JP6572977B2 - Novel taxadiene oxidase and its use - Google Patents

Description

  The present invention relates to a novel taxadiene oxidase gene, a protein encoded by the gene, and use thereof.

  Paclitaxel is one of the important anticancer agents used clinically, and has been used for many years in the treatment of ovarian cancer, non-small cell lung cancer, breast cancer, stomach cancer, endometrial cancer and the like.

  Paclitaxel was first isolated in 1971 from the bark of Taxus brevifolia. Paclitaxel has a structure in which diterpene (C20) is highly oxidized and amino acid side chains are bound, and functions as an anticancer agent by inhibiting microtubule depolymerization activity during cell division. .

  Since the isolation of paclitaxel, research on the biosynthetic pathway of paclitaxel has been actively conducted for the purpose of industrially efficient production and the like. As a result, to date, many enzymes involved in the paclitaxel biosynthetic pathway have been discovered. Examples of enzymes involved in the paclitaxel biosynthetic pathway include an enzyme that oxidizes the 5-position of taxadiene (Non-Patent Document 1), an enzyme that oxidizes the 13-position of 5-hydroxytaxadiene (Non-Patent Document 2), and 5-acetoxy. Enzymes that oxidize position 10 of taxadiene (Non-Patent Document 3) 5,9,10,13-tetraacetoxytaxadiene and 7-hydroxy-5,9,10,13-tetraacetoxytaxadiene Enzyme that oxidizes position 2 (Non-patent Document 4): Oxidizes position 7 of 5,9,10,13-tetraacetoxytaxadiene and 2-hydroxy-5,9,10,13-tetraacetoxytaxadiene Enzymes (Non-patent Document 5) have been reported.

Jennewein S et. Al. (2004) "Cytochrome p450 taxadiene 5alpha-hydroxylase, a mechanistically unusual monooxygenase catalyzing the first oxygenation step of taxol biosynthesis", Chemistry & Biology, Vol.11 (Issue 3), pp.379-387 Jennewein S et. Al. (2001) "Taxol biosynthesis: taxane 13 alpha-hydroxylase is a cytochrome P450-dependent monooxygenase", Proceedings of the National Academy of Sciences of the United States of America, Vol.98 (No.24), pp.13595-13600 Schoendorf A et. Al. (2001) "Molecular cloning of a cytochrome P450 taxane 10 beta-hydroxylase cDNA from Taxus and functional expression in yeast", Proceedings of the National Academy of Sciences of the United States of America, Vol.98 (No .4), pp.1501-1506 Chau M et. Al. (2004) "Molecular cloning and characterization of a cytochrome P450 taxoid 2alpha-hydroxylase involved in Taxol biosynthesis", Archives of Biochemistry and Biophysics, Vol.427 (issue 1), pp.48-57 Chau M et. Al. (2004) "Taxol biosynthesis: Molecular cloning and characterization of a cytochrome P450 taxoid 7 beta-hydroxylase", Chemistry & Biology, Vol.11 (Issue 5), pp.663-672 Edgar S et. Al. (2016) "Mechanistic Insights into Taxadiene Epoxidation by Taxadiene-5α-Hydroxylase", ACS Chemal Biology, Vol.11 (Issue 2), pp.460-469

  However, not all paclitaxel biosynthesis-related genes have been identified, and the paclitaxel biosynthesis pathway has not been fully elucidated.

  Recently, many by-products are generated in the oxidation reaction at the 5-position, which is the first stage of taxadiene oxidation, and it has been reported that the ratio of by-products does not change when the host and culture conditions are changed. (Non-patent document 6), Taxadiene oxidase itself needs to be greatly improved. Therefore, in order to biosynthesize paclitaxel efficiently industrially, further research and development are desired.

  In view of the above circumstances, an object of one embodiment of the present invention is to provide a novel gene involved in the first-stage oxidation of the paclitaxel biosynthesis pathway.

  As a result of intensive studies to solve the above problems, the present inventors have identified a novel gene that synthesizes oxidized taxadiene using taxadiene, which is an intermediate of the paclitaxel biosynthetic pathway, as a substrate, thereby completing the present invention. It came to. That is, the present invention includes the following inventions.

Any gene selected from the group consisting of the following (G1) to (G5):
(G1) a gene encoding a protein consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3;
(G2) The amino acid sequence described in any one of SEQ ID NOs: 1 to 3, consisting of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and / or added, and 10 of taxadiene A gene encoding a protein having an activity of oxidizing the position;
(G3) a gene encoding a protein consisting of an amino acid sequence having 80% or more homology with the amino acid sequence described in any one of SEQ ID NOs: 1 to 3 and having an activity of oxidizing position 10 of taxadiene;
(G4) a gene comprising the base sequence described in any one of SEQ ID NOs: 4 to 7;
(G5) A protein that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to any one of the genes (G1) to (G4) and has an activity to oxidize position 10 of taxadiene The gene to encode.

  According to one embodiment of the present invention, novel taxadiene oxide can be produced.

It is the figure which showed typically a part of conventional paclitaxel biosynthesis pathway, and a part of novel paclitaxel biosynthesis pathway. It is a figure which shows the result of the GC / MS analysis of the substance produced by the transformant which concerns on one Embodiment of this invention. (A) is a structural formula of the novel compound obtained in the examples of the present specification. (B) is a figure which shows the result of having conducted the NMR analysis of the said compound.

  An embodiment of the present invention will be described in detail below. In addition, all of the academic literatures and patent literatures described in this specification are incorporated herein by reference. Unless otherwise specified in this specification, “A to B” representing a numerical range means “A or more (including A and greater than A) and B or less (including B and less than B)”.

  As used herein, the term “gene” is used interchangeably with “polynucleotide”, “nucleic acid” or “nucleic acid molecule” and is intended to be a polymer of nucleotides. Here, the gene may be present in the form of DNA (eg, cDNA or genomic DNA) or RNA (eg, mRNA). DNA or RNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may be a coding strand (sense strand) or a non-coding strand (antisense strand). The gene may be chemically synthesized. In addition, the codon usage may be changed so that the expression of the protein encoded by the gene is improved. Substitutions can be made between codons encoding the same amino acid.

  The term “protein” is also used interchangeably with “peptide” or “polypeptide”. As used herein, the base and amino acid notation uses the one-letter code or three-letter code defined by IUPAC and IUB as appropriate.

  Further, when the terms “oxidation” and the term “hydroxylation” are used for taxadiene, both terms are used interchangeably. That is, “oxidized taxadiene” and “taxadiene hydroxide” are both intended to mean “taxadiene oxidized by being given a hydroxyl group”.

<1. Gene / Protein>
The gene according to one embodiment of the present invention is any polynucleotide selected from the group consisting of the following (G1) to (G5):
(G1) a gene encoding a protein consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3;
(G2) The amino acid sequence described in any one of SEQ ID NOs: 1 to 3, consisting of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and / or added, and 10 of taxadiene A gene encoding a protein having an activity of oxidizing the position;
(G3) a gene encoding a protein consisting of an amino acid sequence having 80% or more homology with the amino acid sequence described in any one of SEQ ID NOs: 1 to 3 and having an activity of oxidizing position 10 of taxadiene;
(G4) a gene comprising the base sequence described in any one of SEQ ID NOs: 4 to 7;
(G5) A protein that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to any one of the genes (G1) to (G4) and has an activity to oxidize position 10 of taxadiene The gene to encode.

The protein according to one embodiment of the present invention is any protein selected from the group consisting of the following (P1) to (P4):
(P1) a protein comprising the amino acid sequence described in any one of SEQ ID NOs: 1 to 3;
(P2) In the amino acid sequence described in any one of SEQ ID NOs: 1 to 3, the amino acid sequence consists of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and / or added, and 10 of taxadiene A protein having the activity of oxidizing the position;
(P3) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence described in any one of SEQ ID NOs: 1 to 3, and having an activity of oxidizing the 10th position of taxadiene;
(P4) A protein encoded by the gene according to any one of (G1) to (G5) above.

  The genes (G1) to (G5) above encode a protein having a function of oxidizing the 10th position of taxadiene. Therefore, novel oxidized taxadiene can be produced by expressing the above gene in plants and microorganisms.

  The gene (G1) will be specifically described. SEQ ID NO: 1 is a kind of paclitaxel biosynthetic pathway-related enzyme derived from a yew tree (Taxus cuspidata). As shown in the upper part of FIG. 1, the activity for oxidizing 10 position of taxadiene (hereinafter referred to as “oxidized taxadiene synthesis”) A protein composed of a total of 483 amino acid residues (sometimes referred to herein as “N11 (native)”). Here, “the activity to oxidize the 10th position of taxadiene” can also be referred to as the activity of introducing a hydroxyl group at the 10th position of taxadiene. It can also be said to be an activity to produce a taxadiene 10-position hydroxide.

  SEQ ID NO: 2 is a protein in which 30 residues (amino acid sequence corresponding to the transmembrane site) have been deleted from the N-terminal side of the amino acid sequence shown in SEQ ID NO: 1 and a Met residue has been added to the N-terminal , Which has oxidized taxadiene synthesis activity and is composed of a total length of 454 amino acid residues (sometimes referred to herein as “Δ30aa N11” or simply “Δ30aa”).

  SEQ ID NO: 3 provides hydrophilicity by deleting 14 residues from the N-terminal side of the amino acid sequence shown in SEQ ID NO: 1 and newly binding 8 residues (MALLLAVF) to the N-terminal side (See Ajikumar PK et. Al. (2010) "Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli", Science, Vol. 330 (Issue 6000), pp. 70-74). It has activity and is composed of a total length of 477 amino acid residues (sometimes referred to herein as “17α-Δ14aa N11” or simply “17α-Δ14aa”).

  The gene of (G2) is a functionally equivalent mutant, derivative, variant, allele, homolog, ortholog, partial peptide, or other protein of the protein having the amino acid sequence shown in any one of SEQ ID NOs: 1 to 3 Alternatively, the specific sequence is not limited as long as it is a fusion protein with a peptide or the like and encodes a protein having oxidized taxadiene synthesis activity. Here, the number of amino acids that may be deleted, substituted or added is not limited as long as the above functions are not lost. However, the number of amino acids may be deleted or replaced by a known introduction method such as site-directed mutagenesis. Or it is a number that can be added, usually within 30 amino acids, preferably within 20 amino acids, more preferably within 10 amino acids, more preferably within 7 amino acids, and even more preferably within 5 amino acids (for example, 5, 4, 3, 2 or 1 amino acid). In the present specification, “mutation” mainly means a mutation artificially introduced by site-directed mutagenesis or the like, but it may be a naturally occurring similar mutation.

  The amino acid residue to be substituted is preferably substituted with another amino acid residue having a common amino acid side chain property. For example, amino acid side chain properties include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G). , H, K, S, T), an amino acid having an aliphatic side chain (G, A, V, L, I, P), an amino acid having a hydroxyl group-containing side chain (S, T, Y), a sulfur atom-containing side Amino acids having chains (C, M), amino acids having carboxylic acid and amide-containing side chains (D, N, E, Q), amino acids having base-containing side chains (R, K, H), aromatic-containing side chains (H, F, Y, W) are included (the parentheses indicate single letter amino acids). It is already known that a polypeptide having an amino acid sequence modified by deletion, addition and / or substitution by other amino acids of one or more amino acid residues to an amino acid sequence maintains its biological activity. ing. Furthermore, it is more preferable that the amino acid residue that is the target of substitution is substituted with an amino acid residue having as many common properties as possible.

  In this specification, “functionally equivalent” means that the target protein has the same (same and / or similar) biological function as the protein consisting of the amino acid sequence described in any one of SEQ ID NOs: 1 to 3. Or it is intended to have a biochemical function. In the present specification, examples of the biological function or biochemical function of the protein consisting of the amino acid sequence shown in any of SEQ ID NOs: 1 to 3 include a function of oxidizing the 10th position of taxadiene. . The biological function may include specificity of a site where the protein is expressed, protein expression level, and the like. Whether a protein into which a mutation has been introduced imparts a desired trait to a host is obtained by obtaining a transformant in which a gene encoding the protein has been introduced and expressed, and this transformant using taxadiene as a substrate. It can be determined by examining whether the position can be oxidized (whether the 10-position can produce oxidized taxadiene).

  Further, as shown in the examples described later, the 30 residues from the N-terminal side of the amino acid sequence represented by SEQ ID NO: 1 is an amino acid sequence corresponding to the transmembrane site, and even when the sequence is deleted, It has been confirmed to have oxidized taxadiene synthesis activity. In addition, when 14 amino acid residues are deleted from the N-terminal side of the amino acid sequence shown in SEQ ID NO: 1 and a sequence for imparting hydrophilicity (8 amino acid residues: MALLLAVF) is added, oxidized taxadiene synthesis The present inventors have confirmed that the activity is improved. From these results, deletion, substitution or addition can be preferably performed in the amino acid sequences shown in SEQ ID NOs: 1 to 3, particularly for the N-terminal sequence. In this case, it is preferable to perform deletion, substitution or addition in terms of deleting the transmembrane site and / or improving the hydrophilicity of the protein.

  The above gene (G3) is also a functionally equivalent variant, derivative, variant, allele, homolog, ortholog, partial peptide, or other protein of the protein having the amino acid sequence shown in any one of SEQ ID NOs: 1 to 3 -As long as it is intended to be a fusion protein with a peptide and encodes a protein having oxidized taxadiene synthesis activity, its specific sequence is not limited.

  Amino acid sequence homology is at least 80% or more, more preferably 90% or more, still more preferably 95% or more (for example, 95%, 96%, 97) of the entire amino acid sequence (or a region necessary for functional expression). %, 98%, 99% or more). Amino acid sequence homology is determined by the BLASTN (nucleic acid level) or BLASTX (amino acid level) program (Altschul SF (1990) "Basic local alignment search tool", Journal of Molecular Biology, Vol. 215 (Issue 3), pp. 403). -410)). The program is based on the algorithm BLAST (Karlin S and Altschul SF (1990) "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes", Proceedings of the National Academy of Sciences of the United States of America by Karlin and Altschul. , Vol.87 (No.6), pp.2264-2268 and Karlin S and Altschul SF (1993) "Applications and statistics for multiple high-scoring segments in molecular sequences", Proceedings of the National Academy of Sciences of the United States of America, Vol. 90 (No. 12), pp. 5873-5877). When the base sequence is analyzed by BLASTN, the parameters are set to score = 100 and wordlength = 12, for example. Further, when the amino acid sequence is analyzed by BLASTX, the parameters are set to score = 50 and wordlength = 3, for example. Moreover, when analyzing an amino acid sequence using the Gapped BLAST program, Altschul SF et. Al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Research, Vol. 25 (Issue 17), pp. 3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known. In order to align the base sequence or amino acid sequence to be compared with each other in an optimal state, addition or deletion (for example, a gap) may be allowed.

  In the present specification, “homology” intends the ratio of the number of amino acid residues having similar properties (homology, positive, etc.), but more preferably the ratio of the number of identical amino acid residues, ie, identity. (Identity). The properties of amino acids are as described above.

  Regarding the gene (G4) above, SEQ ID NOs: 4 and 5 each show the base sequence (Open Reading Frame: ORF) of the gene encoding the polypeptide consisting of the amino acid sequence shown by SEQ ID NO: 1. The base sequence represented by SEQ ID NO: 4 is a base sequence of a natural gene encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 derived from a yew tree (Taxus cuspidata). On the other hand, the base sequence represented by SEQ ID NO: 5 is a sequence obtained by codon-optimizing the base sequence represented by SEQ ID NO: 4 for expression in E. coli. Codon optimization can be performed based on common knowledge in the art. SEQ ID NO: 6 shows the base sequence of the gene encoding the polypeptide consisting of the amino acid sequence shown by SEQ ID NO: 2. SEQ ID NO: 7 shows the base sequence of the gene encoding the polypeptide consisting of the amino acid sequence shown by SEQ ID NO: 3.

The gene (G5) is intended to be a gene that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to any of the genes (G1) to (G4). Here, stringent conditions refer to conditions in which a so-called base sequence-specific double-stranded polynucleotide is formed and a non-specific double-stranded polynucleotide is not formed. In other words, conditions for hybridizing at a temperature ranging from the melting temperature (Tm value) of highly homologous nucleic acids, for example, perfectly matched hybrids, to 15 ° C, preferably 10 ° C, more preferably 5 ° C lower. It can be said. As an example, in a buffer solution (pH 7.2) composed of 0.25M Na ₂ HPO ₄ , 7% SDS, 1 mM EDTA, 1 × Denhardt's solution, the temperature is 60 to 68 ° C., preferably 65 ° C., more preferably. Is hybridized under conditions of 68 ° C. for 16 to 24 hours, and further in a buffer solution (pH 7.2) comprising 20 mM Na ₂ HPO ₄ , 1% SDS, 1 mM EDTA, preferably at a temperature of 60 to 68 ° C., preferably The conditions which wash twice for 15 minutes on 65 degreeC, more preferably 68 degreeC conditions can be mentioned. Other examples include (1) 25% formamide (50% formamide under more severe conditions), 4 × SSC (sodium chloride / sodium citrate), 50 mM Hepes (pH 7.0), 10 × Denhardt's solution, 20 μg / mL Prehybridization is performed overnight at 42 ° C. in a hybridization solution containing denatured salmon sperm DNA, and then a labeled probe is added and hybridization is performed by incubating overnight at 42 ° C. (2) (I) Normally 1 × SSC, 0.1% SDS, about 37 ° C., (ii) More severe conditions than 0.5 × SSC, 0.1% SDS, about 42 ° C. (iii) More severe conditions May include conditions for washing with a washing solution and a temperature of about 0.2 × SSC, 0.1% SDS, about 65 ° C. As the hybridization washing conditions become more severe, hybridization with higher specificity is achieved. However, combinations of the above SSC, SDS and temperature conditions are exemplary, and those skilled in the art will recognize the above or other factors that determine the stringency of hybridization (eg, probe concentration, probe length, hybridization reaction). It is possible to achieve the same stringency as above by appropriately combining the time and the like. This is described, for example, in Joseph Sambrook and David W. Russell, “Molecular cloning: a laboratory manual 3rd Ed.”, Cold Spring Harbor Laboratory Press, 2001, and the like.

  In addition, the gene (G5) is a gene consisting of a DNA comprising the nucleotide sequences set forth in SEQ ID NOs: 4 to 7, wherein 1 to 50 nucleotide sequences are substituted, deleted, inserted and / or added. And a gene comprising a DNA having a nucleotide sequence of SEQ ID NOs: 4 to 7 and having a homology of 90% or more.

  As a method for obtaining the above-mentioned gene / protein, a commonly used polynucleotide modification method may be used. That is, a polynucleotide having the genetic information of a desired recombinant protein can be produced by substituting, deleting, inserting and / or adding a specific base of the polynucleotide having the genetic information of the protein. Specific methods for converting the base of the polynucleotide include, for example, a commercially available kit (KOD-Plus Site-Directed Mutagenesis Kit; manufactured by Toyobo, Transformer Site-Directed Mutagenesis Kit; manufactured by Clontech, QuickChange Site Directed Mutagenesis Kit; manufactured by Stratagene, etc. ) Or use of the polymerase chain reaction (PCR). These methods are known to those skilled in the art.

  The gene may be composed only of a polynucleotide encoding the protein, but may be added with other base sequences. The base sequence to be added is not particularly limited, but includes a label (for example, histidine tag, Myc tag or FLAG tag), a fusion protein (for example, streptavidin, cytochrome, GST, GFP or MBP), a promoter sequence, and Examples thereof include a base sequence encoding a signal sequence (for example, an endoplasmic reticulum transition signal sequence, a secretory sequence, etc.). The site to which these base sequences are added is not particularly limited and may be, for example, the N-terminus or C-terminus of the translated protein.

<2. Recombinant vector, transformant>
One embodiment of the present invention provides a vector containing the gene. This vector includes not only an expression vector for expressing the gene in a host cell to produce a transformant but also a vector used for production of a recombinant protein. The target of transformation is not particularly limited, and examples include bacteria, yeasts (for example, budding yeast, oleaginous yeast, etc.), insects, animals and plants.

  As the base material vector serving as the base of the vector, various commonly used vectors can be used. For example, a plasmid, phage, cosmid or the like can be used, and can be appropriately selected according to the cell to be introduced or the introduction method. That is, the specific type of vector is not particularly limited, and a vector that can be expressed in a host cell may be appropriately selected.

  In order to reliably express the gene, a promoter sequence may be appropriately selected according to the type of host cell, and the promoter sequence and the gene incorporated into various substrate vectors may be used as an expression vector. Such expression vectors include, for example, phage vectors, plasmid vectors, viral vectors, retroviral vectors, chromosomal vectors, episomal vectors and virus-derived vectors (eg, bacterial plasmids, bacteriophages, yeast episomes), yeast chromosomal elements and viruses (eg, Vectors derived from baculoviruses, papovaviruses, vaccinia viruses, adenoviruses, tripox viruses, pseudorabies viruses, herpes viruses, lentiviruses and retroviruses)) and combinations thereof are available (eg cosmids and phagemids).

  In general, plasmid vectors are introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, the vector can be packaged in vitro using an appropriate packaging cell line and then transduced into host cells. A retroviral vector can also be replicable or replication defective. In the latter case, viral propagation generally occurs only in helper cells.

  The vector is preferably a vector containing a cis-acting regulatory region for the gene of interest. Appropriate trans-acting factors can be supplied by the host, supplied by a complementary vector, or supplied by the vector itself upon introduction into the host. A preferred embodiment in this regard is that the vector provides specific expression that may be inducible and / or cell type specific. Particularly preferred among such vectors are vectors that can be induced by environmental factors that are easy to manipulate, such as temperature and nutrient additives.

  Among the preferred vectors for use in bacteria are, for example, pQE-70, pQE-60 and pQE-9 (available from Qiagen); pBS vector, Pagescript vector, Bluescript vector, pNH8A, pNH16A, pNH18A and pNH46A (Stratagene Ptrc99a, pKK223-3, pKK233-3, pDR540 and pRIT5 (available from Addgene); pRSF (available from MERCK); and pAC (available from Nippon Gene) It is. Also, preferred vectors for use in preferred eukaryotes include pWLNE0, pSV2CAT, pOG44, pXT1 and pSG (available from Stratagene); and pSVK3, pBPV, pMSG and pSVL (available from Addgene) It is.

  Various markers may be used to confirm whether or not the gene has been introduced into the host cell, and whether or not the gene has been reliably expressed in the host cell. That is, the vector preferably contains at least one selectable marker. Such selectable markers include, for example, dihydrofolate reductase gene or neomycin resistance gene for eukaryotic cell culture. Examples of the culture in E. coli and other bacteria include drug resistance genes such as a tetracycline resistance gene or an ampicillin resistance gene. In addition, a gene deleted in the host cell may be used as a marker gene. By introducing a plasmid or the like containing the marker gene and the gene according to one embodiment of the present invention into the host cell as an expression vector, the introduction of the gene can be confirmed from the expression of the marker gene. The gene may also be linked to a vector containing a selectable marker for growth in the host cell.

  The gene insert is preferably operably linked to a suitable promoter. Suitable promoters may be those known to those skilled in the art and are not particularly limited, but include, for example, the phage λPL promoter; E. coli lacI promoter, lacZ promoter, T3 promoter and T7 promoter; trp promoter and tac promoter; SV40 early promoter and late promoter; and retroviral LTR promoter.

  When E. coli is used as a host for transformation, “ori” for replication in E. coli and a gene for selecting transformed E. coli (for example, drugs (ampicillin, tetracycline, kanamycin, chloramphenicol, etc.) It is preferable to have a resistance gene) on the vector. Examples of such vectors include M13 vectors, pUC vectors, pBR322, pBluescript, pCR-Script, pGEM-T, pDIRECT, pGEX (GE Healthcare), pET (Promega), pTrc (Invitrogen). ), PT7, pRSF (available from MERCK), pAC (available from Nippon Gene Co., Ltd.), and the like.

  The vector preferably further contains sites for transcription initiation and transcription termination, and a ribosome binding site for translation in the transcription region. The coding portion of the mature transcript expressed by the vector construct will contain a transcription start codon AUG at the beginning of the polypeptide to be translated and a stop codon appropriately located at the end.

  When a higher eukaryote is used as a host in transformation, DNA transcription can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10-300 bp, that serve to increase the transcriptional activity of a promoter in a given host cell type. Enhancers include, for example, SV40 enhancer (which is located 100 to 270 bp downstream of the origin of replication), cytomegalovirus early promoter enhancer, polyoma enhancer and adenovirus enhancer located downstream of the origin of replication. Can be mentioned.

  The host into which the vector is introduced is not particularly limited, but various cells can be suitably used. Representative examples of suitable hosts include cells (eg, E. coli cells, Streptomyces cells and Salmonella typhimurium cells), fungal cells (eg, yeast cells), insect cells (eg, Drosophila S2 cells and Spodoptera Sf9 cells) ), Animal cells (eg, CHO cells, COS cells and Bowes melanoma cells) and plant cells. More specifically, not only mammalian cells such as humans or mice, but also, for example, silkworm-derived cells, insects such as Drosophila melanogaster, bacteria such as Escherichia coli, yeast (Saccharomyces cerevisiae ), Fission yeast (Schizosaccharomyces pombe) and oleaginous yeast (Yarrowia lipolytica, Rhodosporidium toruloides, Xanthophyllomyces dendrorhous, Rhodotorula glutinis, Rhodotorula acheniorum and Lipomyces starkeyi) There is no particular limitation. Appropriate culture media and conditions for the above-described host cells can be used as known in the art.

  A method for introducing the vector into a host cell, ie, a transformation method is not particularly limited, and electroporation method, calcium phosphate method, liposome method, DEAE dextran method, microinjection method, cationic lipid-mediated transfection, electroporation, Conventionally known methods such as transfection, transduction or infection can be preferably used. Such methods are described in many standard laboratory manuals such as Leonard G. Davis et. Al., “Basic methods in molecular biology”, Elsevier, 1986.

  In one embodiment of the present invention, a recombinant expression vector comprising a polynucleotide encoding the partial fragment of the protein and a recombinant expression vector for recombinantly generating the partial fragment (fragment) of the protein are provided. Includes genetically engineered transformants (host cells).

  Further, one aspect of the present invention may include an invention relating to the production of a protein mutant or fragment thereof obtained by the above-described recombinant technique. That is, one embodiment of the present invention may include a method for producing a protein mutant or a fragment thereof using a recombinant technique.

  Protein variants produced by such techniques can be isolated from host cells or extracellular (such as media) and purified as substantially pure and homogeneous proteins. For the separation and purification of the protein, a separation method and a purification method used in normal protein separation and purification may be used. For example, chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, ammonium sulfate precipitation or ethanol precipitation, acid extraction, dialysis and Proteins can be separated and purified by appropriately selecting or combining recrystallization and the like. Furthermore, a plurality of these methods can be combined.

  Specific chromatographic methods include affinity chromatography, anion or cation ion exchange chromatography, hydrophobic chromatography, phosphocellulose chromatography, hydroxyapatite chromatography, lectin chromatography, gel filtration, reverse phase chromatography. And chromatography and adsorption chromatography.

  In addition, when a protein mutant is expressed in a host cell (such as E. coli) as a fusion protein with GST or a recombinant protein to which 6 × His is added, the expressed recombinant protein is added to a glutathione column or nickel. It can be purified using a column. After purification of the fusion protein, if necessary, the region other than the target protein in the fusion protein can be cleaved with thrombin or factor Xa and removed.

  One embodiment of the present invention also includes a transformant containing the above gene or the above vector. Here, “including a gene or vector” means that it has been introduced into a target cell (host cell) so as to be expressed by a known genetic engineering technique (gene manipulation technique). The “transformant” is intended to include not only cells / tissues / organs but also living organisms.

  A method for producing the transformant (production method) includes a method for transforming the above-described vector. In addition, the organism to be transformed is not particularly limited, and examples thereof include various microorganisms exemplified as the host cell. In addition, if a promoter and / or vector is appropriately selected, plants or animals can also be targeted for transformation.

  As a host cell used in one embodiment of the present invention, Escherichia coli can be particularly preferably exemplified. In the examples described later, E. coli B strain BL21 (DE3) was used. However, since various strains such as E. coli K12 strain JM109 (DE3) exist in E. coli, the strain is not limited to BL21 (DE3). In the examples, LB medium and TB medium were used as the culture medium for recombinant Escherichia coli. However, as the culture medium for E. coli, there are many mediums such as 2 × YT medium and M9 medium. It is not limited to TB medium. In addition to the implementation manuals provided by the manufacturers shown in the examples, many manuals exist as genetic recombination experiment methods. For example, Joseph Sambrook and David W. Russell, “Molecular cloning: a laboratory manual 3rd Ed.”, Cold Spring Harbor Laboratory Press, 2001 can be exemplified. This handbook is comprehensive and describes the types of E. coli strains, types of vectors, culture methods, etc. in addition to the usual genetic recombination experiment methods, so that experiments can be conducted with reference.

The transformant according to one embodiment of the present invention is preferably one obtained by introducing and expressing the following genes (g1) to (g4):
(G1) a group of mevalonate pathway genes that synthesize mevalonic acid or mevalonolactone to isopentenyl diphosphate,
(G2) type 1 isopentenyl diphosphate isomerase gene,
(G3) type 2 isopentenyl diphosphate isomerase gene,
(G4) Acetoacetate-coenzyme A ligase gene.

In addition to the genes (g1) to (g4) described above, the transformant according to one embodiment of the present invention is a gene obtained by introducing and expressing the following genes (g5) to (g6). More preferred:
(G5) geranylgeranyl diphosphate synthase gene,
(G6) Taxadiene synthase gene.

In addition to the genes (g1) to (g6) described above, the transformant according to one embodiment of the present invention is more preferably one obtained by introducing and expressing the following gene (g7). :
(G7) NADPH-cytochrome P450 reductase gene.

<3. Method for producing taxadiene oxide>
The method for producing oxidized taxadiene according to one embodiment of the present invention may include a step of culturing the above-described transformant to obtain oxidized taxadiene, and other specific conditions, materials, equipment used, and the like. Is not particularly limited.

  Hereinafter, description will be made by taking, as an example, Escherichia coli, which has the most extensive materials and information among various hosts, and is the most preferable aspect in the production method of one embodiment of the present invention. In addition, this invention is not limited to this, About the host, the various materials described in the above-mentioned <2> column can be used.

  The outline in the case of producing oxidized taxadiene using recombinant Escherichia coli will be described.

  1. Escherichia coli does not have a mevalonate pathway, but is the first isoprenoid due to the non-mevalonate pathway [2-C-methyl-D-erythritol 4-phosphate (hereinafter also referred to as MEP pathway)). The substrate isopentenyl diphosphate (also called isopentenyl pyrophosphate; hereinafter referred to as IPP) is made.

  2. IPP is converted to dimethylallyl diphosphate (hereinafter referred to as DMAPP) by IPP isomerase (hereinafter sometimes referred to as Idi).

  3. DMAPP is condensed with IPP by a farnesyl diphosphate (hereinafter referred to as FPP) synthase (synthase), so that geranyl diphosphate having 10 carbon atoms (hereinafter referred to as GPP), FPP having 15 carbon atoms is obtained. Will be converted sequentially.

  4). FPP is further condensed with IPP by geranylgeranyl diphosphate (hereinafter referred to as GGPP) synthase to synthesize GGPP having 20 carbon atoms.

  5). Taxadiene (sometimes referred to as “Taxa-4,11-diene”) is synthesized from GGPP by taxadiene synthase.

  Since wild-type E. coli does not synthesize the above 4 and 5, in order to synthesize these isoprenoids in E. coli, the biosynthetic enzyme gene (s) responsible for synthesis from mevalonic acid, mevalonolactone, etc. to FPP, FPP It is preferable that a biosynthetic enzyme gene (s) responsible for synthesis from GGPP to GGPP, a biosynthetic enzyme gene (s) responsible for synthesis from GGPP to taxadiene, etc. are introduced into E. coli and expressed. Hereinafter, these genes will be described in detail.

(3-1. Mevalonate pathway gene group)
Examples of mevalonate pathway genes that synthesize mevalonic acid or mevalonolactone to isopentenyl diphosphate include, for example, HMG-CoA synthetase gene, HMG-CoA reductase gene, and HMG-CoA reductase gene. Examples include mevalonate kinase (MVA kinase) gene, phosphomevalonate kinase (PMVA kinase) gene, and diphosphomevalonate kinase (DPMVAkinase gene).

  These mevalonate pathway genes include the mevalonate pathway gene group derived from Streptomyces strain CL190 (Yu F et. Al (2008) "Isolation and functional characterization of a beta-eudesmol synthase, a new sesquiterpene synthase from Zingiber zerumbet See Smith ", FEBS Letters, Vol. 582 (Issue 5), pp. 565-572. INSD accession number: AB037666 can be used, but in addition, mevalon derived from Saccharomyces cerevisiae Acid pathway genes (see Martin VJ et. Al. (2003) “Engineering a mevalonate pathway in Escherichia coli for production of terpenoids”, Nature Biotechnology, Vol. 21 (No. 7), pp. 796-802), bacteria Mevalonate pathway genes from Streptococcus pneumoniae (Yoon SH et. Al. (2006) "Enhanced lycopene production in Escherichia coli engineered to synthesize isopentenyl diphosphate and dimethylallyl d iphosphate from mevalonate ", Biotechnology and Bioengineering, Vol. 94 (Issue 6), pp. 1025-1032) can also be used.

(3-2.1 type / 2 type isopentenyl diphosphate isomerase gene)
Furthermore, in order to increase the supply amount of FPP, it is preferable to use an IPP isomerase (Idi) gene (idi). As the idi gene, an isopentenyl diphosphate isomerase gene derived from Streptomyces genus CL190 (Non-patent Document 4 Accession no AB037666) can be used, but in addition to this, an isopentenyl diphosphate isomerase gene derived from E. coli (Martin) VJ et. Al. (2003) "Engineering a mevalonate pathway in Escherichia coli for production of terpenoids", Nature Biotechnology, Vol.21 (No.7), pp.796-802), derived from Saccharomyces cerevisiae Isopentenyl diphosphate isomerase gene (Kajiwara S et. Al. (1997) "Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid biosynthesis in Escherichia coli", Biochemical Journal, Vol.324 (Issue 2), pp.421- 426), an isopentenyl diphosphate isomerase gene from the green alga Haematococcus pluvialis (Martin VJ et. Al., Op. Ci) t. and Kajiwara S et. al., op. cit.) etc. can also be used.

  In addition, Idi includes a type 1 (type 1) and a type 2 (type 2) having different structures. Although any Idi may be used in one embodiment of the present invention, the inventors used both Idi in the following examples.

(3-3. Acetoacetate-coenzyme A ligase gene)
According to the recombinant Escherichia coli introduced with the mevalonate pathway gene group and Idi gene described above, a large amount of FPP can be obtained by adding mevalonate or mevalonolactone (D-mevalonate lactone) as a substrate in the medium. Can be produced.

  On the other hand, acetoacetate (for example, lithium acetoacetate; LAA), which is cheaper than mevalonolactone, can also be used as a substrate. In order to utilize LAA added to the medium, it is preferable to further introduce an acetoacetate-CoA ligase gene using LAA as a substrate.

  Acetoacetate-CoA ligase is an enzyme that catalyzes conversion to acetoacetyl-CoA using ATP using acetoacetate and CoA as substrates (Stern JR (1971) "A role of acetoacetyl-CoA synthetase in acetoacetate utilization by rat liver cell fractions ", Biochemical and Biophysical Research Communications, Vol.44 (Issue 4), pp.1001-1007 and Bergstrom JD et.al. (1984)" The regulation of acetoacetyl-CoA synthetase activity by modulators of cholesterol synthesis in vivo and the utilization of acetoacetate for cholesterogenesis ", The Journal of Biolgical Chemistry, Vol. 259 (No. 23), pp. 14548-14553). As the acetoacetate-CoA ligase gene, genes derived from mammals such as rats (Rattus norvegicus) and humans, certain bacteria, fungi, and the like are known. Can be used. In addition, a plasmid containing the full-length gene encoding rat-derived acetoacetate-CoA ligase (INSD accession number: BC061803) can be obtained from Invitrogen as a Mammalian Gene Collection cDNA clone (clone ID: 5598532).

  That is, in one embodiment of the present invention, in addition to the gene described in the section <1> above, (g1) a group of mevalonate pathway genes that synthesize mevalonic acid or mevalonolactone to isopentenyl diphosphate, (g2) A medium containing acetoacetate containing a transformant introduced with type 1 isopentenyl diphosphate isomerase gene, (g3) type 2 isopentenyl diphosphate isomerase gene, and (g4) acetoacetate-coenzyme A ligase gene A method for producing taxadiene oxide comprising a step of culturing at 0 to obtain taxadiene oxide is also included.

(3-4. Geranylgeranyl diphosphate synthase gene)
Further, in order to synthesize GGPP from FPP, it is preferable to introduce a geranylgeranyl diphosphate synthase gene. The geranylgeranyl diphosphate synthase gene is not particularly limited as long as the protein expressed from the gene has an activity of converting FPP to GGPP, and the gene sequence may contain a mutation.

  The mutation is preferably a mutation that enhances the activity of geranylgeranyl diphosphate synthase, and examples thereof include, but are not limited to, codon optimization. Codon optimization can be performed based on common knowledge in the art (for example, Burgess-Brown NA et. Al. (2008) "Codon optimization can improve expression of human genes in Escherichia coli: A multi-gene study ", Protein Expression and Purification, Vol. 59 (Issue 1), pp. 94-102). Codon optimization can also be performed using a commercially available artificial gene synthesis service (for example, GenScript artificial gene synthesis service (http://www.genscript.jp/codon_opt.html)). Whether or not a codon has been optimized can be determined by, for example, comparing the expression levels of geranylgeranyl diphosphate synthase before and after the codon optimization process. Codon optimization was successful when geranylgeranyl diphosphate synthase expressed by the sequence after codon optimization was expressed more than geranylgeranyl diphosphate synthase expressed by the sequence before codon optimization Can be determined. The index of codon optimization is not limited to the protein expression level, and can be set as appropriate according to the purpose.

  The origin of the geranylgeranyl diphosphate synthase gene is not particularly limited, and a gene derived from any species can be used. The geranylgeranyl diphosphate synthase gene can be appropriately set in consideration of the relationship with the host cell to be introduced. For example, in the examples of the present specification, Escherichia coli is used as a host, and a geranylgeranyl diphosphate synthase gene (crtE) derived from soil bacteria (Pantoea ananatis) is used as a gene to be introduced.

  In a preferred embodiment of the present invention, the geranylgeranyl diphosphate synthase gene has a sequence number 8 corresponding to the coding region (CDS) of the gene published as INSD accession number: D90087 as shown in the Examples. A gene consisting of the base sequence shown by can be used.

(3-5. Taxadiene synthase gene)
Further, in order to synthesize taxadiene from GGPP, it is preferable to introduce a taxadiene synthase gene. The taxadiene synthase gene is not particularly limited as long as the protein expressed therefrom has an activity of converting GGPP to taxadiene, and the gene sequence may contain a mutation.

  The mutation is preferably a mutation that increases the activity of taxadiene synthase, and examples thereof include, but are not limited to, codon optimization. For details of codon optimization, the above 3-4. It is the same. Whether or not the codon is optimized can be determined by comparing the expression level of taxadiene synthase before and after the codon optimization process, for example. If the taxadiene synthase expressed by the sequence after codon optimization is expressed more than the taxadiene synthase expressed by the sequence before codon optimization, it can be determined that codon optimization has been successful. . The index of codon optimization is not limited to the protein expression level, and can be set as appropriate according to the purpose.

  The origin of the taxadiene synthase gene is not particularly limited, and a gene derived from any species can be used. Further, the taxadiene synthase gene can be appropriately set in consideration of the relationship with the introduced host cell. For example, in the examples of the present specification, E. coli is used as a host, and a taxadiene synthase gene (TXS) derived from Taxus brevifolia is used as a gene to be introduced.

  In a preferred embodiment of the present invention, the taxadiene synthase gene has a codon-optimized gene coding region (CDS) published as INSD accession number: U48796, as shown in the Examples, for E. coli, In addition, a gene consisting of the base sequence represented by SEQ ID NO: 9 designed to remove 60 residues on the N-terminal side and add Met and Gly residues on the N-terminal side may be used.

  That is, in one embodiment of the present invention, in addition to the gene described in the section <1> and the genes (g1) to (g4), (g5) a geranylgeranyl diphosphate synthase gene, and (g6) Also included is a method for producing oxidized taxadiene comprising a step of culturing a transformant introduced with a taxadiene synthase gene in a medium containing acetoacetate to obtain oxidized taxadiene.

(3-6. NADPH-cytochrome P450 reductase gene)
The taxadiene oxidase according to one embodiment of the present invention is a kind of cytochrome P450. Therefore, in order to secure the oxidation reaction by P450, it is preferable to introduce the NADPH-cytochrome P450 reductase gene. The NADPH-cytochrome P450 reductase gene is a flavin enzyme that supplies electrons necessary for P450 to undergo an oxidation reaction, and is a domain to which its own coenzymes FAD and FMN bind (FAD binding domain and FMN binding, respectively) It may be described as a domain). In one embodiment, when the host does not have its own NADPH-cytochrome P450 reductase gene, it is naturally preferable to introduce the gene. In another embodiment, even when the host has its own NADPH-cytochrome P450 reductase gene, the mode of introducing the gene may be preferable. When the host is Escherichia coli, since E. coli itself does not have the NADPH-cytochrome P450 reductase gene, an embodiment in which the gene is introduced may be preferable.

  The origin of the NADPH-cytochrome P450 reductase gene is not particularly limited, and a gene derived from any species can be used. The NADPH-cytochrome P450 reductase gene can be appropriately set in consideration of the relationship between P450 to be simultaneously expressed and the host cell to be introduced. For example, in the examples of the present specification, E. coli is used as the host, and P450 and NADPH-cytochrome P450 reductase gene (TCPR) derived from Japanese yew (Taxus cuspidata) are used as the introduced gene.

  As a preferred embodiment of the present invention, as shown in the Examples, the NADPH-cytochrome P450 reductase gene uses the coding region (CDS) of the gene published as INSD accession number: AY571340.1 for Escherichia coli. A gene consisting of the base sequence shown in SEQ ID NO: 10 designed to optimize codons and remove 74 residues of the N-terminal transmembrane domain can be used.

  That is, in one embodiment of the present invention, in addition to the gene described in the section <1> and the genes (g1) to (g6) described above, (g7) a transformation in which the NADPH-cytochrome P450 reductase gene is introduced. A method for producing oxidized taxadiene comprising a step of culturing a body in a medium containing acetoacetate to obtain oxidized taxadiene is also included.

(3-7. Others)
The gene according to one embodiment of the present invention and the genes (g1) to (g7) described above can be appropriately combined and loaded on a vector and introduced into a host. The combination of genes mounted on the vector is not particularly limited as long as the transformant introduced with the gene produces a desired protein (for example, oxidized taxadiene).

  The number of vectors for introducing the gene according to an embodiment of the present invention and the genes (g1) to (g7) above is not particularly limited, and is, for example, 1, 2, 3, 4, 5 It can be an individual.

  The number of genes mounted on one vector is not particularly limited, and may be, for example, 1, 2, 3, 4, 5, 6, 7, etc.

  In one embodiment of the present invention, for example, a gene involved in the initial stage of oxidized taxadiene synthesis may be loaded on the first vector, and a gene involved in the later stage of oxidized taxadiene synthesis may be loaded on the second vector. In one embodiment of the present invention, preferably, a gene involved in the pathway from LAA to FPP (that is, the gene of (g1) to (g4) above) is mounted on the first vector, and the gene from FPP to oxidized taxadiene is loaded. A gene involved in the pathway (that is, the gene of (g5) to (g7) and the gene according to one embodiment of the present invention) can be mounted on the second vector.

The arrangement order of genes in the same vector is not particularly limited. For example, when the genes (g1) to (g4) are mounted on the same vector, from the N-terminus to the C-terminus,
Harada H et. Al. (2008) "Efficient synthesis of functional isoprenoids from acetoacetate through metabolic pathway-engineered Escherichia coli", Applied Microbiology and Biotechnology, Vol.81 (Issue 5), p.915-925 Can be placed. Specifically, from the N-terminus to the C-terminus, the mevalonate kinase gene (gene (g1))-diphosphomevalonate decarboxylase gene (gene (g1))-phosphomevalonate kinase gene (gene (g1))-2 type IPP Isomerase gene (gene (g3))-HMG-CoA reductase gene (gene (g1))-HMG-CoA synthase gene (gene (g1))-1 type IPP isomerase gene (gene (g2))-acetoacetate-coenzyme A ligase gene (gene (g4)) can be arranged in this order (corresponding to the vector “pAC-Mev / Scidi / Aac1” described in Harada H et. Al., Op. Cit.). For example, when the genes (g5) to (g7) and the gene according to one embodiment of the present invention are mounted on the same vector, the N-terminal is changed to the C-terminal side according to one embodiment of the present invention. It can be arranged in the order of gene-gene (g7) -gene (g5) -gene (g6).

  In a preferred embodiment of the present invention, a first vector (pAC-Mev / Scidi /) in which the genes (g1) to (g4) are arranged in the order described in Harada H et. Al., Op. Cit. Aac1), the genes (g5) to (g7) above and the gene in the present invention from the N-terminus to the C-terminus, gene-gene (g7) -gene (g5) -gene according to one embodiment of the present invention The second vector arranged in the order of (g6) can be introduced into E. coli as a host.

  As described above, one embodiment of the present invention relates to a protein that synthesizes a novel oxidized taxadiene, a gene encoding the protein, and a method for producing oxidized taxadiene using the gene. More specifically, one embodiment of the present invention synthesizes a novel oxidized taxadiene (especially an oxidized taxadiene oxidized at the 10-position of taxadiene) from taxadiene, which is presumed to be an intermediate of the paclitaxel biosynthetic pathway. The present invention relates to a protein, a gene encoding the protein, and a method for producing oxidized taxadiene using the gene.

  According to one embodiment of the present invention, novel oxidized taxadiene can be efficiently produced using recombinant Escherichia coli or the like. The novel oxidized taxadiene that can be produced according to one embodiment of the present invention can be expected to be used not only as an intermediate in paclitaxel biosynthesis but also in various fields.

<4. New compound>
The structure of the substance obtained by the production method in the <3> column can be determined using any technique known in the art. Examples of the technique for determining the structure include, but are not limited to, X-ray diffraction, NMR (nuclear magnetic resonance method), infrared spectroscopy (IR), and MS (mass spectrometry).

  Moreover, the compound obtained by the manufacturing method of <3> column mentioned above can also be included by 1 aspect of this invention. For example, the compound shown in the Example mentioned later may be contained. Although the function of this compound has not been determined, as described above, it is a compound presumed to be involved in the novel biosynthetic pathway of paclitaxel. For this reason, this compound is estimated to be useful as an intermediate for paclitaxel biosynthesis. In addition to pharmaceuticals, functional foods, fragrances, agriculture and horticulture, consumer goods, polymer resin raw materials, catalyst raw materials, chemical raw materials, building materials, fuels, chromatography carriers, filtration carriers, chelating agents, cleaning agents, lubricants It can also be used for chemicals and agricultural chemical raw materials.

<5. Antibody>
One aspect of the present invention includes an antibody that specifically binds to a protein according to one embodiment of the present invention. The present antibody may be an antibody obtained by a known method using the protein described in the section <1> or a partial peptide thereof as an antigen. The form of this antibody is not particularly limited.

  Known methods include, for example, the method described in Ed. Harlow, “Antibodies: a laboratory manual”, Cold Spring Harbor Laboratory, 1988; Ikuzaki Ikuo et al. “Monoclonal antibodies: hybridomas and ELISA”, Kodansha, 1991. It is done. The antibody thus obtained can be used for protein detection and measurement. For example, the protein can be obtained using the sensitizing antigen. The protein used as the sensitizing antigen may be a complete protein or a partial peptide of the protein. Moreover, you may use the cell which expresses protein, its lysate, or the protein chemically synthesize | combined as a sensitizing antigen. A short peptide can be appropriately combined with a carrier protein such as keyhole limpet hemocyanin, bovine serum albumin and egg white albumin to be used as an antigen.

  The mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the parent cell used for cell fusion in the production of a monoclonal antibody. Rodents such as Rabbits, Rabbits such as Rabbits or Primates such as Rhesus monkeys are used.

  Further, one embodiment of the present invention includes a polypeptide having an amino acid sequence of an immunogenic epitope portion in the protein. The polypeptide having the amino acid sequence of the epitope-bearing portion of the protein contains at least the mutation site described in the section <1> and has 6, 7, 8, 9, or 10 amino acids. What is necessary is just to include the part of a peptide.

  In the above protein, the immunogenic epitope part that elicits an antibody response can be identified by methods known in the art. For example, Geysen HM et.al. (1984) "Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid", Proceedings of the National Academy of Sciences of the United States of America, Vol.81 ( No. 13), pp. 3998-4002 describes a procedure for the rapid and simultaneous synthesis of hundreds of amino acids in a sufficiently pure peptide on a solid support to the extent that they can be used in reactions in enzyme-linked immunosorbent assays. Is disclosed. The interaction of the synthetic peptide with the antibody can be easily detected without removing the synthetic peptide from the support. By this method, peptides carrying immunogenic epitopes of the desired protein can be routinely identified by those skilled in the art. For example, an immunologically important immunogenic epitope in the coat protein of foot-and-mouth disease virus is a 7 amino acid sequence by synthesizing an overlapping set of all 208 possible hexapeptides covering the entire 213 amino acid sequence of the protein. It was positioned by Geysen et al. In addition, peptides were synthesized in which each position within the epitope was substituted with all 20 amino acids, and specific amino acids that gave specificity for reaction with antibodies were determined. In this way, peptide analogs of epitope-bearing peptides according to one embodiment of the present invention can be routinely made by this method. Geysen (1987) U.S. Pat. No. 4,708,871 describes this method in more detail for identifying peptides carrying immunogenic epitopes of the desired protein.

  An “immunogenic epitope” is defined as the part of a protein that elicits an antibody response when the entire protein is an immunogen. These immunogenic epitopes are believed to be restricted to a few sites on the molecule. On the other hand, the region of a protein molecule to which an antibody can bind can be defined as an “antigenic epitope”. The number of immunogenic epitopes in a protein is generally less than the number of antigenic epitopes. This is described, for example, in Geysen HM et. Al., Op. Cit.

  Peptides carrying the antigenic epitope of the protein are useful for raising antibodies, particularly monoclonal antibodies, according to one embodiment of the invention. The majority of hybridomas obtained by fusing donor spleen cells immunized with antigenic epitope-bearing peptides generally secrete antibodies reactive with natural proteins. Antibodies raised by antigenic epitope-bearing peptides are useful for detecting mimetic proteins. Antibodies against different peptides can be used to track the end of various regions of the protein precursor that undergo post-translational processing. Because immunoprecipitation assays have shown that even short peptides (eg, about 9 amino acids) can be displaced by binding to longer peptides, peptides and anti-peptide antibodies have various It can be used in qualitative or quantitative assays, such as competitive assays. This is described, for example, in Wilson IA (1984) “The structure of an antigenic determinant in a protein”, Cell, Vol. 37 (Issue 3), pp. 767-778. Antibodies specific for proteins included in one embodiment of the invention are also useful for purification of mimetic proteins (eg, by adsorption chromatography using methods well known in the art).

  The antigenic epitope-bearing peptide derived from the protein designed according to the above guidelines is preferably at least 7, more preferably at least 9, more preferably 15 to 15 included in the amino acid sequence including the mutation site of the protein. Contains a sequence of 30 amino acids. Furthermore, the antigenic epitope-bearing peptide may include 30 to 50 amino acids including a mutation site of the protein, an amino acid sequence having an arbitrary length less than the full length, or a full length amino acid sequence. Also, the amino acid sequence of the antigenic epitope-bearing peptide is selected to be substantially soluble in an aqueous solvent (i.e., the above sequence tends to contain hydrophilic residues, preferably a highly hydrophobic sequence is preferred) It is particularly preferred that the sequence contains a proline residue. In addition, the antigenic epitope-bearing peptide of the protein can be easily obtained by a known recombinant protein production technique or the like.

  The antigenic epitope-bearing peptide can be used to induce antibodies by methods well known in the art. For example, Chow M et.al. (1985) "Synthetic peptides from four separate regions of the poliovirus type 1 capsid protein VP1 induce neutralizing antibodies", Proceedings of the National Academy of Sciences of the United States of America, Vol. .82 (No.3), pp.10-914; and Francis MJ et.al. (1985) "Immunological priming with synthetic peptides of foot-and-mouth disease virus", Journal of General Virology, Vol.66 (Issue 11), pp.2347-2354. In general, animals can be immunized with free peptides. However, anti-protein antibody titers can be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemocyanin (KLH) or tetanus toxoid. For example, a peptide containing cysteine can be coupled to a carrier using a linker such as m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). Other peptides can be coupled to the carrier using more common linking agents such as glutaraldehyde. As an example, animals such as rabbits, rats, and mice can be immunized by intraperitoneal and / or intradermal injection of an emulsion containing about 100 μg of free peptide or carrier-coupled peptide and Freund's adjuvant. Can be For example, one or more booster injections may be performed to provide useful titers of anti-protein antibodies that can be detected by an ELISA assay using free peptide adsorbed on a solid surface. The booster injection is performed, for example, at intervals of about 2 weeks. The titer of anti-protein antibody in serum obtained from immunized animals can be increased by purification of the anti-protein antibody. The purification can be performed, for example, by adsorption of the antibody to the peptide on the solid support and elution of the antibody by methods well known in the art.

Also, as used herein, the term “antibody” includes immunoglobulins (IgA, IgD, IgE, IgG, IgM, and their Fab fragments, F (ab ′) ₂ fragments and Fc fragments). . Examples include, but are not limited to, all classes of polyclonal antibodies, monoclonal antibodies, single chain antibodies, anti-idiotype antibodies, humanized antibodies, and humanized antibodies by gene recombination.

Moreover, as long as this antibody couple | bonds with the said protein, an antibody fragment or an antibody modification thing may be sufficient. That is, the present antibodies include complete molecules and antibody fragments (eg, Fab and F (ab ′) ₂ fragments) that can specifically bind to the proteins described above. Fab and F (ab ′) ₂ fragments lack the Fc portion contained in intact antibodies (the Fc portion can be rapidly removed) and have most of the nonspecific tissue binding found in intact antibodies. (Wahl RL et. Al. (1983) "Improved radioimaging and tumor localization with monoclonal F (ab ') 2", The Journal of Nuclear Medcine, Vol. 24 (No. 4), pp. 316-325) ). Thus, Fab and F (ab ′) ₂ fragments are preferred.

  The antibody can also be produced in a two-step procedure by using an anti-idiotype antibody. Such methods use the fact that antibodies themselves can be antigens, and it is possible to obtain antibodies that bind to secondary antibodies. According to this method, an animal (preferably a mouse) is immunized with the antibody. The animal splenocytes are then used to produce hybridoma cells. The hybridoma cells have the ability to bind to the antibody, and are screened to identify clones that produce antibodies whose binding ability is inhibited by the protein antigen. Such antibodies include anti-idiotypic antibodies to the antibody, and animals can be immunized to induce further formation of the antibody.

Fab and F (ab ′) ₂ and other fragments of the antibodies can be used according to known methods, including those disclosed herein. Such fragments are typically produced by cleavage of the antibody with a proteolytic enzyme such as papain (resulting in a Fab fragment) or pepsin (resulting in an F (ab ′) ₂ fragment). Alternatively, protein binding fragments according to one embodiment of the invention can be produced by application of recombinant DNA technology or chemical synthesis.

  In addition, it is added that the contents described in the items <1> to <5> can be appropriately used in other items. The present invention is not limited to the above-described embodiments, and various modifications can be made within the scope of the claims, and the technical means disclosed in different embodiments can be appropriately combined. Embodiments are also included in the technical scope of the present invention. EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited only to this Example.

[Configuration of the present invention]
The present invention can be configured as follows.

(1) Any gene selected from the group consisting of the following (G1) to (G5):
(G1) a gene encoding a protein consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3;
(G2) The amino acid sequence described in any one of SEQ ID NOs: 1 to 3, consisting of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and / or added, and 10 of taxadiene A gene encoding a protein having an activity of oxidizing the position;
(G3) a gene encoding a protein consisting of an amino acid sequence having 80% or more homology with the amino acid sequence described in any one of SEQ ID NOs: 1 to 3 and having an activity of oxidizing position 10 of taxadiene;
(G4) a gene comprising the base sequence described in any one of SEQ ID NOs: 4 to 7;
(G5) A protein that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to any one of the genes (G1) to (G4) and has an activity to oxidize position 10 of taxadiene The gene to encode.

(2) Any protein selected from the group consisting of the following (P1) to (P4):
(P1) a protein comprising the amino acid sequence described in any one of SEQ ID NOs: 1 to 3;
(P2) In the amino acid sequence described in any one of SEQ ID NOs: 1 to 3, the amino acid sequence consists of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and / or added, and 10 of taxadiene A protein having the activity of oxidizing the position;
(P3) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence described in any one of SEQ ID NOs: 1 to 3, and having an activity of oxidizing the 10th position of taxadiene;
(P4) A protein encoded by the gene according to any one of (G1) to (G5) above.

  (3) A recombinant vector comprising the gene according to (1) above.

  (4) A transformant comprising the gene according to (1) above or the recombinant vector according to (3) above.

(5) The transformant according to (4), wherein the following genes (g1) to (g7) are further introduced and expressed:
(G1) a group of mevalonate pathway genes that synthesize mevalonic acid or mevalonolactone to isopentenyl diphosphate,
(G2) type 1 isopentenyl diphosphate isomerase gene,
(G3) type 2 isopentenyl diphosphate isomerase gene,
(G4) acetoacetate-coenzyme A ligase gene,
(G5) geranylgeranyl diphosphate synthase gene,
(G6) Taxadiene synthase gene,
(G7) NADPH-cytochrome P450 reductase gene.

  (6) The transformant according to (4) or (5), which is a recombinant Escherichia coli.

  (7) A method for producing taxadiene oxide, comprising the step of culturing the transformant according to any one of (4) to (6) above to obtain oxidized taxadiene.

  (8) The following formula (I):

A compound represented by

  (9) An antibody that specifically binds to the protein of (2) above.

[1. N11 gene]
Three types of N11 genes having different N-terminal sequences prepared by artificial synthesis and PCR were prepared. The three types of N11 genes are genes each consisting of a base sequence represented by the following SEQ ID NO.
N11: a gene consisting of the base sequence represented by SEQ ID NO: 5.
Δ30aa N11: a gene consisting of the base sequence represented by SEQ ID NO: 6.
17α-Δ14aa N11: a gene consisting of the base sequence shown in SEQ ID NO: 7.

  The above three types of N11 genes are codon-optimized by the present inventors based on the N11 (native) gene isolated from Japanese yew in order to enhance the expression ability in E. coli.

[2. Construction of expression vector
Each of the above-mentioned three types of N11 genes (N11, Δ30aa N11, and 17α-Δ14aa N11), NADPH-cytochrome P450 reductase gene (TCPR) derived from Japanese yew (Taxus cuspidata), derived from Taxus brevifolia The taxadiene synthase gene (TXS) and geranylgeranyl diphosphate synthase gene (crtE) derived from soil bacterium (Pantoea ananatis) were introduced into a pRSFDuet-1 vector (MERCK) to prepare an E. coli expression plasmid.

  As the TCPR, the coding region (CDS) of the gene published as the accession number: AY571340.1 by INSD is codon-optimized for E. coli, and the transmembrane domain 74 residues on the N-terminal side are removed. The gene consisting of the base sequence shown in SEQ ID NO: 10 designed in (1) was used. In addition, as the TXS, the coding region (CDS) of the gene published as the accession number: U48796 by INSD is codon-optimized for E. coli, 60 residues on the N-terminal side are removed, and the N-terminal is further removed. A gene consisting of the base sequence shown in SEQ ID NO: 9 designed to add Met and Gly residues on the side was used. As the crtE, a gene consisting of the base sequence shown in SEQ ID NO: 8 corresponding to the coding region (CDS) of the gene published as the accession number: D90087 by INSD was used.

[3. Production of taxadiene oxide)
2. The vector obtained by introducing the mevalonate pathway gene group that produces farnesyl diphosphate (FPP) from lithium acetoacetate (LAA) and the expression vector obtained in the above was introduced into Escherichia coli strain BL21 (DE3). As a vector into which a mevalonate pathway gene group for producing farnesyl diphosphate (FPP) from lithium acetoacetate (LAA) was introduced, the previously reported pAC-Mev / Scidi / Aacl was used (Harada H et. al. (2008) "Efficient synthesis of functional isoprenoids from acetoacetate through metabolic pathway-engineered Escherichia coli", Applied Microbiology and Biotechnology, Vol. 81 (Issue 5), p.915-925).

  Further, transformation of E. coli and cultivation of recombinant E. coli, extraction of the product from the cultured E. coli, and identification of the product were performed as follows.

First, the recombinant Escherichia coli introduced with the above two types of vectors was treated with 3 mL of LB medium (12 g / L Bacto Tryptone, 24 g / L Bacto Yeast extract, 90 mM K ₂ HPO ₄ (15.7 g / L) containing Km and Cm. 10 mM KH ₂ PO ₄ (1.4 g / L), 1% (v / v) glycerol) and cultured at 28 ° C. and 180 rpm for 16 hours to prepare a preculture. Subsequently, 1% (v / v) preculture was added to 20 mL of TB medium containing Km, Cm, 5-ALA (LAA) and Fe (Joseph Sambrook and David W. Russell, “Molecular cloning: a laboratory manual 3rd Ed. ", Cold Spring Harbor Laboratory Press, 2001) and cultured at 37 ° C and 180 rpm for 3 to 5 hours. The OD ₆₀₀ value at this time was 0.9. Thereafter, 1 mg / mL LAA, 50 mM IPTG, and 5 mM CuCl ₂ were added, and main culture was performed at 20 ° C. and 180 rpm for 48 hours.

  After completion of the main culture, Escherichia coli was recovered and extracted with ethyl acetate, and then the extract was analyzed using a gas chromatograph mass spectrometer (GC / MS).

  The analysis conditions for GC / MS are as follows. As the apparatus, GCMS-QP2010 (Shimadzu Corporation) was used. For the gas chromatograph, a capillary column HP-5MS (30 m × 0.25 mm, film thickness 0.25 μm: Agilent J & W) was used. The column oven was heated at 180 ° C. for 2 minutes, heated to 280 ° C. at a rate of 4 ° C./minute, and held at 280 ° C. for 10 minutes. The injector temperature was 250 ° C. and the GC interface temperature was 280 ° C. Helium was used as the carrier gas, the flow rate was 0.63 mL / min, the sample injection amount was 1 μL, and the split ratio was 1:10. The mass spectrometer used the electron ionization method (EI), ionization energy was 70 eV, and ion source temperature was 250 degreeC. m / z: 40-600 was scanned once in 0.25 seconds.

  As a result, it was found that transformants into which the N11 gene (N11, Δ30aa N11, and 17α-Δ14aa N11) was introduced produced a novel oxide (see the six peaks in FIG. 2). In addition, it was found that transformants into which 17α-Δ14aa N11 was introduced produced the largest amount of novel oxides (same as above). These results indicate that the N11 gene (N11, Δ30aa N11, and 17α-Δ14aa N11) oxidized taxadiene in the transformant to synthesize a new oxidant.

  Further, when the oxide corresponding to the fifth peak in FIG. 2 was analyzed by NMR, it was found to be Taxa-4,11-diene-10β-ol. The structural formula of Taxa-4,11-diene-10β-ol is shown in FIG. 3 (a), and the analysis result by NMR is shown in FIG. 3 (b). This result indicates that the N11 gene (N11, Δ30aa N11, and 17α-Δ14aa N11) has an activity to directly oxidize the 10 position of taxadiene.

  The gene according to one embodiment of the present invention may be useful in the production of novel oxidized taxadiene and the production of paclitaxel. Moreover, since the novel oxidized taxadiene produced by the gene according to one embodiment of the present invention is presumed to be a new intermediate in the paclitaxel biosynthesis pathway, it can be applied to the efficient production of paclitaxel.

Claims (9)

Any gene selected from the group consisting of the following (G1) to (G5):
(G1) a gene encoding a protein consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3;
(G2) The amino acid sequence described in any one of SEQ ID NOs: 1 to 3, consisting of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and / or added, and 10 of taxadiene A gene encoding a protein having an activity of oxidizing the position;
(G3) a gene encoding a protein consisting of an amino acid sequence having 90 % or more identity with any one of SEQ ID NOs: 1 to 3 and having an activity to oxidize position 10 of taxadiene;
(G4) A gene comprising the base sequence described in any one of SEQ ID NOs: 4 to 7 . Any protein selected from the group consisting of the following (P1) to (P4):
(P1) a protein comprising the amino acid sequence described in any one of SEQ ID NOs: 1 to 3;
(P2) In the amino acid sequence described in any one of SEQ ID NOs: 1 to 3, the amino acid sequence consists of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and / or added, and 10 of taxadiene A protein having the activity of oxidizing the position;
(P3) a protein comprising an amino acid sequence having 90% or more identity with any of the amino acid sequences set forth in SEQ ID NOs: 1 to 3, and having an activity to oxidize position 10 of taxadiene;
(P4) A protein encoded by the gene according to claim 1.   A recombinant vector comprising the gene according to claim 1.   A transformant comprising the gene according to claim 1 or the recombinant vector according to claim 3. The transformant according to claim 4, further comprising the following genes (g1) to (g7) introduced and expressed:
(G1) a group of mevalonate pathway genes that synthesize mevalonic acid or mevalonolactone to isopentenyl diphosphate,
(G2) type 1 isopentenyl diphosphate isomerase gene,
(G3) type 2 isopentenyl diphosphate isomerase gene,
(G4) acetoacetate-coenzyme A ligase gene,
(G5) geranylgeranyl diphosphate synthase gene,
(G6) Taxadiene synthase gene,
(G7) NADPH-cytochrome P450 reductase gene.   The transformant according to claim 4 or 5, which is a recombinant E. coli.   A method for producing taxadiene oxide, comprising the step of culturing the transformant according to any one of claims 4 to 6 to obtain oxidized taxadiene. The following formula (I):

A compound represented by   An antibody that specifically binds to the protein of claim 2.

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