mvk_N (SEQ ID NO: 41) and Mcl_mvk_C (SEQ ID NO: 42) as primers. The reaction solution was prepared according to the composition attached to the kit, and 30 cycles of reaction at 98 ° C. for 10 seconds, 55 ° C. for 5 seconds and 72 ° C. for 1 minute / kb were performed. As a result, a PCR product containing the Mclmvk gene was obtained. Similarly, Prime Star Polymerase (manufactured by Takara Bio Inc.) was prepared using pMW219-Ptac-Ttrp (see WO20133066964A1), synthetic oligonucleotide consisting of the base sequences of PtTt219f (SEQ ID NO: 43) and PtTt219r (SEQ ID NO: 44) as primers. PCR was performed. The reaction solution was prepared according to the composition attached to the kit, and 30 cycles of reaction at 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, and 72 ° C. for 1 minute / kb were performed. As a result, a PCR product containing pMW219-Ptac-Ttrp was obtained. Thereafter, the purified Mclmvk gene and the PCR product of pMW219-Ptac-Trp were ligated using In-Fusion HD Cloning Kit (Clontech). The obtained plasmid for expression of Mclmvk gene was named pMW-Ptac-Mclmvk-Ttrp.

1-5) Construction of an integrative conditional replication plasmid carrying genes upstream and downstream of the mevalonate pathway In order to construct an integrative plasmid carrying genes upstream and downstream of the mevalonate pathway, pAH162-λattL-TcR-λattR vector (Minaeva NI et al., BMC Biotechnol. 2008; 8:63) was used.

The KpnI-SalI fragment of pMW-Para-mvaES-Ttrp was cloned into the SphI-SalI recognition site of pAH162-λattL-TcR-λattR. As a result, E.I. E. coli under the control of the Para Para promoter and repressor gene araC. The pAH162-Para-mvaES plasmid carrying the faecalis-derived mvaES operon was constructed (FIG. 1).

S. The Ecl136II-SalI fragment of the pTrc-KKDyI-ispS (K) plasmid containing the coding portion of the mevalonate kinase, phosphomevalonate kinase, diphosphomevalonate decarboxylase, and IPP isomerase genes from cerevisiae, and the kasp-derived ispS gene, was transformed into pAH162-λattL -Subcloning into the SphI-SalI site of TcR-λattR and the resulting plasmid was named pAH162-KKDyI-ispS (K) (Figure 2).

A BglII-EcoRI fragment of pSTV28-Ptac-ispS-Mamvk containing the ispS (M) and mvk (M.mazei) genes under the control of Ptac is recognized by the BamHI-Ecl136II site of the integrating vector pAH162-λattL-TcR-λattR. Subcloned into. The resulting plasmid pAH162-Ptac-ispS (M) -mvk (Mma) is shown in FIG.

1-6) P. coli harboring attB sites of phi80 phage at different sites in the genome Construction of ananatis SC17 (0) Derivatives P. cerevisiae carrying the attB site of phi80 phage substituted with ampC gene, ampH gene or crt operon. Ananatis SC17 (0) derivatives were constructed (annotated complete genome sequence of P. ananatis AJ13355 is available as PRJDA 162073 or GenBank accession numbers AP01232.1 and AP012033.1). In order to obtain these strains, λRed-dependent integration of a PCR amplified DNA fragment carrying attLphi80-kan-attRphi80 adjacent to a 40 bp region homologous to the target site in the genome was performed by a previously reported method (Katashkina JI et al. , BMC Mol Biol. 2009; 10:34). After electroporation, the cells were cultured on L-agar containing 50 mg / l kanamycin. The DNA fragments used to replace the ampC and ampH genes and the crt operon with attLphi80-kan-attRphi80 were amplified in reactions with oligonucleotides 1 and 2, 3 and 4, and 5 and 6 (Table 3), respectively. The pMWattphi plasmid (Minaeva NI et al., BMC Biotechnol. 2008; 8:63) was used as a template in these reactions. The resulting integrants were named SC17 (0) ΔampC :: attLphi80-kan-attRphi80, SC17 (0) Δamph :: attLphi80-kan-attRphi80, and SC17 (0) Δcrt :: attLphi80-kan-attRphi80. Oligonucleotides 7 and 8, 9 and 10, and 11 and 12 (Table 3) were converted to SC17 (0) ΔampC :: attLphi80-kan-attRphi80, SC17 (0) ΔampHpH :: attLphi80-kan-attRphi80, and SC17, respectively. (0) Δcrt :: attLphi80-kan-attRphi80 strain was used for verification by PCR. The resulting ΔampC :: attLphi80-kan-attRphi80, ΔampH :: attLphi80-kan-attRphi80, and Δcrt :: attLphi80-kan-attRphi80, maps of the genome variants, FIG. 4A), FIG. 5A) and FIG. 6A, respectively. ).

Curing of the constructed strain from the kanamycin resistance marker was performed using the pAH129-cat helper plasmid according to the method of the previous report (Andreeva IG et al., FEMS Microbiol Lett. 2011; 318 (1): 55-60). . Oligonucleotides 7 and 8, 9 and 10, and 11 and 12 (Table 3) were obtained from SC17 (0) ΔampC :: attBphi80, SC17 (0) ΔampH :: attBphi80, and SC17 (0) Δcrt: : Used for verification of attBphi80 strain by PCR.

1-7) Construction of ISP3-S strain The plasmid of pAH162-KKDyI-ispS (K) described in 1-5) was previously reported (Andrewa IG et al., FEMS Microbiol Lett. 2011; 318 (1): 55. According to the procedure of −60), the helper plasmid pAH123-cat was used to integrate into the SC17 (0) ΔampC :: attBphi80 strain described in 1-6). Oligonucleotides 13 and 7, and 14 and 8 (Table 3) were used for PCR verification of the resulting integrants. The obtained SC17 (0) ΔampC :: pAH162-KKDyI-ispS (K) strain was obtained by following the procedure described previously (Katashkina JI et al., BMC Mol Biol. 2009; 10:34). Using the pMWintxis-cat helper plasmid carrying the gene, the vector portion of pAH162-KKDyI-ispS (K) was cured. As a result, an SC17 (0) ΔampC :: KKDyI-ispS (K) strain was obtained. Oligonucleotides 7 and 15 (Table 3) were used for PCR verification of kanamycin sensitive derivatives. The construction of SC17 (0) ΔampC :: KKDyI-ispS (K) is shown in FIG. 4C.

Genomic DNA isolated from the SC17 (0) Δamph :: attLphi80-kan-attRphi80 strain using the GeneElute bacterial genomic DNA kit (Sigma) was previously reported (Katashkina JI et al., BMC Mol Biol. 2009; 10: According to the method of chromosome electroporation of 34), the SC17 (0) ΔampC :: KKDyI-ispS (K) strain was electroporated. Transfer of the ΔampH :: attLphi80-kan-attRphi80 mutation was confirmed by PCR using primers 9 and 10.

The kanamycin resistance marker was removed from the obtained strain using a phi80 Int / Xis-dependent method (Andrewa IG et al., FEMS Microbiol Lett. 2011; 318 (1): 55-60). In the obtained KmS recombinant, the ΔampC :: KKDyI-ispS (K) variant was verified by PCR using primers 7 and 15, and then SC17 (0) ΔampC :: KKDyI-ispS (K) ΔamppH :: attBphi80 A stock was selected.

The pAH162-Para-mvaES plasmid was transformed into SC17 (0) ΔampC :: KKDyI-ispS using the pAH123-cat helper plasmid (Andreweva IG et al., FEMS Microbiol Lett. 2011; 318 (1): 55-60). (K) Incorporated into ΔampH :: attBphi80. Oligonucleotides 13 and 9 and oligonucleotides 14 and 10 (Table 3) were used for PCR verification of the resulting integrants. From this integrant, the vector portion of pAH162-Para-mvaES was removed using a phage λ Int / Xis-dependent technology (Katashkina JI et al., BMC Mol Biol. 2009; 10:34). The removal of the vector from the chromosome was confirmed by PCR using primers 9 and 16. As a result, SC17 (0) ΔampC :: KKDyI-ispS (K) ΔampH :: Para-mvaES containing no marker was obtained. The construct of ΔampH :: Para-mvaES chromosome variant is shown in FIG.

The pAH162-Ptac-ispS (M) -mvk (Mma) plasmid described in 1-5) was prepared using the protocol (Andrewa IG et al., FEMS Microbiol Lett. 2011; 318 (1): 55- 60) was used to integrate into the genome of SC17 (0) Δcrt :: attBphi80. Plasmid integration was confirmed by polymerase chain reaction using primers 11 and 13 and 12 and 14.

The chromosome variant SC17 (0) Δcrt :: pAH162-Ptac-ispS (M) -mvk (Mma) thus constructed has been reported (Katashkina JI et al., BMC Mol Biol. 2009; 10:34). Was transferred to the SC17 (0) ΔampC :: KKDyI-ispS (K) ΔampH :: Para-mvaES strain. The resulting integrant was transformed into pAH162-Ptac-ispS (M) -mvk (Mma) using phage λ Int / Xis dependent technology (Katashkina JI et al., BMC Mol Biol. 2009; 10:34). The vector part was removed. The structure of the final construct Δcrt :: Ptac-ispS (M) -mvk (Mma) (FIG. 6D) was confirmed by PCR using primers 11 and 17.

When the integrative expression cassette introduced into this final strain was verified by PCR, S. It was found that some unexpected rearrangements were made in the 5'-part of the KKDyI operon including the MVK, PMK, MVD and yldI genes from cerevisiae. In order to repair this cassette, SC17 (0) ΔampC :: using GeneElute bacterial genomic DNA kit (Sigma) according to a previously reported method (Katashkina JI et al., BMC Mol Biol. 2009; 10:34). Genomic DNA isolated from the pAH162-KKDyI-ispS (K) strain was electroporated into this strain. The resulting strain contained all the genes necessary for isoprene production.

After removal from the vector portion of pAH162-KKDyI-ispS (K) (see above) in a phage λ Int / Xis dependency, an ISP3-S strain (P. ananatis SC17 (0) ΔampC :: attLphi80- containing no marker) KKDyI-ispS (K) -attRphi80 ΔampH :: attLphi80-Para-mvaES-attRphi80 Δcrt :: attLphi80-Ptac-ispS (M) -mvk (Mma) -attRphi80) was obtained.

1-8) Construction of an integrative plasmid carrying a different mevalonate kinase gene The KpnI-BamHI fragment of pMW-Ptac-Mclmvk-Ttp was subcloned into the KpnI-Ecl136II recognition site of the pAH162-λattL-TcR-λattR integration vector did. As a result, M. coli under the control of the tac promoter. The pAH162-Ptac-Mclmvk plasmid carrying the MVK gene from concili was constructed.

1-9) Construction of AG10265 strain The pAH162-Ptac-Mclmvk plasmid described in 1-8) was used in the protocol of the previously reported (Andrewa IG et al., FEMS Microbiol Lett. 2011; 318 (1): 55-60). Therefore, it was integrated into the genome of SC17 (0) Δcrt :: attBphi80 using the pAH123-cat helper plasmid.

The constructed Δcrt :: pAH123-Ptac-mvk (M.palladicola) chromosomal variant was transformed into ISP3-S via electroporation of genomic DNA isolated from SC17 (0) Δcrt :: pAH162-Ptac-Mclmvk. Strain [see 1-7]. The resulting integrant was designated as AG10265 (P. ananatis SC17 (0) ΔampC :: attLphi80-KKDyI-ispS (K) -attRphi80 ΔamppH :: attLphi80-Para-mvaES-attRphi80 ΔcrtLt80 Ptac-Mclmvk-attRphi80).

Example 2: Preparation of isoprene synthase expression plasmid derived from each plant species 2-1) Pueraria montana var. Chemical synthesis of isobarene synthase from Lovata montana var. The base sequence and amino acid sequence of isoprene synthase from Lobata are already known (ACCESSION: AAQ84170: P. montana var. lobata (kudzu) isoprene synthase (IspS)). The IspS gene was transformed into E. coli. E. coli for efficient expression in intestinal bacteria such as E. coli. An IspS gene that was optimized for E. coli codon usage and further cleaved from the chloroplast translocation signal was designed and named IspSK. The IspSK gene was chemically synthesized and then cloned into pUC57 (GenScript), and the resulting plasmid was named pUC57-IspSK. The sequences of the IspSK gene and plasmid pUC57-IspSM are the same as those disclosed in WO2013 / 179722A1.

2-2) Chemical synthesis of isoprene synthase from Mucuna pururiens (Mucuna) Based on the base sequence of pururiens-derived isoprene synthase, E. An IspS gene optimized for the codon usage of E. coli was designed, and the one obtained by cleaving the chloroplast translocation signal was named IspSM. The IspSM gene was chemically synthesized and then cloned into pUC57 (GenScript), and the resulting plasmid was named pUC57-IspSM. The sequences of the IspSM gene and plasmid pUC57-IspSM are the same as those disclosed in WO2013 / 179722A1.

2-3) Chemical synthesis of isoprene synthase derived from True myrtle Based on the base sequence of myrtle-derived isoprene synthase, E. An IspS gene optimized for the codon usage of E. coli was designed, and the chloroplast translocation signal cleaved (however, having a methionine residue at the N-terminus) was named IspSU. The base sequence of IspSU is shown in SEQ ID NO: 62. Each IspSU gene was chemically synthesized and then cloned into pUC57 (manufactured by GenScript). The resulting plasmid was named pUC57-IspSU.

2-4) Construction of expression plasmid pSTV28-Ptac-Ttrp An expression plasmid pSTV28-Ptac-Ttrp for expressing IspS derived from each plant species in intestinal bacteria such as E. coli was constructed. First, the tac promoter (synonymous: Ptac) region (deBoer, et al., (1983) Proc. Natl. Acad. Sci. USA, 80, 21-25) and E. coli. E. coli-derived tryptophan operon terminator (synonymous Ttrp) region (Wu et al., (1978) Proc. Natl. Acad. Sci. USA, 75, 5442-5446). A DNA fragment (Ptac-Ttrp) having a BamHI site at the 3 ′ end was chemically synthesized. The obtained Ptac-Ttrp DNA fragment was digested with KpnI and BamHI, and similarly ligated with pSTV28 (manufactured by Takara Bio Inc.) digested with KpnI and BamHI by a ligation reaction with DNA ligase. The resulting plasmid was named pSTV28-Ptac-Ttrp. In this plasmid, the expression of the IspS gene can be amplified by cloning the IspS gene downstream of Ptac. Note that pSTV28-Ptac-Ttrp is the same as that disclosed in WO2013 / 179722A1.

2-5) Construction of IspS gene expression plasmid derived from each plant species Plasmids for expressing the IspSK gene, IspSM gene, and IspSU) gene in intestinal bacteria such as E. coli were constructed by the following procedure. Using pUC57-IspSK as a template, a synthetic oligonucleotide consisting of the nucleotide sequences of SEQ ID NO: 63 and SEQ ID NO: 64, using pUC57-IspSM as a template, a synthetic oligonucleotide consisting of the nucleotide sequences of SEQ ID NO: 65 and SEQ ID NO: 66, and further pUC57- PCR was performed using Prime Star polymerase (manufactured by Takara Bio Inc.) using IspSU as a template and a synthetic oligonucleotide consisting of the nucleotide sequences of SEQ ID NO: 67 and SEQ ID NO: 68 as primers. The reaction solution was prepared according to the composition attached to the kit, and 40 cycles of reaction at 98 ° C. for 10 seconds, 54 ° C. for 20 seconds, and 68 ° C. for 120 seconds were performed. As a result, a PCR product containing an IspSK gene, an IspSM gene, and an IspSU gene was obtained. Similarly, pSTV28-Ptac-Ttrp was subjected to PCR using Prime Star polymerase (manufactured by Takara Bio Inc.) using a synthetic oligonucleotide consisting of the nucleotide sequences of SEQ ID NO: 69 and SEQ ID NO: 70 as a primer. The reaction solution was prepared according to the composition attached to the kit, and 40 cycles of reaction at 98 ° C. for 10 seconds, 54 ° C. for 20 seconds, and 68 ° C. for 210 seconds were performed. As a result, a PCR product containing pSTV28-Ptac-Ttrp was obtained. Thereafter, the purified IspSK gene, IspSM gene, and IspSU gene gene fragment and the PCR product of pSTV28-Ptac-Ttp were ligated using In-Fusion HD Cloning Kit (Clontech). The resulting IspSK gene expression plasmid was named pSTV28-Ptac-IspSK, the IspSM gene expression plasmid was named pSTV28-Ptac-IspSM, and the IspSU gene expression plasmid was named pSTV28-Ptac-IspSU. The plasmids pSTV28-Ptac-IspSK and STV28-Ptac-IspSM are the same as those disclosed in WO2013 / 179722A1.

Example 3: Production of isoprene monomer using Pantoea ananatis 3-1) Construction of strain An isoprene-producing bacterium in which isoprene synthase derived from Kudzu, Mucuna and Ginkgo biloba was introduced into the constructed AG10265 according to a conventional method (Table) 4).

3-2) P.I. Effects of introduction of isoprene synthase derived from each plant species in ananasis isoprene-producing bacteria The ability to produce isoprene from glucose in ananatis isoprene-producing bacteria was compared. AG10265 / IspSK, AG10265 / IspSM, and AG10265 / IspSU were uniformly applied to an LB plate containing 60 (mg / L) chloramphenicol and cultured at 34 ° C. for 18 hours. From the obtained plate, 1 platinum loop of microbial cells was inoculated into 1 mL of M9 glucose medium in a headspace vial and sealed with a capbutyl rubber septum for headspace vial (CRIMPS cat # B0104240 manufactured by Perkin Elmer). Incubation was performed at 30 ° C. for 48 hours with a reciprocating shake culture apparatus (120 rpm). The composition of the M9 glucose medium is as shown in Table 5.

After completion of the culture, the isoprene concentration in the head space of the vial was measured by gas chromatography. The OD value was measured at 600 nm with a spectrophotometer (HITACHI U-2900). Table 6 shows the isoprene concentration per OD at the end of the culture of each strain.

The analysis conditions for gas chromatography are described below.
Headspace Sampler (Turbo Matrix 40 manufactured by Perkin Elmer)
Vial insulation temperature 40 ° C
Vial incubation time 30 min
Pressurization time 3.0min
Injection time 0.02min
Needle temperature 70 ℃
Transfer temperature 80 ℃
Carrier gas pressure (high purity helium) 124kPa

Gas chromatography (Shimadzu GC-2010 Plus AF)
Column (Rxi (registered trademark) -1 ms: length 30 m, inner diameter 0.53 mm, liquid phase film thickness 1.5 μm cat # 13370)
Column temperature 37 ° C
Pressure 24.8kPa
Column flow rate 5mL / min
Inflow method Split 1: 0 (actual measurement 1:18)
Transfer flow rate 90mL
GC injection volume 1.8mL (transfer flow rate x injection time)
Sample injection volume to the column 0.1 mL
Inlet temperature 250 ℃
Detector FID (hydrogen 40 mL / min, air 400 mL / min, makeup gas helium 30 mL / min)
Detector temperature 250 ° C

Preparation of isoprene standard sample Reagent isoprene (specific gravity 0.681) was diluted 10, 100, 1000, 10000, and 100,000 times with cooled methanol to prepare a standard solution for addition. Thereafter, 1 μL of each standard solution for addition was added to a headspace vial containing 1 mL of water to prepare a standard sample.

The above strain expresses a combination of IspSK introduced in one copy into the production bacterial genome and any one isoprene synthase (IspSM, IspSK, or IspSU) introduced as a plasmid. As a background, although certain isoprene production derived from IspSK on the production bacterial genome is observed, it is presumed that all three strains are of the same level and have significantly low isoprene production. Therefore, the above value is considered to be derived from the difference in isoprene productivity of the three types of isoprene synthase.

From the results of Table 6, the isoprene production amounts were AG10265 / IspSU, AG10265 / IspSK, and AG10265 / IspSM in descending order. From the above results, P.I. Among the ananatis isoprene-producing bacteria, the strain into which the ginseng-derived isoprene synthase was introduced showed the highest isoprene-producing ability.

Example 4 Comparison of Magnesium Ion (Mg Ion) Concentration Dependence of Isoprene Synthesis Activity The preparation method of the existing species Mucuna-derived isoprene synthase IspS (IspSM) is as described in the prior patent (Patent Document: WO2013 / 179722A1). is there. Here, a method for preparing the ginkgo isoprene synthase IspS (IspSU) will be described.

4-1) Construction of plasmid for expression A plasmid for large-scale expression of isoprene synthase (IspSU) from Ginkgo biloba was constructed according to the following procedure. First, for the vector part, pCold-TF (TaKaRa Bio, # 3365, sequence information is GenBank / EMBL / DDBJ accession ID AB213654) was used as a template, and the synthetic oligonucleotides shown in SEQ ID NO: 71 and SEQ ID NO: 72 were used as primers. Amplified by the PCR method. The DNA fragment encoding IspSU was amplified by PCR using pUC57-IspSU (manufactured by Genescript) as a template and the synthetic oligonucleotides shown in SEQ ID NO: 73 and SEQ ID NO: 74 as primers. PrimeSTAR HS (manufactured by TaKaRa Bio Inc.) was used as a polymerase for PCR. The reaction solution was prepared according to the composition attached to the kit, and the reaction conditions were 95 ° C. for 10 seconds, 55 ° C. for 5 seconds, and 72 ° C. for 6 minutes, and repeated 30 times. The obtained DNA fragments were ligated using In-Fusion HD Cloning Kit (TaKaRa Bio). The constructed plasmid was named pCold-TF-IspSU. The base sequence of pCold-TF-IspSU is shown in SEQ ID NO: 75.
In this expression vector, isoprene synthase was designed to be expressed as a protein fused with a trigger factor (TF) at the N-terminus. The fusion protein of TF and isoprene synthase was named TF-IspS. The fusion protein of Ginkgo biloba isoprene synthase was named TF-IspSU.
pCold-TF-IspSU E. coli BL21 (DE3) (one shot BL21 (DE3), manufactured by Life Technologies) was transformed by the heat shock method. After heat shock at 42 ° C. for 30 seconds, recovery culture using an SOC medium was performed at 37 ° C. for 1 hour at 120 rpm. Thereafter, the entire amount was seeded on an LB plate containing 0.1 mg / L of ampicillin, and statically cultured at 37 ° C. for 14 hours.

4-2) Culture for large-scale preparation of isoprene synthase A colony formed on the plate is picked and inoculated into 5 mL LB medium containing 0.1 mg / L of ampicillin, and then 37 until OD660 = 1.0. The cells were cultured at 200 ° C. and 200 ° C. After confirming that the OD value was reached, the entire amount was inoculated into a 250 mL Sakaguchi flask with 100 mL of LB medium containing ampicillin 100 mg / L, and cultured at 37 ° C. and 120 rpm until OD660 = 1.0 was reached. did. Thereafter, 1M isopropyl-β-thiogalactopyranoside (IPTG: Nacalai Tesque) was added to a final concentration of 1 mM and cultured overnight at 15 ° C. and 100 rpm. After completion of the culture, the cells were collected by centrifugation at 800 × g for 10 minutes, and stored at −80 ° C. until purification.

4-3) Purification of isoprene synthase As described above, TF-IspSU was purified in the same manner as the TF-IspSM purification method described in the literature (patent document: WO2013 / 179722A1). That is, the cells obtained from 100 mL of broth after completion of the culture were suspended in 240 mL of a disruption buffer consisting of 50 mM phosphate buffer (pH 8) and 500 mM NaCl, and an ultrasonic disrupter (Sonifier 250, manufactured by Baransan) was used. Ultrasonic crushing was performed on ice for 8 minutes under the conditions of a duty cycle of 50% and an output control of 6. After ultrasonic crushing, a crushing supernatant was obtained by centrifugation at 20000 × g for 20 minutes. The following purification operations were all performed at 4 ° C. A polyprep column (manufactured by BioRad) was applied with His-select Nickel affinity gel (manufactured by Sigma) corresponding to a bed volume of 2 mL and filled by a gravity method. Next, 10 mL of crushing buffer was applied and equilibrated by the gravity method. The whole amount of the crushed supernatant after ultrasonic pulverization was applied to this column, and TF-IspSU was adsorbed onto the column.
After confirming that all of the applied disruption supernatant had passed through the column, the column was washed with 10 mL of disruption buffer. Further, the column was additionally washed with 10 mL of an activity measurement buffer comprising 50 mM tris-HCl (pH 8.0) and 15 mM MgCl ₂ . Thereafter, 4 mL of a washing solution composed of 50 mM tris-HCl (pH 8.0), 15 mM MgCl ₂ and 10 mM imidazole was applied to the column, and the passing solution was discarded. Finally, an eluate composed of 50 mM tris-HCl (pH 8.0), 15 mM MgCl ₂ and 200 mM imidazole was applied to a 4 mL column to elute TF-IspS. The TF-IspS after elution was concentrated by centrifugal filter filtration (Amicon ultra MWCO 100k (Millipore)).
Subsequently, the TF of the TF-IspSU fusion protein was cleaved. For the cutting, Factor Xa (manufactured by Novagen) was used. The reaction buffer consisted of 50 mM tris-HCl (pH 8.0), 100 mM NaCl, 5 mM CaCl ₂ , 26 U Factor Xa was added, and the reaction was performed at 4 ° C. for 14 hours. By passing the reaction solution after completion of the reaction through a His-Select Nickel Affinity gel, TF was removed from the reaction solution using the fact that only TF was adsorbed on the His-Select Nickel Affinity gel. Subsequently, IspS was concentrated using a gel filtration column (Amicon ultra MWCO 50k, Millipore). When the obtained concentrated solution was developed with NuPAGE 4-12% (manufactured by Life Technologies), a band having the same migration degree as the IspSM band was detected in the lane to which IspSU was applied (FIG. 7).
The IspSU solution thus obtained was stored in a −80 ° C. freezer until the final concentration of 5% glycerol was added to the activity measurement buffer and used for activity measurement.

4-4) Comparison of Mg ion concentration dependence of isoprene synthase The dependence of Mg ion concentration when isoprene synthase catalyzes the reaction was investigated. IspSM and IspSU to be subjected to the reaction were thawed on ice, and then buffer exchange was performed to remove glycerol. The enzyme concentration used for the reaction was quantified by colorimetric determination on SDS-PAGE. The composition of the reaction solution is 50 mM tris-HCl, pH 8.0, 4 mM DMAPP (Dimethylallyl diphosphate: Cayman). In order to examine the Mg ion concentration dependency, 10 mM MgCl ₂ or 20 mM MgCl ₂ was added to the reaction solution as a metal ion. During the reaction, 1 μg of IspS was added to the reaction solution, and the final solution volume was adjusted to 50 μL. 50 μL of the reaction solution thus prepared was placed in a 0.2 mL PCR tube (manufactured by Nippon Genetics), and a hole was made in the lid with a needle (manufactured by Terumo). Next, the tube was put into a 22 mL vial (manufactured by PerkinElmer) and immediately sealed with a cap butyl rubber septum for headspace vial (manufactured by PerkinElmer). The isoprene formation reaction was carried out at 37 ° C. for 1 hour. The amount of generated isoprene was determined according to Example 3. The amount of isoprene generated measured under each condition was normalized with the amount of isoprene generated by IspM when 10 mM Mg ions were added, and is shown in Table 7. From Talbe1, when 20 mM Mg ions were added, IspSU and IspSM had equivalent isoprene conversion capacity per enzyme amount. On the other hand, when 10 mM Mg ion was added, the isoprene conversion ability of IspSU was about 1.9 times that of IspSM under the same conditions. From this, it became clear that IspSU has an excellent isoprene conversion ability under the reaction conditions in which 10 mM Mg ions are added.

The present invention is useful for the production of isoprene monomers that can be used as raw materials for synthetic rubber.

Claims (17)

1. A method for producing an isoprene monomer, comprising producing an isoprene monomer from dimethylallyl diphosphate in the presence of a protein selected from the group consisting of (A) to (F) below:
   (A) a protein comprising the full-length amino acid sequence of SEQ ID NO: 2;
   (B) a protein comprising a partial amino acid sequence consisting of amino acid residues 26 to 581 in the amino acid sequence of SEQ ID NO: 2 and further comprising a starting amino acid residue at the N-terminus;
   (C) a protein comprising an amino acid sequence having 90% or more identity with the full-length amino acid sequence of SEQ ID NO: 2 and having isoprene synthesis activity;
   (D) an amino acid sequence having 90% or more identity with a partial amino acid sequence consisting of amino acid residues 26 to 581 in the amino acid sequence of SEQ ID NO: 2, further comprising a starting amino acid residue at the N-terminus; A protein having isoprene synthesis activity;
   (E) a protein comprising an amino acid sequence in which one or several amino acid residues are deleted, substituted, added or inserted in the full-length amino acid sequence of SEQ ID NO: 2 and having isoprene synthesis activity; and (F) a sequence Including an amino acid sequence in which one or several amino acid residues are deleted, substituted, added or inserted in the partial amino acid sequence consisting of amino acid residues 26 to 581 in the amino acid sequence of No. 2, and at the N-terminus A protein further comprising a starting amino acid residue and having isoprene synthesis activity.
2. The method according to claim 1, wherein the protein is derived from Ginkgo biloba.
3. The method according to claim 1 or 2, wherein isoprene monomer is produced by culturing the transformant producing the protein.
4. The method according to any one of claims 1 to 3, wherein dimethylallyl diphosphate is supplied from a carbon source in the medium by culturing the transformant.
5. The method according to claim 3 or 4, wherein the transformant is a host cell comprising an expression unit comprising a polynucleotide selected from the group consisting of the following (a) to (g):
   (A) a polynucleotide comprising the full-length base sequence of SEQ ID NO: 1;
   (B) a polynucleotide comprising a partial base sequence consisting of nucleotide residues 76 to 1746 in the base sequence of SEQ ID NO: 1 and further comprising a start codon at the 5 ′ end;
   (C) a polynucleotide encoding a protein comprising a base sequence having 90% or more identity with the full-length base sequence of SEQ ID NO: 1 and having isoprene synthesis activity;
   (D) a base sequence having 90% or more identity to a partial base sequence consisting of nucleotide residues 76 to 1746 in the base sequence of SEQ ID NO: 1, further including a start codon at the 5 ′ end, and isoprene A polynucleotide encoding a protein having synthetic activity;
   (E) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the full-length base sequence of SEQ ID NO: 1 and encodes a protein having isoprene synthesis activity;
   (F) hybridizing under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to a partial nucleotide sequence comprising nucleotide residues 76 to 1746 in the nucleotide sequence of SEQ ID NO: 1, and starting codon at the 5 ′ end And (g) a degenerate variant of a polynucleotide selected from the group consisting of (a) to (f).
6. The method according to claim 5, wherein the host has the ability to synthesize dimethylallyl diphosphate through the methyl erythritol phosphate pathway.
7. The method according to claim 6, wherein the host is an Escherichia bacterium.
8. The method according to claim 7, wherein the host is Escherichia coli.
9. The method according to claim 5, wherein the host has the ability to synthesize dimethylallyl diphosphate by the mevalonate pathway.
10. The method according to claim 9, wherein the host is a Pantoea bacterium.
11. The method according to claim 10, wherein the host is Pantoea ananatis.
12. A method for producing an isoprene polymer comprising the following (I) and (II):
    (I) producing an isoprene monomer by the method according to any one of claims 1 to 11;
    (II) polymerizing isoprene monomers to produce isoprene polymers.
13. A protein selected from the group consisting of (A) to (F) below:
    (A) a protein comprising the full-length amino acid sequence of SEQ ID NO: 2;
    (B) a protein comprising a partial amino acid sequence consisting of amino acid residues 26 to 581 in the amino acid sequence of SEQ ID NO: 2 and further comprising a starting amino acid residue at the N-terminus;
    (C) a protein comprising an amino acid sequence having 90% or more identity with the full-length amino acid sequence of SEQ ID NO: 2 and having isoprene synthesis activity;
    (D) an amino acid sequence having 90% or more identity with a partial amino acid sequence consisting of amino acid residues 26 to 581 in the amino acid sequence of SEQ ID NO: 2, further comprising a starting amino acid residue at the N-terminus; A protein having isoprene synthesis activity;
    (E) a protein comprising an amino acid sequence in which one or several amino acid residues are deleted, substituted, added or inserted in the full-length amino acid sequence of SEQ ID NO: 2 and having isoprene synthesis activity; and (F) a sequence Including an amino acid sequence in which one or several amino acid residues are deleted, substituted, added or inserted in the partial amino acid sequence consisting of amino acid residues 26 to 581 in the amino acid sequence of No. 2, and at the N-terminus A protein further comprising a starting amino acid residue and having isoprene synthesis activity.
14. A polynucleotide selected from the group consisting of (a) to (g) below:
    (A) a polynucleotide comprising the full-length base sequence of SEQ ID NO: 1;
    (B) a polynucleotide comprising a partial base sequence consisting of nucleotide residues 76 to 1746 in the base sequence of SEQ ID NO: 1 and further comprising a start codon at the 5 ′ end;
    (C) a polynucleotide encoding a protein comprising a base sequence having 90% or more identity with the full-length base sequence of SEQ ID NO: 1 and having isoprene synthesis activity;
    (D) a base sequence having 90% or more identity to a partial base sequence consisting of nucleotide residues 76 to 1746 in the base sequence of SEQ ID NO: 1, further including a start codon at the 5 ′ end, and isoprene A polynucleotide encoding a protein having synthetic activity;
    (E) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the full-length base sequence of SEQ ID NO: 1 and encodes a protein having isoprene synthesis activity;
    (F) hybridizing under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to a partial nucleotide sequence comprising nucleotide residues 76 to 1746 in the nucleotide sequence of SEQ ID NO: 1, and starting codon at the 5 ′ end And (g) a degenerate variant of a polynucleotide selected from the group consisting of (a) to (f).
15. An expression vector comprising the polynucleotide encoding the protein of claim 13, or the polynucleotide of claim 14, and a heterologous promoter operably linked thereto.
16. A host cell comprising the expression vector according to claim 15.
17. A method for producing a protein, comprising producing the protein using the host cell according to claim 16.

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