Method for producing noroxymorphone and bioactive derivatives thereof

Abstract

The present invention relates to improved scalable methods of preparing noroxymorphone and other opioids, and bioactive derivatives thereof as well as methods for reducing a substrate. The present invention further relates to cell enabling the improved scalable methods, as well as fermentation liquids, and uses thereof.

Description

Case Ref. P322WO IPTector™ Method for producing noroxymorphone and bioactive derivatives thereof Field

[0001] The present invention relates to improved scalable methods of preparing noroxymorphone and bioactive derivatives thereof. The present invention further relates to cell enabling the improved scalable methods, as well as fermentation liquids, and uses thereof.Background

[0002] Noroxymorphone is traditionally produced chemically from poppy-derived opiates. It is an important precursor to the nal-prefix opioids, such as naloxone, naltrexone, and nalmefene (Wicks et al 2021). An important step of the production method is an N-demethylation of the Noroxymorphone precursors. For oripavine and thebaine, N-demethylation toward Noroxymorphpone has been reported using electrochemistry (Sommer et al. 2022). However, difficulties with respect to e.g. selectivity, electrode stability, and scalability have limited its widespread industrial adoption.

[0003] Transformation of morphinone to hydromorphone is traditionally done using catalytic hydrogenation, chemically. Morphinone reductase is an enzyme that converts morphinone to hydromorphone and codeinone to hydrocodone by the saturation of the double bond between C7-C8 of the substrates. Numerous homologs exist but one of the most studied is from Pseudomonas putida (Morb). Other potential substrates for MorB have been contemplated but conversions have not been demonstrated (WO2024073755A). There is still a need in the art of an improved scalable production method of noroxymorphone enabling increased access to pharmaceutically relevant opioids.Summary

[0004] The present inventors have demonstrated an efficient route from oripavine and / or thebaine to noroxymorphone using a combination of chemistry and biocatalysis to enable large scale production of pharmaceutically relevant opioids, such as the nal-prefix opioids, in high yield at significantly lower cost, while reducing impurities generated as well as reducing hazardous waste.

[0005] In one aspect, the present disclosure provides genetically modified host cells and methods for producing noroxymorphone from morphinan substrates. In one aspect, the genetically modified host cells comprise combinations of heterologously expressed enzymes disclosed herein, optionally including one or more demethylases, one or more reductases, and one or more uptake transporters.

[0006] In one aspect, uptake of an exogenously supplied substrate can be increased by expression of an uptake transporter in a genetically modified host cell, as illustrated in at least Examples 14-15. InCase Ref. P322WO IPTector™ one aspect, selection of uptake transporters can influence conversion profiles and / or product levels for fed substrates, as illustrated in at least Examples 14-15.

[0007] In one aspect, selection of a cytochrome P450 reductase in combination with a cytochrome P450 demethylase can influence demethylation performance, as illustrated in Example 13 (Fig. 14).

[0008] In one aspect, the present disclosure provides demethylases, including N-demethylases and O-demethylases, and variants thereof as disclosed herein. In one aspect, the present disclosure provides demethylases belonging to selected cytochrome P450 families and / or demethylases having sequence identity to the demethylases disclosed herein.

[0009] In one aspect, the present disclosure provides morphinone reductases and variants thereof as disclosed herein. In one aspect, morphinone reductases disclosed herein catalyze reduction of morphinan intermediates, as illustrated in at least Example 3, and morphinone reductase variants comprising one or more substitutions are described, as illustrated in at least Example 4.

[0010] In one aspect, the present disclosure provides pathway configurations for converting different morphinan substrates and / or intermediates to noroxymorphone. As illustrated in at least Example 14, host cells disclosed herein convert a fed 14-hydroxycodeinone substrate to noroxymorphone. As illustrated in at least Example 15, host cells disclosed herein convert a fed oxycodone substrate to noroxymorphone, including in a configuration using an O-demethylase (Fig. 18) and in a configuration using an N-demethylase (Fig. 19).

[0011] In one aspect, a genetically modified host cell is provided converting or capable of converting a substrate to noroxymorphone, wherein the cell comprises i) a heterologous polynucleotide encoding a morphinone reductase, and ii) a heterologous polynucleotide encoding an N-demethylase, and optionally iii) a heterologous polynucleotide encoding an O-demethylase.

[0012] In a further aspect, a genetically modified host cell is provided, wherein the cell comprises i) a heterologous polynucleotide encoding a morphinone reductase, andii) a heterologous polynucleotide encoding an N-demethylase,iii) an uptake transporter capable of transporting the substrate into the cell,iv) a polynucleotide encoding a cytochrome P450 reductase;and optionallyv) a heterologous polynucleotide encoding an O-demethylase.

[0013] In a further aspect, a genetically modified host cell is provided converting or capable of converting a substrate to noroxymorphone, wherein the cell comprisesi) a heterologous polynucleotide encoding a morphinone reductase, andii) a heterologous polynucleotide encoding an N-demethylase,iii) an uptake transporter capable of transporting the substrate into the cell,Case Ref. P322WO IPTector™ iv) a polynucleotide encoding a cytochrome P450 reductase;and optionallyv) a heterologous polynucleotide encoding an O-demethylase.

[0014] In a further aspect, a polypeptide is provided selected from the group consisting of: an N-demethylase as defined herein, a morphinone reductase as defined herein, an O-demethylase as defined herein, a cytochrome P450 reductase as defined herein, and an uptake transporter as defined herein.

[0015] In a further aspect, a composition is provided comprising the polypeptide as defined herein, and optionally one or more excipients.

[0016] In a further aspect, a method is provided for reducing a substrate using a morphinone reductase, the method comprising contacting the substrate with the morphinone reductase.

[0017] In one aspect, a method is provided for producing noroxymorphone from a substrate,or a pharmaceutically acceptable salt thereof, wherein the method comprises subjecting the substrate to i) a morphinone reductase, and ii) an N-demethylase, and optionally iii) an O-demethylase.

[0018] In one aspect, a method is provided for producing an opioid from noroxymorphone, the method comprising:a) producing noroxymorphone using a method as defined herein, andb) converting noroxymorphone to the opioid.

[0019] In one aspect, a cell culture is provided comprising the genetically modified host cell as defined herein.Description of drawings

[0020] Fig. 1: The bar diagram shows the production of oxymorphone and noroxymorphone in pM. In all 3 cases (A, B and C) incubation was done using the system described by Duetz et al (2000) in 96-deep-well plates for 3 days in 500ml standard defined yeast medium at pH5.5 containing 600 pM 14-hydroxy morphinone, with shaking at 280rpm in a Kuhner ISF-l-X shaking incubator at 30°C. A:Negative control: incubation as described above of 600 pM 14-hydroxy morphinone in standard defined yeast media without yeast cells. B: Negative control 2 (no N demethylase, no reductase):Case Ref. P322WO IPTector™ Incubation of S. cerevisiae with expression of opioid uptake transporter and CPR in standard defined yeast media containing 600 pM 14-hydroxy morphinone C: Incubation of S. cerevisiae with expression of opioid uptake transporter, CPR, MorB_l and N-demethylase mutant in standard defined yeast media containing 600 pM 14-hydroxymorphinone.

[0021] Fig.2: Reaction schemes showing conversion of 14-OH-Morphinone to NOM.

[0022] Fig.3: Amino acid sequence of MorB with the 16 diverging residues from MorB_2.

[0023] Fig.4: Bar diagram showing the mutants of MorB_2 responsible for introduction of activity of the enzyme for the reduction of 14-hydroxy morphinone and / or 14-hydroxy normorphinone to oxymorphone and / or noroxymorphone. The bar diagram shows the production of oxymorphone and noroxymorphone in pM. In all cases incubation of media and cultures was done using the system described by Duetz et al in 96-deep-well plates for 3 days in 500ml standard defined yeast medium at pH 5.5 containing 600pM 14-hydroxy morphinone, with shaking at 280rpm in a Kuhner ISF-l-X shaking incubator at 30°C. Media controls contain 600uM 14-hydroxy morphinone but no yeast strain. A control with yeast expressing only uptake transporter, CPR, and N-demethylase but no 14-hydroxy morphinone reductase is also shown. The remaining bars show strains expressing of uptake transporter, CPR, and N-demethylase and MorB (MorB_l) or various versions of MorB_2.

[0024] Fig. 5: Bar diagram showing 14-hydroxy morphinone uptake activity of selected uptake transporters by expression in S. cerevisiae and with feeding of 600 pM 14-hydroxy morphinone. In all cases the S. cerevisiae strains SOD1265 in addition to the uptake transporters, had heterologous expression of MorB (MorB_l), HaCPR_E0A3A7_co5 and Hv_CYP_A0A2A4JAM9. The bar diagram shows the production of oxymorphone and noroxymorphone in pM. In all cases incubation was done using the system described by Duetz et al in 96-deep-well plates for 3 days in 500ml standard defined yeast medium at pH5.5 containing 300 pM 14-hydroxy morphinone, with shaking at 280rpm in a Kuhner ISF-l-X shaking incubator at 30°C

[0025] Fig. 6: Conversion of 14-hydroxycodeinone to NOM. Conversion of 14-OH Codeinone to Noroxymorphone requires an O-demethylase, an N-demethylase, and a MorB (reductase) homolog with broad substrate specificity towards BIA substrates but strong regioselectivity for the appropriate double bond. If O-demethylation occurs first, the rest of the catalysis will proceed as shown in earlier examples.

[0026] Fig.7: Comparison of 14-hydroxycodeinone to 14-hydroxymorphinone structure.

[0027] Fig. 8: Morphinan structure numbering. Nal-prefix opioids (Fig. 9) contain the noroxymorphone feature of a 14-hydroxyl group and a reduced 7,8 alkene functionality commonly introduced during 14-hydroxylation of morphinanes containing 8,14 and 6,7 diene bonds.

[0028] Fig.9: General chemistry required for production of "Nals" from Noroxymorphone.Case Ref. P322WO IPTector™

[0029] Fig. 10: Exemplary compounds that can be synthesized from noroxymorphone.

[0030] Fig. 11: Bar diagram showing the effect of S. cerevisiae native aldo-keto reductase deletions on oxymorphone production. Gene names below the bars show which gene has been deleted. Values of oxymorphone are expressed as area under curve of the peaks in LC-MS chromatograms that represent oxymorphone. All S. cerevisiae strains shown, except sOD694, contain heterologous expression of an uptake transporter and MorB (MorB_l). The strain sOD694 is a wild type S. cerevisiae strain with no heterologous genes expressed. In all cases incubation of cultures was done using the system described by Duetz et al in 96-deep-well plates for 3 days in 500ml standard defined yeast medium at pH5.5 containing 600pM 14-hydroxy morphinone, with shaking at 280rpm in a Kuhner ISF-1-X shaking incubator at 30°C.

[0031] Fig. 12: Exemplary de novo route to thebaine from glucose. TYAT is tyrosine aminotransferase, S. cer aro8 and aro9 can serve this function and convert 4-HPP to L-tyrosine endogenously. TYRH or TH is tyrosine hydroxylase. Some tyrosine hydroxylases utilize tetrahydropteridine, others are CYP76 family enzymes. Tyrosine can also be converted to L-DOPA using TYR (tyrosinase). DODC, or dopa decarboxylase, converts DOPA to dopamine. TYDC (EC 4.1.1.25 ) can also catalyze this step (tyrosine decarboxylase). HPPDC is 4-hydroxphenylpyruvate decarboxylase, endogenous arolO in yeast can also serve this function. TYDC can also be used to produce tyramine from L-tyrosine, which can be oxidized by primary amine oxidases to 4-HPAA. Alternately TYRDC-2 (EC 4.1.1.108), 4-hydroxyphenylacetaldehyde synthase can convert L-tyrosine to 4HPAA in one step.

[0032] Fig. 13: Bar diagram showing the production of oxymorphone and Noroxymorphone from 600 .l\ / l 14-hydroxymorphinone using native MorB and mutated homologs. Numbers are in pM.

[0033] Fig. 14: Bar diagram showing the results of Example 13. The bar-diagram production profile is shown in pM. As can be seen in the diagram, expression in yeast of the N-demethylase of SEQ ID NO: 190 and co-enzyme CPR of SEQ ID NO: 192 in SOD1859 gives efficient N-demethylation of 14-hydroxy morphinone and / or oxymorphone which results in high production of Noroxymorphone.

[0034] Fig 15: Schematic illustrating the enzymatic conversion pathway from 14-hydroxycodeinone to noroxymorphone. The schematic details the roles of O-demethylase (ODM), N-demethylase (NDM), and morphinone reductase (morB), and shows potential intermediate products including 14-hydroxynormorphinone, oxymorphone, oxycodone, and noroxycodone.

[0035] Fig. 16: Bar graph showing the results of Example 14. The graph displays data from an uptake transporter screening using 14-hydroxycodeinone as the fed substrate. LC-MS analysis quantified the production of noroxymorphone and 14-hydroxy codeinone, oxycodone, oxymorphone, and noroxycodone to evaluate transporter efficacy. The last bar corresponds to an empty vector (no transporter).Case Ref. P322WO IPTector™

[0036] Fig 17: Bar graph data from uptake transporter screening with oxycodone feeding. LC-MS verification and quantification was done by measuring the LC-MS chromatogram area under curve value of peaks corresponding to 14-hydroxy codeinone, oxycodone, oxymorphone, noroxycodone and noroxymorphone. Reference retention times and fragmentation patterns of the mentioned compounds were obtained by running commercially available standards. The first two bars correspond to media only (no yeast). The last bar corresponds to empty vector without transporter.

[0037] Fig. 18: Bar graph data from uptake transporter screening with oxycodone feeding, using an alternative O-demethylase (SEQ ID NO: 211). LC-MS verification and quantification was done by measuring the LC-MS chromatogram area under curve value of peaks corresponding to 14-hydroxy codeinone, oxycodone, oxymorphone, noroxycodone and noroxymorphone. Reference retention times and fragmentation patterns of the mentioned compounds were obtained by running commercially available standards. The first two bars correspond to media only (no yeast). The last bar corresponds to empty vector without transporter.

[0038] Fig. 19: Bar graph data from uptake transporter screening with oxycodone feeding, using an alternative N-demethylase (SEQ ID NO: 31). LC-MS verification and quantification was done by measuring the LC-MS chromatogram area under curve value of peaks corresponding to 14-hydroxy codeinone, oxycodone, oxymorphone, noroxycodone and noroxymorphone. Reference retention times and fragmentation patterns of the mentioned compounds were obtained by running commercially available standards.Incorporation by reference

[0039] 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 DescriptionDefinitions

[0040] 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, 1-5; 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.Case Ref. P322WO IPTector™

[0041] 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.

[0042] 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.

[0043] The term "in vivo", as used herein refers to within a living cell or organism, including, for example animal, a plant or a microorganism.

[0044] 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.

[0045] 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 demethylation 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.

[0046] Term "endogenous" or "native" as used herein refers to a gene or a polypeptide in a host cell which originates from the same host cell.

[0047] The term "deletion" as used herein refers to manipulation of a gene so that it is no longer expressed in a host cell.Case Ref. P322WO IPTector™

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] The term "non-naturally occurring" as used herein about a substance, refers to any substanceCase Ref. P322WO IPTector™ 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.

[0054] The term "% identity" is used herein about the relatedness between two amino acid sequences or between two nucleotide sequences.

[0055] 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

[0056] 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

[0057] 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:Case Ref. P322WO IPTector™Cost to open gap: default= 5 for nucleotides / 11 for proteinsCost to extend gap: default = 2 for nucleotides / 1 for proteinsPenalty for nucleotide mismatch: default = -3Reward for nucleotide match: default= 1Expect value: default = 10Wordsize: default = 11 for nucleotides / 28 for megablast / 3 for proteins.

[0058] 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.

[0059] 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 candidateCase Ref. P322WO IPTector™ 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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 ofCase Ref. P322WO IPTector™ 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.

[0066] 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.

[0067] The terms "nucleotide sequence and "polynucleotide" are used herein interchangeably.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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, isCase Ref. P322WO IPTector™ based on the total weight of the composition in which the component is included (e.g., on the total amount of the reaction mixture).

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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,Case Ref. P322WO IPTector™ methoxy, ethoxy, propoxy, and isopropoxy.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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,Case Ref. P322WO IPTector™ 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.

[0082] 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, theCase Ref. P322WO IPTector™ 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.

[0083] 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.

[0084] The term "ring system" encompasses monocycles, as well as fused and / or bridged polycycles.

[0085] 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.Case Ref. P322WO IPTector™

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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 group may be used in place of benzyl.

[0090] The term "eukaryotic cytochrome P450 enzyme" as used herein, refers to a polypeptide having cytochrome P450 monooxygenase activity. In some embodiments, the eukaryotic cytochrome P450 enzyme is characterized as a membrane-associated heme-thiolate monooxygenase. In someCase Ref. P322WO IPTector™ embodiments, the eukaryotic cytochrome P450 enzyme comprises a hydrophobic N-terminal anchor for association with the endoplasmic reticulum membrane. In some embodiments, the eukaryotic cytochrome P450 enzyme lacks a fused reductase domain, and the cytochrome P450 monooxygenase activity is functionally dependent on electron transfer from a separate cytochrome P450 reductase polypeptide. In some embodiments, the eukaryotic cytochrome P450 enzyme is provided in a multicomponent cytochrome P450 system comprising a membrane-associated cytochrome P450 and a separate cytochrome P450 reductase. In some embodiments, the eukaryotic cytochrome P450 enzyme comprises a conserved P450 protein fold, which may include a heme-binding motif comprising the amino acid sequence FXXGXRXCXG, an arginine-rich K-helix motif comprising the amino acid sequence EXXR, and / or an l-helix proton transfer groove, wherein X is any amino acid. In some embodiments, the eukaryotic cytochrome P450 enzyme is structurally and functionally distinct from soluble prokaryotic P450 enzymes comprising a fused reductase domain. In some embodiments, the eukaryotic cytochrome P450 enzyme is a demethylase of the present disclosure, such as an N-demethylase and / or an O-demethylase of the present disclosure.

[0091] As used herein, the term "conservative substitution" refers to an amino acid substitution in which an amino acid residue is replaced with another amino acid residue having similar physicochemical properties such that the substitution is expected to be tolerated without abolishing the relevant polypeptide activity. In some embodiments, conservative substitutions include, 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. In some embodiments, examples of tolerated conservative substitutions include substitutions corresponding to l->V, L->M, N->S, and / or R->K. In some embodiments, conservative substitutions may be identified based on sequence alignment software tools, optionally including Clustal W, wherein the software output uses one dot or two dots to indicate the degree of conservation at a position in an alignment. In some embodiments, when a position is described as "corresponding" to a position in a reference sequence, the corresponding position in a candidate sequence is determined by alignment of the candidate sequence to the reference sequence, and the position number in the candidate sequence may differ from the position number in the reference sequence, for example due to amino acid additions, deletions, or extensions.

[0092] As used herein, reference to any particular CYP family, such as CYP6, CYP64 or CYP75 follows the cytochrome P450 (CYP) nomenclature as set out in Nelson, D.R. (2006). Cytochrome P450 Nomenclature, 2004. In: Phillips, I.R., Shephard, E.A. (eds) Cytochrome P450 Protocols. Methods in Molecular Biology, vol 320. Humana Press, Totowa, NJ.Case Ref. P322WO IPTector™

[0093] As used herein, the term "functional homolog" refers to a polypeptide that has sequence similarity to a reference polypeptide and that carries out one or more biochemical function(s) of the reference polypeptide.

[0094] As used herein, the term "functional variant" refers to a polypeptide that has sequence similarity to a reference polypeptide and that carries out one or more biochemical function(s) of the reference polypeptide. In some embodiments, a functional homolog and the reference polypeptide are naturally occurring polypeptides and the sequence similarity is due to evolutionary relatedness, such as orthologs or paralogs. In some embodiments, a functional homolog is a variant of a naturally occurring polypeptide. In some embodiments, functional homologs and / or functional variants include polypeptides created by mutagenesis, including site-directed mutagenesis and / or random mutagenesis, and / or by combining domains from different polypeptides. In some embodiments, a functional homolog and / or functional variant is identified by sequence identity to a reference polypeptide and by retaining the relevant enzymatic activity described herein.Genetically modified host cells

[0095] Microorganisms optimized to produce noroxymorphone or downstream opioids, such as the nal-prefix opioids are in great need.

[0096] In some embodiments, the present disclosure provides a genetically modified host cell converting or capable of converting a substrate to noroxymorphone, wherein the cell comprises i) a heterologous polynucleotide encoding a morphinone reductase, and ii) a heterologous polynucleotide encoding an N-demethylase, and optionally iii) a heterologous polynucleotide encoding an O-demethylase.

[0097] In some embodiments, the present disclosure provides a genetically modified host cell comprising i) a heterologous polynucleotide encoding a morphinone reductase, and ii) a heterologous polynucleotide encoding an N-demethylase, and optionally iii) a heterologous polynucleotide encoding an O-demethylase.

[0098] In some embodiments, the host cell of the present disclosure comprises a polypeptide selected from the group consisting of: an N-demethylase, a morphinone reductase, an O-demethylase, a cytochrome P450 reductase, an uptake transporter, and a combination thereof, wherein at least one of the polypeptides are heterologous to the cell.

[0099] In some embodiments, the cell comprises a heterologous polypeptide selected from the group consisting of: a cytochrome P450 reductase, an uptake transporter, and a combination thereof.

[0100] In some embodiments, the genetically modified host cell, method, or polypeptide disclosed herein is provided wherein the sequence identity is at least 60%, such as at least 61%, such as at leastCase Ref. P322WO IPTector™ 62%, such as at least 63%, such as at least 64%, such as at least 65%, such as at least 66%, such as at least 67%, such as at least 68%, such as at least 69%, such as at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or such as at least 99%, for example 100% sequence identity to the amino acid sequence or polynucleotide sequence defined by a particular SEQ ID NO, for example at least 60%, such as at least 61%, such as at least 62%, such as at least 63%, such as at least 64%, such as at least 65%, such as at least 66%, such as at least 67%, such as at least 68%, such as at least 69%, such as at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or such as at least 99%, for example 100% sequence identity to the amino acid sequence or polynucleotide sequence defined by any one of SEQ ID NO: 1 to SEQ ID NO: 184.

[0101] In some embodiments, the genetically modified host cell, method, polypeptide, and / or polynucleotide disclosed herein is provided wherein the sequence identity is at least 60%, such as at least 61%, such as at least 62%, such as at least 63%, such as at least 64%, such as at least 65%, such as at least 66%, such as at least 67%, such as at least 68%, such as at least 69%, such as at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or such as at least 99%, for example 100% sequence identity to the amino acid sequence or polynucleotide sequence defined by a particular SEQ ID NO, for example at least 60%, such as at least 61%, such as at least 62%, such as at least 63%, such as at least 64%, such as at least 65%, such as at least 66%, such as at least 67%, such as at least 68%, such as at least 69%, such as at least 70%, such as at least 71%,Case Ref. P322WO IPTector™ such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or such as at least 99%, for example 100% sequence identity to the amino acid sequence or polynucleotide sequence defined by any one of SEQ ID NO: 1 to SEQ ID NO: 194.

[0102] In some embodiments, the genetically modified host cell, method, polypeptide, and / or polynucleotide disclosed herein is provided wherein the sequence identity is at least 60%, such as at least 61%, such as at least 62%, such as at least 63%, such as at least 64%, such as at least 65%, such as at least 66%, such as at least 67%, such as at least 68%, such as at least 69%, such as at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or such as at least 99%, for example 100% sequence identity to the amino acid sequence or polynucleotide sequence defined by a particular SEQ ID NO, for example at least 60%, such as at least 61%, such as at least 62%, such as at least 63%, such as at least 64%, such as at least 65%, such as at least 66%, such as at least 67%, such as at least 68%, such as at least 69%, such as at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or such as at least 99%, for example 100% sequence identity to the amino acid sequence or polynucleotide sequence defined by any one of SEQ ID NO: 1 to SEQ ID NO: 212.Substrates

[0103] In the present context, a substrate is understood as the starting material to be converted by the genetically modified host cell as defined herein to noroxymorphone. In some embodiments, theCase Ref. P322WO IPTector™ substrate is fed to the host cell. In some embodiments, the substrate is prepared by one or more precursors, optionally as defined herein, such as prepared de novo, optionally from glucose.

[0104] In some embodiments, the substrate is selected from the group consisting of: 14-hydroxymorphinone, 14-hydroxynormorphinone, oxymorphone, 14-hydroxycodeinone, and a combination thereof. In some embodiments, the substrate is 14-hydroxymorphinone. In some embodiments, the substrate is 14-hydroxycodeinone.

[0105] In some embodiments, the substrate is selected from the group consisting of: 14-hydroxymorphinone, 14-hydroxynormorphinone, oxymorphone, oxycodone, 14-hydroxycodeinone, and a combination thereof. In some embodiments, the substrate is 14-hydroxymorphinone. In some embodiments, the substrate is 14-hydroxycodeinone.

[0106] In addition to the substrates disclosed herein, it has further been demonstrated that oxycodone is also a viable substrate for conversion by the genetically modified host cells of the present disclosure. As shown in Example 15 and the corresponding Figs. 17, 18, and 19, host cells expressing the enzymatic cassette of the invention are capable of converting exogenously supplied oxycodone into downstream products, including noroxymorphone, thereby expanding the utility of the present genetically modified host cells.

[0107] In some embodiments, the host cell comprises the heterologous polynucleotide encoding the O-demethylase.Specific genetically modified host cells

[0108] In some embodiments, the genetically modified host cell is a cell engineered as set out in this section.

[0109] In some embodiments, a cell is provided capable of producing noroxymorphone, wherein the cell comprises an N-demethylase having at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 179, SEQ ID NO: 31, and SEQ ID NO: 33, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% sequence identity thereto.

[0110] In some embodiments, a cell is provided capable of producing noroxymorphone, wherein the cell comprises an N-demethylase comprising the amino acid sequence of SEQ ID NO: 179 and a cytochrome P450 reductase comprising the amino acid sequence of SEQ ID NO: 191.

[0111] In some embodiments, a cell is provided capable of producing noroxymorphone from a 14-hydroxycodeinone substrate, wherein the cell comprises an O-demethylase having at least 50% sequence identity to the polypeptide of SEQ ID NO: 69, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% sequence identity thereto.

[0112] In some embodiments, a cell is provided capable of producing noroxymorphone from a 14-Case Ref. P322WO IPTector™ hydroxycodeinone substrate, wherein the cell comprises an O-demethylase comprising the amino acid sequence of SEQ ID NO: 69 and a cytochrome P450 reductase comprising the amino acid sequence of SEQ ID NO: 67.

[0113] In some embodiments, a cell is provided capable of producing noroxymorphone, wherein the cell comprises a morphinone reductase having at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 35, SEQ ID NO: 193, and SEQ ID NO: 185, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% sequence identity thereto.

[0114] In some embodiments, a cell is provided capable of producing noroxymorphone, wherein the cell comprises an uptake transporter having at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 199, SEQ ID NO: 97, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 91, and SEQ ID NO: 207, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% sequence identity thereto.

[0115] In some embodiments, a cell is provided capable of producing noroxymorphone, wherein the cell comprises an uptake transporter having at least 80% sequence identity to the polypeptide of SEQ ID NO: 205, such as at least 90%, at least 95%, at least 98%, or 100% sequence identity thereto.

[0116] In some embodiments, a cell is provided capable of producing noroxymorphone from a 14-hydroxymorphinone substrate, wherein the cell comprises: a) an N-demethylase having at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 179, SEQ ID NO: 31, and SEQ ID NO: 33; b) a morphinone reductase having at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 35, SEQ ID NO: 193, and SEQ ID NO: 185; c) an uptake transporter having at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 199, SEQ ID NO: 97, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 91, and SEQ ID NO: 207; and d) a cytochrome P450 reductase having at least 50% sequence identity to the polypeptide of SEQ ID NO: 191; wherein the sequence identity for each of a), b), c), and d) may be, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100%.

[0117] In some embodiments, a cell is provided capable of producing noroxymorphone from a 14-hydroxycodeinone substrate, wherein the cell comprises: a) an N-demethylase having at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 179, SEQ ID NO: 31, and SEQ ID NO: 33; b) an O-demethylase having at least 50% sequence identity to the polypeptide of SEQ ID NO: 69; c) a morphinone reductase having at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 35, SEQ ID NO: 193, and SEQ ID NO: 185; d) an uptake transporter having at least 50% sequence identity to a polypeptide selected from the groupCase Ref. P322WO IPTector™ consisting of: SEQ ID NO: 199, SEQ ID NO: 97, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 91, and SEQ ID NO: 207; and e) a cytochrome P450 reductase having at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 191 and SEQ ID NO: 67; wherein the sequence identity for each of a), b), c), d), and e) may be, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100%.

[0118] In some embodiments, a cell is provided capable of producing noroxymorphone from an oxycodone substrate, wherein the cell comprises: a) an N-demethylase having at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 179, SEQ ID NO: 31, and SEQ ID NO: 33; b) a morphinone reductase having at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 35, SEQ ID NO: 193, and SEQ ID NO: 185; c) an uptake transporter having at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 199, SEQ ID NO: 97, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 91, and SEQ ID NO: 207; and d) a cytochrome P450 reductase having at least 50% sequence identity to the polypeptide of SEQ ID NO: 191; wherein the sequence identity for each of a), b), c), and d) may be, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100%.

[0119] In some embodiments, a cell is provided capable of producing noroxymorphone from an oxycodone substrate, wherein the cell comprises:a) an N-demethylase having at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 179, SEQ ID NO: 31, and SEQ ID NO: 33;b) an O-demethylase having at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 69, SEQ ID NO: 71, and SEQ ID NO: 211;c) a morphinone reductase having at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 35, SEQ ID NO: 193, and SEQ ID NO: 185;d) an uptake transporter having at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 195, SEQ ID NO: 89, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 85, SEQ ID NO: 201, SEQ ID NO: 9, SEQ ID NO: 203, SEQ ID NO: 11, SEQ ID NO: 205, SEQ ID NO: 13, SEQ ID NO: 5, SEQ ID NO: 207, and SEQ ID NO: 209; ande) a cytochrome P450 reductase having at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 191 and SEQ ID NO: 67; wherein the sequence identity for each of a), b), c), d), and e) may be, for example, at least 60%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%.

[0120] In some embodiments, a cell is provided capable of producing noroxymorphone from an oxycodone substrate, wherein the cell comprises an N-demethylase and an O-demethylase, and wherein the N-demethylase is also an O-demethylase.Case Ref. P322WO IPTector™

[0121] In some embodiments, a cell is provided capable of producing noroxymorphone from an oxycodone substrate, wherein the cell comprises an N-demethylase having at least 50% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO: 179, SEQ ID NO: 31, and SEQ ID NO: 33, and wherein the N-demethylase is also an O-demethylase, for example, at least 60%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%.

[0122] In some embodiments, a cell is provided capable of producing noroxymorphone from a 14-hydroxycodeinone substrate, wherein the cell comprises: a) an N-demethylase having at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 , SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 77, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, and SEQ ID NO: 189; b) a cytochrome P450 reductase having at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, and SEQ ID NO: 191; c) a morphinone reductase having at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 185, and SEQ ID NO: 193; and d) an uptake transporter having at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 183, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, and SEQ ID NO: 209; and optionally e) an O-demethylase having at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 121, SEQ ID NO: 123, and SEQ ID NO: 125; wherein the sequence identity for each of a), b), c), d), and e) may be, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100%.

[0123] In some embodiments, a cell is provided capable of producing noroxymorphone from a 14-hydroxycodeinone substrate, wherein the cell comprises: a) an N-demethylase having at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 179, SEQ ID NO: 31, and SEQ ID NO: 33; b) an O-demethylase having at least 50% sequence identity to the polypeptide of SEQ ID NO: 69; c) a morphinone reductase having at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 35, SEQ ID NO: 193, and SEQ ID NO: 185; d) an uptake transporter having at least 50% sequence identity to a polypeptide selected from the groupCase Ref. P322WO IPTector™ consisting of: SEQ ID NO: 199, SEQ ID NO: 97, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 91, and SEQ ID NO: 207; and e) a cytochrome P450 reductase having at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 191 and SEQ ID NO: 67; wherein the sequence identity for each of a), b), c), d), and e) may be, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100%.

[0124] In some embodiments, a cell is provided capable of producing noroxymorphone from a substrate as defined herein, wherein the cell comprises heterologous polynucleotides encoding: a) an N-demethylase, wherein the N-demethylase has at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 179, SEQ ID NO: 31, and SEQ ID NO: 33; b) an O-demethylase, wherein the O-demethylase has at least 50% sequence identity to the polypeptide of SEQ ID NO: 69; c) a morphinone reductase, wherein the morphinone reductase has at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 35, SEQ ID NO: 193, and SEQ ID NO: 185; d) an uptake transporter, wherein the uptake transporter has at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 199, SEQ ID NO: 97, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 91, and SEQ ID NO: 207; and e) a cytochrome P450 reductase, wherein the cytochrome P450 reductase has at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 191 and SEQ ID NO: 67; wherein the sequence identity for each of a), b), c), d), and e) may be, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100%.

[0125] In some embodiments, a cell is provided capable of producing noroxymorphone from a substrate as defined herein, wherein the N-demethylase and the O-demethylase are the same polypeptide, and wherein the cell comprises a cytochrome P450 reductase and an uptake transporter as defined herein.

[0126] In some embodiments, a cell is provided wherein:a) the N-demethylase has at least 50% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO: 179, SEQ ID NO: 31, and SEQ ID NO: 33;b) the O-demethylase has at least 50% sequence identity to the polypeptide of SEQ ID NO: 69; c) the morphinone reductase has at least 50% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO: 35, SEQ ID NO: 193, and SEQ ID NO: 185;d) the uptake transporter has at least 50% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO: 199, SEQ ID NO: 97, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 91, and SEQ ID NO: 207; and / ore) the cytochrome P450 reductase has at least 50% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO: 191 and SEQ ID NO: 67;Case Ref. P322WO IPTector™ wherein the sequence identity for each of a), b), c), d), and e) may be, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100%.

[0127] In some embodiments, a host cell is provided comprising a heterologous polynucleotide encoding a morphinone reductase polypeptide having at least 60% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 185, and SEQ ID NO: 193, wherein the sequence identity may be, for example, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, such as 100% sequence identity thereto.

[0128] In some embodiments, a host cell is provided comprising a heterologous polynucleotide encoding a demethylase having at least 60% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 69, SEQ ID NO: 71, and SEQ ID NO: 211, wherein the sequence identity may be, for example, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, such as 100% sequence identity thereto.

[0129] In some embodiments, a host cell is provided comprising: (i) a heterologous polynucleotide encoding a morphinone reductase polypeptide having at least 60% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 185, and SEQ ID NO: 193; and / or (ii) a heterologous polynucleotide encoding a demethylase polypeptide having at least 60% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 69, SEQ ID NO: 71, and SEQ ID NO: 211, wherein the sequence identity of each of (i) and (ii) may be, for example, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, such as 100% sequence identity thereto.

[0130] The host cells specified herein may further include one or more of the polypeptides, proteins, and / or polynucleotides disclosed herein.Morphinone reductase

[0131] In the present context, morphinone reductase catalyzes the reduction of e.g. selected morphinones as supported by at least Example 3 wherein 14-hydroxymorphinone and 14-hydroxy normorphinone are successfully and selectively reduced.

[0132] In some embodiments, the morphinone reductase comprises an amino acid sequence of a homolog mutant that exhibits favourable activity for the production of noroxymorphone.

[0133] In some embodiments, the morphinone reductase has at least 50% sequence identity to an- T1 -Case Ref. P322WO IPTector™ amino acid sequence as defined in any one of: SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, and SEQ ID NO: 63.

[0134] In some embodiments, the morphinone reductase is selected from the group consisting of: SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, and SEQ ID NO: 63.

[0135] In some embodiments, the genetically modified host cell is provided comprising a heterologous polynucleotide encoding a morphinone reductase has at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 64.

[0136] In some embodiments, the heterologous polynucleotide encoding a morphinone reductase is selected from the group consisting of: SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 64.

[0137] In some embodiments, the morphinone reductase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, and SEQ ID NO: 63, SEQ ID NO: 185, and SEQ ID NO: 193.

[0138] In some embodiments, the morphinone reductase is selected from the group consisting of: SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 185, and SEQ ID NO: 193.

[0139] In some embodiments, the heterologous polynucleotide encoding a morphinone reductase has at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 186, and SEQ ID NO: 194.

[0140] In some embodiments, the heterologous polynucleotide encoding a morphinone reductase is selected from the group consisting of: SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 186, and SEQ ID NO:Case Ref. P322WO IPTector™ 194.

[0141] While the morphinone reductase of SEQ ID NO: 35 (MorB) is effective, it has been found that certain modified homologs provide improved performance in the production of noroxymorphone. In some embodiments, the morphinone reductase is a homolog mutant such as that defined by SEQ ID NO: 185 or SEQ ID NO: 193, or variants having at least 60% sequence identity to any of SEQ ID NO: 185 or SEQ ID NO: 193, such as at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identity to said sequences.

[0142] In some embodiments, the morphinone reductase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 185, and SEQ ID NO: 193, such as at least 60% sequence identity to said amino acid sequence, such as at least 80% sequence identity to said amino acid sequence, such as at least 95% sequence identity to said amino acid sequence, such as at least 100% sequence identity to said amino acid sequence, for example wherein the morphinone reductase has at least 90% or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 185 and SEQ ID NO: 193.

[0143] In some embodiments, the morphinone reductase further comprises one or more mutations selected from the group consisting of: L115X, L147X, C191X, and P193X, wherein X is any amino acid other than the corresponding amino acid residue in SEQ ID NO: 35, wherein amino acid position numbering is with reference to SEQ ID NO: 35.

[0144] In some embodiments, the morphinone reductase comprises one or more amino acid residues L115, L147, C191, and / or P193, or any conservative substitutions thereof, at amino acid positions corresponding to 115, 147, 191, and 193 of SEQ ID NO: 35, wherein amino acid position numbering is with reference to SEQ ID NO: 35.

[0145] In some embodiments, the morphinone reductase comprises an amino acid sequence having at least 50% sequence identity to an amino acid sequence defined in SEQ ID NO: 49, and wherein the morphinone reductase comprises a substitution Y191C, or any conservative substitutions thereof, wherein amino acid position numbering is with reference to SEQ ID NO: 49.

[0146] In some embodiments, the morphinone reductase comprises one or more substitutions selected from the group consisting of: M115L, P147L, and L193P, or any conservative substitutions thereof, wherein amino acid position numbering is with reference to SEQ ID NO: 49, for example P147L, Y191C, and L193P, or M115L, P147L, Y191C, and L193P.

[0147] In some embodiments, the morphinone reductase comprises an amino acid sequence encoded by a polynucleotide selected from the group consisting of: SEQ ID NO: 36, SEQ ID NO: 38, SEQCase Ref. P322WO IPTector™ ID NO: 40, SEQ. ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 186, and SEQ ID NO: 194.

[0148] In some embodiments, a composition is provided comprising a morphinone reductase as disclosed herein.

[0149] In some embodiments, a composition is provided comprising a morphinone reductase having at least 60% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 185, and SEQ ID NO: 193, wherein the sequence identity may be, for example, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, such as 100% sequence identity thereto.

[0150] In some embodiments, polypeptides are provided having one or more mutations as specifically disclosed herein or any conservative substitutions thereof.N-demethylase

[0151] In some aspects, the genetically modified host cells of the disclosure express, alone or in combination with other heterologous genes of the disclosure, one or more heterologous genes encoding one or more demethylases capable of removing a methyl group from the secondary amine of selected morphinans, i.e. position 17 as set out in Fig. 8. Importantly, some N-demethylases of the present disclosure are also O-demethylases and hence, the capability of being able to perform a N-demethylation on morphinan position 17 as well as O-demethylation on the phenolic oxygen of morphinans may reside in a single polypeptide. Also, a single heterologous polynucleotide of the present disclosure encoding an N-demethylase may also encode an O-demethylase in a single polypeptide. Consequently, in some embodiments, a host cell is provided wherein the N-demethylase is also an O-demethylase.

[0152] In some preferred embodiments, the N-demethylase is a eukaryotic cytochrome P450 (CYP) enzyme. This class of enzyme is a heme-thiolate monooxygenase that is structurally and functionally distinct from prokaryotic P450s, such as the soluble P450 BM3 family. Unlike their soluble bacterial counterparts, eukaryotic P450s are membrane-associated proteins, typically anchored to the endoplasmic reticulum, and are functionally dependent on a separate, non-fused cytochrome P450 reductase (CPR) protein for the electron transfer required for catalysis. This stands in contrast to bacterial enzymes like P450 BM3, which often contain their own reductase domain within a single polypeptide chain. Accordingly, in some embodiments, the N-demethylase is provided in a multi-Case Ref. P322WO IPTector™ component cytochrome P450 system comprising a membrane-associated cytochrome P450 and a separate cytochrome P450 reductase. Accordingly, in some embodiments, the N-demethylase is of fungal or insect origin, and is in some embodiments not of bacterial, plant, or human origin.

[0153] In some embodiments, the structural and functional characteristics of the eukaryotic P450 N-demethylase provide a clear distinction from other enzyme classes. These enzymes are characterized as membrane-associated heme-thiolate monooxygenases. In certain embodiments, the N-demethylase comprises a hydrophobic N-terminal anchor for association with the endoplasmic reticulum membrane. The catalytic function of these enzymes is, in some embodiments, characterized by a conserved P450 protein fold, which may include a heme-binding motif comprising the amino acid sequence FXXGXRXCXG, an arginine-rich K-helix motif comprising the amino acid sequence EXXR, and / or an l-helix proton transfer groove, wherein X is any amino acid. In some embodiments, the functional dependence on a separate cytochrome P450 reductase for catalytic activity is a further distinguishing feature of these eukaryotic P450 enzymes, such as N-demethylases and / or O-demethylases.

[0154] In some embodiments, the N-demethylase is a membrane-associated heme-thiolate monooxygenase, optionally functionally dependent on a separate cytochrome P450 reductase polypeptide for activity.

[0155] In some embodiments, the N-demethylase comprises a hydrophobic N-terminal anchor for membrane association.

[0156] In some embodiments, the N-demethylase comprises a heme-binding motif comprising the amino acid sequence FXXGXRXCXG, wherein X is any amino acid, such as any naturally occurring (proteinogenic) amino acid.

[0157] In some embodiments, the N-demethylase comprises a K-helix motif comprising the amino acid sequence EXXR, wherein X is any amino acid.

[0158] In some embodiments, the N-demethylase comprises an l-helix proton transfer groove motif.

[0159] In particular embodiments for the production of noroxymorphone, the N-demethylase comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 179, SEQ ID NO: 31, and SEQ ID NO: 33, or a sequence having at least 60% sequence identity to any one of said sequences, such as at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity thereto.

[0160] In some embodiments, the N-demethylase is a eukaryotic cytochrome P450 enzyme.

[0161] In some embodiments, N-demethylase is of, or derived from, an insect.

[0162] In some embodiments, wherein the N-demethylase is of the order Lepidoptera.

[0163] In some embodiments, wherein the N-demethylase is of the genus Spodoptera.Case Ref. P322WO IPTector™

[0164] In some embodiments, the N-demethylase is of a species of the genus Spodoptera.

[0165] In some embodiments, N-demethylase is of the species Spodoptera exigua or Spodoptera frugiperda.

[0166] In some embodiments, the N-demethylase is of, or derived from, an insect or a fungus.

[0167] Also importantly, in some embodiments of the present disclosure, some N-demethylases are excluded. In particular in some embodiments, N-demethylases of the present disclosure do not include N-demethylases having insignificant or no activity towards N-demethylation of morphinan position 17, in particular for example for the substrates 14-hydroxymorphinone and / or oxymorphone.

[0168] In some embodiments, the N-demethylase is not of, or derived from, i) a plant, such as an alkaloid producing plant, for example a poppy, ii) a human, and / or iii) a bacterium.

[0169] In some embodiments, the N-demethylase is not of, or derived from, a mammal and / or a plant.

[0170] In some embodiments, the N-demethylase is of the order Lepidoptera. In some embodiments, the N-demethylase is of the genus Helicoverpa. In some embodiments, the N-demethylase is of the species Helicoverpa armigera. In some embodiments, the N-demethylase is of the genus Heliothis. In some embodiments, the N-demethylase is of the species Heliothis virescens. In some embodiments, the N-demethylase is of the genus Spodoptera. In some embodiments, the N-demethylase is of the species Spodoptera exigua.

[0171] In some embodiments, the N-demethylase is a fungal demethylase.

[0172] In some embodiments, the N-demethylase is a fungal demethylase of a genus selected from Rhizopus, Lichtheimia, Syncephalastrum, Cunninghamella, Mucor, Parasitella, Absidia, Choanephora, Bifiguratus and Choanephora.

[0173] In some embodiments, the N-demethylase is a fungal demethylase of a 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.

[0174] In some embodiments, wherein the N-demethylase has one or more conserved amino acids corresponding to positions G103, Hill, K167, E198, R219, L223, 1256, A259, L273, V284, 1309, L314, Q517, L160, N216, or R443 of any one of: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 , SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 77, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, and SEQ ID NO: 119, or conservative substitutions thereof.Case Ref. P322WO IPTector™

[0175] In some embodiments, the one or more conserved amino acid is / are in or near the active site of the demethylase, optionally corresponding to positions G103, Hill and L314 of any one of: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 , SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 77, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, and SEQ ID NO: 119, or conservative substitutions thereof.

[0176] In some embodiments, wherein the N-demethylase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 , SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 77, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, and SEQ ID NO: 181.

[0177] In some embodiments, the N-demethylase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 , SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, and SEQ ID NO: 77; for example wherein the N-demethylase is selected from the group consisting of: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 , SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, and SEQ ID NO: 77; and optionally wherein the substrate is 14-hydroxymorphinone or oxymorphone.

[0178] In some embodiments, the N-demethylase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, and SEQ ID NO: 181; for example wherein the N-demethylase is selected from the group consisting of: SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, and SEQ ID NO: 181; and optionally wherein the substrate is 14-hydroxymorphinone or oxymorphone.

[0179] In some embodiments, the N-demethylase is selected from the group consisting of: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 , SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 77, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, and SEQ ID NO: 119.

[0180] In some embodiments, the genetically modified host cell of the present disclosure comprises a heterologous polynucleotide encoding a N-demethylase having at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 78, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, and SEQ ID NO: 182.Case Ref. P322WO IPTector™

[0181] In some embodiments, the heterologous polynucleotide encoding a N-demethylase has at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, and SEQ ID NO: 78; for example wherein the heterologous polynucleotide encoding a N-demethylase is selected from the group consisting of: SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, and SEQ ID NO: 78; and optionally wherein the substrate is 14-hydroxymorphinone or oxymorphone.

[0182] In some embodiments, the heterologous polynucleotide encoding a N-demethylase has at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, and SEQ ID NO: 182, for example wherein the heterologous polynucleotide encoding a N-demethylase is selected from the group consisting of: SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, and SEQ ID NO: 182; and optionally wherein the substrate is 14-hydroxymorphinone or oxymorphone.

[0183] In some embodiments, the heterologous polynucleotide encoding a N-demethylase is selected from the group consisting of: SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 78 SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, and SEQ ID NO: 120.

[0184] In some embodiments, the N-demethylase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 , SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 77, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, and SEQ ID NO: 189.

[0185] In some embodiments, the N-demethylase has at least 50% sequence identity to an amino acid sequence as defined in SEQ ID NO: 189. In some embodiments, the heterologous polynucleotide encoding a N-demethylase has at least 50% sequence identity to a polynucleotide as defined in SEQ ID NO: 190.

[0186] In some embodiments, the N-demethylase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 , SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 77, and SEQ ID NO: 189; for example wherein the N-demethylase is selected from the groupCase Ref. P322WO IPTector™ consisting of: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 , SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 77, and SEQ ID NO: 189; and optionally wherein the substrate is 14-hydroxymorphinone or oxymorphone.

[0187] In some embodiments, the N-demethylase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, and SEQ ID NO: 189; for example wherein the N-demethylase is selected from the group consisting of: SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, and SEQ ID NO: 189; and optionally wherein the substrate is 14-hydroxymorphinone or oxymorphone.

[0188] In some embodiments, the N-demethylase is selected from the group consisting of: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 , SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 77, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, and SEQ ID NO: 189.

[0189] In some embodiments, the heterologous polynucleotide encoding a N-demethylase has at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 78, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, and SEQ ID NO: 190.

[0190] In some embodiments, the heterologous polynucleotide encoding a N-demethylase has at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 78, and SEQ ID NO: 190; for example wherein the heterologous polynucleotide encoding a N-demethylase is selected from the group consisting of: SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 78, and SEQ ID NO: 190; and optionally wherein the substrate is 14-hydroxymorphinone or oxymorphone.

[0191] In some embodiments, the heterologous polynucleotide encoding a N-demethylase is selected from the group consisting of: SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 78 SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, and SEQ ID NO: 190.

[0192] In some embodiments, the N-demethylases are found in Tables 5-1, 5-2, 5-3, 5-4, 6-1, 6-2, 41-Case Ref. P322WO IPTector™ 1, 41-2, 41-3, 41-4, 41-5, and 41-6 of WO 2021 / 069714 Al and described in WO 2018 / 229306A1, which are hereby incorporated by reference in their entirety.

[0193] In some embodiments, a composition is provided comprising an N-demethylase as disclosed herein.

[0194] In some embodiments, a composition is provided comprising a demethylase polypeptide having at least 60% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 69, SEQ ID NO: 71, and SEQ ID NO: 211, wherein the sequence identity may be, for example, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, such as 100% sequence identity thereto.CYP families

[0195] In some embodiments, a demethylase is provided, wherein the demethylase is a eukaryotic cytochrome P450 enzyme of a cytochrome P450 family selected from CYP64 and CYP75.

[0196] In some embodiments, the N-demethylase is of family CYP6. In some embodiments, the N-demethylase is a fungal cytochrome P450 enzyme of a cytochrome P450 family selected from CYP64 and CYP75.

[0197] In some embodiments, an O-demethylase is provided, wherein the O-demethylase is a eukaryotic cytochrome P450 enzyme of a cytochrome P450 family selected from CYP64 and CYP75.

[0198] In some embodiments, the O-demethylase is a CYP64 O-demethylase.

[0199] In some embodiments, the O-demethylase is a CYP75 O-demethylase.

[0200] In some embodiments, an O-demethylase is provided, wherein the O-demethylase is a CYP64 O-demethylase having at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 73, SEQ ID NO: 121, and SEQ ID NO: 211, wherein the sequence identity may be, for example, at least 60%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%.

[0201] In some embodiments, an O-demethylase is provided, wherein the O-demethylase is a CYP64 O-demethylase and comprises a polypeptide selected from the group consisting of: SEQ ID NO: 73, SEQ ID NO: 121, and SEQ ID NO: 211.

[0202] In some embodiments, a host cell is provided comprising a heterologous polynucleotide encoding a CYP64 O-demethylase, wherein the CYP64 O-demethylase is encoded by a polynucleotide selected from the group consisting of: SEQ ID NO: 74, SEQ ID NO: 122, and SEQ ID NO: 212.

[0203] In some embodiments, an O-demethylase is provided, wherein the O-demethylase is a CYP75 O-demethylase having at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 125 and SEQ ID NO: 71, wherein the sequence identity may be, for example,Case Ref. P322WO IPTector™ at least 60%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%.

[0204] In some embodiments, an O-demethylase is provided, wherein the O-demethylase is a CYP75 O-demethylase and comprises a polypeptide selected from the group consisting of: SEQ ID NO: 125 and SEQ ID NO: 71.

[0205] In some embodiments, a host cell is provided comprising a heterologous polynucleotide encoding a CYP75 O-demethylase, wherein the CYP75 O-demethylase is encoded by a polynucleotide selected from the group consisting of: SEQ ID NO: 126 and SEQ ID NO: 72.

[0206] In some embodiments, a host cell is provided comprising a heterologous polynucleotide encoding an O-demethylase, wherein the O-demethylase is a cytochrome P450 enzyme of a cytochrome P450 family selected from CYP64 and CYP75, and wherein the O-demethylase has at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 73, SEQ ID NO: 121, SEQ ID NO: 211, SEQ ID NO: 125, and SEQ ID NO: 71, wherein the sequence identity may be, for example, at least 60%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%.

[0207] In some embodiments, a host cell is provided comprising a heterologous polynucleotide encoding an O-demethylase, wherein the O-demethylase is a cytochrome P450 enzyme of a cytochrome P450 family selected from CYP64 and CYP75, and wherein the O-demethylase is encoded by a polynucleotide selected from the group consisting of: SEQ ID NO: 74, SEQ ID NO: 122, SEQ ID NO: 212, SEQ ID NO: 126, and SEQ ID NO: 72.Uptake transporters

[0208] In some embodiments of the present disclosure, of particular relevance when the substrate as defined herein is fed to the host cell, an uptake transporter is provided that enable or increase uptake of the substrate into the host cell. This is supported at least by Example 5 where selected uptake transporters were studied for their ability to transport 14-hydroxy morphinone into the cytosol of S. cerevisiae.

[0209] It has now been further verified that the efficiency of the in vivo bioconversion process is dependent on the effective transport of the fed substrate into the host cell's cytosol if the substrate is not produce de novo. A range of additional heterologous uptake transporters, including those defined by the amino acid sequences of SEQ ID NO: 195, 197, 199, 201, 203, 205, 207, and 209, and encoded by the polynucleotide sequences of SEQ ID NO: 196, 198, 200, 202, 204, 206, 208, and 210 have been identified. As demonstrated in Example 14 and Fig. 16, these transporters are effective for the uptake of 14-hydroxycodeinone. Furthermore, as shown in Example 15 and Figs. 17-19, selected transporters also facilitate the efficient uptake of other morphinan substrates such as oxycodone. Experimental data shows that without the expression of a heterologous transporter, there is noCase Ref. P322WO IPTector™ detectable product formation, demonstrating that the transporter is an important element for enabling an efficient in vivo bioconversion from an exogenously supplied substrate.

[0210] While a range of transporters may be functional, certain transporters have been identified as being particularly effective for the uptake of morphinan substrates for the production of noroxymorphone. The high performance of these specific transporters represents a non-obvious selection from the vast number of available transporter candidates. In some embodiments, the uptake transporter comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 199, SEQ. ID NO: 97, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 91, and SEQ ID NO: 207, or a sequence having at least 50% identity thereto.

[0211] In some embodiments, the cell comprises an uptake transporter having at least 50% sequence identity to an amino acid sequence defined in any one of: SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 183, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, and SEQ ID NO: 209 or a sequence having at least 60% sequence identity to any one of said sequences, such as at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity thereto.

[0212] In some embodiments, the cell comprises an uptake transporter having at least 50% sequence identity to an amino acid sequence defined in any one of: SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, and SEQ ID NO: 183.

[0213] In some embodiments, the cell comprises an uptake transporter having at least 50% sequence identity to an amino acid sequence defined in any one of: SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13; for example wherein the uptake transporter is selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13, and optionally wherein the substrate is 14-hydroxymorphinone or oxymorphone.

[0214] In some embodiments, the uptake transporter is selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, and SEQ ID NO: 183.Case Ref. P322WO IPTector™

[0215] In some embodiments, the cell comprises a heterologous polynucleotide encoding an uptake transporter having at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 184, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, and SEQ ID NO: 210.

[0216] In some embodiments, the cell comprises a heterologous polynucleotide encoding an uptake transporter having at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, and SEQ ID NO: 184.

[0217] In some embodiments, the cell comprises a heterologous polynucleotide encoding an uptake transporter having at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14; for example wherein the polynucleotide encoding an uptake transporter is selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14.

[0218] In some embodiments, the cell comprises a heterologous polynucleotide encoding an uptake transporter having at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, and SEQ ID NO: 184; for example wherein the polynucleotide encoding an uptake transporter is selected from the group consisting of: SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, and SEQ ID NO: 184.

[0219] In some embodiments, the cell comprises a polynucleotide encoding an uptake transporter selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, and SEQ ID NO: 184.

[0220] In some embodiments, the host cell contains an uptake transporter as described in WO 2021 / 069714 Al, W02020078837, or WO2018229306, hereby incorporated by reference in theirCase Ref. P322WO IPTector™ entirety.

[0221] In some embodiments, the present disclosure provides an uptake transporter which is a member of the purine permease (PUP), or NRT1 / PTR (NPF) family.

[0222] In some embodiments, the uptake transporter is a transporter protein belonging to the NRT1 / PTR (NPF) transporter protein family or the purine permease (PUP) transporter family.

[0223] In some embodiments, an uptake transporter is provided, wherein the uptake transporter is an integral membrane protein comprising at least 8 predicted transmembrane segments, such as at least 10 predicted transmembrane segments, such as at least 12 predicted transmembrane segments.

[0224] In some embodiments, an uptake transporter is provided, wherein the uptake transporter is localized to the plasma membrane of the host cell.

[0225] In some embodiments, an uptake transporter is provided, wherein the uptake transporter transports a morphinan substrate by coupling transport to an ion gradient across the plasma membrane.

[0226] In some embodiments, a genetically modified host cell is provided, wherein the host cell produces a detectable amount of noroxymorphone when cultured in the presence of a substrate, and wherein a corresponding host cell lacking the uptake transporter produces no detectable noroxymorphone under otherwise identical conditions.

[0227] In some embodiments, a genetically modified host cell is provided, wherein the host cell comprises an uptake transporter having at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 183, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, and SEQ ID NO: 209.

[0228] In some embodiments, a genetically modified host cell is provided, wherein the host cell comprises an uptake transporter having at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 199, SEQ ID NO: 97, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 91, and SEQ ID NO: 207.

[0229] In some embodiments, the sequence identity in any of the preceding uptake transporter embodiments may be, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100%.O-demethylase

[0230] In some embodiments, a host cell is provided as defined herein comprising an O-demethylase.Case Ref. P322WO IPTector™

[0231] In some embodiments, an O-demethylase is provided, wherein the O-demethylase is a polypeptide capable of catalyzing an oxidative O-demethylation of a morphinan substrate, thereby converting the morphinan substrate to a corresponding O-demethylated morphinan product.

[0232] In some embodiments, an O-demethylase is provided, wherein the O-demethylase is a eukaryotic cytochrome P450 polypeptide.

[0233] In some embodiments, the O-demethylase does not comprise a fused reductase domain.

[0234] In some embodiments, the O-demethylase is also a eukaryotic cytochrome P450 enzyme, exhibiting the characteristic structural features of being membrane-associated and functionally dependent on a separate cytochrome P450 reductase partner. This provides a clear distinction from other O-demethylating enzymes such as plant dioxygenases. In a particularly preferred embodiment for the production of noroxymorphone from 14-hydroxycodeinone, the O-demethylase comprises the amino acid sequence of the mutant defined by SEQ ID NO: 69, or a sequence having at least 60% sequence identity thereto, such as at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity thereto.

[0235] In some embodiments, the O-demethylase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 121, SEQ ID NO: 123, and SEQ ID NO: 125.

[0236] In some embodiments, the O-demethylase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, and SEQ ID NO: 75; for example wherein the O-demethylase is selected from the group consisting of: SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, and SEQ ID NO: 75; and optionally wherein the substrate is 14-hydroxycodeinone.

[0237] In some embodiments, the O-demethylase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, and SEQ ID NO: 75; for example wherein the O-demethylase is selected from the group consisting of: SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, and SEQ ID NO: 75; and optionally wherein the substrate is 14-hydroxycodeinone.

[0238] In some embodiments, the O-demethylase is selected from the group consisting of: SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 121, SEQ ID NO: 123, and SEQ ID NO: 125.

[0239] In some embodiments, the heterologous polynucleotide encoding an O-demethylase has at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, and SEQ ID NO: 212.Case Ref. P322WO IPTector™

[0240] In some embodiments, the heterologous polynucleotide encoding an O-demethylase has at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 76, SEQ ID NO: 122, SEQ ID NO: 124, and SEQ ID NO: 126; for example wherein the heterologous polynucleotide encoding an O-demethylase is selected from the group consisting of: SEQ ID NO: 76, SEQ ID NO: 122, SEQ ID NO: 124, and SEQ ID NO: 126; and optionally wherein the substrate is 14-hydroxycodeinone.

[0241] In some embodiments, the heterologous polynucleotide encoding an O-demethylase is selected from the group consisting of: SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 122, SEQ ID NO: 124, and SEQ ID NO: 126.

[0242] In some embodiments the O-demethylases are found in Tables 6-1 or are the bifunctional demethylases SEQ ID NO: 218, 220, 222, 224, 226, 228, 236, 240, 250, 252, 254 or 268 of WO 2021 / 069714A1, or O-demethylases described in WO 2018 / 229306A1, which are hereby incorporated by reference in their entirety.Bifunctional demethylases

[0243] In some embodiments, a demethylase is provided, for example as part of the host cell defined herein, wherein the demethylase is capable of catalyzing both (i) N-demethylation and (ii) O-demethylation of a morphinan substrate.

[0244] In some embodiments, a genetically modified host cell is provided, wherein the cell comprises a heterologous polynucleotide encoding an N-demethylase, and wherein the N-demethylase is also an O-demethylase.Cytochrome P450 reductase

[0245] The present disclosure also includes a number of "CPRs", namely a cytochrome P450 reductases catalyzing the electron transfer (from NADPH) to selected cytochrome P450 enzymes of the pathway steps disclosed herein.

[0246] As the eukaryotic N-demethylase and O-demethylase enzymes of the present disclosure are generally dependent on electron transfer for catalytic activity, in some embodiments the engineered host cell is provided with a compatible cytochrome P450 reductase (CPR), optionally a heterologous CPR. The choice of CPR can influence the efficiency of demethylation, as illustrated by the data in Fig.14. As shown therein, expression of the N-demethylase of SEQ ID NO: 190 with the CPR of SEQ ID NO: 192 (in strain SOD1859) resulted in higher N-demethylation and higher production of noroxymorphone compared to expression with the CPR of SEQ ID NO: 66 (in strain SOD1829). Accordingly, in some embodiments, selection and / or co-expression of a CPR is provided to improve demethylation and / or product formation. In some embodiments, a CPR for use with an N-Case Ref. P322WO IPTector™ demethylase having at least 50% sequence identity to SEQ ID NO: 15 may comprise SEQ ID NO: 165 or a variant thereof having at least 50% sequence identity to SEQ ID NO: 165.

[0247] In some embodiments, the polynucleotide encoding the cytochrome P450 reductase is heterologous to the host cell.

[0248] In some embodiments, the host cell is engineered to express a selected pairing of an N-demethylase and a CPR. A non-limiting example combination comprises an N-demethylase comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 179, such as at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity thereto, expressed in combination with a cytochrome P450 reductase comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 191, such as at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity thereto. In some embodiments, the cytochrome P450 reductase is of, or derived from, an insect or a fungus, such as of the genus Helicoverpa.

[0249] In some embodiments, the cytochrome P450 reductase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, and SEQ ID NO: 173.

[0250] In some embodiments, the cytochrome P450 reductase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 65, and SEQ ID NO: 67; for example wherein the cytochrome P450 reductase is selected from the group consisting of: SEQ ID NO: 65, and SEQ ID NO: 67.

[0251] In some embodiments, the cytochrome P450 reductase has at least 50% sequence identity to an amino acid sequence as defined in any one of SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, and SEQ ID NO: 173; for example wherein the cytochrome P450 reductase is selected from the group consisting of: SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, and SEQ ID NO: 173.

[0252] In some embodiments, the cytochrome P450 reductase is selected from the group consisting of: SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, and SEQ ID NO: 173.

[0253] In some embodiments, the cell comprises a polynucleotide, optionally heterologous, encoding a cytochrome P450 reductase having at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, and SEQ ID NO: 174.

[0254] In some embodiments, the cell comprises a polynucleotide, optionally heterologous, encoding a cytochrome P450 reductase having at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 66, and SEQ ID NO: 68; for example wherein the polynucleotide encoding aCase Ref. P322WO IPTector™ cytochrome P450 reductase is selected from the group consisting of: SEQ ID NO: 66, and SEQ ID NO: 68.

[0255] In some embodiments, the cell comprises a polynucleotide, optionally heterologous, encoding a cytochrome P450 reductase having at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, and SEQ ID NO: 174; for example wherein the polynucleotide encoding a cytochrome P450 reductase is selected from the group consisting of: SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, and SEQ ID NO: 174.

[0256] In some embodiments, the cell comprises a polynucleotide, optionally heterologous, encoding a cytochrome P450 reductase is selected from the group consisting of: SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, and SEQ ID NO: 174.

[0257] In some embodiments, the cytochrome P450 reductase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, and SEQ ID NO: 191.

[0258] In some embodiments, the cytochrome P450 reductase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 65, SEQ ID NO: 67, and SEQ ID NO: 191; for example wherein the cytochrome P450 reductase is selected from the group consisting of: SEQ ID NO: 65, SEQ ID NO: 67, and SEQ ID NO: 191.

[0259] In some embodiments, the cytochrome P450 reductase has at least 50% sequence identity to an amino acid sequence as defined in any one of SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, and SEQ ID NO: 191; for example wherein the cytochrome P450 reductase is selected from the group consisting of: SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, and SEQ ID NO: 191.

[0260] In some embodiments, the cytochrome P450 reductase is selected from the group consisting of: SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, and SEQ ID NO: 191.

[0261] In some embodiments, the cell comprises a polynucleotide, optionally heterologous, encoding a cytochrome P450 reductase having at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, and SEQ ID NO: 192. In some embodiments, the cytochrome P450 reductase has at least 50% sequence identity to a polynucleotide as defined inCase Ref. P322WO IPTector™ any one of: SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, and SEQ ID NO: 192.

[0262] In some embodiments, the cell comprises a polynucleotide, optionally heterologous, encoding a cytochrome P450 reductase having at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 66, SEQ ID NO: 68, and SEQ ID NO: 192; for example wherein the polynucleotide encoding a cytochrome P450 reductase is selected from the group consisting of: SEQ ID NO: 66, SEQ ID NO: 68, and SEQ ID NO: 192.

[0263] In some embodiments, the cell comprises a polynucleotide, optionally heterologous, encoding a cytochrome P450 reductase having at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, and SEQ ID NO: 192; for example wherein the polynucleotide encoding a cytochrome P450 reductase is selected from the group consisting of: SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, and SEQ ID NO: 192.

[0264] In some embodiments, the cell comprises a polynucleotide, optionally heterologous, encoding a cytochrome P450 reductase is selected from the group consisting of: SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, and SEQ ID NO: 192.

[0265] In some embodiments the demethylase-CPRs are those described in 2021 / 069714A1 or WO 2018 / 229306A1, which are hereby incorporated in their entirety.Decreasing activity of endogenous genes

[0266] In some embodiments, the host cell as defined herein is provided wherein one or more further native or endogenous genes of the cell is attenuated, disrupted and / or deleted to, for example, decrease formation of unwanted byproducts competing with the production of the target morphinan. This is at least demonstrated by Example 7 and Fig. 11.

[0267] In some embodiments, the one or more further native or endogenous genes are selected from the group consisting of: a aldehyde reductase, such as ARI1 (for example SEQ ID NO: 2); and an aldose reductase, such as GRE3 (for example SEQ ID NO: 4), and / or GCY1 (for example SEQ ID NO: 80).

[0268] In some embodiments, the one or more further native or endogenous genes are deleted and selected from the group consisting of: a aldehyde reductase, such as ARI1 (for example SEQ ID NO: 2); and an aldose reductase, such as GRE3 (for example SEQ ID NO: 4), and / or GCY1 (for example SEQ ID NO: 80).

[0269] In some embodiments, the one or more further native or endogenous genes are deleted. InCase Ref. P322WO IPTector™ some embodiments, native genes that are disrupted are those described in WO2018029282 which is hereby incorporated in its entirety.Operative metabolic pathway

[0270] The host cells as defined herein may comprise one or more operative metabolic pathway. Also, if the cell is specified as a particular genus or species that is known to include one or more endogenous operative metabolic pathways, such as the shikimate pathway, specifying the host cell to a particular genus or species implicitly includes endogenous pathways for the specific genus or species.

[0271] In some embodiments, cell comprises an operative metabolic pathway comprising one or more polypeptides producing the substrate from one or more precursors.

[0272] In some embodiments, the one or more precursors are selected from the group consisting of: oripavine, thebaine, and a carbon source, such as glucose.

[0273] In some embodiments, the genetically modified host cell is provided, wherein the one or more precursors comprise oripavine, and the operative metabolic pathway comprises one or more polypeptides selected from:a) A 14-OH hydroxylase, such as a 14-OH hydroxylase capable of converting oripavine to 14-hydroxymorphinone,b) A cytochrome P450 reductase, such as a cytochrome P450 reductase capable of electron transfer to the 14-OH hydroxylase,c) An uptake transporter, such as an uptake transporter capable of transporting oripavine into the cell,d) A morphinone reductase, such as a morphinone reductase as defined herein,e) A N-demethylase, such as a N-demethylase as defined herein, andf) A further cytochrome P450 reductase, such as a cytochrome P450 reductase capable of electron transfer to the N-demethylase.

[0274] In some embodiments, the genetically modified host cell is provided, wherein the one or more precursors comprise thebaine, and the operative metabolic pathway comprises one or more polypeptides selected from:a) A 14-OH hydroxylase, such as a 14-OH hydroxylase capable of converting thebaine to 14-hydroxycodeinone,b) A cytochrome P450 reductase, such as a cytochrome P450 reductase capable of electron transfer to the 14-OH hydroxylase,c) An uptake transporter, such as an uptake transporter capable of transporting thebaine into the cell,Case Ref. P322WO IPTector™ d) A morphinone reductase, such as a morphinone reductase as defined herein,e) A N-demethylase, such as a N-demethylase as defined herein, andf) A further cytochrome P450 reductase, such as a cytochrome P450 reductase capable of electron transfer to the N-demethylase.

[0275] In some embodiments, the genetically modified host cell is provided, wherein the one or more precursors comprise a carbon source, such as glucose, and the operative metabolic pathway comprises one or more polypeptides selected from:a) A tyrosine hydroxylase (TH), such as a tyrosine hydroxylase capable of converting L-tyrosine to L-DOPA;b) A dopa decarboxylase (DODC), such as a dopa decarboxylase capable of converting L-DOPA to dopamine;c) A tyrosine decarboxylase (TYDC), such as a tyrosine decarboxylase capable of converting L-DOPA to dopamine,d) A hydroxyphenylpyruvate decarboxylase (HPPDC), such as a hydroxyphenylpyruvate decarboxylase capable of converting 4-HPP into 4-HPPA;e) A norcoclaurine synthase (NCS), such as a norcoclaurine synthase capable of converting dopamine and 4-HPAA into (S)-norcoclaurine;f) A 6-O-methyltransferase (6-OMT), such as a 6-O-methyltransferase capable of converting (S)-norcoclaurine to (S)-Coclaurine,g) A N-methylcoclaurine 3'-monooxygenase (NMCH, CYP80), such as a N-methylcoclaurine 3'-monooxygenase catalyzing conversion of (S)-Coclaurine to (S)-3'-hydroxycoclaurine and / or (S)-N-Methylcoclaurine to (S)-3'-Hydroxy-N-Methylcoclaurine,h) A coclaurine N-methyltransferase (CNMT), such as a coclaurine N-methyltransferase capable of converting (S)-coclaurine to (S)-N-methylcoclaurine;i) A 3'-hydroxy-N-methyl-(S)-coclaurine 4'-O-methyltransferase (4-OMT), such as a 3'-hydroxy-N-methyl-(S)-coclaurine 4'-O-methyltransferase catalyzing conversion of (S)-3'-Hydroxy-N-Methylcoclaurine to (S)-reticuline;j) A dihydroreticuline synthase / dihydroreticuline reductase (DRS-DRR), such as a dihydroreticuline synthase and reductase capable of converting (S)-reticuline to (R)-reticuline, k) A salutaridine synthase (SAS), such as a salutaridine synthase capable of converting (R)-reticuline to salutaridine,l) A salutaridine reductase (SAR), such as a salutaridine reductase capable of converting salutaridine to salutaridinol,m) A salutaridinol-7-O-acetyltransferase (SAT), such as a salutaridinol-7-O-acetyltransferaseCase Ref. P322WO IPTector™ capable of converting salutaridinol to 7-O-acetylsalutaridinol,n) A thebaine synthase (THS), such as a thebaine synthase capable of converting 7-O-acetylsalutaridinol into thebaine, ando) One or more cytochrome P450 reductases (CPRs), such as a cytochrome P450 reductases capable of electron transfer to the polypeptides.

[0276] In some embodiments, the genetically modified host cell is provided, wherein the one or more precursors comprise a carbon source, such as glucose, and the operative metabolic pathway comprises one or more polypeptides selected from:a) A tyrosine hydroxylase (TH), such as a tyrosine hydroxylase capable of converting L-tyrosine to L-DOPA;b) A dopa decarboxylase (DODC), such as a dopa decarboxylase capable of converting L-DOPA to dopamine;c) A tyrosine decarboxylase (TYDC), such as a tyrosine decarboxylase capable of converting L-DOPA to dopamined) A monoamine oxidase (MAO), such as a monoamine oxidase catalyzing conversion of dopamine to 3,4 DHPAA,e) A norcoclaurine synthase (NCS), such as a norcoclaurine synthase capable of converting dopamine and 3,4 DHPAA into (S)-norlaudanosoline;f) A 6-O-methyltransferase (6-OMT), such as a 6-O-methyltransferase capable of converting (S)-norlaudanosoline into (S)-6-O-methyl- norlaudanosoline,g) A coclaurine N-methyltransferase (CNMT), such as a coclaurine N-methyltransferase capable of converting (S)-6-O-methyl- norlaudanosoline into (S)-3'-Hydroxy-N-Methylcoclaurine or (S)-norreticuline into (S)-reticulineh) A 3'-hydroxy-N-methyl-(S)-coclaurine 4'-O-methyltransferase (4-OMT), such as a 3'-hydroxy-N-methyl-(S)-coclaurine 4'-O-methyltransferase catalyzing conversion of (S)-3'-Hydroxy-N-Methylcoclaurine to (S)-reticuline or (S)-6-O-methyl- norlaudanosoline into (S)-norreticuline;i) A dihydroreticuline synthase / dihydroreticuline reductase (DRS-DRR), such as a dihydroreticuline synthase and reductase capable of converting (S)-reticuline to (R)-reticuline, j) A salutaridine synthase (SAS), such as a salutaridine synthase capable of converting (R)-reticuline to salutaridine,k) A salutaridine reductase (SAR), such as a salutaridine reductase capable of converting salutaridine to salutaridinol,l) A salutaridinol-7-O-acetyltransferase (SAT), such as a salutaridinol-7-O-acetyltransferase capable of converting salutaridinol to 7-O-acetylsalutaridinol,Case Ref. P322WO IPTector™ m) A thebaine synthase (THS), such as a thebaine synthase capable of converting converting 7-0-acetylsalutaridinol into thebaine, andm) One or more cytochrome P450 reductases (CPRs), such as a cytochrome P450 reductases capable of electron transfer to the polypeptides.

[0277] In some embodiments, the polypeptides of the operative metabolic pathway disclosed herein wherein the one or more precursors comprise a carbon source, such as glucose have at least 60% sequence identity, such as at least 61%, such as at least 62%, such as at least 63%, such as at least 64%, such as at least 65%, such as at least 66%, such as at least 67%, such as at least 68%, such as at least 69%, such as at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or such as at least 99%, for example 100% sequence identity to one or more of: SEQ ID NO: SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, and SEQ ID NO: 163.

[0278] In some embodiments, the polynucleotides encoding the polypeptides of the operative metabolic pathway disclosed herein wherein the one or more precursors comprise a carbon source, such as glucose have at least 60% sequence identity, such as at least 61%, such as at least 62%, such as at least 63%, such as at least 64%, such as at least 65%, such as at least 66%, such as at least 67%, such as at least 68%, such as at least 69%, such as at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or such as at least 99%, for example 100% sequence identity to one or more of: SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, and SEQ ID NO: 164.

[0279] In some embodiments, the cell comprises a plurality of the operative metabolic pathways (i.e.Case Ref. P322WO IPTector™ corresponding polypeptides that make up the pathway) as defined herein, and optionally one or more endogenous pathways from the host cell genus or species disclosed herein.

[0280] In some embodiments, the cell further comprises a SAM alkyl transferase, such as a S-adenosylmethionine (SAM) dependent alkyl transferase capable of converting noroxymorphone to an opioid, such as naloxone, naltrexone, or nalmefene. In some embodiments, suitable SAM alkyl transferases can be found in Table 5, Example 16, and Example 20 of W02018075670A1 hereby incorporated by reference in its entirety.

[0281] In some embodiments, the cell further comprises a 14-OH hydroxylase, such as a 14-OH hydroxylase capable of converting oripavine to 14-OH morphinone or converting thebaine to 14-OH codeinone. In some embodiments, suitable 14-OH hydroxylases can be found in WO2024073755A3 hereby incorporated in its entirety.Cells

[0282] In some embodiments, the cell is an eukaryote selected from the group consisting of a mammalian, insect, plant, or fungal cell.

[0283] In some embodiments, the cell is a plant cell of the genus Physcomitrella or Papaver or Nicotiana.

[0284] In some embodiments, the cell is a plant cell of the species Papaver soniferum or Nicotiana benthamiana.

[0285] In some embodiments, the cell is a fungal cell selected from the phylas consisting of Ascomycota, Basidiomycota, Neocallimastigomycota, Glomeromycota, Blastocladiomycota, Chytridiomycota, Zygomycota, Oomycota and Microsporidia.

[0286] In some embodiments, the fungal cell is a yeast selected from the group consisting of ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and Fungi Imperfecti yeast (Blastomycetes).

[0287] In some embodiments, the 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.

[0288] In some embodiments, the fungal cell is a filamentous fungus.

[0289] In some embodiments, the filamentous fungal cell is selected from the phylas consisting of Ascomycota, Eumycota and Oomycota.

[0290] In some embodiments, the filamentous fungal cell is selected from the genera consisting of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Case Ref. P322WO IPTector™ 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.

[0291] In some embodiments, the filamentous fungal cell is 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.Polypeptides and compositions

[0292] In some embodiments, a polypeptide is provided selected from the group consisting of: an N-demethylase as defined herein, a morphinone reductase as defined herein, an O-demethylase as defined herein, a cytochrome P450 reductase as defined herein, and an uptake transporter as defined herein.

[0293] In some embodiments, a composition is provided comprising the polypeptide as defined herein, and optionally one or more excipients.

[0294] In some embodiments, a composition is provided comprising: (i) a morphinone reductase polypeptide having at least 60% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 185, and SEQ ID NO: 193; and / or (ii) a demethylase polypeptide having at least 60% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO: 31, SEQ ID NO:Case Ref. P322WO IPTector™ 33, SEQ ID NO: 69, SEQ ID NO: 71, and SEQ ID NO: 211, wherein the sequence identity of each of (i) and (ii) may be, for example, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, such as 100% sequence identity thereto.MethodsReducing a substrate

[0295] In some embodiments, a method is provided for reducing a substrate using a morphinone reductase, the method comprising contacting the substrate with the morphinone reductase.

[0296] In some embodiments, the substrate is selected from the group consisting of: Morphinone, Oxymorphinone, oxymorphone, Codeinone, Codeine, Morphine, 14-hydroxycodeinone, 14-hydroxy norcodeinone, 14-hydroxymorphinone, 14-hydroxynormorphinone, Neopine, neopinone, Neomorphinone, and Neomorphine.

[0297] In some embodiments, the substrate has been prepared by chemical synthesis.

[0298] In some embodiments, the morphinone reductase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 185, and SEQ ID NO: 193.

[0299] In some embodiments, the morphinone reductase comprises one or more mutations selected from the group consisting of: L115X, L147X, C191X, and P193X, wherein X is any amino acid other than the corresponding amino acid residue in SEQ ID NO: 35, and wherein amino acid position numbering is with reference to SEQ ID NO: 35.

[0300] In some embodiments, the morphinone reductase comprises an amino acid sequence having at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 185, and SEQ ID NO: 193, and wherein the morphinone reductase comprises one or more substitutions at amino acid positions corresponding to 115, 147, 191 and / or 193 of SEQ ID NO: 35, wherein the amino acid residue at each substituted position differs from the amino acid residue at the corresponding position of SEQ ID NO: 35, wherein amino acid position numbering is with reference to SEQ ID NO: 35.

[0301] In some embodiments, the morphinone reductase comprises one or more amino acid residues L147, C191 and P193 at amino acid positions corresponding to 147, 191 and 193 of SEQ ID NO: 35, wherein amino acid position numbering is with reference to SEQ ID NO: 35.Case Ref. P322WO IPTector™

[0302] In some embodiments, the morphinone reductase further comprises amino acid residue L115 at an amino acid position corresponding to 115 of SEQ ID NO: 35, wherein amino acid position numbering is with reference to SEQ ID NO: 35.

[0303] In some embodiments, the morphinone reductase comprises an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 49, and wherein the morphinone reductase comprises a substitution Y191C, wherein amino acid position numbering is with reference to SEQ ID NO: 49.

[0304] In some embodiments, the morphinone reductase further comprises one or more substitutions selected from the group consisting of: M115L, P147L, and L193P, wherein amino acid position numbering is with reference to SEQ ID NO: 49, for example P147L, Y191C, and L193P; or for example M115L, P147L, Y191C, and L193P.

[0305] In some embodiments, the method comprises using the morphinone reductase selected from the group consisting of: SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 185, and SEQ ID NO: 193.Producing noroxymorphone

[0306] In some embodiments, a method is provided for producing noroxymorphone from a substrate,(noroxymorphone),or a pharmaceutically acceptable salt thereof, wherein the method comprises subjecting the substrate to i) a morphinone reductase, and ii) an N-demethylase, and optionally iii) an O-demethylase.

[0307] In some embodiments, the method for producing noroxymorphone disclosed herein comprises a method for reducing a substrate as set out specifically herein, optionally comprising one or more further embodiments disclosed in this section.

[0308] In some embodiments, the substrate is selected from the group consisting of: 14-hydroxymorphinone, 14-hydroxynormorphinone, oxymorphone, 14-hydroxycodeinone, oxycodone, and a combination thereof.

[0309] In some embodiments, a method is provided for producing noroxymorphone from a substrate, wherein the method comprises culturing a cell as defined herein under conditions allowing the cell to convert the substrate to noroxymorphone, and recovering noroxymorphone from theCase Ref. P322WO IPTector™ culture.

[0310] In some embodiments, a method is provided, wherein the method comprises feeding 14-hydroxycodeinone to the cell, and culturing the cell under aerobic conditions.

[0311] In some embodiments, a method is provided, wherein the method comprises performing O-demethylation before N-demethylation.

[0312] In some embodiments, a method is provided, wherein the method comprises performing N-demethylation before O-demethylation.

[0313] In some embodiments, a method is provided, wherein the method comprises recovering noroxymorphone and converting noroxymorphone to an opioid by chemical synthesis.

[0314] In some embodiments, the method further comprises preparing the substrate by chemical synthesis, for example wherein the substrate is a) 14-hydroxymorphinone prepared from oripavine or wherein the substrate is b) 14-hydroxycodeinone prepared from thebaine, such as wherein the chemical synthesis comprises oxidation.

[0315] In some embodiments, the method further comprises a cytochrome P450 reductase, an uptake transporter, or a combination thereof.

[0316] In some embodiments, the morphinone reductase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, and SEQ ID NO: 63.

[0317] In some embodiments, the morphinone reductase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, and SEQ ID NO: 63.

[0318] In some embodiments, the N-demethylase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 , SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 77, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, and SEQ ID NO: 181.

[0319] In some embodiments, the N-demethylase is selected from the group consisting of: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 , SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 77, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, and SEQ ID NO: 181.

[0320] In some embodiments, the method involves contacting the substrate with an uptakeCase Ref. P322WO IPTector™ transporter.

[0321] In some embodiments, the method involves contacting the substrate with an uptake transporter having at least 50% sequence identity to an amino acid sequence defined in any one of: SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 183, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, and SEQ ID NO: 209.

[0322] In some embodiments, the method involves contacting the substrate with an uptake transporter having at least 50% sequence identity to an amino acid sequence defined in any one of: SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, and SEQ ID NO: 183.

[0323] In some embodiments, the uptake transporter is selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, and SEQ ID NO: 183.

[0324] In some embodiments, the method comprises an O-demethylase having at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, and SEQ ID NO: 211.

[0325] In some embodiments, the O-demethylase is selected from the group consisting of: SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, and SEQ ID NO: 211.

[0326] In some embodiments, the method further comprises a cytochrome p450 reductase, for example wherein the cytochrome P450 reductase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, and SEQ ID NO: 173.

[0327] In some embodiments, a method is provided comprising:a) Culturing the genetically modified host cell as defined herein at conditions allowing the cell to produce noroxymorphone; andb) Optionally recovering and / or isolating the noroxymorphone.

[0328] In some embodiments, the method further comprises feeding a substrate to the cell, such asCase Ref. P322WO IPTector™ wherein the substrate is selected from the group consisting of: 14-hydroxymorphinone, 14-hydroxynormorphinone, oxymorphone, 14-hydroxycodeinone, and a combination thereof.

[0329] In some embodiments, the method further comprising one or more elements selected from: a) culturing the cell culture in a nutrient medium;b) culturing the cell culture under aerobic or anaerobic conditionsc) 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; andf) culturing the cell culture for between 10 hours to 30 days.

[0330] In some embodiments, one or more steps of the method are performed in vitro.Producing opioids

[0331] In some embodiments, a method is provided for producing an opioid from noroxymorphone, the method comprising:a) producing noroxymorphone using a method as defined herein, andb) converting noroxymorphone to the opioid.

[0332] In some embodiments, step b) is performed using chemical synthesis.

[0333] In some embodiments, the opioid is selected from the group consisting of: Naloxone, Naltrexone, Nalmefene, Methylnaltrexone halide, such as Methylnaltrexone bromide, Nalfurafine, Naloxegol, and Nalbuphine.

[0334] In some embodiments, the method for producing an opiod disclosed herein comprises a method for reducing a substrate as set out specifically herein, optionally comprising one or more further embodiments disclosed in this section or the following sections.Naloxone

[0335] In some embodiments, a method is provided wherein the opioid is naloxone,(naloxone),and step b) comprises:i. allylation of noroxymorphone by an allylating agent, such as an allylating agent selected from the group consisting of: an allyl halide, for example allyl bromide, allyl chloride, or allyl iodide; allylCase Ref. P322WO IPTector™ acetate; allyl sulfate; allyl sulfonate; allyl carbonate; an allyl alcohol; and an allyl haloformate; such as allyl chloroformate; optionally in the presence of a base, such as triethylamine for example at a temperature of from 50 to 90 °C, such as 70 °C; and / or a catalyst;ii. reductive amination of an allyl aldehyde comprising condensation with an allyl aldehyde, such as acrolein, to form an imine; followed by reduction of the imine to provide naloxone; or ill. peptide coupling of noroxymorphone with a carboxylic acid, for example acrylic acid, to form a peptide, followed by reduction of the peptide to provide naloxone, for example by reduction using LiAIH4.Naltrexone

[0336] In some embodiments, the method is provided wherein the opioid is naltrexone,(naltrexone),and step b) comprises:i. alkylation of noroxymorphone by a cyclopropylmethylating agent, such as a cyclopropylmethyl halide, for example cyclopropylmethyl bromide.

[0337] In some embodiments, step b) further comprises adding a polar aprotic solvent, such as N-ethyl-2-pyrrolidone, and / or toluene.

[0338] In some embodiments, step b) further comprises adding a base, such as potassium bicarbonate, and / or DIPEA.

[0339] In some embodiments, step b) is conducted at a temperature of from 45°C to 80°C, such as from 52° to 60°C.

[0340] In some embodiments, the reaction time of step b) is from 12 hours to 36 hours, such as from 17 to 24 hours.

[0341] In some embodiments, step b) comprises adding a polar aprotic solvent, such as dimethylacetamide (DMA), optionally at a temperature of from 40 °C to 90 °C, such as from 50 °C to 70 °C; and optionally in the presence of a base, such as a bicarbonate, for example as sodium bicarbonate or potassium bicarbonate.Nalmefene

[0342] In some embodiments, the method is provided wherein the opioid is nalmefene,Case Ref. P322WO IPTector™(nalmefene),and the method comprises preparing naltrexone, optionally using a method as defined herein towards preparation of naltrexone, and then in a step bl) converting naltrexone to nalmefene.

[0343] In some embodiments, step bl) comprises performing an olefination reaction on naltrexone, optionally using a methyltriphenylphosphonium halide, such as methyltriphenylphosphonium bromide.Sequences

[0344] The present application contains a Sequence Listing prepared in Patentin ver 3.5 submitted electronically in ST26 format which is hereby incorporated by reference in its entirety. The following sequences are included:Table A: overview of sequences. AA = sequence comprised of amino acids, i.e. peptide; DNA = sequence comprised of deoxyribonucleic acids. In some embodiments herein, an N-demethylase may also be an O-demethylase.Case Ref. P322WO IPTector™Case Ref. P322WO IPTector™Case Ref. P322WO IPTector™Case Ref. P322WO IPTector™Case Ref. P322WO IPTector™ItemsThe present invention further provides the following itemized embodiments:1. A genetically modified host cell converting or capable of converting a substrate to noroxymorphone, wherein the cell comprises i) a heterologous polynucleotide encoding a morphinone reductase, and ii) a heterologous polynucleotide encoding an N-demethylase, and optionally iii) a heterologous polynucleotide encoding an O-demethylase.2. The genetically modified host cell according to item 1, wherein the substrate is selected from the group consisting of: 14-hydroxymorphinone, 14-hydroxynormorphinone, oxymorphone, 14-hydroxycodeinone, and a combination thereof.3. The genetically modified host cell according to any one of the preceding items, wherein the cell comprises a polypeptide selected from the group consisting of: an N-demethylase, a morphinone reductase, an O-demethylase, a cytochrome P450 reductase, an uptake transporter, and a combination thereof, wherein at least one of the polypeptides are heterologous to the cell.4. The genetically modified host cell according to any one of the preceding items, wherein theCase Ref. P322WO IPTector™ cell comprises a heterologous polypeptide selected from the group consisting of: a cytochrome P450 reductase, an uptake transporter, and a combination thereof.5. The genetically modified host cell according to any of the preceding items, wherein the morphinone reductase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 185, and SEQ ID NO: 193.6. The genetically modified host cell according to item 5, wherein the morphinone reductase is selected from the group consisting of: SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 185, and SEQ ID NO: 193.7. The genetically modified host cell according to any of the preceding items, wherein the heterologous polynucleotide encoding a morphinone reductase has at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 186, and SEQ ID NO: 194.8. The genetically modified host cell according to item 7, wherein the heterologous polynucleotide encoding a morphinone reductase is selected from the group consisting of: SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 186, and SEQ ID NO: 194.9. The genetically modified host cell according to any of the preceding items, wherein the N-demethylase is also an O-demethylase.10. The genetically modified host cell according to any of the preceding items, wherein the N-demethylase is of, or derived from, an insect or a fungus.Case Ref. P322WO IPTector™ 11. The genetically modified host cell according to any of the preceding items, wherein the N-demethylase is not of, or derived from, i) a plant, such as an alkaloid producing plant, ii) a human, and / or iii) a bacterium.12. The genetically modified host cell according to any of the preceding items, wherein the N-demethylase is of family CYP6.13. The genetically modified host cell according to any of the preceding items, wherein the N-demethylase is of the order Lepidoptera.14. The genetically modified host cell according to any of the preceding items, wherein the N-demethylase is of the genus Helicoverpa.15. The genetically modified host cell according to any of the preceding items, wherein the N-demethylase is of the species Helicoverpa armigera.16. The genetically modified host cell according to any of the preceding items, wherein the N-demethylase is of the genus Heliothis.17. The genetically modified host cell according to any of the preceding items, wherein the N-demethylase is of the species Heliothis virescens.18. The genetically modified host cell according to any of the preceding items, wherein the N-demethylase is of the genus Spodoptera.13. The genetically modified host cell according to any of the preceding items, wherein the N-demethylase is of the species Spodoptera exigua.19. The genetically modified host cell according to any of the preceding items, wherein the N-demethylase is a fungal demethylase.20. The genetically modified host cell according to any of the preceding items, wherein the N-demethylase is a fungal demethylase of a genus selected from Rhizopus, Lichtheimia, Syncephalastrum, Cunninghamella, Mucor, Parasitella, Absidia, Choanephora, Bifiguratus andCase Ref. P322WO IPTector™ Choanephora.21. The genetically modified host cell according to any of the preceding items, wherein the N-demethylase is a fungal demethylase of a 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.22. The genetically modified host cell according to any of the preceding items, wherein the N-demethylase has one or more conserved amino acids corresponding to positions G103, Hill, K167, E198, R219, L223, 1256, A259, L273, V284, 1309, L314, Q517, L160, N216, or R443 of any one of: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: Tl , SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 77, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, and SEQ ID NO: 119, or conservative substitutions thereof.23. The genetically modified host cell according to item 22, wherein the one or more conserved amino acid is / are in or near the active site of the demethylase, optionally corresponding to positions G103, Hill and L314 of any one of: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: Tl , SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 77, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, and SEQ ID NO: 119, or conservative substitutions thereof.24. The genetically modified host cell according to any of the preceding items, wherein the N-demethylase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: Tl, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 77, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, and SEQ ID NO: 189.25. The genetically modified host cell according to item 24, wherein the N-demethylase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: Tl , SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 77, and SEQ ID NO: 189; for example wherein the N-Case Ref. P322WO IPTector™ demethylase is selected from the group consisting of: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 , SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 77, and SEQ ID NO: 189; and optionally wherein the substrate is 14-hydroxymorphinone or oxymorphone.26. The genetically modified host cell according to item 24, wherein the N-demethylase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, and SEQ ID NO: 189; for example wherein the N-demethylase is selected from the group consisting of: SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, and SEQ ID NO: 189; and optionally wherein the substrate is 14-hydroxymorphinone or oxymorphone.27. The genetically modified host cell according to item 24, wherein the N-demethylase is selected from the group consisting of: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 , SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 77, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, and SEQ ID NO: 189.28. The genetically modified host cell according to any of the preceding items, wherein the heterologous polynucleotide encoding a N-demethylase has at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 78, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, and SEQ ID NO: 190.29. The genetically modified host cell according to item 28, wherein the heterologous polynucleotide encoding a N-demethylase has at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 78, and SEQ ID NO: 190; for example wherein the heterologous polynucleotide encoding a N-demethylase is selected from the group consisting of: SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ IDCase Ref. P322WO IPTector™ NO: 78, and SEQ ID NO: 190; and optionally wherein the substrate is 14-hydroxymorphinone or oxymorphone.30. The genetically modified host cell according to item 28, wherein the heterologous polynucleotide encoding a N-demethylase has at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, and SEQ ID NO: 190, for example wherein the heterologous polynucleotide encoding a N-demethylase is selected from the group consisting of: SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, and SEQ ID NO: 190; and optionally wherein the substrate is 14-hydroxymorphinone or oxymorphone.31. The genetically modified host cell according to item 28, wherein the heterologous polynucleotide encoding a N-demethylase is selected from the group consisting of: SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 78 SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, and SEQ ID NO: 190.32. The genetically modified host cell according to any one of the preceding items, wherein the cell comprises an uptake transporter having at least 50% sequence identity to an amino acid sequence defined in any one of: SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 183, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, and SEQ ID NO: 209.33. The genetically modified host cell according to any one of the preceding items, wherein the cell comprises an uptake transporter having at least 50% sequence identity to an amino acid sequence defined in any one of: SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, and SEQ ID NO: 183.34. The genetically modified host cell according to item 33, wherein the cell comprises an uptakeCase Ref. P322WO IPTector™ transporter having at least 50% sequence identity to an amino acid sequence defined in any one of: SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13; for example wherein the uptake transporter is selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 7 , SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13, and optionally wherein the substrate is 14-hydroxymorphinone or oxymorphone.35. The genetically modified host cell according to item 33, wherein the uptake transporter is selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, and SEQ ID NO: 183.36. The genetically modified host cell according to any of the preceding items, wherein the cell comprises a heterologous polynucleotide encoding an uptake transporter having at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 184, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, and SEQ ID NO: 210.37. The genetically modified host cell according to any of the preceding items, wherein the cell comprises a heterologous polynucleotide encoding an uptake transporter having at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, and SEQ ID NO: 184.38. The genetically modified host cell according to item 37, wherein the cell comprises a heterologous polynucleotide encoding an uptake transporter having at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14; for example wherein the polynucleotide encoding an uptake transporter is selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14.Case Ref. P322WO IPTector™39. The genetically modified host cell according to item 37, wherein the cell comprises a heterologous polynucleotide encoding an uptake transporter having at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, and SEQ ID NO: 184; for example wherein the polynucleotide encoding an uptake transporter is selected from the group consisting of: SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, and SEQ ID NO: 184.40. The genetically modified host cell according to item 37, wherein the cell comprises a polynucleotide encoding an uptake transporter selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, and SEQ ID NO: 184.41. The genetically modified host cell according to any of the preceding items, wherein the O-demethylase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, and SEQ ID NO: 211.42. The genetically modified host cell according to item 41, wherein the O-demethylase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 75, and SEQ ID NO: 211; for example wherein the O-demethylase is selected from the group consisting of: SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 75, and SEQ ID NO: 211; and optionally wherein the substrate is 14-hydroxycodeinone.43. The genetically modified host cell according to item 41, wherein the O-demethylase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, and SEQ ID NO: 75; for example wherein the O-demethylase is selected from the group consisting of: SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, and SEQ ID NO: 75; and optionally wherein the substrate is 14-hydroxycodeinone.Case Ref. P322WO IPTector™44. The genetically modified host cell according to item 41, wherein the O-demethylase is selected from the group consisting of: SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 121, SEQ ID NO: 123, and SEQ ID NO: 125.45. The genetically modified host cell according to any of the preceding items, wherein the heterologous polynucleotide encoding an O-demethylase has at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 122, SEQ ID NO: 124, and SEQ ID NO: 126.46. The genetically modified host cell according to item 45, wherein the heterologous polynucleotide encoding an O-demethylase has at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 76, SEQ ID NO: 122, SEQ ID NO: 124, and SEQ ID NO: 126; for example wherein the heterologous polynucleotide encoding an O-demethylase is selected from the group consisting of: SEQ ID NO: 76, SEQ ID NO: 122, SEQ ID NO: 124, and SEQ ID NO: 126; and optionally wherein the substrate is 14-hydroxycodeinone.47. The genetically modified host cell according to item 45, wherein the heterologous polynucleotide encoding an O-demethylase is selected from the group consisting of: SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 122, SEQ ID NO: 124, and SEQ ID NO: 126.48. The genetically modified host cell according to any one of the preceding items, wherein the cytochrome P450 reductase is of, or derived from, an insect or a fungus, such as of the genus Helicoverpa.49. The genetically modified host cell according to any one of the preceding items, wherein the cytochrome P450 reductase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, and SEQ ID NO: 191.50. The genetically modified host cell according to item 49, wherein the cytochrome P450 reductase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 65, SEQ ID NO: 67, and SEQ ID NO: 191; for example wherein the cytochrome P450 reductase is selected from the group consisting of: SEQ ID NO: 65, SEQ ID NO: 67, and SEQ ID NO: 191.Case Ref. P322WO IPTector™51. The genetically modified host cell according to item 49, wherein the cytochrome P450 reductase has at least 50% sequence identity to an amino acid sequence as defined in any one of SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, and SEQ ID NO: 191; for example wherein the cytochrome P450 reductase is selected from the group consisting of: SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, and SEQ ID NO: 191.52. The genetically modified host cell according to item 49, wherein the cytochrome P450 reductase is selected from the group consisting of: SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, and SEQ ID NO: 191.53. The genetically modified host cell according to any one of the preceding items, wherein the cell comprises a polynucleotide, optionally heterologous, encoding a cytochrome P450 reductase having at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, and SEQ ID NO: 192.54. The genetically modified host cell according to items 53, wherein the cell comprises a polynucleotide, optionally heterologous, encoding a cytochrome P450 reductase having at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 66, SEQ ID NO: 68, and SEQ ID NO: 192; for example wherein the polynucleotide encoding a cytochrome P450 reductase is selected from the group consisting of: SEQ ID NO: 66, SEQ ID NO: 68, and SEQ ID NO: 192.55. The genetically modified host cell according to items 53, wherein the cell comprises a polynucleotide, optionally heterologous, encoding a cytochrome P450 reductase having at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, and SEQ ID NO: 192; for example wherein the polynucleotide encoding a cytochrome P450 reductase is selected from the group consisting of: SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, and SEQ ID NO: 192.56. The genetically modified host cell according to any one of the preceding items, wherein theCase Ref. P322WO IPTector™ cell comprises a polynucleotide, optionally heterologous, encoding a cytochrome P450 reductase is selected from the group consisting of: SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, and SEQ ID NO: 192.57. The genetically modified host cell according to any one of the preceding items, wherein one or more further native or endogenous genes of the cell is attenuated, disrupted and / or deleted.58. The genetically modified host cell according any one of the preceding items, wherein the one or more further native or endogenous genes are selected from the group consisting of: a aldehyde reductase, such as ARI1 (for example SEQ ID NO: 2); and an aldose reductase, such as GRE3 (for example SEQ ID NO: 4), and / or GCY1 (for example SEQ ID NO: 80).59. The genetically modified host cell according to any one of the preceding items, wherein the one or more further native or endogenous genes are deleted and selected from the group consisting of: a aldehyde reductase, such as ARI1 (for example SEQ ID NO: 2); and an aldose reductase, such as GRE3 (for example SEQ ID NO: 4), and / or GCY1 (for example SEQ ID NO: 80).60. The genetically modified host cell according to 58, wherein the one or more further native or endogenous genes are deleted.61. The genetically modified host cell according to any of the preceding items, wherein the cell comprises an operative metabolic pathway comprising one or more polypeptides producing the substrate from one or more precursors.62. The genetically modified host cell according to item 61, wherein the one or more precursors are selected from the group consisting of: oripavine, thebaine, and a carbon source, such as glucose.63. The genetically modified host cell according to any of items 61-62, wherein the one or more precursors comprise oripavine, and the operative metabolic pathway comprises one or more polypeptides selected from:a) A 14-OH hydroxylase, such as a 14-OH hydroxylase capable of converting oripavine to 14- hydroxymorphinone,b) A cytochrome P450 reductase, such as a cytochrome P450 reductase capable of electronCase Ref. P322WO IPTector™transfer to the 14-OH hydroxylase,c) An uptake transporter, such as an uptake transporter capable of transporting oripavine into the cell,d) A morphinone reductase, such as a morphinone reductase as defined in any one of the preceding items,e) A N-demethylase, such as a N-demethylase as defined in any one of the preceding items, andf) A further cytochrome P450 reductase, such as a cytochrome P450 reductase capable of electron transfer to the N-demethylase.64. The genetically modified host cell according to any of items 61-62, wherein the one or more precursors comprise thebaine, and the operative metabolic pathway comprises one or more polypeptides selected from:a) A 14-OH hydroxylase, such as a 14-OH hydroxylase capable of converting thebaine to 14- hydroxycodeinone,b) A cytochrome P450 reductase, such as a cytochrome P450 reductase capable of electron transfer to the 14-OH hydroxylase,c) An uptake transporter, such as an uptake transporter capable of transporting thebaine into the cell,d) A morphinone reductase, such as a morphinone reductase as defined in any one of the preceding items,e) A N-demethylase, such as a N-demethylase as defined in any one of the preceding items, andf) A further cytochrome P450 reductase, such as a cytochrome P450 reductase capable of electron transfer to the N-demethylase.65. The genetically modified host cell according to any of items 61-62, wherein the one or more precursors comprise a carbon source, such as glucose, and the operative metabolic pathway comprises one or more polypeptides selected from:a) A tyrosine hydroxylase (TH), such as a tyrosine hydroxylase capable of converting L-tyrosine to L- DOPA;b) A dopa decarboxylase (DODC), such as a dopa decarboxylase capable of converting L-DOPA to dopamine;c) A tyrosine decarboxylase (TYDC), such as a tyrosine decarboxylase capable of converting L-Case Ref. P322WO IPTector™ DOPA to dopamine,d) A hydroxyphenylpyruvate decarboxylase (HPPDC), such as a hydroxyphenylpyruvate decarboxylase capable of converting 4-HPP into 4-HPPA;e) A norcoclaurine synthase (NCS), such as a norcoclaurine synthase capable of converting dopamine and 4-HPAA into (S)-norcoclaurine;f) A 6-O-methyltransferase (6-OMT), such as a 6-O-methyltransferase capable of converting (S)-norcoclaurine to (S)-Coclaurine,g) A N-methylcoclaurine 3'-monooxygenase (NMCH, CYP80), such as a N-methylcoclaurine 3'- monooxygenase catalyzing conversion of (S)-Coclaurine to (S)-3'-hydroxycoclaurine and / or (S)-N-Methylcoclaurine to (S)-3'-Hydroxy-N-Methylcoclaurine,h) A coclaurine N-methyltransferase (CNMT), such as a coclaurine N-methyltransferase capable of converting (S)-coclaurine to (S)-N-methylcoclaurine;i) A 3'-hydroxy-N-methyl-(S)-coclaurine 4'-O-methyltransferase (4-OMT), such as a 3'- hydroxy-N-methyl-(S)-coclaurine 4'-O-methyltransferase catalyzing conversion of (S)-3'- Hydroxy-N-Methylcoclaurine to (S)-reticuline;j) A dihydroreticuline synthase / dihydroreticuline reductase (DRS-DRR), such as a dihydroreticuline synthase and reductase capable of converting (S)-reticuline to (R)- reticuline,k) A salutaridine synthase (SAS), such as a salutaridine synthase capable of converting (R)- reticuline to salutaridine,l) A salutaridine reductase (SAR), such as a salutaridine reductase capable of converting salutaridine to salutaridinol,m) A salutaridinol-7-O-acetyltransferase (SAT), such as a salutaridinol-7-O-acetyltransferase capable of converting salutaridinol to 7-O-acetylsalutaridinol,n) A thebaine synthase (THS), such as a thebaine synthase capable of converting 7-O- acetylsalutaridinol to thebaine, ando) One or more cytochrome P450 reductases (CPRs), such as a cytochrome P450 reductases capable of electron transfer to the polypeptides.66. The genetically modified host cell according to any of items 61-62, wherein the one or more precursors comprise a carbon source, such as glucose, and the operative metabolic pathway comprises one or more polypeptides selected from:a) A tyrosine hydroxylase (TH), such as a tyrosine hydroxylase capable of converting L-tyrosine to L- DOPA;Case Ref. P322WO IPTector™ b) A dopa decarboxylase (DODC), such as a dopa decarboxylase capable of converting L-DOPA to dopamine;c) A monoamine oxidase (MAO), such as a monoamine oxidase catalyzing conversion of dopamine to 3,4 DHPAA,d) A norcoclaurine synthase (NCS), such as a norcoclaurine synthase capable of converting dopamine and 3,4 DHPAA into (S)-norlaudanosoline;e) A 6-O-methyltransferase (6-OMT), such as a 6-O-methyltransferase capable of converting (S)-norlaudanosoline into form (S)-coclaurine,f) A coclaurine N-methyltransferase (CNMT), such as a coclaurine N-methyltransferase capable of converting (S)-coclaurine into (S)-N-methylcoclaurine;g) A N-methylcoclaurine 3'-monooxygenase (NMCH, CYP80), such as a N-methylcoclaurine 3'- monooxygenase catalyzing conversion of (S)-Coclaurine to (S)-3'-hydroxycoclaurine and / or (S)-N-Methylcoclaurine to (S)-3'-Hydroxy-N-Methylcoclaurine,h) A 3'-hydroxy-N-methyl-(S)-coclaurine 4'-O-methyltransferase (4-OMT), such as a 3'- hydroxy-N-methyl-(S)-coclaurine 4'-O-methyltransferase catalyzing conversion of (S)-3'- Hydroxy-N-Methylcoclaurine to (S)-reticuline;i) A dihydroreticuline synthase / dihydroreticuline reductase (DRS-DRR), such as a dihydroreticuline synthase and reductase capable of converting (S)-reticuline to (R)- reticuline,j) A salutaridine synthase (SAS), such as a salutaridine synthase capable of converting (R)- reticuline to salutaridine,k) A salutaridine reductase (SAR), such as a salutaridine reductase capable of converting salutaridine to salutaridinol,l) A salutaridinol-7-O-acetyltransferase (SAT), such as a salutaridinol-7-O-acetyltransferase capable of converting salutaridinol to 7-O-acetylsalutaridinol,m) A thebaine synthase (THS), such as a thebaine synthase capable of converting 7-O- acetylsalutaridinol to thebaine, andn) One or more cytochrome P450 reductases (CPRs), such as a cytochrome P450 reductases capable of electron transfer to the polypeptides.67. The genetically modified host cell according to any of items 61-66, wherein the cell comprises a plurality of the operative metabolic pathways as defined in any one of items 63-66.68. The genetically modified host cell according to any of items 63-67, wherein the cell furtherCase Ref. P322WO IPTector™ comprises a SAM alkyl transferase, such as a S-adenosylmethionine (SAM) dependent alkyl transferase capable of converting noroxymorphone to an opioid, such as naloxone, naltrexone, or nalmefene.69. The genetically modified host cell according to any one of items 1-68, wherein the cell is an eukaryote selected from the group consisting of a mammalian, insect, plant, or fungal cell.70. The genetically modified host cell of item 69, wherein the cell is a plant cell of the genus Physcomitrella or Papaver or Nicotiana.71. The genetically modified host cell of any of items 69-70, wherein the cell is a plant cell of the species Papaver soniferum or Nicotiana benthamiana.72. The genetically modified host cell of item 69, wherein the cell is a fungal cell selected from the phylas consisting of Ascomycota, Basidiomycota, Neocallimastigomycota, Glomeromycota, Blastocladiomycota, Chytridiomycota, Zygomycota, Oomycota and Microsporidia.73. The genetically modified host cell of item 69, wherein the fungal cell is a yeast selected from the group consisting of ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and Fungi Imperfecti yeast (Blastomycetes).74. The genetically modified host cell of item 73, wherein the yeast cell is selected from the genera consisting of Saccharomyces, Kluveromyces, Candida, Pichia, Debaromyces, Hansenula, Yarrowia, Zygosaccharomyces, and Schizosaccharomyces.75. The genetically modified host cell of item 74, wherein the 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.76. The genetically modified host cell of item 69, wherein the fungal cell is a filamentous fungus.77. The genetically modified host cell of item 76, wherein the filamentous fungal cell is selected from the phylas consisting of Ascomycota, Eumycota and Oomycota.Case Ref. P322WO IPTector™ 78. The genetically modified host cell of item 77 , wherein the filamentous fungal cell is 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 Trichoderma79. The genetically modified host cell of item 78, wherein the filamentous fungal cell is 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.80. A polypeptide selected from the group consisting of: an N-demethylase as defined in any one of the preceding items, a morphinone reductase as defined in any one of the preceding items, an O-demethylase as defined in any one of the preceding items, a cytochrome P450 reductase as defined in any one of the preceding items, and an uptake transporter as defined in any one of the preceding items.81. A composition comprising the polypeptide of item 80, and optionally one or more excipients.82. A method for reducing a substrate using a morphinone reductase, the method comprising contacting the substrate with the morphinone reductase.Case Ref. P322WO IPTector™83. The method of item 82, wherein the substrate is selected from the group consisting of: Morphinone, Oxymorphinone, oxymorphone, Codeinone, Codeine, Morphine, 14-hydroxycodeinone, 14-hydroxy norcodeinone, 14-hydroxymorphinone, 14-hydroxynormorphinone, Neopine, neopinone, Neomorphinone, and Neomorphine.84. A method for producing noroxymorphone from a substrate,(noroxymorphone),or a pharmaceutically acceptable salt thereof, wherein the method comprises subjecting the substrate to i) a morphinone reductase, and ii) an N-demethylase, and optionally iii) an O-demethylase.85. The method according to any of the preceding items, wherein the substrate is selected from the group consisting of: 14-hydroxymorphinone, 14-hydroxynormorphinone, oxymorphone, 14-hydroxycodeinone, and a combination thereof.86. The method according to any one of items 84-85, wherein the method further comprises preparing the substrate by chemical synthesis, for example wherein the substrate is a) 14-hydroxymorphinone prepared from oripavine or wherein the substrate is b) 14-hydroxycodeinone prepared from thebaine, such as wherein the chemical synthesis comprises oxidation.87. The method according to any one of items 84-86, wherein the method further comprises a cytochrome P450 reductase, an uptake transporter, or a combination thereof.88. The method according to any of the preceding items, wherein the morphinone reductase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 35, SEQ. ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, and SEQ ID NO: 63.89. The method according to item 88, wherein the morphinone reductase is selected from theCase Ref. P322WO IPTector™ group consisting of: SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, and SEQ ID NO: 63.90. The method according to any of the preceding items, wherein the N-demethylase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 , SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 77, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, and SEQ ID NO: 181.91. The method according to item 90, wherein the N-demethylase is selected from the group consisting of: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 , SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 77, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, and SEQ ID NO: 181.92. The method according to any one of the preceding items, wherein the method involves contacting the substrate with an uptake transporter.93. The method according to any one of the preceding items, wherein the method involves contacting the substrate with an uptake transporter having at least 50% sequence identity to an amino acid sequence defined in any one of: SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 183, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, and SEQ ID NO: 209.94. The method according to any one of the preceding items, wherein the method involves contacting the substrate with an uptake transporter having at least 50% sequence identity to an amino acid sequence defined in any one of: SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, and SEQ ID NO: 183.Case Ref. P322WO IPTector™95. The method according to item 94, wherein the uptake transporter is selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, and SEQ ID NO: 183.96. The method according to any of the preceding items, wherein the method comprises an O-demethylase having at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, and SEQ ID NO: 211.97. The method according to item 96, wherein the O-demethylase is selected from the group consisting of: SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, and SEQ ID NO: 211.98. The method according to any of the preceding items, wherein the method further comprises a cytochrome p450 reductase, for example wherein the cytochrome P450 reductase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, and SEQ ID NO: 173.99. The method according to any of items 84-98, wherein the method comprises:a) Culturing the genetically modified host cell as defined in any of items 1-78 at conditions allowing the cell to produce noroxymorphone; andb) Optionally recovering recovering and / or isolating the noroxymorphone.100. The method according to item 99, further comprising feeding a substrate to the cell, such as wherein the substrate is selected from the group consisting of: 14-hydroxymorphinone, 14-hydroxynormorphinone, oxymorphone, 14-hydroxycodeinone, and a combination thereof.101. The method according to any of items 99-100, further comprising one or more elements selected from:a) culturing the cell culture in a nutrient medium;Case Ref. P322WO IPTector™ b) culturing the cell culture under aerobic or anaerobic conditionsc) 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; andf) culturing the cell culture for between 10 hours to 30 days.102. The method according to any of items 84-97, wherein one or more steps of the method are performed in vitro.103. A method for producing an opioid from noroxymorphone, the method comprising:a) producing noroxymorphone using a method as defined in any one of items 84-102, andb) converting noroxymorphone to the opioid.104. The method of item 103, wherein step b) is performed using chemical synthesis.105. The method of any of items 103-104, wherein the opioid is selected from the group consisting of: Naloxone, Naltrexone, Nalmefene, Methylnaltrexone halide, such as Methylnaltrexone bromide, Nalfurafine, Naloxegol, and Nalbuphine.106. The method of any of the preceding items, wherein the opioid is naloxone,(naloxone),and step b) comprises:i. allylation of noroxymorphone by an allylating agent, such as an allylating agent selected from the group consisting of: an allyl halide, for example allyl bromide, allyl chloride, or allyl iodide; allyl acetate; allyl sulfate; allyl sulfonate; allyl carbonate; an allyl alcohol; and an allyl haloformate; such as allyl chloroformate; optionally in the presence of a base, such as triethylamine for exampleat a temperature of from 50 to 90 °C, such as 70 °C; and / or a catalyst; ii. reductive amination of an allyl aldehyde comprising condensation with an allyl aldehyde, such as acrolein, to form an imine; followed by reduction of the imine to provide naloxone; orCase Ref. P322WO IPTector™ill. peptide coupling of noroxymorphone with a carboxylic acid, for example acrylic acid, to form a peptide, followed by reduction of the peptide to provide naloxone, for example by reduction using LiAl H4-107. The method of any of the preceding items, wherein the opioid is naltrexone,(naltrexone),and step b) comprises:i. alkylation of noroxymorphone by a cyclopropylmethylating agent, such as a cyclopropylmethyl halide, for example cyclopropylmethyl bromide.108. The method of item 107, wherein step b) further comprises adding a polar aprotic solvent, such as N-ethyl-2-pyrrolidone, and / or toluene.109. The method of any of items 107-108, wherein step b) further comprises adding a base, such as potassium bicarbonate, and / or DIPEA.110. The method of any of items 107-109, wherein step b) is conducted at a temperature of from 45°C to 80°C, such as from 52° to 60°C.111. The method of any of items 107-110, wherein the reaction time of step b) is from 12 hours to 36 hours, such as from 17 to 24 hours.112. The method of item 107, wherein step b) comprises adding a polar aprotic solvent, such as dimethylacetamide (DMA), optionally at a temperature of from 40 °C to 90 °C, such as from 50 °C to 70 °C; and optionally in the presence of a base, such as a bicarbonate, for example as sodium bicarbonate or potassium bicarbonate.113. The method of any of the preceding items, wherein the opioid is nalmefene,Case Ref. P322WO IPTector™(nalmefene),and the method comprises preparing naltrexone, optionally using a method as defined in any of items 107-112, and then in a step bl) converting naltrexone to nalmefene.114. The method of item 113, wherein step bl) comprises performing an olefination reaction on naltrexone, optionally using a methyltriphenylphosphonium halide, such as methyltriphenylphosphonium bromide.115. The genetically modified host cell or method according to any one of the preceding items, wherein the sequence identity is at least 60%, such as at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, for example 100% sequence identity to the amino acid sequence or polynucleotide sequence.Items II1. A genetically modified host cell capable of converting a substrate to noroxymorphone, wherein the cell comprises i) a heterologous polynucleotide encoding a morphinone reductase, and ii) a heterologous polynucleotide encoding an N-demethylase, and an uptake transporter capable of transporting the substrate into the cell, wherein the substrate is selected from the group consisting of: 14-hydroxymorphinone or 14-hydroxycodeinone.2. The genetically modified host cell according to item 1, wherein the substrate is 14- hydroxymorphinone.3. The genetically modified host cell according to item 1, wherein the substrate is 14- hydroxycodeinone and wherein the cell further comprises iii) a heterologous polynucleotide encoding an O-demethylase.Case Ref. P322WO IPTector™4. The genetically modified host cell according to item 3, wherein the O-demethylase has at least 60% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 121, SEQ ID NO: 123, and SEQ ID NO: 125.5. The genetically modified host cell according to any one of items 1-4, wherein the morphinone reductase has at least 60% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, and SEQ ID NO: 63.6. The genetically modified host cell according to any one of items 1-4, wherein the N-demethylase is not of, or derived from, i) a plant, such as an alkaloid producing plant, for example poppy ii) a human, and / or iii) a bacterium.7. The genetically modified host cell according to any one of items 1-5, wherein the N-demethylase has at least 60% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: Tl, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 77, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, and SEQ ID NO: 181.8. The genetically modified host cell according to any one of items 1-6, wherein the uptake transporter has at least 60% sequence identity to an amino acid sequence defined in any one of: SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13; for example wherein the uptake transporter is selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13.9. The genetically modified host cell according to any one of the preceding items, wherein the cell is an eukaryote selected from the group consisting of a mammalian, insect, plant, or fungal cell.10. The genetically modified host cell according to any one of the preceding items, wherein one or more further native or endogenous genes of the cell is attenuated, disrupted and / or deleted; wherein the one or more further native or endogenous genes are selected from the groupCase Ref. P322WO IPTector™ consisting of: an aldehyde reductase, such as ARI1 (for example SEQ ID NO: 2); and an aldose reductase, such as GRE3 (for example SEQ ID NO: 4), and / or GCY1 (for example SEQ ID NO: 80).11. A method for producing noroxymorphone from a substrate,(noroxymorphone),or a pharmaceutically acceptable salt thereof, wherein the method comprises subjecting the substrate to i) a morphinone reductase, and ii) an N-demethylase, and optionally iii) an O- demethylase; and wherein the method further comprises preparing the substrate by chemical synthesis comprising oxidation, wherein the substrate is a) 14-hydroxymorphinone prepared from oripavine or wherein the substrate is b) 14-hydroxycodeinone prepared from thebaine.12. The method according to items 11, wherein the method comprises:a) Culturing a genetically modified host cell capable of converting the substrate to noroxymorphone, wherein the cell comprises i) a heterologous polynucleotide encoding the morphinone reductase, and ii) a heterologous polynucleotide encoding the N-demethylase, and optionally iii) a heterologous polynucleotide encoding the O-demethylase at conditions allowing the cell to produce noroxymorphone; andrecovering and / or isolating the noroxymorphone.13. The method according to any of items 11-12, wherein the genetically modified host cell is as defined in any one of items 1-10.14. A method for producing an opioid from noroxymorphone, the method comprising:a) producing noroxymorphone using a method as defined in any one of items 11-13, and b) converting noroxymorphone to the opioid, wherein the opioid is selected from the group consisting of: Naloxone, Naltrexone, Nalmefene, Methylnaltrexone halide, such as Methylnaltrexone bromide, Nalfurafine, Naloxegol, and Nalbuphine.15. The method of item 14, whereinA) the opioid is naloxone,Case Ref. P322WO IPTector™(naloxone),and step b) comprises:i. allylation of noroxymorphone by an allylating agent, such as an allylating agent selected from the group consisting of: an allyl halide, for example allyl bromide, allyl chloride, or allyl iodide; allyl acetate; allyl sulfate; allyl sulfonate; allyl carbonate; an allyl alcohol; and an allyl haloformate; such as allyl chloroformate; optionally in the presence of a base, such as triethylamine for exampleat a temperature of from 50 to 90 °C, such as 70 °C; and / or a catalyst; ii. reductive amination of an allyl aldehyde comprising condensation with an allyl aldehyde, such as acrolein, to form an imine; followed by reduction of the imine to provide naloxone; or ill. peptide coupling of noroxymorphone with a carboxylic acid, for example acrylic acid, to form a peptide, followed by reduction of the peptide to provide naloxone, for example by reduction using LiAl H4; orB) wherein the opioid is naltrexone,(naltrexone),and step b) comprises:i. alkylation of noroxymorphone by a cyclopropylmethylating agent, such as a cyclopropylmethyl halide, for example cyclopropylmethyl bromide; orC) wherein the opioid is nalmefene,(nalmefene),and the method comprises preparing naltrexone the method as defined in B), and then in a step bl) converting naltrexone to nalmefene using an olefination reaction using a methyltriphenylphosphonium halide.Case Ref. P322WO IPTector™ExamplesMaterials and methods

[0002] Chemicals used in the examples herein, e.g. for buffers and substrates, are commercial products of at least reagent grade. Water utilized in the examples was de-ionized, MilliQ water.Example 1: Analytical methodsA: Analytical methods for Examples 2-13

[0003] Samples containing the analytes 14-hydroxy normorphone, 14-hydroxy morphone, and 14-hydroxy morphinone were injected into an Agilent 1290 Infinity II UHPLC with a binary pump coupled to an Ultivo QqQ mass spectrometer (Agilent Technologies, Palo Alto, CA, USA). Separation was achieved using a Kinetex Biphenyl column (100 x 2.1mm, 1.7pm, 100 A, Phenomenex, Torrance, CA, USA) using 0.1% (v / v) formic acid in H2O and 0.1% (v / v) formic acid in acetonitrile as mobile phases A and B, respectively. The chromatography gradient used is shown in Table 1. Specific conditions for UHPLC and MS described in Tables 2 and 3. 14-hydroxymorphinone, 14-hydroxymorphone, and 14-hydroxynormorphone were detected in multiple reaction monitoring mode (MRM) as specified in Table 4.

[0004] Authentic standards were purchased from Toronto Research Chemicals: 14-hydroxy normorphone Cat. No. N825300 (CAS no. 33522-95-1), 14-hydroxy morphone Cat. No. 0876800 (CAS no. 76-41-5), and 14-hydroxy morphinone Cat. No. 0876730 (CAS no. 41135-98-2).Table 1A: Gradient for Separation on the Kinetix Biphenyl ColumnTable 2A: UHPLC ConditionsCase Ref. P322WO IPTector™Table 3A: Mass Spectrometer Source and Detector Parameters (Agilent Ultivo QqQ)Table 4A: MRM Targets and Conditions (ESI +)B: Analytical methods for Examples 14-15

[0005] Samples containing the analytes 14-hydroxy normorphone, 14-hydroxy morphone, 14-hydroxy morphinone, Noroxycodone, 14-hydroxy norcodeinone, Oxycodone, and 14-hydroxy codeinone were injected into an Agilent 1290 Infinity II UHPLC with a binary pump coupled to an Ultivo QqQ mass spectrometer (Agilent Technologies, Palo Alto, CA, USA). Separation was achieved using a Kinetex Biphenyl column (100 x 2.1mm, 1.7pm, 100 A, Phenomenex, Torrance, CA, USA) using 0.1% (v / v) formic acid in lOmM Ammonium formate (Sigma Aldrich) and 0.1% (v / v) formic acid in acetonitrile as mobile phases A and B, respectively. The chromatography gradient used is shown in Table 1. Specific conditions for UHPLC and MS described in Tables 2 and 3. 14-hydroxymorphinone, 14-hydroxymorphone, 14-hydroxynormorphone, Noroxycodone, 14-hydroxy norcodeinone, Oxycodone, and 14-hydroxy codeinone were detected in multiple reaction monitoring mode (MRM) as specified in Table 4.Case Ref. P322WO IPTector™

[0006] Authentic standards were purchased from Toronto Research Chemicals: 14-hydroxy normorphone Cat. No. N825300 (CAS no. 33522-95-1), 14-hydroxy morphone Cat. No. 0876800 (CAS no. 76-41-5), 14-hydroxy morphinone Cat. No. 0876730 (CAS no. 41135-98-2), Noroxycodone Cat. No. N825150 (CAS no. 57664-96-7), 14-hydroxy norcodeinone Cat. No. H922210 (CAS no. 2302-68-3), Oxycodone Cat no. MM0672.10 (CAS no. 76-42-6), and 14-hydroxy codeinone Cat. No. H922200 (CAS no. 508-54-3).Table IB: Gradient for Separation on the Kinetix Biphenyl ColumnTable 2B: UHPLC ConditionsTable 3B: Mass Spectrometer Source and Detector Parameters (Agilent Ultivo QqQ)Table 4B: MRM Targets and Conditions (ESI +)Case Ref. P322WO IPTector™Example 2 - Synthesis of 14-hydroxy morphinone from oripavine

[0007] 14-hydroxy morphinone can be purchased from Toronto Research Chemicals Cat. No.0876730 (CAS no. 41135-98-2), Dalton Research Molecules (Catalogue #: DC-001198) or other sources. However, the costs are substantial thus it is preferred to synthesize the compound from a common poppy morphinan, oripavine.

[0008] Two grams of 14-hydroxy morphinone were produced from oripavine using chemical oxidation as described by KraRnig et al (1996.) Briefly, 20 grams of oripavine sourced from Alcaliber was reacted with 27% hydrogen peroxide (Thermo Scientific), and 88% formic acid (Sigma) in 0.7% aqueous sulfuric acid (Supelco). After 24h at 40°C, the acidic solution was neutralized and the oxidized product extracted into dichloromethane or ethyl acetate. Pure 14-hydroxy morphinone was obtained as a white powder after recrystallization from ethyl acetate.

[0009] Purity was estimated to be very high based upon area of peaks in LC-UV chromatograms as compared to the peak area corresponding to 14-hydroxy morphinone, and based on the very low response in LC-MS chromatograms to known impurities: 14-hydroxymorphinone N-oxide, oripavine, and oripavine N-oxide.Case Ref. P322WO IPTector™e

[0010] Alternatively, 14-hydroxymorphinone can be synthesized as described in US20100113787. To a 200 mL flask, dried oripavine (10.00 g, 95% wt / wt %) is dissolved in HOAc / H2O (1:4, 100 mL) and cooled to 5° C - 10° C. Peroxyacetic acid, CH 3CO 3H, (9.1 mL, 32% wt / wt %) is added over 3 minutes and the reaction mixture is stirred at 5° C - 10° C for approximately 30 minutes until reaction is completed as judged by HPLC.The reaction mixture is slowly warmed to 25° C (ambient) and incubated for another 30 minutes. HjCzO^S.O g) is added to the reaction mixture, and is stirred at room temperature for approximately 30 minutes and then heated to 50° C for 30 minutes. The solution is assayed by HPLC and >99% conversion can be obtained.

[0011] Hydrochloride salts can also be produced efficiently using the method of GB Kok & PJ Scammels, 2012, disclosed in Example 9, but were not used in the following examples.References

[0012] KraRnig, R., Hederer, C. & Schmidhammer, H. Optimization of the Synthesis of Oxycodone and 5-Methyloxycodone. Arch Pharm (Weinheim) 329, 325-326 (1996).

[0013] Px Wang et al, US20100113787A1, Preparation of Oxymorphone from Oripavine (2010)

[0014] GB Kok & PJ Scammels, Improved synthesis of 14-hydroxy opioid pharmaceuticals and intermediates. RSCAdv., 2012, 2, 11318-11325.Example 3 - Reduction of substrates 14-hydroxy morphinone and 14-hydroxy normorphinone by MorB

[0015] While it is well established in literature that the Morphinone reductase encoded by MorB from Pseudomonas putida M10 can efficiently reduce the carbon-carbon double bond of codeinone and morphinone, this enzyme was not previously shown to accept 14-hydroxy morphinone and / or 14-hydroxy codeinone as substrates.

[0016] The genes encoding MorB (MorB_l, SEQ ID NO: 35), a broad specificity opioid uptake transporter (SEQ ID NO: 5), the thebaine / oripavine N-demethylase SEQ ID NO: 31 and the CYP450 reductase SEQ ID NO: 65 were expressed in a Saccharomyces cerevisiae S288C derived tester strain named SOD1265. WO 2021 / 069714 Al identifies novel insect demethylases and variants thereof that have very high activity towards morphinan substrates, similarly high activity demethylases were also identified in WO 2018 / 229306.Case Ref. P322WO IPTector™

[0017] In the following examples all genes were expressed from constitutively active promoters (pTDH3 and pTEF2) cloned into in either free replicating plasmids (CEN / ARS based plasmids as described by Mumberg et al. (1995) or from copies integrated into the yeast genome as described by Mikkelsen et al (2012) and others.

[0018] The strain described above containing morB was grown in the presence of 600 pM 14-hydroxy morphinone for 3 days at 30°C in a standard synthetic defined yeast media at pH5.5, both oxymorphone and noroxymorphone was produced in significant amounts as shown in Fig. 1. This shows that MorB catalyzes the reduction 14-hydroxy morphinone and 14-hydroxy normorphinone efficiently and that the N-demethylase used in is also active on 14-hydroxy morphinone and / or oxymorphone.

[0019] While the production of oxymorphone must be a direct conversion of 14-hydroxy morphinone to oxymorphone by MorB, the production of noroxymorphone can happen either by N-demethylation of the produced oxymorphone and / or by initial N-demethylation of 14-hydroxy morphinone to 14-hydroxy normorphinone followed by conversion of 14-hydroxy normorphinone to noroxymorphone by MorB (Fig. 2). This work is the first demonstration of insect-derived N-demethylase activity on 14-hydroxylated morphine derivatives, and the first efficient demonstration of the N-demethylation of these compounds in a heterologous cell. Similarly, this is the first description of the use of a heterologous uptake transporter to import these 14-hydroxylated morphine derivatives.References:Mumberg D, Muller R, Funk M. Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene. 1995;156:119-122. doi: 10.1016 / 0378-1119(95)00037-7. M.D. Mikkelsen, L.D. Buron, B. Salomonsen, C.E. Olsen, B.G. Hansen, U.H. Mortensen, B.A.Halkier. Microbial production of indolylglucosinolate through engineering of a multi-gene pathway in a versatile yeast expression platform. Metab. Eng., 14 (2) (2012), pp. 104-111.Duetz WA, Ruedi L, Hermann R, O'Connor K, Buchs J, Witholt B. Methods for intense aeration, growth, storage, and replication of bacterial strains in microtiter plates. Appl Environ Microbiol. 2000 Jun;66(6):2641-6.Example 4 - Identification of specific amino acid residues conferring activity to MorB homologs to improve 14-hydroxy morphinone reductase activity

[0020] Using the amino acid sequence of MorB as a query in a BlastP search (NCBI), a large number of both close and distant homologs of this enzyme were tested for their ability to reduce the C-C double bond of the alpha, beta-unsaturated ketone of 14-hydroxy morphinone. Surprisingly not even the closest homolog MorB_2 (WP_305502777.1, SEQ. ID NO: 49) was active against 14-hydroxyCase Ref. P322WO IPTector™ morphinone when expressed in yeast together with the same broad activity opioid uptake transporter as in the previous Example. To determine which amino acid differences in MorB_2 (shown in Fig. 3) were responsible for the loss of activity in conversion of 14-hydroxy morphinone to oxymorphone, numerous chimeras of MorB and MorB_2 were synthesized at Twist Bioscience (South San Francisco, CA) for expression in S. cerevisiae. These chimeras were designated MorBl_a to MorBl_g (SEQ ID NOs. 37, 39, 41, 43, 45, 47).

[0021] While most chimeras (MorBl_b to MorBl_g) rendered the enzymes fully active, the chimera termed MorB_la had no activity against 14-hydroxy morphinone when expressed in yeast in the tester strain described in Example 3. Thus, one or more of the four amino acid residue changes in MorB_la (L115M, L147P, C191Y and / or P193L) are detrimental to the reduction activity of the enzyme utilizing 14-hydroxy morphinone as a substrate to produce oxymorphone.

[0022] To determine if MorB_2 can be modified to demonstrate measurable reductase activity on the substrate 14-hydroxy morphinone by changing one or more of these four amino acid residues to the residues present in MorB_l, MorB codon optimized mutants MorB_2a to MorB2_g were designed and ordered from Twist Bioscience (SEQ ID Nos: 51, 53, 55, 57 59, 61, and 63). The genes were expressed and assayed in S. cerevisiae as described above, and activity was compared to media only controls, strains with uptake transporter and N-demethylase only (no activity), strains with MorB_2 plus uptake transporter and N-demethylases (no activity), and strains with uptake transporter, N-demethylase and MorB_l (positive control). As can be seen in Fig. 4, these data show that changing an inactive homolog of MorB (MorB_2) by making the mutation Y191C makes it capable of reducing 14-hydroxy morphinone and 14-hydroxy normorphinone. The data also show that a more efficient conversion can be achieved by making additional mutations (L193P, M115L and P147L).Example 5 - Improving uptake of 14-hydroxy morphinone

[0023] Based on previous work (WQ2020078837) with thebaine and oripavine uptake in yeast, a subset of thebaine / oripavine PUP and MFS uptake transporters were tested for their ability to transport 14-hydroxy morphinone into the cytosol of S. cerevisiae. Evaluation of each transporter's ability to transport 14-hydroxy morphinone into yeast was done by measuring the conversion of 14-hydroxy morphinone to oxymorphone and noroxymorphone by yeast expression of heterologous cytosolic enzymes MorB and N-demethylase SEQ ID NO: 17 plus CPR SEQ ID NO: 65. As can be seen in Fig. 5 the preferred uptake transporters are T161_PsPUP3_68 (SEQ ID NO: 9), and the transporter T198_AcoT97_GA (SEQ ID NO: 5).Case Ref. P322WO IPTector™ Example 6 - W-demethylation of 14-hydroxy morphinone and / or oxymorphone

[0024] Additional CYP450 insect N-demethylases with high activity on thebaine and / or oripavine and variants thereof (WO 2021 / 069714 Al) were tested in S. cerevisiae, for their ability to N-demethylate 14-hydroxy morphinone and / or oxymorphone. Ranking of each N-demethylase's ability to N-demethylate 14-hydroxy morphinone and / or oxymorphone was determined by measuring the production of noroxymorphone by yeast strains that in addition to the N-demethylases had heterologous expression of MorB, T198_AcoT97_GA (SEQ ID NO: 5 AA; SEQ ID NO: 6 DNA) and HaCPR_E0A3A7_co5 (SEQ ID NO: 65 AA; SEQ ID NO: 66 DNA). All of seq id no 15-33 were active in the assay, producing between 15-75 uM noroxymorphone, with higher levels of oxymorphone from the morB reaction. Several fungal-sourced N-demethylases identified as having high activity for thebaine and / or oripavine demethylation in WO2018229306 were also tested for noroxymorphone production in the same assay as described above, except Cel_CPR_co (SEQ ID NO: 67) was utilized instead of an insect CPR. Under the conditions tested, SEQ ID NO: 77 displayed clear levels of noroxymorphone production in the same range as the above enzymes.Example 7 - Decreasing unwanted side products made by yeast endogenous enzymes

[0025] When analyzing LC-MS data (Example 1) on samples from yeast that express uptake transporter SEQ ID NO: 5 and MorB_l that have been grown for 3 days in presence of 300-600 pM 14-hydroxy morphinone in the previous examples, byproducts with masses corresponding to 14-hydroxy morphinone + 2 and oxymorphone + 2 were observed. To determine if these unwanted side products were made by yeast endogenous aldo-keto reductases that turn the keto group of 14-hydroxy morphinone / oxymorphone into a hydroxy group, individual deletions of known S. cerevisiae aldo-keto reductases were assayed as described above and compared to strains without the knockouts. An initial large screen suggested that the deletion of ARI1 was beneficial for oxymorphone production in S. cerevisiae, while deletion of the gene GRE3 reduced the area under the curve (AUC) of the peak with the mass of oxymorphone +2. Deletion of GCY1 (also increased production of oxymorphone from 14-hydroxymorphinone (see Fig. 11). This is believed to be caused by lowered reduction of the ketone group of 14-hydroxy morphinone and / or oxymorphone resulting in lower material loss to alcohols 14-hydroxy morphinol and oxymorphol in these two deletion strains. The names of the genes knocked out are based on common S. cerevisiae naming and Saccharomyces Genome Database (SGD) naming (yeastgenome.org). These include ORA1 / YMR226C, YPR1 / YDR368W, GRE3 / YHR104W, GCY1 / YQR120W, GRE2 / YOL151W, YDR541C, YGL039W, ADH6 / YMR318C, ZTA1 / YBR046C, ARI1 / YGL157W, and AAD3 / YCR107W.

[0026] To confirm these initial results, deletions of ARI1 and / or GRE3 in s288C derived strain SOD1265Case Ref. P322WO IPTector™ were done by replacement of these genomic S. cerevisiae gene copies with copies of the KanMX and NatMX yeast selection marker cassettes. Displacement and thereby deletion of GRE3 and ARI1 was confirmed by colony PCR on transformants that grew in presence of Nurseothricin and / or G418.Example 8 - Enzymatic production of Noroxymorphone from 14-hydroxy codeinone

[0027] Having demonstrated the feasibility of converting 14-hydroxymorphinone into Noroxymorphone in the previous examples by expression of various N-demethylases, uptake transporters and MorB variants as well as a CPR in S. cerevisiae and feeding of 14-hydroxy morphinone to the growth medium, it was hypothesized that conversion of 14-hydroxy codeinone would also be possible with the addition of a 3-O-demethylase as shown in Fig. 6. The order of reaction can vary. Strains are constructed by building upon strains described in previous examples, but also contain known thebaine or northebaine O-demethylases as identified in WO2018229306. Numerous O-demethylases known to work on similar substrates are assayed for activity in this reaction such as Codeine-3-O-demethylase (CODM) and for example variants of Ps_CODM_GA (SEQ ID NO: 75) or fungal enzymes (wildtype or variants of) SEQ ID NO: 73, SEQ ID NO: 125, SEQ ID NO: 71, and SEQ ID NO: 69. In some cases enzymes that have both N- and O-demethylase activity are used.

[0028] The strains are assayed for ability to convert 14-hydroxy codeinone into noroxymorphone, by adding 300-600 pM 14-hydroxy codeinone to standard yeast growth medium and growth of the strains using the system described by Duetz et al in 96-deep-well plates for 3 days in 500ml standard defined yeast medium at pH5.5, with shaking at 280rpm in a Kuhner ISF-l-X shaking incubator at 30°C.

[0029] 14-hydroxycodeinone (CAS# 508-54-3) can be purchased from numerous sources such as Boron Molecular (BM2071) or Millipore / Sigma (1485216 or PHR8800). However, it is very expensive and preferably is synthesized as described in Example 9.Example 9 - Synthesis of 14-hydroxy codeinone from thebainePart A

[0030] 14-hydroxycodeinone is similar in structure to 14-hydroxy-morphinone but differs by an O-methyl group c.f. Fig. 7. Therefore, it is more facile to synthesize this molecule from another morphinan derived from poppy, thebaine, rather than oripavine. The procedure described in Example 2 (KraRnig et al in 1996) has been shown to work for production of 14-hydroxycodeinone from thebaine.

[0031] Alternatively, production of 14-hydroxycodeinone from thebaine (and preferably its hydrochloride salt) has been described in the literature as part of a process to synthesize oxycodone (GB Kok & PJ Scammels, 2012).Case Ref. P322WO IPTector™References:GB Kok & PJ Scammels, Improved synthesis of 14-hydroxy opioid pharmaceuticals and intermediates.RSCAdv., 2012, 2, 11318-11325.Part B

[0032] 14-hydroxycodeinone is similar in structure to 14-hydroxy-morphinone but differs by an O-methyl group c.f. Fig. 7. Therefore, it is more facile to synthesize this molecule from another morphinan derived from poppy, thebaine, rather than oripavine. The procedure described in Example 2 (KraRnig et al in 1996) has been shown to work for production of 14-hydroxycodeinone from thebaine.

[0033] Alternatively, production of 14-hydroxycodeinone from thebaine (and preferably its hydrochloride salt) has been described in the literature as part of a process to synthesize oxycodone (GB Kok & PJ Scammels, 2012).

[0034] Synthesis of 14-hydroxycodeinone from thebaine following a procedure published by KraRnig et al (1996).

[0035] One gram of thebaine sourced from Alcaliber was reacted with aqueous hydrogen peroxide (27%), formic acid (88%) and sulfuric acid (0,7%) at 40°C for 24 hours. The solution was neutralized with aqueous ammonia (5%) and the precipitated product collected by centrifugation (12500 rpm, 25°C , 10 min). After washing with water, the product was dried in a vacuum oven at 40°C and produced a white powder (88% crude yield).References:GB Kok & PJ Scammels, Improved synthesis of 14-hydroxy opioid pharmaceuticals and intermediates.RSCAdv., 2012, 2, 11318-11325.R KraRnig, C Hederer & H Schmidhammer, Optimization of the Synthesis of Oxycodone and 5-Methyloxycodone. Archiv Der Pharmazie, 1996, 329 (6), 325-326.Example 10 - Synthesis of Naloxone from noroxymorphone (NOM)

[0036] Numerous pharmaceutical compounds can be produced from noroxymorphone, most notably "nals" compounds including naloxone, naltrexone, and nalmefene. See Fig. 9, and Fig. 10. The numbering is based on the Morphinan structure displayed in Fig. 8.

[0037] There are two main approaches for the alkylation step to lead to Nals compounds fromCase Ref. P322WO IPTector™ noroxymorphone(1) direct alkylation where the required alkyl group is attached to a useful leaving group like bromide or(2) reductive alkylations -- there are multiple examples of this reaction type with different reagents that can be reduced, typically using inorganic catalysts, to leave the required alkyl functional group. Exemplary methods for alkylation chemistry include the following:• Alkylation reactions with bases and alkyl halides, including chloro, bromo and iodo derivatives;• Alkylation reactions using in situ generation of iodo derivatives made of chloro- and bromoderivatives with iodide salts (Finlkelstein type chemistry);• Alkylation reactions with the various forms of activated alcohols (as sulfonates, as Mitsunobu- type intermediates);• Reductive aminations of N with aldehydes and use of various catalysts for the reduction from iminium to amine;• Coupling of nitrogen with carboxylic acids via peptide type coupling reagents, and with carboxylic halides (mostly chloro) followed by reduction (e.g. LiAl H4) of the peptide bond An example of the direct alkylation with the leaving group is described in Tomas Hudlicky. (2015). Recent advances in process development for opiate-derived pharmaceutical agents. Canadian Journal of Chemistry. 93(5): 492-501. In this work they found that simple alkylation of noroxymorphone, with allyl bromide worked better than other methods with a yield of 84%. Allyl bromide is reacted with triethylamine at 70°C for 5-10 hours in a water and N-Methylpyrrolidone (NMP) solvent. These same conditions can be used to synthesize other Nals compounds by choice of a different R-bromide group.

[0038] Specific examples of production of naloxone from noroxymorphone using reductive options can be found, for example, in US9127014B2. Scheme 13 of US9127014B2 describes the use of a transition metal catalyst and N,N-dimethylprop-2-en-l-amine or allyl acetate for the preparation of naloxone from noroxymorphone. Alternatively, the allylation reaction is also carried out using an allyl haloformate, such as allyl chloroformate, generating an allyl carbamate intermediate, and then converting the allyl carbamate to the allyl amine in a transition metal-catalyzed decarboxylation reaction.

[0039] Similar chemistry can be used substituting l-cyclopropenyl-N,N-dimethylmethanamine or cyclopropenylmethyl acetate to provide the corresponding cyclopropenyl-comprising derivative of noroxymorphone that can be hydrogenated to provide naltrexone.Example 11 - Synthesis of Naltrexone and Nalmefene from Noroxymorphone (NOM)

[0040] Example 10 of the present disclosure gave exemplary methods for manufacturing of nals fromCase Ref. P322WO IPTector™ NOM, which apply to naltrexone as well. Patent EP2635586 describes several methods of naltrexone production in detail from noroxymorphone, in example 1-7.Part A

[0041] A mixture of noroxymorphone (approximately 50 g per 200 mL solvent), in equivolume amounts of N-ethyl-2-pyrrolidone and toluene is concentrated under vacuum at 80°C. The mixture is diluted with 0.5 volume of toluene and concentrated again. The suspension is diluted with N-ethyl-2-pyrrolidone (0.25 original volume). Potassium bicarbonate and cyclopropylmethyl bromide ((approximately half mass equivalent each to NOM)) were added and the mixture is heated up to 55°C for approximately 1 day. The composition of the reaction mixture is checked by HPLC (% by area) and typically yields high purity naltrexone > 97% with approximately 1 percent unreacted substrate.

[0042] Noroxymorphone (51.5 g) in N-ethyl-2-pyrrolidone (168 ml) and toluene (100 ml) is concentrated under vacuum at 80-85°C. Toluene is added (200 ml) and vacuum distillation repeated. Potassium bicarbonate (24.4 g) and cyclopropylmethyl bromide (29.3 g) are added and the mixture is heated to approximately 60°C and maintained for ~ 1 day. Further cyclopropylmethyl bromide (2.3 g) can be charged and stirred at 60°C for five additional hours. The composition of the reaction mixture is checked by HPLC and a typical result would be 1-2 % unreacted noroxymorphone with product purity greater than 97%. The reaction mixture can be further treated with HCI 10% (88.9 g) and concentrated under vacuum; cooled and diluted with water (1580 g); followed by ammonium hydroxide 4% in water addition over several hours resulting in a suspension (pH ~ 9.3). The suspension can be stirred and filtered; washed with water and dried under vacuum at 60°C to obtain higher purity naltrexone (>99%) with high yield.

[0043] A mixture of noroxymorphone (52.7 g), potassium bicarbonate (24.4 g) and cyclopropylmethyl bromide (30.5 g) in N-ethyl-2-pyrrolidone (150 ml) is held at 60°C for approximately 17 hours. The composition of the reaction mixture as determined by HPLC area under the curve percentage is typically> 95% naltrexone, with approximately 3% unreacted noroxymorphone.

[0044] Noroxymorphone (52.7 g) in 200 mL equal volumes of N-ethyl-2-pyrrolidone and toluene is concentrated under vacuum. Toluene is added (100 ml) and vacuum distillation is repeated two more times. The mixture is diluted with N-ethyl-2-pyrrolidone. Cyclopropylmethyl bromide (30.5 g) and N,N-diisopropylethylamine (29.2 g) are added and the mixture is heated to 60°C and held for approximately 17 hours. The composition of the reaction mixture as checked by HPLC (% by area under the curve) is ~ 95% product with approximately 3-4% unreacted substrate.

[0045] A mixture of noroxymorphone (60 Kg, 0.209 Kmol), N-ethyl-2-pyrrolidone (180 kg), cyclopropymethyl bromide (36.6 kg) and N,N-diisopropylethylamine (35.1 kg) is heated to 52-57°C for approximately 20 hours. The mixture is diluted with a solution prepared by mixing hydrochloric acidCase Ref. P322WO IPTector™37% (29 kg) and water (79 kg). Low boiling compounds are removed by distillation under vacuum keeping the temperature below 70°C. After cooling to 25-30°C the mixture is diluted with water (1910 kg). Ammonium hydroxide 4% (199 kg) is then added over approximately 3 hours to reach a pH between 9 and 10 to induce precipitation of the product. The solid can be isolated by filtration, washing with water (2x120 kg) and drying under vacuum at 60°C and typically results in a high yield of naltrexone (molar yield > 93% and high purity (approximately 99%).

[0046] A mixture of noroxymorphone (60 Kg, 0.209 Kmol), N-ethyl-2-pyrrolidone (180 kg), cyclopropymethyl bromide (36.6 kg) and N,N-diisopropylethylamine (35.1 kg) is heated to 52-57°C for 18 -19 hours. The mixture is then diluted with a solution of hydrochloric acid 37% (29 kg) and water (79 kg). Volatile components are removed by distillation under vacuum maintaining temperatures below 70°C. After cooling to 25-30°C the mixture is further diluted with water (1910 kg); ammonium hydroxide 4% (199 kg) is then added over approximately 3 hours to reach a pH between 9 and 10 to precipitate the product. The solid is filtered, washed with water (2x120 kg) and dried under vacuum at 60°C obtaining naltrexone at high molar yield, 90% and high purity.

[0047] A mixture of noroxymorphone (62 Kg), N-ethyl-2-pyrrolidone (186 kg), cyclopropymethyl bromide (37.8 kg) and N,N-diisopropylethylamine (36.2 kg) is heated to 52-57°C for approximately 1 day. The mixture is diluted with an aqueous solution of hydrochloric acid 37% (30 kg) and water (82 kg). Volatiles are removed by distillation under vacuum maintaining the temperature below 70°C. After cooling to 25-30°C the mixture is further diluted with water (1975 kg). Ammonium hydroxide 4% (206 kg) is then added over ~ three hours to obtain a pH of 9-10 to precipitate the product. The solid is filtered, washed with water (2x124 kg) and dried under vacuum at 60°C obtaining naltrexone at high yields and purities.Part B

[0048] Naltrexone synthesis from noroxymorphone and 1.5 equivalents bromomethylcyclopropane in DMA (Dimethylacetamide) at 60 C using NaHCO3 (4 eq) base and isolated as follows in Table A. All reactions were verified to reach completion based on LC / MS.noroxymorphone naltrexoneMW = 287 MW = 341Case Ref. P322WO IPTector™Part CNalmefene can be synthesized from naltrexone as well as earlier described methods for nals.

[0049] To methyltriphenylphosphonium bromide (3 eq.) in anhydrous THF, was added t-BuOK portion wise over the course of 1 hour. The mixture was stirred at room temperature for 2 hours. To the mixture, was added naltrexone in anhydrous THF. The reaction was stirred at room temperature until completion. It was then washed with sat. NH4CI and with water. The organic layer was dried over MgSO4, filtered and concentrated in vacuo. The crude material was redissolved with DCM and added 32% HCI (1 eq). The mixture was stirred at room temperature for 2 hours. The precipitate formed was collected by vacuum filtration and washed with cold DCM to deliver the target compound.Example 12 Reduction of substrates 14-hydroxy morphinone and 14-hydroxy normorphinone by MorB and modified homologs therof

[0050] While it is well established in literature that the Morphinone reductase encoded by MorB from Pseudomonas putida M10 can efficiently reduce the carbon-carbon double bond of codeinone and morphinone, this enzyme was not previously shown to accept 14-hydroxy morphinone and / or 14-hydroxy codeinone as substrates.

[0051] The genes encoding MorB (MorB_l, SEQ ID NO: 35), a broad specificity opioid uptake transporter (SEQ ID NO: 5), the thebaine / oripavine N-demethylase SEQ ID NO: 31 and the CYP450 reductase SEQ ID NO: 65 were expressed in a Saccharomyces cerevisiae S288C derived tester strain named SOD1265. WO 2021 / 069714 Al identifies insect demethylases and variants thereof that have very high activity towards morphinan substrates, similarly high activity demethylases were alsoCase Ref. P322WO IPTector™ identified in WO 2018 / 229306.

[0052] In present Example 12 and 13 all genes were expressed from constitutively active promoters (pTDH3 and pTEF2) cloned into either free replicating plasmids (CEN / ARS based plasmids as described by Mumberg et al. (1995) or from copies integrated into the yeast genome as described by Mikkelsen et al (2012) and others.

[0053] The strain described above containing morB was grown in the presence of 600 pM 14-hydroxy morphinone for 3 days at 30°C in a standard synthetic defined yeast media at pH5.5, both oxymorphone and noroxymorphone were produced in significant amounts as shown in Fig. 13. Also shown in Fig. 13 is the activation of two MorB homologs, MorB_2 and MorB_3, by changing three native amino acids to the ones present in MorB, resulting in homologs having amino acids 147L, 191C and 193P, namely SEQ ID NO: 185 (MorB_3_147L_191C_193P) and SEQ ID NO: 193 (MorB_2_147L_191C_193P). While the native MorB_2 and MorB_3 cannot reduce the carbon-carbon double bond of the unsaturated ketone, the mutants are as effective as MorB in doing so, demonstrating that otherwise inactive bacterial homologs of MorB can be turned into active opioid reductases by changing native amino acids to three amino acid of MorB, namely 147L, 191C and 193P.Example 13 / V-demethylation of 14-hydroxy morphinone and / or oxymorphone

[0054] CYP450 insect N-demethylases with high activity on thebaine and / or oripavine and variants thereof (WO 2021 / 069714 Al) were tested in S. cerevisiae, for their ability to N-demethylate 14-hydroxy morphinone and / or oxymorphone. Each N-demethylase's ability to N-demethylate 14-hydroxy morphinone and / or oxymorphone was determined by measuring the production of noroxymorphone by yeast strains that in addition to the N-demethylases had heterologous expression of MorB T198_AcoT97_GA (SEQ ID NO: 5) and the CPR of SEQ ID NO: 66. All of seq id no 15-33 were active in the assay, producing between 15-75 pM noroxymorphone, with higher levels of oxymorphone from the morB reaction. In addition to the already identified insect P450s in previous examples that can efficiently do the N-demethylation of 14-hydroxy morphinone and / or oxymorphone, SEQ ID NO: 189 has now also been shown to be efficient at N-demethylation of 14-hydroxy morphinone and / or oxymorphone.

[0055] The bar diagram in Fig. 14 shows the N-demethylation of 14-hydroxy morphinone and / or oxymorphone by two different S. cerevisiae strains called SOD1829 and SOD1859.SOD1829 express the heterologous genes: MorB, T198_AcoT97_GA (SEQ ID NO: 5), the CPR of SEQ ID NO: 66, and the N-demethylase of SEQ ID NO: 32 while SOD1859 express the heterologous genes: MorB, T198_AcoT97_GA (SEQ ID NO: 5), the CPR of SEQ ID NO: 192 and the N-demethylase of SEQ ID NO: 190.Case Ref. P322WO IPTector™

[0056] These two comparable strains were grown in the presence of 1.5mM 14-hydroxy morphinone for 3 days at 30°C in a standard synthetic defined yeast medium at pH5.5 with shaking. The bardiagram production profile is shown in pM. As can be seen in the diagram, expression in yeast of the CPR of SEQ ID NO: 192 and the N-demethylase of SEQ ID NO: 190 in SOD1859 gives efficient N-demethylation of 14-hydroxy morphinone and / or oxymorphone which results in high production of Noroxymorphone.Example 14 N-demethylation and O-demethylation of 14-hydroxy codeinone and / or oxycodone

[0057] In previous examples it was shown that insect N-demethylases can N-demethylate 14-hydroxy morphinone and / or oxymorphone. In this example we describe that additional expression of an O-demethylase of fungal origin leads to O-demethylation of 14-hydroxy compounds (see Figure 15). A diploid Saccharomyces cerevisiae S288C derivative (sOD1742) was engineered to express the genes CPR (SEQ ID NO: 68), N-demethylase (SEQ ID NO: 190), O-demethylase (SEQ ID NO: 70), MorB_2g (SEQ ID NO: 64), CPR (SEQ ID NO: 166) and MorB_l (SEQ ID NO: 36) from integrated copies and with strong constitutively active promoters driving gene transcription. This engineered strain was termed SQD2006. To test the ability of several opiate uptake transporters to transport 14-hydroxy codeinone fed to the media into the cell cytosol, SQD2006 was transformed with different CEN / ARS based yeast replicating plasmids harbouring expression cassettes for 14 different transporters. The resulting 15 strains (14 strains expressing heterologous uptake transporters in SQD2006 and one termed empty, not expressing any heterologous uptake transporter) were grown in the Duetz 96-deepwell system in the presence of 1.5mM 14-hydroxy codeinone for 3 days at 30°C in a standard synthetic defined yeast medium buffered to pH4.5 with shaking. Samples for analysis were prepared from the culture broth by making appropriate dilutions in 0,1% formic acid in water, followed by heating to 80C for 10 minutes and removal of cell material as a pellet by centrifugation at 3100RCF in an Eppendorf 5810R centrifuge. The cell free supernatant was then subjected to LC-MS analysis.Fig 16 shows the production of oxycodone, noroxycodone and oxymorphone when 1.5mM 14-hydroxy codeinone was fed to a media culture containing SQD2006 transformed to also express the designated uptake transporters or an empty plasmid. As can be seen in the figure all tested transporters could transport 14-hydroxy codeinone from the growth media into the yeast cytosol where it then underwent carbon double bond reduction by MorB_l (SEQ ID NO: 35) and MorB_2g (SEQ ID NO: 63) as well as N or O-demethylation by N-demethylase (SEQ ID NO: 189) with CPR (SEQ ID NO: 191) and O-demethylase (SEQ ID NO: 69) with CPR (SEQ ID NO: 67), respectively. As shown, this experiment resulted in the production of oxycodone, noroxycodone, oxymorphone and possibly 14-hydroxy norcodeinone and 14-hydroxy morphinone.Case Ref. P322WO IPTector™Example 15 N-demethylation and O-demethylation of oxycodone

[0058] To determine whether N-demethylation and O-demethylation are possible if oxycodone is fed to the culture medium, the same 15 S. cerevisiae strains as described in the previous example (sOD2006 plus uptake transporters or an empty plasmid) were tested with 1.5mM oxycodone feeding. Like in the previous example, 14 different uptake transporters were tested for their ability to transport the substrate, in this case oxycodone, from the growth media into the yeast cytosol. As can be seen in Fig 17, feeding of 1.5mM oxycodone results in production of noroxycodone, oxymorphone and noroxymorphone in all cases. If no heterologous transporter is expressed (sOD2006 (empty vector)), then oxycodone uptake, and thereby production of oxymorphone, noroxycodone and noroxymorphone, are negligible. This bar-diagram demonstrates the ability of O-demethylase (SEQ ID NO: 69) with CPR (SEQ ID NO: 67) to O-demethylate oxycodone and possibly noroxycodone to oxymorphone and noroxymorphone respectively. It also demonstrates the ability of N-demethylase (SEQ ID NO: 189) and N-demethylase (SEQ ID NO: 191) to N-demethylate oxycodone and possible oxymorphone to noroxycodone and noroxymorphone respectively. Lastly it demonstrates the ability of a larger number of opiate uptake transporters to also being capable of transporting 14-hydroxy opiate derivatives from the extracellular space into the cytosol when expressed heterologously in S. cerevisiae.

[0059] In all cases production is shown as LC-MS chromatogram area under curve values of peaks corresponding the mentioned compounds as described before.

[0060] To test if yet another O-demethylase (SEQ ID NO: 211) can O-demethylate oxycodone, a diploid Saccharomyces cerevisiae S288C derivative strain SOD1742 was engineered to express the genes CPR (SEQ ID NO: 68), N-demethylase (SEQ ID NO: 190), O-demethylase (SEQ ID NO: 212), MorB_2g (SEQ ID NO: 64), CPR (SEQ ID NO: 192), MorB_l (SEQ ID NO: 36) from integrated copies and with strong constitutively active promoters driving gene transcription. This engineered strain was termed SQD2003. To test the ability of several opiate uptake transporters to transport oxycodone fed to the media into the cell cytosol, SQD2003 was transformed with different CEN / ARS based plasmids harbouring expression cassettes for 14 different transporters. The resulting 15 strains (14 expressing heterologous uptake transporters in SQD2003 and one termed empty, not expressing any heterologous uptake transporter) were grown in the presence of 1.5mM oxycodone for 3 days at 30°C in a standard synthetic defined yeast medium at pH4.5 with shaking. Samples for analysis were prepared from the culture media by making appropriate dilutions in 0,1% formic acid in water, heating to 80 °C for 10 minutes and removing cell material as a pellet by centrifugation at 3100RCF in an Eppendorf 5810R centrifuge. The supernatant was then subjected to LC-MS analysis.Case Ref. P322WO IPTector™

[0061] As can be seen in Fig 18, feeding of 1.5mM oxycodone resulted in production of noroxycodone and oxymorphone in all cases and noroxymorphone in some cases. If no heterologous transporter was expressed (sOD2003 (empty)), then oxycodone uptake, and thereby production of oxymorphone, noroxycodone and noroxymorphone, is negligible. This demonstrates the ability of O-demethylase (SEQ ID NO: 211) and CPR (SEQ ID NO: 67) to O-demethylate oxycodone and possibly noroxycodone to oxymorphone and noroxymorphone respectively. Again, this also demonstrates the ability of N-demethylase (SEQ ID NO: 189) and CPR (SEQ ID NO: 191) to N-demethylate oxycodone and possible oxymorphone to noroxycodone and noroxymorphone respectively. Lastly this again demonstrates the ability of a larger number of opiate uptake transporters to also being capable of transporting 14-hydroxy opiate derivatives from the extracelluar space into the cytosol when expressed heterologously in S. cerevisiae.

Claims

Case Ref. P322WO IPTector™Claims1. A genetically modified host cell converting or capable of converting a substrate to noroxymorphone, wherein the cell comprisesi) a heterologous polynucleotide encodings morphinone reductase, and ii) a heterologous polynucleotide encoding an N-demethylase,iii) an uptake transporter capable of transporting the substrate into the cell, iv) a polynucleotide encoding a cytochrome P450 reductase;and optionallyv) a heterologous polynucleotide encoding an O-demethylase.

2. The genetically modified host cell accordingto claim 1 , wherein the N- demethylase is a eukaryotic cytochrome P450 enzyme.

3. The genetically modified host cell accordingto any one of the preceding claims, wherein the N-demethylase is not of, or derived from, a mammal and / or a plant.

4. The genetically modified host cell accordingto the preceding claim, wherein the N-demethylase is of, or derived from, an insect.

5. The genetically modified host cell accordingto the preceding claim, wherein the N-demethylase is of the order Lepidoptera.

6. The genetically modified host cell accordingto the preceding claim, wherein the N-demethylase is of the genus Spodoptera.

7. The genetically modified host cell accordingto the preceding claim, wherein the N-demethylase is of a species of the genus Spodoptera.

8. The genetically modified host cell according to any of the preceding claims, wherein the N-demethylase is of the species Spodoptera exigua or Spodoptera frugiperda.

9. The genetically modified host cell accordingto any one of the preceding claims, wherein the N-demethylase is a membrane-associated heme-thiolateCase Ref. P322WO IPTector™ monooxygenase, optionally functionally dependent on a separate cytochrome P450 reductase polypeptide for activity.

10. The genetically modified host cell accordingto claim 9, wherein the N- demethylase comprises a hydrophobic N-terminal anchorfor membrane association.

11. The genetically modified host cell according to any one of claims 9 or 10, wherein the N-demethylase comprises a heme-binding motif comprising the amino acid sequence FXXGXRXCXG, wherein X is any amino acid, such as any naturally occurring (proteinogenic) amino acid.

12. The genetically modified host cell according to any one of claims 9-11 , wherein the N-demethylase comprises a K-helix motif comprisingthe amino acid sequence EXXR, wherein X is any amino acid.

13. The genetically modified host cell according to any one of claims 9-12, wherein the N-demethylase comprises an l-helix proton transfer groove motif.

14. The genetically modified host cell according to any one of the preceding claims, wherein the polynucleotide encoding the cytochrome P450 reductase is heterologous to the host cell.

15. The genetically modified host cell accordingto any one of the preceding claims, wherein the O-demethylase is a eukaryotic cytochrome P450 enzyme.

16. The genetically modified host cell according to any one of the preceding claims, wherein the O-demethylase is a membrane-associated heme-thiolate monooxygenase, optionally functionally dependent on a separate cytochrome P450 reductase polypeptide for activity.

17. The genetically modified host cell accordingto any one of the preceding claims, wherein the O-demethylase does not comprise a fused reductase domain.Case Ref. P322WO IPTector™18. The genetically modified host cell according to any one of the preceding claims, wherein the O-demethylase is of, or derived from, an insect or a fungus.

19. The genetically modified host cell according to any one of the preceding claims, wherein the O-demethylase is not of, or derived from, a plant and / or a mammal.

20. The genetically modified host cell according to any one of the preceding claims, wherein the substrate has been prepared by chemical synthesis.

21. The genetically modified host cell according to any one of the preceding claims, wherein the substrate is selected from the group consisting of: 14- hydroxymorphinone, 14-hydroxynormorphinone, oxymorphone, 14- hydroxycodeinone, oxycodone, and a combination thereof.

22. The genetically modified host cell according to claim 20, wherein the substrate is selected from the group consisting of 14-hydroxycodeinone and oxycodone.

23. The genetically modified host cell accordingto the preceding claim, wherein the host cell comprises (v) the heterologous polynucleotide encoding the O- demethylase.

24. The genetically modified host cell according to any one of the preceding claims, wherein the cell comprises a polypeptide selected from the group consisting of: an N-demethylase, a morphinone reductase, an O-demethylase, a cytochrome P450 reductase, an uptake transporter, and a combination thereof, wherein at least one of the polypeptides are heterologous to the cell.

25. The genetically modified host cell accordingto any one of the preceding claims, wherein the cell comprises a heterologous polypeptide selected from the group consisting of: a cytochrome P450 reductase, an uptake transporter, and a combination thereof.Case Ref. P322WO IPTector™26. The genetically modified host cell according to any of the preceding claims, wherein the morphinone reductase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 , SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 185, and SEQ ID NO: 193, such as at least 60% sequence identity to said amino acid sequence, such as at least 80% sequence identity to said amino acid sequence, such as at least 95% sequence identity to said amino acid sequence, such as at least 100% sequence identity to said amino acid sequence, for example wherein the morphinone reductase has at least 90% or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 185 and SEQ ID NO: 193.l. The genetically modified host cell according to any one of the preceding claims, wherein the morphinone reductase further comprises one or more mutations selected from the group consisting of: L115X, L147X, C191 X, and P193X, wherein X is any amino acid otherthan the corresponding amino acid residue in SEQ ID NO: 35, wherein amino acid position numbering is with reference to SEQ ID NO: 35.

28. The genetically modified host cell according to any one of the preceding claims, wherein the morphinone reductase comprises one or more amino acid residues L115, L147, C191, and / or P193 at amino acid positions correspondingto 115, 147, 191, and 193 of SEQ ID NO: 35, wherein amino acid position numbering is with reference to SEQ ID NO: 35.

29. The genetically modified host cell according to any one of the preceding claims, wherein the morphinone reductase comprises an amino acid sequence having at least 50% sequence identity to an amino acid sequence defined in SEQ ID NO: 49, and wherein the morphinone reductase comprises a substitution Y191 C, wherein amino acid position numbering is with reference to SEQ ID NO: 49.

30. The genetically modified host cell according to any one of the preceding claims, wherein the morphinone reductase comprises one or more substitutions selected from the group consisting of: M115L, P147L, and L193P, wherein amino acid position numbering is with reference to SEQ ID NO: 49, for example P147L, Y191 C, and L193P, or M115L, P147L, Y191C, and L193P.Case Ref. P322WO IPTector™31. The genetically modified host cell accordin to claim 26, wherein the morphinone reductase is selected from the group consisting of: SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 , SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 185, and SEQ ID NO: 193.

32. The genetically modified host cell according to any of the preceding claims, wherein the heterologous polynucleotide encoding a morphinone reductase has at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 186, and SEQ ID NO: 194.

33. The genetically modified host cell according to claim 32, wherein the heterologous polynucleotide encoding a morphinone reductase is selected from the group consisting of: SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 186, and SEQ ID NO: 194.

34. The genetically modified host cell according to any of the preceding claims, wherein the morphinone reductase comprises an amino acid sequence encoded by a polynucleotide selected from the group consisting of: SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 186, and SEQ ID NO: 194.

35. The genetically modified host cell according to any of the preceding claims, wherein the N-demethylase is also an O-demethylase.

36. The genetically modified host cell according to any of the preceding claims, wherein the N-demethylase is of, or derived from, an insect or a fungus.Case Ref. P322WO IPTector™ 37. The genetically modified host cell according to any of the preceding claims, wherein the N-demethylase is not of, or derived from, i) a plant, such as an alkaloid producing plant, ii) a human, and / or iii) a bacterium.

38. The genetically modified host cell according to any of the preceding claims, wherein the N-demethylase is of family CYP6.

39. The genetically modified host cell according to any of the preceding claims, wherein the N-demethylase is of the order Lepidoptera.

40. The genetically modified host cell according to any of the preceding claims, wherein the N-demethylase is of the genus Helicoverpa.

41. The genetically modified host cell according to any of the preceding claims, wherein the N-demethylase is of the species Helicoverpa armigera.

42. The genetically modified host cell according to any of the preceding claims, wherein the N-demethylase is of the genus Heliothis.

43. The genetically modified host cell according to any of the preceding claims, wherein the N-demethylase is of the species Heliothis virescens.

44. The genetically modified host cell according to any of the preceding claims, wherein the N-demethylase is of the genus Spodoptera.

45. The genetically modified host cell according to any of the preceding claims, wherein the N-demethylase is a fungal demethylase.

46. The genetically modified host cell according to any one of the preceding claims, wherein the N-demethylase is a fungal cytochrome P450 enzyme of a cytochrome P450 family selected from CYP64 and CYP75.- Ill -Case Ref. P322WO IPTector™47. The genetically modified host cell according to any of the preceding claims, wherein the N-demethylase is a fungal demethylase of a genus selected from Rhizopus, Lichtheimia, Syncephalastrum, Cunninghamella, Mucor, Parasitella, Absidia, Choanephora, Bifiguratus and Choanephora.

48. The genetically modified host cell according to any of the preceding claims, wherein the N-demethylase is a fungal demethylase of a 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.

49. The genetically modified host cell according to any of the preceding claims, wherein the N-demethylase has one or more conserved amino acids corresponding to positions G103, H111 , K167, E198, R219, L223, 1256, A259, L273, V284, 1309, L314, Q517, L160, N216, or R443 of any one of: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 77, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, and SEQ ID NO: 119, or conservative substitutions thereof.

50. The genetically modified host cell according to claim 49, wherein the one or more conserved amino acid is / are in or near the active site of the demethylase, optionally correspondingto positions G103, H111 and L314 of any one of: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 77, SEQ ID NO: 109, SEQ ID NO: 111 , SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, and SEQ ID NO: 119, or conservative substitutions thereof.

51. The genetically modified host cell according to any of the preceding claims, wherein the N-demethylase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 77, SEQ ID NO: 109, SEQ ID NO: 111 , SEQCase Ref. P322WO IPTector™ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, and SEQ ID NO: 189.

52. The genetically modified host cell accordin to claim 51 , wherein the N- demethylase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 77, and SEQ ID NO: 189; for example wherein the N- demethylase is selected from the group consisting of: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 77, and SEQ ID NO: 189; and optionally wherein the substrate is 14-hydroxymorphinone or oxymorphone.

53. The genetically modified host cell accordingto claim 51 , wherein the N- demethylase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, and SEQ ID NO: 189; for example wherein the N- demethylase is selected from the group consisting of: SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, and SEQ ID NO: 189; and optionally wherein the substrate is 14-hydroxymorphinone or oxymorphone.

54. The genetically modified host cell according to claim 51 , wherein the N- demethylase is selected from the group consisting of: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 77, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, and SEQ ID NO: 189.

55. The genetically modified host cell according to any of the preceding claims, wherein the heterologous polynucleotide encoding a N-demethylase has at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 78, SEQCase Ref. P322WO IPTector™ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, and SEQ ID NO: 190.

56. The genetically modified host cell according to claim 55, wherein the heterologous polynucleotide encoding a N-demethylase has at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 78, and SEQ ID NO: 190; for example wherein the heterologous polynucleotide encoding a N-demethylase is selected from the group consisting of: SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 78, and SEQ ID NO: 190; and optionally wherein the substrate is 14-hydroxymorphinone or oxymorphone.

57. The genetically modified host cell according to claim 55, wherein the heterologous polynucleotide encoding a N-demethylase has at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, and SEQ ID NO: 190, for example wherein the heterologous polynucleotide encoding a N-demethylase is selected from the group consisting of: SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, and SEQ ID NO: 190; and optionally wherein the substrate is 14-hydroxymorphinone or oxymorphone.

58. The genetically modified host cell according to claim 55, wherein the heterologous polynucleotide encoding a N-demethylase is selected from the group consisting of: SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 78 SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, and SEQ ID NO: 190.

59. The genetically modified host cell according to any one of the preceding claims, wherein the cell comprises an uptake transporter having at least 50% sequence identity to an amino acid sequence defined in any one of: SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 81, SEQ ID NO:Case Ref. P322WO IPTector™83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91 , SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 183, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, and SEQ ID NO: 211 .

60. The genetically modified host cell according to any one of the preceding claims, wherein the cell comprises an uptake transporter having at least 50% sequence identity to an amino acid sequence defined in any one of: SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91 , SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, and SEQ ID NO: 183.

61. The genetically modified host cell according to claim 60, wherein the cell comprises an uptake transporter having at least 50% sequence identity to an amino acid sequence defined in any one of: SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 , and SEQ ID NO: 13; for example wherein the uptake transporter is selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13, and optionally wherein the substrate is 14-hydroxymorphinone or oxymorphone.

62. The genetically modified host cell according to claim 60, wherein the uptake transporter is selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91 , SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, and SEQ ID NO: 183.

63. The genetically modified host cell according to any of the preceding claims, wherein the cell comprises a heterologous polynucleotide encoding an uptake transporter having at least 50% sequence identity to a polynucleotide as defined in anyone of: SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ IDCase Ref. P322WO IPTector™NO: 184, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, and SEQ ID NO: 210.

64. The genetically modified host cell according to any of the preceding claims, wherein the cell comprises a heterologous polynucleotide encoding an uptake transporter having at least 50% sequence identity to a polynucleotide as defined in anyone of: SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, and SEQ ID NO: 184.

65. The genetically modified host cell according to claim 64, wherein the cell comprises a heterologous polynucleotide encoding an uptake transporter having at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14; for example wherein the polynucleotide encoding an uptake transporter is selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14.

66. The genetically modified host cell according to claim 64, wherein the cell comprises a heterologous polynucleotide encoding an uptake transporter having at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, and SEQ ID NO: 184; for example wherein the polynucleotide encoding an uptake transporter is selected from the group consisting of: SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, and SEQ ID NO: 184.

67. The genetically modified host cell according to claim 64, wherein the cell comprises a polynucleotide encoding an uptake transporter selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ IDCase Ref. P322WO IPTector™ NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, and SEQ ID NO: 184.

68. The genetically modified host cell according to any of the preceding claims, wherein the O-demethylase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 121, SEQ ID NO: 123, and SEQ ID NO: 125.

69. The genetically modified host cell according to claim 68, wherein the O- demethylase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 121 , SEQ ID NO: 123, SEQ ID NO: 125, and SEQ ID NO: 75; for example wherein the O-demethylase is selected from the group consisting of: SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, and SEQ ID NO: 75; and optionally wherein the substrate is 14-hydroxycodeinone.

70. The genetically modified host cell according to claim 68, wherein the O- demethylase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 69, SEQ ID NO: 71 , SEQ ID NO: 73, and SEQ ID NO: 75; for example wherein the O-demethylase is selected from the group consisting of: SEQ ID NO: 69, SEQ ID NO: 71 , SEQ ID NO: 73, and SEQ ID NO: 75; and optionally wherein the substrate is 14-hydroxycodeinone.

71. The genetically modified host cell according to claim 68, wherein the O- demethylase is selected from the group consisting of: SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 121, SEQ ID NO: 123, and SEQ ID NO: 125.

72. The genetically modified host cell according to any of the preceding claims, wherein the heterologous polynucleotide encoding an O-demethylase has at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, and SEQ ID NO: 212.

73. The genetically modified host cell according to claim 72, wherein the heterologous polynucleotide encoding an O-demethylase has at least 50% sequence identity toCase Ref. P322WO IPTector™a polynucleotide as defined in any one of: SEQ ID NO: 76, SEQ ID NO: 122, SEQ ID NO: 124, and SEQ ID NO: 126; for example wherein the heterologous polynucleotide encoding an O-demethylase is selected from the group consisting of: SEQ ID NO: 76, SEQ ID NO: 122, SEQ ID NO: 124, and SEQ ID NO: 126; and optionally wherein the substrate is 14-hydroxycodeinone.

74. The genetically modified host cell according to claim 72, wherein the heterologous polynucleotide encoding an O-demethylase is selected from the group consisting of: SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, and SEQ ID NO: 212.

75. The genetically modified host cell according to any one of the preceding claims, wherein the cytochrome P450 reductase is of, or derived from, an insect or a fungus, such as of the genus Helicoverpa.

76. The genetically modified host cell according to any one of the preceding claims, wherein the cytochrome P450 reductase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, and SEQ ID NO: 191.

77. The genetically modified host cell according to claim 76, wherein the cytochrome P450 reductase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 65, SEQ ID NO: 67, and SEQ ID NO: 191; for example wherein the cytochrome P450 reductase is selected from the group consisting of: SEQ ID NO: 65, SEQ ID NO: 67, and SEQ ID NO: 191 .

78. The genetically modified host cell according to claim 76, wherein the cytochrome P450 reductase has at least 50% sequence identity to an amino acid sequence as defined in any one of SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, and SEQ ID NO: 191; for example wherein the cytochrome P450 reductase is selected from the group consisting of: SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, and SEQ ID NO: 191.Case Ref. P322WO IPTector™ 79. The genetically modified host cell according to claim 76, wherein the cytochrome P450 reductase is selected from the group consisting of: SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, and SEQ ID NO: 191.

80. The genetically modified host cell according to any one of the preceding claims, wherein the cell comprises a polynucleotide, optionally heterologous, encoding a cytochrome P450 reductase having at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, and SEQ ID NO: 192.

81. The genetically modified host cell according to claims 80, wherein the cell comprises a polynucleotide, optionally heterologous, encoding a cytochrome P450 reductase having at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 66, SEQ ID NO: 68, and SEQ ID NO: 192; for example wherein the polynucleotide encoding a cytochrome P450 reductase is selected from the group consisting of: SEQ ID NO: 66, SEQ ID NO: 68, and SEQ ID NO: 192.

82. The genetically modified host cell according to claims 80, wherein the cell comprises a polynucleotide, optionally heterologous, encoding a cytochrome P450 reductase having at least 50% sequence identity to a polynucleotide as defined in any one of: SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, and SEQ ID NO: 192; for example wherein the polynucleotide encoding a cytochrome P450 reductase is selected from the group consisting of: SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, and SEQ ID NO: 192.

83. The genetically modified host cell according to any one of the preceding claims, wherein the cell comprises a polynucleotide, optionally heterologous, encoding a cytochrome P450 reductase is selected from the group consisting of: SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, and SEQ ID NO: 192.Case Ref. P322WO IPTector™ 84. The genetically modified host cell according to any one of the preceding claims, wherein one or more further native or endogenous genes of the cell is attenuated, disrupted and / or deleted.

85. The genetically modified host cell according any one of the preceding claims, wherein the one or more further native or endogenous genes are selected from the group consisting of: a aldehyde reductase, such as ARI1 (for example SEQ ID NO: 2); and an aldose reductase, such as GRE3 (for example SEQ ID NO: 4), and / or GCY1 (for example SEQ ID NO: 80).

86. The genetically modified host cell according to any one of the preceding claims, wherein the one or more further native or endogenous genes are deleted and selected from the group consisting of: a aldehyde reductase, such as ARI1 (for example SEQ ID NO: 2); and an aldose reductase, such as GRE3 (for example SEQ ID NO: 4), and / or GCY1 (for example SEQ ID NO: 80).

87. The genetically modified host cell according to 85, wherein the one or more further native or endogenous genes are deleted.

88. The genetically modified host cell according to any of the preceding claims, wherein the cell comprises an operative metabolic pathway comprising one or more polypeptides producing the substrate from one or more precursors.

89. The genetically modified host cell according to claim 88, wherein the one or more precursors are selected from the group consisting of: oripavine, thebaine, and a carbon source, such as glucose.

90. The genetically modified host cell according to any of claims 88-89, wherein the one or more precursors comprise oripavine, and the operative metabolic pathway comprises one or more polypeptides selected from:a) A 14-OH hydroxylase, such as a 14-OH hydroxylase capable of converting oripavine to 14-hydroxymorphinone,b) A cytochrome P450 reductase, such as a cytochrome P450 reductase capable of electron transfer to the 14-OH hydroxylase,Case Ref. P322WO IPTector™ c) An uptake transporter, such as an uptake transporter capable of transporting oripavine into the cell,d) A morphinone reductase, such as a morphinone reductase as defined in any one of the preceding claims,e) A N-demethylase, such as a N-demethylase as defined in any one of the preceding claims, andf) A further cytochrome P450 reductase, such as a cytochrome P450 reductase capable of electron transfer to the N-demethylase.

91. The genetically modified host cell according to any of claims 88-89, wherein the one or more precursors comprise thebaine, and the operative metabolic pathway comprises one or more polypeptides selected from:a) A 14-OH hydroxylase, such as a 14-OH hydroxylase capable of converting thebaine to 14-hydroxycodeinone,b) A cytochrome P450 reductase, such as a cytochrome P450 reductase capable of electron transfer to the 14-OH hydroxylase,c) An uptake transporter, such as an uptake transporter capable of transporting thebaine into the cell,d) A morphinone reductase, such as a morphinone reductase as defined in any one of the preceding claims,e) A N-demethylase, such as a N-demethylase as defined in any one of the preceding claims, andf) A further cytochrome P450 reductase, such as a cytochrome P450 reductase capable of electron transfer to the N-demethylase.

92. The genetically modified host cell according to any of claims 88-89, wherein the one or more precursors comprise a carbon source, such as glucose, and the operative metabolic pathway comprises one or more polypeptides selected from: a) A tyrosine hydroxylase (TH), such as a tyrosine hydroxylase capable of converting L-tyrosine to L-DOPA;b) A dopa decarboxylase (DODC), such as a dopa decarboxylase capable of converting L-DOPA to dopamine;Case Ref. P322WO IPTector™ c) A tyrosine decarboxylase (TYDC), such as a tyrosine decarboxylase capable of converting L-DOPAto dopamine,d) A hydroxyphenylpyruvate decarboxylase (HPPDC), such as a hydroxyphenylpyruvate decarboxylase capable of convertin 4-HPP into 4-HPPA; e) A norcoclaurine synthase (NCS), such as a norcoclaurine synthase capable of converting dopamine and 4-HPAA into (S)-norcoclaurine;f) A 6-O-methyltransferase (6-OMT), such as a 6-O-methyltransferase capable of converting (S)-norcoclaurine to (S)-Coclaurine,g) A N-methylcoclaurine 3’-monooxygenase (NMCH, CYP80), such as a N- methylcoclaurine 3’-monooxygenase catalyzing conversion of (S)-Coclaurine to (S)-3’-hydroxycoclaurine and / or (S)-N-Methylcoclaurine to (S)-3’-Hydroxy-N- Methylcoclaurine,h) A coclaurine N-methyltransferase (CNMT), such as a coclaurine N- methyltransferase capable of converting (S)-coclaurine to (S)-N- methylcoclaurine;i) A 3’-hydroxy-N-methyl-(S)-coclaurine 4’-O-methyltransferase (4-OMT), such as a 3’-hydroxy-N-methyl-(S)-coclaurine 4’-O-methyltransferase catalyzing conversion of (S)-3’-Hydroxy-N-Methylcoclaurine to (S)-reticuline;j) A dihydroreticuline synthase / dihydroreticuline reductase (DRS-DRR), such as a dihydroreticuline synthase and reductase capable of converting (S)-reticuline to (R)-reticuline,k) A salutaridine synthase (SAS), such as a salutaridine synthase capable of converting (R)-reticuline to salutaridine,l) A salutaridine reductase (SAR), such as a salutaridine reductase capable of converting salutaridine to salutaridinol,m) A salutaridinol-7-O-acetyltransferase (SAT), such as a salutaridinol-7-O- acetyltransferase capable of converting salutaridinol to 7-O-acetylsalutaridinol, n) A thebaine synthase (THS), such as a thebaine synthase capable of converting 7- O-acetylsalutaridinol to thebaine, ando) One or more cytochrome P450 reductases (CPRs), such as a cytochrome P450 reductases capable of electron transfer to the polypeptides.Case Ref. P322WO IPTector™ 93. The genetically modified host cell according to any of claims 88-89, wherein the one or more precursors comprise a carbon source, such as glucose, and the operative metabolic pathway comprises one or more polypeptides selected from: a) A tyrosine hydroxylase (TH), such as a tyrosine hydroxylase capable of converting L-tyrosine to L-DOPA;b) A dopa decarboxylase (DODC), such as a dopa decarboxylase capable of converting L-DOPA to dopamine;c) A monoamine oxidase (MAO), such as a monoamine oxidase catalyzing conversion of dopamine to 3,4 DHPAA,d) A norcoclaurine synthase (NCS), such as a norcoclaurine synthase capable of converting dopamine and 3,4 DHPAA into (S)-norlaudanosoline;e) A 6-O-methyltransferase (6-OMT), such as a 6-O-methyltransferase capable of converting (S)-norlaudanosoline into form (S)-coclaurine,f) A coclaurine N-methyltransferase (CNMT), such as a coclaurine N- methyltransferase capable of converting (S)-coclaurine into (S)-N- methylcoclaurine;g) A N-methylcoclaurine 3’-monooxygenase (NMCH, CYP80), such as a N- methylcoclaurine 3’-monooxygenase catalyzing conversion of (S)-Coclaurine to (S)-3’-hydroxycoclaurine and / or (S)-N-Methylcoclaurine to (S)-3’-Hydroxy-N- Methylcoclaurine,h) A 3’-hydroxy-N-methyl-(S)-coclaurine 4’-O-methyltransferase (4-OMT), such as a 3’-hydroxy-N-methyl-(S)-coclaurine 4’-O-methyltransferase catalyzing conversion of (S)-3’-Hydroxy-N-Methylcoclaurine to (S)-reticuline;i) A dihydroreticuline synthase / dihydroreticuline reductase (DRS-DRR), such as a dihydroreticuline synthase and reductase capable of converting (S)-reticuline to (R)-reticuline,j) A salutaridine synthase (SAS), such as a salutaridine synthase capable of converting (R)-reticuline to salutaridine,k) A salutaridine reductase (SAR), such as a salutaridine reductase capable of converting salutaridine to salutaridinol,l) A salutaridinol-7-O-acetyltransferase (SAT), such as a salutaridinol-7-O- acetyltransferase capable of converting salutaridinol to 7-O-acetylsalutaridinol, m) A thebaine synthase (THS), such as a thebaine synthase capable of converting 7- O-acetylsalutaridinol to thebaine, andCase Ref. P322WO IPTector™ n) One or more cytochrome P450 reductases (CPRs), such as a cytochrome P450 reductases capable of electron transfer to the polypeptides.

94. The genetically modified host cell according to any of claims 88-93, wherein the cell comprises a plurality of the operative metabolic pathways as defined in any one of claims 90-93.

95. The genetically modified host cell according to any of claims 90-94, wherein the cell further comprises a SAM alkyl transferase, such as a S-adenosylmethionine (SAM) dependent alkyl transferase capable of converting noroxymorphone to an opioid, such as naloxone, naltrexone, or nalmefene.

96. The genetically modified host cell according to any one of claims 1 -95, wherein the cell is an eukaryote selected from the group consisting of a mammalian, insect, plant, orfungal cell.

97. The genetically modified host cell of claim 96, wherein the cell is a plant cell of the genus Physcomitrella or Papaver or Nicotiana.

98. The genetically modified host cell of any of claims 96-97, wherein the cell is a plant cell of the species Papaver somniferum or Nicotiana benthamiana.

99. The genetically modified host cell of claim 96, wherein the cell is a fungal cell selected from the phylas consisting of Ascomycota, Basidiomycota, Neocallimastigomycota, Glomeromycota, Blastocladiomycota, Chytridiomycota, Zygomycota, Oomycota and Microsporidia.

100. The genetically modified host cell of claim 96, wherein the fungal cell is a yeast selected from the group consisting of ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and Fungi Imperfect! yeast (Blastomycetes).

101. The genetically modified host cell of claim 100, wherein the yeast cell is selected from the genera consisting of Saccharomyces, Kluveromyces, Candida, Pichia,Case Ref. P322WO IPTector™ Debaromyces, Hansenula, Yarrowia, Zygosaccharomyces, and Schizosaccharomyces.

102. The genetically modified host cell of claim 101, wherein the 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.

103. The genetically modified host cell of claim 96, wherein the fungal cell is a filamentous fungus.

104. The genetically modified host cell of claim 103, wherein the filamentous fungal cell is selected from the phylas consisting of Ascomycota, Eumycota and Oomycota.

105. The genetically modified host cell of claim 104, wherein the filamentous fungal cell is 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 Trichoderma106. The genetically modified host cell of claim 105, wherein the filamentous fungal cell is 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, FusariumCase Ref. P322WO IPTector™ 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.

107. The genetically modified host cell according to any one of the preceding claims, wherein:a) the N-demethylase has at least 50% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO: 179, SEQ ID NO: 31 , and SEQ ID NO: 33;b) the O-demethylase has at least 50% sequence identity to the polypeptide selected from the group consisting of: SEQ ID NO: 69 and SEQ ID NO: 211 ; c) the morphinone reductase has at least 50% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO: 35, SEQ ID NO: 193, and SEQ ID NO: 185;d) the uptake transporter has at least 50% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO: 199, SEQ ID NO: 97, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 91 , and SEQ ID NO: 207; and / or e) the cytochrome P450 reductase has at least 50% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO: 191 and SEQ ID NO: 67;wherein the sequence identity for each of a), b), c), d), and e) may be, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100%.

108. A polypeptide selected from the group consisting of: an N-demethylase as defined in any one of the preceding claims, a morphinone reductase as defined in any one of the preceding claims, an O-demethylase as defined in any one of the preceding claims, a cytochrome P450 reductase as defined in any one of the preceding claims, and an uptake transporter as defined in any one of the preceding claims.Case Ref. P322WO IPTector™109. A composition comprisingthe polypeptide of claim 108, and optionally one or more excipients.

110. A method for reducing a substrate using a morphinone reductase, the method comprising contactingthe substrate with the morphinone reductase.

111. The method of claim 110, wherein the substrate is selected from the group consisting of: Morphinone, Oxymorphinone, oxymorphone, Codeinone, Codeine, Morphine, 14-hydroxycodeinone, 14-hydroxy norcodeinone, 14- hydroxymorphinone, 14-hydroxynormorphinone, Neopine, neopinone, Neomorphinone, and Neomorphine.

112. The method according to any one of claims 100-111 , wherein the morphinone reductase comprises an amino acid sequence having at least 50% sequence identity to a polypeptide selected from the group consisting of: SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 , SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 185, and SEQ ID NO: 193, and wherein the morphinone reductase comprises one or more substitutions at amino acid positions correspondingto 115, 147, 191 and / or 193 of SEQ ID NO: 35, wherein the amino acid residue at each substituted position differs from the amino acid residue at the corresponding position of SEQ ID NO: 35, wherein amino acid position numbering is with reference to SEQ ID NO: 35.

113. The method according to any one of claims 100-112, wherein the morphinone reductase comprises one or more amino acid residues L147, C191 and P193 at amino acid positions correspondingto 147, 191 and 193 of SEQ ID NO: 35, wherein amino acid position numbering is with reference to SEQ ID NO: 35.

114. The method according to any one of claims 100-113, wherein the morphinone reductase further comprises amino acid residue L115 at an amino acid position corresponding to 115 of SEQ ID NO: 35, wherein amino acid position numbering is with reference to SEQ ID NO: 35.

115. The method according to any one of claims 100-114, wherein the morphinone reductase comprises an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 49, and wherein the morphinone reductase comprises aCase Ref. P322WO IPTector™ substitution Y191 C, wherein amino acid position numbering is with reference to SEQ ID NO: 49.

116. The method according to any one of claims 100-115, wherein the morphinone reductase further comprises one or more substitutions selected from the group consisting of: M115L, P147L, and L193P, wherein amino acid position numbering is with reference to SEQ ID NO: 49, for example P147L, Y191 C, and L193P; or for example M115L, P147L,Y191C, and L193P.

117. A method for producing noroxymorphone from a substrate,(noroxymorphone),or a pharmaceutically acceptable salt thereof, wherein the method comprises subjecting the substrate to i) a morphinone reductase, and ii) an N-demethylase, and optionally iii) an O-demethylase.

118. The method according to any of the preceding claims, wherein the substrate is selected from the group consisting of: 14-hydroxymorphinone, 14- hydroxynormorphinone, oxymorphone, 14-hydroxycodeinone, and a combination thereof.

119. The method according to any one of claims 117-118, wherein the method further comprises preparing the substrate by chemical synthesis, for example wherein the substrate is a) 14-hydroxymorphinone prepared from oripavine or wherein the substrate is b) 14-hydroxycodeinone prepared from thebaine, such as wherein the chemical synthesis comprises oxidation.

120. The method according to any one of claims 117-119, wherein the method further comprises a cytochrome P450 reductase, an uptake transporter, or a combination thereof.Case Ref. P322WO IPTector™121 . The method according to any of the preceding claims, wherein the morphinone reductase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 , SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61 , SEQ ID NO: 63, SEQ ID NO: 185, and SEQ ID NO: 193.

122. The method according to any one of the preceding claims, wherein the morphinone reductase comprises one or more mutations selected from the group consisting of: L115X, L147X, C191X, and P193X, wherein X is any amino acid other than the corresponding amino acid residue in SEQ ID NO: 35, and wherein amino acid position numbering is with reference to SEQ ID NO: 35.

123. The method according to any one of claims 121 -122, wherein the morphinone reductase is selected from the group consisting of: SEQ ID: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 , SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 185, and SEQ ID NO: 193.

124. The method according to any of the preceding claims, wherein the N-demethylase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 77, SEQ ID NO: 109, SEQ ID NO: 111 , SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, and SEQ ID NO: 181.

125. The method according to claim 124, wherein the N-demethylase is selected from the group consisting of: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 77, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, and SEQ ID NO: 181.

126. The method according to any one of the preceding claims, wherein the method involves contacting the substrate with an uptake transporter.Case Ref. P322WO IPTector™127. The method according to any one of the preceding claims, wherein the method involves contacting the substrate with an uptake transporter having at least 50% sequence identity to an amino acid sequence defined in anyone of: SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101 , SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 183, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, and SEQ ID NO: 211 .

128. The method according to any one of the preceding claims, wherein the method involves contacting the substrate with an uptake transporter having at least 50% sequence identity to an amino acid sequence defined in anyone of: SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101 , SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, and SEQ ID NO: 183.

129. The method according to claim 128, wherein the uptake transporter is selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91 , SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, and SEQ ID NO: 183.

130. The method according to any of the preceding claims, wherein the method comprises an O-demethylase having at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 69, SEQ ID NO: 71 , SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 121, SEQ ID NO: 123, and SEQ ID NO: 125.131 . The method according to claim 130, wherein the O-demethylase is selected from the group consisting of: SEQ ID NO: 69, SEQ ID NO: 71 , SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 121, SEQ ID NO: 123, and SEQ ID NO: 125.Case Ref. P322WO IPTector™ 132. The method according to any of the preceding claims, wherein the method further comprises a cytochrome p450 reductase, for example wherein the cytochrome P450 reductase has at least 50% sequence identity to an amino acid sequence as defined in any one of: SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, and SEQ ID NO: 173.

133. The method according to any of claims 117-132, wherein the method comprises: a) Culturing the genetically modified host cell as defined in any of claims 1 -107 at conditions allowing the cell to produce noroxymorphone; andb) Optionally recovering and / or isolating the noroxymorphone.

134. The method according to claim 133, further comprising feeding a substrate to the cell, such as wherein the substrate is selected from the group consisting of: 14- hydroxymorphinone, 14-hydroxynormorphinone, oxymorphone, 14- hydroxycodeinone, oxycodone, and a combination thereof.

135. The method according to any of claims 133-134, further comprising one or more elements selected from:a) culturing the cell culture in a nutrient medium;b) culturing the cell culture under aerobic or anaerobic conditionsc) 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; andf) culturing the cell culture for between 10 hours to 30 days.

136. The method according to any of claims 117-131 , wherein one or more steps of the method are performed in vitro.

137. A method for producing an opioid from noroxymorphone, the method comprising: a) producing noroxymorphone using a method as defined in any one of claims 117- 136, andCase Ref. P322WO IPTector™ b) converting noroxymorphone to the opioid.

138. The method of claim 137, wherein step b) is performed using chemical synthesis.

139. The method of any of claims 137-138, wherein the opioid is selected from the group consisting of: Naloxone, Naltrexone, Nalmefene, Methylnaltrexone halide, such as Methylnaltrexone bromide, Nalfurafine, Naloxegol, and Nalbuphine.

140. The method of any of the preceding claims, wherein the opioid is naloxone,(naloxone),and step b) comprises:iv. a llylatio n of noroxymorphone by an a llylati ng agent, such as an a llylating agent selected from the group consisting of: an allyl halide, for example allyl bromide, allyl chloride, or allyl iodide; allyl acetate; allyl sulfate; allyl sulfonate; allyl carbonate; an allyl alcohol; and an allyl haloformate; such as allyl chloroformate; optionally in the presence of a base, such as triethylamine for example at a temperature of from 50 to 90 °C, such as 70 °C; and / or a catalyst;v. reductive amination of an allyl aldehyde comprising condensation with an allyl aldehyde, such as acrolein, to form an imine; followed by reduction of the imine to provide naloxone; orvi. peptide coupling of noroxymorphone with a carboxylic acid, for example acrylic acid, to form a peptide, followed by reduction of the peptide to provide naloxone, for example by reduction using LiAlH4.

141. The method of any of the preceding claims, wherein the opioid is naltrexone,Case Ref. P322WO IPTector™(naltrexone),and step b) comprises:ii. alkylation of noroxymorphone by a cyclopropylmethylating agent, such as a cyclopropylmethyl halide, for example cyclopropylmethyl bromide.

142. The method of claim 141, wherein step b) further comprises adding a polar aprotic solvent, such as N-ethyl-2-pyrrolidone, and / or toluene.

143. The method of any of claims 141-142, wherein step b) further comprises adding a base, such as potassium bicarbonate, and / or DIPEA.

144. The method of any of claims 141-143, wherein step b) is conducted at a temperature of from 45°C to 80°C, such as from 52° to 60°C.

145. The method of any of claims 141-144, wherein the reaction time of step b) is from 12 hours to 36 hours, such as from 17 to 24 hours.

146. The method of claim 141, wherein step b) comprises adding a polar aprotic solvent, such as dimethylacetamide (DMA), optionally at a temperature of from 40 °C to 90 °C, such as from 50 °C to 70 °C; and optionally in the presence of a base, such as a bicarbonate, for example as sodium bicarbonate or potassium bicarbonate.

147. The method of any of the preceding claims, wherein the opioid is nalmefene,Case Ref. P322WO IPTector™(nalmefene),and the method comprises preparing naltrexone, optionally using a method as defined in any of claims 141-146, and then in a step b1) converting naltrexone to nalmefene.

148. The method of claim 147, wherein step b1 ) comprises performing an olefination reaction on naltrexone, optionally using a methyltriphenylphosphonium halide, such as methyltriphenylphosphonium bromide.

149. The genetically modified host cell or method according to any one of the preceding claims, wherein the sequence identity is at least 60%, such as at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, for example 100% sequence identity to the amino acid sequence or polynucleotide sequence.

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