Methods for improving production of morphinan alkaloids and derivatives

Abstract

Disclosed herein are methods useful for the synthesis of benzylisoquinoline alkaloids ("BIA"), such as alkaloid morphinans. The disclosed methods can be used to produce thebaine, eastern papaverine, codeine, morphine, oxycodone, hydrocodone, oxymorphinone, hydromorphinone, naltrexone, naloxone, hydroxycodeinone, neolpinone, and / or buprenorphine.

Description

Cross - Reference to Related Applications

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 377,672, filed Sep. 29, 2022, the entire content of which is incorporated herein by reference for all purposes. Background of the Invention

[0002] Existing manufacturing methods for BIAs (including morphinan alkaloids and their derivatives) suffer from low yields and / or high costs. Some known methods for manufacturing BIAs result in the production of undesired amounts of morphinan alkaloid by - products (see, for example, Rinner, U. and Hudlicky, J., 2012, Top. Cur. Chem. 209:33 - 66). There are no commercial biosynthetic manufacturing methods for BIAs (including morphinan alkaloids and their derivatives). Accordingly, there is a need for improved methods for synthesizing BIAs (including morphinan alkaloids and their derivatives). Summary of the Invention

[0003] The present disclosure provides methods for producing a variety of benzylisoquinoline alkaloids (BIAs) in engineered host cells. In certain embodiments, the engineered host cells are non-plant cells. The present disclosure further provides compositions of a variety of alkaloids produced in engineered host cells. Additionally, the present disclosure provides methods for expressing one or more enzymes that provide C-14 hydroxylase activity in engineered host cells. In certain embodiments, the one or more enzymes that provide C-14 hydroxylase activity are cytochrome P450 proteins (“P450”). In some embodiments, the one or more enzymes that provide C-14 hydroxylase activity are heterologous to the engineered host cells. Additionally, the present disclosure provides methods for expressing one or more engineered P450s that provide C-14 hydroxylase activity in engineered host cells. In particular cases, the present disclosure provides methods for increasing the production of a variety of alkaloid products by using P450s that provide C-14 hydroxylase activity. In some embodiments, the present disclosure provides methods for increasing the production of a variety of alkaloid products by overexpression of formaldehyde dehydrogenase. In some embodiments, the present disclosure provides methods for increasing the production of a variety of alkaloid products by overexpression of alcohol dehydrogenase. In additional embodiments, the present disclosure provides methods for increasing the production of a variety of alkaloid products by preventing the expression of transglutaminase. In some embodiments, the present disclosure provides methods for increasing the production of a variety of alkaloid products in engineered host cells by: converting a precursor morphinan alkaloid having a free hydrogen at carbon C-14 to a product morphinan alkaloid having a hydroxyl group at carbon C-14 via one or more enzymes that provide C-14 hydroxylase activity. In further embodiments, the precursor morphinan alkaloid having a hydrogen at C-14 carbon is produced in the engineered cells via a heterologous biosynthetic pathway that includes a variety of enzymes and starts from simple starting materials such as sugars and / or L-tyrosine. In some embodiments, the present disclosure provides methods for increasing the production of a variety of alkaloid products in engineered host cells by: converting a precursor morphinan alkaloid having a free hydrogen at carbon C-14 to a product morphinan alkaloid having a hydroxyl group at carbon C-14 via one or more enzymes that provide C-14 hydroxylase activity. In certain embodiments, the one or more enzymes that provide C-14 hydroxylase activity are heterologous to the engineered host cells. In other embodiments, the present disclosure provides methods for increasing the production of a variety of alkaloid products by converting codeine to 14-hydroxycodeine via an enzyme that provides C-14 hydroxylase activity.In additional embodiments, the present disclosure provides methods for increasing the production of a variety of alkaloid products by converting codeinone to 14-hydroxycodeinone via an enzyme that provides C-14 hydroxylase activity. In further embodiments, the present disclosure provides methods for increasing the production of a variety of alkaloid products by converting codeine to 14-hydroxycodeine via an enzyme that provides C-14 hydroxylase activity. In some cases, the method further comprises engineering the host cell to contain a variety of heterologous enzymes to produce a variety of benzylisoquinoline alkaloid products from simple starting materials such as sugars and / or L-tyrosine. In some examples, the engineered host cell (e.g., a non-plant cell) contains a plurality of coding sequences, each encoding an enzyme selected from the group of enzymes listed in Table 17. In some examples, the heterologous coding sequences may be operably linked. The operably linked heterologous coding sequences may be within the same pathway for producing a particular benzylisoquinoline alkaloid product via P450 (including one or more enzymes that provide C-14 hydroxylase activity). In some cases, the method further comprises engineering the host cell with a variety of heterologous enzymes to increase the production of BIA precursors including L-tyrosine and 4-HPAA. In some examples, the engineered host cell contains a plurality of coding sequences, each encoding an enzyme selected from the group of enzymes listed in Table 17. In some examples, the engineered host cell further comprises an inactivating mutation in a selected enzyme that results in reduced byproduct production. In some examples, the engineered host cell further comprises heterologous expression or overexpression of a selected enzyme that results in reduced byproduct production. In some examples, the byproducts include formaldehyde, tyrosol, phenethyl alcohol, or methanethiol. In some examples, the engineered host cell further comprises an inactivating mutation in a selected enzyme that results in increased production of a variety of benzylisoquinoline alkaloid products.

[0004] In some embodiments, the present disclosure provides a method for producing a benzylisoquinoline alkaloid (BIA) product in an engineered host cell, the method comprising: (a) expressing in the engineered host cell a heterologous enzyme having 14-hydroxylase activity and (b) optionally expressing in the engineered host cell a heterologous enzyme having cytochrome P450 reductase (CPR) activity, (c) contacting the heterologous enzyme with a BIA precursor substrate that is a morphinan alkaloid having a free hydrogen at carbon C-14, wherein the heterologous enzyme hydroxylates the C-14 carbon on the BIA precursor substrate; and (d) producing the BIA product within the host cell; wherein the engineered host cell produces more BIA product compared to a non-engineered host cell.

[0005] In some embodiments, the method includes producing in the host cell the product of 14-hydroxylation of the BIA precursor substrate described in (c).

[0006] In some embodiments, the method further includes producing in (d) downstream of the molecule additional BIA products by the action of one or more additional enzymes.

[0007] In some embodiments, the engineered host cell produces at least 1.5-fold the amount of the BIA product compared to the same host cell that does not contain enzyme(s) a) and / or b).

[0008] In some embodiments, the engineered host cell produces at least 50% more of the BIA product compared to the same engineered host cell that does not contain enzyme(s) a) and / or b).

[0009] In some embodiments, the method further includes providing the BIA precursor substrate to the engineered host cell.

[0010] In some embodiments, the heterologous enzyme is cytochrome P450.

[0011] In some embodiments, the enzyme is capable of converting codeine to 14-hydroxycodeine.

[0012] In some embodiments, the heterologous enzyme comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO.: 190, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 178, 180, 182, 184, 186, 188, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242 or 244.

[0013] In some embodiments, the heterologous enzyme comprises or consists of the amino acid sequence of SEQ ID NO: 190, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 178, 180, 182, 184, 186, 188, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242 or 244.

[0014] In some embodiments, the host cell is a cell that does not express enzyme a) and / or enzyme b).

[0015] In some embodiments, the BIA precursor substrate is selected from codeine, codeinone, norcodeinone, hydrocodone, noscapine, apomorphine, morphinone, normorphinone, hydromorphone, norhydromorphone, and norhydrocodone.

[0016] In some embodiments, the BIA precursor substrate is codeinone, codeine, hydrocodone, or hydromorphone.

[0017] In some embodiments, the BIA product is noroxymorphone.

[0018] In some embodiments, the BIA product is 14-hydroxycodeine.

[0019] In some embodiments, the engineered host cell expresses cytochrome P450 reductase (CPR).

[0020] In some embodiments, the CPR is heterologous to the engineered host cell.

[0021] In some embodiments, the CPR comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 168, 170, 172, 174, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268 or 270.

[0022] In some embodiments, the CPR comprises or consists of SEQ ID NO: 168, 170, 172, 174, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, or 270.

[0023] In some embodiments, the CPR is a fungal CPR.

[0024] In some embodiments, the CPR is a plant CPR.

[0025] In some embodiments, the CPR is an animal CPR.

[0026] In some embodiments, the cytochrome P450 and the CPR are from the same genus.

[0027] Also provided herein is a host cell that produces a BIA product, the host cell comprising a first heterologous polynucleotide encoding a heterologous enzyme having 14-hydroxylase activity and a second heterologous polynucleotide encoding a cytochrome P450 reductase (CPR).

[0028] In some embodiments, the enzyme having 14-hydroxylase is cytochrome P450.

[0029] In some embodiments, the enzyme having 14-hydroxylase activity comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with 190, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 178, 180, 182, 184, 186, 188, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, or 244.

[0030] In some embodiments, the CPR is a fungal CPR.

[0031] In some embodiments, the CPR is a plant CPR.

[0032] In some embodiments, the CPR is an animal CPR.

[0033] In some embodiments, the CPR comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO.168, 170, 172, 174, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268 or 270.

[0034] In some embodiments, the production of the BIA product comprises hydroxylation of the C-14 carbon on the BIA precursor substrate.

[0035] In some embodiments, the host cell is a microbial cell, a fungal cell or a yeast cell.

[0036] In some embodiments, the host cell comprises a nucleic acid construct containing a promoter.

[0037] Also provided herein is a vector comprising a first polynucleotide encoding an enzyme having 14-hydroxylase activity and a second polynucleotide encoding cytochrome P450 reductase (CPR).

[0038] Also provided herein is a nucleic acid construct comprising a first polynucleotide encoding an enzyme having 14-hydroxylase activity and a second polynucleotide encoding cytochrome P450 reductase (CPR).

[0039] In some embodiments, the first polynucleotide and / or the second polynucleotide are codon-optimized for expression in a host cell.

[0040] In some embodiments, the enzyme having 14-hydroxylase is cytochrome P450.

[0041] In some embodiments, the polynucleotide encoding an enzyme having 14-hydroxylase activity comprises a nucleotide sequence selected from SEQ ID No.191, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 179, 181, 183, 185, 187, 189, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243 or 245.

[0042] In some embodiments, the CPR is a fungal CPR.

[0043] In some embodiments, the CPR is a plant CPR.

[0044] In some embodiments, the CPR is an animal CPR.

[0045] In some embodiments, the polynucleotide encoding the CPR comprises a nucleotide sequence selected from SEQ ID No.169, 171, 173, 175, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, and 271.

[0046] In some embodiments, the enzyme having 14-hydroxylase activity comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO.190, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 178, 180, 182, 184, 186, 188, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, or 244.

[0047] In some embodiments, the CPR comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO.168, 170, 172, 174, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, or 270.

[0048] Also provided herein is a host cell comprising a vector, the vector comprising a first polynucleotide encoding an enzyme having 14-hydroxylase activity and a second polynucleotide encoding cytochrome P450 reductase (CPR).

[0049] In some embodiments, the first polynucleotide and / or the second polynucleotide is codon-optimized for expression in the host cell.

[0050] In some embodiments, the enzyme having 14-hydroxylase is cytochrome P450.

[0051] In some embodiments, the polynucleotide encoding an enzyme having 14-hydroxylase activity comprises a nucleotide sequence selected from SEQ ID No. 191, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 179, 181, 183, 185, 187, 189, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243 or 245.

[0052] In some embodiments, the CPR is fungal CPR.

[0053] In some embodiments, the CPR is plant CPR.

[0054] In some embodiments, the CPR is animal CPR.

[0055] In some embodiments, the polynucleotide encoding the CPR comprises a nucleotide sequence selected from SEQ ID No. 169, 171, 173, 175, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269 and 271.

[0056] In some embodiments, the enzyme having 14-hydroxylase activity comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO. 190, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 178, 180, 182, 184, 186, 188, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242 or 244.

[0057] In some embodiments, the CPR comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO. 168, 170, 172, 174, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268 or 270.

[0058] Also provided herein is a host cell comprising a vector, the vector comprising a first heterologous polynucleotide sequence encoding a heterologous enzyme having formaldehyde dehydrogenase activity and a second heterologous polynucleotide designed to repress the expression of a target gene, wherein the target gene is selected from DUG2 or DUG3.

[0059] In some embodiments, the heterologous enzyme is SFA1.

[0060] In some embodiments, the host cell further comprises a third polynucleotide encoding an enzyme having 14-hydroxylase activity and a fourth polynucleotide encoding cytochrome P450 reductase (CPR).

[0061] Also provided herein is an isolated polypeptide having 14-hydroxylase activity.

[0062] Also provided herein is an engineered polypeptide having 14-hydroxylase activity.

[0063] Also provided herein is an enzyme mixture comprising a polypeptide or engineered polypeptide having 14-hydroxylase activity and a polypeptide having CPR activity.

[0064] The present disclosure also provides an in vitro method for producing benzylisoquinoline alkaloid (BIA) products, the method comprising contacting a BIA precursor substrate with an enzyme mixture, the BIA precursor substrate being a morphinan alkaloid having a free hydrogen at carbon C-14, the enzyme mixture comprising a polypeptide having 14-hydroxylase activity and a polypeptide having CPR activity, wherein the heterologous enzyme having 14-hydroxylase activity hydroxylates the C-14 carbon on the BIA precursor substrate to produce a 14-hydroxylated BIA product. Incorporated by reference

[0065] All publications, patents, and patent applications mentioned in this specification are incorporated herein 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 DESCRIPTION OF THE DRAWINGS

[0066] A better understanding of the features and advantages of the present disclosure will be obtained from the following detailed description and the accompanying drawings that illustrate illustrative embodiments that utilize the principles of the present disclosure, and in the drawings:

[0067] Figure 1 Illustrated are biosynthetic schemes for converting glucose to 4-HPAA, dopamine, 3,4-DHPAA and converting 1-benzylisoquinoline alkaloids to reticuline according to some embodiments of the present disclosure.

[0068] Figure 2 Illustrated are examples of tyrosine hydroxylase activity and the synthesis, recycling, and salvage pathways of tetrahydrobiopterin associated with tyrosine 3-monooxygenase activity according to some embodiments of the present disclosure.

[0069] Figure 3 Illustrated are biosynthetic schemes for converting L-tyrosine to reticuline via norcoclaurine and norlaudanosoline according to some embodiments of the present disclosure.

[0070] Figure 4 Illustrated are biosynthetic schemes for converting L-tyrosine to morphinan alkaloids (including natural and semi-synthetic opioids) according to some embodiments of the present disclosure.

[0071] Figure 5 Illustrated are biosynthetic schemes for producing natural opioids (including isomers of codeine and morphine) according to some embodiments of the present disclosure.

[0072] Figure 6Shows biosynthetic schemes for producing nor - opioids and nal - opioids according to some embodiments of the present disclosure.

[0073] Figure 7 Shows biosynthetic schemes for producing sanguinarine and related pathway metabolites according to some embodiments of the present disclosure.

[0074] Figure 8 Shows biosynthetic schemes for producing berberine and related pathway metabolites according to some embodiments of the present disclosure.

[0075] Figure 9 Shows enzymes with opioid 6 - O - demethylase activity according to some embodiments of the present disclosure.

[0076] Figure 10 Shows enzymes with opioid 3 - O - demethylase activity according to some embodiments of the present disclosure.

[0077] Figure 11 Shows certain substrates and resulting products of enzymes with opioid 14 - hydroxylase activity according to some embodiments of the present disclosure.

[0078] Figure 12 Shows enzymes with opioid alcohol oxidoreductase activity according to some embodiments of the present disclosure.

[0079] Figure 13 Shows enzymes with opioid reductase activity according to some embodiments of the present disclosure.

[0080] Figure 14 Shows enzymes with opioid isomerase activity according to some embodiments of the present disclosure.

[0081] Figure 15 Shows enzymes with N - methyltransferase activity according to some embodiments of the present disclosure.

[0082] Figure 16 Shows yeast platform strains for producing reticuline from L - tyrosine according to some embodiments of the present disclosure.

[0083] Figure 17 Shows yeast strains for producing thebaine and hydrocodone from L - tyrosine according to some embodiments of the present disclosure.

[0084] Figures 18A - 18C Shows morphine alkaloids produced from sugars and L - tyrosine by engineered yeast strains according to some embodiments of the present disclosure.

[0085] Figure 19 Shows an enzyme with norcoclaurine synthase activity according to some embodiments of the present disclosure.

[0086] Figure 20 Depicts a phylogenetic tree of selected plant Bet v I proteins with predicted NCS activity. According to some embodiments of the present disclosure, the representative species are Coptis japonica, Thalictrum flavum, Argemone mexicana, Sinopodophyllum hexandrum, Papaver bracteatum, Papaver somniferum, and Cordalyis saxicola.

[0087] Figure 21 Depicts the N-terminal truncation of CjNCS (SEQ ID NO: 69) and the effect on enzyme activity according to some embodiments of the present disclosure.

[0088] Figure 22 Depicts the key residues identified in the directed evolution screening (Table 6) of NCS (SEQ ID NO: 70) mapped to the crystal structure of TfNCS (PDB: 5N8Q) according to some embodiments of the present disclosure.

[0089] Figure 23 Depicts the key residues for improving norcoclaurine synthase activity in the template NCS parent (SEQ ID NO: 70) and NCS variants from Coptis japonica, Thalictrum flavum, Argemone mexicana, Sinopodophyllum hexandrum, Papaver bracteatum, Papaver somniferum, and Cordalyis saxicola (SEQ ID NOs 69 and 75 - 82 in order of appearance) according to some embodiments of the present disclosure.

[0090] Figure 24 Depicts an engineered NCS variant with enhanced norcoclaurine synthase activity according to some embodiments of the present disclosure.

[0091] Figure 25 Depicts norcoclaurine synthase activity in the presence of increasing dopamine concentrations according to some embodiments of the present disclosure.

[0092] Figure 26 Depicts a biological process for thebaine according to some embodiments of the present disclosure.

[0093] Figure 27 Depicts another biological process for thebaine according to some embodiments of the present disclosure.

[0094] Figure 28 Shows biosynthetic schemes for converting glucose into 4-HPAA, dopamine, 3,4-DHPAA and converting 1-benzylisoquinoline alkaloids into reticuline, according to some embodiments of the present disclosure.

[0095] Figure 29 Shows biosynthetic schemes for converting chorismate into tyrosine and phenylalanine via an arogenate intermediate, according to some embodiments of the present disclosure.

[0097] Figure 30 Shows a biosynthetic scheme for glycolysis, according to some embodiments of the present disclosure, in which phosphoketolase provides a pathway for generating acetyl-CoA.

[0098] Figure 31 Depicts a biological process for thebaine, according to some embodiments of the present disclosure.

[0099] Figure 32 Depicts a biological process for thebaine, according to some embodiments of the present disclosure.

[0100] Figure 33 Depicts a biological process for thebaine, according to some embodiments of the present disclosure.

[0101] Figure 34 Shows a biosynthetic scheme for methionine recycling, according to some embodiments of the present disclosure.

[0102] Figure 35 Depicts a biological process for thebaine, according to some embodiments of the present disclosure.

[0103] Figures 36A - 36D Shows an exemplary biosynthetic scheme for generating morphinan intermediates, involving the removal of a methyl group via oxidation (demethylation) and the formation of the resulting formaldehyde byproduct. Figure 36A Shows a biosynthetic scheme for converting thebaine into codeine, according to some embodiments of the present disclosure, which shows the oxidation of a morphinan intermediate methylated at position 6, producing formaldehyde as a byproduct. In some embodiments, an enzyme providing 14-hydroxylation activity then acts on codeinone, codeine, and / or downstream morphinan intermediates to add a hydroxyl group to the free hydrogen at the C14 position of codeine or a morphinan intermediate. Figure 36BShows biosynthetic schemes for converting thebaine to oripavine according to some embodiments of the present disclosure, which illustrate the oxidation of a morphinan intermediate methylated at position 6 by a 3-O-demethylase and the production of formaldehyde as a byproduct. Figure 36C Shows biosynthetic schemes for converting codeine to morphine according to some embodiments of the present disclosure, which use codeine O-demethylase (CODM) to oxidize a morphinan intermediate and produce formaldehyde as a byproduct. Figure 36D Shows biosynthetic schemes for converting thebaine to nor-thebaine according to some embodiments of the present disclosure, which use an N-demethylase to oxidize a morphinan intermediate and produce formaldehyde as a byproduct.

[0104] Figure 37 Shows a biological process for formaldehyde detoxification using formaldehyde dehydrogenase SFA1 according to some embodiments of the present disclosure. In some embodiments, a biosynthetic scheme for formaldehyde detoxification is provided to enhance the production of morphinan alkaloids.

[0105] Figures 38A - 38H Shows an exemplary biosynthetic scheme for converting thebaine to noroxymorphone according to some embodiments of the present disclosure.

[0106] Figure 39A and Figure 39B Depicts graphs quantifying the production of codeine ( Figure 39A ) and codeinone ( Figure 38B ) in engineered host cells according to some embodiments of the present disclosure.

[0107] Figure 40 Depicts a graph quantifying the production of 14-hydroxycodeine achieved by engineered host cells expressing a P450 variant and a cytochrome P450 reductase (CPR) variant according to some embodiments of the present disclosure.

[0108] Figure 41 Depicts a graph quantifying the production of 14-hydroxycodeine achieved by engineered host cells expressing a P450 variant and a CPR variant according to some embodiments of the present disclosure.

[0109] Figure 42 Depicts a graph quantifying the production of 14-hydroxycodeine achieved by engineered host cells expressing a P450 variant and a CPR variant according to some embodiments of the present disclosure.

[0110] Figure 43 Depicts a graph quantifying the production of 14-hydroxycodeine achieved by engineered host cells expressing a P450 variant and a CPR variant according to some embodiments of the present disclosure.

[0111] Figure 44 A graph depicting the quantification of 14-hydroxycodeine production achieved by engineered host cells expressing P450 variants and CPR variants, according to some embodiments of the present disclosure.

[0112] Figure 45 An exemplary vector for incorporating CPR (here, 14HC_CPR_1) into a microbial strain is depicted.

[0113] Figure 46 An exemplary vector for incorporating P450 (here, 14HC_P450_5) into a microbial strain is depicted.

[0114] Figures 47A - 47D A graph depicting the quantification of thebaine ( Figure 47A ), reticuline ( Figure 47B ), salutaridine ( Figure 47C ), and codeine ( Figure 47D ) production achieved by engineered de novo codeine 14-hydroxylase strains expressing CPR (here, 14HC_CPR_1) and expressing 14-hydroxylase (here, 14HC_P450_5) or an empty vector control.

[0115] Figure 48 A graph depicting the 14-hydroxycodeine titers (nM) produced by expressing different CPRs in YA4997.

[0116] Figure 49 A graph depicting the fold improvement of individual point mutations relative to 14HC_P450_5 in terms of in vivo 14-hydroxycodeine production.

[0117] Figure 50 A graph depicting a comparison of the improvement in in vivo 14-hydroxycodeine titers relative to 14HC_P450_5 after addition of the E58K point mutation.

[0118] Figure 51 A graph depicting the fold improvement in vivo of various combinations of 14HC_P450_5 point mutations relative to 14HC_P450_5 in terms of 14-hydroxycodeine production.

[0119] Figure 52 A graph depicting the fold improvement in vivo of engineered 14HC_P450_5 variants relative to 14HC_P450_5 in terms of 14-hydroxycodeine production (white) and oxycodone production (gray).

[0120] Figures 53A - 53I The 14-hydroxycodeine titers for each mutation normalized for each amino acid position are shown. As Figure 53AAs shown, at position 17, I and L improved 14-hydrocodone production relative to 14HC_P450_36. At position 58, all of the amino acids tested improved 14-hydroxycodone production relative to 14HC_P450_36( Figure 53B ). However, among the amino acids tested, E58K showed the greatest improvement. At position 59, D was the best amino acid among those tested( Figure 53C ). At position 102, L and M improved 14-hydroxycodone production relative to 14HC_P450_36.( Figure 53D ). At position 181, G, I, L, M, P, Q, S, and V showed improvement relative to 14HC_P450_36( Figure 53E ). At position 188, I was the best amino acid among those tested( Figure 53F ). At position 189, V improved 14-hydroxycodone production relative to 14HC_P450_36( Figure 53G ). At position 208, N improved 14-hydroxycodone production relative to 14HC_P450_36( Figure 53H ). At position 325, I, M, and V improved 14-hydroxycodone production relative to 14HC_P450_36( Figure 53I ).

[0121] Figure 54 Shows the in vitro production of 14-hydroxycodeinone when using codeinone as a substrate. When using microsomes from strains expressing 14HC_P450_5 or an empty vector, the level of spontaneous conversion of codeinone to 14-hydroxycodeinone was high. However, significant in vitro 14-hydroxycodeinone production was achieved by 14HC_P450_5 compared to the negative control, especially at 24 hours.

[0122] Figure 55 Shows the in vitro production of 14-hydroxycodone when using codeine as a substrate. Compared to the empty vector negative control, significant 14-hydroxycodone production was achieved by 14HC_P450_5 over time. Note that for this assay, samples were not collected at 3 hours.

[0123] Figure 56 Shows the in vitro production of oxycodone when using hydrocodone as a substrate. Compared to the empty vector negative control, significant oxycodone production was achieved by 14HC_P450_5 over time. There was a background of approximately 4 nM oxycodone, which did not change during the assay.

[0124] Figure 57Depicts the in vitro production of oxymorphone when hydromorphone is used as a substrate. Detailed Description

[0125] The present disclosure provides methods for producing a variety of benzylisoquinoline alkaloids (BIAs) in engineered host cells. The present disclosure further provides compositions of a variety of alkaloids produced in engineered host cells. Additionally, the present disclosure provides methods for expressing one or more proteins that provide C-14 hydroxylase activity in a host cell, the host cell being engineered with a variety of heterologous enzymes to produce a variety of benzylisoquinoline alkaloid products from simple starting materials such as sugars and / or L-tyrosine. In certain embodiments, one or more proteins that provide C-14 hydroxylase activity are heterologous to the engineered host cell. In some embodiments, one or more proteins that provide C-14 hydroxylase activity include cytochrome P450 (P450) proteins. Additionally, the present disclosure provides methods for producing one or more engineered P450 proteins and / or one or more engineered cytochrome P450 reductase (CPR) proteins in engineered host cells. In certain instances, the present disclosure provides methods for increasing the production of a variety of alkaloid products by engineered P450 proteins and / or engineered CPR proteins having specific amino acid mutations with increased activity. In certain instances, the present disclosure provides methods for producing benzylisoquinolines, protomorphinans, morphinans, protoberberines, protopines, benzophenanthridines, secobaberines, phthalideisoquinolines, aporphines, bisbenzylisoquinolines, nal-opioids, nor-opioids, etc. by increasing the conversion of precursor BIAs to benzylisoquinoline alkaloid products in engineered host cells. In additional specific instances, the methods include engineering the host cell with a variety of heterologous enzymes to increase the production of BIA precursors including L-tyrosine and 4-HPAA. In additional specific examples, the engineered host cell further comprises an inactivating mutation in a selected enzyme that results in increased production of a variety of benzylisoquinoline alkaloid products or decreased production of by-products. In additional specific examples, the engineered host cell further comprises heterologous expression or overexpression of a selected enzyme that results in increased production of a variety of benzylisoquinoline alkaloid products or decreased production of by-products. In additional specific instances, the by-products include formaldehyde, tyrosol, phenethyl alcohol, or methanethiol. Objective Benzylisoquinoline alkaloids (BIA)

[0126] Provided are host cells that produce a desired BIA. In some examples, engineered strains of host cells, such as the engineered strains of the present disclosure, provide a platform for the production of benzylisoquinoline alkaloids of interest and their modifications across several structural classes, including but not limited to precursor BIAs, benzylisoquinolines, protomorphenes, morphines, protoberberines, protopines, benzophenanthridines, secobaberines, phthalideisoquinolines, aporphines, bisbenzylisoquinolines, nal - opioids, nor - opioids, and the like. Each of these classes is intended to include biosynthetic precursors, intermediates, and metabolites of any convenient member of the engineered host cell biosynthetic pathway that may produce members of the class. Non - limiting examples of compounds are given below for each of these structural classes. In some cases, the structure of a given example may or may not be characterized as a benzylisoquinoline alkaloid per se. The chemical entities of the present invention are intended to include all possible isomers, including single enantiomers, racemic mixtures, optically pure forms, mixtures of diastereomers, and mixtures of intermediates.

[0127] Benzylisoquinoline alkaloid precursors can include but are not limited to norcoclaurine (NC) and norlaudanosoline (NL) and precursors of NC and NL, such as tyrosine, tyramine, 4 - hydroxyphenylacetaldehyde (4 - HPAA), 4 - hydroxyphenylpyruvic acid (4 - HPPA), L - 3,4 - dihydroxyphenylalanine (L - dopa), 3,4 - dihydroxyphenylacetaldehyde (3,4 - DHPAA), and dopamine. In some embodiments, the one or more BIA precursors are 3,4 - dihydroxyphenylacetaldehyde (3,4 - DHPAA) and dopamine. In certain instances, the one or more BIA precursors are 4 - hydroxyphenylacetaldehyde (4 - HPAA) and dopamine. In particular, NL and NC can be synthesized from precursor molecules via Pictet - Spengler condensation reactions, where the reactions can occur spontaneously or can be catalyzed by any convenient enzyme.

[0128] Benzylisoquinolines can 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.

[0129] Protomorphenes can include but are not limited to salutaridine, salutaridinol, and salutaridinol - 7 - O - acetate.

[0130] The morphinans can include, but are not limited to, thebaine, codeinone, codeine, morphine, morphinone, orientaline, neopinone, neopine, normorphine, hydrocodone, dihydrocodeine, 14-hydroxycodeinone, oxycodone, 14-hydroxycodeine, hydromorphinanone, dihydromorphine, dihydroetorphine, ethylmorphine, etorphine, metopon, buprenorphine, pholcodine, isocodeine, hydroxymorphinanone, norcodeinone, northebaine, orientaline, normorphinone, hydromorphone, normethadone, and norhydrocodone.

[0131] The protopine alkaloids can include, but are not limited to, scoulerine, cheilanthifoline, allocryptopine, nantenine, jatrorrhizine, stepholidine, discretamine, cis-N-methylallocryptopine, tetrahydrocolumbamine, palmatine, tetrahydropalmatine, columbamine, canadine, N-methylcanadine, 1-hydroxycanadine, berberine, N-methyl-corypalmine, 1,13-dihydroxy-N-methylcanadine, and 1-hydroxy-10-O-acetyl-N-methylcanadine.

[0132] The protopines can include, but are not limited to, protopine, 6-hydroxypiopine, allocryptopine, cryptopine, muramine, and thalictricine.

[0133] The benzophenanthridines can include, but are not limited to, dihydrosanguinarine, sanguinarine, dihydrochelerythrine, cheilirubine, dihydromacarpine, macarpine, and chelerythrine.

[0134] The secoberberines can include, but are not limited to, 4'-O-demethylmacrandraldehyde, 4'-O-demethylsalutaridine, 4'-O-demethyl-3-O-acetylsalutaridine, salutaridine, and 3-O-acetylsalutaridine.

[0135] The phthalideisoquinolines can include, but are not limited to, narcotoline hemiacetal, narcotine hemiacetal, narcotoline, narcotine, adlumidine, adlumine, (+) or (-)-dicentrine, canadine, corydalmine, corydaline, corydalmidine, corynoline, 5'-O-demethylnarcotine, (+) or (-)-α or β-hydrastine, and hypecorine.

[0136] The aporphines can include, but are not limited to, magnoflorine, corybulbine, apomorphine, boldine, isoboldine, isothebaine, isocorybulbine, and glaufine.

[0137] Bisbenzylisoquinolines may include but are not limited to berberine, dauricine, menisperine, and liensinine.

[0138] Nal - opioids may include but are not limited to naltrexone, naloxone, nalmefene, nalorphine, norlevorphanol, nalline, naldemedine, naloxol, 6β - naltrexol, naltroxene, methylnaltrexone, methylsamidorphan, alvimopan, opezomib, bevenpran, dinicotinate, levallorphan, samidorphan, buprenorphine, dezocine, etazocine, butorphanol, levorphanol, nalbuphine, pentazocine, phenazocine, nor - naltrexone, and diprenorphine.

[0139] Nor - opioids may include but are not limited to nor - codeine, nor - oxycodone, nor - thebaine, nor - hydrocodone, nor - dihydro - codeine, nor - 14 - hydroxy - codeine, nor - codeinone, nor - 14 - hydroxy - codeinone, nor - morphine, nor - normorphinone, nor - norsalsolinol, nor - dihydromorphinone, nor - dihydro - morphine, nor - 14 - hydroxy - morphine, nor - morphinone, and nor - 14 - hydroxy - morphinone.

[0140] Other compounds that can be produced by the engineered strains of the present disclosure may include but are not limited to rhoeadine, papaverine, isopapaverine, and cularine.

[0141] In certain embodiments, the engineered strains of the present disclosure can provide a platform for producing compounds related to tetrahydrobiopterin synthesis, the compounds including but not limited to dihydroneopterin triphosphate, 6 - pyruvoyltetrahydropterin, 5,6,7,8 - tetrahydrobiopterin, 7,8 - dihydrobiopterin, tetrahydrobiopterin 4a - methanolamine, quinoid dihydrobiopterin, and biopterin. Host cell

[0142] Any convenient cell can be utilized in the subject host cells and methods. In some cases, the host cell is a non-plant cell. In some instances, the host cell can be characterized as a microbial cell. In certain cases, the host cell is an insect cell, a mammalian cell, a bacterial cell, a fungal cell, or a yeast cell. Any convenient type of host cell can be used to produce the subject BIA-producing cells, see, for example, US2008 / 0176754, US2014 / 0273109, PCT / US 2014 / 063738, PCT / US2016 / 030808, PCT / US2015 / 060891, PCT / US2016 / 031506, and PCT / US 2017 / 057237, the disclosures of which are incorporated herein by reference in their entirety. Exemplary host cells include, but are not limited to, bacterial cells (such as Bacillus subtilis, Escherichia coli, Streptomyces, Anabaena, Arthrobacter, Acetobacter, Acetobacterium, Bacillus, Bifidobacterium, Brachybacterium, Brevibacterium, Carnobacterium, Clostridium, Corynebacterium, Enterobacter, Escherichia, Gluconacetobacter, Gluconobacter, Hafnia, Halomonas, Klebsiella, Kocuria, Lactobacillus, Leucononstoc, Macrococcus, Methylomonas, Methylobacter, Methylocella, Methylococcus, Microbacterium, Micrococcus, Microcystis, Moorella, Oenococcus, Pediococcus, Prochlorococcus,Propionibacterium, Proteus, Pseudoalteromonas, Pseudomonas, Psychrobacter, Rhodobacter, Rhodococcus, Rhodopseudomonas, Serratia, Staphylococcus, Streptococcus, Streptomyces, Synechococcus, Synechocystis, Tetragenococcus, Weissella, Zymomonas, and Salmonella typhimuium cells), 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). In some examples, the host cell is a yeast cell or an Escherichia coli (E. coli) cell. In some cases, the host cell is a yeast cell. In some instances, the host cell is from a cell that has been engineered to express one or more heterologous coding sequences,Yeast strains of genes and / or enzymes. As used herein, "heterologous" means that the material is not naturally present in the source in which it is introduced or otherwise present. For example, a heterologous nucleotide sequence is a nucleotide sequence that is not naturally present in a host organism. Such a heterologous nucleotide sequence can be used to express a peptide or protein (e.g., a heterologous enzyme, i.e., one that is not naturally present in the host organism). A heterologous nucleotide sequence can also be a nucleotide sequence that contains nucleotide sequences that are naturally present in the host organism but are configured or arranged in a manner that is not naturally present in the host organism. In some cases, the host cell is from a yeast strain that has been engineered to produce a desired BIA (such as a 14-hydroxylated benzylisoquinoline alkaloid). In some cases, the host cell is from a yeast strain that has been engineered to express a desired enzyme. In some cases, the host cell is from a yeast strain that has been engineered to express an enzyme that provides 14-hydroxylase activity. Additionally, in some embodiments, a heterologous enzyme that provides 14-hydroxylase activity may be able to more efficiently convert a benzylisoquinoline alkaloid to a 14-hydroxylated benzylisoquinoline alkaloid relative to an endogenous enzyme and / or wild-type 14-hydroxylase that provides 14-hydroxylase activity. In some embodiments, a heterologous enzyme that provides 14-hydroxylase activity can be substantially similar to a 14-hydroxylase that is naturally present in another species or organism but is heterologous to the desired host cell. In some cases, a heterologous enzyme that provides 14-hydroxylase activity can have an amino acid sequence that is at least 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 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 14-hydroxylase that is naturally present in another species or organism but is heterologous to the desired host cell. In some cases, the host cell is from a yeast strain that has been engineered to express a CPR enzyme. Additionally, in some embodiments, a host cell that has been engineered to express a CPR enzyme contains a heterologous CPR enzyme. In some cases, a host cell that has been engineered to express a CPR enzyme may be able to more efficiently convert a benzylisoquinoline alkaloid to a 14-hydroxylated benzylisoquinoline alkaloid relative to an endogenous CPR enzyme and / or wild-type CPR enzyme. In some embodiments, a heterologous CPR enzyme can be substantially similar to an endogenous CPR enzyme and / or wild-type CPR enzyme. In some cases, a heterologous CPR enzyme can have an amino acid sequence that is at least 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 81% or more,An amino acid sequence that is 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 endogenous CPR enzyme and / or the wild-type CPR enzyme. In some cases, the host cell is from a yeast strain that has been engineered to express an enzyme providing 14-hydroxylase activity and the CPR enzyme.

[0143] In some cases, the host cell is a fungal cell. In certain embodiments, the fungal cell can belong to Aspergillus species and strains, including 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). In certain embodiments, the fungal cell can belong to Rhizopus species, Trichoderma species, or Penicillium species. In certain embodiments, the fungal cell can be a yeast cell.

[0144] In some cases, the host cell is from a yeast strain engineered to express thebaine synthase. The thebaine synthase may be able to more efficiently convert salutaridinol-7-O-acetate to thebaine relative to the spontaneous reaction. In some cases, the host cell is from a yeast strain engineered to produce an engineered thebaine synthase. In some embodiments, the engineered thebaine synthase can be an engineered fusion enzyme. Additionally, the engineered thebaine synthase may be able to more efficiently convert salutaridinol-7-O-acetate to thebaine relative to the parental thebaine synthase. In some embodiments, the parental thebaine synthase can be a wild-type thebaine synthase. In some embodiments, the parental thebaine synthase can be substantially similar to the wild-type thebaine synthase. In some cases, the parental thebaine synthase that is substantially similar to the wild-type thebaine synthase can have an amino acid sequence that is at least 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 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 the wild-type thebaine synthase. The engineered thebaine synthase can be engineered as a fusion enzyme with another enzyme to more efficiently convert salutaridinol-7-O-acetate to thebaine relative to the parental thebaine synthase.

[0145] In some cases, the host cell is from a yeast strain engineered to produce neopinone isomerase. The neopinone isomerase may be able to convert neopinone to codeinone more efficiently than the spontaneous reaction. In some cases, the host cell is from a yeast strain engineered to produce an engineered neopinone isomerase. In some embodiments, the engineered neopinone isomerase can be an engineered fusion enzyme. Additionally, the engineered neopinone isomerase may be able to convert neopinone to codeinone more efficiently than the parental neopinone isomerase. In some embodiments, the parental neopinone isomerase can be a wild-type neopinone isomerase. In some embodiments, the parental neopinone isomerase can be substantially similar to the wild-type neopinone isomerase. In some cases, the parental neopinone isomerase that is substantially similar to the wild-type neopinone isomerase can have an amino acid sequence that is at least 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 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 the wild-type neopinone isomerase. The engineered neopinone isomerase can be engineered as a fusion enzyme with another enzyme to convert neopinone to codeinone more efficiently relative to the parental neopinone isomerase.

[0146] In some cases, the host cell is from a yeast strain engineered to produce engineered norcoclaurine synthase. Additionally, the engineered norcoclaurine synthase may be able to more efficiently convert 4-HPAA and dopamine to norcoclaurine relative to the parental norcoclaurine synthase. Additionally, the engineered norcoclaurine synthase may be able to more efficiently convert 3,4-DHPA and dopamine to norlaudanosoline relative to the parental norcoclaurine synthase. In some embodiments, the parental norcoclaurine synthase can be a wild-type norcoclaurine synthase. In some embodiments, the parental norcoclaurine synthase can be substantially similar to the wild-type norcoclaurine synthase. In some cases, the parental norcoclaurine synthase that is substantially similar to the wild-type norcoclaurine synthase can have an amino acid sequence that is 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 the wild-type norcoclaurine synthase.

[0147] Any host cell described in US2008 / 0176754, US2014 / 0273109, PCTUS2014 / 063738, PCT / US2016 / 030808, PCT / US2015 / 060891, PCT / US2016 / 031506, PCT / US2017 / 057237, International Patent Application No. WO 2022 / 109194 A1, and US Provisional Application No. 62 / 628,264 by Smolke et al. can be applicable to the subject cells and methods. In certain embodiments, the yeast cell can be of the species Saccharomyces cerevisiae (S. cerevisiae). In certain embodiments, the yeast cell can be of the species Schizosaccharomyces pombe. In certain embodiments, the yeast cell can be of the species Pichia pastoris. Yeast is an interesting host cell because cytochrome P450 proteins can fold properly into the endoplasmic reticulum membrane such that their activity is maintained. In some examples, cytochrome P450 proteins are involved in some biosynthetic pathways of interest. In additional examples, cytochrome P450 proteins are involved in the production of the BIA of interest. In additional examples, cytochrome P450 proteins are involved in the production of the enzyme of interest.

[0148] Yeast strains found to be useful for the purposes of the present disclosure include, but are not limited to, CEN.PK (genotype: MATa / αura3-52 / ura3-52 trp1-289 / trp1-289 leu2-3_112 / leu2-3_112his3Δ1 / his3Δ1MAL2-8C / MAL2-8CSUC2 / SUC2), S288C, W303, D273-10B, X2180, A364A, ∑1278B, AB972, SK1, and FL100. In certain cases, the yeast strain is any one of the following: S288C (MATα; SUC2 mal mel gal2 CUP1flo1flo8-1 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), which are derivatives of the W303-B strain (MATa; ade2-1; his3-11, -15; leu2-3, -112; ura3-1; canR; cyr+), expressing Arabidopsis thaliana NADPH-P450 reductase ATR1 and yeast NADPH-P450 reductase CPR1, respectively. In another embodiment, the yeast cell is W303α (MATα; his3-11,15trp1-1 leu2-3 ura3-1ade2-1). Identification and genotypes of additional useful yeast strains can be found at EUROSCARF (web.uni-frankfurt.de / fb15 / mikro / euroscarf / col_index.html).

[0149] In certain embodiments, the heterologous coding sequence can be codon-optimized for expression in Aspergillus species and expressed by an appropriate promoter. In certain embodiments, the promoter can be selected from the phosphoglycerate kinase promoter (PGK), MbfA promoter, cytochrome c oxidase subunit promoter (CoxA), SrpB promoter, TvdA promoter, malate dehydrogenase promoter (MdhA), β-mannosidase promoter (ManB). In certain embodiments, the terminator can be selected from the glucoamylase terminator (GlaA) or TrpC terminator. In certain embodiments, the expression cassette consisting of the promoter, heterologous coding sequence, and terminator can be expressed from a plasmid or integrated into the genome of the host. In certain embodiments, the selection of cells maintaining the plasmid or integrated cassette can be carried out by antibiotic selection (such as hygromycin) or nitrogen source utilization (such as using acetamide as the sole nitrogen source). In certain embodiments, established transformation methods (such as protoplast transformation, lithium acetate, or electroporation) can be used to introduce the DNA construct into the host cell. In certain embodiments, the cells can be cultured in liquid ME or solid MEA (3% malt extract, 0.5% peptone, and ±1.5% agar) or in Vogel minimal medium with or without selection.

[0150] 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 may be derived include Anabaena, Arthrobacter, Acetobacter, Acetobacter aceti, Bacillus, Bifidobacterium, Brevibacterium, Brevibacterium, Carnobacterium, Clostridium, Corynebacterium, Enterobacter, Escherichia, Gluconacetobacter, Gluconobacter, Hafnia, Halomonas, Klebsiella, Kocuria, Lactobacillus, Leuconostoc, Macrococcus, Methylomonas, Methylobacterium, Methylocella, Methylococcus, Microbacterium, Micrococcus, Microcystis, Moorella, Oenococcus, Pediococcus, Prochlorococcus, Propionibacterium, Proteus, Pseudalteromonas, Pseudomonas, Psychrobacter, Rhodobacter, Rhodococcus, Rhodopseudomonas, Serratia, Staphylococcus, Streptococcus, Streptomyces, Synechococcus, Synechocystis, Tetragenococcus, Weissella, and Zymomonas. Examples of bacterial species that can be used with 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 adolescentis, Brachybacterium tyrofermentans, Brevibacterium linens, Carnobacterium divergens, Corynebacterium flavescens, Enterococcus faecium, Gluconacetobacter europaeus, Gluconacetobacter johannae, Gluconobacter oxydans, Hafnia alvei, Halomonas elongataelongata), Kocuria rhizophila, Lactobacillus acidifarinae, Lactobacillus jensenii, Lactococcus lactis, Lactobacillus yamanashiensis, 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, Streptomyces coelicolor, Lactobacillus plantarum, Lactobacillus sakei, Lactobacillus casei, Pseudoalteromonas citrea, Pseudomonasputida), Clostridium josui / Clostridium aceticum / Clostridium acetobutylicum / Clostridium beijerinckii, and Clostridium thermocellum / Moorella thermoacetica.

[0151] In certain embodiments, the bacterial cell can belong to an Escherichia coli strain. In certain embodiments, the Escherichia coli strain can be selected from BL21, DH5α, XL1-Blue, HB101, BL21, and K12. In certain embodiments, the heterologous coding sequence can be codon-optimized for expression in Escherichia coli and expressed by a suitable promoter. In certain embodiments, the promoter can be selected from the T7 promoter, the tac promoter, the trc promoter, the tetracycline-inducible promoter (tet), the lac operon promoter, and the lacO1 promoter. In certain embodiments, the expression cassette consisting of the promoter, the heterologous coding sequence, and the terminator can be expressed from a plasmid or integrated into the genome. In certain embodiments, the plasmid is selected from pUC19 or pBAD. In certain embodiments, the selection of cells maintaining the plasmid or the integration cassette can be performed using antibiotic selection (such as kanamycin, chloramphenicol, streptomycin, spectinomycin, gentamicin, erythromycin, or ampicillin). In certain embodiments, established transformation methods (such as conjugation, heat shock chemical transformation, or electroporation) can be used to introduce the DNA construct into the host cell. In certain embodiments, the cells can be cultured in liquid Luria-Bertani (LB) medium at about 37°C with or without antibiotics.

[0152] In certain embodiments, the bacterial cell can be a Bacillus subtilis strain. In certain embodiments, the Bacillus subtilis (B. subtilis) strain can be selected from 1779, GP25, RO-NN-1, 168, BSn5, BEST195, 1A382, and 62178. In certain embodiments, the heterologous coding sequence can be codon-optimized for expression in Bacillus species and expressed by a suitable promoter. In certain embodiments, the promoter can be selected from the grac promoter, the p43 promoter, or the trnQ promoter. In certain embodiments, the expression cassette consisting of the promoter, the heterologous coding sequence, and the terminator can be expressed from 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) can be used to integrate the expression cassette into the genome. In certain embodiments, the selection of cells maintaining the plasmid or the integration cassette can be carried out with antibiotics (such as erythromycin, kanamycin, tetracycline, and spectinomycin). In certain embodiments, established transformation methods (such as natural competence, heat shock, or chemical transformation) can be used to introduce the DNA construct into the host cell. In certain embodiments, the cells can be cultured in liquid Luria-Bertani (LB) medium at 37 °C or in M9 medium supplemented with glucose and tryptophan. Genetic modification of host cell

[0153] Host cells can be engineered to include one or more modifications (such as two or more, three or more, four or more, five or more, or even more modifications) that provide for the production of a BIA of interest. Additionally or alternatively, host cells can be engineered to include one or more modifications (such as two or more, three or more, four or more, five or more, or even more modifications) that provide for the production of an enzyme of interest. In some cases, the modifications are genetic modifications, such as mutations, additions, or deletions of genes or fragments thereof, or transcriptional regulation of genes or fragments thereof. As used herein, the term "mutation" refers to the deletion, insertion, or substitution of one or more amino acid residues or one or more nucleotide residues relative to a reference sequence or motif. Mutations can be incorporated into the native gene at the original locus as site-directed mutations. In some cases, mutations can be incorporated as additional copies of a gene introduced by genetic integration at a separate locus or as additional copies on an episomal vector (such as a 2μ or centromeric plasmid). In certain instances, a substrate-inhibited copy of an enzyme is under native cellular transcriptional regulation. In some instances, a substrate-inhibited copy of an enzyme is introduced with engineered constitutive or dynamic regulation of protein expression by placing the substrate-inhibited copy of the enzyme under the control of a synthetic promoter. In some examples, the target of one or more modifications can be a native gene. In some examples, the target of one or more modifications can be a non-native gene. In some examples, a non-native gene can be inserted into a host cell. In additional examples, a non-native gene can be altered by one or more modifications prior to insertion into a host cell.

[0154] Engineered host cells can overproduce one or more BIA of interest. Overproduction means that the cells have an improved or increased production of the BIA molecule of interest relative to control cells (e.g., unmodified cells). Improved or increased production means either of two situations: producing a certain amount of the BIA of interest when the control does not have BIA production of interest, and increasing by 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, including 10-fold or more, when the control has some production of the BIA of interest. Engineered host cells can further overproduce one or more morphinan alkaloids. In some cases, engineered host cells can produce a certain amount of the morphinan alkaloid of interest when the control does not have morphinan alkaloid production, and increase by about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, 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 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 80%, about 100%, about 1% to about 10%, about 5% to about 15%, about 15% to about 25%, about 25% to about 35%, about 35% to about 45%, about 45% to about 55%, about 55% to about 65%, about 65% to about 75%, about 75% to about 85%, about 85% to about 95%, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 110-fold, about 120-fold, about 130-fold, about 140-fold, about 150-fold, about 160-fold, about 170-fold, about 180-fold, about 190-fold, about 200-fold or more than 200-fold when the control host cells have some baseline production of the morphinan alkaloid of interest. In some cases, the morphinan alkaloid is formed from the 1-benzylisoquinoline alkaloid product or its derivatives of the C-14 hydroxylation reaction catalyzed by engineered C-14 hydroxylase and / or engineered CPR in the engineered host cells. Engineered C-14 hydroxylase and / or engineered CPR can comprise two separate enzymes that act to produce the C-14 hydroxylase and / or engineered CPR reaction. Engineered host cells can further overproduce one or more of protomorphinan, noropioid, or morphinan alkaloids.

[0155] In some cases, an engineered host cell is capable of producing an increased amount of thebaine relative to a control host cell lacking said one or more modifications (e.g., as described herein). In some cases, an engineered host cell having thebaine synthase is capable of producing an increased amount of thebaine relative to a host cell lacking thebaine synthase. In some cases, an engineered host cell having an engineered thebaine synthase is capable of producing an increased amount of thebaine relative to a host cell having a wild-type thebaine synthase (e.g., as described herein). In certain instances, the increased amount of thebaine is about 10% or more relative to a control host cell, 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, about 2-fold or more, about 5-fold or more or even about 10-fold or more relative to a control host cell. In some cases, thebaine is a product of a thebaine synthase reaction within the engineered host cell. In some cases, thebaine is a product of a thebaine synthase reaction catalyzed by at least one engineered thebaine synthase within the engineered host cell. In these cases, salutaridinol-7-O-acetate can be a substrate of the thebaine synthase reaction.

[0156] In some cases, an engineered host cell is capable of producing an increased amount of codeinone or a morphinan alkaloid product downstream of codeinone in a biosynthetic pathway relative to a control host cell lacking said one or more modifications (e.g., as described herein). In some cases, an engineered host cell having neopinone isomerase is capable of producing an increased amount of codeinone or a morphinan alkaloid product downstream of codeinone in a biosynthetic pathway relative to a host cell lacking neopinone isomerase. In some cases, an engineered host cell having an engineered neopinone isomerase is capable of producing an increased amount of codeinone or a morphinan alkaloid product downstream of codeinone in a biosynthetic pathway relative to a host cell having a parental neopinone isomerase (e.g., as described herein). In certain instances, the increased amount of codeinone or a morphinan alkaloid downstream of codeinone in the biosynthetic pathway is about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, 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 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 80%, about 100%, about 1% to about 10%, about 5% to about 15%, about 15% to about 25%, about 25% to about 35%, about 35% to about 45%, about 45% to about 55%, about 55% to about 65%, about 65% to about 75%, about 75% to about 85%, about 85% to about 95%, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 110-fold, about 120-fold, about 130-fold, about 140-fold, about 150-fold, about 160-fold, about 170-fold, about 180-fold, about 190-fold, about 200-fold or more than 200-fold relative to the control host cell. In some cases, codeinone is a product of the neopinone isomerase reaction within the engineered host cell. In some cases, codeinone is a product of a neopinone isomerase reaction catalyzed by at least one engineered neopinone isomerase within the engineered host cell. In these cases, neopinone can be a substrate of the neopinone isomerase reaction.

[0157] Alternatively, the engineered host cell can overproduce one or more enzymes of interest. Overproduction means that the cell has improved or increased production of the enzyme of interest relative to a control host cell (e.g., an unmodified cell). Improved or increased production means one of two situations: production of a certain amount of the enzyme of interest when the control does not have production, and 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, including 10-fold or more, when the control host cell has some production of the enzyme of interest.

[0158] The engineered host cell can overproduce one or more thebaine synthases. In some cases, the engineered host cell can produce a certain amount of thebaine synthase when the control host cell does not have thebaine synthase production, and 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, including 10-fold or more, when the control has some thebaine synthase production.

[0159] The engineered host cell can overproduce one or more engineered thebaine synthases. In some cases, the engineered host cell can produce a certain amount of the engineered thebaine synthase when the control host cell does not have thebaine synthase production or when the control host cell has the same level of wild-type thebaine synthase production as the engineered host cell, and 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, including 10-fold or more, when the control has some thebaine synthase production. In some cases, the engineered thebaine synthase can be an engineered fusion enzyme.

[0160] The engineered host cell can overproduce one or more neopinone isomerases. In some cases, the engineered host cell can produce a certain amount of neopinone isomerase when the control does not have neopinone isomerase production, and 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, including 10-fold or more, when the control has some neopinone isomerase production.

[0161] Engineered host cells can overproduce one or more engineered neopinone isomerases. In some cases, engineered host cells can produce a certain amount of engineered neopinone isomerase in the absence of neopinone isomerase production in the control or in the case where the control has the same level of wild-type neopinone isomerase production as the engineered host cell, and increase by 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, including 10-fold or more in the case where the control has some neopinone isomerase production. In some cases, the engineered neopinone isomerase can be an engineered fusion enzyme.

[0162] Engineered host cells can further overproduce one or more enzymes derived from neopinone isomerase. In some cases, engineered host cells can produce a certain amount of enzymes derived from neopinone isomerase in the absence of enzyme production derived from neopinone isomerase in the control, and increase by 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, including 10-fold or more in the case where the control has some enzyme production derived from neopinone isomerase.

[0163] Engineered host cells can overproduce one or more engineered demethylcoclaurine synthases. In some cases, engineered host cells can produce a certain amount of engineered demethylcoclaurine synthase in the absence of demethylcoclaurine synthase production in the control or in the case where the control has the same level of wild-type demethylcoclaurine synthase production as the engineered host cell, and increase by 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, including 10-fold or more in the case where the control has some demethylcoclaurine synthase production.

[0164] Engineered host cells can overproduce one or more enzymes that provide C-14 hydroxylase activity. In some cases, engineered host cells can produce an amount of an enzyme that provides C-14 hydroxylase activity in the absence of enzyme production of a control host cell that provides C-14 hydroxylase activity, and increase by 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, including 10-fold or more, in the presence of some enzyme production of a control host cell that provides C-14 hydroxylase activity. In some embodiments, the enzyme that provides C-14 hydroxylase activity is P450.

[0165] Engineered host cells can overproduce one or more engineered enzymes that provide C-14 hydroxylase activity. In some cases, engineered host cells can produce an amount of an engineered enzyme that provides C-14 hydroxylase activity in the absence of production of an engineered enzyme that provides C-14 hydroxylase activity by a control host cell or in the presence of the same level of production of a wild-type engineered enzyme that provides C-14 hydroxylase activity by a control host cell compared to the engineered host cell, and increase by 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, including 10-fold or more, in the presence of some production of an engineered enzyme that provides C-14 hydroxylase activity by a control host cell. In some cases, the engineered enzyme that provides C-14 hydroxylase activity can be an engineered fusion enzyme. In some embodiments, the enzyme that provides C-14 hydroxylase activity is P450.

[0166] Engineered host cells can further overproduce one or more enzymes derived from an enzyme that provides C-14 hydroxylase activity. In some cases, engineered host cells can produce an amount of an enzyme derived from an enzyme that provides C-14 hydroxylase activity in the absence of production of an enzyme derived from an enzyme that provides C-14 hydroxylase activity by a control host cell, and increase by 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, including 10-fold or more, in the presence of some production of an enzyme derived from an enzyme that provides C-14 hydroxylase activity by a control host cell. In some embodiments, the enzyme that provides C-14 hydroxylase activity is P450.

[0167] Engineered host cells can overproduce one or more CPR enzymes. In some cases, engineered host cells can produce a certain amount of CPR enzyme when control host cells do not have CPR enzyme production, and increase by 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, including 10-fold or more, when control host cells have some CPR enzyme production.

[0168] Engineered host cells can overproduce one or more engineered CPR enzymes. In some cases, engineered host cells can produce a certain amount of engineered CPR enzyme when control host cells do not produce CPR enzyme or when control host cells have the same level of wild-type CPR enzyme production as the engineered host cells, and increase by 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, including 10-fold or more, when control host cells have some CPR enzyme production. In some cases, the engineered CPR enzyme can be an engineered fusion enzyme.

[0169] Engineered host cells can further overproduce one or more enzymes derived from CPR enzymes. In some cases, engineered host cells can produce a certain amount of enzymes derived from CPR enzymes when control host cells do not have enzymes derived from CPR enzyme production, and increase by 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, including 10-fold or more, when control host cells have some enzymes derived from CPR enzyme production.

[0170] In addition, engineered host cells can overproduce one or more 14-hydroxylated morphinan BIA products and / or intermediates. In particular, relative to control host cells lacking one or more of the modifications (e.g., as described herein), engineered host cells are capable of producing increased amounts of C-14-hydroxylated morphinan BIA products and / or intermediates, where the one or more modifications include modifications associated with carrying an engineered C-14 hydroxylase and engineered CPR. In certain instances, relative to control host cells, the increased amount of C-14-hydroxylated morphinan BIA products and / or intermediates is about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, 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 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 80%, about 100%, about 1% to about 10%, about 5% to about 15%, about 15% to about 25%, about 25% to about 35%, about 35% to about 45%, about 45% to about 55%, about 55% to about 65%, about 65% to about 75%, about 75% to about 85%, about 85% to about 95%, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 110-fold, about 120-fold, about 130-fold, about 140-fold, about 150-fold, about 160-fold, about 170-fold, about 180-fold, about 190-fold, about 200-fold or more than 200-fold. In some cases, the one or more C-14-hydroxylated morphinan BIA products and / or intermediates are formed from at least one BIA monomer that is a product or derivative of a C-14 hydroxylation reaction catalyzed by an engineered epimerase within the engineered host cell. The engineered C-14 hydroxylase and engineered CPR can comprise two separate enzymes that act to produce the C-14 hydroxylase reaction. Engineered host cells can further overproduce one or more of codeinone, codeine, morphine, morphinone, oripavine, neopinone, neopine, neomorphine, hydrocodone, dihydrocodeine, 14-hydroxycodeinone, 14-hydroxyisocodeine, oxycodone, 14-hydroxycodeine, hydromorphinone, dihydromorphine, dihydroetorphine, ethylmorphine, etorphine, metopon, buprenorphine, pholcodine, isocodeine, hydroxymorphinone, norhydroxymorphinone, norcodeinone, noscapine, nororipavine, normorphinone, hydromorphone, norhydromorphone, norisocodeine, norhydromorphinone, norhydroxymorphinone, and norhydrocodone.In certain embodiments, the engineered host cell can further overproduce a nor derivative or a 14-hydroxy derivative of morphinan. In certain embodiments, the engineered host cell can further overproduce noroxymorphone. In certain embodiments, the engineered host cell can further overproduce 14-hydroxycodeinone. In certain embodiments, the engineered host cell can further overproduce 14-hydroxycodeine.

[0171] In some cases, the one or more (such as two or more, three or more, or four or more) modifications can be selected from: engineered thebaine synthase modification; engineered neopinone isomerase modification; engineered norcoclaurine synthase modification; enzyme expression modification; inactivation modification; C-14 hydroxylase modification; CPR modification; and byproduct inhibition alleviation modification, or a combination thereof. A cell comprising one or more modifications can be referred to as an engineered cell. Substrate inhibition relief mutation

[0172] In some instances, the engineered host cell is a cell that comprises in one or more of the cell's biosynthetic enzyme genes one or more (such as two or more, three or more, four or more, five or more, or even more) substrate inhibition alleviation mutations. 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 the substrate inhibition control mechanism of the cell.

[0173] Relative to a control cell, a mutation that alleviates substrate inhibition reduces the inhibition of a regulated enzyme in the target cell and provides an increase in the level of the regulated compound or its downstream biosynthetic product. In some cases, alleviating the inhibition of the regulated enzyme means inhibiting the IC 50 is increased by 2-fold or more, such as 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. The increased level means a level of 110% or more of the regulated compound or its downstream product in the control cell, 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, such as at least 3-fold or more, at least 5-fold or more, at least 10-fold or more, or even more, in the engineered host cell.

[0174] In engineered host cells, a variety of substrate inhibition control mechanisms and biosynthetic enzymes that regulate the level of a target BIA or its precursor can be targeted for substrate inhibition alleviation. The engineered host cells can include one or more substrate inhibition alleviating mutations in one or more biosynthetic enzyme genes. The one or more mutations can be located in any convenient biosynthetic enzyme gene in which the biosynthetic enzyme is subject to regulatory control. In some embodiments, the one or more biosynthetic enzyme genes encode one or more tyrosine hydroxylases. In certain instances, the one or more substrate inhibition alleviating mutations are present in the biosynthetic enzyme gene that is TyrH. In some embodiments, the engineered host cells can include one or more substrate inhibition alleviating mutations in one of the biosynthetic enzyme genes (such as those described in Table 11).

[0175] In certain embodiments, the one or more substrate inhibition alleviating mutations are present in the TyrH gene. The TyrH gene encodes tyrosine hydroxylase, which is an enzyme that converts tyrosine to L-DOPA. However, TyrH is inhibited by its substrate tyrosine. Mammalian tyrosine hydroxylase activity (such as that seen in humans or rats) can be improved by mutations in the TyrH gene that alleviate substrate inhibition. In particular, substrate inhibition from tyrosine can be alleviated by the point mutation W166Y in the TyrH gene. The point mutation W166Y in the TyrH gene can also improve the binding of the cosubstrate BH4 to catalyze the reaction of tyrosine to form L-DOPA. Mutants of TyrH, when expressed in yeast strains to produce BIA from sugars (such as those described in U.S. Provisional Patent Application Serial No. 61 / 899,496), can significantly improve the production of BIA.

[0176] Any convenient number and type of mutations can be utilized to alleviate the substrate inhibition control mechanism. In certain embodiments, the engineered host cells of the present disclosure can include 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or even 15 or more substrate inhibition alleviating mutations in one or more biosynthetic enzyme genes within the engineered host cell, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 substrate inhibition alleviating mutations.

[0177] Cofactor recycling promotion mechanism

[0178] In some cases, an engineered host cell is a cell that includes in one or more of the cell's biosynthetic enzyme genes one or more (such as two or more, three or more, four or more, five or more, or even more) cofactor recycling promoting mechanisms. 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 recycling promoting mechanism" refers to a mechanism that promotes the cofactor recycling control mechanism of a cell.

[0179] Multiple cofactor recycling promoting mechanisms and biosynthetic enzymes for the regulation of the level of a target BIA or its precursor in an engineered host cell can be targeted for cofactor recycling promotion. An engineered host cell can include one or more cofactor recycling promoting mechanisms in one or more biosynthetic enzyme genes. In some examples, an engineered host cell 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 recycling BH4, which is a co-substrate of TyrH. In some examples, an engineered host cell can include one or more cofactor recycling promoting mechanisms in one or more biosynthetic enzyme genes (such as one of those genes described in Table 11).

[0180] An important cofactor for the production of a target BIA is S-adenosyl-L-methionine (SAM), which is used by multiple methyltransferases. When SAM is used in this reaction, it is converted to S-adenosyl-L-homocysteine (SAH), homocysteine, methionine, and then back to SAM. This pathway is as Figure 34 shown and can be targeted for modification to increase cofactor recycling. In some examples, an engineered host cell can contain overexpression of native S-adenosyl-L-homocysteine hydrolase (SAH1). In some examples, an engineered host cell can contain overexpression of native methionine synthase (MET6). In some examples, an engineered host cell can contain overexpression of native S-adenosylmethionine synthase (SAM2). When one or more of these genes are overexpressed, the recycling of SAH to SAM can be increased. In some examples, an engineered host cell can contain one or more cofactor recycling genes described in Table 11.

[0181] Any convenient number and type of mechanisms can be utilized to facilitate cofactor recycling control mechanisms. In certain embodiments, the engineered host cells of the present disclosure can include 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 recycling promoting mechanisms, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 cofactor recycling promoting mechanisms, in one or more biosynthetic enzyme genes within the engineered host cell. Cofactor recycling promotion mechanism

[0182] In some cases, the engineered host cell is a cell that includes in one or more of the cell's biosynthetic enzyme genes one or more (such as two or more, three or more, four or more, five or more, or even more) cofactor recycling promoting mechanisms. 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 recycling promoting mechanism" refers to a mechanism that promotes the cofactor recycling control mechanism of a cell.

[0183] Multiple cofactor recycling promoting mechanisms and biosynthetic enzymes targeted for modulation of the level of a desired BIA or its precursor in an engineered host cell can be targeted for cofactor recycling promotion. The engineered host cell can include one or more cofactor recycling promoting mechanisms in one or more biosynthetic enzyme genes. In some examples, the engineered host cell 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 recycling BH4, which serves as a co-substrate for TyrH. In some examples, the engineered host cell can include one or more cofactor recycling promoting mechanisms in one or more biosynthetic enzyme genes (such as one of those genes described in Table 11).

[0184] An important cofactor for the production of a desired BIA is S-adenosyl-L-methionine (SAM), which is used by multiple methyltransferases. When SAM is used in this reaction, it is converted to S-adenosyl-L-homocysteine (SAH), homocysteine, methionine, and then back to SAM. This pathway is as Figure 34As shown, and can be targeted for modification to increase cofactor recycling. In some examples, the engineered host cell can include overexpression of native S-adenosyl-L-homocysteine hydrolase (SAH1). In some examples, the engineered host cell can include overexpression of native methionine synthase (MET6). In some examples, the engineered host cell can include overexpression of native S-adenosylmethionine synthase (SAM2). When one or more of these genes are overexpressed, the recycling of SAH to SAM can be increased. In some examples, the engineered host cell can include one or more cofactor recycling genes described in Table 11.

[0185] Any convenient number and type of mechanisms can be utilized to facilitate the cofactor recycling control mechanism. In certain embodiments, the engineered host cells of the present disclosure can include 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or even 15 or more cofactor recycling promoting mechanisms in one or more biosynthetic enzyme genes within the engineered host cell, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 cofactor recycling promoting mechanisms. Product inhibition relief mutation

[0186] In some cases, the engineered host cell is a cell that includes in one or more biosynthetic enzyme genes of the cell: one or more product inhibition alleviating mutations (such as two or more, three or more, four or more, five or more, even more). 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 alleviating mutation" refers to a mutation that alleviates the short-term and / or long-term product inhibition control mechanism of the engineered host cell. Short-term product inhibition is a control mechanism of the cell where there is competitive binding at the cosubstrate binding site. Long-term product inhibition is a control mechanism of the cell where there is irreversible binding of a compound present away from the desired pathway.

[0187] Relative to a control cell, the mutation that alleviates product inhibition reduces the inhibition of the regulated enzyme in the target cell and provides an increase in the level of the regulated compound or its downstream biosynthetic product. In some cases, alleviating the inhibition of the regulated enzyme means inhibiting IC 50Increased by 2-fold or more, such as 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. The increased level means a level of 110% or more of the regulated compound or its downstream product in the control cells, 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 in the engineered host cells, such as at least 3-fold or more, at least 5-fold or more, at least 10-fold or more, or even more.

[0188] Multiple product inhibition control mechanisms and biosynthetic enzymes that are regulated for a target BIA level in engineered host cells can be targeted for product inhibition alleviation. The engineered host cells can include one or more product inhibition alleviation mutations in one or more biosynthetic enzyme genes. The mutations can be located in any convenient biosynthetic enzyme gene in which the biosynthetic enzyme is under regulatory control. In some embodiments, the one or more biosynthetic enzyme genes encode one or more tyrosine hydroxylases. In certain cases, the one or more product inhibition alleviation mutations are present in the biosynthetic enzyme gene that is TyrH. In some embodiments, the engineered host cells include one or more product inhibition alleviation mutations in one or more biosynthetic enzyme genes (such as one of those genes described in Tables 11 and 17).

[0189] In certain embodiments, the one or more product inhibition alleviation mutations are present in the TyrH gene. The TyrH gene encodes tyrosine hydroxylase, which is an enzyme that converts tyrosine to L-DOPA. TyrH requires tetrahydrobiopterin (BH4) as a cosubstrate for catalyzing the hydroxylation reaction. Some microbial strains (such as Saccharomyces cerevisiae) do not naturally produce BH4, but can be engineered to produce this substrate through a four-enzyme synthesis and recycling pathway, as Figure 2 shown. Figure 2 Examples of the synthesis, recycling, and salvage pathways of tetrahydrobiopterin are shown according to some embodiments of the present disclosure. Figure 2 The uses of the following enzymes are provided: PTPS, pyruvoyl tetrahydropterin synthase; SepR, sepiapterin reductase; PCD, pterin 4a-carbinolamine dehydratase; QDHPR, dihydropteridine reductase; and DHFR, dihydrofolate reductase. Among the enzymes Figure 2 shown, yeast synthesizes endogenous GTP cyclohydrolase I. GTP and dihydroneopterin triphosphate are naturally synthesized in yeast. Additionally, Figure 2 the other metabolites in

[0190] TyrH is inhibited by its product L-DOPA and other catecholamines, particularly dopamine. Mammalian tyrosine hydroxylase activity, such as from humans or rats, can be improved by mutations that relieve product inhibition. For example, short-term product inhibition, such as competitive binding at the cosubstrate binding site, can be relieved by the point mutation W166Y on the TyrH gene. In particular, the point mutation W166Y on the TyrH gene can improve cosubstrate binding. Additionally, short-term product inhibition that relieves competitive binding at the cosubstrate binding site can be improved by the point mutation S40D on the TyrH gene. The combined mutations R37E, R38E on the TyrH gene can also improve short-term product inhibition. In particular, in the presence of dopamine, the R37E, R38E mutations can specifically improve tyrosine hydroxylase activity together.

[0191] Additionally, point mutations on the TyrH gene can relieve long-term product inhibition. Long-term product inhibition relief can include irreversible binding of catecholamines to iron in the active site, such that there is less catecholamine available to act as a product inhibitor of tyrosine hydroxylase activity. The mutations E332D and Y371F in the TyrH gene can relieve long-term product inhibition, respectively.

[0192] Combinations of mutations can be prepared, such as two or three or more mutations at a time, to relieve multiple types of substrate and product inhibition to further improve the activity of TyrH. Mutants of TyrH, when expressed in yeast strains to produce BIAs from sugars, such as those described in U.S. Provisional Patent Application Serial No. 61 / 899,496, can significantly improve BIA production.

[0193] Any convenient number and type of mutations can be utilized to relieve the product inhibition control mechanism. In certain embodiments, the engineered host cells of the present disclosure can include 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or even 15 or more product inhibition relief mutations in one or more biosynthetic enzyme genes within the engineered host cell, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 product inhibition relief mutations. Feedback inhibition relief mutation

[0194] In some cases, an engineered host cell is a cell that contains in one or more of its biosynthetic enzyme genes one or more feedback inhibition alleviating mutations (such as two or more, three or more, four or more, five or more, or even more). 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 examples, the one or more biosynthetic enzyme genes are non-native to the cell. As used herein, the term "feedback inhibition alleviating mutation" refers to a mutation that alleviates the feedback inhibition control mechanism of an engineered host cell. Feedback inhibition is a control mechanism of a cell in which when a regulated compound accumulates to a certain level, the enzymes in the synthesis pathway of the compound are inhibited, thereby balancing the amount of the compound in the cell. A mutation that alleviates feedback inhibition reduces the inhibition of the regulated enzyme in the engineered host cell relative to a control cell. In this way, the engineered host cell provides increased levels of the regulated compound or its downstream biosynthetic products. In some cases, alleviating the inhibition of the regulated enzyme means inhibiting IC 50 by 2-fold or more, such as 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. An increased level means a level that is 110% or more of the regulated compound or its downstream product in the control cell, 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 in the host cell, such as at least 3-fold or more, at least 5-fold or more, at least 10-fold or more, or even more.

[0195] Multiple feedback inhibition control mechanisms and biosynthetic enzymes for the level regulation of a target BIA in a host cell can be targeted for alleviation. The host cell can include one or more feedback inhibition alleviating mutations in one or more biosynthetic enzyme genes that are native to the cell. The one or more mutations can be located in any convenient biosynthetic enzyme gene in which 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-arabino-heptulosonate-7-phosphate (DAHP) synthase and chorismate mutase. In some embodiments, the one or more biosynthetic enzyme genes encode 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase. In some cases, the one or more biosynthetic enzyme genes can encode chorismate mutase. In certain cases, the one or more feedback inhibition alleviating mutations can be present in biosynthetic enzyme genes selected from ARO4 and ARO7. In certain cases, the one or more feedback inhibition alleviating mutations can be present in the biosynthetic enzyme gene that is ARO4. In certain cases, the one or more feedback inhibition alleviating mutations are present in the biosynthetic enzyme gene that is ARO7. In some embodiments, the engineered host cell can include one or more feedback inhibition alleviating mutations in one or more biosynthetic enzyme genes (such as one of those genes described in Table 11).

[0196] Any convenient number and type of mutations can be utilized to alleviate the feedback inhibition control mechanism. As used herein, the term "mutation" refers to a deletion, insertion, or substitution of one or more amino acid residues or one or more nucleotide residues relative to a reference sequence or motif. Mutations can be incorporated as site-directed mutations into the native gene at the original locus. In some cases, mutations can be incorporated as additional copies of a gene introduced by genetic integration at a separate locus or as additional copies on an episomal vector (such as a 2μ or centromeric plasmid). In certain cases, the feedback-inhibited copy of the enzyme is under native cellular transcriptional regulation. In some cases, the feedback-inhibited copy of the enzyme is introduced with engineered constitutive or dynamic regulation of protein expression by placing it under the control of a synthetic promoter.

[0197] In certain embodiments, one or more feedback inhibition alleviating mutations can be present in the ARO4 gene. The ARO4 mutations of interest can include, but are not limited to, replacing the lysine residue at position 229 with leucine, replacing the glutamine residue at position 166 with a lysine residue, or mutations as described in Hartmann M et al. ((2003) Proc Natl Acad Sci U S A 100(3):862 - 867) or Fukuda et al. ((1992) J Ferment Bioeng 74(2):117 - 119). In some cases, the mutations used to confer feedback inhibition can be selected from a mutagenesis library of enzyme mutants. Examples of such selections can include rescuing the growth of o - fluoro - D,L - phenylalanine or aro3 mutant yeast strains in a medium with excess tyrosine, as described in Fukuda et al. ((1990) Breeding of Brewing Yeast Producing a Large Amount of Beta - Phenylethyl Alcohol and Beta - Phenylethyl Acetate. Agr Biol Chem Tokyo 54(1):269 - 271).

[0198] In certain embodiments, the engineered host cells of the present disclosure can contain 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or even 15 or more feedback inhibition alleviating mutations in one or more biosynthetic enzyme genes within the engineered host cell, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 feedback inhibition alleviating mutations. By - product inhibition relief modification

[0199] The host cell can include one or more modifications (such as 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 that are designed to alleviate byproduct inhibition. 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 modification to alleviate the accumulation of key byproducts. As used herein, the term "byproduct inhibition alleviating modification" refers to a modification that reduces the accumulation of key inhibitory byproducts in an engineered host cell. Byproduct inhibition is a mechanism of the cell in which the accumulation of a particular byproduct compound of fermentation inhibits the production of the desired BIA when the compound has accumulated to a certain level. Relative to a control cell, the modification that alleviates byproduct inhibition reduces the accumulation of one or more byproduct compounds in the engineered host cell. In this way, the engineered host cell provides a reduced level of byproduct compounds and / or an increased level of the desired BIA. An increased level means at least about 110% of the level of the desired BIA in the control cell, such as about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, or more than 200%, such as at least about 3-fold, at least about 5-fold, at least about 10-fold the level of the desired BIA in the control cell. A reduced level means that the level of the byproduct compound in the control cell is reduced by at least about 10% or more, such as about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 97%, or about 99%. Modifications to the target host cell process that can be applied to the subject host cell are described in U.S. Publication No. 20140273109 (14 / 211,611) to Smolke et al., the disclosure of which is incorporated herein by reference in its entirety.

[0200] Multiple byproduct inhibition alleviating modifications and biosynthetic enzymes targeting the accumulation modification of the target byproduct level in the engineered host cell can be targeted for modification. The engineered host cell can include one or more byproduct inhibition alleviating mechanisms in one or more biosynthetic enzyme genes. In some examples, the target byproduct is fusel alcohol. In some examples, the byproduct is tyrosol, phenethyl alcohol, or methanethiol. In some examples, the engineered host cell can include one or more byproduct inhibition alleviating mechanisms in one or more biosynthetic enzyme genes (such as one of the genes described in Table 11).

[0201] In some examples, an engineered host cell can comprise one or more heterologous coding sequences encoding one or more biosynthetic enzymes. In some examples, the biosynthetic enzyme is 4-hydroxyphenylacetaldehyde synthase (HPAAS). When HPAAS is expressed, it can convert L-tyrosine to 4-HPAA. In some examples, the biosynthetic enzyme is phosphoketolase (PK). When PK is expressed, it can convert fructose-6-phosphate and xylulose-5-phosphate to acetyl phosphate. In some examples, the biosynthetic enzyme is uridine 5'-diphosphate-glucosyltransferase (UGT). When the UGT enzyme is expressed, it can convert phenol to aryl β-D-glucoside. In the case of expressing UGT, it can be combined with an inactivating mutation in EGH1 to increase the utilization of the substrate UDP-glucose.

[0202] In some examples, an engineered host cell can include one or more inactivating mutations in one or more genes encoding biosynthetic enzymes. In some examples, the one or more inactivating mutations are in ARO8, ARO9, ARO10, PDC1, PDC5, PDC6, ARI1, ATF1, ATF2, EHT1, EEB1, AAD3, YPR1, GRE2, ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, YPR1, YDR541c BAT2, HFD1, TYR1, PHA2, DUG2, SFA1 or DUG3.

[0203] Any convenient number and type of modifications can be utilized to alleviate the byproduct inhibition mechanism. In certain embodiments, the engineered host cells of the present disclosure can include 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or even 15 or more byproduct inhibition alleviating modifications, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 byproduct inhibition alleviating modifications in one or more biosynthetic enzyme genes within the engineered host cell. Transcription regulation modification

[0204] A host cell can include one or more transcriptional regulatory modifications of one or more biosynthetic enzyme genes of the cell (such as two or more, three or more, four or more, five or more or even more modifications). 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 regulation. Transcriptional regulation means regulating the expression of a gene of interest in the modified cell, e.g., increasing or decreasing, enhancing or repressing relative to a control cell (e.g., an unmodified cell). In some cases, transcriptional regulation of the gene of interest includes increasing or enhancing expression. Increasing or enhancing expression means that the expression level of the gene of interest is increased 2-fold or more (such as 5-fold or more) compared to the control (i.e., the expression in the same unmodified cell) and sometimes 25-fold, 50-fold or 100-fold or more and in certain embodiments 300-fold or more or higher (e.g., by using any convenient gene expression assay). Alternatively, in cases where the expression of the gene of interest in the cell is so low as to be undetectable, if the expression is increased to an easily detectable level, the expression level of the gene of interest is considered to have increased. In certain instances, transcriptional regulation of the gene of interest includes decreasing or repressing expression. Decreasing or repressing expression means that the expression level of the gene of interest is decreased 2-fold or more (such as 5-fold or more) compared to the control and sometimes decreased 25-fold, 50-fold or 100-fold or more and in certain embodiments decreased 300-fold or more or higher. In some cases, the expression is decreased to an undetectable level. Modifications to the subject host cell that can be applied to the target host cell process are described in U.S. Publication No. 20140273109 (14 / 211,611) to Smolke et al., the disclosure of which is incorporated herein by reference in its entirety.

[0205] Any convenient biosynthetic enzyme gene can be transcriptionally regulated and includes, but is not limited to, those biosynthetic enzymes described in Figure 1 . In particular, Figure 1 shows a biosynthetic scheme for converting glucose to 4-HPAA, dopamine, and 3,4-DHPAA according to some embodiments of the present disclosure text. Figure 1Examples of the enzymes described in [reference] include ARO3, ARO4, ARO1, ARO7, TYR1, TYR, TyrH, DODC, MAO, ARO10, ARO9, and ARO8. In some cases, the one or more biosynthetic enzyme genes may be selected from ARO10, ARO9, ARO8, and TYR1. In some cases, the one or more biosynthetic enzyme genes may be ARO10. In certain cases, the one or more biosynthetic enzyme genes may be ARO9. In some embodiments, the one or more biosynthetic enzyme genes may be TYR1. In some embodiments, the host cell comprises one or more transcriptional regulatory modifications of one or more genes (such as one of the genes described in Table 11).

[0206] In some embodiments, transcriptional regulatory modifications can include replacing the native promoter of one or more biosynthetic enzyme genes with a strong promoter, or expressing additional copies of one or more genes under the control of a strong promoter. The promoter driving the expression of the gene of interest can be a constitutive promoter or an inducible promoter, provided that the promoter can be active in the host cell. The genes of interest can be expressed from their native promoters. Additionally or alternatively, the genes of interest can be expressed by non-native promoters. Although not required, such promoters can be medium to high strength in the host in which they are used. The promoter can be regulated or constitutive. In some embodiments, a promoter that is not repressed by glucose or is only mildly repressed by the presence of glucose in the culture medium can be used. There are many suitable promoters, examples of which include the promoters of glycolytic genes, such as the promoter of the Bacillus subtilis tsr gene (encoding fructose bisphosphate aldolase) or the GAPDH promoter from Saccharomyces cerevisiae (encoding glyceraldehyde-phosphate dehydrogenase) (Bitter G.A., Meth. Enzymol. 152:673 - 684 (1987)). Other strong promoters of interest include, but are not limited to, the ADH1 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, P.J. et al. eds., Butterworths (1989)), the alkaline phosphatase promoter from Bacillus licheniformis (Lee. J.W.K. et al., J. Gen. Microbiol. 137:1127 - 1133 (1991)), GPD1, and TEF1. Yeast promoters of interest include, but are not limited to, inducible promoters (such as Gal1-10), Gal1, GalL, GalS, repressible promoters Met25, tetO, and constitutive promoters (such as the glyceraldehyde 3-phosphate dehydrogenase promoter (GPD)), alcohol dehydrogenase promoter (ADH), translation elongation factor-1-α promoter (TEF), cytochrome c-oxidase promoter (CYC1), MRP7 promoter, etc. In some cases, the strong promoter is GPD1. In certain cases, the strong promoter is TEF1. Autonomously replicating yeast expression vectors containing promoters that can be induced by hormones such as glucocorticoids, steroids, and thyroid hormones are also known and include, but are not limited to, glucocorticoid response elements (GRE) and thyroid hormone response elements (TRE), see, for example, these promoters described in U.S. Patent No. 7,045,290. Vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used.Alternatively, any convenient promoter / enhancer combination (according to the eukaryotic promoter database EPDB) can be used to drive the expression of the gene of interest. It should be understood that any convenient promoter specific to the host cell can be selected, such as E. coli. In some cases, promoter selection can be used to optimize transcription and thus optimize enzyme levels to maximize yield while minimizing energy resources. Inactivation mutation

[0207] The engineered host cell can include one or more (such as two or more, three or more, four or more, five or more, even more) inactivating mutations of the enzymes or proteins of the cell. Incorporating one or more inactivating mutations can modify the flux of the synthetic pathway of the engineered host cell to increase the level of the desired BIA or the enzymes or precursors required to produce the desired BIA. In some examples, the one or more inactivating mutations target enzymes that are native to the cell. Additionally or alternatively, the one or more inactivating mutations target enzymes that are native to the cell. As used herein, "inactivating mutation" means one or more mutations of a gene or regulatory DNA sequence of a cell, wherein the one or more mutations inactivate the biological activity of the protein expressed by the gene of interest. In some cases, the gene is native to the cell. In some instances, the gene encodes an enzyme that is inactivated and is part of or linked to the synthetic pathway of the desired BIA produced by the host cell. In some instances, the inactivating mutation is located in the regulatory DNA sequence controlling the gene of interest. In certain cases, the inactivating mutation is targeted at the promoter of the gene. Any convenient mutation (e.g., as described herein) can be utilized to inactivate the gene of interest or the regulatory DNA sequence. "Inactivated" or "inactivating" means that the biological activity of the protein expressed by the mutant gene is reduced by 10% or more, such as 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, relative to the control protein expressed by the non-mutated control gene. In some cases, the protein is an enzyme, and the inactivating mutation reduces the activity of the enzyme.

[0208] In some examples, the engineered host cell includes an inactivating mutation in an enzyme or protein that is native to the cell. Any convenient enzyme can be targeted for inactivation. The enzyme of interest can include, but is not limited to, those enzymes described in Table 11, whose role in the synthetic pathway of the engineered host cell tends to reduce the level of the BIA of interest. In some cases, the enzyme has glucose-6-phosphate dehydrogenase activity. In certain embodiments, the enzyme containing the inactivating mutation is ZWF1. In some cases, the enzyme has alcohol dehydrogenase activity. In some embodiments, the enzyme containing the inactivating mutation is selected from ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, and SFA1. In certain embodiments, the enzyme containing one or more inactivating mutations is ADH2. In certain embodiments, the enzyme containing one or more inactivating mutations is ADH3. In certain embodiments, the enzyme containing one or more inactivating mutations is ADH4. In certain embodiments, the enzyme containing one or more inactivating mutations is ADH5. In certain embodiments, the enzyme containing one or more inactivating mutations is ADH6. In certain embodiments, the enzyme containing one or more inactivating mutations is ADH7. In some cases, the enzyme has aldehyde oxidoreductase activity. In certain embodiments, the enzyme containing the inactivating mutation is selected from ALD2, ALD3, ALD4, ALD5, and ALD6. In certain embodiments, the enzyme containing one or more inactivating mutations is ALD2. In certain embodiments, the enzyme containing one or more inactivating mutations is ALD3. In certain embodiments, the enzyme containing one or more inactivating mutations is ALD4. In certain embodiments, the enzyme containing one or more inactivating mutations is ALD5. In certain embodiments, the enzyme containing one or more inactivating mutations is ALD6. In some cases, the enzyme has aldehyde reductase activity. In some embodiments, the enzyme containing the inactivating mutation is ARI1. In some cases, the enzyme has aryl-alcohol dehydrogenase activity. In some embodiments, the enzyme containing the inactivating mutation is selected from AAD4, AAD6, AAD10, AAD14, AAD15, AAD16. In certain embodiments, the enzyme containing one or more inactivating mutations is AAD4. In certain embodiments, the enzyme containing one or more inactivating mutations is AAD6. In certain embodiments, the enzyme containing one or more inactivating mutations is AAD10. In certain embodiments, the enzyme containing one or more inactivating mutations is AAD14. In certain embodiments, the enzyme containing one or more inactivating mutations is AAD15. In certain embodiments, the enzyme containing one or more inactivating mutations is AAD16. In some examples, the engineered host cell includes an inactivating mutation in a transcriptional regulator that is native to the cell. The transcriptional regulator of interest can include, but is not limited to, those proteins described in Table 11. In some cases, the protein has activity as a transcriptional regulator of phospholipid biosynthetic genes.In some embodiments, the transcription regulator comprising an inactivating mutation is OPI1. In some embodiments, the host cell comprises one or more inactivating mutations in one or more genes described in Table 11.

[0209] In some instances, the engineered host cell comprises an inactivating mutation in an enzyme or protein that is native to the cell. The enzyme of interest can include, but is not limited to, those enzymes described in Table 11, which function in the synthetic pathway of the engineered host cell as part of the Erlich pathway for the production of fusel alcohols. In some cases, the enzyme has phenylpyruvate decarboxylase activity. In certain embodiments, the enzyme comprising an inactivating mutation is ARO10. In some cases, the enzyme has pyruvate decarboxylase activity. In some embodiments, the enzyme comprising an inactivating mutation is selected from PDC1, PDC5, or PDC6. In certain embodiments, the enzyme comprising one or more inactivating mutations is PDC1. In certain embodiments, the enzyme comprising one or more inactivating mutations is PDC5. In certain embodiments, the enzyme comprising one or more inactivating mutations is PDC6. In some cases, the enzyme has aromatic aminotransferase activity. In some embodiments, the enzyme comprising an inactivating mutation is selected from ARO8 and ARO9. In certain embodiments, the enzyme comprising one or more inactivating mutations is ARO8. In certain embodiments, the enzyme comprising one or more inactivating mutations is ARO9. In some cases, the enzyme has prephenate dehydrogenase activity. In certain embodiments, the enzyme comprising one or more inactivating mutations is TYR1. In some cases, the enzyme has prephenate dehydratase activity. In certain embodiments, the enzyme comprising one or more inactivating mutations is PHA2. In some embodiments, the host cell comprises one or more inactivating mutations in one or more genes described in Table 11. Epimerization modification

[0210] Some of the 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 can comprise an engineered host cell. In some instances, the conversion of (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids is a key step in the conversion of the substrate to a wide range of alkaloids. In some instances, the conversion of (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids comprises an epimerization reaction via an engineered epimerase. In some cases, the epimerization of the substrate alkaloid can occur by oxidizing the (S)-substrate to the corresponding Schiff base or imine intermediate and then stereospecifically reducing this intermediate to the (R)-product, as Figure 1Provided and generally shown as in Scheme 1. As provided in Scheme 1, R1, R2, R3, and R4 can be H or CH3. R5 can be H, OH, or OCH3. Scheme 1

[0211] In some examples, the conversion of an (S)-substrate to an (R)-product may involve at least one oxidation reaction and at least one reduction reaction. In some cases, the reduction reaction is optionally carried out after the oxidation reaction. In some cases, at least one of the oxidation reaction and the reduction reaction is carried out in the presence of an enzyme. In some cases, at least one of the oxidation reaction and the reduction reaction is catalyzed by an engineered epimerase. In some cases, both the oxidation and reduction reactions are carried out in the presence of an engineered fusion epimerase. In some cases, both the oxidation reaction and the reduction reaction are carried out in the presence of an engineered split epimerase having separately expressed oxidase and reductase components. In some cases, the engineered epimerase can be used to catalyze the oxidation reaction and the reduction reaction. The oxidation reaction and the reduction reaction can be catalyzed by the same engineered epimerase.

[0212] In some of the methods, processes, and systems described herein, the oxidation reaction can be carried out in the presence of an enzyme that is part of an engineered epimerase. In some examples, the engineered epimerase can have an oxidase component. In some cases, the oxidase component can be a component of an engineered fusion epimerase. In some cases, the oxidase component can be expressed independently as part of an engineered split epimerase. The oxidase can use (S)-1-benzylisoquinoline as a substrate. The oxidase can convert the (S)-substrate to the corresponding imine or Schiff base derivative. The oxidase can be referred to as 1,2-dehydroreticuline synthase (DRS). Non-limiting examples of enzymes suitable for oxidizing (S)-1-benzylisoquinoline alkaloids in the present disclosure include cytochrome P450 oxidase, 2-oxoglutarate-dependent oxidase, and flavoprotein oxidase. For example, (S)-tetrahydroprotoberberine oxidase (STOX, E.C 1.3.3.8) can oxidize (S)-nornuciferine and other (S)-1-benzylisoquinoline alkaloids to 1,2-dehydronornuciferine and other corresponding 1,2-dehydro products. In some examples, a protein containing an oxidase domain of any of the foregoing examples can carry out the oxidation. In some examples, the oxidase can catalyze the oxidation reaction within a host cell, such as an engineered host cell, as described herein. In some cases, the oxidase can have one or more components that increase its activity. In some examples, the reduction reaction can occur after the oxidation reaction. The reduction reaction can be carried out by an enzyme that is part of an engineered epimerase. In some examples, 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 an imine or Schiff base derived from an (S)-1-benzylisoquinoline alkaloid include aldo-keto reductase (e.g., codeinone reductase-like enzyme (EC 1.1.1.247)) and short-chain dehydrogenase (e.g., salutaridine reductase-like enzyme (EC 1.1.1.248)). In some examples, a protein containing a reductase domain of any of the foregoing examples can carry out the reduction. In additional embodiments, the reduction is stereospecific. In some examples, the reductase can catalyze the reduction reaction within a host cell, such as an engineered host cell, as described herein.

[0213] Examples of enzymes that can carry out the epimerization reaction include epimerases having an oxidase domain and a reductase domain, which convert an (S)-1-benzylisoquinoline alkaloid to an (R)-1-benzylisoquinoline alkaloid. In particular, the epimerase can have a cytochrome P450 oxidase 82Y2-like domain. Additionally, the epimerase can have a codeinone reductase-like domain. An epimerase having a cytochrome P450 oxidase 82Y2-like domain and also having a codeinone reductase-like domain can be referred to as a DRS-DRR enzyme. In particular, the DRS-DRR enzyme can be a fusion enzyme that is a fusion epimerase. Furthermore, when the DRS-DRR enzyme is modified by at least one activity-enhancing modification, the fusion enzyme can be an engineered fusion epimerase.

[0214] Examples of amino acid sequences of DRS-DRR enzymes that can be used to convert (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids are shown in Table 1. The amino acid sequence of the epimerase utilized in the conversion of (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids can be 50% or more identical to the given amino acid sequence listed in Table 1. For example, the amino acid sequence of such an epimerase can comprise an amino acid sequence that is at least 50% more, 55% more, 60% more, 65% more, 70% more, 75% more, 80% more, 81% more, 82% more, 83% more, 84% more, 85% more, 86% more, 87% more, 88% more, 89% more, 90% more, 91% more, 92% more, 93% more, 94% more, 95% more, 96% more, 97% more, 98% more, or 99% or more identical to the amino acid sequence provided herein. Additionally, in certain embodiments, an "identical" amino acid sequence has at least 80%-99% identity to a specific amino acid sequence at the amino acid level. In some cases, an "identical" amino acid sequence contains at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% and more (in some cases, at least 95%, 96%, 97%, 98% and 99%) identity at the amino acid level. In some cases, for example, the amino acid sequences can be identical, but the nucleic acid sequences encoding the amino acid sequences are altered, such as to optimize codon usage for a host organism.

[0215] Amino acid residues of homologous epimerases can be referenced according to the numbering scheme of SEQ ID NO.16, and this numbering system is used throughout the disclosure to refer to specific amino acid residues of epimerases homologous to SEQ ID NO.16. An epimerase homologous to SEQ ID NO.16 can have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO.16. In some cases, the amino acid called position 50 in a homologous epimerase may not be the 50th amino acid in the homologous epimerase, but will be the amino acid corresponding to the amino acid at position 50 in SEQ ID NO.16 in a protein alignment of the homologous epimerase with SEQ ID NO.16. In some cases, homologous enzymes can be aligned with SEQ ID NO.16 based on primary sequence, secondary structure or tertiary structure.

[0216] Engineered host cells can be provided that produce an engineered epimerase that converts (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids, where the epimerase comprises an amino acid sequence selected from SEQ ID NO: 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 engineered host cells can be recovered and purified to form a biocatalyst. In some cases, the epimerase can be split into one or more enzymes. Additionally, one or more enzymes produced by splitting the epimerase can be recovered from the engineered host cells. These one or more enzymes produced by splitting the epimerase can also be used to catalyze the conversion of (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids. Additionally, the use of engineered split epimerases can be used to increase the production of benzylisoquinoline alkaloid products intracellularly when compared to producing benzylisoquinoline alkaloid products intracellularly using a fusion epimerase.

[0217] In additional cases, one or more enzymes recovered from engineered host cells that produce an epimerase can be used in the process of converting (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids. The process can include contacting the (S)-1-benzylisoquinoline alkaloid with an amount of epimerase sufficient to convert the (S)-1-benzylisoquinoline alkaloid to the (R)-1-benzylisoquinoline alkaloid. In some examples, the (S)-1-benzylisoquinoline alkaloid can be contacted with a sufficient amount of one or more enzymes such that at least 5% of the (S)-1-benzylisoquinoline alkaloid is converted to the (R)-1-benzylisoquinoline alkaloid. In additional examples, the (S)-1-benzylisoquinoline alkaloid can be contacted with a sufficient amount of 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 the (R)-1-benzylisoquinoline alkaloid.

[0218] One or more enzymes capable of converting (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids can be contacted with (S)-1-benzylisoquinoline alkaloids in vitro. Additionally or alternatively, one or more enzymes capable of converting (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids can be contacted with (S)-1-benzylisoquinoline alkaloids in vivo. Additionally, one or more enzymes capable of converting (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids can be provided to a cell that has (S)-1-benzylisoquinoline alkaloids therein, or can be produced within an engineered host cell.

[0219] In some examples, the method provides an engineered host cell that produces an alkaloid product, wherein the epimerization of the (S)-substrate to the (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 (R)-1-benzylisoquinoline alkaloids, including, for example, 4-cycloprotopine and 5-cyclomorphine alkaloids. In another embodiment, the (S)-1-benzylisoquinoline alkaloid is an intermediate in the product of the engineered host cell. In still other embodiments, the alkaloid product is selected from 1-benzylisoquinoline, morphinan, protomorphinan, noropioid, nal-opioid, or bisbenzylisoquinoline alkaloids.

[0220] In some examples, the (S)-substrate is an (S)-1-benzylisoquinoline alkaloid selected from: (S)-nornuciferine, (S)-nuciferine, (S)-tetrahydropapaverine, (S)-demethylcoclaurine, (S)-coclaurine, (S)-N-methylcoclaurine, (S)-3'-hydroxy-N-methylcoclaurine, (S)-nordehydroisocorydine, (S)-dehydroisocorydine, (S)-isocorydine, (S)-norprotosinomenine, (S)-protosinomenine, (S)-nordaurisoline, (S)-laudanosoline, (S)-4'-O-methyllaudanosoline, (S)-6-O-methylnordaurisoline, (S)-4'-O-methylnordaurisoline.

[0221] In some examples, the (S)-substrate is a compound of Formula I: or a salt thereof, wherein: R 1 、R 2 、R 3 and R 4 are independently selected from hydrogen and methyl; and R 5 Selected from hydrogen, hydroxyl, and methoxy.

[0222] In some other examples, R 1 , R 2 , R 3 , R 4 , and R 5 At least one of them is hydrogen.

[0223] In still other examples, the (S)-substrate is a compound of formula II: Or a salt thereof, wherein: R 3 Is selected from hydrogen and C1-C4 alkyl; R 6 , and R 7 Each occurrence is independently selected from hydroxyl, fluorine, chlorine, bromine, formyl, C1-C4 acyl, C1-C4 alkyl, and C1-C4 alkoxy; n is 0, 1, 2, 3, or 4; and n' is 0, 1, 2, 3, 4, or 5.

[0224] When a bond is drawn across a ring, it means that substitution can occur at non-specific ring atoms or positions. For example, in formula II shown above, the hydrogen of any -CH- in the 6-membered ring can be replaced by R 7 To form -CR 7 -.

[0225] In some examples, 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.

[0226] In some examples, the method provides engineered host cells that produce 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). The various alkaloid products can include, but are not limited to, 1-benzylisoquinoline, morphinan, protomorphinan, nor-opioid substances, or nal-opioid alkaloids.

[0227] Any suitable carbon source can be used as a precursor for the epimerization of 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, an unpurified mixture from a renewable feedstock can be used (e.g., corn steep liquor, beet molasses, barley malt, biomass hydrolysate). 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 can be utilized, such as, for example, methylamine, glucosamine, and amino acids (e.g., L-tyrosine). In some examples, the 1-benzylisoquinoline alkaloids can be directly added to the engineered host cells of the present disclosure, which include, for example, norlaudanosoline, laudanosoline, norreticuline, and reticuline. In still further embodiments, the 1-benzylisoquinoline alkaloids can be added to the engineered host cells as a single enantiomer (e.g., (S)-1-benzylisoquinoline alkaloid) or a mixture of enantiomers (including, for example, a racemic mixture).

[0228] In some examples, the method provides for the epimerization of the stereogenic center of an engineered epimerase, a 1-benzylisoquinoline alkaloid, or a derivative thereof. In additional embodiments, the method includes contacting a 1-benzylisoquinoline alkaloid with an engineered epimerase. The engineered epimerase can invert the stereochemistry of the stereogenic center of the 1-benzylisoquinoline alkaloid or a derivative thereof to the opposite stereochemistry. In some examples, the engineered epimerase converts an (S)-1-benzylisoquinoline alkaloid to an (R)-1-benzylisoquinoline alkaloid. In some examples of this conversion of an (S)-1-benzylisoquinoline alkaloid to an (R)-1-benzylisoquinoline alkaloid using an engineered epimerase, the (S)-1-benzylisoquinoline alkaloid is selected from (S)-norreticuline, (S)-reticuline, (S)-tetrahydropapaverine, (S)-nornuciferine, (S)-nuciferine, (S)-N-methylnuciferine, (S)-3'-hydroxy-N-methylnuciferine, (S)-norisocorydine, (S)-isocorydine, (S)-corydine, (S)-nornarceine, (S)-narceine, (S)-4'-O-methyllaudanosoline, (S)-6-O-methylnorlaudanosoline, and (S)-4'-O-methylnorlaudanosoline.

[0229] In still other embodiments, the 1-benzylisoquinoline alkaloids epimerized using an engineered epimerase can contain two or more stereocenters, where only one of the two or more stereocenters is inverted to produce a diastereomer of the substrate (e.g., an (S,R)-1-benzylisoquinoline alkaloid is converted to an (R,R)-1-benzylisoquinoline alkaloid). In examples where only one stereocenter of the 1-benzylisoquinoline alkaloid is inverted when contacted with at least one enzyme, the product is referred to as an epimer of the 1-benzylisoquinoline alkaloid.

[0230] In some examples, the 1-benzylisoquinoline alkaloid is presented to the enzyme as a single stereoisomer. In some other examples, the 1-benzylisoquinoline alkaloid is presented to the enzyme as a mixture of stereoisomers. In still other embodiments, the mixture of stereoisomers can be a racemic mixture. In some other examples, the mixture of stereoisomers can be enriched in one stereoisomer compared to another stereoisomer.

[0231] In some examples, the 1-benzylisoquinoline alkaloid or its derivative is recovered. In some examples, the 1-benzylisoquinoline alkaloid is recovered from a cell culture. In still other embodiments, the recovered 1-benzylisoquinoline alkaloid is enriched in one stereoisomer in terms of enantiomers compared to the original mixture of 1-benzylisoquinoline alkaloid presented to the enzyme. In still other 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%.

[0232] In some examples, protopine or its derivative is recovered. In some examples, protopine is recovered from a cell culture. In still other embodiments, the recovered protopine 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%.

[0233] In some instances, the morphinan or a derivative thereof is recovered. In some instances, the morphinan is recovered from a cell culture. In still other embodiments, the recovered morphinan 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%.

[0234] In some examples, bisbenzyl isoquinoline or a derivative thereof is recovered. In some examples, bisbenzyl isoquinoline is recovered from a cell culture. In still other embodiments, the recovered bisbenzyl isoquinoline is enantiomerically enriched in one stereoisomer compared to the original mixture of bisbenzyl isoquinoline presented to the enzyme. In still other embodiments, the recovered bisbenzyl isoquinoline 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%.

[0235] In some instances, nal-opioids or derivatives thereof are recovered. In some instances, nal-opioids are recovered from cell cultures. In still other embodiments, the recovered nal-opioids have 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%.

[0236] In some examples, nor - opioid substances or their derivatives are recovered. In some examples, nor - opioid substances are recovered from cell cultures. In still further embodiments, the recovered nor - opioid substances have 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%.

[0237] "Isomers" are different compounds with the same molecular formula. "Stereoisomers" are isomers that differ only in the way the 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. "Diastereomers" or "diastereoisomers" are stereoisomers that have at least two asymmetric atoms but are not mirror images of each other. As used herein, the term "epimer" refers to compounds that have the same chemical formula, but have different optical configurations at a specific position. For example, the (R,S) and (S,S) stereoisomers of a compound are epimers of each other. In some examples, 1 - benzylisoquinoline alkaloids are converted to their epimers (e.g., epi - 1 - benzylisoquinoline alkaloids). Absolute stereochemistry is specified according to the Cahn - Ingold - Prelog R - S system. When a compound is a pure enantiomer, the stereochemistry at each chiral carbon can be designated as R or S. A resolved compound of unknown absolute configuration can be designated as (+) or (-) according to the direction (right - or left - handed) in which it rotates plane - polarized light at the sodium D - line wavelength. Certain compounds described herein contain one or more asymmetric centers and can therefore give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined as (R) - or (S) - in terms of absolute stereochemistry. Table 1. Exemplary amino acid sequences of DRS - DRR enzymes, resolved DRS and DRR enzymes, and other nucleotide sequences. Morphinan alkaloid generation modification

[0238] Some methods, processes, and systems provided herein describe the conversion of protomorphinan alkaloids to morphinan alkaloids. Some of these methods, processes, and systems describe the conversion of a tetracyclic scaffold to a pentacyclic scaffold ( Figure 4 ). Some of the methods, processes, and systems can include engineered host cells. In some examples, the production of pentacyclic thebaine or morphinan alkaloids from tetracyclic precursors or protomorphinan alkaloids is described. In some examples, the conversion of protomorphinan alkaloids to thebaine is a key step in the conversion of substrates to a wide range of benzylisoquinoline alkaloids.

[0239] In some examples, the tetracyclic precursor can be salutaridine, salutaridinol, or salutaridinol-7-O-acetate. The tetracyclic precursor can be converted to pentacyclic thebaine by closing the oxide bridge between C-4 and C-5. In some examples, the tetracyclic precursor salutaridine for ring closure can be prepared by stepwise hydroxylation and O-acetylation at C-7. The ring closure can be activated by elimination of the acetate leaving group. In some examples, the allylic elimination and oxide ring closure to form thebaine occur spontaneously. In other examples, factors such as pH or solvent promote the ring closure reaction to form pentacyclic thebaine. In other examples, the ring closure reaction to form thebaine is promoted by contact with a protein or enzyme. These conversion steps are provided in Figure 4 and are generally shown 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 provided in Figure 4 . Scheme 2 In some examples, the first enzyme for preparing the tetracyclic precursor is salutaridine reductase (SalR). In some cases, SalR hydroxylates the substrate salutaridine at the C-7 position (see Formula III). The product of this reaction can be one or more salutaridine alcohol epimers. In some examples, the product is (7S)-salutaridine alcohol. In some examples, salutaridine reductase can catalyze a reduction reaction within a host cell, such as an engineered host cell, as described herein. In some examples, the second enzyme for preparing the tetracyclic precursor is salutaridine 7-O-acetyltransferase (SalAT). In some cases, SalAT transfers an acetyl group from acetyl coenzyme A to the 7-OH of salutaridine alcohol (see Formula IV). In other cases, SalAT can utilize a novel cofactor, such as propionyl coenzyme A, and transfer a propionyl group to the 7-OH of salutaridine alcohol. In some examples, the product of SalAT is (7S)-salutaridine alcohol-7-O-acetate. In some examples, salutaridine 7-O-acetyltransferase can catalyze an acetyl transfer reaction within a host cell, such as an engineered host cell, as described herein.

[0240] In some examples, the tetracyclic precursor of thebaine is (7S)-salutaridine alcohol-7-O-acetate. In some examples, (7S)-salutaridine alcohol-7-O-acetate is unstable and spontaneously eliminates the acetate at C-7 and closes the oxide bridge between C-4 and C-5 to form thebaine (see Formula V). In some examples, the elimination rate of the acetate leaving group is promoted by pH. In some examples, the allylic elimination and oxide bridge closure are catalyzed by an enzyme with thebaine synthase activity or thebaine synthase. In some examples, this enzyme is a Bet v 1 fold protein. In some examples, this enzyme is an engineered thebaine synthase, an engineered SalAT, a dirigent (DIR) protein, or a chalcone isomerase (CHI). In some examples, the enzyme encoding thebaine synthase activity can catalyze a ring closure reaction within a host cell, such as an engineered host cell, as described herein.

[0241] In some examples, the salutaridine reductase can be SalR or a SalR-like enzyme in plants of the order Ranunculales (e.g., Papaver somniferum) that biosynthesize thebaine. In other examples, the enzyme with salutaridine reductase activity can be from mammals or any other vertebrate or invertebrate that biosynthesize endogenous morphine.

[0242] In some examples, salutaridinol 7-O-acetyltransferase can be a SalAT or SalAT-like enzyme in Ranunculales plants (e.g., Papaver somniferum) that biosynthesize thebaine. In other examples, an enzyme having salutaridinol 7-O-acetyltransferase activity can be from a mammal or any other vertebrate or invertebrate that biosynthesizes endogenous morphine.

[0243] In some examples, thebaine synthase (TS) enzyme can be a Bet v 1-fold protein from Ranunculales plants (e.g., Papaver somniferum) that biosynthesize thebaine. In some examples, the Bet v 1 protein includes the following domains in order from the N-terminus to the C-terminus: a beta strand, one or two alpha helices, six beta strands, and one or two alpha helices. The protein is organized such that it has a Bet v 1 fold and an active site that accepts large, bulky hydrophobic molecules such as morphinan alkaloids. This protein can be any plant Bet v 1 protein, pathogenesis-related 10 protein (PR-10), major latex protein (MLP), fruit or pollen allergen, plant hormone-binding protein (e.g., binds to cytokinin or brassinosteroid), plant polyketide cyclase-like protein, or norcoclaurine synthase (NCS)-related protein having a Bet v 1 fold. Other non-plant examples of Bet v 1-fold proteins are polyketide cyclase, Hsp90 ATPase activator homolog 1 (AHA1) protein, SMU440-like protein (e.g., from Streptococcus mutans), PA1206-related protein (e.g., from Pseudomonas aeruginosa), CalC calicheamicin resistance protein (e.g., from Micromonospora echinospora), and CoxG protein from Oligotropha carboxidovorans that metabolizes carbon monoxide. Other examples from the Bet v 1-related family include START lipid transfer proteins, phosphatidylinositol transfer proteins, and cyclooxygenases.

[0244] In some examples, thebaine synthase can be a dirigent protein from Ranunculales plants (e.g., Papaver somniferum) that biosynthesize thebaine. In other examples, the enzyme can be any dirigent protein from a plant.

[0245] In some examples, thebaine synthase can be a chalcone isomerase protein from Ranunculales plants (e.g., Papaver somniferum) that biosynthesize thebaine. In other examples, the enzyme can be any chalcone isomerase protein from a plant.

[0246] In some examples, the thebaine synthase can be a SalAT-like enzyme from a Ranunculales plant (e.g., Papaver somniferum) that biosynthesizes thebaine. In other examples, the enzyme can be any SalAT-like protein from a plant.

[0247] In some examples, the enzyme having thebaine synthase activity can be from a mammal or any other vertebrate or invertebrate that biosynthesizes endogenous morphine.

[0248] In some examples, the combination of the above enzymes with additional accessory proteins can function to convert a variety of tetracyclic precursors into thebaine. In some examples, these enzymes catalyze reactions within a host cell (such as an engineered host cell) as described herein.

[0249] Examples of the amino acid sequences of thebaine synthase activity are shown in Table 2. The amino acid sequence of thebaine synthase utilized in the tetracyclic precursor of thebaine can be 50% or more identical to the given amino acid sequences listed in Table 2. For example, the amino acid sequence of such thebaine synthase can comprise at least 50% more, 55% more, 60% more, 65% more, 70% more, 75% more, 80% more, 81% more, 82% more, 83% more, 84% more, 85% more, 86% more, 87% more, 88% more, 89% more, 90% more, 91% more, 92% more, 93% more, 94% more, 95% more, 96% more, 97% more, 98% more or 99% or more identical to the amino acid sequences provided herein. Additionally, in certain embodiments, an "identical" amino acid sequence has at least 80%-99% identity to a specific amino acid sequence at the amino acid level. In some cases, an "identical" amino acid sequence contains at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% and more (in some cases, at least 95%, 96%, 97%, 98% and 99%) identity at the amino acid level. In some cases, for example, the amino acid sequences can be identical, but the nucleic acid sequences encoding the amino acid sequences are altered, such as to optimize codon usage for a host organism.

[0250] Engineered host cells can be provided that produce salutaridine reductase, salutaridinol 7-O-acetyltransferase, and thebaine synthase that converts a tetracyclic precursor into thebaine, wherein thebaine synthase comprises an amino acid sequence selected from SEQ ID NO:19, 20, 21, 22, 23, 24, 25, and 26 as listed in Table 2. In some cases, thebaine synthase can form a fusion protein with other enzymes. The enzymes produced within the engineered host cells can be recovered and purified to form a biocatalyst. The one or more enzymes can also be used to catalyze the conversion of a tetracyclic protopine precursor into thebaine.

[0251] In other examples, the thebaine synthase comprises an amino acid sequence selected from SEQ ID NO:27, 28, 29, 30, 31, 32, 33, and 34 as listed in Table 2.

[0252] In other cases, one or more enzymes recovered from the engineered host cell can be used in the process of converting the tetrahydroprotoberberine precursor to thebaine. The process can include contacting the tetrahydroprotoberberine precursor with an amount of the recovered enzyme sufficient to convert the tetrahydroprotoberberine precursor to thebaine. In some examples, the tetrahydroprotoberberine precursor can be contacted with a sufficient amount of the one or more enzymes such that at least 5% of the tetrahydroprotoberberine precursor is converted to thebaine. In other examples, the tetrahydroprotoberberine 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 tetrahydroprotoberberine precursor is converted to thebaine.

[0253] In some examples, process conditions are implemented to support the formation of thebaine in the engineered host cell. In some cases, the engineered host cell is grown at pH 3.3 and once a high cell density is reached, the pH is adjusted to pH 8.0 to support continued thebaine production at the higher pH. In some cases, the engineered host cell produces additional enzymes to convert sugars and other simple precursors (such as tyrosine) to thebaine. In some cases, the SalAT enzyme has been engineered to exhibit higher activity at pH 8.0 and is expressed from a late promoter.

[0254] In some examples, one or more enzymes that convert the tetracyclic protopine precursor to thebaine are localized to a cellular compartment. In some examples, SalR, SalAT, and thebaine synthase (TS) can be modified such that they encode targeting sequences that localize them to the endoplasmic reticulum membrane of an engineered host cell. In particular, in certain instances, the host cell can be engineered to increase the production of salutaridinol or thebaine or a product having thebaine as a precursor from reticuline or its precursor by localizing TS and / or SalR and / or SalAT to an organelle in a yeast cell. TS and / or SalR and / or SalAT can be localized to the yeast endoplasmic reticulum to reduce the spatial distance between TS and / or SalR and / or SalAT and CYP2D2 or CYP2D6 or SalSyn or an engineered cytochrome P450 enzyme that catalyzes the conversion of reticuline to salutaridine. An increase in production means either of two situations: producing a certain amount of the desired compound when the control does not produce the desired compound, and increasing by 10% or more, such as 50% or more, including 2-fold or more, for example 5-fold or more, such as 10-fold or more, when the control has some production of the desired compound.

[0255] In other examples, SalAT and TS can be co-localized into a single protein fusion. In some examples, a fusion is created between SalAT and TS by one of several methods, including direct fusion, co-localization to a yeast organelle, or by an enzyme co-localization tool (such as a leucine zipper, a protein scaffold using an adaptor domain, or an RNA scaffold using an aptamer). Co-localizing thebaine synthase can facilitate substrate channeling between the active sites of the enzymes and limit the diffusion of unstable intermediates (such as salutaridinol-7-O-acetate).

[0256] In some examples, an engineered salutaridinol 7-O-acetyltransferase (SalAT) enzyme is used to convert the tetracyclic protopine precursor to thebaine. In some examples, the SalAT enzyme is engineered to combine two functions: (1) transfer of an acyl group from acetyl coenzyme A to the 7-OH of salutaridinol, and (2) subsequent elimination of the acetyl group and closing of the oxide bridge between carbons C4 and C5 to form thebaine.

[0257] In some examples, an enzyme having salutaridinol 7-O-acetyltransferase activity is fused with a peptide having a Bet v 1 fold. In some examples, the salutaridinol 7-O-acetyltransferase and the Bet v 1 fold protein can be fused in any order from N-terminus to C-terminus, C-terminus to N-terminus, N-terminus to N-terminus, or C-terminus to C-terminus. In some examples, the two protein sequences can be directly fused or fused through a peptide linker region.

[0258] In some examples, an enzyme having salutaridine 7-O-acetyltransferase activity is fused to a peptide having 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 within this sequence. In some cases, the resulting fusion protein library is screened for the production of thebaine. 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 cloned. In other cases, this circularly permuted SalAT library is screened for salutaridine 7-O-acetyltransferase activity. In other cases, active variants from the circularly permuted SalAT library are then used to design protein fusions with peptides having a Bet v 1 fold.

[0259] One or more enzymes that can be used to convert a tetracyclic protopine precursor to thebaine can be contacted with the tetracyclic protopine precursor in vitro. Additionally or alternatively, one or more enzymes that can be used to convert a tetracyclic protopine precursor to thebaine can be contacted with the tetracyclic protopine precursor in vivo. Additionally, one or more enzymes that can be used to convert a tetracyclic protopine precursor to thebaine can be provided to a cell in which the tetracyclic protopine precursor is present, or can be produced within an engineered host cell.

[0260] In some examples, the method provides engineered host cells that produce an alkaloid product, wherein the conversion of the tetracyclic protopine precursor to thebaine can constitute a key step in the production of the alkaloid product. In some examples, the alkaloid product is thebaine. In still other embodiments, the alkaloid product is derived from thebaine, including, for example, downstream morphinan alkaloids. In another embodiment, the tetracyclic protopine precursor is an intermediate in the product of the engineered host cell. In still other embodiments, the alkaloid product is selected from morphinan, nor - opioid, or nal - opioid alkaloids.

[0261] In some examples, the substrate of the reduction reaction is a compound of formula III: Formula III, or a salt thereof, wherein: R1, R2, and R3 are independently selected from hydrogen and methyl.

[0262] In some other examples, R1, R2, and R3 are methyl, and the reduction reaction is catalyzed by salutaridine reductase.

[0263] In some examples, the substrate of the carbon chain transfer reaction is a compound of formula IV: Formula IV or a salt thereof, wherein: R1, R2 and R3 are independently selected from hydrogen and methyl.

[0264] In some other examples, R1, R2 and R3 are methyl, and the carbon chain transfer reaction is catalyzed by salutaridine 7-O-acetyltransferase.

[0265] In some examples, the substrate of the thebaine synthase is a compound of formula V: or a salt thereof, wherein: R1, R2 and R3 are independently selected from hydrogen and methyl; and R4 is selected from methyl, ethyl, propyl and other suitable alkyl groups.

[0266] 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. In some examples, the method provides engineered host cells for producing alkaloid products from salutaridine. The conversion of salutaridine to thebaine can constitute a key step in producing various alkaloid products from precursors. In some examples, the precursor is l-tyrosine or a sugar (e.g., glucose). The various alkaloid products can include, but are not limited to, morphinans, nor-opiates or nal-opiates alkaloids. Any suitable carbon source can be used as a precursor for the 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, an unpurified mixture from renewable raw materials (e.g., corn steep liquor, beet molasses, barley malt, biomass hydrolysate) can be used. 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 can be utilized, such as, methylamine, glucosamine and amino acids (e.g., l-tyrosine). In some examples, a 1-benzylisoquinoline alkaloid can be directly added to the engineered host cells of the present disclosure, and the 1-benzylisoquinoline alkaloid includes, for example, norlaudanosoline, laudanosoline, norreticuline and reticuline.

[0267] In some examples, the benzylisoquinoline alkaloid product or its derivative 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-opiate or nal-opiate alkaloid. Table 2. Exemplary amino acid sequences of morphinan alkaloid - generating enzymes. Morphinan alkaloid isomerization modification

[0268] Some of the methods, processes, and systems provided herein describe the production of morphinan alkaloid isomers. Some methods, processes, and systems describe the conversion of a precursor morphinan alkaloid having a carbon-carbon double bond between carbons C-14 and C-8 to a product morphinan alkaloid having a carbon-carbon double bond between carbons C-8 and C-7( Figure 4 ). Some of the methods, processes, and systems can include engineered host cells. In some examples, the conversion of a precursor morphinan alkaloid having a carbon-carbon double bond between carbons C-14 and C-8 to a product precursor morphinan alkaloid having a carbon-carbon double bond between carbons C-8 and C-7 is an important step in converting the precursor to a wide range of benzylisoquinoline alkaloids.

[0269] In some examples, the production of a precursor morphinan alkaloid having a carbon-carbon double bond between carbons C-14 and C-8 occurs within an engineered host cell that contains a plurality of heterologous enzymes for converting simple starting materials to the precursor morphinan alkaloid. In some examples, the simple starting materials are sugars and / or L-tyrosine.

[0270] In some examples, the isomeric precursor morphinan alkaloid can be neopinone, neopine, neomorphine, or neomorphinone. The precursor morphinan alkaloid can be converted to the desired isomer by rearrangement of the carbon-carbon double bonds between carbons C-14 and C-8 and between carbons C-8 and C-7. In some cases, examples of products formed by isomerization can be codeinone, codeine, morphine, or morphinone. In some examples, the rearrangement to form the desired isomer occurs spontaneously. In other examples, the rearrangement to form the desired isomer is facilitated by factors such as pH and solvent. In other examples, the carbon-carbon double bond is transposed by contact with a protein or an enzyme. The isomerization conversion step is provided in Figure 4 and is generally shown in Scheme 3. R1, R2, R3, and R4 can be O, OH, H, CH3, or other suitable alkyl groups. Scheme 3

[0271] In some examples, the first enzyme for generating isomeric precursor morphinan alkaloids is thebaine 6-O-demethylase (T6ODM). In some cases, T6ODM O-demethylates the substrate thebaine at the C-6 position. In some examples, the product of this reaction is neopinone. In some examples, T6ODM can catalyze the O-demethylation reaction within a host cell, such as an engineered host cell, as described herein.

[0272] In some examples, the isomeric precursor morphinan alkaloid is neopinone. In some examples, neopinone undergoes isomerization to codeinone. In some examples, the partitioning of neopinone to codeinone can reach equilibrium in an aqueous solution such that neopinone and codeinone exist at steady-state concentrations. In some examples, the rate of conversion of neopinone to codeinone is promoted by pH. In some examples, the rearrangement of neopinone to codeinone is catalyzed by an enzyme having neopinone isomerase activity. In some examples, this enzyme is a Bet v 1-folded protein. In some examples, this enzyme is neopinone isomerase (NPI). In some examples, this enzyme is an engineered protein with an N-terminal sequence truncation. In some examples, NPI can catalyze the isomerization reaction within a host cell, such as an engineered host cell, as described herein.

[0273] In some examples, the enzyme acting on codeinone is codeinone reductase (COR). In some cases, COR reduces the ketone at the C-6 position of codeinone to form a hydroxyl group. In some examples, the product of this reaction is codeine. In some examples, COR is selected from multiple gene duplication and alternative splicing isoforms to exhibit the highest activity when paired with a protein encoding neopinone isomerase activity. In some examples, COR can catalyze the reduction reaction within a host cell, such as an engineered host cell, as described herein.

[0274] In some examples, the enzyme acting on codeinone is morphinone reductase (morB). In some cases, morB saturates the carbon-carbon double bond between C-7 and C-8 of codeinone. In some examples, the product of this reaction is hydrocodone. In some examples, morB can catalyze the reduction reaction within a host cell, such as an engineered host cell, as described herein.

[0275] In some examples, the thebaine 6-O-demethylase can be a T6ODM or T6ODM-like enzyme from a Ranunculales plant (e.g., Papaver somniferum) that biosynthesizes morphine. In some examples, the T6ODM can be a T6ODM-like enzyme from a plant that biosynthesizes benzylisoquinoline alkaloids (e.g., Papaver bracteatum, Papaver rhoeas, Papaver nudicaule, and Papaver orientale). In some examples, the plant enzyme is a 2-oxoglutarate / Fe(II)-dependent dioxygenase that uses 2-oxoglutarate and oxygen and generates succinate and carbon dioxide when demethylating thebaine to produce neopinone. In some examples, the T6ODM can also demethylate orientaline to produce neomorphinone.

[0276] In other examples, the enzyme having thebaine 6-O-demethylase activity can be from a mammal or another vertebrate or invertebrate that biosynthesizes endogenous morphinan alkaloids.

[0277] In some examples, the neopinone isomerase (NPI) enzyme can be a Bet v 1-folded protein from a Ranunculales plant (e.g., Papaver somniferum) that biosynthesizes morphine. In some examples, the NPI can be an NPI-like enzyme from a plant that biosynthesizes benzylisoquinoline alkaloids (e.g., Papaver bracteatum, Papaver rhoeas, Papaver nudicaule, and Papaver orientale). In some examples, the Bet v 1 protein includes the following domains in order 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. In some examples, truncation is performed at the N-terminus of the enzyme to remove all or part of the first domain. In some examples, the enzyme can have one or more activity-enhancing components as discussed herein and as described in Examples 6 and 7. In some examples, 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. In some examples, the protein can be any plant Bet v 1 protein, pathogenesis-related 10 protein (PR-10), major latex protein (MLP), fruit or pollen allergen, plant hormone-binding protein (e.g., binds to cytokinin or brassinosteroid), plant polyketide cyclase-like protein, or a norcoclaurine synthase (NCS)-related protein having a Bet v 1 fold. In some examples, the function of the Bet v 1-folded protein is to catalyze a reaction that can also occur spontaneously.

[0278] In other examples, the enzyme having neopinone isomerase activity can be from a mammal or another vertebrate or invertebrate that biosynthesizes endogenous morphinan alkaloids.

[0279] In some examples, the codeinone reductase can be a COR or COR-like enzyme from a Ranunculales plant (e.g., Papaver somniferum) that biosynthesizes morphine. In some examples, the COR can be a COR-like enzyme from a plant that biosynthesizes benzylisoquinoline alkaloids (e.g., Papaver bracteatum, Papaver rhoeas, Papaver nudicaule, and Papaver orientale). In some examples, the plant enzyme is an oxidoreductase that uses NADPH as a cofactor in the reversible reduction of codeinone to codeine. In some examples, the COR enzyme is a specific gene duplication or splicing variant that has been selected to have selected kinetic parameters, such as a higher activity rate (K cat ) for one or more reactions, an improved binding affinity (K M ) for one or more substrates, an enhanced specificity for the substrate codeinone relative to neopinone, or enhanced thermal stability. In some examples, the COR enzyme can function to reduce other morphinan alkaloid substrates, such as neopinone, morphinone, neomorphinone, hydrocodone, hydromorphanone, oxycodone, oxymorphanone, 14-hydroxycodeinone, or 14-hydroxymorphinone. In some examples, the products having COR activity are neopine, morphine, neomorphine, dihydrocodeine, dihydromorphine, oxycodol, oxymorphol, 14-hydroxycodeine, or 14-hydroxymorphine.

[0280] In some examples, the morphinone reductase can be morB or a morB-like enzyme from a bacterium of the genus Pseudomonas. In some examples, the morphinone reductase can be an alkene reductase from a Gram-negative bacterium. In some examples, the bacterial enzyme is an α / β-barrel flavoprotein that uses NADH and FMN as cofactors to saturate the carbon-carbon double bond between C-7 and C-8 of codeinone. In some examples, the morB enzyme has selected kinetic parameters, such as a higher activity rate (K cat ) for one or more reactions, an improved substrate binding affinity (K M ) for one or more substrates, an enhanced specificity for one substrate, or enhanced thermal stability. The morB enzyme can also reduce other morphinan substrates, such as morphinone, neomorphinone, codeine, morphine, neopine, neomorphine, 14-hydroxycodeinone, or 14-hydroxymorphinone. Examples of products having morB activity are hydromorphanone, dihydrocodeine, dihydromorphine, oxycodone, or oxymorphanone.

[0281] In other examples, combinations of the above enzymes with additional accessory proteins can function in the production of selected morphinan alkaloid isomers. In some examples, these enzymes catalyze reactions within the host cells described herein, such as engineered host cells.

[0282] Examples of the amino acid sequences of the neopinone isomerase activity are shown in Table 3. The amino acid sequence of the neopinone isomerase for converting a precursor morphinan alkaloid having a carbon-carbon double bond between carbons C-14 and C-8 to a product morphinan alkaloid having a carbon-carbon double bond between carbons C-8 and C-7 can be 50% or more identical to the given amino acid sequence listed in Table 3. For example, the amino acid sequence of such neopinone isomerase can comprise an amino acid sequence that is at least 50% more, 55% more, 60% more, 65% more, 70% more, 75% more, 80% more, 81% more, 82% more, 83% more, 84% more, 85% more, 86% more, 87% more, 88% more, 89% more, 90% more, 91% more, 92% more, 93% more, 94% more, 95% more, 96% more, 97% more, 98% more or 99% or more identical to the amino acid sequence provided herein. Additionally, in certain embodiments, the "identical" amino acid sequence has at least 80%-99% identity to a specific amino acid sequence at the amino acid level. In some cases, the "identical" amino acid sequence contains at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% and more (in some cases, at least 95%, 96%, 97%, 98% and 99%) identity at the amino acid level. In some cases, for example, the amino acid sequences can be identical, but the nucleic acid sequences encoding the amino acid sequences are altered, such as to optimize codon usage for a host organism.

[0283] Engineered host cells can be provided that produce thebaine 6-O-demethylase, neopinone isomerase, and codeinone reductase, which convert a precursor morphinan alkaloid isomer to a desired product morphinan alkaloid isomer by rearrangement of the carbon-carbon double bonds between carbons C-14 and C-8 and between carbons C-8 and C-7, wherein the neopinone isomerase comprises an amino acid sequence selected from SEQ ID NO:54, 55, 56, 57, and 58. In some cases, the neopinone isomerase can physically interact with one or more pathway enzymes. In some cases, the physical interaction can alter the activity of the one or more pathway enzymes. In some cases, the neopinone isomerase can form a fusion protein with one or more other enzymes. The enzymes produced within the engineered host cells can be recovered and purified to form a biocatalyst. These one or more enzymes can also be used to catalyze the conversion of a precursor morphinan alkaloid having a carbon-carbon double bond between carbons C-14 and C-8 to a product morphinan alkaloid having a carbon-carbon double bond between carbons C-8 and C-7.

[0284] In other examples, the neopinone isomerase comprises an amino acid sequence selected from SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, and 58 as listed in Table 3.

[0285] Examples of amino acid sequences with codeinone reductase activity are shown in Table 4. The amino acid sequence of codeinone reductase for reducing the ketone at the C-6 position of morphinan alkaloids to a hydroxyl group at that position can be 50% or more identical to the given amino acid sequences listed in Table 4. For example, the amino acid sequence of such codeinone reductase can comprise an amino acid sequence that is at least 50% more, 55% more, 60% more, 65% more, 70% more, 75% more, 80% more, 81% more, 82% more, 83% more, 84% more, 85% more, 86% more, 87% more, 88% more, 89% more, 90% more, 91% more, 92% more, 93% more, 94% more, 95% more, 96% more, 97% more, 98% more, or 99% or more identical to the amino acid sequences provided herein. Additionally, in certain embodiments, the "identical" amino acid sequence has at least 80%-99% identity to a specific amino acid sequence at the amino acid level. In some cases, the "identical" amino acid sequence contains at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, and more (in certain cases, at least 95%, 96%, 97%, 98%, and 99%) identity at the amino acid level. In some cases, for example, the amino acid sequences can be identical, but the nucleic acid sequences encoding the amino acid sequences are altered, such as to optimize codon usage for a host organism.

[0286] Engineered host cells can be provided that produce thebaine 6-O-demethylase, neopinone isomerase, and codeinone reductase, which convert a precursor morphinan alkaloid isomer to a desired product morphinan alkaloid isomer through the rearrangement of the carbon-carbon double bond between carbon C-14 and C-8 and the reduction of the ketone at the C-6 position to a hydroxyl group, wherein the codeinone reductase comprises an amino acid sequence selected from SEQ ID NO: 59, 60, 61, 62, 63, 64, 65, 66, 67, and 68 as listed in Table 4. In some cases, the codeinone reductase can interact with other enzymes or form a fusion protein with other enzymes. The enzymes produced within the engineered host cells can be recovered and purified to form a biocatalyst. These one or more enzymes can also be used to catalyze the conversion of a precursor morphinan alkaloid having a carbon-carbon double bond between carbon C-14 and C-8 to a product morphinan alkaloid having a carbon-carbon double bond between carbon C-8 and C-7.

[0287] In other cases, one or more enzymes recovered from engineered host cells can be used in the process of converting a precursor morphinan alkaloid having a carbon-carbon double bond between carbons C-14 and C-8 into a product morphinan alkaloid having a carbon-carbon double bond between carbons C-8 and C-7. The process can include contacting the precursor morphinan alkaloid isomer with an amount of the recovered enzyme sufficient to convert the precursor morphinan alkaloid isomer into the desired morphinan alkaloid isomer product. In some examples, the precursor morphinan alkaloid isomer can be contacted with a sufficient amount of the one or more enzymes such that at least 5% of the precursor morphinan alkaloid isomer is converted into the desired product morphinan alkaloid isomer. In other examples, the precursor morphinan alkaloid isomer 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 precursor morphinan alkaloid isomer is converted into the desired product morphinan alkaloid isomer.

[0288] In some examples, process conditions are implemented to support the formation of the desired product morphinan alkaloid isomer in the engineered host cell. In some cases, the engineered host cell is grown at pH 3.3 and once a high cell density is reached, the pH is adjusted to pH 6 - 6.5 to support continued production of the desired product morphinan alkaloid isomer at the higher pH. In some cases, the engineered host cell produces additional enzymes to convert sugars and other simple starting materials (such as tyrosine) into the desired product morphinan alkaloid isomer.

[0289] In some examples, one or more enzymes that convert a precursor morphinan alkaloid having a carbon-carbon double bond between carbon C-14 and C-8 to a product morphinan alkaloid having a carbon-carbon double bond between carbon C-8 and C-7 are localized to a cellular compartment. In some examples, T6ODM, COR, or morB and NPI can be modified such that they encode targeting sequences that localize them to the endoplasmic reticulum membrane of an engineered host cell. In particular, in certain instances, a host cell can be engineered to increase the production of a product morphinan alkaloid isomer or its precursor by localizing NPI and / or T6ODM and / or COR and / or morB to an organelle in a yeast cell. NPI and / or T6ODM and / or COR and / or morB can be localized to the yeast endoplasmic reticulum to reduce the spatial distance between these enzymes. An increase in yield means either of two situations: producing a certain amount of a target compound when the control does not produce the target compound, and increasing by 10% or more, such as 50% or more, including 2-fold or more, for example 5-fold or more, such as 10-fold or more, when the control has some production of the target compound.

[0290] In other examples, T6ODM and NPI can be co-localized into a single protein fusion. In other examples, COR or morB and NPI can be co-localized into a single protein fusion. In some examples, a fusion is created between the proteins by one of several methods, including direct fusion, co-localization to a yeast organelle, or by an enzyme co-localization tool (such as a leucine zipper, a protein scaffold using an adaptor domain, or an RNA scaffold using an aptamer). Co-localizing neopinone isomerase can facilitate substrate channeling between the active sites of the enzymes and limit the diffusion of unstable intermediates (such as neopinone and codeinone).

[0291] In some examples, the T6ODM enzyme is engineered for the conversion between morphinan alkaloid isomers. In some examples, the T6ODM enzyme is engineered to combine two functions: (1) O-demethylation at the C-6 position of thebaine, and (2) rearrangement of the carbon-carbon double bonds between carbon C-14 and C-8 and between carbon C-8 and C-7.

[0292] In some examples, an enzyme having thebaine 6-O-demethylase activity is fused with a peptide having a Betv 1 fold. In some examples, the thebaine 6-O-demethylase and the Bet v 1 fold protein can be fused in any order from N-terminus to C-terminus, C-terminus to N-terminus, N-terminus to N-terminus, or C-terminus to C-terminus. In some examples, the two protein sequences can be directly fused or fused through a peptide linker region.

[0293] In some examples, an enzyme with thebaine 6-O-demethylase activity is fused with a peptide having a Bet v 1 fold by circular permutation. In some cases, the N-terminus and C-terminus of T6ODM are fused, and then the Bet v 1 sequence is randomly inserted within this sequence. In some cases, the resulting fusion protein library is screened to produce the desired morphinan alkaloid isomer product. In other cases, the circularly permuted T6ODM library is first screened for activity in the absence of Bet v 1. In other cases, the N-terminus and C-terminus of T6ODM are fused, and the enzyme is digested and blunt cloned. In other cases, this circularly permuted T6ODM library is screened for thebaine 6-O-demethylase activity. In other cases, then the active variants from the circularly permuted T6ODM library are used to design a protein fusion with a peptide having a Bet v 1 fold.

[0294] In some examples, an engineered COR enzyme or morB enzyme is used for the conversion between morphinan alkaloid isomers. In some examples, the COR or morB enzyme is engineered to combine two functions: (1) the rearrangement of the carbon-carbon double bond between carbon C-14 and C-8 and between carbon C-8 and C-7, and (2) the reduction of the morphinan alkaloid isomer product.

[0295] In some examples, an enzyme with opioid reductase activity is fused with a peptide having a Bet v 1 fold. In some examples, the COR or morB enzyme and the Bet v 1 fold protein can be fused in any order from N-terminus to C-terminus, C-terminus to N-terminus, N-terminus to N-terminus, or C-terminus to C-terminus. In some examples, the two protein sequences can be directly fused or fused through a peptide linker region.

[0296] In some examples, an enzyme with opioid reductase activity is fused with a peptide having a Bet v 1 fold by circular permutation. In some cases, the N-terminus and C-terminus of COR or morB are fused, and then the Bet v 1 sequence is randomly inserted within this sequence. In some cases, the resulting fusion protein library is screened to produce the desired morphinan alkaloid isomer product. In other cases, the circularly permuted COR or morB library is first screened for activity in the absence of Bet v 1. In other cases, the N-terminus and C-terminus of COR or morB are fused, and the enzyme is digested and blunt cloned. In other cases, this circularly permuted COR or morB library is screened for opioid reductase activity. In other cases, then the active variants from the circularly permuted COR or morB library are used to design a protein fusion with a peptide having a Bet v1 fold.

[0297] One or more enzymes that can be used to convert a precursor morphinan alkaloid having a carbon-carbon double bond between carbons C-14 and C-8 into a product morphinan alkaloid having a carbon-carbon double bond between carbons C-8 and C-7 can be contacted with the precursor morphinan alkaloid isomers in vitro. Additionally or alternatively, one or more enzymes that can be used to convert a precursor morphinan alkaloid having a carbon-carbon double bond between carbons C-14 and C-8 into a product morphinan alkaloid having a carbon-carbon double bond between carbons C-8 and C-7 can be contacted with the precursor morphinan alkaloid isomers in vivo. Further, one or more enzymes that can be used to convert a precursor morphinan alkaloid having a carbon-carbon double bond between carbons C-14 and C-8 into a product morphinan alkaloid having a carbon-carbon double bond between carbons C-8 and C-7 can be provided to a cell having the precursor morphinan alkaloid isomers therein, or can be produced within an engineered host cell.

[0298] In some examples, the method provides engineered host cells that produce an alkaloid product, wherein the conversion of a precursor morphinan alkaloid having a carbon-carbon double bond between carbons C-14 and C-8 into a product morphinan alkaloid having a carbon-carbon double bond between carbons C-8 and C-7 can constitute an important step in the production of the alkaloid product. In some examples, the alkaloid product is codeinone. In still other embodiments, the alkaloid product is derived from codeinone, including, for example, downstream morphinan alkaloids. In another embodiment, the precursor morphinan alkaloid having a carbon-carbon double bond between carbons C-14 and C-8 is an intermediate of the product of the engineered host cell. In still other embodiments, the alkaloid product is selected from morphinan, nor-opioid, or nal-opioid alkaloids.

[0299] In some examples, the substrate for the O-demethylation reaction is a compound of formula VI: or a salt thereof, wherein: R1 and R2 are independently selected from hydrogen and methyl.

[0300] In some other examples, R1 and R2 are methyl, and the O-demethylation reaction is catalyzed by thebaine 6-O-demethylase. Other examples of 6-O-demethylation reactions are provided in Figure 9 .

[0301] In some examples, the substrate for the isomerization reaction is a compound of formula VII: or a salt thereof, wherein: R1 and R3 are independently selected from hydrogen and methyl, and R2 is independently selected from hydroxy and oxygen.

[0302] In some other examples, R1 and R3 are methyl, and R2 is oxygen, and the isomerization reaction is catalyzed by neopinone isomerase. Other examples of the isomerization reaction are provided in Figure 14 .

[0303] In some examples, the substrate for the reduction reaction is a compound of formula VIII: or a salt thereof, wherein: R1 and R3 are independently selected from hydrogen and methyl; and R2 is independently selected from hydroxyl and oxygen.

[0304] In some other examples, R1 and R3 are methyl and R2 is oxygen, and the reduction reaction is catalyzed by codeinone reductase. In some other examples, the reduction reaction is catalyzed by morphinone reductase. Other examples of the reduction reaction are provided in Figure 12 and Figure 13 .

[0305] In some examples, the method provides engineered host cells that produce morphinan alkaloids from neopinone. The conversion of neopinone to codeinone can constitute an important step in the production of a variety of morphinan alkaloid products from simple starting materials. In some examples, the simple starting material is l-tyrosine or a sugar (e.g., glucose). The various alkaloid products can include, but are not limited to, morphinans, nor-opiates, or nal-opiate alkaloids.

[0306] In some examples, the engineered host cells are grown by a fed-batch fermentation process, wherein the simple starting material is fed over time and continuously converted to precursor morphinan alkaloids in the engineered host cells over time, thereby providing a constant source of precursor morphinan alkaloids. In some examples, the continuous source of precursor morphinan alkaloids is continuously isomerized to product morphinan alkaloid isomers over time and then converted to downstream alkaloid products by one or more enzymes acting on the morphinan alkaloid isomers in the engineered host cells, thereby providing a constant pull of product isomers to downstream alkaloid products. In some examples, the dynamic system process (e.g., the continuous supply of precursor morphinan alkaloids and the continuous conversion of product morphinan alkaloid isomers to downstream alkaloid products) is a beneficial component for achieving an increased production of the desired alkaloid product through enhanced reversible isomerization reactions.

[0307] In some cases, the pairing of neopinone isomerase with a COR variant that exhibits specific kinetic properties is a beneficial component for achieving an increased production of the desired alkaloid product in the engineered host cells. In some cases, the pairing of neopinone isomerase with a morB variant that exhibits specific kinetic properties is a beneficial component for achieving an increased production of the desired alkaloid product in the engineered host cells.

[0308] Any suitable carbon source can be used as a starting material for morphinan alkaloids. Suitable precursors can include, but are not limited to, simple starting materials such as 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 from renewable feedstocks (e.g., corn steep liquor, beet molasses, barley malt, biomass hydrolysates) can be used. 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 can be utilized, such as, for example, methylamine, glucosamine, and amino acids (e.g., L-tyrosine).

[0309] In some examples, benzylisoquinoline alkaloid products or their derivatives are recovered. In some examples, benzylisoquinoline alkaloid products are recovered from cell cultures. In some examples, the benzylisoquinoline alkaloid product is a morphinan, a nor - opioid, or a nal - opioid alkaloid. Table 3. Exemplary amino acid sequences of morphinan alkaloid isomerases. Table 4. Exemplary amino acid sequences of morphinan alkaloid reductases. Benzylisoquinoline alkaloid generation modification

[0310] Some of the methods, processes, and systems provided herein describe the conversion of BIA precursors to 1 - benzylisoquinoline alkaloids. Some of these methods, processes, and systems can include engineered host cells. In some examples, the production of norcoclaurine or 1 - benzylisoquinoline alkaloids from 4 - HPAA and dopamine or BIA precursors is described. In some examples, the production of norlaudanosoline or 1 - benzylisoquinoline alkaloids from 3,4 - DHPA and dopamine or BIA precursors is described. In some examples, the conversion of BIA precursors to 1 - benzylisoquinoline alkaloids is a key step in the conversion of substrates to a wide range of benzylisoquinoline alkaloids.

[0311] In some examples, the BIA precursor can be 4 - HPAA and dopamine. In some examples, the BIA precursor can be 3,4 - DHPA and dopamine. In some cases, a condensation reaction between two BIA precursors occurs between the amine of the first substrate and the aldehyde of the second substrate to generate an iminium ion, followed by the formation of a carbon - carbon bond between C - 6 of the first substrate and C - 1 of the second substrate, as provided in Figure 1in and generally shown in Scheme 4. As provided in Scheme 4, R1, R2, R3, and R4 can be H or OH. Scheme 4

[0312] In some examples, the condensation of the BIA precursor with the 1-benzylisoquinoline alkaloid product can occur simultaneously. In some examples, the condensation reaction is facilitated by conditions such as pH or solvent. In other examples, the Pictet-Spengler cyclization reaction to generate the 1-benzylisoquinoline alkaloid is by contact with a protein or enzyme having norcoclaurine synthase activity or norcoclaurine synthase. In some examples, this enzyme is a Bet v 1-folded protein. In some examples, this enzyme is an engineered norcoclaurine synthase. In some examples, this enzyme is an engineered norcoclaurine synthase with an N-terminal sequence truncated. In some examples, the enzyme encoding norcoclaurine synthase activity can catalyze the condensation reaction within a host cell (such as an engineered host), as described herein.

[0313] In some examples, the norcoclaurine synthase can be a Bet v 1-folded protein from a Ranunculales plant (e.g., Papaver somniferum) that biosynthesizes thebaine. In some examples, the norcoclaurine synthase can be a Bet v 1-folded protein from a Ranunculales plant (e.g., Camellia japonica or Eschscholzia californica) that biosynthesizes benzylisoquinoline alkaloids. In some examples, the Bet v 1 protein includes the following domains in the order 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. In some examples, truncation is performed at the N-terminus of the enzyme to remove all or part of the first domain. In some examples, the enzyme can have one or more components that increase activity, as discussed herein and as described in Examples 14, 15, and 16. The protein is organized such that it has a Bet v 1 fold and an active site that accepts large, bulky hydrophobic molecules such as 1-benzylisoquinoline alkaloids. This protein can be any plant Bet v 1 protein.

[0314] In some examples, the enzyme having norcoclaurine synthase activity can be from a mammal or any other vertebrate or invertebrate that biosynthesizes endogenous morphine.

[0315] In some examples, the norcoclaurine synthase can be combined with additional accessory proteins and can function to convert any BIA precursor to a 1-benzylisoquinoline alkaloid. In some examples, these enzymes catalyze reactions within a host cell (such as an engineered host), as described herein.

[0316] Examples of the amino acid sequences of norcoclaurine synthase are shown in Table 5. The amino acid sequences of norcoclaurine synthase utilized in the conversion of the BIA precursor to 1-benzylisoquinoline alkaloids can be 75% or more identical to the given amino acid sequences listed in Tables 6, 7, and 8. For example, the amino acid sequences of such norcoclaurine synthase can comprise at least 75% more, 80% more, 81% more, 82% more, 83% more, 84% more, 85% more, 86% more, 87% more, 88% more, 89% more, 90% more, 91% more, 92% more, 93% more, 94% more, 95% more, 96% more, 97% more, 98% more, or 99% or more identical to the amino acid sequences provided herein. Additionally, in certain embodiments, an "identical" amino acid sequence has at least 80%-99% identity to a particular amino acid sequence at the amino acid level. In some cases, an "identical" amino acid sequence contains at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, and more (in some cases, at least 95%, 96%, 97%, 98%, and 99%) identity at the amino acid level. In some cases, for example, the amino acid sequences can be identical, but the nucleic acid sequences encoding the amino acid sequences are altered, such as to optimize codon usage for the host organism.

[0317] Amino acid residues of homologous norcoclaurine synthase can be referenced according to the numbering scheme of SEQ ID NO.70, and this numbering system is used throughout the disclosure to refer to specific amino acid residues of norcoclaurine synthase homologous to SEQ ID NO.70. Norcoclaurine synthase homologous to SEQ ID NO.70 can have at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO.70. In some cases, the amino acid referred to as position 50 in the homologous norcoclaurine synthase may not be the 50th amino acid in the homologous norcoclaurine synthase, but will be the amino acid corresponding to the amino acid at position 50 in SEQ ID NO.70 in a protein alignment of the homologous norcoclaurine synthase with SEQ ID NO.70. In some cases, homologous enzymes can be aligned with SEQ ID NO.70 according to primary sequence, secondary structure, or tertiary structure.

[0318] Engineered host cells can be provided that produce an engineered norcoclaurine synthase that converts a BIA precursor to a 1-benzylisoquinoline alkaloid, wherein the engineered norcoclaurine synthase comprises an amino acid sequence selected from SEQ ID NO: 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, and 82 and has one or more modifications that enhance activity as described in Tables 6, 7, and 8. The engineered norcoclaurine synthase produced in the engineered host cells can be recovered and purified to form a biocatalyst. In some cases, the engineered norcoclaurine synthase can have an N-terminal truncation. These engineered norcoclaurine synthases can also be used to catalyze the conversion of a BIA precursor to a 1-benzylisoquinoline alkaloid. Additionally, the use of the engineered norcoclaurine synthase can be used to increase the production of the 1-benzylisoquinoline alkaloid product intracellularly when compared to the production of the benzylisoquinoline alkaloid product intracellularly using the parental norcoclaurine synthase.

[0319] In additional cases, one or more enzymes recovered from the engineered host cells that produce norcoclaurine synthase can be used in the process of converting a BIA precursor to a 1-benzylisoquinoline alkaloid. The process can include contacting the BIA precursor with the recovered enzymes in an amount sufficient to convert the BIA precursor to a 1-benzylisoquinoline alkaloid. In an example, the BIA precursor can be contacted with a sufficient amount of the one or more enzymes such that at least 5% of the BIA precursor is converted to a 1-benzylisoquinoline alkaloid. In additional examples, the BIA 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 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or 100% of the BIA precursor is converted to a 1-benzylisoquinoline alkaloid.

[0320] In some examples, one or more enzymes that convert BIA precursors to 1-benzylisoquinoline alkaloids are localized to cellular compartments. In some examples, Bet v 1 can be modified such that it encodes a targeting sequence that localizes it to the endoplasmic reticulum membrane of an engineered host cell. In particular, in certain instances, the host cell can be engineered to increase the production of norcoclaurine or norlaudanosoline from BIA precursors or products with norcoclaurine or norlaudanosol as precursors by localizing Bet v 1 to an organelle in a yeast cell. Bet v 1 and / or DODC can be localized to the yeast endoplasmic reticulum to reduce the spatial distance between Bet v 1 and / or DODC. An increase in yield means either of two situations: producing a certain amount of the target compound when the control does not produce the target compound, and increasing by 10% or more, such as 50% or more, including 2-fold or more, for example 5-fold or more, such as 10-fold or more when the control has some production of the target compound.

[0321] In other examples, DODC and Bet v 1 can be co-localized into a single protein fusion. In some examples, a fusion is created between DODC and Bet v 1 by one of several methods, including direct fusion, co-localization to a yeast organelle, or through an enzyme co-localization tool (such as a leucine zipper, a protein scaffold using an adaptor domain, or an RNA scaffold using an aptamer). Co-localizing norcoclaurine synthase can facilitate substrate channeling between the active sites of the enzymes and limit the diffusion of unstable intermediates (such as 4-HPAA).

[0322] In some examples, an enzyme with DODC activity is fused to a peptide with a Bet v 1 fold. In some examples, the DODC enzyme and the Bet v 1-folded protein can be fused in any order from N-terminus to C-terminus, C-terminus to N-terminus, N-terminus to N-terminus, or C-terminus to C-terminus. In some examples, the two protein sequences can be directly fused or fused through a peptide linker region.

[0323] In some examples, an enzyme with DODC 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 DODC 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 the production of 1-benzylisoquinoline alkaloids.

[0324] One or more enzymes that can be used to convert a BIA precursor to a 1-benzylisoquinoline alkaloid can contact the BIA precursor in vitro. Additionally or alternatively, one or more enzymes that can be used to convert a BIA precursor to a 1-benzylisoquinoline alkaloid can contact the BIA precursor in vivo. Additionally, one or more enzymes that can be used to convert a BIA precursor to a 1-benzylisoquinoline alkaloid can be provided to a cell that has the BIA precursor therein, or can be produced within an engineered host cell.

[0325] In some examples, the method provides an engineered host cell that produces an alkaloid product, wherein the condensation of the BIA precursor substrate to the 1-benzylisoquinoline alkaloid product can constitute a key step in the production of the alkaloid product. In some examples, the alkaloid produced is a 1-benzylisoquinoline alkaloid. In still other embodiments, the alkaloid produced is derived from a 1-benzylisoquinoline alkaloid, including, for example, 4-cycloprotopine and 5-cyclomorphinan alkaloids. In another embodiment, the BIA precursor is an intermediate of the engineered host cell product. In still other embodiments, the alkaloid product is selected from 1-benzylisoquinoline, protopine, morphinan, protoberberine, protopine, benzophenanthridine, schizandrin, phthalideisoquinoline, aporphine, bisbenzylisoquinoline, nal-opioid substances or nor-opioid substance alkaloids.

[0326] In some examples, the BIA precursor substrate is selected from 4-HPAA, 3,4-DHPA, and dopamine.

[0327] In some examples, the first BIA precursor substrate or amine substrate is a compound of formula IX: or a salt thereof, wherein: R1 and R2 are independently selected from hydrogen and hydroxyl.

[0328] In some other examples, R1 and R2 are hydroxyl, and the first BIA precursor substrate is dopamine.

[0329] In some examples, the second BIA precursor substrate or aldehyde substrate is a compound of formula X: or a salt thereof, wherein: R3 and R4 are independently selected from hydrogen and hydroxyl.

[0330] In some examples, R3 is hydrogen and R4 is hydroxyl, and the second BIA precursor is 4-HPAA.

[0331] In other examples, R3 and R4 are hydroxyl, and the second BIA precursor is 3,4-DHPAA.

[0332] In some examples, the method provides engineered host cells for producing alkaloid products from BIA precursors. In some cases, the condensation of 4-HPAA and dopamine to norcoclaurine can constitute a key step in producing various alkaloid products from precursors. In some cases, the condensation of 3,4-DHPA and dopamine to norlaudanosoline can constitute a key step in producing various alkaloid products from precursors. In some examples, the precursor is l-tyrosine or a sugar (e.g., glucose). The various alkaloid products can include, but are not limited to, 1-benzylisoquinoline, protopine, morphinan, protoberberine, protopine, benzophenanthridine, secologanin, phthalideisoquinoline, aporphine, bisbenzylisoquinoline, nal-opioid, and nor-opioid alkaloids.

[0333] Any suitable carbon source can be used as a precursor for 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, an unpurified mixture from renewable feedstocks (e.g., corn steep liquor, beet molasses, barley malt, biomass hydrolysate) can be used. 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 can be utilized, such as, methylamine, glucosamine, and amino acids (e.g., l-tyrosine). In some examples, the BIA precursor substrate can be directly added to the engineered host cells of the present disclosure, and the BIA precursor substrate includes, for example, 4-HPAA, 3,4-DHPA, and / or dopamine.

[0334] In some examples, the benzylisoquinoline alkaloid product or its derivative is recovered. In some examples, the benzylisoquinoline alkaloid product is recovered from a cell culture. In some examples, the benzylisoquinoline alkaloid product is 1-benzylisoquinoline, protopine, morphinan, protoberberine, protopine, benzophenanthridine, secologanin, phthalideisoquinoline, aporphine, bisbenzylisoquinoline, nal-opioid, or nor-opioid alkaloid. O-demethylation modification

[0335] Some of the methods, processes, and systems provided herein describe the conversion of a first benzylisoquinoline alkaloid to a second benzylisoquinoline alkaloid by removing an O-linked methyl group. Some of these methods, processes, and systems can include 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 includes a demethylase reaction.

[0336] Figure 10 Shows enzymes with opioid 3-O-demethylase (ODM) activity, according to some embodiments of the present invention. Specifically, the enzyme can act on the morphinan alkaloid structure to remove a methyl group from the oxygen bound to carbon 3.

[0337] Examples of the amino acid sequences of the ODM enzyme are shown in Table 12. The amino acid sequence of the ODM utilized in converting a first alkaloid to a second alkaloid can be 50% or more identical to the given amino acid sequence listed in Table 12. For example, the amino acid sequence of such an epimerase can comprise an amino acid sequence that is at least 50% more, 55% more, 60% more, 65% more, 70% more, 75% more, 80% more, 81% more, 82% more, 83% more, 84% more, 85% more, 86% more, 87% more, 88% more, 89% more, 90% more, 91% more, 92% more, 93% more, 94% more, 95% more, 96% more, 97% more, 98% more, or 99% or more identical to the amino acid sequence provided herein. Additionally, in certain embodiments, an "identical" amino acid sequence has at least 80%-99% identity to a specific amino acid sequence at the amino acid level. In some cases, an "identical" amino acid sequence contains at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% and more (in certain cases, at least 95%, 96%, 97%, 98% and 99%) identity at the amino acid level. In some cases, for example, the amino acid sequences can be identical, but the DNA sequence is altered, such as to optimize codon usage for a host organism.

[0338] Engineered host cells can be provided that produce an O - demethylase (ODM) that converts a first alkaloid to a second alkaloid, where the ODM comprises a given amino acid sequence as listed in Table 12. Engineered host cells can be provided that produce one or more ODM enzymes. The ODM produced within the engineered host cells can be recovered and purified to form a biocatalyst. The process can include contacting the first alkaloid with an amount of the ODM sufficient to convert the first alkaloid to the second alkaloid. In some examples, 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 the second alkaloid. In additional examples, the first alkaloid 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 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 the second alkaloid.

[0339] One or more enzymes useful for converting a first alkaloid to a second alkaloid can contact the first alkaloid in vitro. Additionally or alternatively, one or more enzymes useful for converting a first alkaloid to a second alkaloid can contact the first alkaloid in vivo. In some examples, one or more enzymes useful for converting a first alkaloid to a second alkaloid can be provided to a cell that has the first alkaloid. In some examples, one or more enzymes useful for converting a first alkaloid to a second alkaloid can be produced within an engineered host cell.

[0340] In some examples, the method provides engineered host cells that produce an alkaloid product, where O - demethylation 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 or nal - opioid. In still other embodiments, the alkaloid produced is derived from a nor - opioid or nal - opioid. In another embodiment, the first alkaloid is an intermediate in the product of the engineered host cell. In still other embodiments, the alkaloid product is selected from morphine, oxymorphone, norlaudanosoline, hydromorphinone, dihydromorphine, 14 - hydroxymorphine, morphinone, and 14 - hydroxymorphinone.

[0341] In some examples, the substrate alkaloid is an opioid selected from: codeine, oxycodone, thebaine, hydrocodone, dihydrocodeine, 14 - hydroxycodeine, codeinone, and 14 - hydroxycodeinone. Heterologous coding sequence

[0342] In some cases, the engineered host cell has one or more heterologous coding sequences (such as two or more, three or more, four or more, five or more), which encode activities that enable the engineered host cell to produce a desired target enzyme and / or target BIA, for example, as described herein. As used herein, the term "heterologous coding sequence" is used to indicate any polynucleotide that encodes or ultimately encodes a peptide or protein or its equivalent amino acid sequence (e.g., an enzyme), where the peptide or protein or its equivalent amino acid sequence is not normally present in the host organism and can be expressed in the host cell under appropriate conditions. Thus, a "heterologous coding sequence" includes multiple copies of a coding sequence that is normally present in the host cell such that the cell expresses additional copies of the coding sequence that are not normally present in the cell. The heterologous coding sequence can be RNA or any type thereof, e.g., mRNA, DNA or any type thereof, e.g., cDNA or a hybrid of RNA / DNA. The target coding sequence includes, but is not limited to, a full-length transcription unit that includes features such as a coding sequence, introns, promoter region, 3'-UTR, and enhancer region.

[0343] In some examples, the engineered host cell can contain multiple heterologous coding sequences, each encoding an enzyme, such as those listed in Table 11, Table 17 (e.g., P450), and Table 18 (e.g., CPR). In some examples, the multiple enzymes encoded by the multiple heterologous coding sequences can be different from each other. In some examples, some of the multiple enzymes encoded by the multiple heterologous coding sequences can be different from each other, and some of the multiple enzymes encoded by the multiple heterologous coding sequences can be duplicate copies.

[0344] In some examples, the heterologous coding sequences can be operably linked. The operably linked heterologous coding sequences can be within the same pathway for producing a specific benzylisoquinoline alkaloid product and / or epimerase product. In some examples, the operably linked heterologous coding sequences can be directly contiguous along the pathway for producing a specific benzylisoquinoline alkaloid product and / or epimerase product. In some examples, the operably linked heterologous coding sequences can have one or more native enzymes between one or more of the enzymes encoded by the multiple heterologous coding sequences. In some examples, the heterologous coding sequences can have one or more heterologous enzymes between one or more of the enzymes encoded by the multiple heterologous coding sequences. In some examples, the heterologous coding sequences can have one or more non-native enzymes between one or more of the enzymes encoded by the multiple heterologous coding sequences.

[0345] Engineered host cells can also be modified to have one or more genetic alterations to accommodate heterologous coding sequences. Alterations to the native host genome include, but are not limited to, modifying the genome to reduce or eliminate the expression of specific proteins that may interfere with the desired pathway. The presence of such native proteins can rapidly convert one of the intermediates or the end product of the pathway into metabolites or other compounds that are not available in the desired pathway. Thus, if the activity of the native enzyme is reduced or completely absent, the resulting intermediate will be more readily incorporated into the desired product.

[0346] Heterologous coding sequences include, but are not limited to, sequences encoding enzymes, wild-type or equivalent sequences, which are typically responsible for producing the desired BIA in plants. In some cases, the enzyme encoded by the heterologous sequence can be any enzyme in the 1-benzylisoquinoline alkaloid pathway and can be from any convenient source. The selection and number of enzymes encoded by the heterologous coding sequences for a particular synthetic pathway can be selected based on the desired product. In certain embodiments, the host cells of the present disclosure can include 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or even 15 or more heterologous coding sequences such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 heterologous coding sequences.

[0347] As used herein, the term "heterologous coding sequence" also includes the coding portion of a peptide or enzyme, i.e., the cDNA or mRNA sequence of the peptide or enzyme, and the coding portion of a full-length transcription unit, i.e., a gene containing introns and exons, as well as "codon-optimized" sequences, truncated sequences, or other forms of altered sequences that encode an enzyme or its equivalent amino acid sequence, provided that the equivalent amino acid sequence produces a functional protein. Such equivalent amino acid sequences can have a deletion of one or more amino acids, where the deletion is N-terminal, C-terminal, or internal. Truncated forms are contemplated as long as they have the catalytic ability shown herein. Fusions of two or more enzymes are also contemplated to facilitate the transfer of metabolites in the pathway, provided that catalytic activity is maintained.

[0348] Operable fragments, mutants, or truncated forms can be identified by modeling and / or screening. In some cases, this is achieved by stepwise deletion of, for example, the N-terminal, C-terminal, or internal regions of a protein and then analyzing the activity of the resulting derivative for the desired reaction compared to the original sequence. If the derivative in question functions in such a capacity, it is considered to constitute an equivalent derivative of a suitable enzyme.

[0349] In some examples, when expressed in a recombinant host, some heterologous proteins may show that they are misprocessed. For example, plant proteins (such as cytochrome P450 enzymes) expressed in a microbial production host may be incorrectly processed. In particular, when heterologously expressed in yeast, salutaridine synthase may undergo N-linked glycosylation. This N-linked glycosylation may not be observed in plants, which may indicate incorrect N-terminal sorting of the nascent SalSyn transcript, thereby reducing the enzyme activity in the heterologous microbial host. In such examples, protein engineering aimed at correcting the N-terminal sorting of the nascent transcript to remove the N-linked glycosylation pattern may lead to improved activity of salutaridine synthase in the recombinant production host.

[0350] Aspects of the present disclosure also relate to heterologous coding sequences that encode 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 different from a particular amino acid sequence, but contains at least some amino acid changes (deletions, substitutions, inversions, insertions, etc.) that, when used for the desired purpose, do not substantially affect the biological activity of the protein compared to the similar activity of the particular amino acid sequence. In the case of an epimerase, the biological activity refers to its catalytic activity. Equivalent sequences also mean including those sequences that have been engineered and / or evolved to have properties different from the original amino acid sequence. The desired variable properties include catalytic activity, substrate specificity, selectivity, stability, solubility, localization, etc.

[0351] In some cases, the expression of each type of enzyme is increased by additional gene copies (i.e., multiple copies), which increases the accumulation of intermediates and / or the production of the desired BIA. Some embodiments of the present disclosure include increasing the production of the desired BIA in a host cell by co-expressing multiple species variants of a single or multiple enzymes. In some cases, additional gene copies of a single or multiple enzymes are included in the host cell. Any convenient method can be utilized, including multiple copies of the heterologous coding sequence of the enzyme in the host cell.

[0352] In some examples, the engineered host cell comprises multiple copies of a heterologous coding sequence of an enzyme, such as two or more, three or more, four or more, five or more, or even ten or more copies. In certain embodiments, the engineered host cell comprises multiple copies of a heterologous coding sequence of one or more enzymes, such as multiple copies of two or more, three or more, four or more, etc. In some cases, compared to the host cell, the multiple copies of the heterologous coding sequence of the enzyme are derived from two or more different source organisms. For example, the engineered host cell can comprise multiple copies of a heterologous coding sequence, where each copy is derived from a different source organism. Thus, each copy can include some variation in the explicit sequence based on the interspecies differences of the target enzyme encoded by the heterologous coding sequence.

[0353] In certain embodiments, the engineered host cell comprises multiple copies of a heterologous coding sequence of one or more enzymes, such as multiple copies of two or more, three or more, four or more, etc. In some cases, compared to the host cell, the multiple copies of the heterologous coding sequence of the enzyme are derived from two or more different source organisms. For example, the engineered host cell can comprise multiple copies of a heterologous coding sequence, where each copy is derived from a different source organism. Thus, each copy can include some variation in the explicit sequence based on the interspecies differences of the target enzyme encoded by the heterologous coding sequence.

[0354] The engineered host cell culture medium can be sampled and monitored for the production of the target BIA. Any convenient method can be used to observe and measure the target BIA. Exemplary methods include but are not limited to LC-MS methods (e.g., as described herein), where the target sample is analyzed by comparison with a known amount of a standard compound. Additionally, there are other ways to observe and / or measure the target BIA. Examples of alternative methods for observing and / or measuring BIA include in particular GC-MS, UV-vis spectroscopy, NMR, LC-NMR, LC-UV, TLC, capillary electrophoresis. Identity can be confirmed, for example, by m / z and MS / MS fragmentation patterns, MRM transitions, and quantification or measurement of the compound can be achieved via analysis of LC trace peaks with known retention times and / or EIC MS peaks of corresponding LC-MS analysis with reference to a known amount of a compound standard. In some cases, identity can be confirmed via multiple reaction monitoring using mass spectrometry.

[0355] Additionally, the culture of the engineered host cell can be sampled and monitored for the production of the target enzyme (such as neopinetone isomerase). Any convenient method can be used to observe and measure the target enzyme. Exemplary methods include enzyme activity assays, polyacrylamide gel electrophoresis, carbon monoxide spectroscopy, and Western blot analysis. Formaldehyde toxicity mitigation modification

[0356] The host cell can include one or more modifications of one or more biosynthetic enzyme genes of the cell that are designed to mitigate formaldehyde toxicity (such as two or more, three or more, four or more, five or more, or even more modifications). 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 (e.g., heterologous) to the cell. Any convenient biosynthetic enzyme gene of the cell can be targeted for modification to mitigate formaldehyde accumulation. As used herein, the term "formaldehyde toxicity mitigation modification" refers to a modification that reduces the accumulation of formaldehyde that can be produced as a byproduct of a biosynthetic process in an engineered host cell. Formaldehyde toxicity is a process that occurs in a cell when formaldehyde accumulates in the cell. For example, biosynthetic processes involving methyl oxidation for demethylation produce unwanted byproducts formaldehyde that are toxic to yeast. For example, to produce normorphinone, a yeast strain must also produce intermediates in the normorphinone pathway, particularly benzylisoquinoline alkaloids (BIAs), such as codeinone, codeine, hydrocodone, morphinone, and other morphinan intermediates. A key step in the pathway to produce normorphinone from these morphinan intermediates is the oxidation of an intermediate that is methylated at position 6 of the morphinan intermediate (such as thebaine, oripavine, nor-thebaine, or nor-oripavine or others). This demethylation is known to be catalyzed by a 2-ketoglutarate and Fe(II)-dependent dioxygenase (2ODD) enzyme, thebaine 6-O-demethylase (T6ODM), or any other oxidase with similar activity. Depending on the substrate of the reaction, the products include neopinone, neomorphinone, nor-neopinone (N-demethylated neopinone), nor-neomorphinone (N-demethylated neomorphinone), or others. Regardless of the oxidase, substrate, or product used, the oxidation of the methyl group used for demethylation produces an unwanted byproduct formaldehyde, e.g., Figures 36A - 36D depicts an exemplary metabolic pathway showing that morphinan oxidation results in the production of formaldehyde as a byproduct. Figure 36A depicts an exemplary metabolic pathway showing codeine biosynthesis using the conversion from thebaine to codeine, which involves demethylation of thebaine at position 6 (shown in Figure 36A to be catalyzed by T6ODM). As can be observed from Figure 36A it, formaldehyde is an essential byproduct of this reaction. Figure 36B depicts another exemplary metabolic pathway showing the biosynthesis of a morphinan (here oripavine) from thebaine. In this exemplary pathway, thebaine is oxidized by a 3-O-demethylase to form oripavine, generating formaldehyde as a byproduct of the reaction. Figure 36CDepicts yet another exemplary metabolic pathway showing the biosynthesis of morphinan (here morphine) from thebaine. In this exemplary pathway, thebaine is oxidized by CODM to form morphine, generating formaldehyde as a byproduct of the reaction. Figure 36D Depicts yet another exemplary metabolic pathway showing the biosynthesis of morphinan (here nor-thebaine) from thebaine. In this exemplary pathway, thebaine is oxidized by N-demethylase to form nor-thebaine, generating formaldehyde as a byproduct of the reaction. Without departing from the text of the present disclosure, other demethylations of morphinan can be used (e.g., methylation can occur at position 3 or at the nitrogen group and produce formaldehyde).

[0357] Modifications that alleviate formaldehyde toxicity in engineered host cells reduce the accumulation of formaldehyde in the engineered host cells relative to control cells. In this way, the engineered host cells provide reduced levels of formaldehyde and / or increased levels of the desired BIA. Increased levels mean levels that are 110% or more of the desired BIA in control cells, 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 in control cells, such as at least 3-fold or more, at least 5-fold or more, at least 10-fold or more, or even more. Reduced levels mean a reduction of 10% or more in the level of formaldehyde accumulated in control cells, such as 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, 99% or more, about 10%, about 20%, about 20%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 97%, about 99%, from about 5% to about 10%, from about 10% to about 20%, from about 20% to about 30%, from about 30% to about 40%, from about 40% to about 50%, from about 50% to about 60%, from about 60% to about 70%, from about 70% to about 80%, from about 80% to about 90%, or from about 90% to about 100%.

[0358] Multiple formaldehyde toxicity alleviating modifications and biosynthetic enzymes for the reduction of formaldehyde accumulation in engineered host cells can be targeted for modification. The engineered host cells can include one or more formaldehyde toxicity alleviating modifications in one or more biosynthetic enzyme genes. In some examples, the engineered host cells include a modification that increases the expression of formaldehyde dehydrogenase. In some examples, the formaldehyde dehydrogenase is the enzyme SFA1.

[0359] In some examples, formaldehyde toxicity mitigation modifications can result in unwanted downstream effects. For example, formaldehyde detoxification can deplete the glutathione pool in host cells. For example, FIG. 36 depicts the major detoxification pathway utilizing formaldehyde dehydrogenase SFA1 in yeast. The first step is the spontaneous conjugation of formaldehyde with glutathione (the thiol group of glutathione is specifically shown to demonstrate where the reaction occurs at the molecular level). The second step is the oxidation to S-formylglutathione by SFA1. Glutathione (which is necessary for formaldehyde detoxification via SFA1) can be depleted by catabolism to glutamate and cysteinylglycine catalyzed by DUG2 / 3. In this example, the unwanted downstream effect of depleting the glutathione pool can be mitigated by introducing modifications designed to maintain the glutathione pool. In some examples, the modifications designed to maintain the glutathione pool reduce or knock out the expression of DUG2 and / or DUG3 or any other suitable enzyme.

[0360] Any convenient number and type of modifications can be utilized to mitigate formaldehyde toxicity and / or unwanted downstream effects. In certain embodiments, the engineered host cells of the present disclosure can include 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or even 15 or more modifications that mitigate formaldehyde toxicity and / or unwanted downstream effects, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 modifications, in one or more biosynthetic enzyme genes within the engineered host cell. 14 - Hydroxylation modification

[0361] Some of the methods, processes, and systems provided herein include converting a first morphinan alkaloid to a second morphinan alkaloid by adding a hydroxyl group to the free hydrogen at the C14 position of the first morphinan alkaloid. Exemplary morphinan alkaloids having a free hydrogen at the C14 position are depicted in Formula XI below. Some of these methods, processes, and systems can include engineered host cells. In some examples, the conversion of the first morphinan alkaloid to the second morphinan alkaloid is a key step in the conversion of a substrate to a nor-opioid. In some examples, the conversion of the first morphinan alkaloid to the second morphinan alkaloid includes a hydroxylation reaction. Any suitable enzyme that provides 14-hydroxylase activity can be used to hydroxylate the free hydrogen at the 14 position of the first morphinan alkaloid. In some embodiments, the enzyme is a P450 enzyme.

[0362] Figure 37 A- Figure 37H shows eight exemplary biosynthetic pathways, each involving at least one enzyme having 14-hydroxylation activity, according to some embodiments of the present disclosure. Specifically, the enzyme can act on the morphinan alkaloid structure to add a hydroxyl group to the available C-H group at the 14-position.

[0363] The enzyme that can add a hydroxyl group to the available C-H group at the 14-position of the morphinan alkaloid is an enzyme comprising 14-hydroxylase activity. In certain embodiments, one or more proteins comprising 14-hydroxylase activity are heterologous to the engineered host cell. In some embodiments, one or more proteins comprising C-14 hydroxylase activity include cytochrome p450 (P450) proteins.

[0364] In some embodiments, the cytochrome P450 enzyme comprises one or more mutations relative to the wild-type sequence. In some embodiments, the wild-type sequence is 14HC_P450_5 (SEQ ID NO:179). In some embodiments, the cytochrome P450 comprises one or more mutations at positions 58, 59, 102, 181, 188, 189, 17, 280, 325, and / or 396. In some embodiments, the mutation is at position 17 and comprises an I or L substitution. In some embodiments, the mutation is at position 58 and comprises a K substitution. In some embodiments, the mutation is at position 59 and comprises a D substitution. In some embodiments, the mutation is at position 102 and comprises an L or M substitution. In some embodiments, the mutation is at position 181 and comprises a G, I, L, M, P, O, S, or V substitution. In some embodiments, the mutation is at position 188 and comprises an I substitution. In some embodiments, the mutation is at position 189 and comprises a V substitution. In some embodiments, the mutation is at position 208 and comprises an N substitution. In some embodiments, the mutation is at position 325 and comprises an I, M, or V substitution.

[0365] In some embodiments, the one or more mutations comprise the E58K, A59D, F102L, D181E, L188I, and D189E mutations relative to SEQ ID NO:179. In some embodiments, the cytochrome P450 enzyme comprises SEQ ID NO:190.

[0366] In some embodiments, the one or more mutations comprise the L188I and D189E mutations relative to SEQ ID NO:179. In some embodiments, the cytochrome P450 enzyme comprises SEQ ID NO:184.

[0367] In some embodiments, the one or more mutations include the A59D, L188I, and D189E mutations relative to SEQ ID NO:179. In some embodiments, the cytochrome P450 enzyme comprises SEQ ID NO:186.

[0368] In some embodiments, the cytochrome P450 enzyme has an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO.:190, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 178, 180, 182, 184, 186, 188, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, or 244.

[0369] In some embodiments, the heterologous enzyme comprises or consists of the amino acid sequence of SEQ ID NO:190, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 178, 180, 182, 184, 186, 188, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, or 244.

[0370] In some embodiments, the P450 protein is co-expressed with the CPR enzyme. Examples of amino acid sequences of enzymes having 14-hydroxylase activity and exemplary nucleic acid sequences encoding these amino acid sequences are shown in Table 17.

[0371] In some embodiments, the CPR is fungal CPR. In some embodiments, the CPR is plant CPR. In some embodiments, the CPR is animal CPR.

[0372] In some embodiments, the P450 protein and the CPR are from the same genus. In some embodiments, both the P450 protein and the CPR enzyme are from fungi.

[0373] In some embodiments, the CPR comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 168, 170, 172, 174, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268 or 270.

[0374] In some embodiments, the CPR comprises or consists of SEQ ID NO: 168, 170, 172, 174, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268 or 270.

[0375] In an example, the amino acid sequence of an enzyme having 14-hydroxylation activity for converting morphinan alkaloids into 14-hydroxylated morphinan alkaloids can be 50% or more identical to a given amino acid sequence listed in Table 17. For example, the amino acid sequence of such an enzyme having 14-hydroxylase activity can comprise an amino acid sequence that is at least 50% more, 55% more, 60% more, 65% more, 70% more, 75% more, 80% more, 81% more, 82% more, 83% more, 84% more, 85% more, 86% more, 87% more, 88% more, 89% more, 90% more, 91% more, 92% more, 93% more, 94% more, 95% more, 96% more, 97% more, 98% more, or 99% or more identical to an amino acid sequence provided herein. Additionally, in certain embodiments, an "identical" amino acid sequence has at least 80%-99% identity to a specific amino acid sequence at the amino acid level. In some cases, an "identical" amino acid sequence contains at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% and more (in some cases, at least 95%, 96%, 97%, 98% and 99%) identity at the amino acid level. In some cases, for example, the amino acid sequences can be identical, but the nucleic acid sequences encoding the amino acid sequences are altered, such as to optimize codon usage for a host organism.

[0376] Engineered host cells can be provided that express an enzyme providing 14-hydroxylation activity that converts a first alkaloid to a second alkaloid, wherein the enzyme comprising 14-hydroxylation activity comprises the amino acid sequence provided in Table 17. Engineered host cells can be provided that express an enzyme providing 14-hydroxylation activity and a CPR enzyme, wherein the enzyme providing 14-hydroxylation activity comprises the amino acid sequence provided in Table 17, and / or the CPR enzyme comprises the amino acid sequence provided in Table 18. Engineered host cells can be provided that express one or more enzymes providing 14-hydroxylation activity. The enzyme providing 14-hydroxylation activity produced in the engineered host cells can be recovered and purified to form a biocatalyst. The process can include contacting the first alkaloid with an amount of the enzyme providing 14-hydroxylation activity sufficient to convert the first alkaloid to the second alkaloid. In some examples, 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 the second alkaloid. In additional examples, the first alkaloid 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 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 the second alkaloid.

[0377] One or more enzymes useful for converting a first alkaloid to a second alkaloid can contact the first alkaloid in vitro. Additionally or alternatively, one or more enzymes useful for converting a first alkaloid to a second alkaloid can contact the first alkaloid in vivo. In some examples, one or more enzymes useful for converting a first alkaloid to a second alkaloid can be provided to a cell having the first alkaloid therein. In some examples, one or more enzymes useful for converting a first alkaloid to a second alkaloid can be produced within an engineered host cell.

[0378] In some examples, the method provides engineered host cells that produce an alkaloid product, wherein 14-hydroxylation 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 another embodiment, the first alkaloid is an intermediate of the product of the engineered host cell. In still other embodiments, the alkaloid product is selected from naloxone, naltrexone, and nalmefene.

[0379] In some examples, the substrate alkaloid is an opioid selected from: norcodeine, noroxymorphone, noscapine, norhydrocodone, nordihydrocodeine, nor-14-hydroxycodeine, norcodeinone, nor-14-hydroxycodeinone, normorphine, normorphinone, norlaudanosine, normethorphinone, nordihydromorphine, nor-14-hydroxymorphine, normorphinone, and nor-14-hydroxymorphinone. In some examples, the co-substrate is S-adenosylmethionine, allyl-S-adenosylmethionine, or cyclopropylmethyl-S-adenosylmethionine.

[0380] In some embodiments, the method provides engineered host cells that produce at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least about 2, at least about 3, at least about 4, at least about 5 times more BIA product compared to the same non-engineered host cells.

[0381] In some embodiments, the method provides engineered host cells that produce at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% more BIA product compared to the same non-engineered host cells. Method Method for culturing host cells for BIA production

[0382] As summarized above, some aspects of the present disclosure include methods for preparing a benzylisoquinoline alkaloid (BIA) of interest. Additionally, some aspects of the present disclosure include methods for preparing an enzyme of interest. Accordingly, some aspects of the present disclosure include culturing engineered host cells under conditions in which the one or more host cell modifications (e.g., as described herein) are functionally expressed such that the cells convert a starting compound of interest into an enzyme product and / or BIA of interest. Methods are also provided that include culturing engineered host cells under conditions suitable for protein production such that one or more heterologous coding sequences are functionally expressed and a starting compound of interest is converted into an enzyme product or BIA of interest. In some examples, the method is a method for preparing a benzylisoquinoline alkaloid (BIA) that includes culturing 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 that includes culturing engineered host cells (e.g., as described herein); adding a starting compound to the cell culture; and recovering the enzyme from the cell culture.

[0383] The fermentation medium can contain a suitable carbon substrate. Carbon sources suitable for the methods of the present disclosure can encompass a variety of carbon-containing substrates. Suitable substrates 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 cases, unpurified mixtures from renewable feedstocks (e.g., corn steep liquor, beet molasses, barley malt) can be used. In some cases, the carbon substrate can 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, can be utilized.

[0384] Any convenient method for culturing the engineered host cell can be employed to produce the enzyme of interest and / or the BIA. The specific protocol employed can vary, e.g., depending on the engineered host cell, the heterologous coding sequence, the enzyme of interest, the BIA of interest, etc. The engineered host cell can be present in any convenient environment, such as an environment in which the engineered host cell is capable of expressing one or more functional heterologous enzymes. In some embodiments, the engineered host cell is cultured under conditions that are conducive to enzyme expression and that have available in vivo an appropriate substrate for allowing production of the enzyme of interest and / or the BIA. In some embodiments, the functional enzyme is extracted from the engineered host for production of the enzyme of interest and / or the BIA under in vitro conditions. In some instances, the engineered host cell is returned to a multicellular host organism. The engineered host cell is at any growth stage, including but not limited to the stationary phase and the logarithmic growth phase, etc. Additionally, the culture itself can be a continuous culture or a batch culture.

[0385] The cells can be grown in a suitable fermentation medium at a temperature between 14°C and 40°C. The cells can be grown with agitation at any convenient rate (e.g., 200 rpm). The cells can be grown at a suitable pH. A suitable pH range for fermentation can be between pH 5 and 9. Fermentation can be carried out under aerobic, anaerobic, or microaerophilic conditions. Any suitable growth medium can be used. Suitable growth media can include, but are not limited to, commonly commercially prepared media, such as synthetic defined (SD) minimal medium or medium rich in yeast extract peptone dextrose (YEPD). Any other rich, defined, or synthetic growth medium suitable for the microorganism can be used.

[0386] The cells can be cultured in vessels of essentially any size and shape. Examples of vessels suitable for the methods of the present disclosure can include, but are not limited to, multi-well shake plates, test tubes, flasks (baffled and unbaffled), and bioreactors. The volume of the culture can range from 10 microliters to greater than 10,000 liters.

[0387] It may include adding to the growth medium a reagent known to regulate metabolism in a manner required to produce alkaloids. In a non-limiting example, cyclic adenosine 2’3’-monophosphate can be added to the growth medium to regulate catabolite repression.

[0388] Any convenient cell culture conditions for a particular cell type can be utilized. In certain embodiments, host cells comprising one or more modifications are cultured in standard or readily optimized conditions with standard cell media and supplements. As an example, when no selection pressure for plasmid maintenance is required, the standard growth medium can contain 20 g / L yeast extract, 10 g / L peptone, and 20 g / L dextrose (YPD). Host cells containing plasmids are grown in synthetic complete (SC) medium containing 1.7 g / L yeast nitrogen base without amino acids, 5 g / L ammonium sulfate, and 20 g / L dextrose, supplemented with the appropriate amino acids required for growth and selection. Alternative carbon sources available for inducible enzyme expression include, but are not limited to, sucrose, raffinose, and galactose. In the laboratory, cells are grown in any convenient vessel (e.g., test tubes or flasks in a volume range of 1 - 1000 mL or larger) with shaking at any convenient rate (e.g., 200 rpm) at any convenient temperature (e.g., 30 °C).

[0389] For example, as part of an industrial process, the culture volume can be scaled up to grow in larger fermentors. Industrial fermentation processes can be carried out under closed batch, fed-batch, or continuous chemostat conditions or any suitable fermentation mode. In some cases, engineered host cells can be immobilized on a substrate as whole-cell catalysts and subjected to fermentation conditions for alkaloid production.

[0390] Batch fermentation is a closed system where the composition of the medium is set at the start of fermentation and does not change during the fermentation process. One or more desired organisms are inoculated into the medium at the start of fermentation. In some cases, batch fermentation is run with changes 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 continuously change during fermentation. Cells typically undergo a lag phase, then enter the logarithmic phase (high growth rate), then enter the stationary phase (reduced or stopped growth rate), and ultimately enter the death phase (if not processed). In additional cases, the batch fermentation system can be opened at certain times to add additional substrates for the organisms required for fermentation. In particular, in some cases, the fermentation system can include a fed-batch reactor.

[0391] Continuous fermentation is an open system in which a defined fermentation medium is continuously added to a bioreactor, and an equal amount of the fermentation medium is continuously removed from the vessel for processing. A continuous fermentation system is typically operated to maintain steady-state growth conditions such that cell losses due to medium removal must be balanced by the growth rate in the fermentation. Continuous fermentation is typically operated under conditions where the cells are at a constant high cell density. Continuous fermentation allows for the regulation of one or more factors that affect the concentration of the target product and / or cell growth.

[0392] The liquid medium can include, but is not limited to, an enriched or synthetic defined medium having the additive components as described above. The medium components can be dissolved in water and sterilized by heating, pressure, filtration, radiation, chemicals, or any combination thereof. Several medium components can be prepared separately and sterilized and then combined in the fermentation vessel. The medium can be buffered to help maintain a constant pH throughout the fermentation.

[0393] Process parameters can be monitored or controlled during the fermentation, including temperature, dissolved oxygen, pH, agitation, aeration rate, and cell density. For example, the temperature of the fermentation process can be monitored by a temperature probe immersed in the medium. The culture temperature can be controlled at a set point by adjusting the jacket temperature. Water can be cooled in an external cooler and then flow into the bioreactor control tower and be circulated to the jacket at a temperature required to maintain the set point temperature in the vessel.

[0394] Additionally, gas flow parameters can be monitored during the fermentation. For example, gas can be introduced into the medium through a sparger. Gases suitable for the methods of the present disclosure can include compressed air, oxygen, and nitrogen. The gas flow can be at a fixed rate or regulated to maintain a dissolved oxygen set point.

[0395] The pH of the medium can also be monitored. In some examples, the pH can be monitored by a pH probe immersed in the medium within the vessel. If pH control is effective, the pH can be adjusted by acid and base pumps that add each solution to the medium at a desired rate. The acid solution for controlling the pH can be sulfuric acid or hydrochloric acid. The base solution for controlling the pH can be sodium hydroxide, potassium hydroxide, or ammonium hydroxide.

[0396] Additionally, the dissolved oxygen in the medium can be monitored by a dissolved oxygen probe immersed in the medium. If dissolved oxygen regulation is effective, 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.

[0397] The stirring speed can also be monitored during fermentation. In some examples, a stirrer motor can drive the agitator. The stirrer speed can be set at a consistent rpm throughout the fermentation process or can be dynamically adjusted to maintain a set dissolved oxygen level.

[0398] Additionally, turbidity can be monitored during fermentation. In some examples, a turbidity probe can be used to measure cell density. Alternatively, cell density can be measured by taking samples from the bioreactor and analyzing them in a spectrophotometer. In addition, samples can be taken from the bioreactor at regular time intervals using a sterile sampling device. The alkaloids produced by the host cells in the samples can be analyzed. Other metabolites of the samples and the depletion of sugars, media components, or cell density can also be analyzed.

[0399] In another example, feed parameters can be monitored during fermentation. In particular, a feedstock including sugars and other carbon sources, nutrients, and cofactors can be added to the fermentation using an external pump. Other components can also be added during fermentation, including but not limited to antifoaming agents, salts, chelating agents, surfactants, and organic liquids.

[0400] Any convenient codon optimization technique for optimizing the expression of a heterologous polynucleotide in a host cell can be applied to the subject host cells and methods, see, e.g., Gustafsson, C. et al. (2004) Trends Biotechnol, 22, 346 - 353.

[0401] The subject methods can also include adding a starting compound to the cell culture. Any convenient addition method can be applied to the subject methods. The cell culture can be supplemented with a sufficient amount of the desired starting material (e.g., as described herein), e.g., in mM to μM amounts, such as a starting compound between about 1 - 5 mM. It should be understood that the amount of the starting material added, the time and rate of addition, the form of the material added, etc. can vary depending on a variety of factors. The starting material can be added neat or pre - dissolved in a suitable solvent (e.g., cell culture medium, water, or an organic solvent). The starting material can be added in a concentrated form (e.g., 10x relative to the desired concentration) to minimize dilution of the cell culture upon addition. The starting material can be added in one or more batches or by continuous addition over an extended period of time (e.g., hours or days). Method for isolating products from fermentation broth

[0402] The subject method can also include recovering the enzyme of interest and / or BIA from the cell culture. Any convenient separation and isolation method (e.g., chromatographic methods or precipitation methods) can be applied in the subject method to recover the enzyme of interest and / or BIA from the cell culture. Filtration methods can be used to separate the soluble fraction from the insoluble fraction of the cell culture. In some cases, liquid chromatography (e.g., reverse-phase HPLC, size exclusion, normal-phase chromatography) can 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.) can be used to separate the enzyme of interest and / or BIA from other components of the cell culture.

[0403] Methods known in the art can be used to isolate the produced alkaloids from the fermentation medium. Multiple recovery steps can be performed immediately after fermentation (or in some cases, during fermentation) for the initial recovery of the desired product. Through these steps, the alkaloids (e.g., BIA) can be separated from the engineered host cells, cell debris, and waste, and other nutrients, sugars, and organic molecules can be retained in the spent medium. This process can be used to produce a BIA-rich product.

[0404] In an example, a product stream having a benzylisoquinoline alkaloid (BIA) product is formed by providing engineered yeast cells and a feedstock comprising nutrients and water to a batch reactor. In particular, the engineered yeast cells can be subjected to fermentation by incubating the engineered yeast cells for a period of at least about 5 minutes to produce a solution comprising the BIA product and cell material. Once the engineered yeast cells have been subjected to fermentation, at least one separation unit can be used to separate the BIA product from the cell material to provide a product stream comprising the BIA product. In particular, the product stream can include the BIA product and additional components, such as clarified yeast medium. Additionally, the BIA product can comprise one or more BIA of interest, such as one or more BIA compounds.

[0405] Different methods can be used to remove cells from the bioreactor medium containing the enzyme of interest and / or BIA. In some examples, cells can be removed by precipitation over time. This precipitation process can be accelerated by cooling or by adding a clarifying agent (such as silica). Then, the spent medium can 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, e.g., by continuous-flow centrifugation or by using a continuous extractor.

[0406] If there are some valuable target enzymes and / or BIAs inside the engineered host cells, the engineered host cells can be permeabilized or lysed, and cell debris can be removed by any of the above methods. Reagents for permeabilizing the engineered host cells can include but are not limited to organic solvents (e.g., DMSO) or salts (e.g., lithium acetate). Methods for lysing the engineered host cells can include adding surfactants such as sodium lauryl sulfate, or mechanical disruption by bead milling or sonication.

[0407] The target enzymes and / or BIAs can be extracted from the clarified spent medium by liquid-liquid extraction with an organic liquid immiscible with the aqueous medium. In some examples, 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, ethyl butyrate. The organic liquid can be added at as little as 10% or as much as 100% of the aqueous medium. The organic liquid can be as little as 10% of the volume of the aqueous liquid, can be 100%, can be 200%, can be 300%, can be 400%, can be 500%, can be 600%, can be 700%, can be 800%, can be 900%, can be 1000%, can be greater than 1000%, or can be a percentage between those listed herein.

[0408] In some cases, the organic liquid can be added at the start of fermentation or at any time during fermentation. This extractive fermentation process can increase the yield of the target enzymes and / or BIA from the host cells by continuously transferring the enzymes and / or BIA to the organic phase.

[0409] Agitation can cause the organic phase to form an emulsion with the aqueous medium. Methods for promoting the separation of the two phases into distinct layers can include but are not limited to adding an anti-emulsifier or a nucleating agent, or adjusting the pH. The emulsion can also be centrifuged to separate the two phases, such as by continuous conical plate centrifugation.

[0410] Alternatively, the organic phase can be separated from the aqueous medium such that it can be physically removed after extraction. For example, the solvent can be encapsulated in a membrane.

[0411] In some examples, adsorption methods can be used to extract the target enzymes and / or BIAs from the fermentation medium. In some examples, the target BIA can be extracted from the clarified spent medium by adding a resin (such as XAD4) or another reagent for adsorbing and removing BIA. The target BIA 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.

[0412] The desired BIA can also be extracted from the fermentation broth using filtration. At high pH, the desired BIA can form a crystalline-like precipitate in the bioreactor. This precipitate can be removed by filtration through a filter, membrane, or other porous material. The precipitate can also be collected by centrifugation and / or decantation.

[0413] The above extraction methods can be carried out in situ (in the bioreactor) or ex situ (e.g., in an external loop through which the medium flows out of the bioreactor and contacts the extractant and then recycles back into the vessel). Alternatively, the extraction method can be carried out using the clarified medium taken out from the bioreactor vessel after fermentation termination. Method for purifying products from alkaloid - rich solutions

[0414] Subsequent purification steps can involve treating the post-fermentation solution enriched with one or more desired BIA products using methods known in the art to recover the individual desired product substances to high purity.

[0415] In one example, the desired BIA extracted in the organic phase can be transferred to an aqueous solution. In some cases, the organic solvent can be removed by heating and / or vacuum evaporation, and the resulting powder can be dissolved in an aqueous solution of a suitable pH. In additional examples, the desired BIA can be extracted from the organic phase by adding an aqueous solution of a suitable pH that promotes the extraction of the desired BIA into the aqueous phase. The aqueous phase can then be removed by decantation, centrifugation, or other methods.

[0416] The solution containing BIA can be further treated to remove metals, e.g., by treating with a suitable chelating agent. The solution containing the desired BIA can be further treated to remove other impurities such as proteins and DNA by precipitation. In one example, the solution containing the desired BIA is treated with a suitable precipitant (such as ethanol, methanol, acetone, or isopropanol). In alternative examples, DNA and proteins can be removed by dialysis or other size exclusion methods that can separate the smaller alkaloids from the contaminating biopolymers.

[0417] In additional examples, the solution containing the desired BIA can be extracted to high purity by continuous cross-flow filtration using methods known in the art.

[0418] If the solution contains a mixture of desired BIAs, it can be subjected to acid-base treatment using methods known in the art to produce the individual desired BIA substances. During this process, the pH of the aqueous solution is adjusted to precipitate the individual BIAs.

[0419] For high-purity, small-scale preparation, the BIA can be purified in a single step by liquid chromatography. Liquid chromatography - mass spectrometry (LCMS) method

[0420] The target BIA compounds (including 1-benzylisoquinoline alkaloids, bisbenzylisoquinoline alkaloids, protopine alkaloids, morphinan alkaloids, nal-opioids, and nor-opioids) can be separated using liquid chromatography and detected and quantified using mass spectrometry. Compound identity can be confirmed by characteristic elution times, mass-to-charge ratios (m / z), and fragmentation patterns (MS / MS). Quantification can be performed by comparing the compound peak areas to a standard curve of a known reference standard compound. Additionally, the target BIAs can be detected by alternative methods such as GC-MS, UV-vis spectroscopy, NMR, LC-NMR, LC-UV, TLC, and capillary electrophoresis. Purpald assay method

[0421] For high-throughput screening of demethylation reactions, purpald assays can be used. For example, demethylation catalyzed by 2-oxoglutarate-dependent dioxygenases produces formaldehyde as a product, as shown in the general chemical equation: The Purpald reagent under basic conditions undergoes a color change in the presence of formaldehyde, which can be quantified with a spectrophotometer at 510 nm to concentrations as low as 1 nM. Yeast - derived alkaloid APIs compared to plant - derived APIs

[0422] Clarified yeast culture medium (CYCM) can contain a variety of impurities. The clarified yeast culture medium can be dehydrated by vacuum and / or heating to produce a powder rich in alkaloids. This product is similar to poppy straw concentrate (CPS) or opium, which are exported from poppy-growing countries and purchased by API manufacturers. For the purposes of this disclosure, CPS is a representative example of any type of purified plant extract from which one or more desired alkaloid products can ultimately be further purified. Tables 14 and 15 highlight impurities that may be unique to CYCM or CPS or that may be present in both products. Some BIAs may have pigments as impurities, while other BIAs may themselves be classified as pigments. Thus, these BIAs can be evaluated based on non-pigment impurities. By analyzing a subset of these impurities in a product of unknown origin, one of ordinary skill in the art can determine whether the product is derived from a yeast or plant production host.

[0423] API-level drug components are highly purified molecules. Thus, impurities that may indicate a plant or yeast origin of the API (such as those listed in Tables 14 and 15) may not be present in the API stage of the product. In fact, many API products from yeast strains derived from some embodiments of the present disclosure may be largely indistinguishable from traditional plant-derived APIs. However, in some cases, chemical synthesis methods can be used to subject conventional alkaloid compounds to chemical modification, which may appear as chemical impurities in plant-based products that require such chemical modification. For example, chemical derivatization typically produces a set of impurities associated with the chemical synthesis process. In some cases, these modifications can be carried out biologically in a yeast production platform, thereby avoiding the presence of some impurities associated with chemical sources in yeast-derived products. In particular, these impurities from products of chemical origin may be present in API products produced using chemical synthesis methods, but may not be present in API products produced using yeast-derived products. Alternatively, if a yeast-derived product is mixed with a product of chemical origin, the resulting impurities may be present, but in an amount lower than that expected in an API containing only or predominantly a product of chemical origin. In this example, by analyzing a subset of these impurities in the API product, one of ordinary skill in the art can determine whether the product is derived from a yeast production host or a traditional chemical derivation pathway.

[0424] Non-limiting examples of impurities that may be present in a chemically derived morphinan API but not in a biosynthetic API include codeine-O(6)-methyl ether in the API codeine; 8,14-dihydroxy-7,8-dihydrocodeinone in the API oxycodone; and thebaine in the API hydrocodone. Codeine-O(6)-methyl ether may be formed by chemical over-methylation of morphine. 8,14-dihydroxy-7,8-dihydrocodeinone in the API oxycodone may be formed by chemical over-oxidation of thebaine. Additionally, thebaine in the API hydrocodone may be formed by chemical over-reduction of thebaine.

[0425] However, in cases where both yeast-derived compounds and plant-derived compounds are subjected to chemical modification by chemical synthesis methods, the same impurities associated with the chemical synthesis method can be expected in the product. In such cases, the starting materials (e.g., CYCM or CPS) can be analyzed as described above. Host cell - derived nal - opioids compared to chemically - derived nal - opioids

[0426] Nal - opioids produced by chemical synthesis may contain a variety of impurities. These impurities can be caused by many different reasons (e.g., unreacted starting materials, incomplete reactions, formation of by - products, persistence of intermediates, dimerization or degradation). Examples of unreacted starting materials can be the residual oxymorphone in naltrexone formulations. Examples of impurities produced by incomplete reactions can be 3 - O - methyl buprenorphine resulting from incomplete 3 - O - demethylation of thebaine. Chemical modification may lead to the addition or removal of functional groups at off - target sites. For example, oxidation of C10 during naltrexone synthesis to produce 10 - hydroxy naltrexone and 10 - keto naltrexone, or removal of 6 - O - methyl during buprenorphine synthesis to obtain 6 - O - demethyl buprenorphine. Impurities may arise from the persistence of reaction intermediates, such as N - oxides (like oxymorphone N - oxide) formed during N - demethylation processes. Another source of impurities is dimerization, i.e., the conjugation of two opioid molecules (e.g., two buprenorphine molecules (2,2’ - bisbuprenorphine), two naltrexone molecules (2,2’ - bisnaltrexone) or two naloxone molecules (2,2’ - bisnaloxone)). Impurities may come from the degradation of starting materials, reaction intermediates or reaction products. The extreme physical conditions used in chemical synthesis may make the presence of degradation more likely. Examples of impurities that may be produced by degradation are dehydrobuprenorphine produced under oxidative conditions during buprenorphine synthesis.

[0427] Nal - opioids produced by enzymatic catalysis in host cells can contain different impurities from those produced by chemical synthesis. Nal - opioids produced by enzymatic catalysis in host cells can contain fewer impurities compared to those produced by chemical synthesis. Nal - opioids produced by enzymatic catalysis in host cells may lack certain impurities found in those produced by chemical synthesis. In some examples, key features of enzymatic synthesis can include: (1) the enzyme targets specific substrates and residues with high fidelity; (2) the enzyme reacts under mild physiological conditions within the cell that do not compromise the stability of the molecule; and (3) the enzyme is engineered to be an effective catalyst for driving the reaction to completion.

[0428] Table 16 highlights some impurities that may be specific to chemically produced nal - opioids. Thus, the impurities of nal - opioids can be evaluated to determine if any impurities from Table 16 are present. By analyzing a subset of these impurities in a product of unknown origin, one of ordinary skill in the art can determine whether the product is derived from chemical or enzymatic synthesis. Method for engineering host cells

[0429] Also included are methods of engineering host cells for the purpose of producing enzymes and / or BIAs. Any convenient method can be used to effect insertion of DNA into the host cell. The methods are for inserting a heterologous coding sequence into an engineered host cell such that the host cell functionally expresses the enzyme and converts a starting compound of interest into the product enzyme and / or BIA of interest.

[0430] Any convenient promoter can be utilized in the subject engineered host cells and methods. The promoter driving the expression of the heterologous coding sequence can be a constitutive promoter or an inducible promoter, provided that the promoter is active in the engineered host cell. The heterologous coding sequence can be expressed from its native promoter or a non-native promoter can be used. Such promoters can be low to high 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 repressed or only mildly repressed by the presence of glucose in the medium is used. Exemplary promoters include but are not limited to promoters of glycolytic genes such as the promoter of the Bacillus subtilis tsr gene (the promoter region of the gene encoding fructose bisphosphate aldolase) or the promoter of the Saccharomyces cerevisiae gene encoding glyceraldehyde 3-phosphate dehydrogenase (GPD, GAPDH, or TDH3), the ADH1 promoter of baker's yeast, phosphate starvation-inducible promoters such as the PHO5 promoter of yeast, the alkaline phosphatase promoter from Bacillus licheniformis, yeast inducible promoters such as Gal1-10, Gal1, GalL, GalS, the repressible promoter Met25, tetO, and constitutive promoters such as the glyceraldehyde 3-phosphate dehydrogenase promoter (GPD), the alcohol dehydrogenase promoter (ADH), the translation elongation factor-1-α promoter (TEF), the cytochrome cyclooxygenase promoter (CYC1), the MRP7 promoter, etc. Autonomous replication yeast expression vectors containing promoters that can be induced by hormones such as glucocorticoids, steroids, and thyroid hormones can also be used and include but are not limited to glucocorticoid response elements (GREs) and thyroid hormone response elements (TREs). These and other examples are described in U.S. Patent No. 7,045,290, which is incorporated by reference in its entirety, including the references cited therein. Additional vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used. Additionally, any promoter / enhancer combination (according to the eukaryotic promoter database EPDB) can be used to drive the expression of the gene of interest. Any convenient and suitable promoter can be selected for the host cell (e.g., Escherichia coli). One can also use promoter selection to optimize the transcript and thus the enzyme level to maximize yield while minimizing energy resources.

[0431] Any convenient vector can be utilized in the subject engineered host cells and methods. Desired vectors include vectors for use in yeast and other cells. Types of yeast vectors can be classified into 4 major categories: integrative vectors (YIp), high-copy number vectors that replicate autonomously (YEp or 2μ plasmids), low-copy number vectors that replicate autonomously (YCp or centromeric plasmids), and vectors for cloning large fragments (YAC). Vector DNA can be introduced into prokaryotic or eukaryotic cells via any convenient transformation or transfection technique. Yeast can be engineered by integrating DNA from another source (e.g., a PCR-generated double-stranded DNA product, or a synthetic double-stranded or single-stranded oligonucleotide) into the genome. Any single transformation event can include one or several nucleic acids (vectors, double-stranded or single-stranded DNA fragments) to genetically modify the host cell. Table 10 shows examples of convenient vectors. Utility

[0432] The engineered host cells and methods of the present disclosure (e.g., as described above) can be used in a variety of applications. Desired applications include, but are not limited to: research applications and therapeutic applications. The methods of the present disclosure can be used in a variety of different applications, including any convenient application in which a desired enzyme and / or BIA is produced.

[0433] The subject engineered host cells and methods can be used in a variety of therapeutic applications. Desired therapeutic applications include those in which a pharmaceutical product comprising the desired BIA is prepared. The engineered host cells described herein produce the desired BIA and the desired enzyme. Reticuline is a major desired branch point intermediate in BIA synthesis, including engineering efforts to produce end products such as opioid products. The subject host cells can be utilized to produce the desired BIA from simple and inexpensive starting materials that can be used to produce the desired BIA, including reticuline and BIA end products. Thus, the subject host cells can be used to provide the desired BIA with therapeutic activity.

[0434] In some cases, the engineered host cells and methods can be used to produce commercial-scale amounts of BIA, where the chemical synthesis of these compounds has a low yield and is not a viable way for large-scale production. In certain cases, the host cells and methods are used in a fermentation facility, which will include a bioreactor (fermenter), e.g., having a capacity of 5,000 - 200,000 liters, allowing for rapid production of the desired BIA for therapeutic products. Such applications can include industrial-scale production of the desired BIA from fermentable carbon sources such as cellulose, starch, and free sugars.

[0435] The subject engineered host cells and methods can be used in a variety of research applications. The subject host cells and methods can be used to analyze the effects of a variety of enzymes on the biosynthetic pathways of a variety of target enzymes and / or BIAs. Additionally, the engineered host cells can be engineered to produce a target enzyme and / or BIA, which can be used to test the target bioactivity in unproven therapeutic functions. In some cases, the host cells are engineered to include a variety of heterologous coding sequences encoding a variety of enzymes that elucidate a high-yield biosynthetic pathway to the target enzyme and / or BIA. In certain cases, the research applications include producing a target enzyme and / or BIA for a target therapeutic molecule, and then the target enzyme and / or BIA can be further chemically modified or derivatized into the desired product or used for screening for increased target therapeutic activity. In some instances, host cell lines are used to screen for target enzyme activity in such pathways, which can lead to enzyme discovery via the BIA metabolites produced in these lines.

[0436] The subject engineered host cells and methods can be used as a production platform for plant specialized metabolites. The subject host cells and methods can be used as a platform for drug library development as well as plant enzyme discovery. For example, the subject engineered host cells and methods can be used for the development of a natural product-based drug library by employing yeast strains that produce scaffold molecules of interest (such as menisperine), and further functionalizing the compound structure either by combinatorial biosynthesis or by chemical means. By producing a drug library in this way, any potential drug hits are already associated with a production host suitable for large-scale culture and production. As another example, these subject engineered host cells and methods can be used for plant enzyme discovery. The subject host cells provide a clean background of defined metabolites to express 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. Kits and systems

[0437] Aspects of the present disclosure further include kits and systems, where the kits and systems can contain one or more components employed in the methods of the present disclosure, e.g., engineered host cells, starting compounds, heterologous coding sequences, vectors, media, etc., as described herein. In some embodiments, the subject kits contain an engineered host cell (e.g., as described herein) and one or more components selected from: starting compounds, heterologous coding sequences and / or vectors containing them, vectors, growth ingredients, components suitable for an expression system (e.g., cells, cloning vectors, multiple cloning sites (MCS), bidirectional promoters, internal ribosome entry sites (IRES), etc.), and media.

[0438] Any of the components described herein can be provided in a kit, e.g., including one or more modified host cells, starting compounds, culture media, etc. A variety of components suitable for preparing and using heterologous coding sequences, cloning vectors, and expression systems can be used in the subject kit. The kit can also contain test tubes, buffers, etc., and instructions for use. The various reagent components of the kit can be present in separate containers, or some or all of them can be pre-combined into a reagent mixture in a single container as needed.

[0439] Systems for producing a desired enzyme and / or BIA are also provided, where the systems can include engineered host cells containing one or more modifications (e.g., as described herein), starting compounds, culture media, fermenters, and fermentation equipment (e.g., devices suitable for maintaining host cell growth conditions), sampling and monitoring equipment and components, etc. A variety of components suitable for large-scale fermentation of yeast cells can be used in the subject systems.

[0440] In some cases, the systems include components for large-scale fermentation of engineered host cells and for monitoring and purifying the enzyme 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 where the engineered host cells in the fermenter produce one or more desired BIA products. In some instances, the host cells produce a desired BIA (e.g., as described herein). In certain cases, the desired BIA product is an opioid product, such as thebaine, codeine, neopine, morphine, neomorphine, hydrocodone, oxycodone, hydromorphanone, dihydrocodeine, 14-hydroxycodeine, dihydromorphine, and hydroxymorphanone. In some cases, the desired BIA product is a nal-opioid, such as naltrexone, naloxone, nalmefene, nalorphine, nalorphine, naloxiphene, naldemedine, naloxol, 6β-naltrexol, naltroxone, methylnaltrexone, methylsamidorphan, alvimopan, oprozomib, bivazepam, dinicotinate, levallorphan, samidorphan, buprenorphine, dezocine, etazocine, butorphanol, levorphanol, nalbuphine, pentazocine, phenazocine, nor-naltrexone, and diprenorphine. In some cases, the desired BIA product is a nor-opioid, such as norcodeine, noroxycodone, northebaine, norhydrocodone, nor-dihydro-codeine, nor-14-hydroxy-codeine, norcodeinone, nor-14-hydroxy-codeinone, normorphine, norhydroxymorphanone, nororientaline, norhydromorphanone, nor-dihydro-morphine, nor-14-hydroxy-morphine, normorphinone, and nor-14-hydroxy-morphinone. In some cases, the BIA product is a bisbenzylisoquinoline product, such as berberine, menisperine, dauricine, and liensinine.

[0441] 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 a subject host cell. For example, an LC-MS analysis system, a chromatography system, or any convenient system as described herein in which a sample can be analyzed and compared to a standard, for example, as described herein. The fermentation medium can be monitored by sampling and analysis at any convenient time before and during fermentation. When the conversion of the starting compound to the enzyme of interest and / or BIA product is complete, fermentation can be stopped and the BIA product can be purified. Thus, in some cases, the subject system includes a purification component adapted to purify the enzyme of interest and / or BIA product from the host cell medium producing the enzyme of interest and / or BIA product. The purification component can include any convenient process for purifying the enzyme of interest and / or BIA product produced by fermentation, including 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 system provides for the production and isolation of the enzyme of interest and / or BIA fermentation product after inputting one or more starting compounds into the system.

[0442] The following examples are provided to give the ordinary skilled in the art a complete disclosure and description of how to make and use the present disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure. Discussion of enzyme lists

[0443] Host cells can be engineered to include one or more modifications (such as two or more, three or more, four or more, five or more, or even more modifications) that provide for the production of the BIA of interest and / or the enzyme of interest. Tables 11 and 17 provide a list of exemplary genes that can be modified by one or more modifications to provide for the production of the BIA of interest and / or the enzyme of interest in engineered host cells.

[0444] The genetic modifications provided in Tables 11 and 17 can be used to produce a target BIA from engineered host cells supplied with a minimal medium containing the nutrients required for growth. This minimal medium can contain a carbon source, a nitrogen source, amino acids, vitamins, and salts. For example, the genetic modifications provided in Tables 11 and 17 can be used to produce a target BIA from engineered host cells fed with sugar. Additionally, modifications of one or more genes provided in Tables 11 and 17 can be used to enhance the biosynthetic processes of host cells that can be engineered for drug production.

[0445] Moreover, using these modifications to provide production of a target BIA and / or a target enzyme in engineered host cells is not readily apparent from merely identifying the enzymes that can be produced by the genes. In particular, as described herein, synthetic pathways that have been reconstituted in host cells such as yeast cells include multiple enzymes that do not naturally function together in a single organism. Additionally, some of the enzymes discussed herein are not used for BIA biosynthesis in their native context. Moreover, some of the enzymes described herein have not evolved to function in a particular host cell such as a yeast cell and have not evolved to function together. In these cases, it is not obvious that the enzymes will 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 downstream BIA end products.

[0446] For example, plant enzymes are generally difficult to functionally express in a heterologous microbial host such as yeast. In many cases, the enzymes may be misfolded, not correctly localized within the host cell, and / or incorrectly processed. Differences in protein translation and processing between yeast and plants can result in these enzymes exhibiting significantly reduced to undetectable activity in a yeast host. These challenges typically arise for inner membrane-localized enzymes such as cytochrome P450, which are strongly represented in the BIA pathway. Even reduced enzyme activity can pose a substantial challenge to engineering yeast to produce complex BIAs, which requires sufficient activity at each step to ensure high-level accumulation of the desired BIA product.

[0447] In addition, endogenous enzymes / pathways exist in some host cells (such as yeast) that may act on many of the early precursors in the BIA pathway (i.e., the intermediates from tyrosine to norcoclaurine), and thus, given these competing endogenous pathways, it may not be readily apparent whether there is sufficient flux through the heterologous pathway to achieve substantial 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 converts many of the intermediates in the early BIA pathway into unwanted products and diverts flux from the synthetic pathway.

[0448] Furthermore, as discussed herein and as provided in Tables 11 and 17, many of the enzymes may function under very specific regulatory strategies (including spatial regulation) in natural plant hosts, which may be lost upon transfer to a heterologous yeast host. Additionally, plants present a very different biochemical environment from that of yeast cells (in which the enzymes evolved to function), including pH, redox state, and substrate, cosubstrate, coenzyme, and cofactor availability. Given the differences in biochemical environment and regulatory strategies between the natural host and the heterologous yeast host, it is not obvious that the enzymes will exhibit substantial activity when in the context of the yeast environment, and further it is not obvious that they will function together to direct simple precursors (such as 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), such that if these steps are ineffective, it is expected that the desired downstream products will not accumulate.

[0449] In addition, in natural plant hosts, the relevant metabolites in these pathways can be localized to different cell and tissue types. In several examples, there are cell types that can be dedicated to biosynthesis and cell types that can be used for metabolite accumulation. This type of cell specialization may be lost when expressing these pathways within a heterologous yeast host and may play an important role in controlling the toxicity of these metabolites to the cells. Thus, it is not obvious that yeast can be successfully engineered to biosynthesize and accumulate these metabolites without being harmed by the toxicity of these compounds.

[0450] As an example, in natural plant hosts, the enzyme BBE has been reported to have a dynamic subcellular localization. Specifically, the enzyme BBE initially starts in the ER and is then sorted to the vacuole (Bird and Facchini. 2001. Planta. 213:888-97). It has been proposed that the ER association of BBE in plants (Alcantara et al. 2005. Plant Physiol. 138:173-83) provides the optimal alkaline pH (pH ~8.8) for BBE activity (Ziegler and Facchini. 2008. Annu. Rev. Plant Biol. 59:735-69). As another example, there is evidence that the biosynthesis of sanguinarine occurs in specialized vesicles within plant cells (Amann et al. 1986. Planta. 167:310-20), but only some of the intermediates accumulate in the vesicles. This can occur so as to sequester them from other enzyme activities and / or toxic effects.

[0451] As another example, the biosynthetic enzymes in the morphinan pathway branch are all localized to the phloem, which is part of the vascular tissue in plants. In the phloem, pathway enzymes can be further distinguished between two cell types: sieve elements, which are common to all plants, and laticifers, which are specialized cell types that are only present in certain plants that produce specialized secondary metabolites. The upstream enzymes (i.e., from NCS to SalAT) are mainly in the sieve elements, and the downstream enzymes (i.e., T6ODM, COR, CODM) are mainly in the laticifers (Onoyovwe et al. 2013. Plant Cell. 25:4110 - 22). Additionally, the final steps of the noscapine biosynthetic pathway were found to occur in the laticifers (Chen and Facchini. 2014. Plant J. 77:173 - 84). This compartmentalization is thought to be important for regulating biosynthesis by separating or transporting intermediates, providing optimal pH, and enhancing cofactor supply, although the nature of the opium poppy laticifer microenvironment is still under investigation (Ziegler and Facchini. 2008. Annu. Rev. Plant Biol. 59:735 - 69). Furthermore, several enzymes are predicted to function as multi - enzyme 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). When combining biosynthetic enzymes from different hosts and / or recombinantly expressing them in heterologous yeast cells, it is unclear whether these complexes or channels will form as they do in the native host. In another example, in Coptis japonica, berberine is biosynthesized in the root tissue and then accumulates in the rhizome via the action of specialized ATP - binding cassette transporters (Shitan et al. 2013. Phytochemistry. 91:109 - 16). In the opium poppy, morphinan alkaloids accumulate in the latex (the cytoplasm of laticifer cells) (Martin et al. 1967. Biochemistry. 6:2355 - 63).

[0452] Furthermore, even without these considerations, there are cases where the plant enzymes for several steps in the pathways described herein have not yet been characterized. For example, the conversion of tyrosine to the early benzylisoquinoline alkaloid scaffold norcoclaurine has not been characterized. Thus, for several steps in the pathways described herein, alternative biosynthetic schemes are generated by bringing together enzyme activities that do not normally occur together naturally for the biosynthesis of BIAs or by identifying new enzyme activities from genomic sequence information for use in a reconstructed pathway.

[0453] For example, the two-step conversion of tyrosine to dopamine can be achieved by combining at least 5 mammalian enzymes and 1 bacterial enzyme, where the at least 5 mammalian enzymes and 1 bacterial enzyme do not naturally occur together and have not evolved to function in the context of this pathway or with plant enzymes. In these cases, using these enzymes to biosynthesize compounds that they did not evolve for in nature, and getting these enzymes to function effectively in a heterologous microbial host and in the context of this pathway, may not be obvious.

[0454] Examples of genes that can be modified as targets to produce a target BIA and / or a target enzyme are discussed below. Additionally, genes are discussed in the context of a series of figures that illustrate pathways for generating a target BIA and / or a target enzyme.

[0455] [TKL1] In some examples, an engineered host cell can modify the expression of the enzyme transketolase. Transketolase is encoded by the TKL1 gene. In some examples, the reaction catalyzed by transketolase, such as as outlined in Figure 1 . The engineered host cell can be modified to include constitutive overexpression of the TKL1 gene in the engineered host cell. Additionally or alternatively, the engineered host cell can be modified to synthetically regulate the expression of the TKL1 gene in the engineered host cell. In some examples, the engineered host cell can be modified to incorporate one copy, multiple copies, or additional copies of the TKL1 gene. Additionally or alternatively, the engineered host cell can be modified to incorporate the introduction of a strong promoter element for overexpression of the TKL1 gene within the engineered host cell. The TKL1 gene can be derived from Saccharomyces cerevisiae or other species.

[0456] [ZWF1] In some examples, an 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 some examples, glucose-6-phosphate dehydrogenase catalyzes the reaction of glucose-6-phosphate → 6-phosphogluconolactone, as Figure 1 outlined in

[0457] [ARO4] In some examples, an engineered host cell can modify the expression of the enzyme 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase. DAHP synthase is expressed by the ARO4 gene. In some examples, DAHP synthase catalyzes the reaction of erythrose-4-phosphate + phosphoenolpyruvate → DAHP, as referenced Figure 1。Engineered host cells can modify the ARO4 gene to incorporate one or more feedback inhibition alleviating mutations. In particular, feedback inhibition alleviating mutations (e.g., ARO4 FBR ) can be incorporated as follows: site-directed mutations at the native locus of the native ARO4 gene; additional copies introduced as genetic integrations at separate loci; or additional copies on episomal vectors such as 2-μm or centromeric plasmids. The identifier “FBR” in mutant ARO4 FBR refers to one or more feedback resistance mutations. The feedback-inhibited copy of DAHP synthase can be under native yeast transcriptional regulation, such as when the engineered host cell is a yeast cell. Alternatively, the feedback-inhibited copy of DAHP synthase can be introduced into an engineered constitutive or dynamically regulated engineered host cell with protein expression by placing the feedback-inhibited copy of DAHP synthase under the control of a synthetic promoter. In some cases, the ARO4 gene can be derived from Saccharomyces cerevisiae. Examples of modifications of the ARO4 gene include the feedback inhibition resistance mutations K229L or Q166K.

[0458] [ARO7] In some examples, engineered host cells can modify the expression of chorismate mutase. Chorismate mutase is encoded by the ARO7 gene. In some examples, chorismate mutase catalyzes the reaction chorismate → prephenate, as referenced Figure 1 。Engineered host cells can modify the ARO7 gene to incorporate one or more feedback inhibition alleviating mutations. In particular, feedback inhibition alleviating mutations (e.g., ARO7 FBR ) can be incorporated as follows: site-directed mutations at the native locus of the native ARO7 gene; additional copies introduced as genetic integrations at separate loci; or additional copies on episomal vectors such as 2-μm or centromeric plasmids. The identifier “FBR” in mutant ARO7 FBR refers to one or more feedback resistance mutations. The feedback-inhibited copy of chorismate mutase can be under native yeast transcriptional regulation, such as when the engineered host cell is a yeast cell. Alternatively, the feedback-inhibited copy of chorismate mutase can be introduced into an engineered constitutive or dynamically regulated engineered host cell with protein expression by placing the feedback-inhibited copy of chorismate mutase under the control of a synthetic promoter. In some cases, the ARO7 gene can be derived from Saccharomyces cerevisiae. Examples of modifications of the ARO7 gene include feedback inhibition resistance mutations or T226I.

[0459] [ARO10] In some examples, engineered host cells can modify the expression of the enzyme phenylpyruvate decarboxylase. Phenylpyruvate decarboxylase is encoded by the ARO10 gene. In some examples, phenylpyruvate decarboxylase catalyzes the reaction hydroxyphenylpyruvate → 4-hydroxyphenylacetic acid (4-HPAA), as referenced Figure 1。Engineered host cells can be modified to include constitutive overexpression of the ARO10 gene in the engineered host cell. Additionally or alternatively, engineered host cells can be modified to synthetically regulate the expression of the ARO10 gene in the engineered host cell. In some examples, engineered host cells can be modified to incorporate one copy, multiple copies, or additional copies of the ARO10 gene. Additionally or alternatively, engineered host cells can be modified to incorporate the introduction of a strong promoter element for overexpression of the ARO10 gene within the engineered host cell. The ARO10 gene can be derived from Saccharomyces cerevisiae or another species.

[0460] [ADH2-7, SFA1] In some examples, engineered host cells can modify the expression of alcohol dehydrogenase. Alcohol dehydrogenase can be encoded by one or more of the ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, and SFA1 genes. In some examples, alcohol dehydrogenase catalyzes the reaction 4-HPAA → tyrosol. Engineered host cells can be modified to delete the coding regions of one or ...

Claims

1. A method for producing benzylisoquinoline alkaloid (BIA) products in engineered host cells, the method comprising: (a) expressing a heterologous enzyme having 14-hydroxylase activity in the engineered host cells; (b) expressing a heterologous enzyme having cytochrome P450 reductase (CPR) activity in the engineered host cells, (c) contacting the heterologous enzyme with a BIA precursor substrate, which is a morphinan alkaloid having a free hydrogen at carbon C-14, wherein the heterologous enzyme hydroxylates the C-14 carbon on the BIA precursor substrate; and (d) producing the BIA product within the host cells; wherein the engineered host cells produce more BIA products compared to non-engineered host cells.

2. The method according to claim 1, wherein the enzyme is cytochrome P450.

3. The method according to claim 1 or claim 2, wherein the heterologous enzyme comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO. 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164 or 166.

4. The method according to any one of claims 1-3, wherein the BIA precursor substrate is selected from codeine, codeinone, norcodeinone, hydrocodone, noscapine, orientaline, morphinone, normorphinone, hydromorphine, norhydromorphine and norhydrocodone.

5. The method according to any one of claims 1-4, wherein the BIA product is noroxymorphone.

6. The method according to any one of claims 1-5, wherein the engineered host cells express cytochrome P450 reductase (CPR).

7. The method according to claim 6, wherein the CPR is heterologous to the engineered host cells.

8. The method according to claim 6 or claim 7, wherein the CPR comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO. 168, 170, 172 or 174.

9. The method according to any one of claims 6-8, wherein the CPR is a fungal CPR.

10. A host cell that produces a BIA product, the host cell comprising a first heterologous polynucleotide encoding a heterologous enzyme having 14-hydroxylase activity and a second heterologous polynucleotide encoding cytochrome P450 reductase (CPR).

11. The host cell according to claim 10, wherein the enzyme having 14-hydroxylase is cytochrome P450.

12. The host cell according to claim 10 or claim 11, wherein the enzyme having 14-hydroxylase activity comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO.104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164 or 166.

13. The host cell according to any one of claims 10-12, wherein the CPR is a fungal CPR.

14. The host cell according to any one of claims 10-13, wherein the CPR comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO.168, 170, 172 or 174.

15. The host cell according to any one of claims 10-14, wherein the production of the BIA product comprises hydroxylation of the C-14 carbon on the BIA precursor substrate.

16. A vector, the vector comprising a first polynucleotide encoding an enzyme having 14-hydroxylase activity and a second polynucleotide encoding cytochrome P450 reductase (CPR).

17. The vector according to claim 16, wherein the enzyme having 14-hydroxylase is cytochrome P450.

18. The vector according to claim 16 or claim 17, wherein the polynucleotide encoding the enzyme having 14-hydroxylase activity comprises a nucleotide sequence selected from SEQ ID No.105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165 and 167.

19. The vector according to any one of claims 16-18, wherein the CPR is a fungal CPR.

20. The vector according to any one of claims 16-19, wherein the polynucleotide encoding the CPR comprises a nucleotide sequence selected from SEQ ID No.169, 171, 173 and 175.

21. The vector according to any one of claims 16 - 20, wherein the enzyme having 14-hydroxylase activity comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO.105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165 or 167.

22. The vector according to any one of claims 16 - 21, wherein the CPR comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO.169, 171, 173 or 175.

23. A host cell, the host cell comprising a vector, the vector comprising a first polynucleotide encoding an enzyme having 14-hydroxylase activity and a second polynucleotide encoding cytochrome P450 reductase (CPR).

24. The host cell according to claim 23, wherein the enzyme having 14-hydroxylase is cytochrome P450.

25. The host cell according to claim 23 or claim 24, wherein the CPR is a fungal CPR.

26. The host cell according to any one of claims 23 - 25, wherein the polynucleotide encoding the enzyme having 14-hydroxylase activity comprises a nucleotide sequence selected from SEQ ID No.105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165 and 167.

27. The host cell according to any one of claims 23 - 26, wherein the polynucleotide encoding the CPR comprises a nucleotide sequence selected from SEQ ID No.169, 171, 173 and 175.

28. The host cell according to any one of claims 23 - 27, wherein the enzyme having 14-hydroxylase activity comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO.104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164 or 166.

29. The host cell according to any one of claims 23 - 28, wherein the CPR comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO. 168, 170, 172 or 174.

30. A host cell comprising a vector, the vector comprising a first heterologous polynucleotide sequence encoding a heterologous enzyme having formaldehyde dehydrogenase activity and a second heterologous polynucleotide designed to repress the expression of a target gene, wherein the target gene is selected from DUG2 or DUG3.

31. The host cell according to claim 30, wherein the heterologous enzyme is SFA1.

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