Genetically modified host cells producing benzylisoquinoline alkaloids

Abstract

The invention relate to genetically modified hosts cell comprising a pathway having enhanced production of one or more benzylisoquinoline alkaloids wherein the cell expresses heterologous insect genes encoding insect demethylases converting thebaine into northebaine, thebaine into oripavine, thebaine into nororipavine and / or oripavine into nororipavine.

Description

Cross Reference to Related Applications

[0000] This application is the Australian national phase of international patent application No. 5   PCT / EP2020 / 078496 (publication No. WO / 2021 / 069714 A1) filed on October 9, 2020, which claims benefit of priority to European application No. 19202337.2 filed on October 10, 2019 and European application No. 20169590.5 filed on April 15, 2020, the entire disclosures of which are incorporated herein by reference in their entirety. Field of the Invention 10

[0001] The present disclosure relates to methods of producing benzylisoquinoline alkaloids by use of genetically modified host cells expressing one or more genes in an operative metabolic pathway producing the benzylisoquinoline alkaloids or their precursors as well as optionally subjecting the benzylisoquinoline alkaloids for chemical conversion to produce additional useful benzylisoquinoline alkaloid derivatives. 15 Background of the invention

[0002] Effective production of pharmaceutical opioids by biotransformation, such as wholly or partly by fermentation of genetically engineered strains and / or by bioconversion, requires complex engineering and optimization of metabolic pathways producing the opioids or their precursors and 20 optionally further chemical modifications. Some pharmaceutical opioids such as buprenorphine, naltrexone, naloxone and nalbuphine require demethylation of benzylisoquinoline alkaloids such as thebaine and / or oripavine and an N-alkylation of the demethylated benzylisoquinoline alkaloid. In the art this demethylation step and the subsequent N-alkylation step is achieved chemically and the chemical N-demethylation of benzylisoquinoline alkaloids preceding the N-alkylation is one of the 25 most critical steps in the chemical synthesis of pharmaceutical opioids, as it has low efficiency and produces highly toxic waste.

[0003] Benzylisoquinoline alkaloids for the demethylation step can be provided by production using genetically modified cull cultures comprising the right pathway and / or extraction from plant material. Moreover, genetically modified yeasts comprising certain heterologous fungal Mucorales P450 30 enzymes capable of converting e.g. thebaine into northebaine and / or demethylated reticulin derivatives are known in the art eg. from WO2018229306.

[0004] However, for efficiently producing pharmaceutical opioids there is a continuous desire and need for improving and optimising both pathways in genetically modified microbial strains producing 2020363960   06 Oct 2026 benzylisoquinoline alkaloids as well as critical steps of demethylating benzylisoquinoline alkaloids and improving further chemical modification of benzylisoquinoline alkaloids to produce compounds of particular desireable pharmacological properties with higher efficiency and less waste problems. 5 Summary of the Invention

[0005] Over this background art several improved pathway enzymes as well improvements in auxilliary cellular mechanisms have been identified to be surprisingly efficient at producing highly pure benzylisoquinoline alkaloids as well at demethylating benzylisoquinoline alkaloids thebaine and / or oripavine in host cells into the corresponding northebaine and / or nororipavine - auxilliary cellular 10 mechanisms including transportation of precursors and products, limitation of precursor loss to competing cellular reactions and / or formation of by-products by unspecific enzymes.

[0006] Accordingly, the present invention provides in a first aspect a genetically modified fungal host cell comprising a pathway having enhanced production of northebaine, oripavine, and / or nororipavine wherein the fungal host cell expresses one or more heterologous genes encoding a 15 demethylase converting thebaine into the northebaine, thebaine into the oripavine, thebaine into the nororipavine and / or oripavine into the nororipavine wherein the demethylase comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 140, 142, 146, 148, 152, 154, 156, 158, 160, 162, 166, 168, 170, 172, 174, 176, 178, 182, 184, 186, 188, 190, 192, 194, 196, 827, 829, 831, 833, 835, 837, 839, or 841, and wherein the host cell further comprises a demethylase-CPR 20 capable of reducing and / or regenerating the demethylase enzyme. [0006A] In a further aspect, the invention provides a cell culture, comprising the genetically modified fungal host cell according to any aspect described herein and a growth medium. [0006B] In a further aspect, the invention provides a method for producing northebaine, oripavine, and / or nororipavine comprising 25 a) culturing the cell culture according to any aspect described herein at conditions allowing the cell to produce the northebaine, oripavine, and / or nororipavine; and b) preferably recovering and / or isolating the northebaine, oripavine, and / or nororipavine. [0006C] In a further aspect, the invention provides a mutant demethylase having least 80% identity to the demethylase comprised in SEQ ID NO: 152 and comprising one or more mutations corresponding 30 to A110X, H242X, and / or V224X, optionally A110N, H242P and / or V224I. [0006D] In a further aspect, the invention provides a mutant demethylase having at least 80% identity to the demethylase comprised in SEQ ID NO: 140 and comprising one or more mutations corresponding to A316X and / or D392X, optionally A316G and / or D392E. [0006E] In a further aspect, the invention provides a genetically modified host cell comprising a 2020363960   06 Oct 2026 pathway having enhanced production of one or more benzylisoquinoline alkaloids wherein the cell comprises one or more features selected from: a) expression of one or more heterologous genes encoding a demethylase capable of converting thebaine into northebaine, thebaine into oripavine, thebaine into nororipavine and / or oripavine 5 into nororipavine; b) expression of one or more heterologous genes encoding a tyrosine hydroxylase (TH) converting L-tyrosine into L-dopa, wherein the TH has at least 70% identity to the TH comprised in 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63 or 65; 10 c) reduction or elimination of activity of one or more dehydrogenases native to the host cell comprised in SEQ ID NO: 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703 or 705; d) reduction or elimination of activity of one or more reductases native to the host cell comprised in SEQ ID NO: 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729 or 731; 15 e) expression of one or more heterologous genes encoding a norcoclaurine synthase (NCS) converting Dopamine and 4-HPAA into (S)-norcoclaurine, wherein the NCS has at least 70% identity to the NCS comprised in SEQ ID NO: 73 OR 76; f) expression of one or more heterologous genes encoding i) a fused 1,2-dehydroreticuline synthase-1,2-dehydroreticuline reductase (DRS-DRR) converting 20            (S)-Reticuline into (R)-reticuline, wherein ia) the DRS-DDR has at least 70% identity to the DRS-DRR comprised in SEQ ID NO: 92, 94, 96; or ib) the DRS moiety has at least 70%, identity to the DRS comprised in SEQ ID NO: 98, 100, 102, 104 or 106; and the DRR moiety has at least 70% identity to the DRR comprised in SEQ ID 25              NO: 108 or 110; or ii) a DRS having at least 70% identity to the DRS comprised in SEQ ID NO: 98, 100, 102, 104 or 106; and a DRR having at least 70% identity to the DRR comprised in SEQ ID NO: 108 or 110; iii) a fused 1,2-dehydroreticuline synthase-1,2-dehydroreticuline reductase (DRS-DRR) converting (S)-Reticuline into (R)-reticuline selected from DRS-DDR’s having at least 70% 30           identity to the DRS-DRR comprised in SEQ ID NO: 92, 94, 96; and / or iv) a 1,2-dehydroreticuline synthase (DRS) selected from DRSs having at least 70% identity to the DRS comprised in SEQ ID NO: 98, 100, 102, 104 or 106; and a 1,2-dehydroreticuline reductases (DDR) selected from DDR’s having at least 70% identity to the DRR comprised in SEQ ID NO: 108 or 110; 2020363960   06 Oct 2026 g) expression of one or more heterologous genes encoding a thebaine synthase (THS) converting 7-O-acetylsalutaridinol into thebaine, wherein the THS has at least 70% identity to the THS comprised in SEQ ID NO: 126, 127, 128, 129, 131, 133, 134, 136 or 138; and h) expression of one or more heterologous genes encoding a transporter protein capable of 5 increasing uptake or export in the host cell of a reticuline derivative selected from transporter proteins having at least 70% identity to the transporter protein comprised in SEQ ID NO: 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 10        431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 15        631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 733, 735, 795, 797, 799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823 or 825. In a further aspect the invention provides a polynucleotide construct comprising a polynucleotide sequence encoding a heterologous enzymes or transporter protein of the invention operably linked to one or more control sequences. 20

[0007] In a further aspect the invention provides a cell culture, comprising the host cell of the invention and a growth medium.

[0008] In a further aspect the invention provides a method for producing a benzylisoquinoline alkaloid comprising: a) culturing the cell culture of the invention at conditions allowing the cell to produce the 25          benzylisoquinoline alkaloid; and b) optionally recovering and / or isolating the benzylisoquinoline alkaloid. 2020363960   06 Oct 2026

[0009] In a further aspect the invention provides a fermentation composition comprising the cell culture of the invention and the benzylisoquinoline alkaloid comprised therein.

[0010] In a further aspect the invention provides a composition comprising the fermentation composition of the invention and one or more carriers, agents, additives and / or excipients. 5

[0011] In a further aspect the invention provides a pharmaceutical composition comprising the fermentation composition of the invention and one or more pharmaceutical grade excipient, additives and / or adjuvants.

[0012] In a further aspect the invention provides a method for preparing the pharmaceutical composition of the invention comprising mixing the fermentation composition of the invention with 10 one or more pharmaceutical grade excipient, additives and / or adjuvants.

[0013] In a further aspect the invention provides a method for preventing, treating and / or relieving a disease comprising administering a therapeutically effective amount of the pharmaceutical composition of the invention to a mammal. [0013A] Any discussion of the documents, acts, materials, devices, articles or the like which has been 15 included in the present specification is solely for the purpose of providing a context for the present invention. No admission is made that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application. 20 Description of drawings and figures

[0014] Figure 1 shows the pathway for making the benzylisoquinoline alkaloid precursor tyrosine via the Shikimate pathway and additional steps for producing (s)-norcoclaurine.

[0015] Figure 2 depicts a range of benzylisoquinoline alkaloid compounds having pharmaceutical properties which are derivatives of (S)-norcoclaurine. 25

[0016] Figure 3 shows a schematic representation of the biosynthetic pathway from glucose to thebaine in genetically modified S. cerevisiae strains. Enzymes from NCS to SAT / THS as well as Tyrosine hydroxylase (TH) and DOPA decarboxylase (DODC) are enzymes expressed from heterologous genes.

[0017] Figure 4 shows a stacked bar-diagram made from 3 culture samples analysed by LC-MS. The diagram shows production of reticuline and thebaine in mg / l as described in example 22. 30 Figure 5 shows a bar-diagram made from culture samples analysed by LC-MS. Cultures done and shown in triplicates. Production of thebaine in mg / l in yeast strains as described in example 22. The S-to-R-Reticuline (STORR) enzyme activities were expressed in the yeast strain as native (fused) Papaver somniferum DRS-DRR enzyme SEQ ID NO: 96 (called PsSTORR in figure), as separate Papaver 2020363960   06 Oct 2026 somniferum DRS (SEQ ID NO: 98) and DRR (SEQ ID NO: 108) domains called PsCYP82Y2 + PsAKR in figure), as separate Papaver somniferum DRS and Streptomyces tsukubaensis Imine reductase (SEQ ID NO: 94) enzymes (called PsCYP82Y2 + StIRED in figure), as separate Papaver rhoeas DRS (SEQ ID NO: 101) and Papaver somniferum DRR enzymes (called PrCYP82_AKO60176 + PsAKR in figure), or as 5 [Continued on page 5] separate Papaver somniferum DRS and Papaver rhoeas DRR enzymes ((SEQ ID NO: 110) (called PsCYP82Y2 + PrAKR_AKQ60177 in figure).

[0019] Figure 6 shows a bar-diagram made from culture samples analysed by LC-MS. Cultures done and shown in triplicates. Production of thebaine in mg / l in yeast strains as described in example 22. The bar diagram shows that the three different artificial DRS variants ProlD60 (SEQ. ID NO: 102), ProlD66 (SEQ ID NO: 104) and ProlD79 (SEQ ID NO: 106) all significantly improve production of thebaine as compare to the PsAKR (DRS) (SEQ ID NO: 98) when expressed together with the PsAKR (DRR) in the strain described in example 22. ProlD79 (SEQ ID NO: 106) appears to be the best.

[0020] Figure 7 shows a bar-diagram made from culture samples analysed by LC-MS. Cultures done in triplicates and shown as average of triplicates including standard deviation error bars. Production of thebaine in mg / l in yeast strains as described in example 23. The bar diagram shows that expression of the three different artificial Thebaine synthases called PROths2_138 (SEQ ID NO: 134), PROths2_143 (SEQ ID NO: 136) and PROths2_116 (SEQ ID NO: 13138) improve or show similar production levels of thebaine as compared to the native P. somniferum THS2 enzyme. PROths2_138 show a significant improvement in activity as compare to the native P. somniferum THS2 enzyme.

[0021] Figure 8 shows a bar-diagram made from culture samples analysed by LC-MS. Cultures done and shown in triplicates. Bar diagram showing the production of Northebaine in S. cerevisiae by expression of two different N-demethylases in a Thebaine producing strain as described in example 24. The CYP450 demethylase and CPR of fungal origin are called CYPDN_91 (SEQ ID NO: 251) and CPR gene celCPR (SEQ ID NO: 306). The figure legend MothCYP_CPR means expression of insect (moth) CYP450 demethylase HaCYP6AE15v2 (SEQ ID NO: 141) and CPR gene HaCPR_E7E2N6 (SEQ ID NO: 304).

[0022] Figure 9 shows samples from different timepoints (X-axis, hours) during a fed-batch fermentation with strain expressing the n-demethylase CYPDN_91 (SEQ ID NO: 251) and CPR gene celCPR (SEQ ID NO: 306) in a thebaine producing S. cerevisiae strain as described in example 24. Samples analysed by LC-MS and thebaine and Northebaine production in mg / l is shown as stacked bar-diagram.

[0023] Figure 10 shows thebaine and oripavine production in mg / l demonstrated in a thebaine producer strain (sOD310) as described in example 25. Samples from three cultures were analysed on LC-MS and shown as bars.

[0024] Figure 11 shows the activity of N-terminal variants of HaCYP6AE15v2 expressed in S. cerevisiae and its bioconversion of oripavine to nororipavine in strains expressing N-terminal variants and N-terminal variants combined with single mutations of HaCYP6AE15v2 cytochrome P450 enzyme, grown in DELFT minimal medium at pH 4.5 with 500pM of oripavine. HaCYP6AE15v2 was truncated between amino acids 2 and 21 to generate truncated HaCYP6AE15v2_t. In figure 11 HaCYP6AE15v2 is also referred to as HaCYP6AE15v or HaCYP6AE15.

[0025] Figure 12 shows the activity of N-terminal variants of Hv_CYP_A0A2A4JAM9 expressed in S. cerevisiae ans its bioconversion of oripavine to nororipavine in strains expressing N-terminal of Hv_CYP_A0A2A4JAM9 cytochrome P450 enzyme, grown in DELFT minimal medium at pH 4.5 with 500pM of oripavine. Hv_CYP_A0A2A4JAM9 was truncated between amino acids 2 and 21 to generate truncated Hv_CYP_A0A2A4JAM9_t. In figure 12 Hv_CYP_A0A2A4JAM9 is also referred to as Hv_A0A2A4JAM9 or HvA0A2A4JAM9.

[0026] Figure 13 shows sequence alignment of data set >70% ID to Hv_CYP_A0A2A4JAM9 including HaCYP6AE15v2. The amino acids shaded in grey, represents the different residues compared with the consensus sequence. The residues in the black box correspond to the active site residues, according to modeling predictions. In this alignment the most active sequences Hv_CYP_A0A2A4JAM9 and HaCYP6AE15v2 are provided as the top sequences in the alignment for reference. This multiple sequence alignment was performed locally with Clustal Omega program and alignment visualization with CLC workbench 8.0. In figure 13 Hv_CYP_A0A2A4JAM9 is also be referred to as Hv_CYP_A0A2A4JAM, while HaCYP6AE15v2 is referred to as 15v2. Incorporation by reference

[0027] All publications, patents, and patent applications referred to herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between a term herein and a term in an incorporated reference, the term herein prevails and controls. Detailed Description of the Invention Definitions

[0028] Any EC numbers used herein refers to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, San Diego, California, including 30 supplements 1-5 published in Eur. J. Bio-chem. 1994, 223,15; Eur. J. Biochem. 1995, 232, 1-6; Eur. J. Biochem. 1996, 237, 1-5; Eur. J. Biochem. 1997, 250, 1-6; and Eur. J. Biochem. 1999, 264, 610-650; respectively. The nomenclature is regularly supplemented and updated; see e.g. http: / / enzyme.expasy.org / . The term "PEP" as used herein refers to phosphoenol pyruvate.

[0029] The term "E4P" as used herein refers to erythrose-4-phosphate

[0030] The term "Aro4" as used herein refers to DAHP synthase catalyzing the reaction of PEP and E4P into DAHP.

[0031] The term "DAHP" as used herein refers to 3-deoxy-D-arabino-2-heptulosonic acid 7- phosphate.

[0032] The term "Arol" as used herein refers to EPSP synthase catalyzing conversion of DAHP into EPSP.

[0033] The term "EPSP" as used herein refers to 5-enolpyruvylshikimate-3-phosphate. The term "Aro2" as used herein refers to chorismate synthase catalyzing conversion of EPSP into chorismate.

[0034] The term "Tyrl" as used herein refers to prephenate dehydrogenase catalyzing conversion of prephenate into 4-HPP

[0035] The term "4-HPP" as used herein refers to 4-hydroxyphenylpyruvate

[0036] The term "Aro8" and "Aro9" as used herein refers to aromatic aminotransferase reversibly catalyzing conversion of 4-HPP into L-tyrosine

[0037] The term "ARO10" or HPPDC as used herein refers to hydroxyphenylpyruvate decarboxylase catalyzing 4-HPP into 4-HPAA.

[0038] The term "4-HPAA" as used herein refers to 4-Hydroxyphenylacetaldehyde.

[0039] The term "TH" as used herein refers to a cytochrome P450 enzyme having tyrosine hydroxylase activity and converting L-tyrosine into L-DOPA.

[0040] The term "demethylase" as used herein refers to a P450 enzyme, capabale of demethylating thebaine into northebaine, thebaine into oripavine, thebaine into nororipavine and / or oripavine into nororipavine.

[0041] The term "DRS" as used herein refers to 1,2-dehydroreticuline synthase, a cytochrome P450 enzyme which catalyze conversion of (S)-Reticuline into 1,2-dehydroreticuline.

[0042] The term "DRR" as used herein refers to 1,2-dehydroreticuline reductase which catalyzes conversion of 1,2-dehydroreticuline to (R)-Reticuline.

[0043] The term "DRS-DRR" as used herein refers to 1,2-dehydroreticuline synthase-1,2-dehydroreticuline reductase fused complex catalyzing conversion of (S)-Reticuline into (R)- reticuline. This complex may also be referred to as STORR or REPL DRS-DRR or DRS together with DRR are also categorised as epimerases or isomerases.

[0044] The term "CPR" as used herein refers to a cytochrome P450 reductase catalyzing the electron transfer (from NADPH) to a cytochrome P450 enzyme of the pathway, typically in the endoplasmic reticulum of a eukaryotic cell. For distinction and as disclosed herein CPR's are divided into demethylase-CPR used for CPR's capable of reducing demethylases; DRS-CPR used for CPR's capable of reducing DRS and TH-CPR used for CPR's capable of reducing TH. Demethylase-CPR, DRS-CPR and TH-CPR may be identical or different, depending on the P450 to be reduced.

[0045] The term "Cytochrome P450 enzyme" or "P450 enzymes" or "P450" as used herein interchangeably refers to a family of monooxygenases enzymes containing heme as a cofactor. P450s are also known as "CYPs". For distinction and as disclosed herein P450 enzymes are divided into demethylase P450s; DRS P450s, and TH P450s.

[0046] The term "family CYP6" as used herein about some demethylases refers to demethylases having >40% amino acid sequence identity to any known demethylase belonging to CYP6 family as defined by Nelson 2006, Cytochrome P450 Nomenclature, included herein by reference.

[0047] The term "family CYP76" as used herein about some THs refers to THs having tyrosine hydroxylase activity and capable of catalyzing L-tyrosine into L-DOPA.

[0048] The term "DODC" and TYDC" as used herein refers to L-dopa decarboxylase and tyrosine decarboxylase respectively catalyzing conversion of L-DOPA into dopamine and tyrosine into 4-HPP.

[0049] The term "MAO" as used herein refers to monoamine oxidase catalyzing conversion of dopamine to 3,4 DHPAA

[0050] The term "DHPAA" as used herein refers to 3,4-dihydroxyphenylacetaldehyde.

[0051] The term "NCS" as used herein refers to Norcoclaurine synthase catalyzing conversion of dopamine and 4-HPAA into Norcoclaurine.

[0052] The term "6-OMT" as used herein refers to 6-O-methyltransferase catalyzing conversion of (S)-norcoclaurine to (S)-Coclaurine

[0053] The term "CNMT" as used herein refers to Coclaurine-N-methyltransferase catalyzing conversion of (S)-Coclaurine to (S)-N-Methylcoclaurine and / or (S)-3'-hydroxycoclaurine to (S)-3'-hydroxy-N-methyl-coclaurine.

[0054] The term "NMCH" as used herein refers to N-methylcoclaurine 3'-monooxygenase catalyzing conversion of (S)-Coclaurine to (S)-3'-hydroxycoclaurine and / or (S)-N-Methylcoclaurine to (S)-3'-Hydroxy-N-Methylcoclaurine

[0055] The term "4'-OMT" as used herein refers to 3'-hydroxy-N-methyl-(S)-coclaurine 4'-O-methyltransferase catalyzing conversion of (S)-3'-Hydroxy-N-Methylcoclaurine to (S)-reticuline.

[0056] The term "SAS" as used herein refers to salutaridine synthase catalyzing conversion of (R)-reticuline to Salutaridine.

[0057] The term "SAR" as used herein refers to salutaridine reductase catalyzing conversion of Salutaridine to Salutaridinol.

[0058] The term "SAT" as used herein refers to salutaridinol 7-O-acetyltransferase catalyzing conversion of Salutaridinol to 7-O-acetylsalutaridinol.

[0059] The term "THS" as used herein refers to thebaine synthase catalyzing conversion of 7-O-acetylsalutaridinol into thebaine.

[0060] The term "BIA" or "benzylisoquinoline alkaloid" as used herein refers to a compound of the general formula A: which is the structural backbone of many alkaloids with a wide variety of structures, or to alkaloid products deriving from formula A of the general formula B also known as morphinans: Morphinan

[0061] The terms "heterologous" or "recombinant" or "genetically modified" and their grammatical equivalents as used herein interchangeably refers to entities "derived from a different species or cell". For example, a heterologous or recombinant polynucleotide gene is a gene in a host cell not naturally containing that gene, i.e. the gene is from a different species or cell type than the host cell. The terms as used herein about host cells refers to host cells comprising and expressing heterologous or recombinant polynucleotide genes.

[0062] The term "pathway" or "metabolic pathway" as used herein is intended to mean an enzyme acting in a live cell to convert a chemical substrate into a chemical product. A pathway may include one enzyme or multiple enzymes acting in sequence. A pathway including only one enzyme may also herein be referred to as "bioconversion" in particular relevant for embodiments where the cell of the invention is fed with a precursor or substrate to be converted by the enzyme into a desired benzylisoquinoline alkaloid. Enzymes are characterized by having catalytic activity, which can change the chemical structure of the substrate(s). An enzyme may have more than one substrate and produce more than one product. The enzyme may also depend on cofactors, which can be inorganic chemical compounds or organic compounds (co-factor and / or co-enzymes). The NADPH-dependent cytochrome P450 reductase (CPR) is an electron donor to cytochromes P450 (CYPs). CPR shuttles electrons from NADPH through the Flavin Adenine Dinucleotide (FAD) and Flavin Mononucleotide (FMN) coenzymes into the iron of the prosthetic heme-group of the CYP. The term "operative biosynthetic metabolic pathway" refers to a metabolic pathway that occurs in a live recombinant host, as described herein.

[0063] The term "in vivo", as used herein refers to within a living cell or organism, including, for example animal, a plant or a microorganism.

[0064] The term "in vitro", as used herein refers to outside a living cell or organism, including, without limitation, for example, in a microwell plate, a tube, a flask, a beaker, a tank, a reactor and the like.

[0065] The term "in planta", as used herein refers to within a plant or plant cell.

[0066] The term "substrate" or "precursor", as used herein refers to any compound that can be converted into a different compound. For example, thebaine can be a substrate for P450 and can be converted by demethuylation into Northebaine. For clarity, substrates and / or precursors include both compounds generated in situ by a enzymatic reaction in a cell or exogenously provided compounds, such as exogenously provided organic molecules which the host cell can metabolize into a desired compound.

[0067] Term "endogenous" or "native" as used herein refers to a gene or a polypepetide in a host cell which originates from the same host cell.

[0068] The term "deletion" as used herein refers to manipulation of a gene so that it is no longer expressed in a host cell.

[0069] The term "disruption" as used herein refers to manipulation of a gene or any of the machinery participating in the expression the gene, so that it is no longer expressed in a host cell.

[0070] The term "attenuation" as used herein refers to manipulation of a gene or any of the machinery participating in the expression the gene, so that it the expression of the gene is reduced as compared to expression without the manipulation.

[0071] The terms "substantially" or "approximately" or "about", as used herein refers to a reasonable deviation around a value or parameter such that the value or parameter is not significantly changed. These terms of deviation from a value should be construed as including a deviation of the value where the deviation would not negate the meaning of the value deviated from. For example, in relation to a reference numerical value the terms of degree can include a range of values plus or minus 10% from that value. For example, deviation from a value can include a specified value plus or minus a certain percentage from that value, such as plus or minus 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from the specified value.

[0072] The term "and / or" as used herein is intended to represent an inclusive "or". The wording X and / or Y is meant to mean both X or Y and X and Y. Further the wording X, Y and / or Z is intended to mean X, Y and Z alone or any combination of X, Y, and Z.

[0073] The term "isolated" as used herein about a compound, refers to any compound, which by means of human intervention, has been put in a form or environment that differs from the form or environment in which it is found in nature. Isolated compounds include but is no limited to compounds of the invention for which the ratio of the compounds relative to other constituents with which they are associated in nature is increased or decreased. In an important embodiment the amount of compound is increased relative to other constituents with which the compound is associated in nature. In an embodiment the compound of the invention may be isolated into a pure or substantially pure form. In this context a substantially pure compound means that the compound is separated from other extraneous or unwanted material present from the onset of producing the compound or generated in the manufacturing process. Such a substantially pure compound preparation contains less than 10%, such as less than 8%, such as less than 6%, such as less than 5%, such as less than 4%, such as less than 3%, such as less than 2%, such as less than 1 %, such as less than 0.5% by weight of other extraneous or unwanted material usually associated with the compound when expressed natively or recombinantly. In an embodiment the isolated compound is at least 90% pure, such as at least 91% pure, such as at least 92% pure, such as at least 93% pure, such as at least 94% pure, such as at least 95% pure, such as at least 96% pure, such as at least 97% pure, such as at least 98% pure, such as at least 99% pure, such as at least 99.5% pure, such as 100 % pure by weight.

[0074] The term "non-naturally occurring" as used herein about a substance, refers to any substance that is not normally found in nature or natural biological systems. In this context the term "found in nature or in natural biological systems" does not include the finding of a substance in nature resulting from releasing the substance to nature by deliberate or accidental human intervention. Non-naturally occurring substances may include substances completely or partially synthetized by human intervention and / or substances prepared by human modification of a natural substance.

[0075] The term "% identity" is used herein about the relatedness between two amino acid sequences or between two nucleotide sequences.

[0076] The term "% identity" as used herein about amino acid or nucleotide sequences refers to the degree of identity in percent between two amino acid sequences obtained when using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: identical amino acid residues -------------------------------------------------x 100 Length of alignment — total number of gaps in alignment The term "% identity" as used herein about nucleotide sequences refers to the degree of identity in percent between two nucleotide sequences obtained when using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: identical deoxyribonucleotides -----------------------------------------------------x 100 Length of alignment — total number of gaps in alignment The protein sequences of the present invention can further be used as a "query sequence" to perform a search against sequence databases, for example to identify other family members or related sequences. Such searches can be performed using the BLAST programs. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov). BLASTP is used for amino acid sequences and BLASTN for nucleotide sequences. The BLAST program uses as defaults: Cost to open gap: default= 5 for nucleotides / 11 for proteins Cost to extend gap: default = 2 for nucleotides / 1 for proteins Penalty for nucleotide mismatch: default = -3 Reward for nucleotide match: default= 1 Expect value: default = 10 Wordsize: default = 11 for nucleotides / 28 for megablast / 3 for proteins.

[0077] Furthermore, the degree of local identity between the amino acid sequence query or nucleic acid sequence query and the retrieved homologous sequences is determined by the BLAST program. However only those sequence segments are compared that give a match above a certain threshold. Accordingly, the program calculates the identity only for these matching segments. Therefore, the identity calculated in this way is referred to as local identity. Alternatively, % identity for any candidate nucleic acid or amino acid sequence relative to a reference sequence can be determined as follows. A reference sequence (e.g., a nucleic acid sequence or an amino acid sequence described herein) is aligned to one or more candidate sequences using the computer program Clustal Omega (version 1.2.1, default parameters), which allows alignments of nucleic acid or polypeptide sequences to be carried out across their entire length (global alignment). Chenna et al., 2003, Nucleic Acids Res. 31(13):3497-500.

[0078] Clustal Omega calculates the best match between a reference and one or more candidate sequences, and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a reference sequence, a candidate sequence, or both, to maximize sequence alignments. For fast pairwise alignment of nucleic acid sequences, the following default parameters are used: word size: 2; window size: 4; scoring method: %age; number of top diagonals: 4; and gap penalty: 5. For multiple alignment of nucleic acid sequences, the following parameters are used: gap opening penalty: 10.0; gap extension penalty: 5.0; and weight transitions: yes. For fast pairwise alignment of protein sequences, the following parameters are used: word size: 1; window size: 5; scoring method:%age; number of top diagonals: 5; gap penalty: 3. For multiple alignment of protein sequences, the following parameters are used: weight matrix: blosum; gap opening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps: on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gin, Glu, Arg, and Lys; residue-specific gap penalties: on. The Clustal Omega output is a sequence alignment that reflects the relationship between sequences. Clustal Omega can be run, for example, at the Baylor College of Medicine Search Launcher site on the World Wide Web (searchlauncher.bcm.tmc.edu / multi-align / multi-align.html) and at the European Bioinformatics Institute site at http: / / www.ebi.ac.uk / Tools / msa / clustalo / . To determine a % identity of a candidate nucleic acid or amino acid sequence to a reference sequence, the sequences are aligned using Clustal Omega, the number of identical matches in the alignment is divided by the length of the reference sequence, and the result is multiplied by 100. It is noted that the % identity value can be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.

[0079] The term "mature polypeptide" or "mature enzyme" as used herein refers to a polypeptide in its final active form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. It is known in the art that a host cell may produce a mixture of two of more different mature polypeptides (i.e., with a different C- terminal and / or N-terminal amino acid) expressed by the same polynucleotide.

[0080] The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.

[0081] The term "coding sequence" refers to a nucleotide sequence, which directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0082] The term "control sequence" as used herein refers to a nucleotide sequence necessary for expression of a polynucleotide encoding a polypeptide. A control sequence may be native (i.e., from the same gene) or heterologous or foreign (i.e., from a different gene) to the polynucleotide encoding the polypeptide. Control sequences include, but are not limited to leader sequences, polyadenylation sequence, pro-peptide coding sequence, promoter sequences, signal peptide coding sequence, translation terminator (stop) sequences and transcription terminator (stop) sequences. To be operational control sequences usually must include promoter sequences, transcriptional and translational stop signals. Control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with a coding region of a polynucleotide encoding a polypeptide.

[0083] The term "expression" includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification, and secretion.

[0084] The term "expression vector" refers to a DNA molecule, either single- or double stranded, either linear or circular, which comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression. Expression vectors include expression cassettes for the integration of genes into a host cell as well as plasmids and / or chromosomes comprising such genes.

[0085] The term "host cell" refers to any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. Host cell encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0086] The term "polynucleotide construct" refers to a polynucleotide, either single- or double stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, and which comprises a polynucleotide encoding a polypeptide and one or more control sequences.

[0087] The term "operably linked" refers to a configuration in which a control sequence is placed at an appropriate position relative to the coding polynucleotide such that the control sequence directs expression of the coding polynucleotide.

[0088] The terms "nucleotide sequence and "polynucleotide" are used herein interchangeably.

[0089] The term "comprise" and "include" as used throughout the specification and the accompanying items as well as variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. These words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

[0090] The articles "a" and "an" are used herein refers to one or to more than one (i.e. to one or at least one) of the grammatical object of the article. By way of example, "an element" may mean one element or more than one element.

[0091] Terms like "preferably", "commonly", "particularly", and "typically" are not utilized herein to limit the scope of the itemed invention or to imply that certain features are critical, essential, or even important to the structure or function of the itemed invention. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present invention.

[0092] The term "cell culture" as used herein refers to a culture medium comprising a plurality of host cells of the invention. A cell culture may comprise a single strain of host cells or may comprise two or more distinct host cell strains. The culture medium may be any medium that may comprise a recombinant host, e.g., a liquid medium (i.e., a culture broth) or a semi-solid medium, and may comprise additional components, e.g., a carbon source such as dextrose, sucrose, glycerol, or acetate; a nitrogen source such as ammonium sulfate, urea, or amino acids; a phosphate source; vitamins; trace elements; salts; amino acids; nucleobases; yeast extract; aminoglycoside antibiotics such as G418 and hygromycin B.

[0093] All methods described herein can be performed in any suitable order of steps unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0094] All percentages, ratios and proportions herein are by weight, unless otherwise specified. A weight percent (weight %, also as wt. %) of a component, unless specifically stated to the contrary, is based on the total weight of the composition in which the component is included (e.g., on the total amount of the reaction mixture).

[0095] Terms used herein may be preceded and / or followed by a single dash, " ", or a double dash, "=", to indicate the bond order of the bond between the named substituent and its parent moiety; a single dash indicates a single bond and a double dash indicates a double bond or a pair of single bonds in the case of a spiro-substituent. In the absence of a single or double dash it is understood that a single bond is formed between the substituent and its parent moiety; further, substituents are intended to be read "left to right" with reference to the chemical structure referred to unless a dash indicates otherwise. For example, arylalkyl, arylalkyl-, and alkylaryl indicate the same functionality.

[0096] For simplicity, chemical moieties are defined and referred to throughout primarily as univalent chemical moieties (e.g., alkyl, aryl, etc.). Nevertheless, such terms are also used to convey corresponding multivalent moieties under the appropriate structural circumstances clear to those skilled in the art. For example, while an "alkyl" moiety can refer to a monovalent radical (e.g. CH3-CH2-), in some circumstances a bivalent linking moiety can be "alkyl," in which case those skilled in the art will understand the alkyl to be a divalent radical (e.g., -CH2-CH2-), which is equivalent to the term "alkylene." (Similarly, in circumstances in which a divalent moiety is required and is stated as being "aryl," those skilled in the art will understand that the term "aryl" refers to the corresponding divalent moiety, arylene). All atoms are understood to have their normal number of valences for bond formation (i.e., 4 for carbon, 3 for N, 2 for O, and 2, 4, or 6 for S, depending on the oxidation state of the S). Nitrogens in the presently disclosed compounds can be hypervalent, e.g., an N-oxide or tetrasubstituted ammonium salt. On occasion a moiety may be defined, for example, as -B-(A)a, wherein a is 0 or 1. In such instances, when a is 0 the moiety is -B and when a is 1 the moiety is -B-A.

[0097] As used herein, the term "alkyl" or "alkane" includes a saturated hydrocarbon having a designed number of carbon atoms, such as 1 to 40 carbons (i.e., inclusive of 1 and 40), 1 to 35 carbons, 1 to 25 carbons, 1 to 20 carbons, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18. Alkyl groups or alkanes may be straight or branched and depending on context, may be a monovalent radical or a divalent radical (i.e., an alkylene group). For example, the moiety "-(Cl C6 alkyl) O-" signifies connection of an oxygen through an alkylene bridge having from 1 to 6 carbons and C1-C3 alkyl represents methyl, ethyl, and propyl moieties. Examples of "alkyl" include, for example, methyl, ethyl, propyl, isopropyl, butyl, iso, sec and tert butyl, pentyl, and hexyl. Examples of "alkane" include, for example, methane, ethane, propane, isopropane, butane, isobutane, sec-butane, tert-butane, pentane, hexane, heptane, and octane.

[0098] The term "alkoxy" represents an alkyl group of indicated number of carbon atoms attached to the parent molecular moiety through an oxygen bridge. Examples of "alkoxy" include, for example, methoxy, ethoxy, propoxy, and isopropoxy.

[0099] The term "alkenyl" as used herein, unsaturated hydrocarbon containing from 2 to 10 carbons (i.e., inclusive of 2 and 10), 2 to 8 carbons, 2 to 6 carbons, or 2, 3, 4, 5 or 6, unless otherwise specified, and containing at least one carbon-carbon double bond. Alkenyl group may be straight or branched and depending on context, may be a monovalent radical or a divalent radical (i.e., an alkenylene group). For example, the moiety "-(C2 C6 alkenyl) O-" signifies connection of an oxygen through an alkenylene bridge having from 2 to 6 carbons. Representative examples of alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-l-heptenyl, 3-decenyl, and 3,7-dimethylocta-2,6-dienyl.

[0100] The term "alkynyl" as used herein, unsaturated hydrocarbon containing from 2 to 10 carbons (i.e., inclusive of 2 and 10), 2 to 8 carbons, 2 to 6 carbons, or 2, 3, 4, 5 or 6 unless otherwise specified, and containing at least one carbon-carbon triple bond. Alkynyl group may be straight or branched and depending on context, may be a monovalent radical or a divalent radical (i.e., an alkynylene group). For example, the moiety "-(C2 C6 alkynyl) O-" signifies connection of an oxygen through an alkynylene bridge having from 2 to 6 carbons. Representative examples of alkynyl include, but are not limited to, acetylenyl, 1-propynyl, 2-propynyl, 3-butynyl, 2-pentynyl, and 1-butynyl.

[0101] The term "aryl" represents an aromatic ring system having a single ring (e.g., phenyl) which is optionally fused to other aromatic hydrocarbon rings or non-aromatic hydrocarbon or heterocyclic rings. "Aryl" includes ring systems having multiple condensed rings and in which at least one is carbocyclic and aromatic, (e.g., 1,2,3,4 tetrahydronaphthyl, naphthyl). Examples of aryl groups include phenyl, 1 naphthyl, 2 naphthyl, indanyl, indenyl, dihydronaphthyl, fluorenyl, tetralinyl, and 6,7,8,9-tetrahydro-5H-benzo[a]cycloheptenyl. "Aryl" also includes ring systems having a first carbocyclic, aromatic ring fused to a nonaromatic heterocycle, for example, lH-2,3 dihydrobenzofuranyl and tetrahydroisoquinolinyl. The aryl groups herein are unsubstituted or, when specified as "optionally substituted", can unless stated otherwise be substituted in one or more substitutable positions with various groups as indicated.

[0102] The term "heteroaryl" refers to an aromatic ring system containing at least one aromatic heteroatom selected from nitrogen, oxygen and sulfur in an aromatic ring. Most commonly, the heteroaryl groups will have 1, 2, 3, or 4 heteroatoms. The heteroaryl may be fused to one or more non-aromatic rings, for example, cycloalkyl or heterocycloalkyl rings, wherein the cycloalkyl and heterocycloalkyl rings are described herein. In one embodiment of the present compounds the heteroaryl group is bonded to the remainder of the structure through an atom in a heteroaryl group aromatic ring. In another embodiment, the heteroaryl group is bonded to the remainder of the structure through a non-aromatic ring atom. Examples of heteroaryl groups include, for example, pyridyl, pyrimidinyl, quinolinyl, benzothienyl, indolyl, indolinyl, pyridazinyl, pyrazinyl, isoindolyl, isoquinolyl, quinazolinyl, quinoxalinyl, phthalazinyl, imidazolyl, isoxazolyl, pyrazolyl, oxazolyl, thiazolyl, indolizinyl, indazolyl, benzothiazolyl, benzimidazolyl, benzofuranyl, furanyl, thienyl, pyrrolyl, oxadiazolyl, thiadiazolyl, benzo[l,4]oxazinyl, triazolyl, tetrazolyl, isothiazolyl, naphthyridinyl, isochromanyl, chromanyl, isoindolinyl, isobenzothienyl, benzoxazolyl, pyridopyridinyl, purinyl, benzodioxolyl, triazinyl, pteridinyl, benzothiazolyl, imidazopyridinyl, imidazothiazolyl, benzisoxazinyl, benzoxazinyl, benzopyranyl, benzothiopyranyl, chromonyl, chromanonyl, pyridinyl N-oxide, isoindolinonyl, benzodioxanyl, benzoxazolinonyl, pyrrolyl N-oxide, pyrimidinyl N-oxide, pyridazinyl N-oxide, pyrazinyl N-oxide, quinolinyl N-oxide, indolyl N-oxide, indolinyl N-oxide, isoquinolyl N-oxide, quinazolinyl N-oxide, quinoxalinyl N-oxide, phthalazinyl N-oxide, imidazolyl N-oxide, isoxazolyl N-oxide, oxazolyl N-oxide, thiazolyl N-oxide, indolizinyl N-oxide, indazolyl N-oxide, benzothiazolyl N-oxide, benzimidazolyl N-oxide, pyrrolyl N-oxide, oxadiazolyl N-oxide, thiadiazolyl N-oxide, triazolyl N-oxide, tetrazolyl N-oxide, benzothiopyranyl S oxide, benzothiopyranyl S,S dioxide. Preferred heteroaryl groups include pyridyl, pyrimidyl, quinolinyl, indolyl, pyrrolyl, furanyl, thienyl and imidazolyl, pyrazolyl, indazolyl, thiazolyl and benzothiazolyl. In certain embodiments, each heteroaryl is selected from pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, imidazolyl, isoxazolyl, pyrazolyl, oxazolyl, thiazolyl, furanyl, thienyl, pyrrolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, isothiazolyl, pyridinyl N-oxide, pyrrolyl N-oxide, pyrimidinyl N-oxide, pyridazinyl N-oxide, pyrazinyl N-oxide, imidazolyl N-oxide, isoxazolyl N-oxide, oxazolyl N-oxide, thiazolyl N-oxide, pyrrolyl N-oxide, oxadiazolyl N-oxide, thiadiazolyl N-oxide, triazolyl N-oxide, and tetrazolyl N-oxide. Preferred heteroaryl groups include pyridyl, pyrimidyl, quinolinyl, indolyl, pyrrolyl, furanyl, thienyl, imidazolyl, pyrazolyl, indazolyl, thiazolyl and benzothiazolyl. The heteroaryl groups herein are unsubstituted or, when specified as "optionally substituted", can unless stated otherwise be substituted in one or more substitutable positions with various groups, as indicated.

[0103] The term "heterocycloalkyl" refers to a non-aromatic ring or ring system containing at least one heteroatom that is preferably selected from nitrogen, oxygen and sulfur, wherein said heteroatom is in a non aromatic ring. The heterocycloalkyl may have 1, 2, 3 or 4 heteroatoms. The heterocycloalkyl may be saturated (i.e., a heterocycloalkyl) or partially unsaturated (i.e., a heterocycloalkenyl). Heterocycloalkyl includes monocyclic groups of three to eight annular atoms as well as bicyclic and polycyclic ring systems, including bridged and fused systems, wherein each ring includes three to eight annular atoms. The heterocycloalkyl ring is optionally fused to other heterocycloalkyl rings and / or non-aromatic hydrocarbon rings. In certain embodiments, the heterocycloalkyl groups have from 3 to 7 members in a single ring. In other embodiments, heterocycloalkyl groups have 5 or 6 members in a single ring. In some embodiments, the heterocycloalkyl groups have 3, 4, 5, 6 or 7 members in a single ring. Examples of heterocycloalkyl groups include, for example, azabicyclo[2.2.2]octyl (in each case also "quinuclidinyl" or a quinuclidine derivative), azabicyclo[3.2.1]octyl, 2,5-diazabicyclo[2.2.1]heptyl, morpholinyl, thiomorpholinyl, thiomorpholinyl S oxide, thiomorpholinyl S,S dioxide, 2 oxazolidonyl, piperazinyl, homopiperazinyl, piperazinonyl, pyrrolidinyl, azepanyl, azetidinyl, pyrrolinyl, tetrahydropyranyl, piperidinyl, tetrahydrofuranyl, tetrahydrothienyl, 3,4-dihydroisoquinolin-2(lH)-yl, isoindolindionyl, homopiperidinyl, homomorpholinyl, homothiomorpholinyl, homothiomorpholinyl S,S dioxide, oxazolidinonyl, dihydropyrazolyl, dihydropyrrolyl, dihydropyrazinyl, dihydropyridinyl, dihydropyrimidinyl, dihydrofuryl, dihydropyranyl, imidazolidonyl, tetrahydrothienyl S oxide, tetrahydrothienyl S,S dioxide and homothiomorpholinyl S oxide. Especially desirable heterocycloalkyl groups include morpholinyl, 3,4-dihydroisoquinolin-2(lH)-yl, tetrahydropyranyl, piperidinyl, aza bicyclo[2.2.2]octyl, y butyrolactonyl (i.e., an oxo substituted tetrahydrofuranyl), y butryolactamyl (i.e., an oxo substituted pyrrolidine), pyrrolidinyl, piperazinyl, azepanyl, azetidinyl, thiomorpholinyl, thiomorpholinyl S,S dioxide, 2 oxazolidonyl, imidazolidonyl, isoindolindionyl, piperazinonyl. The heterocycloalkyl groups herein are unsubstituted or, when specified as "optionally substituted", can unless stated otherwise be substituted in one or more substitutable positions with various groups, as indicated.

[0104] The term "cycloalkyl" or "cycloalkane" refers to a non-aromatic carbocyclic ring or ring system, which may be saturated (i.e., a cycloalkyl, a cycloalkane) or partially unsaturated (i.e., a cycloalkenyl). The cycloalkyl ring can be optionally fused to or otherwise attached (e.g., bridged systems) to other cycloalkyl rings. Certain examples of cycloalkyl groups or cycloalkanes present in the disclosed compounds have from 3 to 7 members in a single ring, such as having 5 or 6 members in a single ring. In some embodiments, the cycloalkyl groups have 3, 4, 5, 6 or 7 members in a single ring. Examples of cycloalkyl groups include, for example, cyclohexyl, cyclopentyl, cyclobutyl, cyclopropyl, tetrahydronaphthyl and bicyclo[2.2.1]heptane. Examples of cycloalkanes include, for example, cyclohexane, methylcyclohexane, cyclohexanone, cyclohexanol, cyclopentane, cycloheptane, and cycloctane. The cycloalkyl groups herein are unsubstituted or, when specified as "optionally substituted", may be substituted in one or more substitutable positions with various groups, as indicated.

[0105] The term "ring system" encompasses monocycles, as well as fused and / or bridged polycycles.

[0106] The terms "halogen" or "halo" indicate fluorine, chlorine, bromine, and iodine. In certain embodiments of each and every embodiment described herein, the term "halogen" or "halo" refers to fluorine or chlorine. In certain embodiments of each and every embodiment described herein, the term "halogen" or "halo" refers to fluorine.

[0107] The term "halide" indicates fluoride, chloride, bromide, and iodide. In certain embodiments of each and every embodiment described herein, the term "halide" refers to bromide or chloride.

[0108] The term "substituted," when used to modify a specified group or radical, means that one or more hydrogen atoms of the specified group or radical are each, independently of one another, replaced with the same or different substituent groups as defined below, unless specified otherwise.

[0109] Specific protecting groups may be used to protect reactive functionalities of a starting material or intermediate to prepare a desired product. In general, the need for such protecting groups as well as the conditions necessary to attach and remove such groups will be apparent to those skilled in the art of organic synthesis. An authoritative account describing the many alternatives to the trained practitioner are J. F. W. McOmie, "Protective Groups in Organic Chemistry", Plenum Press, London and New York 1973, in T. W. Greene and P. G. M. Wuts, "Protective Groups in Organic Synthesis", Third edition, Wiley, New York 1999, in "The Peptides"; Volume 3 (editors: E. Gross and J. Meienhofer), Academic Press, London and New York 1981, in "Methoden der organischen Chemie", Houben-Weyl, 4.sup.th edition, Vol. 15 / 1, Georg Thieme Verlag, Stuttgart 1974, in H.-D. Jakubke and H. Jescheit, "Aminosauren, Peptide, Proteine", Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982, and / or in Jochen Lehmann, "Chemie der Kohlenhydrate: Monosaccharide and Derivate", Georg Thieme Verlag, Stuttgart 1974. The protecting groups may be removed at a convenient subsequent stage using methods known from the art.

[0110] As used herein, the term "benzyl" ("Bn") includes unsubstituted (i.e., (C6H5)-CH2-) and substituted benzyl (i.e., benzyl substitututed at the 2-, 3-, and / or 4- position with C1-C8 alkyl or halide). The person of ordinary skill in the art will appreciate that oxygen protecting groups include alkoxycarbonyl, acyl, acetal, ether, ester, silyl ether, alkylsulfonyl, and arylsulfonyl. Exemplary oxygen protecting groups include allyl, triphenylmethyl (trityl or Tr), benzyl, methanesulfonyl, p-toluenesulfonyl, p-methoxybenzyl (PMB), p-methoxyphenyl (PMP), methoxymethyl (MOM), p-methoxyethoxymethyl (MEM), tetrahydropyranyl (THP), ethoxyethyl (EE),methylthiomethyl (MTM), 2-methoxy-2-propyl (MOP), 2-trimethylsilylethoxymethyl (SEM), benzoate (BZ), allyl carbonate, 2.2.2-trichloroethyl carbonate (Troc), 2-trimethylsilylethyl carbonate, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), triphenylsilyl (TPS), t-butyldimethylsilyl (TBDMS), and t-butyldiphenylsilyl (TBDPS). A variety of protecting groups for the oxygen and the synthesis thereof may be found in "Protective Groups in Organic Synthesis" by T. W. Greene and P. G. M. Wuts, John Wiley & Sons, 1999. In certain embodiments, an appropriate oxygen protecting goup may be used in place of benzyl. Genetically modified host cells

[0111] Microorganisms optimized to produce benzylisoquinoline alkaloids are in great need and even more so host cells optimized to demethylate benzylisoquinoline alkaloids such as thebaine and / or oripavine into the corresponding northebaine and / or nororipavine, which are in high demand for chemical conversion into other pharmaceutically relevant benzylisoquinoline alkaloids.

[0112] The invention provides in a first aspect such genetically modified host cell comprising a pathway having enhanced production of one or more benzylisoquinoline alkaloids wherein the cell comprises one or more features selected from: a) expression of one or more heterologous genes encoding a demethylase capable of converting thebaine into northebaine, thebaine into oripavine, thebaine into nororipavine and / or oripavine into nororipavine; b) expression of one or more heterologous genes encoding a tyrosine hydroxylases (TH) converting L-tyrosine into L-dopa selected from TH's having at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the TH comprised in 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63 or 65; c) reduction or elimination of activity of one or more dehydrogenases native to the host cell selected from the dehydrogenases comprised in SEQ ID NO: 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703 or 705; d) reduction or elimination of activity of one or more reductases native to the host cell selected from the reductases comprised in SEQ. ID NO: 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729 or 731; e) expression of one or more heterologous genes encoding a norcoclaurine synthases (NCS) converting Dopamine and 4-HPAA into (S)-norcoclaurine selected from NCS's having at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the NCS comprised in SEQ ID NO: 73 OR 76; f) expression of one or more heterologous genes encoding i) a fused 1,2-dehydroreticuline synthase-l,2-dehydroreticuline reductases (DRS-DRR) converting (S)-Reticuline into (R)-reticuline, wherein ia) the DRS-DDRs has at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the DRS-DRR comprised in SEQ ID NO: 92, 94, 96; or ib) the DRS moiety has at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the DRS comprised in SEQ. ID NO: 98,100,102, 104 or 106; and the DRR moiety has at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the DRR comprised in SEQ ID NO: 108 or 110; or ii) a DRS having at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the DRS comprised in SEQ ID NO: 98, 100, 102, 104 or 106; and a DRR having at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the DRR comprised in SEQ ID NO: 108 or 110; g) expression of one or more heterologous genes encoding a thebaine synthase (THS) converting 7-O-acetylsalutaridinol into thebaine selected from THS's having at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the THS comprised in SEQ ID NO: 126, 127, 128, 129, 131, 133, 134, 136, 138; and h) expression of one or more heterologous genes encoding a transporter protein capable of increasing uptake in the host cell of a reticuline derivative selected from transporter proteins having at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the transporter protein comprised in SEQ ID NO: 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 733, 735, 795, 797, 799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823 or 825. Heterologous Demethylase

[0113] In a further aspect the genetically modified host cells of the invention expresses, alone or in combination with other heterologous genes of the invention, one or more heterologous genes encoding one or more demethylases capable of converting thebaine into northebaine, thebaine into oripavine, thebaine into nororipavine and / or oripavine into nororipavine. The demethylase of the invention can be any suitable demethylase capable of converting thebaine into northebaine, thebaine into oripavine, thebaine into nororipavine and / or oripavine into nororipavine, which is heterologous to the host cell and which cooperates well with the other enzymes of the benzylisoquinoline alkaloid pathway and / or the auxilliary cellular mechanisms.

[0114] In a particular embodiment the demethylase have specificity towards producing the norcompounds and produces less by-products. It has been identified that in particular insect demethylase, when expressed in a genetically modified host cell possess a hitherto unprecedented high product specificity producing a high product:by-product ratio, where the product:by-product is either (Northebaine):(thebaine N-oxide), (Northebaine):(northebaine oxaziridine), (Nororipavine):(oripavine N-oxide) and / or (Nororipavine):(nororipavine oxaziridine). Aside from more effectively converting more thebaine and / or oripaving into the desired corresponding norcompounds, for in vivo conversion the insect demethylase of the invention also produces less N-oxide or oxaziridine by-products and this property provide advantage over the art, since such by-products may impact negatively of the cell function as well as they may interfere with efficiency of any subsequent chemical conversion steps and lower the efficiency of production. Accordingly, in one embodiment the demethylase of the invention have a product:by-product molar ratio of at least 2,0, such as at least 2,25, such as at least 2,5, such as at least 2,75, such as at least 3,0, such as at least 3,25, such as at least 3,5, such as at least 3,75, such as at least 4,0, such as at least 4,5, such as at least 5,0, such as at least 10,0, such as at least 25, such as at least 50, such as at least 75, such as at least 100 and wherein when the product is northebaine then the by-product is thebaine N-oxide and / or northebaine oxaziridine and when the product is nororipavine then the by-product is oripavine N-oxide and / or nororipavine oxaziridine.

[0115] The insect demethylase of the invention remarkably displays N-demethylation activity and / or O-activity, whereby it is capable of converting thebaine of the formula I into northebaine of the 5 formula II: and / or converting oripavine of the formula (III) into nororipavine of formula IV: 10

[0116] Further, the present inventors have found that demethylases derived from insects and in particular demethylases of family CYP6, are remarkably effective in converting thebaine and / or oripavine into the corresponding nor-compounds producing less by-products. Therefore, in one embodiment the demethylase of the invention is derived from an insect and in another embodiment 15 the demethylase of the invention is of family CYP6. Relevant insects include those which feeds on plants with high contents of thebaine and / or oripavine such as poppy and include moths of the order Lepidoptera, such as moths of the genus Helicoverpa, Spodoptera, Cnaphalocrocis, Bombyx and Heliothis. Demethylases from the species Helicoverpa armigera, Spodoptera exigua, Cnaphalocrocis medinalis, Bombyx mandarina and Heliothis virescens, are particularly useful. Without being bound to the theory the present inventors contemplate that insects feeding from plants containing a high level of thebaine and / or oripavine, as a protection mechanism, during evolution have developed enzymes converting these potentially harmful substrates.

[0117] Examples of insect demethylases which works remarkably well in converting thebaine and / or oripavine with low formation of by-products in a heterologous host cell includes the demethylases selected from of SEQ ID NO: 140, 142, 144, 146, 148, 150, 152, 154, 156,158, 160, 162, 164, 166,168, 170,172,174,176,178,180,182,184,186,188, 190, 192,194,196, 827, 829, 831, 833, 835, 837, 839, 841, 843, 845, 847, 849, 851, 853, 855, 857, 859, 861, 863, 865, 867 and 869. No such demethylase nor anyone with any close homology has previously been reported useful in a host and let alone with the remarkably high efficiency. Accordingly, in a further embodiment the demethylase of the invention comprises a polypeptide selected from the group consisting of: a) a demethylase which is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the demethylase comprised in any one of SEQ ID NO: 140,142,144,146,148,150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 827, 829, 831, 833, 835, 837, 839, 841, 843, 845, 847, 849, 851, 853, 855, 857, 859, 861, 863, 865, 867 and 869; b) a demethylase encoded by a polynucleotide which is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to a polynucleotide comprised in any one of SEQ. ID NO: 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 828, 830, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850, 852, 854, 856, 858, 860, 862, 864, 866, 868 and 870 or genomic DNA thereof; and c) a functional variant of the demethylase of (a) or (b) capable of converting thebaine into northebaine, thebaine into oripavine, thebaine into nororipavine and / or oripavine into nororipavine.

[0118] In particular the insect demethylase is a) a demethylase comprised in any one of SEQ ID NO: 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 827, 829, 831, 833, 835, 837, 839, 841, 843, 845, 847, 849, 851, 853, 855, 857, 859, 861, 863, 865, 867 and 869; or b) a demethylase encoded by a polynucleotide comprised in any one of SEQ. ID NO: or genomic DNA thereof encoding the P450 comprised in any one of SEQ ID NO: 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 828, 830, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850, 852, 854, 856, 858, 860, 862, 864, 866, 868 and 870.

[0119] Alternatively, the demethylase of the invention can be derived from a fungus, in particular fungi of a genus selected from Rhizopus, Lichtheimia, Syncephalastrum, Cunninghamella, Mucor, Parasitella, Absidia, Choanephora, Bifiguratus and Choanephora. In a more specific embodiment the P450 may be derived from a fungal species selected from Rhizopus microspores, Rhizopus azygosporus, Rhizopus stolonifera, Rhizopus oryzae, Rhizopus delemar, Lichtheimia corymbifera, Lichtheimia ramose, Syncephalastrum racemosum, Cunninghamella echinulate, Mucor circinelloides, Mucor ambiguous, Parasitella parasitica, Absidia repens, Absidia glauca, Choanephora cucurbitarum, Bifiguratus adelaidae and Choanephora cucurbitarum.

[0120] Examples of fungal demethylases which works well in converting thebaine and / or oripavine with low formation of by-products in a heterologous host cell includes the demethylase selected from SEQ ID NO: 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288 or 290. No such demethylase has previously been reported so effective and useful in a host cell and let alone with the remarkable high efficiency. Accordingly, in a further embodiment the demethylase of the invention comprises a polypeptide selected from the group consisting of: a) a demethylasewhich is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the demethylase comprised in any one of SEQ ID NO: 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288 and 290; b) a demethylase encoded by a polynucleotide which is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the polynucleotide comprised in any one of SEQ ID NO: 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, Til, 279, 281, 283, 285, 287, 289 and 291 or genomic DNA thereof; and c) a functional variant of the demethylase of (a) or (b) capable of converting thebaine into northebaine, thebaine into oripavine, thebaine into nororipavine and / or oripavine into nororipavine.

[0121] In particular the fungal demethylase is: a) the demethylase comprised in any one of SEQ. ID NO: 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288 and 290; or b) the demethylase encoded by a polynucleotide comprised in any one of SEQ ID NO: 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, Til, Tl^, 281, 283, 285, 287, 289 and 291 or genomic DNA thereof.

[0122] A particular demethylase of the invention is one which does not comprise one or more of the amino acids selected from: a) Valine at a position corresponding to V75 of SEQ ID NO: 290; b) Isoleucine at a position corresponding to 179 of SEQ ID NO: 290; c) Isoleucine at a position corresponding to V83 of SEQ ID NO: 290; d) Asparagine at a position corresponding to N84 of SEQ ID NO: 290; e) Arginine at a position corresponding to R86 of SEQ ID NO: 290; f) Aspartic acid at a position corresponding to D87 of SEQ ID NO: 290; g) Glutamic acid at a position corresponding to E126 of SEQ ID NO: 290; h) Threonine at a position corresponding to T145 of SEQ ID NO: 290; i) Asparagine at a position corresponding to N172 of SEQ ID NO: 290; j) Threonine at a position corresponding to T193 of SEQ ID NO: 290; k) Glycine at a position corresponding to G218 of SEQ ID NO: 290; I) Isoleucine at a position corresponding to 1236 of SEQ ID NO: 290; m) Alanine at a position corresponding to A258 of SEQ ID NO: 290; n) Methionine at a position corresponding to M259 of SEQ ID NO: 290; o) Aspartic acid at a position corresponding to D298 of SEQ ID NO: 290; p) Leucine at a position corresponding to L430 of SEQ ID NO: 290; q) Histidine at a position corresponding to H448 of SEQ. ID NO: 290; r) Asparagine at a position corresponding to N503 of SEQ ID NO: 290; s) Proline at a position corresponding to P506 of SEQ ID NO: 290; t) Phenylalanine at a position corresponding to F507 of SEQ ID NO: 290; u) Asparagine at a position corresponding to N508 of SEQ ID NO: 290; and v) Valine at a position corresponding to V509 of SEQ ID NO: 290;

[0123] Further to this embodiment the demethylase may not comprise histidine at a position corresponding to H448 of SEQ ID NO: 290, asparagine at a position corresponding to H508 of SEQ ID NO: 290 and / or valine at a position corresponding to H509 of SEQ ID NO: 290. Still further to this embodiment the demethylase may comprise comprise tyrosine at the position corresponding to position 448 of SEQ ID NO: 290, threonine at the position corresponding to position corresponding to H508 of SEQ ID NO: 290 and / or glycine at the position corresponding to position corresponding to H509 of SEQ ID NO: 290. Within this embodiment the demethylase may specifically be the P450 of SEQ ID NO: 250 or SEQ ID NO: 252.

[0124] The demethylase of SEQ ID NO: 218, 220, 222, 224, 226, 228, 236, 240, 250, 252, 254 and 268 have in addition to N-demethylase activity also O-demethylase activity (ODM) and are capable of demethylating thebaine of the formula I into oripavine of the formula III as described supra.

[0125] In a separate embodiment the cell of the invention further comprises a demethylase-CPR capable of reducing and / or regenerating the demethylase enzyme. The demethylase-CPR may also be heterologous to the cell.

[0126] Some demethylases may work better together with a demethylase-CPR from a related source so in a particular embodiment where the demethylase is an insect demethylase, the demethylase-CPR may also advantageously be an insect demethylase-CPR, such as a demethylase-CPR derived from an insect of the order Lepidoptera, such as the insect demethylase-CPR derived from an insect of the genus helicoverpa, Heliothis or Spodoptera such as demethylase-CPR derived from an insect of the species Helicoverpa armigera, Heliothis virescens or Spodoptera exigua.

[0127] In particular, the insect demethylase-CPR may comprise a polypeptide selected from the group consisting of: a) a demethylase-CPR which is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the demethylase-CPR comprised in SEQ ID NO: 292, 294, 296, 298, 300 or 302; b) a demethylase-CPR encoded by a polynucleotide which is at least 20% identical to the polynucleotide comprised in SEQ ID NO: 293, 295, 297, 299, 301, 303 or 305 or genomic DNA thereof; and c) a functional variant of the demethylase-CPR of (a) or (b) capable of reducing / regenerating the demethylase of the invention.

[0128] In another embodiment where the demethylase is a fungal demethylase the demethylase-CPR may advantageously be a fungal demethylase-CPR. In particular, the fungal demethylase-CPR may comprise a polypeptide selected from the group consisting of: a) a demethylase-CPR which is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the demethylase-CPR comprised in SEQ. ID NO: 305; b) a demethylase-CPR encoded by a polynucleotide which is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the polynucleotide comprised in SEQ ID NO: 306 or genomic DNA thereof; and c) a functional variant of the demethylase-CPR of (a) or (b) capable of reducing / regenerating the demethylase.

[0129] Further suitable Demethylases are disclosed in WO2018 / 229306 or WO2018 / 075670, which is hereby incorporated by reference in their entirety.

[0130] In one embodiment the heterologous demethylase is an artificial mutant. In one type of mutations the naturally occurring leader / signal sequence has been mutated to improve the performance eg. by wholly or partially replacing the the leader / signal sequence with a leader / signal sequence from another enzyme. Examples of such mutations are SEQ ID NOS: 845, 847, 851, 853, 857, 859, 863, 865, 867 and 869.

[0131] In another embodiment the demethylase is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the demethylase comprised in SEQ ID NO 152 (Hv_CYP_A0A2A4JAM9) and has one or more mutations corresponding to A110X, H242X, and / or V224X, such as A110N, 242P and / or V224I, preferably all three mutations A110N+H242P+V224L

[0132] In another embodiment the demethylase is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the demethylase comprised in SEQ ID NO 140 (HaCYP6AE15v2) and has one or more mutations corresponding to A316X and / or D392X, such as A316G and / or D392E preferably both.

[0133] Further the invention provides mutant insect demethylases comprising one or more mutations in the signal sequence of the naturally occurring insect demethylase. In these insect demethylases the signal sequence may have been wholly or partially replaced by a signal sequence from another enzyme. Suitably such mutant demethylases have least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the demethylase comprised in SEQ. ID NO: : 845, 847, 851, 853, 857, 859, 863, 865, 867 or 869. Also mutant insect demethylases are provided having least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the demethylase comprised in SEQ ID NO: 152 and comprising one or more mutations corresponding to A110X, H242X, and / or V224X, optionally A110N, H242P and / or V224I. Still further, mutant insect demethylases are provided having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the demethylase comprised in SEQ ID NO: 140 and comprising one or more mutations corresponding to A316X and / or D392X, optionally A316G and / or D392E.

[0134] Analysis comparing the best performing insect demethylases (see example 41) was shown share structural sequence features in the form of amino acid positions conserved within the active and high perfoming insect demethylases. Accordingly, the insect demethylase of the invention comprise one or more conserved amino acids corresponding to amino acids selected from positions G103, Hill, K167, E198, R219, L223, 1256, A259, L273, V284, 1309, L314, Q517, L160, N216 and / or R443 of SEQ ID NO: 152 (Hv_CYP_A0A2A4JAM9) or any conservative substitutions thereof. In a special embodiment the selected one or more conserved amino acid is / are in or near the active site of the demethylase corresponding to G103, Hill and L314 of SEQ ID NO: 152 or any conservative substitutions thereof. Conservative substitutions which may be considered includes but are not limited to i) aliphatic substitutions, such as between G, A, V, L and I; ii) Hydroxyl or sulfur / selenium-containing substitutions such as between S, C, T and M; iii) aromatic substitutions such as between F, Y, and W, iv) basic substitutions, such as between H, K and R; and v) acidic and amidic substitutions, such as between D, E, N and Q. For example, L160 SEQ ID NO: 152 may also V160 and is considered a conservative substitution (see table 43-3 and figure 13). For clarity as regards positions "corresponding" to conserved positions in SEQ ID NO: 152, such positions also include positions which has a different number in a candidate sequence, but which still corresponds and compares to the conserved position in SEQ ID NO: 152 upon alignment with the candidate sequence. Such shifts in numbers occurs e.g. when making amino acid additions or extensions to a candidate sequence. Additional exemplary conservative substitutions are defined in sequence alignment software tools, for example Clustal W, based on additional structural considerations. The software output uses one dot or two dots in the output to indicate the degree of conservation. Examples of tolerated conservative substitutions are those corresponding to 1256V, L160M, N216S, R443K of SEQ. ID NO: 152. In a particular embodiment the demethylase comprises comprise one or more conserved amino acids corresponding to amino acids selected from positions G103, Hill, K167, E198, R219, L223,1256, A259, L273, V284, 1309, L314, Q517, L160, N216 and / or R443 of SEQ ID NO: 152 (Hv_CYP_A0A2A4JAM9) or any conservative substitutions thereof and comprises a polypeptide which is at least 60% identical to the insect demethylase comprised in SEQ ID NO: 152. Heterologous TH - Tyrosine hydroxylase

[0135] In another aspect the host cell of the invention expresses alone or in combination with other heterologous genes of the invention one or more heterologous genes encoding a tyrosine hydroxylases. The TH of the invention may suitably be any natural or mutant TH capable of catalyzing L-tyrosine into L-DOPA. Particularly, the TH is of the CYP76 family. In a special embodiment the TH has at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identityl to the TH comprised in SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63 or 65. In a separate embodiment the TH has at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the TH comprised in SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25. Further suitable THs are disclosed in PCT / EP2020 / 050610 (unpublished) and WO2016 / 049364, which are hereby incorporated by reference in its entirety. Reducing or eliminating enzymes lowering performance of the benzylisoguinoline alkaloid pathway

[0136] In another aspect the host cell of the invention is genetically modified to reduce or eliminate (knockout) activity of one or more native enzymes, which negatively impacts on the production of benzylisoquinoline alkaloid. Such manipulation may be achieved in several ways, all applicable to the host cell of the invention. Reduction or elimination of enzyme activity may be accomplished by disrupting, deleting and / or attenuating expression of the gene encoding the enzyme and / or the translation of the RNA into the protein, eg. by deleting or mutating the gene. Alternatively, and / or in addition, the the enzyme may also be mutated to a less active or non-active variant. In reducing or eliminating activity activitvity of enzymes native to the host care should be taken to balance the positive impact on production of benzylisoquinoline alkaloid and the potential negative impact on cellular viability and growth for maintain an acceptable level of vital cellular functions. 5

[0137] Reduction or elimination of activity of enzymes native to the host cell, particularly includes reduction or elimination enzymes shunting precursors or products away from the benzylisoquinoline alkaloid pathway, so that they become unavaiable for producing benzylisoquinoline alkaloids. One such group of such enzymes is dehydrogenases native to the host cell and in particular dehydrogenases comprised in SEQ ID NO: 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 10   687, 689, 691, 693, 695, 697, 699, 701, 703 or 705. Another group of such enzymes are reductases native to the host cell and in particular reductases comprised in SEQ ID NO: 707, 709, 711, 713, 715, 717, 719,721, 723,725, 727, 729 or 731. Preferred targets of reduction or elimination are one or more enzymes comprised in SEQ ID NO: 665 (ADH6), 669 (YPR1), 671 (AAD3), 675 (ADH3), 679 (ALD6), 705 (HFD1), 709 (ALD4), 713 (GRE2), 717 (YDR541C), 721 (ARII), 729 (PHA2) or 731 (TRP3). Reduction or 15 elimination of one or more the enzymes comprised in 705 (HFD1), 713 (GRE2) or 721 (ARII), is particularly useful.

[0138] Further useful knockouts include: Gene name Systematic name* Function (ref) ALD2 YMR170C Aldehyde dehydrogenase (1,2) ALD3 YMR169C Aldehyde dehydrogenase (1,2) ALD5 YER073W Aldehyde dehydrogenase (1) ADH1 YOL086C Alcohol dehydrogenase (1) ADH2 YMR303C Alcohol dehydrogenase(1) ADH4 YGL256W Alcohol dehydrogenase(1) ADH5 YBR145W Alcohol dehydrogenase(1) ADH7 YCR105W Alcohol dehydrogenase (1-2) SFA1 YDL168W Formaldehyde dehydrogenase (1,2) YGL039W YGL039W Similar to ARII (1-2) GCY1 YOR120W Similar to ARIl(1-2) AAD14 YNL331C Similar to ARIl(1-2) AAD4 YDL243C Similar to ARIl(1-2) TRP2 YER090W Production of tryptophan(1) WO2019 / 243624(1) and Pyne et al, BioRxiv preprint 2019(2); all hereby incorporated by reference in their entirety. Sequences of the table can also be found in Saccharomyces genome database (https: / / www.yeastgenome.org), incorporated herein by reference. Further suitable knockouts are disclosed in WO2018 / 029282, WO2019 / 157383 Heterologous Norcoclaurine Synthase (NCS)

[0139] In a further aspect the host cell of the invention expresses alone or in combination with other heterologous genes of the invention one or more heterologous gene encoding a norcoclaurine synthase (NCS). The NCS of the invention may suitably be any natural or mutant NCS capable of converting Dopamine and 4-HPAA into (S)-norcoclaurine. In a special embodiment the NCS has at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the NCS comprised in SEQ ID NO: 73 OR 76. In a separate embodiment the NCS has at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the NCS comprised in SEQ ID NO: 73 OR 76. Further suitable NCSs are disclosed in WO2018 / 229305, WO2014 / 143744, WO2019 / 165551 and US2015267233, which is hereby incorporated by reference in its entirety. Heterologous STORR

[0140] In a further aspect the host cell of the invention expresses alone or in combination with other heterologous genes of the invention one or more heterologous genes encoding enzymes capable of epimerizing / isomerizing one benzylisoquinoline alkaloid to a benzylisoquinoline alkaloid isomer, such as for example (S)-Reticuline into (R)- reticuline. In a special embodiment the epimerase is: i) a fused 1,2-dehydroreticuline synthase-l,2-dehydroreticuline reductases (DRS-DRR) converting (S)-Reticuline into (R)-reticuline, wherein ia) the DRS-DDRs has at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the DRS-DRR comprised in SEQ ID NO: 92, 94, 96; or ib) the DRS moiety has at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the DRS comprised in SEQ ID NO: 98,100, 102, 104 or 106; and the DRR moiety has at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the DRR comprised in SEQ ID NO: 108 or 110; or ii) a DRS having at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the DRS comprised in SEQ. ID NO: 98, 100, 102, 104 or 106; and a DRR having at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the DRR comprised in SEQ ID NO: 108 or 110.

[0141] In a partiular embodiment the DRR moiety of the epimerase, whether fused to the DRS or separate an Imine reductase, preferably a StIRED such as the reductases comprised in SEQ ID NO. 108 or 110.

[0142] Further suitable epimerases / isomerases are disclosed in WO2015 / 081437, WO2016 / 183023, WO2015 / 173590, WO2018 / 000089, WO2019 / 028390 and WO2019 / 165551 which are hereby incorporated by reference in their entirety. Heterologous THS

[0143] In another aspect the host cell of the invention expresses alone or in combination with other heterologous genes of the invention one or more heterologous genes encoding a thebaine synthase (THS). The THS of the invention may suitably be any natural or mutant THS capable of converting 7-0-acetylsalutaridinol into thebaine. In a special embodiment the THS has is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the THS comprised in SEQ ID NO: 126, 127, 128, 129, 131, 133,134, 136 or 138. In particular SEQ ID NO: 134 and 136 are very efficient thebaine synthases.

[0144] Further suitable THSs are disclosed in WO2018 / 005553, WO2014 / 143744 and WO2019 / 165551, which are hereby incorporated by reference in their entirety. Heterologous transporters

[0145] In another aspect the host cell of the invention expresses alone or in combination with other heterologous genes of the invention one or more heterologous genes encoding transporter protein. The transporter protein of the invention may suitably be any natural or mutant tranporter protein capable of uptake or export in the host cell of a reticuline derivative, such as thebaine, northebaine, oripavine and / or nororipavine.

[0146] In a special embodiment the transporter protein has at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the transporter protein comprised in SEQ ID NO: 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 733, 735, 795, 797, 799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823 or 825.

[0147] Selecting the optimal transporter for given pathway setup may dependent on choice of other enzymes such as the demethylase. So, in a particular embodiment when incorporating a demethylase, especially an insect demethylases, converting oripavine into nor-oripavine, insect transporters are preferred. In particular transporters T180_McoPUP3_46 (SEQ. ID NO: 595), T193_AanPUP3_55 (SEQ ID NO: 613), T149_AcoPUP3_59 (SEQ ID NO: 537) and / or T165_AcoPUP3_13 (SEQ ID NO: 567) have shown particularly effective. In another particular embodiment when in incorporating a demethylase, especially an insect demethylases, converting thebaine into northebaine, insect transporters are preferred. In particular transporters T193_AanPUP3_55 (SEQ ID NO: 613), T198_AcoT97_GA (SEQ ID NO: 623), T149_AcoPUP3_59 (SEQ ID NO: 537) and / or T122_PsoPUP3_17 (SEQ ID NO: 487) have shown particularly effective. Further suitable transporter proteins are disclosed in WO2020 / 078837, which is hereby incorporated by reference in its entirety.

[0148] In a further separate embodiment, the transporter may be an Equilibrative Nucleoside Transporter (ENT) as described in Boswell-Casteel and Hays, 2017. Equilibrative Nucleoside Transporters including those belonging to the SLC29A / ENT transporter (TC 2.A.57) family (https: / / www.uniprot.org) have been shown herein to be capable of demethylase-mediated bioconversion of methylated benzylisoquinoline alkaloids to the corresponding norbenzylisoquinoline alkaloids - in particular oripavine to nororipavine - in a highly efficient manner. Such improvements in yield are particularly remarkable and represent a significant step forward towards a sustainable, secure, and scalable biosynthetic means of producing these compounds.

[0149] The Equilibrative Nucleoside Transporter may particularly be an insect Equilibrative Nucleoside Transporter, including the transporters having at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the transporter protein comprised in SEQ ID NOS: 795, 797, 799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823 or 825, especially SEQ. ID NOS: 795, 797, 799, 801.

[0150] The useful insect transporters disclosed herein have not hitherto been demonstrated to benefit production of benzylisoquinoline alkaloids when incorporated heterologously in genetically modified microorganisms comprising pathways producing benzylisoquinoline alkaloids. Accordingly, in a separate aspect the invention provides a genetically modified host cell comprising a pathway having enhanced production of one or more benzylisoquinoline alkaloids wherein the cell expresses one or more heterologous genes encoding an insect derived transporter protein increasing the cellular uptake or secretion of a benzylisoquinoline alkaloid precursor, said precursor preferably being a benzylisoquinoline alkaloid itself. Particular insect transporters include transporter proteins from the insect genera of Helicoverpa, Heliothis or Pectinophora, in particular from spieces of Pectinophora gossypiella, Helicoverpa armigera or Heliothis virescens. In particular the transporter proteins have at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the transporter protein comprised in SEQ ID NO: 631, 633, 637, 649, 651, 653, 655, 657 or 659. Moreover, the genetically modified cell of the invention may comprise one or more copies of genes encoding one or more insect transporter proteins such as genes / polynucleotides which is at least 70% identical to the transporter encoding polynucleotide comprised in SEQ ID NO: 632, 634, 638, 652, 654, 656, 658 or 660 or genomic DNA thereof. Further enzymes of the benzylisoguinoline alkaloid pathway

[0151] In another aspect the host cell of the invention expresses in combination with other heterologous genes of the invention one or more further heterologous or native enzymes of the benzylisoquinoline alkaloid pathway. In a particular embodiment the host cell of the invention expresses one or more genes encoding polypeptides selected from: a) a 3-deoxy-D-arabino-2-heptulosonic acid 7-phosphate synthase (DAHP synthase) converting PEP and E4P into DAHP; b) a 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (arol) converting 3-phosphoshikimate and PEP into EPSP; c) an arol polypeptide converting DHAP and PEP into EPSP; d) a chorismate synthase converting EPSP into Chorismate; e) a chorismate mutase converting Chorismate into prephenate; f) a prephenate dehydrogenase (Tyrl) converting prephenate into 4-HPP; g) an aromatic aminotransferase converting 4-HPP into L-Tyrosine; h) a TH-CPR capable of reducing TH; i) a L-dopa decarboxylase (DODC) converting L-dopa into dopamine; j) a Tyrosine decarboxylase (TYDC) converting L-dopa into dopamine; k) a hydroxyphenylpyruvate decarboxylase (HPPDC) converting 4-HPP into 4-HPPA; I) a monoamine oxidase converting dopamine into 3,4-DHPAA; m) a 6-O-methyltransferase (6-OMT) converting (S)-norcoclaurine into (S)-Coclaurine and / or norlaudanosoline into (S / -3'-Hydroxy-coclaurine; n)  a   Coclaurine-N-methyltransferase (CNMT) converting   (S)-Coclaurine   into   (S)-N- Methylcoclaurine and / or (S)-3'-hydroxycoclaurine into (S)-3'-hydroxy-N-methyl-coclaurine; o)  a  N-methylcoclaurine hydroxylase (NMCH) converting  (S)-Coclaurine  into  (S)-3'- hydroxycoclaurine and / or (S)-N-Methylcoclaurine into (S)-3'-Hydroxy-N-Methylcoclaurine; p) a 3'-hydroxy-N-methyl-(S)-coclaurine 4'-O-methyltransferase (4'-OMT) converting (S)-3'-Hydroxy-N-Methylcoclaurine into (S)-Reticuline; q) a DRS-CPR capable of reducing DRS-DRR; r) a salutaridine synthase (SAS) converting (R)- reticuline into Salutaridine; s) a salutaridine reductase (SAR) converting Salutaridine to Salutaridinol; and t) a salutaridinol 7-O-acetyltransferase (SAT) converting Salutaridinol into 7-0-acetylsalutaridinol.

[0152] In a special embodiment the corresponding: a) DAHP synthase has at least 20% , such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the DAHP synthase comprised in SEQ ID NO: 1 b) chorismate mutase has at least 20% , such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the chorismate synthase comprised in SEQ ID NO: 3; c) TH-CPR has at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the TH-CPR comprised in SEQ ID NO: 67; d) DODC has at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the DODC comprised in SEQ ID NO: 69 or 71; e) 6-OMT has at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the 6-OMT comprised in SEQ ID NO: 79 or 81; f) CNMT has at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the CNMT comprised in SEQ ID NO: 82 or 84; g) NMCH has at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the NMCH comprised in EQ ID NO: 85 OR 87; h) 4'-OMT has at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the 4'-OMT comprised in SEQ ID NO: 89 or 91; i) DRS-CPR has at least 20% , such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the DRS-CPR comprised in SEQ ID NO: 112 or 114; j) SAS has at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the SAS comprised in SEQ ID NO: 116 or 118; k) SAR has at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the SAR comprised in SEQ ID NO: 120 or 122; I) SAT has at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the SAT comprised in SEQ. ID NO: 123 or 125; and Further suitable enzymes of the benzylisoquinoline alkaloid pathway are disclosed in US2019100781 and WO2019 / 165551, which is hereby incorporated by reference in their entirety. Additional cell modifications improving production of benzylisoquinoline alkaloids

[0153] During the efforts of improving cellular production of recombinant cellular production of benzylisoquinoline alkaloids, several additional useful modifications to cells improving the cellular performance was discovered. In a first aspect it was found that cytosolic heme levels in a production host cell is a significant limiting factor in production of demethylated nor-benzylisoquinoline alkaloids such as nororipavine and / or northebaine and that modifications to the cell increasing the cytosolic heme levels strongly benefits production of such demethylated nor-benzylisoquinoline alkaloids. Accordingly, in one embodiment the host cell is further modified to increase availability of heme in the cell, in particular by modifying expression of one or more heme expression co-factors in the cell.

[0154] In one embodiment the heme availability can be increased by overexpressing and / or coexpressing one or more rate-limiting enzymes from the heme pathway, including but not limited to HEM2, HEM3 and / or HEM12. Overexpression of such genes can be accomplished for example by increasing the number of copies of integrated genes and / or by using stronger promoters of other factors increase translation or transcription of the gene. Preferably both an increase in copy number and use of an appropriate combination of stronger and weaker promoters are used to increase availability of heme. Useful promoters for these gene include pPYKl, pSEDl, pKEX2, pTEFl, pTDH3 and pPGKl, where pTEFl, pTDH3 and pPGKl are the stronger ones. In another embodiment heme vailability is increased by disrupting, deleting and / or attenuating any heme-down regulating genes, such as HMX1. In another embodiment heme availability is increased by adding a heme production booster agent such as hemin (Protchenko et al., 2003 and Krainer et al., 2015, respectively).

[0155] In a further aspect it was found that overexpressing and / or co-expressing P450 helper genes in a production host cell significantly benefits production of demethylated nor-benzylisoquinoline alkaloids. Such P450 helper genes includes, but is not limited to: a) DAP1, which encodes a heme-binding protein involved in the regulation the function of cytochrome P450 (Hughes et al., 2007); b) HAC1, a transcription factor that modulates the unfolded protein response (Kawahara T, et al., 1997); c) KAR2, HSP82, CNE1, SSA1, CPR6, FES1, HSP104 and STI1 involved in protein processing as well as heat shock response (Yu et al., 2017). In a still further aspect, it was found that increasing cytosolic levels of NADPH by overexpressing and / or co-expressing genes in the pentose metabolic pathway significantly benefits production of demethylated nor-benzylisoquinoline alkaloids. Such genes include but is not limited to ZWF1 and GND1 genes from the pentose phosphate pathway (Stincone et al., 2015).

[0156] In a further aspect it was found that detoxifying the genetically modified cell from formaldehyde, a toxic by-product released during cytochrome P450 N-demethylation reaction (Wehner EP et al., 1993 and Kalasz H et al., 1998), significantly benefits production of demethylated nor-benzylisoquinoline alkaloids. Lowering formation of cytosolic formaldehyde in the cell can be achieved modifying genes encoding factors regulating formaldehyde levels and / or toxicity. Such genes / factors include but is not limited to SFA1, which when overexpressed and / or co-expressed reduce formaldehyde levels and / or toxicity and thereby increase production of demethylated norbenzylisoquinoline alkaloids. Functional homologues

[0157] Functional homologs (also referred herein to as functional variants) of the enzymes / polypeptides described herein are also suitable for use in producing benzylisoquinoline alkaloid in the genetically modified host cell. A functional homolog is a polypeptide that has sequence similarity to a reference polypeptide, and that carries out one or more of the biochemical or physiological function(s) of the reference polypeptide. A functional homolog and the reference polypeptide can be a natural occurring polypeptide, and the sequence similarity can be due to convergent or divergent evolutionary events. As such, functional homologs are sometimes designated in the literature as homologs, or orthologs, or paralogs. Variants of a naturally occurring functional homolog, such as polypeptides encoded by mutants of a wild type coding sequence, can themselves be functional homologs. Functional homologs can also be created via site-directed mutagenesis of the coding sequence for a polypeptide, or by combining domains from the coding sequences for different naturally-occurring polypeptides ("domain swapping"). Techniques for modifying genes encoding functional polypeptides described herein are known and include, inter alia, directed evolution techniques, site-directed mutagenesis techniques and random mutagenesis techniques, and can be useful to increase specific activity of a polypeptide, alter substrate specificity, alter expression levels, alter subcellular location, or modify polypeptide-polypeptide interactions in a desired manner. Such modified polypeptides are considered functional homologs. The term "functional homolog" is sometimes applied to the nucleic acid that encodes a functionally homologous polypeptide. Functional homologs can be identified by analysis of nucleotide and polypeptide sequence alignments. For example, performing a query on a database of nucleotide or polypeptide sequences can identify homologs of benzylisoquinoline alkaloid biosynthesis polypeptides. Sequence analysis can involve BLAST, Reciprocal BLAST, or PSI-BLAST analysis of non-redundant databases using a UGT amino acid sequence as the reference sequence. Amino acid sequence is, in some instances, deduced from the nucleotide sequence. Those polypeptides in the database that have greater than 40% sequence identity are candidates for further evaluation for suitability as a benzylisoquinoline alkaloid biosynthesis polypeptide. Amino acid sequence similarity allows for conservative amino acid substitutions, such as substitution of one hydrophobic residue for another or substitution of one polar residue for another. If desired, manual inspection of such candidates can be carried out to narrow the number of candidates to be further evaluated. Manual inspection can be performed by selecting those candidates that appear to have domains present in benzylisoquinoline alkaloid biosynthesis polypeptides, e.g., conserved functional domains. In some embodiments, nucleic acids and polypeptides are identified from transcriptome data based on expression levels rather than by using BLAST analysis. Methods for conservative substitution is known to the skilled person, see for example https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC1449787 / or https: / / link.springer.com / article / 10.1007 / BF02300754

[0158] Conserved regions can be identified by locating a region within the primary amino acid sequence of a benzylisoquinoline alkaloid biosynthesis polypeptide that is a repeated sequence, forms some secondary structure (e.g., helices and beta sheets), establishes positively or negatively charged domains, or represents a protein motif or domain. See, e.g., the Pfam web site describing consensus sequences for a variety of protein motifs and domains on for example the World Wide Web at sanger.ac.uk / Software / Pfam / and pfam.janelia.org / . The information included at the Pfam database is described in Sonnhammer et al. (1998); Sonnhammer et al. (1997); and Bateman et al. (1999). Conserved regions also can be determined by aligning sequences of the same or related polypeptides from closely related species. Closely related species preferably are from the same family. In some embodiments, alignment of sequences from two different species is adequate to identify such homologs.

[0159] Typically, polypeptides that exhibit at least about 40% amino acid sequence identity are useful to identify conserved regions. Conserved regions of related polypeptides exhibit at least 45% amino acid sequence identity (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% amino acid sequence identity). In some embodiments, a conserved region exhibits at least 92%, 94%, 96%, 98%, or 99% amino acid sequence identity.

[0160] For example, polypeptides suitable for producing benzylisoquinoline alkaloids in a genetically modified hosyt cell include functional homologs of TH's, NCS's, 6-OMT's, CNMT's, NMCH's, 4'-OMT's, DRS-DRR's, SAS's, SAR's, SAT's, THS's, CPR's and demethylating P450's.

[0161] Methods to modify the substrate specificity of benzylisoquinoline alkaloids pathway enzymes are known to those skilled in the art, and include without limitation site-directed / rational mutagenesis approaches, random directed evolution approaches and combinations in which random mutagenesis / saturation techniques are performed near the active site of the enzyme. For example see Osmani et al. (2009).

[0162] A candidate sequence typically has a length that is from 80% to 200% of the length of the reference sequence, e.g., 82, 85, 87, 89, 90, 93, 95, 97, 99,100, 105,110,115,120,130,140,150,160, 170, 180, 190, or 200% of the length of the reference sequence. A functional homolog polypeptide typically has a length that is from 95% to 105% of the length of the reference sequence, e.g., 90, 93, 95, 97, 99,100,105,110,115, or 120% of the length of the reference sequence, or any range between.

[0163] It will be appreciated that functional benzylisoquinoline alkaloids pathway enzymes / polypeptides can include additional amino acids that are not involved in the enzymatic activities carried out by the enzymes. In some embodiments, such enzymes are fusion proteins. The terms "chimera," "fusion polypeptide," "fusion protein," "fusion enzyme," "fusion construct," "chimeric protein," "chimeric polypeptide," "chimeric construct," and "chimeric enzyme" can be used interchangeably herein to refer to proteins engineered through the joining of two or more genes that code for different proteins. In some embodiments, a nucleic acid sequence encoding a benzylisoquinoline alkaloids pathway enzyme / polypeptide can include a tag sequence that encodes a "tag" designed to facilitate subsequent manipulation (e.g., to facilitate purification or detection), secretion, or localization of the encoded enzyme. Tag sequences can be inserted in the nucleic acid sequence encoding the polypeptide such that the encoded tag is located at either the carboxyl or amino terminus of the polypeptide. Non-limiting examples of encoded tags include green fluorescent protein (GFP), human influenza hemagglutinin (HA), glutathione S transferase (GST), polyhistidine-tag (HIS tag), and Flag™ tag (Kodak, New Haven, CT). Other examples of tags include a chloroplast transit peptide, a mitochondrial transit peptide, an amyloplast peptide, signal peptide, or a secretion tag.

[0164] In some embodiments, a fusion protein is a protein altered by domain swapping. As used herein, the term "domain swapping" is used to describe the process of replacing a domain of a first protein with a domain of a second protein. In some embodiments, the domain of the first protein and the domain of the second protein are functionally identical or functionally similar. In some embodiments, the structure and / or sequence of the domain of the second protein differs from the structure and / or sequence of the domain of the first protein. In some embodiments, a benzylisoquinoline alkaloids pathway enzyme / polypeptide is altered by domain swapping. Nucleotides expressed by the host cell

[0165] In another aspect the host cell of the invention expresses one or more polynucleotides or genes selected from: a) one or more polynucleotides which is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the DAHP synthase encoding polynucleotide comprised in SEQ ID NO: 2 or genomic DNA thereof; b) one or more polynucleotides which is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the chorismate mutase encoding polynucleotide comprised in SEQ. ID NO: 4 or genomic DNA thereof; c) one or more polynucleotides which is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the TH encoding polynucleotide comprised in SEQ ID NO: 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64 or 66 or genomic DNA thereof; d) one or more polynucleotides which is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the TH-CPR encoding polynucleotide comprised in SEQ ID NO: 68 or genomic DNA thereof; e) one or more polynucleotides which is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the DODC encoding polynucleotide comprised in SEQ ID NO: 70 or 72 or genomic DNA thereof; f) one or more polynucleotides which is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the NCS encoding polynucleotide comprised in SEQ ID NO: 74 or 77 or genomic DNA thereof; g) one or more polynucleotides which is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the 6-OMT encoding polynucleotide comprised in SEQ. ID NO: 80 or genomic DNA thereof; h) one or more polynucleotides which is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the CNMT encoding polynucleotide comprised in SEQ ID NO: 83 or genomic DNA thereof; i) one or more polynucleotides which is at least20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the NMCH encoding polynucleotide comprised in SEQ ID NO: 86 or 88 or genomic DNA thereof; j) one or more polynucleotides which is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the 4'-OMT encoding polynucleotide comprised in SEQ ID NO: 90 or genomic DNA thereof; k) one or more polynucleotides which is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the DRS-DRR encoding polynucleotide comprised in SEQ ID NO: 93, 95 or 97 or genomic DNA thereof; I) one or more polynucleotides which is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the DRS encoding polynucleotide comprised in SEQ ID NO: 99, 101, 103, 105 or 107 or genomic DNA thereof; m) one or more polynucleotides which is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the DRR encoding polynucleotide comprised in SEQ ID NO: 109 or 111 or genomic DNA thereof; n) one or more polynucleotides which is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the DRS-CPR encoding polynucleotide comprised in SEQ. ID NO: 113 or 115 or genomic DNA thereof; o) one or more polynucleotides which is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the SAS encoding polynucleotide comprised in SEQ ID NO: 117 or 119 or genomic DNA thereof; p) one or more polynucleotides which is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the SAR encoding polynucleotide comprised in SEQ ID NO: 121 or genomic DNA thereof; q) one or more polynucleotides which is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the SAT encoding polynucleotide comprised in SEQ ID NO: 124 or genomic DNA thereof; r) one or more polynucleotides which is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the THS encoding polynucleotide comprised in SEQ ID NO: 130, 132, 135, 137 or 139 or genomic DNA thereof; s) one or more polynucleotides which is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the ODM encoding polynucleotide comprised in SEQ ID NO: 219, 221, 223, 225, 227, 229, 237, 241, 251, 253, 255 and 267 or genomic DNA thereof; t) one or more polynucleotides which is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the demethylase encoding polynucleotide comprised in any one of SEQ. ID NO: 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, Til, 279, 281, 283, 285, 287, 289, 291, 828, 830, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850, 852, 854, 856, 858, 860, 862, 864, 866, 868 and 870 or genomic DNA thereof; u) one or more polynucleotides which is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the demethylase-CPR encoding polynucleotide comprised in any one of SEQ ID NO: 293, 295, 297, 299, 301, 303, 304 or 306 or genomic DNA thereof; and v) one or more polynucleotides which is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the transporter encoding polynucleotide comprised in SEQ ID NO: 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 734 or 736 or genomic DNA thereof.

[0166] Any nucleotides disclosed herein may be codon optimized for expression in a particular selected host using methods available to the skilled person or commercially available from technology providers - see for example Gene Reports Volume 9, December 2017, Pages 46-53: Strategies of codon optimization for high-level heterologous protein expression in microbial expression systems, incorporated herein by reference. Examples of codon optimized genes are those of SEQ. ID NOS: 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791 and 793. Host cells.

[0167] The cell of the invention may be any host cell suitable for hosting and expressing the P450s of the invention and converting thebaine and / or oripavine into the corresponding nor-compounds.

[0168] In particular the cell of the invention may be an eukaryote cell selected from the group consisting of mammalian, insect, plant, or fungal cellsln another embodiment the cell is a fungal cell selected from the phylas consisting of Ascomycota, Basidiomycota, Neocallimastigomycota, Glomeromycota, Blastocladiomycota, Chytridiomycota, Zygomycota, Oomycota and Microsporidia. A particularly useful fungal cell is a yeast cell selected from the group consisting of ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and Fungi Imperfecti yeast (Blastomycetes). Such yeast cells may further be selected from the genera consisting of Saccharomyces, Kluveromyces, Candida, Pichia, Debaromyces, Hansenula, Yarrowia, Zygosaccharomyces, and Schizosaccharomyces. More specifically the yeast cell may be selected from the species consisting of Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, and Yarrowia lipolytica.

[0169] An alternative fungal host cell of the invention is a filamentous fungal cell. Such filamentous fungal cell may be selected from the phylas consisting of Ascomycota, Eumycota and Oomycota, more specifically selected from the genera consisting of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Corio / us, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, and Trichoderma. In important embodiments the filamentous fungal cell may be selected from the species consisting of Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporiuminops, Chrysosporiumkeratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, and Trichoderma viride.

[0170] In one embodiment the cell is a plant cell for example of the genus Physcomitrella or Popover, in particular Popover somniferum. Other plant cells can be of the family Solanaceae, such genuses of Nicotiana, such as Nicotiana benthamiana. In addition to plant cells the invention also provides an isolated plant, e.g., a transgenic plant, plant part comprising the benzylisoquinoline alkaloid pathway polypeptides of the invention and producing the benzylisoquinoline alkaloids of the invention in useful quantities. The compound may be recovered from the plant or plant part. The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous (a monocot). Examples of monocot plants are grasses, such as meadow grass (blue grass, Poa), forage grass such as Festuca, Lolium, temperate grass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum, and maize (corn). Examples of dicot plants are tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and soybean, and cruciferous plants (family Brassicaceae), such as cauliflower, rape seed, and the closely related model organism Arabidopsis thaliana. Examples of plant parts are stem, callus, leaves, root, fruits, seeds, and tubers as well as the individual tissues comprising these parts, e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems. Specific plant cell compartments, such as chloroplasts, apoplasts, mitochondria, vacuoles, peroxisomes and cytoplasm are also considered to be a plant part. Furthermore, any plant cell, whatever the tissue origin, is considered to be a plant part. Likewise, plant parts such as specific tissues and cells isolated to facilitate the utilization of the invention are also considered plant parts, e.g., embryos, endosperms, aleurone and seed coats. Also included within the scope of the present invention is any the progeny of such plants, plant parts, and plant cells. The transgenic plant or plant cells comprising the operative pathway of the invention and produce the compound of the invention may be constructed in accordance with methods known in the art. In short, the plant or plant cell is constructed by incorporating one or more expression vectors of the invention into the plant host genome or chloroplast genome and propagating the resulting modified plant or plant cell into a transgenic plant or plant cell. The expression vector conveniently comprises the polynucleotide construct of the invention. The choice of regulatory sequences, such as promoter and terminator sequences and optionally signal or transit sequences, is determined, for example, on the basis of when, where, and how the pathway polypeptides is desired to be expressed. For instance, the expression of a gene encoding a pathway enzyme polypeptide may be constitutive or inducible, or may be developmental, stage or tissue specific, and the gene product may be targeted to a specific tissue or plant part such as seeds or leaves. Regulatory sequences are, for example, described by Tague et al., 1988, Plant Physiology 86: 506. For constitutive expression, the 358-CaMV, the maize ubiquitin 1, or the rice actin 1 promoter may be used (Franck et al., 1980, Cell 21: 285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhang et al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be, for example, a promoter from storage sink tissues such as seeds, potato tubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or from metabolic sink tissues such as meristems (Ito et al., 1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such as the glutelin, prolamin, globulin, or albumin promoter from rice (Wu et al., 1998, Plant Cell Physiol. 39: 885-889), a Viciafaba promoter from the legumin B4 and the unknown seed protein gene from Vicia faba (Conrad et al., 1998, J. Plant Physiol. 152: 708-711), a promoter from a seed oil body protein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941), the storage protein napA promoter from Brassica napus, or any other seed specific promoter known in the art, e.g., as described in WO 91 / 14772. Furthermore, the promoter may be a leaf specific promoter such as the rbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol. 102: 991-1000), the chlorella virus adenine methyltransferase gene promoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldP gene promoter from rice (Kagaya et al.,1995, Mol. Gen. Genet. 248: 668-674), or a wound inducible promoter such as the potato pin2 promoter (Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promoter may be induced by abiotic treatments such as temperature, drought, or alterations in salinity or induced by exogenously applied substances that activate the promoter, e.g., ethanol, oestrogens, plant hormones such as ethylene, abscisic acid, and gibberellic acid, and heavy metals. A promoter enhancer element may also be used to achieve higher expression in the plant. For instance, the promoter enhancer element may be an intron that is placed between the promoter and the polynucleotide encoding a polypeptide or domain. For instance, Xu et al., 1993, supra, disclose the use of the first intron of the rice actin 1 gene to enhance expression. The selectable marker gene and any other parts of the expression construct may be chosen from those available in the art. The polynucleotide construct or expression vector is incorporated into the plant genome according to conventional techniques known in the art, including Agrobacterium-mediated transformation, virus-mediated transformation, microinjection, particle bombardment, biolistic transformation, and electroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990, Bio / Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274). Agrobacterium tumefaciens-mediated gene transfer is a method for generating transgenic dicots (for a review, see Hooykas and Schilperoort, 1992, Plant Mol. Biol. 19:15-38) and for transforming monocots, although other transformation methods may be used for these plants. A method for generating transgenic monocots is particle bombardment (microscopic gold or tungsten particles coated with the transforming DNA) of embryonic calli or developing embryos (Christou, 1992, Plant J. 2: 275-281; Shimamoto, 1994, Curr. Opin. Biotechnol. 5: 158-162; Vasil etaL, 1992, Bio / Technology 10: 667-674). An alternative method for transformation of monocots is based on protoplast transformation as described by Omirulleh et al., 1993, Plant Mo / . Biol. 21: 415428. Additional transformation methods include those described in U.S. Patent Nos. 6,395,966 and 7,151,204 (both incorporated herein by reference in their entirety). Following transformation, the transformants having incorporated the expression vector or polynucleotide construct of the invention are selected and regenerated into whole plants according to methods well known in the art. Often the transformation procedure is designed for the selective elimination of selection genes either during regeneration or in the following generations by using, for example, co-transformation with two separate T-DNA constructs or site specific excision of the selection gene by a specific recombinase. In addition to direct transformation of a particular plant genotype with a polynucleotide construct of the invention, transgenic plants may be made by crossing a plant comprising the construct to a second plant lacking the construct. For example, a polynucleotide construct encoding a glycosyl transferease of the invention can be introduced into a particular plant variety by crossing, without the need for ever directly transforming a plant of that given variety. Therefore, the invention encompasses not only a plant directly regenerated from cells which have been transformed in accordance with the invention, but also the progeny of such plants. As used herein, progeny may refer to the offspring of any generation of a parent plant prepared in accordance with the present invention. Such progeny may include a polynucleotide construct of the invention. Crossing results in the introduction of a transgene into a plant line by cross pollinating a starting line with a donor plant line. Non-limiting examples of such steps are described in U.S. Patent No. 7,151,204. Plants may be generated through a process of backcross conversion. For example, plants include plants referred to as a backcross converted genotype, line, inbred, or hybrid. Genetic markers may be used to assist in the introgression of one or more transgenes of the invention from one genetic background into another. Marker assisted selection offers advantages relative to conventional breeding in that it can be used to avoid errors caused by phenotypic variations. Further, genetic markers may provide data regarding the relative degree of elite germplasm in the individual progeny of a particular cross. For example, when a plant with a desired trait which otherwise has a non-agronomically desirable genetic background is crossed to an elite parent, genetic markers may be used to select progeny which not only possess the trait of interest, but also have a relatively large proportion of the desired germplasm. In this way, the number of generations required to introgress one or more traits into a particular genetic background is minimized.

[0171]

[0172] The cell of the invention may be even further modified by one or more of a) attenuating, disrupting and / or deleting one or more native or endogenous genes of the cell; b) inserting two or more copies of polynucleotides encoding the P450s, the demethylase-CPR's and / or one or more of the polypeptides comprised in the operative metabolic pathway; c) increasing the amount of a substrate for at least one polypeptide of the operative metabolic pathway; and / or d) increasing tolerance towards one or more substrates, intermediates, or product molecules from the operative metabolic pathway. Polynucleotide constructs and expression vectors

[0173] In a separate aspect the invention also provides a polynucleotide construct comprising a polynucleotide sequence encoding a heterologous enzymes or transporter protein of the invention operably linked to one or more control sequences, which direct expression of the heterologus enzyme or transporter protein in the host cell harbouring the polynucleotide construct. Conditions for the expression should be compatible with the control sequences. In particular, the control sequence is heterologous to the polynucleotide encoding the heterologus enzyme or transporter protein and in one embodiment the polynucleotide sequence encoding the heterologus enzyme or transporter protein and the control sequence are both heterologous to the host cell comprising the construct. In one embodiment the polynucleotide construct is an expression vector, comprising the polynucleotide sequence encoding the heterologus enzyme or transporter protein of the invention operably linked to the one or more control sequences.

[0174] Polynucleotides may be manipulated in a variety of ways allow expression of the heterologus enzyme or transporter protein. Manipulation of the polynucleotide prior to its insertion into an expression vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

[0175] The control sequence may be a promoter, which is a polynucleotide that is recognized by a host cell for expression of a polynucleotide. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. The promoter may also be an inducible promoter.

[0176] Examples of suitable promoters for directing transcription of the nucleic acid construct of the invention in fungal host cell are promoters obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus niger neutral a-amylase, Aspergillus niger acid stable a-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Aspergillus gpdA promoter, Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, A. niger or A. awamori endoxylanase (xlnA) or p-xylosidase (xlnD), Fusarium oxysporum trypsin-like protease (WO 96 / 00787), Fusarium venenatum amyloglucosidase (WO2000 / 56900), Fusarium venenatum Dania (WO 00 / 56900), Fusarium venenatum Quinn (WO 00 / 56900), Rhizomucor miehei lipase, Rhizomucor miehei aspartic proteinase, Trichoderma reesei p-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei p-xylosidase, as well as the NA2-tpi promoter and mutant, truncated, and hybrid promoters thereof. NA2-tpi promoter is a modified promoter from an Aspergillus neutral a-amylase gene in which the untranslated leader has been replaced by an untranslated leader from an Aspergillus triose phosphate isomerase gene. Examples of such promoters include modified promoters from an Aspergillus niger neutral a-amylase gene in which the untranslated leader has been replaced by an untranslated leader from an Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerase gene. Other examples of promoters are the promoters described in W02006 / 092396, W02005 / 100573 and W02008 / 098933, incorporated herein by reference.

[0177] Examples of suitable promoters for directing transcription of the nucleic acid construct of the invention in a yeast host include the glyceraldehyde-3-phosphate dehydrogenase promoter, PgpdA or promoters obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GALI ), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP), Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8: 423-488. Selecting a suitable promoter for expression in yeast is well know and is well understood by persons skilled in the art.

[0178] The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3'-terminus of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell may be used.

[0179] Useful terminators for fungal host cells can be obtained from the genes encoding Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger a-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease; while useful terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra.

[0180] The control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene.

[0181] The control sequence may also be a leader, a non-translated region of an mRNA that is important for translation by the host cell. The leader is operably linked to the 5'-terminus of the polynucleotide encoding the polypeptide. Any leader that is functional in the host cell may be used.

[0182] Useful leaders for fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase, while useful leaders for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae a-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0183] The control sequence may also be a polyadenylation sequence; a sequence operably linked to the 3'-terminus of the polynucleotide and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used. Useful polyadenylation sequences for fungal host cells can be obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger a-glucosidase Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease; while useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

[0184] It may also be desirable to add regulatory sequences that regulate expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. In fungi, the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA a-amylase promoter, and Aspergillus oryzae glucoamylase promoter may be used; while in yeast, the ADH2 system or GAL 1 system may be used. Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals.

[0185] Various nucleotide sequences in addition to the polynucleotide construct of the invention may be joined together to produce a recombinant expression vector, which may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide sequence encoding the P450 of the invention at such sites. The recombinant expression vector may be any vector (e.g., a plasmid or virus or chromosomal) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the P450 encoding polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid (linear or closed circular plasmid), an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring selfreplication. Alternatively, the vector may, when introduced into the host cell, integrate into the genome and replicate together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.

[0186] The vector may contain one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene from which the product provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Useful selectable markers for fungal host cells include amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Useful selectable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.

[0187] The vector may further contain element(s) that permits integration of the vector into genome of the host cell or permits autonomous replication of the vector in the cell independent of the genome. For integration into the host cell genome, the vector may rely on the polynucleotide encoding the P450 or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides for directing integration by homologous recombination into the genome of the host cell at precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, such as 400 to 10,000 base pairs, and such as 800 to 10,000 base pairs, which have a high degree of sequence identity to the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be nonencoding or encoding polynucleotides. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.

[0188] For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term "origin of replication" or "plasmid replicator" refers to a polynucleotide that enables a plasmid or vector to replicate in vivo. Useful origins of replication for fungal cells include AMA 1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Res. 15: 9163-9175; WO 00 / 24883). Isolation of the AMA 1 sequence and construction of plasmids or vectors comprising the gene can be accomplished using the methods disclosed in WO2000 / 24883. Useful origins of replication for yeast host cells are the 2-micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

[0189] As mentioned, supra, more than one copy of a polynucleotide encoding the P450 of the invention may be inserted into a host cell to increase production of the P450. An increase in the copy number can be obtained by integrating one or more additional copies of the enzyme coding sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide, so that cells containing amplified copies of the selectable marker gene - and thereby additional copies of the polynucleotide - can be selected by cultivating the cells in the presence of the appropriate selectable agent. The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present disclosure are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).

[0190] In alignment with the above the vehicles of this disclose also include those comprising a microbial host cell comprising the polynucleotide construct as described, supra. Cultures

[0191] The invention also provides a cell culture, comprising any host cell of the invention and a growth medium. Suitable growth mediums for host cells such as mammalian, insect, plant, fungal and / or yeast cells are known in the art. Methods of producing compounds of the invention.

[0192] The invention also provides a method for producing a benzylisoquinoline alkaloid in particular thebaine, northebaine, oripavine and / or nororipavine and / or a derivative thereof comprising a) culturing the cell culture of the invention at conditions allowing the cell to produce the benzylisoquinoline alkaloid; and b) optionally recovering and / or isolating the benzylisoquinoline alkaloid.

[0193] The cell culture can be cultivated in a nutrient medium and at conditions suitable for production of the northebaine and / or nororipavine of the invention and / or propagating cell count using methods known in the art. For example, the culture may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentations) in laboratory or industrial fermenters in a suitable medium and under conditions allowing the host cells to grow and / or propagate, optionally to be recovered and / or isolated.

[0194] The cultivation can take place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. from catalogues of the American Type Culture Collection). The selection of the appropriate medium may be based on the choice of host cell and / or based on the regulatory requirements for the host cell. Such media are available in the art. The medium may, if desired, contain additional components favoring the transformed expression hosts over other potentially contaminating microorganisms. Accordingly, in an embodiment a suitable nutrient medium comprises a carbon source (e.g. glucose, maltose, molasses, starch, cellulose, xylan, pectin, lignocellolytic biomass hydrolysate, etc.), a nitrogen source (e. g. ammonium sulphate, ammonium nitrate, ammonium chloride, etc.), an organic nitrogen source (e.g. yeast extract, malt extract, peptone, etc.) and inorganic nutrient sources (e.g. phosphate, magnesium, potassium, zinc, iron, etc.).

[0195] The cultivation of the host cell may be performed over a period of from about 0.5 to about 30 days. The cultivation process may be a batch process, continuous or fed-batch process, suitably performed at a temperature in the range of 0-100 °C or 0-80 °C, for example, from about 0 °C to about 50 °C and / or at a pH, for example, from about 2 to about 10. Preferred fermentation conditions for yeast and filamentous fungi are a temperature in the range of from about 25 °C to about 55 °C and at a pH of from about 3 to about 9. The appropriate conditions are usually selected based on the choice of host cell. Accordingly, in an embodiment the method of the invention further comprises one or more elements selected from: a) culturing the cell culture in a nutrient medium; b) culturing the cell culture under aerobic or anaerobic conditions c) culturing the cell culture under agitation; d) culturing the cell culture at a temperature of between 25 to 50 °C; e) culturing the cell culture at a pH of between 3-9; and f) culturing the cell culture for between 10 hours to 30 days.

[0196] In a special embodiment wherein the host cell of the invention express a demethylase converting thebaine to northebaine in the cell , a demethylase-CPR and a transporter, it has been found that for optimal production of northebaines a pH from 6 to 8, such as from 6,5 to 7,5, such as about 7,0 should be maintained for the culturation / fermentation. In another special embodiment wherein the host cell of the invention express a demethylase converting oripavine to nororipavine in the cell, a demethylase-CPR and a transporter, it has been found that for optimal production of nororipavine at a pH from 3,5 to 5,5, such as from 3,0 to 5,0, such as about 4,5 should be maintained for the culturation / fermentation.

[0197] The cell culture of the invention may be recovered and or isolated using methods known in the art. For example, the compound(s) may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, spray-drying, or lyophilization. In a particular embodiment the method includes a recovery and / or isolation step comprising separating a liquid phase of the cell or cell culture from a solid phase of the cell or cell culture to obtain a supernatant comprising the benzylisoquinoline alkaloid, eg. thebaine,northebaine, oripavine and / or nororipavine and subjecting the supernatant to one or more steps selected from: a) contacting the supernatant with one or more adsorbent resins in order to obtain at least a portion of the produced benzylisoquinoline alkaloid, then optionally recovering the benzylisoquinoline alkaloid from the resin in a concentrated solution prior to precipitation or crystallisation of the benzylisoquinoline alkaloid; b) contacting the supernatant with one or more ion exchange or reversed-phase chromatography columns in order to obtain at least a portion of the benzylisoquinoline alkaloid, then optionally recovering the benzylisoquinoline alkaloid from the resin in a concentrated solution prior to precipitation or crystallisation of the benzylisoquinoline alkaloid; c) extracting the benzylisoquinoline alkaloid from the supernatant, such as by liquid-liquid extraction into an immisible solvent, then optionally evaporating the solvent to concentrate and precipitate the benzylisoquinoline alkaloid or performing further liquid-liquid extraction to recover and concentrate benzylisoquinoline alkaloid prior to crystallisation or precipitation or in order to directly perform a further chemical reaction on benzylisoquinoline alkaloid; and d) evaporating the solvent of the supernatant to concentrate or precipitate the benzylisoquinoline alkaloid; thereby recovering and / or isolating the benzylisoquinoline alkaloid benzylisoquinoline alkaloid benzylisoquinoline alkaloid .

[0198] The method of the invention may comprise one or more in vitro steps in the process of producing the benzylisoquinoline alkaloid. It may also comprise one or more in vivo steps performed in another cell, such as a plant cell, for example a cell of Papaver somniferum For example, thebaine and / or oripavine or precursors thereof may be produced in a plant, such as poppy (Papaver somniferum) and isolated therefrom and then fed to a cell culture of the invention for conversionsion into northebaine and / or nororipavine. Accordingly, in one embodiment the method of the invention further comprises feeding the cell culture with exogenous thebaine, oripavine and / or a precursor thereof, and even further where the exogenous thebaine, oripavine and / or precursor thereof is a plant extract.

[0199] In one embodiment of the invention the benzylisoquinoline alkaloid is in particular, the benzylisoquinoline alkaloid is selected from one or more of thebaine, northebaine, oripavine and nororipavine.

[0200] Where thebaine, oripavine, northebaine and / or nororipavine and / or any upstream benzylisoquinoline alkaloid precursors is not the desired end-product further steps may be added to the method of the invention either chemically or biologically modifying the thebaine, northebaine, oripavine and / or nororipavine. Desired end products may be for example buprenorphine, naltrexone, naloxone or nalbuphine. Buprenorphine and other semisynthetic opioids are, or can be, made from thebaine (Hudlicky, Can. J. Chern. 93(5):492-501 (2015)). One route to buprenorphine is made up of 6 major steps, starting from thebaine. (Maehara et aL, Adv. Synth. Catal. 354(4):613-26 (2012); Werner et al., J. Org. Chern. 76(11):4628-34 (2011)). There, the first 3 steps are a Diels-Alder reaction of thebaine with methyl vinyl ketone to form a 4+2 product, hydrogenation of the carbon-carbon double bond of the resultant product, and addition of a tertiary butyl group via a Grignard reaction. The final steps are N- and O- demethylation and cyclopropyl alkylation. The number of steps can increase to 8, if the N- and O-demethylation and N-alkylation steps are performed in 2 stages, rather than 1. The order of the hydrogenation and Grignard steps may be reversed but most, if not all, economically viable preparations include the 3 above-mentioned steps prior to the N-demethylation step.

[0201] The N-demethylation of this known method can involve highly toxic reagents such as cyanogen bromide (von Braun, J. Chern. Ber., 33:1438-1452 (1900)) and chloroformate reagents (Cooley et al., Synthesis, 1:1-7 (1989); Olofson et al., J. Org. Chern., 49:2081-2082 (1984)) or may proceed in low yield, for example, by producing N-oxide intermediates (Polonovski reaction: Kok et al., Adv Synth. Catal., 351:283-286 (2009); Dong et al., J. Org. Chern., 72:9881-9885 (2007)). These methods generate significant amounts of toxic waste. The harsh conditions used for demethylation (e.g., strong bases and high temperatures) generate a significant amount of impurities, requiring additional purification and lowering yields. Attempts to reduce impurities and improve yields have been made by avoiding the O-demethylation step, by using oripavine as starting material, but a principal obstacle to an efficient synthesis remains the N-demethylation step.

[0202] Accordingly, for chemically converting thebaine, oripavine, northebaine and / or nororipavine into buprenophine or other opiate alkaloid derivatives there remains a need for an improved route of synthesis, such as a route that is shorter, more efficient (due to, e.g., improved total yield, decreased impurities), and / or produces less toxic waste. One challenge of known methods for preparation of buprenorphine is the exchange of the N-methyl group for an N-cyclopropyl group. N-demethylation methods can involve highly toxic reagents such as cyanogen bromide (von Braun, J. Chern. Ber., 33:1438-1452 (1900)) and chloroformate reagents (Cooley et al., Synthesis, 1:1-7 (1989); Olofson et al., J. Org. Chern., 49:2081-2082 (1984)) or may proceed in low yield, for example, by producing N-oxide intermediates (Polonovski reaction: Kok et al., Adv Synth. Catal., 351:283-286 (2009); Dong et al., J. Org. Chern., 72:9881-9885 (2007)). These methods generate significant amounts of toxic waste. The harsh conditions used for demethylation (e.g., strong bases and high temperatures) generate a significant amount of impurities, requiring additional purification and lowering yields. Attempts to reduce impurities and improve yields have been made by avoiding the O-demethylation step, by using oripavine as starting material, but a principal obstacle to an efficient synthesis remains the N-demethylation step.

[0203] As disclosed herein the present invention offers 1-2 fewer chemical demethylation steps reducing use or production of environmentally unfriendly chemicals, and it offers yield improvement and time by omittiting those steps.

[0204] In the present invention to increase yields, decrease costs, and stabilize intermediates before further processing, the thebaine, northebaine, oripavine and / or nororipavine produced by fermentation / biotransformation can be subjected to minimal purification steps. For example, fermentation broth can be subjected to a cell removal step (centrifugation or filtration) and a concentration step. Further, if starting from nororipavine, the nororipavine can be addd a protecting group in a first bisbenzylation step using benzyl bromide, to form 3,17-bisbenzylnororipavine using the semipurified nororipavine. Subsequent Diels Alder reaction with methyl vinyl ketone, Grignard reaction using t-butylmagnesium halide, a hydrogenation step using Pd / C, and N-alkylation reaction with cyclopropylmethylbromide as described below can be done to yield buprenorphine.

[0205] Accordingly, in an embodiment the method of the invention includes converting thebaine, oripavine, northebaine and / or nororipavine or alternatively a benzylisoquinoline alkaloid of the general formula Rl-V-H (V): buprenorphine 5 by applying, in sequence, a bis-benzylation step, a Diels-Alder step and a Grignard step.

[0207] In particular for converting nororipavine, HO-V-H (VI), of the general formula:

[0208] into buprenophine the method of the invention comprises steps of: a) in a first solvent system S-l comprising a polar protic solvent, reacting the compound HO-VI- 10            H (VI), with benzyl halide, benzyl sulfonate, or activated benzyl alcohol (e.g. activated with a sulfonate group such as a p-toluene sulfonyl group or a methyl sulfonyl group, or with triphenylphosphine) to provide a compound BnO-VI-Bn (VII) of the general formula: BnO^^ ‘O&i 'i n^ NBn (VII); a preparation of Compound BnO-l-Bn, as an intermediate towards noroxymorphone and 15           ultimately towards naltrexone and naloxone, was described in Helv. Chim. Acta 92:1359-65 (2009); b) in a second solvent system S-2 comprising a polar protic solvent, reacting compound BnO-VI-Bn (VII) with methyl vinyl ketone to provide a compound BnO-VII-Bn (VIII) of the general formula: c) in a third solvent system S-3 comprising a nonpolar solvent, reacting Compound BnO-VII-Bn (VIII) with a tert-butylmagnesium compound to provide a compound BnO-VIIIA-Bn (IX) of the general formula: 10 d) reacting Compound BnO-VIIIA-Bn (IX) with H2 in the presence of a hydrogenation catalyst to provide a compound HO-IX-H (X) of the general formula: ho_ °o& s. JLm' 1           (X); e) reacting Compound HO-IX-H (X) with i) cyclopropane carboxaldehyde followed by a hydride source; or: ii) cyclopropanecarboxylic acid halide followed by a reducing agent; or iii) cyclopropylmethyl halide or activated cyclopropane methanol; to provide buprenorphine. 15 Step a) in the method for converting HO-V-H (VI) into buprenophine

[0209] In some embodiments, the benzyl halide of step a) above is benzyl chloride or benzyl bromide. In some embodiments, the reaction of step a) above is performed in the presence of a strong base, e.g., an alkali metal hydride.

[0210] S-l may comprise at least one protic solvent having a dielectric constant of at least at least about 12, or at least about 13, or at least about 14, or at least about 15, or at least about 16, or at least about 18, or at least about 20.

[0211] In certain embodiments as otherwise described herein, S-l comprises at least about 50 vol.% of at least one protic solvent having a dielectric constant of at least about 12. In various other embodiments, the at least one protic solvent is present in an amount of at least 60 vol.%, or at least 70 vol.%, or at least 75 vol.%, or at least 80 vol.%, or at least 90 vol.%, or at least 95 vol.% , such as at least about 50 vol.%, or at least about 75 vol.%, or at least about 90 vol.% of the said protic solvent.

[0212] In some embodiments, S-l comprises at least one protic solvent having a polarity index of at least about 3, or at least about 3.5, or at least about 3.75, or at least about 4. As used herein, the solvent polarity index of a solvent can be determined according to Snyder, e.g. as reported in Snyder, L.R. "Classification of the Solvent Properties of Common Liquids." J. Chromatogr. (1978) 16:6, 223-234. In various embodiments, S-l comprises at least about 50 vol.%, or at least about 75 vol.%, or at least about 90 vol.% of at least one protic solvent having a polarity index of at least about 3, or at least 3.5, or at least 3.75, or at least 4.

[0213] In some embodiments as otherwise described herein, S-l comprises a C1-C4 alcohol (e.g., methanol, ethanol, n-propanol, isopropanol, n-butanol, or sec-butanol) and optionally water. In various embodiments, S-l comprises about 50-100 vol.% isopropanol and 0-50 vol.% water.

[0214] In some embodiments, the benzyl halide, benzyl sulfonate, or activated benzyl alcohol is reacted at a temperature within the range of about -20°C to about 40°C, e.g., about -20°C to about 35°C, or about -20°C to about 30°C, or about -20°C to about 25°C, or about -20°C to about 20°C, or about -20°C to about 15°C, or about -20°C to about 10°C, or about -20°C to about 5°C, or about -20°C to about 0°C, or about -15°C to about 40°C, or about -10°C to about 40°C, or about -5°C to about 40°C, or about 0°C to about 40°C, or about 5°C to about 20°C, or about 10°C to about 40°C, or about 15°C to about 40°C, or about 20°C to about 40°C, or about -15°C to about 35°C, or about -10°C to about 30°C, or about -5°C to about 25°C, or about 0°C to about 20°C, or about 5°C to about 15°C. In some embodiments, the benzyl halide, benzyl sulfonate, or activated benzyl alcohol is reacted for a period of time within the range of about lh to about 2 days, e.g., 2h to about 2 days, 3h to about 2 days, 6 hours to about 2 days, about 12 hours to about 2 days, or about 18 hours to about 2 days, or about 1 day to about 2 days, or about 1.25 days to about 2 days, or about 1.5 days to about 2 days, or about 6 hours to about 1.75 days, or about 6 hours to about 1.5 days, or about 6 hours to about 1.25 days, or about 6 hours to about 1 day, or about 6 hours to about 18 hours, or about 12 hours to about 1.75 days, or about 18 hours to about 1.5 days, or about lh to about 1 day, or about lh to about 12h, or about lh to about 6h, or about lh to about 4h. Step b) in the method for converting HO-V-H (VI) into buprenophine

[0215] In some embodiments, the methyl vinyl ketone is reacted at a temperature within the range of about 40°C to about 120°C, e.g., about 45°C to about 120°C, or about 50°C to about 120°C, or about 55°C to about 120°C, or about 60°C to about 120°C, or about 65°C to about 120°C, or about 70°C to about 120°C, or about 75°C to about 120°C, or about 80°C to about 120°C, or about 85°C to 120°C, or about 90°C to about 120°C, or about 40°C to about 115°C, or about 40°C to about 110°C, or about 40°C to about 105°C, or about 40°C to about 100°C, or about 40°C to about 95°C, or about 40°C to about 90°C, or about 40°C to about 85°C, or about 40°C to about 80°C, or about 40°C to about 75°C, or about 40°C to about 70°C, or about 45°C to about 115°C, or about 50°C to about 110°C, or about 55°C to about 105°C, or about 60°C to about 100°C, or about 65°C to about 95°C, or about 70°C to about 90°C. In some embodiments, the methyl vinyl ketone is reacted for a period of time within the range of about 2 hours to about 2 days, e.g., about 4 hours to about 2 days, or about 6 hours to about 2 days, or about 12 hours to about 2 days, or about 18 hours to about 2 days, or about 1 days to about 2 days, or about 1.25 days to about 2 days, or about 1.5 days to about 2 days, or about 2 hours to about 1.75 days, or about 2 hours to about 1.5 days, or about 2 hours to about 1.25 days, or about 2 hours to about 1 day, or about 2 hours to about 18 hours, or about 2 hours to about 12 hours, or about 4 hours to about 1.75 days, or about 6 hours to about 1.5 days, or about 12 hours to about 1.25 days, or about 18 hours to about 1 day.

[0216] In certain embodiments as otherwise described herein, the reaction of step b) is carried out under oxygen, e.g., a mixture of inert gas and oxygen having a different composition than that of air. In certain embodiments, the reaction is carried out in an atmosphere wherein inert gas (e.g., argon) is present as greater than 5 vol.% (e.g., greater than 20 vol.%, or greater than 50 vol. %), and and oxygen is present as less than 25 vol.% (e.g., less than 21 vol.%, or less than 20 vol.%, or less than 10 vol.%, or less than 5 vol.%). In certain embodiments, the reaction is carried out in an atmosphere wherein oxygen is present at between 1 vol.% and about 21 vol.%, or between 3 vol.% and 20 vol.%, or between 5 vol. % and 20 vol.%, or between 10 vol.% and 20 vol.%, or between 1 vol.% and 20 vol.%, or between 1 vol.% and 15 vol.%, or between 1 vol.% and 10 vol.%, or between 1 vol.% and 7 vol.%, or between 1 vol.% and 5 vol.%. In certain other embodiments, the reaction is performed in substantially inert atmosphere (e.g., oxygen is present at less than 0.1 vol. %, or less than 0.01 vol. %, or less than 0.001 vol. %).

[0217] In certain other embodiments, the reaction of step b) is carried out under a mixture of gases approximately the same as air (e.g., dry air). In other embodiments, the reaction is carried out wherein the ratio of inert gas to gaseous oxygen is approximately 79 vol.% to 21 vol.%.

[0218] It has been found that the use of some amount of oxygen in the atmosphere of the reaction in stepb) serves to increase the yield of the reaction. Without being bound by theory, it is presently believed that trace oxygen prevents methyl vinyl ketone polymerization, allowing for additional methyl vinyl ketone monomers to be present as reactants. Beyond enhancing the yield, providing a reaction atmosphere containing at least some oxygen generally requires less rigorous reaction condition and equipment, especially at scale, as rigorous oxygen exclusion is no longer required. Together, this development allows for a more efficient synthetic protocol and enhanced reaction yields with lower capital expenditures.

[0219] In some embodiments, the second solvent system S-2 has a dielectric constant of at least about 12, or at least about 13, or at least about 14. In various other embodiments as described herein, the dielectric constant of S-2 is at least about 15, or at least about 16, or at least about 18, or at least about 20.

[0220] In certain embodiments as otherwise described herein, S-2 comprises at least about 50 vol.% of at least one protic solvent having a dielectric constant of at least about 12. In various other embodiments, the at least one protic solvent is present in an amount of at least 60 vol.%, or at least 70 vol.%, or at least 75 vol.%, or at least 80 vol.%, or at least 90 vol.%, or at least 95 vol.%. In certain embodiments, the at least one protic solvent has a dielectric constant of at least 13, or at least 14, or at least 15, or at least 16, or at least 18, or at least 20.

[0221] In some embodiments, S-2 comprises at least one protic solvent having a polarity index of at least about 3, or at least about 3.5, or at least about 3.75, or at least about 4. In various embodiments, S-2 comprises at least about 50 vol.%, or at least about 75 vol.%, or at least about 90 vol.% of at least one protic solvent having a polarity index of at least about 3, or at least 3.5, or at least 3.75, or at least 4.

[0222] In some embodiments as otherwise described herein, S-2 comprises a C1-C4 alcohol (e.g., methanol, ethanol, n-propanol, isopropanol, n-butanol, or sec-butanol) and optionally water. In various embodiments, S-2 comprises about 50-100 vol.% isopropanol and 0-50 vol.% water. In certain desirable embodiments, S-2 has substantially the same composition as S-l, described above.

[0223] In certain desirable embodiments, reactions in steps a) and b) can be performed sequentially, advantageously without intervening purification and without substantial removal of solvent system S-1. In certain embodiments as otherwise described herein, S-2 has substantially the same composition as S-l. In such embodiments, the reactants and purification steps of step b) comprise the crude reaction product of step a). Accordingly, in certain embodiments as otherwise described herein, the methyl vinyl ketone of step b) is added to a crude reaction product of step a), the crude reaction product comprising solvent S-l and BnO-VII-Bn (VIII).

[0224] As noted above, the reaction of b) can be carried out without substantial removal of solvent or purification (e.g. chromatography). However, the person of ordinary skill in the art will appreciate that it may be necessary to adjust the pH of the crude reaction product of step a) before performing step b). The pH may be adjusted with a wide variety of acids known in the art. For example, in certain embodiments, the pH is adjusted with the addition of acetic acid or hydrochloric acid. For example, the pH may be adjusted with a water-diluted acid such as 10% acetic acid, or 10% hydrochloric acid. In various embodiments as otherwise described herein, the pH is adjusted to be about neutral, e.g., between 6 and 8. Step c) in the method for converting HO-V-H (VI) into buprenophine

[0225] In some embodiments, the tert-butylmagnesium compound is a tert-butylmagnesium halide. For example, the tert-butylmagnesium compound is tert-butylmagnesium chloride or tertbutylmagnesium bromide. In some embodiments, the reaction is performed in a solvent comprising a nonpolar solvent, e.g., tert-butylmethyl ether, 2-methyl-tetrahydrofuran, diethyl ether, dimethoxymethane, benzene, toluene, or a mixture of thereof.

[0226] In some embodiments, the tert-butylmagnesium compound is reacted at a temperature within the range of about 15°C to about 100°C, e.g., about 20°C to about 100°C, or about 25°C to about 100°C, or about 30°C to about 100°C, or about 15°C to about 95°C, or about 15°C to about 90°C, or about 15°C to about 85°C, or about 20°C to about 95°C, or about 25°C to about 90°C. In some embodiments, the tert-butylmagnesium halide is reacted for a period of time within the range of about 30 minutes to about 8 hours, e.g., about 1 hours to about 8 hours, or about 1.5 hours to about 8 hours, or about 2 hours to about 8 hours, or about 2.5 hours to about 8 hours, or about 3 hours to about 8 hours, or about 3.5 hours to about 8 hours, or about 4 hours to about 8 hours, or about 4.5 hours to about 8 hours, or about 5 hours to about 8 hours, or about 30 minutes to about 7.5 hours, or about 30 minutes to about 7 hours, or about 30 minutes to about 6.5 hours, or about 30 minutes to about 6 hours, or about 30 minutes to about 5.5 hours, or about 30 minutes to about 5 hours, or about 30 minutes to about 4.5 hours, or about 30 minutes to about 4 hours, or about 30 minutes to about 3.5 hours, or about 1 hour to about 7.5 hours, or about 1.5 hours to about 7 hours, or about 2 hours to about 6.5 hours, or about 2.5 hours to about 6 hours, or about 3 hours to about 5.5 hours.

[0227] As described above, the third solvent system S-3 comprises a nonpolar solvent. In certain embodiments as otherwise described herein, the third solvent system S-3 comprises at least one at least one nonpolar solvent having a dielectric constant of at most about 8, or at most about 7, or at most about 6, or at most about 5, or at most about 4, or at most about 3. For example, in various embodiments, S-3 comprises at least 60 vol.%, or at least 70 vol.%, or at least 75 vol.%, or at least 80 vol.%, or at least 90 vol.%, or at least 95 vol.% of the at least one nonpolar solvent having a dielectric constant of at most 8, or at most 7, or at most 6, or at most 5, or at most 4, or at most 3. In further embodiments, the nonpolar solvent that comprises S-3 has a polarity index of less than 4, or less than 3, or less than 2, or less than 1. For example, in various embodiments, S-3 comprises at least 60 vol.%, or at least 70 vol.%, or at least 75 vol.%, or at least 80 vol.%, or at least 90 vol.%, or at least 95 vol.% of the at least one nonpolar solvent has a polarity index of less than 4, or less than 3, or less than 2, or less than 1.

[0228] In certain desirable embodiments, polar solvents or solvents with large dielectric constants are not substantially present in S-3, or are present in S-3 in a relatively small amount. For example, in certain embodiments, S-3 comprises less than about 20 vol.%, or less than about 10 vol.%, or less than about 5 vol.%, or less than about 1 vol.% of a total amount of solvents having a dielectric constant of greater than 4, or greater than 6, or greater than 8. In various embodiments as otherwise described herein, S-3 comprises less than about 20 vol.%, or less than about 10 vol.%, or less than about 5 vol.%, or less than about 1 vol.% of a total amount of solvents having a polarity index of 2 or greater, or 3 or greater, or 4 or greater.

[0229] In some embodiments, S-3 comprises 30-90 vol.% of one or more C5-C10 alkanes and / or C5-C10 cycloalkanes. In certain embodiments, the alkanes and / or cycloalkanes are substituted (e.g., perfluorocyclohexane, perfluorohexane, etc.). For example, in certain embodiments the one or more alkanes and / or cycloalkanes include cyclohexane. In other embodiments, the one or more alkanes and / or cycloalkanes is cyclohexane. For example, S-3 may comprise 10-50 vol.% toluene (e.g., 20-50 vol.% toluene, or 30-50 vol.% toluene), 30-90 vol.% cyclohexane (e.g., 40-90 vol.% cyclohexane, or 40-70 vol.% cyclohexane), and up to 30 vol% tetrahydrofuran (e.g., up to 20 vol.% tetrahydrofuran, or up to 10 vol.% tetrahydrofuran, or up to 5 vol.% tetrahydrofuran).

[0230] It is known in the art that certain Grignard reagents (e.g., tert-butylmagnesium halide) disproportionate to the bis-alkyl and bis-halide species, with the disproportionation favored under certain reaction and solvent conditions (e.g. a more polar solvent). Without wishing to be bound by theory, the present inventors believe that the reaction conditions of step c) described herein can increase the concentration of the bis adducts present in solution, advantageously improving the yield of BnO-VIIIA-Bn (IX). Accordingly, in certain embodiments, the tert-butylmagnesium compound comprises one or both of a tert-butylmagnesium halide and di-tert-butylmagnesium. For example, in some embodiments, the tert-butylmagnesium compound comprises a tert-butylmagnesium halide and di-tert-butylmagnesium. In certain embodiments, a proportion of the magnesium dihalide (e.g., magnesium dichloride) precipitates from solution. For example, substantially all of the magnesium dihalide may precipitate from solution. Alternatively, in certain other embodiments, there is essentially no precipitate formed from the Grignard reagent. Step d) in the method for converting HO-V-H (VI) into buprenophine

[0231] In some embodiments, the hydrogenation catalyst comprises nickel, palladium, platinum, rhodium, or ruthenium. In some embodiments, the hydrogenation catalyst comprises platinum or palladium, supported on carbon. In some embodiments, the reaction is performed in a solvent comprising a polar protic or aprotic solvent, e.g., n-butanol, isopropanol, ethanol, methanol, N-methylpyrrolidone, tetrahydrofuran, ethyl acetate, acetone, dimethylformamide, acetonitrile, dimethylsulfoxide, propylene carbonate, or a mixture thereof.

[0232] In some embodiments, the hydrogen is reacted at a temperature within the range of about 15°C to about 120°C, e.g., about 20°C to about 120°C, or about 30°C to about 120°C, or about 40°C to about 120°C, or about 15°C to about 115°C, or about 20°C to about 110°C, or about 30°C to about 105°C, or about 40°C to about 115°C, or about 50°C to about 110°C. In some embodiments, the hydrogen is reacted for a period of time within the range of about 6 hours to about 3 days, e.g., about 12 hours to about 3 days, or about 18 hours to about 3 days, or about 1 day to about 3 days, or about 1.25 days to about 3 days, or about 1.5 days to about 3 days, or about 6 hours to about 2.75 days, or about 6 hours to about 2.5 days, or about 6 hours to about 2.25 days, or about 6 hours to about 2 day, or about 6 hours to about 36 hours, or about 12 hours to about 2.5 days, or about 24 hours to about 2 days. In some embodiments, the hydrogen is reacted at a pressure within the range of about 1 atm to about 3 atm, e.g., about 1.25 atm to about 3 atm, or about 1.5 atm to about 3 atm, or about 1.75 atm to about 3 atm, or about 2 atm to about 3 atm, or about 1 atm to about 2.75 atm, or about 1 atm to about 2.5 atm, or about 1 atm to about 2.25 atm, or about 1 atm to about 2 atm, or about 1.25 atm to about 2.75 atm, or about 1.5 atm to about 2.5 atm, or about 1.75 atm to about 2.25 atm. Step e.i) in the method for converting HO-V-H (VI) into buprenophine

[0233] In some embodiments, the hydride source is formic acid, hydrogen, sodium cyanoborohydride, sodium borohydride, or sodium triacetoxy borohydride. In some embodiments, the hydride source is formic acid. In some embodiments, the reaction is catalyzed by a ruthenium(l) complex or a ruthenium(ll) complex, e.g., a dichloro(p-cymene)ruthenium(ll) dimer. In some embodiments, the reaction is performed in a solvent comprising a polar aprotic solvent, e.g., N-methylpyrrolidone, tetrahydrofuran, ethyl acetate, acetone, dimethylformamide, acetonitrile, dimethylsulfoxide, propylene carbonate, or a mixture thereof. In some embodiments, the reaction is performed in the presence of a trialkylamine, e.g., triethylamine, diisopropylethylamine, 4-methyl-morpholine, or N-methyl-piperidine.

[0234] In some embodiments, the cyclopropane carboxaldehyde is reacted at a temperature within the range of about 30°C to about 90°C, e.g., about 35°C to about 90°C, or about 40°C to about 90°C, or about 45°C to about 90°C, or about 50°C to about 90°C, or about 55°C to about 90°C, or about 60°C to about 90°C, or about 65°C to about 90°C, or about 70°C to about 90°C, or about 30°C to about 85°C, or about 30°C to about 80°C, or about 30°C to about 75°C, or about 30°C to about 70°C, or about 30°C to about 65°C, or about 30°C to about 60°C, or about 30°C to about 55°C, or about 30°C to about 50°C, or about 35°C to about 85°C, or about 40°C to about 80°C, or about 45°C to about 75°C, or about 50°C to about 70°C, or about 55°C to about 65°C. In some embodiments, the cyclopropane carboxaldehyde is reacted for a period of time within the range of about 30 minutes to about 5 hours, e.g.,about 1 hour to about 5 hours, or about 1.5 hours to about 5 hours, or about 2 hours to about 5 hours, or about 2.5 hours to about 5 hours, or about 3 hours to about 5 hours, or about 3.5 hours to about 5 hours, or about 4 hours to about 5 hours, or about 30 minutes to about 4.5 hours, or about 30 minutes to about 4 hours, or about 30 minutes to about 3.5 hours, or about 30 minutes to about 3 hours, or about 30 minutes to about 2.5 hours, or about 30 minutes to about 2 hours, or about 30 minutes to about 1.5 hours. Step e.ii) in the method for converting HO-V-H (VI) into buprenophine

[0235] In some embodiments, the cyclopropanecarboxylic acid halide is cyclopropanecarboxylic acid chloride, cyclopropanecarboxylic acid anhydride, cyclopropanecarboxylic acid bromide, or an activated cyclopropanecarboxylic acid (e.g., an activated cyclopropanecarboxylic acid formed by reaction with an alcohol such as pentafluorophenol, 4-nitrophenol, N-hydroxysuccinimide, N-hydroxymaleimide, 1-Hydroxybenzotriazole, or l-hydroxy-7-azabenzotriazole). In some embodiments, the reducing agent is LiAIH4 or NaBH4. In some embodiments, the reaction with cyclopropanecarboxylic acid halide is performed in a solvent comprising a nonpolar solvent, e.g., dichloromethane, chloroform, toluene, 1,4-dioxane, diethyl ether, benzene, or a mixture thereof. In some embodiments, the reaction with a reducing agent is performed in a solvent comprising a polar aprotic solvent, e.g., N-methylpyrrolidone, tetrahydrofuran, ethyl acetate, acetone, dimethylformamide, acetonitrile, dimethylsulfoxide, propylene carbonate, or a mixture thereof.

[0236] In some embodiments, the cyclopropanecarboxylic acid halide is reacted at a temperature within the range of about -20°C to about 40°C, e.g., about -20°C to about 35°C, or about -20°C to about 30°C, or about -20°C to about 25°C, or about -20°C to about 20°C, or about -20°C to about 15°C, or about -20°C to about 10°C, or about -20°C to about 5°C, or about -20°C to about 0°C, or about -15°C to about 40°C, or about -10°C to about 40°C, or about -5°C to about 40°C, or about 0°C to about 40°C, or about 5°C to about 20°C, or about 10°C to about 40°C, or about 15°C to about 40°C, or about 20°C to about 40°C, or about -15°C to about 35°C, or about -10°C to about 30°C, or about -5°C to about 25°C, or about 0°C to about 20°C, or about 5°C to about 15°C. In some embodiments, the cyclopropanecarboxylic acid halide is reacted for a period of time within the range of about 6 hours to about 2 days, e.g., about 12 hours to about 2 days, or about 18 hours to about 2 days, or about 1 day to about 2 days, or about 1.25 days to about 2 days, or about 1.5 days to about 2 days, or about 6 hours to about 1.75 days, or about 6 hours to about 1.5 days, or about 6 hours to about 1.25 days, or about 6 hours to about 1 day, or about 6 hours to about 18 hours, or about 12 hours to about 1.75 days, or about 18 hours to about 1.5 days. In some embodiments, the reducing agent is reacted at a temperature within the range of about 35°C to about 85°C, e.g., about 40°C to about 85°C, or about 45°C to about 85°C, or about 50°C to about 85°C, or about 55°C to about 85°C, or about 60°C to about 85°C, or about 65°C to about 85°C, or about 35°C to about 80°C, or about 35°C to about 75°C, or about 35°C to about 70°C, or about 35°C to about 65°C, or about 35°C to about 60°C, or about 35°C to about 55°C, or about 40°C to about 80°C, or about 45°C to about 75°C, or about 50°C to about 70°C, or about 55°C to about 65°C. In some embodiments, the reducing agent is reacted for a period of time within the range of about 5 minutes to about 3 hours, e.g., or about 10 minutes to about 3 hours, or about 15 minutes to about 3 hours, or about 30 minutes to about 3 hours, or about 45 minutes to about 3 hours, or about 1 hour to about 3 hours, or about 1.25 hours to about 3 hours, or about 1.5 hours to about 3 hours, or about 1.75 hours to about 3 hours, or about 2 hours to about 3 hours, or about 5 minutes to about 2.75 hours, or about 5 minutes to about 2.5 hours, or about 5 minutes to about 2.25 hours, or about 5 minutes to about 2 hours, or about 5 minutes to about 1.75 hours, or about 5 minutes to about 1.5 hours, or about 5 minutes to about 1.25 hours, or about 5 minutes to about 1 hour, or about 10 minutes to about 2.75 hours, or about 15 minutes to about 2.5 hours, or about 30 minutes to about 2.25 hours, or about 45 minutes to about 2 hours, or about 1 hour to about 1.75 hours. Step e.iii) in the method for converting HO-V-H (VI) into buprenophine

[0237] In some embodiments, the cyclopropylmethyl halide is cyclopropylmethyl chloride or cyclopropylmethyl bromide. In some embodiments, the reaction is performed in the presence of a trialkylamine, e.g., triethylamine, diisopropylethylamine, 4-methyl-morpholine, or N-methyl-piperidine. In some embodiments, the reaction is performed in a solvent comprising a polar protic solvent, e.g., n-butanol, isopropanol, ethanol, methanol, water, or a mixture thereof.

[0238] In some embodiments, the cyclopropylmethyl halide or activated cyclopropane methanol is reacted at a temperature within the range of about 40°C to about 120°C, e.g., about 45°C to about 120°C, or about 50°C to about 120°C, or about 55°C to about 120°C, or about 60°C to about 120°C, or about 65°C to about 120°C, or about 70°C to about 120°C, or about 75°C to about 120°C, or about 80°C to about 120°C, or about 85°C to 120°C, or about 90°C to about 120°C, or about 40°C to about 115°C, or about 40°C to about 110°C, or about 40°C to about 105°C, or about 40°C to about 100°C, or about 40°C to about 95°C, or about 40°C to about 90°C, or about 40°C to about 85°C, or about 40°C to about 80°C, or about 40°C to about 75°C, or about 40°C to about 70°C, or about 45°C to about 115°C, or about 50°C to about 110°C, or about 55°C to about 105°C, or about 60°C to about 100°C, or about 65°C to about 95°C, or about 70°C to about 90°C. In some embodiments, the cyclopropylmethyl halide or activated cyclopropane methanol is reacted for a period of time within the range of about 30 minutes to about 6 hours, e.g., about 1 hours to about 6 hours, or about 1.5 hours to about 6 hours, or about 2 hours to about 6 hours, or about 2.5 hours to about 6 hours, or about 3 hours to about 6 hours, or about 3.5 hours to about 6 hours, or about 4 hours to about 6 hours, or about 30 minutes to about 5.5 hours, or about 30 minutes to about 5 hours, or about 30 minutes to about 4.5 hours, or about 30 minutes to about 4 hours, or about 30 minutes to about 3.5 hours, or about 30 minutes to about 3 hours, or about 30 minutes to about 2.5 hours, or about 1 hours to about 5.5 hours, or about 1.5 hours to about 5 hours, or about 2 hours to about 4.5 hours, or about 2.5 hours to about 4 hours.

[0239] A person of ordinary skill in the art will appreciate that additional steps such as, for example, purification (e.g., crystallization) or formation of an addition salt (e.g., formation of buprenorphine-HCI) may be included in the methods of the disclosure as otherwise described herein. Further examples of suitable modifications are disclosed in US provisional application 62 / 962,335 and WO2018229306 and WO2018211331 included herein by reference in their entirety.

[0240] In addition to buprenorphine, other industrially useful products can be synthesized from the thebaine, oripavine, northebaine and / or nororipavine of the invention optionally post fermentation / bioconversion added a protecting group by bisbenzylation as described above for nororipavine. Such useful products include "Nais" compounds, such as naloxone, nalmefene, nalbuphine, naltrexone and methylnaltrexone, which can also be synthesized through a common intermediate noroxymorphone. Thebaine and oripavine of the invention can also be used to produce a range of therapeutically important molecules, including oxycodone, hydrocodone, hydromorphone and oxymorphone, the latter also used as an intermediate for the manufacture of "Nais".

[0241] As shown by A. Sipos, S. Berenyi and S. Antus (Helvetica Chimica Acta Vol 92 (2009) pp 13591365) nororipavine (also known as oripavidine) may be converted to noroxymorphone using a benzylation reaction, an oxidation reaction using peracid, and catalytic hydrogenation. Subsequent N-Alkylation with 3-Bromo-l-propene will yield Naloxone, further alkylation with cyclopropylmethylbromide will yield naltrexone. B. Gutmann et al describe the utility of the intermediate noroxymorphone for the "Nai" compounds, synthesis commencing with thebaine or oripavine rather than nororipavine (European Journal of Organic Chemistry 2017, 914-927). T. Hudlicky (Can. J. Chern 93: 492-501 (2015)) similarly describes methods for production of buprenorphine, naltrexone, naloxone, and nalbuphine from thebaine and oripavine. Numerous patent applications describe similar methods for synthesis of these useful pharmaceutical compounds such as WO 2009 / 003270, WO 2009 / 079013, WO 2010 / 063291, WO 2010 / 136039, WO 2012 / 059103, WO 2012 / 151669, WO 2012 / 149633, WO 2013 / 08365, WO 2013 / 113120, WO 2013 / 164383, WO 2013 / 119886, WO 2005 / 028483 Al, WO 2005 / 084412, WO 2007 / 137782, WO 2009 / 003272, WO 2009 / 152571A1, WO 2009 / 152577A1, WO 2011 / 032214, and WO 2012 / 018872. It is clear that a scalable and stable supply chain of thebaine, oripavine, northebaine or nororipavine made by the fermentation / biotransformation of the invention is useful as starting material for the chemical conversion of the invention as disclosed herein.

[0242] Currently known methods for producing semisynthetic opioids (including oxycodone, hydrocodone, hydromorphone, oxymorphone, naloxone, naltrexone, nalmefene, methylnaltrexone, noroxymorphone, buprenorphine) include production via chemical synthesis from thebaine, oripavine, morphine and codeine, mostly commonly from thebaine or oripavine, all four compounds produced by extraction from the opium poppy (Papaver somniferum). The lack of a commercial supply of nororipavine is in part due to the inability of the opium poppy to produce commercially viable concentrations of nororipavine, which is believed to be due to the lack of a naturally occurring N-demethylase enzyme in the opium poppy. High yielding industrially applicable methods of synthesis of nororipavine have not previously been disclosed and production of commercially relevant quantities of nororipavine have not hitherto been available. Thebaine, oripavine, northebaine and / or in particular nororipavine are attractive for use as a starting material due to their chemical structure and functionality allowing efficient installation of the hydroxy group at C-14 position and / or for performing the Diels-Alder reaction on the methoxydiene moiety to produce the backbone of buprenorphine. Nororipavine produced by fermentation / bioconversion has the additional advantage over thebaine and oripavine that the difficult chemical N-demethylation is already completed further enhancing the utility as a starting material for buprenorphine or "Nais" synthesis. (Maehara et.al. Georg Thieme Verlag Stuttgart • New York — Synthesis 2016, 48, 1803-1813).

[0243] Separation methods for opiates and other alkaloids are well-known in the art. See, for example Tolkachev et al. (1983), Janicot et al. (1988), Barbier (1950), Ramanathan and Chandra (1980), Hosztafi (2014), Hamerslag (1950), Thorton (1992), Heumann (1957), Swiss patent 457433 (1935), GB713689 (1952), GB1586626 (1977), US6723894B2 (2002), and US6054584A (1996) - all incorporate by reference. In further specific embodiments when producing nororipavine by fermentation, either de-novo or by bioconversion of a fed precursor, isolation of the Nororipavine contained in the fermentation broth can be achieved by several routes encompassing the typical unit operations of solid-liquid separation (e.g. ultra and nanofiltration membrane filtration, centrifugal separation, pressure or vacuum filtration through filter membranes or filter aids) residual biomass washing to maximise recovery (with various medium including water, acids, bases and solvents), and the concentration and selective removal of nororipavine from related fermentation products and any residual starting material by direct crystallisation as the Nororipavine base or a selective salt, by adsorption and de-adsorption on solid supports (e.g. ion-exchange resins, molecular imprinted polymers), by liquid-liquid extraction, supercritical fluids extraction, or by reaction with other chemicals to directly form a desired derivative of nororipavine. These downstream reactions could include the addition of new functional groups to add functionality to the secondary nitrogen position to form a tertiary nitrogen (e.g. alkylation) or to the phenolic hydroxide position (e.g. benzylation), to oxidise or reduce to form new products (e.g. introduction of 14-hydroxy-) or reactions directly on diene bond to form new products (e.g. Diels Alder reaction). Incorporating the formation of new products within the processing of the fermentation broth may provide more selective separation from related impurities, improve isolation characteristics such as filtration speed, incorporate a required downstream process step eliminating the need for isolation of Nororipavine as a process intermediate (known as process telescoping), provide a less reactive more stable isolated product and improve overall process yield.

[0244] An exemplary embodiment consists of solid-liquid separation by ultrafiltration of the fermentation broth in order to remove cellular matter and higher molecular weight components, resulting in further concentration of the broth containing Nororipavine. A wash of the clarified solids can then be performed with a dilute acid and can be combined with the clarified broth. A clarified broth can be treated with compounds that form insoluble complexes with divalent cations and clarified by separation, such as filtration or centrifugal separation. Alternatively nanofiltration can be used for partial deionization as well. The pH of the combined clarified broth and water wash may in an embodiment be adjusted, preferably just prior to, or after, being contacted with an immiscible solvent such as toluene, xylene, amyl alcohol, isobutanol, benzyl alcohol or a mixture of similar solvents in order to maximise selective extraction of the Nororipavine into a Nororipavine rich solvent. Contact with the solvent phase may be carried out in batch or continuous mode, optimally as a multi-stage counter current system.

[0245] In an embodiment the Nororipavine rich organic phase can be extracted in another liquidliquid extraction step using either an alkaline or acid aqueous solution to produce a concentrated Nororipavine aqueous solution. Contact with the solvent phase may be carried out in batch or continuous mode optimally as a multi-stage counter current system. The aqueous solution of Nororipavine can optionally be isolated by direct addition of acid or base to precipitate the Nororipavine which is then filtered, washed and dried. In another embodiment the solution can be mixed with solvent prior to reaction with and excess of Benzyl bromide (or similar blocking reactant) to form and precipitate 3,17, bisbenzylnororipavine bisbenzyl. The resultant slurry can be cooled, filtered and washed with water and dried. Fermentation composition

[0246] The invention further provides a fermentation composition comprising the cell culture of the invention and the benzylisoquionoline alkaloid comprised therein.

[0247] In one embodiment at least 10%, 25%, 50%, such as at least 75%, such as at least 95%, such as at least 99% of the cells of the fermentation composition of the invention are lysed. Further in the fermentation composition of the invention at least 10%, 25%, 50%, such as at least 75%, such as at least 95%, such as at least 99% of solid cellular material may have been removed and separated from a liquid phase. Moreover, in addition to benzylisoquionoline alkaloid the fermentation composition of the invention may comprise one or more compounds selected from trace metals, vitamins, salts, yeast nitrogen base, carbon source, YNB, and / or amino acids of the fermentation. In particular the fermentation compositin of the invention comprise a concentration of benzylisoquionoline alkaloid is at least 1 mg / kg composition, such as at least 5 mg / kg, such as at least 10 mg / kg, such as at least 20 mg / kg, such as at least 50 mg / kg, such as at least 100 mg / kg, such as at least 500 mg / kg, such as at least 1000 mg / kg, such as at least 5000 mg / kg, such as at least 10000 mg / kg, such as at least 50000 mg / kg. Compositions and use

[0248] In a further aspect the invention provides a composition comprising the fermentation composition of the invention and one or more carriers, agents, additives and / or excipients. Carriers, agents, additives and / or excipients includes formulation additives, stabilising agent, fillers and the like. The composition may be formulated into a dry solid form by using methods known in the art, such as spray drying, spray cooling, lyophilization, flash freezing, granulation, microgranulation, encapsulation or microencapsulation. The composition may also be formulated into liquid stabilized form using methods known in the art, such as formulation into a stabilized liquid comprising one or more stabilizers such as sugars and / or polyols (e.g. sugar alcohols) and / or organic acids (e.g. lactic acid).

[0249] Still further the invention provides a pharmaceutical composition comprising the fermentation composition of the invention preceding item and one or more pharmaceutical grade excipient, additives and / or adjuvants. The pharmaceutical composition can be in form of a powder, tablet or capsule, or it can be liquid in the form of a pharmaceutical solution, suspension, lotion or ointment. The pharmaceutical composition can also be incorporated into suitable delivery systems such as for buccal administration or as a patch for transdermal administration.

[0250] The invention further provides a method for preparing the pharmaceutical composition of the invention comprising mixing the fermentation composition of the invention with one or more pharmaceutical grade excipient, additives and / or adjuvants.

[0251] The pharmaceutical composition is suitably used as a medicament in a method for treating and / or relieving a disease and / or medical condition, in particular in a mammal. Accordingly, the invention further provides a method for preventing, treating and / or relieving a disease and / or medical condition comprising administering a therapeutically effective amount of the pharmaceutical composition of the invention to a mammal in need of treatment and / or relief. Diseases and / or medical conditions treatable or reliveable by the pharmaceutical composition includes but is not limited to pain, infections, tussive conditions, parasitic conditions, cytotoxic conditions, opiate poisoning conditions and / or cancerous conditions. Appropriate and effective dosages of benzylisoquionoline alkaloids are known in the art. The pharmaceutical preparation can be administered parenterally, such as topically, epicutaneously, sublingually, buccally, nasally, intradermally, intralesionally, (intra)ocularly, intraveneously, intramuscular, intrapulmonary and / or intravaginally. The pharmaceutical composition can also be administered enterally to the gastrointestinal tract. Sequences

[0252] The present application contains a Sequence Listing prepared in Patentin ver 3.5 submitted electronically in ST25 format which is hereby incorporated by reference in its entirety. The following sequences are included: Table A SEQ ID NO: 1 Amino acid sequence of DAHP Aro4fbr From Artificial SEQ ID NO: 2 DNA coding sequence of DAHP Aro4fbr From Artificial SEQ ID NO: 3 Amino acid sequence of chorismate mutase AR07fbr From Artificial SEQ ID NO: 4 DNA coding sequence of chorismate mutase AR07fbr From Artificial SEQ ID NO: 5 Amino acid sequence of Tyrl Tyrl From S. cerevisiae SEQ ID NO: 6 DNA coding sequence of Tyrl Tyrl From S. cerevisiae SEQ ID NO: 7 Amino acid sequence of TH SoCYP76ADr9 From Spinacia oleracea SEQ ID NO: 8 DNA coding sequence of TH SoCYP76ADr9 From Spinacia oleracea SEQ ID NO: 9 Amino acid sequence of TH OfCYP76ADrl2 From Opuntia ficus-indica SEQ ID NO: 10 DNA coding sequence of TH OfCYP76ADrl2 From Opuntia ficus-indica SEQ ID NO: 11 Amino acid sequence of TH FICYP76ADrll From Froelichia latifolia SEQ ID NO: 12 DNA coding sequence of TH FICYP76ADrll From Froelichia latifolia SEQ ID NO: 13 Amino acid sequence of TH BvCYP76ADrlO From Beta vulgaris SEQ ID NO: 14 DNA coding sequence of TH BvCYP76ADrlO From Beta vulgaris SEQ ID NO: 15 Amino acid sequence of TH AnCYP76ADrl7 From Abronia nealleyi SEQ ID NO: 16 DNA coding sequence of TH AnCYP76ADrl7 From Abronia nealleyi SEQ ID NO: 17 Amino acid sequence of TH BvCYP76ADr8 From Beta vulgaris SEQ ID NO: 18 DNA coding sequence of TH BvCYP76ADr8 From Beta vulgaris SEQ ID NO: 19 Amino acid sequence of TH BvCYP76Ar7 From Beta vulgaris SEQ ID NO: 20 DNA coding sequence of TH BvCYP76Ar7 From Beta vulgaris SEQ ID NO: 21 Amino acid sequence of TH BvCYP76ADr6 From Beta vulgaris SEQ ID NO: 22 DNA coding sequence of TH BvCYP76ADr6 From Beta vulgaris SEQ ID NO: 23 Amino acid sequence of TH CbCYP76ADr28 From Cleretum bellidiforme SEQ ID NO: 24 DNA coding sequence of TH CbCYP76ADr28 From Cleretum bellidiforme SEQ ID NO: 25 Amino acid sequence of TH EvCYP76ADr20 From Ercilla volubilis SEQ ID NO: 26 DNA coding sequence of TH EvCYP76ADr20 From Ercilla volubilis SEQ ID NO: 27 Amino acid sequence of TH PdCYP76ADr21 From Phytolacca dioica SEQ ID NO: 28 DNA coding sequence of TH PdCYP76ADr21 From Phytolacca dioica SEQ ID NO: 29 Amino acid sequence of TH AoCYP76ADrl6 From Acleisanthes obtuse SEQ ID NO: 30 DNA coding sequence of TH AoCYP76ADrl6 From Acleisanthes obtuse SEQ ID NO: 31 Amino acid sequence of TH MmCYP76ADrl8 From Mirabilis multiflora SEQ ID NO: 32 DNA coding sequence of TH MmCYP76ADrl8 From Mirabilis multiflora SEQ ID NO: 33 Amino acid sequence of TH AoCYP76ADr24 From Acleisanthes obtuse SEQ ID NO: 34 DNA coding sequence of TH AoCYP76ADr24 From Acleisanthes obtuse SEQ ID NO: 35 Amino acid sequence of TH AnCYP76ADr27 From Abronia nealleyi SEQ ID NO: 36 DNA coding sequence of TH AnCYP76ADr27 From Abronia nealleyi SEQ ID NO: 37 Amino acid sequence of TH PaCYP76ADrl9 From Phytolacca americana SEQ ID NO: 38 DNA coding sequence of TH PaCYP76ADrl9 From Phytolacca americana SEQ ID NO: 39 Amino acid sequence of TH CqCYP76ADr5 From Chenopodium quinoa SEQ ID NO: 40 DNA coding sequence of TH CqCYP76ADr5 From Chenopodium quinoa SEQ ID NO: 41 Amino acid sequence of TH MmCYP76ADr22 From Mirabilis multiflora SEQ ID NO: 42 DNA coding sequence of TH MmCYP76ADr22 From Mirabilis multiflora SEQ ID NO: 43 Amino acid sequence of TH CqCYP76ADr4 From Chenopodium quinoa SEQ ID NO: 44 DNA coding sequence of TH CqCYP76ADr4 From Chenopodium quinoa SEQ ID NO: 45 Amino acid sequence of TH PaCYP76ADrl4 From Phytolacca americana SEQ ID NO: 46 DNA coding sequence of TH PaCYP76ADrl4 From Phytolacca americana SEQ ID NO: 47 Amino acid sequence of TH AnCYP76ADr23 From Abronia nealleyi SEQ ID NO: 48 DNA coding sequence of TH AnCYP76ADr23 From Abronia nealleyi SEQ ID NO: 49 Amino acid sequence of TH SoCYP76ADr2 From Spinacia oleracea SEQ ID NO: 50 DNA coding sequence of TH SoCYP76ADr2 From Spinacia oleracea SEQ ID NO: 51 Amino acid sequence of TH SoCYP76ADr3 From Spinacia oleracea SEQ ID NO: 52 DNA coding sequence of TH SoCYP76ADr3 From Spinacia oleracea SEQ ID NO: 53 Amino acid sequence of TH SoCYP76ADrl From Spinacia oleracea SEQ ID NO: 54 DNA coding sequence of TH SoCYP76ADrl From Spinacia oleracea SEQ ID NO: 55 Amino acid sequence of TH CqCYP76ADrl3 From Chenopodium quinoa SEQ ID NO: 56 DNA coding sequence of TH CqCYP76ADrl3 From Chenopodium quinoa SEQ ID NO: 57 Amino acid sequence of TH SoCYP76ADrl5 From Spinacia oleracea SEQ ID NO: 58 DNA coding sequence of TH SoCYP76ADrl5 From Spinacia oleracea SEQ ID NO: 59 Amino acid sequence of TH MjCYP76ADr26 From Mirabilis jalapa SEQ ID NO: 60 DNA coding sequence of TH MjCYP76ADr26 From Mirabilis jalapa SEQ ID NO: 61 Amino acid sequence of TH MmCYP76ADr25 From Mirabilis multiflora SEQ ID NO: 62 DNA coding sequence of TH MmCYP76ADr25 From Mirabilis multiflora SEQ ID NO: 63 Amino acid sequence of TH BvCYP76ADlVM From Beta vulgaris SEQ ID NO: 64 DNA coding sequence of TH BvCYP76ADlVM From Beta vulgaris SEQ ID NO: 65 Amino acid sequence of TH CYP76ADl_2mut From Artificial SEQ ID NO: 66 DNA coding sequence of TH CYP76ADl_2mut From Artificial SEQ ID NO: 67 Amino acid sequence of CPR'" BvCPRl From Beta vulgaris SEQ ID NO: 68 DNA coding sequence of CPR'" BvCPRl From Beta vulgaris SEQ ID NO: 69 Amino acid sequence of Do DC PpDoDC From Pseudomonas putida SEQ ID NO: 70 DNA coding sequence of Do DC PpDoDC From Pseudomonas putida SEQ ID NO: 71 Amino acid sequence of Do DC PpDoDC From Pseudomonas putida SEQ ID NO: 72 DNA coding sequence of Do DC PpDoDC From Pseudomonas putida SEQ ID NO: 73 Amino acid sequence of NCS dl9CjNCS From Coptis japonica SEQ ID NO: 74 DNA coding sequence of NCS dl9CjNCS From Coptis japonica SEQ ID NO: 75 DNA coding sequence of NCS dl9CjNCS From Coptis japonica SEQ ID NO: 76 Amino acid sequence of NCS HDEL_CjNCS_V152 From Artificial SEQ ID NO: 77 DNA coding sequence of NCS HDEL_CjNCS_V152 From Artificial SEQ ID NO: 78 DNA coding sequence of Integration plasmid pRIV40 From Artificial SEQ ID NO: 79 Amino acid sequence of 6-OMT Ps6OMT_Q6WUCl From Papaver somniferum SEQ ID NO: 80 DNA coding sequence of 6-OMT Ps6OMT_Q6WUCl From Papaver somniferum SEQ ID NO: 81 Amino acid sequence of 6-OMT From Papaver somniferum SEQ ID NO: 82 Amino acid sequence of CNMT CjCNMT From Coptis japonica SEQ ID NO: 83 DNA coding sequence of CNMT CjCNMT From Coptis japonica SEQ ID NO: 84 Amino acid sequence of CNMT From Papaver somniferum SEQ ID NO: 85 Amino acid sequence of NMCH EcNMCH From Eschscholzia californica SEQ ID NO: 86 DNA coding sequence of NMCH EcNMCH From Eschscholzia californica SEQ ID NO: 87 Amino acid sequence of NMCH From Eschscholzia californica SEQ ID NO: 88 DNA coding sequence of NMCH From Eschscholzia californica SEQ ID NO: 89 Amino acid sequence of 4'-0MT Cj4OMT From Coptis japonica SEQ ID NO: 90 DNA coding sequence of 4'-0MT Cj4OMT From Coptis japonica SEQ ID NO: 91 Amino acid sequence of 4'-0MT From Papaver somniferum SEQ ID NO: 92 Amino acid sequence of STORR DRS-DRR From Papaver bracteatum SEQ ID NO: 93 DNA coding sequence of STORR DRS-DRR From Papaver bracteatum SEQ ID NO: 94 Amino acid sequence of STORR StIRED From Streptomyces tsukubaensis SEQ ID NO: 95 DNA coding sequence of STORR StIRED From Streptomyces tsukubaensis SEQ ID NO: 96 Amino acid sequence of STORR PsSTORR From Papaver somniferum SEQ ID NO: 97 DNA coding sequence of STORR PsSTORR From Papaver somniferum SEQ ID NO: 98 Amino acid sequence of STORR P450 PsCYP82Y2 From Papaver somniferum SEQ ID NO: 99 DNA coding sequence of STORR P450 PsCYP82Y2 From Papaver somniferum SEQ ID NO: 100 Amino acid sequence of STORR P450 PrCYP82Y2-like From Papaver rhoeas SEQ ID NO: 101 DNA coding sequence of STORR P450 PrCYP82Y2-like From Papaver rhoeas SEQ ID NO: 102 Amino acid sequence of STORR P450 prolD60 From Artificial SEQ ID NO: 103 DNA coding sequence of STORR P450 prolD60 From Artificial SEQ ID NO: 104 Amino acid sequence of STORR P450 prolD66 From Artificial SEQ ID NO: 105 DNA coding sequence of STORR P450 prolD66 From Artificial SEQ ID NO: 106 Amino acid sequence of STORR P450 prolD79 From Artificial SEQ ID NO: 107 DNA coding sequence of STORR P450 prolD79 From Artificial SEQ ID NO: 108 Amino acid sequence of STORR Reductase PsAKR From Papaver somniferum SEQ ID NO: 109 DNA coding sequence of STORR Reductase PsAKR From Papaver somniferum SEQ ID NO: 110 Amino acid sequence of STORR Reductase PrAKR From Papaver rhoeas SEQ ID NO: 111 DNA coding sequence of STORR Reductase PrAKR From Papaver rhoeas SEQ ID NO: 112 Amino acid sequence of CPR" PsCPR From Papaver somniferum SEQ ID NO: 113 DNA coding sequence of CPR" PsCPR From Papaver somniferum SEQ ID NO: 114 Amino acid sequence of CPR" AtATRl From Arabidopsis thaliana SEQ ID NO: 115 DNA coding sequence of CPR" AtATRl From Arabidopsis thaliana SEQ ID NO: 116 Amino acid sequence of SAS PbSAS From Papaver bracteatum SEQ ID NO: 117 DNA coding sequence of SAS PbSAS From Papaver bracteatum SEQ ID NO: 118 Amino acid sequence of SAS - From Papaver bracteatum SEQ ID NO: 119 DNA coding sequence of SAS - From Papaver bracteatum SEQ ID NO: 120 Amino acid sequence of SAR pbSaIR From Papaver bracteatum SEQ ID NO: 121 DNA coding sequence of SAR pbSaIR From Papaver bracteatum SEQ ID NO: 122 Amino acid sequence of SAR - From Papaver bracteatum SEQ ID NO: 123 Amino acid sequence of SAT PsSAT From Papaver somniferum SEQ ID NO: 124 DNA coding sequence of SAT PsSAT From Papaver somniferum SEQ ID NO: 125 Amino acid sequence of SAT - From Papaver somniferum SEQ ID NO: 126 Amino acid sequence of THS HA BetVIM From Papaver somniferum SEQ ID NO: 127 Amino acid sequence of THS BETV1L HA From Papaver somniferum SEQ ID NO: 128 Amino acid sequence of THS - From Papaver somniferum SEQ ID NO: 129 Amino acid sequence of THS PsTHSl From Papaver somniferum SEQ ID NO: 130 DNA coding sequence of THS PsTHSl From Papaver somniferum SEQ ID NO: 131 Amino acid sequence of THS PsTHS2 From Papaver somniferum SEQ ID NO: 132 DNA coding sequence of THS PsTHS2 From Papaver somniferum SEQ ID NO: 133 Amino acid sequence of THS - From Papaver somniferum SEQ ID NO: 134 Amino acid sequence of THS PROths2_138 From Artificial SEQ ID NO: 135 DNA coding sequence of THS PROths2_138 From Artificial SEQ ID NO: 136 Amino acid sequence of THS PROths2_143 From Artificial SEQ ID NO: 137 DNA coding sequence of THS PROths2_143 From Artificial SEQ ID NO: 138 Amino acid sequence of THS PROths2_116 From Artificial SEQ ID NO: 139 DNA coding sequence of THS PROths2_116 From Artificial SEQ ID NO: 140 Amino acid sequence of P450 HaCYP6AE15v2 protein From Helicoverpa armigera SEQ ID NO: 141 DNA coding sequence of P450 HaCYP6AE15v2 *DNA From Helicoverpa armigera SEQ ID NO: 142 Amino acid sequence of P450 HaCYP6AE19 protein From Helicoverpa armigera SEQ ID NO: 143 DNA coding sequence of P450 HaCYP6AE19 *DNA From Helicoverpa armigera SEQ ID NO: 144 Amino acid sequence of P450 HaCYP6AEll protein From Helicoverpa armigera SEQ ID NO: 145 DNA coding sequence of P450 HaCYP6AEll *DNA From Helicoverpa armigera SEQ ID NO: 146 Amino acid sequence of P450 HaCYP6AE17 protein From Helicoverpa armigera SEQ ID NO: 147 DNA coding sequence of P450 HaCYP6AE17 *DNA From Helicoverpa armigera SEQ ID NO: 148 Amino acid sequence of P450 HaCYP6AE24 protein From Helicoverpa armigera SEQ ID NO: 149 DNA coding sequence of P450 HaCYP6AE24 *DNA From Helicoverpa armigera SEQ ID NO: 150 Amino acid sequence of P450 HaCYP6AE20v2 protein From Helicoverpa armigera SEQ ID NO: 151 DNA coding sequence of P450 HaCYP6AE20v2 *DNA From Helicoverpa armigera SEQ ID NO: 152 Amino acid sequence of P450 Hv_CYP_A0A2A4JAM9 protein From Heliothis virescens SEQ ID NO: 153 DNA coding sequence of P450 Hv_CYP_A0A2A4JAM9 *DNA From Heliothis virescens SEQ ID NO: 154 Amino acid sequence of P450 Hv_CYP_A0A2A4JAK3 protein From Heliothis virescens SEQ ID NO: 155 DNA coding sequence of P450 Hv_CYP_A0A2A4JAK3 *DNA From Heliothis virescens SEQ ID NO: 156 Amino acid sequence of P450 Se_CYP6AE68 protein From Spodoptera exigua SEQ ID NO: 157 DNA coding sequence of P450 Se_CYP6AE68 *DNA From Spodoptera exigua SEQ ID NO: 158 Amino acid sequence of P450 Hv_CYP_A0A2A4J7V4 protein From Heliothis virescens SEQ ID NO: 159 DNA coding sequence of P450 Hv_CYP_A0A2A4J7V4 *DNA From Heliothis virescens SEQ ID NO: 160 Amino acid sequence of P450 CmCYP6_A0A0C5CGV6 protein From Cnaphalocrocis medinalis SEQ ID NO: 161 DNA coding sequence of P450 CmCYP6_A0A0C5CGV6 *DNA From Cnaphalocrocis medinalis SEQ ID NO: 162 Amino acid sequence of P450 BmCYP6AE9_A9QW15 protein From Bombyx mandarina SEQ ID NO: 163 DNA coding sequence of P450 BmCYP6AE9_A9QW15 *DNA From Bombyx mandarina SEQ ID NO: 164 Amino acid sequence of P450 Bm_CYP6AE9 protein From Bombyx mori SEQ ID NO: 165 DNA coding sequence of P450 Bm_CYP6AE9 *DNA From Bombyx mori SEQ ID NO: 166 Amino acid sequence of P450 Se_CYP6AE10 protein From Spodoptera exigua SEQ ID NO: 167 DNA coding sequence of P450 Se_CYP6AE10 *DNA From Spodoptera exigua SEQ ID NO: 168 Amino acid sequence of P450 Sf_CYP_A0A2HlWID4 protein From Spodoptera frugiperda SEQ ID NO: 169 DNA coding sequence of P450 Sf_CYP_A0A2HlWID4 *DNA From Spodoptera frugiperda SEQ ID NO: 170 Amino acid sequence of P450 Sf_CYP_A0A2HlV0E7 protein From Spodoptera frugiperda SEQ ID NO: 171 DNA coding sequence of P450 Sf_CYP_A0A2HlV0E7 *DNA From Spodoptera frugiperda SEQ ID NO: 172 Amino acid sequence of P450 Ha_CYP6AE12 protein From Helicoverpa armigera SEQ ID NO: 173 DNA coding sequence of P450 Ha_CYP6AE12 *DNA From Helicoverpa armigera SEQ ID NO: 174 Amino acid sequence of P450 Sf_CYP6AE44 protein From Spodoptera frugiperda SEQ ID NO: 175 DNA coding sequence of P450 Sf_CYP6AE44 *DNA From Spodoptera frugiperda SEQ ID NO: 176 Amino acid sequence of P450 HaCYP6AE_A0A068F0X7 protein From Helicoverpa armigera SEQ ID NO: 177 DNA coding sequence of P450 HaCYP6AE_A0A068F0X7 *DNA From Helicoverpa armigera SEQ ID NO: 178 Amino acid sequence of P450 DpCYP_Q7YZS2 protein From Depressaria pastinacella SEQ ID NO: 179 DNA coding sequence of P450 DpCYP_Q7YZS2 *DNA From Depressaria pastinacella SEQ ID NO: 180 Amino acid sequence of P450 Sf_CYP_A0A2HlV0E7 protein From Spodoptera frugiperda SEQ ID NO: 181 DNA coding sequence of P450 Sf_CYP_A0A2HlV0E7 *DNA From Spodoptera frugiperda SEQ ID NO: 182 Amino acid sequence of P450 BmCYP6AE2_L0N7C5 protein From Bombyx mori SEQ ID NO: 183 DNA coding sequence of P450 BmCYP6AE2_L0N7C5 *DNA From Bombyx mori SEQ ID NO: 184 Amino acid sequence of P450 BmCYP_ClKJL7 protein From Bombyx mandarina SEQ ID NO: 185 DNA coding sequence of P450 BmCYP_ClKJL7 *DNA From Bombyx mandarina SEQ ID NO: 186 Amino acid sequence of P450 ZfCYP6AE27_D2JLK6 protein From Zygaena filipendulae SEQ ID NO: 187 DNA coding sequence of P450 ZfCYP6AE27_D2JLK6 *DNA From Zygaena filipendulae SEQ ID NO: 188 Amino acid sequence of P450 BmCyp6AE21_B6VFR9 protein From Bombyx mori SEQ ID NO: 189 DNA coding sequence of P450 BmCyp6AE21_B6VFR9 *DNA From Bombyx mori SEQ ID NO: 190 Amino acid sequence of P450 BmCYP6AE7_A4GUB8 protein From Bombyx mori SEQ ID NO: 191 DNA coding sequence of P450 BmCYP6AE7_A4GUB8 *DNA From Bombyx mori SEQ ID NO: 192 Amino acid sequence of P450 CmCYP6_A0A0C5Cll6 protein From Cnaphalocrocis medinalis SEQ ID NO: 193 DNA coding sequence of P450 CmCYP6_A0A0C5Cll6 *DNA From Cnaphalocrocis medinalis SEQ ID NO: 194 Amino acid sequence of P450 SeCYP6_A0A248QEH8 protein From Spodoptera exigua SEQ ID NO: 195 DNA coding sequence of P450 SeCYP6_A0A248QEH8 *DNA From Spodoptera exigua SEQ ID NO: 196 Amino acid sequence of P450 BmCYP6AE9_A5HKMl protein From Bombyx mori SEQ ID NO: 197 DNA coding sequence of P450 BmCYP6AE9_A5HKMl *DNA From Bombyx mori SEQ ID NO: 198 Amino acid sequence of P450 CYPDN_39 protein From Rhizopus microsporus SEQ ID NO: 199 DNA coding sequence of P450 CYPDN_39 gene From Rhizopus microsporus SEQ ID NO: 200 Amino acid sequence of P450 CYPDN_41 protein From Rhizopus microsporus SEQ ID NO: 201 DNA coding sequence of P450 CYPDN_41gene From Rhizopus microsporus SEQ ID NO: 202 Amino acid sequence of P450 CYPDN_43 protein From Lichtheimia corymbifera SEQ ID NO: 203 DNA coding sequence of P450 CYPDN_43 gene From Lichtheimia corymbifera SEQ ID NO: 204 Amino acid sequence of P450 CYPDN_44 protein From Lichtheimia ramosa SEQ ID NO: 205 DNA coding sequence of P450 CYPDN_44 gene From Lichtheimia ramosa SEQ ID NO: 206 Amino acid sequence of P450 CYPDN_45 protein From Rhizopus microsporus SEQ ID NO: 207 DNA coding sequence of P450 CYPDN_45gene From Rhizopus microsporus SEQ ID NO: 208 Amino acid sequence of P450 CYPDN_50 protein From Lichtheimia ramosa SEQ ID NO: 209 DNA coding sequence of P450 CYPDN_50gene From Lichtheimia ramosa SEQ ID NO: 210 Amino acid sequence of P450 CYPDN_51 protein From Lichtheimia ramosa SEQ ID NO: 211 DNA coding sequence of P450 CYPDN_51gene From Lichtheimia ramosa SEQ ID NO: 212 Amino acid sequence of P450 CYPDN_57 protein From Syncephalastrum racemosum SEQ ID NO: 213 DNA coding sequence of P450 CYPDN_57gene From Syncephalastrum racemosum SEQ ID NO: 214 Amino acid sequence of P450 CYPDN_59 protein From Cunninghamella echinulata SEQ ID NO: 215 DNA coding sequence of P450 CYPDN_59 gene From Cunninghamella echinulata SEQ ID NO: 216 Amino acid sequence of P450 CYPDN_61 protein From Rhizopus azygosporus SEQ ID NO: 217 DNA coding sequence of P450 CYPDN_61gene From Rhizopus azygosporus SEQ ID NO: 218 Amino acid sequence of P450 CYPDN_62 protein From Rhizopus azygosporus SEQ ID NO: 219 DNA coding sequence of P450 CYPDN_62 gene From Rhizopus azygosporus SEQ ID NO: 220 Amino acid sequence of P450 CYPDN_63 protein From Rhizopus microsporus SEQ ID NO: 221 DNA coding sequence of P450 CYPDN_63 gene From Rhizopus microsporus SEQ ID NO: 222 Amino acid sequence of P450 CYPDN_64 protein From Mucor circinelloides f. circinelloides SEQ ID NO: 223 DNA coding sequence of P450 CYPDN_64 gene From Mucor circinelloides f. circinelloides SEQ ID NO: 224 Amino acid sequence of P450 CYPDN_65 protein From Mucor ambiguus SEQ ID NO: 225 DNA coding sequence of P450 CYPDN_65 gene From Mucor ambiguus SEQ ID NO: 226 Amino acid sequence of P450 CYPDN_67 protein From Syncephalastrum racemosum SEQ ID NO: 227 DNA coding sequence of P450 CYPDN_67 gene From Syncephalastrum racemosum SEQ ID NO: 228 Amino acid sequence of P450 CYPDN_68 protein From Parasitella parasitica SEQ ID NO: 229 DNA coding sequence of P450 CYPDN_68 gene From Parasitella parasitica SEQ ID NO: 230 Amino acid sequence of P450 CYPDN_69 protein From Syncephalastrum racemosum SEQ ID NO: 231 DNA coding sequence of P450 CYPDN_69 gene From Syncephalastrum racemosum SEQ ID NO: 232 Amino acid sequence of P450 CYPDN_70 protein From Lichtheimia ramosa SEQ ID NO: 233 DNA coding sequence of P450 CYPDN_70 gene From Lichtheimia ramosa SEQ ID NO: 234 Amino acid sequence of P450 CYPDN_74 protein From Lichtheimia corymbifera SEQ ID NO: 235 DNA coding sequence of P450 CYPDN_74 gene From Lichtheimia corymbifera SEQ ID NO: 236 Amino acid sequence of P450 CYPDN_75 protein From Absidia repens SEQ ID NO: 237 DNA coding sequence of P450 CYPDN_75gene From Absidia repens SEQ ID NO: 238 Amino acid sequence of P450 CYPDN_77 protein From Lichtheimia corymbifera SEQ ID NO: 239 DNA coding sequence of P450 CYPDN_77 gene From Lichtheimia corymbifera SEQ ID NO: 240 Amino acid sequence of P450 CYPDN_80 protein From Absidia glauca SEQ ID NO: 241 DNA coding sequence of P450 CYPDN_80 gene From Absidia glauca SEQ ID NO: 242 Amino acid sequence of P450 CYPDN_82 protein From Choanephora cucurbitarum SEQ ID NO: 243 DNA coding sequence of P450 CYPDN_82 gene From Choanephora cucurbitarum SEQ ID NO: 244 Amino acid sequence of P450 CYPDN_84 protein From Absidia glauca SEQ ID NO: 245 DNA coding sequence of P450 CYPDN_84 gene From Absidia glauca SEQ ID NO: 246 Amino acid sequence of P450 CYPDN_85 protein From Absidia repens SEQ ID NO: 247 DNA coding sequence of P450 CYPDN_85 gene From Absidia repens SEQ ID NO: 248 Amino acid sequence of P450 CYPDN_86 protein From Absidia repens SEQ ID NO: 249 DNA coding sequence of P450 CYPDN_86 gene From Absidia repens SEQ ID NO: 250 Amino acid sequence of P450 CYPDN_91 protein From Rhizopus microsporus SEQ ID NO: 251 DNA coding sequence of P450 CYPDN_91gene From Rhizopus microsporus SEQ ID NO: 252 Amino acid sequence of P450 CYPDN_92 protein From Rhizopus azygosporus SEQ ID NO: 253 DNA coding sequence of P450 CYPDN_92 gene From Rhizopus azygosporus SEQ ID NO: 254 Amino acid sequence of P450 CYPDN_93 protein From Rhizopus azygosporus SEQ ID NO: 255 DNA coding sequence of P450 CYPDN_93 gene From Rhizopus azygosporus SEQ ID NO: 256 Amino acid sequence of P450 CYPDN_95 protein From Bifiguratus adelaidae SEQ ID NO: 257 DNA coding sequence of P450 CYPDN_95 gene From Bifiguratus adelaidae SEQ ID NO: 258 Amino acid sequence of P450 CYPDN_98 protein From Rhizopus stolonifer SEQ ID NO: 259 DNA coding sequence of P450 CYPDN_98 gene From Rhizopus stolonifer SEQ ID NO: 260 Amino acid sequence of P450 CYPDN_100 protein From Rhizopus oryzae SEQ ID NO: 261 DNA coding sequence of P450 CYPDN_100 gene From Rhizopus oryzae SEQ ID NO: 262 Amino acid sequence of P450 CYPDN_101 protein From Rhizopus microsporus SEQ ID NO: 263 DNA coding sequence of P450 CYPDN_101gene From Rhizopus microsporus SEQ ID NO: 264 Amino acid sequence of P450 CYPDN_103 protein From Rhizopus delemar RA 99-880 SEQ ID NO: 265 DNA coding sequence of P450 CYPDN_103 gene From Rhizopus delemar RA 99-880 SEQ ID NO: 266 Amino acid sequence of P450 CYPDN_104 protein From Rhizopus stolonifer SEQ ID NO: 267 DNA coding sequence of P450 CYPDN_104 gene From Rhizopus stolonifer SEQ ID NO: 268 Amino acid sequence of P450 CYPDN_105 protein From Rhizopus azygosporus SEQ ID NO: 269 DNA coding sequence of P450 CYPDN_105gene From Rhizopus azygosporus SEQ ID NO: 270 Amino acid sequence of P450 CYPDN_108 protein From Mucor circinelloides f. circinelloides SEQ ID NO: 271 DNA coding sequence of P450 CYPDN_108 gene From Mucor circinelloides f. circinelloides SEQ ID NO: 272 Amino acid sequence of P450 CYPDN_109 protein From Mucor circinelloides f. circinelloides SEQ ID NO: 273 DNA coding sequence of P450 CYPDN_109 gene From Mucor circinelloides f. circinelloides SEQ ID NO: 274 Amino acid sequence of P450 CYPDN_110 protein From Mucor circinelloides f. lusitanicus SEQ ID NO: 275 DNA coding sequence of P450 CYPDN_110gene From Mucor circinelloides f. lusitanicus SEQ ID NO: 276 Amino acid sequence of P450 CYPDN_112 protein From Choanephora cucurbitarum SEQ ID NO: 277 DNA coding sequence of P450 CYPDN_112 gene From Choanephora cucurbitarum SEQ ID NO: 278 Amino acid sequence of P450 CYPDN_115 protein From Lichtheimia corymbifera SEQ ID NO: 279 DNA coding sequence of P450 CYPDN_115 gene From Lichtheimia corymbifera SEQ ID NO: 280 Amino acid sequence of P450 CYPDN_117 protein From Lichtheimia corymbifera SEQ ID NO: 281 DNA coding sequence of P450 CYPDN_117gene From Lichtheimia corymbifera SEQ ID NO: 282 Amino acid sequence of P450 CYPDN_118 protein From Lichtheimia ramosa SEQ ID NO: 283 DNA coding sequence of P450 CYPDN_118gene From Lichtheimia ramosa SEQ ID NO: 284 Amino acid sequence of P450 CYPDN_119 protein From Lichtheimia corymbifera SEQ ID NO: 285 DNA coding sequence of P450 CYPDN_119 gene From Lichtheimia corymbifera SEQ ID NO: 286 Amino acid sequence of P450 CYPDN_120 protein From Lichtheimia ramosa SEQ ID NO: 287 DNA coding sequence of P450 CYPDN_120gene From Lichtheimia ramosa SEQ ID NO: 288 Amino acid sequence of P450 CYPDN_123 protein From Lichtheimia ramosa SEQ ID NO: 289 DNA coding sequence of P450 CYPDN_123 gene From Lichtheimia ramosa SEQ ID NO: 290 Amino acid sequence of P450 CYPDN_8 protein From Rhizopus microsporus SEQ ID NO: 291 DNA coding sequence of P450 CYPDN_8 *DNA From Rhizopus microsporus SEQ ID NO: 292 Amino acid sequence of CPR' HaCPR_E0A3A7 protein From Helicoverpa armigera SEQ ID NO: 293 DNA coding sequence of CPR' HaCPR_E0A3A7 *DNA From Helicoverpa armigera SEQ ID NO: 294 Amino acid sequence of CPR' Se_CPR_FlDI27 From Spodoptera exigua SEQ ID NO: 295 DNA coding sequence of CPR' Se_CPR_FlDI27 From Spodoptera exigua SEQ ID NO: 296 Amino acid sequence of CPR' Bm_CPR_Q9NKV3 From Bombyx mori SEQ ID NO: 297 DNA coding sequence of CPR' Bm_CPR_Q9NKV3 From Bombyx mori SEQ ID NO: 298 Amino acid sequence of CPR' BmCPR_A0FGR6 From Bombyx mandarina SEQ ID NO: 299 DNA coding sequence of CPR' BmCPR_A0FGR6 From Bombyx mandarina SEQ ID NO: 300 Amino acid sequence of CPR' ZfCPR_A0A346M705 From Zygaena filipendulae SEQ ID NO: 301 DNA coding sequence of CPR' ZfCPR_A0A346M705 From Zygaena filipendulae SEQ ID NO: 302 Amino acid sequence of CPR' CmCPR_A0AlS5ZY34 From Cnaphalocrocis medinalis SEQ ID NO: 303 DNA coding sequence of CPR' CmCPR_A0AlS5ZY34 From Cnaphalocrocis medinalis SEQ ID NO: 304 DNA coding sequence of CPR' HaCPR_E7E2N6 From Helicoverpa armigera SEQ ID NO: 305 Amino acid sequence of CPR' CeCPR protein From Cunninghamella elegans SEQ ID NO: 306 DNA coding sequence of CPR' CeCPR gene From Cunninghamella elegans SEQ ID NO: 307 Amino acid sequence of Transporter Tl_CjaMDRl_GA From Camellia japonica SEQ ID NO: 308 DNA coding sequence of Transporter Tl_CjaMDRl_GA From Camellia japonica SEQ ID NO: 309 Amino acid sequence of Transporter T3_NcaNPF_GA From Noccaea caerulescens SEQ ID NO: 310 DNA coding sequence of Transporter T3_NcaNPF_GA From Noccaea caerulescens SEQ ID NO: 311 Amino acid sequence of Transporter T4_EsaGTR_GA From Eutrema salsugineum SEQ ID NO: 312 DNA coding sequence of Transporter T4_EsaGTR_GA From Eutrema salsugineum SEQ ID NO: 313 Amino acid sequence of Transporter T5_AlyPOT_GA From Arabidopsis lyrata subsp. lyrata SEQ ID NO: 314 DNA coding sequence of Transporter T5_AlyPOT_GA From Arabidopsis lyrata subsp. lyrata SEQ ID NO: 315 Amino acid sequence of Transporter T6_CruGTR_GA From Capsella rubella SEQ ID NO: 316 DNA coding sequence of Transporter T6_CruGTR_GA From Capsella rubella SEQ ID NO: 317 Amino acid sequence of Transporter T7_PtrPOT_GA From Populus trichocarpa SEQ ID NO: 318 DNA coding sequence of Transporter T7_PtrPOT_GA From Populus trichocarpa SEQ ID NO: 319 Amino acid sequence of Transporter T8_BnaMFS_GA From Brassica napus SEQ ID NO: 320 DNA coding sequence of Transporter T8_BnaMFS_GA From Brassica napus SEQ ID NO: 321 Amino acid sequence of Transporter T10_BolGTR_GA From Brassica oleracea var. oleracea SEQ ID NO: 322 DNA coding sequence of Transporter T10_BolGTR_GA From Brassica oleracea var. oleracea SEQ ID NO: 323 Amino acid sequence of Transporter Tll_AthGTRl_GA From Arabidopsis thaliana SEQ ID NO: 324 DNA coding sequence of Transporter Tll_AthGTRl_GA From Arabidopsis thaliana SEQ ID NO: 325 Amino acid sequence of Transporter T12_PsoNPFl_GA From Papaver somniferum SEQ ID NO: 326 DNA coding sequence of Transporter T12_PsoNPFl_GA From Papaver somniferum SEQ ID NO: 327 Amino acid sequence of Transporter T14_PsoNPF3_GA From Papaver somniferum SEQ ID NO: 328 DNA coding sequence of Transporter T14_PsoNPF3_GA From Papaver somniferum SEQ ID NO: 329 Amino acid sequence of Transporter T15_PsoNPF4_GA From Papaver somniferum SEQ ID NO: 330 DNA coding sequence of Transporter T15_PsoNPF4_GA From Papaver somniferum SEQ ID NO: 331 Amino acid sequence of Transporter T17_PsoNPF6_GA From Papaver somniferum SEQ ID NO: 332 DNA coding sequence of Transporter T17_PsoNPF6_GA From Papaver somniferum SEQ ID NO: 333 Amino acid sequence of Transporter T18_PsoNPF7_GA From Papaver somniferum SEQ ID NO: 334 DNA coding sequence of Transporter T18_PsoNPF7_GA From Papaver somniferum SEQ ID NO: 335 Amino acid sequence of Transporter T19_RmiPTR2_GA From Rhizopus microsporus SEQ ID NO: 336 DNA coding sequence of Transporter T19_RmiPTR2_GA From Rhizopus microsporus SEQ ID NO: 337 Amino acid sequence of Transporter T20_RmiPTR2_v2_GA From Rhizopus microsporus SEQ ID NO: 338 DNA coding sequence of Transporter T20_RmiPTR2_v2_GA From Rhizopus microsporus SEQ ID NO: 339 Amino acid sequence of Transporter T21_RalPTR2_GA From Rozella allomycis SEQ ID NO: 340 DNA coding sequence of Transporter T21_RalPTR2_GA From Rozella allomycis SEQ ID NO: 341 Amino acid sequence of Transporter T22_CanPOT_GA From Catenaria anguillulae SEQ ID NO: 342 DNA coding sequence of Transporter T22_CanPOT_GA From Catenaria anguillulae SEQ ID NO: 343 Amino acid sequence of Transporter T23_ArePOT_GA From Absidia repens SEQ ID NO: 344 DNA coding sequence of Transporter T23_ArePOT_GA From Absidia repens SEQ ID NO: 345 Amino acid sequence of Transporter T24_SlyPTR2_GA From Stemphylium lycopersici SEQ ID NO: 346 DNA coding sequence of Transporter T24_SlyPTR2_GA From Stemphylium lycopersici SEQ ID NO: 347 Amino acid sequence of Transporter T25_AorPOT_GA From Aspergillus oryzae SEQ ID NO: 348 DNA coding sequence of Transporter T25_AorPOT_GA From Aspergillus oryzae SEQ ID NO: 349 Amino acid sequence of Transporter T26_NfuPOT_GA From Neosartorya fumigata SEQ ID NO: 350 DNA coding sequence of Transporter T26_NfuPOT_GA From Neosartorya fumigata SEQ ID NO: 351 Amino acid sequence of Transporter T27_FoxPOT_GA From Fusarium oxysporum SEQ ID NO: 352 DNA coding sequence of Transporter T27_FoxPOT_GA From Fusarium oxysporum SEQ ID NO: 353 Amino acid sequence of Transporter T28_MciPOT_GA From Mucor circinelloides f. circinelloides SEQ ID NO: 354 DNA coding sequence of Transporter T28_MciPOT_GA From Mucor circinelloides f. circinelloides SEQ ID NO: 355 Amino acid sequence of Transporter T29_AcaPOT_GA From Aspergillus calidoustus SEQ ID NO: 356 DNA coding sequence of Transporter T29_AcaPOT_GA From Aspergillus calidoustus SEQ ID NO: 357 Amino acid sequence of Transporter T30_MlyPQT_GA From Microbotryum lychnidis-dioicae SEQ ID NO: 358 DNA coding sequence of Transporter T30_MlyPQT_GA From Microbotryum lychnidis-dioicae SEQ ID NO: 359 Amino acid sequence of Transporter T31_TgaPOT_GA From Trichoderma gamsii SEQ ID NO: 360 DNA coding sequence of Transporter T31_TgaPOT_GA From Trichoderma gamsii SEQ ID NO: 361 Amino acid sequence of Transporter T32_AarPOT_GA From Aspergillus arachidicola SEQ ID NO: 362 DNA coding sequence of Transporter T32_AarPOT_GA From Aspergillus arachidicola SEQ ID NO: 363 Amino acid sequence of Transporter T33_CcuPTR2_GA From Choanephora cucurbitarum SEQ ID NO: 364 DNA coding sequence of Transporter T33_CcuPTR2_GA From Choanephora cucurbitarum SEQ ID NO: 365 Amino acid sequence of Transporter T34_HvePOT_GA From Hesseltinel la vesiculosa SEQ ID NO: 366 DNA coding sequence of Transporter T34_HvePOT_GA From Hesseltinel la vesiculosa SEQ ID NO: 367 Amino acid sequence of Transporter T35_EcuPOT_GA From Encephalitozoon cuniculi SEQ ID NO: 368 DNA coding sequence of Transporter T35_EcuPOT_GA From Encephalitozoon cuniculi SEQ ID NO: 369 Amino acid sequence of Transporter T36_RnePOT_GA From Rosellinia necatrix SEQ ID NO: 370 DNA coding sequence of Transporter T36_RnePOT_GA From Rosellinia necatrix SEQ ID NO: 371 Amino acid sequence of Transporter T37_OcoPOT_GA From Ordospora colligata SEQ ID NO: 372 DNA coding sequence of Transporter T37_OcoPOT_GA From Ordospora colligata SEQ ID NO: 373 Amino acid sequence of Transporter T38_ScuPTR2_GA From Smittium culicis SEQ ID NO: 374 DNA coding sequence of Transporter T38_ScuPTR2_GA From Smittium culicis SEQ ID NO: 375 Amino acid sequence of Transporter T39_CgrPOT_GA From Colletotrichum graminicola SEQ ID NO: 376 DNA coding sequence of Transporter T39_CgrPOT_GA From Colletotrichum graminicola SEQ ID NO: 377 Amino acid sequence of Transporter T40_EdePQT_GA From Exophiala dermatitidis SEQ ID NO: 378 DNA coding sequence of Transporter T40_EdePQT_GA From Exophiala dermatitidis SEQ ID NO: 379 Amino acid sequence of Transporter T41_CalPTR2_GA From Candida albicans SEQ ID NO: 380 DNA coding sequence of Transporter T41_CalPTR2_GA From Candida albicans SEQ ID NO: 381 Amino acid sequence of Transporter T44_CcaMFS_GA From Cajanus cajan SEQ ID NO: 382 DNA coding sequence of Transporter T44_CcaMFS_GA From Cajanus cajan SEQ ID NO: 383 Amino acid sequence of Transporter T45_PanPOT_GA From Parasponia andersonii SEQ ID NO: 384 DNA coding sequence of Transporter T45_PanPOT_GA From Parasponia andersonii SEQ ID NO: 385 Amino acid sequence of Transporter T46_RchPOT_GA From Rosa chinensis SEQ ID NO: 386 DNA coding sequence of Transporter T46_RchPOT_GA From Rosa chinensis SEQ ID NO: 387 Amino acid sequence of Transporter T47_PbeNPF_GA From Pyrus betulifolia SEQ ID NO: 388 DNA coding sequence of Transporter T47_PbeNPF_GA From Pyrus betulifolia SEQ ID NO: 389 Amino acid sequence of Transporter T48_CcaPOT_GA From Corchoruscapsularis SEQ ID NO: 390 DNA coding sequence of Transporter T48_CcaPOT_GA From Corchoruscapsularis SEQ ID NO: 391 Amino acid sequence of Transporter T49_HanPOT_GA From Helianthus annuus SEQ ID NO: 392 DNA coding sequence of Transporter T49_HanPOT_GA From Helianthus annuus SEQ ID NO: 393 Amino acid sequence of Transporter T50_HimPQT_GA From Handroanthus impetiginosus SEQ ID NO: 394 DNA coding sequence of Transporter T50_HimPQT_GA From Handroanthus impetiginosus SEQ ID NO: 395 Amino acid sequence of Transporter T51_TorPOT_GA From Trema orientalis SEQ ID NO: 396 DNA coding sequence of Transporter T51_TorPOT_GA From Trema orientalis SEQ ID NO: 397 Amino acid sequence of Transporter T52_BmePTR2_GA From Basidiobolus meristosporus SEQ ID NO: 398 DNA coding sequence of Transporter T52_BmePTR2_GA From Basidiobolus meristosporus SEQ ID NO: 399 Amino acid sequence of Transporter T53_EhePOT_GA From Encephalitozoon hellem SEQ ID NO: 400 DNA coding sequence of Transporter T53_EhePOT_GA From Encephalitozoon hellem SEQ ID NO: 401 Amino acid sequence of Transporter T54_MelPOT_GA From Mortierella elongata SEQ ID NO: 402 DNA coding sequence of Transporter T54_MelPOT_GA From Mortierella elongata SEQ ID NO: 403 Amino acid sequence of Transporter T55_NsyNPF_GA From Nicotiana sylvestris SEQ ID NO: 404 DNA coding sequence of Transporter T55_NsyNPF_GA From Nicotiana sylvestris SEQ ID NO: 405 Amino acid sequence of Transporter T56_CanNPF_GA From Capsicum annuum SEQ ID NO: 406 DNA coding sequence of Transporter T56_CanNPF_GA From Capsicum annuum SEQ ID NO: 407 Amino acid sequence of Transporter T57_AcoNPF_GA From Aquilegia coerulea SEQ ID NO: 408 DNA coding sequence of Transporter T57_AcoNPF_GA From Aquilegia coerulea SEQ ID NO: 409 Amino acid sequence of Transporter T59_AmeNPFl_GA From Argemone mexican SEQ ID NO: 410 DNA coding sequence of Transporter T59_AmeNPFl_GA From Argemone mexican SEQ ID NO: 411 Amino acid sequence of Transporter T60_AmeNPF2_GA From Argemone mexican SEQ ID NO: 412 DNA coding sequence of Transporter T60_AmeNPF2_GA From Argemone mexican SEQ ID NO: 413 Amino acid sequence of Transporter T61_TwiNPF_GA From Tripterygium wilfordii SEQ ID NO: 414 DNA coding sequence of Transporter T61_TwiNPF_GA From Tripterygium wilfordii SEQ ID NO: 415 Amino acid sequence of Transporter T62_SmaNPF_GA From Swietenia mahagoni SEQ ID NO: 416 DNA coding sequence of Transporter T62_SmaNPF_GA From Swietenia mahagoni SEQ ID NO: 417 Amino acid sequence of Transporter T63_CfoNPF_GA From Coleus forskoh lii SEQ ID NO: 418 DNA coding sequence of Transporter T63_CfoNPF_GA From Coleus forskoh lii SEQ ID NO: 419 Amino acid sequence of Transporter T64_XsiNPF_GA From Xanthorhiza simplicissima SEQ ID NO: 420 DNA coding sequence of Transporter T64_XsiNPF_GA From Xanthorhiza simplicissima SEQ ID NO: 421 Amino acid sequence of Transporter T66_TelNPF_GA From Tabernaemontana elegans SEQ ID NO: 422 DNA coding sequence of Transporter T66_TelNPF_GA From Tabernaemontana elegans SEQ ID NO: 423 Amino acid sequence of Transporter T67_SdiNPF_GA From Stylophorum diphyllum SEQ ID NO: 424 DNA coding sequence of Transporter T67_SdiNPF_GA From Stylophorum diphyllum SEQ ID NO: 425 Amino acid sequence of Transporter T68_RseNPF_GA From Rauwolfia serpentina SEQ ID NO: 426 DNA coding sequence of Transporter T68_RseNPF_GA From Rauwolfia serpentina SEQ ID NO: 427 Amino acid sequence of Transporter T69_PhoNPF_GA From pelargonium      x hortorum SEQ ID NO: 428 DNA coding sequence of Transporter T69_PhoNPF_GA From pelargonium      x hortorum SEQ ID NO: 429 Amino acid sequence of Transporter T70_CmaNPF_GA From Chelidonium majus SEQ ID NO: 430 DNA coding sequence of Transporter T70_CmaNPF_GA From Chelidonium majus SEQ ID NO: 431 Amino acid sequence of Transporter T71_CchNPF_GA From Corydalis chelanthifolia SEQ ID NO: 432 DNA coding sequence of Transporter T71_CchNPF_GA From Corydalis chelanthifolia SEQ ID NO: 433 Amino acid sequence of Transporter T72_TcoNPF_GA From Tinospora_cordifolia SEQ ID NO: 434 DNA coding sequence of Transporter T72_TcoNPF_GA From Tinospora_cordifolia SEQ ID NO: 435 Amino acid sequence of Transporter T73_PbrNPFl_GA From Papaver bracteatum SEQ ID NO: 436 DNA coding sequence of Transporter T73_PbrNPFl_GA From Papaver bracteatum SEQ ID NO: 437 Amino acid sequence of Transporter T74_PbrNPF2_GA From Papaver bracteatum SEQ ID NO: 438 DNA coding sequence of Transporter T74_PbrNPF2_GA From Papaver bracteatum SEQ ID NO: 439 Amino acid sequence of Transporter T75_PbrNPF3_GA From Papaver bracteatum SEQ ID NO: 440 DNA coding sequence of Transporter T75_PbrNPF3_GA From Papaver bracteatum SEQ ID NO: 441 Amino acid sequence of Transporter T76_AhuNPF_GA From Amsonia hubrichtii SEQ ID NO: 442 DNA coding sequence of Transporter T76_AhuNPF_GA From Amsonia hubrichtii SEQ ID NO: 443 Amino acid sequence of Transporter T77_PocNPF_GA From Platanus occidentals SEQ ID NO: 444 DNA coding sequence of Transporter T77_PocNPF_GA From Platanus occidentals SEQ ID NO: 445 Amino acid sequence of Transporter T78_VofNPF_GA From Valeriana officinalis SEQ ID NO: 446 DNA coding sequence of Transporter T78_VofNPF_GA From Valeriana officinalis SEQ ID NO: 447 Amino acid sequence of Transporter T79_EcaNPF_GA From Eschscholzia californica SEQ ID NO: 448 DNA coding sequence of Transporter T79_EcaNPF_GA From Eschscholzia californica SEQ ID NO: 449 Amino acid sequence of Transporter T80_CroNPF_GA From Catharanthus roseus SEQ ID NO: 450 DNA coding sequence of Transporter T80_CroNPF_GA From Catharanthus roseus SEQ ID NO: 451 Amino acid sequence of Transporter T81_HcaNPF_GA From Hypericum perforatum SEQ ID NO: 452 DNA coding sequence of Transporter T81_HcaNPF_GA From Hypericum perforatum SEQ ID NO: 453 Amino acid sequence of Transporter T82_NsaNPF_GA From Nigella sativa SEQ ID NO: 454 DNA coding sequence of Transporter T82_NsaNPF_GA From Nigella sativa SEQ ID NO: 455 Amino acid sequence of Transporter T83_ScaNPF_GA From Sanguinaria canadensis SEQ ID NO: 456 DNA coding sequence of Transporter T83_ScaNPF_GA From Sanguinaria canadensis SEQ ID NO: 457 Amino acid sequence of Transporter T84_TflNPF_GA From Thalictrum flavum SEQ ID NO: 458 DNA coding sequence of Transporter T84_TflNPF_GA From Thalictrum flavum SEQ ID NO: 459 Amino acid sequence of Transporter T85_GflNPF_GA From Glaucium Flavum SEQ ID NO: 460 DNA coding sequence of Transporter T85_GflNPF_GA From Glaucium Flavum SEQ ID NO: 461 Amino acid sequence of Transporter T97_ScaT14_GA From Sanguinaria canadensis SEQ ID NO: 462 DNA coding sequence of Transporter T97_ScaT14_GA From Sanguinaria canadensis SEQ ID NO: 463 Amino acid sequence of Transporter T101_McoPUP3_l From Macleaya cordata SEQ ID NO: 464 DNA coding sequence of Transporter T101_McoPUP3_l From Macleaya cordata SEQ ID NO: 465 Amino acid sequence of Transporter T102_PsoPUP3_l From Papaver somniferum SEQ ID NO: 466 DNA coding sequence of Transporter T102_PsoPUP3_l From Papaver somniferum SEQ ID NO: 467 Amino acid sequence of Transporter T103_PsoPUP3_2 From Papaver somniferum SEQ ID NO: 468 DNA coding sequence of Transporter T103_PsoPUP3_2 From Papaver somniferum SEQ ID NO: 469 Amino acid sequence of Transporter T104_PsoPUP3_3 From Papaver somniferum SEQ ID NO: 470 DNA coding sequence of Transporter T104_PsoPUP3_3 From Papaver somniferum SEQ ID NO: 471 Amino acid sequence of Transporter T105_PsoPUP-L From Papaver somniferum SEQ ID NO: 472 DNA coding sequence of Transporter T105_PsoPUP-L From Papaver somniferum SEQ ID NO: 473 Amino acid sequence of Transporter T109_GflPUP3_83 From Glaucium Flavum SEQ ID NO: 474 DNA coding sequence of Transporter T109_GflPUP3_83 From Glaucium Flavum SEQ ID NO: 475 Amino acid sequence of Transporter T113_PsoPUP3_32 From Papaver somniferum SEQ ID NO: 476 DNA coding sequence of Transporter T113_PsoPUP3_32 From Papaver somniferum SEQ ID NO: 477 Amino acid sequence of Transporter T114_TorPUP3_40 From Trema orientale SEQ ID NO: 478 DNA coding sequence of Transporter T114_TorPUP3_40 From Trema orientale SEQ ID NO: 479 Amino acid sequence of Transporter T115_CsaPUP3_48 From Cucumis sativus SEQ ID NO: 480 DNA coding sequence of Transporter T115_CsaPUP3_48 From Cucumis sativus SEQ ID NO: 481 Amino acid sequence of Transporter T116_HanPUP3_56 From Helianthus annuus SEQ ID NO: 482 DNA coding sequence of Transporter T116_HanPUP3_56 From Helianthus annuus SEQ ID NO: 483 Amino acid sequence of Transporter T117_MacPUP3_64 From Musa acuminata SEQ ID NO: 484 DNA coding sequence of Transporter T117_MacPUP3_64 From Musa acuminata SEQ ID NO: 485 Amino acid sequence of Transporter T121_NnuPUP3_9 From Nelumbo nucifera SEQ ID NO: 486 DNA coding sequence of Transporter T121_NnuPUP3_9 From Nelumbo nucifera SEQ ID NO: 487 Amino acid sequence of Transporter T122_PsoPUP3_17 From Papaver somniferum SEQ ID NO: 488 DNA coding sequence of Transporter T122_PsoPUP3_17 From Papaver somniferum SEQ ID NO: 489 Amino acid sequence of Transporter T123_PsoPUP3_25 From Papaver somniferum SEQ ID NO: 490 DNA coding sequence of Transporter T123_PsoPUP3_25 From Papaver somniferum SEQ ID NO: 491 Amino acid sequence of Transporter T124_PsoPUP3_33 From Papaver somniferum SEQ ID NO: 492 DNA coding sequence of Transporter T124_PsoPUP3_33 From Papaver somniferum SEQ ID NO: 493 Amino acid sequence of Transporter T125_JcuPUP3_41 From Jatropha curcas SEQ ID NO: 494 DNA coding sequence of Transporter T125_JcuPUP3_41 From Jatropha curcas SEQ ID NO: 495 Amino acid sequence of Transporter T126_CpePUP3_49 From Cucurbita     pepo subsp. pepo SEQ ID NO: 496 DNA coding sequence of Transporter T126_CpePUP3_49 From Cucurbita     pepo subsp. pepo SEQ ID NO: 497 Amino acid sequence of Transporter T127_LsaPUP3_57 From Lactuca sativa SEQ ID NO: 498 DNA coding sequence of Transporter T127_LsaPUP3_57 From Lactuca sativa SEQ ID NO: 499 Amino acid sequence of Transporter T128_PsoPUP3_65 From Papaver somniferum SEQ ID NO: 500 DNA coding sequence of Transporter T128_PsoPUP3_65 From Papaver somniferum SEQ ID NO: 501 Amino acid sequence of Transporter T129_PsoPUP3_73 From Papaver somniferum SEQ ID NO: 502 DNA coding sequence of Transporter T129_PsoPUP3_73 From Papaver somniferum SEQ ID NO: 503 Amino acid sequence of Transporter T130_NdoPUP3_89 From Nandina domestica SEQ ID NO: 504 DNA coding sequence of Transporter T130_NdoPUP3_89 From Nandina domestica SEQ ID NO: 505 Amino acid sequence of Transporter T131_PbrPUP3_81 From Papaver bracteatum SEQ ID NO: 506 DNA coding sequence of Transporter T131_PbrPUP3_81 From Papaver bracteatum SEQ ID NO: 507 Amino acid sequence of Transporter T132_CmiPUP3_10 From Cinnamomum micranthum      f. kanehirae SEQ ID NO: 508 DNA coding sequence of Transporter T132_CmiPUP3_10 From Cinnamomum micranthum      f. kanehirae SEQ ID NO: 509 Amino acid sequence of Transporter T133_PsoPUP3_18 From Papaver somniferum SEQ ID NO: 510 DNA coding sequence of Transporter T133_PsoPUP3_18 From Papaver somniferum SEQ ID NO: 511 Amino acid sequence of Transporter T135_PsoPUP_34 From Papaver somniferum SEQ ID NO: 512 DNA coding sequence of Transporter T135_PsoPUP_34 From Papaver somniferum SEQ ID NO: 513 Amino acid sequence of Transporter T136_RchPUP3_42 From Rosa chinensis SEQ ID NO: 514 DNA coding sequence of Transporter T136_RchPUP3_42 From Rosa chinensis SEQ ID NO: 515 Amino acid sequence of Transporter T137_EguPUP3_50 From Erythranthe guttata SEQ ID NO: 516 DNA coding sequence of Transporter T137_EguPUP3_50 From Erythranthe guttata SEQ ID NO: 517 Amino acid sequence of Transporter T138_AduPUP3_58 From Arachis duranensis SEQ ID NO: 518 DNA coding sequence of Transporter T138_AduPUP3_58 From Arachis duranensis SEQ ID NO: 519 Amino acid sequence of Transporter T139_PsoPUP3_66 From Papaver somniferum SEQ ID NO: 520 DNA coding sequence of Transporter T139_PsoPUP3_66 From Papaver somniferum SEQ ID NO: 521 Amino acid sequence of Transporter T140_PalPUP3_74 From Papaver alpinum SEQ ID NO: 522 DNA coding sequence of Transporter T140_PalPUP3_74 From Papaver alpinum SEQ ID NO: 523 Amino acid sequence of Transporter T141_EcaPUP3_88 From Eschscholzia californica SEQ ID NO: 524 DNA coding sequence of Transporter T141_EcaPUP3_88 From Eschscholzia californica SEQ ID NO: 525 Amino acid sequence of Transporter T142_McoPUP3_4 From Macleaya cordata SEQ ID NO: 526 DNA coding sequence of Transporter T142_McoPUP3_4 From Macleaya cordata SEQ ID NO: 527 Amino acid sequence of Transporter T143_CmiPUP3_ll From Cinnamomum micranthum      f. kanehirae SEQ ID NO: 528 DNA coding sequence of Transporter T143_CmiPUP3_ll From Cinnamomum micranthum      f. kanehirae SEQ ID NO: 529 Amino acid sequence of Transporter T144_PsoPUP3_19 From Papaver somniferum SEQ ID NO: 530 DNA coding sequence of Transporter T144_PsoPUP3_19 From Papaver somniferum SEQ ID NO: 531 Amino acid sequence of Transporter T146_PsoPUP_35 From Papaver somniferum SEQ ID NO: 532 DNA coding sequence of Transporter T146_PsoPUP_35 From Papaver somniferum SEQ ID NO: 533 Amino acid sequence of Transporter T147_MesPUP3_43 From Manihot esculenta SEQ ID NO: 534 DNA coding sequence of Transporter T147_MesPUP3_43 From Manihot esculenta SEQ ID NO: 535 Amino acid sequence of Transporter T148_HimPUP3_51 From Handroanthus impetiginosus SEQ ID NO: 536 DNA coding sequence of Transporter T148_HimPUP3_51 From Handroanthus impetiginosus SEQ ID NO: 537 Amino acid sequence of Transporter T149_AcoPUP3_59 From Aquilegia coerulea SEQ ID NO: 538 DNA coding sequence of Transporter T149_AcoPUP3_59 From Aquilegia coerulea SEQ ID NO: 539 Amino acid sequence of Transporter T150_PsoPUP3_67 From Papaver somniferum SEQ ID NO: 540 DNA coding sequence of Transporter T150_PsoPUP3_67 From Papaver somniferum SEQ ID NO: 541 Amino acid sequence of Transporter T151_PatPUP3_75 From Papaver atlanticum SEQ ID NO: 542 DNA coding sequence of Transporter T151_PatPUP3_75 From Papaver atlanticum SEQ ID NO: 543 Amino acid sequence of Transporter T152_GflPUP3_87 From Glaucium Flavum SEQ ID NO: 544 DNA coding sequence of Transporter T152_GflPUP3_87 From Glaucium Flavum SEQ ID NO: 545 Amino acid sequence of Transporter T153_PsoPUP3_5 From Papaver somniferum SEQ ID NO: 546 DNA coding sequence of Transporter T153_PsoPUP3_5 From Papaver somniferum SEQ ID NO: 547 Amino acid sequence of Transporter T154_CmiPUP3_12 From Cinnamomum micranthum      f. kanehirae SEQ ID NO: 548 DNA coding sequence of Transporter T154_CmiPUP3_12 From Cinnamomum micranthum      f. kanehirae SEQ ID NO: 549 Amino acid sequence of Transporter T156_PsoPUP3_28 From Papaver somniferum SEQ ID NO: 550 DNA coding sequence of Transporter T156_PsoPUP3_28 From Papaver somniferum SEQ ID NO: 551 Amino acid sequence of Transporter T157_RchPUP_36 From Rosa chinensis SEQ ID NO: 552 DNA coding sequence of Transporter T157_RchPUP_36 From Rosa chinensis SEQ ID NO: 553 Amino acid sequence of Transporter T158_DziPUP3_44 From Durio zibethinus SEQ ID NO: 554 DNA coding sequence of Transporter T158_DziPUP3_44 From Durio zibethinus SEQ ID NO: 555 Amino acid sequence of Transporter T159_OeuPUP3_52 From Olea europaea var. sylvestris SEQ ID NO: 556 DNA coding sequence of Transporter T159_OeuPUP3_52 From Olea europaea var. sylvestris SEQ ID NO: 557 Amino acid sequence of Transporter T160_CeuPUP3_60 From Coffea eugenioides SEQ ID NO: 558 DNA coding sequence of Transporter T160_CeuPUP3_60 From Coffea eugenioides SEQ ID NO: 559 Amino acid sequence of Transporter T161_PsoPUP3_68 From Papaver somniferum SEQ ID NO: 560 DNA coding sequence of Transporter T161_PsoPUP3_68 From Papaver somniferum SEQ ID NO: 561 Amino acid sequence of Transporter T162_PmiPUP3_76 From Papaver miyabeanum SEQ ID NO: 562 DNA coding sequence of Transporter T162_PmiPUP3_76 From Papaver miyabeanum SEQ ID NO: 563 Amino acid sequence of Transporter T163_PbrPUP3_86 From Papaver bracteatum SEQ ID NO: 564 DNA coding sequence of Transporter T163_PbrPUP3_86 From Papaver bracteatum SEQ ID NO: 565 Amino acid sequence of Transporter T164_PsoPUP3_78 From Papaver somniferum SEQ ID NO: 566 DNA coding sequence of Transporter T164_PsoPUP3_78 From Papaver somniferum SEQ ID NO: 567 Amino acid sequence of Transporter T165_AcoPUP3_13 From Aquilegia coerulea SEQ ID NO: 568 DNA coding sequence of Transporter T165_AcoPUP3_13 From Aquilegia coerulea SEQ ID NO: 569 Amino acid sequence of Transporter T166_PsoPUP3_21 From Papaver somniferum SEQ ID NO: 570 DNA coding sequence of Transporter T166_PsoPUP3_21 From Papaver somniferum SEQ ID NO: 571 Amino acid sequence of Transporter T168_FvePUP3_37 From Fragaria      vesca subsp. vesca SEQ ID NO: 572 DNA coding sequence of Transporter T168_FvePUP3_37 From Fragaria      vesca subsp. vesca SEQ ID NO: 573 Amino acid sequence of Transporter T169_ZjuPUP3_45 From Ziziphus jujuba SEQ ID NO: 574 DNA coding sequence of Transporter T169_ZjuPUP3_45 From Ziziphus jujuba SEQ ID NO: 575 Amino acid sequence of Transporter T170_LsaPUP3_53 From Lactuca sativa SEQ ID NO: 576 DNA coding sequence of Transporter T170_LsaPUP3_53 From Lactuca sativa SEQ ID NO: 577 Amino acid sequence of Transporter T171_McoPUP3_61 From Macleaya cordata SEQ ID NO: 578 DNA coding sequence of Transporter T171_McoPUP3_61 From Macleaya cordata SEQ ID NO: 579 Amino acid sequence of Transporter T172_AcoPUP3_69 From Aquilegia coerulea SEQ ID NO: 580 DNA coding sequence of Transporter T172_AcoPUP3_69 From Aquilegia coerulea SEQ ID NO: 581 Amino acid sequence of Transporter T173_PnuPUP3_77 From Papaver nudicale SEQ ID NO: 582 DNA coding sequence of Transporter T173_PnuPUP3_77 From Papaver nudicale SEQ ID NO: 583 Amino acid sequence of Transporter T174_PbrPUP3_85 From Papaver bracteatum SEQ ID NO: 584 DNA coding sequence of Transporter T174_PbrPUP3_85 From Papaver bracteatum SEQ ID NO: 585 Amino acid sequence of Transporter T175_PsoPUP3_6 From Papaver somniferum SEQ ID NO: 586 DNA coding sequence of Transporter T175_PsoPUP3_6 From Papaver somniferum SEQ ID NO: 587 Amino acid sequence of Transporter T176_AcoPUP3_14 From Aquilegia coerulea SEQ ID NO: 588 DNA coding sequence of Transporter T176_AcoPUP3_14 From Aquilegia coerulea SEQ ID NO: 589 Amino acid sequence of Transporter T177_PsoPUP3_22 From Papaver somniferum SEQ ID NO: 590 DNA coding sequence of Transporter T177_PsoPUP3_22 From Papaver somniferum SEQ ID NO: 591 Amino acid sequence of Transporter T178_PsoPUP3_30 From Papaver somniferum SEQ ID NO: 592 DNA coding sequence of Transporter T178_PsoPUP3_30 From Papaver somniferum SEQ ID NO: 593 Amino acid sequence of Transporter T179_PyePUP3_38 From Prunus yedoensis var. nudiflora SEQ ID NO: 594 DNA coding sequence of Transporter T179_PyePUP3_38 From Prunus yedoensis var. nudiflora SEQ ID NO: 595 Amino acid sequence of Transporter T180_McoPUP3_46 From Macleaya cordata SEQ ID NO: 596 DNA coding sequence of Transporter T180_McoPUP3_46 From Macleaya cordata SEQ ID NO: 597 Amino acid sequence of Transporter T181_HanPUP3_54 From Helianthus annuus SEQ ID NO: 598 DNA coding sequence of Transporter T181_HanPUP3_54 From Helianthus annuus SEQ ID NO: 599 Amino acid sequence of Transporter T182_CpaPUP3_62 From Carica papaya SEQ ID NO: 600 DNA coding sequence of Transporter T182_CpaPUP3_62 From Carica papaya SEQ ID NO: 601 Amino acid sequence of Transporter T184_PraPUP3_79 From Papaver radicatum SEQ ID NO: 602 DNA coding sequence of Transporter T184_PraPUP3_79 From Papaver radicatum SEQ ID NO: 603 Amino acid sequence of Transporter T186_ScaPUP3_84 From Sanguinaria canadensis SEQ ID NO: 604 DNA coding sequence of Transporter T186_ScaPUP3_84 From Sanguinaria canadensis SEQ ID NO: 605 Amino acid sequence of Transporter T188_AcoPUP3_15 From Aquilegia coerulea SEQ ID NO: 606 DNA coding sequence of Transporter T188_AcoPUP3_15 From Aquilegia coerulea SEQ ID NO: 607 Amino acid sequence of Transporter T189_PsoPUP3_23 From Papaver somniferum SEQ ID NO: 608 DNA coding sequence of Transporter T189_PsoPUP3_23 From Papaver somniferum SEQ ID NO: 609 Amino acid sequence of Transporter T191_MdoPUP3_39 From Malus domestica SEQ ID NO: 610 DNA coding sequence of Transporter T191_MdoPUP3_39 From Malus domestica SEQ ID NO: 611 Amino acid sequence of Transporter T192_CmiPUP3_47 From Cinnamomum micranthum      f. kanehirae SEQ ID NO: 612 DNA coding sequence of Transporter T192_CmiPUP3_47 From Cinnamomum micranthum      f. kanehirae SEQ ID NO: 613 Amino acid sequence of Transporter T193_AanPUP3_55 From Artemisia annua SEQ ID NO: 614 DNA coding sequence of Transporter T193_AanPUP3_55 From Artemisia annua SEQ ID NO: 615 Amino acid sequence of Transporter T194_CchPUP3_63 From Capsicum chinense SEQ ID NO: 616 DNA coding sequence of Transporter T194_CchPUP3_63 From Capsicum chinense SEQ ID NO: 617 Amino acid sequence of Transporter T195_JcuPUP3_71 From Jatropha curcas SEQ ID NO: 618 DNA coding sequence of Transporter T195_JcuPUP3_71 From Jatropha curcas SEQ ID NO: 619 Amino acid sequence of Transporter T196_PtrPUP3_80 From Papaver trinifolium SEQ ID NO: 620 DNA coding sequence of Transporter T196_PtrPUP3_80 From Papaver trinifolium SEQ ID NO: 621 Amino acid sequence of Transporter T197_AcoT97_GA From Aquilegia coerulea SEQ ID NO: 622 DNA coding sequence of Transporter T197_AcoT97_GA From Aquilegia coerulea SEQ ID NO: 623 Amino acid sequence of Transporter T198_AcoT97_GA From Aquilegia coerulea SEQ ID NO: 624 DNA coding sequence of Transporter T198_AcoT97_GA From Aquilegia coerulea SEQ ID NO: 625 Amino acid sequence of Transporter T199_NnuT97_GA From Nelumbo nucifera SEQ ID NO: 626 DNA coding sequence of Transporter T199_NnuT97_GA From Nelumbo nucifera SEQ ID NO: 627 Amino acid sequence of Transporter T200_T97_GA From Prunus yedoensis var. nudiflora SEQ ID NO: 628 DNA coding sequence of Transporter T200_T97_GA From Prunus yedoensis var. nudiflora SEQ ID NO: 629 Amino acid sequence of Transporter T201_HarPUP3_GA From Helicoverpa armigera SEQ ID NO: 630 DNA coding sequence of Transporter T201_HarPUP3_GA From Helicoverpa armigera SEQ ID NO: 631 Amino acid sequence of Transporter T202_PgoPUP3_GA From Pectinophora gossypiella SEQ ID NO: 632 DNA coding sequence of Transporter T202_PgoPUP3_GA From Pectinophora gossypiella SEQ ID NO: 633 Amino acid sequence of Transporter T203_HarPUP3_GA From Helicoverpa armigera SEQ ID NO: 634 DNA coding sequence of Transporter T203_HarPUP3_GA From Helicoverpa armigera SEQ ID NO: 635 Amino acid sequence of Transporter T204_RcoPUP3_GA From Ricinus communis SEQ ID NO: 636 DNA coding sequence of Transporter T204_RcoPUP3_GA From Ricinus communis SEQ ID NO: 637 Amino acid sequence of Transporter T205_HviPUP3_GA From Heliothis virescens SEQ ID NO: 638 DNA coding sequence of Transporter T205_HviPUP3_GA From Heliothis virescens SEQ ID NO: 639 Amino acid sequence of Transporter T206_VviPUP3_3_GA From Vitis vinifera SEQ ID NO: 640 DNA coding sequence of Transporter T206_VviPUP3_3_GA From Vitis vinifera SEQ ID NO: 641 Amino acid sequence of Transporter T207_MprPUP3_GA From Mucuna pruriens SEQ ID NO: 642 DNA coding sequence of Transporter T207_MprPUP3_GA From Mucuna pruriens SEQ ID NO: 643 Amino acid sequence of Transporter T208_McoPUP3_GA From Macleaya cordata SEQ ID NO: 644 DNA coding sequence of Transporter T208_McoPUP3_GA From Macleaya cordata SEQ ID NO: 645 Amino acid sequence of Transporter T209_RcoPUP3_GA From Ricinus communis SEQ ID NO: 646 DNA coding sequence of Transporter T209_RcoPUP3_GA From Ricinus communis SEQ ID NO: 647 Amino acid sequence of Transporter T210_NnuPUP3_GA From Nelumbo nucifera SEQ ID NO: 648 DNA coding sequence of Transporter T210_NnuPUP3_GA From Nelumbo nucifera SEQ ID NO: 649 Amino acid sequence of Transporter T211_HarPUP3_GA From Helicoverpa armigera SEQ ID NO: 650 DNA coding sequence of Transporter T211_HarPUP3_GA From Helicoverpa armigera SEQ ID NO: 651 Amino acid sequence of Transporter T212_HarPUP3_GA From Helicoverpa armigera SEQ ID NO: 652 DNA coding sequence of Transporter T212_HarPUP3_GA From Helicoverpa armigera SEQ ID NO: 653 Amino acid sequence of Transporter T213_HarPUP3_GA From Helicoverpa armigera SEQ ID NO: 654 DNA coding sequence of Transporter T213_HarPUP3_GA From Helicoverpa armigera SEQ ID NO: 655 Amino acid sequence of Transporter T214_HarPUP3_GA From Helicoverpa armigera SEQ ID NO: 656 DNA coding sequence of Transporter T214_HarPUP3_GA From Helicoverpa armigera SEQ ID NO: 657 Amino acid sequence of Transporter T215_HarPUP3_GA From Helicoverpa armigera SEQ ID NO: 658 DNA coding sequence of Transporter T215_HarPUP3_GA From Helicoverpa armigera SEQ ID NO: 659 Amino acid sequence of Transporter T216_HarPUP3_GA From Helicoverpa armigera SEQ ID NO: 660 DNA coding sequence of Transporter T216_HarPUP3_GA From Helicoverpa armigera SEQ ID NO: 661 Amino acid sequence of Transporter T217_AcoPUP3_GA From Aquilegia coerulea SEQ ID NO: 662 DNA coding sequence of Transporter T217_AcoPUP3_GA From Aquilegia coerulea SEQ ID NO: 733 Amino acid sequence of Transporter T65_ljaNPF_GA From Lonicera japonica SEQ ID NO: 734 DNA coding sequence of Transporter T65_ljaNPF_GA From Lonicera japonica SEQ ID NO: 735 Amino acid sequence of Transporter T94_EcrPOT_GA From Emmonsia crescens SEQ ID NO: 736 DNA coding sequence of Transporter T94_EcrPOT_GA From Emmonsia crescens SEQ ID NO: 663 Amino acid sequence of ADH5 Dehydrogenase ADH5 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 664 DNA coding sequence of ADH5 Dehydrogenase ADH5 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 665 Amino acid sequence of ADH6 Dehydrogenase ADH6 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 666 DNA coding sequence of ADH6 Dehydrogenase ADH6 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 667 Amino acid sequence of ADH7 Dehydrogenase ADH7 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 668 DNA coding sequence of ADH7 Dehydrogenase ADH7 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 669 Amino acid sequence of YPR127W Dehydrogenase YPR127W Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 670 DNA coding sequence of YPR127W Dehydrogenase YPR127W Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 671 Amino acid sequence of AAD3 Dehydrogenase AAD3 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 672 DNA coding sequence of AAD3 Dehydrogenase AAD3 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 673 Amino acid sequence of AAD4 Dehydrogenase AAD4 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 674 DNA coding sequence of AAD4 Dehydrogenase AAD4 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 675 Amino acid sequence of ADH3 Dehydrogenase ADH3 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 676 DNA coding sequence of ADH3 Dehydrogenase ADH3 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 677 Amino acid sequence of ADH4 Dehydrogenase ADH4 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 678 DNA coding sequence of ADH4 Dehydrogenase ADH4 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 679 Amino acid sequence of ALD6 Dehydrogenase ALD6 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 680 DNA coding sequence of ALD6 Dehydrogenase ALD6 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 681 Amino acid sequence of BDH1 Dehydrogenase BDH1 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 682 DNA coding sequence of BDH1 Dehydrogenase BDH1 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 683 Amino acid sequence of BDH2 Dehydrogenase BDH2 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 684 DNA coding sequence of BDH2 Dehydrogenase BDH2 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 685 Amino acid sequence of FOX2 Dehydrogenase FOX2 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 686 DNA coding sequence of FOX2 Dehydrogenase FOX2 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 687 Amino acid sequence of GCY1 Dehydrogenase GCY1 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 688 DNA coding sequence of GCY1 Dehydrogenase GCY1 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 689 Amino acid sequence of GPD1 Dehydrogenase GPD1 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 690 DNA coding sequence of GPD1 Dehydrogenase GPD1 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 691 Amino acid sequence of HIS4 Dehydrogenase HIS4 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 692 DNA coding sequence of HIS4 Dehydrogenase HIS4 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 693 Amino acid sequence of IPD1 Dehydrogenase IPD1 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 694 DNA coding sequence of IPD1 Dehydrogenase IPD1 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 695 Amino acid sequence of LYS12 Dehydrogenase LYS12 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 696 DNA coding sequence of LYS12 Dehydrogenase LYS12 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 697 Amino acid sequence of SER33 Dehydrogenase SER33 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 698 DNA coding sequence of SER33 Dehydrogenase SER33 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 699 Amino acid sequence of ZWF1 Dehydrogenase ZWF1 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 700 DNA coding sequence of ZWF1 Dehydrogenase ZWF1 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 701 Amino acid sequence of YPL088W Dehydrogenase YPL088W Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 702 DNA coding sequence of YPL088W Dehydrogenase YPL088W Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 703 Amino acid sequence of ARAI Dehydrogenase ARAI Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 704 DNA coding sequence of ARAI Dehydrogenase ARAI Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 705 Amino acid sequence of HFD1 Dehydrogenase HFD1 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 706 DNA coding sequence of HFD1 Dehydrogenase HFD1 Dehydrogenase From Saccharomyces cerevisiae SEQ ID NO: 707 Amino acid sequence of YPR1 Reductase YPR1 Reductase From Saccharomyces cerevisiae SEQ ID NO: 708 DNA coding sequence of YPR1 Reductase YPR1 Reductase From Saccharomyces cerevisiae SEQ ID NO: 709 Amino acid sequence of ALD4 Reductase ALD4 Reductase From Saccharomyces cerevisiae SEQ ID NO: 710 DNA coding sequence of ALD4 Reductase ALD4 Reductase From Saccharomyces cerevisiae SEQ ID NO: 711 Amino acid sequence of GORI Reductase GORI Reductase From Saccharomyces cerevisiae SEQ ID NO: 712 DNA coding sequence of GORI Reductase GORI Reductase From Saccharomyces cerevisiae SEQ ID NO: 713 Amino acid sequence of GRE2 Reductase GRE2 Reductase From Saccharomyces cerevisiae SEQ ID NO: 714 DNA coding sequence of GRE2 Reductase GRE2 Reductase From Saccharomyces cerevisiae SEQ ID NO: 715 Amino acid sequence of GRE3 Reductase GRE3 Reductase From Saccharomyces cerevisiae SEQ ID NO: 716 DNA coding sequence of GRE3 Reductase GRE3 Reductase From Saccharomyces cerevisiae SEQ ID NO: 717 Amino acid sequence of YDR541C Reductase YDR541C Reductase From Saccharomyces cerevisiae SEQ ID NO: 718 DNA coding sequence of YDR541C Reductase YDR541C Reductase From Saccharomyces cerevisiae SEQ ID NO: 719 Amino acid sequence of YLR460C Reductase YLR460C Reductase From Saccharomyces cerevisiae SEQ ID NO: 720 DNA coding sequence of YLR460C Reductase YLR460C Reductase From Saccharomyces cerevisiae SEQ ID NO: 721 Amino acid sequence of ARII Reductase ARII Reductase From Saccharomyces cerevisiae SEQ ID NO: 722 DNA coding sequence of ARII Reductase ARII Reductase From Saccharomyces cerevisiae SEQ ID NO: 723 Amino acid sequence of YGL039W Reductase YGL039W Reductase From Saccharomyces cerevisiae SEQ ID NO: 724 DNA coding sequence of YGL039W Reductase YGL039W Reductase From Saccharomyces cerevisiae SEQ ID NO: 725 Amino acid sequence of YCR102C Reductase YCR102C Reductase From Saccharomyces cerevisiae SEQ ID NO: 726 DNA coding sequence of YCR102C Reductase YCR102C Reductase From Saccharomyces cerevisiae SEQ ID NO: 727 Amino acid sequence of HMG1 Reductase HMG1 Reductase From Saccharomyces cerevisiae SEQ ID NO: 728 DNA coding sequence of HMG1 Reductase HMG1 Reductase From Saccharomyces cerevisiae SEQ ID NO: 729 Amino acid sequence of PHA2 Dehydratase PHA2 Dehydratase From Saccharomyces cerevisiae SEQ ID NO: 730 DNA coding sequence of PHA2 Dehydratase PHA2 Dehydratase From Saccharomyces cerevisiae SEQ ID NO: 731 Amino acid sequence of TRP3 Synthase TRP3 Synthase From Saccharomyces cerevisiae SEQ ID NO: 732 DNA coding sequence of TRP3 Synthase TRP3 Synthase From Saccharomyces cerevisiae SEQ ID NO: 737 DNA coding sequence of HEME cofactor HEM2 From Saccharomyces cerevisiae SEQ ID NO: 738 Amino acid sequence of HEME cofactor HEM2 From Saccharomyces cerevisiae SEQ ID NO: 739 DNA coding sequence of HEME cofactor HEM3 From Saccharomyces cerevisiae SEQ ID NO: 740 Amino acid sequence of HEME cofactor HEM3 From Saccharomyces cerevisiae SEQ ID NO: 741 DNA coding sequence of HEME cofactor HEM12 From Saccharomyces cerevisiae SEQ ID NO: 742 Amino acid sequence of HEME cofactor HEM12 From Saccharomyces cerevisiae SEQ ID NO: 743 DNA coding sequence of HEME cofactor HMX1 From Saccharomyces cerevisiae SEQ ID NO: 744 Amino acid sequence of HEME cofactor HMX1 From Saccharomyces cerevisiae SEQ ID NO: 745 DNA coding sequence of P450 chaperone KAR2 From Saccharomyces cerevisiae SEQ ID NO: 746 Amino acid sequence of P450 chaperone KAR2 From Saccharomyces cerevisiae SEQ ID NO: 747 DNA coding sequence of P450 chaperone HSP82 From Saccharomyces cerevisiae SEQ ID NO: 748 Amino acid sequence of P450 chaperone HSP82 From Saccharomyces cerevisiae SEQ ID NO: 749 DNA coding sequence of P450 chaperone CNE1 From Saccharomyces cerevisiae SEQ ID NO: 750 Amino acid sequence of P450 chaperone CNE1 From Saccharomyces cerevisiae SEQ ID NO: 751 DNA coding sequence of P450 chaperone SSA1 From Saccharomyces cerevisiae SEQ ID NO: 752 Amino acid sequence of P450 chaperone SSA1 From Saccharomyces cerevisiae SEQ ID NO: 753 DNA coding sequence of P450 chaperone CPR6 From Saccharomyces cerevisiae SEQ ID NO: 754 Amino acid sequence of P450 chaperone CPR6 From Saccharomyces cerevisiae SEQ ID NO: 755 DNA coding sequence of P450 chaperone FES1 From Saccharomyces cerevisiae SEQ ID NO: 756 Amino acid sequence of P450 chaperone FES1 From Saccharomyces cerevisiae SEQ ID NO: 757 DNA coding sequence of P450 chaperone HSP104 From Saccharomyces cerevisiae SEQ ID NO: 758 Amino acid sequence of P450 chaperone HSP104 From Saccharomyces cerevisiae SEQ ID NO: 759 DNA coding sequence of P450 chaperone STI1 From Saccharomyces cerevisiae SEQ ID NO: 760 Amino acid sequence of P450 chaperone STI1 From Saccharomyces cerevisiae SEQ ID NO: 761 DNA coding sequence of P450 regulator DAP1 From Saccharomyces cerevisiae SEQ ID NO: 762 Amino acid sequence of P450 regulator DAP1 From Saccharomyces cerevisiae SEQ ID NO: 763 DNA coding sequence of P450 regulator HAC1 From Saccharomyces cerevisiae SEQ ID NO: 764 Amino acid sequence of P450 regulator HAC1 From Saccharomyces cerevisiae SEQ ID NO: 765 DNA coding sequence of NADPH cofactor ZWF1 From Saccharomyces cerevisiae SEQ ID NO: 766 Amino acid sequence of NADPH cofactor ZWF1 From Saccharomyces cerevisiae SEQ ID NO: 767 DNA coding sequence of NADPH cofactor GND1 From Saccharomyces cerevisiae SEQ ID NO: 768 Amino acid sequence of NADPH cofactor GND1 From Saccharomyces cerevisiae SEQ ID NO: 769 DNA coding sequence of formadehyde toxicity regulator SFA1 From Saccharomyces cerevisiae SEQ ID NO: 770 Amino acid sequence of formadehyde toxicity regulator SFA1 From Saccharomyces cerevisiae SEQ ID NO: 771 DNA coding sequence of Moth       P450 (demethylase) mutant     codon optimized Hv_CYP_A0A2A4JAM9_A110N+H242P+V224l_co6 From Artificial SEQ ID NO: 772 Amino acid sequence of Moth       P450 (demethylase) mutant     codon optimized Hv_CYP_A0A2A4JAM9_A110N+H242P+V224l_co6 From Artificial SEQ ID NO: 773 DNA coding sequence of Transporter codon optimised T149_AcPUP3_59 _co2 From Artificial SEQ ID NO: 774 Amino acid sequence of Transporter T149_AcPUP3_59 _co2 From Aquilegia coerulea SEQ ID NO: 775 DNA coding sequence of Transporter codon optimised T149_AcPUP3_59 _co3 From Artificial SEQ ID NO: 776 Amino acid sequence of Transporter T149_AcPUP3_59 _co3 From Aquilegia coerulea SEQ ID NO: 777 DNA coding sequence of Transporter codon optimised T149_AcPUP3_59 _co4 From Artificial SEQ ID NO: 778 Amino acid sequence of Transporter T149_AcPUP3_59 _co4 From Aquilegia coerulea SEQ ID NO: 779 DNA coding sequence of Transporter codon optimised T180_McPUP3_46_co2 From Artificial SEQ ID NO: 780 Amino acid sequence of Transporter T180_McPUP3_46_co2 From Momordica charantia SEQ ID NO: 781 DNA coding sequence of Transporter codon optimised T180_McPUP3_46_co3 From Artificial SEQ ID NO: 782 Amino acid sequence of Transporter T180_McPUP3_46_co3 From Momordica charantia SEQ ID NO: 783 DNA coding sequence of Transporter codon optimised T180_McPUP3_46_co4 From Artificial SEQ ID NO: 784 Amino acid sequence of Transporter T180_McPUP3_46_co4 From Momordica charantia SEQ ID NO: 785 DNA coding sequence of Transporter codon optimised T180_McPUP3_46_co6 From Artificial SEQ ID NO: 786 Amino acid sequence of Transporter T180_McPUP3_46_co6 From Momordica charantia SEQ ID NO: 787 DNA coding sequence of Transporter codon optimised T193_AanPUP3_55 _co2 From Artificial SEQ ID NO: 788 Amino acid sequence of Transporter T193_AanPUP3_55 _co2 From Artemisia annua SEQ ID NO: 789 DNA coding sequence of Transporter codon optimised T193_AanPUP3_55 _co3 From Artificial SEQ ID NO: 790 Amino acid sequence of Transporter T193_AanPUP3_55 _co3 From Artemisia annua SEQ ID NO: 791 DNA coding sequence of Transporter codon optimised T193_AanPUP3_55 _co5 From Artificial SEQ ID NO: 792 Amino acid sequence of Transporter T193_AanPUP3_55 _co5 From Artemisia annua SEQ ID NO: 793 DNA coding sequence of Transporter codon optimised T193_AanPUP3_55 _co6 From Artificial SEQ ID NO: 794 Amino acid sequence of Transporter T193_AanPUP3_55 _co6 From Artemisia annua SEQ ID NO: 795 Amino acid sequence of Transporter T218_HviENT3_GA From Heliothis virescens SEQ ID NO: 796 DNA coding sequence of Transporter T218_HviENT3_GA From Heliothis virescens SEQ ID NO: 797 Amino acid sequence of Transporter T220_CsuENT3_GA From Chilo suppressalis SEQ ID NO: 798 DNA coding sequence of Transporter T220_CsuENT3_GA From Chilo suppressalis SEQ ID NO: 799 Amino acid sequence of Transporter T221_BmoENT3_GA From Bombyx mori SEQ ID NO: 800 DNA coding sequence of Transporter T221_BmoENT3_GA From Bombyx mori SEQ ID NO: 801 Amino acid sequence of Transporter T227_AcuENT3_GA From Anopheles culicifacies SEQ ID NO: 802 DNA coding sequence of Transporter T227_AcuENT3_GA From Anopheles culicifacies SEQ ID NO: 803 Amino acid sequence of Transporter T234_CsuENT3_GA From Chilo suppressalis SEQ ID NO: 804 DNA coding sequence of Transporter T234_CsuENT3_GA From Chilo suppressalis SEQ ID NO: 805 Amino acid sequence of Transporter T237_PxuENT3_GA From Papilio xuthus SEQ ID NO: 806 DNA coding sequence of Transporter T237_PxuENT3_GA From Papilio xuthus SEQ ID NO: 807 Amino acid sequence of Transporter T238_HviENT3_GA From Heliothis virescens SEQ ID NO: 808 DNA coding sequence of Transporter T238_HviENT3_GA From Heliothis virescens SEQ ID NO: 809 Amino acid sequence of Transporter T239_CmePUP3_GA From Cucumis melo var. makuwa SEQ ID NO: 810 DNA coding sequence of Transporter T239_CmePUP3_GA From Cucumis melo var. makuwa SEQ ID NO: 811 Amino acid sequence of Transporter T240_PpePUP3_GA From Prunus persica SEQ ID NO: 812 DNA coding sequence of Transporter T240_PpePUP3_GA From Prunus persica SEQ ID NO: 813 Amino acid sequence of Transporter T242_AchPUP3_GA From Actinidia chinensis var. chinensis SEQ ID NO: 814 DNA coding sequence of Transporter T242_AchPUP3_GA From Actinidia chinensis var. chinensis SEQ ID NO: 815 Amino acid sequence of Transporter T243_EguPUP3_GA From Erythranthe guttata SEQ ID NO: 816 DNA coding sequence of Transporter T243_EguPUP3_GA From Erythranthe guttata SEQ ID NO: 817 Amino acid sequence of Transporter T244_CcaPUP3_GA From Corchoruscapsularis SEQ ID NO: 818 DNA coding sequence of Transporter T244_CcaPUP3_GA From Corchoruscapsularis SEQ ID NO: 819 Amino acid sequence of Transporter T245_CcaPUP3_GA From Handroanthus impetiginosus SEQ ID NO: 820 DNA coding sequence of Transporter T245_CcaPUP3_GA From Handroanthus impetiginosus SEQ ID NO: 821 Amino acid sequence of Transporter T248_McoPUP3_GA From Macleaya cordata SEQ ID NO: 822 DNA coding sequence of Transporter T248_McoPUP3_GA From Macleaya cordata SEQ ID NO: 823 Amino acid sequence of Transporter T253_AanPUP3_GA From Artemisia annua SEQ ID NO: 824 DNA coding sequence of Transporter T253_AanPUP3_GA From Artemisia annua SEQ ID NO: 825 Amino acid sequence of Transporter T254_CcaPUP3_GA From Cynara cardunculus var. scolymus SEQ ID NO: 826 DNA coding sequence of Transporter T254_CcaPUP3_GA From Cynara cardunculus var. scolymus SEQ ID NO: 827 Amino acid sequence of P450 (demethylase) A0A286QUG7 From Spodoptera exigua SEQ ID NO: 828 DNA coding sequence of P450 (demethylase) A0A286QUG7 From Spodoptera exigua SEQ ID NO: 829 Amino acid sequence of P450 (demethylase) D5L0M5 From Manduca sexta SEQ ID NO: 830 DNA coding sequence of P450 (demethylase) D5L0M5 From Manduca sexta SEQ ID NO: 831 Amino acid sequence of P450 (demethylase) XP026740610 From Trichoplusia ni SEQ ID NO: 832 DNA coding sequence of P450 (demethylase) XP026740610 From Trichoplusia ni SEQ ID NO: 833 Amino acid sequence of P450 (demethylase) W5W4U7 From Lymantria dispar SEQ ID NO: 834 DNA coding sequence of P450 (demethylase) W5W4U7 From Lymantria dispar SEQ ID NO: 835 Amino acid sequence of P450 (demethylase) ACF17813 From Ostrinia furnacalis SEQ ID NO: 836 DNA coding sequence of P450 (demethylase) ACF17813 From Ostrinia furnacalis SEQ ID NO: 837 Amino acid sequence of P450 (demethylase) A0A4C1YMA7 From Eumeta variegata SEQ ID NO: 838 DNA coding sequence of P450 (demethylase) A0A4C1YMA7 From Eumeta variegata SEQ ID NO: 839 Amino acid sequence of P450 (demethylase) MsCPR_XP_030039194 From Manduca sexta SEQ ID NO: 840 DNA coding sequence of P450 (demethylase) MsCPR_XP_030039194 From Manduca sexta SEQ ID NO: 841 Amino acid sequence of P450 (demethylase) HvCPR_A0A2A4IYH3 From Heliothis virescens SEQ ID NO: 842 DNA coding sequence of P450 (demethylase) HvCPR_A0A2A4IYH3 From Heliothis virescens SEQ ID NO: 843 Amino acid sequence of P450 (demethylase) HaCYP6AE15v2_t From Helicoverpa armigera SEQ ID NO: 844 DNA coding sequence of P450 (demethylase) HaCYP6AE15v2_t From Helicoverpa armigera SEQ ID NO: 845 Amino acid sequence of P450 (demethylase) NMCH-HaCYP6AE15v2_t (Amino acid 1-25 -> NMCH N-terminal signal peptide) From Helicoverpa armigera SEQ ID NO: 846 DNA coding sequence of P450 (demethylase) NMCH-HaCYP6AE15v2_t From Helicoverpa armigera SEQ ID NO: 847 Amino acid sequence of P450 (demethylase) EcCFS-SP-HaCYP6AE15v2_t (Amino acid 1-22 -> EcCFS N-terminal signal peptide) From Helicoverpa armigera SEQ ID NO: 848 DNA coding sequence of P450 (demethylase) EcCFS-SP-HaCYP6AE15v2_t From Helicoverpa armigera SEQ ID NO: 849 Amino acid sequence of P450 (demethylase) HaCYP6AE15v2_A316G_t From Helicoverpa armigera SEQ ID NO: 850 DNA coding sequence of P450 (demethylase) HaCYP6AE15v2_A316G_t From Helicoverpa armigera SEQ ID NO: 851 Amino acid sequence of P450 (demethylase) NMCH-HaCYP6AE15v2_A316G_t (Amino acid 1-25 ->NMCH N-terminal signal peptide) From Helicoverpa armigera SEQ ID NO: 852 DNA coding sequence of P450 (demethylase) NMCH-HaCYP6AE15v2_A316G_t From Helicoverpa armigera SEQ ID NO: 853 Amino acid sequence of P450 (demethylase) EcCFS-SP-HaCYP6AE15v2_A316G_t (Amino acid 1 22 -> EcCFS N-terminal signal peptide) From Helicoverpa armigera SEQ ID NO: 854 DNA coding sequence of P450 (demethylase) EcCFS-SP-HaCYP6AE15v2_A316G_t From Helicoverpa armigera SEQ ID NO: 855 Amino acid sequence of P450 (demethylase) HaCYP6AE15v2_D392E_t From Helicoverpa armigera SEQ ID NO: 856 DNA coding sequence of P450 (demethylase) HaCYP6AE15v2_D392E_t From Helicoverpa armigera SEQ ID NO: 857 Amino acid sequence of P450 (demethylase) NMCH-HaCYP6AE15v2_D392E_t (Amino acid 1-25 -> NMCH N-terminal signal peptide) From Helicoverpa armigera SEQ ID NO: 858 DNA coding sequence of P450 (demethylase) NMCH-HaCYP6AE15v2_D392E_t From Helicoverpa armigera SEQ ID NO: 859 Amino acid sequence of P450 (demethylase) EcCFS-SP-HaCYP6AE15v2_D392E _t (Amino acid 1 22 -> EcCFS N-terminal signal peptide) From Helicoverpa armigera SEQ ID NO: 860 DNA coding sequence of P450 (demethylase) EcCFS-SP-HaCYP6AE15v2_D392E_t From Helicoverpa armigera SEQ ID NO: 861 Amino acid sequence of P450 (demethylase) Hv_CYP_A0A2A4JAM9_t From Heliothis virescens SEQ ID NO: 862 DNA coding sequence of P450 (demethylase) Hv_CYP_A0A2A4JAM9_t From Heliothis virescens SEQ ID NO: 863 Amino acid sequence of P450 (demethylase) NMCH-Hv_CYP_A0A2A4JAM9_t (Amino acid 1-25 -> NMCH N-terminal signal peptide) From Heliothis virescens SEQ ID NO: 864 DNA coding sequence of P450 (demethylase) NMCH-Hv_CYP_A0A2A4JAM9_t From Heliothis virescens SEQ ID NO: 865 Amino acid sequence of P450 (demethylase) EcCFS-SP-Hv_CYP_A0A2A4JAM9_t (Amino acid 1 22 -> EcCFS N-terminal signal peptide) From Heliothis virescens SEQ ID NO: 866 DNA coding sequence of P450 (demethylase) EcCFS-SP-Hv_CYP_A0A2A4JAM9_t From Heliothis virescens SEQ ID NO: 867 Amino acid sequence of P450 (demethylase) EcCFS (CYP719A5) (Amino acid 1-22 -> N-terminal signal peptide) From Eschscholzia californica SEQ ID NO: 868 DNA coding sequence of P450 (demethylase) EcCFS (CYP719A5) From Eschscholzia californica SEQ ID NO: 869 Amino acid sequence of P450 (demethylase) EcNMCH (CYP80B2) (Amino acid 1-25 -> N-terminal signal peptide) From Eschscholzia californica SEQ ID NO: 870 DNA coding sequence of P450 (demethylase) EcNMCH (CYP80B2) From Eschscholzia californica Items of the invention The present invention further provides the following itemized embodiments: 1. A genetically modified host cell comprising a pathway having enhanced production of one or more 5 benzylisoquinoline alkaloids wherein the cell comprises one or more features selected from: a) expression of one or more heterologous genes encoding one or more demethylases capable of converting thebaine into northebaine, thebaine into oripavine, thebaine into nororipavine and / or oripavine into nororipavine; b) expression of one or more heterologous genes encoding a tyrosine hydroxylase (TH) converting L-tyrosine into L-dopa, wherein the TH has at least 70% identity to the TH comprised in 7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63 or 65; c) reduction or elimination of activity of one or more dehydrogenases native to the host cell comprised in SEQ ID NO: 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703 or 705; d) reduction or elimination of activity of one or more reductases native to the host cell comprised in SEQ. ID NO: 707, 709, 711, 713, 715, 717, 719, 721, 723, 725,1T1, 729 or 731; e) expression of one or more heterologous genes encoding a norcoclaurine synthase (NCS) converting Dopamine and 4-HPAA into (S)-norcoclaurine, wherein the NCS has at least 70% identity to the NCS comprised in SEQ ID NO: 73 OR 76; f) expression of one or more heterologous genes encoding i) a fused 1,2-dehydroreticuline synthase-l,2-dehydroreticuline reductase (DRS-DRR) converting (S)-Reticuline into (R)- reticuline, wherein ia) the DRS-DDR has at least 70% identity to the DRS-DRR comprised in SEQ ID NO: 92, 94, 96; or ib) the DRS moiety has at least 70%, identity to the DRS comprised in SEQ ID NO: 98, 100, 102, 104 or 106; and the DRR moiety has at least 70% identity to the DRR comprised in SEQ ID NO: 108 or 110; or ii) a DRS having at least 70% identity to the DRS comprised in SEQ ID NO: 98, 100, 102, 104 or 106; and a DRR having at least 70% identity to the DRR comprised in SEQ ID NO: 108 or 110; iii) a fused 1,2-dehydroreticuline synthase-l,2-dehydroreticuline reductase (DRS-DRR) converting (S)-Reticuline into (R)-reticuline selected from DRS-DDR's having at least 70% identity to the DRS-DRR comprised in SEQ ID NO: 92, 94, 96; and / or iv) a 1,2-dehydroreticuline synthase (DRS) selected from DRSs having at least 70% identity to the DRS comprised in SEQ ID NO: 98, 100, 102, 104 or 106; and a 1,2-dehydroreticuline reductases (DDR) selected from DDR's having at least 70% identity to the DRR comprised in SEQ ID NO: 108 or 110; g) expression of one or more heterologous genes encoding a thebaine synthase (THS) converting 7-O-acetylsalutaridinol or 7-O-acetylsalutaridinol acetate into thebaine, wherein the THS has at least 70% identity to the THS comprised in SEQ ID NO: 126, 127, 128, 129, 131,133, 134, 136 or 138; and h) expression of one or more heterologous genes encoding a transporter protein capable of increasing uptake or export in the host cell of a reticuline derivative selected from transporter proteins having at least 70% identity to the transporter protein comprised in SEQ. ID NO: 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 733, 735, 795, 797, 799, 801, 803, 805, 807, 809, 811, 813, 815, 817,819, 821, 823 or 825. 2. The cell of item 1, wherein the sequence identity to any sequence is at least 90 %. 3. The cell of items 1 to 2, wherein the one or more demethylase is an insect demethylase. 4. The cell of item 3, wherein the insect demethylases have a product:by-product molar ratio of at least 2,0, such as at least 2,25, such as at least 2,5, such as at least 2,75, such as at least 3,0, such as at least 3,25, such as at least 3,5, such as at least 3,75, such as at least 4,0, such as at least 4,5, such as at least 5,0, such as at least 10,0 and wherein when the product is northebaine then the by-product is thebaine N-oxide and / or northebaine oxaziridine and when the product is nororipavine then the byproduct is oripavine N-oxide and / or nororipavine oxaziridine. 5. The cell of items 3 to 4, wherein the insect demethylases have N-demethylation activity and / or O-demethylation activity. 6. The cell of items 3 to 5, wherein the insect demethylases are is of family CYP6. 7. The cell of items 3 to 6, wherein the insect is of the order Lepidoptera. 8. The cell of item 7, wherein the insect is of the genus Helicoverpa. 9. The cell of item 8, wherein the insect is of the species Helicoverpa armigera. 10. The cell of item 7, wherein the insect is of the genus Heliothis. 11. The cell of item 10, wherein the insect is of the species Heliothis virescens. 12. The cell of item 7, wherein the insect is of the genus Spodoptera. 13. The cell of item 12, wherein the insect is of the species Spodoptera exigua. 14. The cell of items 3 to 13, wherein the insect demethylase comprises a polypeptide selected from the group consisting of: a) a demethylase which is at least 70% identical to the insect demethylase comprised in any one of SEQ ID NO: 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 827, 829, 831, 833, 835, 837, 839, 841, 843, 845, 847, 849, 851, 853, 855, 857, 859, 861, 863, 865, 867 and 869; b) a demethylase encoded by a polynucleotide which is at least 70% identical to the polynucleotide comprised in any one of SEQ. ID NO: 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 828, 830, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850, 852, 854, 856, 858, 860, 862, 864, 866, 868 and 870 or genomic DNA thereof; and c) a functional variant of the insect demethylase of (a) or (b) capable of converting thebaine into northebaine, thebaine into oripavine, thebaine into nororipavine and / or oripavine into nororipavine. 15. The cell of item 14, wherein the insect demethylase is a) the demethylase comprised in any one of SEQ ID NO: 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 827, 829, 831, 833, 835, 837, 839, 841, 843, 845, 847, 849, 851, 853, 855, 857, 859, 861, 863, 865, 867 and 869; or b) the demethylase encoded by a polynucleotide comprised in any one of SEQ ID NO: 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 828, 830, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850, 852, 854, 856, 858, 860, 862, 864, 866, 868 and 870 or genomic DNA thereof. 16. The cell of any preceding item, wherein the demethylases are artificial mutants comprising one or more mutations in a signal sequence. 17. The cell of item 16, wherein the signal sequence of the demethylases has been wholly or partially been replaced by a signal sequence from another enzyme. 18. The cell of item 17, wherein the demethylases are artificial mutants having least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the demethylase comprised in SEQ. ID NO: : 845, 847, 851, 853, 857, 859, 863, 865, 867 or 869. 19. The cell of item 15, wherein the demethylases are artificial mutants having least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the demethylase comprised in SEQ ID NO: 152 and comprises one or more mutations corresponding to A110X, H242X, and / or V224X, such as A110N, H242P and / or V224I. 20. The cell of item 15, wherein the demethylases are artificial mutants having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the demethylase comprised in SEQ ID NO: 140 and comprises one or more mutations corresponding to A316X and / or D392X, such as A316G and / orD392E. 21. The cell of any preceding item, wherein the demethylase comprises one or more conserved amino acids corresponding to positions G103, Hill, K167, E198, R219, L223, 1256, A259, L273, V284, 1309, L314, Q517, L160, N216, R443 of SEQ ID NO: 152 or conservative substitutions thereof 22. The cell of item 21, wherein the demethylase comprises a polypeptide which is at least 60% identical to the insect demethylase comprised in SEQ ID NO: 15...

Claims

1. A genetically modified fungal host cell comprising a pathway having enhanced production ofnorthebaine, oripavine, and / or nororipavine wherein the fungal host cell expresses one or more heterologous genes encoding a demethylase converting thebaine into the northebaine, thebaine into the oripavine, thebaine into the nororipavine and / or oripavine into the nororipavine wherein the demethylase comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 140, 142, 146, 148, 152, 154, 156, 158, 160, 162, 166, 168, 170, 172, 174, 176, 178, 182, 184, 186, 188, 190, 192, 194, 196, 827, 829, 831, 833, 835, 837, 839, or 841, and wherein the host cell further comprises a demethylase-CPR capable of reducing and / or regenerating the demethylase enzyme.

2. The genetically modified fungal host cell of claim 1, wherein the demethylase has aproduct:by-product molar ratio of at least 2,0 and wherein when the product is northebaine then the by-product is thebaine N-oxide and / or northebaine oxaziridine and when the product is nororipavine then the by-product is oripavine N-oxide and / or nororipavine oxaziridine.

3. The genetically modified fungal host cell of claim 1 or claim 2, wherein the demethylaseis anartificial mutant comprising one or more mutations in a signal sequence, preferably wherein the signal sequence of the demethylases has been wholly or partially been replaced by a signal sequence from another enzyme.

4. The genetically modified fungal host cell of claim 3, wherein the demethylase is an artificialmutant having least preferably 80% identity to the demethylase comprised in SEQ ID NO: 152 and comprises one or more mutations corresponding to A110X, H242X, and / or V224X, such as A110N, H242P and / or V224I.

5. The genetically modified fungal host cell of claim 3, wherein the demethylase is an artificialmutant having at least 80% identity to the demethylase comprised in SEQ ID NO: 140 and comprises one or more mutations corresponding to A316X and / or D392X, such as A316G and / or D392E.

6. The genetically modified fungal host cell of any preceding claim, wherein the demethylasecomprises one or more conserved amino acids corresponding to positions G103, H111, K167, E198, R219, L223, I256, A259, L273, V284, I309, L314, Q517, L160, N216, R443 of SEQ ID NO: 152 or conservative substitutions thereof.2020363960   07 Oct 20267.      The genetically modified fungal host cell of any one of claims 1 to 6, wherein the demethylase-CPR is heterologous to the cell.

8. The genetically modified fungal host cell of any one of claims 1 to 7, wherein the demethylase-CPR comprises a polypeptide which is at least 80% identical to the demethylase-CPR comprised in any one of SEQ ID NO: 292, 294, 296, 298, 300 or 302.

9. The genetically modified fungal host cell of any preceding claim, wherein:(A)     the cell comprises one or more features selected from:a) expression of one or more heterologous genes encoding a tyrosine hydroxylase (TH) converting L-tyrosine into L-dopa;b) reduction or elimination of activity of one or more dehydrogenases native to the host cell;c)  reduction or elimination of activity of one or more reductases native to the host cell;d) expression of one or more heterologous genes encoding a norcoclaurine synthase (NCS) converting Dopamine and 4-HPAA into (S)-norcoclaurine;e) expression of one or more heterologous genes encoding an epimerase;f) expression of one or more heterologous genes encoding a thebaine synthase (THS) converting 7-O-acetylsalutaridinol or 7-O-acetylsalutaridinol acetate into thebaine; andg) expression of one or more heterologous genes encoding a transporter protein capable of increasing uptake or export in the host cell of a reticuline derivative; or(B)    the cell expresses one or more genes encoding polypeptides selected from:a) a 3-deoxy-D-arabino-2-heptulosonic acid 7-phosphate synthase (DAHP synthase) converting PEP and E4P into DAHP;b) a 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (aro1) converting 3-phosphoshikimate and PEP into EPSP;c) an aro1 polypeptide converting DHAP and PEP into EPSP;d) a chorismate synthase converting EPSP into Chorismate;e) a chorismate mutase converting Chorismate into prephenate;f) a prephenate dehydrogenase (Tyr1) converting prephenate into 4-HPP;g) an aromatic aminotransferase converting 4-HPP into L-Tyrosine;h) a TH-CPR capable of reducing TH;i)    a L-dopa decarboxylase (DODC) converting L-dopa into dopamine;j)    a Tyrosine decarboxylase (TYDC) converting L-dopa into dopamine;2020363960   07 Oct 2026k) a hydroxyphenylpyruvate decarboxylase (HPPDC) converting 4-HPP into 4-HPPA;l)    a monoamine oxidase converting dopamine into 3,4-DHPAA;m) a 6-O-methyltransferase (6-OMT) converting (S)-norcoclaurine into (S)-Coclaurine and / or norlaudanosoline into (S)-3’-Hydroxy-coclaurine;n)    a   Coclaurine-N-methyltransferase (CNMT)   converting   (S)-Coclaurine   into   (S)-N-Methylcoclaurine and / or (S)-3’-hydroxycoclaurine into (S)-3’-hydroxy-N-methyl-coclaurine;o)    a  N-methylcoclaurine hydroxylase (NMCH) converting  (S)-Coclaurine  into  (S)-3’-hydroxycoclaurine and / or (S)-N-Methylcoclaurine into (S)-3’-Hydroxy-N-Methylcoclaurine;p) a 3’-hydroxy-N-methyl-(S)-coclaurine 4’-O-methyltransferase (4’-OMT) converting (S)-3’-Hydroxy-N-Methylcoclaurine into (S)-Reticuline;q) a DRS-CPR capable of reducing DRS-DRR;r)     a salutaridine synthase (SAS) converting (R)-reticuline into Salutaridine;s)     a salutaridine reductase (SAR) converting Salutaridine to Salutaridinol; andt) a salutaridinol 7-O-acetyltransferase (SAT) converting Salutaridinol into 7-O-acetylsalutaridinol.

10. The genetically modified fungal host cell of any preceding claim, wherein the cell is further modified to increase cytosolic levels of heme, preferably bya) overexpressing and / or co-expressing one or more rate-limiting proteins in the heme pathway, such as HEM 2, HEM3 and / or HEM12 preferably by increasing the number of copies of the genes integrated in the host cell and / or by linking the genes to a combination of stronger and weaker promoters, such promoters selected from from pPYK1, pSED1, pKEX2, pTEF1, pTDH3 and pPGK1, where pTEF1, pTDH3 and pPGK1; and / orb) disrupting, deleting and / or attenuating any heme-down regulating genes, such as HMX1.

11. The genetically modified fungal host cell of any preceding claim, wherein the cell is further modified by overexpressing and / or co-expressing P450 helper genes, preferably selected from DAP1, HAC1, KAR2, HSP82, CNE1, SSA1, CPR6, FES1, HSP104 and STI1.

12. The genetically modified fungal host cell of any preceding claim, wherein the cell is further modified by overexpressing and / or co-expressing one or more genes in the pentose metabolic pathway, preferably selected from ZWF1 and GND1.

13. The genetically modified fungal host cell of any preceding claim, wherein the cell is further2020363960   07 Oct 2026modified by overexpressing and / or co-expressing one or more genes encoding factors lowering and / or detoxifying cytosolic formaldehyde, preferably selected from SFA1.

14. The genetically modified fungal host cell of claim 1, wherein the fungal cell is a yeastselected from the group consisting of ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and Fungi lmperfecti yeast (Blastomycetes); preferably a yeast cell is selected from the genera consisting of Saccharomyces, Kluveromyces, Candida, Pichia, Debaromyces, Hansenula, Yarrowia, Zygosaccharomyces, and Schizosaccharomyces; preferably a yeast cell is selected from the species consisting of Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, and Yarrowia lipolytica.

15. A cell culture, comprising the genetically modified fungal host cell of any one of claims 1 to 14and a growth medium.

16. A method for producing northebaine, oripavine, and / or nororipavine comprisinga) culturing the cell culture of claim 15 at conditions allowing the cell to produce the northebaine, oripavine, and / or nororipavine; andb) preferably recovering and / or isolating the northebaine, oripavine, and / or nororipavine.

17. The method of claim 16, further comprising chemically or biologically modifying thenorthebaine, oripavine, and / or nororipavine; preferably wherein the modified northebaine, oripavine, and / or nororipavine is selected from one or more of buprenorphine, naltrexone, naloxone and nalbuphine.

18. A mutant demethylase having least 80% identity to the demethylase comprised in SEQ ID NO:152 and comprising one or more mutations corresponding to A110X, H242X, and / or V224X, preferably A110N, H242P and / or V224I.

19. A mutant demethylase having at least 80% identity to the demethylase comprised in SEQ IDNO: 140 and comprising one or more mutations corresponding to A316X and / or D392X, preferably A316G and / or D392E.

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