Method for producing epimerase and benzylisoquinoline alkaloid

Abstract

To provide a method of epimerizing an (S)-1-benzylisoquinoline alkaloid to an (R)-1-benzylisoquinoline alkaloid.SOLUTION: The method comprises contacting the (S)-1-benzylisoquinoline alkaloid with at least one enzyme. Contacting the (S)-1-benzylisoquinoline alkaloid with the at least one enzyme converts the (S)-1-benzylisoquinoline alkaloid to an (R)-1-benzylisoquinoline alkaloid.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] cross reference Under 35 United States Code § 119(e), this application claims priority to the filing dates of U.S. Provisional Patent Application No. 62 / 159,122, filed 8 May 2015, and U.S. Provisional Patent Application No. 62 / 174,475, filed 11 June 2015, the disclosures of which are incorporated herein by reference.

[0002] In addition, this application is published here as US2014-0273109, U.S. Provisional Patent Application No. 14 / 211,611, which was filed on 14 March 2014 and has agent reference number STAN-1018; and PCT Application No. PCT / US2014 / 027833, which is published here as WO2014 / 143744, which was filed on 14 March 2014 and has agent reference number STAN-1018. U.S. Patent Application No. 15 / 031,618, which has N-1018WO; this application was filed on 22 April 2016 and has agent reference number STAN-1078; application number PCT / US2014 / 063738, which is published here as WO2015 / 066642; this application was filed on 3 November 2014 and has agent reference number STAN-1078WO; U.S. Provisional Patent Application No. 6 Application No. 2 / 080,610, filed on 17 November 2014, with agent reference number STAN-1169PRV; Provisional U.S. Patent Application No. 62 / 107,238, filed on 23 January 2015, with agent reference number STAN-1169PRV2; Application No. PCT / US2015 / 060891, filed on 16 November 2015, with agent reference number STA In relation to U.S. Provisional Patent Application No. 62 / 156,701, which has N-1169WO, filed on May 4, 2015, with agent reference number STAN-1221PRV; and Application No. PCT / US2016 / 030808, which has PCT / US2016 / 030808, filed on May 4, 2016, with agent reference number STAN-1221WO, the disclosures of these applications are incorporated herein by reference.

[0003] Description of research funded by the federal government. This invention was made with government support under contract number AT007886, awarded by the National Institutes of Health. The government has certain rights to this invention. [Overview of the Initiative]

[0004] This disclosure provides a method for producing diverse benzylisoquinoline alkaloids (BIAs) in genetically modified host cells. This disclosure further provides compositions of diverse alkaloids produced in genetically modified host cells. In addition, this disclosure provides a method for producing epimerase in genetically modified host cells. In particular cases, this disclosure provides a method for producing diverse alkaloid products in genetically modified host cells through epimerization of (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids. In even more particular cases, this disclosure provides a method for producing diverse alkaloid products through epimerization of (S)-reticulin to (R)-reticulin.

[0005] One aspect of the present invention provides genetically modified non-plant cells having increased tyrosine hydroxylase activity compared to unmodified cells. Another aspect of the present invention provides genetically modified non-plant cells having increased tyrosine hydroxylase activity compared to cells expressing wild-type TyrH. An additional aspect of the present invention provides genetically modified non-plant cells having increased tyrosine hydroxylase activity compared to cells expressing wild-type TyrH without the tyrosine hydroxylase activity-enhancing mutation provided herein. Specifically, the genetically modified non-plant cells have at least one modification selected from the group consisting of substrate inhibition reduction mutations; product inhibition reduction mutations; and cofactor recovery enhancement mechanisms.

[0006] An additional aspect of the present invention provides a method for epimerizing (S)-1-benzylisoquinoline alkaloid to (R)-1-benzylisoquinoline alkaloid. The method comprises contacting (S)-1-benzylisoquinoline alkaloid with at least one enzyme. Contacting (S)-1-benzylisoquinoline alkaloid with at least one enzyme converts (S)-1-benzylisoquinoline alkaloid to (R)-1-benzylisoquinoline alkaloid.

[0007] Another aspect of the present invention provides genetically engineered non-plant cells that produce an epimerase that converts (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids. The epimerase comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15, as shown in Table 1.

[0008] A further aspect of the present invention provides a process for converting an (S)-1-benzylisoquinoline alkaloid to an (R)-1-benzylisoquinoline alkaloid, the process comprising contacting the (S)-1-benzylisoquinoline alkaloid with an epimerase in an amount sufficient to convert the (S)-1-benzylisoquinoline alkaloid to an (R)-1-benzylisoquinoline alkaloid.

[0009] Another aspect of the present invention provides a method for epimerizing the stereocenter of a 1-benzylisoquinoline alkaloid. The method comprises contacting the 1-benzylisoquinoline alkaloid with at least one enzyme. The step of contacting the 1-benzylisoquinoline alkaloid with at least one enzyme inverts the stereochemistry of the stereocenter of the 1-benzylisoquinoline alkaloid to the opposite stereochemistry of the stereocenter of the 1-benzylisoquinoline alkaloid.

[0010] In some cases, genetically modified non-plant cells contain multiple coding sequences, each encoding an enzyme selected from the group of enzymes listed in Table 2. In some cases, heterologous coding sequences may be operably linked. The operably linked heterologous coding sequences may be within the same pathway producing a specific benzylisoquinoline alkaloid product and / or epimerase product.

[0011] In an additional aspect of the present invention, a genetically engineered non-plant cell is provided that produces a bisbenzylisoquinoline alkaloid. The bisbenzylisoquinoline alkaloid is produced using a coupling enzyme present in the genetically engineered non-plant cell. In addition, the genetically engineered non-plant cell contains at least one heterologous coding sequence that encodes at least one enzyme used to produce at least one benzylisoquinoline alkaloid monomer within the genetically engineered non-plant cell. Furthermore, the at least one coupling enzyme dimerizes two benzylisoquinoline alkaloid monomers within the genetically engineered non-plant cell, thereby forming a bisbenzylisoquinoline alkaloid.

[0012] [Invention 1001] A step of contacting an (S)-1-benzylisoquinoline alkaloid with at least one enzyme, wherein the (S)-1-benzylisoquinoline alkaloid is converted to an (R)-1-benzylisoquinoline alkaloid. A method for epimerizing the (S)-1-benzylisoquinoline alkaloid to the (R)-1-benzylisoquinoline alkaloid, comprising the above. [Invention 1002] The method of the present invention 1001, wherein the at least one enzyme is produced by culturing genetically modified non-plant cells having a coding sequence for encoding the at least one enzyme. [Invention 1003] (S)-1-benzylisoquinoline alkaloid added to the cell culture. The method of the present invention 1002, further comprising the above. [Invention 1004] The step of recovering the (R)-1-benzylisoquinoline alkaloid or its derivative from the cell culture. The method of the present invention 1003, further comprising: [Invention 1005] A method according to any one of the present invention 1001 to 1004, wherein the at least one enzyme comprises an oxidase. [Invention 1006] The method according to any one of the present invention 1001 to 1005, wherein the at least one enzyme comprises a reductase. [Invention 1007] The method according to any one of the present invention 1001 to 1006, wherein the at least one enzyme comprises an epimerase. [Invention 1008] The method of the present invention 1007, wherein the epimerase contains an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15. [Invention 1009] The method of the present invention 1007 or 1008, wherein the epimerase comprises an oxidase domain and a reductase domain. [Invention 1010] The method of the present invention 1009, wherein the oxidase domain is a cytochrome P450 oxidase-like domain. [Invention 1011] The method of the present invention 1009, wherein the reductase domain is a codeinone reductase-like domain. [Invention 1012] The method according to any one of the present invention 1007 to 1011, wherein the genetically modified non-plant cells are genetically modified yeast cells. [Invention 1013] Any method 1001 to 1012 of the present invention, wherein the (S)-1-benzylisoquinoline alkaloid is (S)-reticulin. [Invention 1014] Any method 1001 to 1013 of the present invention, wherein the (R)-1-benzylisoquinoline alkaloid is (R)-reticulin. [Invention 1015] The method according to any of the 1001 to 1014 of the present invention, wherein the (S)-1-benzylisoquinoline alkaloid is produced in the genetically modified non-plant cells via a metabolic pathway starting from L-tyrosine. Embedding by reference All publications, patents, and patent applications referenced herein are incorporated by reference to the same extent as each individual publication, patent, or patent application is specifically and individually incorporated by reference. [Brief explanation of the drawing]

[0013] Novel features of the present invention are described in detail in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following embodiments for carrying out the invention and the accompanying drawings, which describe exemplary embodiments in which the principles of the present invention are utilized. [Figure 1] Examples of synthesis, recycling, and regeneration pathways for tetrahydrobiopterin according to embodiments of the present invention are shown. [Figure 2] The present invention illustrates a biosynthetic scheme for the conversion of glucose to 4-HPA, dopamine, and 3,4-DHPA. [Figure 3] A schematic example of (R)-1-benzylisoquinoline alkaloid formation according to an embodiment of the present invention is shown. [Figure 4] The amino acid sequence of the CYP-COR enzyme according to an embodiment of the present invention is shown. [Figure 5] This invention presents a biosynthetic scheme for the conversion of L-tyrosine to reticulin via norcoclaurine, according to an embodiment of the present invention. [Figure 6] This invention presents a biosynthetic scheme for the conversion of L-tyrosine to reticulin via norlaudanotholin, according to an embodiment of the present invention. [Figure 7] The present invention illustrates a biosynthetic scheme for the conversion of L-tyrosine to morphinan alkaloids. [Figure 8]The present invention illustrates a biosynthetic scheme for the conversion of L-tyrosine to protoberberine, phthalidoquinoline, and berberine alkaloid products. [Figure 9] The present invention illustrates a biosynthetic scheme for the conversion of L-tyrosine to noscapine, noscapinoids, and phthalidoisoquinoline alkaloid products. [Figure 10] The present invention illustrates a biosynthetic scheme for the conversion of L-tyrosine to sanguinaline and benzophenanthridine alkaloids. [Figure 11A] The biosynthetic scheme for the conversion of canazine to noscapine according to an embodiment of the present invention is shown. [Figure 11B] This document shows a biosynthetic scheme for the production of semi-synthetic opioids according to an embodiment of the present invention. [Figure 12] The present invention presents a tyrosine hydroxylase mutant that enhances norcoclaurine production from sugars in genetically modified yeast strains according to an embodiment of the present invention. [Figure 13] The present invention presents a tyrosine hydroxylase mutant that enhances reticulin production from sugars in genetically modified yeast strains, according to an embodiment of the present invention. [Figure 14] This embodiment of the present invention shows the co-expression of dihydrofolate reductase (DHFR) in a genetically modified yeast strain that enhances L-DOPA production by tyrosine hydroxylase. [Figure 15] Figure 15(A) shows the addition of an antioxidant to a culture medium that enhances L-DOPA production by tyrosine hydroxylase in a genetically modified yeast strain according to an embodiment of the present invention. Figure 15(B) shows the addition of an antioxidant to a culture medium that increases BH4 levels according to an embodiment of the present invention. [Figure 16] Figure 16(A) shows a biosynthetic scheme for the conversion of L-tyrosine to bisBIA according to an embodiment of the present invention. Figure 16(B) shows a yeast strain genetically engineered to biosynthesize bisBIA according to an embodiment of the present invention. [Figure 17]This shows a phylogenetic tree of a cytochrome P450 oxidase-codeinone reductase-like (CYP-COR) fusion according to an embodiment of the present invention. [Figure 18] Figures 18(A) and (B) show LC-MS / MS analysis of yeast strains genetically engineered to convert (S)-reticulin to saltharidin, according to an embodiment of the present invention. [Figure 19] The following shows chiral LC-MS / MS analysis of a yeast strain genetically engineered to convert raceminol laudanosolin to (R)-reticulin, according to an embodiment of the present invention. [Figure 20] Figures 20(A) and (B) show genetically engineered fusions of saltharidin synthase, which removes N-linked glycosylation of proteins observed when heterologously expressed in yeast rather than plants, according to embodiments of the present invention. [Figure 21] Figures 21(A) and 21(B) show a caylantiforine synthase-saltharidin synthase fusion design according to an embodiment of the present invention. [Figure 22] The present invention presents embodiments of saltharidin synthasecodon-optimized and genetically engineered fusions that enhance activity in yeast. [Figure 23] Figures 23(A) and 23(B) show LC-MS / MS analysis of small-scale batch fermentation in which genetically modified yeast catalyzes the conversion of (R)-reticlin to thebaine and the conversion of rac-norlaudanotholin to thebaine, according to an embodiment of the present invention. [Figure 24] This embodiment of the present invention demonstrates the generation of CODM enzyme variants exhibiting enhanced activity in yeast through random mutagenesis and screening. [Figure 25] Figures 25(A), 25(B), and 25(C) show fermentation optimization for the conversion of (R)-reticulin to thebaine by genetically modified yeast according to an embodiment of the present invention. [Figure 26] This invention presents a yeast platform strain for the production of reticulin, an important branching intermediate from L-tyrosine, according to an embodiment of the present invention. [Figure 27A] This shows thebaine and hydrocodone production in genetically modified yeast strains according to embodiments of the present invention. [Figure 27B] This shows thebaine and hydrocodone production in genetically modified yeast strains according to embodiments of the present invention. [Figure 27C] This shows thebaine and hydrocodone production in genetically modified yeast strains according to embodiments of the present invention. [Figure 27D] This shows thebaine and hydrocodone production in genetically modified yeast strains according to embodiments of the present invention. [Figure 28] According to an embodiment of the present invention, the increased production of the benzylisoquinoline alkaloid reticulin is achieved by increasing the copy number of the NCS gene from two copies to three copies in a genetically modified yeast strain. [Modes for carrying out the invention]

[0014] Detailed description of the invention This disclosure provides a method for producing diverse benzylisoquinoline alkaloids (BIAs) in genetically modified host cells. This disclosure further provides compositions of diverse alkaloids produced in genetically modified host cells. In addition, this disclosure provides a method for producing epimerase in genetically modified host cells. In particular cases, this disclosure provides a method for producing diverse alkaloid products through epimerization of (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids in genetically modified host cells. In even more particular cases, this disclosure provides a method for producing diverse alkaloid products through epimerization of (S)-reticulin to (R)-reticulin.

[0015] The target benzylisoquinoline alkaloid (BIA) A host cell that produces the target BIA is provided. In some examples, a genetically engineered strain of the host cell, e.g., the genetically engineered strain of the present invention, provides a platform for producing the target benzylisoquinoline alkaloid and its variants across several structural classes, including but not limited to precursor BIAs, benzylisoquinoline, protoberberine, protopine, benzophenantholidine, promorphinan, morphinan, secoberberine, phthalidoisoquinoline, aporfin, bisbenzylisoquinoline, and others. Each of these classes is intended to include biosynthetic precursors, intermediates, and metabolites of those classes, of any favorable member of a genetically engineered host cell biosynthetic pathway that may lead to a member of that 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 itself. This chemical entity is intended to include all possible isomers, including single enantiomers, racemic mixtures, optically pure forms, mixtures of diastereomers, and mixtures of intermediates.

[0016] BIA precursors may include, but are not limited to, norcoclaurine (NC) and norlaudanothorine (NL), as well as NC and NL precursors, such as tyrosine, tyramine, 4-hydroxyphenylacetaldehyde (4-HPA), 4-hydroxyphenylpyruvic acid (4-HPPA), L-3,4-dihydroxyphenylalanine (L-DOPA), 3,4-dihydroxyphenylacetaldehyde (3,4-DHPA), and dopamine. In some embodiments, one or more BIA precursors are 3,4-dihydroxyphenylacetaldehyde (3,4-DHPA) and dopamine. In certain specific examples, one or more BIA precursors are 4-hydroxyphenylacetaldehyde (4-HPA) and dopamine. Specifically, NL and NC may be synthesized from precursor molecules, respectively, via a Pictet-Spengler condensation reaction, which may occur spontaneously or be catalyzed by any convenient enzyme.

[0017] Benzylisoquinolines may include, but are not limited to, norcoclaurine, norlaudanotholin, coclaurine, 3'-hydroxycoclaurine, 4'-O-methylnorlaudanotholin, 4'-O-methyl-laudanotholin, N-methylnorcoclaurine, laudanotholin, N-methylcoclaurine, 3'-hydroxy-N-methylcoclaurine, reticulin, norreticulin, papaverine, laudanin, laudanosine, tetrahydropapaverine, 1,2-dihydropapaverine, and orientaline.

[0018] Protoberberine may include, but is not limited to, skourelin, keirantiforine, styropine, nandinine, jatorolizin, stephorizin, discletamine, cis-N-methylstyropine, tetrahydrocolumbamin, palmatine, tetrahydropalmatine, columbamin, canazine, N-methylcanazine, 1-hydroxycanazine, berberine, N-methyl-ophiocarpine, 1,13-dihydroxy-N-methylcanazine, and 1-hydroxy-10-O-acetyl-N-methylcanazine.

[0019] Protopines may include, but are not limited to, protopine, 6-hydroxyprotopine, allocryptopine, cryptopine, muramine, and thalictricin.

[0020] Benzophenanthidines may include, but are not limited to, dihydrosanguinaline, sanguinaline, dihydrocaylirubine, caylirubine, dihydromarcapine, marcapine, and kereritrin.

[0021] Promorphinans may include, but are not limited to, saltharidinol, saltharidinol-7-O-acetate, and saltharidinol-7-O-acetate.

[0022] Morphinan may include, but is not limited to, thebaine, codeinone, codeine, morphine, morphinone, olipavin, neopinone, neopine, neomorphine, hydrocodone, dihydrocodeine, 14-hydroxycodeinone, oxycodone, 14-hydroxycodeine, morphinone, hydromorphone, dihydromorphine, dihydroethorphine, ethylmorphine, etorphine, methopone, buprenorphine, forcozine, heterocodeine, and oxymorphone.

[0023] Secoberberine may include, but is not limited to, 4'-O-desmethylmacrantaldehyde, 4'-O-desmethylpapaveroxin, 4'-O-desmethyl-3-O-acetylpapaveroxin, papaveroxin, and 3-O-acetylpapaveroxin.

[0024] Phthalidoisoquinolines may include, but are not limited to, narcotrin hemiacetal, narcotin hemiacetal, narcotrin, noscapine, capnoidin, adorumin, (+) or (-)-bicuculine, capnoidin, carrumin, collesin, collumidine, dekumbenin, 5'-O-demethylnarcotin, (+) or (-)-α or β-hydrastine, and hypercoumin.

[0025] Aporfin may include, but is not limited to, magnoflorine, colituberine, apomorphine, borzin, isoborzin, isotebaine, isocolituberine, and glaufin.

[0026] Bisbenzylisoquinoline may contain, but is not limited to, berbamnin, guatgaumeline, dauricin, and liensin.

[0027] Other compounds that may be produced by the genetically modified strains of the present invention may include, but are not limited to, roeadin, pavin, isopavin, and clarine.

[0028] In certain embodiments, the genetically engineered strains of the present invention may provide a platform for producing compounds related to tetrahydrobiopterin synthesis, including but not limited to dihydroneopterin triphosphate, 6-pyruboyltetrahydropterin, 5,6,7,8-tetrahydrobiopterin, 7,8-dihydrobiopterin, tetrahydrobiopterin 4α-carbinolamine, quinonoid dihydrobiopterin, and biopterin.

[0029] host cell Any convenient cell may be used as the host cell and method of the subject. In some cases, the host cell is a non-plant cell. In some examples, the host cell may be characterized as a microbial cell. In certain specific cases, the host cell is an insect cell, a mammalian cell, a bacterial cell, or a yeast cell. Any convenient type of host cell may be used in the production of the BIA-producing cell of the subject. See, for example, US2008 / 0176754 and US2014 / 0273109, these disclosures being incorporated in their entirety by reference. Target host cells include, but are not limited to, bacterial cells such as Bacillus subtilis, Escherichia coli, Streptomyces, 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 cases, the host cell is a yeast cell or an Escherichia coli cell. In some cases, the host cell is a yeast cell. In some cases, the host cells are derived from a yeast strain genetically engineered to produce the BIA of the desired type, such as (R)-1-benzylisoquinoline alkaloids. In some cases, the host cells are derived from a yeast strain genetically engineered to produce the enzyme of the desired type. In some cases, the host cells are derived from a yeast strain genetically engineered to produce epimerase. The epimerase may have oxidase and reductase. In addition, the epimerase may be able to convert (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids.Furthermore, epimerase can be separated into smaller enzymes that retain oxidase or reductase activity so that they can be used to convert (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids.

[0030] Any host cell described by Smolke et al. in US2008 / 0176754 and US2014 / 0273109 may be applicable to the use of the cells and methods of the subject. In certain embodiments, the yeast cell may be of the species Saccharomyces cerevisiae (S. cerevisiae). In certain embodiments, the yeast cell may be of the species Schizosaccharomyces pombe. In certain embodiments, the yeast cell may be of the species Pichia pastris. Yeast is the target as a host cell because cytochrome P450 proteins can be properly folded within the endoplasmic reticulum membrane so that their activity is maintained. In some examples, cytochrome P450 proteins are involved in several target biosynthetic pathways. In additional examples, cytochrome P450 proteins are involved in the production of the target BIA. In further examples, cytochrome P450 proteins are involved in the production of the target enzyme, such as epimerase having oxidase and reductase.

[0031] The target yeast strains used in this invention include, but are not limited to, CEN.PK (genotype: MATa / α ura3-52 / ura3-52trp1-289 / trp1-289 leu2-3_112 / leu2-3_112 his3 Δ1 / his3 Δ1 MAL2-8C / MAL2-8C SUC2 / SUC2), S288C, W303, D273-10B, X2180, A364A, Σ1278B, AB972, SK1 and FL100. In certain cases, the yeast strain is S288C (MATα;SUC2 mal mel gal2 CUP1 flo1 flo8-1 hap1), BY4741(MATα;his3Δ1;leu2Δ0;met15Δ0;ura3Δ0), BY4742(MATα;his3Δ1;leu2Δ0;lys2Δ0;ura3Δ0), BY4743(MATa / MATα;his3Δ1 / his3Δ1;leu2Δ0 / leu2Δ0;met15Δ0 / MET15;LYS2 / lys2Δ0;ura3Δ0 / ura3Δ0), and derivatives of WAT11 or W(R), W303-B strain (MATa;ade2-1;his3-11,-15;leu2-3,-112;ura3-1;canR;cyr+), these are from Arabidopsis thaliana. The strain expresses either NADPH-P450 reductase ATR1 or yeast NADPH-P450 reductase CPR1, respectively. In another embodiment, the yeast cell is W303α (MATα; his3-11, 15trp1-1 leu2-3 ura3-1 ade2-1). The identity and genotype of any additional yeast strain of interest can be found on EUROSCARF (web.uni-frankfurt.de / fb15 / mikro / euroscarf / col_index.html).

[0032] Genetic modification of host cells Host cells may be genetically engineered to include one or more modifications (e.g., two or more, three or more, four or more, five or more, or more) that provide the production of the BIA of interest. In addition, or separately, host cells may be genetically engineered to include one or more modifications (e.g., two or more, three or more, four or more, five or more, or more) that provide the production of the enzyme of interest. In some cases, the modifications are genetic alterations, e.g., mutations, additions or deletions of a gene or fragment thereof, or transcriptional regulation of a gene or fragment thereof. As used herein, the term “mutation” refers to the deletion, insertion or substitution of an amino acid residue or nucleotide residue compared to a reference sequence or motif. Mutations may be incorporated as directed mutations to the native gene at the original locus. In some cases, mutations may be incorporated as additional copies of the gene introduced as gene integration at separate loci, or as additional copies on an episomal vector, e.g., a 2μ or kinetochore plasmid. In certain cases, a substrate-inhibited copy of an enzyme is under the control of native cell transcription. In some cases, a substrate-inhibited copy of an enzyme is introduced along with genetically engineered constitutive or dynamic control of protein expression by placing it under the control of a promoter of synthesis. In some cases, one or more modifications may target native genes. In some cases, one or more modifications may target non-native genes. In some cases, non-native genes may be inserted into host cells. In further cases, non-native genes may be mutated by one or more modifications before being inserted into host cells.

[0033] Genetically modified host cells may overproduce one or more BIAs of interest. Overproduction means that the cells produce an increased or improved amount of the BIA molecule of interest compared to control cells (e.g., unmodified cells). Increased or improved production refers to both the production of some amount of the BIA of interest where the control does not produce any, and an increase of about 10% or more, e.g., about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, e.g., more than twice, e.g., more than five times, e.g., more than ten times, in situations where the control produces some of the BIA of interest.

[0034] Genetically modified host cells may overproduce one or more (S)-1-benzylisoquinoline alkaloids. In some cases, genetically modified host cells may produce some amount of the (S)-1-benzylisoquinoline alkaloid of interest when the control does not produce the (S)-1-benzylisoquinoline alkaloid, and may produce it in amounts of about 10% or more, for example, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, for example, more than twice, for example, more than five times, for example, more than ten times, when the control does produce some amount of the (S)-1-benzylisoquinoline alkaloid of interest.

[0035] Genetically modified host cells may further overproduce one or more (R)-1-benzylisoquinoline alkaloids. In some cases, genetically modified host cells may produce some amount of the (R)-1-benzylisoquinoline alkaloid of interest when the control does not produce any (R)-1-benzylisoquinoline alkaloids, and may produce about 10% or more, for example, about 20%, about 30%, about 40%, about 50%, about 60%, about 80%, about 100%, for example, more than twice, for example, more than five times, for example, more than ten times, when the control does produce some (R)-1-benzylisoquinoline alkaloid of interest. Genetically modified host cells may further overproduce one or more of morphinans, protoberberines, noscapinoids, and benzophenanthidine alkaloids.

[0036] In some cases, genetically modified host cells can produce increased amounts of (R)-reticulin compared to control host cells lacking one or more modifications (e.g., as described herein). In certain specific cases, the increased amount of (R)-reticulin is about 10% or more compared to control host cells, for example, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, about 2 times or more, about 5 times or more, or even about 10 times or more compared to control host cells. In some cases, (R)-reticulin is the product of an epimerization reaction in the genetically modified host cell. In these cases, (S)-reticulin may be the substrate for the epimerization reaction.

[0037] In addition, genetically modified host cells may overproduce one or more enzymes of interest. Overproduction means that the cell's production of the enzyme of interest is improved or increased compared to a control cell (e.g., an unmodified cell). Improved or increased production refers to both the production of some amount of the enzyme of interest where the control does not produce it, and an increase of approximately 10% or more, e.g., approximately 20% or more, approximately 30% or more, approximately 40% or more, approximately 50% or more, approximately 60% or more, approximately 80% or more, approximately 100% or more, in a situation where the control produces some of the enzyme of interest.

[0038] Genetically modified host cells may overproduce one or more CYP-COR enzymes. In some cases, genetically modified host cells may produce some amount of CYP-COR enzyme when the control does not produce any, and may increase production by about 10% or more, for example, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, for example, more than twice, for example, more than five times, for example, more than ten times, when the control does produce some amount of CYP-COR enzyme.

[0039] Genetically modified host cells may overproduce one or more enzymes derived from the CYP-COR enzyme. In some cases, genetically modified host cells may produce some amount of the CYP-COR enzyme when the control does not produce any enzyme derived from the CYP-COR enzyme, and may increase the amount to about 10% or more, for example, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, for example, more than twice, for example, more than five times, for example, more than ten times, when the control does produce some amount of the CYP-COR enzyme.

[0040] In addition, genetically modified host cells may overproduce one or more bisbenzylisoquinoline alkaloids (bisBIAs). Specifically, genetically modified host cells may produce increased amounts of bisbenzylisoquinoline alkaloids (bisBIAs) compared to control host cells lacking one or more modifications (e.g., as described herein). In certain cases, the increased amount of bisBIAs may be about 10% or more compared to control host cells, for example, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, about twice or more, about five times or more, or even about ten times or more compared to control host cells. In some cases, the bisBIA is formed from at least one BIA monomer or its derivative, which is the product of an epimerization reaction within the genetically modified host cell. Genetically modified host cells may further overproduce one or more of the following: cepharanthine, fungus quinoline, liensinin, nepheline, tubocurarine, dauricin, tetrandrin, culin, berbamnin, guattegaumeline, 2'-norberbamnin, and berbamin.

[0041] In some cases, one or more modifications (e.g., two or more, three or four or more) may be selected from: substrate inhibition reduction mutations in biosynthetic enzyme genes; product inhibition reduction mutations in biosynthetic enzyme genes; cofactor recovery enhancement mechanisms; feedback inhibition reduction mutations in biosynthetic enzyme genes; transcriptional regulatory modifications of biosynthetic enzyme genes; inactivation mutations in enzyme genes; epimerization modifications; bisBIA generation modifications; and heterologous coding sequences encoding enzymes. Cells containing one or more modifications may be referred to as genetically modified cells.

[0042] Substrate inhibition reduction mutation In some cases, the genetically modified host cell is a cell that contains one or more substrate inhibition reduction mutations (e.g., two or more, three or more, four or more, five or more, or more) in one or more of the cell's biosynthetic enzyme genes. In some cases, one or more biosynthetic enzyme genes are native to the cell (e.g., present in unmodified cells). In some cases, one or more biosynthetic enzyme genes are not native to the cell. As used herein, the term “substrate inhibition reduction mutation” refers to a mutation that reduces the cell’s substrate inhibition regulatory mechanism.

[0043] Mutations that reduce substrate inhibition decrease the inhibition of the regulated enzyme in the target cells compared to control cells, and increase the levels of the regulated compound or its downstream biosynthetic products. In some cases, the reduction of regulated enzyme inhibition leads to increased IC50. 50 However, this means an increase of more than two times, for example, more than three times, more than five times, more than ten times, more than thirty times, more than 100 times, more than three hundred times, more than one thousand hundred times, or more than one thousand hundred times, or even more. An increased level means a level of the regulated compound or its downstream product in the genetically modified host cell that is 110% or more of the level of the regulated compound or its downstream product in the control cell, for example, more than 120%, more than 130%, more than 140%, more than 150%, more than 160%, more than 170%, more than 180%, more than 190%, or more than 200%, for example, at least three times, at least five times, at least ten times, or even more.

[0044] Various substrate inhibition regulatory mechanisms and biosynthetic enzymes in genetically engineered host cells targeting the regulation of the level of a target BIA or its precursor can be targeted for substrate inhibition mitigation. A genetically engineered host cell may contain one or more substrate inhibition mitigation mutations within one or more biosynthetic enzyme genes. These mutations may be located within any convenient biosynthetic enzyme gene in which the biosynthetic enzyme is regulated. In some embodiments, one or more biosynthetic enzyme genes encode one or more tyrosine hydroxylase enzymes. In a particular example, one or more substrate inhibition mitigation mutations reside within a biosynthetic enzyme gene that is TyrH. In some embodiments, a genetically engineered host cell may contain one or more substrate inhibition mitigation mutations within one or more biosynthetic enzyme genes, such as one of the genes listed in Table 2.

[0045] In certain embodiments, one or more substrate inhibition-reducing mutations reside within the TyrH gene. The TyrH gene encodes tyrosine hydroxylase, an enzyme that converts tyrosine to L-DOPA. However, TyrH is inhibited by its substrate, tyrosine. Mammalian tyrosine hydroxylase activity, such as that observed in humans or rats, can be enhanced through mutations in the TyrH gene that mitigate substrate inhibition. Specifically, substrate inhibition from tyrosine can be mitigated by a point mutation W166Y within the TyrH gene. The point mutation W166Y within the TyrH gene may also improve the binding of BH4, a cosubstrate of tyrosine hydroxylase that catalyzes the reaction of tyrosine to L-DOPA. When TyrH mutants are expressed in yeast strains to produce BIA from sugar (such as those described in U.S. Provisional Patent Application No. 61 / 899,496), BIA production can be significantly enhanced.

[0046] The substrate inhibition control mechanism can be mitigated using any convenient number and type of mutations. In certain embodiments, the genetically engineered host cells of the present invention may contain one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, and even fifteen or more substrate inhibition mitigation mutations, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or fifteen substrate inhibition mitigation mutations in one or more biosynthetic enzyme genes within the genetically engineered host cells.

[0047] Cofactor recovery promotion mechanism In some cases, the genetically modified host cell is a cell that contains one or more cofactor recovery-promoting mechanisms (e.g., two or more, three or more, four or more, five or more, or more) within one or more of the cell's biosynthetic enzyme genes. In some cases, one or more biosynthetic enzyme genes are native to the cell (e.g., present in unmodified cells). In some cases, one or more biosynthetic enzyme genes are not native to the cell. As used herein, the term “cofactor recovery-promoting mechanism” refers to a mechanism that promotes the cell’s cofactor recovery regulatory mechanism.

[0048] Various cofactor recovery regulatory mechanisms and biosynthetic enzymes in genetically engineered host cells targeting the regulation of the level of a target BIA or its precursor can be targeted for cofactor recovery enhancement. A genetically engineered host cell may contain one or more cofactor recovery enhancement mechanisms within one or more biosynthetic enzyme genes. For example, a genetically engineered host cell may contain a heterologous coding sequence encoding dihydrofolate reductase (DHFR). When DHFR is expressed, it converts 7,8-dihydrobiopterin (BH2) to tetrahydrobiopterin (BH4), thereby recovering BH4 as a TyrH cosubstrate. In some examples, a genetically engineered host cell may contain one or more cofactor recovery enhancement mechanisms within one or more biosynthetic enzyme genes, such as one of the genes listed in Table 2.

[0049] Cofactor recovery regulatory mechanisms can be promoted using any convenient number and type of mechanisms. In certain embodiments, the genetically engineered host cells of the present invention may contain one or more cofactor recovery promoting mechanisms, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or even 15 cofactor recovery promoting mechanisms, within one or more biosynthetic enzyme genes in the genetically engineered host cells.

[0050] Mutations that reduce product inhibition In some cases, the genetically engineered host cell is a cell that contains one or more product inhibition mitigation mutations (e.g., two or more, three or more, four or more, five or more, or more) within one or more of the cell's biosynthetic enzyme genes. In some cases, one or more of the biosynthetic enzyme genes are native to the cell (e.g., present in unmodified cells). In some cases, one or more of the biosynthetic enzyme genes are not native to the cell. As used herein, the term “product inhibition mitigation mutation” refers to a mutation that reduces the short-term and / or long-term product inhibition regulatory mechanisms of the genetically engineered host cell. Short-term product inhibition is a cellular regulatory mechanism in which competitive binding occurs at the auxiliary substrate binding site. Long-term product inhibition is a cellular regulatory mechanism in which irreversible binding of compounds away from the desired pathway occurs.

[0051] Mutations that reduce product inhibition decrease the inhibition of the regulated enzyme in the target cells compared to control cells, and increase the levels of the regulated compound or its downstream biosynthetic products. In some cases, the reduction of regulated enzyme inhibition leads to increased IC50. 50However, this means an increase of more than two times, for example, more than three times, more than five times, more than ten times, more than thirty times, more than 100 times, more than three hundred times, more than one thousand hundred times, or more than one thousand hundred times, or even more. An increased level means a level of the regulated compound or its downstream product in the genetically modified host cell that is 110% or more of the level of the regulated compound or its downstream product in the control cell, for example, more than 120%, more than 130%, more than 140%, more than 150%, more than 160%, more than 170%, more than 180%, more than 190%, or more than 200%, for example, at least three times, at least five times, at least ten times, or even more.

[0052] Various product inhibition regulatory mechanisms and biosynthetic enzymes in genetically engineered host cells targeting the regulation of the desired BIA level may be targeted for product inhibition mitigation. The genetically engineered host cell may contain one or more product inhibition mitigation mutations within one or more biosynthetic enzyme genes. The mutations may be located within any convenient biosynthetic enzyme gene in which the biosynthetic enzyme is regulated. In some embodiments, one or more biosynthetic enzyme genes encode one or more tyrosine hydroxylase enzymes. In certain specific examples, one or more product inhibition mitigation mutations reside within the biosynthetic enzyme gene TyrH. In some embodiments, the genetically engineered host cell contains one or more product inhibition mitigation mutations within one or more biosynthetic enzyme genes, such as one of the genes listed in Table 2.

[0053] In certain embodiments, one or more product inhibition-reducing mutations reside within the TyrH gene. The TyrH gene encodes tyrosine hydroxylase, an enzyme that converts tyrosine to L-DOPA. TyrH requires tetrahydrobiopterin (BH4) as an auxiliary substrate to catalyze the hydroxylation reaction. While some microbial strains, such as Saccharomyces cerevisiae, do not naturally produce BH4, they can be genetically engineered to produce this substrate through four enzymatic synthesis and recirculation pathways, as shown in Figure 1. Figure 1 illustrates examples of tetrahydrobiopterin synthesis, recirculation, and regeneration pathways according to embodiments of the present invention. Figure 1 provides the use of the enzymes PTPS, pyruvoyltetrahydropterin synthase; SepR, sepiapterin reductase; PCD, pterin 4α-carbinolamine dehydrogenase; QDHPR, dihydropteridine reductase; and DHFR, dihydrofolate reductase. Of the enzymes shown in Figure 1, yeast synthesizes endogenous GTP cyclohydrolase I. GTP and dihydroneopterin triphosphate are synthesized naturally in yeast. In addition, the other metabolites shown in Figure 1 are not produced naturally in yeast.

[0054] TyrH is inhibited by its product, L-dopa, as well as other catecholamines, particularly dopamine. Mammalian tyrosine hydroxylase activity, such as from humans or rats, can be enhanced through mutations that mitigate product inhibition. For example, short-term product inhibition, such as competitive binding at the co-substrate binding site, can be mitigated by the point mutation W166Y on the TyrH gene. Specifically, the point mutation W166Y on the TyrH gene can improve co-substrate binding. In addition, short-term product inhibition that mitigates competitive binding at the co-substrate binding site can be enhanced by the point mutation S40D on the TyrH gene. Short-term product inhibition can also be enhanced by conjugational mutations R37E and R38E on the TyrH gene. Specifically, the R37E and R38E mutations can specifically enhance tyrosine hydroxylase activity together in the presence of dopamine.

[0055] In addition, long-term product inhibition can be mitigated by point mutations in the TyrH gene. Mitigation of long-term product inhibition may involve irreversible binding of catecholamines to iron at the active site, resulting in a reduced presence of catecholamines that act as product inhibitors of tyrosine hydroxylase activity. Long-term product inhibition can be mitigated by mutations E332D and Y371F, respectively, within the TyrH gene.

[0056] By creating combinations of mutations (for example, two, three, or more mutations at once), the activity of TyrH can be further enhanced by mitigating inhibition of multiple types of substrates and products. When TyrH mutants are expressed in yeast strains to produce BIA from sugars (such as those described in U.S. Provisional Patent Application No. 61 / 899,496), BIA production can be significantly enhanced.

[0057] Product inhibition control mechanisms can be mitigated using any favorable number and type of mutations. In certain embodiments, the genetically engineered host cells of the present invention may contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, and even fifteen or more product inhibition mitigation mutations, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or fifteen product inhibition mitigation mutations within one or more biosynthetic enzyme genes of the genetically engineered host cells.

[0058] Feedback inhibition reduction mutation In some cases, the genetically engineered host cell is a cell that contains one or more feedback inhibition mitigation mutations (e.g., two or more, three or more, four or more, five or more, or more) within one or more biosynthetic enzyme genes of the cell. In some cases, one or more biosynthetic enzyme genes are native to the cell (e.g., present in unmodified cells). In addition or / or, in some cases, one or more biosynthetic enzyme genes are not native to the cell. As used herein, the term “feedback inhibition mitigation mutation” refers to a mutation that mitigates the feedback inhibition regulatory mechanism of the genetically engineered host cell. Feedback inhibition is a cellular regulatory mechanism in which an enzyme in the synthesis pathway of a regulated compound is inhibited when the compound accumulates to a certain level, thereby regulating the amount of the compound in the cell. Mutations that mitigate feedback inhibition reduce the inhibition of the regulated enzyme in the genetically engineered host cell compared to control cells. In this way, the genetically engineered host cell increases the level of the regulated compound or its downstream biosynthetic product. In some cases, the mitigation of the inhibition of the regulated enzyme leads to an IC of inhibition. 50 However, this means an increase of more than two times, for example, more than three times, more than five times, more than ten times, more than thirty times, more than one hundred times, more than three hundred times, more than one thousand hundred times, or more than one thousand hundred times, or even more. An increased level means a level of the regulated compound or its downstream product in the host cell that is 110% or more of the level of the regulated compound or its downstream product in the control cell, for example, more than 120%, more than 130%, more than 140%, more than 150%, more than 160%, more than 170%, more than 180%, more than 190%, or more than 200%, for example, at least three times, at least five times, at least ten times, or even more.

[0059] Various feedback inhibition regulatory mechanisms and biosynthetic enzymes targeting the regulation of the desired BIA level may be targeted for mitigation in host cells. Host cells may contain one or more feedback inhibition mitigation mutations within one or more biosynthetic enzyme genes that are native to the cell. The one or more mutations may be located within any convenient biosynthetic enzyme gene in which the biosynthetic enzyme is regulated. In some embodiments, one or more biosynthetic enzyme genes may encode one or more enzymes selected from 3-deoxy-d-arabinose-heptulosone-7-phosphate (DAHP) synthase and colismyate mutase. In some embodiments, one or more biosynthetic enzyme genes encode 3-deoxy-d-arabinose-heptulosone-7-phosphate (DAHP) synthase. In some examples, one or more biosynthetic enzyme genes may encode colismyate mutase. In certain specific examples, one or more feedback inhibition mitigation mutations may be located within biosynthetic enzyme genes selected from ARO4 and ARO7. In certain cases, one or more feedback inhibition reduction mutations may be located within the biosynthetic enzyme gene ARO4. In certain cases, one or more feedback inhibition reduction mutations may be located within the biosynthetic enzyme gene ARO7. In some embodiments, the genetically engineered host cell may contain one or more feedback inhibition reduction mutations within one or more biosynthetic enzyme genes, such as one of the genes listed in Table 2.

[0060] The feedback inhibition control mechanism can be mitigated using any number and type of mutations that are advantageous to the system. As used herein, the term “mutation” refers to the deletion, insertion, or substitution of an amino acid residue or nucleotide residue compared to a reference sequence or motif. Mutations may be incorporated as directed mutations into the native gene at the original locus. In some cases, mutations may be incorporated as additional copies of the gene introduced as gene integration at separate loci, or as additional copies on an episomal vector, e.g., a 2μ or kinetochore plasmid. In certain examples, the feedback-inhibited copy of the enzyme is under native cellular transcriptional control. In some examples, the feedback-inhibited copy of the enzyme is introduced along with genetically engineered constitutive or dynamic regulation of protein expression by placing it under the control of a synthetic promoter.

[0061] In certain embodiments, one or more feedback inhibition reduction mutations may be present within the ARO4 gene. The ARO4 mutation of interest may include, but is not limited to, a substitution of a lysine residue with a leucine residue at position 229, a substitution of a glutamine residue with a lysine residue at position 166, or mutations described by Hartmann M, et al. ((2003) Proc Natl Acad Sci USA 100(3):862-867) or Fukuda et al. ((1992) J Ferment Bioeng 74(2):117-119). In some examples, the mutations to give feedback inhibition may be selected from a mutagenesis library of enzyme mutants. Examples of such options may include supporting the growth of o-fluoro-D,L-phenylalanine or aro3 mutant yeast strains in a medium containing excess tyrosine, as described by 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).

[0062] In certain embodiments, the genetically engineered host cells of the present invention may contain one or more feedback inhibition reduction mutations, for example, 1, 2, 3, 4, 5, 6, 7, 8, 8, 9, 10, 11, 12, 13, 14, or even 15, in one or more biosynthetic enzyme genes within the genetically engineered host cells.

[0063] Transcription regulation modification The host cell may contain one or more transcriptional modifications (e.g., two or more, three or more, four or more, five or more, or more) of one or more biosynthetic enzyme genes of the cell. In some examples, one or more biosynthetic enzyme genes are native to the cell. In some examples, one or more biosynthetic enzyme genes are not native to the cell. Any desirable biosynthetic enzyme gene of the cell may be targeted for transcriptional regulation. Transcriptional regulation means that the expression of the gene of interest in the modified cell is increased or decreased, enhanced or repressed compared to the regulated gene, e.g., control cell (e.g., unmodified cell). In some cases, transcriptional regulation of the gene of interest includes increasing or enhancing its expression. Increasing or enhancing expression means that the expression level of the gene of interest is increased by more than two times, e.g., more than five times, sometimes more than 25, 50, or 100 times, and in certain embodiments more than 300 times, compared to the expression in a control, i.e., an identical unmodified cell (e.g., by using any desirable gene expression assay). Alternatively, if the expression of the gene of interest in a cell is undetectable, the expression level of the gene of interest is considered to have increased if the expression level increases to a level that is easily detectable. In certain cases, transcriptional regulation of the gene of interest includes reducing or repressing its expression. Reducing or repressing expression means that the expression level of the gene of interest is reduced to less than or equal to 1 / 2, e.g., less than or equal to 1 / 5, sometimes 1 / 25, 1 / 50, or 1 / 100, and in certain embodiments less than or equal to 1 / 300, compared to the control. In some cases, the expression is reduced to an undetectable level. Modifications of the host cell process of interest that may be adapted for use in the host cell of the subject are described in U.S. Patent Publication No. 20140273109 (14 / 211,611) by Smolke et al., which disclosure is incorporated herein by reference in its entirety.

[0064] Any convenient biosynthetic enzyme gene may be transcriptionally regulated, including but not limited to the biosynthetic enzymes shown in Figure 2. Specifically, Figure 2 shows a biosynthetic scheme for the conversion of glucose to 4-HPA, dopamine, and 3,4-DHPA according to an embodiment of the present invention. Examples of enzymes shown in Figure 2 include ARO3, ARO4, ARO1, ARO7, TYR1, TYR, TyrH, DODC, MAO, ARO10, ARO9, and TKL. In some examples, one or more biosynthetic enzyme genes may be selected from ARO10, ARO9, and TKL. In some cases, one or more biosynthetic enzyme genes may be ARO10. In certain specific examples, one or more biosynthetic enzyme genes may be ARO9. In some embodiments, one or more biosynthetic enzyme genes may be TKL. In some embodiments, the host cell includes one or more transcriptional regulatory modifications to one or more genes, such as one of the genes listed in Table 2.

[0065] In some embodiments, transcriptional regulatory modifications may include the substitution of a strong promoter for the native promoter of one or more biosynthetic enzyme genes or the expression of an additional copy of one or more genes under the control of a strong promoter. The promoter driving the expression of the gene of interest may be a constitutive or inductive promoter, provided that the promoter can be active in the host cell. The gene of interest may be expressed from its native promoter. In addition, or separately, the gene of interest may be expressed from a non-native promoter. Although not essential, such promoters may be of medium to high intensity in the host in which they are used. The promoter may be controlled or constitutive. In some embodiments, promoters that are not glucose-repressed or are only mildly repressed by the presence of glucose in the culture medium may be used. Many suitable promoters exist, including promoters of glycolysis genes, such as the promoter of the B. subtilis tsr gene (encoding fructose diphosphate aldolase) or the GAPDH promoter from the yeast S. cerevisiae (encoding glyceraldehyde-phosphate dehydrogenase) (Bitter GA, Meth. Enzymol. 152:673 684 (1987)). Other potent promoters for different purposes include, but are not limited to, the baker's yeast ADHI promoter (Ruohonen L., et al, J. Biotechnol. 39:193 203 (1995)), phosphate deficiency-inducible promoters such as the yeast PHO5 promoter (Hinnen, A., et al, in Yeast Genetic Engineering, Barr, PJ, et al. eds, Butterworths (1989), the alkaline phosphatase promoter from B. licheniformis (Lee. JWK, et al., J. Gen. Microbiol. 137:1127 1133 (1991)), GPD1, and TEF1.The yeast promoters of interest include, but are not limited to, inducible promoters such as Gal1-10, Gal1, GalL, GalS, repressive promoters such as Met25 and 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), and MRP7 promoter. In some examples, the potent promoter is GPD1. In certain specific examples, the potent promoter is TEF1. Autonomous replicating yeast expression vectors containing promoters inducible by hormones such as glucocorticoids, steroids, and thyroid hormones are also known, including, but are not limited to, glucocorticoid-responsive elements (GRE) and thyroid hormone-responsive elements (TRE). See, for example, the promoter described in U.S. Patent No. 7,045,290. Vectors containing constitutive or inducible promoters such as α-factor, alcohol oxidase, and PGH may be used. In addition, any promoter / enhancer combination (according to the Eukaryotic Promoter Database EPDB) can be used to promote the expression of the gene of interest. It is understood that any favorable promoter specific to a host cell, e.g., E. coli, may be selected. In some cases, promoter selection may be used to optimize transcription and, therefore, enzyme levels, so as to maximize production while minimizing energy resources.

[0066] Inactivating mutation Genetically engineered host cells may contain one or more inactivating mutations (e.g., two or more, three or more, four or more, five or more, or more) in the cell's enzymes. The presence of one or more inactivating mutations can alter the flux of the synthetic pathway in the genetically engineered host cell to increase the level of the BIA of interest or the desired enzyme or precursor that produces the same event. In some cases, one or more inactivating mutations are to enzymes that are native to the cell. In addition or otherwise, one or more inactivating mutations are to enzymes that are not native to the cell. As used herein, “inactivating mutation” means one or more mutations in a cell's gene or regulatory DNA sequence that inactivates the biological activity of the protein expressed by the gene of interest. In some cases, the gene is native to the cell. In some cases, the gene encodes an enzyme that is part of, or linked to, the synthetic pathway of the BIA of interest that is inactivated and produced by the host cell. In some cases, the inactivating mutation is located in a regulatory DNA sequence that controls the gene of interest. In certain cases, the inactivating mutation is to the gene's promoter. Any desirable mutation (e.g., as described herein) can be used to inactivate a gene or regulatory DNA sequence of interest. “Inactivation” or “deactivation” means that the biological activity of the protein expressed by the mutated gene is reduced by 10% or more, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, or 99% or more, compared to the control protein expressed by an unmutated control gene. In some cases, the protein is an enzyme, and the inactivating mutation reduces the activity of the enzyme.

[0067] In some cases, genetically engineered host cells contain inactivating mutations in enzymes that are native to the cell. Any desirable enzyme can be targeted for inactivation. The enzyme of interest may include, but is not limited to, those listed in Table 2, whose action in the synthetic pathway of the genetically engineered host cell tends to reduce the level of the desired BIA. 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 the inactivating mutation is ADH2. In certain embodiments, the enzyme containing the inactivating mutation is ADH3. In certain embodiments, the enzyme containing the inactivating mutation is ADH4. In certain embodiments, the enzyme containing the inactivating mutation is ADH5. In certain embodiments, the enzyme containing the inactivating mutation is ADH6. In certain embodiments, the enzyme containing the inactivating mutation is ADH7. In some cases, the enzyme has aldehyde oxide reductase 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 the inactivating mutation is ALD2. In certain embodiments, the enzyme containing the inactivating mutation is ALD3. In certain embodiments, the enzyme containing the inactivating mutation is ALD4. In certain embodiments, the enzyme containing the inactivating mutation is ALD5. In certain embodiments, the enzyme containing the inactivating mutation is ALD6. In some embodiments, the host cell contains one or more inactivating mutations in one or more genes listed in Table 2.

[0068] Epimer transformation Several methods, processes, and systems described herein describe the conversion of (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids. Some of these methods, processes, and systems may involve genetically modified host cells. In some examples, the conversion of (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids is a critical step in the conversion of substrates to a diverse range of alkaloids. In some examples, the conversion of (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids involves an epimerization reaction. In some cases, epimerization of the substrate alkaloid may be carried out by oxidizing the (S)-substrate to the corresponding Schiff base or imine intermediate, and then stereospecifically reducing this intermediate to the (R)-product, as shown in Figure 3 and schematically represented in Scheme 1. As shown in Scheme 1, R1, R2, R3, and R4 may be H or CH3. R5 may be H, OH, or OCH3. TIFF0007871023000001.tif35170

[0069] In some cases, the conversion of an (S)-substrate to an (R)-product may necessarily involve at least one oxidation reaction and at least one reduction reaction. In some cases, a reduction reaction may optionally follow an oxidation reaction. In some cases, at least one of the oxidation and reduction reactions is carried out in the presence of an enzyme. In some cases, at least one of the oxidation and reduction reactions is catalyzed by an enzyme. In some cases, both the oxidation and reduction reactions are carried out in the presence of at least one enzyme. In some cases, at least one enzyme is useful for catalyzing the oxidation and reduction reactions. The oxidation and reduction reactions may be catalyzed by the same enzyme.

[0070] In some methods, processes, and systems described herein, the oxidation reaction may be carried out in the presence of an enzyme. In some examples, the enzyme may be an oxidase. The oxidase may use (S)-1-benzylisoquinoline as a substrate. The oxidase may convert the (S)-substrate to the corresponding imine or Schiff base derivative. The oxidase may be referred to as 1,2-dehydroreticulin synthase (DRS). Non-limiting examples of enzymes suitable for the oxidation of (S)-1-benzylisoquinoline alkaloids in this disclosure include cytochrome P450 oxidases, 2-oxoglutarate-dependent oxidases, and flavoprotein oxidases. For example, (S)-tetrahydroprotoberberine oxidase (STOX, E.C1.3.3.8) can oxidize (S)-norreticline and other (S)-1-benzylisoquinoline alkaloids to 1,2-dehydronorreticline and other corresponding 1,2-dehydro products. In some cases, proteins containing the oxidase domain of any one of the preceding examples may perform oxidation. In some cases, oxidases may catalyze oxidation reactions within host cells, such as the genetically engineered host cells described herein.

[0071] In some cases, the reduction reaction may occur after the oxidation reaction. The reduction reaction may be carried out by an enzyme. In some cases, the reductase may use an imine or Schiff base derived from 1-benzylisoquinoline as a substrate. The reductase may convert the imine or Schiff base derivative to (R)-1-benzylisoquinoline. The reductase may be called 1,2-dehydroreticline reductase (DRR). Non-limiting examples of enzymes suitable for the reduction of imines or Schiff bases derived from (S)-1-benzylisoquinoline alkaloids include aldo-ketereductases (e.g., codeinone reductase-like enzymes (EC 1.1.1.247)) and short-chain dehydrogenases (e.g., saltharidin reductase-like enzymes (EC 1.1.1.248)). In some cases, a protein containing any one of the reductase domains of the preceding examples may perform the reduction. In further embodiments, the reduction is stereospecific. In some examples, the reductase may catalyze the reduction reaction within a host cell, such as the genetically engineered host cells described herein.

[0072] An example of an enzyme capable of performing the epimerization reaction that converts (S)-1-benzylisoquinoline alkaloid to (R)-1-benzylisoquinoline alkaloid is an epimerase having an oxidase domain and a reductase domain. Specifically, the epimerase may have a cytochrome P450 oxidase 82Y2-like domain. In addition, the epimerase may have a codeinone reductase-like domain. Furthermore, an epimerase having both a cytochrome P450 oxidase 82Y2-like domain and a codeinone reductase-like domain may be called a CYP-COR enzyme. Specifically, the CYP-COR enzyme may be a fusion enzyme.

[0073] Figure 4 provides an example of an amino acid sequence of a CYP-COR enzyme that may be used to convert (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids. Specifically, Figure 4 shows the amino acid sequence of a CYP-COR enzyme according to an embodiment of the present invention. As seen in Figure 4, underlined text indicates a cytochrome P450 CYP82Y2-like domain (59% identity to AFB74617.1). Dotted underlined text indicates an aldo-ketereductase NADPH-dependent codeinone reductase-like domain (75% identity to ACM44066.1). Additional amino acid sequences of the CYP-COR enzyme are listed in Table 1. The amino acid sequence of the epimerase used to convert (S)-1-benzylisoquinoline alkaloid to (R)-1-benzylisoquinoline alkaloid may be 75% or more identical to a given amino acid sequence listed in Table 1. For example, such an amino acid sequence of epimerase may include an amino acid sequence that is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence provided herein. In addition, in certain embodiments, the “identical” amino acid sequence contains at least 80% to 99% identity at the amino acid level with respect to a particular amino acid sequence. In some cases, “identical” amino acid sequences contain at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, and 94%, and in certain cases even more, at least 95%, 96%, 97%, 98%, and 99% identity at the amino acid level. In some cases, amino acid sequences may be identical, but DNA sequences are mutated, for example, to optimize codon use with respect to the host organism.

[0074] A genetically engineered host cell can be provided that produces an epimerase that converts (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids, wherein the epimerase contains an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15. The epimerase produced in the genetically engineered host cell may be recovered and purified to form a biocatalyst. In some cases, the epimerase may be split into one or more enzymes. In addition, one or more enzymes produced by splitting the epimerase may be recovered from the genetically engineered host cell. These one or more enzymes resulting from the splitting of the epimerase may be used to catalyze the conversion of (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids. Specifically, one or more enzymes recovered from a genetically engineered host cell that produces epimerase may be used in a process for converting (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids. This process may include contacting the (S)-1-benzylisoquinoline alkaloid with epimerase in an amount sufficient to convert the (S)-1-benzylisoquinoline alkaloid to (R)-1-benzylisoquinoline alkaloid. For example, the (S)-1-benzylisoquinoline alkaloid may be contacted with one or more enzymes in sufficient quantities such that at least 5% of the (S)-1-benzylisoquinoline alkaloid is converted to (R)-1-benzylisoquinoline alkaloid.In further examples, the (S)-1-benzylisoquinoline alkaloid may be brought into contact with one or more enzymes in sufficient quantities 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.

[0075] One or more enzymes that may be used to convert (S)-1-benzylisoquinoline alkaloid to (R)-1-benzylisoquinoline alkaloid may be brought into contact with (S)-1-benzylisoquinoline alkaloid in vitro. In addition, one or more enzymes that may be used to convert (S)-1-benzylisoquinoline alkaloid to (R)-1-benzylisoquinoline alkaloid may be brought into contact with (S)-1-benzylisoquinoline alkaloid in vivo. In addition, one or more enzymes that may be used to convert (S)-1-benzylisoquinoline alkaloid to (R)-1-benzylisoquinoline alkaloid may be provided to cells containing (S)-1-benzylisoquinoline alkaloid or produced in genetically modified host cells.

[0076] In some embodiments, the method provides a genetically engineered host cell that produces an alkaloid product, wherein epimerization of the (S)-substrate to the (R)-product may include an important step in the production of the alkaloid product. In some embodiments, the alkaloid produced is a (R)-1-benzylisoquinoline alkaloid. In other embodiments, the alkaloid produced is derived from a (R)-1-benzylisoquinoline alkaloid and includes, for example, 4-ring promorphinane and 5-ring morphinane alkaloids. In yet another embodiment, the (S)-1-benzylisoquinoline alkaloid is an intermediate toward the product of the genetically engineered host cell. In yet another embodiment, the alkaloid product is selected from the group consisting of morphinane, protoberberine, noscapinoid, and benzophenanthidine alkaloids.

[0077] In some examples, the (S)-substrate is an (S)-1-benzylisoquinoline alkaloid selected from the group consisting of (S)-norreticline, (S)-reticline, (S)-tetrahydropapaverine, (S)-norcoclaurine, (S)-coclaurine, (S)-N-methylcoclaurine, (S)-3'-hydroxy-N-methylcoclaurine, (S)-norisoorientaline, (S)-orientaline, (S)-isoorientaline, (S)-norprotosinomenine, (S)-protosinomenine, (S)-norlaudanotholin, (S)-laudanotholin, (S)-4'-O-methyllaudanotholin, (S)-6-O-methylnorlaudanotholin, and (S)-4'-O-methylnorlaudanotholin.

[0078] In some examples, the (S)-substrate is given by formula I: It is a compound or salt of TIFF0007871023000002.tif35128, In the formula, R 1 , R 2 , R 3 , and R 4 It is independently selected from hydrogen and methyl; R 5is selected from hydrogen, hydroxy, and methoxy.

[0079] In some other examples, R 1 , R 2 , R 3 , R 4 and R 5 at least one of which is hydrogen.

[0080] In yet other examples, the (S)-substrate is a compound of formula II: TIFF0007871023000003.tif32128 or a salt thereof, wherein R 3 is selected from hydrogen and C1-C4 alkyl; R 6 and R 7 are each independently selected from hydroxy, fluoro, chloro, bromo, carboxyaldehyde, C1-C4 acyl, C1-C4 alkyl, and C1-C4 alkoxy; n is 0, 1, 2, 3, or 4; n’ is 0, 1, 2, 3, 4 or 5.

[0081] When a bond is depicted across a ring, this means that substitution can occur at any non-specific ring atom or position. For example, in formula II shown above, the hydrogen of any -CH- in the 6-membered ring can be substituted with R 7 to form -CR 7 -.

[0082] 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 yet other embodiments, R 3 is hydrogen or methyl.

[0083] In some other examples, the (S)-substrate is a compound of formula III: TIFF0007871023000004.tif35128 or a salt thereof, wherein R6 and R 7 These are independently selected from hydroxy, fluoro, chloro, bromo, carboxyaldehyde, C1-C4 acyl, C1-C4 alkyl, and C1-C4 alkoxy; n and n' are independently 0, 1, 2, 3, or 4.

[0084] In some examples, R 6 and R 7 n is independently hydroxy, methyl, or methoxy. In some other examples, n and n' are independently 1 or 2. In further embodiments, R 6 and R 7 These are independently fluoro, hydroxy, methyl, or methoxy.

[0085] In some examples, the method provides genetically engineered host cells that produce alkaloid products from (S)-reticulin. Epimerization of (S)-reticulin to (R)-reticulin may involve a key step in the production of diverse alkaloid products from a precursor. In some examples, the precursor is L-tyrosine or a sugar (e.g., glucose). Diverse alkaloid products may include, but are not limited to, morphinans, protoberberines, noscapinoids, and benzophenanthidine alkaloids.

[0086] Any suitable carbon source may be used as a precursor to the epimerized 1-benzylisoquinoline alkaloid. Suitable precursors may include, but are not limited to, monosaccharides (e.g., glucose, fructose, galactose, xylose), oligosaccharides (e.g., lactose, sucrose, raffinose), polysaccharides (e.g., starch, cellulose), or combinations thereof. In some examples, unrefined mixtures from renewable feedstocks can be used (e.g., corn steep liquor, beet molasses, barley malt, biomass hydrolysates). In other embodiments, the carbon precursor may be a monocarbon compound (e.g., methanol, carbon dioxide) or a dicarbon compound (e.g., ethanol). In yet another embodiment, other carbon-containing compounds, such as methylamine, glucosamine, and amino acids (e.g., L-tyrosine), can be used. In some examples, the 1-benzylisoquinoline alkaloids may be added directly to the genetically engineered host cells of the present invention, such as norlaudanotholin, laudanotholin, norreticulin, and reticulin. In further embodiments, the 1-benzylisoquinoline alkaloids may be added to the genetically engineered host cells as a single enantiomer (e.g., (S)-1-benzylisoquinoline alkaloid) or as a mixture of enantiomers, such as a racemic mixture.

[0087] In some examples, the method provides epimerization of the stereocenter of a 1-benzylisoquinoline alkaloid or a derivative thereof. In further embodiments, the method includes contacting a 1-benzylisoquinoline alkaloid with at least one enzyme. The at least one enzyme can invert the stereochemistry of the stereocenter of the 1-benzylisoquinoline alkaloid or a derivative thereof to the opposite stereochemistry. In some examples, the at least one enzyme converts a (S)-1-benzylisoquinoline alkaloid to a (R)-1-benzylisoquinoline alkaloid. In some examples of this conversion of (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinoline alkaloids using at least one enzyme, (S)-1-benzylisoquinoline alkaloids are (S)-norreticulin, (S)-reticulin, (S)-tetrahydropapaverine, (S)-norcoclaurine, (S)-coclaurine, (S)-N-methylcoclaurine, (S)-3'-Hyd Selected from the group consisting of roxy-N-methylcoclaurine, (S)-norisoorientaline, (S)-orientaline, (S)-isoorientaline, (S)-norprotosinomenine, (S)-protosinomenine, (S)-norlaudanosoline, (S)-laudanosoline, (S)-4'-O-methyllaudanosoline, (S)-6-O-methylnorlaudanosoline, and (S)-4'-O-methylnorlaudanosoline.

[0088] In some other examples, at least one enzyme converts 5'R-phthalide isoquinoline alkaloids to 5'S-phthalide isoquinoline alkaloids. In some examples of this conversion of 5'R-phthalide isoquinoline alkaloids to 5'S-phthalide isoquinoline alkaloids using at least one enzyme, the 5'R-phthalide isoquinoline alkaloids are selected from the group consisting of 5'R-narcotrin hemiacetal, 5'R-narcotrin, 5'R-narcotin hemiacetal, and 5'R-noscapine.

[0089] In some cases, the 3S,5'R-phthalide isoquinoline alkaloid is represented by formula IV: It is a compound or salt of TIFF0007871023000005.tif41128, In the formula, R 3 These are selected from hydrogen and C1-C4 alkyl groups; R 8 and R 9 These are independently selected from hydroxy, fluoro, chloro, bromo, carboxyaldehyde, C1-C4 acyl, C1-C4 alkyl, and C1-C4 alkoxy; R 10 It is not present, it is hydrogen, or a C1-C4 alkyl group; Two R's 8 And the carbon atoms to which they are attached optionally form 5- to 8-membered cycloalkyl or heterocycloalkyl groups; Two R's 9 And the carbon atoms to which they are attached optionally form 5- to 8-membered cycloalkyl or heterocycloalkyl groups; n and n' are independently 0, 1, 2, 3, or 4.

[0090] In some other examples, at least one enzyme converts 5'R-secoberberine alkaloids to 5'S-secoberberine alkaloids. In some examples of this conversion of 5'R-secoberberine alkaloids to 5'S-secoberberine alkaloids using at least one enzyme, the 5'R-secoberberine alkaloids are selected from the group consisting of 5'R-4'-O-desmethyl-3-O-acetylpapaberoxin, 5'R-3-O-acetylpapaberoxin, and 5'R-papaberoxin.

[0091] In some examples, 5'R-secoberberine alkaloids are given by formula V: It is a compound or salt of TIFF0007871023000006.tif37128, In the formula, R 3 These are selected from hydrogen and C1-C4 alkyl groups; R 8 and R 9These are independently selected from hydroxy, fluoro, chloro, bromo, carboxyaldehyde, C1-C4 acyl, C1-C4 alkyl, and C1-C4 alkoxy; R 11 It is selected from hydrogen and aldehydes; R 12 It is selected from hydrogen, hydroxyl, and O-methyl; R 13 It is selected from hydroxyl and O-methyl; Two R's 8 And the carbon atoms to which they are attached optionally form 5- to 8-membered cycloalkyl or heterocycloalkyl groups; Two R's 9 And the carbon atoms to which they are attached optionally form 5- to 8-membered cycloalkyl or heterocycloalkyl groups; n and n' are independently 0, 1, 2, 3, or 4.

[0092] In other embodiments, the 1-benzylisoquinoline alkaloid to be epimerized may contain two or more stereocenters, and only one of these stereocenters is inverted to produce a diastereomer of the substrate (for example, (S,R)-1-benzylisoquinoline alkaloid is converted to (R,R)-1-benzylisoquinoline alkaloid). In cases where only one stereocenter of the 1-benzylisoquinoline alkaloid is inverted upon contact with at least one enzyme, the product is referred to as an epimer of the 1-benzylisoquinoline alkaloid.

[0093] In some examples, the 1-benzylisoquinoline alkaloid is supplied to the enzyme as a single stereoisomer. In some other examples, the 1-benzylisoquinoline alkaloid is supplied to the enzyme as a mixture of stereoisomers. In even further embodiments, the mixture of stereoisomers may be a racemic mixture. In some other examples, the mixture of stereoisomers may be enriched in one stereoisomer compared to another.

[0094] In some examples, 1-benzylisoquinoline alkaloids or their derivatives are recovered. In some examples, 1-benzylisoquinoline alkaloids are recovered from cell cultures. In further embodiments, the recovered 1-benzylisoquinoline alkaloids are enantiomer-enriched in one stereoisomer compared to the original mixture of 1-benzylisoquinoline alkaloids provided to the enzyme. In a further embodiment, the recovered 1-benzylisoquinoline alkaloid has an enantiomer 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%.

[0095] "Isomers" are different compounds that have the same molecular formula. "Stereoisomers" are isomers that differ only in the spatial arrangement of their atoms. "Enantiomers" are pairs of stereoisomers that are mirror images of each other and cannot be superimposed. A 1:1 mixture of enantiomer pairs is a "racemic" mixture. "Diastereoisomers" are stereoisomers that have at least two chiral atoms but are not mirror images of each other. The term "epimer," as used herein, refers to compounds that have the same chemical formula but different optical configurations at specific positions. 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 determined according to the Cahn-Ingold-Prelog R-S system. When a compound is a pure enantiomer, the stereochemistry at each chiral carbon can be specified by either R or S. Decomposed compounds whose absolute configuration is unknown can be represented by (+) or (-) depending on the direction in which plane polarization is rotated at the wavelength of the sodium D line (dextrorotatory or levorotatory). Certain compounds described herein contain one or more chiral centers and can therefore give rise to enantiomers, diastereomers and other stereoisomers that can be defined as (R)- or (S)- in terms of absolute stereochemistry.

[0096] (Table 1) Examples of CYP-COR fusion enzyme portions and full-length amino acid sequences TIFF0007871023000007.tif145170TIFF0007871023000008.tif130170TIFF000 7871023000009.tif221170TIFF0007871023000010.tif240170TIFF00078710230 00011.tif255168TIFF0007871023000012.tif245170TIFF0007871023000013.t if246170TIFF0007871023000014.tif225170TIFF0007871023000015.tif123170

[0097] BisBIA generation modification Several methods, processes, and systems provided herein describe the production of bisbenzylisoquinoline alkaloids (bisBIA). BisBIA is a dimeric molecule that can be formed by a coupling reaction between two BIA monomers. In one example, bisBIA may be formed by a carbon-oxygen coupling reaction. In another example, bisBIA may be formed by a carbon-carbon coupling reaction. In some examples, the bisBIA dimeric molecule is a homodimer containing two identical BIA monomers. In one example, a genetically engineered host cell may produce one BIA monomer. In these examples, the BIA monomer may form a homodimer when in contact with one or more coupling enzymes. In another example, the bisBIA dimeric molecule is a heterodimer containing two different BIA monomers. For example, bisBIA may be a heterodimer containing BIA monomers that are enantiomers of each other. In some examples, a genetically engineered host cell may produce two or more BIA monomers. In these examples, BIA monomers can form homodimers and heterodimers when in contact with one or more coupling enzymes.

[0098] Some of these methods, processes, and systems describing the production of bis-BIAs may involve genetically engineered host cells. In some examples, genetically engineered host cells may be genetically engineered to produce BIA monomers, which may then be used as building block molecules for forming bis-BIAs. Examples of BIA monomers that may be used to form bis-BIAs include coclaurine, N-methylcoclaurine, laudanine, norcoclaurine, norlaudanotholin, 6-O-methyl-norlaudanotholin, 3'-hydroxy-N-methylcoclaurine, 3'-hydroxycoclaurine, reticulin, norreticulin, norlaudanine, laudanosine, and papaverine. Specifically, genetically engineered host cells may synthesize BIA monomers from norcoclaurine or norlaudanotholin by expressing heterologous enzymes including O-methyltransferase, N-methyltransferase, and 3'-hydroxylase. Examples of O-methyltransferases include norcoclaurine 6-O-methyltransferase (6OMT) from Thalictrum flavum, Nelumbo nucifera, Populus euphratica, or other species. Further examples of O-methyltransferases include catechol O-methyltransferase (COMT) from Homo sapiens, Mus musculus, Rattus norvegicus, Gorilla gorilla, or other species. Further examples of N-methyltransferases include coclaurine N-methyltransferase (CNMT) from T. flavum, N. nucifera, Aristolochia fimbriata, or other species. Examples of 3'-hydroxylases include N-methylcoclaurine 3'-hydroxylase (CYP80B1) from California poppy (Eschscholzia californica), T. flavum, N. nuciphylla, or other species.

[0099] Genetically modified host cells may produce either the (S) or (R) enantiomer of any given BIA monomer. In addition, or separately, genetically modified host cells may produce a mixture of both enantiomers. The ratio of (S) and (R) enantiomers may be determined by the substrate and product specificity of one or more enzymes that synthesize the BIA monomer. Alternatively, the amount of each enantiomer present may be modified by the expression of one or more additional enzymes that perform epimerization of one stereoisomer to another stereoisomer, as described above.

[0100] These BIA monomers may be fused into a dimeric bis-BIA skeleton. Specifically, BIA monomers may be fused into a dimeric bis-BIA skeleton using one or more enzymes produced by a genetically engineered host cell. In addition, or separately, BIA monomers may be fused into a dimeric bis-BIA skeleton using one or more enzymes supplied to the BIA monomers from a source outside the genetically engineered host cell. One or more enzymes may be used to form carbon-oxygen and / or carbon-carbon coupling reactions to fuse two BIA monomers at one, two, or three positions. In some examples, the two BIA monomers may be linked by an ether crosslink. In some examples, the two BIA monomers may be linked using a direct carbon-carbon bond. In some examples, the bis-BIA formed by fusing two BIA monomers may contain one diphenyl ether bond. In some examples, two BIA monomers may be fused to form a bis-BIA containing two diphenyl ether bonds. In some examples, the bis-BIA formed from two BIA monomers may contain three diphenyl ether bonds. In some cases, the bisBIA may contain one diphenyl ether linkage and one benzylphenyl ether linkage. In some cases, the bisBIA may contain one benzylphenyl ether linkage and two diphenyl ether linkages.

[0101] For example, a BIA monomer may be contacted with one or more enzymes in sufficient quantities to form a coupling reaction for fusing two BIA monomers, such that at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or 100% of the BIA monomer is converted to bisBIA. One or more enzymes that may be used to dimerize the BIA monomer to bisBIA may be contacted with the BIA in vitro. In addition, one or more enzymes that may be used to dimerize the BIA monomer into bisBIA may be brought into contact with BIA in vivo. In addition, one or more bisBIA dimerases may be expressed in a host cell that produces the BIA monomer. Alternatively, the BIA monomer may be provided to a genetically modified host cell that expresses a bisBIA dimerase. Alternatively, one or more bisBIA dimerases may be provided to a cell that contains the BIA monomer.

[0102] In some cases, bisbenzylisoquinoline alkaloids have the formula Va~Vu: It is one of the following compounds or a salt thereof: TIFF0007871023000016.tif122133TIFF0007871023000017.tif200132TIFF0007871023000018.tif118140. In the formula, R 1a , R 1b , R 2a , and R 2b This is independently selected from hydrogen and C1-C4 alkyl groups; R3a , R 3b , R 6a , R 6b , R 8a , and R 8b This is independently selected from hydrogen, hydroxy, fluoro, chloro, bromo, carboxyaldehyde, C1-C4 acyl, C1-C4 alkyl, and C1-C4 alkoxy; R 4a and R 5a is independently selected from hydrogen and C1-C4 alkyl, or R 4a and R 5a They combine to form a methylene bridge; R 4b and R 5b is independently selected from hydrogen and C1-C4 alkyl, or R 4b and R 5b They combine to form a methylene bridge; R 7a , R 7b , and R 9a This is independently selected from hydrogen and C1-C4 alkyl groups.

[0103] In some examples, R 1a and R 1b Each of them is hydrogen; R 2a and R 2b Each of them is methyl; R 3a and R 3b Each of them is hydrogen; R 4a and R 5a R is independently hydrogen or methyl; 4b and R 5b is independently hydrogen or methyl, or R 4b and R 5b They combine to form a methylene bridge; R 6a , R 6b , R 8a , and R 8b Each of them is hydrogen; R 7a , R 7b , and R 9a These are independently hydrogen or methyl.

[0104] As shown above, bis-BIA compounds of formulas Va, Vb, and Vd are formed by fusing two BIA monomers using a carbon-oxygen coupling reaction. In addition, bis-BIA compounds of formulas Vc, Vf, and Vh are formed by fusing two BIA monomers using both carbon-oxygen coupling reactions and carbon-carbon coupling reactions. Furthermore, bis-BIA compounds of formulas Ve, Vg, Vi, Vj, Vk, Vl, Vm, Vo, Vp, and Vq are formed by fusing two BIA monomers using two carbon-oxygen coupling reactions. The bis-BIA compound of formula Vn is formed by fusing two BIA monomers using two carbon-oxygen coupling reactions and one carbon-carbon coupling reaction. In addition, the bis-BIA compound of formula Vr is formed by fusing two BIA monomers using three carbon-oxygen coupling reactions.

[0105] One or more enzymes that may be used to form a coupling reaction may include known cytochrome P450 enzymes, such as Berberis stolonifera CYP80A1, or similar cytochrome P450 enzymes from other plants that naturally synthesize these compounds. Alternatively, the coupling reaction may be carried out by an enzyme that is not a cytochrome P450. One or more enzymes that may be used to form a coupling reaction may be genetically engineered to accept non-natural substrates. Thus, one or more enzymes that may be used to form a coupling reaction may be used to produce non-natural bis-BIA molecules. In one example, one or more enzymes may fuse a natural BIA monomer with a non-natural BIA monomer to produce a non-natural bis-BIA molecule. In another example, one or more enzymes may fuse two non-natural BIA monomers to produce a non-natural bis-BIA molecule. Strategies for genetically engineering enzymes may be used to identify one or more enzymes that may be used to form a coupling reaction that fuses BIA monomers to produce bis-BIA. For example, strategies for genetically modifying enzymes may include site-directed mutagenesis, random mutagenesis and screening, DNA shuffling, and screening.

[0106] Once a bisBIA is formed, the bisBIA may be further derivatized or modified. The bisBIA may be derivatized or modified using one or more enzymes produced by a genetically modified host cell. Specifically, the bisBIA may be derivatized or modified by contacting the bisBIA with one or more enzymes produced by a genetically modified host cell. In addition, or separately, the bisBIA may be derivatized or modified by contacting the bisBIA with one or more enzymes supplied to the bisBIA from a source outside the genetically modified host cell. A tailoring reaction may be carried out using one or more enzymes that may be used to derivatize or modify the bisBIA. Examples of tailoring reactions include oxidation, reduction, O-methylation, N-methylation, O-demethylation, acetylation, methylenedioxy crosslinking, and O,O-demethylenelation. The bisBIA may be derivatized or modified using one or more tailoring reactions.

[0107] Examples of Tayloring reactions are provided in Table 3. In some examples, Tayloring enzymes may be used to catalyze carbon-carbon coupling reactions performed on bisBIA or its derivatives. Examples of Tayloring enzymes that may be used to catalyze carbon-carbon coupling reactions include berberine cross-linking enzyme (BBE) from Papaver somniferum, California poppy, Coptis japonica, Berberis stronifera, Thalictrum flavum, or another species; saltharidin synthase (SalSyn) from Papaver somniferum or another species; and cortuberine synthase (CorSyn) from Coptis japonica or another species. A non-limiting example of reactions that can be catalyzed by Tayloring enzymes is shown in Scheme 2, where R a , R b , R c , and R dR is independently selected from hydrogen, hydroxyl, fluoro, chloro, bromo, carboxyaldehyde, C1-C4 acyl, C1-C4 alkyl, and C1-C4 alkoxy. In some examples, R a , R b , and the carbon atoms to which they are attached optionally form a carbon ring or a heterocycle. In some examples, R c , R d The carbon atoms to which they are attached optionally form a carbocyclic or heterocyclic ring. TIFF0007871023000019.tif89128

[0108] In some cases, tailoring enzymes may be used to catalyze oxidation reactions of bisBIA or its derivatives. Examples of tailoring enzymes that may be used to catalyze oxidation reactions include tetrahydroprotoberberine oxidase (STOX) from Coptis japonica, Argemone mexicana, Berberis wilsonae, or another species; dihydrobenzophenantholidine oxidase (DBOX) from Papaver somnifer or another species; methylstyropine hydroxylase (MSH) from Papaver somnifer or another species; and protopine 6-hydroxylase (P6H) from Papaver somnifer, California poppy, or another species.

[0109] Tayloring enzymes may be used to catalyze the methylenedioxy crosslinking reaction carried out on bisBIA or its derivatives. Examples of Tayloring enzymes that may be used to catalyze the methylenedioxy crosslinking reaction include stylopine synthase (StySyn) from Papaver somnifer, California poppy, thistle poppy, or another species; chelanthorine synthase (CheSyn) from Papaver somnifer, California poppy, thistle poppy, or another species; and canadine synthase (CAS) from Thalictrum flava, Coptis chinensis, or another species.

[0110] In other cases, a tailoring enzyme may be used to catalyze the O-methylation reaction carried out on bisBIA or its derivatives. Examples of tailoring enzymes that may be used to catalyze O-methylation reactions include norcoclaurine 6-O-methyltransferase (6OMT) from Papaver somnifer, Thalictrum flabbum, Coptis japonica, Papaver bracteatum, or another species; 3'hydroxy-N-methylcoclaurine 4'-O-methyltransferase (4'OMT) from Papaver somnifer, Thalictrum flabbum, Coptis japonica, Coptis umbellata, or another species; reticulin 7-O-methyltransferase (7OMT) from Papaver somnifer, California poppy, or another species; and skourelin 9-O-methyltransferase (9OMT) from Papaver somnifer, Thalictrum flabbum, Coptis japonica, Coptis umbellata, or another species.

[0111] In addition, a tailoring enzyme may be used to catalyze the N-methylation reaction of bisBIA or its derivatives. Examples of tailoring enzymes that may be used to catalyze the N-methylation reaction include coclaurine N-methyltransferase (CNMT) from Papaver somnifer, Thalictrum flava, Coptis japonica, or another species; and tetrahydroprotoberberine N-methyltransferase (TNMT) from Papaver somnifer, California poppy, Papaver bracteatum, or another species.

[0112] Furthermore, a tailoring enzyme may be used to catalyze the O-demethylation reaction of bisBIA or its derivatives. Examples of tailoring enzymes that may be used to catalyze the O-demethylation reaction include thebaine demethylase (T6ODM) from Papaver somnifer or another species, and codeine demethylase (CODM) from Papaver somnifer or another species.

[0113] Tayloring enzymes may be used to catalyze reduction reactions carried out on bis-BIA or its derivatives. Examples of Tayloring enzymes that may be used to catalyze reduction reactions include saltharidin reductase (SalR) from Papaver somnifer, Papaver bracteatum, or another species; codeinone reductase (COR) from Papaver somnifer or another species; and sanguinalin reductase (SanR) from California poppy or another species. In other examples, Tayloring enzymes may be used to catalyze acetylation reactions carried out on bis-BIA or its derivatives. Examples of Tayloring enzymes that may be used to catalyze acetylation reactions include saltharidin acetyltransferase (SalAT) from Papaver somnifer or another species.

[0114] Heterogeneous code arrays In some examples, a genetically engineered host cell has one or more heterocoding sequences (e.g., two or more, three or more, four or more, five or more) that encode activity enabling the genetically engineered host cell to produce a desired enzyme and / or a desired BIA, for example, as described herein. As used herein, the term “heterocoding sequence” means any polynucleotide that encodes or ultimately encodes a peptide or protein or its equivalent amino acid sequence, such as an enzyme, which is not normally present in the host organism and can be expressed in the host cell under appropriate conditions. Thus, a “heterocoding sequence” includes multiple copies of a coding sequence normally present in the host cell, so that the cell expresses additional copies of a coding sequence that is not normally present in the cell. The heterocoding sequence may be RNA or any type thereof, e.g., mRNA, DNA or any type thereof, e.g., cDNA, or an RNA / DNA hybrid. The coding sequence of interest includes, but is not limited to, a full-length transcription unit containing features such as a coding sequence, introns, promoter regions, 3'-UTR, and enhancer regions.

[0115] In the example, the genetically modified host cell may contain multiple heterogeneous coding sequences, each encoding an enzyme such as those listed in Table 2. In some examples, the enzymes encoded by the multiple heterogeneous coding sequences may be distinct from one another. In some examples, some of the enzymes encoded by the multiple heterogeneous coding sequences may be distinct from one another, and some of the enzymes encoded by the multiple heterogeneous coding sequences may be duplicate copies.

[0116] In some examples, heterologous coding sequences may be operably linked. The operably linked heterologous coding sequences may be in the same pathway that produces a specific benzylisoquinoline alkaloid product and / or epimerase product. In some examples, the operably linked heterologous coding sequences may be directly sequenced along a pathway that produces a specific benzylisoquinoline alkaloid product and / or epimerase product. In some examples, the operably linked heterologous coding sequences may have one or more native enzymes between one or more enzymes encoded by multiple heterologous coding sequences. In some examples, the heterologous coding sequences may have one or more heterologous enzymes between one or more enzymes encoded by multiple heterologous coding sequences. In some examples, the heterologous coding sequences may have one or more non-native enzymes between one or more enzymes encoded by multiple heterologous coding sequences.

[0117] Genetically modified host cells may be altered to contain one or more gene mutations to accommodate heterologous coding sequences. Mutations in the native host genome include, but are not limited to, altering the genome to reduce or eliminate the expression of specific proteins that may interfere with a desired pathway. The presence of such native proteins may rapidly convert one of the pathway's intermediates or final products into metabolites or other compounds that are not available in the desired pathway. Therefore, if the activity of the native enzyme is reduced or completely absent, the produced intermediates will be more readily available for incorporation into the desired product.

[0118] The heterocoding sequences include, but are not limited to, sequences encoding enzymes that are typically responsible for the production of the BIA of interest in plants, either wild-type or equivalent sequences. In some cases, the enzyme encoded by the heterocoding sequence may be any enzyme in the 1-BIA pathway and may be obtained from any convenient source. The selection and number of enzymes encoded by the heterocoding sequence with respect to a particular synthetic pathway may be selected based on the desired product. In certain embodiments of the present invention, the host cell may contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, and even fifteen or more heterocoding sequences, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or fifteen heterocoding sequences.

[0119] As used herein, the term “heterocoding sequence” also includes the coding portion of a peptide or enzyme, i.e., the cDNA or mRNA sequence of a peptide or enzyme, as well as the coding portion of a full-length transcription unit, i.e., a gene including introns and exons, and “codon-optimized” sequences, truncated sequences, or other forms of mutated sequences that code for or code for an enzyme or an equivalent amino acid sequence thereof, provided that this equivalent amino acid sequence produces a functional protein. Such equivalent amino acid sequences may have one or more amino acid deletions, which may be N-terminal, C-terminal, or intermediate. Truncated forms are assumed insofar as they possess the catalytic activity described herein. Fusions of two or more enzymes are also assumed to facilitate the translocation of metabolites in a pathway, provided that catalytic activity is maintained.

[0120] Operable fragments, mutants, or truncated forms can be identified by modeling and / or screening. In some cases, this is achieved, for example, by stepwise deleting the N-terminus, C-terminus, or internal region of a protein, and then analyzing the resulting derivative for its activity in relation to the desired reaction compared to the original sequence. If the derivative exhibits this capability, the composition of the derivative of the equivalent enzyme is considered appropriate.

[0121] In some examples, we can see that some heterologous proteins may be misprocessed when expressed in recombinant hosts. For example, plant proteins such as cytochrome P450 enzymes expressed in microbial-producing hosts may undergo misprocessing. Specifically, saltharidin synthase may undergo N-linked glycosylation when heterologously expressed in yeast. This N-linked glycosylation may not be observed in plants, but it may be an indicator of incorrect N-terminal sorting of the nascent SalSyn transcript, leading to decreased enzyme activity in heterologous microbial hosts. In such cases, protein manipulation targeting correct N-terminal sorting of the nascent transcript to eliminate the N-linked glycosylation pattern may result in improved activity of the saltharidin synthase enzyme in recombinant-producing hosts. This is further illustrated in Example 10 below.

[0122] Aspects of the present invention also relate to heterocoding 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 not identical to a particular amino acid sequence but rather contains at least some amino acid changes (deletions, substitutions, inversions, insertions, etc.) that, when used for a desired purpose, do not essentially affect the biological activity of the protein compared to the similar activity of the particular amino acid sequence. Biological activity, in the case of epimerase, refers to its catalytic activity. Equivalent sequences are also intended to include sequences that have been genetically engineered and / or evolved to have properties different from the original amino acid sequence. Mutable properties of interest include catalytic activity, substrate specificity, selectivity, stability, solubility, localization, etc.

[0123] In some cases, the expression of each type of enzyme is increased through additional gene copies (i.e., multiple copies), which increases intermediate accumulation and / or the production of the desired BIA. Embodiments of the present invention include increasing the production of the desired BIA in host cells through the co-expression of multiple species variants of one or more enzymes. In some cases, the additional gene copies of one or more enzymes are contained within the host cell. Any convenient method involving multiple copies of heterologous coding sequences of the enzyme in the host cell may be used.

[0124] In some cases, genetically engineered host cells contain multiple copies of heterologous coding sequences for enzymes, e.g., two or more, three or more, four or more, five or even ten or more copies. An example of this is shown in Figure 28. Specifically, Figure 28 shows increased production of the benzylisoquinoline alkaloid reticulin in a genetically engineered yeast strain due to an increase in the copy number of the NCS gene from two copies to three copies.

[0125] In certain embodiments, the genetically engineered host cell contains multiple copies of heterologous coding sequences for one or more enzymes, e.g., two or more, three or more, four or more, etc. In some cases, the multiple copies of the heterologous coding sequences of the enzyme are obtained from two or more different biological sources than the host cell. For example, the genetically engineered host cell may contain multiple copies of a single heterologous coding sequence, each copy obtained from a different biological source. Thus, each copy may contain some variability in its manifest sequence based on interspecies differences in the enzyme of interest encoded by the heterologous coding sequence.

[0126] Genetically modified host cell culture media may be sampled and observed for the production of the BIA of interest. The BIA of interest may be observed and measured using any convenient method. Such methods include, but are not limited to, LC-MS (e.g., as described herein), in which the sample of interest is analyzed by comparison with a known amount of a standard compound. In addition, other methods exist for observing and / or measuring the BIA of interest. Examples of alternative methods for observing and / or measuring BIA include, among others, GC-MS, UV-vis spectroscopy, NMR, LC-NMR, LC-UV, TLC, and capillary electrophoresis. Identity can be confirmed, for example, by m / z and MS / MS fragmentation patterns, and quantification or measurement of the compound can be achieved via EIC MS peak analysis by reference to LC trace peaks with known retention times and / or corresponding LC-MS analysis of a known amount of a standard compound.

[0127] In addition, cultures of genetically modified host cells may be sampled and observed for the production of target enzymes, such as CYP-COR enzymes. The target enzymes may be observed and measured using any convenient method. These methods include enzyme activity assays, polyacrylamide gel electrophoresis, carbon monoxide spectroscopy, and Western blot analysis.

[0128] method Process steps As summarized above, aspects of the present invention include methods for preparing a target benzylisoquinoline alkaloid (BIA). In addition, aspects of the present invention include methods for preparing a target enzyme. Accordingly, aspects of the present invention include culturing genetically engineered host cells under conditions in which one or more host cell modifications (e.g., as described herein) are functionally expressed so that the cells convert a target starting compound to a target product enzyme and / or BIA. Methods are also provided that include culturing genetically engineered host cells under conditions suitable for protein production so that one or more heterologous coding sequences are functionally expressed so that the target starting compound is converted to a target product enzyme or BIA. For example, the method is a method for preparing a benzylisoquinoline alkaloid (BIA), comprising the steps of culturing genetically engineered host cells (e.g., as described herein); adding a starting compound to the cell culture; and recovering the BIA from the cell culture. In some examples, the method is a method for preparing an enzyme, comprising the steps of culturing genetically modified host cells (e.g., as described herein); adding a starting compound to the cell culture; and recovering the enzyme from the cell culture.

[0129] The fermentation medium may contain a suitable carbon substrate. Suitable carbon sources for carrying out the methods of this disclosure may encompass a wide range of carbon-containing substrates. Suitable substrates may include, but are not limited to, monosaccharides (e.g., glucose, fructose, galactose, xylose), oligosaccharides (e.g., lactose, sucrose, raffinose), polysaccharides (e.g., starch, cellulose), or combinations thereof. In some cases, unrefined mixtures from renewable feedstocks may be used (e.g., corn steep liquor, beet molasses, barley malt). In some cases, the carbon substrate may be a monocarbon substrate (e.g., methanol, carbon dioxide) or a dicarbon substrate (e.g., ethanol). In other cases, other carbon-containing compounds, such as methylamine, glucosamine, and amino acids, may be used.

[0130] Any convenient method for culturing genetically modified host cells may be used to produce the enzyme and / or BIA of interest. The specific protocol used may vary depending on, for example, the genetically modified host cells, heterologous coding sequences, the enzyme of interest, the BIA of interest, etc. The cells may be in any convenient environment, for example, an environment in which the cells can express one or more functional heterologous enzymes. In some embodiments, the cells are cultured with appropriate substrates available to enable the production of the enzyme and / or BIA of interest in vivo, under conditions in which enzyme expression is possible. In some embodiments, the functional enzyme is extracted from the genetically modified host for the production of the enzyme and / or BIA of interest under in vitro conditions. In some examples, the genetically modified host cells are returned to a multicellular host organism. The genetically modified host cells are in any growth phase, including but not limited to the quiescent phase and the log growth phase. Furthermore, the culture itself may be continuous culture or batch culture.

[0131] Cells can grow in a suitable fermentation medium at temperatures between 14 and 40°C. Cells can grow with shaking at any convenient rate (e.g., 200 rpm). Cells can grow at a suitable pH. A suitable pH range for fermentation may be between pH 5 and 9. Fermentation can be carried out under aerobic, anaerobic, or microaerophilic conditions. Any suitable growth medium may be used. Suitable growth media may include, but are not limited to, commonly available media such as synthetic normal (SD) minimal medium or yeast extract peptone dextrose (YEPD) rich medium. Any other rich, normal, or synthetic growth medium suitable for the microorganism may be used.

[0132] Cells may be cultured in containers of essentially any size and shape. Suitable containers for carrying out the methods of this disclosure include, but are not limited to, multiwell shaking plates, test tubes, flasks (with and without baffles), and bioreactors. The volume of the culture may range from 10 microliters to more than 10,000 liters.

[0133] This may include the addition of agents to the growth medium that are known to regulate metabolism in a manner desirable for alkaloid production. In a non-limiting example, cyclic adenosine 2'3'-monophosphate may be added to the growth medium to regulate catabolism inhibition.

[0134] Any convenient cell culture conditions for a particular cell type may be used. In certain embodiments, host cells containing one or more modifications are cultured under standard or easily optimized conditions with standard cell culture media and adjuncts. For example, a standard growth medium when selective pressure for plasmid maintenance is not required may contain 20 g / L of yeast extract, 10 g / L of peptone, and 20 g / L of dextrose (YPD). Host cells containing the plasmid are grown in synthetic complete (SC) medium containing 1.7 g / L of yeast nitrogen base, 5 g / L of ammonium sulfate, and 20 g / L of dextrose, supplemented with the appropriate amino acids required for growth and selection. Alternative carbon sources that may be useful for inducible enzyme expression include, but are not limited to, sucrose, raffinose, and galactose. The cells are grown in the laboratory in a container, such as a test tube or flask, in a volume ranging from 1 to 1000 mL or more, at a suitable temperature (e.g., 30°C) and shaken at a suitable speed (e.g., 200 rpm).

[0135] The culture volume may be scaled up, for example, as part of an industrial process, for growth in larger fermentation vessels. The industrial fermentation process may be carried out in closed batches, fed-batch batches, or under continuous chemostat conditions, or in any preferred mode of fermentation. In some cases, cells may be immobilized on a substrate as a whole cell catalyst and subjected to fermentation conditions for alkaloid production.

[0136] Batch fermentation is a closed system in which the composition of the culture medium is set at the start of fermentation and is not changed during the fermentation process. The desired organism is inoculated into the medium at the start of fermentation. In some examples, batch fermentation is carried out with modifications made to the system to control factors such as pH and oxygen concentration (but not carbon). In this type of fermentation system, the biomass and metabolite composition of the system changes continuously throughout the fermentation process. Cells typically progress through an induction phase, then a logarithmic growth phase (high growth rate), then a stationary phase (growth rate decreases or stops), and finally a death phase (if left untreated).

[0137] Continuous fermentation is an open system in which a predetermined fermentation medium is continuously added to a bioreactor and an equal amount of fermentation medium is continuously removed from the container for processing. Continuous fermentation systems are generally operated to maintain steady-state growth conditions so that cell loss due to medium removal balances the growth rate in fermentation. Continuous fermentation is generally operated under conditions where cells are at a constant high cell density. Continuous fermentation allows for the regulation of one or more factors that affect the concentration of a target product and / or cell growth.

[0138] The liquid culture medium may include, but is not limited to, rich or synthetic standard culture media having the above-mentioned additive components. The culture medium components may be dissolved in water and sterilized by heat, pressure, filtration, radiation, chemicals, or any combination thereof. Some culture medium components may be prepared separately, sterilized, and then mixed in the fermentation vessel. The culture medium may be buffered to help maintain a constant pH throughout the fermentation.

[0139] Process parameters, including temperature, dissolved oxygen, pH, agitation, aeration rate, and cell density, can be observed or controlled throughout the fermentation process. For example, the temperature of the fermentation process can be observed by a temperature probe immersed in the culture medium. The culture temperature can be controlled to a set value by adjusting the jacket temperature. Water can be cooled in an external cooler and then flowed into a bioreactor control tower, where it can be circulated through the jacket at the temperature required to maintain the set temperature in the vessel.

[0140] In addition, gas flow parameters can be observed during the fermentation process. For example, gas may flow into the culture medium through a sparger. Suitable gases for the method of this disclosure may include compressed air, oxygen, and nitrogen. The gas flow may be at a fixed rate or may be adjusted to maintain a dissolved oxygen setpoint.

[0141] The pH of the culture medium can also be observed. In the example, the pH can be observed by a pH probe immersed in the culture medium in a container. If pH control is effective, the pH can be adjusted by acidic and basic pumps that add each solution to the culture medium at the required rate. The acidic solution used to control the pH may be sulfuric acid or hydrochloric acid. The basic solution used to control the pH may be sodium hydroxide, potassium hydroxide, or ammonium hydroxide.

[0142] Furthermore, dissolved oxygen can be observed in the culture medium by a dissolved oxygen probe immersed in the medium. If dissolved oxygen control is effective, the oxygen level can be adjusted by increasing or decreasing the stirring speed. The dissolved oxygen level can also be adjusted by increasing or decreasing the gas flow rate. The gas may be compressed air, oxygen, or nitrogen.

[0143] The stirring speed can also be observed during the fermentation process. In this example, a stirring motor may drive the stirrer. The stirring speed may be set to a consistent rpm throughout the fermentation process, or it may be dynamically adjusted to maintain a set dissolved oxygen level.

[0144] In addition, turbidity can be observed during the fermentation process. For example, cell density can be measured using a turbidity probe. Alternatively, cell density can be measured by taking samples from the bioreactor and analyzing them with a spectrophotometer. Furthermore, samples can be removed from the bioreactor at time intervals through a sterile sampling device. Samples can be analyzed for alkaloids produced by host cells. Samples can also be analyzed for other metabolites and sugars, deficiencies of culture medium components, or cell density.

[0145] In another example, feedstock parameters can be observed during the fermentation process. Specifically, feedstocks, nutrients, and cofactors, including sugars and other carbon sources that may be added to the fermentation using an external pump. Other components may also be added during fermentation, including, but are not limited to, antifoaming agents, salts, chelating agents, surfactants, and organic liquids.

[0146] Any convenient codon optimization technique for optimizing the expression of heterologous polynucleotides in host cells may be adapted for use in the subject host cells and methods; see, for example, Gustafsson, C. et al. (2004) Trends Biotechnol, 22, 346-353, which is incorporated herein by reference in its entirety.

[0147] The subject method may also include the step of adding a starting compound to a cell culture. Any convenient method of addition may be adapted for use in the subject method. The cell culture may be supplemented with a sufficient amount of the desired starting material (e.g., those described herein), for example, in mM to μM amounts, e.g., about 1 to 5 mM of the starting compound. It should be understood that the amount of starting material added, the timing and rate of addition, and the form of the material added may vary depending on various factors. The starting material may be added without a solvent, or it may be pre-dissolved in a suitable solvent (e.g., cell culture medium, water, or organic solvent). The starting material may be added in a concentrated form (e.g., 10 times or more of the desired concentration) to minimize the dilution ratio of the cell culture medium at the time of addition. The starting material may be added in one or more batches, or it may be added continuously over a long period of time (e.g., several hours or several days).

[0148] Method for isolating products from fermentation media The subject method may also include a step of recovering the target enzyme and / or BIA from the cell culture. Any convenient separation and isolation method (e.g., chromatography or precipitation) can be adapted for use in the subject method to recover the target enzyme and / or BIA from the cell culture. Filtration can be used to separate the soluble fraction from the insoluble fraction of the cell culture. In some cases, liquid chromatography (e.g., reversed-phase HPLC, size exclusion chromatography, normal-phase chromatography) can be used to separate the target BIA 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 target enzyme and / or BIA from other components of the cell culture.

[0149] The alkaloids produced may be isolated from the fermentation medium using methods known in the art. Several recovery steps may be performed immediately after (or, in some examples, during) fermentation for the initial recovery of the desired product. Through these steps, the alkaloids (e.g., BIA) may be separated from the cells, cell debris, and waste, while other nutrients, sugars, and organic molecules may remain in the used medium. This process may be used to obtain a BIA-enriched product.

[0150] In one example, a product stream containing a benzylisoquinoline alkaloid (BIA) product is formed by supplying genetically modified yeast cells, a feedstock containing nutrients, and water to a batch reactor. Specifically, the genetically modified yeast cells can be subjected to fermentation by incubation for at least about 5 minutes to produce a solution containing the BIA product and cellular material. Once the genetically modified yeast cells have been subjected to fermentation, the BIA product can be separated from the cellular material using at least one separation device to provide a product stream containing the BIA product. Specifically, the product stream may contain the BIA product as well as additional components, such as clarified yeast culture medium. In addition, the BIA product may contain one or more BIAs of interest, such as one or more BIA compounds.

[0151] Cells may be removed from the bioreactor medium containing the enzyme and / or BIA of interest using different methods. In one example, cells may be removed by sedimentation over time. This sedimentation process can be accelerated by cooling or by adding a clarifying agent such as silica. The used medium may then be aspirated from the top of the reactor, or the cells may be decanted from the base of the reactor. Alternatively, cells may be removed by filtration through a filter, membrane, or other porous material. Cells may also be removed by centrifugation, e.g., continuous flow centrifugation, or by using a continuous extractor.

[0152] If several beneficial enzymes and / or BIAs are present inside the cells, the cells may be permeabilized or lysed and the cell debris removed by any of the methods described above. Agents used to permeate the cells include, but are not limited to, organic solvents (e.g., DMSO) or salts (e.g., lithium acetate). Methods for lysing the cells may include the addition of surfactants such as sodium dodecyl sulfate, or mechanical destruction by bead grinding or sonication.

[0153] The target enzyme and / or BIA may be extracted from the clarified used medium by liquid-liquid extraction by adding an organic liquid immiscible with the aqueous medium. In the example, liquid-liquid extraction may be used in addition to other processing steps. Examples of suitable organic liquids include, but are not limited to, isopropyl myristate, ethyl acetate, chloroform, butyl acetate, methyl isobutyl ketone, methyl oleate, toluene, oleyl alcohol, and ethyl butyrate. The organic liquid may be added in amounts ranging from as little as 10% to as much as 100% of the volume of the aqueous medium.

[0154] In some cases, the organic liquid may be added at the start of fermentation or at any time during fermentation. This process of extractive fermentation can increase the yield of the desired enzyme and / or BIA from the host cells by continuously removing the enzyme and / or BIA into the organic phase.

[0155] By stirring, the organic phase can form an emulsion with the aqueous medium. Methods to promote the separation of the two phases into distinct layers include, but are not limited to, the addition of a demulsifying agent or a nucleating agent, or adjustment of the pH. The emulsion may be centrifuged to separate the two phases, for example, by continuous conical plate centrifugation.

[0156] Alternatively, the organic phase may be isolated from the aqueous medium such that it can be physically removed after extraction. For example, the solvent may be encapsulated in a membrane.

[0157] In an example, the enzyme and / or BIA of interest may be extracted from the fermentation medium using an adsorption method. In an example, the BIA of interest may be extracted from the clarified spent medium by the addition of a resin such as Amberlite® XAD4 or another agent that removes BIA by adsorption. The BIA of interest can then be released from the resin using an organic solvent. Examples of suitable organic solvents include, but are not limited to, methanol, ethanol, ethyl acetate, or acetone.

[0158] The BIA of interest may be extracted from the fermentation medium using filtration. At high pH, the BIA of interest may form a crystalline-like precipitate in the bioreactor. This precipitate can be removed directly by filtration through a filter, membrane, or other porous material. The precipitate may be collected by centrifugation and / or decantation.

[0159] The extraction methods described above may be performed either in vivo (inside the bioreactor) or in vitro (e.g., in an external loop through which the medium flows out of the bioreactor, contacts the extractant, and is recycled back into the vessel). Alternatively, the extraction method may be performed after fermentation using the clarified medium removed from the bioreactor vessel.

[0160] Method for purifying products from alkaloid-enriched liquid Subsequent purification steps necessarily involve treating the post-fermentation solution enriched in the desired BIA product using methods known in the art to recover the individual product species of interest to high purity.

[0161] In one example, the desired BIA extracted in the organic phase may be transferred to an aqueous solution. In some cases, the organic solvent may be evaporated by heat and / or vacuum, and the resulting powder may be dissolved in an aqueous solution of a suitable pH. In a further example, the desired BIA may be extracted from the organic phase by addition of an aqueous solution at a suitable pH that promotes extraction of the desired BIA into the aqueous phase. The aqueous phase may then be removed by decantation, centrifugation, or another method.

[0162] The BIA-containing solution may be further treated, for example, to remove metals by treatment with a suitable chelating agent. The desired BIA-containing solution may be further treated to remove other impurities such as proteins and DNA by precipitation. In one example, the desired BIA-containing solution is treated with a suitable precipitating agent such as ethanol, methanol, acetone or isopropanol. In an alternative example, DNA and proteins may be removed by dialysis or other methods of size exclusion that separate smaller alkaloids from contaminating biological macromolecules.

[0163] In a further example, the solution containing the desired BIA may be extracted to high purity by continuous cross-flow filtration using methods known in the art.

[0164] If the solution contains a mixture of desired BIAs, the solution may be subjected to acid-base treatment using methods known in the art to obtain the individual desired species of BIA. In this process, the pH of the aqueous solution is adjusted to precipitate the individual BIAs.

[0165] For high purity small-scale preparations, the BIA may be purified in a single step by liquid chromatography.

[0166] Yeast-derived alkaloid API vs. plant-derived API Clarified yeast culture medium (CYCM) may contain several impurities. The clarified yeast culture medium can be dehydrated by vacuum and / or heating to obtain an alkaloid-enriched powder. This product is similar to poppy straw (CPS) or opium concentrate, which is exported from poppy-growing countries and purchased by API manufacturers. For the purposes of this invention, CPS is a representative example of any type of purified plant extract from which the desired alkaloid product can ultimately be further purified. Tables 4 and 5 highlight impurities in these two products that may be specific to either CYCM or CPS, or may be present in both. Some BIAs may contain pigments as impurities, while others may be classified as pigments themselves. Therefore, these BIAs can be evaluated for impurities based on non-pigment impurities. By analyzing products of unknown origin for a subset of these impurities, those skilled in the art can determine whether the product originates from a yeast-producing host or a plant-producing host.

[0167] API-grade pharmaceutical ingredients are highly purified molecules. Therefore, impurities that may indicate the plant or yeast origin of an API (such as those listed in Tables 4 and 5) may not be present in the API product. In fact, many API products derived from the yeast strains of the present invention are almost indistinguishable from conventional plant-derived APIs. However, in some cases, conventional alkaloid compounds may be chemically modified using chemosynthetic approaches, and such chemical modifications may appear as chemical impurities in plant-derived products. For example, chemical derivatization can often result in a set of impurities associated with the chemosynthetic process. In certain circumstances, these modifications may be carried out biologically on a yeast-producing platform, thereby avoiding the presence of some of the impurities associated with chemical induction in the yeast-derived product. Specifically, these impurities from chemically derived products may be present in API products produced using chemosynthetic processes, but may not be present in API products produced using yeast-derived products. Alternatively, when a yeast-derived product is mixed with a chemically derived product, impurities may be present, but in amounts less than those expected in the chemically derived product alone or in APIs primarily containing it. In this example, by analyzing the API product for a subset of these impurities, a person skilled in the art can determine whether the product originates from a yeast production host or from a conventional chemical derivatization pathway.

[0168] Non-limiting examples of impurities that may be present in chemically derivatized morphinan APIs but would not be present in biosynthesized APIs include codeine-O(6)-methyl ether impurities in codeine APIs; 8,14-dihydroxy-7,8-dihydrocodeinone in oxycodone APIs; and tetrahydrothebaine in hydrocodone APIs. Codeine-O(6)-methyl ether can be formed by chemical hypermethylation of morphine. 8,14-dihydroxy-7,8-dihydrocodeinone in oxycodone APIs can be formed by chemical hyperoxidation of thebaine. In addition, tetrahydrothebaine in hydrocodone APIs can be formed by chemical hyperreduction of thebaine.

[0169] However, when both yeast-derived and plant-derived compounds undergo chemical modification through chemosynthesis, the same impurities associated with the chemosynthesis process may be expected in the product. In such situations, the starting materials (e.g., CYCM or CPS) can be analyzed as described above.

[0170] Methods for genetically modifying host cells This also includes methods for genetically engineering host cells for the purpose of producing the enzyme and / or BIA of interest. Inserting DNA into host cells can be achieved using any convenient method. Using such methods, heterologous coding sequences are inserted into genetically engineered host cells so that the host cells functionally express the enzyme and convert the starting compound of interest into the enzyme and / or BIA product of interest.

[0171] Any suitable promoter may be used in the genetically engineered host cells and methods of the subject. The promoter driving the expression of heterologous coding sequences may be a constitutive or inducible promoter, provided that the promoter is active in the genetically engineered host cells. Heterologous coding sequences can be expressed using their native promoters, or non-native promoters may be used. Such promoters may range in intensity from low to high in the host in which they are used. Promoters may be modulotable or constitutive. In certain embodiments, promoters that are not glucose-repressed or are only mildly repressed by the presence of glucose in the culture medium are used. The promoters of interest include, but are not limited to, promoters of glycolytic genes, such as the promoter of the B. subtilis tsr gene (encoding the promoter region of the fructose diphosphate aldolase gene) or promoters from the yeast S. cerevisiae gene encoding glyceraldehyde 3-phosphate dehydrogenase (GPD, GAPDH, or TDH3), the baker's yeast ADH1 promoter, phosphate deficiency-inducible promoters, such as the yeast PHO5 promoter, the alkaline phosphatase promoter from B. licheniformis, yeast-inducible promoters, such as Gal1-10, Gal1, GalL, GalS, repressive 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. Autonomous replicating yeast expression vectors containing promoters inducible by hormones such as glucocorticoids, steroids, and thyroid hormones may also be used, including, but 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, including the references cited therein.Additional vectors containing constitutive or inductive promoters such as α factor, alcohol oxidase, and PGH may also be used. Furthermore, any promoter / enhancer combination (according to the Eukaryotic Promoter Database EPDB) may also be used to promote gene expression. Any suitable promoter may be selected for a host cell, such as E. coli. Promoter selection may also be used to optimize transcript and, therefore, enzyme levels to maximize production while minimizing energy sources.

[0172] Any suitable vector may be used in the genetically engineered host cells and methods of the subject. The vectors of interest include vectors for use in yeast and other cells. Yeast vector types can be divided into four general classifications: integration vectors (YIp), autonomously replicating high-copy-number vectors (YEp or 2μ plasmids), autonomously replicating low-copy-number vectors (YCp or kinetochore plasmids), and vectors for cloning large fragments (YAC). The vector DNA is introduced into prokaryotic or eukaryotic cells via any suitable transformation or transfection technique. Yeast may be genetically engineered by integration into the genome using DNA from another source (e.g., PCR-generated double-stranded DNA products, or synthetic double-stranded or single-stranded oligonucleotides). Any single transformation event may involve one or more nucleic acids (vectors, double-stranded or single-stranded DNA fragments) to genetically modify the host cell.

[0173] usefulness For example, the genetically modified host cells and methods of the present invention described above can be used for a variety of purposes. These purposes include, but are not limited to, research and therapeutic applications. The methods of the present invention can be used for a variety of different applications, including any convenient applications where the production of enzymes and / or BIAs is the objective.

[0174] The genetically modified host cells and methods described herein are used in a variety of therapeutic applications. These therapeutic applications include those aimed at preparing pharmaceuticals containing biopharmaceutical agents (BIAs). The genetically modified host cells described herein produce the BIA and enzyme of interest. Reticulin is a key branching point intermediate in the synthesis of BIAs, including genetically modified efforts to produce end products such as opioid products. Using the host cells described herein, the BIA of interest can be produced from simple and inexpensive starting materials that can be used in the production of the BIA of interest, e.g., reticulin, and the BIA end product. Therefore, the host cells described herein are used to supply a therapeutically active BIA of interest.

[0175] In some cases, genetically modified host cells and methods are used in the production of BIAs on a commercial scale when the chemical synthesis of these compounds yields low yields and is not a viable means for large-scale production. In certain specific cases, host cells and methods are used in fermentation facilities, which may include, for example, bioreactors (fermenters) with capacities of 5,000 to 200,000 liters, enabling the rapid production of the BIA of interest for therapeutic products. Such applications may include the industrial-scale production of the BIA of interest from fermentable carbon sources such as cellulose, starch, and free sugars.

[0176] The genetically engineered host cells and methods of this subject can be used for a variety of research applications. These host cells and methods can be used to analyze the action of various enzymes on the biosynthetic pathways of various enzymes and / or BIAs of interest. In addition, genetically engineered host cells can be engineered to produce enzymes and / or BIAs of interest for use in testing the biological activity of the target in therapeutic functions that have not yet been elucidated. In some cases, genetically engineering host cells to contain various heterologous coding sequences encoding various enzymes can elucidate high-yield biosynthetic pathways toward the enzymes and / or BIAs of interest. In certain specific cases, research applications include the production of enzymes and / or BIAs of interest for therapeutic molecules of interest that can then be further chemically modified or derivatized into desired products, or for screening for increased therapeutic activity of interest. In some examples, host cell lines are used to screen for enzyme activity of interest in such pathways, which can lead to enzyme discovery through the conversion of BIA metabolites produced in these strains.

[0177] The genetically engineered host cells and methods of the subject can be used as a platform for producing metabolites specialized for plants. The host cells and methods of the subject can also be used as a platform for the development of drug libraries and the discovery of plant enzymes. For example, the genetically engineered host cells and methods of the subject can be used for the development of natural product-based drug libraries, which may be achieved by isolating yeast strains that produce skeletal molecules of interest, such as protopines, and further functionalizing the structure of the compounds through combinatorial biosynthesis or by chemical means. By producing drug libraries in this manner, any potential drug hit has already been associated with a production host that can be applied to large-scale culture and production. As another example, these genetically engineered host cells and methods of the subject can be used for the discovery of plant enzymes. The host cells of the subject express plant EST libraries for identifying novel enzymatic activities, providing a clear background of defined metabolites. The host cells and methods of the subject provide expression methods and culture conditions for the functional expression and increased activity of plant enzymes in yeast.

[0178] Kits and Systems Aspects of the present invention further include kits and systems, which may include one or more components used in the methods of the present invention, such as genetically engineered host cells, starting compounds, heterologous coding sequences, vectors, culture media, etc., as described herein. In some embodiments, the subject kit includes genetically engineered host cells (e.g., as described herein), as well as one or more components selected from starting compounds, heterologous coding sequences and / or vectors containing them, vectors, growth feedstocks, components suitable for use in expression systems (e.g., cells, cloning vectors, multiple cloning sites (MCS), bidirectional promoters, intrasequence ribosome entry sites (IRES), etc.), and culture media.

[0179] Any of the components described herein may be provided in a kit, which may be, for example, a host cell comprising one or more modifications, a starting compound, a medium, etc. Various components suitable for use in the production and use of heterologous coding sequences, cloning vectors, and expression systems may be used in the subject kit. The kit may also include tubes, buffers, etc., and instructions for use. The various reagent components of the kit may, if desired, be present in separate containers or some or all of them may be pre-formulated in a reagent mixture in a single container.

[0180] A system for producing an enzyme and / or BIA of interest, comprising a genetically engineered host cell (e.g., as described herein) comprising one or more modifications, a starting compound, a medium, a fermenter and fermentation equipment, e.g., an apparatus suitable for maintaining growth conditions for the host cell, sampling and monitoring equipment, and components, etc., is also provided. Various components suitable for use in the large-scale fermentation of yeast cells may be used in the subject system.

[0181] In some cases, the system comprises components for the large-scale fermentation of a genetically engineered host cell and for the monitoring and purification of the enzyme and / or BIA compound produced by the fermented host cell. In certain embodiments, one or more starting compounds (e.g., as described herein) are added to the system under conditions such that the genetically engineered host cell in the fermenter produces one or more desired target BIA products. In some examples, the host cell produces the BIA of interest (e.g., as described herein). In certain cases, the target BIA product is an opioid product, e.g., thebaine, codeine, neopine, morphine, neomorphine, hydrocodone, oxycodone, hydromorphone, dihydrocodeine, 14-hydroxycodeine, dihydromorphine, or oxymorphone.

[0182] In some cases, the system includes a process for monitoring and / or analyzing one or more target enzymes and / or BIA compounds produced by the host cells of the subject. For example, a sample can be analyzed and compared to a standard, such as an LC-MS analysis system, a chromatography system, or any other convenient system as described herein. The fermentation medium can be observed by sampling and analysis before and at any convenient time during fermentation. Once the conversion of the starting compounds to the target enzymes and / or BIA products is complete, fermentation may be stopped and the BIA products may be purified. Thus, in some cases, the system of the subject includes a purification component suitable for purifying the target enzymes and / or BIA products from the host cell medium in which the target enzymes and / or BIA products are produced. The purification component may include any convenient means that can be used to purify the target enzymes and / or BIA products produced by fermentation, including, but not limited to, silica chromatography, reversed-phase chromatography, ion-exchange chromatography, HIC chromatography, size exclusion chromatography, liquid extraction, and pH extraction methods. In some cases, the subject system provides the production and isolation of enzymes and / or BIA fermentation products after the introduction of one or more starting compounds into the system.

[0183] The following examples are provided to those skilled in the art to provide a complete disclosure and explanation of how the present invention is made and used, and are not intended to limit the scope of what the inventors consider to be their invention, nor are they intended to represent all or only experiments that have been conducted. Efforts have been made to ensure accuracy with respect to the figures used (e.g., quantity, temperature, etc.), but some experimental error and deviation should be taken into account. Unless otherwise noted, parts are by weight, molecular weight is weight-average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric pressure or near atmospheric pressure.

[0184] Consideration of the enzyme list Host cells may be genetically engineered to include one or more modifications (e.g., two or more, three or more, four or more, five or more, or more modifications) that provide the production of the target BIA and / or the target enzyme. Table 2 provides a table of exemplary genes that may act by one or more modifications to provide the production of the target BIA and / or the target enzyme in genetically engineered host cells.

[0185] Using the gene modifications provided in Table 2, the target BIA can be produced from genetically engineered host cells supplemented with a medium containing the minimum nutrients necessary for growth. This minimal medium may contain a carbon source, a nitrogen source, amino acids, vitamins, and salts. For example, using the gene modifications provided in Table 2, the target BIA can be produced from genetically engineered host cells supplied with sugar. In addition, using one or more gene modifications provided in Table 2, the biosynthetic processes of host cells that can be genetically engineered for drug production can be enhanced.

[0186] Furthermore, the use of these modifications to provide the production of the desired BIA and / or enzyme in genetically engineered host cells is not readily apparent from mere identification of the enzymes that can be produced by the genes. Specifically, the synthetic pathways reconstructed in host cells such as yeast cells, as described herein, involve various enzymes that do not naturally act together within a single organism. In addition, some of the enzymes considered herein do not act for BIA biosynthesis in their natural context. Moreover, some of the enzymes described herein have not evolved to function in or to function together in specific host cells such as yeast cells. In these cases, it would not be obvious that they would exhibit sufficient activity in the context of the synthetic BIA pathway in host cells such as yeast to have sufficient flux through the pathway to produce the downstream BIA end product.

[0187] For example, plant enzymes are often difficult to express functionally in heterologous microbial hosts such as yeast. In many cases, enzymes may be misfolded, not properly localized within host cells, and / or incorrectly processed. Differences in protein translation and processing between yeast and plants can lead to substantially reduced or undetectable activity of these enzymes in the yeast host. These challenges are common to membrane-localized enzymes such as cytochrome P450, which are strongly expressed in the BIA pathway. Even reduced enzyme activity can pose substantial challenges in genetically engineering yeast to produce complex BIAs, where sufficient activity is required at each step to ensure high levels of accumulation of the desired BIA product.

[0188] In addition, there are endogenous enzymes / pathways in some host cells, such as yeast, that can act on many of the early precursors in the BIA pathway (i.e., intermediates from tyrosine to norcoclaurine). Therefore, considering these competing endogenous pathways, it may not be readily apparent that there is sufficient flux to pass through heterologous pathways 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 can divert flux from the synthetic pathway by acting to convert many of the intermediates in the early BIA pathway into undesirable products.

[0189] Furthermore, many of the enzymes discussed herein and those provided in Table 2 may function under highly specific regulatory strategies, including spatial control in native plant hosts, which can be lost upon transfer to heterologous yeast hosts. In addition, plants present a biochemical environment quite different from that of yeast cells, from which the enzymes have evolved to function, including pH, redox conditions, and the availability of substrates, co-substrates, coenzymes, and cofactors. Given the differences in biochemical environments and regulatory strategies between native and heterologous yeast hosts, it is not clear whether enzymes can exhibit substantial activity in a yeast environment, and even less clear whether they function together to direct simple precursors, such as sugars, into complex BIA compounds. Maintaining enzyme activity in the yeast host is particularly important because many pathways involve numerous (more than 10) reaction steps. If these steps are not efficient, the accumulation of the desired downstream products cannot be expected.

[0190] In addition, in natural plant hosts, the relevant metabolites in these pathways can be localized across different cell and tissue types. In some cases, there are cell types that can specialize in biosynthesis and cell types that can synthesize for metabolite accumulation. This type of cellular specialization may be lost when the pathway is expressed in heterologous yeast hosts and may play a crucial role in controlling the toxicity of these metabolites to cells. Therefore, it is not clear that yeast can be successfully genetically engineered to biosynthesize and accumulate these metabolites without being harmed by the toxicity of these compounds.

[0191] As one example, it has been reported that the enzyme BBE has dynamic intracellular localization in native plant hosts. Specifically, BBE is initially initiated in the ER and then sorted into vacuoles (Bird and Facchini. 2001. Planta. 213:888-97). The ER association of BBE in plants (Alcantara, et al. 2005. Plant Physiol. 138:173-83) has been shown to provide an optimal base pH (approximately pH 8.8) for BBE activity (Ziegler and Facchini. 2008. Annu. Rev. Plant Biol. 59:735-69). As another example, it has been demonstrated that sanguinalin biosynthesis occurs in specialized vesicles within plant cells (Amann, et al. 1986. Planta. 167:310-20), although only some of the intermediates accumulate in the vesicles. This may occur to isolate them from other enzyme activities and / or toxic effects.

[0192] As another example, all biosynthetic enzymes in the morphinan pathway branch are localized in the phloem, a part of the plant's vascular tissue. In the phloem, pathway enzymes can be further divided between two cell types: sieve elements, which are common to all plants, and mammary ducts, which are specialized cell types found only in certain plants that produce specialized secondary metabolites. Upstream enzymes (i.e., from NCS to SalAT) are mainly found in sieve elements, while downstream enzymes (i.e., T6ODM, COR, CODM) are mostly found in mammary ducts (Onoyovwe, et al. 2013. Plant Cell. 25:4110-22). In addition, it has been discovered that the final step in the noscapine biosynthesis pathway occurs in the mammary ducts (Chen and Facchini. 2014. Plant J. 77:173-84). This compartmentalization is considered crucial for regulating biosynthesis by isolating or tracking intermediates, providing optimal pH, and enhancing cofactor supply, although the properties of the opium poppy ductal microenvironment are still under investigation (Ziegler and Facchini. 2008. Annu. Rev. Plant Biol. 59:735-69). Furthermore, it is anticipated that some enzymes may 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). It is unclear whether these complexes or channels can be formed similarly to those in native hosts when biosynthetic enzymes are combined from different hosts and / or recombinantly expressed in heterologous yeast cells. In additional examples, in Coptis japonica, berberine is biosynthesized in the root tissue and then accumulated in the rhizome via the action of specialized ATP-binding cassette transport proteins (Shitan, et al. 2013. Phytochemistry. 91:109-16). In poppies, morphinan alkaloids accumulate in the latex (cytoplasm of mammary duct cells) (Martin, et al. 1967. Biochemistry. 6:2355-63).

[0193] Furthermore, even without considering these factors, plant enzymes for some steps in the pathways described herein may not yet be characterized. For example, the conversion of tyrosine to the initial benzylisoquinoline alkaloid skeleton norcoclaurine has not yet been characterized. In addition, the conversion of (S)-reticulin to (R)-reticulin has only recently been characterized, as described herein. Therefore, for some steps in the pathways described herein, alternative biosynthetic schemes were produced by combining or identifying novel enzyme activities from genomic sequence information for use in reconstructed pathways, or by combining enzyme activities that do not normally occur together in nature for BIA biosynthesis.

[0194] For example, the two-step conversion of tyrosine to dopamine can be achieved by combining at least five mammalian enzymes and one bacterial enzyme, which do not exist together in nature and have not evolved to function in this pathway or in plant enzymes. In these examples, it may not be obvious that these enzymes can be used for the biosynthesis of compounds that have not evolved in nature, and that these enzymes can function effectively in heterologous microbial hosts and in this pathway. In another example, the enzyme responsible for the conversion of (S)-reticulin to (R)-reticulin was unknown. The novel enzyme considered herein can carry out this epimerization reaction in yeast and on the synthetic BIA pathway. This represents the discovery of a new enzyme, as it would not have been obvious that this enzyme could be used on the pathway for the synthesis of these BIA compounds.

[0195] Examples of genes targeted for modification to produce the desired BIA and / or enzyme are listed below. In addition, the genes are described in relation to a series of diagrams illustrating the pathways used in the production of the desired BIA and / or enzyme.

[0196] [TLK1] In some cases, genetically modified host cells may have altered expression of the enzyme transketolase. Transketolase is encoded by the TKL1 gene. In some cases, transketolase catalyzes the reaction fructose-6-phosphate + glyceraldehyde-3-phosphate ⇔ xylulose-5-phosphate + erythrose-4-phosphate, as shown in Figure 2. Modification of genetically modified host cells may include constitutive overexpression of the TKL1 gene in the genetically modified host cells. In addition or alternatively, genetically modified host cells may be modified to synthetically regulate the expression of the TKL1 gene in the genetically modified host cells. In some cases, genetically modified host cells may be modified to incorporate one or more copies of the TKL1 gene, or additional copies. In addition or alternatively, genetically modified host cells may be modified to incorporate the introduction of a strong promoter element for overexpression of the TKL1 gene in the genetically modified host cells. The TKL1 gene may originate from Saccharomyces cerevisiae or another species. In some cases, the TKL1 gene may be 100% identical to a naturally occurring gene.

[0197] [ZWF1] In some cases, genetically modified host cells can alter the expression of the enzyme glucose-6-phosphate dehydrogenase. Glucose-6-phosphate dehydrogenase is encoded by the ZWF1 gene. In some cases, glucose-6-phosphate dehydrogenase catalyzes the reaction glucose-6-phosphate → 6-phosphogluconolactone, as shown in Figure 2. Genetically modified host cells can be modified to delete the coding region of the ZWF1 gene in the genetically modified host cells. Alternatively, the functionality of the ZWF1 gene can be disabled by modifying the genetically modified host cells to introduce inactivating mutations, for example.

[0198] In some examples, the genetically engineered host cell can modify the expression of the enzyme 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase. DAHP synthase is encoded by the ARO4 gene. In an example, DAHP synthase catalyzes the reaction of erythrose-4-phosphate + phosphoenolpyruvate → DAHP, as referenced in FIG. 2. The genetically engineered host cell can modify the ARO4 gene to incorporate one or more feedback inhibition reducing mutations. Specifically, the feedback inhibition reducing mutation (e.g., ARO4 FBR ) can be introduced as a site-directed mutation to the native ARO4 gene at its original locus; as an additional copy introduced as a gene integration at another locus; or as an additional copy on an episomal vector, such as a 2-μm or kinetoplast plasmid. The identifier “FBR” in mutant ARO4 FBR refers to feedback resistant mutants and mutations. The feedback inhibitory copy of the DAHP synthase enzyme can be under native yeast transcriptional control, for example, when the genetically engineered host cell is a yeast cell. Alternatively, the feedback inhibitory copy of the DAHP synthase enzyme can be introduced into the genetically engineered host cell with genetically engineered constitutive or dynamic control of protein expression by placing it under the control of a synthetic promoter. In some cases, the ARO4 gene can be derived from Saccharomyces cerevisiae. In some cases, the ARO4 gene can be 100% identical to the naturally occurring gene. Examples of modifications to the ARO4 gene include feedback inhibition resistant mutations, K229L, or Q166K.

[0199] [ARO7] In some cases, genetically engineered host cells may alter the expression of the enzyme colismyate mutase. Colithmyate mutase is encoded by the ARO7 gene. In some cases, colismyate mutase catalyzes the reaction colismyate → prephenate, as shown in Figure 2. Genetically engineered host cells may modify the ARO7 gene to incorporate one or more feedback inhibition reduction mutations. Specifically, feedback inhibition reduction mutations (e.g., ARO7) may be incorporated. FBR Mutant ARO7 can be incorporated as a directed mutation into the native ARO7 gene at its original locus; as an additional copy introduced as gene integration at another locus; or as an additional copy on an episomal vector, e.g., a 2-μm or kinetochore plasmid. FBR The identifier "FBR" in this context refers to feedback-resistant mutants and mutations. Feedback-inhibiting copies of the colismyate mutase enzyme may exist under the transcriptional control of native yeast, for example, when the genetically engineered host cell is a yeast cell. Alternatively, feedback-inhibiting copies of the colismyate mutase enzyme may be introduced into a genetically engineered host cell along with the genetically engineered constitutive or dynamic control of protein expression by placing them under the control of a synthetic promoter. In some cases, the ARO7 gene may originate from Saccharomyces cerevisiae. In some cases, the ARO7 gene may be 100% identical to the naturally occurring gene. An example of modification to the ARO7 gene is the feedback-resistant mutant T226I.

[0200] [ARO10] In some cases, genetically modified host cells may have altered expression of the enzyme phenylpyruvate decarboxylase. Phenyruvate decarboxylase is encoded by the ARO10 gene. In some cases, phenylpyruvate decarboxylase catalyzes the reaction hydroxyphenylpyruvate → 4-hydroxyphenyl acetate (4HPA), as shown in Figure 2. Modification of the genetically modified host cell may include constitutive overexpression of the ARO10 gene in the genetically modified host cell. In addition or alternatively, the genetically modified host cell may be modified to synthetically control the expression of the ARO10 gene in the genetically modified host cell. In some cases, the genetically modified host cell may be modified to incorporate one or more copies, or additional copies, of the ARO10 gene. In addition or alternatively, the genetically modified host cell may be modified to incorporate the introduction of a strong promoter element for overexpression of the ARO10 gene in the genetically modified host cell. The ARO10 gene may be derived from Saccharomyces cerevisiae or another species. In some cases, the ARO10 gene may be 100% identical to the naturally occurring gene.

[0201] [ADH2-7, SFA1] In some cases, genetically modified host cells can alter the expression of the alcohol dehydrogenase enzyme. The alcohol dehydrogenase enzyme may be encoded by one or more of the ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, and SFA1 genes. In some cases, alcohol dehydrogenase catalyzes the 4HPA → tyrosol reaction. Genetically modified host cells can be modified to delete one or more coding regions of the ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, and SFA1 genes in the genetically modified host cells. Alternatively, the function of one or more of the ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, and SFA1 genes can be disabled by modifying the genetically modified host cells, for example, by introducing inactivating mutations.

[0202] [ALD2~6] In some cases, genetically modified host cells can alter the expression of aldehyde oxidase enzymes. Aldehyde oxidase enzymes may be encoded by one or more of the ALD2, ALD3, ALD4, ALD5, and ALD6 genes. In some cases, aldehyde oxidase catalyzes the reaction 4HPA → hydroxyphenylacetic acid. Genetically modified host cells can be modified to delete one or more coding regions of the ALD2, ALD3, ALD4, ALD5, and ALD6 genes in the genetically modified host cells. Alternatively, the function of one or more of the ALD2, ALD3, ALD4, ALD5, and ALD6 genes can be disabled by modifying the genetically modified host cells, for example, by introducing inactivating mutations.

[0203] [ARO9] In some cases, genetically modified host cells may have altered expression of the enzyme aromatic aminotransferase. Aromatic aminotransferase is encoded by the ARO9 gene. In some cases, aromatic aminotransferase catalyzes the reaction hydroxyphenylpyruvic acid + glutamimate → tyrosine + α-ketoglutarate, as shown in Figure 2. Modification of the genetically modified host cell may include constitutive overexpression of the ARO9 gene in the genetically modified host cell. In addition or alternatively, the genetically modified host cell may be modified to synthetically control the expression of the ARO9 gene in the genetically modified host cell. In some cases, the genetically modified host cell may be modified to incorporate one or more copies of the ARO9 gene, or additional copies. In addition or alternatively, the genetically modified host cell may be modified to incorporate the introduction of a strong promoter element for overexpression of the ARO9 gene in the genetically modified host cell. The ARO9 gene may be derived from Saccharomyces cerevisiae or another species. In some cases, the ARO9 gene may be 100% identical to the naturally occurring gene.

[0204] [TYR] In some cases, genetically modified host cells may have altered expression of the enzyme tyrosine kinase. Tyrosinase is encoded by the TYR gene. In one example, tyrosinase catalyzes the tyrosine → L-dopa reaction, as shown in Figure 2. In another example, tyrosinase catalyzes the L-dopa → dopaquinone reaction. Modification of the genetically modified host cell may include constitutive expression of the TYR gene in the genetically modified host cell. In addition or alternatively, the genetically modified host cell may be modified to synthetically control the expression of the TYR gene in the genetically modified host cell. In one example, the genetically modified host cell may be modified to incorporate one or more copies of the TYR gene, or additional copies. In addition or alternatively, the genetically modified host cell may be modified to incorporate the introduction of a strong promoter element for overexpression of the TYR gene in the genetically modified host cell. The TYR gene may originate from Ralstonia solanacearum, Agaricus bisporus, or another species. In some cases, the TYR gene may be 100% identical to a naturally occurring gene.

[0205] [TyrH] In some cases, genetically modified host cells may have altered expression of the enzyme tyrosine hydroxylase. Tyrosine hydroxylase is encoded by the TyrH gene. In some cases, tyrosine hydroxylase catalyzes the tyrosine → L-dopa reaction, as shown in Figures 2 and 5. Modification of the genetically modified host cell may include constitutive expression of the TyrH gene in the genetically modified host cell. In addition or alternatively, the genetically modified host cell may be modified to synthetically control the expression of the TyrH gene in the genetically modified host cell. In some cases, the genetically modified host cell may be modified to incorporate one or more copies of the TyrH gene, or additional copies. In addition or alternatively, the genetically modified host cell may be modified to incorporate the introduction of a strong promoter element for overexpression of the TyrH gene in the genetically modified host cell. The TyrH gene may originate from Homo sapiens, brown rats, mice, or another species. In some cases, the TyrH gene may be 100% identical to a naturally occurring gene.

[0206] [DODC] In some cases, genetically modified host cells may have altered expression of the enzyme L-dopadecarboxylase. L-dopadecarboxylase is encoded by the DODC gene. In some cases, L-dopadecarboxylase catalyzes the L-dopa → dopamine reaction, as shown in Figures 2 and 5. Modification of the genetically modified host cell may include constitutive expression of the DODC gene in the genetically modified host cell. In addition or alternatively, the genetically modified host cell may be modified to synthetically control the expression of the DODC gene in the genetically modified host cell. In some cases, the genetically modified host cell may be modified to incorporate one or more copies of the DODC gene, or additional copies. In addition or alternatively, the genetically modified host cell may be modified to incorporate the introduction of a strong promoter element for overexpression of the DODC gene in the genetically modified host cell. The DODC gene may be derived from Pseudomonas putida, brown rat, or another species. In some cases, DODC genes may be 100% identical to naturally occurring genes.

[0207] [TYDC] In some cases, genetically modified host cells may have altered expression of the enzyme tyrosine / dopadecarboxylase. Tyrosine / dopadecarboxylase is encoded by the TYDC gene. In some cases, tyrosine / dopadecarboxylase catalyzes the L-dopa → dopamine reaction, as shown in Figure 2. Modification of the genetically modified host cell may include constitutive expression of the TYDC gene in the genetically modified host cell. In addition or alternatively, the genetically modified host cell may be modified to synthetically control the expression of the TYDC gene in the genetically modified host cell. In some cases, the genetically modified host cell may be modified to incorporate one or more copies of the TYDC gene, or additional copies. In addition or alternatively, the genetically modified host cell may be modified to incorporate the introduction of a strong promoter element for overexpression of the TYDC gene in the genetically modified host cell. The TYDC gene may originate from Papaver somnifer or another species. In some cases, the TYDC gene may be 100% identical to a naturally occurring gene.

[0208] [MAO] In some cases, genetically modified host cells may have altered expression of the enzyme monoamine oxidase. Monoamine oxidase is encoded by the MAO gene. In some cases, monoamine oxidase catalyzes the dopamine → 3,4-DHPA reaction, as shown in Figure 2. Modification of the genetically modified host cell may include constitutive expression of the MAO gene in the genetically modified host cell. In addition or alternatively, the genetically modified host cell may be modified to synthetically control the expression of the MAO gene in the genetically modified host cell. In some cases, the genetically modified host cell may be modified to incorporate one or more copies of the MAO gene, or additional copies. In addition or alternatively, the genetically modified host cell may be modified to incorporate the introduction of a strong promoter element for overexpression of the MAO gene in the genetically modified host cell. In some cases, the MAO gene may be codon-optimized for expression in Saccharomyces cerevisiae. MAO genes can originate from Escherichia coli, Homo sapiens, Micrococcus luteus, or other species. In some cases, MAO genes are 77% similar to naturally occurring genes.

[0209] [NCS] In some cases, genetically engineered host cells can alter the expression of the enzyme norcoclaurine synthase. Norcoclaurine synthase is encoded by the NCS gene. In some cases, norcoclaurine synthase catalyzes the reaction 4HPA+ dopamine → (S)-norcoclaurine, as shown in Figure 5. Specifically, Figure 5 shows a biosynthetic scheme for the conversion of L-tyrosine to reticulin via norcoclaurine according to an embodiment of the present invention. Figure 5 provides the use of the enzymes TyrH, tyrosine hydroxylase; DODC, dopa decarboxylase; NCS, norcoclaurine synthase, as described herein; 6OMT, 6-O-methyltransferase; CNMT, coclaurine N-methyltransferase; CYP80B1, cytochrome P450 80B1; CPR, cytochrome P450 NADPH reductase; 4'OMT, 3'hydroxy-N-methylcoclaurine 4'-O-methyltransferase; L-dopa, L-3,4-dihydroxyphenylalanine; and 4-HPA, 4-hydroxyphenylacetylaldehyde. Of the enzymes shown in Figure 5, 4-HPA and L-tyrosine are synthesized naturally in yeast. All other metabolites shown are not produced naturally in yeast. In addition, although TyrH is shown to catalyze the conversion of L-tyrosine to L-dopa, this step may be carried out using other enzymes as described herein. For example, tyrosinase may be used to convert L-tyrosine to L-dopa. In addition, other enzymes such as cytochrome P450 oxidase may be used to convert L-tyrosine to L-dopa. Such enzymes may exhibit oxidase activity on relevant BIA precursor compounds containing L-dopa and L-tyrosine.

[0210] In addition, norcoclaurine synthase catalyzes the reaction 3,4-DHPA + dopamine → (S)-norlaudanotholin, as shown in Figure 6. Specifically, Figure 6 shows a biosynthetic scheme for the conversion of L-tyrosine to reticulin via norlaudanotholin according to an embodiment of the present invention. Figure 6 provides the use of the enzymes TyrH, tyrosine hydroxylase; DODC, dopadecarboxylase; maoA, monoamine oxidase; NCS, norcoclaurine synthase; 6OMT, 6-O-methyltransferase; CNMT, coclaurine N-methyltransferase; 4'OMT, 3'hydroxy-N-methylcoclaurine 4'-O-methyltransferase; L-dopa, L-3,4-dihydroxyphenylalanine; and 3,4-DHPA, 3,4-dihydroxyphenylacetaldehyde. Among the enzymes shown in Figure 6, L-tyrosine is synthesized naturally in yeast. The other metabolites shown in Figure 6 are not produced naturally in yeast.

[0211] The genetically modified host cell may be modified to include constitutive expression of the NCS gene in the genetically modified host cell. In addition, or separately, the genetically modified host cell may be modified to synthetically control the expression of the NCS gene in the genetically modified host cell. For example, the genetically modified host cell may be modified to incorporate one or more copies, or additional copies, of the NCS gene. In addition, or separately, the genetically modified host cell may be modified to incorporate the introduction of a strong promoter element for overexpression of the NCS gene in the genetically modified host cell. In addition, norcoclaurine synthase may be shortened by cleaving the N-terminus. In some cases, the NCS gene may be codon-optimized for expression in Saccharomyces cerevisiae. The NCS gene may be derived from Coptis japonica, Papaver somnifer, Papaver bracteatum, Thalictrum flavoum, Corydalis saxicola, or another species. In some cases, NCS genes are 80% similar to naturally occurring genes.

[0212] [6OMT] In some cases, genetically modified host cells may have altered expression of the enzyme norcoclaurine 6-O-methyltransferase. Norcoclaurine 6-O-methyltransferase is encoded by the 6OMT gene. In some cases, norcoclaurine 6-O-methyltransferase catalyzes the norcoclaurine → coclaurine reaction, as shown in Figure 5. In other cases, norcoclaurine 6-O-methyltransferase catalyzes the norlaudanotholine → 3'hydroxycoclaurine reaction, as well as other reactions detailed herein, such as those shown in Figure 6. In addition, genetically modified host cells may include constitutive expression of the 6OMT gene in the genetically modified host cells. In addition or separately, genetically modified host cells may be modified to synthetically control the expression of the 6OMT gene in the genetically modified host cells. In some examples, genetically modified host cells may be modified to incorporate one or more copies, or additional copies, of the 6OMT gene. In addition, or separately, the genetically engineered host cell may be modified to incorporate the introduction of a strong promoter element for overexpression of the 6OMT gene in the genetically engineered host cell. The 6OMT gene may be derived from P. somniferum, T. flavum, Coptis japonica, or another species. In some cases, the 6OMT gene may be 100% identical to a naturally occurring gene.

[0213] [CNMT] In some cases, genetically engineered host cells may have altered expression of the enzyme coclaurine-N-methyltransferase. Coclaurine-N-methyltransferase is encoded by the CNMT gene. In some cases, coclaurine-N-methyltransferase catalyzes the reaction coclaurine → N-methylcoclaurine, as shown in Figure 5. In other cases, the coclaurine-N-methyltransferase enzyme may catalyze the reaction 3'-hydroxycoclaurine → 3'-hydroxy-N-methylcoclaurine. In other cases, coclaurine-N-methyltransferase may catalyze other reactions detailed herein, such as those presented in Figure 6. In addition, genetically engineered host cells may be modified to constitutively express the CNMT gene in the genetically engineered host cells. In addition or otherwise, genetically engineered host cells may be modified to synthetically control the expression of the CNMT gene in the genetically engineered host cells. In some examples, genetically modified host cells may be altered to incorporate one or more copies, or additional copies, of the CNMT gene. In addition, or separately, genetically modified host cells may be altered to incorporate the introduction of a strong promoter element for overexpression of the CNMT gene in the genetically modified host cells. The CNMT gene may be derived from P. somniferum, T. flavum, Coptis japonica, or another species. In some examples, the CNMT gene may be 100% identical to a naturally occurring gene.

[0214] [4'OMT] In some cases, genetically modified host cells may have altered expression of the enzyme 4'-O-methyltransferase. 4'-O-methyltransferase is encoded by the 4'OMT gene. In some cases, 4'-O-methyltransferase catalyzes the reaction 3'-hydroxy-N-methylcoclaurine → reticulin, as shown in Figure 5. In other cases, 4'-O-methyltransferase catalyzes other reactions detailed herein, such as those presented in Figure 6. In addition, genetically modified host cells may include constitutive expression of the 4'OMT gene in the genetically modified host cells. In addition or separately, genetically modified host cells may be modified to synthetically control the expression of the 4'OMT gene in the genetically modified host cells. In some examples, genetically modified host cells may be modified to incorporate one or more copies, or additional copies, of the 4'OMT gene. In addition, or separately, the genetically engineered host cell may be modified to incorporate the introduction of a potent promoter element for overexpression of the 4'OMT gene in the genetically engineered host cell. The 4'OMT gene may be derived from P. somniferum, T. flavum, Coptis japonica, or another species. In some cases, the 4'OMT gene may be 100% identical to a naturally occurring gene.

[0215] [CYP80B1] In some cases, genetically modified host cells may have altered expression of the enzyme cytochrome P450 80B1. Cytochrome P450 80B1 is encoded by the CYP80B1 gene. In some cases, cytochrome P450 80B1 catalyzes the reaction N-methylcoclaurine → 3'-hydroxy-N-methylcoclaurine, as shown in Figure 5. The genetically modified host cells may include constitutive expression of the cytochrome P450 80B1 gene in the genetically modified host cells. In addition, or separately, the genetically modified host cells may be modified to synthetically regulate the expression of the cytochrome P450 80B1 gene in the genetically modified host cells. In some cases, the genetically modified host cells may be modified to incorporate one or more copies, or additional copies, of the cytochrome P450 80B1 gene. In addition, or separately, genetically engineered host cells may be modified to incorporate the introduction of a strong promoter element for overexpression of the cytochrome P450 80B1 gene in the genetically engineered host cells. In some cases, the CYP80B1 gene can be codon-optimized for expression in Saccharomyces cerevisiae. The cytochrome P450 80B1 gene may originate from P. somniferum, California poppy (E. californica), T. flavum, or another species. In some examples, the P450 80B1 gene is 77% similar to the naturally occurring gene.

[0216] [FOL2] In some cases, genetically modified host cells may have altered expression of the enzyme GTP cyclohydrolase. GTP cyclohydrolase is encoded by the FOL2 gene. In some cases, GTP cyclohydrolase catalyzes the reaction GTP → dihydroneopterin triphosphate, as shown in Figure 1. Modification of genetically modified host cells may include constitutive overexpression of the FOL2 gene in the genetically modified host cells. Modification of genetically modified host cells may include intrinsic regulation. In addition or alternatively, genetically modified host cells may be modified to synthetically regulate the expression of the FOL2 gene in the genetically modified host cells. In some examples, genetically modified host cells may be modified to incorporate one or more copies of the FOL2 gene, or additional copies. In addition or alternatively, genetically modified host cells may be modified to incorporate the introduction of a strong promoter element for overexpression of the FOL2 gene in the genetically modified host cells. The FOL2 gene may be derived from Saccharomyces cerevisiae, Homo sapiens, mice, or another species. In some cases, the FOL2 gene may be 100% identical to the naturally occurring gene.

[0217] [PTPS] In some cases, genetically modified host cells may alter the expression of the enzyme 6-pyruvoyltetrahydrobiopterin (PTP) synthase. Pyrvoyltetrahydrobiopterin synthase is encoded by the PTPS gene. In some cases, 6-pyruvoyltetrahydrobiopterin synthase catalyzes the reaction dihydroneopterin triphosphate → PTP, as shown in Figure 1. Modification of genetically modified host cells may include constitutive expression of the PTPS gene in the genetically modified host cells. In addition or alternatively, the genetically modified host cells may be modified to synthetically control the expression of the PTPS gene in the genetically modified host cells. In some cases, the genetically modified host cells may be modified to incorporate one or more copies, or additional copies, of the PTPS gene. In addition or alternatively, the genetically modified host cells may be modified to incorporate the introduction of a strong promoter element for overexpression of the PTPS gene in the genetically modified host cells. In some cases, the PTPS gene may be codon-optimized for expression in Saccharomyces cerevisiae. The PTPS gene can originate from brown rats, Homo sapiens, mice, or other species. In some cases, the PTPS gene is 80% similar to the naturally occurring gene.

[0218] [SepR] In some cases, genetically modified host cells may have altered expression of the enzyme sepiapterin reductase. Sepiapterin reductase is encoded by the SepR gene. In some cases, sepiapterin reductase catalyzes the PTP → BH4 reaction, as shown in Figure 1. Modification of genetically modified host cells may include constitutive expression of the SepR gene in the genetically modified host cells. In addition or alternatively, genetically modified host cells may be modified to synthetically control the expression of the SepR gene in the genetically modified host cells. In some cases, genetically modified host cells may be modified to incorporate one or more copies of the SepR gene, or additional copies. In addition or alternatively, genetically modified host cells may be modified to incorporate the introduction of a strong promoter element for overexpression of the SepR gene in the genetically modified host cells. In some cases, the SepR gene may be codon-optimized for expression in Saccharomyces cerevisiae. The SepR gene can originate from brown rats, Homo sapiens, mice, or other species. In some cases, the SepR gene may be 72% similar to a naturally occurring gene.

[0219] [PCD] In some cases, genetically modified host cells may have altered expression of the enzyme 4α-hydroxytetrahydrobiopterin (pterin-4α-carbinolamine) dehydratase. 4α-hydroxytetrahydrobiopterin dehydratase is encoded by the PCD gene. In some cases, 4α-hydroxytetrahydrobiopterin dehydratase catalyzes the reaction 4α-hydroxytetrahydrobiopterin → H2O + quinonoid dihydropteridine, as shown in Figure 1. Modification of genetically modified host cells may include constitutive expression of the PCD gene in the genetically modified host cells. In addition or alternatively, the genetically modified host cells may be modified to synthetically control the expression of the PCD gene in the genetically modified host cells. In some examples, the genetically modified host cells may be modified to incorporate one or more copies of the PCD gene, or additional copies. In addition or alternatively, the genetically modified host cells may be modified to incorporate the introduction of a strong promoter element for overexpression of the PCD gene in the genetically modified host cells. In some cases, the PCD gene can be codon-optimized for expression in Saccharomyces cerevisiae. The PCD gene may originate from brown rats, Homo sapiens, mice, or another species. In some cases, the PCD gene may be 79% similar to a naturally occurring gene.

[0220] [QDHPR] In some cases, genetically modified host cells may have altered expression of the enzyme quinonoid dihydropteridine reductase. Quinonoid dihydropteridine reductase is encoded by the QDHPR gene. In some cases, quinonoid dihydropteridine reductase catalyzes the quinonoid dihydropteridine → BH4 reaction, as shown in Figure 1. Modification of genetically modified host cells may include constitutive expression of the QDHPR gene in the genetically modified host cells. In addition or alternatively, the genetically modified host cells may be modified to synthetically control the expression of the QDHPR gene in the genetically modified host cells. In some cases, the genetically modified host cells may be modified to incorporate one or more copies of the QDHPR gene, or additional copies. In addition or alternatively, the genetically modified host cells may be modified to incorporate the introduction of a strong promoter element for overexpression of the QDHPR gene in the genetically modified host cells. In some cases, the QDHPR gene may be codon-optimized for expression in Saccharomyces cerevisiae. The QDHPR gene can originate from rats, Homo sapiens, mice, or other species. In some cases, the QDHPR gene may be 75% similar to a naturally occurring gene.

[0221] [DHFR] In some cases, genetically modified host cells may have altered expression of the enzyme dihydrofolate reductase. Dihydrofolate reductase is encoded by the DHFR gene. In some cases, dihydrofolate reductase catalyzes the reaction 7,8-dihydrobiopterin (BH2) → 5,6,7,8-tetrahydrobiopterin (BH4), as shown in Figure 1. This reaction may be useful in recovering BH4 as an auxiliary substrate for the conversion of tyrosine to L-dopa, as shown in Figure 5. Genetically modified host cells may include constitutive expression of the DHFR gene in the genetically modified host cells. In addition, or separately, genetically modified host cells may be modified to synthetically control the expression of the DHFR gene in the genetically modified host cells. In some examples, genetically modified host cells may be modified to incorporate one or more copies, or additional copies, of the DHFR gene. In addition, or alternatively, genetically engineered host cells may be modified to incorporate the introduction of a strong promoter element for overexpression of the DHFR gene in the genetically engineered host cells. In some cases, the DHFR gene can be codon-optimized for expression in Saccharomyces cerevisiae. The DHFR gene may originate from brown rats, Homo sapiens, or another species. In some examples, the DHFR gene is 77% similar to the naturally occurring gene.

[0222] As described above regarding the epimerization of [CYP-COR]1-BIA, genetically engineered host cells can alter the expression of BIA epimerase. BIA epimerase is encoded by the CYP-COR gene (e.g., the CYP82Y2-COR gene). In some cases, CYP-COR may also be referred to as DRS-DRR. In some cases, BIA epimerase catalyzes the conversion of (S)-1-BIA to (R)-1-BIA, as shown in Figure 7. Specifically, Figure 7 shows a biosynthetic scheme for the conversion of L-tyrosine to a morphinan alkaloid according to an embodiment of the present invention. Figure 7 provides the use of the enzymes CPR, cytochrome P450 reductase; CYP-COR, cytochrome P450 CYP82Y1-like codeinone reductase-like fusion; SalSyn, saltharidinol synthase; SalR, saltharidinol reductase; SalAT, saltharidinol 7-O-acetyltransferase; T6ODM, thebaine 6-O-demethylase; COR, codeinone reductase; and CODM, codeine-O-demethylase.

[0223] The genetically modified host cell may be modified to include constitutive expression of the CYP-COR gene in the genetically modified host cell. In addition, or separately, the genetically modified host cell may be modified to synthetically control the expression of the CYP-COR gene in the genetically modified host cell. For example, the genetically modified host cell may be modified to incorporate one or more copies of the CYP-COR gene, or additional copies. In addition, or separately, the genetically modified host cell may be modified to incorporate the introduction of a strong promoter element for overexpression of the CYP-COR gene in the genetically modified host cell. The CYP-COR gene may be derived from Papaver bracteatum, Papaver somnifer, Papaver setigerum, Chelidonium majus, or another species. In some examples, the CYP-COR gene is 77% similar to the naturally occurring gene.

[0224] [CPR] In some cases, genetically modified host cells may have altered expression of the enzyme cytochrome P450 reductase. Cytochrome P450 reductase is encoded by the CPR gene. In some cases, cytochrome P450 reductase catalyzes the (R)-reticulin → saltharidin reaction, as shown in Figure 7. In addition, cytochrome P450 reductase catalyzes other reactions, such as those shown in the figures throughout this specification. Modification of genetically modified host cells may include constitutive expression of the CPR gene in the genetically modified host cells. In addition or alternatively, genetically modified host cells may be modified to synthetically control the expression of the CPR gene in the genetically modified host cells. In some examples, genetically modified host cells may be modified to incorporate one or more copies of the CPR gene, or additional copies. In addition or alternatively, genetically modified host cells may be modified to incorporate the introduction of a potent promoter element for overexpression of the CPR gene in the genetically modified host cells. The CPR gene may originate from California poppy, P. somniferum, H. sapiens, S. cerevisiae, Arabidopsis thaliana, or another species. In some cases, the CPR gene may be 100% identical to a naturally occurring gene.

[0225] [SalSyn] In some cases, genetically modified host cells may alter the expression of the enzyme saltharidin synthase. Saltharidin synthase is encoded by the SalSyn gene. In some cases, saltharidin synthase catalyzes the (R)-reticulin → saltharidin reaction, as shown in Figure 7. Modification of genetically modified host cells may include constitutive expression of the SalSyn gene in the genetically modified host cells. In addition or alternatively, the genetically modified host cells may be modified to synthetically control the expression of the SalSyn gene in the genetically modified host cells. In some cases, the genetically modified host cells may be modified to incorporate one or more copies of the SalSyn gene, or additional copies. In addition or alternatively, the genetically modified host cells may be modified to incorporate the introduction of a strong promoter element for overexpression of the SalSyn gene in the genetically modified host cells. In some cases, the SalSyn gene may be codon-optimized for expression in Saccharomyces cerevisiae. In some cases, SalSyn may be modified at its N-terminus. The SalSyn gene may originate from Papaver somnifer, the genus Papaver (spp.), Chelidonium majas, or another species. In some cases, the SalSyn gene may be 78% similar to a naturally occurring gene.

[0226] [SalR] In some cases, genetically modified host cells may alter the expression of the enzyme saltharidinose reductase. Saltharidinose reductase is encoded by the SalR gene. In some cases, saltharidinose reductase reversibly catalyzes the saltharidinol → saltharidin reaction, as shown in Figure 7. Modification of the genetically modified host cell may include constitutive expression of the SalR gene in the genetically modified host cell. In addition or alternatively, in cases where modification of the genetically modified host cell may synthetically control the expression of the SalR gene in the genetically modified host cell, the genetically modified host cell may be modified to incorporate one or more copies, or additional copies, of the SalR gene. In addition or alternatively, the genetically modified host cell may be modified to incorporate the introduction of a strong promoter element for overexpression of the SalR gene in the genetically modified host cell. In some cases, the SalR gene may be codon-optimized for expression in Saccharomyces cerevisiae. The SalR gene may originate from Papaver somnifer, Papaver bracteatum, the genus Papaver, Chelidonium majas, or another species. In some cases, the SalR gene may be 80–100% similar to the naturally occurring gene.

[0227] [SalAT] In some cases, genetically modified host cells may have altered expression of the enzyme acetyl-CoA:saltalidinol 7-O-acetyltransferase. Acetyl-CoA:saltalidinol 7-O-acetyltransferase is encoded by the SalAT gene. In some cases, acetyl-CoA:saltalidinol 7-O-acetyltransferase catalyzes the reaction acetyl-CoA + saltalidinol → CoA + 7-O-acetylsaltalidinol, as shown in Figure 7. Modification of genetically modified host cells may include constitutive expression of the SalAT gene in the genetically modified host cells. In addition or alternatively, genetically modified host cells may be modified to synthetically control the expression of the SalAT gene in the genetically modified host cells. In some examples, genetically modified host cells may be modified to incorporate one or more copies of the SalAT gene, or additional copies. In addition or alternatively, genetically modified host cells may be modified to incorporate the introduction of a strong promoter element for overexpression of the SalAT gene in the genetically modified host cells. In some cases, the SalAT gene can be codon-optimized for expression in Saccharomyces cerevisiae. The SalAT gene may originate from Papaver somnifer, Papaver bracteatum, Papaver orientale, the genus Papaver, or another species. In some cases, the SalAT gene may be 77–80% similar to a naturally occurring gene.

[0228] [T6ODM] In some cases, genetically modified host cells can alter the expression of the enzyme thebaine 6-O-demethylase. Thebaine 6-O-demethylase is encoded by the T6ODM gene. In some cases, thebaine 6-O-demethylase catalyzes the thebaine → neopinone reaction, as shown in Figure 7. Once neopinone is produced, it can be converted to codeinone. The neopinone → codeinone conversion can occur spontaneously, or it can occur as a result of a catalyzed reaction. In other cases, the T6ODM enzyme can catalyze the O-demethylation of substrates other than thebaine. For example, T6ODM can O-demethylate olipavin to produce morphinone. Alternatively, T6ODM may catalyze the O-demethylation of BIA in 1-benzylisoquinoline, protoberberine, or the protopin class, for example, papaverine, canazine, and allocryptopine, respectively. Modification of the genetically engineered host cell may include constitutive expression of the T6ODM gene in the genetically engineered host cell. In addition, or or otherwise, the genetically engineered host cell may be modified to synthetically control the expression of the T6ODM gene in the genetically engineered host cell. For example, the genetically engineered host cell may be modified to incorporate one or more copies, or additional copies, of the T6ODM gene. In addition, or or otherwise, the genetically engineered host cell may be modified to incorporate the introduction of a strong promoter element for overexpression of the T6ODM gene in the genetically engineered host cell. In some cases, the T6ODM gene may be codon-optimized for expression in Saccharomyces cerevisiae. The T6ODM gene may be derived from Papaver somnifer or another species. In some cases, the T6ODM gene may be 76.2% similar to the naturally occurring gene.

[0229] [COR] In some cases, genetically modified host cells may have altered expression of the enzyme codeinone reductase. Codeinone reductase is encoded by the COR gene. In some cases, codeinone reductase catalyzes the reaction of codeinone to codeine, as shown in Figure 7. In some cases, codeinone reductase can catalyze the reaction of neopinone to neopine. In other cases, COR can catalyze the reduction of other morphinans, including hydrocodon → dihydrocodeine, 14-hydroxycodeinone → 14-hydroxycodeine, and hydromorphone → dihydromorphine. Modification of genetically modified host cells may include constitutive expression of the COR gene in the genetically modified host cells. In addition or separately, modification of genetically modified host cells may include synthetic control of the expression of the COR gene in the genetically modified host cells. In some examples, genetically modified host cells may be modified to incorporate one or more copies, or additional copies, of the COR gene. In addition, or alternatively, genetically engineered host cells may be modified to incorporate the introduction of a strong promoter element for overexpression of the COR gene in the genetically engineered host cells. In some cases, the COR gene may be codon-optimized for expression in Saccharomyces cerevisiae. In addition, or alternatively, the COR gene may be modified by adding targeted sequences to mitochondria, vacuoles, endoplasmic reticulum, or combinations thereof. The COR gene may originate from Papaver somnifer or another species. In some examples, the COR gene may be 76–78% similar to the naturally occurring gene. In other examples, the COR gene may be 76.8%, 77.0%, 77.3%, or 77.7% similar to the naturally occurring gene.

[0230] [CODM] In some cases, genetically engineered host cells may have altered expression of the enzyme codeine O-demethylase. Codeine O-demethylase is encoded by the CODM gene. In some cases, codeine O-demethylase catalyzes the reaction of codeine to morphine, as shown in Figure 7. Codeine O-demethylase can also catalyze the reaction of neopine to neomorphine. Codeine O-demethylase can also catalyze the reaction of thebaine to olipavin. In other cases, CODM may catalyze the O-demethylation of BIA in 1-benzylisoquinoline, aporfin, and the protoberberine class, e.g., reticulin, isocoridine, and skourelin, respectively. In other cases, the CODM enzyme may catalyze the O,O-demethylene reaction, cleaving the methylenedioxy crosslink structure in protopine. Modification of genetically engineered host cells may include constitutive expression of the CODM gene in the genetically engineered host cells. In addition, or alternatively, genetically modified host cells may be altered to synthetically control the expression of the CODM gene in the genetically modified host cells. For example, the genetically modified host cells may be altered to incorporate one or more copies, or additional copies, of the CODM gene. In addition, or alternatively, the genetically modified host cells may be altered to incorporate the introduction of a strong promoter element for overexpression of the CODM gene in the genetically modified host cells. In some cases, the CODM gene may be codon-optimized for expression in Saccharomyces cerevisiae. In addition, or alternatively, the CODM gene may be altered by adding a targeted sequence to mitochondria. The CODM gene may originate from Papaver somnifer, the genus Papaver, or another species. In some examples, the CODM gene may be 75% similar to the naturally occurring gene. In other examples, the CODM gene may be 75.2% similar to the naturally occurring gene.

[0231] [BBE] In some cases, genetically engineered host cells may have altered expression of the enzyme berberine crosslinkase. Berberine crosslinkase is encoded by the BBE gene. In some cases, berberine crosslinkase catalyzes the (S)-reticulin → (S)-scorelin reaction, as shown in Figure 8. Figure 8 shows a biosynthetic scheme for the conversion of L-tyrosine to protoberberine alkaloids according to embodiments of the present invention. Specifically, Figure 8 provides the use of the enzyme BBE, berberine crosslinkase; S9OMT, scorelin 9-O-methyltransferase; CAS, canadine synthase; CPR, cytochrome P450 reductase; and STOX, tetrahydroprotoberberine oxidase. Modification of the genetically engineered host cell may include constitutive expression of the BBE gene in the genetically engineered host cell. In addition, or separately, the genetically engineered host cell may be modified to synthetically control the expression of the BBE gene in the genetically engineered host cell. In some examples, genetically modified host cells may be altered to incorporate one or more copies, or additional copies, of the BBE gene. In addition, or separately, genetically modified host cells may be altered to incorporate the introduction of a strong promoter element for overexpression of the BBE gene in the genetically modified host cells. The BBE gene may originate from Papaver somnifer, Papaver rhoeas, California poppy, Berberis stronifera, Thalictrum flavum subsp. glaucum, Coptis japonica, Papaver species, or another species. In some examples, the BBE gene may be 99% identical to a naturally occurring gene.

[0232] [S9OMT] In some cases, genetically engineered host cells may have altered expression of the enzyme S-adenosyl-L-methionine:(S)-scourelin 9-O-methyltransferase. S-adenosyl-L-methionine:(S)-scourelin 9-O-methyltransferase is encoded by the S9OMT gene. In some cases, S-adenosyl-L-methionine:(S)-scourelin 9-O-methyltransferase catalyzes the reaction S-adenosyl-L-methionine + (S)-scourelin → S-adenosyl-L-homocysteine ​​+ (S)-tetrahydrocolumbamin, as shown in Figure 8. Modification of genetically engineered host cells may include constitutive expression of the S9OMT gene in the genetically engineered host cells. In addition, or separately, modification of genetically engineered host cells may include synthetic control of S9OMT gene expression in the genetically engineered host cells. In some examples, genetically modified host cells may be altered to incorporate one or more copies, or additional copies, of the S9OMT gene. In addition, or separately, genetically modified host cells may be altered to incorporate the introduction of a strong promoter element for overexpression of the S9OMT gene in the genetically modified host cells. In some cases, the S9OMT gene may be codon-optimized for expression in Saccharomyces cerevisiae. The S9OMT gene may originate from Thalictrum flavum subspecies glaucum, Coptis japonica, Coptis japonica, Papaver somnifer, the genera Thalictrum, Coptis spp., Papaver, or another species. In some examples, the S9OMT gene may be 100% identical to the naturally occurring gene. In other examples, the S9OMT gene is 80% identical to the naturally occurring gene.

[0233] [CAS] In some cases, genetically modified host cells may have altered expression of the enzyme (S)-canazine synthase. (S)-canazine synthase is encoded by the CAS gene. In some cases, (S)-canazine synthase catalyzes the reaction (S)-tetrahydrocolumbamin → (S)-canazine, as shown in Figure 8. Genetically modified host cells may be modified to express the CAS gene in the genetically modified host cells. Genetically modified host cells may include constitutive expression of the CAS gene in the genetically modified host cells. In addition or separately, genetically modified host cells may be modified to synthetically control the expression of the CAS gene in the genetically modified host cells. In some examples, genetically modified host cells may be modified to incorporate one or more copies of the CAS gene, or additional copies. In addition, or alternatively, the genetically engineered host cell may be modified to incorporate the introduction of a strong promoter element for overexpression of the CAS gene in the genetically engineered host cell. The CAS gene may originate from Thalictrum flavum subspecies glaucum, Coptis japonica, the genera Thalictrum, Coptis, or another species. In some cases, the CAS gene may be 100% identical to a naturally occurring gene.

[0234] [STOX] In some cases, genetically modified host cells may have altered expression of the enzyme (S)-tetrahydroprotoberberine oxidase. (S)-tetrahydroprotoberberine oxidase is encoded by the STOX gene. In some cases, (S)-tetrahydroprotoberberine oxidase catalyzes the reaction (S)-tetrahydroberberine + 2O2 → berberine + 2H2O2, as shown in Figure 8. Modification of the genetically modified host cell may include constitutive expression of the STOX gene in the genetically modified host cell. In addition or alternatively, the genetically modified host cell may be modified to synthetically control the expression of the STOX gene in the genetically modified host cell. In some cases, the genetically modified host cell may be modified to incorporate one or more copies of the STOX gene, or additional copies. In addition or alternatively, the genetically modified host cell may be modified to incorporate the introduction of a strong promoter element for overexpression of the STOX gene in the genetically modified host cell. In some cases, STOX may be modified at its N-terminus. In some cases, the STOX gene can be codon-optimized for expression in Saccharomyces cerevisiae. The STOX gene may originate from Berberis wilsonae, Coptis japonica, Berberis spp., Coptis genus, or another species. In some cases, the STOX gene may be 78% similar to a naturally occurring gene.

[0235] [TNMT] In some cases, genetically engineered host cells can alter the expression of the enzyme tetrahydroprotoberberine-N-methyltransferase. Tetrahydroprotoberberine-N-methyltransferase is encoded by the TNMT gene. In some cases, tetrahydroprotoberberine-N-methyltransferase catalyzes the canazine → N-methylcanazine reaction, as shown in Figure 9. Figure 9 shows a biosynthetic scheme for the conversion of L-tyrosine to noscapine, noscapinoid, and phthalidoisoquinoline according to an embodiment of the present invention. Specifically, Figure 9 shows the enzymes BBE, berberine cross-linking enzyme; S9OMT, scorelin 9-O-methyltransferase; CAS, canazine synthase; CPR, cytochrome P450 reductase; TNMT, tetrahydroprotoberberine cis-N-methyltransferase; CYP82Y1, N-methylcanazine 1-hydroxylase; CYP82X2, 1-hydroxy-N-methylcanazine 13-hydroxylase; AT1, 1,13-dihydroxy-N-methylcanazine The use of 13-O-acetyltransferase;CYP82X1, 4'-O-desmethyl-3-O-acetylpaparoxin synthase;CXE1, narcotin hemiacetal synthase;NOS (or SDR1), noscapine synthase;MT2, narcotrin-4'-O-methyltransferase 1;MT3, narcotrin-4'-O-methyltransferase 2; and 6OMT, 6-O-methyltransferase is provided.

[0236] In another example, tetrahydroprotoberberine-N-methyltransferase catalyzes the styropine → cis-N-methylstyropine reaction, as shown in Figure 10. Figure 10 shows a biosynthetic scheme for the conversion of L-tyrosine to sanguinaline and benzophenanthridine alkaloids according to embodiments of the present invention. Specifically, Figure 10 provides the use of the enzymes BBE, berberine crosslinking enzyme; CFS, kaylantiphorine synthase; STS, styropine synthase; TNMT, tetrahydroberberine N-methyltransferase; MSH, cis-N-methylstyropine 14-hydroxylase; P6H, protopine 6-hydroxylase; and DBOX, dihydrobenzophenanthride oxidase. Modification of the genetically engineered host cell may include constitutive expression of the TNMT gene in the genetically engineered host cell. In addition, or separately, the genetically engineered host cell may be modified to synthetically control the expression of the TNMT gene in the genetically engineered host cell. In some examples, genetically modified host cells may be altered to incorporate one or more copies, or additional copies, of the TNMT gene. In addition, or separately, genetically modified host cells may be altered to incorporate the introduction of a strong promoter element for overexpression of the TNMT gene in the genetically modified host cells. In some cases, the TNMT gene may be codon-optimized for expression in Saccharomyces cerevisiae. The TNMT gene may be derived from Papaver somnifer, California poppy, Papaver bracteatum, thistle poppy, or another species. In some examples, the TNMT gene may be 100% identical to the naturally occurring gene. In other examples, the TNMT gene may be 81% identical to the naturally occurring gene.

[0237] [CYP82Y1] In some cases, genetically engineered host cells may have altered expression of the enzyme N-methylcanadine 1-hydroxylase. N-methylcanadine 1-hydroxylase is encoded by the CYP82Y1 gene. In some cases, N-methylcanadine 1-hydroxylase catalyzes the reaction N-methylcanadine → 1-hydroxy-N-methylcanadine, as shown in Figure 9. Modification of genetically engineered host cells may include constitutive expression of the CYP82Y1 gene in the genetically engineered host cells. In addition or alternatively, genetically engineered host cells may be modified to synthetically regulate the expression of the CYP82Y1 gene in the genetically engineered host cells. In some cases, genetically engineered host cells may be modified to incorporate one or more copies of the CYP82Y1 gene, or additional copies. In addition or alternatively, genetically engineered host cells may be modified to incorporate the introduction of a strong promoter element for overexpression of the CYP82Y1 gene in the genetically engineered host cells. In some cases, the CYP82Y1 gene can be codon-optimized for expression in Saccharomyces cerevisiae. In some cases, CYP82Y1 may be modified at its N-terminus. The CYP82Y1 gene may originate from Papaver somnifer, the genus Papaver, Plantago arenaria, Rauwolfia heterophylla, Adlumia fungosa, Hydrastis canadensis, Stylomecon heterophylla, the genus Hypecoum, or another species. In some cases, the CYP82Y1 gene may be 70–78% similar to the naturally occurring gene.

[0238] [CYP82X2] In some cases, genetically engineered host cells may have altered expression of the enzyme 1-hydroxy-N-methylcanadine 13-hydroxylase. 1-hydroxy-N-methylcanadine 13-hydroxylase is encoded by the CYP82X2 gene. In some cases, 1-hydroxy-N-methylcanadine 13-hydroxylase catalyzes the reaction 1-hydroxy-N-methylcanadine → 1-hydroxy-N-methylophylocarpine (i.e., 1,13-dihydroxy-N-methylcanadine), as shown in Figure 9. Modification of genetically engineered host cells may include constitutive expression of the CYP82X2 gene in the genetically engineered host cells. In addition, or separately, modification of genetically engineered host cells may synthetically control the expression of the CYP82X2 gene in the genetically engineered host cells. In some examples, genetically engineered host cells may be modified to incorporate one or more copies, or additional copies, of the CYP82X2 gene. In addition, or alternatively, the genetically engineered host cell may be modified to incorporate the introduction of a strong promoter element for overexpression of the CYP82X2 gene in the genetically engineered host cell. In some cases, the CYP82X2 gene may be codon-optimized for expression in Saccharomyces cerevisiae. In some examples, CYP82X2 may be modified at its N-terminus. The CYP82X2 gene may originate from P. somniferum, Papaver, Plantago asiatica, Rauwolfia heterophylla, Adormia fungosa, Hydrastis canadensis, Stylomecon heterophylla, Dactylicapnos torulosa, Glaucium flavum, Berberis laurina, B. vulgaris, Corydalis spp., Fumaria spp., Dactylicapnos spp., or other species. In some cases, the CYP82X2 gene may be 70–77% similar to the naturally occurring gene. In other cases, the CYP82X2 gene may be N-terminally modified.In this example, N-terminal gene manipulation may include shortening of the N-terminal cleavage.

[0239] [CYP82X1] In some cases, genetically engineered host cells may have altered expression of the enzyme 4'-O-desmethyl-3-O-acetylpaparoxine synthase. 4'-O-desmethyl-3-O-acetylpaparoxine synthase is encoded by the CYP82X1 gene. In some cases, 4'-O-desmethyl-3-O-acetylpaparoxine synthase catalyzes the reaction 1-hydroxy-13-O-acetyl-N-methylcanazine → 4'-O-desmethyl-3-O-acetylpaparoxine, as shown in Figure 9. In addition, CYP82X1 catalyzes the reaction 1-hydroxy-N-methylcanazine → 4'-O-desmethylmacrantaldehyde. Modification of genetically engineered host cells may include constitutive expression of the CYP82X1 gene in the genetically engineered host cells. In addition, or separately, the genetically modified host cell may be modified to synthetically control the expression of the CYP82X1 gene in the genetically modified host cell. For example, the genetically modified host cell may be modified to incorporate one or more copies, or additional copies, of the CYP82X1 gene. In addition, or separately, the genetically modified host cell may be modified to incorporate the introduction of a strong promoter element for overexpression of the CYP82X1 gene in the genetically modified host cell. In some cases, the CYP82X1 gene may be codon-optimized for expression in Saccharomyces cerevisiae. In some examples, CYP82X1 may be modified at the N-terminus. The CYP82X1 gene may originate from Papaver somnifer, the genus Papaver, Plantago asiatica, Rauwolfia heterophylla, Adormia fungosa, Hydrastis canadensis, Stylomecon heterophylla, the genus Hypecoum, or another species. In some cases, the CYP82X1 gene may be 71–77% similar to the naturally occurring gene. In other cases, the CYP82X1 gene may undergo N-terminal manipulation. In some cases, N-terminal manipulation may include shortening of the N-terminal cleavage.

[0240] [CFS] In some cases, genetically modified host cells may have altered expression of the enzyme cheirantiforine synthase. Cheirantiforine synthase is encoded by the CFS gene. In some cases, cheirantiforine synthase catalyzes the skourelin → cheirantiforine reaction, as shown in Figure 10. Modification of the genetically modified host cell may include constitutive expression of the CFS gene in the genetically modified host cell. In addition or alternatively, the genetically modified host cell may be modified to synthetically control the expression of the CFS gene in the genetically modified host cell. In some cases, the genetically modified host cell may be modified to incorporate one or more copies of the CFS gene, or additional copies. In addition or alternatively, the genetically modified host cell may be modified to incorporate the introduction of a strong promoter element for overexpression of the CFS gene in the genetically modified host cell. The CFS gene may be derived from P. somniferum, California poppy, A. mexicana, or another species. In some cases, CFS genes may be 77%, 78%, or 79% similar to naturally occurring genes. In addition, CFS genes can be codon-optimized for expression in Saccharomyces cerevisiae.

[0241] [STS] In some cases, genetically modified host cells may have altered expression of the enzyme styropine synthase. Styropine synthase is encoded by the STS gene. In some cases, styropine synthase catalyzes the caylanthofrine → styropine reaction, as shown in Figure 10. Modification of the genetically modified host cell may include constitutive expression of the STS gene in the genetically modified host cell. In addition or alternatively, the genetically modified host cell may be modified to synthetically control the expression of the STS gene in the genetically modified host cell. In some cases, the genetically modified host cell may be modified to incorporate one or more copies of the STS gene, or additional copies. In addition or alternatively, the genetically modified host cell may be modified to incorporate the introduction of a strong promoter element for overexpression of the STS gene in the genetically modified host cell. The STS gene may be derived from P. somniferum, California poppy, thistle poppy, or another species. In some cases, STS genes may be 76%, 78%, or 79% similar to naturally occurring genes. In addition, STS genes can be codon-optimized for expression in Saccharomyces cerevisiae.

[0242] [MSH] In some examples, genetically engineered host cells may have altered expression of the enzyme cis-N-methylstyrope 14-hydroxylase. Cis-N-methylstyrope 14-hydroxylase is encoded by the MSH gene. In some examples, cis-N-methylstyrope 14-hydroxylase catalyzes the cis-N-methylstyrope → protopine reaction, as shown in Figure 10. Modification of genetically engineered host cells may include constitutive expression of the MSH gene in the genetically engineered host cells. In addition or alternatively, genetically engineered host cells may be modified to synthetically regulate the expression of the MSH gene in the genetically engineered host cells. In some examples, genetically engineered host cells may be modified to incorporate one or more copies of the MSH gene, or additional copies. In addition or alternatively, genetically engineered host cells may be modified to incorporate the introduction of a strong promoter element for overexpression of the MSH gene in the genetically engineered host cells. The MSH gene may be derived from P. somniferum or another species. In some cases, the MSH gene can be 79% similar to the naturally occurring gene. In addition, the MSH gene can be codon-optimized for expression in Saccharomyces cerevisiae.

[0243] [P6H] In some cases, genetically engineered host cells may have altered expression of the enzyme protopine-6-hydroxylase. Protopine-6-hydroxylase is encoded by the P6H gene. In some cases, protopine-6-hydroxylase catalyzes the protopine → 6-hydroxyprotopine reaction, as shown in Figure 10. Modification of genetically engineered host cells may include constitutive expression of the P6H gene in the genetically engineered host cells. In addition or alternatively, genetically engineered host cells may be modified to synthetically control the expression of the P6H gene in the genetically engineered host cells. In some cases, genetically engineered host cells may be modified to incorporate one or more copies of the P6H gene, or additional copies. In addition or alternatively, genetically engineered host cells may be modified to incorporate the introduction of a potent promoter element for overexpression of the CFS gene in the genetically engineered host cells. The P6H gene may originate from P. somniferum, California poppy, or another species. In some cases, the P6H gene may be 79% similar to the naturally occurring gene. In addition, the P6H gene can be codon-optimized for expression in Saccharomyces cerevisiae.

[0244] [DBOX] In some examples, genetically modified host cells may have altered expression of the enzyme dihydrobenzophenanthridine oxidase. Dihydrobenzophenanthridine oxidase is encoded by the DBOX gene. In some examples, dihydrobenzophenanthridine oxidase catalyzes the reaction dihydrosanguinaline → sanguinaline, as shown in Figure 10. Modification of the genetically modified host cell may include constitutive expression of the DBOX gene in the genetically modified host cell. In addition or alternatively, the genetically modified host cell may be modified to synthetically control the expression of the DBOX gene in the genetically modified host cell. In some examples, the genetically modified host cell may be modified to incorporate one or more copies of the DBOX gene, or additional copies. In addition or alternatively, the genetically modified host cell may be modified to incorporate the introduction of a strong promoter element for overexpression of the DBOX gene in the genetically modified host cell. The DBOX gene may be derived from P. somniferum or another species. In some examples, the DBOX gene may be 100% identical to a naturally occurring gene. In addition, the DBOX gene can be codon-optimized for expression in Saccharomyces cerevisiae.

[0245] [AT1] In some cases, genetically engineered host cells may have altered expression of the enzyme 1,13-dihydroxy-N-methylkanadine 13-O acetyltransferase. 1,13-dihydroxy-N-methylkanadine 13-O acetyltransferase is encoded by the AT1 gene. In some cases, 1,13-dihydroxy-N-methylkanadine 13-O acetyltransferase catalyzes the reaction 1,13-dihydroxy-N-methylkanadine → 1-hydroxy-13-O-acetyl-N-methylkanadine, as shown in Figure 11A. Figure 11A shows a biosynthetic scheme for the conversion of canadine to noscapine according to an embodiment of the present invention. Modification of the genetically engineered host cell may include constitutive expression of the AT1 gene in the genetically engineered host cell. In addition, or separately, the genetically engineered host cell may be modified to synthetically control the expression of the AT1 gene in the genetically engineered host cell. In some examples, the genetically engineered host cell may be modified to incorporate one or more copies, or additional copies, of the AT1 gene. In addition, or separately, genetically engineered host cells may be modified to incorporate the introduction of a strong promoter element for overexpression of the AT1 gene in the genetically engineered host cells. In some cases, the AT1 gene can be codon-optimized for expression in Saccharomyces cerevisiae. The AT1 gene may originate from P. somniferum, Papaver, Plantago asiatica, Rauwolfia heterophylla, Adormia fungosa, Hydrastis canadensis, Stylomecon heterophylla, Hypecoum leptocarpum, Dactylicapnos torrosa, Glaucium flavum, Berberis laurina, B. vargalis, Corydalis, Humaria, Dactylicapnos, or other species. In some cases, the AT1 gene is 81% similar to the naturally occurring gene.

[0246] [CXE1 or CXE2] In some cases, genetically engineered host cells may have altered expression of the enzyme narcotin hemiacetal synthase. Narcotin hemiacetal synthase is encoded by the CXE1 gene. The enzyme encoded by the CXE2 gene can also function as narcotin hemiacetal synthase. In some cases, narcotin hemiacetal synthase catalyzes the reactions 4'-O-desmethyl-3-O-acetylpapaberoxine → narcotin hemiacetal and 3-O-acetylpapaberoxine → narcotin hemiacetal, as shown in Figure 11A. Modification of genetically engineered host cells may include constitutive expression of the CXE1 or CXE2 gene in the genetically engineered host cells. In addition, or separately, modification of genetically engineered host cells may synthetically regulate the expression of the CXE1 or CXE2 gene in the genetically engineered host cells. In some cases, genetically modified host cells may be altered to incorporate one or more copies, or additional copies, of the CXE1 or CXE2 gene. In addition, or separately, genetically modified host cells may be altered to incorporate the introduction of a strong promoter element for overexpression of the CXE1 or CXE2 gene in the genetically modified host cell. In some cases, the CXE1 or CXE2 gene may be codon-optimized for expression in Saccharomyces cerevisiae. The CXE1 or CXE2 gene may originate from P. somniferum, Papaver, Plantago asiatica, Rauwolfia heterophylla, Adormia fungosa, Hydrastis canadensis, Stylomecon heterophylla, Hypecoum leptocarpum, Dactylicapnos torrosa, Glaucium flavum, Berberis laurina, B. vargaris, Corydalis, Fumaria, Dactylicapnos, or another species. In some cases, the CXE1 or CXE2 gene may be 78% similar to the naturally occurring gene.

[0247] [SDR1] In some cases, genetically modified host cells may have altered expression of the enzyme noscapine synthase. Noscapine synthase is encoded by the SDR1 gene. In some cases, noscapine synthase catalyzes the narcotrin hemiacetal → narcotrin reaction, as shown in Figure 11A. In addition, noscapine synthase catalyzes the narcotrin hemiacetal → noscapine reaction. Modification of genetically modified host cells may include constitutive expression of the SDR1 gene in the genetically modified host cells. In addition or alternatively, genetically modified host cells may be modified to synthetically regulate the expression of the SDR1 gene in the genetically modified host cells. In some examples, genetically modified host cells may be modified to incorporate one or more copies, or additional copies, of the SDR1 gene. In addition or alternatively, genetically modified host cells may be modified to incorporate the introduction of a strong promoter element for overexpression of the SDR1 gene in the genetically modified host cells. In some cases, the SDR1 gene can be codon-optimized for expression in Saccharomyces cerevisiae. The SDR1 gene may originate from P. somniferum, Papaver, Plantago asiatica, Rauwolfia heterophylla, Adormia fungosa, Hydrastis canadensis, Stylomecon heterophylla, Hypecoum leptocarpum, Dactylicapnos torrosa, Glaucium flavum, Berberis laurina, B. vargalis, Corydalis, Humaria, Dactylicapnos, or other species. In some cases, the SDR1 gene may be 79% similar to the naturally occurring gene.

[0248] [MT2 and MT3] In some cases, genetically engineered host cells may have altered expression of the enzyme narcotrin 4'-O-methylase. Narcotrin 4'-O-methylase is a heterodimer formed from O-methyltransferase monomers encoded by the MT2 and MT3 genes. In some cases, narcotrin 4'-O-methylase catalyzes the narcotrin → noscapine reaction, as shown in Figure 11A. In addition, narcotrin 4'-O-methylase catalyzes the narcotrin nehemiacetal → narcotin hemiacetal and the 4'-O-desmethyl-3-O-acetylpapaberoxine → 3-O-acetylpapaberoxine reactions. Modification of the genetically engineered host cells may include constitutive expression of the MT2 and MT3 genes in the genetically engineered host cells. In addition, or alternatively, genetically modified host cells may be altered to synthetically control the expression of the MT2 and MT3 genes in the genetically modified host cells. For example, the genetically modified host cells may be altered to incorporate one or more copies, or additional copies, of the MT2 and MT3 genes. In addition, or alternatively, the genetically modified host cells may be altered to incorporate the introduction of a strong promoter element for overexpression of the MT2 and MT3 genes in the genetically modified host cells. In some cases, the MT2 and MT3 genes may be codon-optimized for expression in Saccharomyces cerevisiae. The MT2 and MT3 genes may be derived from P. somniferum, Papaver, Fumaria parviflora, Plantago asiatica, Rauwolfia heterophylla, or another species. In some cases, the MT2 and MT3 genes may be 80% and 79% similar, respectively, to naturally occurring genes.

[0249] [morA] In some cases, genetically engineered host cells can alter the expression of the enzyme morphine dehydrogenase. Morphine dehydrogenase is encoded by the morA gene. In some cases, morphine dehydrogenase catalyzes the morphine → morphinone reaction, as shown in Figure 11B. In other cases, morphine dehydrogenase catalyzes the codeinone → codeine reaction, as shown in Figure 11B. Figure 11B shows a biosynthetic scheme for the production of a semi-synthetic opioid according to an embodiment of the present invention. Specifically, Figure 11B shows the extended transformation of thebaine in yeast by incorporating morA, morphine dehydrogenase; and morB, morphine reductase.

[0250] The genetically modified host cell may be modified to include constitutive expression of the morA gene in the genetically modified host cell. In addition, or separately, the genetically modified host cell may be modified to synthetically control the expression of the morA gene in the genetically modified host cell. For example, the genetically modified host cell may be modified to incorporate one or more copies, or additional copies, of the morA gene. In addition, or separately, the genetically modified host cell may be modified to incorporate the introduction of a strong promoter element for overexpression of the morA gene in the genetically modified host cell. In some cases, the morA gene may be codon-optimized for expression in Saccharomyces cerevisiae. The morA gene may be derived from Pseudomonas putida or another species. In some cases, the morA gene may be 73.7% similar to the naturally occurring gene.

[0251] [morB] In some cases, genetically modified host cells may have altered expression of the enzyme morphinone reductase. Morphinon reductase is encoded by the morB gene. In some cases, morphinone reductase catalyzes the reaction codeinone → hydrocodone, as shown in Figure 11B. In other cases, morphinone reductase catalyzes the reaction morphinone → hydromorphone, as also shown in Figure 11B. In other cases, morphinone reductase catalyzes the reaction 14-hydroxycodeinone → oxycodone. Modification of the genetically modified host cell may include constitutive expression of the morB gene in the genetically modified host cell. In addition or separately, the genetically modified host cell may be modified to synthetically control the expression of the morB gene in the genetically modified host cell. In some examples, the genetically modified host cell may be modified to incorporate one or more copies, or additional copies, of the morB gene. In addition, or alternatively, genetically engineered host cells may be modified to incorporate the introduction of a strong promoter element for overexpression of the morB gene in the genetically engineered host cells. In some cases, the morB gene can be codon-optimized for expression in Saccharomyces cerevisiae. The morB gene may originate from Pseudomonas putida or another species. In some cases, the morB gene may be 67.2% similar to a naturally occurring gene.

[0252] [CYP80A1] In some cases, genetically engineered host cells may express the enzyme verbamnin synthase. Verbamnin synthase is encoded by the gene for the cytochrome P450 enzyme 80A1 (CYP80A1). In some cases, CYP80A1 catalyzes the reaction (S)-N-methylcoclaurine + (R)-N-methylcoclaurine → verbamnin. In other cases, CYP80A1 catalyzes the reaction (R)-N-methylcoclaurine + (R)-N-methylcoclaurine → guattegaumerine. In other cases, CYP80A1 catalyzes the reaction (R)-N-methylcoclaurine + (S)-coclaurine → 2'norverbamnin. Modification of genetically engineered host cells may include constitutive expression of the CYP80A1 gene in the genetically engineered host cells. In addition, or alternatively, genetically modified host cells may be altered to synthetically control the expression of the CYP80A1 gene in the genetically modified host cells. For example, the genetically modified host cells may be altered to incorporate one or more copies, or additional copies, of the CYP80A1 gene. In addition, or alternatively, the genetically modified host cells may be altered to incorporate the introduction of a strong promoter element for overexpression of the CYP80A1 gene in the genetically modified host cells. In some cases, the CYP80A1 gene may be codon-optimized for expression in Saccharomyces cerevisiae. The CYP80A1 gene may originate from Berberis stronifera or another species. In some cases, the CYP80A1 gene may be 76% similar to the naturally occurring gene.

[0253] [PODA] In some cases, genetically engineered host cells may express the enzyme protopine O-dealkylase. Protopine O-dealkylase is encoded by the gene PODA. In some cases, PODA catalyzes the O,O-demethylenelation of protoberberines and protopines, such as canazine, styropine, berberine, cryptopine, allocryptopine, and protopine. In some cases, PODA catalyzes the O-demethylation of BIAs, including tetrahydropapaverine, tetrahydropalmatine, and cryptopine. Modification of genetically engineered host cells may include constitutive expression of the PODA gene in the genetically engineered host cells. In addition, or separately, modification of genetically engineered host cells may include synthetic control of PODA gene expression in the genetically engineered host cells. In some examples, genetically engineered host cells may be modified to incorporate one or more copies, or additional copies, of the PODA gene. In addition, or separately, the genetically engineered host cell may be modified to incorporate the introduction of a strong promoter element for overexpression of the PODA gene in the genetically engineered host cell. In some cases, the PODA gene can be codon-optimized for expression in Saccharomyces cerevisiae. The PODA gene may be derived from Papaver somnifer or other species. In some cases, the PODA gene may be 70–100% similar to the naturally occurring gene.

[0254] The aforementioned gene examples can be expressed from several different platforms in host cells, including plasmids (2μ, ARS / CEN), YACs, or genomes. In addition, the aforementioned gene sequence examples can be either native or codon-optimized for expression in a desired heterologous host (e.g., Saccharomyces cerevisiae). [Examples]

[0255] The following examples are given for the purpose of illustrating various embodiments of the invention and are not intended to limit the invention in any way. Where shown, expression constructs are understood to include preferred promoters, genes, and terminators, including cases where the exact terminator sequence used is not specified. These examples, together with the methods described herein, represent currently preferred embodiments and are illustrative and are not intended to limit the scope of the invention. Variations and other uses therein, encompassed within the spirit of the invention as defined by the claims, will be conceivable to those skilled in the art.

[0256] Example 1: Tyrosine hydroxylase mutants enhance norcoclaurine production in genetically modified yeast strains. Tyrosine hydroxylase derived from brown rat (R. norvegicus) was synthesized using yeast codon optimization and cloned into low-copy plasmids. Single mutants (W166Y, E332D, S40D, and R37ER38E), double mutants (W166Y and E332D, W166Y and S40D, W166Y and R37ER38E), and one triple mutant (W166Y, R37ER38E, and E332D) were generated through site-directed mutagenesis. Each TyrH mutant was given the following mutation in central metabolism (as described in U.S. Provisional Patent Application No. 61 / 899,496): ARO4 FBRThe genes were expressed from a low-copy plasmid containing the GPD promoter in yeast strains containing ΔZWF1 and GPD-TKL1 promoter substitutions. In addition, the strains expressed a chromosomally integrated copy of dopadecarboxylase (DODC) from P. putida, four chromosomally integrated genes from Norway rats that produce the auxiliary substrate tetrahydrobiopterin (pyruboyltetrahydropterin synthase, PTPS; sepiapterin reductase, SepR; pterin 4α-carbinolamine dehydrogenase, PCD; dihydropteridine reductase, QDHPR), and norcoclaurine synthase (NCS) from Coptis japonica expressed from a low-copy plasmid containing the GPD promoter. Strains containing the TyrH mutant were grown for 96 hours in selectively defined medium (YNB) containing 2% dextrose and lacking tyrosine, and norcoclaurine production was measured in the medium via LC-MS / MS in MRM mode with a transition of 272 m / z to 107 m / z. Figure 12 shows the results of this assay, demonstrating that the TyrH mutant can increase norcoclaurine production by 9 times compared to wild-type TyrH. Thus, Figure 12 shows a tyrosine hydroxylase mutant that enhances norcoclaurine production from sugar in a genetically modified yeast strain according to an embodiment of the present invention.

[0257] Example 2: Tyrosine hydroxylase mutants improve reticulin production in genetically modified yeast strains. Tyrosine hydroxylase derived from brown rats was synthesized, codon-optimized for yeast, and cloned into low-copy plasmids. Single mutants (W166Y, E332D, S40D, and R37ER38E), double mutants (W166Y and E332D, W166Y and S40D, W166Y and R37ER38E), and one triple mutant (W166Y, R37ER38E, and E332D) were generated through site-directed mutagenesis. Each TyrH mutant had the following mutation in central metabolism (as described in U.S. Provisional Patent Application No. 61 / 899,496): ARO4 FBRThe drug was expressed from a low-copy plasmid containing the GPD promoter in yeast strains containing ΔZWF1 and GPD-TKL1 promoter substitutions. In addition, the strain contains a chromosomal copy of dopadecarboxylase (DODC) from P. putida, four chromosomal genes from Norway rats that produce the auxiliary substrate tetrahydrobiopterin (pyruboyltetrahydropterin synthase, PTPS; sepiapterin reductase, SepR; pterin 4α-carbinolamine dehydrogenase, PCD; dihydropteridine reductase, QDHPR), norcoclaurine synthase (NCS) from Coptis japonica expressed from a low-copy plasmid with a GPD promoter, and five genes for the biosynthesis of reticuline from norcoclaurine (P. somniferum 6-O-methyltransferase, Ps6OMT; P. somniferum coclaurine N-methyltransferase, PsCNMT; California poppy cytochrome P450 80B1, EcCYP80B1; P. somniferum cytochrome P450 NADPH reductase (PsCPR) and P. somniferum 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase (Ps4'OMT) were expressed. Strains containing the TyrH mutant were grown for 96 hours in selectively defined medium (YNB) containing 2% dextrose and lacking tyrosine, and reticulin production was measured in the medium via LC-MS / MS in MRM mode with a transition of 330 m / z to 137 m / z. Figure 13 shows the results of this assay, demonstrating that the TyrH mutant can increase reticulin production by 5 times compared to wild-type TyrH. Thus, Figure 13 shows a tyrosine hydroxylase mutant that improves reticulin production from sugar in a genetically modified yeast strain according to an embodiment of the present invention.

[0258] Example 3: DHFR expression enhances tyrosine hydroxylase activity in genetically modified yeast strains. Dihydrofolate reductase (DHFR) derived from Mus musculus was codon-optimized for yeast, synthesized, and cloned into a low-copy plasmid under the control of the GPD promoter. DHFR was co-expressed with wild-type RnTyrH (a low-copy plasmid having the GPD promoter) in a yeast strain containing the following mutations in central metabolism (described in U.S. Provisional Patent Application No. 61 / 899,496): ARO4 FBR ΔZWFl, and the GPD-TKLl promoter substitution. In addition, the strain expressed four genes from Mus musculus integrated into four chromosomes that produce the cofactor tetrahydrobiopterin (pyruvoyl tetrahydropterin synthase, PTPS; sepiapterin reductase, SepR; pterin 4a-carbinolamine dehydratase, PCD; dihydropteridine reductase, QDHPR). Strains expressing DHFR and wild-type RnTyrH were grown for 96 hours in a selective defined medium (YNB) with 2% dextrose and lacking tyrosine, and L-DOPA production was measured in the medium via LC-MS / MS in MRM mode at a transition of l98 m / z to 152 m / z. Expression of DHFR with wild-type RnTyrH increased L-DOPA production 1.8-fold, as shown in Figure 14. Thus, Figure 14 shows co-expression of dihydrofolate reductase (DHFR) to improve L-DOPA production by tyrosine hydroxylase in a genetically engineered yeast strain, according to an embodiment of the present invention.

[0259] Example 4: Addition of an antioxidant to the growth medium improves tyrosine hydroxylase activity in a genetically engineered yeast strain The following mutations in central metabolism (described in U.S. Provisional Patent Application No. 61 / 899,496): ARO4 FBRA yeast strain expressing wild-type RnTyrH from a low-copy plasmid under the control of four chromosomes of Norway rat-derived genes (pyruboyltetrahydropterin synthase, PTPS; sepiapterin reductase, SepR; pterin 4α-carbinolamine dehydrogenase, PCD; dihydropteridine reductase, QDHPR) containing ΔZWF1 and GPD-TKL1 promoter substitutions and producing the auxiliary substrate tetrahydrobiopterin, as well as the GPD promoter, was grown for 96 hours in selectively defined medium (YNB) containing 2% galactose and 2 mM ascorbic acid but lacking tyrosine.

[0260] L-dopa production was measured in culture medium via LC-MS / MS in MRM mode during the transition from 198 m / z to 152 m / z. Addition of 2 mM ascorbic acid increased L-dopa production using wild-type RnTyrH by 1.8 times. In addition, concentrated BH4 intermediates were measured by LC-MS / MS in MRM mode during the following transitions: B, 238 m / z to 178 m / z; BH2, 240 m / z to 165 m / z; and BH4, 242 m / z to 166 m / z. Addition of ascorbic acid also increased BH4 in the culture medium, indicating that the oxidation of BH4 to BH2 was prevented.

[0261] Accordingly, Figure 15 shows, according to embodiments of the present invention, (A) the addition of an antioxidant to a culture medium that enhances L-DOPA production by tyrosine hydroxylase in a genetically modified yeast strain, and (B) the addition of an antioxidant to a culture medium that increases BH4 levels. Specifically, Figure 15A shows that wild-type RnTyrH (expressed from a low-copy plasmid under the control of the GPD promoter) undergoes the following mutation in central metabolism (as described in U.S. Provisional Patent Application No. 61 / 899,496): ARO4 FBRThis indicates that it was expressed in yeast strains containing ΔZWF1 and GPD-TKL1 promoter substitutions. In addition, the strains expressed four rat-derived genes integrated into four chromosomes that produce the auxiliary substrate tetrahydrobiopterin (pyruvoyltetrahydropterin synthase, PTPS; sepiapterin reductase, SepR; pterin 4α-carbinolamine dehydrogenase, PCD; dihydropteridine reductase, QDHPR). Strains expressing wild-type RnTyrH were grown for 96 hours in selectively defined medium (YNB) containing 2% dextrose, lacking tyrosine, and with or without 2 mM ascorbic acid (aa). L-dopa production was measured in the medium via LC-MS / MS in MRM mode with a transition of 198 m / z to 152 m / z. In addition, Figure 15B shows that the concentration of the BH4 intermediate was measured in the culture medium of strains grown with or without 2 mM ascorbic acid (aa) using LC-MS / MS in MRM mode with the following transition: BH4, 242 m / z to 166 m / z, using the same strain as described in Figure 15A.

[0262] Example 5: Yeast strain genetically engineered to produce bisBIA-berbamunin Figure 16 shows (A) a biosynthetic scheme for the conversion of L-tyrosine to bis-BIA and (B) a yeast strain genetically engineered to biosynthesize bis-BIA, according to embodiments of the present invention. Specifically, Figure 16(A) shows the pathway used to produce bis-BIA-verbamunin and guattegaumelin. Figure 16 provides the use of the enzymes ARO9, aromatic aminotransferase; ARO10, phenylpyruvate decarboxlase; TyrH, tyrosine hydroxylase; DODC, dopadecarboxylase; NCS, norcoclaurine synthase; 6OMT, 6-O-methyltransferase; CNMT, coclaurine N-methyltransferase; CYP80A1, cytochrome P450 80A1; and CPR, cytochrome P450 NADPH reductase. Among the metabolites provided in Figure 16, 4-HPA, 4-HPP, and L-tyrosine are synthesized naturally in yeast. The other metabolites shown in Figure 16 are not naturally synthesized in yeast.

[0263] In embodiments of the present invention, bis-BIA-producing yeast strains that produce bis-BIA, such as those generated using the pathway shown in (A), are genetically engineered by incorporating a single construct into the YDR514C locus. In addition, Figure 16(B) provides examples of yeast strains genetically engineered to synthesize bis-BIA. Ps6OMT, PsCNMT, PsCPR, and BsCYP80A1 were incorporated into the yeast genome at a single locus (YDR514C). Each enzyme was expressed from a constitutive promoter. The arrangement of the gene expression cassette and the transcription direction are indicated by arrows in the schematic diagram. These strains convert (R)- and (S)-norcoclaurine to coclaurine, and then to N-methylcoclaurine. In one example, a strain may conjugate one molecule of (R)-N-methylcoclaurine and one molecule of (S)-N-methylcoclaurine to form berbamnin. In another example, the strain may conjugate two molecules of (R)-N-methylcoclaurine to form guattegaumerine. In yet another example, the strain may conjugate one molecule of (R)-N-methylcoclaurine and one molecule of (S)-coclaurine to form 2'-norverbamunin. In yet another embodiment, the strain may be genetically engineered to provide precursors (R)- and (S)-norcoclaurine from L-tyrosine, as shown in Figure 5.

[0264] The construct includes expression cassettes for P. somniferum enzymes 6OMT and CNMT, expressed as their native plant nucleotide sequences. A third enzyme derived from P. somniferum, CPR, is codon-optimized for expression in yeast. PsCPR supports the activity of a fourth enzyme, Berberis stronifera CYP80A1, and is also codon-optimized for expression in yeast. Each expression cassette contains a specific yeast constitutive promoter and terminator. Finally, the incorporated construct includes a LEU2-selective marker adjacent to the loxP site for excision by Cre recombinase.

[0265] Yeast strains expressing Ps6OMT, PsCNMT, BsCYP80A1, and PsCPR are cultured in selective medium for 16 hours at 30°C with shaking. Cells are harvested by centrifugation and resuspended in 400 μL of disruption buffer (100 mM Tris-HCl, pH 7.0, 10% glycerol, 14 mM 2-mercaptoethanol, protease inhibitor cocktail). Cells are physically disrupted by the addition of glass beads and a vortex. The liquid is removed, and the following substrates and cofactors are added to initiate the reaction: 1 mM (R,S)-norcoclaurine, 10 mM S-adenosylmethionine, and 25 mM NADPH. Crude cell lysates are incubated at 30°C for 4 hours, and then quenched by adding ethanol acidified with 0.1% acetic acid in a 1:1 ratio. The reaction products are centrifuged, and the supernatant is analyzed by liquid chromatography-mass spectrometry (LC-MS) to detect the bis-BIA products verbamunin, guattegaumeline, and 2'-norverbamunin based on their retention and mass-charge ratio.

[0266] Example 6. Identification of epimerase enzyme To identify suitable epimerase enzymes for carrying out the epimerization reaction of the method disclosed herein, cytochrome P450 oxidase 82Y1-like domains and codeinone reductase-like domains were identified in a single open reading frame (CYP-COR) from a commercially available plant transcriptome. CYP-COR fusions were identified using blastn, employing queries based on the sequences of previously published COR-silencing VIGS constructs that resulted in reticulin accumulation (Wijekoon and Facchini. 2012. Plant J. 69: 1052-63), obtained from BLAST searches of the 1000 Plants Project (Matasci, et al. 2014. Gigascience. 3:17) and PhytoMetaSyn (Facchini, et al. 2012. Trends Biotechnol. 30:127-31; Xiao, et al. 2013. J. Biotechnol. 166:122-34) transcriptomes. Once a CYP-COR fusion sequence was observed as a hit, its sequence was translated, and its amino acid sequence was used as a query for a second search of both databases using tblastn. Figure 17 shows the phylogenetic tree of the CYP-COR fusion enzyme identified from the database. The sequences were identified based on bioinformatics searches from the 1000 Plants Project and PhytoMetaSyn transcriptome databases. In addition, exemplary amino acid sequences are shown in Figure 4, as described above. Furthermore, Table 1 lists various examples of amino acid sequences identified for this CYP-COR enzyme, which originate from various plants including Papaver somnifer (poppy), Papaver setigerum (poppy of Troy), Papaver bracteatum (bright orchid), and Chelidonium majas (greaterland).

[0267] Example 7. Epimerization of (S)-reticulin to (R)-reticulin in genetically modified non-plant host cells. Non-plant host cells were genetically engineered to heterologously express the enzymes described herein. For example, a yeast strain (Saccharomyces cerevisiae) was genetically engineered to heterologously express the identified epimerases described in Example 6, and their functions in this microbial host were confirmed. Yeast-codon optimized DNA coding sequences for partial amino acid sequences pbr.PBRST1PF_4328 and pbr.PBRST1PF_89405 were synthesized in-frame with the yeast-codon optimized coding sequence for amino acids 1-40 of SSDU-2015634 (Table 1) to produce CYP-COR_4328 and CYP-COR_89405, respectively. These CYP-COR coding sequences were cloned into low-copy plasmids having a URA3 selection marker and expressed from a TDH3 promoter. The plasmids were then expressed using an expression cassette (P) for cytochrome P450 reductase integrated into the chromosome. TEF1 -ATR1 or P TEF1 Yeast strains containing the -PsCPRv2 plasmid were transformed. These yeast strains, each containing two plasmids, were grown in synthetic complete medium with a suitable dropout solution (-Ura-Trp). The yeast strains were supplied with (S)-reticulin, and BIA metabolites were analyzed by LC-MS / MS 72 hours after growth.

[0268] Example 8. Production of saltharidin from (S)-reticulin in genetically modified yeast cells The identified epimerases described in Example 6 were heterologously expressed in a yeast strain (Saccharomyces cerevisiae) through genetic engineering, and their functions in this microbial host were confirmed. Yeast-codon optimized DNA coding sequences for partial amino acid sequences pbr.PBRST1PF_4328 and pbr.PBRST1PF_89405 were synthesized in-frame with the yeast-codon optimized coding sequence for amino acids 1-40 of SSDU-2015634 (Table 1) to generate CYP-COR_4328 and CYP-COR_89405, respectively. These CYP-COR coding sequences were cloned into low-copy plasmids with a URA3 selection marker and expressed via the TDH3 promoter. Saltharidin synthase (SalSyn) coding sequences were cloned into low-copy plasmids with a TRP1 selection marker and expressed via the TDH3 promoter. The plasmids were then expressed using a cytochrome P450 reductase expression cassette (P) integrated into the chromosome. TEF1 -ATR1 or P TEF1 Yeast strains possessing (-PsCPRv2) were transformed. These yeast strains, each containing two plasmids, were grown in synthetic complete medium with a suitable dropout solution (-Ura-Trp). The yeast strains were supplied with (S)-reticulin, and BIA metabolites were analyzed by LC-MS / MS 72 hours after growth. The analysis showed that the genetically modified yeast cells could convert (S)-reticulin to (R)-reticulin, which was then subjected to saltharidin synthase to form saltharidin, a 4-ring promorphinan alkaloid (Figures 7 and 18). It has been previously shown that saltharidin synthase acts on (R)-reticulin, but there is no observable activity on (S)-reticulin (Gesell, et al. 2009. J. Biol. Chem. 284:24432-42).

[0269] As shown in Figure 7, CYP-COR catalyzes the conversion of (S)-reticulin to (R)-reticulin, which is then acted upon by saltharidin synthase to produce the promorphinan alkaloid saltharidin. Figure 18(A) shows chromatographic traces of reticulin and saltharidin for two epimerase variants (CYP-COR_89405, CYP-COR_4328) and a standard. Figure 18(B) also shows the same chromatographic trace for saltharidin in (A), replotted to demonstrate co-elution with the standard. In this experiment, yeast contains two low-copy CEN / ARS plasmids with URA3 and TRP1 selective markers, a TDH3 promoter, and CYP-COR and SalSyn coding sequences. Yeast was grown overnight in 3 mL of selective medium from newly transformed colonies, diluted back to OD 0.8 in 3.5 mL of medium, grown for 7 hours, pelletized, and then resuspended in pH 7.4 HEPES buffer with 100 μM (S)-reticulin (Specs). After 16 hours in a spinner at 30°C, the yeast was pelletized, and the buffer supernatant was analyzed by LC-MS / MS. Each trace is from two representative single samples. Peaks are normalized so that the maximum peak in all chromatograms is 100%.

[0270] Example 9. Production of (R)-reticlin from raceminol laudanotholin in genetically modified non-plant host cells The yeast strain (Saccharomyces cerevisiae) was genetically engineered to heterologously express the identified epimerase described in Example 6, and its function in this microbial host was confirmed. The yeast-codon-optimized DNA coding sequence CYP-COR_89405 described in Example 7 was cloned into a low-copy plasmid containing a URA3 selection marker and expressed from a TDH3 promoter. This plasmid was then expressed using a cytochrome P450 reductase expression cassette (P) integrated into the chromosome. TEF1 -ATR1 or P TEF1-PsCPRv2) and three methyltransferases (Papaver somnifer, norcoclaurine-6-O-methyltransferase, coclaurine N-methyltransferase, and 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase, all P TEF1 Yeast strains containing an expression cassette (expressed from) were transformed. These plasmid-containing yeast strains were grown in synthetic complete medium with a suitable dropout solution (-Ura). Raceminol laudanotholin was supplied to the yeast strains, and BIA metabolites were analyzed by LC-MS / MS 72 hours after growth. For chiral characterization, reticrin was concentrated by pelleting 5 mL of yeast culture from yeast medium, adding 120 mg of XAD-4 resin to 4 mL of the supernatant, incubating overnight on a rotating furnace at room temperature, and eluting with 0.5 mL of methanol. The concentrate was fractionated by reverse-phase HPLC (Pursuit XRs-C18, 5 μm, 50 mm × 10 mm) at a flow rate of 5 mL / min for 6.5 minutes with 15% methanol in a homogeneous solvent with 0.1% formic acid, in injection volumes of 40-50 μL. Peak-based fractions were collected in approximately 4.5 minutes. The fractions were combined, lyophilized, and resuspended in 0.5 mL of isopropanol. Depending on the concentration, 0.5–5 μL were injected into a chiral column (Phenomenex Luxcellulose-1, 3 μm, 150 mm × 2 mm) and separated by detection by MS and 250 nm UV using a homogeneous solvent of 72% N-hexane, 28% isopropanol, and 0.1% diethylamine at a flow rate of 0.3 mL / min. MS detection was performed using an Agilent 6320 ion trap mass spectrometer with an ESI source gas temperature of 350 °C, a gas flow rate of 10 L / min, a nebulizer pressure of 40 PSI, and isolation at a width of 1.0 m / z 330.1. The retention time of the reticline peak was compared to that of true (S)-reticline and (R)-reticline standards. Analysis revealed that genetically modified yeast cells containing the CYP-COR plasmid were able to convert raceminol laudanosolin to (R)-reticulin, while genetically modified yeast cells with an empty plasmid exclusively produced (S)-reticulin (Figure 19).

[0271] Example 10: Genetic manipulation of saltharidin synthase protein to improve the processing and activity of saltharidin synthase when expressed in a microbial host. Heterogeneous proteins can be misprocessed when expressed in recombinant hosts, such as plant proteins like cytochrome P450 enzymes expressed in microbial-producing hosts. For example, saltharidin synthase converts (R)-reticulin to saltharidin, but undergoes N-linked glycosylation when heterogeneously expressed in yeast (Figure 20). The N-linked glycosylation pattern observed on saltharidin synthase is not observed when the enzyme is expressed in plants, indicating incorrect N-terminal sorting of nascent SalSyn transcription, which reduces the enzyme's activity in heterogeneous microbial hosts. Therefore, protein genetic engineering aimed at correcting the N-terminal sorting of nascent transcripts and thereby eliminating the N-linked glycosylation pattern would result in improved activity of the saltharidin synthase enzyme in recombinant-producing hosts.

[0272] For example, the N-terminal α-helix from caylantiphorin synthase (CFS) was used to replace the N-terminal α-helix from saltharidin synthase (SalSyn, Figure 21). The junctions of these fusions were selected based on the secondary structural motifs of CFS and SalSyn, or based on the amino acid alignment of CFS and SalSyn. The fusions were cloned by amplifying the N-terminal fragment from CFS and the C-terminal fragment from SalSyn with 15-40 nucleotides overlapping with the other fragment, and then assembled together and with the vector skeleton by Gibson assembly to form a complete fusion open reading frame (Gibson, et al. 2009. Nat Methods. 6:343-5).

[0273] As another example, the coding sequence of the cytochrome P450 domain from saltharidin synthase was directly placed within the P450 coding region of other stably expressed cytochrome P450 enzymes, such as the BM3 enzyme. For example, the conserved cytochrome P450 domain of saltharidin synthase and the cytochrome P450 domain from a genetically engineered variant of Bacillus megaterium P450 monooxygenase CYP102A1 (BM3, (Michener and Smolke. 2012. Metab. Eng. 14:306-16)) were identified by NCBI conserved domain search. A primer was designed so that the coding sequence of the first few amino acids of BM3 was fused to the coding sequence of the P450 domain of saltharidin synthase, followed by the coding sequence of the BM3 domain at the C-terminus of the P450 domain. As before, this construct was assembled via Gibson assembly.

[0274] Genetically modified saltharidin synthase protein fusions were analyzed by Western blot to confirm alteration or loss of full-length expression and N-linked glycosylation patterns in yeast (Figure 20). The saltharidin synthase enzyme and protein fusions were C-terminus-tagged using a human influenza hemagglutinin (HA) epitope and cloned into expression plasmids appropriate for yeast and plant expression. For yeast, the enzyme coding sequence was cloned into a low-copy yeast / E. coli shuttle vector with a URA3 selection marker and expressed via the TDH3 promoter. For plants, the sequence was cloned into a kanamycin-resistant E. coli / Agrobacterium tumefaciens shuttle vector and into a cauliflower mosaic virus (CaMV) 35S promoter adjacent to the 5' and 3' untranslated regions from cowpea mosaic virus RNA-2 for transient plant expression via Agrobacterium tumefaciens infiltration. Yeast genetically engineered to express saltharidin synthase exhibited a striped pattern indicating N-linked glycosylation. The inventors confirmed that this pattern was due to N-linked glycosylation by site-directed mutagenesis of the glycosylation site. In contrast, as seen in Figure 20(A), plant expression of this enzyme did not result in the striped pattern indicating N-linked glycosylation. As seen in Figure 20(B), the genetically engineered saltharidin synthase protein fusion, while the N-linked glycosylation site remained unmodified, was not N-glycosylated when expressed in yeast. Western blotting demonstrated that the yeast-expressed fusion enzyme existed as a single band, similar to the expression observed for the plant-expressed parent enzyme, indicating that misprocessing of the nascent protein in yeast resulting in N-linked glycosylation was repaired by the genetically engineered fusion.

[0275] We analyzed the improvement in enzyme activity when a genetically modified saltharidin synthase protein fusion was heterologously expressed in yeast. The coding sequences of saltharidin synthase and the genetically modified fusion were cloned into a low-copy plasmid containing a URA3 selection marker and expressed from a TDH3 promoter. Yeast was expressed using P, which was incorporated into the TRP1 locus. TEF1 The study contained a single low-copy plasmid with -PsCPRv2 and a URA3 selection marker, as well as a saltharidin synthase coding sequence with a TDH3 promoter. Yeast was grown overnight in 1 mL of selective medium (-Ura) from newly transformed colonies, and then diluted 1:20 in 0.5 mL of selective medium in 96-well plates with 10 μM (R)-reticulin (Toronto Research Chemicals). After 72–96 hours in a shaking incubator, the yeast was pelleted, and the supernatant was analyzed by LC-MS / MS. Analysis showed that the genetically modified saltharidin synthase enzyme exhibited improved activity compared to the wild-type sequence when heterologously expressed in yeast (Figure 22).

[0276] Figure 22 shows codon-optimized and genetically engineered fusions of saltharidin synthase that enhance activity in yeast according to embodiments of the present invention. As seen in Figure 22, the black bars represent the natural wild-type sequence for saltharidin synthase, PsCYP719B1. The gray bars with black borders are yeast codon-optimized variants from newly identified sequences from Papaver somniferum and Papaver bracteatum. The diagonally patterned bars represent the most improved genetically engineered fusion, which is based on the P. bracteatum sequence. Error bars indicate a range of at least two biological replicates. Natural, synthetic codon-optimized, and / or protein-genetically engineered variants of saltharidin synthase from P. bracteatum, P. somniferum, or P. setigeraum (or related plants) may be used in these genetically engineered strains.

[0277] Genetically modified saltharidinose synthase protein fusions can be used in biosynthetic pathways to increase the production of downstream benzylisoquinoline alkaloid products. For example, yeast was genetically engineered to heterologously express yeast codon-optimized genes encoding the genetically modified saltharidinose synthase fusion, P. bracteatum saltharidinose reductase, and P. somniferum saltharidinol 7-O-acetyltransferase. Three expression cassettes (P TDH3- D94yPsSS, P TPI1 -yPbSalR,P TEF1 -yPsSalAT) was assembled into a yeast artificial chromosome (YAC) containing a TRP1 section marker. The YAC was then used to create an expression cassette (P) of cytochrome P450 reductase incorporated into the chromosome. TEF1 -ATR1 or P TEF1 The yeast strains were introduced into yeast containing (-yPsCPRv2). The yeast strains were grown in synthetic complete medium with a suitable dropout solution (-Trp) and supplied with (R)-reticulin. BIA metabolites were analyzed 96 hours after growth by LC-MS / MS analysis. The analysis showed that yeast strains genetically engineered with saltharidin synthase enzyme and other pathway enzymes produced the morphinan alkaloid thebaine, as shown in Figure 23(A).

[0278] Therefore, Figure 23(A) shows LC-MS / MS analysis of a small batch fermentation in which genetically modified yeast catalyzes the conversion of (R)-reticulin to thebaine, according to an embodiment of the present invention. As shown in Figure 23(A), the yeast strain has P incorporated into the TRP1 locus. TEF1 -Has an ATR1 expression cassette, a TRP1 select marker and three expression cassettes:P TDH3- yEcCFS 1-83 -yPsSS 95-505 , P TPI1 -yPbSalR, and P TEF1The yeast was genetically engineered to contain a single yeast artificial chromosome with -yPsSalAT. The yeast was grown overnight in 3 mL of selective medium from newly transformed colonies, and then diluted 1:20 in 0.5 mL of selective medium (-Trp) with 100 μM (R)-Reticulin (Toronto Research Chemicals) in a culture tube and returned to the culture. After 72 hours in a shaking incubator, the yeast was pelleted, and the supernatant was analyzed by LC-MS / MS. Chromatogram traces show the thebaine produced by this strain, as well as accumulated salthalidinol and saltharidin, along with standards. These traces are representative of the two samples.

[0279] Example 11: Protein gene manipulation of enzymes in downstream morphinan branching to improve morphinan product production from heterologous microbial hosts In one embodiment of the present invention, a pathway enzyme is genetically engineered to exhibit increased activity for increased production of the target BIA. In this example, mutations are introduced into the open reading frame of a specific pathway enzyme by amplification with Mutazyme II (see Table 6). Sufficient template DNA was included in the amplification reaction, resulting in a mutation rate of 1 to 4 nucleotide substitutions per gene. The mutant library was cloned directly into the pYES1L vector in yeast by gap repair. In some examples, the yeast strains selected for library expression contained incorporated copies of genes that produce substrates for the mutagenerated enzymes. For example, a library of CODM variants was transformed into strains having incorporated copies of T6ODM and COR1.3, and thebaine was supplied in culture medium. Expression of T6ODM and COR1.3 in these strains confirmed that codeine and neopine may be available as substrates for each introduced CODM variant. Individual colonies were inoculated into 96-well plates, cultured for 96 hours, and then assayed for product production by liquid chromatography-mass spectrometry (LC-MS). In the example of the CODM library, products were screened for morphine and neomorphine. For each screen, variants with increased BIA production were sequenced and re-cloned for validation. Table 6 contains a summary of the mutant enzyme variants identified through the screens that resulted in increased BIA production in yeast.

[0280] Figure 24 shows data on the validated enhancement of activity of one of these mutants. Specifically, Figure 24 shows the generation of a CODM enzyme variant exhibiting enhanced activity in yeast through random mutagenesis and screening according to an embodiment of the present invention. A library of CODM variants was generated by mutagenesis of the coding region by error-prone PCR. The variant identified by screening this library is CODM. N35S、G335VThe compound was re-cloned and expressed in a yeast strain containing copies incorporating T6ODM and COR1.3. This strain and another control strain expressing wild-type CODM were cultured in liquid medium containing 1 mM thebaine. After 96 hours, the medium was analyzed for CODM activity by LC-MS. Variant CODM N35S、G335V This strain produced 1.4 times more morphine and 2.6 times more neomorphin than strains expressing wild-type CODM.

[0281] Example 12: Optimization of expression and growth conditions to improve benzylisoquinoline alkaloid production from heterologous microbial hosts. The production of bezylisoquinoline alkaloids from genetically modified microbial hosts can be further enhanced by optimizing the expression of pathway enzymes and growth conditions. For example, the expression of saltaridinol 7-O-acetyltransferase can be altered in yeast by expressing the enzyme from a series of different promoters. Yeast was genetically engineered to heterologously express yeast codon-optimization genes encoding P. somniferum saltaridinol 7-O-acetyltransferase from different promoters (shown in Figure 25(A)). Two expression cassettes (P TPI1 -yPbSalR,P X -yPsSalAT) was assembled into a yeast artificial chromosome (YAC) containing the TRP1 section marker. The YAC was placed in yeast, and the cells were grown in synthetic complete medium with a suitable dropout solution (-Trp) and supplied with saltharidin. BIA metabolites were analyzed by LC-MS / MS analysis 72 hours after growth. Optimization of pathway enzyme expression levels can increase the production of the morphinan alkaloid thebaine (shown in Figure 25(A)).

[0282] By optimizing strain culture conditions, including but not limited to sugar sources, growth temperature, and pH, the production of benzylisoquinoline alkaloids from genetically modified yeast strains can be increased (as shown in Figures 25(B) and (C)). In one example, thebaine production from a genetically modified yeast strain was increased by varying the pH. Two expression cassettes (P TPI1- yPbSalR, P TEF1 Saltharidinol (-yPsSalAT) was assembled into a yeast artificial chromosome (YAC) containing the TRP1 section marker. The YAC was added to yeast, and the cells were grown in synthetic medium with a suitable dropout solution (-Trp), resuspended in a buffer at pH 5.7–9, and supplied with saltharidinol. BIA metabolites were analyzed by LC-MS / MS analysis 16 hours after incubation. The levels of the 4-ring promorphinan alkaloid saltharidinol and the 5-ring morphinan alkaloid thebaine increased as a function of increasing pH (shown in Figure 25(B)).

[0283] In another example, thebaine production from genetically modified yeast strains was increased by varying the temperature, sugar, and culture medium buffer content. Three expression cassettes (P TDH3- D94yPsSS, P TPI1 -yPbSalR,P TEF1 -yPsSalAT) was assembled into a yeast artificial chromosome (YAC) containing a TRP1 section marker. The YAC was then used to create an expression cassette (P) of cytochrome P450 reductase incorporated into the chromosome. TEF1 -ATR1 or P TEF1 The yeast strain was introduced into (yPsCPRv2). The yeast strain was grown in synthetic complete medium with a suitable dropout solution (-Trp) and supplied with (R)-reticulin. BIA metabolites were analyzed by LC-MS / MS 72 hours after growth. The analysis showed that microbial production of the morphinan alkaloid thebaine increased under certain culture conditions (interference medium with dextrose at 30°C, shown in Figure 25(C)).

[0284] Accordingly, Figure 25 shows the fermentation optimization for the conversion of (R)-reticulin to thebaine by genetically modified yeast according to an embodiment of the present invention. (A) SalAT promoter variant, (B) SalR and SalAT strains grown under different pH conditions, and (C) LC-MS / MS analysis of whole-cell buffer assays with optimization of sugar source, growth temperature, and medium buffer content. (A) TRP1 selective marker and two expression cassettes using various SalAT promoters:P TPI1 -yPbSalR and P X Yeast strains genetically engineered to contain a single yeast artificial chromosome with -yPsSalAT. Yeast was grown overnight in 3 mL of selective medium from newly transformed colonies, and then diluted 1:20 in 0.5 mL of medium with 100 μM saltharidin (Specs) in culture tubes and returned to the culture tubes. After being placed in a shaking incubator for 72 hours, the yeast was pelleted and the supernatant was analyzed by LC-MS / MS. (B) Yeast strains were genetically engineered to contain a TRP1 selective marker and two expression cassettes: P TPI1 -yPbSalR and P TEF1 Yeast strains were genetically engineered to contain a single yeast artificial chromosome with -yPsSalAT. The yeast was grown overnight in 3 mL of selective medium from newly transformed colonies, diluted back to 3.5 mL of medium to OD 0.8, grown for 7 hours, pelletized, and then resuspended in Tris buffer at pH 7, 8, or 9 with pH 5.7 MOPS or 10 μM saltharidin (Specs). After 16 hours on a spinner at 30°C, the yeast was pelletized, and the buffer supernatant was analyzed by LC-MS / MS. Error bars represent the range between the two samples. (C) Optimization of sugar source, growth temperature, and medium buffer content. In this experiment, the yeast strain was genetically engineered to contain P incorporated into the TRP1 locus. TEF1 -Has ATR1, a TRP1 selective marker and three expression cassettes:P TDH3- yEcCFS 1-83 -yPsSS 95-505 , P TPI1 -yPbSalR, and P TEF1Yeast was genetically engineered to contain a single yeast artificial chromosome with -yPsSalAT. The yeast was grown overnight in 3 mL of selective medium from newly transformed colonies, and then diluted 1:20 in 0.5 mL of medium with 100 μM (R)-reticulin (Toronto Research Chemicals) in a culture tube and returned to the culture tube. After being placed in a shaking incubator for 72 hours, the yeast was pelleted, and the supernatant was analyzed by LC-MS / MS.

[0285] Example 13: Genetically engineered yeast for the production of thebaine from an initial 1-benzylisoquinoline alkaloid skeleton. Yeast strains can be genetically engineered to produce the morphinan alkaloid thebaine from early 1-benzylisoquinoline alkaloids, or morphinan alkaloids derived from thebaine. For example, a genetically engineered yeast strain can produce morphinan alkaloid products from racemic or (S)-norcoclaurine or racemic or (S)-norlaudanotholin (Figures 5, 6, 7, and 23(B)). Yeast strains can be genetically engineered to produce (S)-reticulin from (S)-norcoclaurine or racemic or (S)-norlaudanotholin by incorporating three or five expression cassettes into the yeast genome. To produce (S)-reticulin from racemic or (S)-norlaudanotholin, the incorporated expression cassette encodes Papaver somnifer norcoclaurine 6-O-methyltransferase (Ps6OMT, EC2.1.1.128), 4'-O-methyltransferase (Ps4'OMT, EC2.1.1.116), and coclaurine-N-methyltransferase (CNMT, EC2.1.1.140), each with a TEF1 promoter (Hawkins and Smolke. 2008. Nat. Chem. Biol. 4:564-73). To produce (S)-reticulin from racemic or (S)-norcoclaurine, the strains further possess expression cassettes incorporating yeast codon-optimized California poppy N-methylcoclaurine 3'-hydroxylase (yEcCYP80B1, EC1.14.13.71) and ATR1 or yPsCPRv2 cytochrome P450 reductase (CPR, EC1.6.2.4) expressed from TDH3 or TEF1 promoters. These strains are further genetically engineered to incorporate epimerization-catalysts (e.g., CYP-COR), saltharidinine synthase, saltharidinine reductase, and saltharidinol acetyltransferase to convert racemic or (S)-norcoclaurine or racemic or (S)-norlaudanotholin to the morphinan alkaloid thebaine or thebaine-derived morphinan alkaloids (Figure 7).As an alternative to the expression of epimerization-catalyzing enzymes, 6OMT, 4'OMT, CNMT, and / or CYP80B1 may be genetically engineered so that rac-reticulin is produced from rac-norcoclaurine or rac-norlaudanothorine.

[0286] In one example, a yeast strain is genetically engineered to convert rac-norlaudanotholin to thebaine. The yeast strain has four expression cassettes, each containing a TEF1 promoter, encoding Ps6OMT, Ps4'OMT, CNMT, and yPsCPRv2. TDH3- yEcCFS 1-83 -yPsSS 95-505 , P TPI1 -yPbSalR,P TEF1 -yPsSalAT, P HXT7 -CYP-COR_89405) was assembled into a yeast artificial chromosome (YAC) containing the TRP1 selection marker in this strain. Yeast strains containing the YAC and the incorporated cassette were grown in a synthetic complete medium containing a suitable dropout solution (-Trp) and 1 mM rac-norlaudanotholin substrate. After 96 hours of growth, the medium was analyzed for BIA metabolites by LC-MS / MS analysis. Approximately 200 nM of thebaine was detected (Figure 23 (B)). Other genetically modified saltharidin synthase variants may also be used in this strain (Figure 22, Example 10).

[0287] Example 14: Genetically engineered platform yeast strain for reticulin production from L-tyrosine A platform yeast strain was constructed that produces the important branching point BIA intermediate (S)-reticulin from L-tyrosine (Figure 5). Specifically, four multiple gene expression constructs were incorporated into the genome of the yeast strain. The composition of the four constructs is shown in Figure 26. Each construct consists of four or five genes expressed from a strong constitutive promoter. The genes are located at each locus as a complete expression cassette including the promoter, gene open reading frame, and terminator, as specified in the annotations above the schematic diagram. The schematic diagram indicates the transcription direction of each expression cassette by the direction of the arrows representing the given genes. Selectable markers are italicized in the annotations and represented by gray arrows in the schematic diagram. Each selectable marker is adjacent to a loxP site to allow for the removal of the marker from the locus. In addition, each construct has selectable markers adjacent to loxP sites so that they can be removed by Cre recombinase.

[0288] In the first integration construct, four heterologous genes derived from brown rats are integrated into the YBR197C locus along with the G418 selection marker (KanMX). RnPTPS, RnSepR, RnPCD, and RnQDHPR require the synthesis and regeneration of tetrahydrobiopterin (BH4) from the yeast endogenous folate synthesis pathway. Each gene is codon-optimized for expression in yeast.

[0289] In the second integration construct, four heterologous genes are integrated at the HIS3 locus along with the HIS5 selection marker. The rat tyrosine hydroxylase (RnTyrH) converts tyrosine to L-dopa using the auxiliary substrate BH4 generated by the preceding integration construct. The RnTyrH gene can be either wild-type or an improved mutant that provides enhanced activity (e.g., W166Y, R37E, and R38E, Example 2). The second rat gene, RnDHFR, encodes an enzyme that reduces dihydrobiopterin (an oxidation product of BH4) to BH4, thereby increasing the availability of this auxiliary substrate. Thirdly included in the construct is PpDODC from Pseudomonas putida, an enzyme that converts L-dopa to dopamine. The fourth enzyme is CjNCS, derived from Coptis japonica, which condenses 4-HPA and dopamine to produce norcocrawlin. Each gene has been codon-optimized for expression in yeast.

[0290] In the third integration construct, five heterologous plant-derived genes and a LEU2 selection marker are integrated into the YDR514C locus. Ps6OMT, Ps4'OMT, and PsCNMT are methyltransferases derived from Papaver somnifer and are expressed as native plant nucleotide sequences. The fourth P. somniferum gene, yPsCPRv2, is codon-optimized for yeast and encodes a reductase that supports the activity of EcCYP80A1, a cytochrome P450 derived from California poppy. EcCYP80A1 is expressed as its native plant nucleotide sequence. The enzyme encoded in this construct performs two O-methylation, N-methylation, and hydroxylation to produce reticulin from norcoclaurine produced by the preceding integration construct.

[0291] In the final incorporated construct, an additional copy of the Saccharomyces cerevisiae endogenous gene, ARO4, was added. Q166K ARO7 T226I TKL1 and ARO10 are incorporated into the ARO4 locus along with a hygromycin resistance selection marker.Q166K and ARO7 T226I These are feedback-resistant mutants of ARO4 and ARO10, each encoding a single base pair substitution compared to the wild-type sequence. TKL1 and ARO10 are identical to the native yeast genes but are expressed behind a strong promoter. Aro4p and Aro7p are enzymes in the biosynthesis of aromatic amino acids, including tyrosine. Removing feedback inhibition from these enzymes results in upregulation of endogenous tyrosine biosynthesis. Overexpression of Tkl1p upregulates the pentose phosphate pathway, resulting in an enhanced supply of erythrose 4-phosphate (E4P), a precursor of tyrosine. Overexpression of Aro10p increases the production of 4-HPA.

[0292] The platform yeast strain can be constructed with any number of the four expression cassettes. Specifically, the platform yeast strain was constructed with incorporated constructs 1-4 and incorporated constructs 1-3. In the latter strain, where the tyrosine overproduction construct (construct 4) is excluded, additional tyrosine may be supplied to the culture medium to support reticuline biosynthesis. Additional genetic modifications may be incorporated into the platform strain to support downstream BIA production and increase the flux to BIA biosynthesis.

[0293] Yeast strains were grown at 25 and 30°C in synthetic complete media containing a suitable amino acid dropout solution. BIA metabolites in the media supernatant were analyzed by LC-MS / MS 48 and 96 hours after growth.

[0294] Example 15: Genetically engineered yeast for the production of thebaine and other morphinan alkaloids from L-tyrosine Yeast strains can be genetically engineered to produce the morphinan alkaloid thebaine, or thebaine-derived morphinan alkaloids, from initial precursors such as tyrosine. For example, the platform yeast strain described in Example 14 can be further genetically engineered to produce morphinan alkaloid products from L-tyrosine (Figure 7).

[0295] A platform yeast strain producing (S)-reticulin from L-tyrosine (see description in Example 14) was further genetically engineered to incorporate epimerization-catalyzing enzymes, such as the newly identified CYP-COR, saltharidinose synthase, saltharidinose reductase, and saltharidinol acetyltransferase, to convert the biosynthesized (S)-reticulin to the morphinan alkaloid thebaine, or a morphinan alkaloid derived from thebaine (Figure 7). Three expression cassettes (P TDH3- yEcCFS 1-26 -yPbSS 33-504 , P TPI1 -yPbSalR,P TEF1 -yPsSalAT) was assembled into a yeast artificial chromosome (YAC) directly containing the TRP1 selection marker within the platform yeast strain. Other genetically modified saltharidin synthase variants can also be incorporated into the YAC (Figure 22, Example 10). The resulting yeast strain was then transformed with a low-copy CEN / ARS plasmid containing the URA3 selection marker, TDH3 promoter, and CYP-COR coding sequence.

[0296] Yeast strains containing YAC, low-copy plasmids, and integrated cassettes were grown at 25 and 30°C in synthetic complete medium with a suitable dropout solution (-Ura-Trp). After 96 hours of growth, the medium was analyzed for BIA metabolites by LC-MS / MS. Further optimization of the culture temperature, carbon source, pH conditions, and medium composition improved BIA production.

[0297] Additional gene modifications can be introduced into yeast strains to produce morphinan alkaloids derived from thebaine (Figure 7). In one example, expression cassette P ADH1 -T6ODM-T ADH1 , P HXT7 -COR-T PGK1 , and P TEF1 -CODM-T CYC1This was directly assembled and incorporated into the trp1 gene locus of the thebaine-producing yeast strain (Thodey et al., 2014). In another example, these yeast strains were further genetically engineered to produce an expression cassette P GPD -morA-T CYC1 , P PGK1 -morB-T PHO5 By directly assembling this construct and incorporating it into the ura3 gene locus on the chromosome, additional morphine alkaloids can be produced (Thodey et al., 2014).

[0298] Example 16: Genetically engineered yeast for the production of berberine and other protoberberine alkaloids from L-tyrosine. Yeast strains can be genetically engineered to produce intermediates involved in the formation of berberine alkaloids or berberine from L-tyrosine, as well as from protoberberine, phthalidoquinoline, and berberine (Figures 5, 8). For example, three or four expression cassettes (P PGK1 -PsBBE-T PHO5 , P TEF1 -yPsS9OMT-T CYC1 , P TDH3 -yCjCAS-T ADH1 , P TPI1 -yBwSTOX-T STE2 (with or without) were assembled into a yeast artificial chromosome (YAC) having a TRP1 section marker. The YAC produced (S)-reticulin from L-tyrosine (see Example 14) and an expression cassette of cytochrome P450 reductase (P) was incorporated into the chromosome. TEF1 -ATR1 or P TEF1 -PsCPRv2) was added to the platform yeast strain. TEF1 -S9OMT-T CYC1 The flux to downstream products was improved by transforming this strain with an additional high-copy plasmid containing an expression cassette.

[0299] Yeast strains were grown at 25 and 30°C in synthetic complete media containing a suitable amino acid dropout solution (-Trp). BIA metabolites in the media supernatant were analyzed by LC-MS / MS after 48 and 96 hours of growth.

[0300] Example 17: Genetically engineered yeast for the production of noscapine and other noscapinoid alkaloids from L-tyrosine. Yeast strains can be genetically engineered to produce phthalidoquinoline alkaloids such as narcotrin and noscapine, intermediates involved in the formation of noscapine, or derivatives thereof, from L-tyrosine (Figures 5 and 9).

[0301] For example, expression cassette P ADH1 -CAS-T ADH1 , P HXT7 -CYP82Y1-T PGK1 , P TEF1 -S9OMT-T CYC1 , and P PGK1 -BBE-T PHO5 The expression cassette (P) of cytochrome P450 reductase incorporated into the chromosome is directly assembled to produce (S)-reticulin from L-tyrosine (see Example 14). TEF1 -ATR1 or P TEF1 -PsCPRv2) was added and incorporated into the trp1 gene locus of the platform yeast strain. Then, the expression cassette P GPD -TNMT-T CYC1 , P PGK1 -PsMT2-T PHO5 , P ADH1 -CYP82X1-T GAP1 , and P PYK1 -PsCXE1-T Mf1 The expression cassette P was directly assembled and incorporated into the ura3 gene locus of the yeast strain. HXT7 -CYP82X2-T CYC1 , P GPD -PsAT1-T ADH1 , P TPI1 -PsSDR1-T Ste2 , and P PGK1 -PsMT3-T PHO5The device was directly assembled and incorporated into the leu2 gene locus of the yeast strain. TEF1 -S9OMT-T CYC1 Additional high-copy plasmids and P containing expression cassettes HXT7 -CYP82X2-T CYC1 The flux of downstream products was improved by transforming this yeast strain with an additional low-copy plasmid containing an expression cassette.

[0302] Yeast strains were grown at 25 and 30°C in synthetic complete media containing a suitable amino acid dropout solution (-Trp, Ura). BIA metabolites in the media supernatant were analyzed by LC-MS / MS after 48 and 96 hours of growth.

[0303] Example 18: Genetically engineered yeast for the production of sanguinalin and other benzophenanthridine alkaloids from L-tyrosine. Yeast strains can be genetically engineered to produce benzophenanthidine alkaloids such as protoberberine, protopine, and the final product sanguinaline, as well as intermediates or derivatives involved in the formation of sanguinaline, from L-tyrosine (Figures 5 and 10).

[0304] For example, expression cassette P ADH1 -TNMT-T ADH1 , P HXT7 -EcSTS-T PGK1 , P GPD -EcCFS-T CYC1 , and P PGK1 -BBE-T PHO5 The expression cassette (P) of the cytochrome P450 reductase incorporated into the chromosome is directly assembled to produce (S)-reticulin from L-tyrosine (see Example 14). TEF1 -ATR1 or P TEF1 -PsCPRv2) was added and incorporated into the trp1 gene locus of the platform yeast strain. Then, the expression cassette P GPD -PsMSH-T CYC1 , P PGK1 -PsP6H-T PHO5We directly assembled it and incorporated it into the ura3 gene locus of the yeast strain.

[0305] Yeast strains were grown at 25 and 30°C in synthetic complete media containing a suitable amino acid dropout solution. BIA metabolites in the media supernatant were analyzed by LC-MS / MS after 48 and 96 hours of growth.

[0306] Example 19: Genetically engineered yeast for the production of hydrocodons and other morphinan alkaloids from L-tyrosine. Yeast strains can be genetically modified to produce hydrocodons. As an example, the thebaine-producing yeast strain described in Example 15 can be further genetically modified to produce the active pharmaceutical ingredient hydrocodon. Figure 27 shows thebaine and hydrocodon production in genetically modified yeast strains according to embodiments of the present invention. In Figure 27A, multiple gene constructs were incorporated into a genetically modified yeast strain containing constructs 1-4 (Figure 26). In Figure 27B, the incorporation of construct 5 increased the reticulin titer compared to the strain containing constructs 1-4. In Figure 27C, the incorporation of construct 6 resulted in thebaine production from sugar. Thebaine produced by the genetically modified strains was identified in culture medium by LC-MS / MS analysis and compared with a commercially available reference standard. In Figure 27D, the introduction of construct 7 into YAC resulted in hydrocodon production from sugar. Hydrocodons produced by genetically modified bacterial strains were identified in culture media by LC-MS / MS analysis and compared with commercially available reference standards.

[0307] In one example, a yeast strain producing reticulin from L-tyrosine (see Example 14) was further genetically modified to increase reticulin production, then modified to incorporate the thebaine-producing enzyme described in Example 15, and then modified further to incorporate thebaine demethylase and morphinone reductase to convert the biosynthesized thebaine into hydrocodons. Three multiple gene expression constructs were incorporated into the reticulin-producing yeast strain described in Example 14; two as chromosomal integrations and the third as an episomal YAC construct.

[0308] In the first construct, genes were incorporated to increase the biosynthesis of (S)-reticulin from the strain described in Example 14. To increase (S)-reticulin production, additional copies of CjNCS (to increase the uptake of dopamine into norcocrawulin) and Ps4'OMT (to increase the uptake of 3'-hydroxy-N-methylcocrawulin into (S)-reticulin) were incorporated into the strain. Additional copies of RnTyrH were also included to increase the influx of tyrosine into the heterologous pathway for reticulin biosynthesis. Three expression cassettes (P PGK1 -yCjNCS-T PHO5 , P TEF1 -Ps4'OMT-T CYC1 , and P GPD -RnTyrH WR -T ADH1 The gene YPL250C was incorporated along with a ble selection marker for phleomycin resistance (Figure 27A). The resulting yeast strain showed a fourfold increase in reticulin accumulation (Figure 27B).

[0309] In the second construct, the gene was incorporated to produce thebaine from biosynthesized (S)-reticulin. The four enzymes for thebaine production described in Example 15 were expressed in four expression cassettes (P HXT7 -yPbCYP-COR-T CYC1 , P GPD -yEcCFS 1-83 -yPbSalSyn 92-504 -T ADH1 , P TPI1 -yPbSalR-T STE2 , and P PGK1 -yPsSalAT-T PHO5 The resulting yeast strain was assembled and incorporated the selection marker KlURA3 into the TRP1 locus (Figure 27A). The resulting yeast strain produced thebaine when cultured in standard yeast culture medium (Figure 27C).

[0310] In the third construct, genes were incorporated to produce hydrocodons from biosynthesized thebaine. In this example, thebaine demethylase activity was incorporated as the T6ODM enzyme from P. somniferum to convert thebaine to neopinone, and morphinone reductase activity was incorporated as the morB enzyme from P. putida to convert codeinone to hydrocodon. In this example, the conversion between neopinone and codeinone occurred spontaneously. In another example, an isomerase enzyme is included to enzymatically convert neopinone to codeinone. The T6ODM and morB genes were codon-optimized for yeast and expressed in two expression cassettes (P GPD -yT6ODM-T ADH1 and P PGK1 -yPbmorB-T PHO5 ) was included as and assembled into a TRP1 selection marker and YAC (Figure 27A). In this example, the yeast strain is cultured in a medium supplemented with 50 mM 2-oxoglutarate to support the activity of T6ODM, a 2-oxoglutarate-dependent dioxygenase. In another example, yeast host cells are genetically engineered to accumulate 2-oxoglutarate at levels sufficient to support T6ODM activity. Yeast strains having the described multiple gene constructs and cultured in the described medium biosynthesized hydrocodons from glucose (Figure 27D).

[0311] Example 20: Increasing gene copy number to overcome bottlenecks in pathway flux In some cases, yeast strains can be optimized to enhance the production of benzylisoquinoline alkaloids by increasing the gene copy number for enzymes whose activity is restricted.

[0312] In one example, reticulin production in the reticulin-producing strain described in Example 19 was optimized by adding a third gene copy of NCS (Figure 28). In this example, the parent strain had one copy of NCS incorporated at the YMR206W locus and another copy at the YPL250C locus, both expressed from the PGK1 promoter. The third copy of NCS (expressed from the TDH3 promoter) was incorporated at the BUD9 locus, and this modification resulted in a twofold increase in reticulin titer. In another example, additional copies of other enzymes in the pathway were incorporated into the strain to further increase reticulin titer.

[0313] (Table 2) Enzyme List TIFF0007871023000020.tif193123TIFF0007871023000021.tif193129TIFF0007871023000022.tif193123TIFF0007871023000023.tif193118 TIFF0007871023000024.tif193127TIFF0007871023000025.tif193103TIFF0007871023000026.tif193121TIFF0007871023000027.tif193133

[0314] (Table 3) Tailoring enzymes TIFF0007871023000028.tif105131

[0315] (Table 4) Comparison of impurities that may be present in poppy straw (or opium) concentrate and clarified yeast culture medium. TIFF0007871023000029.tif218126

[0316] (Table 5) Different molecular groups present in clarified yeast culture medium (CYCM) Unlike poppy straw (CPS) or opium concentrates, yeast host strains can be genetically engineered to produce molecules of a specific class of alkaloids in the absence of other classes of alkaloids (i.e., only one biosynthetic pathway per strain). Therefore, this CYCM may contain molecules within a single biosynthetic pathway, including subsets of molecules across one or two columns, as well as subsets of molecules across multiple columns. TIFF0007871023000030.tif22793

[0317] (Table 6) Beneficial mutations of various enzymes identified by mutagenesis screening TIFF0007871023000031.tif175128

[0318] Notwithstanding the attached items, this disclosure is also defined by the following items: 1. Genetically modified non-plant cells having increased tyrosine hydroxylase activity compared to unmodified cells, wherein the genetically modified non-plant cells have at least one modification selected from the group consisting of substrate inhibition reduction mutations; product inhibition reduction mutations; and cofactor recovery promotion mechanisms. 2. A genetically modified non-plant cell as described in item 1, wherein at least one of the modifications includes a substrate inhibition reduction mutation. 3. Genetically modified non-plant cells as described in item 2, wherein the substrate inhibition reduction mutation includes the point mutation W166Y. 4. A genetically modified non-plant cell as described in item 1, wherein the at least one modification comprises at least one product inhibition reduction mutation that mitigates competitive binding at an auxiliary substrate binding site. 5. A genetically engineered non-plant cell as described in item 4, wherein the at least one product inhibition reduction mutation includes the point mutation S40D. 6. A genetically modified non-plant cell as described in item 4, wherein the at least one product inhibition reduction mutation includes conjugate mutations R37E and R38E. 7. Genetically modified non-plant cells as described in item 6, wherein the conjugate mutations R37E and R38E enhance tyrosine hydroxylase activity in the presence of dopamine. 8. A genetically modified non-plant cell as described in item 1, wherein at least one of the modifications includes a product inhibition mitigation mutation that reduces irreversible product inhibition. 9. Genetically modified non-plant cells as described in item 8, wherein the product inhibition reduction mutation includes the point mutation E332D. 10. Genetically modified non-plant cells as described in item 8, wherein the product inhibition reduction mutation includes the point mutation Y371F. 11. Genetically modified non-plant cells as described in item 8, wherein the product inhibition mitigation mutation relaxes the irreversible binding of catecholamines to iron at the active site. 12. A genetically modified non-plant cell as described in item 1, wherein at least one of the modifications includes a cofactor recovery-promoting mechanism. 13. Genetically modified non-plant cells as described in item 12, wherein the cofactor recovery-promoting mechanism includes a heterologous coding sequence encoding dihydrofolate reductase. 14. The genetically modified non-plant cell described in item 13, wherein the dihydrofolate reductase produced by the genetically modified non-plant cell catalyzes the conversion of dihydrobiopterin to tetrahydrobiopterin within the genetically modified non-plant cell. 15. (a) A step of supplying genetically modified non-plant cells having at least one modification selected from the group consisting of substrate inhibition reduction mutations; product inhibition reduction mutations; and cofactor recovery promotion mechanisms, as well as a supply material containing nutrients and water, to a batch reactor. (b) A step of subjecting the genetically modified non-plant cells to fermentation by incubating them in the batch reactor for at least about 5 minutes to produce a solution containing benzylisoquinoline alkaloid products and cytoplasm; and (c) A step of separating the benzylisoquinoline alkaloid product from the cellular material using at least one separation device and providing a product stream containing the benzylisoquinoline alkaloid product. A method for forming the product stream having the benzylisoquinoline alkaloid product, including the benzylisoquinoline alkaloid product. 16. The method according to item 15, wherein at least one process parameter of the batch reactor is modifiable to mutate the resulting benzylisoquinoline alkaloid product composition. 17. The method according to item 16, wherein the at least one modifiable process parameter includes at least one of dissolved oxygen, pH, stirring rate, aeration rate, and cell density. 18. The method according to item 15, wherein the benzylisoquinoline alkaloid product comprises a benzylisoquinoline alkaloid precursor. 19. The method according to item 18, wherein the benzylisoquinoline alkaloid precursor is selected from the group consisting of norcoclaurine, norlaudanothorine, tyrosine, tyramine, 4-hydroxyphenylacetaldehyde, 4-hydroxyphenylpyruvic acid, L-3,4-dihydroxyphenylalanine, 3,4-dihydroxyphenylacetaldehyde, and dopamine. 20. The method according to item 15, wherein the benzylisoquinoline alkaloid product comprises a benzylisoquinoline alkaloid. 21. The method according to item 20, wherein the benzylisoquinoline alkaloid has a structural class selected from the group consisting of benzylisoquinoline, protoberberine, protopine, benzophenanthidine, promorphinan, morphinan, secoberberine, phthalidoisoquinoline, aporfin, and bisbenzylisoquinoline. 22. The method according to item 21, wherein the benzylisoquinoline alkaloid is a benzylisoquinoline selected from the group consisting of cocrawrine, 3'-hydroxycocrawrine, 4'-O-methylnorlaudanotholin, 4'-O-methyl-laudanotholin, N-methylnorcocrawrine, laudanotholin, N-methylcocrawrine, 3'-hydroxy-N-methylcocrawrine, reticuline, norreticuline, papaverine, laudanine, laudanosine, tetrahydropapaverine, 1,2-dihydropapaverine, and orientaline. 23. The method according to item 21, wherein t...

Claims

1. A step of contacting a (S)-1-benzylisoquinoline alkaloid with at least one enzyme in a genetically modified microbial cell, wherein the (S)-1-benzylisoquinoline alkaloid is converted to a (R)-1-benzylisoquinoline alkaloid in the genetically modified microbial cell. A method for epimerizing the (S)-1-benzylisoquinoline alkaloid to the (R)-1-benzylisoquinoline alkaloid in the genetically modified microbial cells, comprising: The enzyme comprises an epimerase including an oxidase domain and a reductase domain, which includes an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 9 and 10. The aforementioned method.

2. The method according to claim 1, wherein the at least one enzyme is produced by culturing the genetically modified microbial cells.

3. (S)-1-benzylisoquinoline alkaloid added to the culture of the microbial cells. The method according to claim 2, further comprising:

4. The step of recovering the (R)-1-benzylisoquinoline alkaloid or its derivative from the cell culture. The method according to claim 3, further comprising:

5. The method according to claim 1, wherein the oxidase domain is a cytochrome P450 oxidase-like domain.

6. The method according to claim 1, wherein the reductase domain is a codeinone reductase-like domain.

7. The method according to any one of claims 5 to 6, wherein the genetically modified microbial cell is a genetically modified yeast cell.

8. The method according to any one of claims 1 to 6, wherein the (S)-1-benzylisoquinoline alkaloid is (S)-reticulin.

9. The method according to any one of claims 1 to 6 and 8, wherein the (R)-1-benzylisoquinoline alkaloid is (R)-reticulin.

10. The method according to any one of claims 1 to 9, wherein the (S)-1-benzylisoquinoline alkaloid is produced in the genetically modified microbial cells via a metabolic pathway starting from L-tyrosine.

11. The method according to claim 1, wherein the epimerase is a fusion epimerase.

12. The method according to claim 1, wherein the epimerase is a split epimerase.

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