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WO2012039438A1 - Procédé de production de benzylisoquinoléine-alcaloïde végétal - Google Patents

Procédé de production de benzylisoquinoléine-alcaloïde végétal Download PDF

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WO2012039438A1
WO2012039438A1 PCT/JP2011/071520 JP2011071520W WO2012039438A1 WO 2012039438 A1 WO2012039438 A1 WO 2012039438A1 JP 2011071520 W JP2011071520 W JP 2011071520W WO 2012039438 A1 WO2012039438 A1 WO 2012039438A1
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Prior art keywords
methyltransferase
host cell
reticuline
recombinant host
gene
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Japanese (ja)
Inventor
中川　明
博道 南
高嶺 片山
英彦 熊谷
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Ishikawa Prefectural University
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Ishikawa Prefectural University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring

Definitions

  • the present invention relates to a method for producing plant benzylisoquinoline alkaloids.
  • Isoquinoline alkaloids are a diverse group of 6,000 kinds of compounds, and contain many useful drugs such as morphine and berberine, and are important secondary metabolites produced by plants. Until now, most of its production has relied on extraction from natural products. However, it is difficult to obtain a high yield by extraction because the accumulation level of secondary metabolites in plant cells is low.
  • Benzylisoquinoline alkaloids such as the analgesic compounds morphine and codeine, and the antibacterial agents berberine and palmatin, from tyrosine (S) in magnoliaceae, buttercupaceae, barberry, poppy, and many other plant species -Synthesized via reticuline.
  • S tyrosine
  • (S) -reticuline is a branch-point intermediate in the biosynthetic pathway of many types of benzylisoquinoline alkaloids. That is, (S) -reticuline is a pharmaceutically important non-narcotic alkaloid useful for the development of antimalarial and anticancer drugs.
  • Non-Patent Documents 1 to 3 A recent report shows that berberine, an antibacterial agent, has cholesterol-lowering activity (Non-Patent Document 4).
  • Non-patent Document 5 Non-patent Document 5
  • enzymatic synthesis is desirable for environmentally friendly and efficient alkaloid production.
  • Plant metabolic engineering has often attempted to increase the amount of alkaloid pathway end products, and selected plant cells can produce secondary metabolites in quantities sufficient for industrial use (non- Patent Document 6).
  • Non- Patent Document 6 Plant metabolic engineering has often attempted to increase the amount of alkaloid pathway end products, and selected plant cells can produce secondary metabolites in quantities sufficient for industrial use.
  • non- Patent Document 6 since the flow of secondary metabolism in plants is complex and strictly controlled, it is very difficult to obtain the desired product, and only a few successful examples of plant metabolic engineering have been reported.
  • Non-Patent Documents 7 and 8 Transformed poppies are suitable for the production of reticuline, but the amount of the product varies greatly depending on the plant and the cultured cell, and the plant and the cultured cell take a long time to grow. In addition, these transformed poppies accumulated some methylated derivatives of reticuline.
  • Non-Patent Documents 10 and 11 Recently, attempts to reconstruct the plant biosynthesis process have been investigated in microbial systems. Since the microbial system does not contain other plant metabolites, it can not only increase the amount of secondary metabolites, but also improve its quality. Microbial systems have several advantages for the biotransformation of chemicals, but especially for plant metabolites, for example, with the disadvantage of limited substrate availability. The combination of microbial and plant-derived genes is useful for establishing an efficient system for the production of various compounds.
  • Patent Document 1 a method for producing reticuline in vitro or in vivo using dopamine as a starting material by reconstructing an isoquinoline alkaloid biosynthetic pathway in a microorganism by combining plant and microbial enzymes.
  • This method synthesizes 3,4-dihydroxyphenylacetaldehyde (hereinafter also referred to as 3,4-DHPAA) from dopamine by using a microorganism-derived monoamine oxidase (hereinafter also referred to as MAO), and the 3,4-dihydroxy.
  • 3,4-DHPAA 3,4-dihydroxyphenylacetaldehyde
  • MAO microorganism-derived monoamine oxidase
  • One feature is that phenylacetaldehyde and dopamine are coupled, and it is possible to efficiently synthesize reticuline only from dopamine while omitting the hydroxylation step in the reticuline biosynthesis pathway.
  • Patent Document 2 the isoquinoline alkaloid biosynthetic pathway is reconstructed in microorganisms and reacted in the presence of dopamine and other amines as substrates to produce various alkaloids in addition to reticuline.
  • Patent Document 2 A method has also been developed (Patent Document 2). However, also in this method, it is essential to use dopamine as a starting material, and when the reaction is performed in vivo, reticuline is obtained as a (R, S) -racemic mixture.
  • the object of the present invention is to establish a system for producing benzylisoquinoline alkaloids from inexpensive and readily available substrates without using relatively expensive starting materials such as dopamine.
  • the present inventor has combined a gene involved in plant and microorganism metabolism and reconstructed the biosynthetic pathway of benzylisoquinoline alkaloid in the microorganism, thereby producing a desired benzylisoquinoline alkaloid.
  • the present inventor has combined a gene involved in plant and microorganism metabolism and reconstructed the biosynthetic pathway of benzylisoquinoline alkaloid in the microorganism, thereby producing a desired benzylisoquinoline alkaloid.
  • the present inventors synthesize (S) -reticuline, which is important as a branching point intermediate in the biosynthetic pathway of benzylisoquinoline alkaloids, by culturing transformed microorganisms that express the enzymes necessary for the biosynthesis. Succeeded.
  • the present invention relates to an isoquinoline alkaloid non-producing cell having a metabolic pathway for producing L-tyrosine, tyrosinase and its adapter protein, L-DOPA-specific decarboxylase, monoamine oxidase, norcoclaurine synthase, norcoclaurine 6.
  • a method for producing (S) -reticuline comprising the step of culturing in a medium containing one or more sugars selected from the group consisting of sucrose, lactose and maltose and / or gly
  • the present invention relates to a non-productive isoquinoline alkaloid-producing cell having a metabolic pathway for producing L-tyrosine, a feedback inhibitory resistant 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, a feedback inhibitory resistant chorismi.
  • the present invention relates to non-quinoline alkaloid-producing cells having a metabolic pathway that produces L-tyrosine, tyrosinase and its adapter protein, L-DOPA-specific decarboxylase, monoamine oxidase, norcoclaurine synthase, norcoclaurine 6.
  • a desired host benzylisoquinoline alkaloid is obtained by culturing a recombinant host cell expressing a gene required for biosynthesis of benzylisoquinoline alkaloid in a medium usually used for culturing microorganisms.
  • a recombinant host cell expressing a gene required for biosynthesis of benzylisoquinoline alkaloid in a medium usually used for culturing microorganisms.
  • benzylisoquinoline alkaloid can be produced from an inexpensive and readily available substrate that constitutes a microorganism medium, and there is no need to use an additional substrate as a starting material.
  • the microbial system of the present invention is a simple and cost-effective system that can dramatically reduce the cost of producing rare benzylisoquinoline alkaloids.
  • a foundation capable of producing a large amount of benzylisoquinoline alkaloid, which is a material for producing various useful compounds, is constructed, and a new drug discovery resource can be developed through further metabolic conversion.
  • the microbial system of the present invention is also useful for developing new pathways for the production of novel isoquinoline alkaloids.
  • the individual steps of the benzylisoquinoline alkaloid biosynthetic pathway reconstructed in the microbial system of the present invention are all known and controllable. Thus, it may be possible to achieve more efficient production by optimizing the expression levels of individual biosynthetic genes and improving pathway flow.
  • FIG. 2 shows the biosynthetic pathway of (S) -reticuline reconstituted in a host cell in the present invention.
  • the tailor-made biosynthetic pathway constructed by the present invention consists of TYR, DODCDO and MAO. Plant biosynthetic pathways after NCS have been modified to avoid CYP80B reactions.
  • E4P erythrose-4-phosphate
  • PEP phosphoenolpyruvate
  • DAHP 3-deoxy-D-arabino-heptulosonate-7-phosphate
  • H HPP 4-hydroxyphenylpyruvic acid
  • 3,4 3,4-DHPAA 3,4-dihydroxyphenylacetaldehyde
  • f fbr-DAHPS feedback inhibitor resistant 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase
  • fbr-CM / PDH feedback inhibitor resistant Chorismate mutase / prephenate dehydrogenase
  • P PEPS phosphoenolpyruvate synthetase
  • TK TKT transketolase
  • TYR tyrosinase
  • DODC L-DOPA specific decarboxylase
  • MAO monoamine oxidase
  • NC NCS norcoclaurine synthase
  • 6OM monoamine oxidase
  • Recombinant production strain A was also cultured in a medium not containing glucose (triangle mark).
  • the cell growth of each strain (right axis, broken line, symbol is the same correspondence as (S) -reticuline production) is shown as absorbance at 600 nm.
  • the arrow indicates the addition of IPTG (50 ⁇ M) at the time it points.
  • the experiment was repeated three times with essentially the same results. Data from one representative experiment is shown in the figure.
  • Selected ion monitoring (SIM) parameters: m / z 330 (reticuline).
  • the biosynthetic pathway of isoquinoline alkaloids from reticuline to magnoflorin or scourelin is shown. Abbreviations are as follows: CN CNMT, coclaurine-N-methyltransferase; CYP80G2, corituberin synthase; BBE, berberine cross-linking enzyme. Analysis of stereoselectivity of reaction products for (R, S) -reticuline.
  • the reaction product (b) obtained by culturing standard (R, S) -reticuline (a) and the recombinant host cell of the present invention (“recombinant production strain A”) is obtained on an Agilent HPLC system equipped with a chiral column. After separation, it was monitored using LC-MS.
  • Selected ion monitoring (SIM) parameters: m / z 330 (reticuline)
  • Media were collected at various time points corresponding to FIGS. 2, 3 and 9 and the concentrations of L-tyrosine (circles), L-DOPA (squares) and dopamine (triangles) were determined.
  • Glycerol consumption (x) was calculated from the concentration in the medium and the amount of glycerol added at each time point. The experiment was repeated three times with essentially the same results. Data from one representative experiment is shown in the figure.
  • pCOLADuet-1 contains the tyrA fbr , aroG fbr , tktA and ppsA genes.
  • pET-21d contains the NCS, ORF378, TYR, DODC and optMAO genes.
  • pACYC184 contains the 6OMT, 4'OMT and CNMT genes. Both genes are controlled by the T7 promoter.
  • tyrA fbr -aroG fbr -tktA-ppsA / pCOLADuet-1 and 6OMT-4'OMT-CNMT / pACYC184 each have a T7 terminator sequence located downstream of the ppsA and CNMT genes, respectively.
  • NCS-ORF378-TYR-DODC-optMAO / pET-21d has two T7 terminator sequences, one located downstream of NCS and the other located downstream of optMAO.
  • T7p T7 promoter
  • T7t T7 terminator
  • Kan r kanamycin resistance gene
  • Amp r ampicillin resistance gene
  • Cm r chloramphenicol resistance gene.
  • Change over time of (S) -reticuline production in a medium containing glycerol as a carbon source left axis, solid line).
  • m / z 330 indicates a peak of (S) -reticuline.
  • pET-21d contains the NCS, RsTYR, DODC and optMAO genes. Both genes are controlled by the T7 promoter.
  • NCS-RsTYR-DODC-optMAO / pET-21d has two T7 terminator sequences, one located downstream of NCS and the other located downstream of optMAO.
  • RsTYR, TYR derived from Ralstonia solanacearum T7p, T7 promoter
  • T7t, T7 terminator Amp r , ampicillin resistance gene.
  • the present invention relates to an isoquinoline alkaloid non-producing cell having a metabolic pathway for producing L-tyrosine, tyrosinase (hereinafter also referred to as TYR) and its adapter protein, L-DOPA-specific decarboxylase (hereinafter also referred to as DODC), Monoamine oxidase (MAO), norcoclaurine synthase (NCS), norcoclaurine 6-O-methyltransferase (6OMT), coclaurine-N-methyltransferase (CNMT) and 3'-hydroxy-N-methylcoclaurine-4 ' -Tyrosinase (TYR) and its adapter protein, L-DOPA-specific decarboxylase, into which at least one gene encoding at least one protein selected from the group consisting of -O-methyltransferase (4'OMT) is introduced (DODC), monoamine oxidase (MAO), norcoclaurine synthase (NCS), Re
  • a recombinant host cell that expresses a specific enzyme is cultured in a medium usually used for culturing microorganisms, so that it can be obtained in vivo from an inexpensive and readily available substrate such as glucose and glycerol (S).
  • S glucose and glycerol
  • -Reticuline can be produced efficiently.
  • the reticuline obtained by the method of the present invention is substantially free of (R) -reticuline.
  • the recombinant host cell used in the method of the present invention preferably has no tyrR gene or lacks its function. Since the product of tyrR gene has a function of suppressing the expression of genes involved in the biosynthesis of aromatic amino acids, for example, by using a host cell that does not have tyrR gene or by losing the function of tyrR gene, Inhibition of ⁇ L-tyrosine biosynthesis, which is important in the biosynthetic pathway reconstructed in the method of the present invention, can be released.
  • the recombinant host cell used in the method of the present invention is a feedback inhibitor-resistant 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (hereinafter also referred to as fbr-DAHPS) and / or feedback inhibitor-resistant chorismate. It preferably expresses mutase / prephenate dehydrogenase (hereinafter also referred to as fbr-CM / PDH). By expressing such an enzyme, the production efficiency of L-tyrosine is improved in the recombinant host cell, and the yield of (S) -reticuline can be increased.
  • fbr-DAHPS feedback inhibitor-resistant 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase
  • fbr-CM / PDH feedback inhibitor-resistant chorismate
  • the recombinant host cell used in the method of the present invention is preferably one that overexpresses transketolase (hereinafter also referred to as TKT) and / or phosphoenolpyruvate synthetase (hereinafter also referred to as PEPS).
  • TKT transketolase
  • PEPS phosphoenolpyruvate synthetase
  • Overexpression of such an enzyme increases the amount of erythrose-4-phosphate (E4P) and / or phosphoenolpyruvate (PEP) that is the starting material of the shikimate pathway in the recombinant host cell, producing L-tyrosine Through increasing amounts, the yield of (S) -reticuline can be increased.
  • E4P erythrose-4-phosphate
  • PEP phosphoenolpyruvate
  • the present invention relates to feedback inhibition resistant 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (fbr-DAHPS), feedback, and non-isoquinoline alkaloid-producing cells having a metabolic pathway that produces L-tyrosine.
  • fbr-DAHPS 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase
  • Inhibition resistant chorismate mutase / prephenate dehydrogenase fbr-CM / PDH
  • transketolase TKT
  • phosphoenolpyruvate synthetase PEPS
  • TERT tyrosinase
  • DODC L-DOPA specific de Carboxylase
  • MAO monoamine oxidase
  • NCS norcoclaurine synthase
  • 6OMT norcoclaurine 6-O-methyltransferase
  • coclaurin-N-methyltransferase CNMT
  • 3'-hydroxy-N-methyl At least selected from the group consisting of coclaurin-4'-O-methyltransferase (4'OMT)
  • a recombinant host cell that is deficient in function and the recombinant host cell is selected from the group consisting of glucose, fructose, galactose, sucrose, lactose and maltose as a carbon source. Culturing in a medium containing sugar and / or glycerol; There is further provided a method for producing (S) -reticuline, comprising:
  • the present invention provides an isoquinoline alkaloid non-producing cell having a metabolic pathway for producing L-tyrosine, fbr-DAHPS, fbr-CM / PDH, TYR and its adapter protein, DODC, MAO, NCS, 6OMT, CNMT and A recombinant host cell into which at least one gene encoding at least one protein selected from the group consisting of 4'OMT is introduced and into which genes encoding TKT and PEPS are introduced, fbr-DAHPS Expresses fbr-CM / PDH, TYR and its adapter proteins, DODC, MAO, NCS, 6OMT, CNMT and 4'OMT, overexpresses TKT and PEPS, and does not have tyrR gene or its function Providing a defective recombinant host cell, and using the recombinant host cell as a carbon source with glucose, fructose, galactose, sucrose, lactose and
  • isoquinoline alkaloid is produced in vivo from an inexpensive and readily available substrate by further introducing into a recombinant host cell a gene encoding an isoquinoline alkaloid biosynthetic enzyme that acts using reticuline as a starting material. It is also possible to do.
  • the present invention acts on isoquinoline alkaloid non-producing cells having a metabolic pathway that produces L-tyrosine, starting from TYR and its adapter proteins, DODC, MAO, NCS, 6OMT, CNMT, 4'OMT and reticuline.
  • TYR and its adapter protein DODC, MAO, NCS, 6OMT, CNMT, 4'OMT, and at least one gene encoding at least one protein selected from the group consisting of isoquinoline alkaloid biosynthetic enzymes
  • One or more selected sugars and Step of culturing in a medium containing / or glycerol, A method for producing isoquinoline alkaloids is provided.
  • isoquinoline alkaloid biosynthetic enzymes that act using reticuline as a starting material include colituberin synthase (hereinafter also referred to as CYP80G2) and berberine cross-linking enzyme (hereinafter also referred to as BBE), and by expressing CYP80G2, Then, magnoflorin can be synthesized, and sverrelin, which is a protoberberine alkaloid, can be synthesized by expressing BBE (FIG. 5).
  • CYP80G2 When CYP80G2 is expressed, it is preferable to express NADPH-cytochrome P450 reductase together.
  • the present invention also provides that isoquinoline alkaloid non-producing cells having a metabolic pathway for producing L-tyrosine are selected from the group consisting of TYR and its adapter proteins, DODC, MAO, NCS, 6OMT, CNMT and 4'OMT.
  • the recombinant host cell of the present invention preferably has one or more of the following properties (i) to (iii): (i) not having the tyrR gene or lacking its function; (ii) express fbr-DAHPS and / or fbr-CM / PDH, (iii) Overexpressing TKT and / or PEPS.
  • properties i) to (iii): (i) not having the tyrR gene or lacking its function; (ii) express fbr-DAHPS and / or fbr-CM / PDH, (iii) Overexpressing TKT and / or PEPS.
  • the present invention relates to isoquinoline alkaloid non-producing cells having a metabolic pathway that produces L-tyrosine, fbr-DAHPS, fbr-CM / PDH, TKT, PEPS, TYR and its adapter proteins, DODC, MAO, NCS, 6OMT.
  • a recombinant host cell into which at least one gene encoding at least one protein selected from the group consisting of CNMT and 4'OMT is introduced comprising fbr-DAHPS, fbr-CM / PDH, TKT, PEPS Further provided are recombinant host cells that express TYR and its adapter proteins, DODC, MAO, NCS, 6OMT, CNMT and 4'OMT, and lack the tyrR gene or lack its function.
  • the present invention relates to non-isoquinoline alkaloid-producing cells having a metabolic pathway that produces L-tyrosine, fbr-DAHPS, fbr-CM / PDH, TYR and its adapter proteins, DODC, MAO, NCS, 6OMT, CNMT and A recombinant host cell into which at least one gene encoding at least one protein selected from the group consisting of 4'OMT is introduced, and into which a gene encoding TKT and PEPS is introduced, fbr-DAHPS Fbr-CM / PDH, TYR and its adapter proteins, DODC, MAO, NCS, 6OMT, CNMTCN and 4'OMT are expressed, TKT and PEPS ⁇ ⁇ are overexpressed, and no tyrR gene is present or its function A defective recombinant host cell is provided.
  • TYR and its adapter protein in place of the above “TYR and its adapter protein”, a TYR that does not require an adapter protein to exhibit its activity, for example, TYR derived from Ralstonia solanacearum can also be used. Accordingly, the present invention relates to isoquinoline alkaloid non-producing cells having a metabolic pathway for producing L-tyrosine, tyrosinase derived from Ralstonia solanacearum (hereinafter also referred to as RsTYR), DODC, MAO, NCS, 6OMT, CNMT and 4′OMT.
  • RsTYR isoquinoline alkaloid non-producing cells having a metabolic pathway for producing L-tyrosine, tyrosinase derived from Ralstonia solanacearum
  • TYR from Ralstonia solanacearum is used instead of “TYR and its adapter protein” in the various methods of producing (S) -reticuline of the invention described herein and in recombinant host cells.
  • RsTYR Ralstonia solanacearum
  • RsTYR means TYR derived from Ralstonia solanacearum, but when it is simply expressed as tyrosinase (TYR), all TYRs are included in the term regardless of its origin. To do.
  • the biosynthetic pathway reconstructed in the method of the present invention synthesizes 3,4-DHPAA from dopamine by using MAO, and couples 3,4-DHPAA and dopamine through the action of NCS.
  • the aromatic L-amino acid decarboxylase when the aromatic L-amino acid decarboxylase is present in the biosynthetic pathway reconstructed in the method of the present invention, the aromatic L-amino acid is decarboxylated by the action of the aromatic L-amino acid decarboxylase, and the aromatic L-amino acid decarboxylase is aromatic.
  • An amine is formed, which is not preferable because it reacts with MAO.
  • L-tyrosine when L-tyrosine is decarboxylated, tyramine is produced, and MAO has a higher selectivity for tyramine than dopamine (ie, it reacts preferentially with tyramine), so the method of the present invention is not intended. HPAA will be generated.
  • the 4-HPAA is coupled with dopamine by the action of NCS to produce (S) -norcoclaurine.
  • NCS NCS
  • CYP80B in addition to 6OMT, CNMT and 4′OMT (FIG. 4), and the CYP80B is cytochrome P450. Because it is an enzyme, it often does not function well in microorganisms.
  • L-DOPA-specific decarboxylase (DODC) is introduced into a host cell and expressed.
  • the method of the present invention avoids the use of an aromatic L-amino acid decarboxylase that decarboxylates L-tyrosine in the reconstruction of (i) benzylisoquinoline alkaloid biosynthetic pathway, instead of (ii ) Tyrosinase (TYR) or tyrosinase (TYR) and its adapter protein are expressed to create a conversion pathway from L-tyrosine to L-DOPA, and (iii) L-DOPA-specific decarboxylase (DODC) is expressed It is characterized by creating a conversion path from L-DOPA to dopamine.
  • the recombinant host cell of the present invention and the recombinant host cell used in the method of the present invention are those into which a gene encoding an aromatic L-amino acid decarboxylase other than L-DOPA-specific decarboxylase has not been introduced. It is preferable.
  • tyramine is not generated from L-tyrosine by the enzyme encoded by the gene to be introduced into the host cell, while dopamine is produced with high efficiency by expression of TYR or TYR and its adapter protein and DODC.
  • the As a result it is possible to efficiently flow metabolic flow to the pathway leading to (S) -reticuline through the production of (S) -norlaudanosoline by coupling of dopamine and 3,4-DHPAA ( FIG. 1).
  • L-DOPA is synthesized mainly from tyrosine hydroxylase (TH, EC 1.14.16.2) from L-tyrosine, and such a reaction requires tetrahydrobiopterin (BH4) as a cofactor.
  • BH4 tetrahydrobiopterin
  • E. coli cells that are preferably used in the method of the present invention cannot synthesize the tetrahydrobiopterin. Therefore, in the method of the present invention, bacterial tyrosinase (TYR, EC 1.14.18.1) does not require a cofactor other than copper and an adapter protein (for example, ORF378) for conversion of L-tyrosine to L-DOPA. ) Is preferably used.
  • TYR derived from Ralstonia solanacearum that does not require an adapter protein can also be suitably used.
  • Recombinant host cells according to the present invention usually include isoquinoline alkaloid non-producing cells having a metabolic pathway that produces L-tyrosine, TYR or TYR and its adapter proteins, DODC, MAO, NCS, 6OMT, CNMT and 4 ′. It can be obtained by introducing at least one gene encoding at least one protein selected from the group consisting of OMT.
  • an isoquinoline alkaloid non-producing cell having a metabolic pathway that produces L-tyrosine is selected from the group consisting of “TYR or TYR and its adapter protein, DODC, MAO, NCS, 6OMT, CNMT and 4'OMT” If you already have one or more genes encoding a protein, you can introduce “TYR or TYR and its adapter protein, DODC, MAO by introducing all the other genes in the group into the cell. , NCS, 6OMT, CNMT and 4′OMT ”can be obtained.
  • non-isoquinoline alkaloid-producing cells that have metabolic pathways that produce L-tyrosine have any of the genes encoding TYR or TYR and its adapter proteins, DODC, MAO, NCS, 6OMT, CNMT and 4'OMT. If not, all of the genes encoding “TYR or TYR and its adapter protein, DODC, MAO, NCS, 6OMT, CNMT and 4′OMT” are introduced into the cells, so that the recombinant of the present invention Host cells can be obtained.
  • the host cell into which the gene is introduced to obtain the recombinant host cell according to the present invention is not particularly limited as long as it is an isoquinoline alkaloid non-producing cell having a metabolic pathway for producing L-tyrosine, but more efficient (S For the production of) -reticuline, a host cell having the ability to produce L-tyrosine in large quantities is preferred.
  • the ability to produce L-tyrosine in large quantities can typically be conferred to a host cell by one or more methods selected from the following (1) to (3): (1) Loss of function of tyrR gene in host cells or suppression of expression of tyrR gene (this loss of function includes complete loss of function and partial loss of function); (2) introduction of one or more genes encoding fbr-DAHPS and / or fbr-CM / PDH into a host cell and expression in the host cell; (3) Introduction of one or more genes encoding TKT and / or PEPS into the host cell and overexpression in the host cell.
  • the function of the tyrR gene can be lost by means known in the art, for example, knock-in / knock-out using homologous recombination, gene disruption using transfer of a transfer factor such as a transposon, and the like.
  • the expression of the tyrR gene can be suppressed by means known in the art, for example, cosuppression, antisense method, RNAi and the like.
  • the recombinant host cell of the present invention which does not have the tyrR gene or lacks its function can be obtained, for example, by the following method: (1) introducing a necessary gene such as TYR into a host cell that does not have the tyrR gene or lacks its function; (2) After losing the function of the tyrR gene possessed by the host cell, introducing a necessary gene such as TYR into the host cell, or (3) After introducing the necessary gene such as TYR into the host cell, Losing the function of the tyrR gene possessed by the host cell.
  • Recombinant host cells of the present invention that express "fbr-DAHPS, fbr-CM / PDH, TKT, PEPS, TYR or TYR and its adapter proteins, DODC, MAO, NCS, 6OMT, CNMT and 4'OMT" It can be obtained by the following method: (1) introducing all these genes into host cells, or (2) "fbr-DAHPS, fbr-CM / PDH, TKT, PEPS, TYR or TYR and their adapter proteins, DODC, MAO, NCS, 6OMT, All other genes included in the group are introduced into a host cell having one or more genes encoding a protein selected from the group consisting of CNMT and 4′OMT ”.
  • the recombinant host cell of the present invention may be transformed into, for example, ⁇ fbr-DAHPS, fbr-CM / PDH, TKT, PEPS, TYR or TYR and its adapter protein, It can be obtained by introducing all of the genes encoding “DODC, MAO, NCS, 6OMT, CNMT and 4′OMT”.
  • the recombinant host cell according to the present invention is obtained not only by the above method but also a mutant strain that has acquired the ability to produce a large amount of L-tyrosine by a naturally occurring mutation or a known mutagenesis method. Therefore, it may be used as a host cell into which a gene is introduced.
  • the host cell into which the gene is introduced in the method of the present invention is not particularly limited as long as it is an isoquinoline alkaloid non-producing cell, and examples thereof include Escherichia coli, yeast, Bacillus subtilis, and filamentous fungi.
  • the gene When introducing a gene into a host cell, the gene may be directly introduced, but it is preferable to introduce a vector into which the gene is incorporated into the host. All transgenes may be incorporated into the same vector, or may be incorporated into two or more separate vectors.
  • a vector constructed for gene recombination from a plasmid or phage capable of autonomous replication in a host cell is suitable.
  • the vector preferably contains a replication origin suitable for the host cell to be introduced, a selectable marker, an expression control sequence such as a promoter, and a terminator sequence.
  • the plasmid vector include a pET vector system, a pQE vector system, and a pCold vector system when expressed in E. coli, and a pYES2 vector system and a pYEX vector system when expressed in yeast.
  • selectable markers include antibiotic resistance genes such as ampicillin resistance gene, kanamycin resistance gene, and streptomycin resistance gene.
  • a vector for expressing the transgene incorporated therein preferably contains an expression control sequence.
  • An expression control sequence when appropriately linked to a DNA sequence, controls the expression of a gene comprising the DNA sequence in a host cell, that is, induces and / or promotes transcription of the DNA sequence to RNA. Or a sequence that can be suppressed.
  • the expression control sequence includes at least a promoter.
  • the promoter may be a constitutive promoter or an inducible promoter.
  • the expression vector preferably contains a transcription termination signal, that is, a terminator sequence.
  • the expression vector used in the present invention can be prepared by adding an appropriate restriction enzyme recognition site to the end of the above gene by a conventional method.
  • a conventionally known method can be used, and examples thereof include a calcium chloride method, an electroporation method, and a heat shock method.
  • the culture conditions of the recombinant host cell are such that the recombinant host cell grows well and the desired group of proteins such as “TYR or TYR and its adapter proteins, DODC, MAO, NCS, 6OMT, CNMT and 4 'OMT' and 'fbr-DAHPS, fbr-CM / PDH, TKT, PEPS, TYR or TYR and their adapter proteins, DODC, MAO, NCS, 6OMT, CNMT and 4'OMT' are all expressed and their functions or There is no particular limitation as long as the enzyme activity is exhibited.
  • the culture conditions may be appropriately selected in consideration of the nutritional physiological properties of the host, and are usually performed in liquid culture.
  • the carbon source of the medium used for culturing the recombinant host cell is not particularly limited as long as it is a substance that can be used by the host cell, and examples thereof include sugar and glycerol, and glycerol is particularly preferable.
  • sugars include monosaccharides such as glucose, fructose, galactose, and disaccharides such as sucrose, lactose, maltose, etc. Among them, glucose is preferred.
  • the production efficiency of (S) -reticuline is higher than when glucose is used.
  • the nitrogen source include ammonium sulfate and casamino acid.
  • salts, specific amino acids, specific vitamins and the like can be used as desired.
  • the medium for culturing E. coli includes LB medium, 2 ⁇ YT medium, and M9 minimal medium
  • the medium for culturing yeast includes SC medium, SD medium, and YPD medium.
  • the culture temperature can be appropriately changed as long as the host cell grows, expresses the target enzyme, and exhibits its activity.
  • the culture conditions of temperature 25 ° C, 80 hours, pH 7.0 ⁇ ⁇ ⁇ are used. be able to.
  • culture conditions of a temperature of 30 ° C., 60 hours, and pH of 5.8 can be used.
  • reaction product and the target isoquinoline alkaloid preparation can be identified by subjecting them to LC-MS and comparing the obtained spectra. It can also be confirmed by comparison of the reaction product with the target isoquinoline alkaloid preparation by NMR analysis.
  • expressing means that a nucleic acid molecule constituting the gene is transcribed to at least an RNA molecule.
  • the gene is constituted.
  • a nucleic acid molecule is transcribed into a RNA molecule, and the RNA molecule is translated into a polypeptide.
  • the gene expression level can be confirmed by methods known in the art, such as Northern blot, quantitative PCR, and the like.
  • expressing means that transcription from the nucleic acid molecule encoding the polypeptide of the enzyme to the RNA molecule and translation from the RNA molecule to the polypeptide are normally performed and have an activity. It means that an enzyme protein is produced and is present inside or outside the cell.
  • the expression level of the enzyme can be confirmed by assaying the enzyme activity. That is, it can be confirmed by assaying the conversion of the target enzyme from the substrate to the product. It can also be confirmed by detecting and quantifying the enzyme protein using a known method such as Western blotting or ELISA.
  • “overexpression” of a specific gene or enzyme means that when the host cell has an endogenous gene or enzyme corresponding to the specific gene or enzyme, the endogenous gene or enzyme A gene or enzyme that exceeds the expression level of the endogenous gene or enzyme in the normal state of the host cell by combining the expression level of the enzyme with the expression level of the introduced specific gene or the enzyme encoded thereby. Achieving an expression level in the host cell.
  • “overexpression” of the specific gene or enzyme means that the introduced specific gene or enzyme For the encoded enzyme, achieving a level of expression in the host cell that can be detected by conventional detection methods.
  • a biosynthetic pathway refers to the 1 necessary to biosynthesize the compound in a host cell that does not naturally produce the desired compound so that the desired compound is obtained. Alternatively, expression of one or more enzymes that catalyze a plurality of reactions to create a metabolic pathway leading to the compound. Thus, a biosynthetic pathway that is “reconstructed” into a host cell is one that the cell does not naturally possess.
  • the desired compound is an isoquinoline alkaloid, particularly (S) -reticuline, and expression of the required enzyme is achieved by introducing a gene encoding the enzyme into a host cell. be able to.
  • the reticuline obtained by the method of the present invention is “substantially free of (R) -reticuline” means that the enantiomeric excess of (S) -reticuline relative to (R) -reticuline is at least 80% or more. , Preferably 90% or more, most preferably 100%.
  • the tyrosinase (TYR) used in the present invention is not particularly limited as long as it has an enzyme activity that catalyzes the reaction of hydroxylating the 3-position of the benzene ring of L-tyrosine and converting it to L-DOPA.
  • the origin of TYR cocoons is not particularly limited, but is preferably derived from microorganisms, and examples include those derived from Streptomyces castaneoglobisporus.
  • TYR derived from S. castaneoglobisporus encoded by the nucleotide sequence shown in SEQ ID NO: 6 can be preferably used.
  • Ralstonia solanacearum-derived TYR can be used as the TYR.
  • RsTYR encoded by the nucleotide sequence shown in SEQ ID NO: 36 or 37 can be preferably used.
  • RsTYR does not require an adapter protein to show its enzymatic activity. Therefore, when RsTYR is used as the TYR, it is not necessary to express the adapter protein described later.
  • TYR usually has the ability to convert L-DOPA or dopamine into its quinone derivative (o-diphenolase activity), but RsTYR encoded by the nucleotide sequence shown in SEQ ID NO: 36 or 37 has such o-diphenolase activity.
  • the tyrosinase (TYR) adapter protein used in the present invention is a protein that assists in transporting divalent copper ions (Cu (II)) to the catalytic center of TYR.
  • TYR tyrosinase
  • an adapter protein is required in order for the TYR to show complete catalytic activity.
  • the TYR gene and ORF378 are arranged in series to constitute the melanin synthase gene operon, and TYR is active when both TYR and ORF378 proteins are expressed (K. Ikeda et al., Appl.
  • TYR adapter proteins include ORF378 protein derived from S. castaneoglobisporus and ORF438 protein derived from Streptomyces antibioticus.
  • ORF378 protein derived from ⁇ S. castaneoglobisporus derived from the nucleotide sequence shown in SEQ ID NO: 7 ORF378 protein can be preferably used.
  • ORF378 As an example, the term ⁇ ORF378 '' means a gene or open reading frame encoding ORF378 protein, which is an adapter protein of TYR, and
  • the term “adapter protein ORF378” or “ORF378 protein” is intended to mean a protein encoded by ORF378.
  • L-DOPA-specific decarboxylase used in the present invention has an enzyme activity that catalyzes a reaction of eliminating a carboxyl group from L-DOPA and converting it to dopamine, and other fragrances other than L-DOPA.
  • the origin of DODC is not particularly limited, and examples thereof include microorganisms such as those derived from Pseudomonas putida. In the method of the present invention, for example, DODC derived from P. putida encoded by the nucleotide sequence shown in SEQ ID NO: 8 can be preferably used.
  • the monoamine oxidase (MAO) used in the present invention is not particularly limited as long as it has an enzyme activity that catalyzes a reaction of converting dopamine to 3,4-DHPAA.
  • Examples of the origin of MAO include microorganisms such as Micrococcus luteus, Escherichia coli, Arthrobacter aurescens, Klebsiella aerogenes, but those derived from Micrococcus luteus are preferably used.
  • MAO derived from M. luteus encoded by the nucleotide sequence shown in SEQ ID NO: 9 can be preferably used.
  • Norcoclaurine synthase (NCS) used in the present invention is a reaction for obtaining 3'-hydroxynorcoclaurine (hereinafter also referred to as norlaudanosoline) from dopamine and 3,4-DHPAA, norcoclaurine 6-O- Methyltransferase (6OMT) produces 3'-hydroxycoclaurine from 3'-hydroxynorcoclaurine, and coclaurin-N-methyltransferase (CNMT) converts 3'-hydroxycoclaurine to 3'-hydroxy-N-methylcocrine.
  • norcoclaurine 6-O- Methyltransferase (6OMT) produces 3'-hydroxycoclaurine from 3'-hydroxynorcoclaurine
  • coclaurin-N-methyltransferase (CNMT) converts 3'-hydroxycoclaurine to 3'-hydroxy-N-methylcocrine.
  • reaction to obtain laurin 3'-hydroxy-N-methylcoclaurine-4'-O-methyltransferase (4'OMT) is an enzyme activity that catalyzes the reaction to obtain reticuline from 3'-hydroxy-N-methylcoclaurine If it has, it will not specifically limit.
  • NCS, 6OMT, CNMTCN and 4'OMT ⁇ ⁇ ⁇ are not particularly limited, but those derived from isoquinoline alkaloid-producing plants are preferred.
  • isoquinoline alkaloid-producing plants include Poppyaceae plants such as red-bellied shrimp, poppy and engosac, Barberry plant such as barberry, Citrus plant such as yellow butterfly, magnoliaceae plant such as beetle, scorpionaceae plant such as Otsuo-rafuji, and oren
  • isoquinoline alkaloid-producing plants such as buttercups such as (Coptis p japonica), and preferably olene.
  • NCS, 6OMT, CNMT and 4'OMT derived from auren all encoded by the nucleotide sequences shown in SEQ ID NOs: 11, 12, 13, and 14, can be preferably used.
  • the isoquinoline alkaloid biosynthetic enzyme that acts as a starting material for reticuline used in the present invention is one or a plurality of enzymes that constitute the pathway for obtaining the desired isoquinoline alkaloid using reticuline as a starting material, and catalyzes the reaction of each pathway. There is no particular limitation as long as it has enzyme activity.
  • the isoquinoline alkaloid biosynthetic enzyme that acts using reticuline as a starting material is not particularly limited, but is preferably derived from an isoquinoline alkaloid-producing plant.
  • NADPH-cytochrome P450 reductase which is preferably used together with ⁇ ⁇ ⁇ ⁇ CYP80G2 (Colituberin synthase), which is a kind of cytochrome P450 as an isoquinoline alkaloid biosynthetic enzyme, is not particularly limited. Mammals.
  • an enzyme that catalyzes the reaction from reticuline to colituberin and an enzyme that catalyzes the reaction from colituberin to magnoflorin may be used, and have an enzyme activity that catalyzes such a reaction. If it is, it will not specifically limit.
  • ⁇ CYP80G2 encoded by the nucleotide sequence shown in SEQ ID NO: 15 can be preferably used as an enzyme that catalyzes the reaction from reticuline to colituberin, and together with this, P450 reductase encoded by the nucleotide sequence shown in SEQ ID NO: 16 is used.
  • P450 reductase encoded by the nucleotide sequence shown in SEQ ID NO: 16 is used.
  • CNMT encoded by the nucleotide sequence shown in SEQ ID NO: 13 can be preferably used.
  • an enzyme that catalyzes the reaction from reticuline to scourelin when producing scourelin, an enzyme that catalyzes the reaction from reticuline to scourelin may be used, and it is not particularly limited as long as it has an enzyme activity that catalyzes such a reaction.
  • BBE encoded by the nucleotide sequence shown in SEQ ID NO: 17 can be preferably used.
  • an enzyme that constitutes a pathway from reticuline to berberine via scourelin may be used, and it is not particularly limited as long as it has an enzyme activity that catalyzes the reaction at each stage.
  • BBE SMT (scourelin-9-O-methyltransferase), CYP719A1 (canazine synthase) and THBO (tetrahydroberberine oxidase) may be used.
  • SMT scourelin-9-O-methyltransferase
  • CYP719A1 canazine synthase
  • THBO tetrahydroberberine oxidase
  • the tyrR gene that can be used to modify host cells in the present invention encodes a DNA-binding transcriptional regulator having a function of regulating the expression of a plurality of genes involved in aromatic amino acid biosynthesis and transport.
  • the transcriptional regulator encoded by the tyrR gene encodes 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS), which functions in the so-called shikimate pathway that biosynthesizes L-tyrosine Suppresses the expression of tyrA gene encoding aroG gene and chorismate mutase / prephenate dehydrogenase (CM / PDH) (AJ Pittard et al., Mol. Microbiol.
  • the tyrR gene that can be used to modify the host cell in the present invention is not particularly limited as long as it has a function of suppressing the expression of aroG gene and / or tyrA gene.
  • Whether or not the host cell used in the present invention has the tyrR gene is determined based on the known tyrR gene sequence information, for example, the tyrR gene sequence information of E. coli K-12 DH10B strain (SEQ ID NO: 1, EMBL accession number ACB02543, KEGG registration number). Based on ECDH10B_1442), it can be estimated by performing a sequence homology search on the genome sequence or the like of the host cell. When the presence of a homologous gene (so-called homolog) is estimated, the presence can be confirmed by techniques such as PCR amplification and sequencing performed based on the sequence information of the homologous gene. Alternatively, a nucleic acid molecule of the tyrR gene can be directly obtained by hybridization with a nucleic acid molecule of the tyrR gene having a known sequence.
  • the homology search can be performed using a known means such as the BLAST algorithm or FASTA algorithm.
  • fbr-DAHPS that can be used to modify host cells is derived from erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP) from 3-deoxy-D-arabino-heptulosonate-
  • E4P erythrose-4-phosphate
  • PEP phosphoenolpyruvate
  • 3-deoxy-D-arabino-heptulosonate- There is no particular limitation as long as it has an enzyme activity that catalyzes a reaction for producing 7-phosphate (DAHP) and is not subject to feedback inhibition by phenylalanine.
  • fbr-DAHPS has an enzyme activity in the presence of phenylalanine that is equal to or greater than that in the absence thereof.
  • fbr-DAHPS is not particularly limited, but is preferably derived from microorganisms, more preferably derived from E. coli. In the present invention, for example, ⁇ fbr-DAHPS derived from E. coli encoded by the nucleotide sequence shown in SEQ ID NO: 2 can be preferably used.
  • fbr-CM / PDH that can be used to modify host cells catalyzes the reaction of producing prefenic acid from chorismic acid and the reaction of producing 4-hydroxyphenylpyruvic acid (HPP) from prefenic acid. If it has the enzyme activity which does not receive the feedback inhibition by tyrosine, it will not specifically limit.
  • “not subject to feedback inhibition by tyrosine (being resistant to feedback inhibition)” means that tyrosine does not inhibit CM and / or PDH enzyme activity at all, or the degree of inhibition of enzyme activity by tyrosine is wild type This means that it is significantly smaller than CM / PDH.
  • fbr-CM / PDH the enzyme activity in the presence of tyrosine is preferably equal to or greater than the enzyme activity in the absence of tyrosine.
  • fbr-CM / PDH is not particularly limited, but is preferably derived from microorganisms, and more preferably derived from E. coli.
  • E. coli-derived fbr-CM / PDH encoded by the nucleotide sequence shown in SEQ ID NO: 3 can be preferably used.
  • TKT is not particularly limited, but is preferably derived from microorganisms, more preferably derived from E. coli. In the present invention, for example, TKT derived from E. coli encoded by the nucleotide sequence shown in SEQ ID NO: 4 can be preferably used.
  • the “PEPS” that can be used to modify the host cell is not particularly limited as long as it has an enzyme activity that catalyzes a reaction for producing phosphoenolpyruvate (PEP) from pyruvate.
  • PEPS is not particularly limited, but is preferably derived from microorganisms, and more preferably derived from E. coli.
  • E. coli-derived PEPS encoded by the nucleotide sequence shown in SEQ ID NO: 5 can be preferably used.
  • the enzymes used in the present invention include, but are not limited to, the following proteins (a) or (b): (a) from the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 36 or 37, 7, 8, 9 or 10, 11, 12, 13, 14, 15, 16 or 17
  • a protein comprising: (b) in the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 36 or 37, 7, 8, 9 or 10, 11, 12, 13, 14, 15, 16 or 17 It consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added, and fbr-DAHPS, fbr-CM / PDH, TKT, PEPS, TYR, ORF378, DODC, MAO, NCS, 6OMT, CNMT , 4'OMT, CYP80G2, P450 reductase or protein having BBE enzyme activity.
  • Examples of the protein used in the present invention also include the following protein (b ′).
  • ⁇ ⁇ ⁇ ⁇ ⁇ protein is ⁇ fbr-DAHPS, fbr-CM / PDH, TKT, PEPS, TYR, ORF378, DODC, MAO, NCS, 6OMT, CNMT, 4'OMT, CYP80G2, P450 reductase or 450BBE It is a protein in which an amino acid mutation (deletion, substitution, addition) has occurred to such an extent that the function of the protein (a) “having enzyme activity” is not lost.
  • Such mutations include artificial mutations in addition to those occurring in nature. Examples of means for causing artificial mutation include, but are not limited to, site-directed mutagenesis (Nucleic Acids Res. 10, 6487-6500, 1982).
  • the number of amino acids mutated (deleted, substituted, added) is not limited as long as the enzyme activity of the protein (a) is not lost, but is preferably within 50 amino acids, more preferably within 30 amino acids. It is.
  • the protein of (b ') is also the enzyme activity of fbr-DAHPS, fbr-CM / PDH, TKT, PEPS, TYR, ORF378, DODC, MAO, NCS, 6OMT, CNMT, 4'OMT, CYP80G2, P450 reductase or BBE
  • the homology is preferably 70% or more, particularly preferably 90% or more.
  • homology means the degree of sequence similarity between two polypeptides or polynucleotides, and is in an optimum state over the region of the amino acid sequence or base sequence to be compared (maximum sequence match). Determined by comparing two sequences aligned to the (state). The homology value (%) determines the number of sites where identical amino acids or bases are present in both aligned (amino acid or base) sequences, and then the number of those sites is compared with the amino acids in the sequence region to be compared. Alternatively, it is calculated by dividing by the total number of bases and multiplying the obtained numerical value by 100.
  • Examples of algorithms for obtaining optimal alignment and homology include various algorithms (for example, BLAST algorithm, FASTA algorithm, etc.) that are usually 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 base sequence homology is determined using software such as BLASTN and FASTA.
  • the protein has the enzymatic activity of fbr-DAHPS, fbr-CM / PDH, TKT, PEPS, TYR, ORF378, DODC, MAO, NCS, 6OMT, CNMT, 4'OMT, CYP80G2, P450 reductase or BBE It can be determined by adding each reaction substrate to the protein preparation and examining whether or not a reaction product of each enzyme has been generated.
  • erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP) were added to produce 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) -Chorismic acid was added for CM / PDH and 4-hydroxyphenylpyruvate (HPP) was produced, or ribose-5-phosphate (R5P) and xylulose-5-phosphate (Xu5P) were used for TKT5.
  • GAP glyceraldehyde-3-phosphate
  • S7P sedheptulose-7-phosphate
  • PEPS pyruvic acid
  • PEP phosphoenolpyruvate
  • L-tyrosine is added for TYR (or TYR and ORF378)
  • L-DOPA is produced
  • L-DOPA is added for DODC
  • dopamine is produced
  • dopamine is added for MAO, 3
  • 4-DHPAA was produced or for NCS dopamine
  • 6OMT was added to 3'-hydroxynorcoclaurine
  • 3'-hydroxycocolaurin was formed, or CNMT was to 3 Check whether 3'-hydroxy-N-methylcoclaurine was formed by adding '-hydroxycoclaurin, or whether reticuline was formed by adding 3'-hydroxy-N-methylcoclaurine for 4'OMT
  • GAP glyceraldehyde-3-phosphate
  • S7P sedheptulose-7-phosphate
  • reticuline was added in the presence of them to determine whether or not colituberin was produced, and for BBE, reticuline was added to determine whether or not scourelin was produced. I can do it.
  • CNMT which catalyzes the reaction of magnoflorin from colituberin
  • CNMT which catalyzes the reaction of 3'-hydroxycoclaurine to 3'-hydroxy-N-methylcoclaurine
  • Whether or not to catalyze the reaction to florin can be determined by adding coritsuberin and examining whether or not magnoflorin has been produced.
  • fbr-DAHPS is resistant to feedback inhibition can be confirmed by measuring the enzyme activity in the presence and absence of phenylalanine and comparing with the enzyme activity of wild-type DAHPS under the same conditions.
  • Kikuchi Y. et al., Appl. Environ. Microbiol. 63, 761-762 (1997).
  • whether fbr-CM / PDHPD is resistant to feedback inhibition is measured by enzyme activity in the presence and absence of tyrosine and compared with the enzyme activity of wild-type CM / PDH under the same conditions.
  • Lutke-Eversloh, T. and Stephanopoulos, G., Appl. Environ. Microbiol. 71, 7224-7228 (2005) See, for example, Lutke-Eversloh, T. and Stephanopoulos, G., Appl. Environ. Microbiol. 71, 7224-7228 (2005)).
  • the reaction products were 3-deoxy-D-arabino-heptulosonic acid-7-phosphate (DAHP), 4-hydroxyphenylpyruvic acid (HPP), glyceraldehyde-3-phosphate (GAP), cedoheptulose-7- Phosphoric acid (S7P), phosphoenolpyruvate (PEP), L-DOPA, dopamine, 3,4-DHPAA, 3'-hydroxynorcoclaurine, 3'-hydroxycoclaurin, 3'-hydroxy-N-methylkoku Whether it is laurin, reticuline or the desired isoquinoline alkaloid (for example, magnoflorin, scourelin, berberine, etc.) can be confirmed by any means known to those skilled in the art.
  • DAHP 3-deoxy-D-arabino-heptulosonic acid-7-phosphate
  • HPP 4-hydroxyphenylpyruvic acid
  • GAP glyceraldehyde-3-phosphate
  • reaction product and 3-deoxy-D-arabino-heptulosonic acid-7-phosphate DAHP
  • 4-hydroxyphenylpyruvic acid HPP
  • GAP glyceraldehyde-3-phosphate
  • S7P Sedoheptulose-7-phosphate
  • PEP phosphoenolpyruvate
  • L-DOPA dopamine
  • 3,4-DHPAA 3'-hydroxynorcoclaurine
  • 3'-hydroxycoclaurine 3'-hydroxycoclaurine
  • 3'-hydroxy- Each sample of N-methylcoclaurine, reticuline, or the desired isoquinoline alkaloid can be identified by subjecting to LC-MS and comparing the resulting spectra. It can also be confirmed by comparison of the product with the corresponding sample by NMR analysis.
  • isoquinoline alkaloid biosynthesis that can be used in the present invention, starting from fbr-DAHPS, fbr-CM / PDH, TKT, PEPS, TYR, ORF378, DODC, MAO, NCS, 6OMT, CNMT, 4'OMT or reticuline
  • DODC DODC
  • MAO NCS
  • 6OMT 6OMT
  • CNMT 4'OMT
  • reticuline The gene encoding the enzyme will be described.
  • fbr-DAHPS fbr-DAHPS
  • fbr-CM / PDH TKT, PEPS, TYR, ORF378, DODC, MAO, NCS, 6OMT, CNMT, 4'OMT, or isoquinoline alkaloid biosynthetic enzymes preferably used in the present invention
  • P450 reductase or BBE for example, SEQ ID NO: 2, 3, 4, 5, 6 or 36 or 37, 7, 8, 9 or 10, 11, 12, 13, 14, Examples thereof include a gene consisting of the nucleotide sequence shown in 15, 16 or 17.
  • the gene used in the present invention is not limited to these, but is preferably a gene that is the following DNA (a) or (b): (a) DNA consisting of the nucleotide sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 36 or 37, 7, 8, 9 or 10, 11, 12, 13, 14, 15, 16 or 17; (b) hybridizes with a DNA comprising a nucleotide sequence complementary to the DNA comprising the nucleotide sequence of (a) under stringent conditions, and fbr-DAHPS, fbr-CM / PDH, TKT, PEPS, TYR, DNA encoding a protein having the enzymatic activity of ORF378, DODC, MAO, NCS, 60MT, CNMT, 4'OMT, CYP80G2, P450 reductase or BBE.
  • examples of the gene used in the present invention include the following genes (c): (c) 70% or more with respect to the nucleotide sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 36 or 37, 7, 8, 9 or 10, 11, 12, 13, 14, 15, 16 or 17 Consisting of a nucleotide sequence having a homology of preferably 80% or more, more preferably 90% or more, and still more preferably 95% or more, and fbr-DAHPS, fbr-CM / PDH, TKT, PEPS, TYR, ORF378, DNA encoding a protein having enzyme activity of DODC, MAO, NCS, 6OMT, CNMT, 4′OMT, CYP80G2, P450 reductase or BBE.
  • stringent conditions refer to conditions in which only specific hybridization occurs and non-specific hybridization does not occur. Such conditions are usually about 6M urea, 0.4% SDS, 0.5xSSC.
  • the DNA obtained by hybridization preferably has a high homology of 70% or more with the DNA comprising the nucleotide sequence (a), and preferably has a homology of 80% or more.
  • “homology” is as described above.
  • the protein encoded by the above gene is ⁇ fbr-DAHPS, fbr-CM / PDH, TKT, PEPS, TYR, ORF378, DODC, MAO, NCS, 6OMT, CNMT, 4'OMT, CYP80G2, P450 reductase or BBE enzyme activity
  • the method for confirming whether or not “has a protein” is as described above for the protein.
  • the gene can be obtained by PCR or hybridization techniques well known to those skilled in the art, or may be artificially synthesized using a DNA synthesizer or the like.
  • the sequence can be determined by a conventional method using a sequencer.
  • tyrA fbr and aroG fbr genes cloned into pUC57 were purchased from GenScript Inc.
  • tyrA fbr was adjacent to the recognition sites for NcoI and EcoRI restriction enzymes, and aroG fbr was linked to the T7 promoter.
  • an EcoRI site was located upstream of the T7 promoter, and a SacI site was located downstream of aroG fbr .
  • the tktA and ppsA genes were extracted from the genomic DNA of Escherichia coli K-12 MG1655 with the primer set of NdeI-tktA-F (SEQ ID NO: 18) and tktA-XhoI-R (SEQ ID NO: 19) and NdeI-ppsA-F (SEQ ID NO: 19), respectively. 20) and ppsA-XhoI-R (SEQ ID NO: 21) primer sets (Table 1).
  • tktA under the control of the T7 promoter was amplified from tktA / pET-41a using primers 5Sac-T7 (SEQ ID NO: 22) and 3NottktA (SEQ ID NO: 23) (Table 1). Then the tktA and T7 promoter, cloned into tyrA fbr -aroG fbr / pCOLADuet-1 of SacI-NotI site to obtain tyrA fbr -aroG fbr -tktA / pCOLADuet- 1.
  • ppsA / pCOLADuet-1 was digested with NotI and XhoI to obtain a fragment containing the ppsA gene and the T7 promoter. Then tyrA fbr -aroG fbr -tktA-ppsA / pCOLADuet-1 The resulting This fragment was cloned into tyrA fbr -aroG fbr -tktA / pCOLADuet- 1 of NotI-XhoI site (Fig. 8), and its sequence was verified.
  • NCS-ORF378-TYR-DODC-optMAO / pKK223-3 To reconstruct the synthesis route of norlaudanosoline, an expression vector containing genes encoding the following four enzymes was constructed: Streptomyces castaneoglobisporus tyrosinase ( TYR) (for the full activity of TYR, co-express TYR and its adapter protein ORF378), Pseudomonas putida L-DOPA-specific decarboxylase (DODC), Micrococcus luteus monoamine oxidase (MAO) and oren (Coptis japonica ) Norcoclaurine synthase (NCS).
  • TYR Streptomyces castaneoglobisporus tyrosinase
  • DODC Pseudomonas putida L-DOPA-specific decarboxylase
  • MAO Micrococcus luteus monoamine oxidase
  • oren
  • ⁇ ⁇ ⁇ MAO SEQ ID NO: 10, hereinafter referred to as optMAO
  • optMAO ⁇ ⁇ ⁇ MAO
  • the optMAO gene cloned into the pGS-21a plasmid was purchased from GenScript Inc., and the MAO gene contained in the NCS-MAO / pKK223-3 was replaced with the optMAO gene driven by the T7 promoter. .
  • the replacement is performed from the purchased pGS-21a plasmid containing the optMAO gene by PCR using primers 5Bam-T7 (SEQ ID NO: 24) and 3oMAO (SEQ ID NO: 25) (Table 1) BamHI- containing the optMAO gene and T7 promoter. This was accomplished by generating a HindIII fragment and incorporating the fragment into the BamHI-HindIII site of NCS-MAO / pKK223-3, resulting in NCS-optMAO / pKK223-3.
  • the gene for L-DOPA-specific decarboxylase (DODC; aromatic L-amino acid decarboxylase belonging to EC 4.1.1.28) of Pseudomonas putida KT2440 strain was extracted from the genomic DNA of the strain 5NdeDODC (SEQ ID NO: 26).
  • 3BamDODC SEQ ID NO: 27 (Table 1).
  • the PCR product was digested with NdeI-BamHI and then ligated to the NdeI-BamHI site of pET-3a (Novagen) to obtain DODC / pET-3a.
  • DODC is a decarboxylase showing the L-DOPA selectivity of greater than 10 3 times as compared with the other aromatic amino acids.
  • the DODC gene with the T7 promoter was amplified from the DODC / pET-3a using primers 5BamSacRVT7 (SEQ ID NO: 28) and 3BamDODC (SEQ ID NO: 27) (Table 1), and this was amplified with NCS-optMAO / pKK223-3 NCS-DODC-optMAO / pKK223-3 was obtained by cloning into the BamHI site.
  • Streptomyces castaneoglobisporus TYR gene (SEQ ID NO: 6) and ORF378 (SEQ ID NO: 7) cloned in series in pGS-21a and each independently driven by the T7 promoter were purchased from GenScript Inc. In this construct, the codon usage of both genes is optimized.
  • a PCR product (SacI-EcoRV fragment) containing the TYR gene and ORF378 was amplified using primers 5Sac-T7 (SEQ ID NO: 22) and 3pGSEcoRV (SEQ ID NO: 29) (Table 1).
  • NCS-DODC-optMAO / pKK223 The NCS-ORF378-TYR-DODC-optMAO / pKK223-3 was obtained by ligation to the SacI-EcoRV site located between -3NCS and DODC.
  • NCS-ORF378-TYR-DODC-optMAO / pET-21d A vector based on the pET-21d (Novagen) plasmid was made to compare production between host cells with plasmids of different backbones.
  • NCS-MAO / pKK223-3 a fragment containing the T7 promoter bound to NCS was amplified from NCS-MAO / pKK223-3 using primers 5BglII-T7 (SEQ ID NO: 30) and 3NCSBamSacRV (SEQ ID NO: 31) (Table 1). The fragment was then ligated to the BglII-BamHI site of pET-21d. The NCS-DODC-optMAO / pKK223-3 ⁇ ⁇ SacI-HindIII fragment containing the DODC and optMAO genes was ligated to the SacI-HindIII site of NCS / pET-21d to obtain NCS-DODC-optMAO / pET-21d .
  • NCS-ORF378-TYR-DODC-optMAO / pKK223-3 and NCS-ORF378-TYR-DODC-optMAO / pET-21d use the same primers
  • Streptomyces castaneoglobisporus TYR gene NCS-RsTYR-DODC-optMAO / pKK223-3 and NCS-RsTYR-DODC-optMAO / pET-21d were prepared using the sequence of the RsTYR gene (SEQ ID NO: 37) instead of ORF378 (FIG. 11, the latter) Only the constructs are shown).
  • NCS-ORF378-TYR-DODC-optMAO / pET-21d and NCS-RsTYR-DODC-optMAO / pET-21d as plasmids for expressing the enzymes of the norlaudanosoline synthesis pathway. did.
  • Typical PCR conditions used in this example are as follows: Initial denaturation step, 94 ° C, 2 min; 94 ° C, 15 sec denaturation, 50 ° C, 30 sec annealing and 68 ° C, 90 sec DNA A cycle consisting of elongation was performed for 30 cycles; and the final elongation at 68 ° C for 5 minutes; for this, KOD-plus DNA polymerase polymerase (Toyobo) was used.
  • PCR was performed by appropriately changing the conditions according to the DNA sequence used as a template, the length of the primer, the Tm value, and the like. Optimizing PCR conditions is a technique well known to those skilled in the art.
  • tyrR gene was achieved by recombination with PCR products using Quick & Easy E.coli Deletion Kit (Gene Bridges). Specifically, using a plasmid having the sequence of FRT-PGK-gb2-neo-FRT included in the kit as a template, a primer d-tyrR-Sm-F having a sequence homologous to the tyrR gene at the 5 'end (sequence No.
  • the Escherichia coli BL21 (DE3) was transformed with an FLP expression vector (708-FLPe; Gene Bridges), and the kanamycin resistance gene cassette was removed by the action of FLP recombinase.
  • the tyrR :: null mutation was confirmed by PCR using the primers 5tyrRKOcheck (SEQ ID NO: 34) and 3tyrRKOcheck (SEQ ID NO: 35) (Table 1).
  • Escherichia coli BL21 (DE3) having the tyrR :: null mutation was transformed with tyrA fbr -aroG fbr -tktA-ppsA / pCOLADuet-1 (FIG. 8) to obtain an L-tyrosine overproducing strain.
  • the L-tyrosine overproducing strain was transformed with NCS-ORF378-TYR-DODC-optMAO / pET-21d and 6OMT-4′OMT-CNMT / pACYC184 (FIG. 8) to obtain a reticuline producing strain. Obtained (this strain is referred to herein as “recombinant production strain A”).
  • this strain is referred to herein as “recombinant production strain A”).
  • coli BL21 (DE3) strain that has not undergone the above modification (introduction of tyrR :: null mutation and tyrA fbr -aroG fbr -tktA-ppsA / pCOLADuet-1) for the production of L-tyrosine is NCS-ORF378-TYR -DODC-optMAO / pET-21d and 6OMT-4'OMT-CNMT / pACYC184 were transformed to obtain a recombinant Escherichia coli strain (this strain is referred to herein as "recombinant production strain B") ). Furthermore, the above L-tyrosine overproducing strain (E.
  • coli BL21 (DE3) having a tyrR :: null mutation and transformed with tyrA fbr -aroG fbr -tktA-ppsA / pCOLADuet-1) was transformed into NCS- RsTYR-DODC-optMAO / pET-21d (FIG. 11) and 6OMT-4′OMT-CNMT / pACYC184 were transformed to obtain a reticuline producing strain (herein, such strain is referred to as “recombinant production”). Referred to as "strain C"). These recombinant production strains A, B and C were used for reticuline production.
  • the pH was maintained at 7.1 by automatic addition of 28% NH 4 OH and 1M HCl.
  • the stirring speed at the time of inoculation was 100 rpm, and the dissolved oxygen level was reduced to 10% oxygen saturation using continuous aeration of 1 v ⁇ v ⁇ 1 ⁇ m ⁇ 1 .
  • the glucose concentration was maintained between 0.1 and 7 g / L by addition of 0.5 g / mL glucose solution.
  • Glycerol concentration was maintained between 0.1 and 6 g / L by addition of 1 g / mL glycerol solution.
  • OD 600 when the OD 600 reaches 10 (for culture in a medium using glucose as a carbon source) or 15 (for culture in a medium using glycerol as a carbon source), it is indicated in the instructions attached to the vector.
  • Expression induction was performed by adding IPTG at the designated or specified concentration (final concentration) to the culture.
  • glucose in the medium was analyzed by the mutarotase-glucose method using glucose CII-Test Wako (Wako Pure Chemical Industries, Ltd.).
  • glycerol in the medium was analyzed using Glycerol Assay Kit (Cayman Chemical Co.).
  • Aromatic compounds other than reticuline in the medium were analyzed by a HPLC equipped with a Discovery HS F5 column (Supelco). The compounds were separated on the column by increasing the acetonitrile concentration from 3% to 20% in 10 mM ammonium formate (pH 3.0) at a flow rate of 0.5 mL / min. Compound elution was monitored by measuring absorbance at 280 nm.
  • the culture supernatant was collected at various time points, and the protein was precipitated using 2% by weight of trichloroacetic acid.
  • the supernatant was separated on an Agilent HPLC system and analyzed by LC-MS (3200 QTRAP, Applied Biosystems Japan Ltd.).
  • the HPLC conditions were as follows: A column, TSKgel ODS-80Ts (4.6 ⁇ 250 mm; Totosoh Corporation); A solvent system, A: 0.1% acetic acid aqueous solution, B: Acetonitrile solution containing 0.1% acetic acid; Gradient mode: 90% A (0-5 minutes), 90% to 60% A (5-20 minutes), 10% A (20-30 minutes); .
  • Reticuline was identified by comparing it to standard reticuline (laboratory stock) for fragmentation spectra in LC-tandem MS (LC-MS / MS). The amount of reticuline was estimated from the calibration curve ( ⁇ mol vs. peak area) using Analyst 1.4.1 software.
  • the reticuline produced was separated on an Agilent HPLC system and then subjected to LC-MS to analyze the stereoselectivity of the reticuline.
  • the conditions of the HPLC column were as follows: column, CHIRALCEL OD-H (4.6 ⁇ 250 mm, Daicel Chemical Industries, Ltd.); solvent system, hexane / 2-propanol / diethylamine (72: 28: 0.1); 0.55 mL / min, temperature: 40 ° C.
  • recombinant production strain B produced (S) -reticuline from glucose in the medium within a period of 80 hours with a yield of 0.53 mg per liter of medium. It was shown that the replacement production strain A produced (S) -reticuline from glucose in the medium in a yield of 2.2 mg per liter of medium within 80 hours (Fig. 2). Moreover, in the medium not containing glucose, the yield of (S) -reticuline by the recombinant production strain A was as extremely low as 80 ⁇ g / L (FIG. 2). These results indicate that (S) -reticuline was obtained from glucose, a simple carbon source.
  • (S) -reticuline was produced by leak expression of the introduced biosynthetic gene without induction by IPTG (FIG. 3). This means that the production cost of (S) -reticuline according to the present invention can be substantially reduced. Furthermore, when recombinant production strain A was cultured using a medium containing glycerol as a carbon source, the yield of (S) -reticuline in 80 hours increased to 6.2 mg per liter of medium (Fig. 9). Was approximately three times higher than the production in the medium using glucose as the carbon source.
  • a dopamine producing strain using RsTYR (a strain in which RsTYR and DODC have been introduced into the above L-tyrosine overproducing strain) is a dopamine producing strain using a TYR of S. castaneoglobisporus (the above L-tyrosine overproducing strain is Since the production of 1.05 ⁇ 0.05g / L (6.85 ⁇ 0.30mM) of dopamine (yield not shown) was about 4 times the yield of TYR and DODC) (data not shown), The possibility of contributing to the increase in reticuline production in the production strain C is considered.
  • reticuline is produced as a racemate, whereas in the method using the above-described recombinant production strain A of the present invention, Produced only (S) -reticuline (FIG. 6). Further, during cultivation of the recombinant production strain A, L-tyrosine accumulated as an intermediate in the stationary phase, but accumulation of L-DOPA and dopamine was not observed throughout the culture period (FIG. 7). No accumulation of dopamine prevents the spontaneous condensation reaction between dopamine and 3,4-DHPAA without the catalysis of NCS, which produces (S) -body specific norlaudanosoline. . As a result, norlaudanosoline is not produced as a (R, S) -racemic mixture, and only (S) -reticuline is considered to be obtained.
  • optically active (S) -reticuline is produced by culturing plant cultured cells or producing a recombinant plant (usually for a period of several months to one year). For example, in 2 to 3 days.
  • a further advantage of the present invention is that (S) -reticuline with almost no contamination with unwanted metabolites (including undesired isoquinoline alkaloids) can be obtained by a simple and efficient purification procedure.
  • (S) -reticuline with almost no contamination with unwanted metabolites (including undesired isoquinoline alkaloids) can be obtained by a simple and efficient purification procedure.
  • HPLC high performance liquid chromatography
  • more than 90% of the purified (S) -reticuline could be recovered (Fig. 10).
  • Such a simple and high-yield purification operation makes the (S) -reticuline production method of the present invention economically feasible.

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Abstract

L'invention concerne un procédé de synthèse d'une (S)-réticuline importante en tant qu'intermédiaire de jonction de voies de biosynthèse d'un benzylisoquinoléine-alcaloïde, mettant en œuvre la culture d'un micro-organisme transformé exprimant un enzyme indispensable à cette biosynthèse.
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