Methods for making nor-opioid and nar-opioid benzylisoquinoline alkaloids

Abstract

Methods for demethylating opioids to nor-opioids are provided. [Solution] A method for demethylating an opioid to a nor-opioid, comprising the following steps: contacting a first opioid with at least one enzyme, where contacting the first opioid with the at least one enzyme converts the first opioid to a second opioid by loss of an O-linked methyl group; and contacting a second opioid with at least one enzyme, where contacting the opioid with the at least one enzyme converts the second opioid to a nor-opioid by loss of an N-linked methyl group.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 409,837, filed October 18, 2016, and U.S. Provisional Patent Application No. 62 / 473,215, filed March 17, 2017, the contents of which are incorporated herein by reference. [Background technology]

[0002] Background of the Invention Medical opioids are used to treat moderate to severe pain but can exhibit addictive properties. The mechanisms by which medical opioids relieve pain make these medications among the most effective painkillers in modern medicine. However, medical opioids are also widely abused. In addressing medical opioid use, policymakers are tasked with balancing the undertreatment of pain with mitigating the risks of opioid abuse. While pharmacotherapy has proven effective in treating and preventing opioid addiction, the high cost of such therapies limits the scope and reach of treatment programs. Summary of the Invention

[0003] The present disclosure provides a method for demethylating a first opioid to a second opioid. The present disclosure further provides a method for demethylating an opioid to a nor-opioid. The present disclosure further provides a method for modifying an opioid to a nar-opioid. The present disclosure also provides a genetically engineered cell for producing a nor-opioid from an opioid present in the genetically engineered cell. The present disclosure also provides a genetically engineered cell for producing a nar-opioid from a nor-opioid present in the genetically engineered cell.

[0004] One aspect of the present invention provides a method for demethylating a first opioid to a second opioid, comprising contacting the first opioid with at least one enzyme, wherein contacting the first opioid with the at least one enzyme converts the first opioid to the second opioid by loss of an O-linked methyl group, wherein the first opioid is not selected from the group consisting of codeine and thebaine.

[0005] Another aspect of the present invention provides a method for demethylating an opioid to a nor-opioid. The method includes contacting a first opioid with at least one enzyme, where contacting the first opioid with the at least one enzyme converts the first opioid to a second opioid by loss of an O-linked methyl group. The method also includes contacting the second opioid with the at least one enzyme, where contacting the opioid with the at least one enzyme converts the second opioid to a nor-opioid by loss of an N-linked methyl group.

[0006] In a further aspect of the present invention, there is provided another method for demethylating an opioid to a nor-opioid, the method comprising contacting the opioid with at least one enzyme, wherein contacting the opioid with the at least one enzyme converts the opioid to a nor-opioid by removal of an N-linked methyl group from the opioid, wherein the opioid is not thebaine when contacted with the at least one enzyme in vitro.

[0007] In a further aspect of the present invention, there is provided a method for converting an opioid into a nar-opioid. The method includes contacting the opioid with at least a first enzyme, where contacting the opioid with the at least a first enzyme converts the opioid into a nor-opioid by removing an N-linked methyl group from the opioid. The method also includes contacting the nor-opioid with at least a second enzyme in the presence of a cofactor, where contacting the nor-opioid with the at least a second enzyme converts the nor-opioid into a nar-opioid by transferring a side chain from the cofactor.

[0008] In another aspect of the present invention, there is provided a method for converting an opioid to a nar-opioid. The method includes contacting a first opioid with at least one enzyme, where contacting the first opioid with the at least one enzyme converts the first opioid to a second opioid by loss of an O-linked methyl group. The method also includes contacting the second opioid with at least a second enzyme, where contacting the opioid with the at least second enzyme converts the second opioid to a nor-opioid by loss of an N-linked methyl group. The method further includes contacting the nor-opioid with at least a third enzyme in the presence of a cofactor, where contacting the nor-opioid with the at least third enzyme converts the nor-opioid to a nar-opioid by transfer of a side chain from the cofactor.

[0009] In a further aspect of the present invention, there is provided a genetically engineered cell that produces a nor-opioid from an opioid present in the genetically engineered cell, the genetically engineered cell comprising a heterologous coding sequence encoding an N-demethylase produced by the genetically engineered cell, wherein the N-demethylase converts the opioid in the genetically engineered cell to the nor-opioid, and the nor-opioid is produced in the genetically engineered cell.

[0010] In a further aspect of the present invention, there is provided a genetically engineered cell that produces a nar-opioid from a nor-opioid present in the genetically engineered cell, the genetically engineered cell comprising a heterologous coding sequence encoding an N-methyltransferase produced by the genetically engineered cell, wherein the N-methyltransferase converts the nor-opioid to the nar-opioid within the genetically engineered cell.

[0011] INCORPORATION BY REFERENCE All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [The present invention 1001] 1. A method for demethylating a first opioid to a second opioid, comprising the steps of: contacting the first opioid with at least one enzyme, wherein contacting the first opioid with the at least one enzyme converts the first opioid to the second opioid by loss of an O-linked methyl group. Including, wherein the first opioid is not selected from the group consisting of codeine and thebaine. The method. [The present invention 1002] 1. A method for demethylating an opioid to a nor-opioid, comprising the steps of: contacting the first opioid with at least one enzyme, wherein contacting the first opioid with the at least one enzyme converts the first opioid to the second opioid by loss of an O-linked methyl group; and contacting the second opioid with at least one enzyme, wherein contacting the opioid with the at least one enzyme converts the second opioid to a nor-opioid by loss of an N-linked methyl group. The method comprising: [The present invention 1003] 1. A method for demethylating an opioid to a nor-opioid, comprising the steps of: contacting the opioid with at least one enzyme, wherein contacting the opioid with the at least one enzyme converts the opioid to a nor-opioid by removal of an N-linked methyl group from the opioid. Including, wherein when the opioid is contacted with the at least one enzyme in vitro, the opioid is not thebaine. The method. [The present invention 1004] 1. A method for modifying an opioid to a nal-opioid, comprising the steps of: contacting the opioid with at least a first enzyme, wherein contacting the opioid with the at least a first enzyme converts the opioid to a nor-opioid by removal of an N-linked methyl group from the opioid; and contacting the nor-opioid with at least a second enzyme, wherein contacting the nor-opioid with the at least a second enzyme in the presence of a cofactor converts the nor-opioid to a nal-opioid by transfer of a side chain from the cofactor. The method comprising: [The present invention 1005] 1. A method for modifying an opioid to a nal-opioid, comprising the steps of: contacting a first opioid with at least one enzyme, wherein contacting the first opioid with the at least one enzyme converts the first opioid to a second opioid by loss of an O-linked methyl group; contacting a second opioid with at least a second enzyme, wherein contacting the opioid with the at least a second enzyme converts the second opioid to a nor-opioid by loss of an N-linked methyl group; and contacting the nor-opioid with at least a third enzyme, wherein contacting the nor-opioid with the at least a third enzyme in the presence of a cofactor converts the nor-opioid to a nal-opioid by transfer of a side chain from the cofactor. The method comprising: [The present invention 1006] The method of claim 1001, wherein the second opioid is produced by culturing genetically engineered cells containing a coding sequence for encoding at least one enzyme. [The present invention 1007] The method of claim 1002, wherein the nor-opioid is produced by culturing genetically engineered cells comprising a coding sequence for encoding the first enzyme. [The present invention 1008] The method of claim 1003, wherein the nor-opioid is produced by culturing genetically engineered cells comprising coding sequences for encoding the first enzyme and the second enzyme. [The present invention 1009] The method of claim 1004, wherein the nal-opioid is produced by culturing genetically engineered cells comprising coding sequences for encoding the first enzyme and the second enzyme. [The present invention 1010] The method of claim 1005, wherein the nor-opioid is produced by culturing genetically engineered cells comprising coding sequences for encoding the first enzyme, the second enzyme, and the third enzyme. [The present invention 1011] Recovering the second opioid from the cell culture The method of the present invention 1006 further comprising: [The present invention 1012] Recovering nor-opioid from the cell culture The method of any of claims 1007 or 1008, further comprising: [The present invention 1013] Recovering the nar-opioid from the cell culture The method of any one of claims 1009 to 1010, further comprising: [The present invention 1014] adding (S)-1-benzylisoquinoline alkaloid to the cell culture Any of the methods of 1006 to 1013 of the present invention, further comprising: [The present invention 1015] Any of the aforementioned methods of the present invention, wherein nor-opioid is produced in the genetically engineered cells by a metabolic pathway beginning with L-tyrosine. [The present invention 1016] Any of the aforementioned methods of the present invention, wherein the nal-opioid is produced in the genetically engineered cells by a metabolic pathway beginning with L-tyrosine. [The present invention 1017] A genetically engineered cell that produces a nor-opioid from an opioid present in the genetically engineered cell, the genetically engineered cell comprising a heterologous coding sequence encoding an N-demethylase produced by the genetically engineered cell, the N-demethylase converting the opioid in the genetically engineered cell to the nor-opioid, the nor-opioid being produced within the genetically engineered cell. [The present invention 1018] 1017. The genetically engineered cell of the present invention further comprising a heterologous coding sequence encoding an N-methyltransferase. [The present invention 1019] 1017. The genetically engineered cell of claim 1017, wherein opioids are produced in the genetically engineered cell by a metabolic pathway beginning with L-tyrosine. [The present invention 1020] A genetically engineered cell that produces a nar-opioid from a nor-opioid present in the genetically engineered cell, the genetically engineered cell comprising a heterologous coding sequence encoding an N-methyltransferase produced by the genetically engineered cell, the N-methyltransferase converting the nor-opioid to the nar-opioid within the genetically engineered cell. [The present invention 1021] The genetically engineered cell of the present invention 1020, wherein a nal-opioid is produced in the genetically engineered cell. [The present invention 1022] 1020. The genetically engineered cell of claim 10, wherein a nal-opioid is produced in the genetically engineered cell by a metabolic pathway beginning with L-tyrosine. [The present invention 1023] Any of the aforementioned methods of the present invention, wherein the genetically engineered cell is a genetically engineered non-plant cell. [The present invention 1024] Any of the aforementioned methods of the present invention, wherein the genetically engineered non-plant cell is a genetically engineered yeast cell. [The present invention 1025] The method of any one of claims 1001 to 1002, wherein the loss of the O-linked methyl group occurs at the 3' position. [The present invention 1026] Nar-opioids produced by genetically engineered cells. [The present invention 1027] A nal-opioid compound that is free from one or more detectable impurities associated with chemical synthesis. [The present invention 1028] The nar-opioid compound of the present invention, wherein the nar-opioid is buprenorphine and one or more impurities incidental to chemical synthesis include 15,16-dehydrobuprenorphine, 17,18-dehydrobuprenorphine, 18,19-demethylbuprenorphine, 19,19'-ethylbuprenorphine, 2,2'-bisbuprenorphine, 3-deshydroxybuprenorphine, 3-O-methylbuprenorphine, 3-O-methyl-N-cyanonorbuprenorphine, 3-O-methyl-N-methylnorbuprenorphine, 6-O-desmethylbuprenorphine, buprenorphine N-oxide, N-but-3-enylnorbuprenorphine, N-but-3-enylnormethylbuprenorphine, N-butylnorbuprenorphine, N-methylbuprenorphine, norbuprenorphine, and tetramethylfuranbuprenorphine. [The present invention 1029] A nar-opioid compound of the present invention 1027, wherein the nar-opioid is oxymorphone and one or more impurities incidental to chemical synthesis include 1-bromoxymorphone, 6-β-oxymorphol, 10-α-hydroxyoxymorphone, 10-ketoxymorphone, 2,2-bisoxymorphone, noroxymorphone, oxymorphone N-oxide, 10-hydroxyoxymorphone, 4-hydroxyoxymorphone, 8-hydroxyoxymorphone, and hydromorphinol. [The present invention 1030] A nal-opioid compound of the present invention 1027, wherein the nal-opioid is naltrexone and one or more impurities incidental to chemical synthesis include 10-hydroxynaltrexone, 10-ketonaltrexone, 14-hydroxy-17-cyclopropylmethylnormorphinone, 2,2'-bisnaltrexone, 3-cyclopropylmethylnaltrexone, 3-O-methylnaltrexone, 8-hydroxynaltrexone, N-(3-butenyl)-noroxymorphone, naltrexone aldol dimer, and N-formyl-noroxymorphone. [The present invention 1031] A nar-opioid compound of the present invention, wherein the nar-opioid is naloxone and one or more impurities incidental to chemical synthesis include 10-α-hydroxynaloxone, 10-β-hydroxynaloxone, 10-ketonaloxone, 3-O-allylnaloxone, 7,8-didehydronaloxone, 2,2'-bisnaloxone, and naloxone N-oxide. [The present invention 1032] A nar-opioid compound of the present invention, wherein the nar-opioid is nalbuphine and one or more impurities incidental to chemical synthesis include the β-epimer of nalbuphine, 2,2'-bisnalbuphine, 6-ketonalbuphine, 10-ketonalbuphine, α-noroxymorphol, N-(cyclobutylcarbonyl)-α-noroxymorphol, and N-formyl-6-α-noroximorphol. [The present invention 1033] Compared to synthetically produced opioids, buprenorphine contains one or more of the following impurities: 15,16-dehydrobuprenorphine, 17,18-dehydrobuprenorphine, 18,19-demethylbuprenorphine, 19,19'-ethylbuprenorphine, 2,2'-bisbuprenorphine, 3-dehydroxybuprenorphine, 3-O-methylbuprenorphine, 3-O-methyl-N-cyanonorbuprenorphine, 3-O-methyl-N-methylnorbuprenorphine, 6-O-desmethylbuprenorphine, Buprenorphine, Buprenorphine N-oxide, N-but-3-enylnorbuprenorphine, N-but-3-enylnormethylbuprenorphine, N-butylnorbuprenorphine, N-methylbuprenorphine, Norbuprenorphine, Tetramethylfuranbuprenorphine, 1-bromoxymorphone, 6-β-oxymorphol, 10-α-hydroxyoxymorphone, 10-ketoxymorphone, 2,2-bisoxymorphone, noroxymorphone, oxymorphone N-oxide, 10-hydroxyoxy Symorphone, 4-hydroxyoxymorphone, 8-hydroxyoxymorphone, hydromorphinol, 10-hydroxynaltrexone, 10-ketonaltrexone, 14-hydroxy-17-cyclopropylmethylnormorphinone, 2,2'-bisnaltrexone, 3-cyclopropylmethylnaltrexone, 3-O-methylnaltrexone, 8-hydroxynaltrexone, N-(3-butenyl)-noroxymorphone, naltrexone aldol dimer, N-formyl-noroxymorphone, 10-α -hydroxynaloxone, 10-β-hydroxynaloxone, 10-ketonaloxone, 3-O-allylnaloxone, 7,8-didehydronaloxone, 2,2'-bisnaloxone, naloxone N-oxide, the β-epimer of nalbuphine, 2,2'-bisnalbuphine, 6-ketonalbuphine, 10-ketonalbuphine, α-noroxymorphol, N-(cyclobutylcarbonyl)-α-noroxymorphol, or N-formyl-6-α-noroxymorphol, or less. [The present invention 1034] The nar-opioid compound of the present invention 1033, wherein the nar-opioid is a buprenorphine compound and contains less of one or more of the following impurities compared to chemically synthesized buprenorphine: 15,16-dehydrobuprenorphine, 17,18-dehydrobuprenorphine, 18,19-demethylbuprenorphine, 19,19'-ethylbuprenorphine, 2,2'-bisbuprenorphine, 3-deshydroxybuprenorphine, 3-O-methylbuprenorphine, 3-O-methyl-N-cyanonorbuprenorphine, 3-O-methyl-N-methylnorbuprenorphine, 6-O-desmethylbuprenorphine, buprenorphine N-oxide, N-but-3-enylnorbuprenorphine, N-but-3-enylnormethylbuprenorphine, N-butylnorbuprenorphine, N-methylbuprenorphine, norbuprenorphine, or tetramethylfuranbuprenorphine. [This invention 1035] A nar-opioid compound of the present invention 1033, wherein the nar-opioid is oxymorphone and contains less of one or more of the following impurities: 1-bromoxymorphone, 6-β-oxymorphol, 10-α-hydroxyoxymorphone, 10-ketoxymorphone, 2,2-bisoxymorphone, noroxymorphone, oxymorphone N-oxide, 10-hydroxyoxymorphone, 4-hydroxyoxymorphone, 8-hydroxyoxymorphone, or hydromorphinol, compared to chemically synthesized oxymorphone. [The present invention 1036] A nal-opioid compound of the present invention 1033, wherein the nal-opioid is naltrexone and contains less of one or more of the following impurities compared to chemically synthesized naltrexone: 10-hydroxynaltrexone, 10-ketonaltrexone, 14-hydroxy-17-cyclopropylmethylnormorphinone, 2,2'-bisnaltrexone, 3-cyclopropylmethylnaltrexone, 3-O-methylnaltrexone, 8-hydroxynaltrexone, N-(3-butenyl)-noroxymorphone, naltrexone aldol dimer, or N-formyl-noroxymorphone. [This invention 1037] A nar-opioid compound of the present invention 1033, wherein the nar-opioid is naloxone and contains less of one or more of the following impurities: 10-α-hydroxynaloxone, 10-β-hydroxynaloxone, 10-ketonaloxone, 3-O-allylnaloxone, 7,8-didehydronaloxone, 2,2'-bisnaloxone, or naloxone N-oxide compared to chemically synthesized naloxone. [The present invention 1038] A nar-opioid compound of the present invention 1033, wherein the nar-opioid is nalbuphine and contains less of one or more of the following impurities: the beta-epimer of nalbuphine, 2,2'-bisnalbuphine, 6-ketonalbuphine, 10-ketonalbuphine, α-noroxymorphol, N-(cyclobutylcarbonyl)-α-noroxymorphol, or N-formyl-6-α-noroximorphol compared to chemically synthesized nalbuphine. [This invention 1039] Genetically engineered cells that produce nal-opioids. [The present invention 1040] Genetically engineered cells that produce nor-opioids. [The present invention 1041] 1041. The method of any one of claims 1026 to 1040, wherein said genetically engineered cell comprises a heterologous coding sequence, said heterologous coding sequence encoding an enzyme selected from the group consisting of N-demethylases. [The present invention 1042] The method of claim 1026, wherein the genetically engineered cell comprises at least first and second heterologous coding sequences, wherein the first heterologous coding sequence encodes an enzyme selected from the group consisting of N-demethylases and the second heterologous coding sequence encodes an enzyme selected from the group consisting of N-methyltransferases. [This invention 1043] 1043. The method of claim 1042, wherein said genetically engineered cell further comprises a third heterologous coding sequence, said third heterologous coding sequence encoding an enzyme selected from the group consisting of O-demethylases. [This invention 1044] 104. The method of claim 1042 or 1043, wherein said genetically engineered cell further comprises at least one enzyme selected from the group consisting of L-DOPA decarboxylase, tyrosine hydroxylase, norcoclaurine synthase, norcoclaurine 6-O-methyltransferase, coclaurine-N-methyltransferase, cytochrome P450 80B1, cytochrome P450 reductase, 4'-O-methyltransferase, 1-benzylisoquinoline alkaloid epimerase, salutaridine synthase, salutaridine reductase, salutaridinol 7-O-acetyltransferase, thebaine 6-O-demethylase, codeinone reductase, codeine O-demethylase, N-methyltransferase, O-demethylase, and N-demethylase. [This invention 1045] The method of any one of claims 1042, 1043 or 1044, wherein said nal-opioid produced by the culture of genetically engineered cells is detectable by LC-MS (liquid chromatography-mass spectrometry). [The present invention 1046] 1. A method for converting a nor-opioid to a nar-opioid, comprising the steps of: contacting the nor-opioid with at least one enzyme, wherein contacting the nor-opioid with the enzyme in the presence of a cofactor converts the nor-opioid to a nal-opioid by transfer of a side chain from the cofactor. The method comprising: [This invention 1047] A method for preparing a nar-opioid from a substrate comprising genetically engineered cells capable of producing the nar-opioid from the substrate. [This invention 1048] 1047. The method of claim 1047, wherein the substrate is a sugar. [This invention 1049] 1047. The method of claim 1047, wherein the substrate is tyrosine. [The present invention 1050] 1047. The method of claim 1047, wherein the substrate is thebaine. [This invention 1051] The method of claim 1050, wherein said thebaine is produced in a genetically engineered cell. [This invention 1052] 1047. The method of claim 1047, wherein said substrate is reticuline. [This invention 1053] 1053. The method of claim 1052, wherein said reticuline is produced in genetically engineered cells. [This invention 1054] 1047. The method of claim 1047, wherein the substrate is an opioid. [This invention 1055] The method of claim 1054, wherein said opioid is produced in a genetically engineered cell. [This invention 1056] 1047. The method of claim 1047, wherein said substrate is a nor-opioid. [This invention 1057] The method of claim 1056, wherein said nor-opioid is produced in a genetically engineered cell. [This invention 1058] The method of claim 1047, further comprising culturing the genetically engineered cells under conditions suitable for protein production, wherein the genetically engineered cells comprise two heterologous coding sequences, the two heterologous coding sequences encoding first and second enzymes, respectively, involved in a metabolic pathway that converts a substrate to a nal-opioid, the first and second enzymes being operably linked along the metabolic pathway. [This invention 1059] The method of claim 1058, wherein each of said first and second enzymes involved in a metabolic pathway producing an N-opioid is selected from the group consisting of an N-demethylase and an N-methyltransferase. [The present invention 1060] The method of claim 1047, further comprising recovering the nal-opioid from the cell culture. [This invention 1061] 1047. The method of claim 1047, wherein the nal-opioid is selected from the group consisting of naltrexone, naloxone, nalmefene, nalorphine, nalorphine, nalodain, naldemedine, naloxegol, 6β-naltrexol, naltolindole, methylnaltrexone, methylsamidophan, alvimopan, axerophthol, bebenprane, dinicotinate, levallorphan, samidophan, buprenorphine, dezocine, eptazocine, butorphanol, levorphanol, nalbuphine, pentazocine, phenazocine, norbinaltorphimine, and diprenorphine. [This invention 1062] A method for preparing a nor-opioid from a substrate comprising genetically engineered cells capable of producing said nor-opioid from said substrate. [The present invention 1063] 1. A method for forming a product stream having a nal-opioid product, comprising the steps of: i. feeding genetically engineered cells and a feedstock comprising nutrients and water into a batch reactor, wherein the genetically engineered cells have at least two heterologous coding sequences comprising an N-demethylase enzyme and an N-methyltransferase enzyme; ii. subjecting the genetically engineered cells to fermentation by incubating the genetically engineered cells in the batch reactor for a period of at least about 5 minutes to produce a solution comprising a nal-opioid and cellular material; and iii. separating the nar-opioid from the cellular material using at least one separation unit to obtain the product stream comprising the nar-opioid. The method comprising: [The present invention 1064] The method of claim 1063, wherein said genetically engineered cell further comprises a third heterologous coding sequence comprising an O-demethylase enzyme. [This invention 1065] 1. A method for forming a product stream having a nor-opioid product, comprising the steps of: i. feeding genetically engineered cells and a feedstock comprising nutrients and water into a batch reactor, wherein the genetically engineered cells comprise at least one heterologous coding sequence comprising an N-demethylase enzyme; ii. subjecting the genetically engineered cells to fermentation by incubating the genetically engineered cells in the batch reactor for a period of at least about 5 minutes to produce a solution comprising a nor-opioid and cellular material; and iii. separating the nor-opioid from the cellular material using at least one separation unit to obtain the product stream comprising the nor-opioid. The method comprising: [The present invention 1066] A method for producing a nar-opioid, comprising the steps of: providing genetically engineered host cells; subjecting the genetically engineered cells to fermentation by incubating the genetically engineered cells for a period of at least about 5 minutes to produce a fermentation reaction mixture; and purifying the nar-opioid from the fermentation reaction mixture. [This invention 1067] A method for producing a nor-opioid, comprising the steps of: providing genetically engineered host cells; subjecting the genetically engineered cells to fermentation by incubating the genetically engineered cells for a period of at least about 5 minutes to produce a fermentation reaction mixture; and purifying the nor-opioid from the fermentation reaction mixture. [Brief explanation of the drawings]

[0012] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0013] [Figure 1] FIG. 1 shows examples of synthesis, recycling, and salvage pathways for tetrahydrobiopterin, according to embodiments of the present invention. [Figure 2] FIG. 2 shows a biosynthetic scheme for the conversion of glucose to 4-HPA, dopamine, and 3,4-DHPA, according to an embodiment of the present invention. [Figure 3] FIG. 3 shows a schematic diagram of an example of (R)-1-benzylisoquinoline alkaloid formation according to an embodiment of the present invention. [Figure 4] FIG. 4 shows the amino acid sequence of a CYP-COR enzyme according to an embodiment of the present invention. [Figure 5] FIG. 5 shows a biosynthetic scheme for the conversion of L-tyrosine to reticuline via norcoclaurine, according to an embodiment of the present invention. [Figure 6] FIG. 6 shows a biosynthetic scheme for the conversion of L-tyrosine to reticuline via norlaudanosoline, according to an embodiment of the present invention. [Figure 7] FIG. 7 shows a biosynthetic scheme for the conversion of L-tyrosine to morphinan alkaloids according to an embodiment of the present invention. [Figure 8] FIG. 8 shows a biosynthetic scheme for the production of semisynthetic opioids according to an embodiment of the present invention. [Figure 9] FIG. 9 shows tyrosine hydroxylase variants that improve reticuline production from sugars in genetically engineered yeast strains, according to an embodiment of the present invention. [Figure 10] FIG. 10 shows co-expression of dihydrofolate reductase (DHFR) to improve L-DOPA production by tyrosine hydroxylase in genetically engineered yeast strains, according to an embodiment of the present invention. [Figure 11] Figure 11A shows the addition of antioxidants to the culture medium to improve L-DOPA production by tyrosine hydroxylase in a genetically engineered yeast strain, and Figure 11B shows the addition of antioxidants to the culture medium to increase BH4 levels, according to an embodiment of the invention. [Figure 12] Figure 12A shows a biosynthetic scheme for the conversion of L-tyrosine to bisBIA, and Figure 12B shows a yeast strain engineered to biosynthesize bisBIA, according to an embodiment of the invention. [Figure 13]FIG. 13 shows a phylogenetic tree of cytochrome P450 oxidase-codeinone reductase-like (CYP-COR) fusions according to an embodiment of the present invention. [Figure 14] Figure 14 shows LC / MS-MS analysis of a yeast strain genetically engineered to convert (S)-reticuline to salutaridine according to an embodiment of the present invention. Figure 14A shows a chromatogram showing reticuline and salutaridine with two epimerase mutants (CYP-COR_89405, CYP-COR_4328) and a standard. Figure 14B shows the same chromatogram of salutaridine in (A) when replotted to show coelution with the standard. [Figure 15] FIG. 15 shows chiral LC / MS-MS analysis of a yeast strain engineered to convert racemic norlaudanosoline to (R)-reticuline, according to an embodiment of the present invention. [Figure 16] Figure 16A shows the N-linked glycosylation status of heterologously expressed salutarideine synthase, and Figure 16B shows an engineered fusion of salutarideine synthase that eliminates the N-linked glycosylation of the protein observed when heterologously expressed in yeast but not in plants, according to an embodiment of the invention. [Figure 17] 17A and 17B show the design of a cheilanthifoline synthase-sultaridine synthase fusion according to an embodiment of the present invention. [Figure 18] FIG. 18 shows codon optimization of salutaridine synthase and engineered fusions to improve activity in yeast, according to an embodiment of the present invention. [Figure 19] 19A and 19B show LC / MS-MS analysis of small-scale batch fermentations in which genetically engineered yeast catalyze the conversion of (R)-reticuline to thebaine and rac-norlaudanosoline to thebaine, according to an embodiment of the invention. [Figure 20] FIG. 20 shows the generation of CODM enzyme variants that exhibit improved activity in yeast by random mutagenesis and screening, according to an embodiment of the invention. [Figure 21] 21A, 21B, and 21C show fermentation optimization for conversion of (R)-reticuline to thebaine by genetically engineered yeast, according to an embodiment of the present invention. [Figure 22] FIG. 22 shows a platform yeast strain for the production of the key branch point intermediate reticuline from L-tyrosine, according to an embodiment of the present invention. [Figure 23] FIG. 23 shows an enzyme having opioid 3-O-demethylase activity, according to an embodiment of the invention. [Figure 24] FIG. 24 shows an enzyme having opioid N-demethylase activity, according to an embodiment of the present invention. [Figure 25] FIG. 25 shows an enzyme with N-methyltransferase activity according to an embodiment of the invention. [Figure 26] FIG. 26 shows a biosynthetic scheme within a microbial cell according to an embodiment of the invention. [Figure 27] FIG. 27 shows functional expression of BM3 mutants according to an embodiment of the invention. [Figure 28] FIG. 28 shows a plasmid / YAC vector for enzyme expression and genetic manipulation according to an embodiment of the present invention. [Figure 29] FIG. 29 shows functional expression of a CODM according to an embodiment of the invention. [Figure 30] FIG. 30 shows a biosynthetic scheme for the production of semisynthetic opioids according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0014] Detailed Description of the Invention Nal-opioids are an important class of pharmacotherapeutic drugs for addressing opioid addiction and opioid-related side effects. Opiates, such as codeine and morphine, are plant molecules derived from the poppy plant that possess a unique pentacyclic structure that allows them to bind to μ-opioid receptors present in the brain, spinal cord, gut, and peripheral sensory organs, mimicking the body's natural pain attenuation. These same opiate molecules can be modified to act as antagonists (e.g., naltrexone and naloxone). In some cases, opioid molecules can be modified by introducing chemical modifications that allow them to bind to opioid receptors but inhibit activation of downstream signaling responses. Other molecules, such as buprenorphine, can bind to opioid receptors and act as mixed partial agonists.

[0015] The set of antagonist and mixed partial agonist opioids, collectively referred to as nal-opioids, may constitute a toolkit of competitive modulators that can occupy receptor binding sites in patients receiving potent opioid agonists. As an example, in addicted patient populations, nal-opioids may be used for: (1) overdose treatment by administering a potent antagonist, e.g., naloxone; (2) detoxification by managing symptoms with a mixed partial agonist, e.g., buprenorphine; and / or (3) maintenance by blocking reward responses, e.g., with a buprenorphine / naloxone combination. For patient populations with severe pain, nal-opioids can be used to reduce side effects by administering: (4) abuse-deterrent agonist / antagonist combination preparations, such as morphine / naltrexone combinations, which can block the euphoria and high that can occur when the drug is misused; and / or (5) peripherally acting antagonists, such as polymer conjugates of naloxone (Movantik™), which can displace opioid agonists from receptors in the intestine, which can cause constipation.

[0016] The starting material for the synthesis of nal-opioids is a natural opiate, such as thebaine, extracted from the opium poppy drug crop. Traditionally, these molecules are then chemically modified into semisynthetic antagonists, weak agonists, and mixed partial agonists through a series of inefficient reaction steps that require the use of catalysts, solvents, reagents, and purification methods to isolate the nal-opioid product from the starting materials and reaction intermediates. Currently used semisynthetic preparation methods are inefficient and add significant costs to the overall process.

[0017] The present disclosure provides methods for the production of diverse nar-opioids in genetically engineered host cells. The present disclosure also provides methods for the production of diverse nor-opioids in genetically engineered host cells. Furthermore, the present disclosure provides methods for the production of O-demethylases and N-demethylases in genetically engineered host cells. In certain cases, the present disclosure provides methods for producing nor-opioid products by demethylating opioids to nor-opioids in genetically engineered host cells. In further particular cases, the present disclosure provides methods for producing diverse nar-opioids by modifying nor-opioids with enzymes capable of adding N-linked side chains, such as N-methyltransferases.

[0018] The present disclosure provides methods for the production of nar-opioid and nor-opioid compounds in genetically engineered host cells. Throughout this disclosure, the term "compound" can be used to refer to something that contains two or more elements, such as a nar-opioid molecule or a nar-opioid composition. A nar-opioid compound can refer to a substantially pure composition of nar-opioid, or a composition of nar-opioid that may or may not contain impurities.

[0019] Nar-opioids of interest Host cells are provided that produce the BIA of interest. In some examples, the host cells are genetically engineered strains, for example, the genetically engineered strains of the embodiments discussed herein, can provide a platform for producing the nal-opioids of interest, such as, but not limited to, naltrexone, naloxone, nalmefene, nalorphine, nalorphine, nalodein, naldemedine, naloxegol, 6β-naltrexol, naltolindole, methylnaltrexone, methylsamidophan, alvimopan, axelopran, bevenpran, dinicotinate, levallorphan, samidophan, buprenorphine, dezocine, eptazocine, butorphanol, levorphanol, nalbuphine, pentazocine, phenazocine, norbinaltorphimine, and diprenorphine.

[0020] Nor-opioids of interest Host cells are provided that produce nor-opioids of interest. In some examples, host cells that are genetically engineered strains, such as the genetically engineered strains of the embodiments discussed herein, can provide a platform for producing nor-opioids of interest, such as, but not limited to, norcodeine, noroxycodone, northebaine, norhydrocodone, nordihydro-codeine, nor-14-hydroxy-codeine, norcodeinone, nor-14-hydroxy-codeinone, normorphine, noroxymorphone, nororipavine, norhydro-morphone, nordihydro-morphine, nor-14-hydroxy-morphine, normorphinone, nor-14-hydroxy-morphinone.

[0021] Benzylisoquinoline Alkaloids (BIA) of Interest Host cells that produce the BIA of interest are provided. In some examples, genetically engineered host cells, such as the genetically engineered strains of the embodiments discussed herein, can provide a platform for producing several structural classes of benzylisoquinoline alkaloids of interest and their modifications, including, but not limited to, precursor BIAs, benzylisoquinolines, promorphinans, morphinans, and the like. Each of these classes is meant to encompass the biosynthetic precursors, intermediates, and metabolites of any convenient member of the biosynthetic pathway of the genetically engineered host cell that can lead to members of that class. Non-limiting examples of compounds are provided below for each of these structural classes. In some cases, the structure of a given example may or may not be characterized as a benzylisoquinoline alkaloid itself. In some cases, the chemical compounds of the present invention are meant to encompass all possible isomers, such as a single enantiomer, a racemic mixture, an optically pure form, a diastereomeric mixture, and a mixture of intermediates.

[0022] BIA precursors may include, but are not limited to, norcoclaurine (NC) and norlaudanosoline (NL), as well as NC and NL precursors, such as tyrosine, tyramine, 4-hydroxyphenylacetaldehyde (4-HPA), 4-hydroxyphenylpyruvic acid (4-HPPA), L-3,4-dihydroxyphenylalanine (L-DOPA), 3,4-dihydroxyphenylacetaldehyde (3,4-DHPA), and dopamine. In some embodiments, one or more of the BIA precursors are 3,4-dihydroxyphenylacetaldehyde (3,4-DHPA) and dopamine. In certain instances, one or more of the BIA precursors are 4-hydroxyphenylacetaldehyde (4-HPA) and dopamine. In particular, NL and NC can be synthesized from their respective precursor molecules via a Pictet-Spengler condensation reaction, which can occur spontaneously or be catalyzed by any convenient enzyme.

[0023] Benzylisoquinolines may include, but are not limited to, norcoclaurine, norlaudanosoline, coclaurine, 3'-hydroxycoclaurine, 4'-O-methylnorlaudanosoline, 4'-O-methyl-laudanosoline, N-methylnorcoclaurine, laudanosoline, N-methylcoclaurine, 3'-hydroxy-N-methylcoclaurine, reticuline, norreticuline, papaverine, laudanine, laudanosine, tetrahydropapaverine, 1,2-dihydropapaverine, and orientaline.

[0024] Promorphinans can include, but are not limited to, salutaridine, salutaridinol, and salutaridinol-7-O-acetate.

[0025] Morphinans may include, but are not limited to, thebaine, codeinone, codeine, morphine, morphinone, oripavine, neopinone, neopine, neomorphine, hydrocodone, dihydrocodeine, 14-hydroxycodeinone, oxycodone, 14-hydroxycodeine, morphinone, hydromorphone, dihydromorphine, dihydroetorphine, ethylmorphine, etorphine, metopon, buprenorphine, pholcodine, and heterocodeine.

[0026] host cell Any convenient cell can be used in the subject host cells and methods. In some cases, the host cell is a non-plant cell. In some examples, the host cell can be characterized as a microbial cell. In some specific cases, the host cell is an insect cell, a vertebrate cell, a mammalian cell, a plant cell, a fungal cell, a bacterial cell, or a yeast cell. Any convenient type of host cell can be used to generate the subject nor-opioid- or nal-opioid-producing cells; see, for example, US2008 / 0176754 and US2014 / 0273109 (the disclosures of which are incorporated by reference in their entirety). Host cells of interest include, but are not limited to, bacterial cells, such as Bacillus subtilis, Escherichia coli, Streptomyces, and Salmonella typhimurium cells, insect cells, such as Drosophila melanogaster S2 and Spodoptera frugiperda Sf9 cells, mammalian cells, such as HeLa and 293 cells, plant cells, such as tobacco BY-2 cells, and yeast cells, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris cells.In some cases, the host cell is a yeast cell or E. coli cell.In some cases, the host cell is a yeast cell. In some examples, the host cell is derived from a yeast strain that is genetically engineered to produce the nor-opioid or nal-opioid BIA of interest, such as northebaine or naloxone.In some examples, the host cell is derived from a yeast strain that is genetically engineered to produce the enzyme of interest.In some examples, the host cell is derived from a yeast strain that is genetically engineered to produce O-demethylase.The O-demethylase can be capable of converting a substrate, such as a first opioid, into a second opioid.In some examples, the host cell is derived from a yeast strain that is genetically engineered to produce N-demethylase.The N-demethylase can be capable of converting a substrate, such as a second opioid, into a nor-opioid.In some examples, the host cell is derived from a yeast strain genetically engineered to produce a methyltransferase. Furthermore, the methyltransferase can be capable of converting nor-opioids to nal-opioids. In some examples, the host cell is derived from a yeast strain genetically engineered to produce an epimerase. The epimerase can have an oxidase and a reductase. Furthermore, the epimerase can be capable of converting (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids. Furthermore, the epimerase can be separated into smaller enzymes that retain oxidase or reductase activity to be used to convert (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids.

[0027] Any host cell described in US2008 / 0176754 and US2014 / 0273109 by Smolke et al. can be adapted for use in the subject cells and methods. In certain embodiments, the yeast cell can be a S. cerevisiae species. In certain embodiments, the yeast cell can be a Schizosaccharomyces pombe species. In certain embodiments, the yeast cell can be a Pichia yeast species. Yeast is of interest as a host cell because cytochrome P450 proteins can be properly folded within the endoplasmic reticulum membrane so that their activity is maintained. In one example, the cytochrome P450 protein is involved in several biosynthetic pathways of interest. In a further example, the cytochrome P450 protein is involved in the production of a BIA of interest, such as naloxone or naltrexone. In a further example, the cytochrome P450 protein is involved in the production of an enzyme of interest, such as an epimerase having an oxidase and a reductase.

[0028] Yeast strains of interest that may find utility herein include, but are not limited to, CEN.PK (genotype: MATa / α ura3-52 / ura3-52 trp1-289 / trp1-289 leu2-3_112 / leu2-3_112 his3 Δ1 / his3 Δ1 MAL2-8C / MAL2-8C SUC2 / SUC2), S288C, W303, D273-10B, X2180, A364A, Σ1278B, AB972, SK1, and FL100. In certain cases, the yeast strains are S288C (MATα; SUC2 mal mel gal2 CUP1 flo1 flo8-1 hap1), BY4741 (MATα; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0), BY4742 (MATα; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0), BY4743 (MATa / MATα; his3Δ1 / his3Δ1; leu2Δ0 / leu2Δ0; met15Δ0 / MET15; LYS2 / lys2Δ0; ura3Δ0 / ura3Δ0), and WAT11 or W(R), respectively, derived from Arabidopsis thaliana ( The yeast cell is either a derivative of the W303-B strain (MATa; ade2-1; his3-11, -15; leu2-3, -112; ura3-1; canR; cyr+) expressing the (A. thaliana) NADPH-P450 reductase ATR1 and the yeast NADPH-P450 reductase CPR1. In another embodiment, the yeast cell is W303α (MATα; his3-11, 15 trp1-1 leu2-3 ura3-1 ade2-1). The identities and genotypes of additional yeast strains of interest can be found at EUROSCARF (web.uni-frankfurt.de / fb15 / mikro / euroscarf / col_index.html).

[0029] In some cases, the host cell is a fungal cell. In certain embodiments, the fungal cell may be of a species of the genus Aspergillus, including strains of Aspergillus niger (ATCC 1015, ATCC 9029, CBS 513.88), Aspergillus oryzae (ATCC 56747, RIB40), Aspergillus terreus (NIH 2624, ATCC 20542), and Aspergillus nidulans (FGSC A4).

[0030] In certain embodiments, the heterologous coding sequence can be codon-optimized for expression in a species of Aspergillus and expressed by an appropriate promoter. In certain embodiments, the promoter can be selected from the group consisting of the phosphoglycerate kinase promoter (PGK), the MbfA promoter, the cytochrome c oxidase subunit promoter (CoxA), the SrpB promoter, the TvdA promoter, the malate dehydrogenase promoter (MdhA), and the β-mannosidase promoter (ManB). In certain embodiments, the terminator can be selected from the glucoamylase terminator (GlaA) or the TrpC terminator. In certain embodiments, an expression cassette consisting of the promoter, the heterologous coding sequence, and the terminator can be expressed by a plasmid or integrated into the genome of the host. In certain embodiments, cells maintaining the plasmid or integrated cassette can be selected by selection with an antibiotic, such as hygromycin, or by the use of a nitrogen source, for example, using acetamide as the sole nitrogen source. In certain embodiments, DNA constructs can be introduced into host cells using established transformation methods, such as protoplast transformation, lithium acetate, or electroporation. In certain embodiments, cells can be cultured in liquid ME or solid MEA (3% malt extract, 0.5% peptone, and ±1.5% agar) or in Vogel's minimal medium, with or without selection.

[0031] In some cases, the host cell is a bacterial cell. The bacterial cell can be selected from any bacterial genus. Examples of genera from which the bacterial cell can be derived include Anabaena, Arthrobacter, Acetobacter, Acetobacterium, Bacillus, Bifidobacterium, Brachybacterium, Brevibacterium, Carnobacterium, Clostridium, Corynebacterium, Enterobacter, Escherichia, Gluconacetobacter, Gluconobacter, Hafnia, Halomonas, Klebsiella, Kocuria, Lactobacillus, Leuconostoc, Macrococcus, Methylomonas, Methylobacter ... Examples of the genera include Tylococcus, Methylococcus, Microbacterium, Micrococcus, Microcystis, Moorella, Oenococcus, Pediococcus, Prochlorococcus, Propionibacterium, Proteus, Pseudoalteromonas, Pseudomonas, Psychrobacter, Rhodobacter, Rhodococcus, Rhodopseudomonas, Serratia, Staphylococcus, Streptococcus, Streptomyces, Synechococcus, Synechocystis, Tetragenococcus, Weissella, and Zymomonas. Examples of bacterial species that can be used in the methods of the present disclosure include Arthrobacter nicotianae, Acetobacter aceti, Arthrobacter arilaitensis, Bacillus cereus, Bacillus coagulans, Bacillus licheniformis, Bacillus pumilus, Bacillus sphaericus, Bacillus stearothermophilus, Bacillus subtilis, Bifidobacterium adressentis, Bacillus spp. ...adolescentis, Brachybacterium tyrofermentans, Brevibacterium linens, Carnobacterium divergens, Corynebacterium flavescens, Enterococcus faecium, Gluconacetobacter europaeus, Gluconacetobacter johannae, Gluconobacter oxydans, Hafnia alvei, Halomonas elongata, Kocuria rhizophylla rhizophila, Lactobacillus acidifarinae, Lactobacillus jensenii, Lactococcus lactis, Lactobacillus yamanashiensis, Leuconostoc citreum, Macrococcus caseolyticus, Microbacterium foliorum, Micrococcus lylae, Oenococcus oeni, Pediococcus acidilactici acidilactici, Propionibacterium acidipropionici, Proteus vulgaris, Pseudomonas fluorescens, Psychrobacterceler, Staphylococcus condimenti, Streptococcus thermophilus, Streptomyces griseus, Tetragenococcus halophilus, Weissella cibaria, Weissella koreensis, Zymomonas mobilis, Corynebacterium glutamicum, Bifidobacterium bifidum / breve / longum, Streptomyces lividans lividans, Streptomyces coelicolor, Lactobacillus plantarum, Lactobacillus sakei, Lactobacillus casei, Pseudoalteromonas citrea, Pseudomonas putida, Clostridium ljungdahlii / aceticum / acetobutylicum / beijerinckii / butyricum, and Moorella themocellum / thermoacetica.

[0032] In certain embodiments, the bacterial cell may be an E. coli strain. In certain embodiments, the E. coli strain may be selected from BL21, DH5α, XL1-Blue, HB101, BL21, and K12. In certain embodiments, the heterologous coding sequence may be codon-optimized for expression in E. coli and expressed by an appropriate promoter. In certain embodiments, the promoter may be selected from the T7 promoter, tac promoter, trc promoter, tetracycline-inducible promoter (tet), lac operon promoter (lac), and lacO1 promoter. In certain embodiments, the expression cassette consisting of the promoter, heterologous coding sequence, and terminator may be expressed by a plasmid or integrated into the genome. In certain embodiments, the plasmid is selected from pUC19 or pBAD. In certain embodiments, cells maintaining the plasmid or integration cassette may be selected by selection with an antibiotic, such as kanamycin, chloramphenicol, streptomycin, spectinomycin, gentamicin, erythromycin, or ampicillin. In certain embodiments, the DNA construct can be introduced into the host cell using established transformation methods, such as conjugation, heat shock chemical transformation, or electroporation. In certain embodiments, the cells can be cultured in liquid Luria-Bertani (LB) medium at 37° C. with or without antibiotics.

[0033] In certain embodiments, the bacterial cell may be a Bacillus subtilis strain. In certain embodiments, the B. subtilis strain may be selected from 1779, GP25, RO-NN-1, 168, BSn5, BEST195, 1A382, and 62178. In certain embodiments, the heterologous coding sequence may be codon-optimized for expression in a species of Bacillus and expressed by an appropriate promoter. In certain embodiments, the promoter may be selected from the grac promoter, p43 promoter, or trnQ promoter. In certain embodiments, an expression cassette consisting of the promoter, heterologous coding sequence, and terminator may be expressed by a plasmid or integrated into the genome. In certain embodiments, the plasmid is selected from pHP13 pE194, pC194, pHT01, or pHT43. In certain embodiments, an integration vector, such as pDG364 or pDG1730, may be used to integrate the expression cassette into the genome. In certain embodiments, cells that maintain the plasmid or integration cassette can be selected by selection with antibiotics, such as erythromycin, kanamycin, tetracycline, and spectinomycin. In certain embodiments, the DNA construct can be introduced into the host cell using established transformation methods, such as natural competence, heat shock, or chemical transformation. In certain embodiments, the cells can be cultured in liquid Luria-Bertani (LB) medium at 37°C or in M9 medium plus glucose and tryptophan.

[0034] Genetic modification of host cells The host cell can be genetically engineered to contain one or more modifications (e.g., two or more, three or more, four or more, five or more, or even more modifications) that result in the production of a BIA of interest. Additionally or alternatively, the host cell can be genetically engineered to contain one or more modifications (e.g., two or more, three or more, four or more, five or more, or even more modifications) that result in the production of an enzyme of interest. In some cases, the modification is a genetic modification, such as a mutation, addition, or deletion of a gene or fragment thereof, or transcriptional regulation of a gene or fragment thereof. As used herein, the term "mutation" refers to the deletion, insertion, or substitution of an amino acid or nucleotide residue relative to a reference sequence or motif. The mutation can be incorporated into the original locus of a native gene as a specific mutation. In some cases, the mutation can be incorporated as an additional copy of the gene introduced as an integrated gene at a separate locus or as an additional copy on an episomal vector, such as a 2μ or centromeric plasmid. In some specific cases, the substrate-inhibiting copy of the enzyme is under the transcriptional control of native cells.In some cases, the substrate-inhibiting copy of the enzyme is introduced with engineered constitutive or dynamic regulation of protein expression by being placed under the control of a synthetic promoter.In some cases, the target of one or more modifications can be a native gene.In some cases, the target of one or more modifications can be a non-native gene.In some cases, a non-native gene can be inserted into host cells.In another example, a non-native gene can be modified by one or more modifications before being inserted into host cells.

[0035] The genetically engineered host cells can overproduce one or more nor-opioid BIA molecules of interest. The genetically engineered host cells can overproduce one or more nar-opioid BIA molecules of interest. By overproduce, it is meant that the cells have improved or increased production of nor-opioid and / or nar-opioid BIA molecules of interest compared to control cells (e.g., unmodified cells). Improved or increased production means both production of some amount of nor-opioid and / or nar-opioid BIA of interest where the control does not have production of nor-opioid and / or nar-opioid BIA of interest, and an increase of about 10% or more, e.g., about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, e.g., 2-fold or more, e.g., 5-fold or more, e.g., 10-fold or more, in situations where the control has production of some nor-opioid and / or nar-opioid BIA of interest.

[0036] The genetically engineered host cells may overproduce one or more nor-opioids. In some cases, the genetically engineered host cells may produce some amount of the nor-opioid of interest when the control does not have nor-opioid production, and may result in an increase of about 10% or more, for example, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, for example, 2-fold or more, for example, 5-fold or more, for example, 10-fold or more, in situations where the control has some nor-opioid production of the nor-opioid of interest.

[0037] The genetically engineered host cells may also overproduce one or more nar-opioids. In some cases, the genetically engineered host cells may produce some amount of the nar-opioid of interest when the control does not have production of the nar-opioid, and may result in an increase of about 10% or more, for example, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, for example, 2-fold or more, for example, 5-fold or more, for example, 10-fold or more, in situations where the control has production of some nar-opioid of interest.

[0038] The engineered host cells can overproduce one or more BIAs of interest. By overproduce, it is meant that the cells have improved or increased production of the BIA molecules of interest compared to control cells (e.g., unmodified cells). Improved or increased production refers to both production of some amount of the BIA of interest when the control does not have production of the BIA of interest, as well as an increase of about 10% or more, e.g., about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, e.g., 2-fold or more, e.g., 5-fold or more, e.g., 10-fold or more, when the control has production of some BIA of interest.

[0039] The genetically engineered host cell may overproduce one or more (S)-1-benzylisoquinoline alkaloids. In some cases, the genetically engineered host cell may produce some amount of the (S)-1-benzylisoquinoline alkaloid of interest when the control does not have production of the (S)-1-benzylisoquinoline alkaloid, and may result in an increase of about 10% or more, e.g., about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, e.g., 2-fold or more, e.g., 5-fold or more, e.g., 10-fold or more, in situations where the control has production of some of the (S)-1-benzylisoquinoline alkaloid of interest.

[0040] The genetically engineered host cells can further overproduce one or more (R)-1-benzylisoquinoline alkaloids. In some cases, the genetically engineered host cells can produce some amount of the (R)-1-benzylisoquinoline alkaloid of interest when the control does not have production of the (R)-1-benzylisoquinoline alkaloid, and in situations where the control has production of some of the (R)-1-benzylisoquinoline alkaloid of interest, the increase can be about 10% or more, for example, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, for example, 2-fold or more, for example, 5-fold or more, for example, 10-fold or more. The genetically engineered host cells can further overproduce one or more of morphinan alkaloids and promorphinan alkaloids.

[0041] In some cases, genetically engineered host cells can produce an increased amount of (R)-reticuline compared to control host cells that do not have one or more modifications (for example, as described herein).In some specific cases, the increased amount of (R)-reticuline is about 10% or more compared to control host cells, for example, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, about 2 times or more, about 5 times or more, or even about 10 times or more compared to control host cells.In some cases, (R)-reticuline is the product of epimerization reaction in genetically engineered host cells.In such cases, (S)-reticuline can be the substrate of epimerization reaction.

[0042] Furthermore, genetically engineered host cells can overproduce one or more enzymes of interest.Overproducing means that the cell has improved or increased production of the enzyme of interest compared to control cells (for example, unmodified cells).Improved or increased production refers to both the production of some amount of the enzyme of interest when the control does not have production, and the increase of about 10% or more, for example, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, for example, 2 times or more, for example, 5 times or more, for example, 10 times or more when the control has some amount of the enzyme of interest.

[0043] The genetically engineered host cell can overproduce one or more O-demethylase (ODM) enzymes. Examples of ODM enzymes that can be used in the embodiments described herein can be found in Table 3. In some cases, the genetically engineered host cell can produce some amount of ODM enzyme when the control does not have ODM enzyme production, and can result in an increase of about 10% or more, for example, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, for example, 2-fold or more, for example, 5-fold or more, for example, 10-fold or more, in situations where the control has some ODM enzyme production.

[0044] The genetically engineered host cell can overproduce one or more N-demethylase (NDM) enzymes. Examples of NDM enzymes that can be used in the embodiments described herein can be found in Table 4. In some cases, the genetically engineered host cell can produce some amount of ODM enzyme when the control does not have NDM enzyme production, and can produce an increase of about 10% or more, for example, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, for example, 2-fold or more, for example, 5-fold or more, for example, 10-fold or more in the situation where the control has some NDM enzyme production.

[0045] The genetically engineered host cell can overproduce one or more N-methyltransferase (NMT) enzymes. Examples of NMT enzymes that can be used in the embodiments described herein can be found in Table 5. In some cases, the genetically engineered host cell can produce some amount of NMT enzyme when the control does not have NMT enzyme production, and can result in an increase of about 10% or more, such as about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, such as 2-fold or more, such as 5-fold or more, for example 10-fold or more, in situations where the control has some NMT enzyme production.

[0046] The genetically engineered host cells may overproduce one or more CYP-COR enzymes. In some cases, the genetically engineered host cells may produce some amount of CYP-COR enzyme when the control does not have CYP-COR enzyme production, and may result in an increase of about 10% or more, e.g., about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, e.g., 2-fold or more, e.g., 5-fold or more, e.g., 10-fold or more, in situations where the control has some CYP-COR enzyme production.

[0047] The genetically engineered host cell may further overproduce one or more enzymes derived from CYP-COR enzymes. In some cases, the genetically engineered host cell may produce some amount of the enzyme derived from CYP-COR enzyme when the control does not have production of the enzyme derived from CYP-COR enzyme, and may result in an increase of about 10% or more, e.g., about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, e.g., 2-fold or more, e.g., 5-fold or more, e.g., 10-fold or more, in situations where the control has some production of the enzyme derived from CYP-COR enzyme.

[0048] In some cases, the one or more (e.g., two or more, three or more, or four or more) modifications can be selected from: substrate inhibition alleviation mutations in biosynthetic enzyme genes; product inhibition alleviation mutations in biosynthetic enzyme genes; cofactor recovery promotion mechanisms; feedback inhibition alleviation mutations in biosynthetic enzyme genes; transcriptional modulation modifications of biosynthetic enzyme genes; inactivating mutations in enzyme genes; epimerization modifications; bisBIA generation modifications; and heterologous coding sequences encoding enzymes. A cell containing one or more modifications can be referred to as a genetically engineered cell.

[0049] Substrate inhibition alleviation mutation In some examples, a genetically engineered host cell is a cell that contains one or more substrate inhibition alleviation mutations (e.g., two or more, three or more, four or more, five or more, or even more) in one or more biosynthetic enzyme genes of the cell. In some examples, the one or more biosynthetic enzyme genes are native to the cell (e.g., present in an unmodified cell). In some examples, the one or more biosynthetic enzyme genes are non-native to the cell. As used herein, the term "substrate inhibition alleviation mutation" refers to a mutation that alleviates a substrate inhibition control mechanism of the cell.

[0050] A mutation that relieves substrate inhibition reduces the inhibition of the target enzyme in the cell of interest compared to a control cell, resulting in increased levels of the target compound or its downstream biosynthetic product. In some cases, relieving inhibition of the target enzyme means reducing the IC of inhibition. 50 By increased level is meant that the level of the compound of interest or its downstream product in the engineered host cell is 110% or more, such as 120% or more, 130% or more, 140% or more, 150% or more, 160% or more, 170% or more, 180% or more, 190% or more, or 200% or more, of that of the compound of interest or its downstream product in the engineered host cell, such as at least 3-fold or at least 5-fold or at least 10-fold or even more.

[0051] Various substrate inhibition control mechanisms and biosynthetic enzymes in genetically engineered host cells directed at regulating the level of a nal-opioid or nor-opioid BIA or its precursor of interest can be targeted for substrate inhibition relief. The genetically engineered host cells can contain one or more substrate inhibition relief mutations in one or more biosynthetic enzyme genes. The one or more mutations can be located in any convenient biosynthetic enzyme gene where the biosynthetic enzyme is subject to regulatory control. In some embodiments, the one or more biosynthetic enzyme genes encode one or more tyrosine hydroxylase enzymes. In some particular cases, the one or more substrate inhibition relief mutations are present in a biosynthetic enzyme gene that is TyrH. In some embodiments, the genetically engineered host cells can contain one or more substrate inhibition relief mutations in one or more biosynthetic enzyme genes, for example, in one of the genes listed in Table 2.

[0052] In certain embodiments, the one or more substrate inhibition-relieving mutations are present in the TyrH gene. The TyrH gene encodes tyrosine hydroxylase, an enzyme that converts tyrosine to L-DOPA. However, TyrH is inhibited by its substrate, tyrosine. Mammalian tyrosine hydroxylase activity, for example, found in humans or rats, can be improved by mutations that relieve substrate inhibition of the TyrH gene. In particular, substrate inhibition by tyrosine can be alleviated by a point mutation W166Y in the TyrH gene. Furthermore, the point mutation W166Y in the TyrH gene can improve the binding of the co-substrate BH4 of tyrosine hydroxylase to catalyze the reaction of tyrosine to L-DOPA. When expressed in yeast strains to produce BIAs from sugars, such mutant forms of TyrH (e.g., those described in U.S. Provisional Patent Application No. 61 / 899,496) can significantly improve the production of BIAs.

[0053] Any convenient number and type of mutations can be used to alleviate substrate inhibition control mechanisms. In certain embodiments, the genetically engineered host cells of the present invention can comprise one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or even fifteen or more substrate inhibition alleviation mutations, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 substrate inhibition alleviation mutations in one or more biosynthetic enzyme genes within the genetically engineered host cell.

[0054] Cofactor recovery promotion mechanism In some examples, the genetically engineered host cell is a cell that contains one or more cofactor restorer mechanisms (e.g., two or more, three or more, four or more, five or more, or even more) within one or more biosynthetic enzyme genes of the cell. In some examples, the one or more biosynthetic enzyme genes are native to the cell (e.g., present in an unmodified cell). In some examples, the one or more biosynthetic enzyme genes are non-native to the cell. As used herein, the term "cofactor restorer mechanism" refers to a mechanism that promotes the cofactor restorer control mechanism of the cell.

[0055] Various regulatory mechanisms for cofactor restoration and biosynthetic enzymes in genetically engineered host cells directed at regulating the levels of a nor-opioid or nal-opioid BIA or its precursor of interest can be targeted for cofactor restoration promotion. The genetically engineered host cells can contain one or more cofactor restoration promotion mechanisms within one or more biosynthetic enzyme genes. In one example, the genetically engineered host cells can contain a heterologous coding sequence encoding dihydrofolate reductase (DHFR). When DHFR is expressed, it can convert 7,8-dihydrobiopterin (BH2) to tetrahydrobiopterin (BH4), thereby restoring BH4 as a TyrH co-substrate. In some examples, the genetically engineered host cells can contain one or more cofactor restoration promotion mechanisms within one or more biosynthetic enzyme genes, for example, within one of the genes listed in Table 2.

[0056] Any convenient number and type of mechanism can be used to promote cofactor restitution control mechanisms. In certain embodiments, the genetically engineered host cells of the present invention can comprise one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or even fifteen or more cofactor restitution promoting mechanisms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 cofactor restitution promoting mechanisms within one or more biosynthetic enzyme genes within the genetically engineered host cell.

[0057] Mutations that relieve product inhibition In some examples, the genetically engineered host cell is a cell that includes one or more product inhibition alleviation mutations (e.g., two or more, three or more, four or more, five or more, or even more) in one or more biosynthetic enzyme genes of the cell. In some examples, the one or more biosynthetic enzyme genes are native to the cell (e.g., present in an unmodified cell). In some examples, the one or more biosynthetic enzyme genes are non-native to the cell. As used herein, the term "product inhibition alleviation mutation" refers to a mutation that alleviates the short-term and / or long-term product inhibition control mechanism of the genetically engineered host cell. Short-term product inhibition is a control mechanism of the cell in which competitive binding of co-substrates to binding sites occurs. Long-term product inhibition is a control mechanism of the cell in which irreversible binding of compounds outside the desired pathway occurs.

[0058] A mutation that relieves product inhibition reduces inhibition of the target enzyme in a cell of interest compared to a control cell, resulting in increased levels of the target compound or its downstream biosynthetic product. In some cases, relieving inhibition of the target enzyme means reducing the IC of inhibition. 50By increased level is meant that the level of the compound of interest or its downstream product in the engineered host cell is 110% or more, such as 120% or more, 130% or more, 140% or more, 150% or more, 160% or more, 170% or more, 180% or more, 190% or more, or 200% or more, of that of the compound of interest or its downstream product in the engineered host cell, such as at least 3-fold or at least 5-fold or at least 10-fold or even more.

[0059] Various control mechanisms of product inhibition and biosynthetic enzymes in genetically engineered host cells directed at regulating the level of nor-opioid or nal-opioid BIA of interest can be targeted for product inhibition relief. The genetically engineered host cells can contain one or more product inhibition relief mutations in one or more biosynthetic enzyme genes. The mutations can be located in any convenient biosynthetic enzyme gene where the biosynthetic enzyme is subject to regulatory control. In some embodiments, the one or more biosynthetic enzyme genes encode one or more tyrosine hydroxylase enzymes. In some particular cases, the one or more product inhibition relief mutations are present in a biosynthetic enzyme gene that is TyrH. In some embodiments, the genetically engineered host cells contain one or more product inhibition relief mutations in one or more biosynthetic enzyme genes, for example, in one of the genes listed in Table 2.

[0060] In certain embodiments, the one or more product inhibition-relieving mutations are present in the TyrH gene. The TyrH gene encodes tyrosine hydroxylase, an enzyme that converts tyrosine to L-DOPA. TyrH requires tetrahydrobiopterin (BH4) as a co-substrate to catalyze the hydroxylation reaction. Some microbial strains, such as Saccharomyces cerevisiae, do not naturally produce BH4 but can be engineered to produce this substrate via a four-enzyme synthesis / recycling pathway, as shown in Figure 1. Figure 1 shows an example of a tetrahydrobiopterin synthesis, recycling, and salvage pathway according to an embodiment of the present invention. Figure 1 illustrates the use of the enzymes PTPS, pyruvoyltetrahydropterin synthase; SepR, sepiapterin reductase; PCD, pterin 4a-carbinolamine dehydratase; QDHPR, dihydropteridine reductase; and DHFR, dihydrofolate reductase. Of the enzymes shown in Figure 1, yeast synthesizes endogenous GTP cyclohydrolase I. GTP and dihydroneopterin triphosphate are naturally synthesized in yeast. Furthermore, the other metabolites in Figure 1 are not naturally produced in yeast.

[0061] TyrH is inhibited by its product, L-DOPA, as well as other catecholamines, particularly dopamine. Mammalian tyrosine hydroxylase activity, e.g., from humans or rats, can be improved by mutations that alleviate product inhibition. For example, short-term product inhibition, such as competitive cosubstrate binding to the binding site, can be alleviated by the point mutation W166Y in the TyrH gene. In particular, the point mutation W166Y in the TyrH gene can improve cosubstrate binding. Furthermore, short-term product inhibition, which alleviates competitive cosubstrate binding to the binding site, can also be improved by the point mutation S40D in the TyrH gene. Short-term product inhibition can also be improved by the joint mutations R37E and R38E in the TyrH gene. In particular, the R37E and R38E mutations together can clearly improve tyrosine hydroxylase activity in the presence of dopamine.

[0062] Furthermore, long-term product inhibition can be alleviated by point mutations in the TyrH gene, which may involve irreversible binding of catecholamines to the iron at the active site, reducing the presence of catecholamines, which act as product inhibitors of tyrosine hydroxylase activity. Long-term product inhibition can be alleviated by mutations E332D and Y371F in the TyrH gene, respectively.

[0063] Combinations of mutations (e.g., two, three, or more mutations at once) can be made to alleviate multiple types of substrate and product inhibition and further improve the activity of TyrH. Such mutant forms of TyrH (e.g., those described in U.S. Provisional Patent Application No. 61 / 899,496) can significantly improve the production of nor-opioid and / or nal-opioid products when expressed in yeast strains to produce nor-opioid and / or nal-opioid BIAs of interest from sugars.

[0064] Any convenient number and type of mutations can be used to alleviate the product inhibition regulatory mechanism. In certain embodiments, the genetically engineered host cells of the present invention can comprise one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or even fifteen or more product inhibition alleviation mutations, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 product inhibition alleviation mutations in one or more biosynthetic enzyme genes within the genetically engineered host cell.

[0065] Feedback inhibition relieving mutations In some examples, the genetically engineered host cell is a cell that contains one or more feedback inhibition relieving mutations (e.g., two or more, three or more, four or more, five or more, or even more) in one or more biosynthetic enzyme genes of the cell. In some cases, the one or more biosynthetic enzyme genes are native to the cell (e.g., present in an unmodified cell). Additionally or alternatively, in some cases, the one or more biosynthetic enzyme genes are non-native to the cell. As used herein, the term "feedback inhibition relieving mutation" refers to a mutation that relieves the feedback inhibition control mechanism of the genetically engineered host cell. Feedback inhibition is a cellular control mechanism in which an enzyme in the synthetic pathway of a regulated compound is inhibited once the compound has accumulated to a certain level, thereby balancing the amount of the compound in the cell. A mutation that relieves feedback inhibition reduces the inhibition of the regulated enzyme in the genetically engineered host cell compared to a control cell. In this way, the genetically engineered host cell results in increased levels of the regulated compound or its downstream biosynthetic product. In some cases, relieving the inhibition of the regulated enzyme refers to reducing the IC of the inhibition. 50 by 2-fold or more, for example, 3-fold or more, 5-fold or more, 10-fold or more, 30-fold or more, 100-fold or more, 300-fold or more, 1000-fold or more, or even more. By increased level, it is meant that the level of the compound to be regulated or its downstream product in the host cell is 110% or more, for example, 120% or more, 130% or more, 140% or more, 150% or more, 160% or more, 170% or more, 180% or more, 190% or more, or 200% or more, of that of the compound to be regulated or its downstream product in the control cell, for example, at least 3-fold or more, at least 5-fold or more, at least 10-fold or even more.

[0066] Various feedback inhibition control mechanisms and biosynthetic enzymes directed at regulating the level of a BIA of interest can be targeted for alleviation in host cells. The host cell can contain one or more feedback inhibition alleviation mutations in one or more biosynthetic enzyme genes native to the cell. The one or more mutations can be located in any convenient biosynthetic enzyme gene where the biosynthetic enzyme is subject to regulatory control. In some embodiments, the one or more biosynthetic enzyme genes can encode one or more enzymes selected from 3-deoxy-d-arabinose-heptulosonate-7-phosphate (DAHP) synthase and chorismate mutase. In some embodiments, the one or more biosynthetic enzyme genes encode 3-deoxy-d-arabinose-heptulosonate-7-phosphate (DAHP) synthase. In some examples, the one or more biosynthetic enzyme genes can encode chorismate mutase. In certain cases, the one or more feedback inhibition alleviation mutations may be present in a biosynthetic enzyme gene selected from ARO4 and ARO7. In certain cases, the one or more feedback inhibition alleviation mutations may be present in a biosynthetic enzyme gene that is ARO4. In certain cases, the one or more feedback inhibition alleviation mutations are present in a biosynthetic enzyme gene that is ARO7. In some embodiments, the genetically engineered host cell may comprise one or more feedback inhibition alleviation mutations in one or more biosynthetic enzyme genes, for example, in one of the genes listed in Table 2.

[0067] Any convenient number and type of mutation can be used to relieve feedback inhibition control mechanism.As used herein, the term "mutation" refers to the deletion, insertion or substitution of amino acid residue or nucleotide residue with respect to a reference sequence or motif.Mutation can be incorporated into the original locus of a native gene as a specific mutation.In some cases, mutation can be incorporated into an additional copy of a gene that is introduced as an integrated gene in a separate locus, or into an additional copy on an episomal vector, for example, 2μ or centromeric plasmid.In some specific cases, the feedback inhibition copy of the enzyme is under the transcriptional control of native cells.In some cases, the feedback inhibition copy of the enzyme is introduced with the constitutive or dynamic genetically engineered regulation of protein expression by being placed under the control of a synthetic promoter.

[0068] In certain embodiments, the one or more feedback inhibition relieving mutations can be present in the ARO4 gene.The ARO4 mutation of interest can include, but is not limited to, the substitution of leucine for the lysine residue at position 229, the substitution of lysine for the glutamine residue at position 166, or the mutations described in Hartmann M, et al. ((2003) Proc Natl Acad Sci USA 100(3):862-867) or Fukuda, et al. ((1992) J Ferment Bioeng 74(2):117-119).In some examples, the mutation for imparting feedback inhibition can be selected from a mutagenized enzyme variant library. Examples of such selection may include the rescue of growth of an aro3 mutant yeast strain or an increase in o-fluoro-D,L-phenylalanine in a medium with excess tyrosine, as described by Fukuda, et al. ((1990) Breeding of Brewing Yeast Producing a Large Amount of β-Phenylethyl Alcohol and β-Phenylethyl Acetate. Agr Biol Chem Tokyo 54(1):269-271).

[0069] In certain embodiments, the genetically engineered host cells of the present invention may comprise one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or even fifteen or more feedback inhibition alleviation mutations, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 feedback inhibition alleviation mutations in one or more biosynthetic enzyme genes within the genetically engineered host cell.

[0070] Transcriptional Modulation A host cell can include one or more transcriptional modulation modifications (e.g., two or more, three or more, four or more, five or more, or even more modifications) of one or more biosynthetic enzyme genes of the cell. In some examples, the one or more biosynthetic enzyme genes are native to the cell. In some examples, the one or more biosynthetic enzyme genes are non-native to the cell. Any convenient biosynthetic enzyme gene of the cell can be targeted for transcriptional modulation. Transcriptional modulation means that the expression of a gene of interest in a modified cell is modulated, e.g., increased or decreased, improved or suppressed, compared to a control cell (e.g., an unmodified cell). In some cases, transcriptional modulation of the gene of interest includes increased or enhanced expression. Increased or enhanced expression means that the expression level of the gene of interest is increased by 2-fold or more, e.g., 5-fold or more, in some cases 25-, 50-, or 100-fold or more, and in certain embodiments 300-fold or more (e.g., by using any convenient gene expression assay) compared to expression in a control, i.e., the same unmodified cell. Alternatively, when the expression of the gene of interest in cells is very low and undetectable, the expression level of the gene of interest is considered to be increased if its expression is increased to a level that can be easily detected.In some particular cases, the transcriptional modulation of the gene of interest includes reducing or suppressing expression.Reducing or suppressing expression means that the expression level of the gene of interest is reduced by 2 times or more, for example, 5 times or more, sometimes 25-, 50- or 100-fold or more, and in certain embodiments, 300 times or more compared to control.In some cases, expression is reduced to an undetectable level.The modification of the host cell process of interest that can be adapted for use in the subject host cell is described in US Patent Application Publication No. 20140273109 (14 / 211,611) by Smolke et al., the disclosure of which is incorporated herein by reference in its entirety.

[0071] Any convenient biosynthetic enzyme gene can be transcriptionally modulated, including, but not limited to, the biosynthetic enzymes set forth in FIG. 2. In particular, FIG. 2 shows a biosynthetic scheme for the conversion of glucose to 4-HPA, dopamine, and 3,4-DHPA according to an embodiment of the present invention. Examples of enzymes set forth in FIG. 2 include ARO3, ARO4, ARO1, ARO7, TYR1, TYR, TyrH, DODC, MAO, ARO10, ARO9, and TKL. In some examples, the one or more biosynthetic enzyme genes can be selected from ARO10, ARO9, and TKL. In some cases, the one or more biosynthetic enzyme genes can be ARO10. In some particular cases, the one or more biosynthetic enzyme genes can be ARO9. In some embodiments, the one or more biosynthetic enzyme genes can be TKL. In some embodiments, the host cell comprises one or more transcriptional modulation modifications to one or more genes, such as one of the genes set forth in Table 2.

[0072] In some embodiments, transcriptional modulation modifications may include replacing the native promoter of one or more biosynthetic enzyme genes with a strong promoter or expressing additional copies of the genes under the control of a strong promoter. The promoter driving the expression of the gene of interest may be a constitutive promoter or an inducible promoter, provided that the promoter is active in the host cell. The gene of interest may be expressed by its native promoter. Additionally or alternatively, the gene of interest may be expressed by a non-native promoter. Although not a requirement, such a promoter may be moderate to high in strength in the host in which it is used. The promoter may be regulated or constitutive. In some embodiments, a promoter that is not glucose-repressible or is only lightly repressed by the presence of glucose in the culture medium may be used. Many suitable promoters exist, including promoters of glycolytic genes, such as the promoter of the Bacillus subtilis tsr gene (encoding fructose biphosphate aldolase) or the GAPDH promoter from Saccharomyces cerevisiae (encoding glyceraldehyde-phosphate dehydrogenase) (Bitter GA, Meth. Enzymol. 152:673-684 (1987)). Other strong promoters of interest include, but are not limited to, the ADHI promoter of baker's yeast (Ruohonen L., et al., J. Biotechnol. 39:193-203(1995)), phosphate-starvation-inducible promoters such as the PHO5 promoter of yeast (Hinnen A., et al., in Yeast Genetic Engineering, Barr PJ, et al. eds., Butterworths(1989)), the alkaline phosphatase promoter from B. licheniformis (Lee JWK, et al., J. Gen. Microbiol. 137:1127-1133(1991)), GPD1, and TEF1.Interesting promoters for yeast include, but are not limited to, inducible promoters such as Gal1-10, Gal1, GalL, GalS, repressible promoters Met25, tetO, and constitutive promoters such as glyceraldehyde 3-phosphate dehydrogenase promoter (GPD), alcohol dehydrogenase promoter (ADH), translation-elongation factor-1-α promoter (TEF), cytochrome c-oxidase promoter (CYC1), MRP7 promoter, etc. In some cases, the strong promoter is GPD1. In some specific cases, the strong promoter is TEF1. Autonomously replicating yeast expression vectors containing promoters inducible by hormones such as glucocorticoids, steroids, and thyroid hormones are also known, including, but not limited to, glucorticoid response elements (GRE) and thyroid hormone response elements (TRE), see, for example, the promoters described in U.S. Patent No. 7,045,290. Vectors containing constitutive or inducible promoters (e.g., for α-factor, alcohol oxidase, and PGH) can be used. Furthermore, any promoter / enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of the gene of interest. It will be understood that any convenient promoter specific to the host cell can be selected, such as the E. coli T7 promoter, lac promoter, or tetO promoter. In some cases, promoter selection can be used to optimize transcription, and therefore enzyme levels, to maximize production and minimize energy resources.

[0073] Inactivating mutations A genetically engineered host cell can contain one or more inactivating mutations (e.g., two or more, three or more, four or more, five or more, or even more) in the enzymes of the cell. By including one or more inactivating mutations, the synthetic pathway flux of the genetically engineered host cell can be modified to increase the level of the desired nor-opioid or nar-opioid BIA or the desired enzyme or precursor that leads to it. In some cases, the one or more inactivating mutations are in an enzyme that is native to the cell. Additionally or alternatively, the one or more inactivating mutations are in an enzyme that is not native to the cell. As used herein, "inactivating mutation" refers to one or more mutations in a gene or regulatory DNA sequence of the cell, which inactivates the biological activity of the protein expressed by the gene of interest. In some cases, the gene is native to the cell. In some cases, the gene is inactivated and encodes an enzyme that is part of or associated with the synthetic pathway of the nor-opioid and / or nal-opioid BIA of interest produced by the host cell. In some cases, the inactivating mutation is placed in the regulatory DNA sequence that controls the gene of interest. In some cases, the inactivating mutation is in the promoter of the gene. Any convenient mutation (e.g., as described herein) can be used to inactivate the gene of interest or regulatory DNA sequence. "Inactivated" or "inactivating" means that the biological activity of the protein expressed by the mutated gene is reduced by 10% or more, for example, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more compared to the control protein expressed by the non-mutated control gene. In some cases, the protein is an enzyme, and the inactivating mutation reduces the activity of the enzyme.

[0074] In some examples, the engineered host cell contains an inactivating mutation in an enzyme native to the cell. Any convenient enzyme can be targeted for inactivation. Enzymes of interest can include, but are not limited to, the enzymes listed in Table 2, in which the action of the engineered host cell in a synthetic pathway tends to reduce the level of the nor-opioid or nal-opioid of interest. In some cases, the enzyme has glucose-6-phosphate dehydrogenase activity. In certain embodiments, the enzyme containing an inactivating mutation is ZWF1. In some cases, the enzyme has alcohol dehydrogenase activity. In some embodiments, the enzyme containing an inactivating mutation is selected from ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, and SFA1. In certain embodiments, the enzyme containing an inactivating mutation is ADH2. In certain embodiments, the enzyme containing an inactivating mutation is ADH3. In certain embodiments, the enzyme containing an inactivating mutation is ADH4. In certain embodiments, the enzyme containing an inactivating mutation is ADH5. In certain embodiments, the enzyme comprising an inactivating mutation is ADH6. In certain embodiments, the enzyme comprising an inactivating mutation is ADH7. In some cases, the enzyme has aldehyde oxidoreductase activity. In certain embodiments, the enzyme comprising an inactivating mutation is selected from ALD2, ALD3, ALD4, ALD5, and ALD6. In certain embodiments, the enzyme comprising an inactivating mutation is ALD2. In certain embodiments, the enzyme comprising an inactivating mutation is ALD3. In certain embodiments, the enzyme comprising an inactivating mutation is ALD4. In certain embodiments, the enzyme comprising an inactivating mutation is ALD5. In certain embodiments, the enzyme comprising an inactivating mutation is ALD6. In some embodiments, the host cell comprises one or more inactivating mutations in one or more genes listed in Table 2.

[0075] Epimerization modification Some methods, processes, and systems provided herein describe the conversion of (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids. Some of these methods, processes, and systems may include genetically engineered host cells. In some examples, the conversion of (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids is a key step in the conversion of substrates to a diverse range of alkaloids. In some examples, the conversion of (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids involves an epimerization reaction. In some cases, epimerization of the substrate alkaloid can be achieved by oxidizing the (S)-substrate to the corresponding Schiff base or imine intermediate, followed by stereospecific reduction of this intermediate to the (R)-product, as shown in FIG. 3 and generally depicted in Scheme 1. As shown in Scheme 1, R1, R2, R3, and R4 can be H or CH3. R5 can be H, OH, or OCH3. TIFF0007730308000001.tif35160

[0076] In some cases, the conversion of an (S)-substrate to an (R)-product may involve at least one oxidation reaction and at least one reduction reaction. In some cases, an oxidation reaction is optionally followed by a reduction reaction. In some cases, at least one of the oxidation reaction and the reduction reaction is carried out in the presence of an enzyme. In some cases, at least one of the oxidation reaction and the reduction reaction is catalyzed by an enzyme. In some cases, both the oxidation reaction and the reduction reaction are carried out in the presence of at least one enzyme. In some cases, the at least one enzyme is useful for catalyzing the oxidation reaction and the reduction reaction. The oxidation reaction and the reduction reaction may be catalyzed by the same enzyme.

[0077] In some methods, processes, and systems described herein, the oxidation reaction can be carried out in the presence of an enzyme. In some examples, the enzyme can be an oxidase. The oxidase can use (S)-1-benzylisoquinoline as a substrate. The oxidase can convert the (S)-substrate to the corresponding imine or Schiff base derivative. The oxidase can be referred to as 1,2-dehydroreticuline synthase (DRS). Non-limiting examples of enzymes suitable for the oxidation of (S)-1-benzylisoquinoline alkaloids in the present disclosure include cytochrome P450 oxidase, 2-oxoglutarate-dependent oxidase, and flavoprotein oxidase. For example, (S)-tetrahydroprotoberberine oxidase (STOX, EC 1.3.3.8) can oxidize (S)-norreticuline and other (S)-1-benzylisoquinoline alkaloids to 1,2-dehydronorreticuline and other corresponding 1,2-dehydro products. In some examples, a protein comprising an oxidase domain from any one of the preceding examples can perform the oxidation. In some examples, the oxidase can catalyze an oxidation reaction in a host cell, e.g., a genetically engineered host cell, as described herein.

[0078] In some cases, the reduction reaction can be followed by an oxidation reaction. The reduction reaction can be performed by an enzyme. In some cases, the reductase can use an imine or Schiff base derived from 1-benzylisoquinoline as a substrate. The reductase can convert the imine or Schiff base derivative to (R)-1-benzylisoquinoline. The reductase can be referred to as 1,2-dehydroreticuline reductase (DRR). Non-limiting examples of enzymes suitable for reducing imines or Schiff bases derived from (S)-1-benzylisoquinoline alkaloids include aldo-keto reductases (e.g., codeinone reductase-like enzymes (EC 1.1.1.247)) and short-chain dehydrogenases (e.g., salutaridine reductase-like enzymes (EC 1.1.1.248)). In some cases, a protein containing any one of the reductase domains in the previous examples can perform the reduction. In a further aspect, the reduction is stereospecific. In some cases, the reductase can catalyze a reduction reaction in a host cell, such as a genetically engineered host cell, as described herein.

[0079] An example of an enzyme capable of epimerizing (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids is an epimerase having an oxidase domain and a reductase domain. In particular, the epimerase may have a cytochrome P450 oxidase 82Y2-like domain. Furthermore, the epimerase may have a codeinone reductase-like domain. Furthermore, an epimerase having a cytochrome P450 oxidase 82Y2-like domain and also a codeinone reductase-like domain may be referred to as a CYP-COR enzyme. In particular, the CYP-COR enzyme may be a fusion enzyme. The CYP-COR enzyme may also be referred to as DRS-DRR (dehydroreticuline synthase-dehydroreticuline reductase).

[0080] An example of the amino acid sequence of a CYP-COR enzyme that can be used to convert (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids is shown in Figure 4. In particular, Figure 4 shows the amino acid sequence of a CYP-COR enzyme according to an embodiment of the present invention. As seen in Figure 4, the underlined letters represent the cytochrome P450 CYP82Y2-like domain (59% identity to AFB74617.1). The dotted underlined letters represent the aldo-keto reductase NADPH-dependent codeinone reductase-like domain (75% identity to ACM44066.1). Additional amino acid sequences of CYP-COR enzymes are shown in Table 1. The amino acid sequence of an epimerase used to convert (S)-1-benzylisoquinoline alkaloid to (R)-1-benzylisoquinoline alkaloid can be 75% or more identical to a given amino acid sequence listed in Table 1. For example, the amino acid sequence of such an epimerase can include an amino acid sequence that is at least 75% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to an amino acid sequence provided herein. Furthermore, in certain embodiments, an "identical" amino acid sequence includes at least 80%-99% identity at the amino acid level with the specific amino acid sequence. In some cases, an "identical" amino acid sequence includes at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or more, and in some particular cases at least 95%, 96%, 97%, 98%, and 99% identity at the amino acid level. In some cases, the amino acid sequence may be identical, but the DNA sequence has been modified, for example, to optimize codon usage for the host organism.

[0081] A genetically engineered host cell can be provided that produces an epimerase that converts (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids, where the epimerase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15. The epimerase produced in the genetically engineered host cell can be recovered and purified to form a biocatalyst. In some cases, the epimerase can be split into one or more enzymes. Furthermore, the one or more enzymes produced by the epimerase split can be recovered from the genetically engineered host cell. The one or more enzymes resulting from the epimerase split can also be used to catalyze the conversion of (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids. In particular, the one or more enzymes recovered from the genetically engineered host cell producing the epimerase can be used in a process for converting an (S)-1-benzylisoquinoline alkaloid to an (R)-1-benzylisoquinoline alkaloid. The process can include contacting the (S)-1-benzylisoquinoline alkaloid with an epimerase in an amount sufficient to convert the (S)-1-benzylisoquinoline alkaloid to an (R)-1-benzylisoquinoline alkaloid. In some examples, the (S)-1-benzylisoquinoline alkaloid can be contacted with the one or more enzymes in an amount sufficient to convert at least 5% of the (S)-1-benzylisoquinoline alkaloid to an (R)-1-benzylisoquinoline alkaloid.In a further example, the (S)-1-benzylisoquinoline alkaloid may be contacted with a sufficient amount of the one or more enzymes such that at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7% or 100% of the (S)-1-benzylisoquinoline alkaloid is converted to (R)-1-benzylisoquinoline alkaloid.

[0082] The one or more enzymes that can be used to convert an (S)-1-benzylisoquinoline alkaloid to an (R)-1-benzylisoquinoline alkaloid may be contacted with the (S)-1-benzylisoquinoline alkaloid in vitro. Additionally or alternatively, the one or more enzymes that can be used to convert an (S)-1-benzylisoquinoline alkaloid to an (R)-1-benzylisoquinoline alkaloid may be contacted with the (S)-1-benzylisoquinoline alkaloid in vivo. Furthermore, the one or more enzymes that can be used to convert an (S)-1-benzylisoquinoline alkaloid to an (R)-1-benzylisoquinoline alkaloid may be supplied to a cell harboring the (S)-1-benzylisoquinoline alkaloid or may be produced in a genetically engineered host cell.

[0083] In some examples, the method provides a genetically engineered host cell that produces an alkaloid product, wherein epimerization of an (S)-substrate to an (R)-product can constitute a key step in the production of the alkaloid product. In some examples, the alkaloid produced is an (R)-1-benzylisoquinoline alkaloid. In still other embodiments, the alkaloid produced is derived from an (R)-1-benzylisoquinoline alkaloid, such as a tetracyclic promorphinan alkaloid or a pentacyclic morphinan alkaloid. In another embodiment, the (S)-1-benzylisoquinoline alkaloid is an intermediate to the product of the genetically engineered host cell. In still other embodiments, the alkaloid product is selected from the group consisting of morphinan alkaloids and promorphinan alkaloids.

[0084] In some examples, the (S)-substrate is a (S)-1-benzylisoquinoline alkaloid selected from the group consisting of (S)-norreticuline, (S)-reticuline, (S)-tetrahydropapaverine, (S)-norcoclaurine, (S)-coclaurine, (S)-N-methylcoclaurine, (S)-3'-hydroxy-N-methylcoclaurine, (S)-norisoorientaline, (S)-orientaline, (S)-isoorientaline, (S)-norprotosinomenine, (S)-protosinomenine, (S)-norlaudanosoline, (S)-laudanosoline, (S)-4'-O-methyllaudanosoline, (S)-6-O-methylnorlaudanosoline, (S)-4'-O-methylnorlaudanosoline.

[0085] In some instances, the (S)-substrate is a compound of Formula I: TIFF0007730308000002.tif36128 or a salt thereof, wherein: R 1 , R 2 , R 3 and R 4 is independently selected from hydrogen and methyl; R 5 is selected from hydrogen, hydroxy and methoxy.

[0086] In some other cases, R 1 , R 2 , R 3 , R 4 and R 5 At least one of the is hydrogen.

[0087] In yet another example, the (S)-substrate is a compound of formula II: TIFF0007730308000003.tif33128 or a salt thereof, wherein: R 3 is selected from hydrogen and C1-C4 alkyl; R 6 and R 7 is independently, at each occurrence, selected from hydroxy, fluoro, chloro, bromo, carboxaldehyde, C1-C4 acyl, C1-C4 alkyl, and C1-C4 alkoxy; n is 0, 1, 2, 3 or 4; n' is 0, 1, 2, 3, 4 or 5.

[0088] When a bond is shown across a ring, this means that the substitution can be at any atom or position within the ring. For example, in Formula II shown above, the hydrogen of any -CH- of the 6-membered ring can be R 7 Replaced by -CR 7 - can be formed.

[0089] In some cases, R 6 and R 7 are independently methyl or methoxy. In some other examples, n and n' are independently 1 or 2. In still other embodiments, R 3 is hydrogen or methyl.

[0090] In some examples, the method provides a genetically engineered host cell that produces alkaloid products from (S)-reticuline.The epimerization of (S)-reticuline to (R)-reticuline can constitute the key step in the production of various alkaloid products from precursors.In some examples, the precursor is L-tyrosine or sugar (e.g., glucose).The various alkaloid products can include, but are not limited to, morphinan alkaloids and promorphinan alkaloids.

[0091] Any suitable carbon source can be used as a precursor to epimerized 1-benzylisoquinoline alkaloids. Suitable precursors can include, but are not limited to, monosaccharides (e.g., glucose, fructose, galactose, xylose), oligosaccharides (e.g., lactose, sucrose, raffinose), polysaccharides (e.g., starch, cellulose), or combinations thereof. In some examples, crude mixtures derived from renewable feedstocks can be used (e.g., corn steep liquor, sugar beet molasses, barley malt, biomass hydrolysis products). In still other embodiments, the carbon precursor can be a one-carbon compound (e.g., methanol, carbon dioxide) or a two-carbon compound (e.g., ethanol). In still other embodiments, other carbon-containing compounds, such as methylamine, glucosamine, and amino acids (e.g., L-tyrosine), can be used. In some examples, 1-benzylisoquinoline alkaloids, such as norlaudanosoline, laudanosoline, norreticuline, and reticuline, can be added directly to engineered host cells. In still further embodiments, the 1-benzylisoquinoline alkaloid can be added to the genetically engineered host cell as a single enantiomer (e.g., (S)-1-benzylisoquinoline alkaloid) or a mixture of enantiomers, such as a racemic mixture.

[0092] In some examples, the method provides epimerization of the stereocenter of a 1-benzylisoquinoline alkaloid or a derivative thereof. In a further aspect, the method includes contacting a 1-benzylisoquinoline alkaloid with at least one enzyme. The at least one enzyme can invert the stereochemical configuration of the stereocenter of the 1-benzylisoquinoline alkaloid or a derivative thereof to the opposite stereochemical configuration. In some examples, the at least one enzyme converts (S)-1-benzylisoquinoline alkaloid to (R)-1-benzylisoquinoline alkaloid. In some examples of the conversion of (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids using the at least one enzyme, the (S)-1-benzylisoquinoline alkaloid is selected from the group consisting of (S)-norreticuline, (S)-reticuline, (S)-tetrahydropapaverine, (S)-norcoclaurine, (S)-coclaurine, (S)-N-methylcoclaurine, (S)-3'-hydro The compound is selected from the group consisting of (S)-oxy-N-methylcoclaurine, (S)-norisoorientaline, (S)-orientaline, (S)-isoorientaline, (S)-norprotosinomenine, (S)-protosinomenine, (S)-norlaudanosoline, (S)-laudanosoline, (S)-4'-O-methyllaudanosoline, (S)-6-O-methylnorlaudanosoline and (S)-4'-O-methylnorlaudanosoline.

[0093] In yet other embodiments, the 1-benzylisoquinoline alkaloid being epimerized can contain two or more stereocenters, and only one of the two or more stereocenters is inverted to produce a diastereomer of the substrate (e.g., (S,R)-1-benzylisoquinoline alkaloid is converted to (R,R)-1-benzylisoquinoline alkaloid). In instances where only one stereocenter of the 1-benzylisoquinoline alkaloid is inverted upon contact with the at least one enzyme, the product is referred to as an epimer of the 1-benzylisoquinoline alkaloid.

[0094] In some examples, 1-benzylisoquinoline alkaloids are presented to the enzyme as one stereoisomer. In other examples, 1-benzylisoquinoline alkaloids are presented to the enzyme as a stereoisomer mixture. In still further embodiments, the stereoisomer mixture can be a racemic mixture. In other examples, the stereoisomer mixture can be enriched in one stereoisomer compared to another stereoisomer.

[0095] In some examples, 1-benzylisoquinoline alkaloids or derivatives thereof are recovered. In some examples, 1-benzylisoquinoline alkaloids are recovered from cell cultures. In yet further embodiments, the recovered 1-benzylisoquinoline alkaloids are enantiomerically enriched in one stereoisomer compared to the original mixture of 1-benzylisoquinoline alkaloids presented to the enzyme. In still further embodiments, the recovered 1-benzylisoquinoline alkaloid has an enantiomeric excess of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7% or 100%.

[0096] "Isomers" are different compounds with the same molecular formula. "Stereoisomers" are isomers that differ only in the way their atoms are arranged in space. "Enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of each other. A 1:1 mixture of a pair of enantiomers is a "racemic" mixture. "Diastereoisomers" or "diastereomers" are stereoisomers that have at least two asymmetric atoms but are not mirror images of each other. The term "epimer," as used herein, refers to compounds that have the same chemical formula but differ in the optical configuration at a specific position. For example, the (R,S) stereoisomer and the (S,S) stereoisomer of a compound are epimers of each other. In some cases, 1-benzylisoquinoline alkaloids are converted to their epimers (e.g., epi-1-benzylisoquinoline alkaloids). Absolute stereochemical configurations are designated according to the Cahn-Ingold-Prelog RS system. When a compound is a pure enantiomer, the stereochemical configuration at each chiral carbon may be designated as either R or S. Resolved compounds of unknown absolute configuration may be designated as (+) or (-) depending on the direction (dextrorotatory or levorotatory) they rotate linearly polarized light of the wavelength of the sodium D line. Certain compounds described herein contain one or more asymmetric centers and may therefore give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be designated as (R)- or (S)- with respect to absolute stereochemical configuration.

[0097] Table 1. Examples of partial and full-length amino acid sequences of CYP-COR fusion enzymes TIFF0007730308000004.tif148170TIFF0007730308000005.tif242166TIFF0007730308000006.tif242166TIFF00077303080 00007.tif242166TIFF0007730308000008.tif242166TIFF0007730308000009.tif242170TIFF0007730308000010.tif199170

[0098] BisBIA generation modification Some methods, processes, and systems provided herein describe the production of bisbenzylisoquinoline alkaloids (bisBIAs). BisBIAs are dimeric molecules that can be formed by a coupling reaction between two BIA monomers. In one example, a bisBIA can be formed by a carbon-oxygen coupling reaction. In another example, a bisBIA can be formed by a carbon-carbon coupling reaction. In some examples, a bisBIA dimeric molecule is a homodimer containing two identical BIA monomers. In one example, a genetically engineered host cell can produce a single BIA monomer. In such an example, the BIA monomer can form a homodimer when contacted with one or more coupling enzymes. In other examples, a bisBIA dimeric molecule is a heterodimer containing two different BIA monomers. For example, a bisBIA can be a heterodimer containing BIA monomers that are enantiomers of each other. In some examples, a genetically engineered host cell can produce more than one BIA monomer. In such instances, the BIA monomers can form homodimers and heterodimers when contacted with one or more coupling enzymes.

[0099] Some such methods, processes, and systems describing the production of bisBIAs may include genetically engineered host cells. In some examples, the genetically engineered host cells may be genetically engineered to produce BIA monomers that can be further used as building block molecules for the formation of bisBIAs. Examples of BIA monomers that can be used to form bisBIAs include coclaurine, N-methylcoclaurine, laudanine, norcoclaurine, norlaudanosoline, 6-O-methyl-norlaudanosoline, 3'-hydroxy-N-methylcoclaurine, 3'-hydroxycoclaurine, reticuline, norreticuline, norlaudanine, laudanosine, and papaverine. In particular, the genetically engineered host cells may synthesize BIA monomers from norcoclaurine or norlaudanosoline by expression of heterologous enzymes, such as O-methyltransferase, N-methyltransferase, and 3'-hydroxylase. Examples of O-methyltransferases include norcoclaurine 6-O-methyltransferase (6OMT) from Thalicrum flavum, Nelumbo nucifera, Populus euphratica, or another species. Further examples of O-methyltransferases include catechol O-methyltransferase (COMT) from humans (Homo sapiens), Mus musculus, Rattus norvegicus, Gorilla, or another species. Further examples of N-methyltransferases include coclaurine N-methyltransferase (CNMT) from T. flavum, N. nucifera, Aristolochia fimbriata, or another species. Examples of 3' hydroxylases may include N-methylcoclaurine 3'-hydroxylase (CYP80B1) from Eschscholzia californica, yellow larch, lotus or another species.

[0100] The genetically engineered host cells can produce either the (S) or the (R) enantiomer of any given BIA monomer. Additionally or alternatively, the genetically engineered host cells can produce a mixture of both enantiomers. The ratio of the (S) and (R) enantiomers can be determined by the substrate and product specificity of the enzyme or enzymes that synthesize the BIA monomer. Alternatively, the amount of each enantiomer present can be modified by expression of additional enzyme(s) that effect epimerization of one stereoisomer to another, as discussed above.

[0101] Such BIA monomers may be fused to a dimeric bisBIA scaffold. In particular, BIA monomers may be fused to a dimeric bisBIA scaffold using one or more enzymes produced by the engineered host cell. Additionally or alternatively, BIA monomers may be fused to a dimeric bisBIA scaffold using one or more enzymes supplied to the BIA monomers from a source external to the engineered host cell. The one or more enzymes may be used to form carbon-oxygen and / or carbon-carbon coupling reactions to fuse two BIA monomers at one, two, or three positions. In some cases, two BIA monomers may be linked by an ether bond. In some cases, a direct carbon-carbon bond may be used to link two BIA monomers. In some cases, the bisBIA formed by fusing two BIA monomers may contain one diphenyl ether bond. In some cases, two BIA monomers may be fused to form a bisBIA containing two diphenyl ether bonds. In some cases, a bisBIA formed from two BIA monomers can include three diphenyl ether linkages. In some cases, a bisBIA can include one diphenyl ether linkage and one benzyl phenyl ether linkage. In some cases, a bisBIA can include one benzyl phenyl ether linkage and two diphenyl ether linkages.

[0102] In one example, BIA monomers can be contacted with a sufficient amount of one or more enzymes that can be used to orchestrate a coupling reaction to fuse two BIA monomers such that at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or 100% of the BIA monomers are converted to bisBIA. The BIA monomers can be contacted in vitro with one or more enzymes that can be used to dimerize the BIA monomers into bisBIAs. Additionally or alternatively, the BIA monomers can be contacted in vivo with one or more enzymes that can be used to dimerize the BIA monomers into bisBIAs. Furthermore, the one or more bisBIA dimerization enzymes can be expressed in a host cell that produces the BIA monomers. Alternatively, the BIA monomers can be provided to a genetically engineered host cell that expresses the bisBIA dimerization enzyme. Alternatively, the one or more bisBIA dimerization enzymes can be provided to a cell that harbors the BIA monomers.

[0103] In some examples, the bisbenzylisoquinoline alkaloid is represented by formula Va-Vu: TIFF0007730308000011.tif242132TIFF0007730308000012.tif201144 or a salt thereof, wherein: R 1a , R 1b , R 2a and R 2b are independently selected from hydrogen and C1-C4 alkyl; R 3a , R 3b , R6a , R 6b , R 8a and R 8b is independently selected from hydrogen, hydroxy, fluoro, chloro, bromo, carboxaldehyde, C1-C4 acyl, C1-C4 alkyl, and C1-C4 alkoxy; R 4a and R 5a are independently selected from hydrogen and C1-C4 alkyl, or R 4a and R 5a are united to form a methylene bond; R 4b and R 5b are independently selected from hydrogen and C1-C4 alkyl, or R 4b and R 5b together form a methylene bond; and R 7a , R 7b and R 9a are independently selected from hydrogen and C1-C4 alkyl.

[0104] In some cases, R 1a and R 1b are each hydrogen; R 2a and R 2b are each methyl; R 3a and R 3b are each hydrogen; R 4a and R 5a are independently hydrogen or methyl; R 4b and R 5b are independently hydrogen or methyl, or R 4b and R 5b are united to form a methylene bond; R 6a , R 6b , R 8a and R 8b are each hydrogen; R 7a , R 7b and R 9a are independently hydrogen or methyl.

[0105] As shown above, bisBIA compounds of formulas Va, Vb, and Vd are formed by fusing two BIA monomers using one carbon-oxygen coupling reaction. Furthermore, bisBIA compounds of formulas Vc, Vf, and Vh are formed by fusing two BIA monomers using both one carbon-oxygen coupling reaction and one carbon-carbon coupling reaction. Furthermore, bisBIA compounds of formulas Ve, Vg, Vi, Vj, Vk, Vl, Vm, Vo, Vp, and Vq are formed by fusing two BIA monomers using two carbon-oxygen coupling reactions. A bisBIA compound of formula Vn is formed by fusing two BIA monomers using two carbon-oxygen coupling reactions and one carbon-carbon coupling reaction. Furthermore, a bisBIA compound of formula Vr is formed by fusing two BIA monomers using a three carbon-oxygen coupling reaction.

[0106] The one or more enzymes that can be used to construct the coupling reaction can include known cytochrome P450s, such as Berberis stolonifera CYP80A1, or similar cytochrome P450 enzymes from other plants that naturally synthesize the compound. Alternatively, the coupling reaction can be performed by a non-cytochrome P450 enzyme. The one or more enzymes that can be used to construct the coupling reaction can be genetically engineered to accept non-natural substrates. Thus, the one or more enzymes that can be used to construct the coupling reaction can be used to generate non-natural bisBIA molecules. In one example, the one or more enzymes can fuse a natural BIA monomer with a non-natural BIA monomer to generate a non-natural bisBIA molecule. In another example, the one or more enzymes can fuse two non-natural BIA monomers to generate a non-natural bisBIA molecule. Engineered enzyme strategies can be used to identify one or more enzymes that can be used to orchestrate coupling reactions that fuse BIA monomers to generate bisBIAs. In one example, enzyme engineering strategies can include site-directed mutagenesis, random mutagenesis and screening, DNA shuffling, and screening.

[0107] Once formed, the bisBIA may be further derivatized or modified. The bisBIA may be derivatized or modified using one or more enzymes produced by the genetically engineered host cell. In particular, the bisBIA may be derivatized or modified by contacting the bisBIA with one or more enzymes produced by the genetically engineered host cell. Additionally or alternatively, the bisBIA may be derivatized or modified by contacting the bisBIA with one or more enzymes provided to the bisBIA from a source external to the genetically engineered host cell. The one or more enzymes that can be used to derivatize or modify the bisBIA can be used to perform a tailoring reaction. Examples of tailoring reactions include oxidation, reduction, O-methylation, N-methylation, O-demethylation, acetylation, methylenedioxy bond formation, and O,O-demethylenation. The bisBIA may be derivatized or modified using one or more tailoring reactions.

[0108] Examples of tailoring reactions are shown in Table 8. In some examples, tailoring enzymes can be used to catalyze carbon-carbon coupling reactions performed on bisBIAs or their derivatives. Examples of tailoring enzymes that can be used to catalyze carbon-carbon coupling reactions include berberine bridge enzyme (BBE) from Papaver somniferum, California bluebell, Coptis japonica, Berberis stolonifer, Thalictrum flavum, or another species; salutaridine synthase (SalSyn) from Papaver somniferum or another species; and corituberine synthase (CorSyn) from Coptis somniferum or another species. Non-limiting examples of reactions that can be catalyzed by tailoring enzymes are shown in Scheme 2, where R a , R b , R c and R dare independently selected from hydrogen, hydroxy, fluoro, chloro, bromo, carboxaldehyde, C1-C4 acyl, C1-C4 alkyl, and C1-C4 alkoxy. a , R b and the carbon atoms to which they are attached optionally form a carbocyclic or heterocyclic ring. c , R d and the carbon atoms to which they are attached optionally form a carbocyclic or heterocyclic ring. TIFF0007730308000013.tif89128

[0109] In some instances, tailoring enzymes can be used to catalyze oxidation reactions carried out on bisBIA or its derivatives. Examples of tailoring enzymes that can be used to catalyze oxidation reactions include tetrahydroprotoberberine oxidase (STOX) from Coptis japonica, Argemone mexicana, Berberis wilsonae, or other species; dihydrobenzophenanthridine oxidase (DBOX) from Papaver somniferum or other species; methylstylopine hydroxylase (MSH) from Papaver somniferum or other species; and protopine 6-hydroxylase (P6H) from Papaver somniferum, California poppy, or other species.

[0110] Tailoring enzymes can also be used to catalyze the methylenedioxy bridge-forming reaction carried out on bisBIAs or their derivatives. Examples of tailoring enzymes that can be used to catalyze the methylenedioxy bridge-forming reaction include stylopine synthase (StySyn or STS) from opium poppy, poppy, poppy, or another species; cheilanthifoline synthase (CheSyn or CFS) from opium poppy, poppy, poppy, or another species; and canadine synthase (CAS) from yellow larch, Coptis chinensis, or another species.

[0111] In other examples, tailoring enzymes can be used to catalyze O-methylation reactions performed on bisBIAs or derivatives thereof. Examples of tailoring enzymes that can be used to catalyze O-methylation reactions include norcoclaurine 6-O-methyltransferase (6OMT) from opium poppy, yellow larch, Coptis chinensis, Papaver bracteatum, or another species; 3'hydroxy-N-methylcoclaurine 4'-O-methyltransferase (4'OMT) from opium poppy, yellow larch, Coptis chinensis, Papaver chinensis, or another species; reticuline 7-O-methyltransferase (7OMT) from opium poppy, yellow larch, Coptis chinensis, Papaver chinensis, or another species; and scourerine 9-O-methyltransferase (9OMT) from opium poppy, yellow larch, Coptis chinensis, Papaver chinensis, or another species.

[0112] Additionally, tailoring enzymes can be used to catalyze the N-methylation reaction carried out on bisBIA or its derivatives. Examples of tailoring enzymes that can be used to catalyze the N-methylation reaction include coclaurine N-methyltransferase (CNMT) from Papaver somniferum, Aubergine japonica, Coptis japonica, or another species; tetrahydroprotoberberine N-methyltransferase (TNMT) from Papaver somniferum, Aubergine poppy, or another species.

[0113] Additionally, tailoring enzymes can be used to catalyze the O-demethylation reaction carried out on bisBIA or its derivatives. Examples of tailoring enzymes that can be used to catalyze the O-demethylation reaction include thebaine demethylase (T6ODM) from opium poppy or another species; and codeine demethylase (CODM) from opium poppy or another species.

[0114] Tailoring enzymes can also be used to catalyze reduction reactions performed on bisBIA or its derivatives. Examples of tailoring enzymes that can be used to catalyze reduction reactions include salutaridine reductase (SalR) from Papaver somniferum, Papaver somniferum, or another species; codeinone reductase (COR) from Papaver somniferum or another species; and sanguinarine reductase (SanR) from California poppy or another species. In another example, tailoring enzymes can be used to catalyze acetylation reactions performed on bisBIA or its derivatives. One example of a tailoring enzyme that can be used to catalyze an acetylation reaction is salutaridine acetyltransferase (SalAT) from Papaver somniferum or another species.

[0115] O-demethylation Some methods, processes, and systems provided herein describe the conversion of a first benzylisoquinoline alkaloid to a second benzylisoquinoline alkaloid by removing an O-linked methyl group. Some such methods, processes, and systems may include genetically engineered host cells. In some examples, the conversion of a first benzylisoquinoline alkaloid to a second benzylisoquinoline alkaloid is a key step in the conversion of a substrate to a nor-opioid or nal-opioid. In some examples, the conversion of a first alkaloid to a second alkaloid involves a demethylase reaction.

[0116] 23 depicts an enzyme with opioid 3-O-demethylase activity according to an embodiment of the present invention, which can act on any morphinan alkaloid structure to remove a methyl group from the oxygen attached to the 3-carbon atom.

[0117] Examples of amino acid sequences of ODM enzymes are shown in Table 3. The amino acid sequence of an ODM used to convert a first alkaloid to a second alkaloid can be 75% or more identical to a given amino acid sequence listed in Table 3. For example, the amino acid sequence of such an epimerase can include an amino acid sequence that is at least 75% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to an amino acid sequence provided herein. Furthermore, in certain embodiments, an "identical" amino acid sequence includes at least 80%-99% identity at the amino acid level with the specific amino acid sequence. In some cases, an "identical" amino acid sequence includes at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or more, and in some particular cases at least 95%, 96%, 97%, 98%, and 99% identity at the amino acid level. In some cases, the amino acid sequence may be identical, but the DNA sequence has been modified, for example, to optimize codon usage for the host organism.

[0118] A genetically engineered host cell can be provided that produces an ODM that converts a first alkaloid into a second alkaloid, wherein the ODM comprises a given amino acid sequence listed in Table 3. A genetically engineered host cell can be provided that produces one or more ODM enzymes. The ODM produced in the genetically engineered host cell can be recovered and purified to form a biocatalyst. The process can include contacting a first alkaloid with an amount of ODM sufficient to convert the first alkaloid into a second alkaloid. In one example, the first alkaloid can be contacted with a sufficient amount of the one or more enzymes such that at least 5% of the first alkaloid is converted into a second alkaloid. In further examples, a first alkaloid may be contacted with a sufficient amount of the one or more enzymes such that at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7% or 100% of the first alkaloid is converted to a second alkaloid.

[0119] The one or more enzymes that can be used to convert a first alkaloid to a second alkaloid may be contacted with the first alkaloid in vitro. Additionally or alternatively, the one or more enzymes that can be used to convert a first alkaloid to a second alkaloid may be contacted with the first alkaloid in vivo. In some examples, the one or more enzymes that can be used to convert a first alkaloid to a second alkaloid may be provided to a cell harboring the first alkaloid. In some examples, the one or more enzymes that can be used to convert a first alkaloid to a second alkaloid may be produced within a genetically engineered host cell.

[0120] In some examples, the method provides a genetically engineered host cell that produces an alkaloid product, where O-demethylation of a substrate to a product can constitute a key step in the production of the alkaloid product. In some examples, the alkaloid produced is a nor-opioid or a nar-opioid. In still other embodiments, the alkaloid produced is derived from a nor-opioid or a nar-opioid. In other embodiments, the first alkaloid is an intermediate to the product of the genetically engineered host cell. In still other embodiments, the alkaloid product is selected from the group consisting of morphine, oxymorphine, oripavine, hydromorphone, dihydromorphine, 14-hydroxymorphine, morphinone, and 14-hydroxymorphinone.

[0121] In some examples, the substrate alkaloid is an opioid selected from the group consisting of codeine, oxycodone, thebaine, hydrocodone, dihydrocodeine, 14-hydroxycodeine, codeinone, and 14-hydroxycodeinone.

[0122] N-demethylation modification Some methods, processes, and systems provided herein describe the conversion of a first alkaloid to a second alkaloid by removing an N-linked methyl group. Some such methods, processes, and systems may include genetically engineered host cells. In some instances, the conversion of a first alkaloid to a second alkaloid is a key step in the conversion of a substrate to a nor-opioid or nal-opioid. In some instances, the conversion of a first alkaloid to a second alkaloid involves a demethylase reaction.

[0123] 24 depicts an enzyme with opioid N-demethylase activity, according to an embodiment of the present invention. Specifically, the enzyme can act on any morphinan alkaloid structure to remove a methyl group from its nitrogen.

[0124] Examples of amino acid sequences of N-demethylase enzymes that can be used to convert a first alkaloid to a second alkaloid are shown in Table 4. The amino acid sequence of an NDM used to convert a first alkaloid to a second alkaloid can be 75% or more identical to a given amino acid sequence listed in Table 4. For example, the amino acid sequence of such an epimerase can include an amino acid sequence that is at least 75% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to an amino acid sequence provided herein. Furthermore, in certain embodiments, an "identical" amino acid sequence includes at least 80%-99% identity at the amino acid level with the specific amino acid sequence. In some cases, an "identical" amino acid sequence includes at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or more, and in some particular cases at least 95%, 96%, 97%, 98%, and 99% identity at the amino acid level. In some cases, the amino acid sequence may be identical, but the DNA sequence has been modified, for example, to optimize codon usage for the host organism.

[0125] Genetically engineered host cells can be provided that produce NDM that converts a first alkaloid to a second alkaloid, wherein the NDM comprises an amino acid sequence listed in Table 4. Genetically engineered host cells can be provided that produce one or more NDM enzymes. The NDM produced in the genetically engineered host cells can be recovered and purified to form a biocatalyst. The process can include contacting a first alkaloid with a sufficient amount of NDM to convert the first alkaloid to a second alkaloid. In one example, the first alkaloid can be contacted with a sufficient amount of the one or more enzymes such that at least 5% of the first alkaloid is converted to a second alkaloid. In further examples, a first alkaloid may be contacted with a sufficient amount of the one or more enzymes such that at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7% or 100% of the first alkaloid is converted to a second alkaloid.

[0126] The one or more enzymes that can be used to convert a first alkaloid to a second alkaloid may be contacted with the first alkaloid in vitro. Additionally or alternatively, the one or more enzymes that can be used to convert a first alkaloid to a second alkaloid may be contacted with the first alkaloid in vivo. In some examples, the one or more enzymes that can be used to convert a first alkaloid to a second alkaloid may be provided to a cell harboring the first alkaloid. In some examples, the one or more enzymes that can be used to convert a first alkaloid to a second alkaloid may be produced within a genetically engineered host cell.

[0127] In some instances, the method provides a genetically engineered host cell that produces an alkaloid product, where N-demethylation of a substrate to a product can constitute a key step in the production of the alkaloid product. In some instances, the alkaloid produced is a nor-opioid or a nal-opioid. In yet other embodiments, the alkaloid produced is derived from a nor-opioid or a nal-opioid. In other embodiments, the first alkaloid is an intermediate to the product of the genetically engineered host cell. In still other embodiments, the alkaloid product is selected from the group consisting of norcodeine, noroxycodone, northebaine, norhydrocodone, nordihydro-codeine, nor-14-hydroxy-codeine, norcodeinone, nor-14-hydroxy-codeinone, normorphine, noroxymorphone, norolipavine, norhydro-morphone, nordihydro-morphine, nor-14-hydroxy-morphine, normorphinone, and nor-14-hydroxy-morphinone.

[0128] In some examples, the substrate alkaloid is an opioid selected from the group consisting of codeine, oxycodone, thebaine, hydrocodone, dihydrocodeine, 14-hydroxycodeine, codeinone and 14-hydroxycodeinone, morphine, oxymorphone, oripavine, hydromorphone, dihydromorphine, 14-hydroxy-morphine, morphinone, or 14-hydroxy-morphinone.

[0129] N-methyltransferase modification Some methods, processes, and systems provided herein describe the conversion of a first alkaloid to a second alkaloid by the addition of an N-linked side group. Some methods, processes, and systems provided herein describe the conversion of a first alkaloid to a second alkaloid by the transfer of a side group from a co-substrate to the first alkaloid. Some such methods, processes, and systems may include genetically engineered host cells. In some examples, the conversion of a first alkaloid to a second alkaloid is a key step in the conversion of a substrate to a n-opioid. In some examples, the conversion of a first alkaloid to a second alkaloid involves a methyltransferase reaction.

[0130] Figure 25 shows an enzyme with N-methyltransferase activity according to an embodiment of the present invention. Specifically, the enzyme can act on any morphinan alkaloid structure, adding a methyl group or other carbon moiety to the nitrogen. S-adenosylmethionine (SAM) can act as a donor of functional groups (methyl, allyl, cyclopropylmethyl, or other).

[0131] Examples of amino acid sequences of NMT enzymes are shown in Table 5. The amino acid sequence of an NMT used to convert a first alkaloid to a second alkaloid can be 75% or more identical to a given amino acid sequence listed in Table 5. For example, the amino acid sequence of such an epimerase can comprise an amino acid sequence that is at least 75% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to an amino acid sequence provided herein. Furthermore, in certain embodiments, an "identical" amino acid sequence comprises at least 80%-99% identity at the amino acid level with the specific amino acid sequence. In some cases, an "identical" amino acid sequence includes at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or more, and in some particular cases at least 95%, 96%, 97%, 98%, and 99% identity at the amino acid level. In some cases, the amino acid sequence may be identical, but the DNA sequence has been modified, for example, to optimize codon usage for the host organism.

[0132] Genetically engineered host cells can be provided that produce an NMT that converts a first alkaloid to a second alkaloid, wherein the NMT comprises an amino acid sequence set forth in Table 5. Genetically engineered host cells can be provided that produce one or more NMT enzymes. The NMT produced in the genetically engineered host cells can be recovered and purified to form a biocatalyst. The process can include contacting a first alkaloid with an amount of NMT sufficient to convert the first alkaloid to a second alkaloid. In one example, the first alkaloid can be contacted with a sufficient amount of the one or more enzymes such that at least 5% of the first alkaloid is converted to a second alkaloid. In further examples, a first alkaloid may be contacted with a sufficient amount of the one or more enzymes such that at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7% or 100% of the first alkaloid is converted to a second alkaloid.

[0133] The one or more enzymes that can be used to convert a first alkaloid to a second alkaloid may be contacted with the first alkaloid in vitro. Additionally or alternatively, the one or more enzymes that can be used to convert a first alkaloid to a second alkaloid may be contacted with the first alkaloid in vivo. In some examples, the one or more enzymes that can be used to convert a first alkaloid to a second alkaloid may be provided to a cell harboring the first alkaloid. In some examples, the one or more enzymes that can be used to convert a first alkaloid to a second alkaloid may be produced within a genetically engineered host cell.

[0134] In some examples, the method provides a genetically engineered host cell that produces an alkaloid product, wherein an N-methyltransferase of a substrate to a product can constitute a key step in the production of the alkaloid product. In some examples, the alkaloid produced is a nor-opioid. In yet other embodiments, the alkaloid produced is a nor-opioid or is derived from a nal-opioid. In other embodiments, the first alkaloid is an intermediate of the genetically engineered host cell to the product. In yet other embodiments, the alkaloid product is selected from the group including naloxone, naltrexone, and nalmefene.

[0135] In some examples, the substrate alkaloid is an opioid selected from the group consisting of norcodeine, noroxycodone, northebaine, norhydrocodone, nordihydro-codeine, nor-14-hydroxy-codeine, norcodeinone, nor-14-hydroxy-codeinone, normorphine, noroxymorphone, norolipavine, norhydro-morphone, nordihydro-morphine, nor-14-hydroxy-morphine, normorphinone, and nor-14-hydroxy-morphinone. In some examples, the co-substrate is S-adenosylmethionine, allyl-S-adenosylmethionine, or cyclopropylmethyl-S-adenosylmethionine.

[0136] Heterologous coding sequences In some examples, the genetically engineered host cell contains one or more heterologous coding sequences (e.g., two or more, three or more, four or more, five or more) encoding an activity(ies) that enables the genetically engineered host cell to produce a desired enzyme of interest and / or a BIA of interest, such as those described herein. As used herein, the term "heterologous coding sequence" refers to any polynucleotide that encodes or ultimately encodes a peptide or protein or its equivalent amino acid sequence, e.g., an enzyme, that is not normally present in the host organism but can be expressed in the cells of the host under appropriate conditions. Thus, a "heterologous coding sequence" includes multiple copies of a coding sequence normally present in the host cell, causing the cell to express additional copies of the coding sequence not normally present in the cell. The heterologous coding sequence can be RNA or any form thereof, such as mRNA, DNA or any form thereof, such as cDNA, or an RNA / DNA hybrid. Coding sequences of interest include, but are not limited to, full-length transcription units containing features such as coding sequences, introns, promoter regions, 3'-UTRs and enhancer regions.

[0137] In addition, genetically engineered host cells can be modified to have one or more genetic modifications to accommodate heterologous coding sequences.Natural host genome modifications include, but are not limited to, genome modifications to reduce or eliminate the expression of specific proteins that can interfere with desired pathways.The presence of such natural proteins can rapidly convert one of the intermediates or final products of the pathway into metabolic products or other compounds that cannot be used in the desired pathway.Therefore, if the activity of natural enzymes is reduced or completely eliminated, the resulting intermediates can be more easily used for incorporation into desired products.

[0138] Heterologous coding sequences include, but are not limited to, sequences encoding enzymes that are either wild-type sequences or equivalent sequences normally responsible for the production of the BIA of interest in plants. In some cases, the enzyme encoded by the heterologous sequence can be any enzyme in the 1-BIA pathway and can be derived from any convenient source. The choice and number of enzymes encoded by the heterologous coding sequence in a particular synthetic pathway can be selected based on the desired product. In certain embodiments, the host cell can contain one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or even fifteen or more heterologous coding sequences, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 heterologous coding sequences.

[0139] As used herein, the term "heterologous coding sequence" also encompasses the coding portion of a peptide or enzyme, i.e., the cDNA or mRNA sequence of the peptide or enzyme, as well as the coding portion of a full-length transcription unit, i.e., a gene including introns and exons, as well as "codon-optimized" sequences, truncated sequences, or other forms of modified sequences that encode an enzyme or an equivalent amino acid sequence, provided that the equivalent amino acid sequence results in the production of a functional protein. Such equivalent amino acid sequences may have one or more amino acid deletions, which may be N-terminal, C-terminal, or internal. Truncated forms are contemplated as long as they retain the catalytic activity set forth herein. Fusions of two or more enzymes to facilitate metabolite transfer within a pathway are also contemplated, provided that catalytic activity is maintained.

[0140] Operable fragments, mutants, or truncated forms can be identified by modeling and / or screening. In some cases, this is done, for example, by stepwise deletion of N-terminal, C-terminal, or internal regions of the protein, followed by analysis of the resulting derivatives for their activity against the native sequence for the desired reaction. If the derivative performs this function, it is considered to constitute an equivalent derivative of the enzyme.

[0141] In some examples, some heterologous proteins may exhibit incorrect processing when expressed in a recombinant host. For example, plant proteins such as cytochrome P450 enzymes expressed in a microbial production host may have incorrect processing issues. In particular, salutaridine synthase may undergo N-linked glycosylation when heterologously expressed in yeast. This N-linked glycosylation is not observed in plants and may indicate incorrect N-terminal selection of the developing SalSyn transcript, reducing the activity of the enzyme in a heterologous microbial host. In such examples, protein engineering directed at correcting the N-terminal selection of the developing transcript to eliminate the N-linked glycosylation pattern may result in improved activity of the salutaridine synthase enzyme in a recombinant production host. This is further illustrated in Example 8 below.

[0142] Some aspects of the present invention also relate to heterologous coding sequences encoding amino acid sequences equivalent to the native amino acid sequences of various enzymes. An "equivalent" amino acid sequence is defined as an amino acid sequence that is not identical to a specific amino acid sequence but contains at least some amino acid changes (deletions, substitutions, inversions, insertions, etc.) that do not essentially affect the biological activity of the protein when used for the desired purpose, compared to the similar activity of the specific amino acid sequence. In the case of an epimerase, biological activity refers to its catalytic activity. Equivalent sequences are also intended to encompass those that have been genetically engineered and / or evolved to have properties different from the original amino acid sequence. Properties of interest that can be varied include catalytic activity, substrate specificity, selectivity, stability, solubility, localization, etc.

[0143] In some instances, expression of each type of enzyme is increased by additional gene copies (i.e., multiple copies), thereby increasing accumulation of intermediates and / or production of the BIA of interest. Some embodiments of the present invention include increasing production of the BIA of interest in a host cell by co-expressing multiple species variants of one or more enzymes. In some cases, additional gene copies of one or more enzymes are included in the host cell. Any convenient method can be used, for example, multiple copies of heterologous coding sequences for the enzymes in the host cell.

[0144] In some cases, the genetically engineered host cell contains multiple copies of the heterologous coding sequence of the enzyme, for example, two or more, three or more, four or more, five or more, or even ten or more copies. In certain embodiments, the genetically engineered host cell contains multiple copies of the heterologous coding sequence of one or more enzymes, for example, two or more, three or more, four or more, etc. In some cases, the multiple copies of the heterologous coding sequence of the enzyme are derived from two or more different organismal sources compared to the host cell. For example, the genetically engineered host cell may contain multiple copies of a single heterologous coding sequence, each of which is derived from a different organismal source. Therefore, each copy may contain some diversity in the explicit sequence based on the interspecies differences of the enzyme of interest encoded by the heterologous coding sequence.

[0145] The culture medium of the genetically engineered host cells can be sampled and monitored for the production of the BIA of interest. The BIA of interest can be observed and measured using any convenient method. Methods of interest include, but are not limited to, LC-MS methods (e.g., as described herein), in which the sample of interest is analyzed by comparison with a standard compound of known quantity. In addition, there are other methods by which the BIA of interest can be observed and / or measured. Examples of alternative methods for observing and / or measuring BIA include, among others, GC-MS, UV-vis spectroscopy, NMR, LC-NMR, LC-UV, TLC, and capillary electrophoresis. The identity can be confirmed, for example, by m / z and MS / MS fragmentation pattern, and the quantification or measurement of the compound can be performed by EIC MS peak analysis by referring to peaks in the LC diagram of known retention times and / or corresponding LC-MS analysis of a standard of known quantity of the compound.

[0146] Furthermore, the culture of genetically engineered host cells can be sampled and monitored for the production of an enzyme of interest, such as a CYP-COR enzyme.The enzyme of interest can be observed and measured using any convenient method.Methods of interest include enzyme activity assay, polyacrylamide gel electrophoresis, carbon monoxide spectroscopy and Western blot analysis.

[0147] method Methods for culturing host cells for BIA production As summarized above, some aspects of the present invention include methods for preparing nor-opioid and nar-opioid BIAs of interest. Additionally, some aspects of the present invention include methods for preparing enzymes of interest. Thus, some aspects of the present invention include culturing genetically engineered host cells under conditions where one or more host cell modifications (e.g., as described herein) are functionally expressed, causing the cells to convert a starting compound of interest into a product, the nor-opioid and / or nar-opioid BIA of interest. Also provided are methods whereby genetically engineered host cells are cultured under conditions suitable for producing one or more heterologous coding sequences, causing the starting compound of interest to be functionally expressed into a product, the enzyme or protein, that converts the starting compound of interest into a nor-opioid and / or nar-opioid BIA of interest. In one example, the method is a method for preparing a BIA of a nor-opioid and / or nar-opioid of interest, comprising culturing a genetically engineered host cell (e.g., as described herein); adding a starting compound to the cell culture; and recovering the nor-opioid and / or nar-opioid from the cell culture. In some examples, the method is a method for preparing an enzyme, comprising culturing a genetically engineered host cell (e.g., as described herein); adding a starting compound to the cell culture; and recovering the enzyme from the cell culture.

[0148] The fermentation medium may contain an appropriate carbon substrate. Carbon sources suitable for carrying out the methods of the present disclosure may include a wide variety of carbon-containing substrates. Suitable substrates may include, but are not limited to, monosaccharides (e.g., glucose, fructose, galactose, xylose), oligosaccharides (e.g., lactose, sucrose, raffinose), polysaccharides (e.g., starch, cellulose), or combinations thereof. In some cases, crude mixtures derived from renewable feedstocks may be used (e.g., corn steep liquor, sugar beet molasses, barley malt). In some cases, the carbon substrate may be a one-carbon substrate (e.g., methanol, carbon dioxide) or a two-carbon substrate (e.g., ethanol). In other cases, other carbon-containing compounds, such as methylamine, glucosamine, and amino acids, may be used.

[0149] Any convenient method for culturing genetically engineered host cells can be used to produce nor-opioid and / or nar-opioid BIAs of interest. The specific protocol used can vary, for example, depending on the genetically engineered host cells, heterologous coding sequences, enzymes of interest, and nor-opioid and / or nar-opioid BIAs of interest. The cells can be present in any convenient environment, for example, an environment in which the cells can express one or more functional heterologous enzymes. In some embodiments, the cells are cultured under conditions that induce enzyme expression and with appropriate substrates available to enable in vivo production of nor-opioid and / or nar-opioid BIAs of interest. In some embodiments, the functional enzymes are extracted from the genetically engineered host for in vitro production of nor-opioid and / or nar-opioid BIAs of interest. In some examples, the genetically engineered host cells are returned to a multicellular host organism. The genetically engineered host cells can be in any growth phase, including but not limited to stationary and logarithmic growth phases, and the culture itself can be continuous or batch culture.

[0150] The cells may be cultured in a suitable fermentation medium at a temperature of 14-40°C. The cells may be cultured with shaking at any convenient speed (e.g., 200 rpm). The cells may be cultured at a suitable pH. A suitable pH range for this fermentation may be pH 5-9. Fermentation may be carried out under aerobic, anaerobic, or microaerobic conditions. Any suitable growth medium may be used. Suitable growth media may include, but are not limited to, common commercially prepared media, such as synthetic defined (SD) minimal medium or yeast extract peptone dextrose (YEPD) enriched medium. Any other enriched, defined, or synthetic growth medium appropriate for the microorganism may also be used.

[0151] Cells can be cultured in vessels of essentially any size and shape. Examples of vessels suitable for carrying out the methods of the present disclosure include, but are not limited to, multi-well shaker plates, test tubes, flasks (baffled and non-baffled), and bioreactors. Culture volumes can range from 10 microliters to more than 10,000 liters.

[0152] The growth medium may include the addition of agents known to modulate metabolism in a manner favorable for alkaloid production. In one non-limiting example, cyclic adenosine 2'3'-monophosphate may be added to the growth medium to modulate catabolite repression.

[0153] Any convenient cell culture conditions for a particular cell type may be used. In certain embodiments, host cells containing one or more modifications are cultured under standard or easily optimized conditions using standard cell culture media and supplements. As an example, a standard growth medium, if no selective pressure for plasmid maintenance is required, may contain 20 g / L yeast extract, 10 g / L peptone, and 20 g / L dextrose (YPD). Plasmid-containing host cells are cultured in synthetic complete (SC) medium containing 1.7 g / L yeast nitrogen base, 5 g / L ammonium sulfate, and 20 g / L dextrose, supplemented with the appropriate amino acids required for growth and selection. Alternative carbon sources that may be useful for inducible enzyme expression include, but are not limited to, sucrose, raffinose, and galactose. Cells are cultured in the laboratory in vessels, e.g., test tubes or flasks, in volumes ranging from 1 to 1000 mL, or in larger volumes, with shaking at any convenient temperature (e.g., 30°C) and at any convenient speed (e.g., 200 rpm).

[0154] The culture volume may be scaled up for cultivation in larger fermentors, for example, as part of an industrial process. Industrial fermentation processes may be carried out under closed-system batch, fed-batch, or continuous constant-compound conditions, or any suitable fermentation mode. In some cases, cells may be immobilized on a substrate as a whole-cell catalyst and subjected to fermentation conditions for alkaloid production.

[0155] Batch fermentation is a closed system in which the composition of the medium is set at the beginning of the fermentation and is not changed during the fermentation process. The desired organisms are inoculated into the medium at the start of the fermentation. In some cases, batch fermentation is performed with changes made to the system to control factors such as pH and oxygen concentration (but not carbon). In this type of fermentation system, the biomass and metabolite composition of the system changes continuously during the fermentation process. Cells typically progress through a lag phase, then a logarithmic phase (high growth rate), then a stationary phase (growth rate slows or stops), and finally a death phase (if left untreated).

[0156] Continuous fermentation is an open system in which a defined fermentation medium is continuously added to a bioreactor and an equal amount of fermentation medium is continuously removed from the vessel for processing. Continuous fermentation systems are typically operated to maintain steady-state growth conditions, so cell loss due to medium removal must be balanced by the growth rate during fermentation. Continuous fermentation is typically operated under conditions where cells are at a high, constant cell density. Continuous fermentation allows for modulation of one or more factors that affect the desired product concentration and / or cell growth.

[0157] Liquid media may include, but are not limited to, enriched or synthetic defined media with additional components as described above. Media components may be dissolved in water and sterilized by heat, pressure, filtration, radiation, chemicals, or any combination thereof. Some media components may be prepared separately, sterilized, and then combined in a fermenter. The culture medium may be buffered to help maintain a constant pH throughout fermentation.

[0158] Process parameters such as temperature, dissolved oxygen, pH, agitation, aeration rate, and cell density can be monitored or controlled during the fermentation process. For example, the temperature of the fermentation process can be monitored by a temperature probe immersed in the culture medium. The culture temperature can be controlled at a set point by adjusting the jacket temperature. Water can be cooled in an external chiller and then flowed into the bioreactor's control tower and circulated through the jacket at the temperature needed to maintain the set point temperature in the vessel.

[0159] Furthermore, gas flow parameters can be monitored during the fermentation process. For example, gas can be flowed through a sparger into the medium. Suitable gases for the method of the present disclosure can include compressed air, oxygen, and nitrogen. The gas flow can be at a fixed rate or can be adjusted to maintain a set point of dissolved oxygen.

[0160] The pH of the culture medium can also be monitored. In one example, the pH can be monitored by a pH probe immersed in the culture medium inside the tank. When pH control is performed, the pH can be adjusted by an acid pump and a base pump that add each solution to the medium at the required rate. The acid solution used to control the pH can be sulfuric acid or hydrochloric acid. The base solution used to control the pH can be sodium hydroxide, potassium hydroxide, or ammonium hydroxide.

[0161] Furthermore, dissolved oxygen in the culture medium can be monitored by a dissolved oxygen probe immersed in the culture medium. When performing dissolved oxygen control, the oxygen level can be adjusted by increasing or decreasing the agitation speed. The dissolved oxygen level can also be adjusted by increasing or decreasing the gas flow rate. The gas can be compressed air, oxygen, or nitrogen.

[0162] The agitation rate can also be monitored during the fermentation process. In one example, an agitator can be driven by a stirrer motor. The agitator speed can be set at a consistent rpm throughout the fermentation or can be dynamically adjusted to maintain a set dissolved oxygen level.

[0163] Additionally, turbidity can be monitored during the fermentation process. In one example, cell density can be measured using a turbidity probe. Alternatively, cell density can be measured by taking samples from the bioreactor and analyzing them with a spectrophotometer. Additionally, samples can be removed from the bioreactor at intervals using a sterile sampling device. Samples can be analyzed for alkaloids produced by the host cells. Samples can also be analyzed for other metabolites and sugars, depletion of culture medium components, or cell density.

[0164] In another example, feedstock parameters can be monitored during the fermentation process, particularly feedstocks such as sugars and other carbon sources, nutrients, and cofactors that can be added to the fermentation using external pumps. Other components can also be added during the fermentation, including, but not limited to, antifoam agents, salts, chelating agents, surfactants, and organic liquids.

[0165] Any convenient codon optimization technique for optimizing expression of heterologous polynucleotides in host cells can be adapted for use in the subject host cells and methods, see, e.g., Gustafsson C., et al. (2004) Trends Biotechnol, 22, 346-353, which is incorporated by reference in its entirety.

[0166] The subject method may also include adding a starting compound to the cell culture. Any convenient method of addition may be adapted for use in the subject method. The cell culture may be supplemented with a sufficient amount of the starting material of interest (e.g., as described herein), e.g., in the mM-μM range, e.g., about 1-5 mM of starting compound. It will be understood that the amount of starting material added, the timing and rate of addition, the form of the added material, etc., may vary depending on various factors. The starting material may be added neat or pre-dissolved in an appropriate solvent (e.g., cell culture medium, water, or an organic solvent). The starting material may be added in a concentrated form (e.g., 10-fold the desired concentration) to minimize dilution of the cell culture medium upon addition. The starting material may be added in one or more batches, or by continuous addition over an extended period of time (e.g., several hours or days).

[0167] Method for isolating a product from a fermentation medium The subject method may also include recovering the nor-opioid and / or nar-opioid BIA of interest from the cell culture. Any convenient method of separation and isolation (e.g., chromatography or precipitation) may be adapted for use in the subject method for recovering the nor-opioid and / or nar-opioid BIA of interest from the cell culture. Filtration methods may be used to separate the soluble fraction of the cell culture from the insoluble fraction. In some cases, liquid chromatography methods (e.g., reverse-phase HPLC, size exclusion, or normal-phase chromatography) may be used to separate the BIA of interest from other soluble components of the cell culture. In some cases, extraction methods (e.g., liquid extraction, pH-based purification, solid-phase extraction, affinity chromatography, ion exchange, etc.) may be used to separate the nor-opioid and / or nar-opioid BIA of interest from other components of the cell culture.

[0168] The produced alkaloids can be isolated from the fermentation medium using methods known in the art. Multiple recovery steps can be performed immediately after (or in some cases during) fermentation with initial recovery of the desired product. Such steps can separate the alkaloids (e.g., nor- or nal-opioids) from cells, cell debris, and waste products, while other nutrients, sugars, and organic molecules may remain in the spent culture medium. This process can be used to obtain nor- or nal-opioid-enriched products.

[0169] In an example, a product stream having a nor-opioid or nar-opioid product is formed by feeding genetically engineered yeast cells and a feedstock comprising nutrients and water into a batch reactor. Specifically, the genetically engineered yeast cells can be subjected to fermentation by incubating the genetically engineered yeast cells for a period of at least about 5 minutes to produce a solution comprising the nor-opioid or nar-opioid product and cellular material. Once the genetically engineered yeast cells have been subjected to fermentation, at least one separation unit can be used to separate the nor-opioid or nar-opioid product from the cellular material, resulting in a product stream comprising the nor-opioid or nar-opioid product. Specifically, the product stream can include the nor-opioid or nar-opioid product and additional components, such as clarified yeast culture medium. Additionally, the nor-opioid or nar-opioid product can include one or more nor-opioids or nar-opioids of interest, such as one or more nor-opioid or nar-opioid compounds.

[0170] Various methods can be used to remove cells from bioreactor media containing the enzyme of interest and / or nor- or nal-opioid. In one example, cells can be removed by settling over time. This settling process can be accelerated by cooling or by adding a clarifying agent, such as silica. The spent culture medium can then be siphoned from the top of the reactor, or the cells can be decanted from the bottom of the reactor. Alternatively, cells can be removed by filtration through a filter, membrane, or other porous material. Cells can also be removed by centrifugation, for example, by continuous flow centrifugation, or by using a continuous extraction device.

[0171] Cells can be removed from a bioreactor medium containing a BIA of interest, such as naloxone or naltrexone, using a variety of methods. In one example, cells can be removed by settling over time. This settling process can be accelerated by cooling or by adding a clarifying agent, such as silica. The spent culture medium can then be siphoned from the top of the reactor, or the cells can be decanted from the bottom of the reactor. Alternatively, cells can be removed by filtration through a filter, membrane, or other porous material. Cells can also be removed by centrifugation, for example, by continuous flow centrifugation, or by using a continuous extraction device.

[0172] If some valuable nor-opioid and / or nal-opioid BIAs of interest are present inside the cell, the cell can be permeabilized or lysed, and cell debris can be removed by any of the above methods.The agent used to permeabilize the cell can include, but is not limited to, organic solvents (e.g., DMSO) or salts (e.g., lithium acetate).Methods for lysing the cell can include adding detergents, such as sodium dodecyl sulfate, or mechanical disruption by bead milling or ultrasonic treatment.

[0173] The nor-opioid and / or nal-opioid BIAs of interest can be extracted from clarified spent culture medium by liquid-liquid extraction using an organic liquid that is immiscible with the aqueous culture medium. In one example, the use of liquid-liquid extraction can be used in addition to other processing steps. Examples of suitable organic liquids include, but are not limited to, isopropyl myristate, ethyl acetate, chloroform, butyl acetate, methyl isobutyl ketone, methyl oleate, toluene, oleyl alcohol, and ethyl butyrate. The organic liquid can be added in as little as 10% or as much as 100% of the volume of the aqueous medium.

[0174] In some cases, the organic liquid may be added at the beginning of fermentation or at any time during fermentation. This extractive fermentation process may increase the yield of the nor-opioid and / or nal-opioid BIA of interest from the host cells by continuously removing the nor-opioid and / or nal-opioid into the organic phase.

[0175] Agitation may cause the organic phase to form an emulsion with the aqueous culture medium. Methods for encouraging separation of the two phases into their respective layers may include, but are not limited to, adding a demulsifier or nucleating agent or adjusting the pH. The emulsion may also be centrifuged, for example, using a continuous conical plate centrifuge, to separate the two phases.

[0176] Alternatively, the organic phase may be isolated from the aqueous culture medium so that it can be physically removed after extraction, for example by encapsulation within a solvent film.

[0177] In one example, the BIA of the nor-opioid and / or nar-opioid of interest can be extracted from the fermentation medium using adsorption methods. In one example, the nor-opioid or nar-opioid of interest can be extracted from the clarified spent culture medium by adding a resin, such as Amberlite® XAD4, or another agent that removes nor-opioids or nar-opioids by adsorption. The nor-opioid or nar-opioid of interest can then be released from the resin using an organic solvent. Examples of suitable organic solvents include, but are not limited to, methanol, ethanol, ethyl acetate, or acetone.

[0178] Alternatively, the nor- or nal-opioid of interest may be extracted from the fermentation medium using filtration. At high pH, ​​the nor- or nal-opioid of interest may form a crystal-like precipitate within the bioreactor. This precipitate may be directly removed by filtration through a filter, membrane, or other porous material. Alternatively, the precipitate may be collected by centrifugation and / or decantation.

[0179] The extraction methods described above can be performed either in situ (within the bioreactor) or ex situ (e.g., in an external loop where the medium flows out of the bioreactor, is contacted with an extractant, and then recycled back into the vessel. Alternatively, the extraction method can be performed using clarified medium removed from the bioreactor vessel after fermentation has terminated.

[0180] Process for purifying products from alkaloid-rich solutions Subsequent purification steps can involve treating the post-fermentation solution enriched in the nor-opioid or nal-opioid product of interest using methods known in the art to recover the individual product species of interest in high purity.

[0181] In one example, the nor-opioid or nal-opioid of interest extracted into the organic phase can be transferred to an aqueous solution. In some cases, the organic solvent can be evaporated by heat and / or vacuum, and the resulting powder can be dissolved in an aqueous solution of an appropriate pH. In a further example, the BIA of interest can be extracted from the organic phase by adding an aqueous solution of an appropriate pH that promotes extraction of the nor-opioid or nal-opioid of interest into the aqueous phase. The aqueous phase can then be removed by decantation, centrifugation, or another method.

[0182] The nor-opioid- or nar-opioid-containing solution can be further treated to remove metals, for example, by treatment with a suitable chelating agent. The nor-opioid- or nar-opioid-containing solution of interest can be further treated by precipitation to remove other impurities, such as proteins and DNA. In one example, the nor-opioid- or nar-opioid-containing solution of interest is treated with a suitable precipitating agent, for example, ethanol, methanol, acetone, or isopropanol. In an alternative example, DNA and proteins can be removed by dialysis or other size-exclusion methods to separate small alkaloids from contaminating biopolymers.

[0183] In a further example, a solution containing a nor-opioid or nal-opioid of interest can be extracted to high purity by continuous cross-flow filtration using methods known in the art.

[0184] If the solution contains a mixture of nor- or nar-opioids of interest, it can be subjected to acid-base treatment to obtain the individual nor- or nar-opioid species of interest using methods known in the art, in which the pH of the aqueous solution is adjusted to precipitate the individual nor- or nar-opioids.

[0185] For high purity small scale preparations, the nor-opioid or nal-opioid can be purified in a single step by liquid chromatography.

[0186] LCMS method: The BIA compounds of interest, such as naloxone or naltrexone, can be separated using liquid chromatography and detected and quantified using mass spectrometry. The identity of the compound can be confirmed by its characteristic elution time, mass-to-charge ratio (m / z) and fragmentation pattern (MS / MS). Quantification can be performed by comparing the peak area of ​​the compound with a standard curve of a known reference standard compound. Furthermore, the BIA compounds of interest can be detected by alternative methods, such as GC-MS, UV-vis spectroscopy, NMR, LC-NMR, LC-UV, TLC and capillary electrophoresis.

[0187] Purpald assay The Purpald assay can be used for high-throughput screening of demethylation reactions. For example, demethylation catalyzed by 2-oxoglutarate-dependent dioxygenases produces formaldehyde as a product, as shown in the general chemical equation: [substrate] + 2-oxoglutarate + O⇔ [product] + formaldehyde + succinate + CO⇔. In alkaline conditions, the Purpald reagent undergoes a color change in the presence of formaldehyde, which can be quantified spectrophotometrically at 510 nm down to concentrations as low as 1 nM.

[0188] Yeast-derived alkaloid APIs vs. plant-derived APIs Clarified yeast culture medium (CYCM) can contain multiple impurities. Clarified yeast culture medium can be dehydrated by vacuum and / or heat to obtain an alkaloid-enriched powder. This product is similar to poppy straw concentrate (CPS), which is exported by poppy-growing countries and purchased by active pharmaceutical ingredient (API) manufacturers. For purposes of this invention, CPS represents any type of purified plant extract from which the desired alkaloid product can ultimately be further purified. Tables 9 and 10 highlight impurities in these two products that may be specific to either CYCM or CPS or may be present in both. Thus, such nor-opioid or nal-opioid impurities can be assessed for impurities based on non-colored impurities. By analyzing a product of unknown origin for a subset of these impurities, one skilled in the art could determine whether the product originated from a yeast-based or plant-based production host.

[0189] API-grade pharmaceutical ingredients are highly purified molecules. As such, impurities that may indicate the plant or yeast origin of the API (e.g., those listed in Tables 9 and 10) may be absent from the API-stage product. Indeed, many of the API products derived from the yeast strains of the present invention may be largely indistinguishable from traditional plant-derived APIs. However, in some cases, conventional alkaloid compounds may be subjected to chemical modification using chemical synthesis approaches, which may result in the appearance of chemical impurities in plant-based products that require such chemical modification. For example, chemical derivatization often generates a set of impurities associated with chemical synthesis processes. In certain circumstances, such modifications may be performed biologically within a yeast-based production platform, thereby avoiding the presence of some of the impurities associated with chemical derivatization in yeast-derived products. Notably, such impurities in chemically derivatized products may be present in API products produced using chemical synthesis processes but absent in API products produced using yeast-derived products. Alternatively, when yeast-derived products are mixed with chemically derived products, the resulting impurities may be present, but in amounts less than would be expected in an API that contains only or primarily chemically derived products. In this example, by analyzing the API product for a subset of such impurities, one skilled in the art could determine whether the product originated from a yeast-based production host or a traditional chemical derivatization route.

[0190] Non-limiting examples of impurities that may be present in chemically derivatized morphinan APIs but not in biosynthetic APIs include the impurity codeine-O(6)-methyl ether in API codeine; 8,14-dihydroxy-7,8-dihydrocodeinone in API oxycodone; and tetrahydrothebaine in API hydrocodone. Codeine-O(6)-methyl ether can be formed by chemical overmethylation of morphine. 8,14-dihydroxy-7,8-dihydrocodeinone in API oxycodone can be formed by chemical overoxidation of thebaine. Additionally, tetrahydrothebaine in API hydrocodone can be formed by chemical overreduction of thebaine.

[0191] However, if both yeast-derived and plant-derived compounds are subjected to chemical modification via a chemical synthesis approach, the same impurities associated with the chemical synthesis process can be expected to be present in the product. In such a situation, the starting material (e.g., CYCM or CPS) can be analyzed as described above.

[0192] Host cell-derived versus chemically derived nal-opioids Multiple impurities may be present in nal-opioids produced by chemical synthesis. Such impurities can arise from many different sources, such as unreacted starting materials, incomplete reactions, by-product formation, residual intermediates, dimerization, or decomposition. An example of an unreacted starting material is oxymorphone remaining in the preparation of naltrexone. An example of an impurity resulting from an incomplete reaction is 3-O-methylbuprenorphine resulting from incomplete 3-O-demethylation of thebaine. Chemical modifications can result in the addition or removal of functional groups at off-target sites. For example, oxidation of C10 in naltrexone synthesis to produce 10-hydroxynaltrexone and 10-ketonaltrexone, or removal of the 6-O-methyl group in buprenorphine synthesis to produce 6-O-desmethylbuprenorphine. Impurities can arise from residual reaction intermediates, such as residual N-oxides, such as oxymorphone N-oxide, formed during the N-demethylation process. Another source of impurities is dimerization, the conjugation of two opioid molecules, such as two buprenorphine molecules (2,2'-bisbuprenorphine), two naltrexone molecules (2,2'-bisnaltrexone), or two naloxone molecules (2,2'-bisnaloxone). Impurities can also arise from the decomposition of starting materials, reaction intermediates, or reaction products. The extreme physical conditions used in chemical synthesis make decomposition more likely. One example of an impurity that can arise from decomposition is dehydrobuprenorphine, which is produced by the oxidative conditions during buprenorphine synthesis.

[0193] Nar-opioids produced by enzyme catalysis in host cells may contain different impurities than nar-opioids produced by chemical synthesis. Nar-opioids produced by enzyme catalysis in host cells may contain fewer impurities than nar-opioids produced by chemical synthesis. Nar-opioids produced by enzyme catalysis in host cells may be free of certain impurities found in nar-opioids produced by chemical synthesis. For example, key features of enzymatic synthesis may include: (1) the enzyme targets specific substrates and residues with high fidelity; (2) the enzyme performs the reaction under mild intracellular physiological conditions that do not compromise the stability of the molecule; and (3) the enzyme is genetically engineered to be an efficient catalyst that drives the reaction to completion.

[0194] Table 11 highlights some impurities that may be specific to chemically produced nar-opioids. Thus, nar-opioids can be evaluated for impurities to determine the presence or absence of any of the impurities in Table 11. By analyzing a product of unknown origin for a subset of these impurities, one skilled in the art could determine whether the product is derived from chemical or enzymatic synthesis.

[0195] Methods for Genetically Manipulating Host Cells Also included are methods for genetically engineering host cells for the purpose of producing nor-opioid and / or nar-opioid BIAs of interest. Insertion of DNA into host cells can be accomplished using any convenient method. The methods are used to insert heterologous coding sequences into the genetically engineered host cells such that the host cells functionally express enzymes that convert the starting compound of interest into the product, the nor-opioid and / or nar-opioid BIA of interest.

[0196] Any convenient promoter can be used in the subject engineered host cells and methods. The promoter driving the expression of the heterologous coding sequence can be a constitutive or inducible promoter, provided that the promoter is active in the engineered host cell. The heterologous coding sequence can be expressed by its native promoter, or a non-native promoter can be used. Such promoters can be low to high in strength in the host in which they are used. The promoter can be regulated or constitutive. In certain embodiments, a promoter that is not glucose-repressible or is only lightly repressed by the presence of glucose in the culture medium is used. Promoters of interest include, but are not limited to, promoters of glycolytic genes, such as the promoter of the Bacillus subtilis tsr gene (encoding the promoter region of the fructose bisphosphate aldolase gene) or promoters from Saccharomyces cerevisiae genes encoding glyceraldehyde 3-phosphate dehydrogenase (GPD, GAPDH, or TDH3), the ADH1 promoter of baker's yeast, phosphate-starvation-inducible promoters, such as the yeast PHO5 promoter, the alkaline phosphatase promoter from B. licheniformis, inducible promoters from yeast, such as Gal1-10, Gal1, GalL, GalS, repressible promoters Met25, tetO, and constitutive promoters, such as the glyceraldehyde 3-phosphate dehydrogenase promoter (GPD), alcohol dehydrogenase promoter (ADH), translation-elongation factor-1-α promoter (TEF), cytochrome c-oxidase promoter (CYC1), MRP7 promoter, and the like. Autonomously replicating yeast expression vectors containing promoters inducible by hormones, such as glucocorticoids, steroids, and thyroid hormones, can also be used, including, but not limited to, the glucocorticoid response element (GRE) and the thyroid hormone response element (TRE). These and other examples are described in U.S. Patent No. 7,045,290, which is incorporated by reference, including the references cited therein.Additional vectors containing constitutive or inducible promoters (e.g., alpha-factor, alcohol oxidase, and PGH) can be used. Furthermore, any promoter / enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive gene expression. Any convenient promoter suitable for the host cell can be selected; examples of promoters that can be used in E. coli cells include the T7, lac, and tetO promoters. Promoter selection can be used to optimize transcript, and therefore enzyme, levels to maximize production and minimize energy resources.

[0197] Any convenient vector can be used in the subject genetically engineered host cells and methods. Vectors of interest include vectors for use in yeast and other cells. Yeast vector types can be divided into four general categories: integrating vectors (YIp), autonomously replicating high copy number vectors (YEp or 2μ plasmids), autonomously replicating low copy number vectors (YCp or centromeric plasmids), and vectors for cloning large fragments (YAC). Vector DNA is introduced into prokaryotic or eukaryotic cells by any convenient transformation or transfection method. DNA from another source (e.g., PCR-generated double-stranded DNA products, or synthetic double- or single-stranded oligonucleotides) can also be used for genetic engineering of yeast by integration into the genome. Any single transformation event can contain one or several nucleic acids (vectors, double- or single-stranded DNA fragments) for genetically modifying the host cell.

[0198] usefulness The genetically engineered host cells and methods disclosed herein, such as those described above, find use in a variety of applications. Applications of interest include, but are not limited to, research and therapeutic applications. The methods disclosed herein find use in a variety of different applications, for example, any convenient application in which the generation of nor-opioid and / or nal-opioid BIAs is of interest.

[0199] The subject genetically engineered host cells and methods find use in a variety of therapeutic applications. Therapeutic applications of interest include those in which the preparation of pharmaceutical formulations containing nor- or nar-opioids is of interest. The genetically engineered host cells described herein produce the nor- or nar-opioid of interest and the enzyme of interest. Reticuline is a key branching point intermediate of interest in the synthesis of BIAs, including genetic engineering operations to produce end products, such as opioid products. The subject host cells can be used to produce the nor- or nar-opioid of interest from simple, inexpensive starting materials, which can find use in producing the BIA of interest (including reticuline and BIA end products, such as nor- or nar-opioids). Thus, the subject host cells find use in providing therapeutically active nor- or nar-opioids of interest.

[0200] In some instances, genetically engineered host cells and methods find use in the production of commercial quantities of nor- or nal-opioids, where chemical synthesis has low yields and is not a viable means for large-scale production. In certain cases, the host cells and methods are used in fermentation facilities that may include, for example, 5,000-200,000 liter bioreactors (fermentors) to enable rapid production of the nor- or nal-opioid of interest for therapeutic formulations. Such applications may include industrial-scale production of the nor- or nal-opioid of interest from fermentable carbon sources, such as cellulose, starch, and free sugars.

[0201] The subject engineered host cells and methods find use in a variety of research applications. The subject host cells and methods can be used to analyze the effects of various enzymes on the biosynthetic pathways of various nor-opioid and / or nar-opioid BIAs of interest. The engineered host cells can also be engineered to produce nor-opioid and / or nar-opioid BIAs of interest that find use in testing for physiological activities of interest in as yet unknown therapeutic functions. In some cases, engineering host cells to include various heterologous coding sequences encoding various enzymes has elucidated high-yield biosynthetic pathways to nor-opioid and / or nar-opioid BIAs of interest. In certain cases, research applications include the production of nor-opioid and / or nar-opioid BIAs of interest for therapeutic molecules of interest that can then be further chemically modified or derivatized into desired products or for screening for increased therapeutic activity of interest. In some instances, host cell lines are used to screen for enzymatic activities of interest in such pathways, which may lead to the discovery of enzymes that convert nor-opioid or nal-opioid metabolites produced in such cell lines.

[0202] The subject engineered host cells and methods can be used as a production platform for plant-specific metabolites. The subject host cells and methods can be used as a platform for drug library development and plant enzyme discovery. For example, the subject engineered host cells and methods can find use in the development of natural product-based drug libraries by employing a yeast strain that produces a scaffold molecule of interest, such as norcodeine or northebaine, and further functionalizing the compound's structure through combinatorial biosynthesis or chemical means. By creating a drug library in this manner, potential drug hits (if any) are already associated with a production host suitable for large-scale culture and production. As another example, such subject engineered host cells and methods can find use in plant enzyme discovery. The subject host cells provide a clean background of defined metabolites for expressing plant EST libraries to identify new enzyme activities. The subject host cells and methods provide expression methods and culture conditions for the functional expression and increased activity of plant enzymes in yeast.

[0203] Kits and Systems Some aspects of the present invention further include kits and systems that can include one or more components used in the methods disclosed herein, e.g., genetically engineered host cells as described herein, starting compounds, heterologous coding sequences, vectors, culture media, etc. In some embodiments, the subject kits include genetically engineered host cells (e.g., as described herein) and one or more components selected from the following: starting compounds, heterologous coding sequences and / or vectors comprising said sequences, vectors, propagation feedstocks, components suitable for use in expression systems (e.g., cells, cloning vectors, multiple cloning sites (MCSs), bidirectional promoters, internal ribosome entry sites (IRESs), etc.), and culture media.

[0204] Any of the components described herein, such as host cells containing one or more modifications, starting compounds, culture media, etc., can be included in the kit. Various components suitable for use in the production and use of heterologous coding sequences, cloning vectors, and expression systems can be utilized in the subject kits. The kits can also include tubes, buffers, etc., and instructions for use. The various reagent components of the kit can be present in separate containers, if desired, or some or all of the components can be pre-combined in a single container to form a reagent mixture.

[0205] Also provided are systems for producing BIAs of nor-opioids and / or nal-opioids of interest, which may include genetically engineered host cells containing one or more modifications (e.g., as described herein), starting compounds, culture media, fermentors and fermentation equipment, e.g., devices suitable for maintaining growth conditions for the host cells, sampling and monitoring equipment and components, etc. A variety of elements suitable for use in large-scale fermentation of yeast cells may find use in the subject systems.

[0206] In some cases, the system includes elements for large-scale fermentation of genetically engineered host cells and monitoring and purification of nor-opioid or nal-opioid compounds produced by the fermented host cells. In certain embodiments, one or more starting compounds (e.g., as described herein) are added to the system under conditions in which one or more desired nor-opioid or nal-opioid products of interest are produced by the genetically engineered host cells in the fermenter. In some examples, the host cells produce the nor-opioid or nal-opioid of interest (e.g., as described herein). In some specific cases, the nor-opioid or nal-opioid product of interest is an opioid antagonist, such as naloxone, naltrexone, nalmefene, or nalorphine. In some specific cases, the nor-opioid or nal-opioid product of interest is an opioid antagonist, such as naltolindole or norbinaltorphimine. In some instances, the nor-opioid or nal-opioid product of interest is a partial agonist, such as buprenorphine.

[0207] In some cases, the system includes a process for monitoring and / or analyzing one or more of the BIA compounds of the nor-opioid and / or nar-opioid of interest produced by the subject host cells. For example, an LC-MS analysis system as described herein, a chromatography system, or any convenient system in which samples can be analyzed and compared to standards, such as those described herein. The fermentation medium can be monitored by sampling and analysis before fermentation and at any convenient time during fermentation. When the conversion of the starting compound to the nor-opioid or nar-opioid product of interest is complete, the fermentation can be stopped, and purification of the nor-opioid or nar-opioid product can be performed. Thus, in some cases, the subject system includes purification elements suitable for purifying the nor-opioid or nar-opioid product of interest from the host cell medium in which it was produced. Purification elements can include any convenient means that can be used to purify the nor-opioid or nal-opioid product of interest produced by fermentation, such as, but not limited to, silica chromatography, reverse-phase chromatography, ion exchange chromatography, HIC chromatography, size exclusion chromatography, liquid extraction, and pH extraction methods. In some cases, the subject systems provide for the production and isolation of an enzyme and / or nor-opioid or nal-opioid fermentation product of interest after charging the system with one or more starting compounds.

[0208] The following examples are put forth to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviation should be accounted for. Unless otherwise noted, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0209] Enzyme List Discussion Host cells can be engineered to contain one or more modifications (e.g., two or more, three or more, four or more, five or more, or even more modifications) that result in the production of a nor-opioid and / or nar-opioid BIA of interest. Table 2 lists exemplary genes that can be affected by one or more modifications to result in the production of a nor-opioid and / or nar-opioid BIA of interest and / or the production of an enzyme of interest in an engineered host cell.

[0210] The genetic modifications shown in Table 2 can be used to produce nor-opioid and / or nal-opioid BIAs of interest from genetically engineered host cells fed a medium containing the minimum nutrients required for growth. This minimal medium can include a carbon source, a nitrogen source, amino acids, vitamins, and salts. For example, the genetic modifications shown in Table 2 can be used to produce nor-opioid and / or nal-opioid BIAs of interest from genetically engineered host cells fed a carbohydrate. Additionally, one or more genetic modifications shown in Table 2 can be used to augment biosynthetic processes in host cells that can be genetically engineered for drug production.

[0211] Furthermore, the use of such modifications to produce nor-opioid and / or nar-opioid BIAs of interest and / or to produce enzymes of interest in genetically engineered host cells is not readily apparent from the mere identification of the enzymes that can be produced by the genes. In particular, the synthetic pathways reconstructed in host cells, such as yeast cells, as described herein contain various enzymes that do not function together in their natural state in a single organism. Moreover, some of the enzymes discussed herein do not function in their natural context for the biosynthesis of nor-opioid and / or nar-opioid BIAs. Furthermore, some of the enzymes described herein have not evolved to function, or function together, in particular host cells, such as yeast cells. Furthermore, some of the nor-opioids or nar-opioids produced do not occur in nature. In such cases, it would not be obvious that the enzyme would have sufficient flux within the pathway and exhibit sufficient activity in the context of a synthetic nor-opioid and / or nal-opioid pathway within a host cell, e.g., yeast, to produce downstream nor-opioid or nal-opioid end products.

[0212] For example, plant enzymes are often difficult to functionally express in heterologous microbial hosts, such as yeast. Often, the enzymes can be misfolded, not properly localized within the host cell, and / or processed incorrectly. Due to differences in protein translation and processing between yeast and plants, the activity of such enzymes in yeast hosts can be very low to undetectable. This challenge generally arises for enzymes localized in the inner membrane, such as cytochrome P450, and is particularly pronounced in the BIA pathway, which produces precursors of nor-opioids or nal-opioids. Even with low enzyme activity, engineering yeast to produce complex BIAs can present significant challenges, as sufficient activity is required at each step to ensure high-level accumulation of the desired BIA product.

[0213] Furthermore, some host cells, such as yeast, have endogenous enzymes / pathways that can act on many of the early precursors in the BIA pathway (i.e., intermediates from tyrosine to norcoclaurine); therefore, given these competing endogenous pathways, it may not be readily apparent that sufficient flux through heterologous pathways will exist to achieve significant BIA production. For example, the Erlich pathway in yeast (Hazelwood, et al. 2008. Appl. Environ. Microbiol. 74: 2259-66; Larroy, et al. 2003. Chem. Biol. Interact. 143-144: 229-38; Larroy, et al. 2002. Eur. J. Biochem. 269: 5738-45) is a major endogenous pathway that may act to convert many of the early BIA pathway intermediates into undesired products, diverting flux away from synthetic pathways.

[0214] Furthermore, many of the enzymes discussed herein and listed in Table 2 may function in their native plant hosts under very specific regulatory strategies, e.g., spatial regulation, that may be lost when transferred to a heterologous yeast host. Plants also present a very different biochemical environment than yeast cells in which the enzymes evolve to function, e.g., pH, redox state, and substrate, cosubstrate, coenzyme, and cofactor availability. Given the differences in biochemical environment and regulatory strategies between native and heterologous yeast hosts, it is not obvious that the enzymes would exhibit significant activity in the context of the yeast environment, let alone that they would operate together to direct simple precursors, e.g., sugars, into complex BIA compounds. Maintaining the activity of the enzymes in the yeast host is particularly important because many of the pathways have many reaction steps (>10), and therefore, if these steps are not efficient, accumulation of the desired downstream product cannot be expected.

[0215] Furthermore, in natural plant hosts, the metabolites associated with such pathways may be localized across different cell and tissue types. In some instances, there are cell types that can be specialized for biosynthesis and cell types that can synthesize for the accumulation of metabolites. This type of cellular specialization may be lost when the pathway is expressed in a heterologous yeast host, and may play an important role in controlling the toxicity of such metabolites to the cell. Therefore, it is not obvious that yeast could be successfully engineered to biosynthesize and accumulate such metabolites without being harmed by the toxicity of such compounds.

[0216] For example, in native plant hosts, the enzyme BBE has been reported to have dynamic subcellular localization. Specifically, BBE is initially localized in the ER and then sorted into the vacuole (Bird and Facchini, 2001, Planta, 213: 888-97). It has been suggested that the ER-association of BBE in plants (Alcantara, et al., 2005, Plant Physiol. 138: 173-83) provides an optimal basic pH (approximately 8.8) for BBE activity (Ziegler and Facchini, 2008, Annu. Rev. Plant Biol. 59: 735-69). Another example is evidence that sanguinarin biosynthesis occurs within specialized vesicles within plant cells (Amann, et al., 1986, Planta, 167: 310-20), although only a portion of the intermediates accumulate within these vesicles. This may occur to sequester the intermediate from other enzymatic activity and / or toxic effects.

[0217] As another example, all biosynthetic enzymes in the morphinan pathway are localized in the phloem, a part of plant vascular tissue. Within the phloem, pathway enzymes can be further divided into two cell types: sieve elements, which are common to all plants, and lacteals, which are specialized cell types present only in certain plants that produce specialized secondary metabolites. The upstream enzymes (i.e., NCS to SalAT) are primarily present in the sieve elements, while the downstream enzymes (i.e., T6ODM, COR, and CODM) are mostly present in the lacteals (Onoyovwe, et al. 2013. Plant Cell. 25: 4110-22). Furthermore, the final step of the noscapine biosynthetic pathway was found to occur within the lacteals (Chen and Facchini 2014. Plant J. 77: 173-84). This compartmentalization is thought to be crucial for regulating biosynthesis by isolating or transporting intermediates, providing optimal pH, and enhancing the supply of cofactors, although the nature of the poppy lactiferous microenvironment is still being explored (Ziegler and Facchini, 2008, Annu. Rev. Plant Biol. 59: 735-69). Furthermore, it is predicted that some of the enzymes may function as multienzyme complexes or metabolic channels common to plant secondary metabolism (Kempe, et al., 2009, Phytochemistry. 70: 579-89; Allen, et al., 2004, Nat. Biotechnol. 22: 1559-66). It is not clear that such complexes or channels will form when biosynthetic enzymes from different hosts are combined and / or recombinantly expressed in heterologous yeast cells, as they do in the native host. In a further example, in Coptis japonica, berberine is biosynthesized in the root tissue and then accumulated in the rhizomes by the action of a special ATP-binding cassette transporter protein (Shitan, et al. 2013. Phytochemistry. 91: 109-16). In poppy, morphinan alkaloids accumulate in the latex (cytoplasm of ductal cells) (Martin, et al. 1967. Biochemistry. 6: 2355-63).

[0218] Furthermore, even without such considerations, plant enzymes may not yet be characterized for some of the steps in the pathways described herein. For example, the conversion of tyrosine to norcoclaurine, the initial benzylisoquinoline alkaloid scaffold, has not yet been characterized. Thus, alternative biosynthetic schemes have been developed for some of the steps in the pathways described herein by combining enzyme activities that do not normally occur together in nature for the biosynthesis of BIAs, or by identifying new enzyme activities from genome sequence information for use in reconstructed pathways.

[0219] For example, the two-step conversion of tyrosine to dopamine can be achieved by combining one bacterial enzyme with at least five mammalian enzymes that do not occur together in nature and that have not evolved to function in the context of this pathway or with plant enzymes. In such cases, it may not be obvious that such an enzyme can be used for the biosynthesis of a compound for which it did not naturally evolve and that it would be able to function effectively in a heterologous microbial host and in the context of this pathway.

[0220] Examples of genes that are the subject of modification to generate nor-opioid and / or nal-opioid BIAs of interest and / or enzymes of interest are discussed below, and the genes are further discussed in the context of a series of diagrams illustrating the pathways used to produce the BIAs of interest and nor-opioid and / or nal-opioid BIAs and / or enzymes of interest.

[0221] [TLK1] In some examples, the genetically engineered host cell can modify the expression of the enzyme transketolase. Transketolase is encoded by the TKL1 gene. In one example, transketolase catalyzes the reaction fructose-6-phosphate + glyceraldehyde-3-phosphate ⇔ xylose-5-phosphate + erythrose-4-phosphate, as shown in Figure 2. The genetically engineered host cell can be modified to contain constitutive overexpression of the TKL1 gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell can be modified to synthetically regulate the expression of the TKL1 gene within the genetically engineered host cell. In one example, the genetically engineered host cell can be modified to incorporate one copy, multiple copies, or additional copies of the TKL1 gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate a strong promoter element for overexpression of the TKL1 gene into the genetically engineered host cell. The TKL1 gene can be from Saccharomyces cerevisiae or another species. In some cases, the TKL1 gene can be 100% similar to the naturally occurring gene.

[0222] [ZWF1] In some examples, the genetically engineered host cell can modify the expression of the enzyme glucose-6-phosphate dehydrogenase. Glucose-6-phosphate dehydrogenase is encoded by the ZWF1 gene. In one example, glucose-6-phosphate dehydrogenase catalyzes the reaction of glucose-6-phosphate → 6-phosphogluconolactone, as shown in Figure 2. The genetically engineered host cell can be modified to delete the coding region of the ZWF1 gene in the genetically engineered host cell. Alternatively, the genetically engineered host cell can be modified to disable the functionality of the ZWF1 gene, for example, by introducing an inactivating mutation.

[0223] [ARO4] In some examples, the genetically engineered host cell may modify the expression of the enzyme 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase. DAHP synthase is encoded by the ARO4 gene. In one example, DAHP synthase catalyzes the reaction erythrose-4-phosphate + phosphoenolpyruvate → DAHP, as shown in Figure 2. The genetically engineered host cell may modify the ARO4 gene to incorporate one or more feedback inhibition alleviation mutations. In particular, feedback inhibition alleviation mutations (e.g., ARO4 FBR ) can be integrated as a specific mutation at the original locus of the native ARO4 gene; as an additional copy introduced as an integrated gene at a separate locus; or as an additional copy on an episomal vector, such as a 2 μm or centromeric plasmid. FBR The designation "FBR" refers to feedback-resistant variants and mutations. The feedback-inhibited copy of the DAHP synthase enzyme can be under the transcriptional regulation of native yeast, for example, when the engineered host cell is a yeast cell. Alternatively, the feedback-inhibited copy of the DAHP synthase enzyme can be introduced into the engineered host cell with constitutive or dynamic engineered regulation of protein expression by placing it under the control of a synthetic promoter. In some cases, the ARO4 gene can be derived from Saccharomyces cerevisiae. In some cases, the ARO4 gene can be 100% similar to the naturally occurring gene. Examples of modifications to the ARO4 gene include the feedback-inhibited mutations K229L or Q166K.

[0224] [ARO7] In some examples, the genetically engineered host cell may modify the expression of the enzyme chorismate mutase. Chorismate mutase is encoded by the ARO7 gene. In one example, chorismate mutase catalyzes the chorismate → prephenate reaction as shown in Figure 2. The genetically engineered host cell may modify the ARO7 gene to incorporate one or more feedback inhibition relieving mutations. In particular, feedback inhibition relieving mutations (e.g., ARO7 FBR) can be integrated as a specific mutation at the original locus of the native ARO7 gene; as an additional copy introduced as an integrated gene at a separate locus; or as an additional copy on an episomal vector, such as a 2 μm or centromeric plasmid. FBR The designation "FBR" refers to feedback-resistant variants and mutations. The feedback-inhibited copy of the chorismate mutase enzyme can be under the transcriptional regulation of native yeast, for example, when the engineered host cell is a yeast cell. Alternatively, the feedback-inhibited copy of the chorismate mutase enzyme can be introduced into the engineered host cell with constitutive or dynamic engineered regulation of protein expression by placing it under the control of a synthetic promoter. In some cases, the ARO7 gene can be derived from Saccharomyces cerevisiae. In some cases, the ARO7 gene can be 100% similar to the naturally occurring gene. Examples of modifications to the ARO7 gene include a feedback-inhibited mutation or T226I.

[0225] [ARO10] In some examples, the genetically engineered host cell may modify the expression of the enzyme phenylpyruvate decarboxylase. Phenylpyruvate decarboxylase is encoded by the ARO10 gene. In one example, phenylpyruvate decarboxylase catalyzes the reaction of hydroxyphenylpyruvate to 4-hydroxyphenylacetate (4HPA), as shown in Figure 2. The genetically engineered host cell may be modified to contain constitutive overexpression of the ARO10 gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell may be modified to synthetically regulate the expression of the ARO10 gene within the genetically engineered host cell. In one example, the genetically engineered host cell may be modified to incorporate one copy, multiple copies, or additional copies of the ARO10 gene. Additionally or alternatively, the genetically engineered host cell may be modified to incorporate a strong promoter element for overexpression of the ARO10 gene within the genetically engineered host cell. The ARO10 gene may be derived from Saccharomyces cerevisiae or another species. In some instances, the ARO10 gene may be 100% similar to the naturally occurring gene.

[0226] [ADH2-7, SFA1] In some examples, genetically engineered host cells can modify the expression of alcohol dehydrogenase enzymes. Alcohol dehydrogenase enzymes can be encoded by one or more of ADH2, ADH3, ADH4, ADH5, ADH6, ADH7 and SFA1 genes. In one example, alcohol dehydrogenase catalyzes the reaction of 4HPA → tyrosol. Genetically engineered host cells can be modified to delete one or more coding regions of ADH2, ADH3, ADH4, ADH5, ADH6, ADH7 and SFA1 genes in the genetically engineered host cells. Alternatively, genetically engineered host cells can be modified to disable the functionality of one or more of ADH2, ADH3, ADH4, ADH5, ADH6, ADH7 and SFA1 genes, for example, by introducing inactivating mutations.

[0227] [ALD2-6] In some examples, the genetically engineered host cell may modify the expression of the aldehyde oxidase enzyme. The aldehyde oxidase enzyme may be encoded by one or more of the ALD2, ALD3, ALD4, ALD5, and ALD6 genes. In one example, aldehyde oxidase catalyzes the reaction 4HPA → hydroxyphenylacetic acid. The genetically engineered host cell may be modified to delete the coding region of one or more of the ALD2, ALD3, ALD4, ALD5, and ALD6 genes within the genetically engineered host cell. Alternatively, the genetically engineered host cell may be modified to disable the functionality of one or more of the ALD2, ALD3, ALD4, ALD5, and ALD6 genes, for example, by introducing an inactivating mutation.

[0228] [ARO9] In some examples, the genetically engineered host cell may be modified to express the enzyme aromatic aminotransferase. Aromatic aminotransferase is encoded by the ARO9 gene. In one example, aromatic aminotransferase catalyzes the reaction hydroxyphenylpyruvate + glutamate → tyrosine + α-ketogluterate, as shown in Figure 2. The genetically engineered host cell may be modified to contain constitutive overexpression of the ARO9 gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell may be modified to synthetically regulate the expression of the ARO9 gene within the genetically engineered host cell. In one example, the genetically engineered host cell may be modified to incorporate one, multiple, or additional copies of the ARO9 gene. Additionally or alternatively, the genetically engineered host cell may be modified to incorporate a strong promoter element for overexpression of the ARO9 gene within the genetically engineered host cell. The ARO9 gene may be derived from Saccharomyces cerevisiae or another species. In some instances, the ARO9 gene may be 100% similar to the naturally occurring gene.

[0229] [TYR] In some examples, the genetically engineered host cell may modify the expression of the enzyme tyrosinase. Tyrosinase is encoded by the TYR gene. In one example, tyrosinase catalyzes the reaction tyrosine → L-DOPA, as shown in Figure 2. In another example, tyrosinase catalyzes the reaction L-DOPA → dopaquinone. The genetically engineered host cell may be modified to contain constitutive expression of the TYR gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell may be modified to synthetically regulate the expression of the TYR gene within the genetically engineered host cell. In one example, the genetically engineered host cell may be modified to incorporate one copy, multiple copies, or additional copies of the TYR gene. Additionally or alternatively, the genetically engineered host cell may be modified to incorporate a strong promoter element for overexpression of the TYR gene into the genetically engineered host cell. The TYR gene can be from Ralstonia solanacearum, Agaricus bisporus, or another species. In some instances, the TYR gene can be 100% similar to the naturally occurring gene.

[0230] [TyrH] In some examples, the genetically engineered host cell can modify the expression of the enzyme tyrosine hydroxylase. Tyrosine hydroxylase is encoded by the TyrH gene. In one example, tyrosine hydroxylase catalyzes the reaction tyrosine → L-DOPA, as shown in Figures 2 and 5. The genetically engineered host cell can be modified to contain constitutive expression of the TyrH gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell can be modified to synthetically regulate the expression of the TyrH gene within the genetically engineered host cell. In one example, the genetically engineered host cell can be modified to incorporate one copy, multiple copies, or additional copies of the TyrH gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate a strong promoter element for overexpression of the TyrH gene into the genetically engineered host cell. The TyrH gene can be derived from humans, Norway rats, house mice, or another species. In some examples, the TyrH gene can be 100% similar to a naturally occurring gene.

[0231] [DODC] In some examples, the genetically engineered host cell may modify the expression of the enzyme L-DOPA decarboxylase. L-DOPA decarboxylase is encoded by the DODC gene. In one example, L-DOPA decarboxylase catalyzes the L-DOPA → dopamine reaction as shown in Figures 2 and 5. The genetically engineered host cell may be modified to contain constitutive expression of the DODC gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell may be modified to synthetically regulate expression of the DODC gene within the genetically engineered host cell. In one example, the genetically engineered host cell may be modified to incorporate one, multiple, or additional copies of the DODC gene. Additionally or alternatively, the genetically engineered host cell may be modified to incorporate a strong promoter element for overexpression of the DODC gene within the genetically engineered host cell. The DODC gene may be derived from Pseudomonas putida, Rattus norvegicus, or another species. In some examples, the DODC gene may be 100% similar to the naturally occurring gene.

[0232] [TYDC] In some examples, the genetically engineered host cell may modify the expression of the enzyme tyrosine / DOPA decarboxylase. Tyrosine / DOPA decarboxylase is encoded by the TYDC gene. In one example, tyrosine / DOPA decarboxylase catalyzes the L-DOPA → dopamine reaction as shown in Figure 2. The genetically engineered host cell may be modified to contain constitutive expression of the TYDC gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell may be modified to synthetically regulate the expression of the TYDC gene within the genetically engineered host cell. In one example, the genetically engineered host cell may be modified to incorporate one, multiple, or additional copies of the TYDC gene. Additionally or alternatively, the genetically engineered host cell may be modified to incorporate a strong promoter element for overexpression of the TYDC gene within the genetically engineered host cell. The TYDC gene may be derived from poppy or another species. In some examples, the TYDC gene may be 100% similar to the naturally occurring gene.

[0233] [MAO] In some cases, the genetically engineered host cell can modify the expression of the enzyme monoamine oxidase. Monoamine oxidase is encoded by the MAO gene. In one example, monoamine oxidase catalyzes the reaction of dopamine → 3,4-DHPA, as shown in Figure 2. The genetically engineered host cell can be modified to contain the constitutive expression of the MAO gene in the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell can be modified to synthetically regulate the expression of the MAO gene in the genetically engineered host cell. In one example, the genetically engineered host cell can be modified to incorporate one copy, multiple copies, or additional copies of the MAO gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate a strong promoter element for overexpression of the MAO gene into the genetically engineered host cell. In some cases, the MAO gene can be codon-optimized for expression in Saccharomyces cerevisiae. The MAO gene can be from E. coli, human, Micrococcus luteus, or another species. In some cases, the MAO gene can be 77% similar to the naturally occurring gene.

[0234] [NCS] In some examples, the genetically engineered host cell may modify the expression of the enzyme norcoclaurine synthase. Norcoclaurine synthase is encoded by the NCS gene. In one example, norcoclaurine synthase catalyzes the reaction 4HPA + dopamine → (S)-norcoclaurine, as shown in Figure 5. In particular, Figure 5 shows a biosynthetic scheme for the conversion of L-tyrosine to reticuline via norcoclaurine, according to an embodiment of the present invention. Figure 5 shows the use of the enzymes TyrH, tyrosine hydroxylase; DODC, DOPA decarboxylase; NCS, norcoclaurine synthase (discussed herein); 6OMT, 6-O-methyltransferase; CNMT, coclaurine N-methyltransferase; CYP80B1, cytochrome P450 80B1; CPR, cytochrome P450 NADPH reductase; 4'OMT, 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase; L-DOPA, L-3,4-dihydroxyphenylalanine; and 4-HPA, 4-hydroxyphenylacetylaldehyde. Of the enzymes shown in Figure 5, 4-HPA and L-tyrosine are naturally synthesized in yeast. All other metabolites shown are not naturally produced in yeast. Furthermore, although TyrH has been shown to catalyze the conversion of L-tyrosine to L-DOPA, other enzymes can also be used to perform this step, as described herein. For example, tyrosinase can also be used to convert L-tyrosine to L-DOPA. Also, other enzymes, such as cytochrome P450 oxidases, can also be used to convert L-tyrosine to L-DOPA. Such enzymes can exhibit oxidase activity toward related BIA precursor compounds, such as L-DOPA and L-tyrosine.

[0235] Furthermore, norcoclaurine synthase catalyzes the reaction 3,4-DHPA + dopamine → (S)-norlaudanosoline, as shown in Figure 6. In particular, Figure 6 shows a biosynthetic scheme for the conversion of L-tyrosine to reticuline via norlaudanosoline, according to an embodiment of the present invention. Figure 6 illustrates the use of the enzymes TyrH, tyrosine hydroxylase; DODC, DOPA decarboxylase; maoA, monoamine oxidase; NCS, norcoclaurine synthase; 6OMT, 6-O-methyltransferase; CNMT, coclaurine N-methyltransferase; 4'OMT, 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase; L-DOPA, L-3,4-dihydroxyphenylalanine; and 3,4-DHPA, 3,4-dihydroxyphenylacetaldehyde. Of the enzymes shown in Figure 6, L-tyrosine is naturally synthesized in yeast. The other metabolites shown in Figure 6 are not naturally produced in yeast.

[0236] The genetically engineered host cell can be modified to contain constitutive expression of the NCS gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell can be modified to synthetically regulate expression of the NCS gene within the genetically engineered host cell. In one example, the genetically engineered host cell can be modified to incorporate one, multiple, or additional copies of the NCS gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate a strong promoter element for overexpression of the NCS gene within the genetically engineered host cell. Furthermore, the norcoclaurine synthase can have an N-terminal truncation. In some cases, the NCS gene can be codon-optimized for expression in Saccharomyces cerevisiae. The NCS gene can be derived from Coptis chinensis, opium poppy, Papaver bracteatum, Thalicitum flavum, Corydalis saxicola, or another species. In some instances, the NCS gene may be 80% similar to the naturally occurring gene.

[0237] [6OMT] In some examples, the genetically engineered host cell can modify the expression of the enzyme norcoclaurine 6-O-methyltransferase. Norcoclaurine 6-O-methyltransferase is encoded by the 6OMT gene. In some examples, norcoclaurine 6-O-methyltransferase catalyzes the reaction of norcoclaurine → coclaurine, as shown in Figure 5. In other examples, norcoclaurine 6-O-methyltransferase catalyzes the reaction of norlaudanosoline → 3'hydroxycoclaurine, as well as other reactions detailed herein, such as those shown in Figure 6. Furthermore, the genetically engineered host cell can be modified to include constitutive expression of the 6OMT gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell can be modified to synthetically regulate the expression of the 6OMT gene within the genetically engineered host cell. In one example, the genetically engineered host cell can be modified to incorporate one copy, multiple copies, or additional copies of the 6OMT gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate a strong promoter element for overexpression of the 6OMT gene into the genetically engineered host cell. The 6OMT gene can be derived from Papaver somniferum, Aubergine japonica, Coptis japonica, or another species. In some cases, the 6OMT gene can be 100% similar to a naturally occurring gene.

[0238] [CNMT] In some examples, the genetically engineered host cell can modify the expression of the enzyme coclaurine-N-methyltransferase. Coclaurine-N-methyltransferase is encoded by the CNMT gene. In some examples, coclaurine-N-methyltransferase catalyzes the reaction coclaurine → N-methylcoclaurine, as shown in Figure 5. In other examples, the coclaurine-N-methyltransferase enzyme can catalyze the reaction 3'hydroxycoclaurine → 3'hydroxy-N-methylcoclaurine. In other examples, coclaurine-N-methyltransferase can catalyze the reaction noroxymorphone → naloxone, as shown in Figure 26. In other examples, coclaurine-N-methyltransferase can catalyze other reactions detailed herein, such as those shown in Figure 6. Furthermore, the genetically engineered host cell can be modified to include constitutive expression of the CNMT gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell can be modified to synthetically regulate the expression of the CNMT gene in the genetically engineered host cell.In one example, the genetically engineered host cell can be modified to incorporate one copy, multiple copies, or additional copies of the CNMT gene.Additionally or alternatively, the genetically engineered host cell can be modified to incorporate a strong promoter element for overexpression of the CNMT gene into the genetically engineered host cell.The CNMT gene can be derived from poppy, yellow larch, coptis or another species.In some examples, the CNMT gene can be 100% similar to the naturally occurring gene.

[0239] [4'OMT] In some examples, the genetically engineered host cell can modify the expression of the enzyme 4'-O-methyltransferase. 4'-O-methyltransferase is encoded by the 4'OMT gene. In some examples, the 4'-O-methyltransferase catalyzes the reaction 3'-hydroxy-N-methylcoclaurine → reticuline, as shown in Figure 5. In other examples, the 4'-O-methyltransferase catalyzes other reactions detailed herein, such as those shown in Figure 6. Furthermore, the genetically engineered host cell can be modified to include constitutive expression of the 4'OMT gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell can be modified to synthetically regulate the expression of the 4'OMT gene within the genetically engineered host cell. In one example, the genetically engineered host cell can be modified to incorporate one, multiple, or additional copies of the 4'OMT gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate a strong promoter element for overexpression of the 4'OMT gene into the genetically engineered host cell. The 4'OMT gene can be derived from Papaver somniferum, Aubergine japonica, Coptis japonica, or another species. In some cases, the 4'OMT gene can be 100% similar to a naturally occurring gene.

[0240] [CYP80B1] In some examples, the genetically engineered host cell can modify the expression of the enzyme cytochrome P450 80B1. Cytochrome P450 80B1 is encoded by the CYP80B1 gene. In one example, cytochrome P450 80B1 catalyzes the reaction N-methylcoclaurine → 3'-hydroxy-N-methylcoclaurine, as shown in Figure 5. The genetically engineered host cell can be modified to contain constitutive expression of the cytochrome P450 80B1 gene in the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell can be modified to synthetically regulate the expression of the cytochrome P450 80B1 gene in the genetically engineered host cell. In one example, the genetically engineered host cell can be modified to incorporate one copy, multiple copies, or additional copies of the cytochrome P450 80B1 gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate a strong promoter element for overexpression of the cytochrome P450 80B1 gene into the genetically engineered host cell. In some cases, the CYP80B1 gene can be codon-optimized for expression in Saccharomyces cerevisiae. The cytochrome P450 80B1 gene can be derived from poppy, E. californica, yellow larch, or another species. In some cases, the P450 80B1 gene can be 77% similar to the naturally occurring gene.

[0241] [FOL2] In some examples, the genetically engineered host cell can modify the expression of the enzyme GTP cyclohydrolase. GTP cyclohydrolase is encoded by the FOL2 gene. In some examples, GTP cyclohydrolase catalyzes the reaction GTP → dihydroneopterin triphosphate, as shown in Figure 1. The genetically engineered host cell can be modified to contain constitutive overexpression of the FOL2 gene within the genetically engineered host cell. The genetically engineered host cell can also be modified to contain natural regulation. Additionally or alternatively, the genetically engineered host cell can be modified to synthetically regulate the expression of the FOL2 gene within the genetically engineered host cell. In one example, the genetically engineered host cell can be modified to incorporate one copy, multiple copies, or additional copies of the FOL2 gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate a strong promoter element for overexpression of the FOL2 gene within the genetically engineered host cell. The FOL2 gene can be derived from Saccharomyces cerevisiae, humans, mice, or another species. In some instances, the FOL2 gene may be 100% similar to the naturally occurring gene.

[0242] [PTPS] In some examples, the genetically engineered host cell can modify the expression of the enzyme 6-pyruvoyltetrahydrobiopterin (PTP) synthase. Pyruvoyltetrahydrobiopterin synthase is encoded by the PTPS gene. In some examples, 6-pyruvoyltetrahydrobiopterin synthase catalyzes the reaction dihydroneopterin triphosphate → PTP, as shown in Figure 1. The genetically engineered host cell can be modified to contain constitutive expression of the PTPS gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell can be modified to synthetically regulate the expression of the PTPS gene within the genetically engineered host cell. In one example, the genetically engineered host cell can be modified to incorporate one copy, multiple copies, or additional copies of the PTPS gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate a strong promoter element for overexpression of the PTPS gene into the genetically engineered host cell. In some cases, the PTPS gene can be codon-optimized for expression in Saccharomyces cerevisiae. The PTPS gene may be from Rattus norvegicus, human, house mouse, or another species. In some instances, the PTPS gene may be 80% similar to the naturally occurring gene.

[0243] [SepR] In some examples, the genetically engineered host cell can modify the expression of the enzyme sepiapterin reductase. Sepiapterin reductase is encoded by the SepR gene. In some examples, sepiapterin reductase catalyzes the PTP → BH4 reaction as shown in Figure 1. The genetically engineered host cell can be modified to contain constitutive expression of the SepR gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell can be modified to synthetically regulate the expression of the SepR gene within the genetically engineered host cell. In one example, the genetically engineered host cell can be modified to incorporate one copy, multiple copies, or additional copies of the SepR gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate a strong promoter element for overexpression of the SepR gene into the genetically engineered host cell. In some cases, the SepR gene can be codon-optimized for expression in Saccharomyces cerevisiae. The SepR gene can be from Rattus norvegicus, human, house mouse, or another species. In some instances, the SepR gene can be 72% similar to the naturally occurring gene.

[0244] [PCD] In some examples, the genetically engineered host cell can modify the expression of the enzyme 4a-hydroxytetrahydrobiopterin (pterin-4α-carbinolamine) dehydratase. 4a-Hydroxytetrahydrobiopterin dehydratase is encoded by a PCD gene. In some examples, 4a-hydroxytetrahydrobiopterin dehydratase catalyzes the reaction 4a-hydroxytetrahydrobiopterin → HO + quinonoid dihydropteridine, as shown in Figure 1. The genetically engineered host cell can be modified to contain constitutive expression of a PCD gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell can be modified to synthetically regulate the expression of a PCD gene within the genetically engineered host cell. In one example, the genetically engineered host cell can be modified to incorporate one copy, multiple copies, or additional copies of a PCD gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate a strong promoter element for overexpression of the PCD gene into the genetically engineered host cell. In some cases, the PCD gene can be codon-optimized for expression in Saccharomyces cerevisiae. The PCD gene can be derived from Rattus norvegicus, human, house mouse, or another species. In some cases, the PCD gene can be 79% similar to a naturally occurring gene.

[0245] [QDHPR] In some examples, the genetically engineered host cell can modify the expression of the enzyme quinonoid dihydropteridine reductase. The quinonoid dihydropteridine reductase is encoded by the QDHPR gene. In some examples, the quinonoid dihydropteridine reductase catalyzes the reaction quinonoid dihydropteridine → BH4, as shown in Figure 1. The genetically engineered host cell can be modified to contain constitutive expression of the QDHPR gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell can be modified to synthetically regulate the expression of the QDHPR gene within the genetically engineered host cell. In one example, the genetically engineered host cell can be modified to incorporate one copy, multiple copies, or additional copies of the QDHPR gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate a strong promoter element for overexpression of the QDHPR gene into the genetically engineered host cell. In some cases, the QDHPR gene can be codon-optimized for expression in Saccharomyces cerevisiae. The QDHPR gene can be from Rattus norvegicus, human, house mouse, or another species. In some instances, the QDHPR gene can be 75% similar to the naturally occurring gene.

[0246] [DHFR] In some examples, the genetically engineered host cell can modify the expression of the enzyme dihydrofolate reductase. Dihydrofolate reductase is encoded by the DHFR gene. In some examples, dihydrofolate reductase catalyzes the reaction 7,8-dihydrobiopterin (BH2) → 5,6,7,8-tetrahydrobiopterin (BH4), as shown in Figure 1. This reaction can be useful for recovering BH4 as a co-substrate for the conversion of tyrosine to L-DOPA, as shown in Figure 5. The genetically engineered host cell can be modified to contain constitutive expression of the DHFR gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell can be modified to synthetically regulate the expression of the DHFR gene within the genetically engineered host cell. In one example, the genetically engineered host cell can be modified to incorporate one, multiple, or additional copies of the DHFR gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate a strong promoter element for overexpression of the DHFR gene into the genetically engineered host cell. In some cases, the DHFR gene can be codon-optimized for expression in Saccharomyces cerevisiae. The DHFR gene can be derived from Rattus norvegicus, human, or another species. In some cases, the DHFR gene can be 77% similar to the naturally occurring gene.

[0247] As discussed above with respect to the epimerization of [CYP-COR]l-BIA, genetically engineered host cells can modify the expression of a BIA epimerase. BIA epimerases are encoded by CYP-COR genes (e.g., CYP82Y2-COR genes). CYP-COR genes may also be referred to as DRS-DRR genes. In some instances, the BIA epimerase catalyzes the conversion of (S)-l-BIA to (R)-l-BIA, as shown in Figure 7. In particular, Figure 7 shows a biosynthetic scheme for the conversion of L-tyrosine to a morphinan alkaloid, according to an embodiment of the present invention. Figure 7 shows the use of the enzymes CPR, cytochrome P450 reductase; CYP-COR, cytochrome P450 CYP82Y1-like codeinone reductase-like fusion; SalSyn, salutaridin synthase; SalR, salutaridinol reductase; SalAT, salutaridinol 7-O-acetyltransferase; T6ODM, thebaine 6-O-demethylase; COR, codeinone reductase; and CODM, codeine-O-demethylase.

[0248] The genetically engineered host cell can be modified to contain constitutive expression of the CYP-COR gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell can be modified to synthetically regulate the expression of the CYP-COR gene within the genetically engineered host cell. In one example, the genetically engineered host cell can be modified to incorporate one copy, multiple copies, or additional copies of the CYP-COR gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate a strong promoter element for overexpression of the CYP-COR gene within the genetically engineered host cell. The CYP-COR gene can be derived from poppy, poppy, silver poppy (Papaver setigerum), celandine (Chelidonium majus), or another species. In some examples, the CYP-COR gene can be 77% similar to a naturally occurring gene.

[0249] [CPR] In some examples, the genetically engineered host cell may modify the expression of the enzyme cytochrome P450 reductase. Cytochrome P450 reductase is encoded by the CPR gene. In some examples, cytochrome P450 reductase catalyzes the reaction (R)-reticuline → salutaridine, as shown in Figure 7. In addition, cytochrome P450 reductase catalyzes other reactions, such as those shown in figures throughout this application. The genetically engineered host cell may be modified to contain constitutive expression of the CPR gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell may be modified to synthetically regulate the expression of the CPR gene within the genetically engineered host cell. In one example, the genetically engineered host cell may be modified to incorporate one, multiple, or additional copies of the CPR gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate a strong promoter element for overexpression of the CPR gene into the genetically engineered host cell. The CPR gene can be derived from California poppy, poppy, human (H. sapiens), Saccharomyces cerevisiae, Arabidopsis thaliana, or another species. In some cases, the CPR gene can be 100% similar to a naturally occurring gene.

[0250] [SalSyn] In some examples, the genetically engineered host cell may modify the expression of the enzyme salutaridine synthase. Salutaridine synthase is encoded by the SalSyn gene. In some examples, salutaridine synthase catalyzes the reaction (R)-reticuline → salutaridine, as shown in Figure 7. The genetically engineered host cell may be modified to contain constitutive expression of the SalSyn gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell may be modified to synthetically regulate the expression of the SalSyn gene within the genetically engineered host cell. In one example, the genetically engineered host cell may be modified to incorporate one copy, multiple copies, or additional copies of the SalSyn gene. Additionally or alternatively, the genetically engineered host cell may be modified to incorporate a strong promoter element for overexpression of the SalSyn gene into the genetically engineered host cell. In some cases, the SalSyn gene may be codon-optimized for expression in Saccharomyces cerevisiae. In some examples, the SalSyn can be N-terminally modified. The SalSyn gene can be derived from poppy, Papaver species, celandine, or another species. In some examples, the SalSyn gene can be 78% similar to the naturally occurring gene.

[0251] [SalR] In some examples, the genetically engineered host cell may modify the expression of the enzyme salutaridin reductase. Salutaridin reductase is encoded by the SalR gene. In some examples, salutaridin reductase reversibly catalyzes the reaction salutaridinol → salutaridin, as shown in Figure 7. The genetically engineered host cell may be modified to contain constitutive expression of the SalR gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell may be modified to synthetically regulate the expression of the SalR gene within the genetically engineered host cell. In one example, the genetically engineered host cell may be modified to incorporate one copy, multiple copies, or additional copies of the SalR gene. Additionally or alternatively, the genetically engineered host cell may be modified to incorporate a strong promoter element for overexpression of the SalR gene into the genetically engineered host cell. In some cases, the SalR gene may be codon-optimized for expression in Saccharomyces cerevisiae. The SalR gene can be from opium poppy, poppy, Papaver somniferum, celandine, or another species. In some instances, the SalR gene can be 80-100% similar to the naturally occurring gene.

[0252] [SalAT] In some examples, the genetically engineered host cell can modify the expression of the enzyme acetyl-CoA:sultaridinol 7-O-acetyltransferase. Acetyl-CoA:sultaridinol 7-O-acetyltransferase is encoded by the SalAT gene. In some examples, acetyl-CoA:sultaridinol 7-O-acetyltransferase catalyzes the reaction acetyl-CoA + salutaridinol → CoA + 7-O-acetylsultaridinol, as shown in Figure 7. The genetically engineered host cell can be modified to contain constitutive expression of the SalAT gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell can be modified to synthetically regulate the expression of the SalAT gene within the genetically engineered host cell. In one example, the genetically engineered host cell can be modified to incorporate one copy, multiple copies, or additional copies of the SalAT gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate a strong promoter element for overexpression of the SalAT gene into the genetically engineered host cell. In some cases, the SalAT gene can be codon-optimized for expression in Saccharomyces cerevisiae. The SalAT gene can be derived from poppy, oriental poppy, oriental poppy (Papaver orientale), a species of the genus Papaver, or another species. In some examples, the SalAT gene can be 77-80% similar to the naturally occurring gene.

[0253] [T6ODM] In some instances, the genetically engineered host cells can modify the expression of the enzyme thebaine 6-O-demethylase. Thebaine 6-O-demethylase is encoded by the T6ODM gene. In some instances, thebaine 6-O-demethylase catalyzes the reaction from thebaine to neopinone, as shown in Figure 7. Once neopinone is produced, it can be converted to codeinone. The neopinone to codeinone conversion can occur spontaneously. Alternatively, the neopinone to codeinone conversion can occur as a result of a catalyzed reaction. In other instances, the T6ODM enzyme can catalyze the O-demethylation of substrates other than thebaine. For example, T6ODM can O-demethylate oripavine to produce morphinone. Alternatively, T6ODM can catalyze the O-demethylation of 1-benzylisoquinoline, protoberberine, or protopine-type BIAs, such as papaverine, canadine, and allocryptopine, respectively. The genetically engineered host cell can be modified to contain constitutive expression of the T6ODM gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell can be modified to synthetically regulate the expression of the T6ODM gene within the genetically engineered host cell. In one example, the genetically engineered host cell can be modified to incorporate one copy, multiple copies, or additional copies of the T6ODM gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate a strong promoter element for overexpression of the T6ODM gene within the genetically engineered host cell. In some cases, the T6ODM gene can be codon-optimized for expression in Saccharomyces cerevisiae. The T6ODM gene can be derived from poppy or another species. In some instances, the T6ODM gene may be 76.2% similar to the naturally occurring gene.

[0254] [COR] In some examples, the genetically engineered host cell can modify the expression of the enzyme codeinone reductase. Codeinone reductase is encoded by the COR gene. In some examples, codeinone reductase catalyzes the reaction of codeinone to codeine, as shown in Figure 7. In some cases, codeinone reductase can catalyze the reaction of neopinone to neopine. In other examples, COR can catalyze the reduction of other morphinans, such as hydrocodone to dihydrocodeine, 14-hydroxycodeinone to 14-hydroxycodeine, and hydromorphone to dihydromorphine. The genetically engineered host cell can be modified to contain constitutive expression of a COR gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell can be modified to synthetically regulate the expression of a COR gene within the genetically engineered host cell. In one example, the genetically engineered host cell can be modified to incorporate one, multiple, or additional copies of a COR gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate a strong promoter element for overexpression of the COR gene. In some cases, the COR gene can be codon-optimized for expression in Saccharomyces cerevisiae. Additionally or alternatively, the COR gene can be modified to include targeting sequences for mitochondria, vacuoles, the endoplasmic reticulum, or a combination thereof. The COR gene can be derived from poppy or another species. In some examples, the COR gene can be 76-78% similar to a naturally occurring gene. In one example, the COR gene can be 76.8%, 77.0%, 77.3%, or 77.7% similar to a naturally occurring gene.

[0255] [CODM] In some examples, the genetically engineered host cell can modify the expression of the enzyme codeine O-demethylase. Codeine O-demethylase is encoded by the CODM gene. In some examples, codeine O-demethylase catalyzes the reaction of codeine to morphine, as shown in Figure 7. Codeine O-demethylase can also catalyze the reaction of neopine to neomorphine. Codeine O-demethylase can also catalyze the reaction of thebaine to oripavine. In other examples, CODM can catalyze the O-demethylation of 1-benzylisoquinoline, aporphine, and protoberberine-type BIAs, such as reticuline, isocolinidine, and scoureline, respectively. In other examples, the CODM enzyme can catalyze the O,O-demethylenation reaction to cleave the methylenedioxy bridge structure in protopine. The genetically engineered host cell can be modified to contain constitutive expression of the CODM gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell can be modified to synthetically regulate the expression of the CODM gene within the genetically engineered host cell. In one example, the genetically engineered host cell can be modified to incorporate one, multiple, or additional copies of the CODM gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate a strong promoter element for overexpression of the CODM gene within the genetically engineered host cell. In some cases, the CODM gene can be codon-optimized for expression in Saccharomyces cerevisiae. Additionally or alternatively, the CODM gene can be modified to have the addition of a mitochondrial targeting sequence. The CODM gene can be derived from poppy, a Papaver species, or another species. In some examples, the CODM gene can be 75% similar to the naturally occurring gene. In one example, the CODM gene can be 75.2% similar to the naturally occurring gene.

[0256] [BBE] In some examples, the genetically engineered host cell can modify the expression of the enzyme berberine bridge enzyme. The berberine bridge enzyme is encoded by the BBE gene. In some examples, the berberine bridge enzyme catalyzes the reaction (S)-reticuline → (S)-scoureline. The genetically engineered host cell can be modified to contain constitutive expression of the BBE gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell can be modified to synthetically regulate the expression of the BBE gene within the genetically engineered host cell. In one example, the genetically engineered host cell can be modified to incorporate one copy, multiple copies, or additional copies of the BBE gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate a strong promoter element for overexpression of the BBE gene into the genetically engineered host cell. The BBE gene can be from Papaver somnifera, Papaver somnifera, California poppy, Berberis stolonifera, Thalictrum flavum subsp. glaucum, Coptis japonica, Papaver somnifera, or another species. In some instances, the BBE gene can be 99% similar to a naturally occurring gene.

[0257] [S9OMT] In some examples, the genetically engineered host cell can modify the expression of the enzyme S-adenosyl-L-methionine:(S)-scourerine 9-O-methyltransferase. S-adenosyl-L-methionine:(S)-scourerine 9-O-methyltransferase is encoded by the S9OMT gene. In some examples, S-adenosyl-L-methionine:(S)-scourerine 9-O-methyltransferase catalyzes the reaction S-adenosyl-L-methionine + (S)-scourerine → S-adenosyl-L-homocysteine ​​+ (S)-tetrahydrocolumbamine. The genetically engineered host cell can be modified to contain constitutive expression of the S9OMT gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell can be modified to synthetically regulate the expression of the S9OMT gene within the genetically engineered host cell. In one example, the genetically engineered host cell can be modified to incorporate one, multiple, or additional copies of the S9OMT gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate a strong promoter element for overexpression of the S9OMT gene into the genetically engineered host cell. In some cases, the S9OMT gene can be codon-optimized for expression in Saccharomyces cerevisiae. The S9OMT gene can be derived from Thalictrum flavum glaucum, Coptis chinensis, Coptis orientalis, Papaver somniferum, Coptis spp., Papaver somniferum, or another species. In some examples, the S9OMT gene can be 100% similar to the naturally occurring gene. In one example, the S9OMT gene can be 80% similar to the naturally occurring gene.

[0258] [CAS] In some examples, the genetically engineered host cell may modify the expression of the enzyme (S)-canadine synthase. (S)-canadine synthase is encoded by the CAS gene. In some examples, (S)-canadine synthase catalyzes the reaction (S)-tetrahydrocolumbamine → (S)-canadine. The genetically engineered host cell may be modified to express the CAS gene within the genetically engineered host cell. The genetically engineered host cell may be modified to contain constitutive expression of the CAS gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell may be modified to synthetically regulate the expression of the CAS gene within the genetically engineered host cell. In one example, the genetically engineered host cell may be modified to incorporate one, multiple, or additional copies of the CAS gene. Additionally or alternatively, the genetically engineered host cell may be modified to incorporate a strong promoter element for overexpression of the CAS gene into the genetically engineered host cell. The CAS gene can be from Thalictrum flavum glaucum, Coptis japonica, Larix spp., Coptis spp., or another species. In some instances, the CAS gene can be 100%.

[0259] [STOX] In some examples, the genetically engineered host cell may modify the expression of the enzyme (S)-tetrahydroprotoberberine oxidase. (S)-tetrahydroprotoberberine oxidase is encoded by the STOX gene. In some examples, (S)-tetrahydroprotoberberine oxidase catalyzes the reaction (S)-tetrahydroberberine + 2 O → berberine + 2 H O. The genetically engineered host cell may be modified to contain constitutive expression of the STOX gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell may be modified to synthetically regulate the expression of the STOX gene within the genetically engineered host cell. In one example, the genetically engineered host cell may be modified to incorporate one copy, multiple copies, or additional copies of the STOX gene. Additionally or alternatively, the genetically engineered host cell may be modified to incorporate a strong promoter element for overexpression of the STOX gene within the genetically engineered host cell. In some examples, STOX may be modified at the N-terminus. In some cases, the STOX gene can be codon-optimized for expression in S. cerevisiae. The STOX gene can be derived from Berberis wilsonii, Coptis japonica, Berberis species, Coptis species, or another species. In some cases, the STOX gene can be 78% similar to the naturally occurring gene.

[0260] [TNMT] In some instances, the genetically engineered host cell may modify the expression of the enzyme tetrahydroprotoberberine-N-methyltransferase. Tetrahydroprotoberberine-N-methyltransferase is encoded by the TNMT gene. In some instances, tetrahydroprotoberberine-N-methyltransferase catalyzes the reaction canadine → N-methylcanadine. In some instances, tetrahydroprotoberberine-N-methyltransferase catalyzes the reaction noroxymorphone → naloxone, as shown in Figure 26.

[0261] In another example, tetrahydroprotoberberine-N-methyltransferase catalyzes the reaction of stylopine to cis-N-methylstylopine. The genetically engineered host cell can be modified to contain constitutive expression of the TNMT gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell can be modified to synthetically regulate the expression of the TNMT gene within the genetically engineered host cell. In one example, the genetically engineered host cell can be modified to incorporate one copy, multiple copies, or additional copies of the TNMT gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate a strong promoter element for overexpression of the TNMT gene within the genetically engineered host cell. In some cases, the TNMT gene can be codon-optimized for expression in Saccharomyces cerevisiae. The TNMT gene can be derived from Papaver somniferum, Poppy japonica, Poppy japonica, or another species. In some examples, the TNMT gene can be 100% similar to a naturally occurring gene. In one example, the TNMT gene may be 81% similar to the naturally occurring gene.

[0262] [CFS] In some examples, the genetically engineered host cell can modify the expression of the enzyme cheilanthifoline synthase. Cheilanthifoline synthase is encoded by the CFS gene. In one example, cheilanthifoline synthase catalyzes the reaction scourerine → cheilanthifoline. The genetically engineered host cell can be modified to contain constitutive expression of the CFS gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell can be modified to synthetically regulate the expression of the CFS gene within the genetically engineered host cell. In one example, the genetically engineered host cell can be modified to incorporate one copy, multiple copies, or additional copies of the CFS gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate a strong promoter element for overexpression of the CFS gene within the genetically engineered host cell. The CFS gene can be derived from poppy, California poppy, American poppy (A. mexicana), or another species. In some instances, the CFS gene may be 77%, 78% or 79% similar to the naturally occurring gene. Additionally, the CFS gene may be codon optimized for expression in S. cerevisiae.

[0263] [STS] In some examples, the genetically engineered host cell can modify the expression of the enzyme stylopine synthase. Stylopine synthase is encoded by the STS gene. In one example, stylopine synthase catalyzes the reaction cheilanthifoline → stylopine. The genetically engineered host cell can be modified to contain constitutive expression of the STS gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell can be modified to synthetically regulate the expression of the STS gene within the genetically engineered host cell. In one example, the genetically engineered host cell can be modified to incorporate one copy, multiple copies, or additional copies of the STS gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate a strong promoter element for overexpression of the STS gene within the genetically engineered host cell. The STS gene can be derived from opium poppy, California poppy, American poppy, or another species. In some instances, the STS gene may be 76%, 78%, or 79% similar to the naturally occurring gene. Additionally, the STS gene may be codon-optimized for expression in S. cerevisiae.

[0264] [MSH] In some examples, the genetically engineered host cell can modify the expression of the enzyme cis-N-methylstylopine 14-hydroxylase. cis-N-methylstylopine 14-hydroxylase is encoded by the MSH gene. In one example, cis-N-methylstylopine 14-hydroxylase catalyzes the reaction cis-N-methylstylopine → protopine. The genetically engineered host cell can be modified to contain constitutive expression of the MSH gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell can be modified to synthetically regulate the expression of the MSH gene within the genetically engineered host cell. In one example, the genetically engineered host cell can be modified to incorporate one copy, multiple copies, or additional copies of the MSH gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate a strong promoter element for overexpression of the MSH gene into the genetically engineered host cell. The MSH gene can be derived from poppy or another species. In some instances, the MSH gene may be 79% similar to the naturally occurring gene. Additionally, the MSH gene may be codon-optimized for expression in S. cerevisiae.

[0265] [P6H] In some examples, the genetically engineered host cell can modify the expression of the enzyme protopine-6-hydroxylase. Protopine-6-hydroxylase is encoded by the P6H gene. In one example, protopine-6-hydroxylase catalyzes the reaction protopine → 6-hydroxyprotopine. The genetically engineered host cell can be modified to contain constitutive expression of the P6H gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell can be modified to synthetically regulate the expression of the P6H gene within the genetically engineered host cell. In one example, the genetically engineered host cell can be modified to incorporate one copy, multiple copies, or additional copies of the P6H gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate a strong promoter element for overexpression of the CFS gene into the genetically engineered host cell. The P6H gene can be derived from poppy, California poppy, or another species. In some examples, the P6H gene can be 79% similar to the naturally occurring gene. Additionally, the P6H gene can be codon-optimized for expression in S. cerevisiae.

[0266] [DBOX] In some examples, the genetically engineered host cell can modify the expression of the enzyme dihydrobenzophenanthridine oxidase. Dihydrobenzophenanthridine oxidase is encoded by the DBOX gene. In one example, dihydrobenzophenanthridine oxidase catalyzes the reaction dihydrosanguinarine → sanguinarine. The genetically engineered host cell can be modified to contain constitutive expression of the DBOX gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell can be modified to synthetically regulate the expression of the DBOX gene within the genetically engineered host cell. In one example, the genetically engineered host cell can be modified to incorporate one copy, multiple copies, or additional copies of the DBOX gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate a strong promoter element for overexpression of the DBOX gene into the genetically engineered host cell. The DBOX gene can be derived from poppy or another species. In some examples, the DBOX gene can be 100% similar to a naturally occurring gene. Furthermore, the DBOX gene can be codon-optimized for expression in Saccharomyces cerevisiae.

[0267] [morA] In some examples, the genetically engineered host cell may modify the expression of the enzyme morphine dehydrogenase. Morphine dehydrogenase is encoded by the morA gene. In some examples, morphine dehydrogenase catalyzes the morphine → morphinone reaction, as shown in Figure 8. In other examples, morphine dehydrogenase catalyzes the codeinone → codeine reaction, also shown in Figure 8. Figure 8 shows a biosynthetic scheme for the production of semisynthetic opioids (opioids) according to embodiments of the present invention. In particular, Figure 8 shows the extended transformation of thebaine in yeast by incorporating morA, morphine dehydrogenase; and morB, morphine reductase. Figure 30 shows further transformation of thebaine according to embodiments of the present invention.

[0268] The genetically engineered host cell can be modified to contain constitutive expression of the morA gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell can be modified to synthetically regulate expression of the morA gene within the genetically engineered host cell. In one example, the genetically engineered host cell can be modified to incorporate one, multiple, or additional copies of the morA gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate a strong promoter element for overexpression of the morA gene within the genetically engineered host cell. In some cases, the morA gene can be codon-optimized for expression in Saccharomyces cerevisiae. The morA gene can be derived from Pseudomonas putida or another species. In some examples, the morA gene can be 73.7% similar to the naturally occurring gene.

[0269] [morB] In some examples, the genetically engineered host cell may modify the expression of the enzyme morphinone reductase. Morphinone reductase is encoded by the morB gene. In some examples, morphinone reductase catalyzes the reaction codeinone → hydrocodone, as shown in Figure 8. In other examples, morphinone reductase catalyzes the reaction morphinone → hydromorphone, also shown in Figure 8. In other examples, morphinone reductase catalyzes the reaction 14-hydroxycodeinone → oxycodone. The genetically engineered host cell may be modified to include constitutive expression of the morB gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell may be modified to synthetically regulate expression of the morB gene within the genetically engineered host cell. In one example, the genetically engineered host cell may be modified to incorporate one, multiple, or additional copies of the morB gene. Additionally or alternatively, the engineered host cell can be modified to incorporate a strong promoter element for overexpression of the morB gene. In some cases, the morB gene can be codon-optimized for expression in S. cerevisiae. The morB gene can be from Pseudomonas putida or another species. In some cases, the morB gene can be 67.2% similar to the naturally occurring gene.

[0270] [CYP80A1] In some examples, the genetically engineered host cell can express the enzyme berbamunine synthase. Berbamunine synthase is encoded by the cytochrome P450 enzyme 80A1 (CYP80A1) gene. In some examples, CYP80A1 catalyzes the reaction (S)-N-methylcoclaurine + (R)-N-methylcoclaurine → berbamunine. In other examples, CYP80A1 catalyzes the reaction (R)-N-methylcoclaurine + (R)-N-methylcoclaurine → guattegaumerine. In other examples, CYP80A1 catalyzes the reaction (R)-N-methylcoclaurine + (S)-coclaurine → 2'-norberbamunine. The genetically engineered host cell can be modified to include constitutive expression of the CYP80A1 gene in the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell can be modified to synthetically regulate the expression of the CYP80A1 gene within the genetically engineered host cell. In one example, the genetically engineered host cell can be modified to incorporate one, multiple, or additional copies of the CYP80A1 gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate a strong promoter element for overexpression of the CYP80A1 gene within the genetically engineered host cell. In some cases, the CYP80A1 gene can be codon-optimized for expression in Saccharomyces cerevisiae. The CYP80A1 gene can be derived from Berberis stolonifera or another species. In some examples, the CYP80A1 gene can be 76% similar to the naturally occurring gene.

[0271] [PODA] In some examples, the genetically engineered host cell can express the enzyme protopine O-dealkylase. Protopine O-dealkylase is encoded by the gene PODA. In some examples, PODA catalyzes the O,O-demethylation of protoberberine and protopine, such as canadine, stylopine, berberine, cryptopine, allocryptopine, and protopine. In some examples, PODA catalyzes the O-demethylation of BIA, such as tetrahydropapaverine, tetrahydropalmatine, and cryptopine. The genetically engineered host cell can be modified to contain constitutive expression of the PODA gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell can be modified to synthetically regulate the expression of the PODA gene within the genetically engineered host cell. In one example, the genetically engineered host cell can be modified to incorporate one copy, multiple copies, or additional copies of the PODA gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate a strong promoter element for overexpression of the PODA gene. In some cases, the PODA gene can be codon-optimized for expression in Saccharomyces cerevisiae. The PODA gene can be derived from poppy or other species. In some cases, the PODA gene can be 70-100% similar to the naturally occurring gene.

[0272] [BM3] In some instances, engineered host cells can express the enzyme BM3. BM3 is a Bacillus megaterium cytochrome P450 involved in monooxygenating fatty acids in the native host. In some cases, BM3 N-demethylates opioids to produce nor-opioids, as shown in Figure 27. It is also readily expressed as an active heterologous enzyme in yeast and bacteria. BM3 has several advantages as a biosynthetic enzyme, such as being soluble, being associated with a fusion reductase partner protein, and being easily engineered to accept new substrates. Additionally, Table 6 shows BM3 N-demethylase variants.

[0273] The exemplary genes described above can be expressed from several different platforms within a host cell, such as plasmids (2μ, ARS / CEN), YACs, or genomes. Additionally, the exemplary gene sequences described above can be either native or codon-optimized for expression in a desired heterologous host (e.g., Saccharomyces cerevisiae). [Example]

[0274] The following examples are presented for the purpose of illustrating various aspects of the present invention and are not meant to limit the invention in any manner. The examples, along with the methods described herein, are presently preferred representative embodiments, are illustrative, and are not intended to limit the scope of the invention. Modifications in the examples and other uses will occur to those skilled in the art that are encompassed within the spirit of the invention, as defined by the scope of the claims.

[0275] Example 1: Tyrosine hydroxylase mutants improve reticuline production in genetically engineered yeast strains Tyrosine hydroxylase from R. norvegicus was codon-optimized for yeast, synthesized, and cloned into a low-copy-number plasmid. Single mutants (W166Y, E332D, S40D, and R37ER38E), double mutants (W166Y and E332D, W166Y and S40D, and W166Y and R37ER38E), and one triple mutant (W166Y, R37ER38E, and E332D) were generated by site-directed mutagenesis. Each TyrH mutant was transfected with a low-copy-number plasmid carrying the GPD promoter, along with the following mutations for central metabolism (described in U.S. Provisional Patent Application No. 61 / 899,496): ARO4 FBR, ΔZWF1, and expressed in yeast strains containing the GPD-TKL1 promoter exchange. This strain also contains a chromosomally integrated copy of DOPA decarboxylase (DODC) from P. putida, four chromosomally integrated genes from Norway rat (pyruvoyltetrahydropterin synthase, PTPS; sepiapterin reductase, SepR; pterin 4a-carbinolamine dehydratase, PCD; and dihydropteridine reductase, QDHPR) that produce the cosubstrate tetrahydrobiopterin, a Coptis japonica norcoclaurine synthase (NCS) expressed by a low-copy-number plasmid carrying the GPD promoter, and five genes for the biosynthesis of reticuline from norcoclaurine (opium poppy 6-O-methyltransferase, Ps6OMT; opium poppy coclaurine N-methyltransferase, PsCNMT; poppy California poppy cytochrome P450 80B1, EcCYP80B1; and poppy California poppy cytochrome P450 80B2). We expressed NADPH reductase, PsCPR; and poppy 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase, Ps4'OMT. Strains harboring the TyrH mutants were cultured in a selective defined medium (YNB) containing 2% dextrose and no tyrosine for 96 hours, and the production of reticuline in the medium was measured by LC-MS / MS in MRM mode for the transition from 330 m / z to 137 m / z. Figure 9 shows the results of this assay, demonstrating that the TyrH mutants can improve reticuline production by as much as five-fold compared to wild-type TyrH. Thus, Figure 9 illustrates tyrosine hydroxylase mutants that improve reticuline production from sugars in engineered yeast strains, according to an embodiment of the present invention.

[0276] Example 2: Expression of DHFR improves tyrosine hydroxylase activity in genetically engineered yeast strains Dihydrofolate reductase (DHFR) from Norway rat was codon-optimized for yeast, synthesized, and cloned into a low-copy-number plasmid under the control of the GPD promoter. DHFR was codon-optimized for yeast, along with wild-type RnTyrH (a low-copy-number plasmid with the GPD promoter) and the following mutations to central metabolism (as described in U.S. Provisional Patent Application No. 61 / 899,496): ARO4 FBR , ΔZWF1, and GPD-TKL1 promoter exchanges were co-expressed in a yeast strain containing the GPD-TKL1 promoter exchange. This strain also expressed four chromosomally integrated genes from Rattus norvegicus (pyruvoyltetrahydropterin synthase, PTPS; sepiapterin reductase, SepR; pterin 4a-carbinolamine dehydratase, PCD; and dihydropteridine reductase, QDHPR) that produce the cosubstrate tetrahydrobiopterin. Strains expressing DHFR and wild-type RnTyrH were grown for 96 hours in selective defined medium (YNB) without tyrosine containing 2% dextrose, and L-DOPA production in the medium was measured by LC-MS / MS in MRM mode for the transition from 198 m / z to 152 m / z. Expression of DHFR with wild-type RnTyrH increased L-DOPA production by 1.8-fold, as shown in Figure 10. Thus, FIG. 10 shows co-expression of dihydrofolate reductase (DHFR) to improve L-DOPA production by tyrosine hydroxylase in a genetically engineered yeast strain, according to an embodiment of the present invention.

[0277] Example 3: Addition of antioxidants to growth medium improves tyrosine hydroxylase activity in genetically engineered yeast strains The following mutations to central metabolism (described in U.S. Provisional Patent Application No. 61 / 899,496): ARO4 FBRYeast strains containing the GPD-TKL1 promoter exchange, ΔZWF1, and GPD-TKL1 promoter exchanges, and expressing four chromosomally integrated genes from Norway rat (pyruvoyltetrahydropterin synthase, PTPS; sepiapterin reductase, SepR; pterin 4a-carbinolamine dehydratase, PCD; dihydropteridine reductase, QDHPR) that produce the cosubstrate tetrahydrobiopterin, as well as wild-type RnTyrH via a low-copy-number plasmid under the control of the GPD promoter, were grown for 96 hours in selective defined medium (YNB) without tyrosine, containing 2% galactose and 2 mM ascorbic acid.

[0278] L-DOPA production in the medium was measured by LC-MS / MS in MRM mode for the transition from 198 m / z to 152 m / z. Addition of 2 mM ascorbic acid improved L-DOPA production by wild-type RnTyrH by 1.8-fold. The concentrations of BH4 intermediates were also measured by LC-MS / MS in MRM mode for the following transitions: B, 238 m / z to 178 m / z; BH2, 240 m / z to 165 m / z; and BH4, 242 m / z to 166 m / z. The addition of ascorbic acid increased BH4 in the medium, indicating that the oxidation of BH4 to BH2 was inhibited.

[0279] Thus, Figure 11A shows the addition of antioxidants to the culture medium to improve L-DOPA production by tyrosine hydroxylase in genetically engineered yeast strains, and (B) the addition of antioxidants to the culture medium to increase BH4 levels, according to an embodiment of the present invention. In particular, Figure 11A shows that wild-type RnTyrH (expressed by a low copy number plasmid under the control of the GPD promoter) exhibits the following mutations to central metabolism (as described in U.S. Provisional Patent Application No. 61 / 899,496): ARO4 FBRThe wild-type RnTyrH was expressed in a yeast strain containing the ΔZWF1, ΔZWF2, and GPD-TKL1 promoter exchanges. This strain also expressed four chromosomally integrated genes from Rattus norvegicus (pyruvoyltetrahydropterin synthase, PTPS; sepiapterin reductase, SepR; pterin 4a-carbinolamine dehydratase, PCD; and dihydropteridine reductase, QDHPR) that produce the cosubstrate tetrahydrobiopterin. The strain expressing wild-type RnTyrH was cultured for 96 hours in selective defined medium (YNB) containing 2% dextrose and tyrosine-free with and without 2 mM ascorbic acid (aa). L-DOPA production in the medium was measured by LC-MS / MS in MRM mode for the transition from 198 m / z to 152 m / z. Furthermore, Figure 11B shows that the concentration of BH4 intermediates in the medium of the same strains shown in Figure 11A grown with and without 2 mM ascorbic acid (aa) was measured in LC-MS / MS MRM mode at the following transition: BH4, 242 m / z to 166 m / z.

[0280] Example 4. Identification of epimerase enzymes To identify suitable epimerase enzymes for carrying out the epimerization reaction of the methods disclosed herein, a cytochrome P450 oxidase 82Y1-like domain and a codeinone reductase-like domain were identified within one open reading frame (CYP-COR) of the publicly available plant transcriptome. This CYP-COR fusion was identified by BLAST searches of the 1000 Plants Project (Matasci, et al. 2014. Gigascience. 3: 17) and PhytoMetaSyn (Facchini, et al. 2012. Trends Biotechnol. 30: 127-31; Xiao, et al. 2013. J. Biotechnol. 166: 122-34) transcriptomes using blastn with a query sequence of a previously reported COR-silencing VIGS construct that leads to reticuline accumulation (Wijekoon and Facchini 2012. Plant J. 69: 1052-63). When a CYP-COR fusion sequence was observed as a hit, the sequence was translated and its amino acid sequence was used as a query for a second search of both databases using tblastn. A phylogenetic tree of CYP-COR fusion enzymes identified from databases is shown in Figure 13. Sequences were identified based on bioinformatics searches from the 1000 Plants Project and PhytoMetaSyn transcriptome databases. Furthermore, an example amino acid sequence, as discussed above, is shown in Figure 4. Furthermore, Table 1 lists various examples of amino acid sequences identified for this CYP-COR enzyme from various plants, such as opium poppy, poppy of Troy, Iranian poppy, and greater celandine.

[0281] Example 5. Epimerization of (S)-Reticuline to (R)-Reticuline in Genetically Engineered Non-Plant Host Cells Non-plant host cells have been genetically engineered to heterologously express the enzymes described herein. For example, a yeast strain (Saccharomyces cerevisiae) was genetically engineered to heterologously express the identified epimerase described in Example 4 and to confirm its function in the context of this microbial host. Yeast-codon-optimized DNA coding sequences for amino acid subsequences pbr.PBRST1PF_4328 and pbr.PBRST1PF_89405 were synthesized in-frame with the yeast-codon-optimized coding sequence for amino acids 1-40 of SSDU-2015634 (Table 1), generating CYP-COR_4328 and CYP-COR_89405, respectively. These CYP-COR coding sequences were cloned into a low-copy-number plasmid with a URA3 selectable marker and expressed under the TDH3 promoter. This plasmid contained a chromosomally integrated expression cassette for cytochrome P450 reductase (P TEF1 -ATR1 or P TEF1 A yeast strain containing the β-reticuline-binding protein (β-PsCPRv2) was transformed. The yeast strain containing the two plasmids was cultured in a synthetic complete medium containing a dedicated dropout solution (-Ura-Trp). The yeast strain was fed with (S)-reticuline, and BIA metabolites were analyzed by LC-MS / MS analysis after 72 hours of culture.

[0282] Example 6. Production of salutaridine from (S)-reticuline in genetically engineered yeast cells A yeast strain (Saccharomyces cerevisiae) was genetically engineered to heterologously express the identified epimerase described in Example 4 and to confirm its function in the context of this microbial host. Yeast-codon-optimized DNA coding sequences for amino acid subsequences pbr.PBRST1PF_4328 and pbr.PBRST1PF_89405 were synthesized in frame with the yeast-codon-optimized coding sequence for amino acids 1–40 of SSDU-2015634 (Table 1), generating CYP-COR_4328 and CYP-COR_89405, respectively. These CYP-COR coding sequences were cloned into a low-copy-number plasmid with a URA3 selectable marker and expressed by the TDH3 promoter. The salutaridine synthase (SalSyn) coding sequence was cloned into a low-copy-number plasmid with a TRP1 selectable marker and expressed by the TDH3 promoter. This plasmid contained a chromosomally integrated expression cassette for cytochrome P450 reductase (P TEF1 -ATR1 or P TEF1 A yeast strain containing the α-reticuline-binding domain (α-PsCPRv2) was transformed. This yeast strain containing the two plasmids was cultured in synthetic complete medium containing a dedicated dropout solution (-Ura-Trp). The yeast strain was fed with (S)-reticuline, and BIA metabolites were analyzed by LC-MS / MS analysis after 72 hours of culture. This analysis demonstrated that the engineered yeast cells were able to convert (S)-reticuline to (R)-reticuline, which was then acted upon by salutaridine synthase to form the tetracyclic promorphinan alkaloid salutaridine (Figures 7 and 14). Salutaridine synthase has previously been shown to act on (R)-reticuline and show no observable activity against (S)-reticuline (Gesell, et al. 2009. J. Biol. Chem. 284: 24432-42).

[0283] As shown in Figure 7, CYP-COR catalyzes the conversion of (S)-reticuline to (R)-reticuline, which is then acted upon by salutaridine synthase to produce the promorphinan alkaloid salutaridine. Figure 14 shows (A) a chromatogram showing reticuline and salutaridine with two epimerase mutants (CYP-COR_89405, CYP-COR_4328) and a standard. Figure 14 also shows (B) the same chromatogram of salutaridine in (A) replotted to show coelution with the standard. In this experiment, yeast contain two low-copy CEN / ARS plasmids carrying URA3 and TRP1 selectable markers, a TDH3 promoter, and CYP-COR and SalSyn coding sequences. Yeast were grown overnight in 3 mL of selective medium from a freshly transformed colony, back-diluted in 3.5 mL of medium to an OD of 0.8, grown for 7 hours, pelleted, and then resuspended in HEPES buffer, pH 7.4, containing 100 μM (S)-reticuline (Specs). After 16 hours at 30°C on a spinner, the yeast were pelleted and the buffer supernatant was analyzed by LC-MS / MS. Each figure is from one representative sample out of two. Peaks are normalized to 100% for the largest peak in all chromatograms.

[0284] Example 7. Production of (R)-reticuline from racemic norlaudanosoline in genetically engineered non-plant host cells A yeast strain (Saccharomyces cerevisiae) was genetically engineered to heterologously express the identified epimerase described in Example 4 and to confirm its function in the context of this microbial host. The yeast-codon-optimized DNA coding sequence CYP-COR_89405 described in Example 5 was cloned into a low copy number plasmid with a URA3 selectable marker and expressed under the TDH3 promoter. This plasmid encodes a chromosomally integrated cytochrome P450 reductase (P TEF1 -ATR1 or P TEF1-PsCPRv2) and an expression cassette for three methyltransferases (norcoclaurine-6-O-methyltransferase, coclaurine N-methyltransferase, and 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase from poppy, all P TEF1A yeast strain was transformed with norlaudanosoline (expressed by ). The yeast strain carrying this plasmid was grown in synthetic complete medium containing a proprietary dropout solution (-Ura). The yeast strain was fed racemic norlaudanosoline, and BIA metabolites were analyzed by LC-MS / MS analysis after 72 hours of growth. For chiral characterization, reticuline was concentrated from the yeast medium by pelleting 5 mL of yeast culture, adding 120 mg of XAD-4 resin to 4 mL of the supernatant, incubating overnight at room temperature on a rotating device, and eluting with 0.5 mL of methanol. The concentrate was fractionated by reverse-phase HPLC (Pursuit XRs-C18, 5 μm, 50 mm x 10 mm) using isocratic 15% methanol containing 0.1% formic acid over 6.5 minutes at a flow rate of 5 mL / min and an injection volume of 40–50 μL. Fractions were collected based on the peak at approximately 4.5 minutes. Fractions were pooled, lyophilized, and resuspended in 0.5 mL of isopropanol. Depending on the concentration, 0.5–5 μL was injected onto a chiral column (Phenomenex Lux Cellulose-1, 3 μm, 150 mm × 2 mm) and separated isocratically using 72% n-hexane, 28% isopropanol, and 0.1% diethylamine at a flow rate of 0.3 mL / min. Detection was performed by mass spectrometry (MS) and UV at 250 nm using an Agilent 6320 Ion Trap mass spectrometer with an ESI source gas temperature of 350 °C, a gas flow of 10 L / min, a nebulizer pressure of 40 PSI, and an isolation band width of 1.0 at m / z 330.1. The retention time of the reticuline peak was compared to that of authentic (S)-reticuline and (R)-reticuline standards. This analysis showed that engineered yeast cells containing the CYP-COR plasmid were able to convert racemic norlaudanosoline to (R)-reticuline, whereas engineered yeast cells carrying the empty plasmid exclusively produced (S)-reticuline (Figure 15).

[0285] Example 8: Protein engineering of salutaridine synthase to improve processing and activity when expressed in a microbial host Heterologous proteins may be processed incorrectly when expressed in a recombinant host (e.g., expression of plant proteins such as cytochrome P450 enzymes in a microbial production host). For example, salutaridine synthase, which converts (R)-reticuline to salutaridine, undergoes N-linked glycosylation when heterologously expressed in yeast (Figures 16A and 16B). The N-linked glycosylation pattern observed for salutaridine synthase is not observed when the enzyme is expressed in plants, indicating incorrect N-terminal sorting of the nascent SalSyn transcript, which reduces the activity of the enzyme in a heterologous microbial host. Therefore, protein engineering directed at correcting the N-terminal sorting of the nascent transcript and thereby removing the N-linked glycosylation pattern will result in improved activity of the salutaridine synthase enzyme in a recombinant production host.

[0286] For example, the N-terminal α-helix of cheilanthifoline synthase (CFS) was used in place of the N-terminal α-helix of salutaridine synthase (SalSyn, Figure 17). The fusion junctions were selected based on the secondary structure motifs of CFS and SalSyn or on the amino acid alignment of CFS and SalSyn. Fusions were cloned by amplifying the N-terminal fragment of CFS and the C-terminal fragment of SalSyn so that they overlapped with each other by 15–40 nucleotides, and then assembled with each other and the vector backbone by Gibson assembly to form the complete fusion open reading frame (Gibson, et al. 2009. Nat Methods. 6: 343–5).

[0287] As another example, the coding sequence for the cytochrome P450 domain of salutaridine synthase was placed directly within the P450-coding region of another stably expressed cytochrome P450, such as the BM3 enzyme. For example, the conserved cytochrome P450 domain of salutaridine synthase and the cytochrome P450 domain of a genetically engineered mutant of Bacillus megaterium P450 monooxygenase CYP102A1 (BM3, (Michener and Smolke, 2012, Metab. Eng. 14: 306-16)) were identified by an NCBI conserved domain search. Primers were designed to fuse the coding sequence for the first few amino acids of BM3 to the coding sequence for the P450 domain of salutaridine synthase, followed by the coding sequence for the BM3 domain C-terminal to the P450 domain. As before, this construct was assembled by Gibson assembly.

[0288] The engineered salutaridine synthase protein fusions were analyzed by Western blot analysis to confirm full-length expression in yeast and modification or loss of N-linked glycosylation patterns (Figures 16A and 16B). The salutaridine synthase enzyme and protein fusions were C-terminally tagged with a human influenza hemagglutinin (HA) epitope and cloned into expression plasmids appropriate for yeast and plant expression. In yeast, the enzyme coding sequence was cloned into a low-copy-number yeast / Escherichia coli shuttle vector with a URA3 selectable marker and expressed under the TDH3 promoter. In plants, the sequence was cloned into an E. coli / Agrobacterium tumefaciens shuttle vector with a cauliflower mosaic virus (CaMV) 35S promoter with kanamycin resistance and flanking 5' and 3' untranslated regions derived from cowpea mosaic virus RNA-2 for transient expression in plants via Agrobacterium tumefaciens infiltration. Yeast engineered to express salutaridine synthase exhibited a banding pattern indicative of N-linked glycosylation. We confirmed that this pattern was due to N-linked glycosylation, which was achieved by site-directed mutagenesis at the glycosylation site. In contrast, plant expression of this enzyme did not result in a banding pattern indicative of N-linked glycosylation, as seen in Figure 16A. Although the N-linked glycosylation site was unmodified, the engineered salutaridine synthase protein fusion was not N-glycosylated when expressed in yeast, as seen in Figure 16B. By Western blot, we demonstrated that the yeast-expressed fusion enzyme appeared as a single band, similar to the expression observed for the plant-expressed parent enzyme, indicating that the engineered fusion had corrected the misprocessing of the protein during production in yeast, which resulted in N-linked glycosylation.

[0289] The improved enzyme activity of engineered salutaradine synthase protein fusions was analyzed when heterologously expressed in yeast. The coding sequences for salutaradine synthase and the engineered fusions were cloned into a low-copy-number plasmid with a URA3 selectable marker and expressed under the TDH3 promoter. Yeast was transformed with P311 integrated into the TRP1 locus. TEF1 The -PsCPRv2 gene contains a single low-copy-number plasmid carrying the URA3 selectable marker and the salutalazine synthase coding sequence with the TDH3 promoter. Yeast were grown overnight in 1 mL of selective medium (-Ura) from a freshly transformed colony and diluted 1:20 back into 0.5 mL of selective medium containing 10∝M(R)-reticuline (Toronto Research Chemicals) in a 96-well plate. After 72–96 h in a shaking incubator, yeast were pelleted and the culture supernatant was analyzed by LC-MS / MS. This analysis demonstrated that the engineered salutalazine synthase enzyme exhibited improved activity compared to that of the wild-type sequence when heterologously expressed in yeast (Figure 18).

[0290] Figure 18 shows codon-optimized and engineered fusions of salutaridine synthase to improve activity in yeast according to an embodiment of the present invention. As seen in Figure 18, the black bars represent the native wild-type sequence of salutaridine synthase PsCYP719B1. The gray bars with black outlines are yeast-codon-optimized mutants from Papaver somniferum and newly identified sequences from Papaver somniferum. The bars with diagonal lines represent the most improved engineered fusions based on the Papaver somniferum sequence. The error bars indicate the range of at least two biological replicates. Natural, synthetic, codon-optimized and / or protein-engineered variants of salutaridine synthase from Papaver somniferum, Papaver somniferum, or Papaver somniferum (or related plants) can be used in such engineered strains.

[0291] Engineered salutaridine synthase protein fusions can be used in the context of biosynthetic pathways to increase production of downstream benzylisoquinoline alkaloid products. In one example, yeast was engineered to heterologously express yeast-codon-optimized genes encoding an engineered salutaridine synthase fusion, a poppy salutaridine reductase, and a poppy salutaridinol 7-O-acetyltransferase. Three expression cassettes (P TDH3- D94yPsSS, P TPI1 -yPbSalR, P TEF1 The YAC was assembled into a yeast artificial chromosome (YAC) containing a TRP1 section marker and an expression cassette for cytochrome P450 reductase (P TEF1 -ATR1 or P TEF1 The yeast strains were cultured in synthetic complete medium containing a proprietary dropout solution (-Trp) and fed with (R)-reticuline. BIA metabolites were analyzed by LC-MS / MS analysis after 96 hours of culture. This analysis shows that the morphinan alkaloid thebaine is produced in yeast strains engineered to contain the engineered salutaridine synthase enzyme and other pathway enzymes, as shown in Figure 19(A).

[0292] Thus, Figure 19 (A) shows an LC / MS-MS analysis of a small-scale batch fermentation in which genetically engineered yeast catalyzed the conversion of (R)-reticuline to thebaine, according to an embodiment of the present invention. As shown in Figure 19 (A), the yeast strain contained a P TEF1 - A single yeast artificial chromosome carrying an ATR1 expression cassette and carrying a TRP1 selection marker, as well as three expression cassettes: TDH3- yEcCFS 1-83 -yPsSS 95-505 , P TPI1 -yPbSalR, and P TEF1The strain was engineered to contain -yPsSalAT. Yeast were grown overnight in 3 mL of selective medium from a freshly transformed colony and diluted 1:20 back into 0.5 mL of selective medium (-Trp) in a culture tube with 100 μM (R)-reticuline (Toronto Research Chemicals). After 72 h in a shaking incubator, the yeast were pelleted and the culture supernatant was analyzed by LC-MS / MS. Chromatograms show the thebaine produced by this strain, as well as the accumulated salutaridinol and salutaridin, along with standards. These figures are representative of two samples.

[0293] Example 9: Protein engineering of downstream morphinan branching enzymes to improve production of morphinan products by heterologous microbial hosts In one embodiment of the present invention, pathway enzymes are engineered to exhibit increased activity to enhance production of a BIA of interest. In this example, mutations were introduced into the open reading frame of a specific pathway enzyme by amplification with Mutazyme II (see Table 11). Sufficient template DNA was included in the amplification reaction to achieve a mutation rate of one to four nucleotide substitutions per gene. The mutagenized library was cloned into the pYES1L vector by direct gap repair in yeast. In some cases, the yeast strain selected for library expression included the integration of a gene copy that provides a substrate for the mutagenized enzyme. For example, a library of CODM mutants was transformed into a strain containing integrated copies of T6ODM and COR1.3, and thebaine was supplied in the culture medium. Expression of T6ODM and COR1.3 in such strains ensured that codeine and neopine were available as substrates for each introduced CODM mutant. Individual colonies were seeded into 96-well plates, grown for 96 hours, and then assayed for their product production by liquid chromatography-mass spectrometry (LC-MS). In the example of the CODM library, the products screened were morphine and neomorphine. In each screen, mutants with improved BIA production were sequenced and recloned for confirmation. Table 11 contains a summary of the mutant enzyme variants identified by the screen that resulted in increased BIA production in yeast.

[0294] Figure 20 shows data on the confirmed improved activity of one of these mutants. In particular, Figure 20 illustrates the generation of CODM enzyme mutants that exhibit improved activity in yeast by random mutagenesis and screening, according to an embodiment of the present invention. A library of CODM mutants was generated by mutagenesis of the coding region by error-prone PCR. Mutant CODMs identified by screening this library were N35S,G335VThe mutant CODM was recloned and expressed in a yeast strain with integrated copies of T6ODM and COR1.3. This strain and another control strain expressing wild-type CODM were grown in liquid medium containing 1 mM thebaine. After 96 hours, the culture medium was analyzed for CODM activity by LC-MS. N35S,G335V produced 1.4-fold more morphine and 2.6-fold more neomorphine than wild-type CODM-expressing strains.

[0295] Example 10: Optimization of expression and growth conditions to improve benzylisoquinoline alkaloid production by heterologous microbial hosts Benzylisoquinoline alkaloid production by genetically engineered microbial hosts can be further improved by optimizing the expression of pathway enzymes and growth conditions. In one example, expression of salutaridinol 7-O-acetyltransferase in yeast was modified by expressing the enzyme from a series of different promoters. Yeast was engineered to heterologously express a yeast-codon-optimized gene encoding poppy salutaridinol 7-O-acetyltransferase from different promoters (as shown in Figure 21A). Two expression cassettes (P TPI1 -yPbSalR, P X The nucleotide sequence (-yPsSalAT) was assembled into a yeast artificial chromosome (YAC) containing a TRP1 section marker. The YAC was placed into yeast, and cells were cultured in synthetic complete medium containing a dedicated dropout solution (-Trp) and fed with salutaridine. BIA metabolites were analyzed by LC-MS / MS analysis after 72 hours of culture. Optimization of the expression levels of enzymes in the pathway can result in increased production of the morphinan alkaloid thebaine (as shown in Figure 21A).

[0296] Optimization of the strain's culture conditions, including but not limited to, sugar source, culture temperature, and pH, can be used to increase the production of benzylisoquinoline alkaloids by the engineered yeast strain (as shown in Figures 21B and 21C). In one example, the pH was altered to increase thebaine production by the engineered yeast strain. Two expression cassettes (P TPI1 -yPbSalR, P TEF1 The nucleotide sequence (-yPsSalAT) was assembled into a yeast artificial chromosome (YAC) containing a TRP1 section marker. This YAC was transfected into yeast, and cells were cultured in synthetic complete medium containing a dedicated dropout solution (-Trp), resuspended in a pH 5.7-9 buffer, and fed with salutaridin. BIA metabolites were analyzed by LC-MS / MS analysis after 16 hours of incubation. Levels of the tetracyclic promorphinan alkaloid salutaridinol and the pentacyclic morphinan alkaloid thebaine increased as a function of increasing pH (as shown in Figure 21(B)).

[0297] In another example, the temperature, sugar, and buffer content of the medium were altered to increase thebaine production by geneti...

Claims

1. 1. A method for demethylating an opioid to a nor-opioid, comprising: contacting the opioid with an N-demethylase enzyme, wherein contacting the opioid with the enzyme converts the opioid to a nor-opioid by removal of an N-linked methyl group from the opioid. Including, the nor-opioid is produced by culturing genetically engineered microbial cells that contain a coding sequence for encoding an N-demethylase enzyme; the amino acid sequence of the N-demethylase enzyme is at least 90% identical to the amino acid sequence of a BM3 variant listed in Table 6: 7A1, 8F11, 4H5, 4H9, or 8C7; the opioid is selected from the group consisting of codeine, oxycodone, thebaine, hydrocodone, dihydrocodeine, 14-hydroxycodeine, codeinone, and 14-hydroxycodeinone, morphine, oxymorphone, oripavine, hydromorphone, dihydromorphine, 14-hydroxy-morphine, morphinone, and 14-hydroxy-morphinone; The method.

2. contacting the first opioid with an O-demethylase enzyme, wherein contacting the first opioid with the O-demethylase enzyme converts the first opioid to a second opioid by loss of an O-linked methyl group; and contacting the second opioid with an N-demethylase enzyme, wherein contacting the second opioid with the N-demethylase enzyme converts the second opioid to a nor-opioid by loss of an N-linked methyl group. further comprising The nor-opioid is produced by culturing genetically engineered cells, the cells further comprising a coding sequence for encoding an O-demethylase enzyme.

10. The method of claim 1.

3. further comprising modifying the opioid to a nal-opioid; contacting the nor-opioid with an N-methyltransferase enzyme in the presence of a cofactor, wherein contacting the nor-opioid with an N-methyltransferase enzyme converts the nor-opioid to a nal-opioid by transfer of a side chain from the cofactor. Including, The nar-opioid is produced by culturing genetically engineered cells, the cells further comprising a coding sequence for encoding an N-methyltransferase enzyme.

3. The method of claim 1 or 2.

4. Recovering nor-opioid from the cell culture 3. The method of claim 1 or claim 2, further comprising:

5. Recovering the nar-opioid from the cell culture 4. The method of claim 3, further comprising:

6. adding (S)-1-benzylisoquinoline alkaloid to the cell culture 6. The method of any one of claims 1 to 5, further comprising:

7. (a) nor-opioids are produced in genetically engineered cells by a metabolic pathway that begins with L-tyrosine; or (b) Nar-opioids are produced in genetically engineered cells by a metabolic pathway that begins with L-tyrosine; 7. The method of any one of claims 1 to 6.

8. A genetically engineered microbial cell that produces a nor-opioid from an opioid present in the genetically engineered cell, the genetically engineered cell comprises a heterologous coding sequence encoding an N-demethylase produced by the genetically engineered cell, the N-demethylase converts the opioid in the genetically engineered cell to the nor-opioid, the nor-opioid being produced within the genetically engineered cell; the amino acid sequence of the N-demethylase enzyme is at least 90% identical to the amino acid sequence of a BM3 variant listed in Table 6: 7A1, 8F11, 4H5, 4H9, or 8C7; the opioid is selected from the group consisting of codeine, oxycodone, thebaine, hydrocodone, dihydrocodeine, 14-hydroxycodeine, codeinone, and 14-hydroxycodeinone, morphine, oxymorphone, oripavine, hydromorphone, dihydromorphine, 14-hydroxy-morphine, morphinone, and 14-hydroxy-morphinone; The genetically engineered microbial cell.

9. (a) further comprising a heterologous coding sequence encoding an N-methyltransferase; and / or (b) Opioids are produced in genetically engineered cells by a metabolic pathway that begins with L-tyrosine; The genetically engineered cell of claim 8.

10. 10. The genetically engineered cell of claim 8 or 9, wherein the cell produces a nar-opioid from a nor-opioid present in the genetically engineered cell, and the genetically engineered cell comprises a heterologous coding sequence encoding an N-methyltransferase produced by the genetically engineered cell, and the N-methyltransferase converts the nor-opioid to the nar-opioid within the genetically engineered cell.

11. (a) nal-opioids are produced in the genetically engineered cells; and / or (b) Nar-opioids are produced in genetically engineered cells by a metabolic pathway that begins with L-tyrosine; The genetically engineered cell of claim 10.

12. 8. The method of any one of claims 1 to 7, wherein the genetically engineered cell is a genetically engineered yeast cell.

13. 13. The method of any one of claims 2-7 or 12, wherein the loss of the O-linked methyl group occurs at the 3' position.

14. 14. The method of any one of claims 1-7 or 12-13, wherein the genetically engineered cell further comprises at least one enzyme selected from the group consisting of L-DOPA decarboxylase, tyrosine hydroxylase, norcoclaurine synthase, norcoclaurine 6-O-methyltransferase, coclaurine-N-methyltransferase, cytochrome P450 80B1, cytochrome P450 reductase, 4'-O-methyltransferase, 1-benzylisoquinoline alkaloid epimerase, salutaridine synthase, salutaridine reductase, salutaridinol 7-O-acetyltransferase, thebaine 6-O-demethylase, codeinone reductase, codeine O-demethylase, N-methyltransferase, O-demethylase, and N-demethylase.

15. preparing a nal-opioid from a substrate; The genetically engineered cells are capable of producing nal-opioids from substrates, 15. The method of any one of claims 3 to 7 or 12 to 14.

16. (a) the substrate is a sugar; (b) the substrate is tyrosine; (c) the substrate is thebaine, and the thebaine is produced in the genetically engineered cell; (d) the substrate is reticuline, and the reticuline is produced in the genetically engineered cell; (e) the substrate is an opioid, and the opioid is produced in the genetically engineered cell; or (f) the substrate is a nor-opioid, and the nor-opioid is produced in the genetically engineered cell; 16. The method of claim 15.

17. (a) the method further comprises culturing the genetically engineered cell under conditions suitable for protein production, wherein the genetically engineered cell comprises two heterologous coding sequences encoding first and second enzymes, respectively, involved in a metabolic pathway that converts a substrate to a nar-opioid, the first and second enzymes being operably linked along the metabolic pathway, and wherein each of the first and second enzymes involved in the metabolic pathway that produces a nar-opioid is selected from the group consisting of an N-demethylase and an N-methyltransferase; or (b) recovering the nal-opioid from the cell culture; 16. The method of claim 15.

Images