JP2017104099A - Method for producing limonene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing limonene efficiently.SOLUTION: The present invention provides a method for producing limonene, comprising the steps of producing limonene by culturing, in a culture medium, microorganisms that express limonene synthase, such as limonene synthase derived from plants of Picea, Abies, Mentha, or Citrus.SELECTED DRAWING: None

Description

  The present invention relates to a method for producing limonene and the like.

  Limonene, known as an aromatic substance, is a type of monocyclic monoterpene contained in essential oils of various plants including mint and lemon. Limonene is used in cosmetics such as perfumes, cosmetics, shampoos and soaps, and is also used as an additive for adding flavor to foods or beverages. Limonene is also an important compound in the pharmaceutical and perfumery industries, for example, as a starting compound for compounds such as carveol, carvone, and perillyl alcohol, or as an organic solvent.

Limonene has enantiomers and is called (+)-limonene (d-limonene, 4R-limonene) and (-)-limonene (l-limonene, 4S-limonene). (+)-Limonene and (-)-limonene are known to have different scents, (+)-limonene has a refreshing citrus-like scent, and (-)-limone has a strong turpentine-like scent. It is said that. The two enantiomers have different odor thresholds, and (+)-limonene is said to show a threshold value about 2.5 times higher than (-)-limonene. In addition, research on the biological activity of limonene has been promoted, and it has become clear that it has relatively many biological activities such as sedation and anticancer activity. In recent years, the need for foods and beverages to which functional plant-derived materials have been added has been increasing due to the growing health consciousness, and the development of products that promote the functions of limonene by adding limonene has actually been carried out. Yes. The need for such foods and beverages is expected to increase in the future, and the demand for limonene is expected to increase accordingly, and it is desired to establish a production method capable of appealing to natural and capable of producing limonene efficiently.
Carvone is contained in caraways and spearmints and used as a fragrance. Chemical and enzymatic conversion of limonene to carvone is known (Non-patent Document 6). For this reason, limonene can be used as an intermediate for useful substances in addition to the usefulness of the raw material itself.

Limonene is synthesized by limonene synthase from geranyl diphosphate (GPP), a common precursor of monoterpenes. GPP is produced by the condensation of isopentenyl diphosphate (IPP) and its isomer, dimethylallyl diphosphate (DMAPP). These biosynthetic pathways include mevalonate pathway and non-mevalonate pathway (MEP). The existence of the route) is known. The mevalonate pathway is present in eukaryotes such as plants, animals, and yeast, and some actinomycetes and archaea. On the other hand, the MEP pathway exists in bacteria and plant plastids.
Conventionally, essential oil components of plants such as limonene are produced by extraction from plants by various extraction methods. As an extraction method, for example, Patent Document 1 describes steam distillation and Patent Document 2 describes a vacuum distillation method. Patent Document 3 describes a method for extracting plant components by subjecting a mixture of a plant material and a solvent to ultra-high temperature treatment. On the other hand, a production method by chemical synthesis has also been established. For example, Patent Document 4 describes a production method of limonene using α-pinene as a raw material by chemical synthesis in a high-temperature and high-pressure water environment. Furthermore, in recent years, limonene production in yeast and prokaryotes using genetic recombination techniques has been reported (Patent Documents 5, 6, 7, 8 and 9, Non-Patent Documents 1, 2, 3, 4 and 5). However, the amount of limonene produced by genetically modified yeast or genetically modified prokaryotic organisms remains at a very small level and is not necessarily an efficient production method.

JP 2005-298580 A JP 2006-291007 A Special table 2011-506713 gazette JP 2009-126721 A International Publication No. 2014/027118 International Publication No. 2015/027209 International Publication No. 2007/139924 International Publication No. 2007/140339 International Publication No. 2002/020815

Behrendorfff J, Vickers CE, Chrysanthopoulos P, Nielsen LK, Microbial Cell Factors, 2013, 12: 76-87. Alonso-Gutierrez J, Chan R, Batth TS, Adams PD, Keasling J D, Petzold C J, Lee TS, Metab. Eng. , 2013, 19, 33-41. Halfmann C, Gu L, Zhou R, Green Chemistry, 2014, 16 (6), 3175-3185. Willrodt C, Hoschek A, Buhler B, Schmid A, Julsing 1 M K, Biotechnol. Bioeng. , 2015, 112 (9), 1738-1750. Willrodt C, David C, Cornelissen S, Buehler B, Julsing M K, Schmid A, Biotechnology Journal, 2014, 9 (8), 1000-1012. MARIUSZ TRYTEK, JAN FIEDUREK, Acta Microbiological Polanica, 2002, 51 (1), 57-62.

  An object of the present invention is to provide an efficient method for producing limonene.

  As a result of earnest research to solve the above problems, the present inventors have cultivated limonene synthase derived from a plant belonging to the genus Spruce, Fir genus, Hakka genus or Citrus genus by culturing limonene in a culture medium. The present inventors have found that it can be produced efficiently and have completed the present invention.

That is, the present invention is as follows.
[1] A method for producing limonene, comprising culturing pantoea bacteria expressing limonene synthase in a culture medium to produce limonene.
[2] The method according to [1], wherein the bacterium comprises a heterologous expression unit comprising a polynucleotide encoding the limonene synthase and a promoter operably linked thereto.
[3] The method according to [2], wherein the polynucleotide is one or more polynucleotides selected from the group consisting of the following (a) to (o).
(A) (i) a base sequence represented by SEQ ID NO: 2, (ii) a base sequence consisting of nucleotide residues 193 to 1905 in the base sequence represented by SEQ ID NO: 2, or (iii) SEQ ID NO: 3 A polynucleotide comprising the base sequence represented by:
(B) a polynucleotide comprising a base sequence having 90% or more identity with the base sequence of (i), (ii) or (iii), and encoding a protein having limonene synthase activity;
(C) a poly which hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence of (i), (ii) or (iii) and which encodes a protein having limonene synthase activity nucleotide;
(D) (iv) a nucleotide sequence represented by SEQ ID NO: 5, (v) a nucleotide sequence consisting of nucleotide residues 205 to 1914 in the nucleotide sequence represented by SEQ ID NO: 5, or (vi) SEQ ID NO: 6 A polynucleotide comprising the base sequence represented by:
(E) a polynucleotide comprising a base sequence having 90% or more identity with the base sequence of (iv), (v) or (vi), and encoding a protein having limonene synthase activity;
(F) a poly which hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence of (iv), (v) or (vi) and which encodes a protein having limonene synthase activity nucleotide;
(G) (vii) a base sequence represented by SEQ ID NO: 8, (viii) a base sequence consisting of nucleotide residues 169 to 1800 in the base sequence represented by SEQ ID NO: 8, or (ix) SEQ ID NO: 9 A polynucleotide comprising the base sequence represented by:
(H) a polynucleotide comprising a base sequence having 90% or more identity with the base sequence of (vii), (viii) or (ix) and encoding a protein having limonene synthase activity;
(I) a poly which hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence of (vii), (viii) or (ix) and which encodes a protein having limonene synthase activity nucleotide;
(J) (x) a base sequence represented by SEQ ID NO: 11, (xi) a base sequence consisting of nucleotide residues 154 to 1827 in the base sequence represented by SEQ ID NO: 11, or (xii) SEQ ID NO: 12 A polynucleotide comprising the base sequence represented by:
(K) a polynucleotide encoding a protein comprising a base sequence having 90% or more identity with the base sequence of (x), (xi) or (xii), and having limonene synthase activity;
(L) a polynucleotide that hybridizes with a polynucleotide comprising a base sequence complementary to the base sequence of (x), (xi) or (xii) under a stringent condition and encodes a protein having limonene synthase activity nucleotide;
(M) (xiii) a nucleotide sequence represented by SEQ ID NO: 14, (xiv) a nucleotide sequence consisting of nucleotide residues 154 to 1821 in the nucleotide sequence represented by SEQ ID NO: 14, or (xv) SEQ ID NO: 15 A polynucleotide comprising the base sequence represented by:
(N) a polynucleotide comprising a base sequence having 90% or more identity with the base sequence of (xiii), (xiv) or (xv) and encoding a protein having limonene synthase activity; or (o) A polynucleotide encoding a protein that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence of (xiii), (xiv) or (xv) and has limonene synthase activity.
[4] The method according to any one of [1] to [3], wherein the limonene synthase is one or more proteins selected from the group consisting of the following (A) to (O).
(A) (i ′) a full-length amino acid sequence represented by SEQ ID NO: 1 or (ii ′) a protein comprising an amino acid sequence consisting of amino acid residues 65 to 634 in the amino acid sequence represented by SEQ ID NO: 1;
(B) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence of (i ′) or (ii ′) and having limonene synthase activity;
(C) a protein having an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence of (i ′) or (ii ′) and having limonene synthase activity;
(D) (iii ′) a full-length amino acid sequence represented by SEQ ID NO: 4 or (iv ′) a protein comprising an amino acid sequence consisting of amino acid residues 69 to 637 in the amino acid sequence represented by SEQ ID NO: 4;
(E) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence of (iii ′) or (iv ′) and having limonene synthase activity; or (F) the (iii ′) or (iv) A protein having a limonene synthase activity, including an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence of ');
(G) (v ′) a full-length amino acid sequence represented by SEQ ID NO: 7 or (vi ′) a protein comprising an amino acid sequence consisting of the 57th to 599th amino acid residues in the amino acid sequence represented by SEQ ID NO: 7;
(H) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence of (v ′) or (vi ′) and having limonene synthase activity;
(I) a protein having an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence of (v ′) or (vi ′) and having limonene synthase activity;
(J) (vii ′) a protein comprising the full-length amino acid sequence represented by SEQ ID NO: 10, or (viii ′) an amino acid sequence consisting of amino acid residues 52 to 608 in the amino acid sequence represented by SEQ ID NO: 10;
(K) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence of (vii ′) or (viii ′) and having limonene synthase activity;
(L) a protein having an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence of (vii ′) or (viii ′) and having limonene synthase activity;
(M) (ix ′) a full-length amino acid sequence represented by SEQ ID NO: 13, or (x ′) a protein comprising an amino acid sequence consisting of amino acid residues 52 to 606 in the amino acid sequence represented by SEQ ID NO: 13; Or (N) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence of (ix ′) or (x ′) and having limonene synthase activity; or (O) the (ix ′) or ( A protein having an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence of x ′) and having limonene synthase activity.
[5] The method according to any one of [1] to [4], wherein the limonene synthase is a limonene synthase derived from a plant belonging to the genus Picea, Abies, Mentha, or Citrus.
[6] The limonene synthase is derived from the American fir (Abies grandis), Alaska spruce (Picea stitchensis), Spearmint (Mentha spicata), Citrus unshu, or lemon (Citrus limon), [1] The method according to any one of [5].
[7] The method according to any one of [1] to [6], wherein the bacterium further expresses geranyl diphosphate synthase.
[8] The method according to any one of [1] to [7], wherein the bacterium has the ability to synthesize dimethylallyl diphosphate or isopentenyl diphosphate by the methylerythritol phosphate pathway.
[9] The method according to any one of [1] to [8], wherein the bacterium has the ability to synthesize dimethylallyl diphosphate or isopentenyl diphosphate by the mevalonate pathway.
[10] The method according to any one of [1] to [9], wherein the bacterium is a microorganism in which a 2-ketogluconic acid production pathway is blocked.
[11] The method according to [10], wherein the 2-ketogluconic acid production pathway is blocked by a decrease in glucose dehydrogenase activity.
[12] The method according to [11], wherein a polynucleotide that is a glucose dehydrogenase gene is disrupted in the bacterium.
[13] The method according to [12], wherein the polynucleotide is one or more polynucleotides selected from the group consisting of the following (x) to (z).
(X) [i] a nucleotide sequence represented by SEQ ID NO: 18, or [ii] a polynucleotide comprising a nucleotide sequence consisting of nucleotide residues 301 to 2691 in the nucleotide sequence represented by SEQ ID NO: 18;
(Y) a polynucleotide comprising a base sequence having 90% or more identity with the base sequence of [i] or [ii] and encoding a protein having glucose dehydrogenase activity; or (z) the above [i ] Or a polynucleotide encoding a protein having a glucose dehydrogenase activity, which hybridizes with a polynucleotide comprising a base sequence complementary to the base sequence of [ii] under stringent conditions.
[14] The method according to [12] or [13], wherein the glucose dehydrogenase is one or more proteins selected from the group consisting of the following (X) to (Z).
(X) a protein comprising the full-length amino acid sequence represented by SEQ ID NO: 19;
(Y) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 19 and having GCD activity; and (Z) one or more in the amino acid sequence represented by SEQ ID NO: 19 or A protein comprising an amino acid sequence in which several amino acids are deleted, substituted, added or inserted, and having GCD activity.
[15] The method according to any one of [1] to [14], wherein the bacterium is Pantoea ananatis.

  According to the present invention, limonene can be produced efficiently.

FIG. E. coli under the control of the E. coli Para promoter and the repressor gene araC. It is a figure which shows the pAH162-Para-mvaES plasmid which carries the mvaES operon derived from faecalis. FIG. 2 is a diagram showing a map of pAH162-mvaES. FIG. 3 is a view showing a plasmid for immobilizing pAH162-MCS-mvaES chromosome. FIG. 4 is a diagram showing a set of chromosome fixing plasmids that retain the mvaES gene under the transcriptional control of (A) P _lldD , (B) P _phoC , or (C) P _pstS . FIG. 5 is a diagram showing an outline of the construction of the pAH162-λattL-KmR-λattR vector. FIG. 6 is a view showing an expression vector for pAH162-Ptac chromosome fixation. FIG. 7 is a diagram showing codon optimization in the KDyI operon obtained by chemical synthesis. FIG. 8 is a diagram showing (A) pAH162-Tc-Ptac-KDyI and (B) pAH162-Km-Ptac-KDyI chromosome-fixing plasmids that retain the codon-optimized KDyI operon. FIG. It is a figure which shows the chromosome fixed plasmid holding the mevalonate kinase gene derived from palidicola. FIG. 10 shows a map of genomic variants of (A) ΔampC :: attB _phi80 , (B) ΔampH :: attB _phi80 , and (C) Δcrt :: attB _phi80 . FIG. 11 shows a map of genomic variants of (A) Δcrt :: pAH162-P _tac -mvk (X) and (B) Δcrt :: P _tac -mvk (X). FIG. 12 shows (A) ΔampH :: pAH162-Km-P _tac -KDyI, (B) ΔampC :: pAH162-Km-P _tac -KDyI, and (C) ΔampC :: P _tac -KDyI. It is a figure which shows a map. FIG. 13 is a diagram showing a map of chromosome modifications of (A) ΔampH :: pAH162-Px-mvaES and (B) ΔampC :: pAH162-Px-mvaES.

  The present invention provides a method for producing limonene.

  The method of the present invention comprises culturing a Pantoea bacterium expressing limonene synthase in a culture medium to produce limonene.

Limonene is one type of isoprenoid compound represented by C ₁₀ H ₁₆ . CAS number is represented by 138-86-3, R-form is represented by 5989-27-5, and S-form is represented by 5989-54-8.

  The method of the present invention comprises culturing a Pantoea bacterium expressing limonene synthase in a culture medium to produce limonene.

  Limonene synthase refers to one or more enzymes involved in the synthesis of limonene from geranyl diphosphate (GPP). Examples of limonene synthase include, for example, Picea genus plants (for example, Picea sitpensis, Picea abies), Abies plants (for example, American fir (Abies grandis)), Mentha (for example, Spearmint (Mentha spicata), peppermint (Mentha x pipeperita), Cannabis plant (eg Cannabis sativa), Solanum plant (eg Tomato lycopersicum plant), Ricco sp. Natans), Perilla genus plants (eg Peril) a fruitescens), Agastache plants (eg, Agastache rugosa), Citrus plants (eg, Citrus unshu), Lemon (i) et al., Schizonepeta, et al. And limonene synthase derived from a plant belonging to the genus Ricinus (for example, Ricinus communis) and a plant from the genus Lavandula (for example, Lavandula angustifolia). Among these, limonene synthase derived from a plant belonging to the genus Picea, Abies, Mentha, or Citrus is preferable, and the American wolf (Abies grandis), Alaska spruce (Picea situchensis), Spearmint (Mentha spicata), and Satsuma mandarin (Mentha spicata) Limonene synthase derived from Citrus unshu and lemon (Citrus limon) is more preferred.

  The phrase “derived from” as used herein for a nucleic acid sequence such as a gene, a promoter, or an amino acid sequence such as a protein, can be naturally or natively synthesized by a microorganism or isolated from a natural or wild-type microorganism. It can mean a nucleotide sequence or an amino acid sequence.

  Examples of the bacteria belonging to the genus Pantoea include Pantoea ananatis, Pantoea stewartii, Pantoea agglomerans, Pantoea citrea, Pantoea pantoea, and Pantoea pantoea. Ananatis (Panthea ananatis) and Pantoea citrea are preferred. Further, as the Pantoea bacterium, strains exemplified in European Patent Application Publication No. 09522121 may be used. As typical strains of the genus Pantoea, for example, Pantoea ananatis AJ13355 strain (FERM BP-6614) and Pantoea ananatis AJ13356 strain (FERM BP-6615), Pantoea ananatis disclosed in European Patent Application No. 0952212 are disclosed. SC17 strain (FERM BP-11901), Pantoea Ananatis SC17 (0) strain (VKPM B-9246) (Katashkina JI et al., BMC Mol Biol., 2009; 10:34).

  Pantoea bacteria expressing limonene synthase can be obtained, for example, by transforming Pantoea bacteria with an expression vector comprising a heterologous expression unit comprising a polynucleotide encoding limonene synthase and a promoter operably linked thereto. it can.

The polynucleotide encoding limonene synthase is preferably one or more polynucleotides selected from the group consisting of the following (a) to (o).
(A) (i) a base sequence represented by SEQ ID NO: 2, (ii) a base sequence consisting of nucleotide residues 193 to 1905 in the base sequence represented by SEQ ID NO: 2, or (iii) SEQ ID NO: 3 A polynucleotide comprising the base sequence represented by:
(B) a polynucleotide comprising a base sequence having 90% or more identity with the base sequence of (i), (ii) or (iii), and encoding a protein having limonene synthase activity;
(C) a poly which hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence of (i), (ii) or (iii) and which encodes a protein having limonene synthase activity nucleotide;
(D) (iv) a nucleotide sequence represented by SEQ ID NO: 5, (v) a nucleotide sequence consisting of nucleotide residues 205 to 1914 in the nucleotide sequence represented by SEQ ID NO: 5, or (vi) SEQ ID NO: 6 A polynucleotide comprising the base sequence represented by:
(E) a polynucleotide comprising a base sequence having 90% or more identity with the base sequence of (iv), (v) or (vi), and encoding a protein having limonene synthase activity;
(F) a poly which hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence of (iv), (v) or (vi) and which encodes a protein having limonene synthase activity nucleotide;
(G) (vii) a base sequence represented by SEQ ID NO: 8, (viii) a base sequence consisting of nucleotide residues 169 to 1800 in the base sequence represented by SEQ ID NO: 8, or (ix) SEQ ID NO: 9 A polynucleotide comprising the base sequence represented by:
(H) a polynucleotide comprising a base sequence having 90% or more identity with the base sequence of (vii), (viii) or (ix) and encoding a protein having limonene synthase activity;
(I) a poly which hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence of (vii), (viii) or (ix) and which encodes a protein having limonene synthase activity nucleotide;
(J) (x) a base sequence represented by SEQ ID NO: 11, (xi) a base sequence consisting of nucleotide residues 154 to 1827 in the base sequence represented by SEQ ID NO: 11, or (xii) SEQ ID NO: 12 A polynucleotide comprising the base sequence represented by:
(K) a polynucleotide encoding a protein comprising a base sequence having 90% or more identity with the base sequence of (x), (xi) or (xii), and having limonene synthase activity;
(L) a polynucleotide that hybridizes with a polynucleotide comprising a base sequence complementary to the base sequence of (x), (xi) or (xii) under a stringent condition and encodes a protein having limonene synthase activity nucleotide;
(M) (xiii) a nucleotide sequence represented by SEQ ID NO: 14, (xiv) a nucleotide sequence consisting of nucleotide residues 154 to 1821 in the nucleotide sequence represented by SEQ ID NO: 14, or (xv) SEQ ID NO: 15 A polynucleotide comprising the base sequence represented by:
(N) a polynucleotide comprising a base sequence having 90% or more identity with the base sequence of (xiii), (xiv) or (xv) and encoding a protein having limonene synthase activity; or (o) A polynucleotide encoding a protein that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence of (xiii), (xiv) or (xv) and has limonene synthase activity.

The base sequence represented by SEQ ID NO: 2 is the full-length base sequence of the Alaska spruce-derived limonene synthase gene. The base sequence represented by SEQ ID NO: 2 encodes the amino acid sequence represented by SEQ ID NO: 1, the base sequence consisting of nucleotide residues 1 to 192 encodes a putative chloroplast transition signal, The base sequence consisting of nucleotide residues 193 to 1905 (1902) can encode the amino acid sequence of mature limonene synthase. The base sequence represented by SEQ ID NO: 3 has a modified codon in the base sequence consisting of nucleotide residues 193 to 1905 in the base sequence represented by SEQ ID NO: 2, and a methionine codon at the 5 ′ end. It consists of an added nucleotide sequence.
The base sequence represented by SEQ ID NO: 5 is the full-length base sequence of the American monkey-derived limonene synthase gene. The base sequence represented by SEQ ID NO: 5 encodes the amino acid sequence represented by SEQ ID NO: 4, the base sequence consisting of nucleotide residues 1 to 204 encodes a putative chloroplast translocation signal, The base sequence consisting of nucleotide residues 205 to 1914 (1911) can encode the amino acid sequence of mature limonene synthase. In the base sequence represented by SEQ ID NO: 6, the codon in the base sequence consisting of nucleotide residues 205 to 1914 in the base sequence represented by SEQ ID NO: 5 is modified, and a methionine codon is further added to the 5 ′ end. It consists of an added nucleotide sequence.
The base sequence represented by SEQ ID NO: 8 is the full-length base sequence of the spearmint-derived limonene synthase gene. The base sequence represented by SEQ ID NO: 8 encodes the amino acid sequence represented by SEQ ID NO: 7, the base sequence consisting of nucleotide residues 1 to 168 encodes a putative chloroplast transition signal, The base sequence consisting of nucleotide residues 169 to 1800 (1797) can encode the amino acid sequence of mature limonene synthase. The base sequence represented by SEQ ID NO: 9 has a modified codon in the base sequence consisting of nucleotide residues 169 to 1800 in the base sequence represented by SEQ ID NO: 8, and a methionine codon at the 5 ′ end. It consists of an added nucleotide sequence.
The base sequence represented by SEQ ID NO: 11 is the full-length base sequence of Limonen synthase gene derived from Citrus unshiu. The base sequence represented by SEQ ID NO: 11 encodes the amino acid sequence represented by SEQ ID NO: 10, the base sequence consisting of nucleotide residues 1 to 153 encodes a putative chloroplast transition signal, The base sequence consisting of nucleotide residues 154 to 1827 (1824) can encode the amino acid sequence of mature limonene synthase. In the base sequence represented by SEQ ID NO: 12, the codon in the base sequence consisting of nucleotide residues 154 to 1827 in the base sequence represented by SEQ ID NO: 11 has been modified, and a methionine codon at the 5 ′ end. It consists of an added nucleotide sequence.
The base sequence represented by SEQ ID NO: 14 is the full-length base sequence of the lemon-derived limonene synthase gene. The base sequence represented by SEQ ID NO: 14 encodes the amino acid sequence represented by SEQ ID NO: 13, the base sequence consisting of nucleotide residues 1 to 153 encodes a putative chloroplast translocation signal, The base sequence consisting of nucleotide residues 154-11821 (1818) can encode the amino acid sequence of mature limonene synthase. The base sequence represented by SEQ ID NO: 15 has a modified codon in the base sequence consisting of nucleotide residues 154 to 1821 in the base sequence represented by SEQ ID NO: 14, and a methionine codon at the 5 ′ end. It consists of an added nucleotide sequence.

  The percent identity with the base sequence may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more. Limonene synthase activity refers to the activity of producing limonene from geranyl diphosphate (GPP) (the same applies hereinafter).

  The percent identity of the base sequence and the percent identity of the amino acid sequence as described below are, for example, the algorithm BLAST by Karlin and Altschul (Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)), FASTA by Pearson (Methods Enzymol). , 183, 63 (1990)). Based on this algorithm BLAST, programs called BLASTP and BLASTN have been developed (see http://www.ncbi.nlm.nih.gov). The% amino acid sequence identity may be calculated. As the homology of amino acid sequences, for example, using GENETYX Ver 7.0.9 of GENETICS, which employs the Lipman-Pearson method, the full length of the polypeptide part encoded by ORF is used. You may use the numerical value at the time of calculating percentity with size to compare = 2 setting. The lowest value among the values derived by these calculations may be adopted as the percent identity between the base sequence and the amino acid sequence.

  “Stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Although it is difficult to quantify such conditions clearly, for example, substantially identical polynucleotides having high identity, for example, polynucleotides having the above-mentioned% identity hybridize. However, this is a condition in which polynucleotides having lower homology do not hybridize with each other. Specifically, such conditions include hybridization at about 45 ° C. in 6 × SSC (sodium chloride / sodium citrate), followed by 50 × 0.2 × SSC in 0.1% SDS. One or more washings at ˜65 ° C. may be mentioned.

  The polynucleotides (a) to (o) may be DNA or RNA obtained from the corresponding DNA by substituting the thymine base with a uracil base, but is preferably DNA.

Limonene synthase is preferably one or more proteins selected from the group consisting of the following (A) to (O).
(A) (i ′) a full-length amino acid sequence represented by SEQ ID NO: 1 or (ii ′) a protein comprising an amino acid sequence consisting of amino acid residues 65 to 634 in the amino acid sequence represented by SEQ ID NO: 1;
(B) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence of (i ′) or (ii ′) and having limonene synthase activity;
(C) a protein having an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence of (i ′) or (ii ′) and having limonene synthase activity;
(D) (iii ′) a full-length amino acid sequence represented by SEQ ID NO: 4 or (iv ′) a protein comprising an amino acid sequence consisting of amino acid residues 69 to 637 in the amino acid sequence represented by SEQ ID NO: 4;
(E) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence of (iii ′) or (iv ′) and having limonene synthase activity; or (F) the (iii ′) or (iv) A protein having a limonene synthase activity, including an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence of ');
(G) (v ′) a full-length amino acid sequence represented by SEQ ID NO: 7 or (vi ′) a protein comprising an amino acid sequence consisting of the 57th to 599th amino acid residues in the amino acid sequence represented by SEQ ID NO: 7;
(H) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence of (v ′) or (vi ′) and having limonene synthase activity;
(I) a protein having an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence of (v ′) or (vi ′) and having limonene synthase activity;
(J) (vii ′) a protein comprising the full-length amino acid sequence represented by SEQ ID NO: 10, or (viii ′) an amino acid sequence consisting of amino acid residues 52 to 608 in the amino acid sequence represented by SEQ ID NO: 10;
(K) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence of (vii ′) or (viii ′) and having limonene synthase activity;
(L) a protein having an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence of (vii ′) or (viii ′) and having limonene synthase activity;
(M) (ix ′) a full-length amino acid sequence represented by SEQ ID NO: 13, or (x ′) a protein comprising an amino acid sequence consisting of amino acid residues 52 to 606 in the amino acid sequence represented by SEQ ID NO: 13; Or (N) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence of (ix ′) or (x ′) and having limonene synthase activity; or (O) the (ix ′) or ( A protein having an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence of x ′) and having limonene synthase activity.

  The amino acid sequence consisting of amino acid residues 1 to 64 in the amino acid sequence represented by SEQ ID NO: 1 can contain a putative chloroplast transition signal. The amino acid sequence consisting of amino acid residues 65 to 634 may include mature limonene synthase. The amino acid sequence consisting of amino acid residues 1 to 68 in the amino acid sequence represented by SEQ ID NO: 4 contains a putative chloroplast transfer signal, and the amino acid sequence consisting of amino acid residues 69 to 637 is Mature limonene synthase may be included. The amino acid sequence consisting of the 1st to 56th amino acid residues in the amino acid sequence represented by SEQ ID NO: 7 contains a putative chloroplast transfer signal, and the amino acid sequence consisting of the 57th to 599th amino acid residues is Mature limonene synthase may be included. The amino acid sequence consisting of amino acid residues 1 to 51 in the amino acid sequence represented by SEQ ID NO: 10 contains a putative chloroplast transition signal, and the amino acid sequence consisting of amino acid residues 52 to 608 is Mature limonene synthase may be included. The amino acid sequence consisting of amino acid residues 1 to 51 in the amino acid sequence represented by SEQ ID NO: 13 contains a putative chloroplast transfer signal, and the amino acid sequence consisting of amino acid residues 52 to 606 is Mature limonene synthase may be included. When expressing mature limonene synthase in a microorganism, a sequence in which a methionine residue is added to the N-terminus is usually used.

  The percent identity with the amino acid sequence may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more.

  Examples of amino acid residue mutations include deletion, substitution, addition and insertion of amino acid residues. The mutation of one or several amino acid residues may be introduced into one region in the amino acid sequence, but may be introduced into a plurality of different regions. The term “one or several” indicates a range in which the three-dimensional structure and activity of the protein are not significantly impaired. The number indicated by the term “one or several” in the case of protein is, for example, 1 to 100, preferably 1 to 80, more preferably 1 to 50, 1 to 30, 1 to 20, 1 to 10 or 1 to 5. The proteins (A) to (O) may have an initiation methionine residue at the N-terminus. The proteins (A) to (O) may have a purification tag such as a histidine tag at the C-terminus.

  When the proteins (B) and (C) are measured under the same conditions, 50% or more and 60% of the limonene synthase activity of the protein comprising any one amino acid sequence of (i ′) or (ii ′) above It is preferable that the activity is 70% or more, 80% or more, 90% or more, or 95% or more. When the proteins of (E) and (F) are measured under the same conditions, 50% or more and 60% of the limonene synthase activity of the protein comprising any one of the amino acid sequences (iii ′) or (iv ′) above It is preferable that the activity is 70% or more, 80% or more, 90% or more, or 95% or more. When the proteins (H) and (I) are measured under the same conditions, 50% or more and 60% of the limonene synthase activity of the protein comprising any one of the amino acid sequences (v ′) or (vi ′) above It is preferable that the activity is 70% or more, 80% or more, 90% or more, or 95% or more. When the proteins of (K) and (L) are measured under the same conditions, 50% or more and 60% of the limonene synthase activity of the protein comprising any one of the amino acid sequences (vii ′) or (viii ′) above It is preferable that the activity is 70% or more, 80% or more, 90% or more, or 95% or more. When the proteins (N) and (O) are measured under the same conditions, 50% or more and 60% of the limonene synthase activity of the protein containing any one of the amino acid sequences (ix ′) or (x ′) above It is preferable that the activity is 70% or more, 80% or more, 90% or more, or 95% or more.

  As long as the said protein can hold | maintain target activity, the variation | mutation may be introduce | transduced into the site | part in a catalytic domain, and sites other than a catalytic domain. Those skilled in the art will know the position of an amino acid residue into which a mutation in a protein that can retain the desired activity may be introduced. Specifically, those skilled in the art will 1) amino acid sequences of a plurality of proteins having the same type of activity (eg, amino acid sequences represented by SEQ ID NOs: 1, 4, 7, 10 or 13 and amino acids of other limonene synthases) Sequence), 2) reveal relatively conserved and relatively unconserved regions, then 3) relatively conserved regions and relatively unconserved From the regions, it is possible to predict regions that can play an important role in the function and regions that can not play an important role in the function, so that the correlation between structure and function can be recognized. Therefore, those skilled in the art can specify the position of an amino acid residue to which a mutation may be introduced in the amino acid sequence of limonene synthase.

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

  The Pantoea bacterium expressing limonene synthase preferably also expresses geranyl diphosphate synthase. Geranyl diphosphate is a precursor of peptidoglycan and electron acceptors (ubiquinone, etc.), and dimethylallyl diphosphate (DMAPP) is known to be essential for the growth of microorganisms (Fujisaki et al. al., J. Biochem., 1986; 99: 1137-1146). Examples of geranyl diphosphate synthase include geranyl diphosphate synthase derived from Escherichia coli. Alternatively, Pantoea ananatis (for example, International Publication No. 2007/029577 A1), Bacillus stearothermophilus (for example, JP 2000-245482 A), Geobacillus stearothermophilus (Geobacillus stearothermophilus), actinomycetes (International Publication No. 2007 / 029577A1) and other microorganism-derived geranyl diphosphate synthase. In addition, American fir (Abies grandis), Peppermint (Mentha × piperita), Spruce (Picea abies), Catharanthus roseus, ul, H. Examples include plant-derived geranyl diphosphate synthase.

The polynucleotide encoding geranyl diphosphate synthase is preferably one or more polynucleotides selected from the group consisting of the following (p), (q) and (r).
(P) (xvi) a nucleotide sequence represented by SEQ ID NO: 16, or (xvii) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 17;
(Q) a polynucleotide comprising a base sequence having 90% or more identity with the base sequence of (xvi) or (xvii) and encoding a protein having geranyl diphosphate synthase activity; or (r) A polynucleotide encoding a protein that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence of (xvi) or (xvii) and has geranyl diphosphate synthase activity.

  The base sequence represented by SEQ ID NO: 16 is the base sequence of the Escherichia coli derived farnesyl diphosphate / geranyl diphosphate synthase gene. The base sequence represented by SEQ ID NO: 17 has a modified codon in the base sequence represented by SEQ ID NO: 16, and the serine codon is mutated to a phenylalanine codon. The protein encoded by this mutant farnesyl diphosphate synthase gene is known as geranyl diphosphate synthase (Reilling KK et al. (2004) Biotechnol Bioeng. 87 (2) 200-212). The polynucleotide encoding geranyl diphosphate synthase is not limited to the S80F mutation described above, and may have a mutation that provides the same effect, and the mutation is not limited to the S80F mutation. The origin of the farnesyl diphosphate synthase gene is not limited to Escherichia coli. For example, in the farnesyl diphosphate synthase derived from Bacillus stearothermophilus, a mutation that increases the concentration of geranyl diphosphate in the cell. (Narita K., et al. (1999) J Biochem 126 (3) 566-571.). Moreover, not only geranyl diphosphate synthase obtained by introducing mutation into the farnesyl diphosphate synthase gene, but also a gene that originally functions as geranyl diphosphate synthase may be used. For example, periwinkle-derived geranyl diphosphate synthase gene (Rai A., et al. (2013) Mol Plant. 6 (5) 1531-49), Arabidopsis derived geranyl diphosphate synthase gene (Camara B,. (2000) Plant) J. 24 (2), 241-252), actinomycete-derived geranyl diphosphate synthase gene (International Publication No. 2007 / 029577A1) and the like may be used. The geranyl diphosphate synthase activity refers to an activity for producing geranyl diphosphate from IPP and DMAPP. Farnesyl diphosphate synthase activity refers to the activity of producing farnesyl diphosphate from geranyl diphosphate (GPP). The identity of the base sequence, stringent conditions, and the definition of the polynucleotide are the same as those described for the polynucleotides (a) to (o) above.

The geranyl diphosphate synthase is preferably one or more proteins selected from the group consisting of the following (P) to (R).
(P) a protein comprising the full-length amino acid sequence represented by SEQ ID NO: 76 or 77;
(Q) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence and having geranyl diphosphate synthase activity; and (R) one or several amino acids are deleted in the amino acid sequence; A protein comprising a substituted, added or inserted amino acid sequence and having geranyl diphosphate synthase activity.

  The amino acid sequence represented by SEQ ID NO: 76 may comprise mature farnesyl diphosphate / geranyl diphosphate synthase. The amino acid sequence represented by SEQ ID NO: 77 may comprise a mutant mature farnesyl diphosphate / geranyl diphosphate synthase. When the proteins of (Q) and (R) are measured under the same conditions, the geranyl diphosphate synthase activity, preferably the geranyl diphosphate synthase activity of the protein comprising the amino acid sequence represented by SEQ ID NO: 76 or 77 And it is preferable to have 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more of the activity of farnesyl diphosphate synthase activity. The definition of deletion, substitution, addition or insertion, amino acid identity, and the like are the same as described in the proteins (A) to (O) above.

  In the expression unit that can be contained in the bacterium used in the present invention, one of the polynucleotide encoding the desired protein and the promoter is not unique to the host cell. Thus, the expression unit can be a heterologous expression unit. Preferably, neither the polynucleotide encoding limonene synthase, the optional geranyl diphosphate synthase, or the promoter is unique to the host cell. The promoter may be homologous or heterologous to the polynucleotide encoding the desired protein. The expression unit may further comprise additional elements such as terminators, ribosome binding sites, and drug resistance genes. The expression unit may be DNA or RNA, but is preferably DNA. The heterologous expression unit may include a gene encoding a protein other than the polynucleotide encoding limonene synthase. Examples of such proteins include, but are not limited to, limonene synthase, mevalonate kinase, isoprene synthase, one or more enzymes involved in the methylerythritol phosphate pathway, and one or more enzymes involved in the mevalonate pathway. Not.

  The bacterium used in the present invention can be obtained, for example, by transformation with the following expression vector: an expression vector comprising a polynucleotide encoding limonene synthase and an expression unit comprising a promoter operably linked thereto; A polynucleotide encoding limonene synthase, an expression vector comprising an expression unit comprising geranyl diphosphate synthase and a promoter operably linked thereto; a polynucleotide comprising a polynucleotide encoding limonene synthase and a promoter operably linked thereto; An expression vector comprising one expression unit and a second expression unit comprising a polynucleotide encoding geranyl diphosphate synthase and a promoter operably linked thereto; and a polynucleotide encoding limonene synthase And combinations of a second expression vector comprising a first expression vector comprising a promoter operably linked to it, the expression unit containing the geranyl diphosphate synthase and a promoter operably linked to it. The expression vector may be an integral vector or a non-integration vector. In the expression vector, the gene encoding limonene synthase can be placed under the control of a constitutive promoter or an inducible promoter. Examples of the constitutive promoter include tac promoter, lac promoter, trp promoter, trc promoter, T7 promoter, T5 promoter, T3 promoter, and SP6 promoter. Examples of the inducible promoter include the growth promoter reverse-dependent promoter described below. The term “operably linked” means that the nucleotide sequence of the regulatory region is expressed (eg, enhanced, improved, constitutive) in the nucleotide sequence of the nucleic acid molecule or gene (ie, polynucleotide). , Basic, anti-transcriptional termination, attenuated, deregulated, reduced or repressed, expression) linked in a manner capable of being thereby encoded by a nucleotide sequence Means that the expression product of is produced.

  Dimethylallyl diphosphate (DMAPP) is a precursor of peptidoglycan and electron acceptors (such as ubiquinone) and is known to be essential for the growth of microorganisms (Fujisaki et al., J. Biochem). , 1986; 99: 1137-1146). The Pantoea bacterium expressing limonene synthase preferably has the ability to synthesize dimethyl diphosphate through the dimethylallyl diphosphate or isopentenyl diphosphate supply pathway. Examples of the dimethylallyl diphosphate or isopentenyl diphosphate supply pathway include a methyl erythritol phosphate (MEP) pathway and a mevalonic acid (MVA) pathway.

  DMAPP, a material for limonene, is usually biosynthesized by either the methylerythritol phosphate pathway or the mevalonate pathway inherent in microorganisms or natively. Therefore, from the viewpoint of supplying DMAPP for efficient production of limonene, the bacterium used in the present invention may have an enhanced methylerythritol phosphate pathway and / or mevalonate pathway as described later.

  The Pantoea bacterium expressing limonene synthase preferably has a dimethylallyl diphosphate or isopentenyl diphosphate supply pathway, and the 2-ketogluconate production pathway is preferably blocked.

  The bacterium used in the present invention is preferably a bacterium in which the 2-ketogluconic acid production pathway is blocked. In the 2-ketogluconic acid production pathway, glucose is oxidized by glucose dehydrogenase to produce gluconic acid, and then gluconic acid is oxidized by gluconate 2-ketodehydrogenase to produce NADPH and 2-ketogluconic acid. The Therefore, a bacterium in which the 2-ketogluconic acid production pathway is blocked is obtained by reducing the activity of one or more enzymes selected from the group consisting of glucose dehydrogenase (GCD) and gluconate 2-keto dehydrogenase. be able to. Preferably, the 2-ketogluconic acid production pathway is blocked by a decrease in enzyme activity. That is, the bacterium used in the present invention has a reduced enzyme activity of one or more enzymes selected from the group consisting of glucose dehydrogenase and gluconate 2-keto-dehydrogenase, whereby a 2-ketogluconate production pathway is formed in the bacterium. Blocked.

  Reduction of enzyme activity in bacteria includes, for example, reduction and complete disappearance of enzyme activity. Moreover, the reduction | decrease of the enzyme activity in bacteria includes the fall of the expression of the enzyme in bacteria, and complete elimination | eradication, for example. This is because a decrease or complete disappearance of the enzyme expression in the bacteria leads to a decrease or complete disappearance of the activity possessed by the bacteria. Reduction of enzyme activity in bacteria is, for example, expression of a gene encoding the enzyme, a gene encoding a factor capable of regulating the expression or activity of the enzyme, or a transcriptional regulatory region, a translational regulatory region, etc. located upstream of these genes. It can be achieved by destroying regulatory regions (eg promoters, Shine-Dalgarno (SD) sequences), untranslated regions. The gene or region can be disrupted by modifying the genomic region corresponding to the gene or region so that the expression or activity of the enzyme is reduced or completely eliminated. Such modifications include, for example, deletion of part or all of the genomic region, insertion of a polynucleotide into the genomic region, and replacement of the genomic region with another polynucleotide. It is not limited.

  Pantoea bacteria expressing limonene synthase are preferably bacteria capable of synthesizing pyrroloquinoline quinone (PQQ) or capable of utilizing PQQ contained in the culture environment.

Pantoea bacteria that express limonene synthase preferably have reduced glucose dehydrogenase activity, and more preferably have reduced glucose dehydrogenase activity using PQQ as a coenzyme.
When a Pantoea bacterium expressing limonene synthase is obtained by transforming a host such as Pantoea bacterium originally having a 2-ketogluconic acid pathway with an expression vector for limonene synthase, the bacterium Preferably, modifications are made to block the 2-ketogluconic acid pathway.
For example, microorganisms belonging to the family Enterobacteriaceae such as Escherichia coli have a gene encoding glucose dehydrogenase and produce GCD apoenzyme, but have no ability to produce PQQ. do not do. However, it is known that when a certain foreign gene is expressed in cells, a substance that substitutes for PQQ is produced and GCD activity is expressed (WO 2006/133898). Microorganisms such as the aforementioned microorganisms belonging to the family Enterobacteriaceae acquired GCD activity are included in the “microorganisms inherently having a 2-ketogluconic acid pathway” in the present invention.

  The modification for blocking the 2-ketogluconic acid production pathway is preferably a modification for reducing glucose dehydrogenase activity, and is a modification for reducing glucose dehydrogenase activity using PQQ as a coenzyme. Preferably there is. The modification can be performed such that the GCD activity per bacterial cell is lower than that of an unmodified strain, such as a strain belonging to the wild type Enterobacteriaceae. For example, the number of GCD molecules per cell of the modified strain or the GCD activity per molecule may be lower than that of the wild strain. The comparison of GCD activity per cell can be performed by, for example, comparing the GCD activity contained in the cell extract of the modified strain and the wild strain cultured under the same conditions. Examples of wild-type Pantoea bacteria that can be used as comparison controls include Pantoea ananatis AJ13355 strain (FERM BP-6614), Pantoea ananatis SC17 strain (FERM BP-11901), Pantoea ananatis SC17 (0) strain (VKPM). B-9246) (Katashkina JI et al., BMC Mol Biol., 2009; 10:34).

  The activity of GCD using PQQ as a coenzyme refers to the activity of catalyzing the following reaction (the same applies hereinafter).

  β-D-glucose + oxidized PQQ → D-δ-gluconolactone + reduced PQQ

  The GCD activity can be measured, for example, based on detection of absorbance at 600 nm by measuring the production of reduced DCPIP through the following reaction (JP 2007-129965; the same applies hereinafter).

D-glucose + oxidized PMS → D-glucono-1,5-lactone + reduced PMS
Reduced PMS + oxidized DCPIP → oxidized PMS + reduced DCPIP
PMS: Phenazine methosulfate DCPIP: 2,6-dichlorophenol-indophenol

  The activity of GCD is reduced by disrupting a gene encoding GCD (gcd gene), a gene encoding a factor capable of regulating GCD expression or activity, or a transcriptional regulatory region located upstream of these genes. Can do.

The gcd gene is preferably one or more polynucleotides selected from the group consisting of the following (x) to (z).
(X) [i] a nucleotide sequence represented by SEQ ID NO: 18, or [ii] a polynucleotide comprising a nucleotide sequence consisting of nucleotide residues 301 to 2691 in the nucleotide sequence represented by SEQ ID NO: 18;
(Y) a polynucleotide comprising a base sequence having 90% or more identity with the base sequence of [i] or [ii] and encoding a protein having GCD activity; or (z) the above [i] or [ ii] a polynucleotide that hybridizes with a polynucleotide comprising a base sequence complementary to the base sequence under stringent conditions and encodes a protein having GCD activity.

  The base sequence shown in SEQ ID NO: 18 is the full-length base sequence of Pantoea ananatis gcd gene. The base sequence represented by SEQ ID NO: 18 encodes the amino acid sequence represented by SEQ ID NO: 19, and the base sequence consisting of the 301st to 2691 (2688) th nucleotide residues can encode the amino acid sequence of GCD. The identity of the base sequence, stringent conditions, and the definition of the polynucleotide are the same as those described for the polynucleotides (a) to (o) above.

GCD is preferably one or more proteins selected from the group consisting of the following (X) to (Z).
(X) a protein comprising the full-length amino acid sequence represented by SEQ ID NO: 19 or the amino acid sequence consisting of the 1st to 796th amino acid residues in the amino acid sequence represented by [ii ′] SEQ ID NO: 1;
(Y) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence of [i ′] or [ii ′] and having GCD activity; and (Z) the above [i ′] or [ii ′ ], A protein having GCD activity, comprising an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted.

  The amino acid sequence represented by SEQ ID NO: 19 can contain GCD. When the proteins of (Y) and (Z) are measured under the same conditions, 50% or more, 60% or more, 70% or more, 80% or more of the GCD activity of the protein comprising the amino acid sequence represented by SEQ ID NO: 19 90% or more, or preferably 95% or more. The definition of deletion, substitution, addition or insertion, amino acid identity, and the like are the same as described in the proteins (A) to (O).

  The gcd gene can be cloned by synthesizing oligonucleotides based on these sequences and performing a PCR reaction using the Pantoea ananatis chromosome as a template. The gcd gene may be disrupted by homologous recombination. In this case, a gene having a certain degree of homology with the gcd gene on the chromosome, for example, 80% or more, preferably 90% or more, more preferably 95% or more may be used. Alternatively, a gene that hybridizes with the gcd gene on the chromosome under stringent conditions may be used. The stringent conditions include, for example, a salt concentration corresponding to 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, and more preferably 2 times. The condition of washing three times is mentioned.

  Disruption of the gcd gene is, for example, deletion of the entire target gene including upstream and downstream sequences of the target gene on the chromosome, and inactivation of the gcd gene is amino acid substitution (missense mutation) in the coding region of the gcd gene on the chromosome , Introduction of a stop codon (nonsense mutation), or introduction of a frameshift mutation that adds or deletes 1 to 2 bases (Journal of Biological Chemistry 272: 8611-8617 (1997) Proceedings of the National Academy of Sciences, USA 95 5511-5515 (1998), Journal of Biological Chemistry 266, 20833-20839 (1991)).

  The disruption of each gene is preferably performed by gene recombination. As a method by gene recombination, for example, homologous recombination is used to delete part or all of the expression regulatory sequence (eg, promoter region, coding region, or non-coding region) of the target gene on the chromosome. Or inserting a polynucleotide into these regions.

  The destruction of the expression regulatory sequence is preferably performed for 1 base or more, more preferably 2 bases or more, and further preferably 3 bases or more. When the coding region is deleted, the region to be deleted may be any of the N-terminal region, internal region, and C-terminal region as long as the function of the protein produced by each gene is reduced. It may be the entire code area. Usually, the longer the region to be deleted, the more reliable the target gene can be destroyed. It is preferable that the reading frames upstream and downstream of the region to be deleted do not match.

  When the polynucleotide is inserted into the coding region, the insertion position may be any region of the target gene, but the longer the inserted sequence, the target gene can be surely destroyed. The sequences before and after the insertion site preferably do not match the reading frame. The polynucleotide is not particularly limited as long as it reduces the function of the protein encoded by the target gene. Examples thereof include a transposon carrying an antibiotic resistance gene or a gene useful for L-amino acid production. .

  Examples of the method for modifying the target gene on the chromosome as described above include the following methods. First, a mutant gene that lacks a partial sequence of the target gene and cannot produce a normally functioning protein is prepared. Next, the target gene on the chromosome is replaced with the mutant gene by transforming bacteria with the DNA containing the gene and causing homologous recombination between the mutant gene and the target gene on the chromosome. Even if the protein encoded by the obtained deletion type target gene is produced, it has a three-dimensional structure different from that of the wild type protein, and its activity is reduced. Gene disruption by gene replacement using such homologous recombination has already been established. For example, a method called “Red-driven integration” (Datsenko, KA, and Wanner, BL Proc. Natl. Acad. Sci. USA. 97: 6640-6645 (2000)), Alternatively, a combination of the Red driven integration method and a λ phage-derived excision system (Cho, EH, Gumpport, RI, Gardner, JFJ Bacteriol. 184: 5200-5203 (2002)). Utilizing linear DNA such as the combined method (WO2005 / 010175), plasmids containing temperature-sensitive replication origins, methods using plasmids with junction transfer ability, and suicide vectors that do not have replication origins in the host Like that method (U.S. Pat. No. 6,303,383 or JP-A 05-007491, JP-).

  Whether or not the transcription of the target gene is decreased can be confirmed by comparing the amount of mRNA transcribed from the target gene with a wild strain or an unmodified strain. Examples of methods for evaluating the amount of mRNA include Northern hybridization, RT-PCR and the like (Molecular cloning (Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001)). The amount of transcription may be reduced as compared with either a wild strain or an unmodified strain. For example, at least 75% or less, 50% or less, 25% or less, or 10% compared to a wild strain or an unmodified strain. It is desirable that it is lowered below, and it is particularly preferable that it is not expressed at all.

  Confirmation that the amount of the protein encoded by the target gene has decreased can be confirmed by Western blotting using an antibody that binds to the protein (Molecular cloning (Cold spring Harbor Press, Cold spring Harbor (USA), 2001). )). The amount of protein may be reduced as long as it is lower than that of a wild strain or an unmodified strain. For example, it is at least 75% lower than that of a wild strain or an unmodified strain as compared to a wild strain or an unmodified strain. In the following, it is desirable that the amount is reduced to 50% or less, 25% or less, or 10% or less, and it is particularly preferable that no protein is produced (activity is completely lost).

  In order to reduce the activity of GCD, in addition to the above-described gene manipulation method, for example, microorganisms belonging to the family Enterobacteriaceae such as bacteria belonging to the genus Pantoea are irradiated with ultraviolet rays or N-methyl-N′-nitro-N-nitroso. Examples include a method of selecting a strain that has been treated with a mutagen such as guanidine (NTG) or nitrous acid, which is usually used for mutagenesis, and has reduced GCD activity.

  The activity of GCD can also be reduced by reducing the ability to synthesize PQQ. The ability to synthesize PQQ can be reduced, for example, by deleting part or all of pqqABCDEF, an operon required for PQQ biosynthesis (J. S. Belterop, P. W. Postma, J. et al. Bacteriology 177 (17): 5088-5098 (1995)).

  Pantoea bacteria expressing limonene synthase may be either aerobic microorganisms or anaerobic microorganisms. When culturing a microorganism under aerobic conditions, the concentration of dissolved oxygen in the medium may be a concentration sufficient for the growth of the microorganism. The concentration of dissolved oxygen in the medium sufficient for the growth of aerobic microorganisms is not particularly limited as long as it is a concentration that can promote the growth of aerobic microorganisms. For example, 1 ppm or more, 3 ppm or more, 5 ppm or more, or 7.22 ppm or more It may be. The concentration of dissolved oxygen for the growth of aerobic microorganisms may also be, for example, 0.3 ppm or less, 0.15 ppm or less, or 0.05 ppm or less.

  The microorganism expressing limonene synthase preferably has the ability to synthesize dimethyl diphosphate through the dimethylallyl diphosphate supply pathway from the viewpoint of supplying IPP and DMAPP for efficient production of limonene. Examples of the dimethylallyl diphosphate supply route include a methyl erythritol phosphate (MEP) route and a mevalonic acid (MVA) route.

  Examples of the enzyme involved in the mevalonate (MVA) pathway include mevalonate kinase (EC: 2.7.1.36; Example 1, Erg12p, ACCESSION ID NP — 013535; Example 2, AT5G27450, ACCESSION ID NP — 001190411), phosphomevalonic acid Kinase (EC: 2.7.4.2; Example 1, Erg8p, ACCESSION ID NP — 013947; Example 2, AT1G31910, ACCESSION ID NP — 001185124), diphosphomevalonate decarboxylase (EC: 4.1.1.33; Example 1, Mvd1p) , ACCESSION ID NP_014441; Example 2, AT2G38700, ACCESSION ID NP_181404; Example 3, AT3G54250, ACCESS ON ID NP_566995), acetyl-CoA-C-acetyltransferase (EC: 2.3.1.9; Example 1, Erg10p, ACCESSION ID NP_015297; Example 2, AT5G47720, ACCESSION ID NP_001032028; Example 3, AT5G48230, 94 ), Hydroxymethylglutaryl-CoA synthase (EC: 2.3.3.10; Example 1, Erg13p, ACCESSION ID NP — 013580; Example 2, AT4G11820, ACCESSION ID NP — 192919; Example 3, MvaS, ACCESSION ID AAG02438), Hydroxymethyl Glutaryl-CoA reductase (EC: 1.1.1.34; Example 1, H Example 2, Hmg1p, ACCESSION ID NP — 013636; Example 3, AT1G76490, ACCESSION ID NP — 177775; Example 4, AT2G17370, ACCESSION ID NP — 17938, EC: 1.1M, P1. ), Acetyl-CoA-C-acetyltransferase / hydroxymethylglutaryl-CoA reductase (EC: 2.3.1.9/1.1.1.34, eg, MvaE, ACCESSION ID AAG02439). In an expression vector, one or more enzymes involved in the mevalonate (MVA) pathway (eg, phosphomevalonate kinase, diphosphomevalonate decarboxylase, acetyl-CoA-C-acetyltransferase / hydroxymethylglutaryl-CoA reductase, hydroxymethylglutaryl) The gene encoding -CoA synthase may be placed under the control of a growth promoter reverse-dependent promoter.

  Examples of the enzyme involved in the methylerythritol phosphate (MEP) pathway include 1-deoxy-D-xylulose-5-phosphate synthase (EC: 2.2.1.7, Example 1, Dxs, ACCESSION ID NP — 414554; Example 2, AT3G21500, ACCESSION ID NP — 567686; Example 3, AT4G15560, ACCESSION ID NP — 193291; Example 4, AT5G11380, ACCESSION ID NP — 0010785570), 1-deoxy-D-xylulose-5-phosphate reduct isomerase (EC: 1.1. 1.267; Example 1, Dxr, ACCESSION ID NP_414715; Example 2, AT5G62790, ACCESSION ID NP_001190600), 4-diphospho Tidyl-2-C-methyl-D-erythritol synthase (EC: 2.7.7.60; Example 1, IspD, ACCESSION ID NP_417227; Example 2, AT2G02500, ACCESSION ID NP_565286), 4-diphosphocytidyl-2-C- Methyl-D-erythritol kinase (EC: 2.7.1.148; Example 1, IspE, ACCESSION ID NP_415726; Example 2, AT2G26930, ACCESSION ID NP_180261), 2-C-methyl-D-erythritol-2,4- Cyclodiphosphate synthase (EC: 4.6.1.12; Example 1, IspF, ACCESSION ID NP — 417226; Example 2, AT1G63970, ACCESSION ID NP — 564819), -Hydroxy-2-methyl-2- (E) -butenyl-4-diphosphate synthase (EC: 1.7.7.7.1; Example 1, IspG, ACCESSION ID NP_417010; Example 2, AT5G60600, ACCESSION ID NP_001119467) 4-hydroxy-3-methyl-2-butenyl diphosphate reductase (EC: 1.17.1.2; Example 1, IspH, ACCESSION ID NP — 414570; Example 2, AT4G34350, ACCESSION ID NP — 567965). In the expression vector, genes encoding these enzymes may be placed under the control of a growth promoter reverse-dependent promoter.

As described above, IPP and DMAPP, which are limonene materials, are usually biosynthesized by either the methylerythritol phosphate pathway or the mevalonate pathway inherent to microorganisms. Therefore, Pantoea bacteria expressing limonene synthase may further have an enhanced pathway for synthesizing dimethylallyl diphosphate (DMAPP), which is a basic material of limonene (eg, a substrate for isoprene synthase).
For such enhancement, the Pantoea bacterium used in the present invention may further express mevalonate kinase in addition to limonene synthase. Therefore, Pantoea bacteria expressing limonene synthase may be transformed with a mevalonate kinase expression vector. Examples of the mevalonate kinase gene include, for example, the genus of Methanosarcina mazei such as Methanosarcina mazei, the genus of Methanocarcaria burium, and the like of Methanocella paricola (Methenocella paricola). Corynebacterium, Methanosaeta conciili and other genus Methanosaeta, and Nitrosopumilus maritimus nitrosopomirus s) include the gene of a microorganism belonging to the genus.
The microorganism expressing limonene synthase may be transformed with one or more enzyme expression vectors involved in the mevalonate pathway or the methylerythritol phosphate pathway. The expression vector may be an integral vector or a non-integration vector. In the expression vector, the gene encoding mevalonate kinase can be placed under the control of a constitutive promoter or an inducible promoter (eg, a growth promoter reverse-dependent promoter). Specifically, the gene encoding mevalonate kinase can be placed under the control of a constitutive promoter. Examples of the constitutive promoter include tac promoter, lac promoter, trp promoter, trc promoter, T7 promoter, T5 promoter, T3 promoter, and SP6 promoter.
Such enzyme expression vectors further express together or individually a plurality of enzymes (eg, 1, 2, 3 or 4 or more) involved in the mevalonate pathway and / or the methylerythritol phosphate pathway. For example, it may be an expression vector for polycistronic mRNA. The origin of one or more enzymes involved in the mevalonate pathway and / or the methylerythritol phosphate pathway may be homologous or heterologous to the host. The origin of the enzyme involved in the mevalonate pathway and / or methylerythritol phosphate pathway is different from the host, for example, the enzyme involved in the mevalonate pathway is derived from fungi (eg, Saccharomyces cerevisiae), actinomycetes, or plants It may be a thing. In addition, when the host inherently produces an enzyme involved in the methylerythritol phosphate pathway, the expression vector introduced into the host may express an enzyme involved in the mevalonate pathway.
The actinomycete example, Streptomyces genus, Kitasatospora spp, Streptacidiphilus genus, Pseudonocardia sp, Actinoalloteichus genus, Actinokineospora genus, Actinomycetospora genus, Actinophytocola genus, Actinosynnema spp, Alloactinosynnema genus, Allokutzneria genus, Amycolatopsis sp, Crossiella genus, Goodfellowiella genus, Haloechinothrix genus , Kibellosporarangium genus, Kutzneria genus, Labedaea genus, Lechevaleria genus, Lentzea genus, Longimiceli m genus Prauserella genus Saccharomonospora genus Saccharopolyspora genus Saccharothrix genus Sciscionella genus Streptoalloteichus genus Tamaricihabitans genus Thermobispora genus Thermocrispum genus Thermotunica genus Umezawaea genus include microorganisms Yuhushiella genus like. As an example of a plant, the specific example of the plant from which the limonene synthase mentioned above originates is mentioned.

  In the expression vector, a gene encoding one or more enzymes involved in the mevalonate (MVA) pathway and / or the methylerythritol phosphate (MEP) pathway may be placed under the control of a growth promoter-independent promoter.

  Further, in order to enhance the methylerythritol phosphate pathway and / or the mevalonate pathway, isopentenyl diphosphate delta isomerase having the ability to convert isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP) May be introduced.

  As isopentenyl diphosphate delta isomerase (EC: 5.3.3.2), for example, Idi1p (ACCESSION ID NP — 015208), AT3G02780 (ACCESSION ID NP — 186927), AT5G16440 (ACCESSION ID NP — 197165), N ). In the expression vector, the gene encoding isopentenyl diphosphate delta isomerase may be placed under the control of a growth promoter reverse-dependent promoter.

  Transformation of a host with an expression vector containing a gene as described above can be performed using one or more known methods. Examples of such a method include a competent cell method using a calcium-treated bacterial cell, an electroporation method, and the like. In addition to a plasmid vector, a phage vector may be used to infect and introduce into a microbial cell.

The method of the present invention preferably includes the following 1) to 3).
1) culturing and growing Pantoea bacteria expressing limonene synthase (hereinafter, Pantoea bacteria expressing limonene synthase may be referred to as “microorganisms”) in the presence of a sufficient concentration of growth promoter;
2) reducing the concentration of the growth promoter to induce the production of limonene by the microorganism; and 3) culturing the microorganism to produce limonene.

  From the viewpoint of efficient production of limonene, the above step 1) corresponding to the growth phase of microorganisms and the above step 3) corresponding to the production phase of limonene are separated. In addition, the above step 2) corresponds to the induction period of limonene production because it shifts from the growth period of microorganisms to the production period of limonene.

  A growth promoting agent is a factor essential for the growth of microorganisms or a factor having a promoting action on the growth of microorganisms that can be consumed by microorganisms. When the amount in the medium is reduced by consumption by microorganisms, the growth of microorganisms is lost or lost. That which reduces. For example, if a certain amount of growth promoter is used, the microorganism will continue to grow until that amount of growth promoter is consumed, but once all of the growth promoter has been consumed, the microorganism cannot grow, or The growth rate can be reduced. Therefore, the degree of microbial growth can be controlled by the growth promoter. Examples of such a growth promoter include substances such as oxygen (gas); minerals such as ions of iron, magnesium, potassium, and calcium; phosphoric acid (eg, monophosphate, diphosphate, polyphosphate) or salts thereof Phosphorus compounds such as ammonia; nitrogen compounds (gas) such as ammonia, nitric acid, nitrous acid, urea; sulfur compounds such as ammonium sulfate and thiosulfuric acid; vitamins (eg, vitamin A, vitamin D, vitamin E, vitamin K, vitamin B1, vitamins) B2, vitamin B6, vitamin B12, niacin, pantothenic acid, biotin, ascorbic acid) and amino acids (eg, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, leucine, isoleucine, lysine, methionine) , Phenylalanine, proline Serine, threonine, tryptophan, tyrosine, valine, including but nutrients selenocysteine), and the like. In the method of the present invention, one type of growth promoter may be used, or two or more types of growth promoters may be used in combination.

  When the method of the present invention includes the above steps 1) to 3), the Pantoea bacterium expressing limonene synthase has the ability to grow depending on the growth promoting agent, and limonene depending on the growth promoting agent-independent promoter. It is preferable that it is a microorganism which has the ability to produce and is provided with the ability to synthesize limonene by enzymatic reaction. Such microorganisms can grow in the presence of a growth promoter at a concentration sufficient for the growth of the microorganisms. Here, “sufficient concentration” may mean that the growth promoter is used at a concentration effective for the growth of microorganisms. The expression “the ability to produce limonene depending on the growth promoter reverse-dependent promoter” means that in the presence of a relatively high concentration of the growth promoter, no limonene can be produced or the production efficiency of limonene is low. However, in the presence of a relatively low concentration of growth promoter or in the absence of a growth promoter, it can mean that limonene can be produced or that the production efficiency of limonene is high. Therefore, the microorganism preferably used in the present invention can grow well in the presence of a sufficient concentration of the growth promoter, but cannot produce limonene or has low production efficiency of limonene. Microorganisms used in the present invention also cannot grow well in the presence of insufficient concentrations of growth promoters or in the absence of growth promoters, but can produce limonene or have high production efficiency of limonene. Is preferred.

  When the method of the present invention includes the above steps 1) to 3), the limonene synthase gene of the Pantoea bacterium expressing limonene synthase can be under the control of a growth promoter reverse-dependent promoter. The expression “growth promoter reverse-dependent promoter” means that in the presence of a relatively high concentration of growth promoter, it has no or low transcriptional activity, but a relatively low concentration of growth promoter. In the presence of or in the absence of a growth promoter, it may mean a promoter having transcription activity or having high transcription activity. Therefore, the growth-promoting agent reverse-dependent promoter can suppress the expression of limonene synthase gene in the presence of a growth-promoting agent at a concentration sufficient for the growth of microorganisms, while the growth-promoting agent at a concentration insufficient for the growth of microorganisms. In the presence of, the expression of the limonene synthase gene can be promoted. Specifically, the microorganism is under the control of a growth promoter reverse-dependent promoter.

  For example, when the growth promoter is a phosphorus compound, a phosphorus deficiency inducible promoter can be used. The expression “phosphorus deficiency inducible promoter” may refer to a promoter that can promote the expression of downstream genes with low concentrations of phosphorus compounds. A low concentration phosphorus compound can refer to conditions with a (free) phosphorus concentration of 100 mg / L or less. The expression “phosphorus” is synonymous with the expression “phosphorus compound” and they may be used in an interchangeable manner. The total phosphorus concentration can be determined by decomposing all phosphorus compounds in the solution into orthophosphoric phosphorus with a strong acid or an oxidizing agent. The total phosphorus concentration under phosphorus-deficient conditions is 100 mg / L or less, 50 mg / L or less, 10 mg / L or less, 5 mg / L or less, 1 mg / L or less, 0.1 mg / L or less, or 0.01 mg / L or less. There may be. Examples of the phosphorus deficiency inducible promoter include a promoter of a gene encoding alkaline phosphatase (eg, phoA), a promoter of a gene encoding acid phosphatase (eg, phoC), and a gene encoding sensor histidine kinase (eg, phoR) Promoter, a gene encoding a response regulator (eg, phoB), and a gene encoding a phosphorus uptake carrier (eg, pstS).

  In the above step 1), Pantoea bacteria expressing limonene synthase are grown in the presence of a sufficient concentration of growth promoter. More specifically, Pantoea bacteria that express limonene synthase can be grown by culturing Pantoea bacteria that express limonene synthase in a medium in the presence of a sufficient concentration of a growth promoter. .

  For example, when a phosphorus compound is used as a growth promoter, Pantoea bacteria that express limonene synthase can grow well in the presence of a sufficient concentration of the phosphorus compound, so that the phosphorus compound is used as a growth promoter. Can act. When the growth promoter is a phosphorus compound, in 1), the concentration of the phosphorus compound sufficient for growth is not particularly limited, but for example, 200 mg / L or more, 300 mg / L or more, 500 mg / L or more, 1000 mg / L or more, Or 2000 mg / L or more may be sufficient. The concentration of the phosphorus compound for growth may also be, for example, 20 g / L or less, 10 g / L or less, or 5 g / L or less.

  In the above step 2), the production of limonene by microorganisms is induced by decreasing the concentration of the growth promoter. More specifically, the concentration of the growth promoter can be reduced by reducing the supply amount of the growth promoter into the medium. The concentration of the growth promoting agent can also be reduced by utilizing the growth of microorganisms even when the supply amount of the growth promoting agent into the medium is kept constant through 1) and 2). At the initial stage of the growth of microorganisms in 1), the microorganisms are not sufficiently grown and the number of microorganisms in the medium is small, so that the consumption of the growth promoter by the microorganisms is relatively small. Therefore, at the early stage of growth, the concentration of the growth promoter in the medium is relatively high. On the other hand, in the later stage of the growth of microorganisms in 1), the microorganisms are sufficiently grown and the number of microorganisms in the medium is large, so that the consumption of the growth promoter by the microorganisms is relatively large. Therefore, at a later stage of growth, the concentration of the growth promoter in the medium is relatively low. Thus, when a constant amount of growth promoter is continuously supplied into the medium through 1) and 2), the concentration of the growth promoter in the medium decreases in inverse proportion to the growth of microorganisms. Using this reduced concentration as a trigger, the production of limonene by the microorganism can be induced.

  For example, when a phosphorus compound or amino acid is used as a growth promoter, the concentration of the phosphorus compound or amino acid in the medium capable of inducing the production of limonene by the microorganism is, for example, 100 mg / L or less, 50 mg / L or less, or 10 mg. / L or less.

  In the above step 3), limonene is produced by culturing microorganisms. More specifically, the production of limonene can be performed by culturing a microorganism in a medium under the condition 2) in which the concentration of the growth promoter is decreased. The concentration of the growth promoter in the medium can be maintained at the concentration as described above in 2) to allow the production of limonene by the microorganism.

  In the method of the present invention, the culture period of the microorganism in 3) can be set longer than that in 1). In the conventional method using an inducer, in order to obtain a larger amount of limonene, it was necessary to culture the microorganism for a long time using the inducer during the limonene production phase. However, since the inducer is decomposed when cultured for a long period of time, the microorganism cannot maintain the limonene production ability. Therefore, it is necessary to continuously add the inducer to the culture medium, but the inducer can be expensive and the production cost of limonene can be increased. Therefore, long-term culture of microorganisms using an inducer during the production period of limonene has a problem that the production cost of limonene may increase depending on the length of the culture period. On the other hand, in the method of the present invention which does not use a specific substance such as an inducer in 3), it is not necessary to consider the decomposition of the specific substance, and long-term culture during the production of limonene causes an increase in limonene production costs. There is no problem. Therefore, unlike the conventional method using an inducer, the method of the present invention can easily set the period of 3) to be long. In the method of the present invention, the longer the period of 3) is set, the more limonene can be produced.

  The method of the present invention may be used in combination with other methods from the viewpoint of further improving the amount of limonene produced. Such methods include, for example, light (Pia Lindberg, Sungsoon Park, Anastasio Melis, Metabolic Engineering 12 (2010): 70-79) or temperature (Normal A Valdez-Cruz, Luis CaspetaT , Maurio A Trujillo-Roldan, Microbial Cell Factors 2010, 9: 1) and other environmental factors, pH change (EP 1233068 A2), addition of surfactant (JP 1100996 A), self-induction method (WO2013) / 151174).

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

Examples of monosaccharides include trioses such as ketotriose (dihydroxyacetone) and aldtriose (glyceraldehyde); tetroses such as ketotetrose (erythrulose) and aldetetrose (erythrose and threose); ketopentose (ribulose and xylulose), aldpentose ( Pentose such as ribose, arabinose, xylose, lyxose), deoxy sugar (deoxyribose); ketohexose (psicose, fructose, sorbose, tagatose), aldohexose (allose, altrose, glucose, mannose, gulose, idose, galactose, talose) ), Deoxy sugars (fucose, fucrose, rhamnose) and other hexoses; heptose such as sedoheptulose; Scan, galactose, C6 sugars such as glucose; xylose, carbohydrates C5 sugars arabinose and the like are preferable.
Examples of the disaccharide include sucrose, lactose, maltose, trehalose, tunulose, and cellobiose, and sucrose and lactose are preferable.
Examples of oligosaccharides include trisaccharides such as raffinose, melezitose, and maltotriose; tetrasaccharides such as acarbose and stachyose; and other oligosaccharides such as fructooligosaccharide (FOS), galactooligosaccharide (GOS), and mannan oligosaccharide (MOS). Can be mentioned.
Examples of the polysaccharide include glycogen, starch (amylose, amylopectin), cellulose, dextrin, and glucan (β-1,3-glucan), and starch and cellulose are preferable.

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

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

  The oil contains one or more C4 or higher saturated and unsaturated fatty acids and can be liquid lipid at room temperature, soy, corn, canola, jatropha, palm, peanut, sunflower, coconut, mustard, cottonseed, palm kernel oil , Olive, safflower, sesame, flaxseed, oily microbial cells, peanut, or a combination of two or more thereof.

Examples of the fatty acid include compounds represented by the formula RCOOH (“R” represents a hydrocarbon group having 2 or more carbon atoms).
An unsaturated fatty acid is a compound having at least one carbon-carbon double bond between two carbon atoms in the above-mentioned group “R”, such as oleic acid, vaccenic acid, linoleic acid, palmitate acid, arachidonic acid, etc. Is mentioned.
The saturated fatty acid is a compound in which “R” is a saturated aliphatic group, and examples thereof include docosanoic acid, icosanoic acid, octadecanoic acid, hexadecanoic acid, tetradecanoic acid, and dodecanoic acid.
Among these, fatty acids containing one or more C2 to C22 fatty acids are preferred, and those containing C12 fatty acids, C14 fatty acids, C16 fatty acids, C18 fatty acids, C20 fatty acids and C22 fatty acids are more preferred.
Examples of the carbon source include salts of these fatty acids, derivatives, and salts of derivatives. Examples of the salt include lithium salt, potassium salt, sodium salt and the like.

  Examples of the carbon source include a combination of lipids, oils, fats and oils, fatty acids, glycerol fatty acid esters and carbohydrates such as glucose.

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

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

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

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

  In addition to a suitable carbon source, the medium includes suitable minerals, salts, cofactors, buffers, and ingredients known to those skilled in the art to be suitable for culture or to promote the production of limonene. It is desirable.

As culture conditions for microorganisms, standard culture conditions can be used.
The culture temperature can be 20 ° C. to 40 ° C., and the pH can be about 4.5 to about 9.5.
Further, it is preferable to perform the culture under aerobic, anoxic, or anaerobic conditions depending on the properties of the host. As a culture method, a known fermentation method such as a batch culture method, a fed-batch culture method, or a continuous culture method can be appropriately used.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

Example 1: Construction of Limonene Synthase Expression Plasmid 1-1) Acquisition of Picea situsis-derived limonene synthase gene Picea situsis-derived limonene synthase (PsLMS) gene base sequence (GenBank accession number: DQ1952751) and amino acid The sequence (GenPept accession number: ABA86248.1.) Is already known. P. The amino acid sequence of the sitensis-derived limonene synthase protein and the base sequence of the gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2. In order to efficiently express the PsLMS gene, the secondary structure is eliminated and P.L. Codons were optimized to be the same as the ananatis codon usage. Furthermore, the chloroplast translocation signal was cleaved. The resulting gene was named opt_PsLMS. The base sequence of opt_PsLMS is shown in SEQ ID NO: 3. After chemically synthesizing the opt_PsLMS gene, it was cloned into pUC57 (GenScript), and the resulting plasmid was named pUC57-opt_PsLMS.

1-2) Acquisition of Limonene Synthase Gene Derived from Abies grandis (American Wolf) Limonene Synthase (AgLMS) Gene Base Sequence (GenBank accession number: AF006193.1), and Amino Acid Sequence (GenPept accessionB: 70) .) Is already known. A. The amino acid sequence and gene sequence of the limonene synthase protein derived from grandis are shown in SEQ ID NO: 4 and SEQ ID NO: 5. In order to efficiently express the AgLMS gene, the secondary structure is eliminated and the P.P. Codons were optimized to be the same as the ananatis codon usage. Furthermore, the chloroplast translocation signal was cleaved. The resulting gene was named opt_AgLMS. The base sequence of opt_AgLMS is shown in SEQ ID NO: 6. After chemically synthesizing the opt_AgLMS gene, it was cloned into pUC57 (GenScript), and the resulting plasmid was named pUC57-opt_AgLMS.

1-3) Acquisition of the limonene synthase gene derived from Mentha spicata (Spearmint) The base sequence (GenBank accession number: L13459.1) of the Mentha spicata-derived limonene synthase (MsLMS) gene, and the amino acid sequence (GenPept accession number AC3736: A3736): Already known. M.M. The amino acid sequence and gene sequence of the spicata-derived limonene synthase protein are shown in SEQ ID NO: 7 and SEQ ID NO: 8. In order to efficiently express the MsLMS gene, the secondary structure is eliminated and the P.P. Codons were optimized to be the same as the ananatis codon usage. Furthermore, the chloroplast translocation signal was cleaved. The resulting gene was named opt_MsLMS. The base sequence of opt_MsLMS is shown in SEQ ID NO: 9. After chemically synthesizing the opt_MsLMS gene, it was cloned into pUC57 (GenScript), and the resulting plasmid was named pUC57-opt_MsLMS.

1-4) Acquisition of Limonene Synthase Gene Derived from Citrus unshiu (Cirrus unshiu) Limonene Synthase Derived from Citrus unshiu (CuLMS) Gene (GenBank accession number: AB110637.1), and Amino Acid Sequence (GenPept. Is already known. C. The amino acid sequence and gene sequence of unshiu-derived limonene synthase protein are shown in SEQ ID NO: 10 and SEQ ID NO: 11. In order to efficiently express the CuLMS gene, the secondary structure is eliminated and P.P. Codons were optimized to be the same as the ananatis codon usage. Furthermore, the chloroplast translocation signal was cleaved. The resulting gene was named opt_CuLMS. The base sequence of opt_CuLMS is shown in SEQ ID NO: 12. After chemically synthesizing the opt_CuLMS gene, it was cloned into pUC57 (GenScript) and the resulting plasmid was named pUC57-opt_CuLMS.

1-5) Acquisition of Limonene Synthase Gene Derived from Citrus limon (Lemon) Base sequence (GenBank accession number: AF5142871) and amino acid sequence (GenPept accession number: AAM539.1) of Aci. Is already known. C. The amino acid sequence and gene sequence of limonene-derived limonene synthase protein are shown in SEQ ID NO: 13 and SEQ ID NO: 14. In order to efficiently express the CLLMS gene, the secondary structure is eliminated and P.P. Codons were optimized to be the same as the ananatis codon usage. Furthermore, the chloroplast translocation signal was cleaved. The resulting gene was named opt_ClLMS. The base sequence of opt_ClLMS is shown in SEQ ID NO: 15. After chemically synthesizing the opt_ClLMS gene, it was cloned into pUC57 (GenScript), and the resulting plasmid was named pUC57-opt_ClLMS.

1-6) Obtaining a mutant farnesyl diphosphate synthase gene derived from Escherichia coli The farnesyl diphosphate synthase of Escherichia coli is encoded by the ispA gene (SEQ ID NO: 16) (Fujisaki S., et al. (1990). J. Biochem. (Tokyo) 108: 995-1000.). In farnesyl diphosphate synthase derived from Bacillus stearothermophilus, a mutation that increases the concentration of geranyl diphosphate in the cells has been clarified (Narita K., et al. (1999) J Biochem 126 (3): 566-571. .). Based on this finding, a similar mutant has also been created in the farnesyl diphosphate synthase of Escherichia coli (Reilling KK et al. (2004) Biotechnol Bioeng. 87 (2) 200-212.). In order to efficiently express the ispA mutant gene (S80F) gene having high geranyl diphosphate production activity, a codon-optimized sequence was designed so as to eliminate the secondary structure, and this was named ispA ^* . The base sequence of ispA ^* is shown in SEQ ID NO: 17. The ispA ^* gene was chemically synthesized and then cloned into pUC57 (GenScript), and the resulting plasmid was named pUC57-ispA ^* .

1-7) Construction of plasmid for simultaneous expression of opt_PsLMS and ispA ^* gene PCR was performed using pUC57-opt_PsLMS as a template using the primer shown in SEQ ID NO: 20 and the primer shown in SEQ ID NO: 21. Next, PCR was performed using the obtained PCR fragment as a template and the primer shown in SEQ ID NO: 22 and the primer shown in SEQ ID NO: 21 to obtain a Ptac-opt_PsLMS fragment. Furthermore, PCR was performed using pUC57-ispA ^* obtained using the primer shown in SEQ ID NO: 23 and the primer shown in SEQ ID NO: 24 as a template to obtain an ispA * fragment. The purified Ptac-opt_PsLMS fragment and the ispA ^* fragment were ligated to pACYC177 (Nippon Gene) digested with restriction enzymes PstI and ScaI using In-Fusion HD cloning kit (Clontech) and pACYC177-Ptac-opt_Ps ispA ^* was constructed.

1-8) Construction of plasmid for simultaneous expression of opt_AgLMS and ispA ^* gene PCR was carried out using pUC57-opt_AgLMS as a template using the primer shown in SEQ ID NO: 25 and the primer shown in SEQ ID NO: 26. Next, PCR was performed using the obtained PCR fragment as a template and the primer shown in SEQ ID NO: 22 and the primer shown in SEQ ID NO: 26 to obtain a Ptac-opt_AgLMS fragment. Furthermore, PCR was performed using pUC57-ispA ^* as a template using the primer shown in SEQ ID NO: 23 and the primer shown in SEQ ID NO: 24 to obtain an ispA * fragment. The purified Ptac-opt_AgLMS fragment and the ispA * fragment were ligated to pACYC177 (Nippon Gene) digested with restriction enzymes PstI and ScaI using In-Fusion HD cloning kit (Clontech), and pACYC177-Ptac-Opt_Ag_Opt_Ag_Opt_Ag_Opt_Ag_Opt_Ag_Opt_Ag ispA ^* was constructed.

1-9) Construction of plasmid for simultaneous expression of opt_MsLMS and ispA ^* gene PCR was carried out using pUC57-opt_MsLMS as a template using the primer shown in SEQ ID NO: 27 and the primer shown in SEQ ID NO: 28. Next, PCR was performed using the obtained PCR fragment as a template and the primer shown in SEQ ID NO: 22 and the primer shown in SEQ ID NO: 28 to obtain a Ptac-opt_MsLMS fragment. Furthermore, PCR was performed using pUC57-ispA ^* as a template using the primer shown in SEQ ID NO: 23 and the primer shown in SEQ ID NO: 24 to obtain an ispA * fragment. The purified Ptac-opt_MsLMS fragment and the ispA * fragment were ligated to pACYC177 (Nippon Gene) digested with restriction enzymes PstI and ScaI using In-Fusion HD cloning kit (Clontech) and pACYC177-Ptac-opt_Ms ispA ^* was constructed.

1-10) Construction of plasmid for simultaneous expression of opt_CuLMS and ispA ^* gene PCR was performed using pUC57-opt_CuLMS as a template using the primer shown in SEQ ID NO: 29 and the primer shown in SEQ ID NO: 30. Next, PCR was performed using the obtained PCR fragment as a template and the primer shown in SEQ ID NO: 22 and the primer shown in SEQ ID NO: 30 to obtain a Ptac-opt_CuLMS fragment. Furthermore, PCR was performed using pUC57-ispA ^* as a template using the primer shown in SEQ ID NO: 23 and the primer shown in SEQ ID NO: 24 to obtain an ispA ^* fragment. The purified Ptac-opt_CuLMS fragment and the ispA * fragment were ligated to pACYC177 (Nippon Gene) digested with restriction enzymes PstI and ScaI using In-Fusion HD cloning kit (Clontech) and pACYC177-Ptac-opt_CuLMS ispA ^* was constructed.

1-11) Construction of plasmid for simultaneous expression of opt_ClLMS and ispA ^* gene PCR was performed using pUC57-opt_ClLMS as a template using the primer shown in SEQ ID NO: 31 and the primer shown in SEQ ID NO: 32. Next, PCR was performed using the obtained PCR fragment as a template and the primer shown in SEQ ID NO: 22 and the primer shown in SEQ ID NO: 32 to obtain a Ptac-opt_ClLMS fragment. Furthermore, PCR was performed using pUC57-ispA ^* as a template using the primer shown in SEQ ID NO: 23 and the primer shown in SEQ ID NO: 24 to obtain an ispA ^* fragment. The purified Ptac-opt_ClLMS fragment and the ispA ^* fragment were ligated to pACYC177 (Nippon Gene) digested with restriction enzymes PstI and ScaI using In-Fusion HD cloning kit (Clontech), and pACYC177-Ptac-opt_ClLMS ispA ^* was constructed.

1-12) Construction of SWITCH-PhoCΔgcd and construction of limonene producing strain The ananatis gcd gene (SEQ ID NO: 18) encodes glucose dehydrogenase (SEQ ID NO: 19). Ananatis is known to accumulate gluconic acid during aerobic growth (Andreeva IG et al., FEMS Microbiol Lett. 2011 May; 318 (1): 55-60).
ΛRed-dependent integration of DNA fragments obtained by PCR using primers gcd-attL and gcd-attR (Table 1) and pMW118-attL-kan-attR plasmid as template (Minaeva NI et al., BMC Biotechnol. 2008; 8: 63) is used to construct the SC17 (0) Δgcd strain in which the gcd gene is disrupted. Primers gcd-t1 and gcd-t2 (Table 1) are used to confirm integrants.

  The genomic DNA of the SC17 (0) Δgcd strain was isolated using Wizard Genomic DNA Purification Kit (Promega), and the previously reported method [Katashkina JI et al., BMC Mol Biol. 2009; 10:34] is electrotransformed into the SWITCH-PphoC strain-free derivative obtained in Reference Example 1 described later. As a result, a SWITCH-PphoC-Δgcd (KmR) strain is obtained. Primers gcd-t1 and gcd-t2 (Table 1) are used for PCR analysis of the resulting integrants. The kanamycin resistance marker gene was obtained by standard λIng / Xis mediated procedures [Katashkina JI et al., BMC Mol Biol. 2009; 10:34]. The resulting strain is named SWITCH-PphoC Δgcd strain.

SWITCH-PphoC competent cells prepared in Δgcd, pACYC177 by electroporation, pACYC177-Ptac-opt_PsLMS-ispA *, pACYC177-Ptac-opt_AgLMS-ispA *, pACYC177-Ptac-opt_MsLMS-ispA *, pACYC177-Ptac-opt_CuLMS -IspA ^* or pACYC177-Ptac-opt_ClLMS-ispA ^* was introduced. The obtained strains were respectively SWITCH-PphoC Δgcd / pACYC177 strain, SWITCH-PphoC Δgcd / PsLMS-ispA ^* strain, SWITCH-PphoC Δgcd / AgLMS-ispA ^* strain, SWITCH-PphoC Δgcd / MsLMS-ISPITa ^* ISCHISP The strains were designated as PphoC Δgcd / CuLMS-ispA ^* strain and SWITCH-PphoC Δgcd / ClLMS-ispA ^* strain.

Example 2: Evaluation of limonene production ability of limonene synthase expression strain derived from SWITCH PphoCΔgcd strain SWITCH-PphoC Δgcd / PsLMS-ispA ^* strain, SWITCH-PphoC Δgcd / AgLMS-ispA ^* strain, SWITCH-PphoC obtained in Example 1 Melt glycerol stocks of -Δgcd / MsLMS-ispA ^* strain, SWITCH-PphoC Δgcd / CuLMS-ispA ^* strain, SWITCH-PphoC Δgcd / ClLMS-ispA ^* strain, and SWITCH-PphoC Δgcd / pACYC177 strain at 50 mg / l The solution was evenly applied to an LB plate containing L kanamycin and cultured at 34 ° C. for 16 hours. The cells on the obtained plate were scraped off in an amount of about 1 loop of 1 μL of Nunc ^™ disposable loop (manufactured by Thermo Fisher Scientific). The scraped bacterial cells are inoculated into 1 mL of a limonene fermentation medium described in Table 2 below containing 50 mg / L kanamycin in a headspace vial (22 mL CLEAR CLIMP TOP VIAL cat # B0104236 manufactured by Perkin Elmer). Was sealed with a cap butyl rubber septum for headspace vials (CRIMPS cat # B0104240 manufactured by Perkin Elmer). SWITCH-PphoC Δgcd / pACYC177 strain, SWITCH-PphoC Δgcd / PsLMS-ispA ^* strain, SWITCH-PphoC Δgcd / AgLMS-ispA ^* strain and SWITCH-PphoMMSgc / Lgc / Lgc / Lgc / Lgc / Lgd / L The -ispA ^* strain was cultured for 48 hours. SWITCH-PphoCΔgcd / pACYC177 strain, SWITCH-PphoCΔgcd / CuLMS-ispA ^* strain and SWITCH-PphoCΔgcd / ClLMS-ispA ^* strain were similarly fermented, and the culture time of these strains was 72 hours.

  After completion of sterilization, the above A, B and C zones were mixed.

  After completion of the culture, the concentration of limonene in the headspace vial was measured using a gas chromatograph mass spectrometer (GCMS-QP2010, Shimadzu Corporation) with a headspace sampler (TurboMatrix 40, manufactured by Perkin Elmer). Details of the analysis conditions are described below. As a GC column, HP-5ms Ultra Inert (manufactured by Agilent) was used, and a limonene standard solution was prepared using a reagent limonene (manufactured by Tokyo Chemical Industry).

Headspace sampler injection time 0.02min
Oven temperature 80 ° C
Needle temperature 80 ℃
Transfer temperature 80 ℃
GC cycle time 5 min
Pressurization time 3min
Lifting time 0.2min
Insulation time 5min
Carrier gas pressure 124kPa

Gas chromatography part Carrier gas He
Vaporization chamber temperature 200.0 ℃
Temperature condition 175 ° C (constant temperature)

Mass Spectrometer Ion source temperature 250 ℃
Interface temperature 250 ℃
Detector voltage 0.1 kV
Detected ion molecular weight 68
Auxiliary molecular weight 93
Filament lighting time 2.0-3.5min

The limonene concentration was shown as a value converted to the amount of the medium. The average value of the results for two vials is shown in Tables 3 and 4. In the control strain introduced with the control vector pACYC177, production of limonene was not observed, but SWITCH-PphoC Δgcd / PsLMS-ispA ^* strain, SWITCH-PphoC Δgcd / AgLMS-ispA ^* strain, SWITCH-PphoC Δgcd / MsLMS-ispA ^* strain Limonene production was confirmed in the SWITCH-PphoCΔgcd / CuLMS-ispA ^* strain and the SWITCH-PphoCΔgcd / ClLMS-ispA ^* strain.

Reference Example 1: Microaerobic inducible isoprenoid compound-producing microorganism (SWITCH-Plld / IspSM), phosphate deficiency-inducing isoprenoid compound-producing microorganism (SWITCH-PphoC / IspSM, SWITCH-PpstS / IspSM), arabinose-derived isoprenoid compound 1. Construction of producing microorganism (SWITCH-Para / IspSM) 1-1) Construction of pMW-Para-mvaES-TTrp 1-1-1) Chemical synthesis of enterococcus faecalis-derived mvaE gene Acetyl-CoA acyltransferase coding and hydroxymethylcodec coding The base sequence of mvaE derived from faecalis and the amino acid sequence are Already known (base sequence ACCESSION numbers: AF2900092.1, (1479..3890), amino acid sequence ACCESSION numbers: AAG02439) (J. Bacteriol. 182 (15), 4319-4327 (2000)). The amino acid sequence of Enterococcus faecalis-derived mvaE protein and the nucleotide sequence of the gene are shown in SEQ ID NO: 37 and SEQ ID NO: 38, respectively. The mvaE gene was transformed into E. coli. E. coli for efficient expression in E. coli. An mvaE gene optimized for E. coli codon usage was designed and named EFmvaE. This base sequence is shown in SEQ ID NO: 39. The mvaE gene was chemically synthesized and then cloned into pUC57 (GenScript), and the resulting plasmid was named pUC57-EFmvaE.

1-1-2) Chemical synthesis of Enterococcus faecalis-derived mvaS gene The base sequence of Enterococcus faecalis-derived mvaS coding for hydroxymethylglutaryl-CoA synthase, and the amino acid sequence are already known (base number: ACCESON. (142 .. 1293), ACCESSION number of amino acid sequence: AAG02438) (J. Bacteriol. 182 (15), 4319-4327 (2000)). The amino acid sequence of Enterococcus faecalis-derived mvaS protein and the base sequence of the gene are shown in SEQ ID NO: 40 and SEQ ID NO: 41, respectively. The mvaS gene was transformed into E. coli. E. coli for efficient expression in E. coli. An mvaS gene optimized for E. coli codon usage was designed and named EFmvaS. This base sequence is shown in SEQ ID NO: 42. The mvaS gene was chemically synthesized and then cloned into pUC57 (GenScript), and the resulting plasmid was named pUC57-EFmvaS.

1-1-3) Construction of arabinose-inducible mvaES expression vector An arabinose-inducible mevalonate pathway upstream gene expression vector was constructed by the following procedure. E. coli by PCR using the plasmid pKD46 as a template and the synthetic oligonucleotides shown in SEQ ID NO: 43 and SEQ ID NO: 44 as primers. A PCR fragment containing Para comprising the araC and araBAD promoter sequences from E. coli was obtained. A PCR fragment containing the EFmvaE gene was obtained by PCR using the plasmid pUC57-EFmvaE as a template and the synthetic oligonucleotides shown in SEQ ID NO: 45 and SEQ ID NO: 46 as primers. A PCR fragment containing the EFmvaS gene was obtained by PCR using the plasmid pUC57-EFmvaS as a template and the synthetic oligonucleotides shown in SEQ ID NO: 47 and SEQ ID NO: 48 as primers. A PCR fragment containing the Ttrp sequence was obtained by PCR using the plasmid pSTV-Ptac-Ttrp (WO20133066964A1) as a template and the synthetic oligonucleotides shown in SEQ ID NO: 49 and SEQ ID NO: 50 as primers. Prime Star polymerase (manufactured by Takara Bio Inc.) was used for PCR to obtain these four PCR fragments. The reaction solution was prepared according to the composition attached to the kit, and 30 cycles of reaction at 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, and 72 ° C. for 1 minute / kb were performed. Synthetic oligonucleotides shown in SEQ ID NO: 43 and SEQ ID NO: 46 using a PCR product containing purified Para and a PCR product containing EFmvaE gene as a template, and a PCR product containing purified EFmvaS gene and PCR product containing Ttrp as a template. PCR was performed using 47 and the synthetic oligonucleotide shown in SEQ ID NO: 50 as primers. As a result, PCR products containing Para and EFmvaE genes, EFmvaS and Ttrp were obtained. Plasmid pMW219 (Nippon Gene) was digested with SmaI according to a conventional method. A PCR product containing pMW219 and purified Para and EFmvaE genes, and a PCR product containing EFmvaS gene and Ttrp after SmaI digestion were ligated using In-Fusion HD Cloning Kit (Clontech). The obtained plasmid was designated as pMW-Para-mvaES-TTrp.

1-2) Construction of an integrated conditional replication plasmid carrying upstream and downstream genes of the mevalonate pathway 1-2-1) Construction of a plasmid containing the mvaES gene under the control of different promoters Upstream and downstream of the mevalonate pathway In order to construct an integrative plasmid carrying the gene, pAH162-λattL-TcR-λattR vector (Minaeva NI et al., BMC Biotechnol. 2008; 8:63) was used.

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

  To obtain a promoter-deficient variant of the operon, the Ecl136II-SalI fragment of pMW219-Para-mvaES-TTrp was subcloned into the same integrative vector. A map of the resulting plasmid is shown in FIG.

  A set of plasmids for chromosomal immobilization carrying the mvaES gene under the control of different promoters was constructed. For this purpose, a polylinker containing I-SceI, XhoI, PstI and SphI recognition sites was inserted into the only HindIII recognition site located upstream of the mvaES gene. To achieve this goal, annealing was performed using primers 1 and 2 (Table 5) and polynucleotide kinase. The obtained double-stranded DNA fragment was 5 'phosphorylated with polynucleotide kinase, and the obtained phosphorylated fragment was inserted into pAH162-mvaES plasmid cleaved with HindIII by ligation reaction. The obtained pAH162-MCS-mvaES plasmid (FIG. 3) is convenient for cloning the promoter while maintaining the desired orientation in front of the mvaES gene. DNA fragments carrying the regulatory regions of the lldD, phoC and pstS genes were obtained from P. cerevisiae. Ananatis SC17 (0) strain (Katashkina JI et al., BMC Mol Biol., 2009; 10:34) as a template, and primers 3 and 4, primers 5 and 6, and primers 7 and 8 (Table 5) ), Respectively, and cloned into the appropriate restriction enzyme recognition sites of pAH162-MCS-mvaES. The resulting plasmid is shown in FIG. The cloned promoter fragment was sequenced and confirmed to correspond exactly to the expected nucleotide sequence.

1-2-2) pAH162-Km-Ptac- KDyI chromosome fixing including construction tetAR genes of the plasmid ^{pAH162-λattL-Tc R -λattR (} Minaeva NI et al, BMC Biotechnol, 2008; 8:.. 63 AatII in) -Replacing the ApaI fragment with the DNA fragment obtained by PCR using primers 9 and 10 (Table 5) and the pUC4K plasmid (Taylor LA and Rose RE., Nucleic Acids Res., 16, 358, 1988) as a template. did. As a result, pAH162-λattL-Km ^R -λattR was obtained (FIG. 5).

The P _tac promoter, ^{pAH162-λattL-Tc} R -λattR vector (Minaeva NI et al, BMC Biotechnol , 2008; 8:.. 63) was inserted into the HindIII-SphI recognition site. As a result, an expression vector pAH162-P _tac for chromosome fixation was constructed. The sequence of the cloned promoter fragment was determined and confirmed to be the sequence as designed. A map of pAH162-P _tac is shown in FIG.

Chemically synthesized by ATG Service Gene (Russia), a rare codon was replaced with a synonymous codon. A DNA fragment (FIG. 7) carrying the cerevisiae-derived PMK, MVD and yldI genes was subcloned into the SphI-KpnI restriction enzyme recognition site of the chromosome fixing vector pAH162-Ptac. The DNA sequence containing the chemically synthesized KDyI operon is shown in SEQ ID NO: 75. The resulting plasmid pAH162-Tc-Ptac-KDyI carrying the Ptac-KDyI expression cassette is shown in FIG. 8 (A). Thereafter, the NotI-KpnI fragment of _pAH162-Tc-P tac -KDyI to hold the tetAR genes for the purpose of replacing the drug resistance marker gene was replaced by the corresponding fragment in pAH162-λattL-KmR-λattR. As a result, a pAH162-Km-Ptac-KDyI plasmid using the kanamycin resistance gene kan as a marker was obtained (FIG. 8B).

A chemically synthesized DNA fragment containing the coding portion of the putative mvk gene from SANAE [see GenBank accession number AP011532 for the complete genomic sequence] linked to the classical SD sequence, the Methanocella pardicola strain. The integrative expression vector pAH162-P _tac was cloned into the PstI-KpnI recognition site. A map of the chromosome-fixed plasmid carrying the mvk gene is shown in FIG.

1-3) Construction of recipient strain SC17 (0) ΔampC :: attB _phi80 ΔampH :: attB _phi80 Δcrt :: P _tac -mvk (M.palladicola) kan gene adjacent to attL _phi80 and attR _phi80 , and target chromosomal site ΛRed dependent integration of a PCR amplified DNA fragment containing a 40 bp sequence homologous to (Katashkina JI et al., BMC Mol Biol., 2009; 10:34), followed by the phage phi80 Int / Xis dependency of the kanamycin resistance marker Do removing (Andreeva IG et al, FEMS Microbiol Lett, 2011; 318 (1):.. 55-60) using a two-step approach comprising, ΔampH :: attB _{ph 80} and _{ΔampC :: attB} phi80 the chromosome modification, P. It was introduced stepwise into the ananatis SC17 (0) strain. SC17 (0) P. ananatis AJ13355 is a λRed resistant derivative (Katashkina JI et al., BMC Mol Biol., 2009; 10:34); The annotated complete genome sequence of ananatis AJ13355 is available as PRJDA 162073 or GenBank accession numbers AP012032.1 and AP012033.1. pMWattphi plasmid [Minaeva NI et al. , BMC Biotechnol. , 2008; 8:63] as templates and primers 11 and 12 and primers 13 and 14 (Table 5) as primers to generate DNA fragments used for incorporation into the ampH and ampC gene regions, respectively. did. Primers 15 and 16 and primers 17 and 18 (Table 5) were used for PCR verification of the resulting chromosome modifications.

In parallel, P.I. a part of the P. ananatis AJ13355 genome that retains the attB site of the phi80 phage in place of the crt operon located on the pEA320 320 kb megaplasmid. A derivative of ananatis SC17 (0) was constructed. To obtain this strain, λRed dependent integration of a PCR amplified DNA fragment _carrying attL _phi80 -kan-attR _phi80 flanked by a 40 bp region homologous to the target site in the genome was performed _using a previously described procedure (Katashkina JI et al., BMC Mol Biol., 2009; 10:34). The DNA fragment used to replace the crt operon with _attL phi80-kan-attR _phi80 was amplified in a reaction using primers 19 and 20 (Table 5). The pMWattphi plasmid (Minaeva NI et al., BMC Biotechnol., 2008; 8:63) was used as a template in this reaction. The resulting _integrant was named SC17 (0) Δcrt :: attL _phi80 -kan-attR _phi80 . Primers 21 and 22 (Table 5) were combined with SC17 (0) Δcrt :: attL _phi80 -kan-attR _phi80 . It was used for PCR verification of the chromosomal structure. Removal of the kanamycin resistance marker from the constructed strain was performed using the pAH129-cat helper plasmid according to a previously reported technique (Andrewa IG et al., FEMS Microbiol Lett., 2011; 318 (1): 55-60). Oligonucleotides 21 and 22 were used for PCR verification of the resulting SC17 (0) Δcrt :: attB _phi80 strain. The maps of the resulting ΔampC :: attB _phi80 , ΔampH :: attB _phi80 and Δcrt :: attB _phi80 genomic variants are shown in FIGS. 10 (A), (B) and (C), respectively.

The pAH162-Ptac-mvk (M. palidicola) plasmid was prepared according to a previously reported protocol (Andrewa IG et al., FEMS Microbiol Lett., 2011; 318 (1): 55-60) SC17 (0) Δcrt :: attB _It was integrated into the attB _phi80 site of _phi80 . Plasmid integration was confirmed by polymerase chain reaction using primers 21 and 23 and primers 22 and 24 (Table 5). As a result, an SC17 (0) Δcrt :: pAH162-P _tac -mvk (M. palidicola) strain was obtained. A map of Δcrt :: pAH162-P _tac -mvk (M. palidicola) modification is shown in FIG. 11 (A).
Subsequently, the genetic traits of SC17 (0) Δcrt :: pAH162-P _tac -mvk (M. palladicola) via genomic DNA electroporation technique (Katashkina JI et al., BMC Mol Biol., 2009; 10:34). _Was transferred to SC17 (0) ΔampC :: attB _phi80 ΔampH :: attB _phi80 . The obtained strain uses the tetracycline resistance gene tetRA as a marker. The vector portion of the pAH162-Ptac-mvk (M. palladicola) integration type plasmid containing the tetRA marker gene was replaced with the previously reported pMW-intxis-cat helper plasmid [Katashkina JI et al. , BMC Mol Biol. , 2009; 10:34]. As a result, a marker gene-deficient strain SC17 (0) ΔampH :: attB _φ80 ΔampC :: attB _φ80 Δcrt :: P _tac -mvk (M. palidicola) was obtained. A map of Δcrt :: P _tac -mvk (M. palidicola) genomic modification is shown in FIG. 11 (B).

1-4) Construction of SWITCH strain set The pAH162-Km-Ptac-KDyI plasmid was prepared according to a previously reported protocol (Andrewa IG et al. FEMS Microbiol Lett. 2011; 318 (1): 55-60), SC17 (0 ) ΔampH :: attB _φ80 ΔampC :: attB _φ80 Δcrt :: P _tac -mvk (M. Palidicola) / pAH123-cat was integrated into the chromosome. Cells were seeded on LB agar containing 50 mg / L kanamycin. Proliferated Km ^R clones were tested in a PCR reaction using primers 11 and 15 and primers 11 and 17 (Table 5). Strains carrying the pAH162-Km-Ptac-KDyI plasmid integrated into ΔampH :: attB _φ80 or ΔampC :: attB _φ80 m were selected. Maps of ΔampH :: pAH162-Km-Ptac-KDyI and ΔampC :: pAH162-Km-Ptac-KDyI chromosome variants are shown in FIGS. 12 (A) and (B).
pAH162-Px-mvaES (where, Px is the one of the following regulatory _{region: araC-P ara (E.coli)} , P lldD, P phoC, P pstS) a previously reported protocol [Andreeva IG et al. , FEMS Microbiol Lett. , 2011; 318 (1): 55-60] using the pAH123-cat helper plasmid SC17 (0) ΔampC :: pAH162-Km-P _tac -KDyI ΔampH pH :: attB _phi80 Δcrt :: P _tac -mvk ( M. palidicola) and SC17 (0) ΔampC :: attB _phi80 ΔampH :: pAH162-Km-P _tac -KDyI Δcrt :: P _tac -mvk (M. palidicola) inserted into the attB _phi80 site of the recipient strain. As a result, two sets of strains respectively named SWITCH-Px-1 and SWITCH-Px-2 were obtained. A map of ΔampH :: pAH162-Px-mvaES and ΔampC :: pAH162-Px-mvaES chromosome modifications is shown in FIG.

1-5) Introduction of isoprene synthase expression plasmid An electrocompetent cell of SWITCH bacteria was prepared according to a conventional method, and pSTV28-Ptac-IspSM (WO2013 / 179722), an expression plasmid for isoprene synthase derived from Mucuna, was introduced by electroporation. . The obtained isoprenoid compound-producing microorganisms were named SWITCH-Para / IspSM, SWITCH-Plld / IspSM, SWITCH-PpstS / IspSM, and SWITCH-PphoC / IspSM, respectively.

  Although the invention has been described in detail with reference to preferred embodiments thereof, it will be apparent to those skilled in the art that various modifications can be made and equivalents can be employed without departing from the scope of the invention. It is. All references cited herein are incorporated by reference as part of this application.

  The method of the present invention is useful for the production of limonene.

Claims (15)

  A method for producing limonene, comprising culturing a Pantoea bacterium expressing limonene synthase in a culture medium to produce limonene.   2. The method of claim 1, wherein the bacterium comprises a heterologous expression unit comprising a polynucleotide encoding the limonene synthase and a promoter operably linked thereto. The method according to claim 2, wherein the polynucleotide is one or more polynucleotides selected from the group consisting of the following (a) to (o).
(A) (i) a base sequence represented by SEQ ID NO: 2, (ii) a base sequence consisting of nucleotide residues 193 to 1905 in the base sequence represented by SEQ ID NO: 2, or (iii) SEQ ID NO: 3 A polynucleotide comprising the base sequence represented by:
(B) a polynucleotide comprising a base sequence having 90% or more identity with the base sequence of (i), (ii) or (iii), and encoding a protein having limonene synthase activity;
(C) a poly which hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence of (i), (ii) or (iii) and which encodes a protein having limonene synthase activity nucleotide;
(D) (iv) a nucleotide sequence represented by SEQ ID NO: 5, (v) a nucleotide sequence consisting of nucleotide residues 205 to 1914 in the nucleotide sequence represented by SEQ ID NO: 5, or (vi) SEQ ID NO: 6 A polynucleotide comprising the base sequence represented by:
(E) a polynucleotide comprising a base sequence having 90% or more identity with the base sequence of (iv), (v) or (vi), and encoding a protein having limonene synthase activity;
(F) a poly which hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence of (iv), (v) or (vi) and which encodes a protein having limonene synthase activity nucleotide;
(G) (vii) a base sequence represented by SEQ ID NO: 8, (viii) a base sequence consisting of nucleotide residues 169 to 1800 in the base sequence represented by SEQ ID NO: 8, or (ix) SEQ ID NO: 9 A polynucleotide comprising the base sequence represented by:
(H) a polynucleotide comprising a base sequence having 90% or more identity with the base sequence of (vii), (viii) or (ix) and encoding a protein having limonene synthase activity;
(I) a poly which hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence of (vii), (viii) or (ix) and which encodes a protein having limonene synthase activity nucleotide;
(J) (x) a base sequence represented by SEQ ID NO: 11, (xi) a base sequence consisting of nucleotide residues 154 to 1827 in the base sequence represented by SEQ ID NO: 11, or (xii) SEQ ID NO: 12 A polynucleotide comprising the base sequence represented by:
(K) a polynucleotide encoding a protein comprising a base sequence having 90% or more identity with the base sequence of (x), (xi) or (xii), and having limonene synthase activity;
(L) a polynucleotide that hybridizes with a polynucleotide comprising a base sequence complementary to the base sequence of (x), (xi) or (xii) under a stringent condition and encodes a protein having limonene synthase activity nucleotide;
(M) (xiii) a nucleotide sequence represented by SEQ ID NO: 14, (xiv) a nucleotide sequence consisting of nucleotide residues 154 to 1821 in the nucleotide sequence represented by SEQ ID NO: 14, or (xv) SEQ ID NO: 15 A polynucleotide comprising the base sequence represented by:
(N) a polynucleotide comprising a base sequence having 90% or more identity with the base sequence of (xiii), (xiv) or (xv) and encoding a protein having limonene synthase activity; or (o) A polynucleotide encoding a protein that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence of (xiii), (xiv) or (xv) and has limonene synthase activity. The method of any one of claims 1 to 3, wherein the limonene synthase is one or more proteins selected from the group consisting of the following (A) to (O).
(A) (i ′) a full-length amino acid sequence represented by SEQ ID NO: 1 or (ii ′) a protein comprising an amino acid sequence consisting of amino acid residues 65 to 634 in the amino acid sequence represented by SEQ ID NO: 1;
(B) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence of (i ′) or (ii ′) and having limonene synthase activity;
(C) a protein having an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence of (i ′) or (ii ′) and having limonene synthase activity;
(D) (iii ′) a full-length amino acid sequence represented by SEQ ID NO: 4 or (iv ′) a protein comprising an amino acid sequence consisting of amino acid residues 69 to 637 in the amino acid sequence represented by SEQ ID NO: 4;
(E) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence of (iii ′) or (iv ′) and having limonene synthase activity; or (F) the (iii ′) or (iv) A protein having a limonene synthase activity, including an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence of ');
(G) (v ′) a full-length amino acid sequence represented by SEQ ID NO: 7 or (vi ′) a protein comprising an amino acid sequence consisting of the 57th to 599th amino acid residues in the amino acid sequence represented by SEQ ID NO: 7;
(H) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence of (v ′) or (vi ′) and having limonene synthase activity;
(I) a protein having an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence of (v ′) or (vi ′) and having limonene synthase activity;
(J) (vii ′) a protein comprising the full-length amino acid sequence represented by SEQ ID NO: 10, or (viii ′) an amino acid sequence consisting of amino acid residues 52 to 608 in the amino acid sequence represented by SEQ ID NO: 10;
(K) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence of (vii ′) or (viii ′) and having limonene synthase activity;
(L) a protein having an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence of (vii ′) or (viii ′) and having limonene synthase activity;
(M) (ix ′) a full-length amino acid sequence represented by SEQ ID NO: 13, or (x ′) a protein comprising an amino acid sequence consisting of amino acid residues 52 to 606 in the amino acid sequence represented by SEQ ID NO: 13; Or (N) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence of (ix ′) or (x ′) and having limonene synthase activity; or (O) the (ix ′) or ( A protein having an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence of x ′) and having limonene synthase activity.   The method according to any one of claims 1 to 4, wherein the limonene synthase is a limonene synthase derived from a plant belonging to the genus Picea, Abies, Mentha, or Citrus.   The limonene synthase is a limonene synthase derived from the American fir (Abies grandis), Alaska spruce (Picea situchensis), Spearmint (Mentha spicata), Citrus unshu, or Lemon (Citrus limon). 6. The method according to any one of 5 above.   The method according to claim 1, wherein the bacterium is a bacterium that further expresses geranyl diphosphate synthase.   The method according to any one of claims 1 to 7, wherein the bacterium has the ability to synthesize dimethylallyl diphosphate or isopentenyl diphosphate through a methylerythritol phosphate pathway.   The method according to any one of claims 1 to 8, wherein the bacterium has the ability to synthesize dimethylallyl diphosphate or isopentenyl diphosphate by the mevalonate pathway.   The method according to claim 1, wherein the bacterium is a microorganism in which a 2-ketogluconic acid production pathway is blocked.   The method according to claim 10, wherein the 2-ketogluconic acid production pathway is blocked by a decrease in glucose dehydrogenase activity.   The method according to claim 11, wherein a polynucleotide that is a glucose dehydrogenase gene is disrupted in the bacterium. The method according to claim 12, wherein the polynucleotide is one or more polynucleotides selected from the group consisting of the following (x) to (z).
(X) [i] a nucleotide sequence represented by SEQ ID NO: 18, or [ii] a polynucleotide comprising a nucleotide sequence consisting of nucleotide residues 301 to 2691 in the nucleotide sequence represented by SEQ ID NO: 18;
(Y) a polynucleotide comprising a base sequence having 90% or more identity with the base sequence of [i] or [ii] and encoding a protein having glucose dehydrogenase activity; or (z) the above [i ] Or a polynucleotide encoding a protein having a glucose dehydrogenase activity, which hybridizes with a polynucleotide comprising a base sequence complementary to the base sequence of [ii] under stringent conditions. The method according to claim 12 or 13, wherein the glucose dehydrogenase is one or more proteins selected from the group consisting of the following (X) to (Z).
(X) a protein comprising the full-length amino acid sequence represented by SEQ ID NO: 19;
(Y) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 19 and having glucose dehydrogenase activity; and (Z) the amino acid sequence represented by SEQ ID NO: 19 A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted, and having glucose dehydrogenase activity.   The method according to claim 1, wherein the bacterium is Pantoea ananatis.

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