Hydroxylase involved in the biosynthesis of benzylisoquinoline alkaloids and its application

Abstract

The invention discloses a hydroxylase involved in the biosynthesis of benzylisoquinoline alkaloids and its application, and relates to the field of medicinal plant genetic engineering, wherein the amino acid sequence is SEQ ID NO.1, or a protein having hydroxylase function obtained by substitution and / or deletion and / or addition of one or more amino acid residues thereof; or a protein having 90% or more identity therewith and having the function of hydroxylating benzylisoquinoline alkaloids; a recombinant expression vector, a recombinant microorganism; a yeast system composed of the enzyme and a reductase StCPR, and the application of the hydroxylase and its recombinant expression vector in the in vitro synthesis of heterologously expressed StCYP80B protein, and the in vivo synthesis of benzylisoquinoline compounds by recombinant microorganisms and the yeast system. The hydroxylase StCYP80B provided by the invention expands the substrate recognition range and is an effective enzymatic tool for hydroxylating monobenzylisoquinoline alkaloids of different structures.

Description

Technical Field

[0001] The invention relates to the field of medicinal plant genetic engineering, in particular to a hydroxylase involved in the biosynthesis of benzylisoquinoline alkaloids and an application thereof. Background Art

[0002] Stephania tetrandra S.Moore is a plant of the genus Stephania in the family Menispermaceae. Its root is a famous traditional Chinese medicine, Stephania tetrandra, which has the effects of diuresis, detumescence, wind and pain relief. Stephania tetrandra is rich in benzylisoquinoline alkaloids (BIAs), mainly including monobenzylisoquinoline type, dibenzylisoquinoline type, aporphine type, protoberberine type, etc. These BIAs have multiple pharmacological effects such as anti-inflammatory, antibacterial, antiviral, and anti-tumor. However, the current wild resources of Stephania tetrandra are scarce, the scale of artificial cultivation is small, the planting cycle is long, and the content of active ingredients is not high, which limits the application and development of active BIAs. Biosynthesis is an important means to solve resource problems. Elucidating the key enzymes in the biosynthesis of BIAs in Stephania tetrandra can provide a theoretical basis for the efficient production of such active ingredients using biotechnology.

[0003] The biosynthesis of BIAs usually starts from the conversion of L-tyrosine into dopamine and 4-hydroxyphenylacetaldehyde, and then S-reticuline is generated under the catalysis of coclaurine synthase (NCS), and then the key intermediate S-reticuline is formed through three methyltransferases (6OMT, CNMT, 4′OMT) and hydroxylase (CYP80B or NMCH), and then various types of BIAs are formed through coupling, isomerization, rearrangement, hydroxylation, methylation, demethylation and other reactions. In the above pathway, from S-reticuline to S-reticuline are monobenzylisoquinoline alkaloids, and the structural modification of this type of compound plays an important role in various types of downstream BIAs, among which hydroxylase CYP80B is one of the key enzymes for structural modification of monobenzylisoquinolines.

[0004] At present, there is no report of CYP80B with clear function in Stephania tetrandra. The CYP80B found in other plants, called N-methylcoclaurine 3′-hydroxylase, can only catalyze the single substrate SN-methylcoclaurine ((S)-N-methylcoclaurine) to form S-3′-hydroxy-N-methylcoclaurine ((S)-3′-OH-N-methylcoclaurine), and cannot catalyze the substrate coclaurine without N-methyl group, and the substrate spectrum is single. Therefore, the discovery of CYP80B that can hydroxylate BIAs of different structures can broaden the substrate spectrum of this type of enzyme, which has important theoretical and practical value for the synthesis of monobenzylisoquinolines and their downstream multi-type BIAs.

[0005] Therefore, those skilled in the art are committed to developing a new hydroxylase with a diverse substrate spectrum to meet the demand for hydroxylation of benzylisoquinoline alkaloids (BIA) of different structures. Summary of the invention

[0006] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is how to develop a hydroxylase with a diverse substrate spectrum to meet the demand for hydroxylation of benzylisoquinoline alkaloids (BIA) of different structures.

[0007] To achieve the above object, the present invention provides a hydroxylase StCYP80B involved in the biosynthesis of benzylisoquinoline alkaloids, and the amino acid sequence of the hydroxylase StCYP80B is:

[0008] (a1) the amino acid sequence of hydroxylase StCYP80B is shown in SEQ ID NO.1;

[0009] or (a2) an amino acid sequence of a protein having hydroxylase function obtained by replacing and / or deleting and / or adding one or more amino acid residues in the amino acid sequence of (a1);

[0010] or (a3) ​​an amino acid sequence of a protein having 90% or more identity with the amino acid sequence of (a1) above and having a hydroxylated benzylisoquinoline alkaloid.

[0011] Furthermore, the nucleotide sequence of hydroxylase StCYP80B is:

[0012] (b1) the nucleotide sequence is shown in SEQ ID NO.2;

[0013] or (b2) a DNA sequence in which the nucleotide sequence of the above (b1) is substituted and / or deleted and / or added with one or more nucleotides;

[0014] or (b3) a DNA molecule having 90% or more identity with the nucleotide sequence shown in the above SEQ ID NO. 2 and encoding a CYP80B protein.

[0015] The invention also provides a recombinant expression vector of the gene of hydroxylase StCYP80B, and the recombinant vector comprises a subcloning vector or a yeast cell expression vector.

[0016] Furthermore, the recombinant vector containing the StCYP80B gene includes a subcloning vector and a microbial cell expression vector such as yeast.

[0017] The present invention also provides a recombinant microorganism of the gene of hydroxylase StCYP80B, and the recombinant microorganism comprises a yeast cell.

[0018] Furthermore, the recombinant microorganism containing the StCYP80B gene includes microbial cells such as yeast.

[0019] The invention also provides a yeast system for co-expressing StCYP80B and StCPR, wherein the yeast system consists of hydroxylase StCYP80B and reductase StCPR.

[0020] Furthermore, the amino acid sequence of the reductase StCPR is shown in SEQ ID NO.3, and its encoding gene is shown in SEQ ID NO.4.

[0021] The present invention also provides an application of hydroxylase StCYP80B in in vitro synthesis of benzylisoquinoline compounds.

[0022] The present invention also provides an application of a recombinant expression vector containing the StCYP80B gene in in vitro synthesis of a heterologously expressed StCYP80B protein.

[0023] The present invention also provides an application of a recombinant microorganism containing the StCYP80B gene in synthesizing benzylisoquinoline compounds in vivo.

[0024] The present invention also provides an application of a yeast system in synthesizing benzylisoquinoline compounds in vivo.

[0025] Furthermore, the hydroxylase protein was derived from Stephania tetrandra and named StCYP80B.

[0026] Furthermore, CYP450 Reductase (CPR) was derived from Stephaniatetrandra and named StCPR.

[0027] Furthermore, the hydroxylation conversion rate of benzylisoquinoline alkaloids in the yeast system co-expressing StCYP80B and StCPR was significantly higher than that in the system expressing only StCYP80B.

[0028] Furthermore, the hydroxylase StCYP80B catalyzes the use of the substrate coclaurine which does not have an N-methyl group.

[0029] Further, application of StCYP80B as hydroxylase.

[0030] Furthermore, the StCYP80B gene is used in the preparation of hydroxylase.

[0031] Furthermore, the recombinant expression vector containing the StCYP80B gene is used in the preparation of hydroxylase.

[0032] Furthermore, the recombinant microorganism containing the StCYP80B gene is used in the preparation of hydroxylase.

[0033] Furthermore, the application of StCYP80B protein in the biosynthesis of benzylisoquinoline alkaloids.

[0034] Furthermore, the protein of StCYP80B is used in the preparation of benzylisoquinoline alkaloids.

[0035] Furthermore, the hydroxylase StCYP80B can catalyze the single substrate SN-methylcoclaurine ((S)-N-methylcoclaurine) to form S-3′-hydroxy-N-methylcoclaurine, and can also catalyze the substrate coclaurine without N-methyl group.

[0036] In the preferred embodiment 1 of the present invention, the discovery process of a benzylisoquinoline alkaloid 3′ hydroxylase (StCYP80B) and its encoding gene in Stephania tetrandra is described in detail;

[0037] In another preferred embodiment 2 of the present invention, the function of StCYP80B verified in vivo in Saccharomyces cerevisiae is described in detail;

[0038] In another preferred embodiment 3 of the present invention, the extraction of StCYP80B microsomal protein and the in vitro catalysis process are described in detail.

[0039] The beneficial technical effects of the present invention are as follows:

[0040] The present invention discloses a novel hydroxylase StCYP80B and a coding gene thereof. StCYP80B can catalyze N-methylcoclaurine to form 3′-hydroxy-N-methylcoclaurine (3′-OH-N-methylcoclaurine), and can also catalyze coclaurine (coclaurine), a substrate without N-methyl, to form 3′-hydroxy-coclaurine (3′-OH-coclaurine). Compared with the reported CYP80B, StCYP80B has a wider substrate recognition range and is an effective enzymatic tool for hydroxylating monobenzylisoquinoline alkaloids of different structures, which is of great significance for the synthesis of monobenzylisoquinolines and their downstream multi-type BIAs.

[0041] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1It is the LC-MS and LC-MS / MS analysis chromatogram of the hydroxylation product formed by (R, S)-N-methylcoclaurine catalyzed by StCYP80B in the yeast system of a preferred embodiment 2 of the present invention;

[0043] Figure 2 It is the LC-MS and LC-MS / MS analysis chromatogram of the hydroxylation product formed by (R, S)-coclaurine catalyzed by StCYP80B in the yeast system of a preferred embodiment 2 of the present invention. DETAILED DESCRIPTION

[0044] The following describes several preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0045] Example 1: Discovery of a benzylisoquinoline alkaloid 3′-hydroxylase (StCYP80B) in Stephania tetrandra and its encoding gene

[0046] The transcriptome database of Stephania tetrandra was obtained by sequencing the transcriptome of root and leaf tissues of Stephania tetrandra. After Blast screening and sequence alignment with sequences from public data such as NCBI and KEGG, 6 candidate sequences of CYP80B were mined. After heterologous expression in Saccharomyces cerevisiae and substrate feeding, it was determined that one of the proteins had the function of catalyzing the 3′ hydroxylation of (R, S)-N-methylcoclaurine and (R, S)-coclaurine. The protein was named StCYP80B, and its amino acids are shown in SEQ ID NO.1 of the sequence table, and it consists of 490 amino acids. The gene encoding the StCYP80B protein was named StCYP80B gene, and its open reading frame cDNA sequence is shown in the sequence SEQ ID NO.2 of the sequence table.

[0047] By the same method, a CYP450 reductase was mined from the transcriptome of Stephania tetrandra, and the protein was named StCPR, which consists of 690 amino acids, and its amino acid sequence is shown in SEQ ID NO.3 of the sequence table. The gene encoding the StCPR protein was named StCPR, and its open reading frame cDNA sequence is shown in SEQ ID NO.4 of the sequence table. In the heterologous expression system of Saccharomyces cerevisiae, the hydroxylation conversion rate of (R, S)-N-methylcoclaurine and (R, S)-coclaurine in the system co-expressing StCYP80B and StCPR was significantly higher than the conversion rate of the system expressing only StCYP80B.

[0048] Example 2: Verification of the function of StCYP80B in Saccharomyces cerevisiae

[0049] 1. Construction of heterologous expression plasmid

[0050] The free plasmid pESC-Leu of Saccharomyces cerevisiae was double digested with restriction endonucleases NotⅠ and SacⅠ, and the linearized plasmid was recovered by gel. Primers (F1 and R1) containing homology arms at both ends of the plasmid were designed, and the StCYP80B gene with homology arms was amplified by PCR from the cDNA sample of Stephania tetrandra, and the PCR product was recovered and purified.

[0051] The primer sequence F1 is as shown in SEQ ID NO.5: 5′-CTCACTAAAGGGCGGCCGCATGGAGATAGTCTCTGC-3′; the primer sequence R1 is as shown in SEQ ID NO.6: 5′-TCACTAAAGGGCGGCCGCATGGATCAAACCATCCTCTC-3′.

[0052] The recombinant plasmid pESC-Leu-StCYP80B was constructed by homologous recombination, and Escherichia coli DH10B was transformed by heat shock method. The ampicillin-resistant transformants were picked and amplified in LB medium. After the plasmid was extracted, PCR, restriction digestion and sequencing were performed in sequence to obtain the recombinant plasmid pESC-Leu-StCYP80B with correct sequencing.

[0053] The free plasmid pESC-Leu-StCYP80B of Saccharomyces cerevisiae was double digested with restriction endonucleases XhoⅠ and NheⅠ, and the linearized plasmid was recovered by gel. Primers (F2 and R2) containing homology arms at both ends of the plasmid were designed, and the StCPR gene with homology arms was amplified by PCR from the cDNA sample of Stephania tetrandra, and the PCR product was recovered and purified.

[0054] The primer sequence F2 is as shown in SEQ ID NO.7: 5′-TTTCCGAAGAAGACCTCGAGATGGCTTCCAAGTACGCGAA-3′; the primer sequence R2 is as shown in SEQ ID NO.8: 5′-TAGAGCGGATCTTAGCTAGCTCACCAAACGTCCCTGAGAT-3′.

[0055] The same method and steps as those for constructing plasmid pESC-Leu-StCYP80B were used to obtain the correctly sequenced recombinant plasmid pESC-Leu-StCYP80B-StCPR.

[0056] 2. Obtaining recombinant yeast strains

[0057] The recombinant plasmid pESC-Leu-StCYP80B or pESC-Leu-StCYP80B-StCPR obtained in step 1 was transformed into the Saccharomyces cerevisiae YPH499 strain using the Frozen-EZ yeast Transformation II kit. The bacterial solution was spread onto the defective medium SD-Leu plate, and a single colony was picked after culturing at 30°C for 2 days. At the same time, the empty plasmid was transformed into the YPH499 strain to obtain a blank control bacterium.

[0058] 3. Yeast Whole Cell Catalysis

[0059] Pick the monoclonal colony grown on the defective plate in step 2 to 15 ml SD-Leu liquid medium, and culture overnight at 30°C and 220 rpm. Control OD600 to 0.8-1.2, centrifuge (3000 rpm, 5 min), remove SD-Leu, replace with induction medium SG-Leu, and centrifuge after 20 hours of induction culture to obtain bacterial cell pellet.

[0060] The cells were washed once with 50 mM PBS and resuspended to a final volume of 500 μl. The substrates (R, S)-N-methylcoclaurine and (R, S)-coclaurine were fed to a final concentration of 0.25 mM. The whole-cell catalysis was carried out at 30°C and 220 rpm for 24 h.

[0061] 4. Reaction Solution Treatment and Detection

[0062] An equal volume of methanol was added to the reaction solution in step 3 to terminate the reaction. After ultrasonic extraction for 15 min, the precipitate was removed by centrifugation (12000 rpm, 10 min). The supernatant was filtered through a 0.22 μm organic filter membrane and analyzed by LC-UV-MS and LC-MS / MS.

[0063] The LC-UV-MS detection conditions are as follows: the liquid phase system is Shimadzu LCMS-2020; the chromatographic column is Shim-pack XR-ODSⅢ (2.0mm×75mm, 1.6μm); the column temperature is room temperature (about 25°C); the flow rate is 0.2ml / min; the mobile phase is A (0.1% formic acid aqueous solution)-B (methanol) for gradient elution, and the time program is: 0min, 5% (v / v) B%; 10min, 45% (v / v) B%; 11min, 95% (v / v) B%; 12min, 95% (v / v) B%; PDA detection, the monitoring wavelength is 282nm; mass spectrometry detection uses an electrospray ionization ion source (ESI) to collect positive ions, and the scanning range is 100-800m / z; the interface voltage is 4.5kv.

[0064] The LC-MS / MS detection conditions were as follows: the liquid phase system was Shimadzu LCMS-8060; the chromatographic column was Venusil XBP PH (2.1×100 mm, 5 μm); the column temperature was 40°C; the flow rate was 0.5 ml / min; the mobile phase was A (0.1% formic acid aqueous solution)-B (acetonitrile), and the time program was: 0 min, 5% (v / v) B%; 5 min, 25% (v / v) B%; 7 min, 95% (v / v) B%; 9 min, 95% (v / v) B%; the mass spectrometry detection adopted ESI to collect positive ions, and the interface voltage was 4.0 kV; the scanning mode was multiple reaction monitoring (MRM), and the following ion pairs were selected for detection: substrate N-methylcoclaurine m / z 300.25→107.15 and its product 3′-hydroxy-N-methylcoclaurine m / z 316.15→123.05, substrate clavaline m / z 286.25→107.15 and its product 3′-hydroxy-clavaline m / z 302.15→123.05.

[0065] The results of the substrate (R, S)-N-methylcoclaurine are as follows Figure 1 As shown, Figure 1 The LC-MS and LC-MS / MS analysis chromatograms of the hydroxylation product formed by (R,S)-N-methylcoclaurine catalyzed by StCYP80B in the yeast system, wherein: Figure 1 The first structural formula in the upper part is 3′-hydroxy-N-methylcoclaurine; the second structural formula is N-methylcoclaurine; Part 1A is the LC-MS extracted ion chromatogram of the culture medium blank control and the enzymatic reaction solution, and Part 1B is the LC-MS / MS chromatogram of the reference substance and the enzymatic reaction solution. The chromatographic peak "1" represents the compound 3′-hydroxy-N-methylcoclaurine, and the chromatographic peak "2" represents the compound N-methylcoclaurine; the culture medium blank control is the culture medium with substrate added but without StCYP80B, and the enzymatic reaction solution is a sample of the substrate catalyzed by the whole cell of recombinant Saccharomyces cerevisiae co-expressing StCYP80B and StCPR. . The mass spectrometry extracted ion chromatogram of LC-MS detection in part 1A shows that Saccharomyces cerevisiae heterologously expressing StCYP80B can catalyze N-methylcoclaurine to produce a hydroxylation product Product 1 with an m / z increase of 16Da; the LC-MS / MS chromatogram in part 1B shows that the ion pair m / z and retention time of Product 1 generated in the enzymatic reaction solution are consistent with those of the 3′-hydroxy-N-methylcoclaurine reference substance, indicating that 3′-hydroxy-N-methylcoclaurine is formed by hydroxylation at the 3′ position.

[0066] The results when the substrate is (R,S)-coclaurine are as follows Figure 2 As shown, Figure 2The LC-MS and LC-MS / MS analysis chromatograms of the hydroxylation product formed by (R,S)-coclaurine catalyzed by StCYP80B in the yeast system, wherein: Figure 2 The first structural formula in the upper part is 3′-hydroxy-coclaurine; the second structural formula is coclaurine; Part 2A is the LC-MS extracted ion chromatogram of the culture medium blank control and the enzymatic reaction solution, and Part 2B is the LC-MS / MS chromatogram of the reference substance and the enzymatic reaction solution. The chromatographic peak "3" represents the compound 3′-hydroxy-coclaurine, and the chromatographic peak "4" represents the compound coclaurine. The culture medium blank control is the culture medium with substrate added but without StCYP80B, and the enzymatic reaction solution is a sample of the substrate catalyzed by the whole cell of recombinant Saccharomyces cerevisiae co-expressing StCYP80B and StCPR. The mass spectrometry extracted ion chromatogram of LC-MS detection in part 2A shows that the Saccharomyces cerevisiae heterologously expressing StCYP80B can catalyze the production of a hydroxylated product Product 2 with an m / z increase of 16Da from rutinine. The LC-MS / MS chromatogram in part 2B shows that the ion pair m / z and retention time of Product 2 generated in the enzymatic reaction solution are consistent with those of the 3′-hydroxy-rutinine reference substance, indicating that 3′-hydroxy-rutinine is formed by hydroxylation at the 3′ position.

[0067] Comparing the two yeast systems transformed with plasmids pESC-Leu-StCYP80B-StCPR and pESC-Leu-StCYP80B, the hydroxylation conversion rate of substrates N-methylcoclaurine and coclaurine in the former was 3-100 times that of the latter, indicating that co-expression of StCPR in yeast can enhance the catalytic effect of StCYP80B.

[0068] The above results indicate that StCYP80B has the function of catalyzing the hydroxylation of N-methylcoclaurine and the 3′ position of coclaurine; StCPR can effectively improve the catalytic activity of StCYP80B in a heterologous expression system.

[0069] Example 3. Extraction of StCYP80B microsomal protein and in vitro catalysis

[0070] 1. Extraction of microsomal proteins

[0071] Obtain bacterial cell precipitation according to the method of steps 1 to 3 in Example 2. Add 1 ml of TESB buffer per gram of bacterial cells, use an oscillating cell disruptor to disrupt the bacterial cells, with an oscillation speed of 6 m / s, a time of 10 s / time, and place on ice for 3 min, repeat 6 times. Centrifuge (10,000g, 4°C, 20min), take the supernatant, ultracentrifuge (100,000g, 4°C, 1h), obtain microsomal precipitation, and dissolve in an appropriate amount of TEG buffer. Use the modified Bradford protein assay kit of Bioengineering to determine the microsomal protein concentration.

[0072] 2. In vitro enzymatic reaction

[0073] Take the microsomal precipitate and prepare the enzymatic reaction system. Each reaction system is 0.5 ml, containing 1 mg of microsomal protein, 0.05 mM FAD, 0.05 mM FMN, 1 mM NADPH, appropriate amount of substrates (R, S)-N-methylcoclaurine and (R, S)-coclaurine, and TEG buffer (PH: 7.5) to 0.5 ml. React at 30°C, 220 rpm for 2 hours to obtain the enzymatic reaction solution.

[0074] 3. Reaction Solution Treatment and Detection

[0075] An equal volume of methanol was added to the enzymatic reaction solution to terminate the reaction, vortexed and mixed, centrifuged (12000 rpm, 4°C, 5 min), and the supernatant was analyzed by LC-MS / MS. The detection conditions were the same as the LC-MS / MS method in step 4 of Example 2.

[0076] The results showed that StCYP80B protein could catalyze the hydroxylation reaction of N-methylcoclaurine and the 3′ carbon atom of coclaurine to generate 3′-OH product.

[0077] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A hydroxylase StCYP80B involved in the biosynthesis of benzylisoquinoline alkaloids, It is characterized in that The amino acid sequence of the hydroxylase StCYP80B is: (a1) The amino acid sequence of the hydroxylase StCYP80B is shown in SEQ ID NO.

1.

2. The hydroxylase StCYP80B according to claim 1, It is characterized in that The nucleotide sequence of the hydroxylase StCYP80B is: (b1) The nucleotide sequence is shown in SEQ ID NO.

2.

3. A recombinant expression vector containing the gene of the hydroxylase StCYP80B as claimed in claim 1 or 2, It is characterized in that The recombinant vector includes a subcloning vector or a yeast cell expression vector.

4. A recombinant microorganism containing the gene of the hydroxylase StCYP80B as claimed in claim 1 or 2, It is characterized in that The recombinant microorganism includes a yeast cell.

5. A yeast system for co-expressing the hydroxylase StCYP80B and the reductase StCPR as claimed in claim 1 or 2, It is characterized in that The amino acid sequence of the reductase StCPR is shown in SEQ ID NO.3, and its encoding gene is shown in SEQ ID NO.

4.

6. Use of the hydroxylase StCYP80B as claimed in claim 1 or 2 in catalyzing N-methylcoclaurine to form 3'-hydroxyN-methylcoclaurine and catalyzing the formation of 3'-hydroxy-coclaurine from the substrate coclaurine without N-methyl in vitro.

7. Use of the recombinant expression vector according to claim 3 in heterologously expressing StCYP80B protein to catalyze N-methylcoclaurine to form 3'-hydroxyN-methylcoclaurine and catalyze the formation of 3'-hydroxy-coclaurine from N-methylcoclaurine in vitro.

8. Use of the recombinant microorganism according to claim 4 in catalyzing N-methylcoclaurine to form 3'-hydroxyN-methylcoclaurine and catalyzing the formation of 3'-hydroxy-coclaurine from the substrate coclaurine without N-methyl group in vivo.

9. Use of the yeast system as claimed in claim 5 in vivo to catalyze N-methylcoclaurine to form 3'-hydroxyN-methylcoclaurine and to catalyze the formation of 3'-hydroxy-coclaurine from the substrate coclaurine without N-methyl group.

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