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WO 2016/149821 Al 29 September 2016 (29.09.2016) P O P C T (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2016/149821 Al 29 September 2016 (29.09.2016) P O P C T (51) International Patent Classification: AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, C12N 9/02 (2006.01) C12N 15/81 (2006.01) BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, C12N 1/19 (2006.01) C12N 9/04 (2006.01) DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, C12N 15/53 (2006.01) CI2P 17/10 (2006.01) HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, C12N 15/54 (2006.01) C12P 17/12 (2006.01) KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, (21) International Application Number: PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, PCT/CA2016/050334 SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, (22) International Filing Date: TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. 23 March 2016 (23.03.2016) (84) Designated States (unless otherwise indicated, for every (25) Filing Language: English kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, (26) Publication Language: English TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, (30) Priority Data: TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, 62/136,912 23 March 2015 (23.03.2015) US DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, (71) Applicant: VALORBEC SOCIETE EN COMMAN¬ SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, DITE [CA/CA]; 355 Peel, Carrefour INGO, Suite 503, GW, KM, ML, MR, NE, SN, TD, TG). Montreal, Quebec H3C 2G9 (CA). Declarations under Rule 4.17 : (72) Inventors: FOSSATI, Elena; 35 rue Marie Chapleau, — as to applicant's entitlement to apply for and be granted a Blainville, Quebec J7C 5Z9 (CA). NARCROSS, Lauren; patent (Rule 4.1 7(H)) 5090 Circle Road, Apt. 308, Montreal, Quebec H3W 2A1 (CA). MARTIN, Vincent; 5205 Beaconsfield, Montreal, Published: Quebec H3X 3R9 (CA). — with international search report (Art. 21(3)) (74) Agent: GOUDREAU GAGE DUBUC; 2000 McGill Col — with sequence listing part of description (Rule 5.2(a)) lege, Suite 2200, Montreal, Quebec H3A 3H3 (CA). (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, (54) Title: METHODS OF MAKING MORPHINAN ALKALOIDS AND ENZYMES THEREFORE (57) Abstract: A method of preparing a morphinan alkaloid (MA) metabolite comprising: (a) culturing a host cell under conditions suitable for MA production including a first fermentation at a pH of between about 7.5 and about 10, said host cell comprising: (i) a o first heterologous coding sequence encoding a first enzyme involved in a metabolite pathway that converts (R)-reticuline into the metabolite; (ii) a second heterologous coding sequence encoding a second enzyme involved in a metabolite pathway that converts (R)-reticuline into the metabolite; and/or (iii) a third heterologous coding sequence encoding a second enzyme involved in a meta bolite pathway that converts (R)-reticuline into the metabolite; (b) adding (R)-reticuline to the cell culture; and (c) recovering the metabolite from the cell culture. Plasmids and host cells encoding the enzymes are also provided. METHODS OF MAKING MORPHINAN ALKALOIDS AND ENZYMES THEREFORE CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application is PCT application Serial No PCT/CA2016/* filed on March 23, 2016 and published in English under PCT Article 2 1(2), which itself claims benefit of U.S. provisional application Serial No. 62/136,912, filed on March 23, 2015. All documents above are incorporated herein in their entirety by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0002] N.A. FIELD OF THE INVENTION [0003] The present invention relates to methods of making morphinan alkaloids and enzymes therefore. More specifically, the present invention is concerned with a recombinant method of making morphinan alkaloids in microbial cells. REFERENCE TO SEQUENCE LISTING [0004] Pursuant to 37 C.F.R. 1.821 (c), a sequence listing is submitted herewith as an ASCII compliant text file named, that was created on March 22, 2016 and having a size of 1080 kilobytes. The content of the aforementioned file named 13234-1 86_ST25 is hereby incorporated by reference in its entirety. BACKGROUND OF THE INVENTION [0005] Morphinan alkaloids are the most powerful narcotic analgesics currently used to treat moderate to severe and chronic pain. They include the opiates codeine and morphine and their semi¬ synthetic derivatives, such as dihydromorphine and hydromorphone as well as thebaine. Thebaine and morphine are the two main opiates extracted from opium poppy latex, meaning that they are the starting precursors for the synthesis of other opioids [3]. The opioids antagonist naloxone and naltrexone, used to treat opiate addiction and overdose, are derived from thebaine. Thebaine is a precursor to codeine and morphine biosynthesis in planta (FIG. 1) and is also the starting precursor for semi-synthetic opioids. For instance, it can be used for the chemical synthesis of the analgesics oxycodone and buprenorphine, which have more favourable side-effect profiles than morphine [1 ,2]. [0006] Morphinan alkaloids belong to a broader class of plant secondary metabolites known as benzylisoquinoline alkaloids (BIAs), with diverse pharmaceutical properties including the muscle relaxant papaverine, the antimicrobials berberine and sanguinarine and the antitussive and potential anticancer drug noscapine [8,9]. Thousands of distinct BIAs have been identified in plants, all derived from a single precursor: (S)-norcoclaurine. BIA synthesis in plants proceeds through the enantioselective Pictet-Spengler condensation of the L-tyrosine derivatives L-dopamine and 4-hydroxyphenylacetaldehyde to produce (S)- norcoclaurine, catalyzed by the enzyme norcoclaurine synthase (NCS; FIG. 2a) [ 10]. (S)-Norcoclaurine can be converted to the branch point intermediate (S)-reticuline via three methylation events (FIG. 2a). In P. somniferum the morphine pathway diverges from other BIA pathways in that it proceeds through (R)- reticuline instead of (S)-reticuline (FIG. 2a). The epimerization of (S)-reticuline to (R)-reticuline has been proposed to proceed via dehydrogenation of (S)-reticuline to 1,2-dehydroreticuline and subsequent enantioselective reduction to (R)-reticuline but the enzyme(s) responsible for this reaction have long remained elusive [ 1 1,12]. [0007] Cultivars of opium poppy improved for optimal opiate production by extensive breeding cycles and mutagenesis are the only commercial source of thebaine and morphine [2,3]. While the supply of morphinan alkaloids from plant extraction currently meets demand [3], efficient production of opiates using microbial platforms could not only contribute to reduce the cost of opiate production, but also offer a versatile platform for the creation of new scaffolds for drug discovery [7]. This refers to both alkaloids that do not accumulate in sufficient quantity and new molecules not yet isolated nor produced from plants. [0008] There remains a need for efficient production of opiates using microbial platforms. [0009] The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety. SUMMARY OF THE INVENTION [0010] Morphinan alkaloids are the most powerful narcotic analgesics currently used to treat moderate to severe and chronic pain. The feasibility of morphinan synthesis in recombinant Saccharomyces cerevisiae starting from the precursor (R,S)-norlaudanosoline and (R)-reticuline was investigated. Chiral analysis of the reticuline produced by the expression of opium poppy methyltransferases showed strict enantioselectivity for (S)-reticuline starting from (R,S)-norlaudanosoline and demonstrated that (R)-reticuline cannot be generated from (R)-norlaudanosoline. In addition, the P. somniferum enzymes salutaridine synthase (PsSAS), salutaridine reductase (PsSAR) and salutaridinol acetyltransferase (PsSAT) (FIG. 1) were functionally co-expressed in S. cerevisiae and optimization of the pH conditions allowed for productive spontaneous rearrangement of salutaridinol-7-O-acetate and synthesis of thebaine from (R)-reticuline. Finally, a 7-gene pathway was reconstituted for the production of codeine and morphine from supplemented precursors in S. cerevisiae. [001 1] Yeast cell feeding assays using (R)-reticuline, salutaridine or codeine as substrates showed that all enzymes were functionally co-expressed in yeast and that activity of salutaridine reductase (SAR) and codeine-O-demethylase (CODM) likely limit flux to morphine synthesis. Poor PsSAR and CODM expression or catalytic properties could all contribute to the low efficiency of this conversion and should be investigated for pathway optimization. Variation of gene expression (through copy number variation for example) [4] and use of orthologues and/or paralogs with better expression and/or catalytic properties are possible approaches to overcome this problem [36]. Also, solutions could be to generate synthetic microbial compartments [34], multi-enzyme scaffolds to channel intermediates to the pathway of interest [35], or alteration of an enzyme's specificity by protein engineering. Salutaridine reductase from Papaver bracteatum (PbSAR), which differs only in 13 amino acids from PsSAR, is known to be substrate inhibited at low concentration of salutaridine ( ,· = 150 µΜ) [29]. A previous mutagenesis study of PbSAR, based on homology modeling, resulted in identification of 2 mutants, F 104A and I275A, with reduced substrate inhibition and increased K , but slightly higher t. The double mutant F104A/I275A showed no substrate inhibition, with a higher K and cat. Therefore, an increased flux in the (R )-reticuline to the thebaine pathway could ostensibly be achieved by incorporating these mutations in PsSAR sequences.
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