WO2012039438A1

WO2012039438A1 - Method for producing plant benzylisoquinoline alkaloid

Info

Publication number: WO2012039438A1
Application number: PCT/JP2011/071520
Authority: WO
Inventors: 中川　明; 博道南; 高嶺片山; 英彦熊谷
Original assignee: 石川県公立大学法人
Priority date: 2010-09-22
Filing date: 2011-09-21
Publication date: 2012-03-29
Also published as: JP5761723B2; JPWO2012039438A1

Abstract

A method for synthesizing (s)-reticulin being important as a junction intermediate in biosynthetic pathway of benzylisoquinoline alkaloid by culturing the transgenic microbes expressing the enzyme necessary for the biosynthesis.

Description

Method for producing plant benzylisoquinoline alkaloids

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) -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.

Recent research also suggests that these alkaloids may be useful as new drugs. For example, an aporphine-type alkaloid, magnoflorin, protects high-density lipoproteins during oxidative stress to prevent the development of arteriosclerotic disease and inhibits human lymphoblastoid cell killing by HIV-1. It has been reported (Non-Patent Documents 1 to 3). A recent report shows that berberine, an antibacterial agent, has cholesterol-lowering activity (Non-Patent Document 4).

Some benzylisoquinoline alkaloids have been chemically synthesized by total synthesis because of great attention in their potential use as pharmaceuticals. For example, the total synthesis of morphine, a narcotic analgesic, has been reported (Non-patent Document 5). However, due to the complex structure and chirality of alkaloids, it is difficult to develop cost-effective production methods. Thus, 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). However, 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.

So far, transformed poppy (opium poppy) plants that have been treated with RNAi on codeinone reductase and transformed California 細胞 poppy cells with RNAi on berberine bridging enzyme (BBE) have been reported to produce reticuline. (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 (Non-patent Document 9). In addition, these transformed poppies accumulated some methylated derivatives of reticuline. Although the transformation approach can be a very powerful tool for metabolic engineering, this technology, including RNAi, still has room for further improvement for use in producing the desired metabolite.

Recently, attempts to reconstruct the plant biosynthesis process have been investigated in microbial systems (Non-Patent Documents 10 and 11). 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.

In the benzylisoquinoline alkaloid pathway, almost all biosynthetic genes from norcoclaurine to berberine have been isolated and their activity has been shown in microbial systems (Non-patent Documents 12 and 13). In the benzylisoquinoline alkaloid pathway, dopamine and 4-hydroxyphenylacetaldehyde (hereinafter also referred to as 4-HPAA) are coupled by norcoclaurine synthase (hereinafter also referred to as NCS), so that (S) -norcoclaurine is It is clear that (S) -reticuline is produced through (S) -Norcoclaurine is then converted to coclaurin by norcoclaurine 6-O-methyltransferase (hereinafter also referred to as 6OMT), which is N-linked by coclaurin-N-methyltransferase (hereinafter also referred to as CNMT). Converted to methylcoclaurine, which is converted to 3′-hydroxy-N-methylcoclaurine (hereinafter also referred to as 6-O-methyllaudanosoline) by P450 hydroxylase (CYP80B), and 3'-hydroxy-N-methylcoclaurine is converted to (S) -reticuline by 3'-hydroxy-N-methylcoclaurine-4'-O-methyltransferase (hereinafter also referred to as 4'OMT) (Figure 4).

The present inventors have so far developed 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 ( Patent Document 1). 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. 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.

However, in such a method, it is essential to add dopamine as a starting material. In addition, when the reaction is performed in vivo, since reticuline is obtained as a (R, S) -racemic mixture, only (S) -reticuline, which is a branch point intermediate in the biosynthetic pathway of many benzylisoquinoline alkaloids, is obtained. For this purpose, it is necessary to perform optical resolution using a chiral column.

In addition, similar to the method of Patent Document 1, 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. 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.

On the other hand, Hawkins and Smolke have succeeded in producing (R, S) -reticuline and its derivatives by recombinant yeast cells using a racemic mixture of norlaudanosoline as a starting material (Non-patent Document 14). .

However, dopamine and norlaudanosoline used as starting materials in the methods described in

Patent Documents

1 and 2 and Non-Patent Document 14 are relatively expensive, and are easily oxidized during culturing, resulting in a brown insoluble polymer. Has the property of forming Therefore, it is difficult to make the above alkaloid production method using such a compound as a starting material an economical system.

Against this background, it is desirable to establish a system that can efficiently produce benzylisoquinoline alkaloids from inexpensive and readily available substrates without using the starting materials such as dopamine and norlaudanosoline. It was.

JP 2009-225669 A International Publication WO2008 / 153094 Pamphlet

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.

This time, 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. Established.

Specifically, 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.

That is, 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. At least one gene encoding at least one protein selected from the group consisting of -O-methyltransferase, coclaurin-N-methyltransferase and 3'-hydroxy-N-methylcoclaurin-4'-O-methyltransferase Tyrosinase and its adapter protein, L-DOPA specific decarboxylase, monoamine oxidase, norcoclaurine synthase, norcoclaurine 6-O-methyltransferase, coclaurin-N-me And a recombinant host cell expressing 3′-hydroxy-N-methylcoclaurine-4′-O-methyltransferase, and using the recombinant host cell as a carbon source with glucose, fructose, galactose, 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 glycerol.

In addition, 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. Acid mutase / prephenate dehydrogenase, transketolase, phosphoenolpyruvate synthetase, tyrosinase and its adapter protein, L-DOPA-specific decarboxylase, monoamine oxidase, norcoclaurine synthase, norcoclaurine 6-O-methyltransferase, At least one gene encoding at least one protein selected from the group consisting of coclaurine-N-methyltransferase and 3′-hydroxy-N-methylcoclaurine-4′-O-methyltransferase A recombinant host cell into which feedback inhibition resistance 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, feedback inhibition resistance chorismate mutase / prephenate dehydrogenase, transketolase, Phosphoenolpyruvate synthetase, tyrosinase and its adapter protein, L-DOPA specific decarboxylase, monoamine oxidase, norcoclaurine synthase, norcoclaurine 6-O-methyltransferase, coclaurine-N-methyltransferase and 3'-hydroxy- Providing a recombinant host cell that expresses N-methylcoclaurine-4'-O-methyltransferase and does not have the tyrR gene or is deficient in its function; and Glucose as a carbon source A method for producing (S) -reticuline comprising the step of culturing in a medium containing one or more sugars and / or glycerol selected from the group consisting of sucrose, fructose, galactose, sucrose, lactose and maltose .

Furthermore, 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. At least one gene encoding at least one protein selected from the group consisting of -O-methyltransferase, coclaurin-N-methyltransferase and 3'-hydroxy-N-methylcoclaurin-4'-O-methyltransferase Tyrosinase and its adapter protein, L-DOPA-specific decarboxylase, monoamine oxidase, norcoclaurine synthase, norcoclaurine 6-O-methyltransferase, coclaurin-N-methyl It provides recombinant host cells expressing transferase and 3'-hydroxy -N- methylcoclaurine cloud phosphorus-4'-O-methyl transferase.

According to the present invention, a desired host benzylisoquinoline alkaloid, particularly (S) 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. -Highly efficient production of reticuline. That is, according to the present invention, 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. Thus, 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.

DETAILED DESCRIPTION OF THE INVENTION According to the present invention, 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. Furthermore, the microbial system of the present invention is also useful for developing new pathways for the production of novel isoquinoline alkaloids.

Also, 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. Abbreviations are as follows: 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; 6OMT , Norcoclaurine 6-O-methyltransferase; CN CNMT, Lauric -N- methyltransferase; 4'OMT, 3'-hydroxy -N- methylcoclaurine cloud phosphorus-4'-O-methyl transferase. Production of (S) -reticuline using recombinant host cells. (A) Change over time of (S) -reticuline production in E. coli cells (left axis, solid line). NCS-ORF378-TYR-DODC-optMAO / pET-21d and 6OMT-4'OMT-CNMT / pACYC184, which overproduces L-tyrosine (maru, "recombinant production strain A") or NCS -ORF378-TYR-DODC-optMAO / pET-21d and 6OMT-4'OMT-CNMT / pACYC184, but the metabolic pathways involved in L-tyrosine production have not been altered (squares, “recombinant production Strain B ″) was cultured in a jar fermenter using a medium containing glucose as a carbon source. 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 μM) 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. (B) LC-MS / MS analysis of the reaction product obtained by culturing the recombinant host cell of the present invention. Selected ion monitoring (SIM) parameters: m / z = 330 (reticuline). Effect of IPTG concentration on (S) -reticuline production in medium containing glucose as a carbon source. An E. coli strain that overproduces L-tyrosine and expresses the reticuline biosynthesis gene (“recombinant production strain A”) was grown to an OD ₆₀₀ of 10 before the addition of IPTG (arrow); IPTG No induction (circle), 50 μM IPTG (square), 150 μM IPTG (triangle), 500 μM IPTG (×). The experiment was repeated three times with essentially the same results. Data from one representative experiment is shown in the figure. Genes involved in benzylisoquinoline alkaloid biosynthesis pathway of the proposed plant and benzylisoquinoline alkaloid biosynthesis from L-tyrosine to (S) -reticuline. Abbreviations are as follows: 4-HPAA, 4-hydroxyphenylacetaldehyde; TH TH, tyrosine hydroxylase; TYDC, tyrosine / DOPA decarboxylase; NCS, norcoclaurine synthase; 6 6OMT, norcoclaurine 6-O-methyltransferase ; CNMT, coclaurine-N-methyltransferase; CYP80B, N-methylcoclaurine 3'-hydroxylase; 4'OMT, 3'-hydroxy-N-methylcoclaurine-4'-O-methyltransferase. 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) The amount of intermediate product present in the medium and glycerol consumption during growth. 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. (S) -Schematic diagram of the plasmid used for reticuline production. 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. Abbreviations: 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). An E. coli strain overproducing L-tyrosine and expressing a reticuline biosynthesis gene (“recombinant production strain A”) was cultured with 50 μM IPTG® (circle) or without IPTG® (square). The cell growth in each case (right axis, broken line, symbol is the same correspondence as (S) -reticuline production) is shown as absorbance at 600 nm. The experiment was repeated three times with essentially the same results. Data from one representative experiment is shown in the figure. Rapid purification of (S) -reticuline from the culture medium of the recombinant E. coli strain of the present invention. The medium before purification (a) and after purification (b) was analyzed by MS in ion scan mode (m / z = 130-530). m / z = 330 indicates a peak of (S) -reticuline. (S) -Schematic diagram of the plasmid used for reticuline production. 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. Abbreviations: RsTYR, TYR derived from Ralstonia solanacearum; T7p, T7 promoter; T7t, T7 terminator; Amp ^r , ampicillin resistance gene. Change over time of (S) -reticuline production in a medium containing glycerol as a carbon source (left axis, solid line). Reticuline production strain (“recombinant production strain A”) using TYR derived from Streptomyces castaneoglobisporus and adapter protein ORF378 (square), and reticuline production strain using TYR derived from Ralstonia solanacearum (“recombinant production strain C”) ( Circles) were cultured. The arrow indicates that IPTG (50 μM) was added at that time. Cell proliferation (right axis, broken line, symbol is the same correspondence as (S) -reticuline production) is shown as absorbance at 600 nm. Error bars indicate the standard deviation of three independent experiments.

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), Recombinant hosts that express lucoclaurine 6-O-methyltransferase (6OMT), coclaurin-N-methyltransferase (CNMT) and 3'-hydroxy-N-methylcoclaurine-4'-O-methyltransferase (4'OMT) Providing a cell, and culturing the recombinant host cell in a medium comprising one or more sugars and / or glycerol selected from the group consisting of glucose, fructose, galactose, sucrose, lactose and maltose as a carbon source. Process,
A method for producing (S) -reticuline is provided.

According to such a method, 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). -Reticuline can be produced efficiently. Preferably, 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 抑制 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.

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). 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.

Therefore, 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. Inhibition resistant chorismate mutase / prephenate dehydrogenase (fbr-CM / PDH), transketolase (TKT), phosphoenolpyruvate synthetase (PEPS), tyrosinase (TYR) and its adapter protein, L-DOPA specific de Carboxylase (DODC), monoamine oxidase (MAO), norcoclaurine synthase (NCS), norcoclaurine 6-O-methyltransferase (6OMT), coclaurin-N-methyltransferase (CNMT) and 3'-hydroxy-N-methyl At least selected from the group consisting of coclaurin-4'-O-methyltransferase (4'OMT) A recombinant host cell into which at least one gene encoding one protein has been introduced, comprising 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (fbr-DAHPS) resistant to feedback inhibition, feedback Inhibition resistant chorismate mutase / prephenate dehydrogenase (fbr-CM / PDH), transketolase (TKT), phosphoenolpyruvate synthetase (PEPS), tyrosinase (TYR) and its adapter protein, L-DOPA specific de Carboxylase (DODC), monoamine oxidase (MAO), norcoclaurine synthase (NCS), norcoclaurine 6-O-methyltransferase (6OMT), coclaurin-N-methyltransferase (CNMT) and 3'-hydroxy-N-methyl Does it express coclaurin-4'-O-methyltransferase (4'OMT) and does not have a tyrR gene? Or providing 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:

In addition, 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 maltose Culturing in a medium containing one or more sugars and / or glycerol selected from the group consisting of
A method for producing (S) -reticuline is provided.

Further, according to the present invention, 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.

That is, 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 Providing a recombinant host cell expressing an isoquinoline alkaloid biosynthetic enzyme that acts as a starting material, and the recombinant host cell is selected from the group consisting of glucose, fructose, galactose, sucrose, lactose and maltose as a carbon source. One or more selected sugars and Step of culturing in a medium containing / or glycerol,
A method for producing isoquinoline alkaloids is provided.

Examples of 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). 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. Recombinant host cells expressing TYR and its adapter proteins, DODC, MAO, NCS, 6OMT, CNMT and 4'OMT, into which at least one gene encoding at least one protein has been introduced.

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.
By having these properties, the productivity of L-tyrosine in the recombinant host cell can be improved, and the yield of (S) -reticuline or other isoquinoline alkaloids can be increased.

Therefore, 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.

In addition, 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 過剰 are overexpressed, and no tyrR gene is present or its function A defective recombinant host cell is provided.

In the present invention, 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. Providing a recombinant host cell expressing RsTYR, DODC, MAO, NCS, 6OMT, CNMT and 4'OMT, into which at least one gene encoding at least one protein selected from the group consisting of: And culturing the recombinant host cell in a medium containing one or more sugars and / or glycerol selected from the group consisting of glucose, fructose, galactose, sucrose, lactose and maltose as a carbon source,
There is further provided a method for producing (S) -reticuline, comprising:

Similarly, TYR from Ralstonia solanacearum (RsTYR) 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. Embodiments are also provided as preferred embodiments of the present invention.

In the present specification, the term 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.

Here, the biosynthetic pathway from L-tyrosine to (S) -reticuline in a plant producing benzylisoquinoline alkaloid is shown in FIG.

On the other hand, 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. This produces (S) -norlaudanosoline (Fig. 1). This eliminates the step of hydroxylation (see FIG. 4) by N-methylcoclaurine 3′-hydroxylase (hereinafter also referred to as CYP80B) in the plant biosynthetic pathway.

However, 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. In particular, 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. In this case, in order to obtain (S) -reticuline from the (S) -norcoclaurin, it is necessary to use 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.

Therefore, in the method of the present invention, L-DOPA-specific decarboxylase (DODC) is introduced into a host cell and expressed. Specifically, 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.

Therefore, 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.

As a result, 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).

In plants and animals, 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. However, 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. Moreover, 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.

For example, 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. Also, 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.
Here, 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. In addition, 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.

In order to obtain a recombinant host cell according to the present invention, a gene expressing fbr-DAHPS and / or fbr-CM / PDH, a cell expressing TKT and / or PEPS at a high level, etc. It may be used as a host cell to be introduced.

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 ”.

In a preferred embodiment, 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”.

In addition, 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.

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.

As the vector into which the transgene is incorporated, 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. Examples of 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.

Examples of 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 (hereinafter referred to as an expression vector) 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. Furthermore, 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.

As a method for transforming an expression vector into a host cell, 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. Specifically, 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. Examples of sugars include monosaccharides such as glucose, fructose, galactose, and disaccharides such as sucrose, lactose, maltose, etc. Among them, glucose is preferred. In addition, when glycerol is used as a carbon source, the production efficiency of (S) -reticuline is higher than when glucose is used. Examples of the nitrogen source include ammonium sulfate and casamino acid. In addition, salts, specific amino acids, specific vitamins and the like can be used as desired.

For example, the medium for culturing E. coli includes LB medium, 2 × YT medium, and M9 minimal medium, and 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. For example, in the case of Escherichia coli, the culture conditions of temperature 25 ° C, 80 hours, pH 7.0 用いる are used. be able to. In the case of yeast, for example, culture conditions of a temperature of 30 ° C., 60 hours, and pH of 5.8 can be used.

Production of the desired isoquinoline alkaloid containing (S) -reticuline can be confirmed by any means known to those skilled in the art. Specifically, the 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.

In the present specification, “expressing” a specific gene means that a nucleic acid molecule constituting the gene is transcribed to at least an RNA molecule. In the case of a gene encoding a polypeptide, 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.

In this specification, “expressing” a specific enzyme 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.

In the present specification, “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. In addition, when the host cell does not have an endogenous gene or enzyme corresponding to a specific gene or enzyme, “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.

As used herein, “reconstructing” 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. In the methods of the present invention, typically 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.

In this specification, 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. In the method of the present invention, for example, TYR derived from S. castaneoglobisporus encoded by the nucleotide sequence shown in SEQ ID NO: 6 can be preferably used.

Also, Ralstonia solanacearum-derived TYR (RsTYR) can be used as the TYR. In the present invention, for example, 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. (Hernandez-Romero, D. et al, FEBS Journal 273 (2006), 257-270). Therefore, by using RsTYR encoded by the nucleotide sequence shown in SEQ ID NO: 36 or 37, the production amount of dopamine can be increased and the production efficiency of (S) -reticuline can be improved.

The tyrosinase (TYR) adapter protein used in the present invention, for example, ORF378 protein, is a protein that assists in transporting divalent copper ions (Cu (II)) to the catalytic center of TYR. At least for TYR derived from bacteria of the genus Streptomyces, an adapter protein is required in order for the TYR to show complete catalytic activity. For example, on the chromosome of S. castaneoglobisporus TY, 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. Microbiol. Biotechnol. 45, 80-85 (1996); Y. Matoba et al., J. Biol. Chem. Vol.281, No.13, pp.8981-8990 (2006)) . Examples of TYR adapter proteins include ORF378 protein derived from S. castaneoglobisporus and ORF438 protein derived from Streptomyces antibioticus. In the present invention, for example, derived from の S. castaneoglobisporus derived from the nucleotide sequence shown in SEQ ID NO: 7 ORF378 protein can be preferably used.

Regarding the terminology used for the adapter protein of TYR in this specification, taking 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 (DODC) 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. An aromatic L-amino acid decarboxylase that exhibits a selectivity of L-DOPA of at least 10 times, preferably 100 times, more preferably 1000 times or more compared to a family L-amino acid. 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. In the method of the present invention, for example, 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. The 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 のもの are not particularly limited, but those derived from isoquinoline alkaloid-producing plants are preferred. Examples of 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 Examples include isoquinoline alkaloid-producing plants such as buttercups such as (Coptis p japonica), and preferably olene. In the method of the present invention, for example, 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.

On the other hand, the origin of NADPH-cytochrome P450 reductase, which is preferably used together with 好ましい CYP80G2 (Colituberin synthase), which is a kind of cytochrome P450 as an isoquinoline alkaloid biosynthetic enzyme, is not particularly limited. Mammals.

When producing magnoflorin in the method of the present invention, 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. For example, 酵素 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. Can be used. Moreover, as an enzyme that catalyzes the reaction from colituberin to magnoflorin, for example, “CNMT” encoded by the nucleotide sequence shown in SEQ ID NO: 13 can be preferably used.

In the method of the present invention, 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. As a specific example, BBE encoded by the nucleotide sequence shown in SEQ ID NO: 17 can be preferably used.

Furthermore, when producing berberine, 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. For example, BBE, SMT (scourelin-9-O-methyltransferase), CYP719A1 (canazine synthase) and THBO (tetrahydroberberine oxidase) may be used. As BBE, those encoded by the nucleotide sequence shown in SEQ ID NO: 17 are preferably used. The sequence information of SMT, CYP719A1, and THBO® can be obtained from “GenBank” (http://www.ncbi.nlm.nih.gov/) provided by NCBI: National Center for Biotechnology Information.

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. 5 (7), 1585-1592 (1991)). Therefore, 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.

If the host cell contains the tyrR gene, the nucleic acid for gene disruption or expression suppression can be obtained by artificial nucleic acid synthesis or mutagenesis after cloning based on the obtained sequence information or nucleic acid molecule of the tyrR gene. The nucleic acid can be used, and the function of the tyrR gene possessed by the host cell can be lost or the expression of the tyrR gene can be suppressed by known means such as homologous recombination or RNAi.

The homology search can be performed using a known means such as the BLAST algorithm or FASTA algorithm.

In the present invention, 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- 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. Here, “does not receive feedback inhibition by phenylalanine (is resistant to feedback inhibition)” means that phenylalanine does not inhibit the enzyme activity of DAHPS at all, or the degree of inhibition of enzyme activity by phenylalanine is compared with wild-type DAHPS It is significantly smaller. It is preferable that fbr-DAHPS has an enzyme activity in the presence of phenylalanine that is equal to or greater than that in the absence thereof. Further, fbr-DAHPS is not particularly limited, but is preferably derived from microorganisms, more preferably derived from E. coli. In the present invention, for example, 大腸菌 fbr-DAHPS derived from E. coli encoded by the nucleotide sequence shown in SEQ ID NO: 2 can be preferably used.

In the present invention, 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. Here, “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. In 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. Further, fbr-CM / PDH is not particularly limited, but is preferably derived from microorganisms, and more preferably derived from E. coli. In the present invention, for example, E. coli-derived fbr-CM / PDH encoded by the nucleotide sequence shown in SEQ ID NO: 3 can be preferably used.

In the present invention, TKT that can be used to modify a host cell transfers a structure containing a carbonyl group, for example, a structure represented by -C (= O) CH ₂ OH from one sugar to another. It does not specifically limit if it has the enzyme activity which catalyzes reaction. 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.

In the present invention, 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. Further, PEPS is not particularly limited, but is preferably derived from microorganisms, and more preferably derived from E. coli. In the present invention, for example, E. coli-derived PEPS encoded by the nucleotide sequence shown in SEQ ID NO: 5 can be preferably used.

That is, 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 ′).
(b ′) 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 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more of the amino acid sequence, 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.

The above (b) タンパク質 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 A protein having homology to the protein of (a) to the extent that the function of the protein of (a) is not lost. The homology is preferably 70% or more, particularly preferably 90% or more.

In the present invention, “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.

Whether 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. For fbr-DAHPS, 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. Added, glyceraldehyde-3-phosphate (GAP) and sedheptulose-7-phosphate (S7P) were produced, or for PEPS, pyruvic acid was added, and phosphoenolpyruvate (PEP) was produced, 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, Whether 4-DHPAA was produced or for NCS dopamine 3,4-DHPAA was added to produce 3'-hydroxynorcoclaurine, 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 Thus, the presence or absence of the activity can be determined. In addition, for CYP80G2 and P450 reductase, 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. As CNMT, which catalyzes the reaction of magnoflorin from colituberin, the same one as CNMT, which catalyzes the reaction of 3'-hydroxycoclaurine to 3'-hydroxy-N-methylcoclaurine, can be used. Whether or not to catalyze the reaction to florin can be determined by adding coritsuberin and examining whether or not magnoflorin has been produced.

Whether 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. (See, for example, Kikuchi, Y. et al., Appl. Environ. Microbiol. 63, 761-762 (1997)). Similarly, 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. (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. Specifically, the reaction product and 3-deoxy-D-arabino-heptulosonic acid-7-phosphate (DAHP), 4-hydroxyphenylpyruvic acid (HPP), glyceraldehyde-3-phosphate (GAP), Sedoheptulose-7-phosphate (S7P), phosphoenolpyruvate (PEP), L-DOPA, dopamine, 3,4-DHPAA, 3'-hydroxynorcoclaurine, 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.

Then, 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 The gene encoding the enzyme will be described.

Specific examples of 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 As a gene encoding CYP80G2, 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.

That is, 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.

Furthermore, 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.

Here, 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. Here, “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.

Materials All synthetic genes were obtained from GenScript Inc. (R, S) -norlaudanosoline was purchased from Acros Organics. L-tyrosine, L-DOPA and dopamine were purchased from Sigma-Aldrich. (R, S) -reticuline used was donated by Mitsui Chemicals.

Construction of expression vector
Construction of tyrA ^fbr -aroG ^fbr -tktA-ppsA / pCOLADuet-1 To create a host cell that overproduces L-tyrosine, an expression vector containing the following four genes was constructed: tyrA ^fbr (fbr-CM / PDH), aroG ^fbr (fbr-DAHPS), tktA (TKT) and ppsA (PEPS).

First, the tyrA ^fbr and aroG ^fbr genes cloned into pUC57 were purchased from GenScript Inc. In the plasmid, tyrA ^fbr was adjacent to the recognition sites for NcoI and EcoRI restriction enzymes, and aroG ^fbr was linked to the T7 promoter. In addition, 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). After restriction enzyme digestion with NdeI-XhoI, the tktA and ppsA genes were individually cloned into the NdeI-XhoI sites of pET-41a (Novagen) and pCOLADuet-1 (Merck), respectively.

In order to construct an expression plasmid containing the above four genes, a DNA fragment containing the tyrA ^fbr gene was excised from tyrA ^fbr / pUC57 using NcoI and EcoRI, and then cloned into the NcoI-EcoRI site of pCOLADuet-1. Next, the aroG ^fbr / pUC57 EcoRI-SacI fragment (including the aroG ^fbr gene linked to the T7 promoter) was cloned into the EcoRI-SacI site of tyrA ^fbr / pCOLADuet-1. Thus, to obtain an expression vector of ^{^{^{tyrA fbr -aroG fbr (tyrA fbr -aroG}}} fbr / pCOLADuet-1).

Next, 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.

Finally, 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.

Construction of 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).

First, according to the method described in Japanese Patent Application Laid-Open No. 2009-225669 and Minami, H. et al. Proc. Natl. Acad. Sci. USA 105, 7393-7398 (2008), the MAO gene (SEQ ID NO: 9) of Micrococcus luteus And an expression vector pKK223-3 (NCS-MAO / pKK223-3) containing the NCS gene (SEQ ID NO: 11) of C. japonica.

Here, since natural MAO has a low expression level in E. coli, we decided to use 用いる MAO (SEQ ID NO: 10, hereinafter referred to as optMAO) with optimized codons. Specifically, 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 (SEQ ID NO: 8) was extracted from the genomic DNA of the strain 5NdeDODC (SEQ ID NO: 26). ) And 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. Such DODC is a decarboxylase showing the L-DOPA selectivity of greater than 10 ³ times as compared with the other aromatic amino acids. Next, 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.

The 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.

Construction of 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.

First, 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 して 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 . Finally, the above PCR product (SacI-EcoRV F fragment) containing the TYR gene and ORF378 is ligated to the SacI-EcoRV site of NCS-DODC-optMAO / pET-21d pET-21d was obtained (FIG. 8).

Construction of NCS-RsTYR-DODC-optMAO / pKK223-3 and NCS-RsTYR-DODC-optMAO / pET-21d Ralstonia solanacearum TYR (RsTYR) gene (SEQ ID NO: 37) cloned in pGS-21a Purchased from Inc. In this construct, the codon usage frequency of the RsTYR gene is optimized. Follow the same procedure as above for constructing NCS-ORF378-TYR-DODC-optMAO / pKK223-3 and NCS-ORF378-TYR-DODC-optMAO / pET-21d (use the same primers), and use 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).

Using shake flask culture to compare productivity between recombinant host cells with pET-21d and pKK223-3 plasmid constructs, recombinant host cells with a plasmid based on pET-21d rather than pKK223-3 However, a slightly higher reticuline production level was obtained (data not shown). Therefore, we selected 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.

Construction of 6OMT-4'OMT-CNMT / pACYC184 In order to reconstruct the pathway for synthesizing reticuline from norlaudanosoline, the aforementioned JP 2009-225669 and Minami, H. et al. Proc. Natl. Acad. According to the method described in Sci. USA 105, 7393-7398 (2008), the expression vector pACYC184 (6OMT-4'OMT-CNMT / pACYC184) containing the 6OMT, 4'OMT and CNMT genes of C. japonica was obtained. (Fig. 8).

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. In this example, 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.

Creation of Reticuline Production Strains First, in order to create host cells that overproduce L-tyrosine, Lutke-Eversloh, T., and Gregory Stephanopoulos, G., Appl. Microbiol. Biotechnol. (2007) 75: 103-110 The described genetic construction of the E. coli strain overproducing L-tyrosine was employed. Specifically, an E. coli strain having a knockout mutation in the tyrR gene and having a plasmid expressing tyrA ^fbr , aroG ^fbr , tktA and ppsA was created.

Deletion of the 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. 32) and d-tyrR-Sm-R (SEQ ID NO: 33) (Table 1) were used to PCR-amplify the kanamycin resistance gene cassette having the FRT sequence, so that tyrR A sputum DNA fragment having a sequence homologous to the gene was obtained. Subsequently, Escherichia coli BL21 (DE3) was transformed with the fragment, and a kanamycin resistance gene cassette having a FRT sequence was introduced into the tyrR gene by homologous recombination. Thereafter, 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. Finally, 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. Next, 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”). In addition, the E. 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 primer sequences used in this example are shown below.

Culture conditions Host cells are cultured overnight in liquid LB medium at 37 ° C, and 10 mL of cell culture is cultured in 1 L of modified conventional medium (3 L of jar fermenter (BMS-03PI, ABLE)) Per liter contains: 47.6 g Turbo Broth ^™ (Athena Enzyme System), 1.6 g NH ₄ Cl, 2.49 mg CuSO ₄ · 5H ₂ O, 3 g glucose or 5 g glycerol, 50 mg Ampicillin, 25 mg kanamycin, and 50 mg chloramphenicol).

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. If necessary, 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.

Detection and quantification of compounds According to the manufacturer's instructions, glucose in the medium was analyzed by the mutarotase-glucose method using glucose CII-Test Wako (Wako Pure Chemical Industries, Ltd.). According to the manufacturer's instructions, 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.

In order to measure the produced reticuline, 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 (μmol 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.

Purification of (S) -reticuline (S) -reticuline was purified from the culture supernatant by Oasis HLB solid phase extraction cartridge (Waters) and HPLC. The cartridge was pre-equilibrated with water before loading the culture supernatant. The cartridge was washed with 5% methanol and then (S) -reticuline was eluted with 100% methanol. The eluate was subjected to HPLC equipped with a TSKgel ODS-80Ts column under the above conditions. The peak fraction corresponding to (S) -reticuline was collected, and then the degree of purification was confirmed by MS analysis.

Results (S) -Reticuline Production by Recombinant Host Cells The two recombinant E. coli strains (tyrR :: null mutations produced as described above) and the expression plasmid tyrA ^fbr -aroG ^fbr -tktA-ppsA / pCOLADuet- 1. Recombinant production strain A with NCS-ORF378-TYR-DODC-optMAO / pET-21d and 6OMT-4'OMT-CNMT / pACYC184, and metabolic pathways involved in L-tyrosine production have been modified Without the expression plasmids NCS-ORF378-TYR-DODC-optMAO / pET-21d and 6OMT-4′OMT-CNMT / pACYC184 using a medium containing glucose as a carbon source. Cultured in a jar fermenter, media was collected at various time points to measure reticuline production.

As a result of liquid chromatography-mass spectrometry (LC-MS), 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 μg / L (FIG. 2). These results indicate that (S) -reticuline was obtained from glucose, a simple carbon source.

In the recombinant production strain A, (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.

As another experiment, the above recombinant production strains A and C were cultured in a medium containing glycerol as a carbon source, and the amount of reticuline produced was measured. As a result, in the recombinant production strain C, the maximum yield of 46 mg per 1 L of medium, which is approximately 7 times the yield of the recombinant production strain A under the same conditions (40.5 ± 4.8 mg as the average of three independent experiments). / L) (S) -reticuline was produced (FIG. 12). Such a result can be said to demonstrate that the use of RsTYR can achieve the production of reticuline without using an adapter protein and can increase the production of reticuline.

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.

In known methods for producing reticuline in vivo (

patent documents

1 and 2, non-patent document 14), 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.

In the method for producing reticuline using the recombinant host cell of the present invention, 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.

Compared with starting materials such as dopamine and norlaudanosoline, substances such as glucose and glycerol that can be used as a carbon source in the method of the present invention are inexpensive and readily available. Therefore, for example, the production cost of (S) -reticuline by the method of the present invention is very low as compared with the conventional production method using microorganisms disclosed in

Patent Documents

1 and 2 and Non-Patent Document 14.

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. For example, as a result of purifying (S) -reticuline from the medium using solid phase extraction and high performance liquid chromatography (HPLC), 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.

Claims

Non-isoquinoline alkaloid-producing cells with metabolic pathways that produce L-tyrosine, and tyrosinase or tyrosinase and its adapter protein, L-DOPA-specific decarboxylase, monoamine oxidase, norcoclaurine synthase, norcoclaurine 6-O-methyl At least one gene encoding at least one protein selected from the group consisting of transferase, coclaurin-N-methyltransferase and 3′-hydroxy-N-methylcoclaurine-4′-O-methyltransferase is introduced. Tyrosinase or its tyrosinase and its adapter protein, L-DOPA specific decarboxylase, monoamine oxidase, norcoclaurine synthase, norcoclaurine 6-O-methyltransferase, rich Providing a recombinant host cell expressing urin-N-methyltransferase and 3′-hydroxy-N-methylcoclaurine-4′-O-methyltransferase, and using the recombinant host cell as a carbon source with glucose, Culturing in a medium containing one or more sugars and / or glycerol selected from the group consisting of fructose, galactose, sucrose, lactose and maltose;
A method for producing (S) -reticuline, comprising:
The method according to claim 1, wherein the recombinant host cell does not have a tyrR gene or lacks its function.
The recombinant host cell is further one which expresses feedback inhibition resistant 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase and / or feedback inhibition resistant chorismate mutase / prephenate dehydrogenase. Item 3. The method according to Item 1 or 2.
The method according to any one of claims 1 to 3, wherein the recombinant host cell further overexpresses transketolase and / or phosphoenolpyruvate synthetase.
3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase resistant to feedback inhibition and chorismate mutase / prefenate resistant to feedback inhibition in non-isoquinoline alkaloid-producing cells with metabolic pathways that produce L-tyrosine Dehydrogenase, transketolase, phosphoenolpyruvate synthetase, tyrosinase or tyrosinase and its adapter proteins, L-DOPA specific decarboxylase, monoamine oxidase, norcoclaurine synthase, norcoclaurine 6-O-methyltransferase, coclaurin-N- Methyltransferase and at least one gene encoding at least one protein selected from the group consisting of 3'-hydroxy-N-methylcoclaurine-4'-O-methyltransferase Recombinant host cells introduced, which are 3-hydroxy-deoxy-D-arabino-heptulosonate-7-phosphate synthase resistant to feedback inhibition, chorismate mutase / prephenate dehydrogenase, transketolase resistant to feedback inhibition, Phosphoenolpyruvate synthetase, tyrosinase or its tyrosinase and its adapter protein, L-DOPA specific decarboxylase, monoamine oxidase, norcoclaurine synthase, norcoclaurine 6-O-methyltransferase, coclaurine-N-methyltransferase and 3'- Providing a recombinant host cell that expresses hydroxy-N-methylcoclaurine-4'-O-methyltransferase and does not have the tyrR gene or lacks its function; and The Culturing glucose, fructose, galactose, sucrose, in a medium containing one or more sugars and / or glycerol selected from the group consisting of lactose and maltose as Motogen,
A method for producing (S) -reticuline, comprising:
The method according to any one of claims 1 to 5, wherein the obtained reticuline is substantially free of (R) -reticuline.
The method according to any one of claims 1 to 6, wherein the isoquinoline alkaloid non-producing cells are selected from the group consisting of Escherichia coli, yeast, Bacillus subtilis and filamentous fungi.
The method according to any one of claims 1 to 7, wherein a gene encoding an aromatic L-amino acid decarboxylase other than L-DOPA-specific decarboxylase has not been introduced into the recombinant host cell.
Non-isoquinoline alkaloid-producing cells with metabolic pathways that produce L-tyrosine, tyrosinase or tyrosinase and its adapter protein, L-DOPA-specific decarboxylase, monoamine oxidase, norcoclaurine synthase, norcoclaurine 6-O-methyl At least one gene encoding at least one protein selected from the group consisting of transferase, coclaurin-N-methyltransferase and 3′-hydroxy-N-methylcoclaurine-4′-O-methyltransferase is introduced. Tyrosinase or tyrosinase and its adapter protein, L-DOPA specific decarboxylase, monoamine oxidase, norcoclaurine synthase, norcoclaurine 6-O-methyltransferase, Kuraurin -N- methyltransferase and 3'-hydroxy -N- methylcoclaurine cloud phosphorus-4'-O-methyl transferase recombinant host cells expressing.
The recombinant host cell according to claim 9, which has no tyrR gene or lacks its function.
Furthermore, it expresses feedback inhibition resistant 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase and / or feedback inhibition resistant chorismate mutase / prephenate dehydrogenase. Recombinant host cell.
The recombinant host cell according to any one of claims 9 to 11, which further overexpresses transketolase and / or phosphoenolpyruvate synthetase.
3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase resistant to feedback inhibition and chorismate mutase / prefenate resistant to feedback inhibition in non-isoquinoline alkaloid-producing cells with metabolic pathways that produce L-tyrosine Dehydrogenase, transketolase, phosphoenolpyruvate synthetase, tyrosinase or tyrosinase and its adapter protein, L-DOPA specific decarboxylase, monoamine oxidase, norcoclaurine synthase, norcoclaurine 6-O-methyltransferase, coclaurin-N- At least one gene encoding a methyltransferase and at least one protein selected from the group consisting of 3'-hydroxy-N-methylcoclaurine-4'-O-methyltransferase Recombinant host cells into which feedback inhibition resistance 3-deoxy-D-arabino-hepturosonate-7-phosphate synthase, feedback inhibition resistance chorismate mutase / prephenate dehydrogenase, transketolase , Phosphoenolpyruvate synthetase, tyrosinase or tyrosinase and its adapter protein, L-DOPA specific decarboxylase, monoamine oxidase, norcoclaurine synthase, norcoclaurine 6-O-methyltransferase, coclaurin-N-methyltransferase and 3 ' A recombinant host cell that expresses hydroxy-N-methylcoclaurine-4'-O-methyltransferase and does not have the tyrR gene or is deficient in its function.
The recombinant host cell according to any one of claims 9 to 13, which is selected from the group consisting of Escherichia coli, yeast, Bacillus subtilis and filamentous fungi.
The recombinant host cell according to any one of claims 9 to 14, wherein a gene encoding an aromatic L-amino acid decarboxylase other than L-DOPA-specific decarboxylase has not been introduced.