Methods for Producing Morphinan Alkaloids and Derivatives - Patent application

Abstract

To provide a method of producing promorphinan, morphine, nal-opioid, and nor-opioid alkaloid products through increased conversion of promorphinan alkaloid into morphinan alkaloid.SOLUTION: A method comprises contacting promorphinan alkaloid with at least one kind of enzyme. The step of contacting the promorphinan alkaloid with the at least one kind of the enzyme converts the promorphinan alkaloid into morphinan alkaloid.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 628,264, filed February 8, 2018, and having attorney docket number 47840-708.801. This application is related to: U.S. Patent Application No. 14 / 211,611, now published as US 2014-0273109, filed March 14, 2014, and having attorney docket number STAN-1018; PCT Application No. PCT / US2014 / 027833, now published as WO 2014 / 143744, filed March 14, 2014, and having attorney docket number STAN-1018WO; U.S. Patent Application No. 15 / 031,618, filed April 22, 2016, and having attorney docket number STAN-1078; now published as WO Application No. PCT / US2014 / 063738, published as PCT Application No. 2015 / 066642, filed November 3, 2014, and having Attorney Docket No. STAN-1078WO; U.S. Provisional Patent Application No. 62 / 080,610, filed November 17, 2014, and having Attorney Docket No. STAN-1169PRV; U.S. Provisional Patent Application No. 62 / 107,238, filed January 23, 2015, and having Attorney Docket No. STAN-1169PRV2; Application No. PCT / US2015 / 060891, filed November 16, 2015, and having Attorney Docket No. STAN-1169WO; and U.S. Provisional Patent Application No. STAN-1221PR, filed May 4, 2015. No. 62 / 156,701, filed May 4, 2016, and having attorney docket number STAN-1221WO; Application No. PCT / US2016 / 031506, filed May 9, 2016; Application No. PCT / US2017 / 057237, filed October 18, 2017; Application No. 62 / 541,038, filed August 3, 2017; and Application No. PCT / US2018 / 045222, filed August 3, 2018; and Application No. 16 / 149,025, filed October 1, 2018. The disclosures of these applications are incorporated herein by reference. Summary of the Invention

[0002] The present disclosure provides methods for the production of diverse benzylisoquinoline alkaloids (BIA) in genetically engineered host cells. Further, the present disclosure provides compositions of diverse alkaloids produced in genetically engineered host cells. Additionally, the present disclosure provides methods for the production of thebaine synthase in genetically engineered host cells. In certain instances, the present disclosure provides methods for producing diverse alkaloid products by the conversion of promorphinan alkaloids to morphinan alkaloids in genetically engineered host cells. In even more particular instances, the present disclosure provides methods for producing diverse alkaloid products by the conversion of salutaridinol-7-O-acetate to thebaine.

[0003] One aspect of the present invention provides genetically engineered non-plant cells having increased tyrosine hydroxylase activity compared to non-genetically engineered cells. Another aspect of the present invention provides genetically engineered non-plant cells having increased tyrosine hydroxylase activity compared to cells expressing wild-type TyrH. A further aspect of the present invention provides genetically engineered non-plant cells having increased tyrosine hydroxylase activity compared to cells expressing wild-type TyrH that do not have a mutation that increases tyrosine hydroxylase activity as provided herein. In particular, the genetically engineered non-plant cells have at least one modification selected from the group consisting of a substrate inhibition relieving mutation; a product inhibition relieving mutation; and a cofactor restoration enhancing mechanism.

[0004] One aspect of the present invention provides genetically engineered plant cells having increased tyrosine hydroxylase activity compared to non-genetically engineered cells. Another aspect of the present invention provides genetically engineered plant cells having increased tyrosine hydroxylase (TyrH) activity compared to cells expressing wild-type TyrH. A further aspect of the present invention provides genetically engineered plant cells having increased tyrosine hydroxylase activity compared to cells expressing wild-type TyrH that do not have a mutation that increases tyrosine hydroxylase activity as provided herein. In particular, the genetically engineered plant cells have at least one modification selected from the group consisting of a substrate inhibition relieving mutation; a product inhibition relieving mutation; and a cofactor restoration enhancement mechanism.

[0005] In some aspects, the disclosure provides methods for increasing the production of diverse alkaloid products by epimerizing (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids via an engineered epimerase in an engineered host cell. In further aspects, the disclosure provides methods for increasing the production of diverse alkaloid products by epimerizing (S)-reticuline to (R)-reticuline via an engineered epimerase comprising two separate enzymes encoding an oxidase and a reductase, compared to the production of diverse alkaloid products by epimerizing (S)-reticuline to (R)-reticuline via a wild-type epimerase.

[0006] The engineered split epimerase can comprise separate oxidase and reductase enzymes derived from a parent or wild-type epimerase, but the engineered epimerase can also comprise separate oxidase and reductase enzymes derived from separate parent or wild-type epimerases. Examples of parent epimerases having oxidase and reductase components comprise 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, 15, and 16 listed in Table 1.

[0007] In some aspects, the disclosure provides methods for increasing the production of diverse alkaloid products by the conversion of promorphinan alkaloids to morphinan alkaloids by thebaine synthase in a genetically engineered host cell. In further aspects, the disclosure provides methods for increasing the production of diverse alkaloid products by the conversion of salutaridinol-7-O-acetate to thebaine by thebaine synthase. Exemplary parent thebaine synthases include: amino acid sequences selected from the group consisting of SEQ ID NOs: 30, 31, 32, 33, 34, 35, 36, and 37 listed in Table 2.

[0008] In some aspects, the disclosure provides methods for increasing the production of diverse alkaloid products by converting promorphinan alkaloids to morphinan alkaloids with an engineered thebaine synthase in an engineered host cell. In further aspects, the disclosure provides methods for increasing the production of diverse alkaloid products by converting salutaridinol-7-O-acetate to thebaine with an engineered thebaine synthase.

[0009] In some embodiments, the engineered thebaine synthase is a fusion enzyme. In further embodiments, the thebaine synthase is fused to an acetyltransferase enzyme. In further embodiments, the thebaine synthase is encoded within an acetyltransferase enzyme. In other embodiments, the thebaine synthase is fused to a reductase enzyme.

[0010] In some cases, the engineered non-plant cell includes multiple coding sequences, each encoding an enzyme selected from the group of enzymes listed in Table 3. In some cases, the heterologous coding sequences can be operably linked. The operably linked heterologous coding sequences can be in the same pathway that produces a particular benzylisoquinoline alkaloid product through thebaine synthase activity or engineered thebaine synthase activity.

[0011] In some embodiments, the present disclosure provides a method for converting a tetracyclic promorphinan precursor to thebaine, the method comprising contacting the tetracyclic promorphinan precursor with at least one enzyme, wherein contacting the tetracyclic promorphinan precursor with the at least one enzyme converts the tetracyclic promorphinan precursor to thebaine. In some cases, the at least one enzyme is produced by culturing a genetically engineered non-plant cell having a coding sequence for encoding the at least one enzyme. In some cases, the method further comprises adding the tetracyclic promorphinan precursor to the cell culture. In some cases, the method further comprises recovering thebaine or a derivative thereof from the cell culture. In some cases, the at least one enzyme comprises thebaine synthase. In some cases, the thebaine synthase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 31, 32, 33, 34, 35, 36, and 37. In some cases, the thebaine synthase is a Bet v 1-fold protein. [The present invention 1001] (i) an epimerase; (ii) thebaine synthase; (iii) at least one modification selected from the group consisting of (a) a mutation that relieves substrate inhibition, (b) a mutation that relieves product inhibition, (c) a mechanism that promotes cofactor recovery, (d) a mutation that relieves feedback inhibition, (e) a transcriptional modulation modification, and (f) an inactivating mutation. 1. A genetically engineered non-plant cell comprising: A genetically engineered non-plant cell, wherein the genetically engineered non-plant cell converts a precursor of a promorphinan molecule into an alkaloid product selected from the group consisting of (i) a morphinan alkaloid, (ii) a nal-opioid alkaloid, and (iii) a nor-opioid alkaloid. [The present invention 1002] 1001. The genetically engineered non-plant cell of the present invention, wherein the epimerase is a genetically engineered epimerase. [The present invention 1003] 1002. The genetically engineered non-plant cell of claim 1002, wherein the genetically engineered epimerase is a split epimerase. [The present invention 1004] 1004. The genetically engineered non-plant cell of claim 1002 or 1003, wherein the genetically engineered epimerase converts an (S)-1-benzylisoquinoline precursor to an (R)-1-benzylisoquinoline product. [The present invention 1005] 1005. The genetically engineered non-plant cell of any of claims 1002 to 1004, wherein the genetically engineered epimerase converts (S)-reticuline to (R)-reticuline. [The present invention 1006] 1004 or 1005, wherein at least 50% of the (S)-1-benzylisoquinoline alkaloid molecules in said genetically engineered non-plant cell are converted to the (R)-1-benzylisoquinoline product. [The present invention 1007] 1001. The genetically engineered non-plant cell of claim 1001, wherein the thebaine synthase is a genetically engineered thebaine synthase. [The present invention 1008] 1001. The genetically engineered non-plant cell of claim 1001, wherein a precursor of a promorphinan molecule is provided to said genetically engineered non-plant cell. [The present invention 1009] 1001. The genetically engineered non-plant cell of claim 1001, wherein a precursor of a promorphinan molecule is produced within said genetically engineered non-plant cell. [The present invention 1010] 1001. The genetically engineered non-plant cell of the present invention, wherein the precursor of the promorphinan molecule is selected from the group consisting of reticuline, 3'hydroxy-N-methylcoclaurine, coclaurine, norcoclaurine, norlaudanosoline, methylnorlaudanosoline, laudanosoline, methylnorlaudanosoline, norreticuline, 3'hydroxy-N-methylcoclaurine, 4'-O'-methyllaudanosoline, L-Dopa, tyrosine, dopamine, 3,4-dihydroxyphenylacetaldehyde (3,4-DHPA), hydroxyphenylpyruvic acid, prephenate, chorismate, 5-enolpyruvylshikimate-3-phosphate (EPSP), 3-deoxy-D-arabinoheptulosonic acid-7-phosphate (DAHP), erythrose-4-phosphate (E4P), phosphoenolpyruvate (PEP), and glucose. [The present invention 1011] 1001. The genetically engineered non-plant cell of claim 1001, wherein at least 50% of the tetracyclic promorphinan precursor molecules in said genetically engineered non-plant cell are converted to thebaine. [The present invention 1012] 1011. The genetically engineered non-plant cell of claim 10, wherein the tetracyclic promorphinan molecule is selected from the group consisting of salutaridine, salutaridinol, or salutaridinol-7-O-acetate. [The present invention 1013] The precursor of the promorphinan molecule is represented by Formula I: TIFF0007730358000001.tif35128 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; The genetically engineered non-plant cell of the present invention. [The present invention 1014] R 1 , R 2 , R 3 , R 4 and R5 1013. The genetically engineered non-plant cell of the present invention, wherein at least one of [The present invention 1015] The precursor of the promorphinan molecule is represented by Formula II: TIFF0007730358000002.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; The genetically engineered non-plant cell of the present invention. [The present invention 1016] Any of the aforementioned genetically engineered non-plant cells of the present invention, wherein the precursor of the promorphinan molecule is tyrosine. [The present invention 1017] Any of the aforementioned genetically engineered non-plant cells of the present invention, wherein the precursor of the promorphinan molecule is a sugar. [The present invention 1018] The genetically engineered non-plant cell of the present invention 1001 further comprising at least one modification selected from the group consisting of: (i) a BIA generation modification, (ii) an O-demethylation modification, (iii) an N-demethylation modification, and (iv) an N-linked modification. [The present invention 1019] 1001. The genetically engineered non-plant cell of claim 1001, wherein the morphinan alkaloid product is 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, heterocodeine, or oxymorphone. [The present invention 1020] 1001. The genetically engineered non-plant cell of the present invention, wherein the nal-opioid alkaloid product is naltrexone, naloxone, nalmefene, nalorphine, nalorphine, nalodeine, naldemedine, naloxegol, 6β-naltrexol, naltolindole, methylnaltrexone, methylsamidophan, alvimopan, axelopran, bevenpran, dinicotinate, levallorphan, samidophan, buprenorphine, dezocine, eptazocine, butorphanol, levorphanol, nalbuphine, pentazocine, phenazocine, norbinaltorphimine, or diprenorphine. [The present invention 1021] 1001. The genetically engineered non-plant cell of the present invention, wherein the nor-opioid alkaloid product is 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, or nor-14-hydroxy-morphinone. [The present invention 1022] The genetically engineered non-plant cell of this invention 1001, which is a bacterial cell or a fungal cell. [The present invention 1023] Bacterial cells of the genera Anabaena, Arthrobacter, Acetobacter, Acetobacterium, Bacillus, Bifidobacterium, Brachybacterium, Brevibacterium, Carnobacterium, and Clostridium are , Corynebacterium, Enterobacter, Escherichia, Gluconacetobacter, Gluconobacter, Hafnia, Halomonas, Klebsiella, Kocuria, Lactobacillus, Leuconostoc ), Macrococcus, Methylomonas, Methylobacter, Methylocella, Methylococcus, Microbacterium, Micrococcus, Microcystis, Moorella, Oenococcus, Pediococcus s), Prochlorococcus, Propionibacterium, Proteus, Pseudoalteromonas, Pseudomonas, Psychrobacter, Rhodobacter, Rhodococcus, Rhodopseudomonas, Serratia,The genetically engineered non-plant cell of the present invention 1022 is derived from a genus selected from the group consisting of Staphylococcus, Streptococcus, Streptomyces, Synechococcus, Synechocystis, Tetragenococcus, Weissella, and Zymomonas. [The present invention 1024] Bacterial cells such as Arthrobacter nicotianae, Acetobacter aceti, Arthrobacter arilaitensis, Bacillus cereus, Bacillus coagulans, Bacillus licheniformis, Bacillus pumilus, Bacillus sphaericus, Bacillus stearothermophilus, Bacillus subtilis, and Bifidobacterium adressentis were detected. 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 yamanasiensisyamanashiensis, Leuconostoc citreum, Macrococcus caseolyticus, Microbacterium foliorum, Micrococcus lylae, Oenococcus oeni, Pediococcus acidilactici, Propionibacterium acidipropionici, Proteus vulgaris, Pseudomonas fluorescens, Psychrobacter celer, 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 casei1022. The genetically engineered non-plant cell of the present invention, wherein the non-plant cell is selected from the group consisting of Pseudoalteromonas casei, Pseudoalteromonas citrea, Pseudomonas putida, Clostridium ljungdahlii / aceticum / acetobutylicum / beijerinckii / butyricum, and Moorella themocellum / thermoacetica. [The present invention 1025] The genetically engineered non-plant cell of claim 1022, wherein the fungal cell is derived from a genus selected from the group consisting of Saccharomyces, Schizosaccharomyces, Pichia, and Aspergillus. [The present invention 1026] 1022. The genetically engineered non-plant cell of claim 1022, wherein the fungal cell is selected from the group consisting of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Aspergillus niger, Aspergillus oryzae, Aspergillus terreus, and Aspergillus nidulans. [The present invention 1027] 1001. The genetically engineered non-plant cell of this invention, which produces at least 50% more alkaloid product than a comparable cell having one or more modifications that are less than said genetically engineered non-plant cell. [The present invention 1028] 1001. The genetically engineered non-plant cell of the present invention, wherein the cell produces at least two times more alkaloid product than a comparable cell having one or more modifications that are less than said genetically engineered non-plant cell. [The present invention 1029] 1. A method for converting a precursor of a promorphinan molecule into thebaine or a derivative thereof, comprising: contacting a precursor of the promorphinan molecule with at least one enzyme, wherein the at least one enzyme comprises thebaine synthase, and wherein the at least one precursor of the promorphinan molecule is produced within the genetically engineered non-plant cell where the conversion occurs; Including, wherein contacting the precursor of the promorphinan molecule with at least one enzyme converts the precursor of the promorphinan molecule into thebaine or a derivative thereof. method. [The present invention 1030] The method of claim 1029, wherein the conversion of the precursor of the promorphinan molecule to thebaine occurs in a genetically engineered non-plant cell. [The present invention 1031] The method of claim 1029, wherein the precursor of the promorphinan molecule is produced in a genetically engineered non-plant cell. [The present invention 1032] 1032. The method of any of claims 1029 to 1031, wherein the genetically engineered non-plant cell is a bacterial cell or a fungal cell. [The present invention 1033] The bacterial cells may be of the genera Anabaena, Arthrobacter, Acetobacter, Acetobacterium, Bacillus, Bifidobacterium, Brachybacterium, Brevibacterium, Carnobacterium, Clostridium, Corynebacterium, Enterobacter, Escherichia, Gluconacetobacter, Gluconobacter, Hafnia, Halomonas, Klebsiella, Kocuria, Lactobacillus, Leuconostoc, Macrococcus, Methylomonas, Methylobacter, Methylococcus, Methylococcus, Microbacterium The method of the present invention 1031, wherein the bacterium is derived from a genus selected from the group consisting of Micrococcus, Microcystis, Moorella, Oenococcus, Pediococcus, Prochlorococcus, Propionibacterium, Proteus, Pseudoalteromonas, Pseudomonas, Psychrobacter, Rhodobacter, Rhodococcus, Rhodopseudomonas, Serratia, Staphylococcus, Streptococcus, Streptomyces, Synechococcus, Synechocystis, Tetragenococcus, Weissella, and Zymomonas. [The present invention 1034] Bacterial cells such as Arthrobacter nicotianae, Acetobacter aceti, Arthrobacter alilactensis, Bacillus cereus, Bacillus coagulans, Bacillus licheniformis, Bacillus pumilus, Bacillus sphaericus, Bacillus stearothermophilus, Bacillus subtilis, Bifidobacterium alesentis, Brachybacterium tyrofermentans, Brevibacterium linens, Carnobacterium divergens, and Corynebacterium flavus were identified. ance, Enterococcus faecium, Gluconacetobacter europaeus, Gluconacetobacter hohannae, Gluconobacter oxydans, Hafnia alvei, Halomonas elongata, Kocuria rhizophila, Lactobacillus acidifarinae, Lactobacillus jensenii, Lactococcus lactis, Lactobacillus yamanashiensis, Leuconostoc citreum, Macrococcus caseolyticus, Microbacterium foliorum, Micrococcus · Leyle, Oenococcus oeni, Pediococcus acidilactici, Propionibacterium acidipropionici, Proteus vulgaris, Pseudomonas fluorescens, Psychrobacter cellarum, Staphylococcus condimentii, Streptococcus thermophilus, Streptomyces griseus, Tetragenococcus halophilus, Weissella tibaria, Weissella coreensis, Zymomonas mobilis, Corynebacterium glutamicum , Bifidobacterium bifidum / breve / longum, Streptomyces lividans, Streptomyces coelicolor, Lactobacillus plantarum, Lactobacillus sakei, Lactobacillus casei, Pseudoalteromonas citrea, Pseudomonas putida, Clostridium ljungdahlii / aceticum / acetobutylicum / beijerinckii / butyricum, and Moorella temocellum / thermoacetica. [This invention 1035] 1031. The method of claim 1031, wherein the fungal cell is from a genus selected from the group consisting of Saccharomyces, Schizosaccharomyces, Pichia, and Aspergillus. [The present invention 1036] 1031. The method of claim 1031, wherein the fungal cell is selected from the group consisting of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Aspergillus niger, Aspergillus oryzae, Aspergillus terreus, and Aspergillus nidulans. [This invention 1037] 1029. The method of claim 1029, wherein the at least one enzyme is produced by culturing a genetically engineered non-plant cell comprising a coding sequence for encoding said at least one enzyme. [The present invention 1038] The method of claim 1029, further comprising the step of adding a precursor of the promorphinan molecule to the cell culture. [This invention 1039] The method of claim 1038, further comprising recovering thebaine or a derivative thereof from the cell culture. [The present invention 1040] 1039. The method of any of claims 1029 to 1039, wherein the at least one enzyme comprises thebaine synthase. [The present invention 1041] 1039. The method of any of claims 1029-1039, wherein the at least one enzyme comprises an engineered thebaine synthase, an engineered SalAT, a morphogenic engineering (DIR) protein, or a chalcone isomerase (CHI). [The present invention 1042] The method of claim 1040, wherein the thebaine synthase enzyme is a Bet v 1 fold protein. [This invention 1043] The method of claim 1040, wherein the thebaine synthase comprises an amino acid sequence having at least 50% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 31, 32, 33, 34, 35, 36, and 37. [This invention 1044] The method of claim 1029, wherein the thebaine synthase converts at least 50% of the precursor promorphinan molecule to thebaine. [This invention 1045] The method of claim 1029, wherein the genetically engineered non-plant cell converts at least 90% of the precursor of the promorphinan molecule to thebaine. [The present invention 1046] (i) an epimerase; (ii) thebaine synthase; (iii) tyrosine hydroxylase (TyrH), L-DOPA-decarboxylase (DODC), norcoclaurine synthase (NCS), norcoclaurine 6-O-methyltransferase (6OMT), coclaurine-N-methyltransferase (CNMT), cytochrome P450 and modification of at least one enzyme selected from the group consisting of CYP80B1 (CYP80B1), cytochrome P450 reductase (CPR), 4'-O-methyltransferase (4'OMT), monoamine oxidase (MAO), transketolase (TKL1), glucose-6-phosphate dehydrogenase (ZWF1), pentafunctional AROM protein (ARO1), bifunctional chorismate synthase (ARO2), 3-deoxy-7-phosphoheptulosonate synthase (ARO3), 3-deoxy-d-arabinose-heptulosonate-7-phosphate synthase (ARO4), chorismate mutase (ARO7), prephenate dehydrogenase (TYR1), aromatic aminotransferase 8 (ARO8), aromatic aminotransferase 9 (ARO9), phenylpyruvate decarboxylase (ARO10), and tyrosinase (TYR). 1. A genetically engineered non-plant cell comprising: A genetically engineered non-plant cell, wherein the genetically engineered non-plant cell converts a precursor of a promorphinan molecule into an alkaloid product selected from the group consisting of (i) a morphinan alkaloid, (ii) a nal-opioid alkaloid, and (iii) a nor-opioid alkaloid. [This invention 1047] 1046. The genetically engineered non-plant cell of the present invention, wherein the epimerase is a genetically engineered epimerase. [This invention 1048] 1047. The genetically engineered non-plant cell of claim 1047, wherein the genetically engineered epimerase is a split epimerase. [This invention 1049] 1047. The genetically engineered non-plant cell of claim 1048, wherein the genetically engineered epimerase converts an (S)-1-benzylisoquinoline precursor to an (R)-1-benzylisoquinoline product. [The present invention 1050] 1049. The genetically engineered non-plant cell of any of claims 1047 to 1049, wherein the genetically engineered epimerase converts (S)-reticuline to (R)-reticuline. [This invention 1051] 1049 or 1050, wherein at least 50% of the (S)-1-benzylisoquinoline alkaloid molecules in said genetically engineered non-plant cell are converted to the (R)-1-benzylisoquinoline product. [This invention 1052] 1046. The genetically engineered non-plant cell of claim 1046, wherein the thebaine synthase is a genetically engineered thebaine synthase. [This invention 1053] 1046. The genetically engineered non-plant cell of claim 1046, wherein a precursor of a promorphinan molecule is provided to said genetically engineered non-plant cell. [This invention 1054] 1046. The genetically engineered non-plant cell of claim 1046, wherein a precursor of a promorphinan molecule is produced within said genetically engineered non-plant cell. [This invention 1055] 1046. The genetically engineered non-plant cell of claim 1046, wherein the precursor of the promorphinan molecule is selected from the group consisting of reticuline, 3'hydroxy-N-methylcoclaurine, coclaurine, norcoclaurine, norlaudanosoline, methylnorlaudanosoline, laudanosoline, methylnorlaudanosoline, norreticuline, 3'hydroxy-N-methylcoclaurine, 4'-O'-methyllaudanosoline, L-Dopa, tyrosine, dopamine, 3,4-dihydroxyphenylacetaldehyde (3,4-DHPA), hydroxyphenylpyruvic acid, prephenate, chorismate, 5-enolpyruvylshikimate-3-phosphate (EPSP), 3-deoxy-D-arabinoheptulosonic acid-7-phosphate (DAHP), erythrose-4-phosphate (E4P), phosphoenolpyruvate (PEP), and glucose. [This invention 1056] 1046. The genetically engineered non-plant cell of claim 1046, wherein at least 50% of the tetracyclic promorphinan precursor molecules in said genetically engineered non-plant cell are converted to thebaine. [This invention 1057] 1056. The genetically engineered non-plant cell of claim 1056, wherein the tetracyclic promorphinan molecule is selected from the group consisting of salutaridine, salutaridinol, or salutaridinol-7-O-acetate. [This invention 1058] The precursor of the promorphinan molecule is represented by Formula I: TIFF0007730358000003.tif35128 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; The genetically engineered non-plant cell of the present invention 1046. [This invention 1059] R 1 , R 2 , R 3 , R 4 and R5 1057. The genetically engineered non-plant cell of claim 1057, wherein at least one of [The present invention 1060] The precursor of the promorphinan molecule is represented by Formula II: TIFF0007730358000004.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; The genetically engineered non-plant cell of the present invention 1046. [The present invention 1061] The genetically engineered non-plant cell of any of claims 1046 to 1060, wherein the precursor of the promorphinan molecule is tyrosine. [The present invention 1062] The genetically engineered non-plant cell of any of claims 1046 to 1060, wherein the precursor of the promorphinan molecule is a sugar. [The present invention 1063] The genetically engineered non-plant cell of the present invention 1046 further comprising at least one modification selected from the group consisting of: (i) a BIA generation modification, (ii) an O-demethylation modification, (iii) an N-demethylation modification, and (iv) an N-linked modification. [The present invention 1064] 1046. The genetically engineered non-plant cell of claim 1046, wherein the morphinan alkaloid product is 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, heterocodeine, or oxymorphone. [This invention 1065] 1046. The genetically engineered non-plant cell of claim 1046, wherein the nal-opioid alkaloid product is naltrexone, naloxone, nalmefene, nalorphine, nalorphine, nalodaine, naldemedine, naloxegol, 6β-naltrexol, naltolindole, methylnaltrexone, methylsamidophan, alvimopan, axerophthol, bebenprane, dinicotinate, levallorphan, samidophan, buprenorphine, dezocine, eptazocine, butorphanol, levorphanol, nalbuphine, pentazocine, phenazocine, norbinaltorphimine, or diprenorphine. [The present invention 1066] 1046. The genetically engineered non-plant cell of claim 1046, wherein the nor-opioid alkaloid product is 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, or nor-14-hydroxy-morphinone. [This invention 1067] The genetically engineered non-plant cell of the present invention 1046, which is a bacterial cell or a fungal cell. [The present invention 1068] The bacterial cells may be of the genera Anabaena, Arthrobacter, Acetobacter, Acetobacterium, Bacillus, Bifidobacterium, Brachybacterium, Brevibacterium, Carnobacterium, Clostridium, Corynebacterium, Enterobacter, Escherichia, Gluconacetobacter, Gluconobacter, Hafnia, Halomonas, Klebsiella, Kocuria, Lactobacillus, Leuconostoc, Macrococcus, Methylomonas, Methylobacter, Methylococcus, Microbacterium ... The genetically engineered non-plant cell of the present invention is derived from a genus selected from the group consisting of Coccus, Microcystis, Moorella, Oenococcus, Pediococcus, Prochlorococcus, Propionibacterium, Proteus, Pseudoalteromonas, Pseudomonas, Psychrobacter, Rhodobacter, Rhodococcus, Rhodopseudomonas, Serratia, Staphylococcus, Streptococcus, Streptomyces, Synechococcus, Synechocystis, Tetragenococcus, Weissella, and Zymomonas. [This invention 1069] Bacterial cells such as Arthrobacter nicotianae, Acetobacter aceti, Arthrobacter alilactensis, Bacillus cereus, Bacillus coagulans, Bacillus licheniformis, Bacillus pumilus, Bacillus sphaericus, Bacillus stearothermophilus, Bacillus subtilis, Bifidobacterium alesentis, Brachybacterium tyrofermentans, Brevibacterium linens, Carnobacterium divergens, Corynebacterium flavescens, Enterococcus faecium, Gluconacetobacter europaeus, Gluconacetobacter hohannae, Gluconobacter oxydans, Hafnia alvei, Halomonas elongata, Kocuria rhizophila, Lactobacillus acidifarinae, Lactobacillus jensenii, Lactococcus lactis, Lactobacillus yamanashiensis, Leuconostoc citreum, Macrococcus caseolyticus, Microbacterium foliorum, Micrococcus leile, Oenococcus oeni, Pediococcus acidilactici, Propionibacterium acidipropionici, Proteus vulgaris, Pseudomonas fluorescens, Psychrobacter cellarum, Staphylococcus condimentii, Streptococcus thermophilus, Streptomyces griseus, Tetragenococcus halophilus, Weissella tibaria, Weissella coreensis, Zymomonas mobilis, Corynebacterium glutamicum, Bifidobacterium diffusum, 1067. The genetically engineered non-plant cell of the present invention, wherein the non-plant cell is selected from the group consisting of Lactobacillus bifidum / breve / longum, Streptomyces lividans, Streptomyces coelicolor, Lactobacillus plantarum, Lactobacillus sakei, Lactobacillus casei, Pseudoalteromonas citrea, Pseudomonas putida, Clostridium ljungdahlii / aceticum / acetobutylicum / beijerinckii / butyricum, and Moorella temocellum / thermoacetica. [The present invention 1070] 1067. The genetically engineered non-plant cell of claim 1067, wherein the fungal cell is derived from a genus selected from the group consisting of Saccharomyces, Schizosaccharomyces, Pichia, and Aspergillus. [This invention 1071] 1067. The genetically engineered non-plant cell of the present invention, wherein the fungal cell is selected from the group consisting of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Aspergillus niger, Aspergillus oryzae, Aspergillus terreus, and Aspergillus nidulans. [This invention 1072] 1046. The genetically engineered non-plant cell of claim 1046, wherein the cell produces at least 50% more alkaloid product than a comparable cell having one or more modifications that are less than said genetically engineered non-plant cell. [This invention 1073] 1046. The genetically engineered non-plant cell of claim 1046, wherein the cell produces at least two times more alkaloid product than a comparable cell having one or more modifications that are less than said genetically engineered non-plant cell.

[0012] 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. [Brief explanation of the drawings]

[0013] 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:

[0014] [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 one example of the formation of an (R)-1-benzylisoquinoline alkaloid according to an embodiment of the present invention. [Figure 4] FIG. 4 shows the amino acid sequence of the parent DRS-DRR enzyme according to an embodiment of the present invention. [Figure 5] FIG. 5 shows the amino acid sequences of the DRS and DRR enzymes, respectively, derived from the parent fusion enzymes shown in FIG. 4, according to an embodiment of the present invention. [Figure 6] FIG. 6 shows an enzyme having opioid 3-O-demethylase activity according to an embodiment of the present invention. [Figure 7] FIG. 7 shows an enzyme having opioid N-demethylase activity according to an embodiment of the present invention. [Figure 8] FIG. 8 shows an enzyme with N-methyltransferase activity according to an embodiment of the present invention. [Figure 9] FIG. 9 shows functional expression of BM3 mutants according to embodiments of the present invention. [Figure 10] FIG. 10 shows a biosynthetic scheme for the conversion of L-tyrosine to a nor-opioid or nal-opioid in a microbial cell, according to an embodiment of the present invention. [Figure 11] FIG. 11 shows a plasmid / YAC vector for enzyme expression and genetic manipulation according to an embodiment of the present invention. [Figure 12] FIG. 12 shows a biosynthetic scheme for the conversion of L-tyrosine to reticuline via norcoclaurine, according to an embodiment of the present invention. [Figure 13] FIG. 13 shows a biosynthetic scheme for the conversion of L-tyrosine to reticuline via norlaudanosoline, according to an embodiment of the present invention. [Figure 14] FIG. 14 shows a biosynthetic scheme for the conversion of L-tyrosine to morphinan alkaloids according to an embodiment of the present invention. [Figure 15] FIG. 15 shows a biosynthetic scheme for the production of semisynthetic opioids according to an embodiment of the present invention. [Figure 16] FIG. 16 shows a biosynthetic scheme for the production of opioids, according to an embodiment of the present invention. [Figure 17] FIG. 17 shows an alignment between PbDRS-DRR, PrDRS and PrDRR according to an embodiment of the present invention. [Figure 18] FIG. 18 shows a platform yeast strain for the production of reticuline from L-tyrosine according to an embodiment of the present invention. [Figure 19] FIG. 19 shows yeast strains for the production of thebaine and hydrocodone from L-tyrosine according to an embodiment of the invention. [Figure 20] FIG. 20 illustrates a general ring-closure reaction for converting a tetracyclic scaffold to a pentacyclic scaffold, according to an embodiment of the present invention. [Figure 21] FIG. 21 shows a phylogenetic tree generated by bioinformatic searches for morphinan alkaloid-producing enzymes, according to an embodiment of the present invention. [Figure 22] FIG. 22 shows the production of the morphinan alkaloid thebaine from sugars and L-tyrosine in an engineered yeast strain according to an embodiment of the invention. [Figure 23] FIG. 23 shows the production of promorphinan alkaloids and the morphinan alkaloid thebaine from sugars and L-tyrosine in genetically engineered yeast strains according to an embodiment of the invention. DETAILED DESCRIPTION OF THE INVENTION

[0015] Detailed Description of the Invention The present disclosure provides methods for the production of various benzylisoquinoline alkaloids (BIA) in genetically engineered host cells. The present disclosure also provides compositions of various alkaloids produced in genetically engineered host cells. Additionally, the present disclosure also provides methods for the production of thebaine synthase in genetically engineered host cells. Additionally, the present disclosure also provides methods for the production of genetically engineered thebaine synthase in genetically engineered host cells. In certain instances, the present disclosure also provides methods for producing promorphinan, morphinan, nar-opioid, and nor-opioid alkaloid products by increasing the conversion of promorphinan alkaloids to morphinan alkaloids in genetically engineered host cells. In further particular instances, the present disclosure also provides methods for producing morphinan, nar-opioid, and nor-opioid alkaloid products by increasing the conversion of promorphinan alkaloids to morphinan alkaloids in genetically engineered host cells. In more particular cases, the present disclosure provides methods for producing diverse alkaloid products by increasing the conversion of promorphinan alkaloids to morphinan alkaloids.

[0016] Benzylisoquinoline Alkaloids (BIA) of Interest Host cells that produce the BIA of interest are provided. In some examples, engineered strains of host cells, such as those of the embodiments discussed herein, can provide a platform for producing benzylisoquinoline alkaloids and variants of interest across several structural classes, including, but not limited to, precursor BIAs, benzylisoquinolines, promorphinans, morphinans, nal-opioids, nor-opioids, and the like. Each of these classes can encompass biosynthetic precursors, intermediates, and metabolites of any convenient member of the engineered host cell's biosynthetic pathway 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 itself be characterized as a benzylisoquinoline alkaloid. In some cases, the chemical compounds of the present invention can encompass all possible isomers, such as single enantiomers, racemic mixtures, optically pure forms, diastereomeric mixtures, and intermediate mixtures.

[0017] 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.

[0018] 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.

[0019] Promorphinans can include, but are not limited to, salutaridine, salutaridinol, and salutaridinol-7-O-acetate.

[0020] 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. In particular, thebaine can be synthesized from salutaridinol-7-O-acetate, where the reaction can occur spontaneously or can be catalyzed by any convenient enzyme.

[0021] Nalu-opioids may include, but are not limited to, naltrexone, naloxone, nalmefene, nalorphine, nalorphine, nalodain, naldemedine, naloxegol, 6β-naltrexol, naltolindole, methylnaltrexone, methylsamidophan, alvimopan, axelopran, bevenpran, dinicotinate, levallorphan, samidophan, buprenorphine, dezocine, eptazocine, butorphanol, levorphanol, nalbuphine, pentazocine, phenazocine, norbinaltorphimine, and diprenorphine.

[0022] Nor-opioids may include, but are not limited to, 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.

[0023] In certain embodiments, the genetically engineered strains of the present invention may provide a platform for producing compounds related to tetrahydrobiopterin synthesis, including, but not limited to, dihydroneopterin triphosphate, 6-pyruvoyltetrahydropterin, 5,6,7,8-tetrahydrobiopterin, 7,8-dihydrobiopterin, tetrahydrobiopterin 4a-carbinolamine, quinonoid dihydrobiopterin, and biopterin.

[0024] 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 mammalian cell, a bacterial cell, or a yeast cell. Any convenient type of host cell can be used to generate the subject BIA-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, both Gram-positive and Gram-negative, insect cells, such as Drosophila melanogaster S2 and Spodoptera frugiperda Sf9 cells, and yeast cells, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris cells. Non-limiting examples of bacterial cells include Bacillus subtilis, Escherichia coli, and the like. coli), Streptomyces, Anabaena, Arthrobacter, Acetobacter, Acetobacterium, Bacillus, Bifidobacterium, Brachybacterium, Brevibacterium um), Carnobacterium, Clostridium, Corynebacterium, Enterobacter, Escherichia, Gluconacetobacter, Gluconobacter, Hafnia, Halomonas,Klebsiella, Kocuria, Lactobacillus, Leuconostoc, Macrococcus, Methylomonas, Methylobacter, Methylocella, Methylococcus, Microbacterium, Micrococcus, Microcystis, Moorella, Oenococcus, Pediococcus, Prochlorococcus, Propionibacterium um, Proteus, Pseudoalteromonas, Pseudomonas, Psychrobacter, Rhodobacter, Rhodococcus, Rhodopseudomonas, Serratia, Staphylococcus, Streptococcus, Streptomyces, Synechococcus, Synechocystis, Tetragenococcus, Weissella, Zymomonas, and Salmonella typhimurium cells. In some cases, the host cell is a yeast cell or an E. coli cell. In some cases, the host cell is a yeast cell or an E. coli cell. In some cases, the host cell is a yeast cell. In some cases, the host cell is derived from a yeast strain that has been genetically engineered to produce a BIA of interest, such as a morphinan alkaloid. In some cases, the host cell is derived from a yeast strain that has been genetically engineered to produce an enzyme of interest. In some cases, the host cell is derived from a yeast strain that has been genetically engineered to produce an enzyme of interest.It is derived from a yeast strain genetically engineered to produce thebaine synthase.

[0025] The thebaine synthase may be capable of converting salutaridinol-7-O-acetate to thebaine more efficiently than the natural reaction. In some cases, the host cell is derived from a yeast strain genetically engineered to produce the genetically engineered thebaine synthase. In some embodiments, the genetically engineered thebaine synthase may be a genetically engineered fusion enzyme. Furthermore, the genetically engineered thebaine synthase may be capable of converting salutaridinol-7-O-acetate to thebaine more efficiently than thebaine synthase. In some embodiments, the thebaine synthase may be a wild-type thebaine synthase. In some embodiments, the thebaine synthase may be substantially similar to the wild-type thebaine synthase. In some cases, a thebaine synthase that is substantially similar to a wild-type thebaine synthase can have 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 similar to the amino acid sequence of a wild-type thebaine synthase. An engineered thebaine synthase may be engineered as a fusion enzyme with another enzyme to more efficiently convert salutaridinol-7-O-acetate to thebaine compared to thebaine synthase.

[0026] Any of the host cells 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 cells can be of the species S. cerevisiae. In certain embodiments, the yeast cells can be of the species Schizosaccharomyces pombe. In certain embodiments, the yeast cells can be of the species Pichia pastoris. 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. In a further example, the cytochrome P450 protein is involved in the production of an enzyme of interest.

[0027] Yeast strains of interest that 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).

[0028] In some cases, the host cell is a fungal cell. In certain embodiments, the fungal cell may be 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).

[0029] 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.

[0030] 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.

[0031] 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 about 37° C. with or without antibiotics.

[0032] 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.

[0033] 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 residue 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.

[0034] 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.

[0035] 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.

[0036] The genetically engineered host cell may further overproduce one or more (R)-1-benzylisoquinoline alkaloids. In some cases, the genetically engineered host cell may 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 may 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 production of some of the (R)-1-benzylisoquinoline alkaloid of interest.

[0037] The genetically engineered host cells can further overproduce one or more morphinan alkaloids. In some cases, the genetically engineered host cells can produce some amount of the morphinan alkaloid of interest when the control does not have the production of the morphinan alkaloid, and in situations where the control has the production of some morphinan alkaloid of interest, 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 times or more, for example, 5 times or more, for example, 10 times or more. The genetically engineered host cells can further overproduce one or more of promorphinan, nor-opioid or nar-opioid alkaloids.

[0038] In some cases, engineered host cells can produce increased amounts of (R)-Reticuline compared to control host cells lacking one or more modifications (e.g., as described herein). In some cases, engineered host cells with engineered split epimerases can produce increased amounts of (R)-Reticuline compared to host cells with fusion epimerases. In some cases, engineered host cells with modifications in the oxidase portion of the engineered epimerase can produce increased amounts of (R)-Reticuline compared to control host cells lacking the one or more modifications in the oxidase portion of the engineered epimerase. In some particular cases, the increase in (R)-Reticuline compared to control host cells is 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, about 2-fold or more, about 5-fold or more, or even about 10-fold or more compared to control host cells. In some cases, (R)-Reticuline is the product of an epimerization reaction in a genetically engineered host cell. In some cases, (R)-Reticuline is the product of an epimerization reaction catalyzed by at least one genetically engineered epimerase in a genetically engineered host cell. In such cases, (S)-Reticuline can be a substrate for this epimerization reaction.

[0039] In some cases, engineered host cells can produce increased amounts of thebaine compared to control host cells lacking one or more modifications (e.g., as described herein). In some cases, engineered host cells with thebaine synthase can produce increased amounts of thebaine compared to host cells lacking thebaine synthase. In some cases, engineered host cells with engineered thebaine synthase can produce increased amounts of thebaine compared to host cells with a non-engineered thebaine synthase (e.g., as described herein). In some cases, the increase in thebaine is about 10% or more compared to control host cells, 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, about 2-fold or more, about 5-fold or more, or even about 10-fold or more compared to control host cells. In some cases, thebaine is the product of the reaction of thebaine synthase in the engineered host cells. In some cases, thebaine is the product of a thebaine synthase reaction catalyzed by at least one engineered thebaine synthase in an engineered host cell. In such cases, salutaridinol-7-O-acetate can be a substrate for the thebaine synthase reaction.

[0040] Furthermore, genetically engineered host cells can overproduce one or more enzymes of interest. By overproducing, it is meant that the cells have improved or increased production of the enzyme of interest compared to control cells (e.g., unmodified cells). By improved or increased production, it is meant both the production of some amount of the enzyme of interest when the control does not have the production of the enzyme of interest, and 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 times or more, for example, 5 times or more, for example, 10 times or more when the control has some production of the enzyme of interest.

[0041] The genetically engineered host cells may overproduce one or more DRS-DRR enzymes. In some cases, the genetically engineered host cells may produce some amount of DRS-DRR enzyme when the control does not have production of the DRS-DRR enzyme, and may 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 DRS-DRR enzyme production.

[0042] The engineered host cell can overproduce one or more engineered DRS-DRR enzymes. In some cases, the engineered host cell can produce some amount of engineered DRS-DRR epimerase when the control does not have DRS-DRR enzyme production or when the control has the same level of wild-type epimerase production as the engineered host cell, 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 when the control has some DRS-DRR enzyme production. In some cases, the engineered DRS-DRR epimerase can be an engineered separate epimerase. In some cases, the engineered DRS-DRR epimerase can be an engineered fusion epimerase.

[0043] The genetically engineered host cell may also overproduce one or more enzymes derived from the DRS-DRR enzyme. In some cases, the genetically engineered host cell may produce some amount of enzyme derived from the DRS-DRR enzyme when the control does not have production of the enzyme derived from the DRS-DRR enzyme, and may 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 production of some enzyme derived from the DRS-DRR enzyme.

[0044] The genetically engineered host cells can overproduce one or more thebaine synthase enzymes. In some cases, the genetically engineered host cells can produce some amount of thebaine synthase enzyme when the control does not have production of thebaine synthase enzyme, and can 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 thebaine synthase enzyme.

[0045] The engineered host cells can overproduce one or more engineered thebaine synthase enzymes. In some cases, the engineered host cells can produce some amount of engineered thebaine synthase when the control does not have thebaine synthase enzyme production or when the control has the same level of wild-type thebaine synthase production as the engineered host cells, and can 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 thebaine synthase enzyme production. In some cases, the engineered thebaine synthase can be an engineered fusion enzyme.

[0046] The genetically engineered host cell may further overproduce one or more enzymes derived from thebaine synthase enzyme. In some cases, the genetically engineered host cell may produce some amount of the enzyme derived from thebaine synthase enzyme when the control does not have production of the enzyme derived from thebaine synthase enzyme, 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 production of the enzyme derived from thebaine synthase enzyme.

[0047] In some cases, 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 modulation; and heterologous coding sequences encoding enzymes. Cells containing one or more modifications can be referred to as genetically engineered cells.

[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 localization mutations; cytochrome P450 reductase interaction mutations; accessibility mutations; activity-enhancing mutations; engineered fusion thebaine synthase modifications; and engineered split epimerase modifications. A cell containing one or more modifications can be referred to as an 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. 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.

[0051] Various substrate inhibition control mechanisms and biosynthetic enzymes in genetically engineered host cells directed at regulating the level of a BIA of interest or its precursor 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 3.

[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, a genetically engineered host cell is a cell that includes 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 enhances 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 BIA of interest or its precursor 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 contains 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. 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.

[0059] Various control mechanisms of product inhibition in genetically engineered host cells, directed at regulating the level of BIA of interest, and biosynthetic enzymes can be targeted for product inhibition relief. The genetically engineered host cell 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 cell contains one or more product inhibition relief mutations in one or more biosynthetic enzyme genes, for example, in one of the genes listed in Table 3.

[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 / salvage pathway, as shown in Figure 1. Figure 1 illustrates an example of a tetrahydrobiopterin synthesis, salvage, 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 BIAs when expressed in yeast strains for producing BIAs 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. 50by 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 3.

[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 alterations 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, ARO8, and TKL. In some examples, the one or more biosynthetic enzyme genes can be selected from ARO10, ARO9, ARO8, 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 3.

[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, e.g., E. coli, can be selected. 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 The 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) to an enzyme of the cell. By including one or more inactivating mutations, the genetically engineered host cell's flux of a synthetic pathway can be modified to increase the level of a BIA of interest or a desirable enzyme or precursor that leads thereto. In some examples, the one or more inactivating mutations are to an enzyme native to the cell. Additionally or alternatively, the one or more inactivating mutations are to an enzyme non-native to the cell. As used herein, "inactivating mutation(s)" refers to one or more mutations to a gene or regulatory DNA sequence of the cell that inactivates the biological activity of a protein expressed by the gene of interest. In some cases, the gene is native to the cell. In some examples, the gene is inactivated and encodes an enzyme that is part of or associated with the synthetic pathway of the BIA of interest that the host cell produces. In some examples, the inactivating mutation(s) is / are placed within a regulatory DNA sequence that controls the gene of interest. In some cases, inactivating mutation is in the promoter of gene.Any convenient mutation (for example, as described herein) can be used to inactivate the gene of interest or regulatory DNA sequence." Inactivated" or "inactivate" 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 with the control protein expressed by the non-mutated control gene.In some cases, the protein is an enzyme, and the activity of the enzyme is reduced by inactivating mutation.

[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 3, in which the action of the engineered host cell in a synthetic pathway tends to reduce the BIA level 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 3.

[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 examples, the conversion of (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids involves an epimerization reaction using a genetically engineered epimerase. 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 Figure 3 and generally 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. Scheme 1 TIFF0007730358000005.tif26170

[0076] In some cases, the conversion of an (S)-substrate to an (R)-product can 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 and reduction reactions is performed in the presence of an enzyme. In some cases, at least one of the oxidation and reduction reactions is catalyzed by an enzyme. In some cases, both the oxidation and reduction reactions are performed in the presence of at least one enzyme. In some cases, at least one enzyme is useful for catalyzing the oxidation and reduction reactions. The oxidation and reduction reactions may be catalyzed by the same enzyme. In some cases, at least one of the oxidation and reduction reactions is catalyzed by an engineered epimerase. In some cases, both the oxidation and reduction reactions are performed in the presence of an engineered fusion epimerase. In some cases, both the oxidation and reduction reactions are performed in the presence of an engineered split epimerase having separately expressed oxidase and reductase components, respectively. In some cases, an engineered epimerase is useful for catalyzing the oxidation and reduction reactions. The oxidation and reduction reactions may be catalyzed by the same engineered epimerase.

[0077] In some methods, processes, and systems described herein, the oxidation reaction may be carried out in the presence of an enzyme. In some cases, the enzyme may be an oxidase. In some cases, the enzyme may be part of an engineered epimerase. In some cases, the engineered epimerase may have an oxidase component. In some cases, the oxidase component may be a component of an engineered fusion epimerase. In some cases, the oxidase component may be expressed independently as part of an engineered split epimerase. The oxidase may use (S)-1-benzylisoquinoline as a substrate. The oxidase may convert the (S)-substrate to the corresponding imine or Schiff base derivative. The oxidase may be referred to as 1,2-dehydroreticuline synthase (DRS). Non-limiting examples of enzymes suitable for oxidizing (S)-1-benzylisoquinoline alkaloids in the present disclosure include cytochrome P450 oxidases, 2-oxoglutarate-dependent oxidases, and flavoprotein oxidases. 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 cases, a protein containing an oxidase domain from any one of the preceding examples can perform the oxidation. In some cases, the oxidase can catalyze the oxidation reaction in a host cell, such as a genetically engineered host cell, as described herein.

[0078] In some cases, the reduction reaction can be followed by an oxidation reaction. In some cases, the reduction reaction can be performed by an enzyme. In some cases, the reduction reaction can be performed by an enzyme that is part of a genetically engineered epimerase. 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 instances, a protein comprising any one of the reductase domains above can perform the reduction. In a further aspect, the reduction is stereospecific. In some instances, 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 epimerization of (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 DRS-DRR enzyme. In particular, the DRS-DRR enzyme may be a fusion enzyme, which is a fusion epimerase. Furthermore, when the DRS-DRR enzyme is modified with at least one activity-enhancing modification, the fusion enzyme may be a genetically engineered fusion epimerase.

[0080] An example of the amino acid sequence of a DRS-DRR 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 DRS-DRR 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 dashed letters represent the aldo-ketoreductase NADPH-dependent codeinone reductase-like domain (75% identity to ACM44066.1). Additional amino acid sequences of DRS-DRR enzymes are shown in Table 1. The amino acid sequence of an epimerase used to convert (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids can be 75% or more identical to a given amino acid sequence listed in Table 1. For example, the amino acid sequence of such epimerases 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 to a 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% and 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 is altered, for example, to optimize codon usage for the host organism.

[0081] A genetically engineered host cell may be provided that produces an epimerase that converts an (S)-1-benzylisoquinoline alkaloid to an (R)-1-benzylisoquinoline alkaloid, the epimerase comprising 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 may be recovered and purified to form a biocatalyst. In some cases, the epimerase may be split into one or more enzymes. Furthermore, one or more enzymes produced by the epimerase split may be recovered from the genetically engineered host cell. The one or more enzymes produced by the epimerase split may also be used to catalyze the conversion of an (S)-1-benzylisoquinoline alkaloid to an (R)-1-benzylisoquinoline alkaloid. 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] An example of an amino acid sequence of a DRS-DRR 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 codon-optimized DRS-DRR enzyme according to an embodiment of the present invention. Additionally, Figure 5 shows the separation of the oxidase and reductase portions of each of the DRS-DRR enzymes of Figure 4. Additional amino acid sequences of DRS-DRR enzymes are shown in Table 1. The amino acid sequence of an epimerase used to convert (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids can be 75% or more identical to a given amino acid sequence listed in Table 1. For example, the amino acid sequence of such epimerases 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 to a 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% and 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 is altered, for example, to optimize codon usage for the host organism.

[0083] Amino acid residues of homologous epimerases may be referred to according to the numbering scheme of SEQ ID NO. 16, and this numbering system is used throughout this disclosure to indicate specific amino acid residues of epimerases homologous to SEQ ID NO. 16. Epimerases homologous to SEQ ID NO. 16 may have at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 16. In some cases, the amino acid designated as position 50 of a homologous epimerase may not be the 50th amino acid in the homologous epimerase, but may be the amino acid corresponding to the 50th amino acid in SEQ ID NO. 16 in a protein alignment of the homologous epimerase with SEQ ID NO. 16. In some cases, homologous enzymes may be aligned with SEQ ID NO. 16 according to either primary sequence, secondary structure, or tertiary structure.

[0084] A genetically engineered host cell may be provided that produces an engineered epimerase that converts (S)-1-benzylisoquinoline alkaloid to (R)-1-benzylisoquinoline alkaloid, wherein 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, 15, 16, 17, and 18, and has one or more activity-enhancing modifications. The epimerase produced in the genetically engineered host cell may be recovered and purified to form a biocatalyst. In some cases, the epimerase may be split into one or more enzymes. Furthermore, one or more enzymes produced by splitting the epimerase may be recovered from the genetically engineered host cell. The enzyme or enzymes resulting from the splitting of the epimerase can also be used to catalyze the conversion of (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids. Furthermore, the use of an engineered split epimerase can be used to increase the production of benzylisoquinoline alkaloid products in cells compared to the production of benzylisoquinoline alkaloid products in cells using a fused epimerase.

[0085] In further cases, 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.

[0086] 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.

[0087] 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 1-benzylisoquinoline, morphinan, promorphinan, nor-opioid, or nal-opioid alkaloids.

[0088] 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.

[0089] In some instances, the (S)-substrate is a compound of Formula I: TIFF0007730358000006.tif35128 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.

[0090] In some other cases, R 1 , R 2 , R 3 , R 4 and R 5 At least one of the is hydrogen.

[0091] In yet another example, the (S)-substrate is a compound of formula II: TIFF0007730358000007.tif33128 or a salt thereof, wherein: R 3 is selected from hydrogen and C1-C4 alkyl; R 6 and R 7is 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.

[0092] 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.

[0093] 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.

[0094] 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 a key step in the production of various alkaloid products from precursors. In some examples, the precursor is L-tyrosine or a sugar (e.g., glucose). Various alkaloid products can include, but are not limited to, 1-benzylisoquinoline alkaloids, morphinan alkaloids, promorphinan alkaloids, nor-opioid alkaloids, or nal-opioid alkaloids.

[0095] 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, unpurified 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 the engineered host cells of the present invention. 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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%.

[0100] In some instances, the promorphinan or a derivative thereof is recovered. In some instances, the promorphinan is recovered from the cell culture.

[0101] In some instances, the morphinan or derivative thereof is recovered. In some instances, the morphinan is recovered from the cell culture.

[0102] In some instances, the nar-opioid or a derivative thereof is recovered. In some instances, the nar-opioid is recovered from a cell culture.

[0103] In some instances, the nor-opioid or a derivative thereof is recovered. In some instances, the nor-opioid is recovered from the cell culture.

[0104] "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 specified as (R)- or (S)- with respect to absolute stereochemical configuration.

[0105] Table 1. Examples of amino acid sequences of DRS-DRR enzymes, split DRS and DRR enzymes, and other nucleotide sequences. TIFF0007730358000008.tif196170TIFF0007730358000009.tif242167TIFF0007730358000010.tif242167TIFF0007730358000011.tif242168TIFF0007730358000012.tif242170TIFF0007730358000013.tif242170TIFF0007730358000014.tif242170TIFF0007730358000015.tif242170TIFF0007730358000016.tif242168TIFF0007730358000017.tif242170TIFF0007730358000018.tif242170TIFF0007730358000019.tif242168TIFF0007730358000020.tif242168TIFF0007730358000021.tif242168TIFF0007730358000022.tif242167TIFF0007730358000023.tif245170TIFF0007730358000024.tif242168TIFF0007730358000025.tif242168TIFF0007730358000026.tif242168TIFF0007730358000027.tif242167TIFF0007730358000028.tif242168TIFF0007730358000029.tif242168TIFF0007730358000030.tif242168TIFF0007730358000031.tif242167TIFF0007730358000032.tif242168TIFF0007730358000033.tif242168TIFF0007730358000034.tif242168TIFF0007730358000035.tif242168TIFF0007730358000036.tif248170TIFF0007730358000037.tif242168TIFF0007730358000038.tif242168TIFF0007730358000039.tif242168TIFF0007730358000040.tif242168TIFF0007730358000041.tif242167TIFF0007730358000042.tif242168TIFF0007730358000043.tif2 42168TIFF0007730358000044.tif242168TIFF0007730358000045.tif242168TIFF0007730358000046.tif80170.

[0106] Modification of morphinan alkaloid production Some methods, processes, and systems provided herein describe the conversion of promorphinan alkaloids to morphinan alkaloids. Some of the methods, processes, and systems describe the conversion of a tetracyclic ring system to a pentacyclic ring system (Figure 20). Some of the methods, processes, and systems may include genetically engineered host cells. In some examples, the production of pentacyclic thebaine or morphinan alkaloids from tetracyclic precursors or promorphinan alkaloids is described. In some examples, the conversion of promorphinan alkaloids to thebaine is a key step in the conversion of substrates to a diverse range of benzylisoquinoline alkaloids.

[0107] In some cases, the tetracyclic precursor can be salutaridinol, salutaridinol, or salutaridinol-7-O-acetate. The tetracyclic precursor can be converted to pentacyclic thebaine by closure of the oxide bridge between C-4 and C-5. In some cases, the tetracyclic precursor salutaridinol can be prepared by stepwise hydroxylation and O-acetylation at C-7 for ring closure. Ring closure can be activated by loss of the acetate leaving group. In some cases, allylic loss and oxide ring closure to produce thebaine occur spontaneously. In other cases, the ring closure reaction to produce pentacyclic thebaine is promoted by factors such as pH or solvent. In other cases, the thebaine-producing ring closure reaction is promoted by contact with a protein or enzyme. These conversion steps are illustrated in Figure 14 and generally depicted in Scheme 2. R1, R2, and R3 can be H or CH3. R4 can be CH3, CH3CH2, CH3CH2CH2, or other suitable alkyl groups. In some cases, R1, R2, R3, and R4 can be CH3, as shown in Figure 14. Scheme 2 TIFF0007730358000047.tif27161

[0108] In some examples, the first enzyme that prepares the tetracyclic precursor is salutaridin reductase (SalR). In some cases, SalR hydroxylates the C-7 position of the substrate salutaridin (see Formula III). The product of this reaction can be one or more salutaridinol epimers. In some examples, the product is (7S)-salutaridinol. In some examples, salutaridin reductase can catalyze the reduction reaction in a host cell described herein, for example, a genetically engineered host.

[0109] In some cases, the second enzyme that prepares the tetracyclic precursor is salutaridinol 7-O-acetyltransferase (SalAT). In some cases, SalAT transfers the acetyl of acetyl-CoA to the 7-OH of salutaridinol (see Formula IV). In other cases, SalAT can utilize a novel cofactor, such as n-propionyl-CoA, to transfer propionyl to the 7-OH of salutaridinol. In some cases, the product of SalAT is (7S)-salutaridinol-7-O-acetate. In some cases, salutaridinol 7-O-acetyltransferase can catalyze the acetyl transfer reaction in the host cell described herein, for example, in a genetically engineered host.

[0110] In some examples, the tetracyclic precursor of thebaine is (7S)-salutaridinol-7-O-acetate. In some examples, (7S)-salutaridinol-7-O-acetate is unstable, and the acetate at C-7 spontaneously disappears, closing the oxide bridge between C-4 and C-5 to form thebaine (see Formula V). In some examples, the rate of acetate leaving group disappearance is accelerated by pH. In some examples, the allylic disappearance and oxide bridge closure are catalyzed by an enzyme having thebaine synthase activity or a thebaine synthase. In some examples, the enzyme is a Bet v 1-fold protein. In some examples, the enzyme is an engineered thebaine synthase, an engineered SalAT, a diversification-induced reversal (DIR) protein, or a chalcone isomerase (CHI). In some examples, an enzyme encoding thebaine synthase activity can catalyze the ring closure reaction in a host cell, e.g., a genetically engineered host, described herein.

[0111] In some examples, the salutaridine reductase enzyme can be a SalR or SalR-like enzyme from a plant of the order Ranunculales, such as Papaver somniferum, that biosynthesizes thebaine. In other examples, the enzyme with salutaridine reductase activity can be from a mammal or any other vertebrate or invertebrate that biosynthesizes endogenous morphine.

[0112] In some examples, the salutaridinol 7-O-acetyltransferase enzyme can be a SalAT or SalAT-like enzyme from a plant of the Ranunculales order, such as the poppy, that biosynthesizes thebaine. In other examples, the enzyme having salutaridinol 7-O-acetyltransferase activity can be from a mammal or any other vertebrate or invertebrate that biosynthesizes endogenous morphine.

[0113] In some examples, the thebaine synthase enzyme can be a Bet v 1-fold protein derived from a plant of the Ranunculaceae order, such as the poppy, which biosynthesizes thebaine. In some examples, the Bet v 1 protein comprises the following domains, from the N-terminus to the C-terminus: a β-strand, one or two α-helices, six β-strands, and one or two α-helices. The protein is organized to have a Bet v 1 fold and an active site that accepts large, bulky hydrophobic molecules, such as morphinan alkaloids. The protein can be any plant Bet v 1 protein, infection-specific 10 protein (PR-10), major latex protein (MLP), fruit or pollen allergen, plant hormone-binding protein (e.g., that binds to cytokinin or brassinosteroids), plant polyketide cyclase-like protein, or norcoclaurine synthase (NCS)-related protein with a Bet v 1 fold. Other examples of non-plant Bet v 1-fold proteins include polyketide cyclases, Hsp90 ATPase homolog activator 1 (AHA1) proteins, SMU440-like proteins (e.g., from Streptococcus mutans), PA1206-related proteins (e.g., from Pseudomonas aeruginosa), the CalC calicheamicin resistance protein (e.g., from Micromonospora echinospora), and the CoxG protein from the carbon monoxide-metabolizing bacterium Oligotropha carboxidovorans. Further examples of Bet v 1-related proteins include the START lipid transfer protein, phosphatidylinositol transfer protein, and ring hydroxylase.

[0114] In some examples, the thebaine synthase enzyme can be a morphogenetic protein from a plant of the Ranunculales order, such as the poppy, that biosynthesizes thebaine. In other examples, the enzyme can be any morphogenetic protein from a plant.

[0115] In some examples, the thebaine synthase enzyme can be a chalcone isomerase protein from a plant of the Ranunculales order, such as the poppy, that biosynthesizes thebaine. In other examples, the enzyme can be any chalcone isomerase protein from a plant.

[0116] In some examples, the thebaine synthase enzyme can be a SalAT-like enzyme from a plant of the Ranunculales order, such as the poppy, that biosynthesizes thebaine. In other examples, the enzyme can be any SalAT-like protein from a plant.

[0117] In some instances, the enzyme having thebaine synthase activity can be derived from a mammal or any other vertebrate or invertebrate that biosynthesizes endogenous morphine.

[0118] In some cases, any combination of the above enzymes along with additional accessory proteins can function to convert any tetracyclic precursor to thebaine, and in some cases, such enzymes catalyze the reaction in the host cells described herein, e.g., genetically engineered hosts.

[0119] Exemplary amino acid sequences for thebaine synthase activity are shown in Table 2. The amino acid sequence of a thebaine synthase used to convert a tetracyclic precursor to thebaine can be 45% or more identical to a given amino acid sequence listed in Table 2. For example, such thebaine synthase amino acid sequences may be at least 45% or more, 46% or more, 47% or more, 48% or more, 49% or more, 50% or more, 51% or more, 52% or more, 53% or more, 54% or more, 55% or more, 56% or more, 57% or more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more identical to the amino acid sequences provided herein. , 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% 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. Furthermore, in some specific embodiments, an "identical" amino acid sequence comprises at least 80%-99% identity at the amino acid level to a 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% and 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 is altered, for example, to optimize codon usage for the host organism.

[0120] A genetically engineered host cell can be provided that produces salutaridin reductase, salutaridinol 7-O-acetyltransferase, and thebaine synthase, which convert a tetracyclic precursor to thebaine, wherein the thebaine synthase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 31, 32, 33, 34, 35, 36, and 37. In some cases, thebaine synthase can form a fusion protein with another enzyme. The enzymes produced in the genetically engineered host cell can be recovered and purified to form a biocatalyst. Such one or more enzymes can also be used to catalyze the conversion of a tetracyclic promorphinan precursor to thebaine.

[0121] In other examples, the thebaine synthase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 and 61.

[0122] In further cases, the one or more enzymes recovered from the genetically engineered host cell can be used in a method for converting a tetracyclic promorphinan precursor to thebaine. The method can include contacting the tetracyclic promorphinan precursor with a sufficient amount of the recovered enzyme to convert the tetracyclic promorphinan precursor to thebaine. In one example, the tetracyclic promorphinan precursor can be contacted with a sufficient amount of the one or more enzymes such that at least 5% of the tetracyclic promorphinan precursor is converted to thebaine. In a further example, the tetracyclic promorphinan precursor can 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 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 tetracyclic promorphinan precursor is converted to thebaine.

[0123] In some cases, process conditions are implemented to support the formation of thebaine in the engineered host cells. In some cases, the engineered host cells are cultured at pH 3.3, and once high cell density is reached, the pH is adjusted to pH 8.0 to support continued production of thebaine at high pH. In some cases, the engineered host cells produce additional enzymes for converting sugars and other simple precursors, such as tyrosine, to thebaine. In some cases, the SalAT enzyme is engineered to be highly active at pH 8.0 and is expressed from a late promoter.

[0124] In some examples, one or more of the enzymes that convert tetracyclic promorphinan precursors to thebaine are localized in intracellular compartments. In some examples, SalR, SalAT, and Bet v 1 can be modified to encode targeting sequences that localize them to the endoplasmic reticulum membrane of genetically engineered host cells (see, for example, WO2014143744). In other examples, SalAT and Bet v 1 can be co-localized in a single protein fusion. In some examples, the fusion of SalAT and Bet v 1 is created by one of several methods, such as direct fusion, co-localization in yeast organelles, or by using enzyme co-localization tools, such as leucine zippers, protein scaffolds using adaptor domains, or RNA scaffolds using aptamers. Co-localizing thebaine synthase can facilitate substrate channeling between the active sites of the enzymes, limiting the diffusion of unstable intermediates, such as salutaridinol-7-O-acetate.

[0125] In some instances, an engineered salutaridinol 7-O-acetyltransferase (SalAT) enzyme is used to convert the tetracyclic promorphinan precursor to thebaine. In some instances, the SalAT enzyme is engineered to combine two functions: (1) the transfer of the acyl group of acetyl-CoA to the 7-OH of salutaridinol, and (2) the subsequent loss of the acetyl group and closure of the oxide bridge between the C4 and C5 carbons to form thebaine.

[0126] In some examples, an enzyme having salutaridinol 7-O-acetyltransferase activity is fused to a peptide having a Bet v 1 fold. In some examples, the salutaridinol 7-O-acetyltransferase enzyme and the Bet v 1 fold protein can be fused N-terminal to C-terminal, C-terminal to N-terminal, N-terminal to N-terminal, or C-terminal to C-terminal in any order. In some examples, the sequences of the two proteins can be fused directly or via a peptide linker region.

[0127] In some cases, an enzyme with salutaridinol 7-O-acetyltransferase activity is fused to a peptide with a Bet v 1 fold by circular permutation. In some cases, the N-terminus and C-terminus of SalAT are fused, and then the Bet v 1 sequence is randomly inserted into this sequence. In some cases, the resulting fusion protein library is screened for thebaine production. In other cases, the circularly permuted SalAT library is first screened for activity in the absence of Bet v 1. In other cases, the N-terminus and C-terminus of SalAT are fused, and the enzyme is digested and blunt-end cloned. In other cases, this library of circularly permuted SalAT is screened for salutaridinol 7-O-acetyltransferase activity. In other cases, active mutants from the circularly permuted SalAT library are then used to design protein fusions with peptides with a Bet v 1 fold.

[0128] The one or more enzymes that can be used to convert a tetracyclic promorphinan precursor to thebaine can be contacted with the tetracyclic promorphinan precursor in vitro. Additionally or alternatively, the one or more enzymes that can be used to convert a tetracyclic promorphinan precursor to thebaine can be contacted with the tetracyclic promorphinan precursor in vivo. Furthermore, the one or more enzymes that can be used to convert a tetracyclic promorphinan precursor to thebaine can be supplied to a cell that has the tetracyclic promorphinan precursor therein or can be produced in a genetically engineered host cell.

[0129] In some examples, the method results in a genetically engineered host cell that produces an alkaloid product, where conversion of a tetracyclic promorphinan precursor to thebaine may constitute a key step in the production of the alkaloid product. In some examples, the alkaloid product is thebaine. In yet other embodiments, the alkaloid product includes, for example, downstream morphinan alkaloids derived from thebaine. In another embodiment, the tetracyclic promorphinan precursor is an intermediate to the product in the genetically engineered host cell. In yet other embodiments, the alkaloid product is selected from the group consisting of morphinan, nor-opioid, or nal-opioid alkaloids.

[0130] In some instances, the substrate for the reduction reaction is of formula III: TIFF0007730358000048.tif32128 or a salt thereof, wherein: R1, R2 and R3 are independently selected from hydrogen and methyl.

[0131] In some other instances, R1, R2, and R3 are methyl and the reduction reaction is catalyzed by salutaridine reductase.

[0132] In some instances, the substrate for the carbon chain transfer reaction is represented by Formula IV: TIFF0007730358000049.tif39128 or a salt thereof, wherein: R1, R2 and R3 are independently selected from hydrogen and methyl.

[0133] In some other instances, R1, R2, and R3 are methyl and the carbon chain transfer reaction is catalyzed by salutaridinol 7-O-acetyltransferase.

[0134] In some instances, the substrate for thebaine synthase has the formula V: TIFF0007730358000050.tif39128 or a salt thereof, wherein: R1, R2, and R3 are independently selected from hydrogen and methyl; R4 is selected from methyl, ethyl, propyl and other suitable alkyl groups.

[0135] In some other examples, R1, R2, R3, and R4 are methyl and the ring closure reaction is catalyzed by thebaine synthase. In some examples, the thebaine synthase is a Bet v 1 protein.

[0136] In some examples, the method results in a genetically engineered host cell that produces alkaloid products from salutardine. The conversion of salutardine to thebaine can constitute a key step in the production of various alkaloid products from precursors. In some examples, the precursor is L-tyrosine or a sugar (e.g., glucose). Various alkaloid products can include, but are not limited to, morphinan, nor-opioid, or nal-opioid alkaloids.

[0137] Any suitable carbon source can be used as a precursor to pentacyclic morphinan 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 the genetically engineered host cells of the present invention.

[0138] In some examples, a benzylisoquinoline alkaloid product or a derivative thereof is recovered. In some examples, the benzylisoquinoline alkaloid product is recovered from the cell culture. In some examples, the benzylisoquinoline alkaloid product is a morphinan, nor-opioid, or nal-opioid alkaloid.

[0139] Table 2: Examples of amino acid sequences of morphinan alkaloid-producing enzymes. TIFF0007730358000051.tif241166TIFF0007730358000052.tif240166TIFF0007730358000053.tif240166 TIFF0007730358000054.tif247166TIFF0007730358000055.tif243166TIFF0007730358000056.tif238166

[0140] BIA generation modification Once formed, the BIA may be further derivatized or modified. The BIA may be derivatized or modified using one or more enzymes produced by the genetically engineered host cell. In particular, the BIA may be derivatized or modified by contacting the BIA with one or more enzymes produced by the genetically engineered host cell. Additionally or alternatively, the BIA may be derivatized or modified by contacting the BIA with one or more enzymes provided to the BIA from a source external to the genetically engineered host cell. The one or more enzymes that can be used to derivatize or modify the BIA may 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 BIA may be derivatized or modified using one or more tailoring reactions.

[0141] Examples of tailoring reactions are shown in Table 9. In some examples, tailoring enzymes can be used to catalyze carbon-carbon coupling reactions performed in BIAs 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 3, where R a , R b , R c and R d are 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. Scheme 3 TIFF0007730358000057.tif79128

[0142] In some examples, tailoring enzymes can be used to catalyze oxidation reactions carried out in BIAs or their 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.

[0143] Tailoring enzymes can also be used to catalyze the methylenedioxy bridge formation reaction carried out in BIAs or their derivatives. Examples of tailoring enzymes that can be used to catalyze the methylenedioxy bridge formation reaction include stylopine synthase (StySyn) from poppy, California poppy, American poppy, or another species; cheilanthifoline synthase (CheSyn) from poppy, California poppy, American poppy, or another species; and canadine synthase (CAS) from yellow larch, Coptis chinensis, or another species.

[0144] In other examples, tailoring enzymes can be used to catalyze O-methylation reactions carried out in BIAs 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.

[0145] Additionally, tailoring enzymes can be used to catalyze N-methylation reactions carried out in BIAs or their derivatives. Examples of tailoring enzymes that can be used to catalyze N-methylation reactions 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.

[0146] Additionally, tailoring enzymes can be used to catalyze the O-demethylation reaction carried out in BIA 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.

[0147] In addition, tailoring enzymes can be used to catalyze reduction reactions performed on BIA 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 other species; codeinone reductase (COR) from Papaver somniferum or other species; and sanguinarine reductase (SanR) from California poppy or other species. In another example, tailoring enzymes can be used to catalyze acetylation reactions performed on BIA or its derivatives. One example of a tailoring enzyme that can be used to catalyze acetylation reactions is salutaridine acetyltransferase (SalAT) from Papaver somniferum or other species.

[0148] O-demethylation modification Some methods, processes, and systems provided herein describe the conversion of a first benzylisoquinoline alkaloid to a second benzylisoquinoline alkaloid by removal of an O-linked methyl group. Some such methods, processes, and systems may include genetically engineered host cells. In some examples, the conversion of the first benzylisoquinoline alkaloid to the 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 the first alkaloid to the second alkaloid involves a demethylase reaction.

[0149] 6 shows 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.

[0150] Examples of amino acid sequences of ODM enzymes are shown in Table 4. 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 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% and 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 altered, for example, to optimize codon usage for the host organism.

[0151] 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 4. 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.

[0152] 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.

[0153] 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 another embodiment, 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.

[0154] 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.

[0155] 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.

[0156] 7 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.

[0157] 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 5. 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 5. 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% and 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 altered, for example, to optimize codon usage for the host organism.

[0158] 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 5. 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.

[0159] 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.

[0160] 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 in the genetically engineered host cell to the product. 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.

[0161] 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.

[0162] N- Combined type 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.

[0163] 8 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).

[0164] Examples of amino acid sequences of NMT enzymes are shown in Table 6. 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 6. 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% and 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 altered, for example, to optimize codon usage for the host organism.

[0165] 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 6. 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.

[0166] 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.

[0167] In some examples, the method provides a genetically engineered host cell that produces an alkaloid product, wherein the N-methyltransferase of the substrate to the 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 in 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.

[0168] 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.

[0169] 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.

[0170] In one example, a genetically engineered host cell can include multiple heterologous coding sequences, each encoding an enzyme, for example, an enzyme listed in Table 3. In some examples, the enzymes encoded by the heterologous coding sequences can differ from one another. In some examples, some of the enzymes encoded by the heterologous coding sequences can differ from one another, and some of the enzymes encoded by the heterologous coding sequences can be duplicate copies.

[0171] In some cases, heterologous coding sequences may be operably linked. Operatively linked heterologous coding sequences may be in the same pathway that produces a particular benzylisoquinoline alkaloid product and / or thebaine synthase product. In some cases, operably linked heterologous coding sequences may be directly sequential along the pathway that produces a particular benzylisoquinoline alkaloid product and / or thebaine synthase product. In some cases, operably linked heterologous coding sequences may have one or more native enzymes between one or more of the enzymes encoded by the multiple heterologous coding sequences. In some cases, a heterologous coding sequence may have one or more heterologous enzymes between one or more of the enzymes encoded by the multiple heterologous coding sequences. In some cases, a heterologous coding sequence may have one or more non-native enzymes between one or more of the enzymes encoded by the multiple heterologous coding sequences.

[0172] In addition, genetically engineered host cells can be modified to have one or more genetic modifications to accommodate heterologous coding sequences.Modifications of natural host genome 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.

[0173] Heterologous coding sequences include, but are not limited to, sequences encoding enzymes that are either wild-type 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 I-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 cells of the invention 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.

[0174] 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.

[0175] Effective 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 original sequence for the desired reaction. If the derivative performs this function, it is considered to constitute an equivalent derivative of the enzyme.

[0176] In some instances, 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 exhibit incorrect processing. 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 instances, 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. See, for example, WO2016183023A1.

[0177] 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 substantially 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 example of thebaine synthase, 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 those of the original amino acid sequence. Properties of interest that can be varied include catalytic activity, substrate specificity, selectivity, stability, solubility, localization, etc.

[0178] 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.

[0179] 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.

[0180] In some specific embodiments, the genetically engineered host cell contains multiple copies of heterologous coding sequences 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 an enzyme are derived from two or more different source organisms 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 source organism. Thus, each copy may contain some diversity in the explicit sequence based on interspecies differences in the enzyme of interest encoded by the heterologous coding sequence.

[0181] 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.

[0182] Furthermore, the culture of the genetically engineered host cells can be sampled and monitored for the production of an enzyme of interest, such as thebaine synthase enzyme. The enzyme of interest can be observed and measured using any convenient method. Methods of interest include enzyme activity assays, polyacrylamide gel electrophoresis, carbon monoxide spectroscopy, and Western blot analysis.

[0183] method Methods for culturing host cells for BIA production As summarized above, some aspects of the present invention include methods for preparing a benzylisoquinoline alkaloid (BIA) of interest. Furthermore, some aspects of the present invention include methods for preparing an enzyme of interest. Thus, some aspects of the present invention include culturing genetically engineered host cells under conditions such that 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 enzyme and / or BIA of interest. Also provided are methods that include culturing genetically engineered host cells under conditions suitable for producing a protein such that one or more heterologous coding sequences are functionally expressed, causing the starting compound of interest to be converted into a product enzyme or BIA of interest. In one example, the method is a method for preparing a benzylisoquinoline alkaloid (BIA), comprising culturing genetically engineered host cells (e.g., as described herein); adding a starting compound to the cell culture; and recovering the BIA 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.

[0184] 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.

[0185] Any convenient method for culturing engineered host cells can be used to produce the enzyme and / or BIA of interest. The specific protocol used can vary, for example, depending on the engineered host cell, heterologous coding sequence, enzyme of interest, BIA of interest, etc. 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 inducible for enzyme expression with an appropriate substrate available to allow for in vivo production of the enzyme and / or BIA of interest. In some embodiments, the functional enzyme is extracted from the engineered host for in vitro production of the enzyme and / or BIA of interest. In some examples, the engineered host cells are returned to a multicellular host organism. The engineered host cells can be in any growth phase, including, but not limited to, stationary and logarithmic growth phases. The culture itself can be continuous or batch culture.

[0186] 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.

[0187] 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.

[0188] This may include the addition to the growth medium 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.

[0189] 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).

[0190] 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.

[0191] 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).

[0192] 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.

[0193] 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.

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 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.

[0200] 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.

[0201] 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.

[0202] The subject method may also include a step of adding a starting compound to the cell culture. Any convenient addition method 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., a mM-μM amount, e.g., about 1-5 mM of the 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 times 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).

[0203] Method for isolating a product from a fermentation medium The subject method may also include recovering the enzyme and / or 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 enzyme and / or 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, 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 enzyme and / or BIA of interest from other components of the cell culture.

[0204] 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., BIAs) 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 a BIA-enriched product.

[0205] In an example, a product stream having a benzylisoquinoline alkaloid (BIA) product is formed by feeding genetically engineered yeast cells and a feedstock containing nutrients and water into a batch reactor. In particular, 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 containing the BIA 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 BIA product from the cellular material, resulting in a product stream containing the BIA product. In particular, the product stream can include the BIA product as well as additional components, such as clarified yeast culture medium. Furthermore, the BIA product can include one or more BIAs of interest, such as one or more BIA compounds.

[0206] Various methods can be used to remove cells from bioreactor media containing the enzyme and / or BIA of interest. In one example, cells can be removed by sedimentation over time. This sedimentation 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.

[0207] If some valuable enzymes of interest and / or BIA are present inside cells, cells can be permeabilized or lysed, and cell debris can be removed by any of the above-mentioned methods.The agent used to permeabilize cells can include, but is not limited to, organic solvents (such as DMSO) or salts (such as lithium acetate).Methods for lysing cells can include adding detergents, such as sodium dodecyl sulfate, or mechanically disrupting by bead milling or ultrasonic treatment.

[0208] The enzyme and / or BIA of interest can be extracted from the clarified spent culture medium by liquid-liquid extraction through the addition of 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.

[0209] In some cases, the organic liquid may be added at the beginning of the fermentation or at any time during the fermentation. This extractive fermentation process may increase the yield of the enzyme and / or BIA of interest from the host cells by continuously removing the enzyme and / or BIA into the organic phase.

[0210] 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.

[0211] 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.

[0212] In an example, the enzyme and / or BIA of interest can be extracted from the fermentation medium using adsorption methods. In one example, the BIA of interest can be extracted from the clarified spent culture medium by adding a resin such as Amberlite® XAD4 or another agent that removes the BIA by adsorption. The BIA 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.

[0213] Alternatively, the BIA of interest may be extracted from the fermentation medium using filtration. At high pH, ​​the BIA 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.

[0214] 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.

[0215] Process for purifying products from alkaloid-rich solutions Subsequent purification steps can involve treating the post-fermentation solution enriched in the BIA products of interest using methods known in the art to recover the individual product species of interest in high purity.

[0216] In one example, the BIA 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 BIA of interest into the aqueous phase. The aqueous phase can then be removed by decantation, centrifugation, or another method.

[0217] The BIA-containing solution may be further processed to remove metals, for example, by treatment with a suitable chelating agent. The BIA-containing solution of interest may be further processed by precipitation to remove other impurities, such as proteins and DNA. In one example, the BIA-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 may be removed by dialysis or other size-exclusion methods that separate small alkaloids from contaminating biopolymers.

[0218] In a further example, a solution containing a BIA of interest can be extracted to high purity by continuous cross-flow filtration using methods known in the art.

[0219] If the solution contains a mixture of BIAs of interest, it can be subjected to acid-base treatment to obtain individual BIA species of interest using methods known in the art, in which the pH of the aqueous solution is adjusted to precipitate the individual BIAs.

[0220] For high purity small scale preparations, the BIA can be purified in a single step by liquid chromatography.

[0221] Liquid Chromatography Mass Spectrometry (LCMS) Method: BIA compounds of interest, including morphinans, nal-opioids, and nor-opioids, 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). Quantitation can be performed by comparing the peak area of ​​the compound with a standard curve of a known reference standard compound. Furthermore, 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.

[0222] 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.

[0223] 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) or opium, which is exported by poppy-growing countries and purchased by 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 10 and 11 highlight impurities in these two products that may be specific to either CYCM or CPS or may be present in both. Some BIAs may have pigments as impurities, while others may themselves be classified as pigments. Therefore, such BIAs can be evaluated for impurities based on non-pigment 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.

[0224] 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 10 and 11) 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.

[0225] 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.

[0226] 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.

[0227] 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.

[0228] 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.

[0229] Table 12 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 12. 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.

[0230] Methods for Genetically Manipulating Host Cells Also included are methods for genetically engineering host cells for the purpose of producing an enzyme and / or BIA of interest. Insertion of DNA into a host cell can be accomplished using any convenient method. The methods are used to insert heterologous coding sequences into the genetically engineered host cell such that the host cell functionally expresses the enzyme and converts the starting compound of interest into the product enzyme and / or BIA of interest.

[0231] 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 from 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, and GalS, repressible promoters Met25 and tetO, and constitutive promoters, such as the glyceraldehyde 3-phosphate dehydrogenase promoter (GPD), alcohol dehydrogenase promoter (ADH), translation-elongation factor-1-alpha promoter (TEF), cytochrome c-oxidase promoter (CYC1), and MRP7 promoter. 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 suitable promoter can be selected for host cells, for example, E. coli. Promoter selection may be used to optimize transcript, and therefore enzyme, levels to maximize production and minimize energy resources.

[0232] 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. Figure 11 shows examples of convenient vectors.

[0233] usefulness The genetically engineered host cells and methods of the invention, 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 of the invention find use in a variety of different applications, for example, any convenient application in which the production of enzymes and / or BIAs is of interest.

[0234] The subject 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 BIAs is of interest. The engineered host cells described herein produce the BIA of interest and the enzyme of interest. Reticuline is a key branching point intermediate of interest in the synthesis of BIAs, including genetic engineering efforts to produce end products, such as opioid products. The subject host cells can be used to produce the BIA of interest from simple, inexpensive starting materials that can find use in producing the BIA of interest (including reticuline and BIA end products). Thus, the subject host cells find use in providing therapeutically active BIAs of interest.

[0235] In some instances, genetically engineered host cells and methods find use in producing commercial quantities of BIAs where chemical synthesis is poorly tolerated and not a viable means for large-scale production. In certain instances, 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 BIAs of interest for therapeutic formulations. Such applications may include industrial-scale production of BIAs of interest from fermentable carbon sources, such as cellulose, starch, and free sugars.

[0236] 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 enzymes and / or BIAs of interest. Engineered host cells can also be engineered to produce enzymes and / or 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 enzymes and / or BIAs of interest. In certain cases, research applications include the production of enzymes and / or 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 examples, host cell lines are used to screen for enzymatic activity of interest in such pathways, which can lead to the discovery of enzymes by conversion of metabolic products of BIAs produced in such cell lines.

[0237] 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 protopine, 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.

[0238] Kits and Systems Aspects of the invention further include kits and systems that can include one or more components used in the methods of the invention, 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.

[0239] 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 used 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.

[0240] Also provided are systems for producing an enzyme and / or BIA 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.

[0241] In some cases, the system includes components for large-scale fermentation of engineered host cells and monitoring and purification of enzymes and / or BIA 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 BIA products of interest are produced by the engineered host cells in the fermentor. In some cases, the host cells produce the BIA of interest (e.g., as described herein). In certain cases, the BIA product of interest is an opioid product, such as thebaine, codeine, neopine, morphine, neomorphine, hydrocodone, oxycodone, hydromorphone, dihydrocodeine, 14-hydroxycodeine, dihydromorphine, or oxymorphone.

[0242] In some cases, the system includes a process for monitoring and / or analyzing one or more enzymes of interest and / or BIA compounds 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. Upon completion of conversion of the starting compound to the enzyme of interest and / or BIA product, fermentation can be stopped and purification of the BIA product can be performed. Thus, in some cases, the subject system includes purification components suitable for purifying the enzyme of interest and / or BIA product from the host cell medium in which it was produced. The purification components can include any convenient means that can be used to purify the enzyme of interest and / or BIA product 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 BIA fermentation product of interest after charging the system with one or more starting compounds.

[0243] 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.

[0244] 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 BIA of interest and / or an enzyme of interest. Table 3 lists exemplary genes that can be affected by one or more modifications that result in the generation of a BIA of interest and / or the production of an enzyme of interest in an engineered host cell.

[0245] The genetic modifications shown in Table 3 can be used to produce a BIA of interest from an engineered host cell 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 3 can be used to produce a BIA of interest from an engineered host cell fed a carbohydrate. Additionally, one or more genetic modifications shown in Table 3 can be used to augment biosynthetic processes in a host cell that can be engineered for drug production.

[0246] Furthermore, the use of such modifications to effect the generation of a BIA of interest and / or the production of an enzyme of interest in a genetically engineered host cell is not readily apparent from the mere identification of the enzyme that can be produced by the gene. In particular, synthetic pathways reconstituted in host cells, such as yeast cells, as described herein include 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 for the biosynthesis of a BIA in their natural context. Furthermore, some of the enzymes described herein have not evolved to function, or function together, in a particular host cell, such as a yeast cell. In such cases, it would not be obvious that the enzymes would exhibit sufficient activity in the context of a synthetic BIA pathway in a host cell, such as yeast, to have sufficient flux through the pathway to produce the downstream BIA end product.

[0247] 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 in 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 that are localized in the inner membrane, such as cytochrome P450, and is particularly pronounced in the BIA pathway. 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 levels of accumulation of the desired BIA product.

[0248] 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.

[0249] Furthermore, many of the enzymes discussed herein and listed in Table 3 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 operate together to direct simple precursors, e.g., sugars, to 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.

[0250] 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.

[0251] 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.

[0252] 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).

[0253] 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. Also, as described herein, the conversion of (S)-reticuline to (R)-reticuline has only recently 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.

[0254] 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 enzymes could be used for the biosynthesis of compounds for which they did not naturally evolve and function effectively in the context of a heterologous microbial host and this pathway. In such cases, it may not be obvious that such enzymes could be used for the biosynthesis of compounds for which they did not naturally evolve and function effectively in the context of a heterologous microbial host and this pathway.

[0255] As another example, until recently, the enzyme responsible for the conversion of (S)-reticuline to (R)-reticuline was unknown. Even when fusion epimerase enzymes were discovered, evolutionary analysis suggested that fusion enzymes of oxidase and reductase for the epimerase reaction occurred in morphine-producing poppies, in contrast to non-morphine-producing poppies, in which the epimerase enzymes were non-fusion. Based on this analysis, some scholars believed that the fusion of oxidase and reductase moieties was necessary to efficiently catalyze the conversion of (S)-reticuline to (R)-reticuline. The novel method using engineered split epimerases discussed herein allows this epimerization reaction to occur in yeast in the context of the BIA synthesis pathway, and this epimerization can be performed with higher efficiency than epimerization performed using wild-type epimerases.

[0256] Examples of genes that may be modified to generate a BIA of interest and / or produce an enzyme 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 BIA of interest and / or enzyme of interest.

[0257] [TLK1] In some examples, the genetically engineered host cell can be modified to express 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 instances, the TKL1 gene can be 100% similar to the naturally occurring gene.

[0258] [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.

[0259] [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. A feedback-inhibited copy of the DAHP synthase enzyme can be under native yeast transcriptional control, for example, when the engineered host cell is a yeast cell. Alternatively, a 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-inhibition-resistant mutations K229L or Q166K.

[0260] [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., ARO7FBR ) 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. A feedback-inhibited copy of the chorismate mutase enzyme can be under native yeast transcriptional control, for example, when the engineered host cell is a yeast cell. Alternatively, a 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-inhibition-resistant mutation or T226I.

[0261] [ARO10] In some examples, the genetically engineered host cell may be modified to express 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 into the genetically engineered host cell. The ARO10 gene can be from Saccharomyces cerevisiae or another species. In some instances, the ARO10 gene can be 100% similar to the naturally occurring gene.

[0262] [ADH2-7, SFA1] In some examples, the genetically engineered host cell can modify the expression of alcohol dehydrogenase enzymes. The alcohol dehydrogenase enzymes can be encoded by one or more of the ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, and SFA1 genes. In one example, alcohol dehydrogenase catalyzes the reaction 4HPA → tyrosol. The genetically engineered host cell can be modified to delete the coding region of one or more of the ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, and SFA1 genes in the genetically engineered host cell. Alternatively, the genetically engineered host cell can be modified to disable the functionality of one or more of the ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, and SFA1 genes, for example, by introducing an inactivating mutation.

[0263] [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.

[0264] [ARO9] In some examples, the 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 + L-alanine ←→ tyrosine + pyruvate, as shown in Figure 2. The engineered host cell may be modified to contain constitutive overexpression of the ARO9 gene within the engineered host cell. Additionally or alternatively, the engineered host cell may be modified to synthetically regulate expression of the ARO9 gene within the engineered host cell. In one example, the engineered host cell may be modified to incorporate one, multiple, or additional copies of the ARO9 gene. Additionally or alternatively, the engineered host cell may be modified to incorporate a strong promoter element for overexpression of the ARO9 gene within the 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.

[0265] [ARO8] In some examples, the genetically engineered host cell may be modified to express the enzyme aromatic aminotransferase. Aromatic aminotransferase is encoded by the ARO8 gene. In one example, aromatic aminotransferase catalyzes the reaction hydroxyphenylpyruvate + glutamate ⇔ tyrosine + alpha-ketogluterate, as shown in Figure 2. The genetically engineered host cell may be modified to contain constitutive overexpression of the ARO8 gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell may be modified to synthetically regulate expression of the ARO8 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 ARO8 gene. Additionally or alternatively, the genetically engineered host cell may be modified to incorporate a strong promoter element for overexpression of the ARO8 gene within the genetically engineered host cell. The ARO8 gene may be derived from Saccharomyces cerevisiae or another species. In some instances, the ARO8 gene may be 100% similar to the naturally occurring gene.

[0266] [TYR1] In some instances, the genetically engineered host cell may alter the expression of the enzyme prephenate dehydrogenase. Prephenate dehydrogenase is encoded by the TYR1 gene. In one example, prephenate dehydrogenase converts prephenate + NADP, as shown in Figure 2. +It catalyzes the reaction: →4-hydroxyphenylpyruvate + CO2 + NADPH. The genetically engineered host cell can be modified to contain constitutive overexpression of the TYR1 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 TYR1 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 TYR1 gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate the introduction of a strong promoter element for overexpression of the TYR1 gene within the genetically engineered host cell. The TYR1 gene can be derived from Saccharomyces cerevisiae or another species. In some examples, the TYR1 gene can be 100% similar to the naturally occurring gene.

[0267] [TYR] In some examples, the genetically engineered host cell may be modified to express the enzyme tyrosinase. Tyrosinase is encoded by the TYR gene. In one example, tyrosinase catalyzes the reaction tyrosine → L-DOPA, as shown in Figures 2, 12, and 13. 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.

[0268] [TyrH] In some examples, the genetically engineered host cell may be modified to express 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, 12, and 13. The genetically engineered host cell may be modified to contain constitutive expression of the TyrH 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 TyrH 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 TyrH gene. Additionally or alternatively, the genetically engineered host cell may be modified to incorporate a strong promoter element for overexpression of the TyrH gene into the genetically engineered host cell. The TyrH gene may be derived from humans, Norway rats, house mice (Mus musculus), or another species. In some instances, the TyrH gene may be 100% similar to the naturally occurring gene.

[0269] [DODC] In some examples, the genetically engineered host cell may be modified to express 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, 12, and 13. 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 instances, the DODC gene may be 100% similar to the naturally occurring gene.

[0270] [TYDC] In some examples, the genetically engineered host cell may be modified to express the enzyme tyrosine / DOPA decarboxylase. Tyrosine / DOPA decarboxylase is encoded by the TYDC gene. In one example, tyrosine / DOPA decarboxylase catalyzes the reaction L-DOPA → dopamine, as shown in Figures 2, 12, and 13. 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 instances, the TYDC gene may be 100% similar to the naturally occurring gene.

[0271] [MAO] In some instances, the genetically engineered host cell may be modified to express the enzyme monoamine oxidase. Monoamine oxidase is encoded by the MAO gene. In one example, monoamine oxidase catalyzes the reaction dopamine → 3,4-DHPA, as shown in Figures 2 and 13. The genetically engineered host cell may be modified to contain constitutive expression of the MAO gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell may be modified to synthetically regulate expression of the MAO 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 MAO gene. Additionally or alternatively, the genetically engineered host cell may be modified to incorporate a strong promoter element for overexpression of the MAO gene within the genetically engineered host cell. In some instances, the MAO gene may 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.

[0272] [NCS] In some examples, the genetically engineered host cell may alter 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 Figures 12 and 13. In particular, Figure 12 shows a biosynthetic scheme for the conversion of L-tyrosine to reticuline via norcoclaurine, according to an embodiment of the present invention. Figure 12 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 12, 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.

[0273] Furthermore, norcoclaurine synthase catalyzes the reaction 3,4-DHPA + dopamine → (S)-norlaudanosoline, as shown in Figure 13. In particular, Figure 13 shows a biosynthetic scheme for the conversion of L-tyrosine to reticuline via norlaudanosoline, according to an embodiment of the present invention. Figure 13 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 13, L-tyrosine is naturally synthesized in yeast. Other metabolites shown in Figure 13 are not naturally produced in yeast.

[0274] 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 from Coptis japonica, Papaver somniferum, Papaver bracteatum, Thalicitum flavum, Corydalis saxicola, or another species. In some instances, the NCS gene can be 80% similar to a naturally occurring gene.

[0275] [6OMT] In some examples, the genetically engineered host cell can be modified to express 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 12. 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 13. Furthermore, the genetically engineered host cell can be modified to contain 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.

[0276] [CNMT] In some examples, the genetically engineered host cell can be modified to express 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 12. 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 other reactions detailed herein, such as those shown in Figure 13. Furthermore, the genetically engineered host cell can be modified to contain 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 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 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.

[0277] [4'OMT] In some examples, the genetically engineered host cell can be modified to express 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 12. In other examples, the 4'-O-methyltransferase catalyzes other reactions detailed herein, such as those shown in Figure 13. Furthermore, the genetically engineered host cell can be modified to contain 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 instances, the 4'OMT gene can be 100% similar to a naturally occurring gene.

[0278] [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 12. 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.

[0279] [FOL2] In some examples, the genetically engineered host cell can be modified to express 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 into the genetically engineered host cell. The FOL2 gene can be from Saccharomyces cerevisiae, human, mouse, or another species. In some instances, the FOL2 gene can be 100% similar to a naturally occurring gene.

[0280] [PTPS] In some examples, the genetically engineered host cell can be modified to express 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 may be codon-optimized for expression in Saccharomyces cerevisiae. The PTPS gene may be from Rattus norvegicus, human, house mouse, or another species. In some cases, the PTPS gene may be 80% similar to the naturally occurring gene.

[0281] [SepR] In some examples, the genetically engineered host cell can be modified to express 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 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, multiple, 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 within 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.

[0282] [PCD] In some examples, the genetically engineered host cell can be modified to express 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 from Rattus norvegicus, human, house mouse, or another species. In some cases, the PCD gene can be 79% similar to the naturally occurring gene.

[0283] [QDHPR] In some examples, the genetically engineered host cell can be modified to express 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 derived from Rattus norvegicus, human, house mouse, or another species. In some instances, the QDHPR gene can be 75% similar to the naturally occurring gene.

[0284] [DHFR] In some examples, the genetically engineered host cell can be modified to express 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 12. 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. In some cases, the DHFR gene can be codon-optimized for expression in Saccharomyces cerevisiae. The DHFR gene can be from Rattus norvegicus, human, or another species. In some cases, the DHFR gene can be 77% similar to the naturally occurring gene.

[0285] As discussed above with respect to the epimerization of [DRS-DRR]l-BIA, genetically engineered host cells can alter the expression of a BIA epimerase. The BIA epimerase is encoded by the DRS-DRR gene. In some instances, DRS-DRR may also be referred to as CYP-COR. In some instances, a BIA epimerase or an engineered split or fusion form of a BIA epimerase catalyzes the conversion of (S)-l-BIA to (R)-l-BIA, as shown in Figure 14. In particular, Figure 14 depicts a biosynthetic scheme for the conversion of L-tyrosine to a morphinan alkaloid according to an embodiment of the present invention. Figure 14 shows the use of the enzymes CPR, cytochrome P450 reductase; DRS-DRR, dehydroreticuline synthase and dehydroreticuline reductase; SalSyn, salutaridin synthase; SalR, salutaridinol reductase; SalAT, salutaridinol 7-O-acetyltransferase; TS, thebaine synthase; T6ODM, thebaine 6-O-demethylase; COR, codeinone reductase; and CODM, codeine-O-demethylase.

[0286] The genetically engineered host cell can be modified to contain the DRS-DRR gene or constitutive expression of the genetically engineered DRS-DRR gene within the genetically engineered host cell. In some cases, the genetically engineered DRS-DRR gene can encode an engineered fusion epimerase. In some cases, the genetically engineered DRS-DRR gene can encode an engineered split epimerase. Additionally or alternatively, the genetically engineered host cell can be modified to synthetically regulate the expression of the DRS-DRR 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 DRS-DRR gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate the introduction of a strong promoter element for overexpression of the DRS-DRR gene within the genetically engineered host cell. The DRS-DRR gene may be derived from Papaver bracteatum, opium poppy, Papaver setigerum, celandine (Chelidonium majus), or another species. In some instances, the DRS-DRR gene may be 77% similar to the naturally occurring gene.

[0287] [CPR] In some examples, the genetically engineered host cell may be modified to express 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 14. 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 a CPR gene into the genetically engineered host cell. The CPR gene can be derived from California poppy, poppy, human (H. sapiens), S. cerevisiae, Arabidopsis (A. thaliana), or another species. In some cases, the CPR gene can be 100% similar to a naturally occurring gene.

[0288] [SalSyn] In some examples, the genetically engineered host cell may be modified to express 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 14. 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 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, multiple, 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.

[0289] [SalR] In some examples, the genetically engineered host cell may be modified to express 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 14. 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 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, multiple, 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 within 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.

[0290] [SalAT] In some examples, the genetically engineered host cell may be modified to express 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 14. The genetically engineered host cell may be modified to contain constitutive expression of the SalAT 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 SalAT 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 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 opium poppy, oriental poppy, oriental poppy (Papaver orientale), a species of the genus Papaver, or another species. In some cases, the SalAT gene can be 77-80% similar to the naturally occurring gene.

[0291] [TS] In some instances, a genetically engineered host cell may modify the expression of the enzyme thebaine synthase. Thebaine synthase is encoded by the TS gene. In some instances, thebaine synthase or a genetically engineered thebaine synthase catalyzes the reaction 7-O-acetylsaltaridinol → thebaine + acetate, as shown in Figure 14. In some instances, the reaction 7-O-acetylsaltaridinol → thebaine + acetate occurs naturally, but thebaine synthase catalyzes part of this reaction. In particular, Figure 14 shows a biosynthetic scheme for the conversion of L-tyrosine to a morphinan alkaloid according to an embodiment of the present invention. Figure 14 shows the use of the enzymes CPR, cytochrome P450 reductase; DRS-DRR, dehydroreticuline synthase and dehydroreticuline reductase; SalSyn, salutaridin synthase; SalR, salutaridinol reductase; SalAT, salutaridinol 7-O-acetyltransferase; TS, thebaine synthase; T6ODM, thebaine 6-O-demethylase; COR, codeinone reductase; and CODM, codeine-O-demethylase.

[0292] The genetically engineered host cell can be modified to contain a TS gene or constitutive expression of the genetically engineered TS gene within the genetically engineered host cell. In some cases, the genetically engineered TS gene can encode an engineered fusion enzyme. Additionally or alternatively, the genetically engineered host cell can be modified to synthetically regulate expression of the TS 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 TS gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate a strong promoter element for overexpression of the TS gene within the genetically engineered host cell. In some cases, the TS gene can be codon-optimized for expression in Saccharomyces cerevisiae. The TS gene can be derived from opium poppy, oriental poppy, oriental poppy (Papaver orientale), a species of the genus Papaver, or another species. In some cases, the TS gene can be 75-80% similar to the naturally occurring gene.

[0293] [T6ODM] In some examples, 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 examples, thebaine 6-O-demethylase catalyzes the reaction from thebaine to neopinone, as shown in Figures 14, 15, and 16. 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 examples, 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 engineered host cell can be modified to contain constitutive expression of the T6ODM gene within the engineered host cell. Additionally or alternatively, the engineered host cell can be modified to synthetically regulate expression of the T6ODM gene within the engineered host cell. In one example, the engineered host cell can be modified to incorporate one, multiple, or additional copies of the T6ODM gene. Additionally or alternatively, the engineered host cell can be modified to incorporate a strong promoter element for overexpression of the T6ODM gene within the 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 opium poppy or another species. In some instances, the T6ODM gene may be 76.2% similar to the naturally occurring gene.

[0294] [COR] In some examples, the genetically engineered host cell can be modified to express 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 Figures 14, 15, and 16. 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 the 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 the naturally occurring gene. In one example, the COR gene can be 76.8%, 77.0%, 77.3%, or 77.7% similar to the naturally occurring gene.

[0295] [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 conversion of codeine to morphine, as shown in Figures 14, 15, and 16. Codeine O-demethylase can also catalyze the conversion of neopine to neomorphine. Codeine O-demethylase can also catalyze the conversion 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 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 contain 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.

[0296] [BBE] In some examples, the genetically engineered host cell can be modified to express 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.

[0297] [S9OMT] In some examples, the genetically engineered host cell can be modified to express 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.

[0298] [CAS] In some examples, the genetically engineered host cell may be modified to express 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% similar to a naturally occurring gene.

[0299] [STOX] In some examples, the genetically engineered host cell may be modified to express 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 into 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 Saccharomyces 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.

[0300] [TNMT] In some instances, the genetically engineered host cell may alter 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.

[0301] 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 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, multiple, 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 opium poppy, California poppy, Indian poppy, 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.

[0302] [CFS] In some examples, the genetically engineered host cell can be modified to express 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 into 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 Saccharomyces cerevisiae.

[0303] In some examples, the genetically engineered host cell may be modified to express 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 may be modified to contain constitutive expression of the STS gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell may be modified to synthetically regulate expression of the STS 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 STS gene. Additionally or alternatively, the genetically engineered host cell may be modified to incorporate a strong promoter element for overexpression of the STS gene within the genetically engineered host cell. The STS gene may be derived from poppy, California poppy, American poppy, or another species. In some examples, the STS gene may be 76%, 78%, or 79% similar to the naturally occurring gene. Additionally, the STS gene can be codon-optimized for expression in Saccharomyces cerevisiae.

[0304] [MSH] In some examples, the genetically engineered host cell can be modified to express 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 cases, the MSH gene can be 79% similar to the naturally occurring gene. Additionally, the MSH gene can be codon-optimized for expression in Saccharomyces cerevisiae.

[0305] [P6H] In some examples, the genetically engineered host cell can be modified to express 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. Furthermore, the P6H gene can be codon-optimized for expression in Saccharomyces cerevisiae.

[0306] [DBOX] In some examples, the genetically engineered host cell can be modified to express 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.

[0307] [morA] In some examples, the genetically engineered host cell may alter 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 FIG. 15. In other examples, morphine dehydrogenase catalyzes the codeinone → codeine reaction, also shown in FIG. 15. FIG. 15 shows a biosynthetic scheme for the production of semisynthetic opioids (opioids) according to embodiments of the present invention. In particular, FIG. 15 shows the extended transformation of thebaine in yeast by incorporating morA, morphine dehydrogenase; and morB, morphine reductase.

[0308] 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 from Pseudomonas putida or another species. In some examples, the morA gene can be 73.7% similar to the naturally occurring gene.

[0309] [morB] In some examples, the genetically engineered host cell can be modified to express 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 15. In other examples, morphinone reductase catalyzes the reaction morphinone → hydromorphone, also shown in Figure 15. In other examples, morphinone reductase catalyzes the reaction 14-hydroxycodeinone → oxycodone. The genetically engineered host cell can be modified to contain constitutive expression of the morB gene within the genetically engineered host cell. Additionally or alternatively, the genetically engineered host cell can be modified to synthetically regulate expression of the morB 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 morB gene. Additionally or alternatively, the genetically engineered host cell can be modified to incorporate a strong promoter element for overexpression of the morB gene into the genetically engineered host cell. In some cases, the morB gene can be codon-optimized for expression in Saccharomyces 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.

[0310] [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 → gategaumerine. In other examples, CYP80A1 catalyzes the reaction (R)-N-methylcoclaurine + (S)-coclaurine → 2'-norberbamunine. The genetically engineered host cell can be modified to contain constitutive expression of the CYP80A1 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 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.

[0311] [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 a...

Claims

1. A method for converting a precursor of a promorphinan molecule via thebaine to an alkaloid product in a genetically engineered yeast host cell, wherein the alkaloid product is selected from the group consisting of a morphinan alkaloid, a nor-opioid alkaloid, or a nal-opioid alkaloid, the method comprising: contacting a precursor of a promorphinan molecule with a plurality of enzymes, wherein the plurality of enzymes includes at least one enzyme that converts the precursor of a promorphinan molecule into a promorphinan molecule, and wherein the plurality of enzymes also includes a thebaine synthase that converts the promorphinan molecule into thebaine, wherein the precursor of a promorphinan molecule is tyrosine and is produced within the engineered yeast host cell where the conversion occurs; Including, thebaine synthase, and The following enzymes: (a) TYR or TyrH, DODC, 6OMT, CNMT, CYP80B1, 4'OMT, DRS-DRR, SalSyn, SalR, and SalAT; or (b) TYR or TyrH, DODC, maoA, 6OMT, CNMT, 4'OMT, DRS-DRR, SalSyn, SalR, and SalAT wherein the plurality of enzymes are produced by culturing the engineered yeast host cell having a plurality of sequences encoding thebaine synthase, and the engineered yeast host cell produces an amount of thebaine that is increased by 10% or more compared to a host cell lacking thebaine synthase. method.

2. The method of claim 1, further comprising a step of recovering the alkaloid product from the cell culture.

3. The method described in claim 1 or 2, wherein the thebaine synthase enzyme is a Bet v 1 fold protein.

4. Thebaine synthase is (a) comprises an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 31, 32, 33, 34, 35, 36, and 37; or (b) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 31, 32, 33, 34, 35, 36, and 37; The method according to any one of claims 1 to 3.

5. A method described in any one of claims 1 to 4, wherein thebaine synthase converts at least 50% of the promorphinan molecules to thebaine.

6. A method described in any one of claims 1 to 5, wherein the genetically engineered yeast host cell converts at least 90% of the promorphinan molecules to thebaine. (a) the genetically engineered yeast host cell produces an amount of thebaine that is increased by at least 20% compared to a host cell lacking thebaine synthase; or (b) the engineered yeast host cells produce at least a two-fold increased amount of thebaine compared to host cells lacking thebaine synthase; The method according to any one of claims 1 to 6.

8. The method of claim 1, wherein the genetically engineered yeast host cell comprises the enzymes CPR and / or NCS.

9. 1. A genetically engineered yeast host cell that overproduces one or more thebaine synthases and produces an alkaloid product from tyrosine via thebaine, wherein the alkaloid product is selected from the group consisting of a morphinan alkaloid, a nor-opioid alkaloid, or a nal-opioid alkaloid, wherein the cell is The following enzymes: (a) TYR or TyrH, DODC, 6OMT, CNMT, CYP80B1, 4'OMT, DRS-DRR, SalSyn, SalR, and SalAT; or (b) TYR or TyrH, DODC, maoA, 6OMT, CNMT, 4'OMT, DRS-DRR, SalSyn, SalR, and SalAT Including, produce an amount of thebaine that is increased by 10% or more compared to a host cell lacking thebaine synthase, Genetically engineered yeast host cells.

10. The genetically engineered yeast host cell of claim 9, comprising the enzymes CPR and / or NCS.

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