NORCOCLAURINE SYNTHASES WITH INCREASED ACITVITY

Abstract

The invention relates to norcoclaurine synthases and substrate binding sites having one or more site-specific mutation which increase the activity, when compared to the wild type synthase, of the condensation of 4-HPAA and dopamine to (S)-norcoclaurine and/or 3,4-DhPAA and dopamine to (S)-norlaudanosoline. The inventors both identified specific mutations corresponding to at position 73, 75, 77, 82, 99, 114, 141, 142, 147, 152, 174 and/or 178 in the count according to SEQ ID No: 1, and sites corresponding to the binding domains defined in SEQ IN NO: 4 and 5, where the mutated increase of the activity may be positioned within these norcoclaurine synthases. These domains are conserved regions.

Claims

1-15. (canceled)

16. A method for preparing a composition of matter comprising a benzylisoquinoline alkaloid (BIA) from a (S)-norcoclaurine and/or (S)-norlaudanosoline substrate comprising the steps of: a) providing a recombinant host cell comprising a variant norcoclaurine synthase capable of catalyzing the condensation of 4-hydroxy-phenylacetaldehyde (4-HPAA) and dopamine into (S)-norcoclaurine and/or 3,4-dihydroxy-acetaldehyde (3,4-DhPAA) and dopamine into (S)-norlaudanosoline, wherein the variant norcoclaurine synthase comprises: i) one or more site-specific substitutions corresponding to positions 73, 77, 99, 114, 142, 152, 174, and 178 of SEQ ID NO: 1; or ii) one or more site-specific substitutions corresponding to positions 73, 77, 99, 114, 142, 152, 174, and/or 178 of SEQ ID NO: 1, and an N-terminal truncation; and b) culturing the recombinant host cell under conditions promoting said catalyzation; and optionally c) isolating the BIA.

17. The method according to claim 16, wherein the variant norcoclaurine synthase comprises a heterologous amino acid sequence which is at least 50% identical to a norcoclaurine synthase comprised in the sequence of SEQ ID NO: 1, or an N-terminal truncation thereof.

18. The method according to claim 16, wherein the one or more site-specific substitutions is one or more of A73P, A77S, A77E, A77T, Q99K, Q99R, K114E, V142I, K152R, V174E, V174G, V174Q, I178A, I178S, I178D, I178N, I178Q, and I178T of SEQ ID NO: 1.

19. The method according to claim 16, wherein the one or more site-specific substitutions are at positions i) 141, 142 and 152; ii), 75 and 178; or iii) 75, 152, 178 of SEQ ID NO: 1.

20. The method according to claim 19, wherein the one or more site-specific substitutions are i) V141I, V142I and K152R; ii) I75L, I178D or I75K, I178D; or iii) I75K, K152R and I178D of SEQ ID NO: 1.

21. The method according to claim 16, wherein the variant norcoclaurine synthase comprises an endoplasmic reticulum (ER) retention signal at the C-terminus of the variant norcoclaurine synthase.

22. The method according to claim 21, wherein the ER retention signal comprises the amino acid sequence HDEL (SEQ ID NO: 22) at the C-terminus of the variant norcoclaurine synthase.

23. The method according to claim 16, wherein the variant norcoclaurine synthase comprises the N-terminal truncation.

24. The method according to claim 23, wherein the N-terminal truncation comprises a substitution or a deletion of one or more amino acids between residues 1-19 of SEQ ID NO: 1; wherein signal peptide function is disrupted.

25. The method according to claim 23, wherein the N-terminal truncation comprises a deletion of residues 1-19 of SEQ ID NO: 1.

26. The method according to claim 16, wherein the variant norcoclaurine synthase has an increased catalyzation of the condensation of 4-hydroxy-phenylacetaldehyde (4-HPAA) and dopamine into (S)-norcoclaurine and/or 3,4-dihydroxy-acetaldehyde (3,4-DhPAA) and dopamine into (S)-norlaudanosoline when compared to a wild-type synthase of SEQ ID NO: 1.

27. The method according to claim 16, wherein the recombinant host cell comprises a nucleic acid sequence encoding a norcoclaurine synthase, wherein the nucleic acid sequence is at least 70% identical to a nucleic acid sequence of SEQ ID NO: 2 encoding a norcoclaurine synthase.

28. The method according to claim 27, wherein the nucleic acid sequence is codon optimized for S. cerevisiae.

29. The method according to claim 27, wherein the nucleic acid sequence is at least 60% identical to the nucleic acid sequence of SEQ ID NO: 3.

30. The method according to claim 29, wherein the nucleic acid sequence is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 3.

31. The method according to claim 16, wherein the recombinant host cell is a yeast cell, a plant cell, a mammalian cell, an insect cell, a fungal cell, a bacterial cell, an algal cell, or a cyanobacterial cell.

32. The method according to claim 31, wherein the recombinant host cell is a yeast cell selected from the group consisting of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Xanthophyllomyces dendrorhous, Candida albicans, Rhodotorula sp., or Rhodospiridium sp.

33. The method according to claim 32, wherein the recombinant host cell is a Saccharomyces.

34. The method according to claim 33, wherein the recombinant host cell is a yeast cell is a Saccharomyces cerevisiae cell.

35. The method according to claim 16, wherein the BIA is selected from the group consisting of Thebaine, Oripavine, Neopinone, Codeinone, Hydrocodone, Morphine, Oxycodone, Codeine, Noscapine, Berberine, Sanguinarine, Tubocurarine, and Papaverine.

36. A benzylisoquinoline alkaloid (BIA) produced by the method of claim 16.

37. The BIA according to claim 36, wherein the BIA is thebaine, oripavine, neopinone, codeinone, hydrocodone, morphine, oxycodone, codeine, noscapine, berberine, sanguinarine, tubocurarine, or papaverine.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0214] FIG. 1Condensation

[0215] NCS catalyzes the condensation of 4-HPAA and dopamine to (S)-norcoclaurine or 3,4-DHPAA and dopamine to (S)-norlaudanosoline.

[0216] FIG. 2pEV3307 plasmid

[0217] Schematic map of pEV3307 plasmid. Vector elements are annotated.

[0218] FIG. 3Norcoclaurine biosynthesis in yeast expressing natural homologs

[0219] Screening of NCS homologs from various plant species. Norcoclaurine titers of the cultures after the 72 hours' cultivation time are represented by bars, optical densities of cultures are shown by dots.

[0220] FIG. 4Norcoclaurine biosynthesis in yeast expressing NCS mutants

[0221] Screening of variants of norcoclaurine synthase from Coptis japonica. Norcolalurine titers of the cultures after the 72 hours' cultivation time are represented by bars, optical densities of cultures are shown by dots.

[0222] FIG. 5Cj NCS homology model

[0223] Cj NCS homology model with active site predicted residues shown (stick form) and reactive substrates shown in space filling format.

[0224] FIG. 6HPAA substrate enhanced interaction with mutation I178D (magenta)

[0225] Modelled interaction between mutant I178D (magenta) and HPAA substrate (cyan). According to this model I178D is predicted to directly interact with aldehyde substrates.

[0226] FIG. 7Disruption of signal peptide function of the C. japonica NCS

[0227] Norcoclaurine production in S. cerevisiae increases dramatically when the first 19 amino acids from the N-terminal of the C. japonica NCS are replaced by a methionine. Duplicate determinations of norcoclaurine production in 4 different S. cerevisiae.

[0228] FIG. 8HDEL (SEQ ID No:22) addition in the C-terminus

[0229] The CjNCS I178D improves the activity of the C. japonica NCS version with the HDEL (SEQ ID No: 22) addition in the C-terminus. Duplicate determinations of norcoclaurine production in 4 different S. cerevisiae strains. A) 11ari1::Aro4.sup.FBR CYP76AD1.sup.W13L, F309L PpDODC CjNCS1co. B) 11ari1: : Aro4FBR CYP76AD1W13L, F309L PpDODC CjNCS1co/178D C) 11ari1::Aro4.sup.FBR CYP76AD1 .sup.W13L, F309L PpDODC CjNCS1co-HDEL. D) 11ari1::Aro4.sup.FBR CYP76AD1W.sup.13L, F309L PpDODC CjNCS1co.sup.I178D-HDEL.

EXAMPLES

Example 1Protein Model of NCS From Coptis japonicaStructural Template

[0230] Protein sequences of Coptis japonica (Cj) and Thalictrum flavum (Tf) NCS were aligned using Clustal Omega (Sievers et al., 2011, Mol Syst Biol. 7 : 539) along with other NCS homologs identified using the BLASTP algorithm up to 40% identity.

[0231] The sequence of Cj NCS has 63% global protein sequence identity to the sequence of Thalictrum flavum (Tf) NCS, indicating that their three-dimensional protein structures are highly likely to be similar. Therefore, a protein model of Cj norcoclaurine synthase (NCS) was constructed using the atomic co-ordinates of the protein structure of NCS from Thalictrum flavum (Tf) (PDB code 2VQ5; Ilari et al. 2009).

[0232] A homology model of Cj NCS was constructed using Homology Model tool in MOE (CCG Inc). A protein modelling software well known and available in the art.

[0233] The Cj NCS model allows key residue-specific interactions at the interface between Cj NCS and substrates dopamine and 4-hydroxyphenylacetylaldehyde to be discerned. Final models were constructed with reactive modelled conformations of dopamine and 4-hydroxyphenylacetylaldehyde [FIG. 5]. These protein models provided the basis to select key residues to mutate in order to affect ligand binding potentials.

[0234] Obtained gene sequences were codon optimized for expression in Saccharomyces cerevisiae, and their synthesis was performed by Thermofischer Scientific Int.

[0235] Synthetized genes were cloned in pEV3307 following the procedure described in Example 3. The mutants showing increased NCS versus the wild type enzyme are summarized in Table 8.

Example 2Cj NCS Activity Increase by Enzyme Engineering ApproachActive Site Model->Design Mutations

[0236] From the constructed homology model, amino acids were selected for in silico mutation studies using the PROTOSCAN methodology as described by example in WO 2013022881. Briefly, selected amino acids are swapped for each of 19 possible amino acid mutations, the coordinates of which are determined by selection of low energy rotamers before calculating both intra and intermolecular energy of interaction versus wild type residue.

[0237] Thus, amino acid mutations can be elucidated, which provide lower binding energy in the context of substrates.

[0238] For example, position 178 of NCS contains Isoleucine. PROTOSCAN ranked energies of other mutations at this position and aspartic acid had the lowest energy, because of enhanced interaction with the para hydroxyl group of HPAA substrate as shown in FIG. 6.

[0239] The mutation was tested and demonstrated higher activity than the wild type protein. Thus, the in silico screen identified enriching mutation selection versus a random selection of mutants and can effectively sample sequence space in particular, where a substrate binding model is available.

Example 3Preparation of Expression Vectors

[0240] NCS gene sequences from different organisms (Table 2) were identified and codon optimized for expression in Saccharomyces cerevisiae.

[0241] The synthesis of the resulting sequences was performed by Thermofisher Scientific Inc. Gene sequences and related organism of origin are shown in Table 2.

[0242] The newly synthesized genes were cloned in the yeast expression plasmid pEV3307 (pRS313 modified with the insertion of PGK1 and TEF1 promoters, CYC1 and ADH 1 terminators. For basic plasmid details see Mumberg et al. 1995, Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds, Gene 156(I):119-22, 1995) carrying the HIS3 auxotrophic marker.

[0243] HindIII/SacII restriction sites were utilized for the insertion of the open reading frames between PGK1 promoter and CYC terminator.

[0244] A schematic map of plasmid pEVE3307 with related annotations is shown in FIG. 2.

Example 4Strain Construction and Expression of NCS Homologs

[0245] EVST25898, a modified version of S288C yeast strain (genotype: MATalpha his3A0 leu20 ura3A0 aro3::pTEFI-ARO4(K229L)-tCYCI::pPGKI-ARO7(T266L)-tADH1::KICAT5-91Met GAL2 ho MIP1-661Thr SAL1-1 YORW22::npBIO1nt-npBIO6nt) was transformed with the constructs described in Example 1.

[0246] Yeast transformation was performed according to conventional methods (see R. D. Gietz and R. Woods, Yeast Transformation by the LiAc/SS Carrier DNA/PEG Method, in Yeast Protocol SE12, vol. 313, W. Xiao, Ed. Humana Press, 2006, pp. 107-120).

[0247] The resulting yeast strains were grown in a 96-deep well plate in appropriate medium (standard SC medium without histidine) for 24 h at 30 C. (pre-culture) with 300 rpm shaking. Cells were then inoculated into fresh SC-HIS medium reaching an initial OD6000.1. Obtained cultures were incubated at 30 C. for 72 h. After 72 h the cells were harvested and the culture supernatants were analyzed according to the methods reported in Examples 6 and 7.

[0248] Norcoclaurine was detected in the supernatant of the strain carrying Coptis japonica NCS, at a concentration of 0.35 mg/l. Results are reported in the graph in FIG. 3-4.

Example 5Screening of Cj_NCS Mutants

[0249] Strain EVST25898 was transformed with the constructs obtained in Example 3 (see Example 2 for genotype) in 96 deep well plate format with a commercially available kit (Froze-EZ Yeast transformation II Kit, Zymo research) according to the manufacturer instructions.

[0250] Transformed cells were grown in SC-HIS medium and incubated for 24 h at 30 C. under mild shaking. 20 l of the transformation cultures were used to inoculate a pre-culture in SC-HIS medium (in 96-well format) and the resulting plate was incubated for 24 h at 30 C. 5 l of pre-cultures were inoculated in fresh SC-HIS standard medium, or with modified composition (0.6 mM ammonium sulfate, 1.4 mM tyrosine, 1.5 mg/mg dopamine) and incubated for 72 h.

[0251] Analysis of culture supernatants was performed according to the method reported in Examples 6 and 7.

[0252] A selection of mutants, including the ones showing increased norcoclaurine production versus the control strain after the first screening, were repeated for experimental confirmation and the results of the analysis are shown in FIG. 4.

[0253] Variants V48, V49, V93, V151, V152, V240, V241, V242, V246, V247, V249, V253, V261 and V273 showed increased norcoclaurine production up to about 1.8-fold.

Example 6Norcoclaurine Chiral Analysis

[0254] Norcoclaurine HCl (also called higenamine, Carbosynth) solution (1 g/L) was prepared in water. A series of calibration solutions at 4 mg/L, 2 mg/L, 1 mg/L, 0.5 mg/L, 0.25 mg/L, 0.125mg/L, 62.5 ug/L and 31.25 ug/L in the culture medium was prepared from this stock solution. Caffeine (Sigma) was added to the samples as internal standard to a concentration of 1mg/L and samples were injected into the UPLC-SQD (Waters).

[0255] The LC-MS method was as follows: Mobile Phase A: water+0.1% formic acid; Mobile Phase B: acetonitrile+0.1% formic acid; Column : ORpakCDBS453 (Shodex).

[0256] The elution gradient is shown in Table 3 and the LC-MS conditions are given in Table 4.

[0257] Table 5 shows the mass spectrometer source and detector parameters. Norcoclaurine and Caffeine were injected analysed in Single lon Monitoring mode with the respective mass over charge ratio (m/z) 227 and 195 corresponding to their protonated ion. (S)-Norcoclaurine eluted at 5.8 min, (R)-Norcoclaurine at 6.1 min and Caffeine at 6.3 min.

TABLE-US-00003 TABLE 3 Gradient for chiral separation Time (min) % B 0 1 5 35 8 100 9 100 9.1 1 10 1

TABLE-US-00004 TABLE 4 LC-MS conditions Injection volume 3 l Column Temperature 30 C. 5 C. Injection method Partial loop Flow 0.4 ml/min Auto sampler temperature 10 C. 2 C. Weak wash 800 l water/acetonitrile 8:2 Strong wash 300 l MeOH Seal wash 5 min with water/acetonitrile 9:1

TABLE-US-00005 TABLE 5 Mass spectrometer source and detector parameters (SQD) Source Parameter Value Ion Source Electrospray Positive Mode (ESI+) Capillary Voltage 3.5 kV Cone Voltage 20 V Extractor 3 V RF lens 0.1 V Source Temperature 150 C. Desolvation temperature 350 C. LM resolution 15 HM resolution 15 Ion Energy 0.5 eV Mode MS LM resolution2 15 HM resolution2 15 Ion Energy2 0.5 API gas 500 L/hour

Example 7Norcoclaurine Non-Chiral Analysis

[0258] Ig/L Higenamine HCl (Carbosynth) solution was prepared in water. A series of calibration solutions at 4 mg/L, 2 mg/L, 1 mg/L, 0.5 mg/L, 0.25 mg/L, 0.125 mg/L, 62.5 g/L and 31.25g/L in the culture medium was prepared from this stock solution. Caffeine (Sigma) was added as internal standard to a concentration of Img/L and samples were injected into the UPLC-SQD (Waters).

[0259] The LC-MS method was as follows: Mobile Phase A: water+0.1% formic acid; Mobile Phase B: acetonitrile+0.1% formic acid; Column: Aquity BEH C181002.1 mm (Waters).

[0260] The elution gradient is shown in Table 6 and the LC-MS conditions are given in Table 7.

[0261] Table 5 shows the mass spectrometer source and detector parameters. Norcoclaurine and Caffeine were injected analysed in Single lon Monitoring mode with the respective mass over charge ratio (m/z) 227 and 195 corresponding to their protonated ion. Retention times of Norcoclaurine and Caffeine were 2.5 min and 3.0 min respectively.

TABLE-US-00006 TABLE 6 Gradient for non-chiral separation Time (min) % B 0 2 5 30 5.1 100 5.9 100 6 2 8 2

TABLE-US-00007 TABLE 7 LC-MS conditions Injection volume 5 l Column Temperature 30 C. 5 C. Injection method Partial loop Flow 0.4 ml/min Auto sampler temperature 10 C. 2 C. Weak wash 800 l water/acetonitrile 8:2 Strong wash 300 l MeOH Seal wash 5 min with water/acetonitrile 9:1

TABLE-US-00008 TABLE 8 Mutations with increased activity Fold increase ID Mutation(s) mutant/WT Cj_NCS_V48 V174G 1.11 Cj_NCS_V49 I178A 1.35 Cj_NCS_V84 K152R 1.28 Cj_NCS_V93 V174E 1.08 Cj_NCS_V114 V141I V142I K152R 1.39 Cj_NCS_V116 D147N K152R T82V 1.08 Cj_NCS_V151 I178S 1.44 Cj_NCS_V152 I178D 2.16 Cj_NCS_V159 K152R V174E 1.51 Cj_NCS_V161 K114E V174E 1.16 Cj_NCS_V162 V174G K152R 1.29 Cj_NCS_V163 V174G K114E 1.37 Cj_NCS_V164 I178A K152R 1.9 Cj_NCS_V240 I178N 1.509 Cj_NCS_V241 I178Q 1.767 Cj_NCS_V242 I178T 1.111 Cj_NCS_V246 I178D I75L 1.469 Cj_NCS_V247 I178D I75K 1.859 Cj_NCS_V248 I178D K152R 2.002 Cj_NCS_V249 I178D A77S 1.567 Cj_NCS_V250 I178D A77S K152R 1.761 Cj_NCS_V251 I178D I75K K152R 1.77 Cj_NCS_V253 I178D V174Q 1.429 Cj_NCS_V258 I178D V174 K152R 1.279 Cj_NCS_V261 A73P A77E 1.036 Cj_NCS_V263 I178D K114E K152R 1.152 Cj_NCS_V267 I178D V174E Q99K 1.257 Cj_NCS_V268 I178D V174Q Q99K 1.786 Cj_NCS_V269 I178D V174E Q99R 1.539 Cj_NCS_V270 I178D V174Q Q99R 1.912 Cj_NCS_V271 I178D V174G K152R A77S 1.937 Cj_NCS_V273 A77T I178D 1.163

Example 8Effect of Norcoclaurine Synthase Localization on Production of Norcoclaurine in S. cerevisiae

[0262] Based on the S. cerevisiae gene integration and expression system developed by Mikkelsen, M D et al (Metab. Eng. 14, Issue 2, 104-111 (2012)), a set of integration/recombination plasmids were designed to simultaneously delete the ORF of the S. cerevisiae gene ARI1 and collectively overexpress: [0263] 1) a feedback resistant variant of ARO4, ARO4.sup.FBR (Luttic, M. A. H et al Metab. Eng. 10, 141-153 (2008), [0264] 2) a Tyrosine hydroxylase based on the Beta vulgaris CYP76AD1 (DeLoache, W. C. et al Nat. Chem. Biol., 11, 465-471 (2015)), [0265] 3) the Pseudomonas putida DOPA decarboxylase (PpDODC), and [0266] 4) several versions of the Coptis japonica Norcoclaurine synthase (CjNCS).

[0267] All genes used for overexpression were codon optimized for expression in S. cerevisiae. The expression cassettes in these integration plasmids were designed to give high expression of the norcoclaurine pathway genes and the following promoters were used: PDC1 promoter (ARO4.sup.FBR), TDH3 promoter (CYP76AD1 .sup.W13L,F309L), TEF2 promoter (PpDODC), and the PGK1 promoter for expression of the NCS variants.

[0268] A laboratory yeast strain (BY4741) transformed with this combined ARI1.

[0269] deletion/norcoclaurine biosynthesis gene overexpression system gives rise to transformants that show high production of norcoclaurine compared to what was reported in literature (DeLoache, W. C. et al Nat. Chem. Biol., 11, 465-471 (2015)).

[0270] Yeast transformants were grown in 96 deep-well plates in 500 L liquid Synthetic Complete media for 3 days at 30 C. with shaking at 230 rpm in a Kuhner Climo-Shaker ISF1-X. Culture samples for LC-MS were prepared by extraction as follows: 96% ethanol and culture sample were mixed 1:1 and incubated on a heating block at 80 C. for 10 min. After heating cells were pelleted in an Eppendorff table top centrifuge by centrifugation and the supernatant was then transferred to a new tube and diluted 1:5 in water.

Example 9Coptis japonica NCS.SUP.I178D

[0271] To test the performance of the NCS.sup.I178D version and the NCS.sup.I178D version with HDEL (SEQ ID No: 22) amino acids added in the C-terminus in this system, new plasmids and yeast strains were constructed.

[0272] Again the system described in Example 8 was applied, i.e. deletion of the ORF of ARI1 with simultaneous overexpression of ARO4.sup.FBR, CYP76AD1 .sup.W13L, F309L, PpDODC and the various versions of the C. japonica NCS.

Example 10LC-MS Protocol

[0273] A Norcoclaurine stock solution was prepared in DMSO at a concentration of 10 mM. Standard solutions were prepared at concentrations of 4 uM, 2 uM, 1 uM, 500 nM, 200 nM, 100 nM, 50 nM, 20 nM and 10 nM from the stock solution. Samples were injected into the Agilent 1290 UPLC coupled to an Ultivo Triple Quadrupole. The LC-MS method was as follows: Mobile Phase A. H2O+0.1% Formic acid; Mobile Phase B: Acetonitrile +0.1% Formic acid; Column: Phenomenex Kinetex 1.7 um XB-C18 IOOA, 2.1100 mm. The elution gradient is shown in Table X and the LC-MS conditions are given in Table X. Table X shows the mass spectrometer source and detector parameters and Table X shows the target compound, retention time, parent ion, transition ions (MRM) as well as dwell time, fragmentor voltag and collision energy used.

TABLE-US-00009 TABLE 9 Gradient for LC-MS Time (min) % B 0 2 0.30 2 3.00 25 3.40 100 3.90 100 4 2 5 2

TABLE-US-00010 TABLE 10 LC-MS conditions Parameter Value Injection volume 2 l Column Temperature 30 C. 4 C. Injection method Flow through needle Flow 0.4 ml/min Auto sampler temperature 10 C. 2 C. Reconditioning wash 2% Acetonitrile (in H.sub.2O), 5 sec Weak wash 20% Methanol (in H.sub.2O), 5 sec Strong wash 30% Acetonitrile, 30% Methanol, 30% 2-Propanol, 10% H.sub.2O, 10 sec Seal wash 20% 2-Propanol (in H.sub.2O)

TABLE-US-00011 TABLE 11 Mass spectrometer source and detector parameters (Ultivo Triple Quadrupole) Parameter Value Ion Source Electrospray Positive Mode (ESI+) Capillary Voltage 3.5 kV Nozzle Voltage 500 V Source Gas Temperature 290 C. Source Gas Flow 12 L/min Source Sheath Gas Temperature 380 C. Source Sheath Gas Flow 12 L/min Nebulizer 30 psi Mode MS/MS Collision See Table 4

TABLE-US-00012 TABLE 12 Multiple reaction monitoring targets and conditions (ESI+) Retention Parent Daughter Dwell Fragment Collision Target time ion ion time or voltage energy compound (min) (m/z) (m/z) (ms) (V) (V) Norcoclaurine 2.29 272 255 200 110 5

REFERENCES

[0274] Bonamore, A., Barba, M., Botta, B., Boffi, A. and Macone, A., 2010. Norcoclaurine synthase: mechanism of an enantioselective pictet-spengler catalyzing enzyme. Molecules, 15(4), pp.2070-2078 [0275] DeLoache, W. C. et al. An enzyme-coupled biosensor enables (S)-reticuline production in yeast from glucose. Nat. Chem. Biol. 11, 465-471 (2015) [0276] Galanie S, Thodey K, Trenchard IJ, Filsinger Interrante M, Smolke CD., 2015. Complete biosynthesis of opioids in yeast. Science. 2015 Sep. 4; 349(6252) : 1095-100. [0277] Ilari, A., Franceschini, S., Bonamore, A., Arenghi, F., Botta, B., Macone, A., Pasquo, A., Bellucci, L. and Boffi, A., 2009. Structural basis of enzymatic (S)-norcoclaurine biosynthesis. Journal of biological chemistry, 284(2), pp.897-904

[0278] 1Lichman, B.R., Gershater, M.C., Lamming, E.D., Pesnot, T., Sula, A., Keep, N.H., Hailes, H. C. and Ward, J.M., 2015. Dopamine-first mechanism enables the rational engineering of the norcoclaurine synthase aldehyde activity profile. FEBS journal, 282(6), pp.1137-1151 [0279] Mumberg D, Muller R, Funk M. Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene. 1995; 156 : 119-122. [0280] Narcross, L., Fossati, E., Bourgeois, L., Dueber, J. E. and Martin, V. J. J. Microbial Factories for the Production of Benzylisoquinoline Alkaloids. Trends Biotechnol. 34, 228-241 (2016) [0281] Mumberg D, Muller R, Funk M. Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene. 156, 119-122 (1995)

SEQUENCE LISTING FREE TEXT

[0282] SEQ ID NO 1: Coptis japonica NCS [0283] SEQ ID NO 2: Coptis japonica NCS nucleotide [0284] SEQ ID NO: 3: NCS 2 nucleotide sequence codon optimized for S. cerevisiae [0285] SEQ ID NO: 4: Coptis japonica NCS wild type loop structure sequence [0286] SEQ ID NO: 5: Coptis japonica NCS wild type helix structure sequence [0287] SEQ ID NO: 6: >CYP76AD1 W13L, F309L [0288] SEQ ID NO: 7: >CYP76AD1 W13L, F309L nucleotide sequence codon optimized for S. cerevisiae [0289] SEQ ID NO: 8: >PpDODC [0290] SEQ ID NO: 9: >PpDODC nucleotide sequence codon optimized for S. cerevisiae [0291] SEQ ID NO: 10: >ARO4.sup.FBR [0292] SEQ ID NO: 11: >ARO4.sup.FBR nucleotide sequence [0293] SEQ ID NO: 12: >CjNCSco [0294] SEQ ID NO: 13: >CjNCSco nucleotide sequence codon optimized for S. cerevisiae [0295] SEQ ID NO: 14: >I 9-CjNCSco [0296] SEQ ID NO: 15: >I 9-CjNCSco nucleotide sequence codon optimized for S. cerevisiae SEQ ID NO: 16: >CjNCSco-HDEL [0297] SEQ ID NO: 17: >CjNCSco-HDEL nucleotide sequence codon optimized for S. cerevisiae [0298] SEQ ID NO: 18: >CjNCSIco.sup.I178D [0299] SEQ ID NO: 19: >CjNCSIco.sup.I178D nucleotide sequence codon optimized for S. cerevisiae [0300] SEQ ID NO: 20: >CjNCSIco.sup.I178D-HDEL [0301] SEQ ID NO: 21: >CjNCSIco.sup.I178D-HDEL nucleotide sequence codon optimized for S. cerevisiae

ITEMS OF THE INVENTION

[0302] 1. A norcoclaurine synthase comprising a substrate binding amino acid sequence, which is at least 50% similar to the substrate binding amino acid sequence SEQ ID NO: 4 and/or SEQ ID NO: 5, and wherein the substrate binding amino acid sequence(s) comprise one or more mutations increasing the norcoclaurine synthase activity compared to wild type. [0303] 2. A norcoclaurine synthase according to item 1, which has the amino acid Proline (P) corresponding to position 2 in the count according to SEQ ID No. 4. [0304] 3. A norcoclaurine synthase according to item 1-2, which has the amino acid Leucine (K) or Lysine (L) corresponding to position 4 in the count according to SEQ ID No. 4. [0305] 4. A norcoclaurine synthase according to item 1-3, which has the amino acid Serine (S), Threonine (T) or Glutamic Acid (E) corresponding to position 6 in the count according to SEQ ID No. 4. [0306] 5. A norcoclaurine synthase according to item 1-4, which has the amino acid Proline (P) corresponding to position 2 and Leucine (K) or Lysine (L) corresponding to position 4 in the count according to SEQ ID No. 4. [0307] 6. A norcoclaurine synthase according to item 1-5, which has the amino acid Proline (P) corresponding to position 2 and Serine (S), Threonine (T) or Glutamic Acid (E) corresponding to position 6 in the count according to SEQ ID No. 4. [0308] 7. A norcoclaurine synthase according to item 1-6, which has the amino acid Leucine (K) or Lysine (L) corresponding to position 4 and Serine (S), Threonine (T) or Glutamic Acid (E) corresponding to position 6 in the count according to SEQ ID No. 4. [0309] 8. A norcoclaurine synthase according to item 1-7, which has the amino acid Proline (P) corresponding to position 2, Leucine (K) or Lysine (L) corresponding to position 4 and Serine (S), Threonine (T) or Glutamic Acid (E) corresponding to position 6 in the count according to SEQ ID No. 4. [0310] 9. A norcoclaurine synthase according to item 1-8 which has the amino acid Glycine (G) or Glutamic Acid (E) corresponding to position 2 in the count according to SEQ ID No. 5. [0311] 10. A norcoclaurine synthase according to item 1-9, which has the amino acid Alanine (A), Serine (S) or Aspartic Acid (D) corresponding to position 6 in the count according to SEQ ID No. 5. [0312] 11. A norcoclaurine synthase according to item 1-10, which has the amino acid Glycine (G) or Glutamic Acid (E) corresponding to position 2 and Alanine (A), Serine (S) or Aspartic Acid (D) corresponding to position 6 in the count according to SEQ ID No. 5. [0313] 12. A norcoclaurine synthase according to any of items 1-11, having an increased catalysation when compared to the wild type synthase of the condensation of 4-HPAA and dopamine to (S)-norcoclaurine and/or 3,4-DhPAA and dopamine to (S)-norlaudanosoline. [0314] 13. A norcoclaurine synthase according to any of items 1-12, wherein the increased catalysation manifest in increased norcoclaurine production of cell cultures after 72 hours' cultivation time compared to norcoclaurine production obtained from a cell comprising SEQ ID NO 1 (WILD TYPE). [0315] 14. A norcoclaurine synthase according to any of items 1-13, wherein the synthase is derived from Coptis japonica. [0316] 15. A nucleic acid encoding a norcoclaurine synthase according to any of items 1-14. [0317] 16. A nucleic acid according to item 15, wherein the nucleic acid sequence is at least 40% identical to the nucleic acid sequence given in SEQ ID No. 2 (DNA). [0318] 17. A nucleic acid according to items 15-16, wherein the nucleic acid is codon optimized for S. cerevisiae. [0319] 18. A nucleic acid according to items 15-16, wherein the nucleic acid sequence is at least 80% identical to the nucleic acid sequence given in SEQ ID No. 3. [0320] 19. A heterologous host cell comprising a nucleic acid according to any one of items 15-18, wherein the nucleic acid is recombinant. [0321] 20. The heterologous host cell according to item 19, wherein the cell is a yeast cell, a plant cell, a mammalian cell, an insect cell, a fungal cell, a bacterial cell, an algal cell, or a cyanobacterial cell. [0322] 21. The heterologous host cell according to any one of items 19-20, wherein the cell is a yeast cell selected from the group consisting of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Xanthophyllomyces dendrorhous, Candida albicans, Rhodotorula sp., or Rhodospiridium sp. [0323] 22. The heterologous host cell according to any one of items 19-21, wherein the host cell is a Saccharomyces. [0324] 23. The heterologous host cell according to any one of items 19-22, wherein the host cell is a yeast cell is Saccharomyces cerevisiae cell. [0325] 24. A method for the preparation of a (S)-norcoclaurine and/or (S)-norlaudanosoline compound, the method comprising the steps of: [0326] providing a recombinant host cell according to any of items 19-23, wherein the host cell is capable of catalysation of the condensation of 4-HPAA and dopamine to (S)-norcoclaurine and/or 3,4-DhPAA and dopamine to (S)-norlaudanosoline, [0327] culturing the cell under conditions promoting said catalysation, and optionally [0328] isolating the (S)-norcoclaurine and/or (S)-norlaudanosoline. [0329] 25. A method for the preparation of a (S)-norcoclaurine and/or (S)-norlaudanosoline compound, comprising contacting compound 4-HPAA and dopamine and/or 3,4-DhPAA and dopamine with a recombinant norcoclaurine synthase according to any of items 1-14, wherein the recombinant norcoclaurine synthase is capable of catalysation of the condensation of 4-HPAA and dopamine to (S)-norcoclaurine and/or 3,4-DhPAA and dopamine to (S)-norlaudanosoline. [0330] 26. The method according to item 25, further comprising cultivating a recombinant host cell of any one of items 19-23 in a culture medium in the presence of 4-HPAA and dopamine and/or 3,4-DhPAA and dopamine, under conditions in which the one or more recombinant genes encoding the recombinant norcoclaurine synthase according to any of items 1-14 is/are expressed in presence of 4-HPAA and dopamine and/or 3,4-DhPAA and dopamine. [0331] 27. The method according to item 25-26, which is performed in vitro. [0332] 28. A norcoclaurine synthase comprising a substrate binding amino acid sequence, which is at least 20% identical to the substrate binding amino acid sequence SEQ ID NO: 4 and/or SEQ ID NO: 5, and wherein the substrate binding amino acid sequence(s) comprise one or more mutations increasing the norcoclaurine synthase activity compared to wild type. [0333] 29. A norcoclaurine synthase according to item 1 or 28, which has the amino acid Proline (P) corresponding to position 2, Leucine (K) or Lysine (L) corresponding to position 4, and/or Serine (S), Threonine (T) or Glutamic Acid (E) corresponding to position 6 in the count according to SEQ ID No: 4. [0334] 30. A norcoclaurine synthase according to item 1 or any of 28-29, which has the amino acid Glycine (G) or Glutamic Acid (E) corresponding to position 2, and/or Alanine (A), Serine (S) or Aspartic Acid (D) corresponding to position 6 in the count according to SEQ ID No: 5. [0335] 31. A norcoclaurine synthase according to any of items 1 or any of 28-30, having an increased catalysation when compared to the wild type synthase of the condensation of 4-HPAA and dopamine to (S)-norcoclaurine and/or 3,4-DhPAA and dopamine to (S)-norlaudanosoline. [0336] 32. A norcoclaurine synthase according to any of items 1 or any of 28-31, which has the amino acid Proline (P) corresponding to position 2 in the count according to SEQ ID No: 4. [0337] 33. A norcoclaurine synthase according to item 1 or any of 28-32, which has the amino acid Leucine (K) or Lysine (L) corresponding to position 4 in the count according to SEQ ID No: 4. [0338] 34. A norcoclaurine synthase according to item 1 or any of 28-33, which has the amino acid Serine (S), Threonine (T) or Glutamic Acid (E) corresponding to position 6 in the count according to SEQ ID No: 4. [0339] 35. A norcoclaurine synthase according to item 1 or any of 28-34, which has the amino acid Proline (P) corresponding to position 2 and Leucine (K) or Lysine (L) corresponding to position 4 in the count according to SEQ ID No: 4. [0340] 36. A norcoclaurine synthase according to item 1 or any of 28-35, which has the amino acid Proline (P) corresponding to position 2 and Serine (S), Threonine (T) or Glutamic Acid (E) corresponding to position 6 in the count according to SEQ ID No: 4. [0341] 37. A norcoclaurine synthase according to item 1 or any of 28-36, which has the amino acid Leucine (K) or Lysine (L) corresponding to position 4 and Serine (S), Threonine (T) or Glutamic Acid (E) corresponding to position 6 in the count according to SEQ ID No: 4. [0342] 38. A norcoclaurine synthase according to item 1 or any of 28-37, which has the amino acid Proline (P) corresponding to position 2, Leucine (K) or Lysine (L) corresponding to position 4 and Serine (S), Threonine (T) or Glutamic Acid (E) corresponding to position 6 in the count according to SEQ ID No: 4 [0343] 39. A nucleic acid encoding a norcoclaurine synthase according to any of items 1 or any of 28-38, wherein the nucleic acid sequence is at least 40% identical to the nucleic acid sequence given in SEQ ID No: 4. [0344] 40. A heterologous host cell comprising a nucleic acid according to any one of items 1 or any of 28-39, wherein the nucleic acid is recombinant. [0345] 41. The heterologous host cell according to item 40, wherein the host cell is a Saccharomyces cerevisiae. [0346] 42. A norcoclaurine synthase comprising an amino acid sequence which is at least 40% identical to the amino acid sequence given in SEQ ID No. 1, and which has one or more site-specific mutations corresponding to position 73, 75, 77, 82, 99, 114, 141, 142, 147, 152, 174 and/or 178 of SEQ ID No. 1. [0347] 43. A norcoclaurine synthase according to item 42, wherein the site-specific mutation is selected from the group consisting of mutations corresponding A73P, I75L, I75K, A77S, A77E, A77T, T82V, Q99K, Q99R, K114E, V1411, V1421, D147N, K152R, V174E, V174G, V174Q, V174E, I178A, I178S, I178D, I178N, I178Q, and I178T of SEQ ID No. [0348] 44. A norcoclaurine synthase according to any of items 42-43, having an increased catalysation when compared to the wild type synthase of the condensation of 4-HPAA and dopamine to (S)-norcoclaurine and/or 3,4-DhPAA and dopamine to (S)-norlaudanosoline. [0349] 45. A norcoclaurine synthase according to any of items 42-44, wherein the synthase is derived from Coptis japonica. [0350] 46. A nucleic acid encoding a norcoclaurine synthase according to any of claims items 42-45. [0351] 47. A nucleic acid according to claim 46, wherein the nucleic acid sequence is at least 40% identical to the nucleic acid sequence given in SEQ ID No. 2. [0352] 48. A nucleic acid according to claims items 46-47, wherein the nucleic acid is codon optimized for S. cerevisiae. [0353] 49. A nucleic acid according to items 46-47, wherein the nucleic acid sequence is at least 60% identical to the nucleic acid sequence given in SEQ ID No. 3. [0354] 50. A heterologous host cell comprising a nucleic acid according to any one of items 46-49, wherein the nucleic acid is recombinant. [0355] 51. The heterologous host cell according to item 50, wherein the cell is a yeast cell, a plant cell, a mammalian cell, an insect cell, a fungal cell, a bacterial cell, an algal cell, or a cyanobacterial cell. [0356] 52. The heterologous host cell according to any one of items 50-51, wherein the cell is a yeast cell selected from the group consisting of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Xanthophyllomyces dendrorhous, Candida albicans, Rhodotorula sp., or Rhodospiridium sp. [0357] 53. The heterologous host cell according to any one of items 50-52, wherein the host cell is a Saccharomyces. [0358] 54. The heterologous host cell according to any one of items 50-53, wherein the host cell is a yeast cell is a Saccharomyces cerevisiae cell. [0359] 55. A method for the preparation of a (S)-norcoclaurine and/or (S)-norlaudanosoline compound, the method comprising the steps of: [0360] providing a recombinant host cell according to any of items 50-54 capable of catalysing of the condensation of 4-HPAA and dopamine to (S)-norcoclaurine and/or 3,4-DhPAA and dopamine to (S)-norlaudanosoline, [0361] culturing the cell under conditions promoting said catalysation, and optionally [0362] isolating the (S)-norcoclaurine and/or (S)-norlaudanosoline. [0363] 56. A method for the preparation of a (S)-norcoclaurine and/or (S)-norlaudanosoline compound, comprising contacting compound 4-HPAA and dopamine and/or 3,4-DhPAA and dopamine with a recombinant norcoclaurine synthase according to any of items 42-49 capable of catalysation of the condensation of 4-HPAA and dopamine to (S)-norcoclaurine and/or 3,4-DhPAA and dopamine to (S)-norlaudanosoline. [0364] 57. The method according to item 56, further comprising cultivating a recombinant host cell of any one of items 50-54 in a culture medium in presence of 4-HPAA and dopamine and/or 3,4-DhPAA and dopamine, under conditions in which the one or more recombinant genes encoding the recombinant norcoclaurine synthase according to any of items 42-49 is/are expressed in presence of 4-HPAA and dopamine and/or 3,4-DhPAA and dopamine. [0365] 58. The method according to claim 56-57, which is performed in vitro. [0366] 59. An opioid selected from the group consisting of Thebaine, Oripavine, Neopinone, Codeinone, Hydrocodone, Morphine, Oxycodone, Codeine, Noscapine, Berberine, Sanguinarine, Tubocurarine and Papaverine obtainable from a method according to any of items 55-58 or 24-27.