Method for producing (R)-Reticuline

Abstract

The present invention relates to a production method of (R)-reticulin comprising: a step for obtaining a recombinant host cell by inserting, to a host cell, a gene 1 which is composed of a nucleotide sequence having at least 70% homology to a nucleotide sequence of SEQ ID NO: 2 and which is DNA encoding a protein having an enzymatic activity of CYP80Y2, and a gene 2 which is composed of a nucleotide sequence having at least 70% homology to a nucleotide sequence of SEQ ID NO: 3 and which is DNA encoding a protein having an enzymatic activity of oxidoreductase; a step for expressing, in the recombinant host cell, the protein having the enzymatic activity of CYP80Y2 and the protein having the enzymatic activity of oxidoreductase; and a step for producing (R)-reticulin from (S)-reticulin by using the recombinant host cell.

Description

[Technical field]

[0001] The present invention relates to a method for producing (R)-reticuline, a plant benzylisoquinoline alkaloid. [Background technology]

[0002] Opioid analgesics, including morphine, are known as medicamentously useful compounds belonging to the benzylisoquinoline alkaloid group. Conventionally, opioid analgesics have been produced by extraction from natural plants. Analgesics obtained by this method, which utilizes secondary metabolites of plants, are expensive compared to analgesics produced by other methods, and therefore an alternative method was desired.

[0003] Some benzylisoquinoline alkaloids, including morphine, can be synthesized chemically, but their complex structures and chirality make low-cost production difficult.

[0004] Therefore, the present inventors have attempted to carry out all steps of the biosynthesis of benzylisoquinoline alkaloids in a microbial system (Patent Document 1, Non-Patent Documents 1 and 2). In this method, which uses microorganisms as host cells, the isoquinoline alkaloid biosynthetic pathway is reconstructed by combining enzymes from plants and microorganisms.

[0005] Incidentally, benzylisoquinoline alkaloids are synthesized from tyrosine in Magnoliaceae, Ranunculaceae, Berberidaceae, Papaveraceae, and many other plant species, and many of them use (S)-reticuline as a biosynthetic intermediate.

[0006] Thebaine, a raw material for opioid analgesics, is obtained through a reaction in which (S)-reticuline is converted to the optical isomer (R)-reticuline. The enzyme that converts (S)-reticuline to (R)-reticuline had not been identified for a long time, but in Non-Patent Document 3, the STORR ((S)-tо-(R)-reticulin) protein, which is the enzyme that converts (S)-reticuline to (R)-reticuline, was identified (Non-Patent Document 3). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2012 / 039438 [Patent Document 2] Special Publication No. 2017-500024 [Non-patent literature]

[0008] [Non-Patent Document 1] Akira N, et al. (2016) Total biosynthesis of opiates by stepwise fermentation using engineered Escherichia coli. Nat Commun. 7: 10390 [Non-Patent Document 2] Akira N, et al. (2011) A bacterial platform for fermentative production of plant alkaloids. Nat Commun. 2: 326 [Non-Patent Document 3] Thilo W, et al. (2015) Morphinan biosynthesis in opium poppy requires a P450-oxidoreductase fusion protein. Science. 349(6245): 309-312 Summary of the Invention [Problem to be solved by the invention]

[0009] However, Non-Patent Document 3 does not disclose a specific method for producing (R)-Reticuline from (S)-Reticuline using a microorganism.

[0010] On the other hand, Patent Document 2 discloses a method for producing (R)-Reticuline using a eukaryote such as yeast as a host cell and utilizing an amino acid sequence identical or similar to STORR (Patent Document 2).

[0011] However, Patent Document 2 does not disclose a method for producing (R)-Reticuline using a prokaryote as a host cell. This is because it is difficult to functionally express P450 enzymes and salutadine synthase (SalS) in prokaryotes. Therefore, in Patent Document 2, the applicable host cells are limited to eukaryotes such as yeast, and it is difficult to produce (R)-Reticuline using a prokaryote (e.g., Escherichia coli) as a host cell.

[0012] In addition, in Patent Document 2, (S)-Reticuline is also mixed with the produced (R)-Reticuline, making it difficult to produce only (R)-Reticuline. Therefore, in the method of Patent Document 2, in order to obtain only (R)-Reticuline, it was necessary to perform optical resolution using a chiral column.

[0013] Therefore, the present invention aims to provide a method for producing (R)-Reticuline from (S)-Reticuline with high conversion efficiency. Another object of the present invention is to provide a method capable of producing (R)-Reticuline with high conversion efficiency even when a prokaryote is used as a host cell. Another object of the present invention is to provide an inexpensive opioid analgesic by constructing a thebaine production system applicable to Escherichia coli, a practically useful prokaryote. [Means for solving the problem]

[0014] With regard to the above-mentioned problems, the present inventors have found that (R)-Reticuline can be obtained at a significantly high conversion efficiency by employing the method of the present invention. They have also found that the method of the present invention makes it possible to produce (R)-Reticuline at a significantly high conversion efficiency even when using prokaryotes as host cells. In the method of the present invention, the amount of (S)-Reticuline was ultimately below the detection limit, and the proportion of (R)-Reticuline produced was apparently 100% relative to the total amount of reticuline.

[0015] The method for producing (R)-Reticuline of the present invention is characterized by comprising the steps of: inserting gene 1 (hereinafter also referred to as "gene encoding CYP80Y2"), which is DNA consisting of a nucleotide sequence having 70% or more homology to the nucleotide sequence of SEQ ID NO: 2 and encoding a protein having the enzyme activity of CYP80Y2, and gene 2 (hereinafter also referred to as "gene encoding oxidoreductase"), which is DNA consisting of a nucleotide sequence having 70% or more homology to the nucleotide sequence of SEQ ID NO: 3 and encoding a protein having the enzyme activity of oxidoreductase, into a host cell to obtain a recombinant host cell; expressing the protein having the enzyme activity of CYP80Y2 and the protein having the enzyme activity of oxidoreductase in the recombinant host cell; and producing (R)-Reticuline from (S)-Reticuline by the recombinant host cell.

[0016] In one aspect, the method for producing (R)-Reticuline of the present invention is characterized by comprising the steps of: dividing gene 3 (hereinafter also referred to as "gene encoding STORR"), which is DNA consisting of a nucleotide sequence having 70% or more homology to the nucleotide sequence of SEQ ID NO: 1 and encoding a protein having the enzyme activity of STORR, into gene 1, which is DNA consisting of a nucleotide sequence having 70% or more homology to the nucleotide sequence of SEQ ID NO: 2 and encoding a protein having the enzyme activity of CYP80Y2, and gene 2, which is DNA consisting of a nucleotide sequence having 70% or more homology to the nucleotide sequence of SEQ ID NO: 3 and encoding a protein having the enzyme activity of oxidoreductase; inserting gene 1 and gene 2 into a host cell to obtain a recombinant host cell; expressing the protein having the enzyme activity of CYP80Y2 and the protein having the enzyme activity of oxidoreductase in the recombinant host cell; and producing (R)-Reticuline from (S)-Reticuline by the recombinant host cell.

[0017] According to the above-mentioned method for producing (R)-Reticuline of the present invention, (R)-Reticuline can be obtained with a significantly high conversion efficiency.

[0018] The above-mentioned method for producing (R)-Reticuline of the present invention may further include at least one of the following steps: a step of deleting the nucleotide sequence encoding the N-terminal hydrophobic region of the protein having the enzyme activity of CYP80Y2 from the gene 1; a step of introducing gene 4 (hereinafter also referred to as "gene encoding 5-aminolevulinic acid synthase 1"), which is a DNA consisting of a nucleotide sequence having 70% or more homology to the nucleotide sequence of SEQ ID NO: 4 and encoding a protein having the enzyme activity of 5-aminolevulinic acid synthase 1, into the host cell to express the protein having the enzyme activity of 5-aminolevulinic acid synthase 1; and a step of introducing gene 5 (hereinafter also referred to as "gene encoding CPR"), which is a DNA consisting of a nucleotide sequence having 70% or more homology to the nucleotide sequence of SEQ ID NO: 5 and encoding a protein having the enzyme activity of CPR, into the host cell to express the protein having the enzyme activity of CPR. This allows for efficient production of (R)-Reticuline even when a prokaryote is used as the host cell.

[0019] In one embodiment, the above-mentioned method for producing (R)-Reticuline of the present invention may further include a step of deleting the nucleotide sequence encoding the N-terminal hydrophobic region of the protein having the enzyme activity of CYP80Y2 from the gene 1, a step of introducing gene 4, which is a DNA consisting of a nucleotide sequence having 70% or more homology to the nucleotide sequence of SEQ ID NO: 4 and encoding a protein having the enzyme activity of 5-aminolevulinic acid synthase 1, into the host cell to express the protein having the enzyme activity of 5-aminolevulinic acid synthase 1, and a step of introducing gene 5, which is a DNA consisting of a nucleotide sequence having 70% or more homology to the nucleotide sequence of SEQ ID NO: 5 and encoding a protein having the enzyme activity of CPR, into the host cell to express the protein having the enzyme activity of CPR. This allows (R)-Reticuline to be produced more efficiently even when a prokaryote is used as the host cell.

[0020] In the method for producing (R)-Reticuline of the present invention, the host cell may be a prokaryote.

[0021] In the method for producing (R)-Reticuline of the present invention, the prokaryote may be Escherichia coli.

[0022] According to the production method of the present invention, the amount of (S)-Reticuline ultimately reaches below the detection limit, and the proportion of produced (R)-Reticuline relative to the total amount of Reticuline appears to be 100%.

[0023] The production method of the present invention is capable of producing (R)-Reticuline with a significantly high conversion efficiency as described above, even when a prokaryote is used as a host cell. Therefore, the production method of the present invention is useful in practice, since it is possible to produce (R)-Reticuline by applying, for example, Escherichia coli, which is a prokaryote, as a host cell. Effect of the Invention

[0024] According to the present invention, a method for producing (R)-Reticuline from (S)-Reticuline with a significantly high conversion efficiency can be provided. In the production method of the present invention, (S)-Reticuline can be finally reduced to an amount below the detection limit. Therefore, in the production method of the present invention, there is no need to perform a process (optical resolution, etc.) for obtaining only (R)-Reticuline. Furthermore, in the production method of the present invention, even if a prokaryote is used as the host cell, it is possible to produce (R)-Reticuline with a high conversion efficiency as described above. Therefore, according to the present invention, for example, a thebaine production system applicable to Escherichia coli, which is a practically useful prokaryote, can be constructed, which enables mass production of (R)-Reticuline, thereby making it possible to supply an inexpensive opioid analgesic. [Brief description of the drawings]

[0025] [Figure 1] 1 shows the biosynthetic pathway of (R)-reticuline that is reconstituted in a host cell in the present invention. [Diagram 2] FIG. 2(a) is a schematic diagram showing a gene encoding STORR in which a domain encoding CYP80Y2 and a domain encoding oxidoreductase are fused together, and FIG. 2(b) is a schematic diagram showing a gene used in the present embodiment in which the domain encoding CYP80Y2 and the domain encoding oxidoreductase are separated. [Diagram 3] 1 shows the results of LC-MS analysis indicating the production of (R)-Reticuline in this embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Hereinafter, an embodiment of the present invention will be described in detail. However, the present invention is not limited to the following embodiment.

[0027] In the present invention, "homology" refers to the degree of similarity between two polypeptides or two polynucleotides, and is determined by comparing two sequences aligned in an optimal state (maximum sequence identity) across the region of the amino acid or base sequences to be compared. The numerical value (%) of homology is calculated by determining the number of sites in both aligned (amino acid or base) sequences where identical amino acids or bases exist, then dividing the number of sites by the total number of amino acids or bases in the region of the sequences to be compared, and multiplying the resulting numerical value by 100. Algorithms for obtaining optimal alignment and homology include various algorithms (e.g., BLAST algorithm, FASTA algorithm) that are commonly available to those skilled in the art. The homology of amino acid sequences is determined using sequence analysis software such as BLASTP and FASTA. The homology of base sequences is determined using software such as BLASTN and FASTA.

[0028] The "host cell" into which a gene is introduced in the method of the present invention is not particularly limited, and examples thereof include prokaryotes such as Escherichia coli and Bacillus subtilis, and eukaryotes such as yeast and filamentous fungi. As the host cell in the present invention, Escherichia coli is preferred.

[0029] When introducing genes into a host cell, the genes may be directly introduced into the genomic DNA of the host cell, but it is preferable to introduce into the host cell a vector in which the genes have been incorporated. The genes to be introduced may all be incorporated into the same vector, or may be divided into two or more separate vectors.

[0030] A vector (expression vector) expresses an introduced gene incorporated therein. A vector for incorporating an introduced gene is preferably one constructed for genetic recombination from a plasmid or phage capable of autonomously replicating in a host cell. A vector preferably contains an origin of replication, a selectable marker, an expression control sequence such as a promoter, and a transcription termination signal (terminator sequence) that is compatible with the host cell to be introduced. Examples of plasmid vectors include pET vectors, pQE vectors, and pCold vectors when expressing in E. coli, and pYES2 vectors and pYEX vectors when expressing in yeast.

[0031] Selectable markers include antibiotic resistance genes such as the ampicillin resistance gene, the kanamycin resistance gene, the streptomycin resistance gene, and the like.

[0032] An expression control sequence means a sequence that, when properly linked to a DNA sequence, can control the expression of a gene consisting of that DNA sequence in a host cell, i.e., can induce and / or promote or suppress the transcription of the DNA sequence into RNA. The expression control sequence includes at least a promoter. The promoter may be a constitutive promoter or an inducible promoter.

[0033] The expression vector used in the present invention can be constructed by adding an appropriate restriction enzyme recognition site to the end of the desired gene by a conventional method.

[0034] Methods for transforming host cells with an expression vector include conventionally known methods, such as the calcium chloride method, electroporation, and heat shock method.

[0035] The culture conditions of the recombinant host cell are not particularly limited as long as the recombinant cell grows well, all of the target proteins are expressed, and each function or enzyme activity is exerted. Specifically, the culture conditions may be appropriately selected in consideration of the nutritional and physiological properties of the host, and are usually performed in liquid culture.

[0036] Carbon sources for the medium used to culture recombinant host cells are not particularly limited as long as they are substances that can be utilized by the host cells, and include sugars and glycerol. Examples of sugars include monosaccharides such as glucose, fructose, and galactose, and disaccharides such as sucrose, lactose, and maltose. Nitrogen sources include ammonium sulfate and casamino acids. In addition, salts, specific amino acids, specific vitamins, and the like can be used as desired.

[0037] Examples of media for culturing E. coli include LB medium, 2xYT medium, and M9 minimal medium. Examples of media for culturing yeast include SC medium, SD medium, and YPD medium.

[0038] The culture temperature can be appropriately changed within a range in which the host cells grow, the target enzyme is expressed, and its activity is exerted. For Escherichia coli, for example, culture conditions of temperature 25°C, 80 hours, pH 7.0 can be used. For yeast, for example, culture conditions of temperature 30°C, 60 hours, pH 5.8 can be used.

[0039] The produced (R)-Reticuline can be confirmed by any means known to those skilled in the art.Specifically, the reaction product and the target (R)-Reticuline standard are subjected to LC-MS, and the obtained spectrum is compared to identify it.It can also be confirmed by comparison by NMR analysis.

[0040] As used herein, "expressing" a particular gene means that the nucleic acid molecule constituting the gene is transcribed at least into an RNA molecule, and in the case of a gene encoding a polypeptide, means that the nucleic acid molecule constituting the gene is transcribed into an RNA molecule and the RNA molecule is translated into a polypeptide.

[0041] The expression level of the gene can be confirmed by methods known in the art, such as Northern blot, quantitative PCR, and the like.

[0042] As used herein, "expressing" a specific enzyme (protein) means that transcription from a nucleic acid molecule encoding the polypeptide of the enzyme into an RNA molecule and translation from the RNA molecule into a polypeptide are carried out normally, producing an active enzyme that is present inside or outside the cell.

[0043] The expression level of the enzyme can be confirmed by detection and quantification using known techniques such as Western blot, ELISA, etc. It can also be confirmed by an enzyme activity assay.

[0044] Next, the "gene encoding STORR" (gene 3), the "gene encoding CYP80Y2" (gene 1), the "gene encoding oxidoreductase" (gene 2), the "gene encoding 5-aminolevulinic acid synthase" (gene 4), and the "gene encoding CPR" (gene 5), which are used in the present invention, will be described.

[0045] STORR is an enzyme derived from Papaver somniferum, and is an epimerase that converts (S)-reticuline to (R)-reticuline. STORR is a fusion polypeptide that contains a CYP80Y2 domain and an oxidoreductase domain. SEQ ID NO: 9 is the amino acid sequence of STORR.

[0046] The "gene encoding STORR" used in the present invention may be, but is not limited to, any one of the DNAs listed in (a) to (c) below: (a) DNA consisting of the nucleotide sequence of SEQ ID NO:1; (b) a DNA that hybridizes under stringent conditions with a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:1 and encodes a protein having the enzyme activity of STORR (e.g., an epimerase activity that converts (S)-reticuline to (R)-reticuline); (c) A DNA consisting of a nucleotide sequence having 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more homology to the nucleotide sequence of SEQ ID NO: 1, and encoding a protein having STORR enzyme activity (e.g., epimerase activity that converts (S)-reticuline to (R)-reticuline).

[0047] In the present invention, as the "gene encoding STORR", of the above (a) to (c), (a) DNA consisting of the nucleotide sequence of SEQ ID NO: 1 is preferably used.

[0048] CYP80Y2 is a P450 enzyme. CYP80Y2 has, for example, the activity of oxidizing (S)-reticuline to generate 1,2-dehydroreticulinium. In one embodiment of the present invention, CYP80Y2 may be, for example, a domain of CYP80Y2 contained in the above-mentioned STORR separated from a domain of oxidoreductase. The amino acid sequence of CYP80Y2 thus obtained (the amino acid sequence of SEQ ID NO: 11 is further deleted from the N-terminal hydrophobic region) is obtained by adding an initiation methionine to the amino acid sequence shown in SEQ ID NO: 11. SEQ ID NO: 10 is the amino acid sequence of the N-terminal hydrophobic region deleted in this embodiment. In SOSUI analysis, the amino acid sequence of the N-terminal hydrophobic region is PTSSVVALLLALVSILSSVVV, but in this embodiment, it is deleted from the initiation codon. By deleting the nucleotide sequence encoding the N-terminal hydrophobic region, inserting an initiation codon sequence (atg), and introducing a termination codon sequence (taa) into the sequence immediately preceding the gene encoding the oxidoreductase, a gene encoding CYP80Y2 having an initiation methionine added to the amino acid sequence shown in SEQ ID NO: 11 can be obtained.

[0049] The "gene encoding CYP80Y2" used in the present invention may be, but is not limited to, any one of the DNAs listed in (a) to (c) below: (a) DNA consisting of the nucleotide sequence of SEQ ID NO:2; (b) a DNA that hybridizes under stringent conditions with a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2 and encodes a protein having the enzyme activity of CYP80Y2 (e.g., the activity of oxidizing (S)-reticuline to produce 1,2-dehydroreticulinium); (c) DNA consisting of a nucleotide sequence having a homology of 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more to the nucleotide sequence of SEQ ID NO: 2, and encoding a protein having the enzymatic activity of CYP80Y2 (e.g., the activity of oxidizing (S)-reticuline to produce 1,2-dehydroreticulinium).

[0050] In the present invention, as the "gene encoding CYP80Y2", of the above (a) to (c), (a) DNA consisting of the nucleotide sequence of SEQ ID NO: 2 is preferably used.

[0051] The "gene encoding CYP80Y2" used in the present invention may be a gene in which the nucleotide sequence encoding the N-terminal hydrophobic region of a protein having the enzymatic activity of CYP80Y2 has been deleted. By deleting (cutting) the base sequence of the N-terminal hydrophobic region, the transmembrane region is deleted.

[0052] The deletion of the nucleotide sequence of the N-terminal hydrophobic region of the "gene encoding CYP80Y2" may be performed by deleting the nucleotide sequence of the N-terminal hydrophobic region of the "gene encoding STORR." That is, since the "gene encoding STORR" has a gene encoding CYP80Y2 on the N-terminus and a gene encoding oxidoreductase on the C-terminus, deleting the nucleotide sequence of the N-terminal hydrophobic region of the "gene encoding STORR" results in the deletion of the nucleotide sequence of the N-terminal hydrophobic region of the gene encoding CYP80Y2.

[0053] In the present invention, the "N-terminal hydrophobic region" of the "gene encoding CYP80Y2" (or the "gene encoding STORR") refers to a hydrophobic region present on the N-terminal side of the "gene encoding CYP80Y2" (or the "gene encoding STORR"), and is, for example, the base sequence shown in SEQ ID NO: 8.

[0054] The oxidoreductase in the present invention is an oxidoreductase having an enzyme activity equivalent to that of the oxidoreductase domain contained in the above-mentioned STORR. In one embodiment of the present invention, the oxidoreductase may be, for example, an oxidoreductase in which the above-mentioned STORR domain is separated from the CYP80Y2 domain. SEQ ID NO: 12 is the amino acid sequence of the oxidoreductase thus obtained.

[0055] The "gene encoding oxidoreductase" used in the present invention may be, but is not limited to, any one of DNAs described in (a) to (c) below: (a) DNA consisting of the nucleotide sequence of SEQ ID NO:3; (b) a DNA that hybridizes under stringent conditions with a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 3 and encodes a protein having an enzyme activity of oxidoreductase (e.g., the activity of reducing 1,2-dehydroreticulinium to produce (R)-reticuline); (c) A DNA consisting of a nucleotide sequence having a homology of 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more to the nucleotide sequence of SEQ ID NO: 3, and encoding a protein having the enzyme activity of oxidoreductase (e.g., the activity of reducing 1,2-dehydroreticulinium to produce (R)-reticuline).

[0056] In the present invention, as the "gene encoding oxidoreductase", of the above (a) to (c), (a) DNA consisting of the nucleotide sequence of SEQ ID NO: 3 is preferably used.

[0057] 5-aminolevulinate synthase 1 is an enzyme that synthesizes 5-aminolevulinate using succinyl-CoA and glycine as substrates and pyridoxal phosphate as a coenzyme.

[0058] The "gene encoding 5-aminolevulinic acid synthase 1" used in the present invention may be, but is not limited to, any one of the DNAs shown in (a) to (c) below: (a) DNA consisting of the nucleotide sequence of SEQ ID NO:4; (b) a DNA that hybridizes under stringent conditions with a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 4 and encodes a protein having the enzyme activity of 5-aminolevulinic acid synthase 1 (e.g., the activity of synthesizing 5-aminolevulinic acid using succinyl-CoA and glycine as substrates and pyridoxal phosphate as a coenzyme); (c) A DNA consisting of a nucleotide sequence having 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more homology to the nucleotide sequence of SEQ ID NO: 4, and encoding a protein having the enzyme activity of 5-aminolevulinic acid synthase 1 (e.g., the activity of synthesizing 5-aminolevulinic acid using succinyl-CoA and glycine as substrates and pyridoxal phosphate as a coenzyme).

[0059] In the present invention, as the "gene encoding 5-aminolevulinic acid synthase 1", of the above (a) to (c), (a) DNA consisting of the nucleotide sequence of SEQ ID NO: 4 is preferably used.

[0060] (a) A gene that is a DNA consisting of the nucleotide sequence of SEQ ID NO: 4 is called the HemA gene. The HemA gene is a gene encoding 5-aminolevulinic acid synthase 1 of Rhodobacter sphaeroides.

[0061] CPR (NADPH-cytochrome P450 reductase 2) is an enzyme that transfers electrons from NADP to cytochrome P450 in microsomes. It also plays a role in transferring electrons to heme oxygenase and cytochrome B5.

[0062] The "gene encoding CPR" used in the present invention may be, but is not limited to, any one of the DNAs described in (a) to (c) below: (a) DNA consisting of the nucleotide sequence of SEQ ID NO:5; (b) a DNA that hybridizes under stringent conditions with a DNA consisting of a nucleotide sequence complementary to the DNA consisting of the nucleotide sequence of SEQ ID NO:5, and encodes a protein having the enzyme activity of CPR (e.g., the activity of transferring electrons from NADP to cytochrome P450 in microsomes); (c) A DNA consisting of a nucleotide sequence having 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more homology to the nucleotide sequence of SEQ ID NO:5, and encoding a protein having the enzyme activity of CPR (e.g., the activity of transferring electrons from NADP to cytochrome P450 in microsomes).

[0063] In the present invention, as the "gene encoding CPR", of the above (a) to (c), (a) DNA consisting of the nucleotide sequence of SEQ ID NO: 5 is preferably used.

[0064] (a) A gene that is a DNA consisting of the nucleotide sequence of SEQ ID NO: 5 is called the ATR2 gene. The ATR2 gene is a gene that encodes the CPR of Arabidopsis thaliana.

[0065] "Stringent conditions" refer to conditions under which only specific hybridization occurs and non-specific hybridization does not occur. Such conditions are usually about 6M urea, 0.4% SDS, and 0.5xSSC. The DNA obtained by hybridization desirably has a high homology of 70% or more with the DNA consisting of the nucleotide sequence of each SEQ ID NO., preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more.

[0066] The above genes can be obtained by PCR or hybridization techniques well known to those skilled in the art. The above genes may also be artificially synthesized using a DNA synthesizer or the like. The sequence can be determined using a sequencer according to conventional methods.

[0067] ((R)-Reticuline Production Method) The method for producing (R)-Reticuline of the present invention will be described.

[0068] FIG. 1 shows the biosynthetic pathway of (R)-reticuline reconstructed in a host cell in the present invention. The brackets indicate the reactions in each strain in each culture step. In the present invention, (R)-reticuline is produced by converting (S)-reticuline to (R)-reticuline. The abbreviations are as follows: TYR, tyrosinase; DDC, dopa decarboxylase; MAO, monoamine oxidase; 3,4-DHPAA, 3,4-dihydroxyphenylacetaldehyde; SalS, salutaridine synthase; SalR, salutaridine reductase; SalAT, salutaridinol acetyltransferase; SPT (spontaneous).

[0069] FIG. 2 is a schematic diagram showing the STORR gene (FIG. 2(a)) and a STORR gene in which the CYP80Y2 domain and the oxidoreductase domain have been split (FIG. 2(b)).

[0070] The method for producing (R)-Reticuline of the present invention involves converting (S)-Reticuline to (R)-Reticuline, and (S)-Reticuline is prepared in advance according to a conventional method. The method for producing (R)-Reticuline of the present invention involves, for example, carrying out the following steps 1 to 4 in this order. Step 1: Dividing a gene encoding STORR (gene 3) into a gene encoding CYP80Y2 (gene 1) and a gene encoding oxidoreductase (gene 2). Step 2: Inserting the gene encoding CYP80Y2 (gene 1) and the gene encoding oxidoreductase (gene 2) into a host cell to obtain a recombinant host cell. Step 3: Expressing a protein having the enzyme activity of CYP80Y2 encoded by gene 1 and a protein having the enzyme activity of oxidoreductase encoded by gene 2 in the recombinant host cell. Step 4: Producing (R)-Reticuline from (S)-Reticuline by the recombinant host cell.

[0071] The above-mentioned step 1 can be omitted. That is, the method for producing (R)-Reticuline of the present invention may, for example, comprise carrying out the following steps 2 to 4 in this order: A step (step 2) of inserting a gene encoding CYP80Y2 (gene 1) and a gene encoding oxidoreductase (gene 2) into a host cell to obtain a recombinant host cell; A step (step 3) of expressing a protein having the enzyme activity of CYP80Y2 encoded by gene 1 and a protein having the enzyme activity of oxidoreductase encoded by gene 2 in the recombinant host cell; and A step (step 4) of producing (R)-Reticuline from (S)-Reticuline by the recombinant host cell.

[0072] When the host cell is a prokaryote (e.g., E. coli), the method for producing (R)-Reticuline is as follows.

[0073] The "gene encoding STORR" (gene 3) has the nucleotide sequence encoding its N-terminal hydrophobic region deleted in advance (Step A). ​​More specifically, the N-terminal hydrophobic region of the gene encoding CYP80Y2 among the "genes encoding STORR" is deleted (cut), thereby deleting the transmembrane region, inserting a start codon sequence (atg), and introducing a stop codon sequence (taa) into the sequence immediately preceding the gene encoding oxidoreductase.

[0074] The "gene encoding STORR" (gene 3) is separated into the "gene encoding CYP80Y2" (gene 1) and the "gene encoding oxidoreductase" (gene 2) (Step B).

[0075] A "gene encoding CYP80Y2" (gene 1), a "gene encoding oxidoreductase" (gene 2), a "gene encoding 5-aminolevulinic acid synthase 1" (gene 4), and a "gene encoding CPR" (gene 5) are introduced into a host cell to obtain a recombinant host cell (Step C). The introduction of these genes does not necessarily have to be performed in this order. They may be introduced simultaneously, or the order may be reversed.

[0076] In the recombinant host cell, a protein having the enzyme activity of CYP80Y2 encoded by gene 1, a protein having the enzyme activity of oxidoreductase encoded by gene 2, a protein having the enzyme activity of 5-aminolevulinic acid synthase 1 encoded by gene 4, and a protein having the enzyme activity of CPR encoded by gene 5 are expressed (Step D). (R)-Reticuline is produced from (S)-Reticuline by the host cell (Step E).

[0077] The above-mentioned steps A and B may be omitted. Alternatively, instead of steps A and B, a step (step X) of deleting a nucleotide sequence encoding an N-terminal hydrophobic region of a protein having the enzymatic activity of CYP80Y2 from the "gene encoding CYP80Y2" (gene 1) may be carried out. EXAMPLES

[0078] An example carried out according to the method of the present invention will now be described. The following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

[0079] (material) All synthetic genes were obtained from GenScript Inc. (S)-reticuline was produced according to the method described in the prior art (e.g., Nakagawa et al., 2011, Nat Commun).

[0080] (Construction of expression vector) In order to reconstruct the pathway for synthesizing (R)-reticuline from (S)-reticuline, multiple expression vectors containing various genes were constructed.

[0081] In this example, a gene encoding CYP80Y2 shown in SEQ ID NO: 2, which was split off from a gene encoding STORR shown in SEQ ID NO: 1 (UniProtKB: P0DKI7) in the sequence listing, and a gene encoding oxidoreductase shown in SEQ ID NO: 3 were used. In addition, a HemA gene (UniProtKB: Q04512), which is a gene encoding 5-aminolevulinic acid synthase 1 shown in SEQ ID NO: 4, and an ATR2 gene (UniProtKB: Q9SUM3), which is a gene encoding CPR shown in SEQ ID NO: 5, were used. The codons of each gene were optimized for E. coli.

[0082] In order to realize the control by the T7 promoter, both genes were inserted into the NdeI-BamHI site of pET23a. When linking genes, the gene to be linked was first inserted into the NdeI-BamHI site of pET23a, and the gene to be linked was inserted into the XhoI site located downstream of the NdeI-BamHI site to create an expression vector. For example, when inserting a gene encoding an oxidoreductase after a gene encoding CYP80Y2, the gene encoding CYP80Y2 was first inserted into the NdeI-BamHI site of pET23a to create an expression vector containing a gene encoding CYP80Y2, CYP80Y2 / pET23a. Then, the gene encoding oxidoreductase was amplified by PCR using primers pr576 (sequence number 6) and pr577 (sequence number 7) shown in Table 1. Then, a gene encoding an oxidoreductase was inserted by infusion into the XhoI site located downstream of the NdeI-BamHI site of CYP80Y2 / pET23a to create CYP80Y2-OxiRed / pET23a, an expression vector containing a gene encoding CYP80Y2 and a gene encoding an oxidoreductase.

[0083] [Table 1]

[0084] In this example, expression vectors CYP80Y2Ncut-OxiRed / pET23a, ATR2 / pCDF23, HemA / pCDF23, and ATR2-HemA / pCDF23 were constructed by a method similar to that described above.

[0085] CYP80Y2Ncut-OxiRed / pET23a is an expression vector based on the pET23a plasmid, which contains a gene encoding CYP80Y2 with the N-terminal hydrophobic region deleted, and a gene encoding oxidoreductase. HemA / pCDF23 is an expression vector based on the pCDF23 plasmid, which contains the HemA gene, which is the gene encoding 5-aminolevulinic acid synthase 1. ATR2 / pCDF23 is an expression vector based on the pCDF23 plasmid, which contains the ATR2 gene, a gene that encodes CPR. ATR2-HemA / pCDF23 is an expression vector based on the pCDF23 plasmid, which contains the HemA gene, which is a gene encoding 5-aminolevulinic acid synthase 1, and the ATR2 gene, which is a gene encoding CPR.

[0086] Next, various E. coli strains, AN2534, AN4415, AN4340, and AN4341, were created. The AN2534 strain was created by introducing CYP80Y2Ncut-OxiRed / pET23a into the BL21DE3 strain, which is an E. coli competent cell. The AN4415 strain was created by introducing the empty vector pCDF23 into the AN2534 strain. The AN4340 strain was created by introducing ATR2 / pCDF23 into the AN2534 strain. The AN4341 strain was created by introducing HemA / pCDF23 into the AN2534 strain. The AN2051 strain was created by introducing ATR2-HemA / pCDF23 into the AN2534 strain.

[0087] Each E. coli strain was inoculated into LB medium (Difco) containing 50 mg / L ampicillin and 100 mg / L spectinomycin, and cultured at 37°C for 12 hours with shaking. The culture was then added to TB medium (per liter: 12 g tryptone (Difco), 24 g yeast extract (Diffco), 9.4 g K2HPO4, 2.2 g K2HPO4 and 4 ml glycerol) containing 50 mg / L ampicillin and 100 mg / L spectinomycin at a volume of 1 / 100. After culturing at 25°C for 12 hours, IPTG was added to a final concentration of 1 mM, and the culture was further cultured at 25°C for 12 hours with shaking. An equal amount of culture supernatant containing 320 μM S-reticuline produced using E. coli (prepared, for example, by the method described in Nakagawa et al., 2011, Nat Commun) was mixed thereto, and the culture was further cultured at 25°C for 12 hours with shaking. The culture supernatant was collected, and the chirality of reticulin was observed by the methods described in Non-Patent Documents 1 and 2. The results are shown in Table 2.

[0088] [Table 2]

[0089] As shown in Table 2, only the AN2051 strain, into which a gene encoding CYP80Y2 with the N-terminal hydrophobic region deleted, a gene encoding an oxidoreductase, the HemA gene encoding 5-aminolevulinic acid synthase 1, and the ATR2 gene encoding CPR have been introduced, is able to produce (R)-reticuline.

[0090] Fig. 3 shows the results of LC-MS analysis showing the production of (R)-reticuline in this embodiment. Fig. 3(a) shows the results when the gene encoding STORR shown in SEQ ID NO: 1 was introduced into a host cell in its full length (i.e., not divided into a gene encoding CYP80Y2 and a gene encoding oxidoreductase). Fig. 3(b) shows the results when the gene encoding STORR shown in SEQ ID NO: 1 was divided into a gene encoding CYP80Y2 and a gene encoding oxidoreductase and then introduced into a host cell. Fig. 3(c) shows the results of overlapping these samples, showing that the peaks in Fig. 3(a) and Fig. 3(b) do not overlap.

[0091] As shown in Figure 3(a), it was shown that (R)-Reticuline cannot be produced even if the full-length gene encoding STORR is introduced into a host cell. And, as shown in Figure 3(b), it was shown that (R)-Reticuline can be obtained by dividing the gene encoding STORR into a gene encoding CYP80Y2 and a gene encoding oxidoreductase and introducing them into a host cell. In other words, when the two domains of STORR are expressed separately, strong activity is obtained, making it possible to produce sufficient amounts (~100 μM) of (R)-Reticuline.

[0092] In addition, the results of Table 2 and Figure 3 show that by introducing a gene encoding CYP80Y2 with the N-terminal hydrophobic region deleted, a gene encoding an oxidoreductase, the HemA gene which is a gene encoding 5-aminolevulinic acid synthase 1, and the ATR2 gene which is a gene encoding CPR into a host cell, (R)-reticuline can be obtained with a significantly high conversion efficiency.

[0093] In this embodiment, prokaryotes were used as host cells, but are not limited thereto. That is, eukaryotes can also be used as host cells. In eukaryotes, P450 enzymes and salutadine synthase (SalS) are functionally expressed, so it is possible to more easily produce (R)-reticuline from (S)-reticuline. For example, when a gene encoding CYP80Y2 and a gene encoding oxidoreductase are introduced into a eukaryote (yeast), it is considered that (R)-reticuline is produced from (S)-reticuline. Naturally, it is also considered that (R)-reticuline is produced from (S)-reticuline when a gene encoding ATR2 and / or a gene encoding HemA are introduced into a eukaryote (yeast) in addition to a gene encoding CYP80Y2 and a gene encoding oxidoreductase. Thus, the present invention is applicable within the scope of its purpose.

Claims

1. A gene 1 is a DNA that is composed of a nucleotide sequence having 90% or more identity to the nucleotide sequence of SEQ ID NO: 2 and encodes a protein having an activity of oxidizing (S)-reticuline to produce 1,2-dehydroreticulinium; a gene 2, which is a DNA consisting of a nucleotide sequence having 90% or more identity to the nucleotide sequence of SEQ ID NO: 3 and encoding a protein having an enzymatic activity of oxidoreductase, into a host cell to obtain a recombinant host cell; expressing in the recombinant host cell a protein having an activity of oxidizing the (S)-reticuline to produce 1,2-dehydroreticulinium and a protein having the enzyme activity of the oxidoreductase; Producing (R)-Reticuline from (S)-Reticuline by the recombinant host cell; Including, A method for producing (R)-reticuline, wherein the host cell is a prokaryote.

2. A gene 3, which is a DNA having a nucleotide sequence having 90% or more identity to the nucleotide sequence of SEQ ID NO: 1 and encoding a protein having the enzyme activity of STORR, A gene 1 is a DNA that is composed of a nucleotide sequence having 90% or more identity to the nucleotide sequence of SEQ ID NO: 2 and encodes a protein having an activity of oxidizing (S)-reticuline to produce 1,2-dehydroreticulinium; a gene 2 consisting of a nucleotide sequence having 90% or more identity to the nucleotide sequence of SEQ ID NO: 3 and being a DNA encoding a protein having an enzymatic activity of oxidoreductase; inserting said gene 1 and said gene 2 into a host cell to obtain a recombinant host cell; a protein having an activity of oxidizing the (S)-reticuline to produce 1,2-dehydroreticulinium in the recombinant host cell; expressing a protein having the enzymatic activity of the oxidoreductase; Producing (R)-Reticuline from (S)-Reticuline by the recombinant host cell; Including, A method for producing (R)-reticuline, wherein the host cell is a prokaryote.

3. a step of deleting a nucleotide sequence encoding an N-terminal hydrophobic region of a protein having an activity of oxidizing the (S)-reticuline to produce 1,2-dehydroreticulinium from the gene 1; introducing gene 4, which is a DNA consisting of a nucleotide sequence having 90% or more identity to the nucleotide sequence of SEQ ID NO: 4 and encoding a protein having the enzyme activity of 5-aminolevulinic acid synthase 1, into the host cell to express the protein having the enzyme activity of 5-aminolevulinic acid synthase 1; A step of introducing gene 5, which is a DNA consisting of a nucleotide sequence having 90% or more identity to the nucleotide sequence of SEQ ID NO: 5 and encoding a protein having CPR enzyme activity, into the host cell to express the protein having CPR enzyme activity, The method for producing (R)-Reticuline according to claim 1 or 2, further comprising at least one of the following steps:

4. a step of deleting a nucleotide sequence encoding an N-terminal hydrophobic region of a protein having an activity of oxidizing the (S)-reticuline to produce 1,2-dehydroreticulinium from the gene 1; introducing gene 4, which is a DNA consisting of a nucleotide sequence having 90% or more identity to the nucleotide sequence of SEQ ID NO: 4 and encoding a protein having the enzyme activity of 5-aminolevulinic acid synthase 1, into the host cell to express the protein having the enzyme activity of 5-aminolevulinic acid synthase 1; introducing gene 5, which is a DNA consisting of a nucleotide sequence having 90% or more identity to the nucleotide sequence of SEQ ID NO:5 and encoding a protein having CPR enzymatic activity, into the host cell to express the protein having CPR enzymatic activity; The method for producing (R)-Reticuline according to claim 1 or 2, further comprising:

5. The method for producing (R)-Reticuline according to claim 4, wherein the prokaryote is Escherichia coli.

6. A gene 3, which is a DNA consisting of a nucleotide sequence having 90% or more identity to the nucleotide sequence of SEQ ID NO: 1 and encoding a protein having the enzyme activity of STORR, A gene 1 is a DNA that is composed of a nucleotide sequence having 90% or more identity to the nucleotide sequence of SEQ ID NO: 2 and encodes a protein having an activity of oxidizing (S)-reticuline to produce 1,2-dehydroreticulinium; a gene 2 consisting of a nucleotide sequence having 90% or more identity to the nucleotide sequence of SEQ ID NO: 3 and being a DNA encoding a protein having an enzymatic activity of oxidoreductase; The gene 1, the gene 2, and A gene 4 which is a DNA having a nucleotide sequence having 90% or more identity to the nucleotide sequence of SEQ ID NO: 4 and encoding a protein having the enzyme activity of 5-aminolevulinic acid synthase 1; A step of inserting gene 5, which is a DNA consisting of a nucleotide sequence having 90% or more identity to the nucleotide sequence of SEQ ID NO:5 and encoding a protein having CPR enzyme activity, into a host cell to obtain a recombinant host cell; a protein having an activity of oxidizing the (S)-reticuline to produce 1,2-dehydroreticulinium in the recombinant host cell; expressing a protein having the enzyme activity of the oxidoreductase, a protein having the enzyme activity of 5-aminolevulinic acid synthase 1, and a protein having the enzyme activity of the CPR; and producing (R)-Reticuline from (S)-Reticuline by the recombinant host cell.

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