Benzylisoquinoline alkaloid (BIA) producing microorganisms and methods of making and using the same

Abstract

To provide host cells engineered to produce benzylisoquinoline alkaloids (BIAs), and methods of making and using the same.SOLUTION: A host cell that produces a BIA compound or a precursor thereof, wherein the host cell comprises multiple copies of one or more heterologous coding sequences for one or more enzymes obtained from a different biological source as compared to the host cell. Includes heterologous coding sequences for various enzymes involved in the host cell's synthetic pathways from starting compounds to BIAs. Also, methods of producing BIAs of interest by culturing host cells under culture conditions that promote expression of the enzymes encoded by the heterologous coding sequences of the host cells.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] Government Rights This invention was made with government support under Contract No. 1066100 awarded by the National Science Foundation and Contract No. AT007886 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS Pursuant to 35 U.S.C. §119(e), this application claims priority to the filing date of U.S. Provisional Patent Application No. 61 / 788,560, filed March 15, 2013; the disclosure of which is incorporated herein by reference. [Background technology]

[0003] Introduction Benzylisoquinoline alkaloids (BIA) are a large group of secondary metabolites from plants and other organisms. These molecules have therapeutic functions in the human organism, ranging from the established analgesic and antitussive properties of morphine and codeine to the novel anticancer and anti-infective activities observed in molecules such as berberine and sanguinarine. There is interest in making all these BIA molecules available to researchers and physicians. The number of synthetic reactions and the need for selective stereochemistry mean that chemical synthesis of BIAs is low-yielding and not an effective means for large-scale production. Instead, opium poppy (Papaver somniferum) has been bred and developed as a crop for the widely used drugs codeine and morphine. Morphine biosynthetic intermediates used as drugs and drug precursors are not accumulated because plant metabolism has evolved to maximize pathway flow to the final opioid. Even for end-product metabolites such as morphine, accumulation occurs only in specialized cells within the shoots and vascular tissue and requires harsh chemical treatment during the extraction process of harvested plant material, which typically yields less than 2% morphine by dry weight. Summary of the Invention

[0004] overview Aspects of the present invention include host cells engineered to produce benzylisoquinoline alkaloids (BIAs). The host cells contain heterologous coding sequences for various enzymes involved in the host cell's synthetic pathway from the starting compound to the BIA. The heterologous coding sequences can be obtained from a different biological source than the host cell, and multiple copies of the heterologous coding sequences can be present in the host cell. In some embodiments, the host cell is selected from a reticuline-producing host cell, a sanguinarine precursor-producing host cell, a protoberberine-producing host cell, a thebaine-producing host cell, and an opiate-producing host cell. Also provided are methods for producing a BIA of interest by culturing the host cell under culture conditions that promote the activity of the enzymes encoded by the heterologous coding sequences of the host cell. Aspects of the present invention further include components, such as host cells, starting compounds, and kits, used in the methods of the present invention. [The present invention 1001] A host cell that produces a BIA compound or a precursor thereof, the host cell comprising multiple copies of one or more heterologous coding sequences for one or more enzymes obtained from a different biological source as compared to the host cell. [The present invention 1002] 1001. A host cell of the present invention comprising two or more enzymes obtained from two or more different biological sources compared to the host cell. [The present invention 1003] The host cell of the present invention 1001, wherein the host cell is capable of producing a BIA compound or a precursor thereof from a starting material via one of the biosynthetic pathways of Figures 2, 3, 4, 11 and 15, and the starting material is selected from norlaudanosoline, norcoclaurine, reticuline and thebaine. [The present invention 1004] The host cell of the present invention 1001 is a yeast strain. [The present invention 1005] A host cell of the present invention 1001 comprising one or more heterologous or endogenous coding sequences for one or more proteins involved in the transport of compounds across the cell membrane. [The present invention 1006] 1001. A host cell of the present invention, wherein one or more enzymes comprise a localization tag and are spatially localized to a compartment within the yeast cell, the compartment being selected from mitochondria, endoplasmic reticulum (ER), Golgi, vacuole, nucleus, plasma membrane, and periplasm. [The present invention 1007] The host cell of the present invention 1001, wherein the BIA compound or precursor thereof is reticuline, and the host cell comprises one or more heterologous coding sequences for one or more methyltransferases selected from 6OMT, CNMT, and 4'OMT. [The present invention 1008] The host cell of the present invention, wherein the BIA compound or precursor thereof is sanguinarine or a sanguinarine precursor, and the host cell comprises one or more heterologous coding sequences for one or more enzymes selected from BBE, CFS, CPR, STS, TNMT, MSH, P6H, and DBOX, and the sanguinarine precursor is selected from cheilanthifoline, stylopine, cis-N-methylstylopine, scoulerine, protopine, and dihydrosanguinarine. [The present invention 1009] The host cell of the present invention, wherein the BIA compound or precursor thereof is a protoberberine alkaloid, and the host cell comprises one or more heterologous coding sequences for one or more enzymes selected from BBE, S9OMT, CAS, and STOX, and the protoberberine alkaloid is represented by one of the following structures: TIFF2026015382000002.tif43128 formula, R1~R 14 are each independently selected from H, alkyl, hydroxyl, or alkoxy. [The present invention 1010] 1001. The host cell of the present invention, wherein the BIA compound or precursor thereof is thebaine, and the host cell comprises one or more heterologous coding sequences for one or more enzymes selected from SalSyn, CYP2D6, CYP2D2, SalR, and SalAT. [The present invention 1011] the BIA compound or its precursor is an opiate compound, and the host cell one or more heterologous coding sequences for one or more enzymes obtained from a different biological source compared to the host cell, selected from T6ODM, COR, and CODM; and one or more heterologous coding sequences for one or more enzymes selected from Pseudomonas putida morA and Pseudomonas putida morB; Including, the opiate compound is selected from codeine, morphine, hydrocodone, hydromorphone, oxycodone, dihydrocodeine, 14-hydroxycodeine, and dihydromorphine; The host cell of the present invention. [The present invention 1012] 1011. A host cell according to the invention, wherein the heterologous coding sequences for the enzymes T6ODM, COR and CODM are present in a ratio of 2:1:3, 1:1:3 or 2:1:2. [The present invention 1013] further comprising an increased amount of one or more of glutamine, 2-oxoglutarate, and glutamic acid compared to a control yeast strain; No production of oripavine or morphinone from thebaine; and can produce a yield of opiate compounds that is 30% or more of total opiates; The host cell of the present invention. [The present invention 1014] A host cell that produces reticuline, wherein the host cell contains multiple copies of one or more heterologous coding sequences for one or more methyltransferases obtained from a different biological source compared to the host cell, and the one or more methyltransferases are selected from 6OMT, CNMT and 4'OMT. [The present invention 1015] A host cell that produces sanguinarine or a sanguinarine precursor, wherein the host cell comprises one or more heterologous coding sequences for one or more enzymes obtained from a different biological source compared to the host cell, wherein the one or more enzymes are selected from BBE, CFS, CPR, STS, TNMT, MSH, P6H and DBOX, and the sanguinarine precursor is selected from cheilanthifoline, stylopine, cis-N-methylstylopine, scoulerine, protopine, and dihydrosanguinarine. [The present invention 1016] 1. A host cell that produces a protoberberine alkaloid, the host cell comprising one or more heterologous coding sequences for one or more enzymes obtained from a different biological source compared to the host cell, the one or more enzymes being selected from BBE, S9OMT, CAS, and STOX, and the protoberberine alkaloid being represented by one of the following structures: TIFF2026015382000003.tif43128 formula, R1~R 14 are each independently selected from H, alkyl, hydroxyl, or alkoxy. [The present invention 1017] A host cell that produces thebaine, wherein the host cell comprises one or more heterologous coding sequences for one or more enzymes obtained from a different biological source compared to the host cell, and the one or more enzymes are selected from SalSyn, CYP2D6, CYP2D2, SalR, and SalAT. [The present invention 1018] A host cell that produces an opiate compound, the host cell comprising one or more heterologous coding sequences for one or more enzymes obtained from a different biological source compared to the host cell, selected from T6ODM, COR, and CODM; and one or more heterologous coding sequences for one or more enzymes selected from Pseudomonas putida morA and Pseudomonas putida morB; Including, the opiate compound is selected from codeine, morphine, hydrocodone, hydromorphone, oxycodone, dihydrocodeine, 14-hydroxycodeine, and dihydromorphine; The host cell. [The present invention 1019] Culturing the host cell of the present invention under conditions suitable for protein production; adding a starting compound to the cell culture; and Harvesting the BIAs from the cell culture Including, The host cells are cultured in a medium containing one or more of glutamine, 2-oxoglutarate, glutamic acid, and 2% or less dimethyl sulfoxide (DMSO); Method for preparing benzylisoquinoline alkaloids (BIA). [Brief explanation of the drawings]

[0005] The invention will be best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to scale. Conversely, the dimensions of the various features have been arbitrarily expanded or reduced for clarity. The drawings include the following figures: [Figure 1] In vivo methylation of norlaudanosoline by methyltransferases from P. somniferum and Thalictrum flavum and detection of the methylated products by liquid chromatography-mass spectrometry (LCMS) are shown. [Figure 2] 1 shows an alternative methylation pathway from norlaudanosoline to reticuline. [Figure 3] 1 shows an alternative methylation pathway from norcoclaurine to reticuline. [Figure 4] 1 shows the biosynthetic steps for producing sanguinarine from reticuline. [Figure 5] 1 shows the biosynthetic steps of interest from reticuline to berberine. [Figure 6]FIG. 1 shows the effect of episomal gene copy number and expression of various cytochrome P450-NADPH reductase enzymes on protoberberine production in yeast cultures. [Figure 7] Microbial production of berberine from norlaudanosoline. [Figure 8] 1 shows the biosynthetic pathway from reticuline to thebaine or its products. [Figure 9] 1 shows the results of combining salutaridin reductase (SalR) and salutaridinol 7-O-acetyltransferase (SalAT) mutants in thebaine-producing strains. [Figure 10] 1 shows the design of gene constructs that enhance promoter activity and prevent instability of two genes that share sequence similarity, namely T6ODM and CODM. [Figure 11] We present a pathway for engineering the biosynthesis of protoberberine and protopine alkaloids from the precursor molecule norlaudanosoline via the branch-point intermediate reticuline in S. cerevisiae. The final engineered protopine-producing strain contains 11 heterologous expression cassettes (seven integrated enzymes and four enzymes expressed from a yeast artificial chromosome), including cytochrome P450 (EcSTS, PsMSH, EcCFS), cytochrome P450 reductase (ATR1), and other enzyme classes (Ps6OMT, PsCNMT, Ps4'OMT, PsBBE, PsTNMT). [Figure 12]Microbial production of (S)-cheilanthifoline. (a) Schematic showing the conversion of norlaudanosoline to (S)-cheilanthifoline. (b) LC-MS analysis of growth medium from yeast strains fed norlaudanosoline shows that the vector control strain (left) produces scourerine (peak 3, m / z = 328). When the EcCFS enzyme is expressed (right), the metabolite cheilanthifoline is detected (m / z = 326, peak 4), confirmed by MS-MS fragmentation (bottom). (c) Pairing of various enzyme mutants with cytochrome P450 NADPH reductase partners results in the production of (S)-cheilanthifoline. (d) P450 expression levels result in distinct ER morphologies. EcCFS C-terminally tagged with GFP on high-copy (top) or low-copy (center) plasmids localizes to the endoplasmic reticulum but displays distinct ER growth morphologies. Wild-type ER (no heterologous P450 expressed) is shown for comparison (bottom). The ratio indicates the GFP-positive rate of yeast. (e) Stable expression of CFS improves cheilanthifoline production and scourerine conversion efficiency. (f) Promoter choice affects CFS activity. EcCFS was expressed from a low-copy plasmid under the control of five different promoters with URA selection. [Figure 13] Optimization of (S)-stylopine production. (A) Schematic showing the conversion of norlaudanosoline to (S)-stylopine. (B) LC-MS analysis of growth medium from yeast strains fed 2 mM norlaudanosoline shows that the vector control strain produces cheilanthifoline (peak 4, m / z = 326). When the EcSTS enzyme is expressed, stylopine (peak 5, m / z = 324) is detected by comparison with a standard. (C) Stylopine production is altered by combining species variants of CFS and STS expressed from separate low-copy plasmids. (D) Growth of engineered yeast strains at 25°C improves STS activity. (E) Gene copy number of CFS and STS affects stylopine production. [Figure 14]Engineering a heterologous protopine biosynthetic pathway. (A) Schematic showing the conversion of norlaudanosoline to protopine. (B) LC-MS analysis of growth medium from a yeast strain fed 2 mM norlaudanosoline shows that the vector control strain produces stylopine (peak 5, m / z = 324). When the TNMT enzyme is expressed, the metabolite cis-N-methylstylopine (m / z = 338, peak 6) is detected, as confirmed by MS-MS fragmentation. (C) When MSH is added, protopine is detected (m / z = 354), as confirmed by comparison with a standard and MS-MS fragmentation. [Figure 15] We demonstrate the engineering of a heterologous morphine biosynthetic pathway in yeast, including the conversion of thebaine by morphine biosynthetic enzymes (thebaine 6-O-demethylase (T6ODM), codeine O-demethylase (CODM), and codeinone reductase (COR) from the opium poppy, P. somniferum). Two pathways to morphine are shown, via the intermediates codeinone and codeine (pathway i) and oripavine and morphinone (pathway ii). Furthermore, a newly identified pathway to neomorphine (iii) exhibits a broader substrate range for COR and CODM. [Figure 16] (a) Methods for engineering host yeast cells and (b) methods for titrating additives in the culture medium to enhance morphine production by providing 2-oxoglutarate as a co-substrate in the morphine biosynthetic pathway are presented. [Figure 17]The design for varying gene copy number to increase pathway flux to morphine is shown. The titers of the target product, morphine (black bars), and the untargeted product, neomorphine (gray bars), were analyzed from strains with varying copy numbers of T6ODM, COR1.3, and CODM. Culture media was analyzed by LC-MS for opiate production after 96 hours of growth in deep-well plates containing 1 mM thebaine. Each strain expressed one copy of T6ODM, COR1.3, and CODM on the pYES1L vector. Additional gene copies were integrated into the host cell genome. A control strain expressing one copy of each gene from the pYES1L vector (1:1:1 gene ratio) produced intermediate levels of morphine and is indicated by the dashed line in the graph. Error bars represent ±1 SD of three biological replicates. [Figure 18]A spatial engineering approach to improve pathway specificity for morphine is shown. (a) Schematic illustrating the principle of using a spatial engineering approach based on enzyme delocalization to improve pathway specificity for the target product, morphine. Localization of COR1.3 to organelles can sequester this enzyme from the nontarget substrate, neopinone (produced by cytoplasmic T6ODM activity), allowing additional time for the spontaneous isomerization of neopinone to the target substrate, codeinone. In this scheme, the heterologous pathway is divided into two parts: (1) cytoplasmic T6ODM (ring) converts thebaine to neopinone, which then rearranges to codeinone at an unknown rate; as a result, (2) the sequestered COR1.3 (ring) is closer to codeinone than to neopinone and converts this substrate to codeine, which is then irreversibly demethylated by CODM (ring) to morphine. The overall effect of this localization scheme is to direct pathway flow toward the target end product, morphine. Design considerations for COR1.3 localization include the ability to direct the enzyme to organelles in one of three configurations: free inside the organelle lumen, membrane-localized with the enzyme extending into the cytoplasm, or membrane-localized with the enzyme extending into the organelle lumen. (b) The organelle routing toolkit allows modular routing of proteins to specific organelles within yeast host cells. A set of modular localization tags (ER1: SEQ ID NO:1, ER2: SEQ ID NO:2, ER3: SEQ ID NO:3 and 4, V1: SEQ ID NO:5, PM1: SEQ ID NO:6, and MT1: SEQ ID NO:7) were designed and fused to any enzyme via a 7-amino acid linker, either N-terminal Gly6SerThr (SEQ ID NO:8) or C-terminal ProGly6 (SEQ ID NO:9). To validate the tags, each was fused to the fluorescent protein GFP and imaged in live yeast cells. The organelle markers KAR2-DsRed-HDEL (SEQ ID NO:4) and COX4-mCherry were included in the organelle routing toolkit as ER3 and MT1, respectively.Untagged COR1.3 (COR1.3-GFP) localized to the cytoplasm in yeast cells. Scale bar, 4 μm. [Figure 19] We demonstrate that the organelle routing toolkit can be used to localize heterologous COR1.3 enzymes to yeast organelles, enhancing potency and selectivity for morphine. The localized COR1.3 mutants were co-expressed with untagged T6ODM and CODM in yeast host cells. The strains were cultured in optimized medium containing 1 mM thebaine and grown for 96 hours. The culture medium was analyzed for morphine (black bars) and neomorphine (gray bars) by LC-MS. [Figure 20] This diagram shows the development of an opiate biosynthetic pathway in an engineered yeast strain by incorporating bacterial enzymes to enable the biological synthesis of semisynthetic opioids. Diagram showing the long conversion of thebaine in yeast by incorporating morA (morphine dehydrogenase) and morB (morphine reductase) from Pseudomonas putida M10 into the heterologous pathway. [Figure 21] Yeast strains optimized for the production of various opioids are shown. (a) Concentration of total opioid molecules in the culture medium after closed-batch fermentation. Yeast strains CSY950, CSY951, and CSY952 (Table 4) were optimized for the production of morphine, hydromorphone, and hydrocodone / oxycodone, respectively. The indicated strains were cultured in closed-batch fermentation in medium supplemented with 1 mM thebaine (equivalent to 311 mg / L). The culture medium was analyzed for a range of opioids by LC-MS at the end of fermentation. (b) Cell density and concentrations of key opioids (hydrocodone, dihydrocodeine, and oxycodone) as a function of fermentation time for yeast strain CSY952. At the indicated time points, samples were withdrawn, diluted, and analyzed for cell density by spectrophotometry and for opioid production by LC-MS. [Figure 22]Liquid chromatography-tandem mass spectrometry (LCMS) of metabolites secreted into the culture medium by an engineered yeast strain expressing Papaver somniferum T6ODM, COR1.3, and CODM was grown for 96 hours in the presence of thebaine. LCMS peaks 1–5 correspond to the MS2 fragmentation patterns for codeinone, codeine, neopine, morphine, and neomorphine. [Figure 23] LCMS analysis of metabolites secreted into the culture medium by engineered yeast strains expressing enzymes from P. somniferum and P. putida M10. Strain CSY946 (expressing T6ODM and morB) and strain CSY945 (expressing T6ODM, CODM, morA, and morB) were cultured for 96 h in the presence of thebaine. LCMS peaks 6–11 correspond to hydrocodone, oxycodone, hydromorphone, dihydrocodeine, 14-hydroxycodeine, and dihydromorphine. DETAILED DESCRIPTION OF THE INVENTION

[0006] Detailed Description As summarized above, aspects of the present invention include host cells engineered to produce benzylisoquinoline alkaloids (BIAs). The host cells contain heterologous coding sequences for various enzymes involved in the host cell's synthetic pathway from a starting compound to a BIA. In some embodiments, the host cell is selected from a reticuline-producing host cell, a sanguinarine precursor-producing host cell, a protoberberine-producing host cell, a thebaine-producing host cell, and an opiate-producing host cell. Also provided are methods for producing a BIA of interest by culturing the host cell under culture conditions that promote expression of the enzymes encoded by the heterologous coding sequences in the host cell.

[0007] Before describing the present invention in more detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. Also, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

[0008] When a series of numerical values ​​is given, it is understood that each intervening value, to one-tenth of the unit of the lower limit, between the upper and lower limits of the range, and any other stated or intervening value within that stated range, is encompassed within the invention, unless the context clearly indicates otherwise. The upper and lower limits of these narrower ranges may independently be included in the narrower ranges, which are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0009] Ranges are presented herein with the term "about" before the numerical values. The term "about" is used herein to provide textual support for the exact number that follows, as well as for numbers that are near or approximately the number that follows the term. In determining whether a number is near or approximately a specifically recited number, the near or approximately unrecited number may be a number that, in the context in which it is presented, provides substantial equivalence to the specifically recited number.

[0010] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, representative exemplary methods and materials are described herein.

[0011] All publications and patents cited herein are incorporated by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are also incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be taken as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0012] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is meant to act as a predicate for the use of exclusive terminology such as "only," "only," and the like in connection with the recitation of claim elements or for the use of a "negative" limitation.

[0013] As will be apparent to those skilled in the art upon reading this disclosure, each individual embodiment described and illustrated herein has distinct components and features that may be readily separated from or combined with the features of any of the other embodiments without departing from the scope or spirit of the invention. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0014] Benzylisoquinoline alkaloids (BIA) Aspects of the present invention include host cells that produce compounds characterized as benzylisoquinoline alkaloids (BIAs), as well as their biosynthetic precursors, intermediates, and metabolites. A variety of BIAs, their biosynthetic precursors, intermediates, and metabolites can be produced by the subject host cells, including, but not limited to, reticuline, sanguinarine, protoberberine, berberine, benzophenanthridine alkaloids, thebaine, opiate compounds, cheilanthifoline, stylopine, cis-N-methylstylopine, saltaridinol, saltaridinol-7-O-acetate, protopine, and dihydrosanguinarine, (S)-canadine, oripavine, codeinone, neopine, neomorphine, morphine, codeine, hydromorphone, hydrocodone, oxycodone, oxymorphone, dihydrocodeine, 14-hydroxycodeine, and dihydromorphine.

[0015] The synthetic pathway that is produced in host cell can start from any convenient compound.The starting compound of interest includes but is not limited to laudanosoline, methyllaudanosoline, norlaudanosoline, methylnorlaudanosoline, norcoclaurine, salutaridine, reticuline, tyramine, dopamine, 4-HPA, 4-HPPA, coclaurine, N-methylcoclaurine, 3'-hydroxy-N-methylcoclaurine, scoureline, tetrahydrocolumbamine, canadine, laudanine, sanguinarine, thebaine, morphine, codeine, codeinone, and dimethyltetrahydroisoquinoline, such as 6,7-dimethyl-1-2-3-4-tetrahydroisoquinoline, or other compounds that may or may not be present in endogenous BIA pathway.In certain embodiments, the starting compound is reticuline, norlaudanosoline, or norcoclaurine. Thus, the starting material may be non-natural, or the starting material may be natural. Other compounds may also be used as starting materials in a desired synthetic pathway based on the synthetic pathway present in the host cell. The source of the starting material may be from the host cell itself (e.g., tyrosine), or the starting material may be added or supplemented to the host cell from an exogenous source. For example, when host cells are grown in liquid culture (an in vivo environment), the cell medium may be supplemented with a starting material, such as tyrosine or norlaudanosoline, which is transported into the cell and converted to the desired product.

[0016] host cell As summarized above, one aspect of the present invention is a host cell that produces one or more BIAs. Any convenient type of host cell may be used to generate the subject BIA-producing cells; see, for example, U.S. Patent No. 2008 / 0176754, the disclosure of which is incorporated herein by reference in its entirety. In some cases, the host cell is yeast. In some cases, the host cell is derived from a yeast strain engineered to produce the BIA of interest. In some embodiments, the host cell is selected from a reticuline-producing host cell, a sanguinarine precursor-producing host cell, a protoberberine-producing host cell, a thebaine-producing host cell, and an opiate-producing host cell.

[0017] Any convenient cell may be used in the subject host cells and methods. In some cases, the host cell is a non-plant cell. In certain cases, the host cell is an insect cell, a mammalian cell, a bacterial cell, or a yeast cell. Host cells of interest include, but are not limited to, bacterial cells such as Bacillus subtilis cells, Escherichia coli cells, Streptomyces cells, and Salmonella typhimurium cells, as well as insect cells such as Drosophila melanogaster S2 cells and Spodoptera frugiperda Sf9 cells. In some embodiments, the host cell is a yeast cell or an E. coli cell. In certain embodiments, the yeast cell can be a S. cerevisiae species. Yeast is an interesting host cell. This is because cytochrome P450 proteins (which are involved in several biosynthetic pathways of interest) can be properly folded within the endoplasmic reticulum membrane, thereby maintaining their activity. Yeast strains of interest for use in the present invention include, but are not limited to, those described by Smolke et al. in U.S. Patent No. 2008 / 0176754 (the disclosure of which is incorporated herein by reference in its entirety), such as CEN.PK (genotype: MATa / α ura3-52 / ura3-52 trp1-289 / trp1-289 leu2-3_112 / leu2-3_112 his3 Δ1 / his3Δ1 MAL2-8C / MAL2-8C SUC2 / SUC2), S288C, W303, D273-10B, X2180, A364A, Σ1278B, AB972, SK1, and FL100.In particular cases, the yeast strains were 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 Arabidopsis thaliana ( The yeast cells are either WAT11 or W(R), which are derivatives of the W303-B strain (MATa; ade2-1; his3-11, -15; leu2-3, -112; ura3-1; canR; cyr+) expressing the (A. thaliana) NADPH-P450 reductase ATR1 and the yeast NADPH-P450 reductase CPR1, respectively. In another embodiment, the yeast cell is W303α (MATα; his3-11,15 trp1-1 leu2-3 ura3-1 ade2-1). The identities and genotypes of additional yeast strains of interest can be found at EUROSCARF (web.uni-frankfurt.de / fb15 / mikro / euroscarf / col_index.html).

[0018] As used herein, the term "host cell" refers to a cell that contains one or more heterologous coding sequences encoding an activity that enables the host cell to produce a desired BIA, for example, as described herein. The heterologous coding sequence can be stably integrated into the genome of the host cell, or the heterologous coding sequence can be transiently inserted into the host cell. As used herein, the term "heterologous coding sequence" refers to any polynucleotide that encodes or ultimately encodes a peptide or protein or its equivalent amino acid sequence, e.g., an enzyme, that is not normally present in the host organism and that can be expressed in the host cell under appropriate conditions. Thus, a "heterologous coding sequence" includes multiple copies of a coding sequence normally present in the host cell, such that the cell expresses additional copies of a coding sequence not normally present in the cell. A heterologous coding sequence can be RNA or any type thereof, e.g., mRNA, DNA or any type thereof, e.g., cDNA, or an RNA / DNA hybrid. Examples of coding sequences include, but are not limited to, full-length transcription units containing features such as a coding sequence, introns, a promoter region, a 3'-UTR, and an enhancer region.

[0019] As used herein, the term "heterologous coding sequence" also includes the coding portion of a peptide or enzyme, i.e., the cDNA or mRNA sequence of the peptide or enzyme, as well as the coding portion of a full-length transcription unit, i.e., a gene including introns and exons, as well as "codon-optimized" sequences, truncated sequences, or other forms of altered sequences that encode an enzyme or its equivalent amino acid sequence (provided that the equivalent amino acid sequence produces a functional protein). Such equivalent amino acid sequences can have one or more amino acid deletions, which may be at the N-terminus, C-terminus, or intermediate. Truncated forms are also contemplated as long as they retain the catalytic activity set forth herein. Fusion of two or more enzymes is also contemplated to facilitate metabolite translocation in a pathway, provided that catalytic activity is maintained.

[0020] Operable fragments, mutants, or truncations can be identified by modeling and / or screening, for example, by stepwise deletion of N-terminal, C-terminal, or internal regions of the protein, followed by analysis of the resulting derivatives for their activity relative to the native sequence for the desired reaction. If the derivative exhibits this ability, the construction of an equivalent enzyme derivative is deemed appropriate.

[0021] Another aspect of the present invention relates to heterologous coding sequences encoding equivalent amino acid sequences to the naturally occurring amino acid sequences corresponding to 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 do not essentially affect the biological activity of the protein when used for the desired purpose, compared to the similar activity of the particular amino acid sequence. Biological activity, in the example of a decarboxylase, refers to its catalytic activity. Equivalent sequences are also meant to include sequences that have been engineered and / or evolved to have properties different from the original amino acid sequence. Mutatable properties of interest include catalytic activity, substrate specificity, selectivity, stability, solubility, localization, etc. In certain embodiments, an "equivalent" amino acid sequence comprises at least 80-99% identity at the amino acid level to a particular amino acid sequence, and in some cases at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or more, and in certain cases at least 95%, 96%, 97%, 98%, and 99% identity at the amino acid level. In some cases, the amino acid sequence may be identical, but the DNA sequence has been modified, for example, to optimize codon usage for the host organism.

[0022] Host cells can also be modified to have one or more genetic changes to accommodate heterologous coding sequences. Alterations to the native host genome include, but are not limited to, modifying the genome to reduce or eliminate expression of specific proteins that may interfere with the desired pathway. The presence of such native proteins may rapidly convert one of the pathway intermediates or the final product into a metabolite or other compound that cannot be used in the desired pathway. Therefore, reducing or completely eliminating the activity of the native enzyme would make the intermediate produced more readily usable for incorporation into the desired product. In some cases, if the host cell is a yeast cell and the desired pathway requires the co-substrate 2-oxoglutarate, expression of the native endogenous glutamate and / or 2-oxoglutarate dehydrogenase enzymes, which can also convert the desired co-substrate (2-oxoglutarate) to glutamate or succinyl-CoA, respectively, may be reduced or eliminated. In some cases, the elimination of protein expression may be of interest for proteins involved in pleiotropic drug responses, including, but not limited to, ATP-binding cassette (ABC) transporters, multidrug resistance (MDR) pumps, and related transcription factors. These proteins are involved in the export of BIA molecules into the culture medium, and thus deletion controls the export of the compounds into the medium, making them more available for incorporation into the desired product. In some embodiments, the deletion of host cell genes of interest includes genes related to the unfolded protein response and endoplasmic reticulum (ER) proliferation. Deletion of such genes can result in improved BIA production. Expression of cytochrome P450 can induce the unfolded protein response and cause ER proliferation. Deletion of these stress response-related genes can control or reduce the overall burden on the host cell and improve pathway performance. Genetic alterations can also include modifying the promoter of an endogenous gene to increase expression and / or introducing additional copies of the endogenous gene. An example of this involves the construction / use of strains that overexpress the endogenous yeast NADPH-P450 reductase CPR1 to increase the activity of a heterologous P450 enzyme.Additionally, endogenous enzymes such as ARO8, 9, and 10, which are directly involved in the synthesis of intermediate metabolites, may also be overexpressed.

[0023] Heterologous coding sequences of interest include, but are not limited to, sequences encoding enzymes (either wild-type or equivalent sequences) normally involved in the production of BIAs in plants. In some cases, the enzyme encoded by the heterologous sequence can be any of the enzymes in the BIA pathway and can be derived from any convenient source. In some cases, cheilanthifoline synthase (CFS; EC 1.14.21.2), found in at least Papaver somniferum, Eschscholzia californica, and Argemone mexicana, is known to synthesize (S)-cheilanthifoline from (S)-scourerine. The choice and number of enzymes encoded by the heterologous coding sequence for a particular synthetic pathway can be selected based on the desired product. In certain embodiments, a host cell of the invention may comprise 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or even 15 or more heterologous coding sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 heterologous coding sequences.

[0024] Unless otherwise specified, heterologous coding sequences are as reported in GENBANK. A list of enzymes of interest is provided in Tables 2 and 3. The host cells of the invention can contain any combination of the listed enzymes from any source. Unless otherwise specified, accession numbers in Table 3 refer to GenBank. Some accession numbers refer to the Saccharomyces Genome Database (SGD), available on the World Wide Web at www.yeastgenome.org.

[0025] In some embodiments, host cells (e.g., yeast strains) are engineered for selective production of a BIA of interest by localizing one or more enzymes to a compartment within the cell. In one embodiment of the invention shown in Figure 18, an enzyme can be localized to the yeast endoplasmic reticulum by fusing an ER2 targeting sequence to the C-terminus of the protein.

[0026] In some cases, an enzyme can be located within a host cell so that the compound produced by the enzyme spontaneously translocates or is converted into a desired metabolite by another enzyme before reaching a localized enzyme that can convert the compound into an undesired metabolite. The spatial distance between the two enzymes can be selected to prevent one enzyme from acting directly on the compound to produce an undesired metabolite and to limit the production of undesired end products (e.g., undesired opioid by-products). In certain embodiments, any of the enzymes described herein, alone or together with a second enzyme, can be localized in any convenient compartment within a host cell, including, but not limited to, organelles, the endoplasmic reticulum, the Golgi, the vacuole, the nucleus, the plasma membrane, or the periplasm (see, for example, Figure 18).

[0027] In some embodiments, the host cell comprises one or more enzymes that contain a localization tag.Any convenient tag can be used.In some cases, the localization tag is a peptide sequence attached to the N-terminus and / or C-terminus of the enzyme.Any convenient method can be used to attach the tag to the enzyme.

[0028] In some cases, localization tags are derived from endogenous yeast proteins, which may provide routes to various yeast organelles, such as the endoplasmic reticulum (ER), mitochondria (MT), plasma membrane (PM), and vacuole (V).

[0029] In certain embodiments, the tag is an ER routing tag (e.g., ER1). In certain embodiments, the tag is a vacuolar tag (e.g., V1). In certain embodiments, the tag is a plasma membrane tag (e.g., P1). In certain cases, the tag comprises or is derived from a transmembrane domain within a tail-anchored protein.

[0030] In some embodiments, the localization tag localizes the enzyme outside of the organelle. In certain embodiments, the localization tag localizes the enzyme inside the organelle.

[0031] In some cases, expression of each type of enzyme is increased through additional gene copies (i.e., multiple copies), thereby increasing the accumulation of intermediates and ultimately the production of BIAs and / or BIA precursors. Embodiments of the invention include increased production of BIAs in host cells through the co-expression of multiple species variants of a single or multiple enzymes. In some cases, additional gene copies of a single or multiple enzymes are included in the host cell. Any convenient method may be used to include multiple copies of the heterologous coding sequence for the enzyme in the host cell.

[0032] In some embodiments, the host cell contains multiple copies of the heterologous coding sequence of the enzyme, for example, two or more, three or more, four or more, five or more, or even ten or more copies. In certain embodiments, the host cell contains multiple copies of the heterologous coding sequence of one or more enzymes, for example, two or more, three or more, four or more copies. In some cases, the multiple copies of the heterologous coding sequence of the enzyme are obtained from two or more different biological sources compared to the host cell. For example, the host cell may contain multiple copies of one type of heterologous coding sequence, each copy being obtained from a different biological source. Thus, each copy may contain some variation in the apparent sequence due to the interspecies differences of the enzyme of interest encoded by the heterologous coding sequence.

[0033] The culture medium of the engineered host cells may be sampled and monitored for the production of the BIA compound of interest. The BIA compound may be observed and measured using any convenient method. Methods of interest include, but are not limited to, LC-MS methods (such as those described herein), in which the sample of interest is analyzed by comparison with a known amount of a standard compound. The identity may be confirmed, for example, by m / z and MS / MS fragmentation pattern, and quantification or measurement of the compound may be achieved through EIC MS peak analysis by reference to LC trace peaks of known retention time and / or corresponding LC-MS analysis of a known amount of a standard compound.

[0034] Reticuline-producing host cells Reticuline is a key branching point intermediate in the synthesis of BIAs, and high yields of this intermediate are of interest when attempting to produce end products such as morphine, sanguinarine, or berberine. In some cases, to produce reticuline from norlaudanosoline, the following three enzymes are expressed in host cells: norcoclaurine 6-O-methyltransferase (6OMT; EC 2.1.1.128), coclaurine N-methyltransferase (CNMT; EC 2.1.1.140), and 3'hydroxy-N-methylcoclaurine 4'-O-methylase (4'OMT; EC 2.1.1.116). Typically, the enzymes are obtained from a different biological source than the host cell. To produce reticuline from norcoclaurine, additional cytochrome P450 enzymes, such as CYP80B3 or CYP80B1 (EC 1.14.13.71), may be expressed along with the three methyltransferases. Engineering S. cerevisiae to produce reticuline may use any convenient optimization method. In some cases, the best reticuline producer is found by expressing all combinations of three methyltransferases from two or more species (e.g., P. somniferum and Thalictrum flavum). In other cases, the optimal combination of methyltransferases (all three from P. somniferum) may be integrated into the yeast chromosome, and expression of each may be gradually increased without affecting reticuline yield. The amount of methyltransferase enzyme expression may be increased or decreased, and the methyltransferases may act on the substrate sequentially, in concert, or a combination of the two.

[0035] Aspects of the present invention include S. cerevisiae strains with improved reticuline production through overexpression of 6OMT, CNMT, and / or 4'OMT genes from several different species. By improved or increased production, it is meant both the production of some amount of reticuline where the control has no reticuline production at all, as well as an increase of about 10% or more, e.g., about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, e.g., 2-fold or more, e.g., 5-fold or more, e.g., 10-fold or more, in situations where the control has some reticuline production. Methyltransferases from different species have slightly different substrate specificities [Choi et al. (2002). J Biol Chem 277, 830-835; Liscombe et al. (2009). Plant J 60, 729-74; Morishige et al., (2000) J Biol Chem 275, 23398-23405; Ounaroon et al. (2003) Plant J 36, 808-819; Sato et al., (1994) Eur J Biochem 225, 125-131]. When methyltransferases from different species are co-expressed in a single strain, it is possible to take advantage of the different substrate specificities and increase the flux through multiple methylation pathways, thereby increasing the yield of reticuline. In some cases, species variants of methyltransferases include, but are not limited to, P. somniferum, Thalictorum flavum, and Coptis japonica (Table 2). In certain cases, species variants of methyltransferases are obtained from P. somniferum. In certain cases, species variants of methyltransferases are obtained from Thalictorum flavum. In some cases, species variants of methyltransferases are obtained from Coptis japonica.

[0036] In some embodiments, the host cell comprises two or more heterologous coding sequences for two or more methyltransferases selected from 6OMT, CNMT, and 4'OMT. In certain cases, the two or more methyltransferases are obtained from two or more different biological sources compared to the host cell.

[0037] In some cases, the host cell comprises heterologous coding sequences for the methyltransferases 6OMT and CNMT. In certain cases, the host cell comprises heterologous coding sequences for the methyltransferases CNMT and 4'OMT.

[0038] In some cases, the host cell contains heterologous coding sequences for the methyltransferases 6OMT and 4'OMT.

[0039] In certain embodiments, the host cell comprises heterologous coding sequences for all of the methyltransferases 6OMT, CNMT and 4'OMT.

[0040] In some cases, expression of each type of methyltransferase is increased through additional gene copies (i.e., multiple copies), thereby increasing the accumulation of intermediates and ultimately the production of reticuline. Aspects of the invention include increased production of reticuline in yeast strains through the co-expression of multiple species variants of single or multiple methyltransferases and the incorporation of additional gene copies of single or multiple methyltransferases.

[0041] In some embodiments, the host cell contains multiple copies of a methyltransferase, for example, two or more, three or more, four or more, five or more, or even ten or more copies. In certain embodiments, the host cell contains multiple copies of one or more methyltransferases, for example, two or more, three or more, four or more, etc. In some cases, the multiple copies of the methyltransferase are obtained from two or more different biological sources compared to the host cell. For example, the host cell contains multiple copies of one heterologous coding sequence, each copy being obtained from a different biological source. Thus, each copy may contain some variation in the expressed sequence based on interspecies differences in the enzyme of interest encoded by the heterologous coding sequence.

[0042] In some cases, the multiple copies are heterologous coding sequences of CNMT. In particular cases, two copies of the heterologous coding sequence of CNMT are included. In some cases, the multiple copies are heterologous coding sequences of 6OMT. In particular cases, two copies of the heterologous coding sequence of 6OMT are included. In some cases, the multiple copies are heterologous coding sequences of 4'OMT. In particular cases, two copies of the heterologous coding sequence of 4'OMT are included.

[0043] In some cases, the host cell can produce an increased amount of reticuline from norcoclaurine compared to a control host cell lacking multiple copies of one or more heterologous coding sequences for one or more methyltransferases. In certain cases, the increased amount of reticuline is about 10% or more compared to the control host cell, 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, 2-fold or more, 5-fold or more, or even 10-fold or more compared to the control host cell.

[0044] In some cases, the host cell can produce an increased amount of reticuline from norlaudanosoline compared to a control host cell lacking multiple copies of one or more heterologous coding sequences for one or more methyltransferases. In certain cases, the increased amount of reticuline is about 10% or more compared to the control host cell, 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, 2-fold or more, 5-fold or more, or even 10-fold or more compared to the control host cell.

[0045] In some embodiments, the host cell is capable of producing a yield of reticuline from norcoclaurine of 10% or more, e.g., a yield of reticuline from norcoclaurine of 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, about 80% or more, or even 90% or more.

[0046] In some embodiments, the host cell is capable of producing a yield of reticuline from norlaudanosoline of 10% or more, e.g., a yield of reticuline from norlaudanosoline of 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or even 90% or more.

[0047] In certain embodiments, the host cell is an engineered strain that contains biosynthetic pathways that incorporate any combination of the following alternative methylation pathways and result in increased production of reticuline. In some cases, the host cell is capable of producing reticuline from norlaudanosoline via the biosynthetic pathway of Figure 2. In certain embodiments, the host cell is capable of producing reticuline from norcoclaurine via the biosynthetic pathway of Figure 3. In some cases, when the starting material of the pathway is norlaudanosoline (Figure 2, (1)), 6OMT, CNMT, and / or 4'OMT can act on this compound to produce three distinctly methylated intermediates: BIA2 is first methylated by 6OMT and then by either CNMT or 4'OMT; BIA3 is first methylated by CNMT and then by either 6OMT or 4'OMT; BIA4 is first methylated by 4'OMT and then by either 6OMT or CNMT; BIA5, previously methylated by 6OMT and 4'OMT, is methylated by CNMT to produce reticuline; BIA6, previously methylated by 6OMT and CNMT, is methylated by 4'OMT to produce reticuline; and BIA7, previously methylated by CNMT and 4'OMT, is methylated by 6OMT to produce reticuline.

[0048] In some cases, when the starting material of the pathway is norcoclaurine, 6OMT or CNMT can act on this compound to produce two distinctly methylated products (Figure 3): BIA10 is first methylated by 6OMT and then by CNMT; BIA11 is first methylated by CNMT and then by 6OMT. BIA12, previously methylated by 6OMT and CNMT, is also known as N-methylcoclaurine, which is then sequentially acted upon by CYP80B1 or CYP80B3 and 4'OMT to produce reticuline.

[0049] In certain cases, the host cell is a yeast strain. In some cases, the yeast strain is S. cerevisiae.

[0050] Sanguinarine and sanguinarine precursor-producing host cells Aspects of the present invention include protoberberine and benzophenanthridine alkaloids (which include cheilanthifoline, stylopine, cis-N-methylstylopine, scourerine, protopine, dihydrosanguinarine, and Sanguinarine Figure 4 illustrates a synthetic pathway present in an embodiment of a host cell according to an embodiment of the present invention. While the pathway may be longer if starting from norlaudanosoline, norcoclaurine, or any other convenient BIA, such as those shown in other figures, this particular pathway depiction begins with reticuline and ends with sanguinarine. The pathway may include fewer enzymes than shown if the desired end result is one of the intermediates in the pathway from norlaudanosoline to sanguinarine. The present invention includes a multi-enzymatic step biosynthetic pathway catalyzed by, for example, the enzymes BBE (EC 1.21.3.3), CFS (EC 1.14.21.2), CPR (EC 1.6.2.4), STS (EC 1.14.21.1), TNMT (EC 2.1.1.122), MSH (EC 1.14.13.37), P6H (EC 1.14.13.55), and DBOX (EC 1.5.3.12) in engineered yeast strains for the production of various protoberberine and benzophenanthridine compounds. Additionally, the present invention includes tools and methods for optimizing the production of protoberberine and benzophenanthridine compounds in conjunction with engineered yeast strains.

[0051] In some embodiments, the host cell is capable of producing sanguinarine or a sanguinarine precursor, and the host cell comprises one or more heterologous coding sequences for one or more enzymes selected from BBE, CFS, CPR, STS, TNMT, MSH, P6H, and DBOX, wherein the one or more enzymes are obtained from a different biological source than the host cell. In certain embodiments, the sanguinarine precursor is protoberberine or a benzophenanthridine alkaloid.

[0052] In certain cases, the biological source is P. somniferum, California poppy, Arabidopsis thaliana, Papaver bracteatum, or Ardisia gracilis. In certain cases, the biological source is P. somniferum. In some cases, the biological source is California poppy. In some cases, the biological source is Arabidopsis thaliana. In certain embodiments, the biological source is California poppy. In some cases, the biological source is Ardisia gracilis.

[0053] In some cases, the one or more enzymes are two or more enzymes obtained from two or more different biological sources as compared to the host cell.

[0054] In some embodiments, host cell comprises multiple copies of one or more heterologous coding sequences.In certain embodiments, the multiple copies of one or more heterologous coding sequences are obtained from two or more different biological sources compared to host cell.For example, host cell can comprise multiple copies of one heterologous coding sequence, and each copy is obtained from a different biological source.Therefore, each copy can comprise some variation in the apparent sequence based on the interspecies difference of the enzyme of interest encoded by the heterologous coding sequence.

[0055] In some cases, the host cell comprises two or more heterologous coding sequences, such as three or more, four or more, five or more, or even more, of two or more enzymes selected from BBE, CFS, CPR, STS, TNMT, MSH, P6H, and DBOX.

[0056] In certain cases, the host cell further comprises one or more gene deletions compared to the native host cell, and the one or more deleted genes are selected from IRE1, HAC1, OPI1, INO1, INO2, INO3, PDR1, STB5, PDR3, PDR5, SNQ2, YOR1, TPO1, TPO2, TPO3, TPO4, PDR10, PDR11, PDR15, PDR16, PDR17, QDR1, QDR2, QDR3, FLR1, AQR1, AQR2 and CIN5.

[0057] In some cases, the host cell is a yeast strain (such as those described herein).

[0058] In certain embodiments, the host cell is capable of producing sanguinarine or a sanguinarine precursor from norlaudanosoline via the biosynthetic pathway of Figure 4. In some cases, the host cell is capable of producing sanguinarine or a sanguinarine precursor from norlaudanosoline via the biosynthetic pathway of Figure 11.

[0059] In some cases, the host cell comprises a heterologous coding sequence for a BBE enzyme. The heterologous coding sequence for the BBE enzyme may be integrated into the host cell chromosome. In some cases, the host cell comprises a heterologous coding sequence for a CFS enzyme. In some cases, the host cell comprises a heterologous coding sequence for a CPR enzyme. In some cases, the host cell comprises a heterologous coding sequence for an STS enzyme. In some cases, the host cell comprises a heterologous coding sequence for a TNMT enzyme. In some cases, the host cell comprises a heterologous coding sequence for an MSH enzyme. In some cases, the host cell comprises a heterologous coding sequence for a P6H enzyme. In some cases, the host cell comprises a heterologous coding sequence for a DBOX enzyme.

[0060] In some cases, the host cell is a scourerine-producing yeast strain, and berberine bridge enzyme (BBE; EC 1.21.3.3) (e.g., from P. somniferum or California poppy) is expressed on a low-copy construct (e.g., low-copy plasmid, YAC, or chromosomal integration) in a yeast strain in which, for example, Ps6OMT, PsCNMT, and / or Ps4'OMT have been chromosomally integrated. Any convenient mutant of the enzyme may be used, for example, one or more of the enzyme mutants shown in Table 2.

[0061] In some cases, the sanguinarine precursor is cheilanthifoline. In certain cases, the host cell contains heterologous coding sequences for cheilanthifoline synthase (CFS; EC 1.14.21.2) and cytochrome P450 NADPH reductase (CPR; EC 1.6.2.4) enzymes. In some cases, the CPR enzyme is an ATR enzyme, such as ATR1. In some cases, the host cell is a yeast strain that produces cheilanthifoline, and cheilanthifoline synthase (CFS) (e.g., from California Poppy (EcCFS), P. somniferum (PsCFS), and / or Argium poppy (AmCFS)) is expressed using a low-copy construct (e.g., a low-copy plasmid, YAC, or chromosomal integration) in a yeast strain in which, for example, Ps6OMT, PsCNMT, Ps4'OMT, PsBBE, and / or ATR1 are integrated into the chromosome. Any convenient variant of the enzyme may be used, for example, one or more of the enzyme variants shown in Table 2.

[0062] In some embodiments, the host cell produces stylopine, and a stylopine synthase (STS; EC 1.14.21.1) (e.g., California Poppy (EcSTS), P. somniferum (PsSTS), and / or Argium poppy (AmSTS)) is expressed in the cheilanthifoline-producing strain host cell on a low-copy construct (e.g., a low-copy plasmid, YAC, or chromosomal integration). Any convenient mutant of the enzyme may be used, for example, one or more of the enzyme mutants shown in Table 2.

[0063] In certain cases, the host cell produces cis-N-methylstylopine, and tetrahydroprotoberberine N-methyltransferase (TNMT; EC 2.1.1.122) (e.g., from P. somniferum (PsTNMT) or California Poppy (EcTNMT)) is expressed in the stylopine-producing host cell on a low-copy construct (e.g., a low-copy plasmid, YAC, or chromosomal integration). Any convenient variant of the enzyme may be used, for example, one of the many enzyme variants shown in Table 2.

[0064] In some cases, the host cell produces protopine. In certain cases, cis-N-methylstylopine 14-hydroxylase (MSH, EC 1.14.13.37) (e.g., from P. somniferum (PsMSH)) is expressed in the host cell of the cis-N-methylstylopine-producing strain on the basis of a low-copy construct (e.g., a low-copy plasmid, YAC, or chromosomal integration). In certain cases, the host cell contains a heterologous coding sequence for the TNMT or MSH enzyme. Any convenient variant of the enzyme may be used, for example, one of the many enzyme variants shown in Table 2.

[0065] In some cases, the host cell produces dihydrosanguinarine and protopine 6-hydroxylase (P6H; EC 1.14.13.55) (e.g., from California Poppy (EcP6H) or P. somniferum (PsP6H)) is expressed from a low-copy construct (e.g., a low-copy plasmid, YAC, or chromosomal integration) in the protopine-producing host cell. Any convenient variant of the enzyme may be used, such as one of the many enzyme variants shown in Table 2.

[0066] In some cases, the host cell produces sanguinarine and dihydrobenzophenanthridine oxidase (DBOX; EC 1.5.3.12) (e.g., from P. somniferum (PsDBOX)) is expressed from a low-copy construct (e.g., a low-copy plasmid, YAC, or chromosomal integration) in the host cell of the dihydrosanguinarine-producing strain. Any convenient variant of the enzyme may be used, for example, one of the many enzyme variants shown in Table 2.

[0067] In some embodiments, the host cell is an engineered strain that produces more cheilanthifoline than the control strain, and additional copies of CFS are expressed in the engineered strain. By more, we mean both the production of some amount of cheilanthifoline when the control does not produce any cheilanthifoline at all, and an increase of about 10% or more, for example, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, for example, 2-fold or more, for example, 5-fold or more, for example, 10-fold or more in situations where the control produces some cheilanthifoline. Changing the expression level of the cytochrome P450 of interest through gene copy number, promoter strength, or promoter regulation can improve the production of target compounds. In certain cases, expression from a high-copy plasmid does not result in measurable cheilanthifoline production. In certain cases, when one or more copies of the CFS gene are expressed from a low-copy construct in the host cell, cheilanthifoline production is greater. In some embodiments, the more copies of the CFS gene contained in the host cell, the higher the levels of cheilanthifolin.

[0068] In certain cases, mutants of the enzymes CFS and STS from different species are combined and expressed in host cells to produce more stylopine. By comparing the engineered host cells with control cells that do not contain the desired expression of the enzyme of interest, increased levels of stylopine can be observed. By increased, we mean both the production of some amount of stylopine when the control does not produce any stylopine at all, and an increase of about 10% or more, for example, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, for example, an increase of 2-fold or more, for example, 5-fold or more, for example, 10-fold or more, when the control produces some stylopine. For example, Figure 13(c) shows the measurement of stylopine production from various CFS mutants expressed together with various STS mutants. Any convenient combination of enzymes can be used to produce increased levels of stylopine.

[0069] In some cases, to produce more cheilanthifoline, host cells (e.g., yeast strains) are engineered to contain chromosomally integrated NADPH cytochrome P450 reductases from various species to optimize the activity of cytochrome P450. For example, measurement of cheilanthifoline production from various cheilanthifoline synthase enzymes expressed together with mutants of cytochrome P450 NADPH reductase enzymes can be performed. Any convenient combination of these enzymes can be used to produce increased levels of cheilanthifoline compared to the control.

[0070] In certain cases, to produce more cheilanthifoline or stylopine, host cells (e.g., yeast strains) that overexpress cytochrome b5 are used to optimize the activity of cytochrome P450. For example, measurements of the production of cheilanthifoline or stylopine from host cells with or without overexpressed cytochrome b5 can be performed. In some cases, the host cells overexpress cytochrome b5 and produce increased levels of cheilanthifoline or stylopine compared to control cells.

[0071] In certain embodiments, to produce more protoberberine alkaloids, host cells are cultured under conditions that confer improved cytochrome P450 activity. Conditions of interest include, but are not limited to, low temperatures (e.g., about 10°C, about 15°C, about 20°C, about 22°C, about 25°C, about 28°C, about 30°C, about 33°C, or about 35°C) and growth in a highly aerated container (e.g., a flask, such as a baffled flask). For example, various protoberberine alkaloids can be measured from host cells grown under various culture conditions. In certain embodiments, host cells are incubated at a reduced temperature (e.g., about 25°C) and under highly aerated conditions (e.g., in a flask). Under such conditions, enzyme levels and / or enzyme activity (e.g., production of cheilanthifoline and stylopine) can be increased compared to the control.

[0072] In some embodiments, host cells produce more protoberberine alkaloids when optimized by deleting genes related to unfolded protein response and endoplasmic reticulum (ER) proliferation to improve BIA production.Genes of interest that are deleted include, but are not limited to, IRE1, HAC1, OPI1, INO1, INO2, and INO3 (Table 3).In some cases, the expression of cytochrome P450 induces unfolded protein response and causes ER proliferation.Deleting genes related to these stress responses can control or reduce the overall burden on host cells and improve pathway performance. By more is meant both the production of some amount of protoberberine alkaloid when the control has no protoberberine alkaloid production at all, and an increase of about 10% or more, e.g., about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, e.g., 2-fold or more, e.g., 5-fold or more, e.g., 10-fold or more, in situations where the control has some protoberberine alkaloid production.

[0073] In certain cases, the host cell comprises one or more heterologous or endogenous coding sequences for one or more proteins involved in compound transport across the cell membrane. In certain cases, the one or more proteins involved in compound transport across the cell membrane are selected from PDR1, PDR5, SNQ2, YOR1, PDR3, CIN5 and PDR3.

[0074] In certain embodiments, genes involved in pleiotropic drug responses in host cells are deleted, including but not limited to ATP-binding cassette (ABC) transporters, multidrug resistance (MDR) pumps and related transcription factors, to reduce the export of BIA molecules into the culture medium.Examples of genes include, but are not limited to, PDR1, STB5, PDR3, PDR5, SNQ2, YOR1, TPO1, TPO2, TPO3, TPO4, PDR10, PDR11, PDR15, PDR16, PDR17, QDR1, QDR2, QDR3, FLR1, AQR1, AQR2 and CIN5.Gene deletions include single deletions or any combination of multiple deletions.In some cases, host cells contain deletions of one or more genes of interest and produce lower levels of reticuline, scourerine, cheilanthifoline or stylopine than control cells that do not contain deletions of one or more genes of interest.

[0075] In another embodiment, in the target host cell, genes involved in pleiotropic drug responses, including but not limited to ATP-binding cassette (ABC) transporters, multidrug resistance (MDR) pumps, and related transcription factors, are placed under the control of a regulated (e.g., inducible or growth phase-dependent) promoter to achieve temporal control of BIA transport. In certain cases, the transporter gene is placed under the control of a stationary phase promoter that keeps the BIA of interest in the cell until the stationary phase. In such host cells, the conversion of starting materials to desired end products can be increased.

[0076] Protoberberine-producing host cells Aspects of the invention include engineered host cells that produce protoberberine alkaloids. In some cases, the protoberberine alkaloids have one of the following structures: TIFF2026015382000004.tif47128 formula, R1~R 14 are each independently -H, alkyl (e.g., lower alkyl such as methyl (CH3) or ethyl), hydroxyl, or alkoxy (OR) (e.g., lower alkoxy such as methoxy or ethoxy).

[0077] In certain cases, protoberberine alkaloids are produced from reticuline or other analogs or derivatives thereof, for example, present in the culture medium (e.g., the culture medium of a reticuline-producing cell, e.g., as described herein) or introduced into a cell lysate or lysate fraction. In certain cases, the host cell may contain one or more heterologous coding sequences for expressing one or more of the following enzymes, or any combination thereof: berberine bridge enzyme (BBE), scoureline 9'-O-methyltransferase (S9OMT), canadine synthase (CAS), and (S)-tetrahydroberberine oxidase (STOX), where the one or more enzymes are obtained from a different biological source than the host cell.

[0078] In some cases, the biological source is P. somniferum, California poppy, Coptis japonica, Thalictrum flavum, Berberis stolonifer, T. flavum subsp. glaucum, Coptis chinensis, Thalictrum spp., Coptis spp., Papaver spp., Berberis wilsonae, Ardisia crenata, or Berberis spp.

[0079] In certain embodiments, host cell comprises multiple copies of one or more heterologous coding sequences.In some cases, the multiple copies of one or more heterologous coding sequences are obtained from two or more different biological sources compared to host cell.For example, host cell can comprise multiple copies of one heterologous coding sequence, and each copy is obtained from different biological sources.Therefore, each copy can comprise some variation in the apparent sequence based on the interspecies difference of the enzyme of interest encoded by heterologous coding sequence.

[0080] In some cases, the host cell comprises two or more heterologous coding sequences for two or more enzymes selected from BBE, S9OMT, CAS, and STOX. In some cases, the host cell comprises three or more heterologous coding sequences for three or more enzymes selected from BBE, S9OMT, CAS, and STOX. In some cases, the host cell comprises a heterologous coding sequence for each of the enzymes BBE, S9OMT, CAS, and STOX.

[0081] In some embodiments, the host cell comprises a heterologous coding sequence for CAS and a heterologous coding sequence for ATR1. In certain embodiments, the host cell comprises a heterologous coding sequence for STOX.

[0082] In some embodiments, the host cell (e.g., an engineered yeast strain) supports a biosynthetic pathway such as that depicted in FIG.

[0083] In some cases, the host cell contains a STOX gene that shares 75% or more (e.g., 78%) nucleic acid sequence identity to the naturally occurring (S)-tetrahydroprotoberberine oxidase gene of B. sieboldii (Table 3), and the gene may be a non-native nucleotide sequence that is codon-optimized for expression in yeast.

[0084] Aspects of the present invention include the functional expression of STOX or its homologues in live yeast cultures. In certain embodiments, the host cells are engineered to produce berberine from its precursor, (S)-canadine.

[0085] In one embodiment of the present invention, the expression levels of the enzymes are relatively low (e.g., CEN / ARS vector or genomic expression) for BBE, CAS, and STOX (see, e.g., Figure 6a), and relatively high (e.g., 2 μm vector or multiple genomic copies) for S9OMT (see, e.g., Figure 6b). Expression levels may be varied using any convenient method. Methods of interest include, but are not limited to, varying the strength of a constitutive promoter, using an inducible promoter, varying the copy number of each gene on the episome or in the genome (see, e.g., Figure 6c), varying the selection marker, and / or culture conditions corresponding to promoter activity or selection.

[0086] In some embodiments, one or more enzymes are recombinantly expressed from a yeast artificial chromosome (eg, Figure 10).

[0087] Aspects of the present invention include the functional expression of CAS or its homologue in live yeast culture as part of a larger biosynthetic pathway. In certain embodiments, the host strain is engineered to produce berberine from norlaudanosoline or its precursor (e.g., according to Figures 5, 7, or Table 2). In some cases, the host cell can produce berberine from reticuline via the biosynthetic pathway of Figure 5. In another embodiment of the present invention, the host cell contains a cytochrome P450 reductase partner for CAS, ATR1, whose co-expression can result in higher CAS activity than California Poppy CPR, Arabidopsis ATR2, P. somniferum CPR, or endogenous yeast CPR (e.g., Figure 6d).

[0088] Aspects of the present invention include functional expression of STOX or a homolog thereof in live yeast culture. In certain embodiments, the host cell is engineered to produce (S)-canadine, a precursor of berberine, from norlaudanosoline. In some embodiments, the host cell is capable of producing (S)-canadine from norlaudanosoline.

[0089] To enhance the accumulation of BIAs in yeast cells, heterologous transporters, including but not limited to plant ATP-binding cassette proteins from BIA-producing plants, may be expressed in the engineered strain. In some embodiments, one or more heterologous coding sequences corresponding to one or more transporters selected from CjABCB1, CjABCB2, and / or CjABCB2 may be included to accumulate berberine in the host cell.

[0090] In some cases, the host cell is a yeast strain.

[0091] Thebaine-producing host cells Aspects of the present invention include engineered host cells that produce thebaine from reticuline or its precursors, either as an intermediate or as an end product. Reticuline or its precursors can be produced by existing strains present in the culture medium, or can be introduced into a cell lysate or lysate fraction. In some cases, the host cells contain one or more heterologous coding sequences for expressing one or more enzymes selected from salutaridine synthase (SalSyn), cytochrome P450 2D6 (CYP2D6), cytochrome P450 2D2 (CYP2D2), salutaridine reductase (SalR), and / or salutaridinol 7-O-acetyltransferase (SalAT). The one or more enzymes can be obtained from a different biological source than the host cell.

[0092] In some cases, the biological source is P. somniferum, Papaver orientale, Papaver species, Homo sapiens, or Rattus norvegicus.

[0093] In certain cases, the host cell is an engineered yeast strain that supports biosynthetic pathways in the host, such as those shown in FIG.

[0094] In certain embodiments, the host cell comprises multiple copies of one or more heterologous coding sequences. In some cases, the multiple copies of one or more heterologous coding sequences are obtained from two or more different biological sources as compared to the host cell.

[0095] In some cases, the host cell includes two heterologous coding sequences. In particular cases, the two heterologous coding sequences are for the enzymes SalR and SalAT.

[0096] In some cases, the host cell contains genes for CYP2D6, CYP2D2 and / or SalSyn, and / or another native or engineered P450 capable of producing salutaridine from reticuline, which may be native or non-native nucleotide sequences that are codon-optimized for expression in yeast.

[0097] In some embodiments, the host cell comprises a cytochrome P450 reductase partner for CYP2D6, CYP2D2 and / or SalSyn that is a mammalian CPR and / or ATR1, co-expression of which can result in higher CYP2D6, CYP2D2 and / or SalSyn activity than California Poppy CPR, Arabidopsis ATR2, P. somniferum CPR, or endogenous yeast CPR.

[0098] In some embodiments, the host cell may include one or more heterologous coding sequences for expressing any one or more SalR mutants listed in Table 2. In some cases, the host cell may express any one or more SalAT mutants listed in Table 2. Additionally, the SalR mutants may include F104A and / or I275A mutations, and / or any other advantageous mutations (e.g., Figure 9). Aspects of the invention include functional expression of both SalR and SalAT, which results in the production of thebaine from salutaridine.

[0099] In one embodiment of the invention, the host cell comprises the following enzyme combination: codon-optimized Papaver somniferum SalR with the F104A or I275A mutation and any codon-optimized SalAT variant (e.g., Figure 9). The SalAT gene can share 80% or less nucleic acid sequence identity with its naturally occurring counterpart (Table 3). These genes can also be non-native nucleotide sequences that are codon-optimized for expression in yeast.

[0100] In another embodiment of the invention, the conversion of salutaridine to thebaine catalyzed by SalR and SalAT occurs in a crude lysate of a subject host cell expressing both enzymes supplemented with the cofactor NADPH. In another embodiment of the invention, SalAT and SalR can be expressed in an engineered host cell expressing any convenient additional enzymes such that the strain produces a product for which thebaine is a precursor. In some cases, the strain can produce oripavine, morphine, codeine, hydromorphone, hydrocodone, oxycodone, and / or oxymorphone from thebaine.

[0101] In some cases, the host cell further comprises heterologous coding sequences for T6ODM and morB (e.g., morB or morB-E160G). In certain cases, the strain can produce one or more opiate compounds, such as hydrocodone.

[0102] In yet another embodiment of the invention, the conversion of reticuline to thebaine catalyzed by SalR and SalAT occurs in a host cell (e.g., an engineered yeast strain) that has been modified to produce increased amounts of NADPH compared to a control host cell (e.g., a native yeast strain) (see, e.g., Table 2).

[0103] In one embodiment of the present invention, SalR and / or SalAT and CYP2D2 or CYP2D6 or SalSyn or an engineered cytochrome P450 enzyme that catalyzes the conversion of reticuline to salutaridine are expressed from a yeast artificial chromosome.

[0104] In some cases, the host cell is a yeast strain. In certain cases, the host cell can be engineered to increase the production of salutaridinol or thebaine, or a product of which thebaine is a precursor, from reticuline or its precursor by localizing SalR and / or SalAT in an organelle within the yeast cell. In order to reduce the spatial distance between SalR and / or SalAT and CYP2D2 or CYP2D6 or SalSyn or engineered cytochrome P450 enzymes that catalyze the conversion of reticuline to salutaridinol, SalR and / or SalAT can be localized in the endoplasmic reticulum of yeast. Increased production refers to both the production of some amount of the compound of interest when the control does not have any production of the compound of interest, and an increase of 10% or more, for example 50% or more, for example 2-fold or more, for example 5-fold or more, for example 10-fold or more, in the situation where the control has some production of the compound of interest.

[0105] Opiate-producing host cells Aspects of the invention include engineered host cells expressing one or more of the following enzymes: thebaine 6-O-demethylase (T6ODM: EC 1.14.11.31), codeinone reductase (COR; EC 1.1.1.247), codeine O-demethylase (CODM: EC 1.14.11.32), morphine dehydrogenase (morA: EC 1.1.1.218 and EC 1.1.1.247), and morphinone reductase (morB: EC 1.3.1.-) (see, e.g., Table 2). The host cells are capable of producing opiate compounds selected from those including codeine, morphine, hydrocodone, and hydromorphone, oxycodone, dihydrocodeine, 14-hydroxycodeine, and dihydromorphine. By "more," we mean both an increase of about 10% or more, for example, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, for example, an increase of 2-fold or more, for example, 5-fold or more, for example, 10-fold or more, in situations where the control has some production of the compound of interest. The host cells can synthesize these products from thebaine provided in the culture medium, produced by the host cells themselves, or produced by one or more strains co-cultured with opiate-producing host cells. In some embodiments, the genetic modifications contained in these host cells can be combined with the genetic modifications of thebaine-producing host cells to produce a master strain capable of biosynthesizing opiates from the upstream intermediate tyrosine or a fermentable carbon source.

[0106] Opiate compounds can be observed and measured by any convenient method.Interesting methods include LC-MS methods (such as those described herein), in which the sample of interest is analyzed by comparison with a known amount of standard opiate compounds.The identity can be confirmed, for example, by m / z and MS / MS fragmentation pattern, and the quantification or measurement of the compound can be achieved through LC and / or EIC MS analysis by referring to the corresponding amount of standard compound.

[0107] In some embodiments, the host cell is capable of producing opiate compounds from thebaine via the biosynthetic pathway of Figure 15.

[0108] In some cases, the host cell does not produce oripavine or morphinone from thebaine at all. In certain cases, the host cell produces one or more of neopine and neomorphine.

[0109] In some cases, the host cell produces an opiate compound yield that is 10% or more, e.g., 20% or more, 30% or more, 40% or more, 50% or more, 50% or more, 50% or more, or even 90% or more of the total opiates in the host cell. In certain cases, the host cell produces an opiate compound yield that is 30% or more, e.g., 50% or more of the total opiates.

[0110] In some embodiments, the host cell produces an opiate compound, and the host cell comprises one or more heterologous coding sequences for one or more enzymes selected from T6ODM, COR, and CODM, wherein the one or more enzymes are obtained from a different biological source than the host cell; the one or more heterologous coding sequences for the one or more enzymes are selected from morA and morB, wherein the one or more enzymes are obtained from a different biological source than the host cell. In certain embodiments, morA is P. putida morA, and morB is P. putida morB. In certain embodiments, the host cell comprises four or more heterologous coding sequences.

[0111] In some cases, the host cell is a cell that produces an opiate compound, and the host cell comprises four or more heterologous coding sequences for four or more enzymes selected from thebaine 6-O-demethylase (T6ODM), codeinone reductase (COR), codeine O-demethylase (CODM), morphine dehydrogenase (morA), and morphinone reductase (morB), wherein the four or more enzymes are obtained from a different biological source as compared to the host cell.

[0112] In particular embodiments, the biological source is P. somniferum, Papaville species, or P. putida.

[0113] In some cases, the host cell comprises heterologous coding sequences for T6ODM, COR, CODM, and morA. In particular cases, the host cell comprises heterologous coding sequences for T6ODM, COR, CODM, and morB. In particular cases, the host cell comprises heterologous coding sequences for T6ODM, CODM, morA, and morB. In some embodiments, the host cell comprises heterologous coding sequences for the enzymes T6ODM, COR, and CODM.

[0114] In certain cases, the host cell contains multiple copies of one or more heterologous coding sequences.The multiple copies of the heterologous coding sequences can be obtained from one or two or more different biological sources compared to the host cell.For example, the host cell can contain multiple copies of one type of heterologous coding sequence, and each copy is obtained from a different biological source.Therefore, each copy may contain some variation in the expressed sequence due to the interspecies differences of the enzyme of interest encoded by the heterologous coding sequence.

[0115] In some cases, the host cell expresses one or more of the thebaine 6-O-demethylase (T6ODM), codeinone reductase (COR), and codeine O-demethylase (CODM) genes. In certain cases, these genes (T6ODM, COR, and CODM) share 76.2%, 76.8%-77.7%, and 75.2% nucleotide sequence similarity, respectively, to the native genes from P. somniferum (see Table 3). In certain cases, the genes expressed in the engineered host cell exhibit non-native nucleotide sequences optimized for codon usage in the S. cerevisiae host cell.

[0116] In some cases, the host cell supports a metabolic pathway such as that shown in Figure 15. In certain cases, in the first step, T6ODM acts on thebaine to produce neopinone. However, some pool of neopinone within the yeast cell is then acted upon by COR to produce neopine, while some other molecules of neopinone spontaneously rearrange to form codeinone. Codeinone is also acted upon by COR to produce codeine. Neopine and codeine are then metabolized by CODM to produce neomorphine and morphine, respectively. This pathway can produce the non-target products neopine and neomorphine, as well as the expected targets codeine and morphine. In some embodiments, the host cell provided a pathway to morphine using three enzymes: T6ODM, COR, and CODM (Figure 15), yet the engineered strain produced little or no oripavine or morphinone.

[0117] In one embodiment of the present invention, the COR enzyme in the pathway is isoform 1.3 from P. somniferum. This isoform of COR can produce similar levels of codeine as other mutants tested, but minimize the amount of non-target neopine.

[0118] In another embodiment, host cells (e.g., yeast strains) are engineered for the selective production of codeine and morphine over neopine and neomorphine by localizing COR to a compartment within the yeast cell. In one embodiment of the present invention, shown in Figure 18, COR is localized to the yeast endoplasmic reticulum by fusing an ER2 targeting sequence to the C-terminus of the protein. T6ODM can be located in the cytoplasm of such host cells so that neopinone produced by this enzyme spontaneously translocates to codeinone before reaching the mitochondrially localized COR enzyme. The spatial distance between the two enzymes can be selected to prevent COR from acting directly on neopinone to produce neopine and limit the production of non-targeted neopine and downstream neomorphine. In another embodiment of the present invention, CODM is colocalized with COR so that codeine produced by COR is converted to morphine. In yet another embodiment, the COR, alone or together with the CODM, is localized to any convenient compartment within the host cell (e.g., yeast cell), including, but not limited to, the endoplasmic reticulum, Golgi, vacuole, nucleus, plasma membrane, and periplasm (see Figure 18).

[0119] In certain embodiments, host cells (for example, yeast strains) have several copies of each heterologous coding sequence present in the cell, which are integrated into chromosomes or in episomal DNA.Any convenient ratio of heterologous coding sequences can be used in the target host cell.In some cases optimized for the production of morphine, the total copy number ratio of the heterologous coding sequences of T6ODM:COR:CODM is 1:1:3, 2:1:2 or 2:1:3 (see, for example, Figure 17).

[0120] The culture medium can be optimized to ensure that excess 2-oxoglutarate is available to support the activity of the 2-oxoglutarate-dependent enzymes T6ODM and CODM. The culture medium can contain any of the following additives, used individually or in combination: 2-oxoglutarate, glutamate, and glutamine (FIG. 16). In certain embodiments, the host cell is an engineered yeast strain capable of producing increased amounts of 2-oxoglutarate compared to a control yeast strain. In other embodiments, 2-oxoglutarate is added directly to the culture medium.

[0121] In some embodiments, the host cell further comprises an increased amount of 2-oxoglutarate compared to a control host cell (e.g., yeast strain), and the increased amount of 2-oxoglutarate is introduced via direct addition to the culture medium of the host cell. In some cases, the host cell further comprises an increased amount of one, two, or three of glutamine, 2-oxoglutarate, and glutamic acid compared to a control yeast strain.

[0122] In some cases, the host cell supports the activity of the 2-oxoglutarate-dependent enzymes T6ODM and CODM, thus making the additional co-substrate 2-oxoglutarate available to heterologous enzymes of the host cell's metabolism (see, e.g., Figure 16). Such modifications of interest to the host cell genotype are shown in Table 3 and may include one or more of the following: (1) overexpression of either a native or heterologous glutamate dehydrogenase enzyme (GDH) to produce 2-oxoglutarate from glutamate, (2) deletion of glutamine synthase (GLN1) to prevent loss of glutamate in glutamine production, and / or deletion of glutamate synthase (GLT1) to prevent loss of 2-oxoglutarate in glutamate production, (3) overexpression of glutamate from 2-oxoglutarate, and (4) overexpression of glutamate from glutamate. (4) deletion of one or several glutamate dehydrogenase genes (GDH1, GDH2, GDH3) to prevent reversible conversion to acid and ammonia; (5) deletion of one or several 2-oxoglutarate dehydrogenase genes (KGD1, KGD2, LPD1) to block the loss of 2-oxoglutarate to succinyl-CoA; and (6) one or several mitochondrial 2-oxoglutarate transporters (including but not limited to ODC1 and ODC2) can be overexpressed or knocked out in the host cell.

[0123] In some cases, the host cell comprises one or more coding sequences for one or more proteins selected from GLN1, GLT1, GDH1, GDH2, GDH3, ODC1, ODC2, KGD1, KGD2, and LPD1.

[0124] In certain cases, promoters that regulate the expression of pathway genes in a production strain are placed adjacent to each other and act in opposite directions, so that the two genes placed side by side are expressed from the sense and antisense strands (for example, as shown in Figure 10). Gene cassettes containing promoters, genes, and terminators can be aligned in pairs using such back-to-back promoter designs to increase expression and result in a corresponding increase in morphine production compared to the same cassettes placed in one direction. The DNA construct of interest with such an arrangement can be integrated into episomal DNA, chromosomal DNA, or both. In some cases, when there is an odd number of gene cassettes, the promoters of the unpaired genes are placed adjacent to the vector or chromosomal DNA.

[0125] In some embodiments, the host cell comprises one or more enzymes that comprise localization tags.In certain embodiments, one or more enzymes in the host cell are spatially localized in a compartment within the host cell.In certain cases, the host cell is a yeast cell.Any convenient location within the host cell can be used for the localization of one or more enzymes.In certain cases, the compartment of the host cell is selected from mitochondria, endoplasmic reticulum (ER), Golgi, vacuole, nucleus, plasma membrane, and periplasm.

[0126] In some cases, one or more enzymes are spatially located outside the compartment in yeast cells.In certain cases, one or more enzymes are spatially located inside the compartment in yeast cells.In some cases, one or more enzymes are COR.In some cases, the COR enzyme is located in mitochondria in yeast cells.In some embodiments, the host cell comprises COR and T6ODM enzymes that are spatially separated from each other in the cell.

[0127] In some cases, when T6ODM and CODM share at least about 80% (for example, 80.1%) similarity with each other and are expressed from the same DNA construct in host cells, the genes are arranged back to back, so that one is expressed from the sense strand and the other is expressed from the antisense strand.This design can enhance the stability of the two sequences that share homology.

[0128] In some cases, uptake or retention can be improved by altering the movement of metabolites between cells and the culture medium. For a single conversion, such as thebaine uptake and its conversion to neopinone by T6ODM (and spontaneous rearrangement to codeinone), including 2% or less dimethyl sulfoxide (DMSO) in the culture medium can enhance metabolite exchange within the culture medium, thereby allowing more substrate to be taken up by the cells and more product to be released. For two or more conversions, plasma membrane transporters in the ATP-binding cassette class can be deleted, allowing intermediates in the heterologous pathway to be retained within the host cells of interest. This supports pathway flow, resulting in enhanced production of the end product. Alternatively, in some cases, transporters can be temporally regulated, such that expression is turned off during exponential growth phase to retain intermediates and then increased during stationary phase to release the end metabolite into the culture medium.

[0129] In certain embodiments, a morphinone reductase (morB) M10 enzyme (e.g., from P. putida) is expressed in a host cell along with T6ODM to generate a host cell strain that produces hydrocodone (see, e.g., Figure 20). The morB enzyme can contain an E160G mutation.

[0130] In another embodiment of the invention, morphine dehydrogenase (morA) (e.g., from P. putida M10) is expressed in a host cell together with morB. Such host cells produce hydromorphone from morphine both in vitro (e.g., in crude lysates) and in vivo (e.g., in live cells). In certain cases, morA contains a C81S mutation. In some embodiments, morA mutants and morB are expressed together in a host cell along with one or more of T6ODM, COR, and CODM, thereby producing hydromorphone from thebaine. In certain embodiments, mutants of one or more of T6ODM, COR, CODM, morA, and morB are expressed in a thebaine-producing strain to form a complete biosynthetic pathway for hydromorphone (Figure 20).

[0131] In another embodiment of the present invention, morA has an increased supply of the cofactors NADP+ / NADPH to support the activity of this enzyme. In certain embodiments, nitrogen metabolism within the host cell is altered so that NADPH-dependent ammonia assimilation is reduced and replaced by NADH-dependent assimilation. Such host cells can contain any one or more of the following genetic modifications: deletion of NADPH-dependent GDH1, deletion of NADPH-dependent GDH3, overexpression of NADH-dependent GDH2, or overexpression of a heterologous glutamate dehydrogenase (Table 3).

[0132] Table 1. Host cell lines of interest TIFF2026015382000005.tif172163

[0133] In some embodiments, the host cell is selected from one of yeast strains 1-7 listed in Table 1. In certain embodiments, the host cell is yeast strain 1, which contains a heterologous coding sequence and is capable of producing a compound as listed in its entry in Table 1. In certain embodiments, the host cell is yeast strain 2, which contains a heterologous coding sequence and is capable of producing a compound as listed in its entry in Table 1. In certain embodiments, the host cell is yeast strain 3, which contains a heterologous coding sequence and is capable of producing a compound as listed in its entry in Table 1. In certain embodiments, the host cell is yeast strain 4, which contains a heterologous coding sequence and is capable of producing a compound as listed in its entry in Table 1. In certain embodiments, the host cell is yeast strain 5, which contains a heterologous coding sequence and is capable of producing a compound as listed in its entry in Table 1. In certain embodiments, the host cell is yeast strain 6, which contains a heterologous coding sequence and is capable of producing a compound as listed in its entry in Table 1. In certain embodiments, the host cell is yeast strain 7, which contains a heterologous coding sequence and is capable of producing a compound as listed in its entry in Table 1. In some embodiments, the host cell is selected from one of the yeast strains listed in Table 4. In particular embodiments, the host cell is yeast strain CSY905 (e.g., as described herein). In particular embodiments, the host cell is yeast strain CSY906 (e.g., as described herein). In particular embodiments, the host cell is yeast strain CSY950 (e.g., as described herein). In particular embodiments, the host cell is yeast strain CSY951 (e.g., as described herein). In particular embodiments, the host cell is yeast strain CSY952 (e.g., as described herein).

[0134] Table 2. Genes used as components of engineered metabolic pathways in yeast. TIFF2026015382000006.tif236146TIFF2026015382000007.tif236160TIFF2026015382000008.tif23760

[0135] Table 3. Modification of host cell metabolic processes TIFF2026015382000009.tif23782TIFF2026015382000010.tif70170

[0136] Table 4. Engineered S. cerevisiae strains used Abbreviations: P, promoter; T, terminator. * CSY907 is repeated in this table to demonstrate its use as a control strain. TIFF2026015382000011.tif110170TIFF2026015382000012.tif190170

[0137] method As summarized above, aspects of the present invention include methods for preparing a benzylisoquinoline alkaloid (BIA) of interest. Accordingly, aspects of the present invention include culturing a host cell under conditions suitable for protein production, such that a heterologous coding sequence is functionally expressed and converts a starting compound of interest into the BIA compound of interest.

[0138] In some cases, the method is a method for preparing a benzylisoquinoline alkaloid (BIA) that includes culturing a host cell (e.g., one described herein) under conditions suitable for protein production; adding a starting compound to the cell culture; and recovering the BIA from the cell culture.

[0139] In some embodiments of the method, the starting compound, BIA product, and host cell are described by one of the entries in Table 1. In certain embodiments, the host cell is described by one of the strains in Table 4. In certain embodiments, the host cell comprises one or more heterologous coding sequences for one or more enzymes listed in Table 2.

[0140] Any convenient method of culturing host cells may be used for producing the BIA of interest. The specific protocol used may vary depending, for example, on the host cell, the heterologous coding sequence, the desired BIA, etc. The cells may be present in any convenient environment, e.g., an environment in which the cells can express one or more functional heterologous enzymes. As used herein, in vitro simply refers to outside of a living cell, regardless of the location of the cells. As used herein, the term in vivo refers to inside a cell, regardless of the location of the cells. In some embodiments, cells are cultured under conditions that allow expression of the enzymes, with appropriate substrates available to enable production of the BIA in vivo. In some embodiments, functional enzymes can be extracted from the host for production of the BIA under in vitro conditions. In some cases, the host cells can be returned to a multicellular host organism. The host cells can be in any growth phase, including, but not limited to, stationary phase and log phase. Furthermore, the culture itself may be a continuous culture, or they may be a batch culture.

[0141] Any convenient cell culture conditions can be used for a particular cell type. In certain embodiments, host cells containing various heterologous coding sequences can be cultured under standard or easily optimized conditions, including standard cell culture media and supplements. As an example, a standard growth medium, when no selective pressure for plasmid maintenance is required, can contain 20 g / L yeast extract, 10 g / L peptone, and 20 g / L dextrose (YPD). Plasmid-containing host cells can be grown in synthetic complete (SC) medium containing 1.7 g / L yeast nitrogen base, 5 g / L ammonium sulfate, and 20 g / L dextrose, supplemented with the appropriate amino acids required for growth and selection. Alternative carbon sources that may be useful for inducible enzyme expression include, but are not limited to, sucrose, raffinose, and galactose. Cells can be grown in the laboratory at any convenient temperature (e.g., 30° C.) in vessels, such as test tubes or flasks, in volumes ranging from 1 to 1000 mL or more, with shaking at any convenient speed (e.g., 200 rpm). Culture volumes can also be scaled up for growth in larger fermentation vessels, for example, as part of an industrial process.

[0142] Any convenient codon optimization technique for optimizing expression of heterologous polynucleotides in host cells may be adapted for use in the subject host cells and methods, see, e.g., Gustafsson, C. et al. (2004) Trends Biotechnol, 22, 346-353, which is incorporated herein by reference in its entirety.

[0143] The subject method also includes adding a starting compound to the cell culture. Any convenient addition method may be adapted for use in the subject method. The cell culture may be supplemented with a sufficient amount of the starting material of interest (e.g., as described herein), e.g., a mM to μM amount, e.g., about 1-5 mM starting compound. It is understood that the amount of starting material added, the timing and rate of addition, the form of the material added, etc., may vary depending on various factors. The starting material may be added neat, e.g., with water, or may be pre-dissolved in an appropriate solvent (e.g., cell culture medium, water, or an organic solvent). The starting material may be added in a concentrated form (e.g., 10 times or more the desired concentration) to minimize dilution of the cell culture medium upon addition. The starting material may be added in one or more batches, or may be added over a longer period of time (e.g., several hours or several days) by continuous addition.

[0144] The subject method also includes recovering BIA from cell culture.Any convenient separation and isolation method (for example, chromatography or precipitation) can be adapted for use in the subject method to recover the BIA of interest from cell culture.Filtration method can be used to separate the soluble fraction from the insoluble fraction of cell culture.In some cases, liquid chromatography method (for example, reverse-phase HPLC, size exclusion chromatography, normal-phase chromatography) is used to separate BIA from other soluble components of cell culture.

[0145] Also included are methods for engineering host cells to produce a BIA of interest. Insertion of DNA into a host cell can be accomplished using any convenient method. The method is used to insert a heterologous coding sequence into the host cell, which then functionally expresses the enzyme and converts the starting compound of interest into the BIA product of interest.

[0146] Any convenient promoter can be used in the subject host cell and method.The promoter that drives the expression of heterologous coding sequence can be a constitutive promoter or an inducible promoter, provided that the promoter is active in host cell.The heterologous coding sequence can be expressed from its native promoter, or a non-native promoter can be used.Such promoters can be low to high in the host they are used in.The promoter can be regulated or constitutive.In certain embodiments, a promoter that is not repressed by glucose or is only lightly repressed by the presence of glucose in the culture medium is used. Promoters of interest include, but are not limited to, promoters of glycolytic genes, such as the promoter of the Bacillus subtilis tsr gene (encoding fructose bisphosphate aldolase), or the GAPDH promoter from the yeast S. cerevisiae (encoding glyceraldehyde phosphate dehydrogenase), the ADH1 promoter from baker's yeast, phosphate-starvation-inducible promoters, such as the yeast PHO5 promoter, the alkaline phosphatase promoter from B. licheniformis, yeast-inducible promoters, such as Gal1-10, Gal1, GalL, and GalS, repressible promoters Met25 and tetO, and constitutive promoters, such as the glyceraldehyde 3-phosphate dehydrogenase promoter (GPD), alcohol dehydrogenase promoter (ADH), translation elongation factor-1-α promoter (TEF), cytochrome c-oxidase promoter (CYC1), and MRP7 promoter. Autonomously replicating yeast expression vectors containing promoters inducible by hormones such as glucocorticoids, steroids, and thyroid hormones can also be used, including, but not limited to, 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 inducible promoters, such as α-factor, alcohol oxidase, and PGH, can also be used. Furthermore, any promoter / enhancer combination (according to the eukaryotic promoter database EPDB) can also be used to drive gene expression. Any convenient, suitable promoter can be selected for the host cell, e.g., E. coli. Promoter selection can also be used to optimize transcript, and therefore enzyme, levels to maximize production while minimizing energy sources.

[0147] Any convenient vector can be used in the target host cell and method.Vector of interest includes the vector for use in yeast and other cells.Yeast vector can be divided into four general categories: integration vector (YIp), autonomously replicating high copy number vector (YEp), autonomously replicating low copy number vector (YCp), and vector for cloning large fragment (YAC).Vector DNA can be introduced into prokaryotic or eukaryotic cells by any convenient transformation or transfection technique.

[0148] usefulness The host cells and methods of the invention, e.g., as described above, find use in a variety of applications. Applications of interest include, but are not limited to, research and therapeutic applications. The methods of the invention find use in a variety of different applications, including any advantageous application in which the production of a BIA is of interest.

[0149] The subject host cells and methods are used in a variety of therapeutic applications. Therapeutic applications of interest include those involving the preparation of pharmaceuticals containing BIAs. Thus, the subject host cells are used to provide therapeutically active BIAs or their precursors. In some cases, the host cells and methods are used to produce commercial-scale quantities of BIAs where chemical synthesis of these compounds is low-yielding and not an effective means for large-scale production. In certain cases, the host cells and methods are used in fermentation facilities containing bioreactors (fermentors) with capacities of, for example, 5,000 to 200,000 liters, enabling rapid production of the BIAs of interest for therapeutic products. Such applications may include industrial-scale production of the BIAs of interest from fermentable carbon sources such as cellulose, starch, and free sugars.

[0150] The subject host cells and methods are used in a variety of research applications. The subject host cells and methods can be used to analyze the effects of various enzymes on the biosynthetic pathways of various BIAs of interest. Furthermore, host cells can be engineered to produce BIAs for use in testing for bioactivity of interest in as-yet-undefined therapeutic functions. In some cases, engineering host cells to contain various heterologous coding sequences encoding various enzymes elucidates high-yield biosynthetic pathways toward the BIA of interest or its precursors. In certain cases, research applications include the production of precursors for therapeutic molecules of interest that can be further chemically modified or derivatized into desired products, or the production of precursors to screen for improved therapeutic activity of interest. In some cases, host cell strains are used to screen for enzymatic activity of interest in such pathways, allowing enzymes to be discovered through the conversion of BIA metabolites produced in these strains.

[0151] The subject host cells and methods can be used as production platforms for plant-specific metabolites.

[0152] The subject host cells and methods can be used as a platform for drug library development and plant enzyme discovery. For example, the subject host cells and methods can be used to develop natural product-based drug libraries by obtaining yeast strains that produce interesting scaffold molecules, such as protopine, and then further functionalizing the compound's structure through combinatorial biosynthesis or chemical means. By producing drug libraries in this manner, all potential hit drugs are already associated with a production host that is amenable to large-scale cultivation and production. As another example, these subject host cells and methods can be used for plant enzyme discovery. The subject host cells provide a clear background of defined metabolites and express plant EST libraries to identify novel enzyme activities. The subject host cells and methods provide expression methods and culture conditions for functional expression and increased activity of plant enzymes in yeast.

[0153] 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 host cells, starting compounds, heterologous coding sequences, vectors, culture media, etc., as described herein. In some embodiments, the subject kits include host cells (as described herein), and one or more components selected from starting compounds, heterologous coding sequences, and / or vectors comprising same, and culture media.

[0154] Any of the components described herein, e.g., host cells containing one or more heterologous coding sequences, starting compounds, components suitable for use in expression systems (e.g., cells, cloning vectors, multiple cloning sites (MCSs), bidirectional promoters, internal ribosome entry sites (IRESs), etc.), culture media, etc., can be provided in a kit. Various components suitable for making and using heterologous coding sequences, cloning vectors, and expression systems can be used in the subject kits. Kits can also include tubes, buffers, etc., and instructions for use. The various reagent components of the kit can be present in separate containers, or, if desired, some or all of them can be precombined into a reagent mixture in a single container.

[0155] In some cases, the kit includes a host cell selected from a reticuline-producing host cell, a sanguinarine precursor-producing host cell, a protoberberine-producing host cell, a thebaine-producing host cell, and an opiate-producing host cell. The host cell may include one or more heterologous coding sequences (e.g., those described herein). In certain cases, the cell expresses a BIA of interest (e.g., those described herein).

[0156] Aspects of the invention include systems for producing a BIA of interest, which may include engineered host cells containing heterologous coding sequences (e.g., those described herein), starting compounds, culture media, fermentors, and fermentation equipment, such as equipment suitable for maintaining growth conditions for the host cells, sampling, and monitoring the equipment and components. A variety of components suitable for use in large-scale fermentation of yeast cells can be used in the subject systems.

[0157] In some cases, the system includes components for large-scale fermentation of engineered host cells and for monitoring and purifying the BIA compounds produced by the fermented host cells. In certain embodiments, one or more starting compounds (e.g., those described herein) are added to the system under conditions in which the engineered host cells in the fermentor produce one or more desired BIA products. In certain cases, the BIA product of interest is an opioid product, such as codeine, neopine, morphine, neomorphine, hydrocodone, oxycodone, hydromorphone, dihydrocodeine, 14-hydroxycodeine, or dihydromorphine.

[0158] In some cases, the system includes a means for monitoring and / or analyzing one or more BIA compounds produced by the subject host cells. For example, samples may be analyzed using an LC-MS analysis system, a chromatography system, or any convenient system described herein, and compared to standards, e.g., as described herein. The fermentation medium may be monitored by sampling and analysis before and at any convenient time during fermentation. Upon completion of conversion of the starting compounds to the BIA product of interest, fermentation may be stopped and purification of the BIA product may occur. Thus, in some cases, the subject system includes purification components suitable for purifying the BIA product of interest from the host cell medium in which the BIA product is produced. The purification components may include any convenient means that can be used to purify the BIA product of fermentation, including, but not limited to, silica chromatography, reverse-phase chromatography, ion exchange chromatography, HIC chromatography, and size exclusion chromatography. In some cases, the subject system provides for the production and isolation of the BIA fermentation product of interest after input of one or more starting compounds into the system.

[0159] The following examples are put forth to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation will occur. Unless otherwise specified, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. [Example]

[0160] experiment I. Reticuline-producing yeast strains We developed S. cerevisiae strains with improved reticuline production through overexpression of 6OMT, CNMT, or 4'OMT genes from several different species.

[0161] Figure 1 shows the in vivo methylation of norlaudanosoline by methyltransferases from P. somniferum and Thalictrum flavum. In each panel, host cell cultures expressing each methyltransferase individually were grown in the presence of norlaudanosoline, and the methylation products were detected using LCMS. The data demonstrate that methyltransferases have broad substrate specificity and support alternative methylation pathways. (a) 6'OMT enzymes from both species exhibit similar methylation activity toward norlaudanosoline. Fragmentation of the methylation product is shown in the inset. (b) PsCNMT exhibits higher methylation activity toward norlaudanosoline than TfCNMT as a substrate. Fragmentation of the methylation product is shown in the inset. (c) 4'OMT enzymes from both species exhibit similar methylation activity toward norlaudanosoline. Fragmentation of the methylation product is shown in the inset.

[0162] Figure 2 shows an alternative methylation pathway from norlaudanosoline to reticuline. If the starting material of the pathway is norlaudanosoline (1), 6OMT, CNMT, or 4'OMT can act on this compound, thereby generating three distinctly methylated intermediates. BIA2, initially methylated by 6OMT, can then be methylated by either CNMT or 4'OMT. Similarly, BIA3, initially methylated by CNMT, can then be methylated by either 6OMT or 4'OMT. Similarly, BIA4, initially methylated by 4'OMT, can then be methylated by either 6OMT or CNMT. BIA5, previously methylated by 6OMT and 4'OMT, can be methylated by CNMT to produce reticuline. BIA6, previously methylated by 6OMT and CNMT, can be methylated by 4'OMT to produce reticuline. BIA7, previously methylated by CNMT and 4'OMT, can be methylated by 6OMT to produce reticuline.

[0163] Figure 3 shows an alternative methylation pathway from norcoclaurine to reticuline. When the starting material of the pathway is norcoclaurine, either 6OMT or CNMT can act on this compound, resulting in two distinctly methylated products. BIA10, initially methylated by 6OMT, can then be methylated by CNMT. BIA11, initially methylated by CNMT, can then be methylated by 6OMT. BIA12, also known as N-methylcoclaurine, previously methylated by 6OMT and CNMT, is then sequentially acted upon by CYP80B1 and 4'OMT to produce reticuline.

[0164] N-methylcoclaurine production: Gradual increase in PsCNMT expression The production of N-methylcoclaurine (BIA 12) was measured when the PsCNMT gene copy number was varied in a strain fed norcoclaurine with either Tf6OMT or Ps6OMT. The production of N-methylcoclaurine (BIA 12) is shown when the PsCNMT gene copy number was varied in a strain fed norcoclaurine with either Tf6OMT or Ps6OMT. Crude cell lysates from expression in host cells containing either Ps6OMT or Tf6OMT and one or two copies of PsCNMT were incubated with norcoclaurine. The doubly methylated product, N-methylcoclaurine, was measured using LCMS. When 2×PsCNMT was used instead of 1×PsCNMT with Ps6OMT, the number of ions was 2×10. 7 to 5.5 x 10 7 When 2×PsCNMT was used instead of 1×PsCNMT with Tf6OMT, the number of ions increased to 1.5×10 7 From 6 x 10 7 The data demonstrate that higher gene copy numbers of the methyltransferase CNMT can increase the production of N-methylcoclaurine.

[0165] II. Sanguinarine precursor-producing yeast strains We developed strains that produce protoberberine and benzophenanthridine alkaloids, including cheilanthifoline, stylopine, cis-N-methylstylopine, protopine, and dihydrosanguinarine.

[0166] Figure 4 shows the synthetic pathway present in a host cell for producing sanguinarine from reticuline. While the pathway can be longer if starting from norlaudanosoline or norcoclaurine, as shown in other figures, this particular pathway depiction begins with reticuline and ends with sanguinarine. The pathway may contain fewer enzymes than those shown if the desired end result is one of the intermediates in the norlaudanosoline to sanguinarine pathway. Addition of multiple enzymatic steps, specifically steps catalyzed by the enzymes CFS, STS, TNMT, MSH, P6H, and DBOX, within engineered yeast strains produces various protoberberine and benzophenanthridine compounds.

[0167] 1. Introduction Microbial production hosts for several protoberberine and protopine alkaloids in the sanguinarine branch of the BIA biosynthetic pathway have been engineered. Specifically, yeast strains were engineered to produce the compounds cheilanthifoline (4), stylopine (5), (S)-cis-N-methylstylopine (6), and protopine (7) from the fed substrate norlaudanosoline (1) (Figure 11). These pathways demonstrate complex plant natural product pathways that have been reconstructed in microbial hosts based on the number and type of heterologous enzymes. In particular, a pathway containing eight enzymatic steps, including three catalyzed by cytochrome P450s, and a total of 11 heterologous expression cassettes was demonstrated. Several pathway optimization strategies were developed for the reconstruction of these complex pathways in yeast to provide an efficient microbial platform for the biosynthesis of plant-specific metabolites. In particular, strategies were used to support the functional expression of multiple plant cytochrome P450 enzymes in the context of many multistep pathways, including balancing expression, harmonizing CPRs, and optimizing culture conditions. These general design strategies can be applied more broadly to aid in the engineering of diverse plant natural product pathways in yeast.

[0168] Figure 11 shows the engineering of protoberberine and protopine alkaloid biosynthesis in S. cerevisiae. An optimized protopine-producing strain is fed the precursor molecule norlaudanosoline, which is converted to the key branch point intermediate reticuline by three chromosomally integrated methyltransferase enzymes (Ps6OMT, PsCNMT, and Ps4'OMT). A chromosomally integrated berberine bridge enzyme (PsBBE) converts (S)-reticuline to (S)-scoureline. Two copies of the cytochrome P450 cheilanthifoline synthase (EcCFS) and stylopine synthase (EcSTS), one chromosomally integrated and one expressed from a yeast artificial chromosome, were co-expressed with one copy of the cytochrome P450 NADPH reductase (ATR1) from Arabidopsis thaliana, to produce (S)-cheilanthifoline and (S)-stylopine compounds, respectively. Tetrahydroprotoberberine-N-methyltransferase (PsTNMT) then converts (S)-stylopine to (S)-cis-N-methylstylopine, and then cytochrome P450 cis-N-methylstylopine 14-hydroxylase (PsMSH) converts cis-N-methylstylopine to protopine. Both PsTNMT and PsMSH were expressed from yeast artificial chromosomes. The final engineered protopine-producing strain contains 11 heterologous expression cassettes (seven integrated enzymes and four enzymes expressed from yeast artificial chromosomes): cytochrome P450 (EcSTS, PsMSH, EcCFS), cytochrome P450 reductase (ATR1), and other enzyme classes (Ps6OMT, PsCNMT, Ps4OMT, PsBBE, PsTNMT).

[0169] 2. Results a. Engineering microbial strains that produce optimized scaffolds for protoberberine and protopine alkaloids In a yeast production host, the reconstruction of the sanguinarine branch of the BIA biosynthetic pathway, which encompasses several protoberberine and protopine alkaloids, was targeted. The sanguinarine branch consists of 10 enzymatic steps (four of which are catalyzed by plant cytochrome P450s) to reach sanguinarine (the pathway's end product) from norlaudanosoline (a commercially available, fed substrate). Several downstream enzymes in this pathway were cloned and characterized, enabling the engineering of microbial production strains for intermediate metabolites along the sanguinarine branch.

[0170] The BIA metabolite scourerine is the backbone from which the protoberberine and protopine alkaloid structures in the sanguinarine branch can be derived. A yeast strain previously engineered to produce scourerine from the fed substrate norlaudanosoline was used (Hawkins, K. & Smolke, C. Production of benzylisoquinoline alkaloids in Saccharomyces cerevisiae. Nat. Chem. Biol. 4, 564-573 (2008)). This strain expresses four plant enzymes: three methyltransferases (Papaver somniferum norcoclaurine 6-O-methyltransferase, Ps6OMT; P. somniferum coclaurine-N-methyltransferase, PsCNMT; and P. somniferum 3'hydroxy-N-methylcoclaurine 4'-O-methyltransferase, Ps4'OMT) contained expression cassettes integrated into the chromosome, and an expression cassette encoding the enzyme that converts the key branch point metabolite reticuline to the scoulerine skeleton (P. somniferum berberine bridge enzyme, PsBBE) was placed on a high-copy plasmid.

[0171] To optimize the strain for increased flux through the sanguinarine branch, the PsBBE expression cassette was integrated into the yeast chromosome to increase the production of scourerine. The strain was fed 4 mM norlaudanosoline and grown for 96 hours. Samples of the growth medium were analyzed using high-pressure liquid chromatography coupled with mass spectrometry (LC-MS), and the production of scourerine was confirmed by comparing the fragmentation pattern (MS-MS) with that reported (Schmidt, J. & Raith, K. Analysis of benzylisoquinoline-type alkaloids by electrospray tandem mass spectrometry and atmospheric pressure photoionization. Eur. J. Mass Spectrom. 11, 325-333 (2005)). By shifting expression of PsBBE from a high-copy plasmid to the chromosome, scourerine production increased threefold to 1.5 mg / L, and the conversion efficiency of reticuline to scourerine improved from 20% to 64%.

[0172] b. Engineering an optimized microbial production host for cheilanthifoline The first step in the sanguinarine branch is the conversion of the backbone molecule, scourerine, to cheilanthifoline by the enzyme cheilanthifoline synthase (CFS), the first cytochrome P450 in this pathway (Figure 12a). Because functional expression of plant cytochrome P450s in microbial hosts has been challenging, various strategies have been explored to support functional heterologous expression of this P450 enzyme and optimize its activity in yeast.

[0173] We first investigated the pairing of various plant cytochrome CPRs with CFS mutants expressed at various levels. Codon-optimized CFS mutants from three native plant hosts were synthesized: California Poppy (EcCFS), American Poppy (AmCFS), and P. somniferum (PsCFS). The CFS mutants were expressed on either high-copy or low-copy plasmids from a strong yeast promoter (pGPD). Three plant CPR expression cassettes encoding Arabidopsis (ATR1), California Poppy (EcCPR), and P. somniferum (PsCPR) CPRs were integrated into yeast chromosomes and tested with each CFS mutant. While EcCPR and PsCPR originate from the same plant species as the CFS mutants, plants possess multiple CPR mutants, and the specific CPR selected may not support CFS activity. Although ATR1 originates from a plant species that does not produce BIA molecules, this CPR was used to support heterologous P450 activity in yeast (Urban, P., Mignotte, C., Kazmaier, M., Delorme, F. & Pompon, D. Cloning, yeast expression, and characterization of the coupling of two distantly related Arabidopsis thaliana NADPH-cytochrome P450 reductases with P450 CYP73A5. J. Biol. Chem. 272, 19176-86 (1997)). Strains harboring all combinations of CPR and CFS mutants were fed 2 mM norlaudanosoline and grown for 96 hours. Samples of the growth medium were analyzed using LC-MS. Cheiranthifoline production was confirmed through comparison of the observed fragmentation pattern with reported fragmentation patterns (Figure 12b).

[0174] The data demonstrate that specific CPR pairing and P450 expression levels can substantially affect the functional activity of plant P450s in heterologous microbial hosts. Specifically, the activity of all CFS mutants supported by ATR1 was 20-50 times better than any other CPR mutant tested, producing 600 μg / L or less of cheilanthifoline (Figure 12c). Native yeast CPRs and EcCPR were able to support low levels of activity from EcCFS and AmCFS, whereas PsCPR did not couple with all expressed P450s. All CFS mutants produced cheilanthifoline when paired with the appropriate CPR, but EcCFS and AmCFS were substantially more active than PsCFS. The activity differences observed between EcCFS and AmCFS mutants in yeast are consistent with the K values ​​reported for these enzymes. m This is supported by the difference in values ​​(EcCFS, 900 nM; AmCFS, 1.9 μM; PsCFS, K m (Not reported.) The data also demonstrate that cheilanthifoline production levels were substantially higher when the CFS mutants were expressed from low-copy plasmids than when they were expressed from high-copy plasmids.

[0175] P450s naturally localize to the endoplasmic reticulum (ER), and overexpression of these enzymes in yeast can trigger a stress response in which the ER membrane proliferates. Activity data suggest that high-level expression of plant P450s in yeast may overwhelm the ER and impair the activity of the enzymes. To examine the concentration and subcellular localization of P450s as a function of expression level in live cells, we tagged the C-terminus of CFS mutants in high- and low-copy plasmids with EGFP and analyzed cells harboring these constructs by confocal microscopy. ER localization of the tagged enzymes was confirmed by colocalization with the ER marker DsRed-Kar2-HDEL (Figure 12d).

[0176] Two major differences were observed between cells expressing tagged CFS variants from high- or low-copy plasmids. First, a significant decrease in the number of GFP-positive cells was observed in cells carrying the P450-GFP fusion construct compared to the GFP-only construct, and the difference was more significant when the fusion was expressed from a high-copy plasmid (27%) than from a low-copy plasmid (56%). The data suggest that the ability of cells to maintain the plasmid expressing the cytochrome P450 enzyme is reduced compared to a plasmid expressing a fluorescent reporter, and that expression of plant P450s from a low-copy plasmid may result in more stable expression across the cell population and, therefore, higher protease activity. Second, the ER morphology was clearly different between cells carrying the P450-GFP expression cassette on a high- or low-copy plasmid. In cells carrying the high-copy plasmid, the P450-GFP fusion protein was generally observed in dense bright patches adjacent to the nucleus or plasma membrane. In contrast, in cells harboring low-copy plasmids, the ER membrane was still distributed throughout the cell, and the GFP fluorescence level was faint, indicating a low concentration of P450. Thus, under conditions in which plant P450s were expressed at low levels, the ER membrane morphology in these engineered cells was more similar to that of wild-type cells. In summary, the confocal microscopy and functional activity data suggest that high-level expression of plant P450s can cause extreme ER proliferation, which is highly stressful for the yeast host and detrimental to the activity of the enzyme. Therefore, the optimal expression strategy for plant P450s in heterologous yeast hosts is from a low-copy plasmid or by stable integration into the chromosome (Figure 12e).

[0177] Finally, we investigated the effects of plant P450 expression level and regulation on cheilanthifoline production in a yeast host. Cheilanthifoline production was examined from engineered pathway mutants in which CFS was expressed under the control of five different promoters on a low-copy plasmid: pGPD (level: strong, regulation: early), pTEF1 (strong, constitutive), pPGK1 (medium, early), pTPI1 (medium, early), and pHXT7 (strong, late). As before, strains harboring the indicated constructs were fed 2 mM norlaudanosoline and grown for 96 h under the indicated conditions. Growth medium samples were analyzed using LC-MS. The GPD promoter resulted in up to a 12-fold improvement in cheilanthifoline production compared to the other promoters tested (Figure 12f). The data indicate that both expression level and regulation strategy play a role in optimizing the activity of plant P450s in heterologous yeast hosts. For example, the TEF promoter exhibited a similar strength but a different regulatory profile to pGPD, and pPGK1 exhibited a similar regulatory profile but a different strength to pGPD; however, each of these promoters resulted in different cheilanthifoline production levels. These results demonstrate that an optimized cheilanthifoline-producing yeast strain expresses the plant P450 EcCFS at low levels (either from a low-copy plasmid under the control of the GPD promoter or integrated into the chromosome), and that this plant P450 has been engineered to pair with the ATR1 CPR. This engineered strain produced up to 600 μg / L of cheilanthifoline and was able to convert up to 38% of it to scourerine.

[0178] Figure 12 shows the microbial production of (S)-cheilanthifoline. (a) Schematic showing the conversion of norlaudanosoline to (S)-cheilanthifoline, highlighting the optimized enzymatic steps. Dashed arrows indicate multiple enzymatic steps. The diagram designation follows that shown in Figure 11. (b) LC-MS analysis of the growth medium of a yeast strain fed with 2 mM norlaudanosoline and grown for 96 hours. The LC-MS trace shows that the vector control strain (left) produces scourerine (peak 3) but no peak at 326 EIC. When the EcCFS enzyme is expressed (right), a peak is detected at 326 EIC (peak 4), and fragmentation (MS-MS, below) confirms the identity of the metabolite as cheilanthifoline. (c) Production of (S)-cheilanthifoline depends on pairing of the enzyme mutant with a cytochrome P450 NADPH reductase partner. Mutant cheilanthifoline synthase (CFS) from California poppy (EcCFS), California poppy (AmCFS), and P. somniferum (PsCFS) were expressed from low-copy plasmids in yeast strains, along with cytochrome P450 reductase enzymes (CPR) from either native yeast or various plant sources (Arabidopsis, California poppy, and P. somniferum) integrated into the TRP locus. (d) P450 expression levels result in distinctly different ER morphologies. EcCFS tagged with GFP at the C-terminus on a high-copy (top) or low-copy (middle) plasmid localizes to the endoplasmic reticulum but exhibits distinctly different ER growth morphologies. Wild-type ER (no heterologous P450 expressed) is shown for comparison (bottom). The ratios indicate the proportion of the yeast population that is GFP-positive under the indicated expression conditions. (e) Stable expression of CFS (low-copy plasmid or chromosomal integration) improves cheilanthifoline production and scourerine transformation efficiency. EcCFS was expressed on a high-copy plasmid (2 μm, TRP selection marker), a low-copy plasmid (CEN / ARS, TRP selection marker), or integrated into the MET15 locus of the yeast chromosome. (f) Promoter choice affects CFS activity.EcCFS was expressed from a low-copy plasmid under the control of five different promoters (GPD, HXT7, PGK1, TEF, TPI1) with URA selection. Data in (c, d) are representative of at least three independent experiments, and data in (e, f) are reported as the mean ± standard deviation of at least three independent experiments.

[0179] c. Engineering an optimized microbial production host for stylopine The next step in the sanguinarine branch is the conversion of cheilanthifoline to stylopine by stylopine synthase (STS), a plant cytochrome P450 closely related to CFS (Figure 13). As a starting point for engineering stylopine-producing strains, we used the P450 optimization strategies elucidated with CFS; specifically, (i) expression of the plant P450 from a low-copy plasmid, (ii) control of P450 expression from the GPD promoter, and (iii) pairing of the P450 with the ATR1 CPR. However, because the biosynthetic pathway to stylopine involves two plant P450s, we investigated additional strategies to further optimize the activity of these enzymes in the context of a multistep pathway.

[0180] Pairings of plant mutants of STS and CFS were first examined to explore any synergistic interactions between specific pairings. Mutant STSs from California poppy (EcSTS), American poppy (AmSTS), and P. somniferum (PsSTS) were synthesized with yeast codon optimization. Yeast strains containing chromosomally integrated ATR1 CPR expression cassettes and various combinations of CFS and STS mutants on low-copy plasmids were fed 2 mM norlaudanosoline and grown for 96 hours. Samples of growth medium were analyzed using LC-MS. Stylopine production was confirmed through comparison of elution times and fragmentation patterns to standards (ChromoDex) and reported fragmentation patterns (Figure 13b). The data demonstrate that heterologous expression of both EcSTS and PsSTS resulted in the production of stylopine in the engineered yeast strains, while AmSTS was not active under these conditions. AmSTS has also been reported to convert scourerine to nandinine, but this product was not detected. The most productive P450 enzyme pair was EcCFS and EcSTS, which produced 14 μg / L of stylopine, an improvement of up to 6-fold over other enzyme combinations (Figure 13c). The observed activity differences between STS mutants in yeast are consistent with the reported K values ​​for these enzymes. m This is supported by the difference in values ​​(EcSTS, 400 nM; AmSTS, 5.2 μM; PsSTS, K m However, even the most productive P450 pair only converted cheilanthifoline to stylopine 25% efficiently.

[0181] Next, we investigated methods to improve the conversion efficiency of cheilanthifoline to stylopine. We investigated the effect of growth temperature on plant P450 activity in our heterologous yeast host. Yeast strains harboring biosynthetic pathways in which EcCFS was paired with each STS mutant were grown at 30°C and 25°C. As before, strains harboring the indicated constructs were fed 2 mM norlaudanosoline and grown under the indicated conditions for 96 hours. Growth medium samples were analyzed using LC-MS. Stylopine production in strains expressing EcCFS and EcSTS increased threefold to a titer of 40 μg / L when the strains were grown at 25°C compared to 30°C (Figure 13d). The data showed that the strains were more productive based on OD, implying that the improved production of stylopine was due to increased activity of the enzyme's bulk substance rather than to higher cell density. The effect on general yeast cellular processes as a result of these changes in conditions is likely to have resulted in improved folding and localization of P450s and therefore improved activity.

[0182] The gene copy number ratio of CFS to STS was examined. Experiments expressing the enzymes from a plasmid showed that a 2:2 ratio of CFS to STS resulted in a three-fold increase in stylopine production compared to a 1:1 ratio (Figure 13e). In summary, the data show that an optimized stylopine-producing yeast strain engineered to express EcCFS and EcSTS at an optimal gene copy number ratio of 2:2 produced stylopine when grown at 25°C and improved the conversion efficiency of scourerine to cheilanthifoline and cheilanthifoline to stylopine.

[0183] Figure 13 shows the optimization of (S)-stylopine production. (A) Diagram showing the conversion of norlaudanosoline to (S)-stylopine, highlighting the optimized enzymatic steps. Dashed arrows indicate multiple enzymatic steps. The diagram designation follows that shown in Figure 11. (B) LC-MS analysis was performed on the growth medium of a yeast strain fed 2 mM norlaudanosoline and grown for 96 hours. The LC-MS trace shows that the vector control strain produces cheilanthifoline (peak 4, m / z = 326) but no peak at m / z = 324 EIC. When the EcSTS enzyme is expressed, a peak is detected at m / z = 324 EIC (peak 5), which elutes at the same time as the stylopine standard. Fragmentation (MS-MS) of the stylopine standard matches that of peak 5, confirming the identity of the metabolite as stylopine. LC-MS traces were representative of at least three independent experiments. (C) Stylopine production varies with the combination of CFS and STS species variants. All pairs of CFS and STS variants were expressed from separate low-copy plasmids. (D) Growth of engineered yeast strains at 25°C improves STS activity. Each STS variant was co-expressed with EcCFS (on separate low-copy plasmids) and grown at either 25°C or 30°C. Gene copy number for CFS and STS influences stylopine production. (E) Gene copy number was varied by integrating an EcCFS expression cassette into the chromosomal MET15 locus and expressing additional copies of EcCFS and EcSTS on low-copy plasmids. All strain variants in this experiment carried three plasmids, regardless of P450 copy number, ensuring consistency in plasmid load and medium composition. The data demonstrate that increasing the copies of the two plant P450s generally increased stylopine production. The highest stylopine production resulted from two copies of EcCFS and two copies of EcSTS, which improved production levels by approximately three-fold compared to the original expression system (one copy of each enzyme).

[0184] d. Engineering a Microbial Production Host for (S)-cis-N-Methylstylopine and Protopine The next step in the pathway is the conversion of stylopine to (S)-cis-N-methylstylopine by the enzyme tetrahydroprotoberberine-N-methyltransferase (TNMT) (Figure 14a). TNMT was added to a YAC containing EcCFS and EcSTS and expressed in a strain with integrated copies of EcCFS and EcSTS. Strains harboring the indicated constructs were fed 2 mM norlaudanosoline and grown at 25°C for 96 hours, and samples of the growth medium were analyzed using LC-MS. Production of cis-N-methylstylopine was confirmed by comparison of the fragmentation pattern with that reported (Figure 14b). When TNMT was expressed in the optimized stylopine-producing strain, cis-N-methylstylopine was produced; stylopine was not detected in the medium, suggesting that TNMT is highly efficient and can reach 100% conversion efficiency.

[0185] The final step in protopine synthesis is the hydroxylation of (S)-cis-N-methylstylopine by the cytochrome P450 (S)-cis-N-methylstylopine 14-hydroxylase (MSH). Optimization techniques for cytochrome P450 were applied to MSH expression, including expression from a stable construct (in this case, a YAC) and pairing with an ATR1 reductase partner. Strains harboring the designated constructs were fed 2 mM norlaudanosoline and grown at 25°C for 96 hours. Protopine production was confirmed through comparison of elution times and fragmentation patterns with standards and reported fragmentation patterns (Figure 14c). The data indicate that MSH is a highly efficient enzyme, and thus the optimized protopine-producing strain produces protopine and achieves the conversion of cis-N-methylstylopine to protopine.

[0186] Figure 14 shows the engineering of a heterologous protopine biosynthetic pathway. (A) Diagram showing the conversion of norlaudanosoline to protopine, highlighting the optimized enzymatic steps. Dashed arrows indicate multiple enzymatic steps. Diagram designations follow those shown in Figure 11. (B) LC-MS analysis was performed on the growth medium of a yeast strain fed 2 mM norlaudanosoline and grown for 96 hours. The LC-MS trace shows that the vector control strain produces stylopine (peak 5, m / z = 324), but no peaks at m / z = 338 or 354 EIC. When the TNMT enzyme is expressed, a peak is detected at m / z = 338 EIC (peak 6), and fragmentation (MS-MS) confirms the identity of the metabolite as cis-N-methylstylopine. (C) Upon addition of MSH, a peak was detected at m / z = 354 in the EIC, which eluted at the same time as the protopine standard. The fragmentation (MS-MS) of the protopine standard matched that of peak 7 (m / z = 354), confirming the identity of the metabolite as protopine. LC-MS traces were representative of at least three independent experiments.

[0187] 5. Optimization of culture conditions for protoberberine and protopine alkaloid production To produce more cheilanthifoline or stylopine, yeast strains were constructed to overexpress cytochrome b5 and optimize cytochrome P450 activity. Cytochrome b5 was co-expressed with CFS and STS. Cheilanthifoline and / or stylopine production was measured from host cells with or without overexpressed cytochrome b5. Cultures were grown in the presence of norlaudanosoline, and products in the medium were detected using LCMS. The data demonstrate that in some cases, levels of cheilanthifoline (e.g., with AmCFS) and / or stylopine (e.g., with EcCFS / AmSTS) increased when cytochrome b5 was expressed.

[0188] To produce more protoberberine alkaloids, the yeast strain was optimized by deleting genes related to the unfolded protein response (UNSP) and endoplasmic reticulum (ER) proliferation to improve BIA production. Examples of gene deletions include IRE1, HAC1, OPI1, INO1, INO2, and INO3 (Table 3). Expression of cytochrome P450 induces the unfolded protein response (UNSP) and causes ER proliferation. Deletion of these stress response-related genes can control or reduce the overall burden on host cells and improve pathway performance.

[0189] Genes involved in pleiotropic drug responses in the host strain, including ATP-binding cassette (ABC) transporters, multidrug resistance (MDR) pumps, and related transcription factors, are deleted to reduce export of BIA molecules into the culture medium. Exemplary genes include PDR1, STB5, PDR3, PDR5, SNQ2, YOR1, TPO1, TPO2, TPO3, TPO4, PDR10, PDR11, PDR15, PDR16, PDR17, QDR1, QDR2, QDR3, FLR1, AQR1, AQR2, and CIN5. Gene deletions include single deletions or multiple deletions in any combination.

[0190] Production of BIA in transporter knockouts Measurements of reticuline, scourelin, cheilanthifoline, and stylopine production from host cells with modifications of various proteins involved in the transport of compounds across the cell membrane were performed: dPDR1, dPDR5, dsNQ2, dYOR1, dPDR3, dClN5, and dPDR1dPDR3 knockouts were compared to WT controls. Cultures were grown in the presence of norlaudanosoline, and products in the medium were detected using LCMS after 96 hours of growth. The data demonstrate that some modifications (e.g., deletions) produce higher levels of reticuline, scourelin, cheilanthifoline, or stylopine than others.

[0191] Genes involved in pleiotropic drug responses, including ATP-binding cassette (ABC) transporters, multidrug resistance (MDR) pumps, and related transcription factors, are placed under regulated (inducible or growth phase-dependent) promoters to allow for temporal control of BIA transport. One example is placing key transporter genes under the control of a stationary phase promoter, which allows BIAs to remain intracellularly until stationary phase, thereby increasing the probability of conversion of starting materials to end products.

[0192] 4. Method a. Plasmid and yeast strain construction Oligonucleotides were synthesized using conventional methods. Cloning was performed in chemically competent E. coli (TOP10, LifeTech, F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(Str R ) endA1 λ - ) was used. E. coli was cultured in Luria-Bertani medium (EMD Chemicals) containing the appropriate antibiotic (100 μg / mL ampicillin (EMD Chemicals) or 50 μg / mL kanamycin (EMD Chemicals)). Plasmids were purified from E. coli cultures using spin columns according to the manufacturer's instructions (Epoch Life Science). Sequencing was performed by Elim Biopharmaceuticals (Hayward, CA). All S. cerevisiae strains described in this study are derived from W303α (MATα leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15). A standard lithium acetate protocol was used for yeast transformation. Yeast were cultured in either YPD or the appropriate synthetic dropout medium, supplemented with 2% dextrose (w / v) for plasmid maintenance.

[0193] The gene sequences corresponding to EcCFS (BAG75113), EcCPR (AAC05022), EcSTS (BAD98250), and AmSTS (ABR14721) were codon-optimized and assembled from oligonucleotides designed using DNAWorks (Hoover, D.M. & Lubkowski, J. DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis. Nucleic Acids Res. 30, e43 (2002)). AmCFS (ABR14722), PsCFS (ADB89213), PsSTS (ADB89214), and PsMSH (AGC92398) were codon-optimized and synthesized by GeneArt (Life Technologies).

[0194] Most yeast expression vectors described in this study were constructed using Gateway Cloning Technology (Life Technologies). Enzymes were PCR amplified using PfuUltraII Fusion HS DNA polymerase (Life Technologies) or Expand High Fidelity polymerase (Roche), cleaned up using a QIAquick PCR purification kit (Qiagen), and cloned into the pENTR vector by either TOPO cloning or BP recombination using BP clonase II and the pDONR221 vector (Life Technologies). All genes were then integrated into selected pAG expression vectors from the Lindquist lab (available from Addgene) using LR clonase II (Life Technologies) (Alberti, S., Gitler, AD & Lindquist, S. A suite of Gateway® cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisiae. 913-919 (2007). doi:10.1002 / yea). Various primers and plasmids were used in this study. For assays testing various promoters with EcCFS, pCS2238 was digested with SacI / SpeI to remove the GPD promoter, and the TEF1, PGK1, TPI1, or HXT7 promoter was ligated into this site using standard ligation techniques.

[0195] Yeast artificial chromosomes (YACs) were constructed using the GeneArt Higher Order Genetic Assembly System (Life Technologies). DNA fragments were generated by PCR using primers designed with DNA Designer for Higher Order Genetic Assembly and Expand High Fidelity polymerase (Roche). DNA fragments were cleaned up using a QIAquick PCR purification kit (Qiagen), and 100 ng of each DNA fragment and the linearized pYES1L vector were transformed via electroporation into an engineered yeast strain containing the appropriate upstream enzymes. Assembled YACs were recovered from yeast cells according to the manufacturer's instructions.

[0196] For chromosomal integration, the gene of interest was integrated into pCS2643 or pCS2644 using LR clonase II (Life Technologies), and the complete integration cassette (gene expression cassette and selectable marker) was PCR amplified using appropriate integration primers and Expand High Fidelity polymerase (Roche) to add approximately 80 nucleotides of homology. PCR products from two 100 μL reactions were precipitated with ethanol and transformed into yeast using a standard lithium acetate procedure. Integration was confirmed through PCR screening across the junction of the target locus and the expression cassette. The integrated selectable marker was flanked by loxP sites to facilitate rescue of the selectable marker, and strains were transformed with pCS277(pSH63), which encodes expression of CRE recombinase (Guldener et al. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res. 24, 2519-24 (1996)). Strains were grown in YPD for 36 hours with 100-fold back-dilutions every 12 hours and then plated to single colonies. Loss of the selectable marker and plasmid was confirmed by restreaking colonies on appropriate selective media.

[0197] b. Imaging of P450 localization in live cells using confocal microscopy Yeast cell cultures were inoculated into 3 mL of selective medium and grown for 8–12 hours to an OD600 of approximately 0.1. 1 mL of culture was pelleted at 6000 rpm for 30 seconds, the supernatant discarded, and the cells resuspended in 25–50 μL of medium. 1–3 μL of culture was plated onto a 2% agarose pad made with the appropriate dropout medium to provide nutrients to the cells and facilitate live cell imaging. Live yeast cells were imaged using a Leica SP5 multiphoton / confocal microscope equipped with a 63.0x glycerol immersion objective.

[0198] c. Growth conditions for the assay Overnight yeast cultures were initiated in dropout medium containing 2% dextrose (w / v) in 3 mL test tube cultures and grown at 260 rpm at 30°C, or in 500 μL cultures in deep-well 96-well plates covered with AeraSeal film and grown at 480 rpm at 30°C and 80% humidity in a Kühner Lab-Therm LX-T 96-well plate shaker.

[0199] Overnight cultures were backdiluted 75-100 times into the appropriate dropout medium supplemented with 2-4 mM norlaudanosoline. More specifically, some assays were performed in 500 μL of culture in deep-well 96-well plates covered with AeraSeal film and grown at 30°C, 480 rpm, and 80% humidity in a Kühner Lab-Therm LX-T 96-well plate shaker. Other assays were performed using 5 mL of culture grown in 125 mL baffled flasks at 25°C and 260 rpm in a New Brunswick Scientific I24 shaking incubator. Unless otherwise noted, cultures were sampled 96 hours after backdilution.

[0200] e. Analysis of metabolite production Aliquots of yeast culture were centrifuged at 6000 rpm for 10 minutes, and growth medium samples were collected for analysis by LC-MS / MS. Samples were run on an Agilent ZORBAX SB-Aq 4.6x50 mm, 5 μm column using 0.1% acetic acid as solvent A and 0.1% acetic acid in methanol as solvent B. The following method for separation of metabolites of interest was used at a constant flow rate of 0.5 mL / min: 0-1 min, 0% to 27.5% B; 1-2 min, 27.5% B; 2-8 min, 27.5% to 60% B; 8-8.5 min, 35-100% B; 8.5-14 min, 100% B; followed by equilibration with 0% solvent B for 6 minutes. After HPLC separation, metabolites were infused into an Agilent 6320 ion trap mass spectrometer for detection and identification.

[0201] Quantitation of metabolites was based on the integrated peak areas of extracted ion chromatogram peaks calculated using data analysis on a 6300 Series Ion Trap LC / MS version 3.4 (Bruker Daltonik GmbH) and reported as mean ± standard deviation. We constructed standard curves for reticuline, stylopine, and berberine, and used the most similar standard chemical structures to estimate the concentrations of intermediates for which standards were unavailable.

[0202] III. Protoberberine-producing yeast strains We have developed S. cerevisiae strains that produce protoberberine alkaloids, including (S)-tetrahydrocolumbamine, (S)-canadine, and berberine, as intermediates or end products from (S)-scourerine or its precursors produced by existing engineered strains present in the culture medium or introduced into cell lysates or lysate fractions. More specifically, these strains express any combination of (S)-scourerine 9-O-methyltransferase (S9OMT), (S)-canadine synthase (CAS), and (S)-tetrahydroprotoberberine oxidase (STOX).

[0203] The structures of protoberberine alkaloids produced by engineered yeast strains are shown above, where -R can be -H, -CH3, -OH, or -OR. These protoberberine alkaloids are produced from reticuline or other similar chemical species produced by existing engineered strains in the culture medium or introduced into cell lysates or lysate fractions. These strains can express any combination of the following enzymes: berberine bridge enzyme (BBE), scourerine 9'-O-methyltransferase (S9OMT), canadine synthase (CAS), and S-tetrahydroprotoberberine oxidase (STOX).

[0204] Figure 5 shows the heterologous biosynthetic pathway assisted by the engineered yeast strain. The STOX gene shares 78% nucleic acid sequence identity with the naturally occurring (S)-tetrahydroprotoberberine oxidase gene from Barberry (Table 2). This gene is a non-native nucleotide sequence that has been codon-optimized for expression in yeast.

[0205] Figure 6 shows the effect of episomal gene copy number on protoberberine production in yeast culture. Preferred expression levels for the enzymes are relatively low (e.g., CEN / ARS vector or genomic expression) for BBE, CAS, and STOX (Figure 6a) and relatively high (e.g., 2 μm vector or multiple genomic copies) for S9OMT (Figure 6b). Expression levels can be varied by changing the strength of the constitutive promoter, by using an inducible promoter, by varying the copy number of each gene on the episome or genome (Figure 6c), by changing the selection marker, and / or by culture conditions corresponding to promoter activity or selection.

[0206] The effect of episomal gene copy number on protoberberine production was determined by assays performed in yeast cultures (Figure 6). (a) For CAS, 4 mM norlaudanosoline was added to the medium of tetrahydrocolumbamine-producing strains containing CAS expressed from either the multicopy 2 μm plasmid or the single-copy CEN / ARS plasmid. For STOX, 250 μM canadine was added to the medium of strains expressed from either the multicopy 2 μm plasmid or the single-copy CEN / ARS plasmid. (b) 4 mM norlaudanosoline was added to the medium of scourerine-producing strains expressing the listed plasmids. For both experiments, after 72 h of growth, yeast were pelleted by centrifugation, and the medium was analyzed by LC-MS. Positive ion electrospray ionization (ESI) mass spectra were obtained using an Agilent 6320 ion trap (electrospray capillary voltage -3.5 kV; heated capillary temperature 350 °C; sheath gas: nitrogen) interfaced to an Agilent 1200 Series HPLC equipped with an Agilent Zorbax SB-Aq column (3.0 x 50 mm, 1.8 μm) and an Agilent Zorbax SB-Aq guard column (2.1 x 12.5 mm, 5 μm). LC separation consisted of a 1-minute isocratic elution with H2O, a 3-minute gradient to 75:25 H2O:CH3OH, a 1-minute gradient to 100% CH3OH, and a final 4-minute isocratic elution, with a flow rate of 0.6 mL / min. Both solvents were 0.1% acetic acid. Extracted ion chromatograms for the products and molecular ions were plotted and manually integrated. Error bars represent standard deviations from three biological replicates. (c) Copy number correlates with expression level and ranking order. Samples were prepared from overnight cultures of yeast strains containing the indicated plasmids as described by Kushnirov et al. (2000). Yeast 16, 857-860, which is incorporated by reference in its entirety.After SDS-PAGE, proteins were transferred to nitrocellulose membranes, which were blocked in 5% BSA for 1 hour and then probed overnight with HRP-conjugated anti-HA antibodies. The membranes were imaged after incubation with enhanced chemiluminescence HRP substrate.

[0207] The effect of cytochrome P450-NADPH reductase partners on CAS activity was examined (Figure 6d). Coexpression of cytochrome P450 reductase ATR1 with CAS resulted in higher CAS activity than that of California Poppy CPR, Arabidopsis ATR2, P. somniferum CPR, or endogenous yeast CPR. Assays were performed in vivo using 4 mM norlaudanosoline in yeast expressing CAS from a multicopy plasmid. Positive ion electrospray ionization (ESI) mass spectra were acquired as described herein. Extracted ion chromatograms for the product standard molecular ions were plotted and manually integrated. Data were normalized to 1 in the absence of CPR.

[0208] Functional expression of STOX in yeast cultures was observed. Assays were performed in vivo using 250 μM canadine (m / z = 340) in yeast expressing STOX from the CEN / ARS plasmid. Positive ion electrospray ionization (ESI) mass spectra were obtained as described in Figure 6. Extracted ion chromatograms for the berberine product (m / z = 336) and the standard molecular ion of 1 μM berberine were plotted and smoothed using the Gaussian processing tool for one cycle with the default smoothing width during data analysis on the 6300 Series Ion Trap LC / MS v. 3.4.

[0209] To enhance the accumulation of BIA in yeast cells, heterologous transporters, such as plant ATP-binding cassette proteins derived from BIA-producing plants, are expressed in the engineered strains. These transporters are CjABCB1, CjABCB2, and / or CjABCB2, which act to accumulate berberine in yeast cells.

[0210] Figure 7 shows the in vivo production of berberine from norlaudanosoline. Cultures of host cells expressing SOMT, CAS, BBE, and STOX from a YAC, harboring a second copy of SOMT on a high-copy 2 μm plasmid, integrating ATR1, and incorporating three enzymes for the conversion of norlaudanosoline to reticuline, demonstrated the production of reticuline, scoureline, tetrahydrocolumbamine, canadine, and berberine. Assays were performed in yeast cultures for 96 hours using 4 mM norlaudanosoline. Products in the medium were identified by LC-MS as described in Figure 6: reticuline m / z 330, scoureline m / z 328, tetrahydrocolumbamine m / z 342, canadine m / z 340, and berberine m / z 336. The MS / MS spectrum for m / z 336 confirms that product 5 is berberine. This is because the spectrum matched that of the berberine standard.

[0211] IV. Thebaine-producing yeast strains Yeast strains have been engineered to produce thebaine as an intermediate or end product from salutaridin or its precursors, either produced by existing engineered strains in the culture medium or introduced into cell lysates or lysate fractions. More specifically, these strains express any combination of salutaridin reductase (SalR) and salutaridinol 7-O-acetyltransferase (SalAT).

[0212] FIG. 8 shows heterologous biosynthetic pathways supported by engineered yeast strains.

[0213] Figure 9 shows the conversion of salutaridine to thebaine in crude lysates of engineered yeast. Functional expression of both SalR and SalAT, resulting in the production of thebaine from salutaridine, was observed by monitoring the ion count (EIC) m / z = 312 peak area. Strains containing P. somniferum SalR produced more thebaine than strains containing P. somniferum SalR. Strains containing SalR with the F104A or I275A mutations produced more thebaine than strains containing SalR without these mutations. Enzyme combinations can also include any of the codon-optimized variants of SalAT listed in Table 2. Assays were performed using 100 μM salutaridine and 50 μM NADPH in crude lysates of yeast expressing SalR and SalAT from the genome. Positive ion electrospray ionization (ESI) mass spectra were obtained as described in Figure 6. Extracted ion chromatograms of the standard molecular ions of the products were plotted and manually integrated.

[0214] SalAT genes share 80% or less nucleic acid sequence identity with their naturally occurring counterparts (Table 3). These genes are non-naturally occurring nucleotide sequences that have been codon-optimized for expression in yeast.

[0215] Conversion of salutaridine to thebaine, catalyzed by SalR and SalAT, occurs in crude lysates of yeast strains expressing both enzymes, supplemented with the cofactor NADPH (Fig. 9 ).

[0216] SalAT and SalR are also expressed in engineered strains expressing additional enzymes such that the strain produces products for which thebaine is a precursor, e.g., the strain can produce oripavine, morphine, codeine, hydromorphone, hydrocodone, oxycodone, and / or oxymorphone.

[0217] The conversion of salutaridine to thebaine, catalyzed by SalR and SalAT, occurs in engineered yeast strains modified to produce increased amounts of NADPH (Table 2).

[0218] Yeast strains are engineered to increase production of salutaridinol or thebaine, or products for which thebaine is a precursor, from reticuline or its precursor by localizing SalR and / or SalAT to organelles within yeast cells. For example, SalR and / or SalAT may be localized to the yeast endoplasmic reticuline to reduce the spatial distance between SalR and / or SalAT and CYP2D6 or SalSyn or engineered cytochrome P450 enzymes that catalyze the conversion of reticuline to salutaridinol.

[0219] V. Opiate-Producing Yeast Strains A. Example 1 1. Introduction The following chapter describes the generation and characterization of yeast strains supporting the final steps of opiate biosynthesis, resulting in strains capable of producing natural opiates and semisynthetic opioids. The results described herein highlight that the loss of native regulatory strategies during the introduction of plant biosynthetic pathways into microbial hosts can result in novel pathway branches that direct flux toward undesired by-products. To reestablish and control pathway specificity, we created a general organelle routing toolkit that directs enzymes to specific native cellular compartments. This toolkit was used in a novel spatial engineering approach to actively delocalize plant pathway enzymes to the yeast inner membrane, thereby increasing the specificity of morphine production over the by-product neomorphine from 44% to 96%. Heterologous morphine biosynthesis was further optimized by increasing the supply of the co-substrate 2-oxoglutarate and gradually increasing the amount of gene copy number to balance pathway flux. By incorporating bacterial enzymes into heterologous pathways, we demonstrated the biosynthesis of a series of valuable semisynthetic opioids, including up to 51 mg / L hydrocodone, 70 mg / L oxycodone, and 1 mg / L hydromorphone, which are typically produced by chemical modification of natural opioids. Optimized and engineered yeast strains produced 31-132 mg / L total opioid products, demonstrating the development of a microbial biomanufacturing platform for supplying natural and semisynthetic opioids to the pharmaceutical industry.

[0220] 2. Results a. Construction of a morphine biosynthetic pathway in yeast The biosynthesis of morphine from thebaine is catalyzed by three enzymes in P. somniferum: 2-oxoglutarate / Fe 2+The enzymes are the NADPH-dependent dioxygenases T6ODM and CODM, and the NADPH-dependent aldo-keto reductase COR. These enzymes form two biosynthetic pathways from thebaine to morphine. One pathway (i) involves a nonenzymatic rearrangement to generate the intermediates neopinone, codeinone (1), codeine (2), and morphine (4) (Figure 15). Based on the reported substrate affinities of T6ODM and CODM, this is the preferred pathway in poppy. The minor pathway (ii) generates oripavine and morphinone as intermediates to morphine (Figure 15).

[0221] Figure 15 shows the engineering of a heterologous morphine biosynthetic pathway in yeast. The diagram shows the observed conversion of thebaine by morphine biosynthetic enzymes from P. somniferum of the poppy (thebaine 6-O-demethylase (T6ODM), codeine O-demethylase (CODM), and codeinone reductase (COR)). Two pathways to morphine occur in the poppy via intermediates codeinone and codeine (pathway i) and oripavine and morphinone (pathway ii). Pathway (i) and the newly identified pathway to neomorphine (iii) occur in heterologous yeast cells, demonstrating a broader substrate range for COR and CODM.

[0222] To reconstruct the morphine biosynthetic pathway in S. cerevisiae, we expressed yeast codon-optimized T6ODM, COR1.3, and CODM, each flanked by a unique yeast promoter and terminator, assembled in a single yeast artificial chromosome (YAC) vector (pYES1L). Of the four characterized P. somniferum COR isoforms, COR1.3 was selected because it had the highest affinity for codeinone. After culturing this strain with thebaine for 96 hours, we observed codeinone, codeine, and morphine in the culture medium, demonstrating that these heterologous plant enzymes can catalyze opiate conversion in yeast (Figure 15). However, the detected opiate levels were low, with morphine production as low as 0.2 mg / L, suggesting that optimization efforts are needed to increase conversion efficiency. Neopinone was not detected in this assay. This is because this intermediate is unstable and likely rearranges to codeinone during the course of the experiment.

[0223] Additional opiates detected in the culture medium indicated differences between the biosynthetic pathways observed in native and heterologous systems. Intermediates from the minor pathway to morphine observed in plants (oripavine and morphinone) were not detected in the engineered yeast strain, suggesting that the low activity of each of these enzymes on their alternative substrates prevented detectable levels. However, two other products were observed in amounts similar to codeine and morphine. The first had the same mass / charge (m / z) ratio as codeine and was determined by MS / MS analysis to be neopine (3). Neopine can be produced by the activity of COR on neopinone, the direct product of T6ODM, before it rearranges to codeinone (Figure 15). The second unknown product had the same m / z ratio as morphine and was determined to be neomorphine (5), produced by CODM-catalyzed demethylation of neopine. Therefore, analysis revealed a novel but undesired opiate pathway (iii) in the engineered yeast strain (Figure 15).

[0224] b. Increased supply of 2-oxoglutarate, a co-substrate for morphine biosynthesis We investigated whether the supply of 2-oxoglutarate, a key cosubstrate, is limited in the heterologous morphine biosynthetic pathway. The dioxygenases T6ODM and CODM require 2-oxoglutarate to accept one oxygen atom in the oxidative demethylation of thebaine and codeine, respectively. Therefore, we investigated whether increasing the supply of 2-oxoglutarate would enhance flux through our engineered biosynthetic pathway.

[0225] In endogenous yeast nitrogen metabolism, the glutamate dehydrogenase (GDH) enzyme catalyzes the interconversion of glutamate and 2-oxoglutarate (Figure 16). Therefore, increasing the supply of glutamate can increase the intracellular 2-oxoglutarate pool. Monosodium glutamate (MSG), a common nitrogen source, was added in small increments to the yeast culture medium. The inclusion of glutamine as a nitrogen source in the culture medium ensured that the cultures were not nitrogen-limited, thus preventing the positive growth effect of MSG supplementation. No differences in final cell density were observed with varying MSG levels. Increasing the MSG concentration from 0 to 2.5 g / L increased morphine production from 0.24 to 0.45 mg / L after 96 h of growth (Figure 16b).

[0226] We next investigated whether adding the co-substrate 2-oxoglutarate directly to the culture medium would further enhance flux through the pathway. 2-oxoglutarate was added in small increments to the culture medium to a concentration of 100 mM. Direct feeding of this co-substrate, in addition to 2.5 g / L MSG, increased the morphine titer to 2.5 mg / L, exceeding the titer observed in standard medium by more than 10-fold (Figure 16b). All subsequent opiate-producing cultures were grown in this optimized culture medium supplemented with 0.5 g / L glutamine, 2.5 g / L MSG, and 50 mM 2-oxoglutarate.

[0227] Figure 16 shows that increased supply of the cosubstrate 2-oxoglutarate increases morphine biosynthesis titer. (a) In yeast metabolism, 2-oxoglutarate is involved in the tricarboxylic acid (TCA) cycle and nitrogen assimilation, where it is reversibly converted to glutamate by the activity of glutamate dehydrogenases Gdh1p, Gdh2p, and Gdh3p. (b) Synthetic complete medium containing 0.5 g / L glutamine as the nitrogen base was supplemented with monosodium glutamate (MSG) to 2.5 g / L and then with 2-oxoglutarate to 100 mM. A yeast strain (CSY907) expressing T6ODM, COR1.3, and CODM from the pYES1L vector was cultured in deep-well plates with 1 mM thebaine for 96 hours in the indicated culture medium composition. Morphine production levels were determined by LC-MS analysis of the culture medium. Error bars represent ±1 SD of three biological replicate experiments.

[0228] c. Increase morphine potency by balancing enzyme expression levels Next, we investigated whether optimizing relative enzyme expression levels would increase pathway flux toward morphine. The COR enzyme catalyzes the reversible reduction of codeinone to codeine in morphine biosynthesis. COR also catalyzes the reversible reduction of neopinone to neopine in yeast (Figure 15). The existence of these reversible reactions suggested that pathway flux toward the production of codeine, and consequently, morphine, could be further increased by gradually increasing the expression levels of pathway enzymes.

[0229] To explore the combinatorial design space surrounding the expression levels of T6ODM, COR, and CODM, strains were constructed with varying gene copy numbers for each enzyme. In all strains, one copy of T6ODM, COR, and CODM was expressed from a YAC vector. Additional copies of one or more genes were integrated into the auxotrophic locus of the host cell genome with a constitutive GPD promoter. Strains were cultured in 96-well plates with 1 mM thebaine in optimized medium (0.5 g / L glutamine, 2.5 g / L MSG, and 50 mM 2-oxoglutarate) for 96 hours. Morphine and neomorphine titers were compared with a control strain containing only the YAC vector (T6ODM:COR:CODM gene ratio 1:1:1).

[0230] Altering the copy number of pathway genes altered total opiate titers and relative morphine and neomorphine levels. Increasing the copy number of COR alone (e.g., 1:3:1) or together with T6ODM (e.g., 2:2:1) decreased morphine production but increased neomorphine production, resulting in similar total opiate production but different end-product ratios (Figure 17). For example, the control 1:1:1 strain produced 2.5 mg / L morphine and 3.7 mg / L neomorphine, for a total of 6.2 mg / L of end-product opiates. In contrast, the 1:3:1 strain produced 2.0 mg / L morphine and 4.1 mg / L neomorphine, resulting in similar overall end-product titers despite the different morphine to neomorphine ratios. Other gene copy number combinations within the design space resulted in increases in morphine and total end-product titers. For example, increased CODM copy number resulted in higher production levels of both morphine and neomorphine (FIG. 17). This effect was enhanced by additional gene copies of T6ODM; thus, one ratio of interest for T6ODM:COR:CODM was 2:1:3, which produced 5.2 mg / L of morphine and 4.8 mg / L of neomorphine, for a total of 10.0 mg / L of end-product opiates in the culture medium (FIG. 17).

[0231] The observed relationship between morphine titer and gene copy number ratio suggested two mechanisms of improvement in the engineered strains. First, providing additional gene copies of the end enzyme CODM increased the conversion rates of, for example, codeine and neopine to morphine and neomorphine, respectively, and thus increased the total amount of end product produced by increasing the forward rate of the reversible COR reaction. Second, changes in expression levels that improved total end product titer also improved specificity for the target gene product morphine over the by-product neomorphine. Specifically, in the low-yielding 1:3:1 strain, morphine constituted 33% of all end-product opiates, whereas in the high-yielding 2:1:3 strain, morphine constituted 52%. Analysis of pathway conversion efficiency indicated that CODM prefers codeine as a substrate, thus biasing the high-copy-number morphine production pathway. In some cases of balanced copy number strains, the non-target neomorphine still accounted for nearly half of the final product.

[0232] d. Improve pathway specificity by developing and implementing a localization toolkit Earlier engineering efforts demonstrated that nearly half of the potential morphine yield was diverted to the undesired byproduct neomorphine (Figure 18), making pathway specificity a key engineering challenge. The conversion efficiency between engineered pathways was examined, and it was determined that one cause of the divergence from morphine to the untargeted neomorphine was a spontaneous step between the reactions catalyzed by T6ODM and COR. An engineering strategy in which T6ODM and COR are spatially separated within the cell may allow additional time for the spontaneous rearrangement of neopinone to codeinone (Figure 18). Specifically, sequestering COR in yeast organelles restricted the enzyme's access to neopinone produced by cytoplasmic T6ODM, providing additional time for neopinone to rearrange to codeinone and ultimately be converted to morphine.

[0233] To address this specificity challenge and enable a broader range of spatial engineering approaches in yeast, we developed a modular organelle routing toolkit. The toolkit consists of six authenticated localization tags derived from endogenous yeast proteins. These tags are directed to various yeast organelles: the endoplasmic reticulum (ER), mitochondria (MT), plasma membrane (PM), and vacuole (V) (Figure 21a, b). The ER routing tag ER1, vacuolar tag V1, and plasma membrane tag P1 were developed based on transmembrane domains derived from three proteins within the tail-anchored protein class. These 31-35 amino acid tags are sufficient to direct the post-transcriptional localization of target proteins, such that the C-terminus is inserted into the inner membrane of the assigned organelle and the protein extends into the cytoplasm. To access the internal environment of the organelle, a second ER routing tag, termed ER2, was designed based on the 28-amino acid transmembrane domain of the integral membrane protein calnexin. Two additional sequences for free protein localization within the ER lumen (ER3) and mitochondrial matrix (MT1) were adopted from established ER and mitochondrial markers, KAR2-DsRed-HDEL and COX4-mCherry, respectively. To confirm targeted localization, each member of the organelle routing toolkit was fused to GFP and examined by confocal microscopy (Figure 18b).

[0234] The organelle routing toolkit was applied to COR enzymes in the morphine pathway to determine whether physical delocalization of pathway enzymes could increase flux to the desired product (morphine) and decrease flux to the undesired product (neomorphine). A control strain in which COR1.3 was untagged and thus colocalized with T6ODM and CODM in the cytoplasm (CYT) produced 2.5 mg / L of morphine with 44% specificity after 96 hours of growth (Figure 19). When the organelle routing toolkit was used to fuse each localization tag to COR to actively target this enzyme to various cellular compartments, increased specificity (relative production of the desired product) and titer (absolute production level of the desired product) for morphine were observed. Localization of COR to the cytoplasm-facing ER (ER1 tag) and vacuole (V1 tag) yielded strains with high morphine titers of approximately 3.5 mg / L. In contrast, a strain with ER3-tagged COR localized to the ER lumen had nearly 100% specificity for morphine but a reduced titer of less than 1 mg / L. Localization of COR to the ER lumen with an ER2 tag provided a balance of enhanced yield and specificity, resulting in a morphine titer of 3.1 mg / L with 86% specificity, which was carried forward for subsequent experiments.

[0235] Table 5 (below) shows the organelle routing toolkit. Modular targeting sequences were used to localize enzymes to organelles in engineered yeast strains. Enzymes selected for localization were fused to the targeting sequence using an intervening 7 amino acid linker, either Gly6SerThr (SEQ ID NO:8) at the N-terminus or ProGly6 (SEQ ID NO:9) at the C-terminus.

[0236] (Table 5) Abbreviations: ER, endoplasmic reticulum; MT, mitochondria; PM, plasma membrane; TM, transmembrane domain; V, vacuole * The unnamed tags did not confer stable modular localization to the predicted compartments. TIFF2026015382000013.tif80169Table 5 continued ER1 (SEQ ID NO: 1) ER2 (SEQ ID NO: 2) ER3 (SEQ ID NO: 3) V1 (SEQ ID NO: 5) PM1 (SEQ ID NO: 6) MT1 (SEQ ID NO: 7) * FIS1 (SEQ ID NO: 10) * PRC1 (SEQ ID NO: 11)

[0237] e. Achieving biological synthesis of semisynthetic opioids by incorporating microbial enzymes The bacterial strain Pseudomonas putida M10, identified in wastewater from a poppy processing plant, performs the enzymatic conversion of opioids. Two characterized enzymes (NADP + The NADH-dependent morphine dehydrogenase (morA) and NADH-dependent morphinone reductase (morB) catalyze many of these reactions. The aldo-keto reductase morA and the α / β-barrel flavoprotein oxidoreductase morB were heterologously expressed in Escherichia coli to convert morphine to hydromorphone.

[0238] We investigated whether morA and morB would extend the biosynthetic capabilities of morphine-producing yeast strains to the valuable end-products hydrocodone, oxycodone, and hydromorphone (Figure 20). One YAC contained the P. somniferum genes T6ODM, COR, and CODM and the P. putida genes morA and morB. Yeast strains transformed with this YAC and cultured with 1 mM thebaine produced trace amounts of hydrocodone and no detectable hydromorphone after 96 hours of growth.

[0239] Engineered yeast strains expressing different combinations of P. somniferum and P. putida M10 enzymes from the pYES1L vector produce different levels of the target opioid. TIFF2026015382000014.tif21158

[0240] An alternative biosynthetic pathway to the semisynthetic opioids hydrocodone and hydromorphone was developed. First, by replacing COR with morA in a YAC encoding morphine production, it was determined whether morA could replace COR in the morphine biosynthetic pathway by reducing codeinone to codeine. Replacement of COR with morA activity resulted in 2.4 mg / L of morphine and 69% selectivity, higher than the selectivity of any untagged COR isoform strain. Four YAC genes, including morB, were generated containing T6ODM, CODM, morA, and morB. Strains carrying this YAC produced 1.3 mg / L of hydrocodone and 0.10 mg / L of hydromorphone. The opioids dihydrocodeine and dihydromorphine were also detected due to morA and morB activity toward hydrocodone and hydromorphone (Figures 20 and 23). 14-hydroxycodeinone was observed, likely resulting from morA activity on 14-hydroxycodeinone (Figure 23). Hydroxylation of codeinone to 14-hydroxycodeinone has been observed to occur spontaneously in vitro. Trace amounts of another 14-hydroxylated product, oxycodone, were observed, likely due to morB activity on 14-hydroxycodeinone. Based on these results, a strain expressing only T6ODM and morB was engineered to increase the flux of morB products into the pathway to hydrocodone and oxycodone. The strain with these two enzymes produced 6.5 mg / L hydrocodone and 2.1 mg / L oxycodone, demonstrating that 14-hydroxylation occurs as part of the pathway (Figure 20).

[0241] Next, we examined the ability of morA and morB mutants to increase flux to hydromorphone. Specifically, we tested morA Cys81Ser, which prevents irreversible product inhibition by morphinone at Cys81 on the enzyme surface. Additionally, we tested an alternative morB amino acid sequence (RCSB PDB: 1GWJ_A) that contains a Glu160Gly mutation compared to the original reported sequence (UniProtKB: Q51990). C81S and morB E160G The mutants were tested individually and in combination in the engineered pathway. The control strain expressing T6ODM, CODM, morA, and morB produced 1.3 mg / L hydrocodone and 0.10 mg / L hydromorphone. C81S Substitution of the mutants reduced the titers of both hydrocodone and hydromorphone to 0.9 mg / L and 0.09 mg / L, respectively. E160G The level of hydromorphone in the culture medium increased when T6ODM, CODM, morA, and morB were replaced with E160G The strain expressing morB produced 0.9 mg / L hydrocodone and 0.14 mg / L hydromorphone. The reduced hydrocodone titer was due to the morB E160G The mutants had reduced activity towards codeinone, suggesting that they redirect flux towards hydromorphone.

[0242] f. Combining strain engineering approaches to produce natural and semisynthetic opioids Using the genetic design elements described herein, three production strains for targeted opioid biosynthesis were constructed. The morphine-producing strain (CSY950) incorporated one integrated copy of T6ODM, two integrated copies of CODM, and YACs encoding COR1.3-ER2, T6ODM, and CODM. This strain used ER-localized COR1.3 to direct pathway flux to morphine, enhancing overall pathway flux with an optimal T6ODM:COR1.3:CODM ratio of 2:1:3. The hydromorphone-producing strain (CSY951) incorporated one integrated copy of T6ODM, two integrated copies of CODM, and T6ODM, CODM, morA, and morB. E160G This hydromorphone-producing strain contained a YAC encoding morB. E160G Mutants were used to direct flux toward hydromorphone and enhance overall production with optimal gene copy number ratios. The hydrocodone / oxycodone-producing strain (CSY952) incorporated two integrated copies of T6ODM and YACs encoding T6ODM and morB. These producers were grown in parallel 0.25 L closed-batch fermentations. Ten key opioid end products (codeine, neopine, morphine, neomorphine, hydrocodone, oxycodone, hydromorphone, dihydrocodeine, 14-hydroxycodeine, and dihydromorphine) were monitored over the course of the fermentation. Target opioids were detected in the culture medium after 24 hours, increased in concentration with increasing cell density, and continued to accumulate during stationary phase. Strains CSY950, CSY951, and CSY952 accumulated 31, 68, and 132 mg / L of the target opioid molecule in the culture medium, respectively, over the course of fermentation (FIG. 24).

[0243] Analysis of the total metabolite profiles between the strains at the final time point revealed significant differences in pathway flux (Figure 24). Strain CSY950, in which COR was localized to the ER, produced lower titers of neopine and neomorphine relative to codeine and morphine (7.7 and 4.7 mg / L vs. 2.6 and 0.76 mg / L), indicating that the spatial engineering approach was still effective in restricting pathway flux toward non-target byproducts in bench-scale fermentation (Figure 24a). However, the overall yield of morphine from this strain was low. Examination of the total BIA profile revealed that the byproduct 14-hydroxycodeine was a major component of the total opioid molecules produced (15 mg / L). In this strain, codeinone can form 14-hydroxycodeinone, which is subsequently reduced by COR to 14-hydroxycodeine. Furthermore, the greater potency of codeine than morphine suggests that factors affecting CODM activity may be disrupting the pathway to morphine.

[0244] Analysis of the fermentation culture medium of CSY951 provided further evidence of a bottleneck in the codeine-to-morphine pathway. The downstream products of CODM (morphine, hydromorphone, and dihydromorphine) accumulated at low levels (0.54, 1.0, and 1.5 mg / L, respectively) compared with other BIA products, suggesting that CODM activity may be limited. This could be due to limited access of CODM to its substrate, codeine, as a result of passive diffusion out of the cell, facilitated by the molecule's moderate polarity, or due to competition for binding sites with thebaine, which is abundant in the culture medium. CSY951 also accumulated low levels of hydrocodone and oxycodone (1.6 and 0.55 mg / L, respectively), which may be due to the morphine-to-morphine pathway being restricted by the morphine-to-morphine pathway. E160GThis is consistent with morA limiting the production of these byproducts and directing the pathway toward hydromorphone. In this strain, morA activity increased the accumulation of the byproducts neopine and neomorphine (21 and 4.4 mg / L, respectively), likely affecting the yield of the target end product, hydromorphone. The data suggest that further optimization of hydromorphone-producing strains can be achieved through dual spatial and temporal regulatory strategies. For example, implementing a spatial engineering approach to localize morA to the ER could support increased production of morphine and limit flux toward the neomorphine branch, as observed in ER-COR1.3. morA expression could be further regulated temporally to "switch on" once adequate levels of morphine have accumulated, subsequently converting this intermediate to morphinone and ultimately hydromorphone. A similar temporal regulatory strategy applied to morB could limit the synthesis of byproducts such as hydrocodone and oxycodone.

[0245] Strain CSY952 was engineered for the production of hydrocodone and oxycodone and has a simple pathway structure. The strain does not incorporate morA / COR or CODM, and therefore does not lose flux to the neomorphine branch or encounter a bottleneck between codeine and morphine. CSY952 converted thebaine to hydrocodone and oxycodone at titers of 51 and 70 mg / L, respectively (Figure 24a). Hydrocodone accumulation was limited by its conversion to dihydrocodeine over the course of fermentation, with a final titer of 11 mg / L (Figure 24). The reduction of hydrocodone to dihydrocodeine could result from a second reduction reaction by morB or could be the result of endogenous yeast enzyme activity. The MS / MS spectrum of dihydrocodeine was consistent with published mass spectra. CSY952 demonstrates that high flux to target compounds can be achieved through a minimally branched pathway of heterologous enzymes with high activity in yeast.

[0246] 3. Discussion S. cerevisiae has been demonstrated as a biosynthetic platform for many valuable BIA target molecules, such as the production host for the conversion of thebaine to opioids, including codeine, morphine, hydrocodone, oxycodone, and hydromorphone. Tools and methods support BIA biosynthesis in yeast by modulating the localization of heterologous pathway enzymes and redirecting pathway flow toward target end products. Gene copy number optimization and enhanced co-substrate supply were also applied to enhance opiate biosynthesis in yeast. For example, three exemplary engineered strains produced 7.7 mg / L codeine and 4.7 mg / L morphine (CSY950), 1 mg / L hydromorphone (CSY951), and 51 mg / L hydrocodone, 11 mg / L dihydrocodeine, and 70 mg / L oxycodone (CSY952) in bench-scale batch fermentations.

[0247] In poppy, thebaine is converted to morphine via two biosynthetic pathways. The first bifurcation occurs when T6ODM or CODM demethylates thebaine at distinct positions. Because both biosynthetic pathways reach morphine in poppy, this bifurcation does not appear to affect in planta yield. However, engineered yeast strains of interest show further bifurcation due to the activity of COR and CODM on neopinone and neopine, respectively, resulting in neomorphine production and reduced morphine production.

[0248] Plant enzymes may function differently when expressed in yeast compared to their native plant hosts due to the absence of native regulatory mechanisms and altered intracellular factors, such as protein processing, protein localization, and the microenvironment. In the engineered yeast strains of interest, production of the intermediates oripavine or morphinone was not observed. In aqueous solution, neopinone rapidly rearranges to codeinone, but this molecule can be stabilized by conditions within yeast cells. Thus, while in vitro conditions favor the production of codeine, in vivo conditions in yeast may slow down neopinone rearrangement, allowing COR to act on this intermediate to produce neopine.

[0249] T6ODM and CODM exhibit broad substrate specificity and catalyze the O-demethylation of nonmorphinan alkaloid substrates, such as scourerine and allocryptopine. These enzymes also catalyze the O-demethylation reaction, cleaving the methylenedioxy bridge in various BIAs, including allocryptopine, cryptopine, and protopine. Other examples of promiscuous BIA enzymes include O- and N-methyltransferases, which methylate many BIA substrates. The absence of natural temporal and spatial regulatory mechanisms in heterologous microbial hosts likely further expands the range of substrates available to BIA biosynthetic enzymes, leading to newly observed pathway branches and metabolites. Furthermore, combining enzymes from different species in a single host cell further increases the number of natural and unnatural substrates available to any given enzyme and the production of various BIA molecules. Targeted reconstruction of microbial BIA biosynthetic pathways can be achieved to manage the flow through highly branched pathways and achieve optimal yields of individual target end products.

[0250] To facilitate spatial engineering of biosynthetic pathways in yeast, we created an organelle routing toolkit that supports the routing of enzymes to selected organelles and endomembrane locations. We applied these spatially engineered tools to limit the production of unwanted byproducts and redirect flux of the target product, morphine. The spontaneous conversion of neopinone to codeinone is a critical branch point in our pathway, with COR activity on neopinone directing flux to neomorphine and its activity on codeinone directing flux to morphine. Sequestration of COR from T6ODM may allow additional time for spontaneous reactions to occur and redirect flux to the morphine branch. The results demonstrate that actively routing COR to various organelles increases pathway specificity to morphine and overall morphine potency, likely through the combined effects of delocalizing pathway enzymes and reducing COR activity, further balancing pathway flux.

[0251] The results demonstrate the construction of the final step of opiate biosynthesis in yeast, converting thebaine to codeine and morphine, and the extension of this pathway to produce semisynthetic drugs such as hydrocodone, oxycodone, and hydromorphone. In combination with the complete microbial biosynthesis of the upstream BIA, reticuline, the subject methods and host cells can be used or adapted to engineer yeast strains capable of producing target opiates from monosaccharide sources. Such strains can contain functional expression of three known enzymes, including one cytochrome P450 that catalyzes the conversion of (R)-reticuline to salutaridine.

[0252] 4. Method a. Plasmid and yeast strain construction Modern molecular microbiology techniques were used to construct plasmids and strains. The S. cerevisiae parent strain (from which the strains described in the Examples were constructed) was haploid W303α (MATα leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15). Yeast synthetic complete (SC) amino acid dropout medium containing 2% dextrose and complex yeast peptone dextrose (YPD) medium containing 200 mg / L G418 sulfate were used for strain construction. Chemically competent E. coli strain TOP10 was used for cloning purposes and grown in LB medium with the indicated antibiotic concentrations. Custom-made oligonucleotides were synthesized using standard methods. All heterologous gene sequences were downloaded from GenBank, codon-optimized for expression in S. cerevisiae using the GeneArt GeneOptimizer program, and synthesized using standard methods. All endogenous promoters, terminators, and organelle targeting sequences were amplified from W303α genomic DNA (Table S3). The polymerase used for PCR was Pfu Hotstart for products less than 2 kb and Expand High Fidelity PCR System for products greater than 2 kb. Plasmids were prepared from E. coli using QIAprep and Econospin columns. Sequencing was performed using standard methods.

[0253] Table S3 shows the YAC expression cassettes. A unique promoter and terminator was paired with each gene to construct an expression cassette for integration into the YES1 vector.

[0254] TIFF2026015382000015.tif25162 a The heterologous gene is codon optimized for expression in S. cerevisiae. bThe COR isoforms that replace COR1.3 in the constructs shown are COR1.1, AAF13736.1, COR1.2, AAF13737.1, and COR1.4, AAF13739.1. c The mutations replacing morA and morB in the constructs shown are morA C81S and morB E160G is.

[0255] To express heterologous genes from P. somniferum and P. putida in W303α, individual expression cassettes containing open reading frames flanked by unique promoters and terminators were constructed (Table S3) and integrated into the pYES1L vector. In the initial construction of the expression cassettes, individual genes were joined to the promoters and terminators by splicing-overlap-extension (SOEing) PCR and integrated into the Gateway vector pDONR221 (BP Clonase II) for sequence verification and conservation. The expression cassettes were then PCR amplified using oligonucleotides designed by the GeneArt High-Order Genetic Assembly online tool, which added homology regions for gap repair in yeast. 100 ng of each expression cassette PCR was combined with 100 ng of the linearized pYES1L and transformed into W303α by electroporation. The pYES1L vector contains the TRP1 and ARS4 / CEN5 regions for selection on tryptophan dropout medium, and therefore each newly constructed vector was maintained as a single-copy episomal plasmid in yeast. pYES1L constructs were confirmed by PCR screening and sequencing. To propagate the plasmid so that it could be transformed into other yeast background strains, the pYES1L vector was isolated from yeast and transformed into TOP10 E. coli, where it was maintained at a single copy and selected on LB medium containing 50 mg / L spectinomycin dihydrochloride pentahydrate. Approximately 2 μg of plasmid, sufficient to transform up to 10 yeast strains, was prepared from a 100 mL overnight E. coli culture.

[0256] To integrate additional gene copies into the yeast genome, expression cassettes containing the gene flanked by promoters and terminators were generated by PCR amplification of pUG vectors modified to allow Gateway cloning (Life Technologies). Vectors pUG6 and pUG73, containing the KanMX and Kluyveromyces lactis LEU2 selectable markers, respectively, were cloned into the yeast genome using the Gateway cassette attR1-ccdB / Cam. R Two new pDEST vectors, designated pCS2643 and pCS2644, were created by modifying the -attR2 vector to contain the GPD promoter and CYC1 terminator flanking the vector. Individual pENTR vectors, each containing a gene open reading frame previously anchored by a Kozak sequence, were recombined with the pDEST vector. From the resulting vector, the gene expression cassette and an adjacent selectable marker (KanMX or LEU2) flanked by loxP sites were PCR amplified using oligonucleotides that added 103 bp homologous to the targeted integration site in the yeast genome. The PCR products were transformed into W303α by standard lithium acetate transformation. Integration events were selected by growth on G418 or leucine dropout medium and confirmed by PCR screening and sequencing of both integration boundaries. The loxP sites were used to remove the selection marker by expression of Cre recombinase (Guldener et al. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res 24, 2519-2524 (1996)).

[0257] Other plasmids were constructed from the Lindquist suite of destination vectors, specifically pAG416GPD-ccdB (Alberti, S., Gitler, AD & Lindquist, S. A suite of Gateway cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisiae. Yeast 24, 913-919 (2007)). Vectors for microscopy were constructed with organelle targeting sequences fused to GFP at the N- or C-terminus, separated by a Gly6SerThr or ProGly6 linker, respectively. The targeting sequences, linkers, and GFP were combined by single-overlay PCR and cloned into pDONR221 using BP clonase II. The resulting entry vector was recombined into pAG416GPD-ccdB or another destination vector using LR clonase II to generate a shuttle vector for expression in yeast. Plasmids containing established markers for mitochondria and endoplasmic reticulum were pHS12-mCherry (Addgene plasmid 25444) and YIPlac204TKC-DsRed-Express2-HDEL (Addgene plasmid 21770), respectively.

[0258] b. Culture and fermentation conditions To assay for opioid production, yeast strains were cultured in 96-well plates containing 0.4 mL of SC growth medium (tryptophan-deficient, 2% dextrose) per well and incubated at 30°C, 480 rpm, on a 1.24 cm orbital diameter shaker with 80% humidity. Strains were first inoculated into SC medium containing 0.5 g / L glutamine, which was used instead of ammonium sulfate as the nitrogen base, and grown for 16 hours. Cultures were then diluted 40-fold back into SC medium containing 0.5 g / L glutamine, 2.5 g / L monosodium glutamate, 50 mM 2-oxoglutarate, and 1 mM thebaine. Strains were grown for 96 hours or until morphine production by the control strain reached approximately 2.5 mg / L. To determine cell density, the final OD 600 (after 10-fold dilution) was measured.

[0259] For enhanced closed-batch cultivation conditions, the strains were cultivated in a 0.5 L Biostat Q-plus bioreactor. The initial medium volume was 250 mL and contained 10x SC tryptophan dropout medium components supplemented with 5 g / L glutamine, 25 g / L MSG, 100 mM 2-oxoglutarate, and 1 mM thebaine. The glucose concentration was 10%, and the medium was further supplemented with 2 g / L adenine hemisulfate. Each vessel was inoculated with 10 mL of a pelleted overnight culture grown in selective medium, and the cells were resuspended in fermentation medium and then added to the vessel. Process parameters were kept constant throughout the fermentation at 30 °C, 200 rpm, and a compressed air flow rate of 2 L / min. At appropriate time points, cell densities from diluted samples measured in cuvettes on a Nanodrop 2000c spectrophotometer were recorded, and additional samples were taken for metabolite analysis.

[0260] c. Analysis of opiate production Opiates secreted into the culture medium by the engineered yeast strain were identified and quantified by liquid chromatography-mass spectrometry (LC-MS). The culture was pelleted by centrifugation, and 5 μL of the supernatant was separated on a Zorbax SB-Aq column (3.0 x 50 mm, 1.8 μM particle size). The column was equilibrated with water, 0.1% acetic acid, and 0.1% methanol (solvent A). The sample was eluted with a mobile phase of methanol and 0.1% acetic acid (solvent B) in the following order: 100% A for 0-1 min, 0-25% B for 1-4 min, and 25% B for 4-7 min. This was followed by washing the column with 100% B and then re-equilibrating with A. The flow rate was kept constant at 0.6 mL / min. Eluted opiates were identified using an Agilent 6320 ion trap mass spectrometer operated in scan mode for total ion monitoring. Extracted ion chromatograms for each metabolite of interest were compared to commercially available standards spiked into the spent yeast culture medium. Fragment ions for the target molecules for both the samples and standards were identified by MS / MS and compared with published spectra to confirm the identity of each opiate. For quantification, peak areas in the extracted ion chromatograms were integrated and compared to standard curves for each molecule. The standards were thebaine, codeine sulfate, morphine sulfate pentahydrate, hydrocodone bitartrate, oxycodone hydrochloride, and hydromorphone hydrochloride.

[0261] For analysis of bioreactor culture media, samples were diluted 2- to 10-fold and separated on a Zorbax SB-Aq column (3.0 x 250 mm, 5 μM particle size). The column was equilibrated with water, 0.1% acetic acid, and 0.1% methanol (solvent A), and the sample was eluted with a mobile phase of methanol and 0.1% acetic acid (solvent B) in the following order: 100% A for 0-10 min, 0-90% B for 10-30 min, followed by a column wash with 100% B and subsequent re-equilibration with A. The flow rate was kept constant at 0.8 mL / min.

[0262] d. Confocal microscope Yeast cells harboring the appropriate plasmids were grown overnight in SC dropout medium, after which 1.5 mL was pelleted and resuspended at a high cell density (in less than 100 μL of medium). Slides were prepared by placing a 2% low-melting-point agarose pad combined with yeast medium on a microscope slide, spotting 1 μl of yeast cells onto the agarose pad, and covering and sealing it with a No. 1 coverslip. Cells were imaged with a Leica TCS SP5 confocal microscope with a 63.0 magnification glycerol immersion objective, a 1.30x numerical aperture, and a digital zoom of 8x or less. The hybrid detector (HyD) smart gain was adjusted between 30 and 200% depending on the sample fluorescence intensity. As an example of fluorescence settings, for monochromatic imaging of GFP, the sample was excited with a 488 nm laser line, and the emitted fluorescence was recorded by the HyD channel in the 500–550 nm range (dichroic mirror = DD 488 / 594). Images were recorded using an Airy 1 pinhole size (108.4 μm), a minimum of two line averaging passes, a pixel size of 30–100 nm, and an optical section thickness of 0.856 μm.

[0263] Aspects of the invention Embodiment 1. A host cell that produces reticuline, wherein the host cell comprises multiple copies of one or more heterologous coding sequences for one or more methyltransferases selected from 6OMT, CNMT, and 4'OMT, and wherein the one or more methyltransferases are obtained from a different biological source compared to the host cell.

[0264] Embodiment 2. The host cell of embodiment 1, wherein the biological source is Papaver somniferum, Thalictorum flavum, or Coptis japonica.

[0265] Embodiment 3. The host cell of embodiment 1, wherein the multiple copies of the one or more methyltransferases are obtained from two or more different biological sources compared to the host cell.

[0266] Embodiment 4. The host cell of embodiment 1, wherein the host cell comprises multiple copies of the heterologous coding sequence of CNMT.

[0267] Embodiment 5. The host cell of embodiment 4, wherein the host cell comprises two copies of the heterologous coding sequence of CNMT.

[0268] Embodiment 6. The host cell of embodiment 1, wherein the host cell is capable of producing increased amounts of reticuline from norcoclaurine compared to a control host cell lacking multiple copies of one or more heterologous coding sequences for one or more methyltransferases.

[0269] Embodiment 7. The host cell of embodiment 6, wherein the increased amount of reticuline is 10% or more as compared to a control host cell.

[0270] Embodiment 8. The host cell of embodiment 6, wherein the host cell is capable of producing reticuline from norcoclaurine.

[0271] Embodiment 9. The host cell of embodiment 1, wherein the host cell comprises two or more heterologous coding sequences for two or more methyltransferases selected from 6OMT, CNMT, and 4'OMT.

[0272] Embodiment 10. The host cell of embodiment 9, wherein the two or more methyltransferases are obtained from two or more different biological sources as compared to the host cell.

[0273] Embodiment 11. The host cell of embodiment 9, wherein the host cell comprises heterologous coding sequences for all of the methyltransferases 6OMT, CNMT, and 4'OMT.

[0274] Embodiment 12. The host cell of embodiment 1, wherein the host cell is capable of producing reticuline from norlaudanosoline via the biosynthetic pathway of Figure 2.

[0275] Embodiment 13. The host cell of embodiment 1, wherein the host cell is capable of producing reticuline from norcoclaurine via the biosynthetic pathway of Figure 3.

[0276] Embodiment 14. The host cell of embodiment 1, wherein the host cell is a yeast strain.

[0277] Embodiment 15. The host cell of embodiment 14, wherein the yeast strain is S. cerevisiae.

[0278] Embodiment 16. A host cell that produces sanguinarine or a sanguinarine precursor, wherein the host cell comprises one or more heterologous coding sequences for one or more enzymes selected from BBE, CFS, CPR, STS, TNMT, MSH, P6H, and DBOX, wherein the one or more enzymes are obtained from a different biological source compared to the host cell.

[0279] Embodiment 17 The host cell of embodiment 16, wherein the sanguinarine precursor is protoberberine or a benzophenanthridine alkaloid.

[0280] Embodiment 18. The host cell of embodiment 16, wherein the sanguinarine precursor is selected from cheilanthifoline, stylopine, cis-N-methylstylopine, scoulerine, protopine, and dihydrosanguinarine.

[0281] Embodiment 19. The host cell according to embodiment 16, wherein the biological source is P. somniferum, California poppy, Arabidopsis thaliana, poppy flower, or poppy flower.

[0282] Embodiment 20. The host cell of embodiment 16, wherein the one or more enzymes are two or more enzymes obtained from two or more different biological sources as compared to the host cell.

[0283] Embodiment 21 The host cell of embodiment 16, wherein the host cell comprises multiple copies of one or more heterologous coding sequences.

[0284] Embodiment 22. The host cell of embodiment 21, wherein the multiple copies of the one or more heterologous coding sequences are obtained from two or more different biological sources as compared to the host cell.

[0285] Embodiment 23 The host cell of embodiment 16, wherein the host cell comprises two or more heterologous coding sequences for two or more enzymes selected from BBE, CFS, CPR, STS, TNMT, MSH, P6H, and DBOX.

[0286] Embodiment 24. The host cell of embodiment 23, wherein the host cell comprises three or more heterologous coding sequences for three or more enzymes selected from BBE, CFS, CPR, STS, TNMT, MSH, P6H, and DBOX.

[0287] Embodiment 25. The host cell of embodiment 23, wherein the host cell comprises four or more heterologous coding sequences for four or more enzymes selected from BBE, CFS, CPR, STS, TNMT, MSH, P6H, and DBOX.

[0288] Embodiment 26. The host cell of embodiment 25, wherein the host cell comprises five or more heterologous coding sequences for five or more enzymes selected from BBE, CFS, CPR, STS, TNMT, MSH, P6H, and DBOX.

[0289] Embodiment 27. The host cell of embodiment 26, wherein the host cell comprises a heterologous coding sequence for each of the enzymes BBE, CFS, CPR, STS, TNMT, MSH, P6H, and DBOX.

[0290] Embodiment 28. The host cell of embodiment 16, wherein the host cell further comprises one or more gene deletions compared to a native host cell, and wherein the one or more deleted genes are selected from IRE1, HAC1, OPI1, INO1, INO2, INO3, PDR1, STB5, PDR3, PDR5, SNQ2, YOR1, TPO1, TPO2, TPO3, TPO4, PDR10, PDR11, PDR15, PDR16, PDR17, QDR1, QDR2, QDR3, FLR1, AQR1, AQR2, and CIN5.

[0291] Embodiment 29 The host cell of embodiment 16, wherein the host cell is a yeast strain.

[0292] Embodiment 30. The host cell of embodiment 16, wherein the host cell is capable of producing sanguinarine or a sanguinarine precursor from norlaudanosoline via the biosynthetic pathway of Figure 4.

[0293] Embodiment 31 The host cell of embodiment 16, wherein the host cell is capable of producing sanguinarine or a sanguinarine precursor from norlaudanosoline via the biosynthetic pathway of Figure 11.

[0294] Embodiment 32 The host cell of embodiment 16, wherein the host cell comprises a heterologous coding sequence for a BBE enzyme.

[0295] Embodiment 33 The host cell of embodiment 16, wherein the heterologous coding sequence for the BBE enzyme is integrated into the host cell chromosome.

[0296] Embodiment 34 The host cell of embodiment 16, wherein the sanguinarine precursor is cheilanthifoline.

[0297] Embodiment 35. The host cell of embodiment 34, wherein the host cell comprises heterologous coding sequences for the CFS and CPR enzymes.

[0298] Embodiment 36 The host cell of embodiment 35, wherein the CPR enzyme is ATR1.

[0299] Embodiment 37. The host cell of embodiment 16, wherein the sanguinarine precursor is scourerine.

[0300] Embodiment 38 The host cell of embodiment 37, wherein the host cell comprises a heterologous coding sequence for an STS enzyme.

[0301] Embodiment 39. The host cell of embodiment 16, wherein the sanguinarine precursor is protopine.

[0302] Embodiment 40 The host cell of embodiment 39, wherein the host cell comprises heterologous coding sequences for the TNMT and MSH enzymes.

[0303] Embodiment 41 The host cell of embodiment 16, wherein the host cell comprises a heterologous or endogenous coding sequence for a cytochrome b5 enzyme.

[0304] Embodiment 42 The host cell of embodiment 16, wherein the host cell comprises one or more heterologous or endogenous coding sequences for one or more proteins involved in compound transport across the cell membrane.

[0305] Embodiment 43. The host cell of embodiment 42, wherein the protein is selected from PDR1, PDR5, SNQ2, YOR1, PDR3, CIN5, and PDR3.

[0306] Embodiment 44. The host cell according to embodiment 16, wherein the host cell is a host cell according to one of embodiments 1 to 15.

[0307] Embodiment 45. The host cell of embodiment 16, wherein the host cell further comprises one or more heterologous coding sequences for one or more methyltransferases selected from 6OMT, CNMT, and 4'OMT, wherein the one or more methyltransferases are obtained from a different biological source compared to the host cell.

[0308] Embodiment 46. A host cell that produces a protoberberine alkaloid, wherein the host cell comprises one or more heterologous coding sequences for one or more enzymes selected from BBE, S9OMT, CAS, and STOX, wherein the one or more enzymes are obtained from a different biological source as compared to the host cell.

[0309] Embodiment 47. The host cell of embodiment 46, wherein the protoberberine alkaloid is represented by one of the following structures: TIFF2026015382000016.tif47128 formula, R1~R 14are each independently selected from H, alkyl, hydroxyl, or alkoxy.

[0310] Aspect 48. The host cell of Aspect 46, wherein the biological source is Papaver somniferum, California poppy, Coptis japonica, Thalictorum flavum, Berberis stolonifera, Thalictorum flavum subsp. glaucum, Coptis orientalis, Thalictorum spp., Coptis spp., Papaver spp., Barberry, Ardisia crenata, or Berberis spp.

[0311] Embodiment 49. The host cell of embodiment 46, wherein the host cell comprises multiple copies of one or more heterologous coding sequences.

[0312] Embodiment 50. The host cell of embodiment 49, wherein the multiple copies of the one or more heterologous coding sequences are obtained from two or more different biological sources as compared to the host cell.

[0313] Embodiment 51 The host cell of embodiment 46, wherein the host cell comprises two or more heterologous coding sequences for two or more enzymes selected from BBE, S9OMT, CAS, and STOX.

[0314] Embodiment 52. The host cell of embodiment 51, wherein the host cell comprises three or more heterologous coding sequences for three or more enzymes selected from BBE, S9OMT, CAS, and STOX.

[0315] Embodiment 53. The host cell of embodiment 52, wherein the host cell comprises a heterologous coding sequence for each of the enzymes BBE, S9OMT, CAS, and STOX.

[0316] Embodiment 54 The host cell of embodiment 46, wherein the host cell comprises heterologous coding sequences for CAS and CPR enzymes.

[0317] Embodiment 55 The host cell of embodiment 54, wherein the CPR enzyme is ATR1.

[0318] Embodiment 56 The host cell of embodiment 46, wherein the host cell comprises a heterologous coding sequence for STOX.

[0319] Embodiment 57. The host cell of embodiment 56, wherein the host cell is capable of producing (S)-canadine from norlaudanosoline.

[0320] Embodiment 58. The host cell of embodiment 46, wherein the host cell further comprises one or more heterologous coding sequences of one or more heterologous transporters selected from CjABCB1, CjABCB2, and CjABCB2.

[0321] Embodiment 59. The host cell of embodiment 46, wherein the host cell is a yeast strain.

[0322] Embodiment 60. The host cell of embodiment 46, wherein the host cell is capable of producing berberine from reticuline via the biosynthetic pathway of Figure 5.

[0323] Embodiment 61. The host cell according to embodiment 46, wherein the host cell is a host cell according to one of embodiments 1 to 15.

[0324] Embodiment 62. The host cell of embodiment 46, wherein the host cell further comprises one or more heterologous coding sequences for one or more methyltransferases selected from 6OMT, CNMT, and 4'OMT, wherein the one or more methyltransferases are obtained from a different biological source compared to the host cell.

[0325] Embodiment 63. A host cell that produces thebaine, wherein the host cell comprises one or more heterologous coding sequences for one or more enzymes selected from SalSyn, CYP2D6, CYP2D2, SalR, and SalAT, wherein the one or more enzymes are obtained from a biological source different from the host cell.

[0326] Embodiment 64. The host cell according to embodiment 63, wherein the biological source is Papaver somniferum, Papaver somniferum, Papaver somniferum, Papaver somniferum, Papaver somniferum, Papaver somniferum, Homo sapiens, or Rattus somniferum.

[0327] Embodiment 65 The host cell of embodiment 63, wherein the host cell comprises multiple copies of one or more heterologous coding sequences.

[0328] Embodiment 66 The host cell of embodiment 65, wherein the multiple copies of the one or more heterologous coding sequences are obtained from two or more different biological sources compared to the host cell.

[0329] Embodiment 67. The host cell of embodiment 63, wherein the host cell comprises a heterologous coding sequence for each of the enzymes SalR and SalAT, and a heterologous coding sequence for one or more enzymes selected from SalSyn, CYP2D2 and CYP2D6.

[0330] Embodiment 68 The host cell of embodiment 67, wherein the host cell comprises a heterologous coding sequence of Papaver somniferum SalR having an F104A or I275A mutation.

[0331] Embodiment 69. The host cell of embodiment 63, wherein the host cell further comprises an enzyme capable of producing, from thebaine, one or more opiate compounds selected from oripavine, morphine, codeine, hydromorphone, hydrocodone, oxycodone, and oxymorphone.

[0332] Embodiment 70 The host cell of embodiment 69, wherein the host cell further comprises heterologous coding sequences for T6ODM and morB-E160G, and wherein the one or more opiate compounds is hydrocodone.

[0333] Embodiment 71. The host cell of embodiment 63, wherein the host cell is an engineered yeast strain that has been modified to produce increased amounts of NADPH compared to a native yeast strain.

[0334] Embodiment 72 The host cell of embodiment 63, wherein the host cell is a yeast strain.

[0335] Embodiment 73. The host cell of embodiment 72, wherein the host cell comprises SalR and SalAT enzymes localized in an organelle within the yeast cell.

[0336] Embodiment 74. The host cell according to embodiment 63, wherein the host cell is a host cell according to one of embodiments 1 to 15.

[0337] Embodiment 75. The host cell of embodiment 63, wherein the host cell further comprises one or more heterologous coding sequences for one or more methyltransferases selected from 6OMT, CNMT, and 4'OMT, and wherein the one or more methyltransferases are obtained from a different biological source compared to the host cell.

[0338] Embodiment 76. A host cell that produces an opiate compound, comprising: one or more heterologous coding sequences for one or more enzymes obtained from a different biological source compared to the host cell, selected from T6ODM, COR, and CODM; and One or more heterologous coding sequences for one or more enzymes selected from Pseudomonas putida morA and Pseudomonas putida morB.

[0339] Embodiment 77. The host cell of embodiment 76, wherein the host cell comprises four or more heterologous coding sequences.

[0340] Embodiment 78. The host cell according to embodiment 76, wherein the biological source is selected from Papaver somniferum, Papaver species, and Pseudomonas putida.

[0341] Embodiment 79. The host cell of embodiment 76, wherein the host cell comprises multiple copies of one or more heterologous coding sequences.

[0342] Embodiment 80 The host cell of embodiment 79, wherein the multiple copies of the heterologous coding sequence are obtained from two or more different biological sources as compared to the host cell.

[0343] Embodiment 81 The host cell of embodiment 76, wherein the host cell comprises heterologous coding sequences for T6ODM, COR, CODM, and morB.

[0344] Embodiment 82 The host cell of embodiment 76, wherein the host cell comprises heterologous coding sequences for the enzymes T6ODM, COR, and CODM.

[0345] Embodiment 83 The host cell of embodiment 82, wherein the heterologous coding sequences for the enzymes T6ODM, COR and CODM are present in a ratio of 2:1:3.

[0346] Embodiment 84. The host cell of embodiment 82, wherein the heterologous coding sequences for the enzymes T6ODM, COR and CODM are present in a ratio of 1:1:3 or 2:1:2.

[0347] Embodiment 85 The host cell of embodiment 76, wherein the opiate compound is selected from codeine, morphine, hydrocodone, hydromorphone, oxycodone, dihydrocodeine, 14-hydroxycodeine, and dihydromorphine.

[0348] Embodiment 86 The host cell of embodiment 76, wherein the host cell is capable of producing an opiate compound from thebaine via the biosynthetic pathway of Figure 15.

[0349] Embodiment 87. The host cell of embodiment 86, wherein the host cell produces little or no oripavine or morphinone from thebaine.

[0350] Embodiment 88. The host cell of embodiment 86, wherein the host cell produces one or more of neopine and neomorphine.

[0351] Embodiment 89 The host cell of embodiment 88, wherein the host cell produces an opiate compound yield that is 30% or more of total opiate.

[0352] Embodiment 90 The host cell of embodiment 89, wherein the host cell produces an opiate compound yield that is 50% or more of total opiate.

[0353] Embodiment 91 The host cell of embodiment 76, wherein the host cell is a yeast strain.

[0354] Embodiment 92. The host cell of embodiment 91, wherein the host cell is an engineered yeast strain capable of producing increased amounts of 2-oxoglutarate compared to a control yeast strain.

[0355] Embodiment 93. The host cell of embodiment 92, wherein the host cell comprises one or more endogenous or heterologous coding sequences for one or more proteins selected from GLN1, GLT1, GDH1, GDH2, GDH3, ODC1, ODC2, KGD1, KGD2, and LPD1.

[0356] Embodiment 94. The host cell of embodiment 93, wherein the host cell comprises a deletion in one or more endogenous coding sequences for one or more proteins selected from GLN1, GLT1, GDH1, GDH2, GDH3, ODC1, ODC2, KGD1, KGD2, and LPD1.

[0357] Embodiment 95. The host cell of embodiment 91, wherein the host cell further comprises an increased amount of 2-oxoglutarate compared to a control yeast strain, and wherein the increased amount of 2-oxoglutarate is introduced via direct addition to a culture medium of the host cell.

[0358] Embodiment 96. The host cell of embodiment 91, wherein the host cell further comprises increased amounts of glutamine, 2-oxoglutarate, and glutamic acid compared to a control yeast strain.

[0359] Embodiment 97 The host cell of embodiment 76, wherein the one or more enzymes comprise a localization tag.

[0360] Embodiment 98. The host cell of embodiment 91, wherein the one or more enzymes are spatially localized to a compartment within the yeast cell, the compartment being selected from mitochondria, endoplasmic reticulum (ER), Golgi, vacuole, nucleus, plasma membrane, and periplasm.

[0361] Embodiment 99. The host cell of embodiment 98, wherein the one or more enzymes are spatially localized outside of a compartment within the yeast cell.

[0362] Embodiment 100. The host cell of embodiment 98, wherein the one or more enzymes are spatially localized inside a compartment within the yeast cell.

[0363] Embodiment 101 The host cell of embodiment 91, wherein the one or more enzymes is COR.

[0364] Embodiment 102. The host cell of embodiment 91, wherein the one or more enzymes is T6ODM.

[0365] Embodiment 103 The host cell of embodiment 91, wherein the host cell comprises a COR enzyme and a T6ODM enzyme that are spatially separated from each other within the cell.

[0366] Embodiment 104 The host cell according to embodiment 101, wherein the COR enzyme is localized to the endoplasmic reticulum within the yeast cell.

[0367] Embodiment 105 The host cell of embodiment 76, wherein the host cell comprises heterologous coding sequences for morB and T6ODM, and the opiate compounds are hydrocodone and oxycodone.

[0368] Embodiment 106 The host cell of embodiment 76, wherein the host cell comprises the enzyme morB, which comprises an E160G mutation.

[0369] Embodiment 107. The host cell of embodiment 76, wherein the host cell comprises heterologous coding sequences for morA, morB, CODM, and T6ODM, and the opiate compound is selected from hydrocodone, oxycodone, and hydromorphone.

[0370] Embodiment 108. The host cell of embodiment 107, wherein the host cell comprises the enzyme morB comprising the E160G mutation.

[0371] Embodiment 109. The host cell according to embodiment 76, wherein the host cell comprises two or more gene cassettes, each cassette comprising a promoter, a gene and a terminator, and the gene cassettes are arranged in pairs with back-to-back promoter designs.

[0372] Embodiment 110. The host cell of embodiment 76, wherein the host cell further comprises one or more gene deletions compared to a native host cell, and the one or more deleted genes are selected from PDR1, STB5, PDR3, PDR5, SNQ2, YOR1, TPO1, TPO2, TPO3, TPO4, PDR10, PDR11, PDR15, PDR16, PDR17, QDR1, QDR2, QDR3, FLR1, AQR1, AQR2, and CIN5.

[0373] Embodiment 111. The host cell according to embodiment 76, wherein the host cell is a host cell according to one of embodiments 1 to 15 and 62 to 75.

[0374] Embodiment 112. The host cell of embodiment 76, wherein the host cell further comprises one or more heterologous coding sequences for one or more methyltransferases selected from 6OMT, CNMT, and 4'OMT, and wherein the one or more methyltransferases are obtained from a different biological source compared to the host cell.

[0375] Embodiment 113. Culturing a host cell selected from a reticuline-producing host cell, a sanguinarine or sanguinarine precursor-producing host cell, a protoberberine-producing host cell, a thebaine-producing host cell, and an opiate-producing host cell under conditions suitable for the production of a protein; adding a starting compound to the cell culture; and Recovering BIAs from the cell culture 1. A method for preparing a benzylisoquinoline alkaloid (BIA), comprising:

[0376] Embodiment 114 The method of embodiment 113, wherein the host cell is a cell according to embodiment 1, the starting compound is selected from norlaudanosoline and norcoclaurine, and the BIA is reticuline.

[0377] Embodiment 115. The method of embodiment 113, wherein the host cell is a cell according to embodiment 16, the starting compound is reticuline, norlaudanosoline, or norcoclaurine, and the BIA is sanguinarine or a sanguinarine precursor.

[0378] Embodiment 116. The method of embodiment 115, wherein the sanguinarine precursor is selected from cheilanthifoline, stylopine, cis-N-methylstylopine, scoureline, protopine, and dihydrosanguinarine.

[0379] Embodiment 117 The method of embodiment 115, wherein the host cell is cultured at low temperature in a highly ventilated container.

[0380] Embodiment 118 The method of embodiment 113, wherein the host cell is a cell of embodiment 46, the starting compound is reticuline, norlaudanosoline, or norcoclaurine, and the BIA is a protoberberine alkaloid.

[0381] Embodiment 119 The method of embodiment 118, wherein the BIA is (S)-canadine and the starting compound is norlaudanosoline.

[0382] Embodiment 120 The method of embodiment 118, wherein the BIA is berberine and the starting compound is reticuline.

[0383] Embodiment 121. The method of embodiment 118, wherein the protoberberine alkaloid is represented by one of the following structures: TIFF2026015382000017.tif46128 formula, R1~R 14 are each independently selected from H, alkyl, hydroxyl, or alkoxy.

[0384] Embodiment 122 The method of embodiment 113, wherein the host cell is the cell of embodiment 63, the starting compound is reticuline, norlaudanosoline, or norcoclaurine, and the BIA is thebaine.

[0385] Embodiment 123 The method of embodiment 122, further comprising producing from thebaine one or more opiate compounds selected from oripavine, morphine, codeine, hydromorphone, hydrocodone, oxycodone, and oxymorphone.

[0386] Embodiment 124 The method of embodiment 113, wherein the host cell is the cell of embodiment 76, the starting compound is reticuline, norlaudanosoline, norcoclaurine, or thebaine, and the BIA is an opiate compound.

[0387] Embodiment 125. The method of embodiment 124, wherein the opiate compound is selected from codeine, morphine, hydrocodone, hydromorphone, oxycodone, dihydrocodeine, 14-hydroxycodeine, and dihydromorphine.

[0388] Embodiment 126 The method of embodiment 124, wherein the host cell produces little or no oripavine or morphinone.

[0389] Embodiment 127. The method of embodiment 124, wherein the host cell is cultured in a medium comprising one or more of glutamine, 2-oxyglutaric acid, and glutamic acid.

[0390] Embodiment 128 The method of embodiment 124, wherein the host cell is cultured in a medium containing about 2% or less dimethyl sulfoxide (DMSO) to enhance the exchange of metabolites with the culture medium.

[0391] Embodiment 129. The method of embodiment 124, wherein the amount of BIA recovered is enhanced compared to host cells cultured in a control medium that excludes one or more of glutamine, 2-oxoglutarate, and glutamate.

[0392] Embodiment 130. The method of embodiment 113, wherein the host cell is the cell of embodiment 75, the starting compound is selected from codeine and morphine, and the BIA is an opioid selected from hydromorphone, hydrocodone, oxycodone, dihydrocodeine, 14-hydroxycodeine, and dihydromorphine.

[0393] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of the present invention that certain changes or modifications can be made thereto without departing from the spirit or scope of the appended claims.

[0394] Thus, the foregoing merely illustrates the principles of the present invention. It will be understood that those skilled in the art will be able to devise various arrangements, not expressly described or shown herein, which embody the principles of the present invention and are within its spirit and scope. Furthermore, the language of all examples and conditions recited herein is primarily intended to aid the reader in understanding the principles of the present invention and concepts provided by the inventors to further the art, and should not be construed as being limited to such specifically recited examples and conditions. Furthermore, all statements herein reciting principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Furthermore, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention are embodied by the appended claims.

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Claims

1. A host cell that produces a BIA compound or a precursor thereof, the host cell comprising multiple copies of one or more heterologous coding sequences for one or more enzymes obtained from a different biological source as compared to the host cell.

2. 10. The host cell of claim 1, comprising two or more enzymes obtained from two or more different biological sources as compared to the host cell.

3. 2. The host cell of claim 1, wherein the host cell is capable of producing a BIA compound or a precursor thereof from a starting material via one of the biosynthetic pathways of Figures 2, 3, 4, 11 and 15, and the starting material is selected from norlaudanosoline, norcoclaurine, reticuline and thebaine.

4. 2. The host cell of claim 1, which is a yeast strain.

5. 10. The host cell of claim 1, comprising one or more heterologous or endogenous coding sequences for one or more proteins involved in the transport of compounds across the cell membrane.

6. 2. The host cell of claim 1, wherein the one or more enzymes comprise a localization tag and are spatially localized to a compartment within the yeast cell, the compartment being selected from mitochondria, endoplasmic reticulum (ER), Golgi, vacuole, nucleus, plasma membrane, and periplasm.

7. The host cell of claim 1, wherein the BIA compound or precursor thereof is reticuline, and the host cell comprises one or more heterologous coding sequences for one or more methyltransferases selected from 6OMT, CNMT, and 4'OMT.

8. 2. The host cell of claim 1, wherein the BIA compound or precursor thereof is sanguinarine or a sanguinarine precursor, and the host cell comprises one or more heterologous coding sequences for one or more enzymes selected from BBE, CFS, CPR, STS, TNMT, MSH, P6H, and DBOX, and the sanguinarine precursor is selected from cheilanthifoline, stylopine, cis-N-methylstylopine, scoulerine, protopine, and dihydrosanguinarine.

9. 2. The host cell of claim 1, wherein the BIA compound or precursor thereof is a protoberberine alkaloid, and the host cell comprises one or more heterologous coding sequences for one or more enzymes selected from BBE, S9OMT, CAS, and STOX, and the protoberberine alkaloid is represented by one of the following structures: In the formula, R 1 ~R 14 are each independently selected from H, alkyl, hydroxyl, or alkoxy.

10. 2. The host cell of claim 1, wherein the BIA compound or precursor thereof is thebaine, and the host cell comprises one or more heterologous coding sequences for one or more enzymes selected from SalSyn, CYP2D6, CYP2D2, SalR, and SalAT.

11. the BIA compound or its precursor is an opiate compound, and the host cell one or more heterologous coding sequences for one or more enzymes obtained from a different biological source compared to the host cell, selected from T6ODM, COR, and CODM; and One or more heterologous coding sequences for one or more enzymes selected from Pseudomonas putida morA and Pseudomonas putida morB. Including, the opiate compound is selected from codeine, morphine, hydrocodone, hydromorphone, oxycodone, dihydrocodeine, 14-hydroxycodeine, and dihydromorphine; The host cell of claim 1.

12. The host cell of claim 11, wherein the heterologous coding sequences for the enzymes T6ODM, COR and CODM are present in a ratio of 2:1:3, 1:1:3, or 2:1:

2.

13. further comprising an increased amount of one or more of glutamine, 2-oxoglutarate, and glutamic acid compared to a control yeast strain; does not produce any oripavine or morphinone from thebaine; and can produce a yield of opiate compounds that is 30% or more of total opiates; The host cell of claim 11.

14. A host cell that produces reticuline, wherein the host cell contains multiple copies of one or more heterologous coding sequences for one or more methyltransferases obtained from a different biological source compared to the host cell, and the one or more methyltransferases are selected from 6OMT, CNMT and 4'OMT.

15. A host cell that produces sanguinarine or a sanguinarine precursor, wherein the host cell comprises one or more heterologous coding sequences for one or more enzymes obtained from a different biological source compared to the host cell, wherein the one or more enzymes are selected from BBE, CFS, CPR, STS, TNMT, MSH, P6H and DBOX, and the sanguinarine precursor is selected from cheilanthifoline, stylopine, cis-N-methylstylopine, scoulerine, protopine, and dihydrosanguinarine.

16. 1. A host cell that produces a protoberberine alkaloid, the host cell comprising one or more heterologous coding sequences for one or more enzymes obtained from a different biological source compared to the host cell, the one or more enzymes being selected from BBE, S9OMT, CAS, and STOX, and the protoberberine alkaloid being represented by one of the following structures: In the formula, R 1 ~R 14 are each independently selected from H, alkyl, hydroxyl, or alkoxy.

17. A host cell that produces thebaine, wherein the host cell comprises one or more heterologous coding sequences for one or more enzymes obtained from a different biological source compared to the host cell, and the one or more enzymes are selected from SalSyn, CYP2D6, CYP2D2, SalR, and SalAT.

18. A host cell that produces an opiate compound, the host cell comprising one or more heterologous coding sequences for one or more enzymes obtained from a different biological source compared to the host cell, selected from T6ODM, COR, and CODM; and one or more heterologous coding sequences for one or more enzymes selected from Pseudomonas putida morA and Pseudomonas putida morB; Including, the opiate compound is selected from codeine, morphine, hydrocodone, hydromorphone, oxycodone, dihydrocodeine, 14-hydroxycodeine, and dihydromorphine; The host cell.

19. Culturing the host cell of claim 1 under conditions suitable for protein production; adding a starting compound to the cell culture; and Harvesting the BIAs from the cell culture Including, The host cells are cultured in a medium containing one or more of glutamine, 2-oxoglutarate, glutamic acid, and 2% or less dimethyl sulfoxide (DMSO); Method for preparing benzylisoquinoline alkaloids (BIA).

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