Microbial cell with improved in vivo conversion of thebaine/oripavine

Abstract

A recombinant microbial host cell having improved in vivo conversion of reticuline and derivatives thereof (such as thebaine and/or oripavine) to relevant downstream opioids (such as neopinone, oripavine, northebaine, nororipavine or morphinone) and related compounds (such as heroin, morphine, codeine, thebaine, oripavine, oxycodone, hydrocodone, hydromorphone, oxymorphone, buprenorphine, naltrexone, naloxone or nalbuphine), wherein the microbial (such as fungal) host cell is heterologously expressing at least one functional transporter protein capable of transporting reticuline or a derivative thereof (such as thebaine and/or oripavine) and a heterologously expressed enzyme capable of acting upon reticuline or a derivative thereof. The invention also relates to uses of the microbial host cells and methods of making an opioid compound and/or opioid precursor compound and/or opioid derivative of interest.

Claims

1. A recombinant yeast cell heterologously expressing a transporter protein, wherein the transporter protein: (i) comprises an amino acid sequence having at least 70% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 112, 42, 56, 76, 78, 92, 94, 96, 98, 102, 106, 108, 116, 120, 124, 128, 130, 136, 138, 140, 142, 144, 148, 154, 156, 158, 160, 168, 172, 182, amino acids 1 to 607 of SEQ ID NO: 8, amino acids 1 to 1289 of SEQ ID NO:4, amino acids 1 to 654 of SEQ ID NO: 6, and amino acids 1 to 583 of SEQ ID NO: 10; and/or (ii) comprises an amino acid sequence having at least 70% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 182, 186, 42, 44, 48, 50, 56, 62, 64, 72, 76, 78, 82, 86, 92, 94, 96, 98, 102, 106, 108, 110, 112, 116, 120, 124, 128, 130, 136, 138, 140, 142, 144, 148, 154, 158, 160, 164, 166, 168, 172, 178, 180, and amino acids 1 to 604 of SEQ ID NO: 31; and wherein heterologous expression of the transporter protein improves the recombinant yeast cell uptake of thebaine or oripavine for in vivo conversion.

2. The recombinant yeast cell of claim 1, wherein: (a) the transporter protein improves the yeast cell uptake of thebaine and comprises an amino acid sequence having at least 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 112, 42, 56, 76, 78, 92, 94, 96, 98, 102, 106, 108, 116, 120, 124, 128, 130, 136, 138, 140, 142, 144, 148, 154, 156, 158, 160, 168, 172, 182, amino acids 1 to 607 of SEQ ID NO:8, amino acids 1 to 1289 of SEQ ID NO:4, amino acids 1 to 654 of SEQ ID NO:6, and amino acids 1 to 583 of SEQ ID NO: 10; and (b) the transporter protein improves the yeast cell uptake of oripavine and comprises an amino acid sequence having at least 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 182, 186, 42, 44, 48, 50, 56, 62, 64, 72, 76, 78, 82, 86, 92, 94, 96, 98, 102, 106, 108, 110, 112, 116, 120, 124, 128, 130, 136, 138, 140, 142, 144, 148, 154, 158, 160, 164, 166, 168, 172, 178, 180, and amino acids 1 to 604 of SEQ ID NO: 31.

3. The recombinant yeast cell of claim 1, which further converts thebaine and/or oripavine into an opioid compound of interest due to the in vivo presence of a heterologously expressed demethylase enzyme.

4. The recombinant yeast cell of claim 3, wherein the heterologously expressed demethylase enzyme is a P450 capable of demethylase activity on thebaine and/or oripavine, and wherein the recombinant yeast cell: (1) converts thebaine into neopinone by in vivo heterologous expression of thebaine 6-O-demethylase; or (2) converts thebaine into oripavine by in vivo heterologous expression of O-demethylase; or (3) converts thebaine into northebaine by in vivo heterologous expression of N-demethylase; or (4) converts oripavine into nororipavine by in vivo heterologous expression of heterologously expressed N-demethylase; or (5) converts oripavine into morphinone by in vivo heterologous expression of oripavine 6-O-demethylase.

5. The recombinant yeast cell of claim 1, wherein the transporter protein comprises an amino acid sequence having at least 70% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 10, 12, 14, 16, 20, 22, 27, and 31.

6. The recombinant yeast cell of claim 1, wherein the transporter protein comprises an amino acid sequence having at least 70% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, and 188.

7. The recombinant yeast cell of claim 1, wherein the transporter protein comprises an amino acid sequence having at least 70% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 42, 50, 96, 106, 136, 140, 164, 180, 182, and 186.

8. The recombinant yeast cell of claim 1, wherein the transporter protein comprises an amino acid sequence having at least 70% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 42, 56, 76, 78, 92, 94, 96, 98, 102, 106, 112, 154, 168, 172, and 182.

9. The recombinant yeast cell of claim 1, wherein the transporter protein comprises an amino acid sequence having at least 70% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 42, 106, 182, and 186.

10. The recombinant yeast cell of claim 1, wherein the recombinant yeast cell is a Saccharomyces cerevisiae cell.

11. A method of producing in vivo a thebaine derivative or an oripavine derivative in a cell culture, comprising culturing the recombinant yeast cell of claim 1 in the cell culture.

12. The method of claim 11, wherein the method further comprises in vivo producing neopinone, oripavine, northebaine, nororipavine or morphinone in the cell culture.

13. The method of claim 11, wherein in vivo production a thebaine derivative or an oripavine derivative is increased compared to a control host yeast cell that is not heterologously expressing the transporter protein.

14. The recombinant yeast cell of claim 3, wherein the opioid compound of interest is neopinone, oripavine, northebaine, nororipavine, or morphinone.

Description

DRAWINGS

(1) FIG. 1: Shows prior art known synthesis pathway for different opioid compounds

DETAILED DESCRIPTION OF THE INVENTION

(2) A Microbial Host Cell

(3) As broadly known in the art, a microbe is a microscopic organism capable of existing in a single-celled form or in a colony of cells. Typically, microbes are capable of rapidly dividing into a relatively homogenous population and may be cultured by those skilled in the art very effectively under relatively simple conditions to quickly produce high densities of cells. Microbial host cells are such microbes suitable for industrial application which may be engineered (e.g. using recombinant DNA technologies) to produce one or more products of interest (such as opioids, their intermediates or derivatives). Suitable microbial host cells may be eukaryotic or prokaryotic cells. Non-limiting examples of suitable eukaryotes for scalable production of opioids, their intermediates or derivatives, include fungi such as a filamentous fungus cell or a yeast cell. Non-limiting examples of suitable prokaryotes for scalable production of opioids, their intermediates or derivatives, include bacteria, such as E. coli, Pseudomonas sp. or Bacillus subtilis. Non-limiting examples of suitable yeast cells for scalable production of opioids, their intermediates or derivatives, include

(4) As broadly known in the art, a fungus host cell may e.g. be a yeast cell or e.g. a filamentous fungus cell.

(5) In some circumstances, the fungus host cell is preferably a yeast cell.

(6) The fungal host cell may e.g. be a filamentous fungus cellsuch as e.g. an Aspergillus sp. cell, Penicillium sp. cell, Trichoderma sp. cell, Talaromyces sp. cell, Asteromyces sp. cell or Neurospora sp. cell.

(7) A preferred filamentous fungus cell is an Aspergillus sp. cell.

(8) For example, suitable filamentous fungus cell species can be Aspergillus nidulans, Aspergillus sydowii, Aspergillus terreus, Aspergillus oryzae, Aspergillus caelatus, Aspergillus chevalieri, Aspergillus longivesica, Aspergillus parvulus, Aspergillus amylovorus, Aspergillus niger, Aspergillus aculeatus, Aspergillus ellipticus, Aspergillus violaceofuscus, Aspergillus brunneoviolaceus, Aspergillus japonicus, Aspergillus brasiliensis, Aspergillus aculeatinus, Aspergillus thermomutatus, Aspergillus implicatus, Aspergillus acristatus, Penicillium bilaiae, Penicillium rubens, Penicillium chrysogenum, Penicillium expansum, Penicillium antarcticum, Trichoderma reesei, Talaromycesatroroseus, Asteromyces cruciatus, or Neurospora crassa.

(9) The yeast cell may e.g. be Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Xanthophyllomyces dendrorhous, Candida albicans, Rhodotorula sp., or Rhodospiridium sp.

(10) In some circumstances, the yeast cell is preferably a Saccharomyces, most preferably, a Saccharomyces cerevisiae cell.

(11) The heterologously expressed enzyme capable of the conversion of thebaine/oripavine of item (i) of the first aspect

(12) Preferably, the enzyme capable of converting thebaine/oripavine into derivatives thereof and/or further intermediates in a pathway for opioid production, is a demethylase.

(13) As discussed above, based on the technical disclosure herein and the prior art knowledge of the skilled person, it is routine work for the skilled person to make a recombinant microbial host (e.g. yeast) capable of: (1): in vivo conversion of thebaine into neopinone due to the in vivo presence of heterologously expressed thebaine 6-O-demethylase; or (2): in vivo conversion of thebaine into oripavine due to the in vivo presence of heterologously expressed O-demethylase; or (3): in vivo conversion of thebaine into northebaine due to the in vivo presence of heterologously expressed N-demethylase; or (4): in vivo conversion of oripavine into nororipavine due to the in vivo presence of heterologously expressed N-demethylase; or (5): in vivo conversion of oripavine into morphinone due to the in vivo presence of heterologously expressed thebaine 6-O-demethylase.

(14) The skilled person knows from the prior art and/or the technical disclosure herein different suitable examples of an enzyme capable of converting thebaine/oripavine into derivatives thereof and/or further intermediates in a pathway for opioid production, which may be heterologously expressed as an enzyme of item (i) of the first aspect such as e.g. the ones explicitly discussed herein.

(15) As discussed above and without being limited by theory, the improved positive yield effect demonstrated herein is probably related to a speculated ability of the herein relevant transporter proteins to transport more thebaine and/or oripavine into the host cell, thereby increasing the intracellular amount of thebaine and/or oripavine (i.e. in vivo) in the host cell.

(16) Consequently, one may obtain the benefit (i.e. improved yield of derivatives enzyme capable of converting thebaine/oripavine into derivatives thereof and/or further intermediates in a pathway for opioid production, thereof and/or further intermediates in a pathway for opioid production) of the present invention independently of the specific enzyme capable of converting thebaine/oripavine/thevinone into, heterologously enzyme usedi.e. one may in principle use any suitable (e.g. prior art known) conversion of thebaine/oripavine heterologously enzyme of interesti.e. this element of the present invention may be seen as an element based on prior art known knowledge for the skilled person.

(17) Examples of suitable conversion of thebaine/oripavine heterologously enzyme may e.g. be: thebaine to neopinone: thebaine 6-O-demethylase encoded by the T6ODM genesee e.g. of WO2018/075670A1; thebaine to oripavine: Codeine O-demethylase encoded by the CODM gene-se e.g. of WO2018/075670A1 thebaine to northebaine: N-demethylase encoded by the Bacillus BM3 genesee e.g. and FIG. 24 of WO2018/075670A1. oripavine into nororipavine: N-demethylase encoded by the Bacillus BM3 genesee e.g. and FIG. 24 of WO2018/075670A1; oripavine into morphinone: thebaine 6-O-demethylase encoded by the T6ODM genesee e.g. of WO2018/075670A1.

(18) In working examples herein were used fungal N-demethylase genes/enzymes that are different from the bacterial N-demethylase (e.g. Bacillus BM3 gene) described in WO2018/075670A1.

(19) The fungal N-demethylase based genes/enzymes used in working Example herein are described in international PCT patent application with number PCT/EP2018/066155, which was filed 18 Jun. 2018 and not published at the filing/priority date of the present application.

(20) The PCT/EP2018/066155 application also describes a number of different fungal O-demethylases that are suitable for the thebaine to oripavine conversion.

(21) Accordingly, in a preferred embodiment the N-demethylase is a N-demethylase selected from the group consisting of: a N-demethylase comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with amino acids 1 to 522 of SEQ ID NO:25 (CYPDN8).

(22) Preferably, the recombinant fungus host cell is capable of: (1): in vivo conversion of thebaine into neopinone due to the in vivo presence of heterologously expressed thebaine 6-O-demethylase; or (2): in vivo conversion of thebaine into oripavine due to the in vivo presence of heterologously expressed O-demethylase; or (3): in vivo conversion of thebaine into northebaine due to the in vivo presence of heterologously expressed N-demethylase.

(23) Most preferably, the recombinant fungus host cell is capable of: (3): in vivo conversion of thebaine into northebaine due to the in vivo presence of heterologously expressed N-demethylase.

(24) It may also be preferred that the recombinant fungus host cell is capable of: (3): in vivo conversion of thebaine into northebaine due to the in vivo presence of heterologously expressed N-demethylase; or (4): in vivo conversion of oripavine into nororipavine due to the in vivo presence of heterologously expressed N-demethylase.

Heterologously Expressing at Least One Functional Transporter Protein Capable of Transporting Reticuline and/or its DerivativesFirst Aspect

(25) It some embodiments it may be preferred that the transporter protein capable of transporting reticuline and/or its derivatives is a transporter protein belonging to the NRT1/PTR (NPF) transporter protein family.

(26) The skilled person may routinely determine whether or not a transporter protein capable of transporting reticuline and/or its derivatives is an NPF transporter protein or not. The two articles: Jrgensen et al. (Origin and evolution of transporter substrate specificity within the NPF fami-ly; eLife 2017; 6: e19466. DOI: 10.7554/eLife. 19466); Jrgensen et al. (A Functional EXXEK Motif is Essential for Proton Coupling and Active Glucosinolate Transport by NPF2.11; Plant Cell Physiol. 56 (12): 2340-2350 (2015)) in detail describe the NPF transporter protein family and based on the definition/description of this NPF family in these articles may the skilled person routinely determine whether or not a transporter protein of interest is an NPF transporter protein or not.

(27) As discussed in the dated 2015 article of Jrgensenthe Functional EXXEK Motif is essential for NPFi.e. in accordance with the art, an NPF transporter protein is a protein comprising this EXXEK Motif.

(28) It some embodiments it may be preferred that the transporter protein capable of transporting reticuline and/or its derivatives is a transporter protein belonging to the Purine Uptake Permease (PUP) transporter protein family. The PUP transporters are believed to be a distinct group of a superfamily of drug and metabolite transporters that evolved in terrestrial plant species. Jelesko J. G. 2012 (An expanding role for purine uptake permease-like transporters in plant secondary metabolism, Front Pnat Sci 2012; 3:78. As used herein, the term capable of PUP activity refers to purine nucleoside transmembrane transporter activity. As used herein, the term PUP transporters refers to uptake transporters capable of enhancing in-vivo concentration of purine nucleobase substrates in the host, and with particular reference to the specific reactions exemplified herein, to increase the uptake of reticuline derivatives, most preferably of thebaine and/or oripavine.

(29) As discussed above, the recombinant host cell of an embodiment of the first aspect is a microbial host cell (such as a yeast cell), wherein the microbial host cell is heterologously expressing at least one functional transporter protein capable of transporting reticuline and/or its derivatives selected from the group consisting of: a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with amino acids 1 to 584 of SEQ ID NO:2 (T14_PsoNPF3_GA); a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with amino acids 1 to 1289 of SEQ ID NO:4 (T1_CjaMDR1_GA); a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with amino acids 1 to 654 of SEQ ID NO:6 (T4_EsaGTR_GA); a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with amino acids 1 to 607 of SEQ ID NO:8 (T7_PtrPOT_GA); a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with amino acids 1 to 583 of SEQ ID NO: 10 (T60_AmeNPF2_GA); a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with amino acids 1 to 594 of SEQ ID NO: 12 (T57_AcoNPF_GA); a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with amino acids 1 to 527 of SEQ ID NO: 14 (T52_BmePTR2_GA); a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with amino acids 1 to 512 of SEQ ID NO: 16 (T38_ScuPTR2_GA); a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with amino acids 1 to 636 of SEQ ID NO: 18 (T11_AthGTR1_GA); a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with amino acids 1 to 565 of SEQ ID NO:20 (T19_RmiPTR2_GA); a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with amino acids 1 to 598 of SEQ ID NO:22 (T70_CmaNPF_GA); a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with amino acids 1 to 515 of SEQ ID NO:24 (T54_MelPOT_GA); a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with amino acids 1 to 593 of SEQ ID NO:27 (T65_ljaNPF_GA); a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with amino acids 1 to 604 of SEQ ID NO:29 (T94_EcrPOT_GA); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with amino acids 1 to 604 of SEQ ID NO:31 (T97_ScaT14_GA); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:32 (T101_McoPUP3_1); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:34 (T102_PsoPUP3_1); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:36 (T103_PsoPUP3_2); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:38 (T104_PsoPUP3_3); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:40 (T105_PsoPUP-L); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:42 (T109_GfIPUP3_83); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:44 (T113_PsoPUP3_32); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:46 (T114_TorPUP3_40); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:48 (T115_CsaPUP3_48); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:50 (T116_HanPUP3_56); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:52 (T117_MacPUP3_64); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:54 (T121_NnuPUP3_9); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:56 (T122_PsoPUP3_17); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:58 (T123_PsoPUP3_25); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:60 (T124_PsoPUP3_33); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:62 (T125_JcuPUP3_41); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:64 (T126_CpePUP3_49); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:66 (T127_LsaPUP3_57); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:68 (T128_PsoPUP3_65); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:70 (T129_PsoPUP3_73); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:72 (T130_NdoPUP3_89); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:74 (T131_PbrPUP3_81); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:76 (T132_CmiPUP3_10); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:78 (T133_PsoPUP3_18); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:80 (T135_PsoPUP_34); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:82 (T136_RchPUP3_42); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:84 (T137_EguPUP3_50); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 86 (T138_AduPUP3_58); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:88 (T139_PsoPUP3_66); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:90 (T140_PalPUP3_74); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:92 (T141_EcaPUP3_88); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:94 (T142_McoPUP3_4); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:96 (T143_CmiPUP3_11); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:98 (T144_PsoPUP3_19); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 100 (T146_PsoPUP_35); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 102 (T147_MesPUP3_43); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 104 (T148_HimPUP3_51); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 106 (T149_AcoPUP3_59); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 108 (T150_PsoPUP3_67); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 110 (T151_PatPUP3_75); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 112 (T152_GfIPUP3_87); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 114 (T153_PsoPUP3_5); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 116 (T154_CmiPUP3_12); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 118 (T156_PsoPUP3_28); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 120 (T157_RchPUP_36); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 122 (T158_DziPUP3_44); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 124 (T159_OeuPUP3_52); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 126 (T160_CeuPUP3_60); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 128 (T161_PsoPUP3_68); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 130 (T162_PmiPUP3_76); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 132 (T163_PbrPUP3_86); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 134 (T164_PsoPUP3_78); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 136 (T165_AcoPUP3_13); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 138 (T166_PsoPUP3_21); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 140 (T168_FvePUP3_37); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 142 (T169_ZjuPUP3_45); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 144 (T170_LsaPUP3_53); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 146 (T171_McoPUP3_61); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 148 (T172_AcoPUP3_69); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 150 (T173_PnuPUP3_77); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 152 (T174_PbrPUP3_85); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 154 (T175_PsoPUP3_6); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 156 (T176_AcoPUP3_14); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 158 (T177_PsoPUP3_22); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 160 (T178_PsoPUP3_30); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 162 (T179_PyePUP3_38); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 164 (T180_McoPUP3_46); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 166 (T181_HanPUP3_54); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 168 (T182_CpaPUP3_62); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 170 (T184_PraPUP3_79); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 172 (T186_ScaPUP3_84); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 174 (T188_AcoPUP3_15); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 176 (T189_PsoPUP3_23); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 178 (T191_MdoPUP3_39); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 180 (T192_CmiPUP3_47); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 182 (T193_AanPUP3_55); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 184 (T194_CchPUP3_63); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 186 (T195_JcuPUP3_71); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 188 (T196_PtrPUP3_80).

(30) It may be preferred that the host cell is a microbial host cell, wherein the microbial host cell is heterologously expressing at least two functional transporter proteins of the first aspectfor instance in working Example 5 the inventors discuss an example of a host cell that is heterologously expressing the six different functional transporter proteins SEQ ID NO:2 (T14_PsoNPF3_GA); SEQ ID NO: 4 (T1_CjaMDR1_GA); SEQ ID NO:10 (T60_AmeNPF2_GA); SEQ ID NO:14 (T52_BmePTR2_GA); SEQ ID NO: 18 (T11_AthGTR1_GA); SEQ ID NO:22 (T70_CmaNPF_GA).

(31) As discussed in for example the Conclusions of Examples 4 and 5 herein, expression of one of the transporter genes T14_PsoNPF3_GA, T1_CjaMDR1_GA, T4_EsaGTR_GA, T7_PtrPOT_GA or T97_ScaT14_GA in combination with a P450 capable of demethylating reticuline and/or its derivatives in a yeast strain was shown to improve bioconversion of thebaine to northebaine in the range of 22-63% in comparison to the control strain. This is objectively a significant improvement.

(32) Accordingly, preferably the recombinant host cell of the first aspect is a microbial host cell, wherein the microbial host cell (such as a yeast host cell) is heterologously expressing a P450 capable of demethylating reticuline and/or its derivatives and also heterologously expressing at least one functional transporter protein capable of transporting reticuline and/or its derivatives selected from the group consisting of: a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with amino acids 1 to 584 of SEQ ID NO:2 (T14_PsoNPF3_GA); a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with amino acids 1 to 1289 of SEQ ID NO:4 (T1_CjaMDR1_GA); a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with amino acids 1 to 654 of SEQ ID NO:6 (T4_EsaGTR_GA); a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with amino acids 1 to 607 of SEQ ID NO:8 (T7_PtrPOT_GA); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with amino acids 1 to 604 of SEQ ID NO:31 (T97_ScaT14_GA); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:32 (T101_McoPUP3_1); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:34 (T102_PsoPUP3_1); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:36 (T103_PsoPUP3_2); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:38 (T104_PsoPUP3_3); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:40 (T105_PsoPUP-L); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:42 (T109_GflPUP3_83); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:44 (T113_PsoPUP3_32); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:46 (T114_TorPUP3_40); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:48 (T115_CsaPUP3_48); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:50 (T116_HanPUP3_56); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:52 (T117_MacPUP3_64); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:54 (T121_NnuPUP3_9); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:56 (T122_PsoPUP3_17); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:58 (T123_PsoPUP3_25); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:60 (T124_PsoPUP3_33); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:62 (T125_JcuPUP3_41); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:64 (T126_CpePUP3_49); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:66 (T127_LsaPUP3_57); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:68 (T128_PsoPUP3_65); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:70 (T129_PsoPUP3_73); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:72 (T130_NdoPUP3_89); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:74 (T131_PbrPUP3_81); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:76 (T132_CmiPUP3_10); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 78 (T133_PsoPUP3_18); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:80 (T135_PsoPUP_34); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:82 (T136_RchPUP3_42); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:84 (T137_EguPUP3_50); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:86 (T138_AduPUP3_58); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:88 (T139_PsoPUP3_66); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:90 (T140_PalPUP3_74); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:92 (T141_EcaPUP3_88); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:94 (T142_McoPUP3_4); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:96 (T143_CmiPUP3_11); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO:98 (T144_PsoPUP3_19); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 100 (T146_PsoPUP_35); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 102 (T147_MesPUP3_43); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 104 (T148_HimPUP3_51); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 106 (T149_AcoPUP3_59); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 108 (T150_PsoPUP3_67); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 110 (T151_PatPUP3_75); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 112 (T152_GfIPUP3_87); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 114 (T153_PsoPUP3_5); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 116 (T154_CmiPUP3_12); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 118 (T156_PsoPUP3_28); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 120 (T157_RchPUP_36); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 122 (T158_DziPUP3_44); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 124 (T159_OeuPUP3_52); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 126 (T160_CeuPUP3_60); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 128 (T161_PsoPUP3_68); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 130 (T162_PmiPUP3_76); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 132 (T163_PbrPUP3_86); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 134 (T164_PsoPUP3_78); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 136 (T165_AcoPUP3_13); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 138 (T166_PsoPUP3_21); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 140 (T168_FvePUP3_37); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 142 (T169_ZjuPUP3_45); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 144 (T170_LsaPUP3_53); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 146 (T171_McoPUP3_61); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 148 (T172_AcoPUP3_69); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 150 (T173_PnuPUP3_77); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 152 (T174_PbrPUP3_85); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 154 (T175_PsoPUP3_6); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 156 (T176_AcoPUP3_14); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 158 (T177_PsoPUP3_22); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 160 (T178_PsoPUP3_30); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 162 (T179_PyePUP3_38); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 164 (T180_McoPUP3_46); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 166 (T181_HanPUP3_54); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 168 (T182_CpaPUP3_62); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 170 (T184_PraPUP3_79); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 172 (T186_ScaPUP3_84); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 174 (T188_AcoPUP3_15); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 176 (T189_PsoPUP3_23); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 178 (T191_MdoPUP3_39); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 180 (T192_CmiPUP3_47); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 182 (T193_AanPUP3_55); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 184 (T194_CchPUP3_63); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 186 (T195_JcuPUP3_71); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with the amino acid sequence of SEQ ID NO: 188 (T196_PtrPUP3_80).

(33) As discussed above and in working examples 4 and 5 herein, T14_PsoNPF3_GA and T97_ScaT14_GA are demonstrated to have a positive in vivo conversion effect for both thebaine and oripavine.

(34) Accordingly, in some embodiments, the recombinant microbial host cell of the first aspect is a microbial cell, wherein the microbial host cell (such as yeast host cell) is heterologously expressing at least one functional transporter protein selected from the group consisting of: a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with amino acids 1 to 584 of SEQ ID NO:2 (T14_PsoNPF3_GA); and a transporter protein comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99%) identity with amino acids 1 to 604 of SEQ ID NO:31 (T97_ScaT14_GA).

A Method of In Vivo Producing a Thebaine Derivative or an Oripavine Derivative (Such as e.g. Neopinone, Oripavine, Northebaine, Nororipavine or Morphinone)Second Aspect

(35) As discussed above, a second aspect of the invention relates to a method of in vivo producing a thebaine derivative or an oripavine derivative in a cell culture, comprising culturing the microbial host cell of the first aspect and/or herein relevant embodiments thereof in the cell culture, under conditions; (A): in which at least one of the heterologously expressed enzyme capable of conversion of thebaine and/or oripavine of the first aspect herein relevant embodiments thereof is thereby present in vivo in the host cell; and (B): in which thebaine and/or oripavine is present in vivo in the host cell; and (C): wherein the thebaine or oripavine of item (B) in vivo is converted into a thebaine derivative or an oripavine derivative due to the presence of the heterologously expressed enzyme of item (A) in order to thereby get in vivo production a thebaine derivative or an oripavine derivative.

(36) A preferred embodiment of the second aspect relates to a method of in vivo producing neopinone, oripavine, northebaine, nororipavine or morphinone in a cell culture, comprising culturing the host cell of the first aspect and/or herein relevant embodiments thereof in the cell culture, under conditions; (A): in which at least one of the conversion of thebaine/oripavine heterologously enzyme of item (1-5) of an embodiment of the first aspect and/or herein relevant embodiments thereof is expressed and thereby present in vivo in the host cell; and (B): in which thebaine and/or oripavine is present in vivo in the host cell; and (C): wherein the thebaine and/or oripavine of item (B) in vivo is converted into neopinone, oripavine, northebaine, nororipavine or morphinone due to the presence of the heterologously expressed enzyme of item (A) in order to thereby get in vivo production of neopinone, oripavine, northebaine, nororipavine or morphinone.

(37) In relation to item (B)thebaine and/or oripavine may be present in vivo in the host cell via e.g.: thebaine and/or oripavine in vivo biosynthesis within the host cell; and/or thebaine and/or oripavine may be present in a cell culture medium or reaction medium and then taken up by the host cell in order for the thebaine and/or oripavine to be present in vivo in the host cell for biotransformation.

(38) As discussed herein-thebaine and/or oripavine in vivo biosynthesis within a microbial host cell (such as a yeast host cell) is well known in the art.

(39) It is also well known (see e.g. working examples herein) how to prepare thebaine and/or oripavine supplemented cell culture medium or reaction medium to be taken up by the yeast host cell in order for the thebaine and/or oripavine to be present in vivo in the microbial host cell.

(40) As discussed above, the improved positive yield effect demonstrated herein is probably related to that the herein relevant transporter proteins increase the intracellular amount of thebaine and/or oripavine (i.e. in vivo) in the fungus host cell because more thebaine and/or oripavine is transported into the host yeast cell. This advantageous effect is also relevant in relation to in vivo biosynthesis within the host cell of thebaine/oripavine, since some of the thebaine/oripavine may be exported out of the host cell and herein relevant transporter proteins can then transport the thebaine/oripavine back into the host cell again.

(41) Using methods known in the art, the in item (C) produced neopinone, oripavine, northebaine, nororipavine or morphinone may be isolated in order to get a substantially pure (e.g. at least 20%, 30%, 50%, 60% or at least 90% pure w/w) composition of the compound(s). Alternatively, they may e.g. in vivo be converted to further herein relevant downstream compounds (see e.g. FIG. 1 herein and the Third Aspect, below).

(42) In short, based on the technical disclosure herein and the prior art knowledge of the skilled personit is routine work for the skilled person to perform the method of the second aspect and/or herein relevant embodiments thereof.

(43) In relation to the second aspectit may be preferred that it is a method for producing neopinone, oripavine or northebaine, wherein (A): at least one of the conversion of thebaine heterologously enzyme of item (1), (2) or (3) of the first aspect and/or herein relevant embodiments thereof is expressed and thereby present in vivo in the host cell; and (B): in which thebaine is present in vivo in the host cell; and (C): wherein the thebaine of item (B) in vivo is converted into neopinone, oripavine or northebaine due to the presence of the heterologously expressed enzyme of item (A) in order to thereby get in vivo production of neopinone, oripavine or northebaine.

(44) In relation to the second aspectit may be preferred that it is a method for producing oripavine or northebaine, wherein (A): at least one of the conversion of thebaine heterologously enzyme of item (2) or (3) of the first aspect and/or herein relevant embodiments thereof is expressed and thereby present in vivo in the host cell; and (B): in which thebaine is present in vivo in the host cell; and (C): wherein the thebaine of item (B) in vivo is converted into oripavine or northebaine due to the presence of the heterologously expressed enzyme of item (A) in order to thereby get in vivo production of oripavine or northebaine.

(45) Most preferably is a method for producing northebaine, wherein (A): at least one of the conversion of thebaine heterologously enzyme of item (3) of the first aspect and/or herein relevant embodiments thereof is expressed and thereby present in vivo in the host cell; and (B): in which thebaine is present in vivo in the host cell; and (C): wherein the thebaine of item (B) in vivo is converted into northebaine due to the presence of the heterologously expressed enzyme of item (A) in order to thereby get in vivo production of northebaine.

(46) Further, and in relation to the second aspect, it may be preferred that it is a method for producing oripavine, northebaine or nororipavine, wherein (A): at least one of the conversion of thebaine heterologously enzyme of item (2), (3) or (4) of the first aspect and/or herein relevant embodiments thereof is expressed and thereby present in vivo in the host cell; and (B): in which thebaine and/or oripavine is present in vivo in the host cell; and (C): wherein the thebaine of item (B) in vivo is converted into oripavine or northebaine or the oripavine of item (B) in vivo is converted into nororipavine due to the presence of the heterologously expressed enzyme of item (A) in order to thereby get in vivo production of oripavine, northebaine or nororipavine.

(47) Preferably, the method of the second aspect and/or herein relevant embodiments thereof is a method, wherein there in item (C) of the second aspect is an increased in vivo conversion of thebaine and/or oripavine due to that the cultured host cell is heterologously expressing at least one functional transporter protein (e.g. T14_PsoNPF3_GA) of the first aspect and/or herein relevant embodiments thereof; and wherein the increased in vivo conversion of thebaine and/or oripavine is understood to be relative to an otherwise identical performed method using an otherwise identical control host cell that is not heterologously expressing at least one functional transporter protein (e.g. T14_PsoNPF3_GA) of the first aspect and/or herein relevant embodiments thereof.

(48) The increased in vivo conversion of thebaine and/or oripavine is understood to be relative to an otherwise identical control host cell, which is not heterologously expressing at least one functional transporter protein (e.g. T14_PsoNPF3_GA) of the first aspect.

(49) The skilled person knows or can easily identify (by e.g. routine genome sequencing) an otherwise identical control host cell with no heterologously expressing of at least one functional transporter protein (e.g. T14_PsoNPF3_GA) of the first aspect.

(50) If for instance the yeast host cell is heterologously expressing e.g. T14_PsoNPF3_GA-then is the method of the second aspect and/or herein relevant embodiments thereof simply performed with the host cell heterologously expressing T14_PsoNPF3_GA and the otherwise identical control host cell with no expressing of T14_PsoNPF3_GA and the amount of in vivo conversion of thebaine and/or oripavine is then measured (e.g. by LC-MS)-if the use of the host cell with expressing of T14_PsoNPF3_GA is giving increased in vivo conversion as compared to the control host cell then it is understood that there is an increased in vivo conversion of thebaine and/or oripavine due to that the host cell is heterologously expressing T14_PsoNPF3_GA.

A Method of Producing an Opioid Compound of InterestThird Aspect

(51) As discussed above, a third aspect of the invention relates to a method of producing an opioid compound of interest, comprising first performing in vivo production of a thebaine derivative or an oripavine derivative (such as e.g. neopinone, oripavine, northebaine, nororipavine or morphinone) according to the second aspect and/or herein relevant embodiments thereof followed by suitable in vivo and/or in vitro synthesis steps in order to obtain the opioid compound of interest.

(52) A preferred embodiment of the third aspect relates to a method of producing an opioid compound of interest, comprising first performing in vivo production of neopinone, oripavine, northebaine, nororipavine or morphinone according to the second aspect and/or herein relevant embodiments thereof followed by suitable in vivo and/or in vitro synthesis steps in order to obtain the opioid compound of interest.

(53) In short, based on the technical disclosure herein and the prior art knowledge of the skilled personit is routine work for the skilled person to perform the method of the third aspect and/or herein relevant embodiments thereof.

(54) As discussed herein, starting from neopinone, oripavine, northebaine, nororipavine or morphinone-suitable in vivo and/or in vitro synthesis steps in order to obtain the opioid compound of interest are well known in the art. See for example WO2018/211331 and Sipos et al. (2009).

(55) As understoodthe term in vitro synthesis steps may e.g. relate to suitable chemical synthesis steps as e.g. illustrated for buprenorphine in FIG. 1 herein.

(56) Preferably, the opioid compound of interest is heroin, morphine, codeine, thebaine, oripavine, oxycodone, hydrocodone, hydromorphone, oxymorphone, buprenorphine, naltrexone, naloxone, nalmefene, noroxymorphone or nalbuphine. In some embodiments, most preferably the opioid compound of interest is buprenorphine, nalmefene or noroxymorphone.

EXAMPLES

(57) As discussed above, the amino acid sequence for P450 CYPDN8 N-demethylase from Rhizopus microspores is shown as SEQ ID NO. 9 herein and discussed in international PCT patent application with number PCT/EP2018/066155, which was filed 18 Jun. 2018.

(58) PCT patent application with number PCT/EP2018/066155 also describes other herein relevant technical details such as e.g. further details in relation to herein referred pOD75 and pOD13 plasmids. Accordingly, based on the technical disclosure herein and the technical disclosure of PCT patent application with number PCT/EP2018/066155the skilled person can routinely carry out the relevant technical matter of the present invention-such as e.g. the relevant working Examples herein.

Example 1: Strain Engineering

(59) Saccharomyces cerevisiae yeast strains were constructed in strain background EVST25898 (genotype MATalpha his30 leu20 ura30 aro3::pTEF1-ARO4 (K229L)-tCYC1::pPGK1-ARO7(T266L)-tADH1::KI CAT5-91Met GAL2 ho MIP1-661Thr SAL1-1 YORW22::npBIO1nt20npBIO6nt).

(60) The EVST25898 with the genotype above corresponds to S288C (genotype MATalpha his30 leu20 ura30). S288C is a publicly available widely used laboratory strain (see the Saccharomyces Genome Database (SGD)). As is known from other works, one would get similar results by use of EVST25898 with genotype above or by use of S288C (genotype MATalpha his30 leu20 ura30) as background/control strains, since these two host phenotypes are substantially identical.

(61) Plasmid Based Gene Expression

(62) Strain was transformed with relevant plasmids using the lithium acetate method (Gietz et al. 2002. Methods Enzymol. Vol 350, p 87-96).

(63) For testing the impact of possible transporter proteins on the bioconversion of Thebaine to Northebaine, the host yeast strain was transformed with a plasmid containing cytochrome P450 gene CYPDN8 N-demethylase from Rhizopus microspores (pOD75) along with a plasmid containing Cel_CPR (co) from Cunninghamella elegans (pOD13) in combination with the various possible transporter proteins. Genes were inserted and expressed using either P413TEF, P415TEF or p416TEF, all described by Mumberg et al., 1995. Gene. April 14; 156 (1): 119-22.

(64) The control strain was constructed by transforming strain EVST25898 with pOD75, pOD13 as well as an empty plasmid: p416TEF.

(65) Table 1 describes the plasmids that were expressed with the yeast strains. Transformants were selected in synthetic complete (SC) agar plates lacking histidine, leucine and uracil. Transformation plates were incubated for 3-4 days at 30 C. until visible colonies were obtained.

(66) TABLE-US-00003 TABLE 1 Plasmids expressed in the corresponding yeast strains Yeast Vector Promoter-Gene- Selection name Backbone Terminator Marker Description pOD13 P413TEF pTEF1-Cel_CPR_co- HIS3 Cel_CPR (co) from tCYC1 Cunninghamella elegans pOD75 P415TEF pTEF1-CYPDN8-tCYC1 LEU2 A0A0C7AZL4 (co) from Rhizopus microsporus P416TEF No gene inserted URA3 Mumberg et al., 1995. Gene. Apr 14; 156(1): 119-22
Gene Expression by Integration.

(67) Strain EVST25898 was modified by genomic integration using the Saccharomyces cerevisiae gene integration and expression system developed by Mikkelsen, M D et al. (Metab. Eng. 14, Issue 2, 104-111 (2012)). The cytochrome P450 gene CYPDN8, N-demethylase from Rhizopus microspores was expressed using the well-known Saccharomyces cerevisiae TEF1 promoter, and the Cel_CPR (co) from Cunninghamella elegans was expressed using the Saccharomyces cerevisiae PGK1 promoter. The expression cassette was integrated in site XII-5 using the Kluyveromyces lactis URA3 marker as selection marker for growth on media lacking uracil (described by Mikkelsen, M D et al. (Metab. Eng. 14, Issue 2, 104-111 (2012)). Subsequently, the transporter genes T11_AthGTR1_GA (SEQ ID NO: 17), T52_BmePTR2_GA (SEQ ID NO: 13), T14_PsoNPF3_GA (SEQ ID NO: 1), T60_AmeNPF2_GA (SEQ ID NO: 9), T1_CjaMDR1_GA (SEQ ID NO: 3) and T70_CmaNPF_GA (SEQ ID NO: 21) were integrated into the site XI-5 of the Saccharomyces cerevisiae strain using the Saccharomyces cerevisiae TEF1, PGK1, TEF2, TDH3, TPI1, and PDC1 promoters respectively. Selection for transformants was done using the well-known Kluyveromyces lactis LEU2 marker available e.g. from EUROSCARF and growth on media lacking leucine. After that, plasmid pOD13 (see Table 1) was transformed with the resulting strain in order to make the strain prototrophic by selecting on media lacking histidine. Transformation plates were incubated for 3-4 days at 30 C. until visible colonies were obtained.

Example 2. Cultivation and Harvest of Yeast Strains

(68) Cultivation. Yeast strains were cultivated in 96-deep-well-plate (DWP) format. Cells were grown in 0.5 ml SC-His-Leu-Ura medium at 30 C. with shaking at 250 rpm in ISF1-X Kuhner shaker for 20-24 hours and utilized as precultures for in vivo bioconversion assays.

(69) 50 l of the overnight cell cultures were grown in 450 l Synthetic complete (SC)-His-Leu-Ura medium (pH 7) or DELFT minimal medium (pH 7) containing 0.5 mM thebaine or oripavine. Both media contain 0.1 M potassium phosphate buffer.

(70) Thebaine (or Oripavine) were added via a 25 mM stock solution in DMSO. Cells were grown for 72 hours with shaking at 250 rpm.

(71) Harvest. 50 l of cell cultures were transferred to a new 96-deep-well-plate containing 50 l of MilliQ water. The harvested 96 well plate was incubated at 80 C. for 10 minutes. Plate was then centrifugated for 10 minutes at 4000 rpm. The supernatants were then diluted in MilliQ water to reach a final dilution of 1:100. Thebaine, northebaine, oripavine and nororipavine contents were analyzed by LC-MS.

Example 3. LC-MS Procedures

(72) For all compounds (Thebaine, Northebaine, Oripavine and Nororipavine) stock solutions were prepared in DMSO at a concentration of 10 mM. Standard solutions were prepared at concentrations of 6 M, 4 M, 2 M, 1 M, 500 nM, 200 nM, 100 nM, 50 nM, 20 nM and 10 nM from the stock solutions. Samples were injected into the Agilent 1290 UPLC coupled to an Ultivo Triple Quadrupole. The LC-MS method was as follows: Mobile Phase A. H.sub.2O+0.1% Formic acid; Mobile Phase B: Acetonitrile+0.1% Formic acid; Column: Phenomenex Kinetex 1.7 m XB-C18 100 , 2.1100 mm. The elution gradient is shown in Table 2 and the LC-MS conditions are given in Table 3. Table 4 shows the mass spectrometer source and detector parameters and Table 5 shows the target compounds, their retention times, their parent ion, transition ions (MRM) as well as dwell times, cone voltages and collision energies used.

(73) TABLE-US-00004 TABLE 2 Gradient for LC-MS Time (min) % B 0 2 0.30 2 4.00 30 4.40 100 4.90 100 5 2 6 2

(74) TABLE-US-00005 TABLE 3 LC-MS conditions Parameter Value Injection volume 2 l Column Temperature 30 C. 4 C. Injection method Flow through needle Flow 0.4 ml/min Auto sampler 10 C. 2 C. temperature Reconditioning wash 2% Acetonitrile (in H.sub.2O), 5 sec Weak wash 20% Methanol (in H.sub.2O), 5 sec Strong wash 30% Acetonitrile, 30% Methanol, 30% 2-Propanol, 10% H.sub.2O, 10 sec Seal wash 20% 2-Propanol (in H.sub.2O)

(75) TABLE-US-00006 TABLE 4 Mass spectrometer source and detector parameters (Ultivo Triple Quadrupole) Source Parameter Value Ion Source Electrospray Positive Mode (ESI+) Capillary Voltage 3.5 kV Nozzle Voltage 500 V Source Gas Temperature 290 C. Source Gas Flow 12 L/min Source Sheath Gas Temperature 380 C. Source Sheath Gas Flow 12 L/min Nebulizer 30 psi Mode MS/MS Collision See Table 4

(76) TABLE-US-00007 TABLE 5 Multiple reaction monitoring targets and conditions (ESI+) Reten- Frag- Colli- tion Parent Daughter Dwell mentor sion Target time ion ion time voltage energy compound (min) (m/z) (m/z) (ms) (V) (V) Northebaine 3.53 298 249 55.03 100 20 Thebaine 3.6 312 58 61.53 110 10 Oripavine 2.59 298 237 64.05 110 5 Nororipavine 2.54 284 218 70.30 110 10

Example 4. Identification of Transporters Capable of Improving Bioconversion of Thebaine and/or Derivatives Thereof

(77) Bioconversion. Expression of transporter genes in a strain containing cytochrome P450 gene CYPDN8 and cytochrome P450 reductase Cel_CPR (co) gave remarkable improvement in bioconversion of thebaine to northebaine for some of the transporter genes, where some exhibited a significant improved bioconversion when strains were grown in presence of 0.5 mM thebaine.

(78) TABLE-US-00008 TABLE 6 Bioconversion of thebaine to northebaine in strains expressing different possible transporter enzymes and improvement in the bioconversion as compared to control strain not expressing any heterologous transporter genes. Percentage Improvement bioconversion of in Thebaine to Thebaine to Northebaine Transporter genes Northebaine (%) bioconversion (%) Growth medium T1_CjaMDR1_GA 12.0 45 SC-his-leu-ura T3_NcaNPF_GA 6.7 19 SC-his-leu-ura T4_EsaGTR_GA 11.3 36 SC-his-leu-ura T5_AlyPOT_GA 6.1 27 SC-his-leu-ura T6_CruGTR_GA 6.4 23 SC-his-leu-ura T7_PtrPOT_GA 13.5 63 SC-his-leu-ura T8_BnaMFS_GA 4.2 49 SC-his-leu-ura T10_BolGTR_GA 6.0 28 SC-his-leu-ura T11_AthGTR1_GA 9.7 17 SC-his-leu-ura T12_PSoNPF1_GA 6.7 19 SC-his-leu-ura T14_PSoNPF3_GA 10.3 24 SC-his-leu-ura T17_PSoNPF6_GA 5.1 39 SC-his-leu-ura Control SC-his-leu-ura 8.3 0.0 SC-his-leu-ura T18_PsoNPF7_GA 10.0 2 DELFT minimal medium T19_RmiPTR2_GA 9.5 13 DELFT minimal medium T20_RmiPTR2_v2_GA 7.9 6 DELFT minimal medium T21_RalPTR2_GA 7.3 13 DELFT minimal medium T22_CanPOT_GA 4.4 48 DELFT minimal medium T23_ArePOT_GA 4.6 45 DELFT minimal medium T24_SlyPTR2_GA 4.0 52 DELFT minimal medium T25_AorPOT_GA 4.1 51 DELFT minimal medium T26_NfuPOT_GA 4.0 52 DELFT minimal medium T28_MciPOT_GA 4.2 50 DELFT minimal medium T29_AcaPOT_GA 5.1 39 DELFT minimal medium T30_MlyPOT_GA 5.6 33 DELFT minimal medium T31_TgaPOT_GA 4.4 48 DELFT minimal medium T32_AarPOT_GA 5.1 39 DELFT minimal medium T33_CcuPTR2_GA 4.6 45 DELFT minimal medium T34_HvePOT_GA 5.5 35 DELFT minimal medium T35_EcuPOT_GA 7.8 7 DELFT minimal medium T36_RnePOT_GA 4.3 49 DELFT minimal medium T37_OcoPOT_GA 4.8 45 DELFT minimal medium T38_ScuPTR2_GA 9.9 18 DELFT minimal medium T39_CgrPOT_GA 5.6 33 DELFT minimal medium T40_EdePOT_GA 6.1 27 DELFT minimal medium T41_CalPTR2_GA 5.7 32 DELFT minimal medium T44_CcaMFS_GA 4.4 48 DELFT minimal medium T45_PanPOT_GA 9.8 0 DELFT minimal medium T46_RchPOT_GA 8.1 4 DELFT minimal medium T47_PbeNPF_GA 4.6 45 DELFT minimal medium T48_CcaPOT_GA 9.7 1 DELFT minimal medium T49_HanPOT_GA 7.7 8 DELFT minimal medium T51_TorPOT_GA 5.5 35 DELFT minimal medium T52_BmePTR2_GA 11.7 19 DELFT minimal medium T53_EhePOT_GA 7.3 13 DELFT minimal medium T54_MelPOT_GA 10.9 11 DELFT minimal medium T55_NsyNPF_GA 3.2 62 DELFT minimal medium T56_CanNPF_GA 8.4 0 DELFT minimal medium T57_AcoNPF_GA 11.7 19 DELFT minimal medium T59_AmeNPF1_GA 5.3 37 DELFT minimal medium T60_AmeNPF2_GA 11.9 21 DELFT minimal medium T61_TwiNPF_GA 8.1 4 DELFT minimal medium T62_SmaNPF_GA 7.5 11 DELFT minimal medium T63_CfoNPF_GA 7.4 12 DELFT minimal medium T64_XsiNPF_GA 6.9 18 DELFT minimal medium T66_TelNPF_GA 8.3 1 DELFT minimal medium T69_PhoNPF_GA 5.4 36 DELFT minimal medium T70_CmaNPF_GA 9.1 8 DELFT minimal medium T72_TcoNPF_GA 8.4 0 DELFT minimal medium T73_PbrNPF1_GA 5.8 31 DELFT minimal medium T74_PbrNPF2_GA 6.6 21 DELFT minimal medium T75_PbrNPF3_GA 7.7 8 DELFT minimal medium T76_AhuNPF_GA 4.9 42 DELFT minimal medium T77_PocNPF_GA 5.5 35 DELFT minimal medium T78_VofNPF_GA 8.5 1 DELFT minimal medium T79_EcaNPF_GA 7.6 10 DELFT minimal medium T80_CroNPF_GA 9.8 0 DELFT minimal medium T82_NsaNPF_GA 8.8 10 DELFT minimal medium Control DELFT 8.4 0.0 DELFT minimal medium Control DELFT 9.8 0.0 DELFT minimal medium Numbers in Italic are relative to Control DELFT of 9.8.
Improvement of Bioconversion:

(79) Expression of one of the transporter genes T14_PsoNPF3_GA, T1_CjaMDR1_GA, T4_EsaGTR_GA or T7_PtrPOT_GA in a yeast strain that contains cytochrome P450 gene CYPDN8 and cytochrome P450 reductase Cel_CPR (co), results in improved bioconversion of thebaine to northebaine in the range of 24-63% in comparison to the control strain.

(80) Further, significant improvement was also seen for the transporter genes T60_AmeNPF2_GA, T57_AcoNPF_GA, T52_BmePTR2_GA, T38_ScuPTR2_GA, T11_AthGTR1_GA, T19_RmiPTR2_GA, T70_CmaNPF_GA or T54_MelPOT_GA.

Conclusions

(81) The results of this Example demonstrate expression of one of the transporter genes T14_PsoNPF3_GA, T1_CjaMDR1_GA, T4_EsaGTR_GA or T7_PtrPOT_GA in a yeast strain that contains cytochrome P450 gene CYPDN8 and cytochrome P450 reductase Cel_CPR (co), results in improved bioconversion of thebaine to northebaine in the range of 24-63% in comparison to the control strain.

(82) Further, significant improvement was also seen for the transporter genes T60_AmeNPF2_GA, T57_AcoNPF_GA, T52_BmePTR2_GA, T38_ScuPTR2_GA, T11_AthGTR1_GA, T19_RmiPTR2_GA, T70_CmaNPF_GA or T54_MelPOT_GA.

(83) Further, Transporters were Tested for Improvement in Conversion of the Thebaine Derivative Oripavine to Nororipavine

(84) Bioconversion. Expression of transporter gene T14_PsoNPF3_GA from Papaver somniferum in a strain containing cytochrome P450 gene CYPDN8 and cytochrome P450 reductase Cel_CPR (co) showed remarkable improvement in bioconversion of oripavine to nororipavine. In an assay where a strain was grown in presence of 0.5 mM oripavine, the strain containing T14_PsoNPF3_GA exhibited 2.3% bioconversion of the oripavine to nororipavine, which corresponds to an improvement in bioconversion of oripavine to nororipavine by 64% in comparison to the control strain.

(85) TABLE-US-00009 TABLE 7 Bioconversion and improvement in oripavine to nororipavine bioconversion compared to the control strain, observed when growing strains expressing different possible transporter proteins. Bioconversion Improvement of oripavine to of oripavine to nororipavine bioconversion as Transporter genes nororipavine (%) compared to control (%) T14_PsoNPF3_GA 2.3 64 Control 1.4 0

Conclusions

(86) The result of this Example demonstrate that expression of transporter gene T14_PsoNPF3_GA gave around 64% more bioconversion of oripavine to nororipavine-which is a remarkable yield improvement.

Example 5. Identification of Further Transporters Capable of Improving Bioconversion of Thebaine and/or Derivatives Thereof

(87) This Example 5 discusses transporter genes that are not explicitly mentioned in corresponding Example 4 above.

(88) Bioconversion. In bioconversion experiments similar to Example 4 above-3 additional transporters have shown to improve bioconversion of thebaine to northebaine.

(89) As shown in Table 8 below, T65_ljaNPF_GA, T94_EcrPOT_GA and T97_ScaT14_GA are able to improve bioconversion of thebaine to northebaine by 29.9%, 31.9% and 21.8%, respectively, when compared to a control strain.

(90) Table 8 also shows a yeast strain which genes CYPDN8 from Rhizopus microspores and Cel_CPR_co from Cunninghamella elegans have been integrated into host strain EVST25898 (Example 1) at Chromosome XII-5 with URA3 from Kluyveromyces lactis as selection marker. Subsequently, 6 different transporters T11_AthGTR1_GA, T52_BmePTR2_GA, T14_PsoNPF3_GA, T60_AmeNPF2_GA, T1_CjaMDR1_GA, and T70_CmaNPF_GA were expressed in the same strain at Chromosome XI-5 with LEU2 from Kluyveromyces lactis as selection marker. Plasmid pOD13 (Table 1) was also expressed in the same strain to make the strain prototrophic. An indication of improvement in the bioconversion of thebaine to northebaine when multiple copies of various transporters were expressed in the same strain.

(91) TABLE-US-00010 TABLE 8 Bioconversion of thebaine to northebaine in strains expressing different possible transporter enzymes and improvement in the bioconversion as compared to control strain not expressing any heterologous transporter genes. Percentage Improvement bioconversion in Thebaine to of Thebaine to Northebaine Transporter genes Northebaine (%) bioconversion (%) Growth medium T65_ljaNPF_GA 10.9 29.9 DELFT minimal medium T94_EcrPOT_GA 11.1 31.9 DELFT minimal medium T97_ScaT14_GA 10.2 21.8 DELFT minimal medium T11_AthGTR1_GA + 11.3 34.6 DELFT minimal T52_BmePTR2_GA + medium T14_PsoNPF3_GA + T60_AmeNPF2_GA + T1_CjaMDR1_GA + T70_CmaNPF_GA Control DELFT 8.4 DELFT minimal medium

(92) When multiple of different genes were expressed in the yeast cell, it is referred to as gene1+gene2, etc.

Conclusions

(93) In bioconversion experiments similar to Example 4 abovethe results of this Example demonstrate that three additional transporters have shown to improve bioconversion of thebaine to northebaine.

(94) As shown in Table 8, T65_ljaNPF_GA, T94_EcrPOT_GA and T97_ScaT14_GA are able to improve bioconversion of thebaine to northebaine by 29.9%, 31.9% and 21.8%, respectively, when compared to a control strain.

(95) Further, a strain comprising a combination of 6 transporter proteins discussed in Example 4 gave a very good improvement of thebaine to northebaine.

(96) Further, Transporters were Tested for Improvement in Conversion of the Thebaine Derivative Oripavine to Nororipavine

(97) Bioconversion. In bioconversion experiments similar to Example 4 abovean additional transporter that is able to help improving bioconversion of oripavine to nororipavine has been identified. As shown in Table 9 below, T97_ScaT14_GA from Sanguinaria canadensis is able to convert close to 5% of oripavine to nororipavine when fed with 0.5 mM oripavine. In comparison to the control strain, expression of T97_ScaT14_GA improves the bioconversion of oripavine to nororipavine by 254.4%.

(98) TABLE-US-00011 TABLE 9 Bioconversion and improvement in oripavine to nororipavine bioconversion compared to the control strain. Bioconversion Improvement of oripavine to of oripavine to nororipavine bioconversion Transporter genes nororipavine (%) as compared to control (%) T97_ScaT14_GA 4.96 254.4 Control 1.4 0

Conclusions

(99) In bioconversion experiments similar to Example 4 above, the results of this Example demonstrate an additional transporter able to help in improving bioconversion of oripavine to nororipavine has been identified.

(100) As shown in Table 9, T97_ScaT14_GA from Sanguinaria canadensis is able to convert close to 5% of oripavine to nororipavine when fed with 0.5 mM oripavine. In comparison to the control strain, expression of T97_ScaT14_GA improves the bioconversion of oripavine to nororipavine by 254.4%.

Example 6. Identification of Purine Uptake Permease (PUP) Transporters Capable of Improving Bioconversion of Thebaine

(101) Bioconversion. The impact of purine uptake permease transporter proteins on bioconversion of thebaine to northebaine was studied by transforming yeast strain with a plasmid containing a cytochrome P450 comparable to the above examples that was capable of acting on reticuline derivatives such as thebaine and/or oripavine using the backbone plasmid p415TEF. A plasmid containing cytochrome P450 reductase (pOD13 from Example 1) was also expressed in combination with various candidate transporter proteins. Yeast strain construction and method of screening for PUP transporters were as previously described in Example 1. Table 10 shows the result of percentage bioconversion from thebaine to northebaine with the expression of various PUP transporters. Table 10 also presents the percentage improvement in the bioconversion when normalized for a control strain expressing P450 but not expressing any heterologous transporter.

(102) TABLE-US-00012 TABLE 10 Percentage P450-mediated bioconversion from Thebaine to Northebaine with the expression of various transporters and percentage improvements in the bioconversion as compared to a control strains not expressing any heterologous transporters. Percentage Percentage Improvement bioconversion in Thebaine to of Thebaine to Northebalne PUP Transporters Northebaine (%) bioconversion (%) T101_McoPUP3_1 7.0 6.7 T102_PsoPUP3_1 8.6 29.8 T103_PsoPUP3_2 7.1 7.9 T104_PsoPUP3_3 7.4 11.4 T105_PsoPUP-L 9.2 39.8 Control 1 6.6 T109_GflPUP3_83 6.4 55.0 T122_PsoPUP3_17 6.1 48.4 T130_NdoPUP3_89 4.9 19.9 T131_PbrPUP3_81 4.9 20.4 T132_CmiPUP3_10 6.6 60.2 T133_PsoPUP3_18 5.9 42.7 T136_RchPUP3_42 4.6 11.1 T137_EguPUP3_50 5.1 24.7 T138_AduPUP3_58 4.6 11.7 T139_PsoPUP3_66 4.9 19.5 T140_PalPUP3_74 5.4 30.5 T141_EcaPUP3_88 6.8 64.7 T142_McoPUP3_4 7.7 88.9 T143_CmiPUP3_11 5.8 41.8 T144_PsoPUP3_19 7.7 87.1 T146_PsoPUP_35 4.6 13.4 T147_MesPUP3_43 6.1 49.8 T148_HimPUP3_51 5.0 21.4 T149_AcoPUP3_59 6.9 69.1 T150_PsoPUP3_67 5.9 43.6 T151_PatPUP3_75 5.7 39.1 T152_GflPUP3_87 8.0 94.0 T153_PsoPUP3_5 4.9 19.1 T154_CmiPUP3_12 7.1 74.2 T157_RchPUP_36 5.8 42.1 T159_OeuPUP3_52 5.8 41.8 T160_CeuPUP3_60 5.4 30.9 T161_PsoPUP3_68 6.2 51.9 T162_PmiPUP3_76 6.4 56.1 T163_PbrPUP3_86 5.1 24.8 T164_PsoPUP3_78 5.2 27.2 T165_AcoPUP3_13 6.5 57.9 T166_PsoPUP3_21 6.6 61.9 T168_FvePUP3_37 6.4 56.5 T169_ZjuPUP3_45 6.6 60.6 T170_LsaPUP3_53 6.7 62.6 T171_McoPUP3_61 5.5 33.8 T172_AcoPUP3_69 6.6 60.2 T174_PbrPUP3_85 5.3 29.4 T175_PsoPUP3_6 6.7 63.9 T176_AcoPUP3_14 5.8 41.5 T177_PsoPUP3_22 6.5 57.4 T178_PsoPUP3_30 6.1 47.6 T180_McoPUP3_46 5.5 35.0 T181_HanPUP3_54 5.3 30.1 T182_CpaPUP3_62 6.9 67.8 T184_PraPUP3_79 5.2 27.9 T186_ScaPUP3_84 7.0 69.8 T188_AcoPUP3_15 4.7 14.7 T189_PsoPUP3_23 4.7 14.8 T191_MdoPUP3_39 5.2 26.5 T192_CmiPUP3_47 5.5 35.0 T193_AanPUP3_55 6.2 51.9 T194_CchPUP3_63 5.7 39.1 T195_JcuPUP3_71 5.3 29.8 T196_PtrPUP3_80 5.7 39.1 Control 2 4.1 Note: Control 1 is used as the control for T101_McoPUP3_1, T102_PsoPUP3_1, T103_PsoPUP3_2, T104_PsoPUP3_3 and T105_PsoPUP-L. Control 2 is used as control for the rest of the PUP transporters. This was done to compensate for any slight variations that may arise between different runs of LC-MS analysis.

(103) Improvement of bioconversion. When compared to a control strain without a heterologous transporter, several strains engineered with PUP transporters exhibited at least 50% greater bioconversion of the 0.5 mM thebaine fed in this assay. Amongst the PUP transporters examined, PUP transporters T152_GflPUP3_87, T149_AcoPUP3_59, T109_GflPUP3_83, T142_McoPUP3_4, T144_PsoPUP3_19, T141_EcaPUP3_88, T182_CpaPUP3_62, T193_AanPUP3_55 and T122_PsoPUP3_17 exhibited improvements in bioconversion of thebaine to northebaine in the range of 48-94% in comparison to the control strain without a heterologous transporter (Table 10). Expression of some PUP transporters, such as T152_GflPUP3_87 from Glaucium flavum, T149_AcoPUP3_59 from Aquilegia coerulea, and T142_McoPUP3_4 from Macleaya cordata, gave remarkable improvements in the P450-mediated bioconversion of thebaine to northebaine.

(104) TABLE-US-00013 TABLE 11 Purine Uptake Permease transporters which have been demonstrated herein to provide especially large improvements in the P450- mediated bioconversion from Thebaine to Northebaine. Latin Name for Origin of PUP Transporters Sourced Genes T152_GflPUP3_87 Glaucium flavum T142_McoPUP3_4 Macleaya cordata T144_PsoPUP3_19 Papaver somniferum T149_AcoPUP3_59 Aquilegia coerulea T109_GflPUP3_83 Glaucium flavum T141_EcaPUP3_88 Eschscholzia californica T182_CpaPUP3_62 Carica papaya T193_AanPUP3_55 Artemisia annua T132_CmiPUP3_10 Cinnamomum micranthum f. kanehirae T186_ScaPUP3_84 Sanguinaria canadensis T175_PsoPUP3_6 Papaver somniferum T122_PsoPUP3_17 Papaver somniferum

Conclusions

(105) Table 11 shows some of the PUP transporters that have been herein demonstrated for the first time to shown very considerable improvements in the bioconversion from Thebaine to Northebaine by P450s. In particular, the results of this Example demonstrate that expression of PUP transporters T152_GflPUP3_87 from Glaucium flavum, T149_AcoPUP3_59 from Aquilegia coerulea, T109_GflPUP3_83 from Glaucium flavum, T142_McoPUP3_4 from Macleaya cordata, T144_PsoPUP3_19 from Papaver somniferum, T141_EcaPUP3_88 from Eschscholzia californica, T182_CpaPUP3_62 from Carica papaya, T193_AanPUP3_55 from Artemisia annua, T132_CmiPUP3_10 from Cinnamomum micranthum f. kanehirae, T186_ScaPUP3_84 from Sanguinaria canadensis, T175_PsoPUP3_6 from Papaver somniferum and T122_PsoPUP3_17 from Papaver somniferum, each stimulated somewhere in the range of 48-94% more bioconversion of thebaine to northebaine. The improvements in yield shown herein are both unexpected and highly valuable given the nature of the opioid-related compounds produced.

Example 7. Identification of Purine Uptake Permease (PUP) Transporters Capable of Improving Bioconversion of Oripavine to Nororipavine

(106) Bioconversion. The impact of purine uptake permease transporter proteins on bioconversion of oripavine to nororipavine was studied by transforming yeast with a plasmid containing a comparable cytochrome P450 that was capable of acting on reticuline derivatives such as thebaine and/or oripavine using the backbone plasmid p415TEF. A plasmid containing cytochrome P450 reductase (pOD13 from Example 1) was also expressed in combination with various possible transporter proteins. Yeast strain construction and method of screening for PUP transporters were as previously described in Example 1. Table 12 shows the result of percentage bioconversion from oripavine to nororipavine with the expression of various PUP transporters. Table 12 also presents the percentage improvement in the bioconversion when normalized for a control strain expressing P450 but not expressing any heterologous transporter.

(107) Improvement of bioconversion. The percentage bioconversion of strains displayed by several PUP transporters exhibited as high as 1600% and greater bioconversion of the 0.5 mM oripavine fed to the assay when compared to a control strain expressing P450 but not expressing transporter. Amongst the transporters examined in this example, PUP transporters T149_AcoPUP3_59, T168_FvePUP3_37, T116_HanPUP3_56, T192_CmiPUP3_47, T109_GflPUP3_83, T180_McoPUP3_46, T193_AanPUP3_55, T165_AcoPUP3_13, T195_JcuPUP3_71 and T143_CmiPUP3_11 exhibited improvements in the P450-mediated bioconversion of oripavine to nororipavine in the range of 1400-1662% in comparison to the control strain expressing P450 but not expressing a heterologous transporter (Table 12). Expression of some PUP transporters, such as T149_AcoPUP3_59 from Aquilegia coerulea, T168_FvePUP3_37 from Fragaria vesca subsp. vesca, and T116_HanPUP3_56 from Helianthus annuus gave particularly remarkable improvements in the P450-mediated bioconversion of oripavine to nororipavine.

(108) TABLE-US-00014 TABLE 12 Percentage of P450-mediated bioconversion from Oripavine to Nororipavine with the expression of various transporters and the percentage improvement in the bioconversion as compared to a control strains not expressing any heterologous transporters. Percentage Percentage Improvement Bioconversion in Oripavine to of Oripavine to Nororipavine PUP Transporters Nororipavine (%) bioconversion (%) T101_McoPUP3_1 3.5 147.7 T102_PsoPUP3_1 10.1 621.4 T103_PsoPUP3_2 1.7 21.9 T104_PsoPUP3_3 8.0 474.8 T105_PsoPUP-L 12.2 771.1 Control 1 1.4 T109_GflPUP3_83 15.5 1447.6 T113_PsoPUP3_32 10.1 912.6 T114_TorPUP3_40 5.9 486.0 T115_CsaPUP3_48 11.7 1065.5 T116_HanPUP3_56 17.5 1653.1 T117_MacPUP3_64 4.2 317.4 T121_NnuPUP3_9 1.5 47.1 T122_PsoPUP3_17 12.5 1149.3 T123_PsoPUP3_25 1.3 32.4 T124_PsoPUP3_33 4.9 393.9 T125_JcuPUP3_41 14.5 1346.2 T126_CpePUP3_49 11.8 1077.1 T127_LsaPUP3_57 5.4 441.7 T128_PsoPUP3_65 4.8 383.9 T129_PsoPUP3_73 6.3 532.3 T130_NdoPUP3_89 14.2 1315.0 T131_PbrPUP3_81 5.0 399.4 T132_CmiPUP3_10 14.8 1383.1 T133_PsoPUP3_18 14.5 1349.6 T135_PsoPUP_34 1.7 73.4 T136_RchPUP3_42 13.0 1197.8 T137_EguPUP3_50 8.4 744.8 T138_AduPUP3_58 14.5 1348.7 T139_PsoPUP3_66 4.4 341.0 T140_PalPUP3_74 3.6 264.7 T141_EcaPUP3_88 11.3 1030.8 T142_McoPUP3_4 15.4 1438.8 T143_CmiPUP3_11 15.8 1483.1 T144_PsoPUP3_19 15.1 1408.2 T146_PsoPUP_35 5.8 478.0 T147_MesPUP3_43 10.5 954.4 T148_HimPUP3_51 7.7 674.8 T149_AcoPUP3_59 17.4 1639.5 T150_PsoPUP3_67 13.4 1240.3 T151_PatPUP3_75 13.2 1223.8 T152_GflPUP3_87 14.9 1394.9 T153_PsoPUP3_5 6.8 583.2 T154_CmiPUP3_12 11.4 1039.5 T156_PsoPUP3_28 6.9 589.7 T157_RchPUP_36 12.2 1123.8 T158_DziPUP3_44 7.7 673.2 T159_OeuPUP3_52 10.0 902.7 T160_CeuPUP3_60 4.0 304.6 T161_PsoPUP3_68 13.4 1237.9 T162_PmiPUP3_76 14.1 1314.8 T163_PbrPUP3_86 3.8 280.2 T164_PsoPUP3_78 5.5 448.3 T165_AcoPUP3_13 15.3 1429.8 T166_PsoPUP3_21 10.3 931.0 T168_FvePUP3_37 17.6 1662.4 T169_ZjuPUP3_45 14.1 1310.9 T170_LsaPUP3_53 14.7 1372.2 T171_McoPUP3_61 3.5 251.3 T172_AcoPUP3_69 12.3 1126.4 T173_PnuPUP3_77 1.9 94.1 T174_PbrPUP3_85 5.5 452.5 T175_PsoPUP3_6 8.7 769.9 T176_AcoPUP3_14 7.4 636.2 T177_PsoPUP3_22 11.3 1029.5 T178_PsoPUP3_30 15.0 1396.5 T179_PyePUP3_38 4.4 344.5 T180_McoPUP3_46 16.8 1580.6 T181_HanPUP3_54 12.6 1160.4 T182_CpaPUP3_62 14.5 1349.5 T184_PraPUP3_79 3.3 234.2 T186_ScaPUP3_84 10.6 962.2 T188_AcoPUP3_15 3.0 197.8 T189_PsoPUP3_23 8.3 729.7 T191_MdoPUP3_39 9.5 849.2 T192_CmiPUP3_47 17.2 1618.5 T193_AanPUP3_55 15.5 1454.4 T194_CchPUP3_63 2.1 110.0 T195_JcuPUP3_71 15.1 1413.6 T196_PtrPUP3_80 10.9 986.6 Control 2 1.0 Note: Control 1 is used as the control for T101_McoPUP3_1, T102_PsoPUP3_1, T103_PsoPUP3_2, T104_PsoPUP3_3 and T105_PsoPUP-L. Control 2 is used as control for the rest of the PUP transporters. This is was done to account for any slight variations that may arise from different runs of LC-MS analysis.

(109) TABLE-US-00015 TABLE 13 Purine Uptake Permease transporters which have demonstrated herein to provide especially large improvements in the P450- mediated bioconversion of Oripavine to Nororipavine. Latin Name for Origin of Transporter Genes Sourced Genes T149_AcoPUP3_59 Aquilegia coerulea T168_FvePUP3_37 Fragaria vesca subsp. vesca T116_HanPUP3_56 Helianthus annuus T192_CmiPUP3_47 Cinnamomum micranthum f. kanehirae T109_GflPUP3_83 Glaucium Flavum T180_McoPUP3_46 Macleaya cordata T193_AanPUP3_55 Artemisia annua T165_AcoPUP3_13 Aquilegia coerulea T195_JcuPUP3_71 Jatropha curcas T143_CmiPUP3_11 Cinnamomum micranthum f. kanehirae

Conclusions

(110) Table 13 shows some of the PUP transporters that have been demonstrated herein for the first time to shown particularly high improvements in the P450-mediated bioconversion of oripavine to nororipavine. Amongst the transporters examined in this example, PUP transporters T149_AcoPUP3_59 from Aquilegia coerulea, T168_FvePUP3_37 from Fragaria vesca subsp. vesca, T116_HanPUP3_56 from Helianthus annuus, T192_CmiPUP3_47 from Cinnamomum micranthum f. kanehirae, T109_GflPUP3_83 from Glaucium flavum, T180_McoPUP3_46 from Macleaya cordata, T193_AanPUP3_55 from Artemisia annua, T165_AcoPUP3_13 from Aquilegia coerulea, T195_JcuPUP3_71 from Jatropha curcas and T143_CmiPUP3_11 from Cinnamomum micranthum f. kanehirae, exhibited improvements in the range of 1400-1662% more P450-mediated bioconversion of thebaine to northebaine in comparison to the control strain expressing P450 but not expressing a heterologous transporter. Such improvements in yield are particularly remarkable and represent a significant step forward towards a sustainable, secure, and scalable biosynthetic means of producing these compounds.

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