JP6735461B2 - Recombinant E. coli and its use - Google Patents

Description

The present invention relates to a recombinant Escherichia coli and its use, and more particularly to a recombinant E. coli and its use for high production of terpene compounds.

Terpenes (also referred to as terpenoids or isoprenoids) are the most complex and diverse compound group in nature, with about 40,000 kinds isolated from plants and microorganisms so far. Various physiological activities have been reported for terpenes and their derivatives, and they are widely distributed in the market as pharmaceuticals, functional foods, flavors and the like. However, since terpenes are abundant in nature and many compounds have complicated structures, it has been expected to develop efficient production technology by modern biotechnology utilizing microorganisms and plants. In recent years, studies aimed at high production of terpenes have been conducted by modifying the basic metabolic pathway of terpenes using Escherichia coli, yeast or plants as hosts.

To date, the present inventors have introduced terpene production into E. coli, which originally has only a non-mevalonate pathway as a basic metabolic pathway, by introducing a mevalonate pathway gene group derived from actinomycetes, which is another basic metabolic pathway. It has been found that the performance is improved ten times or more (Patent Document 1, Non-Patent Document 1).

JP, 2009-207376, A

H. Harada, F. Yu, S. Okamoto, T. Kuzuyama, R. Utsumi, N. Misawa., Appl Microbiol Biotechnol, 81: 915-925, 2009

Although the above-mentioned technology is excellent, it cannot be said to be sufficient by itself, and further development of terpene production technology is desired.

The present invention has been made in view of the above problems, and an object thereof is to provide a novel recombinant Escherichia coli that enables high production of terpenes and a technique relating to the use thereof.

The present inventors have examined whether the conventionally known mevalonate pathway gene group derived from actinomycetes is an optimal selection in Escherichia coli, and further improvement in production amount is necessary for practical use. As a result of intensive studies to achieve the above-mentioned object, it was found that a particularly excellent mevalonate pathway gene group was found in high production of terpenes in recombinant Escherichia coli, and the present invention was completed. .. That is, the present invention includes the following inventions.

(1) Recombinant Escherichia coli introduced with the following three genes derived from Acholeplasma laidlawii, Amphibacillus xylanus, or Streptobacillus moniliformis:
・Mevalonate kinase gene;
-Diphosphomevalonate decarboxylase gene;
-Phosphomevalonate kinase gene.

(2) The recombinant Escherichia coli according to (1), wherein the mevalonate kinase gene is any gene selected from the group consisting of (a) to (e) below:
(A) a gene encoding a protein consisting of the amino acid sequence described in any of SEQ ID NOs: 1 to 3;
(B) In the amino acid sequence described in any 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 having mevalonate kinase activity A gene encoding a protein having:
(C) a gene consisting of an amino acid sequence having 80% or more homology with the amino acid sequence described in any of SEQ ID NOs: 1 to 3, and encoding a protein having mevalonate kinase activity;
(D) a gene comprising the nucleotide sequence described in any of SEQ ID NOs: 4 to 6;
(E) A gene that hybridizes with a polynucleotide having a nucleotide sequence complementary to any of the genes of (a) to (d) under stringent conditions and encodes a protein having mevalonate kinase activity.

(3) The recombinant Escherichia coli according to (1) or (2), wherein the diphosphomevalonate decarboxylase gene is any gene selected from the group consisting of the following (f) to (j):
(F) a gene encoding a protein consisting of the amino acid sequence described in any of SEQ ID NOs: 7 to 9;
(G) A diphosphomevalonate decarboxylase consisting of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and/or added in the amino acid sequence described in any of SEQ ID NOs: 7 to 9. A gene encoding a protein having activity;
(H) a gene consisting of an amino acid sequence having 80% or more homology with the amino acid sequence described in any of SEQ ID NOs: 7 to 9 and encoding a protein having diphosphomevalonate decarboxylase activity;
(I) a gene comprising the base sequence described in any of SEQ ID NOs: 10 to 12;
(J) A gene that hybridizes with a polynucleotide having a nucleotide sequence complementary to any of the genes (f) to (i) under stringent conditions and encodes a protein having diphosphomevalonate decarboxylase activity.

(4) The recombinant Escherichia coli according to any one of (1) to (3), wherein the phosphomevalonate kinase gene is any gene selected from the group consisting of (k) to (o) below:
(K) a gene encoding a protein consisting of the amino acid sequence of any of SEQ ID NOs: 13 to 15;
(L) In the amino acid sequence described in any of SEQ ID NOs: 13 to 15, consisting of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and/or added, and having a phosphomevalonate kinase activity. A gene encoding a protein having:
(M) a gene consisting of an amino acid sequence having 80% or more homology with the amino acid sequence described in any of SEQ ID NOs: 13 to 15 and encoding a protein having phosphomevalonate kinase activity;
(N) a gene comprising the nucleotide sequence described in any of SEQ ID NOs: 16 to 18;
(O) A gene which hybridizes with a polynucleotide having a nucleotide sequence complementary to any of the genes (k) to (n) under stringent conditions and which encodes a protein having phosphomevalonate kinase activity.

(5) The recombinant Escherichia coli according to any one of (1) to (4), further comprising the following three genes introduced:
・HMG-CoA synthase gene;
-HMG-CoA reductase gene;
-Type 1 and/or type 2 isopentenyl diphosphate isomerase genes.

(6) The recombinant Escherichia coli according to any of (1) to (5), further comprising a gene or gene group of an enzyme that synthesizes a terpene from farnesyl diphosphate.

(7) A method for producing a terpene, which comprises culturing the recombinant Escherichia coli according to any one of (1) to (6) above to obtain a terpene.

(8) The method for producing a terpene according to (7), wherein the host Escherichia coli strain of the recombinant Escherichia coli is JM109 or JM101.

According to the present invention, terpenes can be efficiently and highly produced.

It is a figure which shows typically an example of the structure of the recombinant plasmid used in the Example of this invention. It is a figure which shows typically the production procedure of the recombinant plasmid and recombinant E. coli used in the Example of this invention. It is a figure which shows the result of the lycopene production amount in the Example of this invention. It is a figure which shows typically an example of the other structure of the recombinant plasmid used in the Example of this invention. It is a figure which shows typically an example of the other structure of the recombinant plasmid used in the Example of this invention. It is a figure which shows typically the preparation procedure of the recombinant plasmid used in the Example of this invention. It is a figure which shows the result of the taxadiene production amount in the Example of this invention.

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

As used herein, the term "gene" is used interchangeably with "polynucleotide", "nucleic acid" or "nucleic acid molecule" and intends a polymer of nucleotides. Here, the gene may exist in the form of DNA (for example, cDNA or genomic DNA) or RNA (for example, mRNA). The DNA or RNA may be double-stranded or single-stranded. The single-stranded DNA or RNA may be a coding strand (sense strand) or a non-coding strand (antisense strand). In addition, the gene may be chemically synthesized, and the codon usage may be changed so that the expression of the encoded protein is improved. It is also possible to substitute codons that code for the same amino acid. Also, the term "protein" is used interchangeably with "peptide" or "polypeptide." As used herein, the notation of bases and amino acids uses the one-letter or three-letter notations defined by IUPAC and IUB, as appropriate.

<1. Recombinant Escherichia coli introduced with a gene derived from Acholeplasma laidlawii, Amphibacillus xylanus, or Streptobacillus moniliformis>
The recombinant Escherichia coli according to the present invention may be any recombinant Escherichia coli into which the following (1) to (3) genes derived from Acholeplasma laidlawii, Amphibacillus xylanus, or Streptobacillus moniliformis have been introduced and expressed. The configuration is not particularly limited:
(1) Mevalonate kinase gene;
(2) diphosphomevalonate decarboxylase gene;
(3) Phosphomevalonate kinase gene.

As shown in Examples described later, the above-mentioned Acholeplasma laidlawii, Amphibacillus xylanus, or Streptobacillus moniliformis-derived recombinant Escherichia coli introduced mevalonate pathway gene group, compared with those introduced mevalonate pathway gene group derived from other microorganisms. It became clear that the production of terpenes increased remarkably. For example, in the examples, the recombinant Escherichia coli according to the present invention was shown to have a terpene-producing ability several times higher than that in which the mevalonate pathway gene group derived from another microorganism such as actinomycete was introduced. .. Therefore, the recombinant Escherichia coli according to the present invention can achieve high production of terpenes.

An outline of large-scale production of terpenes (isoprenoids) using recombinant Escherichia coli will be described. Escherichia coli does not have the mevalonate pathway and is also called the MEP pathway because it passes through the non-mevalonate pathway [2-C-methyl-D-erythritol 4-phosphate (hereinafter referred to as MEP)] The substrate, isopentenyl diphosphate (also called isopentenyl pyrophosphate; hereinafter described as IPP), is made. IPP is converted to dimethylallyl diphosphate (hereinafter referred to as DMAPP) by IPP isomerase (hereinafter also referred to as Idi), and DMAPP is farnesyl diphosphate (hereinafter referred to as FPP) synthase (farnesyl diphosphate; hereinafter referred to as FPP). Sequential condensation with IPP by synthase) converts geranyl diphosphate having 10 carbon atoms (hereinafter referred to as GPP) to FPP having 15 carbon atoms. It branches from GPP to make a volatile monoterpene. Furthermore, it branches from FPP to produce sesquiterpenes and triterpenes. FPP is further condensed with IPP by geranylgeranyl diphosphate (hereinafter referred to as GGPP) synthase to synthesize GGPP having 20 carbon atoms. By branching from this GGPP, diterpenes and carotenoids (tetraterpenes) are synthesized. Since Escherichia coli does not synthesize the above-mentioned terpenes, in order to synthesize these terpenes in E. coli, it is preferable to introduce the biosynthetic enzyme gene(s) responsible for the synthesis from FPP to the terpene into E. coli and express it. ..

The (1) mevalonate kinase gene is preferably any gene selected from the group consisting of the following (a) to (e):
(A) a gene encoding a protein consisting of the amino acid sequence described in any of SEQ ID NOs: 1 to 3;
(B) In the amino acid sequence described in any 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 having mevalonate kinase activity A gene encoding a protein having:
(C) a gene consisting of an amino acid sequence having 80% or more homology with the amino acid sequence described in any of SEQ ID NOs: 1 to 3, and encoding a protein having mevalonate kinase activity;
(D) a gene comprising the nucleotide sequence described in any of SEQ ID NOs: 4 to 6;
(E) A gene that hybridizes with a polynucleotide having a nucleotide sequence complementary to any of the genes of (a) to (d) under stringent conditions and encodes a protein having mevalonate kinase activity.

The gene (a) above will be specifically described. SEQ ID NO: 1 is a mevalonate kinase derived from Acholeplasma laidlawii ATCC 23206 strain, and is a protein composed of 313 amino acid residues in total length (NCBI accession No. YP_001620791). SEQ ID NO: 2 is a mevalonate kinase derived from Amphibacillus xylanus NBRC15112 strain, and is a protein composed of 321 amino acid residues in full length (NCBI accession No. YP_006844176). SEQ ID NO: 3 is a mevalonate kinase derived from Streptobacillus moniliformis DSM12112 strain, and is a protein composed of 276 amino acid residues in total length (NCBI accession No. YP_003306462).

The gene of (b) above 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 of SEQ ID NOs: 1 to 3. The specific sequence is not limited as long as it encodes a protein having a mevalonate kinase activity, such as a fusion protein with a peptide.

Here, the number of amino acids that may be deleted, substituted or added is not limited as long as the above function is not lost, but the number of amino acids may be deleted, substituted or deleted by a known introduction method such as site-directed mutagenesis. The number that can be added is usually 30 amino acids or less, preferably 20 amino acids or less, more preferably 10 amino acids or less, more preferably 7 amino acids or less, further preferably 5 amino acids or less (for example, 5, 4, 3, 2, or 1 amino acid). Further, in the specification, the “mutation” mainly means a displacement artificially introduced by a site-directed mutagenesis method or the like, but a similar naturally occurring mutation may be used.

The mutated amino acid residue is preferably mutated to another amino acid in which the properties of the amino acid side chain are conserved. For example, the properties of the amino acid side chain include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V) and 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 Chain-containing amino acids (C, M), carboxylic acid and amide-containing side chains (D, N, E, Q), base-containing side chains (R, K, H), aromatic-containing side chains And amino acids (H, F, Y, W) having (in parentheses, all represent one-letter code of amino acid). It is already known that a polypeptide having an amino acid sequence modified by deletion, addition and/or substitution of one or more amino acid residues with respect to an amino acid sequence maintains its biological activity. There is. Furthermore, the target amino acid residue is more preferably mutated to an amino acid residue having as many common properties as possible.

In the present specification, “functionally equivalent” means that the target protein has a biological function or biochemical function equivalent (same and/or similar) to the target protein. The biological property may include the specificity of the site to be expressed, the expression level, and the like.

Whether or not the mutated protein has a desired function can be determined by examining whether or not the mutated protein has mevalonate kinase activity.

The gene (c) is also 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 of SEQ ID NOs: 1 to 3. -It is intended to be a fusion protein with a peptide, etc., and its specific sequence is not limited as long as it encodes a protein having mevalonate kinase activity.

Amino acid sequence homology means at least 80% or more, more preferably 90% or more, still more preferably 95% or more (for example, 95%, 96%, 97) in the entire amino acid sequence (or a region required for expression of function). %, 98%, 99% or more). Amino acid sequence homology can be determined using the BLASTN (nucleic acid level) and BLASTX (amino acid level) programs (Altschul et al. J. Mol. Biol., 215: 403-410, 1990). The program is based on the algorithm BLAST (Proc. Natl. Acad. Sci. USA, 87:2264-2268, 1990, Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993) by Karlin and Altschul. There is. When the nucleotide sequence is analyzed by BLASTN, the parameters are, for example, score=100 and wordlength=12. When the amino acid sequence is analyzed by BLASTX, the parameters are, for example, score=50 and wordlength=3. When the amino acid sequence is analyzed using the Gapped BLAST program, it can be performed as described in Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997). When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known. Additions or deletions (for example, gaps) may be allowed in order to optimally align the base sequence or amino acid sequence to be compared.

In the present specification, “homology” means the proportion of amino acid residues having similar properties (homology, positive, etc.), but more preferably, the proportion of matching amino acid residues, that is, identity. (Identity). The properties of the amino acid are as described above.

Regarding the gene of (d) above, SEQ ID NO: 4 shows the nucleotide sequence (Open Reading Frame: ORF) of the gene encoding the protein consisting of the amino acid sequence shown by SEQ ID NO: 1. Further, SEQ ID NOS: 5 and 6 show the nucleotide sequences (ORFs) of the genes encoding the proteins consisting of the amino acid sequences shown in SEQ ID NOS: 2 and 3, respectively.

The gene (e) is intended to be a gene that hybridizes under stringent conditions with a polynucleotide having a nucleotide sequence complementary to any of the genes (a) to (d). Here, the stringent condition means a condition in which a double-stranded polynucleotide specific to a so-called base sequence is formed and a non-specific double-stranded polynucleotide is not formed. In other words, conditions in which nucleic acids having high homology, such as completely matched hybrids, are hybridized at a temperature ranging from the melting temperature (Tm value) to 15° C., preferably 10° C., and more preferably 5° C. lower. It can also be said. For example, as an example, the temperature in the buffer consisting of 0.25M Na ₂ HPO ₄ , pH 7.2, 7% SDS, 1 mM EDTA, 1 × Denhardt solution 60 ~ 68 ℃, preferably 65 ℃, more preferably Hybridization is carried out for 16 to 24 hours under the condition of 68° C., and the temperature is 60 to 68° C., preferably 65° C., in a buffer consisting of 20 mM Na ₂ HPO ₄ , pH 7.2, 1% SDS and 1 mM EDTA. Preferably, a condition of performing washing for 15 minutes twice at 68° C. can be mentioned. Other examples include 25% formamide, 50% formamide in more stringent conditions, 4x SSC (sodium chloride/sodium citrate), 50 mM Hepes pH 7.0, 10x Denhardt's solution, 20 μg/mL denatured salmon sperm DNA. After performing prehybridization overnight at 42°C in a hybridization solution, a labeled probe is added and the mixture is incubated at 42°C overnight to perform hybridization. The cleaning liquid and temperature conditions in the subsequent cleaning are "1 x SSC, 0.1% SDS, 37°C", more severe conditions are "0.5 x SSC, 0.1% SDS, 42°C", and more stringent conditions are It can be carried out at about "0.2 x SSC, 0.1% SDS, 65°C". Thus, the more stringent the washing conditions for hybridization, the more specific the hybridization becomes. However, the combination of the above SSC, SDS and temperature conditions is an exemplification, and those skilled in the art can determine the above or other factors (eg, probe concentration, probe length, hybridization reaction) that determine the stringency of hybridization. It is possible to realize the same stringency as described above by appropriately combining (such as time). This is described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory (2001).

In addition, in the gene of the above (e), in the DNA consisting of the nucleotide sequence of any of SEQ ID NOs: 4 to 6, 1 to 50 nucleotide sequences are substituted, deleted, inserted and/or added. And a gene consisting of a DNA having 90% or more homology with the DNA consisting of the nucleotide sequence of any one of SEQ ID NOs: 4 to 6.

Further, the (2) diphosphomevalonate decarboxylase gene is preferably any gene selected from the group consisting of the following (f) to (j):
(F) a gene encoding a protein consisting of the amino acid sequence described in any of SEQ ID NOs: 7 to 9;
(G) A diphosphomevalonate decarboxylase consisting of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and/or added in the amino acid sequence described in any of SEQ ID NOs: 7 to 9. A gene encoding a protein having activity;
(H) a gene consisting of an amino acid sequence having 80% or more homology with the amino acid sequence described in any of SEQ ID NOs: 7 to 9 and encoding a protein having diphosphomevalonate decarboxylase activity;
(I) a gene comprising the base sequence described in any of SEQ ID NOs: 10 to 12;
(J) A gene that hybridizes with a polynucleotide having a nucleotide sequence complementary to any of the genes (f) to (i) under stringent conditions and encodes a protein having diphosphomevalonate decarboxylase activity.

Regarding the gene (f) above, SEQ ID NO: 7 is a diphosphomevalonate decarboxylase derived from Acholeplasma laidlawii ATCC 23206 strain, and is a protein composed of 315 amino acid residues in total length (NCBI accession No. YP_001620790). SEQ ID NO: 8 is a diphosphomevalonate decarboxylase derived from Amphibacillus xylanus NBRC15112 strain, and is a protein composed of 326 amino acid residues in total length (NCBI accession No. YP_006844178). SEQ ID NO: 9 is diphosphomevalonate decarboxylase derived from Streptobacillus moniliformis DSM12112 strain, and is a protein composed of 295 amino acid residues in total length (NCBI accession No. YP_003306461).

The gene (g) above 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 of SEQ ID NOs: 7 to 9. The specific sequence is not limited as long as it encodes a protein having a diphosphomevalonate decarboxylase activity, such as a fusion protein with a peptide. Other explanations are omitted since they are common to the gene of (b) above except that they have diphosphomevalonate decarboxylase activity.

The gene of (h) above 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 of SEQ ID NOs: 7 to 9. -A fusion protein with a peptide or the like is intended, and the specific sequence thereof is not limited as long as it encodes a protein having a diphosphomevalonate decarboxylase activity. Other explanations are the same as the gene of (c) above, except that they have diphosphomevalonate decarboxylase activity, and are therefore omitted.

Regarding the gene of (i) above, SEQ ID NOS: 10 to 12 show the base sequences (Open Reading Frame: ORF) of genes encoding proteins consisting of the amino acid sequences shown in any of SEQ ID NOS: 7 to 9, respectively.

The gene (j) is intended to be a gene that hybridizes under stringent conditions with a polynucleotide having a nucleotide sequence complementary to any of the genes (f) to (i). Further, in the gene of (j) above, in the DNA consisting of the nucleotide sequence of any of SEQ ID NOs: 10 to 12, DNA having 1 to 50 nucleotide sequences substituted, deleted, inserted and/or added. And a gene consisting of a DNA having 90% or more homology with the DNA consisting of the nucleotide sequence of any one of SEQ ID NOs: 10 to 12. The other explanations are the same as the gene of (e) above except that they have a diphosphomevalonate decarboxylase activity, and are therefore omitted.

Further, the above (3) phosphomevalonate kinase gene is preferably any gene selected from the group consisting of the following (k) to (o):
(K) a gene encoding a protein consisting of the amino acid sequence of any of SEQ ID NOs: 13 to 15;
(L) In the amino acid sequence described in any of SEQ ID NOs: 13 to 15, consisting of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and/or added, and having a phosphomevalonate kinase activity. A gene encoding a protein having:
(M) a gene consisting of an amino acid sequence having 80% or more homology with the amino acid sequence described in any of SEQ ID NOs: 13 to 15 and encoding a protein having phosphomevalonate kinase activity;
(N) a gene comprising the nucleotide sequence described in any of SEQ ID NOs: 16 to 18;
(O) A gene which hybridizes with a polynucleotide having a nucleotide sequence complementary to any of the genes (k) to (n) under stringent conditions and which encodes a protein having phosphomevalonate kinase activity.

Regarding the gene (k) above, SEQ ID NO: 13 is a phosphomevalonate kinase derived from Acholeplasma laidlawii ATCC 23206 strain, and is a protein composed of 346 amino acid residues in total length (NCBI accession No. YP_001620789). SEQ ID NO: 14 is a phosphomevalonate kinase derived from Amphibacillus xylanus NBRC15112 strain, and is a protein composed of 362 amino acid residues in total length (NCBI accession No. YP_006844177). SEQ ID NO: 15 is a phosphomevalonate kinase derived from Streptobacillus moniliformis DSM12112 strain, and is a protein composed of 288 amino acid residues in total length (NCBI accession No. YP_003306460).

The gene of (l) above 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 of SEQ ID NOs: 13 to 15. The specific sequence is not limited as long as it encodes a protein having a phosphomevalonate kinase activity, such as a fusion protein with a peptide. The other explanations are the same as the gene of (b) above except that they have phosphomevalonate kinase activity, and therefore are omitted.

The gene (m) 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 of SEQ ID NOs: 13 to 15. -A fusion protein with a peptide or the like is intended, and its specific sequence is not limited as long as it encodes a protein having phosphomevalonate kinase activity. Other explanations are the same as the gene of (c) above, except that they have phosphomevalonate kinase activity, and are therefore omitted.

Regarding the above-mentioned gene (n), SEQ ID NOs: 16 to 18 show nucleotide sequences (Open Reading Frame: ORF) of genes encoding proteins consisting of the amino acid sequences shown in SEQ ID NOs: 13 to 15, respectively.

The gene (o) is intended to be a gene that hybridizes under stringent conditions with a polynucleotide having a nucleotide sequence complementary to any of the genes (k) to (n). In addition, in the gene of (o) above, in the DNA consisting of the nucleotide sequence of any of SEQ ID NOs: 16 to 18, DNA having 1 to 50 nucleotide sequences substituted, deleted, inserted and/or added. And a gene consisting of a DNA having 90% or more homology with the DNA consisting of the nucleotide sequence of any one of SEQ ID NOs: 16 to 18. Other explanations are the same as the gene of (e) above except that they have phosphomevalonate kinase activity, and are therefore omitted.

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 prepared 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 bases of the polynucleotide include, for example, commercially available kits (KOD-Plus Site-Directed Mutagenesis Kit; Toyobo, Transformer Site-Directed Mutagenesis Kit; Clontech, QuickChange Site Directed Mutagenesis Kit; Stratagene, etc. ) Or the use of polymerase chain reaction (PCR). These methods are known to those skilled in the art.

The gene may be composed of only the polynucleotide encoding the protein, but may have other base sequences added thereto. 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 include a nucleotide sequence encoding a signal sequence (eg, endoplasmic reticulum translocation signal sequence, secretory sequence, etc.). The site to which these nucleotide sequences are added is not particularly limited and may be, for example, the N-terminal or the C-terminal of the translated protein.

Further, the present invention may include a vector containing the above gene. The present vector includes not only an expression vector for expressing the above gene in a host cell for producing a transformant, but also a vector used for producing a recombinant protein. The subject of transformation is not particularly limited, and examples thereof include bacteria, yeast, insects, animals and plants.

Various commonly used vectors can be used as the base vector that is the base of the above vector. For example, a plasmid, a phage, a cosmid, or the like can be used, and can be appropriately selected according to the cells to be introduced or the method of introduction. 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. Depending on the type of host cell, a promoter sequence may be appropriately selected in order to ensure the expression of the above gene, and this and the above gene may be incorporated into various plasmids or the like and 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 baculovirus). Virus, papovavirus, vaccinia virus, adenovirus, avian pox virus, pseudorabies virus, herpes virus, lentivirus and retrovirus)) and vectors derived from combinations thereof (eg, cosmid and phagemid).

Generally, the plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in complex with a charged lipid. If the vector is a virus, the vector can be packaged in vitro using a suitable packaging cell line and then transduced into host cells. Also, retroviral vectors can be replication-competent or replication-defective. In the latter case, viral propagation generally will occur only in helper cells.

Further, the above vector is preferably a vector containing a cis-acting control region for the gene of interest. Appropriate trans-acting factors can be supplied by the host, supplied by a complementing vector, or supplied by the vector itself upon introduction into the host. In a preferred embodiment in this regard, the vector preferably provides specific expression, which may be inducible and/or cell type specific. Among such vectors, particularly preferred vectors are those inducible 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, Phagescript vector, Bluescript vector, pNH8A, pNH16a, pNH18A and pNH46A (Stratagene). PTrc99a, pKK223-3, pKK233-3, pDR540 and pRIT5 (available from Addgene). Preferred eukaryotic vectors also include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG (available from Stratagene); and pSVK3, pBPV, pMSG and pSVL (available from Addgene).

Various markers may be used in order to confirm whether or not the above-mentioned gene has been introduced into the host cell, and further whether or not the gene is 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 or neomycin resistance for eukaryotic cell culture, E. For culture in E. coli and other bacteria, drug resistance genes such as tetracycline resistance gene or ampicillin resistance gene can be mentioned. Alternatively, a gene deleted in the host cell may be used as a marker. By introducing a plasmid or the like containing this marker and the gene of the present invention into a host cell as an expression vector, the introduction of the above 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 host cells.

It is also preferred that the gene insert is operably linked to a suitable promoter. As other suitable promoters, those known to those skilled in the art can be used, and are not particularly limited, and examples thereof include phage λPL promoter, E. coli lacI promoter, lacZ promoter, T3 promoter and T7 promoter, trp promoter, and Examples include tac promoter, SV40 early promoter and late promoter, and retrovirus LTR promoter.

When Escherichia coli is used as a host in 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. For example, M13 type vector, pUC type vector, pBR322, pBluescript, pCR-Script, pGEM-T, pDIRECT, pGEX (manufactured by GE Healthcare) pET (manufactured by Promega), pTrc (manufactured by Invitrogen), pT7 and the like. To be

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

Also, transcription of DNA by higher eukaryotes can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10-300 bp, that act to increase the transcriptional activity of a promoter in a given host cell type. Examples of the enhancer include SV40 enhancer (which is located at 100 to 270 bp on the late side of the replication origin), cytomegalovirus early promoter enhancer, polyoma enhancer on the late side of the replication origin, and adenovirus enhancer. Are listed.

The host into which the vector is introduced is not particularly limited, but various cells can be preferably used. Representative examples of suitable hosts include bacterial 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 human or mouse but also, for example, cells derived from Bombyx mori, insects such as Drosophila melanogaster, bacteria such as Escherichia coli, yeast (Saccharomyces cerevisiae ) And fission yeast (Schizosaccharomyces pombe)), nematodes (Caenorhabditis elegans), oocytes of Xenopus laevis, and the like, but are not particularly limited. Appropriate culture media and conditions for the above-described host cells are available in the art.

The method for introducing the above vector into a host cell, that is, the transformation method is not particularly limited, and it may be electroporation, calcium phosphate method, liposome method, DEAE dextran method, microinjection method, cationic lipid-mediated transfection, electroporation. Conventionally known methods such as ablation, transduction or infection can be preferably used. Such methods are described in many standard laboratory manuals such as Davis et al., Basic Methods In Molecular Biology (1986).

In the present invention, a recombinant expression vector containing a polynucleotide encoding the partial fragment of the above protein for recombinantly producing the partial fragment of the above protein, and a recombinant expression vector are genetically engineered. And a transformant (host cell).

Furthermore, the present invention may include inventions relating to the production of mutants of the protein or fragments thereof obtained by the above-mentioned recombinant techniques. That is, the present invention may also include a method of producing a mutant of a protein or a fragment thereof by using a recombinant technique.

A protein mutant produced by such a technique can be isolated from a host cell or extracellularly (medium and the like) and purified as a substantially pure and homogeneous protein. The separation and purification of the protein may be carried out by using the separation and purification methods that are commonly used for protein purification. For example, chromatography column, filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, ammonium sulfate precipitation or ethanol precipitation, acid extraction, The protein can be separated and purified by appropriately selecting or combining dialysis and recrystallization. Furthermore, it is also possible to combine a plurality of these columns.

Chromatography includes affinity chromatography, anion or cation ion exchange chromatography, hydrophobic chromatography, phosphocellulose chromatography, hydroxyapatite chromatography, lectin chromatography, gel filtration, reverse phase chromatography and adsorption chromatography. Etc.

When a protein variant is expressed in a host cell (such as Escherichia coli) as a fusion protein with GST or a recombinant protein added with 6×His, the expressed recombinant protein is a glutathione column or nickel. It can be purified using a column. After purification of the fusion protein, the region of the fusion protein other than the protein of interest can be cleaved with thrombin or factor Xa or the like to be removed, if necessary.

The present invention also includes a transformant containing the above gene or the above vector. Here, “including a gene or a vector” means that the gene is introduced into a target cell (host cell) in a expressible manner by a known genetic engineering technique (gene manipulation technique). Further, the above-mentioned “transformant” means not only cells, tissues and organs but also individual organisms.

Examples of the method for producing the present transformant (production method) include a method for transforming the above vector. Further, the organism to be transformed is not particularly limited, and various microorganisms exemplified as the above host cells can be mentioned. In addition, if a promoter or a vector is selected, a plant or an animal can be targeted for transformation.

<2. Recombinant E. coli with other genes introduced>
Furthermore, the present invention includes recombinant E. coli in which the following genes (4) to (6) are introduced and expressed:
(4) HMG-CoA synthase gene;
(5) HMG-CoA reductase gene;
(6) Type 1 and/or type 2 isopentenyl diphosphate isomerase gene.

As a mevalonate pathway gene group for synthesizing mevalonate or mevalonolactone to isopentenyl diphosphate, the genes of (4) and (5) above are recombined in addition to the genes of (1) to (3) above. It is preferably introduced into E. coli.

As the gene group of the above (4) and (5), the mevalonate pathway gene group derived from the Streptomyces genus CL190 strain (Patent Document 1, Accession no AB037666) can be used, but budding yeast (Saccharomyces) is also available. cerevisiae)-derived mevalonate pathway genes (VJJ Martin, DJ Pitera, ST Withers, JD Newman, JD Keasling, Nature Biotechonosy, 21: 796-802, 2003), and the mevalonate pathway from the bacterium Streptococcus pneumoniae. Gene groups (SH Yoon, YM Lee, JE Kim, SH Lee, JH Lee, JY Kim, KH Jung, YC Shin, JD Keasling, SW Kim, Biotechnology & Bioengineering, 94: 1025-1032, 2006) etc. are also preferably used. be able to.

Furthermore, in order to increase the amount of FPP supplied, it is preferable to use (6) type 1 and/or type 2 isopentenyl diphosphate isomerase IPP isomerase (Idi; IPP isomerase) genes. There are type 1 (type 1) and type 2 (type 2) having different structures from each other. In the present invention, either Idi may be used, but most preferably both Idi are used. It is a mode. As the Idi gene, the isopentenyl diphosphate isomerase gene derived from the Streptomyces CL190 strain (Patent Document 1, Accession no AB037666) can be used, but in addition to this, an isopentenyl diphosphate isomerase gene derived from Escherichia coli ( VJJ Martin, DJ Pitera, ST Withers, JD Newman, JD Keasling, Nature Biotechonosy, 21: 796-802, 2003), budding yeast (Saccharomyces cerevisiae)-derived isopentenyl diphosphate isomerase gene (S. Kajiwara, PD Fraser, K. Kondo, N. Misawa, Biochemical Journal, 324: 421-426, 1997), the isopentenyl diphosphate isomerase gene from the green alga Haematococcus pluvialis (JJ Martin et al. S. Kajiwara et al.) can also be used.

According to the recombinant Escherichia coli introduced with the mevalonate pathway gene group of (1) to (4) and (5) and the Idi gene of (6), mevalonate or mevalonolactone (D-mevalonolactone (D -mevalonate lactone)) as a substrate, FPP can be produced in large quantities.

On the other hand, as a substrate, acetoacetate (eg 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-coenzyme A (CoA) ligase (acetoacetate-CoA ligase) gene using it as a substrate.

Acetoacetic acid-CoA ligase is an enzyme that uses acetoacetic acid and CoA as substrates and catalyzes the conversion to acetoacetyl-CoA using ATP (JR Stern, Biochem. Biophys. Res. Commun. 44, 1001-1007. , 1971; Bergstrom, JD; Wong, GA; Edwards, PA; Edmond, J., J. Biol. Chem. 259, 14548-14553, 1984). As the acetoacetic acid-CoA ligase gene, genes derived from mammals such as rat (Rattus norvegicus) and humans, certain bacteria, fungi, etc. are known, and these genes can also be used in the present invention. it can. A plasmid containing the full-length gene (Accession No. BC061803) encoding rat-derived acetoacetate-CoA ligase can be obtained from Invitrogen as a Mammalian Gene Collection cDNA clone (clone ID: 5598532).

<3. Recombinant Escherichia coli into which a gene group for terpene synthesis from FPP has been introduced>
The present invention further includes a recombinant Escherichia coli in which a gene or gene group of an enzyme that synthesizes a terpene (isoprenoid) from farnesyl diphosphate (FPP) is introduced and expressed.

The terpene to be synthesized is not particularly limited, and examples thereof include carotenoid and sesquiterpene. Examples of carotenoids include lycopene, β-carotene, zeaxanthin, and astaxane, and examples of sesquiterpenes include artemisinin, δ-sedinene, δ-selinene, γ-humulene, and (+)- δ-cadinene ((+)-δ-cadinene), 5-epi-aristolochene, vetispiradiene, β-eudesmol, α-humulene -humulene) etc. can be illustrated.

The gene or gene group of the enzyme that synthesizes a terpene from FPP is not particularly limited, and various known genes or gene groups can be preferably used. For example, regarding an enzyme that synthesizes carotenoid from FPP, a known GGPP synthase gene (crtE) that synthesizes GGPP from FPP, a phytoene synthase gene (crtB) that synthesizes phytoene from GGPP, and a phytoene desaturase gene that synthesizes lycopene from phytoene. When (crtI) is introduced into Escherichia coli and expressed therein, Escherichia coli metabolizes FPP in the cells to synthesize lycopene (Japanese Patent Laid-Open No. 8-242861 and the following Reference 2).

Furthermore, by introducing a downstream carotenoid biosynthetic enzyme gene into this recombinant Escherichia coli, carotenoids other than lycopene can be synthesized. For example, when a lycopene cyclase (cyclase) gene (crtY) that synthesizes β-carotene from lycopene is introduced and expressed, β-carotene is synthesized, and β-carotene hydroxy that synthesizes zeaxanthin from β-carotene is further developed. A β-carotene (carotenoid) ketolase gene (ketolase; 4,4) that synthesizes zeaxanthin by introducing and expressing a transferase gene (hydroxylase; 3,3'-hydoroxylase; crtZ) and further synthesizing astaxanthin from zeaxanthin '-oxygenase; crtW) is introduced and expressed in the recombinant E. coli to synthesize astaxanthin (Reference 2, Y. Nishida, K. Adachi, H. Kasai, Y. Shizuri, K. Shindo, A. Sawabe, S. Komemushi, W. Miki, S. Misawa, Appl. Environ. Microbiol., 71; 4286-4296, 2005).

Any of these carotenoid biosynthetic enzyme genes may be used. For example, as the GGPP synthase gene, phytoene synthase gene, and phytoene desaturase gene, the soil bacterium Pantoea ananatis (former name: Erwinia uredovora 20D3) is used. ) Derived from the marine bacterium Paracoccus N81106 strain or Brevundimonas genus SD212 strain can be used as the β-carotene ketolase gene.

Genes of enzymes that synthesize sesquiterpenes from FPP are also known. For example, δ-cedinene synthase gene and γ-humulene synthase gene genes are Little, DB, Croteau, RB, Arch. Biochem. Biohps., 402: 120-135 (2002), (+)-δ-cadinene synthase (cyclase), 5-epi-aristroken synthase, and betispyradiene synthase genes are disclosed in Reference 1. , Β-eudesmol synthase gene (ZSS2) is disclosed in reference 6. In the present invention, these known genes can be used.

<4. Terpene manufacturing method>
The present invention includes a method for producing a terpene, which comprises culturing the above-mentioned recombinant Escherichia coli to obtain a terpene. The method for producing such a terpene may be any method as long as it uses the above-mentioned recombinant Escherichia coli, and other specific configurations are not particularly limited.

Various Escherichia coli strains can be used as the host Escherichia coli strain of the above-mentioned recombinant Escherichia coli, and are not particularly limited. For example, JM109 or JM101 is preferable. With such strains, terpenes can be produced with high efficiency. In particular, when the JM101 strain is produced using a gene group derived from Streptobacillus moniliformis, it is more preferable because the production amount can be increased about twice as much as that of the JM109 strain per culture solution.

The temperature at the time of culturing is not particularly limited, but it is preferably 18 to 30° C., and more preferably 20 to 25° C., according to a conventional method. The culture time is not particularly limited, but it is preferable that the culture is performed for 12 to 72 hours, and more preferably 24 to 48 hours after the transgene expression. The method for collecting the terpene from the culture or the bacterial cells can be performed according to a method commonly used for obtaining a microbial product, and is not particularly limited.

In addition, as described above, acetoacetate is preferable as a substrate to be added when culturing recombinant Escherichia coli because it is inexpensive. Therefore, the method for producing a terpene according to the present invention is preferably a method of culturing the above-mentioned recombinant Escherichia coli in a medium containing acetoacetate to obtain a terpene from the culture or cells.

Examples of the acetoacetate salt include lithium acetoacetate salt and sodium acetoacetate salt, and are not particularly limited. Among them, it is preferable to use lithium acetoacetate salt.

The concentration of acetoacetate in the medium is not particularly limited as long as E. coli can produce a terpene, but is preferably 0.1 to 10 g/L, and more preferably 0.5 to 5 g/L. preferable. The medium components other than acetoacetate may be the same as the components contained in a general E. coli culture medium.

In addition, it is added that the contents described in the above items <1> to <4> can be appropriately applied to other items. Further, the present invention is not limited to the above-described embodiments, but various modifications can be made within the scope of the claims, and the technical means disclosed in the different embodiments can be obtained by appropriately combining them. Embodiments are also included in the technical scope of the present invention. Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the examples.

<References>
[Reference 1] VJJ Martin, Y. Yoshikuni, JD Keasling, Biotechnol. Bioeng. 75: 497-503, 2001
[Reference 2] N. Misawa, Y. Satomi, K. Kondo, A. Yokoyama, S. Kajiwara, T. Saito, T. Ohtani, W. Miki, J. Bacteriol., 177: 6575-6584, 1995
[Reference 3] M. Albrecht, N. Misawa, G. Sandmann, Biotechnol. Lett., 21: 791-795, 1999.
[Reference 4] M. Takagi, T. Kuzuyama, S. Takahashi, H. Seto, J. Bacteriol., 182: 4153-4157, 2000
[Reference 5] K. Kakinuma, Y. Dekishima, Y. Matsushima, T. Eguchi, N. Misawa, M. Takagi, T. Kuzuyama, H. Seto, J. Am. Chem. Soc., 123: 1238- 1239, 2001
[Reference 6] F. Yu, H. Harada, K. Yamasaki, S. Okamoto, S. Hirase, Y. Tanaka, N. Misawa, R. Utsumi, FEBS Lett., 2008 Jan. 30.

[1. Construction of plasmids pAC-AlMev, pAC-SmMev, pAC-AxMev, pAC-LgMev, pAC-SgMev)
Three genes of mevalonate kinase (mevalonate kinase; MVA kinase, AlMVK), diphosphomevalonate decarboxylase (DPMVA decarboxylase, AlDPMVDC), and phosphomevalonate kinase (PMVA kinase, AlPMVK) derived from Acholeplasma laidlawii ATCC 23206 strain. For the 3.5 kbp DNA fragment encoding the 2.9 kbp DNA fragment and the subsequent 3.5 kbp DNA fragment encoding the three genes of IPP isomerase, HMG-CoA synthetase, and HMG-CoA reductase derived from the Streptomyces CL190 strain, the tac promoter (Ptac ) And rrnB terminator (TrrnB) were inserted into the plasmid pAC-AlMev (Fig. 1).

In addition, having a plasmid structure similar to the above, mevalonate kinase, diphosphomevalonate decarboxylase, a DNA fragment encoding three genes of phosphomevalonate kinase, Streptobacillus moniliformis DSM12112 strain-derived DNA fragment of 2.6 kbp (Smmvk, Smdpmvdc, Smpmvk), a 3.0 kbp DNA fragment (Ammvk, Amdpmvdc, Ampmvk) derived from Amphibacillus xylanus NBRC15112 strain, a 3.1 kbp DNA fragment derived from Lactobacillus gasseri ATCC 33323 strain, or a Streptococcus gordonii Challis CH1b strain derived from Streptococcus gordonii Challis CH1b 2. Plasmids replaced with respective DNA fragments, pAC-SmMev, pAC-AxMev, pAC-LgMev, and pAC-SgMev were also prepared.

The specific construction method of each plasmid is as follows. A 2.9 kbp DNA fragment encoding three genes derived from the Acholeplasma laidlawii ATCC 23206 strain was prepared by using Alfw (5'-CAGGAAACACATATGATAGATTTTGGACATGGTGTC-3') and Al Rv (5'-TCTTAGATCCCTTCCTCTTACTCATCGTAATCCCATACCTTT) with genomic DNA purchased from ATCC as a template. It was amplified by PCR using two primers of 3').

A 2.6 kbp DNA fragment encoding 3 genes derived from Streptobacillus moniliformis strain DSM12112 is Sm Fw (5′-CAGGAAACACATATGGCTTACGGTAAAGTAATATTATTTG-3′) and Sm Rv (5′-TCTTAGATCCCTTCCTCAACAATGAA) using genomic DNA purchased from ATCC as a template. It was amplified by PCR using the two primers of').

A 3.0 kbp DNA fragment encoding three genes derived from Amphibacillus xylanus NBRC15112 strain was prepared using Ax Fw (5'-CAGGAAACACATATGATTAATGCATCTAGAAAGAGCGC- as a template, using genomic DNA purchased from National Institute of Technology and Evaluation Biotechnology Center (NBRC) as a template. 3') and Ax Rv (5'-TCTTAGATCCCTTCCTTAGATCATTTGTACACCTACAATTGAAAC-3') were used for amplification by PCR.

A 3.1 kbp DNA fragment encoding three genes derived from the Lactobacillus gasseri ATCC 33323 strain is Lg Fw (5'-CAGGAAACACATATGGACAAACGATATAGACTTAATGAAA-3') and Lg Rv (5'-TCTTAGATCCCTTCCTCTTAAGCAATTGAATGAAT, using the genomic DNA purchased from ATCC as a template. It was amplified by PCR using two primers of 3').

A 2.9 kbp DNA fragment encoding three genes derived from Streptococcus gordonii Challis CH1 strain is Sg Fw (5'-CAGGAAACACATATGACAAAAGAAATTGGTGTCG-3') and Sg Rv (5'-TCTTAGATCCCTTCCTCTTACGATTCTGGCAT using the genomic DNA purchased from ATCC as a template. It was amplified by PCR using two primers of 3').

Next, using the pAC-Mev plasmid (H. Harada, F. Yu, S. Okamoto, T. Kuzuyama, R. Utsumi, N. Misawa, Appl. Microbiol. Biotechnol. 81: 915-925, 2009) as a template, DNA fragments amplified by PCR using two primers, pAC-Mev Fw (5'-GGAAGGGATCTAAGAATGACCAG-3') and pAC-Mev Rv (5'-CATATGTGTTTCCTGTGTGAAATTG-3'), and mevalon amplified from each of the above strains A DNA fragment encoding 3 genes of acid kinase, diphosphomevalonate decarboxylase, and phosphomevalonate kinase was ligated by in vitro homologous recombination method using In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain plasmid pAC. -SmMev, pAC-AxMev, pAC-LgMev, pAC-SgMev were constructed (Fig. 2).

[2. Lycopene production by Escherichia coli carrying plasmids pAC-AlMev, pAC-SmMev, pAC-AxMev, pAC-LgMev(-idi), pAC-SgMev(-idi), pAC-AlMev(+idi))
The plasmid constructed by the method described above was used as a plasmid for producing lycopene pCRT-EIB plasmid (N. Misawa, M. Nakagawa, K. Kobayashi, S. Yamano, Y. Izawa, K. Nakamura, K. Harashima, J. Bacteriol). , 172: 6704-6712, 1990; However, in this document, pCRT-EIB is described as pCAR-ADE) together with E. coli competent cells (ECOS competent E. coli JM109 (Nippon Gene), ECOS competent E. coli BL21(DE3). ) (Manufactured by Nippon Gene Co., Ltd.), C600 (manufactured by a general-purpose method), JM101 (manufactured by a general-purpose method)) and introduced into ampicillin (Ap, final concentration 100 μg/mL) and chloramphenicol (Cm). , A final concentration of 30 μg/mL) was obtained on the LB plate (FIG. 2).

This was inoculated into 2.5 ml of LB medium containing Ap and Cm, precultured at 30° C. for 16 hours, 0.7 mL (1%) was inoculated into 70 ml of 2×YT medium, and 30° C. Main culture was carried out in. When the absorbance at 600 nm reached 0.5, isopropyl β-D-thiogalactopyranoside (IPTG, final concentration 1 mM) and D-mevalonolactone (D-mevalonolactone; MVL, final concentration 0.5 g/l) were added, and 20 Culturing was continued at 72°C for 72 hours. 5 ml of the bacterial cell culture solution after 48 hours and 72 hours was collected by centrifugation (5,000 rpm, 4° C., 5 minutes), and 0.5 to 3 ml of chloroform:methanol=1:1 was mixed with the precipitated bacterial cells. The solution was added to extract the pigment component. The extract was dried in a centrifugal evaporator for 1 to 3 hours, dissolved in 200 μL of ethyl acetate, and quantitatively analyzed by a high performance liquid chromatograph HPLC LC-2000Plus system (manufactured by JASCO Corporation).

A TSK ODS-80Ts column (4.6×150 mm, manufactured by Tosoh Corporation) was used as a separation column, and the flow rate was 1.0 mL/min and the temperature was 25° C. under conditions of solvent A (methanol:water 95:5) and solvent B. (methanol:tetrahydrofuran 7:3) was separated by gradient formation of a two-solvent system and detected by an ultraviolet-visible detector. As a gradient condition, solvent A was kept at 100% for 5 minutes, then a linear gradient from solvent A to solvent B was formed over 5 minutes, and solvent B 100% was kept as it was for 8 minutes. Then, 100% of solvent A was allowed to flow for 12 minutes to equilibrate the inside of the column.

The pigment component was identified based on the retention time at the time of chromatographic separation and the area value prepared using a lycopene standard sample, and the lycopene concentration in the sample was calculated as the amount of lycopene per dry weight of E. coli and per culture solution.

The result is shown in FIG. As shown in the figure, when the mevalonate gene expression plasmids pAC-AlMev and pCRT-EIB derived from the Acholeplasma laidlawii ATCC 23206 strain were introduced, the effect of increasing production was greatest, and 107.9 mg per 1 g dry weight of the cells, the culture solution. 66.1 mg of lycopene was synthesized per liter. This production level was 3.2 times per 1 g dry weight of the bacterial cells and 1.7 times per 1 L of the culture solution, as compared with the case where pAC-Mev and pCRT-EIB were introduced. Also, when pAC-AxMev and pAC-SmMev were introduced, when the production amounts were compared among E. coli strains, about 1.5 times per 1 g dry weight of bacterial cells and about 1.3 times per 1 L of culture solution. An increase in production was seen. When the plasmids of any combination were introduced, the JM109 strain showed high productivity. When pAC-SmMev and pCRT-EIB were introduced into the JM101 strain, the production amount increased twice as much as that of the JM109 strain per 1 L of the culture solution.

Furthermore, when pAC-SmMev was used, stable lycopene production was confirmed also in BL21(DE3) and C600 strains.

From these results, Acholeplasma laidlawii, Amphibacillus xylanus, or Streptobacillus moniliformis-derived mevalonate gene group expression plasmids, the stability in the cells is higher than expression plasmids from other bacterial species, the plasmid in culture. It was shown that falling out is unlikely to occur.

[3. Construction of plasmid pAC-SmMev/Scidi/Aacl)
Streptobacillus moniliformis strain DSM12112-derived mevalonate kinase (MVA kinase, SmMVK), diphosphomevalonate decarboxylase (DPMVA decarboxylase, SmDPMVDC), phosphomevalonate kinase (PMVA kinase), which codes phosphomevalonate kinase; PMV kinase, SmPM. A 2.6 kbp DNA fragment, followed by a 3.5 kbp DNA fragment encoding three genes of IPP isomerase, HMG-CoA synthetase, and HMG-CoA reductase derived from the Streptomyces CL190 strain, and budding yeast downstream thereof. A tac promoter (Ptac) and rrnB terminator for a 2.9 kbp DNA fragment encoding a type 1 isopentenyl diphosphate isomerase gene (Scidi) derived from Saccharomyces cerevisiae and an acetoacetic acid-CoA ligase gene (Aacl) derived from rat Rattus norvegicus. A plasmid pAC-SmMev/Scidi/Aacl having a structure sandwiched by (TrrnB) was prepared (Fig. 4). The specific construction method of the plasmid is as follows.

Restriction enzyme for pAC-Mev/Scidi/Aacl plasmid (H. Harada, F. Yu, S. Okamoto, T. Kuzuyama, R. Utsumi, N. Misawa, Appl. Microbiol. Biotechnol. 81: 915-925, 2009) Digestion with EcoRV and HindIII gave a 2.9 kbp DNA fragment containing Scidi and Aacl. The target pAC-SmMev/Scidi/Aacl plasmid was constructed by ligating this DNA fragment to the EcoRV and HindIII cleavage sites of pAC-SmMev described above by a ligation reaction.

[4. Construction of plasmid pRSF-TXS/crtE]
A 2.4 kbp DNA fragment encoding a taxadiene synthase gene (TXS) derived from Taxus brevifolia, and a geranylgeranyl diphosphate (GGPP) synthase gene (crtE) derived from the soil bacterium Pantoea ananatis. A plasmid pRSF-TXS/crtE having a structure sandwiched between a T7 promoter (PT7) and a T7 terminator (TT7) was constructed for the coding 0.9 kbp DNA fragment (FIG. 5). The specific construction method of the plasmid is as follows.

A 0.9 kbp DNA fragment encoding the geranylgeranyl diphosphate (GGPP) synthase gene (crtE) derived from the soil bacterium Pantoea ananatis is a pCRT-EIB plasmid (N. Misawa, M. Nakagawa, K. Kobayashi, S. Yamano). , Y. Izawa, K. Nakamura, K. Harashima, J. Bacteriol, 172: 6704-6712, 1990) as a template, crtE NdeI Fw (5'-AGGCATATGACGGTCTGCGCAAAA-3', "CATATG" is a restriction enzyme NdeI recognition sequence. ) And crtE PacI Rv (5'-GCGTTAATTAACTGACGGCAGCGAGTT-3', "TTAATTAA" is a restriction enzyme PacI recognition sequence) and it amplified by PCR. The amplified DNA fragment was digested with restriction enzymes NdeI and PacI and ligated to the NdeI and PacI cleavage sites of pRSF-Duet1 plasmid (Merck) by ligation reaction (FIG. 6). For the 2.4 kbp DNA fragment encoding the taxadiene synthase gene (TXS) derived from Taxus brevifolia, the plasmid pUC18Nm-TXShis containing the chemically synthesized TXS sequence was used as a template for TXS NcoI. Two primers were used: Fw (5'-AAACCATGGGCATGAGCAGCTCTACCG-3', "CCATGG" is a restriction enzyme NcoI recognition sequence) and TXS HindIII Rv (5'-TAGAAGCTTCACACCTGAATCGGGTCGATA-3', "AAGCTT" is a restriction enzyme HindIII recognition sequence). It was amplified by PCR. It was constructed by digesting the TXS gene amplified DNA fragment with restriction enzymes NcoI and HindIII, and ligating it to the NcoI and HindIII cleavage sites of the above-mentioned crtE-ligated pRSF-Duet1 plasmid by ligation reaction (FIG. 6).

[5. Production of taxadiene by Escherichia coli harboring plasmids pAC-SmMev/Scidi/Aacl and pRSF-TXS/crtE]
Two plasmids constructed by the above method were introduced into an E. coli competent cell (ECOS competent E. coli BL21(DE3) (Nippon Gene)), and chloramphenicol (Cm, final concentration 30 μg/mL) and Positive clones were obtained on LB plates containing kanamycin (Km, 100 μg/mL). This was inoculated into 3.0 ml of LB medium containing Cm and Km, precultured at 28° C. for 16 hours, and 0.2 mL (1%) was added to 20 ml of TB medium (bactotryptone, final concentration 12 g/ L, yeast extract, final concentration 24 g/L, KH ₂ PO ₄ , final concentration 2.3 g/L, K ₂ HPO ₄ , final concentration 12.4 g/L, and glycerol, final concentration 10 g/L) Main culture was performed at 37°C. When the absorbance at 600 nm reached 0.8, isopropyl β-D-thiogalactopyranoside (IPTG, final concentration 0.1 mM), lithium acetoacetate salt (lithium acetoacetate; LAA, final concentration 1.0 g/L) and dodecane ( dodecane, final concentration 1%) was added, and the culture was continued at 20° C. for 72 hours. After 48 hours and 72 hours, 5 ml of the culture broth was collected by centrifugation (15,000 rpm, 4° C., 5 minutes), and the dodecane layer was collected by a gas chromatograph mass spectrometer GCMS-QP2010 Ultra (manufactured by Shimadzu Corporation). Was analyzed.

As a separation column, HP-5MS (30 m×inner diameter 0.25 mm×0.25 μm thickness, manufactured by Agilent) was used, the vaporization chamber temperature was 250° C., the column flow rate was 0.67 mL/min with helium as a carrier gas, and the split ratio was 10. Separation was performed at 0 and a purge flow rate of 3.0 mL/min. The separation conditions were such that the temperature was held at 100° C. for 2 minutes, the temperature was raised to 280° C. at a rate of 10° C./min, and then the temperature was held for 5 minutes. Ionization was performed by the EI method, and mass spectrometry was performed in a scan mode with an ion source temperature of 250° C., an interface temperature of 280° C., and a mass-to-charge ratio (m/z) of 40 to 600. Taxadiene was identified based on retention time during chromatographic separation, mass-to-charge ratio, and area value prepared using standard samples, and the taxadiene concentration in the sample was calculated as the amount of taxadiene per E. coli dry weight and per culture solution. ..

The analysis result is shown in FIG. 7. As shown in the figure, in the sample after culturing for 72 hours, a peak indicating a main product was detected at a retention time of 16.2 minutes. By comparison with the taxadiene standard sample, the retention times and the MS spectra were in perfect agreement, confirming the production of taxadiene using this production system. As a result of the quantification, 14.0 mg/g DCW per cell dry weight and 146.5 mg/L taxadiene per liter of culture solution were synthesized.

From the above results, it was shown that the present E. coli production system is applicable not only to carotenoids but also to diterpenes, and thus can be applied to various terpenes production.

Terpenes can be expected to have a variety of beneficial bioactivities. Therefore, the present invention can be used in various industries such as pharmaceuticals, functional foods, fragrances, agriculture and horticulture, and consumer goods.

Claims (7)

Recombinant Escherichia coli characterized by introducing the following genes (1) to (3) derived from Amphibacillus xylanus:
(1) Mevalonate kinase gene;
(2) diphosphomevalonate decarboxylase gene;
(3) Phosphomevalonate kinase gene. The recombinant Escherichia coli according to claim 1, wherein the (1) mevalonate kinase gene is any gene selected from the group consisting of the following (a) to (e):
(A) a gene encoding a protein having the amino acid sequence set forth in SEQ ID NO: 2;
(B) A protein consisting of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and/or added in the amino acid sequence of SEQ ID NO: 2 and having a mevalonate kinase activity is encoded. Gene to
(C) a gene consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 2 and encoding a protein having mevalonate kinase activity;
(D) a gene comprising the nucleotide sequence set forth in SEQ ID NO:5;
(E) A gene consisting of a DNA having 90% or more homology with the DNA consisting of the nucleotide sequence of SEQ ID NO: 5 and encoding a protein having mevalonate kinase activity. The recombinant Escherichia coli according to claim 1 or 2, wherein the (2) diphosphomevalonate decarboxylase gene is any gene selected from the group consisting of the following (f) to (j):
(F) a gene encoding a protein consisting of the amino acid sequence of SEQ ID NO: 8;
(G) A protein having an amino acid sequence of SEQ ID NO: 8 in which one or several amino acid residues are substituted, deleted, inserted and/or added, and having a diphosphomevalonate decarboxylase activity. Encoding gene;
(H) a gene consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 8 and encoding a protein having diphosphomevalonate decarboxylase activity;
(I) a gene comprising the nucleotide sequence set forth in SEQ ID NO: 11;
(J) A gene consisting of a DNA having a homology of 90% or more with the DNA consisting of the nucleotide sequence of SEQ ID NO: 11 and encoding a protein having a diphosphomevalonate decarboxylase activity. 4. The (3) phosphomevalonate kinase gene is any gene selected from the group consisting of the following (k) to (o), and the phosphomevalonate kinase gene according to any one of claims 1 to 3. Recombinant E. coli:
(K) a gene encoding a protein consisting of the amino acid sequence of SEQ ID NO: 14;
(L) A protein consisting of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and/or added in the amino acid sequence shown in SEQ ID NO: 14 and coding for a protein having phosphomevalonate kinase activity Gene to
(M) a gene consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 14 and encoding a protein having phosphomevalonate kinase activity;
(N) a gene consisting of the nucleotide sequence set forth in SEQ ID NO: 17;
(O) A gene encoding a protein having a phosphomevalonate kinase activity, comprising a DNA having 90% or more homology with the DNA consisting of the nucleotide sequence of SEQ ID NO: 17 . Further, the recombinant Escherichia coli according to any one of claims 1 to 4, wherein the following genes (4) to (6) are introduced:
(4) HMG-CoA synthase gene;
(5) HMG-CoA reductase gene;
(6) Type 1 and/or type 2 isopentenyl diphosphate isomerase gene. Furthermore, the recombinant Escherichia coli according to any one of claims 1 to 5, wherein a gene or genes of an enzyme that synthesizes a terpene from farnesyl diphosphate is introduced. A method for producing a terpene, which comprises culturing the recombinant Escherichia coli according to any one of claims 1 to 6 to obtain a terpene.

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